Thioridazine aggravates skeletal myositis, systemic and liver inflammation in Trypanosoma cruzi-infected and benznidazole-treated mice.

While thioridazine (Tio) inhibits the antioxidant defenses of Trypanosoma cruzi, the gold standard antitrypanosomal drug benznidazole (Bz) has potent anti-inflammatory and pro-oxidant properties. The combination of these drugs has never been tested to determine the effect on T. cruzi infection. Thus, we compared the impact of Tio and Bz, administered alone and in combination, on the development of skeletal myositis and liver inflammation in T. cruzi-infected mice. Swiss mice were randomized into six groups: uninfected untreated, infected untreated, treated with Tio (80 mg/kg) alone, Bz (50 or 100 mg/kg) alone, or a combination of Tio and Bz. Infected animals were inoculated with a virulent T. cruzi strain (Y) and treated by gavage for 20 days. Mice untreated or treated with Tio alone developed the most intense parasitemia, highest parasitic load, elevated IL-10, IL-17, IFN-γ, and TNF-α plasma levels, increased N-acetylglucosaminidase and myeloperoxidase activity in the liver and skeletal muscle, as well as severe myositis and liver inflammation (P < 0.05). All parameters were markedly attenuated in animals receiving Bz alone (P < 0.05). However, the co-administration of Tio impaired the response to Bz chemotherapy, causing a decrease in parasitological control (parasitemia and parasite load), skeletal muscle and liver inflammation, and increased microstructural damage, when compared to the group receiving Bz alone (P < 0.05). Altogether, our findings indicated that Tio aggravates systemic inflammation, skeletal myositis and hepatic inflammatory damage in T. cruzi-infected mice. By antagonizing the antiparasitic potential of Bz, Tio limits the anti-inflammatory, myoprotectant and hepatoprotective effects of the reference chemotherapy, aggravating the pathological remodeling of both organs. As the interaction of T. cruzi infection, Bz and Tio is potentially toxic to the liver, inducing inflammation and microvesicular steatosis; this drug combination represents a worrying pharmacological risk factor in Chagas disease.

Thioridazine Induces Cardiotoxicity via Reactive Oxygen Species-Mediated hERG Channel Deficiency and L-Type Calcium Channel Activation.

Thioridazine (THIO) is a phenothiazine derivative that is mainly used for the treatment of psychotic disorders. However, cardiac arrhythmias especially QT interval prolongation associated with the application of this compound have received serious attention after its introduction into clinical practice, and the mechanisms underlying the cardiotoxicity induced by THIO have not been well defined. The present study was aimed at exploring the long-term effects of THIO on the hERG and L-type calcium channels, both of which are relevant to the development of QT prolongation. The hERG current (I hERG) and the calcium current (I Ca-L) were measured by patch clamp techniques. Protein levels were analyzed by Western blot, and channel-chaperone interactions were determined by coimmunoprecipitation. Reactive oxygen species (ROS) were determined by flow cytometry and laser scanning confocal microscopy. Our results demonstrated that THIO induced hERG channel deficiency but did not alter channel kinetics. THIO promoted ROS production and stimulated endoplasmic reticulum (ER) stress and the related proteins. The ROS scavenger N-acetyl cysteine (NAC) significantly attenuated hERG reduction induced by THIO and abolished the upregulation of ER stress marker proteins. Meanwhile, THIO increased the degradation of hERG channels via disrupting hERG-Hsp70 interactions. The disordered hERG proteins were degraded in proteasomes after ubiquitin modification. On the other hand, THIO increased I Ca-L density and intracellular Ca2+ ([Ca2+]i) in neonatal rat ventricular cardiomyocytes (NRVMs). The specific CaMKII inhibitor KN-93 attenuated the intracellular Ca2+ overload, indicating that ROS-mediated CaMKII activation promoted calcium channel activation induced by THIO. Optical mapping analysis demonstrated the slowing effects of THIO on cardiac repolarization in mouse hearts. THIO significantly prolonged APD50 and APD90 and increased the incidence of early afterdepolarizations (EADs). In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), THIO also resulted in APD prolongation. In conclusion, dysfunction of hERG channel proteins and activation of L-type calcium channels via ROS production might be the ionic mechanisms for QT prolongation induced by THIO.

In vitro and in vivo toxicity evaluation of non-neuroleptic phenothiazines, antitubercular drug candidates.

