Clinical trial NCT02154477
Conditions: Hypogonadotropic Hypogonadism
Keywords: hypogonadotropic hypogonadism
Sponsor: Texas Tech University Health Sciences Center
Start date: 2014-12-01; Completion date: 2017-12-01
Enrollment: 14 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
Over the last few years, studies have shown that men with type 2 diabetes sometimes develop a condition called hypogonadotrophic hypogonadism (HH). People with this condition produce little or no sex hormones. Obesity and metabolic syndrome are also associated with HH. Research suggests that insulin in the brain may benefit the glands that release these hormones. During this study, subjects will spray a small amount of insulin into their nose.This will increase insulin concentrations in the brain. The purpose of this study is to find out the effect of one dose of intranasal insulin on release of LH (a sex hormone) in obese diabetic men with HH.

Detailed description:
Studies over the last few years have clearly established that at least 25% of men with type 2 diabetes have subnormal free testosterone (T) concentrations in association with inappropriately low leutinizing hormone (LH) and FSH (follicle stimulating hormone) concentrations. These patients thus suffer from hypogonadotrophic hypogonadism (HH). Obesity and metabolic syndrome are also associated with HH. Animal studies and in vitro data have shown that insulin action and insulin responsiveness in the brain are necessary for the maintenance of the functional integrity of the hypothalamo-hypophyseal-gonadal axis. Insulin concentrations can be increased in the brain by delivering insulin intranasally with vianase device. The aim of this project is to study the effect of one dose of intranasal insulin on release of LH in obese diabetic men with HH. 15 obese men with type 2 diabetes and low free T along with normal LH concentrations will be recruited for the study. The study will be carried out at the clinical center of Texas Tech (Odessa campus). There will be two study visits, each comprising of blood tests every 15 minutes over 6 hours. 40 IU of intranasal insulin or saline will be given after a baseline period of blood sampling of 2 hours. Student's t-test will be used to compare the change in LH concentrations after intranasal saline or insulin.

Arms: arm 1: All patient will receive insulin at one visit and saline at another visit arm 2: All patient will receive insulin at one visit and saline at another visit
Interventions: intervention 1: intranasal insulin intervention 2: placebo (intranasal saline)
Intervention descriptions: intervention 1: insulin intervention 2: saline

Protocol documents:
Prot_SAP_000.pdf: 
Protocol version 01-29-2015 
Page 1 of 16 
 
Study Title: Effect of intranasal insulin on LH concentrations in man 
 
 
 
 
 
Principal Investigator 
Department 
School/Campus 
Email Address 
Sandeep Dhindsa 
Medicine 
TTUHSC-PB 
Sandeep.dhindsa@ttuhsc.edu 
 
 
 
 
Sub Investigator 
Department 
School/Campus 
Email Address 
Rama Chemitiganti 
Medicine 
TTUHSC-PB 
Rama.chemitiganti@ttuhsc.edu 
 
 
 
 
 
 
ABSTRACT 
Studies over the last few years have clearly established that at least 25% of men with type 2 diabetes 
have subnormal free testosterone (T) concentrations in association with inappropriately low LH and FSH 
concentrations. These patients thus suffer from hypogonadotrophic hypogonadism (HH).  Obesity and 
metabolic syndrome are also associated with HH. Animal studies and in vitro data have shown that 
insulin action and insulin responsiveness in the brain are necessary for the maintenance of the 
functional integrity of the hypothalamo-hypophyseal-gonadal axis. Insulin concentrations can be 
increased in the brain by delivering insulin intranasally with vianase device. The aim of this project is to 
study the effect of one dose of intranasal insulin on release of LH in diabetic men and healthy menwith a 
normal BMI. 14 men with type 2 diabetes and 14 healthy men will be recruited for the study. The study 
will be carried out at the clinical center of TTUHSC-PB (Odessa campus). There will be two study visits, 
each comprising of blood tests every 15 minutes over 5 hours. 40 IU of intranasal insulin or saline will be 
given after a baseline period of blood sampling of 2 hours. Student’s t-test will be used to compare the 
change in LH concentrations after intranasal saline or insulin. 
 
