Studies on methotrexate and cladribine : Bioanalytical, pharmacokinetic, and clinical aspects

Sammanfattning: The aim of these studies was to delineate the pharmacokinetics of methotrexate (MTX) and cladribine in order to improve the basis for individualization and optimization of treatment with these two antimetabolites. Methodological aspects of MTX assays in clinical routine usage were investigated as plasma MTX determinations are mandatory in the high-dose treatment of childhood acute Iymphoblastic leukemia (ALL). Four clinically commonly used assays (EIA, EMIT, FPIA1, and FPIA2) were compared with HPLC as the reference method. All non-chromatographic procedures gave very reproducible results but suffered from cross-reactivity with plasma metabolites or endogenous substances. 7- hydroxymethotrexate (7-OHMTX), a major metabolite of MTX interfered with all non- chromatographic assays and this has more significance for plasma samples obtained at later points in time when the metabolite concentration exceeds that of the parent substance. 4-diamino-N10- methylpteroic acid (DAMPA), another plasma MTX metabolite, also interfered in clinical assays using antibodies (EMIT and FPIA). However, the variability of the HPLC method was considerably higher than that of clinical assays. Clinical and pharmacokinetic risk factors regarding toxicity after high dose MTX in children with ALL were studied in 13 patients. Oral mucositis was significantly related to high plasma MTX concentration at 28 h after starting the infusion (p=0.013), to a low ratio of plasma 7-OHMTX/MTX at 66 h after starting the infusion (p=0.049), and to a slow clearance of MTX (p=0.048). The risk of leukopenia increased significantly with the course number (p=0.02). Increasing age (p=0.047) and a long exposure to a high MTX concentration in plasma (p=0.009) correlated with liver toxicity. The occurrence of mucositis was significantly related to the ratio between saliva concentration of 7- OHMTX and MTX at 20 h after starting the infusion (p=0.014), but not at later points in time. Cladribine is a chlorinated analogue of deoxyadenosine which is active against chronic Iymphoproliferative disorders. Plasma pharmacokinetic studies showed that subcutaneous administration gave a drug exposure (AUC) similar to intravenous infusion. The bioavailability of oral cladribine was 50%. Food and omeprazole had practically no influence on the bioavailability of cladribine but food reduced the peak concentration and prolonged the time to reach the maximum plasma concentration. In a perused rat liver experiment, cladribine was highly degraded by cleavage of the N-glycosidic bond by liver enzymes. The fluorinated analogue of cladribine, 2-chloro-2'- arabino-fluoro-2'-deoxyadenosine (CAFdA) was more stable against degradation and showed 2-5 fold lower hepatic extraction ratio as compared to cladribine suggesting that CAFdA is a more suitable drug for oral administration than cladribine. An ion-pair HPLC method was developed for monitoring of the pharmacokinetics of cladribine nucleotides in patients. Pharmacokinetics of cladribine, its deglycosylated product, CAde, and cladribine nucleotides (CdAMP, CdADP and CdATP) were studied in 17 patients with CLL after treatment with cladribine. The concentration of CAde was considerably higher after oral administration. The AUC of CdAMP in leukemic cells was generally higher than that of CdATP (median ratio 1.5). In 4 out of 17 patients the reverse relation was seen. The concentration of the 5'-diphosphate was much lower, in most patients undetectable. The terminal phase had a median t1/2 of 14.6 h for CdAMP and 9.7 h for CdATP. No correlation was observed between the concentration of active metabolite (CdATP) and clinical response.