Clinical trial NCT03378635
Conditions: Hypoglycemia Diabetes Mellitus, Type 1
Keywords: Dasiglucagon Glucagon analog
Sponsor: Zealand Pharma
Start date: 2017-12-07; Completion date: 2018-05-25
Enrollment: 170 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The objective of the trial is to demonstrate superiority of dasiglucagon compared to placebo following a single subcutaneous dose administered to subjects with type 1 diabetes mellitus (T1DM) with insulin-induced hypoglycemia. Additionally to compare the glycemic response observed after administration dasiglucagon with that of GlucaGen®.

Detailed description:
This was a global, multicenter, randomized, parallel, and double-blind clinical trial confirming the efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in patients with T1DM. The patients were randomized 2:1:1 to receive a single subcutaneous 0.6 mg dose of dasiglucagon, placebo, or a 1 mg dose of GlucaGen and followed for at least 28 days after receiving treatment.

Arms: arm 1: Single fixed dose (s.c.injection) of dasiglucagon arm 2: Single fixed dose (s.c.injection) of placebo arm 3: Single fixed dose (s.c.injection) of GlucaGen®
Interventions: intervention 1: Dasiglucagon intervention 2: GlucaGen intervention 3: Placebo
Intervention descriptions: intervention 1: Glucagon analog intervention 2: Native glucagon intervention 3: Placebo for dasiglucagon

Protocol documents:
Prot_000.pdf: 
Clinical Trial Report 
08 October 2019 
Protocol: ZP4207-16137 
Page 1 
Final 
CONFIDENTIAL 
 
 
16. APPENDICES 
16.1 
Study Information 
16.1.1 Protocol and Protocol Amendments 
The following protocols are provided in this Appendix:  
Clinical Study Protocols and Protocol Amendments: 
Version 
Date 
Final Version 5.0 
04 Apr 2018 
Final Version 4.0 (Germany only) 
26 Jan 2018 
Final Version 3.0 
06 Oct 2017 
Final Version 2.0 
31 Aug 2017 
Final Version 1.0 
29 Aug 2017 
 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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Page 1 of 82  
CLINICAL TRIAL PROTOCOL 
 
A phase 3, randomized, double-blind, parallel trial to 
confirm the clinical efficacy and safety of dasiglucagon 
in the rescue treatment of hypoglycemia in subjects with 
type 1 diabetes mellitus (T1DM) compared to placebo 
and with reference to GlucaGen® 
 
Sponsor: 
Zealand Pharma A/S 
Sponsor Protocol No.: 
ZP4207-16137 
EudraCT No.: 
2017-002449-31  
IND No: 
127866 
Trial Drug Name: 
Dasiglucagon* injection 
Date of Protocol: 
04-Apr-2018 
 
The trial will be conducted according to the protocol and in compliance with Good 
Clinical Practice (GCP), with the Declaration of Helsinki and with other applicable 
regulatory requirements. 
*Dasiglucagon is the proposed international nonproprietary name for ZP4207. 
Updated protocol, Final version 5.0 including: 
Protocol, Final version 3.0, dated 06-Oct-2017 
Local protocol amendment 1 (Germany), Final version 1.0, dated 26-Jan-2018 
Global protocol amendment 2, Final version 1.0, dated 04-Apr-2018 
The information in this document is confidential and is proprietary to Zealand Pharma. It is understood that 
information in this document shall not be disclosed to any third party, in any form, without prior written 
consent of an authorized officer of Zealand Pharma. 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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Page 2 of 82  
Declaration of sponsor or responsible medical officer 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
This trial protocol was subjected to critical review. The information it contains is 
consistent with current knowledge of the risks and benefits of the investigational product, 
as well as with the moral, ethical and scientific principles governing clinical research as 
set out in the Declaration of Helsinki and the guidelines on Good Clinical Practice (GCP) 
(1). 
 
 
_____________________________________ 
_____________________ 
Name  
 
 
 
Date 
Title 
 
Clinical Trial Manager 
Institution 
Zealand Pharma A/S 
Smedeland 36 
2600 Glostrup, Denmark 
Phone:  
 
 
_____________________________________ 
_____________________ 
Name  
 
 
 
Date 
Title 
 
Medical Officer 
Institution 
Zealand Pharma A/S 
Smedeland 36 
2600 Glostrup, Denmark 
Phone:  
 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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Strictly Confidential  
 
 
Page 3 of 82  
Declaration of the coordinating investigator 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
I have read this protocol. I approve this document and I agree that it contains all 
necessary details for carrying out the trial as described. I will conduct this trial in 
accordance with the design and specific provision of this protocol and will make a 
reasonable effort to complete the trial within the time designated. I will provide copies of 
this protocol and access to all information furnished by Zealand Pharma A/S to trial 
personnel under my supervision. I will discuss this material with them to ensure they are 
fully informed about the trial product and trial procedures. I will let them know that this 
information is confidential and proprietary to Zealand Pharma A/S and that it may not be 
further disclosed to third parties. I understand that the trial may be terminated or 
enrollment suspended at any time by Zealand Pharma A/S, with or without cause, or by 
me if it becomes necessary to protect the best interests of the trial subjects. 
I agree that the trial will be carried out in accordance with GCP (1), with the Declaration 
of Helsinki (with amendments) (2) and with the laws and regulations of the countries in 
which the trial takes place. 
 
 
_____________________________________ 
_____________________ 
Name  
 
 
 
 
 
Date 
Title 
Institution 
Phone: + 
Fax: 
+ 
 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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Strictly Confidential  
 
 
Page 4 of 82  
Declaration of the investigator 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
All documentation for this trial that is supplied to me and that has not been previously 
published will be kept in the strictest confidence. This documentation includes this trial 
protocol, Investigator's Brochure, Case Report Forms, and other scientific data. 
The trial will not be commenced without the prior written approval of a properly 
constituted Institutional Review Board (IRB) or Independent Ethics Committee (IEC). 
No changes will be made to the trial protocol without the prior written approval of the 
sponsor and the IRB or IEC, except where necessary to eliminate an immediate hazard to 
the subjects. 
I have read and understood and agree to abide by all the conditions and instructions 
contained in this protocol. 
 
_____________________________________ 
_____________________ 
Signature 
 
 
 
 
 
Date 
 
_____________________________________ 
Name (block letters) 
 
_____________________________________ 
Title (block letters) 
 
_____________________________________ 
Institution (block letters) 
 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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Table of contents 
DECLARATION OF SPONSOR OR RESPONSIBLE MEDICAL OFFICER .......... 2 
DECLARATION OF THE COORDINATING INVESTIGATOR .............................. 3 
DECLARATION OF THE INVESTIGATOR ............................................................... 4 
TABLE OF CONTENTS .................................................................................................. 5 
1 
SYNOPSIS ............................................................................................................ 11 
2 
INTRODUCTION................................................................................................ 15 
2.1 
Background ...................................................................................................... 15 
2.1.1 
Hypoglycemia ...................................................................................... 15 
2.1.2 
Glucagon .............................................................................................. 16 
2.1.3 
Dasiglucagon........................................................................................ 16 
2.1.3.1 
Pharmacokinetics and pharmacodynamics of dasiglucagon ......... 17 
2.1.3.2 
Safety of dasiglucagon ................................................................... 17 
2.2 
Trial rationale .................................................................................................. 18 
2.3 
Risk-benefit assessment .................................................................................. 18 
3 
TRIAL OBJECTIVES AND ENDPOINTS....................................................... 20 
3.1 
Primary objective ............................................................................................ 20 
3.2 
Secondary objectives ....................................................................................... 20 
3.3 
Primary endpoint ............................................................................................ 20 
3.4 
Key secondary endpoints ................................................................................ 20 
3.5 
Other secondary endpoints ............................................................................. 20 
3.6 
Exploratory endpoint ...................................................................................... 21 
4 
OVERALL DESIGN AND PLAN OF THE TRIAL ........................................ 22 
4.1 
Overview .......................................................................................................... 22 
4.2 
Justification of the trial design ....................................................................... 24 
4.2.1 
Justification for design and parameters................................................ 24 
4.2.2 
Justification for drug, route, dosage and treatment duration ............... 24 
5 
TRIAL POPULATION ....................................................................................... 26 
5.1 
Rationale for trial population ........................................................................ 26 
5.2 
Planned sample size and number of trial centers ......................................... 26 
5.3 
Inclusion criteria ............................................................................................. 26 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
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Protocol Version: Final 5.0 
 
 
 
 
 
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5.4 
Exclusion criteria ............................................................................................ 27 
5.5 
Dosing day exclusion criteria ......................................................................... 28 
5.6 
Premature treatment discontinuation and withdrawal ............................... 29 
5.6.1 
Possible reasons for treatment visit discontinuation ............................ 29 
5.6.2 
Center discontinuation ......................................................................... 30 
5.6.3 
Trial termination .................................................................................. 30 
5.7 
Subject identification and randomization ..................................................... 30 
6 
TRIAL DRUG ...................................................................................................... 32 
6.1 
Identity ............................................................................................................. 32 
6.2 
Treatment assignment and randomization ................................................... 33 
6.3 
Administration ................................................................................................. 34 
6.4 
Packaging and labelling .................................................................................. 34 
6.5 
Storage of study drugs .................................................................................... 34 
6.6 
Blinding and breaking the blind .................................................................... 35 
6.7 
Drug accountability ......................................................................................... 36 
6.8 
Treatment compliance .................................................................................... 36 
6.9 
Prior and concomitant medications ............................................................... 36 
6.9.1 
Prohibited medications......................................................................... 37 
6.10 
Local German requirements for insulin glulisine (Apidra®) ........................ 37 
7 
PARAMETERS AND METHODS OF ASSESSMENT .................................. 38 
7.1 
Efficacy parameters ........................................................................................ 38 
7.1.1 
Pharmacodynamic measurements ........................................................ 38 
7.1.2 
Pharmacokinetic measurements ........................................................... 38 
7.2 
Safety parameters ............................................................................................ 39 
7.2.1 
Adverse events ..................................................................................... 39 
7.2.1.1 
Definitions ...................................................................................... 39 
7.2.1.2 
Collection, recording and reporting of adverse events ................. 42 
7.2.1.3 
Follow-up of adverse events .......................................................... 44 
7.2.1.4 
Clinical laboratory abnormalities and other abnormal 
assessments as adverse events or serious adverse events .............. 44 
7.2.2 
Hypoglycemia ...................................................................................... 45 
7.2.3 
Physical examination ........................................................................... 45 
7.2.4 
Vital signs ............................................................................................ 45 
7.2.5 
Electrocardiogram ................................................................................ 46 
7.2.6 
Local tolerability .................................................................................. 46 
7.2.7 
Clinical laboratory assessments ........................................................... 47 
7.2.8 
Pregnancy ............................................................................................. 48 
7.2.9 
Precautions ........................................................................................... 49 
7.2.10 
Safety committee ................................................................................. 49 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
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Protocol Version: Final 5.0 
 
 
 
 
 
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7.3 
Demography, concomitant illness, medical history and concomitant 
medication ........................................................................................................ 49 
7.3.1 
Demography and body measurements ................................................. 50 
7.3.2 
Concomitant illness and medical history ............................................. 50 
7.3.3 
Diabetes diagnosis and current treatment ............................................ 50 
7.3.4 
Concomitant medication ...................................................................... 51 
7.4 
Other assessments ........................................................................................... 51 
7.4.1 
Immunogenicity ................................................................................... 51 
7.4.2 
Plasma glucose measurements for safety ............................................. 52 
7.4.3 
Plasma insulin measurements .............................................................. 52 
8 
TRIAL CONDUCT .............................................................................................. 53 
8.1 
Schedule of procedures ................................................................................... 53 
8.2 
Procedures by visit .......................................................................................... 53 
8.2.1 
Visit 1 (screening, Day -30 to Day -3) ................................................. 53 
8.2.2 
Instructions to subjects prior to the dosing visit (Visit 2) .................... 55 
8.2.3 
Visit 2 (dosing visit)............................................................................. 56 
8.2.3.1 
Hypoglycemic clamp procedure and administration of trial 
medication ...................................................................................... 57 
8.2.4 
Visit 3 (follow-up visit) ....................................................................... 60 
9 
STATISTICAL METHODS ............................................................................... 61 
9.1 
Determination of sample size ......................................................................... 61 
9.2 
Trial subjects ................................................................................................... 61 
9.2.1 
Analysis samples .................................................................................. 61 
9.2.2 
Disposition of subjects ......................................................................... 62 
9.2.3 
Protocol deviations............................................................................... 62 
9.3 
General considerations ................................................................................... 63 
9.4 
Demographics and baseline characteristics .................................................. 63 
9.5 
Efficacy Analyses ............................................................................................. 63 
9.5.1 
Hierarchical testing procedure ............................................................. 64 
9.5.2 
Primary confirmatory endpoint ............................................................ 64 
9.5.2.1 
Primary analysis ............................................................................ 64 
9.5.3 
Secondary endpoints ............................................................................ 65 
9.5.3.1 
Confirmatory analysis .................................................................... 65 
9.5.4 
Secondary clinical efficacy (PD) endpoints ......................................... 66 
9.5.4.1 
Analysis of secondary clinical efficacy (PD) endpoints ................ 66 
9.5.5 
Exposure (PK) endpoints ..................................................................... 66 
9.5.5.1 
Analysis of exposure (PK) endpoints ............................................. 67 
9.6 
Exploratory analyses ....................................................................................... 67 
9.7 
Safety analyses ................................................................................................. 68 
9.7.1 
Adverse events ..................................................................................... 68 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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9.7.2 
Immunogenicity data ........................................................................... 68 
9.7.3 
Clinical laboratory assessments ........................................................... 68 
9.7.4 
Other safety data .................................................................................. 69 
9.8 
Treatment compliance .................................................................................... 69 
9.9 
Subject withdrawals and missing data .......................................................... 69 
9.10 
Interim analyses .............................................................................................. 70 
10 
ETHICAL, LEGAL, AND ADMINISTRATIVE ASPECTS .......................... 71 
10.1 
Quality assurance ............................................................................................ 71 
10.2 
Electronic case report forms .......................................................................... 72 
10.3 
Access to source data ...................................................................................... 72 
10.4 
Source documentation .................................................................................... 73 
10.5 
Data processing ................................................................................................ 73 
10.6 
Archiving trial records ................................................................................... 73 
10.7 
Good clinical practice ..................................................................................... 73 
10.8 
Informed consent ............................................................................................. 74 
10.9 
Protocol approval and amendment ............................................................... 74 
10.10 Duration of the trial ........................................................................................ 75 
10.11 Premature termination of the trial ................................................................ 75 
10.12 Confidentiality ................................................................................................. 75 
10.13 Other ethical and regulatory issues ............................................................... 76 
10.14 Liability and insurance ................................................................................... 76 
10.15 Publication policy ............................................................................................ 77 
11 
REFERENCE LIST ............................................................................................. 78 
12 
APPENDICES ...................................................................................................... 81 
12.1 
List of trial personnel ...................................................................................... 81 
 
Tables in text 
Table 1 
Description of trial drugs .................................................................................. 32 
Table 2 
Quantities of ingredients in dasiglucagon and placebo injection ...................... 33 
Table 3 
Schedule of procedures ..................................................................................... 54 
Table 4 
Post-treatment blood sampling times ................................................................ 59 
 
Figures in text 
Figure 1 Trial design ........................................................................................................ 23 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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Strictly Confidential  
 
 
Page 9 of 82  
List of abbreviations and definitions of terms 
ADA 
Anti-drug antibody 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALT 
Alanine aminotransferase 
ANCOVA Analysis of covariance 
AST 
Aspartate aminotransferase 
AUC 
Area under the concentration-time curve 
AUC0-inf 
AUC from time zero to infinity 
AUE 
Area under the effect curve  
CFB 
Changes from baseline  
CI 
Confidence interval 
Cmax 
Maximum plasma concentration 
CPH 
Cox proportional hazards 
CRO 
Contract research organization  
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
EU 
European Union 
EudraCT 
European Medicines Agency’s Clinical Trials Database 
FAS 
Full analysis set 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HbA1c 
Glycated hemoglobin 
ICH 
International Conference on Harmonization 
ID card 
Identification card 
IEC 
Independent Ethics Committee 
IM 
Intramuscular(ly) 
IMP 
Investigational medicinal product 
IRB 
Institutional Review Board 
IV 
Intravenous(ly) 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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IWRS 
Interactive Web Response System 
MedDRA 
Medical Dictionary for Regulatory Activities 
NPH 
Neutral protamine Hagedorn 
PD 
Pharmacodynamics 
PK 
Pharmacokinetics 
PP 
Per protocol 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SAS 
Safety analysis set 
SC 
Subcutaneous(ly) 
SUSAR 
Serious unexpected suspected adverse reaction 
T1DM 
Type 1 diabetes mellitus 
T2DM 
Type 2 diabetes mellitus 
TEAE 
Treatment-emergent adverse event 
tmax 
Time to the maximum plasma concentration 
ULN 
Upper limit of the normal range 
US 
United States of America 
 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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1 
SYNOPSIS 
Name of sponsor: 
Zealand Pharma A/S 
Trial ID: 
ZP4207-16137 
Title of the trial: 
A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy and safety of 
dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 1 diabetes mellitus (T1DM) 
compared to placebo and with reference to GlucaGen® 
Trial design:  
The trial is a global, multicenter, randomized, parallel-group, double-blind, clinical trial confirming the 
efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in subjects with type 1 diabetes 
mellitus. The subjects will be randomized 2:1:1 to receive a single fixed subcutaneous 0.6 mg dose of 
dasiglucagon (hereinafter dasiglucagon), placebo for dasiglucagon (hereinafter referred to as placebo), or a 
1 mg dose of GlucaGen® (hereafter referred to as GlucaGen) and followed for at least 28 days after 
treatment. 
Clinical phase of development: 
Phase 3 
Trial centers: 
This trial will be conducted at 4 to 6 sites in the United States of America, Canada, and Europe. 
Planned trial start (first subject first visit): 
Q4/2017 
Planned trial end (last subject last visit): 
Q3/2018  
Trial population:  
Male and female adult subjects with type 1 diabetes mellitus treated with insulin for at least one year  
Key objectives: 
Primary objective:  
 
To demonstrate superiority of dasiglucagon compared to placebo following a single subcutaneous 
0.6 mg dose administered to subjects with type 1 diabetes mellitus with insulin-induced 
hypoglycemia. 
Secondary objective:  
 
To compare the glycemic response observed after dasiglucagon with that of GlucaGen. 
Key endpoints:  
Primary endpoint:  
 
Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase in plasma 
glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic clamp procedure 
without administration of rescue intravenous (IV) glucose.  
Key secondary endpoints: 
 
Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, and within 
10 minutes after study drug injection without administration of rescue IV glucose. 
 
Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection or at the time of rescue. 
Clinical efficacy (Pharmacodynamic) endpoints: 
 
Time to first plasma glucose concentration of ≥70 mg/dL (3.9 mmol/L) without administration of 
rescue IV glucose. 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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Page 12 of 82  
 
Plasma glucose response as area under the curve above baseline from time zero to 30 minutes, 
AUC0-30min. 
Exposure (Pharmacokinetic) endpoints: 
 
Area under the drug concentration curve from time zero to 90 minutes, AUC0-90 min. 
 
Maximum plasma drug concentration (Cmax). 
 
Time to maximum plasma drug concentration (tmax). 
Safety endpoints:  
 
Adverse events, clinical laboratory assessments (biochemistry, hematology, urinalysis), vital signs, 
physical examination, electrocardiogram, and local tolerability. 
 
Administration of rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
Immunogenicity endpoint: 
 
Occurrence of anti-drug antibodies 
Exploratory endpoint: 
 
Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL (1.1 mmol/L) 
within 30 minutes after study drug injection without administration of rescue IV glucose. 
 
Plasma insulin response as area under the curve above baseline from time zero to 60 minutes, 
AUC0-60 min. 
Key inclusion criteria: 
 
Male and female subjects with type 1 diabetes mellitus treated with insulin for at least one year, 
diagnostic criteria as defined by the American Diabetes Association. 
 
Stable insulin treatment 30 days prior to screening, defined as no more than a 10-unit daily variation 
in total daily insulin dose. 
 
Hemoglobin A1c <10%. 
 
Aged between 18 and 75 years, both inclusive. 
Key exclusion criteria: 
 
Previously treated with dasiglucagon. 
 
Known or suspected allergy to trial product(s) or related products. 
 
History of anaphylaxis or symptoms of severe systemic allergy (such as angioedema). 
 
Previous participation (randomization) in this trial. 
Sample size: 
Approximately 156 subjects are intended to complete the trial, with 78 subjects randomized to the 
dasiglucagon group and 39 subjects randomized to each of the placebo and GlucaGen groups.  
Investigational medicinal product: 
Test product: dasiglucagon liquid formulation in pre-filled syringes. 
Comparator products: Placebo and GlucaGen® lyophilized powder. 
Germany only: Insulin glulisine (Apidra®), the challenge agent, will also be defined as an investigational 
medicinal product. 


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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Duration of treatment:  
Subjects will be randomized 2:1:1 to receive a single fixed subcutaneous dose of 0.6 mg dasiglucagon, 
placebo, or 1 mg GlucaGen, and followed for at least 28 days after receiving treatment.  
Assessments:  
The plasma glucose profile for evaluation of the primary and secondary clinical efficacy 
(pharmacodynamic) endpoints will be assessed based on plasma concentration data from samples collected 
at the dosing visit. 
The exposure to trial medication (dasiglucagon, placebo, or GlucaGen) for evaluation of pharmacokinetics 
will also be assessed based on plasma concentration data.  
Statistical methods:  
Due to requirements in the size of the safety database, the sample size is set to 78 subjects treated with 
dasiglucagon 0.6 mg. From phase 2, the median time to an increase of 20 mg/dL of the 0.6 mg dose was 
approximately 10 minutes.With a 2:1:1 randomization ratio for the three treatments, dasiglucagon 0.6 mg, 
placebo, and GlucaGen 1 mg, and assuming an exponential time-to-recovery distributions with median 
times of 10 and at least 20 minutes for dasiglucagon and placebo, respectively, a two-sided log-rank test 
will be able to detect a difference between dasiglucagon and placebo with 90% power with a follow-up time 
of 45 minutes at a 5% significance level with 78 subjects treated with dasiglucagon and 39 subjects with 
placebo. The median time-to-recovery for placebo is expected to be longer than 20 minutes, which means 
that the power will be greater than 90%. 
For the confirmatory analyses, the primary and key secondary endpoints for the dasiglucagon 0.6 mg and 
placebo treated subjects will be compared. A hierarchical procedure will be applied for the control of 
multiplicity in statistical inferences. The primary and secondary endpoints will be evaluated on the Full 
Analysis Set sample. The statistical inference comparisons with placebo will be conducted as superiority 
tests. The comparisons of dasiglucagon versus GlucaGen will be summarized descriptively.    
The primary endpoint will be summarized using Kaplan-Meier (KM) estimates stratified by treatment group 
and injection site. The treatment group difference between dasiglucagon and placebo will be evaluated 
inferentially using a pairwise two-sided log-rank test. 
In the primary analysis, recovery cannot be achieved in those subjects where IV glucose treatment is 
administered. Those subjects who receive IV glucose will be censored (i.e. set to ‘not recovered’) at 45 
minutes after dosing. 
In sensitivity analyses, the time to plasma glucose recovery will be analyzed 1) without censoring for those 
subjects who require rescue IV glucose before 45 minutes and 2) with censoring at the time of 
administration of rescue IV glucose before 45 minutes.   
The primary endpoint will additionally be analyzed using a Cox proportional hazards (CPH) time to event 
statistical model, if the hazards by treatment group can be assumed proportional using a graphical 
comparison of log-minus-log survival curves and no more than two-thirds of the recovery times are 
censored in each treatment group. The CPH model will be used for inferences, with treatment group and 
injection site modeled as categorical effects, and baseline plasma glucose modeled as a continuous 
covariate. The treatment group rate ratios, active versus placebo, will be estimated together with the 95% 
confidence intervals, and pairwise treatment group inferences (dasiglucagon vs placebo, GlucaGen vs 
placebo) will be evaluated using two-sided likelihood ratio tests.  


 
 
Dasiglucagon (ZP4207) 
Date: 04-Apr-2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 5.0 
 
 
 
 
 
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Strictly Confidential  
 
 
Page 14 of 82  
The key secondary incidence variables (plasma glucose recovery within 30 minutes, within 20 minutes, 
within 15 minutes, within 10 minutes, in hierarchical order for inference) will be analyzed using descriptive 
statistics (frequency and percentage) by treatment group. Inferential treatment group comparisons will be 
assessed via pairwise tests of independent binomial proportions (dasiglucagon versus placebo, GlucaGen 
versus placebo) using Fisher’s exact test for each pairwise comparison. 
The key secondary endpoints of plasma glucose changes from baseline (CFB) within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection or at the time of rescue, will 
be analyzed with the plasma glucose CFB at rescue carried forward in those subjects who require rescue IV 
glucose before plasma glucose ≥20 mg/dL recovery. Each of these CFB variables will be analyzed using an 
Analysis of Covariance model, with treatment group modeled as a fixed effect and with the baseline plasma 
glucose modeled as a covariate. The dasiglucagon versus placebo treatment group difference will be 
evaluated inferentially as a least squares means contrast, using a two-sided t-test at the 0.05 significance 
level, first for the 30 minute CFB, and then sequentially for the 20 minute, 15 minute, and 10 minute 
variables, until the first failure to reject. 
The continuous clinical efficacy variables, the exploratory variables, and the pharmacokinetics variables 
will each be summarized descriptively by treatment group. The clinical efficacy variables will be analyzed 
analogous to the plasma glucose CFB variables.   
The safety analyses will include by-treatment-group descriptive summaries of vital sign measurements, 
laboratory measures (including immunogenicity incidence), physical examination assessments, rescue IV 
glucose (incidence and amount of glucose infused), and adverse events. The number and percentage of 
subjects reporting specific events, such as nausea and vomiting, will be presented by body system and 
preferred term. 
Further details will be included in the Statistical Analysis Plan, to be completed before database lock and 
treatment unmasking.   


 
 
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2 
INTRODUCTION 
2.1 
Background 
Zealand Pharma A/S (Zealand Pharma) is developing dasiglucagon, a physically and 
chemically stable peptide analog of human glucagon, in a ready-to-use liquid formulation 
for the acute treatment of severe hypoglycemia in patients with insulin-treated diabetes 
mellitus. Like native glucagon, dasiglucagon is comprised of 29 amino acids, but with 
7 substitutions which improve its physical and chemical stability in aqueous media. 
These amino acid substitutions make dasiglucagon suitable for a liquid formulation, 
while providing similar efficacy and safety as compared with recombinant glucagon in 
the clinical setting of acute treatment of severe hypoglycemia. 
2.1.1 Hypoglycemia 
Hypoglycemia in patients with diabetes is defined as episodes of an abnormally low 
plasma glucose concentration (3). This is a common, unpredictable, and potentially 
dangerous side effect of treatment of diabetes mellitus, especially with insulin or 
sulfonylureas. It is more frequent in patients with profound endogenous insulin 
deficiency, such as occurs in type 1 diabetes mellitus (T1DM) and advanced type 2 
diabetes mellitus (T2DM). Treatment of T2DM with insulin causes hypoglycemia 
progressively and more frequently over time, whereas in T1DM, hypoglycemia is 
experienced throughout the course of established disease (4). 
Symptoms and signs of hypoglycemia are not specific. Patients undergoing a 
hypoglycemic episode experience unpleasant symptoms such as anxiety, sweating, 
hunger, tremors, palpitations, paresthesia, nausea and pallor. Depending on its severity, 
the hypoglycemia may lead to mild confusion, behavioral changes, loss of consciousness, 
seizures, coma, and death (5). 
The incidence of hypoglycemic events or even the fear of hypoglycemia influences 
patients’ adherence to prescribed treatment regimens for diabetes mellitus (6). This leads 
to inadequate glycemic control, which in turn may lead to an increased risk of diabetic 
complications (5). Serious clinically significant hypoglycemia is now defined as plasma 
glucose <54 mg/dL (3.0 mmol/L), while the plasma glucose alert value is defined as 
<70 mg/dL (3.9 mmol/L) (7). When plasma glucose falls below these values, some kind 
of treatment strategy is needed. 


 
 
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2.1.2 Glucagon 
Glucagon is a naturally occurring hormone, secreted from the alpha cells of the 
pancreatic islets. Glucagon plays a central role in the regulation of glucose homeostasis 
and is the counterpart of insulin for controlling blood glucose levels (i.e. it acts in 
opposition to insulin in terms of effects on blood glucose levels) (8,9). Glucagon 
stimulates hepatic glycogenolysis and gluconeogenesis in hypoglycemic states, thereby 
restoring glucose homeostasis. Glucagon receptor agonism has also been shown to exert 
effects on lipid metabolism, energy balance, body adipose tissue mass and food intake 
(10). Insulin decreases blood glucose levels and cases of hypoglycemia can be reversed 
by glucagon. Therefore, glucagon is indicated for the treatment of severe hypoglycemia. 
Besides intravenous (IV) glucose administration, an injectable form of glucagon is given 
as first aid in cases of severe hypoglycemia, when the patient is unconscious or for other 
reasons cannot take glucose orally. The approved glucagon dose for an adult is 1 mg, 
given by intramuscular (IM), IV, or subcutaneous (SC) injection, which quickly raises 
blood glucose levels. As current marketed recombinant glucagon is highly unstable when 
dissolved in solution, the injectable form has to be reconstituted prior to use in a 9-step 
procedure that requires a sterile diluent to be injected into a vial containing lyophilized 
powdered glucagon. When dissolved in a fluid state, glucagon can form amyloid fibrils 
(11), or tightly woven chains of proteins made up of the individual glucagon peptides. 
The reconstitution process makes the use of marketed glucagon products cumbersome 
(12), and a more patient-friendly formulation is needed. Currently, the Food and Drug 
Administration (FDA) approved instructions for commercially available glucagon allow 
only for immediate usage of glucagon after the powder is reconstituted in aqueous 
solution (13). Therefore, a glucagon analog with enhanced biophysical stability may 
represent a leap forward in terms of convenient therapeutic applications. 
2.1.3 Dasiglucagon 
Dasiglucagon (ZP4207) is a stable peptide analog of human glucagon, available in a 
ready-to-use liquid formulation. Dasiglucagon (hereinafter referred to as dasiglucagon) is 
in development for the treatment of severe hypoglycemia in insulin-dependent patients 
with diabetes mellitus. Dasiglucagon is a specific and full glucagon receptor agonist 
designed to mimic the effects of glucagon, having a fast absorption and elimination 
(minutes). It is a peptide of 29 amino acids, with 7 amino acid substitutions compared to 
native glucagon. The main purpose of the substitutions is to increase the physical and 
chemical stability of the glucagon analog compared to marketed glucagon products such 
as Lilly Glucagon or GlucaGen® (hereafter referred to as GlucaGen). Dasiglucagon 
exhibits improved physical and chemical stability and is available in an aqueous solution 
at neutral pH (14). 


 
 
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Three clinical trials have been completed with dasiglucagon: a first-in-human dose trial in 
healthy volunteers and subjects with T1DM (ZP4207-14013), a multiple-dose 
dose-escalation trial (ZP4207-15007) evaluating the safety, tolerability, pharmacokinetics 
(PK) and pharmacodynamics (PD) of dasiglucagon in healthy volunteers, and a phase 2 
crossover trial to assess the PK and PD of a single dose of an optimized formulation of 
dasiglucagon administered SC in subjects with T1DM (ZP4207-15126) (14). 
2.1.3.1 Pharmacokinetics and pharmacodynamics of dasiglucagon 
The results of the phase 1 and 2 clinical trials confirmed dose-proportionality for 
dasiglucagon PK, which is characterized by a fast absorption with a peak plasma 
concentration obtained after 35 minutes. Thereafter, the plasma concentration rapidly 
declines with an average half-life of 28 minutes. The median time to the maximum 
plasma concentration (tmax) was 35 minutes for dasiglucagon compared with 20 minutes 
for GlucaGen.  
At all dose levels in the phase 2 trial, all subjects achieved a plasma glucose level of at 
least 70 mg/dL (3.9 mmol/L) as well as an increase in plasma glucose by at least 
20 mg/dL (1.1 mmol/L) within 30 minutes after dosing. The PD responses to 0.6 mg of 
dasiglucagon and 1 mg of GlucaGen were comparable. 
2.1.3.2 Safety of dasiglucagon 
The safety data for dasiglucagon do not give rise to any safety concerns. No new signals 
were observed, beyond those related to the pharmacological effect of glucagon agonism. 
All adverse events (AEs) recorded in the trials were of mild or moderate severity. The 
most frequently reported systemic AE was nausea, which is a known side effect 
following administration of glucagon. Headache was the next most frequently reported 
event, occurring in all dose groups in the phase 2 trial. Injection site reactions were 
observed only sporadically after administration with either dasiglucagon or GlucaGen 
and all were mild and transient. The most frequent injection site reaction was erythema, 
occurring in all treatment groups, including the placebo group, irrespective of dose. 
Glucagon has been described to exert positive inotropic and chronotropic effects and may 
therefore cause tachycardia and hypertension. In the phase 1 clinical trials, but not the 
phase 2 trial, temporary but clinically significant decreases in blood pressure were 
observed in a few healthy volunteers receiving investigational medicinal product (IMP) 
doses of at least 1 mg (4 with dasiglucagon and 1 with GlucaGen). This is not considered 
a safety concern; however, hemodynamic changes after dosing will be considered an 
adverse event of special interest (AESI). 


 
 
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The phase 1 and 2 results and the safety profile described to date do not give rise to 
specific safety concerns. For further information, please refer to the Investigator’s 
Brochure (14). 
2.2 
Trial rationale 
The aim of the current trial is to confirm the superiority of dasiglucagon for the treatment 
of insulin-induced hypoglycemia in subjects with T1DM as compared to placebo for 
dasiglucagon (hereinafter placebo) and to compare the clinical efficacy and safety of 
dasiglucagon with reference to GlucaGen. A randomized, controlled trial design was 
used. 
See Section 4.2 for justification of the design of this trial. 
2.3 
Risk-benefit assessment  
Non-clinical experience 
The nonclinical development program did not reveal any safety findings that would 
prohibit administration of dasiglucagon to humans. None of the safety pharmacology 
studies, repeated dose toxicity studies or genotoxicity studies revealed any significant 
toxicity findings relevant to the therapeutic use of dasiglucagon. 
Clinical experience 
As glucagon and its analogs belong to a well-known drug class with a known mode of 
action, dasiglucagon is not expected to be a high-risk molecule.  
Treatment with an IMP may result in undesired effects or complaints. Undesired effects 
and complaints such as nausea, vomiting, and diarrhea are known AEs occurring with 
glucagon administration. Similar AEs have also been observed to a limited extent in the 
3 clinical studies conducted to date with dasiglucagon. As with every novel drug 
substance, new and as yet unknown side effects may also occur. 
There are limited data available to assess the immunogenic potential of glucagon 
products available on the market, but the data available indicate that marketed glucagon 
only has a low immunogenic potential.  
Overall, dasiglucagon is judged to be a low-risk molecule, based upon the available 
clinical data. Administration of dasiglucagon may be associated with a risk of allergic 
reactions similar to those observed for other therapeutic peptides or proteins. Subjects 
with known or suspected allergies to the trial medications or related products will be 


 
 
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excluded from the trial. Mild or moderate allergic reactions may include symptoms of 
rash, fever, flu-like symptoms, nausea, headache, and myalgia. Acute generalized 
hypersensitivity reactions are usually very rare, but may include symptoms of flushing, 
sweating, dizziness, change in blood pressure, and difficulties in breathing. No severe 
acute hypersensitivity reactions have been observed in the 3 clinical trials conducted with 
dasiglucagon. Direct access to resuscitation equipment is ensured at the clinical trial 
centers. 
With the exception of medical examinations, a subject participating in this trial is not 
likely to derive any personal health-related benefits. The results of the trial may 
contribute to the future use of dasiglucagon in patients with diabetes mellitus 
experiencing severe hypoglycemic reactions. 
The development program including 141 subjects exposed to dasiglucagon to date has 
demonstrated that administration of dasiglucagon is efficacious and well tolerated, with a 
safety profile that does not give rise to specific safety concerns. Two phase 1 and one 
phase 2 clinical trials have been conducted to investigate the safety, tolerability, PK and 
PD of dasiglucagon after single and multiple dosing to healthy volunteers and subjects 
with T1DM under insulin-induced hypoglycemic conditions. Dasiglucagon has proven to 
have relevant clinical effects in the acute severe hypoglycemia rescue setting and may be 
an effective and reliable emergency treatment for severe hypoglycemia. Overall, the 
anticipated benefits for subjects entering the ZP4207-16137 trial are considered to justify 
the risks. 


 
 
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3 
TRIAL OBJECTIVES AND ENDPOINTS 
3.1 
Primary objective 
 To demonstrate superiority of dasiglucagon compared to placebo following a single 
subcutaneous 0.6 mg dose administered to subjects with type 1 diabetes mellitus with 
insulin-induced hypoglycemia. 
3.2 
Secondary objectives 
 To compare the glycemic response observed after dasiglucagon with that of 
GlucaGen. 
3.3 
Primary endpoint 
 Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase 
in plasma glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic 
clamp procedure without administration of rescue IV glucose.  
3.4 
Key secondary endpoints 
 Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection without administration of rescue IV 
glucose. 
 Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection or at the time of rescue. 
3.5 
Other secondary endpoints 
 Clinical efficacy (PD) endpoints: 
- 
Time to first plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) without 
administration of rescue IV glucose. 
- 
Plasma glucose response as area under the curve (AUC) above baseline from time 
zero to 30 minutes, AUC0-30min. 
 Exposure (PK) endpoints: 
- 
Area under the drug concentration curve from time zero to 90 minutes, 
AUC0-90min. 
- 
 Maximum plasma drug concentration (Cmax). 
- 
Time to maximum plasma drug concentration (tmax). 


 
 
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 Safety endpoints:  
- 
Adverse events, clinical laboratory assessments (biochemistry, hematology, 
urinalysis), vital signs, physical examination, electrocardiogram (ECG), and local 
tolerability. 
- 
Administration of rescue infusion of IV glucose during the hypoglycemic clamp 
procedure. 
- 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp 
procedure. 
 Immunogenicity endpoint: 
- 
Occurrence of anti-drug antibodies 
3.6 
Exploratory endpoint 
 Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL 
(1.1 mmol/L) within 30 minutes after study drug injection without administration of 
rescue IV glucose. 
 Plasma insulin response as area under the curve above baseline from time zero to 
60 minutes, AUC0-60 min. 


 
 
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4 
OVERALL DESIGN AND PLAN OF THE TRIAL 
4.1 
Overview 
This trial is a global, multicenter, randomized, parallel, double-blind, clinical trial 
confirming the efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in 
subjects with T1DM. The subjects will be randomized 2:1:1 to receive a single fixed SC 
0.6 mg dose of dasiglucagon, placebo, or a 1 mg dose of GlucaGen and followed for at 
least 28 days after receiving treatment. A total of 156 subjects with T1DM are expected 
to complete the treatment visit. The trial will be conducted in the European Union (EU) 
and North America. See Figure 1 for an overview of the trial design. 


 
 
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Figure 1 
Trial design 
 
 
 


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4.2 
Justification of the trial design 
4.2.1 Justification for design and parameters 
In order to avoid bias in subject selection and in the evaluation of clinical assessments, 
subjects will be randomly assigned 2:1:1 to either dasiglucagon, placebo, or GlucaGen 
and the trial will be conducted in a double-blinded manner. The randomized parallel 
treatment design with administration of fixed SC doses of dasiglucagon, placebo, or 
GlucaGen to subjects with T1DM and insulin-induced hypoglycemia allows for direct 
comparison of the clinical efficacy of the treatments.  
The trial is double-blind to increase trial validity and to reduce bias during evaluation of 
the treatments. However, since the trial medications are not identical in appearance, the 
handling, preparation and administration of trial medication will be done by unblinded 
trial personnel who will not be involved in any other trial procedures or assessments. See 
Section 6.6 for more information about which assessments are blinded and which are not, 
with reasons.  
A superiority trial design is used because the aim is to show that treatment with 
dasiglucagon is an effective treatment compared to placebo. The secondary objective was 
chosen to allow a comparison between treatment with dasiglucagon and an established 
comparator treatment for severe hypoglycemia, GlucaGen. 
Administration of glucagon is intended to quickly raise blood glucose levels in subjects 
with T1DM with insulin-induced hypoglycemia. Therefore, in order to assess the clinical 
efficacy of dasiglucagon as compared to placebo and GlucaGen following a single SC 
dose, the primary endpoint and secondary efficacy endpoints involve the measurement of 
plasma glucose concentrations at different timepoints. 
4.2.2 Justification for drug, route, dosage and treatment duration 
Dasiglucagon and GlucaGen will be administered as fixed doses independent of body 
weight because this is the intended therapeutic dosing regimen in the emergency 
treatment of hypoglycemia. The selected dose of 1 mg GlucaGen is the approved dose for 
treatment of severe hypoglycemia. Data from the studies conducted to date with 
dasiglucagon, including the phase 2 trial in subjects with T1DM, have been used to 
establish that 0.6 mg of dasiglucagon is an effective dose and also represents a 
therapeutically equivalent dose to 1 mg of GlucaGen (see also Section 6.1). 
Dasiglucagon, placebo, and GlucaGen will be administered in the abdomen, buttocks, or 
thigh by SC injection, as this is one of the intended routes of administration for 
dasiglucagon, besides IM and IV. 


 
 
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Subjects will be followed for at least 28 days after dosing in order to perform an adequate 
immunogenicity evaluation of treatment.  


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5 
TRIAL POPULATION 
5.1 
Rationale for trial population 
Dasiglucagon is in development for the treatment of severe hypoglycemia in 
insulin-dependent patients with diabetes mellitus. In the present trial, subjects with 
T1DM are included in the evaluation of efficacy and safety of dasiglucagon under 
hypoglycemic conditions as this is part of the intended target population. Subjects with 
T1DM are selected to avoid the endogenous glucagon counter-regulatory response to 
insulin-induced hypoglycemia that is present in patients with T2DM. The inclusion and 
exclusion criteria are set to include a trial population that represents the general 
population of subjects with T1DM.  
5.2 
Planned sample size and number of trial centers 
A total number of 156 subjects with T1DM are expected to complete the treatment visit 
(Visit 2). To qualify as completed, the subject must be dosed and have blood drawn for 
the PK and PD assessments described in the protocol. It is expected that up to 
176 subjects will be randomized to have 156 subjects completing Visit 2. Completion of 
156 subjects (78 subjects in the dasiglucagon group and 39 subjects in each of the 
placebo and GlucaGen groups) would be expected to provide adequate power for the 
primary efficacy evaluation, as described in Section 9.1. 
The planned date for first subject first visit is expected to take place in Q4, 2017 and the 
planned date for last subject last visit is expected to take place in Q3, 2018. 
This trial is planned to be conducted at 4 to 6 trial sites in the United States of America 
[US], Canada, and Europe. 
5.3 
Inclusion criteria 
Subjects will be entered into this trial only if they meet all of the following criteria: 
1. Informed consent obtained before any trial-related activities (trial-related
activities are any procedure that would not have been performed during normal
management of the subject).
2. Female or male subjects with T1DM for at least 1 year, diagnostic criteria as
defined by the American Diabetes Association (3).
3. Treated with insulin for T1DM for at least 1 year and with stable insulin treatment
(defined as no more than a 10-unit daily variation in total daily insulin dose)
30 days prior to screening


 
 
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4. Hemoglobin A1c <10%. 
5. Aged between 18 and 75 years, both inclusive. 
6. A female subject must meet one of the following criteria: 
a. Participant is of childbearing potential and agrees to use one of the 
accepted contraceptive regimens throughout the entire duration of the trial 
from screening and until last follow-up visit. An acceptable method of 
contraception includes one of the following: 
i. Abstinence from heterosexual intercourse; 
ii. Systemic contraceptives (birth control pills, injectable/implant/ 
insertable hormonal birth control products, transdermal patch); 
iii. Intrauterine device (with and without hormones); or 
iv. Condom with spermicide; or 
b. Participant is of non-childbearing potential, defined as surgically sterile 
(i.e. has undergone complete hysterectomy, bilateral oophorectomy, or 
tubal ligation), or in a menopausal state (at least 1 year without menses). 
7. A male subject must meet the following criteria: Surgically sterilized or willing to 
refrain from sexual intercourse from screening and until last follow-up visit or, if 
sexually active, uses condom and partner practices contraception during the trial 
from screening and until last follow-up visit. 
5.4 
Exclusion criteria 
Subjects meeting any of the following criteria during screening evaluations will be 
excluded from trial participation: 
1. Previously treated with dasiglucagon (previously referred to as ZP4207). 
2. Known or suspected allergy to trial product(s) or related products. 
3. History of anaphylaxis or symptoms of severe systemic allergy (such as 
angioedema). 
4. Previous participation (randomization) in this trial. 
5. Females who are pregnant according to a positive pregnancy test, are actively 
attempting to get pregnant, or are lactating. 
6. History of hypoglycemic events associated with seizures in the last year prior to 
screening. 
7. History of severe hypoglycemia (defined as plasma glucose <54 mg/dL 
[3.0 mmol/L]) in the last month prior to screening. 
8. Receipt of any investigational drug within 3 months prior to screening. 
9. Active malignancy within the last 5 years. 
10. Congestive heart failure, New York Heart Association class II-IV. 
11. Inadequately treated blood pressure, defined as systolic ≥160 mmHg or diastolic 
≥90 mmHg) at screening (15). 
12. Current bleeding disorder, including anti-coagulant treatment. 


 
 
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13. Known presence or history of pheochromocytoma (i.e. adrenal gland tumor) or 
insulinoma (i.e. insulin secreting pancreas tumor). 
14. Use of a daily systemic beta-blocker drug, indomethacin, warfarin or 
anticholinergic drugs in the previous 28 days before Day 1 of this trial. 
15. Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >2.5 × the 
upper limit of the normal range (ULN), bilirubin >1.5 × ULN, estimated 
glomerular filtration rate <30 mL/min/1.73 m2 according to the Modification of 
Diet in Renal Disease study definition (16), or altered electrolyte values of 
clinical relevance for cardiac conduction, as judged by the investigator. 
16. Clinically significant abnormal ECG at screening as judged by the investigator. 
17. Clinically significant illness within 4 weeks before screening, as judged by the 
investigator. 
18. Donation of blood or plasma in the past month, or in excess of 500 mL within 
12 weeks prior to screening. 
19. Surgery or trauma with significant blood loss within the last 2 months prior to 
screening. 
20. A positive result in the alcohol and/or urine drug screen at the screening visit. 
Significant history of alcoholism or drug abuse as judged by the investigator or 
consuming more than 24 g alcohol per day for men, or more than 12 g alcohol per 
day for women. 
21. Subjects with mental incapacity or language barriers which preclude adequate 
understanding or cooperation, who are unwilling to participate in the trial, or who 
in the opinion of the investigator should not participate in the trial. 
22. Any condition interfering with trial participation or evaluation or that could be 
hazardous to the subject. 
23. The use of prescription or non-prescription medications known to cause QT 
prolongation. 
5.5 
Dosing day exclusion criteria 
Subjects who meet one or more of the following dosing day exclusion criteria at the 
dosing visit will be excluded from the dosing visit, but the visit can be rescheduled 
1-7 days later. The dosing visit can only be rescheduled once. 
1. Atypically strenuous exercise within 4 days prior to dosing, as judged by the 
investigator. Exercise during the trial should follow subject’s typical routine, and 
should not exceed a near maximum intensity for more than 20 minutes per day, or 
moderate intensity for more than 90 minutes per day. 
2. Clinically significant illness within 4 weeks before dosing, as judged by the 
investigator. 
3. Consumption of alcohol within 24 hours prior to dosing visit, or positive results 
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4. Not fasting from 22:00 hours the evening prior to dosing, apart from water. Small
amounts of carbohydrates (up to 20 g) to prevent hypoglycemia are allowed.
5. The use of any non-prescribed systemic or topical medication, except routine
vitamins and occasional use (as judged by the investigator) of acetylsalicylic acid
and paracetamol within 2 weeks prior to dosing. Treatment with insulin, including
analogs, is allowed.
6. Use of insulin Degludec or insulin Glargine U300 within 48 hours prior to dosing;
or use of other long-acting insulins (e.g., insulin Glargine U100 or insulin
Detemir) within 24 hours prior to dosing; or use of insulin Neutral protamine
Hagedorn (NPH) within 16 hours prior to dosing.
7. Use of any short acting (bolus) insulin within 6 hours prior to dosing, except
insulin glulisine (Apidra®).
8. Changes in medical history or concomitant medication resulting in fulfillment of
clinical exclusion criteria, as judged by the investigator.
9. Plasma glucose value <50 mg/dL (2.8 mmol/L) within the last 24 hours or plasma
glucose value <60 mg/dL (3.3 mmol/L) within the last 5 hours prior to initiation
of the hypoglycemic procedure.
5.6 
Premature treatment discontinuation and withdrawal 
Participation in the trial is strictly voluntary. A subject has the right to withdraw from the 
trial at any time and for any reason. If he/she chooses to withdraw, the investigator must 
be informed immediately. The investigator has the right to terminate participation of any 
subject at any time if the investigator deems it in the subject's best interest. The reason 
and circumstances for withdrawal will be documented in the electronic case report form 
(eCRF). 
5.6.1 Possible reasons for treatment visit discontinuation 
A subject will be discontinued from treatment if the following applies: 

Withdrawal of consent by subject.

If a protocol deviation occurs which, in the clinical judgement of the investigator, can
invalidate the trial or endpoints or can interfere pharmacokinetically or
pharmacodynamically with the trial product, the subject will be discontinued by the
investigator.

Adverse events occur which are considered unacceptable by the subject or the
investigator.
If discontinuation occurs following administration of trial medication, every effort should 
be made to have the subject return and participate in the complete follow-up visit on 
Day 28 (see Table 3) to avoid missing data. 


 
 
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If trial participation is terminated due to an AE possibly related to any of the trial 
medications or trial examinations, the subject must be followed up by additional 
examinations according to the medical judgment of the investigator until the abnormal 
condition is resolved or the investigator deems further observations or examinations to be 
no longer medically indicated. 
A total of 156 subjects must complete the treatment visit (Visit 2). To qualify as 
completed, the subject must be dosed and have blood drawn for the PK and PD 
assessments described in the protocol.  
5.6.2 Center discontinuation 
The center can be closed and the trial terminated for the following reasons: 
 The center is unlikely to be able to recruit sufficient subjects within the agreed time 
frame. 
 The center does not respond to trial management requests. 
 Repeat protocol violations. 
5.6.3 Trial termination 
The sponsor reserves the right to modify or terminate the trial at any time. Possible 
reasons for termination are: 
 Safety reasons – the incidence of AEs in this or any other trial using the same trial 
medication indicates a potential health risk for the subjects. 
 New scientific knowledge becomes known that makes the objectives of the trial no 
longer feasible/valid. 
 Unsatisfactory enrolment of subjects. 
5.7 
Subject identification and randomization 
Subjects who have given written informed consent and meet all inclusion and none of the 
exclusion criteria will be randomized in a 2:1:1 ratio to either 0.6 mg dasiglucagon 
(n=78), placebo (n=39), or 1 mg GlucaGen (n=39) via an Interactive Web Response 
System (IWRS). Randomization will continue until 156 subjects have completed Visit 2. 
Subjects with previous exogenic glucagon exposure will not be excluded from the trial, 
but the information on previous glucagon administration will be recorded, to enable 
subgroup analyses.  
In the event of an emergency, e.g. when it becomes necessary for the investigator to 
know which treatment the subject is taking, the subject code can be broken by the 


 
 
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investigator, preferably after consultation with the medical monitor. Emergency code 
breaks can be performed using the IWRS. 


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6 
TRIAL DRUG 
6.1 
Identity 
The following trial drugs will be administered: 

Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in a prefilled syringe.

Placebo, liquid formulation, 0.6 mL delivered in a prefilled syringe.

Recombinant glucagon hydrochloride, 1 mg for reconstitution (GlucaGen®, Novo
Nordisk) in 1 mL sterile water.
The description of the three trial drugs is provided in Table 1. Dasiglucagon is a stable 
peptide analog of human glucagon in a ready-to-use liquid formulation for treatment of 
severe hypoglycemia in insulin-dependent patients with diabetes mellitus. GlucaGen is 
approved in the EU and US and is indicated for treatment of severe hypoglycemic 
reactions, which may occur in the management of insulin-treated children and adults with 
diabetes mellitus. 
Table 1 
Description of trial drugs 
Test product 
Placebo Product 
Comparator product 
Name 
Dasiglucagon 
Placebo 
GlucaGen® 
Active 
substance 
Dasiglucagon 
N/A 
Recombinant 
glucagon 
hydrochloride 
Formulation 
Liquid formulation, 
0.6 mL 
Liquid formulation, 
0.6 mL 
Powder and solvent 
for reconstitution as 1 
mL solution for 
injection 
Strength 
1 mg/mL 
N/A 
1 mg 
Device 
Single use pre-filled 
syringe 
Single use pre-filled 
syringe 
Powder and solvent 
for reconstitution 
packed together in a 
plastic box. A “hypo-
kit”. 
Manufacturer 
Zealand Pharma A/S, 
Glostrup 
(Copenhagen), 
Denmark 
Zealand Pharma A/S, 
Glostrup 
(Copenhagen), 
Denmark 
Novo Nordisk A/S, 
Bagsværd, Denmark 


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Test product 
Placebo Product 
Comparator product 
Storage 
requirements 
Store between 2 and 
8°C 
Store between 2 and 
8°C 
Store between 2 and 
8°C 
The quantities of ingredients for dasiglucagon and placebo are provided in Table 2. 
Table 2 
Quantities of ingredients in dasiglucagon and placebo injection 
Component 
Amount per mL 
(dasiglucagon) 
Amount per mL 
(placebo) 
Function 
Dasiglucagon* 
1.0 mg 
N/A 
Sodium chloride 
10.23 mg 
10.23 mg 
Trometamol/Tromethamine 
6.06 mg 
6.06 mg 
Water for injection 
To make 1 mL 
To make 1 mL 
Sodium hydroxide 
q.s. 
q.s. 
Hydrochloric acid 
q.s. 
q.s. 
*The quantity of drug substance to be used is calculated according to net peptide content and purity.
q.s. = quantum sufficit (quantity required). 
6.2 
Treatment assignment and randomization 
Subjects successfully completing screening and who fulfill entry eligibility and 
randomization criteria will be randomized to one of three treatment groups in a ratio of 
2:1:1: 

Test treatment: Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in a
prefilled syringe.

Placebo treatment: Placebo, liquid formulation, 0.6 mL delivered in a prefilled
syringe.

Reference treatment: Recombinant glucagon hydrochloride, 1 mg for reconstitution.
Randomization will be performed using a fixed-block randomization scheme. The 
randomization scheme will be generated prior to the initiation of the study by an 
independent statistician/programmer who will not be a member of the study team; all 
investigators will not be aware of the block size of the randomization scheme.  
Randomization will be stratified by treatment group and by injection site (abdomen, 
buttocks, or thigh) and controlled via the IWRS.      


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Subjects will be randomized to study treatment using an interactive, automated system 
which has been validated for the intended use under the International Society of 
Pharmaceutical Engineers Good Automated Manufacturing Process guidelines, 21CFR 
Part 11 (FDA regulation for Electronic Records and Electronic Signatures) and the 
International Conference on Harmonization (ICH) Guidance E6 for Industry on Good 
Clinical Practice (GCP).   
6.3 
Administration 
Dasiglucagon, placebo, and GlucaGen will be administered by SC injection in the 
abdomen, buttocks, or thigh.  
An unblinded person (appropriately trained) authorized to prepare the dose and 
administer the treatment in accordance with the randomization will prepare the treatment 
required for each subject on each dosing day. The dose will be administered by the 
unblinded, trained and qualified person. The content of the syringe has to be checked for 
clarity and absence of bubbles.. 
Syringes will be discarded after dose administration. Used GlucaGen vials will be stored 
in a lockable box (separated from unused vials) at ambient temperature. 
6.4 
Packaging and labelling 
The test product will be packed by the sponsor. The information on the labels will be in 
the local language and the product label will be compliant with local laws and 
regulations.  
The study drug label will describe the storage conditions for study drug. The labels will 
supply no information about the subjects. Each treatment kit (pre-filled syringe/vial for 
reconstitution) will have a unique Dispensing Unit Number for drug allocation, drug 
accountability, and traceability purposes. 
Labelling will be performed according to Annex 13 of the Good Manufacturing Practice 
guidelines of the European Commission, ICH GCP guidelines, and local law. 
6.5 
Storage of study drugs 
The investigator must ensure the availability of proper storage conditions. All study drug 
supplies provided for this study will be stored in a secure area with restricted access at the 
study site.  


 
 
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The temperature should be monitored by recording the actual, minimum, and maximum 
temperatures using a calibrated thermometer or thermocouple, or by continuous recording 
using a qualified temperature monitoring system. The temperature should be evaluated 
and documented at least on working days on a temperature log. This log must be included 
in the Investigator Site File upon study termination. 
Dasiglucagon and placebo must be stored in a refrigerator (at a temperature of 2–8°C), 
and should be handled in accordance with guidelines from the sponsor. GlucaGen must 
also be stored in a refrigerator (at a temperature of 2–8°C), and should be handled in 
accordance with the Summary of Product Characteristics (13).  
The unblinded person responsible for study drug handling must contact the unblinded 
monitor in case of temperature deviations outside the acceptable range.  
Please see the Pharmacy Manual for additional information on handling study drug. 
6.6 
Blinding and breaking the blind 
This is a double-blind trial. As the trial products are not identical in appearance, 
dasiglucagon and placebo being available as a liquid formulation and GlucaGen as a 
powder for reconstitution, unblinded trial personnel will be responsible for handling, 
preparing (according to the prescription from the IWRS), and administering the trial 
medication, as well as for keeping the records strictly confidential and accessible only to 
unblinded staff until after the database has been locked. To maintain double-blind 
conditions, all trial assessments at the trial center will be done by blinded trial personnel 
not involved in the administration of trial medications. However, exposure assessments 
and anti-drug antibody (ADA) assessments will be performed by unblinded personnel at 
the special laboratories, to ensure that dasiglucagon, placebo, or GlucaGen administration 
is matched with the applicable bioanalytical assay. 
Treatment assignment will be kept strictly confidential and accessible only to authorized 
persons until after the time of unblinding. Codes with treatment assignment will, 
however, be readily available in the IWRS to the blinded personnel in case of an 
emergency. 
The code for a particular subject may be broken in a medical emergency if knowing the 
identity of the treatment allocation would influence the treatment of the subject. The 
emergency code break can be performed using the IWRS. Whenever a code is broken, the 
person breaking the code must record the time, date and reason as well as his/her initials 
in the source documents. The breaking of blinded codes in case of medical emergency for 
one subject should not unblind the trial personnel to the treatment information of other 


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subjects. The person performing the unblinding should inform as few people as possible 
about the result of the unblinding. All persons unblinded for a specific subject should be 
documented. 
If the trial center needs to break the code, the medical monitor should, if at all possible, 
be contacted prior to breaking the code and the monitor must be notified within 24 hours 
after the code has been broken. 
The pharmacovigilance unit (safety contract research organization [CRO]; see the list of 
trial personnel in Section 12.1) will be able to break the code in case of a serious 
unexpected suspected adverse reaction (SUSAR).  
The central and specialty laboratories will be provided with a copy of the randomization 
list. 
6.7 
Drug accountability 
Handling, preparation and administration of trial medication will be done by unblinded 
trial personnel. Each center will keep accurate records of the trial supplies received, 
stored, and dispensed, using appropriate forms. The trial supplies will be stored under 
appropriate conditions, locked and with restricted access.  
All unused supplies and all empty and partially empty containers of trial medication will 
be stored until the trial closure visit has been performed and then sent for destruction. 
This does not apply to the used syringes as they will be discarded after dose 
administration. Destruction must not take place until approved by the Sponsor.  
6.8 
Treatment compliance 
All trial medications will be prepared and administered by unblinded trial personnel. 
PK assessments will support the surveillance of compliance with IMP administration.  
6.9 
Prior and concomitant medications 
Prior glucagon exposure will be recorded in the eCRF at screening. All concomitant 
medications will be recorded and/or updated in the eCRF at each visit.  
Subjects using any new concomitant medication resulting in fulfillment of a dosing day 
exclusion criterion (see Section 5.5) will be excluded from the dosing visit, but can be 


 
 
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rescheduled to one of the following days (1–7 days later). The dosing visit can only be 
rescheduled once. 
6.9.1 Prohibited medications 
The use of anti-coagulant treatments and medications (prescription and non-prescription) 
that are known to cause QT prolongation are prohibited during the course of the trial. 
Within 28 days prior to dosing, the use of daily systemic beta-blockers, indomethacin, 
warfarin, and anticholinergic drugs is prohibited. 
Within 2 weeks prior to dosing, the use of any non-prescribed systemic or topical 
medication (with the exception of vitamins and the occasional use of acetylsalicylic acid 
and paracetamol) is prohibited.  
Within 48 hours prior to dosing, the use of insulin Degludec or insulin Glargine U300 are 
prohibited. 
Within 24 hours prior to dosing, the use of long-acting insulin (e.g., insulin Glargine 
U100 or insulin Detemir) is prohibited. 
Within 16 hours prior to dosing, the use of insulin NPH is prohibited. 
Within 6 hours prior to dosing, the use of any short acting (bolus) insulin, except insulin 
glulisine (Apidra®) is prohibited.  
During the insulin-induced hypoglycemic procedure, continuous SC insulin infusion must 
be stopped. 
6.10 Local German requirements for insulin glulisine (Apidra®) 
Insulin glulisine (Apidra®), the insulin used to induce hypoglycemia, will according to 
local requirements be defined as an investigational medicinal product when used at 
German sites. The insulin will be provided by the local sites and drug accountability will 
be performed according to local procedures. In contrast to the trial drug for the 
randomized treatment, the insulin will be administered by the blinded trial personnel via 
IV infusion as described in section 8.2.3.1. 
 


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7 
PARAMETERS AND METHODS OF ASSESSMENT 
Overall, approximately 180 mL of blood will be drawn from each subject for PK, PD, 
ADA, and safety laboratory assessments. 
7.1 
Efficacy parameters 
A description of the sample handling and sample processing at the site will be included in 
the laboratory manuals. Validation documentation for the assays must be available prior 
to sample analyses. A bioanalytical report for each analysis of trial PD and PK samples 
will be prepared. 
7.1.1 Pharmacodynamic measurements 
The plasma glucose profile for evaluation of the primary and secondary clinical efficacy 
endpoints will be assessed based on plasma concentration data (AUC0-30min) from samples 
collected at the dosing visit (Visit 2). The samples will be sent to the clinical laboratory 
and analyzed using a sensitive and validated assay for glucose measurements.  
Samples will be collected pre-dose, and at 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 
75, and 90 minutes after dosing (see the schedule of procedures in Table 3). The actual 
time of blood sampling for evaluation of plasma glucose should not deviate from the 
nominal time by more than ±30 seconds until the 20 minute collection time point and by 
more than ±1 minute for the subsequent collection time points. Pre-dose is defined as 
within 2 minutes prior to dosing. 
7.1.2 Pharmacokinetic measurements 
The exposure to trial medication (dasiglucagon or GlucaGen) for evaluation of PK will 
be assessed based on plasma concentration data (AUC0-90 min, Cmax, tmax) from samples 
collected at the dosing visit (Visit 2).  
Samples (including back-up samples) will be collected pre-dose, and at 15, 30, 35, 40, 
50, 60, 90, and 120 minutes after dosing (see the schedule of procedures in Table 3). The 
actual time of blood sampling for exposure to trial medication should not deviate from 
the nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior 
to dosing. 


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7.2 
Safety parameters 
7.2.1 Adverse events 
The investigator is responsible for the detection and documentation of events meeting the 
criteria and definition of an AE or serious adverse event (SAE), as provided in this 
protocol. During the trial, the investigator or center staff will be responsible for reporting 
AEs and SAEs, as detailed in this section of the protocol.  
Based on the investigator’s clinical judgment it will be determined whether an AE is 
related to treatment and of sufficient severity to require the subject’s removal from 
treatment or from the trial. A subject may also voluntarily withdraw from treatment due 
to what he or she perceives as an intolerable AE. If either of these situations arises, the 
subject should be under medical supervision until symptoms cease or the condition 
becomes stable. 
7.2.1.1 Definitions 
Adverse event 
An AE is any untoward medical occurrence in a trial subject given an IMP which does 
not necessarily have a causal relationship with the treatment. An AE can therefore be any 
unfavorable and/or unintended sign (for example, an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of an IMP, whether or not 
considered related to the IMP.  
In this trial, only treatment-emergent adverse events (TEAEs) will be collected and 
reported. TEAEs are events that occur from the first trial-related activity after the subject 
has signed the informed consent form until the end of the post-treatment follow-up 
period.  
AEs include: 

A clinically significant worsening of a concomitant illness.

A clinical laboratory abnormality which is clinically significant, i.e. any abnormality
that suggests a disease and/or organ toxicity and is of a severity that requires active
management. Active management includes active treatment or further investigations,
for example change of medicine dose or more frequent follow-up due to the
abnormality.

Hypoglycemic episodes (see Section 7.2.2).

Injection site reactions (see Section 7.2.6).


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The following should not be recorded as AEs, if recorded at screening (on the Screening 
Form or eCRF): 

Pre-planned procedure, unless the condition for which the procedure was planned has
worsened from the first trial related activity after the subject has signed the informed
consent.

Pre-existing conditions found as a result of screening procedures (pre-existing
conditions should be reported as medical history or concomitant illness).
Serious adverse event 
An SAE is any untoward medical occurrence that at any dose: 

Results in death

Is life-threatening

Requires inpatient hospitalization or prolongation of existing hospitalization

Results in persistent or significant disability or incapacity

Is a congenital anomaly or birth defect

Is medically important*
*Medical judgement must be exercised in deciding whether an AE is believed to be
‘medically important’. Medically important events may not be immediately 
life-threatening or result in death or hospitalization but may jeopardize the subject or 
may require intervention to prevent one of the other outcomes listed in the definition 
above. 
Adverse drug reaction 
All noxious and unintended responses to an investigational product related to any dose 
should be considered adverse drug reactions. The phrase responses to an investigational 
product means that a causal relationship between an investigational product and an 
adverse event is at least a reasonable possibility, i.e. the relationship cannot be ruled out. 
Suspected unexpected serious adverse reactions (SUSARs) 
An AE fulfilling one of the criteria of seriousness and being assessed as related to IMP 
application, the nature or severity of which is not consistent with the applicable reference 
document (e.g. dasiglucagon Investigator’s Brochure or package leaflet/Summary of 
Product Characteristics for GlucaGen). 


 
 
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Adverse event of special interest 
An AESI is an event which, in the evaluation of safety, has a special focus (e.g. required 
by health authorities). In this trial hemodynamic changes, as defined below, are 
considered AESIs: 
 Post-dose clinical signs, or measured vital signs, indicating a clinically significant 
drop in blood pressure including signs of orthostatic hypotension, vasovagal 
responses or bradycardia.   
 Post-dose change in pulse or blood pressure considered an event of hypo- or 
hypertension as judged by the investigator. 
 
Intensity of an adverse event 
The maximum intensity (severity) of all AEs must be assessed by the investigator and 
documented. Severity should be graded when the AE outcome is known: 
Mild:  
A type of AE that is usually transient and may require only minimal 
treatment or therapeutic intervention. The event does not generally 
interfere with usual activities of daily living. 
Moderate:  
A type of AE that is usually alleviated with additional specific therapeutic 
intervention. The event interferes with usual activities of daily living, 
causing discomfort but poses no significant or permanent risk of harm to 
the subject. 
Severe:  
A type of AE that interrupts usual activities of daily living, or significantly 
affects clinical status, or may require intensive therapeutic intervention. 
A ‘severe’ reaction does not necessarily deem the AE as ‘serious’ and an SAE may not 
necessarily be ‘severe’ in nature. 
Causality relationship to trial medication 
The causality of each AE should be assessed by the investigator according to the 
following classification: 
Probable: 
Good reason and sufficient documentation to assume a causal relationship. 
Possible: 
A causal relationship is conceivable and cannot be dismissed. 
Unlikely: 
The event is most likely related to etiology other than the trial product. 
Not related:  No relationship to trial product. 


 
 
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Germany only: The investigator should assess the causality to 
dasiglucacon/placebo/GlucaGen and to insulin glulisine (Apidra®) respectively. A 
special field applicable for Germany only will be present on the AE form in the eCRF to 
capture this data. 
Outcome of an adverse event 
The outcome of all AEs must be assessed by the investigator and documented by his/her 
staff. The following definitions should be used: 
Recovered/resolved:  
The subject has fully recovered, or by medical or 
surgical treatment the condition has returned to the 
level observed at the first trial-related activity after 
the subject signed the informed consent.  
Recovering/resolving: 
The condition is improving and the subject is 
expected to recover from the event. This term is 
only applicable if the subject has completed the 
trial. 
Recovered/resolved with sequelae:  The subject has recovered from the condition, but 
with lasting effect due to a disease, injury, treatment 
or procedure. If a sequela meets an SAE criterion, 
the AE must be reported as an SAE. 
Not recovered/not resolved:  
The condition of the subject has not improved and 
the symptoms are unchanged. 
Fatal:  
This term is only applicable if the subject died from 
a condition related to the reported AE. Outcomes of 
other reported AEs in a subject before he/she died 
should be assessed as "recovered/resolved", 
"recovering/resolving", "recovered/resolved with 
sequelae" or "not recovered/not resolved". An AE 
with fatal outcome must be reported as an SAE. 
Unknown:  
This term is only applicable if the subject is lost to 
follow-up. 
7.2.1.2 Collection, recording and reporting of adverse events 
All events meeting the definition of an AE must be collected and reported from the first 
trial related activity after the subject has signed the informed consent until the end of the 
post-treatment follow-up period. At each contact with the center (visit or telephone, 
excluding visits where the subject is not seeing the investigator or his/her staff [e.g. visits 


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to the laboratory]) the subject must be asked about AEs. All AEs, either observed by the 
investigator or reported by the subject, must be recorded by the investigator and 
evaluated. 
The investigator should record the diagnosis, if possible. If no diagnosis can be made the 
investigator should record each sign and symptom as individual AEs. 
One single Adverse Event Form must be used per AE from start to resolution. For SAEs, 
the Serious Adverse Event form must also be completed. For AESIs, the AESI form must 
also be completed. 
AE information should include the following: 

Date and time of onset and resolution

Date and time of investigator’s first information on the (S)AE

Seriousness

Severity

Causal relationship with IMP

Interruption or withdrawal of treatment during IMP administration and other
measures taken

Outcome.
All AEs are coded; details are described in the trial-specific Data Management Plan. 
If an event classifies as a AESI, the investigator must tick the AESI box on the AE form 
and complete the AESI form. The AESI form will capture if the event was associated 
with any signs or symptoms and capture the highest/lowest blood pressure and pulse 
measured during the event. The investigator must report all AESIs to the sponsor’s 
responsible pharmacovigilance unit (Safety CRO; see the list of trial personnel in 
Section 12.1) immediately (within 24 hours) after obtaining knowledge about the event. 
The investigator must report initial information electronically (e.g. in PDF format) on all 
SAEs to the sponsor’s responsible pharmacovigilance unit (Safety CRO; see the list of 
trial personnel in Section 12.1) immediately (within 24 hours) after obtaining knowledge 
about the event. The Safety CRO will inform the medical monitor and the sponsor about 
the reported SAEs. 
It is the responsibility of the Safety CRO to report all SUSARs (see Section 7.2.1.1) that 
occur in this trial to the Competent Authorities and Institutional Review Boards 
(IRBs)/Independent Ethics Committees (IECs) in accordance with the local requirements 
in force and ICH guideline for GCP. The trial monitor must be informed accordingly. 


 
 
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7.2.1.3 Follow-up of adverse events 
All AEs that are ongoing at the end of the subject’s participation in the trial will be 
followed-up until the event is resolved or reaches a satisfactory outcome as deemed by 
the investigator or until the last visit of the last subject enrolled in the trial, whichever 
occurs first.   
Follow-up actions for all SAEs will be performed according to appropriate clinical care 
practices and may depend on the nature of the event. These will be determined after 
internal review and/or sponsor review. 
The follow-up information should only include new (updated and/or additional) 
information that reflects the situation at the time of the investigator’s signature. 
Follow-up information on (S)AEs will be updated using the (S)AE form. If a non-serious 
event becomes serious during the follow-up the AE form and SAE form have to be used 
and reporting timelines follow those of an SAE. 
The investigator must forward follow-up information on SAEs and if previously 
non-serious AEs become SAEs to the Safety CRO immediately (within 24 hours) after 
obtaining knowledge about the new information. 
The sponsor and/or CROs acting on behalf of the sponsor can upgrade a non-serious AE 
to an SAE. In these situations the investigator will be informed and asked to fill out an 
SAE form and forward to the Safety CRO immediately (within 24 hours). 
7.2.1.4 Clinical laboratory abnormalities and other abnormal assessments as adverse 
events or serious adverse events 
Abnormal laboratory findings (e.g. biochemistry, hematology, urinalysis) or other 
abnormal assessments (e.g. vital signs) that are judged by the investigator as clinically 
significant will be recorded as AEs or SAEs if they meet the definition of an AE or SAE. 
Clinically significant abnormal laboratory findings or other abnormal assessments that 
are detected during the trial or are present at baseline and significantly worsen following 
the start of the trial will be reported as AEs or SAEs. However, clinically significant 
abnormal laboratory findings or other abnormal assessments that are associated with the 
disease being studied, unless judged by the investigator as more severe than expected for 
the subject’s condition, or that are present or detected at the start of the trial and do not 
worsen, will not be reported as AEs or SAEs. 
The investigator will exercise his or her medical and scientific judgment in deciding 
whether an abnormal laboratory finding or other abnormal assessment is clinically 
significant. 


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7.2.2 Hypoglycemia 
Hypoglycemia will be regarded as an AE and recorded and documented on an AE form 
(and SAE form, if applicable). 
Hypoglycemia is defined as a decline in plasma glucose to below 70 mg/dL 
(3.9 mmol/L). However, in the time period from initiation of the hypoglycemic clamp 
procedure (see Section 8.2.3.1) until 45 minutes after dosing, hypoglycemia is defined as 
a decline in plasma glucose to below 45 mg/dL (2.5 mmol/L).  
During the dosing visit, prior to administration of the IMP, a plasma glucose value of 
<45 mg/dL (2.5 mmol/L) will be treated by an IV glucose solution. After administration 
of the IMP in the period from 8 minutes until 44 minutes after dosing, a plasma glucose 
value of <45 mg/dL (2.5 mmol/L) will be treated by an IV glucose solution, and if a 
plasma glucose value of ≥70 mg/dL (3.9 mmol/L) is not achieved within the 45 minutes 
after IMP administration, IV glucose infusion will also be initiated. 
If the subject experiences symptoms of hypoglycemia, a plasma glucose measurement 
should be taken in order to classify the event (please refer to Section 7.4.2 for additional 
details).  
7.2.3 Physical examination 
The physical examination will be carried out at screening (Visit 1) and at the follow-up 
visit (Visit 3; see Table 3).  
The physical examination includes examination of the following body systems: head, 
ears, eyes, nose, throat, including the thyroid gland; heart, lung, chest; abdomen; skin and 
mucosae; musculoskeletal system; nervous system; lymph node; other findings. 
At the screening visit, any abnormality will be recorded and described in the eCRF, 
including the investigator’s assessment of clinical significance (‘abnormal, not clinically 
significant’ or ‘abnormal, clinically significant’). Clinically significant findings at the 
screening visit should be recorded as concomitant illness. 
At subsequent visits, any clinically significant deterioration of a pre-existing condition as 
well as any new clinically significant findings will be recorded as AEs (see 
Section 7.2.1). 
7.2.4 Vital signs 
An examination of the following vital signs will be performed at screening (Visit 1), the 
dosing visit (Visit 2) and at the follow-up visit (Visit 3):  


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
Diastolic and systolic blood pressure (mmHg) will be measured after at least
5 minutes rest in a supine position. At Visit 1, blood pressure will be measured in
both arms. The blood pressure from the arm with the higher systolic value is
transcribed into the eCRF and this arm should be used for all subsequent
measurements of the subject’s blood pressure in this trial.

Pulse (beats per min) measured after at least 5 minutes rest in a supine position.

Body temperature (°C).
At the dosing visit, measurements will be taken prior to the start of the insulin-induced 
hypoglycemic procedure (within 30 minutes) and at 30, 90, and 300 minutes after dosing 
(see Table 3). The actual time of the assessment should not deviate from the nominal 
time by more than ±10 minutes. In addition to the pre-specified assessments as listed in 
Table 3, blood pressure and pulse may be assessed at any time during the trial at the 
discretion of the investigator.  
7.2.5 Electrocardiogram 
A standard 12-lead ECG will be performed at the screening visit (Visit 1), the dosing visit 
(Visit 2; prior to the start of the insulin-induced hypoglycemic procedure [within 
30 minutes]) and at 20, 35, 45, 60, and 300 minutes after dosing) and at the follow-up 
visit (Visit 3; see Table 3). The actual time of the assessment should not deviate from the 
nominal time by more than ±5 minutes.  
ECG parameters (heart rate, PQ, QRS, QT, QTcB) and any abnormality will be recorded 
and described in the eCRF including the investigator’s assessment of clinical significance 
(‘abnormal, not clinically significant’ or ‘abnormal, clinically significant’).  
At subsequent visits, any clinically significant deterioration of a pre-existing condition as 
well as any new clinically significant findings will be recorded as AEs (see 
Section 7.2.1). 
7.2.6 Local tolerability 
Immediately prior to treatment administration, it should be verified that the injection site 
is normal. To ensure all injection site assessments are performed at the injection site, the 
site will be marked with a pen prior to injection. Assessment of local tolerability at the 
injection site will be performed at the dosing visit (Visit 2; at 30, 120 and 300 minutes 
after dosing), and at the follow-up visit (Visit 3) (see Table 3) and more frequently, if 
deemed necessary by the investigator. The actual time of the assessment should not 
deviate from the nominal time by more than ±10 minutes. In the eCRF, the time of 
assessment and any injection site reaction observed will be recorded. In case of an 
observation, the Local Tolerability form will be completed, as well as the (S)AE form. 


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The local tolerability at the injection site will be evaluated by means of the following 
assessments: spontaneous pain, pain on palpation, itching, redness, edema, 
induration/infiltration, and other. Each of these assessments will be reported on a scale of 
0 (none), 1 (mild), 2 (moderate), and 3 (severe). The evaluation and the actual time of the 
assessment will be recorded. The assessments will be performed by a trial physician or 
nurse.  
Digital pictures will be taken of the injection site at the time of identification, and 
thereafter as often as judged necessary by the investigator. The pictures should include a 
subject identifier, visit number, time after dosing, and a ruler for scaling. 
7.2.7 Clinical laboratory assessments 
The safety parameters that will be assessed at the clinical laboratory are listed in Table 3. 
Routine clinical laboratory tests will be performed centrally. Samples for clinical 
laboratory parameters (biochemistry, hematology) will be collected at screening (Visit 1), 
the dosing visit (Visit 2, prior to the start of the insulin-induced hypoglycemic procedure 
[within 30 minutes]) and at 30 and 300 minutes after dosing), and at the follow-up visit 
(Visit 3). The actual time of the assessment should not deviate from the nominal time by 
more than ±10 minutes. Samples for glycated hemoglobin (HbA1c), C-peptide and 
coagulation parameters will be collected at screening only (Visit 1). Samples for 
urinalysis will be collected at screening (Visit 1), at the dosing visit (Visit 2, prior to the 
start of the insulin-induced hypoglycemic procedure [within 120 minutes]) and at the 
follow-up visit (Visit 3). The following parameters will be assessed: 

Clinical biochemistry: sodium, potassium, calcium, glucose, urea, creatinine, total
bilirubin, AST, ALT, gamma-glutamyltransferase, alkaline phosphatase, total protein,
C-reactive protein, HbA1c, C-peptide.

Hematology: hemoglobin, red blood cell count (erythrocytes), hematocrit, platelet
count (thrombocytes), total white blood cell count (leukocytes).

Coagulation: international normalized ratio, fibrinogen (at screening visit only).

Urinalysis: pH, blood (leukocytes and erythrocytes), protein, glucose, ketones, nitrite.
Pregnancy tests will be performed at each visit for women of childbearing potential only. 
A serum pregnancy test will be performed at screening (Visit 1) and urine stick tests will 
be performed at the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure) and the follow-up visit (Visit 3). Test sticks will be provided to 
the trial centers. 
Alcohol breath tests and a urine drug screen will be performed at screening (Visit 1) and 
at the dosing visit (Visit 2, prior to the start of the insulin-induced hypoglycemic 


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procedure). Equipment for the alcohol breath test and uring drug screen will be provided 
to the trial centers.  
Re-assessment of laboratory parameters will be allowed only if handling issues, damaged 
samples, or hemolyzed samples have confounded the measurement results.  
For further details of the clinical laboratory assessments, please refer to the laboratory 
manual.  
7.2.8 Pregnancy 
Female subjects must be instructed to notify the investigator immediately if they become 
pregnant or if they suspect to be pregnant during the trial. The sponsor has a 
responsibility to monitor the outcome of all pregnancies reported during the clinical trial. 
Male subjects must be instructed to notify the investigator immediately if their partner 
becomes pregnant or suspects pregnancy. The sponsor has a responsibility to monitor the 
outcome of all pregnancies reported during the clinical trial. During an information 
meeting at the trial center, the subject’s partner will be fully informed by a physician of 
her participation in capturing the outcome data, and will be asked to provide her written 
consent. 
The investigator must report all information on pregnancies on the Initial Pregnancy 
form. The completed Initial Pregnancy form must be forwarded to the sponsor 
immediately (within 24 hours), according to the procedure stated in Section 7.2.1.2. Any 
(S)AEs in the mother, as well as other pregnancy complications (ectopic pregnancy) 
fulfilling the criteria of an (S)AE, must be reported on the (S)AE form.  
The following must be collected in the Initial Pregnancy form: 

Medical history of the mother

Family history

Course of the pregnancy, including expected delivery date.
The investigator must follow the pregnancy until the pregnancy outcome and follow the 
newborn infant(s) until the age of 1 month. The investigator must collect information on 
the pregnancy and pregnancy complications as well as the pregnancy outcome including 
the health of the newborn infant(s) on the Pregnancy Outcome form. The completed 
Pregnancy Outcome form must be forwarded to the sponsor according to the procedure 
stated in Section 7.2.1.2. Any (S)AEs in the newborn must be reported on the (S)AE 
form. 


 
 
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The SAEs that must be reported include abnormal outcome, such as congenital 
anomalies, fetal death and termination of pregnancy (spontaneous or elective abortion), 
including any anomalies of the fetus observed at gross examination or during autopsy.  
The following must be collected in the Pregnancy Outcome form:  
 Course of the pregnancy 
 Outcome of the pregnancy 
 Condition of the newborn 
 Any AEs in the newborn infant must be followed until the age of 1 month. 
7.2.9 Precautions 
Normal precautions taken for a human trial, including the provision of emergency 
equipment, will be taken during this trial. Qualified and well trained physicians and 
medical staff will instruct the subjects. During a subject’s participation in the trial, the 
investigator should ensure that adequate medical care is provided to the subject for any 
AEs, including clinically significant laboratory values related to the trial. The investigator 
should inform the subject when medical care is needed for intercurrent illnesses of which 
the investigator becomes aware.  
For further information on safety precautions for dasiglucagon and GlucaGen, please 
refer to the current version of the Investigator’s Brochure (14) and the Summary of 
Product Characteristics for GlucaGen (13), respectively. 
7.2.10 Safety committee 
The internal Zealand Pharma Safety Committee is constituted to perform ongoing blinded 
safety surveillance of clinical trials with dasiglucagon, including this trial. 
If safety signals are observed, either based on reported SAEs, periodic review of 
laboratory parameters, review of all AEs reported between the Safety Committee 
meetings, or on notification of significant findings, the Safety Committee will take 
appropriate measures to safeguard the subjects. 
7.3 
Demography, concomitant illness, medical history and concomitant 
medication 
Demographics, body measurements, concomitant illness and medical history will be 
assessed only at screening (Visit 1). Concomitant medication will be assessed at 
screening (Visit 1), the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure) and at the follow-up visit (Visit 3). 


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7.3.1 Demography and body measurements 
Subject demographics and body measurements will include: 

Age

Race, ethnicity

Sex

Height (meters or inch), without shoes

Body weight (kg or lb), only wearing underwear and measured using standard scales

Body mass index (kg/m2) calculated based on height and body weight (body
weight/height2).
7.3.2 Concomitant illness and medical history 
A concomitant illness is any illness that is present at the start of the trial (i.e. at the 
screening visit). Concomitant illnesses present at the start of the trial will be recorded in 
the eCRF at screening.  
Medical history is an account of medical events that the subject has experienced in the 
past, including prior medications. Relevant medical conditions/illnesses in the past 
obtained by asking the subject or by inspecting his/her medical records will be recorded 
in the eCRF at screening. History of alcohol or drug abuse will also be recorded. 
The information collected for concomitant illness and medical history should include 
diagnosis, date of onset, date of resolution or continuation. 
Any change to a concomitant illness should be recorded during the trial, including end 
date, if applicable. A clinically significant worsening of a concomitant illness must be 
reported according to Section 7.2.1. 
All previous and concomitant diseases will be coded with Medical Dictionary for 
Regulatory Activities (MedDRA), the version of which will be provided in the clinical 
trial report. 
7.3.3 Diabetes diagnosis and current treatment 
The date of diagnosis of diabetes will be recorded as will the current diabetes treatment 
(start date, product name(s), dose(s)). 


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7.3.4 Concomitant medication 
A concomitant medication is any medication, other than the trial products and current 
diabetes treatment (including insulin glulisine [Apidra®] for diabetes therapy wash-out), 
which is taken during the trial, including screening and follow-up periods. 
Details of any concomitant medication must be recorded at trial entry (i.e. at screening). 
Any changes in concomitant medication must be recorded at each visit as they occur. The 
information collected for each concomitant medication includes (at a minimum) trade 
name or generic name, indication, start date and stop date or continuation. A change in 
medication due to an AE must be recorded and reported according to Section 7.2.1. If the 
change in medication influences the subject’s eligibility to continue in the trial, the 
sponsor and trial monitor must be informed. 
7.4 
Other assessments 
7.4.1 Immunogenicity 
Antibodies against dasiglucagon/GlucaGen will be measured at the dosing visit (Visit 2) 
and at follow-up (Visit 3). At the dosing visit (Visit 2), samples will be collected prior to 
the start of the insulin-induced hypoglycemic procedure.  
The clinical ADA assays, specific for dasiglucagon and GlucaGen, respectively, have 
been validated in accordance with existing guidelines and recommendations (17-21).  
Confirmed positive anti-dasiglucagon antibody samples (treatment-induced or 
treatment-boosted) from anti-dasiglucagon antibody-positive subjects will be evaluated 
for binding titer neutralizing potential and titer as well as cross-reactivity towards 
endogenous glucagon.  
No further serum sampling will be needed as the ADA samples can be used for 
neutralizing antibody analysis. 
The in vitro neutralizing effect of the antibodies will be measured using an assay based 
on glucagon receptor transfected human embryonic kidney cells (20,22). The calculated 
sensitivity in previous studies was about 51.8 ng/mL. The assay was also validated for 
recombinant glucagon with similar results (21,23). In case of a positive result in the 
neutralizing antibody assay, a titer estimation will be performed. The cell-based 
neutralizing antibody analyses will be performed by a special laboratory, BioAgilytix, 
Durham, NC, USA. 


 
 
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Residual and additional antibody serum samples may be stored until approval of market 
authorization by the health authorities. Further characterization of the antibody response 
may be requested by the health authorities. 
7.4.2 Plasma glucose measurements for safety 
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at 
site. After the start of insulin infusion, plasma glucose should be checked approximately 
every 10 minutes while plasma glucose is above 110 md/dL, and approximately every 5 
minutes once plasma glucose is at or below 110 mg/dL and until after dosing when the 
subject’s plasma glucose is ≥70mg/dL (3.9 mmol/L). Hereafter, plasma glucose should be 
checked approximately every 30 minutes until 300 minutes (5 hours) after dosing. Plasma 
glucose concentrations will be measured using the US FDA-approved glucose analyzer 
YSI 2300, Yellow Springs Instruments, Yellow Springs, OH or the Super GL analyzer, 
Dr. Müller Gerätebau GmbH, Freital, Germany. 
At the discretion of the investigator, additional plasma glucose measurements can be 
taken at any time during the trial, for example when there is a suspicion (e.g. symptoms) 
of a hypoglycemic episode. 
Plasma glucose measurements for safety should only be recorded in the eCRF if they are 
related to an AE (e.g. a hypoglycemic episode). 
In case of persistent post-treatment hypoglycemia, subjects will receive rescue treatment 
with an IV glucose infusion (see Section 8.2.3.1 for details). Blood samples for PD and 
PK assessments should still be taken at the specified timepoints. 
7.4.3 Plasma insulin measurements 
Samples for insulin assessment will be collected at the dosing visit (Visit 2, pre-dose and 
at 30 and 60 minutes after dosing). The actual time of blood sampling for evaluation of 
plasma insulin should not deviate from the nominal time by more than ±1 minute. 
Pre-dose is defined as within 2 minutes prior to dosing. 


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8 
TRIAL CONDUCT 
8.1 
Schedule of procedures 
The schedule of procedures is provided in Table 3. Informed consent will be obtained 
prior to any trial-related procedures; see Section 10.8. 
8.2 
Procedures by visit 
8.2.1 Visit 1 (screening, Day -30 to Day -3) 
Visit 1 will take place between 3 and 30 days before Visit 2, Day -1 to Day 1 (dosing 
day). 
Informed consent can be obtained prior to or at Visit 1, however it must in any case be 
obtained prior to any trial related procedures. During the screening visit, the following 
assessments will take place: 

Informed consent (obtain or check)

Check of subject eligibility (inclusion/exclusion criteria)

Demography

Body measurements

Medical history, diabetes diagnosis, current diabetes treatment

Concomitant illnesses

Concomitant medications

History of alcohol/drug abuse

Physical examination

Vital signs

12-lead ECG

AEs

Biochemistry, hematology, coagulation, HbA1c

Serum pregnancy test (women of childbearing potential only)

Urinalysis

Urine drug screen

Alcohol breath test


 
 
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Table 3 
Schedule of procedures 
Visit number 
V1 
V2 
V3 
Trial day 
-3 
-1 and 1 
28 
Visit type 
Screening 
Dosing 
Follow-up 
Window 
-30 to -3 
 
+5 days 
Subject related information/assessments 
 
 
 
Informed consent 
x 
 
 
Inclusion/exclusion criteria 
x 
x1,2 
 
Demography 
x 
 
 
Body measurements 
x 
 
 
Medical history, diabetes diagnosis, and current diabetes 
treatment 
x 
 
 
Concomitant illnesses 
x 
 
 
Concomitant medications 
x 
x1 
x 
History of alcohol/drug abuse 
x 
 
 
Randomization 
 
x1 
 
Withdrawal criteria 
 
x1 
 
Dosing day exclusion criteria 
 
x1 
 
Insulin-induced hypoglycemia 
 
x 
 
Safety assessments 
 
 
 
Physical examination 
x 
 
x 
Vital signs 
x 
x3 
x 
12-lead ECG 
x 
x4 
x 
Local tolerability  
 
x5 
x 
Adverse events 
x 
x 
x 
Laboratory 
 
 
 
Biochemistry, hematology, coagulation, HbA1c (coagulation 
and HbA1c, at Visit 1 only) 
x 
x6 
x 
Pregnancy test (women of childbearing potential only) 
x7 
x1,8 
x8 
Urinalysis 
x 
x1 
x 
Urine drug screen 
x 
x1 
 
Alcohol breath test 
x 
x1 
 
PK/Clinical efficacy 
 
 
 
Plasma dasiglucagon/GlucaGen 
 
x9 
 
Plasma glucose 
 
x10 
 
Other assessments 
 
 
 
Antibodies against dasiglucagon/GlucaGen 
x1 
x11 
Plasma insulin 
 
x12 
 
Trial material 
 
 
 
Administration of trial product (during hypoglycemic clamp 
procedure) 
 
x 
 
1Prior to the start of the insulin-induced hypoglycemic procedure. 
2Only check of dosing day exclusion criteria and changes between screening visit and Visit 2. 
3Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 30, 90 and 300 minutes 
after dosing. The actual time of the assessment should not deviate from the nominal time by more than ±10 minutes. 


