Determination and quantification of intracellular fludarabine triphosphate, cladribine triphosphate and clofarabine triphosphate by LC-MS/MS in human cancer cells.

Purine nucleoside analogues are widely used in the treatment of haematological malignancies, and their biological activity is dependent on the intracellular accumulation of their triphosphorylated metabolites. In this context, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to study the formation of 5'-triphosphorylated derivatives of cladribine, fludarabine, clofarabine and 2'-deoxyadenosine in human cancer cells. Br-ATP was used as internal standard. Separation was achieved on a hypercarb column. Analytes were eluted with a mixture of hexylamine (5 mM), DEA (0.4%, v/v, pH 10.5) and acetonitrile, in a gradient mode at a flow rate of 0.3mLmin-1. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI-) were used for detection. The application of this method to the quantification of these phosphorylated cytotoxic compounds in a human follicular lymphoma cell line, showed that it was suitable for the study of relevant biological samples.

Effect of gamma-irradiation on cladribine and cladribine-containing biodegradable copolymers.

The aims of this study were to assess the effects of sterilization with gamma-irradiation on (i). bulk cladribine and (ii). cladribine-containing biodegradable copolymers. The stability of cladribine upon irradiation was confirmed by TLC, HPLC, UV, IR, DSC, rentgenography and electron microscopy. The stability of copolymers containing cladribine upon irradiation was assessed by IR, DSC and EPR. In vitro kinetics of nucleoside release from the copolymers before and after irradiation were compared, and only slight changes were found. Results of our study indicate that gamma-irradiation can be safely applied for the sterilization of cladribine or cladribine-containing copolymers for medical purposes.

Efficient syntheses of 2-chloro-2'-deoxyadenosine (cladribine) from 2'-deoxyguanosine(1).

We report efficient syntheses of the clinical agent cladribine (2-chloro-2'-deoxyadenosine, CldAdo), which is the drug of choice against hairy-cell leukemia and other neoplasms, from 2'-deoxyguanosine. Treatment of 3',5'-di-O-acetyl- or benzoyl-2'-deoxyguanosine (1) with 2,4,6-triisopropyl- or 4-methylbenzenesulfonyl chloride gave high yields of the 6-O-arylsulfonyl derivatives 2 or 2'b. Deoxychlorination at C6 of 1 also proceeded to give the 2-amino-6-chloropurine derivative 5 in excellent yields. The nonaqueous diazotization/chloro dediazoniation (acetyl chloride/benzyltriethylammonium nitrite) of 2, 2'b, and 5 gave the 2-chloropurine derivatives 3, 3'b, and 6, respectively. The selective ammonolysis at C6 (arylsulfonate with 3 or chloride with 6) and accompanying deprotection of the sugar moiety gave CldAdo (64-75% overall yield from 1).

Evaluation of purine and pyrimidine analogues in human tumor cells from patients with low-grade lymphoproliferative disorders using the FMCA.

The purine analogues fludarabine and cladribine (CdA) have recently become established to be effective treatment for low-grade non-Hodgkin's lymphoma (NHL). The pyrimidine nucleoside analogue cytarabine (AraC) has an important place in the treatment of acute leukemia, and gemcitabine is a new pyrimidin antimetabolite which has shown clinical activity against solid tumors. We have used the semiautomated fluorometric microculture cytotoxicity assay (FMCA), based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA), to study these drugs. Eighty samples from 60 patients with low-grade NHL were studied. Fifty samples from patients with acute lymphoid leukemia (ALL) and 118 samples from patients with acute myeloid leukemia (AML) were included for comparison. The results indicate that the purine- and pyrimidine nucleoside analogues tested may be as active against low-grade NHL as against acute leukemia. In low-grade NHL, AraC seems to be even more active in comparison to CdA (p=<0.0001) and fludarabine (p=0.001). Untreated patients were more drug sensitive than previously treated patients. Gemcitabine showed the highest correlation with AraC (0.90) whereas CdA showed the highest correlation with fludarabine (0.84). Based on these results we propose that AraC and gemcitabine may have a role in the treatment of low-grade NHL.

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.

Characterization of cladribine and its related compounds by high-performance liquid chromatography/mass spectrometry.

High-performance liquid chromatography/mass spectrometer (HPLC/MS) was used to identify and structurally characterize the modified nucleoside cladribine (2-chloro-2'-deoxy-beta-adenosine) and 13 synthesis-related byproducts in bulk drug. Confirmation of compound identity was accomplished by spectral analysis (1H and 13C NMR spectroscopy, mass spectrometry, and UV absorption spectroscopy) of the related compounds as isolated from crude mixtures of the drug substance and by spiking experiments with authentic standards. The use of on-line mass spectrometric analysis (i.e., LC/MS) to augment UV absorption spectra permitted rapid identification of many of the compounds of interest.