Pathogenomic in silico approach identifies NSP-A and Fe-IIISBP as possible drug targets in Neisseria Meningitidis MC58 and development of pharmacophores as novel therapeutic candidates.

Meningitis creates a life-threatening clinical crisis. Moreover, the administered antibiotics result into multi-drug resistance, thereby necessitating development of alternative therapeutic strategies. This study aimed at identifying novel-drug targets in Neisseria meningitidis and therapeutic molecules which can be exploited for the treatment of meningitis. Novel targets were identified by applying a pathogenomic approach involving protein data-set mining, subtractive channel analysis and subsequent qualitative analysis comprising of in silico pharmacokinetics, molecular docking and pharmacophore generation. Pathogenomic studies revealed Neisserial Surface Protein A (NSP-A) and Iron-III-Substrate Binding Protein (Fe-IIISBP) as potential targets. Two pharmacophore models comprising of 2-(biaryl) carbapenems, efavirenz, praziquantel and pyrimethamine for NSP-A and 2-(biaryl) carbapenems, trimipramine and pyrimethamine for Fe-IIISBP, showed successful docking, followed drug-likeness criteria and generated pharmacophore model with a score of 8.08 and 8.818, respectively, which had further been docked to the target stably. Thus, our study identifies NSP-A and Fe-IIISBP as novel targets in Neisseria meningitidis for which 2-(biaryl) carbapenems, efavirenz, praziquantel, trimipramine and pyrimethamine may be employed for effective treatment of meningitis.

Inhibitory potencies of trimipramine and its main metabolites at human monoamine and organic cation transporters.

RATIONALE: The antidepressant trimipramine shows an atypical pharmacological profile and its mechanism of action is still obscure. OBJECTIVES: The present study investigated whether trimipramine and three of its metabolites interact with targets of other antidepressants, namely, the human monoamine transporters for noradrenaline (hNAT), serotonin (hSERT), and dopamine (hDAT), and with the human organic cation transporters (hOCT1, hOCT2, and hOCT3) which are expressed in the brain and are known to be involved in the uptake of monoamines. METHODS: HEK293 cells heterologously expressing the abovementioned transporters were used to determine the inhibition of [(3)H]MPP(+) uptake by trimipramine and its main metabolites. RESULTS: At concentrations up to 30 µM, all transporters, except hOCT3, were inhibited by all examined substances. With IC(50) values between 2 and 10 µM, trimipramine inhibited hSERT, hNAT, hOCT1, and hOCT2, whereas clearly higher concentrations were needed for half-maximal inhibition of hDAT. Desmethyl-trimipramine showed about the same potencies as trimipramine, whereas 2-hydroxy-trimipramine was less potent at hNAT, hSERT, and hOCT1. Trimipramine-N-oxide preferentially inhibited hSERT. CONCLUSIONS: Neither trimipramine nor its metabolites are highly potent inhibitors of the examined monoamine transporters. However, since at a steady state the sum of the concentrations of the parent compound and its active metabolites is almost two times higher than the plasma concentration of trimipramine and since it is known that tricyclic antidepressants accumulate in the brain (up to tenfold), at least partial inhibition by trimipramine and its metabolites of hSERT and hNAT (but not of hOCT3) may contribute to the antidepressant action of trimipramine.

Role of human UGT2B10 in N-glucuronidation of tricyclic antidepressants, amitriptyline, imipramine, clomipramine, and trimipramine.

The role of human UDP glucuronosyltransferase (UGT) 2B10 in the N-glucuronidation of a number of tricyclic antidepressants was investigated and compared with that of UGT1A4 in both the Sf9 expressed system and human liver microsomes. The apparent K(m) (S(50)) values for the formation of quaternary N-glucuronides of amitriptyline, imipramine, clomipramine, and trimipramine were 2.60, 16.8, 14.4, and 11.2 microM in UGT2B10 and 448, 262, 112, and 258 microM in UGT1A4, respectively. The kinetics of amitriptyline and imipramine glucuronidation in human liver microsomes exhibited a biphasic character, where the high- and low-affinity components were in good agreement with our results in expressed UGT2B10 and UGT1A4, respectively. The kinetics of clomipramine and trimipramine glucuronidation in human liver microsomes were sigmoidal in nature, and the S(50) values were similar to those found for expressed UGT1A4. The in vitro clearances (CL(int) or CL(max)) were comparable between UGT2B10 and UGT1A4 for glucuronidation of imipramine, clomipramine, and trimipramine, whereas CL(int) of amitriptyline glucuronidation by UGT2B10 was more than 10-fold higher than that by UGT1A4. Nicotine was found to selectively inhibit UGT2B10 but not UGT1A4 activity. At a low tricyclic antidepressant concentration, nicotine inhibited their glucuronidation by 33 to 50% in human liver microsomes. Our results suggest that human UGT2B10 is a high-affinity enzyme for tricyclic antidepressant glucuronidation and is likely to be a major UGT isoform responsible for the glucuronidation of these drugs at therapeutic concentrations in vivo.

