11Beta-hydroxysteroid dehydrogenase type 1: purification from human liver and characterization as carbonyl reductase of xenobiotics.

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of 11-oxo glucocorticoids to their 11-hydroxy metabolites, thereby controlling access of glucocorticoid hormones to the glucocorticoid receptor. Interestingly, evidence is emerging that 11beta-HSD1 fulfills an additional role in the metabolism of xenobiotic carbonyl compounds. In our studies, 11beta-HSD1 was identified as a microsomal reductase that initiates the final detoxification of xenobiotics by reducing them to alcohols that are easier to conjugate and eliminate. With its pluripotent substrate specificities for glucocorticoids and xenobiotics, 11beta-HSD1 adds to an expanding list of those hydroxysteroid dehydrogenases which, on the one hand, are capable of catalyzing the carbonyl reduction of non-steroidal carbonyl compounds, and which, on the other hand, exhibit great specificity to their physiological steroid substrates. It is conceivable that large interferences must occur between endogenous steroid metabolism and the detoxification of xenobiotic compounds on the level of hydroxysteroid dehydrogenases.

Use of chiral liquid chromatography for the evaluation of stereospecificity in the carbonyl reduction of potential benzo[c]fluorene antineoplastics benfluron and dimefluron in various species.

Benfluron (B) [5-(2-dimethylaminoethoxy)-7H-benzo[c]fluorene-7-one hydrochloride] is a potential antineoplastic agent. In the organism, B undergoes a rapid phase I biotransformation through oxidative and reductive metabolic pathways. The carbonyl reduction of B leads to reduced benfluron, red-B, this is one of the principal pathways for the deactivation of this compound. The structure of B was modified to suppress its rapid deactivation via the carbonyl reduction on C7. Dimefluron, D (3,9-dimethoxy-benfluron) is one of the derivatives of B, in which an alternative metabolic pathway (O-desmethylation) prevails over the carbonyl reduction. The goal of this study was to develop HPLC methods enabling chiral separations of the red-B and -D enantiomers. The separation of red-B enantiomers was successful done on a Chiralcel OD-R column (250 mm x 4.6 mm ID, 5 microm) using a mobile phase acetonitrile-1 M NaClO4 (40:60, v/v). Another mobile phase, methanol-1 M NaClO4 (75:25, v/v), had to be employed for the sufficient resolution of red-D enantiomers. Flow rate was 0.5 ml min(-1) in both cases. Red-B was detected at 340 nm, red-D at 370 nm. The above chiral HPLC methods were used for the study of the biotransformation of B and D in the microsomal fractions of liver homogenates prepared from various species (rat, rabbit, pig, guinea pig, goat and human). The enantiospecificity of the respective carbonyl reductases was evaluated and discussed for both prochiral compounds, B and D.

Albendazole repeated administration induces cytochromes P4501A and accelerates albendazole deactivation in mouflon (Ovis musimon).

Adult mouflon ewes (Ovis musimon) were treated repeatedly with therapeutic doses of albendazole (ABZ, p.o. 7.5 mg/kg of body weight/day, for five consecutive days). Animals (treated or control) were sacrificed 24 h after the fifth dose of ABZ and liver and small intestine were collected to prepare microsomes. The activities of several biotransformation enzymes were measured in both hepatic and intestinal microsomes. A significant increase in the activity and amount of cytochromes P4501A (CYP1A) was observed in both tissues of ABZ treated mouflons compared to control animals. No other biotransformation enzymes tested were affected by five ABZ doses. The in vitro biotransformation of ABZ was studied in hepatic and intestinal microsomes from ABZ treated and control mouflons. Concentrations of two main ABZ metabolites - pharmacologically active ABZ sulfoxide and pharmacologically inactive ABZ sulfone were analysed using HPLC. A significant increase in rate of formation of ABZ sulfone (which is catalysed by CYP1A) was observed in hepatic as well as in intestinal microsomes from ABZ treated animals. The enhancement of ABZ deactivation by its repeated administration may affect the anthelmintic efficacy of this drug and may contribute to the development of parasite resistance.

Chiral inversion of drugs: coincidence or principle?

