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Research ArticleArticle

Overlapping Substrate Specificities of Cytochrome P450 3A and P-Glycoprotein for a Novel Cysteine Protease Inhibitor

Yuanchao Zhang, Xisheng Guo, Emil T. Lin and Leslie Z. Benet
Drug Metabolism and Disposition April 1998, 26 (4) 360-366;

Abstract

K02 (morpholine-urea-Phe-Hphe-vinylsulfone), a newly developed peptidomimetic, acts as a potent cysteine protease inhibitor, especially of cathepsins B and L (which are associated with cancer progression) and cruzain (a cysteine protease of Trypanosoma cruzi, which is responsible for Chagas’ disease). Here we investigated features of the disposition of K02 using in vitro systems, characterizing the interaction of the drug with human cytochrome P450 (CYP) 3A and P-glycoprotein (P-gp), a mediator of multidrug resistance (MDR) to cancer chemotherapy and a countertransporter in the intestine that limits oral drug bioavailability. P-gp functions as an ATP-dependent drug efflux pump to reduce intracellular cytotoxic concentrations. An HPLC assay was developed to analyze K02 and its metabolites formed in human liver microsomes. Three major primary metabolites were determined by LC/MS/MS to be hydroxylated products of the parent compound. A rabbit anti-CYP3A polyclonal antibody (200 μl antibody/mg microsomal protein) produced 75–94% inhibition of the formation of these three hydroxylated metabolites. Ketoconazole (5 μM), a selective CYP3A inhibitor, produced up to 75% inhibition, whereas other CYP-specific inhibitors,i.e. quinidine (CYP2D6), 7,8-benzoflavone (CYP1A2), and sulfaphenazole (CYP2C9), showed no significant effects. An identical metabolite formation profile for K02 was observed with cDNA-expressed human CYP3A4 (Gentest). These data demonstrate that K02 is a substrate for CYP3A. Formation of 1′-hydroxymidazolam, the primary human midazolam metabolite, was markedly inhibited by K02 viacompetitive processes, which suggests the potential for drug-drug interactions of K02 with other CYP3A substrates. K02 significantly inhibited the photoaffinity labeling of P-gp with azidopine and LU-49888, a photoaffinity analogue of verapamil. Transport studies with [14C]K02, using MDR1-transfected Madin-Darby canine kidney cell monolayers in the Transwell system, demonstrated that the basolateral-to-apical flux of K02 acrossMDR1-transfected Madin-Darby canine kidney cells was markedly greater than the apical-to-basolateral flux (ratio of 63 with 10 μM [14C]K02). This suggests that K02 is also a P-gp substrate. These studies are important for formulating strategies to increase the absorption and/or decrease the elimination of K02 and to optimize its delivery to malignant cells and parasite-infected host cells.

Cancer biology research has demonstrated that proteases are involved in all stages of cancer progression, including growth, angiogenesis, and metastasis (Elliott and Sloane, 1996). A key event necessary for tumor growth and invasion of tumor cells into normal tissues is the removal of extracellular matrix by matrix-degrading proteases, which can be broadly classified into three major groups, i.e. serine proteases, cysteine proteases (e.g. cathepsins B and L), and metalloproteinases (Chen, 1992). Recently, these proteases have become targets for the development of inhibitors, which may serve as potential therapeutic agents (DeClerk and Imren, 1994; Clawson, 1996).

Trypanosoma cruzi, a protozoan parasite, is the etiological agent of American trypanosomiasis or Chagas’ disease. The major proteolytic activity of T. cruzi is a cathepsin L-like cysteine protease, termed cruzain, that is expressed in all life stages of the parasite (Scharfstein et al., 1986; Bontempi et al., 1989; North et al., 1990). Cruzain has been shown to be a promising therapeutic target for antiparasite drug design (McKerrow et al., 1995; McGrath et al., 1995).

K02 (morpholine-urea-Phe-Hphe-vinylsulfone) is one of a class of newly developed peptidomimetics that act as potent cysteine protease inhibitors (Palmer et al., 1995). K02 is an irreversible inhibitor of cysteine proteases, exhibiting nanomolarKi values for cathepsins B and L and cruzain in vitro.1

CYP3A2 is the primary CYP subfamily responsible for phase I metabolism of >50% of drugs administered to humans (Benet et al., 1996a). CYP3A4 is the most prominent CYP in humans, comprising about 30 and 70% of total CYP in liver and intestine, respectively. CYP3A has very broad substrate specificity, encompassing a wide variety of therapeutic agents (Wacheret al., 1995). Many cancer chemotherapeutic drugs, such as epipodophyllotoxins and Vinca alkaloids, are substrates for CYP3A (Relling et al., 1994; Zhou et al., 1993;Zhou-Pan et al., 1993). Human immunodeficiency virus protease inhibitors all appear to be substrates for CYP3A (Prueksaritanont et al., 1994; Kumar et al., 1996; Chiba et al., 1996). Recently, CYP3A has also been found to be expressed in some human neoplasms (Fritz et al., 1993; Kivisto et al., 1995; Murray et al., 1994,1995a,b), which indicates that CYP3A may deactivate cancer drugs within the tumor cells if the drugs are substrates for CYP3A (Wacher et al., 1995). The contribution of intestinal CYP3A-mediated metabolism to poor oral drug bioavailability has been shown to be clinically important for several drugs, such as cyclosporine, tacrolimus, and midazolam (Hebert et al., 1992; Wu et al., 1995; Thummel et al., 1996).

