Pharmacokinetics of pentobarbital enantiomers as determined by enantioselective radioimmunoassay after administration of racemate to humans and rabbits.

Radioimmunoassays were developed for R- and S-pentobarbital. The optical isomers of pentobarbital were individually alkylated to N-crotonic acid analogs that were coupled to bovine serum albumin. Immunization of rabbits with the conjugates, which were enantiomerically pure at the asymmetric' carbon of the pentobarbital moiety, led to formation of antisera that selectively bound the predicted enantiomer. In displacement studies with enantiomerically pure radioligands, the opposite enantiomer showed 1.0 to 1.4% cross-reaction. Similar selective binding was observed for enantiomers of secobarbital, thiopental and thiamylal. Assays were developed and used to determine enantiomer pharmacokinetics in rabbits and humans given racemic pentobarbital. In rabbits, difference in clearance of the two isomers was minimal, the result of a slightly larger volume of distribution of the R-enantiomer combined with a slightly higher value of the elimination rate constant beta for the S-enantiomer. In humans, the volume of distribution was 12% greater for the R-enantiomer, but the value of beta was 14% higher for this isomer as well. Thus, the median clearance of the S-enantiomer (1.96 liters/h) was 25% less than that of the R-isomer (2.58 liters/h). The S-enantiomer was also more strongly protein bound in plasma (73.5% vs 63.4% for the R-enantiomer), which is consistent with its structural congruence to S-warfarin, S-phenprocoumon and S-glifumide.

Identification of in vitro rat metabolites of 1-phenylcyclohexene.

In vitro metabolites of 1-phenylcyclohexene produced by the 10,000g supernatant fraction from rat liver homogenates were identified by a combination of spectrometric, chromatographic, and synthetic techniques. Initial oxidation occurred in the 3-position of 1-phenylcyclohexene to yield 1-phenyl-1-cyclohexen-3-one and 1-phenyl-1-cyclohexen-3-ol. Further allylic oxidation at the 6-position occurred to form 1-phenyl-6-hydroxy-1-cyclohexen-3-one and 1-phenyl-1-cyclohexene-3,6-diol. Trans-1-phenyl-1-cyclohexene-3,4-diol was also found and may have resulted from hydroxylation of 1-phenyl-1-cyclohexen-3-one alpha to the carbonyl to yield 4-hydroxy-1-phenyl-1-cyclohexen-3-one (not isolated) followed by carbonyl reduction. Oxidation of the double bond also occurred to give the cis and trans isomers of 1-phenylcyclohexane-1,2-diol as well as a compound postulated to be 1-phenylcyclohexane-1,2,3-triol.

Immunoassay of racemic drugs: a problem of enantioselective antisera and a solution.

Although it has been demonstrated that immunization with drug-protein conjugates derived from enantiomerically pure drug derivatives leads to stereoselective antisera, the effect of the use of conjugates of racemic drugs on antibody stereoselectivity has received little attention. Antisera were developed by immunizing rabbits with a conjugate (1) of the hemisuccinate of the antimalarial drug 1-(1,3-dichloro-6-trifluoromethyl-9-phenanthryl)-3-N, N-dibutylaminopropan-1-ol (WR 171,669) and bovine thyroglobulin. These antisera in binding competition studies with tritium-labeled racemic WR 171,669 demonstrated different stereoselective for d-, l- and dl-WR 171-669. Immunization with a conjugate (II) made from a nonchiral analog of the drug led to antisera which showed little discrimination between the optical isomers. Hydrolysis of conjugate I showed no excess of one enantiomer over the other, thus indicating that the stereoselectivity was the result of stereoslective processing of the conjugate by the immune system. The use of antisera from racemic conjugates to analyze total drug levels must be examined in each case ot avoid errors. The use of conjugate achiral drug analogs leading to the production of antisera relatively "blind" to enantiomeric differences is one solution to the problem of selective antisera.

Radioimmunoassays for cannabinoids.

The simplicity, sensitivity, and specificity of radioimmunoassay have made it an attractive procedure for the analysis of delta-9-tetrahydrocannabinol (THC) in biological fluids or tissues. The presence of closely related compounds such as metabolites may interfere with radioimmunoassay results. Appropriate design of immunogens may diminish such interference. This work has been directed towards the use of the amyl side chain for linking cannabinoid compounds to proteins to form immunogens. Although the amyl side chain is metabolized to some extent, the metabolites are not quantitatively significant in most cases. 5'-Carboxy-delta-8-THC and 5'-carboxy-delta-9-THC were linked to bovine serum albumin. Immunization of rabbits with the resulting conjugates resulted in the formation of antisera with high selectivity for delta-9-THC vs. its carboxylic acid metabolite, 11-nor-9-carboxy-delta-9-THC. Delta-8-THC radioligands (4',5'-tritium and 5'-iodine-125) could be used with these antisera for analysis of delta-9-THC in plasma. Sensitivity with tritium-labeled material is about 2.5 ng/ml. 5'-Oxo-11-nor-9-carboxy-delta-8-THC was used to prepare an immunogen which led to the generation of an antiserum highly specific for 11-nor-9-carboxy-delta-9-THC. This antiserum and iodine-125-5'-iodo-11-nor-9-carboxy-delta-8-THC were used to develop a highly specific assay for 11-nor-9-carboxy-delta-9-THC in plasma.

Radioimmunoassay for terfenadine in human plasma.

A radioimmunoassay procedure was developed for the antihistamine terfenadine (alpha[4-(1,2-dimethylethyl)phenyl]-4-(hydroxydiphenylmethyl)-1-piperidinebutanol). The keto analog of terfenadine was converted to its O-carboxymethyloxime derivative, which was conjugated to bovine thyroglobulin by a mixed anhydride technique. Rabbits were immunized with the resulting conjugate, and antiserums capable of binding radiolabeled terfenadine were obtained. Tritium-labeled terfenadine was prepared by a combination of exchange and reduction with platinum oxide in the presence of tritium gas, and the procedure yielded a specific activity of 48 Ci/mmole. Plasma containing terfenadine was diluted with sodium carbonate solution and extracted with hexane, and the hexane extracts were evaporated and analyzed. The between-assay coefficient of variation on control samples ranged from 8% at 10 ng/ml to 14% at 1 ng/ml. The lower practical sensitivity limit was at least as low as 0.25 ng/ml (25 pg measured). Two metabolites of terfenadine cross-reacted 16-30% with the antiserum used. However, extraction eliminated essentially all of these compounds. Analysis of plasma samples from human subjects given terfenadine showed marked intersubject variability and low plasma levels.

Warfarin enantiomer disposition: determination by stereoselective radioimmunoassay.

Immunoassay techniques offer promise for the selective analysis of enantiomeric substances without prior separation. In a demonstrationof this selective analysis, radioimmunoassays were developed for R- and S-warfarin. The 4'-carboxyethyl analog of R,S-warfarin was resolved and the enantiomeric acids were coupled to bovine serum albumin. Immunization of rabbits with the conjugates led to formation of antisera which selectively bound the predicted enantiomer. Cross-reactions with various metabolites of warfarin were low (0.1--4%) except for 4'-hydroxywarfarin, as were cross-reactions with the opposite enantiomer (0.3--3%). Assays were developed and used to determine enantiomer half-lives in rats dosed with racemic warfarin. Results were consistent with those found previously by administration of individual enantiomers, thus indicating the utility of the radioimmunoassay method.

Caffeine in plasma and saliva by a radioimmunoassay procedure.

Caffeine was analyzed in human plasma and saliva by a simple, rapid, and sensitive radioimmunoassay procedure. Immunization of rabbits with an antigen prepared by coupling 7-(5-carboxypentyl)-1,3-dimethylxanthine to bovine serum albumin resulted in the formation of antibodies selective for caffeine as opposed to various mono- and dimethylxanthines, mono-, di-, and trimethyluric acids and a variety of common drugs. The radioligand used for competitive binding studies was 7-(2,3-3H2-propyl)-1,3-dimethylxanthine. The procedure permits direct analysis of caffeine in plasma or saliva without extraction. Comparison with a high pressure liquid chromatography method for the analysis of caffeine gave satisfactory results and showed no evidence for interference by metabolites. A caffeine half-life of 4.0 hours determined by the radioimmunoassay was in agreement with previous work. Comparison of human plasma and saliva levels by the radioimmunoassay procedure indicated approximately equal concentrations in the two fluids.

Norethynodrel metabolites in human plasma and urine.

PIP: Norethynodrel metabolites in human plasma and urine were analyzed. Samples were collected from healthy young women over 1 24-hour period after the oral administration of 11.1 mg (50 mcCi) of 6, 7-tritiated norethynodrel. The 3 alpha-hydroxy compound from the reduction of the 3-ketone of norethynodrel was the major free metabolite in plasma, with lesser amounts of its 3 beta-isomer and norethindrone. These compounds were identified by carrier addition analysis and their concentrations were estimated by thin-layer radiochromatography. The bulk of plasma metabolites consisted of conjugates and greater than 95% of urinary metabolites (20% of total dose) were conjugated; 2 of which were beta-glucuronides. Enzymatic hydrolysis enabled the urinary compounds to be identified by gas-lizuid chromatography-mass spectrometry. 70-80% (10% of the dose) of the enzymatically hydrolyzed urinary metabolites were hydroxylated compounds. Triols were the principal hydroxylated compounds isolated. The metabolism of norethynodrel was rapid and extensive.^ieng