Applying the CiPA approach to evaluate cardiac proarrhythmia risk of some antimalarials used off-label in the first wave of COVID-19.

We applied a set of in silico and in vitro assays, compliant with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm, to assess the risk of chloroquine (CLQ) or hydroxychloroquine (OH-CLQ)-mediated QT prolongation and Torsades de Pointes (TdP), alone and combined with erythromycin (ERT) and azithromycin (AZI), drugs repurposed during the first wave of coronavirus disease 2019 (COVID-19). Each drug or drug combination was tested in patch clamp assays on seven cardiac ion channels, in in silico models of human ventricular electrophysiology (Virtual Assay) using control (healthy) or high-risk cell populations, and in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. In each assay, concentration-response curves encompassing and exceeding therapeutic free plasma levels were generated. Both CLQ and OH-CLQ showed blocking activity against some potassium, sodium, and calcium currents. CLQ and OH-CLQ inhibited IKr (half-maximal inhibitory concentration [IC50 ]: 1 µM and 3-7 µM, respectively) and IK1 currents (IC50 : 5 and 44 µM, respectively). When combining OH-CLQ with AZI, no synergistic effects were observed. The two macrolides had no or very weak effects on the ion currents (IC50  > 300-1000 µM). Using Virtual Assay, both antimalarials affected several TdP indicators, CLQ being more potent than OH-CLQ. Effects were more pronounced in the high-risk cell population. In hiPSC-derived cardiomyocytes, all drugs showed early after-depolarizations, except AZI. Combining CLQ or OH-CLQ with a macrolide did not aggravate their effects. In conclusion, our integrated nonclinical CiPA dataset confirmed that, at therapeutic plasma concentrations relevant for malaria or off-label use in COVID-19, CLQ and OH-CLQ use is associated with a proarrhythmia risk, which is higher in populations carrying predisposing factors but not worsened with macrolide combination.

Expression of Indoleamine 2,3-Dioxygenase Gene Is a Feature of Poorly Differentiated Non-muscle-invasive Urothelial Cell Bladder Carcinomas.

AIM: To evaluate indoleamine 2,3-dioxygenase (IDO) gene expression in non-muscle-invasive urothelial cell bladder carcinoma (NMIBC). PATIENTS AND METHODS: Seventy-four patients undergoing surgical treatment for NMIBC were enrolled in the study. IDO gene expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: IDO gene expression was detectable significantly more frequently (48/74, 64.86% vs. 5/21, 23.81%, p<0.001) and to significantly higher extents (p=0.01) in cancer tissues than in normal bladder mucosa. IDO gene expression was observed significantly more frequently in large (p=0.02), high-grade (p=0.05) and stage T1 (p=0.03) than in small, low-grade and stage Ta tumors. Expression levels were also significantly higher in large, high-grade and stage T1 tumors (p<0.01, p=0.05 and p=0.03, respectively). A direct positive correlation between IDO gene expression in tumor tissues and tumor size (R=0.24, p=0.04), grade (R=0.23, p=0.05) and stage (R=0.25, p=0.03) was detected. Multivariate analysis suggested a trend (p=0.08) towards longer overall survival in patients bearing tumors that did not express IDO gene. CONCLUSION: These data indicate that IDO gene expression is a feature of aggressive NMIBC, suggesting a potential immunosuppressive role of IDO.

MAGE-A10 cancer/testis antigen is highly expressed in high-grade non-muscle-invasive bladder carcinomas.

Bladder cancer is a common urinary malignancy and a prevalent cause of cancer-related death. Current therapies of early stage non-muscle-invasive bladder cancer (NMIBC) are frequently associated with undesirable toxicities and recurrence. Active antigen-specific immunotherapy may provide a valid therapeutic option for patients with NMIBC. Cancer-testis antigens (CTA) expressed in various tumour types and in a limited range of healthy tissues may represent potential targets for specific immunotherapy. MAGE-A10 is probably the most immunogenic antigen of the MAGE-A family. We evaluated the expression of MAGE-A10 in NMIBC. Seventy-nine patients undergoing surgical treatment for NMIBC were enrolled in the study. MAGE-A10 gene expression was assessed by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed on paraffin-embedded sections. MAGE-A10 gene was specifically expressed in one-third of NMIBC (n = 24: 32.43%). Gene expression was correlated with high tumour grade. MAGE-A10 protein was exclusively detectable in nuclei of tumour cells. More importantly, MAGE-A10 protein was also more frequently detectable in high-grade tumours (p = 0.0001) and in stage T1 tumours invading subepithelial tissue or lamina propria (p = 0.01). A strong correlation between MAGE-A10 staining score and tumour grade and stage could accordingly be observed. These data indicate that MAGE-A10 expression is a feature of aggressive NMIBC and might be used as a novel target for specific immunotherapy of these cancers.