Effect of co-administration of piperine on pharmacokinetics of f3-lactam antibiotics in rats

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1 Indian Journal of Experimental Biology Vol. 40, March 2002, pp Effect of co-administration of piperine on pharmacokinetics of f3-lactam antibiotics in rats A R Hiwale, J N Dhuley & S R Naik* Research and Development Division of Hindustan Antibiotics Limited, Pimperi, Pune , India Received 4 June 2001; revised 4 Decelnber 2001 Co-administration of piperine, an alkaloid isolated from Piper nigrul1i L. enhanced bioavailability of beta lactam antibiotics, amoxycillin trihydrate and cefotaxime sodium significantly in rats. The improved bioavailability is reflected in various phannacokinetic parameters viz. tnm, Cm"" t y, and AUC, of these antibiotics. The increased bioavailability could be attributed to the effect of piperine on microsomal metabolising enzymes or enzymes system. Piperine (I-piperoyl piperidine) a major component of the Piper Species Piper nigrum L. (Piperaceae) has been used worldwide as spices and therapeutic modality in various traditional systems of Indian medicine '. This compound has shown several Pharmacological activities, viz. anti-asthmatic along with vasaka leaves Adhatoda vasica 2 antifertilit/, CNS depressant and anti-inflammator/- 6, inhibition of hepatic monooxygenase and UDP-glucuronyltransferase 7, 8. In addition piperine has also been shown to enhance the bioavailability of drugs like vascici ne, sparteine, curcumin, barbiturate and oxyphenylbutazone, zoxazolamine, pro-.. I I d h h II'., 5 prana 0 an t eop y me m anima I expenments ', 8 '0 The present communication reports the effect of the concomitant treatment of piperine on the bioavailability of -Iactam antibiotics like amoxycilline trihydrate, cephadroxi I monohydrate and cefotaxime sodium in rats. Materials and Methods Animals-Albino rats weighing g body weight of Hindustan Antibiotic (H.A.) strain of either sex were used for bioavailability studies and albino mice of 20-2Sg body weight of H.A. strain for toxicity studies. The rats and mice were housed at constant temperature (2S0±1 C) and light (10 hr) and dark (14 hr) cycle was maintained throughout the experimental period. All animals were maintained on H.A. diet and water ad libitum. *Present address for correspondence: Department of Pharmacology and Biotechnology, Principal K. M. Kundnani College of Pharmacy, 47, Dr. R. G. Thadani Marg, Worii, Mumbai , India. Kmkcp@bomS.vsnl.nel.in;Kmkcp@vsnl.com Drugs and Chemicals-Drugs, amoxycillin trihydrate from Swet-chem Antibiotics Ltd, Haryana, cefadroxil monohydrate, from Gujrat Lyka Organics, Gujarat and cefotaxime sodium from Chiel Jedang Corporation, Korea were purchased. Piperine was purchased from Sigma Chemical (St. Lauis, MO, USA). Antibiotic assay medium No.1 and 2 were purchased from Himedia Laboratories Pvt. Ltd, Mumbai. Preparation of test drugs and treatment schedule Amoxycillin trihydrate and cefadroxil monohydrate were prepared as suspension in carboxy methyl cellulose (CMC) (O.S% w/v) using pestle and mortar. Cefotaxime sodium solution was prepared by dissolving in normal saline. Rats were randomly divided into 3 groups of 42 rats/group. The first group served as control and received only amoxycillin trihydrate (100 mg/kg) orally. The second group received piperine (10 mg/kg) orally and 30 min later amoxycillin trihydrate (100 mg/kg) orally. The third group received piperine (20 mg/kg) orally and 30 min later amoxycillin trihydrate (100 mg/kg) orally. Similar type of experimental design and treatment schedule was followed for cefadroxil monohydrate and cefotaxime sodium, except cetotaxime sodium dose, which was administered at 10mg/kg ip. Blood collection and determination of blood levels of fjlactam antibiotics -Blood samples of rats treated with various antibiotics alone and with piperine were collected from the heart under light ether anesthesia at different time intervals like IS, 30, 45, 60, 120, 180 and 240 min following the antibiotic treatment. For each time interval 6 rats were used.

