ß-Lactam TRPM8 Antagonists Derived from Phe-Phenylalaninol Conjugates: Structure-Activity Relationships and Antiallodynic Activity.

The protein transient receptor potential melastatin type 8 (TRPM8), a non-selective, calcium (Ca2+)-permeable ion channel is implicated in several pathological conditions, including neuropathic pain states. In our previous research endeavors, we have identified ß-lactam derivatives with high hydrophobic character that exhibit potent and selective TRPM8 antagonist activity. This work describes the synthesis of novel derivatives featuring C-terminal amides and diversely substituted N'-terminal monobenzyl groups in an attempt to increase the total polar surface area (TPSA) in this family of compounds. The primary goal was to assess the influence of these substituents on the inhibition of menthol-induced cellular Ca2+ entry, thereby establishing critical structure-activity relationships. While the substitution of the tert-butyl ester by isobutyl amide moieties improved the antagonist activity, none of the N'-monobencyl derivatives, regardless of the substituent on the phenyl ring, achieved the activity of the model dibenzyl compound. The antagonist potency of the most effective compounds was subsequently verified using Patch-Clamp electrophysiology experiments. Furthermore, we evaluated the selectivity of one of these compounds against other members of the transient receptor potential (TRP) ion channel family and some receptors connected to peripheral pain pathways. This compound demonstrated specificity for TRPM8 channels. To better comprehend the potential mode of interaction, we conducted docking experiments to uncover plausible binding sites on the functionally active tetrameric protein. While the four main populated poses are located by the pore zone, a similar location to that described for the N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist cannot be discarded. Finally, in vivo experiments, involving a couple of selected compounds, revealed significant antinociceptive activity within a mice model of cold allodynia induced by oxaliplatin (OXA).

BTLA dysregulation correlates with poor outcome and diminished T cell-mediated antitumor responses in chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) progressively develop marked immunosuppression, dampening innate and adaptive-driven antitumor responses. However, the underlying mechanisms promoting immune exhaustion are largely unknown. Herein, we provide new insights into the role of BTLA/HVEM axis promoting defects in T cell-mediated responses against leukemic cells. Increased expression of BTLA, an inhibitory immune checkpoint, was detected on the surface of CD4 + and CD8 + T lymphocytes in patients with CLL. Moreover, high levels of BTLA on CD4 + T cells correlated with diminished time to treatment. Signaling through BTLA activation led to decreased IL-2 and IFN-γ production ex vivo, whereas BTLA/HVEM binding disruption enhanced IFN-γ + CD8 + T lymphocytes. Accordingly, BTLA blockade in combination with bispecific anti-CD3/anti-CD19 antibody promoted CD8 + T cell-mediated anti-leukemic responses. Finally, treatment with an anti-BLTA blocking monoclonal antibody alone or in combination with ibrutinib-induced leukemic cell depletion in vitro. Altogether, our data reveal that BTLA dysregulation has a prognostic role and is limiting T cell-driven antitumor responses, thus providing new insights about immune exhaustion in patients with CLL.

Consideraciones sobre integrantes del acto médico tomadas de la literatura / Considerations on Members of the Medical Act Taken from the Literature

Se acepta que la literatura puede ser útil para la enseñanza de la medicina porque ambas disciplinas trabajan con sentimientos de personas y consideran la relación interpersonal como el marco de referencia de su actividad profesional. En este trabajo se aportan una serie de ejemplos integrados por fragmentos de textos narrativos tomados de diferentes obras literarias que inciden en el valor de las descripciones y percepciones de diversos autores sobre aspectos de la interacción entre diferentes actores del acto médico. En síntesis, se refleja la imagen cambiante de la medicina y los médicos, del médico como persona, del efecto curativo de la interacción médico-enfermo, y de la interacción con el sistema sanitario. Si bien se aprecia el carácter altruista y abnegado de la práctica médica, los testimonios vertidos en algunos textos literarios dejan constancia de la necesidad de prestar más atención a la formación en empatía no sólo de los profesionales sanitarios, sino también de cuantas personas realizan su trabajo en el ámbito sanitario. (AU)

