Impact of Timed Bromocriptine-QR Therapy Upon Measures of Sympathetic Tone and Vascular Biology in Type 2 Diabetes Subjects
Phase: PHASE4
Conditions: Diabetic Autonomic Neuropathy
Sponsor: Eastern Virginia Medical School

Veroscience 
 
 
Page 1 
Sponsor 
 VeroScience 
1334 Main Road 
Tiverton Road Island 
CLINICAL STUDY PROTOCOL 
Impact of Timed Bromocriptine-QR Therapy upon Measures of 
Sympathetic Tone and Vascular Biology in Type 2 Diabetes Subjects. 
 
 
Date of Original Protocol    
 
Single center study 
US sites only 
 
Principal Investigator 
Aaron Vinik MD 
 
Co-Investigators 
Henri K. Parson PhD, CCRC  
Carolina M. Casellini MD 
 
 
 
Signatory of the sponsor 
Anthony H Cincotta PhD 
President and Chief Scientific Officer 
Sponsor’s responsible medical expert 
Bindu Chamarthi, MD, MMSc 
Medical Director 
1334 Main Road 
Tiverton, RI 
Telephone: 401 816-0525 
Fax:            401 816-0524 
E-mail: Bindu_Chamarthi@veroscience.com 
 
Sponsor’s Project Manager  
Donna Cowan 
Clinical Trial Operations 
1334 Main Road 
Tiverton, RI 
Telephone: 401 816-0525 
Fax:            401 816-0524 
E-mail: Donna_Cowan@veroscience.com 
 
 
 
 
 
Veroscience Serious Adverse Event Reporting 
inVentiv Health Clinical  
5430 Data Court, Suite 200 
Ann Arbor, MI 48108 
T: 734-757-9731 
F: 866-880-9343 
http://www.inVentivHealthclinical.com 
 

Impact of Timed Bromocriptine-QR Therapy upon Measures of Sympathetic Tone and Vascular Biology 
in Type 2 Diabetes Subjects 
 
 
      
 
   
 
  
Veroscience – Confidential 
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TABLE OF CONTENTS 
  
PROTOCOL OUTLINE ................................................................................................................................... 5 
STUDY SCHEDULE ........................................................................................................................................ 8 
ABBREVIATIONS AND DEFINITIONS .......................................................................................................... 9 
1 
INTRODUCTION AND STUDY RATIONALE..................................................................................... 10 
2 
STUDY OBJECTIVES ........................................................................................................................ 19 
2.1 
PRIMARY OBJECTIVE ....................................................................................................................... 19 
2.2 
SECONDARY OBJECTIVES .............................................................................................................. 19 
3 
STUDY DESIGN, DURATION AND DATES ...................................................................................... 19 
3.1 
STUDY DESIGN ................................................................................................................................. 19 
3.2 
STUDY DURATION AND DATES ....................................................................................................... 22 
4 
SELECTION OF SUBJECTS ............................................................................................................. 23 
4.1 
NUMBER OF SUBJECTS ................................................................................................................... 23 
4.2 
INCLUSION CRITERIA ....................................................................................................................... 23 
4.3 
EXCLUSION CRITERIA ...................................................................................................................... 23 
5 
STUDY TREATMENTS....................................................................................................................... 25 
5.1 
DETAILS OF STUDY TREATMENTS ................................................................................................. 25 
5.2 
SUPPLIES AND ACCOUNTABILITY .................................................................................................. 25 
5.3 
COMPLIANCE ..................................................................................................................................... 25 
5.4 
RUN-IN MEDICATION ........................................................................................................................ 25 
6 
PRIOR AND CONCOMITANT ILLNESSES AND TREATMENTS .................................................... 25 
6.1 
PRIOR AND CONCOMITANT ILLNESSES ........................................................................................ 25 
6.2 
PRIOR AND CONCOMITANT TREATMENTS ................................................................................... 25 
7 
OVERVIEW OF DATA COLLECTION AND STUDY PROCEDURES ............................................... 26 

Impact of Timed Bromocriptine-QR Therapy upon Measures of Sympathetic Tone and Vascular Biology 
in Type 2 Diabetes Subjects 
 
 
      
 
   
 
  
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7.1 
STUDY PROCEDURES AND SCHEDULES ...................................................................................... 26 
7.2 
DESCRIPTION OF STUDY DAYS ...................................................................................................... 26 
7.2.1 Screening - Visit 0 ............................................................................................................................... 26 
7.2.2 Study Days – Treatment Period .......................................................................................................... 27 
7.2.2.1 
Baseline – Visit 1 (Post Visit 0 + <30 days) ................................................................................... 27 
7.2.2.2 
Call 1 (Visit 1 + 7 days) .................................................................................................................. 28 
7.2.2.3 
Call 2 (Call 1 + 7 days) ................................................................................................................... 29 
7.2.2.4 
Call 3 (Call 2 + 7 days) ................................................................................................................... 29 
7.2.2.5 
Visit 2 (Visit 1 + 4 weeks [± 3 days]) .............................................................................................. 29 
7.2.2.6 
Phone call 4 (Visit 2+ 4 weeks) ...................................................................................................... 30 
7.2.2.7 
Visit 3 (Visit 1plus 12weeks [± 3 days]) .......................................................................................... 30 
7.2.2.8 
Phone Call 5 (Visit 3 + 4 weeks [±3 days]) ..................................................................................... 31 
7.2.2.9 
Phone Call 6 (Phone Call 5 + 4 weeks [±3 days]))......................................................................... 31 
7.2.2.10 
Visit 4 (Visit 1 plus 24 weeks [± 3 days] or Early Termination) .................................................... 31 
7.2.3 End of Study Follow up Phone Call (Post Visit 4 + 27-30 days) ......................................................... 32 
7.3 
STATISTICAL METHODS .................................................................................................................. 32 
7.3.1 Primary endpoint ................................................................................................................................. 33 
7.3.2 Secondary endpoint ............................................................................................................................ 33 
7.3.3 Safety Data .......................................................................................................................................... 33 
8 
ADVERSE EVENTS ............................................................................................................................ 33 
8.1 
DEFINITIONS ...................................................................................................................................... 33 
8.1.1 Adverse Event ..................................................................................................................................... 33 
8.1.2 Serious Adverse Event ........................................................................................................................ 34 
8.1.3 Alert Terms and Other Reasons for Expedited Reporting to Pharmacovigilance ............................... 35 
8.2 
PERIOD OF OBSERVATION ............................................................................................................. 35 
8.3 
DOCUMENTATION AND REPORTING OF ADVERSE EVENTS BY INVESTIGATOR .................... 35 
8.4 
IMMEDIATE REPORTING BY INVESTIGATOR TO SPONSOR ....................................................... 36 
9 
WITHDRAWALS ................................................................................................................................. 38 
9.1 
WITHDRAWAL OF SUBJECTS .......................................................................................................... 38 
9.2 
EMERGENCY SPONSOR CONTACT ................................................................................................ 39 
9.3 
EMERGENCY IDENTIFICATION OF INVESTIGATIONAL PRODUCTS ........................................... 39 
9.4 
EMERGENCY TREATMENT .............................................................................................................. 40 
9.5 
ANALYSIS VARIABLES ...................................................................................................................... 40 

Impact of Timed Bromocriptine-QR Therapy upon Measures of Sympathetic Tone and Vascular Biology 
in Type 2 Diabetes Subjects 
 
 
      
 
   
 
  
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9.6 
ANALYSIS POPULATIONS ................................................................................................................ 40 
9.7 
SAMPLE SIZE ..................................................................................................................................... 40 
10 
REGULATORY REQUIREMENTS ..................................................................................................... 41 
10.1 GOOD CLINICAL PRACTICE ............................................................................................................. 41 
10.2 DELEGATION OF INVESTIGATOR DUTIES ..................................................................................... 41 
10.3 SUBJECT INFORMATION AND INFORMED CONSENT .................................................................. 41 
10.4 CONFIDENTIALITY ............................................................................................................................ 42 
10.5 PROTOCOL AMENDMENTS ............................................................................................................. 42 
10.6 APPROVAL OF THE CLINICAL STUDY PROTOCOL AND AMENDMENTS ................................... 42 
10.7 ONGOING INFORMATION FOR INDEPENDENT ETHICS COMMITTEE/ INSTITUTIONAL 
REVIEW BOARD ................................................................................................................................ 43 
10.8 CLOSURE OF THE STUDY ............................................................................................................... 43 
10.9 RECORD RETENTION ....................................................................................................................... 43 
10.10 LIABILITY AND INSURANCE ............................................................................................................. 44 
10.11 FINANCIAL DISCLOSURE ................................................................................................................. 44 
11 
STUDY MONITORING AND AUDITING ............................................................................................ 45 
11.1 STUDY MONITORING AND SOURCE DATA VERIFICATION.......................................................... 45 
11.2 ON-SITE AUDITS ................................................................................................................................ 45 
12 
DOCUMENTATION AND USE OF STUDY FINDINGS ..................................................................... 46 
12.1 DOCUMENTATION OF STUDY FINDINGS ....................................................................................... 46 
12.2 USE OF STUDY FINDINGS ............................................................................................................... 46 
13 
DECLARATIONS OF SPONSOR AND INVESTIGATOR .................................................................. 48 
13.1 DECLARATION OF SPONSOR .......................................................................................................... 48 
13.2 DECLARATION OF INVESTIGATOR ................................................................................................. 48 

 
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PROTOCOL OUTLINE 
 
Study number 
 
Title 
Impact of Timed Bromocriptine-QR Therapy upon Measures of Sympathetic Tone and 
Vascular Biology in Type 2 Diabetes Subjects 
Investigator(s), Study Site(s)  
Aaron  Vinik, M.D. (vinikai@evms.edu ),  The Strelitz Diabetes Center, Norfolk, VA   
 
Sponsor-Funded, Single Center, Investigator Initiated Trial 
 
Study Duration and Dates 
The duration of this study is 
expected to be approximately 
38 months (31-month subject 
recruitment, up to 3 week 
screening phase, and 6-month 
treatment), with subject 
recruitment to start in June 2015 
and the last subject to finish by 
August 2018. 
 
Phase 
IV (Double Blind, Placebo 
Controlled Study of  
Bromocriptine-QR vs 
Placebo added to Usual 
Diabetes Treatment) 
 
Objectives 
Primary Objective:  
 
To demonstrate the effects of dopaminergic activation on the autonomic nervous system in 
subjects with type 2 diabetes.  
 
Secondary Objective:  
 
To demonstrate the effects of dopaminergic activation with bromocriptine-QR on the regulation of 
hypothalamic-pituitary-axis (HPA) hormones and on the plasma levels of markers of inflammation 
and oxidative/nitrosative stress in type 2 diabetes subjects. The study will evaluate treatment 
effects on inflammatory markers, the leptin/adiponectin system, and hormonal levels of renin-
angiotensin system (RAS), aldosterone and cortisol. A co-secondary objective of the study will be 
to assess the impact of bromocriptine-QR vs placebo on measures of insulin resistance and 
glycemic control (e.g., OGTT glucose and insulin, Matsuda index, HOMA-IR, HbA1c). 
 
The treatment effects on all of the above primary and secondary outcomes will also be assessed 
as a function of the duration of diabetes and other baseline demographics such as HbA1c, 
concomitant medications and metabolic status. 
 
 
 

 
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Study Design 
This is a single-site, prospective, interventional, twenty-four week, randomized, double blind, 
placebo-controlled trial with bromocriptine QR in subjects with early diabetes and established 
T2DM to evaluate its effects on the cardiovascular and peripheral autonomic nervous system, as 
well as inflammatory markers, the leptin/adiponectin system, hormonal levels of RAS and HPA 
axis, indices of insulin resistance and glycemic control, and measures of oxidative and nitrosative 
stress. At least twenty (20) but no more than forty (40) early  diabetes subjects and at least forty 
(40) but no more than sixty (60) subjects with established diabetes will be enrolled in the study. 
Entry criteria will be checked at the screening visit. Subjects will be required to be on a stable 
anti-diabetes regimen consisting of  diet and/or oral anti-diabetic agents consisting of metformin 
alone or metformin plus an insulin secretion enhancer (sulfonylurea, DPP4 Inhibitors, or GLP-1 
analogs) for at least 60 days prior to randomization. Following a 2 week lead-in period, subjects 
will be randomized in a 1:1 ratio to receive UDT (usual diabetes therapy) plus bromocriptine-QR 
or UDT plus placebo. The circadian timing of the interventional therapy will be within 2 hours of 
waking in the morning.  
Following randomization, subjects will be titrated to the maximum tolerated dose of the study 
drug (bromocriptine-QR or placebo) over a 4 week period.  
Subjects will return at 4 weeks after randomization, at 12 weeks after randomization, and then at 
study end (week 24 or early termination) for analyses of the primary and secondary endpoints. A 
primary analysis of the study data will be performed after 12 weeks with a secondary analysis 
conducted at 24 weeks. Subjects will be contacted 30 days after stopping the study drug to 
record any adverse events that occurred after cessation. 
All visits will be conducted at The Strelitz Diabetes Center, Norfolk, VA. 
 