The phenothiazine-derived antipsychotic drugs, such as chlorpromazine and thioridazine, are bactericidal against drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis, but produce undesirable side effects at clinically relevant doses. We have previously modified four novel phenothiazines and maintained their antimycobacterial activity. This study evaluated the pharmacological and toxicity profiles of these novel non-neuroleptic phenothiazines, PTZ3, PTZ4, PTZ31 and PTZ32, for their metabolic stability, kinetic solubility and potential cytotoxic effects in vitro. To further support the safet use of these drug candidates, the in vivo pharmacological and toxicity profiles were assessed in C57BL/6 mice via single or repeated oral gavage. In acute toxicity studies, all four modified phenothiazines showed favourable safety in mice. When treated daily with 100 mg/kg of PTZ3 and PTZ4 for 2 weeks, mice displayed no signs of toxicity. Alternatively, treatment with PTZ31 resulted in 20% mortality with no toxicity evident in biochemical or histological analysis, while exposure to PTZ32 resulted in a 45% survival with increased serum concentrations of uric acid and alkaline phosphatase. The combined non-neuroleptic and antimycobacterial effects of the novel phenothiazines PTZ3, PTZ4, PTZ31 and PTZ32 demonstrated favourable pharmacological and toxicity profiles in this study, highlight the potential of these compounds as suitable anti-tuberculosis drug candidates.

Thioridazine Sensitizes Cisplatin Against Chemoresistant Human Lung and Ovary Cancer Cells.

Human lung cancer cell lines A549 and A549/DDP, and ovarian cancer cell lines SKOV3 and SKOV3/DDP were subjected to thioridazine (Thio), cisplatin, or the combination; A549/DDP and SKOV3/DDP were the cisplatin-resistant sublines. Cell viability, apoptosis, and cell cycle were detected; the mitochondrial membrane potential and proteins related to mitochondrial apoptosis were determined. Thio induced cell death, and the combination of Thio and cisplatin led to the highest percentage of dead cells in four cells lines. Thio and the combined modality led to cell apoptosis by inducing G0/G1 arrest. The collapse of mitochondrial membrane potential, activation of caspase 9, upregulation of Bax protein, and downregulation of Bcl-2 protein demonstrated that apoptosis was mitochondria dependent. These data indicated that Thio could be used to modulate cisplatin-based chemotherapeutic regimen in lung and ovary cancers.

Environmental fate and effect assessment of thioridazine and its transformation products formed by photodegradation.

An experimental and in silico quantitative structure-activity relationship (QSAR) approach was applied to assess the environmental fate and effects of the antipsychotic drug Thioridazine (THI). The sunlight-driven attenuation of THI was simulated using a Xenon arc lamp. The photodegradation reached the complete primary elimination, whereas 97% of primary elimination and 11% of mineralization was achieved after 256 min of irradiation for the initial concentrations of 500 µg L(-1) and 50 mg L(-1), respectively. A non-target approach for the identification and monitoring of transformation products (TPs) was adopted. The structure of the TPs was further elucidated using liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proposed photodegradation pathway included sulfoxidation, hydroxylation, dehydroxylation, and S- and N-dealkylation, taking into account direct and indirect photolysis through a self-sensitizing process in the higher concentration studied. The biodegradability of THI and photolytic samples of THI was tested according to OECD 301D and 301F, showing that THI and the mixture of TPs were not readily biodegradable. Furthermore, THI was shown to be highly toxic to environmental bacteria using a modified luminescent bacteria test with Vibrio fischeri. This bacteriotoxic activity of THI was significantly reduced by phototransformation and individual concentration-response analysis confirmed a lowered bacterial toxicity for the sulfoxidation products Thioridazine-2-sulfoxide and Thioridazine-5-sulfoxide. Additionally, the applied QSAR models predicted statistical and rule-based positive alerts of mutagenic activities for carbazole derivative TPs (TP 355 and TP 339) formed through sulfoxide elimination, which would require further confirmatory in vitro validation tests.

Thioridazine in PLGA nanoparticles reduces toxicity and improves rifampicin therapy against mycobacterial infection in zebrafish.

