Influence of Anastrozole on Glimepiride in Experimental Animals

D.K.Suresh^*, Mangesh Gavali, Vivek Ingale, Bhaumik Thakar, Md. Saifuddin K, Raghvendra Rao.

Department of Pharmacology, Luqman College of Pharmacy, Gulbarga- 585 105, Karnataka.

ABSTRACT:

The present study was aimed to find out the effect of multiple dose (7 days) treatment of anastrozole an anticancer drug on the hypoglycaemic activity of glimepiride in normal and diabetic rats. The study was intended to determine the pharmacodynamic parameters of drug interaction between glimepiride and anastrozole in normal and diabetic rats. The studies were conducted using a group of six normal and six diabetic rats of either sex. The experiment was conducted in four stages. Group of six animals were selected for each stage of experiment. These studies were conducted in the four group of the animals and dose of administered drug (glimepiride 0.1 mg/kg and anastrozole 0.004 mg/kg body weight). The drugs were administered orally. The blood samples were collected by tail vein at predetermined time intervals and glucose levels were estimated by GOD/POD method using semi autoanalyer. The results indicated that single and multiple dose treatment of anastrozole an anticancer drug altered the hypoglycaemic activity of glimepiride when administered alone and along with glimepiride in normal and diabetic rats. This may be due to the synergistic effect of anastrozole with glimepiride. The preliminary studies indicate the combination may be unsafe in diabetes associated with cancer.

KEYWORDS:glimepiride, anastrozole, drug interaction, GOD/POD method.

INTRODUCTION:

To obtain a desired therapeutic objective or to treat co-existing diseases many a times it becomes essential for the concomitant use of several drugs together. Simultaneous use of several drugs often leads to drug-drug interactions¹. Diabetes mellitus is a metabolic disorder resulting from deficiency of insulin leading to complications involving many organs. A major threat to future public health resources throughout the world appear due to diabetes and its complications^2,3,4. According to WHO in near future India would maximum diabetes patient⁵. In last two decades several fold increase in the prevalence of type-II diabetes has been revealed by the studies in various urban areas of india^6,7. Lifelong treatment with drugs and diet control is necessary in case of diabetes^2,3,4.

It was recognized by Himsworth in 1930s that two types of diabetes mellitus exit. One due to insufficiency of insulin (type1);the other due to resistance to the action of insulin (type 2);Type 2 diabetes mellitus (T2DM) is the most common endocrine disorder worldwide, characterized by fasting and postprandial hyperglycaemic and relative insulin insufficiency. Untreated hyperglycemia may cause long-term microvascular and macrovascular complication, such as nephropathy, neuropathy, retinopathy, and atherosclerosis and is associated with co morbidities, such as obesity, hypertension, hyperlipidemia .

According to International Diabetes Federation (IDF) the enormity of the T2DM epidemic, disease now affects a staggering 246 million people worldwide, with 46% of all those affected in the 40-59 age group and the total number of people living with diabetes will skyrocket to 380 million within 20 years, if nothing is done⁸.

The natural history of diabetes usually begins with obesity leading to insulin resistence which in turn promotes a state of compensatory hyperinsulinemia leading to other adverse sequelae. Initially, normoglycemia is maintained because of compensatory increase in insulin secretion by β-cell³.

However, gradual loss in drug efficacy over time due to progressive deterioration in β-cell function is the main limitation as most of the observed initial improvement in glycemic control are not sustained. Furthermore, most of these treatments have undesired side effects. sulfonylureas i.e glimepiride, glibenclamide ,gliburide, glipizide, glipizide increase insulin secretion⁹.

Several study suggest that hyperinsulinemia with insulin resistance is a significance risk for breast cancer¹⁰. Comparative cohort studies and case-control studies suggest that type 2 diabetes may be associated with 10-20% excess relative risk of breast cancer¹¹. In such instances necessarily co-administration of anticancer with regular oral antidiabetic drugs. In general, most commonly using anticancer agents in breast cancer are anastrozole, cyclophosphamide, docetaxel etc¹².

Anastrozole, a widely using anticancer drug to treat breast cancer¹³ . Anastrozole is strong inhibitor of isoenzymes CYP19A1, CPYP1A2, CYP2C9 and CYP3A4¹⁴ .Similarly antidiabetic agents like glimepiride are metabolised by isoenzymes CYP2C9 respectively¹⁵. Therefore their may be every possibility of drug interaction of anastrozole with glimepiride. And also drug interaction studies of anastrozole reveals the co-administration of anastrozole with variety of compound for examples antipyrine, cimetidine and warfarin indicate that anastrozole is likely to result in clinically significant drug interactions medicated by cytochrome 450¹⁶. glimepiride shows interactions with phenylbutazone, salicylates, sulphonamides, warfarin, chloramphenicol, propanalol, lithium,theophyline, alcohol etc¹⁷.

