Glucuronidation of 3'-azido-3'-deoxythymidine (zidovudine) by human liver microsomes: relevance to clinical pharmacokinetic interactions with atovaquone, fluconazole, methadone, and valproic acid.

Zidovudine (3'-azido-3'-deoxythymidine [AZT]), an antiviral nucleoside analog effective in the treatment of human immunodeficiency virus infection, is primarily metabolized to an inactive glucuronide form, GAZT, via uridine-5'-diphospho-glucuronosyltransferase (UGT) enzymes. UGT enzymes exist as different isoforms, each exhibiting substrate specificity. Published clinical studies have shown that atovaquone, fluconazole, methadone, and valproic acid decreased GAZT formation, presumably due to UGT inhibition. The effect of these drugs on AZT glucuronidation was assessed in vitro by using human hepatic microsomes to begin understanding in vitro-in vivo correlations for UGT metabolism. The concentrations of each drug studied were equal to those reported with the usual clinical doses and at concentrations at least 10 times higher than would be expected with these doses. High-performance liquid chromatography was used to assess the respective metabolism and formation of AZT and GAZT. All four drugs exhibited concentration-dependent inhibition of AZT glucuronidation. The respective concentrations of atovaquone and methadone which caused 50% inhibition of GAZT were > 100 and 8 micrograms/ml, well above their usual clinical concentrations. Fluconazole and valproic acid exhibited 50% inhibition of GAZT at 50 and 100 micrograms/ml, which are within the clinical ranges of 10 to 100 and 50 to 100 micrograms/ml, respectively. These data suggest that inhibition of AZT glucuronidation may be more clinically significant with concomitant fluconazole and valproic acid. Factors such as inter- and intraindividual pharmacokinetic variability and changes in AZT intracellular concentrations should be considered as other mechanisms responsible for changes in AZT pharmacokinetics with concomitant therapies.

Predicting drug interactions in vivo from experiments in vitro. Human studies with paclitaxel and ketoconazole.

This study was performed to evaluate whether concomitant treatment with ketoconazole could reduce the clearance of paclitaxel given to ovarian cancer patients. Paclitaxel, 175 mg/m2, was given as a 3-hour continuous intravenous infusion and repeated every 21 days. Initially, ketoconazole, 100 to 1600 mg, was given as a single oral dose 3 hours after paclitaxel. Later, ketoconazole, 200 mg, was given perorally 3 hours before paclitaxel. Plasma drug concentrations were measured by high-pressure liquid chromatography (HPLC), and cytochrome P450 3A (CYP3A) activity was measured with the erythromycin breath test (ERMBT). Ketoconazole did not alter plasma concentrations of paclitaxel or its principal metabolite, 6 alpha-hydroxypaclitaxel. Although there was marked inter- and intrapatient variability in ketoconazole pharmacokinetics, peak plasma concentrations in all but one course were below the 50% inhibitory concentration (IC50) point determined for inhibition of paclitaxel metabolism in vitro. Therefore, paclitaxel and ketoconazole can be coadministered safely without dose adjustments. There was no correlation between ERMBT measurements and serial plasma concentrations of paclitaxel. The erythromycin breath-test measurements did correlate with the corresponding ketoconazole plasma concentrations. The erythromycin breath test is a valuable tool for measuring instantaneous CYP3A activity in vivo. This clinical study confirms the results of prior studies with human-derived materials in vitro, reinforcing the notion that such studies are useful predictors of drug pharmacokinetics and interactions in vivo.

Rifampin and rifabutin and their metabolism by human liver esterases.

1. The main metabolites of rifampin and rifabutin in man are their respective 25 deacetylated derivatives, but the enzyme(s) responsible for these biotransformations are not known. 2. In experiments with human liver slices and human liver microsomes, the 25 deacetylated derivatives of these drugs were the main metabolites observed. Slices and microsomes metabolized rifabutin 3-6-fold faster than rifampin, in agreement with their relative clearance in patients. Rifabutin partitioned into slices more avidly than rifampin. 3. In microsomal incubations, deacetylation did not require NADPH, but the amount of metabolite at the end of incubation was affected by NADPH. With NADPH the amount of 25 deacetyl rifabutin decreased, whereas the amount of 25 deacetyl rifampin increased slightly. A panel of liver microsomes from seven donors showed a 3-4-fold difference in the formation of 25 deacetyl rifabutin or 25 deacetyl rifampin, with strong correlation between the production of the two metabolites (r2 = 0.94). 4. The production of 25 deacetyl rifabutin and 25 deacetyl rifampin by human liver microsomes was not significantly affected by 1 microM 4 chloromercuricbenzoic acid or bis-(4-nitrophenyl) phosphate, but was completely inhibited by 1 microM paraoxon or 1 microM diisopropylfluorophosphate. These results indicate that in man rifampin and rifabutin are deacetylated to their main metabolites by B-esterases.