It is well accepted that literature can be useful for the teaching of medicine because both disciplines study people's feelings and consider the interpersonal relationship as the frame of reference for their professional activity. In the present work, a set of examples are provided, made up of fragments of narrative texts taken from different literary works that have impact on the value of the descriptions and perceptions of several authors on aspects of the interaction between different participants in the medical act. Briefly, it reflects the changing picture of medicine and doctors, the doctor as a person, the healing effect of the doctor-patient interaction, and of the interaction with the healthcare system. Whilst the altruistic and selfless nature of medical practice is appreciated, the proofs expressed in some literary texts show the need to pay more attention to training on the empathy not only for health professionals, but also for all the people who work in the healthcare system. (AU)

En torno al concepto de salud y enfermedad. Un dialogo entre la medicina, la literatura y la filosofía / Around the Concept of Health and Disease. A Dialogue between Medicine, Literature and Philosophy

Un problema de la medicina son las incertidumbres epistémicas que le son propias, tanto por sus métodos de elaboración conceptual como por las que toma de las ciencias naturales y sociales en las que se apoya. En consecuencia, la medicina no es una ciencia, aunque sus bases son cada vez más científicas. Dado que en la medicina en general, y en los conceptos de salud y enfermedad, y en la realidad de los enfermos hay componentes biológicos, psicológicos y sociológicos, no tiene nada de particular invocar a la filosofía y a la literatura para la elaboración de las teorías de la medicina. En este artículo planteamos algunos de estos enfoques, se resaltan las limitaciones de la concepción biologicista y reduccionista de la medicina y se estima que adquiere mayor potencia explicativa de los conceptos de salud y enfermedad cuando se consideran los aspectos humanos y sociales de la teoría y la práctica de la medicina. Además, la literatura aporta una imagen social de la medicina y de los enfermos que deberíamos integrar en los conceptos de salud, enfermedad y enfermo. Llamamos, por último, la atención sobre un proceso que parece haber desaparecido de la práctica médica como es la convalecencia y la necesidad de una completa restauración de la salud por el riesgo, como advierte el aforismo hipocrático, de que de los residuos que quedan de las enfermedades, suelen surgir las recidivas. (AU)

A problem of medicine is the epistemic uncertainties that are inherent to it, due to its both conceptual elaboration methods and those it takes from the natural and social sciences on which they are based on. Consequently, medicine is not a science, although its basis are scientifically increasing. Given that there are biological, psychological and sociological aspects in medicine in general, and in the concepts of health and disease, and in the reality of patients, there is nothing special about invoking philosophy and literature for the elaboration of theories of medicine. In the present article we consider some of these approaches, the limitations of the biological and reductionist conception of medicine are highlighted and it is estimated that it acquires greater explanatory power of the concepts of health and disease when the human and social aspects of theory and medicine are considered. In addition, literature provides a social image of medicine and of patients that we should integrate into the concepts of health, disease and patient. Finally, we draw attention to a process that seems to have disappeared from medical practice, such as convalescence and the need for a total restoration of health due to the risk, as the Hippocratic aphorism warns, that the residues that remain from disease, relapse often occurs. (AU)

Valor formativo del cine y la literatura en los estudiantes de Ciencias de la Salud. Aspectos generales / Formative value of cinema and literature in health sciences students. General features

El objetivo de este trabajo es presentar los fundamentos conceptuales de la asignatura optativa Cine, Literatura y Medicina que se imparte en segundo año del grado en Medicina de la Universidad de Oviedo desde el curso académico 2011-2012. Los contenidos que se comentan suponen una introducción general a las formas en las que el cine y la literatura pueden contribuir a la enseñanza de la medicina y de cómo pueden contribuir a la exposición de los estudiantes a aspectos transversales de la medicina dispersos a lo largo de los cursos del Grado. El lenguaje cinematográfico facilita el abordaje de temas complejos en los que las cuestiones éticas y sociales de las enfermedades pueden tratarse mediante la interpretación por actores de forma clara, concisa y dirigida a los elementos nucleares de problemas densos que de otra forma sería dificultoso y lento comprender. La literatura, tanto mediante la escritura como de la lectura, permite aliviar la tensión emocional que produce la enfermedad, interiorizar de forma paciente y reflexiva la experiencia de enfermar, y reorientar la actividad vital en consonancia con la imposición de limitaciones que son propias y características de cada proceso nosológico. (AU)