The present study was planned to find out the effect of multiple dose treatment of anastrozole on blood glucose levels and on hypoglycaemic activity of glimepiride in normal and diabetic rats. If the interaction occurs in normal animals, which represents the conditions of actual use of drugs in humans and to understand the mechanism of drug interaction. Since rat is well established model for preclinical hypoglycaemic activity and is also official model for bioassay and suitable rodent animal model for ease of collection of sufficient volume of blood sample at regular time intervals.

MATERIALS AND METHODS:

Animal:

Inbred adult albino rats of either sex were procured from Mahavir Enterprises, Hyderabad. They were maintained on uniform diet and temperature with 12 h light and dark cycle housed in well ventilated aluminium cages individually for acclimatization. All the animals were given only standard palleted animal diet (Amrut Brand) in adequate quantity, with drinking water ad libitum. The experimental protocol (04_P038_21518) was prior approved by Institutional Animal Ethics committee (IAEC) of Luqman College of Pharmacy, Gulbarga for conduction of experiments. CPCSEA registration number is 346/CPCSEA.

Drugs:

Pure samples of glimepiride and anastrozole were procured as gift samples from Hetro Drugs ltd.(Mumbai) and Nosch Labs Pvt.Ltd. (Hyderabad) respectively. Glucose kit of Pathozyme Diagnostics was used for glucose estimation¹⁸.

Preparation of drug solutions for treatment:

A suspension was prepared by using 2% gum acacia as a suspending agent to represent 7.5mg/ml and 250mg/ml in distilled water for glimepiride and anastrozole, respetively.

Induction of diabetes (Streptozotocin treatment):

Rats of either sex weighing between 150–180 gms were selected and fasted for 18 hr and water ad-libitum. The animals were randomly distributed into different groups. The animals were kept in colony cages at standard husbandry condition. The rats were administered with 50 mg/kg of Streptozotocin Intraperitonially¹⁹. After 48 hrs, the blood samples were collected and analyzed for blood glucose level. It was found that diabetes was induced in about 48 hrs. In our experiment the diabetes was characterized by weight loss and hyperglycaemia. The blood samples were collected and analyzed for seven more days for stabilization of blood glucose levels. These animals were further used for our antidiabetic study.

Experimental procedure in normal and diabetic rats:

six albino rats of either sex weighing between 150-180 gms were selected for the study. Oral route was selected for the administration of drugs since the drugs under study are given generally by oral route in clinical practice. The drugs were administered orally with the help of an oral feeding needle (purchased from Space Labs, Nasik) and a 1ml glass syringe.

The rats were fasted for 18 h prior to the experiment with water ad libitum. During experimentation water also was withdrawn. The experiment was conducted in four stages for both the healthy and diabetic rats.

Stage-I: All the six rats treated with 2% gum acacia suspension, and blood samples were collected at regular time intervals. The blood samples were collected from the tail vein of the rats. The samples were analyzed for blood glucose. This stage served as control without any drug treatment.

Table. 1: Mean percentage blood glucose reduction by anastrozole, glimepiride and their combination in healthy rats.

Group Treatment	Mean percent blood glucose change ± SEM
	Time(h)
	0	^½	1	2	4	8	12	24	48
IControl	0.0	1.48±0.43	2.56±0.54	5.27±0.54	6.02±0.59	6.44±1.12	6.87±0.68	8.33±1.16	8.90±1.12
IIAnastrozole	0.0	2.28±0.13	4.79±0.15	7.22±0.39	10.28±0.58	8.10±0.73	5.79±0.58	3.26±0.45	1.59±0.46
III Glimepiride	0.0	6.44±0.84	13.77±1.22	24.36±0.48	36.91±0.43	30.29±0.46	25.37±0.36	21.34±0.46	7.14±1.96
IVAnastrozole +Glimepiride	0.0	14.94± 2.38***	23.39 ±3.83***	32.65 ±3.81***	48.29± 1.25***	30.33 ±3.81***	28.22 ±3.71***	24.71± 3.56***	21.26 ±3.67***

Number of animals n = 6; Significant at P*<0.02; Highly Significant at P**<0.01; Very highly significant at P***<0.001

SEM- standard error of the mean; Time expressed in hour.