Metabolism of rifabutin and its 25-desacetyl metabolite, LM565, by human liver microsomes and recombinant human cytochrome P-450 3A4: relevance to clinical interaction with fluconazole.

Rifabutin and fluconazole are often given concomitantly as therapy to prevent opportunistic infections in individuals infected with the human immunodeficiency virus. Recent reports have shown increased levels of rifabutin and its 25-desacetyl metabolite, LM565, in plasma when rifabutin is administered with fluconazole. Since fluconazole is known to inhibit microsomal enzymes, this study was undertaken to determine if this rifabutin-fluconazole interaction was due to an inhibition of human hepatic enzymes. The metabolism of both rifabutin and LM565 was evaluated in human liver microsomes and recombinant human cytochrome P-450 (CYP) 3A4 in the presence of fluconazole and other probe drugs known to inhibit CYP groups 1A2, 2C9, 2D6, 2E1, and 3A. The concentrations of rifabutin (1 microg/ml), LM565 (1 microg/ml), and fluconazole (10 and 100 microg/ml) used were equal to those observed in plasma after the administration of rifabutin and fluconazole at clinically relevant doses. High-performance liquid chromatography was used to assess the metabolism of rifabutin and LM565. Rifabutin was readily metabolized to LM565 by human microsomes, but the reaction was independent of NADPH and was not affected by the P-450 inhibitors. No rifabutin metabolism by recombinant CYP 3A4 was found to occur. LM565 was also metabolized by human microsomes to two products, but metabolism was dependent on NADPH and was affected by certain P-450 inhibitors. In addition, LM565 was readily metabolized by the recombinant CYP 3A4 to the same two products found with its metabolism by human microsomes. Therefore, rifabutin is metabolized by human microsomes but not via cytochrome P-450 enzymes, whereas LM565 is metabolized by CYP 3A4.

Small (< or = 3-cm) renal masses: detection with CT versus US and pathologic correlation.

PURPOSE: To determine the sensitivities of computed tomography (CT) and ultrasound (US) for detection and characterization of surgically verified small renal lesions. MATERIALS AND METHODS: Twenty-one patients with von Hippel-Lindau disease or hereditary papillary renal cancer underwent CT and US before partial nephrectomy or enucleation; 205 renal masses were removed (92% were <3 cm). Detection rates and accuracy of CT and US in the characterization of renal morphology were correlated with lesion size. RESULTS: CT and US detection rates for lesions of 0-5 mm were respectively 47% and 0%; 5-10 mm, 60% and 21%; 10-15 mm, 75% and 28%; 15-20 mm, 100% and 58%; 20-25 mm, 100% and 79%; and 25-30 mm, 100% and 100%. Among the lesions 10-35 mm, 80% and 82% were correctly characterized with CT and US, respectively. CONCLUSION: A substantial proportion of lesions under 1 cm were not detected with either modality. Neither CT nor US was superior in the characterization of lesions 3 cm or less. CT and particularly US screening studies in patients with von Hippel-Lindau disease should be interpreted cautiously because missed or mischaracterized small renal lesions are a frequent problem in these patients.

Phase I and pharmacokinetic study of the multidrug resistance modulator dexverapamil with EPOCH chemotherapy.