 
Number Of Subjects 
Planned enrollment; A total of 80 subjects are planned to be enrolled in the study. At least 20 but 
no more than 40 early  (diabetes duration of  < 4 years) diabetes subjects and at least 40 but no 
more than 60 subjects with established diabetes (diabetes duration of ≥ 4 years) will be enrolled 
in the study.  
 
Primary Subject Characteristics at Enrollment 
T2DM male or female subjects between the ages of 30 and 80 years with HbA1c ≤ 10% on a 
stable anti-diabetes regimen of diet and/or metformin alone therapy or metformin plus an insulin 
secretion enhancer (sulfonylureas, DPP4 Inhibitors, GLP-1 analogs) therapy for a 60 day period 
prior to randomization.  Subjects must have a documented C-peptide level of >2 ng/ml from the 
screening visit.  
 
Study Treatments 
Bromocriptine-QR, 0.8 mg/day, with the dose increased by 0.8 mg/day every week to a maximum 
of 3.2 mg/day, or as tolerated to a minimum dose of 1.6 mg/day, or matching placebo, added on 
to usual diabetes therapy consisting of a stable anti-diabetes regimen of diet and/or metformin 

 
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alone therapy or metformin plus an insulin secretion enhancer (sulfonylureas, DPP4 Inhibitors, 
GLP-1 analogs) therapy for a 60 day period prior to randomization. 
_____________________________________________________________________________ 
 
Study Endpoints 
 
The primary endpoint is the effect of bromocriptine-QR vs placebo on changes in autonomic 
function measured by assessing sympathetic and parasympathetic function using conventional 
measures of autonomic function, including power spectral analysis of heart rate as well as 
peripheral autonomic function using sudorimetry and laser scanning of peripheral microvascular 
autonomic control. 
 
Secondary endpoints will be bromocriptine-QR vs placebo effects on inflammatory markers, 
markers of oxidative/nitrosative stress, the leptin/adiponectin system, and hormonal levels of the 
renin-angiotensin system (RAS), aldosterone and cortisol. The following plasma markers of 
inflammation and oxidative/nitrosative stress will be evaluated: 1) CRP, 2) IL6,3) TNF α, 4) PAI1, 
SOD, TBARS and ADMA will be assessed as well as nitrotyrosine and other markers of 
oxidative/nitrosative stress .  
 
 
Statistical Procedures 
We propose that an effect size of 10% mean change from baseline in treated groups represents 
a clinically meaningful difference, and we propose that sample size reflects the ability to detect a 
10% change due to intervention factor.  
We intend to use the parametric statistical procedures ANOVA and MANOVA in these analyses, 
based on our previous experience that the data from these tests is normally distributed in raw 
form or in some cases after simple log-transformations. If data are not normally distributed, the 
Wilcoxon signed-rank (within group) or Mann Whitney (between group) tests may be employed or 
Fisher’s exact test in case of small sample sizes. The level of significance will be set at p<0.05. 
Relationships and/or trends between the effect of Cycloset on neurovascular function and 
diabetic neuropathy will be determined with Spearman’s rank correlation.  
Based on these considerations, we calculate a total of 80 participants will result in a power 
greater than 0.80 for observing statistical significance at the p < 0.05 level. Specifically, we plan 
to recruit approximately 40 in the placebo group and approximately 40 in the treatment group.  In 
total, approximately 120 people will be screened in order to enroll approximately 80 participants 
in this study.  JMP statistical Software version 9.3 will be used to perform all statistical analyses. 
. 
A statistical analysis plan (SAP), providing details of the analyses and presentation structure of 
the results, will be developed and finalized before the database is locked. 
 

 
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    STUDY SCHEDULE 
Data collected and/or 
action 
Screen 
 
Baseline  
Treatment Period (24  weeks) 
Visit 0 
Baseline 
Visit 1 
(Visit 0 + 
<30 days) 
Phone call 
1 
(Visit 2 + 7 
days) 
Phone call 
2 
(Call 1 + 7 
days) 
Phone call 
3 
( Call 2 + 7 
days) 
Visit 2 
Week 4  
Phone call 
4  
Week 8  
Visit 3 
Week 12 
Phone call 
5  
Week 16 
Phone call 
6 
Week 20   
Visit 4 
Week 24 
 (or Early 
Term)  
Phone call 
7 
Visit 4 +30 
days    
 
Random-
ization 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Entry Criteria 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
X 
 
X 
 
 
X 
 
Physical Exam 
X 
 
 
 
 
 
 
 
 
 
X 
 
Neurological Exam  
X 
 
 
 
 
 
 
 
 
 
X 
 
ECG 
X 
 
 
 
 
 
 
 
 
 
X 
 
Hematology and chemistry  
X 
 
 
 
 
 
 
 
 
 
X 
 
Lipids 
 
X 
 
 
 
 
 
X 
 
 
X 
 
HbA1c 
X 
X 
 
 
 
 
 
X 
 
 
X 
 
Urine Microalbumin  
 
X 
 
 
 
 
 
X 
 
 
X 
 
Inflammatory oxidative/ 
nitrosative stress and 
neuroendocrine Markers 
 
X 
 
 
 
X 
 
X 
 
 
X 
 
OGTT (with glucose and 
insulin analyses) 
 
X 
 
 
 
 
 
X 
 
 
X 
 
C-peptide 
X 
 
 
 
 
 
 
X 
 
 
X 
 
HOMA-IR (fasting glucose 
and insulin) 
 
 
 
 
 
X 
 
 
 
 
 
 
Autonomic Function Tests 
 
X 
 
 
 
X 
 
X 
 
 
X 
 
Sudorimetry 
 
X 
 
 
 
X 
 
X 
 
 
X 
 
Skin Blood Flow 
 
X 
 
 
 
X 
 
X 
 
 
X 
 
Quantitative Sensory Tests 
 
X 
 
 
 
X 
 
X 
 
 
X 
 
Study Drug instruction (first 
dose next AM) 
 
X 
 
 
 
 
 
 
 
 
 
 
Study Drug Titration reminder 
 
 
X 
X 
X 
 
 
 
 
 
 
 
AE/SAE query/ Study Drug 
assessment  
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Final follow up for AE/SAE 
resolution  
 
 
 
 
 
 
 
 
 
 
 
X 
Concomitant meds 
 
To be assessed throughout the study 
Adverse Events 
 
To be assessed throughout the study 
Serious Adverse Events 
 
To be assessed throughout the study  Report serious adverse events to sponsor within 24 hours  
Mandatory Contact 
 
Weekly titration phone contacts as described in the protocol 
                    

 
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ABBREVIATIONS AND DEFINITIONS 
 
 
AE 
Adverse Event 
BG 
Blood Glucose 
BMI 
Body Mass Index 
CNS 
Central Nervous System 
CRF 
Case Report Form 
CV 
Cardiovascular 
DM 
Diabetes Mellitus 
FPG 
Fasting Plasma Glucose 
GCP 
Good Clinical Practice 
HbA1c 
Hemoglobin A1C 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
MACE 
Major Adverse Cardiac Event  
QR 
Quick Release 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
T2DM 
Type 2 diabetes mellitus 
UDT 
Usual Diabetes Therapy 
 

 
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1 INTRODUCTION AND STUDY RATIONALE 
A.  Summary  
Type 2 diabetes is a growing health concern and diabetic subjects are at high risk for atherosclerotic 
cardiovascular complications (1). The CNS and the hypothalamus in particular have emerged as 
fundamental regulators of whole body homeostasis, including glucose and lipid metabolism (2). 
Several studies on animal models of type 2 diabetes (T2DM) and insulin resistance have shown 
multiple hypothalamic neurophysiologic derangements characterized mainly by low dopaminergic 
tone, and elevated adrenergic and serotoninergic tone (3, 3A). This cluster of derangements 
contributes to the pituitary hypothalamic release of ACTH and cortisol, but more importantly to the 
overactivity of sympathetic drive to the liver, adipose tissue and cardiovascular system. Increased 
sympathetic drive to adipose tissue stimulates lipolysis with release of free fatty acids (FFA) and 
hepatic synthesis of VLDL, raising triglycerides that cause lipotoxicity with secretion of a number of 
inflammatory cytokines including TNFα and IL6. This induces a state of chronic low grade 
inflammation with increased oxidative/nitrosative stress and resistance to the action of insulin. 
Increased sympathetic drive to the liver induces hepatic glucose production and decreases hepatic 
glucose disposal elevating fasting and postprandial glucose levels (3). Sympathetic overactivity 
affects the cardiovascular system causing vasoconstriction of resistance vessels and inducing 
hypertension. This is a major contributor to cardiovascular disease including tachycardia, 
arrhythmias, sudden death and myocardial cell apoptosis with impaired ventricular function and 
failure. Furthermore, reduction of parasympathetic function with loss of sympathetic/parasympathetic 
balance affects the anti-inflammatory state, levels of anti-inflammatory cytokines such as IL10 and 
the Leptin/Adiponectin ratio, thus increasing the inflammation and insulin resistance in T2DM (4).  
We have shown that loss of sympathovagal balance occurs early in newly diagnosed T2DM and 
may precede the appearance of inflammation and oxidative/nitrosative stress, predicting 
macrovascular damage and cardiovascular events (5). Recently, three prominent studies,  
ACCORD, ADVANCE and VADT, have shown that diabetic cardiac autonomic neuropathy (CAN) 
may predict risk of sudden death with intensification of glycemic control (6-11). While numerous 
likely mechanisms for the elevated incidence of cardiovascular events in these studies have been 
proposed, the presence of autonomic dysfunction is emerging as one of the strongest predictors of 
cardiovascular risk. Furthermore, there are reports that directly associate autonomic dysregulation 
with inflammatory cytokines and adipocytokines, which subsequently increase and promote 
cardiovascular risk. Quick-release (QR) bromocriptine has shown to improve HbA1C, fasting plasma 
glucose and postprandial glucose tolerance in subjects with T2DM (12-15). We have recently 
demonstrated that this drug can reset dopaminergic tone and, in one year, reduce cardiovascular 
death by 39% and major adverse cardiovascular events (MACE) by 52% (see preliminary data). This 
raises the question about the mechanisms implicated for this effect. We hypothesize that 
bromocriptine-QR, a dopamine D2 receptor agonist, resets dopaminergic tone, thus improving the 
sympathovagal balance and mitigating inflammation, oxidative/nitrosative stress and the metabolic 
consequences of dyslipidemia, hyperglycemia and hypertension. In this study, we propose to 
investigate the effects of bromocriptine QR on the unique relationship between autonomic function 
and inflammation in newly diagnosed versus established T2DM subjects.  These studies should 
shed new light on the mechanisms of cardioprotection by resetting autonomic balance. This may 
lead, in time, to a new therapeutic focus for managing cardiovascular risk in T2DM. 