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4Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 20, 35, 45, 60 and 300 
minutes after dosing. The actual time of the assessment should not deviate from the nominal time by more than ±5 
minutes.  
5Local tolerability assessed at 30, 120, and 300 minutes after dosing. The actual time of the assessment should not 
deviate from the nominal time by more than ±10 minutes.  
6Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 30 and 300 minutes after 
dosing. The actual time of the assessment should not deviate from the nominal time by more than ±10 minutes. 
7Serum pregnancy test. 
8Urine stick pregnancy test. 
9Pre-dose, and at 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time of blood sampling should not 
deviate from the nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing. 
10Pre-dose, and at 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, 90 minutes after dosing. The actual time of blood 
sampling should not deviate from the nominal time by more than ±30 seconds until the 20 minute collection time point 
and by more than ±1 minute for the subsequent collection time points. Pre-dose is defined as within 2 minutes prior to 
dosing. 
11Any subject that tests positive for ADA will be monitored until the ADA levels return to baseline levels. 
12Pre-dose, and at 30 and 60 minutes after dosing. The actual time of blood sampling should not deviate from the 
nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing.  
ADA = anti-drug antibodies; ECG = electrocardiogram; HbA1c = glycated hemoglobin. 
Eligible or potentially eligible subjects (laboratory results pending) will be provided with 
an Identification card (ID card), stating that the subject is participating in the trial and 
whom to contact (site address, investigator name and telephone number). The subjects 
should be instructed to return the ID card to the investigator at the last visit or to destroy 
the card after the last visit.  
8.2.2 Instructions to subjects prior to the dosing visit (Visit 2) 
At the screening visit, the investigator will inform the subject about the changes to his/her 
insulin therapy leading up to the start of the insulin-induced hypoglycemic procedure. 
The subject may be provided with insulin NPH in the wash-out period to cover the need 
of basal insulin, if deemed necessary by the investigator. The subject’s current insulin 
therapy will be washed out as defined in Section 5.5.: 48 hours prior to dosing and during 
the dosing visit, treatment with  insulin Degludec and insulin Glargine U300 are not 
allowed; 24 hours prior to dosing and during the dosing visit, treatment with other long-
acting insulins (e.g., insulin Glargine U100 or insulin Detemir) is not allowed; 16 hours 
prior dosing and during the dosing visit treatment with insulin NPH is not allowed; 6 
hours prior to dosing and during the dosing visit, treatment with any short acting (bolus) 
insulin, except insulin glulisine (Apidra®), is not allowed. The basal rate of insulin pumps 
(continuous SC insulin infusion) will be discontinued on the morning of the dosing day 
(if using insulin glulisine [Apidra®]) OR at least 6 hours prior to dosing (if using other 
insulins). 
On the day prior to dosing (Day -1), the subjects will need to attend the clinical center 
and will be required to stay onsite overnight. On the morning of the dosing day (Day 1), 
patients are required to be in a fasting condition, defined as having consumed only water 
since 22:00 hours the night before. However, the subjects are allowed to consume small 


 
 
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amounts (up to 20 g) of carbohydrates to prevent hypoglycemia. The subjects must also 
not consume any alcohol within 24 hours prior to dosing (refer to Section 5.5 for all 
dosing day exclusion criteria). 
8.2.3 Visit 2 (dosing visit) 
Visit 2 will take place on Day -1 to Day 1. 
The subjects will attend the clinical center the day prior to dosing (Day -1) and subject 
eligibility is rechecked (check of changes between the screening visit and Visit 2) and 
those subjects eligible to participate should stay onsite overnight. To target a glucose 
level around 90-110 mg/dL (5.0-6.1 mmol/L) the following morning, subjects may be 
administered insulin glulisine (Apidra®) at the investigator’s discretion either by IV 
infusion, SC bolus or continuous SC insulin infusion. Dosing will take place the 
following morning (Day 1). 
On Day 1 and prior to the start of the insulin-induced hypoglycemic procedure,  those 
subjects eligible to participate will be randomized to treatment with dasiglucagon, 
placebo, or GlucaGen.  
The following assessments will also take place: 
 Document all changes in concomitant medication (prior to the start of the 
insulin-induced hypoglycemic procedure) 
 Check of withdrawal criteria (prior to the start of the insulin-induced hypoglycemic 
procedure) 
 Check of dosing day exclusion criteria (prior to the start of the insulin-induced 
hypoglycemic procedure) 
 Vital signs (prior to the start [within 30 minutes] of the insulin-induced hypoglycemic 
procedure, and at 30, 90 and 300 minutes after dosing). 12-lead ECG (prior to the 
start [within 30 minutes] of the insulin-induced hypoglycemic procedure, and at 20, 
35, 45, 60, and 300 minutes after dosing) 
 Local tolerability (at 30, 120, and 300 minutes after dosing) 
 AEs  
 Biochemistry, hematology (prior to the start of the insulin-induced hypoglycemic 
procedure [within 30 minutes], and at 30 and 300 minutes after dosing) 
 Urine stick pregnancy test (women of childbearing potential only; prior to the start of 
the insulin-induced hypoglycemic procedure) 
 Urinalysis (prior to the start of the insulin-induced hypoglycemic procedure [within 
120 minutes]) 
 Urine drug screen (prior to the start of the insulin-induced hypoglycemic procedure) 
 Alcohol breath test (prior to the start of the insulin-induced hypoglycemic procedure) 


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
Dasiglucagon/GlucaGen plasma concentrations:
- 
Pre-dose, 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time
of blood sampling for exposure should not deviate from the nominal time by more 
than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing. 

Plasma glucose concentrations:
- 
Pre-dose, 4, 6, 8 ,10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, and 90 minutes after
dosing. The actual time for blood sampling for plasma glucose should not deviate 
from the nominal time by more than ±30 seconds until the 20 minute collection 
time point and by more than ±1 minute for the subsequent collection time points. 
Pre-dose is defined as within 2 minutes prior to dosing. 

Antibodies against dasiglucagon/GlucaGen (prior to the start of the insulin-induced
hypoglycemic procedure).

Plasma insulin concentrations:
- 
Pre-dose, 30 and 60 minutes after dosing. The actual time of blood sampling for
plasma insulin should not deviate from the nominal time by more than ±1 minute. 
Pre-dose is defined as within 2 minutes prior to dosing. 
8.2.3.1 Hypoglycemic clamp procedure and administration of trial medication 
The following procedure is based on precendent procedures for hypoglycemia induction 
in patients with T1DM [24, 25]. 
The treatment day (Visit 2, Day 1) will be conducted after an overnight fast, targeting a 
starting plasma glucose around 90 to 110 mg/dL (5.0-6.1 mmol/L).   
Subjects who meet one or more of the dosing day exclusion criteria (Section 5.5) at the 
dosing visit will be excluded from the dosing visit, but the visit can be rescheduled 
1-7 days later. The dosing visit can only be rescheduled once. 
The subject’s current insulin therapy will be washed out as defined in Section 5.5. For 
subjects using multiple daily injections, the date, time and the dose of the last basal 
insulin and the last short-acting insulin (except insulin glulisine [Apidra®]) administration 
prior to dosing will be captured. For subjects using an insulin pump, the time of 
discontinuation of the basal rate will be captured. Any use of insulin glulisine (Apidra®) 
in the last 5 hours prior to initiation of the hypoglycemia induction procedure will also be 
captured.  
At approximately 08:00 hours, an infusion catheter will be inserted into each arm 
(forearm cephalic vein) for the manual glucose clamp procedure, with the glucose 
infusion in one arm and the insulin infusion in the opposite arm (if IV insulin glulisine 
[Apidra®] has been administered during the night the same infusion catheter can be used). 
A third catheter for blood sampling will be placed into a metacarpel vein for blood 


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sampling. This hand will be warmed (55-65ºC) to arterialize venous blood. If there are 
issues with blood sampling from the metacarpal vein for the purpose of glucose 
measurements, a new and more proximal IV access may be used at the discretion of the 
investigator. 
Hypoglycemia will be gradually induced by a fast-acting IV insulin glulisine (Apidra®) 
infusion (15 U [100 U/mL] in 49 mL saline and 1 mL of subject’s blood or plasma), 
initially at 150% of the subject’s usual basal rate and can be increased or decreased over a 
range of 0% to 200% or more as judged necessary by the investigator, to achieve a 
controlled decline in plasma glucose, targeting a plasma glucose level of 55 mg/dL (3.1 
mmol/L).  
Plasma glucose concentrations will be measured using the US FDA-approved glucose 
analyzer YSI 2300, Yellow Springs Instruments, Yellow Springs, OH or the Super GL 
analyzer, Dr. Müller Gerätebau GmbH, Freital, Germany. After the start of the insulin 
infusion, plasma glucose will be measured approximately every 10 minutes while plasma 
glucose is above 110 mg/dL, and approximately every 5 minutes once plasma glucose is 
at or below 110 mg/dL. 
Once the glucose concentration declines to <60 mg/dL (3.3 mmol/L), the insulin infusion 
will be stopped, and 5 min later plasma glucose concentration will be measured at the 
glucose analyzer and blood samples for baseline assessment of plasma glucose, 
dasiglucagon/GlucaGen PK, and insulin PK will be collected. The samples are the 
baseline samples and should be collected within 2 minutes before IMP administration. 

If plasma glucose is ≥45 mg/dL and <60 mg/dL (2.5-3.3 mmol/L), study treatment
(IMP) will be administered, defining time, t=0. The study treatment will be delivered
in the abdomen, buttock, or thigh (according to stratification) via SC injection.

If plasma glucose is <45 mg/dL (2.5 mmol/L), IV glucose solution will be
administered sufficient to raise plasma glucose to within the 45-60 mg/dL target
range. The run-in period will be adequately extended (at least 30 min) until the above
target is achieved and new baseline samples for plasma glucose,
dasiglucagon/GlucaGen PK, and insulin PK will be collected. Glucose should not be
infused within 10 minutes before IMP administration. If plasma glucose is not within
target range after the second attempt, the subject should be rescheduled for a new
treatment visit within 7 days (+ 2 days).
Administration of IMP should not occur earlier than 9:00 hours in the morning or later 
than 12:00 hours. 
As shown in Table 4, serial blood samples for glucose will be collected at t=0, 4, 6, 8, 10, 
12, 15, 17, 20, 25, 30, 40, 50, 60, 75 and 90 minutes post-dosing. Samples for assessing 


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plasma dasiglucagon/GlucaGen concentration will be collected  at t=0, 15, 30, 35, 40, 50, 
60, 90, and 120 minutes. Samples for assessing plasma insulin concentration will be 
collected at t=0, 30, and 60 minutes.  
Table 4 
Post-treatment blood sampling times 
Times 
0 
4 
6 
8 
10 
12 
15 
17 
20 
25 
30 
35 
40 
50 
60 
75 
90 
120 
Plasma glucose 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
PK 
Dasiglucagon/ 
GlucaGen 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
PK Insulin 
Y 
Y 
Y 
Refer to Section 7.2.7 for details of laboratory safety sampling and to Section 7.4.2 for 
details of blood glucose safety sampling.  
When the t=90-minute blood sampling for plasma glucose has been collected the subjects 
are allowed to eat moderately. Drinking of water is allowed ad libitum during the entire 
procedure. 
Hypoglycemia Rescue Provisions 
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at 
site for safety reasons as described in Section 7.4.2. Subjects should receive 
post-treatment rescue glucose infusion to ameliorate persistent hypoglycemia, as follows. 
1. Glucose infusion should be initiated if a subject experiences severe alarming
escalation of symptoms of hypoglycemia (e.g. symptoms suggesting a change in
consciousness) at any time during the trial; glucose infusion should be initiated
targeting a plasma glucose levels >70 mg/dL.
2. If plasma glucose is <45 mg/dL (2.5 mmol/L) between t=8 and t=44 minutes,
rescue glucose infusion (1-2 mg/kg administered IV over about 5 seconds) should
be initiated to maintain plasma glucose between 45 mg/dL and 55 mg/dL
(2.5-3.1 mmol/L). Pause glucose infusion if plasma glucose is >55 mg/dL.
3. If plasma glucose is <70 mg/dL at t=45 minutes, rescue glucose infusion
(2-3 mg/kg administered IV over about 5 seconds) should be initiated to maintain
plasma glucose between 70 mg/dL and 80 mg/dL (3.9-4.4 mmol/L). Pause
glucose infusion if plasma glucose is >75 mg/dL (4.2 mmol/L).
Subjects should remain in bed until completion of the test procedure 300 minutes after 
dosing (bathroom visits are allowed).  


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The IMP will be administered SC according to Section 6.3. The time of IMP 
administration will be recorded. At the timepoint when the insulin infusion is stopped, the 
total insulin dose which was required to induce hypoglycemia will be recorded. 
AEs will be specifically recorded during the procedure at several timepoints. 
The investigator must provide information to the subjects on how to resume their usual 
diabetes treatment. 
The subject may be released from the clinical center if the investigator does not have any 
safety concerns based on the last safety plasma glucose value and the general condition of 
the subject. However, at the discretion of the investigator or on request of the subject, the 
subject may stay at the trial center for a longer period. 
8.2.4 Visit 3 (follow-up visit) 
Visit 3 will take place on Day 28 + 5 days. The subject does not need to be fasting. 
At Visit 3, the following assessments will take place: 

Document all changes in concomitant medication

Physical examination

Vital signs

12-lead ECG

Local tolerability

AEs

Biochemistry, hematology

Urine stick pregnancy test (women of childbearing potential only)

Urinalysis

Antibodies against dasiglucagon/GlucaGen.
After the follow-up visit the End of Trial form must be completed. Even if a subject is not 
able to attend the follow-up visit, the End of Trial form, the eCRF 
Accountability/Affirmation Statement form and the Drug Accountability form must be 
completed. 


 
 
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9 
STATISTICAL METHODS 
Before database lock and treatment unmasking, a separate statistical analysis plan (SAP) 
will be finalized, providing detailed methods for the analyses outlined below. Further 
analysis details may be added or refined in the SAP. 
Any deviations from the planned analyses will be described and justified in the final 
clinical trial report. 
9.1 
Determination of sample size 
Due to requirements in the size of the safety database, the sample size is set to 78 subjects 
treated with dasiglucagon 0.6 mg. From phase 2, the median time to an increase of 20 
mg/dL of the 0.6 mg dose was approximately 10 minutes. With a 2:1:1 randomization 
ratio for the three treatments, dasiglucagon 0.6 mg, placebo, and GlucaGen 1 mg, and 
assuming an exponential time-to-recovery distributions with median times of 10 and at 
least 20 minutes for dasiglucagon and placebo, respectively, a two-sided log-rank test 
will be able to detect a difference between dasiglucagon and placebo with 90% power 
with a follow-up time of 45 minutes at a 5% significance level with 78 subjects treated 
with dasiglucagon and 39 subjects with placebo. The median time-to-recovery for 
placebo is expected to be longer than 20 minutes, which means that the power will be 
greater than 90%. 
9.2 
Trial subjects 
9.2.1 Analysis samples 
For presentation of data and reporting of the statistical analyses, the following analysis 
samples will be used, depending on the context:  
 Safety analysis set (SAS): All randomized subjects who received at least one dose of 
trial medication.  
 Full analysis set (FAS): All randomized subjects who received at least one dose of 
trial medication. 
 Per protocol (PP) set: All subjects of the FAS for whom no relevant protocol 
deviations were documented. This sample will primarily be used for sensitivity 
analysis.  
The analysis of the primary endpoint and secondary endpoints will be based on the FAS. 
A secondary analysis of the primary endpoint will be based on the PP set. All safety 
analyses will be based upon the SAS. 


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The decision regarding whether a protocol deviation is relevant or not for the exclusion of 
subjects from the PP set will be made case-by-case in a data review meeting prior to 
treatment unmasking and database lock (see Section 9.2.3). 
9.2.2 Disposition of subjects 
Subject disposition will be tabulated including the number of screened subjects, screening 
failures, subjects exposed to trial product, subjects completing the trial and subjects in 
each analysis set. 
Subjects withdrawn from the trial will be listed including the primary reason for 
withdrawal. 
9.2.3 Protocol deviations 
Before data are released for statistical analysis, a treatment-masked review of all data will 
take place to identify protocol deviations that may potentially affect the results. This 
review will be performed without revealing to which trial product the subjects were 
assigned. The masking of the trial products will be maintained for everyone involved in 
allocating subjects to the analysis sets until data are released for statistical analysis. 
Furthermore, spurious outliers will be evaluated. In addition, protocol deviations that may 
potentially affect the results will be identified and it will be evaluated if subjects and/or 
data should be excluded from the analysis. Protocol deviations will be classified as minor 
or major in a consistent way. Major deviations from the protocol may lead to the 
exclusion of a subject from the PP set. 
Major protocol deviations may include deviations related to trial inclusion or exclusion 
criteria, conduct of the trial, subject management or subject assessment. Unless explicitly 
decided otherwise during the masked data review, the following will be considered major 
protocol deviations: 

Violation of one or more major inclusion/exclusion criteria

Interruption of administration of trial product

Significant deviation from time windows

Incorrect treatment allocation

Missing primary endpoint.
The violation of several major inclusion/exclusion criteria or the complete absence of 
efficacy data might lead to exclusion of the subject from FAS. In that case, the decision 
should be taken at the masked data review meeting, and the exclusion from efficacy 
analysis justified in the signed notes of the meeting. 


 
 
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Obviously erroneous data points may be excluded from the analyses or re-analyzed (in 
case of e.g. serum concentrations). The decision to re-analyze or exclude data points from 
the statistical analysis is the joint responsibility of the sponsor and the trial statistician.  
The subjects or observations to be excluded and the reason for their exclusion will be 
documented and signed by those responsible prior to database release. The 
documentation will be stored together with the remaining trial documentation. The 
subjects and observations excluded from analysis sets, and the corresponding reasons, 
will be described in the clinical trial report. 
9.3 
General considerations 
All data obtained in this trial and documented in the eCRFs will be listed and 
summarized with statistics or frequency tables as appropriate. In case of termination of 
the trial, all data collected up to that timepoint will be included in the analysis. 
Raw data listings and summary tables will be generated using the software SAS© version 
9.4 or higher.  
Continuous variables will be summarized using means, standard deviations, medians, 
coefficients of variation, and minimum and maximum values.  
Other summaries (e.g. quartiles, 95% confidence intervals [CIs]) may be used as 
appropriate. Categorical efficacy and safety variables will be summarized by counts and 
by percentage of subjects in corresponding categories. 
9.4 
Demographics and baseline characteristics 
Baseline and demographic data will be summarized using descriptive statistics. Baseline 
ADA-positive subjects will be calculated as a percentage of the total number of subjects 
whose baseline samples were tested for ADA. 
All other data obtained in this trial and documented in the eCRF will be listed. 
9.5 
Efficacy Analyses 
For the confirmatory analyses, the primary and key secondary endpoints for the 
dasiglucagon 0.6 mg and placebo treated subjects will be compared. A hierarchical 
procedure will be applied for the control of multiplicity. The primary and secondary 
endpoints will be evaluated on the full analysis set. In the primary analysis, those subjects 


 
 
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who require rescue IV glucose will be censored at the time to plasma glucose recovery.  
In a sensitivity analysis, the time to plasma glucose recovery will be analyzed without 
censoring the subjects who received rescue IV glucose.   
9.5.1 Hierarchical testing procedure 
For the confirmatory analyses, the following a priori defined hierarchical inferential test 
order will be applied for the control of the type 1 error rate across the planned multiple 
comparisons. The primary endpoint and key secondary endpoints will be inferentially 
evaluated within the FAS in the following order, where inference will proceed at the 
two-sided 0.05 criterion significance level until the first failure to reject the null 
hypothesis for a dasiglucagon versus placebo comparison: 
 Primary:  Time to plasma glucose recovery 
 Key secondaries 1-4: Plasma glucose recovery within 30 minutes, within 20 minutes, 
within 15 minutes, and within 10 minutes after study drug injection without 
administration of rescue IV glucose. 
 Key secondaries 5-8:  Plasma glucose changes from baseline (CFB) within 30 
minutes, within 20 minutes, within 15 minutes, and within 10 minutes after study 
drug injection or at the time of rescue. 
The GlucaGen versus placebo comparisons will not be included in the inferential testing 
hierarchy, since the efficacy of GlucaGen is previously established, and these 
comparisons are intended to support the validity of the study for the dasiglucagon versus 
placebo comparisons. 
Analogous supportive sensitivity analyses will be conducted in the PP set, but without 
inference intent. 
9.5.2 Primary confirmatory endpoint 
Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase in 
plasma glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic 
clamp procedure without administration of rescue IV glucose.  
9.5.2.1 Primary analysis 
The primary endpoint will be summarized using Kaplan-Meier (KM) estimates stratified 
by treatment group and injection site. The treatment group difference between 
dasiglucagon and placebo will be evaluated inferentially using a pairwise two-sided 
log-rank test. 


 
 
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In the primary analysis, recovery cannot be achieved in those subjects where IV glucose 
treatment is administered. Those subjects who receive IV glucose will be censored (i.e. 
set to ‘not recovered’) at 45 minutes after dosing. 
In sensitivity analyses, the time to plasma glucose recovery will be analyzed 1) without 
censoring for those subjects who require rescue IV glucose before 45 minutes and 2) with 
censoring at the time of administration of rescue IV glucose before 45 minutes.   
The primary endpoint will additionally be analyzed using a Cox proportional hazards 
(CPH) time to event statistical model, if the hazards by treatment group can be assumed 
proportional using a graphical comparison of log-minus-log survival curves and no more 
than two-thirds of the recovery times are censored in each treatment group. The CPH 
model will be used for inferences, with treatment group and injection site modeled as 
categorical effects, and baseline plasma glucose modeled as a continuous covariate. The 
treatment group rate ratios, active versus placebo, will be estimated together with the 
95% confidence intervals, and pairwise treatment group inferences (dasiglucagon vs 
placebo, GlucaGen vs placebo) will be evaluated using two-sided likelihood ratio tests. 
9.5.3 Secondary endpoints 
 Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection without administration of rescue IV 
glucose. 
 Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection or at the time of rescue. 
9.5.3.1 Confirmatory analysis 
The key secondary incidence variables (plasma glucose recovery within 30 minutes, 
within 20 minutes, within 15 minutes, within 10 minutes, in hierarchical order for 
inference) will be analyzed using descriptive statistics (frequency and percentage) by 
treatment group. Inferential treatment group comparisons will be assessed via pairwise 
tests of independent binomial proportions (dasiglucagon versus placebo, GlucaGen 
versus placebo) using Fisher’s exact test for each pairwise comparison. 
The key secondary endpoints of plasma glucose CFB within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection or at the 
time of rescue, will be analyzed with the plasma glucose CFB at rescue carried forward in 
those subjects who require rescue IV glucose before plasma glucose ≥20 mg/dL recovery. 
Each of these CFB variables will be analyzed using an Analysis of Covariance model, 
with treatment group modeled as a fixed effect and with the baseline plasma glucose 
modeled as a covariate. The dasiglucagon versus placebo treatment group difference will 
be evaluated inferentially as a least squares means contrast, using a two-sided t-test at the 


 
 
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0.05 signficance level, first for the 30 minute CFB, and then sequentially for the 
20 minute, 15 minute, and 10 minute variables, until the first failure to reject. 
9.5.4 Secondary clinical efficacy (PD) endpoints 
 Time to first plasma glucose concentration of ≥70 mg/dL (3.9 mmol/L) without 
administration of rescue IV glucose. 
 Plasma glucose response as area under the curve above baseline from time zero to 
30 minutes, AUC0-30min. 
Secondary clinical efficacy endpoints will be assessed based on plasma concentration 
data from samples collected at the dosing visit (Visit 2).  
Unless otherwise stated, the population base of analysis will be the FAS. 
9.5.4.1 Analysis of secondary clinical efficacy (PD) endpoints 
1. Time to first plasma glucose concentration ≥70 mg/dL from baseline. This 
time-to-event endpoint will be evaluated using a Kaplan-Meier approach, with 
treatment group as a stratification factor, analogous to that used for the primary 
endpoint analysis. Differences between the KM curves (dasiglucagon versus 
placebo, GlucaGen versus placebo) will be evaluated inferentially using pairwise 
two-sided log rank tests. If the ≥70 mg/dL endpoint is not met within 45 minutes 
post-dosing, the time of the last valid plasma glucose measurement up to 45 
minutes will be the censoring time.  
2. The AUC will be calculated as the baseline-adjusted area under the plasma 
glucose profile over time: 
a. AUC0-30min: restricting the time window to the 0 to 30 minutes interval. 
3. The log-transformed AUC endpoint will be analyzed using an analysis of 
covariance (ANCOVA) model with treatment as fixed effect and baseline plasma 
glucose modeled as a covariate. The least squares means treatment group 
differences will be back-transformed (anti-logged) for presentation as a ratio of 
the treatment group geometric means, with their corresponding 95% CI. 
9.5.5 Exposure (PK) endpoints 
 Plasma dasiglucagon and GlucaGen concentrations from time zero to 90 minutes:  
AUC0-90min, Cmax, and tmax. 


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9.5.5.1 Analysis of exposure (PK) endpoints 
AUC will be derived as the area under the individual plasma dasiglucagon/GlucaGen 
concentration profile for PK from 0 to 90 minutes or last valid measurement if this 
measurement is assessed sufficiently close to 90 minutes (decision to be taken at the 
masked data review meeting). The standard trapezoidal method will be used, based on 
actual rather than nominal timepoints.  
Cmax will be determined as the maximum of all valid plasma dasiglucagon/GlucaGen 
concentrations.  
Tmax will be determined as the timepoint where the maximum of all valid plasma 
dasiglucagon/GlucaGen concentration measurements for each measurement series is 
observed. 
The log-transformed PK endpoints AUC and Cmax will be analyzed in the same way as 
the AUC endpoints. 
As tmax is a highly discrete endpoint, Wilcoxon’s rank sum test for unpaired observations 
will be used to assess differences between the two treatment groups. 
9.6 
Exploratory analyses 
Exploratory analyses will include descriptive statistics and modeling analogous to that 
done for key secondary endpoints. However, treatment group comparisons will be 
summarized without inference intent. 

Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL
(1.1 mmol/L) within 30 minutes after study drug injection without administration
of rescue IV glucose.

Plasma insulin response as area under the curve above baseline from time zero to
60 minutes, AUC0-60 min.
Plasma insulin concentrations measured pre-dose and at 30 and 60 minutes after dosing 
(see Table 3) will be presented individually. A summary table per timepoint will be 
provided. The AUC0-60min will be determined and a summary presented.  


 
 
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9.7 
Safety analyses 
9.7.1 Adverse events 
AEs will be coded using the latest available version of the MedDRA, the version of 
which will be provided in the clinical trial report. AE summary tables will include the 
number and percentage of subjects who experienced AEs summarized by system organ 
class and preferred term. 
Germany only: The AE summary for AEs related to insulin glulisine (Apidra®) will be 
presented as an appendix to the clinical trial report, as insulin glulisine (Apidra®) is only 
defined as an IMP in Germany. 
An overall summary table will be provided showing the number and percentage of 
subjects with any: 
 TEAE 
 Severe TEAE 
 Serious TEAE 
 Drug-related TEAE 
 Drug-related severe TEAE 
 Drug-related serious TEAE 
 TEAE leading to withdrawal 
 TEAE with outcome death 
 AESI 
Refer to Section 7.2.1 for the definition of TEAEs. 
9.7.2 Immunogenicity data 
Immunogenicity data will be analyzed descriptively by treatment group. No statistical 
tests are planned. Baseline ADA-positive subjects will be calculated as a percentage of 
the total number of subjects whose baseline samples were tested for ADA. Titer will be 
reported as median and interquartile range. 
9.7.3 Clinical laboratory assessments 
Clinical laboratory test results will be flagged as to whether the result is below, within or 
above the respective reference range. The number of values outside of the reference range 
will be counted. 


 
 
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9.7.4 Other safety data 
Incidence of rescue infusion of IV glucose during the hypoglycemic clamp procedure will 
be analyzed using descriptive statistics (frequency and percentage) by treatment group. 
Inferential treatment group comparisons will be assessed via pairwise tests of 
independent binomial proportions (dasiglucagon versus placebo, GlucaGen versus 
placebo). 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure 
will be evaluated using a KM time to event statistical model, with treatment group and 
injection site as stratification factors. Differences between the KM curves (dasiglucagon 
versus placebo, GlucaGen versus placebo) will be evaluated inferentially using pairwise 
two-sided stratified log-rank tests. If the endpoint is never met, the time of the last plasma 
glucose measurement will be the censoring time. 
Vital signs, physical examination, 12-lead ECG and local tolerability data will be 
summarized using descriptive statistics. 
9.8 
Treatment compliance 
Trained unblinded members of staff will perform all administrations of the IMP at the 
trial center. The administered doses will be recorded in the blinded Drug Accountability 
form in the eCRF. 
PK assessments will support the surveillance of compliance with IMP administration. 
9.9 
Subject withdrawals and missing data 
Failure is defined for the primary time-to-recovery endpoint as censored with time to 
recovery set to the maximum follow-up time, here 45 minutes. Individuals will be set to 
‘failure’ in case of receiving rescue IV glucose, discontinuation due to treatment or 
discontinuation in connection with the induced hypoglycemia state. Only if an 
intermediate assessment is missing independent of rescue treatment, study treatment or an 
adverse event including hypoglycemia, interpolation can be applied to impute a missing 
value. 
The same construct of imputation applies for the analysis of the plasma glucose AUC0-
30min. 


 
 
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In the case of subject withdrawal, no imputation of values for PK or PD measurements 
will be done. Analyses will be done on valid cases only, i.e., no imputation techniques 
such as last observation carried forward will be applied.  
9.10 Interim analyses 
No interim analyses are currently planned. 


 
 
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10 
ETHICAL, LEGAL, AND ADMINISTRATIVE ASPECTS 
10.1  Quality assurance 
The sponsor or designee will conduct a site visit to verify the qualifications of each 
investigator, inspect the facilities, and inform the investigator of responsibilities and the 
procedures for ensuring adequate and correct documentation. 
The investigator is required to prepare and maintain adequate and accurate case histories 
designed to record all observations and other data pertinent to the trial for each trial 
participant. All information recorded on the eCRFs for this trial must be consistent with 
the subjects’ source documentation (i.e. medical records). 
The investigator will permit trial-related monitoring, IRB/IEC review, and regulatory 
inspections, providing direct access to source data /documents. Sponsor-authorized 
quality assurance personnel may carry out audits for which the investigator must provide 
support. 
The trial monitor or a representative of the sponsor will cross-check the data entered in 
the eCRFs with the source data at the trial center and observe the trial procedures in order 
to verify adherence to the trial protocol. Any queries will be resolved by the investigator 
or his/her delegate. 
All of the clinical data will be captured via electronic data capture (EDC) using a 
web-based tool.  
The investigator center staff will enter and edit the data via a secure network, with secure 
access features (username and password). A complete electronic audit trail will be 
maintained. The investigator will approve the data using an electronic signature (Ref: 21 
CFR Part 11), and this approval is used to confirm the accuracy of the data recorded. 
eCRFs will be used for all subjects. The investigator’s data will be accessible from the 
investigator’s center throughout the trial. The eCRFs must be kept current to reflect 
subject status at each phase during the course of the trial. The eCRF will not capture 
personalized data. The investigator must make a separate confidential record of 
personalized details (name and initials) on the subject identification and enrollment log. 
All changes to data are made by the investigator or his/her delegate through the EDC 
system.  
It is the responsibility of the principal investigator of the respective center to ensure that 
all subject discontinuations or changes in trial or other medications entered on the 
subject’s eCRF are also made on the subject’s medical records. 


 
 
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The eCRFs for any subject leaving the trial should be completed at the time of the final 
visit or shortly thereafter. 
10.2  Electronic case report forms 
Remote data capture software will be used for data collection. Following training, trial 
staff will be given access to the software. Access to the software is restricted to staff 
participating in the trial and the extent of access will depend on the participants' user role 
in the trial. 
The subjects enrolled into the trial will be identified in the database by subject number 
and trial identification code. The investigator or delegate will enter subject data into the 
eCRF promptly. All data is to be entered in English. Data recorded in the eCRFs will be 
accessible to the trial staff throughout the trial. 
After data entry, systematic data validation will be performed and data entry 
discrepancies will be presented electronically directly to the center staff. Queries for 
discrepant data may be generated automatically by the software upon entry and/or 
generated manually by the trial monitor or the trial data manager. All queries, whether 
generated by the system or by trial staff, will be in electronic format. 
All sections of the eCRF are to be electronically approved by the investigator or a 
medically qualified delegate after the data has been entered and all queries have been 
resolved. Changes to any eCRF page subsequent to the approval require a new approval 
signature. 
All queries and changes/corrections to the data are documented in the eCRF. 
10.3  Access to source data 
During the course of the trial, a trial monitor will make site visits to review protocol 
compliance, compare eCRFs with individual subject’s medical records, assess drug 
accountability, and ensure that the trial is being conducted according to pertinent 
regulatory requirements. 
Checking of the eCRFs for completeness and clarity, and cross-checking with source 
documents, will be required to monitor the progress of the trial. Moreover, regulatory 
authorities of certain countries, IRBs/IECs, and/or the sponsor may wish to carry out such 
source data checks and/or on-site audit inspections. Direct access to source data will be 
required for these inspections and audits; they will be carried out giving due 
consideration to data protection and medical confidentiality. 


 
 
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10.4  Source documentation 
All source documents from which eCRF entries are derived should be placed in the 
subject’s medical records. If data are to be entered directly into the eCRF this must be 
specified in a source data agreement prior to the start of the trial. 
eCRF entries will be verified with source documentation. The review of medical records 
will be performed in a manner to ensure that subject confidentiality is maintained. 
The trial monitor will check the eCRF for accuracy and completion and perform source 
data verification. The trial monitor will document source data verification of all reviewed 
sections of the eCRF. 
10.5  Data processing 
The trial is run as an EDC trial, i.e. all relevant data is entered by the centers directly into 
the clinical database. The eCRF is designed to capture all required information in 
compliance with GCP standards. 
10.6  Archiving trial records 
According to ICH guidelines, essential documents should be retained for a minimum of 
2 years after the last approval of a marketing application in an ICH region and until there 
are no pending or contemplated marketing applications in an ICH region or at least 
2 years have elapsed since the formal discontinuation of clinical development of the 
investigational product. However, these documents should be retained for a longer period 
if required by the applicable legal requirements. 
Records and documents pertaining to the conduct of the trial and the distribution of the 
investigational product (e.g. informed consent forms, laboratory slips, medication 
inventory records, and other pertinent information) must be retained by the investigator 
according to local requirements. 
10.7  Good clinical practice 
The procedures set out in this trial protocol are designed to ensure that the sponsor and 
investigator abide by the principles of the GCP guidelines of the ICH (1), and of the 
Declaration of Helsinki (2008) (2). The trial also will be carried out in keeping with local 
legal requirements.  


 
 
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10.8  Informed consent 
Before each subject is admitted to the trial, written informed consent will be obtained 
from the subject according to the regulatory and legal requirements of the participating 
country. This consent form must be dated and retained by the investigator as part of the 
trial records. The investigator will not undertake any investigation specifically required 
only for the clinical trial until valid consent has been obtained. The terms of the consent 
and when it was obtained must also be documented in the eCRF. 
The investigator will explain to each subject orally and in writing (subject information 
sheet) the nature, significance, risks and implications of the trial before inclusion. In 
particular, the subjects will be informed about the following: 
 The possibility of withdrawing from the clinical trial at any time by revoking the 
consent and without any resulting disadvantage. 
 How personal and health-related data will be collected and used during the trial. 
 That his/her medical records may be examined by authorized monitors or clinical 
quality assurance auditors appointed by the sponsor, by appropriate IRB/IEC 
members, and by inspectors from regulatory authorities. 
All subjects will receive a copy of the subject information sheet and a copy of their 
signed and dated informed consent form, both of which will be in the subject’s local 
language. 
If a protocol amendment is required, the informed consent form may need to be revised to 
reflect the changes to the protocol. If the consent form is revised, it must be reviewed and 
approved by the appropriate IEC/IRB, and signed by all subjects subsequently enrolled in 
the trial as well as those currently enrolled in the trial. 
10.9  Protocol approval and amendment 
Before the start of the trial, the trial protocol and/or other relevant documents will be 
approved by the IEC/IRB/competent authorities, in accordance with local legal 
requirements. The sponsor must ensure that all ethical and legal requirements have been 
met before the first subject is enrolled in the trial. 
This protocol is to be followed exactly. To alter the protocol, amendments must be 
written, receive approval from the appropriate personnel, and receive IRB/IEC/competent 
authority approval prior to implementation (if appropriate).  


 
 
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Administrative changes (not affecting the subject benefit/risk ratio) may be made without 
the need for a formal amendment. All amendments will be distributed to all protocol 
recipients, with appropriate instructions. 
10.10  Duration of the trial 
The maximum duration of the trial for each subject will be up to 63 days (including up to 
30 days for screening and up to 33 days until the follow-up visit).  
The trial will be closed when all subjects have completed Visit 3. 
10.11  Premature termination of the trial 
If the investigator, the sponsor (e.g. safety committee), or the safety medical monitor 
becomes aware of conditions or events that suggest a possible hazard to subjects if the 
trial continues, the trial may be terminated after appropriate consultation between the 
relevant parties. The trial may also be terminated early at the sponsor’s discretion in the 
absence of such a finding. 
Conditions that may warrant termination include, but are not limited to: 
 The discovery of an unexpected, significant, or unacceptable risk to the subjects 
enrolled in the trial 
 Failure to enroll subjects at an acceptable rate. 
 A decision on the part of the sponsor to suspend or discontinue development of the 
drug. 
The trial can be terminated prematurely by the sponsor at an individual center if: 
 The center cannot comply with the requirements of the protocol. 
 It is not possible for the center to comply with GCP standards. 
10.12  Confidentiality 
All trial findings and documents will be regarded as confidential. The investigator and 
members of his/her research team must not disclose such information without prior 
written approval from the sponsor. 
The anonymity of participating subjects must be maintained. Subjects will be identified 
on eCRFs submitted to the sponsor by their subject number. On the SAE reports and all 
other source documents, the subject will be identified via subject number. Documents not 


 
 
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to be submitted to the sponsor that identify the subject (e.g. the signed informed consent 
form) must be maintained in confidence by the investigator.  
10.13  Other ethical and regulatory issues 
If a significant safety issue is identified, either from an individual case report or review of 
aggregate data, then the sponsor will issue prompt notification to all parties: regulatory 
authorities, investigators and IRB/IECs. 
A significant safety issue is one that has a significant impact on the course of the clinical 
trial or program (including the potential for suspension of the trial program or 
amendments to protocols) or warrants immediate update of informed consent. 
10.14  Liability and insurance 
The sponsor will take out reasonable third-party liability insurance cover in accordance 
with all local legal requirements. The civil liability of the investigator, the persons 
instructed by him and the hospital, practice or institute in which they are employed and 
the liability of the sponsor with respect to financial loss due to personal injury and other 
damage that may arise as a result of the carrying out of this trial are governed by the 
applicable law. 
The sponsor will arrange for liability insurance if subjects should be injured due to the 
participation in the trial and provided that the sponsor is legally liable for that. 
Excluded from the insurance cover are injuries to health and deteriorations of illnesses 
already in existence which would have occurred or continued to exist even if the subject 
had not taken part in the clinical trial. 
The insurance cover is jeopardized if the subject fails to report immediately to the 
investigator or responsible physician any injury to health, which might have resulted 
from participation in the clinical trial, or if he/she undergoes any other medical treatment 
without their consent before the clinical trial has been completely finished in so far as the 
individual subject is concerned. 
Any injury to health, which might have occurred as a result of participation in the clinical 
trial must be reported by the subject to the investigator without delay. The investigator is 
obliged to make such a report in any case. 


 
 
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10.15  Publication policy 
By signing the trial protocol, the investigator agrees with the use of results of the trial for 
the purposes of national and international registration, publication and information for 
medical and pharmaceutical professionals. If necessary, the authorities will be notified of 
the investigator's name, address, qualifications and extent of involvement. 
A clinical trial report will be prepared and reviewed by the sponsor in co-operation with 
the coordinating investigator. The coordinatoing investigator will be appointed by 
Zealand Pharma to review and sign the clinical trial report on behalf of all participating 
investigators. A summary of the clinical trial report (according to CPMP/ICH137/95) 
should be sent to the regulatory authorities and IRB/IEC according to the relevant 
guidelines. 
According to the Declaration of Helsinki (2) investigators and sponsors ‘have ethical 
obligations with regard to the publication and dissemination of the results of research’.   
The trial design and results may be published as one or more original research 
manuscripts/abstracts and presented at a scientific meeting. The original eCRFs and the 
data generated from the eCRFs or otherwise obtained during the trial under this trial 
protocol will become the property of the sponsor. Publication of the results of this trial by 
the investigator is possible only after written consent has been obtained from the sponsor. 
Any material intended for publication will be given to the sponsor at least 4 weeks before 
submission for publication. The sponsor will have the right to comment on the intended 
publication and to take any reasonable measures for patent protection. Authorship on any 
publication(s) resulting from this clinical trial will be assigned according to the 
recommendations of the International Committee of Medicinal Journal Editors (26). 
Participating subjects will not be identified by name in any published reports about the 
clinical trial. 
The sponsor is responsible for trial registration at ClinicalTrials.gov 
(www.clinicaltrials.gov) according to FDA requirements, as well as the European 
Medicines Agency’s Clinical Trials Database (EudraCT).  


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11 
REFERENCE LIST  
1. ICH Harmonised Tripartite Guideline. International Conference on Harmonisation. 
ICH Harmonised Tripartite Guideline. Integrated addendum to ICH E6(R1): 
Guideline for Good Clinical Practice E6 (R2), Step 4. 09-Nov-2016. Available from: 
URL:
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E 
6/E6_R2__Step_4.pdf
2. World Medical Association. Declaration of Helsinki. Ethical Principles for Medical
Research Involving Human Subjects. 52nd WMA General Assembly, Edinburgh,
Scotland, October 2000. Last amended with Note of Clarification on Paragraph 29 by
the WMA General Assembly, Washington 2002, and Note of Clarification on
Paragraph 30 by the WMA General assembly, Tokyo 2004
3. Seaquist ER, Anderson J, Childs B, Cryer P, Dagogo-Jack S, Fish L, et al.
Hypoglycemia and diabetes: a report of a workgroup of the American Diabetes
Association and the Endocrine Society. Diabetes Care 2013 May;36:1384-95
4. Cryer PE. The barrier of hypoglycemia in diabetes. Diabetes 2008;57:3169-76
5. American Diabetes Association Position Statement. Standards of medical care in
diabetes - 2017. Diabetes Care 2017;40(Supplement 1):S1-S135
6. Davies MJ, Gagliardino JJ, Gray LJ, Khunti K, Mohan V, Hughes R. Real-world
factors affecting adherence to insulin therapy in patients with Type 1 or Type 2
diabetes mellitus: a systematic review. Diabetic Medicine 2013;30:512-24
7. International Hypoglycaemia Study Group. Glucose concentrations of less than 3.0
mmol/L (54 mg/dL) should be reported in clinical trials: a joint position statement of
the American Diabetes Association and the European Association for the Study of
Diabetes. Diabetes Care 2017;40(1):155-57
8. Christensen M, Bagger JI, Vilsbøll T, Knop FK. The alpha-cell as target for type 2
diabetes therapy. Rev Diabet Stud 2011;8:369-81
9. Lund A, Bagger JI, Christensen M, Knop FK, Vilsbøll T. Glucagon and type 2
diabetes: the return of the alpha cell. Curr Diab Rep 2014;14:555
10. Habegger KM, Heppner KM, Geary N, Bartness TJ, DiMarchi R, Tschöp MH. The
metabolic actions of glucagon revisited. Nat Rev Endocrinol 2010;6:689-97
11. Onoue S, Ohshima K, Debari K, Koh K, Shioda S, Iwasa S, et al. Mishandling of the
therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils. Pharm Res
2004;21:1274-83


 
 
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12. Fosgerau K, Hoffmann T. Peptide therapeutics: current status and future directions. 
Drug Discovery Today 2015;20:122-8 
13. Novo Nordisk Limited. SPC for GlucaGen Hypokit 1 mg. 2015. 
http://www.medicines.org.uk/emc/medicine/4258/SPC/GlucaGen+Hypokit+1+mg - 
Accessed 13 Mar 2017 . 
14. Zealand Pharma A/S. Dasiglucagon Investigator's Brochure, Edition 3, 12 Dec 2016.  
15. The Task Force for the management of arterial hypertension of the European Society 
of Hypertension (ESH) and of the European Society of Cardiology (ESC). 2013 
ESH/ESC guidelines for the management of arterial hypertension. European Heart 
Journal 2013;34:2159-219 
16. Levey AS, Coresh J, Greene T, Stevens LA, Zhang Y, Hendriksen BA, et al. Using 
standardized serum creatinine values in the modification of diet in renal disease study 
equation for estimating glomerular filtration rate. Annals of Internal Medicine 
2006;145:247-54 
17. Food and Drug Administration (FDA). Guidance for Industry on Bioanalytical 
Method Validation. Food and Drug Administration (FDA) 2001 
18. Viswanathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ, Sailstad J, et al. 
Quantitative bioanalytical methods validation and implementation: best practices for 
chromatographic and ligand binding assays. Pharm Res 2007 Oct;24:1962-73 
19. Shankar G, Devanarayan V, Amaravadi L, Barrett YC, Bowsher R, Finco-Kent D, et 
al. Recommendations for the validation of immunoassays used for detection of host 
antibodies against biotechnology products. J Pharm Biomed Anal 2008;48:1267-81 
20. Zealand Pharma report SS. Commissioning and validation of a method for the 
validation of anti-ZP4207 antibodies in human serum.  2014  
21. Zealand Pharma report SS. Commissioning and validation of a method for the 
validation of anti-glucagon antibodies in human serum.  2014  
22. Bioagilytix Labs. Validation of an assay to detect neutralizing antibodies to ZP4207 
by measuring cyclic adenosine monophosphate (cAMP) in a glucagon receptor 
transfected cell line, BAL-14-206-004-REP.  2016. Report No.: Zealand Pharma, 
study 15-039 
23. Bioagilytix Labs. Validation of an assay to detect neutralizing antibodies to glucagon 
by measuring cyclic adenosine monophosphate (cAMP) in a glucagon receptor 
transfected cell line, BAL-14-206-005.  2016. Report No.: Zealand Pharma, Study 
15-040 


 
 
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24. Amiel SA, Tamborlane WV, Simonson DC, et al. Defective glucose 
counterregulation after strict glycemic control of insulin-dependent diabetes mellitus. 
N Eng J Med 1987; 316: 1376-83. 
25. Rickels MR, Ruedy KJ, Foster NC, et al. Intranasal glucagon for treatment of insulin-
induced hypoglycemia in adults with type 1 diabetes: A randomized crossover 
noninferiority study. Diabetes Care 2016; 39: 264-70. 
26. ICMJE. Recommendations for the conduct, reporting, editing, and publication of 
scholarly work in medical journals. Updated December 2016 
 
 
 
 


 
 
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12 
APPENDICES 
12.1 List of trial personnel 
Sponsor 
Clinical Trial Manager 
 
 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Phone:
 
Medical Officer 
 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Phone: 
 
Contract Research Organization 
Chiltern International (Ltd) 
171 Bath Road 
Slough 
Berkshire 
SL1 4AA 
United Kingdom 
Phone: +44 (0) 175351 2000 
Project Manager 
 
Chiltern International (Ltd) 
171 Bath Road 
Slough 
Berkshire 
SL1 4AA 
United Kingdom 
Phone: 
  
Senior Medical Officer and 
Safety medical monitor 
 
Chiltern International kft 
Canada Square Office House 
Ganz u 


 
 
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12-14, 4 emelet 
1027 Budapest 
Hungary 
Phone: 
 
Pharmacovigilance unit 
Responsible for Serious Adverse 
Event (SAE) Management and 24-
hour SAE reporting 
  
PharmaLex 
Agern Allé 24 
2970 Hørsholm, Denmark 
Phone: 
 (8 a.m. to 4 p.m.) 
Phone: 
 (outside 8 a.m. to 4 p.m.) 
Fax: 
 
email: PV-nordic@pharmalex.com  
Central laboratory 
MLM Laboratory 
Dohrweg 63 
41066 Mönchengladbach 
Germany 
Special laboratory (GlucaGen 
PK, insulin PK) 
MLM Laboratory 
Dohrweg 63 
41066 Mönchengladbach 
Germany 
Special laboratory (dasiglucagon 
PK, dasiglucagon ADA, 
GlucaGen ADA) 
York Bioanalytical Solutions (YBS)  
Cedar House 
Northminster Business Park 
Northfield Lane 
York, YO26 6QR, United Kingdom 
Special laboratory  
(neutralizing antibodies) 
BioAgilytix 
2300 Englert Drive 
Durham, NC, 27713, USA 
A list of all investigators, IECs and IRBs will be provided in a separate document and in 
the clinical trial report. 
 


 
 
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CLINICAL TRIAL PROTOCOL 
 
A phase 3, randomized, double-blind, parallel trial to 
confirm the clinical efficacy and safety of dasiglucagon 
in the rescue treatment of hypoglycemia in subjects with 
type 1 diabetes mellitus (T1DM) compared to placebo 
and with reference to GlucaGen® 
 
Sponsor: 
Zealand Pharma A/S 
Sponsor Protocol No.: 
ZP4207-16137 
EudraCT No.: 
2017-002449-31  
IND No: 
127866 
Trial Drug Name: 
Dasiglucagon* injection 
Date of Protocol: 
26-Jan 2018 
 
The trial will be conducted according to the protocol and in compliance with Good 
Clinical Practice (GCP), with the Declaration of Helsinki and with other applicable 
regulatory requirements. 
*Dasiglucagon is the proposed international nonproprietary name for ZP4207. 
This protocol version 4.0 consists of protocol version 3.0, dated 06-October-2017 and 
local German protocol amendment 1, dated 26-Jan-2018. 
The information in this document is confidential and is proprietary to Zealand Pharma. It is understood that 
information in this document shall not be disclosed to any third party, in any form, without prior written 
consent of an authorized officer of Zealand Pharma. 


 
 
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Declaration of sponsor or responsible medical officer 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
This trial protocol was subjected to critical review. The information it contains is 
consistent with current knowledge of the risks and benefits of the investigational product, 
as well as with the moral, ethical and scientific principles governing clinical research as 
set out in the Declaration of Helsinki and the guidelines on Good Clinical Practice (GCP) 
(1). 
 
 
_____________________________________ 
_____________________ 
Name  
 
 
 
Date 
Title 
 
Clinical Trial Manager 
Institution 
Zealand Pharma A/S 
Smedeland 36 
2600 Glostrup, Denmark 
Phone:  
 
 
_____________________________________ 
_____________________ 
Name  
 
 
 
Date 
Title 
 
Medical Officer 
Institution 
Zealand Pharma A/S 
Smedeland 36 
2600 Glostrup, Denmark 
Phone:  
 


 
 
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Declaration of the coordinating investigator 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
I have read this protocol. I approve this document and I agree that it contains all 
necessary details for carrying out the trial as described. I will conduct this trial in 
accordance with the design and specific provision of this protocol and will make a 
reasonable effort to complete the trial within the time designated. I will provide copies of 
this protocol and access to all information furnished by Zealand Pharma A/S to trial 
personnel under my supervision. I will discuss this material with them to ensure they are 
fully informed about the trial product and trial procedures. I will let them know that this 
information is confidential and proprietary to Zealand Pharma A/S and that it may not be 
further disclosed to third parties. I understand that the trial may be terminated or 
enrollment suspended at any time by Zealand Pharma A/S, with or without cause, or by 
me if it becomes necessary to protect the best interests of the trial subjects. 
I agree that the trial will be carried out in accordance with GCP (1), with the Declaration 
of Helsinki (with amendments) (2) and with the laws and regulations of the countries in 
which the trial takes place. 
 
 
_____________________________________ 
_____________________ 
Name  
 
 
 
 
 
Date 
Title 
Institution 
Phone: + 
Fax: 
+ 
 


 
 
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Declaration of the investigator 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
All documentation for this trial that is supplied to me and that has not been previously 
published will be kept in the strictest confidence. This documentation includes this trial 
protocol, Investigator's Brochure, Case Report Forms, and other scientific data. 
The trial will not be commenced without the prior written approval of a properly 
constituted Institutional Review Board (IRB) or Independent Ethics Committee (IEC). 
No changes will be made to the trial protocol without the prior written approval of the 
sponsor and the IRB or IEC, except where necessary to eliminate an immediate hazard to 
the subjects. 
I have read and understood and agree to abide by all the conditions and instructions 
contained in this protocol. 
 
_____________________________________ 
_____________________ 
Signature 
 
 
 
 
 
Date 
 
_____________________________________ 
Name (block letters) 
 
_____________________________________ 
Title (block letters) 
 
_____________________________________ 
Institution (block letters) 
 


 
 
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Table of contents 
DECLARATION OF SPONSOR OR RESPONSIBLE MEDICAL OFFICER .......... 2 
DECLARATION OF THE COORDINATING INVESTIGATOR .............................. 3 
DECLARATION OF THE INVESTIGATOR ............................................................... 4 
TABLE OF CONTENTS .................................................................................................. 5 
1 
SYNOPSIS ............................................................................................................ 11 
2 
INTRODUCTION................................................................................................ 15 
2.1 
Background ...................................................................................................... 15 
2.1.1 
Hypoglycemia ...................................................................................... 15 
2.1.2 
Glucagon .............................................................................................. 16 
2.1.3 
Dasiglucagon........................................................................................ 16 
2.1.3.1 
Pharmacokinetics and pharmacodynamics of dasiglucagon ......... 17 
2.1.3.2 
Safety of dasiglucagon ................................................................... 17 
2.2 
Trial rationale .................................................................................................. 18 
2.3 
Risk-benefit assessment .................................................................................. 18 
3 
TRIAL OBJECTIVES AND ENDPOINTS....................................................... 20 
3.1 
Primary objective ............................................................................................ 20 
3.2 
Secondary objectives ....................................................................................... 20 
3.3 
Primary endpoint ............................................................................................ 20 
3.4 
Key secondary endpoints ................................................................................ 20 
3.5 
Other secondary endpoints ............................................................................. 20 
3.6 
Exploratory endpoint ...................................................................................... 21 
4 
OVERALL DESIGN AND PLAN OF THE TRIAL ........................................ 22 
4.1 
Overview .......................................................................................................... 22 
4.2 
Justification of the trial design ....................................................................... 24 
4.2.1 
Justification for design and parameters................................................ 24 
4.2.2 
Justification for drug, route, dosage and treatment duration ............... 24 
5 
TRIAL POPULATION ....................................................................................... 26 
5.1 
Rationale for trial population ........................................................................ 26 
5.2 
Planned sample size and number of trial centers ......................................... 26 
5.3 
Inclusion criteria ............................................................................................. 26 


 
 
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5.4 
Exclusion criteria ............................................................................................ 27 
5.5 
Dosing day exclusion criteria ......................................................................... 28 
5.6 
Premature treatment discontinuation and withdrawal ............................... 29 
5.6.1 
Possible reasons for treatment visit discontinuation ............................ 29 
5.6.2 
Center discontinuation ......................................................................... 30 
5.6.3 
Trial termination .................................................................................. 30 
5.7 
Subject identification and randomization ..................................................... 30 
6 
TRIAL DRUG ...................................................................................................... 32 
6.1 
Identity ............................................................................................................. 32 
6.2 
Treatment assignment and randomization ................................................... 33 
6.3 
Administration ................................................................................................. 34 
6.4 
Packaging and labelling .................................................................................. 34 
6.5 
Storage of study drugs .................................................................................... 34 
6.6 
Blinding and breaking the blind .................................................................... 35 
6.7 
Drug accountability ......................................................................................... 36 
6.8 
Treatment compliance .................................................................................... 36 
6.9 
Prior and concomitant medications ............................................................... 36 
6.9.1 
Prohibited medications......................................................................... 37 
7 
PARAMETERS AND METHODS OF ASSESSMENT .................................. 38 
7.1 
Efficacy parameters ........................................................................................ 38 
7.1.1 
Pharmacodynamic measurements ........................................................ 38 
7.1.2 
Pharmacokinetic measurements ........................................................... 38 
7.2 
Safety parameters ............................................................................................ 39 
7.2.1 
Adverse events ..................................................................................... 39 
7.2.1.1 
Definitions ...................................................................................... 39 
7.2.1.2 
Collection, recording and reporting of adverse events ................. 42 
7.2.1.3 
Follow-up of adverse events .......................................................... 44 
7.2.1.4 
Clinical laboratory abnormalities and other abnormal 
assessments as adverse events or serious adverse events .............. 44 
7.2.2 
Hypoglycemia ...................................................................................... 45 
7.2.3 
Physical examination ........................................................................... 45 
7.2.4 
Vital signs ............................................................................................ 45 
7.2.5 
Electrocardiogram ................................................................................ 46 
7.2.6 
Local tolerability .................................................................................. 46 
7.2.7 
Clinical laboratory assessments ........................................................... 47 
7.2.8 
Pregnancy ............................................................................................. 48 
7.2.9 
Precautions ........................................................................................... 49 
7.2.10 
Safety committee ................................................................................. 49 
7.3 
Demography, concomitant illness, medical history and concomitant 
medication ........................................................................................................ 49 


 
 
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7.3.1 
Demography and body measurements ................................................. 50 
7.3.2 
Concomitant illness and medical history ............................................. 50 
7.3.3 
Diabetes diagnosis and current treatment ............................................ 50 
7.3.4 
Concomitant medication ...................................................................... 51 
7.4 
Other assessments ........................................................................................... 51 
7.4.1 
Immunogenicity ................................................................................... 51 
7.4.2 
Plasma glucose measurements for safety ............................................. 52 
7.4.3 
Plasma insulin measurements .............................................................. 52 
8 
TRIAL CONDUCT .............................................................................................. 53 
8.1 
Schedule of procedures ................................................................................... 53 
8.2 
Procedures by visit .......................................................................................... 53 
8.2.1 
Visit 1 (screening, Day -30 to Day -3) ................................................. 53 
8.2.2 
Instructions to subjects prior to the dosing visit (Visit 2) .................... 55 
8.2.3 
Visit 2 (dosing visit)............................................................................. 56 
8.2.3.1 
Hypoglycemic clamp procedure and administration of trial 
medication ...................................................................................... 57 
8.2.4 
Visit 3 (follow-up visit) ....................................................................... 60 
9 
STATISTICAL METHODS ............................................................................... 61 
9.1 
Determination of sample size ......................................................................... 61 
9.2 
Trial subjects ................................................................................................... 61 
9.2.1 
Analysis samples .................................................................................. 61 
9.2.2 
Disposition of subjects ......................................................................... 62 
9.2.3 
Protocol deviations............................................................................... 62 
9.3 
General considerations ................................................................................... 63 
9.4 
Demographics and baseline characteristics .................................................. 63 
9.5 
Efficacy Analyses ............................................................................................. 63 
9.5.1 
Hierarchical testing procedure ............................................................. 64 
9.5.2 
Primary confirmatory endpoint ............................................................ 64 
9.5.2.1 
Primary analysis ............................................................................ 64 
9.5.3 
Secondary endpoints ............................................................................ 65 
9.5.3.1 
Confirmatory analysis .................................................................... 65 
9.5.4 
Secondary clinical efficacy (PD) endpoints ......................................... 66 
9.5.4.1 
Analysis of secondary clinical efficacy (PD) endpoints ................ 66 
9.5.5 
Exposure (PK) endpoints ..................................................................... 66 
9.5.5.1 
Analysis of exposure (PK) endpoints ............................................. 67 
9.6 
Exploratory analyses ....................................................................................... 67 
9.7 
Safety analyses ................................................................................................. 68 
9.7.1 
Adverse events ..................................................................................... 68 
9.7.2 
Immunogenicity data ........................................................................... 68 
9.7.3 
Clinical laboratory assessments ........................................................... 68 


 
 
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9.7.4 
Other safety data .................................................................................. 69 
9.8 
Treatment compliance .................................................................................... 69 
9.9 
Subject withdrawals and missing data .......................................................... 69 
9.10 
Interim analyses .............................................................................................. 69 
10 
ETHICAL, LEGAL, AND ADMINISTRATIVE ASPECTS .......................... 70 
10.1 
Quality assurance ............................................................................................ 70 
10.2 
Electronic case report forms .......................................................................... 71 
10.3 
Access to source data ...................................................................................... 71 
10.4 
Source documentation .................................................................................... 72 
10.5 
Data processing ................................................................................................ 72 
10.6 
Archiving trial records ................................................................................... 72 
10.7 
Good clinical practice ..................................................................................... 72 
10.8 
Informed consent ............................................................................................. 73 
10.9 
Protocol approval and amendment ............................................................... 73 
10.10 Duration of the trial ........................................................................................ 74 
10.11 Premature termination of the trial ................................................................ 74 
10.12 Confidentiality ................................................................................................. 74 
10.13 Other ethical and regulatory issues ............................................................... 75 
10.14 Liability and insurance ................................................................................... 75 
10.15 Publication policy ............................................................................................ 76 
11 
REFERENCE LIST ............................................................................................. 77 
12 
APPENDICES ...................................................................................................... 80 
12.1 
List of trial personnel ...................................................................................... 80 
 
Tables in text 
Table 1 
Description of trial drugs .................................................................................. 32 
Table 2 
Quantities of ingredients in dasiglucagon and placebo injection ...................... 33 
Table 3 
Schedule of procedures ..................................................................................... 54 
Table 4 
Post-treatment blood sampling times ................................................................ 59 
 
Figures in text 
Figure 1 Trial design ........................................................................................................ 23 


 
 
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List of abbreviations and definitions of terms 
ADA 
Anti-drug antibody 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALT 
Alanine aminotransferase 
ANCOVA Analysis of covariance 
AST 
Aspartate aminotransferase 
AUC 
Area under the concentration-time curve 
AUC0-inf 
AUC from time zero to infinity 
AUE 
Area under the effect curve  
CFB 
Changes from baseline  
CI 
Confidence interval 
Cmax 
Maximum plasma concentration 
CPH 
Cox proportional hazards 
CRO 
Contract research organization  
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
EU 
European Union 
EudraCT 
European Medicines Agency’s Clinical Trials Database 
FAS 
Full analysis set 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HbA1c 
Glycated hemoglobin 
ICH 
International Conference on Harmonization 
ID card 
Identification card 
IEC 
Independent Ethics Committee 
IM 
Intramuscular(ly) 
IMP 
Investigational medicinal product 
IRB 
Institutional Review Board 
IV 
Intravenous(ly) 


 
 
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IWRS 
Interactive Web Response System 
MedDRA 
Medical Dictionary for Regulatory Activities 
NPH 
Neutral protamine Hagedorn 
PD 
Pharmacodynamics 
PK 
Pharmacokinetics 
PP 
Per protocol 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SAS 
Safety analysis set 
SC 
Subcutaneous(ly) 
SUSAR 
Serious unexpected suspected adverse reaction 
T1DM 
Type 1 diabetes mellitus 
T2DM 
Type 2 diabetes mellitus 
TEAE 
Treatment-emergent adverse event 
tmax 
Time to the maximum plasma concentration 
ULN 
Upper limit of the normal range 
US 
United States of America 
 


 
 
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1 
SYNOPSIS 
Name of sponsor: 
Zealand Pharma A/S 
Trial ID: 
ZP4207-16137 
Title of the trial: 
A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy and safety of 
dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 1 diabetes mellitus (T1DM) 
compared to placebo and with reference to GlucaGen® 
Trial design:  
The trial is a global, multicenter, randomized, parallel-group, double-blind, clinical trial confirming the 
efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in subjects with type 1 diabetes 
mellitus. The subjects will be randomized 2:1:1 to receive a single fixed subcutaneous 0.6 mg dose of 
dasiglucagon (hereinafter dasiglucagon), placebo for dasiglucagon (hereinafter referred to as placebo), or a 
1 mg dose of GlucaGen® (hereafter referred to as GlucaGen) and followed for at least 28 days after 
treatment. 
Clinical phase of development: 
Phase 3 
Trial centers: 
This trial will be conducted at 4 to 6 sites in the United States of America, Canada, and Europe. 
Planned trial start (first subject first visit): 
Q4/2017 
Planned trial end (last subject last visit): 
Q3/2018  
Trial population:  
Male and female adult subjects with type 1 diabetes mellitus treated with insulin for at least one year  
Key objectives: 
Primary objective:  
 
To demonstrate superiority of dasiglucagon compared to placebo following a single subcutaneous 
0.6 mg dose administered to subjects with type 1 diabetes mellitus with insulin-induced 
hypoglycemia. 
Secondary objective:  
 
To compare the glycemic response observed after dasiglucagon with that of GlucaGen. 
Key endpoints:  
Primary endpoint:  
 
Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase in plasma 
glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic clamp procedure 
without administration of rescue intravenous (IV) glucose.  
Key secondary endpoints: 
 
Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, and within 
10 minutes after study drug injection without administration of rescue IV glucose. 
 
Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection or at the time of rescue. 
Clinical efficacy (Pharmacodynamic) endpoints: 
 
Time to first plasma glucose concentration of ≥70 mg/dL (3.9 mmol/L) without administration of 
rescue IV glucose. 


 
 
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 
Plasma glucose response as area under the curve above baseline from time zero to 30 minutes, 
AUC0-30min. 
Exposure (Pharmacokinetic) endpoints: 
 
Area under the drug concentration curve from time zero to 90 minutes, AUC0-90 min. 
 
Maximum plasma drug concentration (Cmax). 
 
Time to maximum plasma drug concentration (tmax). 
Safety endpoints:  
 
Adverse events, clinical laboratory assessments (biochemistry, hematology, coagulation, urinalysis), 
vital signs, physical examination, electrocardiogram, and local tolerability. 
 
Administration of rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
Immunogenicity endpoint: 
 
Occurrence of anti-drug antibodies 
Exploratory endpoint: 
 
Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL (1.1 mmol/L) 
within 30 minutes after study drug injection without administration of rescue IV glucose. 
 
Plasma insulin response as area under the curve above baseline from time zero to 60 minutes, 
AUC0-60 min. 
Key inclusion criteria: 
 
Male and female subjects with type 1 diabetes mellitus treated with insulin for at least one year, 
diagnostic criteria as defined by the American Diabetes Association. 
 
Stable insulin treatment 30 days prior to screening, defined as no more than a 10-unit daily variation 
in total daily insulin dose. 
 
Hemoglobin A1c <10%. 
 
Aged between 18 and 75 years, both inclusive. 
Key exclusion criteria: 
 
Previously treated with dasiglucagon. 
 
Known or suspected allergy to trial product(s) or related products. 
 
History of anaphylaxis or symptoms of severe systemic allergy (such as angioedema). 
 
Previous participation (randomization) in this trial. 
Sample size: 
Approximately 156 subjects are intended to complete the trial, with 78 subjects randomized to the 
dasiglucagon group and 39 subjects randomized to each of the placebo and GlucaGen groups.  
Investigational medicinal product: 
Test product: dasiglucagon liquid formulation in pre-filled syringes. 
Comparator products: Placebo and GlucaGen® lyophilized powder. 
Germany only: Insulin glulisine (Apidra®), the challenge agent, will also be defined as an investigational 
medicinal product 
Duration of treatment:  


 
 
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Subjects will be randomized 2:1:1 to receive a single fixed subcutaneous dose of 0.6 mg dasiglucagon, 
placebo, or 1 mg GlucaGen, and followed for at least 28 days after receiving treatment.  
Assessments:  
The plasma glucose profile for evaluation of the primary and secondary clinical efficacy 
(pharmacodynamic) endpoints will be assessed based on plasma concentration data from samples collected 
at the dosing visit. 
The exposure to trial medication (dasiglucagon, placebo, or GlucaGen) for evaluation of pharmacokinetics 
will also be assessed based on plasma concentration data.  
Statistical methods:  
Due to requirements in the size of the safety database, the sample size is set to 78 subjects treated with 
dasiglucagon 0.6 mg. With a 2:1:1 randomization ratio for the three treatments, dasiglucagon 0.6 mg, 
placebo, and GlucaGen 1 mg, 78 subjects injected with dasiglucagon and 39 subjects with placebo will 
yield a power of 90% at a 5% two-sided significance level to detect a treatment group difference in recovery 
incidence within 20 minutes, assuming as low as 80% and as high as 50% recovery incidence for the 
dasiglucagon and placebo groups, respectively. 
For the confirmatory analyses, the primary and key secondary endpoints for the dasiglucagon 0.6 mg and 
placebo treated subjects will be compared. A hierarchical procedure will be applied for the control of 
multiplicity in statistical inferences. The primary and secondary endpoints will be evaluated on the Full 
Analysis Set sample. The statistical inference comparisons with placebo will be conducted as superiority 
tests. The comparisons of dasiglucagon versus GlucaGen will be summarized descriptively.    
The primary endpoint will be summarized using Kaplan-Meier (KM) estimates stratified by treatment group 
and injection site. The treatment group difference between dasiglucagon and placebo will be evaluated 
inferentially using a pairwise two-sided log-rank test. 
In the primary analysis, those subjects who require rescue IV glucose will be censored at the time of plasma 
glucose recovery. This approach is based on the assumption that rescue IV glucose administration will 
shorten the time to recovery. This assumption implies that censoring at the time of recovery after rescue 
therapy is valid as a time to recovery without rescue would be longer than the observed duration. If recovery 
has not occurred at 45 minutes after study drug injection, censoring will be applied irrespective of the use of 
rescue IV glucose.  
In sensitivity analyses, the time to plasma glucose recovery will be analyzed 1) without censoring at the 
time of recovery for those subjects who require rescue IV glucose before 45 minutes and 2) with censoring 
at the time of administration of rescue IV glucose before 45 minutes.   
The primary endpoint will additionally be analyzed using a Cox proportional hazards (CPH) time to event 
statistical model, if the hazards by treatment group can be assumed proportional using a graphical 
comparison of log-minus-log survival curves and no more than two-thirds of the recovery times are 
censored in each treatment group. The CPH model will be used for inferences, with treatment group and 
injection site modeled as categorical effects, and baseline plasma glucose modeled as a continuous 
covariate. The treatment group rate ratios, active versus placebo, will be estimated together with the 95% 
confidence intervals, and pairwise treatment group inferences (dasiglucagon vs placebo, GlucaGen vs 
placebo) will be evaluated using two-sided likelihood ratio tests.  
The key secondary incidence variables (plasma glucose recovery within 30 minutes, within 20 minutes, 
within 15 minutes, within 10 minutes, in hierarchical order for inference) will be analyzed using descriptive 


 
 
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statistics (frequency and percentage) by treatment group. Inferential treatment group comparisons will be 
assessed via pairwise tests of independent binomial proportions (dasiglucagon versus placebo, GlucaGen 
versus placebo) using Fisher’s exact test for each pairwise comparison. 
The key secondary endpoints of plasma glucose changes from baseline (CFB) within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection or at the time of rescue, will 
be analyzed with the plasma glucose CFB at rescue carried forward in those subjects who require rescue IV 
glucose before plasma glucose ≥20 mg/dL recovery. Each of these CFB variables will be analyzed using an 
Analysis of Covariance model, with treatment group modeled as a fixed effect and with the baseline plasma 
glucose modeled as a covariate. The dasiglucagon versus placebo treatment group difference will be 
evaluated inferentially as a least squares means contrast, using a two-sided t-test at the 0.05 significance 
level, first for the 30 minute CFB, and then sequentially for the 20 minute, 15 minute, and 10 minute 
variables, until the first failure to reject. 
The continuous clinical efficacy variables, the exploratory variables, and the pharmacokinetics variables 
will each be summarized descriptively by treatment group. The clinical efficacy variables will be analyzed 
analogous to the plasma glucose CFB variables.   
The safety analyses will include by-treatment-group descriptive summaries of vital sign measurements, 
laboratory measures (including immunogenicity incidence), physical examination assessments, rescue IV 
glucose (incidence and amount of glucose infused), and adverse events. The number and percentage of 
subjects reporting specific events, such as nausea and vomiting, will be presented by body system and 
preferred term. 
Further details will be included in the Statistical Analysis Plan, to be completed before database lock and 
treatment unmasking.   


 
 
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2 
INTRODUCTION 
2.1 
Background 
Zealand Pharma A/S (Zealand Pharma) is developing dasiglucagon, a physically and 
chemically stable peptide analog of human glucagon, in a ready-to-use liquid formulation 
for the acute treatment of severe hypoglycemia in patients with insulin-treated diabetes 
mellitus. Like native glucagon, dasiglucagon is comprised of 29 amino acids, but with 
7 substitutions which improve its physical and chemical stability in aqueous media. 
These amino acid substitutions make dasiglucagon suitable for a liquid formulation, 
while providing similar efficacy and safety as compared with recombinant glucagon in 
the clinical setting of acute treatment of severe hypoglycemia. 
2.1.1 Hypoglycemia 
Hypoglycemia in patients with diabetes is defined as episodes of an abnormally low 
plasma glucose concentration (3). This is a common, unpredictable, and potentially 
dangerous side effect of treatment of diabetes mellitus, especially with insulin or 
sulfonylureas. It is more frequent in patients with profound endogenous insulin 
deficiency, such as occurs in type 1 diabetes mellitus (T1DM) and advanced type 2 
diabetes mellitus (T2DM). Treatment of T2DM with insulin causes hypoglycemia 
progressively and more frequently over time, whereas in T1DM, hypoglycemia is 
experienced throughout the course of established disease (4). 
Symptoms and signs of hypoglycemia are not specific. Patients undergoing a 
hypoglycemic episode experience unpleasant symptoms such as anxiety, sweating, 
hunger, tremors, palpitations, paresthesia, nausea and pallor. Depending on its severity, 
the hypoglycemia may lead to mild confusion, behavioral changes, loss of consciousness, 
seizures, coma, and death (5). 
The incidence of hypoglycemic events or even the fear of hypoglycemia influences 
patients’ adherence to prescribed treatment regimens for diabetes mellitus (6). This leads 
to inadequate glycemic control, which in turn may lead to an increased risk of diabetic 
complications (5). Serious clinically significant hypoglycemia is now defined as plasma 
glucose <54 mg/dL (3.0 mmol/L), while the plasma glucose alert value is defined as 
<70 mg/dL (3.9 mmol/L) (7). When plasma glucose falls below these values, some kind 
of treatment strategy is needed. 


 
 
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2.1.2 Glucagon 
Glucagon is a naturally occurring hormone, secreted from the alpha cells of the 
pancreatic islets. Glucagon plays a central role in the regulation of glucose homeostasis 
and is the counterpart of insulin for controlling blood glucose levels (i.e. it acts in 
opposition to insulin in terms of effects on blood glucose levels) (8,9). Glucagon 
stimulates hepatic glycogenolysis and gluconeogenesis in hypoglycemic states, thereby 
restoring glucose homeostasis. Glucagon receptor agonism has also been shown to exert 
effects on lipid metabolism, energy balance, body adipose tissue mass and food intake 
(10). Insulin decreases blood glucose levels and cases of hypoglycemia can be reversed 
by glucagon. Therefore, glucagon is indicated for the treatment of severe hypoglycemia. 
Besides intravenous (IV) glucose administration, an injectable form of glucagon is given 
as first aid in cases of severe hypoglycemia, when the patient is unconscious or for other 
reasons cannot take glucose orally. The approved glucagon dose for an adult is 1 mg, 
given by intramuscular (IM), IV, or subcutaneous (SC) injection, which quickly raises 
blood glucose levels. As current marketed recombinant glucagon is highly unstable when 
dissolved in solution, the injectable form has to be reconstituted prior to use in a 9-step 
procedure that requires a sterile diluent to be injected into a vial containing lyophilized 
powdered glucagon. When dissolved in a fluid state, glucagon can form amyloid fibrils 
(11), or tightly woven chains of proteins made up of the individual glucagon peptides. 
The reconstitution process makes the use of marketed glucagon products cumbersome 
(12), and a more patient-friendly formulation is needed. Currently, the Food and Drug 
Administration (FDA) approved instructions for commercially available glucagon allow 
only for immediate usage of glucagon after the powder is reconstituted in aqueous 
solution (13). Therefore, a glucagon analog with enhanced biophysical stability may 
represent a leap forward in terms of convenient therapeutic applications. 
2.1.3 Dasiglucagon 
Dasiglucagon (ZP4207) is a stable peptide analog of human glucagon, available in a 
ready-to-use liquid formulation. Dasiglucagon (hereinafter referred to as dasiglucagon) is 
in development for the treatment of severe hypoglycemia in insulin-dependent patients 
with diabetes mellitus. Dasiglucagon is a specific and full glucagon receptor agonist 
designed to mimic the effects of glucagon, having a fast absorption and elimination 
(minutes). It is a peptide of 29 amino acids, with 7 amino acid substitutions compared to 
native glucagon. The main purpose of the substitutions is to increase the physical and 
chemical stability of the glucagon analog compared to marketed glucagon products such 
as Lilly Glucagon or GlucaGen® (hereafter referred to as GlucaGen). Dasiglucagon 
exhibits improved physical and chemical stability and is available in an aqueous solution 
at neutral pH (14). 


 
 
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Three clinical trials have been completed with dasiglucagon: a first-in-human dose trial in 
healthy volunteers and subjects with T1DM (ZP4207-14013), a multiple-dose 
dose-escalation trial (ZP4207-15007) evaluating the safety, tolerability, pharmacokinetics 
(PK) and pharmacodynamics (PD) of dasiglucagon in healthy volunteers, and a phase 2 
crossover trial to assess the PK and PD of a single dose of an optimized formulation of 
dasiglucagon administered SC in subjects with T1DM (ZP4207-15126) (14). 
2.1.3.1 Pharmacokinetics and pharmacodynamics of dasiglucagon 
The results of the phase 1 and 2 clinical trials confirmed dose-proportionality for 
dasiglucagon PK, which is characterized by a fast absorption with a peak plasma 
concentration obtained after 35 minutes. Thereafter, the plasma concentration rapidly 
declines with an average half-life of 28 minutes. The median time to the maximum 
plasma concentration (tmax) was 35 minutes for dasiglucagon compared with 20 minutes 
for GlucaGen.  
At all dose levels in the phase 2 trial, all subjects achieved a plasma glucose level of at 
least 70 mg/dL (3.9 mmol/L) as well as an increase in plasma glucose by at least 
20 mg/dL (1.1 mmol/L) within 30 minutes after dosing. The PD responses to 0.6 mg of 
dasiglucagon and 1 mg of GlucaGen were comparable. 
2.1.3.2 Safety of dasiglucagon 
The safety data for dasiglucagon do not give rise to any safety concerns. No new signals 
were observed, beyond those related to the pharmacological effect of glucagon agonism. 
All adverse events (AEs) recorded in the trials were of mild or moderate severity. The 
most frequently reported systemic AE was nausea, which is a known side effect 
following administration of glucagon. Headache was the next most frequently reported 
event, occurring in all dose groups in the phase 2 trial. Injection site reactions were 
observed only sporadically after administration with either dasiglucagon or GlucaGen 
and all were mild and transient. The most frequent injection site reaction was erythema, 
occurring in all treatment groups, including the placebo group, irrespective of dose. 
Glucagon has been described to exert positive inotropic and chronotropic effects and may 
therefore cause tachycardia and hypertension. In the phase 1 clinical trials, but not the 
phase 2 trial, temporary but clinically significant decreases in blood pressure were 
observed in a few healthy volunteers receiving investigational medicinal product (IMP) 
doses of at least 1 mg (4 with dasiglucagon and 1 with GlucaGen). This is not considered 
a safety concern; however, hemodynamic changes after dosing will be considered an 
adverse event of special interest (AESI). 


 
 
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The phase 1 and 2 results and the safety profile described to date do not give rise to 
specific safety concerns. For further information, please refer to the Investigator’s 
Brochure (14). 
2.2 
Trial rationale 
The aim of the current trial is to confirm the superiority of dasiglucagon for the treatment 
of insulin-induced hypoglycemia in subjects with T1DM as compared to placebo for 
dasiglucagon (hereinafter placebo) and to compare the clinical efficacy and safety of 
dasiglucagon with reference to GlucaGen. A randomized, controlled trial design was 
used. 
See Section 4.2 for justification of the design of this trial. 
2.3 
Risk-benefit assessment  
Non-clinical experience 
The nonclinical development program did not reveal any safety findings that would 
prohibit administration of dasiglucagon to humans. None of the safety pharmacology 
studies, repeated dose toxicity studies or genotoxicity studies revealed any significant 
toxicity findings relevant to the therapeutic use of dasiglucagon. 
Clinical experience 
As glucagon and its analogs belong to a well-known drug class with a known mode of 
action, dasiglucagon is not expected to be a high-risk molecule.  
Treatment with an IMP may result in undesired effects or complaints. Undesired effects 
and complaints such as nausea, vomiting, and diarrhea are known AEs occurring with 
glucagon administration. Similar AEs have also been observed to a limited extent in the 
3 clinical studies conducted to date with dasiglucagon. As with every novel drug 
substance, new and as yet unknown side effects may also occur. 
There are limited data available to assess the immunogenic potential of glucagon 
products available on the market, but the data available indicate that marketed glucagon 
only has a low immunogenic potential.  
Overall, dasiglucagon is judged to be a low-risk molecule, based upon the available 
clinical data. Administration of dasiglucagon may be associated with a risk of allergic 
reactions similar to those observed for other therapeutic peptides or proteins. Subjects 
with known or suspected allergies to the trial medications or related products will be 


 
 
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excluded from the trial. Mild or moderate allergic reactions may include symptoms of 
rash, fever, flu-like symptoms, nausea, headache, and myalgia. Acute generalized 
hypersensitivity reactions are usually very rare, but may include symptoms of flushing, 
sweating, dizziness, change in blood pressure, and difficulties in breathing. No severe 
acute hypersensitivity reactions have been observed in the 3 clinical trials conducted with 
dasiglucagon. Direct access to resuscitation equipment is ensured at the clinical trial 
centers. 
With the exception of medical examinations, a subject participating in this trial is not 
likely to derive any personal health-related benefits. The results of the trial may 
contribute to the future use of dasiglucagon in patients with diabetes mellitus 
experiencing severe hypoglycemic reactions. 
The development program including 141 subjects exposed to dasiglucagon to date has 
demonstrated that administration of dasiglucagon is efficacious and well tolerated, with a 
safety profile that does not give rise to specific safety concerns. Two phase 1 and one 
phase 2 clinical trials have been conducted to investigate the safety, tolerability, PK and 
PD of dasiglucagon after single and multiple dosing to healthy volunteers and subjects 
with T1DM under insulin-induced hypoglycemic conditions. Dasiglucagon has proven to 
have relevant clinical effects in the acute severe hypoglycemia rescue setting and may be 
an effective and reliable emergency treatment for severe hypoglycemia. Overall, the 
anticipated benefits for subjects entering the ZP4207-16137 trial are considered to justify 
the risks. 


 
 
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3 
TRIAL OBJECTIVES AND ENDPOINTS 
3.1 
Primary objective 
 To demonstrate superiority of dasiglucagon compared to placebo following a single 
subcutaneous 0.6 mg dose administered to subjects with type 1 diabetes mellitus with 
insulin-induced hypoglycemia. 
3.2 
Secondary objectives 
 To compare the glycemic response observed after dasiglucagon with that of 
GlucaGen. 
3.3 
Primary endpoint 
 Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase 
in plasma glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic 
clamp procedure without administration of rescue IV glucose.  
3.4 
Key secondary endpoints 
 Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection without administration of rescue IV 
glucose. 
 Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection or at the time of rescue. 
3.5 
Other secondary endpoints 
 Clinical efficacy (PD) endpoints: 
- 
Time to first plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) without 
administration of rescue IV glucose. 
- 
Plasma glucose response as area under the curve (AUC) above baseline from time 
zero to 30 minutes, AUC0-30min. 
 Exposure (PK) endpoints: 
- 
Area under the drug concentration curve from time zero to 90 minutes, 
AUC0-90min. 
- 
 Maximum plasma drug concentration (Cmax). 
- 
Time to maximum plasma drug concentration (tmax). 


 
 
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 Safety endpoints:  
- 
Adverse events, clinical laboratory assessments (biochemistry, hematology, 
coagulation, urinalysis), vital signs, physical examination, electrocardiogram 
(ECG), and local tolerability. 
- 
Administration of rescue infusion of IV glucose during the hypoglycemic clamp 
procedure. 
- 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp 
procedure. 
 Immunogenicity endpoint: 
- 
Occurrence of anti-drug antibodies 
3.6 
Exploratory endpoint 
 Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL 
(1.1 mmol/L) within 30 minutes after study drug injection without administration of 
rescue IV glucose. 
 Plasma insulin response as area under the curve above baseline from time zero to 
60 minutes, AUC0-60 min. 


 
 
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4 
OVERALL DESIGN AND PLAN OF THE TRIAL 
4.1 
Overview 
This trial is a global, multicenter, randomized, parallel, double-blind, clinical trial 
confirming the efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in 
subjects with T1DM. The subjects will be randomized 2:1:1 to receive a single fixed SC 
0.6 mg dose of dasiglucagon, placebo, or a 1 mg dose of GlucaGen and followed for at 
least 28 days after receiving treatment. A total of 156 subjects with T1DM are expected 
to complete the treatment visit. The trial will be conducted in the European Union (EU) 
and North America. See Figure 1 for an overview of the trial design. 


 
 
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Figure 1 
Trial design 
 
 
 


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4.2 
Justification of the trial design 
4.2.1 Justification for design and parameters 
In order to avoid bias in subject selection and in the evaluation of clinical assessments, 
subjects will be randomly assigned 2:1:1 to either dasiglucagon, placebo, or GlucaGen 
and the trial will be conducted in a double-blinded manner. The randomized parallel 
treatment design with administration of fixed SC doses of dasiglucagon, placebo, or 
GlucaGen to subjects with T1DM and insulin-induced hypoglycemia allows for direct 
comparison of the clinical efficacy of the treatments.  
The trial is double-blind to increase trial validity and to reduce bias during evaluation of 
the treatments. However, since the trial medications are not identical in appearance, the 
handling, preparation and administration of trial medication will be done by unblinded 
trial personnel who will not be involved in any other trial procedures or assessments. See 
Section 6.6 for more information about which assessments are blinded and which are not, 
with reasons.  
A superiority trial design is used because the aim is to show that treatment with 
dasiglucagon is an effective treatment compared to placebo. The secondary objective was 
chosen to allow a comparison between treatment with dasiglucagon and an established 
comparator treatment for severe hypoglycemia, GlucaGen. 
Administration of glucagon is intended to quickly raise blood glucose levels in subjects 
with T1DM with insulin-induced hypoglycemia. Therefore, in order to assess the clinical 
efficacy of dasiglucagon as compared to placebo and GlucaGen following a single SC 
dose, the primary endpoint and secondary efficacy endpoints involve the measurement of 
plasma glucose concentrations at different timepoints. 
4.2.2 Justification for drug, route, dosage and treatment duration 
Dasiglucagon and GlucaGen will be administered as fixed doses independent of body 
weight because this is the intended therapeutic dosing regimen in the emergency 
treatment of hypoglycemia. The selected dose of 1 mg GlucaGen is the approved dose for 
treatment of severe hypoglycemia. Data from the studies conducted to date with 
dasiglucagon, including the phase 2 trial in subjects with T1DM, have been used to 
establish that 0.6 mg of dasiglucagon is an effective dose and also represents a 
therapeutically equivalent dose to 1 mg of GlucaGen (see also Section 6.1). 
Dasiglucagon, placebo, and GlucaGen will be administered in the abdomen, buttocks, or 
thigh by SC injection, as this is one of the intended routes of administration for 
dasiglucagon, besides IM and IV. 


 
 
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Subjects will be followed for at least 28 days after dosing in order to perform an adequate 
immunogenicity evaluation of treatment.  


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5 
TRIAL POPULATION 
5.1 
Rationale for trial population 
Dasiglucagon is in development for the treatment of severe hypoglycemia in 
insulin-dependent patients with diabetes mellitus. In the present trial, subjects with 
T1DM are included in the evaluation of efficacy and safety of dasiglucagon under 
hypoglycemic conditions as this is part of the intended target population. Subjects with 
T1DM are selected to avoid the endogenous glucagon counter-regulatory response to 
insulin-induced hypoglycemia that is present in patients with T2DM. The inclusion and 
exclusion criteria are set to include a trial population that represents the general 
population of subjects with T1DM.  
5.2 
Planned sample size and number of trial centers 
A total number of 156 subjects with T1DM are expected to complete the treatment visit 
(Visit 2). To qualify as completed, the subject must be dosed and have blood drawn for 
the PK and PD assessments described in the protocol. It is expected that up to 
176 subjects will be randomized to have 156 subjects completing Visit 2. Completion of 
156 subjects (78 subjects in the dasiglucagon group and 39 subjects in each of the 
placebo and GlucaGen groups) would be expected to provide adequate power for the 
primary efficacy evaluation, as described in Section 9.1. 
The planned date for first subject first visit is expected to take place in Q4, 2017 and the 
planned date for last subject last visit is expected to take place in Q3, 2018. 
This trial is planned to be conducted at 4 to 6 trial sites in the United States of America 
[US], Canada, and Europe. 
5.3 
Inclusion criteria 
Subjects will be entered into this trial only if they meet all of the following criteria: 
1. Informed consent obtained before any trial-related activities (trial-related
activities are any procedure that would not have been performed during normal
management of the subject).
2. Female or male subjects with T1DM for at least 1 year, diagnostic criteria as
defined by the American Diabetes Association (3).
3. Treated with insulin for T1DM for at least 1 year and with stable insulin treatment
(defined as no more than a 10-unit daily variation in total daily insulin dose)
30 days prior to screening


 
 
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4. Hemoglobin A1c <10%. 
5. Aged between 18 and 75 years, both inclusive. 
6. A female subject must meet one of the following criteria: 
a. Participant is of childbearing potential and agrees to use one of the 
accepted contraceptive regimens throughout the entire duration of the trial 
from screening and until last follow-up visit. Additionally, if the 
participant is using systemic contraceptives, she must use an additional 
form of acceptable contraception. An acceptable method of contraception 
includes one of the following: 
i. Abstinence from heterosexual intercourse; 
ii. Systemic contraceptives (birth control pills, injectable/implant/ 
insertable hormonal birth control products, transdermal patch); 
iii. Intrauterine device (with and without hormones); or 
iv. Condom with spermicide; or 
b. Participant is of non-childbearing potential, defined as surgically sterile 
(i.e. has undergone complete hysterectomy, bilateral oophorectomy, or 
tubal ligation), or in a menopausal state (at least 1 year without menses). 
7. A male subject must meet the following criteria: Surgically sterilized or willing to 
refrain from sexual intercourse from screening and until last follow-up visit or, if 
sexually active, uses condom and partner practices contraception during the trial 
from screening and until last follow-up visit. 
5.4 
Exclusion criteria 
Subjects meeting any of the following criteria during screening evaluations will be 
excluded from trial participation: 
1. Previously treated with dasiglucagon (previously referred to as ZP4207). 
2. Known or suspected allergy to trial product(s) or related products. 
3. History of anaphylaxis or symptoms of severe systemic allergy (such as 
angioedema). 
4. Previous participation (randomization) in this trial. 
5. Females who are pregnant according to a positive pregnancy test, are actively 
attempting to get pregnant, or are lactating. 
6. History of hypoglycemic events associated with seizures in the last year prior to 
screening. 
7. History of severe hypoglycemia (defined as plasma glucose <54 mg/dL 
[3.0 mmol/L]) in the last month prior to screening. 
8. Receipt of any investigational drug within 3 months prior to screening. 
9. Active malignancy within the last 5 years. 
10. Congestive heart failure, New York Heart Association class II-IV. 


 
 
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11. Inadequately treated blood pressure, defined as systolic ≥160 mmHg or diastolic 
≥90 mmHg) at screening (15). 
12. Current bleeding disorder, including anti-coagulant treatment. 
13. Known presence or history of pheochromocytoma (i.e. adrenal gland tumor) or 
insulinoma (i.e. insulin secreting pancreas tumor). 
14. Use of a daily systemic beta-blocker drug, indomethacin, warfarin or 
anticholinergic drugs in the previous 28 days before Day 1 of this trial. 
15. Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >2.5 × the 
upper limit of the normal range (ULN), bilirubin >1.5 × ULN, estimated 
glomerular filtration rate <30 mL/min/1.73 m2 according to the Modification of 
Diet in Renal Disease study definition (16), or altered electrolyte values of 
clinical relevance for cardiac conduction, as judged by the investigator. 
16. Clinically significant abnormal ECG at screening as judged by the investigator. 
17. Clinically significant illness within 4 weeks before screening, as judged by the 
investigator. 
18. Donation of blood or plasma in the past month, or in excess of 500 mL within 
12 weeks prior to screening. 
19. Surgery or trauma with significant blood loss within the last 2 months prior to 
screening. 
20. A positive result in the alcohol and/or urine drug screen at the screening visit. 
Significant history of alcoholism or drug abuse as judged by the investigator or 
consuming more than 24 g alcohol per day for men, or more than 12 g alcohol per 
day for women. 
21. Subjects with mental incapacity or language barriers which preclude adequate 
understanding or cooperation, who are unwilling to participate in the trial, or who 
in the opinion of the investigator should not participate in the trial. 
22. Any condition interfering with trial participation or evaluation or that could be 
hazardous to the subject. 
23. The use of prescription or non-prescription medications known to cause QT 
prolongation. 
5.5 
Dosing day exclusion criteria 
Subjects who meet one or more of the following dosing day exclusion criteria at the 
dosing visit will be excluded from the dosing visit, but the visit can be rescheduled 
1-7 days later. The dosing visit can only be rescheduled once. 
1. Atypically strenuous exercise within 4 days prior to dosing, as judged by the 
investigator. Exercise during the trial should follow subject’s typical routine, and 
should not exceed a near maximum intensity for more than 20 minutes per day, or 
moderate intensity for more than 90 minutes per day. 


 
 
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2. Clinically significant illness within 4 weeks before dosing, as judged by the 
investigator. 
3. Consumption of alcohol within 24 hours prior to dosing visit, or positive results 
from an alcohol breath test. 
4. Not fasting from 22:00 hours the evening prior to dosing, apart from water. Small 
amounts of carbohydrates (up to 20 g) to prevent hypoglycemia are allowed. 
5. The use of any non-prescribed systemic or topical medication, except routine 
vitamins and occasional use (as judged by the investigator) of acetylsalicylic acid 
and paracetamol within 2 weeks prior to dosing. Treatment with insulin, including 
analogs, is allowed. 
6. Use of insulin Degludec or insulin Glargine U300 within 48 hours prior to dosing; 
or use of other long-acting insulins (e.g., insulin Glargine U100 or insulin 
Detemir) within 24 hours prior to dosing; or use of insulin Neutral protamine 
Hagedorn (NPH) within 16 hours prior to dosing. 
7. Use of any short acting (bolus) insulin within 6 hours prior to dosing, except 
insulin glulisine (Apidra®). 
8. Changes in medical history or concomitant medication resulting in fulfillment of 
clinical exclusion criteria, as judged by the investigator. 
9. Plasma glucose value <50 mg/dL (2.8 mmol/L) within the last 24 hours or plasma 
glucose value <60 mg/dL (3.3 mmol/L) within the last 5 hours prior to initiation 
of the hypoglycemic procedure. 
5.6 
Premature treatment discontinuation and withdrawal 
Participation in the trial is strictly voluntary. A subject has the right to withdraw from the 
trial at any time and for any reason. If he/she chooses to withdraw, the investigator must 
be informed immediately. The investigator has the right to terminate participation of any 
subject at any time if the investigator deems it in the subject's best interest. The reason 
and circumstances for withdrawal will be documented in the electronic case report form 
(eCRF). 
5.6.1 Possible reasons for treatment visit discontinuation 
A subject will be discontinued from treatment if the following applies: 
 Withdrawal of consent by subject. 
 If a protocol deviation occurs which, in the clinical judgement of the investigator, can 
invalidate the trial or endpoints or can interfere pharmacokinetically or 
pharmacodynamically with the trial product, the subject will be discontinued by the 
investigator. 
 Adverse events occur which are considered unacceptable by the subject or the 
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If discontinuation occurs following administration of trial medication, every effort 
should be made to have the subject return and participate in the complete follow-up visit 
on Day 28 (see Table 3) to avoid missing data. 
If trial participation is terminated due to an AE possibly related to any of the trial 
medications or trial examinations, the subject must be followed up by additional 
examinations according to the medical judgment of the investigator until the abnormal 
condition is resolved or the investigator deems further observations or examinations to be 
no longer medically indicated. 
A total of 156 subjects must complete the treatment visit (Visit 2). To qualify as 
completed, the subject must be dosed and have blood drawn for the PK and PD 
assessments described in the protocol.  
5.6.2 Center discontinuation 
The center can be closed and the trial terminated for the following reasons: 

The center is unlikely to be able to recruit sufficient subjects within the agreed time
frame.

The center does not respond to trial management requests.

Repeat protocol violations.
5.6.3 Trial termination 
The sponsor reserves the right to modify or terminate the trial at any time. Possible 
reasons for termination are: 

Safety reasons – the incidence of AEs in this or any other trial using the same trial
medication indicates a potential health risk for the subjects.

New scientific knowledge becomes known that makes the objectives of the trial no
longer feasible/valid.

Unsatisfactory enrolment of subjects.
5.7 
Subject identification and randomization 
Subjects who have given written informed consent and meet all inclusion and none of the 
exclusion criteria will be randomized in a 2:1:1 ratio to either 0.6 mg dasiglucagon 
(n=78), placebo (n=39), or 1 mg GlucaGen (n=39) via an Interactive Web Response 
System (IWRS). Randomization will continue until 156 subjects have completed Visit 2. 


