Pre-activation with TLR7 in combination with thioridazine and loratadine promotes tumoricidal T-cell activity in colorectal cancer.

Colorectal cancer (CRC) is the third most common malignancy worldwide. Our previous studies have shown that combinatorial treatment with thioridazine and loratadine may effectively inhibit CRC. However, the translation of these research findings to clinical practice was impaired by issues related to a lack of therapeutic specificity and to immune evasion. Toll-like receptor (TLR) agonists have been used as adjuvants to enhance the effectiveness of cancer vaccines. The aim of this study was to evaluate the therapeutic efficiency of immunotherapy with thioridazine and loratadine in combination with resiqumiod (R848), a small-molecule TLR7 agonist, in suppressing CRC growth in a mouse model. Twenty-four BALB/c mice were randomly assigned to treatment with PBS, R848, thioridazine + loratadine, or thioridazine + loratadine + R848. Cytokine levels were measured with ELISA. Overall survival, as well as tumor volume and tumor weight, was recorded. Cytotoxicity was measured by counting the numbers of CD8 and CD3-positive (CD8CD3) or CD4 and CD3-positive (CD3CD4) T-cells. The immune response induced by cytokines (as interferon-γ, interleukin-6, and tumor necrosis factor-α) was significantly stronger in mice treated with thioridazine + loratadine + R848. Moreover, thioridazine + loratadine + R848 significantly delayed tumor development and prolonged survival, which was associated with enhanced immune response and dendritic cell maturation. This study suggested that thioridazine + loratadine + R848 combinatorial treatment may be effective in overcoming immune evasion by tumor cells, with promising therapeutic potential in CRC.

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 inhibits autophagy and sensitizes glioblastoma cells to temozolomide.

Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14-15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome-wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late-stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy-lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.

A phase 1 trial evaluating thioridazine in combination with cytarabine in patients with acute myeloid leukemia.

We completed a phase 1 dose-escalation trial to evaluate the safety of a dopamine receptor D2 (DRD2) antagonist thioridazine (TDZ), in combination with cytarabine. Thirteen patients 55 years and older with relapsed or refractory acute myeloid leukemia (AML) were enrolled. Oral TDZ was administered at 3 dose levels: 25 mg (n = 6), 50 mg (n = 4), or 100 mg (n = 3) every 6 hours for 21 days. Intermediate-dose cytarabine was administered on days 6 to 10. Dose-limiting toxicities (DLTs) included grade 3 QTc interval prolongation in 1 patient at 25 mg TDZ and neurological events in 2 patients at 100 mg TDZ (gait disturbance, depressed consciousness, and dizziness). At the 50-mg TDZ dose, the sum of circulating DRD2 antagonist levels approached a concentration of 10 µM, a level noted to be selectively active against human AML in vitro. Eleven of 13 patients completed a 5-day lead-in with TDZ, of which 6 received TDZ with hydroxyurea and 5 received TDZ alone. During this period, 8 patients demonstrated a 19% to 55% reduction in blast levels, whereas 3 patients displayed progressive disease. The extent of blast reduction during this 5-day interval was associated with the expression of the putative TDZ target receptor DRD2 on leukemic cells. These preliminary results suggest that DRD2 represents a potential therapeutic target for AML disease. Future studies are required to corroborate these observations, including the use of modified DRD2 antagonists with improved tolerability in AML patients. This trial was registered at www.clinicaltrials.gov as #NCT02096289.

Targeting Lung Cancer Stem Cells with Antipsychological Drug Thioridazine.

Lung cancer stem cells are a subpopulation of cells critical for lung cancer progression, metastasis, and drug resistance. Thioridazine, a classical neurological drug, has been reported with anticancer ability. However, whether thioridazine could inhibit lung cancer stem cells has never been studied. In our current work, we used different dosage of thioridazine to test its effect on lung cancer stem cells sphere formation. The response of lung cancer stem cells to chemotherapy drug with thioridazine treatment was measured. The cell cycle distribution of lung cancer stem cells after thioridazine treatment was detected. The in vivo inhibitory effect of thioridazine was also measured. We found that thioridazine could dramatically inhibit sphere formation of lung cancer stem cells. It sensitized the LCSCs to chemotherapeutic drugs 5-FU and cisplatin. Thioridazine altered the cell cycle distribution of LCSCs and decreased the proportion of G0 phase cells in lung cancer stem cells. Thioridazine inhibited lung cancer stem cells initiated tumors growth in vivo. This study showed that thioridazine could inhibit lung cancer stem cells in vitro and in vivo. It provides a potential drug for lung cancer therapy through targeting lung cancer stem cells.