Receptor occupancy of mirtazapine determined by PET in healthy volunteers.

RATIONALE: Molecular tools are needed for assessing anti-depressant actions by positron emission tomography (PET) in the living human brain. OBJECTIVES: This study determined whether [(11)C]mirtazapine is an appropriate molecular tool for use with PET to estimate the magnitude of neuroreceptor occupancy produced by daily intake of mirtazapine. METHODS: This study used a randomised, double-blind, placebo-controlled, parallel-group, within-subject design. Eighteen healthy volunteers were PET-scanned twice with [(11)C]mirtazapine; once under baseline condition and again after receiving either placebo or mirtazapine (7.5 or 15 mg) for 5 days. We determined kinetic parameters of [(11)C]mirtazapine in brain regions by the simplified reference region method and used binding potential values to calculate receptor occupancy produced by mirtazapine. RESULTS: Serum concentrations of mirtazapine ranged from 33 to 56 nmol/l after five daily doses of 7.5 mg mirtazapine and were between 41 and 74 nmol/l after 15 mg mirtazapine. Placebo treatment failed to alter the binding potential of [(11)C]mirtazapine from baseline values, whereas daily intake of mirtazapine markedly decreased the binding potential in cortex, amygdala and hippocampus. Receptor occupancy ranged from 74 to 96% in high-binding regions of the brain after five daily doses of 7.5 mg or 15 mg mirtazapine, whereas 17-48% occupancy occurred in low-binding regions. CONCLUSIONS: [(11)C]Mirtazapine together with PET can determine the degree of receptor occupancy produced by daily doses of mirtazapine in regions of the living human brain.

Enantiomers' potential in psychopharmacology--a critical analysis with special emphasis on the antidepressant escitalopram.

Stereochemistry is now influencing most areas of pharmacotherapy, with a growing awareness in the field of psychiatry and, more specifically, depression. This is due to the fact that the enantiomers of many chiral drugs may have distinct pharmacological, pharmacokinetic and/or pharmacogenetic profiles. Consequently, in some instances there may be an advantage in using a single enantiomer over the racemic form-thus providing a basis for the development of new therapeutic agents, as well as the potential to improve current treatments. This review highlights some of the potential advantages and disadvantages that using single enantiomers might offer. The principles are exemplified through reference to the stereoselective properties of several established chiral psychotropic drugs, including thioridazine, methadone, trimipramine, mianserin, mirtazapine, fluoxetine and citalopram. Emphasis is given to the treatment of depression and how the potential of one pure enantiomer-escitalopram, the S-enantiomer of the selective serotonin reuptake inhibitor citalopram-appears to be fulfilling its preclinical promise in the clinic.

Steady state plasma levels of the enantiomers of trimipramine and of its metabolites in CYP2D6-, CYP2C19- and CYP3A4/5-phenotyped patients.

Steady state plasma concentrations of the (L)- and (D)-enantiomers of trimipramine (TRI), desmethyltrimipramine (DTRI), 2-hydroxytrimipramine (TRIOH) and 2-hydroxydesmethyl-trimipramine (DTRIOH) were measured in 27 patients receiving between 300 and 400 mg/day racemic TRI. The patients were phenotyped with dextromethorphan and mephenytoin, and the 8-hour urinary ratios of dextromethorphan/dextrorphan, dextromethorphan/3-methoxymorphinan, and (S)-mephenytoin/(R)mephenytoin were used as markers of cytochrome P-450IID6 (CYP2D6), CYP3A4/5 and CYP2C19 activities, respectively. One patient was a CYP2D6 and one was a CYP2C19 poor metabolizer. A stereoselectivity in the metabolism of TRI has been found, with a preferential N-demethylation of (D)-TRI and a preferential hydroxylation of (L)-TRI. CYP2D6 appears to be involved in the 2-hydroxylation of (L)-TRI, (L)DTRI and (D)-DTRI, but not of (D)-TRI, as significant correlations were measured between the dextromethorphan/dextrorphan ratios and the (L)-TRI/(L)-TRIOH (r = 0.45, p = 0.019), the (L)-DTRI/(L)-DTRIOH (r = 0.47, p = 0.014), and the (D)-DTRI/(D)-DTRIOH (r = 0.51, p = 0.006), but not with the (D)-TRI/(D)-TRIOH ratios (r = 0.29, NS). CYP2C19, but not CYP2D6, appears to be involved in the demethylation pathway, with a stereoselectivity toward the (D)-enantiomer of TRI, as a significant positive correlation was calculated between the mephenytoin (S)/(R) ratios and the concentrations to dose-to-weight ratios of (D)-TRI (r = 0.69, p = 0.00006). CYP3A4/5 appears to be involved in the metabolism of (L)-TRI to a presently not determined metabolite. The CYP2D6 poor metabolizer had the highest (L)-DTRI and (D)-DTRI concentrations to dose-to-weight ratios, and the CYP2C19 poor metabolizer had the highest (L)-TRI and (D)-TRI concentrations to dose-to-weight ratios of the group.