The aim of this work is to present the conceptual BASIS of the optional subject Cinema, Literature and Medicine that is taught in the second year of the degree in Medicine at the University of Oviedo since 2011-2012 school year. The contents discussed are a general introduction to the ways in which film and literature can help to the teaching of medicine and how they can contribute to the exposure of students to cross-cutting aspects of medicine unfocused throughout the whole Degree. Cinematographic language facilitates the approach to complex issues in which the ethical and social aspect of disease can be aced clearly, concisely and directed at the main elements through the interpretation by actors of COMPLEX problems that, OTHERWISE, would be difficult and hard to understand. Literature, both through writing and reading, allows to alleviate the emotional tension caused by the disease, patiently and reflexively internalize the experience of becoming ill, and rearraange vital activity in line with the imposition of limitations that are specific and characteristic of each nosological process. (AU)

ß-Lactam TRPM8 Antagonist RGM8-51 Displays Antinociceptive Activity in Different Animal Models.

Transient receptor potential melastatin subtype 8 (TRPM8) is a cation channel extensively expressed in sensory neurons and implicated in different painful states. However, the effectiveness of TRPM8 modulators for pain relief is still a matter of discussion, since structurally diverse modulators lead to different results, depending on the animal pain model. In this work, we described the antinociceptive activity of a ß-lactam derivative, RGM8-51, showing good TRPM8 antagonist activity, and selectivity against related thermoTRP channels and other pain-mediating receptors. In primary cultures of rat dorsal root ganglion (DRG) neurons, RGM8-51 potently reduced menthol-evoked neuronal firing without affecting the major ion conductances responsible for action potential generation. This compound has in vivo antinociceptive activity in response to cold, in a mouse model of oxaliplatin-induced peripheral neuropathy. In addition, it reduces cold, mechanical and heat hypersensitivity in a rat model of neuropathic pain arising after chronic constriction of the sciatic nerve. Furthermore, RGM8-51 exhibits mechanical hypersensitivity-relieving activity, in a mouse model of NTG-induced hyperesthesia. Taken together, these preclinical results substantiate that this TRPM8 antagonist is a promising pharmacological tool to study TRPM8-related diseases.

Involvement of CD4+ and CD8+ T-lymphocytes in the modulation of nociceptive processing evoked by CCL4 in mice.

AIMS: To explore the mechanisms involved in the transformation of analgesia produced by low doses of CCL4 (pg/kg) to hyperalgesia when higher doses (ng/kg) are administered to mice. MAIN METHODS: The unilateral hot plate test was used to assess thermal nociception. CD3+, CD4+ or CD8+ blood cells were depleted with selective antibodies. Expression of CCR5 and IL-16 in lymphocytes was studied by flow cytometry and IL-16 blood levels were measured by ELISA. IL-16 and CD8 were detected by immunofluorescence. KEY FINDINGS: IL-16 and CCR5 expression were demonstrated in CD4+ and CD8+ T-lymphocytes by flow cytometry. Furthermore, CCL4-induced hyperalgesia was abolished by reducing circulating T-lymphocyte levels or by selectively depleting CD4+ lymphocytes. In contrast, when the anti-CD4 antibody was acutely administered, CCL4 induced analgesia instead of hyperalgesia. A similar response was obtained when administering A-770041, that prevents CD4-mediated CCR5 desensitization by inhibiting p56lck kinase. As occurred with the analgesic effect evoked by low doses of CCL4, analgesia evoked by combining CCL4 and A-770041 was reverted by naloxone, naltrindole or an anti-met-enk antibody. Interestingly, flow cytometry assays showed that the number of CD8+, but not CD4+, T-cells expressing IL-16 is reduced after the acute administration of CCL4, a result compatible with the description that CD8+-lymphocytes can rapidly release preformed IL-16. Accordingly, the rise in IL-16 blood concentration evoked by CCL4 was prevented after CD8+ lymphocyte depletion. SIGNIFICANCE: CCL4-evoked hyperalgesia is related to the desensitization of CCR5 in CD4+ T-cells and to the release of IL-16 from CD8+ lymphocytes.