Encapsulating antibiotics such as rifampicin in biodegradable nanoparticles provides several advantages compared to free drug administration, including reduced dosing due to localized targeting and sustained release. Consequently, these characteristics reduce systemic drug toxicity. However, new nanoformulations need to be tested in complex biological systems to fully characterize their potential for improved drug therapy. Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, requires lengthy and expensive treatment, and incomplete therapy contributes to an increasing incidence of drug resistance. Recent evidence suggests that standard therapy may be improved by combining antibiotics with bacterial efflux pump inhibitors, such as thioridazine. However, this drug is difficult to use clinically due to its toxicity. Here, we encapsulated thioridazine in poly(lactic-co-glycolic) acid nanoparticles and tested them alone and in combination with rifampicin nanoparticles, or free rifampicin in macrophages and in a zebrafish model of tuberculosis. Whereas free thioridazine was highly toxic in both cells and zebrafish embryos, after encapsulation in nanoparticles no toxicity was detected. When combined with rifampicin nanoparticles, the nanoparticles loaded with thioridazine gave a modest increase in killing of both Mycobacterium bovis BCG and M. tuberculosis in macrophages. In the zebrafish, the thioridazine nanoparticles showed a significant therapeutic effect in combination with rifampicin by enhancing embryo survival and reducing mycobacterial infection. Our results show that the zebrafish embryo is a highly sensitive indicator of drug toxicity and that thioridazine nanoparticle therapy can improve the antibacterial effect of rifampicin in vivo.

Dopamine receptor antagonist thioridazine inhibits tumor growth in a murine breast cancer model.

Neuropsychological factors have been shown to influence tumor progression and therapeutic response. The present study investigated the effect of the dopamine receptor antagonist thioridazine on murine breast cancer. The anti­tumor efficacy of thioridazine was assessed using a murine breast cancer model. Cell apoptosis and proliferation were analyzed in vitro using flow cytometry (FCM) and the MTT assay, respectively. Western blot analysis was performed to assess Akt, phosphorylated (p)­Akt, signal transducer and activator of transcription (STAT) 3, p­STAT3 and p­p65 in tumor cells following treatment with thioridazine. The Ki67 index and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)­positive apoptotic cells were assessed in the tumor sections. Thioridazine was found to reduce tumor growth, inhibit tumor cell proliferation and induce apoptosis in a dose­ and time­dependent manner in vitro. Thioridazine was also found to markedly inhibit tumor proliferation and induce tumor cell apoptosis in vivo as shown by the lower Ki67 index and increase in TUNEL­positive cells. In addition, thioridazine was observed to inhibit the activation of the canonical nuclear factor κ­light­chain­enhancer of activated B cells pathway and exert anti­tumor effects by remodeling the tumor stroma, as well as inhibit angiogenesis in the tumor microenvironment. In conclusion, thioridazine was found to significantly inhibit breast tumor growth and the potential for thioridazine to be used in cancer therapy may be re­evaluated and investigated in clinical settings.

Effect of thioridazine on antioxidant status of HEMn-DP melanocytes.

Thioridazine as an antipsychotic agent was extensively used to treat various psychotic disorders, e.g. schizophrenia. However, the therapy with this drug can induce serious side effects such as extrapyramidal symptoms or ocular and skin disorders, which mechanisms are still not fully established. To gain inside the molecular mechanisms underlying thioridazine toxicity, we examined the effect of this drug on cell viability, antioxidant defence system as well as melanogenesis in normal human melanocytes. It was demonstrated that thioridazine induces concentration-dependent loss in cell viability. The value of EC50 was calculated to be 2.24 µM. To study the effect of thioridazine on antioxidant defence system in melanocytes, the level of hydrogen peroxide and the activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were determined. The drug in concentrations of 0.1, 0.25, 1.0 and 2.5 µM caused changes in cellular antioxidant defence system indicating the induction of oxidative stress. It was also shown that the analysed neuroleptic in concentrations of 1.0 and 2.5 µM significantly inhibited melanogenesis. The observed changes in cell viability, antioxidant defence system and melanization in normal human melanocytes after thioridazine treatment may explain an important role of reactive oxygen species as well as melanin in mechanisms involved in this drug side effects directed on pigmented tissues.

Thioridazine pharmacokinetic-pharmacodynamic parameters "Wobble" during treatment of tuberculosis: a theoretical basis for shorter-duration curative monotherapy with congeners.

