Sunitinib does not accelerate tumor growth in patients with metastatic renal cell carcinoma.

Preclinical studies have suggested that sunitinib accelerates metastases in animals, ascribing this to inhibition of the vascular endothelial growth factor receptor or the tumor's adaptation. To address whether sunitinib accelerates tumors in humans, we analyzed data from the pivotal randomized phase III trial comparing sunitinib and interferon alfa in patients with metastatic renal cell carcinoma. The evidence clearly shows that sunitinib was not harmful, did not accelerate tumor growth, and did not shorten survival. Specifically, neither longer sunitinib treatment nor a greater effect of sunitinib on tumors reduced survival. Sunitinib did reduce the tumor's growth rate while administered, thereby improving survival, without appearing to alter tumor biology after discontinuation. Concerns arising from animal models do not apply to patients receiving sunitinib and likely will not apply to similar agents.

Phase 2 trial of romidepsin in patients with peripheral T-cell lymphoma.

Romidepsin (depsipeptide or FK228) is a histone deacetylase inhibitor, one of a new class of agents active in T-cell lymphoma. A phase 2 trial was conducted in cutaneous (CTCL) and peripheral (PTCL) T-cell lymphoma. Major and durable responses in CTCL supported the approval of romidepsin for CTCL. Forty-seven patients with PTCL of various subtypes including PTCL NOS, angioimmunoblastic, ALK-negative anaplastic large cell lymphoma, and enteropathy-associated T-cell lymphoma were enrolled. All patients had received prior therapy with a median of 3 previous treatments (range 1-11); 18 (38%) had undergone stem-cell transplant. All patients were evaluated for toxicity; 2 patients discovered to be ineligible were excluded from response assessment. Common toxicities were nausea, fatigue, and transient thrombocytopenia and granulocytopenia. Complete responses were observed in 8 and partial responses in 9 of 45 patients, for an overall response rate of 38% (95% confidence interval 24%-53%). The median duration of overall response was 8.9 months (range 2-74). Responses were observed in various subtypes, with 6 responses among the 18 patients with prior stem-cell transplant. The histone deacetylase inhibitor romidepsin has single agent clinical activity associated with durable responses in patients with relapsed PTCL.

Phase II multi-institutional trial of the histone deacetylase inhibitor romidepsin as monotherapy for patients with cutaneous T-cell lymphoma.

PURPOSE Romidepsin (depsipeptide or FK228) is a member of a new class of antineoplastic agents active in T-cell lymphoma, the histone deacetylase inhibitors. On the basis of observed responses in a phase I trial, a phase II trial of romidepsin in patients with T-cell lymphoma was initiated. PATIENTS AND METHODS The initial cohort was limited to patients with cutaneous T-cell lymphoma (CTCL), or subtypes mycosis fungoides or Sézary syndrome, who had received no more than two prior cytotoxic regimens. There were no limits on other types of therapy. Subsequently, the protocol was expanded to enroll patients who had received more than two prior cytotoxic regimens. Results Twenty-seven patients were enrolled onto the first cohort, and a total of 71 patients are included in this analysis. These patients had undergone a median of four prior treatments, and 62 patients (87%) had advanced-stage disease (stage IIB, n = 15; stage III, n= 6; or stage IV, n = 41). Toxicities included nausea, vomiting, fatigue, and transient thrombocytopenia and granulocytopenia. Pharmacokinetics were evaluated with the first administration of romidepsin. Complete responses were observed in four patients, and partial responses were observed in 20 patients for an overall response rate of 34% (95% CI, 23% to 46%). The median duration of response was 13.7 months. CONCLUSION The histone deacetylase inhibitor romidepsin has single-agent clinical activity with significant and durable responses in patients with CTCL.

Role of a doxorubicin-containing regimen in relapsed and resistant lymphomas: an 8-year follow-up study of EPOCH.