A major impediment to effective cancer chemotherapy is MDR, a process by which tumor cells rid themselves of anticancer drugs (Patel and Rothenberg, 1994). One important mechanism for MDR is known to involve the overexpression of P-gp in tumor cells in response to drug exposure (Endicott and Ling, 1989; Ling, 1995). P-gp is a member of a superfamily of highly conserved ATP-binding cassette transport proteins (Gottesman and Pastan, 1993). P-gp functions as an ATP-dependent drug efflux pump to reduce intracellular cytotoxic concentrations. In humans, there are two MDR genes, MDR1 and MDR2, with MDR1 conferring drug resistance. MDR1 is expressed on the apical surfaces of many epithelial cells in a number of tissues; for example, it is expressed at high levels in columnar epithelial cells of the jejunum (Thiebaut et al., 1987). Although its exact physiological roles are still under investigation, P-gp is generally considered to play an important detoxification rolevia countertransport. Several pharmacokinetic studies using P-gp-knockout mice demonstrated the importance of P-gp in limiting oral drug bioavailability when drugs are P-gp substrates (Schinkel et al., 1995, 1997; Sparreboom et al., 1997; van Asperenet al., 1996).

A striking overlap of the substrates for CYP3A and P-gp, including a wide variety of hydrophobic therapeutic agents (and many anticancer drugs), and their tissue distributions has been observed (Wacheret al., 1995). These observations led us to suggest that CYP3A and P-gp might play complementary roles in drug disposition by biotransformation (phase I) and antitransport (phase III), especially in the villi of the small intestine, where CYP3A and P-gp act synergistically as an oral drug bioavailability barrier. Several pharmacokinetic studies using CYP3A and/or P-gp inhibitors illustrated the importance of CYP3A and P-gp in drug absorption and disposition (Floren et al., 1997; Gomez et al., 1995; van Asperen et al., 1997). For the present work, we studied the interaction of K02, a promising prototypical cysteine protease inhibitor, with CYP3A and P-gp systems in vitro.

Materials and Methods

Chemicals and Biochemicals.

K02 and [14C]K02 (27.6 Ci/mol) were kindly supplied by Arris Pharmaceutical Corp. (South San Francisco, CA); LU-49888 (28 Ci/mmol), a 3H-labeled photoaffinity analogue of verapamil, was provided by Dr. Xiaodong Qian of BASF (Cambridge, MA). Midazolam was a gift from Hoffmann-LaRoche (Nutley, NJ). Ketoconazole was purchased from United States Pharmacopeial Convention (Rockville, MD). 7,8-Benzoflavone, β-NADPH, quinidine, and sulfaphenazole were obtained from Sigma Chemical Co. (St. Louis, MO). 4-Dimethylamino-4′-(imidazol-1-yl)chalcone was kindly provided by Dr. George L. Kenyon, School of Pharmacy, University of California, San Francisco. Other chemicals were of reagent grade and were also purchased from Sigma. All solvents were of HPLC grade and were obtained from Fisher Scientific (Santa Clara, CA). The Bio-Rad protein assay kit with albumin protein standard was obtained from Bio-Rad (Richmond, CA). The rabbit anti-human CYP3A polyclonal antibody and preimmune IgG were kindly provided by Dr. Steven A. Wrighton, Eli Lilly and Co. (Indianapolis, IN). cDNA-expressed human CYP3A4 (M107r) was purchased from Gentest (Cambridge, MA). Azidopine (44 Ci/mmol) was obtained from Amersham (Arlington Heights, IL).

Human Liver Microsome Preparation.

A human liver sample was obtained under a protocol approved by the Committee on Human Research, University of California, San Francisco. Microsomes were prepared by differential centrifugation as previously described (Bornheim and Correia, 1989). The CYP contents in microsomal suspensions were measured using carbon monoxide difference spectra (Omura and Sato, 1964), and protein concentrations were determined by the method of Lowry et al. (1951).

Incubation Procedure for K02 Metabolism by Human Liver Microsomes and cDNA-Expressed CYP3A4. General Incubation Procedure.

K02 metabolism in human liver microsomes was found to be linear during 20-min incubations with microsomal protein concentrations of 1–2 mg/ml. Incubation mixtures (final volume, 0.5 ml) contained human liver microsomes (1.5 mg protein/ml) or microsomes prepared from a human B-lymphoblastoid cell line (engineered to stably express human CYP3A4 cDNA) (4 mg/ml), in 0.1 M phosphate buffer, plus K02 (10–200 μM) in methanol (≤1%, v/v). After a 5-min preincubation at 37°C, the reaction was initiated by addition of NADPH (1 mM), and the mixture was incubated at 37°C for 10 min. The reaction was stopped by addition of 0.5 ml of ice-cold precipitation reagent (62.5% methanol/37.5% acetonitrile). The internal standard, i.e.4-dimethylamino-4′-(imidazol-1-yl)chalcone in methanol, was added. The resultant mixture was vortex-mixed and centrifuged at 13,400g for 12 min, to precipitate protein. An aliquot of the supernatant (100 μl) was subjected to HPLC analysis.

Chemical Inhibition Study.

