Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans

The kinetics of the glutathione (GSH) conjugation of (+)- and (-)-enantiomers of anti- as well as syn-3,4-dihydroxy-1,2-oxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene (B[c]PDE) catalyzed by murine GSH S-transferase (GST) isoenzymes has been investigated. Murine GSTs exhibited significant differences in their enantioselectivity toward B[c]PDE stereoisomers. For example, while pi class isoenzyme mGSTP1-1 was virtually inactive toward stereoisomers with 1S configuration [(-)-syn-and (+)-anti-B[c]PDE], these stereoisomers were good substrates for alpha class isoenzyme mGSTA1-2. When GST activity was measured as a function of varying B[c]PDE concentration (10-320 microM) at a fixed saturating concentration of GSH (2 mM), each isoenzyme examined obeyed Michaelis-Menten kinetics with all four B[c]PDE stereoisomers. Alpha class isoenzyme mGSTA4-4 exhibited negligible activity toward all four stereoisomers of B[c]PDE. The catalytic efficiency of mGSTA1-2 was approximately 1.5- to 15-fold higher than other murine GSTs in the GSH conjugation of (-)-anti-B[c]PDE, which among the four B[c]PDE stereoisomers is the most potent pulmonary carcinogen in the newborn mouse model and a potent skin tumor-initiator. While alpha class isoenzymes mGSTA3-3 and mGSTA1-2 were equally efficient in the GSH conjugation of (+)-anti-B[c]PDE, their catalytic efficiencies toward this stereoisomer were significantly higher than those of mGSTP1-1 and mGSTM1-1. Likewise, mGSTA1-2 was relatively more efficient than other GSTs in the GSH conjugation of both enantiomers of syn-B[c]PDE. In summary, our results indicate that (a) murine GSTs significantly differ in their enantioselectivity in the GSH conjugation of B[c]PDE stereoisomers, which may partially account for the observed differences in the carcinogenic potency of B[c]PDE stereoisomers, and (b) mGSTA1-2 and mGSTA3-3 play a major role in the detoxification of B[c]PDE.     

Sensitive chiral high-performance liquid chromatographic assay for labetalol in biological fluids

The four stereoisomers of the combined alpha- and beta-adrenoceptor antagonist labetalol were separated and quantified at therapeutic concentrations by normal-phase high-pressure liquid chromatography using a chiral stationary phase and fluorescence detection. Drug in plasma or urine was recovered by solid-phase extraction with 83+/-5% efficiency. Limits of detection from biological samples (3 ml) were between 1.5-1.8 ng ml(-1). Intra-day and inter-day variation at 25 ng ml(-1) were < or = 2.7% and < or = 5.80% respectively for all stereoisomers. The assay was applied to an examination of the disposition of labetalol stereoisomers after a single oral dose of racemate to a human volunteer. Labetalol appears to undergo enantioselective metabolism leading to relatively low plasma concentrations of the pharmacologically active enantiomers.     

Stereoisomers of cyclic urea HIV-1 protease inhibitors: synthesis and binding affinities

We have synthesized stereoisomers of cyclic urea HIV-1 protease inhibitors to study the effect of varying configurations on binding affinities. Four different synthetic approaches were used to prepare the desired cyclic urea stereoisomers. The original cyclic urea synthesis using amino acid starting materials was used to prepare three isomers. Three additional isomers were prepared by synthetic routes utilizing L-tartaric acid and D-sorbitol as chiral starting materials. A stereoselective hydroxyl inversion of the cyclic urea trans-diol was used to prepare three additional isomers. In all 9 of the 10 possible cyclic urea stereoisomers were prepared, and their binding affinities are described.     

Sex pheromone of the oleander scale, Aspidiotus nerii: structural characterization and absolute configuration of an unusual functionalized cyclobutane

The sex pheromone emitted by the female oleander scale, Aspidiotus nerii (Homoptera, Diaspididae), has been isolated and characterized as (1R, 2S)-cis-2-isopropenyl-1-(4'-methyl-4'-penten-1'-yl)cyclobutaneethanol acetate by using advanced MS and NMR spectroscopic methods, as well as a variety of microderivatization sequences. The structure has been confirmed by stereo- and enantioselective synthesis of the four possible stereoisomers. The absolute configuration has been determined by comparison of the activity of the cis (1S,2R) and (1R, 2S) enantiomers with that exhibited by the natural material in greenhouse bioassays and field tests. The structure of this sesquiterpenoid pheromone is new in the coccids and in the pheromone field in general.     

Stereoisomers of 3-(2,3-dihydrobenzofuran-2-yl)quinuclidine: preparation and muscarinic receptor affinities

The four stereoisomers of the antimuscarinic 3-(2,3-dihydrobenzofuran-2-yl)quinuclidine have been prepared by a method involving chromatographic separation of the racemic diastereoisomers as borane complexes. The relative and absolute configurations of the stereoisomers were determined by X-ray crystallographic methods. The crystal structure of (2'R,3R)-3-(2,3-dihydrobenzofuran-2-yl)quinuclidine.HCl.H2O contains two independent molecules with different conformations of both the quinuclidine moiety and the dihydrofuran ring.     

Antigravity suit used for neurosurgical operations in sitting position

Isoproterenol is a chiral catecholamine with a half-life of elimination of less than 10 min. In order to study the pharmacokinetics of this compound using microdialysis sampling, an analytical method was needed which could resolve the individual enantiomers of isoproterenol and required less than 1 microliter of sample. A capillary electrophoretic method using a run buffer containing methyl-O-beta-cyclodextrin as a chiral recognition agent was developed which could resolve the enantiomers of isoproterenol. The detection limits using UV absorbance detection were found to be too high to determine the concentration of isoproterenol in plasma for a sufficient time following administration to establish the pharmacokinetics. The detection limits were decreased three orders of magnitude to 3 ng/ml by using an amperometric detector. The detection limits were decreased to 0.6 ng/ml using an on-column concentration technique in which peak stacking was accomplished by following the sample injection with a plug of acid.     

Local anesthetic effect of carbisocaine and its enantiomers

The optically active isomers of carbisocaine [1-methyl-2-diethylaminoethyl ester of 2-(n)-heptyloxycarbonilic acid] were prepared. The blocking activity of equimolar concentrations of the carbisocaine and its corresponding enantiomers was tested on isolated rat sciatic nerves. There were no significant differences between the anesthetic action of racemic form and enantiomers, however, lower activity for the (--)-enantiomer was observed. The results may indicate negligible stereoselectivity of action of highly lipophilic local anesthetic carbisocaine in the excitable membrane.     

Non-stereoselective formation of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid during cholic acid biosynthesis

Incubation of (25RS)-, (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA, 6, 6a, 6b) and (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid (7) with rat liver mitochondria gave all four stereoisomers (9a,9b,9c,9d) of 3 alpha,7 alpha,12 alpha,24-Tetrahydroxy-5 beta-cholestan-26-oic acid (TeHCA). The corresponding 27-nor analogs (10,11) were also converted non-stereoselectively to a 1:1 mixture of the epimeric 24-hydroxy compounds (12).     

Capillary electrophoretic separation of weak base enantiomers using the single-isomer heptakis-(2,3-dimethyl-6-sulfato)-beta-cyclodextrin as resolving agent and methanol as background electrolyte solvent

The sodium salt of the single-isomer, heptakis-(2,3-dimethyl-6-sulfato)-beta-cyclodextrin (HDMS-betaCD) was used as resolving agent in the capillary electrophoretic (CE) separation of weak base enantiomers in pure methanol background electrolytes (BEs). According to the requirements of the charged resolving agent migration model of CE enantiomer separations (CHARM model), a high buffer-capacity, low pH methanolic BE was created from 25 mM phosphoric acid and 12.5 mM NaOH. In this BE, the solubility of HDMS-betaCD was as high as 50 mM, permitting the realization of very high separation selectivities and short separation times for the fully protonated weak base enantiomers.     

Chiral synthesis and pharmacological evaluation of the enantiomers of SM32, a new analgesic and cognition-enhancing agent

The enantiomers of 3-alpha-tropyl 2-(phenylthio)butyrate (SM32, 1) were prepared by chiral synthesis and tested for analgesic, cognition-enhancing, and ACh-releasing properties. They show enantioselectivity in some of the tests, the eutomer being related in configuration to R-(+)-hyoscyamine.     

Studies on the mechanism of action of a novel anorectic agent, (--)-threo-chlorocitric acid

(--)-threo-Chlorocitric acid, a novel and potent anorectic agent in rats and dogs, decreased food intake to a comparable extent in rats fed chow, low fat, or high fat diets. This inhibition of food intake was not the result of conditioned aversion. Unlike (--)-threo-hydroxycitric acid, a structurally related compound which inhibits fatty acid synthesis, (--)-threo-chlorocitric acid did not suppress fatty acid synthesis in an isolated hepatocyte system or in vivo. (--)-threo-Chlorocitric acid significantly delayed the rate of gastric emptying. Of the four stereoisomers of chlorocitric acid, (--)-threo-chlorocitric acid was the most active both in delaying gastric emptying and producing anorexia in rats. It is suggested that the mechanism by which (--)-threo-chlorocitric acid may suppress food intake is through a reduction of gastric emptying.     

The synthesis and thymidylate synthase inhibitory activity of L-gamma-L-linked dipeptide and L-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583)

Sixteen gamma-linked dipeptide and four L-Glu-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) have been synthesized and evaluated as inhibitors of thymidylate synthase (TS). Z-blocked L-Glu-gamma-L-linked dipeptides and L-Glu-gamma-amides were prepared by condensing alpha-tert-butyl-N-(benzyloxycarbonyl)-L-glutamic acid with the appropriate tert-butyl-protected L-amino acid or amine. The Z group was removed by catalytic hydrogenolysis, and the resulting dipeptides or L-Glu-gamma-amides were condensed with the appropriate pteroic acid analogue trifluoroacetate salt using diethyl cyanophosphoridate as coupling reagent. Deprotection with trifluoroacetic acid in the final step gave the desired quinazoline gamma-linked dipeptides and L-Glu-gamma-amides as their trifluoroacetate salts. Nearly all the dipeptide analogues were potent inhibitors of TS, the best being ICI 198583-gamma-L-2-aminoadipate (IC50 = 2 nM). Several of these dipeptides were found to be susceptible to enzymatic hydrolysis in mice. The quinazoline monocarboxylate L-Glu-gamma-amides, lacking an alpha'-carboxyl group, are less active against TS and L1210 cell growth but are also not susceptible to enzymatic hydrolysis in mice.     

Vancomycin as a chiral selector in capillary electrophoresis: an appraisal of advantages and limitations

The properties of the macrocyclic antibiotic vancomycin, used as a chiral selector, were studied with aminoquinolycarbamate derivatives of amino acids, containing sulfur and selenium, as well as with other organic ions. Vancomycin combines the ability to resolve fully ionized anionic enantiomers, typical of proteins, with excellent separation efficiency, exceeding that of cyclodextrins. It allows better than baseline chiral separations of several anionic analytes within 3-5 min. The resolving power of vancomycin results from its great skill in discriminating enantiomers rather than from high affinities to the separated enantiomers. The association constants of vancomycin are of the same order of magnitude, 10(2) L/mol, as that found for beta-cyclodextrin (beta-CD). The difference in association constants of separated cystine enantiomers with vancomycin, 2 x 10(2) L/mol, is one order of magnitude higher than that of enantiomers separated with beta-CD. Analytically convenient mobility differences up to 1-2 x 10(9) m2V-1s-1, with only one of the enantiomers appreciably decelerated, are obtained at submillimolar vancomycin concentrations. Typical separation efficiencies are close to 250,000 theoretical plates per meter of capillary. Deceleration of various organic ions by millimolar vancomycin implies that chiral separations with vancomycin need not be restricted to carboxylic acids. The vancomycin-analyte interactions are strongly affected by the chemical composition and concentration of the buffer. An additional experimental variable, highly effective in manipulating the separation selectivity of analytes, is the buffer pH.     

Nerve growth factor induced hyperalgesia in the rat hind paw is dependent on circulating neutrophils

Protected N-(2-hydroxyethyl)-N-(nucleobase-acetyl)aminomethanephosphonic+ ++ acid (6a-d) of all four DNA nucleobases have been prepared and oligomerized by solid-phase synthesis. Four DNA decamers containing 1-10 of these 'PPNA' monomers were prepared and evaluated by Tm measurements (medium salt) for binding to their DNA and RNA complements. One central modification reduced the binding strongly (delta Tm = -10 degrees C), but contiguous PPNA monomers gave smaller effects, and the all-PPNA decamer bound to RNA with a delta Tm of -1.2 degrees C per modification. Thus PPNA oligomers are inferior DNA and RNA binders compared to the closely related and strongly binding PNA oligomers.     

Lipopolysaccharide and IL-1 alpha activate CNS pathways as measured by NK cell activity

An outward current (Iout) was produced by stereoisomers of beta-hydroxy-L-glutamic acid (L-BHGA), an L-glutamic acid (L-Glu) derivative, applied by brief pneumatic pressure ejection on an identifiable neurone type, v-LCDN (ventral-left cerebral distinct neurone), of Achatina fulica Férussac. However, L- and D-Glu were almost ineffective on this neurone type. The pharmacological features of this Iout caused by L-BHGA were elucidated in the present study. According to the dose (pressure duration)-response studies on the L-BHGA stereoisomers that produced the Iout, the effective potency of threo-L-BHGA was approximately similar to that of erythro-L-BHGA. The dose (pressure duration)-response curve of quisqualic acid was shifted towards the left direction from those of threo-and erythro-L-BHGA, suggesting that the binding activity of quisqualic acid to the receptors would be stronger than those of the L-BHGA stereoisomers. GABA, glycine and L-homocysteic acid showed an inward current (Iin) on this neurone type, in contrast to the Iout caused by L-BHGA. beta-Alanine and taurine had absolutely no effect. Therefore, no amino acid inhibitory neurotransmitter candidate was found for this neurone type except for L-BHGA. It was assumed that L-BHGA, in either threo-or erythro-configuration, would be an inhibitory neurotransmitter for this neurone type. Mammalian L-Glu receptor antagonists. D(-)-AP-5, (+/-)-CPP, CNQX and L(+)-AP-3, applied by perfusion, showed no effect on the Iout of v-LCDN caused by threo-L-BHGA, indicating that the features of the inhibitory receptor activated by L-BHGA were much different from those of any type of the mammalian L-Glu receptors. Among the inhibitors of ATP-sensitive K+ channel, glipizide significantly inhibited the Iout caused by threo-L-BHGA, whereas tolbutamide did not. Inhibitors of intracellular signal transduction systems, H-7, H-8, H-9, staurosporine, calphostin C, KT5823 and W-7, had no effect on the Iout caused by threo-L-BHGA, suggesting that the receptors activated by threo-L-BHGA would be ionotropic.     

4-Phenyl-4-oxo-butanoic acid derivatives inhibitors of kynurenine 3-hydroxylase

Kynurenine 3-hydroxylase (KYN 3-OHase) is a key enzyme in the kynurenine pathway of tryptophan degradation and its inhibition may be an effective mechanism for counteracting neuronal excitotoxic damage. We present here a new class of inhibitors derived from a structure-activity relationship (SAR) study of the benzoylalanine side-chain of 1. 2-hydroxy-4-(3,4-dichlorophenyl)-4-oxobutanoic acid (8) and 2-benzyl-4-(3,4-dichlorophenyl)-4-oxo-butanoic acid (10) emerged as the most interesting derivatives. Enantiospecific synthesis for both enantiomers of 8 and diastereomeric salt resolution for those of 10 were successfully applied.     

Variation in organophosphate and carbamate insecticide resistance in relation to esterase activity in Culex (c.) quinquefasciatus Say, 1823 (Diptera: Culicidae)

We determined the existence of the resistance mechanism by means of the esterase enzymes in a Culex (C) quinquefasciatus stock established in the laboratory. Bioassays were performed with insecticides such as malathion and temephos (organophosphoric) and propoxur (carbomate); two cycles were completed during one year. Resistance to malathion was higher. The presence of esterase enzymes in this stock was determined by using synergits and starch gel and paper filter electrophoresis techniques.     

Stereoselective metabolism, pharmacokinetics and biliary elimination of phenylethylhydantoin (Nirvanol) in the dog

The influence of stereoisomerism on pharmacokinetics and rates of hepatic drug metabolism was investigated in four dogs using the enantiomers of phenylethylhydantoin (PEH) as model substances. After single i.v. administration of 98 micromoles of the pure enantiomers per kg b.wt., concentrations were measured by gas-liquid chromatography. The l-form exhibited a longer plasma half-life (23.3 +/- S.E. 1.0 hour) than the d-form (16.3 +/- 1.0 hour, P less than .005). Volumes of distribution and renal clearances were practically identical. The differences in plasma half-lives of PEH were explained by stereoselectivity of hepatic hydroxylation: an approximately 10-fold differences was found in urinary excretion of their major metabolities, d- and l-hydroxyphenylethylhydantoin (HPEH). Furthermore, in bile 7.3 +/- 1.6 mumol of of d-HPEH were eliminated within the first 6 hours, whereas l-HPEH could not be detected. The preference in biliary output of d- compared with l-PEH is consistent with the idea that both hepatic uptake and microsomal hydroxylation of PEH contribute to the high degree of stereoselectivity. In view of similar extrahepatic, but different metabolic behavior of these enantiomers, they represent an interesting research tool for in vivo studies of drug metabolism: in otherwise identical conditions, two different rates of PEH hydroxylation may be studied.     

Analysis of the metabolic and ventilatory response to self-paced 12-minute treadmill walking in patients with severe chronic obstructive pulmonary disease

Esterases in normal hamster pancreas and pancreatic tubular adenocarcinoma of ductal origin induced by N-nitrosobis(2-oxopropyl)amine were stained in cryostat sections with mixtures of a diazonium salt (fast blue RR) and with each of the enantiomers of alpha-naphthyl N-methoxycarbonylalaninate, N-methoxycarbonylvalinate, and N-acetylprolinate. Azo coupling of alpha-naphthol formed by enzymatic hydrolysis with the diazonium salt gives an azo dye that indicates the presence and amount of the enzyme activity in situ. Comparison between the color intensities obtained with each of the enantiomers of a chiral alpha-naphthyl ester shows the stereoselectivity, or enantiomeric preference, of the enzyme. Esterases in acinar cells of the normal pancreas showed slight stereoselectivity for N-methoxycarbonylalaninate, while esterases in fat cells scattered throughout the exocrine pancreas showed high stereoselectivity for (R)-N-acetylprolinate. These esterase activities were not found in the tumor, but another prominent esterase activity with high stereoselectivity for (S)-N-methoxycarbonylvalinate was found. Similar results were obtained by staining after polyacrylamide gel electrophoresis showing that the bands of esterases in the adenocarcinoma stained only with the S enantiomer of the N-methoxycarbonylvalinate. The present method is a valuable tool for designing anticancer prodrugs that are activated by tumor-specific esterases.     

Pharmacokinetic stereoselectivity of troglitazone, an antidiabetic agent, in the KK mouse

Troglitazone, an oral antidiabetic agent, is an equal mixture of four stereoisomers involving two asymmetric centres. In the present study, the stereoselectivity of in vitro epimerization in plasma and organ homogenate and in vivo plasma disposition in the KK mouse, an animal model of non-insulin-dependent diabetes, was examined. In the incubation experiments at 37 degrees C, there was a fivefold to eightfold acceleration of epimerization at the 5 position of the thiazolidine ring in KK mouse plasma compared with that in buffer. However, no inversion at the 2 position of the chroman ring was observed. In addition, there was an approximately 1.3-fold difference in the epimerization rates among stereoisomers at the 2 position of the chroman ring. However, there were no differences in the values of the equilibrium constants of epimerization, and the ratio of epimerization among stereoisomers at the 5 position of thiazolidine ring was almost unity. The acceleration of epimerization is thought to be due to the high degree of protein binding because of the relationship between the initial epimerization rate and the dilution ratio of the plasma. Although acceleration of epimerization was also observed in the 20% homogenates of liver, kidney, and intestine of the KK mouse, the degree of stereoselectivity was lower than in plasma. The analysis of the plasma disposition after intravenous administration of troglitazone stereoisomers, using a kinetic model, indicated that the metabolic clearance in the liver showed a 2.5-fold maximum difference among stereoisomers and that the stereoselectivity of epimerization was low.