Theophylline-7ß-d-Ribofuranoside (Theonosine), a New Theophylline Metabolite Generated in Human and Animal Lung Tissue.

While assessing the ability of mammalian lung tissue to metabolize theophylline, a new metabolite was isolated and characterized. The metabolite was produced by the microsomal fraction of lungs from several species, including rat, rabbit, dog, pig, sheep and human tissue. Metabolite production was blocked by boiling the microsomal tissue. This new metabolite, theophylline-7ß-d-ribofuranoside (theonosine), was confirmed by several spectral methods and by comparison to an authentic synthetic compound. Tissue studies from rats, rabbits, dogs, and humans for cofactor involvement demonstrated an absolute requirement for NADP and enhanced metabolite production in the presence of magnesium ion. It remains to be demonstrated whether theonosine may contribute to the known pharmacological effects of theophylline.

Pharmacokinetic disposition of the oral iron chelator deferiprone in the white leghorn chicken.

Deferiprone is a bidentate oral iron chelator used for the treatment of transfusional iron overload in people. The purpose of this study was to determine the pharmacokinetic disposition of deferiprone in the white leghorn chicken as a potential model upon which to base therapeutic regimens for the treatment of iron storage disease (hemochromatosis) in affected avian species. A suspension of deferiprone (DFP) was administered orally at a single dose of 50 mg/kg to 10 birds that were iron-loaded (IL-DFP) and 10 non--iron-loaded control birds (NIL-DFP). After a 30-day washout period, 5 birds from the NIL-DFP group were used for a bioavailability study of deferiprone administered intravenously at the same dose. Blood samples were collected at varying intervals over a 24-hour period and were analyzed for deferiprone by high-performance liquid chromatography, then plasma concentration versus time curves were developed. Deferiprone was rapidly absorbed from the gastrointestinal tract of the chicken, with plasma concentrations effective for iron chelation in humans (>20 micromol/L) maintained for at least 8 hours after oral dosing. The half-life (mean +/- SD) of the orally administered deferiprone in the IL-DFP and NIL-DFP groups was 2.91 +/- 0.78 hours and 3.61 +/- 0.90 hours, respectively, and was 2.42 +/- 0.24 hours for deferiprone administered intravenously. The mean oral bioavailability was 93%. Deferiprone is well absorbed and widely distributed in the chicken, with a longer half-life than reported in mammals.

Pharmacokinetic disposition of the oral iron chelator deferiprone in the domestic pigeon (Columba livia).

Deferiprone is a bidentate oral iron chelator used for the treatment of iron overload in people. The purpose of this study was to determine the pharmacokinetic disposition of deferiprone in the domestic pigeon (Columba livia) and to compare the results with a previous study in the white leghorn chicken. Deferiprone (DFP) was administered orally as a suspension at a single dose of 50 mg/kg to 10 iron-loaded (IL-DFP) pigeons and 10 non--iron-loaded controls (NIL-DFP). Six NIL-DFP birds were also administered deferiprone intravenously to determine the bioavailability of the drug after a 30-day washout period. To evaluate if deferiprone induces its own metabolism, the pharmacokinetic disposition of the drug was also studied in the IL-DFP group after oral therapy with deferiprone at a dosage of 50 mg/kg q12h for 30 days. For each phase, collected blood was analyzed for deferiprone by high-performance liquid chromatography to develop a plasma concentration versus time curve. Deferiprone was rapidly absorbed from the gastrointestinal tract, with plasma concentrations effective for iron chelation maintained for at least 8 hours after administration in iron-loaded birds. The half-life (mean +/- SD) for deferiprone given orally to the IL-DFP and NIL-DFP groups was 2.98 +/- 0.85 hours and 3.26 +/- 1.25 hours, respectively, and when intravenously administered was 3.79 +/- 1.23 hours. The half-life after 30 days of treatment was 3.42 +/- 1.18 hours. Oral bioavailability was 44%. This study demonstrated that oral absorption of deferiprone is acceptable, it does not induce its own metabolism, and the drug was widely distributed in the pigeon, as it was in the chicken, with a longer half-life than that reported in mammals. Minor interspecies variations in the pharmacokinetics of deferiprone exist between chickens and pigeons.

Validated HPLC assay for iron determination in biological matrices based on ferrioxamine formation.

A simple, robust and reproducible HPLC method has been developed and validated for iron determination in biological matrices. It is based on chelation with desferrioxamine (DFO) and the measurement of the chelate ferrioxamine (FO). The method was developed to permit monitoring of iron bio-kinetics and estimation of iron status in experimental animals. The chromatography was performed on a stainless steel XTerra MS C18 column (Waters; 250 mm x 4.6 mm i.d., 5 microm) using a gradient of Tris-HCl buffer (10mM, pH 5) and acetonitrile. The method was validated in terms of selectivity, linearity (0.3-80 nmol on-column), limit of detection (0.2 nmol on-column), low limit of quantification (0.3 nmol on-column), recovery (91-102%), intra- and inter-day reproducibility, stability, and robustness. The method's universal applicability was illustrated by monitoring plasma and heart iron kinetic profiles in rats after a single intraperitoneal (i.p.) injection of 200mg/kg iron dextran.