The reversible inhibitors for CYPs used in this study, i.e.ketoconazole (CYP3A), 7,8-benzoflavone (CYP1A2), quinidine (CYP2D6), and sulfaphenazole (CYP2C9), were added to the incubation mixture at the same time as K02 (50 μM). The incubation protocol was the same as that described above (see General Incubation Procedure).

Immunoinhibition Study.

A designated amount of rabbit anti-CYP3A polyclonal antibody or preimmune rabbit IgG was incubated with microsomes for 30 min at room temperature before the K02 incubation, as described above.

HPLC Assay for K02 and Metabolites.

A Shimadzu LC-600 HPLC system and a Waters 710B WISP autosampler were used with a Shimadzu SPD-10A UV spectrophotometric detector operated at 220 nm. A Du Pont Zorbax SB-C18 column (3.0 × 250 mm; MAC-MOD Analytical, Inc., Chadds Ford, PA) was used with a precolumn 0.2-mm frit. The isocratic mobile phase of 10 mM potassium phosphate, pH 6.0/methanol/acetonitrile (44:35:21, v/v/v) was delivered at 0.5 ml/min. Under these conditions, K02 and the internal standard eluted at 29 and 35 min, respectively. All K02 metabolites eluted between 8 and 24 min. Chromatographic data were collected with a Hewlett Packard 3392A integrator.

Inhibition of Midazolam Oxidation by K02 in Human Liver Microsomes.

The procedure for incubation of midazolam, in the presence or absence of K02, with human liver microsomes was the same as described above. HPLC analysis of midazolam, 4-hydroxymidazolam, and 1′-hydroxymidazolam was carried out following the method of Wrighton and Ring (1994).

MS Analysis.

Mass spectra and tandem mass spectra of K02 and its metabolites were acquired using a PE SCIEX API III triple-quadrupole mass spectrometer (Perkin-Elmer/ABI, Foster City, CA) equipped with a heated Nebulizer inlet (Thornhili, Ontario, Canada). Positive ionization was achieved by atmospheric-pressure chemical ionization. Scans were acquired over the range of m/z 200–700 for MS and m/z 50–600 for MS/MS. Collisional activations were accomplished for MS/MS using 90% argon/10% nitrogen as the collision gas, at an energy of 25 eV.

Photoaffinity Labeling of P-gp and Autoradiography.

Membrane samples containing P-gp (MDR1) prepared from CEM5k cell cultures were kindly supplied by Dr. Xiaodong Wang of BASF (Cambridge, MA). CEM5k is a derived drug-resistant cell line of human leukemic lymphoblasts selected using vinblastine (Beck et al., 1986). Membrane protein (200 μg) was preincubated with 100 nM [3H]LU-49888 (about 2 μCi) (Qian and Beck, 1990) or 50 nM azidopine (2 μCi), in the presence or absence of K02, in a buffer containing 250 mM sucrose and 10 mM Tris-HCl, pH 7.4, at room temperature for 30 min and then was irradiated with UV light (254 nm), at a distance of 10 cm, for 10 min at 4°C. Photolabeled proteins were diluted (1:1) with Laemmli sample buffer and separated by one-dimensional 4–15% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad). After staining with Coomassie blue and destaining, the gels were soaked in Amplify solution (Amersham, Arlington Heights, IL) for 30 min and dried under vacuum at 75°C. The dried gels were exposed to Hyperfilm-ECL (Amersham) for 4–5 days at −80°C and developed. Densitometric readings of autoradiographs were obtained using a Pharmacia LKB UltroScan XL densitometer (Pharmacia LKB, Alameda, CA).

Cell Culture and Transepithelial Transport in the Transwell System. Cell Culture.

The MDR1-MDCK cell line was kindly provided by Dr. Ira Pastan, National Institutes of Health (Pastan et al., 1988). MDR1-MDCK cells were maintained in culture in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 80 ng/ml colchicine. For transport experiments, MDR1-MDCK cells were grown as epithelial layers by seeding onto permeable filter matrices of Transwell inserts (4.7-cm2 growth area; Corning Costar Corp., Cambridge, MA), in six-well cluster plates, for 4–5 days. The integrity of the cell monolayers was assessed by transepithelial electrical resistance measurements with a Millipore Millicell-ERS resistance system (Millipore Corp., Bedford, MA).

Measurement of Bidirectional Transepithelial Fluxes of [14C]K02.

The experimental procedure was as described by Hunter et al.(1993). First, 2.5-ml and 1.5-ml aliquots of serum-free medium were pipetted into insert cups (apical solution) and six-well plates (basolateral solution), respectively. The medium on either the apical or basolateral side of the monolayers contained 10 μM [14C]K02. The monolayers were then incubated at 37°C for up to 3 hr. Fifty microliters of solution were taken from the accumulation side every 30 min and subjected to liquid scintillation counting (Beckman LS1801 scintillation counter; Beckman Instruments, Palo Alto, CA).

Results

Biotransformation of K02 in Human Liver Microsomes.

Fig. 1A illustrates a typical HPLC metabolite profile for K02 in a human liver microsomal incubation. Three major metabolites, i.e. M12, M19, and M20 (numbered with reference to retention times), were chosen for further study to identify the major CYPs involved in biotransformation. The formation of all metabolites was NADPH dependent, and the formation of these three metabolites appeared to follow Michaelis-Menten-type kinetics. The corresponding KM andVmax values are summarized in table1.

