Pharmacokinetics and metabolism of UK-383,367 in rats and dogs: a rationale for long-lived plasma radioactivity.

UK-383,367 (5-{(1R)-4-cyclohexyl-1-[2-(hydroxyamino)-2-oxoethyl]butyl}-1,2,4-oxadiazole-3-carboxamide) is a novel procollagen C-proteinase inhibitor evaluated for the treatment of post-surgical dermal scarring. It is extensively metabolized in rat and dog absorption, distribution, metabolism and excretion studies, and a metabolic pathway for UK-383,367 was determined. A long-lived metabolite was identified in dog plasma. Data indicate that this metabolite results from the oxadiazole ring-cleavage-producing oxamic acid, oxamide and oxalic acid. Ion exclusion chromatography was used to identify these polar metabolites, which were unretained on a standard reversed-phase high-performance liquid chromatography system. The oxamide metabolite was identified as the long-lived radioactivity, which was observed in dog plasma.

A sensitive HPLC-MS-MS assay for quantitative determination of midazolam in dog plasma.

The clinical pharmacokinetics of midazolam have been extensively studied, due to its high clearance by CYP3A4 and sensitivity to drug-drug interactions. In order to investigate the potential to model drug-drug interactions with midazolam in the dog, a selective and sensitive high performance liquid chromatography-tandem mass spectroscopy (HPLC-MS-MS) method has been developed, with sufficient sensitivity to allow analysis of dog plasma samples generated following administration of a clinically relevant dose. The method involves extraction of midazolam and internal standard (flunitrazepam) from dog plasma, using 96-well Oasis MCX solid phase extraction plates. The assay has been validated over a concentration range of 0.1-10 ng/ml and its specificity, accuracy and precision demonstrated. The relative bias of the assay was within +/-15% for all standards with intra- and inter-assay precision (coefficient of variation-%CV) of less than 15%. The assay was applied to the analysis of plasma samples (0.2 ml), generated following intravenous or oral administration of midazolam to male beagle dogs, at a dose level of 0.05 mg/kg, and pharmacokinetic parameters were derived from the resulting data.

The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog, and human.

Voriconazole is a new triazole antifungal agent with potent, wide-spectrum activity. Its pharmacokinetics and metabolism have been studied in mouse, rat, rabbit, dog, guinea pig, and humans after single and multiple administration by both oral and intravenous routes. Absorption of voriconazole is essentially complete in all species. The elimination of voriconazole is characterized by non-linear pharmacokinetics in all species. Consequently, pharmacokinetic parameters are dependent upon dose, and a superproportional increase in area under the curve is seen with increasing dose in rat and dog toxicology studies. Following multiple administration, there is a decrease in systemic exposure. This is most pronounced in mouse and rat, less so in dog, and not observed in guinea pig or rabbit. Repeat-dose toxicology studies in mouse, rat, and dog have demonstrated that induction of cytochrome P450 by voriconazole (autoinduction of metabolism) is responsible for the decreased exposure in these species. Autoinduction of metabolism is not observed in humans, and plasma steady-state concentrations remain constant with time. Voriconazole is extensively metabolized in all species. The major pathways in humans involve fluoropyrimidine N-oxidation, fluoropyrimidine hydroxylation, and methyl hydroxylation. Also, N-oxidation facilitates cleavage of the molecule, resulting in loss of the fluoropyrimidine moiety and subsequent conjugation with glucuronic acid. Major pathways are represented in animal species. The major circulating metabolite in rat, dog, and human is the N-oxide of voriconazole. It is not thought to contribute to efficacy since it is at least 100-fold less potent than voriconazole against fungal pathogens in vitro.

Preliminary study of the metabolism of 17 alpha-methyltestosterone in horses utilizing gas chromatography-mass spectrometric techniques.

Little is known about the metabolism of 17 alpha-alkyl anabolic steroids in horses. In this study, the metabolism of 17 alpha-methyltestosterone is investigated by oral administration of a (1 + 1) mixture of the steroid and its deuteriated analogue. Both compounds were synthesized from dehydroisoandrosterone (DHA), using a Grignard reaction followed by an Oppenauer oxidation. Post-administration urine extracts were analysed by gas chromatography--mass spectrometry (GC-MS) using both electron impact (IE) and chemical ionization (CI). Interpretation of the data was facilitated by observation of the fragment ions present in the mass spectra. Notably, the D-ring fragment ions were indicative of 15- or 16-hydroxylation, where 16-hydroxy metabolites showed ion pairs at m/z 218/221 and at m/z 231/234 while 15-hydroxy compounds gave the 231/234 ion pair alone. Unaltered D-rings showed fragment ions at m/z 143/146. The data showed that the main phase 1 metabolic processes were partial and complete reduction of the 3-oxo-4-ene group, 15-hydroxylation, 16-hydroxylation, 17-epimerization and hydroxylation at at least two other undetermined sites, postulated as the 6 and 11 positions. Phase 2 metabolism, in the form of glucuronide and sulfate formation, was also common. The information provided by this investigation will result in improved effectiveness of confirmatory analytical procedures for 17 alpha-alkyl anabolic steroids.

