Effect of Addition of Losartan to Metformin Alone and in Combination with Glimepiride

Effect of addition of losartan to metformin alone and in combination with glimepiride or repaglinide on fasting blood sugar level, glycatedHb, antioxidant status and lipid profile parameters in type 2 diabetes patients with proteinuria

______

Dr. Momin M A Mujeeb*

Dr. Prakashchandra R Gade**

* Associate Professor of Pharmacology,

Govt. Medical College,Nagpur 440001

**Professor & Head,

Department of Pharmacology,

Govt. Medical College,Nagpur 440001

______

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, hyperlipidimia, negative nitrogen balance and sometimes ketoacidosis. Diabetic nephropathy is one of the common complication of long standing diabetes.1 Non communicable diseases of which coronary artery disease and diabetes top the list, have overtaken communicable diseases with respect to overall mortality even in developing countries like India.2.3,4 Indeed the epidemic of diabetes and coronary artery disease is now spreading to middle and lower income groups in India. The risk for coronary artery disease (CAD) is two to four times higher in diabetics subjects and in Indians, CAD occurs prematurely i.e. one or two decades earlier than in the West.5,6,7 Thus there is an urgent need for studies on CAD in diabetic and non diabetic subjects in India. One of the well accepted predictor of CAD is deranged lipid profile parameter.8

Type 2 diabetes shares several risk factors in common with coronary artery disease such as age, hypertension, dyslipidemia, obesity, physical inactivity, stress.9,10,11 Therefore increase in prevalence of diabetes indirectly implicates an escalating risk of CAD as well. Diabetic subjects are known to have a two to four times higher risk of getting CAD.12,13,14

Diabetes mellitus is associated with increased oxidative stress due to hyperglycemia.15This increased oxidative stress gives rise to micro and macrovascular complications. Long term complications involves almost all vital organs like heart, eyes, kidney, blood vessels and nervous system. These complications will lead to the development of obesity, hypertension, dyslipidemia and insulin resistance.16,17,18 There is close association between complications of diabetes and diabetic dyslipidemia.19,20 Atherosclerosis is a primary cause of death in patients with diabetes.21,22 The pathophysiology of development of atherosclerosis is complex and multifactorial.23,24 Diabetic dyslipidemia accounts for around 80% diabetic death due to cadiovascularcomplications.There is growing body of evidence to show that hyperglycemia and dyslipidemia are connected with excess of cardiovascular risk.25,26

There are currently 150 million people with diabetes in the world, and India leads the world with 60 million people of diabetes.27 Moreover it is projected that by the year 2025, 80.9 million will have diabetes in India.28 The prevalence of diabetes in urban Indians had steadily increased from 2.01%in 1970s to 8.2% in 1980s later climbing to 16%. 29,30 Thus the phenomenon of high prevalence of diabetes reported among migrant Asian Indians has now spread to urban India and is rapidly moving to rural areas as well.31,32 The life expectancy of people with diabetes is reduced by nearly eight years due to increased mortality. Coronary artery disease accounts for more than 80% of all deaths and 75% of all hospitalizations in diabetic subjects. It is also reported that plaques are more vulnerable to rupture among patients with diabetes.33,34

The association between CAD and diabetesis strong despite the fact that there are wide ethnic and geographic variation in their prevalence. The protective female gender effect is lost in diabetic subjects, and indeed women with diabetes are possibly more prone to develop CAD than men with diabetes.35,36 Since 1990, CAD has been the leading cause of death worldwide, and this trend is expected to continue until 2020.Cardiovascular diseases accounted for 30.9% of all deaths in 1998 and 10.3% of disability adjusted life year loss. By 2020, 85% of global cardiovascular disease burden is expected to be borne by developing nations, and the increase in CAD mortality in developing nations is projected to be 120% in women and 137% in men. Thus developing nations would contribute to more than 75% of the global diabetes burden by the year 2025.37Currently holding 15th place in the list of causes of death worldwide, diabetesis expected to affect 300 million people worldwide by 2025.38

As type 2 diabetes is progressive, the repeated appraisal of glycemic control is imperative at all stages. The American Association of Clinical Endocrinologists and American college of Endocrinology advise that monotherapy is effective only when HbA1c levels 7.6% to 8.5% and then progressing to dual and triple terapy. Antidiabetic drugs with different mechanism of action are likely to have the greatest efficacy.48Drug therapy is indicated as an adjunct to diet and exercise. Oxidative streehyperhypocystinemia are risk factors for cardiovascular diseases. The trial was designed to assess the effects of metformin, glimepiride or repaglinide on cardiovascular disease risk factors such as oxidative stress. Total antioxidant status (TAS; combines concentrations of individual antioxidants) were determined and compared before and after therapy.