PURPOSE: Curative up-front regimens for non-Hodgkin's lymphomas contain doxorubicin, vincristine, and cyclophosphamide, whereas salvage regimens generally contain non-cross-resistant agents. We hypothesized that up-front agents may be highly effective for salvage and developed an infusional regimen based on in vitro evidence of increased efficacy. PATIENTS AND METHODS: A prospective phase II study of etoposide, vincristine, and doxorubicin over 96 hours with bolus cyclophosphamide and oral prednisone (EPOCH) was performed in 131 patients with relapsed or resistant lymphoma. RESULTS: Seventy-nine percent of patients had aggressive histologies, 46% were considered high risk by the International Prognostic Index, and 34% had resistant disease. Eighty-eight percent of patients had received at least four of the agents in EPOCH, and 94% had received doxorubicin. In 125 assessable patients, 29 (24%) achieved complete responses and 60 (50%) achieved partial responses. Among 42 patients with resistant disease, 57% responded, and in 28 patients with relapsed aggressive de novo lymphomas, 89% responded with 54% complete responses. With a median follow-up of 76 months, the overall and event-free survivals (EFS) were 17.5 and 7 months, respectively. In 33 patients with sensitive aggressive disease who did not receive stem-cell transplantation, EFS was 19% at 36 months. Toxicity was primarily hematologic, with an 18% incidence of febrile neutropenia. No clinically significant cardiac toxicity was observed, despite no maximum cumulative doxorubicin dose. CONCLUSION: EPOCH is highly effective in patients who had previously received most/all of the same drugs and produces durable remissions in curable subtypes. Salvage regimens need not contain non-cross-resistant agents, and infusional schedules may partially reverse drug resistance and reduce toxicity.

Expression of the multidrug resistance-associated protein gene in refractory lymphoma: quantitation by a validated polymerase chain reaction assay.

Previous work investigating the role of MDR-1 overexpression in relapsed and refractory lymphoma led us to investigate a possible role for multidrug resistance-associated protein (MRP) as a cause of resistance in patients who did not overexpress MDR-1. A quantitative polymerase chain reaction (PCR) method for measuring MRP expression was validated. Immunoblot analysis suggested that no major discrepancy was present between mRNA expression and protein levels. MRP levels were found to be independent of sample tumor content by immunophenotyping, suggesting that the presence of normal cells had no significant impact on measurements of MRP expression. We evaluated MRP in 55 biopsy samples from 40 patients with refractory lymphoma enrolled on a trial of infusional chemotherapy (EPOCH). Pre- and post-EPOCH samples were available from 15 patients. MRP levels were also evaluated in 16 newly diagnosed, untreated lymphoma patient samples. No significant difference in MRP mRNA expression was noted between pre- and post-EPOCH groups. Also, MRP levels in the newly diagnosed patient samples were not significantly different from either pre- or post-EPOCH groups. Two of 15 paired pre- and post-EPOCH patient samples exhibited overexpression of MRP after EPOCH chemotherapy, with measured increases of 10-fold and 18-fold. We conclude that MRP overexpression is not responsible for non-P-glycoprotein (Pgp)-mediated drug resistance in the majority of these patients, although it may be important in a subset of patients. Defining this subset prospectively could aid in the development of clinical trials of MRP modulation in drug-resistant lymphoma.

Identification of mutations at DNA topoisomerase I responsible for camptothecin resistance.

A camptothecin-resistant cell line that exhibits more than 600-fold resistance to camptothecin, designated CPT(R)-2000, was established from mutagen-treated A2780 ovarian cancer cells. CPT(R)-2000 cells also exhibit 3-fold resistance to a DNA minor groove-binding ligand Ho33342, a different class of mammalian DNA topoisomerase I inhibitors. However, CPT(R)-2000 cells exhibit no cross-resistance toward drugs such as Adriamycin, amsacrine, vinblastine, and 4'-dimethyl-epipodophyllotoxin. The mRNA, protein levels, and enzyme-specific activity of DNA topoisomerase I are relatively the same in parental and CPT(R)-2000 cells. However, unlike the DNA topoisomerase I activity of parental cells, which can be inhibited by camptothecin, that of CPT(R)-2000 cells cannot. In addition, parental cells after camptothecin treatment results in a decrease in the level of DNA topoisomerase I, whereas CPT(R)-2000 cells are insensitive to camptothecin treatment. These results suggested that the mechanism of camptothecin resistance is most likely due to a DNA topoisomerase I structural mutation. This notion is supported by DNA sequencing results confirming that DNA topoisomerase I of CPT(R)-2000 is mutated at amino acid residues Gly717 to Val and Thr729 to Ile. We also used the yeast system to examine the mutation(s) responsible for camptothecin resistance. Our results show that each single amino acid change results in partial resistance, and the double mutation gives a synergetic effect on camptothecin resistance. Because both mutation sites are near the catalytic active center, this observation raises the possibility that camptothecin may act at the vicinity of the catalytic active site of the enzyme-camptothecin-DNA complex.

