An Exploratory Study to Evaluate the Effects of Roflumilast on Insulin Sensitivity and Metabolic Parameters in Prediabetic Overweight and Obese Individuals
Phase: PHASE1
Conditions: Obesity
Sponsor: National Heart, Lung, and Blood Institute (NHLBI)

Protocol #:  13-H-0123 
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PI:  Jay Chung, MD, PhD 
Version 20.0 Date: 5/15/2018 
CLINICAL RESEARCH PROJECT  
 
 
 
 
Protocol # 13-H-0123 
 
 
 
 
 
 
 
 
 
 
IND: Exempt 
NHLBI Protocol: 
 
An Exploratory Study to Evaluate the Effects of Roflumilast on Insulin Sensitivity and Metabolic 
Parameters in Pre-diabetic Overweight and Obese Individuals 
 
Short Title: Roflumilast and Insulin Action 
 
Keywords:Roflumilast,Phosphodiesterase-4; Pre-diabetes; Incretins; Insulin Action; and Insulin 
Secretion 
 
Principal Investigator: 
*Jay H. Chung, M.D., Ph.D. 
 
Genetics and Development Biology Center /LBG  
National Heart, Lung, and Blood Institute 
National Institutes of Health 
BG 10 RM 7D14 
Bethesda, MD 20892 
Phone: 301-496-3075 
Email: chungj@nhlbi.nih.gov 
 
Associate Investigators: 
*Ranganath Muniyappa, MD, PhD 
301-451-7702 – 10CRC/5-3671 
NIDDK/DEOB[E] 
*Justin Rodante, PA-C, MPH 
301-451-7128 – 10CRC/3-5330 
NHLBI/OCD [E] 
Vandana Sachdev, MD 
301-496-3015– 10CRC/5-1436 
NHLBI/CVB [E] 
Kong Y. Chen, PhD 
301-451-1636 – 10CRC/5-3671 
NIDDK/HNK[E] 
Ahmed Gharib, MD 
301-451-8982 – 10CRC/3-5340 
NIDDK/BMIB[E] 
Shanna Bernstein, MPH, RD, CDE 
301-496-8170 – 10-CRC/B2-
2426 
CC/NUTR [E] 
Peter J Schmidt, MD 
301-496-6120  - 10-CRC/2-5330 
SBE/NIMH [E] 
Pedro E Martinez, MD 
301-402-0615 - 10-CRC/2-5330 
SBE/NIMH [E] 
Sandra MacDonald, R.N. 
Ijeoma Muo, MD, MPH 
Sung Park, Ph.D. 
Stephanie Mao, BS 
301-451-4899 - 10-CRC/5-1413 
301-451-3343 – 10-7D14 
301-451-3451 – 10-7D14 
301-451-3343– 10-7D13 
NHLBI/OCD [E]  
NHLBI/LOA [C] 
NHLBI/LOA (E) 
NHLBI/LOA (F)  
  The * indicates investigators allowed to obtain informed consent for this protocol. 
 
 
Research Coordinator: 
Sandra MacDonald, R.N. 
301-451-4899 - 10-CRC/5-1413 
NHLBI/CVP [E] 
 
 
 
Subjects in study at NIH: 
Number 
Gender 
Age range 
 
50 
M, F 
30-65 
 
 
Multi-center trial: 
No 
Ionizing Radiation for Research:  
Yes 
Off-Site Project: 
No 
DSMB Involvement: 
No 
Tech Transfer: 
No 
IND/IDE: 
No (FDA IND Exemption 04/19/2012) 

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PI:  Jay Chung, MD, PhD 
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IND Sponsor: 
Not Applicable 
 
Table of Contents 
1. 
Précis ................................................................................................................................................. 3 
2. 
Introduction ....................................................................................................................................... 4 
3. 
Background ........................................................................................................................................ 4 
3.1 
Hypothesis ..................................................................................................................................... 7 
4. 
Objectives and Specific Aims ............................................................................................................ 7 
4.1 
Primary Objective ......................................................................................................................... 7 
4.2 
Secondary Objective ..................................................................................................................... 7 
5. 
Investigational Plan ........................................................................................................................... 7 
5.1 
Study Design and Methods ........................................................................................................... 7 
5.2 
Description of Study Population and Eligibility ........................................................................... 8 
5.3 
Study Procedures .......................................................................................................................... 9 
5.4 
Schedule of Events ...................................................................................................................... 20 
6 
Pharmaceuticals .............................................................................................................................. 24 
6.1 
Roflumilast .................................................................................................................................. 24 
6.2 Stable isotope compound ................................................................................................................. 24 
7 
Data Collection and Management Plan ........................................................................................... 25 
8 
Statistical Considerations ................................................................................................................ 26 
8.1 
Statistical Methods ...................................................................................................................... 26 
8.2 
Sample Size Calculation ............................................................................................................. 26 
9 
Data and Safety Monitoring ............................................................................................................ 27 
9.1 
Safety Monitoring ....................................................................................................................... 27 
9.2 
Adverse Event Characterization and Reporting .......................................................................... 27 
9.3 
Stopping Rules ............................................................................................................................ 30 
9.4 
Protocol Monitoring .................................................................................................................... 30 
10 Human Subjects Protections ............................................................................................................ 30 
10.1 
Rationale for Subject Selection ................................................................................................... 30 
10.2 
Rationale for the Exclusion of Children...................................................................................... 30 
10.3 
Rationale for the Exclusion of Pregnant Women ........................................................................ 30 
10.4 
Risk/Benefit Assessment............................................................................................................. 30 
10.5 
Risks and Discomforts ................................................................................................................ 31 
10.6 
Informed Consent Documentation .............................................................................................. 36 
10.7 
Subject Advocate ........................................................................................................................ 36 
11 Conflict of Interest ........................................................................................................................... 37 
12 Reimbursement for Travel ............................................................................................................... 37 
13 Financial Compensation .................................................................................................................. 37 
14 References ....................................................................................................................................... 38 
Appendix 1:Study drug Diary .................................................................................................................. 40 
Appendix 2:Columbia Suicide Severity Rating Scale ............................................................................. 45 
Appendix 3:DALIRESP® package insert ................................................................................................ 54 
Appendix  4: Quality of Life Enjoyment and Satisfaction Questionnaire ............................................... 68 
 

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1. Précis 
Resveratrol, a polyphenol most notably found in red wine has anti-aging properties in mice fed a high-
fat diet; resveratrol protects against obesity and type 2 diabetes. Several clinical trials have been 
conducted to study the metabolic effects of resveratrol. Although these trials have used different subject 
groups (e.g. obese healthy, type 2 diabetics or older adults with glucose intolerance), they suggest that 
resveratrol may improve insulin sensitivity.  However, the therapeutic potential of resveratrol is 
diminished by the fact that it has a very promiscuous target profile. In order to translate resveratrol 
biology into clinical application, it is helpful to identify the cellular target(s) of resveratrol that mediate 
the desired effects and to develop therapies specific for that target(s).  Recently, we discovered that the 
metabolic effects of resveratrol appear to result from competitive inhibition of cAMP-degrading 
phosphodiesterases (PDEs), which increases cAMP levels. The cAMP-dependent pathways activate 
AMP-activated protein kinase (AMPK), which is essential for the metabolic effects of resveratrol. 
Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including 
protection against diet-induced obesity and an increase in mitochondrial content, fat oxidation, physical 
stamina and glucose tolerance in mice.  Based on results from cellular and preclinical studies, we 
hypothesize that PDE4 inhibition will ameliorate insulin resistance in pre-diabetic individuals.  To test 
these hypotheses, we will conduct an exploratory study on the potential beneficial effects of roflumilast 
(Daxas®), a PDE4 inhibitor, on insulin sensitivity in pre-diabetic individuals. Each study participant 
will receive oral roflumilast (250 μg, once a day for 2 weeks, followed by 500μg once a day for 4 
weeks).  At baseline and after the 6-week treatment period, we will assess insulin sensitivity 
(hyperinsulinemiceuglycemic glucose clamp technique, “glucose clamp”).In addition, β-cell function, 
skeletal muscle mitochondrial function, body composition, and circulating adipocytokine profile will 
be measured at baseline and after treatment to evaluate potential changes that may be related to 
improvements in metabolic function.  Vascular function is not only an indicator of insulin sensitivity, 
but is also important for glucose delivery and metabolism.  If possible, vascular function will be 
assessed along with the other parameters at baseline and after treatment with roflumilast. Regarding 
vascular function, we may measure basal and insulin-stimulated brachial artery blood flow (large 
conduit artery assessed by Doppler ultrasound) as well as capillary recruitment in forearm skeletal 
muscle (small nutritive arterioles assessed by ultrasound with microbubble contrast).  This study will 
explore whether roflumilast is effective at improving insulin sensitivity in pre-diabetic individuals.  
Results from this study may have important implications for the potential use of roflumilast in treating 
type 2 diabetes. 
 
 
 
 
 
 
 
 
 
 
 
 

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2. Introduction 
In the United States, obesity and its related complications such as diabetes and cardiovascular disease 
has risen to near epidemic proportions resulting in significant morbidity, mortality, and health care 
costs. Consequently, there is considerable scientific and public interest in the efficacy of dietary and 
pharmacological interventions that may ameliorate obesity and the associated cardio-metabolic states.  
Phosphodiesterases (PDEs) have a key regulatory role in the cAMP pathway, as they are the only known 
mechanism for inactivating cAMP by its catalysis to 5′-AMP. Genetic ablation or pharmacological 
inhibition of PDE4 reduces inflammation and prevents diet-induced obesity in rodent models. In fact, 
the potent anti-inflammatory actions of PDE4 inhibitors underlie the basis for their therapeutic use in 
exacerbation of chronic obstructive pulmonary disease (COPD). Roflumilast, a selective PDE4 
inhibitor, improves lung function and other clinical outcomes in patients with chronic obstructive 
pulmonary disease (COPD).  An unexpected finding from post-hoc analysis of pooled data from 
previous roflumilast trials was that in patients with COPD and comorbid type 2 diabetes, roflumilast 
reduced fasting blood glucose and hemoglobin A1c (HbA1c) levels (2).  A subsequent double-blind, 
randomized parallel-group study showed that roflumilast decreased blood glucose and HbA1c levels in 
patients aged 35-70 years with newly diagnosed type 2 diabetes who were treatment naïve (3).   Glucose 
decreased by 1.04 mmol/L and HbA1c decreased by 0.79%. As seen in prior trials, roflumilast decreased 
body mass index by -0.26kg/m2 more than with placebo. However, the mechanisms mediating this 
hypoglycemic effect are unknown. Presumably, the glucose lowering effect of roflumilast is mediated 
by cAMP signaling since such a small decrease in body weight would not be expected to decrease 
glucose significantly.  However, the gastrointestinal side-effects associated with roflumilast such as 
diarrhea and nausea may also contribute to the hypoglycemic effects. Even if cAMP signaling is 
responsible for the hypoglycemic effects of roflumilast, the mechanisms by which cAMP signaling can 
decrease plasma glucose levels have not been elucidated. In rodent studies, we have shown that PDE4 
inhibition increases basal metabolic rate, augments mitochondrial function, and improves insulin 
sensitivity (4).The observation that thefasting levels of insulin, adiponectin and leptin were not changed 
by roflumilast does not argue against this hypothesis because insulin sensitivity is reflected by a 
combination of insulin and glucose levels (5). Fasting insulin levels usually reflect hepatic insulin 
sensitivity.  Therefore, it is possible to increase insulin sensitivity and decrease glucose without 
affecting fasting insulin levels if the muscle insulin sensitivity is increased more than hepatic insulin 
sensitivity.  In addition, cAMP signaling has been shown to increase insulin secretion (1), raising the 
possibility that roflumilast may improve glucose tolerance by improving both insulin sensitivity and 
secretion.    Based on these observations, we hypothesize that oral administration of roflumilast will 
increase: insulin sensitivity, β-cell function, vascular function, mitochondrial function and favorably 
alter body composition and circulating adipocytokine profile in prediabetic overweight and obese 
individuals. This population has been chosen as the individuals will have impaired glucose and insulin, 
but will not have been treated with diabetic medications. 
 
