Conditions: Hypoglycemia Diabetes Mellitus, Type 1
Keywords: glucagon
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 45
Healthy volunteers: no

Brief summary:
A randomized, double-blind, parallel-group 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

Detailed description:
Subjects were randomized in a ratio of 3:1 to receive a single subcutaneous dose of dasiglucagon (0.6 mg) or matching placebo. Randomization also determined whether the subject was to be injected in the buttock or deltoid. During the dosing visit (Visit 2), hypoglycemia was induced by a fast-acting insulin glulisine (Apidra®) infusion, and trial drug was administered when the subject's plasma glucose level was 45-60 mg/dL (2.5-3.3 mmol/L). During the insulin-induced hypoglycemia, pharmacokinetic and pharmacodynamic samples were drawn, and plasma glucose levels were monitored closely at site for safety reasons. A rescue glucose infusion could be administered to ameliorate persistent hypoglycemia. Subjects were followed for at least 28 days after dosing to perform an adequate immunogenicity evaluation of treatment.

Arms:
arm 1: single fixed dose (subcutaneous injection) of dasiglucagon arm 2: single fixed dose (subcutaneous injection) of placebo

Interventions: intervention 1: Dasiglucagon intervention 2: Placebo
Intervention descriptions: intervention 1: Glucagon analogue intervention 2: Placebo for dasiglucagon

Protocol document:
Prot_000.pdf: 
Appendix 16.1.1
Protocol and Protocol Amendments 
Clinical Study Report Appendix 16.1.1 
Drug Substance Dasiglucagon 
Protocol Number ZP4207-17145 


 TABLE OF CONTENTS 
Document 
Date of Issue 
Clinical Study Protocol v2.0  
Protocol Approval v2.0  
04 July 
04 July 2018 
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Clinical Study Report Appendix 16.1.1 
Drug Substance Dasiglucagon 
Protocol Number ZP4207-17145 


Clinical trial protocol
A randomized, double-blind, parallel-group 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
Sponsor:
Zealand Pharma A/S
Smedeland 36
2600 Glostrup, Denmark
Sponsor protocol number:
ZP4207-17145
IND number:
127866
Development phase:
Trial drug name:
3
Dasiglucagon
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.
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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Table of contents
Table of contents........................................................................................................2 
List of tables...............................................................................................................5 
List of figures .............................................................................................................6 
List of abbreviations and definition of terms .........................................................7 
1 
Protocol synopsis................................................................................................8 
2 
Introduction......................................................................................................11 
2.1 
Background ...................................................................................................................11 
2.1.1 
Hypoglycemia ..................................................................................................11 
2.1.2 
Glucagon ..........................................................................................................11 
2.1.3 
Dasiglucagon....................................................................................................12 
2.1.3.1 
Pharmacokinetics and pharmacodynamics of dasiglucagon............12 
2.1.3.2 
Safety of dasiglucagon.....................................................................13 
2.2 
Rationale........................................................................................................................13 
2.3 
Risk-benefit assessment ................................................................................................13 
2.3.1 
Nonclinical experience.....................................................................................13 
2.3.2 
Clinical experience...........................................................................................13 
3 
Trial objectives and endpoints........................................................................15 
4 
Overall design and plan of the trial................................................................16 
4.1 
Overview .......................................................................................................................16 
4.2 
Justification of the trial design ......................................................................................17 
4.2.1 
Design and parameters.....................................................................................17 
4.2.2 
Drug, route, dosage and treatment duration.....................................................17 
4.3 
End of trial definition ....................................................................................................17 
5 
Trial population................................................................................................18 
5.1 
Rationale for trial population ........................................................................................18 
5.2 
Planned sample size and number of trial sites...............................................................18 
5.3 
Inclusion criteria............................................................................................................18 
5.4 
Exclusion criteria...........................................................................................................19 
5.5 
Dosing day exclusion criteria........................................................................................20 
5.6 
Premature discontinuation of subjects or trial...............................................................21 
5.6.1 
Subject discontinuation....................................................................................21 
5.6.2 
Site discontinuation..........................................................................................21 
5.6.3 
Trial termination ..............................................................................................21 
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5.7 
Subject identification and randomization......................................................................22 
5.8 
Screening failures..........................................................................................................22 
6 
Trial drug..........................................................................................................23 
6.1 
Identity ..........................................................................................................................23 
6.2 
Dosing and administration ............................................................................................23 
6.3 
Packaging, labelling and storage...................................................................................24 
6.3.1 
Packaging and labelling ...................................................................................24 
6.3.2 
Trial drug storage and stability ........................................................................24 
6.4 
Blinding and breaking the blinding code ......................................................................24 
6.5 
Drug accountability.......................................................................................................25 
6.6 
Drug exposure and compliance.....................................................................................25 
6.7 
Concomitant therapy .....................................................................................................25 
6.7.1 
Previous and concomitant medication .............................................................25 
6.7.2 
Prohibited medication ......................................................................................26 
6.8 
Rescue medication.........................................................................................................26 
7 
Parameters and methods of assessment.........................................................27 
7.1 
Efficacy parameters.......................................................................................................27 
7.1.1 
Pharmacodynamic measurements....................................................................27 
7.1.2 
Pharmacokinetic measurements.......................................................................27 
7.2 
Safety parameters ..........................................................................................................27 
7.2.1 
Adverse events and pregnancies ......................................................................27 
7.2.1.1 
Definitions........................................................................................27 
7.2.1.2 
Collection, recording and reporting of adverse events ....................30 
7.2.1.3 
Follow-up of adverse events ............................................................31 
7.2.1.4 
Hypoglycemia ..................................................................................32 
7.2.1.5 
Pregnancy.........................................................................................33 
7.2.1.6 
Precautions.......................................................................................33 
7.2.1.7 
Safety Committee.............................................................................33 
7.2.2 
Safety laboratory parameters ...........................................................................33 
7.2.2.1 
Clinical chemistry, hematology and urinalysis................................33 
7.2.2.2 
Pregnancy test ..................................................................................34 
7.2.2.3 
Plasma glucose measurements for safety.........................................34 
7.2.2.4 
Insulin measurements.......................................................................34 
7.2.2.5 
Other safety laboratory tests ............................................................35 
7.2.3 
Vital signs ........................................................................................................35 
7.2.4 
Physical examination .......................................................................................35 
7.2.5 
Electrocardiogram............................................................................................35 
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7.2.6 
Local tolerability..............................................................................................36 
7.2.7 
Immunogenicity ...............................................................................................36 
7.3 
Demographics and baseline characteristics...................................................................37 
7.3.1 
Demographics and body measurements...........................................................37 
7.3.2 
Concomitant illness and medical history .........................................................37 
7.3.3 
Diabetes diagnosis and current treatment ........................................................38 
7.3.4 
Concomitant medication ..................................................................................38 
8 
Trial conduct ....................................................................................................39 
8.1 
Schedule of procedures .................................................................................................39 
8.2 
Procedures by visit ........................................................................................................40 
8.2.1 
Visit 1 (screening)............................................................................................40 
8.2.2 
Instructions to subjects prior to the dosing visit (Visit 2)................................41 
8.2.3 
Visit 2 (dosing visit).........................................................................................41 
8.2.3.1 
Hypoglycemic clamp procedure and administration of trial drug ...43 
8.2.3.2 
Hypoglycemia rescue provisions .....................................................44 
8.2.4 
Visit 3 (follow-up visit)....................................................................................45 
9 
Statistical methods ...........................................................................................47 
9.1 
Determination of sample size........................................................................................47 
9.1.1 
Disposition of subjects.....................................................................................47 
9.1.2 
Analysis populations........................................................................................47 
9.2 
General considerations ..................................................................................................48 
9.3 
Demographics, baseline characteristics and concomitant medications.........................48 
9.4 
Treatment compliance...................................................................................................48 
9.5 
Efficacy analyses...........................................................................................................48 
9.5.1 
Hierarchical testing procedure .........................................................................48 
9.5.2 
Analysis of primary endpoint...........................................................................49 
9.5.3 
Analysis of key secondary endpoints...............................................................49 
9.5.4 
Analysis of secondary efficacy endpoints........................................................50 
9.5.5 
Analysis of exposure endpoints .......................................................................50 
9.6 
Safety analyses ..............................................................................................................51 
9.6.1 
Drug exposure..................................................................................................51 
9.6.2 
Adverse events .................................................................................................51 
9.6.3 
Clinical laboratory assessments .......................................................................51 
9.6.4 
Immunogenicity data........................................................................................51 
9.6.5 
Other safety parameters ...................................................................................51 
9.7 
Subject withdrawals and missing data ..........................................................................52 
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9.8 
Interim analyses.............................................................................................................52 
9.9 
Other analyses ...............................................................................................................52 
9.9.1 
Subgroup analyses............................................................................................52 
9.9.2 
Exploratory analyses........................................................................................52 
10 Ethical, legal and administrative aspects.......................................................53 
10.1 Statement of compliance ...............................................................................................53 
10.2 Quality management .....................................................................................................53 
10.2.1 Clinical monitoring ..........................................................................................53 
10.3 Data handling and record keeping.................................................................................54 
10.3.1 Data collection and management .....................................................................54 
10.3.2 Source documentation......................................................................................55 
10.3.3 Access to source data .......................................................................................55 
10.3.4 Archiving of trial records.................................................................................56 
10.4 Subject consent and participation..................................................................................56 
10.4.1 Informed consent..............................................................................................56 
10.4.2 Subject participation card.................................................................................56 
10.5 Protocol requirements ...................................................................................................57 
10.5.1 Protocol deviations...........................................................................................57 
10.6 Confidentiality...............................................................................................................57 
10.7 Other ethical and regulatory issues ...............................................................................57 
10.8 Liability and insurance..................................................................................................58 
10.9 Publication and public disclosure..................................................................................58 
10.9.1 Communication of results ................................................................................58 
10.9.2 Site-specific publication(s) by investigator(s) .................................................59 
10.9.3 Investigator access to data and review of results .............................................59 
11 Reference list ....................................................................................................60 
List of tables
Table 1 
Objectives and endpoints ..............................................................................................15 
Table 2  
Description of trial drug ................................................................................................23 
Table 3  
Quantities of ingredients in dasiglucagon and placebo injection..................................23 
Table 4  
Schedule of procedures .................................................................................................39 
Table 5  
Post-treatment blood sampling times ............................................................................44 
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List of figures
Figure 1 
Trial design....................................................................................................................16 
Figure 2 
Flow of electronic data..................................................................................................55 
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List of abbreviations and definition 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
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
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 Council for Harmonization
IEC
Independent Ethics Committee
IMP
Investigational medicinal product
IRB
Institutional Review Board
IV
Intravenous(ly)
IWRS
Interactive Web Response System
KM
Kaplan-Meier
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
SC
Subcutaneous(ly)
SUSAR
Suspected unexpected serious 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
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1
Protocol synopsis
Title of trial
A randomized, double-blind, parallel-group 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.
Trial site(s)
The trial will be conducted in up to 4 sites in North America.
Clinical phase
3
Objectives
Primary objective
x
To demonstrate superiority of dasiglucagon compared to placebo following a single 
subcutaneous (SC) 0.6 mg dose administered to subjects with T1DM with insulin-induced 
hypoglycemia.
Secondary objectives
x
To evaluate the safety, immunogenicity and PK of dasiglucagon following a single SC dose 
administered to subjects with T1DM with insulin-induced hypoglycemia as compared to placebo.
Trial design
The trial is a multi-center, randomized, parallel-group, double-blind, clinical efficacy and safety trial 
evaluating rescue treatment of insulin-induced hypoglycemia in subjects with T1DM.
Subjects will be randomized to receive a single SC dose of dasiglucagon or matching placebo in a ratio 
of 3:1. Each subject will undergo a screening visit up to 30 days prior to dosing, an overnight in-house 
visit, where dosing will take place, and a follow-up visit. Depending on the development of anti-drug 
antibodies (ADAs) during the trial, additional follow-up visits may be required.
Number of subjects
A total of 40 subjects is planned to complete the dosing visit (Visit 2), with 30 subjects randomized to 
the dasiglucagon group and 10 subjects randomized to the placebo group. It is expected that up to 46 
subjects will be randomized to have 40 subjects completing Visit 2.
.
Criteria for inclusion/exclusion
Key inclusion criteria
x
Female or male subjects with T1DM for at least 1 year; diagnostic criteria as defined by the 
American Diabetes Association (3).
x
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
x
Hemoglobin A1c <10%.
x
Aged between 18 and 75 years, both inclusive.
Key exclusion criteria
x
Previous participation in a clinical trial within the dasiglucagon in the rescue treatment of 
hypoglycemia program.
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x
Known or suspected allergy to trial drug(s) or related products.
x
History of anaphylaxis or symptoms of severe systemic allergy (such as angioedema).
x
Previous participation in this trial. Participation being defined by signing the informed consent 
document.
Investigational medicinal product(s)/trial drugs
Trial drug: dasiglucagon, liquid formulation, 1.0 mg/mL, 0.6 mL delivered in auto-injector.
Comparator drug: Placebo, liquid formulation, 0.6 mL delivered in auto-injector.
Duration of treatment
Subjects will receive one single SC dose of dasiglucagon 0.6 mg or placebo
Duration of participation in the trial
The duration for subjects in this trial, including screening, treatment and follow-up will approximately be  
8 weeks.
Key endpoints
Primary endpoint
x
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
x
Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, and within 10 
minutes after trial drug injection without administration of rescue IV glucose.
x
Plasma glucose changes from baseline at 30 minutes, at 20 minutes, at 15 minutes, and at 10
minutes after trial drug injection or at the time of rescue.
Trial procedures/assessments
A screening visit (Visit 1) will be performed up to 30 days prior to the doing in order to identify eligible 
subjects. Prior to the in-house period (Visit 2), subjects will discontinue their current insulin therapy in 
due time to ensure wash-out. Subjects will check-in to the site on Day -1 for an overnight stay. Subjects 
eligibility to continue will be checked through assessment of withdrawal criteria. On Day 1, after an 
overnight fast, and upon check of eligibility according to Dosing day criteria, subjects will be 
randomized to dasiglucagon or placebo. An insulin-induced hypoglycemic procedure will take place and
the trial drug will be administered when the subject’s plasma glucose level is 45-60 mg/dL. During the 
insulin-induced hypoglycemia, PK and PD samples will be drawn and plasma glucose levels will be 
monitored closely at site for safety reasons. After the end of the procedure, subjects may be released 
from the site if deemed safe by the investigator.
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 drugs (dasiglucagon or placebo) for evaluation of pharmacokinetics will also be 
assessed based on plasma concentration data.
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Statistical methods
From phase 2, the median time to an increase in plasma glucose of 20 mg/dL (1.1 mmol/L) of the 0.6 mg 
dose was approximately 10 minutes. For placebo-treated subjects, it is assumed that the median time to 
recovery will be more than 50 minutes. Assuming an exponential time-to-recovery distribution with 
median times of 10 and 50 minutes, a two-sided log-rank test will be able to detect a difference between 
dasiglucagon and placebo with 92% power with a follow-up time of 45 minutes at a 5% significance 
level with 40 subjects randomized 3 to 1 between active and placebo.
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. 
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 before 45 minutes 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 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) 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) using Fisher’s exact test.
The key secondary endpoints of plasma glucose changes from baseline at 30 minutes, at 20 minutes, at
15 minutes, and at 10 minutes after trial drug 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 ≥20 mg/dL (1.1 mmol/L) recovery. Each of these changes from baseline
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 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.
Further details will be included in the Statistical Analysis Plan, to be completed before database lock and 
treatment unmasking.  
The trial will be conducted in accordance with the Declaration of Helsinki and Good Clinical Practice.
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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 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). Hypoglycemia 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.
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 
13 of 64


