Effect of verapamil, a P-gp inhibitor, on the intestinal absorption of isorhamnetin, a herabl constituent, in rats

Effect of verapamil, a P-gp inhibitor, on the intestinal absorption of isorhamnetin, a herabl constituent, in rats Kaijie Chen a, Zhanguo Wang b, Ke Lan b, * a Department of Pharmacy, Chongqing Cancer Hospital, No. 181, Hanyu Road, Chongqing, 400030, China b West China School of Pharmacy, Sichuan University, Chengdu, China Received 6 May 2009; Revised 26 November 2009; Accepted 4 December 2009 Abstract Purpose: To investigate whether the intestinal absorption of isorhamnetin, a common herbal constituent, was affected by verapamil, a potent and specific MDR1 inhibitor. Method: Caco-2 transport assays and a randomized, two-way crossover pharmacokinetic study in rats were employed in the present investigation. HPLC was used to determine the Caco-2 transport of isorhamnetin. The plasma samples were analysed by LC-MS/MS, following administration of a single oral dose of isorhamnetin (1.0 mg/kg), with or without verapamil, (10.0 mg/kg) to rats. Result: After inhibition by verapamil (100 μm), the rate of absorption of isorhamnetin (50 μm) across a Caco-2 cell monolayer (n = 5) was significantly increased, while the corresponding rate of secretion was significantly decreased. After co-administration with verapamil, the C max, AUC 0-72 and AUC 0- all significantly increased. The biliary excretion of isorhamnetin was not affected by verapamil. Conclusion: The results obtained proved that verapamil significantly enhanced the intestinal absorption of isorhamnetin. The inhibition of intestinal secretion mediated by P-glycoprotein (MDR1) might be the reason for the present herb-drug interaction. Keywords: Herb-drug interaction; Absorption; Pharmacokinetics; Caco-2 cell 1. Introduction Recently, herb-drug interactions have attracted the interest of a number of researchers. However, until now, most of the published studies have involved the influence of herbal components or extracts on the pharmacokinetics of synthetic drugs [1-3]. The known mechanisms of these herb-drug interactions mostly involved the inhibition or induction of transporters and metabolic enzymes [4]. Theoretically, it has been proposed that the pharmacokinetics of herbal constituents might also be altered by synthetic drugs in the same way. Moreover, the components of single herbal extracts can affect the individual pharmacokinetic profiles of each other [5]. Thus, the *Corresponding author. Address: Key Laboratory of Drug Targeting and Novel Drug Delivery Systems, West China School of Pharmacy, Sichuan University, No. 17, the 3rd section, Renminnan Road, Chengdu 610041, Sichuan, China. Tel.: +86-28-85503722; Fax: +86-28-85503722 E-mail: lanwoco@scu.edu.cn; lanwoco@163.com pharmacokinetic interactions between herbal components/extracts and drugs, as well as pharmacokinetic intra- and inter-herbal interactions are of interest to researchers. Isorhamnetin (3 -O-methylquercetin) is a common constituent of a number of herbs, such as gingko leaves [6] and fruits/leaves of Hippophae rhamnoides L. [7]. It is a metabolite of quercetin in mammals [8-11]. The irreversible [12] O-methylation of quercetin is catalyzed by catechol-o-methyltranferase (COMT) [13]. Quercetin and its analogues have been proven to modulate the function and expression of P-glycoprotein (P-gp) [14, 15] and CYP3A4 [16] and, consequently, this can affect the pharmacokinetics of some synthetic drugs. Furthermore, it has been shown that not only quercetin but also isorhamnetin interacts with P-gp [5, 17]. Therefore, based on the above reports and our hypothesis, we carried out the present study to investigate whether the intestinal absorption of isorhamnetin, a common constituent of herbs, was affected by verapamil. 357

2. Materials and methods 2.1. Materials Isorhamnetin (99%) and baicalein (internal standard, 99.9%) were purchased from Tauto Biotech (Shanghai, China). Verapamil was obtained from National Institute for the Control of Pharmaceutical and Biological Products. β-glucuronidase (type H-3, from helix pomatia) and Sulfatase (type H-1, from helix pormatia) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The human colon carcinoma cell line (Caco-2) was obtained from American Type Culture Collection (Rockville, MD). Dulbecco s Modified eagle s medium (DMEM, high glucose, Cat: 61975), Hank's balanced salt solution (HBSS, Cat: 14175), penicillin-streptomycin (Cat: 15070) and trypsin-edta (Cat: 25300) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Fetal cattle serum (FCS, Cat: SH30084) was purchased from Hyclone (Logan, UT, USA). Dimethyl sulfoxide (DMSO) was purchased from Millipore Corp. Methanol, glacial acetic acid and formic acid (HPLC grade) were purchased from the TEDIA Inc (Fairfield, IA, USA). 2.2. Cell culture and transport assay Caco-2 cell culture and transport assay were conducted in accordance with Current Protocols in Pharmacology [18] as described previously [5]. The integrity of the confluent Caco-2 cell monolayers was evaluated using lucifer yellow. Trans-epithelial electric resistance (TEER) was measured (expressed in Ω cm 2 ) using a Millicell-ERS apparatus (Millipore, Bedford, MA, USA). Rhodamine 123 was used as a probe substrate for ensuring P-gp expression in Caco-2 cells. The P-gp expression in cell monolayers was determined by carrying out bidirectional studies using rhodamine 123 at a concentration of 1 μm. The monolayers used for the transport experiments had TEER values greater than 400 Ω cm 2 and the leakage rate of lucifer yellow was less than 1%/h. The permeability of isorhamnetin was measured at 50 μm. Samples (100 μl) were collected from both the apical and basolateral compartments at 20, 40, 60 and 80 min and stored at 70 C until analysis by HPLC. The same procedure was conducted for the verapamil inhibition study except that the transport buffer contained 100 μm verapamil. 2.3. Pharmacokinetic study The animal experiments were approved by the Ethics Committee of the West China Center of Medicine, Sichuan University. A randomized, two-way crossover study design was used. Twelve male Wistar rats (SPF grade, 202.5 ± 10.5 g, Experimental Animals Center, Sichuan University, PR China, Certificate: Chuan Shi Dong Guan-09) aged 12 16 weeks were randomly divided into 2 equal groups and the animals were housed and fed as described in our earlier published paper [12]. Isorhamnetin was dispersed in ethanol at approximately 0.5 mg/ml and subsequently diluted with 1% (v/v) glycerol in water to give intragastric solutions containing 20% ethanol (v/v). Two groups of rats were given either a single oral dose of isorhamnetin (1.0 mg/kg) or a single oral dose of isorhamnetin (1.0 mg/kg) together with verapamil (10.0 mg/kg, equivalent to the daily human dose) during the first study period. During the second study period, the two groups of rats received the other form of administration. The wash-out period was ten days. Blood samples (0.3 0.4 ml of venous blood) were collected from the rats pre-administration and at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 48 and 72 h post-administration from the tail vein. Blood samples were collected into tubes containing lithium-heparin and centrifuged (1500 g for 10 min) within 30 min of collection (EBA21 table centrifuge, Hettich, Germany). Then, 100 µl of the harvested plasma samples was acidified with 10 µl 0.5 M acetic acid containing 2 mg of vitamin C per ml. 2.4. Biliary excretion studies An additional twelve male Wistar rats (SPF grade, 202.5 ± 10.5 g, Experimental Animals Center, Sichuan 358

University, PR China, Certificate: Chuan Shi Dong Guan-09) were randomly divided into 2 equal groups. Verapamil (10.0 mg/kg in water) and control placebo (water) were administrated orally to rats in the pretreatment group (n = 6) and control group (n = 6), 2 h before administration of isorhamnetin (1.0 mg/kg). Bile was collected according to Current Protocols in Pharmacology [19]. After giving isorhamnetin (1.0 mg/kg) to both the pretreatment group and control group, bile was collected at 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 6, 6 8, 8 10 and 10 12 h intervals. The volumes of the bile samples were recorded and the samples were then stored at 70 C until analysis. 2.5. Sample analysis A modified HPLC method based on the report of Wang et al. [17] was used to determine isorhamnetin in the transport buffers. After adding 100 μl methanol, the mixture was vortexed for 1 min at 2000 r/min (Vortex Genius 3, IKA, Germany) and centrifuged for 3 min at 5000 g (EBA21 table centrifuge, Hettich, Germany). A mobile phase of methanol acetonitrile 0.5% acetic acid in water (5:23:72, v/v/v) was delivered at a rate of 1.0 ml/min using a Shimadzu LC-2010C HPLC system. A Shimadzu VP-ODS column (150 nm 4.6 mm, 5 µm) was maintained at 40 C, and the wavelength of the UV detector was set at 370 nm. The ESI + -LC-MS/MS method to determine total isorhamnetin in plasma and bile samples was published previously [12]. After treatment with β-glucuronidase and sulfatase and addition of internal standard (baicalein), samples were extracted with ethyl acetate (redistilled). After centrifugation, the upper phase was evaporated and reconstituted in methanol. The chromatographic separation was performed on a Diamonsil C 18 column with a mobile phase of 2% formic acid-methanol (10: 90, v/v) at a flow rate of 1.00 ml/min, with a split of 200 µl to the mass spectrometer. The mass spectrometer (API3000 LC/MS/MS system, Applied Biosystems, Foster City, CA, USA) was operated in the positive ion mode. The samples were analyzed using the transition of m/z 317 153 amu for isorhamnetin and m/z 271 123 amu for baicalein. 3. Results and discussion 3.1. Transport studies The results of the Caco-2 cell transport studies are shown in Fig. 1 and Table 1. For transport alone, the apparent secretion (basolateral to apical) rate was higher than the apparent absorption (apical to basolateral) rate (P < 0.05, Student s t-test). The P ratio was 0.74, indicating that an efflux mechanism was involved in the transport. These results were consistent with those obtained in a previous report [17]. After inhibition by verapamil (100 μm), the absorption rate (n = 5) of isorhamnetin increased from 2.28 ± 0.11 10-6 cm/s to 3.58 ± 0.19 10-6 cm/s (P = 0.002, Student s t-test), and the corresponding secretion rate (n = 5) decreased from 3.09 ± 0.08 10-6 cm/s to 2.17 ± 0.16 10-6 cm/s (P = 0.007, Student s t-test). The present results listed in Table 1 indicate that verapamil significantly inhibited the efflux of isorhamnetin and, consequently, enhanced the absorption of isorhamnetin across the Caco-2 cell monolayer. 3.2. Pharmacokinetic studies The total plasma concentration of isorhamnetin versus time curve is illustrated in Fig. 2. Consistent with the previous pharmacokinetic studies in humans [20], isorhamnetin showed two or more maximum plasma concentration peaks. This phenomenon was reduced after concomitant oral administration of verapamil. The pharmacokinetic parameters were calculated using DAS 2.0 pharmacokinetic software (Drug and Statistics, Version 2.0) with a non-compartment model. Table 1 Permeability coefficients of isorhamnetin (50 μm) across Caco-2 cell monolayers, with or without verapamil (100 μm) (mean ± SD, n = 5). Permeability coefficients Isorhamnetin (50 μm) Isorhamnetin (50 μm) with verapamil (100 μm) P app(a-b) 10 6 (cm/s) 2.28 ± 0.11 3.58 ± 0.19 P app(b-a) 10 6 (cm/s) 3.09 ± 0.08 2.17 ± 0.16 P ratio 0.74 1.65 359

Isorhamnetin amount ( g) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 + verapamil (100 M) 0 20 40 60 80 Time (min) (A) Isorhamnetin amount ( g) Fig. 1. Increase in isorhamnetin in the receiver compartment during apical to basolateral (A) and basolateral to apical (B) transport of isorhamnetin (50 μm), with or without verapamil (100 μm). 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 + verapamil (100 M) 0 20 40 60 80 Time (min) (B) Table 2 Pharmacokinetic parameters of isorhamnetin after a single oral dose of isorhamentin (1.0 mg/kg) and isorhamnetin (1.0 mg/kg) coadministrated with verapamil (10.0 mg/kg) in male Wistar rats (n = 12). Pharmacokinetic parameters Isorhamnetin (1 mg/kg) Treatment Isorhamnetin (1 mg/kg) + verapamil (10.0 mg/kg) Significance of paired t test C max (ng/ml) 76.7 ± 10.2 108.6 ± 23.7 0.002 t max (h) 7.2 ± 1.8 7.2 ± 1.3 AUC 0-t (μg/l h) 1312.0 ± 215.4 1708.8 ± 280.2 0.001 AUC 0- (μg/l h) 1493.4 ± 257.6 1862.2 ± 321.2 0.007 The t max, C max, AUC 0-72 and AUC 0- of isorhamnetin, with and without co-administration of verapamil, are listed in Table 2. The paired Student s t-test of the logarithmic transformations of C max, AUC 0-72 and AUC 0- showed significant differences between the single dose and combined dose administrations. The Wilcoxon signed rank test of t max found no significant difference (P = 0.951) between the single dose and combined dose. These data indicated that verapamil significantly improved the oral bioavailability of isorhamnetin. The inhibition of intestinal efflux or bile excretion of isorhamnetin and its conjugates by verapamil could contribute to the attenuation of the multiple peak phenomena and the improvement in oral bioavailability. 3.3. Biliary excretion The cumulative biliary excretion profiles for isorhamnetin following oral administration in the presence Plasma concentration of total isorhamnetin (ng/ml) 100 80 60 40 20 0 0 20 40 60 Fig. 2. Mean plasma concentrations of total isorhamnetin over time after oral administration of isorhamentin alone (1.0 mg/kg) and isorhamnetin (1.0 mg/kg) coadministered with verapamil (10 mg/kg) in male Wistar rats (n = 12). All conjugates of isorhamnetin was hydrolyzed with β-glucuronidase and sulfatase before analysis. Isorhamnetin (1 mg/kg) Isorhamnetin (1 mg/kg) + verapamil (10 mg/kg) Time (h) 360

Cumulative biliary excretion of isorhamnetin (ng) 700 600 500 400 300 200 100 0 Isorhamnetin (1 mg/kg) Isorhamnetin (1 mg/kg) + verapamil (10 mg/kg) 0 1 2 3 4 6 8 10 12 Time (h) Fig. 3. Cumulative biliary excretion of total isorhamnetin over time after a single oral dose of isorhamentin (1.0 mg/kg) and isorhamnetin (1.0 mg/kg) following pretreatment with verapamil (10 mg/kg) in male Wistar rats (n = 6). All conjugates of isorhamnetin was hydrolyzed with β-glucuronidase and sulfatase before analysis. or absence of verapamil are shown in Fig. 3. It can be seen that 12 h after dosing, about 0.25% of the isorhamnetin dose was recovered in bile in both the control group and pretreatment groups. No significant difference was found between the control group and the pretreatment group. This result indicates that verapamil did not alter the biliary excretion of isorhamnetin. The absorption interaction contributed to the effect of verapamil on the pharmacokinetics of isorhamnetin significantly more than the biliary excretion interaction did. 4. Conclusion This study shows that verapamil significantly inhibits the secretion and enhances the absorption of isorhamnetin across the Caco-2 cell monolayer, resulting in a significant increase in the oral bioavailability of isorhamnetin, whereas the biliary excretion of isorhamnetin was not affect by verapamil. Verapamil is a well-known P-gp inhibitor, and has also been reported to affect MRP1 (multi-drug resistance associate protein 1) function [21,22]. P-gp has been shown to be expressed on the apical side of Caco-2 cells and intestinal epithelial cells, the canalicular surface of hepatocytes, brain capillary endothelial cells and the apical surface of renal proximal tubules [23], and this might be responsible for the pharmacokinetic interactions observed. Therefore, verapamil has a complex effect on the pharmacokinetics of isorhamnetin. Theoretically, the biliary excretion of isorhamnetin should be inhibited by verapamil, but the result of the present study showed that this was not the case. Considering the long-term effect of verapamil on the biliary excretion of isorhamnetin, further investigations are need to discover if biliary excretion contributes to the present herb-drug interaction. The result of the Caco-2 cell transport assay strongly supported the in vivo results. Therefore, it was confirmed that verapamil could inhibit the intestinal efflux of isorhamnetin and subsequently improve its oral bioavailability. P-gp and MRP1 might play an important role in these pharmacokinetic interactions although the role of other efflux transporters needs further study. Moreover, if verapamil could affect the metabolism of isorhamnetin, it might also alter the pharmacokinetics of isorhamnetin. After being absorbed, isorhamnetin mainly undergoes phase II conjugation metabolism, such as glucuronidation and sulfation [12]. No direct evidence was found that verapamil could influence the activity 361

and expression of UDP-glucurosyltransferase and/or sulfate transferase. Therefore, in the present study, verapamil had no effect on the metabolism of isorhamnetin. The results of the present study proved that the pharmacokinetics of herbal constituents can be altered by synthetic drugs in the same way that herbal constituents affect the pharmacokinetics of synthetic drugs. Verapamil significantly inhibited the secretion, and enhanced the absorption of isorhamnetin across the Caco-2 monolayer, consequently increasing the oral bioavailability of isorhamnetin after oral administration to rats. The inhibition of intestinal secretion of isorhamnetin mediated by P-gp and MRP1 might be one of the main reasons for the present herb-drug interaction. Acknowledgements The authors acknowledge the financial support received from National Natural Science Foundation of China (No. 30572373), for its support and encouragement in carrying out this work. References [1] E. Mills, V. M. Montori, P. 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