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Journal of Clinical Oncology, Vol 19, Issue 6 (March), 2001: 1589-1599
© 2001 American Society for Clinical Oncology

Phase I/II Study of the P-Glycoprotein Modulator PSC 833 in Patients With Acute Myeloid Leukemia

By R. Dorr, C. Karanes, C. Spier, T. Grogan, J. Greer, J. Moore, B. Weinberger, G. Schiller, T. Pearce, M. Litchman, W. Dalton, D. Roe, A. F. List

From the Section of Hematology/Oncology and Bone Marrow Transplantation Program, Arizona Cancer Center, and Departments of Medicine and Pharmacology, University of Arizona College of Medicine, Tucson, AZ; Divisions of Hematology and Medical Oncology, Vanderbilt University Medical Center, Nashville, TN; Wayne State University, Detroit, MI; Duke University Medical Center, Durham, NC; Louisiana State University, Shreveport, LA; University of California at Los Angeles, Los Angeles, CA; and Novartis Pharmaceutical Corporation, East Hanover, NJ.

Address reprint requests to Robert T. Dorr, PhD, Arizona Cancer Center, 1515 N. Campbell Ave, P.O. Box 245024, Tucson, AZ 85724-5024.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To determine the maximum-tolerated dose, pharmacokinetic interaction, and activity of PSC 833 compared with daunorubicin (DNR) and cytarabine in patients with poor-risk acute myeloid leukemia.

PATIENTS AND METHODS: Patients received ara-C 3 g/m2/d on 5 consecutive days, followed by an IV loading dose of PSC 833 (1.5 mg/kg) and an 84-hour continuous infusion escalating from 6, 9, or 10 mg/kg/d. Daunorubicin was administered as a 72-hour continuous infusion at 34 or 45 mg/kg/d. Responding patients received consolidation chemotherapy with DNR pharmacokinetics performed without PSC-833 on day 1, and with PSC-833 on day 4. Response was correlated with expression of P-glycoprotein and lung resistance protein (LRP), and in vitro sensitization of leukemia progenitors to DNR cytotoxicity by PSC 833.

RESULTS: All 43 patients are assessable for toxicity and response. Grade 3 or greater hyperbilirubinemia (70%) was the only dose-dependent toxicity. Four patients (9%) succumbed to treatment-related complications. Twenty-one patients (49%) achieved a complete remission or restored chronic phase, including 10 of 20 patients treated at the maximum-tolerated dose of 10 mg/kg/d of PSC-833 and 45 mg/m2 of DNR. The 95% confidence interval for complete response was 33.9% to 63.7%. Administration of PSC 833 did not alter the mean area under the curve for DNR, although clearance decreased approximately two-fold (P = .04). Daunorubicinol clearance decreased 3.3-fold (P = .016). Remission rates were not effected by mdr-1 expression, but LRP overexpression was associated with chemotherapy resistance.

CONCLUSION: Combined treatment with infused PSC 833 and DNR is well tolerated and has activity in patients with poor risk acute myeloid leukemia. Administration of PSC 833 delays elimination of daunorubicinol, but yields variable changes in DNR systemic exposure.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
APPROXIMATELY 6,000 new cases of acute myeloid leukemia (AML) are reported each year in the United States, with a corresponding disease-related mortality that approaches 80%.1 Although chemotherapy induction regimens that include an anthracycline and cytarabine (ara-C) yield complete remissions in approximately two thirds of patients, the majority of patients ultimately succumb to their disease or complications of treatment.2-9 The failure of conventional chemotherapy relates in part to inherent biologic features of the disease, such as overexpression of the mdr-1 gene product, P-glycoprotein (P-gp).10-12 This transmembrane glycoprotein functions as an ATP-dependent multidrug exporter with broad specificity for natural product antitumor agents.13

Although native expression of P-gp is demonstrable in fewer than 35% of cases of de novo AML, it is detected with greater frequency in leukemic cells at relapse (50%), AML in the elderly or an unfavorable cytogenetic pattern, and secondary AML (50% to 80%).14,15 Anthracycline resistance resulting from P-gp–mediated transmembrane drug extrusion represents a cellular mechanism of resistance potentially amenable to pharmacologic inhibition. Overexpression of P-gp is associated with reduced blast accumulation of anthracyclines12 that is restored in vitro by concurrent exposure to nonselective, or so-called first-generation P-gp antagonists such as verapamil or cyclosporin A (CSA).12,16,17 Cyclosporin A is a potent in vitro modulator of P-gp transport function, but in clinical trials it is associated with significant hyperbilirubinemia and delayed anthracycline elimination.18,19 Other toxicities, such as reversible azotemia, nausea, vomiting, and burning dysesthesias, occur at cyclosporine doses sufficient to inhibit P-gp function.20

