Influence of Piperine on Pioglitazone Metabolism and Pk/Pd: Diabetes Mellitus

In some studies piperine is known to inhibit human CYP2C8, CYP3A4 and P-glycoprotein and the enzymes more important for the metabolism of pioglitazone and transport of xenobiotics [1-3]. Piperine can improve the bioavailability of many drugs and decrease the elimination of the drugs and finally improves the biological effectiveness [2]. Major population from America taking herbal medications without informing their medical doctors [4]. Piperine has been found to have anti diabetic activity per se [5]. However, as the metabolism of pioglitazone is inhibited by piperine there is the chance to improve the therapy with this combination.


Introduction
In some studies piperine is known to inhibit human CYP2C8, CYP3A4 and P-glycoprotein and the enzymes more important for the metabolism of pioglitazone and transport of xenobiotics [1][2][3]. Piperine can improve the bioavailability of many drugs and decrease the elimination of the drugs and finally improves the biological effectiveness [2]. Major population from America taking herbal medications without informing their medical doctors [4]. Piperine has been found to have anti diabetic activity per se [5]. However, as the metabolism of pioglitazone is inhibited by piperine there is the chance to improve the therapy with this combination.
Pioglitazone is metabolised by CYP3A4 [6] and its metabolism is inhibited by piperine. So there is the chance to potential interactions between piperine and pioglitazone combined usage. Pioglitazone can cause several complications during the treatment of the diabetes mellitus and the combination can reduce the dose and also the complications.
Investigators have predicted that India will have the greatest increase in diabetes and will have the largest number of diabetic patient in the world [7]. It has been reported that Asian Indians have an ethnic susceptibility to type 2 diabetes. The literature indicates that the prevalence of the metabolic syndrome and particularly diabetes is high among migrant Asian Indian and is rising very rapidly even within the Indian sub-continent. Recent WHO reports show that, India already has the largest number of the diabetic patient compared to any given company.
analytical grade and HPLC grade acetonitrile, methanol and potassium dihydrogen phosphate supplied by Merck (Mumbai, India).

Chromatographic conditions
Pioglitazone concentration was determined by slight modification of a method reported the mobile phase consists of 25 mM Phosphate buffer (PH adjusted to 3 with orthophosphoric acid), acetonitrile & methanol in a ratio of 55:37.5:7.5 (v/v/v). The mobile phase was degassed & filtered through 0.22 µm membrane filter. The flow rate was 1.2 mL/min & the effluent was monitored at 269 nm. The total run time of the method was set at 10 min.
To a volume of 100 µL of blank rabbit serum, 50 µL of rosiglitazone (2.5 µg in methanol) solution as internal standard & 100 µL 0f acetonitrile were added to precipitate the proteins. The mixture was vortex mixed for 5 min after which it was centrifuged at 10,000 ×g for 10 min. 20 L of the supernatant was injected on to the HPLC system for analysis. The calibration curve was obtained by plotting peak area ratios of pioglitazone to rtosiglitazone (y-axis) against pioglitazone concentration (x-axis).

Preparation of test samples
To a volume of 100 µL of test rabbit serum, 50 µL of rosiglitazone (2.5 µg in methanol) solution as internal standard & 100 µL 0f acetonitrile were added to precipitate the proteins. The mixture was vortex mixed for 5 min after which it was centrifuged at 10,000×g for 10 min. 20 L of the supernatant was injected onto the HPLC system for analysis ( Figure  1).

Limit of detection and Limit of quantification
Three calibration curves were obtained by spiking thrice, the standard dilutionss of pioglitazone in serum samples.  Hence, the limit of detection and limit of quantification were both found to be within the range of the analytic levels in serum samples.

Accuracy and precision
Intra and inter-day precision expressed as percentage of standard deviation (%RSD) and accuracy expressed as percentage of relative error (%RE) obtained from three levels of quality control samples of Pioglitazone. The precision and accuracy of the method was established by using quality control samples at low, medium and high concentrations of 0.1, 1 & 10 μg/mL for Pioglitazone. All the samples were run in three replicates. Intra-day precision data was obtained by analyzing three sets of quality control samples in a single day, while the inter-day data was obtained by analyzing the quality control samples on three consecutive days of assay.
Assay precision was calculated using the formula: Intra and inter day precision and accuracy of the determination of pioglitazone quality control samples (n =4). The assay procedure was found to be precise and accurate.

Calculations of pharmacokinetic parameters
Non compartmental Pharmacokinetic analysis was carried out using Kinetica TM software (version 4.4.1 Thermo Electron Corporation, U.S.A). The following Pharmacokinetic parameters were calculated C max , T max , t ½, AUC 0ton, AUC tot and MRT.

Statistical analysis
Difference in between concentration time profiles; in between pharmacokinetic parameters; in between serum glucose levels and difference between over the entire range tested were analyzed by oneway ANOVA (Bonferoni post-test) (rockvilie1985). The differences were considered to be significant at P<0.05.