DD04107-Derived neuronal exocytosis inhibitor peptides: Evidences for synaptotagmin-1 as a putative target.

The analgesic peptide DD04107 (Pal-EEMQRR-NH2) and its acetylated analogue inhibit α-calcitonin gene-related peptide (α-CGRP) exocytotic release from primary sensory neurons. Examining the crystal structure of the SNARE-Synaptotagmin-1(Syt1) complex, we hypothesized that these peptides could inhibit neuronal exocytosis by binding to Syt1, hampering at least partially its interaction with the SNARE complex. To address this hypothesis, we first interrogate the role of individual side-chains on the inhibition of α-CGRP release, finding that E1, M3, Q4 and R6 residues were crucial for activity. CD and NMR conformational analysis showed that linear peptides have tendency to adopt α-helical conformations, but the results with cyclic analogues indicated that this secondary structure is not needed for activity. Isothermal titration calorimetry (ITC) measurements demonstrate a direct interaction of some of these peptides with Syt1-C2B domain, but not with Syt7-C2B region, indicating selectivity. As expected for a compound able to inhibit α-CGRP release, cyclic peptide derivative Pal-E-cyclo[EMQK]R-NH2 showed potent in vivo analgesic activity, in a model of inflammatory pain. Molecular dynamics simulations provided a model consistent with KD values for the interaction of peptides with Syt1-C2B domain, and with their biological activity. Altogether, these results identify Syt1 as a potential new analgesic target.

Kappa-opioid receptor-mediated thermal analgesia evoked by the intrathecal administration of the chemokine CCL1 in mice.

BACKGROUND: The chemokine CC motif ligand 1 (CCL1) participates in immune cell recruitment and, as other chemokines, is also involved in nociceptive processing. In contrast with previous reports indicating its participation in allodynia and cold hypernociception when spinally administered, its ability to evoke heat thermal analgesia, mediated by circulating leukocytes and endocannabinoids, after systemic administration has recently been reported. OBJECTIVES: Aiming to explore the role played by CCL1 on spinal nociception, we study here the effect of its intrathecal administration on thermal nociception in mice. METHODS: Behavioral nociceptive assays, immunohistochemical experiments, white cell blood depletion procedures and qRT-PCR experiments were performed. RESULTS: The intrathecal administration of CCL1 (0.3-30 ng) produced analgesia as measured by the unilateral hot plate test. This effect peaked 1 h after injection, was prevented by the CCR8 antagonist R243 and was accompanied by a reduction of c-Fos expression in spinal neurons. Whereas blood leukocyte depletion did not modify it, analgesia was abolished by the microglial inhibitor minocycline, but not the astroglial inhibitor aminoadipate. Furthermore, antinociception remained unmodified by the coadministration of cannabinoid type 1 or 2 receptors antagonists. However, it was reversed by naloxone but not by selective blockade of mu- or delta-opioid receptors. The inhibitory effect induced by the selective kappa-opioid receptor antagonist, nor-binaltorphimine, and by an anti-dynorphin A 1-17 antibody indicates that analgesia evoked by spinal CCL1 is mediated by endogenous dynorphins acting on kappa-opioid receptors. CONCLUSIONS: Endogenous dynorphin and microglia behave as key players in heat thermal analgesia evoked by spinal CCL1 in mice.

A capsaicinoid-based soft drug, AG1529, for attenuating TRPV1-mediated histaminergic and inflammatory sensory neuron excitability.