Initial pharmacokinetics and bioavailability of PSC 833, a P-glycoprotein antagonist.

Resistant cancer cells have been shown to overexpress a 170-kd membrane glycoprotein called P-glycoprotein. P-glycoprotein, a product of the multidrug resistance 1 gene, functions as an energy-dependent efflux pump that decreases intracellular drug concentrations. A variety of nonchemotherapeutic agents have been shown to inhibit P-glycoprotein-dependent drug efflux including cyclosporin. PSC 833 is a nonimmunosuppressive derivative of cyclosporin D with the ability to reverse multidrug resistance because of P-glycoprotein overexpression in vitro. As part of early clinical development of PSC 833, the authors investigated the bioavailability of an oral formulation of PSC 833. PSC 833 (3 mg/kg) was administered as a 2-hour intravenous infusion on day 1 of the treatment cycle. Serial blood samples for the determination of PSC 833 whole blood concentrations were obtained after both the intravenous and oral doses. On day 5 of the study, patients received a single oral dose (9 mg/kg) of PSC 833. A total of 14 patients were treated. The intravenous data were best described by a two-compartment open model. The oral data also were described using a two-compartment model, with oral absorption incorporating a lag time to account for possible delays in absorption. There was large intra- and interpatient variability in the pharmacokinetics of PSC 833 in these patients. The absolute bioavailability of PSC 833 was 34% but ranged from 3% to 58% of the administered dose. The clearance (CI) of PSC 833, in general, was consistent between the two dose forms administered. The pharmacokinetic behavior of PSC 833 appears to be similar to that of cyclosporine.

Tubulin from paclitaxel-resistant cells as a probe for novel antimicrotubule agents.

PURPOSE: Treatment with paclitaxel (PTX) can lead to the appearance of drug resistance with accompanying changes in tubulin. The purpose of this study was to develop an assay for microtubule-active agents that are able to circumvent changes in tubulin that result in acquired resistance to paclitaxel. METHODS: The assay measured the promotion of microtubule polymerization when target agents were added to solutions containing tubulin purified from cultured cells. Tubulin was prepared from PTX-sensitive 1A9 ovarian carcinoma cells and from a PTX-resistant clone. Polymerization was monitored spectrophotometrically and validated by electron microscopy. RESULTS: Exposure of tubulin isolated from PTX-sensitive 1A9 ovarian carcinoma cells to substoichiometric PTX resulted in polymerization equivalent to that observed with brain tubulin. In contrast, tubulin from a PTX-resistant 1A9 clone failed to polymerize under identical conditions. If a C-2-modified analog of PTX (2-debenzoyl-2-(m-azidobenzoyl)paclitaxel) was substituted for PTX in the same experiment, the tubulins from both sensitive and resistant cells polymerized as well as brain tubulin. As predicted from these results, the PTX analog was nearly as cytotoxic to the PTX-resistant cells as it was to the parental cells: the relative resistance of the resistant cells compared to the parental is only 3-5-fold for the PTX analog versus 25-30-fold for PTX. CONCLUSION: Polymerization of purified tubulin from the paclitaxel-resistant cells provided an assay for agents able to circumvent the tubulin alterations that result in acquired paclitaxel resistance.

Methods to detect P-glycoprotein-associated multidrug resistance in patients' tumors: consensus recommendations.

