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Phase I/II Trial of the Multidrug-Resistance Modulator Valspodar Combined With Cisplatin and Doxorubicin in Refractory Ovarian Cancer

From the Department of Gynecologic Oncology, Norwegian Radium Hospital, and Department of Clinical Pharmacology, National Hospital, Oslo, Norway; Department of Gynecologic Oncology, St Stephen Hospital, Budapest, Hungary; Department of Medical Oncology, Odense University Hospital, Odense, Denmark; and Novartis Pharmaceuticals, Basel, Switzerland.

Address reprint requests to M. Baekelandt, MD, Department of Gynecologic Oncology, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway; email: mark.baekelandt{at}klinmed.uio.no

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To determine the maximum-tolerated dose (MTD) of doxorubicin when given in combination with cisplatin and the multidrug-resistance (MDR) modulator valspodar and the remission rate induced by this combination in patients with platinum- and anthracycline-resistant ovarian cancer.

PATIENTS AND METHODS: Fifty-nine patients who had failed prior platinum- and anthracycline-based chemotherapy were enrolled. During the dose-finding phase, patients received a loading dose of valspodar (1.5 or 2 mg/kg) via 2-hour intravenous (IV) infusion on day 1 and continuous IV infusion (CIVI) of valspodar (2, 4, or 10 mg/kg/d) over 3 days. Doxorubicin (starting from 20 up to 50 mg/m²) and cisplatin (50 mg/m²) were administered via 15- to 20-minute IV infusions on day 3. During the efficacy phase, patients received at least two treatment cycles unless toxicity was unacceptable, and responding patients and those with stable disease received four to six cycles.

RESULTS: All patients completed at least one cycle of combined treatment. The MTD of doxorubicin was determined to be 35 mg/m² when administered with valspodar at 2 mg/kg loading dose and 10 mg/kg/d CIVI plus 50 mg/m² cisplatin. At these doses, valspodar blood concentrations known to reverse MDR in vitro were reached in all patients. Valspodar was well tolerated at all dose levels. Dose-limiting toxicities of the combination were primarily hematologic and included febrile neutropenia and prolonged leucopenia. The addition of valspodar to the treatment did not worsen cisplatin-related toxicity. Among 33 patients treated at the MTD for doxorubicin, one (3%) had a complete response, and four (12%) had a partial response. An additional seven patients experienced a stabilization of their previously progressive disease. The survival rates at 6 and 12 months were 59% and 19%, respectively.

CONCLUSION: Valspodar can be safely coadministered with doxorubicin and cisplatin. Although the regimen used in this trial produced renewed responses in patients with heavily pretreated, refractory ovarian cancer, the value of valspodar in reversing resistance mediated by P-glycoprotein remains to be determined.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
IN THE UNITED States, about 25,000 women are diagnosed with ovarian cancer annually, the majority of them in an advanced stage of the disease.¹ In up to 80% of the cases, the tumor will initially respond to modern platinum-based combination chemotherapy, but 5-year survival rates in advanced-stage disease remain disappointingly low.² The main reason for these low cure rates is the presence and selection of tumor cell clones that are resistant to cytotoxic drugs. One of the best known mechanisms of drug resistance occurring at the cellular level is the phenomenon of classical multidrug resistance (MDR). MDR, in a strict sense, is the phenotype of cellular cross-resistance to a broad range of structurally and functionally unrelated cytotoxic agents. This type of resistance typically involves natural products, including cytotoxic drugs used in the treatment of ovarian cancer, such as taxanes (paclitaxel), anthracyclines (epirubicin and doxorubicin) and epipodophyllotoxins (etoposide).^3,4 A common feature of the MDR phenotype is the decreased intracellular concentration of the cytotoxic agents administered, resulting from an energy-dependent efflux mechanism. This efflux function can be exerted by P-glycoprotein (Pgp), a 170-kd transmembrane glycoprotein overexpressed in a range of mammalian cell lines selected for MDR.⁵

