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Resumen Objetivo Presentar aspectos sociales del medicamento mediante una asignatura optativa. Metodos La experiencia se ha realizado en los cursos 2016-2017, 2017-2018 y 2018-2019. La actividad consta de 15 h de clases expositivas, 12 h de seminarios y 2 de tutorias. Para la evaluacion se considero la asistencia a clases teoricas (1 punto), a tutorias (1 punto) y a presentaciones expositivas (1 punto); la tarea presentada en la exposicion (3,5 puntos) y un documento escrito sobre ella (3,5 puntos). La satisfaccion se evaluo mediante una encuesta. Resultados Participaron 60 ± 7,1 estudiantes por curso, distribuidos en 16,2 ± 2,2 grupos. El programa teorico recoge las interacciones entre los principales protagonistas de la utilizacion de medicamentos (industria farmaceutica, agencias reguladoras, enfermos y medicos). Los temas de seminarios, de eleccion por los estudiantes, se agrupan en 4 bloques tematicos: a) desigualdad de acceso a los medicamentos, b) condicionantes del uso, c) repercusiones sociales del uso y d) etica y fraude con relacion a los medicamentos. Las calificaciones globales fueron: 7,9 ± 0,15; 7,4 ± 0,2 y 7,8 ± 0,1 en los 3 cursos. Los estudiantes otorgaron 4 o mas puntos sobre 5 a los aspectos organizativos, al programa teorico y a los seminarios. Conclusion La presentacion de aspectos sociales de los medicamentos es bien aceptada por los estudiantes y contribuye a la adquisicion de competencias transversales.

espanolLa vocacion es una cualidad que define al buen medico y esta presente en los profesionales sanitarios de referencia. La vocacion medica puede definirse de varias formas, pero tal vez alcance un amplio consenso la que propone que es una motivacion profunda de servicio al enfermo y a la sociedad que esta determinada por las vivencias y el entorno. Si bien la mayoria de medicos y estudiantes de medicina invocan la vocacion y el atractivo intelectual para su eleccion, 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 vocacion medica como el altruismo o la empatia, asi como algunos estereotipos de la imagen de la profesion. EnglishThe 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 image.

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 β-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 β-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 β-lactams and KPs. β-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.

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.

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.The unilateral hot plate test was used to assess thermal nociception. CD3IL-16 and CCR5 expression were demonstrated in CD4CCL4-evoked hyperalgesia is related to the desensitization of CCR5 in CD4

15 pags, 8 figs, 3 tabs. -- Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioorg.2021.105231., 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., This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO-FEDER), RTI2018-097189-C2 and CTQ2017-84371-P), and the Spanish National Research Council (CSIC, 201880E109, 201980E030). The NMR experiments were performed in the “Manuel Rico” NMR laboratory, LMR, CSIC, a node of the Spanish Large-Scale National Facility ICTS R-LRB. We thank Prof. Josep Rizo and R. Voleti (Dept. Biophysics, Biochemistry and Pharmacology, UT Southwestern Medical Center, Dallas, USA) for providing the clones required for expressing Syt1 and Syt7 proteins. SG-R and AB belong to the Instituto de Investigación Sanitaria del Principado de Asturias (ISPA).

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.

The chemokine CC motif ligand 1 (CCL1) participates in immune cell recruitment and, as for 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. Aiming to explore the possible role played by CCL1 on spinal nociception, we study here the effect of its intrathecal administration on thermal nociception in mice. The intrathecal administration of CCL1 (0.3-30 ng) produced dose-dependent analgesia as measured by the unilateral hot plate test. This analgesia evoked by CCL1 peaked 1 h after injection, was prevented by the CCR8 antagonist R243 and was accompanied by a reduction of c-Fos protein expression in dorsal horn spinal neurons. Blood leukocyte depletion did not modify CCL1-evoked analgesic responses that, in contrast, were abolished by the microglial inhibitor minocycline, but not the astroglial inhibitor aminoadipate, suggesting the involvement of spinal microglial cells. Furthermore, analgesia remained unmodified by the coadministration of cannabinoid type 1 or 2 receptors antagonists (AM251 or SR144285). However, it was reversed by naloxone but not by cyprodime or naltrindole (selective antagonists 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. Endogenous dynorphin and microglia behave as key players in heat thermal analgesia evoked by spinal CCL1 in mice.

