High-performance liquid chromatographic assay for xanomeline, a specific M-1 agonist, and its metabolite in human plasma.

A reversed-phase high-performance liquid chromatographic assay (HPLC) was utilized for monitoring xanomeline (LY246708/NNC 11-0232) and a metabolite, desmethylxanomeline, in human plasma. Xanomeline, desmethylxanomeline and internal standard were extracted from plasm with hexane at basic pH. The organic solvent extract was evaporated to dryness with nitrogen and the dried residue was reconstituted with 0.2 M HCl-methanol (50:50, v/v). A Zorbax CN 150 x 4.6 mm I.D., 5-microns column and mobile phase consisting of 0.5% (5 ml/l) triethylamine (TEA) adjusted to pH 3.0 with concentrated orthophosphoric acid-tetrahydrofuran (THF) (70:30, v/v) produced consistent resolution of analytes from endogenous co-extracted plasma components. Column effluent was monitored at 296 nm/0.008 a.u.f.s. and the assay limit of quantification was 1.5 ng/ml. A linear response of 1.5 to 20 ng/ml was sufficient to monitor plasma drug/metabolite concentrations during clinical trials. HPLC assay validation as well as routine assay quality control (QC) samples indicated assay precision/accuracy was better than +/- 15%.

Determination of xanomeline and active metabolite, N-desmethylxanomeline, in human plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

We have developed a method for the determination of xanomeline and its pharmacologically active N-desmethyl metabolite. The validated method uses hexane to extract xanomeline and its N-desmethyl metabolite from basified plasma. The hexane extract is dried, reconstituted, and analyzed using a liquid chromatographic-atmospheric pressure chemical ionization tandem mass spectrometry system. The method was developed to support phase II clinical trials and has proven to be extremely sensitive, fast, and rugged. The method has a limit of quantitation of 75 and 200 pg/ml plasma for xanomeline and the N-desmethyl metabolite, respectively. Sample analysis times were less than 3 min from one injection to the next.

Determination of xanomeline in human plasma by ion-spray tandem mass spectrometry.

Xanomeline is a muscarinic receptor agonist currently in phase II clinical trials for the treatment of Alzheimer's disease. A fast, sensitive and specific assay has been developed to determine xanomeline plasma concentrations using ion-spray tandem mass spectrometry. Xanomeline and a structural analog, LY282122, were extracted from basifed plasma into hexane. The dried hexane extracts were reconstituted and injected onto a 10 x 1 mm C18 reversed-phase column. A mobile phase of 33 mM ammonium acetate and 0.33% acetic acid in 30/70 (v/v) water-acetonitrile was pumped through the column at 50 microliters min-1. The mobile phase eluant was introduced directly into the ion-spray interface. The mass spectrometer was operated in the positive ion mode for specific detection of the product ions of xanomeline and the internal standard. The method has a linear range of 0.075-5.0 ng xanomeline per milliliter of plasma. Sample run times were 2.5 min from one injection to the next.

Isomazole disposition in man as a function of dose and route of administration.

The disposition of a new cardiotonic agent (isomazole (ISO] was evaluated in healthy volunteers after various single oral (p.o.), intravenous (i.v.), and multiple p.o. doses. Blood samples were collected after dosing in all studies, with urine collected in the single i.v. and multiple dose studies. All biological samples were measured for ISO. In the multiple dose study, samples were collected for analysis after the first and last doses administered. In addition to ISO, several known metabolites (hydroxyisomazole (OHISO), sulfone (SULF), and hydroxysulfone (OHSULF) analogs) were measured after the first and last doses given in the multiple dose study. Pharmacokinetic values compared between doses suggested no saturable processes existed over the entire dose range. The single i.v. dose data showed ISO experienced some extravascular distribution (mean V beta = 1.82 l kg-1), with a high clearance (mean Cls = 18.8 ml min-1 kg-1) and a short half-life (mean t 1/2 = 1.1 h). Elimination was primarily nonrenal (Clr = 3.5 ml min-1 kg-1). Single p.o. data supported these findings and further suggested rapid absorption. ISO data from the first dose of the multiple dose study was in agreement with these data; however, the last dose showed a higher Cls (33.0 ml min-1 kg-1) (p = 0.055). Although not statistically significant, metabolite plasma data and and urinary excretion patterns changed. An increase was observed in plasma AUC and metabolite excretion of SULF and OHSULF, while a decrease was observed in the same parameters for OHISO. These results suggest that multiple dosing of ISO produces autoinduction of ISO metabolism through selective metabolic routes.

14C-isomazole disposition in man after oral administration.

A 50-mg dose containing 50 microCi 14C-isomazole was administered orally to five healthy male volunteers. Blood, plasma, urine, feces, and saliva were collected and measured for total 14C; in addition, all collections except feces were measured for parent drug (ISO) and three metabolites: hydroxyisomazole (OHISO) and sulfone (SULF) and hydroxysulfone (OHSULF) analogues. Urine and fecal recoveries accounted for 97.0% of the drug administered, with 62.6% excreted in urine and 32.4% in feces. Only 47% of the drug recovered in urine could be identified, with ISO the largest constituent. Total plasma 14C peaked at 1.5 hr, indicating rapid absorption, and produced a mean half-life of 3.7 hr. This was similar to the total 14C half-life found in blood (3.1 hr) but longer than in red blood cells (1.8 hr) or saliva (1.4 hr), suggesting that different ISO-related compounds contributed to the results found in each fluid or tissue. An unidentified metabolite(s) composed a large portion of circulating plasma 14C and produced the longer half-life encountered in plasma. ISO exhibited a short half-life (1.35 hr), a high oral clearance (Cls/F; 24.2 ml/min/kg), and some extravascular distribution (V beta; 3.07 L/kg). Total 14C in red blood cells and saliva related very well to plasma ISO disposition, suggesting preferential distribution of parent drug across cellular membranes. The estimated RBC:plasma ISO ratio (1.79) confirmed this hypothesis. Saliva may be used as a noninvasive means to monitor ISO disposition.

Disposition of [14C]pinacidil in humans.

Pinacidil [(+/-)-2-cyano-1-(4-pyridyl)-3-(1,2,2-trimethylpropyl)guanidine monohydrate] is a novel, direct-acting vasodilator antihypertensive agent. The cyano 14C-labeled drug is rapidly and completely absorbed after an oral 12.5-mg dose in solution. The blood:plasma concentration ratios (0.8-0.9) indicate transient penetration of radioactivity into blood cells. Blood and plasma tmax (0.5 h) and t 1/2 (4 h) of [14C]pinacidil equivalents are similar. Pinacidil (51%), pinacidil N-oxide (28%), and unidentified polar metabolites (21%) comprise the plasma radioactivity. The plasma t 1/2 of pinacidil is 2-3 h, and that of pinacidil N-oxide is 4-5 h. Renal excretion of radioactivity is the major route (80-90% dose) of drug elimination; fecal elimination accounted for 4% of the dose. Renal clearance of the N-oxide is 10 times the renal clearance of the parent drug and exceeds the creatinine clearance. Biotransformation products in 0-24-h urine samples include pinacidil (10%), pinacidil N-oxide (60%), and free and conjugated analogues of pinacidil and metabolites (30%). Stereoselective metabolism is not a major biotransformation pathway of pinacidil or the N-oxide metabolite.

Enzyme-linked immunosorbent assay for LY163502, a potent dopamine agonist.

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for the potent selective dopaminergic receptor (D2) agonist, LY163502, is described. The ELISA is a competitive assay in which peroxidase-labeled LY163502 competes with unlabeled LY163502 for binding to solid-phase antibodies specific for LY163502. The limit of detection is 8 pg/mL; intra- and interassay coefficients of variation are 7.2 and 12.3% respectively, for human plasma determinations. Recovery of LY163502 from plasma and urine is quantitative. We found two potential metabolites of LY163502, despropyl-LY163502 and N-oxide LY163502, to be 44 and 0.5% cross-reactive at the ED50 level, respectively. However, LY163502 levels measured by the ELISA and by a specific gas-liquid chromatography (GLC) assay are highly correlated [Pearson's correlation coefficient, r = 0.964]. This suggests that the ELISA assay of biological fluids is not affected by cross-reactivity due to LY163502 metabolites. Application of the ELISA to the measurement of LY163502 in biological fluids is demonstrated.

GLC assay of fenclorac in human plasma.

A simple, sensitive GLC assay for fenclorac is described. Plasma proteins were precipitated with methanol, and the methanolic extract was refluxed with hydrochloric acid to form the methyl esters of fenclorac and the internal standard. The esters were purified by partitioning into benzene. Aliquots of 1 microliter of the concentrated benzene phase were injected into the gas chromatograph and quantitated by a 63Ni-electron-capture detector. Recovery of fenclorac from plasma averaged 82 +/- 1.6%.

Pharmacokinetics and disposition of lidamidine hydrochloride (WHR-1142A), a novel antidiarrheal agent, in rat and monkey.

14C-Labelled 1-(2,6-dimethylphenyl)-3-methylamidinourea hydrochloride (14C-WHR-1142A, lidamidine hydrochloride) was rapidly and quantitatively absorbed from the gastrointestinal tract of rat and monkey after a single oral dose of 5 mg/kg (base). Peak 14C levels occurred within 30 min and the radiolabel was found in both the plasma and cellular components of whole blood. The half-life of the parent compound was 30 min in rat and 1 h in the monkey. The label was essentially cleared from all tissues examined within 24 h in the rat. In both rat and monkey, the compound was extensively metabolized (greater than 90%) prior to excretion and eliminated primarily in the urine (95% of the 14C dose could be accounted for in urine within 24 h in the monkey and 65% within 24 h in the rat); about 15-20% of the dose was recovered in feces within 24 h in the rat. In rat, a significant portion of the dose was eliminated in bile, and enterohepatic recirculation of 14C excreted in bile occurred. In contrast, biliary elimination of 14C was not a major pathway in the monkey.

Comparative metabolism of fenclorac in rat, dog, monkey, and man.

Fenclorac (alpha,m-dichloro-p-cyclohexylphenylacetic acid, diethylammonium salt), a new nonsteroidal anti-inflammatory agent, was rapidly and quantitatively absorbed from the gastrointestinal tract of rat, dog, monkey, and man following oral administration of a solution of 14C-labeled compound. Radiochromatography and mass spectrometry indicated that fenclorac was the principal component in plasma during both the absorption and elimination phases in all species. Small quantities of m-chloro-p-cyclohexylphenylglycolic acid metabolite were also present. Fenclorac and metabolite were confined primarily to the plasma phase of whole blood and were extensively bound to serum albumin. The plasma elimination half-time was species-dependent and varied from 1.6 hr in the rat to 6.5 hr in the dog. The principal tissues of distribution were liver, kidney, and small intestine. There was no significant accumulation or retention of drug or metabolites in any tissue compartment. Fenclorac was completely biotransformed prior to elimination in urine and bile. The major route of elimination was renal in man and monkey, and biliary in the dog. Enterohepatic recirculation of fenclorac metabolites was shown to occur in the rat. The major urinary metabolites were hydroxycyclohexyl analogs of fenclorac and m-chloro-p-cyclohexylphenylglycolic acid. There was no difference in metabolism and biological disposition of fenclorac in normal rats and rats with adjuvant-induced polyarthritis.

Blood levels in methaqualone in man following chronic therapeutic doses.

Human serum was analyzed for methaqualone (MTQ) and hydroxylated metabolites by gas liquid chromatographic (GLC), ultraviolet spectrophotometric (UV) and spectrofluorimetric (SF) procedures. Intact methaqualone was found to be the major circulating drug component after administration of multiple 300 mg daily doses over a 28-day period. Hydroxylated methaqualone metabolites, if present, were estimated to be in extremely low concentrations. After acute ingestion of large quantities of methaqualone (2.4-3.0 g), at least one methaqualone metabolite, [2-methyl-3-(2' hydroxymethylphenyl)-4(3H)-quinazolinone] was present in serum obtained from subjects with a history of chronic drug abuse.