didanosine and 2-fluoro-2--3--dideoxyadenosine

A sensitive precolumn derivatization method has been developed to measure the 5'-triphosphate of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA, lodenosine), a new anti-HIV drug, in human lymphocytes by HPLC using fluorescence detection. Reaction of chloroacetaldehyde with F-ddA triphosphate in extracts from human lymphocytes produces a highly fluorescent etheno adduct. This derivative is then separated and quantitated by reverse-phase paired-ion chromatography. Degradation of natural nucleic acid ribosides, such as ATP, using periodate oxidation simplifies the chromatogram and minimizes interference with detection of the target analyte. This method, modeled using cultured MOLT-4 T-lymphocytes, achieves a linear detector response for peak area measurements over the range 2.5 to 22.5 pmol (50-450 nM using 50 microl sample). Analyte recovery is greater than 90%, and the method achieves a limit of detection and limit of quantitation of 1.4 and 2.5 pmol per HPLC injection (50 microl sample containing cellular extract from 2.5 x 10(6) cells), respectively. Application of this method to measure F-ddATP in peripheral blood mononuclear cells from HIV-infected patients treated with F-ddA at 3.2 mg/kg twice daily for 22 days shows F-ddATP levels which range from 1.5 to 3.5 pmol/10(6) cells.

Topics: Anti-HIV Agents; Calibration; Cells, Cultured; Chromatography, High Pressure Liquid; Didanosine; Dideoxyadenosine; Fluorometry; Humans; Lymphocytes; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Tumor Cells, Cultured

This study examines the central nervous system (CNS) delivery of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA) and 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), acid stable analogues of dideoxyadenosine (ddA) and dideoxyinosine (ddI) having reduced susceptibility to purine salvage pathway enzymes important in the metabolism of ddA and ddI, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), respectively. Their CNS delivery compared to that for ddI provides insight into the role of brain tissue ADA and PNP in these processes.. Brain and cerebrospinal fluid (CSF) concentration-time profiles were obtained for F-ddI during and after intravenous infusions of F-ddI, and for both F-ddA and F-ddI after F-ddA infusions in normal rats or rats pre-treated with the ADA inhibitor 2'-deoxycoformycin (DCF). Rate constants for CNS entry, efflux and metabolism were estimated by computer fits using plasma concentration-time profiles as the driving force functions.. The CNS delivery of F-ddI did not differ significantly from that for ddI. F-ddA, which is more lipophilic than F-ddI, provided higher brain (approximately 8x) and CSF (approximately 11x) concentrations of total dideoxynucleoside (F-ddA and F-ddI) compared to F-ddI. Deamination by brain tissue ADA to form F-ddI reduced CNS levels of intact F-ddA but provided higher brain parenchyma (5x) and CSF/plasma (3x) ratios of F-ddI relative to F-ddI controls. Thus, F-ddA functions in part as a CNS-activated prodrug of F-ddI. DCF pre-treatment inhibited brain tissue ADA, abolishing the prodrug effect, and enhancing F-ddA concentrations in both brain parenchyma (5x) and CSF (6x).. PNP metabolism does not appear to play a role in the low CNS delivery of ddI. On the other hand, deamination of F-ddA by brain tissue ADA is an important process, such that F-ddA functions in part as a CNS-activated prodrug of F-ddI. Enhanced CNS uptake of intact F-ddA can be achieved with ADA inhibition.