We have previously shown that the expression of Pgp is an independent marker of prognosis in a group of untreated Fédération Internationale de Gynécologie et Obstétaique stage III ovarian cancer patients.⁶ Also, we demonstrated, in accordance with Holzmayer et al,⁷ that pretreatment Pgp expression predicted drug resistance in patients with advanced ovarian cancer, if at least one MDR-related compound was used in the actual drug combination. A number of studies indicate the clinical relevance of Pgp expression in a broad range of solid tumor types, but results are difficult to interpret because of methodologic problems in the measurement of Pgp expression and the impossibility of determining its functional status in solid-tumor tissue sections.^8,9

In vitro studies showed that Pgp-related MDR can be reversed by a number of substances, and early clinical trials were performed using such drugs as verapamil and cyclosporine. The serious toxicity, related to the high plasma levels required for MDR reversal with these agents, together with a disappointing efficacy, have prompted the development of more potent and less toxic MDR modulators.^10,11 Valspodar (PSC 833) is an MDR modulator that is designed to reverse drug resistance mediated through Pgp. Valspodar is a cyclosporine D analog that is nonnephrotoxic and nonimmunosuppressive.^12,13 In vitro studies have demonstrated that valspodar is approximately five- to 30-fold more potent than cyclosporine.¹⁴ Whereas cyclosporine seems to exert its modulatory effects on Pgp by competitive inhibition,^15,16 valspodar is a high-affinity, noncompetitive inhibitor of Pgp and a poor substrate for Pgp-mediated transport.¹⁴ Preclinical data demonstrated that valspodar can reverse MDR in leukemias and solid tumors in mice.^17,18 Phase I/II trials have established that whole blood concentrations of valspodar sufficient to reverse MDR in resistant cell lines (1,000 to 2,000 ng/mL) can be achieved and are well tolerated in cancer patients.^19,20 Because Pgp has a physiologic role in tissues normally responsible for the transport and excretion of cytotoxic drugs, treatment with Pgp inhibitors delays the elimination of some anticancer drugs. In phase I studies of cytotoxic drugs administered in combination with valspodar, the dose-normalized area under the curve (AUC) of doxorubicin increased by 61% to 74% and the mean AUC of etoposide increased by 84%.^12,19 Therefor, this pharmacokinetic interaction requires appropriate dose reduction of the cytotoxic drugs that are Pgp substrates to provide effective treatment without increasing toxicity.

In a recently reported study in patients with heavily pretreated refractory ovarian cancer, retreatment with reduced doses of paclitaxel in combination with valspodar produced objective tumor responses in patients with known paclitaxel-refractory disease.²⁰ Interim results indicate that two (4%) complete responses (CRs) and two (4%) partial responses (PRs) were observed in 49 assessable patients. Although the overall response rate was modest, the combination of valspodar and paclitaxel was safe, tolerable, and resulted in renewed responses in some patients with refractory ovarian carcinoma. The objectives of the current study were (1) to determine the safety and maximum-tolerated dose (MTD) of doxorubicin in combination with valspodar and cisplatin, and (2) to determine the efficacy of the combination of valspodar, cisplatin, and doxorubicin (administered at the MTD) in patients with ovarian cancer refractory to the combination of anthracyclines and cisplatin.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Selection
Patients were eligible for enrollment onto the study if they had a histologically confirmed diagnosis of epithelial ovarian cancer, refractory to previous combination treatment with cisplatin and an anthracycline. Patients were between 18 and 70 years old and had measurable disease, a World Health Organization performance status of less than two, and a life expectancy of at least 3 months. The refractory state was defined as either progressive disease during treatment with cisplatin and an anthracycline, or stable disease for at least three cycles of this therapy or a relapse within 6 months after discontinuing it. No routine determination of MDR1 or Pgp expression status was performed. Patients with abnormal left ventricular function (ejection fraction < 40%), significant cardiac disease within the previous 12 months, or impaired gastrointestinal, hepatic, renal, hematologic, or neurologic function were excluded, as were patients who had received MDR-modifying agents together with chemotherapy or myelosuppressive cytotoxic agents within 14 days of study entry. Patients who had had other malignancies (with exception of nonmelanoma skin cancer or in situ cervical carcinoma) within the previous 5 years were not included. Medications known to alter cyclosporine pharmacokinetics were not allowed. Laboratory exclusion criteria included a WBC count of less than 3.0 x 10⁹/L, an absolute neutrophil count (ANC) of less than 1.0 x 10⁹/L, a platelet count of less than 100 x 10⁹/L, serum creatinine and total bilirubin more than 1.5 times the institutional upper limit of normal range, and liver enzymes (AST, ALT, -glutamyltransferase, and alkaline phosphatase) more than three times institutional upper limit of normal. The total dose of previously administered anthracyclines allowed for the administration of six cycles of protocol treatment without exceeding a maximum cumulative doxorubicin dose of 550 mg/m². This protocol was approved by the relevant national regulatory authorities and the ethics and human study committees of the different participating institutions. Before their enrollment, all patients provided a signed informed consent. The study was conducted and monitored according to national good clinical practice guidelines.