PSC 833 (Valspodar; Novartis Pharmaceutical Corporation, East Hanover, NJ) is a selective inhibitor of P-gp that displays high binding affinity with the mdr-1 protein. PSC 833 (3'-keto-Bmt1] [Val2]-cyclosporine) is a structural analog of CSA. It lacks intrinsic renal toxicity and immunosuppressive properties, and is a 10-fold more potent modulator of P-gp transport function than is CSA.20-22 Unlike CSA, PSC 833 does not bind to cyclophilin, which is necessary for the immunosuppressant activity of cyclosporines. Phase I studies investigating PSC 833 as a P-gp modulator have shown that this agent can be administered in doses up to 10 mg/kg/d by continuous infusion for 5 days without excessive toxicity.23 PSC 833 alters the biliary excretion of anthracyclines in animal models, but its effects in humans have not been characterized. In the current trial, four cohorts of patients with poor-risk AML received escalating doses of PSC 833 and daunorubicin (DNR) after ara-C administration. The objectives of this study were to determine the maximum-tolerated doses of PSC 833 and DNR, assess the safety and remitting activity of the combination in patients with poor-risk AML, and delineate the effect of PSC 833 on DNR pharmacokinetics.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Selection
Forty-three patients with histologically confirmed, poor-risk AML were enrolled in the trial. Eligible patients had either AML refractory to induction therapy or in first relapse within 9 months of complete remission; AML in second or later relapse; AML secondary to a myelodysplastic syndrome or previous cytotoxic treatment; unfavorable cytogenetic pattern (-5/5q-, -7/7q-, or complex); or myeloid blast phase of chronic myelogenous leukemia (CML). All patients had a performance status of 2 or less (World Health Organization), serum creatinine less than 2.0 mg/dL, total bilirubin less than twice the upper limit of normal, and serum aspartate transaminase, alanine transaminases, and alkaline phosphatase less than three times the upper limit of normal. Medical conditions such as cirrhosis or hepatitis, which might alter drug clearance, and concurrent administration of medications known to alter blood cyclosporine concentration, including danazol, diltiazem, carbamazepine, erythromycin, fluconazole, itraconazole, methylprednisolone, prednisone, metoclopramide, phenobarbital, phenytoin, nicardipine, rifampin, and verapamil, were excluded. Patients who had received prior resistance-modifying therapies, such as CSA, verapamil, tamoxifen, or trifluoperazine, were excluded from study treatment.

Detection of Multidrug Resistance Proteins
P-glycoprotein. Mononuclear cell fractions were isolated from bone marrow or peripheral blood by Ficoll-Hypaque density gradient centrifugation. P-glycoprotein expression was assessed by flow cytometry using the MRK-16 antibody (Camiya Biomedical Company, Thousand Oaks, CA). Mononuclear cells were incubated with the MRK-16 antibody for 30 minutes at 4°C in 6% heat-inactivated serum to minimize nonspecific antibody binding, then stained with goat antimouse antibody conjugated to fluorescein isothiocyanate. Gating on the blast population was based on linear forward light scatter and right angle side scatter. P-glycoprotein expression was also evaluated by immunocytochemistry in specimens with limited cell recovery. Immunocytochemical detection measured antibody reactivity on cytospin preparations by direct immunoperoxidase activity using a modified biotin-avidin technique as described previously.24,25 Three monoclonal antibodies recognizing different epitopes of the P-gp molecule were used; these included JSB-1 (Sanbio bv, Uden, the Netherlands), C-494 (Centocor, Malvern, PA), and MRK-16. Specimens were considered positive for P-gp if staining with one or more antibodies was detected in at least 20% of blasts and there was P-gp message detected by polymerase chain reaction (PCR). Thus, P-gp expression was confirmed by PCR assay for mdr-1 mRNA message relative to the level in normal bone marrow (see below). Expression of the lung resistance protein (LRP) in leukemic blasts was assessed in cytospin preparations using the monoclonal antibody LRP56 supplied by Dr. Rik Scheper (Free University Hospital, Amsterdam, the Netherlands), as previously described.26

MDR-1 Reverse Transcriptase Polymerase Chain Reaction Assay
mdr-1 mRNA was quantified by reverse transcriptase PCR as previously described.27,28 A total of 300 ng of cellular RNA was isolated from mononuclear cell fractions of patient specimens and normal bone marrow by guanidinium cell lysis and cesium chloride gradient centrifugation.

The PCR products were separated by agarose gel electrophoresis, and bands corresponding to the cellular and synthetic mdr-1 RNA were excised and counted for radioactivity. The initial amount of RNA, both synthetic and cellular was plotted versus the radioactivity incorporated into their PCR products to yield a standard curve for synthetic mdr-1-specific mRNA. The quantity of cellular mdr-1-specific mRNA in each sample was then quantitated by interpolation from this curve.

Daunorubicin Retention Assay
Daunorubicin retention by P-gp–overexpressing 8226/DOX6 cells was evaluated by flow cytometry in the presence of patient plasma obtained on day 1 before treatment (no PSC 833) and day 6 (12 hours after initiation of PSC 833 infusion). Intracellular DNR content was evaluated by mean cellular fluorescence, with the data reported as the percent change in cellular fluorescence in the presence of day 6 plasma (after PSC 833 infusion) compared with day 1 (before PSC 833 infusion).

