Levels of 2′-Deoxycoformycin, Adenosine, and Deoxyadenosine in Patients with Acute Lymphoblastic Leukemia Peter M. Venner, Robert I. Glazer, Julie Blatt, et al. Cancer Res 1981;41:4508-4511. Published online November 1, 1981.
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[CANCER RESEARCH 41, 4508-4511, November 1981] 0008-5472/81 /0041-0000$02.00
Levels of 2'-Deoxycoformycin,
Adenosine, and Deoxyadenosine in
Patients with Acute Lymphoblastic Leukemia Peter M. Venner,1 Robert I. Glazer, Julie Blatt, Stephan Sallan, Gaston Rivera, John S. Holcenberg, Jeffrey Lipton, Sharon B. Murphy, and David G. Poplack Applied Pharmacology Section, Laboratory of Medicinal Chemistry and Biology [P. M. V., R. I. G.]; Pediatrie Oncology Branch [D. G. P., J. B.J National Cancer Institute. Bethesda. Maryland 20205: Sidney Farber Cancer Institute, Boston, Massachusetts 02115 [S. S., J. L.]; St. Jude Children's Research Hospital, Memphis. Tennessee38101 [G. R., S. B. M.]; and Milwaukee Children's Hospital, Milwaukee, Wisconsin 53233 [J. S. H.]
ABSTRACT
2'-Deoxycoformycin (dCF), a potent inhibitor of adenosine deaminase, has recently undergone Phase I clinical trials and has been found to be therapeutically active in acute lymphoblastic leukemia. In this report, levels of dCF in plasma, plasma concentrations of adenosine and deoxyadenosine, and urine levels of deoxyadenosine were measured in leukemic patients undergoing treatment with dCF during a Phase I clinical trial. dCF was administered i.v. at a dose of 0.25 to 1.0 mg/kg (7.5 to 30 mg/sq m) for 3 consecutive days. Plasma drug levels of 2 to 6 ¡IM were observed following the third dose of dCF, and drug accumulation occurred only at the 1-mg/kg dosage. In this limited series of patients, the plasma concentrations of adenosine and deoxyadenosine and the urine concentration of deoxyadenosine did not show an obvious correlation with dCF dose, therapeutic response, or toxicity.
INTRODUCTION
In 1974, dCF ' was isolated as a fermentation product of Streptomyces antibioticus (21) and was subsequently found to be the most potent inhibitor of ADA yet discovered (3). Its inhibitory effect on ADA involves its stoichiometric binding as a transition-state inhibitor with an extremely high association constant (2). The action and therapeutic uses of dCF and related inhibitors have been reviewed recently (7). As a single agent in preclinical testing, dCF was found to have no antitumor activity but to cause lymphocytopenia and to act as an immunosuppressive agent (1, 15, 16). In early clinical trials in England, patients with ALL were found to be responsive to dCF (17). Based on these initial findings, a cooperative Phase I study was initiated in pediatrie patients with ALL (13). The results of this study confirmed the activity of this drug in ALL, a finding also observed recently by other investigators (4, 11-16). In this report, we present the pharmacokinetic data of plasma concentrations of dCF obtained from patients participating in the above mentioned Phase I trial (13). Plasma concentrations of adenosine and dAdo and urine concentrations of dAdo were also assessed in an attempt to 1Recipient of a McEachern Fellowship awarded by the Canadian Cancer Society. To whom requests for reprints should be addressed, at Cancer Research Unit, McEachern Laboratory, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. 2The abbreviations used are: dCF, 2'-deoxycoformycin; ADA, adenosine deaminase; ALL, acute lymphoblastic leukemia; dAdo, 2'-deoxyadenosine; CSF, cerebrospinal fluid; CNS, central nervous system. Received March 5, 1981 : accepted August 11. 1981.