3. Background 
Preliminary data: Resveratrol inhibits PDE4 and increases insulin sensitivity.        
Resveratrol is a natural polyphenol produced by plants in response to environmental stress (6) and is 
present in many plant-based foods, most notably red wine. In mice, long-term administration of 
resveratrol induced gene expression patterns that resembled those induced by calorie-restriction and 
delayed aging-related deterioration (7). Resveratrol also protected against obesity and increased insulin 
sensitivity in rodents fed a high calorie diet (8,9) and in type 2 diabetic patients (10), suggesting that 
the pathway targeted by resveratrol might be important for developing therapies for type 2 diabetes.   
Mitochondrial function, particularly in skeletal muscle, is crucial for insulin sensitivity because 
decreased mitochondrial oxidation of fat leads to accumulation of intramyocellular lipids, which leads 

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to insulin resistance. Individuals with type 2 diabetes have reduced skeletal mitochondrial content and 
function. The anti-diabetes property of resveratrol is mediated by activating 5’-AMP-activated kinase 
(AMPK), particularly in skeletal muscle (8,11-14).  AMPK is a heterotrimeric protein consisting of a 
catalytic subunit and two regulatory subunits, β and γ. Activation of AMPK has been shown to reduce 
fat accumulation and increase glucose tolerance, insulin sensitivity, and mitochondrial function in 
skeletal muscle and physical endurance (15-18). It is believed that AMPK is also a target of metformin, 
a first line type 2 diabetes drug. A hint that cAMP may mediate the metabolic effects of resveratrol, 
including activation of AMPK, was suggested by a previous study reporting that resveratrol increased 
cAMP production in breast cancer cells (19). Therefore, we investigated whether resveratrol increased 
cAMP levels in myotubes. As shown in Figure 1A, cAMP levels increased significantly with 
resveratrol.  Similarly,cAMP levels were elevated inskeletal muscle and white adipose tissue in 
resveratrol treated mice. The possibility that the increase in cAMP production was responsible for the 
activation of AMPK by resveratrol was examined by treating myotubes with resveratrol in the presence 
of the adenylyl cyclase (AC) inhibitor MDL-12,330A (Figure 1B). MDL-12,330A prevented 
resveratrol (50 μM) from increasing the phosphorylation of both AMPK (T172) and the AMPK 
substrate acetyl-CoA carboxylase (ACC) (S79), which are markers of AMPK activity.  These findings 
indicate that cAMP signaling is essential for resveratrol to activate AMPK. 
 
The intracellular levels of cAMP are determined by the activities of AC, which synthesize cAMP from 
ATP and cyclic nucleotide phosphodiesterases (PDEs), which hydrolyze cAMP or cGMP to AMP or 
GMP, respectively. We measured the effect of resveratrol on the activity of AC types 2, 6, and 8, which 
represent the three major subclasses of the ten ACs in mammals. We found that resveratrol had no 
effect on AC activity, either in the basal state or in the activated state (+forskolin), suggesting that 
resveratrol increased cAMP levels by inhibiting PDEs.   
 
The PDE superfamily is comprised of 11 families (PDE1-11) and each family contains a number of 
different isoforms. We measured the activities of recombinant PDE1, 2, 3, 4 and 5 in the presence of 
resveratrol. We found that resveratrol did not inhibit PDE2 or PDE5 but competitively inhibited PDE1 
(IC50∼6 μM), PDE3 (IC50∼10 μM) and PDE4 (IC50∼14 μM) (Figure 2). PDE4 is the predominant PDE 
in skeletal muscle.  If the metabolic effects of resveratrol result from inhibiting PDE4 in skeletal muscle, 
a known PDE inhibitor should produce metabolic effects very similar to those produced by resveratrol. 
To test this hypothesis, we treated mice on high-fat diet with PDE4 inhibitor rolipram (2 mg/kg/d).  
After 12-14 weeks of treatment, we isolated skeletal muscle and measured the mRNA levels of genes 
that are known to be induced by resveratrol and AMPK such as eNOS, PGC-1α and others important 
for mitochondrial biogenesis. We found that rolipram consistently increased the mRNA levels of these 
genes. In agreement with this, treatment with resveratrol, rolipram and cAMP induced mitochondrial 
biogenesis in C2C12 myotubes to comparable levels.  Rolipram and resveratrol also increased 
mitochondrial content to similar levels in mouse skeletal muscle (Figure 3A).  To determine whether 
increased mitochondrial function improved exercise tolerance, we exercised rolipram-treated mice on 
a treadmill.  Rolipram-treated mice ran a significantly greater distance on a treadmill before exhaustion 
than control mice (445±19 m vs. 268±50 m) (Figure 3B).  Taken together, these findings indicate that 
in regard to mitochondrial function in skeletal muscle, rolipram and resveratrol have very similar 
metabolic effects. The similarity between the effects of rolipram and resveratrol also extended to the 
white adipose tissue (WAT).  As was the case with resveratrol-treated mice (13,20), the 
phosphorylation levels of AMPK and ACC were increased in the WAT of rolipram-treated mice. 
C57BL6/J mice treated with rolipram were resistant to weight gain on a high fat diet and had less fat 
content even though their food intake was similar to that of control mice. Decreased weight gain despite 
normal food intake suggests that rolipram increased the metabolic rate.  Indeed, oxygen consumption 
rate was increased inrolipram-treated mice (Figure 3C), but physical activity levels were not affected 
indicating that rolipram increased the basal metabolic rate.  Rolipram-treated mice were more glucose 
tolerant (Figure 4A) and insulin sensitive (Figure 4B) than were control mice Taken together, these 

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findings indicate that PDE4 inhibition reproduces all the metabolic benefits of resveratrol such as 
increased mitochondrial function in skeletal muscle, metabolic rate and insulin sensitivity. Therefore, 
PDE4 inhibitors such as roflumilast may also decrease body weight and lower glucose by increasing 
mitochondrial function and increasing insulin sensitivity. 
 
 
 
 
 
 
 
Figure 1.A.cAMP levels in myotubes 30 min after treatment 
with resveratrol. B. Phosphorylation of ACC, an AMPK 
substrate, and AMPK in myotubes treated with resveratrol in 
the presence of adenylyl cyclase inhibitor MDL-12,330A 
Figure2. Inhibition curve of PDEs with resveratrol. 
Figure 3.  In mice, PDE4 inhibitor rolipram increases A. mitochondrial
density in skeletal muscle as measured by mitochondrial DNA (mtDNA)
content, B. Maximal running distance on treadmill, C.   Metabolic rate. 

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3.1 Hypothesis 
Oral administration of a phosphodiesterase-4 inhibitor, roflumilast for 6 weeks improves insulin 
sensitivity in pre-diabetic overweight/obese individuals. 
 
4. Objectives and Specific Aims 
4.1 Primary Objective 
Determine if oral administration of roflumilast (250 mcg p.o. once a day for 2 weeks followed by 
500 mcg p.o. once a day for 4 weeks) improves insulin sensitivity as assessed by the 
hyperinsulinemic euglycemic glucose clamp technique in prediabetic overweight/obese subjects. 
 
4.2 Secondary Objective 
4.2.1 
Determine if oral administration of roflumilast (250 mcg p.o. once a day for 2 weeks 
followed by 500 mcg p.o. once a day for 4 weeks) improves β-cell function in pre-diabetic 
overweight/obese subjects as assessed by the mixed meal test. 
 
4.2.2 
Determine if oral administration of roflumilast (250 mcg p.o. once a day for 2 weeks 
followed by 500 mcg p.o. once a day for 4 weeks) augments postprandial plasma incretin 
concentrations as assessed by the mixed meal test. 
 
4.2.3 
Determine the effects of oral roflumilast on circulating levels of adipokines, cytokines, and 
inflammatory markers (adiponectin, leptin, hs-CRP, TNF-α, plasma intracellular adhesion 
molecules (ICAM-1) and vascular cell adhesion molecule (VCAM-1)). 
 
4.2.4 
Determine if oral roflumilast alters: total body fat and lean body mass measured by dual-
energy x-ray absorptiometry (DEXA); total abdominal fat mass and its distribution in 
subcutaneous and intra-abdominal compartments measured by MRI/MRS. 
 
4.2.5 
Determine, if possible, if oral roflumilast improves vascular function in prediabetic 
overweight/obese subjects as assessed by vascular ultrasound and contrast-enhanced 
ultrasound (CEU) techniques. 
 
5. Investigational Plan 
5.1 Study Design and Methods 
The exploratory study will utilize an open label, non-randomized, single arm pre-post study design 
to test our hypotheses.  Subjects will receive oral roflumilast (250 mcg p.o. once a day for 2 weeks 
followed by 500 mcg p.o. once a day for 4 weeks) tablets for 6 weeks.  The two-step dose is aimed 
Figure 4.  Rolipram improves A. glucose tolerance and B. 
insulin sensitivity in mice. 

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at maximizing tolerability to roflumilast.  Fasting hepatic glucose production (by 2H-6-glucose 
infusion), glucose clamp, DEXA scan, abdominal MRI/MRS scan, and muscle and adipose tissue 
biopsies (optional) will be performed on all subjects at the beginning of the study and after the 6-
week treatment course.  Muscle and adipose tissue biopsies are optional. The tests for 
vascular function will be optional as well and will depend on whether or not the cardiology 
research team is available to do the vascular studies at the time of the clamp studies, the 
latter which is essential to our primary outcome.  These studies will all be performed in the 
facilities of the NIH Clinical Research Center. 
 
5.2 Description of Study Population and Eligibility 
Fifteen men and women between the ages of 30 and 65 in good general health, except for body 
mass index (BMI) >24.9 and <39.5 kg/m2 with pre-diabetes and currently not on any 
pharmacological regimens or nutritional supplements that affect insulin sensitivity will be 
recruited. The goal will be for 12 subjects to complete all study procedures, thus a sample size of 
15 will allow for dropouts and withdraws.   However, we are requesting to consent and screen on 
this protocol up to 50 participants to obtain the 15 eligible for treatment. Thus, up to 50 participants 
may be enrolled into this protocol. 
 
5.2.1 
Eligibility Criteria 
5.2.1.1 Inclusion Criteria 
• 
Adult, weight- and diet-stable men and women in good general health with no 
significant underlying illnesses and normal or clinically insignificant results 
(medical histories, laboratory profiles, physical examination, and 
electrocardiograms),  
• 
Women must be non-pregnant or post-menopausal, or women of childbearing 
potential must be non-lactating and using an effective form of birth control 
during and for 30 days after the study period (partner's use of condoms or 
partner's vasectomy is not an acceptable contraception method for this study) , 
• 
Must be 30 - 65 years of age, inclusive  
• 
Body Mass Index (BMI) > 24.9 and <39.5 kg/m2with a stable (± 2.5 kg) weight 
for the last 6 months by history, 
• 
Pre-diabetes, as defined by a fasting blood glucose of greater than 100 mg/dL and 
less than 126 mg/dL and/or A1C equal to or greater than 5.7 and less than or equal 
to 6.5 % 
• 
Subjects must be able to understand the protocol and provide written informed 
consent. 
 
5.2.1.2 Exclusion Criteria 
• 
Women will be excluded from our study if they are pregnant, breastfeeding, or if 
they plan to become pregnant prior to the end of the study, 
• 
Cannot be on any medications including multivitamins or nutritional supplements 
that in the investigator’s opinion will affect insulin sensitivity 
• 
Currently taking systemic corticosteroids, insulin, or anticoagulants, anxiolytics, 
ketoconazole, erythromycin, cimetidine, enoxacin, strong CYP 3A4/1A2 inducers 
(e.g., rifampicin, phenobarbital, carbamazepine, phenytoin), birth control pills 
containing gestodene and ethinyl estradiol, use food supplements that cannot be 
discontinued, or any other medication that the investigators deem a 
contraindication. 

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• 
AST or ALT >3 times the upper normal limit 
• 
Hepatitis B antigen, HIV or C positive antibody tests, 
• 
Liver disease, pulmonary disease, renal insufficiency (serum creatinine > 
1.5mg/dl), coronary heart disease, heart failure (New York Heart Association heart 
failure Class III or IV), peripheral vascular disease, coagulopathy. 
• 
History of or current diagnosis of major depressive disorder, or history of or 
current diagnosis of other psychiatric disorders that in the opinion of the 
investigator would make participant unsafe for the participant. 
• 
Currently being treated for any form of cancer or have a history of cancer, that in 
the investigator’s judgment would not make the participant a candidate for the 
study for safety or scientific reasons. 
• 
Claustrophobic, 
• 
On a weight loss program with ongoing weight loss, or a history of eating 
disorders. Actively using tobacco products or have used tobacco products within 
last year (>3 cigarettes/day), regular alcoholic beverage intake of more than two 
drinks per day.  Subjects with any condition that would have made them, in the 
opinion of the principal investigator (PI), unsuitable for the study. 
• 
Subjects with a contraindication for the ultrasound contrast agent. 
 
5.3 Study Procedures 
5.3.1 
Recruitment 
Subjects will be recruited through advertisements in cooperation with the NIH Clinical 
Center Patient Recruitment and Referral Center and/or the Clinical Research Volunteer 
Program.   
 
Advertisements may be placed in local newspapers or other mediums in order to reach a 
wide variety of ethnic and racial groups.  Flyers may also be placed on campus as well as 
the surrounding metropolitan area. 
 