 
 
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Subjects with previous exogenic glucagon exposure will not be excluded from the trial, 
but the information on previous glucagon administration will be recorded, to enable 
subgroup analyses.  
In the event of an emergency, e.g. when it becomes necessary for the investigator to 
know which treatment the subject is taking, the subject code can be broken by the 
investigator, preferably after consultation with the medical monitor. Emergency code 
breaks can be performed using the IWRS. 


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6 
TRIAL DRUG 
6.1 
Identity 
The following trial drugs will be administered: 

Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in a prefilled syringe.

Placebo, liquid formulation, 0.6 mL delivered in a prefilled syringe.

Recombinant glucagon hydrochloride, 1 mg for reconstitution (GlucaGen®, Novo
Nordisk) in 1 mL sterile water.
The description of the three trial drugs is provided in Table 1. Dasiglucagon is a stable 
peptide analog of human glucagon in a ready-to-use liquid formulation for treatment of 
severe hypoglycemia in insulin-dependent patients with diabetes mellitus. GlucaGen is 
approved in the EU and US and is indicated for treatment of severe hypoglycemic 
reactions, which may occur in the management of insulin-treated children and adults with 
diabetes mellitus. 
Table 1 
Description of trial drugs 
Test product 
Placebo Product 
Comparator product 
Name 
Dasiglucagon 
Placebo 
GlucaGen® 
Active 
substance 
Dasiglucagon 
N/A 
Recombinant 
glucagon 
hydrochloride 
Formulation 
Liquid formulation, 
0.6 mL 
Liquid formulation, 
0.6 mL 
Powder and solvent 
for reconstitution as 1 
mL solution for 
injection 
Strength 
1 mg/mL 
N/A 
1 mg 
Device 
Single use pre-filled 
syringe 
Single use pre-filled 
syringe 
Powder and solvent 
for reconstitution 
packed together in a 
plastic box. A “hypo-
kit”. 
Manufacturer 
Zealand Pharma A/S, 
Glostrup 
(Copenhagen), 
Denmark 
Zealand Pharma A/S, 
Glostrup 
(Copenhagen), 
Denmark 
Novo Nordisk A/S, 
Bagsværd, Denmark 


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Test product 
Placebo Product 
Comparator product 
Storage 
requirements 
Store between 2 and 
8°C 
Store between 2 and 
8°C 
Store between 2 and 
8°C 
The quantities of ingredients for dasiglucagon and placebo are provided in Table 2. 
Table 2 
Quantities of ingredients in dasiglucagon and placebo injection 
Component 
Amount per mL 
(dasiglucagon) 
Amount per mL 
(placebo) 
Function 
Dasiglucagon* 
1.0 mg 
N/A 
Sodium chloride 
10.23 mg 
10.23 mg 
Trometamol/Tromethamine 
6.06 mg 
6.06 mg 
Water for injection 
To make 1 mL 
To make 1 mL 
Sodium hydroxide 
q.s. 
q.s. 
Hydrochloric acid 
q.s. 
q.s. 
*The quantity of drug substance to be used is calculated according to net peptide content and purity.
q.s. = quantum sufficit (quantity required). 
6.2 
Treatment assignment and randomization 
Subjects successfully completing screening and who fulfill entry eligibility and 
randomization criteria will be randomized to one of three treatment groups in a ratio of 
2:1:1: 

Test treatment: Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in a
prefilled syringe.

Placebo treatment: Placebo, liquid formulation, 0.6 mL delivered in a prefilled
syringe.

Reference treatment: Recombinant glucagon hydrochloride, 1 mg for reconstitution.
Randomization will be performed using a fixed-block randomization scheme. The 
randomization scheme will be generated prior to the initiation of the study by an 
independent statistician/programmer who will not be a member of the study team; all 
investigators will not be aware of the block size of the randomization scheme.  
Randomization will be stratified by treatment group and by injection site (abdomen, 
buttocks, or thigh) and controlled via the IWRS.      


 
 
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Subjects will be randomized to study treatment using an interactive, automated system 
which has been validated for the intended use under the International Society of 
Pharmaceutical Engineers Good Automated Manufacturing Process guidelines, 21CFR 
Part 11 (FDA regulation for Electronic Records and Electronic Signatures) and the 
International Conference on Harmonization (ICH) Guidance E6 for Industry on Good 
Clinical Practice (GCP).   
6.3 
Administration 
Dasiglucagon, placebo, and GlucaGen will be administered by SC injection in the 
abdomen, buttocks, or thigh.  
An unblinded person (appropriately trained) authorized to prepare the dose and 
administer the treatment in accordance with the randomization will prepare the treatment 
required for each subject on each dosing day. The dose will be administered by the 
unblinded, trained and qualified person. The content of the syringe has to be checked for 
clarity and absence of bubbles.. 
Syringes will be discarded after dose administration. Used GlucaGen vials will be stored 
in a lockable box (separated from unused vials) at ambient temperature. 
6.4 
Packaging and labelling 
The test product will be packed by the sponsor. The information on the labels will be in 
the local language and the product label will be compliant with local laws and 
regulations.  
The study drug label will describe the storage conditions for study drug. The labels will 
supply no information about the subjects. Each treatment kit (pre-filled syringe/vial for 
reconstitution) will have a unique Dispensing Unit Number for drug allocation, drug 
accountability, and traceability purposes. 
Labelling will be performed according to Annex 13 of the Good Manufacturing Practice 
guidelines of the European Commission, ICH GCP guidelines, and local law. 
6.5 
Storage of study drugs 
The investigator must ensure the availability of proper storage conditions. All study drug 
supplies provided for this study will be stored in a secure area with restricted access at the 
study site.  


 
 
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The temperature should be monitored by recording the actual, minimum, and maximum 
temperatures using a calibrated thermometer or thermocouple, or by continuous recording 
using a qualified temperature monitoring system. The temperature should be evaluated 
and documented at least on working days on a temperature log. This log must be included 
in the Investigator Site File upon study termination. 
Dasiglucagon and placebo must be stored in a refrigerator (at a temperature of 2–8°C), 
and should be handled in accordance with guidelines from the sponsor. GlucaGen must 
also be stored in a refrigerator (at a temperature of 2–8°C), and should be handled in 
accordance with the Summary of Product Characteristics (13).  
The unblinded person responsible for study drug handling must contact the unblinded 
monitor in case of temperature deviations outside the acceptable range.  
Please see the Pharmacy Manual for additional information on handling study drug. 
6.6 
Blinding and breaking the blind 
This is a double-blind trial. As the trial products are not identical in appearance, 
dasiglucagon and placebo being available as a liquid formulation and GlucaGen as a 
powder for reconstitution, unblinded trial personnel will be responsible for handling, 
preparing (according to the prescription from the IWRS), and administering the trial 
medication, as well as for keeping the records strictly confidential and accessible only to 
unblinded staff until after the database has been locked. To maintain double-blind 
conditions, all trial assessments at the trial center will be done by blinded trial personnel 
not involved in the administration of trial medications. However, exposure assessments 
and anti-drug antibody (ADA) assessments will be performed by unblinded personnel at 
the special laboratories, to ensure that dasiglucagon, placebo, or GlucaGen administration 
is matched with the applicable bioanalytical assay. 
Treatment assignment will be kept strictly confidential and accessible only to authorized 
persons until after the time of unblinding. Codes with treatment assignment will, 
however, be readily available in the IWRS to the blinded personnel in case of an 
emergency. 
The code for a particular subject may be broken in a medical emergency if knowing the 
identity of the treatment allocation would influence the treatment of the subject. The 
emergency code break can be performed using the IWRS. Whenever a code is broken, the 
person breaking the code must record the time, date and reason as well as his/her initials 
in the source documents. The breaking of blinded codes in case of medical emergency for 
one subject should not unblind the trial personnel to the treatment information of other 


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subjects. The person performing the unblinding should inform as few people as possible 
about the result of the unblinding. All persons unblinded for a specific subject should be 
documented. 
If the trial center needs to break the code, the medical monitor should, if at all possible, 
be contacted prior to breaking the code and the monitor must be notified within 24 hours 
after the code has been broken. 
The pharmacovigilance unit (safety contract research organization [CRO]; see the list of 
trial personnel in Section 12.1) will be able to break the code in case of a serious 
unexpected suspected adverse reaction (SUSAR).  
The central and specialty laboratories will be provided with a copy of the randomization 
list. 
6.7 
Drug accountability 
Handling, preparation and administration of trial medication will be done by unblinded 
trial personnel. Each center will keep accurate records of the trial supplies received, 
stored, and dispensed, using appropriate forms. The trial supplies will be stored under 
appropriate conditions, locked and with restricted access.  
All unused supplies and all empty and partially empty containers of trial medication will 
be stored until the trial closure visit has been performed and then sent for destruction. 
This does not apply to the used syringes as they will be discarded after dose 
administration. Destruction must not take place until approved by the Sponsor.  
6.8 
Treatment compliance 
All trial medications will be prepared and administered by unblinded trial personnel. 
PK assessments will support the surveillance of compliance with IMP administration.  
6.9 
Prior and concomitant medications 
Prior glucagon exposure will be recorded in the eCRF at screening. All concomitant 
medications will be recorded and/or updated in the eCRF at each visit.  
Subjects using any new concomitant medication resulting in fulfillment of a dosing day 
exclusion criterion (see Section 5.5) will be excluded from the dosing visit, but can be 


 
 
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rescheduled to one of the following days (1–7 days later). The dosing visit can only be 
rescheduled once. 
6.9.1 Prohibited medications 
The use of anti-coagulant treatments and medications (prescription and non-prescription) 
that are known to cause QT prolongation are prohibited during the course of the trial. 
Within 28 days prior to dosing, the use of daily systemic beta-blockers, indomethacin, 
warfarin, and anticholinergic drugs is prohibited. 
Within 2 weeks prior to dosing, the use of any non-prescribed systemic or topical 
medication (with the exception of vitamins and the occasional use of acetylsalicylic acid 
and paracetamol) is prohibited.  
Within 48 hours prior to dosing, the use of insulin Degludec or insulin Glargine U300 are 
prohibited. 
Within 24 hours prior to dosing, the use of long-acting insulin (e.g., insulin Glargine 
U100 or insulin Detemir) is prohibited. 
Within 16 hours prior to dosing, the use of insulin NPH is prohibited. 
Within 6 hours prior to dosing, the use of any short acting (bolus) insulin, except insulin 
glulisine (Apidra®) is prohibited.  
During the insulin-induced hypoglycemic procedure, continuous SC insulin infusion must 
be stopped. 
6.10 Local German requirements for insulin glulisine (Apidra®) 
Insulin glulisine (Apidra), the insulin used to induce hypoglycemia, will according to 
local requirements be defined as an investigational medicinal product when used at 
German sites. The insulin will be provided by the local sites and drug accountability will 
be performed according to local procedures. In contrast to the trial drug for the 
randomized treatment, the insulin will be administered by the blinded trial personnel via 
IV infusion as described in section 8.2.3.1. 


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7 
PARAMETERS AND METHODS OF ASSESSMENT 
Overall, approximately 180 mL of blood will be drawn from each subject for PK, PD, 
ADA, and safety laboratory assessments. 
7.1 
Efficacy parameters 
A description of the sample handling and sample processing at the site will be included in 
the laboratory manuals. Validation documentation for the assays must be available prior 
to sample analyses. A bioanalytical report for each analysis of trial PD and PK samples 
will be prepared. 
7.1.1 Pharmacodynamic measurements 
The plasma glucose profile for evaluation of the primary and secondary clinical efficacy 
endpoints will be assessed based on plasma concentration data (AUC0-30min) from samples 
collected at the dosing visit (Visit 2). The samples will be sent to the clinical laboratory 
and analyzed using a sensitive and validated assay for glucose measurements.  
Samples will be collected pre-dose, and at 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 
75, and 90 minutes after dosing (see the schedule of procedures in Table 3). The actual 
time of blood sampling for evaluation of plasma glucose should not deviate from the 
nominal time by more than ±30 seconds until the 20 minute collection time point and by 
more than ±1 minute for the subsequent collection time points. Pre-dose is defined as 
within 2 minutes prior to dosing. 
7.1.2 Pharmacokinetic measurements 
The exposure to trial medication (dasiglucagon or GlucaGen) for evaluation of PK will 
be assessed based on plasma concentration data (AUC0-90 min, Cmax, tmax) from samples 
collected at the dosing visit (Visit 2).  
Samples (including back-up samples) will be collected pre-dose, and at 15, 30, 35, 40, 50, 
60, 90, and 120 minutes after dosing (see the schedule of procedures in Table 3). The 
actual time of blood sampling for exposure to trial medication should not deviate from the 
nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to 
dosing. 


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7.2 
Safety parameters 
7.2.1 Adverse events 
The investigator is responsible for the detection and documentation of events meeting the 
criteria and definition of an AE or serious adverse event (SAE), as provided in this 
protocol. During the trial, the investigator or center staff will be responsible for reporting 
AEs and SAEs, as detailed in this section of the protocol.  
Based on the investigator’s clinical judgment it will be determined whether an AE is 
related to treatment and of sufficient severity to require the subject’s removal from 
treatment or from the trial. A subject may also voluntarily withdraw from treatment due 
to what he or she perceives as an intolerable AE. If either of these situations arises, the 
subject should be under medical supervision until symptoms cease or the condition 
becomes stable. 
7.2.1.1 Definitions 
Adverse event 
An AE is any untoward medical occurrence in a trial subject given an IMP which does 
not necessarily have a causal relationship with the treatment. An AE can therefore be any 
unfavorable and/or unintended sign (for example, an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of an IMP, whether or not 
considered related to the IMP.  
In this trial, only treatment-emergent adverse events (TEAEs) will be collected and 
reported. TEAEs are events that occur from the first trial-related activity after the subject 
has signed the informed consent form until the end of the post-treatment follow-up 
period.  
AEs include: 

A clinically significant worsening of a concomitant illness.

A clinical laboratory abnormality which is clinically significant, i.e. any abnormality
that suggests a disease and/or organ toxicity and is of a severity that requires active
management. Active management includes active treatment or further investigations,
for example change of medicine dose or more frequent follow-up due to the
abnormality.

Hypoglycemic episodes (see Section 7.2.2).

Injection site reactions (see Section 7.2.6).


 
 
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The following should not be recorded as AEs, if recorded at screening (on the Screening 
Form or eCRF): 
 Pre-planned procedure, unless the condition for which the procedure was planned has 
worsened from the first trial related activity after the subject has signed the informed 
consent. 
 Pre-existing conditions found as a result of screening procedures (pre-existing 
conditions should be reported as medical history or concomitant illness). 
Serious adverse event 
An SAE is any untoward medical occurrence that at any dose:  
 Results in death  
 Is life-threatening  
 Requires inpatient hospitalization or prolongation of existing hospitalization 
 Results in persistent or significant disability or incapacity 
 Is a congenital anomaly or birth defect 
 Is medically important* 
*Medical judgement must be exercised in deciding whether an AE is believed to be 
‘medically important’. Medically important events may not be immediately 
life-threatening or result in death or hospitalization but may jeopardize the subject or 
may require intervention to prevent one of the other outcomes listed in the definition 
above. 
Adverse drug reaction 
All noxious and unintended responses to an investigational product related to any dose 
should be considered adverse drug reactions. The phrase responses to an investigational 
product means that a causal relationship between an investigational product and an 
adverse event is at least a reasonable possibility, i.e. the relationship cannot be ruled out. 
 
Suspected unexpected serious adverse reactions (SUSARs) 
An AE fulfilling one of the criteria of seriousness and being assessed as related to IMP 
application, the nature or severity of which is not consistent with the applicable reference 
document (e.g. dasiglucagon Investigator’s Brochure or package leaflet/Summary of 
Product Characteristics for GlucaGen). 


 
 
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Adverse event of special interest 
An AESI is an event which, in the evaluation of safety, has a special focus (e.g. required 
by health authorities). In this trial hemodynamic changes, as defined below, are 
considered AESIs: 
 Post-dose clinical signs, or measured vital signs, indicating a clinically significant 
drop in blood pressure including signs of orthostatic hypotension, vasovagal 
responses or bradycardia.   
 Post-dose change in pulse or blood pressure considered an event of hypo- or 
hypertension as judged by the investigator. 
 
Intensity of an adverse event 
The maximum intensity (severity) of all AEs must be assessed by the investigator and 
documented. Severity should be graded when the AE outcome is known: 
Mild:  
A type of AE that is usually transient and may require only minimal 
treatment or therapeutic intervention. The event does not generally 
interfere with usual activities of daily living. 
Moderate:  
A type of AE that is usually alleviated with additional specific therapeutic 
intervention. The event interferes with usual activities of daily living, 
causing discomfort but poses no significant or permanent risk of harm to 
the subject. 
Severe:  
A type of AE that interrupts usual activities of daily living, or significantly 
affects clinical status, or may require intensive therapeutic intervention. 
A ‘severe’ reaction does not necessarily deem the AE as ‘serious’ and an SAE may not 
necessarily be ‘severe’ in nature. 
Causality relationship to trial medication 
The causality of each AE should be assessed by the investigator according to the 
following classification: 
Probable: 
Good reason and sufficient documentation to assume a causal relationship. 
Possible: 
A causal relationship is conceivable and cannot be dismissed. 
Unlikely: 
The event is most likely related to etiology other than the trial product. 
Not related:  No relationship to trial product. 


 
 
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Germany only: The investigator should assess the causality to 
dasiglucacon/placebo/GlucaGen and to insulin glulisine (Apidra®) respectively. A 
special field applicable for Germany only will be present on the AE form in the eCRF to 
capture this data. 
Outcome of an adverse event 
The outcome of all AEs must be assessed by the investigator and documented by his/her 
staff. The following definitions should be used: 
Recovered/resolved:  
The subject has fully recovered, or by medical or 
surgical treatment the condition has returned to the 
level observed at the first trial-related activity after 
the subject signed the informed consent.  
Recovering/resolving: 
The condition is improving and the subject is 
expected to recover from the event. This term is 
only applicable if the subject has completed the 
trial. 
Recovered/resolved with sequelae:  The subject has recovered from the condition, but 
with lasting effect due to a disease, injury, treatment 
or procedure. If a sequela meets an SAE criterion, 
the AE must be reported as an SAE. 
Not recovered/not resolved:  
The condition of the subject has not improved and 
the symptoms are unchanged. 
Fatal:  
This term is only applicable if the subject died from 
a condition related to the reported AE. Outcomes of 
other reported AEs in a subject before he/she died 
should be assessed as "recovered/resolved", 
"recovering/resolving", "recovered/resolved with 
sequelae" or "not recovered/not resolved". An AE 
with fatal outcome must be reported as an SAE. 
Unknown:  
This term is only applicable if the subject is lost to 
follow-up. 
7.2.1.2 Collection, recording and reporting of adverse events 
All events meeting the definition of an AE must be collected and reported from the first 
trial related activity after the subject has signed the informed consent until the end of the 
post-treatment follow-up period. At each contact with the center (visit or telephone, 
excluding visits where the subject is not seeing the investigator or his/her staff [e.g. visits 


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to the laboratory]) the subject must be asked about AEs. All AEs, either observed by the 
investigator or reported by the subject, must be recorded by the investigator and 
evaluated. 
The investigator should record the diagnosis, if possible. If no diagnosis can be made the 
investigator should record each sign and symptom as individual AEs. 
One single Adverse Event Form must be used per AE from start to resolution. For SAEs, 
the Serious Adverse Event form must also be completed. For AESIs, the AESI form must 
also be completed. 
AE information should include the following: 

Date and time of onset and resolution

Date and time of investigator’s first information on the (S)AE

Seriousness

Severity

Causal relationship with IMP

Interruption or withdrawal of treatment during IMP administration and other
measures taken

Outcome.
All AEs are coded; details are described in the trial-specific Data Management Plan. 
If an event classifies as a AESI, the investigator must tick the AESI box on the AE form 
and complete the AESI form. The AESI form will capture if the event was associated 
with any signs or symptoms and capture the highest/lowest blood pressure and pulse 
measured during the event. The investigator must report all AESIs to the sponsor’s 
responsible pharmacovigilance unit (Safety CRO; see the list of trial personnel in 
Section 12.1) immediately (within 24 hours) after obtaining knowledge about the event. 
The investigator must report initial information electronically (e.g. in PDF format) on all 
SAEs to the sponsor’s responsible pharmacovigilance unit (Safety CRO; see the list of 
trial personnel in Section 12.1) immediately (within 24 hours) after obtaining knowledge 
about the event. The Safety CRO will inform the medical monitor and the sponsor about 
the reported SAEs. 
It is the responsibility of the Safety CRO to report all SUSARs (see Section 7.2.1.1) that 
occur in this trial to the Competent Authorities and Institutional Review Boards 
(IRBs)/Independent Ethics Committees (IECs) in accordance with the local requirements 
in force and ICH guideline for GCP. The trial monitor must be informed accordingly. 


 
 
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7.2.1.3 Follow-up of adverse events 
All AEs that are ongoing at the end of the subject’s participation in the trial will be 
followed-up until the event is resolved or reaches a satisfactory outcome as deemed by 
the investigator or until the last visit of the last subject enrolled in the trial, whichever 
occurs first.   
Follow-up actions for all SAEs will be performed according to appropriate clinical care 
practices and may depend on the nature of the event. These will be determined after 
internal review and/or sponsor review. 
The follow-up information should only include new (updated and/or additional) 
information that reflects the situation at the time of the investigator’s signature. 
Follow-up information on (S)AEs will be updated using the (S)AE form. If a non-serious 
event becomes serious during the follow-up the AE form and SAE form have to be used 
and reporting timelines follow those of an SAE. 
The investigator must forward follow-up information on SAEs and if previously 
non-serious AEs become SAEs to the Safety CRO immediately (within 24 hours) after 
obtaining knowledge about the new information. 
The sponsor and/or CROs acting on behalf of the sponsor can upgrade a non-serious AE 
to an SAE. In these situations the investigator will be informed and asked to fill out an 
SAE form and forward to the Safety CRO immediately (within 24 hours). 
7.2.1.4 Clinical laboratory abnormalities and other abnormal assessments as adverse 
events or serious adverse events 
Abnormal laboratory findings (e.g. biochemistry, hematology, urinalysis) or other 
abnormal assessments (e.g. vital signs) that are judged by the investigator as clinically 
significant will be recorded as AEs or SAEs if they meet the definition of an AE or SAE. 
Clinically significant abnormal laboratory findings or other abnormal assessments that 
are detected during the trial or are present at baseline and significantly worsen following 
the start of the trial will be reported as AEs or SAEs. However, clinically significant 
abnormal laboratory findings or other abnormal assessments that are associated with the 
disease being studied, unless judged by the investigator as more severe than expected for 
the subject’s condition, or that are present or detected at the start of the trial and do not 
worsen, will not be reported as AEs or SAEs. 
The investigator will exercise his or her medical and scientific judgment in deciding 
whether an abnormal laboratory finding or other abnormal assessment is clinically 
significant. 


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7.2.2 Hypoglycemia 
Hypoglycemia will be regarded as an AE and recorded and documented on an AE form 
(and SAE form, if applicable). 
Hypoglycemia is defined as a decline in plasma glucose to below 70 mg/dL 
(3.9 mmol/L). However, in the time period from initiation of the hypoglycemic clamp 
procedure (see Section 8.2.3.1) until 45 minutes after dosing, hypoglycemia is defined as 
a decline in plasma glucose to below 45 mg/dL (2.5 mmol/L).  
During the dosing visit, prior to administration of the IMP, a plasma glucose value of 
<45 mg/dL (2.5 mmol/L) will be treated by an IV glucose solution. After administration 
of the IMP in the period from 8 minutes until 44 minutes after dosing, a plasma glucose 
value of <45 mg/dL (2.5 mmol/L) will be treated by an IV glucose solution, and if a 
plasma glucose value of ≥70 mg/dL (3.9 mmol/L) is not achieved within the 45 minutes 
after IMP administration, IV glucose infusion will also be initiated. 
If the subject experiences symptoms of hypoglycemia, a plasma glucose measurement 
should be taken in order to classify the event (please refer to Section 7.4.2 for additional 
details).  
7.2.3 Physical examination 
The physical examination will be carried out at screening (Visit 1) and at the follow-up 
visit (Visit 3; see Table 3).  
The physical examination includes examination of the following body systems: head, 
ears, eyes, nose, throat, including the thyroid gland; heart, lung, chest; abdomen; skin and 
mucosae; musculoskeletal system; nervous system; lymph node; other findings. 
At the screening visit, any abnormality will be recorded and described in the eCRF, 
including the investigator’s assessment of clinical significance (‘abnormal, not clinically 
significant’ or ‘abnormal, clinically significant’). Clinically significant findings at the 
screening visit should be recorded as concomitant illness. 
At subsequent visits, any clinically significant deterioration of a pre-existing condition as 
well as any new clinically significant findings will be recorded as AEs (see 
Section 7.2.1). 
7.2.4 Vital signs 
An examination of the following vital signs will be performed at screening (Visit 1), the 
dosing visit (Visit 2) and at the follow-up visit (Visit 3):  


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
Diastolic and systolic blood pressure (mmHg) will be measured after at least
5 minutes rest in a supine position. At Visit 1, blood pressure will be measured in
both arms. The blood pressure from the arm with the higher systolic value is
transcribed into the eCRF and this arm should be used for all subsequent
measurements of the subject’s blood pressure in this trial.

Pulse (beats per min) measured after at least 5 minutes rest in a supine position.

Body temperature (°C).
At the dosing visit, measurements will be taken prior to the start of the insulin-induced 
hypoglycemic procedure (within 30 minutes) and at 30, 90, and 300 minutes after dosing 
(see Table 3). The actual time of the assessment should not deviate from the nominal time 
by more than ±10 minutes. In addition to the pre-specified assessments as listed in 
Table 3, blood pressure and pulse may be assessed at any time during the trial at the 
discretion of the investigator.  
7.2.5 Electrocardiogram 
A standard 12-lead ECG will be performed at the screening visit (Visit 1), the dosing visit 
(Visit 2; prior to the start of the insulin-induced hypoglycemic procedure [within 
30 minutes]) and at 20, 35, 45, 60, and 300 minutes after dosing) and at the follow-up 
visit (Visit 3; see Table 3). The actual time of the assessment should not deviate from the 
nominal time by more than ±5 minutes.  
ECG parameters (heart rate, PQ, QRS, QT, QTcB) and any abnormality will be recorded 
and described in the eCRF including the investigator’s assessment of clinical significance 
(‘abnormal, not clinically significant’ or ‘abnormal, clinically significant’).  
At subsequent visits, any clinically significant deterioration of a pre-existing condition as 
well as any new clinically significant findings will be recorded as AEs (see 
Section 7.2.1). 
7.2.6 Local tolerability 
Immediately prior to treatment administration, it should be verified that the injection site 
is normal. To ensure all injection site assessments are performed at the injection site, the 
site will be marked with a pen prior to injection. Assessment of local tolerability at the 
injection site will be performed at the dosing visit (Visit 2; at 30, 120 and 300 minutes 
after dosing), and at the follow-up visit (Visit 3) (see Table 3) and more frequently, if 
deemed necessary by the investigator. The actual time of the assessment should not 
deviate from the nominal time by more than ±10 minutes. In the eCRF, the time of 
assessment and any injection site reaction observed will be recorded. In case of an 
observation, the Local Tolerability form will be completed, as well as the (S)AE form. 


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The local tolerability at the injection site will be evaluated by means of the following 
assessments: spontaneous pain, pain on palpation, itching, redness, edema, 
induration/infiltration, and other. Each of these assessments will be reported on a scale of 
0 (none), 1 (mild), 2 (moderate), and 3 (severe). The evaluation and the actual time of the 
assessment will be recorded. The assessments will be performed by a trial physician or 
nurse.  
Digital pictures will be taken of the injection site at the time of identification, and 
thereafter as often as judged necessary by the investigator. The pictures should include a 
subject identifier, visit number, time after dosing, and a ruler for scaling. 
7.2.7 Clinical laboratory assessments 
The safety parameters that will be assessed at the clinical laboratory are listed in Table 3. 
Routine clinical laboratory tests will be performed centrally. Samples for clinical 
laboratory parameters (biochemistry, hematology, coagulation) will be collected at 
screening (Visit 1), the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure [within 30 minutes]) and at 30 and 300 minutes after dosing), 
and at the follow-up visit (Visit 3). The actual time of the assessment should not deviate 
from the nominal time by more than ±10 minutes. Samples for glycated hemoglobin 
(HbA1c) will be collected at screening only (Visit 1). Samples for urinalysis will be 
collected at screening (Visit 1), at the dosing visit (Visit 2, prior to the start of the 
insulin-induced hypoglycemic procedure [within 120 minutes]) and at the follow-up visit 
(Visit 3). The following parameters will be assessed: 

Clinical biochemistry: sodium, potassium, calcium, glucose, urea, creatinine, total
bilirubin, AST, ALT, gamma-glutamyltransferase, alkaline phosphatase, total protein,
C-reactive protein, HbA1c, C-peptide.

Hematology: hemoglobin, red blood cell count (erythrocytes), hematocrit, platelet
count (thrombocytes), total white blood cell count (leukocytes).

Coagulation: international normalized ratio, fibrinogen (at screening visit only).

Urinalysis: pH, blood (leukocytes and erythrocytes), protein, glucose, ketones, nitrite.
Pregnancy tests will be performed at each visit for women of childbearing potential only. 
A serum pregnancy test will be performed at screening (Visit 1) and urine stick tests will 
be performed at the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure) and the follow-up visit (Visit 3). Test sticks will be provided to 
the trial centers. 
Alcohol breath tests and a urine drug screen will be performed at screening (Visit 1) and 
at the dosing visit (Visit 2, prior to the start of the insulin-induced hypoglycemic 


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procedure). Equipment for the alcohol breath test and uring drug screen will be provided 
to the trial centers.  
Re-assessment of laboratory parameters will be allowed only if handling issues, damaged 
samples, or hemolyzed samples have confounded the measurement results.  
For further details of the clinical laboratory assessments, please refer to the laboratory 
manual.  
7.2.8 Pregnancy 
Female subjects must be instructed to notify the investigator immediately if they become 
pregnant or if they suspect to be pregnant during the trial. The sponsor has a 
responsibility to monitor the outcome of all pregnancies reported during the clinical trial. 
Male subjects must be instructed to notify the investigator immediately if their partner 
becomes pregnant or suspects pregnancy. The sponsor has a responsibility to monitor the 
outcome of all pregnancies reported during the clinical trial. During an information 
meeting at the trial center, the subject’s partner will be fully informed by a physician of 
her participation in capturing the outcome data, and will be asked to provide her written 
consent. 
The investigator must report all information on pregnancies on the Initial Pregnancy 
form. The completed Initial Pregnancy form must be forwarded to the sponsor 
immediately (within 24 hours), according to the procedure stated in Section 7.2.1.2. Any 
(S)AEs in the mother, as well as other pregnancy complications (ectopic pregnancy) 
fulfilling the criteria of an (S)AE, must be reported on the (S)AE form.  
The following must be collected in the Initial Pregnancy form: 

Medical history of the mother

Family history

Course of the pregnancy, including expected delivery date.
The investigator must follow the pregnancy until the pregnancy outcome and follow the 
newborn infant(s) until the age of 1 month. The investigator must collect information on 
the pregnancy and pregnancy complications as well as the pregnancy outcome including 
the health of the newborn infant(s) on the Pregnancy Outcome form. The completed 
Pregnancy Outcome form must be forwarded to the sponsor according to the procedure 
stated in Section 7.2.1.2. Any (S)AEs in the newborn must be reported on the (S)AE 
form. 


 
 
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The SAEs that must be reported include abnormal outcome, such as congenital 
anomalies, fetal death and termination of pregnancy (spontaneous or elective abortion), 
including any anomalies of the fetus observed at gross examination or during autopsy.  
The following must be collected in the Pregnancy Outcome form:  
 Course of the pregnancy 
 Outcome of the pregnancy 
 Condition of the newborn 
 Any AEs in the newborn infant must be followed until the age of 1 month. 
7.2.9 Precautions 
Normal precautions taken for a human trial, including the provision of emergency 
equipment, will be taken during this trial. Qualified and well trained physicians and 
medical staff will instruct the subjects. During a subject’s participation in the trial, the 
investigator should ensure that adequate medical care is provided to the subject for any 
AEs, including clinically significant laboratory values related to the trial. The investigator 
should inform the subject when medical care is needed for intercurrent illnesses of which 
the investigator becomes aware.  
For further information on safety precautions for dasiglucagon and GlucaGen, please 
refer to the current version of the Investigator’s Brochure (14) and the Summary of 
Product Characteristics for GlucaGen (13), respectively. 
7.2.10 Safety committee 
The internal Zealand Pharma Safety Committee is constituted to perform ongoing blinded 
safety surveillance of clinical trials with dasiglucagon, including this trial. 
If safety signals are observed, either based on reported SAEs, periodic review of 
laboratory parameters, review of all AEs reported between the Safety Committee 
meetings, or on notification of significant findings, the Safety Committee will take 
appropriate measures to safeguard the subjects. 
7.3 
Demography, concomitant illness, medical history and concomitant 
medication 
Demographics, body measurements, concomitant illness and medical history will be 
assessed only at screening (Visit 1). Concomitant medication will be assessed at 
screening (Visit 1), the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure) and at the follow-up visit (Visit 3). 


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7.3.1 Demography and body measurements 
Subject demographics and body measurements will include: 

Age

Race, ethnicity

Sex

Height (meters or inch), without shoes

Body weight (kg or lb), only wearing underwear and measured using standard scales

Body mass index (kg/m2) calculated based on height and body weight (body
weight/height2).
7.3.2 Concomitant illness and medical history 
A concomitant illness is any illness that is present at the start of the trial (i.e. at the 
screening visit). Concomitant illnesses present at the start of the trial will be recorded in 
the eCRF at screening.  
Medical history is an account of medical events that the subject has experienced in the 
past, including prior medications. Relevant medical conditions/illnesses in the past 
obtained by asking the subject or by inspecting his/her medical records will be recorded 
in the eCRF at screening. History of alcohol or drug abuse will also be recorded. 
The information collected for concomitant illness and medical history should include 
diagnosis, date of onset, date of resolution or continuation. 
Any change to a concomitant illness should be recorded during the trial, including end 
date, if applicable. A clinically significant worsening of a concomitant illness must be 
reported according to Section 7.2.1. 
All previous and concomitant diseases will be coded with Medical Dictionary for 
Regulatory Activities (MedDRA), the version of which will be provided in the clinical 
trial report. 
7.3.3 Diabetes diagnosis and current treatment 
The date of diagnosis of diabetes will be recorded as will the current diabetes treatment 
(start date, product name(s), dose(s)). 


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7.3.4 Concomitant medication 
A concomitant medication is any medication, other than the trial products and current 
diabetes treatment (including insulin glulisine [Apidra®] for diabetes therapy wash-out), 
which is taken during the trial, including screening and follow-up periods. 
Details of any concomitant medication must be recorded at trial entry (i.e. at screening). 
Any changes in concomitant medication must be recorded at each visit as they occur. The 
information collected for each concomitant medication includes (at a minimum) trade 
name or generic name, indication, start date and stop date or continuation. A change in 
medication due to an AE must be recorded and reported according to Section 7.2.1. If the 
change in medication influences the subject’s eligibility to continue in the trial, the 
sponsor and trial monitor must be informed. 
7.4 
Other assessments 
7.4.1 Immunogenicity 
Antibodies against dasiglucagon/GlucaGen will be measured at the dosing visit (Visit 2) 
and at follow-up (Visit 3). At the dosing visit (Visit 2), samples will be collected prior to 
the start of the insulin-induced hypoglycemic procedure.  
The clinical ADA assays, specific for dasiglucagon and GlucaGen, respectively, have 
been validated in accordance with existing guidelines and recommendations (17-21).  
Confirmed positive anti-dasiglucagon antibody samples (treatment-induced or 
treatment-boosted) from anti-dasiglucagon antibody-positive subjects will be evaluated 
for binding titer neutralizing potential and titer as well as cross-reactivity towards 
endogenous glucagon.  
No further serum sampling will be needed as the ADA samples can be used for 
neutralizing antibody analysis. 
The in vitro neutralizing effect of the antibodies will be measured using an assay based 
on glucagon receptor transfected human embryonic kidney cells (20,22). The calculated 
sensitivity in previous studies was about 51.8 ng/mL. The assay was also validated for 
recombinant glucagon with similar results (21,23). In case of a positive result in the 
neutralizing antibody assay, a titer estimation will be performed. The cell-based 
neutralizing antibody analyses will be performed by a special laboratory, BioAgilytix, 
Durham, NC, USA. 


 
 
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Residual and additional antibody serum samples may be stored until approval of market 
authorization by the health authorities. Further characterization of the antibody response 
may be requested by the health authorities. 
7.4.2 Plasma glucose measurements for safety 
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at 
site. After the start of insulin infusion, plasma glucose should be checked every 
10 minutes while plasma glucose is above 110 md/dL, and every 5 minutes once plasma 
glucose is at or below 110 mg/dL and until after dosing when the subject’s plasma 
glucose is ≥70mg/dL (3.9 mmol/L). Hereafter, plasma glucose should be checked every 
30 minutes until 300 minutes (5 hours) after dosing. Plasma glucose concentrations will 
be measured using a US FDA-approved glucose analyzer (e.g., YSI 2300, Yellow 
Springs Instruments, Yellow Springs, OH). 
At the discretion of the investigator, additional plasma glucose measurements can be 
taken at any time during the trial, for example when there is a suspicion (e.g. symptoms) 
of a hypoglycemic episode. 
Plasma glucose measurements for safety should only be recorded in the eCRF if they are 
related to an AE (e.g. a hypoglycemic episode). 
In case of persistent post-treatment hypoglycemia, subjects will receive rescue treatment 
with an IV glucose infusion (see Section 8.2.3.1 for details). Blood samples for PD and 
PK assessments should still be taken at the specified timepoints. 
7.4.3 Plasma insulin measurements 
Samples for insulin assessment will be collected at the dosing visit (Visit 2, pre-dose and 
at 30 and 60 minutes after dosing). The actual time of blood sampling for evaluation of 
plasma insulin should not deviate from the nominal time by more than ±1 minute. 
Pre-dose is defined as within 2 minutes prior to dosing. 


Dasiglucagon (ZP4207) 
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8 
TRIAL CONDUCT 
8.1 
Schedule of procedures 
The schedule of procedures is provided in Table 3. Informed consent will be obtained 
prior to any trial-related procedures; see Section 10.8. 
8.2 
Procedures by visit 
8.2.1 Visit 1 (screening, Day -30 to Day -3) 
Visit 1 will take place between 3 and 30 days before Visit 2, Day -1 to Day 1 (dosing 
day). 
Informed consent can be obtained prior to or at Visit 1, however it must in any case be 
obtained prior to any trial related procedures. During the screening visit, the following 
assessments will take place: 

Informed consent (obtain or check)

Check of subject eligibility (inclusion/exclusion criteria)

Demography

Body measurements

Medical history, diabetes diagnosis, current diabetes treatment

Concomitant illnesses

Concomitant medications

History of alcohol/drug abuse

Physical examination

Vital signs

12-lead ECG

AEs

Biochemistry, hematology, coagulation, HbA1c

Serum pregnancy test (women of childbearing potential only)

Urinalysis

Urine drug screen

Alcohol breath test


 
 
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Table 3 
Schedule of procedures 
Visit number 
V1 
V2 
V3 
Trial day 
-3 
-1 and 1 
28 
Visit type 
Screening 
Dosing 
Follow-up 
Window 
-30 to -3 
 
+5 days 
Subject related information/assessments 
 
 
 
Informed consent 
x 
 
 
Inclusion/exclusion criteria 
x 
x1,2 
 
Demography 
x 
 
 
Body measurements 
x 
 
 
Medical history, diabetes diagnosis, and current diabetes 
treatment 
x 
 
 
Concomitant illnesses 
x 
 
 
Concomitant medications 
x 
x1 
x 
History of alcohol/drug abuse 
x 
 
 
Randomization 
 
x1 
 
Withdrawal criteria 
 
x1 
 
Dosing day exclusion criteria 
 
x1 
 
Insulin-induced hypoglycemia 
 
x 
 
Safety assessments 
 
 
 
Physical examination 
x 
 
x 
Vital signs 
x 
x3 
x 
12-lead ECG 
x 
x4 
x 
Local tolerability  
 
x5 
x 
Adverse events 
x 
x 
x 
Laboratory 
 
 
 
Biochemistry, hematology, coagulation, HbA1c (HbA1c at 
Visit 1 only) 
x 
x6 
x 
Pregnancy test (women of childbearing potential only) 
x7 
x1,8 
x8 
Urinalysis 
x 
x1 
x 
Urine drug screen 
x 
x1 
 
Alcohol breath test 
x 
x1 
 
PK/Clinical efficacy 
 
 
 
Plasma dasiglucagon/GlucaGen 
 
x9 
 
Plasma glucose 
 
x10 
 
Other assessments 
 
 
 
Antibodies against dasiglucagon/GlucaGen 
x1 
x11 
Plasma insulin 
 
x12 
 
Trial material 
 
 
 
Administration of trial product (during hypoglycemic clamp 
procedure) 
 
x 
 
1Prior to the start of the insulin-induced hypoglycemic procedure. 
2Only check of dosing day exclusion criteria and changes between screening visit and Visit 2. 
3Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 30, 90 and 300 minutes 
after dosing. The actual time of the assessment should not deviate from the nominal time by more than ±10 minutes. 


Dasiglucagon (ZP4207) 
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4Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 20, 35, 45, 60 and 300 
minutes after dosing. The actual time of the assessment should not deviate from the nominal time by more than ±5 
minutes.  
5Local tolerability assessed at 30, 120, and 300 minutes after dosing. The actual time of the assessment should not 
deviate from the nominal time by more than ±10 minutes.  
6Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 30 and 300 minutes after 
dosing. The actual time of the assessment should not deviate from the nominal time by more than ±10 minutes. 
7Serum pregnancy test. 
8Urine stick pregnancy test. 
9Pre-dose, and at 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time of blood sampling should not 
deviate from the nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing. 
10Pre-dose, and at 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, 90 minutes after dosing. The actual time of blood 
sampling should not deviate from the nominal time by more than ±30 seconds until the 20 minute collection time point 
and by more than ±1 minute for the subsequent collection time points. Pre-dose is defined as within 2 minutes prior to 
dosing. 
11Any subject that tests positive for ADA will be monitored until the ADA levels return to baseline levels. 
12Pre-dose, and at 30 and 60 minutes after dosing. The actual time of blood sampling should not deviate from the 
nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing.  
ADA = anti-drug antibodies; ECG = electrocardiogram; HbA1c = glycated hemoglobin. 
Eligible or potentially eligible subjects (laboratory results pending) will be provided with 
an Identification card (ID card), stating that the subject is participating in the trial and 
whom to contact (site address, investigator name and telephone number). The subjects 
should be instructed to return the ID card to the investigator at the last visit or to destroy 
the card after the last visit.  
8.2.2 Instructions to subjects prior to the dosing visit (Visit 2) 
At the screening visit, the investigator will inform the subject about the changes to his/her 
insulin therapy leading up to the start of the insulin-induced hypoglycemic procedure. 
The subject may be provided with insulin NPH in the wash-out period to cover the need 
of basal insulin, if deemed necessary by the investigator. The subject’s current insulin 
therapy will be washed out as defined in Section 5.5.: 48 hours prior to dosing and during 
the dosing visit, treatment with  insulin Degludec and insulin Glargine U300 are not 
allowed; 24 hours prior to dosing and during the dosing visit, treatment with other long-
acting insulins (e.g., insulin Glargine U100 or insulin Detemir) is not allowed; 16 hours 
prior dosing and during the dosing visit treatment with insulin NPH is not allowed; 6 
hours prior to dosing and during the dosing visit, treatment with any short acting (bolus) 
insulin, except insulin glulisine (Apidra®), is not allowed. The basal rate of insulin pumps 
(continuous SC insulin infusion) will be discontinued on the morning of the dosing day 
(if using insulin glulisine [Apidra®]) OR at least 6 hours prior to dosing (if using other 
insulins). 
On the day prior to dosing (Day -1), the subjects will need to attend the clinical center 
and will be required to stay onsite overnight. On the morning of the dosing day (Day 1), 
patients are required to be in a fasting condition, defined as having consumed only water 
since 22:00 hours the night before. However, the subjects are allowed to consume small 


 
 
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Study ID: ZP4207-16137 
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amounts (up to 20 g) of carbohydrates to prevent hypoglycemia. The subjects must also 
not consume any alcohol within 24 hours prior to dosing (refer to Section 5.5 for all 
dosing day exclusion criteria). 
8.2.3 Visit 2 (dosing visit) 
Visit 2 will take place on Day -1 to Day 1. 
The subjects will attend the clinical center the day prior to dosing (Day -1) and subject 
eligibility is rechecked (check of changes between the screening visit and Visit 2) and 
those subjects eligible to participate should stay onsite overnight. To target a glucose 
level around 90-110 mg/dL (5.0-6.1 mmol/L) the following morning, subjects may be 
administered insulin glulisine (Apidra®) at the investigator’s discretion either by IV 
infusion, SC bolus or continuous SC insulin infusion. Dosing will take place the 
following morning (Day 1). 
On Day 1 and prior to the start of the insulin-induced hypoglycemic procedure,  those 
subjects eligible to participate will be randomized to treatment with dasiglucagon, 
placebo, or GlucaGen.  
The following assessments will also take place: 
 Document all changes in concomitant medication (prior to the start of the 
insulin-induced hypoglycemic procedure) 
 Check of withdrawal criteria (prior to the start of the insulin-induced hypoglycemic 
procedure) 
 Check of dosing day exclusion criteria (prior to the start of the insulin-induced 
hypoglycemic procedure) 
 Vital signs (prior to the start [within 30 minutes] of the insulin-induced hypoglycemic 
procedure, and at 30, 90 and 300 minutes after dosing). 12-lead ECG (prior to the 
start [within 30 minutes] of the insulin-induced hypoglycemic procedure, and at 20, 
35, 45, 60, and 300 minutes after dosing) 
 Local tolerability (at 30, 120, and 300 minutes after dosing) 
 AEs  
 Biochemistry, hematology, coagulation (prior to the start of the insulin-induced 
hypoglycemic procedure [within 30 minutes], and at 30 and 300 minutes after dosing) 
 Urine stick pregnancy test (women of childbearing potential only; prior to the start of 
the insulin-induced hypoglycemic procedure) 
 Urinalysis (prior to the start of the insulin-induced hypoglycemic procedure [within 
120 minutes]) 
 Urine drug screen (prior to the start of the insulin-induced hypoglycemic procedure) 
 Alcohol breath test (prior to the start of the insulin-induced hypoglycemic procedure) 


Dasiglucagon (ZP4207) 
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
Dasiglucagon/GlucaGen plasma concentrations:
- 
Pre-dose, 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time
of blood sampling for exposure should not deviate from the nominal time by more 
than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing. 

Plasma glucose concentrations:
- 
Pre-dose, 4, 6, 8 ,10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, and 90 minutes after
dosing. The actual time for blood sampling for plasma glucose should not deviate 
from the nominal time by more than ±30 seconds until the 20 minute collection 
time point and by more than ±1 minute for the subsequent collection time points. 
Pre-dose is defined as within 2 minutes prior to dosing. 

Antibodies against dasiglucagon/GlucaGen (prior to the start of the insulin-induced
hypoglycemic procedure).

Plasma insulin concentrations:
- 
Pre-dose, 30 and 60 minutes after dosing. The actual time of blood sampling for
plasma insulin should not deviate from the nominal time by more than ±1 minute. 
Pre-dose is defined as within 2 minutes prior to dosing. 
8.2.3.1 Hypoglycemic clamp procedure and administration of trial medication 
The following procedure is based on precendent procedures for hypoglycemia induction 
in patients with T1DM [24, 25]. 
The treatment day (Visit 2, Day 1) will be conducted after an overnight fast of at least 
8 hours, targeting a starting plasma glucose around 90 to 110 mg/dL (5.0-6.1 mmol/L).  
Subjects who meet one or more of the dosing day exclusion criteria (Section 5.5) at the 
dosing visit will be excluded from the dosing visit, but the visit can be rescheduled 
1-7 days later. The dosing visit can only be rescheduled once. 
The subject’s current insulin therapy will be washed out as defined in Section 5.5. For 
subjects using multiple daily injections, the date, time and the dose of the last basal 
insulin and the last short-acting insulin (except insulin glulisine [Apidra®]) administration 
prior to dosing will be captured. For subjects using an insulin pump, the time of 
discontinuation of the basal rate will be captured. Any use of insulin glulisine (Apidra®) 
in the last 5 hours prior to initiation of the hypoglycemia induction procedure will also be 
captured.  
At approximately 08:00 hours, an infusion catheter will be inserted into each arm 
(forearm cephalic vein) for the manual glucose clamp procedure, with the glucose 
infusion in one arm and the insulin infusion in the opposite arm (if IV insulin glulisine 
[Apidra®] has been administered during the night the same infusion catheter can be used). 
A third catheter for blood sampling will be placed into a metacarpel vein for blood 


Dasiglucagon (ZP4207) 
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sampling. This hand will be warmed (55-65ºC) to arterialize venous blood. If there are 
issues with blood sampling from the metacarpal vein for the purpose of glucose 
measurements, a new and more proximal IV access may be used at the discretion of the 
investigator. 
Hypoglycemia will be gradually induced by a fast-acting IV insulin glulisine (Apidra®) 
infusion (15 U [100 U/mL] in 49 mL saline and 1 mL of subject’s blood or plasma), 
initially at 150% of the subject’s usual basal rate and can be increased or decreased over a 
range of 0% to 200% as judged necessary by the investigator, to achieve a controlled 
decline in plasma glucose, targeting a plasma glucose level of 55 mg/dL (3.1 mmol/L).  
Plasma glucose concentrations will be measured using a US FDA-approved glucose 
analyzer (e.g., YSI 2300, Yellow Springs Instruments, Yellow Springs, OH). After the 
start of the insulin infusion, plasma glucose will be measured every 10 minutes while 
plasma glucose is above 110 mg/dL, and every 5 minutes once plasma glucose is at or 
below 110 mg/dL. 
Once the glucose concentration declines to <60 mg/dL (3.3 mmol/L), the insulin infusion 
will be stopped, and 5 min later plasma glucose concentration will be measured at the 
glucose analyzer and blood samples for baseline assessment of plasma glucose, 
dasiglucagon/GlucaGen PK, and insulin PK will be collected. The samples are the 
baseline samples and should be collected within 2 minutes before IMP administration. 

If plasma glucose is ≥45 mg/dL and <60 mg/dL (2.5-3.3 mmol/L), study treatment
(IMP) will be administered, defining time, t=0. The study treatment will be delivered
in the abdomen, buttock, or thigh (according to stratification) via SC injection, with
the subject lying in a lateral recumbent position.

If plasma glucose is <45 mg/dL (2.5 mmol/L), IV glucose solution will be
administered sufficient to raise plasma glucose to within the 45-60 mg/dL target
range. The run-in period will be adequately extended (at least 30 min) until the above
target is achieved and new baseline samples for plasma glucose,
dasiglucagon/GlucaGen PK, and insulin PK will be collected. Glucose should not be
infused within 10 minutes before IMP administration. If plasma glucose is not within
target range after the second attempt, the subject should be rescheduled for a new
treatment visit within 7 days (+ 2 days).
Administration of IMP should not occur earlier than 9:00 hours in the morning or later 
than 12:00 hours. 
As shown in Table 4, serial blood samples for glucose will be collected at t=0, 4, 6, 8, 10, 
12, 15, 17, 20, 25, 30, 40, 50, 60, 75 and 90 minutes post-dosing. Samples for assessing 
plasma dasiglucagon/GlucaGen concentration will be collected  at t=0, 15, 30, 35, 40, 50, 


Dasiglucagon (ZP4207) 
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60, 90, and 120 minutes. Samples for assessing plasma insulin concentration will be 
collected at t=0, 30, and 60 minutes.  
Table 4 
Post-treatment blood sampling times 
Times 
0 
4 
6 
8 
10 
12 
15 
17 
20 
25 
30 
35 
40 
50 
60 
75 
90 
120 
Plasma glucose 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
PK 
Dasiglucagon/ 
GlucaGen 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
PK Insulin 
Y 
Y 
Y 
Refer to Section 7.2.7 for details of laboratory safety sampling and to Section 7.4.2 for 
details of blood glucose safety sampling.  
When the t=90-minute blood sampling for plasma glucose has been collected the subjects 
are allowed to eat moderately. Drinking of water is allowed ad libitum during the entire 
procedure. 
Hypoglycemia Rescue Provisions 
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at 
site for safety reasons as described in Section 7.4.2. Subjects should receive 
post-treatment rescue glucose infusion to ameliorate persistent hypoglycemia, as follows. 
1. Glucose infusion should be initiated if a subject experiences severe alarming
escalation of symptoms of hypoglycemia (e.g. symptoms suggesting a change in
consciousness) at any time during the trial; glucose infusion should be initiated
targeting a plasma glucose levels >70 mg/dL.
2. If plasma glucose is <45 mg/dL (2.5 mmol/L) between t=8 and t=44 minutes,
rescue glucose infusion (1-2 mg/kg administered IV over about 5 seconds) should
be initiated to maintain plasma glucose between 45 mg/dL and 55 mg/dL
(2.5-3.1 mmol/L). Pause glucose infusion if plasma glucose is >55 mg/dL.
3. If plasma glucose is <70 mg/dL at t=45 minutes, rescue glucose infusion
(2-3 mg/kg administered IV over about 5 seconds) should be initiated to maintain
plasma glucose between 70 mg/dL and 80 mg/dL (3.9-4.4 mmol/L). Pause
glucose infusion if plasma glucose is >75 mg/dL (4.2 mmol/L).
Subjects should remain in bed until completion of the test procedure 300 minutes after 
dosing (bathroom visits are allowed).  


Dasiglucagon (ZP4207) 
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The IMP will be administered SC according to Section 6.3. The time of IMP 
administration will be recorded. At the timepoint when the insulin infusion is stopped, the 
total insulin dose which was required to induce hypoglycemia will be recorded. 
AEs will be specifically recorded during the procedure at several timepoints. 
The investigator must provide information to the subjects on how to resume their usual 
diabetes treatment. 
The subject may be released from the clinical center if the investigator does not have any 
safety concerns based on the last safety plasma glucose value and the general condition of 
the subject. However, at the discretion of the investigator or on request of the subject, the 
subject may stay at the trial center for a longer period. 
8.2.4 Visit 3 (follow-up visit) 
Visit 3 will take place on Day 28 + 5 days. The subject does not need to be fasting. 
At Visit 3, the following assessments will take place: 

Document all changes in concomitant medication

Physical examination

Vital signs

12-lead ECG

Local tolerability

AEs

Biochemistry, hematology, coagulation

Urine stick pregnancy test (women of childbearing potential only)

Urinalysis

Antibodies against dasiglucagon/GlucaGen.
After the follow-up visit the End of Trial form must be completed. Even if a subject is not 
able to attend the follow-up visit, the End of Trial form, the eCRF 
Accountability/Affirmation Statement form and the Drug Accountability form must be 
completed. 


Dasiglucagon (ZP4207) 
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9 
STATISTICAL METHODS 
Before database lock and treatment unmasking, a separate statistical analysis plan (SAP) 
will be finalized, providing detailed methods for the analyses outlined below. Further 
analysis details may be added or refined in the SAP. 
Any deviations from the planned analyses will be described and justified in the final 
clinical trial report. 
9.1 
Determination of sample size 
Due to requirements in the size of the safety database, the sample size is set to 78 subjects 
treated with dasiglucagon 0.6 mg. With a 2:1:1 randomization ratio for the three 
treatments, dasiglucagon 0.6 mg, placebo, and GlucaGen 1 mg, 78 subjects injected with 
dasiglucagon and 39 subjects with placebo will yield a power of 90% at a 5% two-sided 
significance level to detect a treatment group difference in recovery incidence within 
20 minutes, assuming as low as 80% and as high as 50% recovery incidence for the 
dasiglucagon and placebo groups, respectively. 
9.2 
Trial subjects 
9.2.1 Analysis samples 
For presentation of data and reporting of the statistical analyses, the following analysis 
samples will be used, depending on the context:  

Safety analysis set (SAS): All randomized subjects who received at least one dose of
trial medication.

Full analysis set (FAS): All randomized subjects who received at least one dose of
trial medication and contributed valid information for at least one post-dose endpoint.

Per protocol (PP) set: All subjects of the FAS for whom no relevant protocol
deviations were documented. This sample will primarily be used for sensitivity
analysis.
The analysis of the primary endpoint and secondary endpoints will be based on the FAS. 
A secondary analysis of the primary endpoint will be based on the PP set. All safety 
analyses will be based upon the SAS. 
The decision regarding whether a protocol deviation is relevant or not for the exclusion of 
subjects from the PP set will be made case-by-case in a data review meeting prior to 
treatment unmasking and database lock (see Section 9.2.3). 


 
 
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9.2.2 Disposition of subjects 
Subject disposition will be tabulated including the number of screened subjects, screening 
failures, subjects exposed to trial product, subjects completing the trial and subjects in 
each analysis set. 
Subjects withdrawn from the trial will be listed including the primary reason for 
withdrawal. 
9.2.3 Protocol deviations 
Before data are released for statistical analysis, a treatment-masked review of all data will 
take place to identify protocol deviations that may potentially affect the results. This 
review will be performed without revealing to which trial product the subjects were 
assigned. The masking of the trial products will be maintained for everyone involved in 
allocating subjects to the analysis sets until data are released for statistical analysis. 
Furthermore, spurious outliers will be evaluated. In addition, protocol deviations that may 
potentially affect the results will be identified and it will be evaluated if subjects and/or 
data should be excluded from the analysis. Protocol deviations will be classified as minor 
or major in a consistent way. Major deviations from the protocol may lead to the 
exclusion of a subject from the PP set. 
Major protocol deviations may include deviations related to trial inclusion or exclusion 
criteria, conduct of the trial, subject management or subject assessment. Unless explicitly 
decided otherwise during the masked data review, the following will be considered major 
protocol deviations: 
 Violation of one or more major inclusion/exclusion criteria 
 Interruption of administration of trial product 
 Significant deviation from time windows  
 Incorrect treatment allocation 
 Missing primary endpoint. 
The violation of several major inclusion/exclusion criteria or the complete absence of 
efficacy data might lead to exclusion of the subject from FAS. In that case, the decision 
should be taken at the masked data review meeting, and the exclusion from efficacy 
analysis justified in the signed notes of the meeting. 
Obviously erroneous data points may be excluded from the analyses or re-analyzed (in 
case of e.g. serum concentrations). The decision to re-analyze or exclude data points from 
the statistical analysis is the joint responsibility of the sponsor and the trial statistician.  


 
 
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The subjects or observations to be excluded and the reason for their exclusion will be 
documented and signed by those responsible prior to database release. The 
documentation will be stored together with the remaining trial documentation. The 
subjects and observations excluded from analysis sets, and the corresponding reasons, 
will be described in the clinical trial report. 
9.3 
General considerations 
All data obtained in this trial and documented in the eCRFs will be listed and 
summarized with statistics or frequency tables as appropriate. In case of termination of 
the trial, all data collected up to that timepoint will be included in the analysis. 
Raw data listings and summary tables will be generated using the software SAS© version 
9.4 or higher.  
Continuous variables will be summarized using means, standard deviations, medians, 
coefficients of variation, and minimum and maximum values.  
Other summaries (e.g. quartiles, 95% confidence intervals [CIs]) may be used as 
appropriate. Categorical efficacy and safety variables will be summarized by counts and 
by percentage of subjects in corresponding categories. 
9.4 
Demographics and baseline characteristics 
Baseline and demographic data will be summarized using descriptive statistics. Baseline 
ADA-positive subjects will be calculated as a percentage of the total number of subjects 
whose baseline samples were tested for ADA. 
All other data obtained in this trial and documented in the eCRF will be listed. 
9.5 
Efficacy Analyses 
For the confirmatory analyses, the primary and key secondary endpoints for the 
dasiglucagon 0.6 mg and placebo treated subjects will be compared. A hierarchical 
procedure will be applied for the control of multiplicity. The primary and secondary 
endpoints will be evaluated on the full analysis set. In the primary analysis, those subjects 
who require rescue IV glucose will be censored at the time to plasma glucose recovery.  
In a sensitivity analysis, the time to plasma glucose recovery will be analyzed without 
censoring the subjects who received rescue IV glucose.   


 
 
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9.5.1 Hierarchical testing procedure 
For the confirmatory analyses, the following a priori defined hierarchical inferential test 
order will be applied for the control of the type 1 error rate across the planned multiple 
comparisons. The primary endpoint and key secondary endpoints will be inferentially 
evaluated within the FAS in the following order, where inference will proceed at the 
two-sided 0.05 criterion significance level until the first failure to reject the null 
hypothesis for a dasiglucagon versus placebo comparison: 
 Primary:  Time to plasma glucose recovery 
 Key secondaries 1-4: Plasma glucose recovery within 30 minutes, within 20 minutes, 
within 15 minutes, and within 10 minutes after study drug injection without 
administration of rescue IV glucose. 
 Key secondaries 5-8:  Plasma glucose changes from baseline (CFB) within 30 
minutes, within 20 minutes, within 15 minutes, and within 10 minutes after study 
drug injection or at the time of rescue. 
The GlucaGen versus placebo comparisons will not be included in the inferential testing 
hierarchy, since the efficacy of GlucaGen is previously established, and these 
comparisons are intended to support the validity of the study for the dasiglucagon versus 
placebo comparisons. 
Analogous supportive sensitivity analyses will be conducted in the PP set, but without 
inference intent. 
9.5.2 Primary confirmatory endpoint 
Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase in 
plasma glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic 
clamp procedure without administration of rescue IV glucose.  
9.5.2.1 Primary analysis 
The primary endpoint will be summarized using Kaplan-Meier (KM) estimates stratified 
by treatment group and injection site. The treatment group difference between 
dasiglucagon and placebo will be evaluated inferentially using a pairwise two-sided 
log-rank test. 
In the primary analysis, those subjects who require rescue IV glucose will be censored at 
the time of plasma glucose recovery. This approach is based on the assumption that 
rescue IV glucose administration will shorten the time to recovery. This assumption 
implies that censoring at the time of recovey after rescue therapy is valid as a time to 


 
 
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recovery without rescue would be longer than the observed duration. If recovery has not 
occurred at 45 minutes after study drug injection, censoring will be applied irrespective 
of the use of rescue IV glucose.  
In sensitivity analyses, the time to plasma glucose recovery will be analyzed 1) without 
censoring at the time of recovery for those subjects who require rescue IV glucose before 
45 minutes and 2) with censoring at the time of administration of rescure IV glucose 
before 45 minutes.   
The primary endpoint will additionally be analyzed using a Cox proportional hazards 
(CPH) time to event statistical model, if the hazards by treatment group can be assumed 
proportional using a graphical comparison of log-minus-log survival curves and no more 
than two-thirds of the recovery times are censored in each treatment group. The CPH 
model will be used for inferences, with treatment group and injection site modeled as 
categorical effects, and baseline plasma glucose modeled as a continuous covariate. The 
treatment group rate ratios, active versus placebo, will be estimated together with the 
95% confidence intervals, and pairwise treatment group inferences (dasiglucagon vs 
placebo, GlucaGen vs placebo) will be evaluated using two-sided likelihood ratio tests. 
9.5.3 Secondary endpoints 
 Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection without administration of rescue IV 
glucose. 
 Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection or at the time of rescue. 
9.5.3.1 Confirmatory analysis 
The key secondary incidence variables (plasma glucose recovery within 30 minutes, 
within 20 minutes, within 15 minutes, within 10 minutes, in hierarchical order for 
inference) will be analyzed using descriptive statistics (frequency and percentage) by 
treatment group. Inferential treatment group comparisons will be assessed via pairwise 
tests of independent binomial proportions (dasiglucagon versus placebo, GlucaGen 
versus placebo) using Fisher’s exact test for each pairwise comparison. 
The key secondary endpoints of plasma glucose CFB within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection or at the 
time of rescue, will be analyzed with the plasma glucose CFB at rescue carried forward in 
those subjects who require rescue IV glucose before plasma glucose ≥20 mg/dL recovery. 
Each of these CFB variables will be analyzed using an Analysis of Covariance model, 
with treatment group modeled as a fixed effect and with the baseline plasma glucose 
modeled as a covariate. The dasiglucagon versus placebo treatment group difference will 


 
 
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be evaluated inferentially as a least squares means contrast, using a two-sided t-test at the 
0.05 signficance level, first for the 30 minute CFB, and then sequentially for the 
20 minute, 15 minute, and 10 minute variables, until the first failure to reject. 
9.5.4 Secondary clinical efficacy (PD) endpoints 
 Time to first plasma glucose concentration of ≥70 mg/dL (3.9 mmol/L) without 
administration of rescue IV glucose. 
 Plasma glucose response as area under the curve above baseline from time zero to 
30 minutes, AUC0-30min. 
Secondary clinical efficacy endpoints will be assessed based on plasma concentration 
data from samples collected at the dosing visit (Visit 2).  
Unless otherwise stated, the population base of analysis will be the FAS. 
9.5.4.1 Analysis of secondary clinical efficacy (PD) endpoints 
1. Time to first plasma glucose concentration ≥70 mg/dL from baseline. This 
time-to-event endpoint will be evaluated using a Kaplan-Meier approach, with 
treatment group as a stratification factor, analogous to that used for the primary 
endpoint analysis. Differences between the KM curves (dasiglucagon versus 
placebo, GlucaGen versus placebo) will be evaluated inferentially using pairwise 
two-sided log rank tests. If the ≥70 mg/dL endpoint is not met within 45 minutes 
post-dosing, the time of the last valid plasma glucose measurement up to 45 
minutes will be the censoring time.  
2. The AUC will be calculated as the baseline-adjusted area under the plasma 
glucose profile over time: 
a. AUC0-30min: restricting the time window to the 0 to 30 minutes interval. 
3. The log-transformed AUC endpoint will be analyzed using an analysis of 
covariance (ANCOVA) model with treatment as fixed effect and baseline plasma 
glucose modeled as a covariate. The least squares means treatment group 
differences will be back-transformed (anti-logged) for presentation as a ratio of 
the treatment group geometric means, with their corresponding 95% CI. 
9.5.5 Exposure (PK) endpoints 
 Plasma dasiglucagon and GlucaGen concentrations from time zero to 90 minutes:  
AUC0-90min, Cmax, and tmax. 


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9.5.5.1 Analysis of exposure (PK) endpoints 
AUC will be derived as the area under the individual plasma dasiglucagon/GlucaGen 
concentration profile for PK from 0 to 90 minutes or last valid measurement if this 
measurement is assessed sufficiently close to 90 minutes (decision to be taken at the 
masked data review meeting). The standard trapezoidal method will be used, based on 
actual rather than nominal timepoints.  
Cmax will be determined as the maximum of all valid plasma dasiglucagon/GlucaGen 
concentrations.  
Tmax will be determined as the timepoint where the maximum of all valid plasma 
dasiglucagon/GlucaGen concentration measurements for each measurement series is 
observed. 
The log-transformed PK endpoints AUC and Cmax will be analyzed in the same way as 
the AUC endpoints. 
As tmax is a highly discrete endpoint, Wilcoxon’s rank sum test for unpaired observations 
will be used to assess differences between the two treatment groups. 
9.6 
Exploratory analyses 
Exploratory analyses will include descriptive statistics and modeling analogous to that 
done for key secondary endpoints. However, treatment group comparisons will be 
summarized without inference intent. 

Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL
(1.1 mmol/L) within 30 minutes after study drug injection without administration
of rescue IV glucose.

Plasma insulin response as area under the curve above baseline from time zero to
60 minutes, AUC0-60 min.
Plasma insulin concentrations measured pre-dose and at 30 and 60 minutes after dosing 
(see Table 3) will be presented individually. A summary table per timepoint will be 
provided. The AUC0-60min will be determined and a summary presented.  


 
 
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9.7 
Safety analyses 
9.7.1 Adverse events 
AEs will be coded using the latest available version of the MedDRA, the version of 
which will be provided in the clinical trial report. AE summary tables will include the 
number and percentage of subjects who experienced AEs summarized by system organ 
class and preferred term. 
Germany only: The AE summary for AEs related to insulin glulisine (Apidra®) will be 
presented as an appendix to the clinical trial report, as insulin glulisine (Apidra®) is only 
considered an IMP in Germany. 
 An overall summary table will be provided showing the number and percentage of 
subjects with any: 
 TEAE 
 Severe TEAE 
 Serious TEAE 
 Drug-related TEAE 
 Drug-related severe TEAE 
 Drug-related serious TEAE 
 TEAE leading to withdrawal 
 TEAE with outcome death 
 AESI 
Refer to Section 7.2.1 for the definition of TEAEs. 
9.7.2 Immunogenicity data 
Immunogenicity data will be analyzed descriptively by treatment group. No statistical 
tests are planned. Baseline ADA-positive subjects will be calculated as a percentage of 
the total number of subjects whose baseline samples were tested for ADA. Titer will be 
reported as median and interquartile range. 
9.7.3 Clinical laboratory assessments 
Clinical laboratory test results will be flagged as to whether the result is below, within or 
above the respective reference range. The number of values outside of the reference range 
will be counted. 


 
 
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9.7.4 Other safety data 
Incidence of rescue infusion of IV glucose during the hypoglycemic clamp procedure will 
be analyzed using descriptive statistics (frequency and percentage) by treatment group. 
Inferential treatment group comparisons will be assessed via pairwise tests of 
independent binomial proportions (dasiglucagon versus placebo, GlucaGen versus 
placebo). 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure 
will be evaluated using a KM time to event statistical model, with treatment group and 
injection site as stratification factors. Differences between the KM curves (dasiglucagon 
versus placebo, GlucaGen versus placebo) will be evaluated inferentially using pairwise 
two-sided stratified log-rank tests. If the endpoint is never met, the time of the last plasma 
glucose measurement will be the censoring time. 
Vital signs, physical examination, 12-lead ECG and local tolerability data will be 
summarized using descriptive statistics. 
9.8 
Treatment compliance 
Trained unblinded members of staff will perform all administrations of the IMP at the 
trial center. The administered doses will be recorded in the blinded Drug Accountability 
form in the eCRF. 
PK assessments will support the surveillance of compliance with IMP administration. 
9.9 
Subject withdrawals and missing data 
In the case of subject withdrawal, no imputation of values for PK or PD measurements 
will be done. Analyses will be done on valid cases only, i.e., no imputation techniques 
such as last observation carried forward will be applied. For the primary analysis in the 
FAS, missing values for the primary endpoint will be imputed by a conservative rule 
considering any missing value as a failure. 
9.10 Interim analyses 
No interim analyses are currently planned. 


 
 
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10 
ETHICAL, LEGAL, AND ADMINISTRATIVE ASPECTS 
10.1  Quality assurance 
The sponsor or designee will conduct a site visit to verify the qualifications of each 
investigator, inspect the facilities, and inform the investigator of responsibilities and the 
procedures for ensuring adequate and correct documentation. 
The investigator is required to prepare and maintain adequate and accurate case histories 
designed to record all observations and other data pertinent to the trial for each trial 
participant. All information recorded on the eCRFs for this trial must be consistent with 
the subjects’ source documentation (i.e. medical records). 
The investigator will permit trial-related monitoring, IRB/IEC review, and regulatory 
inspections, providing direct access to source data /documents. Sponsor-authorized 
quality assurance personnel may carry out audits for which the investigator must provide 
support. 
The trial monitor or a representative of the sponsor will cross-check the data entered in 
the eCRFs with the source data at the trial center and observe the trial procedures in order 
to verify adherence to the trial protocol. Any queries will be resolved by the investigator 
or his/her delegate. 
All of the clinical data will be captured via electronic data capture (EDC) using a 
web-based tool.  
The investigator center staff will enter and edit the data via a secure network, with secure 
access features (username and password). A complete electronic audit trail will be 
maintained. The investigator will approve the data using an electronic signature (Ref: 21 
CFR Part 11), and this approval is used to confirm the accuracy of the data recorded. 
eCRFs will be used for all subjects. The investigator’s data will be accessible from the 
investigator’s center throughout the trial. The eCRFs must be kept current to reflect 
subject status at each phase during the course of the trial. The eCRF will not capture 
personalized data. The investigator must make a separate confidential record of 
personalized details (name and initials) on the subject identification and enrollment log. 
All changes to data are made by the investigator or his/her delegate through the EDC 
system.  
It is the responsibility of the principal investigator of the respective center to ensure that 
all subject discontinuations or changes in trial or other medications entered on the 
subject’s eCRF are also made on the subject’s medical records. 


 
 
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The eCRFs for any subject leaving the trial should be completed at the time of the final 
visit or shortly thereafter. 
10.2  Electronic case report forms 
Remote data capture software will be used for data collection. Following training, trial 
staff will be given access to the software. Access to the software is restricted to staff 
participating in the trial and the extent of access will depend on the participants' user role 
in the trial. 
The subjects enrolled into the trial will be identified in the database by subject number 
and trial identification code. The investigator or delegate will enter subject data into the 
eCRF promptly. All data is to be entered in English. Data recorded in the eCRFs will be 
accessible to the trial staff throughout the trial. 
After data entry, systematic data validation will be performed and data entry 
discrepancies will be presented electronically directly to the center staff. Queries for 
discrepant data may be generated automatically by the software upon entry and/or 
generated manually by the trial monitor or the trial data manager. All queries, whether 
generated by the system or by trial staff, will be in electronic format. 
All sections of the eCRF are to be electronically approved by the investigator or a 
medically qualified delegate after the data has been entered and all queries have been 
resolved. Changes to any eCRF page subsequent to the approval require a new approval 
signature. 
All queries and changes/corrections to the data are documented in the eCRF. 
10.3  Access to source data 
During the course of the trial, a trial monitor will make site visits to review protocol 
compliance, compare eCRFs with individual subject’s medical records, assess drug 
accountability, and ensure that the trial is being conducted according to pertinent 
regulatory requirements. 
Checking of the eCRFs for completeness and clarity, and cross-checking with source 
documents, will be required to monitor the progress of the trial. Moreover, regulatory 
authorities of certain countries, IRBs/IECs, and/or the sponsor may wish to carry out such 
source data checks and/or on-site audit inspections. Direct access to source data will be 
required for these inspections and audits; they will be carried out giving due 
consideration to data protection and medical confidentiality. 


 
 
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10.4  Source documentation 
All source documents from which eCRF entries are derived should be placed in the 
subject’s medical records. If data are to be entered directly into the eCRF this must be 
specified in a source data agreement prior to the start of the trial. 
eCRF entries will be verified with source documentation. The review of medical records 
will be performed in a manner to ensure that subject confidentiality is maintained. 
The trial monitor will check the eCRF for accuracy and completion and perform source 
data verification. The trial monitor will document source data verification of all reviewed 
sections of the eCRF. 
10.5  Data processing 
The trial is run as an EDC trial, i.e. all relevant data is entered by the centers directly into 
the clinical database. The eCRF is designed to capture all required information in 
compliance with GCP standards. 
10.6  Archiving trial records 
According to ICH guidelines, essential documents should be retained for a minimum of 
2 years after the last approval of a marketing application in an ICH region and until there 
are no pending or contemplated marketing applications in an ICH region or at least 
2 years have elapsed since the formal discontinuation of clinical development of the 
investigational product. However, these documents should be retained for a longer period 
if required by the applicable legal requirements. 
Records and documents pertaining to the conduct of the trial and the distribution of the 
investigational product (e.g. informed consent forms, laboratory slips, medication 
inventory records, and other pertinent information) must be retained by the investigator 
according to local requirements. 
10.7  Good clinical practice 
The procedures set out in this trial protocol are designed to ensure that the sponsor and 
investigator abide by the principles of the GCP guidelines of the ICH (1), and of the 
Declaration of Helsinki (2008) (2). The trial also will be carried out in keeping with local 
legal requirements.  


 
 
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10.8  Informed consent 
Before each subject is admitted to the trial, written informed consent will be obtained 
from the subject according to the regulatory and legal requirements of the participating 
country. This consent form must be dated and retained by the investigator as part of the 
trial records. The investigator will not undertake any investigation specifically required 
only for the clinical trial until valid consent has been obtained. The terms of the consent 
and when it was obtained must also be documented in the eCRF. 
The investigator will explain to each subject orally and in writing (subject information 
sheet) the nature, significance, risks and implications of the trial before inclusion. In 
particular, the subjects will be informed about the following: 
 The possibility of withdrawing from the clinical trial at any time by revoking the 
consent and without any resulting disadvantage. 
 How personal and health-related data will be collected and used during the trial. 
 That his/her medical records may be examined by authorized monitors or clinical 
quality assurance auditors appointed by the sponsor, by appropriate IRB/IEC 
members, and by inspectors from regulatory authorities. 
All subjects will receive a copy of the subject information sheet and a copy of their 
signed and dated informed consent form, both of which will be in the subject’s local 
language. 
If a protocol amendment is required, the informed consent form may need to be revised to 
reflect the changes to the protocol. If the consent form is revised, it must be reviewed and 
approved by the appropriate IEC/IRB, and signed by all subjects subsequently enrolled in 
the trial as well as those currently enrolled in the trial. 
10.9  Protocol approval and amendment 
Before the start of the trial, the trial protocol and/or other relevant documents will be 
approved by the IEC/IRB/competent authorities, in accordance with local legal 
requirements. The sponsor must ensure that all ethical and legal requirements have been 
met before the first subject is enrolled in the trial. 
This protocol is to be followed exactly. To alter the protocol, amendments must be 
written, receive approval from the appropriate personnel, and receive IRB/IEC/competent 
authority approval prior to implementation (if appropriate).  


 
 
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Administrative changes (not affecting the subject benefit/risk ratio) may be made without 
the need for a formal amendment. All amendments will be distributed to all protocol 
recipients, with appropriate instructions. 
10.10  Duration of the trial 
The maximum duration of the trial for each subject will be up to 63 days (including up to 
30 days for screening and up to 33 days until the follow-up visit).  
The trial will be closed when all subjects have completed Visit 3. 
10.11  Premature termination of the trial 
If the investigator, the sponsor (e.g. safety committee), or the safety medical monitor 
becomes aware of conditions or events that suggest a possible hazard to subjects if the 
trial continues, the trial may be terminated after appropriate consultation between the 
relevant parties. The trial may also be terminated early at the sponsor’s discretion in the 
absence of such a finding. 
Conditions that may warrant termination include, but are not limited to: 
 The discovery of an unexpected, significant, or unacceptable risk to the subjects 
enrolled in the trial 
 Failure to enroll subjects at an acceptable rate. 
 A decision on the part of the sponsor to suspend or discontinue development of the 
drug. 
The trial can be terminated prematurely by the sponsor at an individual center if: 
 The center cannot comply with the requirements of the protocol. 
 It is not possible for the center to comply with GCP standards. 
10.12  Confidentiality 
All trial findings and documents will be regarded as confidential. The investigator and 
members of his/her research team must not disclose such information without prior 
written approval from the sponsor. 
The anonymity of participating subjects must be maintained. Subjects will be identified 
on eCRFs submitted to the sponsor by their subject number. On the SAE reports and all 
other source documents, the subject will be identified via subject number. Documents not 


 
 
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to be submitted to the sponsor that identify the subject (e.g. the signed informed consent 
form) must be maintained in confidence by the investigator.  
10.13  Other ethical and regulatory issues 
If a significant safety issue is identified, either from an individual case report or review of 
aggregate data, then the sponsor will issue prompt notification to all parties: regulatory 
authorities, investigators and IRB/IECs. 
A significant safety issue is one that has a significant impact on the course of the clinical 
trial or program (including the potential for suspension of the trial program or 
amendments to protocols) or warrants immediate update of informed consent. 
10.14  Liability and insurance 
The sponsor will take out reasonable third-party liability insurance cover in accordance 
with all local legal requirements. The civil liability of the investigator, the persons 
instructed by him and the hospital, practice or institute in which they are employed and 
the liability of the sponsor with respect to financial loss due to personal injury and other 
damage that may arise as a result of the carrying out of this trial are governed by the 
applicable law. 
The sponsor will arrange for liability insurance if subjects should be injured due to the 
participation in the trial and provided that the sponsor is legally liable for that. 
Excluded from the insurance cover are injuries to health and deteriorations of illnesses 
already in existence which would have occurred or continued to exist even if the subject 
had not taken part in the clinical trial. 
The insurance cover is jeopardized if the subject fails to report immediately to the 
investigator or responsible physician any injury to health, which might have resulted 
from participation in the clinical trial, or if he/she undergoes any other medical treatment 
without their consent before the clinical trial has been completely finished in so far as the 
individual subject is concerned. 
Any injury to health, which might have occurred as a result of participation in the clinical 
trial must be reported by the subject to the investigator without delay. The investigator is 
obliged to make such a report in any case. 


 
 
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10.15  Publication policy 
By signing the trial protocol, the investigator agrees with the use of results of the trial for 
the purposes of national and international registration, publication and information for 
medical and pharmaceutical professionals. If necessary, the authorities will be notified of 
the investigator's name, address, qualifications and extent of involvement. 
A clinical trial report will be prepared and reviewed by the sponsor in co-operation with 
the coordinating investigator. The coordinatoing investigator will be appointed by 
Zealand Pharma to review and sign the clinical trial report on behalf of all participating 
investigators. A summary of the clinical trial report (according to CPMP/ICH137/95) 
should be sent to the regulatory authorities and IRB/IEC according to the relevant 
guidelines. 
According to the Declaration of Helsinki (2) investigators and sponsors ‘have ethical 
obligations with regard to the publication and dissemination of the results of research’.   
The trial design and results may be published as one or more original research 
manuscripts/abstracts and presented at a scientific meeting. The original eCRFs and the 
data generated from the eCRFs or otherwise obtained during the trial under this trial 
protocol will become the property of the sponsor. Publication of the results of this trial by 
the investigator is possible only after written consent has been obtained from the sponsor. 
Any material intended for publication will be given to the sponsor at least 4 weeks before 
submission for publication. The sponsor will have the right to comment on the intended 
publication and to take any reasonable measures for patent protection. Authorship on any 
publication(s) resulting from this clinical trial will be assigned according to the 
recommendations of the International Committee of Medicinal Journal Editors (26). 
Participating subjects will not be identified by name in any published reports about the 
clinical trial. 
The sponsor is responsible for trial registration at ClinicalTrials.gov 
(www.clinicaltrials.gov) according to FDA requirements, as well as the European 
Medicines Agency’s Clinical Trials Database (EudraCT).  


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11 
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15-040 


 
 
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Study ID: ZP4207-16137 
Protocol Version: Final 4.0 
 
 
 
 
 
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Page 79 of 81  
24. Amiel SA, Tamborlane WV, Simonson DC, et al. Defective glucose 
counterregulation after strict glycemic control of insulin-dependent diabetes mellitus. 
N Eng J Med 1987; 316: 1376-83. 
25. Rickels MR, Ruedy KJ, Foster NC, et al. Intranasal glucagon for treatment of insulin-
induced hypoglycemia in adults with type 1 diabetes: A randomized crossover 
noninferiority study. Diabetes Care 2016; 39: 264-70. 
26. ICMJE. Recommendations for the conduct, reporting, editing, and publication of 
scholarly work in medical journals. Updated December 2016 
 
 
 
 


 
 
Dasiglucagon (ZP4207) 
Date: 26-Jan 2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 4.0 
 
 
 
 
 
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12 
APPENDICES 
12.1 List of trial personnel 
Sponsor 
Clinical Trial Manager 
 
 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Phone:
 
Medical Officer 
 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Phone: 
 
Contract Research Organization 
Chiltern International (Ltd) 
171 Bath Road 
Slough 
Berkshire 
SL1 4AA 
United Kingdom 
Phone: +44 (0) 175351 2000 
Project Manager 
 
Chiltern International (Ltd) 
171 Bath Road 
Slough 
Berkshire 
SL1 4AA 
United Kingdom 
Phone: 
 
Senior Medical Officer and 
Safety medical monitor 
 
Chiltern International kft 
Canada Square Office House 
Ganz u 


 
 
Dasiglucagon (ZP4207) 
Date: 26-Jan 2018 
 
Study ID: ZP4207-16137 
Protocol Version: Final 4.0 
 
 
 
 
 
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Page 81 of 81  
12-14, 4 emelet 
1027 Budapest 
Hungary 
Phone: 
 
Pharmacovigilance unit 
Responsible for Serious Adverse 
Event (SAE) Management and 24-
hour SAE reporting 
  
PharmaLex 
Agern Allé 24 
2970 Hørsholm, Denmark 
Phone: 
 (8 a.m. to 4 p.m.) 
Phone:
 (outside 8 a.m. to 4 p.m.) 
Fax: 
 
email: PV-nordic@pharmalex.com  
Central laboratory 
MLM Laboratory 
Dohrweg 63 
41066 Mönchengladbach 
Germany 
Special laboratory (GlucaGen 
PK, insulin PK) 
MLM Laboratory 
Dohrweg 63 
41066 Mönchengladbach 
Germany 
Special laboratory (dasiglucagon 
PK, dasiglucagon ADA, 
GlucaGen ADA) 
York Bioanalytical Solutions (YBS)  
Cedar House 
Northminster Business Park 
Northfield Lane 
York, YO26 6QR, United Kingdom 
Special laboratory  
(neutralizing antibodies) 
BioAgilytix 
2300 Englert Drive 
Durham, NC, 27713, USA 
A list of all investigators, IECs and IRBs will be provided in a separate document and in 
the clinical trial report. 
 


 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 3.0 
 
 
 
 
 
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Page 1 of 81  
CLINICAL TRIAL PROTOCOL 
 
A phase 3, randomized, double-blind, parallel trial to 
confirm the clinical efficacy and safety of dasiglucagon 
in the rescue treatment of hypoglycemia in subjects with 
type 1 diabetes mellitus (T1DM) compared to placebo 
and with reference to GlucaGen® 
 
Sponsor: 
Zealand Pharma A/S 
Sponsor Protocol No.: 
ZP4207-16137 
EudraCT No.: 
2017-002449-31  
IND No: 
127866 
Trial Drug Name: 
Dasiglucagon* injection 
Date of Protocol: 
06 Oct 2017 
 
The trial will be conducted according to the protocol and in compliance with Good 
Clinical Practice (GCP), with the Declaration of Helsinki and with other applicable 
regulatory requirements. 
*Dasiglucagon is the proposed international nonproprietary name for ZP4207. 
The information in this document is confidential and is proprietary to Zealand Pharma. It is understood that 
information in this document shall not be disclosed to any third party, in any form, without prior written 
consent of an authorized officer of Zealand Pharma. 




 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
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Declaration of the coordinating investigator 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
I have read this protocol. I approve this document and I agree that it contains all 
necessary details for carrying out the trial as described. I will conduct this trial in 
accordance with the design and specific provision of this protocol and will make a 
reasonable effort to complete the trial within the time designated. I will provide copies of 
this protocol and access to all information furnished by Zealand Pharma A/S to trial 
personnel under my supervision. I will discuss this material with them to ensure they are 
fully informed about the trial product and trial procedures. I will let them know that this 
information is confidential and proprietary to Zealand Pharma A/S and that it may not be 
further disclosed to third parties. I understand that the trial may be terminated or 
enrollment suspended at any time by Zealand Pharma A/S, with or without cause, or by 
me if it becomes necessary to protect the best interests of the trial subjects. 
I agree that the trial will be carried out in accordance with GCP (1), with the Declaration 
of Helsinki (with amendments) (2) and with the laws and regulations of the countries in 
which the trial takes place. 
 
 
_____________________________________ 
_____________________ 
Name  
 
 
 
 
 
Date 
Title 
Institution 
Phone: + 
Fax: 
+ 
 


 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 3.0 
 
 
 
 
 
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Page 4 of 81  
Declaration of the investigator 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
All documentation for this trial that is supplied to me and that has not been previously 
published will be kept in the strictest confidence. This documentation includes this trial 
protocol, Investigator's Brochure, Case Report Forms, and other scientific data. 
The trial will not be commenced without the prior written approval of a properly 
constituted Institutional Review Board (IRB) or Independent Ethics Committee (IEC). 
No changes will be made to the trial protocol without the prior written approval of the 
sponsor and the IRB or IEC, except where necessary to eliminate an immediate hazard to 
the subjects. 
I have read and understood and agree to abide by all the conditions and instructions 
contained in this protocol. 
 
_____________________________________ 
_____________________ 
Signature 
 
 
 
 
 
Date 
 
_____________________________________ 
Name (block letters) 
 
_____________________________________ 
Title (block letters) 
 
_____________________________________ 
Institution (block letters) 
 


 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
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Protocol Version: Final 3.0 
 
 
 
 
 
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Table of contents 
DECLARATION OF SPONSOR OR RESPONSIBLE MEDICAL OFFICER .......... 2 
DECLARATION OF THE COORDINATING INVESTIGATOR .............................. 3 
DECLARATION OF THE INVESTIGATOR ............................................................... 4 
TABLE OF CONTENTS .................................................................................................. 5 
1 
SYNOPSIS ............................................................................................................ 11 
2 
INTRODUCTION................................................................................................ 15 
2.1 
Background ...................................................................................................... 15 
2.1.1 
Hypoglycemia ...................................................................................... 15 
2.1.2 
Glucagon .............................................................................................. 16 
2.1.3 
Dasiglucagon........................................................................................ 16 
2.1.3.1 
Pharmacokinetics and pharmacodynamics of dasiglucagon ......... 17 
2.1.3.2 
Safety of dasiglucagon ................................................................... 17 
2.2 
Trial rationale .................................................................................................. 18 
2.3 
Risk-benefit assessment .................................................................................. 18 
3 
TRIAL OBJECTIVES AND ENDPOINTS ...................................................... 20 
3.1 
Primary objective ............................................................................................ 20 
3.2 
Secondary objectives ....................................................................................... 20 
3.3 
Primary endpoint ............................................................................................ 20 
3.4 
Key secondary endpoints ................................................................................ 20 
3.5 
Other secondary endpoints ............................................................................. 20 
3.6 
Exploratory endpoint ...................................................................................... 21 
4 
OVERALL DESIGN AND PLAN OF THE TRIAL ........................................ 22 
4.1 
Overview .......................................................................................................... 22 
4.2 
Justification of the trial design ....................................................................... 24 
4.2.1 
Justification for design and parameters ................................................ 24 
4.2.2 
Justification for drug, route, dosage and treatment duration ............... 24 
5 
TRIAL POPULATION ....................................................................................... 26 
5.1 
Rationale for trial population ........................................................................ 26 
5.2 
Planned sample size and number of trial centers ......................................... 26 
5.3 
Inclusion criteria ............................................................................................. 26 


 
 
Dasiglucagon (ZP4207) 
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Protocol Version: Final 3.0 
 
 
 
 
 
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5.4 
Exclusion criteria ............................................................................................ 27 
5.5 
Dosing day exclusion criteria ......................................................................... 28 
5.6 
Premature treatment discontinuation and withdrawal ............................... 29 
5.6.1 
Possible reasons for treatment visit discontinuation ............................ 29 
5.6.2 
Center discontinuation ......................................................................... 30 
5.6.3 
Trial termination .................................................................................. 30 
5.7 
Subject identification and randomization ..................................................... 30 
6 
TRIAL DRUG ...................................................................................................... 32 
6.1 
Identity ............................................................................................................. 32 
6.2 
Treatment assignment and randomization ................................................... 33 
6.3 
Administration ................................................................................................. 34 
6.4 
Packaging and labelling .................................................................................. 34 
6.5 
Storage of study drugs .................................................................................... 34 
6.6 
Blinding and breaking the blind .................................................................... 35 
6.7 
Drug accountability ......................................................................................... 36 
6.8 
Treatment compliance .................................................................................... 36 
6.9 
Prior and concomitant medications ............................................................... 36 
6.9.1 
Prohibited medications ......................................................................... 37 
7 
PARAMETERS AND METHODS OF ASSESSMENT .................................. 38 
7.1 
Efficacy parameters ........................................................................................ 38 
7.1.1 
Pharmacodynamic measurements ........................................................ 38 
7.1.2 
Pharmacokinetic measurements ........................................................... 38 
7.2 
Safety parameters ............................................................................................ 39 
7.2.1 
Adverse events ..................................................................................... 39 
7.2.1.1 
Definitions ...................................................................................... 39 
7.2.1.2 
Collection, recording and reporting of adverse events ................. 42 
7.2.1.3 
Follow-up of adverse events .......................................................... 43 
7.2.1.4 
Clinical laboratory abnormalities and other abnormal 
assessments as adverse events or serious adverse events .............. 44 
7.2.2 
Hypoglycemia ...................................................................................... 44 
7.2.3 
Physical examination ........................................................................... 45 
7.2.4 
Vital signs ............................................................................................ 45 
7.2.5 
Electrocardiogram ................................................................................ 46 
7.2.6 
Local tolerability .................................................................................. 46 
7.2.7 
Clinical laboratory assessments ........................................................... 47 
7.2.8 
Pregnancy ............................................................................................. 48 
7.2.9 
Precautions ........................................................................................... 49 
7.2.10 
Safety committee ................................................................................. 49 
7.3 
Demography, concomitant illness, medical history and concomitant 
medication ........................................................................................................ 49 


 
 
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Protocol Version: Final 3.0 
 
 
 
 
 
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7.3.1 
Demography and body measurements ................................................. 49 
7.3.2 
Concomitant illness and medical history ............................................. 50 
7.3.3 
Diabetes diagnosis and current treatment ............................................ 50 
7.3.4 
Concomitant medication ...................................................................... 50 
7.4 
Other assessments ........................................................................................... 51 
7.4.1 
Immunogenicity ................................................................................... 51 
7.4.2 
Plasma glucose measurements for safety ............................................. 52 
7.4.3 
Plasma insulin measurements .............................................................. 52 
8 
TRIAL CONDUCT .............................................................................................. 53 
8.1 
Schedule of procedures ................................................................................... 53 
8.2 
Procedures by visit .......................................................................................... 53 
8.2.1 
Visit 1 (screening, Day -30 to Day -3) ................................................. 53 
8.2.2 
Instructions to subjects prior to the dosing visit (Visit 2) .................... 55 
8.2.3 
Visit 2 (dosing visit)............................................................................. 56 
8.2.3.1 
Hypoglycemic clamp procedure and administration of trial 
medication ...................................................................................... 57 
8.2.4 
Visit 3 (follow-up visit) ....................................................................... 60 
9 
STATISTICAL METHODS ............................................................................... 61 
9.1 
Determination of sample size ......................................................................... 61 
9.2 
Trial subjects ................................................................................................... 61 
9.2.1 
Analysis samples .................................................................................. 61 
9.2.2 
Disposition of subjects ......................................................................... 62 
9.2.3 
Protocol deviations ............................................................................... 62 
9.3 
General considerations ................................................................................... 63 
9.4 
Demographics and baseline characteristics .................................................. 63 
9.5 
Efficacy Analyses ............................................................................................. 63 
9.5.1 
Hierarchical testing procedure ............................................................. 64 
9.5.2 
Primary confirmatory endpoint ............................................................ 64 
9.5.2.1 
Primary analysis ............................................................................ 64 
9.5.3 
Secondary endpoints ............................................................................ 65 
9.5.3.1 
Confirmatory analysis .................................................................... 65 
9.5.4 
Secondary clinical efficacy (PD) endpoints ......................................... 66 
9.5.4.1 
Analysis of secondary clinical efficacy (PD) endpoints ................ 66 
9.5.5 
Exposure (PK) endpoints ..................................................................... 66 
9.5.5.1 
Analysis of exposure (PK) endpoints ............................................. 67 
9.6 
Exploratory analyses ....................................................................................... 67 
9.7 
Safety analyses ................................................................................................. 68 
9.7.1 
Adverse events ..................................................................................... 68 
9.7.2 
Immunogenicity data ........................................................................... 68 
9.7.3 
Clinical laboratory assessments ........................................................... 68 


 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
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Protocol Version: Final 3.0 
 
 
 
 
 
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9.7.4 
Other safety data .................................................................................. 68 
9.8 
Treatment compliance .................................................................................... 69 
9.9 
Subject withdrawals and missing data .......................................................... 69 
9.10 
Interim analyses .............................................................................................. 69 
10 
ETHICAL, LEGAL, AND ADMINISTRATIVE ASPECTS .......................... 70 
10.1 
Quality assurance ............................................................................................ 70 
10.2 
Electronic case report forms .......................................................................... 71 
10.3 
Access to source data ...................................................................................... 71 
10.4 
Source documentation .................................................................................... 72 
10.5 
Data processing ................................................................................................ 72 
10.6 
Archiving trial records ................................................................................... 72 
10.7 
Good clinical practice ..................................................................................... 72 
10.8 
Informed consent ............................................................................................. 73 
10.9 
Protocol approval and amendment ............................................................... 73 
10.10 Duration of the trial ........................................................................................ 74 
10.11 Premature termination of the trial ................................................................ 74 
10.12 Confidentiality ................................................................................................. 74 
10.13 Other ethical and regulatory issues ............................................................... 75 
10.14 Liability and insurance ................................................................................... 75 
10.15 Publication policy ............................................................................................ 76 
11 
REFERENCE LIST ............................................................................................. 77 
12 
APPENDICES ...................................................................................................... 80 
12.1 
List of trial personnel ...................................................................................... 80 
 
Tables in text 
Table 1 
Description of trial drugs .................................................................................. 32 
Table 2 
Quantities of ingredients in dasiglucagon and placebo injection ...................... 33 
Table 3 
Schedule of procedures ..................................................................................... 54 
Table 4 
Post-treatment blood sampling times ................................................................ 59 
 
Figures in text 
Figure 1 Trial design ........................................................................................................ 23 


 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 3.0 
 
 
 
 
 
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Page 9 of 81  
List of abbreviations and definitions of terms 
ADA 
Anti-drug antibody 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALT 
Alanine aminotransferase 
ANCOVA Analysis of covariance 
AST 
Aspartate aminotransferase 
AUC 
Area under the concentration-time curve 
AUC0-inf 
AUC from time zero to infinity 
AUE 
Area under the effect curve  
CFB 
Changes from baseline  
CI 
Confidence interval 
Cmax 
Maximum plasma concentration 
CPH 
Cox proportional hazards 
CRO 
Contract research organization  
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
EU 
European Union 
EudraCT 
European Medicines Agency’s Clinical Trials Database 
FAS 
Full analysis set 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HbA1c 
Glycated hemoglobin 
ICH 
International Conference on Harmonization 
ID card 
Identification card 
IEC 
Independent Ethics Committee 
IM 
Intramuscular(ly) 
IMP 
Investigational medicinal product 
IRB 
Institutional Review Board 
IV 
Intravenous(ly) 


 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
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Protocol Version: Final 3.0 
 
 
 
 
 
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IWRS 
Interactive Web Response System 
MedDRA 
Medical Dictionary for Regulatory Activities 
NPH 
Neutral protamine Hagedorn 
PD 
Pharmacodynamics 
PK 
Pharmacokinetics 
PP 
Per protocol 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SAS 
Safety analysis set 
SC 
Subcutaneous(ly) 
SUSAR 
Serious unexpected suspected adverse reaction 
T1DM 
Type 1 diabetes mellitus 
T2DM 
Type 2 diabetes mellitus 
TEAE 
Treatment-emergent adverse event 
tmax 
Time to the maximum plasma concentration 
ULN 
Upper limit of the normal range 
US 
United States of America 
 


 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 3.0 
 
 
 
 
 
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1 
SYNOPSIS 
Name of sponsor: 
Zealand Pharma A/S 
Trial ID: 
ZP4207-16137 
Title of the trial: 
A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy and safety of 
dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 1 diabetes mellitus (T1DM) 
compared to placebo and with reference to GlucaGen® 
Trial design:  
The trial is a global, multicenter, randomized, parallel-group, double-blind, clinical trial confirming the 
efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in subjects with type 1 diabetes 
mellitus. The subjects will be randomized 2:1:1 to receive a single fixed subcutaneous 0.6 mg dose of 
dasiglucagon (hereinafter dasiglucagon), placebo for dasiglucagon (hereinafter referred to as placebo), or a 
1 mg dose of GlucaGen® (hereafter referred to as GlucaGen) and followed for at least 28 days after 
treatment. 
Clinical phase of development: 
Phase 3 
Trial centers: 
This trial will be conducted at 4 to 6 sites in the United States of America, Canada, and Europe. 
Planned trial start (first subject first visit): 
Q4/2017 
Planned trial end (last subject last visit): 
Q3/2018  
Trial population:  
Male and female adult subjects with type 1 diabetes mellitus treated with insulin for at least one year  
Key objectives: 
Primary objective:  
 
To demonstrate superiority of dasiglucagon compared to placebo following a single subcutaneous 
0.6 mg dose administered to subjects with type 1 diabetes mellitus with insulin-induced 
hypoglycemia. 
Secondary objective:  
 
To compare the glycemic response observed after dasiglucagon with that of GlucaGen. 
Key endpoints:  
Primary endpoint:  
 
Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase in plasma 
glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic clamp procedure 
without administration of rescue intravenous (IV) glucose.  
Key secondary endpoints: 
 
Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, and within 
10 minutes after study drug injection without administration of rescue IV glucose. 
 
Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection or at the time of rescue. 
Clinical efficacy (Pharmacodynamic) endpoints: 
 
Time to first plasma glucose concentration of ≥70 mg/dL (3.9 mmol/L) without administration of 
rescue IV glucose. 


 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
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Protocol Version: Final 3.0 
 
 
 
 
 
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Page 12 of 81  
 
Plasma glucose response as area under the curve above baseline from time zero to 30 minutes, 
AUC0-30min. 
Exposure (Pharmacokinetic) endpoints: 
 
Area under the drug concentration curve from time zero to 90 minutes, AUC0-90 min. 
 
Maximum plasma drug concentration (Cmax). 
 
Time to maximum plasma drug concentration (tmax). 
Safety endpoints:  
 
Adverse events, clinical laboratory assessments (biochemistry, hematology, coagulation, urinalysis), 
vital signs, physical examination, electrocardiogram, and local tolerability. 
 
Administration of rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
Immunogenicity endpoint: 
 
Occurrence of anti-drug antibodies 
Exploratory endpoint: 
 
Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL (1.1 mmol/L) 
within 30 minutes after study drug injection without administration of rescue IV glucose. 
 
Plasma insulin response as area under the curve above baseline from time zero to 60 minutes, 
AUC0-60 min. 
Key inclusion criteria: 
 
Male and female subjects with type 1 diabetes mellitus treated with insulin for at least one year, 
diagnostic criteria as defined by the American Diabetes Association. 
 
Stable insulin treatment 30 days prior to screening, defined as no more than a 10-unit daily variation 
in total daily insulin dose. 
 
Hemoglobin A1c <10%. 
 
Aged between 18 and 75 years, both inclusive. 
Key exclusion criteria: 
 
Previously treated with dasiglucagon. 
 
Known or suspected allergy to trial product(s) or related products. 
 
History of anaphylaxis or symptoms of severe systemic allergy (such as angioedema). 
 
Previous participation (randomization) in this trial. 
Sample size: 
Approximately 156 subjects are intended to complete the trial, with 78 subjects randomized to the 
dasiglucagon group and 39 subjects randomized to each of the placebo and GlucaGen groups.  
Investigational medicinal product: 
Test product: dasiglucagon liquid formulation in pre-filled syringes. 
Comparator products: Placebo and GlucaGen® lyophilized powder. 
Duration of treatment:  
Subjects will be randomized 2:1:1 to receive a single fixed subcutaneous dose of 0.6 mg dasiglucagon, 
placebo, or 1 mg GlucaGen, and followed for at least 28 days after receiving treatment.  


 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
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Protocol Version: Final 3.0 
 
 
 
 
 
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Assessments:  
The plasma glucose profile for evaluation of the primary and secondary clinical efficacy 
(pharmacodynamic) endpoints will be assessed based on plasma concentration data from samples collected 
at the dosing visit. 
The exposure to trial medication (dasiglucagon, placebo, or GlucaGen) for evaluation of pharmacokinetics 
will also be assessed based on plasma concentration data.  
Statistical methods:  
Due to requirements in the size of the safety database, the sample size is set to 78 subjects treated with 
dasiglucagon 0.6 mg. With a 2:1:1 randomization ratio for the three treatments, dasiglucagon 0.6 mg, 
placebo, and GlucaGen 1 mg, 78 subjects injected with dasiglucagon and 39 subjects with placebo will 
yield a power of 90% at a 5% two-sided significance level to detect a treatment group difference in recovery 
incidence within 20 minutes, assuming as low as 80% and as high as 50% recovery incidence for the 
dasiglucagon and placebo groups, respectively. 
For the confirmatory analyses, the primary and key secondary endpoints for the dasiglucagon 0.6 mg and 
placebo treated subjects will be compared. A hierarchical procedure will be applied for the control of 
multiplicity in statistical inferences. The primary and secondary endpoints will be evaluated on the Full 
Analysis Set sample. The statistical inference comparisons with placebo will be conducted as superiority 
tests. The comparisons of dasiglucagon versus GlucaGen will be summarized descriptively.    
The primary endpoint will be summarized using Kaplan-Meier (KM) estimates stratified by treatment group 
and injection site. The treatment group difference between dasiglucagon and placebo will be evaluated 
inferentially using a pairwise two-sided log-rank test. 
In the primary analysis, those subjects who require rescue IV glucose will be censored at the time of plasma 
glucose recovery. This approach is based on the assumption that rescue IV glucose administration will 
shorten the time to recovery. This assumption implies that censoring at the time of recovery after rescue 
therapy is valid as a time to recovery without rescue would be longer than the observed duration. If recovery 
has not occurred at 45 minutes after study drug injection, censoring will be applied irrespective of the use of 
rescue IV glucose.  
In sensitivity analyses, the time to plasma glucose recovery will be analyzed 1) without censoring at the 
time of recovery for those subjects who require rescue IV glucose before 45 minutes and 2) with censoring 
at the time of administration of rescue IV glucose before 45 minutes.   
The primary endpoint will additionally be analyzed using a Cox proportional hazards (CPH) time to event 
statistical model, if the hazards by treatment group can be assumed proportional using a graphical 
comparison of log-minus-log survival curves and no more than two-thirds of the recovery times are 
censored in each treatment group. The CPH model will be used for inferences, with treatment group and 
injection site modeled as categorical effects, and baseline plasma glucose modeled as a continuous 
covariate. The treatment group rate ratios, active versus placebo, will be estimated together with the 95% 
confidence intervals, and pairwise treatment group inferences (dasiglucagon vs placebo, GlucaGen vs 
placebo) will be evaluated using two-sided likelihood ratio tests.  
The key secondary incidence variables (plasma glucose recovery within 30 minutes, within 20 minutes, 
within 15 minutes, within 10 minutes, in hierarchical order for inference) will be analyzed using descriptive 
statistics (frequency and percentage) by treatment group. Inferential treatment group comparisons will be 
assessed via pairwise tests of independent binomial proportions (dasiglucagon versus placebo, GlucaGen 


 
 
Dasiglucagon (ZP4207) 
Date: 06 Oct 2017 
 
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Protocol Version: Final 3.0 
 
 
 
 
 
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Page 14 of 81  
versus placebo) using Fisher’s exact test for each pairwise comparison. 
The key secondary endpoints of plasma glucose changes from baseline (CFB) within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection or at the time of rescue, will 
be analyzed with the plasma glucose CFB at rescue carried forward in those subjects who require rescue IV 
glucose before plasma glucose ≥20 mg/dL recovery. Each of these CFB variables will be analyzed using an 
Analysis of Covariance model, with treatment group modeled as a fixed effect and with the baseline plasma 
glucose modeled as a covariate. The dasiglucagon versus placebo treatment group difference will be 
evaluated inferentially as a least squares means contrast, using a two-sided t-test at the 0.05 significance 
level, first for the 30 minute CFB, and then sequentially for the 20 minute, 15 minute, and 10 minute 
variables, until the first failure to reject. 
The continuous clinical efficacy variables, the exploratory variables, and the pharmacokinetics variables 
will each be summarized descriptively by treatment group. The clinical efficacy variables will be analyzed 
analogous to the plasma glucose CFB variables.   
The safety analyses will include by-treatment-group descriptive summaries of vital sign measurements, 
laboratory measures (including immunogenicity incidence), physical examination assessments, rescue IV 
glucose (incidence and amount of glucose infused), and adverse events. The number and percentage of 
subjects reporting specific events, such as nausea and vomiting, will be presented by body system and 
preferred term. 
Further details will be included in the Statistical Analysis Plan, to be completed before database lock and 
treatment unmasking.   


 
 
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2 
INTRODUCTION 
2.1 
Background 
Zealand Pharma A/S (Zealand Pharma) is developing dasiglucagon, a physically and 
chemically stable peptide analog of human glucagon, in a ready-to-use liquid formulation 
for the acute treatment of severe hypoglycemia in patients with insulin-treated diabetes 
mellitus. Like native glucagon, dasiglucagon is comprised of 29 amino acids, but with 
7 substitutions which improve its physical and chemical stability in aqueous media. 
These amino acid substitutions make dasiglucagon suitable for a liquid formulation, 
while providing similar efficacy and safety as compared with recombinant glucagon in 
the clinical setting of acute treatment of severe hypoglycemia. 
2.1.1 Hypoglycemia 
Hypoglycemia in patients with diabetes is defined as episodes of an abnormally low 
plasma glucose concentration (3). This is a common, unpredictable, and potentially 
dangerous side effect of treatment of diabetes mellitus, especially with insulin or 
sulfonylureas. It is more frequent in patients with profound endogenous insulin 
deficiency, such as occurs in type 1 diabetes mellitus (T1DM) and advanced type 2 
diabetes mellitus (T2DM). Treatment of T2DM with insulin causes hypoglycemia 
progressively and more frequently over time, whereas in T1DM, hypoglycemia is 
experienced throughout the course of established disease (4). 
Symptoms and signs of hypoglycemia are not specific. Patients undergoing a 
hypoglycemic episode experience unpleasant symptoms such as anxiety, sweating, 
hunger, tremors, palpitations, paresthesia, nausea and pallor. Depending on its severity, 
the hypoglycemia may lead to mild confusion, behavioral changes, loss of consciousness, 
seizures, coma, and death (5). 
The incidence of hypoglycemic events or even the fear of hypoglycemia influences 
patients’ adherence to prescribed treatment regimens for diabetes mellitus (6). This leads 
to inadequate glycemic control, which in turn may lead to an increased risk of diabetic 
complications (5). Serious clinically significant hypoglycemia is now defined as plasma 
glucose <54 mg/dL (3.0 mmol/L), while the plasma glucose alert value is defined as 
<70 mg/dL (3.9 mmol/L) (7). When plasma glucose falls below these values, some kind 
of treatment strategy is needed. 


 
 
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2.1.2 Glucagon 
Glucagon is a naturally occurring hormone, secreted from the alpha cells of the 
pancreatic islets. Glucagon plays a central role in the regulation of glucose homeostasis 
and is the counterpart of insulin for controlling blood glucose levels (i.e. it acts in 
opposition to insulin in terms of effects on blood glucose levels) (8,9). Glucagon 
stimulates hepatic glycogenolysis and gluconeogenesis in hypoglycemic states, thereby 
restoring glucose homeostasis. Glucagon receptor agonism has also been shown to exert 
effects on lipid metabolism, energy balance, body adipose tissue mass and food intake 
(10). Insulin decreases blood glucose levels and cases of hypoglycemia can be reversed 
by glucagon. Therefore, glucagon is indicated for the treatment of severe hypoglycemia. 
Besides intravenous (IV) glucose administration, an injectable form of glucagon is given 
as first aid in cases of severe hypoglycemia, when the patient is unconscious or for other 
reasons cannot take glucose orally. The approved glucagon dose for an adult is 1 mg, 
given by intramuscular (IM), IV, or subcutaneous (SC) injection, which quickly raises 
blood glucose levels. As current marketed recombinant glucagon is highly unstable when 
dissolved in solution, the injectable form has to be reconstituted prior to use in a 9-step 
procedure that requires a sterile diluent to be injected into a vial containing lyophilized 
powdered glucagon. When dissolved in a fluid state, glucagon can form amyloid fibrils 
(11), or tightly woven chains of proteins made up of the individual glucagon peptides. 
The reconstitution process makes the use of marketed glucagon products cumbersome 
(12), and a more patient-friendly formulation is needed. Currently, the Food and Drug 
Administration (FDA) approved instructions for commercially available glucagon allow 
only for immediate usage of glucagon after the powder is reconstituted in aqueous 
solution (13). Therefore, a glucagon analog with enhanced biophysical stability may 
represent a leap forward in terms of convenient therapeutic applications. 
2.1.3 Dasiglucagon 
Dasiglucagon (ZP4207) is a stable peptide analog of human glucagon, available in a 
ready-to-use liquid formulation. Dasiglucagon (hereinafter referred to as dasiglucagon) is 
in development for the treatment of severe hypoglycemia in insulin-dependent patients 
with diabetes mellitus. Dasiglucagon is a specific and full glucagon receptor agonist 
designed to mimic the effects of glucagon, having a fast absorption and elimination 
(minutes). It is a peptide of 29 amino acids, with 7 amino acid substitutions compared to 
native glucagon. The main purpose of the substitutions is to increase the physical and 
chemical stability of the glucagon analog compared to marketed glucagon products such 
as Lilly Glucagon or GlucaGen® (hereafter referred to as GlucaGen). Dasiglucagon 
exhibits improved physical and chemical stability and is available in an aqueous solution 
at neutral pH (14). 


 
 
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Three clinical trials have been completed with dasiglucagon: a first-in-human dose trial in 
healthy volunteers and subjects with T1DM (ZP4207-14013), a multiple-dose 
dose-escalation trial (ZP4207-15007) evaluating the safety, tolerability, pharmacokinetics 
(PK) and pharmacodynamics (PD) of dasiglucagon in healthy volunteers, and a phase 2 
crossover trial to assess the PK and PD of a single dose of an optimized formulation of 
dasiglucagon administered SC in subjects with T1DM (ZP4207-15126) (14). 
2.1.3.1 Pharmacokinetics and pharmacodynamics of dasiglucagon 
The results of the phase 1 and 2 clinical trials confirmed dose-proportionality for 
dasiglucagon PK, which is characterized by a fast absorption with a peak plasma 
concentration obtained after 35 minutes. Thereafter, the plasma concentration rapidly 
declines with an average half-life of 28 minutes. The median time to the maximum 
plasma concentration (tmax) was 35 minutes for dasiglucagon compared with 20 minutes 
for GlucaGen.  
At all dose levels in the phase 2 trial, all subjects achieved a plasma glucose level of at 
least 70 mg/dL (3.9 mmol/L) as well as an increase in plasma glucose by at least 
20 mg/dL (1.1 mmol/L) within 30 minutes after dosing. The PD responses to 0.6 mg of 
dasiglucagon and 1 mg of GlucaGen were comparable. 
2.1.3.2 Safety of dasiglucagon 
The safety data for dasiglucagon do not give rise to any safety concerns. No new signals 
were observed, beyond those related to the pharmacological effect of glucagon agonism. 
All adverse events (AEs) recorded in the trials were of mild or moderate severity. The 
most frequently reported systemic AE was nausea, which is a known side effect 
following administration of glucagon. Headache was the next most frequently reported 
event, occurring in all dose groups in the phase 2 trial. Injection site reactions were 
observed only sporadically after administration with either dasiglucagon or GlucaGen 
and all were mild and transient. The most frequent injection site reaction was erythema, 
occurring in all treatment groups, including the placebo group, irrespective of dose. 
Glucagon has been described to exert positive inotropic and chronotropic effects and may 
therefore cause tachycardia and hypertension. In the phase 1 clinical trials, but not the 
phase 2 trial, temporary but clinically significant decreases in blood pressure were 
observed in a few healthy volunteers receiving investigational medicinal product (IMP) 
doses of at least 1 mg (4 with dasiglucagon and 1 with GlucaGen). This is not considered 
a safety concern; however, hemodynamic changes after dosing will be considered an 
adverse event of special interest (AESI). 


 
 
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The phase 1 and 2 results and the safety profile described to date do not give rise to 
specific safety concerns. For further information, please refer to the Investigator’s 
Brochure (14). 
2.2 
Trial rationale 
The aim of the current trial is to confirm the superiority of dasiglucagon for the treatment 
of insulin-induced hypoglycemia in subjects with T1DM as compared to placebo for 
dasiglucagon (hereinafter placebo) and to compare the clinical efficacy and safety of 
dasiglucagon with reference to GlucaGen. A randomized, controlled trial design was 
used. 
See Section 4.2 for justification of the design of this trial. 
2.3 
Risk-benefit assessment  
Non-clinical experience 
The nonclinical development program did not reveal any safety findings that would 
prohibit administration of dasiglucagon to humans. None of the safety pharmacology 
studies, repeated dose toxicity studies or genotoxicity studies revealed any significant 
toxicity findings relevant to the therapeutic use of dasiglucagon. 
Clinical experience 
As glucagon and its analogs belong to a well-known drug class with a known mode of 
action, dasiglucagon is not expected to be a high-risk molecule.  
Treatment with an IMP may result in undesired effects or complaints. Undesired effects 
and complaints such as nausea, vomiting, and diarrhea are known AEs occurring with 
glucagon administration. Similar AEs have also been observed to a limited extent in the 
3 clinical studies conducted to date with dasiglucagon. As with every novel drug 
substance, new and as yet unknown side effects may also occur. 
There are limited data available to assess the immunogenic potential of glucagon 
products available on the market, but the data available indicate that marketed glucagon 
only has a low immunogenic potential.  
Overall, dasiglucagon is judged to be a low-risk molecule, based upon the available 
clinical data. Administration of dasiglucagon may be associated with a risk of allergic 
reactions similar to those observed for other therapeutic peptides or proteins. Subjects 
with known or suspected allergies to the trial medications or related products will be 


 
 
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excluded from the trial. Mild or moderate allergic reactions may include symptoms of 
rash, fever, flu-like symptoms, nausea, headache, and myalgia. Acute generalized 
hypersensitivity reactions are usually very rare, but may include symptoms of flushing, 
sweating, dizziness, change in blood pressure, and difficulties in breathing. No severe 
acute hypersensitivity reactions have been observed in the 3 clinical trials conducted with 
dasiglucagon. Direct access to resuscitation equipment is ensured at the clinical trial 
centers. 
With the exception of medical examinations, a subject participating in this trial is not 
likely to derive any personal health-related benefits. The results of the trial may 
contribute to the future use of dasiglucagon in patients with diabetes mellitus 
experiencing severe hypoglycemic reactions. 
The development program including 141 subjects exposed to dasiglucagon to date has 
demonstrated that administration of dasiglucagon is efficacious and well tolerated, with a 
safety profile that does not give rise to specific safety concerns. Two phase 1 and one 
phase 2 clinical trials have been conducted to investigate the safety, tolerability, PK and 
PD of dasiglucagon after single and multiple dosing to healthy volunteers and subjects 
with T1DM under insulin-induced hypoglycemic conditions. Dasiglucagon has proven to 
have relevant clinical effects in the acute severe hypoglycemia rescue setting and may be 
an effective and reliable emergency treatment for severe hypoglycemia. Overall, the 
anticipated benefits for subjects entering the ZP4207-16137 trial are considered to justify 
the risks. 


 
 
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3 
TRIAL OBJECTIVES AND ENDPOINTS 
3.1 
Primary objective 
 To demonstrate superiority of dasiglucagon compared to placebo following a single 
subcutaneous 0.6 mg dose administered to subjects with type 1 diabetes mellitus with 
insulin-induced hypoglycemia. 
3.2 
Secondary objectives 
 To compare the glycemic response observed after dasiglucagon with that of 
GlucaGen. 
3.3 
Primary endpoint 
 Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase 
in plasma glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic 
clamp procedure without administration of rescue IV glucose.  
3.4 
Key secondary endpoints 
 Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection without administration of rescue IV 
glucose. 
 Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection or at the time of rescue. 
3.5 
Other secondary endpoints 
 Clinical efficacy (PD) endpoints: 
- 
Time to first plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) without 
administration of rescue IV glucose. 
- 
Plasma glucose response as area under the curve (AUC) above baseline from time 
zero to 30 minutes, AUC0-30min. 
 Exposure (PK) endpoints: 
- 
Area under the drug concentration curve from time zero to 90 minutes, 
AUC0-90min. 
- 
 Maximum plasma drug concentration (Cmax). 
- 
Time to maximum plasma drug concentration (tmax). 


 
 
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 Safety endpoints:  
- 
Adverse events, clinical laboratory assessments (biochemistry, hematology, 
coagulation, urinalysis), vital signs, physical examination, electrocardiogram 
(ECG), and local tolerability. 
- 
Administration of rescue infusion of IV glucose during the hypoglycemic clamp 
procedure. 
- 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp 
procedure. 
 Immunogenicity endpoint: 
- 
Occurrence of anti-drug antibodies 
3.6 
Exploratory endpoint 
 Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL 
(1.1 mmol/L) within 30 minutes after study drug injection without administration of 
rescue IV glucose. 
 Plasma insulin response as area under the curve above baseline from time zero to 
60 minutes, AUC0-60 min. 


 
 
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4 
OVERALL DESIGN AND PLAN OF THE TRIAL 
4.1 
Overview 
This trial is a global, multicenter, randomized, parallel, double-blind, clinical trial 
confirming the efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in 
subjects with T1DM. The subjects will be randomized 2:1:1 to receive a single fixed SC 
0.6 mg dose of dasiglucagon, placebo, or a 1 mg dose of GlucaGen and followed for at 
least 28 days after receiving treatment. A total of 156 subjects with T1DM are expected 
to complete the treatment visit. The trial will be conducted in the European Union (EU) 
and North America. See Figure 1 for an overview of the trial design. 


 
 
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Figure 1 
Trial design 
 
 
 


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4.2 
Justification of the trial design 
4.2.1 Justification for design and parameters 
In order to avoid bias in subject selection and in the evaluation of clinical assessments, 
subjects will be randomly assigned 2:1:1 to either dasiglucagon, placebo, or GlucaGen 
and the trial will be conducted in a double-blinded manner. The randomized parallel 
treatment design with administration of fixed SC doses of dasiglucagon, placebo, or 
GlucaGen to subjects with T1DM and insulin-induced hypoglycemia allows for direct 
comparison of the clinical efficacy of the treatments.  
The trial is double-blind to increase trial validity and to reduce bias during evaluation of 
the treatments. However, since the trial medications are not identical in appearance, the 
handling, preparation and administration of trial medication will be done by unblinded 
trial personnel who will not be involved in any other trial procedures or assessments. See 
Section 6.6 for more information about which assessments are blinded and which are not, 
with reasons.  
A superiority trial design is used because the aim is to show that treatment with 
dasiglucagon is an effective treatment compared to placebo. The secondary objective was 
chosen to allow a comparison between treatment with dasiglucagon and an established 
comparator treatment for severe hypoglycemia, GlucaGen. 
Administration of glucagon is intended to quickly raise blood glucose levels in subjects 
with T1DM with insulin-induced hypoglycemia. Therefore, in order to assess the clinical 
efficacy of dasiglucagon as compared to placebo and GlucaGen following a single SC 
dose, the primary endpoint and secondary efficacy endpoints involve the measurement of 
plasma glucose concentrations at different timepoints. 
4.2.2 Justification for drug, route, dosage and treatment duration 
Dasiglucagon and GlucaGen will be administered as fixed doses independent of body 
weight because this is the intended therapeutic dosing regimen in the emergency 
treatment of hypoglycemia. The selected dose of 1 mg GlucaGen is the approved dose for 
treatment of severe hypoglycemia. Data from the studies conducted to date with 
dasiglucagon, including the phase 2 trial in subjects with T1DM, have been used to 
establish that 0.6 mg of dasiglucagon is an effective dose and also represents a 
therapeutically equivalent dose to 1 mg of GlucaGen (see also Section 6.1). 
Dasiglucagon, placebo, and GlucaGen will be administered in the abdomen, buttocks, or 
thigh by SC injection, as this is one of the intended routes of administration for 
dasiglucagon, besides IM and IV. 


 
 
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Subjects will be followed for at least 28 days after dosing in order to perform an adequate 
immunogenicity evaluation of treatment.  


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5 
TRIAL POPULATION 
5.1 
Rationale for trial population 
Dasiglucagon is in development for the treatment of severe hypoglycemia in 
insulin-dependent patients with diabetes mellitus. In the present trial, subjects with 
T1DM are included in the evaluation of efficacy and safety of dasiglucagon under 
hypoglycemic conditions as this is part of the intended target population. Subjects with 
T1DM are selected to avoid the endogenous glucagon counter-regulatory response to 
insulin-induced hypoglycemia that is present in patients with T2DM. The inclusion and 
exclusion criteria are set to include a trial population that represents the general 
population of subjects with T1DM.  
5.2 
Planned sample size and number of trial centers 
A total number of 156 subjects with T1DM are expected to complete the treatment visit 
(Visit 2). To qualify as completed, the subject must be dosed and have blood drawn for 
the PK and PD assessments described in the protocol. It is expected that up to 
176 subjects will be randomized to have 156 subjects completing Visit 2. Completion of 
156 subjects (78 subjects in the dasiglucagon group and 39 subjects in each of the 
placebo and GlucaGen groups) would be expected to provide adequate power for the 
primary efficacy evaluation, as described in Section 9.1. 
The planned date for first subject first visit is expected to take place in Q4, 2017 and the 
planned date for last subject last visit is expected to take place in Q3, 2018. 
This trial is planned to be conducted at 4 to 6 trial sites in the United States of America 
[US], Canada, and Europe. 
5.3 
Inclusion criteria 
Subjects will be entered into this trial only if they meet all of the following criteria: 
1. Informed consent obtained before any trial-related activities (trial-related
activities are any procedure that would not have been performed during normal
management of the subject).
2. Female or male subjects with T1DM for at least 1 year, diagnostic criteria as
defined by the American Diabetes Association (3).
3. Treated with insulin for T1DM for at least 1 year and with stable insulin treatment
(defined as no more than a 10-unit daily variation in total daily insulin dose)
30 days prior to screening


 
 
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4. Hemoglobin A1c <10%. 
5. Aged between 18 and 75 years, both inclusive. 
6. A female subject must meet one of the following criteria: 
a. Participant is of childbearing potential and agrees to use one of the 
accepted contraceptive regimens throughout the entire duration of the trial 
from screening and until last follow-up visit. Additionally, if the 
participant is using systemic contraceptives, she must use an additional 
form of acceptable contraception. An acceptable method of contraception 
includes one of the following: 
i. Abstinence from heterosexual intercourse; 
ii. Systemic contraceptives (birth control pills, injectable/implant/ 
insertable hormonal birth control products, transdermal patch); 
iii. Intrauterine device (with and without hormones); or 
iv. Condom with spermicide; or 
b. Participant is of non-childbearing potential, defined as surgically sterile 
(i.e. has undergone complete hysterectomy, bilateral oophorectomy, or 
tubal ligation), or in a menopausal state (at least 1 year without menses). 
7. A male subject must meet the following criteria: Surgically sterilized or willing to 
refrain from sexual intercourse from screening and until last follow-up visit or, if 
sexually active, uses condom and partner practices contraception during the trial 
from screening and until last follow-up visit. 
5.4 
Exclusion criteria 
Subjects meeting any of the following criteria during screening evaluations will be 
excluded from trial participation: 
1. Previously treated with dasiglucagon (previously referred to as ZP4207). 
2. Known or suspected allergy to trial product(s) or related products. 
3. History of anaphylaxis or symptoms of severe systemic allergy (such as 
angioedema). 
4. Previous participation (randomization) in this trial. 
5. Females who are pregnant according to a positive pregnancy test, are actively 
attempting to get pregnant, or are lactating. 
6. History of hypoglycemic events associated with seizures in the last year prior to 
screening. 
7. History of severe hypoglycemia (defined as plasma glucose <54 mg/dL 
[3.0 mmol/L]) in the last month prior to screening. 
8. Receipt of any investigational drug within 3 months prior to screening. 
9. Active malignancy within the last 5 years. 
10. Congestive heart failure, New York Heart Association class II-IV. 


 
 
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11. Inadequately treated blood pressure, defined as systolic ≥160 mmHg or diastolic 
≥90 mmHg) at screening (15). 
12. Current bleeding disorder, including anti-coagulant treatment. 
13. Known presence or history of pheochromocytoma (i.e. adrenal gland tumor) or 
insulinoma (i.e. insulin secreting pancreas tumor). 
14. Use of a daily systemic beta-blocker drug, indomethacin, warfarin or 
anticholinergic drugs in the previous 28 days before Day 1 of this trial. 
15. Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >2.5 × the 
upper limit of the normal range (ULN), bilirubin >1.5 × ULN, estimated 
glomerular filtration rate <30 mL/min/1.73 m2 according to the Modification of 
Diet in Renal Disease study definition (16), or altered electrolyte values of 
clinical relevance for cardiac conduction, as judged by the investigator. 
16. Clinically significant abnormal ECG at screening as judged by the investigator. 
17. Clinically significant illness within 4 weeks before screening, as judged by the 
investigator. 
18. Donation of blood or plasma in the past month, or in excess of 500 mL within 
12 weeks prior to screening. 
19. Surgery or trauma with significant blood loss within the last 2 months prior to 
screening. 
20. A positive result in the alcohol and/or urine drug screen at the screening visit. 
Significant history of alcoholism or drug abuse as judged by the investigator or 
consuming more than 24 g alcohol per day for men, or more than 12 g alcohol per 
day for women. 
21. Subjects with mental incapacity or language barriers which preclude adequate 
understanding or cooperation, who are unwilling to participate in the trial, or who 
in the opinion of the investigator should not participate in the trial. 
22. Any condition interfering with trial participation or evaluation or that could be 
hazardous to the subject. 
23. The use of prescription or non-prescription medications known to cause QT 
prolongation. 
5.5 
Dosing day exclusion criteria 
Subjects who meet one or more of the following dosing day exclusion criteria at the 
dosing visit will be excluded from the dosing visit, but the visit can be rescheduled 
1-7 days later. The dosing visit can only be rescheduled once. 
1. Atypically strenuous exercise within 4 days prior to dosing, as judged by the 
investigator. Exercise during the trial should follow subject’s typical routine, and 
should not exceed a near maximum intensity for more than 20 minutes per day, or 
moderate intensity for more than 90 minutes per day. 


 
 
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2. Clinically significant illness within 4 weeks before dosing, as judged by the 
investigator. 
3. Consumption of alcohol within 24 hours prior to dosing visit, or positive results 
from an alcohol breath test. 
4. Not fasting from 22:00 hours the evening prior to dosing, apart from water. Small 
amounts of carbohydrates (up to 20 g) to prevent hypoglycemia are allowed. 
5. The use of any non-prescribed systemic or topical medication, except routine 
vitamins and occasional use (as judged by the investigator) of acetylsalicylic acid 
and paracetamol within 2 weeks prior to dosing. Treatment with insulin, including 
analogs, is allowed. 
6. Use of insulin Degludec or insulin Glargine U300 within 48 hours prior to dosing; 
or use of other long-acting insulins (e.g., insulin Glargine U100 or insulin 
Detemir) within 24 hours prior to dosing; or use of insulin Neutral protamine 
Hagedorn (NPH) within 16 hours prior to dosing. 
7. Use of any short acting (bolus) insulin within 6 hours prior to dosing, except 
insulin glulisine (Apidra®). 
8. Changes in medical history or concomitant medication resulting in fulfillment of 
clinical exclusion criteria, as judged by the investigator. 
9. Plasma glucose value <50 mg/dL (2.8 mmol/L) within the last 24 hours or plasma 
glucose value <60 mg/dL (3.3 mmol/L) within the last 5 hours prior to initiation 
of the hypoglycemic procedure. 
5.6 
Premature treatment discontinuation and withdrawal 
Participation in the trial is strictly voluntary. A subject has the right to withdraw from the 
trial at any time and for any reason. If he/she chooses to withdraw, the investigator must 
be informed immediately. The investigator has the right to terminate participation of any 
subject at any time if the investigator deems it in the subject's best interest. The reason 
and circumstances for withdrawal will be documented in the electronic case report form 
(eCRF). 
5.6.1 Possible reasons for treatment visit discontinuation 
A subject will be discontinued from treatment if the following applies: 
 Withdrawal of consent by subject. 
 If a protocol deviation occurs which, in the clinical judgement of the investigator, can 
invalidate the trial or endpoints or can interfere pharmacokinetically or 
pharmacodynamically with the trial product, the subject will be discontinued by the 
investigator. 
 Adverse events occur which are considered unacceptable by the subject or the 
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If discontinuation occurs following administration of trial medication, every effort should 
be made to have the subject return and participate in the complete follow-up visit on Day 
28 (see Table 3) to avoid missing data. 
If trial participation is terminated due to an AE possibly related to any of the trial 
medications or trial examinations, the subject must be followed up by additional 
examinations according to the medical judgment of the investigator until the abnormal 
condition is resolved or the investigator deems further observations or examinations to be 
no longer medically indicated. 
A total of 156 subjects must complete the treatment visit (Visit 2). To qualify as 
completed, the subject must be dosed and have blood drawn for the PK and PD 
assessments described in the protocol.  
5.6.2 Center discontinuation 
The center can be closed and the trial terminated for the following reasons: 

The center is unlikely to be able to recruit sufficient subjects within the agreed time
frame.

The center does not respond to trial management requests.

Repeat protocol violations.
5.6.3 Trial termination 
The sponsor reserves the right to modify or terminate the trial at any time. Possible 
reasons for termination are: 

Safety reasons – the incidence of AEs in this or any other trial using the same trial
medication indicates a potential health risk for the subjects.

New scientific knowledge becomes known that makes the objectives of the trial no
longer feasible/valid.

Unsatisfactory enrolment of subjects.
5.7 
Subject identification and randomization 
Subjects who have given written informed consent and meet all inclusion and none of the 
exclusion criteria will be randomized in a 2:1:1 ratio to either 0.6 mg dasiglucagon 
(n=78), placebo (n=39), or 1 mg GlucaGen (n=39) via an Interactive Web Response 
System (IWRS). Randomization will continue until 156 subjects have completed Visit 2. 


 
 
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Subjects with previous exogenic glucagon exposure will not be excluded from the trial, 
but the information on previous glucagon administration will be recorded, to enable 
subgroup analyses.  
In the event of an emergency, e.g. when it becomes necessary for the investigator to 
know which treatment the subject is taking, the subject code can be broken by the 
investigator, preferably after consultation with the medical monitor. Emergency code 
breaks can be performed using the IWRS. 


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6 
TRIAL DRUG 
6.1 
Identity 
The following trial drugs will be administered: 

Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in a prefilled syringe.

Placebo, liquid formulation, 0.6 mL delivered in a prefilled syringe.

Recombinant glucagon hydrochloride, 1 mg for reconstitution (GlucaGen®, Novo
Nordisk) in 1 mL sterile water.
The description of the three trial drugs is provided in Table 1. Dasiglucagon is a stable 
peptide analog of human glucagon in a ready-to-use liquid formulation for treatment of 
severe hypoglycemia in insulin-dependent patients with diabetes mellitus. GlucaGen is 
approved in the EU and US and is indicated for treatment of severe hypoglycemic 
reactions, which may occur in the management of insulin-treated children and adults with 
diabetes mellitus. 
Table 1 
Description of trial drugs 
Test product 
Placebo Product 
Comparator product 
Name 
Dasiglucagon 
Placebo 
GlucaGen® 
Active 
substance 
Dasiglucagon 
N/A 
Recombinant 
glucagon 
hydrochloride 
Formulation 
Liquid formulation, 
0.6 mL 
Liquid formulation, 
0.6 mL 
Powder and solvent 
for reconstitution as 1 
mL solution for 
injection 
Strength 
1 mg/mL 
N/A 
1 mg 
Device 
Single use pre-filled 
syringe 
Single use pre-filled 
syringe 
Powder and solvent 
for reconstitution 
packed together in a 
plastic box. A “hypo-
kit”. 
Manufacturer 
Zealand Pharma A/S, 
Glostrup 
(Copenhagen), 
Denmark 
Zealand Pharma A/S, 
Glostrup 
(Copenhagen), 
Denmark 
Novo Nordisk A/S, 
Bagsværd, Denmark 


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Test product 
Placebo Product 
Comparator product 
Storage 
requirements 
Store between 2 and 
8°C 
Store between 2 and 
8°C 
Store between 2 and 
8°C 
The quantities of ingredients for dasiglucagon and placebo are provided in Table 2. 
Table 2 
Quantities of ingredients in dasiglucagon and placebo injection 
Component 
Amount per mL 
(dasiglucagon) 
Amount per mL 
(placebo) 
Function 
Dasiglucagon* 
1.0 mg 
N/A 
Sodium chloride 
10.23 mg 
10.23 mg 
Trometamol/Tromethamine 
6.06 mg 
6.06 mg 
Water for injection 
To make 1 mL 
To make 1 mL 
Sodium hydroxide 
q.s. 
q.s. 
Hydrochloric acid 
q.s. 
q.s. 
*The quantity of drug substance to be used is calculated according to net peptide content and purity.
q.s. = quantum sufficit (quantity required). 
6.2 
Treatment assignment and randomization 
Subjects successfully completing screening and who fulfill entry eligibility and 
randomization criteria will be randomized to one of three treatment groups in a ratio of 
2:1:1: 

Test treatment: Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in a
prefilled syringe.

Placebo treatment: Placebo, liquid formulation, 0.6 mL delivered in a prefilled
syringe.

Reference treatment: Recombinant glucagon hydrochloride, 1 mg for reconstitution.
Randomization will be performed using a fixed-block randomization scheme. The 
randomization scheme will be generated prior to the initiation of the study by an 
independent statistician/programmer who will not be a member of the study team; all 
investigators will not be aware of the block size of the randomization scheme.  
Randomization will be stratified by treatment group and by injection site (abdomen, 
buttocks, or thigh) and controlled via the IWRS.       


 
 
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Subjects will be randomized to study treatment using an interactive, automated system 
which has been validated for the intended use under the International Society of 
Pharmaceutical Engineers Good Automated Manufacturing Process guidelines, 21CFR 
Part 11 (FDA regulation for Electronic Records and Electronic Signatures) and the 
International Conference on Harmonization (ICH) Guidance E6 for Industry on Good 
Clinical Practice (GCP).   
6.3 
Administration 
Dasiglucagon, placebo, and GlucaGen will be administered by SC injection in the 
abdomen, buttocks, or thigh.  
An unblinded person (appropriately trained) authorized to prepare the dose and 
administer the treatment in accordance with the randomization will prepare the treatment 
required for each subject on each dosing day. The dose will be administered by the 
unblinded, trained and qualified person. The content of the syringe has to be checked for 
clarity and absence of bubbles.. 
Syringes will be discarded after dose administration. Used GlucaGen vials will be stored 
in a lockable box (separated from unused vials) at ambient temperature. 
6.4 
Packaging and labelling 
The test product will be packed by the sponsor. The information on the labels will be in 
the local language and the product label will be compliant with local laws and 
regulations.  
The study drug label will describe the storage conditions for study drug. The labels will 
supply no information about the subjects. Each treatment kit (pre-filled syringe/vial for 
reconstitution) will have a unique Dispensing Unit Number for drug allocation, drug 
accountability, and traceability purposes. 
Labelling will be performed according to Annex 13 of the Good Manufacturing Practice 
guidelines of the European Commission, ICH GCP guidelines, and local law. 
6.5 
Storage of study drugs 
The investigator must ensure the availability of proper storage conditions. All study drug 
supplies provided for this study will be stored in a secure area with restricted access at the 
study site.  


 
 
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The temperature should be monitored by recording the actual, minimum, and maximum 
temperatures using a calibrated thermometer or thermocouple, or by continuous recording 
using a qualified temperature monitoring system. The temperature should be evaluated 
and documented at least on working days on a temperature log. This log must be included 
in the Investigator Site File upon study termination. 
Dasiglucagon and placebo must be stored in a refrigerator (at a temperature of 2–8°C), 
and should be handled in accordance with guidelines from the sponsor. GlucaGen must 
also be stored in a refrigerator (at a temperature of 2–8°C), and should be handled in 
accordance with the Summary of Product Characteristics (13).  
The unblinded person responsible for study drug handling must contact the unblinded 
monitor in case of temperature deviations outside the acceptable range.  
Please see the Pharmacy Manual for additional information on handling study drug. 
6.6 
Blinding and breaking the blind 
This is a double-blind trial. As the trial products are not identical in appearance, 
dasiglucagon and placebo being available as a liquid formulation and GlucaGen as a 
powder for reconstitution, unblinded trial personnel will be responsible for handling, 
preparing (according to the prescription from the IWRS), and administering the trial 
medication, as well as for keeping the records strictly confidential and accessible only to 
unblinded staff until after the database has been locked. To maintain double-blind 
conditions, all trial assessments at the trial center will be done by blinded trial personnel 
not involved in the administration of trial medications. However, exposure assessments 
and anti-drug antibody (ADA) assessments will be performed by unblinded personnel at 
the special laboratories, to ensure that dasiglucagon, placebo, or GlucaGen administration 
is matched with the applicable bioanalytical assay. 
Treatment assignment will be kept strictly confidential and accessible only to authorized 
persons until after the time of unblinding. Codes with treatment assignment will, 
however, be readily available in the IWRS to the blinded personnel in case of an 
emergency. 
The code for a particular subject may be broken in a medical emergency if knowing the 
identity of the treatment allocation would influence the treatment of the subject. The 
emergency code break can be performed using the IWRS. Whenever a code is broken, the 
person breaking the code must record the time, date and reason as well as his/her initials 
in the source documents. The breaking of blinded codes in case of medical emergency for 
one subject should not unblind the trial personnel to the treatment information of other 


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subjects. The person performing the unblinding should inform as few people as possible 
about the result of the unblinding. All persons unblinded for a specific subject should be 
documented. 
If the trial center needs to break the code, the medical monitor should, if at all possible, 
be contacted prior to breaking the code and the monitor must be notified within 24 hours 
after the code has been broken. 
The pharmacovigilance unit (safety contract research organization [CRO]; see the list of 
trial personnel in Section 12.1) will be able to break the code in case of a serious 
unexpected suspected adverse reaction (SUSAR).  
The central and specialty laboratories will be provided with a copy of the randomization 
list. 
6.7 
Drug accountability 
Handling, preparation and administration of trial medication will be done by unblinded 
trial personnel. Each center will keep accurate records of the trial supplies received, 
stored, and dispensed, using appropriate forms. The trial supplies will be stored under 
appropriate conditions, locked and with restricted access.  
All unused supplies and all empty and partially empty containers of trial medication will 
be stored until the trial closure visit has been performed and then sent for destruction. 
This does not apply to the used syringes as they will be discarded after dose 
administration. Destruction must not take place until approved by the Sponsor.  
6.8 
Treatment compliance 
All trial medications will be prepared and administered by unblinded trial personnel. 
PK assessments will support the surveillance of compliance with IMP administration.  
6.9 
Prior and concomitant medications 
Prior glucagon exposure will be recorded in the eCRF at screening. All concomitant 
medications will be recorded and/or updated in the eCRF at each visit.  
Subjects using any new concomitant medication resulting in fulfillment of a dosing day 
exclusion criterion (see Section 5.5) will be excluded from the dosing visit, but can be 


 
 
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rescheduled to one of the following days (1–7 days later). The dosing visit can only be 
rescheduled once. 
6.9.1 Prohibited medications 
The use of anti-coagulant treatments and medications (prescription and non-prescription) 
that are known to cause QT prolongation are prohibited during the course of the trial. 
Within 28 days prior to dosing, the use of daily systemic beta-blockers, indomethacin, 
warfarin, and anticholinergic drugs is prohibited. 
Within 2 weeks prior to dosing, the use of any non-prescribed systemic or topical 
medication (with the exception of vitamins and the occasional use of acetylsalicylic acid 
and paracetamol) is prohibited.  
Within 48 hours prior to dosing, the use of insulin Degludec or insulin Glargine U300 are 
prohibited. 
Within 24 hours prior to dosing, the use of long-acting insulin (e.g., insulin Glargine 
U100 or insulin Detemir) is prohibited. 
Within 16 hours prior to dosing, the use of insulin NPH is prohibited. 
Within 6 hours prior to dosing, the use of any short acting (bolus) insulin, except insulin 
glulisine (Apidra®) is prohibited.  
During the insulin-induced hypoglycemic procedure, continuous SC insulin infusion must 
be stopped. 


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7 
PARAMETERS AND METHODS OF ASSESSMENT 
Overall, approximately 180 mL of blood will be drawn from each subject for PK, PD, 
ADA, and safety laboratory assessments. 
7.1 
Efficacy parameters 
A description of the sample handling and sample processing at the site will be included in 
the laboratory manuals. Validation documentation for the assays must be available prior 
to sample analyses. A bioanalytical report for each analysis of trial PD and PK samples 
will be prepared. 
7.1.1 Pharmacodynamic measurements 
The plasma glucose profile for evaluation of the primary and secondary clinical efficacy 
endpoints will be assessed based on plasma concentration data (AUC0-30min) from samples 
collected at the dosing visit (Visit 2). The samples will be sent to the clinical laboratory 
and analyzed using a sensitive and validated assay for glucose measurements.  
Samples will be collected pre-dose, and at 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 
75, and 90 minutes after dosing (see the schedule of procedures in Table 3). The actual 
time of blood sampling for evaluation of plasma glucose should not deviate from the 
nominal time by more than ±30 seconds until the 20 minute collection time point and by 
more than ±1 minute for the subsequent collection time points. Pre-dose is defined as 
within 2 minutes prior to dosing. 
7.1.2 Pharmacokinetic measurements 
The exposure to trial medication (dasiglucagon or GlucaGen) for evaluation of PK will 
be assessed based on plasma concentration data (AUC0-90 min, Cmax, tmax) from samples 
collected at the dosing visit (Visit 2).  
Samples (including back-up samples) will be collected pre-dose, and at 15, 30, 35, 40, 50, 
60, 90, and 120 minutes after dosing (see the schedule of procedures in Table 3). The 
actual time of blood sampling for exposure to trial medication should not deviate from 
the nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior 
to dosing. 


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7.2 
Safety parameters 
7.2.1 Adverse events 
The investigator is responsible for the detection and documentation of events meeting the 
criteria and definition of an AE or serious adverse event (SAE), as provided in this 
protocol. During the trial, the investigator or center staff will be responsible for reporting 
AEs and SAEs, as detailed in this section of the protocol.  
Based on the investigator’s clinical judgment it will be determined whether an AE is 
related to treatment and of sufficient severity to require the subject’s removal from 
treatment or from the trial. A subject may also voluntarily withdraw from treatment due 
to what he or she perceives as an intolerable AE. If either of these situations arises, the 
subject should be under medical supervision until symptoms cease or the condition 
becomes stable. 
7.2.1.1 Definitions 
Adverse event 
An AE is any untoward medical occurrence in a trial subject given an IMP which does 
not necessarily have a causal relationship with the treatment. An AE can therefore be any 
unfavorable and/or unintended sign (for example, an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of an IMP, whether or not 
considered related to the IMP.  
In this trial, only treatment-emergent adverse events (TEAEs) will be collected and 
reported. TEAEs are events that occur from the first trial-related activity after the subject 
has signed the informed consent form until the end of the post-treatment follow-up 
period.  
AEs include: 

A clinically significant worsening of a concomitant illness.

A clinical laboratory abnormality which is clinically significant, i.e. any abnormality
that suggests a disease and/or organ toxicity and is of a severity that requires active
management. Active management includes active treatment or further investigations,
for example change of medicine dose or more frequent follow-up due to the
abnormality.

Hypoglycemic episodes (see Section 7.2.2).

Injection site reactions (see Section 7.2.6).


 
 
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The following should not be recorded as AEs, if recorded at screening (on the Screening 
Form or eCRF): 
 Pre-planned procedure, unless the condition for which the procedure was planned has 
worsened from the first trial related activity after the subject has signed the informed 
consent. 
 Pre-existing conditions found as a result of screening procedures (pre-existing 
conditions should be reported as medical history or concomitant illness). 
Serious adverse event 
An SAE is any untoward medical occurrence that at any dose:  
 Results in death  
 Is life-threatening  
 Requires inpatient hospitalization or prolongation of existing hospitalization 
 Results in persistent or significant disability or incapacity 
 Is a congenital anomaly or birth defect 
 Is medically important* 
*Medical judgement must be exercised in deciding whether an AE is believed to be 
‘medically important’. Medically important events may not be immediately 
life-threatening or result in death or hospitalization but may jeopardize the subject or 
may require intervention to prevent one of the other outcomes listed in the definition 
above. 
Adverse drug reaction 
All noxious and unintended responses to an investigational product related to any dose 
should be considered adverse drug reactions. The phrase responses to an investigational 
product means that a causal relationship between an investigational product and an 
adverse event is at least a reasonable possibility, i.e. the relationship cannot be ruled out. 
 
Suspected unexpected serious adverse reactions (SUSARs) 
An AE fulfilling one of the criteria of seriousness and being assessed as related to IMP 
application, the nature or severity of which is not consistent with the applicable reference 
document (e.g. dasiglucagon Investigator’s Brochure or package leaflet/Summary of 
Product Characteristics for GlucaGen). 


 
 
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Adverse event of special interest 
An AESI is an event which, in the evaluation of safety, has a special focus (e.g. required 
by health authorities). In this trial hemodynamic changes, as defined below, are 
considered AESIs: 
 Post-dose clinical signs, or measured vital signs, indicating a clinically significant 
drop in blood pressure including signs of orthostatic hypotension, vasovagal 
responses or bradycardia.   
 Post-dose change in pulse or blood pressure considered an event of hypo- or 
hypertension as judged by the investigator. 
 
Intensity of an adverse event 
The maximum intensity (severity) of all AEs must be assessed by the investigator and 
documented. Severity should be graded when the AE outcome is known: 
Mild:  
A type of AE that is usually transient and may require only minimal 
treatment or therapeutic intervention. The event does not generally 
interfere with usual activities of daily living. 
Moderate:  
A type of AE that is usually alleviated with additional specific therapeutic 
intervention. The event interferes with usual activities of daily living, 
causing discomfort but poses no significant or permanent risk of harm to 
the subject. 
Severe:  
A type of AE that interrupts usual activities of daily living, or significantly 
affects clinical status, or may require intensive therapeutic intervention. 
A ‘severe’ reaction does not necessarily deem the AE as ‘serious’ and an SAE may not 
necessarily be ‘severe’ in nature. 
Causality relationship to trial medication 
The causality of each AE should be assessed by the investigator according to the 
following classification: 
Probable: 
Good reason and sufficient documentation to assume a causal relationship. 
Possible: 
A causal relationship is conceivable and cannot be dismissed. 
Unlikely: 
The event is most likely related to etiology other than the trial product. 
Not related:  No relationship to trial product. 


 
 
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Outcome of an adverse event 
The outcome of all AEs must be assessed by the investigator and documented by his/her 
staff. The following definitions should be used: 
Recovered/resolved:  
The subject has fully recovered, or by medical or 
surgical treatment the condition has returned to the 
level observed at the first trial-related activity after 
the subject signed the informed consent.  
Recovering/resolving: 
The condition is improving and the subject is 
expected to recover from the event. This term is 
only applicable if the subject has completed the 
trial. 
Recovered/resolved with sequelae:  The subject has recovered from the condition, but 
with lasting effect due to a disease, injury, treatment 
or procedure. If a sequela meets an SAE criterion, 
the AE must be reported as an SAE. 
Not recovered/not resolved:  
The condition of the subject has not improved and 
the symptoms are unchanged. 
Fatal:  
This term is only applicable if the subject died from 
a condition related to the reported AE. Outcomes of 
other reported AEs in a subject before he/she died 
should be assessed as "recovered/resolved", 
"recovering/resolving", "recovered/resolved with 
sequelae" or "not recovered/not resolved". An AE 
with fatal outcome must be reported as an SAE. 
Unknown:  
This term is only applicable if the subject is lost to 
follow-up. 
7.2.1.2 Collection, recording and reporting of adverse events 
All events meeting the definition of an AE must be collected and reported from the first 
trial related activity after the subject has signed the informed consent until the end of the 
post-treatment follow-up period. At each contact with the center (visit or telephone, 
excluding visits where the subject is not seeing the investigator or his/her staff [e.g. visits 
to the laboratory]) the subject must be asked about AEs. All AEs, either observed by the 
investigator or reported by the subject, must be recorded by the investigator and 
evaluated. 
The investigator should record the diagnosis, if possible. If no diagnosis can be made the 
investigator should record each sign and symptom as individual AEs. 


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One single Adverse Event Form must be used per AE from start to resolution. For SAEs, 
the Serious Adverse Event form must also be completed. For AESIs, the AESI form must 
also be completed. 
AE information should include the following: 

Date and time of onset and resolution

Date and time of investigator’s first information on the (S)AE

Seriousness

Severity

Causal relationship with IMP

Interruption or withdrawal of treatment during IMP administration and other
measures taken

Outcome.
All AEs are coded; details are described in the trial-specific Data Management Plan. 
If an event classifies as a AESI, the investigator must tick the AESI box on the AE form 
and complete the AESI form. The AESI form will capture if the event was associated 
with any signs or symptoms and capture the highest/lowest blood pressure and pulse 
measured during the event. The investigator must report all AESIs to the sponsor’s 
responsible pharmacovigilance unit (Safety CRO; see the list of trial personnel in 
Section 12.1) immediately (within 24 hours) after obtaining knowledge about the event. 
The investigator must report initial information electronically (e.g. in PDF format) on all 
SAEs to the sponsor’s responsible pharmacovigilance unit (Safety CRO; see the list of 
trial personnel in Section 12.1) immediately (within 24 hours) after obtaining knowledge 
about the event. The Safety CRO will inform the medical monitor and the sponsor about 
the reported SAEs. 
It is the responsibility of the Safety CRO to report all SUSARs (see Section 7.2.1.1) that 
occur in this trial to the Competent Authorities and Institutional Review Boards 
(IRBs)/Independent Ethics Committees (IECs) in accordance with the local requirements 
in force and ICH guideline for GCP. The trial monitor must be informed accordingly. 
7.2.1.3 Follow-up of adverse events 
All AEs that are ongoing at the end of the subject’s participation in the trial will be 
followed-up until the event is resolved or reaches a satisfactory outcome as deemed by 
the investigator or until the last visit of the last subject enrolled in the trial, whichever 
occurs first.   


 
 
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Follow-up actions for all SAEs will be performed according to appropriate clinical care 
practices and may depend on the nature of the event. These will be determined after 
internal review and/or sponsor review. 
The follow-up information should only include new (updated and/or additional) 
information that reflects the situation at the time of the investigator’s signature. 
Follow-up information on (S)AEs will be updated using the (S)AE form. If a non-serious 
event becomes serious during the follow-up the AE form and SAE form have to be used 
and reporting timelines follow those of an SAE. 
The investigator must forward follow-up information on SAEs and if previously 
non-serious AEs become SAEs to the Safety CRO immediately (within 24 hours) after 
obtaining knowledge about the new information. 
The sponsor and/or CROs acting on behalf of the sponsor can upgrade a non-serious AE 
to an SAE. In these situations the investigator will be informed and asked to fill out an 
SAE form and forward to the Safety CRO immediately (within 24 hours). 
7.2.1.4 Clinical laboratory abnormalities and other abnormal assessments as adverse 
events or serious adverse events 
Abnormal laboratory findings (e.g. biochemistry, hematology, urinalysis) or other 
abnormal assessments (e.g. vital signs) that are judged by the investigator as clinically 
significant will be recorded as AEs or SAEs if they meet the definition of an AE or SAE. 
Clinically significant abnormal laboratory findings or other abnormal assessments that 
are detected during the trial or are present at baseline and significantly worsen following 
the start of the trial will be reported as AEs or SAEs. However, clinically significant 
abnormal laboratory findings or other abnormal assessments that are associated with the 
disease being studied, unless judged by the investigator as more severe than expected for 
the subject’s condition, or that are present or detected at the start of the trial and do not 
worsen, will not be reported as AEs or SAEs. 
The investigator will exercise his or her medical and scientific judgment in deciding 
whether an abnormal laboratory finding or other abnormal assessment is clinically 
significant. 
7.2.2 Hypoglycemia 
Hypoglycemia will be regarded as an AE and recorded and documented on an AE form 
(and SAE form, if applicable). 


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Hypoglycemia is defined as a decline in plasma glucose to below 70 mg/dL 
(3.9 mmol/L). However, in the time period from initiation of the hypoglycemic clamp 
procedure (see Section 8.2.3.1) until 45 minutes after dosing, hypoglycemia is defined as 
a decline in plasma glucose to below 45 mg/dL (2.5 mmol/L).  
During the dosing visit, prior to administration of the IMP, a plasma glucose value of 
<45 mg/dL (2.5 mmol/L) will be treated by an IV glucose solution. After administration 
of the IMP in the period from 8 minutes until 44 minutes after dosing, a plasma glucose 
value of <45 mg/dL (2.5 mmol/L) will be treated by an IV glucose solution, and if a 
plasma glucose value of ≥70 mg/dL (3.9 mmol/L) is not achieved within the 45 minutes 
after IMP administration, IV glucose infusion will also be initiated. 
If the subject experiences symptoms of hypoglycemia, a plasma glucose measurement 
should be taken in order to classify the event (please refer to Section 7.4.2 for additional 
details).  
7.2.3 Physical examination 
The physical examination will be carried out at screening (Visit 1) and at the follow-up 
visit (Visit 3; see Table 3).  
The physical examination includes examination of the following body systems: head, 
ears, eyes, nose, throat, including the thyroid gland; heart, lung, chest; abdomen; skin and 
mucosae; musculoskeletal system; nervous system; lymph node; other findings. 
At the screening visit, any abnormality will be recorded and described in the eCRF, 
including the investigator’s assessment of clinical significance (‘abnormal, not clinically 
significant’ or ‘abnormal, clinically significant’). Clinically significant findings at the 
screening visit should be recorded as concomitant illness. 
At subsequent visits, any clinically significant deterioration of a pre-existing condition as 
well as any new clinically significant findings will be recorded as AEs (see 
Section 7.2.1). 
7.2.4 Vital signs 
An examination of the following vital signs will be performed at screening (Visit 1), the 
dosing visit (Visit 2) and at the follow-up visit (Visit 3):  

Diastolic and systolic blood pressure (mmHg) will be measured after at least
5 minutes rest in a supine position. At Visit 1, blood pressure will be measured in
both arms. The blood pressure from the arm with the higher systolic value is


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transcribed into the eCRF and this arm should be used for all subsequent 
measurements of the subject’s blood pressure in this trial.  

Pulse (beats per min) measured after at least 5 minutes rest in a supine position.

Body temperature (°C).
At the dosing visit, measurements will be taken prior to the start of the insulin-induced 
hypoglycemic procedure (within 30 minutes) and at 30, 90, and 300 minutes after dosing 
(see Table 3). The actual time of the assessment should not deviate from the nominal 
time by more than ±10 minutes. In addition to the pre-specified assessments as listed in 
Table 3, blood pressure and pulse may be assessed at any time during the trial at the 
discretion of the investigator.  
7.2.5 Electrocardiogram 
A standard 12-lead ECG will be performed at the screening visit (Visit 1), the dosing visit 
(Visit 2; prior to the start of the insulin-induced hypoglycemic procedure [within 
30 minutes]) and at 20, 35, 45, 60, and 300 minutes after dosing) and at the follow-up 
visit (Visit 3; see Table 3). The actual time of the assessment should not deviate from the 
nominal time by more than ±5 minutes.  
ECG parameters (heart rate, PQ, QRS, QT, QTcB) and any abnormality will be recorded 
and described in the eCRF including the investigator’s assessment of clinical significance 
(‘abnormal, not clinically significant’ or ‘abnormal, clinically significant’).  
At subsequent visits, any clinically significant deterioration of a pre-existing condition as 
well as any new clinically significant findings will be recorded as AEs (see 
Section 7.2.1). 
7.2.6 Local tolerability 
Immediately prior to treatment administration, it should be verified that the injection site 
is normal. To ensure all injection site assessments are performed at the injection site, the 
site will be marked with a pen prior to injection. Assessment of local tolerability at the 
injection site will be performed at the dosing visit (Visit 2; at 30, 120 and 300 minutes 
after dosing), and at the follow-up visit (Visit 3) (see Table 3) and more frequently, if 
deemed necessary by the investigator. The actual time of the assessment should not 
deviate from the nominal time by more than ±10 minutes. In the eCRF, the time of 
assessment and any injection site reaction observed will be recorded. In case of an 
observation, the Local Tolerability form will be completed, as well as the (S)AE form. 
The local tolerability at the injection site will be evaluated by means of the following 
assessments: spontaneous pain, pain on palpation, itching, redness, edema, 


 
 
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induration/infiltration, and other. Each of these assessments will be reported on a scale of 
0 (none), 1 (mild), 2 (moderate), and 3 (severe). The evaluation and the actual time of the 
assessment will be recorded. The assessments will be performed by a trial physician or 
nurse.  
Digital pictures will be taken of the injection site at the time of identification, and 
thereafter as often as judged necessary by the investigator. The pictures should include a 
subject identifier, visit number, time after dosing, and a ruler for scaling. 
7.2.7 Clinical laboratory assessments 
The safety parameters that will be assessed at the clinical laboratory are listed in Table 3. 
Routine clinical laboratory tests will be performed centrally. Samples for clinical 
laboratory parameters (biochemistry, hematology, coagulation) will be collected at 
screening (Visit 1), the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure [within 30 minutes]) and at 30 and 300 minutes after dosing), 
and at the follow-up visit (Visit 3). The actual time of the assessment should not deviate 
from the nominal time by more than ±10 minutes. Samples for glycated hemoglobin 
(HbA1c) will be collected at screening only (Visit 1). Samples for urinalysis will be 
collected at screening (Visit 1), at the dosing visit (Visit 2, prior to the start of the 
insulin-induced hypoglycemic procedure [within 120 minutes]) and at the follow-up visit 
(Visit 3). The following parameters will be assessed: 
 Clinical biochemistry: sodium, potassium, calcium, glucose, urea, creatinine, total 
bilirubin, AST, ALT, gamma-glutamyltransferase, alkaline phosphatase, total protein, 
C-reactive protein, HbA1c, C-peptide. 
 Hematology: hemoglobin, red blood cell count (erythrocytes), hematocrit, platelet 
count (thrombocytes), total white blood cell count (leukocytes). 
 Coagulation: international normalized ratio, fibrinogen (at screening visit only).  
 Urinalysis: pH, blood (leukocytes and erythrocytes), protein, glucose, ketones, nitrite. 
Pregnancy tests will be performed at each visit for women of childbearing potential only. 
A serum pregnancy test will be performed at screening (Visit 1) and urine stick tests will 
be performed at the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure) and the follow-up visit (Visit 3). Test sticks will be provided to 
the trial centers. 
Alcohol breath tests and a urine drug screen will be performed at screening (Visit 1) and 
at the dosing visit (Visit 2, prior to the start of the insulin-induced hypoglycemic 
procedure). Equipment for the alcohol breath test and uring drug screen will be provided 
to the trial centers.  


 
 
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Re-assessment of laboratory parameters will be allowed only if handling issues, damaged 
samples, or hemolyzed samples have confounded the measurement results.  
For further details of the clinical laboratory assessments, please refer to the laboratory 
manual.  
7.2.8 Pregnancy 
Female subjects must be instructed to notify the investigator immediately if they become 
pregnant or if they suspect to be pregnant during the trial. The sponsor has a 
responsibility to monitor the outcome of all pregnancies reported during the clinical trial. 
Male subjects must be instructed to notify the investigator immediately if their partner 
becomes pregnant or suspects pregnancy. The sponsor has a responsibility to monitor the 
outcome of all pregnancies reported during the clinical trial. During an information 
meeting at the trial center, the subject’s partner will be fully informed by a physician of 
her participation in capturing the outcome data, and will be asked to provide her written 
consent. 
The investigator must report all information on pregnancies on the Initial Pregnancy 
form. The completed Initial Pregnancy form must be forwarded to the sponsor 
immediately (within 24 hours), according to the procedure stated in Section 7.2.1.2. Any 
(S)AEs in the mother, as well as other pregnancy complications (ectopic pregnancy) 
fulfilling the criteria of an (S)AE, must be reported on the (S)AE form.  
The following must be collected in the Initial Pregnancy form:  
 Medical history of the mother 
 Family history 
 Course of the pregnancy, including expected delivery date. 
The investigator must follow the pregnancy until the pregnancy outcome and follow the 
newborn infant(s) until the age of 1 month. The investigator must collect information on 
the pregnancy and pregnancy complications as well as the pregnancy outcome including 
the health of the newborn infant(s) on the Pregnancy Outcome form. The completed 
Pregnancy Outcome form must be forwarded to the sponsor according to the procedure 
stated in Section 7.2.1.2. Any (S)AEs in the newborn must be reported on the (S)AE 
form. 
The SAEs that must be reported include abnormal outcome, such as congenital 
anomalies, fetal death and termination of pregnancy (spontaneous or elective abortion), 
including any anomalies of the fetus observed at gross examination or during autopsy.  


 
 
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The following must be collected in the Pregnancy Outcome form:  
 Course of the pregnancy 
 Outcome of the pregnancy 
 Condition of the newborn 
 Any AEs in the newborn infant must be followed until the age of 1 month. 
7.2.9 Precautions 
Normal precautions taken for a human trial, including the provision of emergency 
equipment, will be taken during this trial. Qualified and well trained physicians and 
medical staff will instruct the subjects. During a subject’s participation in the trial, the 
investigator should ensure that adequate medical care is provided to the subject for any 
AEs, including clinically significant laboratory values related to the trial. The investigator 
should inform the subject when medical care is needed for intercurrent illnesses of which 
the investigator becomes aware.  
For further information on safety precautions for dasiglucagon and GlucaGen, please 
refer to the current version of the Investigator’s Brochure (14) and the Summary of 
Product Characteristics for GlucaGen (13), respectively. 
7.2.10 Safety committee 
The internal Zealand Pharma Safety Committee is constituted to perform ongoing blinded 
safety surveillance of clinical trials with dasiglucagon, including this trial. 
If safety signals are observed, either based on reported SAEs, periodic review of 
laboratory parameters, review of all AEs reported between the Safety Committee 
meetings, or on notification of significant findings, the Safety Committee will take 
appropriate measures to safeguard the subjects. 
7.3 
Demography, concomitant illness, medical history and concomitant 
medication 
Demographics, body measurements, concomitant illness and medical history will be 
assessed only at screening (Visit 1). Concomitant medication will be assessed at 
screening (Visit 1), the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure) and at the follow-up visit (Visit 3). 
7.3.1 Demography and body measurements 
Subject demographics and body measurements will include: 


 
 
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 Age  
 Race, ethnicity 
 Sex 
 Height (meters or inch), without shoes 
 Body weight (kg or lb), only wearing underwear and measured using standard scales 
 Body mass index (kg/m2) calculated based on height and body weight (body 
weight/height2). 
7.3.2 Concomitant illness and medical history 
A concomitant illness is any illness that is present at the start of the trial (i.e. at the 
screening visit). Concomitant illnesses present at the start of the trial will be recorded in 
the eCRF at screening.  
Medical history is an account of medical events that the subject has experienced in the 
past, including prior medications. Relevant medical conditions/illnesses in the past 
obtained by asking the subject or by inspecting his/her medical records will be recorded 
in the eCRF at screening. History of alcohol or drug abuse will also be recorded. 
The information collected for concomitant illness and medical history should include 
diagnosis, date of onset, date of resolution or continuation. 
Any change to a concomitant illness should be recorded during the trial, including end 
date, if applicable. A clinically significant worsening of a concomitant illness must be 
reported according to Section 7.2.1. 
All previous and concomitant diseases will be coded with Medical Dictionary for 
Regulatory Activities (MedDRA), the version of which will be provided in the clinical 
trial report. 
7.3.3 Diabetes diagnosis and current treatment 
The date of diagnosis of diabetes will be recorded as will the current diabetes treatment 
(start date, product name(s), dose(s)). 
7.3.4 Concomitant medication 
A concomitant medication is any medication, other than the trial products and current 
diabetes treatment (including insulin glulisine [Apidra®] for diabetes therapy wash-out), 
which is taken during the trial, including screening and follow-up periods. 


 
 
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Details of any concomitant medication must be recorded at trial entry (i.e. at screening). 
Any changes in concomitant medication must be recorded at each visit as they occur. The 
information collected for each concomitant medication includes (at a minimum) trade 
name or generic name, indication, start date and stop date or continuation. A change in 
medication due to an AE must be recorded and reported according to Section 7.2.1. If the 
change in medication influences the subject’s eligibility to continue in the trial, the 
sponsor and trial monitor must be informed. 
7.4 
Other assessments 
7.4.1 Immunogenicity 
Antibodies against dasiglucagon/GlucaGen will be measured at the dosing visit (Visit 2) 
and at follow-up (Visit 3). At the dosing visit (Visit 2), samples will be collected prior to 
the start of the insulin-induced hypoglycemic procedure.  
The clinical ADA assays, specific for dasiglucagon and GlucaGen, respectively, have 
been validated in accordance with existing guidelines and recommendations (17-21).  
Confirmed positive anti-dasiglucagon antibody samples (treatment-induced or 
treatment-boosted) from anti-dasiglucagon antibody-positive subjects will be evaluated 
for binding titer neutralizing potential and titer as well as cross-reactivity towards 
endogenous glucagon.  
No further serum sampling will be needed as the ADA samples can be used for 
neutralizing antibody analysis. 
The in vitro neutralizing effect of the antibodies will be measured using an assay based 
on glucagon receptor transfected human embryonic kidney cells (20,22). The calculated 
sensitivity in previous studies was about 51.8 ng/mL. The assay was also validated for 
recombinant glucagon with similar results (21,23). In case of a positive result in the 
neutralizing antibody assay, a titer estimation will be performed. The cell-based 
neutralizing antibody analyses will be performed by a special laboratory, BioAgilytix, 
Durham, NC, USA. 
Residual and additional antibody serum samples may be stored until approval of market 
authorization by the health authorities. Further characterization of the antibody response 
may be requested by the health authorities. 


 
 
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7.4.2 Plasma glucose measurements for safety 
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at 
site. After the start of insulin infusion, plasma glucose should be checked every 
10 minutes while plasma glucose is above 110 md/dL, and every 5 minutes once plasma 
glucose is at or below 110 mg/dL and until after dosing when the subject’s plasma 
glucose is ≥70mg/dL (3.9 mmol/L). Hereafter, plasma glucose should be checked every 
30 minutes until 300 minutes (5 hours) after dosing. Plasma glucose concentrations will 
be measured using a US FDA-approved glucose analyzer (e.g., YSI 2300, Yellow 
Springs Instruments, Yellow Springs, OH). 
At the discretion of the investigator, additional plasma glucose measurements can be 
taken at any time during the trial, for example when there is a suspicion (e.g. symptoms) 
of a hypoglycemic episode. 
Plasma glucose measurements for safety should only be recorded in the eCRF if they are 
related to an AE (e.g. a hypoglycemic episode). 
In case of persistent post-treatment hypoglycemia, subjects will receive rescue treatment 
with an IV glucose infusion (see Section 8.2.3.1 for details). Blood samples for PD and 
PK assessments should still be taken at the specified timepoints. 
7.4.3 Plasma insulin measurements 
Samples for insulin assessment will be collected at the dosing visit (Visit 2, pre-dose and 
at 30 and 60 minutes after dosing). The actual time of blood sampling for evaluation of 
plasma insulin should not deviate from the nominal time by more than ±1 minute. 
Pre-dose is defined as within 2 minutes prior to dosing. 


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8 
TRIAL CONDUCT 
8.1 
Schedule of procedures 
The schedule of procedures is provided in Table 3. Informed consent will be obtained 
prior to any trial-related procedures; see Section 10.8. 
8.2 
Procedures by visit 
8.2.1 Visit 1 (screening, Day -30 to Day -3) 
Visit 1 will take place between 3 and 30 days before Visit 2, Day -1 to Day 1 (dosing 
day). 
Informed consent can be obtained prior to or at Visit 1, however it must in any case be 
obtained prior to any trial related procedures. During the screening visit, the following 
assessments will take place: 

Informed consent (obtain or check)

Check of subject eligibility (inclusion/exclusion criteria)

Demography

Body measurements

Medical history, diabetes diagnosis, current diabetes treatment

Concomitant illnesses

Concomitant medications

History of alcohol/drug abuse

Physical examination

Vital signs

12-lead ECG

AEs

Biochemistry, hematology, coagulation, HbA1c

Serum pregnancy test (women of childbearing potential only)

Urinalysis

Urine drug screen

Alcohol breath test


 
 
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Table 3 
Schedule of procedures 
Visit number 
V1 
V2 
V3 
Trial day 
-3 
-1 and 1 
28 
Visit type 
Screening 
Dosing 
Follow-up 
Window 
-30 to -3 
 
+5 days 
Subject related information/assessments 
 
 
 
Informed consent 
x 
 
 
Inclusion/exclusion criteria 
x 
x1,2 
 
Demography 
x 
 
 
Body measurements 
x 
 
 
Medical history, diabetes diagnosis, and current diabetes 
treatment 
x 
 
 
Concomitant illnesses 
x 
 
 
Concomitant medications 
x 
x1 
x 
History of alcohol/drug abuse 
x 
 
 
Randomization 
 
x1 
 
Withdrawal criteria 
 
x1 
 
Dosing day exclusion criteria 
 
x1 
 
Insulin-induced hypoglycemia 
 
x 
 
Safety assessments 
 
 
 
Physical examination 
x 
 
x 
Vital signs 
x 
x3 
x 
12-lead ECG 
x 
x4 
x 
Local tolerability  
 
x5 
x 
Adverse events 
x 
x 
x 
Laboratory 
 
 
 
Biochemistry, hematology, coagulation, HbA1c (HbA1c at 
Visit 1 only) 
x 
x6 
x 
Pregnancy test (women of childbearing potential only) 
x7 
x1,8 
x8 
Urinalysis 
x 
x1 
x 
Urine drug screen 
x 
x1 
 
Alcohol breath test 
x 
x1 
 
PK/Clinical efficacy 
 
 
 
Plasma dasiglucagon/GlucaGen 
 
x9 
 
Plasma glucose 
 
x10 
 
Other assessments 
 
 
 
Antibodies against dasiglucagon/GlucaGen 
x1 
x11 
Plasma insulin 
 
x12 
 
Trial material 
 
 
 
Administration of trial product (during hypoglycemic clamp 
procedure) 
 
x 
 
1Prior to the start of the insulin-induced hypoglycemic procedure. 
2Only check of dosing day exclusion criteria and changes between screening visit and Visit 2. 
3Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 30, 90 and 300 minutes 
after dosing. The actual time of the assessment should not deviate from the nominal time by more than ±10 minutes. 


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4Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 20, 35, 45, 60 and 300 
minutes after dosing. The actual time of the assessment should not deviate from the nominal time by more than ±5 
minutes.  
5Local tolerability assessed at 30, 120, and 300 minutes after dosing. The actual time of the assessment should not 
deviate from the nominal time by more than ±10 minutes.  
6Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 30 and 300 minutes after 
dosing. The actual time of the assessment should not deviate from the nominal time by more than ±10 minutes. 
7Serum pregnancy test. 
8Urine stick pregnancy test. 
9Pre-dose, and at 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time of blood sampling should not 
deviate from the nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing. 
10Pre-dose, and at 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, 90 minutes after dosing. The actual time of blood 
sampling should not deviate from the nominal time by more than ±30 seconds until the 20 minute collection time point 
and by more than ±1 minute for the subsequent collection time points. Pre-dose is defined as within 2 minutes prior to 
dosing. 
11Any subject that tests positive for ADA will be monitored until the ADA levels return to baseline levels. 
12Pre-dose, and at 30 and 60 minutes after dosing. The actual time of blood sampling should not deviate from the 
nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing.  
ADA = anti-drug antibodies; ECG = electrocardiogram; HbA1c = glycated hemoglobin.
Eligible or potentially eligible subjects (laboratory results pending) will be provided with 
an Identification card (ID card), stating that the subject is participating in the trial and 
whom to contact (site address, investigator name and telephone number). The subjects 
should be instructed to return the ID card to the investigator at the last visit or to destroy 
the card after the last visit.  
8.2.2 Instructions to subjects prior to the dosing visit (Visit 2) 
At the screening visit, the investigator will inform the subject about the changes to his/her 
insulin therapy leading up to the start of the insulin-induced hypoglycemic procedure. 
The subject may be provided with insulin NPH in the wash-out period to cover the need 
of basal insulin, if deemed necessary by the investigator. The subject’s current insulin 
therapy will be washed out as defined in Section 5.5.: 48 hours prior to dosing and during 
the dosing visit, treatment with  insulin Degludec and insulin Glargine U300 are not 
allowed; 24 hours prior to dosing and during the dosing visit, treatment with other long-
acting insulins (e.g., insulin Glargine U100 or insulin Detemir) is not allowed; 16 hours 
prior dosing and during the dosing visit treatment with insulin NPH is not allowed; 6 
hours prior to dosing and during the dosing visit, treatment with any short acting (bolus) 
insulin, except insulin glulisine (Apidra®), is not allowed. The basal rate of insulin pumps 
(continuous SC insulin infusion) will be discontinued on the morning of the dosing day 
(if using insulin glulisine [Apidra®]) OR at least 6 hours prior to dosing (if using other 
insulins). 
On the day prior to dosing (Day -1), the subjects will need to attend the clinical center 
and will be required to stay onsite overnight. On the morning of the dosing day (Day 1), 
patients are required to be in a fasting condition, defined as having consumed only water 
since 22:00 hours the night before. However, the subjects are allowed to consume small 


 
 
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amounts (up to 20 g) of carbohydrates to prevent hypoglycemia. The subjects must also 
not consume any alcohol within 24 hours prior to dosing (refer to Section 5.5 for all 
dosing day exclusion criteria). 
8.2.3 Visit 2 (dosing visit) 
Visit 2 will take place on Day -1 to Day 1. 
The subjects will attend the clinical center the day prior to dosing (Day -1) and subject 
eligibility is rechecked (check of changes between the screening visit and Visit 2) and 
those subjects eligible to participate should stay onsite overnight. To target a glucose 
level around 90-110 mg/dL (5.0-6.1 mmol/L) the following morning, subjects may be 
administered insulin glulisine (Apidra®) at the investigator’s discretion either by IV 
infusion, SC bolus or continuous SC insulin infusion. Dosing will take place the 
following morning (Day 1). 
On Day 1 and prior to the start of the insulin-induced hypoglycemic procedure,  those 
subjects eligible to participate will be randomized to treatment with dasiglucagon, 
placebo, or GlucaGen.  
The following assessments will also take place: 
 Document all changes in concomitant medication (prior to the start of the 
insulin-induced hypoglycemic procedure) 
 Check of withdrawal criteria (prior to the start of the insulin-induced hypoglycemic 
procedure) 
 Check of dosing day exclusion criteria (prior to the start of the insulin-induced 
hypoglycemic procedure) 
 Vital signs (prior to the start [within 30 minutes] of the insulin-induced hypoglycemic 
procedure, and at 30, 90 and 300 minutes after dosing). 12-lead ECG (prior to the 
start [within 30 minutes] of the insulin-induced hypoglycemic procedure, and at 20, 
35, 45, 60, and 300 minutes after dosing) 
 Local tolerability (at 30, 120, and 300 minutes after dosing) 
 AEs  
 Biochemistry, hematology, coagulation (prior to the start of the insulin-induced 
hypoglycemic procedure [within 30 minutes], and at 30 and 300 minutes after dosing) 
 Urine stick pregnancy test (women of childbearing potential only; prior to the start of 
the insulin-induced hypoglycemic procedure) 
 Urinalysis (prior to the start of the insulin-induced hypoglycemic procedure [within 
120 minutes]) 
 Urine drug screen (prior to the start of the insulin-induced hypoglycemic procedure) 
 Alcohol breath test (prior to the start of the insulin-induced hypoglycemic procedure) 


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
Dasiglucagon/GlucaGen plasma concentrations:
- 
Pre-dose, 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time
of blood sampling for exposure should not deviate from the nominal time by more 
than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing. 

Plasma glucose concentrations:
- 
Pre-dose, 4, 6, 8 ,10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, and 90 minutes after 
dosing. The actual time for blood sampling for plasma glucose should not deviate 
from the nominal time by more than ±30 seconds until the 20 minute collection 
time point and by more than ±1 minute for the subsequent collection time points. 
Pre-dose is defined as within 2 minutes prior to dosing. 

Antibodies against dasiglucagon/GlucaGen (prior to the start of the insulin-induced
hypoglycemic procedure). 

Plasma insulin concentrations:
- 
Pre-dose, 30 and 60 minutes after dosing. The actual time of blood sampling for 
plasma insulin should not deviate from the nominal time by more than ±1 minute. 
Pre-dose is defined as within 2 minutes prior to dosing. 
8.2.3.1 Hypoglycemic clamp procedure and administration of trial medication 
The following procedure is based on precendent procedures for hypoglycemia induction 
in patients with T1DM [24, 25]. 
The treatment day (Visit 2, Day 1) will be conducted after an overnight fast of at least 
8 hours, targeting a starting plasma glucose around 90 to 110 mg/dL (5.0-6.1 mmol/L).  
Subjects who meet one or more of the dosing day exclusion criteria (Section 5.5) at the 
dosing visit will be excluded from the dosing visit, but the visit can be rescheduled 1-7 
days later. The dosing visit can only be rescheduled once. 
The subject’s current insulin therapy will be washed out as defined in Section 5.5. For 
subjects using multiple daily injections, the date, time and the dose of the last basal 
insulin and the last short-acting insulin (except insulin glulisine [Apidra®]) administration 
prior to dosing will be captured. For subjects using an insulin pump, the time of 
discontinuation of the basal rate will be captured. Any use of insulin glulisine (Apidra®) 
in the last 5 hours prior to initiation of the hypoglycemia induction procedure will also be 
captured.  
At approximately 08:00 hours, an infusion catheter will be inserted into each arm 
(forearm cephalic vein) for the manual glucose clamp procedure, with the glucose 
infusion in one arm and the insulin infusion in the opposite arm (if IV insulin glulisine 
[Apidra®] has been administered during the night the same infusion catheter can be used). 
A third catheter for blood sampling will be placed into a metacarpel vein for blood 


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sampling. This hand will be warmed (55-65ºC) to arterialize venous blood. If there are 
issues with blood sampling from the metacarpal vein for the purpose of glucose 
measurements, a new and more proximal IV access may be used at the discretion of the 
investigator. 
Hypoglycemia will be gradually induced by a fast-acting IV insulin glulisine (Apidra®) 
infusion (15 U [100 U/mL] in 49 mL saline and 1 mL of subject’s blood or plasma), 
initially at 150% of the subject’s usual basal rate and can be increased or decreased over a 
range of 0% to 200% as judged necessary by the investigator, to achieve a controlled 
decline in plasma glucose, targeting a plasma glucose level of 55 mg/dL (3.1 mmol/L).  
Plasma glucose concentrations will be measured using a US FDA-approved glucose 
analyzer (e.g., YSI 2300, Yellow Springs Instruments, Yellow Springs, OH). After the 
start of the insulin infusion, plasma glucose will be measured every 10 minutes while 
plasma glucose is above 110 mg/dL, and every 5 minutes once plasma glucose is at or 
below 110 mg/dL. 
Once the glucose concentration declines to <60 mg/dL (3.3 mmol/L), the insulin infusion 
will be stopped, and 5 min later plasma glucose concentration will be measured at the 
glucose analyzer and blood samples for baseline assessment of plasma glucose, 
dasiglucagon/GlucaGen PK, and insulin PK will be collected. The samples are the 
baseline samples and should be collected within 2 minutes before IMP administration. 

If plasma glucose is ≥45 mg/dL and <60 mg/dL (2.5-3.3 mmol/L), study treatment
(IMP) will be administered, defining time, t=0. The study treatment will be delivered
in the abdomen, buttock, or thigh (according to stratification) via SC injection, with
the subject lying in a lateral recumbent position.

If plasma glucose is <45 mg/dL (2.5 mmol/L), IV glucose solution will be
administered sufficient to raise plasma glucose to within the 45-60 mg/dL target
range. The run-in period will be adequately extended (at least 30 min) until the above
target is achieved and new baseline samples for plasma glucose,
dasiglucagon/GlucaGen PK, and insulin PK will be collected. Glucose should not be
infused within 10 minutes before IMP administration. If plasma glucose is not within
target range after the second attempt, the subject should be rescheduled for a new
treatment visit within 7 days (+ 2 days).
Administration of IMP should not occur earlier than 9:00 hours in the morning or later 
than 12:00 hours. 
As shown in Table 4, serial blood samples for glucose will be collected at t=0, 4, 6, 8, 10, 
12, 15, 17, 20, 25, 30, 40, 50, 60, 75 and 90 minutes post-dosing. Samples for assessing 
plasma dasiglucagon/GlucaGen concentration will be collected  at t=0, 15, 30, 35, 40, 50, 


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60, 90, and 120 minutes. Samples for assessing plasma insulin concentration will be 
collected at t=0, 30, and 60 minutes.  
Table 4 
Post-treatment blood sampling times 
Times 
0 
4 
6 
8 
10 
12 
15 
17 
20 
25 
30 
35 
40 
50 
60 
75 
90 
120 
Plasma glucose 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
PK 
Dasiglucagon/ 
GlucaGen 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
PK Insulin 
Y 
Y 
Y 
Refer to Section 7.2.7 for details of laboratory safety sampling and to Section 7.4.2 for 
details of blood glucose safety sampling.  
When the t=90-minute blood sampling for plasma glucose has been collected the subjects 
are allowed to eat moderately. Drinking of water is allowed ad libitum during the entire 
procedure. 
Hypoglycemia Rescue Provisions 
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at 
site for safety reasons as described in Section 7.4.2. Subjects should receive 
post-treatment rescue glucose infusion to ameliorate persistent hypoglycemia, as follows. 
1. Glucose infusion should be initiated if a subject experiences severe alarming
escalation of symptoms of hypoglycemia (e.g. symptoms suggesting a change in
consciousness) at any time during the trial; glucose infusion should be initiated
targeting a plasma glucose levels >70 mg/dL.
2. If plasma glucose is <45 mg/dL (2.5 mmol/L) between t=8 and t=44 minutes,
rescue glucose infusion (1-2 mg/kg administered IV over about 5 seconds) should
be initiated to maintain plasma glucose between 45 mg/dL and 55 mg/dL
(2.5-3.1 mmol/L). Pause glucose infusion if plasma glucose is >55 mg/dL.
3. If plasma glucose is <70 mg/dL at t=45 minutes, rescue glucose infusion
(2-3 mg/kg administered IV over about 5 seconds) should be initiated to maintain
plasma glucose between 70 mg/dL and 80 mg/dL (3.9-4.4 mmol/L). Pause
glucose infusion if plasma glucose is >75 mg/dL (4.2 mmol/L).
Subjects should remain in bed until completion of the test procedure 300 minutes after 
dosing (bathroom visits are allowed).  


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The IMP will be administered SC according to Section 6.3. The time of IMP 
administration will be recorded. At the timepoint when the insulin infusion is stopped, the 
total insulin dose which was required to induce hypoglycemia will be recorded. 
AEs will be specifically recorded during the procedure at several timepoints. 
The investigator must provide information to the subjects on how to resume their usual 
diabetes treatment. 
The subject may be released from the clinical center if the investigator does not have any 
safety concerns based on the last safety plasma glucose value and the general condition of 
the subject. However, at the discretion of the investigator or on request of the subject, the 
subject may stay at the trial center for a longer period. 
8.2.4 Visit 3 (follow-up visit) 
Visit 3 will take place on Day 28 + 5 days. The subject does not need to be fasting. 
At Visit 3, the following assessments will take place: 

Document all changes in concomitant medication

Physical examination

Vital signs

12-lead ECG

Local tolerability

AEs

Biochemistry, hematology, coagulation

Urine stick pregnancy test (women of childbearing potential only)

Urinalysis

Antibodies against dasiglucagon/GlucaGen.
After the follow-up visit the End of Trial form must be completed. Even if a subject is not 
able to attend the follow-up visit, the End of Trial form, the eCRF 
Accountability/Affirmation Statement form and the Drug Accountability form must be 
completed. 


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9 
STATISTICAL METHODS 
Before database lock and treatment unmasking, a separate statistical analysis plan (SAP) 
will be finalized, providing detailed methods for the analyses outlined below. Further 
analysis details may be added or refined in the SAP. 
Any deviations from the planned analyses will be described and justified in the final 
clinical trial report. 
9.1 
Determination of sample size 
Due to requirements in the size of the safety database, the sample size is set to 78 subjects 
treated with dasiglucagon 0.6 mg. With a 2:1:1 randomization ratio for the three 
treatments, dasiglucagon 0.6 mg, placebo, and GlucaGen 1 mg, 78 subjects injected with 
dasiglucagon and 39 subjects with placebo will yield a power of 90% at a 5% two-sided 
significance level to detect a treatment group difference in recovery incidence within 
20 minutes, assuming as low as 80% and as high as 50% recovery incidence for the 
dasiglucagon and placebo groups, respectively.
9.2 
Trial subjects 
9.2.1 Analysis samples 
For presentation of data and reporting of the statistical analyses, the following analysis 
samples will be used, depending on the context:  

Safety analysis set (SAS): All randomized subjects who received at least one dose of
trial medication.

Full analysis set (FAS): All randomized subjects who received at least one dose of
trial medication and contributed valid information for at least one post-dose endpoint.

Per protocol (PP) set: All subjects of the FAS for whom no relevant protocol
deviations were documented. This sample will primarily be used for sensitivity
analysis.
The analysis of the primary endpoint and secondary endpoints will be based on the FAS. 
A secondary analysis of the primary endpoint will be based on the PP set. All safety 
analyses will be based upon the SAS. 
The decision regarding whether a protocol deviation is relevant or not for the exclusion of 
subjects from the PP set will be made case-by-case in a data review meeting prior to 
treatment unmasking and database lock (see Section 9.2.3). 