 
BACKGROUND 
   Studies over the last few years have clearly established that at least 25% of men with type 2 diabetes 
have subnormal free testosterone (T) concentrations in association with inappropriately low LH and FSH 
concentrations. These patients thus suffer from hypogonadotrophic hypogonadism (HH).  Another 4% 
have subnormal testosterone concentrations with elevated LH and FSH concentrations. Obesity and 
metabolic syndrome are also associated with HH.  
Association of type 2 diabetes, obesity and metabolic syndrome with low testosterone 
    Subnormal free testosterone concentrations in association with inappropriately low LH and FSH 
concentrations in men with type 2 diabetes were first described in 2004(1). These abnormalities were 
independent of the duration and severity of hyperglycemia (HbA1c). Magnetic resonance imaging  in 
these hypogonadal patients  showed no abnormality in brain or the pituitary(1). The response of LH and 
FSH to GnRH injection was normal. It has now been shown that kisspeptin injection also leads to an 
increase in gonadotropin and T concentrations in men with type 2 diabetes(2) (discussed later in more 
detail). 
    This association of HH with type 2 diabetes has now been confirmed in several studies and is present in 
25-40% of these men (3-6). In this context, it is important that the Endocrine Society now includes type 2 
diabetes in the list of conditions known to have a high prevalence of hypogonadism and suggests screening 
men with type 2 diabetes for hypogonadism(7). Younger patients with type 2 diabetes (between the ages of 
18 and 35 years) also have a similarly high prevalence of HH(8). In all of the above studies, total T and free T 
concentrations were inversely related to BMI and age. However, the presence of low T concentration was 


Protocol version 01-29-2015 
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not entirely dependent upon obesity since 25% of non-obese patients(31% of lean and 21% of overweight) 
also had HH(1).  HH is relatively rare in type 1 diabetes, and, therefore, is not a function of diabetes or 
hyperglycemia per se(9). Thus, in view of the inverse relationship between BMI and T concentrations in both 
type 1 and type 2 diabetes, HH is probably related to insulin resistance (1, 5, 9). Previous studies have shown 
that hypogonadism is associated with upper abdominal adiposity, insulin resistance and the metabolic 
syndrome (10, 11). We presented preliminary data from a study comparing the insulin resistance in type 2 
diabetic men with subnormal or normal T concentrations at the annual meetings of the ADA, the Endocrine 
Society and AACE this year (12). 81 men with type 2 diabetes had their insulin sensitivities measured by 
hyperinsulinemic euglycemic clamps. 39 men had HH and 42 were eugonadal. Men with HH had greater BMI 
(39 vs 34 kg/m2, p=0.002) and fat mass (46 vs 34 kg, p<0.001) but similar age (54 vs. 52 years, p=0.4) and 
HbA1c (7.1 vs. 7.2%, p=0.5) as compared to eugonadal men. The glucose infusion rate was 30% lower in men 
with HH as compared to eugonadal men, even after adjustment for the difference in fat mass among the 
two groups (p=0.02). Treatment of systemic insulin resistance by rosiglitazone leads to a modest increase in 
T concentrations in men with type 2 diabetes(13), without the restoration of T concentrations to normal.  
    Many studies have shown free T concentrations to be also low in the obese, especially in those with 
BMI≥40kg/m2.(14, 15) Vermeulen et al (15) compared 35 obese men (mean BMI 41.1 kg/m2) with 54 
lean men. The FT concentrations were 26% lower in the obese. The Free T concentrations correlated 
inversely with BMI. The authors also compared LH pulsatility over 12 hours in 8 obese and lean men and 
found that the mean integrated LH levels over 12 hours were significantly lower in obese men. Free T 
concentrations correlated positively with the sum of LH pulse amplitudes in each individual(15). A study 
from Netherlands in 160 obese men (mean age 58 years) found a 35.6% prevalence of HH (16). Nielsen 
et al(17) examined T concentrations of 615 non-obese and 70 obese young Danish men (20-29 years of 
age) in association with subcutaneous and visceral fat mass, measured by DEXA and MRI.  Total and 
bioavailable T concentrations were negatively related to all measures of fat mass. 23% of obese young 
men had subnormal total T concentrations, while 10% had subnormal total and bioavailable T 
concentrations. The largest study in this regard compared the prevalence of low testosterone 
concentrations in obese and diabetic men (mean age 60 years; range: 45-96 years)(18): 44% of diabetic 
and 33% of age matched non-diabetic men had subnormal free testosterone concentrations 
respectively. 40% of obese men and 50% of obese diabetic men had subnormal free testosterone 
concentrations. Thus, obesity is associated with a high prevalence of hypogonadism and the presence of 
diabetes adds to that risk. However, this study was restricted to a relative elderly population (age >45 
years). Furthermore, these studies did not utilize the current gold standard method of measuring 
testosterone, liquid chromatography tandem mass spectrometry (LC-MS/MS). Therefore the actual 
prevalence of hypogonadism in obesity may be lower. 
    We have recently studied the impact of obesity on T concentrations in boys at the completion of 
puberty(19). We compared the T, LH and FSH concentrations of 25 lean and 25 obese boys in tanner 
stage 4 and 5. The free T concentrations (measured by equilibrium dialysis followed by LC-MS/MS) of 
obese boys were 40% lower than those of lean boys. The gonadotropin concentrations of lean and 
obese boys were similar. The T concentrations were inversely related to BMI, HOMA-IR (homeostatic 
model assessment- insulin resistance) and to C-reactive protein concentrations. 
    Similar to the negative impact of obesity on T concentrations in men, studies have highlighted the 
association of metabolic syndrome(MetS) with hypogonadism(20). Kaplan et al studied 864 men (mean 
age 52 yrs) and demonstrated that aging men with obesity and the MetS have a significantly decreased 
total T (150 and 300 ng/dl less in obese and severely obese respectively) compared to aging, 
metabolically healthy men(21). Free T concentrations decrease proportionately with increasing number 
of components of metabolic syndrome(22). In a population based study of 1896 middle aged Finnish 
males (345 of whom had MetS), Laaksonen et al showed that Free T and SHBG were respectively 11% 