As recently described, the administration of extremely low doses (pg/kg) of CCL4 (Macrophage inflammatory protein 1β, MIP-1β) can induce antinociceptive effects in mice (Garcia-Dominguez 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.

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.

espanolObjetivo. Identificar las razones por las cuales los estudiantes acceden al Grado en Medicina. Sujetos y metodos.Estudiantes de primer curso de Grado en Medicina de los anos academicos 2014-2015, 2015-2016 y 2017-2018 realizaron una descripcion abierta de sus razones de acceso al grado. Estas se identificaron y clasificaron para su analisis, y se calculo el numero de razones aducidas por el conjunto y en cada ano academico, y su distribucion segun sexo. Se clasificaron en categorias para su comparacion 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 estadisticamente significativa. Los estudiantesjustificaron su acceso por una media de 2,5 ± 0,06 razones, sin diferencias de sexo ni entre los tres anos academicos. Se identificaron 23 razones diferentes para acceder al grado, que se agruparon en categorias, siendo las principales el altruismo, el cono-cimiento cientifico, los motivos instrumentales y las razones personales. Aumento significativamente el acceso por adqui-sicion de conocimiento entre los cursos 2014-2015 y 2017-2018 y se redujeron los accesos por vocacion. La consideracion social de la medicina o la imagen social del medico se invocaron con muy poca frecuencia. Las mujeres refieren con mas frecuencia los motivos altruistas y los relacionados con el conocimiento para el acceso, pero sin diferencias significativas. Conclusion.Los valores altruistas continuan siendo los mas frecuentes motivos de acceso al Grado en Medicina. La voca-cion se reduce y la adquisicion de conocimientos cientificos aumenta como motivos de acceso. EnglishAim. 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.

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-agonist 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 i.p.), an oxaliplatin-induced cold allodynia (at 10-30 μg s.c.), and CCI-induced thermal hyperalgesia (at 11.5 mg/kg i.p.) mice models. These results confirm the tryptophan moiety as a solid pharmacophore template for the design of highly potent modulators of TRPM8-mediated activities.

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 Ca(2+) entry in HEK-293 cells stably expressing human TRPM8 (hTRPM8) with an IC(50) of 2.4 ± 1.0 nM, while in whole-cell patch-clamp recordings this analogue inhibits menthol-evoked currents with a hTRPM8 IC(50) 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.

Despite being an old molecule, capsaicin is still a hot topic in the scientific community, and the development of new capsaicinoids is a promising pharmacological approach in the management of skin disorders related to inflammation and pruritus. Here we report the synthesis and the evaluation of capsaicin soft drugs that undergo deactivation by the hydrolyzing activity of skin esterases. The implanting of an ester group in the lipophilic moiety of capsaicinoids by the Passerini multicomponent reaction affords both agonists and antagonists that retain transient receptor potential vanilloid 1 channel (TRPV1) modulating activity and, at the same time, are susceptible to hydrolysis. The most promising antagonist identified shows in vivo anti-nociceptive activity on pruritus and hyperalgesia without producing hyperthermia, thus validating it as novel treatment for dermatological conditions that implicate TRPV1 channel dysfunction.

α7 Nicotinic acetylcholine receptors (nAChRs) are ion channels implicated in a number of CNS pathological processes, including pain and psychiatric, cognitive and inflammatory diseases. Comparing with orthosteric agonism, positive allosteric modulation of these channels constitutes an interesting approach to achieve selectivity versus other nicotinic receptors. We have recently described new chalcones and 1,3-diphenylpropanones as positive allosteric modulators (PAMs) of α7 nAChRs, which proved to have good analgesic activities but poor pharmacokinetic properties. Here we report the preparation of amino acid and peptide derivatives as prodrugs of these modulators with the aim of improving their in vivo biological activity. While the valine derivative showed very short half life in aqueous solutions to be considered a prodrug, Val-Val and Val-Pro-Val are suitable precursors of the parent 1,3-diphenylpropanones, via chemical and enzymatic transformation, respectively. Compounds 19 (Val-Val) and 21 (Val-Pro-Val), prodrugs of the 2′,5′,4-trihydroxy-1,3-diphenylpropan-1-one 3, showed significant antinociceptive activity in in vivo assays. The best compound, 21, displayed a better profile in the analgesia test than its parent compound 3, exhibiting about the same potency but long-lasting effects., Funding: This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) SAF2011-22802, BFU2015- 70067-REDC and SAF2015-66275-C2-R, and the Generalitat Valenciana, PROMETEO/2014/011. BBP thanks the CSIC for a predoctoral fellowship (JAE-Predoc from Junta para la Ampliación de Estudios, co-financed by FSE).

