Synthesis, Characterization, and Biological Evaluation of Cu(II) Complexes Containing Triflupromazine with Glycine and Histidine.

Two novel copper (II) complexes [Cu(TFP)(Gly)Cl] ⋅ 2H2 O complex (1) and [Cu(TFP)(His)Cl] ⋅ 2H2 O complex (2) are synthesized, where TFP stands for trifluropromazine, Gly. represents glycine, and His. is histidine. Chemical composition, IR, mass spectra, and magnetic susceptibility tests are performed. Complex binding with macromolecules was investigated using UV-vis, viscosity, gel electrophoresis, and fluorescence quenching. Fluorescence spectroscopy revealed that each complex could replace ethidium bromide (EB). These complexes exhibit grooved, non-covalent, and electrostatic interactions with CT-DNA. Spectroscopy analysis of the BSA interaction showed that complexes bind to protein (Kb values for (1) is 5.89×103  M-1 and for (2) is 9.08×103  M-1 ) more strongly than CT-DNA (Kb values for (1) is 5.43×103  M-1 and for (2) is 7.17×103  M-1 ). Molecular docking analysis and spectral absorption measurements showed high agreement. Antimicrobial, antioxidant, and anti-inflammatory properties were tested in vitro. The druggability of complex (2) should be tested in vivo as it is more biologically active.

Inhibition of human UDP-glucuronosyltransferase enzyme by Dabrafenib: Implications for drug-drug interactions.

Dabrafenib is a novel small molecule tyrosine kinase inhibitor (TKI) which is used to treat metastatic melanoma. The aim of this research was to survey the effects of dabrafenib on human UDP-glucuronosyltransferases (UGTs) and to evaluate the risk of drug-drug interactions (DDIs). The formation rates for 4-methylumbelliferone (4-MU) glucuronide and trifluoperazine-glucuronide in 12 recombinant human UGT isoforms with or without dabrafenib were measured and HPLC was used to investigate the inhibitory effects of dabrafenib on UGTs. Inhibition kinetic studies were also conducted. In vitro-in vivo extrapolation approaches were further used to predict the risk of DDI potentials of dabrafenib via inhibition of UGTs. Our data indicated that dabrafenib had a broad inhibitory effect on 4-MU glucuronidation by inhibiting the activities of UGTs, especially on UGT1A1, UGT1A7, UGT1A8, and UGT1A9, and dabrafenib could increase the area under the curve of co-administered drugs. Dabrafenib is a strong inhibitor of several UGTs and the co-administration of dabrafenib with drugs primarily metabolized by UGT1A1, 1A7, 1A8 or 1A9 may induce potential DDIs.

High content phenotypic screening identifies serotonin receptor modulators with selective activity upon breast cancer cell cycle and cytokine signaling pathways.

Heterogeneity in disease mechanisms between genetically distinct patients contributes to high attrition rates in late stage clinical drug development. New personalized medicine strategies aim to identify predictive biomarkers which stratify patients most likely to respond to a particular therapy. However, for complex multifactorial diseases not characterized by a single genetic driver, empirical approaches to identifying predictive biomarkers and the most promising therapies for personalized medicine are required. In vitro pharmacogenomics seeks to correlate in vitro drug sensitivity testing across panels of genetically distinct cell models with genomic, gene expression or proteomic data to identify predictive biomarkers of drug response. However, the vast majority of in vitro pharmacogenomic studies performed to date are limited to dose-response screening upon a single viability assay endpoint. In this article we describe the application of multiparametric high content phenotypic screening and the theta comparative cell scoring method to quantify and rank compound hits, screened at a single concentration, which induce a broad variety of divergent phenotypic responses between distinct breast cancer cell lines. High content screening followed by transcriptomic pathway analysis identified serotonin receptor modulators which display selective activity upon breast cancer cell cycle and cytokine signaling pathways correlating with inhibition of cell growth and survival. These methods describe a new evidence-led approach to rapidly identify compounds which display distinct response between different cell types. The results presented also warrant further investigation of the selective activity of serotonin receptor modulators upon breast cancer cell growth and survival as a potential drug repurposing opportunity.