Study Design and Treatment
The study was a prospective, multicenter, open-label, phase I/II trial of valspodar combined with doxorubicin plus cisplatin. A sequential design was adopted in which patients with confirmed refractory ovarian cancer continued on treatment with cisplatin and doxorubicin with the addition of valspodar, and also with dose adaptations as described later. The study consisted of a dose-finding phase that was run at the Norwegian Radium Hospital and a multi-institutional efficacy phase. Patients were stratified into one of four groups (A, B, C, or D) based on a dose-finding protocol. Patients in group A received valspodar (1.5 mg/kg loading dose, 2 mg/kg/d continuous intravenous infusion [CIVI]), doxorubicin 50 mg/m²/d, and cisplatin 50 mg/m²/d. Patients in group B received valspodar (1.5 mg/kg loading dose, 4 mg/kg/d CIVI dose), doxorubicin 50 mg/m²/d (dose reduction to 20 mg/m²/d as needed), and cisplatin 50 mg/m²/d. Patients in group C received valspodar (1.5 mg/kg loading dose, 10 mg/kg/d CIVI dose), doxorubicin 20 mg/m²/d (dose escalation to 25, 30, or 35 mg/m²/d), and cisplatin 50 mg/m²/d. Patients in group D received valspodar (2 mg/kg loading dose, 10 mg/kg/d CIVI dose), doxorubicin 30 mg/m²/d (dose escalation to 35 or 40 mg/m²/d), and cisplatin 50 mg/m²/d (Table 1).

The dose-finding protocol used to determine the MTD of doxorubicin is listed in Table 1. Initially, three patients were entered into group A (cohort 1) and were treated with the lowest dose of valspodar plus cisplatin and 50 mg/m²/d doxorubicin. If none of the three patients in cohort 1 experienced DLT (defined as prolonged grade 4 neutropenia lasting more than 8 days or any nonhematologic toxicity higher than grade 3), then the next three patients enrolled in the trial would be stratified to group B (cohort 2). If only one of the three patients in group A experienced DLT, then three additional patients would be stratified to group A (for a total of six patients). If only one of these six patients experienced DLT, however, then the next three patients that enrolled would be stratified to the first cohort of group B.

For patients stratified to group B, if no patients in cohort 2 experienced DLT, then the next three patients enrolled would be stratified to group C (cohort 4). If one of the three in cohort 2 experienced DLT, an additional three patients would repeat cohort 2. If only one of these six experienced DLT, then the next three enrolled patients would be stratified to group C (cohort 4). If two or more of these six experienced DLT, then the next three enrolled patients would be stratified to cohort 3 and receive a reduced dose of doxorubicin. Patient stratification beyond cohort 3 and into groups C (cohorts 4 to 7) and D (cohorts 9 and 10) was governed by the following criteria. If no patients in a given cohort experienced DLT, then the next three patients were stratified to the next sequential cohort. If one of three patients experienced DLT, then three additional patients repeated the same cohort. If only one of these six patients experienced DLT, then the next three patients were stratified to the next sequential cohort. If two or more of these six patients experienced DLT and the dose of doxorubicin was more than 20 mg/m², then the previous dose of doxorubicin was defined as the MTD. A complete treatment cycle consisted of 28 days. All patients received a minimum of two treatment cycles, unless toxicity was unacceptable following the first cycle. Responding patients who achieved a CR or PR after four treatment cycles received two additional cycles. Patients with progressive disease were withdrawn from the study after more than two cycles. Patients were observed until January 2000 or death.