Protocol version 01-29-2015 
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and 18% lower in MetS than in normal subjects (11). Men with Free T in the lowest third tertile were 1.7 
times (after adjustment for age and BMI) more likely to have MetS. Low T and low SHBG concentrations 
were associated with MetS and its components, independently of BMI. Total T, Free T and SHBG were 
inversely associated with concentrations of insulin, glucose, triglycerides and CRP, and positively 
associated with HDL. Obesity might be the most important contributor to the presence of hypogonadism 
in MetS, and the linear inverse relationship of T with BMI is preserved in the presence of MetS(8; 10). 
Thus from the preceding discussion, it appears that insulin resistance in men is associated with HH. 
     
Possible pathophysiological mechanisms underlying HH in type 2 diabetes/obesity 
a) Role of Estradiol: Since testosterone and androstenedione in the male can be converted to estradiol 
and estrone respectively through the action of aromatase in the mesenchymal cells and pre-adipocytes 
of adipose tissue, it has been suggested that excessive estrogen secretion due to aromatase activity in 
the obese may potentially suppress the hypothalamic secretion of GnRH(23). It therefore follows that 
the oestradiol concentrations in men with HH and type 2 diabetes /obesity should be elevated to 
account for the suppression of gonadotropin secretion. However, we have shown that this widely 
believed presumption is not true(24).  We measured the estradiol concentrations in 240 type 2 diabetic 
men with and without HH. Total estradiol concentrations were measured by immunoassay and free 
estradiol concentrations were calculated, using SHBG.  Total and free estradiol concentrations in men 
with HH were significantly lower than in those without HH(24). To confirm these findings, total estradiol 
concentrations were measured in a subset of 102 men by the LC-MS/MS assay and free estradiol 
concentrations were measured by equilibrium dialysis (24). Estradiol concentrations were 25% lower in 
men with HH. Free estradiol concentrations were directly related free T concentrations, irrespective of 
age or BMI. As mentioned above, our recent data on young pubertal and post-pubertal males (14-20 
years of age) have shown that free testosterone concentrations are approximately 40% lower in obese 
adolescents as compared to lean adolescents(19). Similar to our study in middle-aged men with type 2 
diabetes, we found that estradiol concentrations were also significantly lower in adolescents with lower 
free testosterone as compared to those with normal T. The estradiol concentrations related directly to 
testosterone concentrations but not to BMI. Population based studies such as European Male Aging 
Study also showed that estradiol concentrations are lower in hypogonadal men as compared to 
eugonadal men, regardless of whether the hypogonadism is primary or secondary (25). In fact, no study 
has shown increased concentrations on estradiol in hypogonadal men as compared to eugonadal men 
with obesity or type 2 diabetes. The direct relationship between plasma estradiol and T concentrations 
is consistent with the concept that estradiol concentrations are dependent upon testosterone, the 
substrate for estradiol synthesis. Clomiphene (estrogen antagonist) and aromatase inhibitors (which 
decrease estradiol concentrations) have been shown to increase testosterone concentrations in obese 
men with low testosterone(26). This, however, cannot be taken as evidence of estradiol as the cause of 
low testosterone in those men because eugonadal men also respond to aromatase inhibition by an 
increase in testosterone concentrations. This is because estradiol has an inhibitory effect upon 
gonadotropin secretion even in a normal physiological setting. A decrease in estradiol action or 
concentration would lower its inhibitory effect upon gonadotropins and eugonadal men may achieve 
even supranormal LH, FSH and T concentrations (27).  Elevated gonadotropin concentrations are also 
seen in men with congenital aromatase deficiency or absence of estrogen receptor-α (28, 29). Clearly, 
the suppression of LH, FSH and T in type 2 diabetes and obesity is not induced by circulating estradiol 
concentrations.  
 