Pharmacokinetic studies of a novel 1,2,4-thiadiazole derivative, inhibitor of Factor XIIIa, in the rabbit by a validated HPLC method.

Activated Factor XIII (FXIIIa) stabilizes fibrin clot by covalent cross-linking of fibrin strands in the fibrin, making it resistant to physiological and pharmacologically induced fibrinolysis. Inhibition of Factor XIIIa offers a novel approach to treatment of thrombosis. Selected derivatives of 1,2,4-thiadiazoles, presently in discovery and development, may offer new treatment strategies as inhibitors of Factor XIIIa. In order to evaluate its pharmacokinetic (PK) profile and to facilitate the selection of drug candidates for drug discovery and development process, we developed and validated a simple and selective reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection for the determination of N-[6-(imidazo[1,2-d][1,2,4]thiadiazol-3-ylamino)hexyl]-2-nitrobenzensulfonamide (5624) in rabbit plasma. The plasma protein precipitation and sample preparation was achieved by using acetonitrile, followed by organic phase evaporation to dryness and the residue reconstitution in the mobile phase. The 5624 recovery from the plasma was about 90%. Chromatography was performed on a C18 column using a gradient of acetonitrile in water as a mobile phase. A chemically related compound, N-[6-(imidazo[1,2-d][1,2,4]thiadiazol-3-ylamino)hexyl]naphthalene-1-sulfonamide (5422), was used as an internal standard. Limit of detection (LOD), based on signal to noise ratio>3, was 0.2 microM (on-column amount of about 7 ng), while limit of quantification (LOQ), based on signal to noise ratio>10, was 0.5 microM (on-column amount of about 20 ng). The plasma samples for the PK study were collected at defined time points during and after 5624 slow intravenous infusion (25 mg/kg) to male White New Zealand rabbits and analyzed by RP-HPLC method. The PK parameters, such as half-life, volume of distribution, total clearance, elimination rate constant etc., were determined. The PK profile of 5624 offered insights in the design and development of additional new compounds, derivatives of 1,2,4-thiadiazole, with desired PK properties.

Metabolic and pharmacokinetic evaluation of a novel 3-hydroxypyridinone iron chelator, CP502, in the rat.

A recently synthesized 3-hydroxypyridinone derivative with an amido function at the 2-position, CP502 (1,6-dimethyl-3-hydroxy-4-(1H)-pyridinone-2-carboxy-(N-methyl)-amide hydrochloride), exhibited high in vitro iron chelating potency (pFe3+ =21.7). It was targeted as a new iron-chelating candidate for further development in early pre-clinical testing. To evaluate its pharmacokinetics, including oral bioavailability, metabolic and disappearance profiles, studies were conducted in Sprague Dawley male rats. A single 150 mg/kg intravenous and oral dose was given to male Sprague Dawley rats (N=6, B.Wt. 250g). The rats were placed in metabolic cages and fasted overnight before the dosing. Venous blood samples (200 microL per withdrawal) were collected at defined time points before (blank) and up to 28 h post administration. Urine and feces were collected before dosing (blank) and in 24 h intervals up to 72 h post administration. Plasma CP502 concentration versus time profiles were consistent with two-compartment distribution, and the oral bioavailability approached 100%. Total clearance and mean residence time (i.v.) were 1.02 L/kg/h and 1.10 h, respectively. Simultaneous computer fitting yielded V1 and Vss estimates of 0.96 L/kg and 1.74 L/kg, respectively. CP502 was mainly excreted unchanged via urine (45.29+/-9.40 % of total dose) or as glucuronide (6.46+/-1.22% of total dose). High iron chelation potential and favorable pharmacokinetic and metabolic profiles indicate that CP502 is a promising candidate for further development.

Improved high-performance liquid chromatographic method for the pharmacokinetic studies of a novel iron chelator, CP502, in rats.

An improved reverse-phase high-performance liquid chromatographic method (RP-HPLC) for the determination of a novel iron chelator CP502 (1,6-dimethyl-3-hydroxy-4-(1H)-pyridinone-2-carboxy-(N-methyl)-amide hydrochloride) in rat plasma, urine and feces was developed and validated. The separation was performed on a polymeric column using a mobile phase composed of 1mM ethylenediaminetetra-acetic acid disodium salt (EDTA), acetonitrile, methanol and methylene chloride. Separation of CP502 from plasma, urine or feces endogenous compounds was achieved by gradient elution. Retention times of CP502 and its major metabolite (glucuronide) were about 13 and 4 min, respectively. The method was validated in terms of limit of detection (LOD), limit of quantification (LOQ), selectivity (endogenous from plasma, urine or feces), linearity, extraction recovery, robustness (column selection, mobile phase composition, detection mode, internal standard (IS) selection, analyte stability), day-to-day reproducibility and system suitability (repeatability, peak symmetry and resolution). The method is applicable to bioavailability and pharmacokinetic studies of CP502 in rats.