Influence of atazanavir on the pharmacodynamics and pharmacokinetics of gliclazide in animal models

1. Introduction 

The study of mechanisms of drug interaction is of much value in selecting drug concentrations to provide rational therapy. Drug interaction studies assume much importance, especially for drugs that have a narrow margin of safety, and where the drugs are used for a prolonged period of time. Diabetes mellitus is one such metabolic disorder that needs treatment for prolonged periods, and maintenance of normal blood glucose level is very important in this condition, since both hyperglycemia, as well as hypoglycemia, is unwanted phenomenon [1].

Diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose levels and disturbances in carbohydrate, fat and protein metabolism, and an increased risk of complications from vascular disease. Diabetes may be due to a decrease in the synthesis of insulin (type-1) or a decrease in the secretion of insulin (type-2) from the β-cells of islets of Langerhans of the pancreas. There are an estimated 143 million people world wide sufferings from diabetes [2] and the number may well double by the year 2030 [3]. In India, the prevalence rate of diabetes is estimated to be 1–5%.

Insulin resistance, impaired glucose tolerance and type-2 diabetes are conditions that are increasingly described in HIV-1 infected subjects receiving highly active antiretroviral therapy (HAART), especially with protease inhibitors (PIs) [4], [5]. Atazanavir is a commonly prescribed protease inhibitor, due to its once-daily dosing regimen, favorable metabolic profile and low frequency of adverse effects [6]. However, its effect on oral antidiabetic therapy is not known.

Oral hypoglycemic agents are used in the treatment of type-2 diabetes, among which gliclazide, a second generation sulphonylurea derivative, is preferred in therapy because of its selective inhibitory activity towards pancreatic K+ ATP channels [7], antioxidant property [8], low incidence of producing severe hypoglycemia [9] and other haemobiological effects. Gliclazide is known to act mainly by releasing insulin by blocking K+ channels in the pancreatic β-cells [10].

Atazanavir is a substrate and potent inhibitor of the cytochrome P450 (CYP) system, in particular CYP3A4 and CYP2C9 and affect the metabolism of several drugs [11]. Because atazanavir can inhibit CYP3A4 and CYP2C9-mediated drug metabolism and gliclazide is reported to be metabolized by CYP2C9 primarily and partly by CYP3A4 [10], [12], it is important to study the possible effects of atazanavir on the pharmacokinetics and pharmacodynamics of gliclazide. However, there seem to be no published studies on the effects of enzyme inhibition on the pharmacokinetics of gliclazide.

Since there is every possibility for the combined use of gliclazide and atazanavir in chronic diabetics with associated HIV infection, the study is planned to investigate the effect of atazanavir on the activity of gliclazide in normal and diabetic rats, to evaluate the safety and effectiveness of the combination. Also the study is planned to find the pharmacodynamics and pharmacokinetics of gliclazide in the presence of atazanavir in rabbits, to evaluate the mechanisms of interaction if they occur.

2. Material and methods 

3. Results 

3.1. Pharmacodynamic interaction study in normal and diabetic rats 

Gliclazide produced hypoglycemic activity with maximum biphasic reduction of 40.88±0.57% and 39.01±0.73% in normal rats, and 42.95±1.74% and 44.14±1.78% in diabetic rats at 2h and 8h, respectively. Atazanavir has no significant effect on the blood glucose levels in normal and diabetic rats. In combination, atazanavir produced enhanced hypoglycemic effect of gliclazide with maximum blood glucose reduction of 48.27±1.04% & 45.69±1.53% and 50.14±0.87% & 47.54±1.00% at 2h and 8h, following single dose and multiple dose administration of atazanavir, respectively, in normal rats (Table 1). In combination, atazanavir produced enhanced hypoglycemic effect of gliclazide with maximum blood glucose reduction of 44.99±1.13% & 48.00±1.26% and 48.89±1.18% & 50.25±0.87% at 2h and 8h, following single dose and multiple dose administration of atazanavir, respectively, in diabetic rats (Table 1). The enhancement in gliclazide effect is more with the multiple dose treatment of atazanavir than single dose treatment.

Table 1. Mean percent blood glucose reduction in normal and diabetic rats (N=6).

