Pharmacodynamics of TRPV1 Agonists in a Bioassay Using Human PC-3 Cells

Altres ajuts: La Fundació Marató de TV3Purpose. TRPV1 is a multimodal channel mainly expressed in sensory neurons. We aimed to explore the pharmacodynamics of the TRPV1 agonists, capsaicin, natural capsaicinoids, and piperine in an in vitro bioassay using human PC-3 cells and to examine desensitization and the effect of the specific antagonist SB366791. Methods. PC-3 cells expressing TRPV1 were incubated with Fluo-4. Fluorescence emission changes following exposition to agonists with and without preincubation with antagonists were assessed and referred to maximal fluorescence following the addition of ionomycin. Concentration-response curves were fitted to the Hill equation. Results. Capsaicin and piperine had similar pharmacodynamics (E 204.8 ± 184.3% piperine versus 176.6 ± 35.83% capsaicin, P = 0.8814, Hill coefficient 0.70 ± 0.50 piperine versus 1.59 ± 0.86 capsaicin, P = 0.3752). In contrast, capsaicinoids had lower E (40.99 ± 6.14% capsaicinoids versus 176.6 ± 35.83% capsaicin, P < 0.001). All the TRPV1 agonists showed significant desensitization after the second exposition and their effects were strongly inhibited by SB366791. Conclusion. TRPV1 receptor is successfully stimulated by capsaicin, piperine, and natural capsaicinoids. These agonists present desensitization and their effect is significantly reduced by a TRPV1-specific antagonist. In addition, PC-3 cell bioassays proved useful in the study of TRPV1 pharmacodynamics

Natural history of swallow function during the three-month period after stroke

Oropharyngeal dysphagia is a prevalent complication following stroke (PS-OD), and one that is sometimes spontaneously recovered. This study describes the natural history of PS-OD between admission and three months post-stroke, and the factors associated with its prevalence and development. PS-OD was assessed with the volume- iscosity swallow test (V-VST) in all stroke patients on admission and at the three-month follow-up. We analyzed clinical, demographic, and neuroanatomical factors of 247 older post-stroke patients (National Institute of Health Stroke Scale (NIHSS) = 3.5 ± 3.8), comparing among those with PS-OD the ones with and without spontaneous recovery. PS-OD prevalence on admission was 39.7% (34.0% impaired safety; 30.8%, efficacy) and 41.7% (19.4% impaired safety; 39.3%, efficacy) at three months. Spontaneous swallow recovery occurred in 42.4% of patients with unsafe and in 29.9% with ineffective swallow, associated with younger age and optimal functional status. However, 26% of post-stroke patients developed new signs/symptoms of ineffective swallow related to poor functional, nutritional and health status, and institutionalization. PS-OD prevalence on admission and at the three-month follow-up was very high in the study population. PS-OD is a dynamic condition with some spontaneous recovery in patients with optimal functional status, but also new signs/symptoms can appear due to poor functionality. Regular PS-OD monitoring is needed to identify patients at risk of nutritional and respiratory complications

Targeting patient recovery priorities in degenerative cervical myelopathy:design and rationale for the RECEDE-Myelopathy trial-study protocol

Introduction: Degenerative cervical myelopathy (DCM) is a common and disabling condition of symptomatic cervical spinal cord compression secondary to degenerative changes in spinal structures leading to a mechanical stress injury of the spinal cord. RECEDE-Myelopathy aims to test the disease-modulating activity of the phosphodiesterase 3/phosphodiesterase 4 inhibitor Ibudilast as an adjuvant to surgical decompression in DCM. Methods and analysis: RECEDE-Myelopathy is a multicentre, double-blind, randomised, placebo-controlled trial. Participants will be randomised to receive either 60-100 mg Ibudilast or placebo starting within 10 weeks prior to surgery and continuing for 24 weeks after surgery for a maximum of 34 weeks. Adults with DCM, who have a modified Japanese Orthopaedic Association (mJOA) score 8-14 inclusive and are scheduled for their first decompressive surgery are eligible for inclusion. The coprimary endpoints are pain measured on a visual analogue scale and physical function measured by the mJOA score at 6 months after surgery. Clinical assessments will be undertaken preoperatively, postoperatively and 3, 6 and 12 months after surgery. We hypothesise that adjuvant therapy with Ibudilast leads to a meaningful and additional improvement in either pain or function, as compared with standard routine care. Study design: Clinical trial protocol V.2.2 October 2020. Ethics and dissemination: Ethical approval has been obtained from HRA - Wales. The results will be presented at an international and national scientific conferences and in a peer-reviewed journals.Trial registration number: ISRCTN Number: ISRCTN16682024.</p