Subjects may also find information about trials through the NIH, Clinicaltrials.gov, and 
NHLBI Internet recruitment sites. 
 
The contact information provided in all advertisements will be the NIH Clinical Center 
Patient Recruitment and Referral Center. 
 
When potential participants contact the NIH Clinical Center Patient Recruitment and 
Referral Center about the study, they are asked questions concerning basic eligibility for 
the study.  Participants that appear to be suitable candidates will be referred to the study 
team.   
 
The study team will contact the potential participant.  The study team will schedule a 
screening appointment at the NIH CC to obtain informed consent and perform the 
eligibility assessment.  If the participant requests a copy of the consent prior to the 
screening appointment, the consent may be faxed, emailed, or mailed to the potential 
participant. 
 
Based on recruitment data from prior studies, we propose to screen 50 participants to obtain 
the 12 eligible for treatment.  Thus, we propose to have a recruitment ceiling of 50. 
 
5.3.2 
Informed Consent 

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Informed consent will be obtained prior to eligibility assessment procedures being 
performed.  Up to 50 participants will be consented under this protocol and screened in 
order to ensure that 15 participants receive treatment.  Thus, up to 50 may be enrolled into 
this protocol.  See section 10.6 for detailed information on the process. 
 
5.3.3 
Eligibility Assessment 
Screening procedures will be performed prior to the start of treatment.  Screening 
procedures for enrollment will include the following: 
 
 
Medical History and Physical Examination 
 
A thorough medical history (including queries regarding psychosocial history, 
diet, physical activity and the use of nutritional supplements or complementary 
and alternative medicine therapies, prior history of patent foramen ovale (the 
latter which would be a contraindication to the ultrasound contrast agent)), and a 
physical examination. 
 
BMI assessment 
 
Baseline electrocardiogram 
 
Clinical Laboratory tests 
 
CBC w/differential 
 
Electrolytes (Sodium (Na), Potassium (K), Chloride (Cl), Bicarb (HCO3)) 
 
liver function tests (Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, 
Direct Bilirubin),  
 
Creatinine and BUN 
 
urinalysis,  
 
plasma glucose, 
 
plasma insulin,  
 
lipid profile (Total Cholesterol, Triglycerides, HDL Cholesterol, LDL 
Cholesterol (calculated)),  
 
C-peptide,  
 
HbA1C,  
 
free fatty acids (FFA),  
 
highly sensitive C-reactive protein, 
 
thyroid stimulating hormone function tests 
 
HIV, and hepatitis B and C antibody tests 
 
Serum pregnancy test 
 
Psychiatric Evaluation of Suicidal Ideation, Major Depression, Quality of Life, 
and other disorders 
 
Structured Clinical Interview for DSM Disorders (SCID) 
The SCID will be administered by a trained AI from NIMH.  The presence or 
absence of past and current episodes of major depression will be evaluated by 
administering a structured psychiatric interview called SCID-IV.{American 
Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders: 
DSM-IV 1994} Completion of the psychiatric interview usually takes 40 minutes, 
but can take as long as two hours. If a psychiatric disorder is found during the 
evaluation, the patient will be referred to the NIH consultation service or, if he/she 
prefers, they will be referred to a provider in the community. 
 
Columbia Suicide Severity Rating Scale (C-SSRS) 
The Columbia Suicide Severity Rating Scale (C-SSRS) will be employed to 

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evaluate suicidal ideation.  A baseline assessment will be performed prior to 
administration of investigational product at screening.  Then the subjects will be 
evaluated at each visit to the clinical center (21-22).  This scale is being 
administered due to roflumilast’s black box warning of suicidal ideation. 
The C-SSRS is a validated measure on suicidal ideation (21-22) and is 
recommended by the FDA for use in clinical trials using drugs that have been 
shown to potentially cause suicidal ideation (22). 
If the result of the C-SSRS, at any point in this study, indicates the participant is 
having thoughts of suicide and has the potential to act of those thoughts, the 
participant will be removed from the study and be referred for a psychiatry 
consult. Additionally, the participant will be followed to make sure the 
appropriate inventions are provided to resolve the event. 
 
Quality of Life Enjoyment and Satisfaction Questionnaire (Q-Les-Q) 
The Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES-Q) is a 
self-report measure designed to measure the degree of enjoyment and satisfaction 
experienced by participants (25). 
 
Best efforts will be made to admit research subjects within three months after the initial 
screening visit.  If more than three months have elapsed between the initial clinic screening 
visit and the planned inpatient admission date, research subject will be asked to repeat 
screening laboratory tests prior to the proposed inpatient admission.  These laboratory tests 
will be repeated to ensure that there is no interval change in the test results and will be 
performed within two days prior to the proposed admission date.  Clinically significant 
deviations from the initial screening results will lead to exclusion of the subject from the 
inpatient admission at that time. Decision on whether or not to re-consider the subject for 
continued participation in the study in the near future will be according to the clinical 
judgement of the research team. The research team may choose to evaluate the subject in 
clinic within two days prior to the planned admission to facilitate decision making about 
whether or not to proceed with the admission.  
 
Participants who do not undergo their first inpatient admission within six months after the 
initial screening visit will be removed from the study. 
    
• 
Diet and Exercise Assessment:  
For one day before admission (day -1 or -2 before admission) and the 3 days during 
admission to the NIH Metabolic Clinical Research Unit (MCRU) for visits 1 and 4, 
each subject will be provided with a weight-maintenance diet using a standard diet 
composition of 55% of energy from carbohydrate (minimum of 150 grams of 
carbohydrate), 30% of energy from fat, and 15% of energy from protein on an out-
patient basis. The food will be supplied by the Clinical Center Metabolic Kitchen and 
subjects may eat one meal per day on the MCRU if their outpatient visit occurred the 
day prior to admission. The rest of the meals and snacks will be packed out for them 
to take home. If the subject’s outpatient visit occurred 2 days prior to admission, then 
the meals for the next day (that is 1 day prior to admission) would be packed for them 
to take home. The daily caloric content of the diet during the out-patient segment will 
be determined by the Mifflin-St Jeor equation based on age, gender, height, weight, 
and an activity factor of 1.4. 
During screening, a dietitian will provide detailed instruction regarding the accurate 

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completion of 3-day food records (2 weekdays and 1 weekend day) to each subject. 
Subjects will be instructed to record their intake for the same three days that they wear 
the accelerometers just the week prior to each visit. Dietetic professionals will review 
all food records using three-dimensional food models with participants at visits 1 and 
4 before coding the food records into Nutrition Data System for Research (NDSR, 
Minneapolis, MN).   
5.3.4 
Baseline – Visit 1 
Baseline procedures will be conducted over four days, to be designated as (Day -2 or Day 
-1), Day 1, Day 2 and Day 3.  The first day (Day -2 or Day -1), participants will report to 
the MCRU as outpatients. The reason for having participants report on either 1 or 2 days 
prior to admission is to provide flexibility in scheduling for the participants and the 
research coordinator.  For the second part of visit 1 (Day 1), participants will be admitted 
to the NIH CC MCRU as inpatients for 3 days and 2 overnights.  The following procedures 
will be performed during this time period:  
• 
Diet and Exercise Assessment (Day -2/-1): 
For one day before admission to the NIH for inpatient visits 1 and 4, and for the three 
days during each inpatient admission, each subject will be provided with a weight-
maintenance diet using a standard diet composition of 55% of energy from 
carbohydrate (minimum of 150 grams of carbohydrate), 30% of energy from fat, and 
15% of energy from protein on an out-patient basis. The food will be supplied by the 
Clinical Center Metabolic Kitchen and subjects may eat one meal per day on the 
MCRU; the rest of the meals and snacks will be packed out for them to take home. The 
daily caloric content of the diet during the out-patient segment will be determined by 
the Mifflin-St Jeor equation based on age, gender, height, weight, and an activity factor 
of 1.4.Weight and BMI will be measured as part of this assessment. 
• 
Assessment of hepatic and peripheral insulin sensitivity (Day +2): 
The hepatic insulin-resistance index is the product of hepatic glucose production 
(HGP) and the corresponding plasma insulin concentration. In order to measure the 
differences in HGP we plan to assess fasting glucose kinetics with stable isotope 
technique. Basal endogenous glucose production will be determined using deuterium 
glucose as tracer. [6,62H2] glucose will be administered intravenously initially as a 
bolus (5.1 mg/kg) then at the rate of 0.072 mg/kg/min. After a three-hour infusion, 
blood samples will be collected at times 0, 10’, 20’, and 30’ minutes (+/- 5 minutes for 
all time points), and [6,62H2] -glucose levels will be determined by mass spectrometry. 
HGP will be assessed pre- and post-treatment intervention after an overnight fast.  
Hyperinsulinemic Euglycemic Glucose Clamp (Day +2) - This is the gold-standard test 
to determine insulin sensitivity. Urinary pregnancy test will be done to exclude 
pregnancy where applicable.  Using a four way parallel port, lines for infusing glucose 
(D20W), insulin, potassium phosphate (0.09 mEq/ml) and normal saline will be 
connected to a 20 gauge intravenous cannula placed in one arm.  Alaris pumps will be 
used to control the rates of infusion for glucose, potassium phosphate, and saline.  
Insulin will be infused at a constant rate (40 mU/m²/min), following a loading dose at 
double the rate for the first 8 min.  To assess suppression of hepatic glucose production 
during clamp, [6,62H2]-glucose will be infused at a rate of 0.0182 mg/kg/min for 
approximately 3 hr.  Potassium phosphate will be infused up to a maximum rate of 10 
mmol/hr (15 mEq K+/hr) to prevent hypokalemia.  The glucose infusion rate will be 
adjusted to maintain blood glucose at the fasting level for each patient.  The rate of 

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potassium phosphate infusion will be adjusted throughout the clamp period, according 
to empiric judgment of the patient’s insulin sensitivity (as demonstrated by the ongoing 
changes in glucose infusion rate), not to exceed the maximum rate specified above.  A 
second peripheral intravenous catheter will be placed in the opposite arm and will be 
used for obtaining blood samples for glucose determination. The arm used for sampling 
will be warmed with a heating blanket in order to arterialize the blood.  0.5 ml blood 
samples for measuring blood glucose will be obtained on the following schedule: 10 
and 5 minutes prior to beginning the insulin infusion; every 5 - 10 minutes until 
completion of the study (approximately 3 additional hours).  Blood samples will be 
analyzed immediately for determination of blood glucose concentration using a YSI 
2700 dual chamber blood glucose analyzer.  In addition, 1 ml blood samples will be 
taken for insulin determination 10 and 5 minutes before insulin infusion, at initiation 
of insulin infusion, and at 20 - 30 minute intervals after the beginning of insulin 
administration.  1 ml blood samples for potassium levels are also taken before initiation 
of clamp infusions and 1 hour after insulin infusion has been stopped.  After the clamp 
portion of the study, the insulin infusion will be turned off and the patient will be 
weaned from the glucose infusion over the next 2 hours while also eating a high 
carbohydrate, high protein meal.   
• 
Measurement of plasma incretins, circulating inflammatory markers and 
adipocytokines: 
Plasma incretin, adiponectin, leptin, adhesion molecules, TNF-α, and MCP-1 levels 
will be measured by Millipore, Billerica, MA. 
• 
Measurements of brachial artery flow and forearm capillary recruitment (Day +2): 
When administratively possible to schedule, brachial artery flow and forearm capillary 
recruitment will be assessed at baseline and during the steady-state period of the 
glucose clamp (2 h after initiation of insulin infusion) as previously described (Am J 
ClinNutr. 2008 Dec;88(6):1685-96).  For measurement of brachial artery flow, 
Doppler ultrasound will be used.  An ultrasound system with a linear array transducer 
with a transmit frequency of 7.5 MHz will be used for 2-D imaging in long-axis.  
Brachial artery diameter will be measured using on-line video calipers.  Measurements 
of capillary recruitment will be assessed by using the contrast-enhanced ultrasound 
(CEU) technique during continuous microbubble infusion.  Definity microbubbles that 
are FDA approved (octafluoropropane gas encapsulated in an outer lipid shell; Bristol 
Myers Squibb, N. Billerica, MA) will be used for all of the studies.  One vial of 
microbubbles (1.3 ml) will be used for baseline measurement of capillary recruitment 
while a second vial will be used to assess capillary recruitment at 2 hours after initiation 
of the insulin infusion for the glucose clamp.  Microbubbles will be intravenously 
infused using a Medfusion syringe pump at a rate of 150 ml/hr for approximately 10 
min.  Deep flexor muscles of the right forearm will be imaged in two dimensions at a 
transaxial plane 5 cm distal to the antecubital fossa.  Power imaging will be performed. 
Once Definity microbubbles reach a steady-state concentration (2 min after initiation 
of microbubble infusion), imaging at pulsing intervals of 0.2 to 15 seconds will be 
conducted.  This allows for progressively greater replenishment of microbubbles in the 
ultrasound beam.  The digitally acquired images will be transferred to an offline 
computer for further analysis.  Data analysis will include estimation of the rate of 
increase in capillary density and the absolute capillary density both at baseline and at 
two hours after initiation of the insulin infusion.  
 
