Evaluation of the Effect of Piperine on Bioavailability and Pharmacokinetics of Macrolides in Rats

 

Samir Shah*, Kintu Patel, Mohsin Pathan

Department of Pharmacology, Sardar Patel College of Pharmacy, Bakrol - 388315. Dist. Anand. Gujarat.

 

ABSTRACT:

Objective: In this study attempt was made to evaluate the effect of piperine on the bioavailability and pharmacokinetic of macrolides using experimental animals. Material and Methods: Albino Wistar rats (250-300 g body weight) were used for bioavailability study. All the rats were divided into four groups. First and third group of animals received only azithromycin (10mg/kg, p.o.) and erythromycin (50mg/kg, p.o.) respectively. Second and fourth group of animals received piperine (20mg/kg, p.o.) 30 minute before administration of drugs. About 2.5 ml of blood samples were collected at different time intervals from each rat. Plasma was separated from blood and plasma concentration of macrolides was measured by High performance liquid chromatography (HPLC). Pharmacokinetic parameters such as area under curve (AUC), peak plasma concentration (Cmax), time to occur peak plasma concentration (Tmax)were calculated and compared between macrolides with and without piperine.  Results: Animals were treated with piperine followed by macrolides shown significantly (P<0.05) increased AUC as compared to animal administered only macrolides. The Cmax of azithromycin without and with piperine (20 mg/kg) was found to be 233.3±1.313 ng/l and 350.6±0.751ng/l respectively. Peak time at which maximum drug concentration of azithromycin in plasma without and with piperine (20mg/kg) was found to be 4 and 8 hrs respectively. The Cmax of erythromycin without and with piperine (20 mg/kg) was found to be 192.4±0.836 ng/l and 203.1±1.224 ng/l respectively. Peak time at which maximum drug concentration of erythromycin in plasma without and with piperine (20mg/kg) was found unchanged at 1.5 hrs.Conclusion: The result of present investigation shows that piperine significantly increased bioavailability of macrolides. Possible reason of increase in bioavailability by piperine may be due to inhibition of CYP3A4 enzyme drug metabolizing system.

 

 

 

 

 

 

 

INTRODUCTION:

Drugs that are easily absorbed from the gastrointestinal tract and having a short half life are eliminated quickly from the blood circulation. However, the intestinal epithelium may constitute a permeability barrier for the absorption of orally administered drugs. This problem stimulated a search for new strategies to overcome mucosal barriers¹.The bioavailability of a drug is determined by its absorption, metabolism, distribution and excretion. These processes are mainly mediated by nuclear receptor-mediated detoxification system involving a variety of CYPs. The cytochrome P450(CYP) enzyme consists of a superfamily of hemeproteins that catalyze the oxidative metabolism of a wide variety of exogenous chemicals including drugs, carcinogens, toxins and endogenous compounds such as steroids, fatty acids and prostagladins².

 

Piperine ,a major alkaloid of black and long pepper is a trans isomer of 1, piperoyl piperidine. This alkaloid is also reported to possess several pharmacological actions and in addition enhance the bioavailability of several drugs³. The selected marketed herbal formulation is a Trikatuchurna which contains, Piper longum, Piper nigrum, and Zingiber officinalis, and Pimpalichoorna which contain Piper longum. Pipli (Piper longum) is a pungent alkaloidal drug; well known for its bioavailability enhancing properties⁴. Piperine has previously been shown to inhibit several cytochrome P450-mediated pathways and phase II reactions in animal models. With regard to the effect of piperine or black pepper on drug disposition in humans, there are only very limited data.The mechanisms for the bio-enhancer activity of piperine have been proposed including DNA receptor binding, modulation of cell signal transduction and inhibition of drug metabolism. Piperine has been demonstrated to increase the serum levels and lengthen the serum half lives of some nutritional substances, such as coenzyme Q10 and beta-carotene⁵.

 

Drug-drug interactions (DDIs) are a concern for patients and providers, as multiple medication use becoming more common to manage complex diseases. The consequences of DDIs can range from no untoward effects to drug-related morbidity and mortality⁶.Very recently, it was shown that a single administration of 1g of black pepper more than doubled area under the plasma concentration-time curve and elimination half-life of phenytoin⁷. The mechanisms underlying these drug inter-actions in humans have not been investigated so far.In spite of a large number of evidences indicating the role of piperine as a bio enhancer, one of the studies has indicated a decline in the bioavailability of diclofenac sodium in rats⁸.