b) Role of kisspeptin: It is now known that kisspeptin, a hypothalamic neuropeptide encoded by the 
KISS1 gene, and the presence of kisspeptin receptors on the GnRH neurons (G protein-coupled receptor 


Protocol version 01-29-2015 
Page 4 of 16 
 
54) is obligate for the release of GnRH(30, 31). Humans with absence of either kisspeptin gene or its 
receptor (GPR54) have HH(31, 32). Data from animal studies show that factors associated with 
decreased GnRH secretion, such as inflammation and estrogen, decrease KISS1 gene expression in the 
hypothalamus(33, 34). Intravenous administration of kisspeptin increases LH and testosterone 
concentrations in men with type 2 diabetes and HH(2), thus suggesting that the hypothalamic-pituitary-
gonadal axis is intact per se in men with HH and type 2 diabetes. However, the (presumably) metabolic 
insult in insulin resistance that results in hypogonadotropism is yet to be defined. Kisspeptin neurons 
express both leptin and insulin receptors, thus possibly accumulating evidence of metabolic health and 
translating it into reproductive health. 
 
c) Role of inflammatory mediators: Tumor necrosis factor α and interleukin-1β have been shown to 
suppress hypothalamic GnRH and LH secretion in experimental animals and in vitro (35, 36). It is 
therefore relevant that C-reactive protein(CRP) concentrations are markedly increased in hypogonadal 
type 2 diabetic men as compared to men with type 2 diabetes and normal testosterone(6.5 mg/l versus 
3.2 mg/l) (37). These data were confirmed by another study from Australia in which the median CRP 
concentration in type 2 diabetic patients with low total T was 7.7 mg/l as compared to 4.5 mg/l in men 
with normal testosterone(5). Free T concentrations were inversely related to CRP concentrations. It is 
thus possible that inflammatory mediators may contribute to the suppression of the hypothalamo-
hypophyseal axis and the syndrome of HH in type 2 diabetes. The presence of inflammation may also 
contribute to insulin resistance since several inflammation related mediators, such as suppressor of 
cytokine signaling-3, IκB kinase β and c-Jun N-terminal kinase-1 interfere with insulin signal transduction 
(38, 39) and contribute to insulin resistance. These mediators are also known to be increased in 
obesity(40).   
 
d) Role of Leptin: Leptin is known to play a permissive role in the regulation of reproductive axis. Leptin 
appears to serve as a signal of signal of energy reserves to regulate the hypothalamo-pituitary-gonadal 
axis in relation to nutritional status (41). Men and women with anorexia nervosa have low levels of 
leptin and hypogonadotropism. Absence of leptin gene or leptin signaling in humans results in HH (42). 
While there are extremely uncommon forms of human obesity due to lack of leptin gene, almost all 
obesity in humans is associated with leptin resistance and high leptin concentrations. It is possible that 
leptin resistance in hypothalamic neurons or in some other neurons is responsible for the 
hypogonadotropism seen in obesity. Direct evidence in humans supporting or disproving this reasoning 
is, however, lacking. 
 
e) Role of insulin resistance: The selective deletion of the insulin receptor from neurons leads to a 
reduction in LH concentrations by 60%-90% and low T concentrations (43). These animals respond to 
GnRH challenge by normal or supra- normal release of LH. In addition, these animals had atrophic 
seminiferous tubules with markedly impaired or absent spermatogenesis. In addition, it is known that 
the incubation of hypothalamic neurons with insulin results in the facilitation of secretion of GnRH(44, 
45). Thus, insulin action and insulin responsiveness in the brain are necessary for the maintenance of the 
functional integrity of the hypothalamo-hypophyseal-gonadal axis. However, it is unlikely that the site of 
action of insulin on reproductive axis is the GnRH neuron. Isolated knock out of insulin receptor in GnRH 
neuron does not lead to a decrease in LH concentrations or in fertility in either male or female mice(46). 
The loss of insulin receptors in proopiomelanocortin- or agouti-related peptide-expressing (POMC- or 
AgRP-expressing) neurons — neurons involved in the regulation of appetite and peripheral metabolism 
of glucose and fat — also did not affect fertility(47). However, the selective deletion of insulin receptors 
from kisspeptin neurons resulted in a delay in puberty in both male and female mice, although LH 


Protocol version 01-29-2015 
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concentrations, T concentrations and fertility during adulthood were not affected (48). Thus, the site or 
sites) of hypogonadtropism seen in neuronal insulin receptor knock-out mice is still not clear. 
    
