Patient-Specific Dose Rate for Continuous Infusion High-Dose Cytarabine in Relapsed Acute Myelogenous Leukemia

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1 Patient-Specific Dose Rate for Continuous Infusion High-Dose Cytarabine in Relapsed Acute Myelogenous Leukemia By Volker Heinemann, Elihu Estey, Michael J. Keating, and William Plunkett We hypothesized that the steady-state concentration of intracellular cytarabine 5'-triphosphate (ara-ctp,.) in leukemia cells is proportional to the dose rate of cytarabine (ara-c) during continuous infusion. To evaluate this possibility, patients with acute myelogenous leukemia in relapse were treated with two sequential schedules of serially increasing ara-c dose rates over a total of 36 hours. Schedule I consisted of serial infusions of 250, 500, and 750 mg/m 2 each over 12 hours. Subsequently, patients entered on schedule II received 500, 1,000, and 1,500 mg/m 2 serially, each over 12 hours. Steady-state levels of ara-ctp were achieved within four hours after beginning ara-c infusion and, in separate studies of a single ara-c dose rate, were shown to be maintained beyond 36 hours. Four patients treated with schedule I and two patients treated with schedule II showed a linear dose C YTARABINE (ara-c) is one of the most effective drugs in the treatment of adult acute myelogeneous leukemia.1' Although continuous infusion high-dose ara-c (CIHDAC) has recently been introduced as a successful regimen in the treatment of newly diagnosed and relapsed acute leukemia, 4 ' 5 the optimal dose rate and schedule of ara-c remains in question. The pro-drug ara-c is transported into the cell by carrier-mediated facilitated diffusion 68 and is subsequently phosphorylated to the active drug cytarabine 5'-triphosphate (ara-ctp). 9 " Cellular accumulation and retention of ara-ctp are determinants of ara-c-mediated cytotoxicityl2-14 and of clinical response." 15 8 Earlier studies have demonstrated marked interpatient heterogeneity in steady-state ara-ctp concentrations (ara- From the Departments of Medical Oncology and Hematology, The University of Texas M.D. Anderson Cancer Center, Houston. Submitted August 25, 1988; accepted December 27, Supported by Grant No. CA32839 from the National Cancer Institute, Department of Health and Human Services. Address reprint requests to William Plunkett, PhD, Department of Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX by American Society of Clinical Oncology X/89/ $3.00/0 rate-dependent increase of ara-ctp, at all three dose rates. The cells of one patient on schedule I and two patients on schedule II had a dose rate-dependent ara-ctp, increase only over the first two dose levels, while no increase or lower ara-ctp, was observed at the third dose rate. The ara-ctp, of one patient on schedule II did not change. These results suggest that there is a proportionality between the continuous infusion dose rate of ara-c and the ara-ctp, in circulating leukemia cells within the dose range of 250 to 1,000 mg/m 2 over 12 hours. This opens the possibility that pharmacologic determinations may be used to redirect the ara-c dose rate to achieve a desired ara-ctp, level in leukemia blasts during therapy. J Clin Oncol 7: by American Society of Clinical Oncology. CTPss) during CIHDAC. 4,16,19, 20 The likelihood that antileukemic activity is related to minimal levels of ara-ctp maintained during therapy prompted efforts to understand the relationships between continuous infusion ara-c dose rates, ara-ctp,, in leukemia blasts, and clinical response.16,19 In an initial approach, the ability of leukemia blast cells to accumulate and retain ara-ctp was determined in vivo after a test dose of 3 g/m 2 infused over two hours. The continuous infusion dose rate necessary to achieve a target ara-ctp,, was calculated assuming a linear relationship between plasma ara-c and cellular ara-ctp.1' 6 20 However, it became evident that a strictly linear relationship between plasma ara-c pharmacokinetics and ara-ctp metabolism did not exist in circulating leukemia cells. 2 1 ' 2 2 Furthermore, saturation of ara-ctp accumulation occurred at plasma ara-c concentrations well below those achieved by the test dose. 21 "' The lack of proportionality between ara-c dose rate and the kinetics of ara-ctp accumulation by leukemia cells resulted in over-estimates of the ara-c dose rates required to achieve a target ara-ctp,,.' 6 In the present study we have attempted to define patient-specific ara-ctp accumulation characteristics under conditions where the rate of cellular ara-ctp accumulation is not saturated. Under these conditions we hypothesized 622 Journal of Clinical Oncology, Vol 7, No 5 (May), 1989: pp

