Clinical Trial to Assess the Effects of Candesartan on the Carbohydrate Metabolism of Obese Subjects
Clinical Trial to Assess the Effects of Candesartan on the Carbohydrate Metabolism of Obese Subjects
Hypothesis:

 The use of candesartan 16-32 mg/d for 6 months improves the carbohydrate metabolism, and
 decreases the plasmatic levels of adipocytokines and oxidative stress markers, in non
 diabetic, non hypertensive subjects with dysglycemia and abdominal obesity, and these effects
 are independent of the changes in arterial blood pressure.

 General Objectives:

 The objective is to study the impact of the treatment with candesartan in the carbohydrate
 metabolism and the plasmatic levels of adipocytokines and oxidative stress markers, in non
 diabetic, non hypertensive subjects with dysglycemia and abdominal obesity.

 Study Design:

 This is a randomized, double blind, cross-over, placebo-controlled, clinical trial to assess
 the effects of candesartan (up to 32 mg/d for 6 months), over the carbohydrate metabolism,
 plasma levels of adipocytokines and concentrations of oxidative stress markers in non
 diabetic, non hypertensive, dysglycemic and obese subjects from Colombia. The total duration
 of the study is 36 months.

 Population:

 One hundred non diabetic, dysglycemic and obese, subjects of both genders, over 18 years old,
 will be included. To be included subjects should have blood pressure values under 140/90 mmHg
 and should be receiving no antihypertensive medical treatment.

 Procedures:

 Subjects whom fulfill all selection criteria will be included in a run-in period of 15 days
 with placebo and hygiene-dietary measures (MHD) including educational, nutritional and
 exercise support. The patients that during this "Run in" phase have a compliance equal to or
 greater than 80% will be randomized to one of the two treatment groups ("Group A" receiving
 candesartan 16/32 mg/d for 6 months and then placebo for 6 months, or "Group B" receiving
 placebo during the first 6 months and then candesartan 16/32 mg/d during the last 6 months)
 in a 1:1 proportion by blocks of 4 subjects. Randomization will be performed by the
 AstraZeneca clinical department. Both groups will concurrently receive the standard treatment
 with MHD. Control visits will be programmed every month. Metabolic parameters, including
 C-reactive protein (CRP), interleukin-6 (IL-6), adiponectin, leptin, insulin, malonaldehyde
 and 8-isoprostanes, will be evaluated every 6 months (at the beginning and end of each
 treatment).

 Statistical Analysis:

 The analysis strategy will be performed by intention-to-treat. In a descriptive analysis, the
 averages and proportions will be obtained with their corresponding 95% confidence intervals
 for the clinically relevant variables during the baseline evaluation. In order to evaluate
 the differences between the groups, the Student's t test, Mann-Whitney and Fischer's exact
 tests will be used according to the nature of the study variables. Multiple lineal regression
 will be used with the purpose of comparing the treatment groups from baseline and its changes
 up to the 6th month of treatment.

 Ethical Aspects:

 The study will be conducted according to the Helsinki declaration, the good clinical
 practices guidelines and the Colombian legislation. Prior to entering the study, patients
 must sign a written informed consent that has been approved by the Institutional Ethics
 Committee of Fundación Cardiovascular de Colombia.
Background:

 During the last years, the worldwide prevalence of diabetes mellitus type II (DM2) has
 increased dramatically, impacting the cardiovascular morbidity and mortality. It has been
 estimated that more than 171 million people suffer this disease (2.8% of the worldwide
 population) and it's predicted that it will increase to 366 million (6.5 %) in 2030, from
 which 298 million will be from developing countries. Currently, in Latin America, the DM2
 prevalence ranges are between 1.2% and 8%, and it is expected to increase 38% during the next
 10 years, with higher levels in the urban zones.

 Recently, we have demonstrated that the Colombian population with a lower abdominal
 circumference than those reported in Caucasian populations presented an increased risk of
 developed metabolic syndrome and coronary artery disease. Moreover, abdominal obesity in our
 population is associated with higher levels of inflammatory markers, as C-reactive protein
 (CRP) and proinflammatory cytokines. Nowadays, the relationship between abdominal obesity,
 inflammation, insulin resistance, diabetes mellitus type 2, metabolic syndrome and
 cardiovascular disease is a crucial aim of research, especially in populations as the
 Colombian, that is in high risk of being affected by the epidemic of diabetes mellitus and
 cardiovascular disease. The occurrence of DM2 is associated with a 2 to 4 fold increase in
 the risk of developing coronary disease. The diabetic patients that present unstable angina
 have a greater risk of developing acute myocardial infarct (AMI) and the diabetic patients
 with AMI have more risk of death than the non-diabetic patients. Additionally, the subjects
 with DM2 have an increased risk of experiencing cardiovascular events (1.5 to 3 times), and
 greater recurrence and mortality for these causes. The incidence and severity of the
 peripheral arterial disease are also increased from 2 to 4 times in diabetic patients.