Treatment of type 2 diabetes requires drugs which in addition to maintaining the blood sugar level within normal limits they should have beneficial effects on dyslipidemia, hypertension, obesity, hyperinsulinemia and insulin resistance.39,40Jin H M et al2 pointed out that losartanangiotensin 1 receptor antagonist halts progression of diabetic nephropathy and produces beneficial changes in serum creatinine and albumin excretion rate and is highly effective in controlling proteinuria. Losartan does not increase kinin levels and therefore no side effect of cough like ACE inhibitor. Angioedema, urticaria and taste disturbances are also rare with losartan. 34VM Raoet al 3 showed that beneficial effects of losartan on diabetic nephropathy is seen only when patient had good glycemic control. Therfore to get the benefit of losartan on diabetic nephropathy, we must first control the blood sugar level. Therapeutic approaches directing at reducing proteinuria are under development. Approximately 20% of diabetic nephropathy enter maintenance dialysis. Epidemiological studies have shown that albumin excretion rate represents a strong and independent predictor of renal outcome in patients with diabetic nephropathy.2

D Rodbard et al 5 , M Broulee et al 7 reported that metformin alone and combination with repaglinide has favourable changes on serum urea and serum creatinine and halts progression of diabetic nephropathy. D Einhorn et al 29 , A Gokcellet al 30 repoted that metformin, a biguanide produces good glycemic control, had favourable effect on antioxidant status and showed significant reduction in total cholesterol, TG, LDL-C but it does not produce significant change in HDL-C. J B Buseet al 31 reported that metformin at higher dose reduces total cholesterol and it does not have significant effect on other lipids.Ralph et al 28,A Cerielloet al 8 found that metformin produces significant reduction in total cholesterol, triglycerides, LDL-C with significant increase in HDL-C.Peter H Benett et al 14 showed that metforminrepaglinide combination has beneficial effects on blood sugar level, antioxiddant status, lipid profile and on progression of renal failure.MJasket al 36 ,Viswanathan M et al 9 reported that glimepiride produces significant reduction in total cholesterol and TG.Simultaneously, T Allavoineet al 40 reported that metformin + glimepirde combination produces good glycemic control, improves antioxidant status and has effect on total cholesterol, HDL-C, LDL-C but it does not have effect on serum triglycerides.KP Singh et al 29 reported that metformin + glimepirde combination produces good glycaemic control and halts the progression of renal failure in patients with proteinuria.On the contrary studies carried out by V Sheshiah et al 32 has reported that repaglinide has no effect on proteinuria and renal function tests of individual and does not produce any change in total cholesterol, TG, HDL-C, LDL-C.Studies carried out by Richard C et al34 showed that glimepiride or repaglinide alone and in combination with metformin have no effect on antioxidant status, lipid profile or proteinuria.Simultaneously Chen K W et al 33 reported that glimepiride or repaglinide in combination with losartan has no effect on progression of nephropathy and proteinuria.

Taking into account the above studies and their contradictory findings the present study was carried out to find out the effect of addition of losartan to metformin alone and in combination with glimepiride or repaglinide on antioxidant status, lipid profile, glycatedHb and proteinuria in patients with type-2 diabetes with proteinuria.

AIMS AND OBJECTIVES

1. To study the effect of Metformin + Losartan on glycemic control, lipid profile, antioxidant status and progression of diabetic nephropthy before and after therapy by fasting blood sugar,glycatedHb, lipid profile, antioxidant status, renal function tests and proteinuria.

2. To study the effect of Metformin + Glimepiride+ Losartan on glycemic control, lipid profile, antioxidant status and progression of diabetic nephropathy before and after therapy by fasting blood sugar,glycatedHb, lipid profile, antioxidant status, renal function tests and proteinuria.

3.To study the effect of metformin + Repaglinide+ Losartan on glycemic control, lipid profile, antioxidant status and progression of diabetic nephropathy before and after therapy by fasting blood sugar,glycatedHb, lipid profile, antioxidant status, renal function tests and proteinuria.

4. To compare the changes of fasting blood glucose, glycatedHb, lipid profile, antioxidant status, progression of diabetic nephropathy and proteinuria in three groups.