take glucose orally. The approved glucagon dose for an adult is 1.0 mg, given by intramuscular,
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 (hereafter referred to as dasiglucagon) is in development for the 
treatment of severe hypoglycemia in 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).
A total of five clinical trials have been conducted with dasiglucagon to date. Four trials were 
conducted using a 1.0 mg/mL dasiglucagon formulation: two clinical pharmacology trials and two
phase 2 trials. The fifth trial (ZP4207-16098) was a phase 2 trial using a 4.0 mg/mL dasiglucagon 
formulation.
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.
No major differences in PD responses were observed between dasiglucagon doses under 
euglycemic or hypoglycemic conditions in the phase 2 trial ZP4207-16098. Under euglycemic
conditions, all subjects achieved an increase in PG of at least 20 mg/dL (1.1 mmol/L) within 30
minutes after dosing with 0.08 mg dasiglucagon and above, while only the 0.2 mg dose of 
marketed glucagon (Lilly Glucagon™) reached this target. Under hypoglycemic conditions, 
dasiglucagon met the target at doses of 0.2 mg and above. The PD response over the entire 
observation time of 240 minutes was significantly higher with dasiglucagon than with Lilly 
Glucagon at the same doses.
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2.1.3.2
Safety of dasiglucagon
No safety concerns were raised for dasiglucagon at the doses investigated in the clinical trials. The 
most frequently reported adverse event (AE) in each of the trials was nausea, which is a known 
side-effect following administration of glucagon and appeared at a similar frequency to marketed 
glucagon, which was used as active comparator in some of the trials. No local tolerability issues 
occurred. Injection site reactions were observed only sporadically after administration with 
dasiglucagon, placebo or marketed glucagon in trials with SC or IM administration, and all events 
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 and bradycardia were 
observed in a few healthy volunteers receiving investigational medicinal product (IMP) doses of 
at least 1.0 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) and information will be collected for this type of adverse events, to support 
the evaluation of the data at the end of the trial.
For further information on the safety of dasiglucagon, please refer to the Investigator’s Brochure 
(14).
2.2
Rationale
The aim of this phase 3 trial is to confirm the superiority of dasiglucagon for the treatment of 
hypoglycemia in subjects with T1DM as compared to placebo.
2.3
Risk-benefit assessment
2.3.1
Nonclinical 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.
2.3.2
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 clinical studies 
conducted to date with dasiglucagon. As with every novel drug substance, new and as yet 
unknown side effects may also occur.
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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. Based on the 4 clinical studies completed with dasiglucagon to date (see 
Section 2.1.3), no anti-dasiglucagon or anti-glucagon antibodies have been detected, except for 
trial ZP4207-16098, in which there was one transient low titer anti-drug antibody (ADA)
incidence showing reactivity towards both dasiglucagon and glucagon. The subject was tested 
positive for both anti-dasiglucagon and anti-glucagon antibodies at the follow-up visit, 24 days 
after the last drug exposure (titer: 35.4 and 33.8, respectively). The subject was additionally found 
to be positive for dasiglucagon and glucagon in vitro neutralizing activities. The titer of anti-
dasiglucagon activity was equal to the assay minimum required dilution. Due to the crossover 
nature of the trial, it was impossible to establish the presence of ADA in association with dosing
of dasiglucagon or glucagon. Testing performed 3.5 months after last dosing, confirmed a positive 
finding for anti-dasiglucagon antibodies (titer of 38.8), while the test was negative for anti-
glucagon antibodies. At a final follow-up visit performed 7 months after last dosing, the subject
was negative for anti-dasiglucagon antibodies. There was no evidence for altered PK, PD, or 
safety profile for this subject.
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 drugs 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 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 clinical trials conducted with 
dasiglucagon. Direct access to resuscitation equipment is ensured at the clinical trial sites.
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.
Two phase 1 and three 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. These trials have demonstrated that SC and intramuscular administration of
dasiglucagon is efficacious and well tolerated, with a safety profile that does not give rise to 
specific safety concerns (14). 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 benefit-risk balance for patients participating in the trial is considered acceptable.
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3
Trial objectives and endpoints
The objectives and endpoints of this trial are shown in Table 1.
Table 1
Objectives and endpoints
Objectives
Endpoints
Primary objective
To demonstrate superiority of 
dasiglucagon compared to placebo 
following a single SC 0.6 mg dose 
administered to subjects with T1DM
with insulin-induced hypoglycemia.
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.
Key secondary endpoints:
x Plasma glucose recovery within 30 minutes, within 20 
minutes, within 15 minutes, and within 10 minutes after trial
drug injection without administration of rescue IV glucose.
x Plasma glucose changes from baseline at 30 minutes, at 20
minutes, at 15 minutes, and at 10 minutes after trial drug 
injection or at the time of rescue.
Secondary objectives
Secondary endpoints (other)
To evaluate the safety, 
immunogenicity and PK of 
dasiglucagon following a single SC 
dose administered to subjects with 
T1DM with insulin-induced 
hypoglycemia as compared to 
placebo.
Clinical efficacy (PD) endpoints:
x Time to first plasma glucose concentration ≥70 mg/dL (3.9 
mmol/L) without administration of rescue IV glucose.
x Plasma glucose response as area under the effect curve 
(AUE) above baseline from time zero to 30 minutes, 
AUE0-30min.
Exposure (PK) endpoints:
x Area under the drug concentration curve from time zero to 90 
minutes, AUC0-90min
x Maximum plasma drug concentration (Cmax).
x Time to maximum plasma drug concentration (tmax).
Safety endpoints: 
x Adverse events.
x Clinical laboratory assessments (biochemistry, hematology, 
urinalysis).
x Local tolerability.
x Number of rescue infusions of IV glucose after trial drug 
administration.
x Time to first rescue infusion of IV glucose after trial drug 
administration.
Immunogenicity endpoint:
x Occurrence of anti-drug-antibodies.
Tertiary/Exploratory objective(s)
Tertiary/Exploratory endpoints
Tertiary/exploratory objective(s) 
serve as a basis for explaining or 
supporting findings of primary 
analyses and for suggesting further 
hypotheses for later research.
x Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or 
increase of ≥20 mg/dL (1.1 mmol/L) within 30 minutes after 
trial drug injection without administration of rescue IV 
glucose.
x 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
The trial is a multi-center, randomized, parallel-group, double-blind, clinical efficacy and safety 
trial evaluating rescue treatment of insulin-induced hypoglycemia in subjects with T1DM. The 
design of the trial is shown in Figure 1.
The subjects will be randomized 3:1 to receive a single fixed SC dose of dasiglucagon or placebo 
and will be followed for at least 28 days after dosing.
Prior to the dosing visit (Visit 2), the subjects will discontinue their current insulin therapy in due 
time to ensure wash-out. At the dosing visit, hypoglycemia will be gradually induced by a 
controlled IV infusion of a fast-acting insulin, i.e. insulin glulisine (Apidra®), targeting a plasma 
glucose level of 55 mg/dL (3.1 mmol/L). Trial drug will be administered when the subject’s 
plasma glucose level is 45-60 mg/dL (2.5-3.3 mmol/L).
During the insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at site 
for safety reasons.
Figure 1
Trial design
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4.2
Justification of the trial design
4.2.1
Design and parameters
In order to avoid bias in subject selection and in the evaluation of clinical assessments, subjects 
will be randomly assigned 3:1 to either dasiglucagon or placebo and the trial will be conducted in 
a double-blinded manner.
A superiority trial design is used because the aim is to show that treatment with dasiglucagon is an 
effective treatment compared to placebo.
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 following a single SC dose, the primary endpoint and 
secondary efficacy endpoints involve the measurement of plasma glucose concentrations at 
different time points.
Subjects will be followed for at least 28 days after dosing in order to perform an adequate 
immunogenicity evaluation of treatment. Depending on the development of ADAs during the trial, 
additional follow-up visits may be performed.
4.2.2
Drug, route, dosage and treatment duration
Dasiglucagon will be administered as a single fixed dose of 0.6 mg independent of body weight 
because this is the intended therapeutic dosing regimen in the emergency treatment of 
hypoglycemia. Data from the trials 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.
Dasiglucagon or placebo will be administered in the buttock or deltoid by SC injection, as this is 
one of the intended routes of administration for dasiglucagon, besides intramuscular and IV.
4.3
End of trial definition
A subject is considered to have completed the trial if he or she has completed the 28 days follow-
up visit or the last scheduled procedure shown in Table 4.
The end of the trial is defined as completion of the last visit or procedure shown in the Table 4.
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5
Trial population
5.1
Rationale for trial population
Dasiglucagon is in development for the treatment of severe hypoglycemia in patients with 
diabetes mellitus. In the present trial, subjects with T1DM are selected to avoid the endogenous 
glucagon counter-regulatory response to insulin induced hypoglycemia that may be 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 sites
A total of 40 subjects is planned to complete the dosing visit (Visit 2). It is expected that up to 46
subjects will be randomized to have 40 subjects completing Visit 2.
The trial will be conducted at up to 4 sites in North America.
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%.
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)
iv) Condom with spermicide
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).
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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 are not eligible:
1. Previous participation in a clinical trial within the dasiglucagon in the rescue treatment of 
hypoglycemia program.
2. Known or suspected allergy to trial drug(s) or related products.
3. History of anaphylaxis or symptoms of severe systemic allergy (such as angioedema).
4. Previous participation in this trial. Participation being defined by signing the informed consent 
document.
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 (an episode requiring assistance from another person) in the 
last month prior to screening.
8. Receipt of any investigational medicinal product 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.
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 at 
screening.
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.
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19. Surgery or trauma with significant blood loss within the last 2 months prior to screening.
20. Significant history of alcoholism or non-prescribed opioid misuse as judged by the 
investigator.
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 within 7 (+2) 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 since screening, 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.
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5.6
Premature discontinuation of subjects or trial
5.6.1
Subject discontinuation
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).
A subject will be discontinued from dosing and/or trial if the following applies:
x
Withdrawal of consent by subject.
x
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 drug, the subject will be discontinued by the 
investigator.
x
Adverse events occur which are considered unacceptable by the subject or the investigator.
If discontinuation occurs following administration of the trial drug, every effort should be made to 
have the subject return and participate in the complete follow-up visit on Day 28 (see Table 4) to 
avoid missing data.
If trial participation is terminated due to an AE possibly related to the trial drug 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.
5.6.2
Site discontinuation
The site can be closed for the following reasons:
x
The site is unlikely to be able to recruit sufficient subjects within the agreed time frame
x
The site does not respond to trial management requests
x
Repeat protocol violations
x
The site cannot comply with the requirements of the protocol
x
It is not possible for the site to comply with GCP standards
x
The site is unable to comply with the timely entry of data in the eCRF
5.6.3
Trial termination
If the investigator, the sponsor 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.
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Conditions that may warrant termination include, but are not limited to:
x
The discovery of an unexpected, significant or unacceptable risk to the subjects enrolled in 
the trial
x
New scientific knowledge that makes the objectives of the trial no longer feasible/valid
x
Failure to enroll subjects at an acceptable rate
x
A decision on the part of the sponsor to suspend or discontinue development of the drug
5.7
Subject identification and randomization
Subjects who have given written informed consent will be provided with a subject number. Once 
assigned, the subject number will be used throughout the trial and must not be reused by any other 
subject. 
Subjects who meet all inclusion and none of the exclusion criteria or dosing day exclusion criteria 
will be assigned a subject randomization number and randomized in a 3:1 ratio to 0.6 mg 
dasiglucagon (n=30) or placebo (n=10) via an Interactive Web Response System (IWRS). This
randomization will also determine whether the subject is to be injected in the buttock or deltoid. 
Randomization will continue until a total of 40 subjects have completed Visit 2.
If the subject fails to be randomized or to start treatment for any reason, the reason will be entered 
into the screening disposition page. The IWRS must be notified within 2 days that the subject was 
not randomized.
In the event of an emergency, e.g. when it becomes necessary for the investigator to know which 
trial drug 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,
see Section 6.4.
5.8
Screening failures
Screening failures are defined as subjects who consent to participate in the clinical trial but do not 
meet the inclusion or exclusion criteria at the screening visit (Visit 1). A minimal set of screen 
failure information is required to ensure transparent reporting of screen failure subjects, to meet 
the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to 
respond to queries from regulatory authorities. Minimal information includes demography, screen 
failure details, eligibility criteria and any serious adverse event (SAE).
Re-screening is not allowed.
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6
Trial drug
6.1
Identity
The following trial drugs will be administered:
x
Dasiglucagon, liquid formulation, 1 mg/mL, 0.6 mL delivered in an auto-injector.
x
Placebo, liquid formulation, 0.6 mL delivered in an auto-injector.
The description of the trial drugs is provided in Table 2. Dasiglucagon is a stable peptide analog 
of human glucagon in a ready-to-use liquid formulation for treatment of severe hypoglycemia in
patients with diabetes mellitus.
The quantities of ingredients for dasiglucagon and placebo are provided in Table 3.
Table 2
Description of trial drug
Name of active 
ingredient
Dasiglucagon
Placebo
Active substance
Dasiglucagon
N/A
Formulation
Liquid formulation
Liquid formulation
Strength
1.0 mg/mL
N/A
Device
Single use auto-injector
Single use auto-injector
Manufacturer
Zealand Pharma A/S, Glostrup (Copenhagen), Denmark
Storage
Store between 2 and 8°C
N/A not applicable
Table 3
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 1.0 mL
To make 1.0 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
Dosing and administration
The trial drug (dasiglucagon or placebo) will be administered by SC injection in the buttock or
deltoid (as determined by randomization).
All doses will be administered by staff at the trial site.
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6.3
Packaging, labelling and storage
6.3.1
Packaging and labelling
The trial drug 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 trial drug label will describe the storage conditions for the trial drug. The labels will supply 
no information about the subjects. Each auto-injector will have a unique Dispensing Unit Number 
for drug allocation, drug accountability and traceability purposes, and is labelled in a blinded 
fashion.
Labelling will be performed according to Annex 13 of the Good Manufacturing Practice 
guidelines of the European Commission, ICH GCP guidelines and local law.
6.3.2
Trial drug storage and stability
The investigator must ensure the availability of proper storage conditions. All trial drug supplies 
provided for this trial will be stored in the refrigerator in a secure area with restricted access at the 
trial site. The trial drugs ready for use should be separated from any other clinical supplies, to 
avoid medication errors.
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 trial termination.
The trial drug (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.
The person responsible for trial drug handling must contact the monitor in case of temperature 
deviations outside the acceptable range.
Please see the Trial Materials Manual/Pharmacy Manual for additional information on handling 
the trial drug.
6.4
Blinding and breaking the blinding code
In this double-blind trial, the trial drug (dasiglucagon and placebo) are identical liquid 
formulations in auto-injectors.
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 
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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 site 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]) will be able to break 
the code in case of a suspected unexpected serious adverse reaction (SUSAR).
The central and specialty laboratories will be provided with a copy of the randomization list.
6.5
Drug accountability
Handling, preparation and administration of the trial drug will be done by trial personnel. The site 
will keep accurate records of the trial supplies received, stored and dispensed, using appropriate 
forms. The trial supplies must be stored under appropriate conditions, locked and with restricted 
access.
All unused supplies and all empty packaging will be stored until the trial closure visit has been 
performed and then sent for destruction. Destruction must not take place until approved by the 
sponsor.
6.6
Drug exposure and compliance
All trial drugs will be administered by trial personnel. Time and date of the administered doses 
will be recorded in the eCRF.
PK assessments will support the surveillance of compliance with trial drug administration.
6.7
Concomitant therapy
6.7.1
Previous and concomitant medication
Concomitant medication is any medication apart from the trial drug that is used during the 
subject’s participation in the trial. All concomitant medication used must be recorded from the 
time of signature of the informed consent form (ICF) and to the end of the trial. The investigator 
or delegate must seek information on concomitant medication use at each trial visit. The 
information collected for concomitant medication must include:
x
Medication name (preferably generic name)
x
Indication
x
Dosage
x
Frequency
x
Route of administration
x
Start and stop date or continuation
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6.7.2
Prohibited medication
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) 
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.
6.8
Rescue medication
During the hypoglycemic clamp procedure, it may be necessary to administer a rescue glucose
infusion. Provisions for hypoglycemia rescue are described in Section 8.2.3.2.
Use of rescue glucose is to be recorded in the eCRF.
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7
Parameters and methods of assessment
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 (AUE0-30min) 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, 45, 50, 60, 75 and 
90 minutes after dosing (see the schedule of procedures in Table 4). The actual time of blood 
sampling for evaluation of plasma glucose should not deviate from the nominal time by more than 
±1 minute. Pre-dose is defined as within 2-5 minutes prior to dosing.
7.1.2
Pharmacokinetic measurements
The exposure to dasiglucagon 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 4. The actual time of blood 
sampling for exposure to trial drug should not deviate from the nominal time by more than ±1 
minute. Pre-dose is defined as within 2-5 minutes prior to dosing.
7.2
Safety parameters
7.2.1
Adverse events and pregnancies
Timely, accurate and complete reporting and analysis of safety information from clinical trials are 
crucial for the protection of subjects, investigators and the sponsor, and are mandated by 
regulatory agencies worldwide.
7.2.1.1
Definitions
7.2.1.1.1 Adverse events
An adverse event (AE) is any untoward medical occurrence in a clinical trial subject administered 
a medicinal (investigational or non-investigational) product and which does not necessarily have a 
causal relationship with this treatment. An AE can therefore be any unfavorable and unintended 
sign (including an abnormal laboratory finding), symptom or disease temporarily associated with 
the use of a product, whether or not related to the product.
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AEs include:
x
A clinically significant worsening of a concomitant illness.
x
A clinical laboratory adverse event: a clinical 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, e.g. change of dose or more frequent follow-up due to the abnormality.
The following should not be considered as AEs:
x
Pre-existing conditions, including those found as a result of screening procedures (pre-
existing conditions should be reported as medical history or concomitant illness).
x
Pre-planned procedures, 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.
When assessing an adverse event, the following definitions are used:
Severity:
x
Mild: No or transient symptoms, no interference with the subject’s daily activities.
x
Moderate: Marked symptoms, moderate interference with the subject’s daily activities.
x
Severe: Considerable interference with the subject’s daily activities, which the subject 
finds unacceptable. A severe reaction does not necessarily deem the AE as serious and an 
SAE are not always severe in nature.
Causality:
x
Probable: Good reason and sufficient documentation to assume a causal relationship.
x
Possible: A causal relationship is conceivable and cannot be dismissed.
x
Unlikely: The event is most likely related to etiology other than the product.
x
Not Related: An AE that does not follow a reasonable temporal sequence from 
administration of a drug and/or that can reasonably be explained by other factors, such as 
underlying diseases, complications, concomitant drugs and concurrent treatments.
Outcome:
x
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 ICF.
x
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 or has 
died from another AE.
x
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.
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x
Not recovered/not resolved: The condition of the subject has not improved, and the 
symptoms are unchanged, or the outcome is not known.
x
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.
x
Unknown: This term is only applicable if the subject is lost to follow-up.
7.2.1.1.2 Treatment-emergent adverse events (TEAEs)
A treatment emergent adverse event (TEAE) is defined as an AE with an onset at the time of or 
following the start of treatment with the trial drug. 
7.2.1.1.3 Serious adverse event
A serious adverse event (SAE) is any untoward medical experience that at any dose result in any 
of the following:
x
Results in death.
x
Is life-threatening.
x
Requires inpatient hospitalization or prolongation of existing hospitalization.
x
Results in persistent or significant disability or incapacity.
x
Is a congenital anomaly or birth defects.
x
Is otherwise medically important, that may not result in death, be life threatening or 
require hospitalization may be considered an SAE when (based on appropriate medical 
judgement) it may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed in the definition of SAE. Examples could be 
emergency room or home treatment of allergic bronchospasm or convulsion.
7.2.1.1.4 Other important adverse event
The following events must always be reported to the sponsor according to SAE timelines, 
regardless of whether the event is non-serious or serious: 
x
Risk of liver injury defined as ALT or AST > 3x upper limit of normal (ULN) and total 
bilirubin > 2x ULN, where no alternative etiology exists.
x
Suspicion of transmission of infectious agents via the trial drug.
x
Overdose of the trial drug.
x
Medication error involving the trial drug.
x
Inadvertent or accidental exposure to the trial drug.
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7.2.1.1.5 Non-serious adverse events
A non-serious AE is any AE which does not fulfil the definition of an SAE.
7.2.1.1.6 Adverse event of special interest
In this trial the following adverse events are considered adverse events of special interest (AESI),
with data collected under a specific eCRF form:
x
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.
x
Post-dose change in pulse or blood pressure considered an event of hypo- or hypertension 
as judged by the investigator.
7.2.1.1.7 Suspected unexpected serious adverse reactions
An AE is considered a SUSAR if the nature or severity is not consistent with the applicable 
product Reference Safety Information (RSI). For dasiglucagon, the expectedness of an AE will be 
determined by whether or not it is listed in the RSI section of the Investigator’s Brochure.
7.2.1.1.8 Adverse events associated with the use of the drug
An AE is considered associated with the use of the drug if the causality assessment is possible or 
probable by the definition listed in Section 7.2.1.1.1.
7.2.1.2
Collection, recording and reporting of adverse events
All AEs, whether serious or non-serious, will be reported from the time a signed and dated ICF is 
obtained until the end of the post-treatment follow-up period (which may include contacts for 
follow-up of safety). In addition, subjects will be observed for any signs or symptoms and asked 
about their condition by open questioning, such as “How have you been feeling since you were 
last asked?”, at each contact with the trial site (visit or telephone). Subjects will also be 
encouraged to spontaneously report AEs occurring at any other time during the trial.
All AEs, regardless of seriousness, severity, or presumed relationship to trial drug, must be 
recorded and evaluated by the investigator. Whenever possible, diagnoses should be given when 
signs and symptoms are due to a common etiology. If no diagnosis can be made, the investigator 
should record each sign and symptom as individual AEs. Investigators must record their opinion 
concerning the relationship of the AE to the trial drug. All assessments completed during AE 
evaluation must be recorded in the source document and reported according to sponsor 
instructions.
Each AE must be reported on an AE eCRF within 5 calendar days of the investigator becoming 
aware of the event.
AESI will be reported by adding a tick-mark in the appropriate box on the AE and/or SAE form 
and by completing 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 
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the event. The investigator must report all AESIs to the sponsor’s responsible pharmacovigilance 
unit immediately (within 24 hours) after obtaining knowledge about the event.
AE information should as a minimum include the following:
x
Date and time of onset
x
Date and time of investigator’s first information about the AE
x
Seriousness
x
Severity
x
Causal relationship with trial drug
x
Measures taken due to AE
x
Interruption or withdrawal of treatment during IMP administration
x
Date and time of resolution and final outcome
SAEs, including those spontaneously reported to the investigator within 30 days after the last dose 
of trial drug, must be reported within 24 hours after obtaining knowledge about the event by 
completing the SAE form in the Electronic Data Capture (EDC) system. Once additional 
information becomes available, a follow-up SAE form must be reported. For each SAE, a separate 
SAE form should be completed.
All SAEs will be reported in EDC, and for each reported event a system-generated email will be 
sent to the safety CRO (PharmaLex), the medical monitor, sponsor medical director, and trial 
manager
Pharmacovigilance for this trial is outsourced to PharmaLex Denmark A/S.
It is the responsibility of the Safety CRO to report all SUSARs that occur in this trial to competent 
authorities. It is the responsibility of clinical CRO to report all SUSARs that occur in this trial to
IRBs in accordance with the local requirements in force and ICH guideline for GCP.
7.2.1.3
Follow-up of adverse events
The investigator must record follow-up information on the eCRF for non-serious AEs, on the 
AESI form for AESIs, and on the SAE form for SAEs during the follow-up periods as described 
below. Follow-up questions to investigators regarding SAEs are queried directly by PharmaLex to 
the investigator.
Follow-up information must be reported according to the following:
x
SAEs: All SAEs must be followed until the outcome of the events is “recovered/resolved”, 
“recovered/resolved with sequelae” or “fatal” and until all queries have been resolved. 
Cases of chronic conditions, cancer or AEs ongoing at time of death (where death is due to 
another AE) may be closed with the outcome “recovering/resolving” or “not recovered/not 
resolved”. Cases can be closed with the outcome of “recovering/resolving” when the 
subject has completed the follow-up period and is expected by the investigator to recover.
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The SAE follow-up information should only include new (e.g. corrections or additional) 
information and must be reported within 24 hours of the investigator’s first knowledge of 
the information. This is also the case for previously non-serious AEs which subsequently 
become SAEs.
x
Non-serious AEs: Non-serious AEs must be followed until the outcome of the event is 
“recovering/resolving”, “recovered/resolved” or “recovered/resolved with sequelae” or 
until the end of the follow-up period stated in the protocol, whichever comes first, and 
until all queries related to these AEs have been resolved. Cases of chronic conditions, 
cancer or AEs ongoing at time of death (where death is due to another AE) may be closed 
with the outcome of “recovering/resolving” or “not recovered/not resolved”. Cases can be 
closed with the outcome of “recovering/resolving” when subject has completed the follow-
up period and is expected by the investigator to recover.
If a potential hypersensitivity reaction is observed, additional blood samples may be required to 
further characterize the potential hypersensitivity reaction. If an anaphylactic shock is suspected, 
samples may be taken for the measurement of tryptase. In this case, a blood sample should be 
taken 3 to 4 hours after the event and again approximately 1 to 2 weeks later to determine tryptase 
baseline levels. In addition, assessments for elevated histamine levels may be considered.
The investigator must ensure that the maximum severity of an event is kept throughout the trial,
starting at the time of exposure to trial drug. A worsening of an unresolved AE must be reported 
as follow up with re-assessment of severity of the event.
Queries or follow-up requests must be responded within 14 calendar days from the date of receipt
of the request, unless otherwise specified in the follow-up request.
7.2.1.4
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 trial drug, a plasma glucose value of <45 
mg/dL (2.5 mmol/L) will be treated by an IV glucose solution. After administration of the trial 
drug 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 trial drug 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.
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7.2.1.5
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. All initial reports of pregnancy in female subjects 
must be reported to the sponsor by the trial site personnel within 24 hours of their knowledge of 
the event using the appropriate pregnancy form. Abnormal pregnancy outcomes (e.g. spontaneous 
abortion, fetal death, stillbirth, congenital anomalies, ectopic pregnancy) are considered SAEs and 
must be reported using the SAE form. If a subject becomes pregnant during the trial, a 
determination regarding the trial drug discontinuation must be made by the investigator.
Because the effect of the trial drug on sperm is unknown, pregnancies in partners of male subjects 
must be reported by the trial site personnel within 24 hours of their knowledge of the event using 
the appropriate pregnancy form. Male subjects must be instructed to notify the investigator 
immediately if their partner becomes pregnant or suspects to be pregnant.
The investigator must follow the pregnancy until the pregnancy outcome is known and the 
newborn infant is one month of age. The investigator must report information about the 
pregnancy, pregnancy outcome and health of the newborn infant(s). Any AEs in connection with 
the pregnancy or AEs in the fetus and newborn infant must be reported.
7.2.1.6
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 subjects 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, refer to the current version of the 
Investigator’s Brochure (14).
7.2.1.7
Safety Committee
An internal Zealand Pharma Safety Committee is constituted to perform ongoing safety 
surveillance of clinical trials with dasiglucagon, including this trial.
If safety signals or concerns are observed, whether based on reported SAEs, review of all AEs and 
laboratory parameters reported or any other notification of significant findings, the Safety 
Committee will respond appropriately to protect the safety of the subjects. The Safety Committee 
meets quarterly and additionally on ad-hoc basis as needed.
7.2.2
Safety laboratory parameters
7.2.2.1
Clinical chemistry, hematology and urinalysis
Routine clinical laboratory tests will be performed centrally.
Samples for clinical laboratory parameters (biochemistry, hematology) and urinalysis will be 
collected at screening (Visit 1), the dosing visit (Visit 2) and the follow-up visit (Visit 3). Samples 
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for glycated hemoglobin (HbA1c), C-peptide and coagulation parameters will be collected at 
screening only (Visit 1). The following parameters will be assessed:
x
Clinical biochemistry: sodium, potassium, calcium, glucose, urea, creatinine, total 
bilirubin, AST, ALT, gamma-glutamyl transferase, alkaline phosphatase, total protein, 
C-reactive protein, C-peptide, HbA1c.
x
Hematology: hemoglobin, red blood cell count (erythrocytes), hematocrit, platelet count 
(thrombocytes), total white blood cell count (leukocytes).
x
Coagulation: international normalized ratio, fibrinogen (only Visit 1).
x
Urinalysis: pH, blood (leukocytes and erythrocytes), protein, glucose, ketones, nitrite.
At the dosing visit, samples for clinical laboratory parameters (biochemistry, hematology) will be 
collected prior 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. The sample for urinalysis will be collected prior to the 
start of the insulin-induced hypoglycemic procedure (within 120 minutes).
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.
7.2.2.2
Pregnancy test
Pregnancy tests will be performed at each visit for women of childbearing potential. A serum 
pregnancy test will be performed at screening (Visit 1) and urine stick tests will be performed at 
the trial site at the dosing visit (Visit 2) and the follow-up visit (Visit 3). Test sticks will be 
provided to the trial site.
7.2.2.3
Plasma glucose measurements for safety
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at site. 
After the start of the IV insulin infusion, plasma glucose should be checked approximately every 
10 minutes while plasma glucose is above 110 mg/dL (6.1 mmol/L), and approximately every 5 
minutes once plasma glucose is at or below 110 mg/dL (6.1 mmol/L) and until after dosing when 
the subject’s plasma glucose is ≥70 mg/dL (3.9 mmol/L). Plasma glucose will continue to be 
checked every 5 minutes after dosing until the 45 min time point. 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.
7.2.2.4
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 insulin should 
not deviate from the nominal time by more than ±1 minute. Pre-dose is defined as within 2-5
minutes prior to dosing.
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7.2.2.5
Other safety laboratory tests
Alcohol breath tests and a urine drug screen (12 panel drug test; including benzodiazepines,
barbiturates, cocaine, marijuana, metamphetamine, morphine, methadone, buprenorphine,
oxycodone, methylendiaxymetamphetamine, phencyclidine, amphetamine) 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 urine drug screen will be 
provided to the trial site.
7.2.3
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):
x
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 assessment of Exclusion Criteria #11, and this arm should 
be used for all subsequent measurements of the subject’s blood pressure in this trial.
If blood pressure measurements are within 10 mmHg in both arms, one arm will be chosen 
and be used for all subsequent measurements of the subject’s blood pressure in this trial.
x
Pulse (beats per min) measured after at least 5 minutes rest in a supine position.
x
Body temperature aural (°C).
At the dosing visit vital signs will be measured pre-dose (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.
7.2.4
Physical examination
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.
7.2.5
Electrocardiogram
A standard 12-lead ECG (safety ECG) will be performed after 5 minutes in a supine position at 
the screening visit (Visit 1), the dosing visit (Visit 2) and the follow-up visit (Visit 3).
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At the dosing visit single recordings will be made pre-dose (within 30 minutes) and at 20, 35, 45, 
60 and 300 minutes. The actual time of the assessment should not deviate from the nominal time 
by more than ±5 minutes.
Safety ECGs will be printed and assessed locally by the investigator or a qualified designee.
ECG parameters (heart rate, PQ, QRS, QTcF [corrected using the Fridericia correction]) 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’).
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), at the follow-up
visit (Visit 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.
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 in the eCRF. The 
assessments will be performed by a trial physician or nurse. In case of an observation, the Local 
Tolerability form will be completed, as well as the (S)AE form.
7.2.7
Immunogenicity
Antibodies against dasiglucagon 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 injection. The ADA 
samples will be analyzed at a special laboratory, York Bioanalytical Solutions.
The clinical ADA assay specific for dasiglucagon has been validated in accordance with existing 
guidelines and recommendations (17-20).
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. Any anti-
dasiglucagon antibody-positive subject must be followed up every 3 months at extra visits, where 
a blood sample for the assessment of ADAs and plasma glucose must be collected until the ADA 
levels return to baseline levels.
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, 21). The calculated sensitivity 
in previous studies was about 51.8 ng/mL. 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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In vivo neutralizing effect will be evaluated by comparing PK and PD endpoints between ADA 
positive and ADA negative subjects, further investigation may be performed by correlating ADA 
titer with PK and PD endpoints.
The results from the neutralizing antibody assay may be included in the clinical trial report (CTR)
or reported separately pending availability of the results.
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.
7.3
Demographics and baseline characteristics
Demographics, body measurements, concomitant illness and medical history will only be assessed 
at screening (Visit 1). Concomitant medication will be assessed at screening (Visit 1), the dosing 
visit (Visit 2, prior to dosing) and at the follow-up visit (Visit 3).
7.3.1
Demographics and body measurements
Subject demographics and body measurements consist of:
x
Age
x
Race and ethnicity
x
Sex
x
Height (meters or inches), without shoes
x
Body weight (kg), wearing light clothing and measured using standard scales
x
Body mass index (kg/m2) calculated by the system based on height and body weight
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 as an AE.
All previous and concomitant diseases will be coded with Medical Dictionary for Regulatory 
Activities (MedDRA), the version of which will be provided in the CTR.
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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 drugs, 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 7.2.1.2. If the change in medication influences the 
subject’s eligibility to continue in the trial, the sponsor and trial monitor must be informed.
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x
Physical examination
x
Vital signs
x
12-lead ECG
x
AEs
x
Biochemistry, hematology, coagulation, HbA1c
x
Serum pregnancy test (women of childbearing potential only)
x
Urinalysis
x
Urine drug screen
x
Alcohol breath test
Eligible or potentially eligible subjects (laboratory results pending) will be provided with an 
Identification 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 
Identification 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 up to Visit 
2: 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 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 site 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
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mmol/L) the following morning, subjects may be administered insulin glulisine (Apidra®) at the 
investigator’s discretion by IV 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 or placebo.
The following assessments will take place:
x
Document all changes in concomitant medication (prior to the start of the insulin induced 
hypoglycemic procedure)
x
Check of withdrawal criteria (prior to the start of the insulin-induced hypoglycemic
procedure)
x
Check of dosing day exclusion criteria (prior to the start of the insulin-induced 
hypoglycemic procedure)
x
Vital signs (prior to the start [within 30 minutes] of the insulin-induced hypoglycemic
procedure, and at 30, 90 and 300 minutes after dosing).
x
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)
x
Local tolerability (at 30, 120, and 300 minutes after dosing)
x
AEs 
x
Biochemistry, hematology (prior to the start of the insulin-induced hypoglycemic
procedure [within 30 minutes], and at 30 and 300 minutes after dosing)
x
Urine stick pregnancy test (women of childbearing potential only; prior to the start of the 
insulin-induced hypoglycemic procedure)
x
Urinalysis (prior to the start of the insulin-induced hypoglycemic procedure [within 120 
minutes])
x
Urine drug screen (prior to the start of the insulin-induced hypoglycemic procedure)
x
Alcohol breath test (prior to the start of the insulin-induced hypoglycemic procedure)
x
Dasiglucagon 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 2-5 minutes prior to dosing (baseline).
x
Plasma glucose concentrations:

Pre-dose, 4, 6, 8,10, 12, 15, 17, 20, 25, 30, 40, 45, 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 ±1 minute. Pre-dose is defined as within 2-5 minutes 
prior to dosing (baseline).
x
Antibodies against dasiglucagon (prior to the start of the insulin induced hypoglycemic
procedure).
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x
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 2-5 minutes prior to dosing.
8.2.3.1
Hypoglycemic clamp procedure and administration of trial drug
The following procedure is based on precedent procedures for hypoglycemia induction in patients 
with T1DM (22, 23).
The dosing 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 within 7 (+2) 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 metacarpal dorsal hand vein for blood 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 (but distal to 
the infusion vein) may be used at the discretion of the investigator. A third catheter for blood 
sampling will be placed into a metacarpal dorsal hand vein for blood 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
(but distal to the infusion vein) 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. 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.
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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 PK, and insulin PK 
will be collected. The samples are the baseline samples and should be collected within 2 minutes 
before trial drug administration.
x
If plasma glucose is ≥45 mg/dL and <60 mg/dL (2.5-3.3 mmol/L), trial drug will be 
administered, defining time, t=0. The trial drug will be delivered in the buttock or
deltoid (according to stratification) via SC injection.
x
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 (2.5-3.3 mmol/L) target 
range. The run-in period will be adequately extended (at least 30 minutes) until the above 
target is achieved and new baseline samples for plasma glucose, dasiglucagon, and insulin 
PK will be collected. Glucose should not be infused within 10 minutes before trial drug 
administration. If plasma glucose is not within target range after the second attempt, the 
subject should be rescheduled for a new dosing visit within 7 days (+ 2 days).
Administration of trial drug should not occur later than 12:00 hours.
As shown in Table 5, serial blood samples for plasma glucose will be collected at t=0, 4, 6, 8, 10, 
12, 15, 17, 20, 25, 30, 40, 45, 50, 60, 75 and 90 minutes post-dosing. Samples for assessing 
plasma dasiglucagon 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 5
Post-treatment blood sampling times
Times
0
4
6
8
10
12
15
17
20
25
30
35
40
45
50
60
75
90
120
Plasma glucose
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
PK dasiglucagon
Y
Y
Y
Y
Y
Y
Y
Y
Y
PK insulin
Y
Y
Y
PK pharmacokinetics; Y yes
Refer to Section 7.2.2 for details of laboratory safety sampling and to Section 7.2.2.3 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 a light snack. Drinking of water is allowed ad libitum during the entire procedure.
8.2.3.2
Hypoglycemia rescue provisions
During insulin-induced hypoglycemia, plasma glucose levels will be monitored closely at site for 
safety reasons as described in Section 7.2.2.3. 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 (3.9 mmol/L).
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2. If plasma glucose is <45 mg/dL (2.5 mmol/L) between t=8 and t=44 minutes (both inclusive),
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 (3.1 mmol/L).
3. If plasma glucose is <70 mg/dL (3.9 mmol/L) 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 trial drug will be administered SC according to Section 6.2. The time of trial drug 
administration will be recorded. At the time point when the insulin infusion is stopped, the total 
insulin dose which was required to induce hypoglycemia will be recorded.
During the procedure, the Investigator will assess whether any subjective AEs have occurred. 
Subjects may report AEs occurring at any time during the procedure.
The investigator must provide information to the subjects on how to resume their usual diabetes 
treatment.
The subject may be released from the clinical site 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 site for a longer period.
8.2.4
Visit 3 (follow-up visit)
Visit 3 will take place on Day 28. The subject does not need to be fasting.
At Visit 3, the following assessments will take place:
x
Document all changes in concomitant medication 
x
Physical examination
x
Vital signs
x
12-lead ECG
x
Local tolerability
x
AEs 
x
Biochemistry, hematology
x
Urine stick pregnancy test (women of childbearing potential only)
x
Urinalysis
x
Antibodies against dasiglucagon
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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 treatment unblinding, a separate statistical analysis plan (SAP) will be finalized, providing 
detailed methods for the analyses outlined below. 
Any deviations from the planned analyses will be described and justified in the final clinical trial 
report.
9.1
Determination of sample size
From phase 2, the median time to an increase in plasma glucose of 20 mg/dL (1.1 mmol/L) of the 
0.6 mg dose was approximately 10 minutes. For placebo-treated subjects, we assume that the 
median time to recovery will be more than 50 minutes. Assuming an exponential time-to-recovery 
distribution with median times of 10 and 50 minutes, a two-sided log-rank test will be able to 
detect a difference between dasiglucagon and placebo with 92% power with a follow-up time of 
45 minutes at a 5% significance level with 40 subjects randomized 3 to 1 between active and 
placebo.
9.1.1
Disposition of subjects
All randomized subjects will be accounted for. All post-randomization discontinuations will be 
summarized by reason for discontinuation. The number and characteristics of subjects screened 
but not found eligible will be stated in the CTR, together with a summary of reasons and types of 
failures. A detailed listing of these subjects will be provided.
The number of subjects in each analysis set will be summarized by frequency and percentage in a 
subject disposition table.
9.1.2
Analysis populations
For presentation of data and reporting of the statistical analyses, the following analysis sets will be 
used, depending on the context: 
x
Safety analysis set: All randomized subjects who received at least one dose of trial drug.
x
Full analysis set (FAS): All randomized subjects who received at least one dose of trial 
drug.
x
Per protocol (PP) analysis set: All subjects of the FAS for whom no relevant protocol 
deviations were documented. 
The decision to exclude a subject or exclude specific data from a subject should be taken at the
blinded data review meeting before unblinding, and the exclusion from efficacy or other analyses 
justified in the signed notes of the meeting.
The analysis of the primary endpoint and secondary endpoints will be based on the FAS. A 
supportive analysis of the primary endpoint will be based on the PP. All safety analyses will be 
based upon the safety analysis set.
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9.2
General considerations
All statistical analysis of the trial will be performed using the statistical software SAS for 
Windows Version 9.4. In all calculations, zero will be substituted for concentrations below the 
quantification limit of the assay. All data collected from all subjects will be presented in data 
listings. Both absolute values and change from baseline values for each subject will be given 
where applicable. All continuous data will be listed with the same precision as will be presented 
in the database. Data listings will be sorted by treatment, subject ID and time point. A missing 
value will be represented by an empty cell and no imputation will be made.
Continuous data will be summarized in tables using number of subjects (n), mean, median, 
standard deviation (SD), standard error (SE), 95% confidence interval (CI; based on a t-
distribution if not otherwise stated), minimum and maximum by trial time point.  Categorical data 
will be summarized in two ways, by subject and by time point. Subject data will be summarized 
using the count of distinct subjects that fall in the category and the percentage of the total number 
of subjects. Time point data will be summarized using the count of the assessments that fall into 
the category and the percentage of the total number of assessments. Population counts (either 
number of subjects or number of time points at the assessment) will be used as the denominator in 
the calculation of percentages unless otherwise specified.
9.3
Demographics, baseline characteristics and concomitant medications
Baseline and demographic data will be summarized using descriptive statistics.
9.4
Treatment compliance
Treatment compliance will be summarized and listed for the safety analysis set.
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 
FAS. 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.
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:
x
Primary: Time to plasma glucose recovery
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x
Key secondary endpoints 1-4: Plasma glucose recovery within 30 minutes, within 20 
minutes, within 15 minutes, and within 10 minutes after trial drug injection without 
administration of rescue IV glucose.
x
Key secondary endpoints 5-8: Plasma glucose changes from baseline at 30 minutes, at 20
minutes, at 15 minutes, and at 10 minutes after trial drug injection or at the time of rescue.
Analogous supportive analyses will be conducted in the PP set.
9.5.2
Analysis of primary endpoint
The primary endpoint is 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.
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 before 45 minutes 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 1) without censoring 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 ratio (dasiglucagon versus 
placebo) will be estimated together with the 95% confidence intervals, and the treatment group 
inference (dasiglucagon vs placebo) will be evaluated using the two-sided likelihood ratio test.
9.5.3
Analysis of key secondary endpoints
The key secondary endpoints are in this order:
x
Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, and 
within 10 minutes after trial drug injection without administration of rescue IV glucose.
x
Plasma glucose changes from baseline at 30 minutes, at 20 minutes, at 15 minutes, and at
10 minutes after trial drug injection or at the time of rescue.
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) using Fisher’s exact test.
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The key secondary endpoints of plasma glucose changes from baseline at 30 minutes, at 20
minutes, at 15 minutes, and at 10 minutes after trial drug 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 ≥20 mg/dL (1.1 mmol/L) recovery. 
Each of these changes from baseline 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 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.
9.5.4
Analysis of secondary efficacy endpoints
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.
Time to first plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) from baseline will be 
evaluated using a KM 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) will be evaluated inferentially using pairwise two-sided log rank tests. If the ≥70 mg/dL 
(3.9 mmol/L) 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.
The AUE0-30min will be calculated as the baseline-adjusted area under the plasma glucose profile 
from time zero to 30 minutes. The log-transformed AUE endpoint will be analyzed using an 
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
Analysis of exposure endpoints
AUC will be derived as the area under the individual plasma dasiglucagon 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 time points.
Cmax will be determined as the maximum of all valid plasma dasiglucagon concentrations. 
Tmax will be determined as the time point where the maximum of all valid plasma dasiglucagon 
concentration measurements for each measurement series is observed.
These exposure endpoints will be summarized descriptively for each treatment group.
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9.6
Safety analyses
9.6.1
Drug exposure
Drug exposure will be summarized and listed for the safety analysis set.
9.6.2
Adverse events
AEs will be coded using the latest available version of the MedDRA, the version of which will be 
provided in the CTR.
An overall summary table will be provided showing the number and percentage of subjects with 
any:
x
Treatment-emergent adverse event (TEAE)
x
Severe TEAE
x
Treatment-emergent SAE
x
Drug-related TEAE
x
Drug-related severe TEAE
x
Treatment-emergent drug-related SAE
x
TEAE leading to withdrawal
x
TEAE with outcome death
x
AESI
9.6.3
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.
9.6.4
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.
9.6.5
Other safety parameters
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).
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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) 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.
9.7
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, trial drug 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 AUE0-30min.
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. 
9.8
Interim analyses
No interim analyses are currently planned.
9.9
Other analyses
9.9.1
Subgroup analyses
Summary tables describing the primary endpoint will be created based on age, sex, race/ethnicity 
or other demographic characteristic(s).
9.9.2
Exploratory analyses
Exploratory analyses cannot be used as confirmatory proof for registration trials. 
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10 Ethical, legal and administrative aspects
10.1
Statement of compliance
The information contained within this protocol is consistent with the current risk/benefit 
evaluation of the IMP as well as with the ethical and scientific principles governing clinical 
research as set out in the current version of the Declaration of Helsinki (1) and the guidelines on 
GCP (2).
10.2
Quality management
The sponsor or designee will conduct a site visit to verify the qualifications of each investigator, 
inspect the site facilities and inform the investigator of responsibilities and the procedures for 
ensuring adequate and correct documentation.
Each clinical site will perform internal quality management of trial conduct, data and biological 
specimen collection, documentation and completion, according to GCP guidelines.
Quality control procedures will be implemented beginning with the data entry system and data 
quality checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution.
Following written standard operating procedures (SOPs), the monitors will verify that the clinical 
trial is conducted, and data are generated, and biological specimens are collected, documented 
(recorded) and reported in compliance with the protocol, GCP and applicable regulatory 
requirements, Good Manufacturing Practices (GMP)).
The investigational site will provide direct access to all trial related sites, source data/documents 
and reports for the purpose of monitoring and auditing by the sponsor and inspection by local and 
regulatory authorities.
10.2.1
Clinical monitoring
Clinical site monitoring is conducted to ensure that the rights and well-being of trial subjects are 
protected, that the reported trial data are accurate, complete and verifiable, and that the conduct of 
the trial is in compliance with the currently approved protocol/amendment(s), with GCP and with 
applicable regulatory requirement(s).
During the course of the trial, the monitor will visit the trial site to ensure that the protocol and 
GCP are adhered to, that all issues have been recorded, to perform source data verification and to 
monitor drug accountability. The monitoring visit intervals will depend on the trial site’s 
recruitment rate and the compliance of the trial site with the protocol and GCP.
In order to perform their role effectively, monitors and persons involved in quality assurance and 
inspections will need direct access to source data, e.g. medical records, laboratory reports, 
appointment books, etc. If the electronic medical record does not have a visible audit trail, the 
investigator must provide the monitor with signed and dated printouts. In addition, relevant site 
staff should be available for discussions at monitoring visits and between monitoring visits (e.g. 
by telephone).
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Details of clinical site monitoring are documented in a monitoring manual. The manual describes 
in detail who will conduct the monitoring, at what frequency monitoring will be done, at what 
level of detail monitoring will be performed and the distribution of monitoring reports.
10.3
Data handling and record keeping
10.3.1
Data collection and management 
Data will be collected by means of EDC. 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 investigator or staff authorized by the investigator will enter subject data into eCRFs. Data 
recorded in the eCRFs will be accessible to the trial site and sponsor personnel immediately after 
entry. The eCRFs must be maintained in an up-to-date state at the trial site at all times.
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the 
site investigator. The investigator is responsible for ensuring the accuracy, completeness, 
legibility and timeliness of the data reported. All source documents should be completed in a neat, 
legible manner to ensure accurate interpretation of data. The investigator must sign all eCRFs 
used. This signature information will be kept in the audit trail and cannot be altered. Any 
correction(s) made by the (sub)investigator or authorized site staff to the eCRF after original entry 
will be documented in the audit trail. Changes to data already approved will require the re-
signature by the investigator. The person making the change to the data, and the date, time and 
reason for the change will be identified in the audit trail.
Data integrity must be secured through the complete data life cycle, from data creation to data 
deletion. Data integrity means that data must be attributable, legible, contemporaneous, original or 
a true copy, accurate, complete, consistent, enduring and available (ALCOA+).
Figure 2 illustrates the flow of data collected for this trial.
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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 subject confidentiality.
10.3.4
Archiving of trial records
The investigator is responsible for maintaining and archiving all trial records during and after the 
trial in compliance with GCP guidelines.
According to the GCP 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 IMP. However, these documents 
should be retained for a longer period if required by the applicable legal requirements.
No trial site document may be destroyed without prior written agreement between the investigator 
and sponsor. Should the investigator elect to assign the trial documents to another party, or move 
them to another location, the sponsor must be notified.
10.4
Subject consent and participation
10.4.1
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.
If a protocol amendment is required, the ICF 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.
10.4.2
Subject participation card
The subjects will be provided with a subject participation card bearing the following information:
x
That he/she is participating in a blinded clinical trial, including the trial ID
x
That he/she is treated with dasiglucagon or placebo
x
The address, name and phone number of the investigator
The subjects will be asked to keep the subject participation card in their possession at all times 
during the trial and to destroy or return it at the end-of-trial visit.
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10.5
Protocol requirements
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 l 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.
10.5.1
Protocol deviations
Deviations from the protocol should be avoided. A protocol deviation is any noncompliance with 
the clinical trial protocol or ICH GCP requirements. The noncompliance may be either on the part 
of the participant, the investigator or the trial site staff. As a result of deviations, corrective and 
preventive actions are to be developed by the site and implemented promptly.
It is the responsibility of the investigator to use continuous vigilance to identify and report 
deviations. The investigator must inform the site monitor. All deviations must be addressed in trial 
source documents and reported to sponsor. The implications of the deviation must be reviewed 
and discussed.
10.6
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) must be maintained in 
confidence by the investigator.
10.7
Other ethical and regulatory issues
If a significant safety issue is identified, either from an individual case report or from 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.
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10.8
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 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 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 insofar as the individual subject is concerned.
Any injury to health, which might have occurred as 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.
10.9
Publication and public disclosure
The information obtained during the conduct of this trial is considered confidential and may be 
used by or on behalf of Zealand Pharma for regulatory purposes as well as for the general 
development of the trial drug. All information supplied by Zealand Pharma in connection with 
this trial shall remain the sole property of Zealand Pharma and is to be considered confidential 
information.
No confidential information shall be disclosed to others without prior written consent from 
Zealand Pharma. Such information shall not be used except in the performance of this trial. The 
information obtained during this trial may be made available to other physicians who are 
conducting other clinical trials with the trial drug, if deemed necessary by Zealand Pharma. 
Provided that certain conditions are fulfilled, Zealand Pharma may grant access to information 
obtained during this trial to researchers who require access for research projects studying the same 
disease and/or trial drug studied in this trial. 
One investigator will be appointed to review and sign the clinical trial report (signatory 
investigator) on behalf of all participating investigators.
10.9.1
Communication of results
No permission to publish shall be granted to any CRO involved in the trial. 
The results of this trial will be subject to public disclosure on external web sites according to 
international and national regulations.
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Zealand Pharma reserves the right to defer the release of data until specified milestones are 
reached, for example when the clinical trial report is available. This includes the right not to 
release the results of interim analyses, because the release of such information may influence the 
results of the entire trial.
At the end of the trial, one or more scientific publications (including abstracts, posters and 
presentations) may be prepared collaboratively by the investigator(s) and Zealand Pharma. 
Zealand Pharma reserves the right to postpone publication and/or communication for up to 60 
days to protect intellectual property. In all cases the trial results will be reported in an objective, 
accurate and balanced manner. In the event of any disagreement on the content of any publication, 
the opinions of both the investigators and Zealand Pharma will be fairly and sufficiently 
represented in the publication.
Authorship of publications should be in accordance with the Uniform Requirements of the 
International Committee of Medical Journal Editors (sometimes referred to as the Vancouver 
Criteria).
10.9.2
Site-specific publication(s) by investigator(s)
For a multi-centre clinical trial, analyses based on single-site data usually have significant 
statistical limitations and frequently do not provide meaningful information for healthcare 
professionals or trial patients, and therefore may not be supported by Zealand Pharma. It is a 
Zealand Pharma policy that such individual reports do not precede the primary manuscript and 
should always reference the primary manuscript of the trial. Zealand Pharma reserves the right to 
prior review of such publications. Further to allow for the primary manuscript to be published as 
the first, Zealand Pharma asks for deferment of publication of individual site results until the 
primary manuscript is accepted for publication. As Zealand Pharma wants to live up to the 
industry publication policy, submission of a primary publication will take place no later than 18 
months after trial completion.
The results of this trial may be published or presented at scientific meetings. If this is foreseen, the 
investigator agrees to submit all manuscripts or abstracts to the sponsor before submission. This 
allows the sponsor to protect proprietary information and to provide comments.
10.9.3
Investigator access to data and review of results
As owner of the trial database, Zealand Pharma has the discretion to determine who will have 
access to the database. Individual investigators will have their own research subjects' data and will 
be provided with the randomization code after results are available.
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11 Reference list
1. WMA Declaration of Helsinki – Ethical Principles for Medical Research Involving Human
Subjects. Available from URL: https://www.wma.net/policies-post/wma-declaration-of-
helsinki-ethical-principles-for-medical-research-involving-human-subjects/
2. 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.
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;40:155-57.
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.
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 4, 25 Oct 2017.
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
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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;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. 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.
22. 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.
23. 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.
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SAP_001.pdf: 




