Clinical trial NCT02206152
Conditions: Type 1 Diabetes Hypoglycemia Unawareness
Keywords: Diabetes Hypoglycemia Unawareness HAAF
Sponsor: University of Minnesota
Start date: 2015-02-01; Completion date: 2017-12-01
Enrollment: 22 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
This is a single center, double blind randomized cross over design trial that will compare the impact of N-acetyl cysteine (200 mg) vs. saline infusion during experimental hypoglycemia on day one on the responses to experimental hypoglycemia on day two. 18 participants will be studied twice, 8 weeks apart. On each occasion they will undergo a 2 hour hypoglycemic clamp (target 50 mg/dl) in the morning and in the afternoon on day one and then again on the morning of day 2 and day 3. During the morning clamps, samples will be collected for later measurement of serum epinephrine levels, plasma and red blood cell NAC, cysteine, and glutathione concentrations and GSH/GSSG ratios (redox status), and participants will be asked to complete a hypoglycemia symptom questionnaire

Detailed description:
This study will consist of a screening visit and two 2-day intervention visits separated by approximately 8 weeks.

At the screening visit, informed consent will be obtained, a standardized form will be used to ensure subjects meet inclusion/exclusion criteria, and baseline hemoglobin A1c will be obtained. On arrival, two IVs will be placed and they will be randomized to receive NAC or saline infusion. Baseline blood samples will be drawn for glucose, epinephrine, norepinephrine, cortisol, cysteine and glutathione measurements. Subjects will then be given 25 mg IV diphenhydramine (Benadryl) followed by a 60 minute infusion of NAC (150 mg) or a similar volume of saline between 8 am and 9 am, followed by a four hour infusion of 50 mg of NAC. Thirty minutes after the start of the NAC infusion, a hyperinsulinemic (2.0 mu/kg/min) hypoglycemic (target = 50 mg/dl) clamp protocol will be started. During the morning study, blood samples will be collected every 5 minutes for monitoring of blood glucose levels and every 15 minutes for later measurement of serum epinephrine, norepinephrine, cortisol. Plasma and red blood cells samples will also be collected every 15 minutes for later measurement of NAC, cysteine, glutathione, and GSH/GSSG ratios (redox status). During the final 15 minutes of the morning clamp, subjects will be asked to quantitate their symptoms using a standardized method (13). At the completion of the morning clamp, glucose will be given to return the participant to euglycemia. 2 hours after the end of the morning clamp, a second hyperinsulinemic (2.0 mu/kg/min) hypoglycemic (target = 50 mg/dl) clamp protocol will be followed over two hours. . The afternoon clamp study will proceed as in the morning except that no serum or plasma will be collected except for the monitoring of glucose and collection of samples for subsequent measurement of NAC, cysteine, glutathione, and GSH/GSSG ratios. After the completion of the afternoon clamps, subjects will be returned to euglycemia and fed a meal. They will be discharged home.

At 7 AM the following morning they will under a single 2 hour hyperinsulinemic hypoglycemic stepped clamp (75, 65, 55,45 mg/dl targets) during which blood samples will be collected as on the morning of day 1. Symptom scores will be collected in the final 15 minutes of each step in the clamp.

At the completion of the study on day 2 of Part 1, they will be scheduled to return for Part 2 in 8 weeks. This timing is selected to ensure that female participants are studied at the same phase of the menstrual cycle. During Part 2 they will receive the treatment not provided during Part 1 in a blinded fashion. The rest of the study protocol will be the same as in Part 1.

Arms: arm 1: Normal Saline IV infusion given during a controlled hyperinsulinemic hypoglycemic insulin clamp arm 2: N-acetyl cysteine IV infusion given as a 150 mg/kg loading dose over the first hour and then follow that with a 50 mg/kg maintenance dose infused over the next 4 hours during a controlled hyperinsulinemic hypoglycemic insulin clamp
Interventions: intervention 1: N-acetyl cysteine intervention 2: Placebo
Intervention descriptions: intervention 1: N-acetyl cysteine (or saline) IV infusion given as a 150 mg/kg loading dose over the first hour and then follow that with a 50 mg/kg maintenance dose infused over the next 4 hours during a controlled hyperinsulinemic hypoglycemic insulin clamp intervention 2: None

Protocol documents:
Prot_SAP_000.pdf: 
A randomized double blinded study to examine the use of N-acetyl cysteine for the 
prevention and treatment of HAAF in patients with type 1 diabetes. 
Regulatory Sponsor: 
Elizabeth Seaquist MD 
University of Minnesota 
612-626-4833
Funding Sponsor: 
JDRF
Study Product: 
NAC 
Protocol Number: 
NA 
IND/IDE Number: 
122861 
Date of Protocol Updated: September 17, 2015
Date of IRB Approval for Protocol Version: October 12, 2015 
CONFIDENTIAL 
This document is confidential and the property of the University of Minnesota.  No 
part of it may be transmitted, reproduced, published, or used by other persons 
without prior written authorization from the study sponsor. 


 
 
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Table of Contents 
STUDY SUMMARY .................................................................................................................................................... 1 
1 
INTRODUCTION ............................................................................................................................................... 3 
1.1 
BACKGROUND .............................................................................................................................................. 3 
1.2 
INVESTIGATIONAL AGENT............................................................................................................................. 3 
1.3 
PRECLINICAL DATA ...................................................................................................................................... 3 
1.4 
CLINICAL DATA TO DATE ............................................................................................................................. 3 
1.5 
DOSE RATIONALE AND RISK/BENEFITS ....................................................................................................... 4 
2 
STUDY DESIGN ................................................................................................................................................ 5 
2.1 
GENERAL DESIGN ........................................................................................................................................ 5 
2.2 
PRIMARY STUDY ENDPOINTS ...................................................................................................................... 5 
2.3 
SECONDARY STUDY ENDPOINTS ................................................................................................................. 5 
2.4 
PRIMARY SAFETY ENDPOINTS ..................................................................................................................... 5 
3 
SUBJECT SELECTION AND WITHDRAWAL............................................................................................. 5 
3.1 
INCLUSION CRITERIA .................................................................................................................................... 5 
3.2 
EXCLUSION CRITERIA .................................................................................................................................. 6 
3.3 
SUBJECT RECRUITMENT AND SCREENING .................................................................................................. 6 
3.4 
EARLY WITHDRAWAL OF SUBJECTS ............................................................................................................ 6 
3.4.1 
When and How to Withdraw Subjects ........................................................................................... 6 
3.4.2 
Data Collection and Follow-up for Withdrawn Subjects .............................................................. 6 
4 
STUDY DRUG ................................................................................................................................................... 7 
4.1 
DESCRIPTION ............................................................................................................................................... 7 
4.2 
TREATMENT REGIMEN ................................................................................................................................. 7 
4.3 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ................................................................. 7 
4.4 
SUBJECT COMPLIANCE MONITORING .......................................................................................................... 7 
4.5 
PRIOR AND CONCOMITANT THERAPY .......................................................................................................... 7 
4.6 
PACKAGING .................................................................................................................................................. 7 
4.7 
BLINDING OF STUDY DRUG .......................................................................................................................... 7 
4.8 
RECEIVING, STORAGE, DISPENSING AND RETURN ..................................................................................... 7 
4.8.1 
Receipt of Drug Supplies ................................................................................................................. 7 
4.8.2 
Storage ............................................................................................................................................... 8 
4.8.3 
Dispensing of Study Drug ................................................................................................................ 8 
4.8.4 
Return or Destruction of Study Drug .............................................................................................. 8 
5 
STUDY PROCEDURES ................................................................................................................................... 8 
6 
STATISTICAL PLAN ...................................................................................................................................... 10 
6.1 
SAMPLE SIZE DETERMINATION .................................................................................................................. 10 
6.2 
STATISTICAL METHODS ............................................................................................................................. 10 
6.3 
SUBJECT POPULATION(S) FOR ANALYSIS ................................................................................................. 11 
7 
SAFETY AND ADVERSE EVENTS ............................................................................................................. 11 
7.1 
DEFINITIONS ............................................................................................................................................... 11 
7.2 
RECORDING OF ADVERSE EVENTS............................................................................................................ 13 
7.3 
REPORTING OF SERIOUS ADVERSE EVENTS ............................................................................................ 13 
7.3.1 
Study Sponsor Notification by Investigator ................................................................................. 13 
7.3.2 
IRB Notification by Investigator .................................................................................................... 14 
7.3.3 
FDA Notification by Sponsor ......................................................................................................... 14 
7.4 
UNBLINDING PROCEDURES........................................................................................................................ 15 
7.5 
STOPPING RULES ...................................................................................................................................... 15 
7.6 
MEDICAL MONITORING............................................................................................................................... 16 
7.6.1 
Internal Data and Safety Monitoring Board ................................................................................. 16 


