D-400, a herbomineral preparation has proven antidiabetic activity in experimental models as well as in clinical trials. The possibility of concomitant use of this drug with sulphonylureas was explored in animal models. D-400 has been investigated for its interaction with oral hypoglycaemic agents namely, tolbutamide and glibenclamide in alloxan-induced diabetic rabbits. Administration of D-400 at a dose of 1 g/kg for 15 days significantly elevated plasma tolbutamide and glibenclamide concentrations with simultaneous reduction of blood glucose. Plasma tolbutamide and glibenclamide concentrations with simultaneous reduction of blood glucose. Plasma tolbutamide and glibenclamide concentrations were significantly lowered after withdrawal of D-400 treatment. Elevation of plasma concentration of tolbutamide was observed only for the first 4 h after which it declined towards normal levels and no significant difference between D-400 treated and control group was observed at the end of 8 h. Significant elevation of plasma glibenclamide levels was observed at 2, 4 and 8 h with D-400 treatment. Incubation of D-400 with tolbutamide in plasma resulted in a significant increase in free tolbutamide levels.

Keywords: D-400; Tolbutamide; Glibenclamide; Drug interaction

Diabetes mellitus is one of the challenges faced by modern medicine. Non-Insulin-Dependent Diabetes Mellitus (NIDDM) is one of the most common disorders in developing countries. While there are oral hypoglycaemic agents (OHAs) like biguanides and sulphonylureas to treat diabetes mellitus, none have been successful in maintaining euglycaemia and avoiding late stage complications of diabetes. In about one-quarter of the patients these drugs lose their effectiveness, though the initial response has been good.

About 15-20% of the patients with newly diagnosed NIDDM present little or no response to sulphonylureas (Berhhard, 1965; Balodimos et al., 1966). About 50% will attain acceptable or normal glycaemic control and the remainder will attain between acceptable and poor glycaemic control. Every year, about 3-5% of the patients with NIDDM who have attained acceptable or better glycaemic control are said to lose their responsiveness to sulphonylureas (Cervantes Amecua et al., 1965; Powell and Howells, 1966; Muller et al., 1969; Turner et al., 1989; Groop et al., 1986). Adverse effects or oral hypoglycaemic agents occur in roughly 3% of the patients. Hypoglycaemia is often one of the most important and fatal effects of sulphonylureas.

Ancient Indian medicine mentions several indigenous plants and mineral preparations for the treatment of diabetes mellitus. D-400 is one such herbomineral formulation containing plants and minerals known for their antidiabetic action (Table 1). Several experimental trials have proved significant lowering of blood sugar levels with this formulation (Anturlikar et al., 1995; Dubey et al., 1994; Mitra et al., 1996). This is also said to have a beneficial effect on lipid profile, kidney profile and glycated haemoglobin in streptozotocin-induced diabetic rats (Mitra et al., 1995).

A study was conducted keeping perspective the above information to evaluate the efficacy of the herbomineral formulation D-400, in diabetic rabbits treated with oral hypoglycaemic agents. The objective of the study was to obtain better glycaemic control with a low dose of hypoglycaemic and thereby to reduce the adverse effects and tolerance to oral hypoglycaemic agents.

Plant Materials
The constituent plants of the formulation were procured from authentic sources and were identified by Dr. S. Farooq, botanist of The Himilaya Drug Company. A voucher sample of the formulation was deposited in the herbarium of our R and D Centre, Bangalore (Fa 201-223). All plant powders were individually weighed and mixed. The drug was administered as an oral aqueous suspension.

Experimental animals
Sixteen adult male rabbits of New Zealand white strain weighing between 1.6 – 2.0 kg were selected for the study. The animals were housed at a room temperature of 26 ± 2° C under a 12 h light, 12-h dark cycle, and were fed a synthetic diet (Lipton India Ltd. pellets). Water was allowed ad libitum. Diabetes was induced by a single i. p. injection of alloxan monohydrate (150mg/kg). After 96 h, basal blood glucose was estimated. Twelve rabbits which showed fasting, blood sugar (FBS) more than 250mg/dl were selected for the study. The animals were divided into two groups consisting of six animals each. Group I received tolbutamide at the dose of 250mg/kg once a day orally for 7 days and on day seven, blood was collected from the marginal ear vein after 2, 4, 6 and 8 h for blood glucose and plasma tolbutamide estimation. From day 8 to day 21, D-400 was administered orally as an aqueous suspension at a dose of 1 g/kg along with tolbutamide. At the end of this treatment period, the rabbits were fasted overnight and blood collected for glucose and plasma tolbutamide estimation as mentioned above. From day 16, D-400 treatment was withdrawn but tolbutamide treatment was continued for another 15 days at the end of which blood glucose and plasma tolbutamide levels were estimated as mentioned above.