 
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B.  Detailed Background  
Obesity, insulin resistance, and T2DM are growing health concerns, and the incidence and 
prevalence of these diseases are increasing worldwide (1).  The CNS has been identified as a key 
regulator of whole body homeostasis. Evidence indicates that, within the brain, the hypothalamus in 
particular is responsible for the day-to-day regulation of a number of factors including body 
temperature, blood pressure, thirst, and hunger; and is a fundamental structure for the integration of 
the nervous and endocrine systems. Over the past decade, it has been shown that the CNS senses: 
1) hormones, namely insulin, leptin, and glucagon-like peptide (GLP)-1, and 2) nutrients, namely 
fatty acids and glucose, to regulate both energy and glucose homeostasis (2).   
Quick-Release (QR) Bromocriptine: Bromocriptine is an ergot derivative that exerts 
activities as a sympatholytic dopamine D2 receptor agonist, adrenergic alpha-1 antagonist, alpha-2 
agonist, and also as a modulator of serotonin and prolactin levels. It activates D2 receptors, which 
results in reduced norepinephrine secretion and suppression of sympathetic nervous system activity 
(16).  A QR formulation has recently been approved by the FDA for the treatment of T2DM.  Based 
on animal and human studies, bromocriptine-QR administration within 2 h of awakening is believed 
to augment low hypothalamic dopamine activity and inhibit excessive sympathetic tone within the 
CNS. This results in reduced postprandial plasma glucose levels due to improvements in impaired 
hypothalamic fuel sensing mechanisms and subsequent impaired insulin- and non-insulin mediated 
glucose disposal and enhanced suppression of hepatic glucose production. Such treatment also 
reduces fasting and postprandial plasma free fatty acid (FFA) and triglyceride levels. Studies have 
shown that, in subjects with poorly controlled T2DM treated with diet alone, metformin, 
sulfonylureas, or thiazolidinediones, the addition of bromocriptine produces a 0.5–0.91 decrement in 
HbA1c and improves fasting plasma glucose and postprandial glucose tolerance (12-14) (see 
preliminary data).  A prospective 1-year study in type 2 diabetic subjects demonstrated a significant 
40% relative risk reduction among bromocriptine-QR treated subjects of a composite pre-specified 
cardiovascular end point that included ischemic (myocardial infarction and stroke) and non-ischemic 
(hospitalizations for unstable angina, congestive heart failure and revascularization) related 
endpoints (15). In a further analysis it reduced MACE by 39% and mortality by 50% within a year of 
treatment (see preliminary data). This is the first drug approved for the treatment of hyperglycemia 
that has shown a reduction on CV events. The mechanism of the drug’s beneficial effect on 
cardiovascular events has not been clearly delineated. 
Mechanism of action of bromocriptine: Bromocriptine’s effects are mediated via resetting 
of dopaminergic and sympathetic tone within the CNS. Mammalian species living in the wild have 
the ability to alter their metabolism from the insulin-sensitive/glucose-tolerant state to the insulin-
resistant/glucose-intolerant state seasonally for survival.  Such metabolic changes are governed by 
changes in monoaminergic concentrations/activity in the suprachiasmatic nuclei (SCN) of the 
hypothalamus and in the ventromedial hypothalamus (VMH) (3). Numerous studies implicate shifts 
in the circadian phase relationship between endogenous dopaminergic and serotonergic activity 
rhythms in SCN in the transition from the insulin-sensitive to insulin-resistant state (reviewed in 3A). 
The VMH plays a pivotal role in modulating autonomic nervous system function, hormonal secretion, 
peripheral glucose/lipid metabolism, and feeding behavior (17,18). Importantly, Humans also do 
manifest circadian oscillations and seasonal changes in metabolism (3B).  Available evidence 
suggests that the same circadian neuroendocrine events that control peripheral metabolism and 
cardiovascular biology in species of all the major vertebrate classes is also operative in humans (3B, 

 
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 20 October 2017 
3C).  Hypothalamic centers that regulate these circadian rhythms receive inputs via numerous 
centers throughout the CNS, neurogenic stimuli from peripheral tissues and gastrointestinal tract, 
hormonal signals, and signals from circulating metabolites. Interventions, such as timed daily 
bromocriptine administration to coincide with the daily peak in endogenous CNS dopaminergic 
activity which is diminished in insulin resistant states and, which alter monoamine neurotransmitter 
levels within these hypothalamic circadian centers, can exert significant improvements on glucose 
and lipid dysmetabolism (19).  It is believed that type 2 diabetic subjects have an early morning 
diminution  in dopaminergic tone, which leads to increased sympathetic activity (16).  In lean, normal 
individuals, plasma prolactin concentrations peak at night during sleep and CNS dopaminergic 
activity peaks at the onset of waking. These neuroendocrine rhythms along with others function to 
entrain a circadian pattern of normal fuel metabolism.  In contrast, obese insulin-resistant individuals 
have elevated day time plasma prolactin levels (20), and  reduced morning dopaminergic tone (21).  
Administration of bromocriptine-QR reduces the elevated day-time prolactin levels (12,20,22) 
and restores central dopaminergic activity, thereby reducing sympathetic activity, hepatic 
gluconeogenesis, postprandial plasma glucose, triglycerides, and FFA concentrations, all 
without increasing plasma insulin levels (Figure 2) (23).  
Cardiovascular risk in DM and bromocriptine: It is well known that type 2 diabetic subjects are at 
high risk for atherosclerotic cardiovascular complications. More than 50% of adults with T2DM have 
coronary artery disease (CAD), and death from CAD is two to five-times more likely in a diabetic 
than in a nondiabetic patient (24). Although hyperglycemia is a risk factor for cardiovascular events, 
it is relatively weak when compared with other risk factors such as dyslipidemia, hypertension, 
obesity, insulin resistance and metabolic syndrome (25). However, even after correction of these 
more established risk factors, type 2 diabetic subjects still remain at high risk for atherosclerotic 
cardiovascular complications (26). Moreover, a number of recent trials (27) including ADVANCE (6) 
and VADT (7), which assessed the impact on cardiovascular events of intensive glucose-lowering 
therapy, were not able to demonstrate a significant reduction of cardiovascular events in the 
intensive group as compared to the standard group. The primary endpoints in these studies were 
similar, mainly MACE including: non-fatal MI, stroke, PVD, CABG and sudden death. None of these 
studies showed a significant reduction in MACE. Furthermore, in ACCORD, the study with the most 
ambitious goal (HbA1c < 6%), the overall and cardiovascular mortality was greater in the intensive 
group, with a 22% increase in the likelihood of sudden death. The presence of autonomic 
dysfunction increased the risk of an event 2.8 times and the presence of peripheral neuropathy 
raised the risk to 4.4. Similarly, the presence of autonomic dysfunction combined with peripheral 
neuropathy, was the strongest predictor of cardiovascular events in the DIAD study. In the first three 
studies, the hypoglycemic risk was indeed increased in the intensive group.  However, hypoglycemia 
was not the cause of sudden death in the ACCORD study but a marker for susceptibility to the event 
(8).  It now seems that autonomic imbalance coupled with peripheral nerve dysfunction are lead 
candidates conferring susceptibility to CV risk (11). Therefore, anti-diabetic agents that not only 
improve glycemia but also reduce cardiovascular risk by altering 
sympathetic/parasympathetic balance are highly desirable (3). 
Diabetic CAN is a serious complication found in one fourth of type 1 and one third of type 2 diabetic 
subjects. It has long been recognized that cardiac autonomic neuropathy is associated with 
increased cardiovascular events and mortality in diabetes and may have greater predictive power 
than traditional risk factors for cardiovascular events. Moreover, recent studies have shown that 
autonomic imbalance may be a predictor for risk of sudden death with intensification of glycemic 
control (4,7,11).  This can be attributable to autonomic imbalance between the sympathetic and 

 
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 20 October 2017 
parasympathetic nervous system regulation of cardiovascular function (4,28). Chang et al. 
demonstrated that cardiac autonomic dysfunction (as measured by spectral analysis and expiratory-
inspiratory ratio) may occur prior to the development of insulin resistance in individuals with 1 or 2 
components of the metabolic syndrome (29). The Finnish Diabetes Prevention Study showed that 
cardiovascular autonomic dysfunction was common in subjects with impaired glucose tolerance and 
metabolic syndrome, especially in the overweight, obese subgroup (30). We recently demonstrated 
the presence of cardiac autonomic dysfunction both in newly diagnosed and established diabetic 
subjects (see preliminary data) (5). Thus, parasympathetic tone may decline with an autonomic 
imbalance shifting toward augmented sympathetic tone during the development from normal glucose 
tolerance to impaired glucose tolerance and finally diabetes (31). It is then not surprising to find 
abnormalities in autonomic function in the early stages of the disease, but without established 
autonomic neuropathy, suggesting a potential reversibility at the stage of newly diagnosed diabetes. 
In the cardiovascular safety trial (15), bromocriptine-QR reduced HbA1c, blood pressure, heart rate, 
and plasma triglycerides. However, these changes were modest and do not seem to explain the 
40% decrease in composite cardiovascular outcome. We propose that bromocriptine’s beneficial 
effect on cardiac events is that it decreases overactivity of the sympathetic nervous system and 
attenuates overactivity of the HPA axis and the RAS. In animal studies, bromocriptine has been 
shown to attenuate the effect of CNS sympathetic overactivity on the vasculature (32,33). 
Furthermore, in humans, bromocriptine has been shown to decrease plasma norepinephrine levels 
in the resting state as well as with maneuvers that increase catecholamine levels, such as tilting (34-
37). Although bromocriptine is no longer circulating or bound to D2 receptors throughout the 24-h 
period, changes in catecholamine levels persist because of the appropriately timed dopamine pulse. 
As increased sympathetic activity is associated with both sustained ventricular arrhythmias and heart 
failure, the reduction in sympathetic activity that occurs with bromocriptine could improve ventricular 
function and decrease cardiac arrhythmias in the diabetic patient who has a higher prevalence of 
these cardiac complications (38). Thus, bromocriptine- QR’s potential to restore a diurnal 
variation in sympathetic activity in addition to its ability to decrease total sympathetic 
nervous system activity may explain its cardioprotective effect. Yet, this has not been further 
investigated. 
 
Postprandial hyperglycemia, which is decreased by QR bromocriptine, has been shown to be 
associated with an increased risk of CV events in subjects with and without T2DM (39). Elevations of 
postprandial glucose especially when accompanied by potentially more important factors such as 
increased triglyceride and FFA levels, lead to inflammation, oxidative stress and endothelial 
dysfunction, which in turn may lead not only to increases in the volume of arterial atheroma but also 
to increased cardiac events. Pharmacological lowering of postprandial glucose has been shown to 
decrease cardiac events in some studies (STOP-NIDDM trial) (40-42) but not in others 
(NAVIGATOR trial) (43,44). Bromocriptine-QR significantly lowers postprandial glucose. This places 
it in a select group of drugs (α-glucosidase inhibitors, incretin mimetics, DPP4-inhibitors, pramlitide, 
thiazolidinediones and fast-acting insulins) that have been shown to effectively lower postprandial 
glucose. Therefore, the decrease in cardiac events seen with this drug could also be due to its 
effects on postprandial glucose and lipid levels (postprandial dysmetabolism) (38). However the 
mechanisms whereby these agents lower postprandial glucose may be very different. Here we will 
examine the contribution of changes in autonomic balance on postprandial blood glucose 
responses. 
Inflammation and Autonomic Imbalance: IL6, IL12 and C reactive protein (CRP) have been 
associated with reduced heart rate variability (HRV) and sympathovagal balance (4). Other markers 

 
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of inflammation such as TNF alpha, ADMA, and adipose-derived hormones have also been linked to 
CAN (4). It has been hypothesized that inflammation alters HRV, but the opposite directional 
relationship suggesting that autonomic changes could be pro-inflammatory is also conceivable (45). 
Experimental data support both possibilities. IL6 and CRP were found to be associated with reduced 
heart rate variability (HRV) in a study of 264 middle-age male twins free of symptomatic coronary 
artery disease (45). The results of this cross-sectional study suggest that autonomic dysregulation 
may lead to inflammation providing a pathway through which traditional risk factors promote the 
development of cardiovascular disease. Both exposure to acetylcholine and direct vagal stimulation 
inhibits release of cytokines by macrophages (46). Conversely, sympathetic activation is pro-
inflammatory. In isolated adipocytes, β-adrenergic stimulation increases IL6 (47), whereas β –
blockers dampen the IL6 increase normally seen in response to stress in rats (48). Given that 
cause–effect relationships cannot be determined from cross-sectional studies; prospective studies 
are needed to determine whether autonomic dysfunction mediates the inflammatory process or if 
autonomic imbalance is a consequence of inflammation. In the study performed in our lab we 
demonstrated an association between cardiac autonomic dysfunction and markers of inflammation 
(IL6, PAI1)  both in newly diagnosed and established diabetic subjects (see preliminary data) (5). An 
association between both CRP and IL6 and diabetic polyneuropathy has also recently been 
demonstrated (49). 
Oxidative/Nitrosative Stress and Autonomic Dysfunction: Growing evidence suggests that 
enhanced oxidative⁄nitrosative stress and, in particular, increased production of the potent oxidant 
peroxynitrite is a characteristic feature of both experimental and clinical diabetes mellitus (50). 
Peroxynitrite causes damage to a variety of tissues, including peripheral nerve, spinal cord, dorsal 
root ganglion neurons, and vasa nervorum (51). Several markers of oxidative stress in plasma, 
including superoxide and peroxynitrite, are elevated in subjects with diabetes and cardiac autonomic 
neuropathy (52). These findings suggest the presence of peroxynitrite cytotoxicity at both early and 
advanced stages of diabetes and, furthermore, at the pre-diabetic stage. Here we will examine the 
relationship between modulation of autonomic balance and the amelioration of oxidative and 
nitrosative stress.  
Leptin and Autonomic Dysfunction: It is well documented that leptin, secreted by the adipose 
tissue, plays a critical role in the regulation of energy, body weight and glucose homeostasis (2). 
Recent observations strongly suggest that leptin, just as insulin, can also regulate glucose 
homeostasis independent of its effects on weight loss (53). Leptin’s glucose homeostatic regulation 
also has a central component and the hypothalamic arcuate nucleus (ARC) has been spotlighted as 
the key CNS site.  Hyperleptinaemia may be an important player in the activation of the sympathetic 
nervous system in humans. Leptin levels were associated with a shift of sympathovagal balance 
toward increased sympathetic activation in a study of 120 non-obese adults (54). Leptin receptor-
deficient db ⁄ db mice develop T2DM, hypertension and obesity, with a disrupted circadian blood 
pressure, higher resting heart rates as well as loss of heart rate variability (55).  In our study (5) we 
showed a fall in the adiponectin/leptin ratio in newly diagnosed diabetes with further progression in 
established diabetes which accompanied the changes in autonomic balance (see preliminary data). 
This evidence shows that alterations in leptin homeostasis may have important 
consequences in the balance of the autonomic nervous system and changes in leptin in 
relation to autonomic balance with bromocriptine QR treatment will be monitored.  
Adiponectin and Autonomic Balance: Another adipocyte-derived protein that may be regulated by 
the sympathetic nervous system is adiponectin. In humans with autonomic imbalance (i.e. 
predominant sympathetic activation), low levels of circulating adiponectin have been demonstrated 