 
 
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8 
DATA HANDLING AND RECORD KEEPING ........................................................................................... 17 
8.1 
CONFIDENTIALITY ....................................................................................................................................... 17 
8.2 
SOURCE DOCUMENTS ............................................................................................................................... 17 
8.3 
CASE REPORT FORMS ............................................................................................................................... 17 
8.4 
RECORDS RETENTION ............................................................................................................................... 18 
9 
STUDY MONITORING, AUDITING, AND INSPECTING.......................................................................... 18 
9.1 
STUDY MONITORING PLAN ........................................................................................................................ 18 
9.2 
AUDITING AND INSPECTING ........................................................................................................................ 18 
10 
ETHICAL CONSIDERATIONS ..................................................................................................................... 18 
11 
STUDY FINANCES ......................................................................................................................................... 19 
11.1 
FUNDING SOURCE...................................................................................................................................... 19 
11.2 
CONFLICT OF INTEREST ............................................................................................................................. 19 
11.3 
SUBJECT STIPENDS OR PAYMENTS ........................................................................................................... 19 
12 
PUBLICATION PLAN .................................................................................................................................... 19 
13 
REFERENCES ................................................................................................................................................ 20 
14 
ATTACHMENTS ............................................................................................................................................. 20 
 


 
 
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List of Abbreviations 
 
 
List all abbreviations used in protocol 
 
 
AE 
 
Adverse Event 
 
CFR  
Code of Federal Regulations 
 
CRF  
Case Report Form 
 
 
DSMB  
Data and Safety Monitoring Board 
 
DSMP  
Data and Safety Monitoring Plan 
 
FDA  
Food and Drug Administration 
 
GCP  
Good Clinical Practice 
 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 
 
ICH 
 
International Conference on Harmonisation 
 
IDS 
 
Investigational Drug Services 
 
IRB 
 
Institutional Review Board 
 
PHI 
 
Protected Health Information 
 
SAE  
Serious Adverse Event 
 
 
 


Clinical Research Protocol Template 
 
Page 1 
Version 3210.22.14 
Study Summary 
Title 
A randomized double blinded study to examine the use of N-acetyl 
cysteine for the prevention and treatment of HAAF in patients with type 
1 diabetes                                                         
Short Title 
NAC for HAAF 
Protocol Number 
NA 
Phase 
I/II 
Methodology 
This is a single center, double blind randomized cross over design trial 
that will compare the impact of N-acetyl cysteine (200 mg/kg) vs. saline 
infusion during experimental hypoglycemia on day one on the 
responses to experimental hypoglycemia on day two.  18 participants 
will be studied twice, 8 weeks apart.  On each occasion they will 
undergo a 2 hour hypoglycemic clamp (target 50 mg/dl) in the morning 
and in the afternoon on day one and then again on the morning of day 
2. During the morning clamps, samples will be collected for later 
measurement of serum epinephrine levels, plasma and red blood cell 
NAC, cysteine, and glutathione concentrations and GSH/GSSG ratios 
(redox status), and participants will be asked to complete a 
hypoglycemia symptom questionnaire 
Study Duration 
10-12 weeks per subject 
Study Center(s) 
Single Center 
Objectives 
Primary endpoint will be the within person difference in epinephrine 
secretion during the morning episodes of hypoglycemia on days one 
and two under the two treatment conditions.  Epinephrine secretion 
during hypoglycemia is assessed by collecting blood samples for 
measurement of epinephrine concentrations at baseline and every 15 
minutes during the period of hypoglycemia (starting at point where 
blood glucose is first < 55 mg/dl) in the clamp studies done in the 
mornings of days 1, 2, and 3 of both parts 1 and 2.   
Number of 
Subjects 
36 
Diagnosis and 
Main Inclusion 
Criteria 
Healthy adults ages 18-65 years with hemoglobin A1c < 6.0%. 
Exclusion criteria will include history of diabetes, hyperglycemia, stroke, 
seizures, arrhythmias, active cardiac disease; pregnancy or plan to 
become pregnant during the study period; diagnosis of asthma; use of 
anti-oxidants or drugs that can alter glucose metabolism; and 
concomitant medical problems that may prevent the subject from 
successfully completing the protocol. 
Study Product, 
Dose, Route, 
Regimen 
N-acetyl cysteine 200 mg/kg (or saline) IV infusion (given as  a 150 
mg/kg loading dose over the first hour and then followed by a 50 mg/kg 
maintenance dose infused over the next 4 hours on day 1 of each of 
the two parts of the study) 
Duration of 
administration 
5 hours on day one 


 
 
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Reference therapy 
NAC will be tested against placebo. 
Statistical 
Methodology 
Our primary outcome is the peak epinephrine response during the third 
hypoglycemic clamp of each infusion experiment, thus each participant 
will have two observations: one from their NAC infusion experiment and 
one from their saline infusion experiment. We will fit a general linear 
mixed model with fixed effects for treatment (NAC vs. saline), treatment 
order (NAC first vs. saline first), and period (Part 1 vs. Part 2) and a 
random effect for participant. We will also consider adjustment for 
important baseline characteristics, such as age, gender, and duration of 
diabetes. Diagnostics will be examined to assess model assumptions. 
Secondary outcomes include: the within-person difference in peak 
epinephrine response between the first hypoglycemic clamp (when an 
infusion is given) and the third hypoglycemic clamp (when no infusion is 
given) of each infusion experiment; symptom scores during the third 
hypoglycemic clamp of each infusion experiment; and the within-person 
difference in symptom scores between the first hypoglycemic clamp 
and the third hypoglycemic clamp of each infusion experiment 
 


 
 
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1 Introduction 
 
This document is a protocol for a human research study. This study will be conducted 
according to US and international standards of Good Clinical Practice (FDA Title 21 part 
312 or 812 and International Conference on Harmonisation guidelines), applicable 
government regulations and Institutional research policies and procedures.  
1.1 Background 
The purpose of this project is to evaluate N-acetyl cysteine (NAC) as a therapy for the 
prevention and treatment of hypoglycemia associated autonomic failure (HAAF) in 
patients with type 1 diabetes. Impaired awareness of hypoglycemia occurs in 
approximately 25% of adults with type 1 diabetes and is associated with a six fold 
higher risk of having severe hypoglycemia that requires the assistance of another to 
recognize and treat. HAAF is also the primary barrier to achieving the level of glucose 
control necessary to prevent the microvascular complications of the disease and 
contributes to the mortality seen in patients with T1DM. To fully evaluate NAC as a 
therapy, it is imperative that its action in humans be examined. 
1.2 Investigational Agent 
N-acetyl cysteinev(or saline) IV infusion given as a 150 mg/kg loading dose over the 
first hour and then follow that with a 50 mg/kg maintenance dose infused over the next 4 
hours 
1.3 Preclinical Data 
In animal models, administration of N-acetyl cysteine (NAC) during episodes of 
hypoglycemia prevents the development of HAAF, possibly by preventing the 
hypoglycemia induced rise in hypothalamic reactive oxygen species known to occur 
during acute episodes of hypoglycemia . 
1.4 Clinical Data to Date 
The sponsor investigator completed a randomized placebo controlled trial of 
naltrexone as a therapy of patients with type 1 diabetes hypoglycemia unawareness 
in which 30 subjects enrolled over 3 years. One third were unable to complete the 
study: one because of rising liver function tests during the drug treatment and the 
rest for technical reasons related to the magnetic resonance imaging part of the 
study. In another study in which the default mode network was evaluated during 
hypoglycemia, we enrolled 11 people with type 1 diabetes and awareness of 
hypoglycemia, 9 people with type 1 diabetes and unawareness’ of hypoglycemia, 
and 11 controls over an 18 months period.  NAC has not been used in humans as a 
treatment of hypoglycemia unawareness in type 1 diabetes. 
 