Caracterización de nuevas dianas terapéuticas para el tratamiento farmacológico de la disfagia orofaríngea asociada a enfermedades neurológicas y al envejecimiento

La disfagia orofaríngea (DO) se describe como la alteración de la correcta función deglutoria y como la dificultad para transportar el bolo alimentario y los líquidos de la boca al esófago de forma segura y eficaz. La DO es muy prevalente en ancianos, pacientes que han tenido un ictus, pacientes con enfermedades neurodegenerativas o distrofias musculares y pacientes que han padecido enfermedades o han tenido intervenciones otorrinolaringológicas. Actualmente el tratamiento de estos pacientes se basa en la compensación con espesantes, posturas y maniobras y la rehabilitación logopédica, pero no existen tratamientos farmacológicos que permitan mejorar la fisiología alterada de estos pacientes. En esta Tesis hemos desarrollado las bases para una nueva línea de tratamiento farmacológico específico para la DO mediante investigación traslacional, partiendo de la investigación básica (histología y biología molecular) hasta llegar al ensayo de distintos tratamientos en estudios clínicos, pasando por el estudio farmacodinámico de estos tratamientos. Estos nuevos tratamientos están basados en la estimulación sensorial con agonistas farmacológicos de los receptores que se encuentran en la orofaringe humana. La estrategia se basa en añadir estos agonistas al bolo alimentario para mejorar la deglución promoviendo una respuesta motora orofaríngea (RMO) más rápida y una mayor fuerza de propulsión lingual. La estructura de la mucosa orofaríngea humana a nivel histológico es diferente en la lengua (mucosa especializada) respecto a la faringe y la epiglotis (mucosa de recubrimiento). La inervación sensorial de la mucosa orofaríngea es generalmente subepitelial, aunque también hay terminaciones nerviosas que penetran el epitelio. TRPV1 y TRPA1 se expresan en las regiones inervadas por los NC V, IX y X, pero mientras TRPV1 se localiza principalmente en la membrana de las células epiteliales, TRPA1 se localiza principalmente por debajo del epitelio. TRPM8 no se expresa en la orofaringe humana, pero se localiza en las terminaciones nerviosas que inervan la mucosa. ASIC3 se expresa en las regiones inervadas por los NC V, IX y X y se localiza en las terminaciones nerviosas que inervan la mucosa. La capsaicina y la piperina en un mayor grado y los capsaicinoides naturales en un grado menor estimulan los canales de TRPV1. Una segunda exposición a estos agonistas disminuye su efecto sobre TRPV1, lo que sugiere procesos de desensitización. Además, la exposición a un antagonista específico de TRPV1 reduce de forma significativa el efecto de estos agonistas, lo que nos indica que su mecanismo de acción está mediado de forma específica por este receptor. La estimulación TRPV1 con capsaicinoides tiene mayor efecto terapéutico sobre los signos clínicos de la disfagia y la RMO que la estimulación dual TRPV1/A1 con piperina o la estimulación de TRPM8 con mentol. Mientras que la estimulación de receptores TRP aumenta la velocidad del bolo y acelera la RMO, aumentar la viscosidad con espesantes reduce la velocidad del bolo y retrasa aún más la RMO. Esta Tesis sienta las bases para el desarrollo de una nueva generación de tratamientos para la DO basados en la estimulación de la deglución a través de agonistas de los receptores encontrados en orofaringe humana. En el futuro, el tratamiento de pacientes con DO asociada a las enfermedades neurológicas y al envejecimiento se realizará de forma personalizada, con un estudio integral de la fisiopatología del paciente que nos permita iniciar el mejor tratamiento; desde la compensación de los tratamientos actuales a la rehabilitación de la función deglutoria de los tratamientos futuros.Oropharyngeal dysphagia (OD) is described as a disorder of the swallowing function and as difficulty or discomfort while safely and efficiently transporting the alimentary bolus and liquids from the mouth to the esophagus. OD is highly prevalent among older people, stroke patients, neurodegenerative or neuromuscular disease patients and patients with head and neck diseases or that undergo head and neck surgery. The management of these patients is currently based in compensating with thickeners, postures and maneuvers and the speech therapy, but there is no specific pharmacological treatment that improves the impaired physiology of these patients. With this Thesis we have set the bases for a new line of specific pharmacological treatments for OD by means of translational research, from basic research (histology and molecular biology) to clinical studies in which we assayed different treatments, by way of pharmacodynamic studies of these treatments. These new treatments are based in the sensory stimulation with pharmacological agonists of receptors found in the human oropharynx. This strategy aims to improve swallow by speeding the oropharyngeal swallow response (OSR) and strengthening lingual propulsion adding those agonists into the alimentary bolus. The human oropharyngeal mucosa structure is histologically different in the tongue (specialized mucosa) compared to the pharynx and epiglottis (lining mucosa). The oropharyngeal mucosa sensory innervation is generally found under the epithelium, although there are also nerve ends that penetrate the epithelium. TRPV1 and TRPA1 are expressed in the regions innervated by the CN V, IX and X, but while TRPV1 is mainly found on the epithelial cell membrane, TRPA1 is mainly found below the epithelium. TRPM8 is not expressed in the human oropharynx, but it is found on the nerve ends innervating the mucosa. ASIC3 is expressed in the regions innervated by the CN V, IX and X and it is found on the nerve ends innervating the mucosa. Capsaicin and piperine to a greater degree and natural capsaicinoids to a lesser degree successfully stimulate the TRPV1 channel. Repeated exposition of these agonists decreases the effect on TRPV1, suggesting desensitization. Moreover, their effect is significantly reduced by a TRPV1-specific antagonist, showing their action to be specific to this receptor. Supplementing nectar viscosity boluses with natural capsaicinoids has the greatest therapeutic effect on VFS signs and OSR compared with the dual TRPV1/A1 agonist, piperine, or the TRPM8 agonist, menthol. While TRP stimulants increased bolus velocity and reduced OSR times, thickeners reduce bolus velocity and further delay the OSR. This Thesis sets the bases for the development of a new generation of treatments for OD based in the stimulation of deglutition through the use of agonist of receptors found in the human oropharynx. In the future, the treatment for patients with OD caused by neurological disease and ageing will be personalized, involving the study of the physiopathology to select the best treatment; from compensation with current OD management to the rehabilitation of swallow function with future treatments