PURPOSE: Dexverapamil is a competitive inhibitor of the P-glycoprotein (Pgp) efflux pump, a potent mechanism of multidrug resistance (mdr-1) in vitro. We performed a phase I study to determine the maximum-tolerated dose (MTD) and pharmacokinetics of dexverapamil with etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (EPOCH) chemotherapy. PATIENTS AND METHODS: Eligible patients had relapsed or refractory lymphoma or sarcoma. Patients initially received EPOCH alone, and those with stable or progressive disease were crossed-over to received dexverapamil on subsequent cycles of EPOCH. Dexverapamil was administered orally for 6 days and escalated over eight dose levels ranging from 240 to 1,200 mg/m2/d. Pharmacokinetics of dexverapamil and its active metabolite, nor-dexverapamil, were obtained in most patients. In seven patients, pharmacokinetics of doxorubicin, doxorubicinol, and etoposide were determined on paired cycles of EPOCH with or without dexverapamil. RESULTS: Sixty-five patients received 130 cycles of dexverapamil/EPOCH chemotherapy. The MTD of dexverapamil was 150 mg/m2 every 4 hours (900 mg/m2/d), and hypotension was the principal dose-limiting toxicity. The dexverapamil area under the curve (AUC) increased proportionally with dexverapamil dose, but significant interpatient variation occurred. At the MTD, the median plasma average concentrations of dexverapamil and nor-dexverapamil were 1.2 and 1.4 mumol/L, respectively. Dexverapamil did not affect the steady-state concentration (Css) of etoposide, but increased the Css of doxorubicin and doxorubicinol nearly twofold. The absolute neutrophil and platelet nadirs were significantly lower on the dexverapamil cycles compared with cycles of EPOCH alone, but other chemotherapy-related toxicities did not change. CONCLUSION: The phase II recommended dose of dexverapamil with EPOCH is 150 mg/m2 every 4 hours. This dose was well tolerated on an outpatient basis and achieved plasma concentrations of dexverapamil and nor-dexverapamil within the effective range for Pgp inhibition in vitro. Although dexverapamil increased the hematopoietic toxicity of EPOCH, it was mild, readily reversible, and offset by EPOCH dose reductions. Dexverapamil should be considered for further study.

Metabolism of taxol by human and rat liver in vitro: a screen for drug interactions and interspecies differences.

Human liver slices, human liver microsomes, and rat liver microsomes were used to investigate the metabolism of 3H-taxol. The effects of drugs frequently coadministered with taxol and the effects of several cytochrome P450 system probes were studied. In all, 16 compounds were screened. After incubation with liver slices or with microsomal protein, 3H-taxol was converted into several radioactive species resolved by HPLC. There were qualitative and quantitative species differences in the metabolism of taxol. The pattern of metabolism was similar for both human-derived preparations, with 6 alpha-hydroxytaxol being the major metabolite peak. In drug interaction studies performed with human liver microsomes, cimetidine 80 microM, and diphenhydramine 200 microM, had little or no effect on 6 alpha-hydroxytaxol formation. Quinidine, ketoconazole, dexamethasone and Cremophor EL inhibited 6 alpha-hydroxytaxol formation with IC50 values of 36 microM, 37 microM, 16 microM and 1 microliter/ml, respectively, but these concentrations exceed the usual clinical range. Cremophor EL also inhibited microsomal metabolism of taxol, but at 2 microliters/ml it had little or no effect on 6 alpha-hydroxytaxol production by human liver slices. These results suggest that: (1) taxol is metabolized by the cytochrome P450 system; (2) taxol metabolism is different in humans than in rats; (3) taxol metabolism in humans is unlikely to be altered by cimetidine, dexamethasone, or diphenhydramine, drugs regularly coadministered with taxol; (4) taxol metabolism can be indirectly affected by Cremophor EL, the formulation vehicle; (5) taxol metabolism may be altered by concentrations of ketoconazole achievable in humans only at very high doses; and (6) taxol metabolism and drug interaction studies of clinical relevance can be performed in vitro with human liver microsomes and human liver slices, but not with rat liver preparations.

Effect of cimetidine, probenecid, and ketoconazole on the distribution, biliary secretion, and metabolism of [3H]taxol in the Sprague-Dawley rat.