MATERIALS & METHODS

The study was carried out after permission of institutional ethics committee.Patients studied as per National Cholesterol Education Programme low risk desirable lipid levels are:16

Total Serum Cholesterol <200mg/dl

Serum triglycerides <200mg/dl

HDL Cholesterol >75mg/dl

LDL Cholesterol <100 mg/dl

Patients having lipid levels above low risk desirable level according to National Cholesterol Education Programme were studied.

WRITTEN INFORMED CONSENT

Written informed consent in local language andenglish was taken from each patient after explaining the full details regarding treatment.

INCLUSION CRITERIA

Patients with type 2 diabetes with dyslipidemia (as per National Cholesterol Education Programme)17,18,obesity,significantproteinuria

Type 2 diabetes patients with proteinuria are particularly included in the trial to assess the effect of losartan on prerenal failure condition and to study the drug effects on progression of renal failure and diabetic nephropathy.

EXCLUSION CRITERIA

1. Patients with type 1 diabetes

2. Patients requiring insulin for diabetic control

3.Patients known allergic to these drugs

4.Patients who has taken insulin in past

5.Patients with deranged liver function tests

6.Patients with rapidly progressive retinopathy/ neuropathy requiring insulin

7.Patients not willing to give informed consent

8.Pregnancy & lactation

Study Design

Randomized comparative prospective trial. Randomization was made by using random number table.

Total number of patients studied

Three hundred (n=300) patients of type-2 diabetes with dyslipidemia (deranged lipid profile according to National Cholesterol Education Programme) were studied and was followed up for 6 month.

GROUPS

Group-1 Metformin+ Losartan

Group-2 Metformin+ Glimepiride + Losartan

Group-3 Metformin+ Repaglinide + Losartan

Hundred patients was assigned to each group.

DOSAGE SCHEDULE

1.Tab. Metformin 500 mg b.i.d.

2.Tab.Glimepiride 2mg o.d.

3.Tab.Repaglinide 2mg t.d.s.

4. Tab.Losartan 100 mg o.d.

Groups / Drug dosage / Duration
Group 1 (M+losartan) / Metformin 500mg b.i.d.
Losatan 100 mg od / 6 month
Group 2 (M+gimepiride+losartan) / Metformin 500 mg b.i.d.
Glimepiride 2 mg od
Losartan 100 mg od / 6 month
Group 3 (M+repaglinide+losartan) / Metformin 500 mg b.i.d.
Repaglinide 2mg 8hrly
Losartan 100 mg od / 6 month

INVESTIGATIONS BEFORE ENROLLMENT & ON COMPLETION OF TREATMENT

Hemogram, blood pressure measurement, liver function tests,body mass index,blood sugar fasting,glycatedHb, lipid profile parameters, total antioxidant status, plasma malondialdehyde level, serum urea level (mg/dl),serum creatinine level (mg/dl),albumin excretion rate (mg/dl)

FOLLOW UP

On weekly follow up patients are examined for blood sugar fasting, postmeal, blood pressure (systolic and diastolic), appearance of any new symptom or sign, adverse effects, haemogram for anemia or leucopenia.

PROCEDURE

The study protocol was approved by institutional ethics committee.According to National Cholesterol Education Programme, three hundred type-2 diabetes patients (n=300) with deranged lipid profile parameters and deranged antioxidant status were enrolled in the syudy.Patients were explained about the study pattern and related hazards.Informed written consent was obtained from the patient. Those included also examined by complete blood count, liver function test, kidney function test & fundoscopy. Enrolled patients were divided into three groups of hundred each according to random number table.Each patient in respective group was provided free samples throughout the study period and was asked to visit weekly diabetic clinic.Forfollowup and for collection of drugs. At each follow up visit, patients were assessed for glycaemiccontrol,history pertaining to adverse effects was asked. All patients was given diet and exercise suggestions.

Collection of blood samples

Patients was asked to come fasting for follow up, 2ml of venous blood sample was collected in plain bulb. The collected blood samples were centrifuged by centrifuge machine at 10,000 r.p.m. for 10 min and serum was separated.The separated serum was analyzed for lipid profile parameters on semiautoanalyser.

Sample analysis

Semiautoanalyser method was used.Lipid profile parameters like serum cholesterol, serum TG, HDL-C was calculated in concentration on linear mode of semiautoanalyser.