2 PATIENT-SPECIFIC DOSE RATE FOR CYTARABINE that, during continuous infusion, the ara-ctp, would be proportional to the ara-c infusion rate. The objective of the present investigation was to evaluate this hypothesis and to determine the range of ara-c dose rates over which the proportionality might exist. Patients and Therapy PATIENTS AND METHODS Ten patients with relapsed acute myelogeneous leukemia were entered on the study after informed consent was obtained. There were six women and four men. The median age was 32 years (range, 23 to 61). All patients were refractory to a variety of previous treatment protocols. Patient characteristics are shown in Table 1. Treatment with continuous infusion ara-c was performed at three different dose rates in each patient. A scheme of the investigational infusion strategy is shown in Fig 1. Two infusion schedules (I and II) were each studied in five patients. Schedule I consisted of successive treatment of patients A through E at serial dose rates of 250, 500, and 750 mg/m2; schedule II consisted of successive treatment of patients F through K at dose rates of 500, 1,000, and 1,500 mg/m2. Each dose was infused over 12 hours for a total of 36 hours during the pharmacology studies. However, in patients F and H the change of dose rates was performed at four-hour intervals. Subsequent to the stepwise dose increases, all patients received continuous infusion ara-c at a dose rate of 1,500 mg/m2/d for four days. This protocol (DM ) for treatment had been reviewed and approved by the Surveillance Committee of the University of Texas Cellular Pharmacology Study. Duplicate peripheral blood samples were taken at two and one hours before the end of each ara-c infusion to determine the ara-ctp, concentration in circulating leukemia blasts (Fig 1). Peripheral blood samples of 10 ml were drawn into heparinized tubes containing the cytidine deaminase inhibitor tetrahydrouridine at a final concentration of 500 #jmol/l a I C- ra Schedule I Schedule II Hours g//s Fig 1. Strategy for the determination of individualized target-specific ara-c dose rates. Concentrations of ara-ctp, were determined at three different successively applied dose rates. Schedule 1, 250, 500, 750 mg/m 2 over 12 hours; schedule II, 500, 1,000, 1,500 mg/m2 over 12 hours. Linear regression analysis was used to define the proportionality between ara-c dose rate and ara-ctp,. and were immediately placed in an ice-water bath. After separation of plasma and cells by centrifugation, mononuclear cells (> 90% leukemia blasts) were isolated by stepgradient centrifugation of Ficoll/Hypaque." 6 Cell number and cell volume were determined with a model ZM Coulter Counter equipped with a model C-1000 Channelizer (Coulter Inc, Hialeah, FL). The cells were extracted with HC104 and the acid-soluble fraction was analyzed for ara-ctp by ionexchange high-pressure liquid chromatography as previously described." 6 The amount of ara-ctp in a given number of cells was related to the volume of cell water and, assuming uniform ara-ctp distribution in the cell water, was used to calculate the intracellular ara-ctp concentration. Plasma Pharmacology Plasma ara-c concentrations were measured by radioimmunoassay according to the method of Piall et al and h Table 1. Patient Characteristics Patient Age/Sex FAB % Blasts Prior Therapy (CR duration, mo) Schedule I A 50/F ND 77 TAD (4); TAD; HDAC B 28/M M4 93 AdOAP (28); DFMO + IFN x 3; AdOAP (19); HHT; DHAD + HDAC (10); AMSA x 2; Mithra C 33/F ND 87 AdOAP x 2 D 61/F M4 94 HDAC x 2 (5) E 38/F M2 97 AMSA-OAP 21; DHAD + HDAC (9); HHT x 2 Schedule II F 29/F M5 92 HDAC + OP (4) G 31/M M4 96 HDAC + OP (6) H 37/M AUL 96 A + D; HDAC (7); DHAD + VP-16 I 25/F M4 72 A + D; HDAC + L-Asp (3); HDAC + L-Asp K 23/F M2 90 HDAC + OP x 2 (2); CTX + VP-16/TBI/AIIoBMT (5) Abbreviations: AlloBMT, allogeneic bone marrow transplantation; A, cytarabine; Ad, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH); CTX, cyclophosphamide; D, daunorubicin; DHAD, mitoxantrone; HDAC, high-dose ara-c; HHT, homoharringtonine; L-Asp, asparaginase; Mithra, plicamycin; ND, not determined; O, Oncovin (vincristine; Eli Lilly and Co, Indianapolis); P, prednisone; TBI, total body irradiation; VP-16, etoposide.