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.

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.

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 Aβ, 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., This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (MINECO-FEDER) grant number SAF2015-66275-C2-R and RTI2018-097189-C2 to RGM and AFM, and RTI2018-095793-B-I00 to MGL, the Comunidad de Madrid/European Union Ref S2017/BMD-3827 to MGL and the CSIC, grant number 201980E030 to RGM.

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.

Illness is a traumatic experience full of uncertainty which checks our confrontation strategies against adversity. When disease is severe and is loaded with suffering premonitions, it can be felt as an emotional cataclysm where narration might be disclosed as an invaluable help. The narrative process requires solitude, introspection and reflection prior to the writing action, thus allowing to analyze from the distance the new condition the patient is exposed to. The curative effect of narration is produced by the catharsis provided by the reflection, by the fears and negative thoughts transferred onto paper, as well as the alleviation, relief and the comfort provided by the narrative extraversion. Given the curative power of narration, it seems necessary that medicine graduate students know the advantages that writing might provide/add to their education besides the convenience of prescribing writing to their patients because they will feel, at least, relief from the emotional distress that the diagnosis and prognostic uncertainties frequently produce.

Chemokines are chemotactic cytokines whose involvement in nociceptive processing is being increasingly recognized. Based on the previous description of the involvement of CC chemokine receptor type 1 (CCR1) in pathological pain, we have assessed the participation of CCR1 and its endogenous ligands CCL3 and CCL5 in hyperalgesia and allodynia in mice after acute inflammation with carrageenan and chronic inflammation with complete Freund's adjuvant (CFA). The subcutaneous administration of the CCR1 antagonist J113863 (3-30 mg/kg; 30 min. before) dose dependently inhibited carrageenan- and CFA-evoked thermal hyperalgesia and mechanical allodynia produced by CFA, but not by carrageenan. The maximal dose of J113863 did not modify the increase in paw thickness induced by carrageenan or CFA. An almost ten times augmentation of CCL3 levels was detected by ELISA assays in both carrageenan and CFA paws, but not in spinal cords of inflamed mice, whereas CCL5 concentrations remained unaltered. Accordingly, a marked increase of CCL3 mRNA expression was observed in inflamed paws, with CCL3 protein detected in neutrophils and macrophages by immunohistochemical experiments. The intraplantar administration of an anti-CCL3 antibody (0.3-3 μg) blocked thermal hyperalgesia in carrageenan- and CFA-inflamed mice as well as CFA-evoked mechanical allodynia. Our data suggest that the increased concentrations of CCL3 present in inflamed tissues can be involved in acute and chronic inflammatory hyperalgesia as well as in chronic mechanical allodynia, and that these hypernociceptive symptoms can be counteracted by its neutralization with an antibody or by the blockade of CCR1 receptors.

Chronic pain is one of the most debilitating human diseases and represents a social and economic burden for our society. Great efforts are being made to understand the molecular and cellular mechanisms underlying the pathophysiology of pain transduction. It is particularly noteworthy that some types of chronic pain, such as migraine, display a remarkable sex dimorphism, being up to three times more prevalent in women than in men. This gender prevalence in migraine appears to be related to sex differences arising from both gonadal and genetic factors. Indeed, the functionality of the somatosensory, immune, and endothelial systems seems modulated by sex hormones, as well as by X-linked genes differentially expressed during development. Here, we review the current data on the modulation of the somatosensory system functionality by gonadal hormones. Although this is still an area that requires intense investigation, there is evidence suggesting a direct regulation of nociceptor activity by sex hormones at the transcriptional, translational, and functional levels. Data are being accumulated on the effect of sex hormones on TRP channels such as TRPV1 that make pivotal contributions to nociceptor excitability and sensitization in migraine and other chronic pain syndromes. These data suggest that modulation of TRP channels' expression and/or activity by gonadal hormones provide novel pathways for drug intervention that may be useful for targeting the sex dimorphism observed in migraine.

TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of N, N'-dibenzyl tryptamine 1, a potent TRPM8 antagonist, was prepared and screened using a fluorescence-based in vitro assay based on menthol-evoked calcium influx in TRPM8 stably transfected HEK293 cells. The tryptophan derivative 14 was identified as a potent (IC50 0.2 ± 0.2 nM) and selective TRPM8 antagonist. In vivo, 14 showed significant target coverage in both an icilin-induced WDS (at 1-30 mg/kg s.c.) and oxaliplatin-induced cold allodynia (at 0.1-1 μg s.c.) mice models. Molecular modeling studies identified the putative binding mode of these antagonists, suggesting that they could influence an interaction network between the S1-4 transmembrane segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new pharmacophoric scaffold for the design of highly potent modulators of TRPM8-mediated pain.

Novel painkillers are urgently needed. The activation of opioid receptors in peripheral inflamed tissue can reduce pain without central adverse effects such as sedation, apnoea, or addiction. Here, we use an unprecedented strategy and report the synthesis and analgesic efficacy of the standard opioid morphine covalently attached to hyperbranched polyglycerol (PG-M) by a cleavable linker. With its high-molecular weight and hydrophilicity, this conjugate is designed to selectively release morphine in injured tissue and to prevent blood-brain barrier permeation. In contrast to conventional morphine, intravenous PG-M exclusively activated peripheral opioid receptors to produce analgesia in inflamed rat paws without major side effects such as sedation or constipation. Concentrations of morphine in the brain, blood, paw tissue, and in vitro confirmed the selective release of morphine in the inflamed milieu. Thus, PG-M may serve as prototype of a peripherally restricted opioid formulation designed to forego central and intestinal side effects. DOI: http://dx.doi.org/10.7554/eLife.27081.001

BACKGROUND: This study examined the development of chronic pain, a cardinal symptom of rheumatoid arthritis (RA), in mice with antigen- and collagen-induced arthritis (ACIA). Since the role of CD8(+) T cells in arthritis is controversial, we investigated the consequences of CD8-depletion on arthritis development and opioid modulation of pain in this novel model of chronic autoimmune arthritis. METHODS: Disease severity in control and CD8-depleted animals was determined by histological assessment of knee-joint sections and measurement of autoantibody formation. Pain was evaluated by measuring mechanical allodynia and thermal hyperalgesia in von Frey and Hargreaves tests, respectively. The production and release of endogenous opioids and inflammatory cytokines was assessed in immunoassays. RESULTS: In ACIA, mice display persistent mechanical allodynia and thermal hyperalgesia for more than 2 months after induction of arthritis. The blockade of peripheral opioid receptors with naloxone-methiodide (NLXM) transiently increased thermal hyperalgesia, indicating that endogenous opioid peptides were released in the arthritic joint to inhibit pain. CD8(+) T cell depletion did not affect autoantibody formation or severity of joint inflammation, but serum levels of the pro-inflammatory cytokines TNFα and IL-17 were increased. The release of opioid peptides from explanted arthritic knee cells and the NLXM effect were significantly reduced in the absence of CD8(+) T cells. CONCLUSIONS: We have successfully modeled the development of chronic pain, a hallmark of RA, in ACIA. Furthermore, we detected a yet unknown protective role of CD8(+) T cells in chronic ACIA since pro-inflammatory cytokines rose and opioid peptide release decreased in the absence of these cells.