 
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Table of Contents 
1 INTRODUCTION ..................................................................................................................................... 6 
2 STUDY OBJECTIVES .............................................................................................................................. 6 
2.1 PRIMARY OBJECTIVE .................................................................................................................... 6 
2.2 SECONDARY OBJECTIVES ............................................................................................................ 6 
2.3 TERTIARY/EXPLORATORY OBJECTIVES .................................................................................. 6 
3 STUDY OVERVIEW ................................................................................................................................ 7 
3.1 STUDY DESIGN ................................................................................................................................ 7 
3.2 TRIAL PROCEDURES ...................................................................................................................... 8 
3.3 SAMPLE SIZE CONSIDERATION ................................................................................................ 10 
4 STUDY ENDPOINTS ............................................................................................................................. 11 
4.1 PRIMARY ENDPOINTS ................................................................................................................. 11 
4.2 SECONDARY ENDPOINTS ........................................................................................................... 11 
4.3 SAFETY ENDPOINTS .................................................................................................................... 11 
4.4 TERTIARY/EXPLORATORY ENDPOINTS ................................................................................. 12 
5 ANALYSIS POPULATIONS ................................................................................................................. 12 
5.1 ENROLLED ..................................................................................................................................... 12 
5.2 FULL ANALYSIS SET (FAS) ......................................................................................................... 12 
5.3 SAFETY ........................................................................................................................................... 12 
5.4 PER-PROTOCOL (PP) ..................................................................................................................... 13 
6 INTERIM ANALYSIS ............................................................................................................................ 13 
7 STUDY SUBJECTS ................................................................................................................................ 13 
7.1 SUBJECT DISPOSITION ................................................................................................................ 13 
7.2 DEMOGRAPHICS AND BASELINE CHARACTERISTICS ........................................................ 13 
7.3 CONCOMITANT ILLNESS AND MEDICAL HISTORY ............................................................. 14 
7.4 INCLUSION/EXCLUSION CRITERIA .......................................................................................... 14 
7.5 STUDY DRUG ADMINISTRATION ............................................................................................. 14 
7.6 CONCOMITANT MEDICATION ................................................................................................... 14 
7.7 PROTOCOL DEVIATIONS ............................................................................................................ 15 
8 STATISTICAL METHODS OF DATA ANALYSES ............................................................................ 15 
8.1 GENERAL CONSIDERATIONS .................................................................................................... 15 
8.1.1 Statistical Notation and Presentation ......................................................................................... 15 
8.1.2 Hypothesis Testing and Multiplicity Adjustment ...................................................................... 16 