 


 
 
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1.5 Dose Rationale and Risk/Benefits 
This is a proof of principle experiment in which the maximal dose of NAC approved for 
human use will be intravenously infused to ensure that high serum concentrations of the 
drug are achieved.  We propose a 200 mg/kg dose in which we will infuse a 150 mg/kg 
loading dose over the first hour followed by a 50 mg/kg maintenance dose infused over 
the next 4 hours, as is recommended for acetaminophen overdose,   
 
In the package insert, the manufacturer includes a table that lists the incidence of drug 
related adverse events following drug administration.  Data from 180 individuals are 
included in the table; 10 experienced mild anaphylactoid reactions (6%), 16 experienced 
moderate reactions (9%) and 2 experienced severe reactions (1%).  All of these 
reactions occurred within the first 2 hours following the drug administration, following a 
dosage schedule we plan to use for this protocol.  The package insert also includes a 
table that summarizes the adverse events observed in 76 published articles in which 
NAC was intravenously infused.  The Rocky Mountain Poison and Drug Center 
prepared this table from information collected on 2040 subjects.  1.67% were noted to 
have urticara, 1.47% had vasodilatation and rash, 0.78% had hypotension.  Our 
protocol excludes individuals with asthma or with a history of bronchospasm in an effort 
to reduce the risk of severe anaphylactoid reactions.  We believe the small risk of 
adverse events from NAC exposure are reasonable given the impact of hypoglycemia 
unanwareness on patients with diabetes. The first sign that these patients have 
hyogycmeia is confusion or loss of consciousness, both of which can lead to injury from 
accidents.  If untreted, this hypogycmeia could cause seizures and death.  The number 
of patients with type 1 diabetes who experience hypoglycemia unawareness has been 
estimated to be as high as 25%. 
 
Primary Objective 
Primary endpoint will be the within person difference in epinephrine secretion during the 
morning episodes of hypoglycemia on days one and two under the two treatment 
conditions.  Epinephrine secretion during hypoglycemia is assessed by collecting blood 
samples for measurement of epinephrine concentrations at baseline and every 15 
minutes during the period of hypoglycemia (starting at point where blood glucose is first 
< 55 mg/dl) in the clamp studies done in the mornings of days 1 and 2 of both parts 1 
and 2. 
 
Secondary Objective 
Secondary endpoint will be within person difference in symptom scores collected during 
the morning episodes of hypoglycemia on days one and two under the two treatment 
conditions.  Symptom scores during hypoglycemia will be quantified using a previously 
validated questionnaire at baseline and at the end of the hypoglycemic clamp studies 
done in the mornings of days 1 and 2 of both parts 1 and 2.. 


 
 
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2 Study Design 
2.1 General Design 
This is a single center, double blind randomized cross over design trial that will compare 
the impact of NAC vs. saline infusion during experimental hypoglycemia on day one on 
the responses to experimental hypoglycemia on day two. Figure 1 shows the protocol in 
schematic form.  We intend to enroll 18 individuals to obtain the complete data sets 
from 15 participants.  
 
Expected duration of subject participation is 10-12 weeks. This study will consist of a 
screening visit and two 2-day intervention visits separated by approximately 8 weeks. 
 
2.2 Primary Study Endpoints 
Primary endpoint will be the within person difference in epinephrine secretion during the 
morning episodes of hypoglycemia on days one and two under the two treatment 
conditions.  Epinephrine secretion during hypoglycemia is assessed by collecting blood 
samples for measurement of epinephrine concentrations at baseline and every 15 
minutes during the period of hypoglycemia (starting at point where blood glucose is first 
< 55 mg/dl) in the clamp studies done in the mornings of days 1 and 2 of both parts 1 
and 2. 
2.3 Secondary Study Endpoints 
Secondary endpoint will be within person difference in symptom scores collected during 
the morning episodes of hypoglycemia on days one and two under the two treatment 
conditions.  Symptom scores during hypoglycemia will be quantified using a previously 
validated questionnaire at baseline and at the end of the hypoglycemic clamp studies 
done in the mornings of days 1 and 2 of both parts 1 and 2. 
2.4 Primary Safety Endpoints 
The safety endpoint is ascertainment of symptoms related to allergic reactions (pruritus, 
swelling/tingling of mouth or tongue, shortness of breath) at baseline and at 30, 60, and 
90 minutes after the start of the NAC/saline infusion.  Participants’ symptoms will be 
ascertained by the use of a standardized questionnaire. 
3 Subject Selection and Withdrawal 
3.1 Inclusion Criteria 
• Healthy volunteer 
• Age 18 – 65 years 
• Hemoglobin A1c < 6.0% 


 
 
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3.2 Exclusion Criteria 
• History of diabetes, hyperglycemia, stroke, seizures, arrhythmias, active 
cardiac disease  
• Pregnancy or plan to become pregnant during the study period 
• Diagnosis of asthma (increases risk of hypersensitivity reactions to NAC) 
• Use of anti-oxidants or drugs that can alter glucose metabolism 
• Concomitant medical problems that may prevent the subject from successfully 
completing the protocol 
• Unwillingness to avoid exercise during the 7 days before each part of the study 
3.3 Subject Recruitment and Screening 
Subjects will be recruited from a registry of normal volunteers who are interested in 
participating in research that is maintained by the investigator.  In addition, subjects will 
be drawn from the community using IRB approved public notices.  The only laboratory 
testing necessary to meet inclusion/exclusion criteria will be a hemoglobin A1c done in 
the last month. 
3.4 Early Withdrawal of Subjects 
3.4.1 When and How to Withdraw Subjects 
Subjects will be withdrawn at any point during the study if they remove their 
consent or if they develop any of the exclusion criteria after enrollment. In 
addition, any subjects who experience the symptoms of an allergic reaction to 
the NAC severe enough that the infusion is stopped early will be withdrawn 
from the study at the time the infusion is stopped (before unblinding that 
participant’s treatment assignment). Procedures for withdrawal include 
collecting the necessary information to provide them with the compensation 
due them for the time they devoted to the study. At the end of the study, after 
treatment assignments are unblinded, a letter will be sent to the any subject 
withdrawn because of symptoms of an allergic reaction to NAC informing 
them of the treatment they were given. If they had been given NAC, we will 
instruct them to share the letter with their personal physician so the allergy 
could be noted in the clinical record. 
3.4.2 Data Collection and Follow-up for Withdrawn Subjects 
Data will be collected per the protocol up until the point the subject is 
withdrawn from the study. No additional data will be collected from subjects 
after their withdrawal unless they were withdrawn due to early stopping of the 
infusion.  For such participants, a follow-up phone call within 1-2 days will 
ascertain their status and if hospitalized we will request consent to access 
hospitalization records. NAC is already used in clinical practice for other 
indications and it does not have an effect on survival. 
 