A). Serum cholesterol reagent 16

Reagent 1

Pipes buffer 50 m.mol/l

Phenol 21.3 m.mol/l

Reagent 2

Cholesterol esterase >200 unit/l

Cholesterol oxidase >200 unit/l

Peroxidase >1000 unit/l

P-amino antipyrene 0.5 m.mol/l

Cholesterol 5.17 m.mol/l or 200mg/dl

In the presence of cholesterol esterase , cholesterol esters are dissociated into cholesterol and fatty acids, cholesterol oxidase then converts the cholesterol into hydrogen peroxide and cholestenone. In the presence of peroxidase hydrogen peroxide reacts with p-aminoantipyrene and phenol to form a red quinoneimine dye.

Assay procedure

Wave length 500 (492-550) nm

Cuvette 1 cm light path

Temperature 37OC

Blank / Standard / Sample
Working reagent / 0.5 ml / 1 ml / 1 ml
Sample / -- / -- / 0.01ml
Standard / -- / 0.01ml / 0.01ml

In a clean K.J.tube take 0.01 ml of serum sample.Add 0.5 ml cholesterol woking reagent. Mix and incubate for 10 min at 37 o C. The measure of absorbance (A) of thesample and standard against the bank.

b). Serum triglyceride reagent 166

Reagent 1

Pipes buffer 55 m.mol/l

ESPAS 1m.mol/l

Reagent 2

GPO >2500unit/l

Lipase >100m.mol/l

Detergent > 0.1gm/l

PAP 0.3 m.mol/l

POD 350u/l

GK 1200u/l

Filling material 3%

Magnesium acetate 5m.mol/l

Amino antipyrene 0.7 m.mol/l

ATP 0.3m.mol/l

Standard triglyceride 200mg/l or 2.29 m.mol/l

In the presence of lipoprotein lipase triglycerides are split into glycerol and fatty acids. In the presence of ATP and glycerokinase, glycerol is converted into glycerol-3 phosphate and ADP. Glycerol phosphate oxidase dissociates glycerol-3-phosphate into dihydroxy acetone phosphate and hydrogen peroxide reacts with p-aminoantipyrene and ESPAS to form a violet colouredquinoneimine as indicator.

ESPAS: N ethyl N sulfopropyl n anisidine

Assay procedure

Wavelength 546 (505-550) nm

Cuvette 1 cm length path

Temperature 370C

Blank / Standard / Sample
Workng reagent / 1ml / 1ml / 1ml
Sample / -- / -- / 0.01ml
Standard / -- / 0.01ml / --

In a clean K.J. tube take 1m of serum sample. Add 1ml triglyceride working reagent. Mix and incubate for 5 min at37 0 C. Measure the absorbance (A) of the sample and standard against the blank within 60 minutes.

C) High Density Lipoprotein Cholesterol47

Reagent: Phosphotungstate

Magnesium chloride

Standard HDL Cholesterol 50 mg/dl

The chylomicrons, VLDL (Very low density lipoprotein) and LDL (low density lipoprotein)are precipitated by addition of phosphotungstic acid and magnesium chloride. After centrifugation the supernatent fluid contains the HDL fraction which isassayedfoer HDL cholesterol with the cholesterol reagent.

Assay procedure 48

Precipitation

Sample 0.3 ml

HDL reagent 0.3 ml

Mix well and allow it to stand for 10 minutes at room temperature, mix again and centrifuge for 10 minutes at 4000 rpm.After centrifugation separate the clear supernatent from the precipitate within 1 hour and determine the cholesterol concentration using the cholesterol reagent.

Wavelength 500 to 532 nm

Cuvette 1 cm light path

In a clean K.J. tube tke 1ml of serum sample. Add 0.01 ml of HDL precipitating reagent. Centrifuge it for 5 minutes. Take 50 microlitreof supernatent in a K.J. tube. Add 1 ml of cholesterol reagent. Incubate it at room temperature for 15 minutes.Take reading on semiautoanalyser.The actual HDL cholesterol is thereading multiplied by two.

D). Low Density Lipoprotein Cholesterol

LDL Cholesterol= Total Cholesterol-( HDL+ Serum triglycerides)

5

= Total Cholesterol-HDL-VLDL

(Friedwalds formula)

E). Very Low Density Lipoprotein Cholesterol

VLDL= Serum triglycerides

5

Fasting blood glucose

Fasting blood glucose concentrtionswas measured by enzymatic colorimetric method.

HbA1c

GlycatedHb (HbA1c) levels were determined by Abott architect c16000 system before and after therapy.

Proteinuria

Proteinuria was determined by Albumin excretion rate in urine (mg/dl) before and after therapy.

