Many P-glycoprotein (P-gp) inhibitors studied in vitro and in vivo are also known or suspected to be substrates and/or inhibitors of cytochrome P-450 3A (CYP3A). Such overlap raises the question of whether CYP3A inhibition is an intrinsic characteristic of P-gp inhibitors, a matter of concern in the development and rational use of such agents. Thus, the purpose of the present study was to determine whether the ability to inhibit P-gp and CYP3A is, in fact, linked and whether specific P-gp inhibitors with limited ability to inhibit CYP3A can be identified. Therefore, the potency of a series of 14 P-gp inhibitors was assessed by measuring their inhibition of the transepithelial flux across Caco-2 cells of digoxin, a prototypical P-gp substrate. CYP3A inhibition was determined from the impairment of nifedipine oxidation by human liver microsomes. Determination of the apparent Ki values for CYP3A inhibition and the IC50s for P-gp and CYP3A inhibition allowed comparison of the relative inhibitory potency of the compounds on the two proteins’ function. The IC50s for P-gp inhibition ranged from 0.04 to 3.8 μm. All compounds inhibited CYP3A with apparent Ki values of between 0.3 and 76 μm and IC50s between 1.5 and 50 μm. However, no correlation was found between the extent of P-gp inhibition and CYP3A inhibition, and the ratio of the IC50 for CYP3A inhibition to the IC50 for P-gp inhibition varied from 1.1 to 125. These results demonstrate that, although many P-gp inhibitors are potent inhibitors of CYP3A, a varying degree of selectivity is present. The development and use of P-gp inhibitors with minimal or absent CYP3A inhibitory effects should decrease the impact of drug interactions on the therapeutic use of such compounds.

P-gp3 is an ATP-dependent efflux drug transporter that is constitutively expressed in normal tissues including the gastrointestinal epithelium, the canalicular membrane of the liver, the kidney (1, 2), and capillary endothelial cells in the central nervous system (3, 4). Because of such tissue localization and its broad substrate specificity, P-gp appears to play a key role in absorption, distribution, and elimination of many drugs including anticancer agents (5, 6, 7). In addition, overexpression in malignant cells has been associated with the development of the multidrug resistance phenomenon for many anticancer drugs (8, 9). In vitro, the efficacy of such chemotherapeutic agents can be restored by pharmacological inhibition of P-gp (10, 11, 12), and many chemicals have been shown to possess such an effect. Clinically, however, definitive benefit of P-gp inhibition in anticancer drug treatment still remains to be established and is complicated by the fact that many chemomodulators also result in interactions with drug-metabolizing enzymes (13, 14, 15, 16, 17, 18, 19). For example, plasma concentrations of doxorubicin almost doubled during PSC833 administration (18), and similar increases in plasma concentration of etoposide are also produced by this P-gp inhibitor (19).

In addition to their effects as P-gp inhibitors, many chemomodulating agents also inhibit cytochrome P-450 3A (CYP3A) activity (20, 21), which is the most abundant cytochrome P-450 enzyme present in human liver and intestine and is known to be involved in the metabolism of a large number of drugs including anticancer agents (22). Some anticancer drugs such as taxol are metabolized by CYP3A to products that lack antitumor activity (23); in contrast, CYP3A-mediated metabolism of tamoxifen, methoxymorpholinyldoxorubicin, cyclophosphamide, and ifosfamide (CYP3A/2B) produce metabolites, some of which have antitumor activity greater than the parent compound (24, 25, 26). For drugs requiring activation, the administration of P-gp inhibitors, which are also CYP3A inhibitors, may result in a reduced therapeutic effect, despite the P-gp inhibition-mediated, enhanced intracellular accumulation of the parent drug. Conversely, for drugs, the elimination of which is dependent on CYP3A, inhibition of CYP3A by P-gp inhibitors may cause excessive drug accumulation and increased toxicity, resulting in the need to reduce the dose of chemotherapeutic agent (13, 14, 15, 16, 17, 18, 19). Accordingly, an understanding of the likelihood of CYP3A interactions with P-gp inhibitors is essential for their rational use.