Table. 2:Mean percentage blood glucose reduction by anastrozole, glimepiride and their combination in diabetic rats.

Group Treatment	Mean percent blood glucose change ± SEM
	Time(h)
	0	½	1	2	4	8	12	18	24
IControl	0.0	1.08±0.0.31	1.92±0.26	7.64±4.88	7.71±4.89	7.53±4.73	6.76±4.65	6.26±5.10	0.21±7.69
IIAnastrozole	0.0	0.30±0.21	5.99±3.81	6.10±4.51	6.09±4.78	6.21±4.72	5.93±4.53	5.92±4.90	5.33±4.80
IIIGlimepiride	0.0	3.55±0.69	12.96±1.78	22.27±4.36	35.41±4.14	29.82±4.15	22.76±2.43	20.89±2.11	9.68±2.83
IVAnastrozole +Glimepiride	0.0	15.31±1.05***	22.99±0.81***	32.51±0.97***	47.15±2.88***	39.05±1.74***	33.87±0.83***	25.67±0.47***	22.89±0.59**

Number of animals n = 6; Significant at P*<0.02; Highly Significant at P**<0.01; Very highly significant at P***<0.001 SEM- standard error of the mean; Time expressed in hour.

Stage-II: Group II was treated with therapeutic dose of anastrozole (0.004mg/ kg body weight) and samples were collected at regular time intervals. The samples were analyzed for blood glucose. Stage-III: Group III was treated with therapeutic dose of glimepiride (0.1 mg/ kg body weight) and samples were collected at regular time intervals. The samples were analyzed for blood glucose. Stage- IV: Group IV was treated with suspension of anastrozole (0.004 mg/ kg body weight) through oral route for 7 days, on 7^th day, 6 h after the dose anastrozole the animals were fasted for 18 h. This fasting continued till the end of experiments. On 8^th day 1 h after the dose administration glimepiride 0.1 mg/kg was administered to the same animals and the samples were collected at regular time intervals. The samples were analysed for blood glucose.

The blood samples were collected into eppendorff tubes containing a small quantity of anticoagulant (sodium fluoride and potassium oxalate, 1:3) at regular time intervals (0,1/2, 1, 2, 4, 8, 12, and 24 h). Sodium fluoride was added to prevent in vitro glycolysis in the blood samples collected. The above samples were centrifuged and plasma was collected after separation. The blood glucose was estimated by glucose kit (GOD/POD method) using semi auto analyzer²⁰.

STATISTICAL SIGNIFICANCE:

The data are presented as mean percent blood glucose change ± SEM. The significance of the observed differences in percentage reduction in blood glucose level were calculated by applying paired Student’s t-test in normal rats. The ‘P’ values <0.05 were considered as significant.

RESULTS:

In multiple dose study, the mean percent blood glucose reduction by amiodarone, pioglitazone and their combination in healthy and diabetic rats are given in table.1and 2 respectively.

In this study the pre-treatment with of amiodarone has significantly enhanced the onset of hypoglycemia the peak hypoglycaemia and duration of hypoglycemia induced by pioglitazone in both healthy and diabetic rats

DISCUSSION:

Drug-drug interactions may occur in patients where in several drugs are used concomitantly to treat a single disease or multiple diseases in a single patient. In the present study the possible interaction between the two different classes of drugs used to treat two different pathophysiological conditions like diabetes mellitus and cancer is investigated.

Diabetes mellitus is a disease caused due to destruction\ defective of β cells of langerhans and is of two types one is (IDDM) insulin dependent diabetes mellitus and the other is (NIDDM) non insulin dependent diabetes mellitus.

About 5% of the diabetic patients suffer with insulin dependent diabetes mellitus and requires insulin to control the blood glucose levels whereas remaining 95% are suffering from non-insulin dependent diabetes mellitus, which is controlled by using oral hypoglycemic agents. In the present study one important drugs of anti diabetic agents such as glimepirides are being taken to evaluate the interaction of these agents with a relatively newer anticancer agent such as anastrozole.

Specially in case of diabetes mellitus regulation of blood glucose level is highly essential and important. But when a drug potentiates the effect of antidiabetic agent, the severe hypoglycemia may be developed or if it inactivates the antidiabetic agents then the doses may be ineffective. There are large number of diabetic patients also suffer with breast cancer. In such case, in those patients if, antidiabetic agents like glimepiride are administered along with anticancer agent like anastrozole is given, then there may be possibility of drug drug interactions.

There are several reports that anticancer agents inhibit the isoenzymes of CYP-450 enzyme system. The isoenzymes that are affected by anastrozole belongs to a anticancer agents are CYP2C9 and CYP3A4. There is a possibility that drug-drug interaction may occur between anastrozole and the drugs metabolised by these enzymes²¹. nateglinide and glimepiride are metablised by CYP2C9 ²¹. Therefore, there is a possibility of development of drug-drug interaction between these two types of therapeutic agents. There are no reports regarding the interaction of these drugs but those are not enough to confirm the interaction. Hence, in the present study glimepiride are oral antidiabetic agents and anastrozole is an anticancer agent are being used to understand, evaluate and confirm the drug-drug interaction between them. In this project interaction between the above mentioned classes of drugs were assessed in healthy albino rats, and diabetic rats.

In the first phase of study per se effect of anastrozole on blood glucose level on healthy albino rats was evaluated. It was observed that anastrozole dose (0.004mg/kg) has failed to influence the blood glucose, indicating anastrozole does not possess any hypoglycaemic activity in healthy albino rats, .Therefore the possible interactions with oral hypoglycaemic agents is not pharmacodynamic type.

In the second phase of this experiment influence of glimepiride (0.1mg/kg) were administered and extent of hypoglycemia, duration of hypoglycemia and onset of hypoglycemia were assessed, then to the same animals after 15 days, anastrozole (0.004 mg/kg) was given for 7 days and then on the 8^th day influence of this pre-treatment on the hypoglycemia produced by nateglinide and glimepiride were studied.

Whereas the pre-treatment with of anastrozole (0.004mg/kg for seven days) has significantly enhanced the onset of hypoglycemia (i.e. from 2 hrs 24.36 ± 0.48% to 1 hr 23.39 ± 3.83% p<0.0001, p<0.0001) and significantly enhanced the peak effect of hypoglycemia (i.e. 36.91 ± 0.43% reduction before treatment to 48.29 ± 1.25% reduction after treatment p<0.0001, p< 0.0001) and duration of hypoglycemia was increased from18 hrs to more than 24 hrs (i.e. 21.34 ± 0.46%, before treatment and 21.26 ± 3.67% after treatment p<0.0001) induced by glimepiride

Our studies in healthy rats suggested that dug-drug interaction occurs between anastrozole and glimepiride when they are used concomitantly in healthy conditions. However the interaction in the pathophysiological conditions like in diabetes was not clear. Hence, in the third phase of our study the diabetic rats (Streptozotocin induced diabetic rats) were used, glimepiride was given to diabetic animals and the onset of hypoglycemia, duration of hypoglycemia and peak antidiabetic effect was determined. To the same animal The pre-treatment with of anastrozole (0.004 mg/kg for seven days) has significantly enhanced the onset of hypoglycemia (i.e. from 2 hrs 22.27 ± 4.36% to 1 hr 22.99 ± 0.81% p< 0.0001, p< 0.0001) and significantly enchanced the peak effect of hypoglycemia (i.e. 35.41± 4.14% reduction before treatment to 47.15 ± 2.88% reduction after treatment p< 0.0001, p< 0.0001) and duration of hypoglycemia was increased from 18hrs to more than to 24 hrs (i.e. 20.89 ± 2.11% before treatment and 22.89 ± 0.59% after treatment p< 0.0001, p<0.001) induced by glimepiride.

It was observed in healthy rats and diabetic rats that, drug-drug interaction occur, when anastrozole and glimepiride are administered concomitantly. Since the anastrozole has shown significant effect on onset of hypoglycemia, it may be inferred that anastrozole interferes with absorption of glimepiride. However anastrozole have significantly enhanced the hypoglycemia induced by glimepiride. This may be due to fact that anastrozole mainly inhibit CYP2C9 and CYP3A4, which is involved in the metabolism of glimepiride.

The above observations suggest that the interaction between anastrozole and oral antidiabetic agents are very intense and it demands the readjustment of dose and frequency of oral antidiabetic agents when they are used concomitantly.

ACKNOWLEDGEMENTS:

Authors are thankful to the authorities of Luqman College of Pharmacy, Gulbarga for providing facilities to carry out this study. We are grateful HETRO Drugs ltd. (Mumbai) and Nosch Labs Pvt. Ltd. (Hyderabad) for providing the gift samples of glimepiride and anastrozole, respectively.