Structural requirements for the antitubercular quaternized triflupromazine pharmacophore.

Quaternized triflupromazine derivatives (QTDs) must possess benzyl groups attached to the quaternary nitrogen in order to have significant antitubercular potency. Replacing the quaternary amine with a triazole abolishes antitubercular activity. A modest halogen substitution effect exists, with the 4-bromophenyl QTD 3 having the best selectivity index (>21). All N-benzyl QTDs 1-4 similarly inhibit non-replicating, persistent Mycobacterium tuberculosis with MIC<8 µM, and compounds 1-3 were all nontoxic to mammalian cells in vitro (IC(50)>128 µM).

The identification of inhibitors of Schistosoma mansoni miracidial transformation by incorporating a medium-throughput small-molecule screen.

In Schistosoma mansoni, the miracidium-to-primary sporocyst transformation process is associated with many physiological, morphological, transcriptional and biochemical changes. In the present study, we use a medium-throughput small-molecule screen to identify chemical compounds inhibiting or delaying the in vitro transformation of miracidia to the sporocyst stage. The Sigma-Aldrich Library of Pharmacologically Active Compounds (LOPAC) contains 1280 well-characterized chemical compounds with various modes of action including enzyme inhibitors, antibiotics, cell-cycle regulators, apoptosis inducers and GPCR ligands. We identified 47 compounds that greatly reduce or delay this transformation process during a primary screen of live miracidia. The majority of compounds inhibiting larval transformation were from dopaminergic, serotonergic, ion channel and phosphorylation classes. Specifically, we found that dopamine D2-type antagonists, serotonin reuptake inhibitors, voltage-gated calcium channel antagonists and a PKC activator significantly reduced in vitro miracidial transformation rates. Many of the targets of these compounds regulate adenylyl cyclase activity, with the inhibition or activation of these targets resulting in increased cAMP levels in miracidia and concomitant blocking/delaying of larval transformation.

Monitoring the (photo)genotoxicity of photosensitizer drugs: direct quantitation of single-strand breaks in deoxyribonucleic acid using an oligonucleotide chip.

Oligonucleotide chip-based assays can be a sample-thrifty, time-saving, routine tool for evaluation of chemical-induced DNA strand breaks. This article describes a novel approach using an oligonucleotide chip to determine photosensitizer-induced DNA single-strand breaks. Surface coverage of fluorophore-labeled oligonucleotides on silicon dioxide chip surfaces was determined on alkaline phosphatase digestion. Fluorescence maxima (at 520 nm) of the solutions were converted to molar concentrations of the fluorescein-modified oligonucleotide by interpolation from a predetermined standard linear calibration curve. The photosensitizing activity of chlorpromazine and triflupromazine toward DNA single-strand breaks was then studied at different drug doses and also as a function of photoirradiation time. Photoinduced single-strand breaks calculated using the method described here agreed with values predicted by theoretical extrapolation of the single-strand breaks obtained for plasmid DNAs from agarose gel electrophoresis, and thereby indirectly validated the chip-based assays. Under UV irradiation (>or=93.6 kJ/m2) chlorpromazine (>or=0.08 mM) was found to have significant photogenotoxicity. However, triflupromazine did not exhibit any (photo)genotoxicity over the concentration range studied (0.04-0.20mM). The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of breaks in DNA.

Modification of the thermal unfolding pathways of myoglobin upon drug interaction in different aqueous media.