The purpose of the present study was, therefore, to determine the quantitative relationship between P-gp and CYP3A inhibition of a series of compounds with established P-gp inhibitory properties.

Materials.

With the exception of PSC833, which was a gift from Novartis (Basel, Switzerland), the remaining 13 compounds studied (Fig. 1) were provided by Pfizer (Groton, CT). All compounds were dissolved in DMSO. [3H]Digoxin (specific activity, 15 Ci/mmol; radiochemical purity, >97%) was obtained from DuPont-New England Nuclear (Boston, MA). Nifedipine and quinidine were purchased from Sigma Chemical Co. (St. Louis, MO). All tissue culture media and reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD).

Transport in Cultured Caco-2 Cells.

Caco-2 cells were grown with DMEM (high glucose) supplemented with 10% FCS, 2 mml-glutamine, 100 units penicillin/ml, 100 μg of streptomycin/ml, and 1% nonessential amino acids (Life Technologies, Inc.), and incubated at 37°C under 5% CO2. Caco-2 cells were plated at a density of 1 × 105 cells/12 mm well on 0.4 μm size polycarbonate membrane filters (Transwell; Costar Corp., Cambridge, MA). Cells were supplemented with fresh media every 2 days and used in the transport studies on the fourth day after plating. Transepithelial resistance was measured in each well using a Millicell ERS ohm meter (Millipore, Bedford, MA); wells registering a resistance of 200 ohms or greater, after correcting for the resistance obtained in control blank wells, were used. Transport experiments were carried out using the same protocol described previously (27). Briefly, 1–2 h before the start of the transport experiments, the medium in each compartment was replaced with a serum-free medium (Optimem; Life Technologies, Inc.). Then, digoxin transport in the basal-to-apical direction as well as the apical-to-basal direction was measured in two corresponding wells by replacing the media with 700 μl of serum-free medium (Optimem) containing [3H]digoxin (5 μm) in either the apical compartment of the first well or in the basal compartment of the corresponding second well. The digoxin concentration corresponded to about one-third of the reported KM for P-gp-mediated transport (28). [3H]Digoxin appearing in the opposite compartment after 0.5, 1, 2, and 2.5 h was quantified in 25-μl aliquots taken from each compartment by liquid scintillation counting, following the addition of 5 ml of scintillation fluid (ScintiVerse BD; Fisher Scientific, Fair Lawn, NJ), and expressed as percentage of added radioactivity. Inhibition of digoxin translocation was determined after the addition of the test compound in equal concentrations to both the apical and basal compartments. The DMSO concentration in both incubations with and without inhibitor was 10%; preliminary experiments demonstrated that 10% DMSO in Caco-2 cells did not affect the translocation process (data not shown). The extent of inhibition was estimated using the assumption that complete inhibition of P-gp-mediated digoxin transport would result in the loss of net basal-to-apical versus apical-to-basal translocation differences. Accordingly, the degree of inhibition at each time point was determined from the relationship:

\[\mathrm{\%\ inhibition}\ {=}\ \mathrm{1}\ {-}\ \frac{(i_{\mathrm{basal-to-apical}}\ {-}\ \mathrm{1}_{\mathrm{apical-to-basal}})}{a_{\mathrm{basal-to-apical}}\ {-}\ a_{\mathrm{apical-to-basal}}}\ {\times}\ \mathrm{100}\]

where i and a represent the percentage of digoxin transport in the presence and absence of an inhibitor, respectively. Because digoxin transport during the studied time points was linear, the percentage of inhibition calculated at each time point was averaged. The extent of inhibition at various inhibitor concentrations (0.001, 0.01, 0.1, 1, 5, 10, 20, and 100 μm) was used to determine the IC50 according to the Hill equation (Prism program; Graphpad, San Diego, CA). Experiments were performed in triplicate, with each experiment carried out on a different day.

Measurement of CYP3A Activity.

