Metabolites of probenecid. Chemical, physical, and pharmacological studies

Abstract

Probenecid, its metabolites (including 14C labeled), and several analogs were synthesized. The uv spectral properties, p.Ka's, and partition coefficients were determined. The binding of probenecid and metabolites to human plasma, human albumin, and dog plasma was measured. Pmr parameters of probenecid, its metabolites, and 9 other analogs were obtained. There was no correlation between the nature of the alkyl side-chain substituents or the partition coefficient and the chemical shifts of the aromatic ring protons. In rats all the metabolites with a single exception were terminal metabolites; similar results were obtained in vitro. Propionic acid was identified as one of the metabolic products of probenecid. The effects on urate clearance of probenecid, the various metabolites, and the piperidyl analog were determined in Dalmatian and mongrel dogs. In Dalmatians the clearance decreased while in mongrels it increased. Our studies suggest that the metabolites of probenecid may very well play a significant role in the overall uricosuric effect of the parent drug.The structures of essentially all the metabolites of probenecid in rat bile1 and human urine2'4 have been elucidated.The major routes of biotransformation are oxidation of the side chain (Figure 1, Table I) and glucuronide conjugation. In man, formation of the acyl glucuronide accounts for the disposition of about 0.2 of the drug.2'3 Incubation of probenecid-^C with reinforced rat liver preparations leads to the same metabolites as found in vivo.5The purpose of the present study was (1) to synthesize the metabolites (labeled and unlabeled) of probenecid, (2) to learn whether they could contribute to the overall pharmacologic activity of the parent drug, and (3) to extend previous investigations of structure-activity relationships in this series.6 The latter studies included detailed analyses of high-resolution proton magnetic resonance (pmr) spectra, pAa determinations, and measurement of drug-protein binding. Experimental SectionA. Synthesis.Our general procedure for synthesis of probenecid analogs (adapted from Miller7) was: to an ice-cold soln of the appropriate secondary amine (0.12 mole) in 25 ml of anhyd MeOH 8.8 g (0.040 mole) of p-(chlorosulfonyl)benzoic acid (1)1 was added and the mixt stirred at room temp overnight. The MeOH was removed in vacuo and the oily residue taken up in 25 ml of H20. The pH was adjusted to 1 with coned HC1 and the resulting ppt collected by filtration, then dissolved in a slight excess of 0.1 N NaOH (pH >10). After one extn with Et20 and repptn with HC1, the compd was crystd from EtOH-H20, with prior charcoal treatment, and dried at 110°in vacuo. Purity of the analogs was detd by capillary mp in a Thomas-Hoover Uni-Melt, by known Rf values on tic,2 and by elemental analysis. The 14C compds# were synthesized by a modification of the procedure of Motoichi, et al.8 Ring 14C-labeled probenecid and some nonlabeled analogs were a gift from Dr.

An Orphan Esterase ABHD10 Modulates Probenecid Acyl Glucuronidation in Human Liver

“…A major metabolic pathway of probenecid, which contains a carboxylic acid, is acyl glucuronidation in humans (Israili et al, 1972). Severe allergic or anaphylactoid reactions are known as adverse effects of probenecid (Hillecke, 1965).…”

“…Reported side effects of probenecid include severe allergic, anaphylactoid reactions, and massive liver necrosis associated with the hypersensitivity reaction (Hillecke, 1965). Probenecid is extensively metabolized in humans via acyl glucuronidation and oxidation of alkyl side chains (Israili et al, 1972). Approximately 40% of the dose is eliminated as probenecid acyl glucuronide (PRAG) in urine within 48 hours, and excretion of the other metabolites, including 2-hydroxylprobenecid (7.2-12.5%), 3-hydroxyprobenecid (1.6-3.7%), carboxyprobenecid (6.3-9.2%), and N-depropylprobenecid (4.6-8.0%) as well as unchanged probenecid (,4%), was lower than that of PRAG (Perel et al, 1970) (Fig.…”

Observations on the Disposition of Probenecid in Patients Receiving Allopurinol

“…Argon was the carrier; a hydrogen flame detector and temperature programming (up to 250 °C) was used. Reference standards of probenecid and the metabolites were synthesized (7,8) and converted to their respective methyl esters for calibration.…”

“…Plates were developed (benzene, EtOAc, HOAc, 7:3:1 v/v) dried and sprayed with H ,0 for visualization (Rf values: probenecid 0.70, N-depropyl 0.58, COOH 0.54, 2-OH 0.46, 3-OH 0.36, conjugates <0.1). The silica gel zones were scraped and counted in 18 ml liquid scintillation fluid (prepared by mixing 3 1 toluene with 21 g PPO, 1.08 g POPOP and 600 ml BBS-3) (8).…”

Hepatic and Renal Clearances of Probenecid in the Rat

“…Previous studies have reported dose-dependent elimination of probenecid [Selen et al, 1982;Dayton et al, 1963;Benet, 1981, 1984;Emanuelsson and Paal zow, 1988;Emanuelsson et al, 1987], Inves tigations of different elimination pathways have been performed both in vivo and in vitro [Israili et al, 1972;Guarino et al, 1969;Perel et al, 1970Perel et al, , 1971Dayton and Perel, 1971;Dayton et al, 1973;Cunningham et al, 1977Cunningham et al, , 1981, and hepatic metabolism has been found to be the major pathway with oxidation of one of the alkyl side chains as the favoured step. The renal elimination of unchanged probenecid in man and dog seems to be responsible for only 5% of the administered dose [Perel et al, 1970;Beyer et al, 1951;Melethil and Conway, 1976], In a previous study the kinetics of pro benecid in the rat has been evaluated after intravenous bolus doses [Emanuelsson and Paalzow, 1988], and the results indicated both saturable protein binding and saturable elimination.…”