Figure 1
Figure 1

Typical HPLC chromatographs of K02 and its metabolites formed using in vitro incubation systems.

A, Human liver microsomes; B, cDNA-expressed human CYP3A4 (Gentest). The K02 concentration was 100 μM in both cases.

View this table:
Table 1

Kinetic parameters for primary metabolites (M12, M19, and M20) of K02 in incubations with human liver microsomes and cDNA-expressed CYP3A4

Determination of Metabolite Structure by Tandem MS.

M12, M19, and M20 were determined by LC/MS to be single-hydroxylated products of the parent compound K02. Analysis of tandem mass spectra of these three major primary metabolites revealed the possible hydroxylation sites. The deduced structures of M12, M19, and M20 and the corresponding MS/MS spectra, together with that for K02, are illustrated in fig. 2. Other K02 metabolites shown in fig. 1A were determined to be isomers of M12, M19, and M20, in terms of the corresponding hydroxylation regions, and to be sequentially hydroxylated products (data not shown).

Figure 2
Figure 2

Tandem mass spectra of K02 and its three major primary hydroxylated metabolites, with corresponding deduced structures.

A, K02; B, M12; C, M19;D, M20.

CYP3A4, a Primary Enzyme Responsible for K02 Biotransformation in Human Liver Microsomes. Chemical Inhibition Study.

Ketoconazole, a reversible inhibitor of CYP3A4, strongly inhibited the formation of M12, M19, and M20, with IC50 values in the range of 1–4 μM (fig.3A). Quinidine, 7,8-benzoflavone, and sulfaphenazole, selective chemical inhibitors of CYP2D6, CYP1A2, and CYP2C9, respectively, at their recommended inhibitor concentrations (Halpert et al., 1994) had no significant effects on the formation of M12, M19, and M20 (fig.4).

Figure 3
Figure 3

Effects of ketoconazole and rabbit anti-human CYP3A polyclonal antibody on the formation in human liver microsomes of the three major primary metabolites of K02.

A, Ketoconazole; B, rabbit anti-human CYP3A polyclonal antibody. Values are mean ± SD from duplicate incubations.

Figure 4
Figure 4

Effects of various selective inhibitors of CYPs on the formation in human liver microsomes of the three major primary metabolites of K02.

Values are mean ± SD of duplicate incubations.

Immunoinhibition Study.

Rabbit anti-CYP3A polyclonal antibody (Gorski et al., 1994), which is known to cross-react with human CYP3A4, exhibited concentration-dependent inhibitory effects on M12, M19, and M20 formation. At a concentration of 200 μl/mg microsomal protein, this antibody showed 75–94% inhibitory effects on M12, M19, and M20 formation (fig. 3B).

Metabolism by cDNA-Expressed CYP3A4.

The HPLC metabolite profile of K02 in a cDNA-expressed human CYP3A4 system (with CYP reductase coexpression) is shown in fig.1B. Identical metabolite profiles were observed in HPLC chromatographs obtained for K02 incubations with human liver microsomes and cDNA-expressed human CYP3A4 (fig. 1). The formation of M12, M19, and M20 appeared to follow Michaelis-Menten kinetics with the cDNA-expressed enzyme. The correspondingKM and Vmaxvalues are summarized in table 1.

Inhibition of 1′-Hydroxymidazolam Formation by K02 in Human Liver Microsomes.

The Lineweaver-Burk plot shown in fig. 5demonstrates that K02 competitively inhibited formation of 1′-hydroxymidazolam, a major metabolite formed by CYP3A4 in humans. The concentration range of midazolam was 12.5–150 μM. TheKM for 1′-hydroxymidazolam was determined to be 11.9 ± 0.6 μM and the Ki for K02 was found to be 12.1 ± 0.2 μM by nonlinear regression analysis using PCNONLIN version 4.0 (Statistical Consultants Inc., Lexington, KY), based on a simple competitive-inhibition Michaelis-Menten model.

Figure 5
Figure 5

Lineweaver-Burk plot showing that K02 competitively inhibits 1′-hydroxymidazolam formation in human liver microsomes.

Midazolam concentrations were in the range of 12.5–100 μM. □, 0 μM K02; ⋄, 10 μM K02; ○, 20 μM K02. Solid lines, fit of the data by nonlinear regression analysis using PCNONLIN version 4.0 (Statistical Consultants), based on a simple competitive-inhibition Michaelis-Menten model. The corresponding kinetic parameters were calculated to beVmax = 2.6 ± 0.1 nmol/min/mg protein,KM = 11.9 ± 0.6 μM, andKi = 12.1 ± 0.2 μM. Values are means of duplicate incubations. Kinetic parameters are mean ± SD values from corresponding nonlinear regression-fitted values.

Inhibition by K02 of Photoaffinity Labeling of P-gp.

K02 significantly inhibited the photoaffinity labeling of P-gp with azidopine and LU-49888, a photoaffinity analogue of verapamil, as depicted in the autoradiograph shown in fig.6. Densitometric readings for labeled P-gp bands in the autoradiographs, in the absence and presence of K02 (10 μM), yielded 80 and 74% (fig. 6, lanes 2 and6) inhibition of LU-49888 and azidopine labeling, respectively, compared with controls (0 μM K02) (fig. 6, lanes 1 and 5).