CYP3A activity was determined in human liver microsomes prepared from human liver sample HL110 (Nashville Regional Organ Procurement Agency, Nashville, TN) as described (29) with the total cytochrome P-450 content measured as described by Omura and Sato (30). CYP3A activity was assessed in duplicate by the formation rate of the nifedipine metabolite (dehydronifedipine). Briefly, the incubation medium consisted of microsomes containing 100 pmol cytochrome P-450, 1.5 mm NADPH, nifedipine at various concentrations (15, 20, 30, 60, and 200 μm), and 10 μl of various inhibitor concentrations diluted in DMSO, resulting in final inhibitor concentrations between 0.5 and 40 μm in a total volume of 500 μl 0.1 m phosphate buffer (pH 7.4). Control incubations without addition of inhibitor were also performed in the presence of 2% DMSO. At this concentration of DMSO, nifedipine oxidation was reduced by 25% (data not shown). To avoid variability in CYP3A activity due to differing DMSO concentrations, 2% DMSO was used in all incubation procedures. Incubations were performed at 37°C and stopped after 10 min by the addition of 1 ml of CH2Cl2. The nifedipine metabolite was measured by HPLC-UV as described elsewhere (31) with an interassay variability of <5%. The Michaelis-Menten kinetics (apparent Vmax, apparent Km) were calculated using a nonlinear regression computer program (“kcat”; BioMetallics, Princeton, NJ). The type of inhibition and the inhibition constant (apparent Ki) were derived from appropriate replotting of the Lineweaver-Burk plot (32). IC50s were calculated by the same procedure as for P-gp inhibition, using a nifedipine concentration of 20 μm.

Statistics.

Significance of inhibition for CYP3A and P-gp were determined using a Students’ t test or Mann-Whitney U test, with P < 0.05 being taken as the minimum level of significance accepted. Data are expressed as means ± SD.

P-gp Inhibition.

The effect of P-gp inhibition on the basal-to-apical and apical-to-basal digoxin translocation across Caco-2 cells is shown in Fig. 2, which also illustrates the extremes of potency found for P-gp inhibition with CP114416 and CP99542. The IC50s for inhibition of digoxin transport for the various compounds are presented in Fig. 3 A.

CYP3A Inhibition.

The apparent Km and apparent Vmax values for nifedipine oxidation in the presence of 2% DMSO without any inhibitor were 30 μm and 7 nmol/nmol cytochrome P-450/min, respectively [which are similar to data previously reported in the literature (31)]. All of the compounds except the cyclosporine derivatives acted as mixed-function inhibitors, whereas the cyclosporine derivatives were competitive inhibitors. The apparent Ki values ranged from 0.32 to 76 μm (Table 1), and the IC50 values ranged from 1.5 to 50 μm (Fig. 3 A).

Except for CP117227, which produced hyperbolic inhibition, all compounds exhibited linear inhibition. CP117227 inhibited CYP3A by 35% at 40 μm. Because of its weak CYP3A inhibitory effects and limited solubility in DMSO, the IC50 for verapamil could not be determined, but at concentrations up to 40 μm, the maximal inhibition by verapamil of CYP3A-mediated nifedipine oxidation was 30%. On the basis of the inhibition produced by the range of verapamil concentrations, the concentration required to produce 25% inhibition was calculated.

Comparison of P-gp with CYP3A Inhibition.

The relative inhibition ratio of IC50 (CYP3A):IC50 (P-gp) varied widely (1.06–125; Fig. 3 B). However, a statistically significant correlation of the CYP3A and P-gp inhibition IC50s was not observed (r2 = 0.17, P = 0.16; n = 12).