Phenothiazines are being repurposed for treatment of tuberculosis. We examined time-kill curves of thioridazine and first-line drugs against log-growth-phase and semidormant bacilli under acidic conditions and nonreplicating persistent Mycobacterium tuberculosis. While both the potency and the efficacy of first-line drugs declined dramatically as M. tuberculosis replication rates decreased, those of thioridazine improved. The mutation prevalence to 3 times the thioridazine MIC was <1 × 10(-11), better than for ≥2 first-line drugs combined. Hollow fiber system studies revealed that the relationship between sterilizing effect and pharmacodynamic indices (PDI) was characterized by an r(2) of 0.88 for peak/MIC, an r(2) of 0.47 for the area under the concentration-time curve (AUC) to MIC, and an r(2) of 0.14 for the cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) at the end of the first week. However, the PDI linked to effect "wobbled" as the duration of therapy increased, so that by the fourth week the r(2) was 0.88 for AUC/MIC, 0.78 for %TMIC, and 0.72 for peak/MIC. This "wobble" has implications on general pharmacokinetic/pharmacodynamic theory, whereby efficacy is linked to only one of the three PDIs in deterministic models. The potency changed 8.9-fold from the first to the fourth weeks. The non-protein-bound AUC/MIC associated with maximal kill at the end of therapy was 50.53 (protein binding = 99.5%). This thioridazine exposure was calculated to extinguish all three M. tuberculosis metabolic populations in human lungs in only 42.9 days of monotherapy. However, this concentration exceeds the 2- to 8-mg/liter thioridazine concentration in serum known to be lethal to humans. Therefore, the way forward for phenothiazine monotherapy that also reduces therapy duration is via synthesis of less toxic congeners.

Effects of diverse psychopharmacological substances on the activity of brain prolyl oligopeptidase.

Prolyl oligopeptidase (POP) has been connected to learning, memory and mood. Changes in serum or plasma POP activity have been linked to psychiatric disorders. POP has been thought to interfere in these conditions by cleaving neuroactive peptides or via the phosphatidylinositol second messenger system. However, little is known about the possible POP inhibition of commonly used psychoactive drugs. In this study, we measured the effects of various psychotropic drugs, including antidepressants, antipsychotics, mood stabilisers and anxiolytics, on the activity of the rat brain homogenate POP. Of the 38 compounds tested, 18 inhibited POP by at least 20% at 10 µM (buspirone, chlorpromazine, citalopram, clozapine, desipramine, duloxetine, escitalopram, flupenthixol, imipramine, ketanserin, lamotrigine, levomepromazine, prazosin, prochlorperazine, promazine, risperidone ritanserin and thioridazine). Thioridazine and valproate (VPA) acted at therapeutic plasma levels. Kinetically, VPA was a competitive inhibitor, thioridazine a non-competitive inhibitor and ketanserin a mixed type inhibitor. Being lipophilic, many of the psychoactive compounds are present in the brain at several-times higher concentrations than in plasma. At concentrations reported to be reached in the brain, chlorpromazine, clozapine, desipramine, imipramine, prochlorperazine and promazine inhibited POP by 30-50% suggesting that they could inhibit POP in vivo. However, when studied ex vivo, a single dose of 10 mg/kg thioridazine caused a deep sedation in the mice but did not inhibit the activity of POP. In conclusion, compared with conventional POP inhibitors, all psychopharmacological compounds tested are very weak inhibitors in vitro, and we doubt that their POP inhibition would be therapeutically meaningful.

Interferences of the autonomic nervous system with drug induced QT prolongation: a point to consider in non-clinical safety studies.

INTRODUCTION: QT interval assessment by telemetry has become one of the most useful models in testing strategies adopted for detection of drug induced QT prolongation in non-clinical safety pharmacology studies. This study reports experimental data showing that the autonomic nervous system might influence drug induced QT prolongation. METHODS: Animals were instrumented with telemetric transmitters and epicardial ECG leads. Effects on QT interval of reference drugs such as thioridazine and terfenadine were analysed with different approaches, the Holzgrefe's probabilistic method, the QT shift method and an individual analysis of beat-to-beat QT/RR pair distribution visualised as points-cloud. RESULTS: Two cases of unexpected absence of QT interval prolongation are reported with thioridazine and terfenadine in conscious beagle dogs under conditions of concomitant tachycardia. The pro-arrhythmic properties of these two molecules were unmasked by co-treatment with sympatholytic agents, atenolol and clonidine respectively suggesting that sympathetic activation and/or parasympathetic withdrawal might impair a drug induced QT prolongation. DISCUSSION: The apparent absence of changes in the QT interval due to novel drug candidates should be interpreted cautiously under conditions of concomitant tachycardia or elevated heart rate levels in non-clinical safety studies.