Safety and Efficacy Evaluation
After determination of the MTD, the safety and efficacy of combination valspodar plus doxorubicin and cisplatin were assessed. Safety was evaluated by review of adverse events, physical examinations, vital signs, concomitant medication, and selected laboratory tests. The severity of adverse events was graded according to common toxicity criteria. Efficacy end points included response rate and overall survival. The World Health Organization’s criteria of CR, PR, and stable (SD) or progressive disease (PD) were applied to measurable lesions and used to characterize tumor response. CA-125 levels were measured at baseline and before each cycle. Accepted 50% (four samples) and 75% (three samples) CA-125 response criteria²¹ were used as a supplementary assessment of possible antitumor activity. Overall survival was described using Kaplan-Meier survival analysis. Descriptive summary statistics were used to evaluate efficacy variables.

Pharmacokinetic Analysis
Valspodar blood concentrations were measured at the end of the loading dose (ie, 2 hours) and during administration of the CIVI dose (ie, 3 and 19 hours). This analysis was used to determine the optimal dosing regimen necessary to achieve concentrations of valspodar adequate to inhibit Pgp. Blood samples (3 mL) were drawn at the indicated times and stored in ethylenediaminetetraacetic-acid–coated tubes at -20°C. Valspodar concentrations were measured using a valspodar radio-immunoassay kit provided by ANAWA Trading SA (Wangen-Zürich, Switzerland). Originally, a secondary objective was to include a monitoring of doxorubicin pharmacokinetic parameters with and without valspodar given at MTD. Because patients thus would have to receive an extra cycle of treatment with cisplatin and doxorubicin for the purpose of pharmacokinetic monitoring, it was deemed unethical to include patients with known PD in this part of the study. Among the patients with SD, two were recruited for a 10-sample doxorubicin pharmacokinetic profile. Samples were drawn at the end of the doxorubicin infusion and at 2, 20, 40, and 60 minutes and at 2, 4, 6, 12, and 24 hours afterward. Analysis of doxorubicin plasma concentrations was performed according to a previously described high-performance liquid chromatography method.²²

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Characteristics
The characteristics of the 59 patients enrolled onto the study between November 1996 and July 1998 are listed in Table 1. The patients were entered in 10 cohorts: three patients each in cohorts 1, 3, 4, 5, 6, 7, and 8; four patients each in cohorts 2 and 10; and 30 patients in cohort 9. Cohorts were well balanced for age, height, and weight, with mean values of 52.2 years, 66.2 kg, and 166 cm, respectively. At the time of inclusion in the study, the number of different, previously administered chemotherapy regimens was one for 20 patients, two for 21 patients, three for 10 patients, four for five patients, five for two patients and seven for another two patients. Thirty-two patients had PD while on their cisplatin and anthracycline therapy, 24 had SD, and three were included after they had relapsed within 6 months after discontinuing such therapy.