relate the toxic or therapeutic activity of dCF to altered metab olite levels arising from the blockade of ADA. MATERIALS
AND METHODS
Patients. Twenty-six patients (19 male and 7 female) with ALL, ranging from 3 to 23 years of age (median age, 10 years), were entered into the study (13). These patients were evaluated for clinical response and toxicity as described previously (13). Of this number of patients, 19 were available for pharmacokinetic studies, although not all analy ses were performed on every patient. dCF was administered i.v. for 30 min for 3 consecutive days starting at a dose of 0.25 mg/kg (9 courses) and then escalating the dose to 0.5 mg/kg (7 courses), 0.75 mg/kg (19 courses), and 1.0 mg/kg (7 courses). Venous blood samples were collected at 1, 4, and 24 hr after each dose of dCF and placed in heparinized tubes. Plasmaobtained following centrifugation was stored at -20°. Twenty-four-hr urine collections were adjusted to pH 7, and aliquots were stored at —¿20°. CSF obtained from diagnostic lumbar punctures was stored at -20°. dCF Analysis. The concentrationof dCF in plasmaand urine was determined by the enzyme inhibition assay described previously (5). The plasma was prepared for analysis by centrifugation in Amicon (Amicon Corp., Lexington, Mass.) Centriflo CF-25 Ultrafiltration mem brane cones (molecular weight 25,000 exclusion limit) at 750 x g for 25 min. Recovery of known concentrations of dCF, adenosine, and dAdo was greater than 80%. The enzyme inhibition assay was deter mined with 0.00625 unit of ADA (calf intestine; Sigma Chemial Co., St. Louis, Mo.) preincubated with appropriate dilutions of plasma in 0.05 M potassium phosphate buffer, pH 7.4, for 10 min and followed by the addition of this mixture to a 1-ml cuvet containing 0.1 mw adenosine. Dilutions of plasma were prepared to obtain reaction rates of 50 to 85% of the uninhibited control reaction rates. Urine and CSF samples were similarly diluted but were not processed by ultrafiltration. A standard enzyme inhibition curve was prepared with dCF standards of 0.2 to 0.6 HMand was determined for each dilution of enzyme used in the assay. Urine concentrations of dCF could not be determined accurately because of substances which interfered with the enzyme inhibition assay and were not further characterized. Adenosine and dAdo Analyses. The concentrationsof adenosine and dAdo in plasma and urine were determined by HPLC using a reversed-phase column (/i Bondapak C,H;Waters Associates, Milford, Mass.) (8). Plasma and urine samples were depleted of protein by centrifugation in Amicon membrane cones as described under "dCF Analysis." Analyses of urine were carried out in 25-/il aliquots with a Waters 204-W high-pressure liquid chromatography apparatus using a linear gradient of 0.02 M KH2PO4,pH 5.6, to 15% (v/v) methanol in water over a period of 30 min at a flow rate of 1.5 ml/min. Sample volumes were 25 /tl. Plasma samples were analyzed similarly with a Spectra Physics 8000 high-pressure liquid chromatography system utilizing a linear gradient of 0.02 M KH2PO„, pH 5.6, to 18% (v/v) methanol in water over a period of 30 min at a flow rate of 1.0 ml/min. Adenosine and dAdo peaks were identified by their respective retention times and coelution with known standards. Concentrations were deter-
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CANCER
Downloaded from cancerres.aacrjournals.org on July 13, 2011 Copyright © 1981 American Association for Cancer Research
RESEARCH
VOL. 41
Clinical Pharmacology of dCF mined by comparison of peak height or peak area using adenosine and dAdo standards (2.5 to 12.5 nmol). RESULTS
Plasma concentrations of dCF are shown in Chart 1 and Table 1. Levels of dCF were generally dose dependent, partic ularly at 1 hr after each dose, and the disappearance of drug from the plasma approximated first-order kinetics. Plasma levels of 2 to 6 ¡IM were attained 1 hr after i.v. injection of dCF, and no accumulation of drug was noted after 3 consecutive daily doses except at the 1-mg/kg dosage. The disappearance kinetics of drug from plasma was similar in all patients regard less of toxicity or clinical response states. CSF samples were available for study in 2 patients. In one patient, the CSF concentration of dCF 4 hr after the first dose of 0.25 mg/kg was 0.07 JUM,while the plasma level of drug was 0.6 JIM.In a second patient receiving 3 consecutive 1.0mg/kg doses of dCF, the drug concentration in the CSF was 0.11 juM1 day after the last dose, and the plasma concentration of drug was 0.21 /IM. Adenosine and dAdo concentrations in the plasma at 1, 4, 24, 48, and 72 hr following the initiation of dCF therapy were measured in 7 patients (Chart 2). Prior to each course of
1X10-5 -
treatment, neither adenosine nor dAdo were detectable. Fol lowing therapy, the levels of adenosine ranged from undetectable (