	Time (h)	Normal rats				Diabetic rats
		G	A	A+G (SDT)	A+G (MDT)	G	A	A+G (SDT)	A+G (MDT)
	1	31.96±0.60	−06.30±0.66	36.15±1.42⁎	38.55±1.88⁎	33.43±1.89	−06.55±2.64	37.58±1.40⁎⁎	39.16±1.18⁎⁎
	2	40.88±0.57	−04.90±0.96	48.27±1.04⁎	50.14±0.87⁎	42.95±1.74	−04.82±2.59	44.99±1.13	48.89±1.18⁎⁎
	3	28.62±0.58	−03.37±0.80	35.00±2.02⁎	36.71±1.49⁎	30.89±2.01	−03.12±2.71	36.86±1.92⁎⁎	37.78±1.76⁎⁎
	4	23.40±0.81	−02.28±1.02	28.82±1.46⁎	33.04±1.48⁎	24.81±1.99	−02.69±2.41	30.07±1.31⁎⁎	32.74±0.94⁎⁎
	6	30.10±0.84	−01.13±0.51	38.01±1.35⁎	42.68±1.24⁎	36.76±1.63	04.74±1.87	40.07±1.44⁎⁎	42.59±1.15⁎⁎
	8	39.01±0.73	06.72±0.68	45.69±1.53⁎	47.54±1.00⁎	44.14±1.78	06.03±2.22	48.00±1.26⁎⁎	50.25±0.87⁎⁎
	10	26.76±0.53	08.94±0.72	32.85±1.43⁎	35.25±1.25⁎	28.77±2.09	08.10±1.37	31.75±1.47⁎⁎	32.54±1.27⁎⁎
	12	13.74±0.62	09.27±0.71	25.15±1.57⁎	28.21±0.74⁎	24.63±2.36	04.62±1.76	26.17±1.92	28.56±1.77⁎⁎

G: gliclazide – 2mg/kgbd.wt. treated group. A: atazanavir – 36mg/kgbd.wt. treated group. A+G (SDT): atazanavir+gliclazide (single dose treatment). A+G (MDT): atazanavir+gliclazide (multiple dose treatment).

⁎ Significant at P<0.05 compared to gliclazide control (normal rats). ⁎⁎ Significant at P<0.05 compared to gliclazide control (diabetic rats).

3.2. Pharmacodynamic interaction study in normal rabbits 

Gliclazide produced hypoglycemic activity with maximum reduction of 35.22±1.09% at 3h in normal rabbits. Atazanavir has no significant effect on the blood glucose levels in normal rabbits. Atazanavir produced enhanced hypoglycemic effect of gliclazide with maximum reduction of 43.66±0.39% and 45.72±0.94% in the blood glucose in normal rabbits at 3h following single dose and multiple dose treatment of atazanavir, respectively (Table 2). The enhancement in gliclazide effect is more with the multiple dose treatment of atazanavir than the single dose treatment. The serum insulin levels were increased with atazanavir treatment in normal rabbits (Fig. 1).

Table 2. Mean percent blood glucose reduction in normal rabbits (N=6).

	Time (h)	Gliclazide (5.6mg/1.5kgbd.wt.)	Atazanavir (28mg/1.5kgbd.wt.)	Atazanavir+gliclazide (single dose treatment)	Atazanavir+gliclazide (multiple dose treatment)
	1	20.43±1.80	−06.56±0.82	28.87±1.03⁎	30.48±1.05⁎
	2	26.59±0.66	−05.30±1.40	34.15±0.52⁎	36.87±0.51⁎
	3	35.22±1.09	−04.59±1.47	43.66±0.39⁎	45.72±0.94⁎
	4	27.63±1.76	−04.57±1.24	35.90±0.64⁎	37.55±1.28⁎
	6	25.52±0.75	−03.17±1.77	34.85±0.80⁎	36.53±1.27⁎
	8	19.70±2.44	08.47±1.43	28.87±2.08⁎	30.82±2.63⁎
	12	12.54±1.54	06.38±1.81	20.74±2.29⁎	24.04±2.48⁎
	16	07.16±4.01	04.95±1.42	16.90±1.72⁎	20.17±2.05⁎
	20	06.79±1.34	04.93±1.76	12.32±0.99⁎	15.92±1.35⁎
	24	03.92±1.41	03.18±0.90	10.20±083⁎	12.75±1.08⁎

⁎ Significant at P<0.05 compared to gliclazide control.

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Fig. 1. Effect of atazanavir/gliclazide and their combination on serum insulin levels in normal rabbits (N=6).

3.3. Pharmacokinetic interaction study in normal rabbits 

The serum gliclazide levels were increased, and pharmacokinetic parameters of gliclazide like Cmax, Tmax, AUC, AUMC, Kel and T1/2 were altered significantly with single- and multiple-dose treatments of atazanavir in normal rabbits (Table 3). The percent increase of serum gliclazide level is 22.73% and 24.26% following single dose and multiple dose administration of atazanavir, respectively.

Table 3. Mean pharmacokinetic parameters of gliclazide before and after atazanavir administration in rabbits.

