INTRODUCTION:

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia, altered metabolism of lipids, carbohydrates, proteins and an increased risk of complications from vascular diseases^1,2,3.Diabetes occur either due to decreased synthesis of insulin (Type-1) or due to defective secretion of insulin from beta cells of islets of langerhans (Type-2)⁴.Literature study reveals that diabetic patients develop multiple pathology such as fungal infection, cardiovascular disorders, nephropathy, retinopathy, neuropathy, sexual impotence, hyperacidity and respiratory tract infections⁵. Pharmacological stimulation of insulin secretion by sulfonylurea drugs has for many years been a tool in the treatment of Type-2 diabetic patients^6,7,8.Hypertension which coexists with diabetes mellitus is not only an indicator of increased risk of mortality but also a contributory factor to the development of diabetic complications⁹.To treat the coexisting disease, multi-drug therapy is inevitable and there is every possibility for a drug interaction to occur when drugs are concomitantly used.

Drug interactions have been reported to be the fourth to sixth leading cause of death in hospitalized patients in United States¹⁰.Patients receiving therapy for diabetes may also be potential candidates for anti-hypertension therapy. Gliclazide is a second generation sulfonylurea anti-diabetic drug. It is generally well tolerated, is associated with beneficial effects beyond the reduction of blood glucose¹¹.Gliclazide is exclusively metabolized by cytochrome P450 (CYP) 2C9¹².Irbesartan is a potent-long acting Angiotensin II receptor antagonist with high selectivity for the AT₂ receptor. Currently it is indicated for the treatment of essential hypertension, alone or in combination with other anti-hypertensive agents. Irbesartan is indicated for the treatment of type-2 diabetes mellitus patients with hypertension based on the results of the Irbesartan Diabetic Nephropathy Trial (IDNT). Irbesartan undergoes primary oxidation via the cytochrome P450 (CYP) 2C9 isoenyme and negligible metabolism by CYP 3A4^13,14,15. Earlier studies revealed that Irbesartan has the potential to exhibit in-vitro inhibition of CYP 2C9¹⁶.Hence the study is planned in such a way to bring the possible influence of Irbesartan on the pharmacokinetics of gliclazide on rabbits as both involve the utilization of CYP 2C9 isoenzyme for the metabolism.

MATERIALS AND METHODS:

Drugs and Chemicals:

Gliclazide was obtained from Aurobindo Pharmaceuticals, Hyderabad. Irbesartan and Glipizide were obtained from Sun Pharmaceuticals Ltd, Mumbai, India. Acetonitrile (HPLC) grade was procured from Qualigens chemicals, Mumbai, India. Orthophosphoric acid of HPLC grade was procured from SD. Fine chemicals, Mumbai, India. Double distilled water was prepared and used in the laboratory.

Animals:

Adult albino rabbits weighing between 1.3-1.5kg obtained from the animal house of Bapatla College of Pharmacy (1032/ac/07/CPCSEA); Bapatla, were maintained at a constant temperature of 26 ± 2^oC and humidity 30-40% with 12 h light/dark cycle, throughout the experiments. The animals were housed in clean rabbit cages in an air conditioned animal house and were fed with commercial rabbit feed and sterile water. The experimental protocol (IAEC/I/BCOP/07-08) was approved by the institutional animal ethics committee (IAEC) of Bapatla College of Pharmacy; Bapatla and was in accordance with the guidelines of the committee for the purpose of control and supervision of experimentation on animals.

Figure-1 Serum Concentration Profile of Gliclazide at Various Conditions

Experimental Procedure:

Adult healthy albino rabbits were used for the study (n=5). All the animals were fasted for a period of 18 hours prior to the experiment and water was fed ad libitum. The experimental part was conducted in three phases.

First Phase (Single Gliclazide treatment):

In the first phase, the pharmacokinetics of gliclazide 3.7 mg /kg, p.o was studied. The dose used in the first phase was based upon the preliminary study data of gliclazide. The blood samples were collected from the marginal ear vein in separate eppendorf tubes at time intervals of 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24 hours. The samples were then centrifuged at 4000 rpm (Micro centrifuge, Remi) to separate the serum and the separated serum samples were then subjected to analysis of gliclazide concentration by HPLC method as described by Mohammad et al¹⁷ and modified suitably to laboratory conditions.

Second Phase (Single dose interaction):

In this phase, the influence of irbesartan on the pharmacokinetics of gliclazide was studied. After a washout period of one week, the animals of first phase were used for second phase study and were administered with irbesartan 14 mg/kg, p.o. After a time interval of 30 min gliclazide 3.7mg/kg, p.o was administered. The blood samples were collected from the marginal ear vein at predetermined time intervals and analyzed for gliclazide concentration by HPLC method as mentioned in first phase.

