Bioanalyses and Pharmacokinetics of Nafronyl in the Dog

Abstract

Improved specific and sensitive reverse-phase HPLC assays of nafronyl (I) and its acidic metabolite and hydrolysis product (II) in biological fluids were developed with sensitivities of 3–6 ng/mL using fluorometric detection with 225 nm excitation and 330 nm emission wavelengths. There were no significant differences in the stabilities and assays of I and II in plasma obtained using heparin, citrate phosphate dextrose solution, EDTA, citrate, or oxalate as anticoagulant. Inordinately high membrane binding did not permit the quantification of the high plasma protein binding of I by ultrafiltration; its instability precluded the use of equilibrium dialysis. Plasma protein binding of II by ultrafiltration was 76.4% and was not concentration dependent. The apparent red blood cell-plasma partition coefficients for I and II were 2.00 and 0.49, respectively, with almost all anticoagulants; the red blood cell-plasma water partition coefficient for II was 2.08 when corrected for plasma protein binding. Thus, both I and II had erythrocyte binding sites in addition to simple volume partitioning. Only heparin-treated blood gave anomalously low erythrocyte-plasma partition coefficients, indicating that heparin inhibited the partitioning of I and II into red blood cells from plasma water. The total body clearance of nafronyl (I) referenced to total plasma concentration [1295 ± 65(SEM) mL/min] was dose independent (35–70-mg range) and showed biphasic plasma half-lives (intravenous) of 12 and 100 min. Only 34% of the nafronyl appears as systemically circulating II in the plasma. Apparent volumes of distribution similarly referenced were 39.8 and 163 L for the central compartment and total body, respectively. Renal clearances referenced to total plasma concentration were 8.3 and 0.18 mL/min for I and II, respectively. The respective total urinary excretions of I, II, and the glucuronide of II (III) were 0.48, 0.021, and 0.32% of the administered intravenous doses. The respective total urinary excretions of I, II, and III for a bilecannulated dog were 0.005, 0.16, and 0.40%. The total body clearance of intravenously administered II was 225 mL/min, with a renal clearance of 0.057 mL/min referenced to total plasma concentration. The respective total urinary excretions of II and III were 0.027 and 0.44% of the intravenous dose of II. Respective plasma half-lives of II (intravenous) were 2.5, 10.9, and 225 min. The apparent volume of distribution referenced to total plasma concentration was 2.2 L (9.1 L referenced to plasma water concentration). The apparent overall volume of distribution referenced to plasma concentration was 73 L. The bioavailability of nafronyl (I) was 0.3–2.7% of the oral 250-mg dose of I. The bioavailability of II was 10–17% of the oral 250-mg dose of I, indicating high presystemic and/or first-pass metabolism of I and II. Additional metabolites of nafronyl, probably the major ones, could be chromatographically identified and quantified. Their extraction and fluorescent properties indicated that they were hydroxylated naphthalenic compounds containing carboxylic acid groups and their glucuronides. Only one major unconjugated metabolite (IV) was found in significant quantities in plasma. In addition to IV, other organic extractable nonconjugated compounds (V, VI, and VII) appeared in urine and bile. Conjugates of VII were also present in urine and bile.