Drug-drug interaction between voriconazole and oral hypoglycemic agents in diabetic rats.

OBJECTIVE: The objective was to study the of drug-drug interaction between voriconazole and oral hypoglycemic agents in normal and alloxan induced diabetic rats.
MATERIALS AND METHODS: The study was designed in two phases. In the first phase, influence of glibenclamide (0.45 mg/kg, p.o.) and pioglitazone (2.7 mg/kg, p.o. once daily) on blood glucose levels in normoglycemic rats was studied and then influence of voriconazole (18 mg/kg, p.o. twice daily.) pre-treatment on the hypoglycemic activity studied. Simultaneously the influence of voriconazole treatment for seven consecutive days (per se effect) on blood glucose levels was also studied in normoglycemic rats. In the second phase of the study alloxan-induced diabetic rats were used to find out the influence of voriconazole pre-treatment on glibenclamide and pioglitazone induced hypoglycemic effect in pathophysiological condition. Blood samples were collected from retro orbital plexus at regular intervals of 0.0, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 18.0 and 24.0 h after drug treatment. All the blood samples were analyzed for plasma glucose by glucose oxidase peroxidase method (GOD/POD).
RESULTS: The therapeutic dose of voriconazole potentiates the hypoglycemic activity of glibenclamide and pioglitazone both in normoglycemic and diabetic rats respectively.
CONCLUSION: The results indicate that the dose of oral hypoglycemic agents needs to be adjusted if co-administered with voriconazole.

Introduction

The drug-drug interaction is the qualitative or quantitative change in the effect of a drug by concurrent administration of two or more drugs. To treat the co-existing diseases in diabetic subjects poly-pharmacy has become mandatory and certain classes of drugs like anti-fungal and antimicrobials are frequently co-prescribed either to manage or treat either the secondary complications or other diseases. Since diabetics are immuno compromised, they are easily susceptible to a number of opportunistic fungal infections.[1-3] and a wide range of anti-fungal drugs are available among which the voriconazole is a relatively newer anti-fungal drug gaining priority in prescription for anti-fungal therapy in diabetic subjects.[3-5]

It is not always true that the repercussions due to co-administered drugs are harmful or fatal; however, impact cannot be ignored, since a considerable number of co-administered drugs alter the glycemic regulation achieved due to a particular oral hypoglycemic therapy.[6-8] Hence to safeguard the delicate health of diabetic patients it is appreciable to have accurate information on glycemic regulation achieved by commonly prescribed oral hypoglycemic drugs such as glibenclamide[9] and pioglitazone,[10] when co-administered with voriconazole. In this investigation, it was designed to evaluate the effect of voriconazole on glycemic regulation achieved by glibenclamide and pioglitazone in normal and alloxan induced diabetic rats.

Materials and Methods

Drugs

Voriconazole was a gifted by Ranbaxy Labs Pvt., Ltd, Gurgaon, Glibenclamide and Pioglitazone were procured from Maithri Labs Pvt., Ltd Hyderabad. Alloxan was purchased from SD fine chemicals, Mumbai All chemicals used were of analytical grade.

Experimental Animals

Healthy Wistar albino rats of either sex weighing 200-250 g were procured from the central animal house of the institution. The animals were housed in polypropylene cages and allowed to acclimatize for 7 days. All the animals were maintained under standard husbandry conditions of 12:12 h light: dark cycle at a temperature of 24 ± 2°C and a relative humidity of 30-70% with free access to water and standard laboratory rat pellet diet. Prior approval for animal experimentation was obtained from the institutional animal ethics committee. All the animal handling and blood with drawl techniques were carried out in accordance with the Committee for the purpose of control and supervision on experimental animals (CPCSEA) guidelines (Regd No. 576/02/bc/CPCSEA).

Preparation of Drugs

In clinical practice, voriconazole, glibenclamide and pioglitazone in therapeutic doses administered orally. Hence, human therapeutic doses extrapolated to rats based on the body surface area were administered orally.[11] Voriconazole, glibenclamide and pioglitazone were individually suspended in 2% w/v acacia suspension in distilled water. The suspension of any particular drug to be administered was freshly prepared just before treatment.

Induction of Diabetes

Diabetes was induced by using freshly prepared solution of alloxan monohydrate dissolved in normal saline to 18 h fasted rats in two doses (i.e., 100 mg/kg and 50 mg/kg; i.p.) for two consecutive days.[12] Hyperglycemia was confirmed by elevated glucose level in plasma determined at 48 h after injection. Rats with blood-glucose levels above 250 mg/dL were considered as diabetic and selected for the study.

