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1735-2657/07/62-171-176 IRANIAN JOURNAL OF PHARMACOLOGY & THERAPEUTICS Copyright 2006 by Razi Institute for Drug Research (RIDR) IJPT 6:171-176, 2007 1 ESEARCH ARTICLE fect of Honey on CYP3A4 Enzyme and Activity in Healthy Human Volunteers MAS, KESAVAN RAMASAMY, RAJAN SUNDARAM and ADITHAN 6 or author affiliations, see end of text. ed May 25, 2006; Revised August 6, 2007; Accepted August 14, 2007 This paper is available online at http://ijpt.iums.ac.ir The activity of cytochro me p450 isozyme 3A4 (CYP3A4) enzyme and P-glycoprotein (P-gp) is modulated ruit juice and herbal drugs. CYP3A4 is the major phase I drug metabolizing enzyme and P-gp is endent drug efflux pump that regulates the intestinal absorption of orally administered drugs. ey is commonly consumed as a dietary supplement. However, its influence on human CYP3A4 and ctivity is not yet well documented. Therefore, we investigated the influence of a 10-day honey ad- n CYP3A4 and P-gp activity in healthy volunteers using carbamazepine and digoxin as their e drugs respectively. A within-group pharmacokinetic study was done in 12 healthy volunteers. They stered single oral dose of carbamazepine (200 mg) and digoxin (0.5 mg) before and after 10 of honey (10 ml twice daily) intake. Blood samples (5ml) were collected at 0, 0.25, 0.5, 0.75, 1.0, 8, 12, 24, 48 and 72 h after drug administration. Concentration of carbamazepine and digoxin in a was measured by HPLC and RIA method respectively. Ten days of honey administration did not cantly alter the C max, Tmax and AUC (0-t) of carbamazepine (probe drug for CYP3A4) and digoxin
drug for P-gp). Our results suggest that honey may not significantly modulate the CYP3A4 enzyme -glycoprotein activity. The coadministration of honey with drugs may not result in significant drug words: Honey, CYP3A4, P-glycoprotein, carbamazepine, digoxin
Honey is a natural saccharine product made by hon- failed to show any significant effect on CYP3A4 [ 7]. the nectar of flowers [ 1]. Being a natural The effect of m ultiple doses of honey on CYP3A4 in rce of fructose and glucose with some oligosaccha- ans has not been reported to date. It has been well 29 des, proteins, vitamins and minerals, honey has be- umented that the CYP3A4 enzyme is involved in the e a dietary supplement for healthy individuals [ 2]. metabolis m and elimination of carbamazepine [ 8]. The ney is also consumed by many patients with diabetes, rmacokinetics of carbamazepine is influenced by rtension and epilepsy who receive drugs for their alteratio ns in the catalytic activity of CYP3A4 [ 33 ments. This increases the possibility of honey-drug ce, carbamazepine is used as a probe drug for 34 teraction. Most of the herb-drug interactions occur at ssing the CYP3A4 enzyme activity in our 35 he level of metabolism and drug transport mediated by P 450 group of drug metabolizing enzymes and P- 57 P-glycoprotein (P-gp) is an ATP dependent drug ef- 37 lycoprotein (P-gp) respectively [ 3]. p. It plays an important role as a secretory sys- Among the CYP group of drug metabolizing en- 59 m in the intestinal barrier and regulates the intestinal 39 mes, CYP3A4 is the major phase I drug metabolizing orption of orally administered drugs [ 40 zyme. It is present in the liver, jejunum, colon and 61 inically important drugs viz., digoxin, losartan, eryth- reas. It has broad substrate specificity and is re- ycin and rifampin are substrates for P-gp. Some of nsible for metabolism of more than 50% of adminis- besides being a substrate also induce or inhibit the 43 ered drugs [ 4]. There are few studies showing the effect p activity. Drugs like fexofenadine, digoxin and lop- oney on CYP3A4. Animal studies have shown that eram ide are used as probe drugs to assess P-gp activity multiple doses of honey induced CYP3A4 activity [ 5, 6]. 11]. Among them, digoxin is most commonly used 46 n a study done in humans, single oral dose of honey 67 12]. The effect of various dietary derivatives and herbal IJPT July 2007 vol. 6 no. 2 171-176