2-arylpropionic acid derivatives are probably the most frequently cited drugs exhibiting the phenomenon that is best known as chiral inversion. One enantiomer of drug is converted into its antipode either in the presence of a solvent or more often in inner environment of an organism. Mechanistic studies of the metabolic chiral inversion were carried out for several drugs from NSAIDs, and a model of this inversion was suggested and subsequently confirmed. The chiral inversion of NSAIDs has been intensively studied in the context of the pharmacological and toxicological consequences. However, the group of NSAIDs is not the sole group of drugs in which the inversion phenomenon can be observed. There exist several other drugs that also display chiral inversion of one or even both of their enantiomers. These drugs belong to different pharmacotherapeutic groups as monoamine oxidase inhibitors, antiepileptic drugs, drugs used in the treatment of hyperlipoproteinemia or drugs that are effective in the treatment of leprosy. Moreover, some chiral or prochiral drugs are metabolized to give chiral metabolites that undergo chiral inversion too, which can have direct impact on pharmacological properties or toxicity of the drug. As the process of chiral inversion is affected by several factors, so the intensity of chiral inversion of individual substances and at different conditions can differ considerably. Interspecies differences and types of tissue are reported to be the main factors that were recognized to play the key role in the process of chiral inversion. Some of more recent studies have revealed that several other factors, such as the route of administration or interaction with other xenobiotics, can influence the enantiomeric conversion, too. Chiral inversion does not seem to be a phenomenon connected with only several drugs from some unique group of 2-arylpropionic acid derivatives: it is also observed in drugs with rather different chemical structures and is much more frequent than it can be realized.

3-Phenyl-5-acyloxymethyl-2H,5H-furan-2-ones: synthesis and biological activity of a novel group of potential antifungal drugs.

3-(Substituted phenyl)-5-acyloxymethyl-2H,5H-furan-2-ones related to the natural product (-)incrustoporine were synthesized and their in vitro antifungal activity evaluated. The compounds with halogen substituents on the phenyl ring displayed much higher antifungal effect against Aspergillus fumigatus than selected representatives of azole antifungal drugs. In particular, the activity (1.34 microg/mL) of the most promising derivative, 3-(3,4-dichlorophenyl)-5-pivaloyloxymethyl-2H,5H-furan-2-one, was comparable to that of amphotericin B (0.5 microg/mL). Preliminary evaluation of the toxicity of the compound was carried out as well. Considering the size and properties of these molecules in comparison with those of amphotericin B, further development of this novel group of antifungals may lead to substances with better pharmacological profiles than that of the standard anti-Aspergillus drug.

High-performance liquid chromatography study of stereospecific microsomal enzymes catalysing the reduction of a potential cytostatic drug, oracin. Interspecies comparison.

One of the main metabolites of oracin (I) ¿6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2- c] isoquinoline¿, a potential cytostatic drug, is 11-dihydrooracin (II) ¿(+),(-)-6-[2-(2-hydroxyethyl)aminoethyl]-5-oxo-11-hydroxy-5,6-dihydro-1 1H- indeno[1,2-c]isoquinoline¿, a metabolite formed by the reduction of oracin's pro-chiral centre on C 11. This metabolite has been found in all laboratory species in vitro and in vivo and it constitutes the main metabolite in man. The stereospecificity of reducing enzymes participating in the oracin biotransformation pathway was investigated using microsomal preparations from standard laboratory animals. Enzyme stereospecificity has been defined as preferential formation by the enzyme of the (+) or (-) stereoisomer of II. Significant interspecies differences were observed in the stereospecificity of the respective biotransformation enzymes. HPLC quantitative determinations of both enantiomers were performed using a Chiralcel OD-R column as chiral stationary phase with excellent resolution and stability.

Effect of substituents on microsomal reduction of benzo(c)fluorene N-oxides.

