Therapeutic Potential of Fingolimod and Dimethyl Fumarate in Non-Small Cell Lung Cancer Preclinical Models.

New therapies are required for patients with non-small cell lung cancer (NSCLC) for which the current standards of care poorly affect the patient prognosis of this aggressive cancer subtype. In this preclinical study, we aim to investigate the efficacy of Fingolimod, a described inhibitor of sphingosine-1-phosphate (S1P)/S1P receptors axis, and Dimethyl Fumarate (DMF), a methyl ester of fumaric acid, both already approved as immunomodulators in auto-immune diseases with additional expected anti-cancer effects. The impact of both drugs was analyzed with in vitro cell survival analysis and in vivo graft models using mouse and human NSCLC cells implanted in immunocompetent or immunodeficient mice, respectively. We demonstrated that Fingolimod and DMF repressed tumor progression without apparent adverse effects in vivo in three preclinical mouse NSCLC models. In vitro, Fingolimod did not affect either the tumor proliferation or the cytotoxicity, although DMF reduced tumor cell proliferation. These results suggest that Fingolimod and DMF affected tumor progression through different cellular mechanisms within the tumor microenvironment. Fingolimod and DMF might uncover potential therapeutic opportunities in NSCLC.

Blinding and Randomization.

Most, if not all, guidelines, recommendations, and other texts on Good Research Practice emphasize the importance of blinding and randomization. There is, however, very limited specific guidance on when and how to apply blinding and randomization. This chapter aims to disambiguate these two terms by discussing what they mean, why they are applied, and how to conduct the acts of randomization and blinding. We discuss the use of blinding and randomization as the means against existing and potential risks of bias rather than a mandatory practice that is to be followed under all circumstances and at any cost. We argue that, in general, experiments should be blinded and randomized if (a) this is a confirmatory research that has a major impact on decision-making and that cannot be readily repeated (for ethical or resource-related reasons) and/or (b) no other measures can be applied to protect against existing and potential risks of bias.

Proarrhythmic risk assessment using conventional and new in vitro assays.

Drug-induced QT prolongation is a major safety issue in the drug discovery process. This study was conducted to assess the electrophysiological responses of four substances using established preclinical assays usually used in regulatory studies (hERG channel or Purkinje fiber action potential) and a new assay (human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs)-field potential). After acute exposure, moxifloxacin and dofetilide concentration-dependently decreased IKr amplitude (IC50 values: 102 µM and 40 nM, respectively) and lengthened action potential (100 µM moxifloxacin: +23% and 10 nM dofetilide: +18%) and field potential (300 µM moxifloxacin: +76% and 10 nM dofetilide: +38%) durations. Dofetilide starting from 30 nM induced arrhythmia in hiPSC-CMs. Overnight application of pentamidine (10 and 100 µM) and arsenic (1 and 10 µM) decreased IKr, whereas they were devoid of effects after acute application. Long-term pentamidine incubation showed a time- and concentration-dependent effect on field potential duration. In conclusion, our data suggest that hiPSC-CMs represent a fully functional cellular electrophysiology model which may significantly improve the predictive validity of in vitro safety studies. Thereafter, lead candidates may be further investigated in patch-clamp assays for mechanistic studies on individual ionic channels or in a multicellular Purkinje fiber preparation for confirmatory studies on cardiac conduction.

Treatment of patients with carcinomas in advanced stages with 5-fluorouracil, folinic acid and pyridoxine in tandem.

The effect of high-dose pyridoxine (PN) on activity of 5-fluorouracil (FUra) and folinic acid (FA)-containing regimens was studied in 50 patients including 14 with digestive tract, and 36 with breast carcinomas (BC) in advanced stages with poor prognostic characteristics. Patients with colorectal, and pancreas adenocarcinoma received oxaliplatin, irinotecan, FUra, FA (Folfirinox), and patients with squamous cell carcinoma of the esophagus had paclitaxel, carboplatin, FUra, FA (TCbF). Patients with BC received AVCF (doxorubicin, vinorelbine, cyclophosphamide, FUra, FA) followed by TCbF or TCbF only, and patients who overexpressed HER2 received TCbF plus trastuzumab and pertuzumab. PN (1000-3000 mg/day iv) preceded each administration of FUra and FA. 47 patients (94%) responded, including 16 (32%) with CR. Median tumor reduction was 93%. Median event-free survival (EFS) was 37.7 months. The 25 patients with tumor shrinkage ≥ 91% had EFS of 52% from 42 months onwards. Unexpected toxicity did not occur. PN enhances potency of chemotherapy regimens comprising FUra and FA.