Topics: Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Anti-HIV Agents; Brain; Brain Chemistry; Didanosine; Dideoxyadenosine; Enzyme Inhibitors; Infusions, Intravenous; Male; Pentostatin; Purine-Nucleoside Phosphorylase; Rats; Rats, Sprague-Dawley

Enzymes of the purine salvage pathway play an important role in altering the in vivo pharmacokinetics of 2',3'-dideoxypurine nucleosides. This study examines the pharmacokinetics of enzyme-resistant 2'-beta-fluoro analogues of 2',3'-dideoxyinosine (ddI) and 2',3'-dideoxyadenosine (ddA). 2'-beta-Fluoro-2',3'-dideoxyinosine (F-ddI) is an acid-stable analogue of ddI that is highly resistant to purine nucleoside phosphorylase, the principal enzyme in ddI metabolism. 2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA), an acid-stable and purine nucleoside phosphorylase-resistant analogue of ddA, is converted in vivo to F-ddI by adenosine deaminase (ADA) but is a much poorer substrate for this enzyme than is ddA. Both F-ddA and F-ddI have been shown to have activity against human immunodeficiency virus in vitro, and F-ddA has been selected by the National Cancer Institute for clinical trials as a new human immunodeficiency virus reverse transcriptase inhibitor. The pharmacokinetics of F-ddI and ddI were compared at equivalent doses in chronically catheterized rats. Because ddI and F-ddI are isosteres having nearly identical lipophilicity, this comparison is likely to reflect primarily metabolic differences. The clearance of F-ddI was substantially reduced, in comparison with that of ddI (27.3 ml/min/kg vs. 90.9 ml/min/kg), resulting in higher systemic concentrations at steady state and prolonged retention of F-ddI after termination of infusions, consistent with a significant metabolic component in the clearance of ddI. Concentrations of F-ddA and F-ddI during and after infusions of F-ddA were determined in both untreated and 2'-deoxycoformycin-pretreated rats. In untreated rats, F-ddA was rapidly eliminated from plasma, with a total clearance of 68.5 ml/kg/min. Metabolic clearance of F-ddA to F-ddI accounted for 58% of this value (bioconversion t1/2 = 9.8 +/- 1.9 min). Pretreatment with 2'-deoxycoformycin, an ADA inhibitor, reduced the clearance of F-ddA to 23.8 ml/min/kg, leading to 2.9 +/- 0.4-fold higher steady-state plasma concentrations of F-ddA, in agreement with a 2.5-fold enhancement predicted by a compartmental model assuming complete ADA inhibition.

Topics: Adenosine Deaminase Inhibitors; Animals; Anti-HIV Agents; Didanosine; Dideoxyadenosine; Enzyme Inhibitors; Male; Pentostatin; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Inhibitors

The acute cardiotoxic potential of single dosages of FddA (2'-fluoro-2',3'-dideoxyadenosine) and FddI (2'-fluoro-2',3'-dideoxyinosine) was investigated in 6- to 9-week-old rats. Both nucleoside analogs were administered orally at 1000 and 2000 mg/kg and intravenously at 500 or 1000 mg/kg. For comparative purposes, additional groups of rats received 2'-deoxyadenosine or the 2-fluororibose moiety common to both the FddA and FddI molecules. The effects of two adenosine receptor antagonists, caffeine and theophylline, on the cardiotoxicity induced by FddA were also investigated. Deaths occurred within a few hours to a few days in FddA-treated rats given 2000 mg/kg orally or 500 mg/kg intravenously and in FddI-treated rats given 1000 mg/kg intravenously. Microscopic examination of the hearts revealed myocardial degeneration and necrosis for all rats that died and myocardial fibrosis for many survivors. No deaths or cardiac lesions were observed after administration of 2'-deoxyadenosine or the 2-fluororibose moiety. FddA was more cardiotoxic than FddI in rats at equivalent dosages administered either orally or intravenously. Based on the anatomic findings, all deaths were attributed to cardiac lesions. The administration of high, oral dosages of caffeine and theophylline accentuated the acute cardiotoxicity of FddA in rats.

Topics: Administration, Oral; Animals; Antiviral Agents; Behavior, Animal; Body Weight; Didanosine; Dideoxyadenosine; Dose-Response Relationship, Drug; Heart Diseases; Injections, Intravenous; Male; Myocardium; Necrosis; Rats; Rats, Sprague-Dawley; Xanthines