 
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(56). Adiponectin acts in the hypothalamic paraventricular nucleus to coordinate neuroendocrine and 
autonomic functions (57). Parasympathetic input to adipose tissue has been demonstrated by Kreier 
et al (58), illustrating that adipose tissue does receive dual autonomic control. This is important in the 
regulation of cytokine release, as well as in the control of release of FFA and the development of 
oxidative/nitrosative stress. In our study we showed decreased adiponectin/leptin ratios in both 
newly diagnosed and established diabetes and a significant correlation between these ratios and 
abnormalities in the ANS function (see preliminary data) (5). There appears to be a closed-loop 
system, wherein the adipose tissue mass regulates the hypothalamic autonomic system, with both 
the sympathetic and parasympathetic nervous system impacting the metabolic and inflammatory 
potential of adipose tissue. 
Significance of the proposal: Reduced HRV, the hallmark of  autonomic dysfunction, has been 
shown to have consequences in terms of morbidity (e.g. progression of coronary atherosclerosis) 
and mortality (59), independent of traditional cardiovascular risk factors in various populations, 
including those with pre-diabetes and diabetes (60,61). With a growing understanding of bi-
directional interactions between the sympathetic and parasympathetic efferent pathways at different 
levels of the neuro-axis and at target organs involved in inflammation (62), it is possible that this 
neuroinflammatory pathway  may be a key component involved in both the etiology and the clinical 
course of cardiovascular disease (4). The question arises as to what the mechanism could be. A 
number of biochemical and molecular abnormalities involved in the development of atherosclerosis, 
as well as multiple cardiovascular risk factors (hyperglycemia, hypertriglyceridemia, elevated FFA 
and hypertension) have been shown to improve with bromocriptine-QR therapy. Actually, it is not 
clear whether bromocriptine’s reduction on cardiovascular events is due to the drug’s 
beneficial effect on any of these pathologic processes since the response is so rapid and of 
such large magnitude.  Recent preclinical studies suggest that anti-inflammatory 
mechanisms in liver, other metabolic tissues, and the vessel wall itself as well as an effect to 
reduce reactive oxygen/nitrogen species generation in the vessel will contribute to the 
effects of bromocriptine-QR to reduce CVD outcomes (62A, 62B) Further mechanistic studies 
are necessary to establish the mechanism of action providing cardiovascular protective 
benefit of this therapy. Trials with bromocriptine have been performed in subjects with more 
established diabetes. We propose to conduct an interventional study with bromocriptine-QR in 
subjects with newly diagnosed versus established T2DM to evaluate its effects on cardiovascular 
autonomic function, HPA axis hormones and inflammatory markers of disease. This will aid into 
further understanding of the mechanisms by which this drug improves glucose tolerance and 
reduces cardiovascular risk in these subjects. The possibility of reversing autonomic imbalance by 
early intervention may have important impact in the development of new potential therapeutic 
strategies to abrogate the CV complications of diabetes.  
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2 STUDY OBJECTIVES 
2.1 PRIMARY OBJECTIVE 
To demonstrate the effects of dopaminergic activation on the autonomic nervous system in subjects 
with. The primary endpoint is the effect of bromocriptine-QR on changes in autonomic function 
measured by assessing sympathetic and parasympathetic function using conventional measures of 
autonomic function, including power spectral analysis of heart rate as well as peripheral autonomic 
function using sudorimetry and laser scanning of peripheral microvascular autonomic control. 
 
2.2 SECONDARY OBJECTIVES 
To demonstrate the effects of dopaminergic activation with bromocriptine-QR on the regulation of 
plasma neuroendocrine factors such as the hypothalamic-pituitary-axis (HPA) hormones, and on the 
plasma levels of markers of inflammation and oxidative/nitrosative stress in type 2 diabetes subjects. 
The study will evaluate treatment effects on inflammatory markers, the leptin/adiponectin system, 
and hormonal levels of renin-angiotensin system (RAS), aldosterone and cortisol. Specifically, we 
will evaluate the following markers of inflammation and oxidative/nitrosative stress: 1) CRP, 2) IL6, 
3) TNF α, 4) PAI1, SOD, TBARS and ADMA and nitrotyrosine as well as other markers of 
oxidative/nitrosative stress.  The study will evaluate the leptin/adiponectin system by measuring total 
adiponectin (TA), high molecular weight (HMW) adiponectin, leptin, and their ratios (TA/leptin and 
HMW adiponectin/leptin).  A co-secondary objective of the study will be to assess the impact of 
bromocriptine-QR vs Placebo on measures of insulin resistance and glycemic control (e.g., OGTT 
glucose and insulin, Matsuda index, HOMA-IR, HbA1c). 
The treatment effects on all of the above primary and secondary outcomes will also be assessed as 
a function of the duration of diabetes and other baseline demographics such as HbA1c, concomitant 
medications and metabolic status. 
3 STUDY DESIGN, DURATION AND DATES 
3.1 STUDY DESIGN 
This is an interventional, twenty-four week, randomized, double blind, placebo-controlled trial with 
bromocriptine-QR  in subjects with early diabetes and established T2DM to evaluate its effects on 
the cardiovascular and peripheral autonomic nervous system, as well as on inflammatory markers, 
the leptin/adiponectin system, hormonal levels of RAS and HPA, indices of insulin resistance and 
glycemic control, and measures of oxidative and nitrosative stress. A total of 80 subjects are planned 
to be enrolled in the study. At least 20 and no more than 40 early diabetes subjects and at least 40 
but no more than 60 subjects with established diabetes will be enrolled in the study and each 
randomized to treatment with bromocriptine-QR or placebo. Subjects will be required to be on a 
stable anti-diabetes regimen consisting of either diet and/or oral anti-diabetic agents consisting of 

 
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metformin alone or metformin plus an insulin secretion enhancer (sulfonylurea, DPP4 Inhibitors, or 
GLP-1 analogs) for at least 60 days prior to randomization. Following a 2 week lead-in period, 
subjects will be randomized in a 1:1 ratio to receive UDT (usual diabetes therapy) plus 
bromocriptine-QR or UDT plus placebo. Subjects must have a documented C-peptide level of >2 
ng/ml from the screening visit.  The circadian timing of the interventional therapy will be within 2 
hours of waking in the morning.  Following randomization subjects will be titrated to the maximum 
tolerated dose of the study drug over a four-week period. During these first 4 weeks, the daily dose 
of the study drug will be titrated up by one tablet (0.8 mg bromocriptine-QR or 1 matching placebo 
tablet) per day on a weekly basis until a maximal tolerated dose of at least two tablets (1.6 mg/day 
bromocriptine-QR or 2 placebo tablets) and no more than four tablets (3.2 mg/per day b-QR or 4 
placebo tablets) is achieved. During the first 4 weeks of the study, subjects will be called weekly to 
monitor for possible adverse events. Subjects will be maintained at their maximum tolerated dose of 
between two to four tablets per day (1.6 to 3.2 mg bromocriptine-QR per day or 2 to 4 placebo 
tablets) for the duration of the study.  Subjects unable to tolerate two tablets per day of study drug 
will be terminated from the study and any further analyses.  Subjects will be seen at 4 weeks after 
randomization, at 12 weeks after randomization, and then at study end (week 24 or early 
termination). A primary analysis of the primary and secondary endpoints will be performed after 12 
weeks with a secondary analysis conducted on these study endpoints at 24 weeks. Subjects will be 
contacted 30 days after stopping the study drug to record any adverse events that occurred after 
cessation. During the study period subjects will be advised to perform home blood glucose 
monitoring at least on a daily basis. Subjects will be required to continue their usual anti-diabetes 
regimen during the study but will be allowed to alter the dosages of these medications and/or add or 
subtract medications as deemed necessary by the Principal Investigator to optimize blood glucose 
control to avoid hypoglycemia or persistent hyperglycemia. In any case, rescue therapy for 
hyperglycemia will be administered if a subject has two consecutive fasting glucose measurements 
>270 mg/dL spaced ≤7 days apart before week 12, or two consecutive fasting glucose 
measurements >250 mg/dL spaced ≤7 days apart or a HbA1c >11% at or after week 12.  The choice 
of rescue therapy will be at the discretion of the Principal Investigator and may include adjustments 
in the dosages of the medications that the subject is already on and/or addition of other glucose 
lowering therapies.  The subjects with concurrent antidiabetes medication changes may remain "on 
study" for the remainder of the study until week 24 undergoing all evaluations if in the opinion of the 
investigator this does not pose any serious risk to the subject or the study objectives unless the 
rescue therapy requires insulin, in which case the subject will be withdrawn from the study as per the 
exclusion criteria for the study. Subjects will receive a complete physical and neurological exam and 
clinical laboratory assessments at baseline and at the completion of the study.  The primary and 
secondary endpoints will be evaluated at baseline, week 4, week 12, and week 24 or at early 
termination. Subjects will be evaluated using the procedures listed below. 
STUDY PROCEDURES 
 
 
Fasting blood chemistries- We will obtain blood samples using standard venipuncture 
techniques for determination of:  Fasting serum glucose, HbA1c, and C-peptide (C-peptide may be 
non-fasting); lipid profile including total serum cholesterol, HDL, LDL, FFA and triglycerides; 
comprehensive metabolic panel including creatinine, AST and ALT; TSH, renin, angiotensin, 
aldosterone, ACTH, cortisol, prolactin, and serotonin. Oral glucose tolerance tests and HOMA 
measure of insulin resistance and beta cell function will also be determined. All of these assays are 
performed routinely by Sentara Healthcare System. We will also collect samples to assess markers 

 
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of inflammation and oxidative/nitrosative stress which include 1) CRP, 2) IL6, 3) TNF alpha, 4) PAI1, 
SOD, TBARS and ADMA and nitrotyrosine as well as other measures of oxidative/nitrosative stress. 
We will measure total adiponectin (TA), high molecular weight (HMW) adiponectin, leptin, and their 
ratios (TA/leptin and HMW adiponectin/leptin).  The analyses of plasma markers of inflammation, 
oxidative/nitrosative stress, neuroendocrine factors and the adiponectin/leptin ratio will be conducted 
at Vero Science LLC (Tiverton, RI).    
 
