Introduction

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Quinine is recommended for the treatment of chloroquinine-resistant Plasmodium falciparum malaria and is an important drug of choice for the treatment of complicated and/or cerebral malaria (Tracy and Webster, 1996; Hien et al., 1996; Boele van Hensbroek et al., 1996). However, its most common use outside of countries with endemic malaria is as treatment for leg cramps (Dyer et al., 1994). It has been known that quinine has a relatively low therapeutic index with some adverse reactions such as cinchonism, hypoglycemia, and cardiac arrhythmias (White, 1988). Thus, quinine-drug interactions seem to be of clinical importance.

The primary route of the systemic elimination of quinine in humans is known to be via extensive hepatic metabolism with less than 20% of the drug excreted unchanged in urine (Tracy and Webster, 1996; White, 1992; Krishna and White, 1996). Despite the fact that quinine is one of the oldest drugs (at least 350 years) in the pharmacopoeia and the most widely used antimalarial drug, the detailed metabolism of quinine and cytochrome P450(CYP) isoform(s) involved have been only recently elucidated: the formation of 3-hydroxyquinine from quinine is the major metabolic pathway (Wanwimolruk et al., 1995; Wanwimolruk et al., 1996). It is catalyzed mainly by CYP3A4 (Zhao et al., 1996; Zhang et al., 1997) and to a minor extent by CYP2C19 (Zhao et al., 1996) in human liver microsomes. Our recent study has shown that CYP3A/Cyp3a also plays a dominant role in the formation of 3-hydroxyquinine from quinine in mouse, rat, and dog liver microsomes, and 3-hydroxyquinine is the main metabolite of quinine in these animal livers (Zhao and Ishizaki, in press).

On the other hand, etoposide, a commonly used anticancer agent with a broad range of antitumor activity, is claimed to be metabolized largely via CYP3A4 by 3-demethylation in human liver microsomes and its metabolite has some antitumor activity (Relling et al., 1992). Like quinine, etoposide also possesses a relatively low therapeutic index with some adverse reactions in cancer patients (e.g. leukopenia) (Kobayashi and Ratain, 1994). Therefore, from a theoretical point of view, a drug-drug interaction might occur when quinine and etoposide are co-administered in patients with cancer who are living in malaria endemic areas such as Southeast Asia, South America, and East Africa (i.e. 300 to 500 million new cases of malaria every year). More importantly, if a mutual inhibition between quinine and etoposide in human liver microsomes would occur, it should provide further evidence for the role of CYP3A4 involvement in the metabolism of both quinine and etoposide. Based on the background as discussed above, we conducted this study to assess the mutual interaction potential of quinine and etoposide in vitro, as well as to confirm further that etoposide and quinine are metabolized mainly via CYP3A in human liver microsomes.

	Materials and Methods

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Drugs and Chemicals. Synthetic 3-hydroxyquinine was a generous gift from Dr. P. Winstanley (University of Liverpool, Liverpool, UK). Quinine, ketoconazole, and troleandomycin (TAO) were purchased from Sigma Chemical Co. (St. Louis, MO). Etoposide, 3-demethyletoposide and the internal standard (4-demethylepi-podophyllotoxin-9-(4,6-0-propyliodone--D-glucopyranoside) were obtained from Nippon Kayaku Co., Ltd. (Tokyo, Japan). Acetonitrile, methanol, and other reagents of analytical grade were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). NADP⁺, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase were obtained from Oriental Yeast (Tokyo, Japan).

Preparation of Human Liver Microsomes. Six histologically normal liver samples were obtained from Japanese patients with hepatic metastatic cancer (from colon or rectum) who underwent a partial hepatectomy at the Division of General Surgery, International Medical Center of Japan, Tokyo, as excess material that was removed during surgery on the liver as reported previously (Chiba et al., 1993; Echizen et al., 1993). Ethical approval for the study had been granted by the Institutional Ethics Committee of the International Medical Center of Japan. Washed microsomes were prepared by classical differential centrifugation technique (Chiba et al., 1993; Echizen et al., 1993). After the determination of microsomal protein by the method of Lowry et al. (Lowry et al., 1951), aliquots of the individual microsomal samples were stored at 80^oC until used.