Evaluation of NADPH oxidases as drug targets in a mouse model of familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by progressive loss of motor neurons, gliosis, neuroinflammation and oxidative stress. The aim of this study was to evaluate the involvement of NADPH oxidases (NOX) in the oxidative damage and progression of ALS neuropathology. We examined the pattern of NOX expression in spinal cords of patients and mouse models of ALS and analyzed the impact of genetic deletion of the NOX1 and 2 isoforms as well as pharmacological NOX inhibition in the SOD1(G93A) ALS mouse model. A substantial (10-60 times) increase of NOX2 expression was detected in three etiologically different ALS mouse models while up-regulation of some other NOX isoforms was model-specific. In human spinal cord samples, high NOX2 expression was detected in microglia. In contrast to previous publications, survival of SOD1(G93A) mice was not modified upon breeding with constitutive NOX1 and NOX2 deficient mice. As genetic deficiency of a single NOX isoform is not necessarily predictive of a pharmacological intervention, we treated SOD1(G93A) mice with broad-spectrum NOX inhibitors perphenazine and thioridazine. Both compounds reached in vivo CNS concentrations compatible with NOX inhibition and thioridazine significantly decreased superoxide levels in the spinal cord of SOD1(G93A) mice in vivo. Yet, neither perphenazine nor thioridazine prolonged survival. Thioridazine, but not perphenazine, dampened the increase of microglia markers in SOD1(G93A) mice. Thioridazine induced an immediate and temporary enhancement of motor performance (rotarod) but its precise mode of action needs further investigation. Additional studies using specific NOX inhibitors will provide further evidence on the relevance of NOX as drug targets for ALS and other neurodegenerative disorders.

EAG2 potassium channel with evolutionarily conserved function as a brain tumor target.

Over 20% of the drugs for treating human diseases target ion channels, but no cancer drug approved by the US Food and Drug Administration (FDA) is intended to target an ion channel. We found that the EAG2 (Ether-a-go-go 2) potassium channel has an evolutionarily conserved function for promoting brain tumor growth and metastasis, delineate downstream pathways, and uncover a mechanism for different potassium channels to functionally cooperate and regulate mitotic cell volume and tumor progression. EAG2 potassium channel was enriched at the trailing edge of migrating medulloblastoma (MB) cells to regulate local cell volume dynamics, thereby facilitating cell motility. We identified the FDA-approved antipsychotic drug thioridazine as an EAG2 channel blocker that reduces xenografted MB growth and metastasis, and present a case report of repurposing thioridazine for treating a human patient. Our findings illustrate the potential of targeting ion channels in cancer treatment.

Co-delivery of thioridazine and doxorubicin using polymeric micelles for targeting both cancer cells and cancer stem cells.

In this study, thioridazine (THZ), which was reported to kill cancer stem cells, was used in a combination therapy with doxorubicin (DOX) to eradicate both cancer cells and DOX-resistant cancer stem cells to mitigate the reoccurrence of the disease. Both THZ and DOX were loaded into micelles with sizes below 100 nm, narrow size distribution and high drug content. The micelles were self-assembled from a mixture of acid-functionalized poly(carbonate) and poly(ethylene glycol) diblock copolymer (PEG-PAC) and urea-functionalized poly(carbonate) (PUC) and PEG diblock copolymer (PEG-PUC). The drug-loaded mixed micelles (MM) were used to target both cancer cells and stem cells via co-delivery. Cancer stem cells were sorted by a side population assay from BT-474 and MCF-7 human breast cancer cell lines, and identified by CD44+/CD24- phenotype. The cytotoxicity of various formulations was evaluated on the sorted cancer stem cells (side population SP cells), sorted non-stem-like cancer cells (non-side population NSP cells) and unsorted cancer cells. Antitumor activity was also evaluated on BT-474 xenografts in nude mice. As compared with NSP cells, DOX suppressed SP cell growth less effectively, while THZ and THZ-MM were more effective in the inhibition of SP cells. A stronger inhibitory effect was observed on SP cells with the co-delivery of free DOX and THZ or DOX-MM and THZ-MM as compared to free DOX or DOX-MM. THZ and THZ-MM were capable of lowering the population of SP cells in unsorted cells. In the BT-474 xenografts, the co-delivery of DOX-MM and THZ-MM produced the strongest antitumor efficacy, and both THZ and THZ-MM showed strong activity against cancer stem cells. This combination therapy may provide a promising strategy for breast cancer treatment by targeting both cancer cells and cancer stem cells.