In this work, we have analyzed the influence of two structurally related phenothiazine drugs, promazine and triflupromazine hydrochlorides, when bound to myoglobin, a model protein, and how the drug concentration and solution conditions may affect the denaturation process of this protein. In this manner, we derive the thermodynamic quantities of the unfolding process by using a spectroscopic technique such as UV-vis spectroscopy at different drugs concentrations and at pH 2.5, 5.5, and 9.0. To do this, a thermodynamic model was used which included experimental data corresponding to the pre- and post-transition into the observable transition. It has been found that both drugs play a destabilizing role for the protein, at least at low concentrations. In addition, at acidic pH and higher drug concentrations, a stabilizing effect can be observed, which may be related to the formation of some type of protein refolding, subsequent aggregation, or both. The reason for this behavior has been suggested to be the different protein conformations at acidic pH, the increase of solvent-exposed hydrophobic and hydrophilic residues after denaturation and/or binding, and the different strength of drug-protein interactions when changing the solution conditions. For this reason, thermodynamic quantities such as Gibbs energies, DeltaG, and entropies of unfolding, DeltaS(m), increase as the solution pH increases provided that additional solvent-exposed hydrophobic residues are present, which were previously buried at room temperature. Moreover, the larger binding affinity at pH 9.0 due to enhanced electrostatic interactions between protein and drug molecules (drug and protein differ in their net electrical charge) additionally collaborates to this residue exposition to solvent as a consequence of the alteration of protein conformation as due to drug binding. Comparison of thermodynamic data between promazine and triflupromazine hydrochlorides also shows that drug-protein affinity and hydrophobicity also affect the thermodynamic denaturation parameters.

Dissociation constants of phenothiazine drugs incorporated in phosphatidylcholine bilayer of small unilamellar vesicles as determined by carbon-13 nuclear magnetic resonance spectrometric titration.

The dissociation constants (pKms) of the phenothiazine drugs promazine, chlorpromazine, and triflupromazine, incorporated in the phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUV), were investigated by a 13C nuclear magnetic resonance (NMR) titration method employing their N-13CH3 (ionizable group) labelled derivatives. Use of the labelled drugs enabled direct observations of the ionization equilibrium of the N-dimethyl group. A second derivative spectrophotometric study proved that 95-98% of the phenothiazine species in the sample solutions (200 microM phenothiazine in the presence of 27 mM PC SUV) were incorporated into the PC bilayer, which simplified the calculation of pKm values by allowing that the phenothiazines in the aqueous phase could be neglected. The pKm values were calculated from the chemical shift dependence of the N-dimethyl 13C NMR signal on the pH value of sample solutions. The pKm values obtained were smaller than those measured in aqueous solutions by about one unit. The existence of cholesterol (30 mol%) in the PC bilayer showed little effect on the pKm values, suggesting that cholesterol in the bilayer does not largely affect the interfacial region where the N-dimethyl group of the incorporated phenothiazines is located. The results offered clear evidence for the pKm decrease and provided their precise values.

Trans-cellular proliferating cell nuclear antigen gene activation in cerebral vascular smooth muscle by endothelial oxidative injury in vivo.

OBJECTIVE: This study was undertaken to assess the role of vascular smooth muscle cell (VSMC) Ca2+ channels and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in gene regulation after oxidative endothelial injury (OEI). METHODS AND RESULTS: OEI was produced by infusion of Na fluorescein (NaFluo) photoactivated by UV light immediately before intravenous injection. Posterior cerebral arteries were studied using immunofluorescence imaging, Western blotting, or patch clamping of isolated cells. After infusion of photoactivated NaFluo, but not NaFluo, (1) superoxide dismutase-1 (SOD-1) was upregulated in endothelium, consistent with oxidant stress; (2) the fraction of VSMC nuclei labeled for proliferating cell nuclear antigen (PCNA) increased 7-fold at 6 hours, preceded by a several-fold increase in nuclear phospho-cAMP-response element binding protein, with PCNA upregulation prevented by pretreatment with polyethylene glycol (PEG)-SOD; (3) in VSMCs, phospho-CaMKII increased 20-fold 5 minutes after OEI, with a 2-fold increase in peak Ca2+ channel currents; and (4) changes in cAMP-response element binding protein and PCNA were blocked by systemic administration of lipophilic (nifedipine) or hydrophilic (amlodipine) 1,4-dihydropyridine Ca2+ channel blockers, the calmodulin inhibitor trifluoperazine, or the CaMKII inhibitor KN-93, with none of these agents preventing SOD-1 upregulation in endothelium. CONCLUSIONS: Activation of VSMC Ca2+ channels and CaMKII is a key early signaling event required for upregulation of PCNA gene expression in VSMCs after oxidative injury to endothelium.