Metabolism of trimipramine in vitro by human CYP2D6 isozyme.

In vitro metabolism of the tricyclic antidepressant trimipramine using a commercial preparation of human CYP2D6 isozyme expressed in a human cell line is described. 2-Hydroxytrimipramine and a previously unreported metabolite, 2,10- or 2,11-dihydroxytrimipramine were isolated. Their structures were determined by gas chromatography/mass spectroscopy of underivatized and derivatized extracts. Acetylation of the new metabolite resulted in dehydration at C10 to give 10,11-dehydro-2-acetoxytrimipramine. No N-dealkylation of trimipramine was observed. Prior administration of quinidine produced a large reduction in the metabolic oxidation of trimipramine with CYP2D6 while prior administration of quinine had no effect. The use of this CYP2D6 isozyme preparation in vitro is of value in the identification of possible in vivo substrates for the human CYP2D6 isozyme.

Plasma levels of trimipramine and metabolites in four patients: determination of the enantiomer concentrations of the hydroxy metabolites.

A two-step high-performance liquid chromatography method is described, using a CN column and an alpha 1-acid glycoprotein column, which allows the measurement of the enantiomers of the hydroxy metabolites of trimipramine in plasma of trimipramine-treated patients. Of the four patients analyzed, three showed approximately equimolar concentrations of the (D)- and (L)-enantiomers of the hydroxy metabolites (2-hydroxy-trimipramine and 2-hydroxy desmethyltrimipramine), and one was found to have roughly twice as much of the (L)-form and of the (D)-form of 2-hydroxy trimipramine and 2-hydroxy desmethyltrimipramine. From the data available on the pharmacological effects of the enantiomers of trimipramine, it is postulated that this interindividual variability in its pharmacokinetics is another factor that could contribute to the interindividual variability in its pharmacodynamics.

Effect of iprindole on the metabolism of trimipramine in the rat.

Major metabolites of trimipramine in young male Sprague-Dawley rats are the result of alicyclic and aromatic ring oxidation. The four major urinary metabolites have been identified as 10-oxotrimipramine, 2-hydroxytrimipramine, 2-hydroxynortrimipramine, and 2-hydroxy-10-oxotrimipramine. When iprindole was administered to rats prior to trimipramine, the effect on trimipramine metabolism was profound. The formation of both 10-oxo metabolites was virtually completely inhibited; the production of 2-hydroxytrimipramine was significantly reduced while the metabolic formation of 2-hydroxynortrimipramine was increased. It is apparent from these preliminary results that metabolic alicyclic and aromatic hydroxylations are catalyzed by different cytochrome P450 isozymes and more than one P450 isozyme is involved in the aromatic ring oxidation of trimipramine and nortrimipramine.

Metabolism of antidepressants: urinary metabolites of trimipramine in the rat.

1. Studies on the metabolism of the tricyclic antidepressant trimipramine (TMP) in the rat are described. 2. Twenty metabolites of TMP were isolated from rat urine after enzymatic hydrolysis and their structures were determined by a gas chromatographic-mass spectrometric (GC-MS) method. 3. Twelve TMP metabolites were the result of alicyclic (C10 or C11) oxidation in addition to the other metabolic pathways.

The metabolism of trimipramine in the rat.

Studies on the metabolism of the tricyclic antidepressant trimipramine, 5-(3-dimethylamino-2-methylpropyl)-10,11-dihydro-5H-dibenz[b,f]azepine, in the rat are described. Many metabolites of trimipramine (TMP) were isolated from rat urine after enzymatic hydrolysis and their structures were identified by a gas chromatographic/mass spectrometric method, before and after appropriate chemical derivatization. Besides unchanged TMP, 20 metabolites were characterized (underivatized, and after acetylation), of which 12 had undergone alicyclic (C10 or C11) oxidation. This is a hitherto unreported metabolic pathway for TMP in the rat.

Metabolism of trimipramine in man.