Male Sprague-Dawley rats had their bile ducts cannulated and were dosed with [3H]taxol (2 mg/kg, 68-77 microCi/mg) as a continuous intravenous infusion for 6 hr so that the plasma concentrations, tissue distribution, metabolism, and biliary secretion of taxol could be studied. Defining potential drug-drug interactions of taxol with cimetidine (90 mg/kg), probenecid (360 mg/kg), and ketoconazole (50 mg/kg) was motivated by frequent concomitant clinical use or the potential to reduce clearance of taxol so that lower doses could be used. At 6 hr, rats were killed. Samples of blood (plasma), lung, spleen, liver, kidney, heart, skeletal muscle, brain, testes, and fat were obtained. Taxol and metabolites were measured by total radioactivity counting and HPLC separation using on-line radioactivity detection. Concentrations of taxol in plasma increased to 0.19 microM in the control rats and did not reach steady-state by 6 hr. Lung, spleen, liver, and kidneys had the greatest tissue taxol concentrations [4.7-5.7 nmol/g (microM)] and were > 25-fold higher than the simultaneous 6-hr plasma taxol concentration. Taxol concentrations in brain and testes were negligible, 0.06 and 0.07 nmol/g, respectively. Radioactive metabolites were not found in plasma or most tissues. Only liver had appreciable concentrations of taxol and metabolites; however, > 80% of hepatic radioactivity was parent taxol. Through 6 hr of collection, 24% of the dose was secreted in the bile approximately 38% of which was as parent taxol. Cimetidine had no effect on the distribution, metabolism, or elimination of [3H]taxol. Probenecid did not effect tissue distribution or plasma concentrations of taxol.(ABSTRACT TRUNCATED AT 250 WORDS)

Steady-state plasma concentrations and effects of taxol for a 250 mg/m2 dose in combination with granulocyte-colony stimulating factor in patients with ovarian cancer.

Taxol, a natural product initially isolated from the stem bark of the western yew Taxus brevifolia, is undergoing phase II and III evaluation due to its reported activity against a variety of tumors. Previous studies have described correlations between plasma concentrations and toxicity when taxol is given (a) at lower doses, (b) for shorter infusion times, and (c) without granulocyte-colony-stimulating factor. Because the 24-h infusion schedule is most commonly used in current clinical trials, we attempted to correlate steady-state plasma concentrations of taxol achieved with a 24-h continuous i.v. infusion with toxicities and responses. Plasma samples from 48 refractory ovarian cancer patients were obtained 1-2 h prior to the end of the first taxol infusion. Taxol concentrations were measured by high-performance liquid chromatography (HPLC). Interpatient variation of taxol plasma concentrations was small (mean +/- SD, 0.85 +/- 0.21 microM. Total taxol body clearance was 256 +/- 72 ml min-1 m-2 (mean +/- SD). Taxol plasma protein binding was 88.4% +/- 1.3% (mean +/- SD, n = 9). Grade 3-4 hematologic toxicity, mainly leukopenia, occurred in 92% of the patients. The leukopenia was transient and did not warrant a reduction in the dose of taxol. Grade 3-4 nonhematologic toxicity occurred in 8% of the patients. No severe hypersensitivity reaction or grade 3-4 neurotoxicity was observed. Correlations of plasma concentrations and toxicities were not feasible due to the high frequency of hematologic effects and the low frequency of nonhematologic toxicity. The low degree of interpatient variation in plasma concentrations hindered the development of correlations with response.

Effect of suramin on human prostate cancer cells in vitro.

Suramin, a polyanionic compound with known antiparasitic activity, has been shown to be adrenocorticolytic in primates and to have clinical efficacy in the treatment of patients with metastatic prostate cancer refractory to conventional hormonal manipulation. To better characterize the activity of suramin on prostate cancer biology, we studied the effect of the drug on plasma adrenal androgens of patients and on the human prostate adenocarcinoma cell lines PC-3, DU 145 and LNCaP-FGC. Five cancer patients treated with suramin had an approximate 40% decline in circulating androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate levels. The drug inhibited the colony formation in two of the three cell lines at concentrations clinically achievable in humans without excessive drug-related toxicity. The presence of suramin 300 micrograms./ml. partially inhibited the growth stimulatory effect of testosterone and basic fibroblast growth factor, but not that of epidermal growth factor. The cellular concentration of suramin following exposure to a single dose increases linearly over time in each of the cell lines with LNCaP-FGC accumulating the highest levels of the drug; cellular levels of suramin, not androgen or growth factor sensitivity, correlated with the sensitivity to the drug. The concentrations of prostatic acid phosphatase and prostatic specific antigen released by LNCaP-FGC cells in cell culture medium declined in the presence of increasing levels of suramin in a manner which exceeded the decrease in cell number. We conclude that suramin, aside from decreasing circulating androgens through its adrenocorticolytic effect, is also capable exerting a direct inhibitory effect on cell proliferation of prostate cancer cells, and interfere at a cellular level with the growth stimulatory effects of exogenous testosterone and basic fibroblast growth factor.

