Effect of Androgel on Atherogenesis, Inflammation, Cardiovascular Risk Factors And Adiposity in Type 2 Diabetic Males With Hypogonadotrophic Hypogonadism.: a Prospective, Randomized and Controlled-Study
Phase: NA
Conditions: Type 2 Diabetic Male With Hypogonadotrophic Hypogonadism
Sponsor: University at Buffalo

 
  
University at Buffalo Institutional Review Board (UBIRB)  
Office of Research Compliance | Clinical and Translational Research Center Room 5018  
875 Ellicott St. | Buffalo, NY 14203  
UB Federalwide Assurance ID#: FWA00008824  
  
 
Effect of Androgel on Atherogenesis, Inflammation, 
Cardiovascular Risk Factors And Adiposity in Type 2 Diabetic 
Males with Hypogonadotrophic Hypogonadism.: a Prospective, 
Randomized and Controlled-Study 
 
NCT00467987  
 
 Document Date: November 16, 2017 
 
 

 
  
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IRB Version: JAN2016  
 
Complete Research Protocol (HRP-503)  
  
Table of Contents  
Template Instructions.......................................................................................................... 2 
1.0 Objectives ................................................................................................................ 4 
2.0 Scientific Endpoints ................................................................................................. 6 
3.0 Background .............................................................................................................. 7 
4.0 Study Design .......................................................................................................... 25 
5.0 Local Number of Subjects ..................................................................................... 25 
6.0 Inclusion and Exclusion Criteria ............................................................................ 26 
7.0 Vulnerable Populations .......................................................................................... 27 
8.0 Eligibility Screening .............................................................................................. 28 
9.0 Recruitment Methods ............................................................................................. 29 
10.0 Procedures Involved ............................................................................................... 30 
11.0 Study Timelines ..................................................................................................... 38 
12.0 Setting .................................................................................................................... 38 
13.0 Community-Based Participatory Research ............................................................ 39 
14.0 Resources and Qualifications ................................................................................. 39 
15.0 Other Approvals ..................................................................................................... 40 
16.0 Provisions to Protect the Privacy Interests of Subjects .......................................... 40 
17.0 Data Management and Analysis ............................................................................ 41 
18.0 Confidentiality ....................................................................................................... 42 
A. Confidentiality of Study Data ............................................................................ 42 
B. Confidentiality of Study Specimens ................................................................... 43 
19.0 Provisions to Monitor the Data to Ensure the Safety of Subjects .......................... 44 
20.0 Withdrawal of Subjects .......................................................................................... 46 
21.0 Risks to Subjects .................................................................................................... 47 
22.0 Potential Benefits to Subjects ................................................................................ 49 
23.0 Compensation for Research-Related Injury ........................................................... 49 
24.0 Economic Burden to Subjects ................................................................................ 49 25.0 
Compensation for Participation ............................................................................. 49 
26.0 Consent Process ..................................................................................................... 50 
27.0 Waiver or Alteration of Consent Process ............................................................... 54 
28.0 Process to Document Consent ............................................................................... 54 
29.0 Multi-Site Research (Multisite/Multicenter Only) ................................................. 55 
30.0 Banking Data or Specimens for Future Use .......................................................... 56 
31.0 Drugs or Devices.................................................................................................... 57 
32.0 Humanitarian Use Devices .................................................................................... 58 
  
    
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IRB Version: JAN2016  
Template Instructions  
Sections that do not apply:  
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added to the template as responses.    
o If an N/A checkbox is present, select the appropriate justification from 
the list.    
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checkboxes apply to your study, you must write in your own 
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• 
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Update the version date or number on Page 3.  
    
PROTOCOL TITLE:  
Include the full protocol title.  
Response:  

 
  
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Effect of Androgel on Atherogenesis, Inflammation, Cardiovascular Risk Factors 
And Adiposity in Type 2 Diabetic Males with Hypogonadotrophic Hypogonadism.: 
a Prospective, Randomized and Controlled-Study  
  
  
PRINCIPAL INVESTIGATOR:  
Name  
Department  
Telephone Number  
Email Address  
Response: Paresh Dandona, M.B.B.S., Ph.D., F.R.C.P, F.A.C.P, F.A.C.C  
Director, Diabetes-Endocrinology Center of Western NY  
           SUNY Distinguished Professor Chief of 
Endocrinology, SUNY at Buffalo  
1000 Youngs Road, Suite 15  
          Williamsville NY 14221  
Telephone: (716) 55-1850  
Fax: (716) 639-1200  
E-mail:pdandona@kaleidahealth.org  
Co-Investigators: Husam Ghanim, PhD  
 
  
  
Manav Batra, MD  
  
VERSION:  
Include the version date or number.  
Response: 11/16/2017  
  
  
GRANT APPLICABILITY:  
Indicate whether this protocol is funded by a grant (e.g. NIH, foundation grant).  
For a grant with multiple aims, indicate which aims are covered by this research 
proposal.  
NOTE: This question does not apply to studies funded by a sponsor contract.  
 Include a copy of the grant proposal with your submission.      
Response:   
Fully funded by Abbvie Incorporated   
  
RESEARCH REPOSITORY:  
Indicate where the research files will be kept, including when the study has been 
closed.  The repository should include, at minimum, copies of IRB 
correspondence (approval, determination letters) as well as signed consent 
documents.  This documentation should be maintained for 3 years after the study 
has been closed.   
 
Response: Diabetes Endocrinology Center of WNY  

 
  
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Location: 1000 Youngs Road, Suite 105  
Williamsville NY 14221  
Department: Diabetes Endocrinology & Metabolism   
  
  
  
    
1.0 Objectives  
1.1 Describe the purpose, specific aims, or objectives of this research.  
Response: We have shown that the prevalence of hypogonadotrophic hypogonadism (HH) 
in type 2 diabetic (DM) patient population may be around 35%. We have also shown that 
in an age and glycemia matched group of type 1 diabetic patients, there is no significant 
hypogonadotrophic hypogonadism. HH in type 2 diabetics is associated with a normal 
release of LH and FSH following GnRH administration. Thus, the defect in testosterone 
secretion is specific to type 2 diabetes and is likely to be at the hypothalamic level.   
Since insulin may facilitate GnRH secretion from hypothalamic neurons and interference 
with insulin signaling may result in a reduction in GnRH secretion, it is possible that insulin 
resistance in type 2 diabetes may play a key role in the pathogenesis of HH. Furthermore, 
as insulin resistance may be mediated by pro-inflammatory factors which are also known 
to reduce GnRH and gonadotrophin secretion, it is possible that there may be an increase 
in pro-inflammatory mediators in association with low testosterone concentrations in type 
2 DM. We have shown that there is an inverse relationship between CRP and testosterone 
concentrations and that patients with HH have CRP concentrations which are twice as high 
(6.5mg/L) as those in patients with normal cFT and TT concentrations (3.2 mg/L). Since 
CRP concentrations of 3mg/L or greater are predictive of cardiovascular disease, patients 
with HH and type 2 DM would be in the highest risk category. Low testosterone 
concentrations constitute a significant atherogenic risk as shown in an epidemiological 
study from Britain. However, the combination of a low T and type 2 diabetes mellitus was 
not examined in this study.   
Our work has recently also demonstrated that in patients with type 2 D.M. and HH, 
hematocrit and bone mineral density are significantly lower while upper abdominal 
adiposity is significantly greater than normal subjects and those with type 2 DM without 
HH.    
In view of these observations, it is necessary to investigate the cardiovascular risk in 
patients with HH and type 2 DM prospectively and to examine the effect of Testosterone 
replacement on atherogenesis, endothelial function, inflammatory and oxidative stress 
mediators, hematocrit and body composition in this cohort of patients  
1.2 Describe the purpose, specific aims, or objectives.  
Response:  To compare the carotid IMT of type 2 diabetic males with and without HH at 
baseline.   
Aim 1.2: To compare the carotid IMT of type 2 diabetic males with HH before and after 
treatment with Androgel 1% or placebo daily for 24 months. Measurements will be done 
at baseline and at 6, 12, 18 and 24 months.  
Aim 2.1: To compare the brachial FMD% of type 2 diabetic males with and without HH 
at baseline.   
Aim 3.1:  To compare the serum concentrations and expression in adipose tissue of 
Tumor  
Necrosis 
Factor 
alpha 
(TNF- ), 
Interleukin-6 
(IL-6), 
Intracellular 
adhesion 
molecule1(ICAM-1), plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-

 
  
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9 (MMP9), C-reactive protein (CRP), serum Amyloid A (SAA) and Monocyte 
Chemoattractant protein-1 (MCP-1) in type 2 diabetic males with and without HH at 
baseline.   
Aim 3.2:  To compare the levels of inflammatory mediators in aim 3.1 before and after 
therapy with Androgel 1% or placebo daily for 24 months in type 2 diabetic males with 
HH. Serum will be obtained at baseline , 6, 12, 18 and 24 months. Adipose tissue and 24 
hour urine will be obtained at baseline, 12 and 24 months.  
Aim 4.1: To compare the abdominal adipose tissue mass of type 2 diabetic males with and 
without HH as measured by MRI. Abdominal MRI will be done to measure hepatic, 
visceral and subcutaneous fat at baseline.   
Aim 4.2: To compare the hepatic, visceral and subcutaneous abdominal fat mass of type 2 
diabetic males with HH as measured by MRI, before and after treatment with Androgel 1% 
or placebo daily for 24 months. Measurements will be done at baseline, 12 and 24 months. 
Aim 5.1: to compare the hematocrit of type 2 diabetic males with and without HH at 
baseline.  
Aim 5.2:  To compare hematocrit levels before and after therapy with Androgel 1% or 
placebo daily for 24 months in type 2 diabetic males with HH. Measurements will be done 
at baseline and at 6, 12, 18 and 24 months.  
Aim 6.1: to compare the aromatase enzyme expression in adipose tissue of type 2 
diabetic males with and without HH at baseline.  
 Aim 6.2:  To compare aromatase enzyme expression in adipose tissue before and after 
therapy with Androgel 1% or placebo daily for 24 months in type 2 diabetic males with 
HH. Measurements will be done at baseline, 12 and 24 months.  
Aim 7.1: to compare the APP expression in adipose tissue of type 2 diabetic males with 
and without HH at baseline.  
Aim 7.2: To compare APP expression in adipose tissue before and after therapy with 
Androgel 1% or placebo daily for 24 months in type 2 diabetic males with HH. 
Measurements will be done at baseline, 12 and 24 months  
  
1.3 State the hypotheses to be tested, if applicable.  
NOTE:  A hypothesis is a specific, testable prediction about what you expect to 
happen in your study that corresponds with your above listed objectives.  
Response: Hypothesis 1: HH in type 2 diabetic males is associated with increased 
atherogenesis as assessed by carotid intima media thickness (IMT) compared to age and 
BMI matched type 2 diabetic males without hypogonadism. Testosterone replacement in 
type 2 diabetic males with HH reduces atherogenesis.  
Hypothesis 2: HH in type 2 diabetic males is associated with impaired endothelial 
function as assessed by brachial artery Flow mediated dilatation (FMD%) compared to 
age and BMI  matched type 2 diabetic males without hypogonadism. Testosterone 
replacement in type 2 diabetic males with HH improves endothelial function.  
Hypothesis 3: HH in type 2 diabetic males is associated with increased inflammatory 
mediators as compared to age and BMI matched type 2 diabetic males without 
hypogonadism. Testosterone replacement in type 2 diabetic males with HH decreases 
inflammatory mediators in serum and adipose tissue.  
Hypothesis 4: HH in the diabetic patients is associated with increased abdominal adipose 
tissue mass as compared to age and BMI matched type 2 diabetic males. Testosterone 
replacement in type 2 diabetic males with HH decreases abdominal adipose tissue mass 
as compared to age matched type 2 diabetic patients with untreated hypogonadism.  

 
  
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Hypothesis 5: HH in type 2 diabetic males is associated with lower hematocrit compared 
to age matched type 2 diabetic males without hypogonadism.  
Hypothesis 6: HH in type 2 diabetic men is associated with an increase in aromatase 
enzyme expression in adipose tissue. Testosterone therapy for 24 months will decrease the 
expression of aromatase enzyme.  
Hypothesis 7: HH is type 2 diabetic men is associated with an increase in amyloid 
precursor protein (APP) expression in adipose tissue. Testosterone therapy for 24 months 
will decrease the expression of APP.  
Aim 7.1: to compare the APP expression in adipose tissue of type 2 diabetic males with 
and without HH at baseline.  
 .  
  
2.0 Scientific Endpoints  
  
2.1    Describe the scientific endpoint(s), the main result or occurrence under 
study.    
  
NOTE:  Scientific endpoints are outcomes defined before the study begins to determine 
whether the objectives of the study have been met and to draw conclusions from the data.  
Include primary and secondary endpoints.  Some example endpoints are:  reduction of 
symptoms, improvement in quality of life, or survival.  Your response should not be a 
date.    
Response: The focus of the proposed research is to evaluate the effect of hypogonadotrophic 
hypogonadism on atherogenesis, endothelial function, inflammatory mediators and oxidative stress 
in type 2 diabetic patients and the effect of testosterone replacement on these parameters. The 
similarities between the study groups, baseline values for subject’s demographics, carotid IMT, 
FMD%, body composition, hematocrit and inflammatory markers will be compared using 
appropriate parametric tests. Transformations of the data in order to meet statistical assumptions 
may be considered. The PRIMARY ENDPOINT of the study is to detect a difference in 
atherogenesis as measured by carotid IMT between type 2 diabetic subjects with and without HH 
after 24 months of testosterone replacement with Androgel. There are no prior studies looking at 
these two populations in humans in vivo, however studies assessing the effect of thiazolidinediones 
(insulin sensitizers with anti-inflammatory properties) on carotid IMT in type 2 diabetics have 
shown a reduction in IMT by 0.033mm at 12 weeks and 0.054mm at 24 weeks (178).  In a study of 
154 diabetic patients, serum free testosterone (F-tes) concentrations were found to be inversely 
correlated with mean IMT. Patients with low concentrations of FT (<10 pg/ml) had greater mean 
IMT (1.01 ± 0.29 mm) than those with high concentrations of FT(0.91 ± 0.26 mm) (179). On the 
basis of these previous observations, we have conservatively estimated a difference in carotid IMT 
of 0.020mm at 24 months between the type 2 DM with HH and the type 2 DM with HH who is on 
androgel replacement. A sample size of 30 patients per group (assuming a drop-out rate of 5%) will 
provide adequate power (  = 0.8) to detect a significant difference of 0.020mm in carotid IMT (  
= 0.05), provided the standard deviation of the residuals is not equal to or greater than the mean 
difference. The results will be computed as mean ± SD. Comparisons for endpoints will be made 
using repeated measures ANOVA, with Tukey’s test used for pair wise comparisons. Thus there 
will be 30 HH type 2 diabetic subjects each in placebo and treatment group of diabetic subjects with 
HH (total 60 subjects with HH). 20 eugonadal type 2 diabetic subjects will be recruited to serve as 
controls. SECONDARY END POINTS: The secondary endpoints for the study will be comparison 
of the relative change from baseline in FMD%, inflammatory mediators, abdominal fat, waist and 
hip ratio, after either testosterone or placebo. Comparison of these parameters at baseline will also 

 
  