 
Autonomic function tests- ANS function will be assessed by the measurement of heart rate 
variability (HRV), which is reduced early in the development of autonomic imbalance in 
diabetic subjects, and has been associated with increased risk for death after myocardial 
infarction. Power spectral analysis of HRV will be assessed using ANSAR (ANX 3.0 software; 
ANSAR Group, Inc., Philadelphia, PA). The ANSAR Parasympathetic and Sympathetic (P&S) 
monitoring method was developed and validated by the Harvard Medical School and Massachusetts 
Institute of Technology (MIT) Biomedical Engineering department (Akselrod and Aysin). The P&S 
monitoring method received US FDA Market Clearance in 1995. Time- and frequency-domain 
analyses will be performed. Time-domain analysis provides a measure of the sympathetic and 
parasympathetic control of the heart beat (the R-R interval on an electrocardiogram) recorded with 
maneuvers including deep breathing, Valsalva, and standing from the supine position while 
frequency-domain analysis is performed under resting conditions. Given their complementary nature 
both will be utilized in this study. Specifically, total spectral power (TSP) is the average low 
frequency plus high frequency for each phase during baseline, deep breathing, Valsalva, and 
postural stimulations. The sample difference of the beat to beat (NN) intervals measures heart rate 
variability, and was significantly impaired in both diabetic groups when compared to healthy controls 
in our prior study. The TSP will be calculated, as well as the standard deviation of all normal R-R 
intervals (sdNN), a measure of both sympathetic and parasympathetic action on HRV, and the root-
mean square of the difference of successive R-R intervals (rmSSD), a measure primarily of 
parasympathetic activity. It is also a measure of changing heart rate variability. These time and 
frequency domain analyses will allow quantification of ANS components, including 
sympathetic/parasympathetic balance before and after treatment with bromocriptine-QR. We 
anticipate that autonomic balance, lost early in T2DM subjects, will be re-established with 
bromocriptine-QR therapy. 
 
 
Sudorimetry- Sweat glands have a preganglionic sympathetic innervation that is regulated by 
acetylcholine and neuropeptidergic activity. The functional impairment of this system can 
cause abnormal sweating. The Sudoscan (Impeto Medical, Paris, France) will be used to measure 
the impact of autonomic imbalance on the sweat gland system using reverse iontophoresis. The 
Sudoscan will employ sudorimetry to assess sweat gland nerve fiber function. Test-retest reliability 
in 112 healthy controls before and after VO2max test was excellent for the feet (Correlation 
coefficient of 0.8, p<0.0001). Changes in peripheral autonomic function have now been shown 
to correlate with indices of insulin resistance and inflammation. We will determine if 
abnormal peripheral autonomic function occurs in newly diagnosed vs. established diabetes 
and if these are modified by bromocriptine-QR. 
 

 
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Skin blood flow (SkBF): methods and procedures- Since activation of the sympathetic arm of 
the autonomic nervous system has profound effects on microvascular function we will 
quantify this using laser Doppler techniques. We anticipate that impaired microvascular 
perfusion seen in newly diagnosed and established diabetes will be correctible with 
bromocriptine-QR. Continuous laser Doppler (CLD) will be used to assess skin blood flow in 
response to several stimuli and allows us to evaluate sympathetic versus neuropeptidergic control of 
neurovascular perfusion. CLD measurements are a reliable index of SkBF and are uninfluenced by 
blood flow in the underlying muscle (63). Several pieces of equipment in our lab will be used:  the 
Periflux Master Unit PF4001-2, the Peritemp heating module with sensor PF4005-3, and the 
(Pressure Unit) (all from Perimed, Inc., Smithtown, NY). After a 30-minute acclimation period 10 
minutes of baseline blood flow will be recorded.  Following this ten minute period heat will be applied 
to stimulate WT and NOCI blood flow.  WT stimulation will be achieved at 40°C and NOCI 
stimulation will be achieved by heating the skin to 44°C for 10 and 40 minutes respectively. Skin 
perfusion will also be measured during cholinergic stimulation. 
 
Laser Doppler Skin Blood Flow Imaging- Laser Doppler imaging (Moor Instruments Inc., 
Wilmington, DE) is a standard non-invasive, non-contact technique used to monitor and measure 
blood flow in very small blood vessels of the microvasculature.  Sympathetic overactivity affects the 
cardiovascular system causing vasoconstriction of small, resistance vessels and we will use this 
technique as a measure of peripheral sympathetic activity. Skin blood flow images will be recorded 
using the Moor LDI2-VR imager.  A visible red laser beam will be placed 14 inches from the exposed 
site and it will take a few minutes to scan over each site being measured.  The information collected 
by the laser is digitally processed into a color coded image displaying the skin blood flow at that site. 
 
Quantitative sensory tests (QST) - Sympathetic activation can be measured by quantifying the 
sensory perception of heat and cold stimuli. Enhanced responses to these stimuli (hyperesthesia 
and hyperalgesia) reflect increases in sympathetic tone and will be used as a measure of enhanced 
peripheral sympathetic activity mediated by the loss of central dopaminergic tone and correctible 
with bromocriptine. We will use our previously published methods and algorithms for measuring 
small fiber somatosensory function, including cold and warm thermal sensation, and cold- and heat-
induced pain thresholds at the dominant great toe, forearm, and finger in all subjects (64).  Generally 
for each of these non-noxious sensations we will use the method of limits, 4 ascending trials with an 
inter-stimulus interval randomly varying from 4 to 20 seconds using the Medoc TSA 2001 / VSA 
3000 (Medoc Advanced Medical, Minneapolis, MN).  Threshold is calculated as the mean stimulus 
intensity level over all 4 responses.  
 
3.2 STUDY DURATION AND DATES 
The duration of this study is expected to be approximately 38 months, with subject recruitment 
proposed to start in July 2015 and end by August 2018.  The actual overall study duration or subject 
recruitment period may vary.

 
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4 SELECTION OF SUBJECTS 
4.1  NUMBER OF SUBJECTS 
A total of up to 80 subjects are planned to be enrolled; At least twenty and no greater than 
forty early diabetes subjects (diabetes duration of   < 4 years) and at least 40 but no greater 
than 60 subjects with established diabetes (diabetes duration of  ≥4 years) will be enrolled in the 
study.  
4.2 INCLUSION CRITERIA 
 
1. 
Diagnosis of Type 2 Diabetes (as defined by the 2004 American Diabetes Association 
guidelines) 
2. 
Age 30–80 years 
3. 
HbA1c at screening ≤ 10 
4. 
Male or Female (female of child bearing age must use definitive contraceptive therapy) 
5. 
Type 2 Diabetes Mellitus subjects on a stable anti-diabetes regimen of diet and/or 
metformin alone therapy or on metformin plus an insulin secretion enhancer 
(sulfonylureas, DPP4 Inhibitors, GLP-1 analogs) therapy for a 60 day period prior to 
randomization.  Subjects must have a documented C-peptide level (either fasting or 
random) of > 2 ng/ml from the screening visit.  
 
4.3 EXCLUSION CRITERIA 
1. 
Presence of type 1 diabetes mellitus.  
2. 
Type 2 diabetes mellitus subjects on insulin. 
3. 
Use of prescription sympathomimetics, ergot alkaloid derivatives, or anti-migraine 
medications, dopamine2 (D2)-like receptor antagonists (e.g. metoclopramide, domperidone) 
or systemic corticosteroids 
4. 
Uncontrolled hypertension (systolic BP >160 or diastolic BP > 100 at screening) or a history 
of orthostatic hypotension 
5. 
History of significant gastroparesis  
6. 
Presence of diabetic retinopathy that is more severe than “background” level 
7. 
Presence of clinically significant peripheral or autonomic neuropathy that is clearly of non-diabetic origin 
8. 
Presence of renal impairment defined by serum creatinine > 1.4 mg/dl if female taking 
metformin, >1.5 mg/dl. if male taking metformin, and >1.6 mg/dl if not taking metformin 
9. 
History of major macrovascular events such as myocardial infarction or cerebrovascular event such as 
stroke within the past 6 months. Other exclusions include coronary artery bypass graft or 
coronary angioplasty in the previous 3 months, unstable angina pectoris (chest pain at rest, 
worsening chest pain, or admission to the ER or hospital for chest pain) within the previous 3 
months, or seizure disorders. 
10. 
Active infection (e.g., HIV, hepatitis), or a history of severe infection during the 30 days prior 
to screening 

 
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11. 
Major surgical operation during the 30 days prior to screening 
12. 
Cancer, other than non-melanoma skin or non-metastatic prostate cancer, within the past 5 
years 
13. 
Uncontrolled or untreated hypothyroidism as evidenced by TSH concentrations >4.8 uU/ml 
14. 
Other serious medical conditions which, in the opinion of the investigator, would compromise the 
subject’s participation in the study, including any concurrent illness, other than diabetes mellitus, 
not controlled by a stable therapeutic regimen, or conditions or abnormalities (e.g., blindness) 
that might interfere with interpretation of safety or efficacy data, or history of non-compliance 
15. 
Clinically significant abnormalities on screening laboratory evaluation, unless approved by the Sponsor 
16. 
Abnormalities of liver function defined as any liver enzymes (AST, ALT, SGPT, SGOT) greater than 3 
times the upper limit of normal  
17. 
History of NYHA Class III-IV congestive heart failure. 
18. 
Concurrent participation in another clinical trial with use of an experimental drug or device within 30 days 
of study entry. 
19. 
History (within 3 years) of alcohol or substance abuse, or dementia  
20. 
Pregnant or lactating women. Women of childbearing potential must have a negative 
pregnancy test at screening. Women who become pregnant will be discontinued from the 
study.  
21. 
Known hypersensitivity to any of the formulation components 
22. 
Working rotating, varying or night shifts 
23. 
Use of unapproved herbal supplements that may be associated with a risk of cardiovascular 
events (such as ephedra, yohimbe etc) 
24. 
Subjects who have started therapy with an erectile dysfunction drug within 2 weeks prior to 
screening; subjects may not begin treatment with an erectile dysfunction drug during the 
study period; subjects currently taking erectile dysfunction drugs should do so only under 
medical supervision. 
25. 
Donation of blood in the previous 30 days. Blood donation is also not allowed during the 
study or for 30 days after completion of the study.  
 
 
 

 
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5 STUDY TREATMENTS 
5.1 DETAILS OF STUDY TREATMENTS 
Bromocriptine-QR, 0.8 mg/day, with the dose increased by 0.8 mg/day every week to a maximum of 
3.2 mg/day, or as tolerated to a minimum dose of 1.6 mg/day, or matching placebo, added on to 
usual diabetes therapy consisting of a stable anti-diabetes regimen of diet and/or metformin alone 
therapy or metformin plus an insulin secretion enhancer (sulfonylureas, DPP4 Inhibitors, GLP-1 
analogs) therapy for a 60 day period prior to randomization.  
5.2 SUPPLIES AND ACCOUNTABILITY      
Bromocriptine-QR tablets and matching placebo will be provided by the Sponsor.  Accountability will 
be performed at the study site at the end of the study.  
5.3 COMPLIANCE 
Subjects will be instructed to bring their current, used and unused (vials) of study drug to every visit.  
5.4 RUN-IN MEDICATION 
 
There is no run-in medication prior to enrollment. Subjects will remain on their current regimen of 
diabetes therapy.   
6 PRIOR AND CONCOMITANT ILLNESSES AND TREATMENTS 
6.1 PRIOR AND CONCOMITANT ILLNESSES 
Additional illnesses present at the time informed consent is given are regarded as concomitant 
illnesses and must be documented in the case report form. Relevant past illnesses must also be 
documented in the case report form. Illnesses first occurring or detected during the study, and 
worsening of a concomitant illness during the study, are to be regarded as adverse events and must 
be documented as such in the case report form. 
6.2 PRIOR AND CONCOMITANT TREATMENTS 
All treatments being taken by the subjects on entry to the study or at any time during the study in 
addition to the study drug are regarded as concomitant treatments and must be documented on the 
appropriate pages of the case report form.  Concomitant medications should be kept to a minimum 
during the study and adhere to the inclusion/exclusion criteria described above.  However, if these 
are considered necessary for the subject's welfare and are unlikely to interfere with the 
investigational products, they may be given at the discretion of the investigator and recorded in the 
case report form. Subjects on systemic corticosteroids will be excluded.  

 
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7 OVERVIEW OF DATA COLLECTION AND STUDY PROCEDURES 
 
7.1 STUDY PROCEDURES AND SCHEDULES 
The study will consist of a screening visit, followed by visits at week 0 (baseline), week 0 + 1-3 days 
for study drug initiation, week 4, week 12 and week 24 or early termination.  There will be a final 
telephone contact 30 days after discontinuation of study drug to assess for new adverse events or 
for resolution of ongoing adverse events.  
 
7.2 DESCRIPTION OF STUDY DAYS 
 
7.2.1 Screening - Visit 0  
Screening of subjects will occur at Visit 0. Potential subjects will be instructed to arrive at the 
screening visit having fasted from midnight the night before. Consent form must be signed prior to 
conduct of any study procedures including screening tests for eligibility. 
 