TRPV1, a member of the transient receptor potential (TRP) family, is a nonselective calcium permeable ion channel gated by physical and chemical stimuli. In the skin, TRPV1 plays an important role in neurogenic inflammation, pain and pruritus associated to many dermatological diseases. Consequently, TRPV1 modulators could represent pharmacological tools to respond to important patient needs that still represent an unmet medical demand. Previously, we reported the design of capsaicinoid-based molecules that undergo dermal deactivation (soft drugs), thus preventing their long-term dermal accumulation. Here, we investigated the pharmacological properties of the lead antagonist, 2-((4-hydroxy-2-iodo-5-methoxybenzyl) amino)-2-oxoethyl dodecanoate (AG1529), on heterologously expressed human TRPV1 (hTRPV1), on nociceptor excitability and on an in vivo model of acute pruritus. We report that AG1529 competitively blocked capsaicin-evoked activation of hTRPV1 with micromolar potency, moderately affected pH-induced gating, and did not alter voltage- and heat-mediated responses. AG1529 displays modest receptor selectivity as it mildly blocked recombinant hTRPA1 and hTRPM8 channels. In primary cultures of rat dorsal root ganglion (DRG) neurons, AG1529 potently reduced capsaicin-evoked neuronal firing. AG1529 exhibited lower potency on pH-evoked TRPV1 firing, and TRPA1-elicited nociceptor excitability. Furthermore, AG1529 abolished histaminergic and inflammation mediated TRPV1 sensitization in primary cultures of DRG neurons. Noteworthy, dermal wiping of AG1529, either in an acetone-based formulation or in an anhydrous ointment, dose-dependently attenuated acute histaminergic itch in a rodent model. This cutaneous anti-pruritic effect was devoid of the normal nocifensive action evoked by the burning sensation of capsaicin. Taken together, these preclinical results unveil the mode of action of AG1529 on TRPV1 channels and substantiate the tenet that this capsaicinoid-based soft drug is a promising candidate for drug development as a topical anti-pruritic and anti-inflammatory medication.

Razones por las cuales los estudiantes justifican la elección del Grado en Medicina / Reasons why students justify the choice of Medical Degree

OBJETIVO: Identificar las razones por las cuales los estudiantes acceden al Grado en Medicina. SUJETOS Y MÉTODOS: Estudiantes de primer curso de Grado en Medicina de los años académicos 2014-2015, 2015-2016 y 2017-2018 realizaron una descripción abierta de sus razones de acceso al grado. Éstas se identificaron y clasificaron para su análisis, y se calculó el número de razones aducidas por el conjunto y en cada año académico, y su distribución según sexo. Se clasificaron en categorías para su comparación con otros estudios. RESULTADOS: Participaron 367 estudiantes (252 mujeres y 115 hombres). La diferencia en el porcentaje medio de respuesta entre mujeres (83,71 ± 4,8%) y hombres (82,4 ± 5,1%) no fue estadísticamente significativa. Los estudiantes justificaron su acceso por una media de 2,5 ± 0,06 razones, sin diferencias de sexo ni entre los tres años académicos. Se identificaron 23 razones diferentes para acceder al grado, que se agruparon en categorías, siendo las principales el altruismo, el conocimiento científico, los motivos instrumentales y las razones personales. Aumentó significativamente el acceso por adquisición de conocimiento entre los cursos 2014-2015 y 2017-2018 y se redujeron los accesos por vocación. La consideración social de la medicina o la imagen social del médico se invocaron con muy poca frecuencia. Las mujeres refieren con más frecuencia los motivos altruistas y los relacionados con el conocimiento para el acceso, pero sin diferencias significativas. CONCLUSIÓN: Los valores altruistas continúan siendo los más frecuentes motivos de acceso al Grado en Medicina. La vocación se reduce y la adquisición de conocimientos científicos aumenta como motivos de acceso