The potential benzo(c)fluorene antineoplastic agent benfluron (B) displays high activity against a broad spectrum of experimental tumours in vitro and in vivo. In order to suppress some of its undesirable properties, its structure has been modified. Benfluron N-oxide (B N-oxide) is one of benfluron derivatives tested. The main metabolic pathway of B N-oxide is its reduction to tertiary amine B. A key role of cytochrome P4502B and P4502E1 in B N-oxide reduction has been proposed in the rat. Surprisingly, B N-oxide is reduced also in the presence of oxygen although all other N-oxides undergo reduction only under anaerobic conditions. With the aim to determine the influence of the N-oxide chemical structure and its redox potential on reductase affinity, activity and oxygen sensitivity five relative benzo(c)fluorene N-oxides were prepared. A correlation between the redox potential measured and the non-enzymatic reduction ability of the substrate was found, but no effect of the redox potential on reductase activity was observed. Microsomal reductases display a high affinity to B N-oxide (apparent K(m) congruent with0. 2 mM). A modification of the side-chain or nitrogen substituents has led to only a little change in apparent K(m) values, but a methoxy group substitution on the benzo(c)fluorene moiety induced a significant K(m) increase (ten-fold). Based on kinetic study results, the scheme of mechanism of cytochrome P450 mediated benzo(c)fluorene N-oxides reduction have been proposed. All benzo(c)fluorene N-oxides under study were able to be reduced in the presence of oxygen. Changes in the B N-oxide structure caused an extent of anaerobic conditions preference. The relationship between the benzo(c)fluorene N-oxide structure and the profile of metabolites in microsomal incubation was studied and important differences in the formation of individual N-oxide metabolites were found.

Effect of ivermectin on activities of cytochrome P450 isoenzymes in mouflon (Ovis musimon) and fallow deer (Dama dama).

Ivermectin is an antiparasitic drug widely used in veterinary and human medicine. We have found earlier that repeated treatments of rats with high doses of this drug led to significant increase of cytochrome P450-dependent 7-methoxyresorufin O-demethylase (MROD) and 7-ethoxyresorufin O-deethylase (EROD) activities in hepatic microsomes. In the present study, the effects of ivermectin on cytochrome P450 (CYP) activities were investigated in mouflon (Ovis musimon) and fallow deer (Dama dama). This study was conducted also to point out general lack of information on both basal levels of CYP enzymes and their inducibilities by veterinary drugs in wild ruminants. Liver microsomes were prepared from control animals, mouflons, after single or repeated (six doses in six consecutive days) treatments with therapeutic doses of ivermectin (0.5 mg kg(-1) of body weight), and fallow deer exposed to repeated doses of ivermectin under the same conditions. Alkyloxyresorufins, testosterone and chlorzoxazone were used as the specific substrate probes of activities of the CYP isoenzymes. A single therapeutic dose of ivermectin significantly induced (300-400% of the control group) the activities of all alkyloxyresorufin dealkylases tested in mouflon liver microsomes. Repeated doses of ivermectin also caused an increase of these activities, but due to fair inter-individual differences, this increase was not significant. The administration of ivermectin led to an induction (170-210% of the control) of the testosterone 6beta- and 16alpha-hydroxylase activities in mouflon liver but no significant modulation of chlorzoxazone hydroxylase (CZXOH) activity was found in mouflon liver. CYP-dependent activities in hepatic microsomes were generally higher in fallow deer than in mouflons. However, with the exception of slight increase in the 7-benzyloxyresorufin O-dealkylase (BROD) activities, no significant modulation of the other activities was observed. The induction of CYP3A-like isoenzyme was confirmed by immunoblotting only in the microsomes from mouflons administered with repeated doses of ivermectin; however, no significant increase of CYP1A isoenzymes was observed due to a weak cross-reactivity of anti-rat CYP1A1/2 polyclonal antibodies used in the study. The results indicate that ivermectin should be considered as an inducer of several cytochrome P450 isoenzymes, including CYP1A, 2B and 3A subfamilies, in mouflons. The comparison of induction effect of ivermectin in rat, mouflon and fallow deer also demonstrates the inter-species differences in inducibility of CYP enzymes.

Reduction of the potential anticancer drug oracin in the rat liver in-vitro.