    In summary, it is likely that there are several inter-linked causative mechanisms underlying HH in men 
with type 2 diabetes. It is possible that delivery of insulin to the hypothalamus may enhance the 
secretion of LH (probably by increasing kisspeptin stimulated GnRH secretion). The following paragraphs 
briefly describe the literature on central effects of insulin, with emphasis on human studies. The human 
experiments have used an intranasal approach to increase insulin levels in the brain. 
 
CNS actions of insulin 
     Havrankova et al. showed in 1978 that insulin receptors are present throughout the rat CNS, followed 
closely by the demonstration that insulin receptors are also expressed in the human brain(49, 50). The 
insulin receptor is found in particularly high densities in brain regions like the olfactory bulb, the 
cerebellum, the dentate gyrus, the pyriform cortex, the hippocampus, the choroid plexus and the 
arcuate nucleus of the hypothalamus. It is assumed that peripheral insulin crosses the blood–brain 
barrier (BBB) by a saturable, receptor-mediated transport mechanism and by binding to brain insulin 
receptors affects functions as diverse as energy and glucose homeostasis, reproduction and cognition 
(43, 51-53). There are good data in rodents that brain insulin signaling suppresses hepatic glucose 
output (54). Lack of brain insulin receptors leads to mild diabetic phenotype in mice, along with HH as 
mentioned above (43). Studies also suggest that IN insulin reduces sympathetic outflow to adipose 
tissue and decrease free fatty acid concentrations(55). There are only a few human studies on intranasal 
insulin’s effects on metabolism. Acute administration of intranasal insulin reduces food intake while 
chronic administration reduces fat mass (56, 57). These effects are more pronounced in men than in 
women for unclear reasons. Acute intranasal administration also decreased HOMA-IR(58).  A recent 
study showed that intranasal insulin increased insulin sensitivity measured by hyperinsulinemic 
euglycemic clamp (gold standard for measuring insulin sensitivity) by 41% in lean men(59). This was 
accompanied by an increase in activity of parasympathetic  nervous system and an increase in cerebral 
blood flow in the hypothalamic region (as assessed by functional magnetic resonance imaging). There 
was no change in insulin sensitivity of overweight/obese men. Presumably, there is “brain insulin 
resistance” in obese men but there is no direct evidence for this. Intranasal insulin has been shown to 
improve cerebral blood flow, cortical activity, memory and cognition in healthy subjects, diabetics and in 
patient with Alzhemier’s disease(53, 60). 
In the proposed project, we will study the effect of intranasal insulin on LH concentrations in lean 
healthy men as well as in men with type 2 diabetes.  
Pharmacokinetics of intranasal (IN) insulin: Born et al measured concentrations of insulin in CSF and 
serum for 80 minutes after IN administration(61). 27 men and 9 women were studied. After IN dose of 
40IU regular insulin, CSF insulin concentrations increased rapidly and remained elevated above baseline 
for the duration of the study (figure above). The area under curve increased from 603 to 
1091pmol/l*min. There was no change in serum insulin concentrations. Results were similar with ACTH 
or vasopressin administration.  
It is assumed that after IN administration, insulin travels extracellularly through patent intercellular 
clefts in the olfactory epithelium to diffuse into the subarachnoid space. This appears more plausible 
that the alternate intracellular route which would carry a greater risk of proteolysis and would take 
hours to reach the olfactory bulb(62).  
 


Protocol version 01-29-2015 
Page 6 of 16 
 
    
 
CSF 
 
 
 
 
 
 
 
Serum 
 
 
Vianase device: Vianase is a liquid drug delivery system based on controlled particle dispersion 
technology. It is manufactured by Kurve technology (www.kurvetech.com). The device is being donated 
by Kurve technology for this research project. They usually donate one device to a center (to begin with) 
after a “material service agreement” has been reached. The device has a disposable nose piece and 
hence can be re-used. The insulin (regular human insulin) is reconstituted for each application. The 
device delivers a metered dose of insulin into the chamber that covers the subject’s nose. Using the 
principal of vortical flow, Controlled Particle Dispersion effectively disrupts inherent nasal cavity airflows 
to deliver formulations to the entire nasal cavity. By varying control parameters, CPD can target specific 
nasal regions including the olfactory region and the paranasal sinuses while minimizing peripheral 
deposition to the lungs and stomach. 
 