Caracterización de nuevas dianas terapéuticas para el tratamiento farmacológico de la disfagia orofaríngea asociada a enfermedades neurológicas y al envejecimiento /

Premi Extraordinari de Doctorat concedit pels programes de doctorat de la UAB per curs acadèmic 2016-2017La disfagia orofaríngea (DO) se describe como la alteración de la correcta función deglutoria y como la dificultad para transportar el bolo alimentario y los líquidos de la boca al esófago de forma segura y eficaz. La DO es muy prevalente en ancianos, pacientes que han tenido un ictus, pacientes con enfermedades neurodegenerativas o distrofias musculares y pacientes que han padecido enfermedades o han tenido intervenciones otorrinolaringológicas. Actualmente el tratamiento de estos pacientes se basa en la compensación con espesantes, posturas y maniobras y la rehabilitación logopédica, pero no existen tratamientos farmacológicos que permitan mejorar la fisiología alterada de estos pacientes. En esta Tesis hemos desarrollado las bases para una nueva línea de tratamiento farmacológico específico para la DO mediante investigación traslacional, partiendo de la investigación básica (histología y biología molecular) hasta llegar al ensayo de distintos tratamientos en estudios clínicos, pasando por el estudio farmacodinámico de estos tratamientos. Estos nuevos tratamientos están basados en la estimulación sensorial con agonistas farmacológicos de los receptores que se encuentran en la orofaringe humana. La estrategia se basa en añadir estos agonistas al bolo alimentario para mejorar la deglución promoviendo una respuesta motora orofaríngea (RMO) más rápida y una mayor fuerza de propulsión lingual. La estructura de la mucosa orofaríngea humana a nivel histológico es diferente en la lengua (mucosa especializada) respecto a la faringe y la epiglotis (mucosa de recubrimiento). La inervación sensorial de la mucosa orofaríngea es generalmente subepitelial, aunque también hay terminaciones nerviosas que penetran el epitelio. TRPV1 y TRPA1 se expresan en las regiones inervadas por los NC V, IX y X, pero mientras TRPV1 se localiza principalmente en la membrana de las células epiteliales, TRPA1 se localiza principalmente por debajo del epitelio. TRPM8 no se expresa en la orofaringe humana, pero se localiza en las terminaciones nerviosas que inervan la mucosa. ASIC3 se expresa en las regiones inervadas por los NC V, IX y X y se localiza en las terminaciones nerviosas que inervan la mucosa. La capsaicina y la piperina en un mayor grado y los capsaicinoides naturales en un grado menor estimulan los canales de TRPV1. Una segunda exposición a estos agonistas disminuye su efecto sobre TRPV1, lo que sugiere procesos de desensitización. Además, la exposición a un antagonista específico de TRPV1 reduce de forma significativa el efecto de estos agonistas, lo que nos indica que su mecanismo de acción está mediado de forma específica por este receptor. La estimulación TRPV1 con capsaicinoides tiene mayor efecto terapéutico sobre los signos clínicos de la disfagia y la RMO que la estimulación dual TRPV1/A1 con piperina o la estimulación de TRPM8 con mentol. Mientras que la estimulación de receptores TRP aumenta la velocidad del bolo y acelera la RMO, aumentar la viscosidad con espesantes reduce la velocidad del bolo y retrasa aún más la RMO. Esta Tesis sienta las bases para el desarrollo de una nueva generación de tratamientos para la DO basados en la estimulación de la deglución a través de agonistas de los receptores encontrados en orofaringe humana. En el