P-gp inhibition has a potential for therapeutic benefit in the treatment of drug-resistant cancer. In addition, we have shown recently that the disruption of P-gp at the blood/brain barrier results in the enhanced penetration of HIV protease inhibitors and possibly other drugs into the central nervous system (27). Optimal P-gp inhibitor therapy will require drugs that are sufficiently potent to inhibit P-gp at therapeutically achievable plasma concentrations while producing minimal inhibition of CYP3A at these concentrations. To identify such drugs, it is necessary to compare their relative potency as P-gp and CYP3A inhibitors to define their relative selectivity. Drugs that are relatively selective for P-gp will be less likely to produce metabolically determined drug interactions (15). In the past, little attention has been given to the potential for inhibitors of P-gp to also inhibit CYP3A activity (16), despite the potential for such inhibition to alter the therapeutic efficacy of anticancer drugs in at least two ways: (a) through reduced cytotoxicity of anticancer agents, which are activated by CYP3A; and (b) through increased systemic toxicity of anticancer drugs, the elimination of which is mediated by CYP3A (19, 22). In addition, because of the dual expression of P-gp and CYP3A in the gastrointestinal epithelial cells, P-gp inhibition combined with CYP3A inhibition may produce a substantial increase in oral bioavailability of drugs over and above that produced by P-gp inhibition alone. Thus, an understanding of the relative effects of putative P-gp inhibitors on CYP3A activity is essential for rational development and therapy with these compounds.

This study demonstrates that there is a large range in the potency of these compounds as both P-gp and CYP3A inhibitors. All of the compounds exhibited some inhibitory effect on CYP3A. However, an important finding was that no significant correlation exists between the ability of the compounds to inhibit P-gp and their ability to inhibit CYP3A. Although some of the most potent P-gp inhibitors were also the most potent CYP3A inhibitors (e.g., CP114416), conversely, some relatively potent CYP3A inhibitors were relatively poor P-gp inhibitors (e.g., CP99542; Fig. 3 A). Thus, the molecular recognition sites of P-gp and CYP3A differ in ways that result in differential effects of compounds at the two sites.

From a scientific standpoint, P-gp inhibitory potency would appear to be the most important factor in developing a P-gp inhibitor. However, with regard to therapeutic utility, this may not be the case if such inhibition is also accompanied by a significant CYP3A interaction. A less potent P-gp inhibitor but one without or with minimal CYP3A inhibitory effect might be more desirable, provided adequate concentrations can be achieved in vivo. Such selectivity can be assessed by the ratio of IC50 (CYP3A):IC50 (P-gp). Given that the higher the IC50 the higher the drug concentration required for inhibition, those drugs with the highest ratios will be those with the greatest selectivity for P-gp inhibition. Of the drugs studied, CP100356 has the highest selectivity ratio (Fig. 3 B), although it was not the most potent P-gp inhibitor. Interestingly, the cyclosporine derivative PSC833 showed a high selectivity index of about 90, in contrast to the low ratio (2.3) for cyclosporine itself, implying that compounds with similar chemical structures can substantially differ in their ability to inhibit P-gp and CYP3A. By the use of the selectivity index, it should be possible to screen compounds for their relative effects on transport and drug metabolism. This will allow the selection of those compounds that produce the greatest inhibition of drug transport and, hence, reversal of multidrug resistance, minimizing, at the same time, the increase in plasma concentrations due to inhibition of drug metabolism and loss of drug efficacy due to reduction in metabolic activation.

The disappointing therapeutic effects of P-gp inhibitors in cancer therapy thus far may reflect the fact that in many of the studies, the P-gp inhibition strategy used drugs like verapamil and cyclosporine, which are established and readily available. However, as can be seen in Fig. 3, these are drugs with relatively low potency and low selectivity indices, implying that the high concentrations required to inhibit P-gp would also produce CYP3A inhibition. Concentrations of these drugs achieved clinically range up to 10 μm for quinidine, 0.4 μm for cyclosporine, and 0.4 μm for verapamil. The duality of effects of these P-gp inhibitors on both P-gp and CYP3A will result in multiple effects at different tissue sites so that inhibition of P-gp will increase tumor concentration, increase oral absorption, and increase central nervous system penetration. In addition, inhibition of CYP3A will further enhance oral absorption and decrease hepatic metabolism with consequent further increase in plasma concentration. The therapeutic/toxic effects of the changes will depend on whether CYP3A is involved in activation or detoxification of the drug in question (16, 17, 18, 19).