Device Information provided by Kurve Technology, Inc. 
ViaNase is a Class I electrically powered atomizer operating under Code CFR 21 and JPW 8774.5220 
Device 
Pump, nebulizer, electrically powered 


Protocol version 01-29-2015 
Page 7 of 16 
 
Regulation Description 
Ear, nose, and throat drug administration device. 
Regulation Medical Specialty 
Ear Nose & Throat 
Review Panel 
Ear Nose & Throat  
Product Code 
JPW 
Submission Type 
510(K) Exempt  
Regulation Number 
874.52206  
Device Class 
1  
Total Product Life Cycle (TPLC) 
TPLC Product Code Report7  
GMP Exempt? 
Yes  
Note: This device is also exempted from the GMP regulation, except for general 
requirements concerning records (820.180) and complaint files (820.198), as long 
as the device is not labeled or otherwise represented as sterile. 
Note: FDA has exempted almost all class I devices (with the exception of reserved 
devices8) from the premarket notification requirement, including those devices 
that were exempted by final regulation published in the Federal Registers of 
December 7, 1994, and January 16, 1996. It is important to confirm the exempt 
status and any limitations that apply with 21 CFR Parts 862-8929. Limitations of 
device exemptions are covered under 21 CFR XXX.9, where XXX refers to Parts 
862-892. 
 
If a manufacturer's device falls into a generic category of exempted class I devices 
as defined in 21 CFR Parts 862-89210, a premarket notification application and 
FDA clearance is not required before marketing the device in the U.S. however, 
these manufacturers are required to register their establishment. Please see the 
Device Registration and Listing website11 for additional information.  
Third Party Review 
 
Not Third Party Eligible 


Protocol version 01-29-2015 
Page 8 of 16 
 
 
(Continued) 
TITLE 21--FOOD AND DRUGS 
CHAPTER I--FOOD AND DRUG ADMINISTRATION 
DEPARTMENT OF HEALTH AND HUMAN SERVICES 
SUBCHAPTER H--MEDICAL DEVICES 
PART 874 -- EAR, NOSE, AND THROAT DEVICES 
Subpart F--Therapeutic Devices  
Sec. 874.5220 Ear, nose, and throat drug administration device.  
(a)Identification. An ear, nose, and throat drug administration device is one of a group of ear, nose, and 
throat devices intended specifically to administer medicinal substances to treat ear, nose, and throat 
disorders. These instruments include the powder blower, dropper, ear wick, manual nebulizer pump, 
and nasal inhaler. 
(b)Classification. Class I (general controls). The device is exempt from the premarket notification 
procedures in subpart E of part 807 of this chapter subject to the limitations in 874.9. If the device is not 
labeled or otherwise represented as sterile, it is exempt from the current good manufacturing practice 
requirements of the quality system regulation in part 820 of this chapter, with the exception of 820.180, 
with respect to general requirements concerning records, and 820.198, with respect to complaint files. 
[51 FR 40389, Nov. 6, 1986, as amended at 59 FR 63009, Dec. 7, 1994; 66 FR 38801, July 25, 2001]  
 
The following are some of the studies that have used the Vianase device to deliver intranasal insulin: 
Claxton et al; Sex and ApoE genotype differences in treatment response to two doses of intranasal 
insulin (20IU or 40IU) in adults with mild cognitive impairment or Alzheimer's disease. J Alzheimers Dis. 
2013;35(4):789-97.  


Protocol version 01-29-2015 
Page 9 of 16 
 
Craft et al; Intranasal insulin therapy for Alzheimer disease and amnestic mild cognitive impairment: a 
pilot clinical trial. Arch Neurol. 2012 Jan;69(1):29-38. Subjects used 10IU bid or 20IU bid for 4 months. 
As per authors: “No treatment-related severe adverse events occurred during our study, and most 
adverse events were minor, such as mild rhinitis. The adverse events that occurred more than 5% of the 
time in any group are listed in table”:-  
 
Novak et al; Enhancement of Vasoreactivity and Cognition by Intranasal Insulin in Type 2 Diabetes. 
Diabetes Care. 2013 Oct 25. Dose used was 40IU (given once for the study).  
This study is very similar to this protocol because we intend to give patients one dose of 40IU. As per 
authors, in this study, “there were no hypoglycemic episodes, nasal irritation or allergic reactions to 
insulin. Glucose levels and vital signs were stable and similar across insulin and placebo conditions in 
both groups”. 
Significance: HH is one of the most common conditions associated with insulin resistance state in men. 
Very little is known about its pathophysiology. This study will attempt to evaluate the effect of 
increasing insulin concentrations in the brain on release of LH.  
 