Studies on the metabolism of the potential cytostatic drug oracin have shown that a principal metabolite of oracin is 11-dihydrooracin (DHO). We conducted in-vitro experiments to investigate the extent of oracin carbonyl reduction in microsomal or cytosolic fractions and to find out the enzymes involved under these conditions. Among several inducers of rat cytochrome P450 only 3-methylcholanthrene caused a significant (P < 0.01) stimulation (1.9 times) of DHO production in microsomal fraction and the specific P4501A inhibitor alpha-naphthoflavone significantly (P < 0.01) decreased (twice) the induced reduction activity. Cytochrome P4501A participates in oracin reduction in microsomes. 18beta-Glycyrrhetinic acid, a specific inhibitor of hydroxysteroid dehydrogenase, significantly (P < 0.01) inhibited the production of DHO in the microsomal fraction (>95% inhibition) in comparison with the non-inhibited reaction. Statistically significant (P < 0.01) inhibition (95%) of DHO formation was caused by metyrapone, which is also the substrate of 11-hydroxysteroid dehydrogenase. The main microsomal enzyme which catalyses the carbonyl reduction of oracin is probably 11beta-hydroxysteroid dehydrogenase. Important oracin reduction to DHO in the cytosolic fraction was found. According to its specific sensitivity towards quercitrin (inhibition by 99%, P < 0.01), the enzyme responsible for DHO formation in the rat liver cytosol is postulated to be carbonyl reductase.

Induction and inhibition of cytochrome P450-catalysed reduction of biologically active benfluron N-oxide.

Benfluron N-oxide [5-(2-N-oxo-2-N,N'-dimethylaminoethoxy)-7-oxo-7-H-benzo[c]fluorene] is a biologically active substance which displays a cytostatic effect on several experimental tumour cells. The main metabolic pathway of benfluron N-oxide in vitro and in vitro--its reduction to the parent tertiary amine benfluron--and the role of cytochrome P450 in this reduction were studied. The value of the benfluron N-oxide/benfluron redox potential as a criterion of suitability of the substrate for cytochrome P450 reductase activity was determined. Results of induction and inhibition studies on rats suggest that cytochromes P4502B and P4502E1 participate in microsomal reduction of benfluron N-oxide. Unlike most cytochrome P450 catalysed reactions, the reduction of benfluron N-oxide also occurs under aerobic conditions. Microsomes induced by phenobarbital, ethanol or beta-naphthoflavone showed no significantly greater inhibitory effect of oxygen on benfluron N-oxide reduction.

Inter-species comparison of microsomal reductive transformation of biologically active benfluron N-oxide.

Benfluron N-oxide is an anti-neoplastic active metabolite of benfluron (B) /1/. It is generated by flavine-monooxygenase-catalysed reactions /2/ and immediately undergoes subsequent metabolic transformations, the most important of which are reductive reactions /3/. The products of reductive pathways catalysed by two different microsomal enzymatic systems are the tertiary amine benfluron (i.e. the original parent compound) and/or 7-dihydrobenfluron N-oxide. Our studies on the reductive transformation of B N-oxide in rat, mouse, guinea-pig, rabbit, mini-pig and human microsomes have revealed significant species differences both in the yields of respective reduced metabolites and in the conditions essential for the activity of the reductases involved. While B, the original tertiary amine, is the main product of aerobic incubation of B N-oxide with NADPH in rat, mouse and mini-pig, significantly higher activities of the enzymes catalysing the formation of 7-dihydro-B N-oxide have been detected in rabbit and human microsomes. In rat, mouse and mini-pig, NADPH rather than NADH is the preferred coenzyme for B formation, and NADPH is also the preferred coenzyme for the formation of 7-dihydro-B N-oxide in most of the species used. The yield of tertiary amine B is higher in anaerobic rather than aerobic conditions in most experimental species studied. Aerobic or anaerobic incubating conditions have an insignificant effect on the formation of 7-dihydro-B N-oxide. Based on the inhibitory effect of CO on the reductive transformation of B N-oxide, cytochromes P450 can be assumed to participate in the formation of B both in rat and mini-pig, and, in mini-pig only, also in the formation of 7-dihydro-B N-oxide. Inter-species comparison of the properties of the reductases participating in the transformation of B N-oxide shows that the rabbit is a suitable model to study reductive transformation of B N-oxide in man.

Comparison of in vitro activities of biotransformation enzymes in pig, cattle, goat and sheep.