 
 
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4 Study Drug  
4.1 Description 
N-acetyl cysteine (or saline) IV infusion given as a 150 mg/kg loading dose over the 
first hour and then follow that with a 50 mg/kg maintenance dose infused over the 
next 4 hours 
4.2 Treatment Regimen 
A 150 mg/kg loading dose of NAC or saline over the first hour followed by a 50 mg/kg 
maintenance dose infused over the next 4 hours on days one of both parts of the study. 
4.3 Method for Assigning Subjects to Treatment Groups 
The investigational pharmacy at the University of Minnesota will be responsible for 
implementing the randomization order in which participants are given NAC or 
saline, prepared by the study statistician. This information will be blinded to 
investigators and participants and they will be unblinded only at the end of the 
study. 
4.4 Subject Compliance Monitoring 
Treatment will be witnessed by the investigators, so compliance will be assumed.  
4.5 Prior and Concomitant Therapy 
No restrictions 
4.6 Packaging 
• See Section 5.1 
4.7 Blinding of Study Drug 
The investigational pharmacy at the University of Minnesota Medical Center will prepare 
and dispense NAC or saline in blinded fashion.  The solutions will be prepared to look 
the same so blinding is maintained.  Half normal saline (0.45% NaCl) will be used as the 
dilutent in the solution.  Only the investigational pharmacy will have record of the drug 
assignment link to participant IDs. 
4.8 Receiving, Storage, Dispensing and Return 
4.8.1 Receipt of Drug Supplies 
 
Upon receipt of the study treatment supplies, an inventory will be performed and a 
drug receipt log filled out and signed by the person accepting the shipment.  The 
designated study staff will count and verify that the shipment contains all the items 
noted in the shipment inventory.  Any damaged or unusable study drug in a given 
shipment (active drug or comparator) will be documented in the study files.   


 
 
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4.8.2 Storage 
See section 5.91 
4.8.3 Dispensing of Study Drug 
The investigational pharmacy at the University of Minnesota Medical Center will 
dispense the study drug or saline and will follow standard pharmaceutical protocols 
to reconcile the stock. 
4.8.4 Return or Destruction of Study Drug 
At the completion of the study, there will be a final reconciliation of drug shipped, 
drug consumed, and drug remaining.  This reconciliation will be logged on the drug 
reconciliation form, signed and dated.  Any discrepancies noted will be 
investigated, resolved, and documented prior to return or destruction of unused 
study drug.  Drug destroyed on site will be documented in the study files. 
5 Study Procedures 
This study will consist of a screening visit and two 3-day intervention visits 
separated by approximately 8 weeks. 
 
At the screening visit (approximately 2-4 days prior to study day one) informed 
consent will be obtained, the clinical history will be reviewed to be certain a 
volunteer meets the inclusion critieria and baseline hemoglobin A1c will be 
obtained.  
 
Subjects will be asked to report to the research center by 7 am on the first day of the 
study after fasting overnight. On arrival, intravenous lines will be placed in each arm 
(one will be used for infusions and the other for blood sampling).  A minimum of 30 
minutes after the IVs are placed, baseline blood samples will be drawn for glucose, 
epinephrine, norepinephrine, cortisol, cysteine and glutathione measurements. Subjects 
will then be given diphenhydramine 25 mg IV push immediately priot to the start of a 60 
minute infusion of NAC (150 mg) or a similar volume of saline between 8 am and 9 am, 
followed by a four hour infusion of 50 mg of NAC.  Thirty minutes after the start of the 
NAC infusion, a hyperinsulinemic (2.0 mu/kg/min) hypoglycemic (target = 50 mg/dl) 
clamp protocol will be started.  During the morning study, blood samples will be 
collected every 5 minutes for monitoring of blood glucose levels and every 15 minutes 
for later measurement of serum epinephrine, norepinephrine, cortisol. Plasma and red 
blood cells samples will also be collected every 15 minutes for later measurement of 
NAC, cysteine, glutathione, and GSH/GSSG ratios (redox status). During the final 15 
minutes of the morning clamp, subjects will be asked to quantitate their symptoms using 
a standardized method (13). At the completion of the morning clamp, glucose will be 
given to return the participant to euglycemia. 2 hours after the end of the morning 
clamp, a second hyperinsulinemic (2.0 mu/kg/min) hypoglycemic (target = 50 mg/dl) 
clamp protocol  will be followed over two hours.  The afternoon clamp study will proceed 
as in the morning except that no serum or plasma will be collected except for the 
monitoring of glucose and collection of samples for subsequent measurement of NAC, 


 
 
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cysteine, glutathione, and GSH/GSSG ratios. After the completion of the afternoon 
clamps, subjects will be returned to euglycemia and fed a meal.  They will then be 
allowed to leave the research center with instructions to return the next morning. At 7 
AM on day 2 they will under a single 2 hour hyperinsulinemic hypoglycemic stepped 
clamp (75, 65, 55, 45 mg/dl targets) during which blood samples will be collected as on 
the morning of day 1. Symptom scores will be collected in the final 15 minutes of each 
step in the clamp.  At the end of the study, infusions will be stopped, they will be fed and 
meal and then be sent home after they are scheduled to return for Part 2 in 8 weeks. 
This timing is selected to ensure that female participants are studied at the same phase 
of the menstrual cycle; males will return for Part 2 in 8±2 weeks. During Part 2 they will 
receive the treatment not provided during Part 1 in a blinded fashion. The rest of the 
study protocol will be the same as in Part 1. 
A description of the study visits is below: 
Visit description 
Expected activities 
Screening 
• Confirm subject qualification 
• Obtain informed consent 
• Baseline A1c 
 
Part 1 Day 1 
• Randomization 
• Morning (8 – 10 AM) hyperinsulinemic hypoglycemic clamp 
study with infusion of NAC or saline depending on 
randomization assignment, collection of counterregulatory 
hormones and hypoglycemia symptom scores 
• Afternoon (1 – 3 PM) hyperinsulinemic hypoglycemic clamp 
study with infusion of NAC or saline depending on 
randomization assignment 
 
Part 1 Day 2 
• Hyperinsulinemic stepped hypoglycemic clamp study with 
collection of counterregulatory hormones and hypoglycemia 
symptom scores 
• Lunch 
8 week wash out period Usual activities 
Part 2 Day 1 
• Morning (8 – 10 AM) hyperinsulinemic hypoglycemic clamp 
study with infusion of NAC or saline depending on 
randomization assignment, collection of counterregulatory 
hormones and hypoglycemia symptom scores 
• Afternoon (1 – 3 PM) hyperinsulinemic hypoglycemic clamp 
study with infusion of NAC or saline depending on 
randomization assignment 
 
Part 2 Day 2 
Hyperinsulinemic hypoglycemic clamp study with collection of 
counterregulatory hormones and hypoglycemia symptom 
scores 
• Lunch 


 
 
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6 Statistical Plan 
6.1 Sample Size Determination 
We propose a two-period two-treatment cross-over study, where each participant 
gets NAC infusion and saline infusion, in randomized order as shown in Figure 
under 2.1. Our primary outcome is the peak epinephrine response during the 
third hypoglycemic clamp of each infusion experiment, thus each participant will 
have two observations: one epinephrine response from their NAC infusion 
experiment and one epinephrine response from their saline infusion experiment. 
We expect to recruit 18 individuals in order to complete 15 studies. Additional 
participants will be recruited if needed to have 15 completed studies. If NAC 
prevents HAAF, then the epinephrine response during the third hypoglycemic 
clamp after NAC infusion will still be high, while the epinephrine response during 
the third hypoglycemic camp after saline infusion will be blunted. Therefore this 
within-person difference in epinephrine response will be large and positive. 
Assuming a one-sided type I error of 5% and a power of 85%, we can detect an 
epinephrine response difference between NAC and saline of at least 154 units. 
This magnitude of difference is comparable to the difference between healthy 
controls and subjects with T1DM and hypoglycemia unawareness that we found 
in a previous study. In that study, participants underwent a hyperinsulinemic 
hypoglycemic clamp and the epinephrine response during hypoglycemia in the 
subjects with T1DM was 121 ± 168 pg/ml vs. 278 ± 134 pg/ml in controls.  
6.2 Statistical Methods 
Our primary outcome is the peak epinephrine response during the third 
hypoglycemic clamp of each infusion experiment, thus each participant will have 
two observations: one from their NAC infusion experiment and one from their 
saline infusion experiment. We will fit a general linear mixed model with fixed 
effects for treatment (NAC vs. saline), treatment order (NAC first vs. saline first), 
and period (Part 1 vs. Part 2) and a random effect for participant. We are 
confident that our study design, with a washout period of 8 weeks between Parts 
1 and 2, has minimized the possibility of a treatment effect from Part 1 that 
lingers into Part 2. We will also consider adjustment for important baseline 
characteristics, such as age, gender, and duration of diabetes. Diagnostics will 
be examined to assess model assumptions. Secondary outcomes include: the 
within-person difference in peak epinephrine response between the first 
hypoglycemic clamp (when an infusion is given) and the third hypoglycemic 
clamp (when no infusion is given) of each infusion experiment; symptom scores 
during the third hypoglycemic clamp of each infusion experiment; and the within-
person difference in symptom scores between the first hypoglycemic clamp and 
the third hypoglycemic clamp of each infusion experiment. 
 