An Update on Stiripentol Mechanisms of Action: A Narrative Review.

Stiripentol (Diacomit®) (STP) is an orally active antiseizure medication (ASM) indicated as adjunctive therapy, for the treatment of seizures associated with Dravet syndrome (DS), a severe form of childhood epilepsy, in conjunction with clobazam and, in some regions valproic acid. Since the discovery of STP, several mechanisms of action (MoA) have been described that may explain its specific effect on seizures associated with DS. STP is mainly considered as a potentiator of gamma-aminobutyric acid (GABA) neurotransmission: (i) via uptake blockade, (ii) inhibition of degradation, but also (iii) as a positive allosteric modulator of GABAA receptors, especially those containing α3 and δ subunits. Blockade of voltage-gated sodium and T-type calcium channels, which is classically associated with anticonvulsant and neuroprotective properties, has also been demonstrated for STP. Finally, several studies indicate that STP could regulate glucose energy metabolism and inhibit lactate dehydrogenase. STP is also an inhibitor of several cytochrome P450 enzymes involved in the metabolism of other ASMs, contributing to boost their anticonvulsant efficacy as add-on therapy. These different MoAs involved in treatment of DS and recent data suggest a potential for STP to treat other neurological or non-neurological diseases.

Nonhuman primates: translational models for predicting antipsychotic-induced movement disorders.

Repeated haloperidol treatment administered to nonhuman primates (NHPs) over several months or even years leads to the gradual appearance of drug-induced dystonic reactions in the orofacial region (mouth opening, tongue protrusion or retraction, bar biting) and in the whole body (writhing of the limbs and trunk, bar grasping). The propensity of antipsychotics to induce dystonia in NHPs is not correlated with their propensity to induce catalepsy in rodents, suggesting that the two types of effects are dissociated and may represent distinct aspects of the extrapyramidal symptoms induced by antipsychotics. In view of the clear homology to clinically observed phenomena, antipsychotic-induced dystonias in antipsychotic-primed NHPs would appear to possess a high degree of translational validity. These NHP phenomena could therefore serve as a useful model for predicting the occurrence of similar abnormal movements with novel substances developed for the treatment of schizophrenia or other psychotic disorders. Moreover, the NHP dystonia model could possibly serve as a biomarker for substances that will eventually cause tardive dyskinesia in patients.

In vitro safety cardiovascular pharmacology studies: impact of formulation preparation and analysis.

Collection of formulation samples is required for GLP in vitro studies to check the exposure of the test system and allow reliable determinations of safety margins. In vitro studies conducted in-house were investigated to evaluate problems of solubility, stability and adsorption of the formulations. Terfenadine was used as reference substance to illustrate the purpose. Lowered target concentrations of test substances in in vitro studies can be attributed to the solubility limitation in the superfusion medium, the low stability under frozen conditions (24% of the final solutions stable at -20 °C) and/or the adsorption on the superfusion tubing (30% of the studies). Terfenadine also showed a limited solubility (measured concentrations ranging from 0.597 µM to 0.833 µM instead of 1 µM) and a loss of substance through the superfusion tubing from -30.2% to -39.2% with dimethylsulfoxide, ethanol or methanol. Terfenadine solubility was improved with 2-hydroxypropyl-ß-cyclodextrin, no adsorption was observed, but its capacity to block the hERG channel was decreased. It is recommended to determine the substance solubility in appropriate buffers, to evaluate possible adsorption during method validation (formulation samples collected after superfusion), and to prepare fresh formulation each testing day with immediate analysis in absence of stability data. This strategy clearly favors single-site as opposed to multi-site studies.

Evaluation of Temozolomide and Fingolimod Treatments in Glioblastoma Preclinical Models.