It is not the aim of this study to diagnose, cure or treat a disease condition (such as HH). The aim is to 
understand the role of insulin signaling in brain in the release of LH. 
 
Future directions: Leptin has a permissive role in reproductive axis. Once available, intranasal leptin 
alone or in combination with insulin also needs to be studied. 
 


Protocol version 01-29-2015 
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Hypothesis: One dose of 40 IU intranasal insulin administration increases serum concentrations of LH in 
healthy men and in men with type 2 Diabetes. 
 
Objective 1: To measure the concentrations of LH every 15 minutes for 3 hours after administration of 
40 IU of intranasal insulin or saline. 
 
 
STUDY DESIGN AND METHODS: 
Study population: 14 men with type 2 diabetes and 14 healthy men with a normal BMIwill be recruited for the 
study.  
 
Screening Day: After informed consent is obtained, all subjects will complete the following procedures: 
1) Medical History  
2) Physical Exam 
3) Baseline HbA1c (in type 2 diabetic men only). If this been done in the last 6 weeks by patient’s health 
care provider, and the results are available, then they will not be re-drawn. This can be done by finger-
prick. 
 
Randomization Method: Subjects who qualify for the study will be assigned a number by a 
computerized random number generation program (Microsoft office – Excel) and will be randomized to 
receive either intranasal insulin or saline at study visit 1 (randomized, cross-over design). This number 
will be used as the study subject ID.  A master list of subject name and study ID will be kept under lock 
and key to protect patient identifiers.  For visit 2 subjects who received intranasal insulin will receive 
intranasal saline and vice versa.  
The subjects and key study personnel, including the PI, will be blinded to the treatment. The study 
coordinators, the study subjects and laboratory personnel will be blinded. The sub investigators (Dr. 
Chemitiganti or Dr. Bilkis) will be responsible for preparation of the drug for vianase device and hence 
will not be blinded. 
Study visit 1: Subjects will come fasting to the clinical center of TTUHSC-Odessa campus between 8 and 
9 AM.  For the diabetic group, they will not take their anti-diabetic pills in morning. If they are on long 
acting insulin in AM, that will be withheld as well. Diabetic subjects can take their anti-diabetic pills and 
long acting insulin the night before the study. In order to avoid the confounding effects of poorly 
controlled diabetes, patients with HbA1c >8.5% will be excluded. 
 
A peripheral intravenous cannula will be placed for intermittent blood draws.  
 
Blood sample will be drawn every 15 minutes for 2 hours, starting at 9AM.  
 
At 11AM (after the 2 hour samples have been drawn), subjects will spray 40 IU of insulin or 
equal volume of saline into their nostril once with the vianase device.  
 
Blood sugar will be checked just before the administration of insulin/saline, and after 30, 60, 120 
and 180 minutes. Blood sugar will be checked in the venous blood that is drawn at all these time 
points (see next tab). 
 
Blood sample will be drawn every 15 minutes for the next 3 hours.  
 
Last blood sample will be collected at 2 PM. IV line will then be removed and subjects will be 
discharged home. 
 


Protocol version 01-29-2015 
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Study visit 2: This visit will be scheduled for approximately one week after visit 1. Visit 2 will not be done 
sooner than 5 days after visit 1 or later than 2 months. The same procedure will be followed as listed for 
visit 1. Subjects who received intranasal insulin will receive intranasal saline and vice versa. 
 
About 150ml of blood will be drawn during each study visit. 
 
 
Inclusion Criteria: 
 
Males age 18 to 75 years 
 
Type 2 diabetes OR normal (BMI 19-24.9 kg/m2) healthy men 
 
Exclusion Criteria:-  
 
HbA1c>8.5% 
 
Use of testosterone currently or in the past 4 months 
 
Use of over the counter health supplements which contain androgens 
 
Use of corticosteroids or narcotics in the past 3 months 
 
Coronary event or procedure(myocardial infarction, unstable angina, coronary artery bypass, 
surgery or coronary angioplasty) in the previous twelve weeks  
 
Type I Diabetes 
 
Currently suffering from foot ulcer, significant periodontal disease or any other chronic 
infectious or inflammatory condition  
 
Known history of hepatic disease (transaminase > 3 times normal) or cirrhosis 
 
Known history of renal impairment (defined as GFR<30) 
 