• 
Radiologic Assessments (Day -2/-1 or on Day +1, +3): 

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 
Dual energy x-ray absorptiometry (DEXA) - With this technique, one can 
determine total and regional body fat and fat-free masses and can estimate 
appendicular muscle mass.   
 
Assessment of visceral, liver, and skeletal muscle fat content and 
distribution - Magnetic resonance imaging (MRI) and magnetic resonance 
spectroscopy (MRS) are used to quantify triglyceride content in the 
abdominal cavity, liver and muscle.  Multi-echo imaging using the 3.0 T 
magnet will be employed to measure ectopic fat. 
• 
Optional Skeletal Muscle Biopsy (Day -2/-1 or Day +1): 
To directly evaluate mitochondrial respiratory function (respiration and respiratory 
coupling and mitochondrial biogenesis genomic and proteomic analysis) skeletal 
muscle biopsies will be performed by general surgeons at the NIH Clinical Center, 
before and after roflumilast. The muscle biopsy is a routine outpatient procedure that 
is performed using local anesthesia.  Under sterile conditions, an excisional biopsy of 
400 mg of muscle will be obtained from the subject’s calf or thigh.  Each subject will 
undergo a total of two muscle biopsies: one at baseline and one after therapy with 
roflumilast. 
The muscle biopsy specimen will be divided into three parts. The first part will be 
snap frozen in liquid nitrogen for subsequent genomic, proteomic and enzymatic 
analysis. The other half will be transported to the laboratory in an ice-cold buffer (KE) 
containing 0.18M KCl and 0.01 M EDTA (pH 7.4) immediately following the biopsy. 
This latter sample will be used for mitochondrial biochemical analysis. 
To evaluate whether the genomic control of mitochondrial respiration is altered in 
response to roflumilast, mitochondrial DNA copy number and the expression of genes 
known to regulate mitochondrial biogenesis and function.  
A sample of the skeletal muscle will be stored at -80 degrees C for subsequent 
proteomic assessment.  
All identifying data in stored specimens including name, social security number, 
medical record number or date of birth will be removed. Only the study subject ID 
will be kept at the NHLBI will link the specimen to the subjects in the study.   
• 
Optional Adipose tissue biopsy (Day -2/-1 or Day +1): 
To measure mRNA levels of "beige fat" markers, including Prdm16, Cox8b and 
UCP1 by using real-time PCR (23), adipose tissue biopsies will be performed by 
general surgeons at the NIH Clinical Center, before and after roflumilast.  
Subcutaneous adipose tissue (3-5 g) will be removed from the abdominal region or 
thigh by aspiration with a 16-gauge needle under local anesthesia (2% xylocaine). 
Connective tissue will be removed. A representative portion of the tissues will be 
formalin fixed, paraffin embedded, sectioned, and stained using standard histological 
methods. Slides will be examined visually to assess cell populations/tissue 
composition to insure that morphologically similar tissue biopsies were sampled over 
time. The remaining tissue will be separated into aliquots of approximately 500 mg 
from adipose tissue specimens for RNA isolation and protein extraction, flash frozen 
in liquid nitrogen and stored at -80ºC for future studies. 
• 
PBMC collection: 
Before the beginning of each clamp study, the subjects will have 10 mL blood drawn 

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to collect PMBCs to look at the mitochondrial biogenesis gene profile. 
• 
Blood Samples for Future Biochemical Studies: 
Before the beginning of each clamp study, the subjects will have between 10 - 20 mL 
blood drawn to be stored so that determination of plasma analytes that are not 
currently anticipated may be made in the future. 
• 
Clinical Labs: 
 
Electrolytes (Na, K, Cl, HCO3) 
 
Liver Function Tests (Alkaline Phosphatase, ALT/GPT, AST/GOT, Total 
Bilirubin, Direct Bilirubin) 
 
CBC w/differential 
 
Serum creatinine 
 
BUN 
 
urinalysis,  
 
plasma glucose, 
 
plasma insulin,  
 
lipid profile: (Total Cholesterol, Triglycerides, HDL Cholesterol, LDL 
Cholesterol (calculated)),  
 
lipoprotein profile: Plasma lipid analysis, including VLDL, LDL and HDL 
particle size and number are measured by nuclear magnetic resonance (NMR) 
spectroscopy using the Vantera Clinical Analyzer (LipoScience Inc., Raleigh, 
North Carolina) in the Clinical Center. This clinical test which 
quantifies VLDL-P, LDL-P and HDL-P based on lipoprotein particle size using 
the amplitudes of their distinct lipid methyl group NMR signals to calculate the 
different lipoproteins.  Lipoprotein particles is further subdivided into large, 
medium, and small particles based on the mean particle sizes as the weighted 
average of related subclasses. The rationale for adding this test is that cyclic 
AMP (cAMP) signaling in the liver decreases lipid synthesis.  Therefore, it is 
possible that roflumilast, by increasing cAMP and activating protein kinase A, 
may decrease hepatic lipid production. 
 
C-peptide,  
 
free fatty acids (FFA), Hemoglobin A1C 
 
highly sensitive C-reactive protein, 
 
Serum pregnancy test 
 
The following procedures will be performed on the third day of the inpatient visit. 
• 
Mixed Meal Stimulation Test (Day +3):  
On the third day of hospitalization, the effect of a standardized liquid meal of standard 
macronutrient contents on glucose, insulin, and gut peptides will be assessed 
following an overnight fast. During the mixed meal stimulation test, we will 
administer a liquid meal of known macronutrient composition. Blood samples will be 
obtained at baseline, 15, 30, 45, 60, 120 and 180, 240 and 300 minutes (+/- 5 minutes 
for all time points) after consuming the liquid meal. 
• 
Drug Dispensing and first dose (Day +3, first inpatient visit) 
The pharmacy will dispense 2 weeks and 2 days of tablets containing 250 mcg of 
roflumilast (half of commercially available 500 mcg tablet).  The subject will be 
instructed to take the first dose from the supply provided by the pharmacy while still 
in the clinical center and will be observed for approximately two hours after the taking 

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the first dose. 
• 
Study Drug Diary: 
Each participant will be provided a study drug diary and instructed to record on a in 
the diary each time they take the study drug.  The study drug diary will be reviewed at 
each visit to ensure compliance. 
 
5.3.5 
Week 2, Visit 2 Procedures: (+/- 2 days) 
The following procedures will be performed at this visit. 
• 
AE Assessment 
• 
Pill count and review of study drug diary 
• 
Dose Escalation 
 
Subjects’ dose will be increased to 500 mcg p.o.  Subjects will be instructed to 
take one 500 mcg tablet a day. 
• 
Dispensing of enough drug to last the participant 4 weeks plus 4 days 
 
5.3.6 
Week 3, Visit 3- Procedures (+/- 4 days) 
The following procedures will be performed at this visit. 
• 
AE Assessment 
• 
ECG 
• 
Clinical Labs: 
 
Electrolytes (Na, K, Cl, HCO3) 
 
Liver Function Tests (Alkaline Phosphatase, ALT/GPT, AST/GOT, Total 
Bilirubin, Direct Bilirubin) 
 
Serum creatinine, BUN 
 
Pregnancy test 
• 
Pill count and review of study drug diary 
 
5.3.7 
Week 5 - Accelerometer and Food Record 
Participants will complete the 3-day food records (2 week days and 1 weekend day) and 
wear the accelerometer on the same 3 days one week prior to Visit 4.  Participants will 
complete these procedures at home.  A reminder to complete the procedures will be 
included in the drug diary. 
 
5.3.8 
Week 6, Visit 4 –End of Study Procedures: (+/- 2 days) 
Visit 4, Week 6 procedures will be conducted over four days.  The visit will include one 
day as outpatient visit to the MRCU for the meals (as was done in visit 1) and three days 
inpatient days, with participants staying overnight for 2 nights in the inpatient unit.  The 
one outpatient visit to the MRCU will be performed within the 2 days prior to prior to the 
inpatient admission.  The below evaluations can be performed within these 4 days:  
• 
MCRU meals and assessments within 2 days prior to inpatient admission. Dietary 
and exercise assessment may be conducted on this day, if this is more conducive to 
the subject’s and research team’s schedule. 
• 
Diet and Exercise Assessment – collection and review of accelerometer and food 
record as well as weight and BMI measurement. The assessment may be conducted 
on the day that the participants come into the MCRU to pick up their metabolic diet. 
• 
C-SSRS 
• 
Q-Les-Q 

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• 
AE Assessment 
• 
ECG 
• 
Clinical Labs: 
 
Electrolytes (Na, K, Cl, HCO3) 
 
Liver Function Tests (Alkaline Phosphatase, ALT/GPT, AST/GOT, Total 
Bilirubin, Direct Bilirubin) 
 
CBC w/differential 
 
Serum creatinine 
 
BUN 
 
urinalysis,  
 
plasma glucose, 
 
plasma insulin,  
 
lipid profile: (Total Cholesterol, Triglycerides, HDL Cholesterol, LDL 
Cholesterol (calculated)),  
 
Apolipoprotein panel 
 
lipoprotein profile: Plasma lipid analysis, including VLDL, LDL and HDL 
particle size and number are measured by nuclear magnetic resonance (NMR) 
spectroscopy using the Vantera Clinical Analyzer (LipoScience Inc., Raleigh, 
North Carolina) in the Clinical Center. This clinical test quantifies VLDL-P, 
LDL-P and HDL-P based on lipoprotein particle size using the amplitudes of 
their distinct lipid methyl group NMR signals to calculate the different 
lipoproteins.  Lipoprotein particles is further subdivided into large, medium, 
and small particles based on the mean particle sizes as the weighted average of 
related subclasses. Results of this test are reported in the CRIS electronic 
medical record system. The rationale for adding this test is that cyclic AMP 
(cAMP) signaling in the liver decreases lipid synthesis.  Therefore, it is 
possible that roflumilast, by increasing cAMP and activating protein kinase A, 
may decrease hepatic lipid production. 
 
C-peptide,  
 
HbA1C,  
 
free fatty acids (FFA),  
 
highly sensitive C-reactive protein, 
 
thyroid stimulating hormone function tests 
 
Serum pregnancy test 
 
Day 2 of Admission 
• 
Assessment of hepatic insulin sensitivity 
• 
Euglycemic clamp study 
• 
Measurements of Brachial Artery Flow and Forearm Capillary Recruitment (if the 
research team was able to schedule any of these procedures) 
• 
Measurement of Plasma Incretins, circulating inflammatory markers and 
adipocytokines 
• 
PBMC collection 
• 
Research Blood Samples for Future Biochemical Studies 
 
Day -2/-1 or Day 1/3: 
• 
Radiologic Assessments 
 
DEXA 
 
Day -2/-1 or Day 1: 
 
MRI and MRS (Assessment of visceral, liver, and skeletal muscle fat content 

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and distribution) 
 
Day -2/-1 or Day 1: 
• 
Optional Muscle biopsy  
• 
Optional Adipose tissue biopsy 
• 
Pill count and review of study drug diary (Day 1, 2, or 3) 
 
Day 3 (Day of discharge) 
• 
Mixed Meal Stimulation Test 
• 
Radiological assessment (if not obtained earlier) 
 
5.3.9 
Week 7, Visit 5- Follow-up visit procedures (+/- 2 days) 
The following procedures will be performed at this visit. 
• 
AE evaluation 
• 
Weight will be measured 
 
5.3.10 General Procedure Considerations for each study visit 
5.3.10.1 Medical History and Physical Examination 
Medical history (including medication history involving over-the-counter and herbal 
medications), physical examination, and laboratory values will be completed on each 
subject for every admission while on the study. Vital signs and biophysical data 
(including blood pressure, heart rate, respiratory rate, temperature, height, and weight) 
will also be collected at each visit. These measurements will be made per NIH Clinical 
Center inpatient protocols. 
 
5.3.10.2 Laboratory Tests and Parameters 
Any laboratory test result that the Investigator considers clinically significant will be 
repeated at the discretion of the investigator to rule out laboratory error. For tests where 
a persistent abnormality is considered to be drug related, repeat analyses will be 
performed until the cause is determined and either a return to normality occurs or the 
investigator deems the abnormality to be of no clinical significance. Any values judged 
by the investigator to represent a clinically significant, abnormal change from baseline 
during the treatment phase will be recorded as an adverse event. Per Clinical Center 
policy, no more than 550 mL of blood will be collected over an 8-week period. 
 