This paper describes studies on the metabolism of the tricyclic antidepressant 5-(3-dimethylamino-2-methylpropyl)-10,11-dihydro-5H-dibenz[b,f]azepine (trimipramine, Stangyl) in man. The metabolites were identified after cleavage of conjugates, extraction and derivatisation by acetylation in human urine using gas chromatography-mass spectrometry. Besides the unchanged trimipramine (T), the following 15 metabolites could be identified: mono- and dihydroxy-T, hydroxy-methoxy-T; iminodibenzyl (I), mono- and dihydroxy-I, hydroxy-methoxy-I; nor-T (NT), mono- and dihydroxy-NT, hydroxy-methoxy-NT; bis-nor-T (BNT), mono- and dihydroxy-BNT and hydroxy-methoxy-BNT. Therefore 3 overlapping metabolic pathways can be postulated: N-dealkylation of the nitrogen in the iminodibenzyl ring, 1- and 2-fold N-demethylation of the nitrogen in the side chain and 1- and 2-fold aromatic hydroxylation of the iminodibenzyl ring. The dihydroxy metabolites are partly methylated at one of the hydroxy groups. The hydroxy metabolites are conjugated additionally.

[Drugs from the classes of tricyclic antidepressives and antiepileptics, nitrosatable under simulated human gastric conditions]. / Unter simulierten Bedingungen des menschlichen Magens nitrosierbare Arzneimittel aus den Gruppen der trizyklischen Antidepressiva und Antiepileptika.

The nitrosatability of Pryleugan (imipramine), Herphonal (trimipramine), and Finlepsin (carbamazepine) was investigated under simulated human gastric conditions using a colorimetric measuring method. All of them proved to be nitrosatable even at very low nitrite concentrations. In the presence of ascorbic acid, the formation of N-nitroso compounds under model conditions was inhibited markedly. N-nitroso-dihydrodibenzazepine and N-nitroso-dibenzazepine could be identified by thin layer chromatography as main products. The biological effects of these N-nitroso compounds are not known up to now.

[Microbial mutagenicity testing of N-nitrosoiminostilbene and N-nitrosoiminodibenzyl, the nitrosation products of the drugs carbamazepine and trimipramine hydrochloride]. / Mikrobielle Mutagenitätstestungen von N-Nitroso-iminostilben und N-Nitroso-iminodibenzyl, den Nitrosierungsprodukten der Arzneimittelwirkstoffe Carbamazepin und Trimipraminhydrochlorid.

The active agents of the drugs Finlepsin and Herphonal, carbamazepine and trimipramine, were nitrosated under simulated human gastric conditions. For both formed N-nitroso compounds, N-nitroso iminostilbene (N-nitroso-5H-dibenz(b,f)azepine) and N-nitroso iminodibenzyl (N-nitroso-10,11-dihydro-5H-dibenz(b,f)azepine), tests for mutagenic potency in the Ames-test gave negative results.

Types of interaction of amphiphilic drugs with phospholipid vesicles.

Binding characteristics of nine amphiphilic drugs, which induce pulmonary phospholipidosis, to L-alpha-dipalmitoyl phosphatidylcholine (DPPC) vesicles were studied using fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalene sulfonate (ANS) for hydrophobic and hydrophilic interactions, respectively. Drug binding to DPPC was quantitated using Scatchard analysis. The tested drugs bound to DPPC with different capacities. The order of binding capacity to hydrophobic site of DPPC using 1,6-diphenyl-1,3,5-hexatriene as fluorescence probe was promethazine greater than amiodarone greater than chlorpromazine greater than chloramphenicol greater than imipramine greater than trimipramine greater than propranolol much greater than chloroquine and chlorphentermine. Two binding affinities were evident for amiodarone, chlorpromazine, imipramine, trimipramine and promethazine. The order of binding strength at high affinity site was amiodarone greater than trimipramine greater than chlorpromazine greater than promethazine greater than imipramine. The order of drug binding capacity using ANS as fluorescence probe was chlorphentermine greater than trimipramine greater than propranolol much greater than amiodarone, chloroquine and chloramphenicol. Each of these drugs displayed a single binding affinity. Imipramine and chlorpromazine at 1 mM and higher concentrations showed intense fluorescence with ANS (5-20 microM) in the absence of DPPC indicating an interaction of these drugs with ANS. Chloroquine did not bind to either sites on DPPC. The binding of these drugs and their interactions with hydrophobic or hydrophilic sites of DPPC were correlated with their capacity to induce pulmonary phospholipidosis. These results indicate that not all the drugs which bind to DPPC in vitro induce phospholipidosis in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Selectivity in the binding of psychotropic drugs to the variants of alpha-1 acid glycoprotein.

The S- and F-forms of alpha-1 acid glycoprotein (AAG) variants have been isolated by isoelectric focusing with immobilines from commercially available AAG. In equilibrium dialysis experiments using a multicompartmental system, a higher affinity for various basic drugs has been found with S- in comparison with F-AAG: Amitriptyline, nortriptyline, imipramine, desipramine, trimipramine, methadone, thioridazine, clomipramine, desmethylclomipramine, and maprotiline. The selectivity (binding to S- vs. F-AAG) is the most pronounced for methadone and the lowest for thioridazine, while it is absent for the acidic drug mephenytoin.