Calmodulin antagonists stimulate renin release from isolated rat glomeruli.

Effects of calmodulin antagonists on renin release from isolated rat glomeruli were examined. The calmodulin antagonists used were N-(6-aminohexyl)-5-chloro-naphthalene-1-sulfonamide (W-7), triflupromazine and trifluoperazine. These drugs induced renin release from isolated glomeruli in a dose-dependent manner. The threshold concentration for renin release in the calcium-containing medium was 50 microM for W-7, 5 microM for triflupromazine and 2 microM for trifluoperazine respectively. The threshold concentrations were 2-5 times less in the calcium-free medium. The maximum levels of renin release by the three antagonists were similar in both calcium-containing and calcium-free media. In the absence of these antagonists, the basal rate of renin release in the calcium-free medium was markedly higher than in the calcium-containing medium. These results suggest that the calcium-calmodulin system inhibits renin release and that renin release is regulated by a mechanism different from the calcium-stimulated exocytotic mechanism by which many hormones are released.

Acute exacerbation of paranoid psychotic state after long-term abstinence in patients with previous methamphetamine psychosis.

From a population of 111 patients with chronic methamphetamine (MAP) psychosis, who were treated at ten mental hospitals during the past 3 years, 21 patients were selected for study. Sixteen patients who experienced MAP psychosis again used MAP one or more times after long-term abstinence and experienced acute exacerbation of a paranoid psychotic state which was almost identical to the initial psychotic episode. Four of these patients relapsed following a single MAP reuse of an amount less than that initially used, and one relapsed without evidence of MAP reuse. In eight patients, small doses of neuroleptics, e.g., 3 mg per day of haloperidol, prevented the acute provocation of a psychotic state by MPA reuse. Subsequently, three of these relapsed into a psychotic state following MAP reuse without concurrent haloperidol medication. The clinical data were compared with animal experiments which indicate that chronic MAP use can induce a long-term susceptibility to sensitization to MAP. The positive prophylactic effect of small doses of haloperidol on the acute exacerbation may suggest the participation of dopaminergic supersensitivity as a mechanism for the paranoid psychotic state.

In vitro cross-linking of bovine lens proteins photosensitized by promazines.

Promazine derivatives induce cross-linking of bovine lens crystallins in vitro by irradiation with near-ultraviolet (UV) light in the presence of O2, as revealed by electrophoresis after denaturation. With the five derivatives tested (promazine [PZ], chlorpromazine [CPZ], triflupromazine [ TFPZ ], methoxypromazine [ MTPZ ], and acepromazine [ ACPZ ] ), single-hit kinetics are observed. Evidence implicating the cation radicals of the PZ derivatives as the causative agent of this in vitro effect is presented. Hydroxyl radicals do not appear to be involved in the photo-cross-linking reaction. Sodium ascorbate protects against damage induced either by PZ derivatives plus light or by PZ cation radicals in the dark. These findings are discussed with respect to development of cataracts induced by these drugs in vivo.

A kinetic study of the generation and decomposition of some phenothiazine free radicals formed during enzymatic oxidation of phenothiazines by peroxidase-hydrogen peroxide.

A kinetic study of the oxidation of four different phenothiazines (Pts) by peroxidase-hydrogen peroxide was carried out. The free radical formed during the enzymatic oxidation suffers a non-enzymatic breakdown and the overall system was analysed and characterized. The non-enzymatic breakdown of the cation radical does not occur through a disproportionation mechanism but through a more complex mechanism. The kinetic parameters of the overall system were determined for the different Pts. These experimental data may serve in the understanding of the pharmacological action of Pts.

Structure-activity relationships of phenothiazines in inhibiting lymphocyte motility as determined by a novel flow cytometric assay.