Daunorubicin accumulation in the 8226/DOX6 multidrug-resistant subline in the presence and absence of patient plasma samples was determined by flow cytometric analysis. Control tubes consisted of 1 x 106 DOX6 cells in normal human plasma with and without 1 µmol/L PSC 833 and experimental tubes consisted of 1 x 106 DOX6 cells in patient plasma. Cells were incubated at 37°C for 15 minutes. Daunorubicin, 12 µmol/L, was added and incubated at 37°C for an additional 60 minutes. The reaction was quenched with 4°C phosphate-buffered saline solution, and cells were resuspended in 0.5 mL of 4°C phosphate-buffered saline solution, placed on ice, and covered for immediate flow analysis (FACScan, Becton Dickinson) with excitation measured at 488 nm and emission measured at 585 nm. The 8226 parent cell line, 8226/S, was also used as a positive control for maximum DNR accumulation.

Leukemia Colony Inhibition Assay
In vitro sensitivity of clonogenic leukemia progenitors to DNR in the presence and absence of PSC 833 was evaluated using a modification of methods previously described.29 Briefly, cryopreserved, T-cell–depleted leukemia specimens were thawed and washed three times, and dead cells were removed by Ficoll-Hypaque density-gradient centrifugation. Viability was assessed by trypan blue dye exclusion. After washing in Isacove’s modified Dulbecco’s medium, 105 cells are incubated in alpha-medium containing 10% fetal bovine serum with the following drug exposures: (1) DNR at concentrations of 0.0001 to 5.0 µg/mL for 48 hours in the presence of PSC 833 vehicle, (2) DNR preceded 30 minutes by 1.0 µmol/L PSC 833, (3) 1.0 µmol/L PSC 833 alone, or (4) PSC 833 vehicle alone. After the 48-hour incubation, cells were pelleted and washed twice in alpha medium and plated. Twenty thousand cells were plated in methylcellulose (0.8%), 20% fetal bovine serum, and 10% phytohemagglutinin-stimulated lymphocyte-conditioned medium in 1-mL microwells. Microwells were plated in triplicate for each drug exposure and incubated in a moist atmosphere with 6% CO2. Aggregates of greater than 20 cells were counted with an inverted microscope after a 7-day culture, and the percent inhibition of leukemia colony-forming units (CFU-L) was calculated by comparison with growth in control plates. Sensitivity to DNR was characterized by the concentration of DNR causing 50% inhibition of colony growth (IC50). The ability of PSC 833 to potentiate DNR colony inhibition was assessed by the ratio of vehicle IC50 divided by PSC 833 IC50 (ie, sensitization factor [SF]).

Study Design and Treatment
This was an open-label, phase I/II trial to evaluate the safety, tolerability, and efficacy of the resistance modulator, PSC 833 (Valspodar), combined with ara-C and DNR in patients with poor-risk AML. In the dose escalation portion of the study, patients were assigned to one of four induction cohorts to receive escalating doses of DNR and PSC 833 ( Table 1). Cytarabine was administered at a dose of 3 g/m2 daily on days 1 through 5 as a 3-hour IV infusion. Patients received dexamethasone eye drops four times a day during ara-C administration. PSC 833 was administered as an 84-hour continuous IV infusion concurrently with a loading dose of 1.5 mg/kg IV for 2 hours beginning on day 5 of the induction regimen. Daunorubicin was administered as a continuous IV infusion 12 hours after initiation of PSC 833, at doses of 34 mg/m2/d (cohorts 1 and 2) or 45 mg/m2/d (cohorts 3 and 4) on days 6, 7, and 8 (for a total of 72 hours). Bone marrow response was evaluated on day 14 of induction therapy. Patients with persistent leukemia who experienced a greater than 50% reduction in blast percentage received a second course of induction therapy on or before day 21. Patients with 50% or less reduction in blast percentage were considered treatment failures and were removed from the study. Hematopoietic growth factor support was not required, but was permitted at the discretion of the investigator.


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  		Table 1. Dose Levels for Phase I Induction Cycle
 
Patients who achieved a complete remission were eligible for consolidation therapy. The consolidation regimen was designed to permit comparative analysis of DNR pharmacokinetics in the presence or absence of concurrent PSC 833 administration. Eligible patients received ara-C (3 g/m2), days 1 through 3, and DNR was administered as a 20-minute IV infusion on 3 days: day 1 (without PSC 833), and days 4 and 6 (with PSC 833). The day 4 dose of DNR was administered 12 hours after initiation of the PSC 833 84-hour continuous infusion. The dosage of DNR and PSC 833 infusion and load were the same as that administered during remission induction.

Valspodar (PSC 833 or Amdray) was supplied in 1.0-mL ampules containing 50 mg of drug in Cremophor EL by Novartis Pharmaceutical Corporation (Basel, Switzerland). The drug was diluted in 5% dextrose in water or 0.9% sodium chloride for injection, U.S.P. at final ratios between 1:20 and 1:50 with a 24-hour stability time for each diluted PSC 833 solution. Daunorubicin (Cerubidine) was obtained from Immunex Laboratories (Seattle, WA), and ara-C (Cytosar) was obtained from Pharmacia Upjohn, (Kalamazoo, MI).