Third Phase (Multiple dose interaction):

In this phase the influence of multiple dose of irbesartan on the pharmacokinetics of gliclazide was studied. The animals were administered with irbesartan 14 mg/kg, p.o for 7 consecutive days post second phase. During this period the animals had free access to food and water. On the 7^th day, 6 hours after irbesartan administration food was deprived and water was fed ad libitum. On the 8^th day 30 minutes after irbesartan administration, the animals received gliclazide 3.7mg/kg. The blood samples were collected at predetermined time intervals as mentioned in first phase and analyzed for gliclazide concentration by HPLC method.

Table-1 Serum Concentration Data of Gliclazide Obtained at Various Conditions.

Time (h)	Mean concentration (μg/ml) + S.E.M
Time (h)	First Phase	Second Phase	Third Phase
0	-	-	-
0.5	0.321+0.007	0.322+0.003	0.320+0.004
1	0.413+0.016	0.414+0.012	0.413+0.012
2	0.603+0.021	0.602+0.024	0.602+0.023
3	1.190+0.026	1.161+0.028	1.165+0.027
4	1.080+0.038	1.049+0.036	1.052+0.036
6	0.967+0.028	0.964+0.026	0.963+0.023
8	0.887+0.032	0.903+0.037	0.865+0.022
10	0.781+0.042	0.825+0.065	0.792+0.041
12	0.712+0.048	0.749+0.063	0.705+0.044
16	0.612+0.029	0.627+0.031	0.602+0.026
20	0.508+0.024	0.509+0.024	0.509+0.021
24	0.411+0.020	0.414+0.019	0.412+0.026

The values are mean±SEM of five observations at each time interval. First Phase (Gliclazide 3.7mg/kg). Second Phase (Gliclazide 3.7mg/kg +Irbesartan 14 mg/kg) - Single dose interaction. Third Phase (Gliclazide 3.7mg/kg +Irbesartan 14 mg/kg) - Multiple dose interaction.

Data analysis:

The pharmacokinetic parameters of gliclazide at all the three phases were estimated by non-compartmental methods, with the use of PK Solutions 2.0, USA. The various pharmacokinetic parameters estimated were K_e (1/hr), t_1/2(hr), K_a (1/hr), AUC _(0-α)(µg-hr/ml), MRT (hr), C_max (µg/ml), T_max (hr).

Statistical significance:

The data are presented as Mean ± SEM. The significance of the observed difference in the pharmacokinetic parameter of gliclazide before and after treatment with irbesartan was assessed by students unpaired‘t’ test. A value of P<0.05 was considered to be statistically significant.

RESULTS:

The mean serum concentration profiles of gliclazide at various phases of treatment are presented in Table1, Fig1. The log concentration profiles are illustrated in Fig 2. The Pharmacokinetic parameters estimated are presented in Table 2. The pharmacokinetic parameters such as K_e (1/hr), t_1/2(hr), K_a (1/hr), AUC _(0-α)(µg-hr/ml), MRT (hr), C_max (µg/ml), T_max (hr) estimated in the first phase were not altered significantly by Irbesartan in the second and third phase of study. Statistical treatment of the pharmacokinetic data of the second and third phase in comparison with the first phase showed no significant change.

FIGURE-2 Log concentration profile of gliclazide at various conditions

DISCUSSION:

The practice of prescribing several drugs simultaneously is common. Thus, an awareness of possible drug-drug interaction is essential to avoid catastrophic synergistic effects and chemical, enzymic and pharmacokinetic interations that may produce toxic side effects¹⁸. Diabetic patients are likely to suffer with hypertension and hence anti-hypertensives are co-administered along with oral anti-diabetic drugs more frequently. Commonly prescribed anti-hypertensives belong to the class of Angiotensin receptor blockers and Angiotensin converting enzyme inhibitors. Preclinical studies are mandatory before any clinical evaluation; hence the kinetic interactions study was carried as no previous data was published on the kinetic interaction between gliclazide and irbesartan. The pharmacokinetic interaction study was carried out in rabbits, a suitable animal model for the evaluation of pharmacokinetic data. The CYP 450 functions as a multi-component electron transport system responsible for the oxidative metabolism of a variety of endogenous substrates. The CYP 2C subfamily is the most complex family consisting of CYP2C9, metabolizing approximately 25% of the clinically important drugs. CYP2C9 is found primarily in the liver and intestine¹⁸. Literature survey shows that angiotensin receptor blocker, irbesartan have the potential to inhibit cytochrome P450 isoenzymes invitro¹⁶, which means that there is a probable pharmacokinetic interaction between gliclazide and irbesartan. Literature also states that both the drugs are metabolized by 2C9 isoenzyme^12,13. Though both drugs are reported to be metabolized by the same isoenzyme, the pharmacokinetic data did not show any interference of irbesartan on the kinetics of gliclazide. Previous studies in our laboratory showed pharmacodynamic potentiation of the gliclazide effect by irbesartan. Hence based on the results it is concluded that irbesartan is well tolerated with gliclazide therapy in the management of hypertension associated with diabetes and requires frequent monitoring of blood glucose levels.

Table-2 Pharmacokinetic Data of Gliclazide at Various Conditions.