2 278 INDIAN J EXP BIOL, MARCH 2002 Serum was separated from each blood sample after keeping for one hour at room temperature and then centrifuged at 2000 rpm for 20 min and then used for microbiological assay. Microbiological assay (in vitro) -The agar-well diffusion assay method was used for antibiotic assay using Sarcina leutea (ATCC 9341) for amoxycillin trihydrate and cefadroxil monohydrate, and Bacillus subtilis (ATCC 6633) for cefotaxime sodium Toxicity studies -Acute toxicity studies in mice were also carried out by administering piperine 30 min prior to antibiotics, amoxycillin, cefadroxil and cefotaxime sodium treatment to find out whether piperine potentiate the toxicity of antibiotics. Statistical analysis-the apparent elimination rate constant (Kel) for antibiotics was determined by nonlinear regression analysis of the serum concentration versus time data of the terminal phase. Elimination half-life, ty, of antibiotics was calculated from relationship tl/2 = 0.693/Ke" while area under the curve (AUC) was calculated using the trapezoidal rule. The significance between the respective treatment group was calculated by paired Student's 't' test. LDso was calculated by the method of Litchfield and Wilcoxon l 3. Results clfect of piperine on bioavailability of amoxycillin in rats -The serum concentration versus time profile of amoxycillin administered with and without piperine is outlined in Table I. The data fitted to a one compartment open model with first order kinetics and other pharmacokinetic pattern derived from these data are summarised in Table 2. In both the treatment absorption process was complete with median tmax of 1.5 ± 0.35, 1.0 ± 0.22 and 1.1 ± 0.3 hr for amoxycihin (100 mg/kg) orally, piperine (10 mg/kg po)+ amoxycillin (loomg/kg, po) and piperine (20 mg/kg, po)+amoxycillin (loomg/kg, po) respectively. Piperine pretreatment induced a significant (P<0.05) shift in tmax of amoxycillin which was statistically significant (P < 0.05). The distribution phase was fairly Sh0l1 in the groups a concentration decay being evident within 2 hr of drug administration. Comparison of other parameters showed significant differences in peak antibiotic levels attained (ClllUJ or mean elimination half - life (tll2)' Comparison of AUC showed that higher serum levels of amoxycillin were achieved in the group treated with piperine+amoxycillin. Comparison of various pharmacokinetic parameters between the two different doses of piperine with amoxycillin treated groups also showed a significant difference (see Table 2). Effect of piperine on bioavailability of cefotaxime sodium in rats-the mean serum concentration versus time profile of cefotaxime administration with and without piperine is shown in Table 3. In both the treatment category the absorption process was complete with median tmax of 0.5 ± 0.28, 0.55 ± 0.35, 0.6±0.31 hr for cefotaxime (10 mg/kg, ip) alone, piperine (10 mg/kg, po) + cefotaxime (10 mg/kg, ip) and piperine (20 mg/kg, po)+cefotaxime (10 mg/kg, ip) respectively (Table 4). The piperine induced shift in tmax of cefotaxime was significant (P < 0.05). The distribution phase was short in both the groups, a concentration decay being evident within Ihr of administration. Comparison of other parameters showed significant difference in peak drug levels (CllOX) and the mean elimination of half-life (t1/2)' Both the doses of piperine with cefotaxime treatment also showed dose related effect of piperine on the serum concentration of cefotaxime. Effect of piperine on bioavailability of cefadroxil The result outlined in Table 5 show the mean serum concentration versus time profile of cefadroxil administered alone and in combination with piperine. In both the treatment the absorption process was complete with median tmax of 1.0±0.13,. 1.0±O.l5 and 1.0±0.0Ihr for the cefadroxil (100 mg/kg, po), piperine (10 mg/kg, po)+cefadroxil (100 mg/kg, po) and piperine (20 mg/kg, po)+cefadroxil (100 mg/kg, po) groups respectively (Table 6). The piperine induced shift in the tmax cefadroxil was not statistically significant P > The distribution phase was short in both the groups, a concentration decay being evident within 2hr of cefadroxil administration. Comparison of the other parameters failed to show significant differences in either peak drug levels (C,llax) or mean elimination half-life (tl/2) attained. There is no significant difference between respective AUC values. LDso studies on amoxycillin cefadroxil and cefotaxime with and without piperine co-administration. LDso results show that piperine co-administration did not alter LDso values of amoxycillin. However, coadministration of piperine with cefotaxime sodium decreased LDso of cefotaxime sodium insignificantly (Table 7) LDso studies with cefadroxil was not carried out. 1