Induction of thymidylate synthase associated with multidrug resistance in human breast and colon cancer cell lines.

A series of Adriamycin-resistant human breast MCF-7 and human colon DLD-1 cancer cell lines were established by stepwise selection. The concentration of Adriamycin required to inhibit cell proliferation by 50% (IC50) in the parent breast line (MCF-7), Adriamycin-resistant lines (MCF-Ad5 and MCF-Ad10), and a 5-fluorouracil (5-FU)-revertant line (MCF-R) was 0.005, 3.3, 6, and 4.9 microM, respectively. The Adriamycin IC50 value for the resistant colon line (DLD-Ad) was 8.2 microM, 68-fold higher than that for its parent line (DLD-1) (IC50 = 0.12 microM). The MCF-Ad5 and MCF-Ad10 cells were cross-resistant to 5-FU, with respective 5-FU IC50 values of 11.7 and 22.5 microM, or 7.3- and 14-fold less sensitive than their parent MCF-7 (IC50 = 1.6 microM) line. The MCF-R line completely reverted in sensitivity to 5-FU, with an IC50 of 1.7 microM. The resistant DLD-Ad line was 3.5-fold more resistant to 5-FU than was the parent DLD-1 line. Using both the 5-fluoro-2'-deoxyuridine-5'-monophosphate binding and catalytic assays for measurement of thymidylate synthase (TS) activity, there was significantly increased TS activity in the resistant MCF-Ad5 (2.4- and 2.5-fold), MCF-Ad10 (11.5- and 6.8-fold), and DLD-Ad (4.8- and 10.7-fold) lines, for binding and catalytic assays, respectively, compared with their parent MCF-7 and DLD-1 lines. The level of TS in cytosolic extracts, as determined by Western immunoblot analysis, was markedly increased for the resistant MCF-Ad5 (31-fold), MCF-Ad10 (46-fold), and DLD-Ad (52-fold) cells. Measurement of TS mRNA levels by Northern analysis revealed elevation of TS mRNA in the resistant MCF-AD5 (16.7-fold), MCF-Ad10 (31-fold), and DLD-Ad (55-fold) cells. Southern analysis showed that this increase in TS mRNA was not accompanied by any major rearrangements or amplification of the TS gene. Incorporation of 5-FU into the RNA and DNA of the resistant MCF-Ad10 cells was not significantly different, compared with that for parent MCF-7 cells. These studies suggest that exposure of human breast and human colon cancer cells to Adriamycin leads to overexpression of TS, with concomitant development of resistance to 5-FU.

Suramin in adrenal cancer: modulation of steroid hormone production, cytotoxicity in vitro, and clinical antitumor effect.

Suramin, a drug known to have antiparasitic effects, has been previously shown to have adrenocorticolytic activity in primates. We now confirm preferential accumulation of this compound in the normal adrenal gland, evaluate its in vitro effect against two human adrenocortical carcinoma cell lines (SW-13 and NCI-H295), and report the clinical activity of suramin in 17 patients with metastatic adrenocortical carcinoma. Inhibition of colony formation occurred in both adrenal cell lines in vitro at concentrations that are clinically achievable in humans. In addition, suramin concentrations as low as 100 micrograms/mL were able to inhibit glucocorticoid, mineralocorticoid, and androgen production by the NCI-H295 cell line. Of 16 patients with adrenocortical carcinoma now evaluable for tumor response, 2 achieved a partial response, 2 had a minor response, and 5 remained with stable disease for periods ranging from 3-10 months; the remainder progressed. One of 7 patients with excessive steroid hormone production achieved a partial normalization of her steroid levels for the duration of suramin therapy in the setting of radiographic disease stabilization. An additional patient treated off-study for lack of radiographically measurable disease, achieved complete normalization of plasma aldosterone levels. We conclude that suramin preferentially accumulates in adrenal cells, induces cytotoxicity and significant down-regulation of steroid hormone production in vitro, and has some therapeutic efficacy as a single agent in patients with metastatic adrenocortical carcinoma.