 
 
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6.3 Subject Population(s) for Analysis 
In this version of the protocol, we intend to only study healthy volunteers.  In the 
original version, we intended to enroll humans with type 1 diabetes of less than 
20 years duration, with A1c 6.9-8.2%, who were aware of hypoglycemia but were 
unable to reach our recruitment goals with this population.  More than 45 
potential participants were contacted over 14 months and only one subject was 
able to complete the entire protocol.  The reasons potential participants refused 
enrollment included strong preferences to not experience hypoglycemia and 
inability to commit the time necessary to complete the study.  Previously our 
group has had no trouble enrolling enough normal volunteers to participate in 
studies in which they were asked to experience several periods of hypoglycemia.  
Thus, we believe that we will meet our recruitment goals by changing the 
population to normal volunteers.  In addition, this is a proof of principle study in 
which the impact of NAC on hypoglycemia induced blunting of the 
counterregulatory response will be measured.  Normal volunteers who participate 
in this protocol are expected to have the same degree of hypoglycemia induced 
blunting in their counterregulatory response as patients with type 1 diabetes and 
normal awareness of hypoglycemia.  Therefore, this redesign will allow us to 
determine if NAC works under controlled conditions.  If it does, it will provide 
rationale to use in in patient populations in the future.  
 
Exclusion criteria will include history of diabetes, hyperglycemia, stroke, seizures, 
arrhythmias, active cardiac disease, pregnancy or plan to become pregnant 
during the study period; diagnosis of asthma (increases risk of hypersensitivity 
reactions to NAC); use of anti-oxidants or drugs that can alter glucose 
metabolism; concomitant medical problems that may prevent the subject from 
successfully completing the protocol; and an unwillingness to avoid exercise 
during the 7 days before each part of the study. 
7 Safety and Adverse Events 
7.1 Definitions 
Adverse Event 
An adverse event (AE) is any symptom, sign, illness or experience that develops 
or worsens in severity during the course of the study.  Intercurrent illnesses or 
injuries will be regarded as adverse events.  Abnormal results of diagnostic 
procedures are considered to be adverse events if the abnormality: 
• results in study withdrawal 
• is associated with a serious adverse event 
• is associated with clinical signs or symptoms 
• leads to additional treatment or to further diagnostic tests 
• is considered by the investigator to be of clinical significance. 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.  A serious adverse 
event is any AE that is:  


 
 
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• fatal 
• life-threatening 
• requires or prolongs a hospital stay 
• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• an important medical event. 
 
Important medical events are those that may not be immediately life threatening, 
but are clearly of major clinical significance.   They may jeopardize the subject, and 
may require intervention to prevent one of the other serious outcomes noted 
above.  For example, drug overdose or abuse, a seizure that did not result in in-
patient hospitalization, or intensive treatment of bronchospasm in an emergency 
department would typically be considered serious.  
 
All adverse events that do not meet any of the criteria for serious should be 
regarded as non-serious adverse events.  
Adverse Event Reporting Period 
The study period during which adverse events must be reported is normally 
defined as the period from the initiation of any study procedures to the end of the 
study treatment follow-up.  For this study, the study treatment follow-up is defined 
as 30 days following the last administration of study treatment.  
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A preexisting 
condition will be recorded as an adverse event if the frequency, intensity, or the 
character of the condition worsens during the study period. 
General Physical Examination Findings 
At screening, any clinically significant abnormality will be recorded as a preexisting 
condition. 
 
At 
the 
end 
of 
the 
study, 
any 
new 
clinically 
significant 
findings/abnormalities that meet the definition of an adverse event will also be 
recorded and documented as an adverse event.  
Post-study Adverse Event 
All unresolved adverse events will be followed by the investigator until the events 
are resolved, the subject is lost to follow-up, or the adverse event is otherwise 
explained.  At the last scheduled visit, the investigator will instruct each subject to 
report any subsequent event(s) that the subject, or the subject’s personal 
physician, believes might reasonably be related to participation in this study.   
 
Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an adverse event if any 
one of the following conditions is met:  
• The laboratory abnormality is not otherwise refuted by a repeat test to 
confirm the abnormality. 
• The abnormality suggests a disease and/or organ toxicity. 


 
 
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• The abnormality is of a degree that requires active management; e.g. 
change of dose, discontinuation of the drug, more frequent follow-up 
assessments, further diagnostic investigation, etc. 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization will be 
documented and reported as a serious adverse event unless specifically instructed 
otherwise in this protocol.  Any condition responsible for surgery should be 
documented as an adverse event if the condition meets the criteria for an adverse 
event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are 
reported as an adverse event in the following circumstances: 
• Hospitalization or prolonged hospitalization for diagnostic or elective 
surgical procedures for a preexisting condition.  Surgery should not be 
reported as an outcome of an adverse event if the purpose of the surgery 
was elective or diagnostic and the outcome was uneventful. 
• Hospitalization or prolonged hospitalization required to allow efficacy 
measurement for the study. 
• Hospitalization or prolonged hospitalization for therapy of the target disease 
of the study, unless it is a worsening or increase in frequency of hospital 
admissions as judged by the clinical investigator. 
7.2 Recording of Adverse Events 
At each contact with the subject, the investigator must seek information on adverse 
events by specific questioning and, as appropriate, by examination.  Information on all 
adverse events will be recorded immediately in the source document, and also in the 
appropriate adverse event module of the case report form (CRF).  All clearly related 
signs, symptoms, and abnormal diagnostic procedures results will be recorded in the 
source document, though should be grouped under one diagnosis. 
 
All adverse events occurring during the study period must be recorded.  The clinical 
course of each event will be followed until resolution, stabilization, or until it has been 
determined that the study treatment or participation is not the cause.  Serious adverse 
events that are still ongoing at the end of the study period must be followed up to 
determine the final outcome.  Any serious adverse event that occurs after the study 
period and is considered to be possibly related to the study treatment or study 
participation should be recorded and reported immediately. 
7.3 Reporting of Serious Adverse Events 
7.3.1 Study Sponsor Notification by Investigator 
A serious adverse event must be reported to the data safety and monitoring 
board within 24 hours of finding out of the event using a Serious Adverse Event 
(SAE) form. The investigator will keep a copy of this SAE form on file at the study 
site.   


 
 
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At the time of the initial report, the following information should be provided: 
• Study identifier 
• Study Center 
• Subject number 
• A description of the event 
• Date of onset 
• Current status 
• Whether study treatment was 
discontinued 
• The reason why the event is 
classified as serious 
• Investigator assessment of the 
association between the event 
and study treatment 
 
Within the following 48 hours, the investigator must provide further information on 
the serious adverse event in the form of a written narrative.  This should include 
a copy of the completed Serious Adverse Event form, and any other diagnostic 
information that will assist the understanding of the event.  Significant new 
information of ongoing serious adverse events should be provided promptly to 
the data and safety monitoring board. 
7.3.2 IRB Notification by Investigator 
Reports of all serious adverse events (including follow-up information) must be 
submitted to the IRB within 10 working days if it falls under the UPIRTSO 
guidelines.  Copies of each report and documentation of IRB notification and 
receipt will be kept in the Clinical Investigator’s binder. 
7.3.3 FDA Notification by Sponsor 
The study sponsor shall notify the FDA by telephone or by facsimile transmission 
of any unexpected fatal or life-threatening experience associated with the use of 
the drug as soon as possible but no later than 7 calendar days from the data and 
safety monitoring board’s original receipt of the information. 
 
If a previous adverse event that was not initially deemed reportable is later found 
to fit the criteria for reporting, the investigator will submit the adverse event in a 
written report to the FDA as soon as possible, but no later than 15 calendar days 
from the time the determination is made. 
 