3 624 HEINEMANN ET AL others 2t ' 23 ' 2 5 using an anti-ara-c rabbit serum purchased from the University of Surrey (Guilford, England). Duplicate determinations in dual dilutions were made in a range of 2 to 20 nmol/l; the coefficient of variation did not exceed 20% for any sample. RESULTS The strategy of the study was to define the patient-specific ara-ctp accumulation characteristics by measurements of ara-ctp, concentrations at three different ara-c infusion dose rates. A range of infusion rates from 250 to 1,500 mg/m 2 over 12 hours was selected as clinically efficacious and tolerable. It was expected that within this range plasma ara-c levels would be below those required to saturate ara-ctp accumulation. 23, 24 To assure proper sampling times of ara-ctp,, concentrations, it was necessary to define the kinetics of intracellular ara-ctp accumulation during continuous infusion of ara-c. The ara- CTP concentrations in the circulating leukemia cells of four patients treated with continuous infusion ara-c at a dose rate of 500 mg/m 2 over 12 hours were analyzed (Fig 2). It could be demonstrated that, independent of the marked interpatient variability of cellular ara-ctp accumulation, ara-ctp., concentrations were reached three hours after the beginning of the infusion. Therefore, it appeared appropriate to define ara-ctps, by measurements taken at times three hours or more after the beginning of the continuous infusion. Separate studies demonstrated that ara-ctp, was maintained for more than 48 hours.' 6 a. I-- t Ca 0. C ara-c Infusion Rate. mg/m/i12 h Fig 3. Relationship between the CIHDAC dose rate and the cellular ara-ctp, concentrations in five patients treated according to schedule 1(250,500, 750 mg/m 2 over 12 hours). Each ara-ctp, value is the mean of four independent determinations. Letters correspond to patients presented in Table 1. The relationship between ara-c dose rate and ara-ctp, was investigated in ten patients. Four of five patients infused with schedule I showed a dose-dependent increase of cellular ara-ctp,, in circulating leukemia cells at all three dose rates (Fig 3). The ara-ctp, levels in the blasts of the remaining patient who received schedule I (patient C) appeared to have reached a plateau or declined slightly at the dose rate of 750 mg/m2 over 12 hours. The range of ara-c dose rates was doubled for the patient group entered on schedule II, starting at 500 mg/m 2 over 12 hours followed by 1,000 mg/m 2 over 12 hours and ending at the final dose rate of 1,500 mg/m2 over Inn C., Lr L a, QI O a C-, oel F g Sd) Hours Fig 2. Kinetic analysis of cellular ara-ctp accumulation during CIHDAC at a dose rate of 500 mg/m 2 over 12 hours. Each ara-ctp, value is the mean of two determinations. Letters correspond to the patients presented in Table 1. -* I- C. I m C- 0O ara-c Infusion Rate. mg/m 2 /12 h Fig 4. Relationship between the CIHDAC dose rate and the cellular ara-ctp, concentrations in five patients treated according to schedule II (500, 1,000, 1,500 mg/m 2 over 12 hours). Each ara-ctp. value is the mean of four independent determinations. Letters correspond to the patients presented in Table 1.