5.3.10.3 Procedures 
All tests will follow NIH Clinical Center policies and clinical consent forms for 
individual tests requiring these will be obtained in addition to the study protocol 
consent. Sedation for tissue biopsy sampling will be offered in accordance with 
Clinical Center policies. Indicated and recognized pharmacologic compounds that are 
routinely employed to increase the sensitivity and specificity of certain tests may be 
employed in these tests in compliance with Clinical Center policies.  With the 
exception of the pregnancy test, any of the listed procedures or laboratory tests 
performed within 4 weeks prior to screening visit in this protocol may/may not be 
repeated. However, if these procedures are not repeated, the prior studies (within the 
last 4 weeks) will be reviewed in CRIS as part of the protocol.  If data from another 
Clinical Center protocol is used, this will be clearly documented in the source 
document.  The purpose for not repeating study procedures is to prevent unnecessary 

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inconveniences, risks or discomforts for study participants.  The investigator will 
determine based on the scientific objectives of the protocol as well as participant safety, 
which results will be used. 
 
5.3.10.4 Off Treatment and Off Study Procedures 
Participants taken off treatment due to serious adverse events, such as liver toxicities 
(elevated AST/ALT and bilirubin), major depression, suicidal ideation, and/or >10% 
weight loss, will be monitored to verify that the appropriate interventions are 
performed and until the event resolves in severity to baseline, at which time they will 
be taken off study. 
Participants may opt to discontinue treatment at their request.  The risks of opting to 
discontinue treatment will be discussed, and we will request permission to follow-up 
with the participant for one more week to ensure patient safety and well-being. 
 
Participants who request going off study on their own accord will be queried as to 
why they are withdrawing, and we will request permission to follow-up with the 
participant for one additional week to ensure patient safety and then take them off 
study.

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5.4 Schedule of Events 
Screening 
Visit 1 
(Days -2, 
or -1); 
Day1, 2,3) 
Visit 2  
(Wk2/ Day 
15) (+/-2 
days) 
Visit 3 
(Wk3/ Day 
22)  (+/- 4 
days) 
Week 5 
Visit 4 
(Wk6/ Days 
42) (+/-2 days) 
Visit 5 
(Wk7)  
(+/- 2 
days) 
General Assessments 
  
  
  
  
 
  
 
informed Consent 
X 
 
 
 
 
 
 
medical history  
X 
 
 
 
 
 
 
physical examination 
X 
 
 
 
 
 
 
electrocardiogram 
X 
 
 
X 
 
X 
 
Psychiatric Evaluations 
 
 
 
 
 
 
 
C-SSRS 
X 
 
 
 
 
X 
 
SCID 
X 
 
 
 
 
 
 
Q-Les-Q 
X 
 
 
 
 
X 
 
Diet and Exercise 
Assessment 
 
 
 
 
 
 
 
MCRU Meals 
 
X 
 
 
 
X 
 
 3-Day Food Records 
X 
X 
 
 
X 
X 
 
Accelerometer 
X 
X 
 
 
X 
X 
 
 collection and review of 
accelerometer and food 
record 
 
X 
 
 
 
X 
 
Weight and/or BMI 
X 
X 
X 
X 
 
X 
X 
Drug Administration 
 
 
 
 
 
 
 
Dispensing of Medication 
 
X B 
X 
 
 
 
 
Observation of 1st dose 
 
X B 
 
 
 
 
 
Dose Escalation 
 
 
X 
 
 
 
 
Pill count/study diary 
review 
 
 
X 
X 
 
X 
 
Adverse Event 
Assessment 
 
 
 
 
 
 
 
Assessment 
 
X B 
X 
X 
 
X 
X 

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Screening 
Visit 1 
(Days -2, 
or -1); 
Day1, 2,3) 
Visit 2  
(Wk2/ Day 
15) (+/-2 
days) 
Visit 3 
(Wk3/ Day 
22)  (+/- 4 
days) 
Week 5 
Visit 4 
(Wk6/ Days 
42) (+/-2 days) 
Visit 5 
(Wk7)  
(+/- 2 
days) 
Clinical Lab tests 
 
 
 
 
 
 
 
CBC w/ differential 
X 
X 
 
 
 
x 
 
Electrolytes [Sodium 
(Na), Potassium (K), 
Chloride (Cl), Bicarb 
(HCO3)] 
X 
X 
 
X 
 
X 
 
Liver Function Tests 
[Alkaline Phosphatase,  
ALT/GPT, AST/GOT,  
Total Bilirubin, Direct 
Bilirubin] 
X 
X 
 
X 
 
X 
 
Serum creatinine 
X 
X 
 
X 
 
X 
 
BUN 
X 
X 
 
X 
 
X 
 
plasma glucose 
X 
X 
 
 
 
X 
 
plasma insulin 
X 
X 
 
 
 
X 
 
Lipid profile [Total 
Cholesterol, 
Triglycerides, HDL 
Cholesterol, LDL 
Cholesterol] 
X 
X 
 
 
 
X 
 
Plasma lipid analysis 
(lipoprotein profile, 
apolipoprotein panel) 
 
X 
 
 
 
X 
 
C-peptide 
X 
X 
 
 
 
X 
 
free fatty acids (FFA) 
X 
X 
 
 
 
X 
 
highly sensitive C-
reactive protein 
X 
X 
 
 
 
X 
 
Hemoglobin A1C 
(HbA1C) 
X 
X 
 
 
 
X 
 
Thyroid Function Test 
X 
 
 
 
 
X 
 
Anti-HIV-1/2 
X 
 
 
 
 
 
 
Anti-HCV Antibody 
X 
 
 
 
 
 
 

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Screening 
Visit 1 
(Days -2, 
or -1); 
Day1, 2,3) 
Visit 2  
(Wk2/ Day 
15) (+/-2 
days) 
Visit 3 
(Wk3/ Day 
22)  (+/- 4 
days) 
Week 5 
Visit 4 
(Wk6/ Days 
42) (+/-2 days) 
Visit 5 
(Wk7)  
(+/- 2 
days) 
Anti-HBc (Total IgG and 
IgM) 
X 
 
 
 
 
 
 
Urinalysis 
X 
X 
 
 
 
X 
 
Serum PregnancyC 
X 
X 
 
X 
 
X 
 
Other Laboratory 
Assessments 
 
 
 
 
 
 
 
Assessment of hepatic 
insulin sensitivity  
 
X A 
 
 
 
X 
 
Glucose clamp 
 
X A 
 
 
 
X 
 
Mixed Meal Stimulation 
Test 
 
X B 
 
 
 
X 
 
Plasma incretin, 
adiponectin, leptin, 
adhesion molecules, TNF-
α, & MCP-1 levels  
 
X A 
 
 
 
X 
 
 PBMC collection 
 
X 
 
 
 
X 
 
Radiologic Assessments: 
 
 
 
 
 
 
 
 DEXA 
 
X A 
 
 
 
X 
 
MRI/MRS (Assessment 
of visceral, liver, and 
skeletal muscle fat 
content and distribution) 
 
X A 
 
 
 
X 
 
Vascular Function by 
Ultrasound 
 
X 
 
 
 
X 
 
Optional Biopsy 
Assessments 
 
 
 
 
 
 
 
muscle biopsy 
 
X A 
 
 
 
X 
 
Adipose Tissue 
 
X A 
 
 
 
X 
 
Research Samples 
 
 
 
 
 
 
 
Blood Samples for Future 
Biochemical Studies  
 
X D 
 
 
 
X 
 

Protocol #:  13-H-0123 
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PI:  Jay Chung, MD, PhD 
Version 20.0 Date: 5/15/2018 
 
Screening 
Visit 1 
(Days -2, 
or -1); 
Day1, 2,3) 
Visit 2  
(Wk2/ Day 
15) (+/-2 
days) 
Visit 3 
(Wk3/ Day 
22)  (+/- 4 
days) 
Week 5 
Visit 4 
(Wk6/ Days 
42) (+/-2 days) 
Visit 5 
(Wk7)  
(+/- 2 
days) 
A These assessments can be performed on either on the outpatient visit just prior to admission or during 1st day of admission of visit 1, 
with the exception of DEXA which can also be performed on Day 3 of admission 
B These assessments will be performed on third inpatient day of visit prior to release from the clinical center 
C May be performed at any time pregnancy is suspected. 
D This assessment can be performed on either the first or second inpatient day of Visit 1 
 
VISIT #1 and #4 - SAMPLE INPATIENT VISITS 
Day -1 or Day -2 prior 
to admit 
Inpatient Day 1 
Inpatient Day #2 
Inpatient Day #3 
 
Pick up metabolic 
meals 
 
MRI/MRS (if subject 
cannot be scheduled as 
inpatient)  
 
DEXA (if subject 
cannot get as inpatient) 
 
Optional: Muscle or 
adipose biopsy (for 
subjects who consent)  
 
 
 
Metabolic diet 
Collection of food diary 
and accelerometer 
Admission to inpatient 
metabolic unit at 2 PM. 
Assessment of venous 
access;  
 
History and physical 
 
Metabolic meal 
 
DEXA (if not done on 
Inpatient Day #3) 
 
Optional: Muscle & 
adipose biopsy 
 
MRI/MRS (if subject 
cannot be scheduled as 
inpatient)  
 
PBMC collection,  10 mL 
blood for future research, 
clinical lab collection 
 
Glucose tracer study (takes 
approximately 6 hours) 
 
Glucose clamp (takes 
approximately 3 hours) 
 
Doppler Ultra Sound and 
Brachial artery test at bedside 
during clamp  
 
  
DEXA (if not done on 
Inpatient Day #1) 
 
Mixed meal test 
 
Drug dispensing, then 
observe for 2 hours (for visit 
1 only) 
 
Discharge home.  

Protocol #:  13-H-0123 
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PI:  Jay Chung, MD, PhD 
Version 20.0 Date: 5/15/2018 
6 Pharmaceuticals 
6.1 Roflumilast 
• 
Other names: 
 
DALIRESP® in US 
 
 
 
 
 
• 
Classification: 
 
Selective phosphodiesterase 4 (PDE4) inhibitor 
 
• 
Route of Administration: Oral 
 
• 
Roflumilast Dose 
The dose (500 mcg once daily) was chosen based on double-blind, randomized parallel-group 
study that showed roflumilast administration for 12 week decreased blood glucose and HbA1c 
levels in patients aged 35-70 years with newly diagnosed type 2 diabetes who were treatment 
naïve when compared with placebo. However, due to gastrointestinal side effects associated 
with the 500 mcg dose, we will start with 250 mcg once daily for the first two weeks before 
increasing the dose to 500 mcg once daily for 4weeks. 
 
Drug Supply: 
• 
Roflumilast will be obtained from commercial sources as a 500mcg tablet.  Patients will take: 
250 mcg (one-half tablet) daily for 14 days, followed by 500 mcg (1 tablet) daily for 4 weeks 
 
• 
Stability/Storage: Controlled Room Temperature at 20-25 C (68-77 F); excursions 
permitted to 15-30C (59 – 86 F). 
 
• 
Regulatory Status:   
On February 28, 2011 the FDA approved Forest Research Institute, Inc.’s New Drug 
Application (N022522) for roflumilast, 500mcg tablets, for the treatment of COPD. An IND 
determination request was submitted to the FDA and the FDA issued a letter, included with 
submission, on April 19, 2012 stating the use of roflumilast in this study is exempt from IND 
requirements as stated in 21 CFR 312.2 (b) (1) (i-v).  Specifically, the use is exempt because: 
1. The investigation is not intended to be reported to FDA as a well-controlled study in 
support of a new indication for use, nor intended to be used to support any other significant 
change in the labeling for the drug. 
2. The investigation is not intended to support a significant change in the advertising for a 
prescription drug product. 
3. The investigation does not involve a change in route of administration, dosage level, or 
patient population, or other factor that significantly increases the risks (or decreases the 
acceptability of risks) associated with use of the drug product. 
4. The investigation is conducted in compliance with the requirements for institutional review 
(21 CFR 56) and informed consent (21 CFR 50). 
5. The investigation is conducted in compliance with the requirements of 21 CFR 312.7, i.e., 
the drug may not be represented as safe or effective, nor may it be commercially 
distributed, for the purposes for which it is under investigation. 
 
6.1.1 
Dispensing Study Drug: 
The NIH Pharmacy will dispense roflumilast medication in dosages of 250 mcg (one half tablet of 
500mcg) and 500 mcg (one tablet) to the study participants. 
 