 The current criteria for DM2 diagnosis established by the American Association of Diabetes is
 a glucose level in fasting >126 mg/dl, which has been established based on the risk of
 suffering ophthalmic and renal micro-vascular complications with values superior to this
 limit. Nevertheless, several works have shown that patients with altered fasting glucose
 levels (≥100 mg/dl - <126 mg/dl) have an increased risk of cardiovascular morbidity and
 mortality.

 We recently reported in Colombia, the existence of a strong association between the presence
 of cardiovascular risk factors and altered fasting plasma glucose, this association being
 greater with the presence of abnormal glucose blood levels after the glucose overload test.
 This association has been explained since the hyperglycemia per-se may be implicated in the
 development of atherosclerosis due to metabolic and structural changes at the endothelial
 level. At long term it may result in irreversible alterations; a "non-returning" point
 leading to cardiovascular complications typical of diabetes. According to these observations,
 our group has recently shown that patients with altered glycemia in fasting, regardless of
 other classic factors of cardiovascular risk, present a greater risk of coronary disease,
 supporting the hypothesis that the hyperglycemia leads to structural changes in the
 endothelial wall.

 It is well known that people with insulin resistance present less vasodilatation mediated by
 insulin and an altered endothelium dependent vasodilatation. Additionally, it has been
 described that insulin causes a physiologic vasodilatation in the skeletal muscle of healthy
 subjects, an effect that is blocked in insulin-resistance obesity. In our population, we find
 that obesity, hypercholesterolemia and diabetes are related with a flow-mediated
 vasodilatation reduction. The endothelial dysfunction is strongly related with the insulin
 resistance syndrome. At the same time, the endothelial dysfunction worsens the resistance to
 insulin, increases the vascular reactivity and predisposes to cardiovascular disease. It has
 been proposed that the vasodilator effect of the insulin is primarily due to a greater
 expression of mRNA for endothelial nitric oxide synthase (eNOS) due to a probable increase of
 the transcriptional rate. There are elements to support the fact that this activity is
 modulated by C-kinase protein (PKC). The inhibition of PKC increases the mRNA levels for
 eNOS. Prolonged incubation of endothelial cells with a selective inhibitor of the PKC beta
 isoform increase the mRNA levels of eNOS. This observation may have important clinical
 implications since the PKC activation in the vasculature of diabetic subjects in alteration
 of the vascular wall. The existing relationship between endothelial dysfunction and insulin
 resistance is dependent on multiple factors. Obesity generates an alteration of the
 endothelial function in the metabolically active capillary bed, altering the lipase
 lipoprotein (LPL) that is linked to the endothelium by glycosaminoglycans. The ultimate loss
 of these caused by aggressive factors such as smoking and oxygen free radicals, alters the
 endothelial function and also impairs the action of the LPL causing hypertriglyceridemia
 which is an insulin resistance factor. At the same time, the alteration of endothelial
 function in the capillary beds reduces the interstitial flow, carrying less insulin or
 delaying its delivery to the muscular tissue. Additionally, it has been proposed that there
 is less surface of endothelium functionally normal in the vessels that are irrigating the
 skeletal muscle. It has also been shown that hyperinsulinemia predicts the appearance of
 atherosclerosis and cardiovascular events, independently of other risk factors.