AIM: To identify the reasons why students access the Degree in Medicine. SUBJECTS AND METHODS: First-year medical students in 2014-15, 2015-16 and 2017-18 provided an open description of their reasons for access to the degree. These were identified and classified for analysis, the number of reasons given by the whole and in each academic year, as well as their distribution according to gender was calculated. They were classified into categories for comparison with other studies. RESULTS: A total of 367 students (252 women and 115 men) participated. The distribution of women (83.71 ± 4.8%) and men (82.4 ± 5.1%) was not statistically significant. The students justified their access for an average of 2.5 ± 0.06 reasons, with no gender differences or between the three academic years. Twenty-three different reasons for accessing the degree were identified and grouped into categories, the main ones being altruism, scientific knowledge, instrumental motives and personal reasons. There was a significant increase in access through knowledge acquisition between the 2014-15 and 2017-18 academic years, and a reduction in access by vocation. The social consideration of medicine or the social image of the doctor is rarely invoked by students as a reason for accessing the degree. Women refer altruistic and knowledge-related motives for access more frequently than men, but without significant differences. CONCLUSION: Altruistic values continue to be the most frequent reasons for accessing to the Degree in Medicine. The vocation is reduced and the acquisition of scientific knowledge increases as reasons for access

Sobre la vocación médica a través de opiniones tomadas de la literatura médica y de ficción / The medical vocation through literatura

La vocación es una cualidad que define al buen médico y está presente en los profesionales sanitarios de referencia. La vocación médica puede definirse de varias formas, pero tal vez alcance un amplio consenso la que propone que es una motivación profunda de servicio al enfermo y a la sociedad que está determinada por las vivencias y el entorno. Si bien la mayoría de médicos y estudiantes de medicina invocan la vocación y el atractivo intelectual para su elección, no es menos cierto que el azar o la pertenencia a un determinado grupo social pueden ser decisivos en algunos casos. Los literatos, por su parte, suelen atribuir a sus personajes valores tradicionales de la medicina y de la vocación médica como el altruismo o la empatía, así como algunos estereotipos de la imagen de la profesión

The vocation is a value intangible that defines the good doctor and is present in the reference healthcare professionals. The medical vocation can be defined in several ways, a broad consensus of acceptance for the one that proposes it as a deep motivation of service to the patient and society which is determined by the experiences and the environment. While most physicians and medical students invoke the vocation and intellectual appeal for their choice, it is not less true that the chance or being part of certain social groups can be decisive in some cases. The writers, on the other hand, usually attribute traditional values of medicine and medical vocation to their characters such as altruism or empathy, as well as some stereotypes of the profession's

A low pKa ligand inhibits cancer-associated pain in mice by activating peripheral mu-opioid receptors.

The newly designed fentanyl derivative [( ±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide] (NFEPP) was recently shown to produce analgesia selectively via peripheral mu-opioid receptors (MOR) at acidic pH in rat inflamed tissues. Here, we examined the pH-dependency of NFEPP binding to brain MOR and its effects on bone cancer-induced pain in mice. The IC50 of NFEPP to displace bound [3H]-DAMGO was significantly higher compared to fentanyl at pH 7.4, but no differences were observed at pH 5.5 or 6.5. Intravenous NFEPP (30-100 nmol/kg) or fentanyl (17-30 nmol/kg) inhibited heat hyperalgesia in mice inoculated with B16-F10 melanoma cells. The peripherally-restricted opioid receptor antagonist naloxone-methiodide reversed the effect of NFEPP (100 nmol/kg), but not of fentanyl (30 nmol/kg). The antihyperalgesic effect of NFEPP was abolished by a selective MOR- (cyprodime), but not delta- (naltrindole) or kappa- (nor-binaltorphimine) receptor antagonists. Ten-fold higher doses of NFEPP than fentanyl induced maximal antinociception in mice without tumors, which was reversed by the non-restricted antagonist naloxone, but not by naloxone-methiodide. NFEPP also reduced heat hyperalgesia produced by fibrosarcoma- (NCTC 2472) or prostate cancer-derived (RM1) cells. These data demonstrate the increased affinity of NFEPP for murine MOR at low pH, and its ability to inhibit bone cancer-induced hyperalgesia through peripheral MOR. In mice, central opioid receptors may be activated by ten-fold higher doses of NFEPP.

Exploration of TRPM8 Binding Sites by ß-Carboline-Based Antagonists and Their In Vitro Characterization and In Vivo Analgesic Activities.