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Table 1. Pharmacokinetic parameters of carbamazepine (200 mg single oral dose) before and after 10 days of honey administration Pharmacokinetic parameters C max (µg.ml -1) AUC (0-72) (µg.h.ml -1) Values are shown as mean ± SEM. (n =12) 68 oducts on the P-gp activity has also been studied. In 108 xin intake, seizures and drug allergy were also ex- in vitro study using various fruit extracts, it was hat extracts of strawberry, orange, apricot and Study de t inhibited the intestinal P-gp [ 13]. In another in vi-111 On day 1, single oral dose of 200 mg carbamazepine study using rat small intestine, extracts of grapefruit rital, Novartis [India] Limited) and 0.5 mg digoxin 73 uice and orange juice inhibited the transport activity of oxin, Burrough's Wellcome, [India] Limited) were 74 gp [ 14]. In a study done in humans, grapefruit juice inistered to the volunteers at 7 AM who were fasted 75 ad no effect on P-gp activity [ 15]. Another human rnight. They were not allowed to take food for fur- 76 udy revealed that St. John's Wort, an herbal product 116 er 2 h. Blood samples were collected from indwelling 77 duced P-gp activity [ 16]. This shows that P-gp is a ous catheter using heparinised disposable syringes ential target for drug interactions exhibited by herbal 118 st before and at 0.25, 0.5, 0.75, 1.0, 1.5, 2, 4, 8, 12, 24, pounds. The effect of honey on P-gp activity has 119 , 72 h after administration of drugs. A standardized been studied so far. kfast and lunch were given to all the volunteers. Since we wanted to know whether honey, a natural day 5 to day 14, the volunteers were administered 82 ietary supplement, will interact with concomitantly l of honey (Periyakulam Sarwodaya Sangh, Khadi 83 ministered drugs, we investigated the effect of multi ralaya, Theni District, Tamilnadu, South India; Lot e administration of honey on CYP3A4 and P-gp ac- /2002) twice daily in empty stomach with 200 ml ans using carbamazepine and digoxin as water. On day 15, the volunteers were given single respectively. Carbamazepine is a CYP3A4 substrate but it is not a substrate for P-gp [ 17]. dose of 200 mg carbamazepine and 0.5 mg digoxin. blood samples were collected as mentioned before. he other hand, digoxin is a substrate for P-gp only aration of the plasma, the samples were stored 89 d not a substrate for CYP3A4 [ 18]. Hence any change 129 −20 0C till the drug assays were done. The study pro-
rmacokinetic profile of carbamazepine and 130 col is shown as a flow chart in Figure 1. 91 goxin due to honey administration may reflect the The honey used in the present study was tested for 92 ange in the activity of CYP3A4 and P-gp respec- urity in Public Health Laboratory, Pondicherry, 133 dia. It was found to be within PFA (Prevention of 134 od adulteration act-1955, India) values. It was com- ed of reducing sugar 71.6%, moisture 24%, sucrose MATERIALS AND METHODS
136 4% and ash 0.3%. The fructose/glucose ratio was A within group pharmacokinetic study was done in healthy male volunteers (Age 20-45 years). The Dru mean age of the vol unteers was 27.4 ± 1.96 yrs (mean ± 139 Serum carbamazepine concentration was estimated ) and their mean body mass index was 23.2 ± 0.94 140 sing a HPLC method [ 19]. The plasma sample (900 μl) (mean ±SEM). The study was approved by insti- andard (900 μl) were taken in a 2 ml mi- 100 tional ethics committee. A written informed consent ntrifuge tube. After vortex mixing, 600 μl was aken from all the volunteers. The health of the vol- 143 ransferred to a conical flask, into which 4:1 mixture of eers was assessed by doing a thorough physical ex- oroform: methanol was added. After mixing in an ination and by performing ECG, liver and kidney o 145 rbital shaker, the contents of conical flask were trans- 104 nction tests. Volunteers suffering from chronic dis- red to centrifuging tubes. After centrifugation at 2500 r taking concomitant medications were excluded rpm for 10 min, the upper protein layer was transferred 106 om the study. Similarly, regular users of alcohol i 148 nto evaporating tubes for evaporation at 50 0C. The
or tobacco, those with history of vomiting after di- 149 ried evaporated samples were reconstituted in 400 μl Table 2. Pharmacokinetic parameters of digoxin (0.5 mg single oral dose) before and after 10 days of honey administration Pharmacokinetic parameters C max (µg.ml -1) AUC (0-4) (ng.h.ml -1) AUC (0-72) (µg.h.ml -1) Values are shown as mean ± SEM. (n =12)

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Fig 1. The study plan described as a flow chart.
