Phenothiazines and thioxanthenes inhibit multidrug efflux pump activity in Staphylococcus aureus.
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Efflux-related multidrug resistance (MDR) is a significant means by which bacteria can evade the effects of selected antimicrobial agents. Genome sequencing data suggest that Staphylococcus aureus may possess numerous chromosomally encoded MDR efflux pumps, most of which have not been characterized. Inhibition of these pumps, which may restore clinically relevant activity of antimicrobial agents that are substrates for them, may be an effective alternative to the search for new antimicrobial agents that are not substrates. The inhibitory effects of selected phenothiazines and two geometric stereoisomers of the thioxanthene flupentixol were studied using strains of S. aureus possessing unique efflux-related MDR phenotypes. These compounds had some intrinsic antimicrobial activity and, when combined with common MDR efflux pump substrates, resulted in additive or synergistic interactions. For S. aureus SA-1199B, which overexpresses the NorA MDR efflux pump, and for two additional strains of S. aureus having non-NorA-mediated MDR phenotypes, the 50% inhibitory concentration (IC(50)) for ethidium efflux for all tested compounds was between 4 and 15% of their respective MICs. Transport of other substrates was less susceptible to inhibition; the prochlorperazine IC(50) for acriflavine and pyronin Y efflux by SA-1199B was more than 60% of its MIC. Prochlorperazine and trans(E)-flupentixol were found to reduce the proton motive force (PMF) of S. aureus by way of a reduction in the transmembrane potential. We conclude that the mechanism by which phenothiazines and thioxanthenes inhibit efflux by PMF-dependent pumps is multifactorial and, because of the unbalanced effect of these compounds on the MICs and the efflux of different substrates, may involve an interaction with the pump itself and, to a lesser extent, a reduction in the transmembrane potential. (+info)
Dose-related effects of propericiazine in rats.
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We evaluated the effects of the neuroleptic agent propericiazine on animal models of anxiety and memory. Adult male Wistar rats (250 to 350 g) received intraperitoneal injections of propericiazine (0.05, 0.075 and 0.1 mg/kg), diazepam (1 mg/kg), saline, or diazepam vehicle (20% propylene glycol and 80% saline) 30 min prior to the experimental procedure. Animals (10-15 for each task) were tested for step-down inhibitory avoidance (0.3-mA footshock) and habituation to an open-field for memory assessment, and submitted to the elevated plus-maze to evaluate the effects of propericiazine in a model of anxiety. Animals treated with 0.075 mg/kg propericiazine showed a reduction in anxiety measures (P<0.05) similar to that observed in those treated with diazepam. Propericiazine at the doses of 0.05 and 0.1 mg/kg had no significant anxiolytic effects (P>0.05) in the elevated plus-maze model of anxiety. Memory was not affected by propericiazine in any of the tests, but was impaired by diazepam. The results indicate a dose-related, inverse U-shaped effect of propericiazine in an anxiety model, but not on memory tasks, perhaps reflecting involvement of the dopaminergic system in the mechanisms of anxiety. (+info)
Contribution of human cytochrome p-450 isoforms to the metabolism of the simplest phenothiazine neuroleptic promazine.