Assay with Human Liver Microsomes. The basic incubation medium contained 0.1 mg/ml human liver microsomes, 0.5 mM NADP⁺, 2.0 mM glucose-6-phosphate dehydrogenase, 4 mM MgCl₂, 0.1 mM EDTA, 100 mM potassium phosphate buffer (pH 7.4), and 100 µM of quinine or 50 µM of etoposide in a final volume of 250 µl. All the reactions, except for TAO, were initiated by the addition of the NADPH-generating system without preincubation, and the mixture was incubated at 37^oC in a shaking water bath for 15 min. After the reaction was stopped by the addition of 500 µl ice-cold methanol for quinine metabolism or 100 µl ice-cold acetonitrile for etoposide metabolism, 100 µl of 1M sodium-phosphate buffer (pH 3.0) as well as 50 µl of 5 µM internal standard was added to the etoposide metabolism mixture. The mixture was centrifuged at 10,000g for 10 min and the supernatant was injected onto a high-performance liquid chromatography (HPLC) system as described below.

Inhibition Study. The drugs tested for a possible inhibitory effect on quinine 3-hydroxylation or etoposide 3-demethylation were dissolved in methanol. To identify the respective IC₅₀ (50% inhibition of quinine 3-hydroxylation or etoposide 3-demethylation compared with the respective control values), various test drug concentrations, ranging from 0.001 to 100 µM, were chosen. Quinine and etoposide concentrations were set at 100 µM and 50 µM, respectively, according to the respective apparent K_m values determined previously by Zhao et al. (1996) and Kawashiro et al. (unpublished data) in our laboratory. TAO was preincubated with microsomes and the NADPH-generating system for 15 min at 37^oC (for determining IC₅₀ values) before the reaction was started by addition of the substrate quinine or etoposide. Fifty milliliters of each drug dissolved in methanol was evaporated to dryness before the addition of the other reaction constituents. Three to four different microsomal samples were used in the experiments, and the assays were carried out in duplicate. In all cases the inhibited activities were compared with those from the respective control incubations. The IC₅₀ values were calculated from plots of percentage activity remaining vs inhibitor concentration using a regression method by a MULTI software (Yamaoka et al., 1981).

Correlation Study. Different concentrations of quinine, ranging from 25-400 µM, and those of etoposide, ranging from 5-125 µM, were used in the experiments for assessing the respective apparent kinetic parameters for quinine 3-hydroxylation and etoposide 3-demethylation in six different human liver microsomes. The apparent kinetic parameters (i.e. apparent K_m and V_max) were estimated according to the Eadie-Hofstee equation by use of the nonlinear least-squares regression analysis program, MULTI (Yamaoka et al., 1981). Then, the apparent intrinsic clearance for the two substrates was estimated as the V_max/K_m. Correlation between the quinine 3-hydroxylase and etoposide 3-demethylase activities was assessed from the apparent V_max/K_m values as described above.

Statistical Analysis. All values are expressed as means ± SD. Correlation between the quinine 3-hydroxylation and etoposide 3-demethylation activities was determined by the least-squares linear regression analysis. A p of < 0.05 was considered statistically significant.

	Results and Discussion

Top Abstract Introduction Materials & Methods Results & Discussion References

Chromatograms and Assessment of Incubation Conditions. With the described chromatographic conditions, no interfering peaks for 3-hydroxyquinine, 3-demethyletoposide, or the internal standard were present in the incubation mixture. The formation of both 3-hydroxyquinine from quinine and 3-demethyletoposide from etoposide were time-, NADPH- and microsome-dependent, suggesting the possible involvement of P450(s) in their metabolism. P450 involvement was later confirmed by the inhibition study using specific inhibitors/substrates of CYPs as well as by recombinant studies for quinine (Zhao et al., 1996) and etoposide (Kawashiro et al., unpublished data) metabolism. Preliminary studies revealed that the 3-hydroxylation of quinine with human liver microsomes was linear with regard to the incubation time from 5 to 60 min when 100 µM (around K_m value in human liver microsomes) of quinine was incubated with microsomes equivalent to 0.1 mg of protein/ml. A linear relationship was also observed between the rate of the metabolite production for up to 15 min and protein concentration for up to 0.25 mg/ml. On the other hand, the formation rate of 3-demethyletoposide was linear at 37^oC for up to 15 min when 50 µM (around K_m value in human liver microsomes) of etoposide and 0.1 mg/ml microsomal protein were present. A linear relationship was also observed between the rate of metabolite production at 15 min and protein concentration for up to 0.2 mg/ml. Accordingly, the subsequent inhibition studies were performed with a 15-min incubation and a microsomal protein content of 0.1 mg/ml for both quinine 3-hydroxylation and etoposide 3-demethylation.