Study in Normoglycemic Rats

Wistar rats of either sex weighing 200-250 g were randomly allocated to three groups (n = 6). The animals were fasted for 18 h before the scheduled time of experimentation provided water ad libitum and the fasting was continued till the end of the experimentation. All the group I animals received freshly prepared suspension of voriconazole (18 mg/kg; p.o. twice daily) for seven consecutive days and animals of group II and group III received suspensions of glibenclamide (0.45 mg/kg; p.o.) and pioglitazone (2.7 mg/kg; p.o. once daily for 7 days) for assessing the per se effect. Blood samples were collected from retro orbital plexus of all rats at regular time intervals of 0.0, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 18.0 and 24.0 h after drug administration.[1314] All the blood samples were analyzed for plasma glucose by glucose oxidase peroxidase (GOD/POD) method.[15] Blood glucose levels were expressed as mg/100 mL of blood. After 7 days of wash out period, the same animals of group I and group II received voriconazole (18 mg/kg; p.o. twice daily) for seven consecutive days. On the 7th day, 6 h after treatment of voriconazole, all the animals were fasted for 18 h and provided water ad libitum. On the 8th day after 1 h of voriconazole treatment all the animals of group I received suspension of glibenclamide and animals of group II received suspension of pioglitazone. Blood samples were collected thereafter at above-mentioned time intervals and blood glucose levels were estimated.

Study in Diabetic Rats

The diabetic rats (n = 6) of either sex were selected for the study. The animals were fasted for 18 h with water ad libitum and the fasting was continued till the end of the experimentation. After fasting the blood samples were collected for blood glucose estimation. In the first part of this study group I animals received suspension of glibenclamide (0.45 mg/kg) and group II animals received pioglitazone (2.7 mg/kg) through oral route. Blood samples were collected from retro orbital plexus of diabetic rats at regular time intervals of 0.0, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 18.0 and 24.0 h after drug administration. All the blood samples were analyzed for plasma glucose by GOD/POD method. In the next part of this study after 7 days of the wash out period, the same animals of group I and group II received voriconazole (18 mg/kg, p.o. twice daily) for 1 week. On the 7th day, 6 h after administration of voriconazole all the rats were fasted for 18 h with water ad libitum. On the 8th day 1 h after the administration of voriconazole all the group I animals received glibenclamide and group II animals received pioglitazone. Blood samples were collected thereafter at above mentioned time intervals and blood glucose levels were estimated.

Statistical Analysis

The data were expressed as mean ± standard error of the mean. The significance was determined by Paired Student's t test. Data were computed for statistical analysis using Graph Pad Instat and prism software. The results were considered statistically significant if P < 0.05.

Results

The results of our study are compiled in the Table 1 and Figures 1–4. The onset of action (a minimum of 20% reduction in fasting blood sugar level), peak effect (highest percentage of reduction in fasting blood sugar level recorded in the sampling schedule), duration of action (the total time period during which there is reduction of fasting blood sugar by at least 20% or above) were considered to interpret the study results.

Table 1

Effect of voriconazole (18 mg/kg; p.o.) administration on blood glucose levels in normoglycemic rats (per se effect)

Figure 1

Effect of Voriconazole (18 mg/kg) on Glibenclamide (0.45 mg/kg) induced hypoglycemia in normoglycemic rats

Figure 4

Effect of Voriconazole (18 mg/kg) on Pioglitazone (2.7 mg/kg) induced hypoglycemia in diabetic rats

Voriconazole (18 mg/kg, p.o.) per se administration for seven consecutive days did not influence the basal blood glucose levels in normoglycemic rats [Table 1]. This indicates that voriconazole do not possess any significant hypoglycemic effect by itself. The onset of action was at 2nd h in normoglycemic rats when administered with glibenclamide (0.45 mg/kg; p.o.). The peak hypoglycemic effect was observed at the 4th h and the duration of hypoglycemia was within 12 h [Figure 1]. However, the same animals after washout period were pre-treated with voriconazole (18 mg/kg; p.o.) and again when glibenclamide (0.45 mg/kg, p.o) was administered on 8th day, the parameters were significantly changed. After pre-treatment with voriconazole (18 mg/kg, p.o.) there was no significant change in the onset of action. The peak hypoglycemic effect was enhanced from 28.85 ± 4.3% at 4th h (before voriconazole treatment) to 58.91 ± 4.3% at 8th h (after voriconazole treatment), which is both statistically and clinically significant (P < 0.01). Further, in this case the duration of hypoglycemia was prolonged for more than 18 h.

When pioglitazone (2.7 mg/kg, p.o.) alone was administered to normoglycemic rats, the onset of hypoglycemic action was observed at 2nd h [Figure 2]. In this case, the peak hypoglycemic effect was observed at 8th h and the duration of hypoglycemic action was within 18 h. After 1 week of washout period the same animals when pre-treated with voriconazole (18 mg/kg; p.o.) and then challenged with pioglitazone (2.7 mg/kg, p.o) the onset of hypoglycemic action was postponed for more than 2 h and the peak hypoglycemic effect was enhanced from 35.07 ± 6.5% at 8th h (before voriconazole treatment) to 45.93 ± 6.5% at 4th h (after voriconazole treatment), which is both statistically and clinically significant (P < 0.01). However, there was prolongation of duration of hypoglycemia up to 18 h.