4 PATIENT-SPECIFIC DOSE RATE FOR CYTARABINE 12 hours (Fig 4). Only two of five patients (patients F and I) on schedule II responded with increments of ara-ctp, throughout three dose rates. The ara-ctp, reached plateau values at the initial dose rate of 500 mg/m 2 over 12 hours in patient K and at 1,000 mg/m2 over 12 hours in patient H, whereas a decrease of the ara-ctp, was observed during the 1,500 mg/m 2 over 12 hours infusion in patient G. When the proportionality between the ara-c infusion rate and ara-ctp, was evaluated for patients treated with both schedules, a median correlation coefficient of (range, to 0.991) was determined for patients who showed an increase of ara-ctp, concentrations throughout only two (n = 3) or all three (n = 6) dose rate increments. Comparing schedules I and II, it was evident that dose rates > 1,000 mg/m 2 over 12 hours were more likely to saturate incremental increases in the ara-ctp, than infusion rates below this threshold. However, a proportionality between ara-c dose rate and the ara-ctp,, was found in 80% of the patients who were analyzed at infusion rates in the range of 250 to 1,000 mg/m 2 over 12 hours. The relationship between ara-c dose rate and plasma ara-c concentrations has been reported in populations in which each patient received a single ara-c infusion.19,' The present study afforded the opportunity to document this relationship in individuals who received ara-c at multiple dose rates. The relationship between ara-c dose rate and plasma ara-c, concentrations was investigated in patient G (Fig 5). The stepwise increase of ara-c infusion rates from 500 to 1,000 to 1,500 mg/m 2 over 12 hours was accompanied by a linear increase of the steadystate plasma ara-c (ara-c,) concentration (r =.998). A plasma ara-c, of was determined at a dose rate of 1,500 mg/m 2 over 12 hours. Because our previous studies have shown that cellular ara-ctp accumulation is saturated at plasma ara-c concentrations in the range of 5 to 7 gmol/l,"' this may provide an explanation for the failure of the cells of this patient and those of two others treated on schedule II to show an increase in ara-ctp, at this dose. DISCUSSION The hypothesis underlying this investigation was that the steady-state concentration of the U Cj I cc1 C- (0 (U (U 0a ara-c Dose Rate mg/m 2 /12h 625 Fig 5. Plasma ara-c, concentrations as a function of ara-c dose rate. Plasma ara-c, values measured in patient G are the means of two determinations. active metabolite ara-ctp in circulating leukemia blasts was proportional to the dose rate of the pro-drug ara-c during high-dose continuous infusion. Furthermore, it is known that the accumulation of ara-ctp by leukemia blasts may be saturated at plasma ara-c concentrations > 10 Amol/L. 23 ' 24 Therefore, we sought to determine the range of dose rates over which such a proportionality was operative. The clinical plan for evaluation of this hypothesis was to infuse patients with relapsed acute myelogenous leukemia serially at three different ara-c dose rates, each for 12 hours. The pharmacologic approach was to quantitate the concentration of ara-ctp in circulating leukemia blasts and to relate these values to the different ara-c dose rates. The first step in this plan was to define the time required for ara-ctp to reach a steady state in leukemia blasts. This occurred relatively rapidly; steady state was achieved by three hours in the cells of four patients infused with 500 mg/m2 over 12 hours (Fig 2). This is consistent with our earlier experience in a similar group of patients who received continuous infusions of 300 to 3,000 mg/m 2 /d over 12 hours after a bolus infusion of 3 g/m 2 of ara-c.' 6 More extensive

5 626 experience in previously untreated adults with acute myelogenous leukemia receiving 1,500 mg/m2/d of ara-c by continuous infusion also supports these findings. 5 Moreover, these investigations demonstrated that circulating leukemia cells from both previously untreated and relapsed patients are capable of maintaining ara-ctp,, for more than 36 hours. Thus, the study design of measuring ara-ctp., at ten to 12 hours over the 36-hour serial-infusion duration is supported by these investigations. The proportionality between ara-c dose rate and ara-ctp, in blasts was initially investigated at infusion rates of 250, 500, and 750 mg/m2 over 12 hours, each infused serially in five patients. Four of these individuals showed successive increases in ara-ctp. as the dose rate was increased (Fig 3). Subsequently the range of dose rates was increased to 500, 1,000, and 1,500 mg/m 2 over 12 hours to evaluate the upper limit of the proportionality. Only two of the five patients evaluated on this schedule