Figure 6
Figure 6

Effects of K02 on photoaffinity labeling of P-gp by azidopine and LU-49888.

Lanes 1, 2, 3, and4, LU-49888; lanes 5, 6,7, and 8, azidopine. Lanes 1 and 5, 0 μM K02; lanes 2 and6, 10 μM K02; lanes 3 and7, 50 μM K02; lanes 4 and8, 100 μM K02.

Bidirectional Transepithelial Transport Studies Using MDR1-MDCK Cell Monolayers in the Transwell System.

As depicted in fig. 7, the basolateral-to-apical flux of 10 μM [14C]K02 across MDR1-MDCK cell monolayers was markedly greater than the apical-to-basolateral flux (ratio, 63). This specific basolateral-to-apical transport was significantly inhibited by vinblastine, an avid P-gp substrate (48% inhibition at 25 μM).

Figure 7
Figure 7

Bidirectional transepithelial transport of K02 across MDR1-MDCK cell monolayers.

The [14C]K02 concentration was 10 μM. Solid lines, in the absence of vinblastine; □, basolateral to apical; ⋄, apical to basolateral. Dashed lines, in the presence of 25 μM vinblastine in the basolateral solution; ○, basolateral to apical; ▵, apical to basolateral. Values are mean ± SD of triplicate measurements.

Discussion

K02 is the first member of a newly developed class of vinylsulfone peptidomimetics, which act as potent mechanism-based cysteine protease inhibitors, to be subjected to biotransformation studies using human liver microsomal preparations and transport studies using MDR1-MDCK cell monolayers in the Transwell system. K02 and other compounds in its class act as potent inhibitors of cathepsins B and L (two cancer-associated cysteine proteases) and cruzain (the major cysteine protease present in T. cruzi). The study described here demonstrates that K02 is a CYP3A substrate and CYP3A plays a primary role in the oxidative biotransformation of K02 in human liver microsomal preparations. These conclusions are based on the results of an integrated in vitro approach using 1) selective CYP chemical inhibitors, 2) immunoinhibition with an anti-CYP3A antibody, and 3) cDNA-expressed human CYP3A4. Both ketoconazole, a CYP3A-selective inhibitor, and rabbit anti-CYP3A antibody demonstrated potent and concentration-dependent inhibitory effects on the formation of three major primary hydroxylated metabolites of K02, designated M12, M19, and M20. The IC50 values for ketoconazole were in the range of 1–4 μM, which are typical values for this potent reversible CYP3A inhibitor. Representative chemical inhibitors of other major CYP enzymes responsible for human drug metabolism,i.e. quinidine (CYP2D6), 7,8-benzoflavone (CYP2C9), and sulfaphenazole (CYP1A2), at their recommended concentrations showed no significant effects on K02 metabolite formation. Incubation of K02 with cDNA-expressed human CYP3A4 produced a metabolite profile almost identical to that obtained with human liver microsomes. However, theKM and Vmaxvalues obtained in these two incubation systems were not exactly comparable, with differences of up to 4-fold. We speculate that the CYP conformations and/or the interactions of NADPH-CYP reductase with CYP are not exactly the same in these two systems, because of differing conditions of the microsomal membranes. K02 also competitively inhibited the formation of 1′-hydroxymidazolam, which is the major midazolam metabolite in humans and is produced by CYP3A4 (Thummelet al., 1994a,b). This suggests the potential for drug-drug interactions of K02 with other CYP3A substrates when the drugs are administered concomitantly.

K02 strongly inhibited the photoaffinity labeling of P-gp with azidopine and LU-49888, a photoaffinity analogue of verapamil. Verapamil is a documented inhibitor and substrate of P-gp and has been clinically coadministered with many anticancer drugs as a P-gp inhibitor, to overcome or alleviate MDR in cancer chemotherapy (Futscher et al., 1996). Bidirectional transepithelial transport studies of K02 with MDR1-MDCK cell monolayers in the Transwell system clearly demonstrated that K02 is a substrate of P-gp.

These data are consistent with our hypothesis that there is substrate specificity overlap between CYP3A and P-gp for hydrophobic protease inhibitors used as therapeutic agents. K02 is the first example to be examined using in vitro systems. The substrate specificity overlap between CYP3A and P-gp for K02 provides valuable information for early drug discovery and development among this class of vinylsulfone peptidomimetics, including formulation of strategies to increase the absorption and decrease the elimination of K02 by coadministration of CYP3A and/or P-gp inhibitors with K02 (Benetet al., 1996b). Our K02 pharmacokinetic studies conducted in male Sprague-Dawley rats demonstrate that K02 is a compound with poor oral bioavailability and that coadministration of K02 with ketoconazole, a dual inhibitor of CYP3A and P-gp, can significantly increase K02 oral bioavailability (Zhang Y, Hsieh Y, Isumi T, Lin ET and Benet LZ. Effects on ketoconazole on the intestinal metabolism, transport, and oral bioavailability of KO2, a novel vinylsulfone peptidomimetic cysteine protease inhibitor and a P4503A, P-glycoprotein dual substrate, in male Sprague Dawley rats. Submitted for publication in J Pharmacol Exp Ther). The studies described here may serve as a template for in vitro characterization of interactions of new investigational drugs with CYP3A and P-gp.

Footnotes

  • Send reprint requests to: Dr. Leslie Z. Benet, Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, 513 Parnassus Avenue, San Francisco, CA 94143-0446.