We show here that the intraplantar administration of CCL5 in mice produces hyperalgesia at low doses but activates compensatory antinociceptive mechanisms at doses slightly higher. Thus, the injection of 3–10 ng of CCL5 evoked thermal hyperalgesia through the activation of CCR1 and CCR5 receptors, as demonstrated by the inhibitory effect exerted by the selective antagonists J113863 (0.01–0.1 μg) and DAPTA (0.3–3 μg), respectively. The prevention of this hyperalgesia by diclofenac (1–10 μg), the inhibitors of COX-1 SC-560 (0.1–1 μg) or COX-2 celecoxib (1–5 μg), the TRPV1 antagonist capsazepine (0.03–0.3 μg) or the TRPA1 antagonist HC030031 (10–50 μg) demonstrates the involvement of prostaglandin synthesis and TRP sensitization in CCL5-evoked hyperalgesia. Doses of CCL5 higher than 17 μg did not evoke hyperalgesia. However, this effect was restored by the administration of naloxone-methiodide (5 μg), nor-binaltorphimine (10 mg/kg) or an anti-dynorphin A antibody (0.62–2.5 ng). The administration of 30 ng of CCL5 also induced hyperalgesia in mice with reduced number of circulating white blood cells in response to cyclophosphamide or with selective neutrophil depletion induced by an anti-Ly6G antibody. In fact, the number of neutrophils present in paws treated with 30 ng of CCL5 was greater than in paws receiving the administration of the hyperalgesic dose of 10 ng. Finally, the expression of the endogenous opioid peptide dynorphin A was demonstrated by double immunofluorescence assays in these neutrophils attracted by CCL5. These results support previous data describing the hyperalgesic properties of CCL5 and constitute the first indication that a chemokine of the CC group can activate endogenous analgesic mechanisms.

Background The first line pharmacological treatment of cancer pain is morphine and surrogates but a significant pain relief and a reduction of the side-effects of these compounds makes it necessary to combine them with other drugs acting on different targets. The aim of this study was to measure the antinociceptive effect on cancer-induced bone pain resulting from the association of the endogenous opioids enkephalin and non-opioid analgesic drugs. For this purpose, PL265 a new orally active single dual inhibitor of the two degrading enkephalins enzymes, neprilysin (NEP) and aminopeptidase N (APN) was used. It strictly increased the levels of enkephalin at their sites of releases. The selected non-opioid compounds are: gabapentin, A-317491 (P2X3 receptor antagonist), ACEA (CB1 receptor antagonist), AM1241 (CB2 receptor antagonist), JWH-133 (CB2 receptor antagonist), URB937 (FAAH inhibitor), and NAV26 (Nav1.7 channel blocker). Methods Experiments. Experiments were performed in 5–6 weeks old (26–33g weight) C57BL/6 mice. Cell culture and cell inoculation. B16-F10 melanoma cells were cultured and when preconfluent, treated and detached. Finally related cells were resuspended to obtain a concentration of 2×106 cells/100μL. Then 105 cells were injected into the right tibial medullar cavity. Control mice were treated by killed cells by freezing. Behavioural studies. Thermal withdrawal latencies were measured on a unilatered hot plate (UHP) maintained at 49±0.2 °C.Mechanical threshold values were obtained by performing the von Frey test using the “up and down” method. To evaluate the nature (additive or synergistic) of the interactions between PL265 and different drugs, an isobolographic analysis following the method described by Tallarida was performed. Results The results demonstrate the ability of PL265, a DENKI that prevents the degradation of endogenous ENKs, to counteract cancer-induced bone thermal hyperalgesia in mice, by exclusively stimulating peripheral opioid receptors as demonstrated by used of an opioid antagonist unable to enter the brain. The development of such DENKIs, endowed with druggable pharmacokinetic characteristics, such as good absorption by oral route, can be considered as an important step in the development of much needed novel antihyperalgesic drugs. Furthermore, all the tested combinations resulted in synergistic antihyperalgesic effects. As shown here, the greatest synergistic antinociceptive effect (doses could be lowered by 70%) was produced by the combination of PL265 with the P2X3 receptor antagonist (A-317491), cannabinoid CB1 receptor agonist (exogenous, ACEA and endogenous URB937-protected-AEA) and Nav1.7 blocker (NAV26) whose mechanism of action involves the direct activation of the enkephalinergic system. Conclusions These multi-target-based antinociceptive strategies using combinations of non-opioid drugs with dual inhibitors of enkephalin degrading enzymes may bring therapeutic advantages in terms of efficacy and safety by allowing the reduction of doses of one of the compounds or of both, which is of the utmost interest in the chronic treatment of cancer pain. Implications This article presents synergistic antinociceptive effect produced by the combination of PL265 with non-opioid analgesic drugs acting via unrelated mechanisms. These multi-target-based antinociceptive strategies may bring therapeutic advantages by allowing the reduction of doses, which is of great interest in the chronic treatment of cancer pain.