Molecular basis for methoxyamine-initiated mutagenesis: 1H nuclear magnetic resonance studies of oligonucleotide duplexes containing base-modified cytosine residues.

Methoxyamine, N4-methoxycytidine and its 2'-deoxyribo analogue are transition mutagens. The mechanism by which the latter acts after incorporation into or generation within DNA has been ascribed to the ability of the base analogue to pair effectively with both adenine and guanine. To obtain a detailed understanding of these interactions, the solution structures of the self-complementary octanucleotide d(CGGATCCG) and its analogues d(CGGATTCG), d(CGGATMCG) and d(CGGATPCG) (designated 8mer-GC, -GT, -GM and -GP, respectively) were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2] oxazin-7-one. A variable temperature study showed the order of stability as 8mer GC > GP > GT > GM. Nuclear Overhauser spectroscopy permitted the assignment of the base, anomeric and H2'/H2" protons in these 8mers. All had spectra consistent with regular B-DNA duplex structures. Imino proton spectra showed that the 8mers GC, GP and GM involved Watson-Crick base-pairing but that the G.P and to a greater extent G.M base-pairs were in slow exchange on the nuclear magnetic resonance time-scale with the wobble configuration. Indeed, the G.M pair showed an additional exchange process interpreted in terms of the presence of syn and anti conformers of the methoxy group in the wobble pair. This accounts for the destabilization of M compared with the P-containing duplex. The observations are compared with those made earlier on the corresponding AT, AP and AM octamers. It is evident that M and P can form stable base-pairs with both A and G with essentially Watson-Crick geometry. This confirms the earlier, although unsubstantiated explanation for the transition mutational propenstty of methoxyamine which, in turn, was based on the fact that methoxycytosine bases have tautomeric constants (KT) much nearer to unity than the normal bases. The same general explanation for hydroxylamine and hydrazine-induced mutations is correspondingly rendered more certain.

Molecular basis for methoxyamine initiated mutagenesis. 1H nuclear magnetic resonance studies of base-modified oligodeoxynucleotides.

In order to reach a more detailed understanding of the mechanism of the mutagenic action of methoxyamine and of N4-methoxycytidine and its 2'-deoxyribo-analogue, the solution structures of the self-complementary octanucleotide, d(CGAATTCG) and its analogues, d(CGAATCCG), d(CGAATMCG) and d(CGAATPCG) (designated 8mer-AT, 8mer-AC, 8mer-AM, and 8mer-AP, respectively), were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2]oxazin-7-one, in which the N-O bond is held in the anti configuration with respect to N3 of the cytosine ring. Correlated spectroscopy and nuclear Overhauser spectroscopy allowed assignment of the base, anomeric and H2'/H2" protons in 8mers-AT, -AM and -AP, and showed that all three had features consistent with a regular B-DNA duplex structure. Duplex-to-coil transition temperatures were determined to be 52(+/- 2) degrees C (8mer-AT), 51(+/- 2) degrees C (8mer-AP), 32(+/- 2) degrees C (8mer-AM); on the chemical shift timescale, the melting transition was fast for 8mer-AT and 8mer-AP, but slow for 8mer-AM. Imino proton spectra were indicative of Watson-Crick base-pairing in 8mers-AT, -AP and -AM. The 8mer-AP duplex had a structure and melting characteristics virtually identical with those of the 8mer-AT duplex. The preferred syn configuration of the methoxyl group in M had a destabilising effect on the 8mer-AM duplex. At low temperatures, the A.M base-pair was in fast equilibrium between Watson-Crick and wobble configurations, with the methoxyl function anti-oriented, but the melting transition was accompanied by isomerization of the methoxyl group to the syn conformation. This syn-anti isomerization was the rate-determining step in the duplex-to-coil transition. The 8mer-AC oligomer did not form a stable duplex.