Marked Decrease of Cyclosporin Absorption Caused by Phellamurin in Rats

• Abstract
• Introduction
• Materials and Methods
• Chemicals
• Everted rat intestine study
• Drug administration and blood collection
• Quantitation of blood cyclosporin concentrations
• Data analysis
• Results and Discussion
• References

Abstract

Phellamurin is a flavonoid glycoside that is abundant in the leaves of Phellodendron wilsonii Hayata et Kanehira (Rutaceae). In vitro everted rat intestine study indicated that phellamurin inhibited intestinal P-glycoprotein in a dose-dependent manner. In order to investigate the effect of phellamurin on cyclosporin absorption and disposition, rats were given cyclosporin (5 mg/kg) with or without phellamurin in a parallel design. Fluorescence polarization immunoassay was used to determine the blood concentration of cyclosporin. Unanticipatedly, our results indicated that the coadministration of phellamurin significantly decreased the C_max of cyclosporin by 77 % and reduced the AUC_0-∞ of cyclosporin by 56 %. This indicated that a serious interaction occurred between phellamurin with cyclosporin. To ensure the efficacy of cyclosporin, we suggest that the coadministration of phellamurin or Phellodendron wilsonii with cyclosporin should be avoided.

Introduction

Flavonoids have attracted much attention in recent years because of their beneficial pharmacological activities including antioxidation [1], free radical scavenging [2], anticancer [3], antivirus [4] etc. and furthermore, their additional abilities to modulate both CYP 3A4 and P-glycoprotein (Pgp), the product of mdr (multidrug - resistance) genes (5 - 8). CYP3A4 is mainly present in the intestine and liver. The significant role of CYP 3A4 for drug-drug interactions was well recognized. Pgp is expressed in various normal human tissues such as small intestine, kidney, liver and capillary endothelial cells of brain and testes [9] [10] [11], and its significant roles for chemoprevention of organisms and drug - drug interaction had been proposed [12] [13] [14].[]

Phellamurin is 3,4′,5,7-tetrahydroxy-8-isoprenylflavanone-7-glucoside, a flavanone glycoside present in great abundance in the leaves (ca. 10 % in fresh leaves) of Phellodendron wilsonii Hayata et Kanehira (Rutaceae) [15] which is common in Taiwan and the bark is often used as a bitter stomachic and bacteriostatic in clinical Chinese medicine. Cyclosporin is a widely used immunosuppressant with a narrow therapeutic range. Cyclosporin is a substrate of both CYP 3A4 (16) and Pgp [17]. In a recent report, inhibition of P-glycoprotein was proposed to be a more important mechanism for enhanced cyclosporin absorption than inhibition of CYP 3A4 [18]. In vitro everted rat intestine study indicated that phellamurin significantly inhibited the Pgp of jejunum and ileum as shown in Fig 1. Therefore, this study attempted to investigate the effect of phellamurin on the absorption and disposition of cyclosporin in rats.

Materials and Methods

Chemicals

Cyclosporin (Sandimmun Neoral®, 100 mg/ml) was kindly offered by Novartis (Taiwan) Co. Ltd. Phellamurin was isolated by the second author from the leaves of Phellodendron wilsonii and the structure was identified by spectroscopic methods [15]. The purity of phellamurin was 95 % determined by HPLC coupled with photodiode array detector. Rhodamine 123 was purchased from Aldrich (Milw. WI, USA). Glycofurol and medium 199 were supplied by Sigma (St. Louis, MI, USA). Dimethylacetamide was obtained from Wako (Osaka, Japan). PEG 400 and hydrochloric acid were purchased from Merck (Germany). Milli-Q plus Water (Millipore, Bedford, MA, USA) was used for all preparation.

Everted rat intestine study

Nine female Sprague-Dawley rats were sacrificed. The jejunum (30 cm long from stomach) and ileum (30 cm long from the ileocecum) were isolated. After flushing with ice-cold saline, each segment was everted and both ends were ligated tightly to prepare a 25 cm long everted rat intestine. Then the rat intestine was immersed into 50 ml medium 199 prewarmed at 37 °C and preoxygenated with 95 % O₂/5 % CO₂. After incubating for 20 min, 3 ml rhodamine 123 solution (20 μg/ml in medium 199) was introduced into the everted rat intestine (serosal side). Under bubbling with the 95 % O₂/5 % CO₂ mixture gas, the transport of rhodamine 123 solution from serosal to mucosal surfaces across the intestine was measured by sampling the mucosal medium every 20 min until 100 min. On the other hand, phellamurin was dissolved with glycofurol and added to the mucosal medium in order to give designated final concentrations of 200 and 400 μM. The transport of rhodamine 123 in the absence (control) or presence of phellamurin was measured fluorometrically using a Luminescence Spectrometer LS-50B (Perkin Elmer, USA). The animal study adhered to the ”Principles of Laboratory Animal Care” (NIH publication #85 - 23, revised 1985).

Drug administration and blood collection

Eleven male Sprague-Dawley rats weighing 200 - 300 g were fasted for 12 h before drug administration. Six of them were given 5 mg/kg cyclosporin which had been prepared by diluting Neoral^® with deionized water to 2.5 mg/ml. The other 5 rats were administered a single dose of 100 mg/kg phellamurin dissolved in water : PEG : dimethylacetamide (5 : 4 : 1, 40 mg/ml) 30 sec before cyclosporin via gastric gavage. Blood samples (0.3 ml) were withdrawn via cardiopuncture at 0, 0.33, 0.66, 1, 3, 5, 7, 9, 12 and 24 h after drug administration. The blood was collected into small plastic vials containing EDTA and assayed within one week.

Quantitation of blood cyclosporin concentrations

Cyclosporin concentration in blood was measured by using a specific monoclonal fluorescence polarization immunoassay (Abbott, Abbott Park, Ill, USA). The assay was calibrated for concentrations from 25.0 to 1500.0 ng/ml.

Data analysis

Pharmacokinetic parameters of cyclosporin were calculated by using noncompartment model of WINNONLIN (version 1.1, SCI software, Statistical Consulting, Inc., Apex, NC). Unpaired Student’s t-test was used for statistical comparison taking p < 0.05 as significant.

Results and Discussion

Fig. [1] shows the effect of phellamurin on the efflux transport of rhodamine 123 from the serosal side to the mucosal side. The result indicated that phellamurin significantly inhibited the function of intestinal Pgp in a dose-dependent manner for both jejunum and ileum. This prompted us to investigate the effect of phellamurin on the absorption and disposition of cyclosporin. Fig. [2] depicts the blood profiles of cyclosporin after administration of cyclosporin alone and coadministration with a single dose of phellamurin. The pharmacokinetic parameters of cyclosporin for both treatments are given in Table [1]. Our results showed that phellamurin significantly decreased the C_max of cyclosporin by 77 % and reduced the total exposure of cyclosporin by 56 %. The peak time was significantly delayed for 2.2 h by phellamurin, whereas the elimination half life was not significantly affected. It indicated that coadministration of phellamurin markedly inhibited the absorption of cyclosporin. The outcome could not be explainable by the inhibition of intestinal Pgp by phellamurin as observed in the in vitro study. Modulation of Pgp is an important mechanism of drug interaction. However, absorption and disposition of a drug and its metabolites frequently is not only determined by Pgp, but also by metabolizing enzymes and possibly by other transporters, e. g., MRP (multidrug resistance protein) [14].

Our previous pharmacokinetic study showed that phellamurin was not absorbed at all and it was hydrolyzed, absorbed and glucuronidated rapidly to reach the C_max of neophellamuretin glucuronides at 30 min after dosing. The total exposure of neophellamuretin glucuronides was 4 times that of the aglycone - neophellamuretin [19]. The metabolic fate of phellamurin was in good agreement with most flavonoid glycosides which were absorbed as their aglycones and then were mainly circulating as glucuronides in the plasma [20]. Therefore, the glucuronides of aglycone derived in vivo from phellamurin might play a more important role in drug interaction than the parent glycoside or the aglycone. The significant role of the glucuronidated metabolites of flavonoids for drug-drug interaction is worthy of further investigations.

The present pharmacokinetic interaction study was initially carried out in a crossover design. However, after one week wash-out, the blood levels of cyclosporin demonstrated prominent carry-over effect from the first dose of cyclosporin. The comparisons of AUC_{0 - 36} and C_max between two control groups as well as two treatment groups in the first and second runs are shown in Table [2]. Because the elimination rate constants of some rats at the second run could not be estimated, AUC_{0 - 36} was calculated for comparison instead of AUC_0-∞. The AUC_{0 - 36} and C_max of the control group in the second run were significantly reduced by 72 % and 79 %, respectively, when compared to the corresponding data of the first run. Regarding the treatment groups, the AUC_{0 - 36} and C_max of the second run were significantly reduced by 49 % and 46 %, respectively, when compared to the corresponding data of the first run. This result clearly indicated that drug resistance developed after only a single dose of cyclosporin in rats. Therefore, the data analysis of this study was calculated based on the comparisons between two parallel groups. It is suggested that when using the rat as the model for cyclosporin (Sandimmun Neoral®) study, a parallel design is preferable to a crossover design.

In recent years, many transplant recipients were reported to show subtherapeutic cyclosporin concentrations after they started self-medicating with St. John’s wort (Hypericum perforatum) [21] [22] [23]. Hypericum extracts are likely to act as potent inducers of hepatic enzymes. Most in vitro studies have agreed that Hypericum extracts double the metabolic activity of CYP 3A4. Other cytochrome P-450 isoenzymes as well as Pgp, may be affected by Hypericum [24]. It is proposed that herbs represent a potential and possibly an overlooked cause for drug interaction in transplant recipients.

In summary, phellamurin markedly decreased the absorption of cyclosporin. Because cyclosporin is a substrate of both Pgp and CYP 3A4, we suggest that the coadministration of phellamurin or phellamurin-containing herb with cyclosporin or other medications whose absorption and metabolism are mediated by Pgp and/or CYP 3A4 should require close monitoring. Inquiries regarding the usage of herbal medicines should be an integral component of a transplant recipient’s medication history.

References

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  CrossrefPubMedSearch in Google Scholar

Prof. Pei-Dawn Lee Chao

Dept. of Pharmacy

China Medical College

Taichung

Taiwan 404

R. O. C

Fax: +886-4-22031028

Email: pdlee@mail.cmc.edu.tw

References

• 1 da Silva E L, Tsushida T, Terao J. Inhibition of mammalian 15-lipoxygenase-dependent lipid peroxidation in low-density lipoprotein by quercetin and quercetin monoglucosides.  Archchives of Biochemistry and Biophysics. 1998;  349 313-20
  PubMedSearch in Google Scholar
• 2 Brown J E, Khodr H, Hider R C, RiceEvans C A. Structural dependence of flavonoid interactions with Cu²⁺ ions: Implications for their antioxidant properties.  Biochemical Journal. 1998;  330 1173-8
  PubMedSearch in Google Scholar
• 3 Yoshida M, Yamamoto M, Nikaido T. Quercetin arrests human leukemic T-cell in late G1 phase of the cell cycle.  Cancer Research. 1992;  52 6676-81
  PubMedSearch in Google Scholar
• 4 Kitamura K, Honda M, Yoshizaki H, Yamamoto S, Nakane H, Fukushima M, Ono K, Tokunaga T. Baicalin, an inhibitor of HIV-1 production in vitro .  Antiviral Research. 1998;  37 131-40
  CrossrefPubMedSearch in Google Scholar
• 5 Miniscalco A, Landahl J, Regardh C G, Edgar B, Eriksson U G. Inhibition of dihydropyridine in rat and human liver microsomes by flavonoids found in grapefruit juice.  Journal of Pharmacology and Experimental Therapeutics. 1992;  261 1195-8
  PubMedSearch in Google Scholar
• 6 Scambia G, Ranelletti F O, Panici P B, De Vincenzo R, Bonanno G, Ferrandina G, Piantelli M, Bussa S, Rumi C, Cianfriglia M ,. et al . Quercetin potentiates the effect of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: P-glycoprotein as a possible target.  Cancer Chemotherapy and Pharmacology. 1994;  34 459-64
  PubMedSearch in Google Scholar
• 7 Critchfield J W, Welsh C J, Phang J M, Yeh G C. Modulation of adriamycin accumulation and efflux by flavonoids in HCT-15 colon cells. Activation of P-glycoprotein as a putative mechanism.  Biochemical Pharmacology. 1994;  48 437-45
  PubMedSearch in Google Scholar
• 8 Bosch I, Croop J. P-glycoprotein, multidrug resistance and cancer.  Biochimica et Biophysica Acta. 1996;  1288 F37-54
  PubMedSearch in Google Scholar
• 9 Sugawara I, Kataoka I, Morishita Y. Tissue distribution of P-glycoprotein encoded by a multidrug-resistant gene as revealed by a monoclonal antibody, MRK 16.  Cancer Research. 1998;  48 1926-9
  PubMedSearch in Google Scholar
• 10 Thiebaut F, Tsuruo T, Hamada H, Gottesman M M, Pastan I, Willingham M C. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues.  Proceedings of the National Academy of Sciences of USA. 1987;  84 7735-8
  CrossrefPubMedSearch in Google Scholar
• 11 Thiebaut F, Tsuruo T, Hamada H, Gottesman M M, Pastan I, Willingham M C. Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein p170: evidence for localization in brain capillaries and cross reactivity of one antibody with a muscle protein.  Journal of Histochemistry and Cytochemistry. 1989;  37 159-64
  PubMedSearch in Google Scholar
• 12 Gottesman M M, Pastan I, Ambudkar S V. P-glycoprotein and multidrug resistance.  Current Opinion in Genetic Development. 1996;  6 610-7
  CrossrefPubMedSearch in Google Scholar
• 13 Schinkel A H, Wagenaar E, Mol C AAM, van Deemter L. P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs.  Journal of Clinical Investigation. 1996;  97 2517-24
  PubMedSearch in Google Scholar
• 14 Fromm M F. P-glycoprotein: a defense mechanism limiting oral bioavailability and CNS accumulation of drugs.  International Journal of Clinical Pharmacology and Therapeutics. 2000;  38 69-74
  PubMedSearch in Google Scholar
• 15 Wu T S. Constituents of Formosan folk medicine. IV. The constituents of the leaves of Phellodendron wilsonii Hayata et Kanehira.  Journal of Chinese Chemical Society. 1979;  26 25-8
  PubMedSearch in Google Scholar
• 16 Kolars J C, Awni W M, Merion R M, Watkins P B. First-pass metabolism of cyclosporin by the gut.  Lancet. 1991;  338 1488-90
  CrossrefPubMedSearch in Google Scholar
• 17 Lown K S, Mayo R R, Leichtman A b, Hsiao H L, Turgeon D K, Schmiedlin R, Brown M B, Guo W, Rossi S J, Benet L Z, Watkins P B. Role of intestinal P-glycoprotein (mdr 1) in interpatient variation in the oral bioavailability of cyclosporin.  Clinical Pharmacology and Therapeutics. 1997;  62 248-60
  CrossrefPubMedSearch in Google Scholar
• 18 Edward D J, Fitzsimmons M E, Schuetz E G, Yasuda K, Ducharme M P, Warbasse L H, et al. 6′,7′-Dihydroxybergamottin in grapefruit juice and Seville orange juice: Effects on cyclosporin disposition, enterocyte CYP 3A4, and P-glycoprotein.  Clinical Pharmacology and Therapeutics. 1999;  65 237-44
  PubMedSearch in Google Scholar
• 19 Chen H Y, Wu T S, Wang J P, Kuo S C, Chao P DL. The fate of phellamurin in rats.  The Chinese Pharmaceutical Journal. 2001;  53 37-44
  PubMedSearch in Google Scholar
• 20 Scalbert A, Williamson G. Dietary intake and bioavailability of polyphenols.  Journal of Nutrition. 2000;  130 2073S-85S
  PubMedSearch in Google Scholar
• 21 Bon S, Hartmann F, Kuhn H. Johanisskraut: Ein enzyminducer?.  Schweitzer Apothekerzeitug. 1999;  16 535-6
  PubMedSearch in Google Scholar
• 22 Ruschitzka F, Meier P J, Turina M, Luscher T F, Noll G. Acute heart transplant rejection due to St. John’s wort.  Lancet. 2000;  355 48-9
  CrossrefPubMedSearch in Google Scholar
• 23 Ernst E. Second thoughts about safety of St. John’s wort.  Lancet. 1999;  354 2015-6
  PubMedSearch in Google Scholar
• 24 Biffignandi P M, Bilia A R. The growing knowledge of St. John’s wort (Hypericum perforatum L) drug interactions and their clinical significance.  Current Therapeutic Research. 2000;  61 389-94
  CrossrefPubMedSearch in Google Scholar

Prof. Pei-Dawn Lee Chao

Dept. of Pharmacy

China Medical College

Taichung

Taiwan 404

R. O. C

Fax: +886-4-22031028

Email: pdlee@mail.cmc.edu.tw