Parameter	First Phase	Second Phase	Third Phase
Elimination Rate (k_e) (1/hr)	0.052+0.0010	0.052+0.0010Ns	0.051+0.0008Ns
Elimination Half-life (t_1/2) (hr)	13.16+0.2434	13.45+0.2860Ns	13.44+0.2079Ns
Absorption Rate (k_a) (1/hr)	0.273+0.0251	0.346+0.0459Ns	0.452+0.1084Ns
AUC (0-¥) (µg-hr/ml)	24.44+0.9922	24.86+1.1539Ns	24.58+0.9112Ns
MRT	21.04+0.2767	21.36+0.2619Ns	21.48+0.4554Ns
Cmax (µg/ml)	1.18+0.0374	1.16+0.0379Ns	1.18+0.0374Ns
Tmax (hr)	3.00+0.000	3.00+0.000Ns	3.0+0.0000Ns

The values are mean±SEM of five observations at each time interval. First Phase (Gliclazide 3.7mg/kg). Second Phase (Gliclazide 3.7mg/kg +Irbesartan 14 mg/kg) – Single dose interaction. Third Phase (Gliclazide 3.7mg/kg +Irbesartan 14 mg/kg)- Multiple dose interaction.

Ns = Non significant,* = P< 0.05 when compared to kinetics of first phase.

Acknowledgements:

The authors are thankful to the management of Bapatla Educational Society for providing all facilities to conduct this experimental work. The authors are thankful to Aurobindo and Sun Pharmaceuticals for providing the gratis samples.

REFERENCES:

1. Murthy TEGK, et al. Study of interaction between Amlodipine Besylate and Gliclazide in healthy rats. International Journal of Pharmacology and Biological Sciences.2008; 2(1):139-142.

2. Swami AM, et al. A study on drug-drug interaction of Roxithromycin and anti-diabetic drugs. Indian Drugs.2005; 42(12):808-813.

3. Bastaki S. Diabetes mellitus and its treatment. International Journal of Diabetes and Metabolism.2005; 13:111-134.

4. Satyanarayana S, et al. Influence of Quinidine, Selegiline and Amphotericin-B on the pharmacokinetics and pharmacodynamics of Tolbutamide in rabbits. Indian Drugs.1998; 35(10):640-644.

5. Rambhimaiah S, et al. Influence of Metronidazole on the hypoglycemic affect of Tobutamide in healthy albino rabbits. Indian Drugs.2003; 40(9):535-538.

6. Groop LC. Sulfonylureas in NIDDM. Diabetes Care 1992; 15:737-754.

7. Lebovitz HE. Stepwise and combination drug therapy for the treatment of NIDDM. Diabetes Care.1994; 17:1542-1544.

8. Melander A, Lebovitz HE and Faber OK. Sulfonylureas: why, which and how?. Diabetes care.1990; 13(3):18-25.

9. Goyal RK, Joshi SS and Shah TS. Effects of chronic treatment with Nitrendipine in streptozotocin- induced diabetic rats. Indian Journal of Pharmaceutical Sciences.1996; 58(3):100-105.

10. Ramachandra SS, et al. Influence of Itraconazole on sulfonylureas-induced hypoglycemia in diabetic rats. Indian Journal of Pharmaceutical Sciences.2005; 67(6):677-680.

11. Palmer KJ and Brogden RN. An update of tits pharmacological properties and therapeutic efficacy in non-insulin-dependent diabetes mellitus. Drugs.1993; 46:92-125.

12. Ji YP, et al. Effect of rifampin on the pharmacokinetics and pharmacodynamics of gliclazide. Clin Pharmcol Ther. 2003; 74:334-40.

13. Mangold B, Gielsdorf W and Marino MR. Irbesaran does not affect the steady-state pharmacodynamics and pharmacokinetics of warfarin. Eur. J clin pharmacol. 1999; 55:593-598.

14. Sujata V, et al. Pharmacokinetics of the oral direct rennin inhibitor Aliskiren alone and in combination with Irbesartan in renal impairment. Clin Pharmacokinet.2007; 46(8):661-675.

15. Maria RM, Nimish NV and Ophelia WH. Irbesartan does not affect the pharmacokinetics of Simvastatin in healthy subjects. J Clin Pharmacol.2000; 40:875-879.

16. Taavitsainen P, Kiukaannieme K and Pelkonen O. In vitro inhibition screening of human hepatic P[450] enzymes by five Angiotensin-II receptor antagonists. Eur.j.clin.pharmacol.2000;56:135-140.

17. Mohammad RR, Afshin M and Mohammad HT. A simple and sensitive HPLC method for determination of gliclazide in human serum. J.Chromatogr B.2003; 785:383-386.

18. David AW. Drug Metabolism. In Foye’s Principles of Medicinal Chemistry, Edited by Thomas LL and David AW. Lippincott Williams and Wilkins, Philadelphia. 2008;6^th ed:pp.253-326.

Received on 12.08.2008 Modified on 22.08.2008

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008;Page 418-421

Pharmacokinetics of Gliclazide Alone and in Combination with Irbesartan in Rabbits.