Daunorubicin Pharmacokinetics
Plasma samples were obtained on days 1 and 4 of consolidation therapy to determine the effect of PSC 833 on DNR and daunorubincinol (DNR-ol) pharmacokinetics. Sample times (after the end of the 20-minute DNR infusion) were at 5, 15, and 30 minutes, and 1, 4, 8, 12, and 24 hours. A reference (pre-DNR) plasma sample was obtained just before initiation of the DNR infusion on day 4. Plasma samples were stored at -80°C before being thawed and analyzed by reverse-phase high-pressure liquid chromatography (HPLC) using a method modified after Peng et al.30 Samples were first washed with water and extracted with 0.1 mol/L methanolic HCl on a stationary column of octadecanoyl silane (Bond-Elut C-18 columns, Varian Associates, Harbor City, CA). The HPLC procedure used an isocratic elution, pumped at 2.0 mL/min with a mobile phase consisting of 70% (v/v) 0.02 mol/L ammonium acetate, pH 4.0, in 30% (vol/vol) HPLC-grade acetonitrile. A Water’s C-18, 125A column was used having a nominal particle size of 10 µm. The internal standard was doxorubicin (Pharmacia Laboratories, Kalamazoo, MI). Detection was by fluorescence (Perkin-Elmer LS-1 Detector) with excitation and emission set at 480 nm and 550 nm, respectively. The chromatographic data was analyzed and stored using a PE Nelson 900 Series Interface with Nelson Turbochrom version 4.1 software supported by a Gateway 200, P5-90 computer. For parent drug (DNR) and the alcohol metabolite (DNR-ol), the day 1 and day 4 samples were analyzed to determine the median, mean, minimum, maximum, and SD of the following pharmacokinetic variables: maximal concentraton (Cmax), rate constant (Ke), half-life (t1/2), the area under the plasma concentrations times the time curve (AUC), the volume of distribution (Vd), and clearance. For half-life and clearance, the harmonic mean and jackknife estimation of the SD were calculated.

PSC 833 Pharmacokinetics
Whole-blood samples were collected on days 5 through 21 of induction therapy and days 3 through 21 of consolidation therapy for measurement of PSC 833 concentration by radioimmunoassay (ANAWA Laboratories, Zurich, Switzerland).

Response and Toxicity Assessment
Response to induction therapy was assessed using previously described response criteria.31 Complete remission (CR) was defined as less than 5% blasts in a marrow aspirate of adequate cellularity with a peripheral blood neutrophil count of at least 1,500/µL, absence of circulating blasts, an untransfused platelet count of at least 100,000/µL, and no extramedullary disease for at least 28 days. A partial remission (PR) was defined by the peripheral blood criteria for CR accompanied by a marrow aspirate containing 5% to 25% myeloblasts, or less than 5% marrow blasts associated with persistent thrombocytopenia (50,000 to 100,000/µL). For patients with CML, a restored chronic phase was defined as less than 10% blasts, and less than 20% blasts plus promyelocytes in the marrow or peripheral blood. Patients who failed to respond to study treatment were considered treatment failures and classified according to the following modified Preisler criteria: absolute drug resistance (patient survives 7 days after treatment but fails to achieve hypocellular marrow by day 14 with less than 50% reduction in blasts or persistent leukemia in peripheral blood or at an extramedullary site); relative drug resistance (patient survives greater than 7 days after treatment and achieves severe marrow hypoplasia [less than 5%] by day 14, but marrow repopulates with leukemic blasts within 30 days of hematologic recovery); regeneration failure (persistence of severe marrow hypoplasia [5%] beyond 40 days after completion of therapy); aplasia death (patient survives at least 7 days with cytopenic death, hypoplastic bone marrow but void of persistent leukemic blasts).32 Relapse from CR was defined as reappearance of leukemic blasts in the peripheral blood or greater than 5% blasts in the bone marrow (not related to recovery), or appearance of extramedullary disease. Nonhematologic toxicity was graded according to the National Cancer Institute common toxicity criteria. Standard criteria for dose-limiting toxicity were used for this study with dose-escalation halted for grade 4 nonhematologic toxicity (NCI criteria) occurring in (1) one third of patients (three more patients then added), or (2) in at least two of six patients at any dose level. In addition, patients with the following toxicities would be discontinued from further protocol treatment: (1) a minimal 15% decrease in cardiac ejection fraction (by multiple gated aquisition scan); (2) grade 3 or 4 neurocentral or neuromuscular toxicity; (3) a creatinine clearance decrease to less than 30 mL/min; and (4) patients whose transaminase levels increase to greater than two times baseline and do not return to less than two times baseline before the next course of therapy.

Statistical Methods
Disease-free survival, remission duration, or time to treatment failure were calculated from the date of remission to the date of marrow relapse or death by any cause. Survival curves were estimated by the methods of Kaplan and Meier.33 Treatment outcome comparisons between subgroups were performed by log-rank analysis.34 Toxicity comparisons among subgroups were performed by analysis of variance (ANOVA). For the DNR pharmacokinetics, a paired t-test was used to compare the day 1 and day 4 pharmacokinetic variables for parent drug, DNR, and its alcohol metabolite, DNR-ol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Demographics
The pretreatment characteristics of the 43 patients enrolled in this study are summarized in Table 2. The median age was 57 years, and the largest group comprised patients with relapsed AML (n = 23), the majority of whom (n = 20) were in first relapse within 9 months of primary treatment. There were 11 patients with treatment-induced AML: five from prior chemotherapy and six evolving from an antecedent myelodysplastic or myeloproliferative syndrome. Five patients had CML in previously untreated myeloid blast phase. The number of patients enrolled on each treatment cohort is summarized in Table 1.


