Desarrollo preclínico de antagonistas de canales TRPM8 para el tratamiento de la alodinia asociada a quimioterapia antitumoral

Zusammenfassung

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a dysfrunction of the peripheral system nerves, which is developed in many patients receiving standard doses of Chemotherapy, and in almost all patients treated with high doses. To date, there are no effective therapeutic strategies to combat this pathology, which causes pain, cold sensitivity, parestesia, inflammation and muscle weakness, normally starting by hands and feet. There are several experimental evidences that implicate TRPM8 channels in the cold hypersensitivity caused by chemotherapy, so the modulation of these channels could be considered a therapeutic alternative to treat CIPN. A few years ago, our research group discovered a series of β-lactam compounds that behave as TRPM8 channel antagonists (patent WO2017005950). After the interest of Alodia Farmacéutica SL in these compounds, the company licensed the patent, and we started the industrial PhD, program described in this report. Two main objectives were proposed: 1) To develop a β-lactam, selected among the patent compounds, as a neurocosmetic ingredient to alleviate cold allodynia induced by chemotherapeutic treatments. 2) To start the preclinical development of this family of compounds in order to find a topical treatment for this pathology. To achive the first objective, in Chapter 1, we describe the scale-up synthesis of the selected β-lactam, initially based on the patent procedure, and subsequently carried out under GLPs (Good Laboratory Practice), through modifications in some reaction steps. Next, the in vitro characterization of the pure selected compound, including activity on rat and human TRPM8 channels, selectivity against other pain-related receptors, and dermal irritancy and mutagenicity were performed. Finally, β-lactam was formulated into cosmetic preparations (hand cream and foot cream) and the dermal absortion, termal stability and the ability of the preservative against microbial contamination were studied. This part of the work concluded in the approval of the selected β-lactam as a neurocosmetic ingredient and in two creams that will be lanched to the market soon. As for to the second objective, in Chapter 2, we describe new β-lactam derivatives, prepared for stablishing structure-activity relationships (>50 new derivatives). Preliminary evaluation, identified 4-carboxamide derivatives as more potent compounds than the model β-lactam. Alternative synthesis strategies for the formation of these 2-azetidinones were also explored: while the combination of multicomponent UGI and cyclization reactions led to product mixtures, and solid-phase methodologies using a “Safety Catch”-resin did not allow cleavable compounds, a benzyl-type Wang-resin allowed the preparation of 4-carboxylate intermediates, without any intermediate purificaction. Moreover, studies on the influence of the configuration on the TRPM8 antagonist activity showed the best results for 3R,4R,2’R isomers. Finally, a photoswitchable β-lactam allowed the control of the TRPM8 antagonist activity by light. In Chapter 3, the pharmacological characterization of a selection of these new compounds was detailed. Initially, the preliminary activity was confirmed by electrophysiology (Patch-Clamp) and in HEK cells expressing the human hTRPM8 channels, showing that the compounds are also active. In addition, we performed selectivity studies, by measuring the activity against other pain-related channels and receptors, and evaluation of ADME pharmacokinetis properties. Finally, three animal pain models were used for the in vivo preclinical assays. Despite a fast metabolism in liver microsomes, the selected β-lactams were able to reduce cold allodynia in mice with peripheral neuropathy after chemotherapy (i.pl.), and cold and mechanical allodynia in a rat model of neurophatic pain (CCI, i.pl. and i.p.). One of these β-lactams also reduced migraineous pain in male mice and, preferentially, in female mice after i.v. administration.