Joseph Gal

Effect of ritonavir/saquinavir on stereoselective pharmacokinetics of methadone : Results of AIDS clinical trials group (ACTG) 401

The effect of ritonavir 400 mg/saquinavir 400 mg twice daily on the stereoselective pharmacokinetics of methadone was examined in 12 HIV‐infected, methadone‐using study subjects. Design: A 24‐hour methadone pharmacokinetic study was performed before antiretroviral therapy was begun and after 15 days of therapy. Methadone concentration was measured by a chiral plasma assay because the drug is administered as a racemic mixture of R‐ and S‐methadone, but only the R‐isomer is active. Both changes in plasma protein binding and changes in objective and subjective opioid effect were monitored. Results: Ritonavir/saquinavir administration was associated with 40% decrease in total S‐methadone AUC0‐24hr and 32% decrease in R‐methadone area under the curve (AUC)0‐24hr, and both changes were statistically significant (p = .001 for both). When AUC was corrected for the changes in protein binding induced by ritonavir/saquinavir, R‐methadone free AUC0‐24hr decreased 19.6% whereas the S‐methadone decreased 24.6%, neither of these changes was statistically significant (p = .129 and p = .0537, respectively). This change in methadone exposure was not associated with any evidence of withdrawal from narcotics and no modification of methadone dose was required. Conclusions: Our data indicate that ritonavir/saquinavir administration is associated with induction of metabolism of methadone but this is greater for the inactive S‐methadone. However, approximately 37% of the decrease in the total R‐methadone exposure can be explained by protein binding displacement. Ritonavir/saquinavir can be used in HIV‐infected people taking methadone without routine dose adjustments.

Computational Discovery of Novel Low Micromolar Human Pregnane X Receptor Antagonists

Very few antagonists have been identified for the human pregnane X receptor (PXR). These molecules may be of use for modulating the effects of therapeutic drugs, which are potent agonists for this receptor (e.g., some anticancer compounds and macrolide antibiotics), with subsequent effects on transcriptional regulation of xenobiotic metabolism and transporter genes. A recent novel pharmacophore for PXR antagonists was developed using three azoles and consisted of two hydrogen bond acceptor regions and two hydrophobic features. This pharmacophore also suggested an overall small binding site that was identified on the outer surface of the receptor at the AF-2 site and validated by docking studies. Using computational approaches to search libraries of known drugs or commercially available molecules is preferred over random screening. We have now described several new smaller antagonists of PXR discovered with the antagonist pharmacophore with in vitro activity in the low micromolar range [S-p-tolyl 3′,5-dimethyl-3,5′-biisoxazole-4′-carbothioate (SPB03255) (IC50, 6.3 μM) and 4-(3-chlorophenyl)-5-(2,4-dichlorobenzylthio)-4H-1,2,4-triazol-3-ol (SPB00574) (IC50, 24.8 μM)]. We have also used our computational pharmacophore and docking tools to suggest that most of the known PXR antagonists, such as coumestrol and sulforaphane, could also interact on the outer surface of PXR at the AF-2 domain. The involvement of this domain was also suggested by further site-directed mutagenesis work. We have additionally described an FDA approved prodrug, leflunomide (IC50, 6.8 μM), that seems to be a PXR antagonist in vitro. These observations are important for predicting whether further molecules may interact with PXR as antagonists in vivo with potential therapeutic applications.

R-α-Methylbenzyl isothiocyanate, a new and convenient chiral derivatizing agent for the separation of enantiomeric amino compounds by high-performance liquid chromatography

R-α-Methylbenzyl isothiocyanate (R-AMBI), a commercially available chiral compound, was evaluated as a chiral derivatizing agent for the separation of enantiomeric amines by reversed-phase liquid chromatography. Ephedrine, propranolol, phenylglycine, phenylglycinol, phenylalanine methyl ester and 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane were derivatized with R-AMBI, and the derivatives chromatographed on octadecylsilane columns using aqueous methanol or acetonitrile as mobile phase. The separation factor, α, for the resolution of the enantiomers ranged between 1.05 and 1.24, and peak resolution, R, ranged between 1.29 and 3.28. In some applications, R-AMBI may have advantages over other similar reagents.

Enantiospecific drug analysis via the ortho-phthalaldehyde/homochiral thiol derivatization method

Pre-column derivatization with o-phthaldialdehyde and an optically active thiol has hitherto been used mainly for liquid-chromatographic chiral separation of amino acids. Chiral separation of non-amino-acid primary amines, especially of pharmaceuticals, via this approach has been largely ignored. We have therefore examined the applicability of the method to the chiral resolution of several pharmaceutical amines. o-Phthaldialdehyde and four commercially available homochiral thiols were used to study the separation of the enantiomers of amphetamine, p-hydroxyamphetamine, p-chloroamphetamine, 3-amino- 1-phenylbutane, 3-amino-1-(4-hydroxyphenyl)-butane, mexiletine, tocainide, tranylcypromine and rimantadine. The resulting highly fluorescent isoindole derivatives were resolved on a Waters Nova-Pak C18 column using mobile phases consisting of mixtures of methanol, a sodium acetate buffer and acetonitrile, and the column effluent was monitored using fluorescence or UV detection. In some cases the fluorescence and/or the UV absorbance of the two diastereomers were unequal. It was found that the resolution of most of the amines could be optimized by varying the homochiral thiol in the derivatization step. This method of chiral separation may have wide applicability in enantiospecific drug analysis of non-amino-acid primary amines due to its simplicity and the high sensitivity it provides.

High-performance liquid chromatographic resolution of enantiomers of 1-phenyl-2-aminopropanes (amphetamines) with four chiral reagents.

High-performance liquid chromatography (HPLC) was employed for resolution of enantiomers of chiral ring-substituted 1-phenyl-2- aminopropanes (amphetamines) and 1-phenylethylamine following derivatization with four chiral reagents: (R)-(+)-1-phenylethyl isocyanate ( PEIC ), (-)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride ( MTPA X Cl), 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate ( GITC ), and 2,3,4-tri-O-acetyl-alpha-D- arabinopyranosyl isothiocyanate ( AITC ). Reactions were accomplished under mild conditions (25-70 degrees C) and were complete for all substrates within 60 min. Derivatization with the sugar isothiocyanates ( GITC and AITC ) and the acyl chloride ( MTPA . Cl) was carried out in methylene chloride or acetonitrile in the presence of a base catalyst while derivatization with the isocyanate ( PEIC ) was performed in methylene chloride. The diastereomeric derivatives were separated by reversed-phase HPLC (C18) with a methanol-water mobile phase. In general, HPLC resolution of the diastereomeric reaction products of GITC or AITC , and MTPA .Cl with the amine substrates was more complete (usually greater than 98% baseline separation) than HPLC resolution of the diastereomeric reaction products of PEIC .

Serum Binding of Diltiazem in Humans

Abstract: Diltiazem binding was measured by equilibrium dialysis in the serum of five healthy male subjects, aged 22 to 34 years, at seven serum diltiazem concentrations ranging from 0.03 to 2.06 μg/ml. The percentage of unbound diltiazem ranged from 19.6 ± 3.1 to 22.6 ± 4.1 (mean ± S.D.) and was independent of serum diltiazem concentration. Serum diltiazem binding was also examined in the presence of six drugs that might be used concurrently with diltiazem in patients being treated for cardiac disease. The percentage of unbound diltiazem was not influenced by digoxin, hydrochlorothiazide, phenylbutazone, propranolol, salicylic acid, or warfarin at therapeutic concentrations of these drugs over the therapeutic range of diltiazem.

Determination of the Enantiomeric Composition of Chiral Aminoalcohols Using Chiral Derivatization and Reversed-Phase Liquid Chromatography

Abstract A variety of racemic aminoalcohols were derivatized with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate. The resulting two diastereomeric derivatives of each racemate were separated on octadecylsilane liquid-chromatographic columns using methanol-water mixtures as mobile phase and detection at 254 nm. The procedure is simple, rapid and convenient, and may be generally applicable to the determination of the enantiomeric composition of aminoalcohols.

The Metabolism of Amphetamine in vitro by Rabbit Liver Preparations: A Comparison of R(−) and S(+) Enantiomers

1. Incubation of R(-), S(+) and RS(+/-) amphetamines with rabbit liver 9000 g supernatant indicated that R(-) was metabolized at a faster rate than S(+), but that racemic amphetamine was metabolized at the same rate as S(+) during one hour incubations. 2. N-Hydroxyamphetamine and 1-phenyl-2-propranol were the major compounds detected in both R(-) and S(+) amphetamine incubations. 3. Phenylacetone oxime was detected in significant quantities after 3 h incubations of R(-) amphetamine, but only in minor quantities from S(+). 4. A fall in the amount of N-hydroxyamphetamine present in R(-) amphetamine incubations after a 3 h period as compared to a 1 h incubation, paralleled by a rise in the amount of phenylacetone oxime during 3 h suggested that the oxime was derived as a secondary metabolics from N-hydroxyamphetamine. 5. R(-) and S(+) N-hydroxyamphetamines were both metabolized to phenylacetone oxime by rabbit liver 9000 g supernatant, but the R(-) enantiomer was converted at a faster rate than S(+).

Determination of the enantiomeric composition of salsolinol in biological samples by high-performance liquid chromatography with electrochemical detection

The enantiomers of salsolinol were completely separated as diastereoisomeric derivatives, after reaction with S-1-(1-naphthyl)ethyl isothiocyanate, by reversed-phase high-performance liquid chromatography and quantified by electrochemical detection. Good calibration curves were obtained for the quantification and determination of the enantiomeric composition of salsolinol in human urine. The sensitivity and specificity to the assay also permit the determination of the enantiomeric composition of salsolinol in food such as dried bananas and chocolate.

Determination of acetazolamide in biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay for acetazolamide is presented. A 100-microliter sample is mixed with an aliquot of the internal standard solution and the mixture, buffered at pH 4.5, is extracted with ethyl acetate. The extract is evaporated to dryness and the residue is analyzed by HPLC, using a reverse-phase octadecylsilane column. The wavelength of the detection is 254 nm. The coefficient of variation (CV) in the within-day analysis of replicate 10-microgram/ml acetazolamide samples in human blood plasma was 6.5%, while the between-day CV was 7.1%. The procedures was developed for the 1-25 microgram/ml acetazolamide concentration range. The internal standard used is similar in chemical structure to acetazolamide and can be readily prepared in one step from a commercially available precursor. In addition to blood serum or plasma, the assay can also use aqueous and vitreous humor samples. Theophylline and acetaminophen interfere in the assay. The technique was used to determine the concentration of acetazolamide in the blood serum of human volunteers after an oral dose of the drug, and in the aqueous and vitreous humors of rabbits after an intravenous dose of acetazolamide.