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  		Table 2. Patient Demographics
 
Nonhematologic Toxicities
Table 3 summarizes the nonhematologic toxicities greater than or equal to grade 3 reported for all treatment cohorts. Transient hyperbilirubinemia was an expected effect of PSC 833 administration as previously reported.34 There was no evidence of an interaction between PSC 833 dose and magnitude of bilirubin elevation among the treatment cohorts (P = .48 by ANOVA). The maximum serum bilirubin ranged from 1.5 to 6.8 mg/dL (mean, 2.69 mg/dL; median, 2.2 mg/dL), reaching a peak elevation 24 hours after termination of the PSC 833 infusion on day 10 of the induction course. Hepatic toxicity as assessed by elevation in serum transaminases was uncommon (4%). Nausea and vomiting during the PSC 833 infusion occurred in 20% of patients. Eight (18%) patients developed asymptomatic hypocalcemia (7.7 mg/dL [1.94 mmol/L] or less). There were nine documented infections caused by bacteria (seven patients) or fungal pathogens (two patients), which contributed to two septic deaths during aplasia on dose level three. One heavily pretreated patient with primary refractory AML enrolled in cohort three developed congestive heart failure associated with a 29% reduction in left ventricular ejection fraction that complicated a pulmonary infection (pretreatment, 55%; postinduction 39%). This patient achieved a CR, and left ventricular ejection fraction returned to baseline on subsequent evaluation, suggesting transient suppression of myocardial contractile function exacerbated by sepsis or study treatment. One patient in cohort four developed a subdural hematoma, complicating grade 4 thrombocytopenia. Central nervous system toxicity was suspected in two patients who experienced transient confusion. In one patient from cohort one, symptoms were reported after premedication for PSC 833 with lorazepam, whereas in the second patient, transient cerebellar dysfunction was manifested by ataxia, which occurred during the PSC 833 infusion. Symptomatic mucositis was uncommon and was noted in only five patients treated at various dose levels.


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  		Table 3. Nonhematologic Toxicity of at Least Grade 3
 
Hematologic Toxicity and Treatment Outcome
Profound myelosuppression is an expected effect of leukemia induction therapy. To determine whether concurrent treatment with PSC 833 augments the myelotoxicity of the treatment regimen, we assessed the interval to hematologic recovery in remitting patients enrolled on each cohort ( Table 4). There was no evidence of dose-related effect of PSC 833 on the interval to neutrophil recovery greater than 500/µL. However, myeloid growth factors were frequently administered at the discretion of individual investigators. In contrast, there was an incremental prolongation in the interval to recovery of a sustained (greater than 5 days) untransfused platelet count greater than 20,000/µL with each dose escalation in PSC 833 and DNR. This effect on platelet recovery approached statistical significance when cohort four (32 days to recovery) is compared with all other cohorts (median time to platelet recovery, 24 to 29 days; P = .051 by ANOVA).


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  		Table 4. Response and Days to Hematologic Recovery by Study Cohort
 
Response Twenty-one (49%) of the 43 assessable patients (95% confidence interval, 33.9% to 63.7%), achieved a CR or restored chronic phase, and three patients achieved a PR ( Table 5). Only two patients, one each in cohorts two and three, received two induction courses because of persistent disease at day 14. The CR rate was comparable among each treatment cohort (Table 4). However, the number of remitting patients within each cohort is small, limiting formal statistical comparison. Response by disease category is detailed in Table 5. There were no responses among the four patients with primary refractory AML, whereas nine (82%) of 11 patients with secondary AML and 12 (52%) of 23 patients with relapsed AML achieved a CR or PR. Thirteen patients (30%) failed induction therapy because of resistant disease, whereas six patients succumbed either to infectious complications during aplasia (four patients) or regeneration failure (two patients). The median time to treatment failure by disease category in patients achieving CR was 330 days for relapsed AML, 297 days for secondary AML, and greater than 140 days for patients with CML-blast phase. Nine patients underwent bone marrow transplantation as consolidation therapy and were therefore excluded from the pharmacokinetic studies. Thus, only 12 of 21 patients received study-directed consolidation chemotherapy, and eight of these patients had full pharmacokinetic studies performed.