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be done. Comparison for each endpoint will be made using ANOVA, with Tukey’s test used for 
pairwise comparisons.  
3.0 Background  
3.1 Provide the scientific or scholarly background, rationale, and significance of 
the research based on the existing literature and how it will contribute to 
existing knowledge.  Describe any gaps in current knowledge.  Include 
relevant preliminary findings or prior research by the investigator.   
Response: Hypogonadism is associated with increased adipose tissue mass, decreased 
muscle and bone mass, decreased insulin sensitivity and increased inflammatory stress. 
Testosterone replacement in nondiabetic males is associated with an improvement in some 
of these parameters and also sexual function and mood (1-4).  It has recently been 
recognized that hypogonadotrophic hypogonadism occurs frequently in type 2 diabetes 
mellitus(5). We have shown that HH in type 2 DM is associated with increased 
inflammation compared to eugonadal type 2 DM. It is therefore important to study the 
effect of HH on cardiovascular risk prospectively and the effect of Testosterone (T) 
replacement on these parameters in this cohort of patients. If HH is found to be associated 
with increased cardiovascular risk and T replacement is shown to reduce this risk, this will 
provide the rationale for treatment of this common defect in type 2 DM.  
Hypogonadism and Type 2 Diabetes  
Total testosterone concentrations are determined to a large extent by circulating sex 
hormone binding globulin (SHBG) concentrations. In the blood of normal men, around 
44% of total testosterone (T) is bound to SHBG, 2% is unbound (free T) and 54% circulates 
bound to albumin and other proteins.(6; 7) Circulating SHBG concentrations are decreased 
in obesity and increase with aging. A complete assessment of hypogonadism should 
therefore include measurement of free and/or bioavailable testosterone. Equilibrium 
Dialysis (ED) is considered to be the gold standard for measuring free testosterone. Free 
testosterone can also be calculated from SHBG and T using the method of Vermeulen et 
al.(8) This calculated free testosterone (cFT) is reliable (9) and has been shown to correlate 
very well (r=0.92) with free testosterone measured by ED.(5; 10; 11) Bioavailable 
testosterone can also be calculated from the equation of Vermeulen et al(8). For proposed 
research project, hypogonadism will be defined as calculated free testosterone below 
6.5ng/dL.  
We have recently observed that a significant proportion of type 2 diabetic patients have low 
free testosterone (measured by ED or calculated using T and SHBG) and the nature of 
hypogonadism in type 2 diabetics is hypogonadotrophic(5; 12) (see preliminary data). This 
condition is a potential candidate for being the commonest complication of type 2 diabetes 
(through association rather than causality at this time) and requires further assessment in 
terms of the etiology of the defect and the possible consequences, complications and 
treatment. Another recent study (ADA, 2005) has confirmed the high prevalence of 
hypogonadotrophic hypogonadism in an older group of type 2 diabetic patients, aged over 
45 years. The patients with HH had greater prevalence of hypertension, hyperlipidemia, 
obesity and inadequate sexual function and libido than patients with normal gonadal 
function. These findings have also been confirmed in type 2 diabetic subjects attending a 
tertiary clinic for the assessment of erectile dysfunction(13). Low Testosterone in type 2 
diabetics in this study was also associated with a reduction in blood flow in the cavernous 
arteries when assessed by Doppler ultrasound.   
Hypogonadism and insulin sensitivity  
Insulin sensitivity is closely correlated with intramuscular, intramyocellular and 
intraabdominal adipose tissue. Hypogonadism is associated with increased subcutaneous 

 
  
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(both trunk and appendicular), intraabdominal and intermuscular adipose tissue(14; 15). 
Epidemiological studies have suggested an inverse relationship between testosterone 
concentrations and insulin sensitivity, probably mediated by total or abdominal adiposity 
(16). Metabolic syndrome, insulin resistance and visceral obesity have all been associated 
with low SHBG and low total testosterone levels in men.(17; 18) It is believed that the low 
total testosterone in obesity is due to low SHBG concentrations. However, free testosterone 
levels have also been found to correlate inversely with body mass index (BMI)(19). 
Vermeulen et al(20) compared LH pulsatility over 12 hours in obese and lean men and 
found that the mean integrated LH levels over 12 hours were significantly lower in obese 
men. Free testosterone (FT) levels correlated positively with the sum of LH pulse 
amplitudes in each individual.(20)  Other studies showed a rise in LH or FSH levels after 
weight loss(21; 22). It appears that in obesity induced hypogonadism, FT levels fall into 
hypogonadal range only when the obesity is severe (BMI>40kg/m2)(23). Elevated estradiol 
and leptin levels observed in obesity probably mediate part of the hypogonadotrophic effect 
of obesity , but they do not account for the full effect of obesity on hypothalamus/pituitary 
(22; 24; 25).   
As mentioned above, type 2 diabetes is associated with hypogonadotrophic hypogonadism 
(HH). FT correlates inversely with BMI in type 2 diabetic patients (5). We have recently 
also shown that BMI is inversely related to FT in Type 1 DM patients although these 
patients do not have HH. The existence of a hypothalamic defect resulting in HH in type 2 
diabetes is of interest in view of its association with insulin resistance. It is known that 
neuron specific insulin receptor knock out (NIRKO) mice exhibit HH (26).  Plasma LH 
levels are decreased by 60-90% in NIRKO mice as compared to controls. When these mice 
were injected with lupron, a GnRH receptor agonist, they displayed a normal to twofold 
increase in LH levels as compared to control mice. Thus, the pituitary reserve in these 
animals was normal. These mice also had increased adipose tissue and insulin resistance. 
A recent study demonstrated an association between insulin sensitivity (as measured by 
hyperinsulinemic euglycemic clamp) and hCG stimulated testicular response among 
humans with varying degrees of glucose tolerance(27). Thus data from literature suggests 
that obesity/insulin resistance and type 2 diabetes are associated with hypogonadism, this 
relationship 
may 
be 
bidirectional, 
and 
may 
occur 
at 
multiple 
levels 
of 
hypothalamicpituitary-gonadal axis (28-30). It is interesting that type 1 diabetes mellitus is 
not associated with hypogonadism (31; 32)  (preliminary data).  
Testosterone replacement leads to a dose-dependent decrease in adipose tissue and increase 
in muscle mass and strength(14; 33; 34). This effect appears to be more pronounced if the 
population studied has higher amounts of adipose tissue. The mechanism by which 
testosterone replacement produces these effects is not well understood. Inhibition of 
lipoprotein lipase activity in intraabdominal adipose tissue and the differentiation of 
pluripotent mesenchymal precursor cells preferentially into myogenic lineage instead of 
adipocytic lineage may play a role(35; 36). Marin et al demonstrated a decrease in visceral 
adipose tissue and improvement in insulin sensitivity with oral and transdermal testosterone 
treatment in middle aged obese non-diabetic men (37; 38). The subjects were not 
hypogonadal but insulin sensitivity improved more in subjects whose testosterone 
concentrations were in the low normal range at the start of the study. The improvement in 
insulin sensitivity and plasma testosterone concentrations were inversely related. In one of 
these studies, oral testosterone undecanoate 80mg twice a day (which increased serum 
testosterone concentrations to 500-600 ng/dL for a few hours after the dosing) or placebo 
given for 8 months reduced visceral body fat (measured by CT scan) and total body fat  by 
~6%. Subcutaneous adipose tissue did not change. Glucose disposal rate (HE clamp) 
increased by 20%. Change in glucose disposal rate was related to baseline testosterone 
levels (r= -0.65, P<0.05). In another publication using a similar population, transdermal 

 
  
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testosterone for 3 months (which doubled T concentrations from 418 ng/dL at baseline to 
820 ng/dL) increased glucose uptake in HE clamp by 17% and decreased waist/hip 
circumference. Relatively hypogonadal men had more demonstrable effects. However, 
other studies have failed to show an improvement in insulin sensitivity(39-41). It is possible 
that the lack of a low testosterone prior to treatment was responsible for the lack of change 
in insulin sensitivity in these studies. The effect of testosterone on adipose tissue, muscle 
mass and insulin sensitivity seems to depend on the population studied, dose of testosterone 
replacement and duration of therapy. It is possible that the improvement in insulin 
sensitivity after testosterone replacement is due to an increase in lean body mass and a 
decrease in adipose tissue mass, or more specifically by a decrease in visceral or 
intramyocellular adipose tissue, and that this effect is more pronounced in the hypogonadal 
patients. No studies have been done to evaluate the effect of testosterone on insulin 
sensitivity in type 2 diabetic patients. We intend to study the effects of testosterone 
replacement in type 2 diabetic subjects, a population that is likely to benefit from a 
reduction in adipose tissue and an increase in muscle mass. We have recently demonstrated 
that HH in type 2 DM is associated with an increase in total, appendicular and truncal fat 
(preliminary data).  
  
Obesity, diabetes and inflammation   
Both obesity and diabetes have been shown to be associated with oxidative stress (4245) 
and inflammation (46-50). The understanding of mechanisms leading to oxidative stress 
and inflammation is important.  One possible reason why obesity and type 2 diabetes are 
associated with oxidative stress and inflammation is the state of insulin resistance. This is 
due to the fact that 1) resistance is associated with the presence of pro-inflammatory factors 
including cytokines (51); 2) insulin has been shown to exert an anti-inflammatory and anti-
oxidant effects at the cellular and molecular level both, in vitro, and in vivo (43; 52-54). A 
low dose infusion of insulin (2.5 IU/h) has been shown to reduce ROS generation by 
mononuclear cells (MNC), suppress NADPH oxidase expression and intranuclear NFκB 
binding, induce IκB expression and suppress plasma ICAM-1 and MCP-1 concentrations 
(43). It also suppresses intranuclear Egr-1, plasma tissue factor (TF), PAI1 and MCP-1 
concentrations (52). An interruption of insulin signal transduction would prevent the anti-
inflammatory effect of insulin from being exerted. The anti-inflammatory and pro-
fibrinolytic effects of insulin have also been confirmed in the setting of acute ST segment 
elevation myocardial infarction(55).   
We have previously demonstrated that macronutrient intake (glucose, protein and 
lipids) induce ROS generation and cause oxidative stress in humans. Glucose or a mixed 
meal intake cause an increase in NF B and a fall in total cellular I B , the two cardinal 
indices of inflammation at the cellular level (56-58). Mixed meal also induces an increase 
in C-reactive protein (CRP) (56).  Glucose intake has further been shown to increase the 
ratio of phosphorylated I B  to native non-phosphorylated I B  and to induce an increase 
in I B  kinase (IKK ) and IKK .  At a cellular level, the changes that occur in the MNC 
following a glucose challenge are very similar to those which occur after endotoxin 
challenge.   
It is, thus, possible that obesity is a pro-oxidative and pro-inflammatory state resulting 
from chronically increased macronutrient intake. An increasing amount of epidemiologic 
data show that persistent, low-grade inflammation is an independent predictor of 
ischemic heart disease (59; 60), stroke (61; 62), diabetes (63; 64), and all-cause mortality 
(65; 66). In addition to these epidemiologic findings, experimental evidence shows that 
markers of chronic inflammation, such as pro-inflammatory cytokines IL-6 and TNFα 
and the acute-phase reactant CRP, play a direct role in the etiology of atherosclerosis and 

 
  
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insulin resistance (67; 68). Studies are beginning to show that diet-induced weight loss 
decreases concentrations of CRP, IL-6, and TNFα  (46; 69-74).  It is also relevant that 
caloric restriction in the obese and a fast in normal subjects result in a reduction in 
oxidative stress and inflammatory mediators (43; 46; 75) (Tables 1 & 2). In this regard, it 
is interesting to note that TNF-α and IL-1β have been shown to reduce hypothalamic 
GnRH and LH secretion in animals and in vitro.(76; 77) It is therefore possible that 
inflammatory cytokines may partly mediate HH of obesity and type 2 diabetes.  
  
  
  
  
  
Baseline  
24 hr  
48 hr  
ROS by PMN (% change from the baseline)  
100  
66 ± 20  
46 ± 23§  
ROS by MNC  (% change from the baseline)  
100  
62 ± 17  
48 ± 17§  
p47phox Subunit  (% change from the baseline)  
100  
41 ± 13   
32 ± 16§  
o-tyrosine  
3.26± 0.05  
3.23 ± 0.06  
3.03 ± 0.05*  
m-tyrosine  
3.79 ± 0.62  
3.80 ± 0.62  
3.59 ± 0.66*  
  
Table 1: ROS generation, p47phox Subunit  protein expression, o- and m-tyrosine following 2 day fast; §: P<0.001; *: 
P<0.05(75).  
   
  
Lean  
Obese (0 Week)  
4 Weeks   
ROS by PMN (mV)  
220 ± 60  
236 ± 96  
103± 36 §  
ROS by MNC  (mV)  
165 ± 80  
188 ± 75  
75 ± 32 §  
TBARS ( M; lipid peroxide)  
1.29 ± 0.12 *  
1.68 ± 0.17  
1.47 ± 0.13 *   
13-HODE( mol/L)  
2.04 ± 0.72 §  
6.67 ± 3.85  
4.15 ± 5.62 *  
9-HODE( mol/L)  
1.97 ± 0.84 §  
7.10 ± 3.88  
4.50 ± 5.70 *  
Carbonylated proteins ( mol/mg protein)  
0.60 ± 0.10 §  
1.39 ± 0.27  
1.17 ± 0.12 *  
o-tyrosine/phenyalanine ratio (mmol/mol)  
0.28 ± 0.02 §  
0.42 ± 0.03  
0.36 ± 0.02 *  
m-tyrosine/phenyalanine ratio (mmol/mol)  
0.27 ± 0.03 §  
0.45 ± 0.04  
0.40 ± 0.03 *  
  
Table 2: Oxidative stress markers in lean vs. obese subjects and  the effect of diet restriction for 4 weeks (1000 
kcal/day);§:P<0.001; *: P<0.05 when compared with obese at baseline  (43):   
Measuring Inflammation and Oxidative Stress  
Inflammation at the cellular level can be described as an increase in the pro-inflammatory 
transcription factor, NFκB, in the nucleus and with a concomitant decrease in its inhibitors IκB  
and/or IκB . NF B is a heterodimer, and usually consists of two proteins, a p65 (RelA) subunit 
and a p50 subunit. In the basal state NF B is bound to its inhibitor protein I B, which restricts 
NF B to the cytoplasm. Stimulation of cells by cytokines like TNF  or IL-1 or endotoxin results 
in phosphorylation of I B, unbinding of NF B from I B and activation of NF B with its 
subsequent translocation into the nucleus (78; 79). This in turn induces the transcription of 
proinflammatory cytokines (such as TNFα, IL-1 and IL-6), adhesion molecules (ICAM, VCAM), 
chemokines (such as MCP-1 and MIF), metalloproteinases (MMP-1, and MMP-9) and many other 
genes that regulate transcription, apoptosis and cell proliferation.  
 Oxidative stress can be defined as an increase in ROS generation. The term ROS is a collective 
term that includes not only oxygen-centered radicals such as superoxide (Oꞏ2 ) and hydroxyl (OH), 
but also some non-radical derivative of oxygen such as hydrogen peroxide (H2O2), singlet oxygen 
and hypochlorous acid (HOCL). A critical balance exists between generation and detoxification of 
ROS in respiring cells of normal individuals. The various risk factors for atherosclerosis, including 
hyperlipidemia, hypertension, and diabetes, have in common the generation of oxidative stress. 
ROS are able to break off cell membrane proteins, fuse membrane lipid and proteins, hardening the 

 
  