 

Thus, we tried to find out the effect of combining piperine, an antioxidant and a neuroprotective agent⁹ with macrolides in rats. Among various animals, rats are widely used in non-clinical studies, such as toxicity and Absorption, Distribution, Metabolism and Excretion (ADME) and pharmacological studies and are considered to be the most convenient animal¹⁰.

Similar studies have been carried out of various drugs including Insulin, Simvastatin and Gliclazide^11,12,13.

 

MATERIAL AND METHODS:

PROCURMENT OF PLANT MATERIAL:

Piper nigrum seeds was obtained from commercial sources. The plantmaterials were authenticated by Medicinal Plant Survey and Collection Unit, Government Agriculture University, Anand, India. (Letter No.SPCP/CORRS/1573/13)

 

EXTRACTION OF PIPERINE^:

10 g black pepper was grounded to a fine powder with a mortar and pestle. Extract was thenprepared using finely ground pepper with 200 mL 95% ethanol in a soxhlet extraction apparatus for 4 hours. Solution was allowed to cool and then filter through whatman #1 filter paper. Solution was concentrated in vacuum on a 50 ºC water bath to remove most of the ethanol solvent. The final volume was about 5 mL, then, about 10 mL 10% alcoholic KOH was added to the residue and left it standing for 1 hour. The solution was decanted from the insoluble residue.  The alcoholic solution was allow to stand undisturbed overnight; long yellow needles of piperine were  deposited. (The crystals may take 24–48 hours to form.) The yield is approximately 0.3 g. The yellow needles were collected by vacuum filtration and washed with a minimum volume of95% ethanol. The crystals were allowed to air dry and weighed.

 

DRUG AND CHEMICALS

Table1. Procurement of Drug, Chemicals and their source

Drugs and Chemicals	Source
Azithromycin and Erythromycin	Rajat Pharmaceuticals Ltd. Ankelweshwar,Gujarat,INDIA
Ditilled water, Acetonitrile(HPLC Grade > 99.93%), Tetrahydrofuran, Methanol, Ethanol, H2SO4, NaOH, CHCl3, Alcoholic KOH	Dutt Chemicals, Anand,Gujarat,INDIA.

 

EXPERIMENTAL DESIGN

Experimental Animals

Four groups of Adult male Wistar Albino rats (each group consist of 6 rats having initial weight 200-250 gm were selected.nAll animals were kept in house for at least 2 days before experiment in a clean room and were be given food and water before experiment. The experimental Protocol Number of this project at IAEC was SPCP/IAEC/RP-004/2012-13.

 

Experimental Groups

At the start of the study, all the animal were divided four groupstest groups were given solution of piperine i.e. 20 mg/kg,dose required for blockage of CYP3A4 enzymes in body, afterwards 30 minutes, each animal were given respective drugs.

 

 

Group 1:- 10 mg/kg of azithromycin only. (Azithromycin)

Group 2:- 20 mg/kg of piperine solution 30 minutes before the administration of 10 mg/kg of azithromycin. (AZO + Piperine)

Group 3:- 50 mg/kg of erythromycin. (Erythromycin)

Group 4:- 20mg/kg of piperine solution 30 minutes before administration of 50mg/kg of erythromycin. (ERY + Piperine)

 

Collection of blood sample:

0.10-0.25 ml samples were drawn at 0, 0.5, 1, 2, 4, 8, 12, 16, 24 hrs for azithromycin and 0,0.25,0.5,1,1.5,2,4,8 hrs intervals as per their respective half-life.After sampling, centrifuge it immediately at 5000 rpm for 4 min and plasma store at -20°C until analysis^14,15.

 

Sample preparation for measurement of plasma drug concentration:-

For azithromycin, Solvent mixture in the quantity of 25 ml containing water/THF/ Methanol will be added to the plasma sample in the ratio of 2:1:2 v/v. Then it will be shaken for at least 30 minutes. The sample was extracted with ether. The organic layer was removed and it was evaporated to dryness under nitrogen conditions. After that it was dissolved in mobile phase and standard solution will be added to it. Finally 20 ul of aliquot will be injected into the column and For erythromycin,in brief, to 200 µ l of plasma sample, 400 µ l 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. Then 20 µ l of the supernatant was injected to the HPLC system for analysis using UV detector was set at 220 nm for the present analysis.

 

Pharmacokinetic parameters Evaluted

Ø  AUC

Ø  Cmax

Ø  Tmax

AUC (Area under Curve)

 

Figure 1.Plasma drug concentrations vs. time profile

AUC was calculated by using the peak area of macrolides with and without extract which were obtained from HPLC analysis. Peak areas were converted to concentration (ng/l) range of macrolides at 0-24 hrs time interval of each animal. All values were plotted as plasma drug concentration vs. time profile. After plotting the graph AUC was calculated by using trapezoidal rule¹⁶.

