Jeri S. Roth

Determination of 2'-β. -fluoro-2', 3' -dideoxyadenosine, an experimental anti-AIDS drug, in human plasma by high-performance liquid chromatography

Abstract 2′-β-Fluoro-2′,3′-dideoxyadenosine (F-ddA, lodenosine) is an experimental anti-AIDS drug currently being evaluated in a Phase I clinical trial. A simple and specific HPLC method with UV detection, suitable for use in clinical studies, has been developed to determine both F-ddA and its deaminated catabolite, 2′-β-fluoro-2′,3′-dideoxyinosine (F-ddI) in human plasma. After inactivation of plasma HIV by 0.5% Triton X-100, the compounds of interest are isolated and concentrated using solid-phase extraction. Processed samples are separated by use of a pH 4.8 buffered methanol gradient on a reversed-phase phenyl column. The method has a linear range of 0.05–5 μg/ml (0.2–20 μ M ) and intra-assay precision is better than 8%. Analyte recovery is quantitative and plasma protein binding is minimal. In addition, drug and metabolite levels measured in Triton-treated human plasma remain stable for at least 5 months when samples are stored frozen without further treatment. Compound concentrations determined after samples are processed and then frozen for up to 1 month before analysis are also unchanged.

Gas Chromatographic Determination of Hexamethylene Bisacetamide in Plasma and Urine

Abstract A rapid, simple and specific gas chromatographic method has been developed to measure the differentiating agent hexamethylene bisacetamide (HMBA) in biological samples. Addition of a homolog as an internal standard, ultrafiltration and then direct packed column GC-FID analysis of the ultrafiltrate gives a detection limit of less than 50 μg/ml (0.25 mM) for HMBA in plasma or urine. Ultrafiltration is quantitative and assay precision is better than 4.3% for the 1–5 mM range. This method has been applied to determine the bolus dose pharmacokinetics and disposition of HMBA in a single small animal such as a rat. The developed assay should be suitable for therapeutic monitoring of human patients undergoing HMBA treatment.

Determination of the Anti-HIV Drug 2′-β-Fluoro-2′, 3′-Dideoxyadenosine in Biological Fluids by Reversed-Phase HPLC

Abstract 2′-β-Fluoro-2′, 3′-dideoxyadenosine (F-ddA) is a synthetic dideoxynucleoside analogue that has been designed to overcome the acid stability problems of the anti-AIDS drug didanosine. F-ddA is also a clinical candidate and will be tested in AIDS patients upon completion of its preclinical evaluation. We have developed a straightforward reversed-phase HPLC method to measure both F-ddA and its deaminated metabolite, 2′-β-fluoro-2′, 3′-dideoxyinosine, in plasma and urine. This method employs an adenosine deaminase inhibitor to prevent sample degradation, an internal standard for quantitation, and C18 solid-phase extraction to isolate and concentrate the fluorinated dideoxynucleosides. Gradient HPLC analysis on a reversed-phase phenyl column with UV detection at 260 nm gives a limit of quantitation of 50 ng/ml (0.2 μM) for both analytes. This assay has been applied to preclinical studies in rats and monkeys to determine drug stability, disposition, metabolism and plasma kinetics.

Distribution, elimination, metabolism and bioavailability of hexamethylenebisacetamide in rats

SummaryHexamethylenebisacetamide (HMBA), an in vitro differentiating agent, was studied for its pharmaco-dynamic actions in animals. Plasma stability, organ distribution, excretion, oral bioavailability, and estimates of pharmacokinetic parameters and acute lethality were determined in rats. The single dose intraperitoneal LD50 was greater than 3000 mg/kg in both mice and rats. The drug was stable in plasma from several different species during an 8 h in vitro incubation at 37°C. Following a single intravenous (iv) bolus injection (1000 mg/kg) to rats, HMBA was removed from the plasma with a half time of 2.2 ± 0.5 h, and 65 ± 8% of the dose was excreted unchanged in the urine during the first 24 h after dosing. During an 8 h iv infusion, plasma concentrations of 4 mM were easily maintained with no apparent adverse effects. Drug was uniformly distributed, with highest concentrations found in thymus, kidney, liver, and lymph node throughout the first 24 h after a single iv bolus dose. In vivo metabolism was very small, and the presence of apparent metabolites was undetectable until 48 h after iv administration. Oral bioavailability was good (32%), with peak plasma concentrations of 2 mM achieved one hour after oral administration. After oral dosing urinary excretion and plasma decay were comparable to similar data obtained after iv dosing.

Reverse phase HPLC determination and murine pharmacokinetics of arabinosyl-5-azacytosine

Abstract A sensitive and specific reverse phase HPLC assay has been developed to measure the new antitumor agent arabinosyl-5-aza-cytosine (ara-AC) in biological fluids at concentrations as low as 50 ng/ml (0.2 μM). This assay also detects arabinosyl-N-formyl-guanylurea (AGU-CHO), the initial hydrolytic metabolite of ara-AC. 2′-Deoxy-5-azacytidine, an analogue with similar chemical stability, is used as an internal standard. Chromatographically interfering plasma ribosides are removed by solid phase extraction on a phenyl boronic acid cartridge. Separation of ara-AC, AGU-CHO and internal standard is then accomplished isocratically (1% CH3CN in 10 mM pH 6.8 phosphate buffer) on fully carbon loaded and end-capped C8 and C18 columns connected in tandem. The compounds of interest are detected by UV absorption at 240 nm and total analysis time is 20 min. This assay has been used to determine bolus dose plasma kinetics in male BDF1 mice given 200 mg/kg ara-AC as a tail vein injection. Plasma elimination of the ...

Simultaneous measurement of the cell-differentiating agent hexamethylene bisacetamide and its metabolites by gas chromatography

Hexamethylene bisacetamide (HMBA) is a potent in vitro differentiating agent that has clinical potential as an anticancer drug both as a single agent and as a component of combination therapy. A sensitive and efficient GC method for the isolation, derivatization, and measurement of both HMBA and its two major metabolites in plasma and urine in a single analysis is described. In situ carbamylation of the biological sample with diethylpyrocarbonate forms the urethane derivative of the basic N-acetyl diaminohexane metabolite and allows analyte isolation and concentration by solid-phase extraction. Subsequent formation of the n-butyl ester of 6-acetamidohexanoic acid, the major metabolite, provides a derivatized biological extract that can be rapidly analyzed by temperature-programmed GC. The quantitative extraction and the efficient derivatization steps provide a limit of quantitation of 0.05 mM (10 micrograms/ml) for all analytes with a precision better than 8% for the range of in vitro activity (0.1-2.0 mM). This method is amenable to automation and is well-suited for the analysis of clinical samples.