New approaches to augment fungal biotransformation

The possibility of using solid supports and intermittent substrate feeding to manipulate biotransformation by fungi was examined, with amoxapine as a model compound. Cunninghamella elegans ATCC 8688a grown as free cells in six-well plates showed 7-hydroxyamoxapine as the major metabolite of amoxapine biotransformation. However, when cells were grown in the presence of activated carbon, N-formyl-7-hydroxyamoxapine was formed as the major metabolite. Intermittent feeding of amoxapine also favored the formation of N-formyl-7-hydroxyamoxapine.     

Fungal Transformation of the Tricyclic Antidepressant Amoxapine: Identification of N-Carbomethoxy Compounds Formed as Artifacts by Phosgene in Chloroform used for the Extraction of Metabolites

The biotransformation of the antidepressant drug amoxapine by Cunninghamella elegans formed three metabolites, 7-hydroxyamoxapine, N-formyl-7-hydroxyamoxapine, and N-formylamoxapine; two other compounds were only present when chloroform was used in the extraction process. All five of the compounds were separated by reversed-phase HPLC, then analyzed by ¹H NMR and mass spectrometry, and by ¹³C NMR when sample quantities permitted. The artifacts were identified as N-carbomethoxy-7-hydroxyamoxapine and N-carbomethoxyamoxapine. Phosgene is a decomposition product of chloroform that can form carbomethoxy compounds at the secondary nitrogen of a piperazine ring in an alcoholic solution. Since N-carbomethoxy compounds were not observed when ethyl acetate was used for extraction of the culture medium, they were considered artifacts and not metabolites. These findings suggest that chloroform should be tested for the formation of phosgene before using it to extract any compound with a piperazine ring or any other amine-containing structure.     

Amoxapine inhibition of GABA-stimulated chloride conductance: investigations of potential sites of activity

Amoxapine inhibits GABA-stimulated chloride conductance by acting on the GABAA-receptor chloride-ionophore complex which can be studied using membrane vesicles prepared from rat cerebral cortex. Amoxapine produces a right shift in the GABA concentration-response curve for the stimulation of 36Cl- uptake into these vesicles with no apparent change in the maximum response. Schild analysis of these data gave a pA2 value of 5.52 with a slope of 0.79. Amoxapine inhibits the binding of the GABAA receptor selective antagonist [3H]SR 95531 with an IC50 value of 3.45 microM and a pseudo Hill coefficient of 0.83. In contrast, 10 microM amoxapine inhibits [3H]flunitrazepam binding by less than 25% while the benzodiazepine antagonist Ro 15-1788 reduces the amoxapine inhibition of GABA-stimulated chloride conductance only at high concentrations. These data suggest that amoxapine does not inhibit chloride conductance by acting as a benzodiazepine inverse agonist and either acts directly on the GABAA receptor as an antagonist or blocks GABA activity at a site closely coupled to it. The ability of amoxapine to inhibit GABA-stimulated chloride conductance is a likely explanation for its proconvulsant activity observed at high doses.     

Development and validation of a sensitive spectrofluorimetric method for the determination of amoxapine in human plasma and urine

A selective and sensitive spectrofluorimetric method was developed and validated for the determination of amoxapine in human plasma and urine. The developed method is based on labeling with 5‐dimethylaminonaphthalene‐1‐sulfonyl chloride (dansyl chloride) and monitoring at 397 nm (excitation)/514 nm (emission). The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, recovery and robustness. The calibration curves were linear over a concentration range of 250–2500 and 50–1250 ng/mL for plasma and urine, respectively. The LOD values were calculated to be 13.31 and 13.17 ng/mL for plasma and urine, respectively. The proposed method was applied to study of amoxapine in human plasma and urine. Copyright © 2013 John Wiley & Sons, Ltd.     

Unusual acute effects of antidepressants and neuroleptics on S2-serotonergic receptors

The antidepressants mianserin and amoxapine, and the neuroleptic loxapine caused significant decreases in the number of rat frontal cortex S2-serotonergic receptors after a single acute injection. The affinity of serotonin for this site was also decreased after acute mianserin. Daily injections of loxapine and amoxapine for 2, 7 or 28 days resulted in decreased receptor density but no change in Kd. Down-regulation of S2 sites by mianserin was not dependent on endogenous serotonin stores or occupation of the S2 recognition site since chronic PCPA or acute ketanserin preadministration did not affect the mianserin-induced decreases. The results suggest that mianserin may be acting on other sites which it does not share in common with other S2-antagonists such as ketanserin.     