Agency 
Criteria for 
Reporting 
Timeframe 
Form to Use  
Submission 
address/fax 
numbers  
U of MN 
IRB 
SAE: fatal, life-
threatening or 
serious, 
unexpected, at 
least possibly 
related  
10 working 
days 
UMCC SAE 
MMC 820 
FDA 
SAE: fatal, life-
threatening, 
7 calendar days  FDA prefers 
MedWatch 
Fax: 1 (800) FDA - 
0178 


 
 
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unexpected, at 
least possible 
related 
3500a Form but 
alternative 
formats are 
acceptable (e.g. 
summary letter) 
SAE: serious, 
unexpected, at 
least possibly 
related 
15 calendar 
days 
 
7.4 Unblinding Procedures 
Unblinding of participants and investigators will be done at the end of the study and a 
letter will be sent to any subject withdrawn because of symptoms of an allergic reaction 
to NAC informing them of the treatment they were given. If they had been given NAC, 
we will instruct them to share the letter with their personal physician so the allergy could 
be noted in the clinical record. 
7.5 Stopping Rules  
Individuals will be withdrawn from the trial if they experience any symptoms of 
allergy to NAC severe enough to require early stopping of the infusion, including 
feeling itchy on their skin, having tingling or swelling in their mouth or tongue, or 
having trouble breathing.  
 
A Data Safety Monitoring Board (DSMB) will be assembled at the start of the 
study to regularly monitor patient safety and study documentation of adverse and 
serious adverse events. This board will consist of the study statistician Dr. Lynn 
Eberly, endocrinologist Dr. Lisa Chow (who is independent of the study team), 
investigational pharmacist Darlette Luke, RPharm, and endocrinologist Dr. 
Anthony McCall (who is independent of the study team) from the University of 
Virginia.  DSMB members will be unblinded to treatment assignments. They will 
review (1) incidence of early stopping of an infusion and (2) incidence of any 
adverse or serious adverse events on at least a monthly basis. After an 
experiment where the infusion was stopped early and an adverse or serious 
adverse event was reported, they will review that experiment’s data within two 
weeks. 
 
Participant accrual will be halted if an excessively high rate of participants with 
allergic symptoms requiring early stopping of the infusion is seen. Statistical 
sequential boundaries on the cumulative number of participants with allergic 
symptoms will be used to monitor the rate. If allergic symptoms requiring early 
stopping of the infusion are noted during an experiment, the participant will be 
withdrawn from the trial; the withdrawal will be carried out by blinded study staff, 
thus without knowledge of whether the infusion given was NAC or saline. The 
DSMB will then review the unblinded participant data within two weeks of the 
experiment. If the infusion given was saline, the reaction will be denoted in 
DSMB records as a placebo reaction, and that participant will not be counted 
towards the cumulative number of participants with allergic symptoms to NAC. If 


 
 
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the infusion given was NAC, and in the DSMB’s judgment the reaction was likely 
related to NAC, then the reaction will be denoted in DSMB records as an allergic 
reaction to NAC, and that participant will be counted towards the cumulative 
number of participants with allergic symptoms to NAC. The DSMB will inform the 
PI that there was an allergic reaction to NAC, and at study’s end the project PI 
will notify the participant that they should inform their personal care provider that 
an allergic reaction to NAC was observed. 
 
Specifically, the trial will be stopped early if the number of participants with an 
allergic reaction to NAC is equal to or exceeds the boundary number (bn) out of n 
participants who have completed at least the Part 1 infusion (Table below). This 
is a Pocock-type stopping boundary that yields the probability of crossing the 
boundary at most 0.05 when the rate of allergic symptoms is equal to the 
acceptable rate of 10%.  
 
Cumulative early stopping rule for allergic symptoms to NAC with 10% as the 
maximum allowable rate of allergic symptoms. 
 
Number 
who have 
completed 
at least 
one Part 
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 
Boundary, 
bn 
- 
2 3 3 3 3 4 4 4 4 
4 
4 
5 
5 
5 
5 
5 
6 
 
7.6 Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the study at 
his/her site.  This safety monitoring will include careful assessment and appropriate 
reporting of adverse events as noted above, as well as the construction and 
implementation of a site data and safety-monitoring plan (see section 9 Auditing, 
Monitoring and Inspecting).  Medical monitoring will include a regular assessment of the 
number and type of serious adverse events. 
7.6.1 Internal Data and Safety Monitoring Board 
We have formed a DSMB that will consists of the study statistician Dr. Lynn Eberly, 
endocrinologist Dr. Lisa Chow (chair), endocrinologist Dr. Anthony McCall from the 
University of Virginia, and investigational pharmacist Darlette Luke. They will review the 
data collected at each experiment on a monthly basis and more frequently if adverse 
events are reported (as described above in 7.5). In addition, we will file an IND with the 
FDA for the use of N-acetyl cysteine for the purpose outlined in this application. Past 
INDs filed by Dr. Lisa Coles for the IV use of N-acetyl cysteine in the study of 
neurological disease were given an exempt status so we anticipate the FDA will give the 
IND for this study and exempt status as well, but if not it will be monitored by the staff of 


 
 
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the Clinical and Translational Sciences Institute at the University of Minnesota. They 
have extensive experience with the monitoring of investigator-initiated trials at the 
University of Minnesota, including for Dr. Seaquist who recently completed trial of 
naltrexone as a therapy for hypoglycemia unawareness in type 1 diabetes. Adverse 
events will be filed with the University of Minnesota IRB according to requirements. 
8 Data Handling and Record Keeping 
8.1 Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 
(HIPAA).  Those regulations require a signed subject authorization informing the subject 
of the following:  
• What protected health information (PHI) will be collected from subjects in this 
study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of their PHI.  
In the event that a subject revokes authorization to collect or use PHI, the investigator, 
by regulation, retains the ability to use all information collected prior to the revocation of 
subject authorization.  For subjects that have revoked authorization to collect or use 
PHI, attempts should be made to obtain permission to collect at least vital status (i.e. 
that the subject is alive) at the end of their scheduled study period. 
8.2 Source Documents 
Source data is all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  
Source data are contained in source documents   Examples of these original 
documents, and data records include: hospital records, clinical and office charts, 
laboratory notes, memoranda, subjects’ diaries or evaluation checklists, pharmacy 
dispensing records, recorded data from automated instruments, copies or transcriptions 
certified after verification as being accurate and complete, microfiches, photographic 
negatives, microfilm or magnetic media, x-rays, subject files, and records kept at the 
pharmacy, at the laboratories, and at medico-technical departments involved in the 
clinical trial. 
 
Data generated by the methods described in the protocol will be recorded in the 
subjects' medical records and/or study progress notes. Data may be transcribed legibly 
on CRFs supplied for each subject or directly inputted into an electronic system or any 
combination thereof. 
8.3 Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for the study.  
All data requested on the CRF must be recorded.  All missing data must be explained.  


 
 
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If a space on the CRF is left blank because the procedure was not done or the question 
was not asked, write “N/D”.  If the item is not applicable to the individual case, write 
“N/A”.  All entries should be printed legibly in black ink.  If any entry error has been 
made, to correct such an error, draw a single straight line through the incorrect entry 
and enter the correct data above it.  All such changes must be initialed and dated.  DO 
NOT ERASE OR WHITE OUT ERRORS.  For clarification of illegible or uncertain 
entries, print the clarification above the item, then initial and date it. 
8.4 Records Retention 
It is the investigator’s responsibility to retain study essential documents for at least 6 
years after the last approval of a marketing application in their country and until there 
are no pending or contemplated marketing applications in their country or at least 6 
years have elapsed since the formal discontinuation of clinical development of the 
investigational product.  These documents will be retained for a longer period if required 
by an agreement with the sponsor.  In such an instance, it is the responsibility of the 
sponsor to inform the investigator/institution as to when these documents no longer 
need to be retained.  
9 Study Monitoring, Auditing, and Inspecting 
9.1 Study Monitoring Plan 
This study will be monitored according to FDA/GCP guidelines.  The investigator will 
allocate adequate time for such monitoring activities.  The Investigator will also ensure 
that the monitor or other compliance or quality assurance reviewer is given access to all 
the above noted study-related documents and study related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.), and has adequate space to conduct the monitoring visit. 
9.2 Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, 
the sponsor, government regulatory bodies, and University compliance and quality 
assurance groups of all study related documents (e.g. source documents, regulatory 
documents, data collection instruments, study data etc.).  The investigator will ensure 
the capability for inspections of applicable study-related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection 
by government regulatory authorities and applicable University compliance and quality 
assurance offices. 
10 Ethical Considerations 
This study is to be conducted according to US and international standards of Good 
Clinical Practice (FDA Title 21 part 312 and International Conference on Harmonization 
guidelines), applicable government regulations and Institutional research policies and 
procedures. 
 


