Phase I trial of doxorubicin with cyclosporine as a modulator of multidrug resistance.

PURPOSE: To study the effects of cyclosporine (CsA), a modulator of multidrug resistance (MDR), on the pharmacokinetics and toxicities of doxorubicin. PATIENTS AND METHODS: Nineteen patients with incurable malignancies entered this phase I trial. Initially patients received doxorubicin alone (60 or 75 mg/m2) as a 48-hour continuous intravenous (i.v.) infusion. Patients whose tumors did not respond received CsA as a 2-hour loading dose of 6 mg/kg and a 48-hour continuous infusion of 18 mg/kg/d with doxorubicin. Target CsA levels were 3,000 to 4,800 ng/mL (2.5 to 4.0 mumol/L). Doxorubicin doses were reduced to 40% of the prior dose without CsA, and then escalated until myelosuppression equivalent to that resulting from doxorubicin alone was observed. Doxorubicin pharmacokinetics were analyzed with and without CsA. RESULTS: Thirteen patients received both doxorubicin alone and the combination of doxorubicin and CsA. Mean CsA levels were more than 2,000 ng/mL for all cycles and more than 3,000 ng/mL for 68% of cycles. Dose escalation of doxorubicin with CsA was stopped at 60% of the doxorubicin alone dose, as four of five patients at this dose level had WBC nadirs equivalent to those seen with doxorubicin alone. Nonhematologic toxicities were mild. Reversible hyperbilirubinemia occurred in 68% of doxorubicin/CsA courses. The addition of CsA to doxorubicin increased grade 1 and 2 nausea (87% v 47%) and vomiting (50% v 10%) compared with doxorubicin alone. There was no significant nephrotoxicity. Paired pharmacokinetics were studied in 12 patients. The addition of CsA increased the dose-adjusted area under the curve (AUC) of doxorubicin by 55%, and of its metabolite doxorubicinol by 350%. CONCLUSION: CsA inhibits the clearance of both doxorubicin and doxorubicinol. Equivalent myelosuppression was observed when the dose of doxorubicin with CsA was 60% of the dose of doxorubicin without CsA. Understanding these pharmacokinetic interactions is essential for the design and interpretation of clinical trials of MDR modulation, and should be studied with more potent MDR modulators.

Alteration of etoposide pharmacokinetics and pharmacodynamics by cyclosporine in a phase I trial to modulate multidrug resistance.

PURPOSE: To determine the effects of high-dose cyclosporine (CsA) infusion on the pharmacokinetics of etoposide in patients with cancer. PATIENTS AND METHODS: Sixteen patients were administered 20 paired courses of etoposide and CsA/etoposide. Etoposide was administered daily for three days, alone or with CsA, which was delivered by a loading dose and 3-day infusion. Etoposide was measured by high-performance liquid chromatography (HPLC) and serum CsA by nonspecific immunoassay. Etoposide pharmacokinetics included area under the concentration-time curve (AUC), total and renal clearance (CL), half-life (T1/2), and volume of distribution at steady state (Vss). RESULTS: CsA concentrations more than 2,000 ng/mL produced an increase in etoposide AUC of 80% (P less than .001), a 38% decrease in total CL (P < .01), a > twofold increase in T1/2 (P < .01), and a 46% larger Vss (P = .01) compared with etoposide alone. CsA levels ranged from 297 to 5,073 ng/mL. Higher CsA levels (< 2,000 ng/mL v > 2,000 ng/mL) resulted in greater changes in etoposide kinetics: Vss (1.4% v 46%) and T1/2 (40% v 108%). CsA produced a 38% decrease in renal and a 52% decrease in nonrenal CL of etoposide. Etoposide with CsA levels > 2,000 ng/mL produced a lower WBC count nadir (900/mm3 v 1,600/mm3) compared with baseline etoposide cycles. CONCLUSIONS: High-dose CsA produces significant increases in etoposide systemic exposure and leukopenia. These pharmacokinetic changes are consistent with inhibition by CsA of the multidrug transporter P-glycoprotein in normal tissues. Etoposide doses should be reduced by 50% when used with high-dose CsA in patients with normal renal and liver function. Alterations in the disposition of other multidrug resistance (MDR)-related drugs should be expected to occur with modulation of P-glycoprotein function in clinical trials.