Induction of diabetes in rabbits
Experimental diabetes in rabbits (2-3 kg) was induced by intravenous [10] injecting alloxan monohydrate (80 mg/kg) freshly disolve in normal saline in to marginal ear vein to the overnight fasted rabbits. 20% glucose solution was injected after 4-6 hrs. Rabbits were kept for the next 24 hrs on 5% oral glucose solution, in their cages to prevent hypoglycemia. On the 5th day blood glucose level is increased the levels are measured by GOD POD method [11]. The levels of 230mg/dl were considered as diabetic and control groups were given the same volume of normal saline.

Pharmacokinetic & pharmacodynamic interaction study in normal rabbits
Following an overnight fast, rabbits were divided in to 3 groups each group contain 4 rabbits (n=4).

Pharmacokinetic & pharmacodynamic interaction study in diabetic rabbits
Following an overnight fasted, rabbits were divided in to 3 groups each group contain 4 rabbits (n = 4). Blood samples were collected from marginal ear vein at time intervals at 0 ,0.5, 1, 2, 4, 8, 24 hrs in to eppendroff tubes the serum was separated by centrifugation using biofuge 13 (Heraeus instruments, germany) at 3000g/15min separate serum and store at -20°c until further analysis.

Calculations of pharmacokinetic and pharmacodynamic parameters
Non compartmental pharmacokinetic analysis was carried out using Kinetica TM software (version 4.4.1 Thermo Electron Corporation, U.S.A). The following Pharmacokinetic parameters were calculated: C max , T max , AUC 0ton , AUC tot , AUMC 0ton , AUMC tot , t 1/2 , MRT, Cl, Vd and Vdss. Mean glucose levels and percentage reduction in blood glucose concentrations were determined for the pharmacodynamic data.

Statistical analysis
The results expressed as mean ± SD. The difference in between concentration time profiles; in between pharmacokinetic parameters, in between serum glucose levels and difference between over the entire range tested were analyzed by one-way ANOVA (Bonferoni post-test). The differences were considered to be significant at P<0.05.

Pharmacokinetic data
Pharmacokinetics of pioglitazone in normal rabbits with piperine: The serum concentration levels of pioglitazone in normal rabbits were read out by substituting the peak area ratio values of each sample in the equation obtained from the calibration curve of pioglitazone. The mean values along with standard deviation were calculated for each time point (Table 1) and a concentration versus time curve was obtained by plotting the mean concentration of pioglitazone on x-axis against the time points on y-axis ( Figure 2). The pharmacokinetic parameters for pioglitazone were calculated and showed a Cmax of 2.03049 ± 0.49 µg/ml, T max of 0.5 hrs and AUC 0ton of 9.21 ± 1.12 µg/hr ml (Table 2) in normal rabbits.

Pharmacokinetics of pioglitazone under piperine pretreatment in normal rabbits
The mean serum concentration levels of pioglitazone in normal rabbits under piperine pretreatment were also read out (Table 3), a comparative concentration versus time curves of all groups was obtained ( Figure 3) and the pharmacokinetic parameters of all groups were also compared ( Table 4). In normal rabbits, pretreatment of piperine resulted in an increase in mean serum concentration of pioglitazone at 1 hr in single dose interaction group (from 1.03 ± 0.32 to 2.04 ± 0.34; p<0.05); at 1 & 2 hr in multiple dose interaction group (from 1.03 ± 0.32 to 2.06 ± 0.44 & from 0.70 ± 0.21 to 1.18 ± 0.45 respectively; p<0.05 Table 2) and significant changes in pharmacokinetic parameters. The T max of pioglitazone extended from 0.5 hr to 1 hr under piperine pretreatment.

Pharmacokinetics of pioglitazone in non diabetic and diabetic rabbits with piperine
The pharmacokinetic parameters for pioglitazone were calculated and showed a C max of 2.01 ± 0.87 µg/ml, T max of 0.5 hrs and AUC 0ton of 12.65 ± 1.97 µg/hr ml in normal rabbits. The T max of pioglitazone extended from 0.5 hr to 1 hr under piperine pretreatment. In diabetic rabbits, multiple dose pretreatment of piperine resulted in an increase in mean serum concentration levels of pioglitazone at 2 hr (from 0.81 ± 0.34 to 1.36 ± 0.19; p<0.05 Table 3) and a significant increase in AUC 0 to n (from 12.65 ± 2.97 to 19.70 ± 3.16; p<0.05 Table 3     were observed in pharmacokinetic parameters including decrease in clearance and increase in t 1/2 under multiple dose exposure. There was an increase in mean serum levels of pioglitazone in both normal and diabetic rabbits and also on the pharmacokinetic parameters under piperine pretreatment.