Glioblastomas are malignant brain tumors which remain lethal due to their aggressive and invasive nature. The standard treatment combines surgical resection, radiotherapy, and chemotherapy using Temozolomide, albeit with a minor impact on patient prognosis (15 months median survival). New therapies evaluated in preclinical translational models are therefore still required to improve patient survival and quality of life. In this preclinical study, we evaluated the effect of Temozolomide in different models of glioblastoma. We also aimed to investigate the efficacy of Fingolimod, an immunomodulatory drug for multiple sclerosis also described as an inhibitor of the sphingosine-1-phosphate (S1P)/S1P receptor axis. The effects of Fingolimod and Temozolomide were analyzed with in vitro 2D and 3D cellular assay and in vivo models using mouse and human glioblastoma cells implanted in immunocompetent or immunodeficient mice, respectively. We demonstrated both in in vitro and in vivo models that Temozolomide has a varied effect depending on the tumor type (i.e., U87MG, U118MG, U138MG, and GL261), demonstrating sensitivity, acquired resistance, and purely resistant tumor phenotypes, as observed in patients. Conversely, Fingolimod only reduced in vitro 2D tumor cell growth and increased cytotoxicity. Indeed, Fingolimod had little or no effect on 3D spheroid cytotoxicity and was devoid of effect on in vivo tumor progression in Temozolomide-sensitive models. These results suggest that the efficacy of Fingolimod is dependent on the glioblastoma tumor microenvironment. Globally, our data suggest that the response to Temozolomide varies depending on the cancer model, consistent with its clinical activity, whereas the potential activity of Fingolimod may merit further evaluation.

A Face-To-Face Comparison of Tumor Chicken Chorioallantoic Membrane (TCAM) In Ovo with Murine Models for Early Evaluation of Cancer Therapy and Early Drug Toxicity.

Ethical considerations, cost, and time constraints have highlighted the need to develop alternatives to rodent in vivo models for evaluating drug candidates for cancer. The tumor chicken chorioallantoic membrane (TCAM) model provides an affordable and fast assay that permits direct visualization of tumor progression. Tumors from multiple species including rodents and human cell lines can be engrafted. In this study, we engrafted several tumor models onto the CAM and demonstrated that the TCAM model is an alternative to mouse models for preliminary cancer drug efficacy testing and toxicity analysis. Tumor cells were deposited onto CAM, and then grown for up to an additional 10 days before chronic treatments were administered. The drug response of anticancer therapies was screened in 12 tumor cell lines including glioblastoma, melanoma, breast, prostate, colorectal, liver, and lung cancer. Tumor-bearing eggs and tumor-bearing mice had a similar chemotherapy response (cisplatin and temozolomide) in four human and mouse tumor models. We also demonstrated that lethality observed in chicken embryos following chemotherapies such as cisplatin and cyclophosphamide were associated with corresponding side-effects in mice with body weight loss. According to our work, TCAM represents a relevant alternative model to mice in early preclinical oncology screening, providing insights for both the efficacy and the toxicity of anticancer drugs.

Anti-CTLA-4 and anti-PD-1 immunotherapies repress tumor progression in preclinical breast and colon model with independent regulatory T cells response.

The recent development of immunotherapy represents a significant breakthrough in cancer therapy. Several immunotherapies provide robust efficacy gains in a wide variety of cancers. However, in some patients the immune checkpoint blockade remains ineffective due to poor therapeutic response and tumor relapse. An improved understanding of the mechanisms underlying tumor-immune system interactions can improve clinical management of cancer. Here, we report preclinical data evaluating two murine antibodies corresponding to recent FDA-approved antibodies for human therapy, e.g. anti-CTLA-4 and anti-PD-1. We demonstrated in two mouse syngeneic grafting models of triple negative breast or colon cancer that the two antibodies displayed an efficient anticancer activity, which is enhanced by combination treatment in the breast cancer model. We also demonstrated that CTLA-4 targeting reduced metastasis formation in the colon cancer metastasis model. In addition, using cytometry-based multiplex analysis, we showed that anti-CTLA-4 and anti-PD-1 affected the tumor immune microenvironment differently and in particular the tumor immune infiltration. This work demonstrated anti-cancer effect of CTLA-4 or PD-1 blockade on mouse colon and triple negative breast and on tumor-infiltrating immune cell subpopulations that could improve our knowledge and benefit the breast and colon cancer tumor research community.