Structures of the 5-HT2A receptor in complex with the antipsychotics risperidone and zotepine.

Many drugs target the serotonin 2A receptor (5-HT2AR), including second-generation antipsychotics that also target the dopamine D2 receptor (D2R). These drugs often produce severe side effects due to non-selective binding to other aminergic receptors. Here, we report the structures of human 5-HT2AR in complex with the second-generation antipsychotics risperidone and zotepine. These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket, the movements of which are important for receptor activation. 5-HT2AR is structurally similar to 5-HT2CR but possesses a unique side-extended cavity near the orthosteric binding site. A docking study and mutagenic studies suggest that a highly 5-HT2AR-selective antagonist binds the side-extended cavity. The conformation of the ligand-binding pocket in 5-HT2AR significantly differs around extracellular loops 1 and 2 from that in D2R. These findings are beneficial for the rational design of safer antipsychotics and 5-HT2AR-selective drugs.

Synthesis of targeted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines as potential lead molecules with promising anti-breast cancer activity.

A targeted library of substituted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines (prototypes I, II and III), and structurally analogous to tamoxifen have been synthesized as a new class of anti-breast cancer agents. All the prototype molecules exhibited potential antiproliferative activity against ER + ve and ER-ve breast cancer cell lines. Dibenzo[b,f]thiepine prototypes were found to be more active. Of all the compound tested, 14b exhibited potent in-vitro antiproliferative activity at 1.33 µM and 5 µM concentration in MCF-7 and MDA-MB-231 cell lines and was devoid of any cytotoxicity in normal HEK cells even at 50 µM. Cell cycle analysis showed that the compound 14b inhibited cell proliferation due to G0/G1 arrest in MCF-7 cells. Annexin-V FITC and PI staining experiments confirmed that the cell inhibition was primarily due to apoptosis and not by necrosis, which was also supported by LDH release assay experiment. Molecular docking studies showed better binding interaction of the new dibenzo[b,f]thiepine analogue 14b with the estrogen receptor (ER) as compared to 4-hydroxy-tamoxifen and this enhanced binding might be responsible for its estrogen antagonistic activity that induces cell cycle arrest, apoptosis and inhibition of breast cancer cells.

XenoSite server: a web-available site of metabolism prediction tool.

UNLABELLED: Cytochrome P450 enzymes (P450s) are metabolic enzymes that process the majority of FDA-approved, small-molecule drugs. Understanding how these enzymes modify molecule structure is key to the development of safe, effective drugs. XenoSite server is an online implementation of the XenoSite, a recently published computational model for P450 metabolism. XenoSite predicts which atomic sites of a molecule--sites of metabolism (SOMs)--are modified by P450s. XenoSite server accepts input in common chemical file formats including SDF and SMILES and provides tools for visualizing the likelihood that each atomic site is a site of metabolism for a variety of important P450s, as well as a flat file download of SOM predictions. AVAILABILITY AND IMPLEMENTATION: XenoSite server is available at http://swami.wustl.edu/xenosite.

Radiolabelling and PET brain imaging of the α1-adrenoceptor antagonist Lu AE43936.

Cerebral α1-adrenoceptors are a common target for many antipsychotic drugs. Thus, access to positron emission tomography (PET) brain imaging of α1-adrenoceptors could make important contributions to the understanding of psychotic disorders as well as to the pharmacokinetics and occupancy of drugs targeting the α1-adrenoceptors. However, so far no suitable PET radioligand has been developed for brain imaging of α1-adrenoceptors. Here, we report the synthesis of both enantiomers of the desmethyl precursors of the high affinity α1-adrenoceptor ligand (1). The two enantiomers of 1 were subsequently [¹¹C] radiolabelled and evaluated for brain uptake and binding by PET imaging in Danish Landrace pigs. (S)-[¹¹C]-1 and (R)-[¹¹C]-1 showed very limited brain uptake. Pre-treatment with cyclosporine A (CsA) resulted in a large increase in brain uptake, indicating that (R)-[¹¹C]-1 is a substrate for active efflux-transporters. This was confirmed in Madin Darby canine kidney (MDCK) cells overexpressing permeability glycoprotein (Pgp). In conclusion, the limited brain uptake of both (S)-[¹¹C]-1 and (R)-[¹¹C]-1 in the pig brain necessitates the search for alternative radioligands for in vivo PET brain imaging of α1-adrenoceptors.

Identification of cytochrome P450 enzymes involved in the metabolism of zotepine, an antipsychotic drug, in human liver microsomes.