Agonists of μ-opioid receptors are currently used in the management of cancer pain. However, several data suggest that the analgesic effect of morphine can diminish during the development of experimental tumors. By using a thermal test, we have studied whether the analgesic effect evoked by morphine is altered in mice bearing two painful bone tumors. The analgesic effect evoked by systemic morphine remained unaltered after the intratibial inoculation of B16-F10 melanoma cells and was potentiated after the inoculation of NCTC 2472 osteosarcoma cells. Although the number of spinal μ-opioid receptors measured by western blot studies was not augmented in osteosarcoma-bearing mice, the analgesia evoked by intrathecal (i.t.) morphine was also enhanced. The analgesic response produced by the spinal administration of the Gi/o protein activator mastoparan was amplified, whereas the analgesic response evoked by the i.t. administration of the N-type calcium channel blocker ω-conotoxin remained unaltered. The efficacy of the GIRK channel blocker tertiapin-Q to antagonize the analgesic effect produced by a maximal dose of morphine was also increased in osteosarcoma-bearing mice. Our results seem to indicate that the analgesic effect of morphine on thermal nociception can be enhanced in response to the development of particular bone tumors in mice, being this potentiation probably related to a greater efficacy of the transduction system driven by Gi/o proteins and GIRK channels.

Although previous studies describe the up-regulation of purinergic P2X(3) receptors expressed at peripheral nociceptive fibers in experimental painful neoplastic processes, the analgesic efficacy of P2X(3) receptor antagonists has not been tested in these settings. We study here the effect of the P2X(3) receptor antagonist, A-317491, on thermal hyperalgesia produced by the intratibial inoculation of NCTC 2472 fibrosarcoma cells to C3H/HeJ mice. The peritumoral administration of A-317491 (10-100 microg) dose-dependently attenuated osteosarcoma-induced thermal hyperalgesia without modifying thermal latencies measured in the contralateral paws. This antihyperalgesic effect was inhibited by the coadministration of naloxone-methiodide (0.1-1 microg) or the systemic injection of the selective mu-opioid receptor antagonist cyprodime (1 mg/kg), demonstrating the involvement of peripheral mu-opioid receptors. Furthermore, the antihyperalgesic effect induced by A-317491, was antagonised by the coadministration of an anti-enkephalin antibody supporting the participation of endogenous enkephalins. Consistent with this result, the antihyperalgesic effect induced by A-317491 was dramatically enhanced by the administration of an enkephalin-degrading inhibitor, Debio 0827, as demonstrated by isobolographic analysis. This synergism opens the theoretical possibility that the combination of both types of drugs could be useful to counteract some nociceptive symptoms derived from tumor development.

The analgesic effect induced by opiates is often potentiated during experimental inflammatory processes. We describe here that lower doses of systemic morphine are necessary to increase thermal withdrawal latencies measured in both hind paws of mice acutely inflamed with carrageenan than in healthy ones. This bilateral potentiation seems mediated through spinal opioid receptors since it is inhibited by the intrathecal (i.t.), but not intraplantar (i.pl.) administration of the opioid receptor antagonist naloxone-methiodide, and also appears when morphine is i.t. administered. Furthermore, the i.pl. administration of the nitric oxide (NO) synthase inhibitor, L-NMMA, or the K (ATP) (+) -channel blocker, glibenclamide, to carrageenan-inflamed mice inhibits the enhanced effect of systemic morphine in the paw that receives the injection of the drug, without affecting the potentiation observed in the contralateral one. The i.pl. administration of L-NMMA also partially antagonised the analgesic effect induced by i.t. morphine in inflamed mice. Finally, the increased analgesic effect evoked by the i.pl. administration of the NO donor SIN-1 either in the inflamed or in the contralateral paw of carrageenan-inflamed mice suggests that enhanced responsiveness to the peripheral analgesic effect of NO may be also underlying the bilateral potentiation of morphine-induced analgesia in acutely inflamed mice.