HIV or Hepatitis C positive status 
 
Any other life-threatening, non-cardiac disease 
 
History of untreated severe obstructive sleep apnea(defined as apnea-hypopnea index ≥30)  
 
currently suffering from symptomatic depression, with or without treatment 
 
Use of an investigational agent or therapeutic regimen within 30 days of study 
 
Participation in any other concurrent clinical trial 
 
Pituitary tumor/damage/ other trophic hormone deficiency 
 
Laboratory measurements  
Since LH is secreted in a pulsatile manner, LH concentrations will be measured at 15 minute intervals 
throughout the study (for about 5 hours). The 2 hour period prior to intranasal treatment will serve as 
the baseline period. Normal men secrete LH pulses at the rate of 0.8 pulses/hr(63).  It is likely that the 
secretion in our study population (with HH) will be lower than normal men. 
 
Apart from the lab tests mentioned above, 10ml of blood will be drawn at time 0h, 2h and 5h and stored 
for future analysis. We will consider measuring testosterone concentrations in the stored samples if the 
study shows a rise in LH concentrations. 
 
Statistical analysis: The primary endpoint of the study is to compare the LH concentrations after 
intranasal insulin as compared to placebo (intranasal saline). Area under curve (AUC) of LH 
concentrations in the 3 hours following intervention will be compared by paired t-test or appropriate 
non-parametric equivalent. Type I error (α) will be set at 0.05 and Type II error (β) at 0.2. Assuming that 
there will be a 50% increase in LH concentrations, 12 subjects will be needed for the experiment 


Protocol version 01-29-2015 
Page 12 of 16 
 
provided that the standard deviation of the residuals is no more than the mean change. Assuming a 
dropout rate of 15%, 14 patients will be recruited. 14 healthy men will also be recruited. 
 
Secondary end points: A secondary endpoint of the study is to compare the change in LH concentrations 
after intranasal insulin as compared to placebo (intranasal saline). Change will be defined as the 
difference between baseline LH concentrations (average of the 9 samples collected in the two hours 
prior to intervention) and average LH concentrations following intervention (average of the 16 samples 
collected in the four hours following intervention). Paired t-test or the appropriate non-parametric 
equivalent will be used for comparisons. Another secondary end-point will be comparing the change in 
LH concentrations by repeated measures ANOVA.  Finally, we will compare the change in LH 
concentrations between diabetic men and healthy men. 
 
Method of identifying and recruiting subjects: Subjects will be recruited from the TTUHSC clinics. 
Compensation given to subjects: Subjects will receive $200 for completing the study. In case they 
complete only one study visit, they will receive $100. They will not be compensated for screening visits.  
Describe the site(s) where study will be done: The study will be conducted at TTUHSC Odessa campus. 
Blood tests will be analyzed by LabCorp. 
Risks:  Placement of IV cannulas may result in pain, a feeling of faintness, irritation of the vein, bruising, 
or bleeding and possible infection at the site of puncture.  Systemic absorption of intranasal insulin is 
not expected at the dose used in the study, hence hypoglycemia is not anticipated and blood sugars will 
not be monitored intensively. However, a fingerstick blood sugar will be checked 30 minutes after taking 
the insulin/saline. Subjects will also be monitored for any symptoms suggestive of hypoglycemia. If there 
are symptoms at any time, fingerstick blood glucose will be checked. If finger stick is below 60, orange 
juice will be provided. Patient will then be rechecked in 15 minutes. If still below 60, the patient will be 
given orange juice and the study will be terminated.  Data collected till that time will be stored for 
analysis. 
About 150ml of blood will be drawn during each study visit; this amount is not typically associated with 
adverse effects. 
Although the vianase device is not approved to market, the device and the dose of IH intranasal 
insulin chosen for this study has been shown to be safe in numerous prior studies which have been 
referenced in this protocol.  It is not the aim of this study to diagnose, cure or treat a disease 
condition (such as HH). The aim is to understand the role of insulin signaling in brain in the release of 
LH. 
 
Possible benefits to subjects: Subjects may experience the altruistic benefits of participating in 
research. They will not have any direct benefit from the study. 
Confidentiality measures: All subjects who qualify and agree to be in the study will receive a 
randomization number. They will then be identified during data collection by their initials and 
randomization number only. Data will be stored in locked office and password protected computer. 
Funding: 


Protocol version 01-29-2015 
Page 13 of 16 
 
This study is funded by the  School of Medicine Regional Dean’s Office at the Permian Basin.  The 
Vianase device has been donated to TTUHSC by Kurve technology Inc. for research. 
 
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