Photodegradation and phototoxicity of thioridazine and chlorpromazine evaluated with chemical analysis and aquatic organisms.

The photochemical behaviour of chlorpromazine (CPZ) and thioridazine (THR) incubated under VIS light and a UV-A lamp was investigated with a high-performance liquid chromatography photodiode array detector (HPLC-PAD) and two bioassays. VIS light caused the decrease of CPZ and THR to 25% and 34% of the initial level, respectively, while UV-A degraded the drugs almost totally. CPZ and THR were very toxic to the protozoan Spirostomum ambiguum (Spirotox) and anostracan crustacean Thamnocephalus platyurus (Thamnotoxkit F) with 24-h LC50 values of around 0.5 mg l(-1). In spite of the drastic decrease of the concentration of the drugs, the irradiated samples were toxic to the protozoan, especially when a sublethal end-point was taken into consideration. Contrary to the protozoan the crustacean was not sensitive to the products of photodegradation. Mass spectrometry analysis showed the presence of dimers and trimers of the CPZ and mono-, di-, and tri-oxygenated derivatives of THR. The presented data give a strong indication of the importance of the investigation of the environmental fate of drugs, especially those known to be phototoxic.

Antipsychotic medication dispensing and associated odds ratios of death in elderly veterans and war widows, 2001.

OBJECTIVES: To explore the odds ratios (ORs) of death associated with antipsychotic (AP) medications dispensed to elderly subjects. METHOD: Subjects were veterans and war widows 65 years and older dispensed an AP drug in 2001 in NSW or ACT. For all subjects, dispensing records for AP medication, benzodiazepines, lithium, carbamazepine, sodium valproate and antidepressant medication were extracted and combined with age, gender and date of death. A study date was allocated, either the date of death or a random date from 1.5.01 to 31.12.01. Subjects dispensed an AP in 2001, but not dispensed an AP or other psychotropic medication in the 120 days prior to their study date, formed a reference group. Psychotropic dispensing in the 120 days prior to the study date was analysed using nested logistic regression models to produce ORs of death associated with various AP drugs. The ORs for risperidone, olanzapine and pericyazine were compared. Haloperidol ORs were established for those dispensed the drug 0-30 days prior to study date or 31-120 days prior to the study date. RESULTS: The ORs associated with haloperidol, olanzapine, risperidone, pericyazine, thioridazine and chlorpromazine were significant when compared with the reference group. Odds ratios for all three haloperidol periods were significant when compared with olanzapine, risperidone and pericyazine 120 day ORs. Although there was a trend favouring olanzapine when compared with risperidone, the difference in the ORs failed to reach significance (p=0.066). CONCLUSIONS: Haloperidol is associated with significantly higher mortality rates than other AP medication but it is not clear whether this represents drug toxicity or the medical conditions for which it was dispensed. There was no evidence that the conventional AP pericyazine was associated with a higher mortality rate than olanzapine or risperidone.

Effects of antipsychotic drugs on I(to), I (Na), I (sus), I (K1), and hERG: QT prolongation, structure activity relationship, and network analysis.

PURPOSE: To evaluate in vitro and computationally model the effects of selected antipsychotic drugs on several ionic currents that contribute to changes in the action potential in cardiac tissue. METHODS: Fourteen antipsychotic drugs or metabolites were examined to determine whether QT interval prolongation could be accounted for by an effect on one or more myocardial ion channels [I(to), I(Na), I(sus), I(K1), and human ether-a-go-go related gene (hERG)]. Using the patch clamp technique, drug effects on these human cardiac currents were tested. RESULTS: All molecules had little inhibitory effect on ion channels (blocking at concentrations >5 microM) other than hERG. A significant correlation was observed between the estimated hERG blockade and the increase in corrected QT for five of the antipsychotics. Molecular modeling identified hydrophobic features related to the interaction with hERG and correctly rank-ordered the test set molecules olanzapine and its metabolites. A network analysis of ligand and protein interactions around hERG using MetaCore (GeneGo Inc., St. Joseph, MI, USA) was used to visualize antipsychotics with affinity for this channel and their interactions with other proteins in this database. CONCLUSION: The antipsychotics do not inhibit the ion channels I(to), I(Na), I(sus), I(K1) to any appreciable extent; however, blockade of hERG is a likely mechanism for the prolongation of the QT interval.