obile phase composed of acetonitrile: methanol: (T ) were read directly from the actual plasma con- 151 osphate buffer (12.5:25:62.5, v/v/v) and injected into . The area under the plasma concentration he inter-day coefficient of variation for car- us time curve [AUC ] was calculated by trape- azepine HPLC assay was less than 7%. The digoxin concentration in plasma was measured o the manufacturer's directions, in duplicate Statistical an ng RIA kits (Orion diagnostics, Finland; Lot No. 157 88501). Into the appropriate labeled test tubes, 25 μl 179 Pharmacokinetic data was expressed as mean ± ors, plasma samples (unknown concentration SEM . The normality of the data was assessed by the goxin) and 100 μl of antiserum solution were Kol mogorov –Smirnov test. The C max and AUC
d. All the tubes were mixed on a vortex mixer and (0-72)
were analysed by paired Student's ‘t' test. All the 161 en incubated for 1 h at room temperature. One ml of statistical analyses were ca rried out by using GraphPad aration reagent was added to all the test tubes and Inst at (version 3.05, 2000, San Diego, USA) software 163 ixed on a vortex mixer. They were centrifuged for 15- m. p<0.05 was considered statistically significant. in at 2000 g. After centrifugation, the supernatant was decanted and the head of each tube was tapped ly against absorbent paper. Radioactivity in each 167 ube was counted using gamma counter for 1 min. The rement range of the kit was 0.5-8.0 nmol/l. The Effect of honey on 169 etection limit of the kit was 0.1nmol/l. The plasma carbamazepine concentration measured culation of pharmacokinetic parameters: 190 p to 72 h was not significantly altered by honey ad- The pharmacokinetic analysis was done using model istration (Figure 2). After ten days of honey admini- 172 ndependent formulae. The peak plasma concentration n, there was no statistically significant change in ) and the time to reach peak plasma concentration max or AUC (0-72)

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Fig 2. Concentration versus time profile of plasma carbamazepine (AUC 0-72) before and after honey. Values are shown as mean
Effect of honey on di goxin pharmacokinetics 204 tracts of certain herbs used in traditional Chinese 205 edicine like Angelica dahurica [ 24], Angelica sinensis The plasma digoxin concentrations measured up to [ 206 25] and Glycyrrhiza glabra [ 26] modulate the CYP3A4 196 h were not significantly altered by honey administra- ity. Herbal extracts of Curcumin [ 27], hawthorn n (Figure 3). There was no statistically significant [ 208 28], ginseng [ 29], green tea [ 30], milk thistle [ 31], 32], and grapefruit juice [ 14], orange juice 210 14] and St. John's Wort [ 22] modulate P-gp activity. 211 Flavonoids present in herbs have been found to in- DISCUSSION
212 eract with CYP3A4 and P-gp [ 3]. Honey is a natural Herbal extracts of garlic [ 20], grapefruit juice [ 21], sacchari ne product rich in sugars and phytochemicals. 202 . John's Wort [ 22] and milk thistle [ 23] modulate the flavonoids present in honey are pinocembrine, pi- ity of CYP3A4 resulting in drug interactions. The no 215 banskin, chrysin, galangin, quercetin, luteolin and Fig 3. Concentration versus time profile of plasma digoxin (AUC 0-72) before and after honey. Values are shown as mean ± SEM.
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2]. Studies in rabbits have shown that 3.
Evans AM. Influence of dietary components on the gastrointes- 217 oney induced the metabolism of diltiazem [ 5] and car-277 tinal metabolism and transport of drugs. Ther Drug Monit 2000; azepine [ 6]. In a human study, where the effect of Zhang Y, Benet LZ. The gut as a barrier to drug absorption: 219 ngle dose of honey on CYP3A4 was investigated us-280 Combined role of cytochrome P450 3A and P-glycoprotein. Clin rbamazepine as a probe drug, honey failed to 281 Pharmacokinet 2001; 40:159-68. show statistically signifi cant effect on carbamazepine 5.
Koumaravelou K, Adithan C, Shashindran CH, Asad M, Abra- rmacokinetic parameters like C ham BK. Influence of honey on orally and intravenously admin- [ 7]. Hence, we studied the effect of multiple doses 284 istered diltiazem kinetics in rabbits. Indian J Exp Biol 2002; carbamazepine pharmacokinetics. In our dy, multiple doses of honey failed to significantly Koumaravelou K, Adithan C, Shashindran CH, Asad M, Abra- ham BK. Effect of honey on carbamazepine kinetics in rabbits. the pharmacokinetics of carbamazepine. Hence we 288 Indian J Exp Biol 2002; 40: 560-3. me that flavonoids present in honey may not have 7.
