Pharmacokinetics of cladribine in plasma and its 5'-monophosphate and 5'-triphosphate in leukemic cells of patients with chronic lymphocytic leukemia.

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 separates 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 micromol/liter x h) than the AUC of CdATP (median, 22 micromol/liter x h); 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 (t(1/2)) of CdAMP was 15 h, and that of CdATP was 10 h. The median terminal t(1/2) 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 great heterogeneity among patients in terms of AUC and t(1/2) 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.

Interspecies differences in the kinetic properties of deoxycytidine kinase elucidate the poor utility of a phase I pharmacologically directed dose-escalation concept for 2-chloro-2'-deoxyadenosine.

2-Chloro-2'-deoxyadenosine (CdA, Cladribine), is a purine antimetabolite currently under investigation in phase II clinical trials for the treatment of lymphoid malignancies. Significant differences in CdA toxicity between mice and humans were observed during phase I clinical evaluation. For the elucidation of interspecies differences in drug toxicity the pharmacokinetics of CdA after subcutaneous injection and the kinetic properties of the CdA-phosphorylating enzyme, deoxycytidine kinase (dCK), were compared in mice and humans. The ratio of the dose lethal to 10% of mice (LD10) to the maximum tolerated dose (MTD) in humans was 50 and the ratio of the area under the curve obtained at approximately one-half the LD10 (AUCapprox. one-half the LD10)/AUC(MTD) was 49. A significant interspecies difference was observed in the kinetic properties of dCK, the main CdA-activating enzyme. With CdA as a substrate, the Michaelis constant (Km) of dCK in crude extracts of mouse thymus was 10 times higher than that in human thymus. An approximately 9-fold interspecies difference in maximum velocity (Vmax)/Km indicated a higher efficiency of dCK for CdA in humans than in mice. The peak plasma concentration was 210 times higher and exceeded the Km in mice. Initial and terminal half-lives were approximately 7 times shorter in mice and trough levels were similar in mice and humans. Thus, the differences in AUCs at equitoxic doses are largely explained by differences in the target enzyme properties and the pharmacokinetic pattern. The observed lower tolerance for CdA in humans as compared with mice confirms the view that antimetabolites may not be good candidates for pharmacokinetically guided dose-escalation schemes unless detailed information on interspecies variability in drug bioactivation is available.

Decrease in intensity of DNA fluorescence caused by interaction between DNA and platinum complexes.

The decrease of DNA fluorescence caused by an impaired capacity of ethidium bromide to intercalate into the DNA reflected structural changes caused in the DNA molecule by its interaction with platinum complexes. This fall in DNA fluorescence was proportional to the length of exposure of DNA to the platinum complexes, and depended on the environment in which the interaction took place. The therapeutically active cisplatinum (cis-DDP) was more efficient to inhibit fluorescence in a solution of 4 X 10(-3) mol NaCl than its therapeutically inactive trans-isomer (trans-DDP). For comparison, the inhibition of DNA fluorescence was also studied in a solution of 10(-2) mol NaClO4. The inhibitory effect was elicited more rapidly, but no difference was found between the two isomers. We concluded that the larger effect of cis-DDP on DNA was induced by the 4 X 10(-3) mol concentration of NaCl. Since also the intracellular concentration of chloride ions is 4 X 10(-3) mol, it cannot be ruled out that the interaction between DNA and cis-DDP and trans-DDP in vivo might be influenced by the intracellular environment.

HPLC analysis of platinum cytostatics.

High performance liquid chromatography (HPLC) with diode array detector (DAD) was applied for the separation of platinum cytostatics (cisplatin, carboplatin and oxo-carboplatin). Their stabilities were studied in water and the influence of chloride anions, pH, temperature and time was discussed and rate constants of the aquation reactions at different conditions were calculated. Ligand exchange reactions cisplatin----carboplatin were evaluated using HPLC-DAD system. Detection limits for all studied cytostatics were determined and extraction recoveries for carboplatin in clinical samples (serum) were demonstrated for a wide range of concentrations together with the relative standard deviations. The solid-phase extraction procedure was recommended for clinical sample analysis represented by a model mixture and real patients' samples.

HPLC determination of methotrexate and its metabolite in serum.