Multidrug resistance (MDR), especially that associated with overexpression of MDR1 and its product, P-glycoprotein (Pgp), is thought to play a role in the outcome of therapy for some human tumors; however, a consensus conclusion has been difficult to reach, owing to the variable results published by different laboratories. Many factors appear to influence the detection of Pgp in clinical specimens, including its low and heterogeneous expression; conflicting definitions of detection end points; differences in methods of sample preparation, fixation, and analysis; use of immunological reagents with variable Pgp specificity and avidity and with different recognition epitopes; use of secondary reagents and chromogens; and differences in clinical end points. Also, mechanisms other than Pgp overexpression may contribute to clinical MDR. The combined effect of these factors is clearly important, especially among tumors with low expression of Pgp. Thus, a workshop was organized in Memphis, Tennessee, to promote the standardization of approaches to MDR1 and Pgp detection in clinical specimens. The 15 North American and European institutions that agreed to participate conducted three preworkshop trials with well-characterized MDR myeloma and carcinoma cell lines that expressed increasing amounts of Pgp. The intent was to establish standard materials and methods for a fourth trial, assays of Pgp and MDR1 in clinical specimens. The general conclusions emerging from these efforts led to a number of recommendations for future studies: (a) although detection of Pgp and MDR1 is at present likely to be more reliable in leukemias and lymphomas than in solid tumors, accurate measurement of low levels of Pgp expression under most conditions remains an elusive goal; (b) tissue-specific controls, antibody controls, and standardized MDR cell lines are essential for calibrating any detection method and for subsequent analyses of clinical samples; (c) use of two or more vendor-standardized anti-Pgp antibody reagents that recognize different epitopes improves the reliability of immunological detection of Pgp; (d) sample fixation and antigen preservation must be carefully controlled; (e) multiparameter analysis is useful in clinical assays of MDR1/Pgp expression; (f) immunostaining data are best reported as staining intensity and the percentage of positive cells; and (g) arbitrary minimal cutoff points for analysis compromise the reliability of conclusions. The recommendations made by workshop participants should enhance the quality of research on the role of Pgp in clinical MDR development and provide a paradigm for investigations of other drug resistance-associated proteins.

bcl-2 protein downregulation is not required for differentiation of multidrug resistant HL60 leukemia cells.

Parental and multidrug resistant HL60 leukemia cell lines were used to study coupling of expression of apoptotic/cytostatic (bcl-2, bax, bclxL, p21/Waf1, and c-myc) genes during differentiation. The multidrug resistant HL60 cell line, HL60/ADR, was less sensitive than parental cells to cytostatic activity of low (0.4-2 ng/ml) doses of PMA. However, during treatment with standard differentiating doses of PMA (10 ng/ml), no difference between the two cell lines in cytostasis and differentiation was found. Downregulation of c-myc and upregulation of p21/Waf1 proteins showed the same time-course in both cell lines. The bcl-2 mRNA was rapidly downregulated while bax and bclxL gene expression was not altered in both differentiating HL60 and HL60/ADR cells. Significant downregulation of bcl-2 protein occurred only in parental HL60 cells. In HL60/ADR, despite rapid cessation of bcl-2 protein synthesis, almost no change in steady-state bcl-2 protein level was found. The lack of bcl-2 protein downregulation was a result of the prolonged half-life of this protein in HL60/ADR cells. Thus, although downregulation of bcl-2 mRNA is coupled to differentiation, actual loss of bcl-2 protein is not required for accomplishment of the differentiation program.

Transfection of a human topoisomerase II alpha gene into etoposide-resistant human breast tumor cells sensitizes the cells to etoposide.

The etoposide-resistant human breast cancer cell line MDA-VP was derived from MDA-parent cells by sequential selection in increasing concentrations of etoposide. MDA-VP cells express a lower amount of topoisomerase II alpha mRNA than the MDA-parent does, have mutations in topoisomerase II alpha (topo II) cDNA, and show cross-resistance to doxorubicin and amsacrine. We investigated whether transfer of a normal human topoisomerase II alpha (H-topo II) gene into MDA-VP cells could overcome their resistance to etoposide. H-topo II in a mammalian expression vector containing a glucocorticoid-inducible mouse mammary tumor virus (MMTV) promoter (pMAMneo) was transfected into MDA-VP cells (MDA-VP-hTOP2MAM). These H-topo II-transfected cells showed increased H-topo II mRNA expression and protein levels compared with MDA-VP parental cells or with MDA-VP cells transfected with the control pMAM vector (MDA-VP-MAM). Following cell exposure to dexamethasone, DNA-protein cleavable complex formation and cytotoxicity induced by etoposide, doxorubicin, and amsacrine were increased in the MDA-VP-hTOP2MAM cells compared with MDA-VP-MAM cells. However, these changes were short-lived, and by 24 h, cytotoxicity, cleavable DNA-protein complex formation, and H-topo II protein levels returned to baseline values. These results indicate that sensitivity of MDA-VP cells correlated with changes in cellular H-topo II. The gene transfer of a normal H-topo II gene can sensitize MDA-VP cells to the actions of multiple antineoplastic agents that target topo II.

