Key structural features of ligands for activation of human pregnane X receptor.

The ligand-binding domain of human pregnane X receptor (hPXR) is highly hydrophobic and flexible, allowing promiscuity in accepting structurally diverse ligands. However, little information is available regarding the critical substituents of compounds involved in the activation of hPXR. The aim of this study was to determine the structure-activity relationships for hPXR-mediated transactivation by barbiturates, hydantoins, and macrolide antibiotics. Most of the barbiturates studied (mephobarbital, pentobarbital, phenobarbital, etc.) activated hPXR. However, barbital, which has a low hydrophobic moiety at the 5-position, and primidone, which has no carbonyl moiety at the 2-position, did not activate hPXR. Therefore, a hydrophobic moiety at the 5-position and a hydrogen-bond acceptor being sufficiently separated from the phenyl-ring are responsible for activation of hPXR by barbiturates. In the case of hydantoins, only mephenytoin and ethotoin, which have an alkylchain at the R1-position, strongly activated hPXR at 300 microM. Phenytoin and 5-(4-methylphenyl)-5-phenylhydantoin, which contain a phenyl or methylphenyl group at both R2- and R3-positions, also activated hPXR, whereas 5-(4-hydroxyphenyl)-5-phenylhydantoin did not activate the receptor. These results suggest that the presence of an alkyl-chain at the R1-position and the presence of bulky and hydrophobic moieties at both R2- and R3-positions are important factors for activation of hPXR by hydantoins. In the case of macrolide antibiotics, troleandomycin, but not oleandomycin, showed significant activation of hPXR. Therefore, triacetate esterification of oleandomycin might increase the hydrophobicity and enhance the activation of hPXR. These findings suggest that hydrophobicity of the ligand and adequate distance between the hydrogen-bond acceptor and the hydrophobic group are important for hPXR activation.

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography-mass spectrometry.

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography-mass spectrometry (GC-MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C18 disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol-water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform-isopropanol (3:1, v/v) from the cartridge. The eluate (1 microl) was injected into a GC-MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 microg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.

Relative potencies for barbiturate binding to the Torpedo acetylcholine receptor.

1. The structural requirements of an allosteric barbiturate binding site on acetylcholine receptor-rich membranes isolated from Torpedo electroplaques have been characterized by the ability of fourteen barbiturates to displace [14C]-amobarbitone binding. 2. The barbiturates could be grouped into two classes with ten barbiturates producing a strong inhibition of [14C]-amobarbitone binding (class one) and with four exerting minimal effects (class two). 3. Eight of the ten class one barbiturates displaced essentially all of the [14C]-amobarbitone from its binding site, while, at their respective aqueous solubility limits, two of these barbiturates (thiopentone and dimethylbutylbarbitone (DMBB) inhibited [14C]-amobarbitone binding by nearly 80%. The apparent inhibition constants (KI) for the class one barbiturates ranged from 13 microM for amobarbitone to 2.8 mM for barbitone with the other eight agents lying in the range 100-600 microM, and having the rank order pentobarbitone approximately secobarbitone greater than thiopentone greater than DMBB greater than butabarbitone approximately phenobarbitone greater than aprobarbitone greater than allylbarbitone. 4. By contrast, the class two barbiturates had minimal effects even at close to saturating concentrations. [14C]-amobarbitone binding was reduced slightly (less than 30%) by hexobarbitone, mephobarbitone and methohexitone and was enhanced slightly (less than 20%) by metharbitone. 5. All of the class two, but none of the class one barbiturates, were N-methylated.

A multivariate analysis of the infrared spectra of drugs of abuse.

The infrared (IR) spectra of 15 drugs of abuse were analyzed for similarity by using techniques of numerical taxonomy. The study included six barbiturates (amobarbital, barbital, butabarbital, phentobarbital, phenobarbital, and secobarbital), four amphetamine-related compounds (amphetamine, ephedrine, methamphetamine, and phentermine), and five other drugs (cocaine, heroin, phencyclidine, phendimetrazine, and diazepam). Three character sets were based on increasing numbers (10, 24, and 36) of IR peaks. The cluster analysis, principal component analysis, and nonmetric multidimensional scaling elements of the program system NT-SYS were used to structure taxonomic distances between drugs. Best results were obtained from the 36-peak data set; ordination diagrams proved to be more visually informative than phenograms. Preliminary results from our analysis of this set of drugs indicate that an expanded multivariate approach to drug classification may be useful.