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.

Determination of intracellular fludarabine triphosphate in human peripheral blood mononuclear cells by LC-MS/MS.

Fludarabine is a nucleoside analog routinely used in conditioning regimens of pediatric allogeneic stem cell transplantation to promote stem cell engraftment. In children, it remains a challenge to accurately and precisely quantify the active intracellular triphosphate species of fludarabine in vivo, primarily due to limitations on blood volume and inadequate assay sensitivity. Here we report a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of fludarabine triphosphate in human peripheral blood mononuclear cells (PBMC). PBMC (∼5 million cells) were collected and lysed in 1mL 70% methanol containing 1.2mM tris buffer (pH 7.4). The lysate (80µL) was mixed with internal standard (2-chloro-adenosine triphosphate, 150ng/mL, 20µL) and injected onto an API5000 LC-MS/MS system. Separation was achieved on a hypercarb column (100mm×2.1mm, 3µm) eluted with 100mM ammonium acetate (pH 9.8) and acetonitrile in a gradient mode at a flow rate of 0.4mL/min. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI(-)) were used for detection. The ion pairs 524.0/158.6 for the drug and 540.0/158.8 for the IS were selected for quantification and 524.0/425.7 used for confirmation. Retention time was 3.0 and 3.4min for fludarabine triphosphate and the IS, respectively. The concentration range for the calibration curve was 1.52-76nM. Our method is simple, fast, and has been successfully applied in a clinical dose-concentration study in children to quantify intracellular fludarabine in low volume clinical samples. The median concentration was 1.03 and 3.19pmole/million PBMC at trough and peak time points, respectively. Fludarabine triphosphate is degraded in water within hours but relatively stable in 70% methanol-tris (1.2mM, pH 7.4). One limitation is that the hypercarb column takes a longer time to equilibrate than conventional reverse phase columns, and peaks become broad and distorted if the column is not washed and stored properly.

Simple and sensitive method for quantification of fludarabine triphosphate intracellular concentration in leukemic cells using isocratic liquid chromatography.

A simple, isocratic HPLC method was newly developed for quantitating intracellular fludarabine triphosphate (F-ara-ATP). Samples (500 microl) were injected onto an anion-exchange column and eluted isocratically with phosphate-acetonitrile buffer (flow rate: 0.7 ml/min) at an ambient temperature. F-ara-ATP was quantitated according to its peak area at the absorbance of 261 nm. The standard curve was linear with minimal within-day and inter-day variability. The low and high quantification limits were 50 pmol and 20 nmol, respectively. The method was capable of measuring F-ara-ATP generated in cultured leukemic cells in vitro. Thus, our method will be useful because of its sensitivity and simplicity as well as applicability to biological materials.

High-performance liquid chromatography method for the determination and quantitation of arabinosylguanine triphosphate and fludarabine triphosphate in human cells.

A gradient anion-exchange high-performance liquid chromatographic assay was developed for the simultaneous determination and quantitation of the cytotoxic triphosphates of arabinosylguanine (ara-GTP) and fludarabine (F-ara-ATP). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy using authentic standards. To test this assay in a more complex biological matrix, perchloric acid extracts of circulating human leukemia cells spiked with known concentrations of ara-GTP and F-ara-ATP were examined. Finally, to assess the clinical utility of our method, perchloric acid extracts of circulating human leukemia cells isolated from patients treated with fludarabine and nelarabine were analyzed. The range of quantitation was 0.0125-10 nmol for the ara- and native NTPs in cellular extracts. This assay should be helpful in establishing the mechanistic rationales for drug scheduling and combinations of nelarabine and fludarabine, and for correlating the therapeutic efficacy and levels of the cytotoxic triphosphates in target cells.

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.

Antivirais / Antivirals

Doenças causadas por vírus representam dificuldades terapêuticas. O conjunto de medicamentos antivirais disponíveis é ainda relativamente pequeno, mas tem crescido nos últimos anos em razäo de enormes investimentos materiais e de talentos: a síndrome da imunodeficiência adquirida é, certamente, o motivo principal dessa procura, mas näo o único. Com a presente atualizaçäo, dedicada a oftalmologistas, o modo de açäo, as aplicaçöes, os efeitos adversos, as apresentaçöes e a posologia dos principais quimioterápicos desse grupo, assim como novidades e perspectivas, säo aqui resumidas

Vidarabine-loaded nanoparticles: a physicochemical study.

