Disposition of [14C]velnacrine maleate in rats, dogs, and humans.

This study describes the disposition of [14C]velnacrine maleate in rats, dogs, and humans, and the isolation and identification of metabolites in dog urine. Following oral administration of [14C]velnacrine maleate, drug-related material was well absorbed in all three species, with the majority of the dose recovered in the urine. Fecal elimination of radioactivity accounted for the remainder of the dose. The majority of the radioactivity was eliminated within 24 hr. Pharmacokinetic parameters for the elimination of radioactivity from the plasma of rats and dogs were similar after oral dosing compared with intravenous dosing. In humans, the plasma and urinary levels of velnacrine maleate were substantially lower, and the elimination half-life shorter than for total radioactivity, indicating the presence of one or more metabolites with a longer half-life than the parent compound. Preliminary TLC analysis of urine, plasma, and feces showed that metabolism appeared to be similar in the three species investigated. Velnacrine maleate was extensively metabolized with only approximately 10%, 19%, and 33% of the dose appearing in the urine as unchanged drug in humans, dogs, and rats, respectively. Isolation and identification of dog urinary metabolites was conducted. The identity of the isolated metabolites was determined by GC/MS and proton NMR. One of the main metabolic routes was found to be via hydroxylation of the tetrahydroaminoacridine ring with other minor hydroxylated and dihydroxylated metabolites being detected. In addition two dihydrodiol metabolites were also identified. Phase II metabolism did not appear to be a significant route.

Studies on the biochemical effects of the aldose reductase inhibitor 2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione (Al 1576, HOE 843). Detection of D-glucaric and D-glucuronic acid excretion by high resolution 1H and 13C NMR spectroscopy.

The effects of two aldose reductase inhibitors on the biochemical composition of rat urine were investigated using high resolution 1H and 13C NMR spectroscopy. We report the elevated excretion of D-glucaric acid (DGA) and D-glucuronic acid (GCA) following treatment with 2,7-difluorospirofluorene-9,5'-imidazolidine-2'4'-dione (Imirestat, IM, Al 1576, HOE 843) at 50 mg/kg/day for 1 month, but not with 3-4-bromo-2-fluorobenzyl-4-oxo-3-phthalazine-1-ylacetic acid (Ponalrestat, Statil), dosed at 50 mg/kg/day for 2 weeks. Sugar aciduria was also detected following treatment with the cytochrome P450 inducer phenobarbitone (PB) at 45 mg/kg/day for 1 month, although the qualitative and quantitative pattern of excretion of sugar acids differed greatly between the IM and PB treatment groups. The levels of GCA excreted are elevated 11-fold by IM treatment from 19.0 to 210.0 mumol/24 hr, but only 2.5-fold by PB, from 9.7 to 23.9 mumol/24 hr. DGA was not detectable in control urine, although levels did increase by 30% during the study from 7.5 to 10.9 mumol/24 hr, between day 8 and day 29, with IM treatment, and by 60% from 1.7 to 4.9 mumol/24 hr following PB administration for the same time period. This predominant elevation of DGA and GCA caused by IM treatment far exceeds previous records. In contrast, PB treatment resulted in an increase in intensity of a number of partially resolved sugar resonances, but at a much lower level than resulted from IM treatment. A raised level of DGA and GCA is usually associated with hepatic P450 induction; however, we report here profound DGA and GCA uria as a result of the inhibition of the aldehyde reductase, hexonate dehydrogenase (EC 1.1.1.19, EC 1.1.1.20). This mechanism is not closely linked to P450 induction, corroborating the current view that elevated excretion of DGA is not a reliable indicator of hepatic enzyme induction. This study further demonstrates the use of high resolution NMR spectroscopy in the detection of a novel biochemical effect which may go unnoticed during routine clinical chemistry tests.

Application of 19F-n.m.r. spectroscopy to the identification of dog urinary metabolites of imirestat, a spirohydantoin aldose reductase inhibitor.

1. Urine from a dog dosed orally at 20 mg/kg with 14C-imirestat, a spirohydantoin aldose reductase inhibitor, contained 17.7 and 12.5% of the administered radioactivity at 0-48 and 48-72 h respectively. 2. Radio-h.p.l.c. of the 0-48 h urine revealed a complex mixture of metabolites and a small proportion of parent drug (1.6% of dose). Direct 19F-n.m.r. spectroscopy of this urine showed the fluoride ion, numerous metabolites which were predominantly glucuronide conjugates and, as a minor component, the parent drug. 3. After incubation with beta-glucuronidase the 0-48 h urine gave a 19F-n.m.r. spectrum showing fewer signals. This finding is consistent with aromatic ring hydroxylation followed by glucuronidation being the major metabolite pathways. 4. Deconjugated urine was analysed by proton-coupled 19F-n.m.r. and two-dimensional 19F-19F correlated spectroscopy. Results indicate that major components included three monohydroxy metabolites, a diphenol with both phenolic functions in the same ring, and a phenolic metabolite containing only one fluorine atom. 5. Semi-preparative h.p.l.c. of 0-48 h dog urine gave individual glucuronides isolated as mixtures of C-9 epimers. These fractions were hydrolysed and purified a second time by h.p.l.c. to give aglycones which were analysed by multi-nuclear n.m.r. and g.l.c.-mass spectrometry. The 3- and 4-hydroxy derivatives of imirestat were identified, as was the 2-hydroxy product obtained during or following defluorination. The other major aglycone was postulated to be the 3-fluoro-2-hydroxy metabolite. This represents a novel 'NIH-shift' type pathway for the metabolism of fluorobenzenes.

19F and 1H magnetic resonance strategies for metabolic studies on fluorinated xenobiotics: application to flurbiprofen [2-(2-fluoro-4-biphenylyl)propionic acid].

Strategies for the use of 1H and 19F nuclear magnetic resonance (NMR) spectroscopy as an aid to the study of the metabolic fate of fluorinated drugs are discussed with reference to the application of these methods to flurbiprofen metabolism in man. 1H and 19F NMR analysis of untreated urine enabled the detection of two major and eight minor metabolites of the drug. The two major metabolites were identified using a combination of NMR spectroscopy, solid-phase extraction chromatography with 19F and 1H NMR detection and chemical hydrolysis to a flurbiprofen glucuronide and the glucuronide of the 4-hydroxy metabolite. 1H-19F 2D shift correlated spectroscopy and spin-echo difference experiments are discussed in relation to their use in the structural identification of drug metabolites.