Transient receptor potential melastatin 8 (TRPM8) ion channel represents a valuable pharmacological option for several therapeutic areas. Here, a series of conformationally restricted derivatives of the previously described TRPM8 antagonist N,N'-dibenzyl tryptophan 4 were prepared and characterized in vitro by Ca2+-imaging and patch-clamp electrophysiology assays. Molecular modeling studies led to identification of a broad and well-defined interaction network of these derivatives inside the TRPM8 binding site, underlying their antagonist activity. The (5R,11aS)-5-(4-chlorophenyl)-2-(4-fluorobenzyl)-5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione (31a) emerged as a potent (IC50 = 4.10 ± 1.2 nM), selective, and metabolically stable TRPM8 antagonist. In vivo, 31a showed significant target coverage in an icilin-induced WDS (at 11.5 mg/kg ip), an oxaliplatin-induced cold allodynia (at 10-30 µg sc), and CCI-induced thermal hyperalgesia (at 11.5 mg/kg ip) mice models. These results confirm the tryptophan moiety as a solid pharmacophore template for the design of highly potent modulators of TRPM8-mediated activities.

Highly functionalized ß-lactams and 2-ketopiperazines as TRPM8 antagonists with antiallodynic activity.

The cool sensor transient receptor potential melastatin channel 8 (TRPM8) is highly expressed in trigeminal and dorsal root ganglia, playing a key role in cold hypersensitivity associated to different peripheral neuropathies. Moreover, these channels are aberrantly expressed in different cancers, and seem to participate in tumor progression, survival and invasion. Accordingly, the search for potent and selective TRPM8 modulators attracted great interest in recent years. We describe new heterocyclic TRPM8 antagonist chemotypes derived from N-cloroalkyl phenylalaninol-Phe conjugates. The cyclization of these conjugates afforded highly substituted ß-lactams and/or 2-ketopiperazine (KP) derivatives, with regioselectivity depending on the N-chloroalkyl group and the configuration. These derivatives behave as TRPM8 antagonists in the Ca2+ microfluorometry assay, and confirmed electrophysiologically for the best enantiopure ß-lactams 24a and 29a (IC50, 1.4 and 0.8 µM). Two putative binding sites by the pore zone, different from those found for typical agonists and antagonists, were identified by in silico studies for both ß-lactams and KPs. ß-Lactams 24a and 29a display antitumor activity in different human tumor cell lines (micromolar potencies, A549, HT29, PSN1), but correlation with TRPM8 expression could not be established. Additionally, compound 24a significantly reduced cold allodynia in a mice model of oxaliplatin-induced peripheral neuropathy.

Dual dose-related effects evoked by CCL4 on thermal nociception after gene delivery or exogenous administration in mice.

As recently described, the administration of extremely low doses (pg/kg) of CCL4 (Macrophage inflammatory protein 1ß, MIP-1ß) can induce antinociceptive effects in mice (García-Domínguez et al., 2019b). We describe here that hydrodynamic delivery of a plasmid containing CCL4 cDNA provokes a biphasic response consisting in an initial thermal hyperalgesic reaction for 8 days followed by analgesia at days 10-12, being both responses blocked after the administration of the CCR5 antagonist DAPTA. Both the luminiscence evoked in liver after the administration of a plasmid containing CCL4 and luciferase cDNAs and the hepatic concentration of CCL4 measured by ELISA were maximal 4 days after plasmid administration and markedly diminished at day 10. A dose-effect curve including a wide dose range of exogenous CCL4 revealed thermal analgesia after the administration of 10-100 pg/kg whereas 1000 times higher doses (30-100 ng/kg) induced, instead, thermal hyperalgesia inhibited by DAPTA. This hyperalgesia was absent in mice with reduced white blood cells after cyclophosphamide treatment, thus supporting the involvement of circulating leukocytes. A multiarray bioluminescent assay revealed increased plasma levels of IL-1α, CCL2, CXCL1, CXCL13, IL-16 and TIMP-1 in mice treated with 100 ng/kg of CCL4. The hyperalgesic response evoked by CCL4 was prevented by IL-1R, CXCR2 or CCR2 antagonists or by the neutralization of CXCL13 or IL-16, but not TIMP-1, with selective antibodies. The administration of the anti-IL-16 antibody was the unique treatment able to convert hyperalgesia evoked by 100 ng/kg of CCL4 in an analgesic effect. The ability of IL-16 to evoke hypernociception was confirmed by studying the response to its exogenous administration (10-30 ng/kg). In summary, the present results demonstrate that CCL4 induces a dual modulation of nociception and describe some mechanisms involved in the hyperalgesic response evoked by this chemokine.