 

Trapezoidal rule: Area of trapezoid with the first time interval

 

AUC₁= C₀+C₁ / 2 X time _1-0

 

Calculate the trapezoid with all-time interval and sum of all trapezoids gives AUC_0-tlast

 

For AUC_0-inf = AUC_C0-C6 + AUC_C6-inf

                         AUC_C6-inf = C₆/K

Cmax and Tmax:

 

Figure 2.Cmax and Tmax

 

 

By plotting the values of plasma drug concentration of macrolides vs. time at which blood sample were collected from each animal at 0-24 hrs intervals. Cmax and Tmax were directly observed from plotted graph as plasma concentration vs. time profile. Cmax was represented maximum drug concentration of macrolides in plasma and Tmax represented time at which maximum drug concentration of macrolides in plasma reached¹⁷.

 

RESULT:

EFFECT OF PIPERINE ON AUC OF AZITHROMYCIN

AUC analysis of azithromycin

The graph shows Plasma concentration of azithromycin vs Time at different time intervals 0-24 hrs.

 

Figure 3. Plasma conc-time profiles in rats after oral dose of azithromycin (10mg/kg) was given to rats . Data are expressed as MEAN±SEM (n = 6) rats.  

 

 

AUC analysis of Azithromycin with Piperine ( AZO + Piperine)

The graph shows Plasma concentration of Azithromycin vs Time at different time intervals 0-24 hrs.

 

 

 

Figure 4.  Plasma conc-time profiles in rats after oral dose of azithromycin (10mg/kg) with Piperine (20 mg/kg) was given to rats . Data are expressed as MEAN±SEM (n = 6) rats.

 

 

 

 

Comparison of Plasma drug concentration of Azithromycin without and with Piperine (20 mg / kg)

 

Figure  5. Plasma concentration-time profiles in rats after oral dose of azithromycin was given to rats in the following treatment groups: Azithromycin and AZO + Piperine (20mg/kg). All data are expressed  as MEAN ± SEM ( n = 6 animal group).

 

 

 

 

 

 

 

 

 PHARMACOKINETIC ANALYSIS

Table 2. Determination of AUC,Cmax,Tmax of azithromycin without and with Piperine(20 mg/kg)

Parameter	Azithromycin	AZO + Piperine
	Mean ± SEM	Mean ± SEM
AUC	3222±15.49*	3836±15.36*
Cmax	233.3±1.313*	350.6±0.751*
Tmax	4±0	8±0

All Values are expressed as MEAN ± S.E.M, (n = 6) on One way ANOVA followed by dunnet’s test.

*(p < 0.05) Azithromycin vs Azothromycin with Piperine (AZO + Piperine)

 

Area under curve (AUC) of Azithromycin and with piperine (AZO + Piperine) were found to be 3222 ± 15.49 and 4773 ± 15.36 respectively. Animals were treated with Piperine followed by received azithromycin shown significantly increased AUC  as compared to animal administered only azithromycin as summarized in Table 2.

 

Cmax of Azithromycin and with piperine (AZO + Piperine) were found to be 233±1.313, 350.6±0.751 respectively.

 

Animals were treated with extract followed by received Azithromycin shown significantly increased Cmax  as compared to animal administered only Azithromycin as summarized in Table 2.

 

Tmax of azithromycin and with piperine (Azo + Piperine) were found at 4 hrs and 8hrs respectively as summarized in Table 2.

 

EFFECT OF PIPERINE ON AUC OF ERYTHROMYCIN

AUC analysis of Erythromycin

The graph shows Plasma concentration of erythromycin vs Time at different time intervals 0-8 hrs.

 

Figure 6. Plasma conc-time profiles in rats after oral dose of erythromycin (50mg/kg) was given to rats . Data are expressed as MEAN±SEM (n = 6) rats.   

 

AUC analysis of Erythromycin with piperine ( ERY + Piperine)

The graph shows Plasma concentration of erythromycin vs Time at different time intervals 0-8 hrs.

 

Figure 7. Plasma conc-time profiles in rats after oral dose of erythromycin (50mg/kg) with piperine (20 mg/kg) was given to rats. Data are expressed as MEAN±SEM (n = 6) rats.

 

Comparison of Plasma drug concentration of erythromycin without and with Extract (20 mg/kg)

 

Figure 8. Plasma conc-time profiles in rats after oral dose of erythromycin was given to rats in the following treatment groups: Erythromycin and ERY + Piperine. All data are expressed  as MEAN ± SEM ( n = 6 animal group).