 The increase of adipocytes at the abdominal level is directly related with a condition of
 insulin resistance and hyperinsulinism. The hyperinsulinemia promotes the release of free
 fatty acids by the adipocyte and its later hepatic transformation to oxidized LDL, which have
 a great atherogenic potential. Also, the abdominal adipocytes in response to the increase of
 free fatty acids, oxidized LDL or any other non-well defined metabolic factor increase the
 production and release of proinflammatory cytokines such as the tumor necrosis factor alpha
 (TNF-α) and interleukin-6 (IL-6), which have shown to be able to reduce the expression and
 activity of the nitric oxide synthase (eNOS) in human cultivated umbilical endothelium cells,
 suggesting that this could be the mechanism by which the abdominal obesity is related with
 endothelial dysfunction. In this sense, we have recently demonstrated that in cultured
 endothelial cells, the angiotensin II, through receptor AT1, stimulates the TNF-α production,
 which at the same time activates the metalloproteinase 2, enzyme which induces changes in the
 endothelium structure and in the stability of the atherosclerotic plaque. This effect of
 angiotensin II is mediated by the AT1 receptor, since we have shown that the CANDESARTAN
 inhibited the production of TNF-α induced by Angiotensin II.

 The increase in the production and storage of free fatty acids may be the mechanism by which
 angiotensin II relates with the development of insulin resistance. It has been demonstrated
 that the elevated levels of free fatty acids induce insulin resistance through the inhibition
 of the transport and phosphorylation of glucose at a muscular level, followed by a reduction
 of the glycogen synthesis and glucose oxidation. Additionally, it has been suggested that
 free fatty acids may interfere in the stimulation of insulin in the GLUT 4 and hexokinase
 activity. Also, angiotensin II has a stimulating effect on the transcription rate of the ob
 gene in human adipocytes. The ob gene is in charge of codifying the leptin protein, which
 inhibits the appetite and regulates the thermogenesis. The chronic and sustained increase in
 leptin levels leads to a leptin-resistance condition, a condition in which the hormone loses
 its physiological actions. The increase in the leptin levels produces a greater expression of
 UPC 2 (Uncoupling protein 2) in an action mediated by Peroxisome Proliferator-Activated
 Receptors (PPARs), which interferes with the mitochondrial respiration chain in the
 pancreatic beta cells, getting to a reduction in the ATP generation and blocking the first
 peak of insulin secretion. This offsetting mechanism seems to be opposed to the insulin's
 lipogenic effect and avoids greater lipid storage. Unfortunately, the derived consequences of
 this alteration in the insulin secretion are a lower efficacy to maintain the euglycemia and
 to maintain a glucidic homeostasis. On the other hand, the hyperleptinemia, by mediating the
 sympathetic activity and increasing the sodium renal resorption, leads to the increases of
 blood pressure, which adds to the greater expression of the angiotensinogen produced by
 hypertrophied lipid cells. These cells generate angiotensin II to activate the adipocyte
 differentiation and to regulate the fat storage in response to nutritional changes.

 Recently, some clinical trials have demonstrated that the angiotensin converting enzyme
 inhibitors (ACEIs) and ARA II, reduce the risk of presenting new cases of DM2 as compared
 with other antihypertensive therapies. However, no clinical trials have addressed
 specifically the study of the effects of the ARA II in the improvement of the dysglycemia and
 in the prevention of the diabetes type 2. Additionally, the treatment with ACEI and ARA II
 has shown to improve the resistance to peripheral insulin both in animal models and clinical
 studies. The mechanism by which the renin-angiotensin-aldosterone system blockade has a
 beneficial effect on the responsiveness to insulin has not been totally cleared. In obese
 Zucker-type rats, it was demonstrated that the chronic treatment with a selective ARA II
 receptor produced a significant increase in the GLUT 4 transporter expression in skeletal
 muscle and a reduction in the concentrations of plasma fatty acids associated with an
 improvement of the responsiveness to insulin.

 Although the mechanisms are still speculative, the beneficial effects of the
 renin-angiotensin system blockade, demonstrated in several studies such as HOPE, LIFE, VALUE,
 on the responsiveness to insulin and in the prevention of the development of new cases of
 DM2, suggest that angiotensin II produced in the adipocytes of obese subjects is associated
 to the insulin resistance syndrome and supports the execution of clinical trials oriented to
 establish the beneficial effect of ARA II in the prevention of DM2 in susceptible individuals
 coming from populations at high risk of developing CVD and diabetes mellitus, as the
 Colombian one.

 Hypothesis:

 In non-diabetic non-hypertensive subjects with dysglycemia and abdominal obesity:

 The treatment with candesartan 16/32 mg/d during 6 months improves carbohydrate metabolism
 measured by HOMA insulin sensibility index, oral glucose tolerance Test (OGTT), fasting
 plasma glucose levels, and glycosylated hemoglobin (HbA1c).

