Pharmacokinetics of Cladribine in Plasma and Its 5’-Monophosphate and 5’-Triphosphate in Leukemic Cells of Patients with Chronic Lymphocytic Leukemia1 Freidoun Albertioni,2 Synn#{246}ve Lindemalm,

The pharmacokinetic parameters of cladribine (CdA) in patient plasma and its intracellular nucleotides CdA 5’monophosphate (CdAMP) and CdA 5’-triphosphate (CdATP) were delineated in circulating leukemia cells in 17 patients with chronic lymphocytic leukemia, after the last dose intake and up to 72 h thereafter. Patients were treated with 10 mg/m2 CdA p.o. on 3 consecutive days. A novel and specific ion-pair liquid chromatographic method, which sep. arates the intracellular CdA nucleotides, was used. The area under the concentration versus time curve (AUC) of CdAMP in leukemia cells was generally higher (median, 47 jimol/ Hterh) than the AUC of CdATP (median, 22 tmol/literh); however, in some patients (3 of 17), the reverse relationship was seen. The median ratio between the AUC values for CdATP and CdAMP was 0.60 (95% confidence interval, 0.4-1.0). The median half-life (t112) of CdAMP was 15 h, and that of CdATP was 10 h. The median terminal t112 of CdA in plasma was 21 h. A significant correlation was found between the maximum plasma CdA and cellular CdAMP concentrations (r 0.56, P 0.02). There was no correlation between the AUC values of cellular CdAMP and CdATP (r 0.224, P = 0.55). No correlation was found between deoxycytidine kinase activity and intracellular pharmacokinetic parameters of CdAMP or CdATP. The response to treatment was not significantly related to intracellular concentration of CdAMP or active metabolite CdATP. There is Received 10/14/97; revised 12/22/97: accepted 12/24/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This study was supported by grants from the Swedish Cancer Foundation, the Swedish Children Cancer Foundation, the Swedish Medical Research Council, and the Jenny Nordqvist Foundation. 2 To whom requests for reprints should be addressed. Phone: 46-8517 75832; Fax: 46-8-331343; E-mail: [email protected]. great heterogeneity among patients in terms of AUC and t ,2 of CdAMP and CdATP. Furthermore, the results emphasize the differences between the pharmacokinetics of plasma CdA and those of the metabolites in circulating leukemic cells. INTRODUCTION The antimetabolite 2-chboro-2’-deoxyadenosine (CdA3 or Leustatin) is a purine analogue that is resistant to adenosine deaminase due to protonation at the N-7 position (1). In dividing cells, CdA is incorporated into DNA and arrests cells in S phase (2). CdA also inhibits ribonucleotide reductase (3), thus causing an imbalance in the intracellular pool of deoxyribonucleotides. In nondividing cells, CdA causes an accumulation of DNA strand breaks, which activates poly(ADP) ribosylation, leading to depletion of NAD and apoptosis (4, 5). CdA is the drug of choice for HCL (6). with about an 85% complete response. It is also a promising drug for the treatment of other lymphoproliferative disorders, such as CLL (7-9), low-grade non-Hodgkin’s bymphoma (10), and Waldenstr#{246}m’s macrogbobulinemia ( 1 1 ). CdA is also effective in acute myeboid leukemia (12, 13), Langerhans cell histiocytosis (14), multiple sclerosis, and other autoimmune diseases (15, 16). CdA is a prodrug and intracellular phosphorybation is needed for cytostatic effect. Interindividual variability in intracellular phosphorylation may explain differences in response to the treatment. Investigation of the cellular pharmacokinetics of the active metabolites is, therefore, warranted. The metabolic pathways of CdA include degradation to CAde (17), an apparently inactive metabolite (18), and intracellular phosphorylation of CdA by dCK to CdAMP (19) and, further, to CdATP by relatively abundant nucleotide kinases. In contrast to other nucleoside analogues, the phosphorybation of CdA by dCK to its 5’-monophosphate, CdAMP, is not the rate-limiting step in the anabolism of the CdA to CdAIP. CdATP is generally considered to be responsible for the cytotoxic effects of CdA. However, previous pharmacokinetic studies suggest that there is a direct relationship between plasma pharmacokinetics and the clinical outcome in HCL (20). The cellular pharmacokinetics of total CdA nucleotides were described previously (21). In that study, the concentrations 3 The abbreviations used are: CdA, cladribine; HCL, hairy cell leukemia; CLL, chronic lymphocytic leukemia; CAde, 2-chloroadenine; dCK, deoxycytidine kinase; CdAMP, CdA 5’-monophosphate; CdATP, CdA 5’-triphosphate; HPLC, high-performance liquid chromatography; AUC, area under the concentration versus time curve; CI. confidence interval. Research. on September 22, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 654 Intracellular Pharmacokinetics of CdA Nucleotides Table 1 Patient characteristics Characteristic Median Range Total no. of patients 17 Sex (no. of patients) Male 9 Female 8 Age (yr) 71 45-90 Weight (kg) 70 54-106 Height (cm) 172 155-182 Body surface area (m2) 1.8 1.5-2.0 Lymphocytes (X 109/liter) 69 19-S 12 Hemoglobin (g/liter) 1 1 1 68-137 Platelets (X 109/liter) 131 46-281 Serum creatinine (p.mollliter) 91 76-142 Bilirubin (p.mollliter) 10 6-32 Transaminase (no. of patients) Alanine aminotransferase (p.katallliter) 0.4 0.2-0.8 Aspartate aminotransferase (p.katallliter) 0.5 0.5-0.9 Prior therapy (no. of patients) 0 regimens 1 1 