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  		Table 5. Response by Disease Category
 
Multidrug Resistance Phenotype
Thirty-eight patients had sufficient pathologic material for evaluation of P-gp expression, and 36 of these patients were assessable for both LRP and P-gp expression. Among the three antibodies used for detecting multidrug resistance (MDR), the C-494 antibody was most frequently positive at 50%, followed by MRK-16 (31%) and JSB-1 (19%). The C-494 antibody was singly positive in 16% of the MDR-positive patients. No other antibody was singly positive in the MDR-positive patients. For patients in whom two antibodies were positive, the percentage of MDR-positive patients was 63% for the combination of MRK-16 plus C-494, 20% for JSB-1 plus MRK-16, and only 10% for C-494 plus JSB-1. The relation between MDR phenotype and response to induction chemotherapy is summarized in Table 6. Immunodetection of P-gp did not adversely impact response to the study treatment. Twelve of 19 (63%) patients with P-gp+ AML achieved a CR or PR, compared with nine of 19 (47%) of patients without detectable P-gp overexpression (not statistically significant). The frequency of treatment failure (32%), was the same in each of the P-gp phenotypic groups. In contrast, LRP overexpression was associated with an inferior response rate of 47% (nine of 19), compared with 71% (12 of 17) in the LRP-negative group (P = .165, t-test). There was also a significantly higher frequency of treatment failure in the LRP-positive group, nine of 19, or 47%, compared with only two of 17, or 12%, in the LRP-negative group.


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  		Table 6. Response to Study Treatment by P-Glycoprotein and Lung Resistance Protein Phenotype
 
To determine whether study treatment suppressed or impacted the emergence of mdr-1 gene overexpression, mdr-1 mRNA expression was quantitated by reverse transcriptase PCR in leukemia specimens obtained before induction therapy and after treatment failure, and the level of gene message expressed as units relative to normal bone mononuclear cells. Sequential specimens were available from eight patients for analysis, including seven patients achieving a CR or PR with study treatment, and one patient with resistant disease. Figure 2 summarizes the reverse transcriptase PCR results. Four of these patients had a 4.2- to 21-fold mdr-1 gene overexpression relative to normal bone marrow. In each of the patients who remitted with study treatment, mdr-1 gene message at relapse was either undetectable or equivalent to the level in normal bone marrow. One patient refractory to study treatment maintained comparable levels of mdr-1 gene overexpression: 4.2 at pretreatment versus 4.9 at progression.



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  		Fig 1. Mean ± SD whole blood concentration of PSC 833 by PSC 833 dose level. Diamonds denote 6 mg/kg/d; boxes, 9 mg/kg/d; and triangles, 10 mg/kg/d.

 
Daunorubicin and Daunorubicinol Pharmacokinetics
Nine patients had blood samples drawn for DNR and DNR-ol pharmacokinetics in the consolidation phase to measure the effect of PSC 833 on the disposition of DNR and its less active alcohol metabolite, DNR-ol. One of those patients did not finish the second sampling phase and was therefore unassessable for comparative DNR pharmacokinetics. Three other patients undergoing consolidation did not have blood samples drawn. Noncompartmental analysis was used on the resultant eight patients’ data set with day 1 (without PSC 833) compared with day 4 (24 hours after starting PSC 833). Table 7 summarizes the pharmacokinetic parameters for these two DNR dosing days. For the parent compound, there was a significant decrease in clearance on day 4 (P = .04). Despite this mean change in clearance by about one-half, two patients (nos. 04-202 and 05-407) exhibited no changes in DNR clearance with the addition of PSC 833 ( Table 8). Because of large interpatient variations, the eight-fold increase in mean DNR AUC with PSC-833 was not significant. In contrast, clearance of the alcohol metabolite, DNR-ol, was significantly reduced from day 1 to day 4 by a mean of three-fold with PSC 833 (P = .016). The mean AUC of DNR-ol increased four-fold with PSC 833 (P = .015). These results show that PSC 833 reduces clearance and increases systemic exposure to DNR-ol by three- to four-fold. There were no changes in the volume of distribution for either the parent drug or the metabolite.


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  		Table 7. Summary Pharmacokinetics for Daunorubicin and Daunorubicinol on Consolidation Arm
 

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  		Table 8. Effect of PSC 833 on Mean Daunorubicin Pharmacokinetics
 
Daunorubicin Cytotoxicity and Drug Retention
The in vitro sensitivity of clonogenic leukemia cells to DNR was evaluated in pretreatment marrow specimens from 18 patients using the CFU-L assay. Daunorubicin concentrations yielding 50% inhibition of leukemic colony growth ranged from 0.0003 to 1.47 µg/mL, with a median IC50 of 0.0053 µg/mL. Excluding the early deaths, a comparison of IC50 values for responding patients (mean IC50 0.020 ± 0.02 µg/mL; n = 11) and nonresponders (IC50 0.370 ± 0.732 µg/mL; n = 5) revealed no statistically significant difference (P = .146 by t-test). Next, the differences in cellular sensitization to DNR by PSC 833 were evaluated. The magnitude of CFU-L sensitization to DNR by PSC 833 was determined by the change in DNR IC50 concentration with the addition of 1 µmol/L PSC 833 to the culture medium. The mean sensitization ± SD was significantly higher in responders, 6.49-fold ± 9.61, compared with 0.59 ± 0.35 for nonresponders (P = .039 by t-test). Analysis by P-gp phenotype showed greater sensitization by PSC 833 in P-gp+ patients, (10.21-fold ± 12.39) compared with PGP- patients (0.53-fold ± 0.27).