 
 
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This material is the property of the University of Minnesota.  Do not disclose or use except as authorized in writing by the study sponsor 
This protocol and any amendments will be submitted to a properly constituted 
independent Institutional Review Board (IRB), in agreement with local legal 
prescriptions, for formal approval of the study conduct.  The decision of the IRB 
concerning the conduct of the study will be made in writing to the investigator and a 
copy of this decision will be provided to the sponsor before commencement of this 
study.  The investigator should provide a list of IRB members and their affiliate to the 
sponsor. 
 
All subjects for this study will be provided a consent form describing this study and 
providing sufficient information for subjects to make an informed decision about their 
participation in this study.  The consent form will be submitted with the protocol for 
review and approval by the IRB for the study.  The formal consent of a subject, using 
the IRB-approved consent form, must be obtained before that subject is submitted to 
any study procedure.  This consent form must be signed by the subject or legally 
acceptable surrogate, and the investigator-designated research professional obtaining 
the consent.  
11 Study Finances 
11.1 Funding Source 
This study is funded by  the Juvenile Diabetes Research Foundation. 
11.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, 
royalties, or financial gain greater than the minimum allowable by their institution, etc.) 
must refer to the Regents Policies on Individual Conflict of Interest Policy or Institutional 
Conflict of Interest Policy. These policies require University Faculty and staff to report 
external professional activities and business and significant financial interests related to 
his or her University activities by submitting a REPA (Report of External Professional 
Activities) at least once per year. Faculty and staff should also file a REPA when 
substantial changes in business or financial interests occur, when an activity that 
presents a potential conflict of interest is anticipated, or when submitting an application 
for research support or technology transfer, submitting research protocols to the IRB, or 
receiving financial contributions. All University of Minnesota investigators will follow the 
University conflict of interest policy. 
11.3 Subject Stipends or Payments 
Subjects will be paid $25 for the screening visit, $150 per day for each day of Part one 
they complete, and $200 for each day of Part Two they complete to cover travel 
expenses and time lost from work for each day of study. 
12 Publication Plan 
The results of the study will be published. 


 
 
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13 References 
14 Attachments 

ICF_001.pdf: 
 
IRB No. 1406M51341 
Page 1 of 5 
IRB APVD: 06.30.2017 
Diphenhydramine Pretreatment Study 
 v06.15.2017 
CONSENT FORM 
A randomized double blinded study to examine the use of 
N-acetyl cysteine for the prevention and treatment of HAAF in patients with type 1 diabetes.   
 
Diphenhydramine pretreatment study 
 
(June 15, 2017) 
 
You are invited to participate in a study designed to investigate the effects of N-acetyl cysteine on the 
prevention and treatment of impaired awareness of hypoglycemia in patients with type 1 diabetes. You 
were selected as a possible participant because you are a healthy volunteer. We ask that you read this 
form and ask any questions you may have before agreeing to be in the study. This study is being 
conducted by: 
Elizabeth R. Seaquist, M.D. 
Department of Medicine 
(612) 626-4833 
University of Minnesota Medical School 
Minneapolis, MN 55455 
 
 
Why is this study being done? 
Low blood sugar is also called hypoglycemia. Usually, it is mild and can be treated quickly and easily by 
eating or drinking a small amount of a sugar-rich food. If low blood sugar is left untreated, it can get 
worse and cause confusion, clumsiness or fainting. Severe hypoglycemia can lead to seizures, coma, 
and even death. 
 
Some people with diabetes do not have early warning signs of low blood sugar. This condition is called 
hypoglycemia unawareness. It happens when the body stops reacting to low blood sugar levels and the 
person does not realize that they need to treat their hypoglycemia. This can lead to more severe and 
dangerous hypoglycemia. 
 
The purpose of this early study is to see if a drug called N-acetyl cysteine should be studied more in 
people with Type I diabetes and hypoglycemia unawareness, impaired awareness of hypoglycemia, or 
hypoglycemia associated autonomic failure (HAAF). This study will show whether N-acetyl cysteine 
(NAC) could be used to prevent and treat hypoglycemia unawareness. 
 
How long will the study last? 
Each person in this study will participate for approximately 12 weeks. The study will be done in two 
parts separated by 8 weeks. 
 
What is involved in the study? 
If you agree to participate in this study, this is what you will be asked to do: 
 
Screening visit 
At this visit, you will be asked questions about your medical history to make sure you are eligible to be 
in this study. You will also have some blood collected for measurement of a hemoglobin A1c to make 
certain you do not have diabetes. If you are a female who could become pregnant, a pregnancy test will 
also be done. The amount of blood that will be collected at this visit is approximately three teaspoons. 
This visit is estimated to last approximately one hour. 
 
 


 
IRB No. 1406M51341 
Page 2 of 5 
IRB APVD: 06.30.2017 
Diphenhydramine Pretreatment Study 
 v06.15.2017 
Part one 
On day one, you will be asked to come to the Masonic Clinical Research Unit (MCRU) by 7AM after 
fasting overnight.  Two IV catheters will be placed in your arms. This will allow for giving insulin, 
glucose and potassium, N-acetyl cysteine or saline during the test and for the collection of blood 
samples. The arm used for blood drawing may be placed in a heated blanket for the duration of study to 
speed up the flow of blood through vein and for easy withdrawing of blood. 
 
30 minutes after the catheters are in place, you will be given an intravenous infusion of 
diphenhydramine (common name Benadryl) followed by 5 hour intravenous infusion of NAC or saline. 
Which drug you will be given will be determined in the pharmacy and neither you nor the study 
personnel will know which you have been assigned. The assignment will be randomly made, like the flip 
of a coin. 30-90 minutes after the start of the NAC or saline, infusions of insulin and potassium will be 
started. Insulin is a hormone that your body makes naturally and it causes your blood sugar to 
decrease. Potassium is a salt-like substance that is present in the blood. Your glucose will be checked 
every five minutes throughout the study and we will let it fall to a value of 50 mg/dl. Once it gets that low 
we will keep it there by infusing glucose. We will continue the insulin and potassium infusions for 120 
minutes and then give you glucose to return the sugar to normal. During the study blood will be drawn 
every 5 minutes for measurement of glucose and other hormones and chemicals. You will also be 
asked to complete a questionnaire about any symptoms of hypoglycemia you may be experiencing at 
the start of the day and at the end of the hypoglycemic period. You will be asked about whether you 
have symptoms of itching, tingling, swelling of your mouth or tongue, or are having trouble breathing 
since these are rare side effects of NAC. 
 
120-240 minutes after the insulin infusion is stopped, infusions of insulin and potassium will be started 
again. Your glucose will be checked every five minutes throughout the study and we will let it fall to a 
value of 50 mg/dl. Once it gets that low we will keep it there by infusing glucose. We will continue the 
insulin and potassium infusions for 120 minutes and then give you glucose to return the sugar to 
normal. During the study blood will be drawn every 5 minutes for measurement of glucose and other 
hormones and chemicals. You will also be asked to complete a questionnaire about any symptoms of 
hypoglycemia you may be experiencing at the start of the day and at the end of the hypoglycemic 
period. You will be asked about whether you have symptoms of itching, tingling, swelling of your mouth 
or tongue, or are having trouble breathing since these are rare side effects of NAC. At the completion of 
the study, you will be given a meal and then allowed to leave the research center with the plan to return 
the next morning. 
 