Structure-Based Design of Novel Biphenyl Amide Antagonists of Human Transient Receptor Potential Cation Channel Subfamily M Member 8 Channels with Potential Implications in the Treatment of Sensory Neuropathies.

Structure-activity relationship studies of a reported menthol-based transient receptor potential cation channel subfamily M member 8 channel (TRPM8) antagonist, guided by computational simulations and structure-based design, uncovers a novel series of TRPM8 antagonists with >10-fold selectivity versus related TRP subtypes. Spiro[4.5]decan-8-yl analogue 14 inhibits icilin-evoked Ca2+ entry in HEK-293 cells stably expressing human TRPM8 (hTRPM8) with an IC50 of 2.4 ± 1.0 nM, while in whole-cell patch-clamp recordings this analogue inhibits menthol-evoked currents with a hTRPM8 IC50 of 64 ± 2 nM. Molecular dynamics (MD) simulations of compound 14 in our homology model of hTRPM8 suggest that this antagonist forms extensive hydrophobic contacts within the orthosteric site. In the wet dog shakes (WDS) assay, compound 14 dose-dependently blocks icilin-triggered shaking behaviors in mice. Upon local administration, compound 14 dose dependently inhibits cold allodynia evoked by the chemotherapy oxaliplatin in a murine model of peripheral neuropathy at microgram doses. Our findings suggest that 14 and other biphenyl amide analogues within our series can find utility as potent antagonist chemical probes derived from (-)-menthol as well as small molecule therapeutic scaffolds for chemotherapy-induced peripheral neuropathy (CIPN) and other sensory neuropathies.

1-(2',5'-Dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (RGM079): A Positive Allosteric Modulator of α7 Nicotinic Receptors with Analgesic and Neuroprotective Activity.

Acetylcholine α7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain as well as in neuropsychiatric, neurodegenerative, and inflammatory processes. Positive allosteric modulators (PAMs) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here, we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the α7 receptors and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in Alzheimer's disease (AD)-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aß1-42, with cell death almost completely prevented at 10 and 30 µM, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the complete Freund's adjuvant (CFA)-induced paw inflammation model after intraperitoneal administration.

Is TRPA1 Burning Down TRPV1 as Druggable Target for the Treatment of Chronic Pain?

Over the last decades, a great array of molecular mediators have been identified as potential targets for the treatment of chronic pain. Among these mediators, transient receptor potential (TRP) channel superfamily members have been thoroughly studied. Namely, the nonselective cationic channel, transient receptor potential ankyrin subtype 1 (TRPA1), has been described as a chemical nocisensor involved in noxious cold and mechanical sensation and as rivalling TRPV1, which traditionally has been considered as the most important TRP channel involved in nociceptive transduction. However, few TRPA1-related drugs have succeeded in clinical trials. In the present review, we attempt to discuss the latest data on the topic and future directions for pharmacological intervention.

In Vivo Methods to Study ThermoTRP Channels in Rodents.

Ion channels participate in several biological processes. Among these channels, the ionotropic TRP family is the most prominent group being TRPV1 the most studied. The activation of these channels can elicit pain sensation; thus, the development of blockers for these channels is receiving increasing attention. TRP channels are the responsible for thermonociception but also, they are involved in osmolarity, taste, and chemical substances perception such as capsaicin or menthol which can evoke pain. The needed of testing new compounds implies the use of animal models of pain and nociceptive tests in order to evaluate their potential efficacy for the treatment of painful symptoms. Several methods have been developed. Here, I describe the standard, current, and available tests to explore nociception in rodents, especially when thermal or mechanical stimuli are applied.