Group II received glibenclamide at a dose of 3mg/kg once a day orally for 7 days. On day 7, the animals were fasted overnight and blood was collected from the marginal ear vein for fasting blood sugar estimation. Glibenclamide was administered orally in the dose of 3mg/kg for 7 days and blood was collected after 1,2, 4 and 8 h for glucose and plasma glibenclamide for 15 days. At the end of this period, D-400 treatment was withdrawn and blood was collected from overnight fasted rabbits for blood glucose and plasma glibenclamide levels were estimated again as mentioned above.

Biological assays
Plasma concentration of oral hypoglycaemics (tolbutamide and glibenclamide) was estimated by HPLC using a Spectra physics model P2000 pump with UV1000 detector. A Versapak C₁₈ 10 m (4.1 mm i. d. and 250 mm length) column was used. The mobile phase for the estimation of tolbutamide consisted of acetonitrile/phosphoric acid (0.05%), 38:62 at a flow rate of 0.9ml/min. Chlorpropamide was used as an internal standard for the estimation of tolbutamide. The mobile phase for the determination of glibenclamide consisted of acetonitrile/phosphoric acid (0.05%), 50:50 at a flow rate of 1ml/min. Tolbutamide was used as an internal standard for the estimation of glibenclamide. The eluent was monitored at 228 nm. Tolbutamide was extracted according to the method of Keal et al. (1986). Glibenclamide was extracted according to the method of Potter and Hulm (1983). In both cases, samples were evaporated and reconstituted with acetonitrile and 20 m l of the sample was injected into the column.

An in vitro experiment was conducted in order to study the displacement of tolbutamide and glibenclamide from the protein binding sites by D-400 (Miller et al., 1978). In this case the HPLC method was adopted as mentioned above instead of the RIA method for estimation of tolbutamide and glibenclamide. Blood glucose was estimated using the GOD-PAP kit of Boehringer Mannheim.

Statistical analysis
The data were expressed as mean ± SEM and Student’s t-test was used to check the level of significance.

Administration of D-400 at a dose of 1 g/kg for 15 days (Day-21) significantly increased the plasma level of tolbutamide at the 2nd and 4th h (Table 2). Withdrawal of the D-400 treatment for 15 days significantly reduced the plasma concentration of tolbutamide when compared with the tolbutamide + D-400 treatment. Blood sugar also reduced significantly with D-400 + tolbutamide treatment as compared to tolbutamide treatment alone. D-400 withdrawal resulted in increased blood sugar level as compared to combination treatment (Table 2).

In the present study, when D-400 was added to tolbutamide therapy, there was a significant reduction in blood sugar levels substantiated by a fourfold increase in plasma tolbutamide levels. This increase in plasma was observed after 2 h and at the end of 8 h. no significant difference was observed in the plasma level of tolbutamide. This indicates that the D-400 has either inhibited the hepatic cytochrome P-450 or might have displaced tolbutamide from the protein binding sites resulting in a fourfold increase in plasma tolbutamide level (which was observed in an in vitro study, where incubation of tolbutamide along with D-400 resulted in more free tolbutamide levels). That D-400 was responsible for the increase in plasma tolbutamide was further proved by the fact that when it was withdrawn for 15 days there was a decrease in plasma tolbutamide level with a rise in blood sugar level.

In the glibenclamide treated group, the significant increase in plasma levels of glibenclamide was observed at the end of 2 h and remained significantly high even at the end of 8 h with a reduction in blood sugar levels. There was no free glibenclamide available in the in vitro study which could be due to strong protein binding capacity of glibenclamide. It is premature at the present juncture to conclude the mechanism of interaction of D-400 with glibenclamide.

It can thus be concluded that D-400 not only reduces blood sugar levels in diabetes mellitus but may also be a good adjuvant to oral hypoglycaemic agents. This will help in reducing the dosage of OHAs and thereby alleviate the side effects.