Mutual Inhibition between Quinine and Etoposide. The effect of etoposide on the 3-hydroxylation of quinine is shown in fig. 1. Etoposide inhibited the microsomal metabolism of quinine 3-hydroxylation in a concentration-dependent manner by human liver microsomes with a mean IC₅₀ value of 65 µM (fig. 1). The mean maximum inhibition produced by etoposide (100 µM) on the 3-hydroxylation of quinine was about 60% compared with the control values, indicating that etoposide is a weak inhibitor of quinine 3-hydroxylation in vitro.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of etoposide on quinine 3-hydroxylation in human liver microsomes. The plotted data are the mean ± SD values of experiments performed with microsomes obtained from four different livers.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of quinine on etoposide 3-demethylation in human liver microsomes. The plotted data are the mean ± SD values of experiments performed with microsomes obtained from four different livers.

Correlation Study. Fig. 3 shows an excellent correlation (r = 0.947, p < 0.01) between the quinine 3-hydroxylase and etoposide 3-demethylase activities in six different human liver microsomes, indicating that the respective metabolic pathways of quinine and etoposide were catalyzed mainly by one same human CYP isoform. However, the linear regression line of the formation rate of 3-hydroxyquinine vs 3-demethyletoposide gave a small x-intercept (when y = 0), suggesting the possibility that other minor enzyme(s) may also be involved in the metabolism of both the drugs tested herein in human liver microsomes. This result is in a good agreement with our previous findings that both CYP3A4 (major) and CYP2C19 (minor) are involved in quinine 3-hydroxylation in human liver microsomes (Zhao et al., 1996). In addition, our previous study showed that CYP3A4 is the main, but not the sole, isoenzyme involved in etoposide 3-demethylation. Several other minor CYP isoforms also seem to catalyze this pathway in human liver microsomes (Kawashiro et al., unpublished data).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Correlation between quinine 3-hydroxylase and etoposide 3-demethylase activities in six different human liver microsomes.

Inhibition Study by Ketoconazole and TAO. To confirm further that CYP3A4 is a major isoenzyme involved in quinine 3-hydroxylation and etoposide 3-demethylation, we employed two well-known CYP3A4 inhibitors, ketoconazole and TAO (Newton et al., 1995; Watkins et al., 1985), to perform an inhibition study in human liver microsomes. Although ketoconazole was regarded as a general inhibitor of several CYP subfamilies (e.g., CYP3A4, CYP2C9, and CYP2C19) (Glue and Banfield, 1996), studies have shown its specificity toward CYP3A4 when present at a low concentration (< 2 µM) (Newton et al., 1995; Maurice et al., 1995). Thus, the concentrations of ketoconazole used in this study were < 2 µM (i.e. 0.1 and 1.0 µM). The effects of co-incubation with the inhibitors on quinine 3-hydroxylation and etoposide 3-demethylation are shown in fig. 4. The plots showed that both quinine 3-hydroxylation and etoposide 3-demethylation were markedly inhibited by ketoconazole or TAO. However, the magnitude of inhibition differed between the data derived from the mutual inhibition (a maximal inhibition of 60%) and chemical inhibition experiments presented here (a maximal inhibition of 90%). Although we do not know the exact reason(s) for this observation, the possibility cannot be ignored that their respective binding constants to the protein may differ, and therefore a different extent of the mutual vs chemical inhibition might have been observed. Furthermore, the different inhibitory capacities can also be explained in this case because quinine and etoposide are not solely metabolized by CYP3A4. Indeed, other minor CYP isoforms like CYP2C19 for quinine metabolism (Zhao et al., 1996) and CYP1A2 and 2E1 for etoposide metabolism (Kawashiro et al., unpublished data) are also involved in their major metabolic pathways. Nevertheless, these inhibition results by ketoconazole and TAO provided further strong evidence that CYP3A4 is the main isoform involved in both quinine 3-hydroxylation and etoposide 3-demethylation. This is in accordance with the published results (Zhao et al., 1996; Zhang et al., 1997; Zhao and Ishizaki, in press; Relling et al., 1992).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effects of TAO and ketoconazole on quinine 3-hydroxylation () and etoposide 3-demethylation () in human liver microsomes. Data are the mean ± SD values of experiments performed with microsomes obtained from three to four different livers.