 

 

PHARMACEUTICAL ANALYSIS

Table 3. Determination of AUC,Cmax,Tmax of erythromycin and with Piperine(20 mg/kg)

Parameter	Erythromycin	ERY + Piperine
	Mean ± SEM	Mean ± SEM
AUC	551.8±7.761*	599.5±6.001*
Cmax	192.4±0.836*	203.1±1.224*
Tmax	1.5±0	1.5±0

All Values are expressed as MEAN ± S.E.M, (n = 6) on One way ANOVA followed by dunnet’s test.

*(p < 0.05) erythromycin vs erythromycin with piperine (ERY + Piperine)

 

AUC of erythromycin and with piperine (ERY+ Piperine) were found to be 551.8 ± 4.761 and 599.5 ± 6.001 respectively. Animals were treated with extract followed by received erythromycin shown significantly increased AUC  as compared to animal administered only erythromycin as summarized in Table 3.

 

Cmax of erythromycin and with piperine (ERY+ Piperine) were found to be 192.4±0.836, 203.1±1.224  respectively.

Animals were treated with piperine followed by received erythromycin shown  increased Cmax  as compared to animal administered only erythromycin as summarized in Table 3.

 

Tmax of erythromycin and with piperine (ERY + Piperine) were found at 1.5hr and 1.5hrs respectively as summarized in Table 3.

 

DISCUSSUION:

With the development of new drugs, dosage forms and better formulations, bioavailability seems to be an important part of drug formulation and performance of drugs. Literature data suggest that piperine inhibits CYP3A4 and other enzymes.

 

Black pepper, Piper nigrum, is a popular spice, but has been found to be have broad effect on drug dispositions. Its major components, the piperine was shown to increase the bioavaibility,Cmax and AUC of several drugs including propranolol, theophylline, phenytoin and rifampicin^18-21. The mechanism of action was demonstrated to involve inhibition of CYP3A4 and P-gp.

 

As in previous studies the pharmacokinetics of azithromycin was demonstrated that azithromycin attains peak plasma concentration in 4.2± 1.1h was about 0.23±0.09 µg/ml after giving oral dose at dose 10mg/kg²². In the present study, it has been observed that azithromycin peak plasma concentration was achieved at 233.3±1.313 ng/L at 4.0±0.0 h and with piperine 3836±15.36 ng/L at 8.0±0.0 h.

 

 

In previous studies the pharmacokinetics of erythromycin was demonstrated that azithromycin attins peak plasma concentration in 2.653±0.021 h was about 2.725±0.027 µg/ml after giving oral drug of dose 25 mg/kg²³. In present study, it has been observed that erythromycin peak plasma concentration achieved was 192.4±4.761 ng/L at 1.5±0.0 hr and with piperine was 203.1±1.224 ng/L at 1.5±0.0 hr.

 

Recently, there have been many reports regarding food-drug interaction by the inhibition of drug metabolism mediated by CYP3A subfamily. Because Piperine (an alkaloid), a major constituent of Black Pepper strongly inhibits the CYP3A4 activity in vitro¹⁶, wondered whether co-administration of Piperine with Macrolides, might alter the bioavailability and pharmacokinetic parameters of Macrolides. This is in line with limited number of clinical studies showing an effect of Piperine intake on drug dipostion. Therefore, the present investigation was done to investigate the effect of piperine on Macrolides bioavailability and pharmacokinetics in rats.

 

The result of present investigation shows that Piperine might have influence the pharmacokinetics of CYP3A4 mediated drugs in humans. So far,  there has been no clinic report suggesting the adverse food-drug reaction caused  by the intake of Black pepper. It has been chemical observed in rats may not necessarily observed in humans. However, it is difficult to extrapolate results, which were obtained in rats, to humans. Quantitative evaluation of Pipeirne interactions in humans needed to be verified in humans. Therefore, further studies in humans are needed to clarify the impact of this nutrient on intake of orally adminstratered drug for the therapeutic benefits.

 

CONCULSION:

The aim of the present study was to evaluate the effect of Piperine on bioavailability of Azithromycin and Erythromycin in rats. These studies were carried out in rats as a parallel design study.

 

After the analysis of blood samples, it has been concluded thatpiperine, major component of Black Pepper might be inhibits the CYP3A4 mediated metabolism of the drugs. Futhersome,Piperine has an influence on the pharmacokinetics of both the drugs in rats.Bioavailability of azithromycin and erythromycin was increased after giving with Piperine due to inhibition of enzyme CYP3A4.However detailed enzyme inhibition studies are required substantiate the hypothesis.

 

 

 

 

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