	Pharmacokinetic parameter	Gliclazide	Atazanavir+Gliclazide (single dose treatment)	Atazanavir+Gliclazide (multiple dose treatment)
	Cmax (ng/ml)	346.79±4.21	428.21±8.68⁎	431.63±8.61⁎
	Tmax (h)	3.00±0.00	3.00±0.00⁎	3.00±0.00⁎
	AUC0–24 (ng/ml/h)	4013.84±147.00	4913.80±112.05⁎	4969.46±11.53⁎
	AUC0–inf (ng/ml/h)	4777.04±206.58	6048.95±176.75⁎	6143.66±174.7⁎
	AUC% Extrapolation	15.82±1.20	18.62±1.51⁎	18.98±1.45⁎
	AUMC0–24 (ng/ml/h⁎h)	38787.60±1809.59	46863.51±1440.81⁎	47517.88±1426.66⁎
	AUMC0–inf (ng/ml/h⁎h)	66332.61±4644.81	89966.53±5680.50⁎	92221.81±5612.05⁎
	AUMC% Extrapolation	40.82±2.54	47.07±2.93⁎	47.71±2.78⁎
	MRT0–24 (h)	9.64±0.13	9.53±0.09	9.55±0.09
	MRT0–inf (h)	13.82±0.49	14.81±0.62⁎	14.95±0.60⁎
	Kel (h−1)	0.08±0.00	0.07±0.00	0.07±0.00
	T1/2 (h)	8.15±0.53	9.41±0.58⁎	9.50±0.56⁎

⁎ Significant at P<0.05 compared to gliclazide control.

4. Discussion 

Drug interactions are usually seen in clinical practice, and the mechanisms of interactions are evaluated usually in animal models (rodent and non-rodent). We studied the influence of atazanavir on the pharmacodynamics and pharmacokinetics of gliclazide in rats (rodents) and rabbits (non-rodent). The normal rat model served to quickly identify the interaction and the diabetic rat model served to validate the same response in the actually used condition of the drug. The rabbit model is another dissimilar species to validate the occurrence of the interaction. The multiple dose effect of atazanavir on gliclazide activity was also studied for the influence of long term treatment with atazanavir, since both are used for chronic period.

Rats are known to be more sensitive to gliclazide response. So we have conducted the dose effect-relationship study of gliclazide to select the oral dose, which produces approximately 35% of blood glucose reduction in rats. Gliclazide produced biphasic response in rat model when administered alone, which may be due to its biliary excretion and entero hepatic cycling. Such an effect is not seen in the rabbit model. The enterohepatic recirculation observed in our study was consistent with the previous reports in animal models [1], [12] and humans [19]. As per the recent clinical study [19], the extent of mean enterohepatic recirculation observed in humans was consistent with data from animals. This consistency will address the probable correlation of preclinical studies to human subjects.

In our study, atazanavir alone did not produce any significant activity on blood glucose levels of rats (normal and diabetic) and rabbits, and our results were consistent with earlier reports from in vitro [20] and in vivo studies [6], [21]. This suggested that protease inhibitors effects were drug specific but not class specific, as other protease inhibitors have a significant impact on glucose homeostasis. Interestingly, however, the gliclazide hypoglycemic and antidiabetic activity was significantly enhanced by atazanavir, following a single and multiple dose treatment in rat and rabbit models, and this confirmed the presence of potential interaction between gliclazide and atazanavir. Further, the presence of interaction was supported by an increase in serum insulin levels with atazanavir treatment. It is clear that since atazanavir did not alter blood glucose levels on its own, the increase in the effect of gliclazide on blood glucose may be due to improved blood gliclazide level in the presence of atazanavir, as it was confirmed by pharmacokinetic interaction study in rabbits.

There was a significant rise in serum gliclazide levels and an alteration in pharmacokinetic parameters like Cmax, Tmax, AUC, AUMC, Kel and T1/2 of gliclazide with single- and multiple-dose treatments of atazanavir. The increase in AUC and AUMC indicates improved availability of gliclazide in presence of atazanavir. There might not be interaction at absorption level, since oral absorption of atazanavir is not high. Gliclazide is a highly protein bound drug (85–99%) [22], whereas atazanavir is bound to proteins to the extent of 86–89% [23]. Hence, the possibility of displacing gliclazide from protein bound sites by atazanavir was low. Moreover, the rise of gliclazide blood levels in the presence of atazanavir might be other than improved absorption and altered distribution.

The altered Kel and T1/2 indicates alteration either in metabolism or the excretion process. Atazanavir is reported to be a potent inhibitor of CYP3A4 and CYP2C9 [11], and there is more possibility of atazanavir for inhibition of metabolism of gliclazide, which is also metabolized by both CYP2C9 and CYP3A4 [10], [12]. Further gliclazide is eliminated through renal (80%) and biliary (20%) routes [22], [24]. The major elimination pathway of atazanavir is the biliary route. Atazanavir is eliminated by 13% being in urine and 79% in feacal matter [23]. Hence, there is also a possibility for interaction between atazanavir and gliclazide at biliary excretion. However, the drug atazanavir did not change the pattern of biphasic response of gliclazide, indicating that it did not interfere with the reabsorption of gliclazide in its enterohepatic circulation in rats. Hence, the interaction at hepatic metabolism with reduced gliclazide metabolism by atazanavir, leading to raised serum levels, remains possible.

5. Conclusions 

The interaction appears to be pharmacokinetic interaction at metabolic level. Since the interaction was seen in two dissimilar species, it is likely to occur in humans also leading to increased activity of gliclazide, which may need dosage adjustment. Hence, care should be taken when the combination is prescribed for clinical benefit in diabetic patients. However, the present study warrants further studies to find out the relevance of this interaction in human beings.