 
 
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9.2.2 Disposition of subjects 
Subject disposition will be tabulated including the number of screened subjects, screening 
failures, subjects exposed to trial product, subjects completing the trial and subjects in 
each analysis set. 
Subjects withdrawn from the trial will be listed including the primary reason for 
withdrawal. 
9.2.3 Protocol deviations 
Before data are released for statistical analysis, a treatment-masked review of all data will 
take place to identify protocol deviations that may potentially affect the results. This 
review will be performed without revealing to which trial product the subjects were 
assigned. The masking of the trial products will be maintained for everyone involved in 
allocating subjects to the analysis sets until data are released for statistical analysis. 
Furthermore, spurious outliers will be evaluated. In addition, protocol deviations that may 
potentially affect the results will be identified and it will be evaluated if subjects and/or 
data should be excluded from the analysis. Protocol deviations will be classified as minor 
or major in a consistent way. Major deviations from the protocol may lead to the 
exclusion of a subject from the PP set. 
Major protocol deviations may include deviations related to trial inclusion or exclusion 
criteria, conduct of the trial, subject management or subject assessment. Unless explicitly 
decided otherwise during the masked data review, the following will be considered major 
protocol deviations: 
 Violation of one or more major inclusion/exclusion criteria 
 Interruption of administration of trial product 
 Significant deviation from time windows  
 Incorrect treatment allocation 
 Missing primary endpoint. 
The violation of several major inclusion/exclusion criteria or the complete absence of 
efficacy data might lead to exclusion of the subject from FAS. In that case, the decision 
should be taken at the masked data review meeting, and the exclusion from efficacy 
analysis justified in the signed notes of the meeting. 
Obviously erroneous data points may be excluded from the analyses or re-analyzed (in 
case of e.g. serum concentrations). The decision to re-analyze or exclude data points from 
the statistical analysis is the joint responsibility of the sponsor and the trial statistician.  


 
 
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The subjects or observations to be excluded and the reason for their exclusion will be 
documented and signed by those responsible prior to database release. The 
documentation will be stored together with the remaining trial documentation. The 
subjects and observations excluded from analysis sets, and the corresponding reasons, 
will be described in the clinical trial report. 
9.3 
General considerations 
All data obtained in this trial and documented in the eCRFs will be listed and 
summarized with statistics or frequency tables as appropriate. In case of termination of 
the trial, all data collected up to that timepoint will be included in the analysis. 
Raw data listings and summary tables will be generated using the software SAS© version 
9.4 or higher.  
Continuous variables will be summarized using means, standard deviations, medians, 
coefficients of variation, and minimum and maximum values.  
Other summaries (e.g. quartiles, 95% confidence intervals [CIs]) may be used as 
appropriate. Categorical efficacy and safety variables will be summarized by counts and 
by percentage of subjects in corresponding categories. 
9.4 
Demographics and baseline characteristics 
Baseline and demographic data will be summarized using descriptive statistics. Baseline 
ADA-positive subjects will be calculated as a percentage of the total number of subjects 
whose baseline samples were tested for ADA. 
All other data obtained in this trial and documented in the eCRF will be listed. 
9.5 
Efficacy Analyses 
For the confirmatory analyses, the primary and key secondary endpoints for the 
dasiglucagon 0.6 mg and placebo treated subjects will be compared. A hierarchical 
procedure will be applied for the control of multiplicity. The primary and secondary 
endpoints will be evaluated on the full analysis set. In the primary analysis, those subjects 
who require rescue IV glucose will be censored at the time to plasma glucose recovery.  
In a sensitivity analysis, the time to plasma glucose recovery will be analyzed without 
censoring the subjects who received rescue IV glucose.   


 
 
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9.5.1 Hierarchical testing procedure 
For the confirmatory analyses, the following a priori defined hierarchical inferential test 
order will be applied for the control of the type 1 error rate across the planned multiple 
comparisons. The primary endpoint and key secondary endpoints will be inferentially 
evaluated within the FAS in the following order, where inference will proceed at the 
two-sided 0.05 criterion significance level until the first failure to reject the null 
hypothesis for a dasiglucagon versus placebo comparison: 
 Primary:  Time to plasma glucose recovery 
 Key secondaries 1-4: Plasma glucose recovery within 30 minutes, within 20 minutes, 
within 15 minutes, and within 10 minutes after study drug injection without 
administration of rescue IV glucose. 
 Key secondaries 5-8:  Plasma glucose changes from baseline (CFB) within 30 
minutes, within 20 minutes, within 15 minutes, and within 10 minutes after study 
drug injection or at the time of rescue. 
The GlucaGen versus placebo comparisons will not be included in the inferential testing 
hierarchy, since the efficacy of GlucaGen is previously established, and these 
comparisons are intended to support the validity of the study for the dasiglucagon versus 
placebo comparisons. 
Analogous supportive sensitivity analyses will be conducted in the PP set, but without 
inference intent. 
9.5.2 Primary confirmatory endpoint 
Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase in 
plasma glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic 
clamp procedure without administration of rescue IV glucose.  
9.5.2.1 Primary analysis 
The primary endpoint will be summarized using Kaplan-Meier (KM) estimates stratified 
by treatment group and injection site. The treatment group difference between 
dasiglucagon and placebo will be evaluated inferentially using a pairwise two-sided 
log-rank test. 
In the primary analysis, those subjects who require rescue IV glucose will be censored at 
the time of plasma glucose recovery. This approach is based on the assumption that 
rescue IV glucose administration will shorten the time to recovery. This assumption 
implies that censoring at the time of recovey after rescue therapy is valid as a time to 


 
 
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recovery without rescue would be longer than the observed duration. If recovery has not 
occurred at 45 minutes after study drug injection, censoring will be applied irrespective 
of the use of rescue IV glucose.  
In sensitivity analyses, the time to plasma glucose recovery will be analyzed 1) without 
censoring at the time of recovery for those subjects who require rescue IV glucose before 
45 minutes and 2) with censoring at the time of administration of rescure IV glucose 
before 45 minutes.   
The primary endpoint will additionally be analyzed using a Cox proportional hazards 
(CPH) time to event statistical model, if the hazards by treatment group can be assumed 
proportional using a graphical comparison of log-minus-log survival curves and no more 
than two-thirds of the recovery times are censored in each treatment group. The CPH 
model will be used for inferences, with treatment group and injection site modeled as 
categorical effects, and baseline plasma glucose modeled as a continuous covariate. The 
treatment group rate ratios, active versus placebo, will be estimated together with the 
95% confidence intervals, and pairwise treatment group inferences (dasiglucagon vs 
placebo, GlucaGen vs placebo) will be evaluated using two-sided likelihood ratio tests. 
9.5.3 Secondary endpoints 
 Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection without administration of rescue IV 
glucose. 
 Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection or at the time of rescue. 
9.5.3.1 Confirmatory analysis 
The key secondary incidence variables (plasma glucose recovery within 30 minutes, 
within 20 minutes, within 15 minutes, within 10 minutes, in hierarchical order for 
inference) will be analyzed using descriptive statistics (frequency and percentage) by 
treatment group. Inferential treatment group comparisons will be assessed via pairwise 
tests of independent binomial proportions (dasiglucagon versus placebo, GlucaGen 
versus placebo) using Fisher’s exact test for each pairwise comparison. 
The key secondary endpoints of plasma glucose CFB within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection or at the 
time of rescue, will be analyzed with the plasma glucose CFB at rescue carried forward in 
those subjects who require rescue IV glucose before plasma glucose ≥20 mg/dL recovery. 
Each of these CFB variables will be analyzed using an Analysis of Covariance model, 
with treatment group modeled as a fixed effect and with the baseline plasma glucose 
modeled as a covariate. The dasiglucagon versus placebo treatment group difference will 


 
 
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be evaluated inferentially as a least squares means contrast, using a two-sided t-test at the 
0.05 signficance level, first for the 30 minute CFB, and then sequentially for the 
20 minute, 15 minute, and 10 minute variables, until the first failure to reject. 
9.5.4 Secondary clinical efficacy (PD) endpoints 
 Time to first plasma glucose concentration of ≥70 mg/dL (3.9 mmol/L) without 
administration of rescue IV glucose. 
 Plasma glucose response as area under the curve above baseline from time zero to 
30 minutes, AUC0-30min. 
Secondary clinical efficacy endpoints will be assessed based on plasma concentration 
data from samples collected at the dosing visit (Visit 2).  
Unless otherwise stated, the population base of analysis will be the FAS. 
9.5.4.1 Analysis of secondary clinical efficacy (PD) endpoints 
1. Time to first plasma glucose concentration ≥70 mg/dL from baseline. This 
time-to-event endpoint will be evaluated using a Kaplan-Meier approach, with 
treatment group as a stratification factor, analogous to that used for the primary 
endpoint analysis. Differences between the KM curves (dasiglucagon versus 
placebo, GlucaGen versus placebo) will be evaluated inferentially using pairwise 
two-sided log rank tests. If the ≥70 mg/dL endpoint is not met within 45 minutes 
post-dosing, the time of the last valid plasma glucose measurement up to 45 
minutes will be the censoring time.  
2. The AUC will be calculated as the baseline-adjusted area under the plasma 
glucose profile over time: 
a. AUC0-30min: restricting the time window to the 0 to 30 minutes interval. 
3. The log-transformed AUC endpoint will be analyzed using an analysis of 
covariance (ANCOVA) model with treatment as fixed effect and baseline plasma 
glucose modeled as a covariate. The least squares means treatment group 
differences will be back-transformed (anti-logged) for presentation as a ratio of 
the treatment group geometric means, with their corresponding 95% CI. 
9.5.5 Exposure (PK) endpoints 
 Plasma dasiglucagon and GlucaGen concentrations from time zero to 90 minutes:  
AUC0-90min, Cmax, and tmax. 


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9.5.5.1 Analysis of exposure (PK) endpoints 
AUC will be derived as the area under the individual plasma dasiglucagon/GlucaGen 
concentration profile for PK from 0 to 90 minutes or last valid measurement if this 
measurement is assessed sufficiently close to 90 minutes (decision to be taken at the 
masked data review meeting). The standard trapezoidal method will be used, based on 
actual rather than nominal timepoints.  
Cmax will be determined as the maximum of all valid plasma dasiglucagon/GlucaGen 
concentrations.  
Tmax will be determined as the timepoint where the maximum of all valid plasma 
dasiglucagon/GlucaGen concentration measurements for each measurement series is 
observed. 
The log-transformed PK endpoints AUC and Cmax will be analyzed in the same way as 
the AUC endpoints. 
As tmax is a highly discrete endpoint, Wilcoxon’s rank sum test for unpaired observations 
will be used to assess differences between the two treatment groups. 
9.6 
Exploratory analyses 
Exploratory analyses will include descriptive statistics and modeling analogous to that 
done for key secondary endpoints. However, treatment group comparisons will be 
summarized without inference intent. 

Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL
(1.1 mmol/L) within 30 minutes after study drug injection without administration
of rescue IV glucose.

Plasma insulin response as area under the curve above baseline from time zero to
60 minutes, AUC0-60 min.
Plasma insulin concentrations measured pre-dose and at 30 and 60 minutes after dosing 
(see Table 3) will be presented individually. A summary table per timepoint will be 
provided. The AUC0-60min will be determined and a summary presented.  


 
 
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9.7 
Safety analyses 
9.7.1 Adverse events 
AEs will be coded using the latest available version of the MedDRA, the version of 
which will be provided in the clinical trial report. AE summary tables will include the 
number and percentage of subjects who experienced AEs summarized by system organ 
class and preferred term. 
An overall summary table will be provided showing the number and percentage of 
subjects with any: 
 TEAE 
 Severe TEAE 
 Serious TEAE 
 Drug-related TEAE 
 Drug-related severe TEAE 
 Drug-related serious TEAE 
 TEAE leading to withdrawal 
 TEAE with outcome death 
 AESI 
Refer to Section 7.2.1 for the definition of TEAEs. 
9.7.2 Immunogenicity data 
Immunogenicity data will be analyzed descriptively by treatment group. No statistical 
tests are planned. Baseline ADA-positive subjects will be calculated as a percentage of 
the total number of subjects whose baseline samples were tested for ADA. Titer will be 
reported as median and interquartile range. 
9.7.3 Clinical laboratory assessments 
Clinical laboratory test results will be flagged as to whether the result is below, within or 
above the respective reference range. The number of values outside of the reference range 
will be counted. 
9.7.4 Other safety data 
Incidence of rescue infusion of IV glucose during the hypoglycemic clamp procedure will 
be analyzed using descriptive statistics (frequency and percentage) by treatment group. 
Inferential treatment group comparisons will be assessed via pairwise tests of 


 
 
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independent binomial proportions (dasiglucagon versus placebo, GlucaGen versus 
placebo). 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure 
will be evaluated using a KM time to event statistical model, with treatment group and 
injection site as stratification factors. Differences between the KM curves (dasiglucagon 
versus placebo, GlucaGen versus placebo) will be evaluated inferentially using pairwise 
two-sided stratified log-rank tests. If the endpoint is never met, the time of the last plasma 
glucose measurement will be the censoring time. 
Vital signs, physical examination, 12-lead ECG and local tolerability data will be 
summarized using descriptive statistics. 
9.8 
Treatment compliance 
Trained unblinded members of staff will perform all administrations of the IMP at the 
trial center. The administered doses will be recorded in the blinded Drug Accountability 
form in the eCRF. 
PK assessments will support the surveillance of compliance with IMP administration. 
9.9 
Subject withdrawals and missing data 
In the case of subject withdrawal, no imputation of values for PK or PD measurements 
will be done. Analyses will be done on valid cases only, i.e., no imputation techniques 
such as last observation carried forward will be applied. For the primary analysis in the 
FAS, missing values for the primary endpoint will be imputed by a conservative rule 
considering any missing value as a failure. 
9.10 Interim analyses 
No interim analyses are currently planned. 


 
 
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10 
ETHICAL, LEGAL, AND ADMINISTRATIVE ASPECTS 
10.1  Quality assurance 
The sponsor or designee will conduct a site visit to verify the qualifications of each 
investigator, inspect the facilities, and inform the investigator of responsibilities and the 
procedures for ensuring adequate and correct documentation. 
The investigator is required to prepare and maintain adequate and accurate case histories 
designed to record all observations and other data pertinent to the trial for each trial 
participant. All information recorded on the eCRFs for this trial must be consistent with 
the subjects’ source documentation (i.e. medical records). 
The investigator will permit trial-related monitoring, IRB/IEC review, and regulatory 
inspections, providing direct access to source data /documents. Sponsor-authorized 
quality assurance personnel may carry out audits for which the investigator must provide 
support. 
The trial monitor or a representative of the sponsor will cross-check the data entered in 
the eCRFs with the source data at the trial center and observe the trial procedures in order 
to verify adherence to the trial protocol. Any queries will be resolved by the investigator 
or his/her delegate. 
All of the clinical data will be captured via electronic data capture (EDC) using a 
web-based tool.  
The investigator center staff will enter and edit the data via a secure network, with secure 
access features (username and password). A complete electronic audit trail will be 
maintained. The investigator will approve the data using an electronic signature (Ref: 21 
CFR Part 11), and this approval is used to confirm the accuracy of the data recorded. 
eCRFs will be used for all subjects. The investigator’s data will be accessible from the 
investigator’s center throughout the trial. The eCRFs must be kept current to reflect 
subject status at each phase during the course of the trial. The eCRF will not capture 
personalized data. The investigator must make a separate confidential record of 
personalized details (name and initials) on the subject identification and enrollment log. 
All changes to data are made by the investigator or his/her delegate through the EDC 
system.  
It is the responsibility of the principal investigator of the respective center to ensure that 
all subject discontinuations or changes in trial or other medications entered on the 
subject’s eCRF are also made on the subject’s medical records. 


 
 
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The eCRFs for any subject leaving the trial should be completed at the time of the final 
visit or shortly thereafter. 
10.2  Electronic case report forms 
Remote data capture software will be used for data collection. Following training, trial 
staff will be given access to the software. Access to the software is restricted to staff 
participating in the trial and the extent of access will depend on the participants' user role 
in the trial. 
The subjects enrolled into the trial will be identified in the database by subject number 
and trial identification code. The investigator or delegate will enter subject data into the 
eCRF promptly. All data is to be entered in English. Data recorded in the eCRFs will be 
accessible to the trial staff throughout the trial. 
After data entry, systematic data validation will be performed and data entry 
discrepancies will be presented electronically directly to the center staff. Queries for 
discrepant data may be generated automatically by the software upon entry and/or 
generated manually by the trial monitor or the trial data manager. All queries, whether 
generated by the system or by trial staff, will be in electronic format. 
All sections of the eCRF are to be electronically approved by the investigator or a 
medically qualified delegate after the data has been entered and all queries have been 
resolved. Changes to any eCRF page subsequent to the approval require a new approval 
signature. 
All queries and changes/corrections to the data are documented in the eCRF. 
10.3  Access to source data 
During the course of the trial, a trial monitor will make site visits to review protocol 
compliance, compare eCRFs with individual subject’s medical records, assess drug 
accountability, and ensure that the trial is being conducted according to pertinent 
regulatory requirements. 
Checking of the eCRFs for completeness and clarity, and cross-checking with source 
documents, will be required to monitor the progress of the trial. Moreover, regulatory 
authorities of certain countries, IRBs/IECs, and/or the sponsor may wish to carry out such 
source data checks and/or on-site audit inspections. Direct access to source data will be 
required for these inspections and audits; they will be carried out giving due 
consideration to data protection and medical confidentiality. 


 
 
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10.4  Source documentation 
All source documents from which eCRF entries are derived should be placed in the 
subject’s medical records. If data are to be entered directly into the eCRF this must be 
specified in a source data agreement prior to the start of the trial. 
eCRF entries will be verified with source documentation. The review of medical records 
will be performed in a manner to ensure that subject confidentiality is maintained. 
The trial monitor will check the eCRF for accuracy and completion and perform source 
data verification. The trial monitor will document source data verification of all reviewed 
sections of the eCRF. 
10.5  Data processing 
The trial is run as an EDC trial, i.e. all relevant data is entered by the centers directly into 
the clinical database. The eCRF is designed to capture all required information in 
compliance with GCP standards. 
10.6  Archiving trial records 
According to ICH guidelines, essential documents should be retained for a minimum of 
2 years after the last approval of a marketing application in an ICH region and until there 
are no pending or contemplated marketing applications in an ICH region or at least 
2 years have elapsed since the formal discontinuation of clinical development of the 
investigational product. However, these documents should be retained for a longer period 
if required by the applicable legal requirements. 
Records and documents pertaining to the conduct of the trial and the distribution of the 
investigational product (e.g. informed consent forms, laboratory slips, medication 
inventory records, and other pertinent information) must be retained by the investigator 
according to local requirements. 
10.7  Good clinical practice 
The procedures set out in this trial protocol are designed to ensure that the sponsor and 
investigator abide by the principles of the GCP guidelines of the ICH (1), and of the 
Declaration of Helsinki (2008) (2). The trial also will be carried out in keeping with local 
legal requirements.  


 
 
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10.8  Informed consent 
Before each subject is admitted to the trial, written informed consent will be obtained 
from the subject according to the regulatory and legal requirements of the participating 
country. This consent form must be dated and retained by the investigator as part of the 
trial records. The investigator will not undertake any investigation specifically required 
only for the clinical trial until valid consent has been obtained. The terms of the consent 
and when it was obtained must also be documented in the eCRF. 
The investigator will explain to each subject orally and in writing (subject information 
sheet) the nature, significance, risks and implications of the trial before inclusion. In 
particular, the subjects will be informed about the following: 
 The possibility of withdrawing from the clinical trial at any time by revoking the 
consent and without any resulting disadvantage. 
 How personal and health-related data will be collected and used during the trial. 
 That his/her medical records may be examined by authorized monitors or clinical 
quality assurance auditors appointed by the sponsor, by appropriate IRB/IEC 
members, and by inspectors from regulatory authorities. 
All subjects will receive a copy of the subject information sheet and a copy of their 
signed and dated informed consent form, both of which will be in the subject’s local 
language. 
If a protocol amendment is required, the informed consent form may need to be revised to 
reflect the changes to the protocol. If the consent form is revised, it must be reviewed and 
approved by the appropriate IEC/IRB, and signed by all subjects subsequently enrolled in 
the trial as well as those currently enrolled in the trial. 
10.9  Protocol approval and amendment 
Before the start of the trial, the trial protocol and/or other relevant documents will be 
approved by the IEC/IRB/competent authorities, in accordance with local legal 
requirements. The sponsor must ensure that all ethical and legal requirements have been 
met before the first subject is enrolled in the trial. 
This protocol is to be followed exactly. To alter the protocol, amendments must be 
written, receive approval from the appropriate personnel, and receive IRB/IEC/competent 
authority approval prior to implementation (if appropriate).  


 
 
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Administrative changes (not affecting the subject benefit/risk ratio) may be made without 
the need for a formal amendment. All amendments will be distributed to all protocol 
recipients, with appropriate instructions. 
10.10  Duration of the trial 
The maximum duration of the trial for each subject will be up to 63 days (including up to 
30 days for screening and up to 33 days until the follow-up visit).  
The trial will be closed when all subjects have completed Visit 3. 
10.11  Premature termination of the trial 
If the investigator, the sponsor (e.g. safety committee), or the safety medical monitor 
becomes aware of conditions or events that suggest a possible hazard to subjects if the 
trial continues, the trial may be terminated after appropriate consultation between the 
relevant parties. The trial may also be terminated early at the sponsor’s discretion in the 
absence of such a finding. 
Conditions that may warrant termination include, but are not limited to: 
 The discovery of an unexpected, significant, or unacceptable risk to the subjects 
enrolled in the trial 
 Failure to enroll subjects at an acceptable rate. 
 A decision on the part of the sponsor to suspend or discontinue development of the 
drug. 
The trial can be terminated prematurely by the sponsor at an individual center if: 
 The center cannot comply with the requirements of the protocol. 
 It is not possible for the center to comply with GCP standards. 
10.12  Confidentiality 
All trial findings and documents will be regarded as confidential. The investigator and 
members of his/her research team must not disclose such information without prior 
written approval from the sponsor. 
The anonymity of participating subjects must be maintained. Subjects will be identified 
on eCRFs submitted to the sponsor by their subject number. On the SAE reports and all 
other source documents, the subject will be identified via subject number. Documents not 


 
 
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to be submitted to the sponsor that identify the subject (e.g. the signed informed consent 
form) must be maintained in confidence by the investigator.  
10.13  Other ethical and regulatory issues 
If a significant safety issue is identified, either from an individual case report or review of 
aggregate data, then the sponsor will issue prompt notification to all parties: regulatory 
authorities, investigators and IRB/IECs. 
A significant safety issue is one that has a significant impact on the course of the clinical 
trial or program (including the potential for suspension of the trial program or 
amendments to protocols) or warrants immediate update of informed consent. 
10.14  Liability and insurance 
The sponsor will take out reasonable third-party liability insurance cover in accordance 
with all local legal requirements. The civil liability of the investigator, the persons 
instructed by him and the hospital, practice or institute in which they are employed and 
the liability of the sponsor with respect to financial loss due to personal injury and other 
damage that may arise as a result of the carrying out of this trial are governed by the 
applicable law. 
The sponsor will arrange for liability insurance if subjects should be injured due to the 
participation in the trial and provided that the sponsor is legally liable for that. 
Excluded from the insurance cover are injuries to health and deteriorations of illnesses 
already in existence which would have occurred or continued to exist even if the subject 
had not taken part in the clinical trial. 
The insurance cover is jeopardized if the subject fails to report immediately to the 
investigator or responsible physician any injury to health, which might have resulted 
from participation in the clinical trial, or if he/she undergoes any other medical treatment 
without their consent before the clinical trial has been completely finished in so far as the 
individual subject is concerned. 
Any injury to health, which might have occurred as a result of participation in the clinical 
trial must be reported by the subject to the investigator without delay. The investigator is 
obliged to make such a report in any case. 


 
 
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10.15  Publication policy 
By signing the trial protocol, the investigator agrees with the use of results of the trial for 
the purposes of national and international registration, publication and information for 
medical and pharmaceutical professionals. If necessary, the authorities will be notified of 
the investigator's name, address, qualifications and extent of involvement. 
A clinical trial report will be prepared and reviewed by the sponsor in co-operation with 
the coordinating investigator. The coordinatoing investigator will be appointed by 
Zealand Pharma to review and sign the clinical trial report on behalf of all participating 
investigators. A summary of the clinical trial report (according to CPMP/ICH137/95) 
should be sent to the regulatory authorities and IRB/IEC according to the relevant 
guidelines. 
According to the Declaration of Helsinki (2) investigators and sponsors ‘have ethical 
obligations with regard to the publication and dissemination of the results of research’.   
The trial design and results may be published as one or more original research 
manuscripts/abstracts and presented at a scientific meeting. The original eCRFs and the 
data generated from the eCRFs or otherwise obtained during the trial under this trial 
protocol will become the property of the sponsor. Publication of the results of this trial by 
the investigator is possible only after written consent has been obtained from the sponsor. 
Any material intended for publication will be given to the sponsor at least 4 weeks before 
submission for publication. The sponsor will have the right to comment on the intended 
publication and to take any reasonable measures for patent protection. Authorship on any 
publication(s) resulting from this clinical trial will be assigned according to the 
recommendations of the International Committee of Medicinal Journal Editors (26). 
Participating subjects will not be identified by name in any published reports about the 
clinical trial. 
The sponsor is responsible for trial registration at ClinicalTrials.gov 
(www.clinicaltrials.gov) according to FDA requirements, as well as the European 
Medicines Agency’s Clinical Trials Database (EudraCT).  


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11 
REFERENCE LIST 
1. ICH Harmonised Tripartite Guideline. International Conference on Harmonisation. 
ICH Harmonised Tripartite Guideline. Integrated addendum to ICH E6(R1): 
Guideline for Good Clinical Practice E6 (R2), Step 4. 09-Nov-2016. Available from: 
URL:
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E 
6/E6_R2__Step_4.pdf
2. World Medical Association. Declaration of Helsinki. Ethical Principles for Medical
Research Involving Human Subjects. 52nd WMA General Assembly, Edinburgh,
Scotland, October 2000. Last amended with Note of Clarification on Paragraph 29 by
the WMA General Assembly, Washington 2002, and Note of Clarification on
Paragraph 30 by the WMA General assembly, Tokyo 2004
3. Seaquist ER, Anderson J, Childs B, Cryer P, Dagogo-Jack S, Fish L, et al.
Hypoglycemia and diabetes: a report of a workgroup of the American Diabetes
Association and the Endocrine Society. Diabetes Care 2013 May;36:1384-95
4. Cryer PE. The barrier of hypoglycemia in diabetes. Diabetes 2008;57:3169-76
5. American Diabetes Association Position Statement. Standards of medical care in
diabetes - 2017. Diabetes Care 2017;40(Supplement 1):S1-S135
6. Davies MJ, Gagliardino JJ, Gray LJ, Khunti K, Mohan V, Hughes R. Real-world
factors affecting adherence to insulin therapy in patients with Type 1 or Type 2
diabetes mellitus: a systematic review. Diabetic Medicine 2013;30:512-24
7. International Hypoglycaemia Study Group. Glucose concentrations of less than 3.0
mmol/L (54 mg/dL) should be reported in clinical trials: a joint position statement of
the American Diabetes Association and the European Association for the Study of
Diabetes. Diabetes Care 2017;40(1):155-57
8. Christensen M, Bagger JI, Vilsbøll T, Knop FK. The alpha-cell as target for type 2
diabetes therapy. Rev Diabet Stud 2011;8:369-81
9. Lund A, Bagger JI, Christensen M, Knop FK, Vilsbøll T. Glucagon and type 2
diabetes: the return of the alpha cell. Curr Diab Rep 2014;14:555
10. Habegger KM, Heppner KM, Geary N, Bartness TJ, DiMarchi R, Tschöp MH. The
metabolic actions of glucagon revisited. Nat Rev Endocrinol 2010;6:689-97
11. Onoue S, Ohshima K, Debari K, Koh K, Shioda S, Iwasa S, et al. Mishandling of the
therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils. Pharm Res
2004;21:1274-83


 
 
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12. Fosgerau K, Hoffmann T. Peptide therapeutics: current status and future directions. 
Drug Discovery Today 2015;20:122-8 
13. Novo Nordisk Limited. SPC for GlucaGen Hypokit 1 mg. 2015. 
http://www.medicines.org.uk/emc/medicine/4258/SPC/GlucaGen+Hypokit+1+mg - 
Accessed 13 Mar 2017 . 
14. Zealand Pharma A/S. Dasiglucagon Investigator's Brochure, Edition 3, 12 Dec 2016.  
15. The Task Force for the management of arterial hypertension of the European Society 
of Hypertension (ESH) and of the European Society of Cardiology (ESC). 2013 
ESH/ESC guidelines for the management of arterial hypertension. European Heart 
Journal 2013;34:2159-219 
16. Levey AS, Coresh J, Greene T, Stevens LA, Zhang Y, Hendriksen BA, et al. Using 
standardized serum creatinine values in the modification of diet in renal disease study 
equation for estimating glomerular filtration rate. Annals of Internal Medicine 
2006;145:247-54 
17. Food and Drug Administration (FDA). Guidance for Industry on Bioanalytical 
Method Validation. Food and Drug Administration (FDA) 2001 
18. Viswanathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ, Sailstad J, et al. 
Quantitative bioanalytical methods validation and implementation: best practices for 
chromatographic and ligand binding assays. Pharm Res 2007 Oct;24:1962-73 
19. Shankar G, Devanarayan V, Amaravadi L, Barrett YC, Bowsher R, Finco-Kent D, et 
al. Recommendations for the validation of immunoassays used for detection of host 
antibodies against biotechnology products. J Pharm Biomed Anal 2008;48:1267-81 
20. Zealand Pharma report SS. Commissioning and validation of a method for the 
validation of anti-ZP4207 antibodies in human serum.  2014  
21. Zealand Pharma report SS. Commissioning and validation of a method for the 
validation of anti-glucagon antibodies in human serum.  2014  
22. Bioagilytix Labs. Validation of an assay to detect neutralizing antibodies to ZP4207 
by measuring cyclic adenosine monophosphate (cAMP) in a glucagon receptor 
transfected cell line, BAL-14-206-004-REP.  2016. Report No.: Zealand Pharma, 
study 15-039 
23. Bioagilytix Labs. Validation of an assay to detect neutralizing antibodies to glucagon 
by measuring cyclic adenosine monophosphate (cAMP) in a glucagon receptor 
transfected cell line, BAL-14-206-005.  2016. Report No.: Zealand Pharma, Study 
15-040 


 
 
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24. Amiel SA, Tamborlane WV, Simonson DC, et al. Defective glucose 
counterregulation after strict glycemic control of insulin-dependent diabetes mellitus. 
N Eng J Med 1987; 316: 1376-83. 
25. Rickels MR, Ruedy KJ, Foster NC, et al. Intranasal glucagon for treatment of insulin-
induced hypoglycemia in adults with type 1 diabetes: A randomized crossover 
noninferiority study. Diabetes Care 2016; 39: 264-70. 
26. ICMJE. Recommendations for the conduct, reporting, editing, and publication of 
scholarly work in medical journals. Updated December 2016 
 
 
 
 


 
 
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12 
APPENDICES 
12.1 List of trial personnel 
Sponsor 
Clinical Trial Manager 
 
 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Phone: 
 
Medical Officer 
 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Phone: 
 
Contract Research Organization 
Chiltern International (Ltd) 
171 Bath Road 
Slough 
Berkshire 
SL1 4AA 
United Kingdom 
Phone: +44 (0) 175351 2000 
Project Manager 
 
Chiltern International (Ltd) 
171 Bath Road 
Slough 
Berkshire 
SL1 4AA 
United Kingdom 
Phone: 
 
Senior Medical Officer and 
Safety medical monitor 
 
Chiltern International kft 
Canada Square Office House 
Ganz u 


 
 
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12-14, 4 emelet 
1027 Budapest 
Hungary 
Phone: 
 
Pharmacovigilance unit 
Responsible for Serious Adverse 
Event (SAE) Management and 24-
hour SAE reporting 
  
PharmaLex 
Agern Allé 24 
2970 Hørsholm, Denmark 
Phone: 
 (8 a.m. to 4 p.m.) 
Phone: 
 (outside 8 a.m. to 4 p.m.) 
Fax: 
 
email: PV-nordic@pharmalex.com  
Central laboratory 
MLM Laboratory 
Dohrweg 63 
41066 Mönchengladbach 
Germany 
Special laboratory (GlucaGen 
PK, insulin PK) 
MLM Laboratory 
Dohrweg 63 
41066 Mönchengladbach 
Germany 
Special laboratory (dasiglucagon 
PK, dasiglucagon ADA, 
GlucaGen ADA) 
York Bioanalytical Solutions (YBS)  
Cedar House 
Northminster Business Park 
Northfield Lane 
York, YO26 6QR, United Kingdom 
Special laboratory  
(neutralizing antibodies) 
BioAgilytix 
2300 Englert Drive 
Durham, NC, 27713, USA 
A list of all investigators, IECs and IRBs will be provided in a separate document and in 
the clinical trial report. 
 


 
 
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CLINICAL TRIAL PROTOCOL 
 
A phase 3, randomized, double-blind, parallel trial to 
confirm the clinical efficacy and safety of dasiglucagon 
in the rescue treatment of hypoglycemia in subjects with 
type 1 diabetes mellitus (T1DM) compared to placebo 
and with reference to GlucaGen® 
 
Sponsor: 
Zealand Pharma A/S 
Sponsor Protocol No.: 
ZP4207-16137 
EudraCT No.: 
2017-002449-31  
IND No: 
127866 
Trial Drug Name: 
Dasiglucagon* injection 
Date of Protocol: 
31 Aug 2017  
 
The trial will be conducted according to the protocol and in compliance with Good 
Clinical Practice (GCP), with the Declaration of Helsinki and with other applicable 
regulatory requirements. 
*Dasiglucagon is the proposed international nonproprietary name for ZP4207. 
The information in this document is confidential and is proprietary to Zealand Pharma. It is understood that 
information in this document shall not be disclosed to any third party, in any form, without prior written 
consent of an authorized officer of Zealand Pharma. 




 
 
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Declaration of the coordinating investigator 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
I have read this protocol. I approve this document and I agree that it contains all 
necessary details for carrying out the trial as described. I will conduct this trial in 
accordance with the design and specific provision of this protocol and will make a 
reasonable effort to complete the trial within the time designated. I will provide copies of 
this protocol and access to all information furnished by Zealand Pharma A/S to trial 
personnel under my supervision. I will discuss this material with them to ensure they are 
fully informed about the trial product and trial procedures. I will let them know that this 
information is confidential and proprietary to Zealand Pharma A/S and that it may not be 
further disclosed to third parties. I understand that the trial may be terminated or 
enrollment suspended at any time by Zealand Pharma A/S, with or without cause, or by 
me if it becomes necessary to protect the best interests of the trial subjects. 
I agree that the trial will be carried out in accordance with GCP (1), with the Declaration 
of Helsinki (with amendments) (2) and with the laws and regulations of the countries in 
which the trial takes place. 
 
 
_____________________________________ 
_____________________ 
Name  
 
 
 
 
 
 
Date 
Title 
Institution 
Phone: + 
Fax: 
+ 
 


 
 
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Declaration of the investigator 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
All documentation for this trial that is supplied to me and that has not been previously 
published will be kept in the strictest confidence. This documentation includes this trial 
protocol, Investigator's Brochure, Case Report Forms, and other scientific data. 
The trial will not be commenced without the prior written approval of a properly 
constituted Institutional Review Board (IRB) or Independent Ethics Committee (IEC). 
No changes will be made to the trial protocol without the prior written approval of the 
sponsor and the IRB or IEC, except where necessary to eliminate an immediate hazard to 
the subjects. 
I have read and understood and agree to abide by all the conditions and instructions 
contained in this protocol. 
 
_____________________________________ 
_____________________ 
Signature 
Date 
 
_____________________________________ 
Name (block letters) 
 
_____________________________________ 
Title (block letters) 
 
_____________________________________ 
Institution (block letters) 
 


 
 
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Table of contents 
DECLARATION OF SPONSOR OR RESPONSIBLE MEDICAL OFFICER .......... 2 
DECLARATION OF THE COORDINATING INVESTIGATOR .............................. 3 
DECLARATION OF THE INVESTIGATOR ............................................................... 4 
TABLE OF CONTENTS .................................................................................................. 5 
1 
SYNOPSIS ............................................................................................................ 11 
2 
INTRODUCTION................................................................................................ 15 
2.1 
Background ...................................................................................................... 15 
2.1.1 
Hypoglycemia ...................................................................................... 15 
2.1.2 
Glucagon .............................................................................................. 16 
2.1.3 
Dasiglucagon........................................................................................ 16 
2.1.3.1 
Pharmacokinetics and pharmacodynamics of dasiglucagon ......... 17 
2.1.3.2 
Safety of dasiglucagon ................................................................... 17 
2.2 
Trial rationale .................................................................................................. 18 
2.3 
Risk-benefit assessment .................................................................................. 18 
3 
TRIAL OBJECTIVES AND ENDPOINTS ...................................................... 20 
3.1 
Primary objective ............................................................................................ 20 
3.2 
Secondary objectives ....................................................................................... 20 
3.3 
Primary endpoint ............................................................................................ 20 
3.4 
Key secondary endpoints ................................................................................ 20 
3.5 
Other secondary endpoints ............................................................................. 20 
3.6 
Exploratory endpoints .................................................................................... 21 
4 
OVERALL DESIGN AND PLAN OF THE TRIAL ........................................ 22 
4.1 
Overview .......................................................................................................... 22 
4.2 
Justification of the trial design ....................................................................... 24 
4.2.1 
Justification for design and parameters ................................................ 24 
4.2.2 
Justification for drug, route, dosage and treatment duration ............... 24 
5 
TRIAL POPULATION ....................................................................................... 26 
5.1 
Rationale for trial population ........................................................................ 26 
5.2 
Planned sample size and number of trial centers ......................................... 26 
5.3 
Inclusion criteria ............................................................................................. 26 


 
 
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5.4 
Exclusion criteria ............................................................................................ 27 
5.5 
Dosing day exclusion criteria ......................................................................... 28 
5.6 
Premature treatment discontinuation and withdrawal ............................... 29 
5.6.1 
Possible reasons for treatment visit discontinuation ............................ 29 
5.6.2 
Center discontinuation ......................................................................... 30 
5.6.3 
Trial termination .................................................................................. 30 
5.7 
Subject identification and randomization ..................................................... 30 
6 
TRIAL DRUG ...................................................................................................... 31 
6.1 
Identity ............................................................................................................. 31 
6.2 
Treatment assignment and randomization ................................................... 32 
6.3 
Administration ................................................................................................. 33 
6.4 
Packaging and labelling .................................................................................. 33 
6.5 
Storage of study drugs .................................................................................... 33 
6.6 
Blinding and breaking the blind .................................................................... 34 
6.7 
Drug accountability ......................................................................................... 35 
6.8 
Treatment compliance .................................................................................... 35 
6.9 
Prior and concomitant medications ............................................................... 35 
6.9.1 
Prohibited medications ......................................................................... 36 
7 
PARAMETERS AND METHODS OF ASSESSMENT .................................. 37 
7.1 
Efficacy parameters ........................................................................................ 37 
7.1.1 
Pharmacodynamic measurements ........................................................ 37 
7.1.2 
Pharmacokinetic measurements ........................................................... 37 
7.2 
Safety parameters ............................................................................................ 38 
7.2.1 
Adverse events ..................................................................................... 38 
7.2.1.1 
Definitions ...................................................................................... 38 
7.2.1.2 
Collection, recording and reporting of adverse events ................. 41 
7.2.1.3 
Follow-up of adverse events .......................................................... 42 
7.2.1.4 
Clinical laboratory abnormalities and other abnormal 
assessments as adverse events or serious adverse events .............. 43 
7.2.2 
Hypoglycemia ...................................................................................... 43 
7.2.3 
Physical examination ........................................................................... 44 
7.2.4 
Vital signs ............................................................................................ 44 
7.2.5 
Electrocardiogram ................................................................................ 45 
7.2.6 
Local tolerability .................................................................................. 45 
7.2.7 
Clinical laboratory assessments ........................................................... 46 
7.2.8 
Pregnancy ............................................................................................. 47 
7.2.9 
Precautions ........................................................................................... 48 
7.2.10 
Safety committee ................................................................................. 48 
7.3 
Demography, concomitant illness, medical history and concomitant 
medication ........................................................................................................ 48 


 
 
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7.3.1 
Demography and body measurements ................................................. 48 
7.3.2 
Concomitant illness and medical history ............................................. 49 
7.3.3 
Diabetes diagnosis and current treatment ............................................ 49 
7.3.4 
Concomitant medication ...................................................................... 49 
7.4 
Other assessments ........................................................................................... 50 
7.4.1 
Immunogenicity ................................................................................... 50 
7.4.2 
Plasma glucose measurements for safety ............................................. 50 
7.4.3 
Plasma insulin measurements .............................................................. 51 
8 
TRIAL CONDUCT .............................................................................................. 52 
8.1 
Schedule of procedures ................................................................................... 52 
8.2 
Procedures by visit .......................................................................................... 52 
8.2.1 
Visit 1 (screening, Day -30 to Day -3) ................................................. 52 
8.2.2 
Instructions to subjects prior to the dosing visit (Visit 2) .................... 54 
8.2.3 
Visit 2 (dosing visit)............................................................................. 55 
8.2.3.1 
Hypoglycemic clamp procedure and administration of trial 
medication ...................................................................................... 56 
8.2.4 
Visit 3 (follow-up visit) ....................................................................... 58 
9 
STATISTICAL METHODS ............................................................................... 60 
9.1 
Determination of sample size ......................................................................... 60 
9.2 
Trial subjects ................................................................................................... 60 
9.2.1 
Analysis samples .................................................................................. 60 
9.2.2 
Disposition of subjects ......................................................................... 61 
9.2.3 
Protocol deviations ............................................................................... 61 
9.3 
General considerations ................................................................................... 62 
9.4 
Demographics and baseline characteristics .................................................. 62 
9.5 
Efficacy Analyses ............................................................................................. 62 
9.5.1 
Hierarchical testing procedure ............................................................. 63 
9.5.2 
Primary confirmatory endpoint ............................................................ 63 
9.5.2.1 
Primary analysis ............................................................................ 63 
9.5.3 
Secondary endpoints ............................................................................ 64 
9.5.3.1 
Confirmatory analysis .................................................................... 64 
9.5.4 
Secondary clinical efficacy (PD) endpoints ......................................... 65 
9.5.4.1 
Analysis of secondary clinical efficacy (PD) endpoints ................ 65 
9.5.5 
Exposure (PK) endpoints ..................................................................... 65 
9.5.5.1 
Analysis of exposure (PK) endpoints ............................................. 66 
9.6 
Exploratory analyses ....................................................................................... 66 
9.7 
Safety analyses ................................................................................................. 67 
9.7.1 
Adverse events ..................................................................................... 67 
9.7.2 
Immunogenicity data ........................................................................... 67 
9.7.3 
Clinical laboratory assessments ........................................................... 67 


 
 
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9.7.4 
Other safety data .................................................................................. 67 
9.8 
Treatment compliance .................................................................................... 68 
9.9 
Subject withdrawals and missing data .......................................................... 68 
9.10 
Interim analyses .............................................................................................. 68 
10 
ETHICAL, LEGAL, AND ADMINISTRATIVE ASPECTS .......................... 69 
10.1 
Quality assurance ............................................................................................ 69 
10.2 
Electronic case report forms .......................................................................... 70 
10.3 
Access to source data ...................................................................................... 70 
10.4 
Source documentation .................................................................................... 71 
10.5 
Data processing ................................................................................................ 71 
10.6 
Archiving trial records ................................................................................... 71 
10.7 
Good clinical practice ..................................................................................... 71 
10.8 
Informed consent ............................................................................................. 72 
10.9 
Protocol approval and amendment ............................................................... 72 
10.10 Duration of the trial ........................................................................................ 73 
10.11 Premature termination of the trial ................................................................ 73 
10.12 Confidentiality ................................................................................................. 73 
10.13 Other ethical and regulatory issues ............................................................... 74 
10.14 Liability and insurance ................................................................................... 74 
10.15 Publication policy ............................................................................................ 74 
11 
REFERENCE LIST ............................................................................................. 76 
12 
APPENDICES ...................................................................................................... 79 
12.1 
List of trial personnel ...................................................................................... 79 
 
Tables in text 
Table 1 
Description of trial drugs .................................................................................. 31 
Table 2 
Quantities of ingredients in dasiglucagon and placebo injection ...................... 32 
Table 3 
Schedule of procedures ..................................................................................... 53 
Table 4 
Post-treatment blood sampling times ................................................................ 57 
 
Figures in text 
Figure 1 Trial design ........................................................................................................ 23 


 
 
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List of abbreviations and definitions of terms 
ADA 
Anti-drug antibody 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALT 
Alanine aminotransferase 
ANCOVA Analysis of covariance 
AST 
Aspartate aminotransferase 
AUC 
Area under the concentration-time curve 
AUC0-inf 
AUC from time zero to infinity 
AUE 
Area under the effect curve  
CFB 
Changes from baseline  
CI 
Confidence interval 
Cmax 
Maximum plasma concentration 
CPH 
Cox proportional hazards 
CRO 
Contract research organization  
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
EU 
European Union 
EudraCT 
European Medicines Agency’s Clinical Trials Database 
FAS 
Full analysis set 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HbA1c 
Glycated hemoglobin 
ICH 
International Conference on Harmonization 
ID card 
Identification card 
IEC 
Independent Ethics Committee 
IM 
Intramuscular(ly) 
IMP 
Investigational medicinal product 
IRB 
Institutional Review Board 
IV 
Intravenous(ly) 


 
 
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IWRS 
Interactive Web Response System 
MedDRA 
Medical Dictionary for Regulatory Activities 
NPH 
Neutral protamine Hagedorn 
PD 
Pharmacodynamics 
PK 
Pharmacokinetics 
PP 
Per protocol 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SAS 
Safety analysis set 
SC 
Subcutaneous(ly) 
SUSAR 
Serious unexpected suspected adverse reaction 
T1DM 
Type 1 diabetes mellitus 
T2DM 
Type 2 diabetes mellitus 
TEAE 
Treatment-emergent adverse event 
tmax 
Time to the maximum plasma concentration 
ULN 
Upper limit of the normal range 
US 
United States of America 
 


 
 
Dasiglucagon (ZP4207) 
Date: 31 Aug 2017 
 
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1 
SYNOPSIS 
Name of sponsor: 
Zealand Pharma A/S 
Trial ID: 
ZP4207-16137 
Title of the trial: 
A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy and safety of 
dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 1 diabetes mellitus (T1DM) 
compared to placebo and with reference to GlucaGen® 
Trial design:  
The trial is a global, multicenter, randomized, parallel-group, double-blind, clinical trial confirming the 
efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in subjects with type 1 diabetes 
mellitus. The subjects will be randomized 2:1:1 to receive a single fixed subcutaneous 0.6 mg dose of 
dasiglucagon (hereinafter dasiglucagon), placebo for dasiglucagon (hereinafter referred to as placebo), or a 
1 mg dose of GlucaGen® (hereafter referred to as GlucaGen) and followed for at least 28 days after 
treatment. 
Clinical phase of development: 
Phase 3 
Trial centers: 
This trial will be conducted at 4 to 6 sites in the United States of America, Canada, and Europe. 
Planned trial start (first subject first visit): 
Q4/2017 
Planned trial end (last subject last visit): 
Q3/2018  
Trial population:  
Male and female adult subjects with type 1 diabetes mellitus treated with insulin for at least one year  
Key objectives: 
Primary objective:  
 
To demonstrate superiority of dasiglucagon compared to placebo following a single subcutaneous 
0.6 mg dose administered to subjects with type 1 diabetes mellitus with insulin-induced 
hypoglycemia. 
Secondary objective:  
 
To compare the glycemic response observed after dasiglucagon with that of GlucaGen. 
Key endpoints:  
Primary endpoint:  
 
Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase in plasma 
glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic clamp procedure 
without administration of rescue intravenous (IV) glucose.  
Key secondary endpoints: 
 
Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, and within 
10 minutes after study drug injection without administration of rescue IV glucose. 
 
Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection or at the time of rescue. 
Clinical efficacy (Pharmacodynamic) endpoints: 
 
Time to first plasma glucose concentration of ≥70 mg/dL (3.9 mmol/L) without administration of 
rescue IV glucose. 


 
 
Dasiglucagon (ZP4207) 
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 
Plasma glucose response as area under the curve above baseline from time zero to 30 minutes, 
AUC0-30min. 
Exposure (Pharmacokinetic) endpoints: 
 
Area under the drug concentration curve from time zero to 90 minutes, AUC0-90 min. 
 
Maximum plasma drug concentration (Cmax). 
 
Time to maximum plasma drug concentration (tmax). 
Safety endpoints:  
 
Adverse events, clinical laboratory assessments (biochemistry, hematology, coagulation, urinalysis), 
vital signs, physical examination, electrocardiogram, and local tolerability. 
 
Administration of rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
Immunogenicity endpoint: 
 
Occurrence of anti-drug antibodies 
Exploratory endpoints: 
 
Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL (1.1 mmol/L) 
within 30 minutes after study drug injection without administration of rescue IV glucose. 
 
Plasma insulin response as area under the curve above baseline from time zero to 60 minutes, 
AUC0-60 min. 
Key inclusion criteria: 
 
Male and female subjects with type 1 diabetes mellitus treated with insulin for at least one year, 
diagnostic criteria as defined by the American Diabetes Association. 
 
Stable insulin treatment 30 days prior to screening, defined as no more than a 10-unit daily variation 
in total daily insulin dose. 
 
Hemoglobin A1c <10%. 
 
Aged between 18 and 75 years, both inclusive. 
Key exclusion criteria: 
 
Previously treated with dasiglucagon. 
 
Known or suspected allergy to trial product(s) or related products. 
 
History of anaphylaxis or symptoms of severe systemic allergy (such as angioedema). 
 
Previous participation (randomization) in this trial. 
Sample size: 
Approximately 156 subjects are intended to complete the trial, with 78 subjects randomized to the 
dasiglucagon group and 39 subjects randomized to each of the placebo and GlucaGen groups.  
Investigational medicinal product: 
Test product: dasiglucagon liquid formulation in pre-filled syringes. 
Comparator products: Placebo and GlucaGen® lyophilized powder. 
Duration of treatment:  
Subjects will be randomized 2:1:1 to receive a single fixed subcutaneous dose of 0.6 mg dasiglucagon, 
placebo, or 1 mg GlucaGen, and followed for at least 28 days after receiving treatment.  


 
 
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Assessments:  
The plasma glucose profile for evaluation of the primary and secondary clinical efficacy 
(pharmacodynamic) endpoints will be assessed based on plasma concentration data from samples collected 
at the dosing visit. 
The exposure to trial medication (dasiglucagon, placebo, or GlucaGen) for evaluation of pharmacokinetics 
will also be assessed based on plasma concentration data.  
Statistical methods:  
Due to requirements in the size of the safety database, the sample size is set to 78 subjects treated with 
dasiglucagon 0.6 mg. With a 2:1:1 randomization ratio for the three treatments, dasiglucagon 0.6 mg, 
placebo, and GlucaGen 1 mg, 78 subjects injected with dasiglucagon and 39 subjects with placebo will 
yield a power of 90% at a 5% two-sided significance level to detect a treatment group difference in recovery 
incidence within 20 minutes, assuming as low as 80% and as high as 50% recovery incidence for the 
dasiglucagon and placebo groups, respectively. 
For the confirmatory analyses, the primary and key secondary endpoints for the dasiglucagon 0.6 mg and 
placebo treated subjects will be compared. A hierarchical procedure will be applied for the control of 
multiplicity in statistical inferences. The primary and secondary endpoints will be evaluated on the Full 
Analysis Set sample. The statistical inference comparisons with placebo will be conducted as superiority 
tests. The comparisons of dasiglucagon versus GlucaGen will be summarized descriptively.    
The primary endpoint will be summarized using Kaplan-Meier (KM) estimates stratified by treatment group 
and injection site. The treatment group difference between dasiglucagon and placebo will be evaluated 
inferentially using a pairwise two-sided log-rank test. 
In the primary analysis, those subjects who require rescue IV glucose will be censored at the time of plasma 
glucose recovery. This approach is based on the assumption that rescue IV glucose administration will 
shorten the time to recovery. This assumption implies that censoring at the time of recovery after rescue 
therapy is valid as a time to recovery without rescue would be longer than the observed duration. If recovery 
has not occurred at 45 minutes after study drug injection, censoring will be applied irrespective of the use of 
rescue IV glucose.  
In sensitivity analyses, the time to plasma glucose recovery will be analyzed 1) without censoring at the 
time of recovery for those subjects who require rescue IV glucose before 45 minutes and 2) with censoring 
at the time of administration of rescue IV glucose before 45 minutes.   
The primary endpoint will additionally be analyzed using a Cox proportional hazards (CPH) time to event 
statistical model, if the hazards by treatment group can be assumed proportional using a graphical 
comparison of log-minus-log survival curves and no more than two-thirds of the recovery times are 
censored in each treatment group. The CPH model will be used for inferences, with treatment group and 
injection site modeled as categorical effects, and baseline plasma glucose modeled as a continuous 
covariate. The treatment group rate ratios, active versus placebo, will be estimated together with the 95% 
confidence intervals, and pairwise treatment group inferences (dasiglucagon vs placebo, GlucaGen vs 
placebo) will be evaluated using two-sided likelihood ratio tests.  
The key secondary incidence variables (plasma glucose recovery within 30 minutes, within 20 minutes, 
within 15 minutes, within 10 minutes, in hierarchical order for inference) will be analyzed using descriptive 
statistics (frequency and percentage) by treatment group. Inferential treatment group comparisons will be 
assessed via pairwise tests of independent binomial proportions (dasiglucagon versus placebo, GlucaGen 
versus placebo) using Fisher’s exact test for each pairwise comparison. 
The key secondary endpoints of plasma glucose changes from baseline (CFB) within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection or at the time of rescue, will 


 
 
Dasiglucagon (ZP4207) 
Date: 31 Aug 2017 
 
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Protocol Version: Final 2.0 
 
 
 
 
 
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Page 14 of 80  
be analyzed with the plasma glucose CFB at rescue carried forward in those subjects who require rescue IV 
glucose before plasma glucose ≥20 mg/dL recovery. Each of these CFB variables will be analyzed using an 
Analysis of Covariance model, with treatment group modeled as a fixed effect and with the baseline plasma 
glucose modeled as a covariate. The dasiglucagon versus placebo treatment group difference will be 
evaluated inferentially as a least squares means contrast, using a two-sided t-test at the 0.05 significance 
level, first for the 30 minute CFB, and then sequentially for the 20 minute, 15 minute, and 10 minute 
variables, until the first failure to reject. 
The continuous clinical efficacy variables, the exploratory variables, and the pharmacokinetics variables 
will each be summarized descriptively by treatment group. The clinical efficacy variables will be analyzed 
analogous to the plasma glucose CFB variables.   
The safety analyses will include by-treatment-group descriptive summaries of vital sign measurements, 
laboratory measures (including immunogenicity incidence), physical examination assessments, rescue IV 
glucose (incidence and amount of glucose infused), and adverse events. The number and percentage of 
subjects reporting specific events, such as nausea and vomiting, will be presented by body system and 
preferred term. 
Further details will be included in the Statistical Analysis Plan, to be completed before database lock and 
treatment unmasking.   


 
 
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2 
INTRODUCTION 
2.1 
Background 
Zealand Pharma A/S (Zealand Pharma) is developing dasiglucagon, a physically and 
chemically stable peptide analog of human glucagon, in a ready-to-use liquid formulation 
for the acute treatment of severe hypoglycemia in patients with insulin-treated diabetes 
mellitus. Like native glucagon, dasiglucagon is comprised of 29 amino acids, but with 
7 substitutions which improve its physical and chemical stability in aqueous media. 
These amino acid substitutions make dasiglucagon suitable for a liquid formulation, 
while providing similar efficacy and safety as compared with recombinant glucagon in 
the clinical setting of acute treatment of severe hypoglycemia. 
2.1.1 Hypoglycemia 
Hypoglycemia in patients with diabetes is defined as episodes of an abnormally low 
plasma glucose concentration (3). This is a common, unpredictable, and potentially 
dangerous side effect of treatment of diabetes mellitus, especially with insulin or 
sulfonylureas. It is more frequent in patients with profound endogenous insulin 
deficiency, such as occurs in type 1 diabetes mellitus (T1DM) and advanced type 2 
diabetes mellitus (T2DM). Treatment of T2DM with insulin causes hypoglycemia 
progressively and more frequently over time, whereas in T1DM, hypoglycemia is 
experienced throughout the course of established disease (4). 
Symptoms and signs of hypoglycemia are not specific. Patients undergoing a 
hypoglycemic episode experience unpleasant symptoms such as anxiety, sweating, 
hunger, tremors, palpitations, paresthesia, nausea and pallor. Depending on its severity, 
the hypoglycemia may lead to mild confusion, behavioral changes, loss of consciousness, 
seizures, coma, and death (5). 
The incidence of hypoglycemic events or even the fear of hypoglycemia influences 
patients’ adherence to prescribed treatment regimens for diabetes mellitus (6). This leads 
to inadequate glycemic control, which in turn may lead to an increased risk of diabetic 
complications (5). Serious clinically significant hypoglycemia is now defined as plasma 
glucose <54 mg/dL (3.0 mmol/L), while the plasma glucose alert value is defined as 
<70 mg/dL (3.9 mmol/L) (7). When plasma glucose falls below these values, some kind 
of treatment strategy is needed. 


 
 
Dasiglucagon (ZP4207) 
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2.1.2 Glucagon 
Glucagon is a naturally occurring hormone, secreted from the alpha cells of the 
pancreatic islets. Glucagon plays a central role in the regulation of glucose homeostasis 
and is the counterpart of insulin for controlling blood glucose levels (i.e. it acts in 
opposition to insulin in terms of effects on blood glucose levels) (8,9). Glucagon 
stimulates hepatic glycogenolysis and gluconeogenesis in hypoglycemic states, thereby 
restoring glucose homeostasis. Glucagon receptor agonism has also been shown to exert 
effects on lipid metabolism, energy balance, body adipose tissue mass and food intake 
(10). Insulin decreases blood glucose levels and cases of hypoglycemia can be reversed 
by glucagon. Therefore, glucagon is indicated for the treatment of severe hypoglycemia. 
Besides intravenous (IV) glucose administration, an injectable form of glucagon is given 
as first aid in cases of severe hypoglycemia, when the patient is unconscious or for other 
reasons cannot take glucose orally. The approved glucagon dose for an adult is 1 mg, 
given by intramuscular (IM), IV, or subcutaneous (SC) injection, which quickly raises 
blood glucose levels. As current marketed recombinant glucagon is highly unstable when 
dissolved in solution, the injectable form has to be reconstituted prior to use in a 9-step 
procedure that requires a sterile diluent to be injected into a vial containing lyophilized 
powdered glucagon. When dissolved in a fluid state, glucagon can form amyloid fibrils 
(11), or tightly woven chains of proteins made up of the individual glucagon peptides. 
The reconstitution process makes the use of marketed glucagon products cumbersome 
(12), and a more patient-friendly formulation is needed. Currently, the Food and Drug 
Administration (FDA) approved instructions for commercially available glucagon allow 
only for immediate usage of glucagon after the powder is reconstituted in aqueous 
solution (13). Therefore, a glucagon analog with enhanced biophysical stability may 
represent a leap forward in terms of convenient therapeutic applications. 
2.1.3 Dasiglucagon 
Dasiglucagon (ZP4207) is a stable peptide analog of human glucagon, available in a 
ready-to-use liquid formulation. Dasiglucagon (hereinafter referred to as dasiglucagon) is 
in development for the treatment of severe hypoglycemia in insulin-dependent patients 
with diabetes mellitus. Dasiglucagon is a specific and full glucagon receptor agonist 
designed to mimic the effects of glucagon, having a fast absorption and elimination 
(minutes). It is a peptide of 29 amino acids, with 7 amino acid substitutions compared to 
native glucagon. The main purpose of the substitutions is to increase the physical and 
chemical stability of the glucagon analog compared to marketed glucagon products such 
as Lilly Glucagon or GlucaGen® (hereafter referred to as GlucaGen). Dasiglucagon 
exhibits improved physical and chemical stability and is available in an aqueous solution 
at neutral pH (14). 


 
 
Dasiglucagon (ZP4207) 
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Three clinical trials have been completed with dasiglucagon: a first-in-human dose trial in 
healthy volunteers and subjects with T1DM (ZP4207-14013), a multiple-dose 
dose-escalation trial (ZP4207-15007) evaluating the safety, tolerability, pharmacokinetics 
(PK) and pharmacodynamics (PD) of dasiglucagon in healthy volunteers, and a phase 2 
crossover trial to assess the PK and PD of a single dose of an optimized formulation of 
dasiglucagon administered SC in subjects with T1DM (ZP4207-15126) (14). 
2.1.3.1 Pharmacokinetics and pharmacodynamics of dasiglucagon 
The results of the phase 1 and 2 clinical trials confirmed dose-proportionality for 
dasiglucagon PK, which is characterized by a fast absorption with a peak plasma 
concentration obtained after 35 minutes. Thereafter, the plasma concentration rapidly 
declines with an average half-life of 28 minutes. The median time to the maximum 
plasma concentration (tmax) was 35 minutes for dasiglucagon compared with 20 minutes 
for GlucaGen.  
At all dose levels in the phase 2 trial, all subjects achieved a plasma glucose level of at 
least 70 mg/dL (3.9 mmol/L) as well as an increase in plasma glucose by at least 
20 mg/dL (1.1 mmol/L) within 30 minutes after dosing. The PD responses to 0.6 mg of 
dasiglucagon and 1 mg of GlucaGen were comparable. 
2.1.3.2 Safety of dasiglucagon 
The safety data for dasiglucagon do not give rise to any safety concerns. No new signals 
were observed, beyond those related to the pharmacological effect of glucagon agonism. 
All adverse events (AEs) recorded in the trials were of mild or moderate severity. The 
most frequently reported systemic AE was nausea, which is a known side effect 
following administration of glucagon. Headache was the next most frequently reported 
event, occurring in all dose groups in the phase 2 trial. Injection site reactions were 
observed only sporadically after administration with either dasiglucagon or GlucaGen 
and all were mild and transient. The most frequent injection site reaction was erythema, 
occurring in all treatment groups, including the placebo group, irrespective of dose. 
Glucagon has been described to exert positive inotropic and chronotropic effects and may 
therefore cause tachycardia and hypertension. In the phase 1 clinical trials, but not the 
phase 2 trial, temporary but clinically significant decreases in blood pressure were 
observed in a few healthy volunteers receiving investigational medicinal product (IMP) 
doses of at least 1 mg (4 with dasiglucagon and 1 with GlucaGen). This is not considered 
a safety concern; however, hemodynamic changes after dosing will be considered an 
adverse event of special interest (AESI). 


 
 
Dasiglucagon (ZP4207) 
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The phase 1 and 2 results and the safety profile described to date do not give rise to 
specific safety concerns. For further information, please refer to the Investigator’s 
Brochure (14). 
2.2 
Trial rationale 
The aim of the current trial is to confirm the superiority of dasiglucagon for the treatment 
of insulin-induced hypoglycemia in subjects with T1DM as compared to placebo for 
dasiglucagon (hereinafter placebo) and to compare the clinical efficacy and safety of 
dasiglucagon with reference to GlucaGen. A randomized, controlled trial design was 
used. 
See Section 4.2 for justification of the design of this trial. 
2.3 
Risk-benefit assessment  
Non-clinical experience 
The nonclinical development program did not reveal any safety findings that would 
prohibit administration of dasiglucagon to humans. None of the safety pharmacology 
studies, repeated dose toxicity studies or genotoxicity studies revealed any significant 
toxicity findings relevant to the therapeutic use of dasiglucagon. 
Clinical experience 
As glucagon and its analogs belong to a well-known drug class with a known mode of 
action, dasiglucagon is not expected to be a high-risk molecule.  
Treatment with an IMP may result in undesired effects or complaints. Undesired effects 
and complaints such as nausea, vomiting, and diarrhea are known AEs occurring with 
glucagon administration. Similar AEs have also been observed to a limited extent in the 
3 clinical studies conducted to date with dasiglucagon. As with every novel drug 
substance, new and as yet unknown side effects may also occur. 
There are limited data available to assess the immunogenic potential of glucagon 
products available on the market, but the data available indicate that marketed glucagon 
only has a low immunogenic potential.  
Overall, dasiglucagon is judged to be a low-risk molecule, based upon the available 
clinical data. Administration of dasiglucagon may be associated with a risk of allergic 
reactions similar to those observed for other therapeutic peptides or proteins. Subjects 
with known or suspected allergies to the trial medications or related products will be 
excluded from the trial. Mild or moderate allergic reactions may include symptoms of 
rash, fever, flu-like symptoms, nausea, headache, and myalgia. Acute generalized 


 
 
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hypersensitivity reactions are usually very rare, but may include symptoms of flushing, 
sweating, dizziness, change in blood pressure, and difficulties in breathing. No severe 
acute hypersensitivity reactions have been observed in the 3 clinical trials conducted with 
dasiglucagon. Direct access to resuscitation equipment is ensured at the clinical trial 
centers. 
With the exception of medical examinations, a subject participating in this trial is not 
likely to derive any personal health-related benefits. The results of the trial may 
contribute to the future use of dasiglucagon in patients with diabetes mellitus 
experiencing severe hypoglycemic reactions. 
The development program including 141 subjects exposed to dasiglucagon to date has 
demonstrated that administration of dasiglucagon is efficacious and well tolerated, with a 
safety profile that does not give rise to specific safety concerns. Two phase 1 and one 
phase 2 clinical trials have been conducted to investigate the safety, tolerability, PK and 
PD of dasiglucagon after single and multiple dosing to healthy volunteers and subjects 
with T1DM under insulin-induced hypoglycemic conditions. Dasiglucagon has proven to 
have relevant clinical effects in the acute severe hypoglycemia rescue setting and may be 
an effective and reliable emergency treatment for severe hypoglycemia. Overall, the 
anticipated benefits for subjects entering the ZP4207-16137 trial are considered to justify 
the risks. 


 
 
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3 
TRIAL OBJECTIVES AND ENDPOINTS 
3.1 
Primary objective 
 To demonstrate superiority of dasiglucagon compared to placebo following a single 
subcutaneous 0.6 mg dose administered to subjects with type 1 diabetes mellitus with 
insulin-induced hypoglycemia. 
3.2 
Secondary objectives 
 To compare the glycemic response observed after dasiglucagon with that of 
GlucaGen. 
3.3 
Primary endpoint 
 Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase 
in plasma glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic 
clamp procedure without administration of rescue IV glucose.  
3.4 
Key secondary endpoints 
 Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection without administration of rescue IV 
glucose. 
 Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection or at the time of rescue. 
3.5 
Other secondary endpoints 
 Clinical efficacy (PD) endpoints: 
- 
Time to first plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) without 
administration of rescue IV glucose. 
- 
Plasma glucose response as area under the curve (AUC) above baseline from time 
zero to 30 minutes, AUC0-30min. 
 Exposure (PK) endpoints: 
- 
Area under the drug concentration curve from time zero to 90 minutes, 
AUC0-90min. 
- 
Maximum plasma drug concentration (Cmax). 
- 
Time to maximum plasma drug concentration (tmax). 


 
 
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 Safety endpoints:  
- 
Adverse events, clinical laboratory assessments (biochemistry, hematology, 
coagulation, urinalysis), vital signs, physical examination, electrocardiogram 
(ECG), and local tolerability. 
- 
Administration of rescue infusion of IV glucose during the hypoglycemic clamp 
procedure. 
- 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp 
procedure. 
 Immunogenicity endpoint: 
- 
Occurrence of anti-drug antibodies 
3.6 
Exploratory endpoints 
 Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL 
(1.1 mmol/L) within 30 minutes after study drug injection without administration of 
rescue IV glucose. 
 Plasma insulin response as area under the curve above baseline from time zero to 
60 minutes, AUC0-60 min. 


 
 
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4 
OVERALL DESIGN AND PLAN OF THE TRIAL 
4.1 
Overview 
This trial is a global, multicenter, randomized, parallel, double-blind, clinical trial 
confirming the efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in 
subjects with T1DM. The subjects will be randomized 2:1:1 to receive a single fixed SC 
0.6 mg dose of dasiglucagon, placebo, or a 1 mg dose of GlucaGen and followed for at 
least 28 days after receiving treatment. A total of 156 subjects with T1DM are expected 
to complete the treatment visit. The trial will be conducted in the European Union (EU) 
and North America. See Figure 1 for an overview of the trial design. 


 
 
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Figure 1 
Trial design 
 
 
 


 
 
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4.2 
Justification of the trial design 
4.2.1 Justification for design and parameters 
In order to avoid bias in subject selection and in the evaluation of clinical assessments, 
subjects will be randomly assigned 2:1:1 to either dasiglucagon, placebo, or GlucaGen 
and the trial will be conducted in a double-blinded manner. The randomized parallel 
treatment design with administration of fixed SC doses of dasiglucagon, placebo, or 
GlucaGen to subjects with T1DM and insulin-induced hypoglycemia allows for direct 
comparison of the clinical efficacy of the treatments.  
The trial is double-blind to increase trial validity and to reduce bias during evaluation of 
the treatments. However, since the trial medications are not identical in appearance, the 
handling, preparation and administration of trial medication will be done by unblinded 
trial personnel who will not be involved in any other trial procedures or assessments. See 
Section 6.6 for more information about which assessments are blinded and which are not, 
with reasons.  
A superiority trial design is used because the aim is to show that treatment with 
dasiglucagon is an effective treatment compared to placebo. The secondary objective was 
chosen to allow a comparison between treatment with dasiglucagon and an established 
comparator treatment for severe hypoglycemia, GlucaGen. 
Administration of glucagon is intended to quickly raise blood glucose levels in subjects 
with T1DM with insulin-induced hypoglycemia. Therefore, in order to assess the clinical 
efficacy of dasiglucagon as compared to placebo and GlucaGen following a single SC 
dose, the primary endpoint and secondary efficacy endpoints involve the measurement of 
plasma glucose concentrations at different timepoints. 
4.2.2 Justification for drug, route, dosage and treatment duration 
Dasiglucagon and GlucaGen will be administered as fixed doses independent of body 
weight because this is the intended therapeutic dosing regimen in the emergency 
treatment of hypoglycemia. The selected dose of 1 mg GlucaGen is the approved dose for 
treatment of severe hypoglycemia. Data from the studies conducted to date with 
dasiglucagon, including the phase 2 trial in subjects with T1DM, have been used to 
establish that 0.6 mg of dasiglucagon is an effective dose and also represents a 
therapeutically equivalent dose to 1 mg of GlucaGen (see also Section 6.1). 
Dasiglucagon, placebo, and GlucaGen will be administered in the abdomen, buttocks, or 
thigh by SC injection, as this is one of the intended routes of administration for 
dasiglucagon, besides IM and IV. 


 
 
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Subjects will be followed for at least 28 days after dosing in order to perform an adequate 
immunogenicity evaluation of treatment.  


 
 
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5 
TRIAL POPULATION 
5.1 
Rationale for trial population 
Dasiglucagon is in development for the treatment of severe hypoglycemia in 
insulin-dependent patients with diabetes mellitus. In the present trial, subjects with 
T1DM are included in the evaluation of efficacy and safety of dasiglucagon under 
hypoglycemic conditions as this is part of the intended target population. Subjects with 
T1DM are selected to avoid the endogenous glucagon counter-regulatory response to 
insulin-induced hypoglycemia that is present in patients with T2DM. The inclusion and 
exclusion criteria are set to include a trial population that represents the general 
population of subjects with T1DM.  
5.2 
Planned sample size and number of trial centers 
A total number of 156 subjects with T1DM are expected to complete the treatment visit 
(Visit 2). To qualify as completed, the subject must be dosed and have blood drawn for 
the PK and PD assessments described in the protocol. It is expected that up to 
176 subjects will be randomized to have 156 subjects completing Visit 2. Completion of 
156 subjects (78 subjects in the dasiglucagon group and 39 subjects in each of the 
placebo and GlucaGen groups) would be expected to provide adequate power for the 
primary efficacy evaluation, as described in Section 9.1. 
The planned date for first subject first visit is expected to take place in Q4, 2017 and the 
planned date for last subject last visit is expected to take place in Q3, 2018. 
This trial is planned to be conducted at 4 to 6 trial sites in the United States of America 
[US], Canada, and Europe. 
5.3 
Inclusion criteria 
Subjects will be entered into this trial only if they meet all of the following criteria: 
1. Informed consent obtained before any trial-related activities (trial-related 
activities are any procedure that would not have been performed during normal 
management of the subject). 
2. Female or male subjects with T1DM for at least 1 year, diagnostic criteria as 
defined by the American Diabetes Association (3). 
3. Treated with insulin for T1DM for at least 1 year and with stable insulin treatment 
(defined as no more than a 10-unit daily variation in total daily insulin dose) 
30 days prior to screening 
4. Hemoglobin A1c <10%. 


 
 
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5. Aged between 18 and 75 years, both inclusive. 
6. A female subject must meet one of the following criteria: 
a. Participant is of childbearing potential and agrees to use one of the 
accepted contraceptive regimens throughout the entire duration of the trial 
from screening and until last follow-up visit. Additionally, if the 
participant is using systemic contraceptives, she must use an additional 
form of acceptable contraception. An acceptable method of contraception 
includes one of the following: 
i. Abstinence from heterosexual intercourse; 
ii. Systemic contraceptives (birth control pills, injectable/implant/ 
insertable hormonal birth control products, transdermal patch); 
iii. Intrauterine device (with and without hormones); or 
iv. Condom with spermicide; or 
b. Participant is of non-childbearing potential, defined as surgically sterile 
(i.e. has undergone complete hysterectomy, bilateral oophorectomy, or 
tubal ligation), or in a menopausal state (at least 1 year without menses). 
7. A male subject must meet the following criteria: Surgically sterilized or willing to 
refrain from sexual intercourse from screening and until last follow-up visit or, if 
sexually active, uses condom and partner practices contraception during the trial 
from screening and until last follow-up visit. 
5.4 
Exclusion criteria 
Subjects meeting any of the following criteria during screening evaluations will be 
excluded from trial participation: 
1. Previously treated with dasiglucagon (previously referred to as ZP4207). 
2. Known or suspected allergy to trial product(s) or related products. 
3. History of anaphylaxis or symptoms of severe systemic allergy (such as 
angioedema). 
4. Previous participation (randomization) in this trial. 
5. Females who are pregnant according to a positive pregnancy test, are actively 
attempting to get pregnant, or are lactating. 
6. History of hypoglycemic events associated with seizures in the last year prior to 
screening. 
7. History of severe hypoglycemia (defined as plasma glucose <54 mg/dL 
[3.0 mmol/L]) in the last month prior to screening. 
8. Receipt of any investigational drug within 3 months prior to screening. 
9. Active malignancy within the last 5 years. 
10. Congestive heart failure, New York Heart Association class II-IV. 
11. Inadequately treated blood pressure, defined as systolic ≥160 mmHg or diastolic 
≥90 mmHg) at screening (15). 


 
 
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12. Current bleeding disorder, including anti-coagulant treatment. 
13. Known presence or history of pheochromocytoma (i.e. adrenal gland tumor) or 
insulinoma (i.e. insulin secreting pancreas tumor). 
14. Use of a daily systemic beta-blocker drug, indomethacin, warfarin or 
anticholinergic drugs in the previous 28 days before Day 1 of this trial. 
15. Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >2.5 × the 
upper limit of the normal range (ULN), bilirubin >1.5 × ULN, estimated 
glomerular filtration rate <30 mL/min/1.73 m2 according to the Modification of 
Diet in Renal Disease study definition (16), or altered electrolyte values of 
clinical relevance for cardiac conduction, as judged by the investigator. 
16. Clinically significant abnormal ECG at screening as judged by the investigator. 
17. Clinically significant illness within 4 weeks before screening, as judged by the 
investigator. 
18. Donation of blood or plasma in the past month, or in excess of 500 mL within 
12 weeks prior to screening. 
19. Surgery or trauma with significant blood loss within the last 2 months prior to 
screening. 
20. A positive result in the alcohol and/or urine drug screen at the screening visit. 
Significant history of alcoholism or drug abuse as judged by the investigator or 
consuming more than 24 g alcohol per day for men, or more than 12 g alcohol per 
day for women. 
21. Subjects with mental incapacity or language barriers which preclude adequate 
understanding or cooperation, who are unwilling to participate in the trial, or who 
in the opinion of the investigator should not participate in the trial. 
22. Any condition interfering with trial participation or evaluation or that could be 
hazardous to the subject. 
23. The use of prescription or non-prescription medications known to cause QT 
prolongation. 
5.5 
Dosing day exclusion criteria 
Subjects who meet one or more of the following dosing day exclusion criteria at the 
dosing visit will be excluded from the dosing visit, but the visit can be rescheduled 
1-7 days later. The dosing visit can only be rescheduled once. 
1. Atypically strenuous exercise within 4 days prior to dosing, as judged by the 
investigator. Exercise during the trial should follow subject’s typical routine, and 
should not exceed a near maximum intensity for more than 20 minutes per day, or 
moderate intensity for more than 90 minutes per day. 
2. Clinically significant illness within 4 weeks before dosing, as judged by the 
investigator. 


 
 
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3. Consumption of alcohol within 24 hours prior to dosing visit, or positive results 
from an alcohol breath test. 
4. Not fasting from 22:00 hours the evening prior to dosing, apart from water. Small 
amounts of carbohydrates (up to 20 g) to prevent hypoglycemia are allowed. 
5. The use of any non-prescribed systemic or topical medication, except routine 
vitamins and occasional use (as judged by the investigator) of acetylsalicylic acid 
and paracetamol within 2 weeks prior to dosing. Treatment with insulin, including 
analogs, is allowed. 
6. Use of any basal insulin within 16 hours prior to dosing. 
7. Use of any short acting (bolus) insulin within 6 hours prior to dosing, except 
insulin glulisine (Apidra®). 
8. Changes in medical history or concomitant medication resulting in fulfillment of 
clinical exclusion criteria, as judged by the investigator. 
9. Plasma glucose value <50 mg/dL (2.8 mmol/L) within the last 24 hours or plasma 
glucose value <60 mg/dL (3.3 mmol/L) within the last 5 hours prior to dosing. 
5.6 
Premature treatment discontinuation and withdrawal 
Participation in the trial is strictly voluntary. A subject has the right to withdraw from the 
trial at any time and for any reason. If he/she chooses to withdraw, the investigator must 
be informed immediately. The investigator has the right to terminate participation of any 
subject at any time if the investigator deems it in the subject's best interest. The reason 
and circumstances for withdrawal will be documented in the electronic case report form 
(eCRF). 
5.6.1 Possible reasons for treatment visit discontinuation 
A subject will be discontinued from treatment if the following applies: 
 Withdrawal of consent by subject. 
 If a protocol deviation occurs which, in the clinical judgement of the investigator, can 
invalidate the trial or endpoints or can interfere pharmacokinetically or 
pharmacodynamically with the trial product, the subject will be discontinued by the 
investigator. 
 Adverse events occur which are considered unacceptable by the subject or the 
investigator. 
If discontinuation occurs following administration of trial medication, every effort should 
be made to have the subject return and participate in the complete follow-up visit on 
Day 28 (see Table 3) to avoid missing data. 
If trial participation is terminated due to an AE possibly related to any of the trial 
medications or trial examinations, the subject must be followed up by additional 


 
 
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examinations according to the medical judgment of the investigator until the abnormal 
condition is resolved or the investigator deems further observations or examinations to be 
no longer medically indicated. 
A total of 156 subjects must complete the treatment visit (Visit 2). To qualify as 
completed, the subject must be dosed and have blood drawn for the PK and PD 
assessments described in the protocol.  
5.6.2 Center discontinuation 
The center can be closed and the trial terminated for the following reasons: 
 The center is unlikely to be able to recruit sufficient subjects within the agreed time 
frame. 
 The center does not respond to trial management requests. 
 Repeat protocol violations. 
5.6.3 Trial termination 
The sponsor reserves the right to modify or terminate the trial at any time. Possible 
reasons for termination are: 
 Safety reasons – the incidence of AEs in this or any other trial using the same trial 
medication indicates a potential health risk for the subjects. 
 New scientific knowledge becomes known that makes the objectives of the trial no 
longer feasible/valid. 
 Unsatisfactory enrolment of subjects. 
5.7 
Subject identification and randomization 
Subjects who have given written informed consent and meet all inclusion and none of the 
exclusion criteria will be randomized in a 2:1:1 ratio to either 0.6 mg dasiglucagon 
(n=78), placebo (n=39), or 1 mg GlucaGen (n=39) via an Interactive Web Response 
System (IWRS). Randomization will continue until 156 subjects have completed Visit 2. 
Subjects with previous exogenic glucagon exposure will not be excluded from the trial, 
but the information on previous glucagon administration will be recorded, to enable 
subgroup analyses.  
In the event of an emergency, e.g. when it becomes necessary for the investigator to 
know which treatment the subject is taking, the subject code can be broken by the 
investigator, preferably after consultation with the medical monitor. Emergency code 
breaks can be performed using the IWRS. 


 
 
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6 
TRIAL DRUG 
6.1 
Identity 
The following trial drugs will be administered: 
 Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in a prefilled syringe. 
 Placebo, liquid formulation, 0.6 mL delivered in a prefilled syringe. 
 Recombinant glucagon hydrochloride, 1 mg for reconstitution (GlucaGen®, Novo 
Nordisk) in 1 mL sterile water. 
The description of the three trial drugs is provided in Table 1. Dasiglucagon is a stable 
peptide analog of human glucagon in a ready-to-use liquid formulation for treatment of 
severe hypoglycemia in insulin-dependent patients with diabetes mellitus. GlucaGen is 
approved in the EU and US and is indicated for treatment of severe hypoglycemic 
reactions, which may occur in the management of insulin-treated children and adults with 
diabetes mellitus. 
Table 1 
Description of trial drugs 
 
Test product 
Placebo Product 
Comparator product 
Name 
Dasiglucagon 
Placebo  
GlucaGen® 
Active 
substance 
Dasiglucagon 
N/A 
Recombinant 
glucagon 
hydrochloride 
Formulation 
Liquid formulation, 
0.6 mL 
Liquid formulation, 
0.6 mL 
Powder and solvent 
for reconstitution as 1 
mL solution for 
injection 
Strength 
1 mg/mL 
N/A 
1 mg 
Device 
Single use pre-filled 
syringe 
Single use pre-filled 
syringe 
Powder and solvent 
for reconstitution 
packed together in a 
plastic box. A “hypo-
kit”. 
Manufacturer 
Zealand Pharma A/S, 
Glostrup 
(Copenhagen), 
Denmark 
Zealand Pharma A/S, 
Glostrup 
(Copenhagen), 
Denmark 
Novo Nordisk A/S, 
Bagsværd, Denmark 


 
 
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Test product 
Placebo Product 
Comparator product 
Storage 
requirements 
Store between 2 and 
8°C 
Store between 2 and 
8°C 
Store between 2 and 
8°C 
The quantities of ingredients for dasiglucagon and placebo are provided in Table 2. 
Table 2 
Quantities of ingredients in dasiglucagon and placebo injection 
Component 
Amount per mL 
(dasiglucagon) 
Amount per mL 
(placebo) 
Function 
Dasiglucagon* 
1.0 mg 
N/A 
Sodium chloride 
10.23 mg 
10.23 mg 
Trometamol/Tromethamine 
6.06 mg 
6.06 mg 
Water for injection 
To make 1 mL 
To make 1 mL 
Sodium hydroxide 
q.s. 
q.s. 
Hydrochloric acid 
q.s. 
q.s. 
*The quantity of drug substance to be used is calculated according to net peptide content and purity.  
q.s. = quantum sufficit (quantity required). 
6.2 
Treatment assignment and randomization 
Subjects successfully completing screening and who fulfill entry eligibility and 
randomization criteria will be randomized to one of three treatment groups in a ratio of 
2:1:1: 
 Test treatment: Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in a 
prefilled syringe. 
 Placebo treatment: Placebo, liquid formulation, 0.6 mL delivered in a prefilled 
syringe. 
 Reference treatment: Recombinant glucagon hydrochloride, 1 mg for reconstitution. 
Randomization will be performed using a fixed-block randomization scheme. The 
randomization scheme will be generated prior to the initiation of the study by an 
independent statistician/programmer who will not be a member of the study team; all 
investigators will not be aware of the block size of the randomization scheme.  
Randomization will be stratified by treatment group and by injection site (abdomen, 
buttocks, or thigh) and controlled via the IWRS.       


 
 
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Subjects will be randomized to study treatment using an interactive, automated system 
which has been validated for the intended use under the International Society of 
Pharmaceutical Engineers Good Automated Manufacturing Process guidelines, 21CFR 
Part 11 (FDA regulation for Electronic Records and Electronic Signatures) and the 
International Conference on Harmonization (ICH) Guidance E6 for Industry on Good 
Clinical Practice (GCP).   
6.3 
Administration 
Dasiglucagon, placebo, and GlucaGen will be administered by SC injection in the 
abdomen, buttocks, or thigh.  
An unblinded person (appropriately trained) authorized to prepare the dose and 
administer the treatment in accordance with the randomization will prepare the treatment 
required for each subject on each dosing day. The dose will be administered by the 
unblinded, trained and qualified person. The content of the syringe has to be checked for 
clarity and absence of bubbles. To ensure proper administration, the unblinded person 
should administer the SC injection at a 90 degree angle by grasping skin between the 
thumb and first finger. The injection should be given within 5 seconds. 
Syringes will be discarded after dose administration. Used GlucaGen vials will be stored 
in a lockable box (separated from unused vials) at ambient temperature. 
6.4 
Packaging and labelling 
The test product will be packed by the sponsor. The information on the labels will be in 
the local language and the product label will be compliant with local laws and 
regulations.  
The study drug label will describe the storage conditions for study drug. The labels will 
supply no information about the subjects. Each treatment kit (pre-filled syringe/vial for 
reconstitution) will have a unique Dispensing Unit Number for drug allocation, drug 
accountability, and traceability purposes. 
Labelling will be performed according to Annex 13 of the Good Manufacturing Practice 
guidelines of the European Commission, ICH GCP guidelines, and local law. 
6.5 
Storage of study drugs 
The investigator must ensure the availability of proper storage conditions. All study drug 
supplies provided for this study will be stored in a secure area with restricted access at the 
study site.  


 
 
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The temperature should be monitored by recording the actual, minimum, and maximum 
temperatures using a calibrated thermometer or thermocouple, or by continuous recording 
using a qualified temperature monitoring system. The temperature should be evaluated 
and documented at least on working days on a temperature log. This log must be included 
in the Investigator Site File upon study termination. 
Dasiglucagon and placebo must be stored in a refrigerator (at a temperature of 2–8°C), 
and should be handled in accordance with guidelines from the sponsor. GlucaGen must 
also be stored in a refrigerator (at a temperature of 2–8°C), and should be handled in 
accordance with the Summary of Product Characteristics (13).  
The unblinded person responsible for study drug handling must contact the unblinded 
monitor in case of temperature deviations outside the acceptable range.  
Please see the Pharmacy Manual for additional information on handling study drug. 
6.6 
Blinding and breaking the blind 
This is a double-blind trial. As the trial products are not identical in appearance, 
dasiglucagon and placebo being available as a liquid formulation and GlucaGen as a 
powder for reconstitution, unblinded trial personnel will be responsible for handling, 
preparing (according to the prescription from the IWRS), and administering the trial 
medication, as well as for keeping the records strictly confidential and accessible only to 
unblinded staff until after the database has been locked. To maintain double-blind 
conditions, all trial assessments at the trial center will be done by blinded trial personnel 
not involved in the administration of trial medications. However, exposure assessments 
and anti-drug antibody (ADA) assessments will be performed by unblinded personnel at 
the special laboratories, to ensure that dasiglucagon, placebo, or GlucaGen administration 
is matched with the applicable bioanalytical assay. 
Treatment assignment will be kept strictly confidential and accessible only to authorized 
persons until after the time of unblinding. Codes with treatment assignment will, 
however, be readily available in the IWRS to the blinded personnel in case of an 
emergency. 
The code for a particular subject may be broken in a medical emergency if knowing the 
identity of the treatment allocation would influence the treatment of the subject. The 
emergency code break can be performed using the IWRS. Whenever a code is broken, the 
person breaking the code must record the time, date and reason as well as his/her initials 
in the source documents. The breaking of blinded codes in case of medical emergency for 
one subject should not unblind the trial personnel to the treatment information of other 
subjects. The person performing the unblinding should inform as few people as possible 


 
 
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about the result of the unblinding. All persons unblinded for a specific subject should be 
documented. 
If the trial center needs to break the code, the medical monitor should, if at all possible, 
be contacted prior to breaking the code and the monitor must be notified within 24 hours 
after the code has been broken. 
The pharmacovigilance unit (safety contract research organization [CRO]; see the list of 
trial personnel in Section 12.1) will be able to break the code in case of a serious 
unexpected suspected adverse reaction (SUSAR).  
The central and specialty laboratories will be provided with a copy of the randomization 
list. 
6.7 
Drug accountability 
Handling, preparation and administration of trial medication will be done by unblinded 
trial personnel. Each center will keep accurate records of the trial supplies received, 
stored, and dispensed, using appropriate forms. The trial supplies will be stored under 
appropriate conditions, locked and with restricted access.  
All unused supplies and all empty and partially empty containers of trial medication will 
be stored until the trial closure visit has been performed and then sent for destruction. 
This does not apply to the used syringes as they will be discarded after dose 
administration. Destruction must not take place until approved by the Sponsor.  
6.8 
Treatment compliance 
All trial medications will be prepared and administered by unblinded trial personnel. 
PK assessments will support the surveillance of compliance with IMP administration.  
6.9 
Prior and concomitant medications 
Prior glucagon exposure will be recorded in the eCRF at screening. All concomitant 
medications will be recorded and/or updated in the eCRF at each visit.  
Subjects using any new concomitant medication resulting in fulfillment of a dosing day 
exclusion criterion (see Section 5.5) will be excluded from the dosing visit, but can be 
rescheduled to one of the following days (1–7 days later). The dosing visit can only be 
rescheduled once. 


 
 
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6.9.1 Prohibited medications 
The use of anti-coagulant treatments and medications (prescription and non-prescription) 
that are known to cause QT prolongation are prohibited during the course of the trial. 
Within 28 days prior to dosing, the use of daily systemic beta-blockers, indomethacin, 
warfarin, and anticholinergic drugs is prohibited. 
Within 2 weeks prior to dosing, the use of any non-prescribed systemic or topical 
medication (with the exception of vitamins and the occasional use of acetylsalicylic acid 
and paracetamol) is prohibited.  
Within 16 hours prior to dosing, the use of any basal insulin is prohibited. 
Within 6 hours prior to dosing, the use of any short acting (bolus) insulin, except insulin 
glulisine (Apidra®) is prohibited.  
During the insulin-induced hypoglycemic procedure, continuous SC insulin infusion must 
be stopped. 


 
 
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7 
PARAMETERS AND METHODS OF ASSESSMENT 
Overall, approximately 180 mL of blood will be drawn from each subject for PK, PD, 
ADA, and safety laboratory assessments. 
7.1 
Efficacy parameters 
A description of the sample handling and sample processing at the site will be included in 
the laboratory manuals. Validation documentation for the assays must be available prior 
to sample analyses. A bioanalytical report for each analysis of trial PD and PK samples 
will be prepared. 
7.1.1 Pharmacodynamic measurements 
The plasma glucose profile for evaluation of the primary and secondary clinical efficacy 
endpoints will be assessed based on plasma concentration data (AUC0-30min) from samples 
collected at the dosing visit (Visit 2). The samples will be sent to the clinical laboratory 
and analyzed using a sensitive and validated assay for glucose measurements.  
Samples will be collected pre-dose, and at 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 
75, and 90 minutes after dosing (see the schedule of procedures in Table 3). The actual 
time of blood sampling for evaluation of plasma glucose should not deviate from the 
nominal time by more than ±30 seconds until the 20 minute collection time point and by 
more than ±1 minute for the subsequent collection time points. Pre-dose is defined as 
within 2 minutes prior to dosing. 
7.1.2 Pharmacokinetic measurements 
The exposure to trial medication (dasiglucagon or GlucaGen) for evaluation of PK will 
be assessed based on plasma concentration data (AUC0-90 min, Cmax, tmax) from samples 
collected at the dosing visit (Visit 2).  
Samples (including back-up samples) will be collected pre-dose, and at 15, 30, 35, 40, 50, 
60, 90, and 120 minutes after dosing (see the schedule of procedures in Table 3). The 
actual time of blood sampling for exposure to trial medication should not deviate from 
the nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior 
to dosing. 


 
 
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7.2 
Safety parameters 
7.2.1 Adverse events 
The investigator is responsible for the detection and documentation of events meeting the 
criteria and definition of an AE or serious adverse event (SAE), as provided in this 
protocol. During the trial, the investigator or center staff will be responsible for reporting 
AEs and SAEs, as detailed in this section of the protocol.  
Based on the investigator’s clinical judgment it will be determined whether an AE is 
related to treatment and of sufficient severity to require the subject’s removal from 
treatment or from the trial. A subject may also voluntarily withdraw from treatment due 
to what he or she perceives as an intolerable AE. If either of these situations arises, the 
subject should be under medical supervision until symptoms cease or the condition 
becomes stable. 
7.2.1.1 Definitions 
Adverse event 
An AE is any untoward medical occurrence in a trial subject given an IMP which does 
not necessarily have a causal relationship with the treatment. An AE can therefore be any 
unfavorable and/or unintended sign (for example, an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of an IMP, whether or not 
considered related to the IMP.  
In this trial, only treatment-emergent adverse events (TEAEs) will be collected and 
reported. TEAEs are events that occur from the first trial-related activity after the subject 
has signed the informed consent form until the end of the post-treatment follow-up 
period.  
AEs include: 
 A clinically significant worsening of a concomitant illness. 
 A clinical laboratory abnormality which is clinically significant, i.e. any abnormality 
that suggests a disease and/or organ toxicity and is of a severity that requires active 
management. Active management includes active treatment or further investigations, 
for example change of medicine dose or more frequent follow-up due to the 
abnormality. 
 Hypoglycemic episodes (see Section 7.2.2). 
 Injection site reactions (see Section 7.2.6). 


 
 
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The following should not be recorded as AEs, if recorded at screening (on the Screening 
Form or eCRF): 
 Pre-planned procedure, unless the condition for which the procedure was planned has 
worsened from the first trial related activity after the subject has signed the informed 
consent. 
 Pre-existing conditions found as a result of screening procedures (pre-existing 
conditions should be reported as medical history or concomitant illness). 
Serious adverse event 
An SAE is any untoward medical occurrence that at any dose:  
 Results in death  
 Is life-threatening  
 Requires inpatient hospitalization or prolongation of existing hospitalization 
 Results in persistent or significant disability or incapacity 
 Is a congenital anomaly or birth defect 
 Is medically important* 
*Medical judgement must be exercised in deciding whether an AE is believed to be 
‘medically important’. Medically important events may not be immediately 
life-threatening or result in death or hospitalization but may jeopardize the subject or 
may require intervention to prevent one of the other outcomes listed in the definition 
above. 
Adverse drug reaction 
All noxious and unintended responses to an investigational product related to any dose 
should be considered adverse drug reactions. The phrase responses to an investigational 
product means that a causal relationship between an investigational product and an 
adverse event is at least a reasonable possibility, i.e. the relationship cannot be ruled out. 
 
