Binding of (+)- and (-)-isomers and racemate of etomoxir to human serum albumin and effect of stearic acid and stanozolol.

Binding of (+)- and (-)-isomers and the racemate of sodium 2[6-(4-chlorophenoxy)-hexyl]-oxiran-2-carboxylate dihydrat (etomoxir) to the human serum albumin (HSA) was studied by the gel filtration method. The experimental results are presented graphically using the method of Scatchard. Measurements revealed the following data on the binding: (a) for either of the isomers there are two independent and nonequivalent classes of binding sites on the HSA molecule; (b) the binding constants calculated for both isomers were of the same order of magnitude (K1/n approximately 20 x 10(5) L.mol-1 for the concentration range 3.48-4.0 x 10(-5) mol.L-1, and K2/n approximately 2 x 10(5) L.mol-1 for the concentration range 4.28-10 x 10(-5) mol.L-1, for the high and low affinity binding sites, respectively); (c) statistically significant difference (p < or = 0.05) between the low affinity binding constant estimated for the (+)-isomer K2 = 1.9 +/- 0.1 x 10(5) L.mol-1) compared with the constants evaluated for the (-)-isomer and racemic etomoxir (2.6 +/- 0.1 and 2.9 +/- 0.2 x 10(5) L.mol-1, respectively); and (d) both isomers are bound into a high extent to the HSA molecule (i.e., at a ligand concentration of 3.48 x 10(-5) mol.L-1, the percent of binding was approximately 95% for the compound tested. When plotting the percent binding (% Cb) against the total concentration (Ctot), a statistically significant difference (p < or = 0.05) was obtained between the slope of the straight line for the (+)-isomer and those for other two compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereochemical characterization of interactions of chiral 1,4-benzodiazepine-2-ones with liver microsomes.

Microsomal P-450 cytochrome are stereoselective toward 7-Chloro-1,3-dihydro-3(S)-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one. (S)-1, and 7-chloro-1,3-dihydro-3(S)-isopropyl-5-phenyl-2H-1,4-benzodiazepin-2-one, (S)-2. (S)-enantiomers bind to a substrate binding site which accomodates the stable M-conformation (Ks =0.01 to 0.018 mmol/l). (R)-enantiomers, however, undergo a ligand type of interaction (Ks = 0.036 to 0.12 mmol/l). Prochiral desmethyldiazepam behaves similarly to the (S)-enantiomers of the above compounds. The ligand binding site does not differentiate between M- and P-conformers.

Biotransformations and plasma-level curves of chiral 1,4-benzodiazepine-2-ones.

Biotransformations of chiral 1,4-benzodiazepine-2-ones, (S)- and (R)-1 (7-chloro-1,3-dihydro-3 (S and R)-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) in untreated and phenobarbital-pretreated rats were investigated. In urine, a 4'-oxygenated metabolite (compound 2) was identified as the biotransformation product from both enantiomers, (S)-2 being present in much higher amounts than (R)-2. Unchanged parent compounds were not found in urine. In plasma, 3'- and 4'-oxygenated metabolites were identified after administration of (S)-1 and (R)-1, respectively. The metabolite possessing an R-configuration was present in much lower amounts. The maximum concentrations of (R)-1 in plasma, following a single dose, was about 6 time as high as the maximum plasma concentration of (S)-1. Faster biotransformation and elimination of (S)-1 is assumed to be the explanation of these findings.

Urinary excretion and metabolism of orally administered mefenorex.

Metabolic pathways and the pharmacokinetic profile of mefenorex ((+/-)N-(3-chloropropyl)-1-methyl-2-phenylethylamine), and its main metabolite amphetamine (1-methyl-2-phenylethylamine) have been studied in two healthy volunteers, after a single oral dose of mefenorex (1.2 mg/kg body weight for a male subject and 2.4 mg/kg body weight for a female subject). Urinary concentrations were determined by gas chromatography (GC) and metabolite structure was identified by GC/MS following derivatization of urine extracts. The ratio of this metabolite to unchanged drug in urine samples, collected up to 5 h following administration, was essentially the same after either of the administered doses. The calculated Kel for mefenorex after the higher dose was in the range of 0.191-0.272 h-1, with a biological half life (t1/2) of 3.98-2.55 h, depending on the method of calculation used. The elimination of amphetamine was much slower with a Kel ranging from 0.039-0.073 h-1 and a t1/2 from 9.5-17.8 h. Depending on the dose administered, the rate constant of metabolite formation was 0.129 and 0.685 h-1 for low and high doses, respectively. Urinary excretion of Rondimen amounted to 11.9% within 72 h after administration. Of this amount, 1.5% represented unchanged drug and 10.4% represented metabolites. In addition to amphetamine 3 other metabolites were identified: p-hydroxy mefenorex, p-hydroxy amphetamine and p-hydroxy-m-methoxy mefenorex.

Cimetidine and ranitidine: their interaction with human and pig liver microsomes and with purified cytochrome P-450.

Cimetidine and ranitidine interact with microsomes from human and pig liver and with purified cytochrome P-450 in the ligand-type manner. The affinity for cimetidine is about 10 times as high as that for ranitidine. Accordingly amplitudes of the specta are much higher for cimetidine. These results are in accordance with those obtained earlier with rat liver microsomes. The inhibitory potency of either compound with regard to dealkylation of 7-ethoxycoumarin appears to be less in the human preparation.

Update information on drug metabolism systems--2009, part II: summary of information on the effects of diseases and environmental factors on human cytochrome P450 (CYP) enzymes and transporters.

The present paper is an update of the data on the effects of diseases and environmental factors on the expression and/or activity of human cytochrome P450 (CYP) enzymes and transporters. The data are presented in tabular form (Tables 1 and 2) and are a continuation of previously published summaries on the effects of drugs and other chemicals on CYP enzymes (Rendic, S.; Di Carlo, F. Drug Metab. Rev., 1997, 29(1-2), 413-580., Rendic, S. Drug Metab. Rev., 2002, 34(1-2), 83-448.). The collected information presented here is as stated by the cited author(s), and in cases when several references are cited the latest published information is included. Inconsistent results and conclusions obtained by different authors are highlighted, followed by discussion of the major findings. The searchable database is available as an Excel file, for information about file availability contact the corresponding author.

Drug interactions of H2-receptor antagonists involving cytochrome P450 (CYPs) enzymes: from the laboratory to the clinic.

This paper reviews the main steps in the research of the interactions of H2-receptor antagonist drugs with cytochrome P450 (CYP) enzymes. Cimetidine, ranitidine, and related compounds are used as examples. The results from in vitro studies are related to the observed clinically significant in vivo drug-drug and drug-chemical interactions. Uses of the in vitro results are discussed for the interpretation and possible prediction of drug-drug interactions, which may be important in developing new drugs. Other approach in the use of the in vitro data is to prevent undesirable and toxic actions of drugs related to the catalytic activity of CYP enzymes. In the case of H2-receptor antagonists, the inhibition of the metabolic reactions due to the binding of the drugs with the enzymes was used to avoid side effects of co-administered drugs. The in vitro metabolic studies using recombinant human as well as animal CYP enzymes are now widely used as model syste ms for designing new drugs with improved therapeutic properties.

Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes. Gas chromatographic-mass spectrometric determination of metabolites.

Metabolism of steroid hormones with anabolic properties was studied in vitro using human recombinant CYP3A4, CYP2C9 and 2B6 enzymes. The enzyme formats used for CYP3A4 and CYP2C9 were insect cell microsomes expressing human CYP enzymes and purified recombinant human CYP enzymes in a reconstituted system. CYP3A4 enzyme formats incubated with anabolic steroids, testosterone, 17alpha-methyltestosterone, metandienone, boldenone and 4-chloro-1,2-dehydro-17alpha-methyltestosterone, produced 6beta-hydroxyl metabolites identified as trimethylsilyl (TMS)-ethers by a gas chromatography-mass spectrometry (GC-MS) method. When the same formats of CYP2C9 were incubated with the anabolic steroids, no 6beta-hydroxyl metabolites were formed. Human lymphoblast cell microsomes expressing human CYP2B6 incubated with the steroids investigated produced traces of 6beta-hydroxyl metabolites with testosterone and 17alpha-methyltestosterone only. We suggest that the electronic effects of the 3-keto-4-ene structural moiety contribute to the selectivity within the active site of CYP3A4 enzyme resulting in selective 6beta-hydroxylation.

Characterization of pirenzepine interaction with cytochrome P-450.

Pirenzepine interacts with the haem iron of cytochrome P-450 from rat-and pig-liver microsomes, to give absorption spectra with max. at 424-429 nm, and min. at 391-399 nm. Binding to cytochrome P-450 was not detected with human-liver microsomes. Inhibition of 7-ethoxycoumarin dealkylation by pirenzepine using rat-liver microsomes gave values of I50 = 5 mM and Kis = 0.53 mM. E.p.r. spectra showed that pirenzepine probably interacts with the haem iron through the pirenzepine N-4(1) tertiary amine group.

Characterization of cimetidine, ranitidine, and related structures' interaction with cytochrome P-450.

Cimetidine (I) interacts with the hemin iron of cytochrome P-450 from rat liver microsomes, with its imidazole and cyano coordinating groups. Ranitidine (II) interacts through its nitronic acid oxygen and its amine nitrogen, as shown by optical difference and ESR-spectra. I, N-cyano-N'[2-[[[5-(dimethylamino)-methyl-2-furanyl]methyl] thio]-ethyl]-N"-methyl guanidine (IV), 4(5)-hydroxymethyl-5(4)-methyl imidazole (VII), 4(5)-methyl-5(4)-[(2-aminoethyl)-thiomethyl]-imidazole hydrochloride (IX), 2-[[[(5-dimethylamino)-methyl-2-furanyl]-methyl]-thio]ethene amine dihydrochloride (X) and imidazole (XI) inhibit 7-ethoxycoumarin dealkylation competitively. In I both imidazole and cyano groups contribute to the inhibitory activity, the latter group being more effective according to electron spin resonance. Mixed type inhibition was observed with II, desmethylranitidine (VIII) and N-[[2-(5-methylimidazol-4-yl)methylthio]-ethyl]-N'-methyl-2-nitro-1, 1-ethenediamine (III). These compounds inhibited the reaction to a small extent; ranitidine S-oxide (VI) did not interact at all with microsomes from phenobarbital-pretreated rats. Using microsomes from 3-methylcholanthrene-pretreated rats, the affinities of interaction and the amplitudes of optical difference spectra were higher with VIII than with its parent, compound II.

[Prevention by metyrapone of halothane induced liver necrosis in rats (author's transl)]. / Hemmung Halothan-induzierter Leberzellnekrosen bei Ratten durch Metyrapon.

Liver necrosis and an increase of serum sorbitol dehydrogenase activity can be produced in rats by halothane anesthesia (1% v/v in oxygen) following pretreatment with polychlorinated biphenyls. Using this model, it was shown that administration of metyrapone (2-methyl-1,2-bis(3-pyridyl)-1-propanone) (100 mg/kg b.w.) 1 h prior to anesthesia prevents liver necrosis and the concomitant increase in serum sorbitol dehydrogenase activity. No significant differences were observed when halothane was applied in air instead of oxygen, but the protective effect of metyrapone was abolished.

Interaction of ranitidine with liver microsomes.

1. Ranitidine interacts with liver microsomes from rats pretreated with different inducers of cytochrome P-450 to produce substrate difference optical spectra with a peak at 426-429 nm and a trough at 390-400 nm. 2. Cytochrome P-450 reduced with dithionite in the presence of ranitidine produced substrate difference spectra with a peak at 447 nm. 3. Ks values for the interaction of ranitidine with cytochrome P-450 (not reduced), calculated from double reciprocal plots, were in the range 1.4-2.8 mM. 4. The O-dealkylation of 7-ethoxycoumarin and of p-nitroanisole was inhibited by the presence of ranitidine and the inhibition was of a mixed type. Kii and Kis values were: for inhibition of 7-ethoxycoumarin dealkylation, 0.8 to 9 mM, and 0.16 to 0.67 mM, respectively; for inhibition of p-nitroanisole dealkylation, 5.8 to 13.7 mM, and 1 to 4.5 mM, respectively. 5. The I50 values for 7-ethoxycoumarin dealkylation was 1.8 mM and for p-nitroanisole dealkylation about 7.2 mM (microsomes from phenobarbital-pretreated rats). 6. The e.p.r. spectra of cytochrome P-450 from phenobarbital-pretreated rats, in the presence of ranitidine, reveal two types of interaction depending on the ranitidine concentration. At lower concentrations of ranitidine, a ligand exchange reaction with an oxygen atom is indicated, and at higher concentrations are with nitrogenous or thioether ligand of ranitidine.

Study of the sporulation of Bacillus thuringiensis var. thuringiensis.

During the submerged cultivation of Bacillus thuringiensis var. thuringiensis in 300- and 3000-liter fermentors, lysis occurred at the end of the exponential phase of growth. New vegetative cells were subsequently formed which usually sporulated. At time of lysis, the amount of soluble sugar was 1-12 g/liter, pH value dropped to 5.3-5.8 from the original PH 6.8 and started to rise after all the cells had lysed. The proteolytic activity was low during the lysis and increased as the sporulation commenced.