Preliminary in vitro assessment of pharmacokinetic drug-drug interactions of EST64401 and EST64514, two sigma-1 receptor antagonists.

EST64401 and EST64514 are two selective sigma-1 receptor ligands that showed a good profile in a lead optimization process for oral pain treatment. Their potential for pharmacokinetic-based drug-drug interactions was assessed to anticipate clinical interactions.Both compounds showed a low potential for CYP inhibition with percentages of inhibition <50% at 1 µM in recombinant human CYPs (CYP1A2, 2C9, 2C19, 2D6 and 3A4) and IC50 ≥75 µM for CYP3A4 and 2D6 in human liver microsomes.No CYP induction was observed for CYP1A2, 2B6 and 3A4 at concentrations ≤25 µM (EST64401) or ≤50 µM (EST64514) in human hepatocytes using as endpoints CYP activities and mRNA levels.More than one enzyme participated in compound metabolism. The main enzymes involved were CYP3A4 for EST64401 and CYP2D6 besides CYP3A4 for EST64514.Neither EST64401 nor EST64514 seemed to be substrates of P-gp or BCRP in Caco-2 cells (efflux ratio ≤2). Transporter inhibition was observed at concentrations ≥20 µM; EST64401 only inhibiting P-gp at higher concentrations (≥125 µM).Preliminary in vitro interaction studies suggest a similar profile for EST64401 and EST64514. Therefore, other properties will have to be considered for compound differentiation and selection for further development.

Preliminary in vitro approach to evaluate the drug-drug interaction potential of EST73502, a dual µ-opioid receptor partial agonist and σ1 receptor antagonist.

The potential for drug-drug interactions (DDI) of EST73502 was preliminary explored in vitro. EST73502 is a new chemical entity intended for oral pain treatment with dual sigma-1 receptor (σ1R) antagonism and µ-opioid receptor (MOR) partial agonism, that presents a promising potent analgesic activity.Several enzymes were involved in EST73502 metabolism catalysing the formation of different metabolites, CYP3A4 and CYP2D6 being the main ones.Fraction unbound was determined due to its impact in interactions, a considerable proportion of EST73502 being available.EST73502 showed a low potential for CYP inhibition, except for CYP2D6 that showed time-dependent inhibition.No induction potential was found for CYP1A2 and 3A4, while CYP2B6 was induced at high concentration.EST73502 seemed to be a potential efflux transporter substrate (efflux ratio ≥ 2) but a negligible in vivo impact would be expected due to its high solubility and permeability in Caco-2 cells. P-gp inhibition was observed while no BCRP inhibition was detected.Preliminary in vitro interaction studies suggested that neither CYPs nor efflux transporters interactions would preclude further development of EST73502 to thoroughly assess the clinical relevance of these findings.

Preliminary in Vitro Assessment of the Potential of EST64454, a Sigma-1 Receptor Antagonist, for Pharmacokinetic Drug-Drug Interactions.

EST64454 is a selective sigma-1 receptor ligand intended for orally administered pain treatment that showed a promising profile in the lead optimization process. As part of the preliminary compound profiling, the potential for future drug-drug interactions was explored in vitro. Both direct and time-dependent CYP inhibition for CYP1A2, 2C9, 2C19, 2D6 and 3A4 was studied in human liver microsomes. EST64454 showed a low potential for CYP inhibition (IC50 between 100 and 1000 µM) and as time-dependent inhibitor (IC50 shift mainly around 1). CYP induction studies with HepaRG™ cells revealed no CYP induction at concentrations ≤50 µM, as shown by the CYP1A2, 3A4 and 2B6 activities measured. Reaction phenotyping was assessed after incubation with recombinant human enzymes. Although a very low metabolism was observed, several enzymes catalyzed the formation of metabolites, including CYP3A4, 2C19 and flavin monooxygenases (FMO) 1 and 3. EST64454 was not a P-glycoprotein (P-gp) substrate and was highly permeable in Caco-2 cells. P-gp inhibition was only observed at 200 µM, the highest concentration studied. Preliminary studies suggest that neither CYP nor P-gp interaction of EST64454 would be of any concern for further development. At later stages, the interaction kinetics and the clinical relevance of these findings will be thoroughly evaluated.