  • This study was supported in part by National Institutes of Health Grants GM26691 and CA72006. This work was presented in part at the 10th Annual Meeting of the American Association of Pharmaceutical Scientists (Seattle, WA, October 27–31, 1996).

  • ↵1 McKerrow JH (Department of Pathology, Veterans Affairs Medical Center, San Francisco, CA), personal communication.

  • Abbreviations used are::
    CYP
    cytochrome P450
    P-gp
    P-glycoprotein
    MDR
    multidrug resistance
    MDCK
    Madin-Darby canine kidney
    • Received May 12, 1997.
    • Accepted December 18, 1997.

References

  1. ↵
    1. Beck WT,
    2. Cirtain MC,
    3. Look AT,
    4. Ashmun RA
    (1986) Reversal of Vinca alkaloid resistance but not multiple drug resistance in human leukemic cells by verapamil. Cancer Res 46:778–784.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Hardman JH,
    2. Limbird LE,
    3. Molinoff PB,
    4. Ruddon RW,
    5. Gilman AG
    1. Benet LZ,
    2. Kroetz DL,
    3. Sheiner LB
    (1996a) Pharmacokinetics: The dynamics of drug absorption, distribution, and elimination. in Goodman & Gilman’s The Pharmacological Basis of Therapeutics, eds Hardman JH, Limbird LE, Molinoff PB, Ruddon RW, Gilman AG (McGraw-Hill, New York), 9th Ed. pp 3–28.
  3. ↵
    1. Benet LZ,
    2. Wu CY,
    3. Hebert MF,
    4. Wacher VJ
    (1996b) Intestinal drug metabolism and antitransport processes: A potential paradigm shift in oral drug delivery. J Controlled Release 39:139–143.
  4. ↵
    1. Bontempi E,
    2. Martinez J,
    3. Cazzulo JJ
    (1989) Subcellular localization of a cysteine proteinase from Trypanosoma cruzi. Mol Biochem Parasitol 33:43–48.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Bornheim LM,
    2. Correia MA
    (1989) Effect of cannabidiol on cytochrome P-450 isozymes. Biochem Pharmacol 38:2789–2794.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Chen WT
    (1992) Membrane proteases: Roles in tissue remodeling and tumour invasion. Curr Opin Cell Biol 4:802–809.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Chiba M,
    2. Hensleigh M,
    3. Nishime JA,
    4. Balani SK,
    5. Lin JH
    (1996) Role of cytochrome P450 3A in human metabolism of MK-639, a potent human immunodeficiency virus protease inhibitor. Drug Metab Dispos 24:307–314.
    OpenUrlAbstract
  8. ↵
    1. Clawson GA
    (1996) Protease inhibitors and carcinogenesis: A review. Cancer Invest 14:597–608.
    OpenUrlPubMed
  9. ↵
    1. DeClerk YA,
    2. Imren S
    (1994) Protease inhibitors: Role and potential therapeutic use in human cancer. Eur J Cancer 30A:2170–2180.
    OpenUrlCrossRef
  10. ↵
    1. Elliott E,
    2. Sloane BF
    (1996) The cysteine protease cathepsin B in cancer. Perspect Drug Discovery Design 6:12–32.
  11. ↵
    1. Endicott JA,
    2. Ling V
    (1989) The biochemistry of P-glycoprotein mediated multidrug resistance. Annu Rev Biochem 58:137–171.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Floren LC,
    2. Bekersky I,
    3. Benet LZ,
    4. Mekki Q,
    5. Dressler D,
    6. Lee JW,
    7. Roberts JP,
    8. Hebert MF
    (1997) Tacrolimus oral bioavailability doubles with coadministration of ketoconazole. Clin Pharmacol Ther 62:41–49.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Fritz P,
    2. Behrle E,
    3. Beaune P,
    4. Eichelbaum M,
    5. Kroemer HK
    (1993) Differential expression of drug metabolizing enzymes in primary and secondary liver neoplasm: Immunohistochemical characterization of cytochrome P450 3A and glutathione-S- transferase. Histochemistry 99:443–451.
    OpenUrlPubMed
  14. ↵
    1. Futscher BW,
    2. Foley NE,
    3. Gleason-Guzman MC,
    4. Meltzer PS,
    5. Sullivan DM,
    6. Dalton WS
    (1996) Verapamil suppresses the emergence of P-glycoprotein-mediated multi-drug resistance. Int J Cancer 66:520–525.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Gomez DY,
    2. Wacher VJ,
    3. Tomlanovich SJ,
    4. Hebert MF,
    5. Benet LZ
    (1995) The effects of ketoconazole on the intestinal metabolism and bioavailability of cyclosporine. Clin Pharmacol Ther 58:15–19.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Gorski JC,
    2. Jones DR,
    3. Wrighton SA,
    4. Hall SD
    (1994) Characterization of dextromethorphan N-demethylation by human liver microsomes: Contribution of the cytochrome P450 3A (CYP3A) subfamily. Biochem Pharmacol 48:173–182.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Gottesman MM,
    2. Pastan I
    (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 62:385–427.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Halpert JR,
    2. Guengerich FP,
    3. Bend JR,
    4. Correia MA
    (1994) Selective inhibitors of cytochromes P450. Toxicol Appl Pharmacol 125:163–175.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Hebert MF,