The ability of patient plasma containing PSC 833, obtained 12 hours after initiation of the PSC 833 infusion, to augment DNR retention in the 8226/DOX6 P-gp+ cell line was compared with the effect of plasma obtained before study treatment. Daunorubicin intracellular accumulation was evaluated by flow cytometry using mean cellular fluorescence. Plasma samples from 26 patient samples were analyzed, including 13 responding patients, 11 nonresponders, and two early death patients. Day 6 plasma augmented DNR accumulation relative to pretreatment plasma in 8226/DOX6 cells by a median value of 88% ± 101% (range, 11% to 501%). The median change in DNR retention increased with escalation in PSC 833 dosage (6 mg/kg, 40% ± 22%; 9 mg/kg, 82% ± 121%; 10 mg/kg, 132% ± 57%) and corresponded to changes in mean PSC 833 whole blood concentrations on day 9 (6 mg/kg, 1,213 ng/mL; 9 mg/kg, 1,840 ng/mL; and 10 mg/kg; 2,238 ng/mL; Fig 2). However, there were no differences in DNR uptake between responding and nonresponding patients. The mean change in 8226 cell DNR fluorescence using plasma from responding patients was 65.9% ± 15.5%, and in the nonresponders (including early deaths) the mean change in percent fluorescence was 120.5% ± 39.88% (P = .21 by t-test).



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  		Fig 2. Reverse transcriptase polymerase chain reaction quantitation of cellular mdr-1 message in sequential specimens obtained from eight patients before study treatment (PreTx) and after disease progression, relative to expression level in normal bone marrow (BM). Triangles denote patients responding to study treatment; squares, a patient with primary resistance. Abbreviations: CR, complete remission; PR, partial remission.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this phase I/II trial, patients with poor-risk AML received sequential treatment with ara-C and continuous infusions of DNR and PSC 833. Dose-limiting toxicity was not observed, permitting escalation to the highest doses of PSC 833 (10 mg/kg/d) and DNR (45 mg/m2) in the fourth treatment cohort. This dosage of PSC 833 yielded a mean whole blood steady-state concentration of 2,238 ng/mL, which exceeds the target threshold for P-gp blockade of 1,000 ng/mL in all patients (range, 1,220 to 3,710 ng/mL). Ex vivo exposure of MDR cells to plasma from patients treated with 10 mg/kg/d of PSC 833 increased mean cellular retention of DNR by 132% ± 57%. These data indicate that PSC 833 administered at a dosage of 10 mg/kg/d yields pharmacologically active blood concentrations in vivo that are sufficient for inhibition of P-gp drug export function.

Transient hyperbilirubinemia was the most common nonhematologic toxicity associated with PSC 833 administration (Table 3). This is consistent with reports by other investigators.24,35-37 Moderate elevations in serum bilirubin (median, 2.2 mg/dL; range, 1.5 to 6.8 mg/dL) were observed in 31 (70%) patients, but these returned to baseline within 72 hours of termination of the PSC 833 infusion. Cerebellar toxicity, which manifested as truncal ataxia, a common toxicity with oral PSC 833, occurred at the end of the PSC 833 infusion in only one patient and was rapidly reversible. Symptomatic stomatitis was uncommon and occurred in only five (11%) patients. This low frequency of mucosal toxicity contrasts sharply with the results of other resistance-modulation studies in AML for which mucositis was dose limiting, often necessitating significant dose reductions of the anticancer drugs modulated by CSA.24,35,38-40 Eligibility for these previous trials was similar to the present study: restricted to either older patients with AML or patients with poor-risk acute leukemia. However, there are notable differences with these prior studies of PSC 833 modulation. Although the targeted P-gp substrate in the study by Sonneveld et al38 was DNR, the other studies combined DNR or mitoxantrone with etoposide, which is weakly modulated by PSC-833. Furthermore, in each of these prior studies, the antineoplastic agent was administered as a rapid IV infusion. This may explain the differences with the current study, which used continuous infusion of DNR.

We used a 24-hour DNR infusion based on preclinical studies demonstrating that continuous exposure to cytotoxic drugs reduced the influence of P-gp.41 The prolonged infusion schedule may also account for the low prevalence of gastrointestinal and, especially, mucosal toxicities. Furthermore, preclinical studies have shown that sensitization of MDR tumor cell lines to anthracyclines and other natural products by a P-gp modifier is both concentration- and time-dependent, and it is optimized by prolonged exposure to the targeted anticancer drug.41-45 In addition, clinical trials investigating extended schedules of anthracycline administration have shown that prolonging the duration of drug infusion reduces mucosal and cardiac toxicity without compromising antitumor activity.46-49 Extending the duration of DNR infusion may offer additional therapeutic benefit by favorably impacting cellular pharmacodynamics. In a small comparative study of 4-hour or less versus 24-hour DNR infusions in patients with AML, Paul et al50 reported greater cellular accumulation of DNR with the extended schedule of drug infusion. It is important to consider that this enhanced drug retention occurred despite a significant reduction in peak drug concentrations. Similar findings with continuous DNR infusion have been described in a recent preliminary report of a Southwest Oncology Group randomized phase III trial testing CSA modulation of anthracycline resistance. Using the current trial design in patients with poor-risk AML, this study showed a reduction in the frequency of resistant disease, and a significant improvement in disease-free and overall survival that correlated to achieving high steady-state DNR plasma concentrations.51 The results of both trials are encouraging and suggest that both the concentration and the duration of anticancer drug exposure are important determinants of treatment outcome with a P-gp modulator. Therefore, an extended infusion schedule may not only optimize P-gp modulation, but also decrease gastrointestinal and cardiac toxicities to facilitate safe delivery of the maximal dose of the targeted antineoplastic agent.