Potential repurposing of known drugs as potent bacterial β-glucuronidase inhibitors

The active metabolite of the chemotherapeutic irinotecan, SN-38, is detoxified through glucuronidation and then excreted into the gastrointestinal tract. Intestinal bacteria convert the glucuronidated metabolite back to the toxic SN-38 using β-glucuronidase (GUS), resulting in debilitating diarrhea. Inhibiting GUS activity may relieve this side effect of irinotecan. In this study, we sought to determine whether any known drugs have GUS inhibitory activity. We screened a library of Food and Drug Administration-approved drugs with a cell-free biochemical enzyme assay using purified bacterial GUS. After triage, five drugs were confirmed to inhibit purified bacterial GUS. Three of these were the monoamine oxidase inhibitors nialamide, isocarboxazid, and phenelzine with average IC(50) values for inhibiting GUS of 71, 128, and 2300 nM, respectively. The tricyclic antidepressant amoxapine (IC(50) = 388 nM) and the antimalarial mefloquine (IC(50) = 1.2 μM) also had activity. Nialamide, isocarboxazid, and amoxapine had no significant activity against purified mammalian GUS but showed potent activity for inhibiting endogenous GUS activity in a cell-based assay using living intact Escherichia coli with average IC(50) values of 17, 336, and 119 nM, respectively. Thus, nialamide, isocarboxazid, and amoxapine have potential to be repurposed as therapeutics to reduce diarrhea associated with irinotecan chemotherapy and warrant further investigation for this use.     

Pharmacological properties of glycine uptake in the developing rat retina

A pharmacological characterization of glycine transport was performed in the rat retina at different postnatal ages. The uptake of 3H-glycine increased during the first 2 weeks of postnatal age, reaching maximum values at 12 days; then it decreased sharply to the adult values. We found a Na+ -dependent and high-affinity transport system with a Km of 100 microM. The Na+ Hill coefficient for glycine uptake was 1.76 +/- 0.07. Although glycine uptake was insensitive to staurosporine and phorbol ester, it was reduced 40-50% by sarcosine and ALX5407. Besides, amoxapine inhibited glycine uptake by 40 and 70% in adult and immature retina, respectively. These results suggest that the Glyt1 transporter was concentrated in the nerve terminals. In addition to the presence of Glyt1 in the retina, our results provided evidence of the occurrence of Glyt2 and/or another isoform of glycine transporter, which might have had a role in the retina development.     

Chemical composition and biological activity of Nepeta parnassica oils and isolated nepetalactones

Essential oils of Nepeta parnassica, collected at different developmental stages, were analyzed by means of GC/MS. From the fifty-five identified constituents in samples A and B, representing 94.8% and 98.7% of the oils respectively, 4a(alpha),7alpha,7alpha(beta)-nepetalactone (22.0%), 1,8-cineole (21.1%), alpha-pinene (9.5%) and 4a(alpha),7,beta,7alpha(beta)-nepetalactone (7.9%) were the major components of sample A (vegetative stage), whereas in sample B (flowering stage) the main contributors were 1,8-cineole (34.6%), 4a(alpha),7alpha,7a(alpha)-nepetalactone (17.3%), alpha-pinene (11.4%) and 4a(alpha),7alpha,7alpha(beta)-nepetalactone (8.9%). The oils were tested on human health important insects such as the Pogonomyrmex sp. ants and the Culex pipiens molestus mosquitoes with promising results on insect repellency/toxicity.     

Pharmacological Properties of Glycine Transport in the Frog Retina

The high-affinity glycine transport in neurons and glial cells is the primary means for inactivating synaptic glycine. Two different glycine transporter genes, Glyt-1 and Glyt-2, have been cloned. Glyt-1 has been reported to occur in the retina, but there is no evidence for expression of the Glyt-2 transporter. We have pharmacologically characterized glycine transport in the frog retina. 3H-Glycine uptake in the retina was insensitive to modulation by phorbol esters or changes in cAMP levels, and was partially inhibited by sarcosine. Differential sensitivity of glycine transport to sarcosine was exhibited by synaptosomal fractions from the inner and outer plexiform layers of the frog retina. The Na+ Hill coefficient of glycine uptake was 2.0, as has been reported for Glyt-2. In addition, amoxapine, a specific inhibitor of the Glyt-2a isoform, reduced by 60% glycine uptake by P2 synaptosomal fraction. Our results indicate the presence of different glycine transporter isoforms in the frog retina, acting mainly through the classical inhibitory glycine system.     