Treatment
All 59 patients completed at least one course of combined cisplatin, doxorubicin, and valspodar treatment and were evaluated for toxicity and response. In the dose-finding part of the study, a total of 84 combined cisplatin, doxorubicin and valspodar treatment cycles were administered, with a median of two cycles per patient (range, one to six cycles), whereas 112 cycles were given in the phase 2 part of the study (median, two cycles; range, one to eight cycles). At the inception of this study, the aim was to give the highest possible dose of doxorubicin and escalate the dose of valspodar. When patients were treated in group B, the profound pharmacologic interaction between doxorubicin and valspodar became obvious. Based on the toxicity observed at this level, together with pharmacokinetic data that were made available to us from other studies, we decided to administer valspodar at its MTD and escalate the doxorubicin dose. This necessitated a reduction in doxorubicin dose to 20 mg/m² in cohort 3. Based on the observed DLT, the MTD of doxorubicin was determined to be 35 mg/m² (given in cohorts 7 and 9). The MTD for the combination doxorubicin/cisplatin and valspodar was identified as a loading dose of valspodar of 2.0 mg/kg over 2 hours; a valspodar CIVI of 10 mg/kg/d; doxorubicin 35 mg/m²; and cisplatin 50 mg/m². The observed DLTs were hematologic; three patients in cohort 2, one in cohort 9, and one in cohort 10 experienced febrile neutropenia. In another patient in cohort 10, a grade 4 thrombocytopenia and prolonged neutropenia occurred. One patient in cohort 1 was diagnosed with febrile neutropenia in the third course of her protocol treatment. Doxorubicin dose reductions were performed six times in four patients (once in two cohort 2 patients and twice in one cohort 1 and one cohort 2 patient). All dose reductions were due to severe hematologic toxicity. Five patients discontinued the study treatment because of adverse events. In only one patient, who accidentally received an overdose of valspodar, was the adverse event considered to be treatment-related (paresthesias, dizziness, and nervousness). In the others, adverse events were related to the underlying disease or technical procedures (eg, central venous catheter thrombosis).

Toxicity
The main toxicity observed was hematologic, with grade 3 or 4 leucopenia occurring in at least one treatment course in 61% of all patients. In more than half of the patients treated at the MTD, at least one episode of grade 3 or 4 leucopenia was noted. In the dose-finding part of the study this toxicity was clinically relevant in six patients who developed febrile neutropenia. One patient (3%) treated at the MTD experienced a grade 4 neutropenia and died of a Staphylococcus aureus–related meningitis and septic shock on day 9 of her second treatment course. This was the only treatment-related death in the study. Grade 3 or 4 thrombocytopenia was fairly frequent at doxorubicin levels around the MTD, but no patients experienced any episodes of abnormal bleeding. Anemia of any grade was observed in 86% of all patients, and was severe, necessitating blood transfusions, in 22%. However, anemia was most often present already at the time patients were enrolled after previous cisplatin/doxorubicin treatment. There was a tendency for anemia and thrombocytopenia to be cumulative and worsen along with the number of cycles given. Hyperbilirubinemia, a valspodar-related toxicity, was observed in 37% of patients, with grade 3 or 4 toxicity occurring in 12%. Hyperbilirubinemia occurred at the beginning of each subsequent cycle, and was often accompanied by a slight and transient increase in transaminase activity. Typically, bilirubin and liver enzymes became normal again within the week after valspodar administration. There was a tendency to more pronounced increases in bilirubin levels with increasing doses of valspodar. Laboratory abnormalities of more than or equal to National Cancer Institute common toxicity criteria (CTC) grade 3 are listed in Table 2. Among the nonhematologic toxicities, listed in Table 3, headache was frequent, but was most probably related to the administration of 5HT3 inhibitors to control nausea and vomiting. Neurological toxicity was mostly limited to transient paresthesias, and occurred most frequently in patients treated at the MTD. Typically, perioral paresthesias or numbness occurred during the simultaneous IV infusion of the valspodar loading and first maintenance doses, and disappeared a few hours after the loading dose was given. Pre-existing low-grade, cisplatin-related, peripheral neuropathy did not worsen during combined valspodar/doxorubicin/cisplatin treatment. No instances of oto- or renal toxicity were reported.