Chemokine CCL2, also known as monocyte chemoattractant protein-1 (MCP-1), is a molecule that in addition to its well-established role in chemotaxis can also act as nociceptor sensitizer. The upregulation of this chemokine in inflamed tissues could suggest its involvement in inflammatory hypernociception. Thus, we have measured CCL2 levels in mice with acute or chronic inflammation due to the intraplantar (i.pl.) injection of carrageenan or complete Freund's adjuvant (CFA), respectively, and we have studied whether inflammatory hyperalgesia or allodynia could be attenuated by blocking CCR2 receptors or neutralizing CCL2 with an anti-CCL2 antibody. A remarkable increase in CCL2 concentration was detected by ELISA in paw homogenates coming from carrageenan- or CFA-inflamed mice, being its expression mainly localized in macrophages, as shown by immunohistochemical assays. The s.c. (0.3-3 mg/kg) or i.pl. (0.3-3 μg) administration of the CCR2 antagonist, RS 504393, dose dependently inhibited thermal hyperalgesia measured in acutely or chronically inflamed mice, whereas s.c. administration of this drug did not reduce inflammatory mechanical allodynia. Furthermore, the inhibition of inflammatory hyperalgesia after the administration of an anti-CCL2 antibody (0.1-1 μg; i.pl.) suggests that CCL2 could be the endogenous chemokine responsible for CCR2-mediated hyperalgesic effects. Besides, the acute administration of the highest antihyperalgesic dose of RS 504393 assayed did not reduce paw tumefaction or modify the presence of inflammatory cells. These results indicate that the blockade of the CCL2/CCR2 system can counteract inflammatory hyperalgesia, being this antinociceptive effect unrelated to a decrease in the inflammatory reaction.

During inflammation, several mediators directly or indirectly induce pain including pro-inflammatory cytokines and there is evidence that the JAK-STAT pathway is involved in the formation of pronociceptive cytokines. The same pathway, however, is also of importance for anti-inflammatory cytokines such as IL-4 to counteract the inflammatory reaction and—as it seems based on the current literature—nociceptive symptoms. Current therapeutic approaches targeting molecules of the JAK-STAT signaling cascade are auspicious but as this review demonstrates, more experimental and clinical studies are required to decipher the specific contribution of this pathway in the modulation of pain.

The hypernociceptive role played by the chemokine CCL2, and its main receptor, CCR2, in pathological settings is being increasingly recognized. We aimed to characterize the involvement of spinal CCL2 in the hyperalgesia due to the intratibial inoculation of fibrosarcoma NCTC 2472 cells in mice. The intrathecal (i.t.) administration of the CCR2 antagonist RS 504393 (1–3 μg) or an anti-CCL2 antibody inhibited tumoral hyperalgesia. No change in the expression of spinal CCR2 was detected by western blot, whereas immunohistochemical experiments demonstrated increased CCL2 staining at the superficial laminae of the spinal cord ipsilateral to the tumor. This spinal CCL2 does not seem to be released from nociceptors since CCL2 mRNA and CCL2 levels in DRGs, as measured by RT-PCR and ELISA, remain unmodified in tumor-bearing mice. In contrast, immunohistochemical assays demonstrated the spinal up-regulations of GFAP and Iba-1, respective markers of astroglia and microglia, and the expression of CCL2 in both types of glial cells at the superficial laminae of the spinal cord of tumor-bearing mice. Finally, since CCL2 could induce astroglial or microglial activation, we studied whether the blockade of CCR2 could inhibit the increased spinal glial expression. GFAP, but not Iba-1, up-regulation was reduced in tumor-bearing mice treated for 3 days with i.t. RS 504393, indicating that spinal CCL2 acts as an astroglial activator in this setting. The participation at spinal level of CCL2/CCR2 in tumoral hypernociception, together with its previously described involvement at periphery, makes attractive the modulation of this system for the alleviation of neoplastic pain.