Clinical reversal of multidrug resistance.

Reversal of drug resistance offers the hope of increasing the efficacy of conventional chemotherapy. We tested dexverapamil as a P-glycoprotein antagonist in combination with EPOCH chemotherapy in refractory non-Hodgkin's lymphoma. In a cross-over design, dexverapamil was added to EPOCH after disease stabilization or progression occurred. Objective responses were observed in 10 of 41 assessable patients. Biopsies for mdr-1 were obtained before EPOCH treatment and at the time of cross-over to dexverapamil. Levels of mdr-1 were low before EPOCH, but increased four-fold or more in 42% of patients in whom serial samples were obtained. Pharmacokinetic analysis revealed median peak concentrations of dexverapamil and its metabolite, nor-dexverapamil, of 1.66 mumol/l and 1.58 mumol/l, respectively. Since both are comparable antagonists, a median peak total reversing concentration of 3.24 mumol/l was achieved. Pharmacokinetic analysis of doxorubicin and etoposide levels confirmed a delay in the clearance of doxorubicin ranging from 5% to 24%; no change in the pharmacokinetics of etoposide was observed. This study provides sufficient rationale for testing dexverapamil in a randomized clinical trial.

Controlled trial of dexverapamil, a modulator of multidrug resistance, in lymphomas refractory to EPOCH chemotherapy.

PURPOSE: Overexpression of the multidrug resistance gene (mdr-1) is present in up to 60% of relapsed lymphomas. To study its role in lymphomas, we conducted a controlled trial of dexverapamil, an inhibitor of the mdr-1 gene product, P-glycoprotein (Pgp), in lymphomas refractory to etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (EPOCH) chemotherapy. PATIENTS AND METHODS: Eligible patients had recurrent Hodgkin's (HD) or non-Hodgkin's lymphomas (NHL) and measurable disease. Patients initially received EPOCH alone and those with stable tumor over two cycles or progressive disease crossed over to receive dexverapamil and EPOCH on subsequent cycles. Dexverapamil was escalated eight dose levels, from 240 to 1,200 mg/m2/d. When possible, serial biopsies were obtained to measure mdr-1 expression by quantitative polymerase chain reaction (PCR). RESULTS: Of 154 patients entered onto the trial, 109 had NHL and 45 had HD. The median age was 44 years, 67% had stage IV disease, and the median number of prior regimens was two (range, one to 12) in NHL and one (range, one to four) in HD. Sixty-four patients (42%) crossed over, of which eight were not assessable. The maximum-tolerated dose of dexverapamil was 900 mg/m2/d. Among 41 NHL patients (excluding mycosis fungoides), there were three complete responses (CRs) and two partial responses (PRs) (12%) and five minor responses (MRs); two of 10 HD patients achieved PRs. The mdr-1 level was measured in 44 biopsies from 19 patients. Pretherapy, mdr-1 was low (median, 2.5 U) but increased (median, 12.2 U) at crossover. Of six patients with mdr-1 levels greater than 15 U, three responded to dexverapamil, while only one of eight patients with mdr-1 levels less than 15 U responded. EPOCH and dexverapamil were well tolerated, but compared with EPOCH alone, produced more hematologic toxicity. CONCLUSION: These results suggest that Pgp plays a role in clinical drug resistance of lymphomas. However, they also suggest that mechanisms other than Pgp are prominent in heavily pretreated patients and that, although Pgp inhibition may be necessary, it is probably insufficient. Earlier intervention with dexverapamil may be more effective and warrants further study.

Phase II study of paclitaxel in relapsed non-Hodgkin's lymphomas.