3 HIWALE el oj.: EFFECT OF PIPERINE ON PHARMACOKINETICS OF -LACTAM 279 Table 1-Effect of co-administration of piperine on serum concentration of Hmoxycillin in rats [Values expressed as g/ml, are mean ± SO] Time (hr) Amoxycillin Piperine (10 mg/kg, po) + Piperine (20 mg/kg, po) + (IOOmg/kg, po) Amoxycillin (I00mg/kg, po) Amoxyeillin (IOOmg/kg, po) ± OA7 4.1 ± ± 0.23 * ± ± A ± 0.27 *P<O.OOI 4.2 ± ± OAO 8.16 ± 0.29 *P< ±_ ± ± 0.57 * ± 0.5 I 2.52 ± A8 ± OAI * ± ± ± 0.11 **P< ± ±O.II 1.24 ± 0.3 * 1.5 *Compared with piperine (10 mg/kg, po) + amoxyeillin (100 mg/kg, po) treated group. ** Compared with amoxycillin (I OOmg/kg, po) alone treated group. Table 2- Pharmacokinetic parameters of amoxycillin alone and in combination with piperine [Values are mean ± SO] Treatment Tmax Cmax t'h (hr) g/ml (hr) AUC ( g/ml/hr) Amocycillin (I00mg/kg, po) 1.5 ± ± ± OA8 Piperine (IOmg/kg, po) +Amoxycillin (I OOmg/kg, po) 8.0 ± ± ± ± 0.39 Piperine (20mg/kg, po) +Amoxycillin (I00mg/kg, po) 1.1 ± ± ± ± OAI 22.18±OA9 * Compared with piperine (10 mg/kg, po) + amoxycillin (IOOmg/kg, po) treated group. ** Compared with amoxycillin (100 mg/kg, po) alone treated group. Table 3 -Effect of co-administration of piperine on serum concentration of cefotaxime in rats [Values expressed as g/ml, are mean ± SO] >0 Time (hrs) Cefotaxime Piperine (I0mg/kg, po) + Piperine (20mg/kg, po) + ( 10mg/kg, i p) Cefotaxime (IOmg/kg, ip) Cefotaxime (IOmg/kg, ip) ± OA ± ± OAO P<0.05** ± ± 0.35 *35.13±0.51 P< O.OOI ** ± A6 ± ± 0.50 P<O.OOI** ± ± OA ± ± ± ± A± ± ± 0.25 P<O.OOI** 4.0 2A5 ± A±OAI 8.9 ± 0.23 P<0.03** *Compared with piperine (I0mg/kg, po) + cefotaxime (IOmg/kg, ip) group ** Compared with cefotaxime (IOmg/kg, ip) alone treated group.

4 280 INDIAN J EX? BIOL, MARCH 2002 Discussion The present experimental findings in animal experiments clearly demonstrated that co-administration of piperine enhanced the bioavailability of amoxycillin and cefotaxime significantly. The analysis of various pharmacokinetic parameters tmax, Cmax, tl/2 and AUC point out that piperine might influence the dif- ferent physiological process like alteration in gastric milieu and/or transport or absorption process 10. However, it can not be ruled out direct inhibitory effect of piperine on microsomal metabolising enzymes which are involved in amoxycillin or cefotaxime metabolism because piperine is shown to inhibit microsomal enzymes and enzymes systems 7. S. IO. 14. Further more Table 4-Pharmacokinetic parameters of cefotaxime alone and in combination with piperine [Values expressed as IAg/ml, are mean ± SD] Treatment lmax emilx. (hr) (lag / ml) t 112 (hr) AUC (lag /ml/hr) Cefotaxime (lomg/kg, ip) 0.5 ± ± 0.3 Piperine (lomg/kg, po) ± ± 0.32 Cefotaxime (I Omg/kg, ip) P<O.OI ** Piperine (20mglkg, po) ±0.25 Cefotaxime (IOmg/kg, ip) 0.6 ± 0.31 P<O.OOI** 1.08 ± ± ± ± 0.45 P<0.05** P<O.OOI ** 1.78 ± ± 0.42 P<0.05 ** P<O.OOI ** *Compared with Piperine (10 mg/kg, po.) + Cefotaxime (I Omg/kg, ip.) group. ** Comparead with cefotaxime (10 mg/kg, ip) alone treated group. Table 5 -Effect of co-administration piperine on serum concentration of cefadroxil [Values expressed as IAg/ml, are mean ± SDl Time (hr) Cefadroxil (I OOmg/kg, po) Piperine (lomg/kg, po) + Pi peri ne (20mg/kg, po) + Cefadroxil (IOOmg/kg, po) Cefadroxil (IOOmg/kg. po) 33.5 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table 6- Pharmacokinetic parameters of cefadroxil alone and in combination with Piperine [Values expressed as IAg/ml, are mean ± SD] Treatment lmax Cmax ty, AVC (hr) lag / ml (hr) (lag /mllhr) Cefadroxil (loomg/kg, po) I ± ± ± ± 1.4 Piperine (lomg/kg, po) +Cefadroxil (IOmg/kg, po) I ± ± ± ± 1.35 Piperine (20mg/kg, po) +Cefadroxil (IOOmg/kg, po) 1.0 ± ± ± ± 1.55 Table 7-LD5o Studies on amoxycillin and cefotaxime with and without piperine in mice Drug Route Dose LD o with 95% confidence Behavioural (mg/kg) interval in respective asses changes A mox yci Iii n Oral 5000,10000, Mild depression, > mg/kg Mild diarrhoea Piperine 5000, 10000, Mild depre sion, (20mg/kg, po) +Amoxycillin Oral > mg/kg Mild diarrhoea 10000, 12000, (12000 to Loss of gait, mild depression, followed by Cefotaxime ip ) mg/kg convulsions prior to death Piperine 10000, 12000, (I 1500 to 15000) mg/kg Loss of gait, mild depression, followed by (20mg/kg, po) +Cefotaxime ip convulsions prior to death. Toxicity studies on cefadroxil were not performed with and without piperine as its results were not significant in various pharmacokinetic parameters.