In vitro activities of cytochromes P450 (7-alkyl/aryloxyresorufin dealkyl(aryl)ases, testosterone hydroxylase/oxidase, 6-chlorzoxazone hydroxylase, 7-methoxy-4-trifluoromethyl-coumarin demethylase, and lauric acid hydroxylases), reductases of carbonyl group (toward metyrapone, daunorubicin, glyceraldehyde, and 4-pyridine-carboxaldehyde) and conjugation enzymes (p-nitrophenol-UDP-glucuronosyl transferase, 1-chloro-2,4-dinitrobenzene glutathione-S-tranferase) in young adults, males, non-castrated (N=6) farm animals were studied and compared. Presence of proteins cross-reacting with anti-human CYP3A4, CYP2C9, and CYP2E1 IgG was detected in all farm species. Bovine microsomes differed from other microsomes of farm species in very high 7-ethoxyresorufin-O-deethylase activity (CYP1A1/2). Significantly higher 7-methoxy-4-trifluoromethyl-coumarin demethylase (2-3 times) and 12-lauric acid hydroxylases (4-10 times) activities (probably corresponding to CYP2C and CYP4A, respectively) were found in ovine microsomes. The highest 6beta-testosterone hydroxylase activity, which is usually considered to be a CYP3A activity marker, was found in pig. Reductases of all farm animals display considerable ability to reduce carbonyl group of xenobiotics. Significant differences in level and activity of many biotransformation enzymes tested suggest that extrapolation of pharmacokinetic data obtained in one species to another (even related) could be misleading.

Role of cytochrome P4501A in biotransformation of the potential anticancer drug oracin.

Metabolic fate of the potential anticancer drug oracin (I), was studied at microsomal level in rat using enzyme induction and inhibition. One of the main metabolites arising during incubation of hepatic microsomal fraction with oracin is 3-hydroxyoracin (III). Cytochromes P450 non-specific inhibitors (carbon monoxide, aminobenzotriazole, 1-benzylimidazole, proadifen hydrochloride, n-octylamine) diminished amount of III. Among several specific inducers of rat cytochromes P450 isoforms used, only 3-methylcholanthrene, inducer of cytochrome P4501A, caused a significant stimulation of 3-hydroxyoracin production. The amount of III was decreased to the level of controls when the microsomes prepared from 3-methylcholanthrene treated rats were incubated with substrate in the presence of specific P4501A inhibitor alpha-naphthoflavone. From the above mentioned results we can assume that metabolite III is formed from oracin by cytochrome P450 belonging to subfamily 1A.

Use of rat hepatocytes immobilized in agarose gel threads for biosynthesis of metabolites of potential cytostatics.

The aim of this work was to evaluate possibility of use of the rat isolated hepatocytes immobilized in agarose gel and continuously perfused for production of needed metabolites of two potential cytostatics, benfluron (5-(2-dimethylamino-ethoxy)-7-oxo-7H-benzo[c]fluorene) and oracin (6-[2-(hydroxyethyl) amino-ethyl]-5,11-dioxo-5,6-dihydro-11H-indeno [1,2-c]isoquinoline). The rat isolated hepatocytes obtained by two-step collagenase perfusion method were immobilized in agarose threads and perfused in a small bioreactor under a recirculation regimes. Biosynthesis of 9-hydroxybenfluron and 3-hydroxyoracin in immobilized rat hepatocytes was studied. Yields of the metabolites of interest in hepatocytes in immobilized and perfused rat hepatocytes was compared to production of metabolites in hepatocyte suspension and in rats in vivo. 9-hydroxybenfluron was presented during perfusion of immobilized rat hepatocytes in a relatively high amounts but total recovery all forms of benfluron was very low due to especially high binding to components of the perfusion system. More effective method remains the production of 9-hydroxybenfluron in rats in vivo. A considerable biosynthesis of 3-hydroxyoracin by immobilized rat hepatocytes in the bioreactor was found. Concentration of the metabolite in the perfusate rose continuously during 6 hours of perfusion. 3-hydroxyoracin production was increased several times with use of immobilized hepatocytes from rats treated for three days with methylcholanthrene. The yield of 3-hydroxyoracin in rats in vivo was comparably high but an advantage of in vitro synthesis is a much shorter interval to obtain the same amount of the metabolite of interest. In spite of some limitations in compounds exerting high trapping in the perfusion system, the method of the immobilized and perfused hepatocytes can be very useful and effective for production of some drug metabolites in biochemistry and pharmacology.

Metabolic pathways of flobufen-a new antirheumatic and antiarthritic drug. Interspecies comparison.