Column liquid chromatographic determination of clozapine and N-desmethylclozapine in human serum using solid-phase extraction

A 0.5-ml aliquot of a serum sample, after the addition of a 50-μl aliquot of a 5 μg/ml solution of amoxapine as the internal standard, is vortex-mixed with 0.5 ml of acetonitrile and centrifugated. The supernatant is applied to a 1-ml BondElut C₁₈ silica extraction column-conditioned with subsequent washings with 1 M HCl, methanol and water. After passing the sample at a slow rate, the column is washed twice with water and once with acetonitrile. The desired compounds are then eluted with a 0.25-ml aliquot of 35% perchloric acid-methanol (1:100, v/v). A 15-μl aliquot of the eluate is injected onto a 150 × 4.6 mm I.D. column packed with 5-μm C₈ silica particles and eluted at ambient temperature with a mobile phase of 0.1% tetramethylammonium perchlorate-acetonitrile (73:27, v/v) adjusted to pH 4.2 with 10% perchloric acid. The peaks are detected with an absorbance detector at 245 nm.     

Novel two-stage screening procedure leads to the identification of a new class of transfection enhancers

BACKGROUND: Non-viral gene transfer efficiency is low as compared to viral vector systems. Here we describe the discovery of new drugs that are capable of enhancing non-viral gene transfer into mammalian cells using a novel two-stage screening procedure. METHODS: First, potential candidates are preselected from a molecular library at various concentrations by a semi-automated yeast transfection screen (YTS). The maximal transfection efficiency of every positive drug is subsequently determined in independent experiments at the optimal concentration and compared to the inhibitory effect of the drug on cell growth (IC50). In a subsequent mammalian cell transfection screen (MTS), the maximal transfection efficiency and the IC50 are determined for all preselected drugs using a human cell line and a luciferase reporter gene construct. RESULTS: Employing our novel system we have been able to identify a new class of transfection enhancers, the tricyclic antidepressants (i.e. doxepin, maprotiline, desipramine and amoxapine). All positive drugs enhanced gene transfer in both yeast and human cell lines, but lower concentrations were sufficient for mammalian cells. With a triple combination of doxepin, amoxapine and chloroquine we obtained a transfection efficiency that exceeded that of chloroquine, one of the best-known transfection enhancers of mammalian cells, by nearly one order of magnitude. CONCLUSIONS: Non-viral gene transfer efficiency can be increased significantly using new transfection enhancers that are identified by a novel, semi-automated two-stage screening system employing yeast cells in the first and specific human target cells in the second round.     

Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 μm porous microspherical silica gel

A high-performance liquid chromatographic method has been developed for the forensic analysis of eleven frequently used cyclic antidepressant drugs (ADSs) (amitriptyline, amoxapine, clomipramine, desipramine, dosulepine, doxepin, imipramine, maprotiline, melitracen, mianserine and nortriptyline) using a recently developed reversed-phase column with 2 μm particles for the analysis of biological samples. The separation was carried out using two different C₈ reversed-phase columns (column 1: 100 mm × 4.6 mm I.D., particle size 2 μm, TSK gel Super-Octyl; column 2: 100 mm × 4.6 mm I.D., particle size 5 μm, Hypersil MOS-C₈) for comparison. The mobile phase was composed of methanol-20 mM KH₂PO₄ (pH 7) (60:40, v/v) and the flow-rate was 0.6 ml/min for both columns. The absorbance of the eluent was monitored at 254 nm. When the eleven drugs were determined, the sensitivity with the 2 μm particles was about five times greater than with the 5 μm particles. Retention times on column 1 were shorter than those on column 2. These results show that the new ODS column packing with a particle size of 2 μm gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.     