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cell membrane and exposing genetic material in the nucleus, leaving the DNA open for mutation or 
destruction. They may damage the endothelium of blood vessels, thereby leading to atherosclerosis. 
ROS-mediated lipid peroxidation is a cardinal step in the initiation of atherosclerosis (80; 81).  
NF B is one of the transcription factors that may be controlled by the redox status of the cell (82). 
NADPH oxidase complex, composed of cytoplasmic (p47phox p67phox, small GTP-binding p21rac) 
and plasma membrane componenets (heterodimer of gp91 & p22phox)  is a key enzyme involved in 
the production of ROS.(83) Oxidative stress can also be measured by the presence of isoprostanes 
in urine.  
Lipid Peroxidation and Protein Oxidation: Lipid peroxidation is an autocatalytic free 
radicalmediated chemical mechanism in which polyunsaturated fatty acids (PUFA) undergo 
oxidation to form lipid hydroperoxide (LHP)(84; 85). Levels of 15-isoprostane F2 in urine are 
useful for the non-invasive assessment of oxidant stress in vivo (86). There is evidence that 
oxidative modification of proteins may be physiologically important, serving as a “marking” step 
for initiating protein degradation. Oxidation of proteins by microsomal oxidases and metal-
catalyzed oxidation treatment is known to produce carbonyl modifications of amino acids (87). Phe 
reacts with the hydroxyl radical (OH) to form the hydroxylated products ortho-, meta-, and para-
tyrosine (o-, m-, and p-tyr). The aromatic hydroxylation of Phe can be used to detect OH formation 
and is considered as a marker of protein oxidation (88).   
Hypogonadism and Inflammation  
It has been suggested that hypogonadism is associated with an increase in inflammatory mediators 
and treatment of hypogonadism leads to a reduction in inflammation(89). In vitro studies have 
demonstrated that testosterone induces an inhibition of IL-6 production by human monocytes(90). 
Testosterone treatment of human aortic endothelial cells results in an inhibition of TNF-α induced 
VCAM-1 mRNA expression and NF-κB activation.(91) Intramuscular testosterone replacement in 
hypogonadal males results in a decrease in pro-inflammatory cytokines(IL-1 and TNF-α) and an 
increase in anti-inflammatory cytokine(IL-10) levels in serum(92). However, some studies were 
unable to demonstrate an anti-inflammatory effect of testosterone(39; 93). It is possible that the 
effects of testosterone replacement on inflammation depend on the population studied. A 
comprehensive study on inflammation and testosterone replacement needs to be done to clarify the 
issue.   
We have demonstrated clearly for the first time that low plasma TT and cFT concentrations are 
associated with elevated CRP concentrations in patients with type 2 diabetes (Diabetes Care 2006 
and Preliminary data). In addition, there is an inverse relationship between plasma TT and plasma 
CRP concentrations. This relationship is independent of the known relationship between BMI and 
CRP, which has previously been observed by others and by us during studies on obesity (47-50; 
94-97). It is noteworthy that this relationship between CRP and testosterone concentrations was 
observed in spite of the intake of statins, ACE-inhibitors, ARBs and aspirin that have 
antiinflammatory effects.   
While the establishment of this important relationship does not provide us with a causal link 
between HH and inflammation, it provides us with several potential novel hypotheses and avenues 
of investigation. Firstly, it is possible that a low T contributes to the overall inflammatory state of 
a patient with type 2 diabetes. This can be tested by administering T to hypogonadal patients to 
determine whether CRP concentrations and other indices of inflammation fall as proposed in this 
investigation. Secondly, it is possible that the pro-inflammatory state, of which elevated CRP 
concentration is an indicator, may lead to the suppression of GnRH secretion by the hypothalamic 
neurons and thus lead to HH. The pro-inflammatory cytokine, TNF , known to be elevated in type 
2 diabetes and obesity(47; 63; 94; 98; 99), suppresses the secretion of hypothalamic hormones 
including gonadotropins.(76; 100-102).  
In a previous study on young, non-obese healthy men in whom endogenous testosterone production 
was suppressed by giving GnRH analogue and graded doses of testosterone were given (39), the 
circulating levels of CRP did not change with testosterone administration, nor were they related to 
T concentrations. This can be probably explained by the low level of inflammatory mediators 
including CRP in younger healthy subjects with lower fat mass. Furthermore, the study probably 

 
  
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did not allow a sufficient period of hypogonadism for a change in CRP concentration to emerge. It 
is relevant that specific insulin receptor deletion in neurons has recently been shown to result in 
hypogonadotrophic hypogonadism in mice (26). It would, therefore appear that insulin has a 
facilitatory role in gonadotrophin secretion, possibly through GnRH secretion in the hypothalamus. 
Recently, insulin has been shown to exert other specific effects on the hypothalamus. For example, 
it mediates satiety(103; 104) and also regulates gluconeogenesis and hepatic glucose production 
through the hypothalamus and its vagal outflow(105). It is possible that insulin also has a 
facilitatory, trophic effect on the release of GnRH from hypothalamic neurons and that 
inflammatory mediators like TNF  and IL-6, known to interfere with insulin signaling(106-108), 
thus suppress GnRH secretion in conditions associated with insulin resistance like obesity and the 
metabolic syndrome. These conditions are characterized by insulin resistance at least in part due to 
pro-inflammatory mediators like TNF  and IL-6 which interfere with insulin signal 
transduction(109; 110). Furthermore, glucose also induces oxidative stress (111; 112)and 
inflammation, especially if endogenous insulin secretion is  impaired or inadequate and may thus 
contribute to insulin resistance.   
In this context, it is also important that insulin has recently been shown to exert a powerful and 
rapid, anti-inflammatory effect with a suppression of pro-inflammatory transcription factors 
(NFkB, Egr-1), chemokines (MCP-1), adhesion molecules (ICAM-1), matrix metalloproteinases 
and CRP(52; 54; 108; 113-115). Resistance to the action of insulin would therefore, facilitate a 
proinflammatory state, which in turn would further interfere with insulin signal transduction. Since 
the patients with hypogonadism had a higher BMI, tended to have lower HDL apart from having 
diabetes and a higher CRP, they probably had the features of the metabolic syndrome. Hypertension 
cannot be commented on since blood pressure was controlled with anti-hypertensive drugs. This 
makes it likely that hypogonadism may be a feature of the metabolic syndrome. Indeed,  nondiabetic 
patients with metabolic syndrome, have been shown to have low testosterone concentrations.(95; 
116-118). Whether these low testosterone concentrations in patients with metabolic syndrome are 
associated with low LH and FSH concentrations as in type 2 diabetes has not yet been investigated.  
Since hypogonadism promotes obesity(19; 22; 24; 25), it is possible that the increase in obesity 
secondary to a low T, further promotes inflammation and an increase in CRP. It is possible that T 
administration may both reduce obesity and decrease inflammation and oxidative stress.  Recent 
work has shown that a low testosterone/cortisol ratio may be a risk factor for an increase in 
atherosclerotic cardiovascular events(119). Our data provides a potential mechanism for this 
increased risk through inflammation since atherosclerosis is a chronic inflammation of the arterial 
wall(120-124). Clearly, we need to investigate whether testosterone administration will not only 
reduce inflammation (CRP) but also retard the rate of atherogenesis. Elevated CRP concentrations 
are known to be associated with an increased risk of cardiovascular events(125; 126). There is also 
the evidence that CRP may induce the release of pro-inflammatory factors like plasminogen 
Activator Inhibitor-1 and intracellular adhesion molecule-1(ICAM-1) from endothelial cells in 
vitro, through the activation of FcII receptor on the endothelium(127-130). Infusion of CRP has 
recently been shown to induce an increase in pro-inflammatory mediators in humans, in vivo(131). 
The markedly elevated plasma CRP concentrations (mean: 6.13 mg/l) in the hypogonadal type 2 
DM thus would place them at a much higher risk for cardiovascular disease than the eugonadal 
group (mean CRP 3.2 mg/L). One previous study in non-diabetic subjects has shown that 
cardiovascular risk factors like higher BMI, systolic hypertension, high blood glucose and insulin 
concentrations occur more frequently with low testosterone concentrations(132).  
It is of interest that testosterone has been shown to be protective of endothelial function in 
experimental animals(133). Thus, bilateral orchidectomy in rats is associated with a reduction in 
NO generation by the aorta while administration of testosterone to orchidectomized animals leads 
to partial prevention of the loss of NO generation(134). Since NO exerts vasodilatory, platelet 
antiaggregatory, anti-inflammatory and anti-atherogenic actions, it is possible that testosterone is 
also anti-atherogenic in the male. Orchidectomy in rats result in insulin resistance which can be 
reversed by testosterone administration at physiological doses(135; 136). The loss of testosterone 
in human male is associated with a loss in muscle mass and an increase in plasma free fatty acid 

 
  
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concentration(15; 137; 138) (16; 17; 117). Free fatty acids are known to interfere with insulin 
signaling and to induce inflammation and oxidative stress in association with insulin 
resistance(139-141). Oral and transdermal testosterone administration to obese, hypogonadal 
patients leads to a fall in blood pressure(38). Conversely, androgen deprivation in patients with 
prostatic cancer leads to increased arterial stiffness(142),(143). A Japanese study has shown that 
low testosterone concentration predicts abdominal adiposity(144).    
    Inflammation is known to be a causative factor in inducing atherosclerosis(145; 146). Adherence 
of circulating monocytes and lymphocytes to the arterial endothelial lining is one of the earliest 
detectable events in human and experimental atherosclerosis. The initial entrapment of the 
monocyte to the endothelium is dependent upon the increased expression of the adhesion molecules, 
P-selectin, VCAM-1 and ICAM-1, on the endothelium (147).  This expression is mediated by the 
activation of NF B which modulates the transcription of pro-inflammatory cytokines, TNF , 
IL1 , IL-6; adhesion molecules ICAM-1, VCAM-1; chemokines like MCP-1; and enzymes 
generating ROS such as NADPH oxidase (78).  NF B is now considered cardinal in the 
pathogenesis of both acute inflammation and chronic inflammation like that seen in atherosclerosis 
(82).  
Since hypogonadism may be a pro-inflammatory state, it is worth mentioning that hypogonadism 
is associated with increased atherosclerosis(148). In this regard, it is interesting that testosterone 
has been shown to have anti-anginal properties in humans(149-151) Animal studies have shown 
that high physiological doses of androgens (testosterone or DHEA) decrease aortic atherosclerosis 
in cholesterol-fed castrated rabbits.(152) It is possible that treatment of hypogonadism can lead to 
decreased atherosclerosis in humans, but long-term studies looking at this effect have not been 
done. This study therefore presents a unique opportunity to study the effect of testosterone 
replacement on inflammatory and oxidative stress in type 2 diabetes, a known pro-inflammatory, 
pro-oxidative and pro-atherosclerotic state.    
  
Hypogonadism and Hematocrit  
We have recently demonstrated a significantly lower hematocrit in hypogonadal males and a direct 
relationship between the hematocrit and testosterone in type 2 diabetics (JCEM 2006 and 
preliminary data). This suggests that a low testosterone may contribute to the pathogenesis of the 
mild anemia in these patients. Testosterone is known to stimulate erythropoiesis in the bone marrow 
and to increase the hematocrit.(153)   
We also showed a significant inverse relationship between CRP concentrations and the hematocrit 
which suggests that inflammatory processes in type 2 diabetes probably regulate the hematocrit in 
a negative fashion. CRP has for long been considered a marker/indicator of systemic inflammation 
and therefore, it is also a prognosticator of cardiovascular events(125; 126; 154). However, there is 
now evidence that CRP may be a mediator of inflammation(127-129). It induces ICAM-1 and 
MCP-1 in endothelial cells, in vitro, and probably exerts this effect through the FcII receptor(130). 
More recently it has been shown to exert a pro-inflammatory effect following an injection into 
normal human subjects, in vivo(131). Thus the relationship of CRP with hematocrit may either be 
through the direct action of CRP or through other inflammatory mediators associated with CRP or 
both.  Inflammatory mechanisms may affect the hematocrit in two ways. Firstly, they may suppress 
erythropoietin secretion(155) and secondly, there may be increased apoptotic death of red cell 
precursors resulting in no increase in erythropoiesis in spite of elevated erythropoietin 
levels(155158). These mechanisms are relevant to the pathogenesis of anemia of chronic 
inflammatory disease. Thus, the mild anemia of type 2 diabetes may in part be attributable to 
processes similar to those involved in chronic inflammatory disease in addition to the contribution 
by low testosterone concentrations. The overwhelming predominance of normocytic normochromic 
picture in the anemic patients is also consistent with marrow suppression secondary to inflammatory 
mechanisms. Elevated erythropoietin levels associated with anemia, suggest an inadequate response 
to erythropoietin, a feature of anemia of chronic disease.The relationship of inflammatory 
mechanisms with hematocrit becomes even more intricate since these mechanisms may be involved 
in the pathogenesis of hypogonadotrophic hypogonadism itself in patients with type 2 diabetes as 

 
  
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discussed above. Thus, both a low testosterone and inflammatory mechanisms play an important 
role in the pathogenesis of the low grade anemia observed in patients with type 2 diabetes.  Type 2 
diabetes, HH and Estrogen  
The patients with HH tend to be obese, and, there is an inverse relationship between BMI and total 
and free testosterone concentrations(5). Since adipose tissue expresses the enzyme aromatase which 
converts testosterone to estradiol, it has been suggested that the state of HH in these patients may 
be due to an excessive aromatase dependent conversion of testosterone into estradiol(159162). 
Elevated concentrations of estradiol may in turn suppress hypothalamic GnRH and gonadotrophin 
secretion from the pituitary gland (163; 164). This would then explain the pathogenesis of HH. We, 
therefore, hypothesize that the plasma concentration of estradiol and aromatase expression in 
adipose tissue in patients with type 2 diabetes and HH is elevated when compared with those who 
have normal testosterone concentrations. We will also evaluate the aromatase expression after 
Androgel therapy.  
Type 2 diabetes, Alzheimer’s disease and Hypogonadism  
With a progressively aging population, the total load of Alzheimer’s disease (AD) and the 
characteristic dementia, the burden to society is likely to be phenomenally large. AD is a clinical 
diagnosis characterized by progressive dementia. A definitive diagnosis can only be made by 
histopathological examination of brain tissue. MRI findings suggestive of AD (amyloid plaques; 
reduced brain volumes in hippocampus, amygdala and entorhinal cortex) are detected when the 
disease is well established. There is a great need for biochemical tests that can detect AD early in 
its course. We have recently discovered that mononuclear cells (MNC) express amyloid precursor 
protein (APP). APP (a transmembrane protein) is sequentially cleaved by β- and γ-secretases in the 
neuronal cells to form amyloid-β (Aβ) peptide, the principal component of senile plaques.   
Type 2 diabetes and male hypogonadism are both known to independently increase the risk of AD 
markedly (165; 166). Since the pathogenesis of AD is dependent upon oxidative and inflammatory 
stress, patients with HH and DM would be expected to have a markedly enhanced risk of AD. 
Consistent with this, our preliminary data show that type 2 diabetic men with low free T have 
increased expression of APP in their peripheral blood MNC. Treatment of these patients with 
testosterone for 8 weeks suppresses APP. We now want to study the expression of APP and 
correlate it with the expression of inflammatory mediators in the adipose tissue  
  
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atherosclerosis in men with type 2 diabetes. Diabetes Care 26:1869-1873, 2003  
   
  
4.0 Study Design  
4.1 Describe and explain the study design (e.g. case-control, cross-sectional, 
ethnographic, experimental, interventional, longitudinal, observational).  