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1. The aim of the present study was to identify human cytochrome p-450 isoforms (CYPs) involved in 5-sulphoxidation and N-demethylation of the simplest phenothiazine neuroleptic promazine in human liver. 2. The experiments were performed in the following in vitro models: (A). a study of promazine metabolism in liver microsomes-(a). correlations between the rate of promazine metabolism and the level and activity of CYPs; (b). the effect of specific inhibitors on the rate of promazine metabolism (inhibitors: CYP1A2-furafylline, CYP2D6-quinidine, CYP2A6+CYP2E1-diethyldithiocarbamic acid, CYP2C9-sulfaphenazole, CYP2C19-ticlopidine, CYP3A4-ketoconazole); (B). promazine biotransformation by cDNA-expressed human CYPs (Supersomes 1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2E1, 3A4); (C). promazine metabolism in a primary culture of human hepatocytes treated with specific inducers (rifampicin-CYP3A4, CYP2B6 and CYP2C inducer, 2,3,7,8-tetrachlordibenzeno-p-dioxin (TCDD)-CYP1A1/1A2 inducer). 3. In human liver microsomes, the formation of promazine 5-sulphoxide and N-desmethylpromazine was significantly correlated with the level of CYP1A2 and ethoxyresorufin O-deethylase and acetanilide 4-hydroxylase activities, as well as with the level of CYP3A4 and cyclosporin A oxidase activity. Moreover, the formation of N-desmethylpromazine was correlated well with S-mephenytoin 4'-hydroxylation. 4. Furafylline (a CYP1A2 inhibitor) and ketoconazole (a CYP3A4 inhibitor) significantly decreased the rate of promazine 5-sulphoxidation, while furafylline and ticlopidine (a CYP2C19 inhibitor) significantly decreased the rate of promazine N-demethylation in human liver microsomes. 5. The cDNA-expressed human CYPs generated different amounts of promazine metabolites, but the rates of CYP isoforms to catalyse promazine metabolism at therapeutic concentration (10 microM) was as follows: 1A1>2B6>1A2>2C9>3A4>2E1>2A6>2D6>2C19 for 5-sulphoxidation and 2C19>2B6>1A1>1A2>2D6>3A4>2C9>2E1>2A6 for N-demethylation. The highest intrinsic clearance (V(max)/K(m)) was found for CYP1A subfamily, CYP3A4 and CYP2B6 in the case of 5- sulphoxidation, and for CYP2C19, CYP1A subfamily and CYP2B6 in the case of N-demethylation. 6. In a primary culture of human hepatocytes, TCDD (a CYP1A subfamily inducer), as well as rifampicin (mainly a CYP3A4 inducer) induced the formation of promazine 5-sulphoxide and N-desmethylpromazine. 7. Regarding the relative expression of various CYPs in human liver, the obtained results indicate that CYP1A2 and CYP3A4 are the main isoforms responsible for 5-sulphoxidation, while CYP1A2 and CYP2C19 are the basic isoforms that catalyse N-demethylation of promazine in human liver. Of the other isoforms studied, CYP2C9 and CYP3A4 contribute to a lesser degree to promazine 5-sulphoxidation and N-demethylation, respectively. The role of CYP2A6, CYP2B6, CYP2D6 and CYP2E1 in the investigated metabolic pathways of promazine seems negligible. (+info)
Sensing phenothiazine drugs at a gold electrode co-modified with DNA and gold nanoparticles.
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DNA and gold nanoparticles are co-immobilized at a gold electrode through elaborate self-assembly processes. This configuration has proven to be useful as a sensor for phenothiazine drugs, taking advantage of the well-known, relatively large surface area of gold nanoparticles and the strong intercalation between dsDNA and phenothiazine drugs. This modified electrode has demonstrated good sensitivity and stability towards the oxidation of two model phenothiazine drugs: promethazine and chlorpromazine. A linear dependence between the concentration of phenothiazine drugs and the peak current is observed, with a concentration range of 2.0 x 10(-5)-1.6 x 10(-4) M and 1.0 x 10(-5)-1.2 x 10(-4) M, and a detection limit of 1.0 x 10(-5) M and 7.0 x 10(-6) M, for promethazine and chlorpromazine, respectively. (+info)
Two new polymorphisms of the FMO3 gene in Caucasian and African-American populations: comparative genetic and functional studies.