Preclinical and phase I studies have shown that PSC 833, like many other P-gp modulators, alters the hepatobiliary disposition of many anticancer drugs.20,24,36,37,52 In these trials, PSC 833 has been shown to delay etoposide elimination and increase systemic drug exposure by 66% to 89%.24,36 However, only one other study has examined the effect of PSC 833 on anthracycline pharmacokinetics, in this case, doxorubicin. Giaccone et al37 reported that PSC 833 increased the mean AUC for doxorubicin by 54%, and the AUC of the primary metabolite, doxorubicinol, increased 2.7-fold. The results of our pharmacokinetic analysis of DNR plus PSC 833 are similar. In the current study, the addition of PSC 833 on day 4 of the consolidation cycle significantly decreased both DNR and DNR-ol clearance by two-fold and three-fold, respectively. There was also a significant increase in the DNR-ol AUC (four-fold). Interestingly, this did not correlate with PSC 833 blood concentrations. Furthermore, two of eight patients showed no substantial change in DNR AUC with PSC 833. The latter finding reflects the wide individual variability in the effect of PSC 833 on DNR elimination (Table 8). The clinical impact of an increased systemic exposure to DNR-ol is unknown, but is believed to be minimal, based on prior laboratory and clinical investigations. For example, DNR-ol produces approximately 20-fold lower in vitro toxicity against both normal bone marrow progenitors and tumor cell lines compared with the parent compound.53-55 In addition, DNR-ol exhibited much lower antitumor potency in a single clinical trial.56 The lack of a consistent effect of PSC 833 on DNR pharmacokinetics in all patients in the current trial is similar to the effect of PSC 833 on mitoxantrone pharmacokinetics reported by Kornblau et al23 Thus, although our findings indicate that PSC 833 may significantly increase systemic exposure to DNR in approximately 50% of patients, an empiric reduction in DNR dose risks the potential for substantial anthracycline underdosing in those patients who do not experience a significant change in DNR pharmacokinetics.

Overall, this chemotherapy induction regimen was well tolerated and had significant remitting activity in this relatively poor-risk AML population. Forty-nine percent of assessable patients achieved a CR of AML or restoration of chronic phase CML. Only 12 (28%) patients displayed primary resistance to the study treatment, and four (9%) patients succumbed to treatment-related complications during drug-induced aplasia. Of particular interest, P-gp overexpression did not adversely impact response to induction chemotherapy in this trial, suggesting that the regimen had preserved activity in P-gp+ AML. Indeed, we found that 1 µmol/L PSC 833 yielded greater potentiation of DNR cytotoxicity in clonogenic leukemia cells (CFU-L) from patients with P-gp+ leukemia, and in responding patients. Similarly, quantitation of mdr-1 gene message by reverse transcriptase PCR in paired specimens from seven patients who remitted with study treatment revealed the absence of detectable gene overexpression at the time of disease progression (Fig 2). These data suggest that the addition of PSC 833 to this induction regimen is active in P-gp+ AML, and may eliminate or prevent emergence of mdr-1 overexpressing clonogenic cells. Alternatively, it is possible that the 5-day high-dose ara-C therapy contributed substantial antitumor efficacy to the regimen, thereby minimizing the role of P-gp modulation by PSC 833 in the overall outcome. Although the observation that treatment outcome was not impacted by P-gp phenotype in this study is encouraging, overexpression of LRP was associated with a lower probability of induction response (LRP+, 47% versus LRP-, 71%; P = .018). Expression of LRP, also known as the major vault protein, identifies a non–P-gp–mediated MDR phenotype that is closely associated with overexpression of the multidrug transmembrane transporter, termed breast cancer resistance protein.27,57 The lack of benefit of drug modulation in LRP+ AML is consistent with our previous findings58 and with the minor in vitro effects of P-gp modulators on such alternate MDR phenotypes.27,55,57,58

From the results of these studies we conclude that combined treatment with PSC 833, 10 mg/kg/d, and infused DNR administered at a standard dose as an infusion is well tolerated and yields pharmacologically active blood concentrations of both agents. This regimen has promising remitting activity in patients with poor-risk AML. PSC 833 significantly delays the clearance of DNR and its metabolite, but yields variable changes in systemic drug exposure. The absence of dose-limiting toxicity in this trial suggests that continuous infusion of the targeted anthracycline may obviate the need for empiric dose-reductions and thereby maximize the potential anthracycline exposure when administered in conjunction with PSC 833.


   ACKNOWLEDGMENTS
 
Supported by National Cancer Institute grants nos. CA 17094 and CA 43043.


   NOTES
 
The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
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Submitted April 5, 2000; accepted December 7, 2000.




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