 
• 
Each subject will be assigned a study subject number*  
 
• 
Assessment of inclusion/exclusion criteria will be performed* 
 
• 
Medical history, including demographic and background assessments will be documented in the 
CRF* 
 
• 
Routine physical examination will be performed* 
  
• 
ECG test will be performed* 
 
• 
Blood will be taken for the determination of HbA1c, clinical chemistry variables (and serum 
pregnancy test for women of child-bearing potential), hematology, (see study schedule).  
 
• 
A C-peptide level (fasting or random) will be measured and must be > 2 ng/ml for continuation in 
the trial.*  
 
• 
Total amount of blood drawn at this visit will be approximately 30 ml. 
 
* These procedures may be performed at the same visit the consent form is signed or at Screening Visit 
0.  
 

 
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7.2.2 Study Days – Treatment Period 
7.2.2.1 Baseline – Visit 1 (Post Visit 0 + <30 days) 
Subjects will be instructed to arrive having fasted from the night before this visit.  
 
 
• 
Sudorimetry  
o Sudoscan  
• 
Autonomic Function Tests 
o Heart Rate Variability (HRV) 
 
• 
Blood will be taken for the determination of HbA1c, lipids, neuroendocrine factors, 
inflammatory markers and oxidative/nitrosative stress markers 
 
• 
Urine will be obtained for measurement of microalbumin  
 
 
• 
An OGTT will be administered and OGTT glucose and insulin levels will be obtained at 0, 30, 
60, 90 and 120 minutes post-glucose challenge.  
o  
• 
Skin Blood Flow (SkBF) 
o Continuous Laser Doppler 
• 
Quantitative Sensory Tests (QST)  
 
• 
Total amount of blood drawn at this visit will be approximately 50 ml. 
 
• 
Subjects will be randomized per Eastern Virginia Medical School SOP II-805:  
 
• 
Subjects will be provided with instructions on starting study drug the morning after this visit.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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• Sub-investigator 
• Research coordinator 
 
Randomize and blind an investigational drug by 
assigning each patient/subject a study number in the order 
they are recruited.  The patients are randomized using a 
randomizing web site at (www.randomizer.org).  The 
randomized patient numbers are recorded in the grid in 
Attachment A, Blinding/Randomization Code.  This code 
is then placed in an envelope and sealed.  Place a label 
(Attachment B, Blinding/Randomization Individual 
Subject Envelope Label) on the envelope.  This envelope 
should remain sealed until the study is concluded. 
 
 
• Sub-investigator 
• Research coordinator 
 
 
 
Complete a blinding/randomization patient form, 
(Attachment C) for each patient and seal the individual 
envelopes.  The outside of the envelopes should contain 
patient numbers for identification purposes.  This allows 
the research coordinator to break the individual blind in 
emergency situations without compromising the rest of 
the blinding data.   
Document all circumstances appropriately and place in 
the study binder. 
Report all serious adverse events to the FDA and IRB.  
More general guidelines on adverse event reporting can 
be found in SM-404 and II-806 of these SOPs. 
• Sub-investigator 
• Research coordinator 
 
The randomization code and the individual patient forms 
should reside in the research coordinator’s office in one 
large envelope labeled with the Blinding/Randomization 
Envelope Label (Attachment D). 
7.2.2.2 Call 1 (Visit 1 + 7 days) 
• 
Study site will contact the subject by telephone to remind them to increase the dose of 
bromocriptine-QR to 2 tablets and assess tolerance. Subjects will be instructed to contact the 
study site if they experience intolerable nausea or vomiting. 
• 
Assessment for any AE/SAE will be completed 

 
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7.2.2.3 Call 2 (Call 1 + 7 days) 
• 
Study site will contact the subject by telephone to remind them to increase the dose of 
bromocriptine-QR to 3 tablets and assess tolerance. Subjects will be instructed to contact the 
study site if they experience intolerable nausea or vomiting. 
• 
Assessment for any AE/SAE will be completed 
7.2.2.4 Call 3 (Call 2 + 7 days) 
• 
Study site will contact the subject by telephone to remind them to increase the dose of 
bromocriptine-QR to 4 tablets and assess tolerance. Subjects will be instructed to contact the 
study site if they experience intolerable nausea or vomiting. 
• 
If at any time a subject is not able tolerate the next higher dose of bromocriptine-QR, they 
may stay at the current dose, providing it is at least 2 tablets per day.  
• 
Assessment for any AE/SAE will be completed 
• 
Subjects will schedule an appointment for the week 4 follow up visit on day 28 ± 3 days 
7.2.2.5 Visit 2 (Visit 1 + 4 weeks [± 3 days]) 
Subjects will be instructed to arrive having fasted from the night before this visit and having taken 
their morning dose of the study drug, with time taken noted. 
• 
Assessment for any AE/SAE will be completed 
• 
Medical history will be taken 
• 
Sudorimetry  
o Sudoscan 
 
• 
Autonomic Function Tests 
o Heart Rate Variability (HRV) 
 
• 
Blood will be taken for the determination of HbA1c, HOMA-IR (FPG and insulin), 
neuroendocrine factors, inflammatory markers and oxidative/nitrosative stress (see study 
schedule).  
 
• 
Total amount of blood drawn at this visit will be approximately 25 ml. 
 
• 
Skin Blood Flow (SkBF) 
o Continuous Laser Doppler 

 
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• 
Quantitative Sensory Tests (QST)  
 
7.2.2.6  Phone call 4 (Visit 2+ 4 weeks) 
 
• 
AE/SAE Query 
 
• 
Study Drug Compliance Assessment  
 
7.2.2.7 Visit 3 (Visit 1plus 12weeks [± 3 days]) 
Subjects will be instructed to arrive having fasted from the night before this visit and having taken 
their morning dose of the study drug with time taken noted. 
• 
Assessment for any AE/SAE will be completed 
• 
Medical history will be taken 
 
• 
Sudorimetry  
o Sudoscan 
 
• 
Autonomic Function Tests 
o Heart Rate Variability (HRV) 
 
• 
Blood will be taken for the determination of HbA1c, lipids, c-peptide, neuroendocrine factors, 
inflammatory markers and oxidative/nitrosative stress (see study schedule).  
 
• 
Total amount of blood drawn at this visit will be approximately 55-60 ml. 
 
• 
Urine will be obtained for measurement of microalbumin  
 
 
• 
An OGTT will be administered and OGTT glucose and insulin levels will be obtained at 0, 30, 
60, 90 and 120 minutes post-glucose challenge.   
 
• 
Skin Blood Flow (SkBF) 
o Continuous Laser Doppler 
 
 
• 
Quantitative Sensory Tests (QST)  

 
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7.2.2.8 Phone Call 5 (Visit 3 + 4 weeks [±3 days]) 
 
• 
AE/SAE Query 
 
• 
Study Drug Compliance Assessment  
7.2.2.9 Phone Call 6 (Phone Call 5 + 4 weeks [±3 days])) 
 
• 
AE/SAE Query 
 
• 
Study Drug Compliance Assessment  
 
7.2.2.10   Visit 4 (Visit 1 plus 24 weeks [± 3 days] or Early Termination)  
Subjects will be instructed to arrive having fasted from the night before this visit and having taken 
their morning dose of the study drug with time taken noted. 
• 
Assessment for any AE/SAE will be completed 
• 
Medical history will be taken 
 
• 
Routine physical examination will be performed  
  
• 
ECG test will be performed 
 
• 
Study drug will be discontinued  
 
• 
Sudorimetry  
o Sudoscan 
 
• 
Autonomic Function Tests 
o Heart Rate Variability (HRV) 
o  
• 
Blood will be taken for the determination of HbA1c, lipids, clinical chemistry variables, 
hematology, c-peptide, inflammatory markers and oxidative/nitrosative stress (see study 
schedule).  
 
• 
Total amount of blood drawn at this visit will be approximately 55-60 ml. 
 
• 
Urine will be obtained for measurement of microalbumin  
 

 
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• 
An OGTT will be administered and OGTT glucose and insulin levels will be obtained at 0, 30, 
60, 90 and 120 minutes post-glucose challenge.   
 
• 
Skin Blood Flow (SkBF) 
o Continuous Laser Doppler 
 
 
• 
Quantitative Sensory Tests (QST)  
7.2.3 End of Study Follow up Phone Call (Post Visit 4 + 27-30 days) 
 
• 
 Sites will contact all subjects to assess for new AE/SAE since study drug was discontinued 
or to resolve any AE/SAE that were ongoing at final visit.   
 
 
 
7.3 STATISTICAL METHODS 
Statistical analyses will be conducted on between group (bromocriptine-QR vs. placebo) differences 
in change from baseline of all primary and secondary endpoints.  The treatment effects on all of the 
primary and secondary outcomes will also be assessed as a function of the duration of diabetes and 
other baseline demographics such as HbA1c, concomitant medications and metabolic status. Mean 
differences of glycemic control, various derivative autonomic function values, and inflammatory 
marker levels will be compared first using standard Analysis of Variance (ANOVA), with diagnostic 
group entered as the predictor variable.  Within-group analysis for change in primary and secondary 
endpoints after 12 weeks of treatment will be done using repeated measures MANOVA (multivariate 
analysis of variance). For glycemic control analysis patients will be stratified according to post-
prandial insulin response. If data are not normally distributed, the Wilcoxon signed-rank (within 
group) or Mann Whitney (between group) tests may be employed or Fisher’s exact test in case of 
small sample sizes. The level of significance will be set at p<0.05. Relationships and/or trends 
between the effect of Cycloset on neurovascular function and diabetic neuropathy will be determined 
with Spearman’s rank correlation.  
A statistical analysis plan (SAP), providing details of the analyses and presentation structure of the 
results, will be developed and finalized before the database is locked. 
A local laboratory will be utilized for collection and analysis of the laboratory tests associated with 
efficacy, and safety. Standardized guidelines for preparation, collection, and centralized analysis of 
blood specimens will provide uniformity of the data and will avoid potential inter-laboratory variability 
due to inconsistent methodology.  
The analyses of plasma markers of inflammation, oxidative/nitrosative stress, neuroendocrine 
factors, and the adiponectin/leptin ratio will be conducted at VeroScience (Tiverton, RI).    

 
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7.3.1 Primary endpoint 
The primary endpoint is the effect of bromocriptine-QR on changes in autonomic function measured 
by assessing sympathetic and parasympathetic function using conventional measures of autonomic 
function, including power spectral analysis of heart rate as well as peripheral autonomic function 
using sudorimetry and laser scanning of peripheral microvascular autonomic control. 
7.3.2 Secondary endpoint 
Secondary endpoints will be the evaluation of bromocriptine-QR’s effects on inflammatory markers, 
neuroendocrine factors, markers of oxidative/nitrosative stress, the leptin/adiponectin system, and 
hormonal levels of renin-angiotensin system (RAS), aldosterone and cortisol.  A co-secondary 
endpoint will be the impact of bromocriptine-QR vs Placebo on measures of insulin resistance and 
glycemic control (e.g., OGTT glucose and insulin, Matsuda index, HOMA-IR, HbA1c). 
7.3.3 Safety Data 
Safety will be monitored throughout the trial by active and passive collection of reports of AE/SAE. 
 
Adverse Events 
AE monitoring will be conducted at all study visits as well as all titration contacts. (Please refer to 
Section 8 for additional information regarding AEs.) 
 
8 ADVERSE EVENTS 
8.1 DEFINITIONS 
8.1.1 Adverse Event 
The term adverse event covers any unfavorable and unintended sign, symptom, syndrome, or 
illness that develops or worsens during the period of observation in the clinical study.  Clinically 
relevant abnormal results of diagnostic procedures including abnormal laboratory findings (e.g., 
requiring unscheduled diagnostic procedures or treatment measures, or resulting in withdrawal from 
the study) are considered to be adverse events. 
Worsening of a sign or symptom of the condition under treatment will normally be measured by 
efficacy parameters.  However, if the outcome fulfills the definition of “serious adverse event”, it must 
be recorded as such (see Section 8.1.2). 
The adverse event may be: 
• 
A new illness 

 
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• 
Worsening of a concomitant illness 
• 
An effect of the study medication, including comparator 
• 
A combination of two or more of these factors 
No causal relationship with the study medication or with the clinical study itself is implied by the use 
of the term “adverse event”. 
Adverse events fall into the categories “non-serious” and “serious” (see Section 8.1.2). 
Surgical procedures themselves are not adverse events; they are therapeutic measures for 
conditions that require surgery.  The condition for which the surgery is required is an adverse event, 
if it occurs or is detected during the study period. Planned surgical measures permitted by the 
clinical study protocol and the condition(s) leading to these measures are not adverse events, if the 
condition(s) was (were) known before the start of study treatment.  In the latter case the condition 
should be reported as medical history. 
8.1.2 Serious Adverse Event 
A serious adverse event is one that at any dose (including overdose): 
• 
Results in death 
• 
Is life-threatening
1 
• 
Requires in subject hospitalization or prolongation of existing hospitalization 
• 
Results in persistent or significant disability or incapacity
2 
• 
Is a congenital anomaly or birth defect 
• 
Is an important medical event
3 
1“Life-threatening” means that the subject was at immediate risk of death at the time of the serious 
adverse event; it does not refer to a serious adverse event that hypothetically might have caused 
death if it were more severe. 
2“Persistent or significant disability or incapacity” means that there is a substantial disruption of a 
person’s ability to carry out normal life functions. 
3Medical and scientific judgment should be exercised in deciding whether expedited reporting is 
appropriate in situations where no outcomes listed above occurred.  Important medical events that 
may not be immediately life-threatening or result in death or hospitalization but may jeopardize the 
subject or may require intervention to prevent one of the other outcomes listed above should also 
usually be considered serious.  Examples of such events include allergic bronchospasm requiring 
intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not 
result in in-subject hospitalization, or the development of drug dependency or drug abuse.  A 
diagnosis of cancer during the course of a treatment should be considered as medically important.  