 
  
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Response:. The proposed study will prospectively investigate, in a randomized controlled 
manner, the changes in carotid intima-media thickness (IMT), brachial flow mediated dilatation 
(FMD%), inflammatory  mediators, and adiposity over a period of 24 months in hypogonadotrophic 
hypogonadal type 2 diabetics (HHDM, n=60) randomized equally to either placebo or Androgel 
1% (50mg daily). Fasting blood samples will be collected and carotid IMT, brachial FMD%, MRI 
measurements will be taken at baseline and at selected time points up to 24 months. 20 subjects 
with type 2 diabetes who are eugonodal will be recruited as controls for comparison of carotid IMT, 
brachial FMD%, inflammatory mediators and adiposity at baseline with the HHDM group. The 
study will be approved by the State University of New York at Buffalo School of Medicine 
Institutional Review Board.  
Study Design  
  
 
Response: 80  
5.2 If applicable, indicate how many subjects you expect to screen to reach your 
target sample (i.e. your screen failure rate).   
Response: 100-110. All screened and qualified patients will be enrolled and randomized 
up to 80 enrolled patients.  
5.3 Justify the feasibility of recruiting the proposed number of eligible subjects 
within the anticipated recruitment period. For example, how many potential 
subjects do you have access to? What percentage of those potential subjects 
do you need to recruit?  
Response: The Diabetes and Endocrinology Center of WNY is the largest Diabetes 
center in the WNY area, seeing between 100 to 120 type 2 diabetes patients every week. 
5%-10% of the patients will be males with Hypogonadism who might qualify for this 
study. Therefore, majority of recruited patients are our Kaleida Health and UBMD clinic 
patients. We will also recruit patients through advertisement, Buffalo Research Registry 
and researchmatch.org. These sources will suffice to recruit the needed number to 
subjects.   
  
  
  
  
  
  
  
  
  
  
  
  
  
  
5.0   Local Number of Subjects  
5.1   Indicate the total number of subjects that will be enrolled or records that will  
be reviewed locally.  
Male subjects  
N =80  
Eugonadal DM  
N=20  
HHDM  
N =60   
Baseline  
measurements  
Placebo  
N=30  
Androgel 1%  
N=30  
Baseline and  
follow up  
measurements  
at 6,12, 18  
and 24  
months.  

 
  
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6.0 Inclusion and Exclusion Criteria  
6.1 Describe the criteria that define who will be included in your final study 
sample.   
NOTE:  This may be done in bullet point fashion.  
Response:   
1- Type 2 diabetes  
2- males  
3- Age 30-60 years inclusive. The lower age limit was decided on the fact that in our 
study on hypogonadotrophic hypogonadism in type 2 diabetic patients, the 
youngest subject was 31 years old. The upper age limit has been restricted to 60 to 
avoid including subjects with significant age-related declines in testosterone 
concentrations. 4- PSA < 4 ng/ml 5- IPSS ≤ 19.  
6- Subjects on thiazolidinediones, statins, ACE inhibitors, angiotensin receptor blockers 
or antioxidants will be allowed as long as they are on stable doses (for last 3 months) 
of these compounds and the dosage in not changed during the study. Subjects on 
insulin, metformin or sulfonylureas can participate in the study, provided that minimal 
changes are made to the doses during the study.   
  
   
6.2 Describe the criteria that define who will be excluded from your final study 
sample.    
NOTE:  This may be done in bullet point fashion.   
Response:    
1)Coronary event or procedure (myocardial infarction, unstable angina, coronary artery 
bypass, surgery or coronary angioplasty) in the previous 6 months   
2)Hemoglobin A1c >10%   
3)h/o prostate carcinoma  
4)Hepatic disease (transaminase > 3 times normal) or cirrhosis   
5)Renal impairment (defined as GFR<30)   
6)HIV or Hepatitis C positive status  
7)Participation in any other concurrent clinical trial  
8)Any other life-threatening, non-cardiac disease   
9)Use of over the counter health supplements which contain androgens   
10)Use of an investigational agent or therapeutic regimen within 30 days of study.  
11) Hematocrit > 50%.  
12) PSA > 4 ng/ml  
 
6.3 Indicate specifically whether you will include any of the following special 
populations in your study using the checkboxes below.    
  
NOTE:  Members of special populations may not be targeted for enrollment in 
your study unless you indicate this in your inclusion criteria.  
Response:  None of the below populations will be enrolled  
☐  
Adults unable to consent  
  
  

 
  
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☐  
Individuals who are not yet adults (infants, children, teenagers)  
☐  
Pregnant women  
☐  
Prisoners  
6.4 Indicate whether you will include non-English speaking individuals in your 
study.  Provide justification if you will exclude non-English speaking 
individuals.   
In order to meet one of the primary ethical principles of equitable selection 
of subjects, non-English speaking individuals may not be routinely excluded 
from research as a matter of convenience.  
In cases where the research is of therapeutic intent or is designed to 
investigate areas that would necessarily require certain populations who may 
not speak English, the researcher is required to make efforts to recruit and 
include non-English speaking individuals.  However, there are studies in 
which it would be reasonable to limit subjects to those who speak English.  
Some examples include pilot studies, small unfunded studies with validated 
instruments not available in other languages, studies with numerous 
questionnaires, and some non-therapeutic studies which offer no direct 
benefit.  
Response: We have no non-English speaking patients in this population.  We 
have patients that English is a second language, but they are able to read, 
write and understand it.  This population is less than 10% of the total 
population.  
7.0 Vulnerable Populations  
If the research involves special populations that are considered vulnerable, 
describe the safeguards included to protect their rights and welfare.    
NOTE: You should refer to the appropriate checklists, referenced below, to ensure you 
have provided adequate detail regarding safeguards and protections. You do not, 
however, need to provide these checklists to the IRB.  
7.1 For research that involves pregnant women, safeguards include: NOTE 
CHECKLIST: Pregnant Women (HRP-412)  
Response: We will not be using subjects from vulnerable populations  
☒  
N/A:  This research does not involve pregnant women.  
7.2 For research that involves neonates of uncertain viability or non-viable 
neonates, safeguards include:  
NOTE CHECKLISTS: Non-Viable Neonates (HRP-413), or Neonates of Uncertain 
Viability (HRP-414)  
Response:   
☒  N/A:  This research does not involve non-viable neonates or neonates of 
uncertain viability.  
 
7.3  
For research that involves prisoners, safeguards include:  
NOTE CHECKLIST: Prisoners (HRP-415)  
Response:   

 
  
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☒  
N/A:  This research does not involve prisoners.  
7.4 For research that involves persons who have not attained the legal age for 
consent to treatments or procedures involved in the research 
(“children”), safeguards include:    
NOTE CHECKLIST: Children (HRP-416)  
Response:   
☒  
N/A:  This research does not involve persons who have not attained the 
legal age for consent to treatments or procedures (“children”).  
7.5 For research that involves cognitively impaired adults, safeguards include:  
NOTE CHECKLIST: Cognitively Impaired Adults (HRP-417)  
Response:   
☒  
N/A:  This research does not involve cognitively impaired adults.  
7.6 Consider if other specifically targeted populations such as students, employees 
of a specific firm, or educationally or economically disadvantaged persons 
are vulnerable.  Provide information regarding their safeguards and 
protections, including safeguards to eliminate coercion or undue 
influence.  
Response: No specific populations or vulnerable groups will be targeted. All 
subjects enrolled in this study will be of legal adult consenting age with the ability 
to speak, read and interrupt the English language. Patients will have the ability to 
speak with the research team regarding any questions or concern they have before 
signing the consent. Patients are made aware that this study is voluntary and they 
are able to stop participating at any time they feel uncomfortable. Patients are not 
be pressured into participating and their clinic standard of care will remain the 
same if they participate or choose not to participate.   
8.0 Eligibility Screening   
 
8.1  Describe screening procedures for determining subjects’ eligibility.   
Screening refers to determining if prospective participants meet inclusion and 
exclusion criteria.   
 Include all relevant screening documents with your submission (e.g. screening 
protocol, script, questionnaire).   
Response: :   
Prospective participants will be asked to read and understand the consent and any questions 
they may have regarding the protocol will be answered. If the subject wants to participate in 
the study, they will be asked to sign the informed consent form. Each participant will 
complete a physical exam including Digital Rectal Examination, Medical History and 
baseline labs to measure CBC, SMA, HbA1c, lipid profile, PSA, testosterone, SHBG,  
Estradiol, LH and FSH. A total of 80 male subjects will be recruited (60 type 2 diabetics with 
Hypogonadotrophic hypogonadism, 20 eugonadal type 2 diabetics). There will be two groups in the 
study: -    
Hypogonadal group: Sixty type 2 diabetic male subjects with hypogonadotrophic hypogonadism (as 
defined below) will be recruited into the study. Hypogonadotrophic hypogonadism (HH) will be 
defined as low calculated free testosterone (below 6.5ng/dL) with normal or low LH and FSH 
concentrations.  

 
  
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Eugonadal group: Twenty type 2 diabetic male patients with normal calculated free testosterone 
concentrations will also be recruited in the study to serve as an age-matched comparison group. 
They will not be treated with testosterone.  
☐  
N/A:  There is no screening as part of this protocol.  
9.0 Recruitment Methods  
☐  
N/A:  This is a records review only, and subjects will not be 
recruited.  NOTE:  If you select this option, please make sure that 
all records review procedures and inclusion/exclusion screening are 
adequately described in other sections.  
 
9.1  Describe when, where, and how potential subjects will be recruited.   
NOTE:  Recruitment refers to how you are identifying potential participants and 
introducing them to the study.  Include specific methods you will use (e.g. 
searching charts for specific ICD code numbers, Research Participant Groups, 
posted advertisements, etc.).  
Response: Participants will be identified by prescreening clinical charts, patient 
doctor interaction at the time of their visits, flyers advertisements and 
researchmatch.org. Diabetes Endocrinology Center of WNY Locations include:  
1. 1020 Youngs Road, Williamsville NY 14221  
2. 705 Maple Road, Williamsville NY 14221  
3. 1000 Youngs Road, Suite 105, Williamsville NY 14221  
4. 462 Grider Street, Buffalo NY 14215   
The study clinical team will evaluate their clinic patients for possible participation 
in this study according to the inclusion and exclusion criteria at the Diabetes and 
Endocrinology Center of WNY. Patients that may qualify for the study are 
referred to the research team for further eligibility evolution. Patients meeting the 
inclusion and exclusion criteria based on preliminary phone evaluation will be 
invited to participate in the study.  
9.2 Describe how you will protect the privacy interests of prospective subjects 
during the recruitment process.    
NOTE:  Privacy refers to an individual’s right to control access to him or herself.    
Response: Patient charts will be screened according to the study inclusion and 
exclusion criteria by our trained clinical staff and physicians. If the patient 
qualifies and is of consenting age, the physicians will speak to them about their 
interests in participating in research. If the patient agrees, their information will be 
given to the research coordinator to be contacted for further evaluation. All 
personal information will be kept confidential and locked in the coordinator 
office.   
9.3 Identify any materials that will be used to recruit subjects.   
NOTE:  Examples include scripts for telephone calls, in person announcements / 
presentations, email invitations.   
For advertisements, include the final copy of printed advertisements with your 
submission. When advertisements are taped for broadcast, attach the final  

 
  
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audio/video tape.  NOTE:  You may submit the wording of the advertisement prior 
to taping to ensure there will be no IRB-required revisions, provided the IRB also 
reviews and approves the final version.  
Response: In addition to screening clinical charts, participations will be identified 
through; flyer advertisement and researchmatch.org  
 
10.0  
Procedures Involved  
10.1 Provide a description of all research procedures or activities being 
performed and when they are performed once a subject is screened and 
determined to be eligible. Provide as much detail as possible.    
NOTE:  This should serve as a blueprint for your study and include enough detail 
so that another investigator could pick up your protocol and replicate the research.  
For studies that have multiple or complex visits or procedures, consider the 
addition of a schedule of events table in in your response.  
Response:   
Screening Day (week -1)  
Each subject will have completed the following procedures prior to participating in the study.  
1) Informed consent;   
2) Physical Exam including Digital Rectal examination.   
3) Medical History;   
4) Baseline lab draws to measure CBC, SMA, HbA1c, lipid profile, PSA, testosterone, SHBG, 
estradiol, LH and FSH.   
Study design for the hypogonadal group  
After the screening visit, subjects will be randomized to receive Androgel 1% (5grams) daily (starting 
at week 0) or placebo for 24 months.   
Randomization Method: Patients who qualify for the study will be assigned a number by a 
computerized randomization program (Random Allocation Software) and will be therefore 
randomized to either of the two study groups. Randomization will be done in blocks of 4 and 
there will thus be 15 blocks. The subjects will be blinded to the treatment.  
The subjects in the hypogonadal group will have following visits during the study:-  
1. Baseline visit 1:  Vitals and blood pressure, MRI will be done estimate total body and regional 
adiposity and lean body mass. Waist and Hip measurements will be taken to determine the waist 
to hip ratio. Subjects will be given a container for 24-hour urine collection for measurement of 
isoprostanes.   
2. Baseline visit 2: This visit will occur within 30 days of visit 1. The participants will have blood 
drawn for research lab (1.5 tablespoons (23ml)) and Vitals and blood pressure. Carotid IMT will 
be measured and brachial artery flow mediated dilatation will be done as per methodology 
described in the protocol. A fat biopsy will be obtained. Subjects will also receive instructions on 
standardized diet and exercise. Subjects will turn in their 24-hour urine collection container.  
3. Titration: Visit 3, 4, (week 4, week 10): A blood sample will be taken to measure calculated free 
testosterone concentration (1.5 tablespoons (23ml)) and Vitals and blood pressure. During the 
study, we will attempt to keep the free testosterone concentrations in the range of 14-17 ng/dL for 
subjects randomized to drug group. This range has been chosen because it falls in middle of the 
normal range for lean healthy young men. Dose response studies of testosterone replacement have 
shown that in the mid-normal range of testosterone, subjects have demonstrable beneficial effects 
on lean body mass (34). If the free testosterone concentration is less than 14ng/dL in subjects 
randomized to drug group, then the dose of Androgel 1% will be increased to 7.5 grams at week 6 
and if still lower than 14 ng/dl at week 10, the dose will be increased to Androgel 1% (10grams) 
at week 12. If the total testosterone concentration is more than 17ng/dL in subjects randomized to 
drug group, then the dose of Androgel 1% will be decreased by 2.5 mg starting from week 6.  On 

 
  
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visit 4, blood samples will be taken for PSA and subjects will be questioned for BPH symptoms to 
estimate the IPSS.   
4. Visit 5, 6, 7 (month 6, 12, 18): The participants will have to come fasting and blood will be drawn 
for research labs (1.5 tablespoons (23ml)) and Vitals and blood pressure. CBC, PSA, testosterone, 
SHBG and liver function test will be done at each visit and subjects will be questioned for BPH 
symptoms to estimate the IPSS. Carotid IMT will be measured and brachial artery flow mediated 
dilatation will be done as per methodology described in the protocol. On visit 6 (month 12), fat 
biopsy and MRI will be done. Subjects will also receive instructions on standardized diet and 
exercise at visit 5 and 6. At every visit, compliance with study drug will be assessed and a 180 day 
supply of study drug will be dispensed and we will adjust the dose 2 week after each visit as 
mentioned above. Subjects will give 24-hour urine at month 12. At each of these visits, dose of 
Androgel will be adjusted (as above) if needed.   
5. End of study Visit 8 (month 24): The participants will come fasting and blood will be collected for 
research labs(1.5 tablespoons (23ml)) and Vitals and blood pressure. The subjects will be 
questioned for BPH symptoms to estimate the IPSS. Carotid IMT will be measured and brachial 
artery flow mediated dilatation will be done as per methodology described in the protocol. A MRI 
will be done to estimate total body and regional adiposity and waist and hip measurements will be 
taken. Blood for CBC, SMA, HbA1c, PSA, testosterone, estradiol, SHBG, LH and FSH and lipid 
profile will also be drawn. Subjects will turn in their 24-hour urine collection container.  A fat 
biopsy will be obtained.  
  
Study design for Eugonadal Diabetic group   
1. Baseline visit 1(day 0): 24 hour urine will be given and MRI will be done to estimate total body and 
regional adiposity and lean body mass.   
1.2. Baseline visit 2 (day 1): The participants will have blood drawn for research labs(1.5 
tablespoons (23ml)), Waist and Hip measurements will be taken to determine the waist to hip 
ratio. Carotid IMT will be measured and brachial artery flow mediated dilatation will be done 
as per methodology described in the protocol. Fat Biopsy will be obtained. Subjects will turn 
in their 24-hour urine collection container. This visit will be the end of study visit for this 
group  
LABORATORY PROCEDURES   
2. Plasma concentrations of inflammatory mediators: Concentrations of TNF- , IL-6, ICAM1, 
MMP-9, PAI-1, MCP-1 are measured in plasma using sensitive and high sensitive ELISA 
kits (R&D Systems, MN).  CRP concentrations will be measured using an ELISA kit from 
Alpha Diagnostics International Inc. (San Antonio, TX). SAA will be measured in serum 
using an ELISA kit from Biosource (CA).   
3. Plasma glucose, insulin and free fatty acids measurements: Insulin levels are determined 
using an ELISA kit from Diagnostic Systems Laboratories Inc. (Webster, TX). Glucose 
levels are measured in plasma by YSI 2300 STAT Plus glucose analyzer (Yellow Springs, 
Ohio). Free fatty acid levels are measured by a colorimetric assay (Wako, Richmond, VA).  
4. Serum testosterone, SHBG, estradiol, LH and FSH, CBC, CMP, PSA and HbA1c 
measurements: All these tests will be done by Quest Laboratories. Total testosterone and 
estradiol will be done by liquid chromatography followed mass spectrometry. LH, FSH, 
SHBG, CBC, CMP, PSA and hbA1c will be done well established clinical assays. Free 
testosterone will be calculated from SHBG and T using the method of Vermeulen et al (8).   
5. MRI: Multislice MRI of abdomen will be obtained (14). Abdominal subcutaneous adipose 
tissue, visceral adipose tissue (adipose tissue in the intraabdominal cavity delineated at the 
innermost aspect of the abdominal and oblique muscle walls surrounding the cavity and the 
anterior aspect of the vertebral body) and hepatic fat will be analyzed in a blinded fashion 
using customized software (167).MRI measurements will be expressed as volume in cm3.  
6. Carotid Intima-media thickness (IMT): Carotid IMT is used to assess subclinical and clinical 
atherosclerosis. The carotid arteries are most suitable for IMT measurements given their 
superficial location, size and limited movement (168). IMT is measured in both the right and 

 
  
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left common carotid arteries and a 7.5 MHz linear array transducer is used for the 
purpose(169). All vascular images will be acquired using a Hewlett Packard (HP) Image 
Point Hx Multispecialty Ultrasound System. Following imaging by B-mode Duplex 
scanning, IMT measurement is defined by two parallel echogenic lines, corresponding to the 
interfaces between the lumen-intima and the media-adventitia. While imaging is done of both 
the near and far walls, these interfaces are best defined in the far wall of the carotid 
arteries(170). The IMT thus is the combined thickness of both the intima and the media.   
Subjects are positioned with a wedge of approximately 35 degrees to reduce respiratory variation and 
motion of the jugulars. Initially, the transducer is swept in cross-section to note the position and 
orientation of the carotid artery bifurcation. Following this, the transducer is applied in the 
longitudinal plane and images acquired at two angles, 90- and 45-degrees. The 90-degree imaging 
plane is a frontal plane of the head of the common carotid artery. In each plane, the transducer is 
manipulated till the best image of the far wall of the distal 1 cm of the common carotid is obtained 
(171). Images will be digitized using a frame-grabber card and a PC and specialized software 
(IntiMate, Pixen Technologies, India) will be used for edge detection and subsequent IMT 
measurement. This technique is being applied to the assessment of atherosclerosis in several of 
our studies in obesity and diabetes.  
7. Brachial flow-mediated dilatation (FMD): FMD is the capacity of blood vessels to respond 
to an increase in flow, or more precisely shear stress, by dilating. Endothelium-derived nitric 
oxide (NO) is a principal mediator of FMD. A 7.5 – 10 MHz broadband multi-frequency 
linear array transducer will be used to acquire images. The subject is positioned supine with 
the arm in a comfortable position for imaging the brachial artery. To create a flow stimulus, 
a blood pressure cuff is placed on the forearm. After acquiring a baseline rest image, arterial 
occlusion is created by cuff inflation to suprasystolic pressure. To occlude arterial inflow for 
a standardized length of time, the cuff is inflated to at least 50 mm Hg above systolic pressure. 
The resultant ischemia triggers dilatation of downstream resistance vessels via autoregulatory 
mechanisms. Following 5 minutes of occlusion, the cuff is deflated. This results in reactive 
hyperemia (a brief high-flow state) in the brachial artery to accommodate the dilated 
resistance vessels. The shear stress that results causes dilatation of the brachial artery. The 
recording of the arterial image is done continuously from 30 seconds before to 2 minutes 
following cuff deflation (172). This procedure has been utilized in several studies carried out 
in our unit, including the first studies describing abnormalities in African Americans(173), 
brachial reactivity improvements related to reversal of oxidative stress in the obese and type 
2 diabetics following troglitazone (174), rosiglitazone (submitted for publication) and 
pioglitazone therapy; and gender differences in brachial reactivity and the effect of hormone 
replacement therapy (HRT) in post-menopausal women (175).  
8. Fat aspiration procedure: Adipocytes will be harvested from the subjects by methods 
previously described in literature (176; 177). Subcutaneous fat tissue aspiration will be 
performed on abdomen at a 10 cm distance from umbilicus. The subjects will not have taken 
aspirin or NSAIDS in the last 72 hours. If that is the case, then the procedure will not be 
done. The skin will first be prepared with povidone-iodine (Betadine) and alcohol. A sterile 
drape will be placed around the appropriate area. 3 cc of 1% lidocaine will then be 
administered subcutaneously. After adequate anesthesia has been achieved, approximately 
20-50cc of 0.5% lidocaine will be injected in the adipose tissue. Dose of lidocaine will not 
exceed 4.5mg/kg body weight. Aspiration of fat tissue will then be performed with a 3-holed 
cannula (Tulip Instrumentation, length: 15cm, diameter: 2.1mm) fixed to a 10mL syringe. 
More than one attempt at aspiration can be done at the same site during the procedure to get 
adequate sample. After getting adequate fat tissue (500mg-3g), the puncture site will be 
pressed for at least ten minutes before the patient rises up from supine position (to minimize 
bruising). The study subjects will then be discharged home. The adipose tissue will be 
centrifuged to remove blood and fluid contaminants. The upper adipose tissue will be 
collected into a separate sterile tube, washed twice with cold sterile Phosphate Buffered 
Saline (PBS) and centrifuged to remove the PBS. The adipose tissue sample will be weighed 

 
  
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and approximately 500 mg transferred to a separate tube for analysis. Total RNA, nuclear 
extracts and total cell lysates will be prepared from the adipose tissue.  
9. During the study visits subjects would be screened for side effects .Blood test results would 
be reviewed by physician and appropriate adjustments would be made as per protocol.   
  
10.2 Describe what data will be collected.    
NOTE:  For studies with multiple data collection points or long-term follow up, 
consider the addition of a schedule or table in your response.  
Response:. Study 1: Type 2 diabetes and hypogonadism(5)  
Table 1   
  
Hypogonadal 
Eugonadal 
 n  
34 
69 
Age(years)  
57.2±2.4 
53.5±1.5 
BMI(kg/m2)  
35.7±1.7 
31.7±1.0 
T (nmol/L)  
8.07±0.65 
14.58±0.62# 
FT(nmol/L)  
0.146±0.011 
0.306±0.015# 
cFT(nmol/L)  
0.172±0.007 
0.326±0.013# 
LH (MIU/mL)  
3.15±0.26 
3.91±0.24* 
FSH (MIU/mL)  
4.25±0.45 
5.53±0.40* 
PRL (MIU/mL)  
6.69±0.58 
6.69±0.46 
SHBG (nmol/L)  
28.87±2.79 
27.31±1.96 
HbA1c%  
8.5±0.3 
8.42±0.3 
Duration of diabetes(years)  
9.03±1.31 
7.12±0.97 
# 
P<0.001 versus Hypogonadal group *P<0.05 versus Hypogonadal group.   
To convert testosterone from SI units (nmol/L) into conventional units (ng/dL) multiply by 28.8. We 
systematically investigated 103 consecutive male patients with type 2 diabetes mellitus who had 
been referred to our center (Diabetes Endocrinology Center of Western New York) by measuring 
total testosterone, free testosterone, SHBG, LH, FSH and prolactin (PRL) in order to determine the 
prevalence of hypogonadism (as defined by a low free testosterone) in type 2 diabetes and to 
differentiate whether the nature of hypogonadism was hypogonadotrophic or hypergonadotrophic 
(5) (please see attached manuscript). All patients had either free testosterone (FT) measured by 
Equilibrium Dialysis (ED) or calculated FT (free testosterone calculated using SHBG and T). 
Hypogonadism was defined as low FT or cFT. Out of a total of 103 patients who had either FT or 
cFT measured, 34 patients (33%) were hypogonadal. Table 1 depicts the clinical and biochemical 
features of patients with normal or low free (FT or cFT) testosterone. As shown above, LH 
concentrations and FSH concentrations were significantly lower in the hypogonadal group. LH 
levels in our study correlated significantly and positively with FT concentrations (r=0.287, P<0.05). 
FT correlated inversely with weight (r = -0.413, P<0.01) and BMI (r = -0.382, P<0.01).  
Study 2: Comparative study of hypogonadism in type 1 and type 2 diabetes(32)  
We examined the testosterone, sex hormone binding globulin (SHBG) and gonadotrophin 
concentrations in patients with type 1 diabetes and compared with those in age matched type 2 
diabetes. Type 1 diabetics had normal total and free testosterone (measured by equilibrium dialysis 
or calculated using total testosterone and SHBG) concentrations. Both were significantly higher 
than those in type 2 diabetes (Table 2). None of the type 1 diabetics had subnormal total testosterone 
concentrations while 48% of type 2 diabetic patients had low total testosterone concentrations. 6% 
of type 1 and 26% of type 2 diabetic patients (P<0.01 vs. type 1) had hypogonadism (low free 

 
  
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testosterone concentrations). SHBG concentrations were at the upper end of the normal range and 
were significantly higher in type 1 diabetics (49.32±2.83 nmol/L) when compared with those in 
type 2 diabetes (20.44±1.68 nmol/L, P<0.001 vs. type 1). Serum LH and prolactin concentrations 
were in the normal range in type 1 diabetes but were significantly higher than those in type 2 
diabetics with hypogonadism. Thus, type 1 diabetics have a normal function of 
hypothalamicohypophysio-gonadal axis but with elevated SHBG concentrations. In contrast, type 
2 diabetics have hypogonadotrophic hypogonadism. The normal but significantly lower prolactin 
concentrations in type 2 diabetics may point to a further hypothalamico-hypophyseal defect in the 
type 2 diabetic patients. We conclude that the pathogenesis of frequent hypogonadotrophic 
hypogonadism in type 2 diabetes is not related to diabetes or hyperglycemia per se but may be 
related to a combination of insulin resistance and hyperglycemia. Table 2  
  
Type 1 
Diabetes 
Type 2 
Diabetes 
P (vs type 1 
diabetes) 
Type 2 diabetes 
with HH  
 P (vs type 1 
Diabetes) 
subjects(n)  
50 
50 
  
12  
  
Hypogonadal subjects (%)  
3 (6%) 
13 (26%) 
  
12 (100%)  
  
Age (years)  
42.78±1.4 
43.74±0.8 
0.261 
42.85±1.4  
0.982 
BMI (kg/m2)  
26.09±0.75 
34.91±1.26 
<0.001 
37.53±3.7  
<0.001 
Total T (nmol/L)  
22.97±0.99 
11.20±0.60 
<0.001 
6.76±0.65  
<0.001 
Free Testosterone(nmol/L)  0.382±0.025 0.262±0.022 
0.001 
0.144±0.021  
<0.001 
cFT (nmol/L)  
0.398±0.019 0.278±0.0.017 
<0.001 
0.171±0.010  
<0.001 
Bioavailable T (nmol/L)  
9.28±0.44 
6.46±0.43 
<0.001 
4.08±0.19  
<0.001 
LH (IU/L)  
4.12±0.28 
3.94±0.33 
0.39 
2.79±0.37  
0.04 
FSH(IU/L)  
4.46±0.51 
5.57±0.61 
0.121 
3.84±0.52  
0.79 
Prolactin( g/L)  
11.21±2.1 
6.20±0.54 
<0.001 
5.78±1.13  
0.038 
SHBG (nmol/L)  
49.32±2.83 
20.44±1.68 
<0.001 
20.43±3.54  
<0.001 
HbA1c%  
7.57±0.20 
8.40±0.25 
0.024 
8.95±0.52  
0.015 
  
Study 3: Type 2 diabetes, hypogonadism and inflammation:  
We examined the relationship of hsCRP with total testosterone (TT) in 50 subjects with type 2 
diabetes mellitus. Hypogonadism was defined as TT < 300 ng/dl. Hs CRP was significantly higher 
in the hypogonadal (4.6 ±0.8 mg/l) compared to the eugonadal group (2.4 ±0.5 mg/l). There was 
also an inverse relationship between TT and hsCRP which was significant after adjusting for BMI 
on multivariate analysis (Figure 2 and 3). These preliminary findings suggest that the hypogonadal 
type 2 diabetics may have greater inflammatory stress than the eugonadal type 2 diabetic 
(Hypothesis 2).  
  

 
  
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Figure 3: HsCRP (mg/L) in type 2 diabetes  
Figure 2: Correlations: HsCRP (mg/l), TT.  
 
  Pearson correlation of HsCRP (mg/l) and TT = - 
(hypogonadal TT < 300ng/dl vs eugonadal  
 
  0.402. P-Value = 0.005  
TT > 300ng/dl. * p=0.003 by students t-test  
  
Study 4: Relationship of low testosterone and high CRP to hematocrit in type 2 Diabetes Mellitus.  
Background: We explored the biological relevance of HH in another direction by evaluating its 
effect on hemoglobin/hematocrit since testosterone increases hemoglobin concentrations and the 
hematocrit.  
Methods: In 70 consecutive male patients with type 2 diabetes mellitus (DM), fasting blood 
samples were collected to measure Hemoglobin, Hematocrit, serum Total Testosterone (TT), Sex 
Hormone Binding Globulin (SHBG), LH, FSH, Prolactin, glucose, glycosylated hemoglobin and 
hs C-Reactive protein (CRP). Calculated free Testosterone (cFT) was calculated by the method of 
Vermuelen et al [described in the grant application]. Hypogonadotophic Hypogonadism (HH) was 
defined as cFT < 6.5 ng/dl, with normal LH and FSH. Anemia was defined as hemoglobin < 13g/dl 
or hematocrit (Hct) < 39% as per WHO definition.   
Results:  cFT concentrations were shown to be significantly related to hematocrit (r = 0.46; p<  
0.0001) [Figure 1] and HH patients with type 2 DM had significantly lower hematocrit (40.6 ± 1.1% 
vs. 43.3 ± 0.72%; p =0.01) than those with normal cFT (figure 2). Hematocrit was also related 
inversely to CRP (r = -0.41; p < 0.0004) [fig 3]. Anemia was found in 37. 8% of men with CRP > 
3mg/L, compared to 15.8 % of men with CRP < 3mg/L (fig 4), consistent with the suppressive 
effect of chronic inflammation on hematocrit. On multivariate analysis, the effect of testosterone 
and CRP on hematocrit was independent of each other.  
 