 
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8.1.3 Study Baseline ........................................................................................................................... 18 
8.1.4 Handling of Multiple Observations ............................................................................................ 18 
8.1.5 Handling of Missing or Partial Dates ......................................................................................... 19 
8.1.5.1 Adverse Event Date Imputations ........................................................................................ 19 
8.1.5.2 Medication Date Imputations .............................................................................................. 20 
8.1.6 Handling of Missing Efficacy Data and Subject Withdrawals .................................................. 20 
8.1.7 Censored Data ............................................................................................................................ 21 
8.2 EFFICACY ANALYSES ................................................................................................................. 22 
8.2.1 Primary Endpoint ....................................................................................................................... 22 
8.2.1.1 Sensitivity Analyses to the Primary Endpoint .................................................................... 23 
8.2.1.2 Additional Analyses for Inferences ..................................................................................... 23 
8.2.2 Key Secondary Endpoints .......................................................................................................... 23 
8.2.3 Analysis of Clinical Efficacy Endpoints .................................................................................... 24 
8.2.4 Analysis of Exposure Endpoints ................................................................................................ 25 
8.3 SAFETY ANALYSES ...................................................................................................................... 27 
8.3.1 Drug Exposure ........................................................................................................................... 27 
8.3.2 Adverse Events .......................................................................................................................... 27 
8.3.3 Clinical Laboratory Assessments ............................................................................................... 28 
8.3.4 Local Tolerability ....................................................................................................................... 28 
8.3.5 Other Safety Parameters............................................................................................................. 28 
8.3.6. Vital Signs ................................................................................................................................. 28 
8.3.7. Physical Examination ................................................................................................................ 28 
8.3.8. 12-Lead ECG ............................................................................................................................ 29 
8.3.9. Immunogenicity Data ................................................................................................................ 29 
8.4 OTHER ANALYSES ....................................................................................................................... 29 
8.4.1 Subgroup Analyses .................................................................................................................... 29 
8.4.2 Exploratory Analyses ................................................................................................................. 29 
9. CHANGES TO PLANNED ANALYSIS IN THE PROTOCOL ........................................................... 30 
10. STATISTICAL SOFTWARE ............................................................................................................... 30 
 