Lymphocyte motility is highly dependent on rapid changes in cell shape. The human T-lymphoma cell line, MOLT-4, is constitutively shape-changing and motile, and both of these properties can be inhibited by the phenothiazine, chlorpromazine, as assessed by video analysis and migration across polycarbonate filters. In this paper, the light-scattering facility of a flow cytometer has been used to establish a simpler and more quantitative means of measuring changes in shape. By this method, the structure activity relationship (SAR) of phenothiazines and related compounds has been determined. The most active compounds had the tricyclic phenothiazine nucleus with a constrained dialkylaminoalkyl substituent at the nitrogen. The SAR for inhibition of lymphocyte motility differs from those reported for neuroleptic effects and for inhibition of PKC or calmodulin. Phenothiazine concentrations that inhibited lymphocyte shape-changing resulted in reduced F-actin concentrations. This indicates that the probable mode of action is disruption of mechanisms regulating actin polymerisation.

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.

Quantitative NMR imaging study of the mechanism of drug release from swelling hydroxypropylmethylcellulose tablets.

NMR imaging has been used to study the release behavior of two model drugs, triflupromazine-HCl and 5-fluorouracil, from swelling hydroxypropylmethylcellulose (HPMC) tablets. Preliminary experiments were performed on equilibrium mixtures of drug, polymer and water to determine how properties such as NMR relaxation parameters and self-diffusion were affected by the drug and polymer concentrations. The tablet swelling was restricted to one dimension and distributions of the water and model drugs were obtained by 1H and 19F imaging, respectively. The HPMC distribution at each time in the swelling process was determined indirectly from its effect on the relaxation parameters of the water and the drugs. In the one-dimensional swelling tablet, distributions of drug and polymer were compared to determine what factors influenced the release of drug from the swelling tablet. The distributions for triflupromazine-HCl and HPMC paralleled each other and the drug was only released at the eroding edge of the tablet where the HPMC concentration dropped below 10%. In contrast, 5-fluorouracil was released much more rapidly from the tablet and appeared to escape by diffusion from regions as high as 30% HPMC. An empirical measure of the rate of tablet edge movement can be obtained from plots of the edge position as a function of the square root of time. For HPMC, the rate of tablet expansion was determined in this way to be (2.4+/-0.8)x10(-6) cm(2) s(-1). The self-diffusion of triflupromazine-HCl in equilibrated mixtures of similar composition to the eroding tablet edge is approximately 3x10(-6) cm(2) s(-1) while the self-diffusion coefficient of 5-fluorouracil remained higher than this value until the HPMC concentration reached about 30%. This comparison of 'diffusion' properties may be useful in predicting the mechanism of drug release from other swelling hydrophilic matrix systems.

Enchancement of the antiplaque value of antibacterial agents by enamel-conditioning methods. I. Rationale, mechanism, and initial findings.

A method is proposed to induce chemically the incorporation of bacterial agents inot enamel and thus rende this tissue resistant to bacterial colonization. It consists of promoting a slight initial dissolution of enamel, followed immediately by tis reprecipitation. During this latter phase, antibacterial agents included in the test formulations would become trapped in the reprecipitating enamel. That this happens is proved by the chemical detection of antibacterial agents in treated enamel, as well as by the development of a remarkable resistance of treated specimens to grow in vitro plaque.

The anti-emetic properties of 1-sulpiride in a ground-based model of space motion sickness.

L-sulpiride, at a dose of 4 mg/kg, essentially abolished motion-induced emesis in a group of 6 squirrel monkeys undergoing horizontal rotation at 25 rpm, a terrestrial model of space motion sickness (SMS). Extrapyramidal side effects were not observed. In the absence of the drug, the usual emetic response returned. In comparison while typical neuroleptics were also strongly anti-emetic, they produced a considerable degree of rigidity and akinesia.

The present lack of evidence for unique rodent germ-cell mutagens.

