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%.

Characterization of impurities in sulfasalazine.

The chemical structures of four impurities isolated from sulfasalazine were determined. Three impurities are the by-products of the reaction process in drug synthesis, i.e., during diazotization of sulfapyridine and during coupling with salicylic acid. Only one contaminant was identified as a starting material, sulfapyridine, in the drug synthesis. The four impurities were characterized as 2-[[p-(2-pyridylsulfamoyl)-phenyl]azo]hydroxybenzene (I), 3-[[P-(2-pyridylsulfamoyl) phenyl]-azo]salicylic acid (II), 5-[[p-[4-(2-pyridylanilino)]-N-phenyl]azo]salicylic acid (III), and sulfapyridine (IV). Compounds I-III are novel molecules, and IV is the precursor of sulfasalazine. The isolation of the impurities was accomplished by TLC and liquid extraction procedures. The methods used to characterize the impurities were a combination of IR, UV, and NMR spectroscopy, mass spectrometry, and TLC. For I and III, comparisons also were made with the synthesized materials to supplement the evaluation.

Hydrolytic degradation of 2,6-dichlorobenzylthiopseudourea hydrochloride.

Degradation of 2,6-dichlorobenzylthiopseudourea hydrochloride was followed in basic medium (pH 7.5) to isolate and characterize all possible degradation products. IR, Raman, and NMR spectroscopy, TLC, and elemental analysis were used to identify the products. Degradation of base-hydrolyzed 2,6-dichlorobenzylthiopseudourea hydrochloride produced 2,6-dichlorobenzylthiol and cyanamide and was followed by oxidation (air) to produce bis(2,6-dichlorobenzyl) disulfide, dimerization to give cyanoguanidine, and hydrolysis to yield urea. The kinetics of hydrolysis at 22.5 degrees (pH 7.0 and 7.5) and at 37 degrees (pH 7.0) revealed a pseudo-first-order reaction with respect to the substrate. Apparent first-order rate constants and energy of activation, entropy of activation, and half-life values were determined.

The inhibition of prostaglandin synthetase in vivo by nonsteroidal anti-inflammatory agents.

The effects of two nonsteroidal anti-inflammatory compounds, fenclorac and indomethacin, on arachidonate and prostaglandins (PG) E1- and E2-induced vasodepressor responses were determined in the spontaneously hypertensive male rat. A 0.4 to 2 mg/kg intravenous dose of fenclorac blocked the arachidonate-induced vasodepressor response and had no effect on PGE1- and E2-induced hypotension. Similar responses were observed after indomethacin. These results were indicative of in vivo inhibition of PG synthetase by fenclorac and indomethacin. Administration of propranolol did not alter the arachidonate or PG responses; regitine reduced the arachidonate and PG response, suggesting that alpha-adrengergic receptors in vascular smooth muscle may play a role in the prostaglandin-induced hypotension.

Quantitative determination of fenclorac in serum.

A spectrophotometric method for the analysis of fenclorac and its metabolite, 3-chloro-4-cyclohexylbenzeneglycolic acid, in human serum was developed. The parent compound represented at least 90% of the total species present in blood; the metabolite was present to the extent of about 10%, primarily in the elimination phase. The basic procedure consists of extraction of both compounds from serum, further extraction to remove interfering substances, alkaline conversion of fenclorac to the alpha-hydroxy acid metabolite, oxidation of this metabolite to the corresponding benzaldehyde derivative, and spectrophotometric measurement of the absorbance of the aldehyde at 252 nm. A comparison of serum concentrations obtained by this method with concentrations calculated from 14C-data following oral administration of 1-14C-fenclorac to eight normal adult volunteers indicated a 90% correlation between methodologies over a range of 1.4-25.5 microgram of fenclorac/ml of serum.

Kinetics of hydrolysis of fenclorac.

The kinetics of hydrolysis of fenclorac were studied to determine its stability in aqueous solution at different pH's and temperatures. For this study, a stability-specific liquid chromatographic assay method was developed to separate fenclorac from its hydrolysis product, alpha-hydroxy-3-chloro-4-cyclohexylbenzeneacetic acid. The k-pH profile in the 0-12 pH range in various buffer solutions shows that fenclorac is stable in its undissociated form in strongly acidic media and is unstable in neutral and alkaline media. The instability of fenclorac in aqueous solution is proportional to the degree of ionization of the carboxyl group in the 1-4 pH range and is independent of pH above 4. The rate-determining step in the mechanism of hydrolysis of fenclorac involves ionization of the carbon-chlorine bond. The ionization is catalyzed by an intramolecular necleophilic attack on the alpha-carbon by the dissociated carboxyl group, resulting in the formation of an unstable intermediate, a three-membered ring lactone. This unstable intermediate rapidly hydrolyzes to the final hydrolysis product. This mechanism is supported by experimental evidence such as the medium effect, positive salt effect, common ion effect, and substituent effect. Arrhenius parameters for the hydrolysis of fenclorac and its 3-nitro substituted analog were obtained.

The antiphlogistic, antinociceptive and antipyretic properties of fenclorac.

Fenclorac (a,m-dichloro-p-cyclohexlphenylacetic acid, diethylammonium salt) is a potent nonsteroidal anti-inflammatory agent with significant analgesic and antipyretic activity. Fenclorac had an ED50 of 7.9 mg/kg in the carrageenan paw edema assay and had a duration of action of 18-22 hours. Comparative tests in the carrageenan paw edema assay in the rat indicated that the potency of fenclorac was 13 times that of aspirin, 3.4 times phenylbutazone, 3 times ibuprofen and 0.3 times indomethacin. Fenclorac was less potent than indomethacin, but more potent than phenylbutazone or aspirin in treatment of developing or established adjuvant arthritis. The anti-inflammatory effectiveness of fenclorac did not depend upon the integrity of the adrenopituitary axis and was not affected by the route of administration or sex of the test animal. Fenclorac was 77 times more potent than aspirin and more than twice as potent as indomethacin in reducing fever in rats rendered hyperthermic with brewer's yeast. Fenclorac did not affect normal body temperatures. Fenclorac did not interfere with cellular immune mechanisms as measured by its lack of effectiveness in experimental allergic encephalomyelitis. Antinociceptive testing indicated that fenclorac had peripheral but not central analgesic activity. Fenclorac had an acute oral LD50 in rats and mice of 285 and 430 mg/kg, respectively. The acute gastric lesion UD50 for fenclorac was 7 mg/kg in the fasted rat. Studies using 51Cr-tagged erythrocytes indicated that fenclorac did not produce significant fecal blood loss in the rat at twice the therapeutic ED50 dose for up to 12 days after dosing. Extensive and prolonged fecal blood loss was observed with a corresponding dose of indomethacin for up to nine days after administration. Comparison of the anti-inflammatory pharmacology, Therapeutic Ratio and the data obtained from the 51Cr-fecal blood loss studies indicated that fenclorac was well tolerated after acute or subacute administration to the rat.

GLC assay for fenclorac.

A rapid stability-indicating GLC method is described for the determination of fenclorac as the diethylamine salt in dosage formulations. The procedure is also applied in detecting and quantitating the weight percent of 3-chloro-4-cyclohexylphenylglycolic acid and alpha-chloro-4-cyclohexylphenylacetic acid impurities associated with the synthesis of the fenclorac drug substance. The former compound, in addition to being an impurity, is also the degradation product of fenclorac. The procedure involves preparations of the silyl derivatives, addition of an internal standard (triphenylethylene), and use of a hydrogen flame-ionization detector. The column, with a phenyl methyl silicone liquid phase and operated at about 195degrees, is well suited for the separation. The silyl derivatives were characterized by GLC-mass spectrometry.

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.

Correlation of in vitro and in vivo methodology for evaluation of antacids.

The rate and extent of acid consumption of an antacid suspension and tablet were evaluated by in vitro and in vivo techniques. Four different test procedures were used to estimate in vitro antacid reactivity. In vivo effects were determined in the fasted and postcibal states in normal human subjects by a radiotelemetry procedure. The duration of elevation of intragastric pH greater than 3 was in agreement with in vitro estimates of total acid consumption of the antacid. There was also good correlation between onset, extent, and duration of in vivo antacid activity and a modified in vitro Beekman antacid test procedure. There was no significant difference in antacid activity of the tablet or suspension in either in vitro or in vivo test procedures. A wide variation in antacid activity was observed between subjects and also in the fasted versus postcibal states. These studies emphasize the requirements for standardization of antacid products by comparactive in vitro and in vivo evaluations to facilitate individualized dose titration of the antacid in each patient and correlation of the acid secretion rate in various types of GI disease with the antacid dose.

Correlation between dissolution characteristics and absorption of methaqualone from solid dosage forms.

A methaqualone tablet in two strengths, 150 and 300 mg, was developed. The dissolution rate of an experimental formulation in pH 7.0 phosphate buffer, measured by the resin flask method, was shown to correlate with bioavailability in humans. The dissolution rate criterion was used to develop the final tablet formulation. Bioavailability of this formulation in two strengths was compared with a commercial capsule formulation and a slowly dissolving tablet formulation. Correlation between dissolution rate and bioavailability was shown in freshly prepared methaqualone tablet formulations. Bioavailability of tablets under accelerated stability testing conditions remained unaltered, whereas the dissolution rates in pH 7 phosphate buffer decreased, using the resin flask method. A rotating-flask method was developed, and dissolution in 0.1 N HCl at 2 rpm correlated with the bioavailability of both new and aged tablet formulations.

Induction of hepatic enzymes by methaqualone and effect on warfarin-induced hypoprothrombinemia.

The effect of methaqualone on the induction of hepatic enzymes was evaluated in rats and compared with that of phenobarbital by measuring effects on hexobarbital and methaqualone hypnosis, plasma and tissue levels of methaqualone, hepatic aniline hydroxylase and aminopyrine demethylase activity and warfarin-induced hypoprothrombinemia. Maximal reductions in hexobarbital hypnosis occurred 3 days after daily administration of 60 mg of methaqualone per kg per day. At this time, the activities of aniline hydroxylase and aminopyrine demethylase were increased 60 and 139%, respectively, and hepatic microsomal proteins increased 15% above controls in methaqualone-pretreated animals. Methaqualone altered its own metabolism as demonstrated by a 48% reduction in methaqualone hypnosis in pretreated animals. The extent and duration of induction by phenobarbital was considerably greater than methaqualone in all experiments. Methaqualone pretreatment did not affect warfarin-induced hypoprothrombinemia, whereas phenobarbital-pretreated animals showed a 32 to 64% reduction in response to the anticoagulant. These studies indicate that methaqualone is a relatively weak inducer of hepatic drug-metabolizing enzymes and has no effect on the anticoagulant acitivty of warfarin.

Age differences affecting induction of hepatic drug metabolizing enzymes by methaqualone and phenobarbital in the rat.

Methaqualone pretreatment for 3 or 6 days caused an induction of hepatic enzymes in the young male rat as measured by a reduction in hexobarbital-hypnosis. However, methaqualone pretreatment had no effect on the hexobarbital-hypnotic response in older male rats. Phenobarbital was a more potent enzyme inducer than methaqualone, and caused induction of liver enzymes in both age groups.