In conclusion, this study has demonstrated the considerable overlap in the activity of inhibitors of P-gp on CYP3A. The use of first generation P-gp inhibitors such as verapamil and cyclosporine with poor P-gp selectivity may be complicated by metabolic drug interactions with the anticancer drug regimen. Even small structural modifications, such as cyclosporine to PSC833, can profoundly alter the selectivity of the drug. Therefore, if potent P-gp-selective agents can be developed that minimize the risk of CYP3A-mediated metabolic interactions with the anticancer drug regimen, this should result in improved therapeutic specificity and efficacy.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

      
1

Supported by USPHS Grants GM 31304, CA 44353, and ES 00267.

            
3

The abbreviation used is: P-gp, P-glycoprotein.

Fig. 1.

Chemical structures of the various compounds examined.

Fig. 1.

Chemical structures of the various compounds examined.

Close modal
Fig. 2.

Transepithelial translocation of [3H]digoxin (5 μm) across a Caco-2 cell culture monolayer in the absence or presence of varying concentrations of CP114416 and CP99542. ▴ and solid line, translocation from basal to apical compartments; ▪ and dotted line, translocation from apical to basal compartments. Data are means from three or more experiments; bars, SD.

Fig. 2.

Transepithelial translocation of [3H]digoxin (5 μm) across a Caco-2 cell culture monolayer in the absence or presence of varying concentrations of CP114416 and CP99542. ▴ and solid line, translocation from basal to apical compartments; ▪ and dotted line, translocation from apical to basal compartments. Data are means from three or more experiments; bars, SD.

Close modal
Fig. 3.

A, IC50 for P-gp and CYP3A inhibition [for CP117227 and verapamil, inhibitor concentrations that produced 25% CYP3A inhibition (IC25), respectively]. B, the selectivity ratio for CYP3A to P-gp inhibition [IC50 (CYP3A):IC50 (P-gp)]. The higher the ratio, the less risk for CYP3A-mediated drug interactions with a similar P-gp inhibitory effect.

Fig. 3.

A, IC50 for P-gp and CYP3A inhibition [for CP117227 and verapamil, inhibitor concentrations that produced 25% CYP3A inhibition (IC25), respectively]. B, the selectivity ratio for CYP3A to P-gp inhibition [IC50 (CYP3A):IC50 (P-gp)]. The higher the ratio, the less risk for CYP3A-mediated drug interactions with a similar P-gp inhibitory effect.

Close modal
Table 1

Mechanism of CYP 3A inhibition and respective apparent Ki of the 14 compounds studied