 
 


 
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List of Abbreviations 
Abbreviation Definition 
Abbreviation Definition 
ADA  
Anti-drug antibody 
AE  
Adverse event 
AESI 
Adverse events of special interest 
ANCOVA  
Analysis of covariance 
AUC  
Area under the concentration-time curve 
AUE  
Area under the effect curve 
BLQ 
Below the limit of quantitation 
CI  
Confidence interval 
Cmax  
Maximum plasma concentration 
CPH  
Cox proportional hazards 
ECG  
Electrocardiogram 
FAS  
Full analysis set 
HbA1c  
Glycated hemoglobin 
IV  
Intravenous(ly) 
KM  
Kaplan-Meier 
MedDRA  
Medical Dictionary for Regulatory Activities 
PD  
Pharmacodynamics 
PK  
Pharmacokinetics 
PP  
Per-protocol 
SAE  
Serious adverse event 
SAP  
Statistical analysis plan 
SC  
Subcutaneous(ly) 
T1DM  
Type 1 diabetes mellitus 
TEAE  
Treatment emergent adverse event 
tmax  
Time to the maximum plasma concentration 
 
 


 
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1 INTRODUCTION 
This document describes the statistical methods and procedures to be implemented in the 
confirmation of clinical safety and efficacy of dasiglucagon in the rescue treatment of 
hypoglycemia in subjects with type 1 diabetes mellitus (T1DM) compared to placebo. This 
statistical analysis plan is based on study protocol ZP4207-17145 (Version 2.0, 04 July 2018). If 
the data suggest and warrant it, deviations from this plan will be considered. However, any 
deviations from this SAP must be substantiated by sound statistical rationale and documented in 
the final clinical study report. 
2 STUDY OBJECTIVES 
2.1 PRIMARY OBJECTIVE 
The primary objective of this study is: 
• To demonstrate superiority of dasiglucagon compared to placebo following a single 
subcutaneous (SC) 0.6 mg dose administered to subjects with T1DM with insulin-
induced hypoglycemia. 
2.2 SECONDARY OBJECTIVES 
Secondary objectives of this study are: 
• To evaluate the safety, immunogenicity and PK of dasiglucagon following a single SC 
dose administered to subjects with T1DM with insulin-induced hypoglycemia as 
compared to placebo. 
2.3 TERTIARY/EXPLORATORY OBJECTIVES 
Tertiary/Exploratory objective of this study is: 
• To serve as a basis for explaining or supporting findings of primary analyses and for 
suggesting further hypotheses for later research. 


 
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3 STUDY OVERVIEW 
3.1 STUDY DESIGN 
The trial is a multi-center, randomized, parallel-group, double-blind, clinical efficacy and safety 
trial evaluating rescue treatment of insulin-induced hypoglycemia in subjects with T1DM. 
Subjects will be randomized to receive a single SC dose of dasiglucagon 0.6 mg or matching 
placebo in a ratio of 3:1, as shown in Table 1. It is expected that up to 46 subjects will be 
randomized to have 40 subjects completing the dosing visit (Visit 2).  
Table 1. Randomized Treatment Groups and Dose Regimens 
Treatment Group 
N 
Dose Regimen and Injection Site 
Dasiglucagon (liquid formulation, 1.0 mg/mL) 
30 
0.6 mL delivered in auto-injector 
Placebo (liquid formulation) 
10 
0.6 mL delivered in auto-injector 
 
Each subject will undergo a screening visit (Visit 1) up to 30 days prior to dosing, an overnight 
in-house visit (Visit 2), where dosing will take place, and a follow-up visit (Visit 3); the duration 
of participation for the subjects in this trial will approximately be 8 weeks. Additional follow-up 
visits may be required in case of the development of anti-drug antibodies (ADA). The trial will 
be conducted at up to 3 sites in North America. The following Figure 1 provides an overview of 
the trial design. 
 
 


 
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Figure 1. Trial design 
 
3.2 TRIAL PROCEDURES 
A screening visit (Visit 1) will be performed up to 30 days prior to the doing in order to identify 
eligible subjects. Prior to the in-house period (Visit 2), subjects will discontinue their current 
insulin therapy in due time to ensure wash-out. Subjects will check-in to the site on Day -1 for an 
overnight stay. Subjects’ eligibility to continue will be checked through assessment of 
withdrawal criteria. On Day 1, after an overnight fast, and upon check of eligibility according to 
Dosing Day Criteria, subjects will be randomized to dasiglucagon or placebo. Hypoglycemic 
induction procedure will take place and the trial drug will be administered when the subject’s 
plasma glucose level is 45-60 mg/dL. During the insulin-induced hypoglycemia, PK and PD 
samples will be drawn and plasma glucose levels will be monitored closely at site for safety 






 
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4 STUDY ENDPOINTS 
4.1 PRIMARY ENDPOINTS 
The primary endpoint is: 
• 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 without administration of 
rescue intravenous (IV) glucose. 
4.2 SECONDARY ENDPOINTS 
Key secondary endpoints are: 
• Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, and 
within 10 minutes after trial drug injection without administration of rescue IV glucose. 
• Plasma glucose changes from baseline at 30 minutes, at 20 minutes, at 15 minutes, and at 
10 minutes after trial drug injection or at the time of rescue. 
Clinical efficacy (PD) endpoints are: 
• 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 effect curve (AUE) above baseline from time 
zero to 30 minutes, AUE0-30min. 
Exposure (PK) endpoints are: 
• Area under the drug concentration curve from time zero to 90 minutes, AUC0-90min. 
• Area under the drug concentration curve from time zero to 120 minutes, AUC0-120min. 
• Maximum plasma drug concentration (Cmax). 
• Time to maximum plasma drug concentration (tmax). 
4.3 SAFETY ENDPOINTS 
Safety endpoints are: 
• Adverse events. 
• Clinical laboratory assessments (biochemistry, hematology, urinalysis). 


 
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•  Local tolerability. 
•  Number of rescue infusions of IV glucose after trial drug administration. 
• Time to first rescue infusion of IV glucose after trial drug administration. 
Immunogenicity endpoint is: 
• Occurrence of anti-drug-antibodies. 
4.4 TERTIARY/EXPLORATORY ENDPOINTS 
Tertiary/Exploratory endpoints include: 
• Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL (1.1 
mmol/L) within 30 minutes after trial 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. 
5 ANALYSIS POPULATIONS 
The following analysis populations will be used in study summaries and analyses: Enrolled, Full 
analysis set (FAS), Safety, and Per-Protocol (PP). 
5.1 ENROLLED 
The Enrolled population will consist of all enrolled subjects. The Enrolled population will be 
used for data listings. 
5.2 FULL ANALYSIS SET (FAS) 
The Full analysis set (FAS) population will consist of all randomized subjects who receive at 
least one dose of trial drug. The FAS population will be used for the analysis of the primary 
endpoint, secondary endpoints, and tertiary endpoints which will be analyzed as randomized 
(using planned treatment).  
5.3 SAFETY 
The Safety population will consist of all randomized subjects who receive at least one dose of 
trial drug. The Safety population will be used for all safety analyses. Summary tables of baseline 


 
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and demographic data will also be calculated for the Safety population. Safety analyses will be 
performed by actual treatment received. 
5.4 PER-PROTOCOL (PP) 
The Per-Protocol (PP) population will consist all subjects of the FAS for whom no relevant 
protocol deviations were documented. The decision to exclude a subject or exclude specific data 
from a subject should be taken at the blinded data review meeting before unblinding, and the 
exclusion from efficacy or other analyses justified in the signed notes of the meeting or 
documented in a memo that will be finalized prior to database lock.  Supportive analyses of the 
primary and key secondary efficacy endpoints will be based on the PP population. Additional 
exclusion and handling of data for the PP analysis is discussed in section 8.1.6. 
6 INTERIM ANALYSIS 
No interim analyses are planned. 
7 STUDY SUBJECTS 
7.1 SUBJECT DISPOSITION 
Subject disposition will be presented for all randomized subjects. Counts and percentages of 
subjects who completed or discontinued from the study will be summarized by treatment group. 
Reasons for early discontinuation include the following categories: safety concern at discretion 
of investigator, withdrawal of consent by subject, study discontinuation by sponsor and other.  
The number and percentage of randomized subjects will also be summarized by investigator site 
and treatment group. 
7.2 DEMOGRAPHICS AND BASELINE CHARACTERISTICS 
Demographics and baseline characteristics will only be assessed at screening (Visit 1). 
Demographics and baseline characteristics data will be summarized using descriptive statistics 
for, including but not limited to, age, sex, race and ethnicity, height, body weight, and body mass 
index (BMI). These summaries will be based upon the Safety analysis population. 


 
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7.3 CONCOMITANT ILLNESS AND MEDICAL HISTORY 
Concomitant illness and medical history will be summarized with descriptive statistics 
(frequencies and percentages) by concomitant illness (or medical history) code and treatment 
group for the Safety population. 
The date of diagnosis of diabetes will be recorded as will the current diabetes treatment (start 
date, product name(s), dose(s)) and will be listed. 
7.4 INCLUSION/EXCLUSION CRITERIA 
Inclusion and exclusion criteria failures will not be summarized but will be listed. 
7.5 STUDY DRUG ADMINISTRATION 
Subjects will receive their assigned single dose as SC injections. Study drug administration will 
be summarized and listed for the safety analysis set. 
7.6 CONCOMITANT MEDICATION 
The WHO DRUG Dictionary (September 2017) will be used to categorize verbatim descriptions 
of non-study medications into the Anatomic Therapeutic Chemistry (ATC) classification system. 
Each verbatim name will be classified by anatomical main group (ATC level 1), therapeutic 
subgroup (ATC level 2), pharmacological subgroup (ATC level 3), chemical subgroup (ATC 
level 4), and trade name.  
The number and percentage of subjects receiving prior concomitant, new concomitant, and any 
concomitant medications will be summarized by treatment group and ATC classification (ATC 
level 2 and level 4) for the Safety Population. An additional table for concomitant medications 
summarized by ATC classification and generic drug name will be provided. In order to 
characterize the use and the change in the use of anti-diabetic medications, separate summaries 
will be provided for number of anti-diabetic medications (e.g., 0, 1, 2, ≥3) the subjects received 
at baseline, and number of subjects who have an increase, decrease, and no change in their 
diabetes treatment regimen during the 8-week treatment period.  
Pre-treatment medications that start and stop prior to receiving randomized trial drug  will be 
listed. 


 
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7.7 PROTOCOL DEVIATIONS 
Protocol deviations (frequencies and percentages) will be summarized by treatment group for 
Safety population. All protocol deviations will be reviewed by clinical and statistical personnel 
to identify notable deviations (those anticipated to have an impact on efficacy or safety findings) 
prior to database lock. The identified protocol deviations may be used as one of the criteria to 
determine the subject’s eligibility for inclusion in the PP Population. 
8 STATISTICAL METHODS OF DATA ANALYSES 
8.1 GENERAL CONSIDERATIONS 
8.1.1 Statistical Notation and Presentation 
In all calculations, zero will be substituted for concentrations below the quantification limit of 
the assay. All data collected from all subjects will be presented in data listings. Both absolute 
values and change from baseline values for each subject will be given where applicable. All 
continuous data will be listed with the same precision as will be presented in the database. Data 
listings will be sorted by treatment, subject ID and time point. A missing value will be 
represented by an empty cell and no imputation will be made. 
Continuous data will be summarized in tables using number of subjects (n), mean, median, 
standard deviation (SD), standard error (SE), 95% confidence interval (CI; based on a t 
distribution if not otherwise stated), minimum and maximum by trial time point. For logarithm-
transformed data, the geometric mean and standard error of the geometric mean will also be 
provided. Negative values and values of zero will be treated as missing when calculating the 
geometric statistics since these values are undefined when log-transformed. Categorical data will 
be summarized in two ways, by subject and by time point. Subject data will be summarized using 
the count of distinct subjects that fall in the category and the percentage of the total number of 
subjects. Time point data will be summarized using the count of the assessments that fall into the 
category and the percentage of the total number of assessments. Population counts (either 
number of subjects or number of time points at the assessment) will be used as the denominator 
in the calculation of percentages unless otherwise specified. 


 
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Min and max values will use the precision of the original value. Means, least squares (LS) 
means, and medians will be rounded to one decimal place greater than the precision of the 
original value. SDs, SEs, and 95% confidence intervals (CIs) will be rounded to two decimal 
places greater than the precision of the original value. Derived PK and PD data, such as Cmax, 
AUC, and AUE, will be presented with three significant figures. Percentages will be rounded to 
the nearest tenth. P-values will be presented with four decimal places and values less than 0.0001 
will be presented as <.0001.  
8.1.2 Hypothesis Testing and Multiplicity Adjustment 
Define mediandasi and medianplacebo as the median time to an increase in plasma glucose of 20 
mg/dL of the 0.6 mg dose of dasiglucagon and placebo respectively. The hypotheses to be tested 
for the primary endpoint are: 
H0: mediandasi = medianplacebo 
Ha: mediandasi ≠ medianplacebo 
Define Ρdasi,i and Ρplacebo,i, i=30, 20, 15, 10, as the proportion of subjects of dasiglucagon and 
placebo respectively with plasma glucose recovery within i minutes after trial drug injection 
without administration of rescue IV glucose. The hypotheses to be tested for the key secondary 
endpoints 1-4 are: 
H0i: Ρdasi,i = Ρplacebo,i 
Hai: Ρdasi,i ≠ Ρplacebo,i 
where i=30, 20, 15, 10. 
Define µdasi,i and µplacebo,i, i=30, 20, 15, 10, as the mean plasma glucose change from baseline of 
dasiglucagon and placebo respectively at i minutes after trial drug injection or at the time of 
rescue. The hypotheses to be tested for the key secondary endpoints 5-8 are: 
H0i: µdasi,i = µplacebo,i 
Hai: µdasi,i ≠ µplacebo,i 


 
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where i=30, 20, 15, 10. 
A hierarchical procedure will be applied for the control of multiplicity. For the confirmatory 
analyses, the following a priori defined hierarchical inferential test order will be applied for the 
control of the type I 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 inferential statistical testing 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 secondary endpoints 1-4: Plasma glucose recovery within 30 minutes, within 20 
minutes, within 15 minutes, and within 10 minutes after trial drug injection without 
administration of rescue IV glucose. 
• Key secondary endpoints 5-8: Plasma glucose changes from baseline at 30 minutes, at 20 
minutes, at 15 minutes, and at 10 minutes after trial drug injection or at the time of 
rescue. 
The process is presented graphically in the following flow chart (Figure 2). 
 
 


 
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Figure 2. The Hierarchical Procedure of Key Secondary Endpoints 
 
 
8.1.3 Study Baseline 
For a particular study measurement, each subject’s baseline value for that measurement will be 
determined by sorting all scheduled or unscheduled values prior to the dosing of randomized trial 
drug using the date (or date and collection time for laboratory values). The last value collected in 
chronological order will be defined as the baseline value for that measurement.  
8.1.4 Handling of Multiple Observations 
For scheduled visit such as follow-up, the latest measurement within the visit window will be 
used for data summary and analysis. All measurements will be listed. 
 


 
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8.1.5 Handling of Missing or Partial Dates 
Date imputation will only be used for computational purposes; e.g., treatment-emergent status or 
identifying concomitant medications. Actual data values as they appear in the clinical database 
will be shown in the subject data listings. 
8.1.5.1 Adverse Event Date Imputations 
In cases of incomplete dates for adverse events (AEs), the missing component(s) will be assumed 
as the most conservative value(s) possible. For example, the imputation rule is to conservatively 
capture AEs with missing start dates as treatment emergent AEs (TEAEs) unless information of 
AE stop date/time was pre-treatment, which would automatically disqualify the treatment 
emergent status. Imputation of start dates for AEs with partial start dates is done for the purpose 
of identifying which AEs are treatment-emergent and to define what their onset dates are for the 
purpose of time to event analyses. The original values with no imputation applied will be 
presented in the data listings. The imputation rules follow. 
• If “day” is the only missing field, impute the “day” as the randomized dose date if the 
month and year of randomization are equal to the AE start month and year; otherwise, 
impute the “day” as the first day of the AE start month. 
• If “day” and “month” are missing, impute the “day” and “month” as the day and month 
of the randomized dose date if “year” is the same as the year of randomization; otherwise, 
impute January 1 of the non-missing year. 
• Missing time will not be imputed. If “time” is missing and the start date is the same as the 
dose date, then the time is assumed to be after the dose time so that the event will be 
classified as treatment emergent. 
•  If the start date is completely missing: 
O and from the end date (either complete or partial date) it can be deduced to be prior     
to the dose date, then the AE will not be assigned as a TEAE. 
     O and from the end date (either complete or partial date) it cannot be deduced to be prior  
            to the dose date, then the AE will be assigned as a TEAE. 


 
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8.1.5.2 Medication Date Imputations 
Imputation of start and end dates for medications partial dates is done for the purpose of 
classifying medications. The original values with no imputation applied will be presented in the 
data listings. The imputation rules follow. 
For medications with partial start dates: 
• If “day” is the only missing field, impute the “day” as the first of the month. 
• If “day” and “month” are the missing fields, impute the “day” and “month” to January 1. 
• Missing time will not be imputed. If the medication “time” is missing and the medication 
start date is the same as: 
o first dose date of randomized trial drug, then the medication is a new concomitant 
medication. 
For a medication that is not checked as ongoing and the start date is completely missing: 
• and from the end date (either complete or partial date) it can be deduced to be prior to the 
dose date of randomized trial drug, then medication is a pre-randomization medication. 
8.1.6 Handling of Missing Efficacy Data and Subject Withdrawals 
Missing safety data will be treated as missing with no imputation with the exception of date and 
time information for concomitant medications and adverse events as described in Section 8.1.5.1 
and 8.1.5.2. 
Missing efficacy data may occur when individuals 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, trial drug or an adverse 
event including hypoglycemia, linear interpolation can be applied to impute a missing value. The 
same construct of imputation applies for the analysis of the plasma glucose AUE0-30min. 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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Prior to database lock, the data will be reviewed during a blinded data review meeting where the 
decision to exclude subjects or exclude specific data will be made for the per protocol analysis. 
For the per protocol analysis, if a post dose pharmacodynamic glucose record during the first 45 
minutes is out of window by more than 1 minute (i.e. ±2 minutes), then the record will be 
flagged and the deviation will be identified as an important deviation.  
In the situation where data had been collected out of window by more than 1 minute, the 
following will be applied for the Per Protocol analysis only: 
• Out of window records that exceed the window by more than 1 minute will be treated as 
missing and will be imputed with linear interpolation 
• If the out of window record was taken later than expected, then the out of window record 
and the closest record collected just prior to the scheduled time of the record being 
imputed may be used for linear interpolation 
• If the out of window record was taken earlier than expected, then the out of window 
record and the closest record collected after the scheduled time of the record being 
imputed may be used for linear interpolation 
• The time point of the record being imputed must be within the time points of the records 
used for imputation 
For the primary analysis using the FAS, the out of window records will be used as observed and 
will not be linearly interpolated. 
8.1.7 Censored Data 
Based on the analysis, there are multiple censoring methods to consider during analyses: 
• In the primary analysis, subjects who require rescue IV glucose will be censored and time 
set to 45 min. 
o Sensitivity Analysis 1: subjects requiring rescue IV glucose will not be censored 
o Sensitivity Analysis 2: subjects requiring rescue IV glucose will be censored and 
time set to the administration of rescue IV glucose. 
o If recovery has not occurred at 45 minutes after study drug injection, censoring 
will be applied irrespective of the use of rescue IV glucose 