Preparation and biodegradability of chitin derivatives having mercapto groups

Cell walls of glasswort (Salicornia ramosissima Woods), a halophytic Chenopodiaceae, prepared as alcohol-insoluble solids, were found to be rich in arabinose, galacturonic acid, glucose and proteins, and contained 0·7% ferulic acid and 3·8% acetic acid. Pectic and hemicellulosic polysaccharides were extracted by cyclohexanediaminotetraacetic acid, hot dilute acid, cold dilute alkali and concentrated alkali (twice), with yields of 2·9, 19·1, 4·7, 7·4 and 1·9% of the alcohol-insoluble solids, respectively. Protein-rich material precipitated upon dialysis. The dialysed fractions were fractionated by ion-exchange chromatography, and the main fractions were analysed by gel-filtration and glycosyl linkage analysis. The hot acid extract contained 46·2% arabinose and 28·9% galacturonic acid, with high degrees of methylation and acetylation (65 and 45, respectively). It could be fractionated into a low-molecular-weight arabinan rich in ferulic acid, and a pectic fraction still relatively rich in neutral sugars. The concentrated alkali extracts were rich in xylose (33·4 and 23·6%, respectively). They were separated by ion-exchange chromatography into a fucogalactoxyloglucan and a glucuronoarabinoxylan.     

Assay of microsomal oxysterol 7alpha-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols

A method of assaying hepatic cytochrome P-450, oxysterol 7alpha-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27-(3)H]hydroxycholesterol as a substrate and hydroxypropyl-beta-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7alpha,25-dihydroxycholesterol, into 3-oxo-7alpha,25-dihydroxy-4-cholesten by the activity of 3beta-hydroxy-Delta(5)-C(27) steroid oxydoreductase, a microsomal NAD(+) and NADP(+) dependent enzyme of bile acid metabolism. A great improvement was reached by using a continuous NADPH generating system which constantly re-transforms NADP(+) into NADPH, thus inhibiting this activity. This improved CYP7B assay, comparable to our previously described assay for cholesterol 7alpha-hydroxylase (CYP7A), allowed a 3-fold increase of the apparent enzyme activity. The possibility to simultaneously measure CYP7A and CYP7B activities on the same microsomal preparation was investigated. A marked decrease (-33%) in the CYP7B activity was noticed, while that of CYP7A remained unchanged. The CYP7B activity was observed to be inhibited by cholesterol (-30%) and also by the oxysterols 7alpha-hydroxycholesterol (-21%), 7beta-hydroxycholesterol (-25%) and epicoprostanol (-20%), and by cyclosporin A (-26%). It can be concluded that this sensible and easy to perform CYP7B assay allows to observe, at least in vitro, a modulation of the enzyme activity by oxysterols.     

Isolation and biochemical studies of enriched populations of spermatogonia and early primary spermatocytes from rat testes

A method to obtain several highly enriched populations of testis cell types from rats of a single age is described. Single cell suspensions from immature rat testes were prepared after enzymatic removal of interstitial cells. Cells were separated on the basis of size into four fractions (bulk preparations) or eight fractions (analytical preparations) by centrifugal elutriation. These elutriator fractions were further separated by equilibrium density centrifugation in Percoll gradients. In this manner, populations of 2 X 10(7) type A spermatogonia (51% purity), 3 X 10(7) type B spermatogonia (76% purity), 5 X 10(7) zygotene/early pachytene spermatocytes (56% purity), 3 X 10(7) midpachytene spermatocytes (70% purity), and 4 X 10(7) Sertoli cells (89% purity) could be obtained from 50 immature rats within 6 h after killing. Purities, determined by examination of cytologic smears, were verified by Coulter volume and flow cytometric DNA determinations. These separation methods were used to obtain cell populations for characterization of levels and synthesis of high mobility group proteins in the early stages of spermatogenesis.     

Induction of detoxification enzymes by quercetin in the silkworm

Quercetin is one of the most abundant flavonoids and the defense secondary metabolites in plants. In this study, the effect of quercetin on the growth of the silkworm larvae was investigated. Cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), and carboxylesterases (COE) were assayed after exposure to different concentrations of quercetin for 3 d (short-term) and 7 d (long-term), respectively. The results showed that the weight gain of the silkworm larvae significantly decreased after the larvae were treated by different concentrations of quercetin except for the treatment with 0.5% quercetin. Activities of P450, GST, and COE were induced by 0.5 or 1% concentration of quercetin. In the midgut, the induction activity of P450s was reached to the highest level (2.3-fold) by 1% quercetin for 7 d, the highest induction activities of GSTs toward CHP and CDNB were 4.1-fold and 2.6-fold of controls by 1% quercetin after 7 d exposure, respectively. For COEs, the highest activity (2.3-fold) was induced by 0.5% quercetin for 7 d. However, P450s in whole body were higher inducible activities in short-term treatment than those in long-term treatment. The responses of eight cytochrome P450 (CYP) genes belonged to CYP6 and CYP9 families and seven GST genes were detected with real-time polymerase chain reaction. In addition, the genes induced by quercetin significantly were confirmed by qRT-PCR. CYP6AB5, CYP6B29, and GSTe8 were identified as inducible genes, of which the highest induction levels were 10.9-fold (0.5% quercetin for 7 d), 6.2-fold (1% quercetin for 7 d), and 7.1-fold (1% quercetin for 7 d), respectively.     