    2. Roberts JP,
    3. Prueksaritanont T,
    4. Benet LZ
    (1992) Bioavailability of cyclosporine with concomitant rifampin administration is markedly less than predicted by hepatic enzyme induction. Clin Pharmacol Ther 52:453–457.
    OpenUrlPubMed
  20. ↵
    1. Hunter J,
    2. Jepson MA,
    3. Tsuruo T,
    4. Simmons NL,
    5. Hirst BH
    (1993) Functional expression of P-glycoprotein in apical membrane of human intestinal Caco-2 cells. J Biol Chem 268:14991–14997.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Kivisto KT,
    2. Fritz P,
    3. Linder A,
    4. Friedel G,
    5. Beaune P,
    6. Kroemer HK
    (1995) Immunohistochemical localization of cytochrome P450 3A in human pulmonary carcinomas and normal bronchial tissue. Histochem Cell Biol 103:25–29.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Kumar GN,
    2. Rodrigues AD,
    3. Buko AM,
    4. Denissen JF
    (1996) Cytochrome P450-mediated metabolism of the HIV-1 protease inhibitor ritonavir (ABT-538) in human liver microsomes. J Pharmacol Exp Ther 277:423–431.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Ling V
    (1995) P-glycoprotein: Its role in drug resistance. Am J Med 99:31S–34S.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Lowry OH,
    2. Rosebrough NJ,
    3. Farr AL,
    4. Randall RJ
    (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275.
    OpenUrlFREE Full Text
  25. ↵
    1. McGrath ME,
    2. Eakin AE,
    3. Engel JC,
    4. McKerrow JH,
    5. Craik CS,
    6. Fletterick RJ
    (1995) The crystal structure of cruzain: A therapeutic target for Chagas’ disease. J Mol Biol 247:251–259.
    OpenUrlCrossRefPubMed
  26. ↵
    1. McKerrow JH,
    2. McGrath ME,
    3. Engel JC
    (1995) The cysteine protease of Trypanosoma cruzi as a model for antiparasite drug design. Parasitol Today 11:279–282.
  27. ↵
    1. Murray GI,
    2. Shaw D,
    3. Weaver RJ,
    4. McKay JA,
    5. Ewen SW,
    6. Melvin WT,
    7. Burke MD
    (1994) Cytochrome P450 expression in oesophageal cancer. Gut 35:599–603.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Murray GI,
    2. Taylor VE,
    3. McKay JA,
    4. Weaver RJ,
    5. Ewen SW,
    6. Melvin WT,
    7. Burke MD
    (1995a) The immunohistochemical localization of drug-metabolizing enzymes in prostate cancer. J Pathol 177:147–152.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Murray GI,
    2. Taylor VE,
    3. McKay JA,
    4. Weaver RJ,
    5. Ewen SW,
    6. Melvin WT,
    7. Burke MD
    (1995b) Expression of xenobiotic metabolizing enzymes in tumours of the urinary bladder. Int J Exp Pathol 76:271–276.
    OpenUrlPubMed
  30. ↵
    1. North M, Jr,
    2. Mottram JC,
    3. Coombs CH
    (1990) Cysteine proteinases of parasitic protozoa. Parasitol Today 6:270–275.
  31. ↵
    1. Omura T,
    2. Sato R
    (1964) The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties. J Biol Chem 239:2370–2378.
    OpenUrlFREE Full Text
  32. ↵
    1. Palmer JT,
    2. Rasnick D,
    3. Klaus JL,
    4. Bromme D
    (1995) Vinyl sulfones as mechanism-based cysteine protease inhibitors. J Med Chem 38:3193–3196.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Pastan I,
    2. Gottesman MM,
    3. Ueda K,
    4. Lovelace E,
    5. Rutherford AV,
    6. Willingham MC
    (1988) A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells. Proc Natl Acad Sci USA 85:4486–4490.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Patel NH,
    2. Rothenberg ML
    (1994) Multidrug resistance in cancer chemotherapy. Invest New Drugs 12:1–13.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Prueksaritanont T,
    2. Dwyer LM,
    3. Balani SK,
    4. Theoharides AD
    (1994) In vitro metabolism of L-696,229, an HIV-1 reverse transcriptase inhibitor in rats and humans: Hepatic and extrahepatic metabolism and identification of enzymes involved in the hepatic metabolism. Drug Metab Dispos 22:281–288.
    OpenUrlAbstract
  36. ↵
    1. Qian XD,
    2. Beck WT
    (1990) Binding of an optically pure photoaffinity analogue of verapamil, LU-49888, to P-glycoprotein from multidrug-resistance human leukemic cell lines. Cancer Res 50:1132–1137.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Relling MV,
    2. Nemec J,
    3. Schuetz EG,
    4. Schuetz JD,
    5. Gonzalez FJ,
    6. Korzekwa KR
    (1994) O-Demethylation of epipodophyllotoxins is catalyzed by human cytochrome P450 3A4. Mol Pharmacol 45:352–358.
    OpenUrlAbstract
  38. ↵
    1. Scharfstein J,
    2. Schechter M,
    3. Senna M,
    4. Peralta JM,
    5. Mendonca-Previato L,
    6. Miles MA
    (1986) Trypanosoma cruzi: Characterization and isolation of a 57/51,000 m.w. surface glycoprotein (GP57/51) expressed by epimastigotes and bloodstream trypomastigotes. J Immunol 137:1336–1341.