Metabolic transformations of flobufen, [4-(2',4'-difluoro-biphenyl-4-y1)-4-oxo-2-methylbutanoic acid], a non-steroid antiinflammatory agent, were studied in vitro using the following biological models and species: rat and mouse liver homogenates and liver subcellular fractions (5 000 g and 100 000 g supernatant, mitochondria); rat, mouse, rabbit, guinea-pig and mini-pig liver microsomes; isolated rat hepatocytes; perfused rat liver and 5000 g rat muscle tissue supernatant. Reduced flobufen [4-(2',4'-difluorobiphenyl-4-yl)-4-hydroxy-2-methylbutanoic acid] is the major metabolite generated by the subcellular fractions (in the mild acidic extraction conditions during subsequent laboratory processing is converted to its lactone form). It was detected upon the incubation of flobufen with liver microsomes isolated from all the animals tested. Maximum yield of reduced flobufen in experiments with rat and mouse liver microsomes was found after anaerobic incubation with NADPH. This finding combined with the knowledge of subcellular distribution of enzymes suggest that metabolite formation depends on the activity of microsomal reductases and, probably, also on the activity of the important microsomal reductase, cytochrome P-450. Another flobufen metabolite, arylacetic acid [(2',4'-difluorobiphenyl-4-yl)ethanoic acid], is generated from the reduced metabolite by the cleavage of its side chain, and was detected in isolated hepatocytes - it was the only metabolite found in urine and faeces upon oral administration of the drug. All these metabolites were identified and quantified.

The potential of non-adrenergic, non-cholinergic targets in the treatment of interstitial cystitis/painful bladder syndrome.

Regulation of bladder function involves both divisions of the autonomic nervous system. However, in addition to the classical autonomic transmitters, noradrenaline and acetylcholine, other autonomic transmitters and other signalling components play important roles in physiology and pathophysiology of the lower urinary tract. Several substances of neuronal non-adrenergic, non-cholinergic (NANC) systems have already proven to considerably influence functional responses in the inflamed urinary bladder. Interstitial cystitis (IC) or painful bladder syndrome (PBS) is a chronic inflammatory bladder disease, characterized by urinary frequency, urgency and pelvic pain. IC/PBS is difficult to diagnose, especially because the etiology of the condition is largely unknown. Despite the unclear nature of the cause and manifestation of IC/PBS, it has been shown that the disease involves a significant NANC component. Here, we review the possible roles of ATP, adenosine, nitric oxide, vasoactive intestinal polypeptide, substance P, and pituitary adenylate cyclase-activating peptide in the contribution to IC/PBS development and manifestation of IC/PBS symptoms.

Coupled nitric oxide and autonomic receptor functional responses in the normal and inflamed urinary bladder of the rat.

Both divisions of the autonomic nervous system are involved in regulation of urinary bladder function. Several substances, other than noradrenaline and acetylcholine, seem to play important roles in physiology and pathophysiology of lower urinary tract. In the current study, we aimed to examine if there exist interplays between nitric oxide (NO) and autonomic transmitters and if such interactions vary in different parts of the urinary bladder in healthy and cyclophosphamide (CYP)-induced cystitic rats; when administered to the animals (100 mg/kg; i.p.), the cytotoxic CYP metabolite acrolein induces bladder inflammation. In the current study a series of in vitro functional studies were performed on detrusor muscle strip preparations. Stimulation with electrical field stimulation (EFS), methacholine, adenosine 5´-triphosphate (ATP), and adrenaline evoked contractile responses in isolated bladder preparations that were significantly reduced in cyclophosphamide (CYP)-treated rats. While the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA; 10(-4) M) did not affect contractile responses in normal, healthy strip preparations, it significantly increased the contractile responses to EFS, methacholine and adrenaline, but not to ATP, in the bladders from the CYP-treated rats. In the CYP-treated rats, the ATP-evoked relaxatory part of its dual response (an initial contraction followed by a relaxation) was 6-fold increased in comparison with that of normal preparations, whereas the isoprenaline relaxation was halved in the CYP-treated. While L-NNA (10(-4) M) had no effect on the isoprenaline-evoked relaxations, it reduced the ATP-evoked relaxations in strip preparations from the bladder body of CYP-treated rats. Stimulation of beta(2)- and beta(3)-adrenoceptors evoked relaxations and both responses were reduced in cystitis, the latter to a larger extent. In the trigone, the reduced ATP-evoked contractile response in the inflamed strips was increased by L-NNA, while L-NNA had no effect on the ATP-evoked relaxations, neither on the relaxations in healthy nor on the larger relaxations in the inflamed trigone. The study shows that both contractile and relaxatory functions are altered in the state of inflammation. The parasympathetic nerve-mediated contractions of the body of the bladder, evoked by the release of ATP and acetylcholine, were substantially reduced in cystitis. The relaxations to beta-adrenoceptor and purinoceptor stimulation were also reduced but only the ATP-evoked relaxation involved NO.