  
  
HsCRP (mg/l) 
14 
12 
10 
8 
6 
4 
2 
0 
1200 
1000 
800 
600 
400 
200 
0 
Scatterplot of TT vs HsCRP (mg/l) 
TT<300ng/dl 
TT>300ng/dl 
0 
2 
4 
6 
8 
*  

 
  
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Figure 1: Correlation of Hct with cFT                             Figure 2: Hct in subjects with and 
without HH  
  
 
Figure 3: Correlation of Hct with CRP                     Figure 4: Anemia in subjects with 
CRP < or > 3mg/L                       
  
Study 5  
We therefore hypothesized that the expression of amyloid precursor protein (APP) is increased in 
patients with type 2 diabetes and hypogonadism. We used the peripheral blood mononuclear cell 
(MNC) for the expression of APP since we have shown in our previous work that it functions as an 
excellent representative cell for the assessment of oxidative stress and inflammation. Our 
preliminary data show that the expression of APP in MNC is significantly increased in diabetic 
hypogonadal men when compared with either eugonadal diabetic subjects (Figure 1). This increase 
is consistent with the potential dual increase in the risk of AD as suggested by the concomitant 
presence of diabetes and hypogonadism. Although the increased expression of APP in MNC does 
not necessarily represent an increase in the expression of this protein in the brain, it is an indication 
that the marked pro-inflammatory state of hypogonadism with diabetes is conducive to an increase 
in APP expression. The significance of the increase in the expression of APP in hypogonadal 
diabetic men is amplified further by our data showing that T treatment (5g Androgel/day) 
suppresses the expression of APP (Figure 2). The suppression was observed within 4 weeks of the 
initiation of T therapy and persisted for the 8 weeks of treatment with T. Clearly, thus, the lack of 
T in the male results in the propensity to accumulate APP while the replenishment of this hormone 
reverses this process.   
  
 
  
0.00 
0.05 
0.10 
0.15 
0.20 
0.25 
0.30 
APP 
* 
Time (Weeks) 
0 
4 
8 
16 
50 
60 
70 
80 
90 
100 
110 
120 
130 
Androgel (5g/day) 
Placebo 
* # 
* 
Figure 1: Basal APP mRNA expression in  Figure 2:  APP mRNA expression in MNC in hypogonadal  

 
  
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MNC of euogonadal and hypogonadal DM  DM patients following 8 weeks of testosterone therapy patients 
(n=23 vs. 33) *  P<0.05 by t-test. (androgel 5g/day) vs. placebo (n=13 vs. 13) * P<0.05 by One  
 
  
Way Repeated Measures ANOVA for comparison versus the # 
baseline,  P<0.05 by Two Way Repeated Measures ANOVA  
 
  
for comparison versus control group.  
.  
10.3 List any instruments or measurement tools used to collect data (e.g.  
questionnaire, interview guide, validated instrument, data collection 
form).    
  
Include copies of these documents with your submission.  
Response: Source documents will be used to collect patient information. Patients 
,, BP, weight are monitored every visit to ensure safety. The subjects will be 
questioned for BPH symptoms to estimate the IPSS. .  
10.4 Describe any source records that will be used to collect data about subjects 
(e.g. school records, electronic medical records).  
Response: electronic medical records and research files.  
10.5 Indicate whether or not individual subject results, such as results of 
investigational diagnostic tests, genetic tests, or incidental findings will be 
shared with subjects or others (e.g., the subject’s primary care physician) 
and if so, describe how these will be shared.  
Response: Individual participant lab results will be disclosed to the participant 
upon their request. If the participant requests documentation be shared with 
another physician, physician office or hospital the participant must come to the 
research center to collect said documentation or the documentation can be mailed 
to their given home address.  
10.6 Indicate whether or not study results will be shared with subjects or others, 
and if so, describe how these will be shared.  
Response: Not Applicable. Study results will not be shared with the subjects. 
However, unidentifiable study results could be published in the form of a 
manuscript or abstract and will be reported to Abbvie and to clinicaltrilas.gov.  
 
11.0  
Study Timelines  
11.1 Describe the anticipated duration needed to enroll all study subjects.  
Response: 36 months  
11.2 Describe the duration of an individual subject’s participation in the study. 
Include length of study visits, and overall study follow-up time.  
Response: After the screening visit, subjects who meet the inclusion and exclusion 
criteria will be expected to participate from 1-24 months depending on which part 
of the study the participant is randomized into..  
11.3 Describe the estimated duration for the investigators to complete this study 
(i.e. all data is collected and all analyses have been completed).  
Response: 60 months  

 
  
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12.0  
Setting  
12.1 Describe all facilities/sites where you will be conducting research 
procedures.  Include a description of the security and privacy of the facilities 
(e.g. locked facility, limited access, privacy barriers).  Facility, department, 
and type of room are relevant.  Do not abbreviate facility names.    
NOTE:  Examples of acceptable response may be:  “A classroom setting in the 
Department of Psychology equipped with a computer with relevant survey 
administration software,” “The angiogram suite at Buffalo General Medical Center, 
a fully accredited tertiary care institution within New York State with badge 
access,” or, “Community Center meeting hall.”  
Response: Research will be conducted at the Diabetes Endocrinology Research  
Center of WNY, located at 1000 Youngs Road, Suite 105, Williamsville NY  
14221 and at the CTRC located in 875 Ellicott St. Buffalo NY14203. The Diabetes 
Research Center has facilities and exam rooms available for insulin pump 
download, CGM device download, meal and infusion studies and presence of 
study coordinator and registered nurse for data collection and blood work at all 
times. One of the investigators will be available at all times to address patients’ 
related issues. CTRC location is a fully equipped laboratory with equipment 
include ultra-low freezers for sample storage, centrifuges, microscopes for sample 
preparation, infusion pumps, ELISA, PCR and immunoblotting instrumentation.   
12.2 For research conducted outside of UB and its affiliates, describe:  
• 
Site-specific regulations or customs affecting the research  
• 
Local scientific and ethical review structure  
NOTE:  This question is referring to UB affiliated research taking place outside 
UB, i.e. research conducted in the community, school-based research, international 
research, etc.  It is not referring to multi-site research.  UB affiliated institutions 
include Kaleida Health, ECMC, and Roswell Park Cancer Institute.    
Response:   
☒  
N/A:  This study is not conducted outside of UB or its affiliates.  
 
13.0  
Community-Based Participatory Research  
13.1 Describe involvement of the community in the design and conduct of the 
research.   
NOTE:  Community-Based Participatory Research (CBPR) is a collaborative 
approach to research that equitably involves all partners in the research process and 
recognizes the unique strengths that each brings.  CBPR begins with a research 
topic of importance to the community, has the aim of combining knowledge with 
action and achieving social change to improve health outcomes and eliminate 
health disparities.  
Response:   
☒  
N/A:  This study does not utilize CBPR.  
13.2  
Describe the composition and involvement of a community advisory 
board.  
Response:   

 
  
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☒  
N/A:  This study does not have a community advisory board.  
 
14.0  
Resources and Qualifications  
14.1 Describe the qualifications (e.g., education, training, experience, expertise, or 
certifications) of the Principal Investigator and staff to perform the research.  
When applicable describe their knowledge of the local study sites, culture, 
and society.  Provide enough information to convince the IRB that you have 
qualified staff for the proposed research.   
NOTE:  If you specify a person by name, a change to that person will require prior 
approval by the IRB.  If you specify a person by role (e.g., coordinator, research 
assistant, co-investigator, or pharmacist), a change to that person will not usually 
require prior approval by the IRB, provided that the person meets the qualifications 
described to fulfill their roles.  
Response: All study personnel are educated, trained, and licensed as required for 
their delegated role in this study. All study personnel have also received the 
required university training and will be trained by the PI before the study starts.  
Describe other resources available to conduct the research.   
14.2 Describe the time and effort that the Principal Investigator and research staff 
will devote to conducting and completing the research.  
NOTE:  Examples include the percentage of Full Time Equivalents (FTE), hours 
per week.  The question will elicit whether there are appropriate resources to 
conduct the research.  
Response: The principal investigator supervises the research project and weekly research 
meetings are conducted to discuss the recruitment rate, resolve and discuss issues related 
to the conduct, safety, analysis of the study and related publications. PI is expected to 
spend 5% of his academic time on this research. The co-investigators and study 
coordinator provide coverage to the research related activity for 365 days a year.  
14.3 Describe the availability of medical or psychological resources that subjects 
might need as a result of anticipated consequences of the human research, if 
applicable.  
NOTE:  One example includes: on-call availability of a counselor or psychologist for 
a study that screens subjects for depression.  
Response: Available medical literature will be provided as deemed appropriate or 
requested by patient through UB libraries, Pubmed, Google scholar as all the 
investigators have access to medical literature through listed resources above  
The patient will also have access to physician (Investigators and Co-Investigators) who 
will be available to address any adverse effects or other questions during the course of the 
study who will be available to address any adverse effects or other questions during the 
course of the study  
14.4 Describe your process to ensure that all persons assisting with the research 
are adequately informed about the protocol, the research procedures, and 
their duties and functions.  
Response: Education through training, meetings, conferences and discussions.  

 
  
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15.0  
Other Approvals  
15.1 Describe any approvals that will be obtained prior to commencing the 
research (e.g., school, external site, funding agency, laboratory, radiation 
safety, or biosafety).  
Response:   
☒  
N/A:  This study does not require any other approvals.  
 
16.0  
Provisions to Protect the Privacy Interests of Subjects  
  
16.1 Describe how you will protect subjects’ privacy interests during the course of 
this research.  
NOTE:  Privacy refers to an individual’s right to control access to him or herself.  
Privacy applies to the person.  Confidentiality refers to how data collected about 
individuals for the research will be protected by the researcher from release.  
Confidentiality applies to the data.    
Examples of appropriate responses include:  “participant only meets with a study 
coordinator in a classroom setting where no one can overhear”, or “the participant 
is reminded that they are free to refuse to answer any questions that they do not feel 
comfortable answering.”    
Response: Our clinical providers involved in the study will identify potential patients for 
recruitment from the Diabetes-Endocrinology Center of WNY Clinics according to the 
inclusions and exclusion criteria and through advertisements. Patient who qualify will be 
asked in private during their one on one consultation time with the physician if they wish 
to participate in the research study. If the patient agrees, the research coordinator will 
contact them for a telephone screening privately. The patients who call for potential 
participation in the study due to advertisement flyers will be screened over the phone 
with the research coordinator, using our telephone screening form.   
When the patient is being seen at our clinics for the first time they sign the “Consent to 
use and disclosure of protected health information” form which clearly states that their 
protected health information (PHI) can be used for review in preparation for possible 
research.  
If the patient passes the telephone screening, they will be asked to make an appointment 
to review and sign the consent. Patient will do this in a private, screen off area of the 
research department and will be allowed to discuss the consent in detail with the research 
coordinator and or study doctor. Patient will be no notified that it is completely voluntary 
to participate in the research study and can withdraw at any time.  
We will not be accessing any medical information of the patients for whom the services 
are not provided by our clinic providers.  
16.2 Indicate how the research team is permitted to access any sources of 
information about the subjects.    
NOTE:  Examples of appropriate responses include:  school permission for review 
of records, consent of the subject, HIPAA waiver.  This question does apply to 
records reviews.  
Response: Consent of the subject and partial HIPAA waiver.  

 
  
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17.0  
Data Management and Analysis  
17.1 Describe the data analysis plan, including any statistical procedures.  This 
section applies to both quantitative and qualitative analysis.   
Response: The focus of the proposed research is to evaluate the effect of hypogonadotrophic 
hypogonadism on atherogenesis, endothelial function, inflammatory mediators and oxidative 
stress in type 2 diabetic patients and the effect of testosterone replacement on these parameters. 
The similarities between the study groups, baseline values for subject’s demographics, carotid 
IMT, FMD%, body composition, hematocrit and inflammatory markers will be compared using 
appropriate parametric tests. Transformations of the data in order to meet statistical assumptions 
may be considered. The PRIMARY ENDPOINT of the study is to detect a difference in 
atherogenesis as measured by carotid IMT between type 2 diabetic subjects with and without HH 
after 24 months of testosterone replacement with Androgel. There are no prior studies looking at 
these two populations in humans in vivo, however studies assessing the effect of 
thiazolidinediones (insulin sensitizers with anti-inflammatory properties) on carotid IMT in type 
2 diabetics have shown a reduction in IMT by 0.033mm at 12 weeks and 0.054mm at 24 weeks 
(178).  In a study of 154 diabetic patients, serum free testosterone (F-tes) concentrations were 
found to be inversely correlated with mean IMT. Patients with low concentrations of FT (<10 
pg/ml) had greater mean IMT (1.01 ± 0.29 mm) than those with high concentrations of FT(0.91  
± 0.26 mm) (179). On the basis of these previous observations, we have conservatively estimated 
a difference in carotid IMT of 0.020mm at 24 months between the type 2 DM with HH and the 
type 2 DM with HH who is on androgel replacement. A sample size of 30 patients per group 
(assuming a drop-out rate of 5%) will provide adequate power (  = 0.8) to detect a significant 
difference of 0.020mm in carotid IMT (  = 0.05), provided the standard deviation of the residuals 
is not equal to or greater than the mean difference. The results will be computed as mean ± SD. 
Comparisons for endpoints will be made using repeated measures ANOVA, with Tukey’s test 
used for pair wise comparisons. Thus there will be 30 HH type 2 diabetic subjects each in placebo 
and treatment group of diabetic subjects with HH (total 60 subjects with HH). 20 eugonadal type 
2 diabetic subjects will be recruited to serve as controls. SECONDARY END POINTS: The 
secondary endpoints for the study will be comparison of the relative change from baseline in 
FMD%, inflammatory mediators, abdominal fat, waist and hip ratio, after either testosterone or 
placebo. Comparison of these parameters at baseline will also be done. Comparison for each 
endpoint will be made using ANOVA, with Tukey’s test used for pairwise comparisons  
17.2 If applicable, provide a power analysis.    
NOTE:  This may not apply to certain types of studies, including chart/records 
reviews, survey studies, or observational studies.  This question is asked to elicit 
whether the investigator has an adequate sample size to achieve the study 
objectives and justify a conclusion.   
Response: There are no prior studies looking at these two populations in humans in vivo, 
however studies assessing the effect of thiazolidinediones (insulin sensitizers with 
antiinflammatory properties) on carotid IMT in type 2 diabetics have shown a reduction in 
IMT by 0.033mm at 12 weeks and 0.054mm at 24 weeks (178). In a study of 154 diabetic 
patients, serum free testosterone (F-tes) concentrations were found to be inversely correlated 
with mean IMT. Patients with low concentrations of FT (<10 pg/ml) had greater mean IMT 
(1.01 ± 0.29 mm) than those with high concentrations of FT(0.91 ± 0.26 mm) (179). On the 
basis of these previous observations, we have conservatively estimated a difference in carotid 
IMT of 0.020mm at 24 months between the type 2 DM with HH and the type 2 DM with HH 
who is on androgel replacement. A sample size of 30 patients per group (assuming a drop-out 
rate of 5%) will provide adequate power (  = 0.8) to detect a significant difference of 
0.020mm in carotid IMT (  = 0.05), provided the standard deviation of the residuals is not 
equal to or greater than the mean difference.  

 
  
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17.3 Describe any procedures that will be used for quality control of collected 
data.  
Response: Three investigators and research nurse will double check the accuracy 
of collected data. All laboratory testing will be standardized using references and 
standards.  
 
18.0  
Confidentiality  
  
A. Confidentiality of Study Data 
  
Describe the local procedures for maintenance of confidentiality of study data 
and any records that will be reviewed for data collection.    
  