1. Studies using human liver microsomes and recombinant human cytochrome P450 (P450) enzymes and flavin-containing monooxygenase (FMO) were performed to identify the enzymes responsible for the formation of zotepine metabolites in man. 2. Human liver microsomes produced four metabolites and a tentative order of importance was: norzotepine, 3-hydroxyzotepine, zotepine S-oxide and 2-hydroxyzotepine. Zotepine N-oxide was also detected, but it could not be quantified. 3. The rates of formation of the major metabolite, norzotepine, and zotepine S-oxide (at a substrate concentration of 20 microM) were significantly correlated with the testosterone 6beta-hydroxylase activities and CYP3A4 contents of the 12 different human liver microsomal samples. Inhibition studies with P450 enzyme selective inhibitors and anti-rat CYP3A2 antibodies also indicated a predominant role of CYP3A4 in the formation of norzotepine and zotepine S-oxide. Furafylline and sulphaphenazole inhibited the N-demethylation of zotepine by up to approximately 30%. 4. Correlation and inhibition data for the 2- and 3-hydroxylation of zotepine were consistent with the predominant role of CYP1A2 and 2D6 in the formation of these metabolites, respectively. 5. Recombinant CYP1A1, 1A2, 2B6, 2C19, 3A4 and 3A5 efficiently catalysed N-demethylation of zotepine. CYP1A1, 1A2, 2B6 and 3A4 were also active for S-oxidation. CYP1A2 and 2D6*1-Val374 efficiently produced 2-hydroxyzotepine and 3-hydroxyzotepine, respectively. Recombinant human FMO3 did not catalyse zotepine S-oxidation. 6. These results suggest that both the N-demethylation and S-oxidation of zotepine are mediated mainly by CYP3A4, and that CYP1A2 and 2D6 play an important role in the 2- and 3-hydroxylation of zotepine, respectively.

Pharmacological profile of neuroleptics at human monoamine transporters.

Using radioligand binding techniques, we determined the equilibrium dissociation constants (K(D)) for 37 neuroleptics and one metabolite of a neuroleptic (haloperidol metabolite) for the human serotonin, norepinephrine, and dopamine transporters with [3H]imipramine, [3H]nisoxetine, and [3H]WIN35428, respectively. Among neuroleptics, the four most potent compounds at the human serotonin transporter were triflupromazine, fluperlapine, chlorpromazine, and ziprasidone (K(D) 24-39 nM); and at the norepinephrine transporter, chlorpromazine, zotepine, chlorprothixene, and promazine (K(D) 19-25 nM). At the human dopamine transporter, only pimozide (K(D) = 69+/-3) ziprasidone (K(D) = 76+/-5) had notable potency. These data may be useful in predicting therapeutic and adverse effects, including drug interactions of neuroleptics.

The moderate affinity of clozapine at H3 receptors is not shared by its two major metabolites and by structurally related and unrelated atypical neuroleptics.

We determined the affinity and/or potency of two metabolites of clozapine (clozapine-N-oxide and N-desmethylclozapine) and of five atypical neuroleptics, chemically related (olanzapine) or unrelated to clozapine (remoxipride, risperidone, thioridazine, zotepine), at H3 receptors. The specific binding of 3H-N alpha-methylhistamine to rat brain cortex homogenates was inhibited by the seven compounds; the pKi values were: N-desmethylclozapine (5.33); clozapine-N-oxide (4.18); olanzapine (5.45); thioridazine (4.91); zotepine (4.75); remoxipride (4.51) and risperidone (4.43). Three compounds were examined in a functional H3 receptor model as well. The electrically evoked tritium overflow from superfused mouse brain cortex slices, which represents quasi-physiological noradrenaline release, was not affected by N-desmethylclozapine (3.2 and 10 microM), clozapine-N-oxide (3.2-100 microM) and olanzapine (3.2-32 microM). On the other hand, the three compounds shifted to the right the concentration-response curve of histamine for its inhibitory effect on the evoked overflow; the apparent pA2 values were 5.84, 4.21 and 5.80, respectively. The present study shows that five atypical neuroleptics of different chemical classes and the two major metabolites of clozapine possess a lower affinity and/or antagonistic potency at H3 receptors than clozapine itself (pKi 6.15, pA2 6.33; Kathmann M, Schlicker E, Göthert M (1994). Psychopharmacology 116: 464-468).

[Metabolic N- and S-oxygenation of dokloxytepin in vitro]. / Metabolická N- a S-oxygenace dokloxytepinu in vitro.

Dokloxytepin in the medium of the induced monooxygenase system of the microsomal fraction of the liver of the rat, rabbit and mice is metabolized into three metabolites: two identical, i.e., N-oxide and 5-sulfoxide, and a third different one. In the rat and rabbit it is the hitherto unknown metabolite M1, and in the mouse S,N-dioxide of dokloxytepin. The metabolites were identified by thin-layer chromatography by comparing with synthetic standards.

[The effect of cofactors on microsomal S-oxygenation of oxyprothepine]. / Vplyv kofaktorov na mikrozomálnu S-oxygenáciu oxyprotepínu.