Expression of multidrug resistance gene mdr1 mRNA in a subset of normal bone marrow cells.

The multidrug resistance gene mdr1, encoding P-glycoprotein (P-gp), can be expressed at high levels in tumour cells derived from normal tissues with constitutive high expression of this gene. In myelogenous leukaemia, the incidence of increased expression of mdr1 gene contrasts with the low expression of this gene in normal bone marrow (b.m.). To detect cells expressing mdr1 gene in normal and post-chemotherapy b.m., we used in situ RNA hybridization and RNA phenotyping by the polymerase chain reaction for mdr1 mRNA detection. The presence of P-gp was evaluated by immunocytochemistry with MRK16. Fifteen b.m. (eight normal and seven post chemotherapy) were tested by in situ hybridization and either PCR (three b.m.) or immunocytochemistry (11 b.m.) or both (one b.m.). With in situ mRNA hybridization, a subset (7.7% +/- 3.1%) of b.m. cells expressed mdr1 mRNA in all cases tested, with no significant differences between normal b.m. and post chemotherapy b.m. 18% of myeloid recognizable cells and 7% of the cells with lymphoid morphology expressed mdr1 mRNA. By RNA phenotyping, the four samples tested for in situ hybridization and two additional post chemotherapy b.m. expressed mdr1. MRK16 was unable to detect a significant number of cells expressing P-gp either by immunocytochemistry in the 12 b.m. tested for in situ hybridization (0% in nine cases; 0.4%, 1% and 3% of positive cells in three cases), or by flow cytometry in six additional normal b.m. (0-1.4% positive cells).

Multifactorial mechanisms associated with broad cross-resistance of ovarian carcinoma cells selected by cyanomorpholino doxorubicin.

The cyanomorpholino derivative of doxorubicin (MRA-CN) is a DNA intercalator and alkylator that is a highly potent cytotoxin, non-cross-resistant in multidrug-resistant cells, and noncardiotoxic in comparison with doxorubicin. To further examine mechanisms of action and resistance to MRA-CN, a cell line resistant to MRA-CN, ES-2R, was established by growing a human ovarian carcinoma cell line, ES-2, in increasing concentrations of the drug. The resistant subline was 4-fold resistant to MRA-CN and cross-resistant to other DNA cross-linking agents, cisplatin (7-fold) and carmustine (3-fold), as well as to the DNA strand-breaking agents etoposide (6-fold), doxorubicin (2-fold), bleomycin (5-fold), and ionizing radiation (2-fold). In contrast, ES-2R cells were not cross-resistant to vinblastine. Several months of additional growth of ES-2R cells in MRA-CN did not yield higher, stable levels of drug resistance. A low level of P-glycoprotein was detectable in the ES-2R cells. However, the extent of intracellular accumulation of [3H]MRA-CN by this resistant cell line was identical to that of the sensitive line. The number of DNA cross-links formed by cisplatin in ES-2R was only 50% of that of the ES-2 cells and was associated with a 50% increase in the rate of repair of these cross-links in the resistant cells. Ionizing radiation induced similar amounts of single- and double-strand breaks in the ES-2 line as well as in the ES-2R cells. There was no apparent difference between the two cell lines in the rate and extent of repair of these DNA breaks. Thus, enhanced DNA repair cannot explain the phenomenon of cross-resistance to radiation. Comparisons of glutathione (GSH) content and the enzymes involved in GSH homeostasis showed significant differences. Resistant cells contained 1.5-fold more GSH, a 2.2-fold increase in gamma-glutamyltranspeptidase activity, and a 2.4-fold increase in GSH reductase compared with ES-2 cells (all P less than 0.05). Total glutathione-S-transferase (GST) activity was 2.6-fold higher (P less than 0.01) in the ES-2R line. The pi-class GST subunit by Western blotting and GST activity toward ethacrynic acid were increased 2-fold in the resistant cells. Depletion of GSH levels in ES-2R cells by buthionine sulfoximine restored the sensitivity of ES-2R to MRA-CN. These findings implicate a role for GSH metabolism in the resistance phenotype of ES-2R cells. We have previously reported that these cells have an increased generation time and decreased topoisomerase II content. Thus, the ES-2R cell line exhibits a complex phenotype of broad cross-resistance, which is likely to involve multiple mechanisms, and includes enhanced DNA repair and increased GSH content and GST activity.