The chemical reaction of vidarabine (VIDA) with isohexyl cyanoacrylate nanoparticles in a pH-dependent fashion occurs only in the presence of dioctylsulfosuccinate (DOSS). The formation of an ion pair with DOSS allows a better contact of VIDA with the monomer during the polymerization process taking place in micelles. On the basis of molecular weight profiles of the polymer, determined by gel permeation chromatography (GPC), it is proposed that VIDA induces the polymerization of cyanoacrylic monomers through a zwitterionic pathway. This mechanism allows the covalent linkage of the drug with the polymer, which is consistent with NMR experiments. The present study illustrates the need for physicochemical studies in the design of new colloidal drug delivery formulations.

High-pressure liquid chromatographic methods for determining arabinosyladenine-5'-monophosphate, arabinosyladenine, and arabinosylhypoxanthine in plasma and urine.

New high-pressure liquid chromatographic methods for determining concentrations of arabinosyladenine (Ara-A), its 5'-monophosphate (Ara-AMP), and arabinosylhypoxanthine (Ara-H) in plasma and urine are presented. A fluorescence detector is used for Ara-A and Ara-AMP, which are first converted to highly fluorescent derivatives with chloroacetaldehyde. This increases sensitivity greatly over previous methods. The sensitivities of the methods (in micrograms per milliliter) are as follows: in plasma, Ara-AMP, 0.002; Ara-A, 0.0015; and Ara-H, 0.35; and in urine, 9 times these values, respectively. Drug concentration data are also presented, which were obtained after doses of Ara-AMP were given intramuscularly to two patients treated with this drug for severe herpes zoster. One patient was given 13 mg of Ara-AMP per kg of body weight once daily, and the other was given 6.5 mg/kg twice daily. Peak Ara-AMP and Ara-A levels in plasma occurred within 1 h after the doses, and neither exceeded 2 micrograms/ml. Ara-AMP and Ara-A concentrations in plasma fell to less than 0.01 micrograms/ml in both patients by 4 to 6 h after the doses. Peak Ara-H concentrations in plasma occurred within 1 to 2 h after doses and were 21 micrograms/ml in patient 1 and 2. The highest concentration of Ara-AMP in urine was 0.09 micrograms/ml. The highest Ara-A concentration in urine was 62 micrograms/ml, and the highest Ara-H concentration in urine was 1,080 micrograms/ml. An interfering substance of unknown nature, cochromatographing with Ara-H, was encountered sporadically in urine samples. An algorithm based on differential spectrophotometry to identify and correct for this problem is described. Estimates of the renal clearances of Ara-AMP, Ara-A, and Ara-H are also given.

Hydrolysis and solvent-dependent 2'----5'and 3'----5' acyl migration in prodrugs of 9-beta-D-arabinofuranosyladenine.

As a prerequisite to quantitative in vivo studies to further explore the promising topical activity of the 2',3'-di-O-acetyl derivative of 9-beta-D-arabinofuranosyladenine (ara-A) against herpes virus infections, the kinetics of solution degradation of the 2',3'-di-O-acetyl derivative and the 2'-,3'-, and 5'-monoacetates were investigated. The rates of aqueous solution hydrolysis were found to be consistent with rank order predictions based on a consideration of substituent effects. Preliminary in vivo hydrolysis data, however, do not correlate with such predictions, indicating a need for more systemic studies of the effect of molecular structure on enzyme-catalyzed hydrolysis. An important reaction of the 2'-3'-diester and the 3'-monoester in aqueous solution, in addition to ester hydrolysis, is 3'----5' acyl migration. 2'----5' Acyl migration does not occur in water but is the predominant migration pathway in organic solvents, as verified by studies in acetonitrile. 1H NMR spectroscopy was employed to study the dependence of the conformation of the sugar ring on the solvent environment. Although a change in the equilibrium between the C(2')endo and C(3')endo conformational states does occur, it is not a dramatic change and cannot explain the solvent selectivity observed in the acyl migration kinetics.