Malhotra S, Garg SK, Dixit RK. Effect of concomitantly admin- 228 y significant effect on human CYP3A4 activity. istered honey on the pharmacokinetics of carbamazepine in Since honey did not change the pharmacokinetics of 291 healthy volunteers. Methods Find Exp Clin Pharmacol 2003; 230 goxin, it is assumed that the flavonoids present in 231 ney may not have any significant effect on P-gp also. Kerr BM, Thummel KE, Wurden CJ, Klein SM, Kroetz DL, Gonzalez FJ et al. Human liver carbamazepine metabolism. Role 232 ecquemont et al investigated the effect of grapefruit 295 of CYP3A4 and CYP2C8 in 10, 11-epoxide formation. Biochem 233 uice on P-gp activity in 12 healthy volunteers using 296 Pharmacol 1994; 47:1969-79. 234 goxin as a probe drug. It was found that grapefruit Spina E, Arena D, Scordo MG, Fazio A, Pisani F, Perucca E. 235 uice did not significantly inhibit the intestinal P-gp ac-298 Elevation of plasma carbamazepine concentrations by ketocona- ity [ 15]. Although the C zole in patients with epilepsy. Ther Drug Monit 1997;19:535-8. 237 goxin did not change significantly, there was a statis- 300 Pan G, Wang G, Fawcett P. The role of P-glycoprotein in the ant increase in AUC of digoxin (i.e. in 301 intestinal barrier. Asian J Drug Metab Pharmacokinet 2002; 239 rst 4 h) following co-administration with grapefruit 303 Marzolini C, Paus E, Buclin T, Kim RB. Polymorphisms in orrelates with observations made by West-304 human MDR1(P-glycoprotein): Recent advances and clinical et al that P-gp inhibitors alter the early digoxin 305 relevance. Clin Pharmacol Ther 2004; 75 :13-33. rmacokinetics by interfering with the absorption of 12.
306 de Lannoy AI, Silverman M. The MDR1 gene product, P- 243 goxin [ 33]. In our study, 10 days of honey administra-307 glycoprotein, mediates the transport of the cardiac glycoside, di- n did not alter even the early absorption pharmacoki-308 goxin. Biochem Biophys Res Commun 1992; 189: 551-7. 245 etics (AUC ) of digoxin. 309 Deferme S, Van GJ, Augustijns P. Inhibitory effect of fruit ex- Honey and its various derivatives are natural dietary 310 tracts on P-glycoprotein related efflux carriers: An in vitro screening. J Pharm Pharmacol 2002; 54:1213-9. plements consumed commonly all over the world. 312 Tian R, Koyabu N, Takanaga H, Matsuo H, Ohtani H, Sawada y individuals prefer honey to maintain their Y. Effects of grapefruit juice and orange juice on the intestinal 249 ealth and patients with chronic illness take honey along 314 efflux of P-glycoprotein substrates, Pharm Res 2002; 19: 802-9. 250 th other medications. Hence the possibility of honey 315 Becquemont L, Verstuyft C, Kerb R, Brinkmann U, Lebot, M, 251 rug interactions cannot be ruled out. Apart from con-316 Jaillon P et al. Effect of grapefruit juice on digoxin pharmacoki- ing honey as a single dose along with drugs, some 317 netics in humans, Clin Pharmacol Ther 2001; 70: 311-6. ients take honey daily as a nutritional and healthy 16.
318 Johne A, Brockmoller J, Bauer S, Maurer A, Langheinrich M, 254 ietary supplement. Roots I. Pharmacokinetic interaction of digoxin with an herbal extract from St John's wort (Hypericum perforatum). Clin Phar- Since, in vitro and in vivo studies have reported that 321 macol Ther 1999; 66: 338-45. bal extracts may modulate CYP3A4 and P-gp activ- 322 Owen A, Pirmohamed M, Tettey JN, Morgan P, Chadwick D, lting in various types of herb drug interactions; 323 Park BK. Carbamazepine is not a substrate for P-glycoprotein. 258 he safety of coadministration of honey with drugs Br J Clin Pharmacol 2001; 51:345-9. 259 eeds to be studied. This study is an attempt to investi- 325 Greiner B, Eichelbaum M, Fritz P, Kreichgauer HP, von Richter 260 ate the same. To the best of our knowledge, this is the 326 O, Zundler J et al. The role of intestinal P-glycoprotein in the in- teraction of digoxin and rifampin. J Clin Invest 1999; 104:147- 261 rst study in humans where the effect of multi dose 328 262 oney administration on CYP3A4 and P-gp activity has Gerson B, Bell F, Chan S. Antiepileptic agents - primidone, nvestigated. Based upon the present study, it can 330 phenobarbital, phenytoin and carbamazepine by reversed-phase oncluded that honey does not affect the CYP3A4 331 liquid chromatography. Clin Chem 1984; 30:105-8. 265 ediated metabolism and P-gp mediated transport of 20.
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