CompoundMechanism of CYP 3A inhibitionapparent Kim)
CP114146 Linear, mixed function 0.32 
CP100356 Linear, mixed function 13.0 
CP114769 Linear, mixed function 2.0 
CP101556 Linear, mixed function 5.9 
FK506 Linear, competitive 1.1 
CP99542 Linear, competitive 1.6 
Cyclosporin Linear, competitive 4.9 
PSC833 Linear, competitive 16 
Verapamil Linear, mixed function 76 
CP12379 Linear, mixed function 13 
CP69042 Linear, mixed function 23 
CP117227 Hyperbolic, mixed function 3.0 
CP147478 Linear, mixed function 0.75 
Quinidine Linear, mixed function 51 
CompoundMechanism of CYP 3A inhibitionapparent Kim)
CP114146 Linear, mixed function 0.32 
CP100356 Linear, mixed function 13.0 
CP114769 Linear, mixed function 2.0 
CP101556 Linear, mixed function 5.9 
FK506 Linear, competitive 1.1 
CP99542 Linear, competitive 1.6 
Cyclosporin Linear, competitive 4.9 
PSC833 Linear, competitive 16 
Verapamil Linear, mixed function 76 
CP12379 Linear, mixed function 13 
CP69042 Linear, mixed function 23 
CP117227 Hyperbolic, mixed function 3.0 
CP147478 Linear, mixed function 0.75 
Quinidine Linear, mixed function 51 
1
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.
Proc. Natl. Acad. Sci. USA
,
84
:
7735
-7738,  
1987
.
2
Sugawara I., Kataoka I., Morishita Y., Hamada H., Tsuruo T., Hoyama S., Mori S. Tissue distribution of P-glycoprotein encoded by a multidrug-resistant gene as revealed by a monoclonal antibody, MRK 16.
Cancer Res.
,
48
:
1926
-1929,  
1988
.
3
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 crossreactivity of one antibody with a muscle protein.
J. Histochem. Cytochem.
,
37
:
159
-164,  
1989
.
4
Cordon-Cardo C., O’Brien J. P., Casals D., Rittman-Grauer L., Biedler J. L., Melamed M. R., Bertino J. R. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites.
Proc. Natl. Acad. Sci. USA
,
86
:
695
-698,  
1989
.
5
Leveque D., Jehl F. P-glycoprotein and pharmacokinetics.
Anticancer Res.
,
15
:
231
-336,  
1995
.
6
Schinkel A. H., Wagenaar E., van Deemter L., Mol C. A. A. M., Borst P. Absence of the mdr 1a P-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin and cyclosporin A.
J. Clin. Invest.
,
96
:
1698
-1705,  
1995
.
7
Relling M. V. Are the major effects of P-glycoprotein modulators due to altered pharmacokinetics of anticancer drugs?.
Ther Drug Monit.
,
18
:
350
-356,  
1996
.
8
Gottesman M. M., Pastan I., Ambudkar S. V. P-glycoprotein and multidrug resistance.
Curr. Opin. Genet. Dev.
,
6
:
610
-617,  
1996
.
9
Ling V. Multidrug resistance. Molecular mechanisms and clinical relevance.
Cancer Chemother. Pharmacol.
,
40 (Suppl.)
:
S3
-S8,  
1997
.
10
Tsuruo T., Iida H., Tsukagoshi S., Sakurai Y. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil.
Cancer Res.
,
41
:
1967
-1972,  
1981
.
11
Ford J. M., Bruggemann E. P., Pastan I., Gottesman M. M., Hait W. N. Cellular and biochemical characterization of thioxanthenes for reversal of multidrug resistance in human and murine cell lines.
Cancer Res.
,
50
:
1748
-1756,  
1990
.
12
Boesch D., Gaveriaux C., Jachez B., Pourtier-Manzanedo A., Bollimger P., Loor F. In vitro circumvention of P-glycoprotein-mediated multidrug resistance of tumor cells with SDZ PSC 833.
Cancer Res.
,
51
:
4226
-4233,  
1991
.
13
Raderer M., Scheithauer W. Clinical trials of agents that reverse multidrug resistance.
Cancer (Phila.)
,
72
:
3553
-3563,  
1993
.
14
Ford J. M., Hait W. N. Pharmacology of drugs that alter multidrug resistance in cancer: effects of chemosensitizers in human trials.
Pharmacol. Rev.
,
42
:
181
-183,  
1990
.
15
Bates S. E., Wilson W. H., Fojo A. T., Alvarez M., Zhan Z., Regis J., Robey R., Hose C., Monks A., Kang Y. K., Chabner B. Clinical reversal of multidrug resistance.
Stem Cells
,
14
:
56
-63,  
1996
.
16