The following morning (day 2) you will arrive at the MCRU by 7 AM after fasting overnight.  Two IV lines 
will be placed in your arms and infusions of insulin and potassium will be started. Your glucose will be 
checked every five minutes throughout the study and we will let it fall to a value of 45 mg/dl over 120 
minutes. We will stop the insulin and potassium infusions and give you glucose to return the sugar to 
normal. During the study blood will be drawn every 5 minutes for measurement of glucose and other 
hormones and chemicals. You will also be asked to complete questionnaires about any symptoms of 
hypoglycemia you experience during the hypoglycemic period. At the end of the study you will be given 
a meal, and you will then be sent home. 
 
 
Part two 
Part two will be done approximately 8 weeks later, exactly like part one, except if you were given NAC 
during part one you will now be given saline. If you were given saline during part one, you will now be 
given NAC. 
 
 
 


 
IRB No. 1406M51341 
Page 3 of 5 
IRB APVD: 06.30.2017 
Diphenhydramine Pretreatment Study 
 v06.15.2017 
Risks and Benefits of Being in the Study: 
The study has several risks, which are understood, and measures to minimize the risks have been 
established: 
1. Infusions/blood drawing: Occasionally individuals may have some bruises afterwards where the 
catheter was inserted. Some people experience a burning or cramping in their arm when glucose 
or potassium is infused. This feeling goes away when we reduce the rate at which the substance 
is given to you. Rarely an individual may experience inflammation of the vein (phlebitis). If this 
happens, your arm may feel sore for a couple of weeks and your vein may feel lumpy. These 
symptoms usually go away with local heat and elevation of the extremity. Participants will be 
observed closely for these complications, and if these complications occur, care will be promptly 
and appropriately administered. The maximum amount of blood that will be collected during the 
study will be 30-40 tablespoons. We will not allow you to be in the study if you have given or 
expect to give a similar amount of blood in the month before or after the research procedures. 
2. Hypoglycemia (low blood sugar): Symptoms of hypoglycemia include sweating, shakiness, 
confusion, increased heartbeat and feeling "low". It is reversed within minutes by stopping the 
insulin infusion and by raising your blood sugar by increasing the rate of glucose infusion. When 
you are participating in the low blood glucose protocol of the study we expect you will develop 
some of these symptoms during the study. To be certain that your blood sugar level does not drop 
less than 30 mg/dl, we will check your blood every five minutes during the study and adjust the 
amount of sugar we give you by vein to keep your blood sugar at the correct level. If the blood 
sugar drops below 20 mg/dl, confusion, seizures, coma or abnormalities in heart rhythm can 
occur. With careful monitoring, it is very unlikely that your blood sugar will drop to less than 30 
mg/dl.  
3. N-acetyl cysteine: NAC is a therapy that has long been FDA approved for the treatment of 
acetaminophen overdose is available as a nutritional supplement, and has been used to treat a 
variety of human conditions. The most common adverse reactions (incidence > 2%) are rash, 
facial flushing and itching. Less common reactions include nausea and vomiting. These reactions 
usually occur 30-60 minutes after initiating the infusion, and often resolve spontaneously despite 
continued infusion of NAC. Serious anaphylactic reactions, including death in a patient with 
asthma, have been reported in patients given intravenous NAC. These reactions include 
symptoms such as rash, hypotension, wheezing and/or shortness of breath. To make sure you 
are tolerating the NAC, we will ask you about symptoms like itching, nausea, tingling or swollen in 
your mouth or tongue, and breathing four times during the infusion. If you develop symptoms the 
infusion will be stopped and the doctor will monitor you until the symptoms are gone. If the 
symptoms are severe, we will have a kit at the bedside that can treat allergic reactions 
(medications we have available include diphenhydramine, ondansetron, epinephrine) or we can 
call upon the emergency help available in the hospital where the MCRU is located. 
4. Diphenhydramine:  Diphenhydramine will be given immediately before you are given the NAC or 
saline infusion to minimize your chance of developing side effects to the NAC.  This drug is 
commonly used to treat allergies and is available over the counter.  Side effects of 
diphenhydramine include drowsiness, stomach upset, dizziness and headache.  
There are no direct benefits of participation to you other than the satisfaction you may have in helping 
us answer important as yet unanswered questions about human glucose metabolism. 
 
Research Related Injury 
In the event that this research activity results in an injury, treatment will be available, including first aid, 
emergency treatment, and follow-up care as needed. Care for such injuries will be billed in the ordinary 
manner to you or your insurance company. If you think that you have suffered a research related injury, 
let the study physicians know right away. 
 
 


 
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Compensation: 
You will be paid $25 for the screening visit, $150 per day for each day of Part one you complete, and 
$200 for each day of Part Two you complete to cover travel expenses and time lost from work for each 
day of study, up to a total of $725. Because the amount of money paid to you may exceed $600, we will 
be obligated to ask you to complete a W9 tax form.  If the money paid to you does exceed $600 in a 
calendar year, this information will be reported to the Internal Revenue Service (IRS), and you will be 
issued a Form 1099-MISC from the University of Minnesota to use when completing your personal 
income tax. 
 
Confidentiality: 
Efforts will be made to limit the use and disclosure of your personal information, including research 
study and medical records, to people who have a need to review this information. We cannot promise 
complete privacy. Organizations that may inspect and copy your information include the IRB and other 
representatives of this institution, including those that have responsibilities for monitoring or ensuring 
compliance. 
 
The sponsor, monitors, auditors, the IRB, the University of Minnesota Research Compliance Office and 
other University compliance units, the US Office of Research Integrity (ORI), the US Office for Human 
Research Protections (OHRP), and the US Food and Drug Administration (FDA) may be granted direct 
access to your medical records to conduct and oversee the research.  By signing this document you 
are authorizing this access.  We may publish the results of this research. However, we will keep your 
name and other identifying information confidential. 
 
A description of this clinical trial is available at http://www.ClinicalTrials.gov, as required by U.S. Law. 
This Web site will not include information that can identify you. At most, the Web site will include a 
summary of the results. You can search this Web site at any time. 
 
Protected Health Information (PHI): 
Your PHI created or received for the purposes of this study is protected under the federal regulation 
known as HIPAA.  Refer to the attached HIPAA authorization for details concerning the use of this 
information. 
 
Voluntary Nature of the Study: 
Your decision whether or not to participate will not affect your current or future relations with the 
University. If you decide to participate, you are free to withdraw at any time without affecting those 
relationships. 
 
New Information: 
If during the course of this research study, there are significant new findings discovered which might 
influence your willingness to continue, the researchers will inform you of those developments. 
 
Contacts and Questions: 
The researcher conducting this study is Elizabeth R. Seaquist MD. You may ask any questions you 
have now. If you have questions later, you may contact her at (612) 624-9176. 
 
This research has been reviewed and approved by an Institutional Review Board (IRB) within the 
Human Research Protections Program (HRPP) at the University of Minnesota. To share feedback 
privately with the HRPP about your research experience, call the Research Participants’ Advocate Line 
at 612-625-1650 or go to www.irb.umn.edu/report.html. You are encouraged to contact the HRPP if:  
 
● Your questions, concerns, or complaints are not being answered by the research team. 
● You cannot reach the research team. 


 
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● You want to talk to someone besides the research team. 
● You have questions about your rights as a research participant. 
● You want to get information or provide input about this research. 
 
 
Future Contact for Other Research Studies: 
The following research activities are optional, meaning that you do not have to agree to them in order to 
participate in the research study. Please indicate your willingness to participate in these optional 
activities by placing your initials next to each activity. 
 
I agree 
I disagree 
 
 
 
 
 
 
 
The researchers may contact me in the future to see whether I am 
interested in participating in other research studies conducted by the 
Division of Diabetes and Endocrinology at the University of Minnesota. 
 
Statement of Consent: 
I have read the above information, asked questions and have received answers, and I consent to 
participate in the study. 
 
You will be given a copy of this form to keep for your records. 
Signature:  
 
 Date:  
 
Signature of Investigator:  
 Date: