Comparison of the toxicity of fluconazole and other azole antifungal drugs to murine and human granulocyte-macrophage progenitor cells in vitro.

We studied the inhibitory effects on colony formation by granulocyte-macrophage colony forming units (cfu-gm) of eight azole antifungal agents in vitro. All agents, except fluconazole, inhibited colony formation dose-dependently with 50% inhibitory concentrations (IC50) in the range of 0.78-49 micromol/L in cultures of murine and human bone marrow. For human cells, the IC50 values were 0.553 mg/L for itraconazole, 1.24 mg/L for saperconazole, 2.58 mg/L for clotrimazole, 5.33 mg/L for miconazole, 6.17 mg/L for econazole, 6.27 mg/L for ketoconazole and 8.38 mg/L for oxiconazole. The IC50 of itraconazole for human cfu-gm in vitro was similar to the plasma level of this drug recommended for systemic antifungal therapy (>0.5 mg/L) thus indicating the potential clinical relevance of our data. The IC50 of ketoconazole for human cfu-gm in vitro may be exceeded by plasma levels produced in vivo by high (> or =400 mg) doses, whereas fluconazole failed to reduce colony formation by 50% even at 100 mg/L, a concentration not reached in vivo even after extremely high doses (2000 mg/day). To most of the drugs studied, murine progenitor cells seemed to be less sensitive than the human ones. There was, however, a close correlation between the murine and human log IC50 values of the drugs (r2 = 0.964, P< 0.001), suggesting that cultures of murine bone marrow may be suitable to predict the in-vitro toxicity of azole antifungals to human cfu-gm.

Central benzodiazepine receptors: in vitro efficacies and potencies of 3-substituted 1,4-benzodiazepine stereoisomers.

3-Acyloxy-, 3-methoxy-, and 3-alkyl-substituted derivatives of the benzodiazepine (BZ) agonist desmethyl-diazepam (DMD) were resolved, and the stereochemical properties of binding to central BZ receptors were investigated in synaptosomal membrane preparations of rat brain. Decreasing potency and stereoselectivity of 3-methyl, 3-ethyl, and 3-isopropyl derivatives in displacement of [3H]diazepam binding can be attributed to differential susceptibilities for steric hindrance of 3-axial versus 3-equatorial substituents of the binding conformation M. Chirality in the alpha-methyl-beta-phenyl-propionic acyl moiety of oxazepam, the 3-OH-derivative of DMD, was noncritical in binding, whereas the beta-phenyl substituent selectively increased the binding of the 3S-stereoisomer. Changing the pH from 7.4 to 5.6 significantly increased the IC50 of (3R)-oxazepam acetate but not those of (3R)-methyl-DMD and diazepam. Binding data led to a steric model of the BZ binding site with the postulation of an additional hydrogen-bond-donating moiety, probably histidine in the "ceiling" of the receptor cavity, that binds the 3-carbonyloxy groups and hinders the 3-alkyl ones. In vitro efficacies of 3-substituted BZs were estimated by allosteric binding interactions within the gamma-aminobutyric acidA (GABAA) receptor-ionophore complex. Non-equilibrium enhancement of t-butyl-bicyclophosphoro[35S]thionate binding by the BZ agonist oxazepam was stereoselectively antagonized by (3S)-oxazepam-(S)-alpha-methyl-beta-phenyl-propionate, suggesting a mixed agonist-antagonist character. GABA enhanced the [3H]diazepam-displacing potencies of the 3S-enantiomers of the acetate, hemisuccinate, and (S)-alpha-methyl-beta-phenyl-propionate esters of oxazepam by a factor of about 1.5-1.6, whereas the GABA shifts for 3R-esters were about 1.2. UV affinity labeling with flunitrazepam resulted in a significantly smaller decrease in the displacing potency of (3R)-oxazepam acetate than in that of the 3S-enantiomer. GABA shifts of successively 3-methylated DMD derivatives were also compared. The GABA shifts of DMD and its (3S)-methyl and 3,3-dimethyl derivatives were all characteristic of full agonists (2.4-2.7), whereas that of (3R)-methyl-DMD was 1.5. The 3-methoxy enantiomers of DMD displayed stereoselectivity and GABA shift values intermediate between those of 3-methyl and 3-acetoxy derivatives. These allosteric interactions suggest that 3-carbonyloxy derivatives in general, as well as (3R)-BZ enantiomers bound with axial 3-alkyl and 3-alkyloxy groups, decrease the agonist efficacies of 1,4-BZs to modulate the GABAA receptor complex.

Demonstration of stereoselective disposition of warfarin enantiomers by whole-body autoradiography.

The oral anticoagulant warfarin and its 14C-labelled derivative are commercially available in racemic forms. Two methods for the chromatographic resolution of the radiolabel were used to investigate the distribution of radioactivity of radioactivity of individual enantiomers in the rat by whole-body autoradiography. Computer-assisted quantification of autoradiograms indicated the average disappearance of levo-warfarin and its metabolites to be substantially slower than that of dextro-warfarin and its metabolites from the following organs: liver, pancreas, kidney, lung, blood, intestines.

Studies on the pharmacokinetics and metabolism of N(4)-carbamoyl-1,3,4,5-dihydro-diazepam (Uxepam(R)) in rats, dogs and man.

The pharmacokinetics and metabolism of Uxepam(R), a new minor tranquillizer, have been investigated in rats [2], dogs and man. For the experiments in rats [2] separation of metabolites of 2-(14)C-uxepam was achieved by thin-layer chromatography. In the experiments on dogs and man, Extrelut microcolumns were used for preseparation. Recovery was 95% +/- 12.77 (S.D.) determined by radioactive tracer experiments. The compounds were separated and determined by reversed-phase HPLC with UV detection at 240 nm. The limit of detection for uxepam was 10 ng ml(-1). The metabolises were identified by mass spectrometry. The main metabolites in the rat were desmethyl-uxepam, 5-hydroxy-phenyl-desmethyl-uxepam and diazepam. Desmethyl-carbamoyl-dihydro-diazepam, diazepam and desmethyl-diazepam were found in human plasma. In dogs only one metabolite, desmethyl-uxepam, was detected in plasma. Enterohepatic recycling was observed in dogs and in humans.

Stereoselective binding of 3-acetoxy-, and 3-hydroxy-1,4-benzodiazepine-2-ones to human serum albumin. Selective allosteric interaction with warfarin enantiomers.

Stereoselective binding of oxazepam, lorazepam, temazepam and methyl lorazepam as well as of their acetates to human serum albumin was investigated by different techniques. The 2'-chlorine and the N(1)-methyl substitution exert opposite effects on the antipodes. Enantiomers of oxazepam acetate (OAc) and lorazepam acetate (LAc) displace diazepam. Allosteric interactions with warfarin were manifested by either mutually increased or decreased binding depending on the structure of benzodiazepine and on the configuration of both benzodiazepine and warfarin. The most remarkable effect could be observed in the simultaneous binding of (S)-lorazepam acetate and (S)-warfarin.

Oxazepam esters. 3. Intrinsic activity, selectivity, and prodrug effect.

Antimetrazol and muscle-relaxant activities of 11 aliphatic esters of oxazepam were studied as a function of time in mice. The esters given intravenously retained antimetrazol activity, while muscle-relaxant activity was generally decreased. The administration of a dose equivalent to the antimetrazol ED50 resulted in constant oxazepam brain levels for most esters; therefore, the intrinsic anticonvulsant activity of the intact ester is insignificant. The dimethylphenylpropionyl ester appeared to antagonize the effect of oxazepam, since it elevated the free oxazepam level required to achieve the ED50 in the antimetrazol assay. The administration of doses equivalent to the muscle-relaxant ED50 values resulted in no correlation with total brain benzodiazepine levels, suggesting that changes in the selectivity of action are the consequence of different sites of action.

Specific binding of racemic oxazepam esters to rat brain synaptosomes and the influence of bioactivation by esterases.

Six aliphatic esters of oxazepam were investigated for 3H-diazepam displacing activity and hydrolysis in rat in the brain P2-fraction treated with diisopropylfluorophosphate. The esters are prodrugs in relation to oxazepam. Increasing size of the 3-substituent and steric hindrance increased the value of IC50. Controversial reports on oxazepam hemisuccinate could be explained by its stereoselective hydrolysis in vivo.

Comparison of dihydrodiazepam enantiomers: metabolism, serum binding and brain receptor binding.

Dihydrodiazepam is a diazepam prodrug, as shown by its in vitro metabolism by rat and mouse liver and brain microsomal fractions, and its displacing activity on brain diazepam binding. The mechanism of bioactivation is discussed. Stereoselectivity of metabolism and of binding to specific benzodiazepine binding sites in brain synpatosomes and serum albumin were studied.

Structural parameters in the microsomal hydrolysis of 3-acyloxy-1, 4-benzodiazepines and the multiplicity of the esterases involved.

The biotransformation of several prodrug-type esters of centrally acting 1, 4-benzodiazepines was studied. Their rates of hydrolysis catalyzed by the hepatic microsomal fraction of mice were measured by pH-stat. The heterogeneity of the microsomal esterases was investigated with induction by phenobarbital and with inhibition by DFP. The resulting changes in esterase activity indicated that the phenyl-substituted esters separate from the homogenous sets of oxazepam and lorazepam esters. Regression analysis of the relative hydrolysis rates of the homogenous ester sets revealed a similar dependence on the steric ES the polar sigma* and hydrophobic deltaRM terms of the acyl moiety. The role of the polar term shows that a nucleophilic attack of the acyl moiety determines the hydrolysis. The role of hydrophobicity can be attributed to its interrelation with the steric parameter. The common equations for the aliphatic sters of oxazepam and larazepam suggest the similar nature of the esterases in question and the same catalytic mechanism. Different 3-acetoxy-1, 4-benzodiazepines were also synthetised and their maximal hydrolysis rates were quite different. This excludes the possibility that the deacylation step of the enzymes is rate-determining. Instead, our data suggest that acylation of the microsomal esterases is rate-limiting for the hydrolysis of the aliphatic esters of 3-OH-benzodiazepines.

Oxazepam esters. 1. Correlation between hydrolysis rates and brain appearance of oxazepam.

Esters of the centrally acting oxazepam were investigated to find quantitative correlations between the pharmacokinetics of the parent drug and in vitro biotransformation rates and physicochemical properties of its prodrugs. The 14C-labeled aliphatic and omega-phenyl-substituted esters were administered intravenously to mice. Brain levels of the esters and oxazepam were determined and the latter was fitted to a simplified exponential equation. In vitro hydrolysis rate of the esters catalyzed by the hepatic microsomal fraction was measured with a pH stat. Pharmacokinetic constants characterizing the rising part of oxazepam brain levels correlate well with the chromatographic RM values and with in vitro maximal hydrolysis rates of the esters. The hydrolysis is capacity limited in the liver. In a closely related set of aliphatic esters, oxazepam brain penetration also correlates with the steric constant (ES) of its esters.

Oxazepam esters. 2. Correlation of hydrophobicity with serum binding, brain penetration, and excretion.

Pharmacokinetics of a series of prodrug-type oxazepam esters were studied in mice. The effect of hydrophobicity was investigated in relation to serum binding, brain penetration, tissue storage, and excretion. Binding to mouse serum and to human serum albumin was measured by equilibrium dialysis, and the changes in binding free energy were correlated with RM values. Brain-blood partition of the esters did not change parallel with their serum binding. An indirect correlation exists between RM of the esters and oxazepam brain accrual. Brain-blood concentration ratios of oxazepam prove that hydrolysis precedes brain penetration and hydrophobicity might primarily influence the hydrolysis rate. The amount of tissue storage and total excretion rates also correlate with hydrophobicity.

Stereospecificity of esterases hydrolyzing oxazepam acetate.

Esterases hydrolyzing the racemic acetate ester of the centrally acting drug oxazepam in mice were examined. Radiolabeled ester administered intravenously was hydrolyzed rapidly in the liver, kidneys, and brain. The distribution of the enzyme activity of liver and brain subcellular fractions was measured. Kinetic data and structure investigation of partially hydrolyzed racemic ester pointed to the stereoselectivity of liver and brain esterases. The preferred hydrolysis of the (R)-(-)-isomer in liver homogenates was attributed mainly to microsomal enzymes, while that of the (S)-(+)-isomer in brain was considered to be due to the mitochondrial fraction. This phenomenon was a common property of all species tested.

Kinetic investigations of liver microsomal esterases with oxazepam esters.

Hepatic microsomal esterases of the mouse responsible for the bioactivation of inactive (prodrug) esters of the centrally acting oxazepam were studied. The enzymes are situated on the cytoplasmic side of the microsomes. The esterases are partly solubilized and partly inactivated by homogenization in aqueous glycerol and treatment with deoxycholate. There is good correlation between the rates of hydrolysis and steric constants of the acyl moiety. Substrate binding increases to an optimum with the number of carbon atoms in the acyl moiety and is of hydrophobic nature. An attempt has been made to classify the esterases on the basis of the effect of inhibitors and activators.

Metabolism of levorotary 4,5-dihydrodiazepam in the rat.

The metabolism of (-)-4,5-dihydrodiazepam (7-chloro-1,3,4,5-tetrahydro-1-methyl-5-phenyl-2H-1,4-benzo[2-14C]diazepin-2-one) (I) was investigated in rats. Metabolites from urine and bile extracts purified by thin-layer chromatography were identified by mass spectrometry. Diazepam and its main metabolites were found among the biotransformation products of I. Based on these findings, the most important identified metabolic route of the compound studied appears to be the formation of an unsaturated bond between the N4 and C5 atoms, and further transformation of the diazepam formed.