High-dose megestrol acetate therapy of ovarian carcinoma: a phase II study by the Northern California Oncology Group.

The activity of high-dose megestrol acetate was studied in 47 patients with epithelial ovarian cancers after failure of initial chemotherapy. The dose of megestrol acetate was 800 mg/d orally (PO) for 4 weeks and then 400 mg/d until tumor progression. Patients generally had far-advanced disease. Prior therapy included cisplatin, doxorubicin, and cyclophosphamide (PAC) or other cisplatin-containing regimens in 37, other combinations in eight, and single agents in only two patients. Seventeen patients (36%) developed intestinal obstructions within the first 2 months on study. Tumor histology was serous in 37, endometrioid in six, and clear-cell in two. Two thirds of the tumors were histologic grade 3, and the others were grade 2. Complete remission was obtained in one patient, with time to progression of 4 months. There were three partial remissions, with times to progression of 4, 5, and 18 months. The overall response rate (complete and partial) was 8%. Three additional patients had minor remissions (3, 5, and 8 months), and five had stable disease, for 3, 4, 5, 6, and 9 months. There was no correlation of response with grade, histologic type, or site of disease, but responding patients had a longer survival from diagnosis to protocol entry and from protocol failure to death than did nonresponding patients. The major side effect of megestrol acetate was increased appetite, which caused one patient to withdraw from the study, and resulted in a 10- to 20-kg weight gain in five patients. Plasma levels of megestrol acetate averaged 600 ng/mL in the first month of therapy and decreased to approximately 400 ng/mL at 8 and 12 weeks, after the drug dosage had been reduced. Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were markedly lower during megestrol therapy compared with pretreatment values. Megestrol acetate at 1 microgram/mL in vitro inhibited soft agar colony formation from one of 17 specimens of ovarian carcinomas. We conclude that megestrol acetate in high doses has modest, but definite, palliative effects in some patients with advanced ovarian carcinoma in whom chemotherapy has failed. A controlled trial of megestrol plus combination chemotherapy as first-line treatment of advanced ovarian carcinoma should be considered.

Dissociation of antitumor potency from anthracycline cardiotoxicity in a doxorubicin analog.

The search for new congeners of the leading anticancer drug doxorubicin has led to an analog that is approximately 1000 times more potent, noncardiotoxic at therapeutic dose levels, and non-cross-resistant with doxorubicin. The new anthracycline, 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin (MRA-CN), is produced by incorporation of the 3' amino group of doxorubicin in a new cyanomorpholinyl ring. The marked increase in potency was observed against human ovarian and breast carcinomas in vitro; it was not accompanied by an increase in cardiotoxicity in fetal mouse heart cultures. Doxorubicin and MRA-CN both produced typical cardiac ultrastructural and biochemical changes, but at equimolar concentrations. In addition, MRA-CN was not cross-resistant with doxorubicin in a variant of the human sarcoma cell line MES-SA selected for resistance to doxorubicin. Thus antitumor efficacy was dissociated from both cardiotoxicity and cross-resistance by this modification of anthracycline structure.