futuro, el tratamiento de pacientes con DO asociada a las enfermedades neurológicas y al envejecimiento se realizará de forma personalizada, con un estudio integral de la fisiopatología del paciente que nos permita iniciar el mejor tratamiento; desde la compensación de los tratamientos actuales a la rehabilitación de la función deglutoria de los tratamientos futuros.Oropharyngeal dysphagia (OD) is described as a disorder of the swallowing function and as difficulty or discomfort while safely and efficiently transporting the alimentary bolus and liquids from the mouth to the esophagus. OD is highly prevalent among older people, stroke patients, neurodegenerative or neuromuscular disease patients and patients with head and neck diseases or that undergo head and neck surgery. The management of these patients is currently based in compensating with thickeners, postures and maneuvers and the speech therapy, but there is no specific pharmacological treatment that improves the impaired physiology of these patients. With this Thesis we have set the bases for a new line of specific pharmacological treatments for OD by means of translational research, from basic research (histology and molecular biology) to clinical studies in which we assayed different treatments, by way of pharmacodynamic studies of these treatments. These new treatments are based in the sensory stimulation with pharmacological agonists of receptors found in the human oropharynx. This strategy aims to improve swallow by speeding the oropharyngeal swallow response (OSR) and strengthening lingual propulsion adding those agonists into the alimentary bolus. The human oropharyngeal mucosa structure is histologically different in the tongue (specialized mucosa) compared to the pharynx and epiglottis (lining mucosa). The oropharyngeal mucosa sensory innervation is generally found under the epithelium, although there are also nerve ends that penetrate the epithelium. TRPV1 and TRPA1 are expressed in the regions innervated by the CN V, IX and X, but while TRPV1 is mainly found on the epithelial cell membrane, TRPA1 is mainly found below the epithelium. TRPM8 is not expressed in the human oropharynx, but it is found on the nerve ends innervating the mucosa. ASIC3 is expressed in the regions innervated by the CN V, IX and X and it is found on the nerve ends innervating the mucosa. Capsaicin and piperine to a greater degree and natural capsaicinoids to a lesser degree successfully stimulate the TRPV1 channel. Repeated exposition of these agonists decreases the effect on TRPV1, suggesting desensitization. Moreover, their effect is significantly reduced by a TRPV1-specific antagonist, showing their action to be specific to this receptor. Supplementing nectar viscosity boluses with natural capsaicinoids has the greatest therapeutic effect on VFS signs and OSR compared with the dual TRPV1/A1 agonist, piperine, or the TRPM8 agonist, menthol. While TRP stimulants increased bolus velocity and reduced OSR times, thickeners reduce bolus velocity and further delay the OSR. This Thesis sets the bases for the development of a new generation of treatments for OD based in the stimulation of deglutition through the use of agonist of receptors found in the human oropharynx. In the future, the treatment for patients with OD caused by neurological disease and ageing will be personalized, involving the study of the physiopathology to select the best treatment; from compensation with current OD management to the rehabilitation of swallow function with future treatments