Six chemicals, diethylhexyl phthalate (DEHP), ethanol, cyclohexylamine (CHA), sodium saccharin (NaS), cadmium chloride (CdCl2) and triflupromazine (TFP), were suggested to be unique germ-cell mutagens (Auletta and Ashby, 1988) by the GeneTox Workgroups of the U.S. Environmental Protection Agency (EPA). If this is a correct classification it would have major consequences when screening for mutagenicity and when labelling genotoxic substances. However, our re-evaluation of the GeneTox literature, including some more recent publications, has failed to find substantive evidence that any of these chemicals have been unequivocally established as having unique mutagenic activity in germ cells. For DEHP, NaS and TFP the evidence for genotoxic/mutagenic effects is questionable, in both germinal and somatic cells. Ethanol and CdCl2 showed clastogenic activity, but it was not restricted to germ cells. Both, ethanol and cadmium salts, appear to induce aneuploidy. The unconfirmed clastogenic effect of CHA was restricted to rats, but it occurred in both bone marrow and spermatogonia. Therefore, the general observation that rodent germ-cell mutagens are also genotoxic in somatic cells in vivo (Brusick, 1980; Holden, 1982) remains valid.

Killing of endothelial cells and release of arachidonic acid. Synergistic effects among hydrogen peroxide, membrane-damaging agents, cationic substances, and proteinases and their modulation by inhibitors.

51Chromium-labeled rat pulmonary artery endothelial cells (EC) cultivated in MEM medium were killed, in a synergistic manner, by mixtures of subtoxic amounts of glucose oxidase-generated H2O2 and subtoxic amounts of the following agents: the cationic substances, nuclear histone, defensins, lysozyme, poly-L-arginine, spermine, pancreatic ribonuclease, polymyxin B, chlorhexidine, cetyltrimethyl ammonium bromide, as well as by the membrane-damaging agents phospholipases A2 (PLA2) and C (PLC), lysolecithin (LL), and by streptolysin S (SLS) of group A streptococci. Cytotoxicity induced by such mixtures was further enhanced by subtoxic amounts either of trypsin or of elastase. Glucose-oxidase cationized by complexing to poly-L-histidine proved an excellent deliverer of membrane-directed H2O2 capable of enhancing EC killing by other agonists. EC treated with rabbit anti-streptococcal IgG were also killed, in a synergistic manner, by H2O2, suggesting the presence in the IgG preparation of cross-reactive antibodies. Killing of EC by the various mixtures of agonists was strongly inhibited by scavengers of hydrogen peroxide (catalase, dimethylthiourea, MnCl2), by soybean trypsin inhibitor, by polyanions, as well as by putative inhibitors of phospholipases. Strong inhibition of cell killing was also observed with tannic acid and by extracts of tea, but less so by serum. On the other hand, neither deferoxamine, HClO, TNF, nor GTP gamma S had any modulating effects on the synergistic cell killing. EC exposed either to 6-deoxyglucose, puromycin, or triflupromazin became highly susceptible to killing by mixtures of hydrogen peroxide with several of the membrane-damaging agents. While maximal synergistic EC killing was achieved by mixtures of H2O2 with either PLA2, PLC, LL, or with SLS, a very substantial release of [3H]arachidonic acid (AA), PGE2, and 6-keto-PGF occurred only if a proteinase was also added to the mixture of agonists. The release of AA from EC was markedly inhibited either by scavengers of H2O2, by proteinase inhibitors, by cationic agents, by HClO, by tannic acid, and by quinacrin. We suggest that cellular injury induced in inflammatory and infectious sites might be the result of synergistic effects among leukocyte-derived oxidants, lysosomal hydrolases, cytotoxic cationic polypeptides, proteinases, and microbial toxins, which might be present in exudates. These "cocktails" not only kill cells, but also solubilize AA and several of its metabolites. However, AA release by the various agonists can be also achieved following attack by leukocyte-derived agonists on dead cells. It is proposed that treatment by "cocktails" of adequate antagonists might be beneficial to protect against cellular injury in vivo.