18.1  
A.  Where and how will all data and records be stored?  Include 
information about:  password protection, encryption, physical controls, 
authorization of access, and separation of identifiers and data, as applicable.  
Include physical (e.g. paper) and electronic files.  
Response: All data records will be stored on password protected computers and or 
in locked cabinets within the research department. These files will only be 
accessible by authorized study personnel.  
 
18.2  A.  How long will the data be stored?  
Response: Data and specimens storage has no expiration date and will be stored for a 
minimum of 7 years. The researchers may continue to rely on this for future use in 
research study  
 
18.3  A.  Who will have access to the data?  
Response: Those physicians, nurses, and laboratory staff that are on all 
documentation for the study will have access to the data and specimens  
 
18.4  A.  Who is responsible for receipt or transmission of the data?  
Response: Those physicians, nurses, and laboratory staff that are on all 
documentation for the study will have access to the data and specimens and can 
handle transfer of data and samples  
 
18.5  A.  How will the data be transported?  
Response: All data are stored at one location and is not transported unless it is 
being archived. At that point files will be transferred to Iron Mountain for storage 
and archiving. Samples that are transported will be done so using dry ice in a 
properly labeled Styrofoam container by the laboratory technician.  
B. Confidentiality of Study Specimens  
  
Describe the local procedures for maintenance of confidentiality of study 
specimens.    
  
 
☐  
N/A:  No specimens will be collected or analyzed in this research.    
(Skip to Section 19.0)  

 
  
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18.6  
B.  Where and how will all specimens be stored?  Include information 
about:  physical controls, authorization of access, and labeling of specimens, as 
applicable.    
Response: The data and specimens will be stored in the laboratory located at 1000 
Youngs Road, Suite 105, Williamsville NY 14221 and at the CTRC located in 875 
Ellicott St. Buffalo NY14203. Samples will be stored in a locked -80o C freezer. Data 
will be stored on computers that are password protected.  
 
18.7  B.  How long will the specimens be stored?  
Response: Data and specimens storage has no expiration date and will be stored for a 
minimum of 7 years. The researchers may continue to rely on this for future use in 
research study  
 
18.8  B.  Who will have access to the specimens?  
Response: Those physicians, nurses, and laboratory staff that are on all 
documentation for the study will have access to the data and specimens.  
 
18.9  B.  Who is responsible for receipt or transmission of the specimens?  
Response: Those physicians, nurses, and laboratory staff that are on all 
documentation for the study will have access to the data and specimens and can 
handle transfer of data and samples  
18.10 B.  How will the specimens be transported?  
Response: All data are stored at one location and is not transported unless it is 
being archived. At that point files will be transferred to Iron Mountain for storage 
and archiving. Samples that are transported will be done so using dry ice in a 
properly labeled Styrofoam container by the laboratory technician  
  
19.0  Provisions to Monitor the Data to Ensure the Safety of Subjects  
☐  N/A:  This study is not enrolling subjects, or is limited to records review 
procedures only.  This section does not apply.  
  
NOTE:  Minimal risk studies may be required to monitor subject safety if the 
research procedures include procedures that present unique risks to subjects that 
require monitoring.  Some examples include:  exercising to exertion, or instruments 
that elicit suicidality or substance abuse behavior.  In such cases, N/A is not an 
acceptable response.  
  
19.1 Describe the plan to periodically evaluate the data collected regarding both 
harms and benefits to determine whether subjects remain safe.  
Response: The principal investigator Paresh Dandona, MD, PhD and 
coinvestigators Husam Ghanim, PhD and Manav Batra, MD will review the data 
every 3 months to assess the safety of the participants. Furthermore they will also 
assess other risks including the physical, psychological, social, legal and 
economic harm to these patients. The investigators listed above will carefully 
watch for any invasion of privacy and breach of confidentiality  

 
  
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1- Testosterone levels, PSA, CBC would be checked periodically as mentioned in 
study protocol.  
 
2-  
Dose of  Androgel would be adjusted as follows:  
 
 If the free testosterone concentration is less than 14ng/dL in subjects randomized to drug 
group, then the dose of Androgel 1% will be increased to 7.5 grams at week 6 and if still 
lower than 14 ng/dl at week 10, the dose will be increased to Androgel 1% (10grams) at 
week 12. If the total testosterone concentration is more than 17ng/dL in subjects randomized 
to drug group, then the dose of Androgel 1% will be decreased by 2.5 mg starting from week 
6.Blood samples will be taken for PSA and subjects will be questioned for BPH symptoms to 
estimate the IPSS.  
The dose of androgel 1% will be decreased if the hematocrit is >50%. If PSA concentration 
rises by more than 1.5 ng/ml in 12 month period or if subjects complain of symptoms 
suggestive of severe BPH (IPSS > 19), the subjects will be excluded from the study and 
referred to their physician. The patient will remain blinded to the study drug or dose.   
3-  
Study subjects would routinely be evaluated for any side effects during study 
visits as per protocol.  
19.2 Describe what data are reviewed, including safety data, untoward events, and 
efficacy data.  
Response:   
Safety data: All reports from patients or from diagnostic labs will be reviewed for 
safety. Any skin reaction to the gel or other side effects reported by patients, 
laboratory safety tests including PSA or Hct will be documented. Any new FDA or 
NIH guidelines and warnings regarding the drug and testosterone will be reviewed 
and adopted.  
Efficacy data: The effect of testosterone replacement on IMT, FMD%, body 
composition, hematocrit, endothelial function, inflammatory mediators and oxidative 
stress  
19.3 Describe any safety endpoints.  
Response: This study will not be evaluating any safety endpoints. However safety 
of all participating subjects will be monitored   
19.4 Describe how the safety information will be collected (e.g., with case report 
forms, at study visits, by telephone calls with participants).  
Response: The safety information will be collected at the time of the participants 
visit, and or during telephone calls with the participant  
19.5 Describe the frequency of safety data collection.  
Response: The data collection will be done at all study visits which will be at 
intervals of either one or two weeks depending on the number of study visit. The 
patients, however, will be asked to report any adverse event or safety related 
information via phone as soon as it occurs and it will be reviewed the same day.  
19.6 Describe who will review the safety data.  
Response: The principal investigator Paresh Dandona, MD, PhD and 
coinvestigators, Manav Batra, MD and Husam Ghanim, PhD will review the data 
at the completion of all visits by each subject and every 3 months to assess the 
safety and any potential risks to the participants. Furthermore they will also assess 
other risks including the physical, psychological, social, legal and economic harm 

 
  
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to these patients. The investigators listed above will carefully watch for any 
invasion of privacy and breach of confidentiality.  The principal investigator will 
be sharing the results of safety analysis of the study with the sponsor (Abbvie) and 
with the IRB. The study groups will remain blinded. If there are any safety 
concerns then co-investigator Husam Ghanim, PhD who is not directly involved 
with the study participants will unblind the study groups on the discretion of 
principal investigator and the team will assess potential harm to the patients and 
inform the IRB and sponsor of this potential harm. The corrective actions will 
then be taken and research subjects will be withdrawn from the study if risks 
outweigh the benefits. The IRB will be kept well-informed at all times.  
19.7 Describe the frequency or periodicity of review of cumulative safety data.  
Response: Safety data will be reviewed every 3 months. Study endpoint data will 
be reviewed once after half of the recruited patients have completed the study and 
then at the end of the study  
19.8 Describe the statistical tests for analyzing the safety data to determine 
whether harm is occurring.  
Response: The statistical analysis will be carried out using student t-test, 
Chisquare and Wilcoxson’s test for paired data  
19.9 Describe any conditions that trigger an immediate suspension of the research.  
Response:   
1- New information about the safety of the used drug (Testosterone)  2- 
Sponsor suspension of the funds.  
3- Significant high incidence of SAE and events leading to withdrawal of 
subjects determined based on the continuous review by the investigators.  
 
  
 
20.0  
Withdrawal of Subjects  
 
☐  
N/A:  This study is not enrolling subjects.  This section does not apply.  
  
20.1 Describe anticipated circumstances under which subjects may be withdrawn 
from the research without their consent.  
Response: Drug intolerability (severe nausea and/or vomiting) developing any of 
the exclusionary condition listed in the inclusion and exclusion criteria.  
The principal investigator of the study can remove a participant from the research 
study without their approval if for any reason he/she feels is appropriate, including: 
severe side effect, injury or medical condition which may place the patient at risk 
of further complications if patient continues to participate, failure to take the 
medication as instructed, failure to keep your scheduled appointments, cancellation 
of the study by the sponsor, or other administrative reasons.  
Participation in this research study is voluntary. Subjects have the right to refuse to 
participate or to withdraw from participation at any time for any reason.  Refusal to 
participate or withdrawal from the study will involve no penalty or loss of entitled 
benefits, nor affect the subjects ongoing medical care  
20.2 Describe any procedures for orderly termination.    

 
  
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NOTE:  Examples may include return of study drug, exit interview with clinician.  
Include whether additional follow up is recommended for safety reasons for 
physical or emotional health.  
Response:  If a subject withdraws from the research, the data collected to 
that point will be used toward the research finding. If applicable the subject 
will have to bring back any unused research drug. If necessary, they will be 
asked to complete an end of study visit for their safety.    
20.3 Describe procedures that will be followed when subjects withdraw from the 
research, including retention of already collected data, and partial 
withdrawal from procedures with continued data collection, as applicable.  
Response: If a subject withdraws from the research, the data collected to that point 
will be used toward the research finding. Efficacy and safety data will continue to 
be collected for the parts of study that patients agree to participate in.  
 
21.0  
Risks to Subjects  
21.1 List the reasonably foreseeable risks, discomforts, hazards, or inconveniences 
to the subjects related to their participation in the research. Consider 
physical, psychological, social, legal, and economic risks.  Include a 
description of the probability, magnitude, duration, and reversibility of the 
risks.   
NOTE:  Breach of confidentiality is always a risk for identifiable subject data.  
 Response:    
1- Subjects will arrive after having fasted overnight. A fasting blood sample will be taken. Blood 
samples will be drawn by venipuncture (60 mL/ visit). This study requires that blood be drawn 
by venipuncture. Drawing blood may result in pain, a feeling of faintness, irritation of the vein, 
bruising, or bleeding at the site of puncture. MRI study should not pose any risks. Testosterone 
replacement can lead to increases in hemoglobin, liver enzymes and PSA. These will be 
monitored by lab tests before, during and after the study. Serum testosterone concentrations will 
be checked during the study and dose adjustments will be made. Mood changes (such as 
aggressiveness) and an increased incidence of sleep apnea may also occur after testosterone 
replacement. Patients will be asked to report any changes.  
2- There have been some post marketing reports of CVD after testosteorone replacement .FDA has 
reviewed the data and agreed that the CV signal is weak and advised the need for more 
randomized studies to identify the risk. We will follow the study subjects closely for any CV 
event. 3-   
4- There have been post marketing reports of clots in legs and lungs, in patients using testosterone 
products. Subjects would be advised to report symptoms of pain, swelling, warmth and redness 
in the legs or acute shortness of breath and discontinue treatment with AndroGel 1% and inform 
doctor immediately.   
5- With large doses of exogenous androgens, including AndroGel 1%, sperm production may be 
suppressed through feedback inhibition of pituitary hormones which could possibly lead to 
adverse effects on semen parameters including sperm count.  
6- Androgens, including AndroGel 1%, may promote retention of sodium and water. Alcohol based 
products, including AndroGel 1%, are flammable; therefore, patients would be advised to avoid 
fire, flame or smoking until the AndroGel 1% has dried. Androgel can transfer from body  
to others. This can happen if other people come in contact with the area where Androgel was 
applied. This can be harmful to the people who get exposed. Subjects will be explained the 
precautions to take to avoid this from happening.   
  

 
  
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7- The vascular or blood vessel studies of the neck and arm should not present any risks. Sodium 
nitroglycerin that will be used as part of the brachial reactivity studies may cause headache, 
lightheadedness and a lowering of blood pressure. There may be mild discomfort when the 
ultrasound probe is placed on neck. Moderate discomfort or pain may result from prolonged 
blood pressure cuff inflation on forearm.  
8- MRI may involve some discomfort as subject has to lie still for several minutes at a time and 
may feel “closed in” during the procedure.  MRI study should not pose any risks as MRI does 
not use any radiation. Ultrasound again does not use any radiation.   
9- Fat tissue aspiration will probably lead to a bruise at the site of aspiration. The site will therefore 
maybe painful for 1-2 weeks. Subjects are advised to call us if they have a lot of pain or swelling 
at the site after the procedure. Rarely some people have side effects such as low blood pressure 
or heart rate and allergic reaction to lidocaine including swelling of the throat.   
In addition to the risks listed above, the study drugs and procedures may have unknown, 
unforeseen or unanticipated side effects, including life-threatening reactions. There is 
always the possibility that may have a reaction that is currently not known and not expected. 
It is important for subjects to report all symptoms or reactions to the study doctor as well as 
to their personal doctor. Subjects will be monitored for side effects by study staff and study 
doctor may decide to withdrawn from the study as per protocol.  
21.2 Describe procedures performed to lessen the probability or magnitude of 
risks, including procedures being performed to monitor subjects for safety.  
Response:   
Monitoring of testosterone replacement : The dose of androgel 1% will be decreased if the 
hematocrit is >50%. If PSA concentration rises by more than 1.5 ng/ml in 12 month period or 
if subjects complain of symptoms suggestive of severe BPH (IPSS > 19), the subjects will be 
excluded from the study and referred to their physician. The patient will remain blinded to the 
study drug or dose. CBC, CMP, lipid profile would be periodically monitored and necessary 
adjustments would be made as per study protocol.  
  
21.3 If applicable, indicate which procedures may have risks to the subjects that 
are currently unforeseeable.  
Response: Any adverse effects of Androgel that are not currently known may be 
some of the unforeseeable risks.  
21.4 If applicable, indicate which research procedures may have risks to an 
embryo or fetus should the subject be or become pregnant.  
Response: Not applicable  
21.5 If applicable, describe risks to others who are not subjects.  
Response: Androgel can transfer from body to others. This can happen if other 
people come in contact with the area where Androgel was applied. This can be 
harmful to the people who get exposed. Participants will be explained the 
precautions to take to avoid this from happening  
 
22.0  
Potential Benefits to Subjects  
22.1 Describe the potential benefits that individual subjects may experience by 
taking part in the research.  Include the probability, magnitude, and duration 
of the potential benefits.  Indicate if there is no direct benefit.    
NOTE:  Compensation cannot be stated as a benefit.  

 
  
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Response: Testosterone replacement in hypogonadism may improve energy, 
stamina, decrease adiposity, increase muscle mass, decrease insulin resistance and 
decrease inflammatory markers.  
 
23.0  
Compensation for Research-Related Injury  
☐  
N/A:  The research procedures for this study do not present risk of 
research related injury (e.g. survey studies, records review studies).  This 
section does not apply.    
23.1  
If the research procedures carry a risk of research related injury, 
describe the available compensation to subjects in the event that such injury 
should occur.    
Response: Routinely, Buffalo General Hospital, Erie County Medical Center, 
and/or the University at Buffalo, State University of New York, its agents, or its 
employees do not compensate for or provide free medical care for human 
subjects/participants in the event that any injury results from participation in a 
human research project.  In the unlikely event that they become ill or injured as a 
direct result of participating in this study, they may receive medical care, that will 
be covered by study.  
23.2  
Provide a copy of contract language, if any, relevant to compensation for 
research related injury.  
 NOTE:  If the contract is not yet approved at the time of this submission, submit the 
current version here.  If the contract is later approved with different language regarding 
research related injury, you must modify your response here and submit an amendment 
to the IRB for review and approval.  
Response: No contract injury language available.  
  