Suspected unexpected serious adverse reactions (SUSARs) 
An AE fulfilling one of the criteria of seriousness and being assessed as related to IMP 
application, the nature or severity of which is not consistent with the applicable reference 
document (e.g. dasiglucagon Investigator’s Brochure or package leaflet/Summary of 
Product Characteristics for GlucaGen). 


 
 
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Adverse event of special interest 
An AESI is an event which, in the evaluation of safety, has a special focus (e.g. required 
by health authorities). In this trial hemodynamic changes, as defined below, are 
considered AESIs: 
 Post-dose clinical signs, or measured vital signs, indicating a clinically significant 
drop in blood pressure including signs of orthostatic hypotension, vasovagal 
responses or bradycardia.   
 Post-dose change in pulse or blood pressure considered an event of hypo- or 
hypertension as judged by the investigator. 
 
Intensity of an adverse event 
The maximum intensity (severity) of all AEs must be assessed by the investigator and 
documented. Severity should be graded when the AE outcome is known: 
Mild:  
A type of AE that is usually transient and may require only minimal 
treatment or therapeutic intervention. The event does not generally 
interfere with usual activities of daily living. 
Moderate:  
A type of AE that is usually alleviated with additional specific therapeutic 
intervention. The event interferes with usual activities of daily living, 
causing discomfort but poses no significant or permanent risk of harm to 
the subject. 
Severe:  
A type of AE that interrupts usual activities of daily living, or significantly 
affects clinical status, or may require intensive therapeutic intervention. 
A ‘severe’ reaction does not necessarily deem the AE as ‘serious’ and an SAE may not 
necessarily be ‘severe’ in nature. 
Causality relationship to trial medication 
The causality of each AE should be assessed by the investigator according to the 
following classification: 
Probable: 
Good reason and sufficient documentation to assume a causal relationship. 
Possible: 
A causal relationship is conceivable and cannot be dismissed. 
Unlikely: 
The event is most likely related to etiology other than the trial product. 
Not related:  No relationship to trial product. 


 
 
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Outcome of an adverse event 
The outcome of all AEs must be assessed by the investigator and documented by his/her 
staff. The following definitions should be used: 
Recovered/resolved:  
The subject has fully recovered, or by medical or 
surgical treatment the condition has returned to the 
level observed at the first trial-related activity after 
the subject signed the informed consent.  
Recovering/resolving: 
The condition is improving and the subject is 
expected to recover from the event. This term is 
only applicable if the subject has completed the 
trial. 
Recovered/resolved with sequelae:  The subject has recovered from the condition, but 
with lasting effect due to a disease, injury, treatment 
or procedure. If a sequela meets an SAE criterion, 
the AE must be reported as an SAE. 
Not recovered/not resolved:  
The condition of the subject has not improved and 
the symptoms are unchanged. 
Fatal:  
This term is only applicable if the subject died from 
a condition related to the reported AE. Outcomes of 
other reported AEs in a subject before he/she died 
should be assessed as "recovered/resolved", 
"recovering/resolving", "recovered/resolved with 
sequelae" or "not recovered/not resolved". An AE 
with fatal outcome must be reported as an SAE. 
Unknown:  
This term is only applicable if the subject is lost to 
follow-up. 
7.2.1.2 Collection, recording and reporting of adverse events 
All events meeting the definition of an AE must be collected and reported from the first 
trial related activity after the subject has signed the informed consent until the end of the 
post-treatment follow-up period. At each contact with the center (visit or telephone, 
excluding visits where the subject is not seeing the investigator or his/her staff [e.g. visits 
to the laboratory]) the subject must be asked about AEs. All AEs, either observed by the 
investigator or reported by the subject, must be recorded by the investigator and 
evaluated. 
The investigator should record the diagnosis, if possible. If no diagnosis can be made the 
investigator should record each sign and symptom as individual AEs. 


 
 
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One single Adverse Event Form must be used per AE from start to resolution. For SAEs, 
the Serious Adverse Event form must also be completed. For AESIs, the AESI form must 
also be completed. 
AE information should include the following: 
 Date and time of onset and resolution 
 Date and time of investigator’s first information on the (S)AE 
 Seriousness 
 Severity 
 Causal relationship with IMP 
 Interruption or withdrawal of treatment during IMP administration and other 
measures taken 
 Outcome. 
All AEs are coded; details are described in the trial-specific Data Management Plan.  
If an event classifies as a AESI, the investigator must tick the AESI box on the AE form 
and complete the AESI form. The AESI form will capture if the event was associated 
with any signs or symptoms and capture the highest/lowest blood pressure and pulse 
measured during the event. The investigator must report all AESIs to the sponsor’s 
responsible pharmacovigilance unit (Safety CRO; see the list of trial personnel in 
Section 12.1) immediately (within 24 hours) after obtaining knowledge about the event. 
The investigator must report initial information electronically (e.g. in PDF format) on all 
SAEs to the sponsor’s responsible pharmacovigilance unit (Safety CRO; see the list of 
trial personnel in Section 12.1) immediately (within 24 hours) after obtaining knowledge 
about the event. The Safety CRO will inform the medical monitor and the sponsor about 
the reported SAEs. 
It is the responsibility of the Safety CRO to report all SUSARs (see Section 7.2.1.1) that 
occur in this trial to the Competent Authorities and Institutional Review Boards 
(IRBs)/Independent Ethics Committees (IECs) in accordance with the local requirements 
in force and ICH guideline for GCP. The trial monitor must be informed accordingly. 
7.2.1.3 Follow-up of adverse events 
All AEs that are ongoing at the end of the subject’s participation in the trial will be 
followed-up until the event is resolved or reaches a satisfactory outcome as deemed by 
the investigator or until the last visit of the last subject enrolled in the trial, whichever 
occurs first.   


 
 
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Follow-up actions for all SAEs will be performed according to appropriate clinical care 
practices and may depend on the nature of the event. These will be determined after 
internal review and/or sponsor review. 
The follow-up information should only include new (updated and/or additional) 
information that reflects the situation at the time of the investigator’s signature. 
Follow-up information on (S)AEs will be updated using the (S)AE form. If a non-serious 
event becomes serious during the follow-up the AE form and SAE form have to be used 
and reporting timelines follow those of an SAE. 
The investigator must forward follow-up information on SAEs and if previously 
non-serious AEs become SAEs to the Safety CRO immediately (within 24 hours) after 
obtaining knowledge about the new information. 
The sponsor and/or CROs acting on behalf of the sponsor can upgrade a non-serious AE 
to an SAE. In these situations the investigator will be informed and asked to fill out an 
SAE form and forward to the Safety CRO immediately (within 24 hours). 
7.2.1.4 Clinical laboratory abnormalities and other abnormal assessments as adverse 
events or serious adverse events 
Abnormal laboratory findings (e.g. biochemistry, hematology, urinalysis) or other 
abnormal assessments (e.g. vital signs) that are judged by the investigator as clinically 
significant will be recorded as AEs or SAEs if they meet the definition of an AE or SAE. 
Clinically significant abnormal laboratory findings or other abnormal assessments that 
are detected during the trial or are present at baseline and significantly worsen following 
the start of the trial will be reported as AEs or SAEs. However, clinically significant 
abnormal laboratory findings or other abnormal assessments that are associated with the 
disease being studied, unless judged by the investigator as more severe than expected for 
the subject’s condition, or that are present or detected at the start of the trial and do not 
worsen, will not be reported as AEs or SAEs. 
The investigator will exercise his or her medical and scientific judgment in deciding 
whether an abnormal laboratory finding or other abnormal assessment is clinically 
significant. 
7.2.2 Hypoglycemia 
Hypoglycemia will be regarded as an AE and recorded and documented on an AE form 
(and SAE form, if applicable). 


 
 
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Hypoglycemia is defined as a decline in plasma glucose to below 70 mg/dL 
(3.9 mmol/L). However, in the time period from initiation of the hypoglycemic clamp 
procedure (see Section 8.2.3.1) until 45 minutes after dosing, hypoglycemia is defined as 
a decline in plasma glucose to below 45 mg/dL (2.5 mmol/L).  
During the dosing visit, prior to administration of the IMP, a plasma glucose value of 
<45 mg/dL (2.5 mmol/L) will be treated by an IV glucose solution. After administration 
of the IMP in the period from 8 minutes until 44 minutes after dosing, a plasma glucose 
value of <45 mg/dL (2.5 mmol/L) will be treated by an IV glucose solution, and if a 
plasma glucose value of ≥70 mg/dL (3.9 mmol/L) is not achieved within the 45 minutes 
after IMP administration, IV glucose infusion will also be initiated. 
If the subject experiences symptoms of hypoglycemia, a plasma glucose measurement 
should be taken in order to classify the event (please refer to Section 7.4.2 for additional 
details).  
7.2.3 Physical examination 
The physical examination will be carried out at screening (Visit 1) and at the follow-up 
visit (Visit 3; see Table 3).  
The physical examination includes examination of the following body systems: head, 
ears, eyes, nose, throat, including the thyroid gland; heart, lung, chest; abdomen; skin and 
mucosae; musculoskeletal system; nervous system; lymph node; other findings. 
At the screening visit, any abnormality will be recorded and described in the eCRF, 
including the investigator’s assessment of clinical significance (‘abnormal, not clinically 
significant’ or ‘abnormal, clinically significant’). Clinically significant findings at the 
screening visit should be recorded as concomitant illness. 
At subsequent visits, any clinically significant deterioration of a pre-existing condition as 
well as any new clinically significant findings will be recorded as AEs (see 
Section 7.2.1). 
7.2.4 Vital signs 
An examination of the following vital signs will be performed at screening (Visit 1), the 
dosing visit (Visit 2) and at the follow-up visit (Visit 3):  
 Diastolic and systolic blood pressure (mmHg) will be measured after at least 
5 minutes rest in a supine position. At Visit 1, blood pressure will be measured in 
both arms. The blood pressure from the arm with the higher systolic value is 


 
 
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transcribed into the eCRF and this arm should be used for all subsequent 
measurements of the subject’s blood pressure in this trial.  
 Pulse (beats per min) measured after at least 5 minutes rest in a supine position. 
 Body temperature (°C). 
At the dosing visit, measurements will be taken prior to the start of the insulin-induced 
hypoglycemic procedure (within 30 minutes) and at 30, 90, and 300 minutes after dosing 
(see Table 3). The actual time of the assessment should not deviate from the nominal time 
by more than ±10 minutes. In addition to the pre-specified assessments as listed in 
Table 3, blood pressure and pulse may be assessed at any time during the trial at the 
discretion of the investigator.  
7.2.5 Electrocardiogram 
A standard 12-lead ECG will be performed at the screening visit (Visit 1), the dosing visit 
(Visit 2; prior to the start of the insulin-induced hypoglycemic procedure [within 
30 minutes]) and at 20, 35, 45, 60, and 300 minutes after dosing) and at the follow-up 
visit (Visit 3; see Table 3). The actual time of the assessment should not deviate from the 
nominal time by more than ±5 minutes.  
ECG parameters (heart rate, PQ, QRS, QT, QTcB) and any abnormality will be recorded 
and described in the eCRF including the investigator’s assessment of clinical significance 
(‘abnormal, not clinically significant’ or ‘abnormal, clinically significant’).  
At subsequent visits, any clinically significant deterioration of a pre-existing condition as 
well as any new clinically significant findings will be recorded as AEs (see 
Section 7.2.1). 
7.2.6 Local tolerability 
Immediately prior to treatment administration, it should be verified that the injection site 
is normal. To ensure all injection site assessments are performed at the injection site, the 
site will be marked with a pen prior to injection. Assessment of local tolerability at the 
injection site will be performed at the dosing visit (Visit 2; at 30, 120 and 300 minutes 
after dosing), and at the follow-up visit (Visit 3) (see Table 3) and more frequently, if 
deemed necessary by the investigator. The actual time of the assessment should not 
deviate from the nominal time by more than ±10 minutes. In the eCRF, the time of 
assessment and any injection site reaction observed will be recorded. In case of an 
observation, the Local Tolerability form will be completed, as well as the (S)AE form. 
The local tolerability at the injection site will be evaluated by means of the following 
assessments: spontaneous pain, pain on palpation, itching, redness, edema, 


 
 
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induration/infiltration, and other. Each of these assessments will be reported on a scale of 
0 (none), 1 (mild), 2 (moderate), and 3 (severe). The evaluation and the actual time of the 
assessment will be recorded. The assessments will be performed by a trial physician or 
nurse.  
Digital pictures will be taken of the injection site at the time of identification, and 
thereafter as often as judged necessary by the investigator. The pictures should include a 
subject identifier, visit number, time after dosing, and a ruler for scaling. 
7.2.7 Clinical laboratory assessments 
The safety parameters that will be assessed at the clinical laboratory are listed in Table 3. 
Routine clinical laboratory tests will be performed centrally. Samples for clinical 
laboratory parameters (biochemistry, hematology, coagulation) will be collected at 
screening (Visit 1), the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure [within 30 minutes]) and at 30 and 300 minutes after dosing), 
and at the follow-up visit (Visit 3). The actual time of the assessment should not deviate 
from the nominal time by more than ±10 minutes. Samples for glycated hemoglobin 
(HbA1c) will be collected at screening only (Visit 1). Samples for urinalysis will be 
collected at screening (Visit 1), at the dosing visit (Visit 2, prior to the start of the 
insulin-induced hypoglycemic procedure [within 120 minutes]) and at the follow-up visit 
(Visit 3). The following parameters will be assessed: 
 Clinical biochemistry: sodium, potassium, calcium, glucose, urea, creatinine, total 
bilirubin, AST, ALT, gamma-glutamyltransferase, alkaline phosphatase, total protein, 
C-reactive protein, HbA1c, C-peptide. 
 Hematology: hemoglobin, red blood cell count (erythrocytes), hematocrit, platelet 
count (thrombocytes), total white blood cell count (leukocytes). 
 Coagulation: international normalized ratio, fibrinogen (at screening visit only).  
 Urinalysis: pH, blood (leukocytes and erythrocytes), protein, glucose, ketones, nitrite. 
Pregnancy tests will be performed at each visit for women of childbearing potential only. 
A serum pregnancy test will be performed at screening (Visit 1) and urine stick tests will 
be performed at the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure) and the follow-up visit (Visit 3). Test sticks will be provided to 
the trial centers. 
Alcohol breath tests and a urine drug screen will be performed at screening (Visit 1) and 
at the dosing visit (Visit 2, prior to the start of the insulin-induced hypoglycemic 
procedure). Equipment for the alcohol breath test and uring drug screen will be provided 
to the trial centers.  


 
 
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Re-assessment of laboratory parameters will be allowed only if handling issues, damaged 
samples, or hemolyzed samples have confounded the measurement results.  
For further details of the clinical laboratory assessments, please refer to the laboratory 
manual.  
7.2.8 Pregnancy 
Female subjects must be instructed to notify the investigator immediately if they become 
pregnant or if they suspect to be pregnant during the trial. The sponsor has a 
responsibility to monitor the outcome of all pregnancies reported during the clinical trial. 
Male subjects must be instructed to notify the investigator immediately if their partner 
becomes pregnant or suspects pregnancy. The sponsor has a responsibility to monitor the 
outcome of all pregnancies reported during the clinical trial. During an information 
meeting at the trial center, the subject’s partner will be fully informed by a physician of 
her participation in capturing the outcome data, and will be asked to provide her written 
consent. 
The investigator must report all information on pregnancies on the Initial Pregnancy 
form. The completed Initial Pregnancy form must be forwarded to the sponsor 
immediately (within 24 hours), according to the procedure stated in Section 7.2.1.2. Any 
(S)AEs in the mother, as well as other pregnancy complications (ectopic pregnancy) 
fulfilling the criteria of an (S)AE, must be reported on the (S)AE form.  
The following must be collected in the Initial Pregnancy form:  
 Medical history of the mother 
 Family history 
 Course of the pregnancy, including expected delivery date. 
The investigator must follow the pregnancy until the pregnancy outcome and follow the 
newborn infant(s) until the age of 1 month. The investigator must collect information on 
the pregnancy and pregnancy complications as well as the pregnancy outcome including 
the health of the newborn infant(s) on the Pregnancy Outcome form. The completed 
Pregnancy Outcome form must be forwarded to the sponsor according to the procedure 
stated in Section 7.2.1.2. Any (S)AEs in the newborn must be reported on the (S)AE 
form. 
The SAEs that must be reported include abnormal outcome, such as congenital 
anomalies, fetal death and termination of pregnancy (spontaneous or elective abortion), 
including any anomalies of the fetus observed at gross examination or during autopsy.  


 
 
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The following must be collected in the Pregnancy Outcome form:  
 Course of the pregnancy 
 Outcome of the pregnancy 
 Condition of the newborn 
 Any AEs in the newborn infant must be followed until the age of 1 month. 
7.2.9 Precautions 
Normal precautions taken for a human trial, including the provision of emergency 
equipment, will be taken during this trial. Qualified and well trained physicians and 
medical staff will instruct the subjects. During a subject’s participation in the trial, the 
investigator should ensure that adequate medical care is provided to the subject for any 
AEs, including clinically significant laboratory values related to the trial. The investigator 
should inform the subject when medical care is needed for intercurrent illnesses of which 
the investigator becomes aware.  
For further information on safety precautions for dasiglucagon and GlucaGen, please 
refer to the current version of the Investigator’s Brochure (14) and the Summary of 
Product Characteristics for GlucaGen (13), respectively. 
7.2.10 Safety committee 
The internal Zealand Pharma Safety Committee is constituted to perform ongoing blinded 
safety surveillance of clinical trials with dasiglucagon, including this trial. 
If safety signals are observed, either based on reported SAEs, periodic review of 
laboratory parameters, review of all AEs reported between the Safety Committee 
meetings, or on notification of significant findings, the Safety Committee will take 
appropriate measures to safeguard the subjects. 
7.3 
Demography, concomitant illness, medical history and concomitant 
medication 
Demographics, body measurements, concomitant illness and medical history will be 
assessed only at screening (Visit 1). Concomitant medication will be assessed at 
screening (Visit 1), the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure) and at the follow-up visit (Visit 3). 
7.3.1 Demography and body measurements 
Subject demographics and body measurements will include: 


 
 
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 Age  
 Race, ethnicity 
 Sex 
 Height (meters or inch), without shoes 
 Body weight (kg or lb), only wearing underwear and measured using standard scales 
 Body mass index (kg/m2) calculated based on height and body weight (body 
weight/height2). 
7.3.2 Concomitant illness and medical history 
A concomitant illness is any illness that is present at the start of the trial (i.e. at the 
screening visit). Concomitant illnesses present at the start of the trial will be recorded in 
the eCRF at screening.  
Medical history is an account of medical events that the subject has experienced in the 
past, including prior medications. Relevant medical conditions/illnesses in the past 
obtained by asking the subject or by inspecting his/her medical records will be recorded 
in the eCRF at screening. History of alcohol or drug abuse will also be recorded. 
The information collected for concomitant illness and medical history should include 
diagnosis, date of onset, date of resolution or continuation. 
Any change to a concomitant illness should be recorded during the trial, including end 
date, if applicable. A clinically significant worsening of a concomitant illness must be 
reported according to Section 7.2.1. 
All previous and concomitant diseases will be coded with Medical Dictionary for 
Regulatory Activities (MedDRA), the version of which will be provided in the clinical 
trial report. 
7.3.3 Diabetes diagnosis and current treatment 
The date of diagnosis of diabetes will be recorded as will the current diabetes treatment 
(start date, product name(s), dose(s)). 
7.3.4 Concomitant medication 
A concomitant medication is any medication, other than the trial products and current 
diabetes treatment (including insulin glulisine [Apidra®] for diabetes therapy wash-out), 
which is taken during the trial, including screening and follow-up periods. 


 
 
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Details of any concomitant medication must be recorded at trial entry (i.e. at screening). 
Any changes in concomitant medication must be recorded at each visit as they occur. The 
information collected for each concomitant medication includes (at a minimum) trade 
name or generic name, indication, start date and stop date or continuation. A change in 
medication due to an AE must be recorded and reported according to Section 7.2.1. If the 
change in medication influences the subject’s eligibility to continue in the trial, the 
sponsor and trial monitor must be informed. 
7.4 
Other assessments 
7.4.1 Immunogenicity 
Antibodies against dasiglucagon/GlucaGen will be measured at the dosing visit (Visit 2) 
and at follow-up (Visit 3). At the dosing visit (Visit 2), samples will be collected prior to 
the start of the insulin-induced hypoglycemic procedure.  
The clinical ADA assays, specific for dasiglucagon and GlucaGen, respectively, have 
been validated in accordance with existing guidelines and recommendations (17-21).  
Confirmed positive anti-dasiglucagon antibody samples (treatment-induced or 
treatment-boosted) from anti-dasiglucagon antibody-positive subjects will be evaluated 
for binding titer neutralizing potential and titer as well as cross-reactivity towards 
endogenous glucagon.  
No further serum sampling will be needed as the ADA samples can be used for 
neutralizing antibody analysis. 
The in vitro neutralizing effect of the antibodies will be measured using an assay based 
on glucagon receptor transfected human embryonic kidney cells (20,22). The calculated 
sensitivity in previous studies was about 51.8 ng/mL. The assay was also validated for 
recombinant glucagon with similar results (21,23). In case of a positive result in the 
neutralizing antibody assay, a titer estimation will be performed. The cell-based 
neutralizing antibody analyses will be performed by a special laboratory, BioAgilytix, 
Durham, NC, USA. 
Residual and additional antibody serum samples may be stored until approval of market 
authorization by the health authorities. Further characterization of the antibody response 
may be requested by the health authorities. 
7.4.2 Plasma glucose measurements for safety 
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at 
site. After the start of insulin infusion, plasma glucose should be checked every 


 
 
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10 minutes while plasma glucose is above 110 md/dL, and every 5 minutes once plasma 
glucose is at or below 110 mg/dL and until after dosing when the subject’s plasma 
glucose is ≥70mg/dL (3.9 mmol/L). Hereafter, plasma glucose should be checked every 
30 minutes until 300 minutes (5 hours) after dosing. Plasma glucose concentrations will 
be measured using a US FDA-approved glucose analyzer (e.g., YSI 2300, Yellow 
Springs Instruments, Yellow Springs, OH). 
At the discretion of the investigator, additional plasma glucose measurements can be 
taken at any time during the trial, for example when there is a suspicion (e.g. symptoms) 
of a hypoglycemic episode. 
Plasma glucose measurements for safety should only be recorded in the eCRF if they are 
related to an AE (e.g. a hypoglycemic episode). 
In case of persistent post-treatment hypoglycemia, subjects will receive rescue treatment 
with an IV glucose infusion (see Section 8.2.3.1 for details). Blood samples for PD and 
PK assessments should still be taken at the specified timepoints. 
7.4.3 Plasma insulin measurements 
Samples for insulin assessment will be collected at the dosing visit (Visit 2, pre-dose and 
at 30 and 60 minutes after dosing). The actual time of blood sampling for evaluation of 
plasma insulin should not deviate from the nominal time by more than ±1 minute. 
Pre-dose is defined as within 2 minutes prior to dosing. 


 
 
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8 
TRIAL CONDUCT 
8.1 
Schedule of procedures 
The schedule of procedures is provided in Table 3. Informed consent will be obtained 
prior to any trial-related procedures; see Section 10.8. 
8.2 
Procedures by visit 
8.2.1 Visit 1 (screening, Day -30 to Day -3) 
Visit 1 will take place between 3 and 30 days before Visit 2, Day -1 to Day 1 (dosing 
day). 
Informed consent can be obtained prior to or at Visit 1, however it must in any case be 
obtained prior to any trial related procedures. During the screening visit, the following 
assessments will take place: 
 Informed consent (obtain or check) 
 Check of subject eligibility (inclusion/exclusion criteria) 
 Demography  
 Body measurements 
 Medical history, diabetes diagnosis, current diabetes treatment 
 Concomitant illnesses 
 Concomitant medications 
 History of alcohol/drug abuse 
 Physical examination 
 Vital signs 
 12-lead ECG 
 AEs  
 Biochemistry, hematology, coagulation, HbA1c 
 Serum pregnancy test (women of childbearing potential only) 
 Urinalysis  
 Urine drug screen 
 Alcohol breath test 


 
 
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Table 3 
Schedule of procedures 
Visit number 
V1 
V2 
V3 
Trial day 
-3 
-1 and 1 
28 
Visit type 
Screening 
Dosing 
Follow-up 
Window 
-30 to -3 
 
+5 days 
Subject related information/assessments 
 
 
 
Informed consent 
x 
 
 
Inclusion/exclusion criteria 
x 
x1,2 
 
Demography 
x 
 
 
Body measurements 
x 
 
 
Medical history, diabetes diagnosis, and current diabetes 
treatment 
x 
 
 
Concomitant illnesses 
x 
 
 
Concomitant medications 
x 
x1 
x 
History of alcohol/drug abuse 
x 
 
 
Randomization 
 
x1 
 
Withdrawal criteria 
 
x1 
 
Dosing day exclusion criteria 
 
x1 
 
Insulin-induced hypoglycemia 
 
x 
 
Safety assessments 
 
 
 
Physical examination 
x 
 
x 
Vital signs 
x 
x3 
x 
12-lead ECG 
x 
x4 
x 
Local tolerability  
 
x5 
x 
Adverse events 
x 
x 
x 
Laboratory 
 
 
 
Biochemistry, hematology, coagulation, HbA1c (HbA1c at 
Visit 1 only) 
x 
x6 
x 
Pregnancy test (women of childbearing potential only) 
x7 
x1,8 
x8 
Urinalysis 
x 
x1 
x 
Urine drug screen 
x 
x1 
 
Alcohol breath test 
x 
x1 
 
PK/Clinical efficacy 
 
 
 
Plasma dasiglucagon/GlucaGen 
 
x9 
 
Plasma glucose 
 
x10 
 
Other assessments 
 
 
 
Antibodies against dasiglucagon/GlucaGen 
x1 
x11 
Plasma insulin 
 
x12 
 
Trial material 
 
 
 
Administration of trial product (during hypoglycemic clamp 
procedure) 
 
x 
 
1Prior to the start of the insulin-induced hypoglycemic procedure. 
2Only check of dosing day exclusion criteria and changes between screening visit and Visit 2. 
3Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 30, 90 and 300 minutes 
after dosing. The actual time of the assessment should not deviate from the nominal time by more than ±10 minutes. 


 
 
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4Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 20, 35, 45, 60 and 300 
minutes after dosing. The actual time of the assessment should not deviate from the nominal time by more than ±5 
minutes.  
5Local tolerability assessed at 30, 120, and 300 minutes after dosing. The actual time of the assessment should not 
deviate from the nominal time by more than ±10 minutes.  
6Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 30 and 300 minutes after 
dosing. The actual time of the assessment should not deviate from the nominal time by more than ±10 minutes. 
7Serum pregnancy test. 
8Urine stick pregnancy test. 
9Pre-dose, and at 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time of blood sampling should not 
deviate from the nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing. 
10Pre-dose, and at 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, 90 minutes after dosing. The actual time of blood 
sampling should not deviate from the nominal time by more than ±30 seconds until the 20 minute collection time point 
and by more than ±1 minute for the subsequent collection time points. Pre-dose is defined as within 2 minutes prior to 
dosing. 
11Any subject that tests positive for ADA will be monitored until the ADA levels return to baseline levels. 
12Pre-dose, and at 30 and 60 minutes after dosing. The actual time of blood sampling should not deviate from the 
nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing.  
ADA = anti-drug antibodies; ECG = electrocardiogram; HbA1c = glycated hemoglobin. 
Eligible or potentially eligible subjects (laboratory results pending) will be provided with 
an Identification card (ID card), stating that the subject is participating in the trial and 
whom to contact (site address, investigator name and telephone number). The subjects 
should be instructed to return the ID card to the investigator at the last visit or to destroy 
the card after the last visit.  
8.2.2 Instructions to subjects prior to the dosing visit (Visit 2) 
At the screening visit, the investigator will inform the subject about the changes to his/her 
insulin therapy leading up to the start of the insulin-induced hypoglycemic procedure. 
The subject’s current insulin therapy will be washed out as defined in Section 5.5: 
16 hours prior to dosing and during the dosing visit, treatment with any basal insulin is 
not allowed; 6 hours prior to dosing and during the dosing visit, treatment with any short 
acting (bolus) insulin, except insulin glulisine (Apidra®), is not allowed. The basal rate of 
insulin pumps (continuous SC insulin infusion) will be discontinued on the morning of 
the dosing day OR 6 hours prior to dosing. 
On the day prior to dosing (Day -1), the subjects will need to attend the clinical center 
and will be required to stay onsite overnight. On the morning of the dosing day (Day 1), 
patients are required to be in a fasting condition, defined as having consumed only water 
since 22:00 hours the night before. However, the subjects are allowed to consume small 
amounts (up to 20 g) of carbohydrates to prevent hypoglycemia. The subjects must also 
not consume any alcohol within 24 hours prior to dosing (refer to Section 5.5 for all 
dosing day exclusion criteria). 


 
 
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8.2.3 Visit 2 (dosing visit) 
Visit 2 will take place on Day -1 to Day 1. 
The subjects will attend the clinical center the day prior to dosing (Day -1) and should 
stay onsite overnight. Dosing will take place the following morning (Day 1). 
On Day 1 and prior to the start of the insulin-induced hypoglycemic procedure, subject 
eligibility is rechecked (check of changes between the screening visit and Visit 2) and 
those subjects eligible to participate will be randomized to treatment with dasiglucagon, 
placebo, or GlucaGen.  
The following assessments will also take place: 
 Document all changes in concomitant medication (prior to the start of the 
insulin-induced hypoglycemic procedure) 
 Check of withdrawal criteria (prior to the start of the insulin-induced hypoglycemic 
procedure) 
 Check of dosing day exclusion criteria (prior to the start of the insulin-induced 
hypoglycemic procedure) 
 Vital signs (prior to the start [within 30 minutes] of the insulin-induced hypoglycemic 
procedure, and at 30, 90 and 300 minutes after dosing). Blood pressure should be 
measured first in sitting and then in standing. 
 12-lead ECG (prior to the start [within 30 minutes] of the insulin-induced 
hypoglycemic procedure, and at 20, 35, 45, 60, and 300 minutes after dosing) 
 Local tolerability (at 30, 120, and 300 minutes after dosing) 
 AEs  
 Biochemistry, hematology, coagulation (prior to the start of the insulin-induced 
hypoglycemic procedure [within 30 minutes], and at 30 and 300 minutes after dosing) 
 Urine stick pregnancy test (women of childbearing potential only; prior to the start of 
the insulin-induced hypoglycemic procedure) 
 Urinalysis (prior to the start of the insulin-induced hypoglycemic procedure [within 
120 minutes]) 
 Urine drug screen (prior to the start of the insulin-induced hypoglycemic procedure) 
 Alcohol breath test (prior to the start of the insulin-induced hypoglycemic procedure) 
 Dasiglucagon/GlucaGen plasma concentrations: 
- 
Pre-dose, 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time 
of blood sampling for exposure should not deviate from the nominal time by more 
than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing. 
 Plasma glucose concentrations:  
- 
Pre-dose, 4, 6, 8 ,10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, and 90 minutes after 
dosing. The actual time for blood sampling for plasma glucose should not deviate 


 
 
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from the nominal time by more than ±30 seconds until the 20 minute collection 
time point and by more than ±1 minute for the subsequent collection time points. 
Pre-dose is defined as within 2 minutes prior to dosing. 
 Antibodies against dasiglucagon/GlucaGen (prior to the start of the insulin-induced 
hypoglycemic procedure). 
 Plasma insulin concentrations: 
- 
Pre-dose, 30 and 60 minutes after dosing. The actual time of blood sampling for 
plasma insulin should not deviate from the nominal time by more than ±1 minute. 
Pre-dose is defined as within 2 minutes prior to dosing. 
8.2.3.1 Hypoglycemic clamp procedure and administration of trial medication 
The following procedure is based on precendent procedures for hypoglycemia induction 
in patients with T1DM [24, 25]. 
The treatment day (Visit 2, Day 1) will be conducted after an overnight fast of at least 
8 hours, targeting a starting plasma glucose around 90 to 110 mg/dL (5.0-6.1 mmol/L).   
Subjects who meet one or more of the dosing day exclusion criteria (Section 5.5) at the 
dosing visit will be excluded from the dosing visit, but the visit can be rescheduled 
1-7 days later. The dosing visit can only be rescheduled once. 
The subject’s current insulin therapy will be washed out as defined in Section 5.5. For 
subjects using multiple daily injections, the date, time and the dose of the last basal 
insulin administration prior to dosing will be captured. 
At approximately 08:00 hours, an infusion catheter will be inserted into each arm 
(forearm cephalic vein) for the manual glucose clamp procedure, with the glucose 
infusion in one arm and the insulin infusion in the opposite arm. A third catheter for 
blood sampling will be placed into a metacarpel vein for blood sampling. This hand will 
be warmed (55-65ºC) to arterialize venous blood.   
Hypoglycemia will be gradually induced by a fast-acting IV insulin glulisine (Apidra®) 
infusion (15 U [100 U/mL] in 49 mL saline and 1 mL of subject’s blood or plasma), 
initially at 150% of the subject’s usual basal rate and can be increased or decreased over a 
range of 75% to 200% as judged necessary by the investigator, to achieve a controlled 
decline in plasma glucose, targeting a plasma glucose level of 55 mg/dL (3.1 mmol/L).  
Plasma glucose concentrations will be measured using a US FDA-approved glucose 
analyzer (e.g., YSI 2300, Yellow Springs Instruments, Yellow Springs, OH). After the 
start of the insulin infusion, plasma glucose will be measured every 10 minutes while 


 
 
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plasma glucose is above 110 mg/dL, and every 5 minutes once plasma glucose is at or 
below 110 mg/dL. 
Once the glucose concentration declines to <60 mg/dL (3.3 mmol/L), the insulin infusion 
will be stopped, and 5 min later plasma glucose concentration will be measured at the 
glucose analyzer and blood samples for baseline assessment of plasma glucose, 
dasiglucagon/GlucaGen PK, and insulin PK will be collected. The samples are the 
baseline samples and should be collected within 2 minutes before IMP administration. 
 If plasma glucose is >45 mg/dL and <60 mg/dL (2.5-3.3 mmol/L), study treatment 
(IMP) will be administered, defining time, t=0. The study treatment will be delivered 
in the abdomen, buttock, or thigh (according to stratification) via SC injection, with 
the subject lying in a lateral recumbent position. 
 If plasma glucose is <45 mg/dL (2.5 mmol/L), IV glucose solution will be 
administered sufficient to raise plasma glucose to within the 45-60 mg/dL target 
range. The run-in period will be adequately extended (at least 30 min) until the above 
target is achieved and new baseline samples for plasma glucose, 
dasiglucagon/GlucaGen PK, and insulin PK will be collected. Glucose should not be 
infused within 10 minutes before IMP administration. If plasma glucose is not within 
target range after the second attempt, the subject should be rescheduled for a new 
treatment visit within 7 days (+ 2 days). 
Administration of IMP should not occur earlier than 9:00 hours in the morning or later 
than 12:00 hours. 
As shown in Table 4, serial blood samples for glucose will be collected at t=0, 4, 6, 8, 10, 
12, 15, 17, 20, 25, 30, 40, 50, 60, 75 and 90 minutes post-dosing. Samples for assessing 
plasma dasiglucagon/GlucaGen concentration will be collected  at t=0, 15, 30, 35, 40, 50, 
60, 90, and 120 minutes. Samples for assessing plasma insulin concentration will be 
collected at t=0, 30, and 60 minutes.  
Table 4 
Post-treatment blood sampling times 
Times 
0 
4 
6 
8 
10 
12 
15 
17 
20 
25 
30 
35 
40 
50 
60 
75 
90 
120 
Plasma glucose 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
 
Y 
Y 
Y 
Y 
Y 
 
PK 
Dasiglucagon/ 
GlucaGen 
Y 
 
 
 
 
 
Y 
 
 
 
Y 
Y 
Y 
Y 
Y 
 
Y 
Y 
PK Insulin 
Y 
 
 
 
 
 
 
 
 
 
Y 
 
 
 
Y 
 
 
 
Refer to Section 7.2.7 for details of laboratory safety sampling and to Section 7.4.2 for 
details of blood glucose safety sampling.  


 
 
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When the t=90-minute blood sampling for plasma glucose has been collected the subjects 
are allowed to eat moderately. Drinking of water is allowed ad libitum during the entire 
procedure. 
Hypoglycemia Rescue Provisions 
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at 
site for safety reasons as described in Section 7.4.2. Subjects should receive 
post-treatment rescue glucose infusion to ameliorate persistent hypoglycemia, as follows. 
1. Glucose infusion should be initiated if a subject experiences severe alarming 
escalation of symptoms of hypoglycemia (e.g. symptoms suggesting a change in 
consciousness) at any time during the trial; glucose infusion should be initiated 
targeting a plasma glucose levels >70 mg/dL. 
2. If plasma glucose is <45 mg/dL (2.5 mmol/L) between t=8 and t=44 minutes, 
rescue glucose infusion (1-2 mg/kg administered IV over about 5 seconds) should 
be initiated to maintain plasma glucose between 45 mg/dL and 55 mg/dL 
(2.5-3.1 mmol/L). Pause glucose infusion if plasma glucose is >55 mg/dL. 
3. If plasma glucose is <70 mg/dL at t=45 minutes, rescue glucose infusion 
(2-3 mg/kg administered IV over about 5 seconds) should be initiated to maintain 
plasma glucose between 70 mg/dL and 80 mg/dL (3.9-4.4 mmol/L). Pause 
glucose infusion if plasma glucose is >75 mg/dL (4.2 mmol/L). 
Subjects should remain in bed until completion of the test procedure 300 minutes after 
dosing (bathroom visits are allowed).  
The IMP will be administered SC according to Section 6.3. The time of IMP 
administration will be recorded. At the timepoint when the insulin infusion is stopped, the 
total insulin dose which was required to induce hypoglycemia will be recorded. 
AEs will be specifically recorded during the procedure at several timepoints. 
The investigator must provide information to the subjects on how to resume their usual 
diabetes treatment. 
The subject may be released from the clinical center if the investigator does not have any 
safety concerns based on the last safety plasma glucose value and the general condition of 
the subject. However, at the discretion of the investigator or on request of the subject, the 
subject may stay at the trial center for a longer period. 
8.2.4 Visit 3 (follow-up visit) 
Visit 3 will take place on Day 28 + 5 days. The subject does not need to be fasting. 


 
 
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At Visit 3, the following assessments will take place: 
 Document all changes in concomitant medication  
 Physical examination 
 Vital signs 
 12-lead ECG 
 Local tolerability 
 AEs  
 Biochemistry, hematology, coagulation 
 Urine stick pregnancy test (women of childbearing potential only) 
 Urinalysis 
 Antibodies against dasiglucagon/GlucaGen. 
After the follow-up visit the End of Trial form must be completed. Even if a subject is not 
able to attend the follow-up visit, the End of Trial form, the eCRF 
Accountability/Affirmation Statement form and the Drug Accountability form must be 
completed. 


 
 
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9 
STATISTICAL METHODS 
Before database lock and treatment unmasking, a separate statistical analysis plan (SAP) 
will be finalized, providing detailed methods for the analyses outlined below. Further 
analysis details may be added or refined in the SAP. 
Any deviations from the planned analyses will be described and justified in the final 
clinical trial report. 
9.1 
Determination of sample size 
Due to requirements in the size of the safety database, the sample size is set to 78 subjects 
treated with dasiglucagon 0.6 mg. With a 2:1:1 randomization ratio for the three 
treatments, dasiglucagon 0.6 mg, placebo, and GlucaGen 1 mg, 78 subjects injected with 
dasiglucagon and 39 subjects with placebo will yield a power of 90% at a 5% two-sided 
significance level to detect a treatment group difference in recovery incidence within 
20 minutes, assuming as low as 80% and as high as 50% recovery incidence for the 
dasiglucagon and placebo groups, respectively. 
9.2 
Trial subjects 
9.2.1 Analysis samples 
For presentation of data and reporting of the statistical analyses, the following analysis 
samples will be used, depending on the context:  
 Safety analysis set (SAS): All randomized subjects who received at least one dose of 
trial medication.  
 Full analysis set (FAS): All randomized subjects who received at least one dose of 
trial medication and contributed valid information for at least one post-dose endpoint. 
 Per protocol (PP) set: All subjects of the FAS for whom no relevant protocol 
deviations were documented. This sample will primarily be used for sensitivity 
analysis.  
The analysis of the primary endpoint and secondary endpoints will be based on the FAS. 
A secondary analysis of the primary endpoint will be based on the PP set. All safety 
analyses will be based upon the SAS. 
The decision regarding whether a protocol deviation is relevant or not for the exclusion of 
subjects from the PP set will be made case-by-case in a data review meeting prior to 
treatment unmasking and database lock (see Section 9.2.3). 


 
 
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9.2.2 Disposition of subjects 
Subject disposition will be tabulated including the number of screened subjects, screening 
failures, subjects exposed to trial product, subjects completing the trial and subjects in 
each analysis set. 
Subjects withdrawn from the trial will be listed including the primary reason for 
withdrawal. 
9.2.3 Protocol deviations 
Before data are released for statistical analysis, a treatment-masked review of all data will 
take place to identify protocol deviations that may potentially affect the results. This 
review will be performed without revealing to which trial product the subjects were 
assigned. The masking of the trial products will be maintained for everyone involved in 
allocating subjects to the analysis sets until data are released for statistical analysis. 
Furthermore, spurious outliers will be evaluated. In addition, protocol deviations that may 
potentially affect the results will be identified and it will be evaluated if subjects and/or 
data should be excluded from the analysis. Protocol deviations will be classified as minor 
or major in a consistent way. Major deviations from the protocol may lead to the 
exclusion of a subject from the PP set. 
Major protocol deviations may include deviations related to trial inclusion or exclusion 
criteria, conduct of the trial, subject management or subject assessment. Unless explicitly 
decided otherwise during the masked data review, the following will be considered major 
protocol deviations: 
 Violation of one or more major inclusion/exclusion criteria 
 Interruption of administration of trial product 
 Significant deviation from time windows  
 Incorrect treatment allocation 
 Missing primary endpoint. 
The violation of several major inclusion/exclusion criteria or the complete absence of 
efficacy data might lead to exclusion of the subject from FAS. In that case, the decision 
should be taken at the masked data review meeting, and the exclusion from efficacy 
analysis justified in the signed notes of the meeting. 
Obviously erroneous data points may be excluded from the analyses or re-analyzed (in 
case of e.g. serum concentrations). The decision to re-analyze or exclude data points from 
the statistical analysis is the joint responsibility of the sponsor and the trial statistician.  


 
 
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The subjects or observations to be excluded and the reason for their exclusion will be 
documented and signed by those responsible prior to database release. The 
documentation will be stored together with the remaining trial documentation. The 
subjects and observations excluded from analysis sets, and the corresponding reasons, 
will be described in the clinical trial report. 
9.3 
General considerations 
All data obtained in this trial and documented in the eCRFs will be listed and 
summarized with statistics or frequency tables as appropriate. In case of termination of 
the trial, all data collected up to that timepoint will be included in the analysis. 
Raw data listings and summary tables will be generated using the software SAS© version 
9.4 or higher.  
Continuous variables will be summarized using means, standard deviations, medians, 
coefficients of variation, and minimum and maximum values.  
Other summaries (e.g. quartiles, 95% confidence intervals [CIs]) may be used as 
appropriate. Categorical efficacy and safety variables will be summarized by counts and 
by percentage of subjects in corresponding categories. 
9.4 
Demographics and baseline characteristics 
Baseline and demographic data will be summarized using descriptive statistics. Baseline 
ADA-positive subjects will be calculated as a percentage of the total number of subjects 
whose baseline samples were tested for ADA. 
All other data obtained in this trial and documented in the eCRF will be listed. 
9.5 
Efficacy Analyses 
For the confirmatory analyses, the primary and key secondary endpoints for the 
dasiglucagon 0.6 mg and placebo treated subjects will be compared. A hierarchical 
procedure will be applied for the control of multiplicity. The primary and secondary 
endpoints will be evaluated on the full analysis set. In the primary analysis, those subjects 
who require rescue IV glucose will be censored at the time to plasma glucose recovery.  
In a sensitivity analysis, the time to plasma glucose recovery will be analyzed without 
censoring the subjects who received rescue IV glucose.   


 
 
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9.5.1 Hierarchical testing procedure 
For the confirmatory analyses, the following a priori defined hierarchical inferential test 
order will be applied for the control of the type 1 error rate across the planned multiple 
comparisons. The primary endpoint and key secondary endpoints will be inferentially 
evaluated within the FAS in the following order, where inference will proceed at the 
two-sided 0.05 criterion significance level until the first failure to reject the null 
hypothesis for a dasiglucagon versus placebo comparison: 
 Primary:  Time to plasma glucose recovery 
 Key secondaries 1-4: Plasma glucose recovery within 30 minutes, within 20 minutes, 
within 15 minutes, and within 10 minutes after study drug injection without 
administration of rescue IV glucose. 
 Key secondaries 5-8:  Plasma glucose changes from baseline (CFB) within 30 
minutes, within 20 minutes, within 15 minutes, and within 10 minutes after study 
drug injection or at the time of rescue. 
The GlucaGen versus placebo comparisons will not be included in the inferential testing 
hierarchy, since the efficacy of GlucaGen is previously established, and these 
comparisons are intended to support the validity of the study for the dasiglucagon versus 
placebo comparisons. 
Analogous supportive sensitivity analyses will be conducted in the PP set, but without 
inference intent. 
9.5.2 Primary confirmatory endpoint 
Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase in 
plasma glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic 
clamp procedure without administration of rescue IV glucose.  
9.5.2.1 Primary analysis 
The primary endpoint will be summarized using Kaplan-Meier (KM) estimates stratified 
by treatment group and injection site. The treatment group difference between 
dasiglucagon and placebo will be evaluated inferentially using a pairwise two-sided 
log-rank test. 
In the primary analysis, those subjects who require rescue IV glucose will be censored at 
the time of plasma glucose recovery. This approach is based on the assumption that 
rescue IV glucose administration will shorten the time to recovery. This assumption 
implies that censoring at the time of recovey after rescue therapy is valid as a time to 


 
 
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recovery without rescue would be longer than the observed duration. If recovery has not 
occurred at 45 minutes after study drug injection, censoring will be applied irrespective 
of the use of rescue IV glucose.  
In sensitivity analyses, the time to plasma glucose recovery will be analyzed 1) without 
censoring at the time of recovery for those subjects who require rescue IV glucose before 
45 minutes and 2) with censoring at the time of administration of rescure IV glucose 
before 45 minutes.   
The primary endpoint will additionally be analyzed using a Cox proportional hazards 
(CPH) time to event statistical model, if the hazards by treatment group can be assumed 
proportional using a graphical comparison of log-minus-log survival curves and no more 
than two-thirds of the recovery times are censored in each treatment group. The CPH 
model will be used for inferences, with treatment group and injection site modeled as 
categorical effects, and baseline plasma glucose modeled as a continuous covariate. The 
treatment group rate ratios, active versus placebo, will be estimated together with the 
95% confidence intervals, and pairwise treatment group inferences (dasiglucagon vs 
placebo, GlucaGen vs placebo) will be evaluated using two-sided likelihood ratio tests. 
9.5.3 Secondary endpoints 
 Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection without administration of rescue IV 
glucose. 
 Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection or at the time of rescue. 
9.5.3.1 Confirmatory analysis 
The key secondary incidence variables (plasma glucose recovery within 30 minutes, 
within 20 minutes, within 15 minutes, within 10 minutes, in hierarchical order for 
inference) will be analyzed using descriptive statistics (frequency and percentage) by 
treatment group. Inferential treatment group comparisons will be assessed via pairwise 
tests of independent binomial proportions (dasiglucagon versus placebo, GlucaGen 
versus placebo) using Fisher’s exact test for each pairwise comparison. 
The key secondary endpoints of plasma glucose CFB within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection or at the 
time of rescue, will be analyzed with the plasma glucose CFB at rescue carried forward in 
those subjects who require rescue IV glucose before plasma glucose ≥20 mg/dL recovery. 
Each of these CFB variables will be analyzed using an Analysis of Covariance model, 
with treatment group modeled as a fixed effect and with the baseline plasma glucose 
modeled as a covariate. The dasiglucagon versus placebo treatment group difference will 


 
 
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be evaluated inferentially as a least squares means contrast, using a two-sided t-test at the 
0.05 signficance level, first for the 30 minute CFB, and then sequentially for the 
20 minute, 15 minute, and 10 minute variables, until the first failure to reject. 
9.5.4 Secondary clinical efficacy (PD) endpoints 
 Time to first plasma glucose concentration of ≥70 mg/dL (3.9 mmol/L) without 
administration of rescue IV glucose. 
 Plasma glucose response as area under the curve above baseline from time zero to 
30 minutes, AUC0-30min. 
Secondary clinical efficacy endpoints will be assessed based on plasma concentration 
data from samples collected at the dosing visit (Visit 2).  
Unless otherwise stated, the population base of analysis will be the FAS. 
9.5.4.1 Analysis of secondary clinical efficacy (PD) endpoints 
1. Time to first plasma glucose concentration ≥70 mg/dL from baseline. This 
time-to-event endpoint will be evaluated using a Kaplan-Meier approach, with 
treatment group as a stratification factor, analogous to that used for the primary 
endpoint analysis. Differences between the KM curves (dasiglucagon versus 
placebo, GlucaGen versus placebo) will be evaluated inferentially using pairwise 
two-sided log rank tests. If the ≥70 mg/dL endpoint is not met within 45 minutes 
post-dosing, the time of the last valid plasma glucose measurement up to 45 
minutes will be the censoring time.  
2. The AUC will be calculated as the baseline-adjusted area under the plasma 
glucose profile over time: 
a. AUC0-30min: restricting the time window to the 0 to 30 minutes interval. 
3. The log-transformed AUC endpoint will be analyzed using an analysis of 
covariance (ANCOVA) model with treatment as fixed effect and baseline plasma 
glucose modeled as a covariate. The least squares means treatment group 
differences will be back-transformed (anti-logged) for presentation as a ratio of 
the treatment group geometric means, with their corresponding 95% CI. 
9.5.5 Exposure (PK) endpoints 
 Plasma dasiglucagon and GlucaGen concentrations from time zero to 90 minutes:  
AUC0-90min, Cmax, and tmax. 


 
 
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9.5.5.1 Analysis of exposure (PK) endpoints 
AUC will be derived as the area under the individual plasma dasiglucagon/GlucaGen 
concentration profile for PK from 0 to 90 minutes or last valid measurement if this 
measurement is assessed sufficiently close to 90 minutes (decision to be taken at the 
masked data review meeting). The standard trapezoidal method will be used, based on 
actual rather than nominal timepoints.  
Cmax will be determined as the maximum of all valid plasma dasiglucagon/GlucaGen 
concentrations.  
Tmax will be determined as the timepoint where the maximum of all valid plasma 
dasiglucagon/GlucaGen concentration measurements for each measurement series is 
observed. 
The log-transformed PK endpoints AUC and Cmax will be analyzed in the same way as 
the AUC endpoints. 
As tmax is a highly discrete endpoint, Wilcoxon’s rank sum test for unpaired observations 
will be used to assess differences between the two treatment groups. 
9.6 
Exploratory analyses 
Exploratory analyses will include descriptive statistics and modeling analogous to that 
done for key secondary endpoints. However, treatment group comparisons will be 
summarized without inference intent. 
 Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL 
(1.1 mmol/L) within 30 minutes after study drug injection without administration 
of rescue IV glucose. 
 Plasma insulin response as area under the curve above baseline from time zero to 
60 minutes, AUC0-60 min. 
Plasma insulin concentrations measured pre-dose and at 30 and 60 minutes after dosing 
(see Table 3) will be presented individually. A summary table per timepoint will be 
provided. The AUC0-60min will be determined and a summary presented.  


 
 
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9.7 
Safety analyses 
9.7.1 Adverse events 
AEs will be coded using the latest available version of the MedDRA, the version of 
which will be provided in the clinical trial report. AE summary tables will include the 
number and percentage of subjects who experienced AEs summarized by system organ 
class and preferred term. 
An overall summary table will be provided showing the number and percentage of 
subjects with any: 
 TEAE 
 Severe TEAE 
 Serious TEAE 
 Drug-related TEAE 
 Drug-related severe TEAE 
 Drug-related serious TEAE 
 TEAE leading to withdrawal 
 TEAE with outcome death 
 AESI 
Refer to Section 7.2.1 for the definition of TEAEs. 
9.7.2 Immunogenicity data 
Immunogenicity data will be analyzed descriptively by treatment group. No statistical 
tests are planned. Baseline ADA-positive subjects will be calculated as a percentage of 
the total number of subjects whose baseline samples were tested for ADA. Titer will be 
reported as median and interquartile range. 
9.7.3 Clinical laboratory assessments 
Clinical laboratory test results will be flagged as to whether the result is below, within or 
above the respective reference range. The number of values outside of the reference range 
will be counted. 
9.7.4 Other safety data 
Incidence of rescue infusion of IV glucose during the hypoglycemic clamp procedure will 
be analyzed using descriptive statistics (frequency and percentage) by treatment group. 
Inferential treatment group comparisons will be assessed via pairwise tests of 


 
 
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independent binomial proportions (dasiglucagon versus placebo, GlucaGen versus 
placebo). 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure 
will be evaluated using a KM time to event statistical model, with treatment group and 
injection site as stratification factors. Differences between the KM curves (dasiglucagon 
versus placebo, GlucaGen versus placebo) will be evaluated inferentially using pairwise 
two-sided stratified log-rank tests. If the endpoint is never met, the time of the last plasma 
glucose measurement will be the censoring time. 
Vital signs, physical examination, 12-lead ECG and local tolerability data will be 
summarized using descriptive statistics. 
9.8 
Treatment compliance 
Trained unblinded members of staff will perform all administrations of the IMP at the 
trial center. The administered doses will be recorded in the blinded Drug Accountability 
form in the eCRF. 
PK assessments will support the surveillance of compliance with IMP administration. 
9.9 
Subject withdrawals and missing data 
In the case of subject withdrawal, no imputation of values for PK or PD measurements 
will be done. Analyses will be done on valid cases only, i.e., no imputation techniques 
such as last observation carried forward will be applied. For the primary analysis in the 
FAS, missing values for the primary endpoint will be imputed by a conservative rule 
considering any missing value as a failure. 
9.10 Interim analyses 
No interim analyses are currently planned. 


 
 
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10 
ETHICAL, LEGAL, AND ADMINISTRATIVE ASPECTS 
10.1  Quality assurance 
The sponsor or designee will conduct a site visit to verify the qualifications of each 
investigator, inspect the facilities, and inform the investigator of responsibilities and the 
procedures for ensuring adequate and correct documentation. 
The investigator is required to prepare and maintain adequate and accurate case histories 
designed to record all observations and other data pertinent to the trial for each trial 
participant. All information recorded on the eCRFs for this trial must be consistent with 
the subjects’ source documentation (i.e. medical records). 
The investigator will permit trial-related monitoring, IRB/IEC review, and regulatory 
inspections, providing direct access to source data /documents. Sponsor-authorized 
quality assurance personnel may carry out audits for which the investigator must provide 
support. 
The trial monitor or a representative of the sponsor will cross-check the data entered in 
the eCRFs with the source data at the trial center and observe the trial procedures in order 
to verify adherence to the trial protocol. Any queries will be resolved by the investigator 
or his/her delegate. 
All of the clinical data will be captured via electronic data capture (EDC) using a 
web-based tool.  
The investigator center staff will enter and edit the data via a secure network, with secure 
access features (username and password). A complete electronic audit trail will be 
maintained. The investigator will approve the data using an electronic signature (Ref: 21 
CFR Part 11), and this approval is used to confirm the accuracy of the data recorded. 
eCRFs will be used for all subjects. The investigator’s data will be accessible from the 
investigator’s center throughout the trial. The eCRFs must be kept current to reflect 
subject status at each phase during the course of the trial. The eCRF will not capture 
personalized data. The investigator must make a separate confidential record of 
personalized details (name and initials) on the subject identification and enrollment log. 
All changes to data are made by the investigator or his/her delegate through the EDC 
system.  
It is the responsibility of the principal investigator of the respective center to ensure that 
all subject discontinuations or changes in trial or other medications entered on the 
subject’s eCRF are also made on the subject’s medical records. 


 
 
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The eCRFs for any subject leaving the trial should be completed at the time of the final 
visit or shortly thereafter. 
10.2  Electronic case report forms 
Remote data capture software will be used for data collection. Following training, trial 
staff will be given access to the software. Access to the software is restricted to staff 
participating in the trial and the extent of access will depend on the participants' user role 
in the trial. 
The subjects enrolled into the trial will be identified in the database by subject number 
and trial identification code. The investigator or delegate will enter subject data into the 
eCRF promptly. All data is to be entered in English. Data recorded in the eCRFs will be 
accessible to the trial staff throughout the trial. 
After data entry, systematic data validation will be performed and data entry 
discrepancies will be presented electronically directly to the center staff. Queries for 
discrepant data may be generated automatically by the software upon entry and/or 
generated manually by the trial monitor or the trial data manager. All queries, whether 
generated by the system or by trial staff, will be in electronic format. 
All sections of the eCRF are to be electronically approved by the investigator or a 
medically qualified delegate after the data has been entered and all queries have been 
resolved. Changes to any eCRF page subsequent to the approval require a new approval 
signature. 
All queries and changes/corrections to the data are documented in the eCRF. 
10.3  Access to source data 
During the course of the trial, a trial monitor will make site visits to review protocol 
compliance, compare eCRFs with individual subject’s medical records, assess drug 
accountability, and ensure that the trial is being conducted according to pertinent 
regulatory requirements. 
Checking of the eCRFs for completeness and clarity, and cross-checking with source 
documents, will be required to monitor the progress of the trial. Moreover, regulatory 
authorities of certain countries, IRBs/IECs, and/or the sponsor may wish to carry out such 
source data checks and/or on-site audit inspections. Direct access to source data will be 
required for these inspections and audits; they will be carried out giving due 
consideration to data protection and medical confidentiality. 


 
 
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10.4  Source documentation 
All source documents from which eCRF entries are derived should be placed in the 
subject’s medical records. If data are to be entered directly into the eCRF this must be 
specified in a source data agreement prior to the start of the trial. 
eCRF entries will be verified with source documentation. The review of medical records 
will be performed in a manner to ensure that subject confidentiality is maintained. 
The trial monitor will check the eCRF for accuracy and completion and perform source 
data verification. The trial monitor will document source data verification of all reviewed 
sections of the eCRF. 
10.5  Data processing 
The trial is run as an EDC trial, i.e. all relevant data is entered by the centers directly into 
the clinical database. The eCRF is designed to capture all required information in 
compliance with GCP standards. 
10.6  Archiving trial records 
According to ICH guidelines, essential documents should be retained for a minimum of 
2 years after the last approval of a marketing application in an ICH region and until there 
are no pending or contemplated marketing applications in an ICH region or at least 
2 years have elapsed since the formal discontinuation of clinical development of the 
investigational product. However, these documents should be retained for a longer period 
if required by the applicable legal requirements. 
Records and documents pertaining to the conduct of the trial and the distribution of the 
investigational product (e.g. informed consent forms, laboratory slips, medication 
inventory records, and other pertinent information) must be retained by the investigator 
according to local requirements. 
10.7  Good clinical practice 
The procedures set out in this trial protocol are designed to ensure that the sponsor and 
investigator abide by the principles of the GCP guidelines of the ICH (1), and of the 
Declaration of Helsinki (2008) (2). The trial also will be carried out in keeping with local 
legal requirements.  


 
 
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10.8  Informed consent 
Before each subject is admitted to the trial, written informed consent will be obtained 
from the subject according to the regulatory and legal requirements of the participating 
country. This consent form must be dated and retained by the investigator as part of the 
trial records. The investigator will not undertake any investigation specifically required 
only for the clinical trial until valid consent has been obtained. The terms of the consent 
and when it was obtained must also be documented in the eCRF. 
The investigator will explain to each subject orally and in writing (subject information 
sheet) the nature, significance, risks and implications of the trial before inclusion. In 
particular, the subjects will be informed about the following: 
 The possibility of withdrawing from the clinical trial at any time by revoking the 
consent and without any resulting disadvantage. 
 How personal and health-related data will be collected and used during the trial. 
 That his/her medical records may be examined by authorized monitors or clinical 
quality assurance auditors appointed by the sponsor, by appropriate IRB/IEC 
members, and by inspectors from regulatory authorities. 
All subjects will receive a copy of the subject information sheet and a copy of their 
signed and dated informed consent form, both of which will be in the subject’s local 
language. 
If a protocol amendment is required, the informed consent form may need to be revised to 
reflect the changes to the protocol. If the consent form is revised, it must be reviewed and 
approved by the appropriate IEC/IRB, and signed by all subjects subsequently enrolled in 
the trial as well as those currently enrolled in the trial. 
10.9  Protocol approval and amendment 
Before the start of the trial, the trial protocol and/or other relevant documents will be 
approved by the IEC/IRB/competent authorities, in accordance with local legal 
requirements. The sponsor must ensure that all ethical and legal requirements have been 
met before the first subject is enrolled in the trial. 
This protocol is to be followed exactly. To alter the protocol, amendments must be 
written, receive approval from the appropriate personnel, and receive IRB/IEC/competent 
authority approval prior to implementation (if appropriate).  
Administrative changes (not affecting the subject benefit/risk ratio) may be made without 
the need for a formal amendment. All amendments will be distributed to all protocol 
recipients, with appropriate instructions. 


 
 
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10.10  Duration of the trial 
The maximum duration of the trial for each subject will be up to 63 days (including up to 
30 days for screening and up to 33 days until the follow-up visit).  
The trial will be closed when all subjects have completed Visit 3. 
10.11  Premature termination of the trial 
If the investigator, the sponsor (e.g. safety committee), or the safety medical monitor 
becomes aware of conditions or events that suggest a possible hazard to subjects if the 
trial continues, the trial may be terminated after appropriate consultation between the 
relevant parties. The trial may also be terminated early at the sponsor’s discretion in the 
absence of such a finding. 
Conditions that may warrant termination include, but are not limited to: 
 The discovery of an unexpected, significant, or unacceptable risk to the subjects 
enrolled in the trial 
 Failure to enroll subjects at an acceptable rate. 
 A decision on the part of the sponsor to suspend or discontinue development of the 
drug. 
The trial can be terminated prematurely by the sponsor at an individual center if: 
 The center cannot comply with the requirements of the protocol. 
 It is not possible for the center to comply with GCP standards. 
10.12  Confidentiality 
All trial findings and documents will be regarded as confidential. The investigator and 
members of his/her research team must not disclose such information without prior 
written approval from the sponsor. 
The anonymity of participating subjects must be maintained. Subjects will be identified 
on eCRFs submitted to the sponsor by their subject number. On the SAE reports and all 
other source documents, the subject will be identified via subject number. Documents not 
to be submitted to the sponsor that identify the subject (e.g. the signed informed consent 
form) must be maintained in confidence by the investigator.  


 
 
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10.13  Other ethical and regulatory issues 
If a significant safety issue is identified, either from an individual case report or review of 
aggregate data, then the sponsor will issue prompt notification to all parties: regulatory 
authorities, investigators and IRB/IECs. 
A significant safety issue is one that has a significant impact on the course of the clinical 
trial or program (including the potential for suspension of the trial program or 
amendments to protocols) or warrants immediate update of informed consent. 
10.14  Liability and insurance 
The sponsor will take out reasonable third-party liability insurance cover in accordance 
with all local legal requirements. The civil liability of the investigator, the persons 
instructed by him and the hospital, practice or institute in which they are employed and 
the liability of the sponsor with respect to financial loss due to personal injury and other 
damage that may arise as a result of the carrying out of this trial are governed by the 
applicable law. 
The sponsor will arrange for liability insurance if subjects should be injured due to the 
participation in the trial and provided that the sponsor is legally liable for that. 
Excluded from the insurance cover are injuries to health and deteriorations of illnesses 
already in existence which would have occurred or continued to exist even if the subject 
had not taken part in the clinical trial. 
The insurance cover is jeopardized if the subject fails to report immediately to the 
investigator or responsible physician any injury to health, which might have resulted 
from participation in the clinical trial, or if he/she undergoes any other medical treatment 
without their consent before the clinical trial has been completely finished in so far as the 
individual subject is concerned. 
Any injury to health, which might have occurred as a result of participation in the clinical 
trial must be reported by the subject to the investigator without delay. The investigator is 
obliged to make such a report in any case. 
10.15  Publication policy 
By signing the trial protocol, the investigator agrees with the use of results of the trial for 
the purposes of national and international registration, publication and information for 
medical and pharmaceutical professionals. If necessary, the authorities will be notified of 
the investigator's name, address, qualifications and extent of involvement. 


 
 
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A clinical trial report will be prepared and reviewed by the sponsor in co-operation with 
the coordinating investigator. The coordinatoing investigator will be appointed by 
Zealand Pharma to review and sign the clinical trial report on behalf of all participating 
investigators. A summary of the clinical trial report (according to CPMP/ICH137/95) 
should be sent to the regulatory authorities and IRB/IEC according to the relevant 
guidelines. 
According to the Declaration of Helsinki (2) investigators and sponsors ‘have ethical 
obligations with regard to the publication and dissemination of the results of research’.   
The trial design and results may be published as one or more original research 
manuscripts/abstracts and presented at a scientific meeting. The original eCRFs and the 
data generated from the eCRFs or otherwise obtained during the trial under this trial 
protocol will become the property of the sponsor. Publication of the results of this trial by 
the investigator is possible only after written consent has been obtained from the sponsor. 
Any material intended for publication will be given to the sponsor at least 4 weeks before 
submission for publication. The sponsor will have the right to comment on the intended 
publication and to take any reasonable measures for patent protection. Authorship on any 
publication(s) resulting from this clinical trial will be assigned according to the 
recommendations of the International Committee of Medicinal Journal Editors (26). 
Participating subjects will not be identified by name in any published reports about the 
clinical trial. 
The sponsor is responsible for trial registration at ClinicalTrials.gov 
(www.clinicaltrials.gov) according to FDA requirements, as well as the European 
Medicines Agency’s Clinical Trials Database (EudraCT).  


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11 
REFERENCE LIST 
1. ICH Harmonised Tripartite Guideline. International Conference on Harmonisation. 
ICH Harmonised Tripartite Guideline. Integrated addendum to ICH E6(R1): 
Guideline for Good Clinical Practice E6 (R2), Step 4. 09-Nov-2016. Available from: 
URL:
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E 
6/E6_R2__Step_4.pdf
2. World Medical Association. Declaration of Helsinki. Ethical Principles for Medical
Research Involving Human Subjects. 52nd WMA General Assembly, Edinburgh,
Scotland, October 2000. Last amended with Note of Clarification on Paragraph 29 by
the WMA General Assembly, Washington 2002, and Note of Clarification on
Paragraph 30 by the WMA General assembly, Tokyo 2004
3. Seaquist ER, Anderson J, Childs B, Cryer P, Dagogo-Jack S, Fish L, et al.
Hypoglycemia and diabetes: a report of a workgroup of the American Diabetes
Association and the Endocrine Society. Diabetes Care 2013 May;36:1384-95
4. Cryer PE. The barrier of hypoglycemia in diabetes. Diabetes 2008;57:3169-76
5. American Diabetes Association Position Statement. Standards of medical care in
diabetes - 2017. Diabetes Care 2017;40(Supplement 1):S1-S135
6. Davies MJ, Gagliardino JJ, Gray LJ, Khunti K, Mohan V, Hughes R. Real-world
factors affecting adherence to insulin therapy in patients with Type 1 or Type 2
diabetes mellitus: a systematic review. Diabetic Medicine 2013;30:512-24
7. International Hypoglycaemia Study Group. Glucose concentrations of less than 3.0
mmol/L (54 mg/dL) should be reported in clinical trials: a joint position statement of
the American Diabetes Association and the European Association for the Study of
Diabetes. Diabetes Care 2017;40(1):155-57
8. Christensen M, Bagger JI, Vilsbøll T, Knop FK. The alpha-cell as target for type 2
diabetes therapy. Rev Diabet Stud 2011;8:369-81
9. Lund A, Bagger JI, Christensen M, Knop FK, Vilsbøll T. Glucagon and type 2
diabetes: the return of the alpha cell. Curr Diab Rep 2014;14:555
10. Habegger KM, Heppner KM, Geary N, Bartness TJ, DiMarchi R, Tschöp MH. The
metabolic actions of glucagon revisited. Nat Rev Endocrinol 2010;6:689-97
11. Onoue S, Ohshima K, Debari K, Koh K, Shioda S, Iwasa S, et al. Mishandling of the
therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils. Pharm Res
2004;21:1274-83


 
 
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12. Fosgerau K, Hoffmann T. Peptide therapeutics: current status and future directions. 
Drug Discovery Today 2015;20:122-8 
13. Novo Nordisk Limited. SPC for GlucaGen Hypokit 1 mg. 2015. 
http://www.medicines.org.uk/emc/medicine/4258/SPC/GlucaGen+Hypokit+1+mg - 
Accessed 13 Mar 2017 . 
14. Zealand Pharma A/S. Dasiglucagon Investigator's Brochure, Edition 3, 12 Dec 2016.  
15. The Task Force for the management of arterial hypertension of the European Society 
of Hypertension (ESH) and of the European Society of Cardiology (ESC). 2013 
ESH/ESC guidelines for the management of arterial hypertension. European Heart 
Journal 2013;34:2159-219 
16. Levey AS, Coresh J, Greene T, Stevens LA, Zhang Y, Hendriksen BA, et al. Using 
standardized serum creatinine values in the modification of diet in renal disease study 
equation for estimating glomerular filtration rate. Annals of Internal Medicine 
2006;145:247-54 
17. Food and Drug Administration (FDA). Guidance for Industry on Bioanalytical 
Method Validation. Food and Drug Administration (FDA) 2001 
18. Viswanathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ, Sailstad J, et al. 
Quantitative bioanalytical methods validation and implementation: best practices for 
chromatographic and ligand binding assays. Pharm Res 2007 Oct;24:1962-73 
19. Shankar G, Devanarayan V, Amaravadi L, Barrett YC, Bowsher R, Finco-Kent D, et 
al. Recommendations for the validation of immunoassays used for detection of host 
antibodies against biotechnology products. J Pharm Biomed Anal 2008;48:1267-81 
20. Zealand Pharma report SS. Commissioning and validation of a method for the 
validation of anti-ZP4207 antibodies in human serum.  2014  
21. Zealand Pharma report SS. Commissioning and validation of a method for the 
validation of anti-glucagon antibodies in human serum.  2014  
22. Bioagilytix Labs. Validation of an assay to detect neutralizing antibodies to ZP4207 
by measuring cyclic adenosine monophosphate (cAMP) in a glucagon receptor 
transfected cell line, BAL-14-206-004-REP.  2016. Report No.: Zealand Pharma, 
study 15-039 
23. Bioagilytix Labs. Validation of an assay to detect neutralizing antibodies to glucagon 
by measuring cyclic adenosine monophosphate (cAMP) in a glucagon receptor 
transfected cell line, BAL-14-206-005.  2016. Report No.: Zealand Pharma, Study 
15-040 


 
 
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24. Amiel SA, Tamborlane WV, Simonson DC, et al. Defective glucose 
counterregulation after strict glycemic control of insulin-dependent diabetes mellitus. 
N Eng J Med 1987; 316: 1376-83. 
25. Rickels MR, Ruedy KJ, Foster NC, et al. Intranasal glucagon for treatment of insulin-
induced hypoglycemia in adults with type 1 diabetes: A randomized crossover 
noninferiority study. Diabetes Care 2016; 39: 264-70. 
26. ICMJE. Recommendations for the conduct, reporting, editing, and publication of 
scholarly work in medical journals. Updated December 2016 
 
 
 
 


 
 
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12 
APPENDICES 
12.1 List of trial personnel 
Sponsor 
Clinical Trial Manager 
 
 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Phone: 
 
Medical Officer 
 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Phone: 
 
Contract Research Organization 
Chiltern International (Ltd) 
171 Bath Road 
Slough 
Berkshire 
SL1 4AA 
United Kingdom 
Phone: +44 (0) 175351 2000 
Project Manager 
 
Chiltern International (Ltd) 
171 Bath Road 
Slough 
Berkshire 
SL1 4AA 
United Kingdom 
Phone: 
 
Senior Medical Officer and 
Safety medical monitor 
 
Chiltern International kft 
Canada Square Office House 
Ganz u 


 
 
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12-14, 4 emelet 
1027 Budapest 
Hungary 
Phone: 
 
Pharmacovigilance unit 
Responsible for Serious Adverse 
Event (SAE) Management and 24-
hour SAE reporting 
  
PharmaLex 
Agern Allé 24 
2970 Hørsholm, Denmark 
Phone:
 (8 a.m. to 4 p.m.) 
Phone: 
 (outside 8 a.m. to 4 p.m.) 
Fax: 
 
email: PV-nordic@pharmalex.com  
Central laboratory 
MLM Laboratory 
Dohrweg 63 
41066 Mönchengladbach 
Germany 
Special laboratory (GlucaGen 
PK, insulin PK) 
MLM Laboratory 
Dohrweg 63 
41066 Mönchengladbach 
Germany 
Special laboratory (dasiglucagon 
PK, dasiglucagon ADA, 
GlucaGen ADA) 
York Bioanalytical Solutions (YBS)  
Cedar House 
Northminster Business Park 
Northfield Lane 
York, YO26 6QR, United Kingdom 
Special laboratory  
(neutralizing antibodies) 
BioAgilytix 
2300 Englert Drive 
Durham, NC, 27713, USA 
A list of all investigators, IECs and IRBs will be provided in a separate document and in 
the clinical trial report. 
 


 
 
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CLINICAL TRIAL PROTOCOL 
 
A phase 3, randomized, double-blind, parallel trial to 
confirm the clinical efficacy and safety of dasiglucagon 
in the rescue treatment of hypoglycemia in subjects with 
type 1 diabetes mellitus (T1DM) compared to placebo 
and with reference to GlucaGen® 
 
Sponsor: 
Zealand Pharma A/S 
Sponsor Protocol No.: 
ZP4207-16137 
EudraCT No.: 
2017-002449-31  
IND No: 
127866 
Trial Drug Name: 
Dasiglucagon* injection 
Date of Protocol: 
29 Aug 2017 
 
The trial will be conducted according to the protocol and in compliance with Good 
Clinical Practice (GCP), with the Declaration of Helsinki and with other applicable 
regulatory requirements. 
*Dasiglucagon is the proposed international nonproprietary name for ZP4207. 
The information in this document is confidential and is proprietary to Zealand Pharma. It is understood that 
information in this document shall not be disclosed to any third party, in any form, without prior written 
consent of an authorized officer of Zealand Pharma. 


Dasiglucagon (ZP4207) 
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ZEALAND PHARMA 
Declaration of sponsor or responsible medical officer 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (Tl DM) compared to placebo and with reference to GlucaGen® 
This trial protocol was subjected to critical review. The information it contains is 
consistent with current knowledge of the risks and benefits of the investigational product, 
as well as with the moral, ethical and scientific principles governing clinical research as 
set out in the Declaration ofHelsinki and the guidelines on Good Clinical Practice (GCP) 
(1 ). 
Title 
Institution 
Clinical Trial Manager 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Institution 
Phone: 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Strictly Confidential 
Date 
Page 2 of79 


 
 
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Declaration of the coordinating investigator 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
I have read this protocol. I approve this document and I agree that it contains all 
necessary details for carrying out the trial as described. I will conduct this trial in 
accordance with the design and specific provision of this protocol and will make a 
reasonable effort to complete the trial within the time designated. I will provide copies of 
this protocol and access to all information furnished by Zealand Pharma A/S to trial 
personnel under my supervision. I will discuss this material with them to ensure they are 
fully informed about the trial product and trial procedures. I will let them know that this 
information is confidential and proprietary to Zealand Pharma A/S and that it may not be 
further disclosed to third parties. I understand that the trial may be terminated or 
enrollment suspended at any time by Zealand Pharma A/S, with or without cause, or by 
me if it becomes necessary to protect the best interests of the trial subjects. 
I agree that the trial will be carried out in accordance with GCP (1), with the Declaration 
of Helsinki (with amendments) (2) and with the laws and regulations of the countries in 
which the trial takes place. 
 
 
_____________________________________ 
_____________________ 
Name  
 
 
 
 
 
 
Date 
Title 
Institution 
Phone: + 
Fax: 
+ 
 


 
 
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Declaration of the investigator 
Title: A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy 
and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 
1 diabetes mellitus (T1DM) compared to placebo and with reference to GlucaGen® 
All documentation for this trial that is supplied to me and that has not been previously 
published will be kept in the strictest confidence. This documentation includes this trial 
protocol, Investigator's Brochure, Case Report Forms, and other scientific data. 
The trial will not be commenced without the prior written approval of a properly 
constituted Institutional Review Board (IRB) or Independent Ethics Committee (IEC). 
No changes will be made to the trial protocol without the prior written approval of the 
sponsor and the IRB or IEC, except where necessary to eliminate an immediate hazard to 
the subjects. 
I have read and understood and agree to abide by all the conditions and instructions 
contained in this protocol. 
 
_____________________________________ 
_____________________ 
Signature 
Date 
 
_____________________________________ 
Name (block letters) 
 
_____________________________________ 
Title (block letters) 
 
_____________________________________ 
Institution (block letters) 
 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
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Table of contents 
DECLARATION OF SPONSOR OR RESPONSIBLE MEDICAL OFFICER .......... 2 
DECLARATION OF THE COORDINATING INVESTIGATOR .............................. 3 
DECLARATION OF THE INVESTIGATOR ............................................................... 4 
TABLE OF CONTENTS .................................................................................................. 5 
1 
SYNOPSIS ............................................................................................................ 11 
2 
INTRODUCTION................................................................................................ 15 
2.1 
Background ...................................................................................................... 15 
2.1.1 
Hypoglycemia ...................................................................................... 15 
2.1.2 
Glucagon .............................................................................................. 16 
2.1.3 
Dasiglucagon........................................................................................ 16 
2.1.3.1 
Pharmacokinetics and pharmacodynamics of dasiglucagon ......... 17 
2.1.3.2 
Safety of dasiglucagon ................................................................... 17 
2.2 
Trial rationale .................................................................................................. 18 
2.3 
Risk-benefit assessment .................................................................................. 18 
3 
TRIAL OBJECTIVES AND ENDPOINTS....................................................... 20 
3.1 
Primary objective ............................................................................................ 20 
3.2 
Secondary objectives ....................................................................................... 20 
3.3 
Primary endpoint ............................................................................................ 20 
3.4 
Key secondary endpoints ................................................................................ 20 
3.5 
Other secondary endpoints ............................................................................. 20 
3.6 
Exploratory endpoint ...................................................................................... 21 
4 
OVERALL DESIGN AND PLAN OF THE TRIAL ........................................ 22 
4.1 
Overview .......................................................................................................... 22 
4.2 
Justification of the trial design ....................................................................... 22 
4.2.1 
Justification for design and parameters................................................ 22 
4.2.2 
Justification for drug, route, dosage and treatment duration ............... 23 
5 
TRIAL POPULATION ....................................................................................... 24 
5.1 
Rationale for trial population ........................................................................ 24 
5.2 
Planned sample size and number of trial centers ......................................... 24 
5.3 
Inclusion criteria ............................................................................................. 24 


 
 
Dasiglucagon (ZP4207) 
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5.4 
Exclusion criteria ............................................................................................ 25 
5.5 
Dosing day exclusion criteria ......................................................................... 26 
5.6 
Premature treatment discontinuation and withdrawal ............................... 27 
5.6.1 
Possible reasons for treatment visit discontinuation ............................ 27 
5.6.2 
Center discontinuation ......................................................................... 28 
5.6.3 
Trial termination .................................................................................. 28 
5.7 
Subject identification and randomization ..................................................... 28 
6 
TRIAL DRUG ...................................................................................................... 30 
6.1 
Identity ............................................................................................................. 30 
6.2 
Treatment assignment and randomization ................................................... 31 
6.3 
Administration ................................................................................................. 32 
6.4 
Packaging and labelling .................................................................................. 32 
6.5 
Storage of study drugs .................................................................................... 33 
6.6 
Blinding and breaking the blind .................................................................... 33 
6.7 
Drug accountability ......................................................................................... 34 
6.8 
Treatment compliance .................................................................................... 34 
6.9 
Prior and concomitant medications ............................................................... 35 
6.9.1 
Prohibited medications......................................................................... 35 
7 
PARAMETERS AND METHODS OF ASSESSMENT .................................. 36 
7.1 
Efficacy parameters ........................................................................................ 36 
7.1.1 
Pharmacodynamic measurements ........................................................ 36 
7.1.2 
Pharmacokinetic measurements ........................................................... 36 
7.2 
Safety parameters ............................................................................................ 37 
7.2.1 
Adverse events ..................................................................................... 37 
7.2.1.1 
Definitions ...................................................................................... 37 
7.2.1.2 
Collection, recording and reporting of adverse events ................. 40 
7.2.1.3 
Follow-up of adverse events .......................................................... 41 
7.2.1.4 
Clinical laboratory abnormalities and other abnormal 
assessments as adverse events or serious adverse events .............. 42 
7.2.2 
Hypoglycemia ...................................................................................... 42 
7.2.3 
Physical examination ........................................................................... 43 
7.2.4 
Vital signs ............................................................................................ 43 
7.2.5 
Electrocardiogram ................................................................................ 44 
7.2.6 
Local tolerability .................................................................................. 44 
7.2.7 
Clinical laboratory assessments ........................................................... 45 
7.2.8 
Pregnancy ............................................................................................. 46 
7.2.9 
Precautions ........................................................................................... 47 
7.2.10 
Safety committee ................................................................................. 47 
7.3 
Demography, concomitant illness, medical history and concomitant 
medication ........................................................................................................ 47 


 
 
Dasiglucagon (ZP4207) 
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7.3.1 
Demography and body measurements ................................................. 47 
7.3.2 
Concomitant illness and medical history ............................................. 48 
7.3.3 
Diabetes diagnosis and current treatment ............................................ 48 
7.3.4 
Concomitant medication ...................................................................... 48 
7.4 
Other assessments ........................................................................................... 49 
7.4.1 
Immunogenicity ................................................................................... 49 
7.4.2 
Plasma glucose measurements for safety ............................................. 50 
7.4.3 
Plasma insulin measurements .............................................................. 50 
8 
TRIAL CONDUCT .............................................................................................. 51 
8.1 
Schedule of procedures ................................................................................... 51 
8.2 
Procedures by visit .......................................................................................... 51 
8.2.1 
Visit 1 (screening, Day -30 to Day -3) ................................................. 51 
8.2.2 
Instructions to subjects prior to the dosing visit (Visit 2) .................... 53 
8.2.3 
Visit 2 (dosing visit)............................................................................. 54 
8.2.3.1 
Hypoglycemic clamp procedure and administration of trial 
medication ...................................................................................... 55 
8.2.4 
Visit 3 (follow-up visit) ....................................................................... 57 
9 
STATISTICAL METHODS ............................................................................... 59 
9.1 
Determination of sample size ......................................................................... 59 
9.2 
Trial subjects ................................................................................................... 59 
9.2.1 
Analysis samples .................................................................................. 59 
9.2.2 
Disposition of subjects ......................................................................... 60 
9.2.3 
Protocol deviations............................................................................... 60 
9.3 
General considerations ................................................................................... 61 
9.4 
Demographics and baseline characteristics .................................................. 61 
9.5 
Efficacy Analyses ............................................................................................. 61 
9.5.1 
Hierarchical testing procedure ............................................................. 62 
9.5.2 
Primary confirmatory endpoint ............................................................ 62 
9.5.2.1 
Primary analysis ............................................................................ 62 
9.5.3 
Secondary endpoints ............................................................................ 63 
9.5.3.1 
Confirmatory analysis .................................................................... 63 
9.5.4 
Secondary clinical efficacy (PD) endpoints ......................................... 63 
9.5.4.1 
Analysis of secondary clinical efficacy (PD) endpoints ................ 64 
9.5.5 
Exposure (PK) endpoints ..................................................................... 64 
9.5.5.1 
Analysis of exposure (PK) endpoints ............................................. 64 
9.6 
Exploratory analyses ....................................................................................... 65 
9.7 
Safety analyses ................................................................................................. 65 
9.7.1 
Adverse events ..................................................................................... 65 
9.7.2 
Immunogenicity data ........................................................................... 66 
9.7.3 
Clinical laboratory assessments ........................................................... 66 


 
 
Dasiglucagon (ZP4207) 
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9.7.4 
Other safety data .................................................................................. 66 
9.8 
Treatment compliance .................................................................................... 67 
9.9 
Subject withdrawals and missing data .......................................................... 67 
9.10 
Interim analyses .............................................................................................. 67 
10 
ETHICAL, LEGAL, AND ADMINISTRATIVE ASPECTS .......................... 68 
10.1 
Quality assurance ............................................................................................ 68 
10.2 
Electronic case report forms .......................................................................... 69 
10.3 
Access to source data ...................................................................................... 69 
10.4 
Source documentation .................................................................................... 70 
10.5 
Data processing ................................................................................................ 70 
10.6 
Archiving trial records ................................................................................... 70 
10.7 
Good clinical practice ..................................................................................... 70 
10.8 
Informed consent ............................................................................................. 71 
10.9 
Protocol approval and amendment ............................................................... 71 
10.10 Duration of the trial ........................................................................................ 72 
10.11 Premature termination of the trial ................................................................ 72 
10.12 Confidentiality ................................................................................................. 72 
10.13 Other ethical and regulatory issues ............................................................... 73 
10.14 Liability and insurance ................................................................................... 73 
10.15 Publication policy ............................................................................................ 74 
11 
REFERENCE LIST ............................................................................................. 75 
12 
APPENDICES ...................................................................................................... 78 
12.1 
List of trial personnel ...................................................................................... 78 
 
Tables in text 
Table 1 
Description of trial drugs .................................................................................. 30 
Table 2 
Quantities of ingredients in dasiglucagon and placebo injection ...................... 31 
Table 3 
Schedule of procedures ..................................................................................... 52 
Table 4 
Post-treatment blood sampling times ................................................................ 56 
 
Figures in text 
Figure 1 Trial design ........................................................................................................ 22 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
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Protocol Version: Final 1.0 
 
 
 
 
 
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List of abbreviations and definitions of terms 
ADA 
Anti-drug antibody 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALT 
Alanine aminotransferase 
ANCOVA Analysis of covariance 
AST 
Aspartate aminotransferase 
AUC 
Area under the concentration-time curve 
AUC0-inf 
AUC from time zero to infinity 
AUE 
Area under the effect curve  
CI 
Confidence interval 
Cmax 
Maximum plasma concentration 
CPH 
Cox proportional hazards 
CRO 
Contract research organization  
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
EU 
European Union 
EudraCT 
European Medicines Agency’s Clinical Trials Database 
FAS 
Full analysis set 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HbA1c 
Glycated hemoglobin 
ICH 
International Conference on Harmonization 
ID card 
Identification card 
IEC 
Independent Ethics Committee 
IM 
Intramuscular(ly) 
IMP 
Investigational medicinal product 
IRB 
Institutional Review Board 
IV 
Intravenous(ly) 
IWRS 
Interactive Web Response System 


 
 
Dasiglucagon (ZP4207) 
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MedDRA 
Medical Dictionary for Regulatory Activities 
NPH 
Neutral protamine Hagedorn 
PD 
Pharmacodynamics 
PK 
Pharmacokinetics 
PP 
Per protocol 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SAS 
Safety analysis set 
SC 
Subcutaneous(ly) 
SUSAR 
Serious unexpected suspected adverse reaction 
T1DM 
Type 1 diabetes mellitus 
T2DM 
Type 2 diabetes mellitus 
TEAE 
Treatment-emergent adverse event 
tmax 
Time to the maximum plasma concentration 
ULN 
Upper limit of the normal range 
US 
United States of America 
 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 1.0 
 
 
 
 
 
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1 
SYNOPSIS 
Name of sponsor: 
Zealand Pharma A/S 
Trial ID: 
ZP4207-16137 
Title of the trial: 
A phase 3, randomized, double-blind, parallel trial to confirm the clinical efficacy and safety of 
dasiglucagon in the rescue treatment of hypoglycemia in subjects with type 1 diabetes mellitus (T1DM) 
compared to placebo and with reference to GlucaGen® 
Trial design:  
The trial is a global, multicenter, randomized, parallel-group, double-blind, clinical trial confirming the 
efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in subjects with type 1 diabetes 
mellitus. The subjects will be randomized 2:1:1 to receive a single fixed subcutaneous 0.6 mg dose of 
dasiglucagon (hereinafter dasiglucagon), placebo for dasiglucagon (hereinafter referred to as placebo), or 
a 1 mg dose of GlucaGen® (hereafter referred to as GlucaGen) and followed for at least 28 days after 
treatment. 
Clinical phase of development: 
Phase 3 
Trial centers: 
This trial will be conducted at 4 to 6 sites in the United States of America, Canada, and Europe. 
Planned trial start (first subject first visit): 
Q4/2017 
Planned trial end (last subject last visit): 
Q3/2018  
Trial population:  
Male and female adult subjects with type 1 diabetes mellitus treated with insulin for at least one year  
Key objectives: 
Primary objective:  
 
To demonstrate superiority of dasiglucagon compared to placebo following a single subcutaneous 
0.6 mg dose administered to subjects with type 1 diabetes mellitus with insulin-induced 
hypoglycemia. 
Secondary objective:  
 
To compare the glycemic response observed after dasiglucagon with that of GlucaGen. 
Key endpoints:  
Primary endpoint:  
 
Time to plasma glucose ≥20 mg/dL recovery, defined as first increase in plasma glucose of 
≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic clamp procedure. In the primary 
analysis, those subjects who require rescue intravenous (IV) glucose will be censored at the time 
to plasma glucose recovery. In a sensitivity analysis, the time to plasma glucose recovery will be 
analyzed without censoring those subjects who require rescue IV glucose before 45 minutes.   
Key secondary endpoints: 
 
Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 15 minutes, 
and within 10 minutes after study drug injection or at the time of rescue. 
 
Incidences of plasma glucose recovery (achieving a plasma glucose concentration increase of ≥20 
mg/dL [1.1 mmol/L]) within 30 minutes, within 20 minutes, within 15 minutes, and within 10 
minutes after study drug injection. 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 1.0 
 
 
 
 
 
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Clinical efficacy (Pharmacodynamic) endpoints: 
 
Time to first plasma glucose concentration of ≥70 mg/dL (3.9 mmol/L). 
 
Plasma glucose response as area under the curve above baseline from time zero to 30 minutes, 
AUC0-30min. 
Exposure (Pharmacokinetic) endpoints: 
 
Area under the drug concentration curve from time zero to 90 minutes, AUC0-90 min. 
 
Maximum plasma drug concentration (Cmax). 
 
Time to maximum plasma drug concentration (tmax). 
Safety endpoints:  
 
Adverse events, clinical laboratory assessments (biochemistry, hematology, coagulation, 
urinalysis), vital signs, physical examination, electrocardiogram, and local tolerability. 
 
Incidence of rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
Immunogenicity endpoint: 
 
Incidence of anti-drug antibodies 
Exploratory endpoint: 
 
Incidence of plasma glucose recovery (achieving a plasma glucose concentration ≥70 mg/dL [3.9 
mmol/L] or an increase of ≥20 mg/dL [1.1 mmol/L]) within 30 minutes after study drug 
injection. 
 
Plasma insulin response as area under the curve above baseline from time zero to 60 minutes, 
AUC0-60 min. 
Key inclusion criteria: 
 
Male and female subjects with type 1 diabetes mellitus treated with insulin for at least one year, 
diagnostic criteria as defined by the American Diabetes Association. 
 
Stable insulin treatment 30 days prior to screening, defined as no more than a 10-unit daily 
variation in total daily insulin dose. 
 
Hemoglobin A1c <10%. 
 
Aged between 18 and 75 years, both inclusive. 
Key exclusion criteria: 
 
Previously treated with dasiglucagon. 
 
Known or suspected allergy to trial product(s) or related products. 
 
History of anaphylaxis or symptoms of severe systemic allergy (such as angioedema). 
 
Previous participation (randomization) in this trial. 
Sample size: 
Approximately 156 subjects are intended to complete the trial, with 78 subjects randomized to the 
dasiglucagon group and 39 subjects randomized to each of the placebo and GlucaGen groups.  
Investigational medicinal product: 
Test product: dasiglucagon liquid formulation in pre-filled syringes. 
Comparator products: Placebo and GlucaGen® lyophilized powder. 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
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Duration of treatment:  
Subjects will be randomized 2:1:1 to receive a single fixed subcutaneous dose of 0.6 mg dasiglucagon, 
placebo, or 1 mg GlucaGen, and followed for at least 28 days after receiving treatment.  
Assessments:  
The plasma glucose profile for evaluation of the primary and secondary clinical efficacy 
(pharmacodynamic) endpoints will be assessed based on plasma concentration data from samples 
collected at the dosing visit. 
The exposure to trial medication (dasiglucagon, placebo, or GlucaGen) for evaluation of pharmacokinetics 
will also be assessed based on plasma concentration data.  
Statistical methods:  
Due to requirements in the size of the safety database, the sample size is set to 78 subjects treated with 
dasiglucagon 0.6 mg. With a 2:1:1 randomization ratio for the three treatments, dasiglucagon 0.6 mg, 
placebo, and GlucaGen 1 mg, 78 subjects injected with dasiglucagon and 39 subjects with placebo will 
yield a power of 90% at a 5% two-sided significance level to detect a treatment group difference in 
recovery incidence within 20 minutes, assuming as low as 80% and as high as 50% recovery incidence for 
the dasiglucagon and placebo groups, respectively. 
For the confirmatory analyses, the primary and key secondary endpoints for the dasiglucagon 0.6 mg and 
placebo treated subjects will be compared. A hierarchical procedure will be applied for the control of 
multiplicity in statistical inferences. The primary and secondary endpoints will be evaluated on the Full 
Analysis Set sample. The statistical inference comparisons with placebo will be conducted as superiority 
tests. The comparisons of dasiglucagon versus GlucaGen will be summarized descriptively.    
The primary endpoint of time to plasma glucose ≥20 mg/dL recovery, is defined as first increase from 
baseline in plasma glucose of ≥20 mg/dL (1.1 mmol/L) during the hypoglycemic clamp procedure. If the 
hazards by treatment group can be assumed proportional using a graphical comparison of the Nelson-
Aalen estimates, a Cox proportional hazards (CPH) model will be used for inferences, with treatment 
group and injection site modeled as categorical effects, and baseline plasma glucose modeled as a 
continuous covariate. The treatment group rate ratios, active versus placebo, will be estimated together 
with the 95% confidence intervals, and pairwise treatment group inferences (dasiglucagon vs placebo, 
GlucaGen vs placebo) will be evaluated using two-sided likelihood ratio tests.   
If the PH assumption for treatment groups is not met, the primary endpoint will be analyzed using a 
Kaplan-Meier (KM) time to event statistical model, with treatment group and injection site as 
stratification factors. Treatment group differences between the KM curves (dasiglucagon vs placebo, 
GlucaGen vs placebo) will be evaluated inferentially using pairwise two-sided log-rank tests. 
In the primary analysis, subjects who require rescue IV glucose will be censored at the time to plasma 
glucose ≥20 mg/dL recovery. In a sensitivity analysis, the time to plasma glucose ≥20 mg/dL recovery 
will be analyzed without censoring the subjects who received rescue IV glucose before 45 minutes.   
The key secondary endpoints of plasma glucose changes from baseline (CFB) within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection or at the time of rescue, 
will be analyzed with the plasma glucose CFB at rescue carried forward in those subjects who require 
rescue IV glucose before plasma glucose ≥20 mg/dL recovery. Each of these CFB variables will be 
analyzed using an Analysis of Covariance model, with treatment group modeled as a fixed effect and with 
the baseline plasma glucose modeled as a covariate. The dasiglucagon versus placebo treatment group 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 1.0 
 
 
 
 
 
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Page 14 of 79  
difference will be evaluated inferentially as a least squares means contrast, using a two-sided t-test at the 
0.05 significance level, first for the 30 minute CFB, and then sequentially for the 20 minute, 15 minute, 
and 10 minute variables, until the first failure to reject.  
The key secondary incidence variables (plasma glucose recovery within 30 minutes, within 20 minutes, 
within 15 minutes, within 10 minutes, in hierarchical order for inference) will be analyzed using 
descriptive statistics (frequency and percentage) by treatment group. Inferential treatment group 
comparisons will be assessed via pairwise tests of independent binomial proportions (dasiglucagon vs 
placebo, GlucaGen vs placebo).    
The continuous clinical efficacy variables, the exploratory variables, and the pharmacokinetics variables 
will each be summarized descriptively by treatment group. The clinical efficacy variables will be analyzed 
analogous to the plasma glucose CFB variables.   
The safety analyses will include by-treatment-group descriptive summaries of vital sign measurements, 
laboratory measures (including immunogenicity incidence), physical examination assessments, rescue IV 
glucose (incidence and amount of glucose infused), and adverse events. The number and percentage of 
subjects reporting specific events, such as nausea and vomiting, will be presented by body system and 
preferred term. 
Further details will be included in the Statistical Analysis Plan, to be completed before database lock and 
treatment unmasking.   