 
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• In the analysis of secondary efficacy endpoints, if the ≥70 mg/dL (3.9 mmol/L) 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. 
8.2 EFFICACY ANALYSES 
8.2.1 Primary Endpoint 
The primary endpoint will be evaluated on the FAS. The primary endpoint is 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 without administration of rescue IV glucose. This will be 
summarized using Kaplan-Meier (KM) estimates stratified by treatment group and injection site.  
In the primary analysis, recovery cannot be achieved in those subjects where IV glucose 
treatment is administered prior to recovery. Those subjects who receive IV glucose before 45 
minutes will be censored (i.e. set to 'not recovered’) at 45 minutes after dosing. Define T as the 
actual time to plasma glucose recovery of each subject. Assuming the data of the study are right-
censored. 
A Kaplan-Meier plot will be used to summarize the time to plasma glucose recovery analysis 
with the proportion of subjects at risk on the y-axis and time in minutes on the x-axis. Censored 
observations will be marked on the Kaplan-Meier curve.  
The treatment group difference between dasiglucagon and placebo will be evaluated inferentially 
using a pairwise two-sided log-rank test. 
As a supportive analysis to the stratified log-rank test for the time to plasma glucose recovery, 
dasiglucagon will be compared to placebo using a Wilcoxon test stratified by injection site. 
To graphically describe recovery in time, a histogram showing the proportions of patients that 
recover 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. 


 
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8.2.1.1 Sensitivity Analyses to the Primary Endpoint 
In sensitivity analyses, the time to plasma glucose recovery will be analyzed 1) without 
censoring 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. 
8.2.1.2 Additional Analyses for Inferences 
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.  
Under the proportional hazard assumption, the primary endpoint will be analyzed using a 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.  
The treatment group rate ratio (dasiglucagon versus placebo) will be estimated together with the 
95% profile confidence intervals. 
Treatment group inferences (dasiglucagon vs placebo) will be evaluated using the two-sided 
likelihood ratio test. 
Due to the discrete nature of blood sampling in time, the true recovery time is unmeasured. The 
method using “exact discrete” partial likelihood for tied failure times will be applied. The factor 
of injection site will be evaluated in the proportional hazards model by a type 3 test. 
To obtain an estimate of the true, but unmeasured, recovery times, a linear interpolation between 
the 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 analyzed in the same 
manner as for the discrete times. 
8.2.2 Key Secondary Endpoints 
The key secondary endpoints will be evaluated on the FAS. The key secondary endpoints are in 
this order: 


 
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(1) Plasma glucose recovery within 30 minutes, within 20 minutes, within 15 minutes, and 
within 10 minutes after trial drug injection without administration of rescue IV glucose. 
(2) Plasma glucose changes from baseline at 30 minutes, at 20 minutes, at 15 minutes, and at 
10 minutes after trial drug injection or at the time of rescue. 
(1) 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 (frequencies and percentages) by treatment group. 
Inferential treatment group comparisons will be assessed via pairwise tests of independent 
binomial proportions (dasiglucagon versus placebo) using Fisher’s exact test. 
(2) The key secondary endpoints of plasma glucose changes from baseline at 30 minutes, at 20 
minutes, at 15 minutes, and at 10 minutes after trial drug 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 the change from baseline in plasma glucose 
reached ≥20 mg/dL (1.1 mmol/L). Each of these changes from baseline variables will be 
analyzed using an analysis of covariance (ANCOVA) model, with treatment group (dasiglucagon 
and placebo) 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 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. 
In addition, line plots of 1) individual and 2) by treatment group mean plasma glucose 
concentration will be generated. 
Sensitivity analyses for the key secondary endpoints will be performed using the PP population 
using imputation rules described in Section 8.1.6. 
8.2.3 Analysis of Clinical Efficacy Endpoints 
The secondary clinical efficacy endpoints will be evaluated on the FAS (unless otherwise stated). 
Secondary clinical efficacy endpoints will be assessed based on plasma concentration data from 
samples collected at the dosing visit (Visit 2). 


 
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Time to first plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) from baseline will be 
evaluated using a KM 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) will be evaluated inferentially using pairwise two-sided log-rank tests. Those subjects 
whose time to first plasma glucose concentration  ≥70 mg/dL (3.9 mmol/L) is not met within 45 
minutes post-dosing will be censored, at the time of the last valid plasma glucose measurement 
up to 45 minutes. The mathematical procedures and SAS code are the same as that used for the 
primary endpoint analysis in 8.2.1, except the difference that here it is stratified only by 
treatment group. 
The AUE0-30min will be calculated as the baseline-adjusted area under the plasma glucose profile 
from time zero to 30 minutes. Subjects who received rescue IV glucose before 30 minutes will 
not have their AUE0-30min calculated. Calculation is determined by Linear Trapezoidal Method: 
AUE 0−30min = ෍1
2 [(𝐶𝐶𝑖𝑖−1 −𝐶𝐶0) + (𝐶𝐶𝑖𝑖−𝐶𝐶0)](𝑡𝑡𝑖𝑖−𝑡𝑡𝑖𝑖−1)
10
𝑖𝑖=1
 
where i from 1 to 10 represent the timepoints of PD sampling within 30 minutes, i.e., at 4, 6, 8, 
10, 12, 15, 17, 20, 25, 30 minutes after dosing. C0 is the baseline plasma glucose concentration 
sampling at t0 (pre-dose, defined as within 2-5 minutes prior to dosing), and Ci is the plasma 
glucose concentration at ti. t0 is assumed as 0 minutes, and ti is timepoints of PD sampling 
represented by i. 
The log-transformed AUE endpoint will be analyzed using an ANCOVA model with treatment 
group as fixed effect and baseline plasma glucose modeled as a covariate. The least squares 
means treatment group differences (and SE) will be back transformed (anti logged) for 
presentation as a ratio of the treatment group geometric means, with their corresponding 95% CI 
and p-value. 
8.2.4 Analysis of Exposure Endpoints 
Blood sampling for PK of ZP4207-17145 are to be collected at pre-dose, and at 15, 30, 35, 40, 
50, 60, 90, and 120 minutes after dosing. Pre-dose is defined as within 2-5 minutes prior to 


 
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dosing. The actual time of blood sampling should not deviate from the nominal time by more 
than ±1 minute. The exposure endpoints are given as below: 
• AUC will be derived as the area under the individual plasma dasiglucagon 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 time points. Calculation is determined by: 
AUC 0−90min = ෍1
2 (𝐶𝐶𝑖𝑖−1 + 𝐶𝐶𝑖𝑖)(𝑡𝑡𝑖𝑖−𝑡𝑡𝑖𝑖−1)
7
𝑖𝑖=0
 
where i from 0 to 7 represent the timepoints of blood sampling for PK from 0 to 90 minutes, i.e., 
at pre-dose, and at 15, 30, 35, 40, 50, 60 and 90 minutes after dosing. Ci is the individual plasma 
dasiglucagon concentration at i. ti is timepoints of PK sampling represented by i.  
• AUC 0 to 120 minutes or last valid measurement if this measurement is assessed 
sufficiently close to 120 minutes (decision to be taken at the masked data review 
meeting) will also be calculated. 
• Cmax will be determined as the maximum of all valid plasma dasiglucagon 
concentrations. 
• tmax will be determined as the time point where the maximum of all valid plasma 
dasiglucagon concentration measurements for each measurement series is observed. 
For these exposure endpoints calculations, concentration values that are below the limit of 
quantitation (BLQ) will be as follows:  
• Any post dose BLQ value will be set to missing for the purposes of PK parameter 
calculation.  
• Concentration at pre-dose that are BLQ will be defined as zero for calculation of AUC.  
These exposure endpoints will be summarized descriptively for each treatment group. For the 
descriptive statistics summary. In addition, line plots of 1) individual and 2) by treatment group 
mean plasma dasiglucagon concentration will be generated. 


 
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8.3 SAFETY ANALYSES 
8.3.1 Drug Exposure 
Drug exposure will be summarized and listed for the safety analysis set. 
8.3.2 Adverse Events 
AEs will be coded using the latest available version of the MedDRA, the version of which will 
be provided in the CTR. An overall summary table will be provided showing the number and 
percentage of subjects with any: 
• Treatment-emergent adverse event (TEAE) 
• Severe TEAE 
• Treatment-emergent SAE 
• Drug-related TEAE 
• Drug-related severe TEAE 
 
• Treatment-emergent drug-related SAE 
• TEAE leading to withdrawal 
• TEAE with outcome death 
• Adverse Events of Special Interest (AESI) 
o 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 
o Post-dose changes in pulse or blood pressure considered an event of hypo- or 
hypertension as judged by the investigator. 
The assessment should be based on signs and symptoms and Investigator clinical judgement. 
The following definitions can be used as a guidance  
o Orthostatic hypotension: decrease in systolic BP at least 20 mmHg (at least 30 
mmHg in patients with hypertension) and/or a fall in diastolic blood pressure of 
at least 10 mmHg and/or increase of heart rate at least 20 BPM within 3 minutes 
of standing associated with clinical symptoms such as dizziness or fainting  
o Vasovagal response: A sudden and clinically significant drop of blood pressure 
and/or heart rate associated with clinical symptoms such as pale skin and 
sweating, altered vision, dilated pupils or fainting  
o Bradycardia: a drop in heart rate more than 10 BPM from the last pre-dose 
resting value and a heart rate of less than 50 BPM 
 


 
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8.3.3 Clinical Laboratory Assessments 
Clinical laboratory test results will be flagged as to whether the result is below, within or above 
the respective reference range (Low, Normal, High, and missing). The number of values outside 
of the reference range will be counted. Observed values and change from baseline will be 
summarized for continuous assessments. Shift tables will also be generated. 
8.3.4 Local Tolerability 
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).  An overall summary table will be provided 
showing the number and percentage of these assessments. 
8.3.5 Other Safety Parameters 
Incidence of rescue infusion of IV glucose after trial drug administration will be analyzed using 
descriptive statistics (frequency and percentage) by treatment group. Inferential treatment group 
comparisons will be assessed via Fisher’s exact test (dasiglucagon versus placebo). 
Time to first rescue infusion of IV glucose after trial drug administration 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) 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.  
In addition, a KM plot of time to first rescue will be generated. 
A summary of total glucose infused prior to trial drug administration will be summarized by 
treatment group. 
8.3.6. Vital Signs 
Vital signs data will be summarized using descriptive statistics by treatment group. 
8.3.7. Physical Examination 
Physical examination data will be summarized using descriptive statistics by treatment group. 


 
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8.3.8. 12-Lead ECG 
12-lead ECG data will be summarized using descriptive statistics by treatment group. Shift tables 
will also be generated. 
8.3.9. 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. Post-baseline ADA-positive subjects 
will be calculated as a percentage of the total number tested for ADA at the follow-up visit. Titer 
levels will be reported as median and interquartile range. 
8.4 OTHER ANALYSES 
8.4.1 Subgroup Analyses 
Summary tables and KM plots describing the primary endpoint may be created based on age 
(≤40 years and >40 years), sex or other demographic characteristic(s) may be generated. 
8.4.2 Exploratory Analyses 
Plasma glucose concentration ≥70 mg/dL (3.9 mmol/L) or increase of ≥20 mg/dL (1.1 mmol/L) 
within 30 minutes after trial drug injection without administration of rescue IV glucose will be 
analyzed using descriptive statistics (frequency and percentage) by treatment group.  
Plasma insulin response as area under the curve above baseline from time zero to 60 minutes, 
AUC0-60 min, will be summarized by treatment group. The standard trapezoidal method will be 
used, based on actual rather than nominal time points. Calculation is determined by: 
AUC 0−60min = ෍1
2 [(𝐶𝐶𝑖𝑖−1 −𝐶𝐶0) + (𝐶𝐶𝑖𝑖−𝐶𝐶0)](𝑡𝑡𝑖𝑖−𝑡𝑡𝑖𝑖−1)
2
𝑖𝑖=1
 
where i from 1 to 2 represent the timepoints of Plasma insulin collecting within 60 minutes, i.e., 
at 30 and 60 minutes after dosing. C0 is the baseline plasma insulin collecting at t0 (pre-dose, 
defined as within 2-5 minutes prior to dosing), and Ci is the plasma insulin at ti. t0 is assumed as 
0 minutes, and ti is timepoints of plasma insulin response represented by i. 


 
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In addition, line plots of 1) individual and 2) by treatment group mean plasma insulin 
concentration will be generated. 
9. CHANGES TO PLANNED ANALYSIS IN THE PROTOCOL 
Added PK exposure endpoint, AUC 0-120 minutes. 
Added a descriptive analysis of total insulin infused and total glucose infused after study drug 
administration. 
Added actual time analysis of primary endpoint using interpolation. 
10. STATISTICAL SOFTWARE 
All statistical analyses will be performed using SAS® version 9.4.