 
24.0  
Economic Burden to Subjects  
24.1 Describe any costs that subjects may be responsible for because of 
participation in the research.    
NOTE:  Some examples include transportation or parking.  
Response: All research expenses will be covered. Participants will not be 
subjected to any out of pocket cost.  
☐  
N/A:  This study is not enrolling subjects, or is limited to records review 
procedures only.  This section does not apply.  
 
25.0  
Compensation for Participation  
25.1  Describe the amount and timing of any compensation to subjects, 
including monetary, course credit, or gift card compensation.  
Response:   

 
  
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Men with low testosterone: $25.00 for each blood sample drawn (of which  
there are 7), $25.00 for carotid and brachial ultrasound (of which 
there are 5), $ 25.00 for each 24-hour urine collection (of which 
there are 3) and $25.00 for each MRI (of which there are 3) and 
$50.00 for each fat biopsy (of which there are 3). Total of up to 
$600.00 for the study. The study drug will be provided to free of 
cost. Subjects will not be paid for the screening visit  
☐  
N/A:  This study is not enrolling subjects, or is limited to records 
review procedures only.  This section does not apply.  
☐  
N/A:  There is no compensation for participation.  This section 
does not apply.  
 
26.0  
Consent Process  
26.1 Indicate whether you will be obtaining consent.    
NOTE:  This does not refer to consent documentation, but rather whether you will 
be obtaining permission from subjects to participate in a research study.   Consent 
documentation is addressed in Section 27.0.  
 
☒  
Yes  
(If yes, Provide responses to each question in this Section)  
 
☐  
No  
(If no, Skip to Section 27.0)  
  
26.2 Describe where the consent process will take place.  Include steps to 
maximize subjects’ privacy.  
Response: All participants will come to the research department to be consented. 
Participants will be placed in a private, screened off area and or room where they 
can review the consent. Participant questions and or concerns will be address with 
a member of the study team or research doctor if applicable. The research 
coordinator will discuss in length the participants requests for privacy of their 
PHI.   
26.3 Describe how you will ensure that subjects are provided with a sufficient 
period of time to consider taking part in the research study.    
NOTE:  It is always a requirement that a prospective subject is given sufficient time 
to have their questions answered and consider their participation.  See “SOP: 
Informed Consent Process for Research (HRP-090)” Sections 5.5 and 5.6.  
Response: participants will be made aware that participating in research is 
completely voluntary, and they may withdraw at any time with no consequence to 
their routine clinic care. If the patients requires time to decide and or discuss 
partaking in a research study, the subject will be given said time.  
26.4 Describe any process to ensure ongoing consent, defined as a subject’s 
willingness to continue participation for the duration of the research study.    
Response: The research coordinator and study team are available to answer any 
question or concerns with the patient during the duration of the research trial. At 
each study visit, the patient is asked a series of questions to ensure they are on 
task with the study visits and feel comfortable. Upon departing from their study 
visit, the patients are told of their next visit and given detail instruction for their 
next visit. If study is revised or amendment or new information becomes available 

 
  
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about drug safety that may affect patients participation, the patient may be 
reconsented to ensure patient ongoing consent,  
26.5 Indicate whether you will be following “SOP: Informed Consent Process for 
Research (HRP-090).” If not, or if there are any exceptions or additional 
details to what is covered in the SOP, describe:  
• 
The role of the individuals listed in the application who are involved in 
the consent process  
• 
The time that will be devoted to the consent discussion  
• 
Steps that will be taken to minimize the possibility of coercion or 
undue influence  
• 
Steps that will be taken to ensure the subjects’ understanding  
Response:   
☒  
We have reviewed and will be following “SOP: Informed Consent Process 
for Research (HRP-090).”  
  
Non-English Speaking Subjects   
☒ N/A:  This study will not enroll Non-English speaking subjects.   (Skip 
to Section 26.8)  
26.6 Indicate which language(s) other than English are likely to be 
spoken/understood by your prospective study population or their legally 
authorized representatives.  
  
NOTE: The response to this Section should correspond with your response to Section 
6.4 of this protocol.  
Response:   
  
26.7 If subjects who do not speak English will be enrolled, describe the process 
to ensure that the oral and written information provided to those subjects 
will be in that language.  Indicate the language that will be used by those 
obtaining consent.  
NOTE:  Guidance is provided on “SOP:  Informed Consent Process for Research 
(HRP-090).”  
Response:   
  
Cognitively Impaired Adults  
 
☒  N/A:  This study will not enroll cognitively impaired adults.    
(Skip to Section 26.9)  
26.8 Describe the process to determine whether an individual is capable of 
consent.   
Response:   

 
  
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Adults Unable to Consent  
 
☒  
N/A:  This study will not enroll adults unable to consent.   
(Skip to Section 26.13)  
When a person is not capable of consent due to cognitive impairment, a legally 
authorized representative should be used to provide consent (Sections 26.9 and 
26.10) and, where possible, assent of the individual should also be solicited 
(Sections 26.11 and 26.12).  
26.9 Describe how you will identify a Legally Authorized Representative  
(LAR).  Indicate that you have reviewed the “SOP: Legally Authorized 
Representatives, Children, and Guardians (HRP-013)” for research in New 
York State.   
NOTE:  Examples of acceptable response includes: verifying the electronic medical 
record to determine if an LAR is recorded.  
Response:   
☐  
We have reviewed and will be following “SOP: Legally Authorized 
Representatives, Children, and Guardians (HRP-013).”   
26.10 For research conducted outside of New York State, provide information 
that describes which individuals are authorized under applicable law to 
consent on behalf of a prospective subject to their participation in the 
research.  One method of obtaining this information is to have a legal 
counsel or authority review your protocol along with the definition of 
“legally authorized representative” in “SOP: Legally Authorized 
Representatives, Children, and Guardians (HRP-013).”  
Response:   
26.11 Describe the process for assent of the adults:  
• 
Indicate whether assent will be obtained from all, some, or none of the 
subjects.  If some, indicate which adults will be required to assent and 
which will not.  
Response:   
• 
If assent will not be obtained from some or all subjects, provide an 
explanation of why not.  
Response:   
26.12Describe whether assent of the adult subjects will be documented and the 
process to document assent.    
NOTE:  The IRB allows the person obtaining assent to document assent on the 
consent document using the “Template Consent Document (HRP-502)” Signature 
Block for Assent of Adults who are Legally Unable to Consent.  
Response:   
  

 
  
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Subjects who are not yet Adults (Infants, Children, and Teenagers)  
 
☒  N/A:  This study will not enroll subjects who are not yet adults.    
(Skip to Section 27.0)  
26.13Describe the criteria that will be used to determine whether a prospective 
subject has not attained the legal age for consent to treatments or 
procedures involved in the research under the applicable law of the 
jurisdiction in which the research will be conducted (e.g., individuals under 
the age of 18 years).  For research conducted in NYS, review “SOP: 
Legally Authorized Representatives, Children, and Guardians (HRP-013)” 
to be aware of which individuals in the state meet the definition of 
“children.”   
NOTE:  Examples of acceptable responses include: verification via electronic 
medical record, driver’s license or state-issued ID, screening questionnaire.  
Response:  
26.14For research conducted outside of New York State, provide information 
that describes which persons have not attained the legal age for consent to 
treatments or procedures involved the research, under the applicable law of 
the jurisdiction in which research will be conducted.  One method of 
obtaining this information is to have a legal counsel or authority review your 
protocol along the definition of “children” in “SOP: Legally Authorized 
Representatives, Children, and Guardians (HRP-013).”  
Response:   
26.15 Describe whether parental permission will be obtained from:  
Response: N/A  
☐  One parent even if the other parent is alive, known, competent, reasonably 
available, and shares legal responsibility for the care and custody of the 
child.  
☐  Both parents unless one parent is deceased, unknown, incompetent, or not 
reasonably available, or when only one parent has legal responsibility for 
the care and custody of the child.  
☐  Parent permission will not be obtained.  A waiver of parent permission is 
being requested.  
NOTE:  The requirement for parent permission is a protocol-specific determination  
made by the IRB based on the risk level of the research.  For guidance, review the  
“CHECKLIST: Children (HRP-416).”   
26.16Describe whether permission will be obtained from individuals other than 
parents, and if so, who will be allowed to provide permission.  Describe 
your procedure for determining an individual’s authority to consent to the 
child’s general medical care.  
Response:   

 
  
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26.17 Indicate whether assent will be obtained from all, some, or none of the 
children.  If assent will be obtained from some children, indicate which 
children will be required to assent.  
Response:   
26.18 When assent of children is obtained, describe how it will be documented.  
Response:  
 
27.0  
Waiver or Alteration of Consent Process  
Consent will not be obtained, required information will not be disclosed, or 
the research involves deception.  
 
☒  
N/A:  A waiver or alteration of consent is not being requested.  
27.1 If the research involves a waiver or alteration of the consent process, please 
review the “CHECKLIST: Waiver or Alteration of Consent Process 
(HRP410)” to ensure that you have provided sufficient information for the 
IRB to make the determination that a waiver or alteration can be granted.    
NOTE:  For records review studies, the first set of criteria on the “CHECKLIST: 
Waiver or Alteration of Consent Process (HRP-410)” applies.   
Response:   
27.2 If the research involves a waiver of the consent process for planned 
emergency research, please review the “CHECKLIST: Waiver of Consent 
for Emergency Research (HRP-419)” to ensure you have provided sufficient 
information for the IRB to make these determinations.  Provide any 
additional information necessary here:  
Response:   
 
28.0  
Process to Document Consent  
 
☐  
N/A:  A Waiver of Consent is being requested.    
(Skip to Section 29.0)  
28.1 Indicate whether you will be following “SOP: Written Documentation of 
Consent (HRP-091).” If not or if there are any exceptions, describe whether 
and how consent of the subject will be obtained including whether or not it 
will be documented in writing.    
NOTE:  If your research presents no more than minimal risk of harm to subjects 
and involves no procedures for which written documentation of consent is normally 
required outside of the research context, the IRB will generally waive the 
requirement to obtain written documentation of consent.  This is sometimes 
referred to as ‘verbal consent.’  Review “CHECKLIST: Waiver of Written 
Documentation of Consent (HRP-411)” to ensure that you have provided sufficient 
information.   
If you will document consent in writing, attach a consent document with your 
submission.  You may use “TEMPLATE CONSENT DOCUMENT (HRP- 
502)”.  If you will obtain consent, but not document consent in writing, attach the 
script of the information to be provided orally or in writing (i.e. consent script or 
Information Sheet).    
Response:   

 
  
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☒  
We will be following “SOP:  Written Documentation of Consent” (HRP-
091).  
 
29.0  
Multi-Site Research (Multisite/Multicenter Only)  
☒  
N/A:  This study is not an investigator-initiated multi-site study.  This 
section does not apply.  
  
29.1 If this is a multi-site study where you are the lead investigator, describe the 
processes to ensure communication among sites, such as:  
• 
All sites have the most current version of the IRB documents, 
including the protocol, consent document, and HIPAA authorization.  
• 
All required approvals have been obtained at each site (including 
approval by the site’s IRB of record).  
• 
All modifications have been communicated to sites, and approved 
(including approval by the site’s IRB of record) before the 
modification is implemented.  
• 
All engaged participating sites will safeguard data as required by local 
information security policies.  
• 
All local site investigators conduct the study appropriately.  
• 
All non-compliance with the study protocol or applicable requirements 
will be reported in accordance with local policy.  
Response:   
29.2 Describe the method for communicating to engaged participating sites:  
• 
Problems  
• 
Interim results  
• 
Study closure  
Response:   
29.3 
Indicate the total number of subjects that will be enrolled or records that 
will be reviewed across all sites.  
Response:  
29.4 
If this is a multicenter study for which UB will serve as the IRB of 
record, and subjects will be recruited by methods not under the control of the local 
site (e.g., call centers, national advertisements) describe those methods.   
Response:   
 
30.0  
Banking Data or Specimens for Future Use  
☐  
N/A:  This study is not banking data or specimens for future use or 
research outside the scope of the present protocol.  This section does not 
apply.  
30.1 If data or specimens will be banked (stored) for future use, that is, use or 
research outside of the scope of the present protocol, describe where the 
data/specimens will be stored, how long they will be stored, how the 

 
  
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data/specimens will be accessed, and who will have access to the 
data/specimens.   
NOTE:  Your response here must be consistent with your response at the “What 
happens if I say yes, I want to be in this research?” Section of the Template 
Consent Document (HRP-502).  
Response: The study data/specimens will be stored in a locked closet or 80 
freezer at the research facility of the Diabetes and Endocrinology Center of 
WNY for up to 7 years.   
The research staff (study personnel including coordinator) only will be 
authorized to access data and or specimens  
30.2 List the data to be stored or associated with each specimen.  
Response: Patient ID number, study visit information and date of collection will 
be stored with specimen. Other data stored will include record files of all patients 
participating in the study, including data collection sheets andlab results.  
30.3 Describe the procedures to release banked data or specimens for future uses, 
including: the process to request a release, approvals required for release, 
who can obtain data or specimens, and the data to be provided with 
specimens.  
Response: The copy of the individual patient data collected during the study 
period will be provided to these individual patients who can choose to hand 
carry it to their respective physicians and a copy will be faxed to their 
respective clinical providers upon verbal request from the patient. The data 
provided will include the insulin pump or CGM data or any of the lab results 
obtained during the study period. The results of the completed study will be 
made available to the patients if requested through published manuscript.  
 
31.0  
Drugs or Devices  
☐  
N/A:  This study does not involve drugs or devices.  This section does not 
apply.  
31.1 If the research involves drugs or devices, list and describe all drugs and 
devices used in the research, the purpose of their use, and their regulatory 
approval status.   
Response:   
Investigational 
product  
Dosage form 
and strength  
Approval status  
Manufacturer  
Androgel 1% (50grams)  
gel  
Approved for 
testosterone 
treatment  
Abbive  
Matching placebo for 
Androgel  
gel  
N/A  
Abbive  
31.2 Describe your plans to store, handle, and administer those drugs or devices 
so that they will be used only on subjects and be used only by authorized 
investigators.  

 
  
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Response: Drugs will be stored in a locked cabinet and temperature controlled 
refrigerator at 4C at the research facility of the Diabetes and Endocrinology 
Center of WNY.  
If the drug is investigational (has an IND) or the device has an IDE or a claim 
of abbreviated IDE (non-significant risk device), include the following 
information:  
31.3 Identify the holder of the IND/IDE/Abbreviated IDE.  
Response: IND holder: Dr Paresh Dandona. FDA has issued IND exempt and this 
is on file with IRB  
31.4 Explain procedures followed to comply with FDA sponsor requirements for 
the following:    
  
  
Applicable to:  
FDA Regulation  
IND Studies  
IDE studies  
Abbreviated 
IDE studies  
21 CFR 11  
X  
X  
  
21 CFR 54  
X  
X  
  
21 CFR 210  
X  
  
  
21 CFR 211  
X  
  
  
21 CFR 312  
X  
  
  
21 CFR 812  
  
X  
X  
21 CFR 820  
  
X  
  
Response: All FDA sponsor requirements have been reviewed and will be 
followed during the study procedures.  
 
32.0  
Humanitarian Use Devices  
☒  
N/A:  This study does not involve humanitarian use devices.  This does 
not apply.  
32.1  
For Humanitarian Use Device (HUD) uses provide a description of the 
device, a summary of how you propose to use the device, including a description 
of any screening procedures, the HUD procedure, and any patient follow-up 
visits, tests or procedures.  
Response:   
32.2  
 For HUD uses provide a description of how the patient will be informed 
of the potential risks and benefits of the HUD and any procedures associated with 
its use.  
Response: