In Vitro interactionsof plant phenolic compounds and antiparasitic drugs with drug metabolising enzymes and transporters

Abstract

Drug metabolising enzymes and transporters play a critical role in the pharmacokinetics and hence, absorption, distribution, metabolism, and excretion (ADME) of compounds and medicines. The objective of this thesis was to apply in vitro systems used in ADME studies to investigate interactions of pure compounds isolated from plants and of antiparasitic drugs with drug metabolising enzymes and transporters. In addition, drug metabolising enzymes and

transporters are often studied in isolation and though this is simpler, the two biological systems are often coupled, thus, the Caco-2 cell line was also used to study enzyme-transporter coupling. Simple in vitro experimental systems were also compared to complex in vitro and in vivo systems.

In this study, the inhibitory effects of 19 purified compounds isolated from plants, including diospyrin, geshoidin, and some flavonoids, on cytochrome P450s (CYPs) 1A2, 3A4, 2C9, 2C19 and 2D6 and glutathione transferases (GSTs) A1-1, M1-1 and P1-1, as well as p-glycoprotein (Pgp), was investigated in vitro. The effect of 21 antiparasitic drugs, such as chloroquine, cycloguanil and amodiaquine, on Pgp was also investigated.

Inhibition of heterologously expressed human CYPs and GSTs was investigated using spectrophotometric, fluorescence and liquid chromatography-based assays. Eleven out of the 19

plant compounds inhibited at least one of the GSTs. Diospyrin was the most potent inhibitor with IC50 values in the range 0.1 - 0.5 μM. Diospyrin and geshoidin were further investigated and it was found that the predominant mode of GST inhibition was noncompetitive with respect to both glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Diospyrin, however, competitively inhibited A1-1 and M1-1 with respect to GSH and geshoidin displayed mixed inhibition toward A1-1 with respect to GSH. The Ki values for diospyrin with respect to both GSH and CDNB were in the range 0.08–0.6 μM and those for geshoidin were in the range 16– 173 μM. Diospyrin and geshoidin were also found to inactivate GSTP1-1 with diospyrin being a potent inactivator with KI of 0.7 μM, whereas geshoidin had a KI of 47 μM. Eight of the 19 plant

compounds inhibited at least one of the CYPs. Diospyrin was found once again to be the most potent inhibitor with IC50 values in the range 0.4 - 2 μM. Diospyrin showed mixed inhibition toward CYPs 3A4, 2C9 and 2D6, with Ki values in the range 0.25 – 2 μM. For CYP1A2, the inhibition was non competitive with Ki 0.8 μM, compared to mixed inhibition of rat recombinant CYP1A with Ki 0.34 μM. Diospyrin was further investigated for its in vivo CYP1A inhibition properties in rats, and no evidence for in vivo CYP1A inhibition was found.

The plant compounds and antiparasitic drugs were screened for interaction with Pgp based on inhibition of Pgp mediated [3H]-taxol transport in Caco-2 cells. Bidirectional transport of selected inhibitors was further evaluated to identify potential Pgp substrates using the Caco-2 cells. Of 21 antiparasitics tested, 14 were found to inhibit Pgp mediated [3H]-taxol with Kiapp values in the range 4–2000 μM. The antimalarial quinine was the most potent inhibitor with a

Kiapp of 4 μM. Of the 12 natural compounds tested, 3 inhibited [3H]-taxol transport with Kiapp values in the range 50–400 μM. Quinine, amodiaquine, chloroquine, flavone, genistein, praziquantel, quercetin and thiabendazole were further investigated in bidirectional transport assays to determine whether they were substrates for Pgp. Transport of quinine in the secretory direction exceeded that in the absorptive direction and was saturable, suggesting quinine being a Pgp substrate. The rest of the compounds inhibiting Pgp showed no evidence of being Pgp substrates. Amodiaquine was further investigate because the transport experiments in Caco-2 cells showed low recovery of 30 % and rapid disappearance of the compound from the apical chamber. Compounds structurally similar to amodiaquine, and those affecting non-specific binding of amodiaquine or pH of the system, were tested to unravel the mechanism behind these

observations. Chloroquine and ammonium chloride increased the transmonolayer permeability of amodiaquine and decreased its accumulation in Caco-2 cells, whereas bovine serum albumin

(BSA) had no effect. Chloroquine and BSA decreased plastic binding whereas ammonium chloride had no effect. This suggests that amodiaquine is trapped in acidic cell compartments such as lysosomes. Amodiaquine was also trapped in rat intestinal tissue with permeability from the apical to basolateral direction significantly higher than in the opposite direction, suggesting an active uptake over the apical membrane of the rat tissue. Transport in rat jejunum was asymmetric with apparent active apical uptake. Rapid apical uptake in Caco-2 cells appeared to support this theory. However, screening of a range of potential inhibitors as a first step to identifying the potential transporter was not successful and further studies will be required.

To study enzyme-transporter coupling in Caco-2 cells, the GST substrate monochlorobimane (MCB) was used. The Caco-2 cells displayed GST activity by metabolising MCB to the fluorescent conjugate glutathione-bimane (GSB). The cells were loaded with MCB which they conjugated to GSB, and the latter was effluxed into the apical and basolateral compartments with a greater amount being effluxed into the apical compartment. The multidrug resistance protein (MRP) inhibitor benzbromarone, inhibited GSB efflux at both the apical and

basolateral membranes of the Caco-2 cells indicating the presence of GSB-transporting MRPs at both membranes. Ethacrynic acid, which inhibits both GSTs and MRPs reduced both apical and basolateral efflux as well as GSB formation. Diospyrin and ellagic acid which had been seen to be potent inhibitors of recombinant GSTs had no effect on MCB conjugation to GSB or on GSB efflux. In another experiment, Caco-2 cells were loaded with bromosulfophthalein (BSP) a

known MRP substrate, and BSP was effluxed preferentially into the apical compartment.

Addition of the MRP inhibitor benzbromarone resulted in decrease in apical efflux of BSP, and a corresponding increase in the basolateral efflux. This indicates a transporter-transporter interaction whereby the basolateral MRP has a lower affinity for BSP than the apical MRP, and upon inhibition of the apical MRP, the basolateral MRP takes over the efflux of BSP.

In conclusion, a number of purified compounds isolated from plants and a number of antiparasitic drugs, in a range of concentrations, were found to be capable of inhibiting the major drug metabolising CYPs and the major drug transporter Pgp as well as GSTs, in vitro. Drug metabolising enzymes and transporters play an important role in pharmacokinetics. Interaction of the purified plant isolates and antiparasitic drugs with CYPs, GSTs and Pgp has implications for potentially harmful drug-drug interactions where the plant isolates contained in, for instance, a herbal medicine, may be taken concomitantly with prescribed medicines and where antiparasitic drugs may be taken in combination due to emerging resistance. The GST and Pgp inhibitors may also be potential chemomodulators in the case of multidrug resistance in cancer therapy. In the case of amodiaquine, the compound was trapped in acidic cell compartments due to its basicity,

and the use of ammonium chloride rather than BSA in transport experiments with similar compounds is recommended for better prediction of permeability. Comparison of the different experimental methods used, i.e. in vitro recombinant human CYPs vs in vitro recombinant rat CYP; in vitro recombinant rat CYP vs in vivo rat CYP; in vitro recombinant GST vs cellular GST, and membrane transport in cells vs membrane transport in excised tissue, indicated that scaling of in vitro data to more complex in vivo effects should be done with caution. The research work also demonstrated the applicability of in vitro ADME assays routinely used in pharmaceutical companies to the evaluation of natural products and antiparasitic drugs.