 
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The List of Critical Terms (1998 adaptation of WHO Adverse Reaction Terminology Critical Terms 
List, provided in the “Instructions for completing the ‘Serious Adverse Event/Expedited Report from a 
Clinical Trial’ form”) should be used as guidance for adverse events that may be considered serious 
because they are medically important. 
Clarification 
of 
the 
difference 
in 
meaning 
between 
“severe” 
and 
“serious” 
The term “severe” is often used to describe the intensity (severity) of a specific event (as in mild, 
moderate, or severe myocardial infarction); the event itself, however, may be of relatively minor 
medical significance (such as severe headache).  This is not the same as “serious”, which is based 
on the outcome or action criteria usually associated with events that pose a threat to life or 
functioning.  Seriousness (not severity) serves as a guide for defining regulatory reporting 
obligations. 
8.1.3 Alert Terms and Other Reasons for Expedited Reporting to Pharmacovigilance 
No special events are subject to reporting as alert terms in this study. 
However, cases in which a “significant overdose” of the investigational product was taken and a non-
serious adverse event or no adverse event occurred are to be reported to the sponsor in an 
expedited manner on a “Serious Adverse Event/Expedited Report from a Clinical Trial” form. 
In addition, any pregnancy diagnosed in a female subject or in the female partner of a male subject 
during treatment with the investigational product must be reported to the sponsor immediately.  
Information related to the pregnancy must be given on a “Drug Exposure via Parent – Data 
Collection” form that will be provided by the sponsor. 
 
8.2 PERIOD OF OBSERVATION 
For the purposes of this study, the period of observation for collection of adverse events extends 
from the time the subject gives informed consent until 30 days (± 3 days) after the last dose of study 
medication was given.  
If the investigator detects a serious adverse event in a study subject after the end of the period of 
observation, and considers the event possibly related to prior study treatment, he or she should 
contact the sponsor to determine how the adverse event should be documented and reported. 
8.3 DOCUMENTATION AND REPORTING OF ADVERSE EVENTS BY INVESTIGATOR 
All adverse events that occur during the observation period set in this protocol (see Section 8.2) 
must be documented on the pages provided in the case report form in accordance with the 
instructions for the completion of adverse event reports in clinical studies.  These instructions are 
provided in the investigator’s study file and in the case report form itself. 
The following approach will be taken for documentation: 

 
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• 
All adverse events (whether serious or non-serious, or considered as an alert term) must be 
documented on the “Adverse Event” page of the case report form. 
• 
If the adverse event is serious (see Section 8.1.2), the investigator must complete that 
section of the Adverse Event case report form. 
• 
This form must be completed and faxed to the sponsor’s Pharmacovigilance 
department within 24 hours.  
• 
If the adverse event is listed as an alert term (see Section 8.1.3) even if the “alert term” is 
non-serious, the investigator must complete, in addition to the “Adverse Event” page in the 
case report form, a “Serious Adverse Event/Expedited Report from a Clinical Trial” form at 
the time the adverse event is detected.   
• 
This form must be completed and faxed to the sponsor’s Pharmacovigilance 
department within 24 hours. 
• 
When a “significant overdose” of the investigational product occurs without an adverse event 
or in other situations where the sponsor requires an expedited report without an adverse 
event (see Section 8.1.3), the investigator should only complete a “Serious Adverse 
Event/Expedited Report from a Clinical Trial” form.  Instructions on where to send this form 
will be provided by the sponsor.  In this case, there is no need to complete the “Adverse 
Event” page in the case report form. 
Every attempt should be made to describe the adverse event in terms of a diagnosis.  If a clear 
diagnosis has been made, individual signs and symptoms will not be recorded unless they represent 
atypical or extreme manifestations of the diagnosis, in which case they should be reported as 
separate events. If a clear diagnosis cannot be established, each sign and symptom must be 
recorded individually. 
All subjects who have adverse events, whether considered associated with the use of the 
investigational products or not, must be monitored to determine the outcome.  The clinical course of 
the adverse event will be followed up according to accepted standards of medical practice, even 
after the end of the period of observation, until a satisfactory explanation is found or the investigator 
considers it medically justifiable to terminate follow-up. Should the adverse event result in death, a 
full pathologist’s report should be supplied, if possible. 
All questions on the completion and supply of adverse event report forms and any further forms 
issued to the investigator at a later date to clarify unresolved issues should be addressed to the 
sponsor. 
8.4 IMMEDIATE REPORTING BY INVESTIGATOR TO SPONSOR 
Serious adverse events and adverse events that fulfill a reason for expedited reporting to 
Pharmacovigilance (alert term and/or “significant overdose”, as defined in Section 8.1.3) must be 
documented on the Adverse Event form. 
• 
This form must be completed and faxed to the sponsor’s Pharmacovigilance department within 
24 hours. 

 
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The initial report must be as complete as possible, including details of the current illness and 
(serious) adverse event, and an assessment of the causal relationship between the event and the 
investigational product(s). 
Information not available at the time of the initial report (e.g., an end date for the adverse event or 
laboratory values received after the report) must be documented on a follow-up Adverse Event form. 

 
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9 WITHDRAWALS 
9.1 WITHDRAWAL OF SUBJECTS 
Subjects may be withdrawn from the study (i.e. from any further study medication or study 
procedure) for the following reasons:  
• 
At their own request or at the request of their legally authorized representative* 
• 
If, in the investigator's opinion, continuation in the study would be detrimental to the subject's 
well-being 
• 
At the specific request of the sponsor 
* “Legally authorized representative” means an individual or judicial or other body authorized under 
applicable law to consent on behalf of a prospective subject to the subject’s participation in the 
procedure(s) involved in the research. 
Subjects must be withdrawn from the investigational product under the following circumstances: 
• 
Women who become pregnant 
• 
Women of childbearing potential who discontinue contraception with the intention of 
becoming pregnant. 
In all cases, the reason for and date of withdrawal must be recorded in the case report form and in 
the subject's medical records.  The subject must be followed up to establish whether the reason was 
an adverse event, and, if so, this must be reported in accordance with the procedures in Section 8. 
The investigator must make every effort to contact subjects lost to follow-up.  Attempts to contact 
such subjects must be documented in the subject’s records (e.g., times and dates of attempted 
telephone contact, receipt for sending a registered letter). 
Subject withdrawal due to deterioration of glycemic control 
If a subject has a HbA1c of > 12% or fasting glucose readings of greater than 275 mg/dl on two 
consecutive days, then the subject’s regimen must be adjusted to include medications offering 
stricter glycemic control. As per the exclusion criteria, subjects requiring insulin must be withdrawn 
from the study. If the subject's HbA1c remains > 12%, he/she should cease study treatment to be 
stabilized on alternative medications.  Subjects that are withdrawn will be referred to their primary 
care physician or endocrinologist for further diabetes medication adjustments as needed.   
Replacement of subjects 
Subjects that are withdrawn within 4 weeks following completion of study drug titration will be 
replaced. As far as possible, for subjects who are withdrawn from the study, all safety examinations 
scheduled for the final study day will be performed prior to withdrawal.  

 
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9.2 EMERGENCY SPONSOR CONTACT 
In emergency situations, the investigator should contact the sponsor by telephone at the number 
given on the title page of the protocol. 
 
9.3 EMERGENCY IDENTIFICATION OF INVESTIGATIONAL PRODUCTS 
If during the course of the study, it becomes vital for the safety of the study subject to have her/his 
randomization code broken then the following Eastern Virginia Medical School SOP II-805 is to be 
followed. 
 
• Sub-investigator 
• Research coordinator 
 
Randomize and blind an investigational drug by 
assigning each patient/subject a study number in the order 
they are recruited.  The patients are randomized using a 
randomizing web site at (www.randomizer.org).  The 
randomized patient numbers are recorded in the grid in 
Attachment A, Blinding/Randomization Code.  This code 
is then placed in an envelope and sealed.  Place a label 
(Attachment B, Blinding/Randomization Individual 
Subject Envelope Label) on the envelope.  This envelope 
should remain sealed until the study is concluded. 
 
• Sub-investigator 
• Research coordinator 
 
 
 
Complete a blinding/randomization patient form, 
(Attachment C) for each patient and seal the individual 
envelopes.  The outside of the envelopes should contain 
patient numbers for identification purposes.  This allows 
the research coordinator to break the individual blind in 
emergency situations without compromising the rest of 
the blinding data.   
Document all circumstances appropriately and place in 
the study binder. 
Report all serious adverse events to the FDA and IRB.  
More general guidelines on adverse event reporting can 
be found in SM-404 and II-806 of these SOPs. 
• Sub-investigator 
• Research coordinator 
 
The randomization code and the individual patient forms 
should reside in the research coordinator’s office in one 
large envelope labeled with the Blinding/Randomization 
Envelope Label (Attachment D). 

 
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9.4 EMERGENCY TREATMENT 
During and after a subject's participation in the trial, the investigator and/or institution should ensure 
that adequate medical care is provided to a subject for any adverse events, including clinically 
significant laboratory values, related to the trial.  The investigator and/or institution should inform a 
subject when medical care is needed for intercurrent illness(es) of which the investigator becomes 
aware.  
9.5 ANALYSIS VARIABLES 
A statistical analysis plan (SAP), providing details of the analyses and presentation structure of the 
results, will be developed and finalized before the database is locked. 
 
9.6 ANALYSIS POPULATIONS 
The primary analysis of the study will be the assessment of the primary and secondary study 
endpoints after 12 weeks of study drug treatment.  The secondary analysis of the study will be the 
assessment of the primary and secondary study endpoints after 24 weeks of study drug treatment.  
Dropouts will be replaced as described in section 9.1.  
 
9.7 SAMPLE SIZE    
We propose that an effect size of 10% mean change from baseline in treated groups represents a 
clinically meaningful difference, and we propose that sample size reflects the ability to detect a 10% 
change due to intervention factor.  
We intend to use the parametric statistical procedures ANOVA and MANOVA in these analyses, 
based on our previous experience that the data from these tests is normally distributed in raw form 
or in some cases after simple log-transformations. If data are not normally distributed, the Wilcoxon 
signed-rank (within group) or Mann Whitney (between group) tests may be employed or Fisher’s 
exact test in case of small sample sizes. The level of significance will be set at p<0.05. Relationships 
and/or trends between the effect of Cycloset on neurovascular function and diabetic neuropathy will 
be determined with Spearman’s rank correlation.  
Based on these considerations, we calculate a total of 80 participants will result in a power greater 
than 0.80 for observing statistical significance at the p < 0.05 level. Specifically, we plan to recruit 
approximately 40 in the placebo group and approximately 40 in the treatment group.  In total, 
approximately 120 people will be screened in order to enroll approximately 80 participants in this 
study. JMP statistical Software version 9.3 will be used to perform all statistical analyses. 
 