Pharmacodynamic data
Effects of piperine on the pharmacodynamics of pioglitazone in non diabetic and diabetic rabbits: The mean blood glucose levels for each time point in normal rabbits were calculated using glucose oxidase peroxidise method and the percentage glucose reduction at each time point compared to the mean glucose levels at 0 hr was calculated. The mean glucose levels and percentage glucose reduction was compared in piperine pretreatment against control group in normal rabbits. Pretreatment of piperine resulted in an increase in percentage glucose reduction more in 8

Discussion
Diabetes is a chronic metabolic disorder and needs prolonged      treatment for maintenance of normal blood glucose levels. Diabetes poses several complications like retinopathy, nephropathy, peripheral neuropathy and an underlying high oxidant stress. There is a wide use of herbal medications in diabetes condition, not only for controlling or slowing the progress of the disease, but also for most of its complications as they had antioxidant potential. In the context of diabetes and a high prevalence of herbal usage in diabetes; any of the herbal constituents having an influence on the disposition of antidiabetic agents may influence the control of blood glucose levels. Herb drug interactions are usually under-reported as the practitioner is unaware of concomitant use of herbs. Herbs not only in the form of alternative medicinal preparations, but also as dietary constituents can influence the safety and efficacy of a drug. Piperine is a constituent of black pepper and black pepper is widely used as spice. There is an increasing incidence of diabetes, and pioglitazone is a widely prescribed antidiabetic agent either alone or in combination showing better safety profile among thiazolidinedioes with significantly lower risk of cardiovascular damage [12]. Pioglitazone is known to be metabolised by CYP 2C8, 3A4 enzymes [13] and even the active metabolites of pioglitazone M III & M IV (keto & enol derivatives of pioglitazone) were found to be metabolised by CYP 2C8, 3A4 enzymes and hence, found to have improved bioavailability in presence of CYP inhibitors in both, rats [14] and in human liver microsomes in-vitro [15]. A biochemical evidence has long been reported for piperine as a potent inhibitor of drug metabolism [16]. In a study performed in rats, piperine was found to inhibit p-gp and CYP 3A4 activity. The influence of piperine on human hepatic metabolism can be further appreciated in a study on human volunteers, where piperine pretreatment significantly increased the bioavailability of metronidazole, a substrate of CYP 2C8, 2C9 & 3A4 enzymes [17]. Thus the present study was planned to investigate the influence of piperine on the pharmacokinetics and pharmacodynamics of pioglitazone in rabbits in-vivo, in both normal and alloxan induced diabetic condition. We studied the influence of piperine on the pharmacokinetic & pharmacodynamics of pioglitazone in rabbits. The pharmacokinetic parameters were found to alter in curcumin pre-treatment, but were statistically more significant in multiple doses pre-treatment. In normal rabbits, pre-treatment of piperine resulted in an increase in AUC tot by 0.59 folds (p<0.05), AUMC tot by 1.09 folds (p<0.01), t 1/2 by 0.34 folds (p<0.05), MRT by 0.31 folds (p<0.05) and a decrease in Cl by 0.36 folds (p<0.05). In diabetic rabbits, multiple dose pretreatment of piperine resulted in an increase in AUC tot by 0.82 folds (p<0.01), AUMC tot by 1.56 folds (p<0.01), t 1/2 by 0.43 folds (p<0.01), MRT by 0.40 folds (p<0.05) and a decrease in Cl by 0.45 folds (p<0.05). Maximum percentage glucose reduction was observed at 4 hr in all groups of normal rabbits and any changes were less significant. In diabetic rabbits also maximum percentage glucose reduction was observed at 4 hr and the influence of piperine was found to be statistically significant in both single dose pretreatment (at 4, 8, 24 hr by 0.38, 0.23, 1.52 folds respectively; p<0.05) and multiple dose pretreatment (at 2, 4, 8, 24 hr by 0.73, 0.67, 0.50, 3.14 folds respectively; p<0.01). The increase in AUC only in multiple dose treatment suggests an inhibitory influence of piperine on hepatic metabolism. Also, influence of piperine being effective in improving pharmacodynamics (glycemic control) only in diabetic rabbits indicates that the alteration might be partly because of improved pharmacokinetics of pioglitazone and partly because of antihyperglycemic activity of piperine. Thus, the improved pharmacokinetic parameters of pioglitazone was more observed in the multiple dose treatment groups, and the improvement of pharmacodynamics was significant in only diabetic rabbits under multiple dose treatment, thus showing the significance of influence of piperine in multiple dose exposure.

Conclusions
The results of increased AUC of pioglitazone under piperine exposure suggest an interaction which may be due to decreased metabolism of pioglitazone as a result of CYP 3A4 and 2C8 inhibition. Since the alterations are more pronounced in multiple dose treatment groups, it indicates the significance of long term exposure of piperine in diabetic condition being controlled by pioglitazone in rabbits, and thus it may apply to diabetic patients under pioglitazone treatment. Hence, the combination has a beneficial effect in diabetic condition, but therapeutic drug monitoring has to be observed in view of side effects of pioglitazone. Hence the present investigation warrants further studies to find out the relevance of this interaction in human beings and postulates the exact mechanism involved.