Figure 2

Effect of Voriconazole (18 mg/kg) on Pioglitazone (2.7 mg/kg) induced hypoglycemia in normoglycemic rats

In alloxan-induced diabetic rats, voriconazole (18 mg/kg; p.o.) pre-treatment has no effect on the onset of hypoglycemic action of glibenclamide (0.45 mg/kg, p.o.). The peak hypoglycemic activity was enhanced from 44.65 ± 2.1% at 2nd h (before voriconazole treatment) to 62.35 ± 1.3% at 4th h (after voriconazole treatment) which is clinically and statistically significant (P < 0.001) [Figure 3]. The duration of anti-diabetic effect of glibenclamide was within 18 h both before and after treatment. Most of the animals exhibited prognostic signs of hypoglycemia, characterized by lack of interest in the surroundings and reduced physical activity indicating the occurrence of drug-drug interaction between voriconazole and glibenclamide. Voriconazole (18 mg/kg; p.o.) pre-treatment altered the onset of glucose lowering action of pioglitazone (2.7 mg/kg; p.o.) which is within 2nd h before voriconazole treatment and is at1 h after voriconazole pre-treatment [Figure 4]. The peak hypoglycemic effect was enhanced from 28.89 ± 3.7% at 2nd h (before voriconazole treatment) to 50.41 ± 2.6% at 4th h (after voriconazole treatment), which is both clinically and statistically significant (P < 0.001). Further, in this case, the hypoglycemic effect was prolonged up to 18 h after voriconazole pre-treatment.

Figure 3

Effect of Voriconazole (18 mg/kg) on Glibenclamide (0.45 mg/kg) induced hypoglycemia in diabetic rats

Discussion

The new chemical entities are finding the place for pharmacotherapy. The co-administration of these drugs may complicate the pharmacotherapy and the consequence may be useful or harmful. Hence, it is essential to evaluate the possibility of drug-drug interactions among the multiple drug prescriptions. In this regard, the current study involving evaluation of drug-drug interaction between voriconazole and oral hypoglycemic drugs is essential. Among the oral hypoglycemics of different categories, the most mostly prescribed drugs were insulin, sulfonyl ureas (glibenclamide) and thiazolidinediones. Of the letters, pioglitazone was prescribed most frequently and in this study these oral hypoglycemics were selected for evaluation.[910] Drug-drug interactions that usually occur in clinical practice are mostly evaluated by using animal models (rodent or non-rodent species). In our early study, we have revealed the effect of Voriconazole on glycemic regulation achieved by Glibenclamide and Pioglitazone in New Zealand rabbits[16] and this study is planned to study the effect of voriconazole on the anti-diabetic drugs in normal and alloxan-induced diabetic rats. The normoglycemic rat model served to quickly identify the interaction and the diabetic rat model served to validate the same response in pathophysiological condition in which our study drugs glibenclamide and pioglitazone are used. When two or more drugs are co-administered with oral anti-diabetic drugs, the instances of hypoglycemia are due to occurrence of either pharmacokinetic or pharmacodynamics type of drug interactions. The possibility of additive type of drug-drug interactions between the study drugs is ruled out; since, voriconazole by itself is devoid of any hypoglycemic activity. However, several documented reports reveal that oral hypoglycemic drugs when co-administered with oral anti-fungal drugs or any other drugs for the rational treatment of co-morbidities, it resulted in severe hypoglycemia.[717-19] The isoenzymes cytochrome (CYP), CYP2C9, CYP2C19 and CYP3A4 are responsible for metabolism of glibenclamide,[20] Similarly the other oral anti-diabetic drug pioglitazone is metabolized by microsomal isoenzymes CYP2C8, CYP3A4 and to lesser extent by CYP2C9.[2122] Documented reports reveal that, the study drug voriconazole is an inhibitor of CYP enzymes particularly CYP2C19, CYP2C9, CYP3A4. The affinity of voriconazole is highest for CYP2C19, followed by CYP2C9, and lower for CYP3A4.[523] In vitro studies also show that the major metabolite of voriconazole, voriconazole N-oxide, inhibits the metabolic activity of CYP2C9 and CYP3A4 to a greater extent than that of CYP2C19. Therefore, there is potential for voriconazole and its major metabolite to increase the systemic exposure (plasma concentrations) of other drugs metabolized by these CYP450 enzymes.[24] As peak hypoglycemic activity of glibenclamide is enhanced significantly after voriconazole pre-treatment in every case it is inferred that, the interaction at the metabolic phase has played a significant role in the effect. The prolongation of hypoglycemic action of pioglitazone may be due to inhibition of specific enzymes responsible for its metabolism by voriconazole. Based on blood sugar levels both before and after voriconazole treatment it is suggested that, when an anti-fungal drug voriconazole is co-administered with routine oral hypoglycemic drugs such as glibenclamide or pioglitazone for specified period, there occurs pharmacokinetic type of drug-drug interaction in both normal and diabetic animals.

In conclusion the drug-drug interaction is evident between oral hypoglycemic and anti-fungal drugs. This suggest that co-administration of these drugs should be done with caution as it may lead to severe hypoglycemia. This is particularly significant with pioglitazone and voriconazole.