A Comparative Study on the Effect of Acute Pharyngeal Stimulation with TRP Agonists on the Biomechanics and Neurophysiology of Swallow Response in Patients with Oropharyngeal Dysphagia

Fluid thickening is the main compensatory strategy for patients with oropharyngeal dysphagia (OD) associated with aging or neurological diseases, and there is still no pharmacological treatment. We aimed to compare the effects of increasing bolus viscosity with that of acute stimulation with TRPV1, TRPA1 or TRPM8 agonists on the biomechanics and neurophysiology of swallow response in patients with OD. We retrospectively analyzed seven studies from our laboratory on 329 patients with OD. The effect of increasing shear viscosity up to 3682 mPa&middot;s was compared by videofluoroscopy and pharyngeal sensory evoked potentials (pSEP) with that of adding to the bolus: capsaicin (TRPV1, 150 &mu;M/10 &mu;M), piperine (TRPA1/V1, 1 mM/150 &mu;M), menthol (TRPM8, 1 mM/10 mM), cinnamaldehyde-zinc (TRPA1, 100 ppm&ndash;70 mM), citral (TRPA1, 250 ppm) or citral-isopulegol (TRPA1-TRPM8, 250 ppm&ndash;200 ppm). Fluid thickening improved the safety of swallow by 80% (p &lt; 0.0001) by delaying bolus velocity by 20.7 &plusmn; 7.0% and time to laryngeal vestibule closure (LVC) by 23.1 &plusmn; 3.7%. Capsaicin 150&mu;M or piperine 1 mM significantly improved safety of swallow by 50% (p &lt; 0.01) and 57.1% (p &lt; 0.01) by speeding time to LVC by 27.6% (p &lt; 0.001) and 19.5% (p &lt; 0.01) and bolus velocity by 24.8% (p &lt; 0.01) and 16.9% (p &lt; 0.05), respectively. Cinnamaldehyde-zinc shortened the P2 latency of pSEPs by 11.0% (p &lt; 0.01) and reduced N2-P2 amplitude by 35% (p &lt; 0.01). In conclusion, TRPV1 and TRPV1/A1 agonists are optimal candidates to develop new pharmacological strategies to promote the recovery of brain and swallow function in patients with chronic OD

Pharmacodynamics of TRPV1 Agonists in a Bioassay Using Human PC-3 Cells

Altres ajuts: La Fundació Marató de TV3Purpose. TRPV1 is a multimodal channel mainly expressed in sensory neurons. We aimed to explore the pharmacodynamics of the TRPV1 agonists, capsaicin, natural capsaicinoids, and piperine in an in vitro bioassay using human PC-3 cells and to examine desensitization and the effect of the specific antagonist SB366791. Methods. PC-3 cells expressing TRPV1 were incubated with Fluo-4. Fluorescence emission changes following exposition to agonists with and without preincubation with antagonists were assessed and referred to maximal fluorescence following the addition of ionomycin. Concentration-response curves were fitted to the Hill equation. Results. Capsaicin and piperine had similar pharmacodynamics (E 204.8 ± 184.3% piperine versus 176.6 ± 35.83% capsaicin, P = 0.8814, Hill coefficient 0.70 ± 0.50 piperine versus 1.59 ± 0.86 capsaicin, P = 0.3752). In contrast, capsaicinoids had lower E (40.99 ± 6.14% capsaicinoids versus 176.6 ± 35.83% capsaicin, P < 0.001). All the TRPV1 agonists showed significant desensitization after the second exposition and their effects were strongly inhibited by SB366791. Conclusion. TRPV1 receptor is successfully stimulated by capsaicin, piperine, and natural capsaicinoids. These agonists present desensitization and their effect is significantly reduced by a TRPV1-specific antagonist. In addition, PC-3 cell bioassays proved useful in the study of TRPV1 pharmacodynamics