View this table: [in this window] [in a new window]   Table 2. Laboratory Abnormalities of CTC Grade 3

Treatment Efficacy
All patients included in the study had measurable disease, and response was evaluated after at least two cycles of protocol treatment. The three patients who only received a single course are included in the efficacy analysis and considered treatment failures. Of the 33 patients treated at the MTD, one (3%) had a CR and four (12%) a PR, giving an overall response rate of 15%. One additional patient had a 40% decrease in total tumor load of her different measurable lesions for 4 months, and one had a mixed response with disappearance of a pelvic tumor, but SD of an upper abdominal lesion. In the dose-finding part of the study, one patient from cohort 2 had a partial response for 6 months. The median duration of response was 6.5 months (range, 6 months to 4.5 years). In Table 4, disease status at entry is compared with the best treatment effect obtained after at least two cycles of combined therapy. Seven patients (22%) of 32 who had PD while on cisplatin/anthracycline treatment experienced a stabilization of their disease for more than 2 months after the addition of valspodar to their therapy, whereas four of the six responders had PD before their inclusion in the study. When CA-125 response criteria were applied, World Health Organization responses were confirmed in those responding patients who had an elevated pretreatment CA-125. Additionally, four patients met the 50% and one the 75% CA-125 response definitions. When evaluated by World Health Organization criteria, three of these patients had SD both at inclusion and response evaluation and two had a clinical stabilization of PD. Of these five additional CA-125 responders, three were treated at valspodar/doxorubicin doses below the MTD. If one allows a CA-125 response as a sign of treatment effect, then, for the whole group, 11 (18.5%) out of 59 fulfilled the criteria of either a standard World Health Organization or a CA-125 response definition. For the whole study group (phase 1 and 2), Kaplan-Meier survival analysis showed a median survival time of 218 days (95% CI, 177 to 260 days), with 6-, 9-, and 12-month survival rates of 59%, 29%, and 19% respectively.

View this table: [in this window] [in a new window]   Table 4. Comparison of Disease Status Before Enrollment Versus Best Response After Two Cycles of Protocol Treatment for the Whole Patient Group (N = 59)

View larger version (109K): [in this window] [in a new window]   Fig 1. (A) Computed tomography scan of the pelvis demonstrating a tumor (white arrow) in the left pararectal space. Cytologic examination after fine-needle aspiration revealed it to be a relapse of a serous papillary ovarian carcinoma. (B) Computed tomography scan after six cycles of combined cisplatin/doxorubicin and valspodar therapy, showing complete disappearance of tumor.

View larger version (23K): [in this window] [in a new window]   Fig 2. The pharmacokinetics of doxorubicin 50 mg/m2 single-drug administration (dots) compared with doxorubicin 35 mg/m2 combined with valspodar CIVI (line) in patient no. 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

The MTD for doxorubicin in combination with valspodar given as a CIVI was at 35 mg/m², a finding confirming previous studies.^27,28 The toxicity encountered at that level was mainly hematologic, and, although no formal comparison was made in this study, considerably increased as compared with similar treatment without valspodar. This enhanced myelosuppression is mostly related to the pharmacologic interaction between the modulator and the MDR-related anticancer drug (ACD), and has been demonstrated for most modulators currently tested.²⁹ This interaction may be situated at the level of ACD excretion, as valspodar has been shown to inhibit doxorubicin biliary excretion in rats, possibly by inhibiting Pgp in the hepatic canaliculus.³⁰ Also, in MDR1 knockout mice, the excretion of vinblastine in liver, gut and kidney was delayed, resulting in a significant increase in its AUC.³¹ Alternative explanations are increased ACD reabsorption or an interaction with the cytochrome P -450 system.⁸ The nature of the pharmacokinetic interactions suggests that the different mechanisms operate together.³² Although anecdotal in nature, the increase in doxorubicin elimination t_1/2 and AUC observed in our patients was confirmed in other studies using valspodar or cyclosporine combined with doxorubicin.^27,33 Although this pharmacokinetic interaction can explain much of the increased toxicity of the ACDs, it is also critical in terms of defining the mechanism of drug resistance modulation: does any observed activity result form blocking Pgp, or is it merely the result of an increase in dose-intensity? The only way to answer the question in solid tumors is to perform a phase III trial with equivalent dose exposures and expected toxicities in both arms. When valspodar is used as a modulator, this could be obtained by dose reductions of the MDR-related ACD in the 50% to 60% range.¹¹ A recent study comparing both cellular and plasma pharmacokinetics in patients with Pgp-positive acute myeloid leukemia showed that the addition of valspodar to daunorubicin caused a proportionally greater increase in intracellular daunorubicin AUC than in plasma AUC, and this, to a degree that could be clinically relevant.³⁴ Though not being an evaluation of any clinical benefit, the results of that study are a mechanistic proof of the concept of MDR reversal in vivo.