PURPOSE: To assess the efficacy and toxicity of paclitaxel administered as a 96-hour infusion to patients with relapsed non-Hodgkin's lymphomas (NHLs). PATIENTS AND METHODS: Eligible patients had relapsed NHL and measurable disease and were considered incurable. Paclitaxel was infused at a dose of 140 mg/m2 every 3 weeks. Premedications to prevent paclitaxel hypersensitivity reactions were not administered and no patients received corticosteroids. Expression of the multidrug resistance (mdr-1) gene was determined in tumor from 17 patients by mRNA quantitative polymerase chain reaction (PCR). RESULTS: Thirty-one patients received a total of 99 cycles of paclitaxel. Two patients were not assessable for response. The median age was 50 years, 71% had stage IV disease, and intermediate/high-grade histology was present in 65% of patients. Patients had received a median of three prior chemotherapy regimens, and 68% of patients had responded to the previous chemotherapy (chemotherapy-sensitive). Of 29 assessable patients, five (17%) achieved a partial response (PR). With a median potential follow-up time of 17 months, the median event-free and overall survival durations were 1.6 and 7.5 months, respectively. No correlation was found between response to paclitaxel and extent of prior treatment or response. The mdr-1 gene was easily detectable in 14 of 17 tumor biopsies, but was low in all but one sample. The most serious toxicity was grade 4 neutropenia, which occurred during 14% of cycles. CONCLUSION: Paclitaxel was well tolerated, but had a low response rate in patients with relapsed NHLs. There was no clear association between response to paclitaxel and extent of our response to prior treatment. Most patients had chemotherapy-sensitive disease, which suggests that the low response rate to paclitaxel was probably not due to general chemotherapy resistance. Paclitaxel provided good palliation in a minority of patients and is a reasonable agent to consider for use in patients who have failed to respond to standard chemotherapy.

Modulation of multidrug resistance by dexverapamil in EPOCH-refractory lymphomas.

We conducted a controlled trial of dexverapamil, an inhibitor of Pgp, in 45 Hodgkin's (HD) and 154 Non-Hodgkin's (NHL) lymphomas refractory to EPOCH chemotherapy. A total of 154 patients initially received EPOCH alone and (4.2%) with stable disease over two cycles or progressive disease "crossed over" to receive dexverapamil with EPOCH. Dexverapamil was escalated 8 dose levels, from 240 to 1200 mg/m2 per day. When possible, serial biopsies were obtained to measure MDR-1 expression by quantitative polymerase chain reaction. Median age was 44 years, 67% had stage IV disease, and median (range) prior regimens were 2 (1-12) in NHL and 1 (1-4) in HD. The maximum tolerated dose of dexverapamil was 900 mg/m2/day, and median plasma average concentrations of dexverapamil and nor-dexverapamil were 1.2 and 1.4 microM, respectively. There were 3 complete and 2 partial responses (12%) and 5 minor responses in NHL, and 2 of 10 HD patients achieved partial responses. MDR-1 was measured in 44 biopsies from 19 patients. Pre-therapy, MDR-1 was low (median 2.5 U) but increased (median 12.2 U) at cross-over. Among 6 patients with MDR-1 > 15, 3 responded to dexverapamil whereas only 1/8 patients with MDR-1 < 15 responded. EPOCH and dexverapamil were well tolerated. This study suggests that MDR-1 plays a role in clinical drug resistance of lymphomas, but also suggests that non-MDR-1 mechanisms are present in such patients. Earlier intervention with dexverapamil may be more effective and warrants further study.

Expression of a 95 kDa membrane protein is associated with low daunorubicin accumulation in leukaemic blast cells.

A 95 kDa membrane protein (P-95) has been previously noted to be overexpressed in a doxorubicin-resistant subline of the MCF-7 breast cancer line and in clinical samples obtained from patients with solid tumours refractory to doxorubicin. We performed Western blotting on blast cell lysates from adults with acute myeloid leukaemia, using antisera to P-95. Concomitant flow cytometric assays measured daunorubicin accumulation and retention. Blasts from 16/46 patient samples had detectable P-95 and had reduced accumulation of daunorubicin compared with the negative marrows. Experiments with the P-95 positive MCF-7 multidrug-resistant subline demonstrated decreased daunorubicin accumulation and retention relative to the sensitive parent line. AML blast cells positive for P-95 also demonstrated greater overall in vitro survival in the presence of daunorubicin relative to the P-95-negative samples. The expression of P-95 did not correlate with failure to achieve an initial complete remission with daunorubicin and cytarabine induction chemotherapy. We conclude that the P-95 protein may possess an efflux transporter function, and may represent another mechanism responsible for anthracycline resistance in acute myeloid leukaemia.