 The effects of the treatment with candesartan over carbohydrate metabolism is related to a
 reduction in the plasma concentration of adipocytokines such as IL-6, CRP, Leptin and
 Adiponectin; and oxidative stress markers such as plasma concentrations of Malonaldehyde and
 urinary concentration of 8-Isoprostanes.

 These effects over carbohydrate metabolism, inflammatory adipocytokines or oxidative stress
 markers are independent of changes over arterial blood pressure.

 Study Objectives:

 General Objectives:

 To study the impact of the treatment with candesartan over the carbohydrate metabolism,
 inflammatory adipocytokines and levels and oxidative stress markers in non-diabetic
 non-hypertensive subjects with dysglycemia and abdominal obesity.

 Specific Objectives:

 In non-diabetic non-hypertensive subjects with abdominal obesity and dysglycemia:

 To establish the impact of the treatment with candesartan (16/32 mg/d) in the carbohydrate
 metabolism assessed through the HOMA index, fasting glucose plasma levels, OGTT, and HbA1c
 levels.

 To study the effects of candesartan 16/32 mg/d during 6 months over fasting plasma levels of
 adipocytokines such as Leptin, Adiponectin, IL-6 and CRP.

 To determine if the treatment with candesartan (16/32 mg/d during 6 months) decreases the
 concentration of oxidative stress markers such as plasma levels of malonaldehyde and urinary
 levels of 8-Isoprostanes.

 To determine if the effects of candesartan on the carbohydrate metabolism, adipocytokines and
 oxidative stress marker concentrations, are independent of its effect upon the blood
 pressure.

 Study Design:

 A randomized, double blind, placebo-controlled, cross-over clinical trial, to assess the
 effects of candesartan (16/32 mg/d during 6 months) over metabolic, oxidative, and
 inflammatory parameters, in non-diabetic non-hypertensive subjects with dysglycemia and
 abdominal obesity.

 Study treatments:

 Treatment A: Candesartan 16 mg (one tablet per day) taken with breakfast during 4 weeks,
 depending on the subject's tolerance the dosage will be increased to 32 mg/d (two tablets)
 during the next 20 weeks.

 Treatment B: Placebo tablets administered similarly to treatment A (one tablet during 4 weeks
 and then 2 tablets per day during the next 20 weeks).

 All subjects will be included in a hygiene-dietary measures program (MHD; educational,
 nutritional and exercise support) during the study.

 Study groups:

 The study embraces two arms

 Group 1: Will receive treatment A first during the first 24 weeks and then treatment B during
 the last 24 weeks.

 Group 2: Will receive treatment B during the first 24 weeks and then treatment A during the
 last 24 weeks.

 Population:

 The study is going to be integrated by non-diabetic non-hypertensive subjects of both
 genders, older than 18 years with abdominal obesity and dysglycemia .

 Abdominal obesity is defined as a waist diameter greater than 90 cms in men and 80 cms in
 women.

 Dysglycemia is defined as having plasma glucose levels in fasting between 100 and 125 mg/dL
 and/or on glucose tolerance test at 2 hours between 140 mg/dL and 200 mg mg/dL.

 Size of sample:

 The size of the sample was estimated considering a crossover, clinical trial design and
 following the proposal by Hills and Armitage accepting a type I error of 0.05, a power of
 90%, and assuming a difference of 20% in the HOMA index after 6 months of treatment with
 candesartan (3 to 2.4) and a maximum standard deviation of 1.5, sample size of 84 subjects
 was estimated. By adjusting the rate of losses of 8%, the final sample size is 100 subjects
 (50 in each group).

 This sample size ensures a power of 90% to detect differences in the fasting glycemia of at
 least 8mg/dL (0.44mmol/L), with a standard deviation (SD) of 20mg/dL. (1.1mmol/L) or a
 difference of 14mg/dL (0.77mmol/L) in the 2 hours post load glycemia SD of 40mg/dl
 (2.2mmol/L).

 Statistical Analysis:

 The study is fundamentally set forth as an efficacy study on the prevention of the
 development of diabetes mellitus and on the changes of the carbohydrate metabolism. The
 averages and proportions with their corresponding 95% confidence intervals for clinically
 relevant variables measured during the baseline evaluation will be obtained in a descriptive
 analysis. In order to evaluate the presence of differences between the groups, the Student's
 t test, the Mann-Whitney test, the CHI2 test or the Fisher´s exact test according to the
 variable under study will be used. A Linear multiple regression will be used with the purpose
 of comparing the treatment groups from baseline and its changes up to month end 6 of each
 treatment. The possibility of performing adjustments by required baseline parameters and risk
 factors and prior treatment in each one of the treatment groups is considered.