Early evolution of BRAFV600 status in the blood of melanoma patients correlates with clinical outcome and identifies patients refractory to therapy.

Serial analysis of BRAF mutations in circulating-free DNA (cfDNA) could be of prognostic value in melanoma patients. We collected blood samples from 63 advanced BRAFV600E/K melanoma patients and determined BRAFV600E/K status in cfDNA using a quantitative 5'-nuclease PCR-based assay. Levels of BRAF mutation in pre-cfDNAs were associated significantly with tumour burden, progression-free survival and overall survival. Changes in BRAF status in cfDNA after initiation of treatment (early-cfDNA) had a significant correlation with outcome. In patients with persistent BRAF mutations (n=12), progression-free survival and overall survival were 3.5 months [95% confidence interval (CI): 1.6-4.6] and 5.3 months (95% CI: 3.4-8.1) compared with 16.6 months (95% CI: 8.2-22.3) and 21.9 months (95% CI: 10.2-NR) in patients with BRAF negativization (n=16), and 15.1 months (95% CI: 2.3-NR) and NR (95% CI: 5.1-NR) in patients who maintained their initial negative status (n=12) (P<0.0001). The median duration of response in patients with radiological response, but persistence of BRAFV600 in early-cfDNA (n=5) was 4 months. Our study indicates that serial BRAF testing in the blood of advanced melanoma identifies patients refractory to therapy.

Usefulness of circulating free DNA for monitoring epidermal growth factor receptor mutations in advanced non-small cell lung cancer patients: a case report.

Genomic analysis of circulating tumor DNA (ctDNA) released from cancer cells into the bloodstream has been proposed as a useful method to capture dynamic changes during the course of the disease. In particular, the ability to monitor epidermal growth factor receptor (EGFR) mutation status in cell-free circulating DNA (cfDNA) isolated from advanced non-small cell lung cancer (NSCLC) patients EGFR can help to the correct management of the disease and overcome the challenges associated with tumor heterogeneity and insufficient biopsied material to perform key molecular diagnosis. Here, we report a case of long term monitorization of EGFR mutation status in cfDNA from peripheral blood in an NSCLC patient in, with excellent correlation with clinical evolution.

Liquid Biopsy in Non-Small Cell Lung Cancer.

Liquid biopsy analyses are already incorporated in the routine clinical practice in many hospitals and oncology departments worldwide, improving the selection of treatments and monitoring of lung cancer patients. Although they have not yet reached its full potential, liquid biopsy-based tests will soon be as widespread as "standard" biopsies and imaging techniques, offering invaluable diagnostic, prognostic, and predictive information. This review summarizes the techniques available for the isolation and analysis of circulating free DNA and RNA, exosomes, tumor-educated platelets, and circulating tumor cells from the blood of cancer patients, presents the methodological challenges associated with each of these materials, and discusses the clinical applications of liquid biopsy testing in lung cancer.

Synthesis and structure-activity relationship study of a new series of selective σ(1) receptor ligands for the treatment of pain: 4-aminotriazoles.

The synthesis and pharmacological activity of a new series of 4-aminotriazoles as potent σ1 receptor (σ1R) ligands are reported. The compounds were prepared using a 4-5-step process, involving as a key step a click chemistry reaction between ynamides and azides. The most active compounds exhibited nanomolar potency for the σ1R, and the selectivity over the σ2R was improved on decreasing the central amine basicity. It was concluded that in order to achieve good σ1R potency a minimum lipophilicity was required, while limiting to a defined range of cLogP avoided human ether-a-go-go-related gene channel inhibition. This made the most interesting derivatives to be concentrated in a narrow margin of lipophilicity. Among them, compound 13g exhibited the most potent in vivo antinociceptive properties, which are indicative of its antagonist character.