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To characterize the contribution of the human flavin-containing monooxygenase form 3 (FMO3) in the metabolism and disposition of drugs and xenobiotics, we determined the single nucleotide polymorphisms in the coding region and adjacent splice junctions of FMO3 in 134 African Americans and 120 Caucasians from the United States. In the regions examined, DNA resequencing or high throughput MassEXTEND studies coupled with mass spectrometric genotyping showed that 12 sites of variation were present. Three variants encoding synonymous mutations and four polymorphisms were observed in the noncoding region. Another three variants, Lys158-FMO3, Met257-FMO3 and Gly308-FMO3, previously reported in similar populations, were prominent polymorphisms. Two new polymorphisms, His132-FMO3 and Pro360-FMO3, were identified in this study. Both variants were found only in African Americans. To evaluate the effect of the amino acid substitutions on the function of FMO3, each amino acid substitution was introduced by site-directed mutagenesis into a wild-type FMO3 cDNA. Selective functional activity was studied with methimazole, trimethylamine, and 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl) phenothiazine. Both His132-FMO3 and Pro360-FMO3 variants were able to metabolize the substrates examined. Compared with wild-type FMO3, the His132-FMO3 was less catalytically efficient. The His132-FMO3 variant moderately altered the catalytic efficiency of FMO3 (decrease of 30%, 60% and 6% with methimazole, trimethylamine and 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine, respectively). The Pro360-FMO3 variant was more catalytically efficient than wild-type FMO3. Pro360-FMO3 oxygenated methimazole, trimethylamine and 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine, respectively, 3-, 5- and 2-fold more efficiently than wild-type FMO3. Based on the functional activity of the variant FMO3 enzymes, it is likely that population differences exist for compounds primarily metabolized by FMO3. (+info)
Thermodynamics of partitioning of phenothiazine drugs between phosphatidylcholine bilayer vesicles and water studied by second-derivative spectrophotometry.
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The partition coefficients (Kps) of phenothiazine drugs (trifluoperazine, triflupromazine, chlorpromazine and promazine) between phosphatidylcholine (PC) small unilamellar vesicles (SUV) and water were determined over the temperature range of 10-40 degrees C by a second-derivative spectrophotometric method. The second derivative spectra of each drug solution containing various amounts of SUV showed distinct derivative isosbestic points confirming the entire elimination of the residual background signal effects of the SUV. The Kp values were calculated from the derivative intensity change of the drugs induced by the addition of SUV to the drug buffer solutions (pH 7.4) and obtained with the R.S.D. below 10% (n=3). The van't Hoff analysis of the temperature dependence of Kp values revealed negative deltaH(w-->l) and positive deltaS(w-->l), suggesting an enthalpy/entropy driven mechanism for the phenothiazine partitioning. The negative deltaH(w-->l) implies that the electrostatic interaction, positively charged alkyl amino groups of phenothiazine drugs with negatively charged phosphate groups on the surface of PC SUV, partly contributes to the partitioning. The existence of halogen atom(s) on the phenothiazine ring at position C-2 enhanced the Kp value (Hl) value (Hl) increase is considered to be enhancement of disorder in the hydrophobic acyl chain regions of PC SUV membranes derived from the phenothiazine ring insertion and thus depends on the bulkiness of the substituent. The enthalpy-entropy correlation analysis yielding a good linear relationship also suggests that the phenothiazine drugs studied have identically an enthalpy-entropy compensation mechanism for the partitioning. (+info)
Functional evidence of a transmembrane channel within the Ca2+ transport ATPase of sarcoplasmic reticulum.
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Ca2+ efflux can be studied conveniently following dilution of sarcoplasmic reticulum (SR) vesicles preloaded with 45Ca2+ by active transport. The rates of efflux are highly dependent on ATPase substrates and cofactors (Pi, Mg2+, Ca2+ and ADP) in the efflux medium. On the other hand, phenothiazines stimulate efflux through a passive permeability channel with no coupled catalytic events. Efflux activation by manipulation of catalytically active ATPase ligands, as well as by the catalytically inactive phenothiazines, can be prevented by thapsigargin, which is a highly specific inhibitor of the Ca(2+)-ATPase. This demonstrates that the passive channel activated by phenothiazines is an integral part of the ATPase, and can operate either uncoupled or coupled to catalytic events. (+info)
Rationally designed selective inhibitors of trypanothione reductase. Phenothiazines and related tricyclics as lead structures.
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Trypanothione reductase, an essential component of the anti-oxidant defences of parasitic trypanosomes and Leishmania, differs markedly from the equivalent host enzyme, glutathione reductase, in the binding site for the disulphide substrate. Molecular modelling of this region suggested that certain tricyclic compounds might bind selectively to trypanothione reductase without inhibiting host glutathione reductase. This was confirmed by testing 30 phenothiazine and tricyclic antidepressants, of which clomipramine was found to be the most potent, with a K(i) of 6 microM, competitive with respect to trypanothione. Many of these compounds have been noted previously to have anti-trypanosomal and anti-leishmanial activity and thus they can serve as lead structures for rational drug design. (+info)