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 1.0 
 
 
 
 
 
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2 
INTRODUCTION 
2.1 
Background 
Zealand Pharma A/S (Zealand Pharma) is developing dasiglucagon, a physically and 
chemically stable peptide analog of human glucagon, in a ready-to-use liquid formulation 
for the acute treatment of severe hypoglycemia in patients with insulin-treated diabetes 
mellitus. Like native glucagon, dasiglucagon is comprised of 29 amino acids, but with 
7 substitutions which improve its physical and chemical stability in aqueous media. 
These amino acid substitutions make dasiglucagon suitable for a liquid formulation, 
while providing similar efficacy and safety as compared with recombinant glucagon in 
the clinical setting of acute treatment of severe hypoglycemia. 
2.1.1 Hypoglycemia 
Hypoglycemia in patients with diabetes is defined as episodes of an abnormally low 
plasma glucose concentration (3). This is a common, unpredictable, and potentially 
dangerous side effect of treatment of diabetes mellitus, especially with insulin or 
sulfonylureas. It is more frequent in patients with profound endogenous insulin 
deficiency, such as occurs in type 1 diabetes mellitus (T1DM) and advanced type 2 
diabetes mellitus (T2DM). Treatment of T2DM with insulin causes hypoglycemia 
progressively and more frequently over time, whereas in T1DM, hypoglycemia is 
experienced throughout the course of established disease (4). 
Symptoms and signs of hypoglycemia are not specific. Patients undergoing a 
hypoglycemic episode experience unpleasant symptoms such as anxiety, sweating, 
hunger, tremors, palpitations, paresthesia, nausea and pallor. Depending on its severity, 
the hypoglycemia may lead to mild confusion, behavioral changes, loss of consciousness, 
seizures, coma, and death (5). 
The incidence of hypoglycemic events or even the fear of hypoglycemia influences 
patients’ adherence to prescribed treatment regimens for diabetes mellitus (6). This leads 
to inadequate glycemic control, which in turn may lead to an increased risk of diabetic 
complications (5). Serious clinically significant hypoglycemia is now defined as plasma 
glucose <54 mg/dL (3.0 mmol/L), while the plasma glucose alert value is defined as 
<70 mg/dL (3.9 mmol/L) (7). When plasma glucose falls below these values, some kind 
of treatment strategy is needed. 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 1.0 
 
 
 
 
 
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2.1.2 Glucagon 
Glucagon is a naturally occurring hormone, secreted from the alpha cells of the 
pancreatic islets. Glucagon plays a central role in the regulation of glucose homeostasis 
and is the counterpart of insulin for controlling blood glucose levels (i.e. it acts in 
opposition to insulin in terms of effects on blood glucose levels) (8,9). Glucagon 
stimulates hepatic glycogenolysis and gluconeogenesis in hypoglycemic states, thereby 
restoring glucose homeostasis. Glucagon receptor agonism has also been shown to exert 
effects on lipid metabolism, energy balance, body adipose tissue mass and food intake 
(10). Insulin decreases blood glucose levels and cases of hypoglycemia can be reversed 
by glucagon. Therefore, glucagon is indicated for the treatment of severe hypoglycemia. 
Besides intravenous (IV) glucose administration, an injectable form of glucagon is given 
as first aid in cases of severe hypoglycemia, when the patient is unconscious or for other 
reasons cannot take glucose orally. The approved glucagon dose for an adult is 1 mg, 
given by intramuscular (IM), IV, or subcutaneous (SC) injection, which quickly raises 
blood glucose levels. As current marketed recombinant glucagon is highly unstable when 
dissolved in solution, the injectable form has to be reconstituted prior to use in a 9-step 
procedure that requires a sterile diluent to be injected into a vial containing lyophilized 
powdered glucagon. When dissolved in a fluid state, glucagon can form amyloid fibrils 
(11), or tightly woven chains of proteins made up of the individual glucagon peptides. 
The reconstitution process makes the use of marketed glucagon products cumbersome 
(12), and a more patient-friendly formulation is needed. Currently, the Food and Drug 
Administration (FDA) approved instructions for commercially available glucagon allow 
only for immediate usage of glucagon after the powder is reconstituted in aqueous 
solution (13). Therefore, a glucagon analog with enhanced biophysical stability may 
represent a leap forward in terms of convenient therapeutic applications. 
2.1.3 Dasiglucagon 
Dasiglucagon (ZP4207) is a stable peptide analog of human glucagon, available in a 
ready-to-use liquid formulation. Dasiglucagon (hereinafter referred to as dasiglucagon) is 
in development for the treatment of severe hypoglycemia in insulin-dependent patients 
with diabetes mellitus. Dasiglucagon is a specific and full glucagon receptor agonist 
designed to mimic the effects of glucagon, having a fast absorption and elimination 
(minutes). It is a peptide of 29 amino acids, with 7 amino acid substitutions compared to 
native glucagon. The main purpose of the substitutions is to increase the physical and 
chemical stability of the glucagon analog compared to marketed glucagon products such 
as Lilly Glucagon or GlucaGen® (hereafter referred to as GlucaGen). Dasiglucagon 
exhibits improved physical and chemical stability and is available in an aqueous solution 
at neutral pH (14). 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 1.0 
 
 
 
 
 
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Page 17 of 79  
Three clinical trials have been completed with dasiglucagon: a first-in-human dose trial in 
healthy volunteers and subjects with T1DM (ZP4207-14013), a multiple-dose 
dose-escalation trial (ZP4207-15007) evaluating the safety, tolerability, pharmacokinetics 
(PK) and pharmacodynamics (PD) of dasiglucagon in healthy volunteers, and a phase 2 
crossover trial to assess the PK and PD of a single dose of an optimized formulation of 
dasiglucagon administered SC in subjects with T1DM (ZP4207-15126) (14). 
2.1.3.1 Pharmacokinetics and pharmacodynamics of dasiglucagon 
The results of the phase 1 and 2 clinical trials confirmed dose-proportionality for 
dasiglucagon PK, which is characterized by a fast absorption with a peak plasma 
concentration obtained after 35 minutes. Thereafter, the plasma concentration rapidly 
declines with an average half-life of 28 minutes. The median time to the maximum 
plasma concentration (tmax) was 35 minutes for dasiglucagon compared with 20 minutes 
for GlucaGen.  
At all dose levels in the phase 2 trial, all subjects achieved a plasma glucose level of at 
least 70 mg/dL (3.9 mmol/L) as well as an increase in plasma glucose by at least 
20 mg/dL (1.1 mmol/L) within 30 minutes after dosing. The PD responses to 0.6 mg of 
dasiglucagon and 1 mg of GlucaGen were comparable. 
2.1.3.2 Safety of dasiglucagon 
The safety data for dasiglucagon do not give rise to any safety concerns. No new signals 
were observed, beyond those related to the pharmacological effect of glucagon agonism. 
All adverse events (AEs) recorded in the trials were of mild or moderate severity. The 
most frequently reported systemic AE was nausea, which is a known side effect 
following administration of glucagon. Headache was the next most frequently reported 
event, occurring in all dose groups in the phase 2 trial. Injection site reactions were 
observed only sporadically after administration with either dasiglucagon or GlucaGen 
and all were mild and transient. The most frequent injection site reaction was erythema, 
occurring in all treatment groups, including the placebo group, irrespective of dose. 
Glucagon has been described to exert positive inotropic and chronotropic effects and may 
therefore cause tachycardia and hypertension. In the phase 1 clinical trials, but not the 
phase 2 trial, temporary but clinically significant decreases in blood pressure were 
observed in a few healthy volunteers receiving investigational medicinal product (IMP) 
doses of at least 1 mg (4 with dasiglucagon and 1 with GlucaGen). This is not considered 
a safety concern; however, hemodynamic changes after dosing will be considered an 
adverse event of special interest (AESI). 


 
 
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The phase 1 and 2 results and the safety profile described to date do not give rise to 
specific safety concerns. For further information, please refer to the Investigator’s 
Brochure (14). 
2.2 
Trial rationale 
The aim of the current trial is to confirm the superiority of dasiglucagon for the treatment 
of insulin-induced hypoglycemia in subjects with T1DM as compared to placebo for 
dasiglucagon (hereinafter placebo) and to compare the clinical efficacy and safety of 
dasiglucagon with reference to GlucaGen. A randomized, controlled trial design was 
used. 
See Section 4.2 for justification of the design of this trial. 
2.3 
Risk-benefit assessment  
Non-clinical experience 
The nonclinical development program did not reveal any safety findings that would 
prohibit administration of dasiglucagon to humans. None of the safety pharmacology 
studies, repeated dose toxicity studies or genotoxicity studies revealed any significant 
toxicity findings relevant to the therapeutic use of dasiglucagon. 
Clinical experience 
As glucagon and its analogs belong to a well-known drug class with a known mode of 
action, dasiglucagon is not expected to be a high-risk molecule.  
Treatment with an IMP may result in undesired effects or complaints. Undesired effects 
and complaints such as nausea, vomiting, and diarrhea are known AEs occurring with 
glucagon administration. Similar AEs have also been observed to a limited extent in the 
3 clinical studies conducted to date with dasiglucagon. As with every novel drug 
substance, new and as yet unknown side effects may also occur. 
There are limited data available to assess the immunogenic potential of glucagon 
products available on the market, but the data available indicate that marketed glucagon 
only has a low immunogenic potential.  
Overall, dasiglucagon is judged to be a low-risk molecule, based upon the available 
clinical data. Administration of dasiglucagon may be associated with a risk of allergic 
reactions similar to those observed for other therapeutic peptides or proteins. Subjects 
with known or suspected allergies to the trial medications or related products will be 


 
 
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excluded from the trial. Mild or moderate allergic reactions may include symptoms of 
rash, fever, flu-like symptoms, nausea, headache, and myalgia. Acute generalized 
hypersensitivity reactions are usually very rare, but may include symptoms of flushing, 
sweating, dizziness, change in blood pressure, and difficulties in breathing. No severe 
acute hypersensitivity reactions have been observed in the 3 clinical trials conducted with 
dasiglucagon. Direct access to resuscitation equipment is ensured at the clinical trial 
centers. 
With the exception of medical examinations, a subject participating in this trial is not 
likely to derive any personal health-related benefits. The results of the trial may 
contribute to the future use of dasiglucagon in patients with diabetes mellitus 
experiencing severe hypoglycemic reactions. 
The development program including 141 subjects exposed to dasiglucagon to date has 
demonstrated that administration of dasiglucagon is efficacious and well tolerated, with a 
safety profile that does not give rise to specific safety concerns. Two phase 1 and one 
phase 2 clinical trials have been conducted to investigate the safety, tolerability, PK and 
PD of dasiglucagon after single and multiple dosing to healthy volunteers and subjects 
with T1DM under insulin-induced hypoglycemic conditions. Dasiglucagon has proven to 
have relevant clinical effects in the acute severe hypoglycemia rescue setting and may be 
an effective and reliable emergency treatment for severe hypoglycemia. Overall, the 
anticipated benefits for subjects entering the ZP4207-16137 trial are considered to justify 
the risks. 


 
 
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3 
TRIAL OBJECTIVES AND ENDPOINTS 
3.1 
Primary objective 
 To demonstrate superiority of dasiglucagon compared to placebo following a single 
subcutaneous 0.6 mg dose administered to subjects with type 1 diabetes mellitus with 
insulin-induced hypoglycemia. 
3.2 
Secondary objectives 
 To compare the glycemic response observed after dasiglucagon with that of 
GlucaGen. 
3.3 
Primary endpoint 
 Time to plasma glucose ≥20 mg/dL recovery, defined as first increase in plasma 
glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic clamp 
procedure. In the primary analysis, those subjects who require rescue intravenous (IV) 
glucose will be censored at the time to plasma glucose recovery. In a sensitivity 
analysis, the time to plasma glucose recovery will be analyzed without censoring 
those subjects who require rescue IV glucose before 45 minutes.     
3.4 
Key secondary endpoints 
 Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection or at the time of rescue. 
 Incidences of plasma glucose recovery (achieving a plasma glucose concentration 
increase of ≥20 mg/dL [1.1 mmol/L]) within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection. 
3.5 
Other secondary endpoints 
 Clinical efficacy (PD) endpoints: 
- 
Time to first plasma glucose concentration ≥70 mg/dL (3.9 mmol/L). 
- 
Plasma glucose response as area under the curve (AUC) above baseline from time 
zero to 30 minutes, AUC0-30min. 
 Exposure (PK) endpoints: 
- 
Area under the drug concentration curve from time zero to 90 minutes, 
AUC0-90min. 
- 
 Maximum plasma drug concentration (Cmax). 


 
 
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- 
Time to maximum plasma drug concentration (tmax). 
 Safety endpoints:  
- 
Adverse events, clinical laboratory assessments (biochemistry, hematology, 
coagulation, urinalysis), vital signs, physical examination, electrocardiogram 
(ECG), and local tolerability. 
- 
Incidence of rescue infusion of IV glucose during the hypoglycemic clamp 
procedure. 
- 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp 
procedure. 
 Immunogenicity endpoint: 
- 
Incidence of anti-drug antibodies 
3.6 
Exploratory endpoint 
 Incidences of plasma glucose recovery (achieving a plasma glucose concentration 
≥70 mg/dL [3.9 mmol/L] or an increase of ≥20 mg/dL [1.1 mmol/L]) within 
30 minutes after study drug injection. 
 Plasma insulin response as area under the curve above baseline from time zero to 
60 minutes, AUC0-60 min. 


 
 
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4 
OVERALL DESIGN AND PLAN OF THE TRIAL 
4.1 
Overview 
This trial is a global, multicenter, randomized, parallel, double-blind, clinical trial 
confirming the efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in 
subjects with T1DM. The subjects will be randomized 2:1:1 to receive a single fixed SC 
0.6 mg dose of dasiglucagon, placebo, or a 1 mg dose of GlucaGen and followed for at 
least 28 days after receiving treatment. A total of 156 subjects with T1DM are expected 
to complete the treatment visit. The trial will be conducted in the European Union (EU) 
and North America. 
Figure 1 
Trial design 
 
Patients with type T1DM 
made hypoglycemic by 
controlled IV insulin 
Dasiglucagon 0.6 mg
N=78
Placebo
N=39
GlucaGen 1 mg
N=39
N=156
 
4.2 
Justification of the trial design 
4.2.1 Justification for design and parameters 
In order to avoid bias in subject selection and in the evaluation of clinical assessments, 
subjects will be randomly assigned 2:1:1 to either dasiglucagon, placebo, or GlucaGen 
and the trial will be conducted in a double-blinded manner. The randomized parallel 
treatment design with administration of fixed SC doses of dasiglucagon, placebo, or 
GlucaGen to subjects with T1DM and insulin-induced hypoglycemia allows for direct 
comparison of the clinical efficacy of the treatments.  
The trial is double-blind to increase trial validity and to reduce bias during evaluation of 
the treatments. However, since the trial medications are not identical in appearance, the 


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handling, preparation and administration of trial medication will be done by unblinded 
trial personnel who will not be involved in any other trial procedures or assessments. See 
Section 6.6 for more information about which assessments are blinded and which are not, 
with reasons.  
A superiority trial design is used because the aim is to show that treatment with 
dasiglucagon is an effective treatment compared to placebo. The secondary objective was 
chosen to allow a comparison between treatment with dasiglucagon and an established 
comparator treatment for severe hypoglycemia, GlucaGen. 
Administration of glucagon is intended to quickly raise blood glucose levels in subjects 
with T1DM with insulin-induced hypoglycemia. Therefore, in order to assess the clinical 
efficacy of dasiglucagon as compared to placebo and GlucaGen following a single SC 
dose, the primary endpoint and secondary efficacy endpoints involve the measurement of 
plasma glucose concentrations at different timepoints. 
4.2.2 Justification for drug, route, dosage and treatment duration 
Dasiglucagon and GlucaGen will be administered as fixed doses independent of body 
weight because this is the intended therapeutic dosing regimen in the emergency 
treatment of hypoglycemia. The selected dose of 1 mg GlucaGen is the approved dose for 
treatment of severe hypoglycemia. Data from the studies conducted to date with 
dasiglucagon, including the phase 2 trial in subjects with T1DM, have been used to 
establish that 0.6 mg of dasiglucagon is an effective dose and also represents a 
therapeutically equivalent dose to 1 mg of GlucaGen (see also Section 6.1). 
Dasiglucagon, placebo, and GlucaGen will be administered in the abdomen, buttocks, or 
thigh by SC injection, as this is one of the intended routes of administration for 
dasiglucagon, besides IM and IV. 
Subjects will be followed for at least 28 days after dosing in order to perform an adequate 
immunogenicity evaluation of treatment.  


 
 
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5 
TRIAL POPULATION 
5.1 
Rationale for trial population 
Dasiglucagon is in development for the treatment of severe hypoglycemia in 
insulin-dependent patients with diabetes mellitus. In the present trial, subjects with 
T1DM are included in the evaluation of efficacy and safety of dasiglucagon under 
hypoglycemic conditions as this is part of the intended target population. Subjects with 
T1DM are selected to avoid the endogenous glucagon counter-regulatory response to 
insulin-induced hypoglycemia that is present in patients with T2DM. The inclusion and 
exclusion criteria are set to include a trial population that represents the general 
population of subjects with T1DM.  
5.2 
Planned sample size and number of trial centers 
A total number of 156 subjects with T1DM are expected to complete the treatment visit 
(Visit 2). To qualify as completed, the subject must be dosed and have blood drawn for 
the PK and PD assessments described in the protocol. It is expected that up to 
176 subjects will be randomized to have 156 subjects completing Visit 2. Completion of 
156 subjects (78 subjects in the dasiglucagon group and 39 subjects in each of the 
placebo and GlucaGen groups) would be expected to provide adequate power for the 
primary efficacy evaluation, as described in Section 9.1. 
The planned date for first subject first visit is expected to take place in Q4, 2017 and the 
planned date for last subject last visit is expected to take place in Q3, 2018. 
This trial is planned to be conducted at 4 to 6 trial sites in the United States of America 
[US], Canada, and Europe. 
5.3 
Inclusion criteria 
Subjects will be entered into this trial only if they meet all of the following criteria: 
1. Informed consent obtained before any trial-related activities (trial-related 
activities are any procedure that would not have been performed during normal 
management of the subject). 
2. Female or male subjects with T1DM for at least 1 year, diagnostic criteria as 
defined by the American Diabetes Association (3). 
3. Treated with insulin for T1DM for at least 1 year and with stable insulin treatment 
(defined as no more than a 10-unit daily variation in total daily insulin dose) 
30 days prior to screening 


 
 
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4. Hemoglobin A1c <10%. 
5. Aged between 18 and 75 years, both inclusive. 
6. A female subject must meet one of the following criteria: 
a. Participant is of childbearing potential and agrees to use one of the 
accepted contraceptive regimens throughout the entire duration of the trial 
from screening and until last follow-up visit. Additionally, if the 
participant is using systemic contraceptives, she must use an additional 
form of acceptable contraception. An acceptable method of contraception 
includes one of the following: 
i. Abstinence from heterosexual intercourse; 
ii. Systemic contraceptives (birth control pills, injectable/implant/ 
insertable hormonal birth control products, transdermal patch); 
iii. Intrauterine device (with and without hormones); or 
iv. Condom with spermicide; or 
b. Participant is of non-childbearing potential, defined as surgically sterile 
(i.e. has undergone complete hysterectomy, bilateral oophorectomy, or 
tubal ligation), or in a menopausal state (at least 1 year without menses). 
7. A male subject must meet the following criteria: Surgically sterilized or willing to 
refrain from sexual intercourse from screening and until last follow-up visit or, if 
sexually active, uses condom and partner practices contraception during the trial 
from screening and until last follow-up visit. 
5.4 
Exclusion criteria 
Subjects meeting any of the following criteria during screening evaluations will be 
excluded from trial participation: 
1. Previously treated with dasiglucagon (previously referred to as ZP4207). 
2. Known or suspected allergy to trial product(s) or related products. 
3. History of anaphylaxis or symptoms of severe systemic allergy (such as 
angioedema). 
4. Previous participation (randomization) in this trial. 
5. Females who are pregnant according to a positive pregnancy test, are actively 
attempting to get pregnant, or are lactating. 
6. History of hypoglycemic events associated with seizures in the last year prior to 
screening. 
7. History of severe hypoglycemia (defined as plasma glucose <54 mg/dL 
[3.0 mmol/L]) in the last month prior to screening. 
8. Receipt of any investigational drug within 3 months prior to screening. 
9. Active malignancy within the last 5 years. 
10. Congestive heart failure, New York Heart Association class II-IV. 


 
 
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11. Inadequately treated blood pressure, defined as systolic ≥160 mmHg or diastolic 
≥90 mmHg) at screening (15). 
12. Current bleeding disorder, including anti-coagulant treatment. 
13. Known presence or history of pheochromocytoma (i.e. adrenal gland tumor) or 
insulinoma (i.e. insulin secreting pancreas tumor). 
14. Use of a daily systemic beta-blocker drug, indomethacin, warfarin or 
anticholinergic drugs in the previous 28 days before Day 1 of this trial. 
15. Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >2.5 × the 
upper limit of the normal range (ULN), bilirubin >1.5 × ULN, estimated 
glomerular filtration rate <30 mL/min/1.73 m2 according to the Modification of 
Diet in Renal Disease study definition (16), or altered electrolyte values of 
clinical relevance for cardiac conduction, as judged by the investigator. 
16. Clinically significant abnormal ECG at screening as judged by the investigator. 
17. Clinically significant illness within 4 weeks before screening, as judged by the 
investigator. 
18. Donation of blood or plasma in the past month, or in excess of 500 mL within 
12 weeks prior to screening. 
19. Surgery or trauma with significant blood loss within the last 2 months prior to 
screening. 
20. A positive result in the alcohol and/or urine drug screen at the screening visit. 
Significant history of alcoholism or drug abuse as judged by the investigator or 
consuming more than 24 g alcohol per day for men, or more than 12 g alcohol per 
day for women. 
21. Subjects with mental incapacity or language barriers which preclude adequate 
understanding or cooperation, who are unwilling to participate in the trial, or who 
in the opinion of the investigator should not participate in the trial. 
22. Any condition interfering with trial participation or evaluation or that could be 
hazardous to the subject. 
23. The use of prescription or non-prescription medications known to cause QT 
prolongation. 
5.5 
Dosing day exclusion criteria 
Subjects who meet one or more of the following dosing day exclusion criteria at the 
dosing visit will be excluded from the dosing visit, but the visit can be rescheduled 
1-7 days later. The dosing visit can only be rescheduled once. 
1. Atypically strenuous exercise within 4 days prior to dosing, as judged by the 
Investigator. Exercise during the trial should follow subject’s typical routine, and 
should not exceed a near maximum intensity for more than 20 minutes per day, or 
moderate intensity for more than 90 minutes per day. 


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2. Clinically significant illness within 4 weeks before dosing, as judged by the
investigator.
3. Consumption of alcohol within 24 hours prior to dosing visit, or positive results
from an alcohol breath test.
4. Not fasting from 22:00 hours the evening prior to dosing, apart from water. Small
amounts of carbohydrates (up to 20 g) to prevent hypoglycemia are allowed.
5. The use of any non-prescribed systemic or topical medication, except routine
vitamins and occasional use (as judged by the investigator) of acetylsalicylic acid
and paracetamol within 2 weeks prior to dosing. Treatment with insulin, including
analogs, is allowed.
6. Use of any basal insulin within 24 hours prior to dosing.
7. Use of any short acting (bolus) insulin within 12 hours prior to dosing.
8. Changes in medical history or concomitant medication resulting in fulfillment of
clinical exclusion criteria, as judged by the investigator.
9. Plasma glucose value <50 mg/dL (2.8 mmol/L) within the last 24 hours or plasma
glucose value <60 mg/dL (3.3 mmol/L) within the last 5 hours prior to dosing.
5.6 
Premature treatment discontinuation and withdrawal 
Participation in the trial is strictly voluntary. A subject has the right to withdraw from the 
trial at any time and for any reason. If he/she chooses to withdraw, the investigator must 
be informed immediately. The investigator has the right to terminate participation of any 
subject at any time if the investigator deems it in the subject's best interest. The reason 
and circumstances for withdrawal will be documented in the electronic case report form 
(eCRF). 
5.6.1 Possible reasons for treatment visit discontinuation 
A subject will be discontinued from treatment if the following applies: 

Withdrawal of consent by subject.

If a protocol deviation occurs which, in the clinical judgement of the investigator, can
invalidate the trial or endpoints or can interfere pharmacokinetically or
pharmacodynamically with the trial product, the subject will be discontinued by the
investigator.

Adverse events occur which are considered unacceptable by the subject or the
investigator.
If discontinuation occurs following administration of trial medication, every effort should 
be made to have the subject return and participate in the complete follow-up visit on Day 
28 (see Table 3) to avoid missing data. 


 
 
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If trial participation is terminated due to an AE possibly related to any of the trial 
medications or trial examinations, the subject must be followed up by additional 
examinations according to the medical judgment of the investigator until the abnormal 
condition is resolved or the investigator deems further observations or examinations to be 
no longer medically indicated. 
A total of 156 subjects must complete the treatment visit (Visit 2). To qualify as 
completed, the subject must be dosed and have blood drawn for the PK and PD 
assessments described in the protocol.  
5.6.2 Center discontinuation 
The center can be closed and the trial terminated for the following reasons: 
 The center is unlikely to be able to recruit sufficient subjects within the agreed time 
frame. 
 The center does not respond to trial management requests. 
 Repeat protocol violations. 
5.6.3 Trial termination 
The sponsor reserves the right to modify or terminate the trial at any time. Possible 
reasons for termination are: 
 Safety reasons – the incidence of AEs in this or any other trial using the same trial 
medication indicates a potential health risk for the subjects. 
 New scientific knowledge becomes known that makes the objectives of the trial no 
longer feasible/valid. 
 Unsatisfactory enrolment of subjects. 
5.7 
Subject identification and randomization 
Subjects who have given written informed consent and meet all inclusion and none of the 
exclusion criteria will be randomized in a 2:1:1 ratio to either 0.6 mg dasiglucagon 
(n=78), placebo (n=39), or 1 mg GlucaGen (n=39) via an Interactive Web Response 
System (IWRS). Randomization will continue until 156 subjects have completed Visit 2. 
Subjects with previous exogenic glucagon exposure will not be excluded from the trial, 
but the information on previous glucagon administration will be recorded, to enable 
subgroup analyses.  
In the event of an emergency, e.g. when it becomes necessary for the investigator to 
know which treatment the subject is taking, the subject code can be broken by the 


 
 
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investigator, preferably after consultation with the medical monitor. Emergency code 
breaks can be performed using the IWRS. 


 
 
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6 
TRIAL DRUG 
6.1 
Identity 
The following trial drugs will be administered: 
 Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in a prefilled syringe. 
 Placebo, liquid formulation, 0.6 mL delivered in a prefilled syringe. 
 Recombinant glucagon hydrochloride, 1 mg for reconstitution (GlucaGen®, Novo 
Nordisk) in 1 mL sterile water. 
The description of the three trial drugs is provided in Table 1. Dasiglucagon is a stable 
peptide analog of human glucagon in a ready-to-use liquid formulation for treatment of 
severe hypoglycemia in insulin-dependent patients with diabetes mellitus. GlucaGen is 
approved in the EU and US and is indicated for treatment of severe hypoglycemic 
reactions, which may occur in the management of insulin-treated children and adults with 
diabetes mellitus. 
Table 1 
Description of trial drugs 
 
Test product 
Placebo Product 
Comparator product 
Name 
Dasiglucagon 
Placebo  
GlucaGen® 
Active 
substance 
Dasiglucagon 
N/A 
Recombinant 
glucagon 
hydrochloride 
Formulation 
Liquid formulation, 
0.6 mL 
Liquid formulation, 
0.6 mL 
Powder and solvent 
for reconstitution as 1 
mL solution for 
injection 
Strength 
1 mg/mL 
N/A 
1 mg 
Device 
Single use pre-filled 
syringe 
Single use pre-filled 
syringe 
Powder and solvent 
for reconstitution 
packed together in a 
plastic box. A “hypo-
kit”. 
Manufacturer 
Zealand Pharma A/S, 
Glostrup 
(Copenhagen), 
Denmark 
Zealand Pharma A/S, 
Glostrup 
(Copenhagen), 
Denmark 
Novo Nordisk A/S, 
Bagsværd, Denmark 


 
 
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Test product 
Placebo Product 
Comparator product 
Storage 
requirements 
Store between 2 and 
8°C 
Store between 2 and 
8°C 
Store between 2 and 
8°C 
The quantities of ingredients for dasiglucagon and placebo are provided in Table 2. 
Table 2 
Quantities of ingredients in dasiglucagon and placebo injection 
Component 
Amount per mL 
(dasiglucagon) 
Amount per mL 
(placebo) 
Function 
Dasiglucagon* 
1.0 mg 
N/A 
Sodium chloride 
10.23 mg 
10.23 mg 
Trometamol/Tromethamine 
6.06 mg 
6.06 mg 
Water for injection 
To make 1 mL 
To make 1 mL 
Sodium hydroxide 
q.s. 
q.s. 
Hydrochloric acid 
q.s. 
q.s. 
*The quantity of drug substance to be used is calculated according to net peptide content and purity.  
q.s. = quantum sufficit (quantity required). 
6.2 
Treatment assignment and randomization 
Subjects successfully completing screening and who fulfill entry eligibility and 
randomization criteria will be randomized to one of three treatment groups in a ratio of 
2:1:1: 
 Test treatment: Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in a 
prefilled syringe. 
 Placebo treatment: Placebo, liquid formulation, 0.6 mL delivered in a prefilled 
syringe. 
 Reference treatment: Recombinant glucagon hydrochloride, 1 mg for reconstitution. 
Randomization will be performed using a fixed-block randomization scheme. The 
randomization scheme will be generated prior to the initiation of the study by an 
independent statistician/programmer who will not be a member of the study team; all 
investigators will not be aware of the block size of the randomization scheme.  
Randomization will be stratified by treatment group and by injection site (abdomen, 
buttocks, or thigh) and controlled via the IWRS.       


 
 
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Subjects will be randomized to study treatment using an interactive, automated system 
which has been validated for the intended use under the International Society of 
Pharmaceutical Engineers Good Automated Manufacturing Process guidelines, 21CFR 
Part 11 (FDA regulation for Electronic Records and Electronic Signatures) and the 
International Conference on Harmonization (ICH) Guidance E6 for Industry on Good 
Clinical Practice (GCP).   
6.3 
Administration 
Dasiglucagon, placebo, and GlucaGen will be administered by SC injection in the 
abdomen, buttocks, or thigh.  
An unblinded person (appropriately trained) authorized to prepare the dose and 
administer the treatment in accordance with the randomization will prepare the treatment 
required for each subject on each dosing day. The dose will be administered by the 
unblinded, trained and qualified person. The content of the syringe has to be checked for 
clarity and absence of bubbles. To ensure proper administration, the unblinded person 
should administer the SC injection at a 90 degree angle by grasping skin between the 
thumb and first finger. The injection should be given within 5 seconds. 
Syringes will be discarded after dose administration. Used GlucaGen vials will be stored 
in a lockable box (separated from unused vials) at ambient temperature. 
6.4 
Packaging and labelling 
The test product will be packed by the sponsor. The information on the labels will be in 
the local language and the product label will be compliant with local laws and 
regulations.  
The study drug label will describe the storage conditions for study drug. The labels will 
supply no information about the subjects. Each treatment kit (pre-filled syringe/vial for 
reconstitution) will have a unique Dispensing Unit Number for drug allocation, drug 
accountability, and traceability purposes. 
Labelling will be performed according to Annex 13 of the Good Manufacturing Practice 
guidelines of the European Commission, ICH GCP guidelines, and local law. 


 
 
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6.5 
Storage of study drugs 
The Investigator must ensure the availability of proper storage conditions. All study drug 
supplies provided for this study will be stored in a secure area with restricted access at the 
study site.  
The temperature should be monitored by recording the actual, minimum, and maximum 
temperatures using a calibrated thermometer or thermocouple, or by continuous recording 
using a qualified temperature monitoring system. The temperature should be evaluated 
and documented at least on working days on a temperature log. This log must be included 
in the Investigator Site File upon study termination. 
Dasiglucagon and placebo must be stored in a refrigerator (at a temperature of 2–8°C), 
and should be handled in accordance with guidelines from the sponsor. GlucaGen must 
also be stored in a refrigerator (at a temperature of 2–8°C), and should be handled in 
accordance with the Summary of Product Characteristics (13).  
The unblinded person responsible for study drug handling must contact the unblinded 
monitor in case of temperature deviations outside the acceptable range.  
Please see the Pharmacy Manual for additional information on handling study drug. 
6.6 
Blinding and breaking the blind 
This is a double-blind trial. As the trial products are not identical in appearance, 
dasiglucagon and placebo being available as a liquid formulation and GlucaGen as a 
powder for reconstitution, unblinded trial personnel will be responsible for handling, 
preparing (according to the prescription from the IWRS), and administering the trial 
medication, as well as for keeping the records strictly confidential and accessible only to 
unblinded staff until after the database has been locked. To maintain double-blind 
conditions, all trial assessments at the trial center will be done by blinded trial personnel 
not involved in the administration of trial medications. However, exposure assessments 
and anti-drug antibody (ADA) assessments will be performed by unblinded personnel at 
the special laboratories, to ensure that dasiglucagon, placebo, or GlucaGen administration 
is matched with the applicable bioanalytical assay. 
Treatment assignment will be kept strictly confidential and accessible only to authorized 
persons until after the time of unblinding. Codes with treatment assignment will, 
however, be readily available in the IWRS to the blinded personnel in case of an 
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The code for a particular subject may be broken in a medical emergency if knowing the 
identity of the treatment allocation would influence the treatment of the subject. The 
emergency code break can be performed using the IWRS. Whenever a code is broken, the 
person breaking the code must record the time, date and reason as well as his/her initials 
in the source documents. The breaking of blinded codes in case of medical emergency for 
one subject should not unblind the trial personnel to the treatment information of other 
subjects. The person performing the unblinding should inform as few people as possible 
about the result of the unblinding. All persons unblinded for a specific subject should be 
documented. 
If the trial center needs to break the code, the medical monitor should, if at all possible, 
be contacted prior to breaking the code and the monitor must be notified within 24 hours 
after the code has been broken. 
The pharmacovigilance unit (safety contract research organization [CRO]; see the list 
of trial personnel in Section 12.1) will be able to break the code in case of a serious 
unexpected suspected adverse reaction (SUSAR).  
The central and specialty laboratories will be provided with a copy of the randomization 
list. 
6.7 
Drug accountability 
Handling, preparation and administration of trial medication will be done by unblinded 
trial personnel. Each center will keep accurate records of the trial supplies received, 
stored, and dispensed, using appropriate forms. The trial supplies will be stored under 
appropriate conditions, locked and with restricted access.  
All unused supplies and all empty and partially empty containers of trial medication will 
be stored until the trial closure visit has been performed and then sent for destruction. 
This does not apply to the used syringes as they will be discarded after dose 
administration. Destruction must not take place until approved by the Sponsor.  
6.8 
Treatment compliance 
All trial medications will be prepared and administered by unblinded trial personnel. 
PK assessments will support the surveillance of compliance with IMP administration.  


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6.9 
Prior and concomitant medications 
Prior glucagon exposure will be recorded in the eCRF at screening. All concomitant 
medications will be recorded and/or updated in the eCRF at each visit.  
Subjects using any new concomitant medication resulting in fulfillment of a dosing day 
exclusion criterion (see Section 5.5) will be excluded from the dosing visit, but can be 
rescheduled to one of the following days (1–7 days later). The dosing visit can only be 
rescheduled once. 
6.9.1 Prohibited medications 
The use of anti-coagulant treatments and medications (prescription and non-prescription) 
that are known to cause QT prolongation are prohibited during the course of the trial. 
Within 28 days prior to dosing, the use of daily systemic beta-blockers, indomethacin, 
warfarin, and anticholinergic drugs is prohibited. 
Within 2 weeks prior to dosing, the use of any non-prescribed systemic or topical 
medication (with the exception of vitamins and the occasional use of acetylsalicylic acid 
and paracetamol) is prohibited.  
Within 24 hours prior to dosing, the use of any basal insulin is prohibited. 
Within 12 hours prior to dosing, the use of any short acting (bolus) insulin, except insulin 
glulisine (Apidra®) is prohibited.  
During the insulin-induced hypoglycemic procedure, continuous SC insulin infusion must 
be stopped. 


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7 
PARAMETERS AND METHODS OF ASSESSMENT 
Overall, approximately 180 mL of blood will be drawn from each subject for PK, PD, 
ADA, and safety laboratory assessments. 
7.1 
Efficacy parameters 
A description of the sample handling and sample processing at the site will be included in 
the laboratory manuals. Validation documentation for the assays must be available prior 
to sample analyses. A bioanalytical report for each analysis of trial PD and PK samples 
will be prepared. 
7.1.1 Pharmacodynamic measurements 
The plasma glucose profile for evaluation of the primary and secondary clinical efficacy 
endpoints will be assessed based on plasma concentration data (AUC0-30min) from samples 
collected at the dosing visit (Visit 2). The samples will be sent to the clinical laboratory 
and analyzed using a sensitive and validated assay for glucose measurements.  
Samples will be collected pre-dose, and at 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 
75, and 90 minutes after dosing (see the schedule of procedures in Table 3). The actual 
time of blood sampling for evaluation of plasma glucose should not deviate from the 
nominal time by more than ±30 seconds until the 20 minute collection time point and by 
more than ±1 minute for the subsequent collection time points. Pre-dose is defined as 
within 2 minutes prior to dosing. 
7.1.2 Pharmacokinetic measurements 
The exposure to trial medication (dasiglucagon or GlucaGen) for evaluation of PK will 
be assessed based on plasma concentration data (AUC0-90 min, Cmax, tmax) from samples 
collected at the dosing visit (Visit 2).  
Samples (including back-up samples) will be collected pre-dose, and at 15, 30, 35, 40, 50, 
60, 90, and 120 minutes after dosing (see the schedule of procedures in Table 3). The 
actual time of blood sampling for exposure to trial medication should not deviate from the 
nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to 
dosing. 


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7.2 
Safety parameters 
7.2.1 Adverse events 
The investigator is responsible for the detection and documentation of events meeting the 
criteria and definition of an AE or serious adverse event (SAE), as provided in this 
protocol. During the trial, the investigator or center staff will be responsible for reporting 
AEs and SAEs, as detailed in this section of the protocol.  
Based on the investigator’s clinical judgment it will be determined whether an AE is 
related to treatment and of sufficient severity to require the subject’s removal from 
treatment or from the trial. A subject may also voluntarily withdraw from treatment due 
to what he or she perceives as an intolerable AE. If either of these situations arises, the 
subject should be under medical supervision until symptoms cease or the condition 
becomes stable. 
7.2.1.1 Definitions 
Adverse event 
An AE is any untoward medical occurrence in a trial subject given an IMP which does 
not necessarily have a causal relationship with the treatment. An AE can therefore be any 
unfavorable and/or unintended sign (for example, an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of an IMP, whether or not 
considered related to the IMP.  
In this trial, only treatment-emergent adverse events (TEAEs) will be collected and 
reported. TEAEs are events that occur from the first trial-related activity after the subject 
has signed the informed consent form until the end of the post-treatment follow-up 
period.  
AEs include: 

A clinically significant worsening of a concomitant illness.

A clinical laboratory abnormality which is clinically significant, i.e. any abnormality
that suggests a disease and/or organ toxicity and is of a severity that requires active
management. Active management includes active treatment or further investigations,
for example change of medicine dose or more frequent follow-up due to the
abnormality.

Hypoglycemic episodes (see Section 7.2.2).

Injection site reactions (see Section 7.2.6).


 
 
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The following should not be recorded as AEs, if recorded at screening (on the Screening 
Form or eCRF): 
 Pre-planned procedure, unless the condition for which the procedure was planned has 
worsened from the first trial related activity after the subject has signed the informed 
consent. 
 Pre-existing conditions found as a result of screening procedures (pre-existing 
conditions should be reported as medical history or concomitant illness). 
Serious adverse event 
An SAE is any untoward medical occurrence that at any dose:  
 Results in death  
 Is life-threatening  
 Requires inpatient hospitalization or prolongation of existing hospitalization 
 Results in persistent or significant disability or incapacity 
 Is a congenital anomaly or birth defect 
 Is medically important* 
*Medical judgement must be exercised in deciding whether an AE is believed to be 
‘medically important’. Medically important events may not be immediately 
life-threatening or result in death or hospitalization but may jeopardize the subject or 
may require intervention to prevent one of the other outcomes listed in the definition 
above. 
Adverse drug reaction 
All noxious and unintended responses to an investigational product related to any dose 
should be considered adverse drug reactions. The phrase responses to an investigational 
product means that a causal relationship between an investigational product and an 
adverse event is at least a reasonable possibility, i.e. the relationship cannot be ruled out. 
 
Suspected unexpected serious adverse reactions (SUSARs) 
An AE fulfilling one of the criteria of seriousness and being assessed as related to IMP 
application, the nature or severity of which is not consistent with the applicable reference 
document (e.g. dasiglucagon Investigator’s Brochure or package leaflet/Summary of 
Product Characteristics for GlucaGen). 


 
 
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Adverse event of special interest 
An AESI is an event which, in the evaluation of safety, has a special focus (e.g. required 
by health authorities). In this trial hemodynamic changes, as defined below, are 
considered AESIs: 
 Post-dose clinical signs, or measured vital signs, indicating a clinically significant 
drop in blood pressure including signs of orthostatic hypotension, vasovagal 
responses or bradycardia.   
 Post-dose change in pulse or blood pressure considered an event of hypo- or 
hypertension as judged by the investigator. 
 
Intensity of an adverse event 
The maximum intensity (severity) of all AEs must be assessed by the investigator and 
documented. Severity should be graded when the AE outcome is known: 
Mild:  
A type of AE that is usually transient and may require only minimal 
treatment or therapeutic intervention. The event does not generally 
interfere with usual activities of daily living. 
Moderate:  
A type of AE that is usually alleviated with additional specific therapeutic 
intervention. The event interferes with usual activities of daily living, 
causing discomfort but poses no significant or permanent risk of harm to 
the subject. 
Severe:  
A type of AE that interrupts usual activities of daily living, or significantly 
affects clinical status, or may require intensive therapeutic intervention. 
A ‘severe’ reaction does not necessarily deem the AE as ‘serious’ and an SAE may not 
necessarily be ‘severe’ in nature. 
Causality relationship to trial medication 
The causality of each AE should be assessed by the investigator according to the 
following classification: 
Probable: 
Good reason and sufficient documentation to assume a causal relationship. 
Possible: 
A causal relationship is conceivable and cannot be dismissed. 
Unlikely: 
The event is most likely related to etiology other than the trial product. 
Not related:  No relationship to trial product. 


 
 
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Outcome of an adverse event 
The outcome of all AEs must be assessed by the investigator and documented by his/her 
staff. The following definitions should be used: 
Recovered/resolved:  
The subject has fully recovered, or by medical or 
surgical treatment the condition has returned to the 
level observed at the first trial-related activity after 
the subject signed the informed consent.  
Recovering/resolving: 
The condition is improving and the subject is 
expected to recover from the event. This term is 
only applicable if the subject has completed the 
trial. 
Recovered/resolved with sequelae:  The subject has recovered from the condition, but 
with lasting effect due to a disease, injury, treatment 
or procedure. If a sequela meets an SAE criterion, 
the AE must be reported as an SAE. 
Not recovered/not resolved:  
The condition of the subject has not improved and 
the symptoms are unchanged. 
Fatal:  
This term is only applicable if the subject died from 
a condition related to the reported AE. Outcomes of 
other reported AEs in a subject before he/she died 
should be assessed as "recovered/resolved", 
"recovering/resolving", "recovered/resolved with 
sequelae" or "not recovered/not resolved". An AE 
with fatal outcome must be reported as an SAE. 
Unknown:  
This term is only applicable if the subject is lost to 
follow-up. 
7.2.1.2 Collection, recording and reporting of adverse events 
All events meeting the definition of an AE must be collected and reported from the first 
trial related activity after the subject has signed the informed consent until the end of the 
post-treatment follow-up period. At each contact with the center (visit or telephone, 
excluding visits where the subject is not seeing the investigator or his/her staff [e.g. visits 
to the laboratory]) the subject must be asked about AEs. All AEs, either observed by the 
investigator or reported by the subject, must be recorded by the investigator and 
evaluated. 
The investigator should record the diagnosis, if possible. If no diagnosis can be made the 
investigator should record each sign and symptom as individual AEs. 


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One single Adverse Event Form must be used per AE from start to resolution. For SAEs, 
the Serious Adverse Event form must also be completed. For AESIs, the AESI form must 
also be completed. 
AE information should include the following: 

Date and time of onset and resolution

Date and time of investigator’s first information on the (S)AE

Seriousness

Severity

Causal relationship with IMP

Interruption or withdrawal of treatment during IMP administration and other
measures taken

Outcome.
All AEs are coded; details are described in the trial-specific Data Management Plan. 
If an event classifies as a AESI, the investigator must tick the AESI box on the AE form 
and complete the AESI form. The AESI form will capture if the event was associated 
with any signs or symptoms and capture the highest/lowest blood pressure and pulse 
measured during the event. The investigator must report all AESIs to the sponsor’s 
responsible pharmacovigilance unit (Safety CRO; see the list of trial personnel in 
Section 12.1) immediately (within 24 hours) after obtaining knowledge about the event. 
The investigator must report initial information electronically (e.g. in PDF format) on all 
SAEs to the sponsor’s responsible pharmacovigilance unit (Safety CRO; see the list of 
trial personnel in Section 12.1) immediately (within 24 hours) after obtaining knowledge 
about the event. The Safety CRO will inform the medical monitor and the sponsor about 
the reported SAEs. 
It is the responsibility of the Safety CRO to report all SUSARs (see Section 7.2.1.1) that 
occur in this trial to the Competent Authorities and Institutional Review Boards 
(IRBs)/Independent Ethics Committees (IECs) in accordance with the local requirements 
in force and ICH guideline for GCP. The trial monitor must be informed accordingly. 
7.2.1.3 Follow-up of adverse events 
All AEs that are ongoing at the end of the subject’s participation in the trial will be 
followed-up until the event is resolved or reaches a satisfactory outcome as deemed by 
the investigator or until the last visit of the last subject enrolled in the trial, whichever 
occurs first.   


 
 
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Follow-up actions for all SAEs will be performed according to appropriate clinical care 
practices and may depend on the nature of the event. These will be determined after 
internal review and/or sponsor review. 
The follow-up information should only include new (updated and/or additional) 
information that reflects the situation at the time of the investigator’s signature. 
Follow-up information on (S)AEs will be updated using the (S)AE form. If a non-serious 
event becomes serious during the follow-up the AE form and SAE form have to be used 
and reporting timelines follow those of an SAE. 
The investigator must forward follow-up information on SAEs and if previously 
non-serious AEs become SAEs to the Safety CRO immediately (within 24 hours) after 
obtaining knowledge about the new information. 
The sponsor and/or CROs acting on behalf of the sponsor can upgrade a non-serious AE 
to an SAE. In these situations the investigator will be informed and asked to fill out an 
SAE form and forward to the Safety CRO immediately (within 24 hours). 
7.2.1.4 Clinical laboratory abnormalities and other abnormal assessments as adverse 
events or serious adverse events 
Abnormal laboratory findings (e.g. biochemistry, hematology, urinalysis) or other 
abnormal assessments (e.g. vital signs) that are judged by the investigator as clinically 
significant will be recorded as AEs or SAEs if they meet the definition of an AE or SAE. 
Clinically significant abnormal laboratory findings or other abnormal assessments that 
are detected during the trial or are present at baseline and significantly worsen following 
the start of the trial will be reported as AEs or SAEs. However, clinically significant 
abnormal laboratory findings or other abnormal assessments that are associated with the 
disease being studied, unless judged by the investigator as more severe than expected for 
the subject’s condition, or that are present or detected at the start of the trial and do not 
worsen, will not be reported as AEs or SAEs. 
The investigator will exercise his or her medical and scientific judgment in deciding 
whether an abnormal laboratory finding or other abnormal assessment is clinically 
significant. 
7.2.2 Hypoglycemia 
Hypoglycemia will be regarded as an AE and recorded and documented on an AE form 
(and SAE form, if applicable). 


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Hypoglycemia is defined as a decline in plasma glucose to below 70 mg/dL 
(3.9 mmol/L). However, in the time period from initiation of the hypoglycemic clamp 
procedure (see Section 8.2.3.1) until 45 minutes after dosing, hypoglycemia is defined 
as a decline in plasma glucose to below 45 mg/dL (2.2 mmol/L).  
During the dosing visit, prior to administration of the IMP, a plasma glucose value of 
<45 mg/dL (2.5 mmol/L) will be treated by an IV glucose solution. After administration 
of the IMP in the period from 8 minutes until 44 minutes after dosing, a plasma glucose 
value of <45 mg/dL (2.5 mmol/L) will be treated by an IV glucose solution, and if a 
plasma glucose value of ≥70 mg/dL (3.9 mmol/L) is not achieved within the 45 minutes 
after IMP administration, IV glucose infusion will also be initiated. 
If the subject experiences symptoms of hypoglycemia, a plasma glucose measurement 
should be taken in order to classify the event (please refer to Section 7.4.2 for additional 
details).  
7.2.3 Physical examination 
The physical examination will be carried out at screening (Visit 1) and at the follow-up 
visit (Visit 3; see Table 3).  
The physical examination includes examination of the following body systems: head, 
ears, eyes, nose, throat, including the thyroid gland; heart, lung, chest; abdomen; skin and 
mucosae; musculoskeletal system; nervous system; lymph node; other findings. 
At the screening visit, any abnormality will be recorded and described in the eCRF, 
including the investigator’s assessment of clinical significance (‘abnormal, not clinically 
significant’ or ‘abnormal, clinically significant’). Clinically significant findings at the 
screening visit should be recorded as concomitant illness. 
At subsequent visits, any clinically significant deterioration of a pre-existing condition as 
well as any new clinically significant findings will be recorded as AEs (see 
Section 7.2.1). 
7.2.4 Vital signs 
An examination of the following vital signs will be performed at screening (Visit 1), the 
dosing visit (Visit 2) and at the follow-up visit (Visit 3):  

Diastolic and systolic blood pressure (mmHg) will be measured after at least
5 minutes rest in a supine position. At Visit 1, blood pressure will be measured in
both arms. The blood pressure from the arm with the higher systolic value is


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transcribed into the eCRF and this arm should be used for all subsequent 
measurements of the subject’s blood pressure in this trial.  

Pulse (beats per min) measured after at least 5 minutes rest in a supine position.

Body temperature (°C).
At the dosing visit, measurements will be taken prior to the start of the insulin-induced 
hypoglycemic procedure (within 30 minutes) and at 30, 90, and 300 minutes after dosing 
(see Table 3). The actual time of the assessment should not deviate from the nominal time 
by more than ±10 minutes. In addition to the pre-specified assessments as listed in 
Table 3, blood pressure and pulse may be assessed at any time during the trial at the 
discretion of the investigator.  
7.2.5 Electrocardiogram 
A standard 12-lead ECG will be performed at the screening visit (Visit 1), the dosing visit 
(Visit 2; prior to the start of the insulin-induced hypoglycemic procedure [within 
30 minutes]) and at 20, 35, 45, 60, and 300 minutes after dosing) and at the follow-up 
visit (Visit 3; see Table 3). The actual time of the assessment should not deviate from the 
nominal time by more than ±5 minutes.  
ECG parameters (heart rate, PQ, QRS, QT, QTcB) and any abnormality will be recorded 
and described in the eCRF including the investigator’s assessment of clinical significance 
(‘abnormal, not clinically significant’ or ‘abnormal, clinically significant’).  
At subsequent visits, any clinically significant deterioration of a pre-existing condition as 
well as any new clinically significant findings will be recorded as AEs (see 
Section 7.2.1). 
7.2.6 Local tolerability 
Immediately prior to treatment administration, it should be verified that the injection site 
is normal. To ensure all injection site assessments are performed at the injection site, the 
site will be marked with a pen prior to injection. Assessment of local tolerability at the 
injection site will be performed at the dosing visit (Visit 2; at 30, 120 and 300 minutes 
after dosing), and at the follow-up visit (Visit 3) (see Table 3) and more frequently, if 
deemed necessary by the investigator. The actual time of the assessment should not 
deviate from the nominal time by more than ±10 minutes. In the eCRF, the time of 
assessment and any injection site reaction observed will be recorded. In case of an 
observation, the Local Tolerability form will be completed, as well as the (S)AE form. 
The local tolerability at the injection site will be evaluated by means of the following 
assessments: spontaneous pain, pain on palpation, itching, redness, edema, 


 
 
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induration/infiltration, and other. Each of these assessments will be reported on a scale of 
0 (none), 1 (mild), 2 (moderate), and 3 (severe). The evaluation and the actual time of the 
assessment will be recorded. The assessments will be performed by a trial physician or 
nurse.  
Digital pictures will be taken of the injection site at the time of identification, and 
thereafter as often as judged necessary by the investigator. The pictures should include a 
subject identifier, visit number, time after dosing, and a ruler for scaling. 
7.2.7 Clinical laboratory assessments 
The safety parameters that will be assessed at the clinical laboratory are listed in Table 3. 
Routine clinical laboratory tests will be performed centrally. Samples for clinical 
laboratory parameters (biochemistry, hematology, coagulation) will be collected at 
screening (Visit 1), the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure [within 30 minutes]) and at 30 and 300 minutes after dosing), 
and at the follow-up visit (Visit 3). The actual time of the assessment should not deviate 
from the nominal time by more than ±10 minutes. Samples for glycated hemoglobin 
(HbA1c) will be collected at screening only (Visit 1). Samples for urinalysis will be 
collected at screening (Visit 1), at the dosing visit (Visit 2, prior to the start of the 
insulin-induced hypoglycemic procedure [within 120 minutes]) and at the follow-up visit 
(Visit 3). The following parameters will be assessed: 
 Clinical biochemistry: sodium, potassium, calcium, glucose, urea, creatinine, total 
bilirubin, AST, ALT, gamma-glutamyltransferase, alkaline phosphatase, total protein, 
C-reactive protein, HbA1c, C-peptide. 
 Hematology: hemoglobin, red blood cell count (erythrocytes), hematocrit, platelet 
count (thrombocytes), total white blood cell count (leukocytes). 
 Coagulation: international normalized ratio, fibrinogen (at screening visit only).  
 Urinalysis: pH, blood (leukocytes and erythrocytes), protein, glucose, ketones, nitrite. 
Pregnancy tests will be performed at each visit for women of childbearing potential only. 
A serum pregnancy test will be performed at screening (Visit 1) and urine stick tests will 
be performed at the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure) and the follow-up visit (Visit 3). Test sticks will be provided to 
the trial centers. 
Alcohol breath tests and a urine drug screen will be performed at screening (Visit 1) and 
at the dosing visit (Visit 2, prior to the start of the insulin-induced hypoglycemic 
procedure). Equipment for the alcohol breath test and uring drug screen will be provided 
to the trial centers.  


 
 
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Re-assessment of laboratory parameters will be allowed only if handling issues, damaged 
samples, or hemolyzed samples have confounded the measurement results.  
For further details of the clinical laboratory assessments, please refer to the laboratory 
manual.  
7.2.8 Pregnancy 
Female subjects must be instructed to notify the investigator immediately if they become 
pregnant or if they suspect to be pregnant during the trial. The sponsor has a 
responsibility to monitor the outcome of all pregnancies reported during the clinical trial. 
Male subjects must be instructed to notify the investigator immediately if their partner 
becomes pregnant or suspects pregnancy. The sponsor has a responsibility to monitor the 
outcome of all pregnancies reported during the clinical trial. During an information 
meeting at the trial center, the subject’s partner will be fully informed by a physician of 
her participation in capturing the outcome data, and will be asked to provide her written 
consent. 
The investigator must report all information on pregnancies on the Initial Pregnancy 
form. The completed Initial Pregnancy form must be forwarded to the sponsor 
immediately (within 24 hours), according to the procedure stated in Section 7.2.1.2. Any 
(S)AEs in the mother, as well as other pregnancy complications (ectopic pregnancy) 
fulfilling the criteria of an (S)AE, must be reported on the (S)AE form.  
The following must be collected in the Initial Pregnancy form:  
 Medical history of the mother 
 Family history 
 Course of the pregnancy, including expected delivery date. 
The investigator must follow the pregnancy until the pregnancy outcome and follow the 
newborn infant(s) until the age of 1 month. The investigator must collect information on 
the pregnancy and pregnancy complications as well as the pregnancy outcome including 
the health of the newborn infant(s) on the Pregnancy Outcome form. The completed 
Pregnancy Outcome form must be forwarded to the sponsor according to the procedure 
stated in Section 7.2.1.2. Any (S)AEs in the newborn must be reported on the (S)AE 
form. 
The SAEs that must be reported include abnormal outcome, such as congenital 
anomalies, fetal death and termination of pregnancy (spontaneous or elective abortion), 
including any anomalies of the fetus observed at gross examination or during autopsy.  


 
 
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The following must be collected in the Pregnancy Outcome form:  
 Course of the pregnancy 
 Outcome of the pregnancy 
 Condition of the newborn 
 Any AEs in the newborn infant must be followed until the age of 1 month. 
7.2.9 Precautions 
Normal precautions taken for a human trial, including the provision of emergency 
equipment, will be taken during this trial. Qualified and well trained physicians and 
medical staff will instruct the subjects. During a subject’s participation in the trial, the 
investigator should ensure that adequate medical care is provided to the subject for any 
AEs, including clinically significant laboratory values related to the trial. The investigator 
should inform the subject when medical care is needed for intercurrent illnesses of which 
the investigator becomes aware.  
For further information on safety precautions for dasiglucagon and GlucaGen, please 
refer to the current version of the Investigator’s Brochure (14) and the Summary of 
Product Characteristics for GlucaGen (13), respectively. 
7.2.10 Safety committee 
The internal Zealand Pharma Safety Committee is constituted to perform ongoing blinded 
safety surveillance of clinical trials with dasiglucagon, including this trial. 
If safety signals are observed, either based on reported SAEs, periodic review of 
laboratory parameters, review of all AEs reported between the Safety Committee 
meetings, or on notification of significant findings, the Safety Committee will take 
appropriate measures to safeguard the subjects. 
7.3 
Demography, concomitant illness, medical history and concomitant 
medication 
Demographics, body measurements, concomitant illness and medical history will be 
assessed only at screening (Visit 1). Concomitant medication will be assessed at 
screening (Visit 1), the dosing visit (Visit 2, prior to the start of the insulin-induced 
hypoglycemic procedure) and at the follow-up visit (Visit 3). 
7.3.1 Demography and body measurements 
Subject demographics and body measurements will include: 


 
 
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 Age  
 Race, ethnicity 
 Sex 
 Height (meters or inch), without shoes 
 Body weight (kg or lb), only wearing underwear and measured using standard scales 
 Body mass index (kg/m2) calculated based on height and body weight (body 
weight/height2). 
7.3.2 Concomitant illness and medical history 
A concomitant illness is any illness that is present at the start of the trial (i.e. at the 
screening visit). Concomitant illnesses present at the start of the trial will be recorded in 
the eCRF at screening.  
Medical history is an account of medical events that the subject has experienced in the 
past, including prior medications. Relevant medical conditions/illnesses in the past 
obtained by asking the subject or by inspecting his/her medical records will be recorded 
in the eCRF at screening. History of alcohol or drug abuse will also be recorded. 
The information collected for concomitant illness and medical history should include 
diagnosis, date of onset, date of resolution or continuation. 
Any change to a concomitant illness should be recorded during the trial, including end 
date, if applicable. A clinically significant worsening of a concomitant illness must be 
reported according to Section 7.2.1. 
All previous and concomitant diseases will be coded with Medical Dictionary for 
Regulatory Activities (MedDRA), the version of which will be provided in the clinical 
trial report. 
7.3.3 Diabetes diagnosis and current treatment 
The date of diagnosis of diabetes will be recorded as will the current diabetes treatment 
(start date, product name(s), dose(s)). 
7.3.4 Concomitant medication 
A concomitant medication is any medication, other than the trial products and current 
diabetes treatment (including insulin glulisine [Apidra®] for diabetes therapy wash-out), 
which is taken during the trial, including screening and follow-up periods. 


 
 
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Details of any concomitant medication must be recorded at trial entry (i.e. at screening). 
Any changes in concomitant medication must be recorded at each visit as they occur. The 
information collected for each concomitant medication includes (at a minimum) trade 
name or generic name, indication, start date and stop date or continuation. A change in 
medication due to an AE must be recorded and reported according to Section 7.2.1. If the 
change in medication influences the subject’s eligibility to continue in the trial, the 
sponsor and trial monitor must be informed. 
7.4 
Other assessments 
7.4.1 Immunogenicity 
Antibodies against dasiglucagon/GlucaGen will be measured at the dosing visit (Visit 2) 
and at follow-up (Visit 3). At the dosing visit (Visit 2), samples will be collected prior to 
the start of the insulin-induced hypoglycemic procedure.  
The clinical ADA assays, specific for dasiglucagon and GlucaGen, respectively, have 
been validated in accordance with existing guidelines and recommendations (17-21).  
Confirmed positive anti-dasiglucagon antibody samples (treatment-induced or 
treatment-boosted) from anti-dasiglucagon antibody-positive subjects will be evaluated 
for binding titer neutralizing potential and titer as well as cross-reactivity towards 
endogenous glucagon.  
No further serum sampling will be needed as the ADA samples can be used for 
neutralizing antibody analysis. 
The in vitro neutralizing effect of the antibodies will be measured using an assay based 
on glucagon receptor transfected human embryonic kidney cells (20,22). The calculated 
sensitivity in previous studies was about 51.8 ng/mL. The assay was also validated for 
recombinant glucagon with similar results (21,23). In case of a positive result in the 
neutralizing antibody assay, a titer estimation will be performed. The cell-based 
neutralizing antibody analyses will be performed by a special laboratory, BioAgilytix, 
Durham, NC, USA. 
Residual and additional antibody serum samples may be stored until approval of market 
authorization by the health authorities. Further characterization of the antibody response 
may be requested by the health authorities. 


 
 
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7.4.2 Plasma glucose measurements for safety 
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at 
site. After the start of insulin infusion, plasma glucose should be checked every 
10 minutes while plasma glucose is above 110 md/dL, and every 5 minutes once plasma 
glucose is at or below 110 mg/dL and until after dosing when the subject’s plasma 
glucose is ≥70mg/dL (3.9 mmol/L). Hereafter, plasma glucose should be checked every 
30 minutes until 300 minutes (5 hours) after dosing. Plasma glucose concentrations will 
be measured using a US FDA-approved glucose analyzer (e.g., YSI 2300, Yellow 
Springs Instruments, Yellow Springs, OH). 
At the discretion of the investigator, additional plasma glucose measurements can be 
taken at any time during the trial, for example when there is a suspicion (e.g. symptoms) 
of a hypoglycemic episode. 
Plasma glucose measurements for safety should only be recorded in the eCRF if they are 
related to an AE (e.g. a hypoglycemic episode). 
In case of persistent post-treatment hypoglycemia, subjects will receive rescue treatment 
with an IV glucose infusion (see Section 8.2.3.1 for details). Blood samples for PD and 
PK assessments should still be taken at the specified timepoints. 
7.4.3 Plasma insulin measurements 
Samples for insulin assessment will be collected at the dosing visit (Visit 2, pre-dose and 
at 30 and 60 minutes after dosing). The actual time of blood sampling for evaluation of 
plasma insulin should not deviate from the nominal time by more than ±1 minute. 
Pre-dose is defined as within 2 minutes prior to dosing. 


 
 
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8 
TRIAL CONDUCT 
8.1 
Schedule of procedures 
The schedule of procedures is provided in Table 3. Informed consent will be obtained 
prior to any trial-related procedures; see Section 10.8. 
8.2 
Procedures by visit 
8.2.1 Visit 1 (screening, Day -30 to Day -3) 
Visit 1 will take place between 3 and 30 days before Visit 2, Day -1 to Day 1 (dosing 
day). 
Informed consent can be obtained prior to or at Visit 1, however it must in any case be 
obtained prior to any trial related procedures. During the screening visit, the following 
assessments will take place: 
 Informed consent (obtain or check) 
 Check of subject eligibility (inclusion/exclusion criteria) 
 Demography  
 Body measurements 
 Medical history, diabetes diagnosis, current diabetes treatment 
 Concomitant illnesses 
 Concomitant medications 
 History of alcohol/drug abuse 
 Physical examination 
 Vital signs 
 12-lead ECG 
 AEs  
 Biochemistry, hematology, coagulation, HbA1c 
 Serum pregnancy test (women of childbearing potential only) 
 Urinalysis  
 Urine drug screen 
 Alcohol breath test 


 
 
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Table 3 
Schedule of procedures 
Visit number 
V1 
V2 
V3 
Trial day 
-3 
-1 and 1 
28 
Visit type 
Screening 
Dosing 
Follow-up 
Window 
-30 to -3 
 
+5 days 
Subject related information/assessments 
 
 
 
Informed consent 
x 
 
 
Inclusion/exclusion criteria 
x 
x1,2 
 
Demography 
x 
 
 
Body measurements 
x 
 
 
Medical history, diabetes diagnosis, and current diabetes 
treatment 
x 
 
 
Concomitant illnesses 
x 
 
 
Concomitant medications 
x 
x1 
x 
History of alcohol/drug abuse 
x 
 
 
Randomization 
 
x1 
 
Withdrawal criteria 
 
x1 
 
Dosing day exclusion criteria 
 
x1 
 
Insulin-induced hypoglycemia 
 
x 
 
Safety assessments 
 
 
 
Physical examination 
x 
 
x 
Vital signs 
x 
x3 
x 
12-lead ECG 
x 
x4 
x 
Local tolerability  
 
x5 
x 
Adverse events 
x 
x 
x 
Laboratory 
 
 
 
Biochemistry, hematology, coagulation, HbA1c (HbA1c at 
Visit 1 only) 
x 
x6 
x 
Pregnancy test (women of childbearing potential only) 
x7 
x1,8 
x8 
Urinalysis 
x 
x1 
x 
Urine drug screen 
x 
x1 
 
Alcohol breath test 
x 
x1 
 
PK/Clinical efficacy 
 
 
 
Plasma dasiglucagon/GlucaGen 
 
x9 
 
Plasma glucose 
 
x10 
 
Other assessments 
 
 
 
Antibodies against dasiglucagon/GlucaGen 
x1 
x11 
Plasma insulin 
 
x12 
 
Trial material 
 
 
 
Administration of trial product (during hypoglycemic clamp 
procedure) 
 
x 
 
1Prior to the start of the insulin-induced hypoglycemic procedure. 
2Only check of dosing day exclusion criteria and changes between screening visit and Visit 2. 
3Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 30, 90 and 300 minutes 
after dosing. The actual time of the assessment should not deviate from the nominal time by more than ±10 minutes. 


 
 
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4Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 20, 35, 45, 60 and 300 
minutes after dosing. The actual time of the assessment should not deviate from the nominal time by more than ±5 
minutes.  
5Local tolerability assessed at 30, 120, and 300 minutes after dosing. The actual time of the assessment should not 
deviate from the nominal time by more than ±10 minutes.  
6Prior to the start of the insulin-induced hypoglycemic procedure (within 30 minutes), and at 30 and 300 minutes after 
dosing. The actual time of the assessment should not deviate from the nominal time by more than ±10 minutes. 
7Serum pregnancy test. 
8Urine stick pregnancy test. 
9Pre-dose, and at 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time of blood sampling should not 
deviate from the nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing. 
10Pre-dose, and at 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, 90 minutes after dosing. The actual time of blood 
sampling should not deviate from the nominal time by more than ±30 seconds until the 20 minute collection time point 
and by more than ±1 minute for the subsequent collection time points. Pre-dose is defined as within 2 minutes prior to 
dosing. 
11Any subject that tests positive for ADA will be monitored until the ADA levels return to baseline levels. 
12Pre-dose, and at 30 and 60 minutes after dosing. The actual time of blood sampling should not deviate from the 
nominal time by more than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing.  
ADA = anti-drug antibodies; ECG = electrocardiogram; HbA1c = glycated hemoglobin; NPH = neutral protamine 
Hagedorn. 
Eligible or potentially eligible subjects (laboratory results pending) will be provided with 
an Identification card (ID card), stating that the subject is participating in the trial and 
whom to contact (site address, investigator name and telephone number). The subjects 
should be instructed to return the ID card to the investigator at the last visit or to destroy 
the card after the last visit.  
8.2.2 Instructions to subjects prior to the dosing visit (Visit 2) 
At the screening visit, the investigator will inform the subject about the changes to his/her 
insulin therapy leading up to the start of the insulin-induced hypoglycemic procedure. 
The subject’s current insulin therapy will be washed out as defined in Section 5.5: 
24 hours prior to dosing and during the dosing visit, treatment with any basal insulin is 
not allowed; 12 hours prior to dosing and during the dosing visit, treatment with any short 
acting (bolus) insulin, except insulin glulisine (Apidra®), is not allowed. The basal rate of 
insulin pumps (continuous SC insulin infusion) will be discontinued on the morning of 
the dosing day. 
On the day prior to dosing (Day -1), the subjects will need to attend the clinical center 
and will be required to stay onsite overnight. On the morning of the dosing day (Day 1), 
patients are required to be in a fasting condition, defined as having consumed only water 
since 22:00 hours the night before. However, the subjects are allowed to consume small 
amounts (up to 20 g) of carbohydrates to prevent hypoglycemia. The subjects must also 
not consume any alcohol within 24 hours prior to dosing (refer to Section 5.5 for all 
dosing day exclusion criteria). 


 
 
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8.2.3 Visit 2 (dosing visit) 
Visit 2 will take place on Day -1 to Day 1. 
The subjects will attend the clinical center the day prior to dosing (Day -1) and should 
stay onsite overnight. Dosing will take place the following morning (Day 1). 
On Day 1 and prior to the start of the insulin-induced hypoglycemic procedure, subject 
eligibility is rechecked (check of changes between the screening visit and Visit 2) and 
those subjects eligible to participate will be randomized to treatment with dasiglucagon, 
placebo, or GlucaGen.  
The following assessments will also take place: 
 Document all changes in concomitant medication (prior to the start of the 
insulin-induced hypoglycemic procedure) 
 Check of withdrawal criteria (prior to the start of the insulin-induced hypoglycemic 
procedure) 
 Check of dosing day exclusion criteria (prior to the start of the insulin-induced 
hypoglycemic procedure) 
 Vital signs (prior to the start [within 30 minutes] of the insulin-induced hypoglycemic 
procedure, and at 30, 90 and 300 minutes after dosing). Blood pressure should be 
measured first in sitting and then in standing. 
 12-lead ECG (prior to the start [within 30 minutes] of the insulin-induced 
hypoglycemic procedure, and at 20, 35, 45, 60, and 300 minutes after dosing) 
 Local tolerability (at 30, 120, and 300 minutes after dosing) 
 AEs  
 Biochemistry, hematology, coagulation (prior to the start of the insulin-induced 
hypoglycemic procedure [within 30 minutes], and at 30 and 300 minutes after dosing) 
 Urine stick pregnancy test (women of childbearing potential only; prior to the start of 
the insulin-induced hypoglycemic procedure) 
 Urinalysis (prior to the start of the insulin-induced hypoglycemic procedure [within 
120 minutes]) 
 Urine drug screen (prior to the start of the insulin-induced hypoglycemic procedure) 
 Alcohol breath test (prior to the start of the insulin-induced hypoglycemic procedure) 
 Dasiglucagon/GlucaGen plasma concentrations: 
- 
Pre-dose, 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time 
of blood sampling for exposure should not deviate from the nominal time by more 
than ±1 minute. Pre-dose is defined as within 2 minutes prior to dosing. 
 Plasma glucose concentrations:  
- 
Pre-dose, 4, 6, 8 ,10, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, and 90 minutes after 
dosing. The actual time for blood sampling for plasma glucose should not deviate 


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from the nominal time by more than ±30 seconds until the 20 minute collection 
time point and by more than ±1 minute for the subsequent collection time points. 
Pre-dose is defined as within 2 minutes prior to dosing. 

Antibodies against dasiglucagon/GlucaGen (prior to the start of the insulin-induced
hypoglycemic procedure). 

Plasma insulin concentrations:
- 
Pre-dose, 30 and 60 minutes after dosing. The actual time of blood sampling for 
plasma insulin should not deviate from the nominal time by more than ±1 minute. 
Pre-dose is defined as within 2 minutes prior to dosing. 
8.2.3.1 Hypoglycemic clamp procedure and administration of trial medication 
The following procedure is based on precendent procedures for hypoglycemia induction 
in patients with T1DM [24, 25]. 
The treatment day (Visit 2, Day 1) will be conducted after an overnight fast of at least 
8 hours, targeting a starting plasma glucose around 90 to 110 mg/dL (5.0-6.1 mmol/L).  
Subjects who meet one or more of the dosing day exclusion criteria (Section 5.5) at the 
dosing visit will be excluded from the dosing visit, but the visit can be rescheduled 
1-7 days later. The dosing visit can only be rescheduled once. 
For participants using an insulin pump, the continuous subcutaneous insulin infusion will 
be suspended during the procedure, while participants using multiple daily injections of 
insulin will take their last long-acting insulin dose at least 24 hours before testing.  
At approximately 08:00 hours, an infusion catheter will be inserted into each arm 
(forearm cephalic vein) for the manual glucose clamp procedure, with the glucose 
infusion in one arm and the insulin infusion in the opposite arm. A third catheter for 
blood sampling will be placed into a metacarpel vein for blood sampling. This hand will 
be warmed (55-65ºC) to arterialize venous blood.   
Hypoglycemia will be gradually induced by a fast-acting IV insulin glulisine (Apidra®) 
infusion (15 U [100 U/mL] in 49 mL saline and 1 mL of subject’s blood or plasma), 
initially at 150% of the subject’s usual basal rate and can be increased or decreased over a 
range of 75% to 200% as judged necessary by the investigator, to achieve a controlled 
decline in plasma glucose, targeting a plasma glucose level of 55 mg/dL (3.0 mmol/L).  
Plasma glucose concentrations will be measured using a US FDA-approved glucose 
analyzer (e.g., YSI 2300, Yellow Springs Instruments, Yellow Springs, OH). After the 
start of the insulin infusion, plasma glucose will be measured every 10 minutes while 


 
 
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plasma glucose is above 110 mg/dL, and every 5 minutes once plasma glucose is at or 
below 110 mg/dL. 
Once the glucose concentration declines to <60 mg/dL (3.3 mmol/L), the insulin infusion 
will be stopped, and 5 min later plasma glucose concentration will be measured at the 
glucose analyzer and blood samples for baseline assessment of plasma glucose, 
dasiglucagon/GlucaGen PK, and insulin PK will be collected. The samples are the 
baseline samples and should be collected within 2 minutes before IMP administration. 
 If plasma glucose is >45 mg/dL and <60 mg/dL (3.0-3.3 mmol/L), study treatment 
(IMP) will be administered, defining time, t=0. The study treatment will be delivered 
in the abdomen, buttock, or thigh (according to stratification) via SC injection, with 
the subject lying in a lateral recumbent position. 
 If plasma glucose is <45 mg/dL (2.8 mmol/L), IV glucose solution will be 
administered sufficient to raise plasma glucose to within the 45-60 mg/dL target 
range. The run-in period will be adequately extended (at least 30 min) until the above 
target is achieved and new baseline samples for plasma glucose, 
dasiglucagon/GlucaGen PK, and insulin PK will be collected. Glucose should not be 
infused within 10 minutes before IMP administration. If plasma glucose is not within 
target range after the second attempt, the subject should be rescheduled for a new 
treatment visit within 7 days (+ 2 days). 
Administration of IMP should not occur earlier than 9:00 hours in the morning or later 
than 12:00 hours. 
As shown in Table 4, serial blood samples for glucose will be collected at t=0, 4, 6, 8, 10, 
12, 15, 17, 20, 25, 30, 40, 50, 60, 75 and 90 minutes post-dosing. Samples for assessing 
plasma dasiglucagon/GlucaGen concentration will be collected  at t=0, 15, 30, 35, 40, 50, 
60, 90, and 120 minutes. Samples for assessing plasma insulin concentration will be 
collected at t=0, 30, and 60 minutes.  
Table 4 
Post-treatment blood sampling times 
Times 
0 
4 
6 
8 
10 
12 
15 
17 
20 
25 
30 
35 
40 
50 
60 
75 
90 
120 
Plasma glucose 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
Y 
 
Y 
Y 
Y 
Y 
Y 
 
PK 
Dasiglucagon/ 
GlucaGen 
Y 
 
 
 
 
 
Y 
 
 
 
Y 
Y 
Y 
Y 
Y 
 
Y 
Y 
PK Insulin 
Y 
 
 
 
 
 
 
 
 
 
Y 
 
 
 
Y 
 
 
 
Refer to Section 7.2.7 for details of laboratory safety sampling and to Section 7.4.2 for 
details of blood glucose safety sampling.  


 
 
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When the t=90-minute blood sampling for plasma glucose has been collected the subjects 
are allowed to eat moderately. Drinking of water is allowed ad libitum during the entire 
procedure. 
Hypoglycemia Rescue Provisions 
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at 
site for safety reasons as described in Section 7.4.2. Subjects should receive 
post-treatment rescue glucose infusion to ameliorate persistent hypoglycemia, as follows. 
1. Glucose infusion should be initiated if a subject experiences severe alarming 
escalation of symptoms of hypoglycemia (e.g. symptoms suggesting a change in 
consciousness) at any time during the trial; glucose infusion should be initiated 
targeting a plasma glucose levels >70 mg/dL. 
2. If plasma glucose is <45 mg/dL (2.5 mmol/L) between t=8 and t=44 minutes, 
rescue glucose infusion (1-2 mg/kg administered IV over about 5 seconds) should 
be initiated to maintain plasma glucose between 45 mg/dL and 55 mg/dL 
(2.5-3.0 mmol/L). Pause glucose infusion if plasma glucose is >55 mg/dL. 
3. If plasma glucose is <70 mg/dL at t=45 minutes, rescue glucose infusion 
(2-3 mg/kg administered IV over about 5 seconds) should be initiated to maintain 
plasma glucose between 70 mg/dL and 80 mg/dL (3.9-4.4 mmol/L). Pause 
glucose infusion if plasma glucose is >75 mg/dL (4.2 mmol/L). 
Subjects should remain in bed until completion of the test procedure 300 minutes after 
dosing (bathroom visits are allowed).  
The IMP will be administered SC according to Section 6.3. The time of IMP 
administration will be recorded. At the timepoint when the insulin infusion is stopped, the 
total insulin dose which was required to induce hypoglycemia will be recorded. 
AEs will be specifically recorded during the procedure at several timepoints. 
The investigator must provide information to the subjects on how to resume their usual 
diabetes treatment. 
The subject may be released from the clinical center if the investigator does not have any 
safety concerns based on the last safety plasma glucose value and the general condition of 
the subject. However, at the discretion of the investigator or on request of the subject, the 
subject may stay at the trial center for a longer period. 
8.2.4 Visit 3 (follow-up visit) 
Visit 3 will take place on Day 28 + 5 days. The subject does not need to be fasting. 


 
 
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At Visit 3, the following assessments will take place: 
 Document all changes in concomitant medication  
 Physical examination 
 Vital signs 
 12-lead ECG 
 Local tolerability 
 AEs  
 Biochemistry, hematology, coagulation 
 Urine stick pregnancy test (women of childbearing potential only) 
 Urinalysis 
 Antibodies against dasiglucagon/GlucaGen. 
After the follow-up visit the End of Trial form must be completed. Even if a subject is not 
able to attend the follow-up visit, the End of Trial form, the eCRF 
Accountability/Affirmation Statement form and the Drug Accountability form must be 
completed. 


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9 
STATISTICAL METHODS 
Before database lock and treatment unmasking, a separate statistical analysis plan (SAP) 
will be finalized, providing detailed methods for the analyses outlined below. Further 
analysis details may be added or refined in the SAP. 
Any deviations from the planned analyses will be described and justified in the final 
clinical trial report. 
9.1 
Determination of sample size 
Due to requirements in the size of the safety database, the sample size is set to 78 subjects 
treated with dasiglucagon 0.6 mg. With a 2:1:1 randomization ratio for the three 
treatments, dasiglucagon 0.6 mg, placebo, and GlucaGen 1 mg, 78 subjects injected with 
dasiglucagon and 39 subjects with placebo will yield a power of 90% at a 5% two-sided 
significance level to detect a treatment group difference in recovery incidence within 
20 minutes, assuming as low as 80% and as high as 50% recovery incidence for the 
dasiglucagon and placebo groups, respectively. 
9.2 
Trial subjects 
9.2.1 Analysis samples 
For presentation of data and reporting of the statistical analyses, the following analysis 
samples will be used, depending on the context:  

Safety analysis set (SAS): All randomized subjects who received at least one dose of
trial medication.

Full analysis set (FAS): All randomized subjects who received at least one dose of
trial medication and contributed valid information for at least one post-dose endpoint.

Per protocol (PP) set: All subjects of the FAS for whom no relevant protocol
deviations were documented. This sample will primarily be used for sensitivity
analysis.
The analysis of the primary endpoint and secondary endpoints will be based on the FAS. 
A secondary analysis of the primary endpoint will be based on the PP set. All safety 
analyses will be based upon the SAS. 
The decision regarding whether a protocol deviation is relevant or not for the exclusion of 
subjects from the PP set will be made case-by-case in a data review meeting prior to 
treatment unmasking and database lock (see Section 9.2.3). 


 
 
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9.2.2 Disposition of subjects 
Subject disposition will be tabulated including the number of screened subjects, screening 
failures, subjects exposed to trial product, subjects completing the trial and subjects in 
each analysis set. 
Subjects withdrawn from the trial will be listed including the primary reason for 
withdrawal. 
9.2.3 Protocol deviations 
Before data are released for statistical analysis, a treatment-masked review of all data will 
take place to identify protocol deviations that may potentially affect the results. This 
review will be performed without revealing to which trial product the subjects were 
assigned. The masking of the trial products will be maintained for everyone involved in 
allocating subjects to the analysis sets until data are released for statistical analysis. 
Furthermore, spurious outliers will be evaluated. In addition, protocol deviations that may 
potentially affect the results will be identified and it will be evaluated if subjects and/or 
data should be excluded from the analysis. Protocol deviations will be classified as minor 
or major in a consistent way. Major deviations from the protocol may lead to the 
exclusion of a subject from the PP set. 
Major protocol deviations may include deviations related to trial inclusion or exclusion 
criteria, conduct of the trial, subject management or subject assessment. Unless explicitly 
decided otherwise during the masked data review, the following will be considered major 
protocol deviations: 
 Violation of one or more major inclusion/exclusion criteria 
 Interruption of administration of trial product 
 Significant deviation from time windows  
 Incorrect treatment allocation 
 Missing primary endpoint. 
The violation of several major inclusion/exclusion criteria or the complete absence of 
efficacy data might lead to exclusion of the subject from FAS. In that case, the decision 
should be taken at the masked data review meeting, and the exclusion from efficacy 
analysis justified in the signed notes of the meeting. 
Obviously erroneous data points may be excluded from the analyses or re-analyzed (in 
case of e.g. serum concentrations). The decision to re-analyze or exclude data points from 
the statistical analysis is the joint responsibility of the sponsor and the trial statistician.  


 
 
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The subjects or observations to be excluded and the reason for their exclusion will be 
documented and signed by those responsible prior to database release. The 
documentation will be stored together with the remaining trial documentation. The 
subjects and observations excluded from analysis sets, and the corresponding reasons, 
will be described in the clinical trial report. 
9.3 
General considerations 
All data obtained in this trial and documented in the eCRFs will be listed and 
summarized with statistics or frequency tables as appropriate. In case of termination of 
the trial, all data collected up to that timepoint will be included in the analysis. 
Raw data listings and summary tables will be generated using the software SAS© version 
9.4 or higher.  
Continuous variables will be summarized using means, standard deviations, medians, 
coefficients of variation, and minimum and maximum values.  
Other summaries (e.g. quartiles, 95% confidence intervals [CIs]) may be used as 
appropriate. Categorical efficacy and safety variables will be summarized by counts and 
by percentage of subjects in corresponding categories. 
9.4 
Demographics and baseline characteristics 
Baseline and demographic data will be summarized using descriptive statistics. Baseline 
ADA-positive subjects will be calculated as a percentage of the total number of subjects 
whose baseline samples were tested for ADA. 
All other data obtained in this trial and documented in the eCRF will be listed. 
9.5 
Efficacy Analyses 
For the confirmatory analyses, the primary and key secondary endpoints for the 
dasiglucagon 0.6 mg and placebo treated subjects will be compared. A hierarchical 
procedure will be applied for the control of multiplicity. The primary and secondary 
endpoints will be evaluated on the full analysis set. In the primary analysis, those subjects 
who require rescue IV glucose will be censored at the time to plasma glucose recovery.  
In a sensitivity analysis, the time to plasma glucose recovery will be analyzed without 
censoring the subjects who received rescue IV glucose.   


 
 
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9.5.1 Hierarchical testing procedure 
For the confirmatory analyses, the following a priori defined hierarchical inferential test 
order will be applied for the control of the type 1 error rate across the planned multiple 
comparisons. The primary endpoint and key secondary endpoints will be inferentially 
evaluated within the FAS in the following order, where inference will proceed at the 
two-sided 0.05 criterion significance level until the first failure to reject the null 
hypothesis for a dasiglucagon versus placebo comparison: 
 Primary:  Time to plasma glucose ≥20 mg/dL recovery 
 Secondaries 1-4: Plasma glucose changes from baseline within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection or at 
the time of rescue. 
 Secondaries 5-8:  Incidences of plasma glucose recovery within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drug injection. 
The GlucaGen versus placebo comparisons will not be included in the inferential testing 
hierarchy, since the efficacy of GlucaGen is previously established, and these 
comparisons are intended to support the validity of the study for the dasiglucagon versus 
placebo comparisons. 
Analogous supportive sensitivity analyses will be conducted in the PP set, but without 
inference intent. 
9.5.2 Primary confirmatory endpoint 
 Time to plasma glucose ≥20 mg/dL recovery, defined as first increase in plasma 
glucose of ≥20 mg/dL (1.1 mmol/L) from baseline during the hypoglycemic clamp 
procedure.  
9.5.2.1 Primary analysis 
The primary endpoint of time to plasma glucose ≥20 mg/dL recovery will be analyzed 
using a Cox proportional hazards (CPH) time to event statistical model, if the hazards by 
treatment group can be assumed proportional using a graphical comparison of the 
Nelson-Aalen estimates. The CPH model will be used for inferences, with treatment 
group and injection site modeled as categorical effects, and baseline plasma glucose 
modeled as a continuous covariate. The treatment group rate ratios, active versus placebo, 
will be estimated together with the 95% confidence intervals, and pairwise treatment 
group inferences (dasiglucagon vs placebo, GlucaGen vs placebo) will be evaluated using 
two-sided likelihood ratio tests.  


 
 
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If the PH assumption for treatment groups is not met, the primary endpoint will be 
analyzed using a Kaplan-Meier (KM) time to event statistical model, with treatment 
group and injection site as stratification factors. Treatment group differences between the 
KM curves (dasiglucagon vs placebo, GlucaGen vs placebo) will be evaluated 
inferentially using pairwise two-sided log-rank tests. 
In the primary analysis, those subjects who require rescue IV glucose will be censored at 
the time to plasma glucose recovery. In a sensitivity analysis, the time to plasma glucose 
recovery will be analyzed without censoring those subjects who require rescue IV 
glucose before 45 minutes.   
9.5.3 Secondary endpoints 
 Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection or at the time of rescue. 
 Incidences of plasma glucose recovery (achieving a plasma glucose concentration 
increase of ≥20 mg/dL [1.1 mmol/L]) within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection. 
9.5.3.1 Confirmatory analysis 
The key secondary endpoints of plasma glucose changes from baseline (CFB) within 
30 minutes, within 20 minutes, within 15 minutes, and within 10 minutes after study drug 
injection or at the time of rescue, will be analyzed with the plasma glucose CFB at rescue 
carried forward in those subjects who require rescue IV glucose before plasma glucose 
≥20 mg/dL recovery. Each of these CFB variables will be analyzed using an Analysis of 
Covariance model, with treatment group modeled as a fixed effect and with the baseline 
plasma glucose modeled as a covariate. The dasiglucagon versus placebo treatment group 
difference will be evaluated inferentially as a least squares means contrast, using a 
two-sided t-test at the 0.05 signficance level, first for the 30 minute CFB, and then 
sequentially for the 20 minute, 15 minute, and 10 minute variables, until the first failure 
to reject. 
The key secondary incidence variables (plasma glucose recovery within 30 minutes, 
within 20 minutes, within 15 minutes, within 10 minutes, in hierarchical order for 
inference) will be analyzed using descriptive statistics (frequency and percentage) by 
treatment group. Inferential treatment group comparisons will be assessed via pairwise 
tests of independent binomial proportions (dasiglucagon versus placebo, GlucaGen 
versus placebo).    
9.5.4 Secondary clinical efficacy (PD) endpoints 
 Time to first plasma glucose concentration of ≥70 mg/dL (3.9 mmol/L). 


 
 
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 Plasma glucose response as area under the curve above baseline from time zero to 
30 minutes, AUC0-30min. 
Secondary clinical efficacy endpoints will be assessed based on plasma concentration 
data from samples collected at the dosing visit (Visit 2).  
Unless otherwise stated, the population base of analysis will be the FAS. 
9.5.4.1 Analysis of secondary clinical efficacy (PD) endpoints 
1. Time to first plasma glucose concentration ≥70 mg/dL from baseline. This 
time-to-event endpoint will be evaluated using a KM time to event statistical 
model, with treatment group as a stratification factor, analogous to that used for 
the primary endpoint analysis. Differences between the KM curves (dasiglucagon 
versus placebo, GlucaGen versus placebo) will be evaluated inferentially using 
pairwise two-sided stratified log rank tests. If the ≥70 mg/dL endpoint is not met 
within 45 minutes post-dosing, the time of the last valid plasma glucose 
measurement up to 45 minutes will be the censoring time.  
2. The AUC will be calculated as the baseline-adjusted area under the plasma 
glucose profile over time: 
a. AUC0-30min: restricting the time window to the 0 to 30 minutes interval. 
3. The log-transformed AUC endpoint will be analyzed using an analysis of 
covariance (ANCOVA) model with treatment as fixed effect and baseline plasma 
glucose modeled as a covariate. The least squares means treatment group 
differences will be back-transformed (anti-logged) for presentation as a ratio of 
the treatment group geometric means, with their corresponding 95% CI. 
9.5.5 Exposure (PK) endpoints 
 Plasma dasiglucagon and GlucaGen concentrations from time zero to 90 minutes:  
AUC0-90min, Cmax, and tmax. 
9.5.5.1 Analysis of exposure (PK) endpoints 
AUC will be derived as the area under the individual plasma dasiglucagon/GlucaGen 
concentration profile for PK from 0 to 90 minutes or last valid measurement if this 
measurement is assessed sufficiently close to 90 minutes (decision to be taken at the 
masked data review meeting). The standard trapezoidal method will be used, based on 
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Cmax will be determined as the maximum of all valid plasma dasiglucagon/GlucaGen 
concentrations.  
Tmax will be determined as the timepoint where the maximum of all valid plasma 
dasiglucagon/GlucaGen concentration measurements for each measurement series is 
observed. 
The log-transformed PK endpoints AUC and Cmax will be analyzed in the same way as 
the AUC endpoints. 
As tmax is a highly discrete endpoint, Wilcoxon’s rank sum test for unpaired observations 
will be used to assess differences between the two treatment groups. 
9.6 
Exploratory analyses 
Exploratory analyses will include descriptive statistics and modeling analogous to that 
done for key secondary endpoints. However, treatment group comparisons will be 
summarized without inference intent. 

Incidence of plasma glucose recovery (achieving a plasma glucose concentration
≥70 mg/dL [3.9 mmol/L] or an increase of ≥20 mg/dL [1.1 mmol/L]) within
30 minutes after study drug injection.