 The analysis will be performed by the intention-to-treat approach. A p value under 0.05 will
 be considered as statistically significant.The primary endpoint for analysis will be made on
 the change in HOMA index value, baseline glucose, and post-charge glucose plasma levels. The
 secondary endpoint for analyses will include the possible changes in serum insulin, leptin
 and adiponectin, inflammatory markers and oxidative stress markers .

 Treatment safety register and analysis will be made through the clinical review and
 statistics of the adverse events reported.

 Safety Committee and Events Assignation Committee:

 A safety and events assignation committee will be created, according to the Harmonized
 Tripartite Guidelines of the International Conference of Harmonization for Good Clinical
 Practices.

 Ethical Aspects:

 The clinical trial will be conducted according to the Helsinki's Declaration, Good Clinical
 Practice Guidelines and the Colombian legislation (Resolution 8430-93 of the Ministry of
 Health). The patient will provide written informed consent in a form designed for such a
 purpose.

 The information generated by the study will be confidential and strictly used for the
 purposes stipulated within the protocol.

 Finally, the patient may refuse to continue participating in the study at any moment after
 providing his/her consent. The study will be approved by the FVC ethics committee. All
 assessments will be performed by trained staff. The blood samples will be collected in
 aseptic conditions by an expert bacteriologist.

 Study Timeline:

 The study period will be 36 months. The initiation will be defined by the project's financial
 approval.
Inclusion Criteria:

 The subjects that fulfill the following inclusion criteria will be eligible to participate
 in the study:

 -  Men and women older than 18 years of age.

 -  Waist perimeter > 90 cm in males or > 80 cm in females

 -  Have plasma glucose levels in fasting between 100 and 125 mg/dL and/or on glucose
 tolerance test at 2 hours > 140 mg/dL and < 200 mg/dL.

 -  Having a treatment compliance of over 80% at the end of the run-in phase.

 -  All women with childbearing potential must have a secure contraceptive method. A
 secure method will be considered as sterilization by surgical methods, postmenopausal
 condition with an age greater than 45 years and a menopausal period equal to or
 greater than two years. In premenopausal women, the use of two barrier contraceptive
 methods including 1 month after the conclusion of the active phase of study treatment.

 Exclusion Criteria:

 Individuals with any of the following characteristics will be excluded:

 -  Prior diagnosis of type 1 or 2 diabetes mellitus, chronic or acute renal
 insufficiency, coronary disease clinically evident (acute myocardial infarction, chest
 angina, myocardial revascularization) or cardiac insufficiency, or history of prior
 cardiovascular events (AMI, CVD, or CABG).

 -  Significant chronic disease (terminal stage cirrhosis or hepatic disease or cancer)
 that affects the survival of patients at 12 months.

 -  Chronic inflammatory diseases of obesity (lupus erythematosus, rheumatoid arthritis,
 etc.)

 -  Infectious acute or chronic processes of any etiology with an occurrence within the 4
 weeks prior to the beginning of the study.

 -  Use of steroid hormones or NSAIDs 1 month prior to the beginning of the study.

 -  The patient is participating in a program or under treatment to lose weight during the
 8 weeks prior to the study entry.

 -  The patient requires (for any circumstance) treatment with immunosuppressive agents.

 -  Has participated in a clinical trial in the 8 weeks prior to the study entry.

 -  At the study entry, the patient is considering the possibility of a surgical procedure
 during the next 12 months.

 -  History of severe chronic gastritis or any condition of the gastrointestinal tract
 that may affect the absorption and/or distribution of any drug administered orally.

 -  Alteration of the hepatic function tests. The maximum value for ALT or AST will be
 considered as > 2 times the upper normal limit.

 -  Triglycerides > 600 mg/dl.

 -  History of the use of psychoactive drugs or abuse of alcohol.

 -  Positive pregnancy test in the screening visit.

 -  Concomitant treatment with any other antihypertensive drug.

 -  Contraindication to receive treatment with candesartan.

 -  Pathological alterations of aortic or mitral cardiac valves (stenosis or
 insufficiency) or hypertrophic cardiomyopathy.

 -  Denial to sign informed consent, or any mental condition that makes the patient part
 of a susceptible population