Antioxidant status

Totl antioxidant statu (TAS : combine concentration of indiviidual antioxidants like vitamin C, vitamin E, beta carotene and thiol group) plasma malonyldialdehyde level (MDA) was determined before and after therapy. TAS is sensitive to the changes in plasma antioxidant level and degrees of oxidative stress. 67 Plasmamalonyldialdehyde is a marker of lipid peroxidation and increases in oxidative stress.43

OBSERVATIONS

Table No.1 Group I

EFFECT OF ADDITION OFLOSARTAN TO METFORMIN

BEFORE AND AFTER THERAPY

Parameter / Before therapy / After therapy / P value
Fasting blood sugar (FBS) mg/dl / 169 + 32.16 / 164.62 + 22.49 / NS
HbA1c % / 8.25 + 0.55 / 7.92 + 0.59 / NS
Total antioxidant status mmol/l / 0.98 + 0.03 / o.97 + 0.18 / NS
MDA nmol/l / 7.30 + 1.49 / 6.76 + 1.74 / NS
Total cholesterol (mg/dl) / 238.72 + 16.24 / 234.34 + 18.74 / NS
LDL cholesterol (mg/dl) / 181.71 + 15.52 / 172.12 + 18.56 / NS
VLDL cholesterol (mg/dl) / 44.68 + 5.34 / 41.41 + 3.86 / NS
Triglycerides (mg/dl) / 221.32 + 26.27 / 212.54 + 14.23 / NS
HDL cholesterol (mg/dl) / 36.42 + 5.34 / 39.41 + 3.56 / NS
Serum urea (mg/dl) / 38.16 + 3.93 / 29.43+ 2.64 / NS
Serum creatinine (mg/dl) / 2.4+ 0.35 / 1.97 + 0.56 / NS
Albumin excretion rate (mg/dl) / 104.67 + 12.43 / 87.45 + 8.23 / NS

Table No.2

Group II

EFFECT OF ADDITION OF LOSARTAN TO METFORMIN + GLIMEPIRIDE

Parameter / Before therpy / After therapy / P value
Fasting blood sugar (FBS) mg/dl / 171 + 27.23 / 99.17 +18.63 / <0.05
HbA 1 c (%) / 8.28 + 0.43 / 6.48 + 0.62 / <0.05
Total antioxidant status (mmol/l) / 0.94 + 0.04 / 1.30 +0.23 / < 0.05
MDA (nmol/l) / 7.31 + 2.01 / 3.44 + 1.43 / <0.05
Total cholesterol (mg/dl) / 242.32+ 11.32 / 200 + 22.04 / < 0.05
LDL cholesterol (mg/dl) / 176 + 16.21 / 158.62 + 16.43 / < 0.05
Triglycerides (mg/dl) / 222 + 24.24 / 208+17.23 / < 0.05
VLDLcholesterol (mg/dl) / 46.32 + 4.86 / 42.56 + 3.41 / < 0.05
HDLcholesterol (mg/dl) / 36.81+ 1.89 / 42.32 + 4.32 / < 0.05
Serum urea (mg/dl) / 43.83+ 2.39 / 17.76+ 1.92 / P < 0.05
Serum creatinine (mg/dl) / 2.5+ 0.78 / 0.96 + 0.43 / P < 0.05
Albumin excretion rate (mg/dl) / 126.26 + 10.73 / 38.25 + 7.63 / P < 0.05

Table No.3 Group III

EFFECT OF ADDITION OF LOSARTAN TO METFORMIN + REPAGLINIDE

Parameter / Before therpy / After therapy / P value
Fasting blood sugar (FBS) mg/dl / 170 + 20.97 / 96.03 + 14.03 / <0.001
HbA 1 c (%) / 8.27 + 0.60 / 6.23 + 0.51 / <0.001
Total antioxidant status (mmol/l) / 0.96 + 0.08 / 1.38 + 0.12 / < 0.001
MDA (nmol/l) / 7.28 + 1.98 / 3.21 + 1.26 / <0.001
Total cholesterol (mg/dl) / 240.74 + 58 / 193 + 23.67 / < 0.001
LDL cholesterol (mg/dl) / 172.25 + 20.42 / 142.81 + 18.69 / < 0.001
Triglycerides (mg/dl) / 226+ 24.46 / 198+16.42 / < 0.001
VLDL cholesterol (mg/dl) / 48.06 + 3.88 / 40.86 + 3.97 / < 0.001
Albumin excretion rate (mg/dl) / 126.26 + 10.73 / 38.25 + 7.63 / P < 0.05
Albumin excretion rate (mg/dl) / 109.12 + 11.23 / 21.35 + 6.12 / P < 0.001

TABLE IV