espanolIntroduccion. La lectura y el analisis de textos narrativos pueden contribuir a la adquisicion de las competencias humanisticas que deben adquirir los estudiantes de medicina. Este trabajo presenta una actividad realizada con este fin. Sujetos y metodos. La actividad se ha realizado durante cinco anos dentro de una asignatura obligatoria del primer curso del grado de Medicina en la Universidad de Oviedo, en dos seminarios (cuatro horas). Los estudiantes (150-155/ano) realizan la actividad en subgrupos de cuatro-cinco. En el primer seminario, el profesor comenta dos poemas para iniciar a los estudiantes en la identifi cacion de terminos relacionados con la medicina. En el segundo, cada subgrupo expone el analisis realizado sobre un relato asignado por el profesor. Se evaluo el contenido y la organizacion del trabajo, asi como la exposicion oral. La evaluacion de la satisfaccion de los estudiantes se realizo mediante una encuesta. Resultados. Se han utilizado 79 relatos, de los que 33 han sido comentados en dos-tres cursos academicos. Las puntuaciones obtenidas por los estudiantes en la actividad han variado entre 3,59 ± 0,11 y 3,85 ± 0,05 sobre cuatro en los diferentes cursos. Los estudiantes han mostrado su satisfaccion con la actividad y resaltan (sobre 5) que la formacion humanista (3,9), la descripcion de enfermedades (4), la relacion medico-paciente (3,7), la dimension social (3,8) y la actitud del paciente ante la enfermedad (3,8) pueden adquirirse mediante el analisis de textos literarios. Conclusion. La lectura y el analisis de textos literarios permiten aproximar a los estudiantes a aspectos relevantes de la practica profesional ausente durante el grado. EnglishIntroduction. Reading and analysis of narrative texts may contribute to the acquisition of humanistic skills required for medical students. This article presents an activity performed to this aim. Subjects and methods. The activity has been performed over fi ve academic years, in the form of two seminars (four hours) as a part of an obligatory subject in the fi rst year of the Medicine degree of the University of Oviedo. Students (150-155 per year) perform this activity in subgroups of four-fi ve. At the fi rst seminar, the teacher comments two poems in order to initiate the students in the identifi cation of medical terms. During the second of the seminars, every group presented an analysis of a short story assigned by the teacher. Contents, structure and oral presentations were evaluated. Student satisfaction was measured in a survey. Results. We used 79 stories whose 33 were commented in two-three academic courses. Students’ mean scores on this activity ranged from 3.59 ± 0.11 to 3.85 ± 0.05 out of 4 in the diff erent courses. Students expressed satisfaction with the activity and stressed (out of 5 points) that the humanistic formation (3.9), the description of diseases (4), the doctor-patient relationship (3.7), the social dimension (3.7) and the patient’s attitude towards disease (3.8) may be acquired through the analysis of literary texts. Conclusion. Reading and analysis of literary texts allow students to approach relevant aspects of the professional practice absent in the curriculum of the degree.

The participation of the chemokine CCL2 (monocyte chemoattractant protein-1) in inflammatory and neuropathic pain is well established. Furthermore, the release of CCL2 from a NCTC 2472 cells-evoked tumor and its involvement in the upregulation of calcium channel α2δ1 subunit of nociceptors was demonstrated. In the present experiments, we have tried to determine whether the increase in CCL2 levels is a common property of painful tumors and, in consequence, the administration of a chemokine receptor type 2 (CCR2) antagonist can inhibit tumoral hypernociception. CCL2 levels were measured by ELISA in the tumoral region of mice intratibially inoculated with NCTC 2472 or B16-F10 cells, and the antihyperalgesic and antiallodynic effects evoked by the administration of the selective CCR2 antagonist RS 504393 were assessed. Cultured NCTC 2472 cells release CCL2 and their intratibial inoculation evokes the development of a tumor in which CCL2 levels are increased. Moreover, the systemic or peritumoral administration of RS 504393 inhibited thermal and mechanical hyperalgesia, but not mechanical allodynia evoked after the inoculation of these cells. Thermal hyperalgesia was also inhibited by the peritumoral administration of a neutralizing CCL2 antibody. In contrast, no change in CCL2 levels was observed in mice inoculated with B16-F10 cells, and RS 504393 did not inhibit the hypernociceptive reactions evoked by their intratibial inoculation. The peripheral release of CCL2 is involved in the development of thermal and mechanical hyperalgesia, but not mechanical allodynia evoked by the inoculation of NCTC 2472 cells, whereas this chemokine seems unrelated to the hypernociception induced by B16-F10 cells.