 
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10 REGULATORY REQUIREMENTS 
10.1 GOOD CLINICAL PRACTICE 
This study is to be conducted according to globally accepted standards of good clinical practice (as 
defined in the ICH E6 Guideline for Good Clinical Practice, 1 May 1996), in agreement with the 
Declaration of Helsinki and in keeping with local regulations. 
10.2 DELEGATION OF INVESTIGATOR DUTIES 
The investigator will ensure that all persons assisting with the trial are adequately qualified, informed 
about the protocol, any amendments to the protocol, the study treatments, and their trial-related 
duties and functions. 
The investigator will maintain a list of sub investigators and other appropriately qualified persons to 
whom he or she has delegated significant trial-related duties. 
10.3 SUBJECT INFORMATION AND INFORMED CONSENT 
Before being enrolled in the clinical study, subjects must consent to participate after the nature, 
scope, and possible consequences of the clinical study have been explained in a form 
understandable to them. 
An informed consent document that includes information about the study will be prepared and given 
to the subject.  This document will contain all the elements required by the ICH E6 Guideline for 
Good Clinical Practice and any additional elements required by local regulations.  The document 
must be in a language understandable to the subject and must specify who informed the subject.  
Where required by local law, the person who informs the subject must be a physician. 
After reading the informed consent document, the subject must give consent in writing.  The 
subject's consent must be confirmed at the time of consent by the personally dated signature of the 
subject and by the personally dated signature of the person conducting the informed consent 
discussions. 
If the subject is unable to read, oral presentation and explanation of the written informed consent 
form and information to be supplied to subjects must take place in the presence of an impartial 
witness.  Consent must be confirmed at the time of consent orally and by the personally dated 
signature of the subject or by a local legally recognized alternative (e.g., the subject's thumbprint or 
mark).  The witness and the person conducting the informed consent discussions must also sign and 
personally date the consent document.   
A copy of the signed consent document must be given to the subject.  The original signed consent 
document will be retained by the investigator. 
 

 
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“Legally authorized representative” means an individual or judicial or other body authorized under 
applicable law to consent on behalf of a prospective subject to the subject’s participation in the 
procedure(s) involved in the research. 
The investigator will not undertake any measures specifically required only for the clinical study until 
valid consent has been obtained. 
It is recommended that the investigator inform the subject’s primary physician about the subject’s 
participation in the trial if the subject has a primary physician and if the subject agrees to the primary 
physician being informed. 
10.4 CONFIDENTIALITY 
Subject names will not be supplied to the sponsor.  Only the subject number and subject initials will 
be recorded in the case report form, and if the subject name appears on any other document (e.g., 
laboratory report), it must be obliterated on the copy of the document to be supplied to the sponsor.  
Study findings stored on a computer will be stored in accordance with local data protection laws.  
The subjects will be informed that representatives of the sponsor, independent ethics committee 
(IEC)/ institutional review board (IRB), or regulatory authorities may inspect their medical records to 
verify the information collected, and that all personal information made available for inspection will 
be handled in strictest confidence and in accordance with local data protection laws. 
The investigator will maintain a personal subject identification list (subject numbers with the 
corresponding subject names) to enable records to be identified. 
10.5 PROTOCOL AMENDMENTS  
Neither the investigator nor the sponsor will alter this clinical study protocol without obtaining the 
written agreement of the other.  Once the study has started, amendments should be made only in 
exceptional cases.  The changes then become part of the clinical study protocol.  
10.6 APPROVAL OF THE CLINICAL STUDY PROTOCOL AND AMENDMENTS 
Before the start of the study, the clinical study protocol, informed consent document, and any other 
appropriate documents will be submitted to the IEC/IRB with a cover letter or a form listing the 
documents submitted, their dates of issue, and the site (or region or area of jurisdiction, as 
applicable) for which approval is sought.  If applicable, the documents will also be submitted to the 
authorities, in accordance with local legal requirements. 
Investigational products can only be supplied to the investigator after documentation on all ethical 
and legal requirements for starting the study has been received by the sponsor.  This documentation 
must also include a list of the members of the IEC/IRB and their occupation and qualifications.  If the 
IEC/IRB will not disclose the names of the committee members, it should be asked to issue a 
statement confirming that the composition of the committee is in accordance with GCP.  Formal 
approval by the IEC/IRB should preferably mention the study title, study code, study site (or region 

 
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or area of jurisdiction, as applicable), amendment number where applicable, and any other 
documents reviewed.  It must mention the date on which the decision was made and must be 
officially signed by a committee member. 
Before the first subject is enrolled in the study, all ethical and legal requirements must be met. 
The IEC/IRB and, if applicable, the authorities must be informed of all subsequent protocol 
amendments and administrative changes, in accordance with local legal requirements.  
Amendments must be evaluated to determine whether formal approval must be sought and whether 
the informed consent document should also be revised. 
The investigator must keep a record of all communication with the IEC/IRB and, if applicable, 
between a coordinating investigator and the IEC/IRB.  This also applies to any communication 
between the investigator (or coordinating investigator, if applicable) and the authorities. 
10.7 ONGOING INFORMATION FOR INDEPENDENT ETHICS COMMITTEE/ 
INSTITUTIONAL REVIEW BOARD 
Unless otherwise instructed by the IEC/IRB, the investigator must submit to the IEC/IRB: 
• 
Information on serious or unexpected adverse events from the investigator’s site, as soon as 
possible 
• 
Expedited safety reports from the sponsor, as soon as possible 
• 
Periodic reports on the progress of the study 
10.8 CLOSURE OF THE STUDY 
The study must be closed at the site on completion.  Furthermore, the sponsor or the investigator 
has the right to close this study site at any time.  As far as possible, premature closure should occur 
after mutual consultation.  Depending on local legislation, it may be necessary to inform IEC/IRB and 
the regulatory authorities when the study site is closed. 
Study materials must be returned, disposed of or retained as directed by the sponsor. 
10.9 RECORD RETENTION 
The investigator must obtain approval in writing from the sponsor before destruction of any records. 
Essential documents should be retained until at least 2 years after the last approval of a marketing 
application in an ICH region and until there are no pending or contemplated marketing applications 
in an ICH region, or at least 2 years have elapsed since the formal discontinuation of clinical 
development of the investigational product.  However, because of international regulatory 
requirements, the sponsor may request retention for a longer period. 
Essential documents include: 
• 
Signed informed consent documents for all subjects 

 
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• 
Subject identification code list, screening log (if applicable) and enrollment log 
• 
Record of all communications between the investigator and the IEC/IRB 
• 
Composition of the IEC/IRB (or other applicable statement as described in Section 12.6) 
• 
Record of all communications between the investigator and sponsor (or CRO) 
• 
List of subinvestigators and other appropriately qualified persons to whom the investigator 
has delegated significant trial-related duties, together with their roles in the study and their 
signatures 
• 
Copies of case report forms and of documentation of corrections for all subjects 
• 
Investigational product accountability records 
• 
Record of any body fluids or tissue samples retained 
• 
All other source documents (subject medical records, hospital records, laboratory records, 
etc.) 
• 
All other documents as listed in section 8 of the ICH E6 Guideline for Good Clinical Practice 
(Essential Documents for the Conduct of a Clinical Trial) 
Normally, these records will be held in the investigator's archives.  If the investigator is unable to 
meet this obligation, he or she must ask the sponsor for permission to make alternative 
arrangements.  Details of these arrangements should be documented. 
10.10 LIABILITY AND INSURANCE 
Liability and insurance provisions for this study are given in separate agreements. 
10.11 FINANCIAL DISCLOSURE 
Before the start of the study, the investigator will disclose to the sponsor any proprietary or financial 
interests he or she might hold in the investigational products or the sponsor company as outlined in 
the financial disclosure form provided by the sponsor.  The investigator agrees to update this 
information in case of significant changes during the study or within one year of its completion.  The 
investigator also agrees that, where required by law or regulation, the sponsor may submit this 
financial information to domestic or foreign regulatory authorities in applications for marketing 
authorizations. 
Similar information will be provided by each sub-investigator to whom the investigator delegates 
significant study related responsibilities. 

 
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11 STUDY MONITORING AND AUDITING 
Monitoring and auditing procedures will be followed according to study site policy in order to comply 
with GCP guidelines.   
11.1 STUDY MONITORING AND SOURCE DATA VERIFICATION 
Monitoring will be done by personal visits from a representative of the sponsor (study monitor) that 
will check the case report forms for completeness and clarity, and crosscheck them with source 
documents.  In addition to the monitoring visits, frequent communications (letter, telephone, and fax), 
by the study monitor will ensure that the investigation is conducted according to protocol design and 
regulatory requirements. 
Study close-out will be performed by the study monitor upon completion of the study. 
11.2 ON-SITE AUDITS 
Domestic and foreign regulatory authorities, the IEC/IRB, and an auditor authorized by the sponsor 
may request access to all source documents, case report forms, and other study documentation for 
on-site audit or inspection.  Direct access to these documents must be guaranteed by the 
investigator, who must provide support at all times for these activities.  Medical records and other 
study documents may be copied during audit or inspection provided that subject names are 
obliterated on the copies to ensure confidentiality. 

 
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12 DOCUMENTATION AND USE OF STUDY FINDINGS 
12.1 DOCUMENTATION OF STUDY FINDINGS 
 
A case report form will be provided for each subject. 
All protocol-required information collected during the study must be entered by the investigator, or 
designated representative, in the case report form.  Details of case report form completion and 
correction will be explained to the investigator.  If the investigator authorizes other persons to make 
entries in the case report form, the names, positions, signatures, and initials of these persons must 
be supplied to the sponsor. 
The investigator, or designated representative, should complete the case report form pages as soon 
as possible after information is collected, preferably on the same day that a study subject is seen for 
an examination, treatment, or any other study procedure.  Any outstanding entries must be 
completed immediately after the final examination.  An explanation should be given for all missing 
data. 
A source data location list will be prepared prior to study start.  This list will be filed in both the trial 
master file and the investigator study file and updated as necessary. 
The completed case report form must be reviewed and signed by the investigator named in the 
clinical study protocol or by a designated sub-investigator. 
The sponsor will retain the originals of all case report forms.  The investigator will retain a copy of all 
completed case report form pages. 
  
12.2 USE OF STUDY FINDINGS 
All information concerning the product as well as any matter concerning the operation of the 
sponsor, such as clinical indications for the drug, its formula, methods of manufacture and other 
scientific data relating to it, that have been provided by the sponsor and are unpublished, are 
confidential and must remain the sole property of the sponsor.  The investigator will agree to use the 
information only for the purposes of carrying out this study and for no other purpose unless prior 
written permission from the sponsor is obtained. 
The sponsor has full ownership of the original case report forms completed as part of the study. 
By signing the clinical study protocol, the investigator agrees that the results of the study may be 
used for the purposes of national and international registration, publication, and information for 
medical and pharmaceutical professionals.  The authorities will be notified of the investigator's name, 
address, qualifications, and extent of involvement.  

 
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The sponsor will ensure that a final report on the study is prepared. 
 
The investigator (or coordinating investigator) will be required to sign a statement that he or she 
confirms that, to the best of his or her knowledge, it accurately describes the conduct and results of 
the study. 
All materials, documents and information supplied by the sponsor to the investigator, and all 
materials, documents and information prepared or developed in the course of the study to be 
performed under this protocol, shall be the sole and exclusive property of the sponsor.  Subject to 
obligations of confidentiality, the investigator reserves the right to publish only the results of the work 
performed pursuant to this protocol, provided, however, that the investigator provides an authorized 
representative of the sponsor with a copy of any proposed publication for review and comment at 
least 45 days in advance of its submission for publication.  In addition, if requested, the investigator 
will withhold publication an additional 90 days to allow for filing a patent application or taking such 
other measures as sponsor deems appropriate to establish and preserve its proprietary rights. 
It is agreed that, consistent with scientific standards, publication of the results of the study shall be 
made only as part of a publication of the results obtained by the investigator performing the protocol. 

 
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13 DECLARATIONS OF SPONSOR AND INVESTIGATOR 
13.1 DECLARATION OF SPONSOR 
This clinical study protocol was subject to critical review and has been approved by the sponsor.  
The information it contains is consistent with: 
• 
The current risk-benefit evaluation of the investigational product 
• 
The moral, ethical, and scientific principles governing clinical research as set out in the 
Declaration of Helsinki and the principles of GCP as described in the US Code of Federal 
Regulations, part 50, 54, 56, and 312, as well as in the ICH Guidelines, May 9, 1997. 
• 
The investigator will be supplied with details of any significant or new findings, including adverse 
events, relating to treatment with the investigational product. 
 
Sponsor Representative 
Date: ________  
Signature: _________________________________  
 
Name (block letters): _________________________  
13.2 DECLARATION OF INVESTIGATOR 
I confirm that I have read the above protocol.  I understand it, and I will work according to the 
principles of GCP as described in 21 CFR parts 50, 54, 56, and 312 and according to applicable 
local requirements. 
Investigator 
Date: __________  
Signature: ______________________________________  
 
Name (block letters): _____________________________