Natural history of swallow function during the three-month period after stroke

Oropharyngeal dysphagia is a prevalent complication following stroke (PS-OD), and one that is sometimes spontaneously recovered. This study describes the natural history of PS-OD between admission and three months post-stroke, and the factors associated with its prevalence and development. PS-OD was assessed with the volume- iscosity swallow test (V-VST) in all stroke patients on admission and at the three-month follow-up. We analyzed clinical, demographic, and neuroanatomical factors of 247 older post-stroke patients (National Institute of Health Stroke Scale (NIHSS) = 3.5 ± 3.8), comparing among those with PS-OD the ones with and without spontaneous recovery. PS-OD prevalence on admission was 39.7% (34.0% impaired safety; 30.8%, efficacy) and 41.7% (19.4% impaired safety; 39.3%, efficacy) at three months. Spontaneous swallow recovery occurred in 42.4% of patients with unsafe and in 29.9% with ineffective swallow, associated with younger age and optimal functional status. However, 26% of post-stroke patients developed new signs/symptoms of ineffective swallow related to poor functional, nutritional and health status, and institutionalization. PS-OD prevalence on admission and at the three-month follow-up was very high in the study population. PS-OD is a dynamic condition with some spontaneous recovery in patients with optimal functional status, but also new signs/symptoms can appear due to poor functionality. Regular PS-OD monitoring is needed to identify patients at risk of nutritional and respiratory complications

Targeting patient recovery priorities in degenerative cervical myelopathy: design and rationale for the RECEDE-Myelopathy trial-study protocol.

Peer reviewed: TrueAcknowledgements: Research in the senior author’s laboratory is supported by the Cambridge NIHR Brain Injury MedTech Cooperative. MRNK is supported by a NIHR Clinician Scientist Award.INTRODUCTION: Degenerative cervical myelopathy (DCM) is a common and disabling condition of symptomatic cervical spinal cord compression secondary to degenerative changes in spinal structures leading to a mechanical stress injury of the spinal cord. RECEDE-Myelopathy aims to test the disease-modulating activity of the phosphodiesterase 3/phosphodiesterase 4 inhibitor Ibudilast as an adjuvant to surgical decompression in DCM. METHODS AND ANALYSIS: RECEDE-Myelopathy is a multicentre, double-blind, randomised, placebo-controlled trial. Participants will be randomised to receive either 60-100 mg Ibudilast or placebo starting within 10 weeks prior to surgery and continuing for 24 weeks after surgery for a maximum of 34 weeks. Adults with DCM, who have a modified Japanese Orthopaedic Association (mJOA) score 8-14 inclusive and are scheduled for their first decompressive surgery are eligible for inclusion. The coprimary endpoints are pain measured on a visual analogue scale and physical function measured by the mJOA score at 6 months after surgery. Clinical assessments will be undertaken preoperatively, postoperatively and 3, 6 and 12 months after surgery. We hypothesise that adjuvant therapy with Ibudilast leads to a meaningful and additional improvement in either pain or function, as compared with standard routine care. STUDY DESIGN: Clinical trial protocol V.2.2 October 2020. ETHICS AND DISSEMINATION: Ethical approval has been obtained from HRA-Wales.The results will be presented at an international and national scientific conferences and in a peer-reviewed journals. TRIAL REGISTRATION NUMBER: ISRCTN Number: ISRCTN16682024

The construction of the L3 experiment

The L3 experiment is one of the six large detectors designed for the new generation of electron-positron accelerators. It is the only detector that concentrates its efforts on limited goals of measuring electrons, muons and photons. By not attempting to identify hadrons, L3 has been able to provide an order of magnitude better resolution for electrons, muons and photons. Vertices and hadron jets are also studied. The construction of L3 has involved much state of the art technology in new principles of vertex detection and in new crystals for large scale electromagnetic shower detection and ultraprecise muon detection. This paper presents a summary of the construction of L3.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28628/3/0000442.pd