1-2 regimens 6 of the total CdA nucleotides were measured indirectly from the concentrations of the parent nucleoside, CdA, following dephosphorylation by treating CdA nucleotides with alkaline phosphatase. The cellular kinetics of CdA metabobites (specifically, CdAMPs and CdATPs) have not been determined previously in vivo. Here, we describe the intracellular pharmacokinetics of CdAMP and CdATP during the treatment of CLL, with a comparison to the plasma pharmacokinetics using a specific ion-pair HPLC method. A preliminary report of part of this work was reported elsewhere in abstract form (22). PATIENTS AND METHODS Chemicals and Reagents. CdATP, synthesized by Sierra Bioresearch (Tucson, AZ), was provided by Dr. William Plunkett (M. D. Anderson Cancer Center, Houston, TX). CdA and CAde were synthesized by Dr. Zygmunt Kazimierczuk at the Foundation for the Development of Diagnostics and Therapy (Warsaw, Poland). The molar absorbtivities were 15,000 and 12,600 absorbtivity units/mol for CdA and CAde, respectively, at 264 nm (pH 7.0). Guaneran was generously donated by Dr. Gertrude Ebion (Wellcome Foundation, Research Triangle Park, NC). Triethybamine was purchased from Fluka (Buchs, Switzerland), and Lymphoprep was purchased from Nycomed (Oslo, Norway). Other compounds were of analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO). Patients and Treatment. Seventeen patients with (Bcell) CLL participated in the study, having given their informed consent prior to initiation of the study (Table 1). The study was approved by the local Ethics Committee at the Karolinska Institute (Dnr 94:369) and the Swedish Medical Products Agency (Dnr 15 1 : 1603/94). CdA was administered over 3 consecutive days. The dose for iv. (2-h infusion) administration was 5 mg/m2. The corresponding p.o. dose was 10 mg/m2, administered after overnight fasting. The solutions (2 mg/mI) were taken with 150 ml of water. No food was allowed until 2 h after dosage. Patients receiving the CdA treatment in the study were required to have normal renal and hepatic function. Response to treatment was evaluated according to guidelines for clinical protocols for CLL, as recommended by the National Cancer Institute (23). Responders are patients who had partial or complete remissions, and nonresponders are patients who had stable or progressive disease. Blood Sampling. Venous blood samples (10 ml) were collected from an indwelling catheter into heparmnized Venoject tubes before drug intake at the following time points: before and 1, 2, 3, 4, 6, 9, 12, 16, 24, 36, 48, 60, and 72 h after dosing on day 3. In the first two patients, three additional blood samples were also taken at 0.25, 1.5, and 20 h after dosing. The blood was stored on ice. The plasma and leukemic cells were isolated within 6 h after sampling and frozen immediately at -20#{176}C.The analyses were performed within 2 months for plasma and within 3 weeks for cellular CdA metabolite measurements. Leukemic Cell Isolation. Mononuclear cells were isolated by Ficoll-Hypaque density centrifugation. After separation on Lymphoprep and washing with PBS, the cell pellet was dissolved in 2 ml of ice-cold distilled water. One ml of 2.7% NaCl was added, and after 30 s, the volume was adjusted to 50 ml with PBS. The number ofcells in the samples and the median cell volume of the samples analyzed were determined by a Coubter Mubtisizer (Coulter Electronics, Luton, United Kingdom). Determination of CdA, CAde, and CdA Nucleotides. A previously published (17) reversed-phase HPLC method was used to quantitate CdA and CAde in plasma. CdA and CAde were determined at 265 nm, with a limit of quantitation of 1 n using 1 ml of plasma. CdA nucleotides were extracted from leukemic cells by a previously described HPLC method (24). In brief, a 200-pA volume of ice-cold 0.4 M perchloric acid containing 0.08 M triethylammonium phosphate was added to the cell pellet (1 X l08 3 x 108), mixed on a vortex mixer, and brought to pH 6.2 by addition of 100 pA of ice-cold 1.2 M potassium hydroxide-0.4 M ammonium dihydrogenphosphate. The supernatant was collected after vortex mixing and centrifugation at 14,500 X g for S mm at 4#{176}C.A 90-pA aliquot was injected onto the HPLC column directly, or the sample was stored at 20#{176}Cuntil analysis. The column was an Ultrasphere ODS (250 X 4.6 mm; 5 jim), purchased from Beckman Instruments (Fullerton, CA) and equipped with the Guard-Pak precolumn (p.Bondapak Cl8; Millipore, Milford, MA). The mobile phase consisted of triethybammonium phosphate buffer (0.08 M, pH 6.1) and methanol (89: 1 1, v/v). The elution was carried out at flow rates of 1.5 and 1.8 mb/mm, respectively, at 265 nm and at the ambient temperature (22#{176}C).The temperature of the autosampler was maintamed at 8#{176}C. The concentration of the CdA nucleotides was measured by comparing the peak area of the CdAMP and CdATP concentrations obtained with those of the standard substance, CdATP. Measurement of dCK. dCK activities were measured in extracts of leukemic cells according to a previously published procedure (25) using CdA as a substrate. The dCK activities were expressed as pmol/mg of cellular protein/mm (pmol/mg/ mm). Pharmacokinetic Variables. Pharmacokinetic variables were calculated for each subject from the plasma and cell Research. on September 22, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from