Inactivation and reactivation of intracellular S-adenosylhomocysteinase in the presence of nucleoside analogues in rat hepatocytes.

Several nucleoside analogues, some of which are potent inactivators of isolated S-adenosylhomocysteinase (AdoHcyase), were tested with respect to their effect on intracellular AdoHcyase and S-adenosylhomocysteine (AdoHcy) catabolism in intact rat hepatocytes. Among the analogues tested, 9-beta-D-arabinofuranosyladenine and 9-beta-D-arabinofuranosyl-3-deazaadenine were the most potent inactivators of intracellular AdoHcyase. Compounds like 2-chloroadenosine and carbocyclic adenosine are extremely efficient inactivators of the isolated enzyme, but these nucleosides exerted only a limited effect on the enzyme in intact hepatocytes. Only a moderate effect was observed with 2-chloro-3-deazaadenosine and 2'-deoxyadenosine, and a high concentration of 5'-deoxy-5'-methylthioadenosine was required to inactivate the enzyme. There was a correlation between inactivation and accumulation of AdoHcy. Carbocyclic-3-deazaadenosine caused no inactivation of AdoHcyase in liver cells but caused a massive accumulation of AdoHcy, suggesting that this analogue functions as a reversible inhibitor of the enzyme. Residual enzyme activity was observed with all the nucleoside analogues tested. The intracellular enzyme inactivated in the presence of 2'-deoxyadenosine and 9-beta-D-arabinofuranosyladenine was readily recovered when the cells were transferred to fresh medium containing adenosine deaminase, but reactivation of the enzyme activity after treatment of the cells with 2-chloroadenosine and 5'-deoxy-5'-methylthioadenosine was also observed. The inactivation of intracellular enzyme induced by 2-chloro-3-deazaadenosine, 9-beta-D-arabinofuranosyl-3-deazaadenine, and carbocyclic adenosine was essentially irreversible under the conditions of the experiments. Factors determining the effect of nucleoside analogues on intracellular AdoHcyase are discussed.

Formulation of a stable vidarabine phosphate injection.

An injection fluid with vidarabine phosphate (37.5 mg/ml, pH 7.4) was formulated. After steam sterilization for 20 min at 120 degrees C a 6.1% degradation was observed. No significant (p greater than 0.05) degradation could be observed after heating in steam for 30 min at 100 degrees C and after storage for four months at 4 degrees C. From the results of an investigation of the degradation rate at elevated temperatures a negligible loss of content after a three years' storage period at 4 degrees C could be predicted.

9-beta-D-Arabinofuranosyl-8-n-butylaminoadenine, a C-8 substituted nucleoside in the anti conformation. Crystallographic and NMR studies.

The protein NMR spectrum of 9-beta-D-arabinofuranosyl-8-n-butylaminoadenine shows an unusually low-field 5'-hydroxyl proton resonance, which has been interpreted in terms of an anti glycosidic conformation together with an 05' ... N8 intramolecular hydrogen bond. Confirmatory evidence for this was obtained by an X-ray crystallographic study; in the crystal, the glycosidic angle chi is 52.7 degrees and the sugar pucker is C3' endo-C4' exo.

Stability-indicating assay for vidarabine.

A physicochemical procedure for the analysis of vidarabine in aqueous parenteral formulations was needed to assure potency and to define stability. Concurrent with the development of this method, its decomposition products and route in aqueous solution were determined. A quantitative procedure was developed to determine intact drug in the presence of decomposition products, and the results obtained were validated by microbial assay. Spectral (UV and polarimetric) and TLC evidence indicated that, in aqueous solution, hydrolysis without racemization occurs, yielding adenine and arabinose. The sensitivity of the method to decomposition is improved by ion-exchange separation of adenine and drug before UV measurement. Analysis of partially decomposed solutions of the drug by both ion-exchange and microbiological methods gave comparable results.