Iontophoretic studies on rat hippocampus with some novel GABA antagonists

Twelve substances which appear to be GABA antagonists, judging by their ability to reverse the inhibitory effect of GABA on 35S-TBPS binding to rat brain membranes, were tested iontophoretically on population spikes in the rat hippocampus. Eight of them, including seven which completely reversed the inhibitory action of GABA on 35S-TBPS binding, caused a marked enhancement of population spikes, with slow onset and long duration and they antagonized the inhibition of population spikes by GABA. These effects were similar to those produced by bicuculline. Electrophysiologically, the most potent of the "complete reversers" were bathophenanthroline disulfonate and brucine. In vitro, amoxapine and brucine most effectively reversed the inhibitory action of GABA on 35S-TBPS binding. Of the five substances which only partly reversed the inhibitory effect of GABA on 35S-TBPS binding, four depressed the population spikes and potentiated the inhibitory action of GABA. The fifth "partial reverser", pipazethate, potently increased the population spikes, like the "complete reversers". Although other interpretations are possible the results are consistent with the existence of several GABA-A receptor types in brain, only some of which are blocked by certain partial reversers.     

Further Characterization of 5-HT1A Receptors in the Goldfish Retina: Role of Cyclic AMP in the Regulation of the In Vitro Outgrowth of Retinal Explants

The presence of serotonin 5-HT_1A receptors and their physiological role were further characterized in the goldfish retina. The effects of the 5-HT_6/7 receptor antagonists pimozide, fluphenazine and amoxapine, the 5-HT_1A receptor antagonist WAY-100,135, and the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, on the 5-HT_1A receptor agonist [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes, were evaluated. In addition, the effects of serotonin, 8-hydroxy-2-(di-n-propylamino)tetralin, WAY-100,135, the adenylate cyclase inhibitors SQ22536 and MDL12330A, and the cyclic AMP analog 8-bromoadenosine-3:5 cyclic monophosphate were also studied on neuritic outgrowth from retinal explants. WAY-100,135 but not 5-HT_6/7receptor antagonists inhibited [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline decreased [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding sites up to 70%, while receptor turnover was similar to that reported in other tissues. Serotonin and 8-hydroxy-2-(di-n-propylamino)tetralin stimulated cyclic AMP production, both ex vivo and in vitro, and these increases were related to inhibition of neuritic outgrowth. The inhibitory effect was reduced by SQ22536 and by WAY-100,135, and was mimicked by 8-bromoadenosine-3:5cyclic monophosphate. This study supports previous findings about the role of serotonin as a regulator of axonal outgrowth during in vitro regeneration of the goldfish retina and demonstrates that this effect is mediated, at least in part, by 5-HT_1A receptors through a mechanism which involves an increase of cyclic AMP levels.     

Characterisation of the extractable pectins and hemicelluloses of the cell wall of glasswort, Salicornia ramosissima

Cell walls of glasswort (Salicornia ramosissima Woods), a halophytic Chenopodiaceae, prepared as alcohol-insoluble solids, were found to be rich in arabinose, galacturonic acid, glucose and proteins, and contained 0·7% ferulic acid and 3·8% acetic acid. Pectic and hemicellulosic polysaccharides were extracted by cyclohexanediaminotetraacetic acid, hot dilute acid, cold dilute alkali and concentrated alkali (twice), with yields of 2·9, 19·1, 4·7, 7·4 and 1·9% of the alcohol-insoluble solids, respectively. Protein-rich material precipitated upon dialysis. The dialysed fractions were fractionated by ion-exchange chromatography, and the main fractions were analysed by gel-filtration and glycosyl linkage analysis. The hot acid extract contained 46·2% arabinose and 28·9% galacturonic acid, with high degrees of methylation and acetylation (65 and 45, respectively). It could be fractionated into a low-molecular-weight arabinan rich in ferulic acid, and a pectic fraction still relatively rich in neutral sugars. The concentrated alkali extracts were rich in xylose (33·4 and 23·6%, respectively). They were separated by ion-exchange chromatography into a fucogalactoxyloglucan and a glucuronoarabinoxylan.