Application of gold nanoparticles to microencapsulation of thioridazine.

Thioridazine-containing ethyl cellulose (EC) microcapsules were prepared in the presence of gold nanoparticles via the W/O/W emulsification solvent-evaporation method. The gold nanoparticles have been verified as human safe and the nondestructive physisorption of thioridazine on gold nanoparticles was corroborated with the time-of-flight second ion mass spectrometry measurements. The morphology of the formed microcapsules (ETA, containing EC, Thioridazine and Au) changed substantially because of the presence of gold nanoparticles. In addition to a prolonged controlled release, these ETA microcapsules had an enhanced thioridazine encapsulation with an efficiency over one and half times that of the microcapsules (ET) containing no nanogold particles. While data of the release kinetics for ET microcapsules fitted the apparent first-order model, corresponding data for ETA microcapsules agreed better with the Higuchi model indicating a uniform distribution of thioridazine in the monolithic-type microcapsules.

In vitro phototoxicity of phenothiazines: involvement of stable UVA photolysis products formed in aqueous medium.

This paper reports the results of an in vitro evaluation of the phototoxic potential of stable photoproducts formed by UVA photolysis of three phenothiazines, perphenazine, fluphenazine, and thioridazine, in a water environment. Perphenazine gave a single product due to dechlorination. From thioridazine, the two major products formed; the endocyclic sulfoxide and the endocyclic N-oxide in which the 2-SCH3 substituent was replaced by a hydroxy group were tested. From fluphenazine, two products have been examined as follows: an exocyclic N-piperazine oxide and a carboxylic acid arising from hydrolysis of the 2-CF3 group. The phototoxicity of the isolated photoproducts has been studied in order to determine their possible involvement in the photosensitizing effects exhibited by the parent drugs, using hemolysis and 3T3 fibroblasts viability as in vitro assays. As fluphenazine, perphenazine, and thioridazine did, some photoproducts proved phototoxic. In particular, the perphenazine dechlorinated photoproduct and the thioridazine N-oxide were found to exert phototoxic properties similar to the parent compounds. Therefore, our data suggest that some phenothiazine photoproducts may play a role in the mechanism of photosensitivity of these drugs. Because some of these photoproducts correspond to metabolic products of phenothiazines found in humans, it cannot be ruled out that metabolites of phenothiazines can be phototoxic in vivo.

Mitochondria and plasma membrane as targets of UVA-induced toxicity of neuroleptic drugs fluphenazine, perphenazine and thioridazine.

In order to gain insights into the mechanism of phototoxicity of the neuroleptic drugs fluphenazine, perphenazine and thioridazine in cultured cells, studies were performed with murine 3T3 fibroblasts, aimed at identifying some cellular targets responsible for photoinduced cell death and possible cytotoxic reactive species involved in the photosensitization process. 3T3 fibroblasts incubated with 5 microM drugs and irradiated with UVA light (up to 8 J/cm2) underwent cell death, the extent of which depended on light dose. Of the three drugs, fluphenazine exhibited the highest phototoxicity and 100% cell death was achieved with a light dose of 5 J/cm2. Superoxide dismutase and alpha-tocopherol exerted a dose-dependent protective effect against drug phototoxicity, whereas N-acetylcysteine failed to do so. These findings indicate that superoxide anion and other free radical intermediates, generated in lipophilic cellular environments, play a role in photoinduced toxicity. Phototreatment of drug-loaded cells induces release of the cytosolic enzyme lactate dehydrogenase and causes loss of activity of mitochondrial NADH dehydrogenase, indicating that plasma membrane and mitochondria are among the targets of the phototoxicity of these drugs.

Characterization of phenothiazine-induced apoptosis in neuroblastoma and glioma cell lines: clinical relevance and possible application for brain-derived tumors.