REFERENCES :

1. Steel KK, Gertman PM, Cresiene C, Anderson J. Iatrogenic illness on a general Medical service at a university hospital . N Eng J Med 1981; 34: 638-64

2. Zimmet PZ. Diabetes epidemiology as a tool to trigger diabetes research and care. Diabetologia 1999; 42: 499-518.

3. King H, Aubert RE, Herman WH. Global burden of diabetes 1995-2025. Prevalence, numerical estimates and protection. Diabetes care 1998; 2: 1414-31.

4. Songer TJ, Zimmet PZ, Epidemiology of type-II diabetes on international perspective. Pharmacometrics 8(Supl): 1995; 1-11.

5. King H, Rewers M . Diabetes in adult is now aThird World problem. The WHO Ad Hoc Diabetes Reporting Group, Bull. World Health Organ 1991; 69: 643-48.

6. Ramachandran A, Jali MV, Mohan V, Snehalatha C, Viswanathan M. High prevalence of diabetes in an urban population in india. BMJ 1998; 297: 587-590.

7. Ramaiya KL, Kodali VRR, Alberti KGMN. Epidemiology of diabetes in Asians of the Indian Sub-continent. Diabetes Metab Rev. 1990; 6: 125-146.

8. Satyanarayana S, Nitin M, Prasand K Eswarkumar K.Pharmacodynamic Drug interaction of Tolbutamide and Amlodipine in Rats, Advances in Pharmacology and Toxicology, 2003; 4(2): 55-59.

9. American Diabetes Association, Standards of Medical Care in Diabetes –2007. Diabetes Care 2007; 30: 4-41.

10. Bruning PF, Bonfrer JM, Van Noord PA, Hart AA, De Jony-Bakker M, Noorijem WJ. Insulin resistance and breast cancer risk. Int J Cancer. The Netherlands cancer institute Amsterdam. 1992 oct 21; 52(4): 511-6.

11. Wolf I, Sadetzki S, Catane R, Karasik A, Kaufman B. Diabetes mellitus and breast cancer. Lancet Oncol, institute of oncology Chaim Sheba medical center, Tel- Hashomer, Israel. Wolf@cshs.org. Feb 2005; 6(2): 103-11.

12. http://en.wikipedia.org/wiki/Breast_cancer.Breast cancer from Wikipedia the Free encyclopedia.

13. K.D Tripathi. Essential of medical pharmacology, 6^th edition, Japee Brothers Medical publishers (p) ltd. New Delhi 2008;306.

14. http:// drugbank.ca/drugs/DB01217.

15. William R Brian. Lippincott Williams and Wilks, Metabolic Drug interactions; 529-543.

16. Dowsett M, Tobias JS, Howell A, Blackman GM, Welch H, King N,Ponzone R, Von Euler M and Baum M. The effect of anastrozole on thePharmacokinetic of tamoxifen in post menopausal women with early breastCancer. Academic department of biochemistry. Royal Marsden hospital, London SW36JJ, UK, Meyerstein institute, Middlesex hospital, London W1N8AA, UK, CRC department of medical oncology, Christi hospital NHS trust, Wilmslow road, Manchester M20 9BX UK, Zeneca pharmaceutical, Mereside, Alderley park , Macclesfieid, Cheshire SK 104TG, UK, Institute of surgical Studies, Charles Bell House, W1P 7LD, UK. British Journal of cancer (1999) 79 (2), 311-315.

17. Jack De Uiterd. Overview of the antidiabetic agents Endcorine Pharmacotherapy Module, spring, 2003.

18. http://en.wikipedia.org/wiki/Drug_interaction

19. Labana PG, Mehta HR, Ramani AK, Labana PG, Shah SK. Liraglutide: a Molecule for treatment of type II DMRJBCS 2010; 1(2): 193.

20. http://en.wikipedia.org/wiki/Breast_cancer.Breast cancer from Wikipedia the Free encyclopedia.

21. Nelson, Koymans L, Kamataki, Stegeman J J, Feyereisen R, Waxman D J et al. P450 Super Family: Update on New Sequence, Gene Mapping, Accession Numbers and Nomenclature. Parmacogenetics 1996; 6:1 42.

22. William R Brian. Lippincott Williams and Wilks, Metabolic Drug interactions; 529-543.

Received on 30.05.2012

Modified on 05.06.2012

Accepted on 07.06.2012

© A&V Publication all right reserved

Research J. Pharmacology and Pharmacodynamics. 4(4): July –August, 2012, 236-240