exhibited elevations in ara-ctp,, through the highest infusion rate. In contrast, the cells from four of the five patients increased their ara-ctp, levels when the infusion rate was raised from 500 to 1,000 mg/m2 over 12 hours. These results suggest that an infusion rate of 1,000 mg/m2 over 12 hours is likely to approach the upper limit of proportionality with ara-ctp,, in circulating leukemia blasts. While multiple factors, eg, ara-c uptake and plasma ara-c concentrations, may have contributed to this decreasing proportionality at higher ara-c dose rates, saturation of the rate of ara-ctp synthesis is assumed to be the major effector. Indeed, consistent with previous findings,6' 12, 26, 27 our in vitro studies demonstrated that ara-ctp accumulation in circulating leukemia cells from six of seven patients was saturated by exogenous ara-c concentrations of 10 gmol/l or less (data not shown). The plasma ara-c concentration during infusion of 1,500 mg/m 2 over 12 hours in patient G, whose blasts did not show an increase of ara-ctp,, after increasing the dose rate from 1,000 to 1,500 mg/m 2 over 12 hours, was 6.5,imol/L. This value is within the range at which saturation of ara-ctp synthesis has been demonstrated in human leukemia cells during therapy 23, 2 4 and in vitro. 6 ' 12 ' 2 6 ' 27 Substantial heterogeneity in plasma ara-c,, concentrations has HEINEMANN ET AL been demonstrated in patients during continuous infusion of high doses of ara-c.28s31 It may be that patients receiving 1,500 mg/m 2 over 12 hours whose blasts did increase ara-ctp, (patients F and I) had plasma ara-c levels that did not saturate the rate of ara-ctp accumulation. Further studies will be required to define more precisely the relationship between ara-c dose rate, plasma ara-c levels, and the intracellular ara-ctp,,. The next phase of these investigations would be a prospective test to determine if the proportionality relationship can be used to individualize the ara-c dose rate to achieve a target ara-ctp, in blasts. The question arises as to what the target ara-ctp,, should be. Our past findings indicated a therapeutic range that extended between 75 and 200 imol/l" 9 in patients with relapsed leukemia. Recent experience includes the finding of a significant difference in the ara-ctp,, in responding (119 Amol/L, n = 39) compared with resistant (64 Amol/L, n = 7) patients to induction therapy with previously untreated acute myelogenous leukemia'; (Estey and Plunkett, unpublished data, January 1989). Based on this background, it is reasonable to assume that 150 Amol/L would be in the range of a therapeutically efficacious ara-ctp, level in leukemia blasts. This approach has been attempted in a 65- year-old woman with acute promyelocytic leukemia in relapse. The patient was infused at a test rate of 250 mg/m 2 over 12 hours after which the ara-ctp,, was determined to be 24 imol/l with a plasma ara-c,, of 1.4 umol/l. Subsequently, in an attempt to raise the ara-ctp,, concentration to 150 Amol/L the ara-c infusion rate was increased to 1,500 mg/m 2 over 12 hours. While the increase of plasma ara-c,, concentration was proportional to the increment in the dose rate, ie, 1.4 to 9.8 Amol/L, the augmentation of the ara-ctp, to 94 gmol/l was less than expected. Previous studies of CIHDAC did not predict for such high plasma ara-cs, concentrations at these dose rates.23,24,28.30 The failure to achieve the target level of 150 gmol/l may be explained by the likelihood that the plasma ara-c, of 9.8 Amol/L, determined after a 12-hour infusion of 1,500 mg/m 2 over 12 hours, could have saturated the rate of synthesis of ara-ctp. 23 ' 24 While this is a plausible explanation for our findings, it is clear

6 PATIENT-SPECIFIC DOSE RATE FOR CYTARABINE that multiple factors regulate the interpatient variability that we hope to exploit for improved therapy. This approach to pharmacologic direction of leukemia therapy will be pursued in the future. ACKNOWLEDGMENT 627 The authors are grateful for the dedicated and expert technical assistance of Glen McMullen and Billie Nowak throughout these investigations, and to Susan Wilkinson for editorial assistance in the preparation of the manuscript. 1. Keating MJ, McCredie KB, Bodey GP, et al: Improved prospects for long term survival in adults with acute myelogeneous leukemia. JAMA 248: , Gahrton G: Treatment of acute leukemia-advances in chemotherapy, immunotherapy and bone marrow transplantation. Adv Cancer Res 40: , Clarkson BD, Gee T, Arlin ZA, et al: Current status of treatment of acute leukemia in adults: An overview of the Memorial Sloan-Kettering experience and review of literature. 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