Plasma insulin response as area under the curve above baseline from time zero to
60 minutes, AUC0-60 min.
Plasma insulin concentrations measured pre-dose and at 30 and 60 minutes after dosing 
(see Table 3) will be presented individually. A summary table per timepoint will be 
provided. The AUC0-60min will be determined and a summary presented.  
9.7 
Safety analyses 
9.7.1 Adverse events 
AEs will be coded using the latest available version of the MedDRA, the version of 
which will be provided in the clinical trial report. AE summary tables will include the 
number and percentage of subjects who experienced AEs summarized by system organ 
class and preferred term. 
An overall summary table will be provided showing the number and percentage of 
subjects with any: 


 
 
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 TEAE 
 Severe TEAE 
 Serious TEAE 
 Drug-related TEAE 
 Drug-related severe TEAE 
 Drug-related serious TEAE 
 TEAE leading to withdrawal 
 TEAE with outcome death 
 AESI 
Refer to Section 7.2.1 for the definition of TEAEs. 
9.7.2 Immunogenicity data 
Immunogenicity data will be analyzed descriptively by treatment group. No statistical 
tests are planned. Baseline ADA-positive subjects will be calculated as a percentage of 
the total number of subjects whose baseline samples were tested for ADA. Titer will be 
reported as median and interquartile range. 
9.7.3 Clinical laboratory assessments 
Clinical laboratory test results will be flagged as to whether the result is below, within or 
above the respective reference range. The number of values outside of the reference range 
will be counted. 
9.7.4 Other safety data 
Incidence of rescue infusion of IV glucose during the hypoglycemic clamp procedure will 
be analyzed using descriptive statistics (frequency and percentage) by treatment group. 
Inferential treatment group comparisons will be assessed via pairwise tests of 
independent binomial proportions (dasiglucagon versus placebo, GlucaGen versus 
placebo). 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure 
will be evaluated using a KM time to event statistical model, with treatment group and 
injection site as stratification factors. Differences between the KM curves (dasiglucagon 
versus placebo, GlucaGen versus placebo) will be evaluated inferentially using pairwise 
two-sided stratified log-rank tests. If the endpoint is never met, the time of the last plasma 
glucose measurement will be the censoring time. 
Vital signs, physical examination, 12-lead ECG and local tolerability data will be 
summarized using descriptive statistics. 


 
 
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9.8 
Treatment compliance 
Trained unblinded members of staff will perform all administrations of the IMP at the 
trial center. The administered doses will be recorded in the blinded Drug Accountability 
form in the eCRF. 
PK assessments will support the surveillance of compliance with IMP administration. 
9.9 
Subject withdrawals and missing data 
In the case of subject withdrawal, no imputation of values for PK or PD measurements 
will be done. Analyses will be done on valid cases only, i.e., no imputation techniques 
such as last observation carried forward will be applied. For the primary analysis in the 
FAS, missing values for the primary endpoint will be imputed by a conservative rule 
considering any missing value as a failure. 
9.10 Interim analyses 
No interim analyses are currently planned. 


 
 
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10 
ETHICAL, LEGAL, AND ADMINISTRATIVE ASPECTS 
10.1  Quality assurance 
The sponsor or designee will conduct a site visit to verify the qualifications of each 
investigator, inspect the facilities, and inform the investigator of responsibilities and the 
procedures for ensuring adequate and correct documentation. 
The investigator is required to prepare and maintain adequate and accurate case histories 
designed to record all observations and other data pertinent to the trial for each trial 
participant. All information recorded on the eCRFs for this trial must be consistent with 
the subjects’ source documentation (i.e. medical records). 
The investigator will permit trial-related monitoring, IRB/IEC review, and regulatory 
inspections, providing direct access to source data /documents. Sponsor-authorized 
quality assurance personnel may carry out audits for which the investigator must provide 
support. 
The trial monitor or a representative of the sponsor will cross-check the data entered in 
the eCRFs with the source data at the trial center and observe the trial procedures in order 
to verify adherence to the trial protocol. Any queries will be resolved by the investigator 
or his/her delegate. 
All of the clinical data will be captured via electronic data capture (EDC) using a 
web-based tool.  
The investigator center staff will enter and edit the data via a secure network, with secure 
access features (username and password). A complete electronic audit trail will be 
maintained. The investigator will approve the data using an electronic signature (Ref: 21 
CFR Part 11), and this approval is used to confirm the accuracy of the data recorded. 
eCRFs will be used for all subjects. The investigator’s data will be accessible from the 
investigator’s center throughout the trial. The eCRFs must be kept current to reflect 
subject status at each phase during the course of the trial. The eCRF will not capture 
personalized data. The investigator must make a separate confidential record of 
personalized details (name and initials) on the subject identification and enrollment log. 
All changes to data are made by the investigator or his/her delegate through the EDC 
system.  
It is the responsibility of the principal investigator of the respective center to ensure that 
all subject discontinuations or changes in trial or other medications entered on the 
subject’s eCRF are also made on the subject’s medical records. 


 
 
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The eCRFs for any subject leaving the trial should be completed at the time of the final 
visit or shortly thereafter. 
10.2  Electronic case report forms 
Remote data capture software will be used for data collection. Following training, trial 
staff will be given access to the software. Access to the software is restricted to staff 
participating in the trial and the extent of access will depend on the participants' user role 
in the trial. 
The subjects enrolled into the trial will be identified in the database by subject number 
and trial identification code. The investigator or delegate will enter subject data into the 
eCRF promptly. All data is to be entered in English. Data recorded in the eCRFs will be 
accessible to the trial staff throughout the trial. 
After data entry, systematic data validation will be performed and data entry 
discrepancies will be presented electronically directly to the center staff. Queries for 
discrepant data may be generated automatically by the software upon entry and/or 
generated manually by the trial monitor or the trial data manager. All queries, whether 
generated by the system or by trial staff, will be in electronic format. 
All sections of the eCRF are to be electronically approved by the investigator or a 
medically qualified delegate after the data has been entered and all queries have been 
resolved. Changes to any eCRF page subsequent to the approval require a new approval 
signature. 
All queries and changes/corrections to the data are documented in the eCRF. 
10.3  Access to source data 
During the course of the trial, a trial monitor will make site visits to review protocol 
compliance, compare eCRFs with individual subject’s medical records, assess drug 
accountability, and ensure that the trial is being conducted according to pertinent 
regulatory requirements. 
Checking of the eCRFs for completeness and clarity, and cross-checking with source 
documents, will be required to monitor the progress of the trial. Moreover, regulatory 
authorities of certain countries, IRBs/IECs, and/or the sponsor may wish to carry out such 
source data checks and/or on-site audit inspections. Direct access to source data will be 
required for these inspections and audits; they will be carried out giving due 
consideration to data protection and medical confidentiality. 


 
 
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10.4  Source documentation 
All source documents from which eCRF entries are derived should be placed in the 
subject’s medical records. If data are to be entered directly into the eCRF this must be 
specified in a source data agreement prior to the start of the trial. 
eCRF entries will be verified with source documentation. The review of medical records 
will be performed in a manner to ensure that subject confidentiality is maintained. 
The trial monitor will check the eCRF for accuracy and completion and perform source 
data verification. The trial monitor will document source data verification of all reviewed 
sections of the eCRF. 
10.5  Data processing 
The trial is run as an EDC trial, i.e. all relevant data is entered by the centers directly into 
the clinical database. The eCRF is designed to capture all required information in 
compliance with GCP standards. 
10.6  Archiving trial records 
According to ICH guidelines, essential documents should be retained for a minimum of 
2 years after the last approval of a marketing application in an ICH region and until there 
are no pending or contemplated marketing applications in an ICH region or at least 
2 years have elapsed since the formal discontinuation of clinical development of the 
investigational product. However, these documents should be retained for a longer period 
if required by the applicable legal requirements. 
Records and documents pertaining to the conduct of the trial and the distribution of the 
investigational product (e.g. informed consent forms, laboratory slips, medication 
inventory records, and other pertinent information) must be retained by the investigator 
according to local requirements. 
10.7  Good clinical practice 
The procedures set out in this trial protocol are designed to ensure that the sponsor and 
investigator abide by the principles of the GCP guidelines of the ICH (1), and of the 
Declaration of Helsinki (2008) (2). The trial also will be carried out in keeping with local 
legal requirements.  


 
 
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10.8  Informed consent 
Before each subject is admitted to the trial, written informed consent will be obtained 
from the subject according to the regulatory and legal requirements of the participating 
country. This consent form must be dated and retained by the investigator as part of the 
trial records. The investigator will not undertake any investigation specifically required 
only for the clinical trial until valid consent has been obtained. The terms of the consent 
and when it was obtained must also be documented in the eCRF. 
The investigator will explain to each subject orally and in writing (subject information 
sheet) the nature, significance, risks and implications of the trial before inclusion. In 
particular, the subjects will be informed about the following: 
 The possibility of withdrawing from the clinical trial at any time by revoking the 
consent and without any resulting disadvantage. 
 How personal and health-related data will be collected and used during the trial. 
 That his/her medical records may be examined by authorized monitors or clinical 
quality assurance auditors appointed by the sponsor, by appropriate IRB/IEC 
members, and by inspectors from regulatory authorities. 
All subjects will receive a copy of the subject information sheet and a copy of their 
signed and dated informed consent form, both of which will be in the subject’s local 
language. 
If a protocol amendment is required, the informed consent form may need to be revised to 
reflect the changes to the protocol. If the consent form is revised, it must be reviewed and 
approved by the appropriate IEC/IRB, and signed by all subjects subsequently enrolled in 
the trial as well as those currently enrolled in the trial. 
10.9  Protocol approval and amendment 
Before the start of the trial, the trial protocol and/or other relevant documents will be 
approved by the IEC/IRB/competent authorities, in accordance with local legal 
requirements. The sponsor must ensure that all ethical and legal requirements have been 
met before the first subject is enrolled in the trial. 
This protocol is to be followed exactly. To alter the protocol, amendments must be 
written, receive approval from the appropriate personnel, and receive IRB/IEC/competent 
authority approval prior to implementation (if appropriate).  


 
 
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Administrative changes (not affecting the subject benefit/risk ratio) may be made without 
the need for a formal amendment. All amendments will be distributed to all protocol 
recipients, with appropriate instructions. 
10.10  Duration of the trial 
The maximum duration of the trial for each subject will be up to 63 days (including up to 
30 days for screening and up to 33 days until the follow-up visit).  
The trial will be closed when all subjects have completed Visit 3. 
10.11  Premature termination of the trial 
If the investigator, the sponsor (e.g. safety committee), or the safety medical monitor 
becomes aware of conditions or events that suggest a possible hazard to subjects if the 
trial continues, the trial may be terminated after appropriate consultation between the 
relevant parties. The trial may also be terminated early at the sponsor’s discretion in the 
absence of such a finding. 
Conditions that may warrant termination include, but are not limited to: 
 The discovery of an unexpected, significant, or unacceptable risk to the subjects 
enrolled in the trial 
 Failure to enroll subjects at an acceptable rate. 
 A decision on the part of the sponsor to suspend or discontinue development of the 
drug. 
The trial can be terminated prematurely by the sponsor at an individual center if: 
 The center cannot comply with the requirements of the protocol. 
 It is not possible for the center to comply with GCP standards. 
10.12  Confidentiality 
All trial findings and documents will be regarded as confidential. The investigator and 
members of his/her research team must not disclose such information without prior 
written approval from the sponsor. 
The anonymity of participating subjects must be maintained. Subjects will be identified 
on eCRFs submitted to the sponsor by their subject number. On the SAE reports and all 
other source documents, the subject will be identified via subject number. Documents not 


 
 
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to be submitted to the sponsor that identify the subject (e.g. the signed informed consent 
form) must be maintained in confidence by the investigator.  
10.13  Other ethical and regulatory issues 
If a significant safety issue is identified, either from an individual case report or review of 
aggregate data, then the sponsor will issue prompt notification to all parties: regulatory 
authorities, investigators and IRB/IECs. 
A significant safety issue is one that has a significant impact on the course of the clinical 
trial or program (including the potential for suspension of the trial program or 
amendments to protocols) or warrants immediate update of informed consent. 
10.14  Liability and insurance 
The sponsor will take out reasonable third-party liability insurance cover in accordance 
with all local legal requirements. The civil liability of the investigator, the persons 
instructed by him and the hospital, practice or institute in which they are employed and 
the liability of the sponsor with respect to financial loss due to personal injury and other 
damage that may arise as a result of the carrying out of this trial are governed by the 
applicable law. 
The sponsor will arrange for liability insurance if subjects should be injured due to the 
participation in the trial and provided that the sponsor is legally liable for that. 
Excluded from the insurance cover are injuries to health and deteriorations of illnesses 
already in existence which would have occurred or continued to exist even if the subject 
had not taken part in the clinical trial. 
The insurance cover is jeopardized if the subject fails to report immediately to the 
investigator or responsible physician any injury to health, which might have resulted 
from participation in the clinical trial, or if he/she undergoes any other medical treatment 
without their consent before the clinical trial has been completely finished in so far as the 
individual subject is concerned. 
Any injury to health, which might have occurred as a result of participation in the clinical 
trial must be reported by the subject to the investigator without delay. The investigator is 
obliged to make such a report in any case. 


 
 
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10.15  Publication policy 
By signing the trial protocol, the investigator agrees with the use of results of the trial for 
the purposes of national and international registration, publication and information for 
medical and pharmaceutical professionals. If necessary, the authorities will be notified of 
the investigator's name, address, qualifications and extent of involvement. 
A clinical trial report will be prepared and reviewed by the sponsor in co-operation with 
the coordinating investigator. The coordinatoing investigator will be appointed by 
Zealand Pharma to review and sign the clinical trial report on behalf of all participating 
investigators. A summary of the clinical trial report (according to CPMP/ICH137/95) 
should be sent to the regulatory authorities and IRB/IEC according to the relevant 
guidelines. 
According to the Declaration of Helsinki (2) investigators and sponsors ‘have ethical 
obligations with regard to the publication and dissemination of the results of research’.   
The trial design and results may be published as one or more original research 
manuscripts/abstracts and presented at a scientific meeting. The original eCRFs and the 
data generated from the eCRFs or otherwise obtained during the trial under this trial 
protocol will become the property of the sponsor. Publication of the results of this trial by 
the investigator is possible only after written consent has been obtained from the sponsor. 
Any material intended for publication will be given to the sponsor at least 4 weeks before 
submission for publication. The sponsor will have the right to comment on the intended 
publication and to take any reasonable measures for patent protection. Authorship on any 
publication(s) resulting from this clinical trial will be assigned according to the 
recommendations of the International Committee of Medicinal Journal Editors (26). 
Participating subjects will not be identified by name in any published reports about the 
clinical trial. 
The sponsor is responsible for trial registration at ClinicalTrials.gov 
(www.clinicaltrials.gov) according to FDA requirements, as well as the European 
Medicines Agency’s Clinical Trials Database (EudraCT).  


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11 
REFERENCE LIST  
1. ICH Harmonised Tripartite Guideline. International Conference on Harmonisation. 
ICH Harmonised Tripartite Guideline. Integrated addendum to ICH E6(R1): 
Guideline for Good Clinical Practice E6 (R2), Step 4. 09-Nov-2016. Available from: 
URL:
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E 
6/E6_R2__Step_4.pdf
2. World Medical Association. Declaration of Helsinki. Ethical Principles for Medical
Research Involving Human Subjects. 52nd WMA General Assembly, Edinburgh,
Scotland, October 2000. Last amended with Note of Clarification on Paragraph 29 by
the WMA General Assembly, Washington 2002, and Note of Clarification on
Paragraph 30 by the WMA General assembly, Tokyo 2004
3. Seaquist ER, Anderson J, Childs B, Cryer P, Dagogo-Jack S, Fish L, et al.
Hypoglycemia and diabetes: a report of a workgroup of the American Diabetes
Association and the Endocrine Society. Diabetes Care 2013 May;36:1384-95
4. Cryer PE. The barrier of hypoglycemia in diabetes. Diabetes 2008;57:3169-76
5. American Diabetes Association Position Statement. Standards of medical care in
diabetes - 2017. Diabetes Care 2017;40(Supplement 1):S1-S135
6. Davies MJ, Gagliardino JJ, Gray LJ, Khunti K, Mohan V, Hughes R. Real-world
factors affecting adherence to insulin therapy in patients with Type 1 or Type 2
diabetes mellitus: a systematic review. Diabetic Medicine 2013;30:512-24
7. International Hypoglycaemia Study Group. Glucose concentrations of less than 3.0
mmol/L (54 mg/dL) should be reported in clinical trials: a joint position statement of
the American Diabetes Association and the European Association for the Study of
Diabetes. Diabetes Care 2017;40(1):155-57
8. Christensen M, Bagger JI, Vilsbøll T, Knop FK. The alpha-cell as target for type 2
diabetes therapy. Rev Diabet Stud 2011;8:369-81
9. Lund A, Bagger JI, Christensen M, Knop FK, Vilsbøll T. Glucagon and type 2
diabetes: the return of the alpha cell. Curr Diab Rep 2014;14:555
10. Habegger KM, Heppner KM, Geary N, Bartness TJ, DiMarchi R, Tschöp MH. The
metabolic actions of glucagon revisited. Nat Rev Endocrinol 2010;6:689-97
11. Onoue S, Ohshima K, Debari K, Koh K, Shioda S, Iwasa S, et al. Mishandling of the
therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils. Pharm Res
2004;21:1274-83


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 1.0 
 
 
 
 
 
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Strictly Confidential  
 
 
Page 76 of 79  
12. Fosgerau K, Hoffmann T. Peptide therapeutics: current status and future directions. 
Drug Discovery Today 2015;20:122-8 
13. Novo Nordisk Limited. SPC for GlucaGen Hypokit 1 mg. 2015. 
http://www.medicines.org.uk/emc/medicine/4258/SPC/GlucaGen+Hypokit+1+mg - 
Accessed 13 Mar 2017 . 
14. Zealand Pharma A/S. Dasiglucagon Investigator's Brochure, Edition 3, 12 Dec 2016.  
15. The Task Force for the management of arterial hypertension of the European Society 
of Hypertension (ESH) and of the European Society of Cardiology (ESC). 2013 
ESH/ESC guidelines for the management of arterial hypertension. European Heart 
Journal 2013;34:2159-219 
16. Levey AS, Coresh J, Greene T, Stevens LA, Zhang Y, Hendriksen BA, et al. Using 
standardized serum creatinine values in the modification of diet in renal disease study 
equation for estimating glomerular filtration rate. Annals of Internal Medicine 
2006;145:247-54 
17. Food and Drug Administration (FDA). Guidance for Industry on Bioanalytical 
Method Validation. Food and Drug Administration (FDA) 2001 
18. Viswanathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ, Sailstad J, et al. 
Quantitative bioanalytical methods validation and implementation: best practices for 
chromatographic and ligand binding assays. Pharm Res 2007 Oct;24:1962-73 
19. Shankar G, Devanarayan V, Amaravadi L, Barrett YC, Bowsher R, Finco-Kent D, et 
al. Recommendations for the validation of immunoassays used for detection of host 
antibodies against biotechnology products. J Pharm Biomed Anal 2008;48:1267-81 
20. Zealand Pharma report SS. Commissioning and validation of a method for the 
validation of anti-ZP4207 antibodies in human serum.  2014  
21. Zealand Pharma report SS. Commissioning and validation of a method for the 
validation of anti-glucagon antibodies in human serum.  2014  
22. Bioagilytix Labs. Validation of an assay to detect neutralizing antibodies to ZP4207 
by measuring cyclic adenosine monophosphate (cAMP) in a glucagon receptor 
transfected cell line, BAL-14-206-004-REP.  2016. Report No.: Zealand Pharma, 
study 15-039 
23. Bioagilytix Labs. Validation of an assay to detect neutralizing antibodies to glucagon 
by measuring cyclic adenosine monophosphate (cAMP) in a glucagon receptor 
transfected cell line, BAL-14-206-005.  2016. Report No.: Zealand Pharma, Study 
15-040 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 1.0 
 
 
 
 
 
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Strictly Confidential  
 
 
Page 77 of 79  
24. Amiel SA, Tamborlane WV, Simonson DC, et al. Defective glucose 
counterregulation after strict glycemic control of insulin-dependent diabetes mellitus. 
N Eng J Med 1987; 316: 1376-83. 
25. Rickels MR, Ruedy KJ, Foster NC, et al. Intranasal glucagon for treatment of insulin-
induced hypoglycemia in adults with type 1 diabetes: A randomized crossover 
noninferiority study. Diabetes Care 2016; 39: 264-70. 
26. ICMJE. Recommendations for the conduct, reporting, editing, and publication of 
scholarly work in medical journals. Updated December 2016 
 
 
 
 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 1.0 
 
 
 
 
 
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Strictly Confidential  
 
 
Page 78 of 79  
12 
APPENDICES 
12.1 List of trial personnel 
Sponsor 
Clinical Trial Manager 
 
 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Phone: 
 
Medical Officer 
 
Zealand Pharma 
Smedeland 36 
2600 Glostrup, Denmark 
Phone: 
 
Contract Research Organization 
Chiltern International (Ltd) 
171 Bath Road 
Slough 
Berkshire 
SL1 4AA 
United Kingdom 
Phone: +44 (0) 175351 2000 
Project Manager 
 
Chiltern International (Ltd) 
171 Bath Road 
Slough 
Berkshire 
SL1 4AA 
United Kingdom 
Phone: 
 
Senior Medical Officer and 
Safety medical monitor 
 
Chiltern International kft 
Canada Square Office House 
Ganz u 


 
 
Dasiglucagon (ZP4207) 
Date: 29 Aug 2017 
 
Study ID: ZP4207-16137 
Protocol Version: Final 1.0 
 
 
 
 
 
________________________________________________________________________ 
Strictly Confidential  
 
 
Page 79 of 79  
12-14, 4 emelet 
1027 Budapest 
Hungary 
Phone: 
 
Pharmacovigilance unit 
Responsible for Serious Adverse 
Event (SAE) Management and 24-
hour SAE reporting 
  
PharmaLex 
Agern Allé 24 
2970 Hørsholm, Denmark 
Phone: 
 (8 a.m. to 4 p.m.) 
Phone: 
 (outside 8 a.m. to 4 p.m.) 
Fax: 
 
email: PV-nordic@pharmalex.com  
Central laboratory 
MLM Laboratory 
Dohrweg 63 
41066 Mönchengladbach 
Germany 
Special laboratory (GlucaGen 
PK, insulin PK) 
MLM Laboratory 
Dohrweg 63 
41066 Mönchengladbach 
Germany 
Special laboratory (dasiglucagon 
PK, dasiglucagon ADA, 
GlucaGen ADA) 
York Bioanalytical Solutions (YBS)  
Cedar House 
Northminster Business Park 
Northfield Lane 
York, YO26 6QR, United Kingdom 
Special laboratory  
(neutralizing antibodies) 
BioAgilytix 
2300 Englert Drive 
Durham, NC, 27713, USA 
A list of all investigators, IECs and IRBs will be provided in a separate document and in 
the clinical trial report. 
 

SAP_001.pdf: 
Clinical Trial Report 
08 October 2019 
Protocol: ZP4207-16137 
Page 1 
Final 
CONFIDENTIAL 
 
 
16.1.9  Documentation of Statistical Methods 
The following documents are provided in this section: 
Statistical Analysis Plan: 
Version 1 Final 1 
20 Aug 2018 
 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma A/S 
Protocol 
Number: 
Zealand Pharma A/S 
ZP4207-16137 
STATISTICAL ANALYSIS PLAN 
Protocol No.: ZP4207-16137 
EudraCT No.: 2017-002449-31 
Treatment: Dasiglucagon injection 
A phase 3, randomized, double-blind, parallel trial to confirm the clinical 
efficacy and safety of dasiglucagon in the rescue treatment of hypoglycemia 
in subjects with type 1 diabetes mellitus (TlDM) compared to placebo and 
with reference to GlucaGen® 
Author: 
Document Status: 
Version Dates: 
Pages: 
, MS, MBA 
Version I, Final I 
Version I, Final 1: 20AUG2018 
36 
STATEMENT OF CONFIDENTIALITY 
CONFIDENTIAL 
SAP Version 1, Final 1 
Page 1of36 
20AUG2018 


, II 
STATISTICAL ANALYSIS PLAN SIGNATURE FORM 
Sponsor. 
Zealand Pharma 
Protocol Number: 
ZP4207-16137 
CIL Project Number: 
40337 
Project Manager 
 
I 
S1at1stical Analysis Plan 
SAP Version 1, Final 1 20AUG2018 
Version I Date: 
_J 
The Project Manager 1s signing below to confirm they have reviewed and approved the 
Statistical Analysis Plan 1n accordance with the study protocol and CRF 
Approval Signature 
Print Name 
Oat e o 
m>I 
O 
Not applicable - No Chiltern PM assigned 
The Lead Statistician is signing below to confirm they have authored/reviewed and 
approved the Statistical Analysis Plan in accordance w1tt1 the study protocol and CRF. 
Approva1 Signalure 
P11nl Name 
Date1"""'""'·11i">> 
The Support Statistician 1s signing below to confirm they have reviewed and approved the 
Statistical Analysis Plan in accordance with the study protocol and CRF 
Approval Signature 
P11nt Name 
Date toonm"'Yl'YYl 
The Sponsor Representative 1s signing below to confirm they have reviewed and approved 
the Statistical Analysis Plan 1n accordance w1t11 the study protocol, CRF, and any other study 
requirements. 
Approval Signature 
;:>rmt Name 
Job Title 
Date oo-nrrn'f'/'(fl 
FBS007 Version 7 
(based on TPOA036 Vers on 2) 
Page 1 of 1 
Effect111e Date 9" March 2017 
Prior Effective Dale 3"' November 2016 
I 
I 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma A/S 
Contents 
Protocol 
Number: 
ZP4207-16137 
CHILTERN 
ABBREVIATIONS .................................................................................................................... 4 
1 
INTRODUCTION ............................................................................................................. 6 
2 
STUDY OBJECTIVES ...................................................................................................... 6 
2.1 
PRIMARY ENDPOINTS .................................................................................................... 6 
2.2 
SECONDARY ENDPOINTS ............................................................................................... 6 
KEY SECONDARY ENDPOINTS ............................................................................................. ....... 6 
OTHER SECONDARY ENDPOINTS ................................................................................................ 7 
EXPLORATORY ENDPOINTS ....................................................................................................... 7 
2.3 
SAFETY ENDPOINTS ....................................................................................................... 7 
3 
STUDY DESIGN ................................................................................................................ 8 
3.1 
DISCUSSION OF STUDY DESIGN ..................................................................................... 8 
3.2 
STUDYTREATMENT ...................................................................................................... 8 
3.3 
STUDY SCHEDULE ................................... ...................................................................... 8 
3.4 
CONCOMITANT MEDICATION ...................................................................................... 11 
3.5 
STUDY ANALYSIS SAMPLES ........................................................................................ 11 
3.6 
WITHDRAWN SUBJECTS .............................................................................................. 11 
3.7 
RANDOMIZATION ........................................................................................................ 12 
3.8 
BLINDING AND BREAKING THE BLIND ........................................................................ 12 
3 .9 
SAMPLE SIZE ............................................................................................................... 13 
4 
STATISTICAL METHODOLOGY .............................................................................. 13 
4.1 
PLANNED ANALYSES .................................................................................................. 13 
4.2 
I NTERIM ANALYSIS ..................................................................................................... 14 
4.3 
DISPOSITION OF SUBJECTS .......................................................................................... 14 
4.4 
BASELINE AND DEMOGRAPHIC CHARACTERISTICS ..................................................... 14 
4.5 
EXPOSURE ................................................................................................................... 14 
4.6 
PRIOR AND CONCOMITANT MEDICATION .................................................................... 15 
4.7 
EFFICACY/ PRIMARY AND SECONDARY ANALYSIS ..................................................... 15 
4.8 
EXPLORATORY ANALYSIS ........................................................................................... 21 
4.9 
SAFETY ANALYSIS ......................... ............................................................................. 22 
4.9.1 
Adverse events ....................................................................................................... 22 
4.9.1.1 
Subsets ........................................................................................................... 23 
4.10 
ADJUSTMENT FOR COVARIATES .................................................................................. 25 
4.10.1 
Center/Study Site Effects .................................................................................. 26 
4.11 
PROTOCOL VIOLATIONS OR D EVIATIONS .................................................................... 26 
4.12 
SUBJECT W!THDRA WALS AND MISSING DAT A .......... .................................................. 27 
METHODS FOR H ANDLING MISSING DA TES ............................................................................ 28 
4.13 
DEVIATIONS FROM SAP .............................................................................................. 29 
4.14 
CHANGES IN CONDUCT OR PLANNED ANALYSES FROM THE PROTOCOL ..................... 29 
CONFIDENTIAL 
SAP Version 1, Final 1 
Page 2 of 36 
20AUG2018 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma A/S 
Protocol 
Number: 
ZP4207-16137 
CHll1FRN 
4.15 
ALGORITHMS/SAS CODES .......................................................................................... 29 
5 
TABLES AND LISTINGS .............................................................................................. 32 
5.1 
TABLE FORMAT .......................................................................................................... 32 
5.2 
CONVENTIONS ............................................................................................................ 33 
5.3 
TABLES ....................................................................................................................... 33 
5.3.1 Section 14.1: Baseline a11d Demographic Data ................................................... 33 
5.3.2 Section 14.2: Efficacy A11alysis Data ................................................................... 34 
5.3.3 Section 14.2: Primary Co11firmat01y E11dpoi11t .................................................... 34 
5.3.4 Section 14.2: Secondary Endpoints ...................................................................... 34 
5.3.5 Section 14.2: Exposure (PD/PK) Endpoints ........................................................ 34 
5.3. 6 Section 14.2: Exploratory ..................................................................................... 34 
5.3. 7 Section 14.3: Safety Analysis Data ....................................................................... 34 
5.3.7.1 
Adverse events ............................................................................................... 34 
5.3.7.2 
Laboratory results .......................................................................................... 34 
5.3.7.3 
Vital signs ...................................................................................................... 35 
5.3.7.4 
ECG ............................................................................................................... 35 
5.3.7.5 
Other safety data ............................................................................................ 35 
5.4 
LISTINGS ..................................................................................................................... 35 
5.5 
FIGURES ...................................................................................................................... 36 
5.6 
APPENDICES ................................................................................................................ 36 
5.7 
REFERENCES ............................................................................................................... 36 
CONFIDENTIAL 
SAP Version 1, Final 1 
Page 3 of 36 
20AUG2018 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma A/S 
Protocol 
Number: 
Abbreviations 
ADA 
Anti-drug antibody 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALT 
Alanine aminotransferase 
ANCOVA 
Analysis of covariance 
AST 
Aspartate aminotransferase 
AUC 
Area under the concentration-time curve 
AUCo-inf 
AUC from time zero to infinity 
AUE 
Area under the effect curve 
CI 
Confidence interval 
Cmax 
Maximum plasma concentration 
CPH 
Cox proportional hazards 
CRO 
Contract research organization 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
EU 
European Union 
CHll TERN 
ZP4207-16137 
EudraCT 
European Medicines Agency's Clinical Trials Database 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HbA 1c 
Glycated hemoglobin 
ICH 
International Conference on Harmonization 
ID card 
Identification card 
IEC 
Independent Ethics Committee 
IM 
lntramuscular(ly) 
IMP 
Investigational medicinal product 
IRB 
Institutional Review Board 
IV 
Intravenous(ly) 
IWRS 
Interactive Web Response System 
MedDRA 
Medical Dictionary for Regulatory Activities 
CONFIDENTIAL 
SAP Version 1, Final 1 
Page 4 of 36 
20AUG2018 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma A/S 
Protocol 
Number: 
NPH 
Neutral protamine Hagedorn 
PD 
Pha1macodynamics 
PK 
Pharmacokinetics 
PP 
Per protocol 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SC 
Subcutaneous(ly) 
SU SAR 
Serious unexpected suspected adverse reaction 
TIDM 
Type 1 diabetes mellitus 
T2DM 
Type 2 diabetes mellitus 
TEAE 
Treatment-emergent adverse event 
tmax 
Time to the maximum plasma concentration 
ULN 
Upper limit of the normal range 
us 
United States of America 
CONFIDENTIAL 
SAP Version 1, Final 1 
Page 5 of 36 
CHIL 1 ERN 
ZP4207-16137 
20AUG2018 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma NS 
1 Introduction 
Protocol 
Number: 
ZP4207-16137 
CH!l 1ERN 
This document presents the statistical analysis plan (SAP) for Zealand Pharma A/S, Protocol 
No. ZP4207-16137: A phase 3, randomized, double-blind, parallel trial to confirm the clinical 
efficacy and safety of dasiglucagon in the rescue treatment of hypoglycemia in subjects with 
type I diabetes mellitus (Tl DM) compared to placebo and with reference to GlucaGen®. 
This analysis plan is based on the final clinical trial protocol (Final, v3.0) dated 060CT2017 and 
global protocol amendment 2 (Final, vl.0) dated 04APR2018. The SAP will be finalized and 
approved prior to the unblinding of the database. 
The SAP provides the description of the statistical analysis for the final analyses. 
2 Study Objectives 
The objectives of this study are: 
• 
Primary: To demonstrate superiority of dasiglucagon compared to placebo following a single 
subcutaneous 0.6 mg dose administered to subjects with type I diabetes mellitus with 
insulin-induced hypoglycemia. 
• 
Secondary: To compare the glycemic response observed after dasiglucagon with that of 
GlucaGen. 
2.1 Primary endpoints 
The primary endpoint of this study is: 
• 
Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase in 
plasma glucose of 
mg/dL (1.1 mmol/L) from baseline during the hypoglycemic clamp 
procedure without administration of rescue IV glucose. 
2.2 Secondary endpoints 
The secondary endpoints of this study are: 
Key secondary endpoints 
• 
Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, and 
within I 0 minutes after study drug injection without administration of rescue IV glucose. 
CONFIDENTIAL 
SAP Version 1, Final 1 
Page 6 of 36 
20AUG2018 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma A/S 
Protocol 
Number: 
ZP4207-16137 
CHll TFRN 
• 
Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 
15 minutes, and within I 0 minutes after study drug injection or at the time of rescue. 
Other secondary endpoints 
• 
Clinical efficacy (PD) endpoints: 
Time to first plasma glucose concentration 
mg/dL (3.9 mmol/L) without 
administration of rescue IV glucose. 
Plasma glucose response as area under the curve (AUC) above baseline from time zero 
to 30 minutes, A UCo-30min. 
• 
Exposure (PK) endpoints: 
Area under the drug concentration curve from tin1e zero to 90 minutes, AUCo-90min. 
Maximum plasma drug concentration (Cmax). 
Time to maximum plasma drug concentration (tmax). 
• 
Immunogenicity endpoint: 
Occurrence of anti-drug antibodies 
Exploratory endpoints 
• 
Plasma glucose concentration 
mg/dL (3.9 mmol/L) or increase of 
mg/dL 
(I. I mmol/L) within 30 minutes after study drug injection without administration of rescue 
IV glucose. 
• 
Plasma insulin response as area under the curve above baseline from time zero to 60 minutes, 
AUCo-60 min. 
2.3 Safety endpoints 
The safety endpoints of this study are summaries of the following: 
• 
Adverse events, clinical laboratory assessments (biochemistry, hematology, urinalysis), 
vital signs, physical examination, electrocardiogram (ECG), and local tolerability. 
• 
Administration of rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
• 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure. 
CONFIDENTIAL 
SAP Version 1, Final 1 
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20AUG2018 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma A/S 
3 Study Design 
3.1 Discussion of Study Design 
Protocol 
Number: 
ZP4207-16137 
CHIL TFRN 
This trial is a global, multicenter, randomized, parallel, double-blind, clinical trial confirming 
the efficacy and safety of dasiglucagon for insulin-induced hypoglycemia in subjects with 
TI DM. The subjects will be randomized 2: I: I to receive a single fixed SC 0.6 mg dose of 
dasiglucagon, placebo, or a I mg dose of GlucaGen and followed for at least 28 days after 
receiving treatment. A total of 156 subjects with TIDM are expected to complete the treatment 
visit. This trial is planned lo be conducted at 4 to 6 trial sites in the United States of America 
[US], Canada, and Europe. 
3.2 Study Treatment 
Subjects successfully completing screening and who fulfill entry eligibility and randomization 
criteria will be randomized to one of three treatment groups in a 2: I: 1 ratio: 
• 
Test treatment: Dasiglucagon, liquid formulation, I mg/mL, 0.6 mL delivered in a prefilled 
syringe. 
• 
Placebo treatment: Placebo, liquid formulation, 0.6 mL delivered in a prefilled syringe. 
• 
Recombinant glucagon hydrochloride, 1 mg for reconstitution (GlucaGen®, Novo Nordisk) 
in I mL sterile water. 
Subjects will attend the study site at Trial Day -3 (Screening Visit I, Day -30 to Day - I) for 
informed consent, Trial Day -1 lo Day 1 for dosing (Dosing Visit 2, Day I), and followed for at 
least 28 days after dosing in order to perfom1 an adequate immunogenicity evaluation of 
treatment (Safety Follow-up Visit, Day 28 + 5). 
3.3 Study Schedule 
The schedule of procedures is provided in Protocol Table 3. 
3.3.l 
Visit l (screening, Day -30 to Day -3) 
Visit I will take place between 3 and 30 days before Visit 2, Day-I to Day I (dosing day). 
Informed consent can be obtained prior to or at Visit I, however it must in any case be obtained 
prior to any trial related procedures. During the screening visit, the following assessments will 
take place: 
• 
Informed consent (obtain or check) 
• 
Check of subject eligibility (inclusion/exclusion criteria) 
• 
Demography 
CONFIDENTIAL 
SAP Version 1, Final 1 
Page 8 of 36 
20AUG2018 


CHILTERN 
ZEALAND PHARMA 
Sponsor: 
Zealand Pharma A/S 
Protocol 
Number: 
ZP4207-16137 
• 
Body measurements 
• 
Medical history, diabetes diagnosis, cuJTent diabetes treatment 
• 
Concomitant illnesses 
• 
Concomitant medications 
• 
History of alcohol/drug abuse 
• 
Physical examination 
• 
Vital signs 
• 
12-lead ECG 
• 
Adverse Events 
• 
Biochemistry, hematology, coagulation, HbA1c, C-peptide 
• 
Serum pregnancy test (women of childbearing potential only) 
• 
Urinalysis 
• 
Urine drug screen 
• 
Alcohol breath test 
3.3.2 
Visit 2 (dosing visit) 
The subjects will attend the clinical center the day prior to dosing (Day -I ) and should stay onsite 
overnight. Dosing will take place the following morning (Day I). 
On Day I and prior to the start of the insulin-induced hypoglycemic procedure, subject eligibility 
is rechecked (check of changes between the screening visit and Visit 2) and those subjects 
eligible to participate will be randomized to treatment with dasiglucagon, placebo, or GlucaGen. 
The following assessments will also take place: 
• 
Document all changes in concomitant medication (prior to the start of the insulin-induced 
hypoglycemic procedure) 
• 
Check of withdrawal criteria (prior to the start of the insulin-induced hypoglycemic 
procedure) 
• 
Check of dosing day exclusion criteria (prior to the start of the insulin-induced 
hypoglycemic procedure) 
• 
Vital signs (prior to the start [within 30 minutes] of the insulin-induced hypoglycemic 
procedure, and at 30, 90 and 300 minutes after dosing). Blood pressure should be measured 
first in sitting and then in standing. 
• 
12-lead ECG (p1ior to the start [within 30 minutes] of the insulin-induced hypoglycemic 
procedure, and at 20, 35, 45, 60, and 300 minutes after dosing) 
• 
Local tolerability (at 30, 120, and 300 minutes after dosing) 
• 
AEs 
• 
Biochemistry and hematology (prior to the start of the insulin-induced hypoglycemic 
procedure [within 30 minutes], and at 30 and 300 minutes after dosing) 
CONFIDENTIAL 
SAP Version 1, Final 1 
Page 9 of 36 
20AUG2018 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma NS 
Protocol 
Number: 
ZP4207-16137 
CHllTFRN 
• 
Urine stick pregnancy test (women of childbearing potential only; prior to the start of the 
insulin-induced hypoglycemic procedure) 
• 
Urinalysis (prior to the start of the insulin-induced hypoglycemic procedure [within 120 
minutes]) 
• 
Urine drug screen (prior to the sta1t of the insulin-induced hypoglycemic procedure) 
• 
Alcohol breath test (prior to the start of the insulin-induced hypoglycemic procedure) 
• 
Dasiglucagon/GlucaGen plasma concentrations: 
Pre-dose, 15, 30, 35, 40, 50, 60, 90, and 120 minutes after dosing. The actual time of 
blood sampling for exposure should not deviate from the nominal time by more than 
± I minute. Pre-dose is defined as within 2 minutes prior to dosing. 
• 
Plasma glucose concentrations: 
Pre-dose, 4, 6, 8, l 0, 12, 15, 17, 20, 25, 30, 40, 50, 60, 75, and 90 minutes after dosing. 
The actual time for blood sampling for plasma glucose should not deviate from the 
nominal time by more than ±30 seconds until the 20 minute collection time point and 
by more than ± 1 minute for the subsequent collection time points. Pre-dose is defined 
as within 2 minutes prior to dosing. 
• 
Antibodies against dasiglucagon/GlucaGen (prior to the start of the insulin-induced 
hypoglycemic procedure). 
• 
Plasma insulin concentrations: 
Pre-dose, 30 and 60 minutes after dosing. The actual time of blood sampling for plasma 
insulin should not deviate from the nominal time by more than ± I minute. Pre-dose is 
defined as within 2 minutes prior to dosing. 
3.3.3 
Visit 3 (follow-up visit) 
Visit 3 will take place on Day 28 + 5 days. The subject does not need to be fasting. 
At Visit 3, the following assessments will take place: 
• 
Document all changes in concomitant medication 
• 
Physical examination 
• 
Vital signs 
• 
12-lead ECG 
• 
Local tolerability 
• 
AEs 
• 
Biochemistry, hematology 
• 
Urine stick pregnancy test (women of childbearing potential only) 
• 
Urinalysis 
• 
Antibodies against dasiglucagon/GlucaGen. 
CONFIDENTIAL 
SAP Version 1, Final 1 
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20AUG2018 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma A/S 
3.4 Concomitant Medication 
Protocol 
Number: 
ZP4207-16137 
CHIL TFRN 
A concomitant medication is any medication, other than the trial products and current diabetes 
treatment (including insulin glulisine [Apidra®] for diabetes therapy wash-out), which is taken 
during the trial, including screening and follow-up periods. 
Details of any concomitant medication must be recorded at trial entry (i.e., at screening). Any 
changes in concomitant medication must be recorded at each visit as they occur. The information 
collected for each concomitant medication includes (at a minimum) trade name or generic name, 
indication, start date and stop date or continuation. A change in medication due to an AE must 
be recorded and reported according to Protocol Section 7.2. I " Adverse Events". If the change in 
medication influences the subject's eligibility to continue in the trial, the sponsor and trial 
monitor must be informed. 
3.5 Study Analysis Samples 
For presentation of data and reporting of the statistical analyses, the following analysis samples 
will be used, depending on the context: 
• 
Safety analysis set: All randomized subjects who received at least one dose of trial 
medication. 
• 
Full analysis set: All randomized subjects who received at least one dose of trial 
medication. 
• 
Per protocol (PP) set: All subjects of the Full analysis set for whom no relevant protocol 
deviations were documented. This sample will primarily be used for sensitivity analysis. 
The analysis of the primary endpoint and secondary endpoints will be based on the Full 
analysis set. A secondary analysis of the primary endpoint will be based on the PP set. All 
safety analyses will be based upon the Safety analysis set, which is identical to the Full 
analysis set, but will be labelled as Safety analysis set. 
The decision regarding whether a protocol deviation is relevant or not for the exclusion of 
subjects from the PP set will be made case-by-case in a blinded data review meeting prior to 
treatment unmasking and database lock (see Protocol Section 9.2.3 " Protocol Deviations"). 
3.6 Withdrawn Subjects 
Subjects withdrawn from the trial will be listed including the primary reason for withdrawal. 
Any subject who withdraws after randomization will not be replaced. Withdrawn subjects will 
not be re-entered into the study. 
CONFIDENTIAL 
SAP Version 1, Final 1 
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20AUG2018 


ZEALAND PHARMA 
Sponsor: 
Zealand Pharma A/S 
3. 7 Randomization 
Protocol 
Number: 
ZP4207-16137 
CHILTERN 
Subjects who have given written informed consent and meet all inclusion and none of the 
exclusion criteria will be randomized to one of three treatment groups in a 2: I: I ratio: 
• 
Test treatment: Dasiglucagon, liquid formulation, I mg/mL, 0.6 mL delivered in a prefilled 
syringe. 
• 
Placebo treatment: Placebo, liquid fommlation, 0.6 mL delivered in a prefilled syringe. 
• 
Reference treatment: Recombinant glucagon hydrochloride, 1 mg for reconstitution. 
Randomization will be performed using a fixed-block randomization scheme. The 
randomization scheme will be generated prior to the initiation of the study by an independent 
statistician/programmer who will not be a member of the study team; all investigators will not 
be aware of the block size of the randomization scheme. Randomization will be stratified by 
treatment group and by injection site (abdomen, buttocks, or thigh) and controlled via the IWRS. 
3.8 Blinding and Breaking the Blind 
This is a double-blind trial. As the trial products are not identical in appearance, dasiglucagon 
and placebo being available as a liquid formulation and GlucaGen as a powder for reconstitution, 
unblinded trial personnel will be responsible for handling, preparing (according to the 
prescription from the IWRS), and administering the trial medication, as well as for keeping the 
records strictly confidential and accessible only to unblinded staff until after the database has 
been locked. To maintain double-blind conditions, all trial assessments at the trial center will be 
done by blinded trial personnel not involved in the administration of trial medications. However, 
exposure assessments and anti-drug antibody (ADA) assessments will be performed by 
unblinded personnel at the special laboratories, to ensure that dasiglucagon, placebo, or 
GlucaGen administration is matched with the applicable bioanalytical assay. 
Treatment assignment will be kept strictly confidential and accessible only to authorized persons 
until after the time of unblinding. Codes with treatment assignment will, however, be readily 
available in the IWRS to the blinded personnel in case of an emergency. 
The code for a particular subject may be broken in a medical emergency if knowing the identity 
of the treatment allocation would influence the treatment of the subject. The emergency code 
break can be perfonned using the IWRS. Whenever a code is broken, the person breaking the 
code must record the time, date and reason as well as his/her initials in the source documents. 
The breaking of blinded codes in case of medical emergency for one subject should not unblind 
the trial personnel to the treatment infonnation of other subjects. The person performing the 
unblinding should inform as few people as possible about the result of the unblinding. All 
persons unblinded for a specific subject should be documented. 
If the trial center needs to break the code, the medical monitor should, if at all possible, be 
contacted prior to breaking the code and the monitor must be notified within 24 hours after the 
code has been broken. 
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The phannacovigilance unit (safety contract research organization [CRO]; see Protocol Section 
12.1 " List of Trial Personnel") will be able to break the code in case of a serious unexpected 
suspected adverse reaction (SUSAR). 
The central and specialty laboratories will be provided with a copy of the randomization list. 
3.9 Sample Size 
Due to requirements in the size of the safety database, the sample size is set to 78 subjects treated 
with dasiglucagon 0.6 mg. From phase 2, the median time to an increase of20 mg/dL of the 0.6 
mg dose was approximately I 0 minutes. With a 2: 1: I randomization ratio for the three 
treatments, dasiglucagon 0.6 mg, placebo, and GlucaGen I mg, and assuming an exponential 
time-to-recovery distributions with median times of I 0 and at least 20 minutes for dasiglucagon 
and placebo, respectively, a two-sided log-rank test will be able to detect a difference between 
dasiglucagon and placebo with 90% power with a follow-up time of 45 minutes at a 5% 
significance level with 78 subjects treated with dasiglucagon and 39 subjects with placebo. The 
median time-to-recovery for placebo is expected to be longer than 20 minutes, which means that 
the power will be greater than 90%. 
4 Statistical Methodology 
4.1 Planned Analyses 
Before database lock and treatment unmasking, the statistical analysis plan (SAP) will be 
finalized, providing detailed methods for the analyses outlined below. Further analysis details 
may be added or refined in the SAP. 
The analysis of the primary endpoint and secondary endpoints will be based on the Full analysis 
set. A secondary analysis of the primary endpoint will be based on the PP set. All safety analyses 
will be based upon the Safety analysis set. 
The decision regarding whether a protocol deviation is relevant or not for the exclusion of 
subjects from the PP set will be made case-by-case in a blinded data review meeting prior to 
treatment unmasking and database lock (see Protocol Section 9.2.3 .. Protocol Deviations"). 
All data obtained in this trial and documented in the eCRFs will be listed and summarized with 
statistics or frequency tables as appropriate. In case of tennjnation of the trial, all data collected 
up to that timepoint will be included in the analysis. 
Raw data listings and summary tables will be generated using the software SAS© version 9.4 or 
higher. 
Continuous variables will be summarized using means, standard deviations, medians, 
coefficients of variation, and minimum and maximum values. Other summaries (e.g. quartiles, 
95% confidence intervals [Cls]) may be used as appropriate. 
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Categorical efficacy and safety variables will be summa1ized by counts and by percentage of 
subjects in corresponding categories. 
4.2 Interim Analysis 
No interim analysis is planned for this study. 
4.3 Disposition of Subjects 
Subject disposition will be tabulated including the number of screened subjects, screening 
failures, subjects exposed to trial product, subjects completing the trial and subjects in each 
analysis set. A CONSORT diagram will be included to display the disposition of subjects. 
Subjects withdrawn from the trial will be listed including the primary reason for withdrawal. 
4.4 Baseline and Demographic Characteristics 
Baseline assessments are defined as the last non-missing result prior to administration of the first 
dose of study medication. In cases where there are multiple such values, the non-missing value 
closest to the start of study treatment will be selected. If time is available for an assessment, it 
will be compared with dosing time of the first dose of study medication to define the baseline 
value. 
Baseline and demographic data will be summarized using descriptive statistics. Baseline ADA-
positive subjects will be calculated as a percentage of the total number of subjects whose baseline 
samples were tested for ADA. 
Notes: 
• 
Age will be calculated as (screening date - date of birth + I )/365.25 and presented to 
decimal place. Age will be rounded down to the nearest integer before analysis. 
• 
BMI will be calculated as weight(kg)/height(m2) 
• 
Medical history will be coded according to MedDRA, version 19. 1. 
• 
Active medical history is defined as histories marked as active at time of screening. 
• 
Relevant medical histories are all medical histories recorded in the eCRF. 
• 
Any duration [days] = end date - onset date + 1 
4.5 Exposure 
The exposure to trial medication (dasiglucagon or GlucaGen) for evaluation of pharmacokinetics 
will be assessed based on plasma concentration data from samples collected at the dosing visit 
(Visit 2): 
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• 
Area under the drug concentration curve from time zero to 90 minutes, AUCo.90 min. 
• 
Maximum plasma drug concentration (Cmax). 
• 
Time to maximum plasma drug concentration (tmax). 
Samples (including back-up samples) will be collected pre-dose, and at 15, 30, 35, 40, 50, 60, 
90, and 120 minutes after dosing. The actual time of blood sampling for exposure to trial 
medication should not deviate from the nominal time by more than ± 1 minute. Pre-dose is 
defined as within 2 minutes prior to dosing. 
All data collected related to study drug administration will be provided in the data listings. 
4.6 Prior and Concomitant Medication 
Prior glucagon exposure will be recorded in the eCRF at screening. All concomitant medications 
will be recorded and/or updated in the eCRF at each visit, i.e., at screening (Visit 1), the dosing 
visit (Visit 2, prior to the start of the insulin-induced hypoglycemic procedure) and at the follow-
up visit (Visit 3). 
Subjects using any new concomitant medication resulting in fulfillment of a dosing day 
exclusion criterion (see Protocol Section 5.5 " Dosing Day Exclusion Criteria") will be excluded 
from the dosing visit, but can be rescheduled to one of the following days ( 1-7 days later). The 
dosing visit can only be rescheduled once. 
Prior and concomitant medications will be coded using the World Health Organization (WHO) 
Drug Dictionary, version 2017Dec. 
Incidence of prior and concomitant medication will be presented by treatment, drug class, and 
preferred drug name using patients in the Safety analysis set. 
Prior medications are those that started and stopped before exposure to study medication; 
concomitant medications are all medications taken during the study period, including those 
started before but ongoing at first dose of study drug. 
Where a medication start date is partially or fully missing, and it is unclear as to whether the 
medication is prior or concomitant, it will be assumed that it is concomitant. 
4. 7 Efficacy I Primary and Secondary Analysis 
For the confirmatory analyses, the primary and key secondary endpoints for the dasiglucagon 
0.6 mg and placebo treated subjects will be compared. A hierarchical procedure will be applied 
for the control of multiplicity. The primary and secondary endpoints will be evaluated on the 
Full analysis set. In the primary analysis, recovery cannot be achieved in those subjects where 
IV glucose treatment is administered. Those subjects who receive IV glucose will be censored 
(i.e., set to ·not recovered') at 45 minutes after dosing. In sensitivity analyses, the time to plasma 
glucose recovery will be analyzed I) without censoring for those subjects who require rescue IV 
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glucose before 45 minutes and 2) with censoring at the time of administration of rescue IV 
glucose before 45 minutes. 
4.7.1. Hierarchical testing procedure 
For the confirmatory analyses, the following a priori defined hierarchical inferential test order 
will be applied for the control of the type 1 error rate across the planned multiple comparisons. 
The primary endpoint and key secondary endpoints will be inferentially evaluated within the 
Full analysis set in the following order, where inference will proceed at the two sided 0.05 
c1iterion significance level until the first failure to reject the null hypothesis for a dasiglucagon 
versus placebo comparison: 
• 
Primary: Time to plasma glucose recovery 
• 
Key secondaries 1-4: Plasma glucose recovery within 30 minutes, within 20 minutes, within 
15 minutes, and within 10 minutes after study drug injection without administration of rescue 
IV glucose. 
• 
Key secondaries 5-8: Plasma glucose changes from baseline within 30 minutes, within 20 
minutes, within 15 minutes, and within 10 minutes after study drug injection or at the time 
of rescue. 
The GlucaGen versus placebo comparisons will not be included in the inferential testing 
hierarchy, since the efficacy of GlucaGen is previously established, and these comparisons are 
intended to support the validity of the study for the dasiglucagon versus placebo comparisons. 
Analogous supportive sensitivity analyses will be conducted in the PP set, but without inference 
intent. 
4.7.2. Primary endpoint 
• 
Time to plasma glucose recovery. Plasma glucose recovery is defined as first increase in 
plasma glucose of 2:20 mg/dL ( I.I mmol/L) from baseline during the hypoglycemic clamp 
procedure without administration of rescue IV glucose. 
The planned time points at 0, 6, 8, 10, 12, 15, 17, 20, 25, 30, 40, 50 and 60 minutes will be used 
in the analyses. 
4. 7.2.1 Primary analysis 
The primary endpoint will be summarized using Kaplan-Meier (KM) estimates stratified by 
treatment group and injection site. The treatment group difference between dasiglucagon and 
placebo will be evaluated inferentially using a two-sided log-rank test stratified by injection site. 
Time to plasma glucose recovery will also be summarized using Kaplan-Meier estimates 
stratified by treatment group alone. 
Time to plasma glucose recovery will be summarized using Kaplan-Meier estimates stratified 
by treatment group and investigational site. 
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In the primary analysis, recovery cannot be achieved in those subjects where IV glucose 
treatment is administered. Those subjects who receive IV glucose will be censored (and set to 
' not recovered') at 45 minutes after dosing. 
In sensitivity analyses, the time to plasma glucose recovery will be analyzed I) without 
censoring for those subjects who require rescue IV glucose before 45 minutes and 2) with 
censoring at the time of administration of rescue TV glucose before 45 minutes. 
As a supportive analysis to the stratified log-rank test for the time to plasma glucose recovery 
described above, dasiglucagon will be compared to placebo using a Wilcoxon test stratified by 
injection site. 
To graphically desc1ibe recovery in time, a histogram showing the propo11ion of patients that 
recovered within each time interval will be created, one for each treatment group. 
If the site of injection does not appear to have an effect on time to recovery, the factor can be 
disregarded and further presentations will not be stratified by injection site. 
The primary endpoint will additionally be analyzed using a Cox propo11ional hazards (CPH) 
time to event statistical model, if the hazards by treatment group can be assumed proportional 
using a graphical comparison of log-minus-log survival curves and no more than two-thirds of 
the recovery times are censored in each treatment group. The CPH model will be used for 
descriptive purposes, with treatment group and injection site modeled as categorical effects, and 
baseline plasma glucose modelled as a continuous covariate. The treatment group rate ratios, 
active versus placebo, will be estimated together with the 95% confidence intervals, and pairwise 
treatment group inferences (dasiglucagon vs placebo, GlucaGen vs placebo) will be evaluated 
using two-sided likelihood ratio tests. 
Due to the discrete nature of blood sampling in time, and the fact that there must be a true, but 
unmeasured, ordering of the recove1y times, the method using the ·'exact discrete" partial 
likelihood for tied failure times will be applied. In SAS, this is implemented in the p h reg 
procedure with the option ties=discrete. The influence of injection site on time to recovery 
will also be evaluated in the proportional hazards model by a 'type 3' test in the phreg 
procedure. 
To obtain an estimate of these true, but unassessed, patient recovery times, a linear 
interpolation (between the two time points before and after recovery is observed) will be 
carried out to estimate the patients' actual time of recovery. These derived recovery times will 
be described and analysed in the same manner as for the discrete times above. 
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Table 4-1: Definition of Events, Censoring, and Populations for Time to Event Analyses 
Time to 
Initiating 
Terminating 
Censoring Event(s) 
Analysis Study Endpoint 
Event 
Event 
Event(s) 
/Competing Risk(s) 
Sample 
/Statistical 
Endpoint 
Analyses 
Time to 
Administration First increase in 
Rescue IV glucose 
Full 
Primary/ 
plasma 
of IMP 
plasma glucose 
administration will be 
analysis Pairwise two-sided 
glucose 
of2:20 mg/dL 
censored at 45 minutes after 
set, 
log rank test 
recovery 
(1.1 mmol/ L) 
IMP dosing 
pp 
from baseline 
w/out rescue IV 
glucose admin. 
4. 7.2.2 Primary analysis: Missing Baseline Glucose Value 
In a blind review of anonymised plasma glucose values, it was observed that the baseline glucose 
value, which is used for evaluation of the primary endpoint, is missing for one subject. Since 
plasma glucose levels were monitored at site by using an US FDA-approved glucose analyzer 
(Yellow Springs Instrument, YSI), a substitute baseline value can be obtained for this subject. 
Even though some differences in the plasma glucose level are expected between local YSI 
measures obtained at site and clinical laboratory measures, it has been decided to use the baseline 
plasma glucose value from the YSI in the analysis for this subject. The actual pre-dose YSI value 
used for this subject will be reported in the C linical Trial Report. 
In a sensitivity analysis, the time to plasma glucose recovery will be analyzed without the subject 
missing the baseline clinical laboratory plasma value. 
4.7.3 
Secondary endpoints 
• 
Plasma glucose recovery within 30 minutes, within 20 minutes, within I 5 minutes, and 
within 10 minutes after study drug injection without administration of rescue IV glucose. 
• 
Plasma glucose changes from baseline within 30 minutes, within 20 minutes, within 15 
minutes, and within 10 minutes after study drug injection or at the time of rescue. 
4. 7.3. J Confirmatory analysis 
The key secondary incidence variables (plasma glucose recovery within 30 minutes, within 20 
minutes, within 15 minutes, within I 0 minutes, in hierarchical order for inference) will be 
analyzed using descriptive statistics (frequency and percentage) by treatment group. Inferential 
treatment group comparisons will be assessed via pairwise tests of independent binomial 
proportions (dasiglucagon versus placebo, GlucaGen versus placebo) using Fisher's exact test 
for each pairwise comparison. 
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The key secondary endpoints of plasma glucose changes from baseline within 30 minutes, within 
20 minutes, within 15 minutes, and within 10 minutes after study drng injection or at the time of 
rescue, will be analyzed with the plasma glucose changes from baseline at rescue carried forward 
in those subjects who require rescue IV glucose before plasma glucose 2:20 mg/dL recovery. 
Each of these changes from baseline variables will be analyzed using an Analysis of Covariance 
model, with treatment group modelled as a fixed effect and with the baseline plasma glucose 
modelled as a covariate. The dasiglucagon versus placebo treatment group difference will be 
evaluated inferentially as a least squares means contrast, using a two sided t-test at the 0.05 
significance level, first for the 30 minute changes from baseline, and then sequentially for the 
20 minute, 15 minute, and I 0 minute variables, until the first failure to reject. 
4.7.4 Secondary clinical efficacy (PD) endpoints 
• 
Time to first plasma glucose concentration of 2:70 mg/dL (3.9 mmol/L) without 
administration of rescue IV glucose. 
• 
Plasma glucose response as area under the curve above baseline from time zero to 30 
minutes, AUCo-30min· 
Secondary clinical efficacy endpoints will be assessed based on plasma concentration data from 
samples collected at the dosing visit (Visit 2). 
Unless otherwise stated, the population base of analysis will be the Full analysis set. 
4.7.4.1 Analysis ofsecond{//y clinical efficacy (PD) endpoints 
I. 
Time to first plasma glucose concentration 2:70 mg/dL from baseline. This time to-event 
endpoint will be evaluated using a Kaplan-Meier approach, with treatment group as a 
stratification factor, analogous to that used for the primary endpoint analysis. Differences 
between the KM curves (dasiglucagon versus placebo, GlucaGen versus placebo) will 
be evaluated inferentially using pairwise two-sided log rank tests. If the 2:70 mg/dL 
endpoint is not met within 45 minutes post-dosing, the time of the last valid plasma 
glucose measurement up to 45 minutes will be the censoring time. 
2. 
The AUC will be calculated as the baseline-adjusted area under the plasma glucose 
profile over time: 
a. AUCo-30min: restricting the time window to the 0 to 30 minutes interval. 
The log-transformed AUC endpoint will be analyzed using an analysis of covariance 
(ANCOV A) model with treatment as fixed effect and baseline plasma glucose modelled 
as a covariate. The least squares means treatment group differences will be back 
transformed (anti logged) for presentation as a ratio of the treatment group geometric 
means, with their corresponding 95% Cl. 
4.7.5 
Exposure (PK) endpoints 
• 
Plasma dasiglucagon and GlucaGen concentrations from time zero to 90 minutes: 
AUCo-90min, Cmax, and tmax. 
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AUC will be derived as the area under the individual plasma dasiglucagon/GlucaGen 
concentration profile for PK from 0 to 90 minutes or last valid measurement if this measurement 
is assessed sufficiently close to 90 minutes (decision to be taken at the blinded data review 
meeting). The standard trapezoidal method will be used, based on actual rather than nominal 
timepoints. 
Cmax will be determined as the maximum of all valid plasma dasiglucagon/GlucaGen 
concentrations. 
Tmax will be detennined as the timepoint where the maximum of all valid plasma 
dasiglucagon/GlucaGen concentration measurements for each measurement series is observed. 
The log-transformed PK endpoints AUC and Cmax will be analyzed in the same way as the 
AUC endpoints. 
As tmax is a highly discrete endpoint, Wilcoxon's rank sum test for unpaired observations will 
be used to assess differences between the two treatment groups. 
Exposure endpoints will be summarised for the Full analysis set, which is identical to the Safety 
analysis set. 
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Table 4-2: Overview of Efficacy Analyses 
Efficacy Endpoint 
Endpoints with binary response 
outcomes: 
• 
Plasma glucose recovery within 30 
minutes, within 20 minutes, within 15 
minutes, and within 10 minutes after 
study drug injection without 
administration of rescue IV glucose. 
Endpoints with continuous outcomes: 
• 
Plasma glucose changes from baseline 
changes from baseline within 30 
minutes, within 20 minutes, within 15 
minutes, and within I 0 minutes afier 
study drug injection or at the time of 
rescue. 
Endpoints with time-to-event outcomes: 
• 
Time to plasma 
glucose recovery 
Time to plasma 
glucose recovery 
without censoring at 
the time of recovery 
for those subjects 
who require rescue 
IV glucose before 45 
minutes 
• 
Time to plasma 
glucose recovery 
with censoring at the 
time of 
administration of 
rescue IV glucose 
before 45 minutes 
4.8 Exploratory Analysis 
Statistical Analvses 
• 
Fisher's exact test for each pairwise 
comparison 
• 
Changes 
from 
baseline: 
Analysis 
of 
Covariance 
model, with treatment group 
modeled as a fixed effect and 
with 
the baseline plasma 
glucose 
modeled 
as 
a 
covariate 
• 
Changes from baseline: T\\O sided I-lest 
at the 0.05 significance level 
• 
Kaplan-Meier (KM) estimates stratified 
by treatment group and injection site 
• 
Pairwise two-sided log rank test 
• 
Cox proportional hazards (CPH) time 
to event statistical model with 
treatment group and injection site 
modeled as categorical effects, and 
baseline plasma glucose modeled as a 
continuous covariate 
• 
Kaplan-Meier plots, both stratified by 
injection site and without stratifying 
by injection site 
• 
SAS Procedure 
• 
FREQ 
• 
LOGISTIC 
GLM 
LIFETEST 
• 
PH REG or 
TPHREG 
Exploratory analyses will include descriptive statistics and modeling analogous to that done for 
key secondary endpoints. However, treatment group comparisons will be summarized without 
inference intent. 
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• 
Plasma glucose concentration 
mg/dL (3.9 mmol/L) or increase of 
mg/dL ( I. I 
mrnol/L) within 30 minutes after study drug injection without administration of rescue IV 
glucose. 
• 
Plasma insulin response as area under the curve above baseline from time zero to 60 minutes, 
AUCo-60 min· 
Plasma insulin concentrations measured pre-dose and at 30 and 60 minutes after dosing (see 
Protocol Table 3 '·Schedule of Procedures") will be presented individually. A summary table per 
timepoint will be provided. The AUCo-60min will be detennined and a summary presented. 
4.9 Safety Analysis 
4.9.1 Adverse events 
Adverse events and serious adverse events will be summarised separately by treatment by 
presenting the number and percentage of subjects having any event, having a related event, 
having an event leading to withdrawal, having an event in each MedDRA system organ class 
and prefeffed tenn, having each individual event and the severity, relationship and outcome of 
each event. Number of events is also presented. Missing severity, relationship or outcome will 
be classed as unknown. 
A subject with more than one occuffence of the same adverse event in a particular system organ 
class will be counted only once in the total of those experiencing adverse event in that particular 
system organ class. If a subject experiences the same adverse event at more than one severity, or 
with more than one relationship to investigational product, the most severe rating or the stronger 
causal relationship to investigational product will be given precedence. Any missing severity, 
causality, or outcome will not be imputed and classed as unknown. 
Summaries classifying both events according to severity and relationship will be presented. 
AEs will be coded according to MedDRA, version 19.1. AE summary tables will include the 
number and percentage of subjects who experienced A Es summarized by system organ class and 
prefe1Ted term. 
Only treatment-emergent adverse events (TEAEs) will be reported. TEAEs are undesirable 
events not present prior to medical treatment, or an already present event that worsens in 
intensity following the treatment. 
Related events are defined as events that are probably or possibly related to study medication or 
with an unknown relationship. 
Adverse event of special interest (AESI) is an event which, in the evaluation of safety, has a 
special focus (e.g. required by health authorities). Jn this trial hemodynamic changes, as defined 
below, are considered AES!s: 
• 
Post-dose clinical signs, or measured vital signs, indicating a clinically significant drop in 
blood pressure including signs of orthostatic hypotension, vasovagal responses or 
bradycardia. 
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• 
Post-dose change in pulse or blood pressure considered an event of hypo- or hypertension as 
judged by the investigator. 
4.9.1.1 Subsets 
An overall summary table will be provided showing the number and percentage of subjects with 
any: 
• TEAE 
• Severe TEAE 
• Serious TEAE 
• Drug-related TEAE 
• Drug-related severe TEAE 
• Drug-related serious TEAE 
• TEAE leading to withdrawal 
• TEAE with outcome death 
• AESI 
All other information collected (e.g., action taken) will be listed as appropriate. 
Only treatment-emergent adverse events (commencing during or after exposure to study 
treatment) will be included in the adverse and serious adverse event summaries. Non-treatment-
emergent events (starting prior to exposure to study treatment) will be included in the subject 
listings and flagged but not included in the above summaries. Where an AE start date is partially 
or fully missing, and it is unclear as to whether the AE is treatment emergent, it will be assumed 
that it is. 
4.9.2 
Immunogenicity data 
lmmunogenicity data will be analyzed descriptively by treatment group. No statistical tests are 
planned. Baseline ADA-positive subjects will be calculated as a percentage of the total number 
of subjects whose baseline samples were tested for ADA. Titer will be reported as median and 
interquartile range. 
4.9.3 
Clinical laboratory assessments 
Clinical laboratory test results will be flagged as to whether the result is below, within, or above 
the respective reference range. The number of values outside of the reference range will be 
counted. 
Results from the following laboratory parameters, recorded at the Screening, Dosing, and 
Follow-up visits will be summarised by treatment and time point: 
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• 
Clinical biochemistry: sodium, potassium, calcium, glucose, urea, creatinine, total 
bilirnbin, AST, ALT, gamma-glutamyltransferase, alkaline phosphatase, total protein, C-
reactive protein. HbA I c and C-peptide at screening visit only. 
• 
Hematology: hemoglobin, red blood cell count (erythrocytes), hematocrit, platelet count 
(thrombocytes), total white blood cell count (leukocytes). 
• 
Coagulation: international normalized ratio, fibrinogen (at screening visit only). 
• 
Urinalysis: pH, blood (leukocytes and erythrocytes), protein, glucose, ketones, nitrite. 
Categorical and numeric variables will be presented separately. 
Shift tables for the hematology and biochemistry laboratory parameters comparing values below, 
within, and above the normal reference range will be presented using standard reference ranges. 
Abnom1al laboratory findings (e.g. biochemistry, hematology, and urinalysis) that are judged by 
the investigator as clinically significant will be recorded as AEs or SAEs if they meet the 
definition of an AE or SAE. Clinically significant abnormal laboratory findings or other 
abnormal assessments that are detected during the trial or are present at baseline and significantly 
worsen following the start of the trial will be reported as A Es or SAEs. 
4.9.4 Other safety data 
Incidence of rescue infusion of IV glucose during the hypoglycemic clamp procedure will be 
analyzed using descriptive statistics (frequency and percentage) by treatment group. Inferential 
treatment group comparisons will be assessed via pairwise tests of independent binomial 
proportions (dasiglucagon versus placebo, GlucaGen versus placebo). 
Time to first rescue infusion of IV glucose during the hypoglycemic clamp procedure will be 
evaluated using a KM time to event statistical model, with treatment group and injection site 
(abdomen, buttock, or thigh) as stratification factors. Differences between the KM curves 
(dasiglucagon versus placebo, GlucaGen versus placebo) will be evaluated inferentially using 
pairwise two-sided stratified log-rank tests. If the endpoint is never met, the time of the last 
plasma glucose measurement will be the censoring time. 
Vital signs, physical examination, 12-lead ECG and local tolerability data will be summarized 
using descriptive statistics. 
4.9.5 Vital signs 
The following vital signs will be summarized for the Safety analysis set subjects: 
• 
Systolic and diastolic blood pressure (mmHg) 
• 
Pulse rate (beats/min) 
• 
Body temperature (
0 C) 
At the dosing visit, measurements will be taken prior to the start of the insulin-induced 
hypoglycemic procedure (within 30 minutes) and at 30, 90, and 300 minutes after dosing. The 
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actual time of the assessment should not deviate from the nominal time by more than ±10 
minutes. In addition to the pre-specified assessments, blood pressure and pulse may be assessed 
at any time during the trial at the discretion of the investigator. 
Vital signs data and changes from baseline in vital signs will be summarized by treatment group 
and time point where appropriate using standard descriptive statistics. 
4.9.6 
Physical examination 
A complete physical examination will be perfonned at Screening and Follow-up visits; after 
study drng has been administered any clinically significant changes in physical examination 
findings will be recorded as AEs. Physical examination data will be summarized using 
descriptive statistics and will be provided in the listings. 
4.9.7 
12-Lead ECG 
At the dosing visit, measurements will be taken prior to the start of the insulin-induced 
hypoglycemic procedure (within 30 minutes), and at 20, 35, 45, 60 and 300 minutes after dosing. 
The actual time of the assessment should not deviate from the nominal time by more than ±5 
minutes. 
The quantitative ECG assessments (heart rate, PQ, QRS, QT, QTcB) will be summarised using 
descriptive statistics for actual values and changes from baseline to each time point by treatment 
and time. 
4.9.8 
Local tolerability 
At the dosing visit, immediately p1ior to treatment administration, it should be verified that the 
injection site is normal. To ensure all injection site assessments are performed at the injection 
site, the site will be marked with a pen prior to injection. Local tolerability at the injection site 
will be assessed at 30, 120, and 300 minutes after dosing. The actual time of the assessment 
should not deviate from the nominal time by more than± 10 minutes. 
The local tolerability at the injection site will be evaluated by means of the following 
assessments: 
spontaneous 
pain, 
pain 
on 
palpation, 
itching, 
redness, 
edema, 
induration/infiltration, and other. Each of these assessments will be reported on a scale of 0 
(none), l (mild), 2 (moderate), and 3 (severe). The evaluation and the actual time of the 
assessment will be recorded. Local tolerability data will be summarized using descriptive 
statistics by injection site and overall, and will be provided in the listings. 
4.10 Adjustment for Covariates 
The primary endpoint will be additionally analyzed using a Cox Proportional Hazards (CPH) 
time to event statistical model. The CPH model will be used for inferences, with treatment group 
and injection site (abdomen, buttock, or thigh) modelled as categorical effects, and baseline 
plasma glucose modelled as a continuous covariate. The treatment group rate ratios, active 
versus placebo, will be estimated together with the 95% confidence intervals, and pairwise 
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treatment group inferences (dasiglucagon vs placebo, GlucaGen vs placebo) will be evaluated 
using two-sided likelihood ratio tests. 
The key secondary endpoints of plasma glucose changes from baseline changes from baseline 
within 30 minutes, within 20 minutes, within 15 minutes, and within I 0 minutes after study drug 
injection or at the time ofrescue, will be analyzed with the plasma glucose changes from baseline 
at rescue carried forward in those subjects who require rescue IV glucose before plasma glucose 
mg/dL recovery. Each of these changes from baseline variables will be analyzed using an 
Analysis of Covariance model, with treatment group modelled as a fixed effect and with the 
baseline plasma glucose modelled as a covariate. The dasiglucagon versus placebo treatment 
group difference will be evaluated inferentially as a least squares means contrast, using a two-
sided t-test at the 0.05 significance level, first for the 30 minute changes from baseline, and then 
sequentially for the 20 minute, 15 minute, and 10 minute variables, until the first failure to reject. 
4.10.l Center/Study Site Effects 
This study will be conducted in 4 countries at 5 study sites. Subjects from all study sites will be 
pooled. No adjustment for study site will be carried out. 
Number of 
Total 
Re2ion 
Country 
Study Sites 
Subjects 
Europe 
Austria 
1 
80 
N. America 
Canada 
1 
40 
Europe 
Germany 
2 
35 
N. America 
USA 
1 
30 
4.11 Protocol Violations or Deviations 
Before data are released for statistical analysis, a treatment-masked review of all data will take 
place to identify protocol deviations that may potentially affect the results. This review will be 
perfom1ed without revealing to which trial product the subjects were assigned. The masking of 
the trial products will be maintained for everyone involved in allocating subjects to the analysis 
sets until data are released for statistical analysis. Furthermore, spurious outliers will be 
evaluated. In addition, protocol deviations that may potentially affect the results will be 
identified and it will be evaluated if subjects and/or data should be excluded from the analysis. 
Protocol deviations will be classified as minor or major in a consistent way. Major deviations 
from the protocol may lead to the exclusion of a subject from the PP set. 
Major protocol deviations may include deviations related to trial inclusion or exclusion criteria, 
conduct of the trial, subject management or subject assessment. Unless explicitly decided 
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otherwise during the masked data review, the following will be considered major protocol 
deviations: 
Violation of one or more major inclusion/exclusion criteria 
Intenuption of administration of trial product 
Significant deviation from time windows 
Incorrect treatment allocation 
Missing primary endpoint. 
The violation of several major inclusion/exclusion criteria or the complete absence of efficacy 
data might lead to exclusion of the subject from Full analysis set. In that case, the decision should 
be taken at the blinded data review meeting, and the exclusion from efficacy analysis justified 
in the signed notes of the meeting. 
Obviously erroneous data points may be excluded from the analyses or reanalyzed (in case of 
e.g., serum concentrations). The decision to reanalyze or exclude data points from the statistical 
analysis is the joint responsibility of the sponsor and the trial statistician. 
The subjects or observations to be excluded and the reason for their exclusion will be 
documented and signed by those responsible prior to database release. The documentation will 
be stored together with the remaining trial documentation. The subjects and observations 
excluded from analysis sets, and the corresponding reasons, will be described in the clinical trial 
report. 
4.12 Subject Withdrawals and Missing Data 
Failure is defined for the primary time-to-recovery endpoint as censored with time to recovery 
set to the maximum follow-up time, here 45 minutes. Individuals will be set to ' failure' in case 
of receiving rescue IV glucose, discontinuation due to treatment or discontinuation in connection 
with the induced hypoglycemia state. Only if an intermediate assessment is missing independent 
of rescue treatment, study treatment or an adverse event including hypoglycemia, a linear 
interpolation can be applied to impute a missing value. 
The same construct of imputation for missing values applies for the analysis of the plasma 
glucose AUC0-30min. 
In the case of subject withdrawal, no imputation of values for PK or PD measurements will be 
done. Analyses will be done on valid cases only, i.e., no imputation techniques such as last 
observation carried forward will be applied. 
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Methods for Handling Missing Dates 
All analyses and descriptive summaries will be based on the observed data. For the subject data 
listings, no imputation of incomplete dates will be applied. The listings will present the 
incomplete dates without any change. 
Missing or Partial Death Dates 
• 
If the entire date is missing, the death date will be imputed as the day after the date of 
last contact. 
• 
lf the day or both day and month is missing, the death date will be imputed to the 
maximum of the full (non-imputed) day after the date of last contact and the following: 
If day is missing, day will be 151 of the month 
If both day and month are missing, death month and day will be January I si. 
Missing Dates in Adverse Events/Concomitant Therapies 
Dates missing the day or both the day and month of the year will adhere to the following 
conventions: 
• 
The missing day of onset of an AE or start date of a therapy will be set to: 
first day of the month that the event occurred, if the onset yyyy-mm is after the yyyy-
mm of first study treatment 
the day of the first study treatment, if the onset yyyy-mm is the same as yyyy-mm of 
the first study treatment 
the date of informed consent, if the onset yyyy-mm is before the yyyy-mm of the first 
treatment 
• 
The missing day of resolution of an AE or end date of a therapy will be set to: 
the last day of the month of the occurrence. If the subject died in the same month, 
then set the imputed date as the death date 
• 
If the onset date of an AE or start date of a therapy is missing both the day and month, 
the onset date will be set to: 
January I of the year of onset, if the onset year is after the year of the first study 
treatment 
the date of the first treatment, if the onset year is the same as the year of the first 
study treatment 
the date of informed consent, if the onset year is before the year of the first 
treatment 
• 
If the resolution date of an AE or end date of a therapy is missing both the day and month, 
the date will be set to: 
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December 31 of the year of occurrence. If the subject died in the same year, then set 
the imputed date as the death date 
• 
If date is completely missing, then no imputation will be done and the event will be 
considered as treatment emergent (for AEs) or concomitant (for medications) unless the 
end date rules out the possibility. 
4.13 Deviations from SAP 
Any deviations from the statistical analysis plan will be described and justified in the final 
clinical trial report. 
4.14 Changes in Conduct or Planned Analyses from the Protocol 
There have been no changes in planned analyses from those defined in the protocol. 
If any analysis, definitions, or summaries that have been written in the protocol have been 
amended for the purpose of the analysis, these will be described and justified in the final clinical 
trial report. This may include: 
• 
Changes to populations 
• 
Additional analysis 
• 
Changes to equations 
• 
Additional data to analyze 
4.15 Algorithms/SAS Codes 
• 
Tables that need descriptive statistics - continuous variables: 
PROC UNIVARIATE DATA =dset NOPRJNT; 
VAR var] var2 var3 ... varn; 
BY byvar; (optional) 
OUTPUT OUT=outname 
N=n MEAN=mean MIN=min MAX=max MEDJAN=median STD=std; 
RUN; 
• 
Tables that need frequency counts: 
PROC FREQ DATA =dset NO PRINT; 
BY byvar; (optional) 
TABLES var] *var2; 
OUTPUT OUT=outname; 
RUN; 
• 
Tables that need exact or asymptotic 95% Cls between groups for proportions: 
PROC FREQ DA TA =dset; 
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BY byvar; (optional) 
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TABLES varl * var2 I MEASURES JUSKDIFF ALPHA=0.05; 
EXACT MEASURES; 
RUN; 
Notes: I Estimates are computed for 2x2 tables only 
CHILTI RN 
2 This code also gives exact 95% Cls within group for binomial proportions 
• 
Tables that need 95% Cls within group for binomial proportions: 
PROC FREQ DA TA =dset; 
BY byvar; (optional) 
TABLES varl; 
EXACT BINOMIAL; 
RUN; 
• 
Tables that need 95% Cls within group for continuous variables: 
DATA outdata; 
SET outname; 
LCL=mean-(TJNV(0.975,n-l)*(std/SQRT(n))); 
UCL=mean+(T!NV(0.975,n-l)*(std/SQRT(n))}; 
RUN; 
• 
Tables that require generalised linear modelling, including 95% Cls: 
PROC GENMOD DATA= dset; 
CLASS class variables; 
MODEL 'Responses I Al/empts'= <efect> < treatment> IDJST=B!N; 
LSMEANS treatment I DIFF CL; 
ESTJMATE Drug 1 vs. Placebo' treatment 1 0 0 - 1 I EXP; 
ESTIMATE Drug 2 vs. Placebo' treatment 0 1 0 -1 /EXP; 
ESTIMATE 'Drug 3 vs. Placebo' treatment 0 0 I -I /EXP; 
BY byvar; (optional) 
WHERE wherever; (optional) 
RUN; 
Note: (Treatment order: I =drug 1.2= drug 2.3= drug 3. etc ... 5= placebo) 
• 
Tables that require analysis of (co)variance and 95/90% Cls between arms for 
continuous variables: 
PROC GLM DATA= dset OUTSTAT=outset; 
CLASS class variables; 
MODEL response = <effect> <treatment> I SOLUTION; 
LSMEANS treatment I STD ERR PD/FF CL; *ADD: ALPHA =0.1 for 90% Cls; 
ESTIMATE 'Tl - T2' treatment 1 -1 O; *or 'T2- Tl' treatment -/ 1 0 if applic.; 
EST/MA TE 'T 1 - T3' treatment 1 0 -1; 
ESTIMATE 'T2 - T3' treatment 0 1 -! ; 
BY byvar; (optional) 
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WHERE wherever; (optional) 
RUN; 
QUIT; 
Note: (Treatment order: I =drug 1,2= drug 2.3= drug 3, etc .. . 5= placebo) 
• 
Tables that present Fisher's Exact or CMH: 
PROC FREQ DATA=dset NOPRINT; 
BY byvar; (optional) 
TABLES varl *var2/CMH score=MODRIDIT EXACT; 
OUTPUT OUT=outname CMH EXACT; 
RUN; 
• 
Tables that need to use WILCOXON: 
PROC NPARJ WAY DATA=dset WILCOXON; 
CLASS class variables; 
VAR variable; 
RUN; 
• 
Tables that need a regression slope and 90 % CI: 
PROC MIXED DA TA =dset NOCLPRINT NOITPRINT; 
MODEL lnauc=lntrt IS CL ALPHA=. l; 
MAKE 'SOLUTIONF' OUT=outstats NOPRINT; 
RUN; 
• 
Tables that need number of events/censored and probabilities of failure/survival at cut 
off times: 
PROC LIFETEST DATA=dset OUTSURV=LJFE METHOD=KM JNTERVALS=l2, 24,; 
TIME duration *censor (0 or l); 
ID subject; 
STRATA treatment; 
RUN; 
• 
Tables that need life table with estimates of survival, with Cls and log rank test: 
PROC LIFETEST DATA=dset OUTSURV=LIFE METHOD=KM; 
TIME duration*censor (0 or l); 
ID subject; 
STRATA treatment; 
RUN; 
• 
The calculation of AUC using trapezoidal method (Area of segment (ab) = (b-a)*(f(a) + 
f(b))/2: 
DATA auc (KEEP= pid area); 
RETAIN A FA AREA O; 
SETdset; 
BY pid time; 
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IF FIRST.pid THEN DO; 
A = TIME; 
FA = <FEVI>; 
AREA = O; 
END; 
ELSE DO; 
Protocol 
Number: 
AREA = AREA+ (TIME-A)*((FA +<FEVJ >)/2); 
A 
=TIME; 
FA =VALUE; 
END; 
IF LAST.pid THEN OUTPUT; 
RUN; 
5 Tables and Listings 
5.1 Table Format 
ZP4207-16137 
All output will be produced using SAS version 9.4 or a later version. 
CHll 1CRN 
In the top left portion of each table/listing, a table/listing number followed by the title of the 
table/listing will be presented. After the title line, optional sub-title or population information 
can be presented. Horizontal lines will appear before and after the column heading of the 
table/listing. Footnotes will be put under the main body of text at the bottom of the page. 
The sponsor name, protocol number, programmers User ID, status of the table/listing (i.e. draft 
or final) and SAS program name will appear bottom left in a string and the page number will 
appear on the bottom right comer of each table/listing. The date and time of creation of 
table/listing will appear bottom left under the sponsor name. The source listing number will 
appear bottom left. 
A landscape layout is proposed for both table and listing presentations. 
The lefl and right margins of all tables and listings will be a minimum of 2.1 cm from the left 
and I .9cm from the right. The top and bottom margins will be a minimum 2.92cm. Header and 
footer will be both 1.27 cm. 
There is no special requirement of.font type and size, but an 8-point font size for tables and 7or 
8-point for listings is proposed using Courier New font. A maximum SAS line size=l4 l and 
page size=44 for 8-point font size, and line size= l61 and page size=50 for 7-point will be used 
so as to fit on both UK and US paper sizes. 
In a listing, in the case that a subject" s record has been continued to the next page, an appropriate 
identification (e.g., the subject ID number) must be presented at the beginning of that page. 
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5.2 Conventions 
Protocol 
Number: 
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Unless othexwise specified, in summa1y tables of continuous variables, the mtrumum and 
maximum values will be displayed to the same number of decimal places as the raw data, the 
mean and median will be presented to one extra decimal place compared to the raw data, and the 
standard deviation will be displayed to two extra decimal places compared to the raw data. PK 
and PD data will be repo11ed with 3 significant digits. Wherever possible data will be decimal 
aligned. 
Unless othexwise specified frequency tabulations will be presented by number and percentage, 
where the percentage is presented in brackets to I decimal place. 
P-values, if applicable, will be presented to 3 decimal places. If a p-value is less than 0.05 but is 
greater than or equal to 0.01, then an asterisk (*) will be added next to this value. If a p-value is 
less than 0.01 but is greater than or equal to 0.001 , then two asterisks(**) will be added next to 
this value. Finally, if the p-value is less than 0.00 I then three asterisks (***) will be added next 
to this value and it will be presented as <0.001. If the rounded result is a value of 1.000, it will 
be displayed as >0.999. Any date information in the listing will use the date9. fonnat, for 
example, 060CT2017. In the listing, a unit associated with a variable will be presented only 
once within parentheses either below or next to that variable in the heading portion. If a 
parameter has multiple units, each unit will be displayed only once, as applicable. 
All tables will have their source listing referenced in a footnote. Listings should be sorted by 
treatment group, subject and visit and have the source data received by data management 
referenced in a footnote. All tables and listings will be converted into Microsoft Word documents 
and collated into two complete documents. 
The following conventions apply for calculation of PK parameters: 
Any BLQ (<LLOQ) values that occur before the first quantifiable concentration will be 
replaced with zero. 
• If a BLQ (<LLOQ) value occurs after a quantifiable concentration in a profile and is followed 
by a value of LLOQ or above, then the BLQ (<LLOQ) will be replaced with LLOQ/2. 
• 
If two BLQ ( <LLOQ) values occur in succession, the profile will be deemed to have 
terminated at the final quantifiable concentration and subsequent LLOQ or above values will 
be treated as 'missing'. 
5.3Tables 
5.3.1 Section 14.1: Baseline and Demographic Data 
Table 14. l . l 
Patient Disposition 
Table 14.1.2 Discontinuation and Drop-outs 
Table 14.1.3 Demographic and Baseline Characteristics 
Table 14.1.4 Medical History/Concomitant Illness 
Table 14.1.5 Summary of Diabetes Duration 
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Table 14.1.6 Concomitant Medication 
Protocol 
Number: 
Table 14.1.7 Histo1y of Alcohol/Drug Abuse 
5.3.2 
Section 14.2: Efficacy Analysis Data 
Table 14.2.l 
Summary of Plasma Glucose Concentrations 
Table 14.2.2 Summary of Plasma Dasiglucagon Concentrations 
Table 14.2.3 Summary of Plasma GlucaGen Concentrations 
5.3.3 
Section 14.2: Primary Confirmatory Endpoint 
Table 14.2.4 Time to Plasma Glucose Recovery 
CHll l C-Rf\J 
ZP4207-16137 
Table 14.2.5 Time to Plasma Glucose Recovery without Censoring Rescue IV Glucose 
Subjects 
Table 14.2.6 Time to Plasma Glucose Recovery with Censoring at the Time of Rescue IV 
Glucose Administration 
5.3.4 Section 14.2: Secondary Endpoints 
Table 14.2.7 Plasma Glucose Recovery within Defined Time Points 
Table 14.2.8 Plasma Glucose Changes from Baseline within Defined Time Points 
Table 14.2.9 Time to First Plasma Glucose Concentration of >/=70 mg/dL 
5.3.5 Section 14.2: Exposure (PD/PK) Endpoints 
Table 14.2.10 Summary Statistics of Phamrncodynamics Endpoints 
Table 14.2.11 
Statistical Analysis of Phannacodynamics Parameters 
Table 14.2.12 Summary Statistics of Phannacokinetic Endpoints 
Table 14.2.13 Statistical Analysis of Phannacokinetic Parameters 
5.3.6 Section 14.2: Exploratory 
Table 14.2.14 Time to First Plasma Glucose Concentration of >/=70 mg/dL or Increase of 
>/= 20 mg/dL within 30 Minutes 
Table 14.2.15 Summary Statistics of Plasma Insulin Response 
5.3.7 
Section 14.3: Safety Analysis Data 
5.3.7.1 
Adverse events 
Table 14.3. 1.1 
Overview of Incidence of TEAEs 
Table 14.3.1.2 
Summary ofTEAEs by MedDRA System Organ Class and Preferred Term 
Table 14.3.1.3 
Summary of TEAEs by Severity, Relationship, Seriousness, and Outcome 
Table 14.3 .1 .4 Summary of TE AES Is by Severity, Relationship, Seriousness and Outcome 
5.3.7.2 
Laboratory results 
Table 14.3.2.1 Summary of Hematology Data -
Actual and Change from Pre-Dose 
Measurement 
Table 14.3.2.2 
Summary of Biochemistry Data -
Actual and Change from Pre-Dose 
Measurement 
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Table 14.3.2.3 
Summary of Coagulation Data -
Actual and Change from Pre-Dose 
Measurement 
Table 14.3.2.4 Summary of Urinalysis Data -
Actual and Change from Pre-Dose 
Measurement 
Table 14.3.2.5 Frequencies of Urinalysis Data 
Table 14.3.2.6 Shifts Pre-Dose versus Post-Dose of Interpretation of Hematology Data 
Table 14.3.2. 7 
Shifts Pre-Dose versus Post-Dose of Interpretation of Biochemistry Data 
5.3. 7.3 
Vital signs 
Table 14.3.3.1 
Summary of Vital Signs Data -
Actual and Change from Pre-Dose 
Measurement 
Table 14.3.3.2 Vital Signs - Overall Interpretation 
Table 14.3.3.3 
Shifts Pre-Dose versus Post-Dose of Interpretation of Vital Signs Data 
5.3.7.4 
ECG 
Table 14.3.4.1 
Summary of 12-Lead ECG Data -
Actual and Change from Pre-Dose 
Measurement 
12-Lead ECG - Overall Interpretation 
Table 14.3.4.2 
Table 14.3.4.3 
Shifts Pre-Dose versus Post-Dose oflnterpretation of 12-Lead ECG Data 
5.3.7.5 
Other safety data 
Table 14.3.5 
Physical Examination 
Table 14.3.6 
Summary of Local Tolerability 
Table 14.3.7 
Frequencies of Urine Drug Screen Data 
Table 14.3.8 
Frequencies of Alcohol Breath Test Data 
Table 14.3.9 
Summary of Pregnancy Tests 
Table 14.3.10 
Frequencies of Anti-Drug Antibodies Test Data 
Table 14.3.11 
Summary of Total Insulin and Glucose Dose Required for Insulin-Induced 
Hypoglycemic Clamp Procedure 
Table 14.3.12 
Summary of Time to First Rescue Jnfusion of IV Glucose During the 
Hypoglycemic Clamp Procedure 
5.4 Listings 
Listing 16.1 .1 
Listing 16.2. 1.1 
Listing 16.2.1.2 
Listing 16.2.1.3 
Listing 16.2.2 
Listing 16.2.3.1 
Listing 16.2.3.2 
Listing 16.2.4.1 
SAP Version 1, Final 1 
Randomization 
Patient Disposition 
Dosing Day Exclusion Criteria 
Important Protocol Deviations 
Demographics and Baseline Characteristics 
Medical History/Concomitant Illness 
Concomitant Medication 
Adverse Events 
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Listing 16.2.4.2 
Listing 16.2.5.1 
Listing 16.2.5.2 
Listing 16.2.5.3 
Listing 16.2.5.4 
Listing 16.2.6 
Listing 16.2. 7 
Listing 16.2.8 
Listing 16.2.9 
Listing 16.2.10 
Listing 16.2.11 
Listing 16.2.12 
5.5Figures 
Adverse Events of Special Interest 
Abnormal Laboratory Results: Hematology 
Abnormal Laboratory Results: Biochemistry 
Abnormal Laborato1y Results: Coagulation 
Abnormal Laboratory Results: Urinalysis 
Vital Signs 
12-Lead ECG 
Physical Examination 
Local Tolerability 
Urine Drug Screen 
Alcohol Breath Test 
Pregnancy Test 
Individual PD Plasma Glucose Profile of Each Treatment Group 
Individual PD Plasma Glucose Profile by Treatment Group 
Individual PD Plasma Glucose Profile of Each Subject 
Individual PK Dasiglucagon Profile of Each Treatment Group 
Individual PK Dasiglucagon Profile by Treatment Group 
Individual PK Dasiglucagon Profile of Each Subject 
Individual PK GlucaGen Profile of Each Treatment Group 
Individual PK GlucaGen Profile by Treatment Group 
Individual PK GlucaGen Profile of Each Subject 
Mean Dasiglucagon/GlucaGen Profiles 
CHIL 1 FRN 
Figure 14.2.X.X 
Figure 14.2.X.X 
Figure 14.2.X.X 
Figure 14.2.X.X 
Figure 14.2.X.X 
Figure 14.2.X.X 
Figure 14.2.X.X 
Figure 14.2.X.X 
Figure 14.2.X.X 
Figure 14.2.X.X 
Figure 14.3.X.X 
Box-and-Whisker Plots of Pharmacodynamics Endpoints vs. 
Treatment 
Figure 14.4.X.X 
Box-and-Whisker Plots of Pharmacokinetic Endpoints 
vs. 
Treatment 
5.6 Appendices 
All analyses will require raw SAS output, but may or may not be required to be collated into an 
appendix document. 
5. 7 References 
1. 
Allison, Paul D. 20 I 0. Survival Analysis Using SAS®: A Practical Guide, Second Edition. 
Cary, NC: SAS Institute Inc. 
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