The hyperbilirubinemia associated with the administration of valspodar is not related to an inhibition of Pgp, but of the canalicular multiple organic anion transporter. This bile acid transporter is highly expressed in the liver,³⁵ and its inhibition by valspodar and cyclosporine can lead to hyperbilirubinemia.¹¹ We observed an asymptomatic grade 3 or 4 hyperbilirubinemia in only 12% of the patients. Bilirubin and liver enzyme changes typically normalized within the week after valspodar administration. Bartlett et al³³ noted an incidence of hyperbilirubinemia of 68% for the combination of doxorubicin and cyclosporine, supporting the notion that cyclosporine is a more potent inhibitor of canalicular multiple organic anion transporter than valspodar. An interesting observation from our study was that valspodar did not increase cisplatin-related oto-, neuro-, or nephrotoxicity. No cases of cerebellar ataxia, a potential and dose-limiting toxicity of valspodar itself, were observed here. This toxicity is related to peak serum levels of valspodar, and is, for that reason, more frequent after oral administration of the drug.²⁷ No cases of cerebellar ataxia were reported in the studies in which an IV dosing scheme identical to ours was used.^19,28,36 In the study by Boote et al,¹⁹ cases of ataxia were noted only when IV valspodar maintenance doses of 12 mg/kg/d or higher were administered.

Significant (CTC grade 2) decreases in LVEF were observed in 8.5% of the patients. The only case of clinically significant cardiac toxicity occurred several months after treatment was discontinued and may have been related to a pre-existing mild, but untreated, arterial hypertension in this specific patient. The cardiac toxicity of doxorubicin is attributed largely to its metabolite doxorubicinol.³⁷ Other studies have shown increases of 2.7 to 3.5 times in doxorubicinol AUC after the addition of valspodar or cyclosporine.^27,33 For this reason, cardiac function should be closely monitored when valspodar is used in combination with doxorubicin. In view of the presence of Pgp in most normal tissues and its possible physiologic role as a transporter and part of the xenobiotic detoxification and excretion system, there has been a theoretical concern that use of Pgp-blocking modulators would unveil a range of new toxicities. For the time being, this concern has not been realized. Experiments with MDR1 knockout mice have produced phenotypically normal animals, that have an increased sensitivity for MDR-related toxins.³¹ Recent experiments in a rat model suggest that the pharmacokinetic interaction between doxorubicin and valspodar is significantly lower when doxorubicin is encapsulated in sterically stabilized liposomes.³⁸ Although such observations still are at the preclinical level, this might be a way of further reducing the toxicity and possibly increasing the efficacy of the combination.