5 HIWALE et al.. : EFFECT OF PIPERINE ON PHARMACOKINETICS OF -LACTAM 281 increased blood concentration levels of sulfadiazine and tetracycline hydrochloride in Piper choba, (a piper species) treated dogs was also reported 15 which has been attributed to an increased absorption and/or inhibition of metabolising enzyme system. The noneffectiveness of piperine on cefadroxil bioavailability is attributed to its non-metabolic nature and shown to be excreted as such in urine l6. In conclusion it is stated that piperine coadministration with amoxycillin and cefotaxime sodium improved the bioavailability which is also reflected in various pharmacokinetic parameters studied. Since piperine is a natural product used by many in day today food preparation with less toxicological consequences piperine might prove to be useful as an adjuvant with some of -lactam antibiotics dosage preparation and would cut down the frequency of administration. However, in depth bioavailability studies are necessary with different -lactam antibiotics with piperine on human volunteers to establish prior to such a claim in clinical medicine. References I Atal C K, Zutrshi U & Rao P G, Scientific evidence on the role of Ayurvedic herbals on Bioavailability of drug. J. Ethanopharmacol. 4, (1981) Wealth of India Vol VIII, (Publication and Information Directorate, CSIR, New Delhi) 1969,96. 3 Piyachaturwat P, Glinsukon T & Peugvicha P, Post-coital antifertility effect of piperine. Contraception, 26 (1982) Lee E B, Shin K H & Woo W S. Central nervous system depressant and anti-inflammatory activity of piperine. Arch. Phannac. Res, 7 (1984) Majumdar A M, Dhuley J N, Deshmukh V K, Raman P H & Naik S R, Antiinflammatory activity of pipeprine. Japan J. Med Sc Bioi. 43 (1990) Dhuley J N, Raman PH, Mujumdar AM & Naik S.R, (1993) Inhibition.of lipid peroxidation by piperine during experimental inflammation in rats. Illdiall J Exp. Biol, 31 (1993) Atal C K, Dubey R K & Singh J, Biochemical basis of enchaced drug bioavailability by piperine-evidence is apotent inhibtor of drug metabolism. Pharmacal Exp Ther, 232, (1985) Shobha G, Joseph T, Majeed M, Rajendran R & Srinivas P S S R, Influence on the pharmacokinetics of curcumin in animals and human volunteers. Planta Med, 64 (1998) Mujumdar A M, Dhuley J N, Deshmukh V K. Raman P H, Tharat S L & Naik S R. Effect of piperine on a pentobarbitore induced hypnosis in rats Indian J Exp Bioi, 28 (1990) Mujumdar A M, Dhuley J N, Deshmukh V K & Naik S R, II Effect of piperine on Bioavailability of Oxyphenylbutazone in rats. Indian Drugs, 36 (1999) 123. Victor Lorian, M D. Antibiotics in laboratory medicine, (Waverly Press Inc. NY) 1980, Indian Pharmacopoeia. (Ministry of Health and Family Welfare, Govt. of India) (1996) Appendix 9.1, A-I Litchfield J T & Wilcoxon F W. A simplified method of evaluating dose effect experiments. 1. Pharmacal Expt. Ther, 96 (1949) Singh J, Dubey R K & Atal C K. Piperine-mediated inhibition of glucuronidation activity in isolated epithelial cells of the guinea-pig small intestine: Evidence that piperine lowers the endogenous UDP-glucuronic acid content lowers. J Pharmacal Expt. Ther, 236 (1986) Atal C K, Manavalan R, Nighojkar R, Sareen A N & Gupta o P. Studies on piperchaba as a bioavailable agent, Indian Drugs, 17 (1980) Munson P.L. Principles of Pharmacology, Basic Concept and Clinical Applications, (Chapman and Hall, USA) 1995, 39.

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