Lum B. L., Kaubisch S., Yahanda A. M., Adler K. M., Jew L., Ehsan M. N., Brophy N. A., Halsey J., Gosland M. P., Sikic B. I. Alteration of etoposide pharmacokinetics and pharmacodynamics by cyclosporine in a Phase I trial to modulate multidrug resistance.
J. Clin. Oncol.
,
10
:
1635
-1642,  
1992
.
17
Tolcher A. W., Cowan K. H., Solomon D., Ognibene F., Goldspiel B., Chang R., Noone M. H., Denicoff A. M., Barnes C. S., Gossard M. R., Fetsch P. A., Berg S. L., Balis F. M., Venzon D. J., O’Shaughnessy J. A. Phase I crossover study of paclitaxel with R-verapamil in patients with metastatic breast cancer.
J. Clin. Oncol.
,
14
:
1173
-1184,  
1996
.
18
Sonneveld P., Marie J. P., Huisman C., Vekhoff A., Schoester M., Faussat A. M., Vankapel J., Groenewegen A., Charnick S., Zizzoun R., Lowenberg B. Reversal of multidrug-resistance by SDZ PSC-833, combined with VAD (vincristine, doxorubicin, dexamethasone) in refractory multiple myeloma-a Phase I study.
Leukemia (Baltimore)
,
10
:
1741
-1750,  
1996
.
19
Boote D. J., Dennis I. F., Twentyman P. R., Osborne R. J., Laburte C., Hensel S., Smyth J. F., Brampton M. H., Bleehen N. M. Phase I study of etoposide with SDZ PSC 833 as a modulator of multidrug-resistance in patients with cancer.
J. Clin. Oncol.
,
14
:
610
-618,  
1996
.
20
Wacher V. J., Wu C. Y., Benet L. L. Overlapping substrate specificities and tissue distribution of cytochrome P4503A and P-glycoprotein: implications for drug delivery and activity in cancer chemotherapy.
Mol. Carcinog.
,
13
:
129
-134,  
1995
.
21
Kim R. B., Wandel C., Leake B., Cvetkovic M., Fromm M. F., Dempsey P. J., Roden M. M., Belas F., Chaudhary A. K., Roden D. M., Wood A. J. J., Wilkinson G. R. Interrelationship between inhibitors and substrates of human CYP 3A and P-glycoprotein.
Pharmacol. Res.
,
16
:
408
-414,  
1999
.
22
Kivistoe K. T., Kroemer H. K., Eichelbaum M. The role of human cytochrome P450 enzymes in the metabolism of anticancer agents: implications for drug interactions.
Br. J. Clin. Pharmacol.
,
40
:
523
-530,  
1995
.
23
Harris J. W., Rahman A., Kim B-R., Guengerich F. P., Collins J. M. Metabolism of Taxol by human hepatic microsomes and liver slices: participation of cytochrome P450 3A4 and of an unknown P450 enzyme.
Cancer Res.
,
54
:
4026
-4035,  
1994
.
24
Jacolot F., Simon I., Dreano Y., Beaune P., Riche C., Berthou F. Identification of the cytochrome P450IIIA family as the enzymes involved in the N-demethylation of tamoxifen in human liver microsomes.
Biochem. Pharmacol.
,
41
:
1911
-1919,  
1991
.
25
Lau D. H., Duran G. E., Lewis A. D., Sikic B. I. Metabolic conversion of methoxymorpholinyl doxorubicin: from a DNA strand breaker to a DNA cross-linker.
Br. J. Cancer
,
70
:
79
-84,  
1994
.
26
Ren S., Yang J-S., Kalhorn T. F., Slattery J. T. Oxidation of cyclophosphamide to 4-hydroxycyclophosphamide and deschloroethylcyclophosphamide in human liver microsomes.
Cancer Res.
,
57
:
4229
-4235,  
1997
.
27
Kim R. B., Fromm M. F., Wandel C., Leake B., Wood A. J. J., Roden D. M., Wilkinson G. R. The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors.
J. Clin. Invest.
,
101
:
289
-294,  
1998
.
28
Ito S., Koren G., Harper P. A., Silverman M. Energy-dependent transport of digoxin across renal tubular cell monolayers (LLC-PK1).
Can. J. Physiol. Pharmacol.
,
71
:
40
-47,  
1993
.
29
Guengerich F. P. Analysis and characterization of enzymes Ed. 3 Hayes A. W. eds. .
Principles and Methods of Toxicology
,
:
1259
-1313, Raven Press New York  
1994
.
30
Omura T., Sato R. The carbon monoxide-binding pigment of liver microsomes.
J. Biol. Chem.
,
239
:
2370
-2378,  
1964
.
31
Guengerich F. P., Martin M. V., Beune P. H., Kremers P., Wolff T., Waxman D. J. Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism.
J. Biol. Chem.
,
261
:
5051
-5060,  
1988
.
32
Segel I. H. Enzyme Kinetics Wiley New York  
1975
.