Rhodamine efflux patterns predict P-glycoprotein substrates in the National Cancer Institute drug screen.

Fifty-eight cell lines in the National Cancer Institute drug screen were analyzed for their ability to efflux the fluorescent dye rhodamine 123 as a functional assay for P-glycoprotein (Pgp). Using flow cytometry, the rhodamine fluorescence was measured for each cell line under four incubation conditions, i.e., after accumulation in the presence or absence of the Pgp antagonist cyclosporin A and after efflux in rhodamine-free medium in the presence or absence of cyclosporin A. The results in some cell lines were compatible with Pgp-mediated efflux. There was a significant correlation between mdr-1 expression and rhodamine efflux in the 58 cell lines (r = 0.788, p = 0.0001). Using the rhodamine efflux data as a seed for COMPARE analysis with the cytotoxicity data on > 30,000 compounds in the National Cancer Institute drug screen database, hundreds of compounds with high correlation coefficients were identified. Selected compounds were tested for reversal of cross-resistance in a multidrug-resistant cell line. A high degree of reversibility, up to 10,000-fold, for some of the compounds was noted in the presence of the Pgp antagonist PSC 833. This finding suggested that compounds with predominately Pgp-mediated resistance were being identified. Using these compounds as seeds for COMPARE analysis against a more restricted database of 187 standard agents, a series of standard compounds were repeatedly identified as having high correlation coefficients with the newly identified Pgp substrates. These standard agents, including phyllanthoside, bisantrene, and homoharringtonine, constitute an mdr-1 profile. New agents identified as being highly correlated with these compounds may benefit from clinical trials with Pgp antagonists.

Paclitaxel in doxorubicin-refractory or mitoxantrone-refractory breast cancer: a phase I/II trial of 96-hour infusion.

PURPOSE: A phase I study of paclitaxel infused over 96-hours was performed to determine toxicity, maximum-tolerated dose (MTD), and pharmacokinetics in patients with incurable lymphomas and solid tumors. A phase II study was performed at the MTD of paclitaxel in patients with doxorubicin/mitoxantrone-refractory metastatic breast cancer. PATIENTS AND METHODS: In the phase I study, paclitaxel dose levels ranged from 120 to 160 mg/m2, administered on a 21-day cycle. Patients with metastatic breast cancer who had either no response or a partial response (PR) to doxorubicin or mitoxantrone and had measurable disease were eligible for the phase I and II studies. Expression of the multidrug resistance (mdr-1) gene was determined in tumor biopsies by mRNA quantitative polymerase chain reaction. RESULTS: Twelve patients received a total of 73 cycles of paclitaxel on the phase I study. Dose-limiting mucositis and/or grade IV granulocytopenia was reached at 160 mg/m2, and 140 mg/m2 was selected as the phase II dose. Thirty-six consecutive patients with metastatic breast cancer were treated, of whom three were not assessable. The median age was 49 years, with disease in the liver and/or lung in 76%. Patients received a median of two prior regimens for metastatic disease, and 73% had no response to prior doxorubicin or mitoxantrone. Of 33 patients treated with paclitaxel, 16 patients (48%) achieved a PR and five (15%) achieved a minor response (MR). With a median potential follow-up duration of 60 weeks, the median progression-free and overall survival durations were 27 and 43 weeks, respectively. No correlation was found between extent of prior treatment or prior response to doxorubicin/mitoxantrone, and response to paclitaxel. Paclitaxel pharmacokinetics showed a correlation between both granulocyte and mucosal toxicity, and serum steady-state concentrations (Css) more than 0.07 mumol/L. Patients with liver metastases had significantly decreased paclitaxel clearance and higher paclitaxel Css. Levels of mdr-1 were uniformly low in all tumor biopsies studied. CONCLUSION: The recommended phase II dose of paclitaxel is 140 mg/m2 in patients without liver metastases and 105 mg/m2 in patients with liver metastases. Ninety-six-hour infusions of paclitaxel were effective and well tolerated in patients with doxorubicin/mitoxantrone-refractory breast cancer. Prolonged infusion schedules may be more effective than shorter schedules and deserve further study.