In this study we aimed to (1). screen phenothiazines for cytotoxic activity in glioma, neuroblastoma, and primary mouse brain tissue; and (2). determine the mechanism of the cytotoxic effect (apoptosis, necrosis) and the roles of calmodulin inhibition and sigma receptor modulation. Rat glioma (C6) and human neuroblastoma (SHSY-5Y) cell lines were treated with different phenothiazines. All agents induced a dose-dependent decrease in viability and proliferation, with the highest activity elicited by thioridazine. Sensitivity to thioridazine of glioma and neuroblastoma cells was significantly higher (p < 0.05) than that of primary mouse brain culture (IC50 11.2 and 15.1 microM vs 41.3 microM, respectively). The N-mustard fluphenazine induced significantly lower cytotoxicity in glioma cells, compared to fluphenazine. The sigma receptor selective ligand (+)-SK&F10047 increased viability slightly while combined with fluphenazine; SK&F10047 did not alter fluphenazine activity. Flow cytometry of propidium iodide (PI)-stained glioma cells treated with thioridazine, fluphenazine, or perphenazine (6-50 microM) resulted in a concentration-dependent increase of fragmented DNA up to 94% vs 3% in controls by all agents. Thioridazine (12.5 microM)-treated glioma cells costained with PI and Hoechst 33342 revealed a red fluorescence of fragmented nuclei in treated cells and a blue fluorescence of intact control nuclei. After 4-h exposure to thioridazine (25 and 50 microM), a 25- to 30-fold increase in caspase-3 activity in neuroblastoma cells was noted. Overall, the marked apoptotic effect of phenothiazines in brain-derived cancer cells, and the low sensitivity of primary brain tissue suggest the potential use of selected agents as therapeutic modalities in brain cancer.

The effects of sodium bicarbonate on thioridazine-induced cardiac dysfunction in the isolated perfused rat heart.

To determine the site of thioridazine-induced cardiotoxicity and investigate the effectiveness of sodium bicarbonate (NaHCO3) therapy, isolated rat hearts were perfused with Krebs-Henseleit-Bicarbonate buffer (KHB) at a constant coronary flow of 10 mL/min and electrically paced at 300 bpm. Experimental protocol included 15 min intervals of KHB, thioridazine (TDZ), TDZ + NaHCO3, KHB. Left ventricular (LV) pressure was measured with a balloon-tipped catheter placed in the LV via the mitral valve. Coronary perfusion pressure was monitored continuously as an index of coronary vascular resistance (CVR). LV generated pressure (LVGP) was used as our index of cardiac function and was calculated by subtracting LV end diastolic pressure (LVEDP) from LV peak systolic pressure (LVPSP). TDZ at 7,500 ng/mL was chosen as the toxic dose. NaHCO3 treatment was at an approximate sodium = 155 mM and pH = 7.60. Hearts perfused with TDZ resulted in a progressive decrease in LVGP. After 15 min of TDZ perfusion, LVGP decreased by 50%, and 75% at 30 min (n = 5). TDZ increased LVEDP and decreased LVPSP. TDZ perfusion increased CVR by 83%. In another experiment, hearts were perfused with TDZ for 15 min and then for an additional 15 min with TDZ + NaHCO3. NaHCO3 treatment transiently (approximately 5 min) increased LVGP by 23% (n=5). During NaHCO3 treatment, LVPSP increased and LVEDP and CVR decreased during the first 5 min. During the remainder of the NaHCO3 protocol, the hearts failed, similar to TDZ alone. TDZ diminished left ventricular function and promoted coronary artery vasoconstriction. NaHCO3 temporariy reversed these toxic effects.

A combined biochemical and cytogenetic study of thioridazine-induced damage to nucleic acids.

In this work the biochemical effects of thioridazine, a commonly used phenothiazine, have been studied upon native double- and single-stranded DNA and also upon a supercoiled plasmid. The results indicate that thioridazine causes damage and scissions to these nucleic acids but only at concentrations much higher than the one used in our cytogenetic experiments and that the damage seems to depend on the concentrations used. Furthermore, we studied the action of thioridazine alone or in combination with caffeine and/or melphalan upon human lymphocytes in vitro. Thioridazine and caffeine (a well-known inhibitor of cellular repair mechanisms) were shown to act synergistically to potentiate the cytogenetic effect of melphalan on human lymphocytes. It is suggested that thioridazine alone or in combination with caffeine may exert its synergistic effect on melphalan cytotoxicity to cultured human lymphocytes not only indirectly, i.e. as a strong calmodulin inhibitor by facilitating the intracellular retention of melphalan, but also directly by reaction with nucleic acids and by causing scissions in and damage to them. Therefore, thioridazine (as chlorpromazine) has some potential as an adjuvant chemotherapeutic agent for the treatment of human cancer.