Oxyprothepine in the medium of the microsomal fraction of the rat liver is biotransformed to the corresponding metabolites originating by oxidation of both sulfur atoms. The formation of S-oxygenation products is bound to the presence of NADPH, with a marked synergism with NADH. Induction with phenobarbital increases the formation of the individual metabolites.

Inhibition of post-decapitation convulsions in the rat by dibenzothiepin neuroleptics via alpha 1-adrenoceptor blockade.

The mechanisms involved in inhibitory effects of isofloxythepin, a newly synthesized dibenzothiepin neuroleptic, on post-decapitation convulsions were studied in rats. Isofloxythepin (0.05-2.0 mg/kg s.c.) inhibited post-decapitation convulsions in a dose-dependent manner as shown by the decrease in the incidence and the shortening of the duration of convulsions. The convulsions were also inhibited by oxyprothepin, zotepine or chlorpromazine but not by haloperidol. Prazosin and bunazosin, both alpha 1-adrenoceptor antagonists, suppressed the post-decapitation convulsions but a non-selective alpha 2-adrenoceptor agonist, tolazoline, was without effect. The convulsions were inhibited dose dependently by clonidine, an alpha 2-adrenoceptor agonist, but were prolonged in duration by yohimbine, an alpha 2-adrenoceptor antagonist. Yohimbine antagonized the inhibitory effects of isofloxythepin, prazosin and clonidine. The noradrenaline-induced contraction of rat vas deferens was inhibited by isofloxythepin, prazosin or chlorpromazine. Isofloxythepin bound to alpha 1-receptors as did chlorpromazine in the rat brain cortex. The results imply that post-decapitation convulsions seem to be inhibited by a block of postsynaptic alpha 1-adrenoceptors, enhanced by a block of presynaptic alpha 2-adrenoceptors and reduced by isofloxythepin via the blocking of postsynaptic alpha 1-adrenoceptors. The convulsions thus could serve as a good model for studying the actions of drugs on the central nervous system alpha-adrenoceptors.

Simultaneous determination of tropatepine and its major metabolites by high-performance liquid chromatographic-mass spectrometric identification. Application to metabolic and kinetic studies.

Tropatepine is used to combat against extrapyramidal syndrome induced by neuroleptic drugs. A high-performance liquid chromatographic method was proposed for the simultaneous determination of tropatepine and its potential metabolites in biological fluids. After double extraction of compounds in hexane and back-extraction in hydrochloric acid, the chromatographic separation was performed on a reversed-phase column with an acetonitrile--perchlorate buffer mixture as mobile phase. Compounds were detected at 229 nm and the detection limit was about 15 ng/ml. The method was applied to bile and urine samples collected in rats, after a single high oral dose of 100 mg/kg of tropatepine hydrochloride. Gas chromatography-mass spectrometry was used for identification of the potential metabolites. Nortropatepine and tropatepine S-oxide were identified in this way, and it seemed that tropatepine was subjected to a large and intense metabolic process. The analytical procedure and the results of the metabolic investigation were applied to a preliminary pharmacokinetic study in patients undergoing long-term oral therapy with tropatepine.

Pharmacokinetics of dothiepin in humans: a single dose dose-proportionality study.

Dothiepin hydrochloride (N,N-dimethyldibenzo[b,e]thiepin-delta 11(6 H), gamma-propylamine hydrochloride) is a tricyclic antidepressant which is structurally similar to amitriptyline. Twenty-seven healthy men received three single oral doses of 50-, 100-, and 150-mg dothiepin hydrochloride capsules in a three-way randomized, crossover dose-proportionality study. Plasma concentration-time profiles of dothiepin (1) were described by both one- and two-compartment models with first-order absorption. The total intrinsic clearance of dothiepin decreased from 165.5 to 121.1 L/h as the dose was increased from 50 to 150 mg, but there was no significant effect on the terminal half-life (approximately 20 h). Plasma concentration-time profiles of the three major metabolites of dothiepin, the S-oxide derivative of dothiepin, N,N-dimethyl[b,e]thiepin-delta 11(6 H), gamma-propylamine 5-oxide (2), the demethyl derivative, N-methyldibenzo[b,e]thiepin-delta 11(6 H), gamma-propylamine (3) and the demethyl S-oxide derivative N-methyldibenzo[b,e]thiepin-delta 11(6 H), gamma-propylamine 5-oxide (4), were described by a one-compartment model with apparent first-order formation. The AUC infinity values of the S-oxide 2 and the demethyl S-oxide 4 increased proportionally with dose. The dose proportionality of the demethyl metabolite 3 may not be ascertained from the data in this study. The corresponding half-lives of the three metabolites, which are dose independent, were approximately 24, 28, and 40 h, respectively.