Liver microsomal biotransformation of albendazole in deer, cattle, sheep and pig and some related wild breeds.

Albendazole (ABZ) biotransformation was studied in vitro in liver microsomes of adult noncastrated male farm animals (ram, buck, bull and boar), castrated adult males (wether, billy and hog), and free living males (fallow buck, red deer stag, mouflon ram, roe buck and wild boar). Liver microsomal fractions were incubated with either ABZ or racemic albendazole sulphoxide (ABZSO). ABZ was extensively metabolized to the (+) and (-) enantiomers of ABZSO, whereas ABZSO underwent a slow oxidation to albendazole sulphone (ABZSO2) in all species. In all species both ABZSO enantiomers were detected. The chiral ratio, (+)-ABZSO/(-)-ABZSO, was greater than one in farm animals, mouflon and wild boar, and less than one in three species of deer. For total ABZ sulphoxidation, deer like species had lower values compared to the other species. Mouflon ram and ram had lower total sulphoxidation rates compared to wethers, as well as ABZ suphoxidation towards (+)-ABZSO. No significant difference occurred comparing ABZSO formation in mouflon ram and ram, but ABZSO2 formation rate in mouflon ram was higher than in rams and wethers. Roe deer stag, fallow buck and red deer stag did not differ in both total-ABZSO and (-)-ABZSO synthesis rates and roe deer stag and fallow buck did not differ in synthesis rates of (+)-ABZSO and ABZSO2. The bull differed from other species in all metabolites studied, except for red deer stag and boar in (-)-ABZSO synthesis rate. The extent of ABZSO sulphonation to ABZSO2 in bull microsomes was more than twice that of other species.

High-performance liquid chromatographic assay for the separation and characterization of metabolites of the potential cytostatic drug oracine.

Oracine (I), a potential cytostatic drug, is enzymically converted to a number of metabolites whose formation has been studied in vitro and in vivo. The metabolites were separated by reversed-phase HPLC and characterized by UV spectra. Preparative TLC served for the isolation of the individual metabolites to allow their identification. Two metabolites were identified by Fourier transform NMR as 11-dihydrooracine (II) and a phenolic product (III). Two further metabolites (IV,V) were characterized. Some minor, presumably 11-dihydro metabolites and an 11-oxo metabolite produced in vitro and in vivo were revealed.

A comparison between stereospecificity of oracin reduction and stereoselectivity of oxidation of 11-dihydrooracin enantiomers in vitro in rat and guinea pig.

11-dihydrooracin (DHO) arises from the potential cytostatic drug oracin through the metabolic conversion of its prochiral centre (C11). The participation of reduction enzymes on production of DHO enantiomer under various incubation conditions were tested in rat and guinea pig microsomal and cytosolic fractions. Interesting differences in stereospecificity of oracin reduction enzymes were found. Reduction stereospecificity was further studied on rat and guinea pig isolated hepatocytes. The enantiomers were detected in rat and guinea pig hepatocytes in the (+)/(-) ratio 63/37 and 32/68 respectively. As the differences in the amounts of DHO enantiomers can be caused not only by stereospecificity of oracin reduction but also by subsequent conversion of the enantiomer, stereoselectivity of DHO oxidation to oracin was investigated. Synthetically prepared pure (+)- and (-)-DHO were incubated with rat or guinea pig microsomes and cytosol and with various coenzymes under aerobic or anaerobic conditions. Significant oxidation of DHO to oracin was observed in rat microsomes. This oxidation depends on NADPH and O2 and is stereoselective for (+)-DHO. The formation of oracin in the guinea pig was greater in cytosol than microsomes, but no significant preference for a particular DHO enantiomer was found.