6.1.2 
Treatment Compliance monitoring: 

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PI:  Jay Chung, MD, PhD 
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Subjects will be prescribed and given the drug initially at the NIH Clinical Center. Treatment 
compliance will be monitored by reviewing the study drug diary.  Participants will instructed to 
complete the medication diary each day at the time they take the study drug.   Patients with 
questions regarding any adverse effects will have access to trained medical staff including the study 
nurse and physician at all times. Records of follow-up contact to verify subject compliance with 
drug intake will also be documented in the individual research chart. 
Participants will return any unused roflumilast tablets at the completion of each visit. At this time 
a pill count will be performed and the study drug diary will be reviewed. 
In instances where a subject misses a dose, the subject will be instructed to take the study drug as 
soon as he/she remembers. If it is almost time for the next dose, subjects will be instructed to skip 
the missed dose and take the medicine at the next regularly scheduled time. Extra medicine should 
not be taken to make up the missed dose. Follow-up phone calls may be placed as often as daily 
while the subject is on study drug. If subjects become unable or unwilling to continue treatment 
they will be removed from the study. The subjects will be instructed to return remaining drug, in 
original drug containers, to NIH at each applicable study visit for a drug count, or if all the drug 
has been taken, empty drug containers will be returned. Any missed doses of the study drug will be 
returned to the NIH pharmacy for disposal. 
 
6.2 Stable isotope compound: 6,6 D2-Glucose 
Drug Supply: Pine Pharmaceutical Company 
6.2.1 
Dispensing Study Drug: 
The NIH Pharmacy will dispense the stable isotope compound to the study investigators. 
 
7 Data Collection and Management Plan 
Research Samples for Future Use- Before the beginning of each glucose clamp study, the subjects will 
have 10 ml blood drawn to be stored so that determination of plasma analytes that are not currently 
anticipated may be made.  Additionally, the expression levels of the PGC-1a, PGC-1b, UCP, MCAD, 
ERRa genes may be measured.  These samples are immediately centrifuged for 5 minutes at 3,000 
rpm in a clinical centrifuge and the plasma collected and stored at -80˚C.  Until the final analysis is 
completed for this protocol, samples will be coded with access to the sources kept in a security 
protected database, protected.  When data analysis has been completed, the code will be stripped of 
all identifiers by use of an arbitrary or random alphanumeric code and the key to the code will be 
destroyed, thus making it impossible for anyone to link the samples to the sources.  The NHLBI IRB 
will be notified in writing when this is done.     
 
Data will be kept on the NHLBI “P” drive, accessible through password-protected computers.  Only 
the members of the research team will have access to the samples and data.   
 
All human subjects personally identifiable information (PII) as defined in accordance to the Health 
Insurance Portability and Accountability, eligibility and consent verification will be recorded in DIR’s 
Clinical Data System (CDS) database.  Primary data obtained during the conduct of the protocol will 
be kept in secure network drives that comply with NIH security standards.  Primary and final analyzed 
data will have identifiers so that research data can be attributed to an individual participant. 
At the completion of the protocol (termination), samples and data will either be destroyed, or after 
IRB approval, transferred to another existing protocol or a repository. 
End of study procedures:  Data will be stored in locked cabinets and in a password protected network 

Protocol #:  13-H-0123 
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PI:  Jay Chung, MD, PhD 
Version 20.0 Date: 5/15/2018 
servers until it is no longer of scientific value.  
Loss or destruction of data:  Should we become aware that a major breech in our plan to protect 
subject confidentiality and trial data has occurred, the IRB will be notified. 
Publication Policy:  Investigators will publish and present the results of the study.   In all publications 
and presentations resulting from this research project the anonymity of the research subjects will be 
protected. 
Privacy and Confidentiality:  All efforts, within reason, will be made to keep subjects’ protected 
health information (PHI) private.  Using or sharing (“disclosure”) such data must follow federal 
privacy rules.  Under certain circumstances, the United States Office of Human Research Protections 
(OHRP) and the NHLBI Institutional Review Board (IRB) will be able to inspect and copy confidential 
study-related records which identify participants by name.  Therefore, absolute confidentiality cannot 
be guaranteed.  Authorized personnel from NIH, NHLBI, NIDDK, the NHLBI IRB, or other 
appropriate authorities, may have access to our research files in order to verify that the rights of the 
research subjects have been safeguarded. 
 
Transmission of Data to Outside Investigators: Data will not be sent outside NIH without IRB 
notification and an executed agreement. 
 
8 Statistical Considerations 
 
8.1 Statistical Methods 
 
For this exploratory study, primary and secondary outcomes will be considered significant at 
p<0.05. All tests will be two-sided. Differences from baseline to the end of 6weeks in the outcome 
measures will be evaluated using paired t-tests and linear regression if additional variables are 
needed in the models. Data will be checked for normality and appropriate transformations will be 
applied if necessary.  Nonparametric analysis methods will be performed if needed. 
 
Normality in the response variable will be assessed using the Shapiro-Wilk statistic and normal 
probability plots. Tukey's re-expression ladder will be used to identify transformations such that 
the relationships between the response and the covariates are linear.  Non-parametric tests, such as 
the Wilcoxon Signed Rank test for paired data and the Mann-Whitney (Wilcoxon Rank Sum) test 
for unpaired data, will be used in place of the paired and unpaired Student t-tests, respectively, if 
the normality assumption of the data is in question. 
 
8.2 Sample Size Calculation 
The primary outcome in this study is changes in insulin sensitivity as measured by glucose clamp. 
Insulin sensitizing interventions such as weight loss and pharmacotherapy, improve insulin-
mediated glucose disposal by 10~30% or more.  Based on mean values of insulin sensitivity using 
the clamp method, a sample size of n=15 will be sufficient to detect a 20% difference pre- and 
post-treatment with 80% power (alpha = 0.05, 2-sided test, with SD change of 2.5 units)(Magkos, 
F et alInt J Obes (Lond). 2011 September; 35(9): 1233–1240).  A 20% difference was chosen 
because it represents a clinically meaningful difference that is expected based on published data 
with other insulin sensitizing interventions, such as weight loss and pharmacotherapy.  The Power 
Tables below indicate the minimal required sample size to be 12 to cover the most hypotheses. 
 
*** Power Table *** 
mean.null 
sd1 
mean.alt 
delta 
alpha 
power 
n1 

Protocol #:  13-H-0123 
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PI:  Jay Chung, MD, PhD 
Version 20.0 Date: 5/15/2018 
0 
2.5 
2 
2 
0.05 
0.8 
12 
*** Power Table *** 
mean.null 
sd1 
mean.alt 
delta 
alpha 
power 
n1 
0 
2.5 
2.2 
2.2 
0.05 
0.8 
11 
 
*** Power Table *** 
mean.null 
sd1 
mean.alt 
delta 
alpha 
power 
n1 
0 
2.5 
2.4 
2.4 
0.05 
0.8 
9 
 
We propose to have a recruitment ceiling of 50 subjects in order to obtain n = 15. This allows for 
a drop-out rate of 20%. 
 
9 Data and Safety Monitoring 
9.1 Safety Monitoring 
Accrual and safety data will be monitored by the Principal Investigator who will provide oversight 
to the conduct of this study. 
Accrual and safety data will be monitored and reviewed annually by the Institutional Review Board 
(IRB).  Prior to implementation of this study, the protocol and the proposed subject informed 
consent document will be reviewed and approved by the properly constituted IRB operating 
according to the 45 CRF 46 and 21 CFR 56.  This committee must approve all amendments to the 
protocol or informed consent document, and conduct continuing annual review so long as the 
protocol is open to accrual or follow up of subjects. 
 
9.2 Adverse Event Characterization and Reporting 
9.2.1 
Definitions 
Adverse Event (AE):  Any untoward or unfavorable medical occurrence in a human subject, 
including any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or 
disease, temporally associated with the subject’s participation in the research, whether or not 
considered related to the research. 
 
Serious Adverse Event (SAE):  A serious adverse event that: 
• 
results in death; 
• 
is life-threatening (places the subject at immediate risk of death from the event as it occurred); 
• 
results in in-patient hospitalization or prolongation of existing hospitalization; 
• 
results in a persistent or significant incapacity; 
• 
results in a congenital anomaly/birth defect; or 
• 
based upon appropriate medical judgment, may jeopardize the subject’s health and may require 
medical or surgical intervention to prevent one of the other outcomes listed in this definition. 
 
Suspected adverse reaction: Suspected adverse reaction means any adverse event for which there 
is a reasonable possibility that the drug caused the adverse event. For the purposes of safety 
reporting, ‘reasonable possibility’ means there is evidence to suggest a causal relationship between 
the drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty 
about causality than adverse reaction, which means any adverse event caused by a drug. 
 
Unexpected adverse reaction: An adverse event or suspected adverse reaction is considered 
“unexpected” if it is not listed in the package insert or is not listed at the specificity or severity that 
has been observed; or is not consistent with the risk information described in the protocol or 
elsewhere in the current application. "Unexpected”, also refers to adverse events or suspected 
adverse reactions that are mentioned in the investigator brochure as occurring with a class of drugs 

Protocol #:  13-H-0123 
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or as anticipated from the pharmacological properties of the drug, but are not specifically mentioned 
as occurring with the particular drug under investigation. 
 
Unanticipated Problem (UP):  Any incident, experience, or outcome that is: 
1. unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research protocol and informed 
consent document; Investigator’s Brochure or other study documents; and 
b. the characteristics of the subject population being studied; and 
2. related or possibly related to participation in the research; and 
3. places subjects or others at a greater risk of harm (including physical, psychological, economic, 
or social harm) than was previously known or recognized. 
 
Unanticipated Problem that is not an Adverse Event:  An unanticipated problem that does not fit 
the definition of an adverse event, but which may, in the opinion of the investigator, involves risk 
to the subject, affect others in the research study, or significantly impact the integrity of research 
data.  For example, report occurrences of breaches of confidentiality, accidental destruction of 
study records, or unaccounted-for study drug. 
 
Protocol Deviation (PD):  Any change, divergence, or departure from the IRB approved study 
procedures in a research protocol that does not have a major impact on the subject's rights, safety 
or well-being, or the completeness, accuracy and reliability of the study data. 
 
9.2.2 
Adverse Event Recording 
The principal investigator (and medical advisory investigator) will be responsible for assessing 
adverse events. Information on adverse events will be solicited from subjects through questions 
from study personnel and information volunteered by the subject. All patients will be specifically 
counseled to report physical symptoms or changes experienced while on treatment. Inquiries will 
be made to subjects at least once every 7 days to inquire about potential adverse effects. Liver 
function tests will be performed at visit 3, and 4 after the initiation of roflumilast. 
Adverse event recording will start after the initial dose of the drug is administered.   
All AEs, including clinically significant abnormal findings on laboratory evaluations, regardless 
of severity, will be recorded and followed until satisfactory resolution. AEs should be reported up 
to the end of the subject’s participation in the study. AEs that are considered treatment related, 
expected, continuing, but not resolvable by the end of the subject’s participation in the study will 
not be followed after the end of the subject’s participation in the study. 
An abnormal laboratory value will be considered an AE if the laboratory abnormality is 
characterized by any of the following: 
• 
Results in discontinuation from the study 
• 
Is associated with clinical signs or symptoms 
• 
Requires treatment or any other therapeutic intervention 
• 
Is associated with death or another serious adverse event, including hospitalization 
• 
Is judged by the Investigator to be of significant clinical impact 
• 
If any abnormal laboratory result is considered clinically significant, the investigator will 
provide details about the action taken with respect to the test drug and about the patient’s 
outcome. 

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The intensity of adverse events will be graded as follows: 
Mild: 
Awareness of sign or symptom, but easily tolerated.  Not expected to have a 
clinically significant effect on the patients overall health and well-being.  Not 
likely to require medical attention. 
Moderate: 
Discomfort enough to cause interference with usual activity or affects clinical 
status.  May require medical intervention. 
Severe: 
Incapacitating or significantly affecting clinical status.  Likely requires medical 
intervention and/or close follow-up. 
 
9.2.3 
Event Reporting 
Reporting Timeframes to IRB Chair, Clinical Director, and/or NHLBI IRB 
 
Serious Events 
Reports to the IRB and CD: The PI must report Serious UPs, and Serious PDs to the IRB and CD 
as soon as possible but not more than 7 days after the PI first learns of the event using the NIH 
Problem Report Form. 
 
Reports to the IRB Chair and CD: The PI must report all SAEs that do not meet the definition of 
UP to the IRB chair and CD not more than 14 days after the PI first learns of the event, using the 
SAE submission form. 
 
Non-serious Events   
Reports to the IRB and CD: The PI must report all UPs that are not Serious to the IRB and CD, 
and PDs that are not Serious to the IRB, not more than 14 days after the PI first learns of the 
event using the NIH Problem Report Form. 
 
Deaths 
The PI must report all deaths (that are not UPs) to the CD as soon as possible, but not more than 7 
days after the PI first learns of the event. 
 