Determination of methotrexate (MTX) residues in biological samples (serum, cerebrospinal fluid) was worked out using liquid-liquid and solid-phase extractions and high performance liquid chromatography (HPLC) with UV detection. Clinical samples from patients treated by low and high doses of MTX were analyzed using reversed-phase and ion-pair liquid chromatography, and buffer-methanolic mobile phases. Chromatographic conditions were optimized for the simultaneous determination of MTX and its metabolite and the minimal analysis time was recommended. The results were statistically evaluated, elimination curves and chromatograms have been demonstrated. Solid-phase extraction recovery was 93.1 +/- 1.5% and the determination limit for methotrexate in serum samples was 5.10(-7) mol/l and after the preconcentration of samples 5.10(-8) mol/l.

Comparison of some biochemical parameters of arabinosylcytosine and cyclocytidine in L1210 murine leukemia cells.

The basic biochemical characteristics of cyclocytidine hydrochloride (cC.HCl) and arabinosylcytosine (araC) were compared. It was demonstrated that despite different lipophilicity and different pK (4.15 for araC and 6.60 for cC.HCl), the mechanism of inhibition of DNA synthesis by both compounds is the same (ID50 for araC was 0.048 mumol/l and for cC. HCl 0.23 mumol/l). The compounds had a different mechanism of inhibition of RNA synthesis (ID50 for araC was 2.69 mmol/l and for cC.HCl 1.08 mmol/l) and showed a marginal effect on protein synthesis. Hydrolysis of the 0(2),2'-anhydro bond in cC.HCl and formation of araC in vivo was characterized by a Km = 280 mumol/l using HPLC. Deamination of araC in vivo was studied in healthy mice (Km = 247 mumol/l), 8.6% of arabinosyluracil 15 minutes after araC administration) and in mice with sensitive and araC resistant leukemia L1210 (15.5% and 8.5% of arabinosyluracil 15 minutes after araC administration, respectively). On the basis of different physico-chemical properties of cC.HCl and different mechanisms of inhibition of RNA synthesis it can be assumed that cC.HCl, when therapeutically used, may have its own mechanism of biological effect(s) and that its application may be therapeutically advantageous in some aspects as compared to araC.

Determination of 2-chloro-2'-deoxyadenosine nucleotides in leukemic cells by ion-pair high-performance liquid chromatography.

A specific isocratic ion-pair HPLC method for the quantitation of mono-, di- and triphosphates of 2-chloro-2'-deoxyadenosine (CdA) in leukemic cells from patients is described. The method is based on an extraction of nucleotides from cells with a solution of perchloric acid containing triethylammonium phosphate followed by an isocratic separation on an Ultrasphere ODS column (250 x 4.6 mm, 5 microns) with a mixture of 89% triethylammonium phosphate buffer (0.08 M, pH 6.1) and 11% methanol as the eluent. UV absorbance at 265 nm was used. The limit of detection was 65 nM. Standard curves for the CdA triphosphate (CdATP) were linear within the concentration range of 200 nM to 12 microM. The mean overall recovery of CdATP was 90% within a concentration range of standard curves. The within-day and day-to-day coefficients of variation at concentrations of 1.44 microM and 6.25 microM CdATP were < 10%. The applicability of the method was demonstrated by in vitro studies of the accumulation of CdA mono-, di- and triphosphates in CCRF-CEM cells and by determination of the cellular pharmacokinetics of CdA nucleotides in leukemic cells from a patient treated with CdA.

Evaluation of synergism of drugs cis-diamminedichloroplatinum (II) and arabinosylcytosine on the level of chemical interaction with DNA and on the growth of mouse leukemia.

1. Cytotoxic synergism of drugs cis-diamminedichloroplatinum(II) (cis-DDP) and arabinosylcytosine (araC) was studied both on the level of interaction with DNA in chemically determined conditions and on leukemia L1210 bearing mice. 2. AraC and its structural natural precursor cytidine were tested for the modulation of kinetics of bifunctional adducts production induced by cis-DDP in DNA. 3. This process plays the basic role in cytotoxic mechanism and antitumor activity of cis-DDP. 4. No interaction was seen between cis-DDP and araC. Further, presence of araC in reaction mixture had no effect on cis-DDP-DNA interaction. 5. Therefore, cytotoxic synergism does not arise in the araC-cis-DDP-DNA interaction and its origin is different. 6. Finding that cytidine has no synergistic effect on life span of leukemia L1210 bearing mice when administered together with cis-DDP it shows the difference between cytidine and araC. 7. The small structural difference between cytidine and araC is very important for synergism of cytotoxicity.