The observed efficacy in this study, although modest in proportion of patients fulfilling standard World Health Organization response definitions, should be considered in the challenging clinical context of refractory disease in heavily pretreated patients. Although an approach such as ours can serve as a proof of principle, it is clear that once drug resistance has developed, it most often will be multifactorial and cannot be overcome solely by modulating Pgp-related resistance.^8,39 Overcoming drug resistance in this context would require the development of multifunctional drug-resistance modulators or the combination of different modulators aimed at different resistance mechanisms. Separate lines of evidence suggest that a more appropriate strategy is to focus efforts on modulating drug resistance at the time of first-line treatment. First, Beketic-Oreskovic et al⁴⁰ showed that by coselecting human MES-SA sarcoma cells with valspodar, the mutation rate for doxorubicin-selected resistance was reduced 10-fold.⁴⁰ The emergence of MDR1 mutants was prevented, and resistant cell clones that emerged after valspodar and doxorubicin treatment exhibited altered topo-isomerase II expression, but no Pgp overexpression. Similar results were reported with verapamil,⁴¹ whereas both valspodar and cyclosporine were shown to prevent the induction of MDR1 gene expression after exposure of a leukemia cell line to epirubicin.⁴² These different observations suggest that modulators of Pgp should be used at the time of first-line treatment. In an elegant study by Abolhoda et al,⁴³ five patients underwent isolated lung perfusion with doxorubicin for treatment of unresectable metastatic sarcoma. Tumor and normal tissue control biopsies were taken before treatment and 20 and 50 minutes after doxorubicin perfusion. In four of five patients, a three- to 15-fold increase in MDR1 RNA levels was detected after 50 minutes in tumor tissue but not in normal tissue. The gene activation was reversible and expression levels decreased over time after drug removal. This was the first in vivo demonstration of the acute and transient induction of MDR1 expression, a phenomenon of possibly profound significance for treatment outcome. Apart from all the methodologic problems involved in measuring Pgp expression and defining Pgp positivity, the reversible nature of the changes observed in the study by Abolhoda et al⁴³ is an important argument against limiting the inclusion in solid tumor drug resistance trials to patients with Pgp-positive tumors only. In agreement with these observations, an international randomized phase III trial was performed in the first-line treatment of patients with advanced ovarian cancer. Patients were randomized between standard treatment with six cycles of carboplatin and paclitaxel in one arm and carboplatin, paclitaxel (dose-reduced) and oral valspodar in the other. The primary end point was progression-free survival, a more relevant end point than response rate for studies of resistance modulation. Results of this trial are awaited and will be critical in determining the clinical usefulness of valspodar in advanced ovarian cancer.

Finally, and further complicating the picture, recent experiments have suggested that valspodar, in addition to its MDR-modulating effect, has other mechanisms of action. Lehne et al⁴⁴ showed that exposure to 1 µmol/L valspodar resulted in growth inhibition, cytokinesis failure and apoptosis of Pgp-rich leukemia cells. The inhibition of Pgp resulted in a selective cytotoxicity in the Pgp-expressing cell lines. Other studies have shown that valspodar activates the synthesis of ceramide,^45,46 a lipid messenger that mediates apoptosis, the final common pathway in cell kill by ACDs. In these cell line experiments, treatment with valspodar alone^45,46 or in combination with doxorubicin⁴⁶ resulted in an increase in cellular ceramide along with a progressive decrease in cell survival. Analysis of DNA in cells treated with valspodar showed the characteristic changes of apoptosis. The possible contribution of these actions to the efficacy of valspodar should be further explored in future studies.

In conclusion, we have determined the MTD of doxorubicin together with cisplatin and valspodar given as a CIVI in patients with refractory ovarian cancer. Myelotoxicity, although enhanced as expected from the pharmacokinetic interaction between doxorubicin and valspodar, was manageable, and no unexpected nonhematologic toxicity was observed. Although the overall efficacy of the combination in this challenging clinical setting was only modest, the addition of valspodar to the treatment of cisplatin and doxorubicin refractory ovarian cancer patients produced renewed responses. The preclinical and phase II evidence available at this time suggests that further studies should focus on the role of valspodar and other modulators in the first-line treatment of essentially chemosensitive malignancies that have a high rate of relapse.

	ACKNOWLEDGMENTS

 
We thank Dr Ignace Vergote from the Department of Gynecological Oncology, Gasthuisberg University Hospital, Leuven, Belgium, and Dr Tamás Pulay from the National Institute of Oncology, Budapest, Hungary, for their participation in this trial. We also thank Marit Hallberg and Björg Sinding-Larsen for excellent technical assistance with the analyses of doxorubicin in plasma.

	NOTES

 
B.G. is medical advisor for the Nordic countries with Novartis Pharmaceuticals. None of the other authors has any potential financial conflicts of interest in relation to Novartis Pharmaceuticals or any of the compounds used in this study.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted August 1, 2000; accepted March 22, 2001.

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