We request a waiver from reporting the following commonly occurring, mild to moderate, and 
expected events to the IRB unless they reach the threshold of an SAE, at which time these events 
will also be reported to the IRB as UP: 
 
• 
Emergence or worsening of insomnia, anxiety, weight loss of < 10% of baseline total body 
weight 
• 
Bruising and possible inflammation at intravenous catheter sites 
• 
Nausea with emesis or headache 
• 
Symptoms specifically mentioned in the consent form that do not threaten patient safety. 
• 
Asymptomatic correctable hypoglycemia 
 
The following adverse events will be listed in the consent and reported to the IRB at the time of 
continuing review: 
 
• 
Subject withdrawal from study before completion related to intolerance of medication regime 
or other non-serious adverse events 
• 
Suicidal ideation and new onset depression. 

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• 
moderate weight loss (≥ 10% of baseline total body weight) 
 
9.3 Stopping Rules 
Monitoring of Subjects and Criteria for Withdrawal of Subjects 
Participants may choose to withdraw from this study at any time for any reason.  Participants who 
withdraw will be compensated for their time per guidelines stated in this protocol. Participants will 
be withdrawn from the study if they cannot continue with the study regimen or: a) develop major 
depression or suicidal ideation, b) if there is suspected hypersensitivity to the study medication; or 
c) develop significant weight loss (> 10% of initial body weight), or c) die.  If serum transaminases 
(ALT/AST) or bilirubin exceed 3 times the upper limit of normal and/or if clinical signs and 
symptoms suggest the onset of hepatic dysfunction, participants will be withdrawn from the study 
and treatment discontinued. Participants will be followed until the resolution of abnormalities with 
appropriate medical management. Participants who become pregnant or diabetic during the study 
will be withdrawn and referred to their primary care doctor.  
 
Criteria for suspending study 
If 2 out of 15 participants experience major depression or suicidal ideation serious adverse events, 
recruitment and randomization will be suspended to determine if after review of the data, the study 
should be stopped.  The IRB will be informed if this occurs. 
 
9.4 Protocol Monitoring 
As per ICH-GCP 5.18 clinical protocols are required to be adequately monitored by the study 
sponsor.  The Principal Investigator and/or designee will have primary responsibility for monitoring 
the clinical trial data. 
The investigator (and/or designee) will make study documents (e.g., consent forms and pertinent 
hospital or clinical records readily available for inspection by the local IRB, the site monitors, and 
the NHLBI staff for confirmation of the study data. 
 
10 Human Subjects Protections 
10.1 Rationale for Subject Selection 
Subjects of both genders will be considered for inclusion in this study.  There will be no racial, 
ethnic, or gender discrimination.  Presence of metabolic dysfunction and insulin resistance is firmly 
established in all races and genders.  Therefore, it is appropriate to study the effects of roflumilast 
to modulating insulin resistance and pancreatic β-cell function in these populations.  Cognitively 
impaired and institutionalized persons will not participate in this study.  Criteria for exclusion or 
withdrawal from the study are based on the presence of other disease processes that may be harmful 
to the health of subjects. 
 
10.2 Rationale for the Exclusion of Children 
Subjects under 18 years of age will not be considered for inclusion in this protocol because 
roflumilast is not approved for use in children and there is no direct medical benefit from 
participation in this study. 
10.3 Rationale for the Exclusion of Pregnant Women 
The effects of roflumilast on pregnant women have not been evaluated to date in a controlled 
clinical trial.  Thus, given this and the lack of known direct medical benefit to participants of this 
study, pregnant women will be excluded from the study. 
 
10.4 Risk/Benefit Assessment 

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There are no direct benefits for the subjects participating in this study although information 
obtained may help to establish the health benefits of roflumilast for subjects with prediabetes.  In 
addition, the screening physical exam and laboratory tests may provide information that is useful 
for the subjects.  Abnormal values will be discussed with the study volunteers and forwarded to 
their primary care physicians. 
 
The level of risk to the adult research participants is greater than minimal risk and involves no 
prospect of direct benefit to individual subjects, but likely to yield generalizable knowledge to 
further society’s understanding of the disorder or condition under study.  This assessment is in 
accordance with 45 CFR 46.102 (h)(i). 
 
As of June 28, 2017, this study is closed to accrual, and is now in data and/or sample analysis 
only.  Therefore, the level of risk is now minimal. 
 
10.5 Risks and Discomforts 
10.5.1 Roflumilast-related toxicity: 
AdverseReactionsReportedby≥2%ofPatients 
Treatedwithroflumilast500mcgdailyandGreaterThanPlacebo 
 
Treatment 
Adverse Reactions  
(Preferred Term)  
DALIRESP 
Placebo 
(N=4438) 
(N=4192) 
n (%) 
n (%) 
Diarrhea 
420( 9.5) 
113 (2.7) 
Weight decreased  
331 (7.5) 
89 (2.1) 
Nausea  
 
209 (4.7) 
60 (1.4) 
Headache  
195 (4.4) 
87 (2.1) 
Back pain  
142 (3.2) 
92 (2.2) 
Influenza  
124 (2.8) 
112 (2.7) 
Insomnia  
105 (2.4) 
41 (1.0) 
Dizziness  
92 (2.1) 
45 (1.1) 
Decreased appetite  
91 (2.1) 
15 (0.4) 
 
Adverse reactions that occurred in the roflumilast group at a frequency of 1 to 2% where 
rates exceeded that in the placebo group include: 
• 
Gastrointestinal disorders - abdominal pain, dyspepsia, gastritis, vomiting 
• 
Infections and infestations - rhinitis, sinusitis, urinary tract infection, 
• 
Musculoskeletal and connective tissue disorders - muscle spasms 
• 
Nervous system disorders - tremor 
• 
Psychiatric disorders - anxiety, depression 
 
Psychiatric Events including Suicidality: 
Treatment with roflumilast is associated with an increase in psychiatric adverse reactions. 
In 8 controlled clinical trials 5.9% (263) of patients treated with roflumilast 500 mcg daily 
reported psychiatric adverse reactions compared to 3.3% (137) treated with placebo. The 
most commonly reported psychiatric adverse reactions were insomnia, anxiety, and 
depression which were reported at higher rates in those treated with roflumilast 500 mcg 
daily (2.4%, 1.4%, and 1.2% for roflumilast versus 1.0%, 0.9%, and 0.9% for placebo, 

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respectively) [see [package insert] Adverse Reactions (6.1)]. Instances of suicidal ideation 
and behavior, including completed suicide, have been observed in clinical trials. Three 
patients’ experienced suicide-related adverse reactions (one completed suicide and two 
suicide attempts) while receiving roflumilast compared to one patient (suicidal ideation) 
who received placebo. 
Weight Decrease 
Weight loss was a common adverse reaction in roflumilast clinical trials and was reported 
in 7.5% (331) of patients treated with roflumilast 500 mcg once daily compared to 2.1% 
(89) treated with placebo Adverse Reactions (6.1)]. In addition to being reported as adverse 
reactions, weight was prospectively assessed in two placebo-controlled clinical trials of 
one year duration. In these studies, 20% of patients receiving roflumilast experienced 
moderate weight loss (defined as between 5-10% of body weight) compared to 7% of 
patients who received placebo. In addition, 7% of patients who received roflumilast 
compared to 2% of patients receiving placebo experienced severe (>10% body weight) 
weight loss. During follow-up after treatment discontinuation, the majority of patients with 
weight loss regained some of the weight they had lost while receiving roflumilast. Patients 
treated with roflumilast should have their weight monitored regularly. If unexplained or 
clinically significant weight loss occurs, weight loss should be evaluated, and 
discontinuation of roflumilast should be considered. 
 
10.5.2 Glucose clamp study: 
The major potential risk in this study is the development of hypoglycemia during the 
glucose clamp studies.  This is extremely unlikely because blood glucose levels are 
monitored at 5 to 10 minute intervals throughout the study and for at least one hour after 
the insulin infusion has been stopped.  Venous access is available to give appropriate 
glucose infusions should significant hypoglycemia occur. The YSI glucose analyzer we 
use meets CLIA requirements for Quality Assurance in patient testing.  The administration 
of insulin can theoretically result in low blood glucose.  With low blood glucose, people 
get hungry, have a headache, feel shaky or dizzy or may become drowsy or sweat. If low 
blood glucose does occur, the test will be stopped and the subject will be given juice or 
glucose by vein to raise the blood glucose to normal. Hypokalemia is avoided by 
establishing baseline potassium levels before starting the study and monitoring during and 
at the completion of the insulin infusion.  A potassium phosphate infusion is given during 
the glucose clamp procedure to maintain serum potassium levels in the normal range.  The 
Clinical Pathology Laboratory at NIH gives results within 15 minutes for monitoring of 
patient serum potassium levels.   
10.5.3 Dual Energy X-ray absorptiometry (DEXA): 
Although DEXA is a safe procedure, it is associated with a small amount of ionizing 
radiation.  The radiation exposure is less than 0.3.0mrem per study, for total exposure of 
0.6.0mrem.  In some subjects, lying on the table may result in some discomfort due to back 
pain or claustrophobia. 
 
 
 
 
 

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ORGAN 
RADIATION DOSE (rem) 
Per single 
Administration 
 
ICRP 103 
weighting factor Per Year (12 mos.) 
Adrenals 
  
 
 
Brain 
  
 
 
Breasts 
  
 
 
Esophagus 1 
  
 
 
Gallbladder Wall 
  
 
  
GI-tract:  Lower Large Intestine 
  
 
 
     Small Intestine 
  
 
 
     Stomach 
  
 
 
     Upper Large Intestine 
  
 
 
           Colon 2 
 
 
 
Heart Wall 
  
 
 
Kidneys 
  
 
 
Liver 
  
 
 
Lungs 
  
 
 
Muscle 
 
 
 
Ovaries 
  
 
 
Pancreas 
  
 
 
Red Marrow 
  
 
 
Bone Surfaces 
 
 
 
Skin 
0.3  
0.01 
0.6 
Spleen 
  
 
 
Testes 
  
 
 
Thymus 
  
 
 
Thyroid 
  
 
 
Urinary Bladder Wall 3 
  
 
 
Lens of Eye 
 
 
 
Uterus 
  
 
 
     REMAINDER AVE 
 
 
 
        EFFECTIVE DOSE 
0.003 
0.01 
0.006 
NOTES:   
1 Since no dose is explicitly tabulated for esophagus, thymus dose is used (as per ICRP 80) 
2 Colon Dose estimated by [0.57 (DoseULI) + 0.43 (DoseLLI)] (as per ICRP 80). 
 
3  Dynamic urinary bladder model used; void interval of ___ hours 
 

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10.5.4 MRI/MRS assessment of visceral, liver and muscle fat: 
There are no long-term risks or consequences of MRI/MRS scans without gadolinium 
contrast in patients.  In the 3T scanner, it is possible that the patient may experience 

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peripheral nerve stimulation resulting in a sensation of mild twitching, warmth or vibration.  
The duration of the study is 90 minutes to 2 hours during which the subject will need to lie 
on the table.  The tight space may prohibit patients of a particular body habitus to 
participate and may cause claustrophobic symptoms in susceptible individuals. 
 
10.5.5 Blood drawing: 
The placement of intravenous catheters may cause transient pain and bruising and rarely 
infection at the insertion site or phlebitis. 
 
10.5.6 Stable isotopes: 
The stable isotopes, deuterium is not associated with toxicity or abnormalities in 
metabolism. 
 
10.5.7 Psychiatric Evaluations: 
Participants may experience psychological discomfort or distress when answering 
questions pertaining to suicide, depression, quality of life, and their mental health in 
general. 
 
10.5.8 Biopsy: 
The major risks/discomforts include pain, bruising, hematoma, infection, scarring, and 
localized lipodystrophy. The degree of obesity will not add to the risk of this procedure.  
The procedure will be performed under sterile technique to minimize the chances of 
infection. Local anesthetic will be used to minimize pain.  Ice will be applied to the site 
immediately after the procedure to limit bruising, swelling and tenderness. After 
subcutaneous tissue biopsy, patients will be monitored by nurses. Post-biopsy the incision 
site will be cleaned and closed with adhesive wound closures and covered with gauze and 
translucent dressing tape. Study participants will be instructed to report to the clinical staff 
any changes at the biopsy site including bleeding, secretion, erythema, pain, and signs and 
symptoms of infection. Study participants will be instructed to self-monitor the incision 
site after discharge from the Clinical Center. 
 
10.5.9 Dietary assessments: 
Participants may find the wearing the accelerometer for 3 days prior to each visit 
uncomfortable.  Participants may not like the meals they are asked to eat.  Participants 
may find writing down everything she/he eats for 3 days prior to each visit time 
consuming and bothersome. 
 