Analysis of 2-chloro-2'-deoxyadenosine in human blood plasma and urine by high-performance liquid chromatography using solid-phase extraction.

A reversed-phase high-performance liquid chromatographic (HPLC) method for the simultaneous determination of a new and promising anticancer drug, 2-chloro-2'-deoxyadenosine (CdA), and its metabolite, 2-chloroadenine (CAde), in plasma and urine was developed. A solid-phase extraction procedure with guaneran as internal standard (IS) was used. Plasma (1 ml) or diluted urine (1/100) mixed with 1 ml of phosphate buffer (10 mM, pH 6.5) was applied on a C8 isolute cartridge, which was prewashed with acetonitrile and phosphate buffer. The cartridge was further washed with 2.5 ml of 1% acetonitrile/phosphate buffer and 2.5 ml of hexane/dichloromethane (50/50). The compounds were eluted from the cartridge with 2.5 ml 5% MeOH in ethyl acetate. Chromatographic separation was achieved on C18 column eluted isocratically with phosphate buffer (10 mM, pH 3.0) containing 11% MeOH and 7% acetonitrile, and ultraviolet (UV) detection at 265 nm. Recoveries of CdA and CAde at 100 nmol/L were 90.6 +/- 4.9 and 98.7 +/- 7.8%, respectively. Recovery of IS was 96.1 +/- 6.1% at 250 nmol/l. The inter- and intraday coefficients of variation (CV) were < 10% at different concentrations within the range 1-500 nmol/L for both substances. In plasma, limits of detection of CdA and CAde were 1 and 2 nmol/L, respectively. In urine, the limit of detection was 100 nmol/L for both compounds. Standard curves were linear up to 50 and 500 nmol/L for urine and plasma, respectively. The present method will be a useful tool for further investigations of the pharmacokinetics of CdA in patients treated with different routes of administration.

Biotransformation and in vitro activity of an arabinosylcytosine 5'-chloro-5'-deoxy analog.

5'-Chloro-5'-deoxyarabinosylcytosine (5'-chloro-araC), a lipophilic and cytidine-deaminase resistant analog of the cytotoxic agent arabinosylcytosine (araC) was evaluated in terms of bioactivation, transformation and its cytotoxic activity in vitro. 5'-Chloro-araC interferes with DNA synthesis (IC50 = 2.8 mumol/l) and inhibits the growth of L1210 cells in suspension culture (IC50 = 1.05 mumol/l) and in the soft agar assay (IC50 = 0.65 mumol/l). Being phosphorylated to the triphosphate of araC-araCTP (5'-triphosphate of araC), 5'-chloro-araC has the same mechanism of action as arabinosylcytosine. In alkaline solutions 5'-chloro-araC is transformed to another (cytostatically inactive) araC analog--2',5'-anhydroarabinosylcytosine--but at physiological pH and temperature conditions, it has sufficient stability to be phosphorylated and thus activated. A lower rate of araCTP formation from 5'-chloro-araC explains the somewhat lower cytotoxic effect of this compound against various established cell lines in vitro compared to araC. Lipophilicity that would allow an oral drug formulation and certain other superior physico-chemical and biochemical characteristics of 5'-chloro-araC make this compound an interesting candidate for further investigations.

The disposition of new arabinosylcytosine derivative-- 5'-chloro-5'-deoxy-arabinosylcytosine--in rats.

1. Pharmacokinetic properties of a new derivative of the widely used and very potent antileukemic agent arabinosylcytosine (araC)--5'chloro-5'-deoxy-arabinosylcytosine (5'-Cl-araC)--were investigated after intraperitoneal (i.p.) and oral routes of administration in rats and compared with the equimolar dose of araC administered orally. 2. It was found that substitution of the hydroxyl group at position 5' resulted in a change of pharmacokinetic parameters. 3. There is a large difference in average serum concentrations of 5'-Cl-araC administered by the i.p. and oral routes; the average serum concentration obtained after i.p. injection being several times higher in comparison to those after oral administration. 4. However, the latter are, at the same time, lower than the average serum concentrations of araC administered by the same route in an equimolar dose. 5. On the other hand, the apparent volume of distribution is much larger, and the area under the curve of serum concentration of 5'-Cl-araC is smaller, after oral as compared to the i.p. route of administration indicating more extensive tissue distribution together with higher tissue binding of 5'-Cl-araC when compared to the parental drug araC.