    OpenUrlAbstract
  39. ↵
    1. Schinkel AH,
    2. Mayer U,
    3. Wagenaar E,
    4. Mol CA,
    5. van Deemter L,
    6. Smit JJ,
    7. van der Valk MA,
    8. Voordouw AC,
    9. Spits H,
    10. van Tellingen O,
    11. Zijlmans JM,
    12. Fibbe WE,
    13. Borst P
    (1997) Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc Natl Acad Sci USA 94:4028–4033.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Schinkel AH,
    2. Wagenaar E,
    3. van Deemter L,
    4. Mol VA,
    5. Borst P
    (1995) Absence of the mdr1a P-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J Clin Invest 96:1698–1705.
  41. ↵
    1. Sparreboom A,
    2. van Asperen J,
    3. Mayer U,
    4. Schinkel AH,
    5. Smit JW,
    6. Meijer DK,
    7. Borst P,
    8. Nooijen WJ,
    9. Beijnen JH,
    10. van Tellingen O
    (1997) Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine. Proc Natl Acad Sci USA 94:2031–2035.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Thiebaut F,
    2. Tsuruo T,
    3. Hamada H,
    4. Gottesman MM,
    5. Pastan I,
    6. Willingham MC
    (1987) Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA 84:7735–7738.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Thummel KE,
    2. O’Shea D,
    3. Paine MF,
    4. Shen DD,
    5. Kunze KL,
    6. Perkins JD,
    7. Wilkinson GR
    (1996) Oral first-pass elimination of midazolam involves both gastrointestinal and hepatic CYP3A-mediated metabolism. Clin Pharmacol Ther 59:491–502.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Thummel KE,
    2. Shen DD,
    3. Podoll TD,
    4. Kunze KL,
    5. Trager WF,
    6. Bacchi CE,
    7. Marsh CL,
    8. McVicar JP,
    9. Barr DM,
    10. Perkins JD,
    11. Carithers RL
    (1994a) Use of midazolam as a human cytochrome P450 3A probe. II. Characterization of inter- and intraindividual hepatic CYP3A variability after liver transplantation. J Pharmacol Exp Ther 271:557–566.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Thummel KE,
    2. Shen DD,
    3. Podoll TD,
    4. Kunze KL,
    5. Trager WF,
    6. Hartwell PS,
    7. Raisys VA,
    8. Marsh CL,
    9. McVicar JP,
    10. Barr DM,
    11. Perkins JD,
    12. Carithers RL
    (1994b) Use of midazolam as a human cytochrome P450 3A probe. I. In vitro-in vivo correlations in liver transplant patients. J Pharmacol Exp Ther 271:549–556.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. van Asperen J,
    2. Schinkel AH,
    3. Beijnen JH,
    4. Nooijen WJ,
    5. Borst P,
    6. van Tellingen O
    (1996) Altered pharmacokinetics of vinblastine in mdr1a P-glycoprotein-deficient mice. J Natl Cancer Inst 88:994–999.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. van Asperen J,
    2. van Tellingen O,
    3. Sparreboom A,
    4. Schinkel AH,
    5. Borst P,
    6. Nooijen WJ,
    7. Beijnen JH
    (1997) Enhanced oral bioavailability of paclitaxel in mice treated with the P-glycoprotein blocker SDZ PSC 833. Br J Cancer 76:1181–1183.
    OpenUrlPubMed
  48. ↵
    1. Wacher VJ,
    2. Wu CY,
    3. Benet LZ
    (1995) Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: Implications for drug delivery and activity in cancer chemotherapy. Mol Carcinog 13:129–134.
    OpenUrlPubMed
  49. ↵
    1. Wrighton SA,
    2. Ring BJ
    (1994) Inhibition of human CYP3A-catalyzed 1′-hydroxy midazolam formation by ketoconazole, nifedipine, erythromycin, cimetidine, and nizatidine. Pharm Res 11:921–924.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Wu CY,
    2. Benet LZ,
    3. Hebert MF,
    4. Gupta SK,
    5. Rowland M,
    6. Gomez DY,
    7. Wacher VJ
    (1995) Differentiation of absorption and first-pass gut and hepatic metabolism in humans: Studies with cyclosporine. Clin Pharmacol Ther 58:492–497.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Zhou XJ,
    2. Zhou-Pan XR,
    3. Gauthier T,
    4. Placidi M,
    5. Maurel P,
    6. Rahmani R
    (1993) Human liver microsomal cytochrome P450 3A isozymes mediated vindesine biotransformation. Biochem Pharmacol 45:853–861.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Zhou-Pan XR,
    2. Seree E,
    3. Zhou XJ,
    4. Placidi M,
    5. Maurel P,
    6. Barra Y,
    7. Rahmani R
    (1993) Involvement of human liver cytochrome P450 3A in vinblastine metabolism: Drug interactions. Cancer Res 53:5121–5126.
    OpenUrlAbstract/FREE Full Text
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Overlapping Substrate Specificities of Cytochrome P450 3A and P-Glycoprotein for a Novel Cysteine Protease Inhibitor

Yuanchao Zhang, Xisheng Guo, Emil T. Lin and Leslie Z. Benet
Drug Metabolism and Disposition April 1, 1998, 26 (4) 360-366;
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