10.5.10 Microbubble contrast administration: 
Contrast agents have very few side effects in most people. Minor side effects  
occur in 5-8% of subjects and include headache, nausea, flushing and dizziness. These 
symptoms are usually short lived and do not require treatment. Allergic reactions have 
been seen with contrast agents, but they are very rare.  In October 2007, the FDA issued a 
warning about serious complications with the use of contrast in critically ill  
subjects and in those with severe lung disease. In July 2008, the FDA removed several of 
the contraindications after studies in thousands of subjects showed that the benefits from 
diagnostic information outweighed the risks even for subjects at high risk for 
complications. 
 
Definity is approved by the FDA for use with echocardiography for the purpose of 
endocardial definition and opacification of the left ventricle. It will be used in this study 
as an off-label investigational use of a marketed drug. According to the FDA website, 

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http://www.fda.gov/RegulatoryInformation/Guidances/ucm126486.htm the clinical 
investigation of a marketed drug does not require submission of an IND if all six of the 
following conditions are met: 
i. 
it is not intended to be reported to FDA in support of a new indication for use or to 
support any other significant change in the labeling for the drug [correct] 
ii. 
it is not intended to support a significant change in the advertising for the product 
[correct] 
iii. 
it does not involve a route of administration or dosage level, use in a subject 
population, or other factor that significantly increases the risks (or decreases the 
acceptability of the risks) associated with the use of the drug product [It will be 
given intravenously; there should be no change in the risk profile. Our 
collaborator, Dr. Lindner, has published extensively on this technique and has 
used microbubble agents with perfusion imaging for over 10 years with no 
evidence of increased risk.] 
iv. 
it is conducted in compliance with the requirements for IRB review and informed 
consent [21 CFR parts 56 and 50, respectively] [correct] 
v. 
it is conducted in compliance with the requirements concerning the promotion and 
sale of drugs [21 CFR 312.7] [not applicable] 
vi. 
it does not intend to invoke 21 CFR 50.24. [This regulation refers to exception 
from informed consent requirements for emergency research and is not applicable 
to this study.] 
 
10.5.11 Brachial artery blood flow imaging: 
There are no known risks to the ultrasound imaging. 
 
10.6 Informed Consent Documentation 
Each participant will receive an oral and written explanation of the goals, procedures, and risks of 
this study.  The Principal Investigator and those Associate Investigators who are listed on the cover 
page of the protocol with an asterisk next to their name may obtain informed consent from research 
participants.  Consent will be obtained at the NIH Clinical Center.  The original, signed informed 
consent document will be placed in the medical record, and the subject will receive a signed copy 
of the informed consent document. 
 
If there is an unexpected enrollment of a research participant for which there is no translated extant 
IRB approved consent document, the principal investigator and/or those authorized to obtain 
informed consent will use the Short Form Oral Consent Process as described in MAS Policy M77-
2, 45 CFR 46.117 ( b) (2). The summary that will be used is the English version of the extant IRB 
approved consent document.  
 
We request prospective IRB approval of the use of the short form for up to a maximum of 5 
participants in a given language and will notify the IRB at the time of continuing review of the 
frequency of the use of the Short Form.  Should we reach the threshold of 5, we will notify the IRB 
of the need for an additional use of the Short Form and that we will have that consent document 
translated into the given inherent language. 
 
10.7 Subject Advocate 
A subject’s rights representative is available to subjects on this protocol.  The representative can 
be reached at 301-496-2626 and is located in Building 10.  Subjects may ask any questions about 
the study and may withdraw their consent at any time. 
 
 

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11 Conflict of Interest 
The National Institutes of Health reviews NIH staff researchers at least yearly for conflicts of interest. 
The following link contains details on this process http://ethics.od.nih.gov/forms/Protocol-Review-
Guide.pdf.None of the members of the research team reported a potential conflict of interest. 
 
12 Reimbursement for Travel 
Reimbursement for local travel, US travel, food, and lodging will not be provided under this protocol. 
 
13 Financial Compensation 
There are no direct benefits for the subjects participating in this study although information obtained 
may help to establish the mechanism of action of roflumilast in overweight glucose-intolerant subjects.  
In addition, the screening physical exam and laboratory tests may provide information that is useful for 
the subjects.  The screening visit will not be compensated but results of lab work will be made available 
to subjects and to their primary physician. Remuneration for participation in this study is based on 
existing NIH guidelines based on inconvenience, to offset potential loss of earnings and travel expenses.  
The amount paid will be prorated based upon participation.  Participants may not participate in all tests 
and therefore an estimate of total potential compensation is not included.  Participants will be 
compensated as described in the table below: 
 
Description of tests or procedures 
Maximum Possible 
Compensation for each test 
1. Daily payment for visits. $50/day X 5maximum paid visits    
$250 
2. Mixed meal test (2 unit) ($50 x 2) 
$100 
3. Glucose clamp (2 unit) ($100 x 2) 
$200 
4. Ultrasound Imaging (2 Unit) (($50 x 2) 
$60 
5. Stable isotope studies (1 unit) ($30 x 2) 
$100 
6. DEXA Scan - Body composition (1 unit) ($50 x2) 
$100 
7. MRI/MRS Scan ($50 x2) 
$100 
8. Optional Muscle biopsy ($200 x 2) 
$400 
9. Optional Adipose Tissue biopsy ($50 x2) 
$100 
10. In patient ($40 x 4) 
$160 
Maximum possible compensation 
$1570 
 
 
 

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14 References 
 
1. 
Yajima, H., Komatsu, M., Schermerhorn, T., Aizawa, T., Kaneko, T., Nagai, M., Sharp, G. W., and 
Hashizume, K. (1999) Diabetes 48, 1006-1012 
2. 
Wouters, E. T., P.; Brose, M.; Fabbri, L. M.; Rabe, K. F.;. (2010) Eur Respir J 
http://www.ersnet.org/learning_resources_player/abstract_print_10/main_frameset.htm., 
P4002 
3. 
Wouters, E. F. et al. Effect of the phosphodiesterase 4 inhibitor roflumilast on glucose 
metabolism in patients with treatment-naive, newly diagnosed type 2 diabetes mellitus. J Clin 
Endocrinol Metab 97, E1720-1725 (2012). 
4. 
Park, S. J., Ahmad, F., Philp, A., Baar, K., Williams, T., Luo, H., Ke, H., Rehmann, H., Taussig, R., 
Brown, A. L., Kim, M. K., Beaven, M. A., Burgin, A. B., Manganiello, V., and Chung, J. H. (2012) 
Cell 148, 421-433 
5. 
Katz, A., Nambi, S. S., Mather, K., Baron, A. D., Follmann, D. A., Sullivan, G., and Quon, M. J. 
(2000) J Clin Endocrinol Metab 85, 2402-2410 
6. 
Signorelli, P., and Ghidoni, R. (2005) The Journal of nutritional biochemistry 16, 449-466 
7. 
Pearson, K. J., Baur, J. A., Lewis, K. N., Peshkin, L., Price, N. L., Labinskyy, N., Swindell, W. R., 
Kamara, D., Minor, R. K., Perez, E., Jamieson, H. A., Zhang, Y., Dunn, S. R., Sharma, K., Pleshko, 
N., Woollett, L. A., Csiszar, A., Ikeno, Y., Le Couteur, D., Elliott, P. J., Becker, K. G., Navas, P., 
Ingram, D. K., Wolf, N. S., Ungvari, Z., Sinclair, D. A., and de Cabo, R. (2008) Cell Metab 8, 157-
168 
8. 
Baur, J. A., Pearson, K. J., Price, N. L., Jamieson, H. A., Lerin, C., Kalra, A., Prabhu, V. V., Allard, J. 
S., Lopez-Lluch, G., Lewis, K., Pistell, P. J., Poosala, S., Becker, K. G., Boss, O., Gwinn, D., Wang, 
M., Ramaswamy, S., Fishbein, K. W., Spencer, R. G., Lakatta, E. G., Le Couteur, D., Shaw, R. J., 
Navas, P., Puigserver, P., Ingram, D. K., de Cabo, R., and Sinclair, D. A. (2006) Nature 444, 337-
342 
9. 
Lagouge, M., Argmann, C., Gerhart-Hines, Z., Meziane, H., Lerin, C., Daussin, F., Messadeq, N., 
Milne, J., Lambert, P., Elliott, P., Geny, B., Laakso, M., Puigserver, P., and Auwerx, J. (2006) Cell 
127, 1109-1122 
10. 
Brasnyo, P., Molnar, G. A., Mohas, M., Marko, L., Laczy, B., Cseh, J., Mikolas, E., Szijarto, I. A., 
Merei, A., Halmai, R., Meszaros, L. G., Sumegi, B., and Wittmann, I. (2011) The British journal of 
nutrition 106, 383-389 
11. 
Dasgupta, B., and Milbrandt, J. (2007) Proc Natl Acad Sci U S A 104, 7217-7222 
12. 
Zang, M., Xu, S., Maitland-Toolan, K. A., Zuccollo, A., Hou, X., Jiang, B., Wierzbicki, M., 
Verbeuren, T. J., and Cohen, R. A. (2006) Diabetes 55, 2180-2191 
13. 
Um, J. H., Park, S. J., Kang, H., Yang, S., Foretz, M., McBurney, M. W., Kim, M. K., Viollet, B., and 
Chung, J. H. (2010) Diabetes 59, 554-563 
14. 
Canto, C., Jiang, L. Q., Deshmukh, A. S., Mataki, C., Coste, A., Lagouge, M., Zierath, J. R., and 
Auwerx, J. (2010) Cell Metab 11, 213-219 
15. 
Bergeron, R., Ren, J. M., Cadman, K. S., Moore, I. K., Perret, P., Pypaert, M., Young, L. H., 
Semenkovich, C. F., and Shulman, G. I. (2001) Am J Physiol Endocrinol Metab 281, E1340-1346 
16. 
Narkar, V. A., Downes, M., Yu, R. T., Embler, E., Wang, Y. X., Banayo, E., Mihaylova, M. M., 
Nelson, M. C., Zou, Y., Juguilon, H., Kang, H., Shaw, R. J., and Evans, R. M. (2008) Cell 134, 405-
415 
17. 
Viollet, B., Andreelli, F., Jorgensen, S. B., Perrin, C., Geloen, A., Flamez, D., Mu, J., Lenzner, C., 
Baud, O., Bennoun, M., Gomas, E., Nicolas, G., Wojtaszewski, J. F., Kahn, A., Carling, D., Schuit, F. 
C., Birnbaum, M. J., Richter, E. A., Burcelin, R., and Vaulont, S. (2003) J Clin Invest 111, 91-98 
18. 
Zong, H., Ren, J. M., Young, L. H., Pypaert, M., Mu, J., Birnbaum, M. J., and Shulman, G. I. (2002) 

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Proc Natl Acad Sci U S A 99, 15983-15987 
19. 
El-Mowafy, A. M., and Alkhalaf, M. (2003) Carcinogenesis 24, 869-873 
20. 
Baur, J. A., and Sinclair, D. A. (2006) Nat Rev Drug Discov 5, 493-506 
21. 
Gassmann-Mayer, C., Jiang, K., McSorley, P.,  Arani, R., DuBrava, S., Suryawanshi, S.,  Webb, 
D.M., & Nilsson, M. (2011).  Clinical and Statistical Assessment of Suicidal Ideation and Behavior 
in Pharmaceutical Trials.  Clinical Pharmacology & Therapeutics 90 4, 554–560.  
22. 
Food and Drug Administration.  (2010). Guidance for Industry Suicidality: Prospective 
Assessment of Occurrence in Clinical Trials DRAFT GUIDANCE.  ucm225130.  Retrieved from 
http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/uc
m225130.pdf 
23. 
Wu et al., Beige Adipocytes Are a Distinct Type of Thermogenic Fat Cell in Mouse and Human, 
Cell (2012), in press. 
24 
 Avila, C, Huang, R. J., Stevens, M. V., Aponte , A. M., Tripodi, D., Kim, K. Y., &  Sack, M. N. (2012).  
Platelet Mitochondrial Dysfunction is Evident in Type 2 Diabetes in Association with Modifi 
cations of Mitochondrial Anti-Oxidant Stress Proteins.  Exp Clin Endocrinol Diabetes; 120: 248–
251 
25 
Endicott J, Nee J, Harrison W, Blumenthal R. (1993).  Quality of Life Enjoyment and Satisfaction 
Questionnaire: a new measure.  Pharmacol Bull. 29(2):321-6. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Appendix 1: Study drug Diary 

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Appendix 2: Columbia Suicide Severity Rating Scale 
 

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Appendix 3:DALIRESP® package insert 
 

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Appendix  4: Quality of Life Enjoyment and Satisfaction Questionnaire 
 
 
 
 
 
 
 
 
 
 
 

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