A gene signature-based approach identifies thioridazine as an inhibitor of phosphatidylinositol-3'-kinase (PI3K)/AKT pathway in ovarian cancer cells.

OBJECTIVE: Thioridazine, a derivative of phenothiazine, has been reported to have antiproliferative activity on tumor cells. However, the mechanism has not been well defined. METHODS: Using in-silico gene signature based approach, we have demonstrated that thioridazine could inhibit phosphatidylinositol-3'-kinase (PI3K)/Akt pathway, and thus exert cytotoxicity in ovarian cancer cells. RESULTS: The Connectivity Map indicated that thioridazine induces gene signature similar to that of Akt inhibition. Moreover, preexisting inhibitors of PI3K/Akt pathway were also found to reveal similar signature. In SKOV-3 cells, immunoblot using p85 antibody showed that thioridazine could inhibit PI3K signal. In addition, thioridazine was found to inhibit p-Akt (Ser 473) in a dose-dependent manner. Furthermore, thioridazine was found to decrease cell viability and induce apoptosis. Exposure to thioridazine induced G(0)/G(1) arrest and down-regulated the cell cycle regulator, Cyclin D1 and CDK4, and up-regulated p21, p16, and p-CDC25A. Finally, additive cytotoxicity was observed when cisplatin and thioridazine were treated simultaneously. CONCLUSIONS: The current study indicated that in-silico approach, such as Connectivity Map, is a potentially useful method to identify the unknown cellular function among the drugs already in use in clinic. Owing to the property of Akt inhibition and additive cytotoxicity observed with the platinum compound, further research should be focused on this drug.

Factors affecting drug concentrations and QT interval during thioridazine therapy.

The objective of this study was to investigate factors affecting steady-state plasma concentrations of thioridazine. A cross-sectional study of patients receiving chronic thioridazine was employed. Common allelic variants of CYP2D6 and CYP2C19, as well as thioridazine and metabolite concentrations and QTc intervals, were determined. In 97 patients, dose-corrected plasma concentrations (C/Ds) of thioridazine and metabolites were correlated with age but not sex or CYP2C19 genotype. Patients with no functional CYP2D6 alleles (n=9) had significantly higher C/D for thioridazine (P=0.017) and the ring sulfoxide metabolite and a significantly higher thioridazine/mesoridazine ratio compared with those with >/=1 functional CYP2D6 allele (n=82). Smokers had significantly lower C/D for thioridazine, mesoridazine, and sulforidazine and significantly lower thioridazine/ring sulfoxide ratios than non-smokers. QTc interval was not significantly affected by CYP2D6 or CYP2C19 genotypes. Plasma concentrations of thioridazine are influenced by age, smoking, and CYP2D6 genotype, but CYP2D6 genotype does not appear to influence on-treatment QTc interval.

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.

A fulminant case of neuroleptic malignant syndrome.

We report a fulminant case of Neuroleptic Malignant Syndrome in a 31-year-old male schizophrenic on haloperidol, thioridazine, benzhexol and flurazepam who presented with rigidity, fever, stupor and autonomic instability. He succumbed rapidly over 6 days to rhabdomyolysis, acute renal failure, status epilepticus and disseminated intravascular coagulopathy despite treatment with dantrolene and bromocriptine at the outset.

Neuroleptic malignant syndrome and hypothyroidism.

Two patients with primary hypothyroidism who developed neuroleptic malignant syndrome (NMS) are described. Thyroid disease might predispose to NMS by altering brain dopamine metabolism.

Polypharmacy in the psychiatric treatment of elderly hospitalized patients: a survey of 12 Veterans Administration Hospitals.

Polypharmacy with psychoactive drugs was surveyed in 1276 elderly psychiatric patients from 12 Veterans Administration hospitals. One out of every six patients received two or more psychoactive agents. No specific combination of drugs was overly popular. The most frequently administered combinations, thioridazine plus phenobarbital and chlorpromazine plus phenobarbital, were administered to only 13 patients each (1% of the sample). The large majority of the combinations involved antipsychotic agents. One antipsychotic drug, thioridazine, was a component of nearly one-third of the combinations. The most frequent pairings were an antipsychotic drug plus and antidepressant, two antipsychotic drugs, and an antipsychotic drug plus an antianxiety agent or sedative-hypnotic. The use of polypharmacy was significantly related to patient age. One out of every four patients 60 to 65 years of age received two or more psychoactive drugs compared to only one out of eight over 75 years of age. The implications of these findings for treatment and research are discussed.