RESUMEN
BACKGROUND: Respiratory syncytial virus (RSV) is a highly infectious disease that poses a significant clinical and medical burden, as well as social disruption and economic costs, recognized by the World Health Organization as a public health issue. After several failed attempts to find preventive candidates (compounds, products, including vaccines), new alternatives might be available, one being nirsevimab, the first and only option approved for RSV prevention in neonates and infants during their first RSV season. The objective of this study was to develop a novel multi-criteria decision analysis (MCDA) framework for RSV antibody-based preventive alternatives and to use it to assess the value of nirsevimab vs. placebo as a systematic immunization approach to prevent RSV in neonates and infants during their first RSV season in Spain. METHODS: Based on a pre-established model called Vaccinex, an ad-hoc MCDA framework was created to reflect relevant attributes for the assessment of current and future antibody-based preventive measures for RSV. The estimated value of nirsevimab was obtained by means of an additive linear model combining weights and scores assigned by a multidisciplinary committee of 9 experts. A retest and three sensitivity analyses were conducted. RESULTS: Nirsevimab was evaluated through a novel framework with 26 criteria by the committee as a measure that adds value (positive final estimated value: 0.56 ± 0.11) to the current RSV scenario in Spain, by providing a high efficacy for prevention of neonates and infants. In addition, its implementation might generate cost savings in hospitalizations and to the healthcare system and increase the level of public health awareness among the general population, while reducing health inequities. CONCLUSIONS: Under a methodology with increasing use in the health field, nirsevimab has been evaluated as a measure which adds value for RSV prevention in neonates and infants during their first RSV season in Spain.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Recién Nacido , Lactante , Humanos , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/prevención & control , Antivirales , España , Técnicas de Apoyo para la DecisiónRESUMEN
Here, we investigate the effects of obesity induced by monosodium glutamate (MSG) on cognitive impairment and whether this model induces any alteration in the affinity, density, and subtypes of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Healthy rats were used as controls, and MSG-obese rats were selected via the Lee index > 0.300. The effects of MSG-induced obesity on hippocampal spatial learning and memory processes were evaluated by using the working memory versions of the Morris' water maze task and the evaluation of mAChRs by binding assay and their subtypes by immunoprecipitation assays. [ 3 H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (K D ) did not differ between control and MSG, indicating that affinity is not affected by obesity induced by MSG. The maximum number of binding sites (B max ) obtained in MSG subjects was lower than that obtained from control rats, indicating a decrease in the expression of total mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M 1 subtype of MSG when compared with control rats (M 2 to M 5 subtypes did not differ between control and MSG). We also observed that MSG promotes a disruption of the spatial working memory which was accompanied by a decrease in the M 1 mAChR subtype in rat hippocampus, thus suggesting deleterious long-term effects besides the obesity. In conclusion, these findings provide new insights into how obesity can influence spatial learning and memory that is hippocampal-dependent. The data suggest that the M 1 mAChR subtype protein expression is a potential therapeutic target.
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Receptores Muscarínicos , Glutamato de Sodio , Ratas , Animales , Glutamato de Sodio/efectos adversos , Glutamato de Sodio/metabolismo , Ratas Wistar , Receptores Muscarínicos/metabolismo , Obesidad , HipocampoRESUMEN
Psoriasis is a chronic, systemic inflammatory disease that affects the skin, with a high impact on patients' quality of life. The aim of this study was to identify and determine the relative importance of unmet needs in the management of moderate-to-severe psoriasis in Spain, from a multi-stakeholder perspective. A mixed method-approach was used to collect information, design a questionnaire and a discrete-choice exercise, and elicit the unmet needs through a multidisciplinary committee composed of 12 experts. A total of 65 unmet needs were identified and categorized into 4 areas: clinical, patient-related, decision-making process, and social. Decision-making process unmet needs were perceived as the most pressing ones, followed by social, clinical and patient-related. Individually, the need to incorporate outcomes that are important to the patients and to have treatments that achieve total clearance with a rapid onset of action and long-term persistence were the most important unmet needs.
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Psoriasis , Calidad de Vida , Ejercicio Físico , Humanos , Psoriasis/diagnóstico , Psoriasis/tratamiento farmacológico , España/epidemiología , Encuestas y CuestionariosRESUMEN
BACKGROUND: Pulmonary Arterial Hypertension (PAH) is a rare, debilitating, and potentially fatal disease. This study aims to quantify the economic burden of PAH in Spain. METHODS: The study was conducted from a societal perspective, including direct and indirect costs associated with incident and prevalent patients. Average annual costs per patient were estimated by multiplying the number of resources consumed by their unit cost, differentiating the functional class (FC) of the patient. Total annual costs per FC were also calculated, taking the 2020 prevalence and incidence ranges into account. An expert committee validated the information on resource consumption and provided primary information on pharmacological consumption. Unit costs were estimated using official tariffs and salaries in Spain. A deterministic sensitivity analysis was conducted to test the uncertainty of the model. RESULTS: The average annual total cost was estimated at 98,839 per prevalent patient (FC I-II: 65,233; FC III: 103,736; FC IV: 208,821), being 42,110 for incident patients (FC I-II: 25,666; FC III: 44,667; FC IV: 95,188). The total annual cost of PAH in Spain, taking into account a prevalence between 16.0 and 25.9 cases per million adult inhabitants (FC I-II 31.8%; FC III 61.3%; FC IV 6.9%) and an incidence of 3.7, was estimated at 67,891,405 to 106,131,626, depending on the prevalence considered. Direct healthcare costs accounted for 64% of the total cost, followed by indirect costs (24%), and direct non-healthcare costs (12%). The total costs associated with patients in FC I-II ranged between 14,161,651 and 22,193,954, while for patients in FC III costs ranged between 43,763,019 and 68,391,651, and for patients in FC IV between 9,966,735 and 15,546,021. In global terms, patients with the worst functional status (FC IV) account for only 6.9% of the adults suffering from PAH in Spain, but are responsible for 14.7% of the total costs. CONCLUSIONS: PAH places a considerable economic burden on patients and their families, the healthcare system, and society as a whole. Efforts must be made to improve the health and management of these patients since the early stages of the disease.
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Hipertensión Arterial Pulmonar , Adulto , Atención a la Salud , Estrés Financiero , Costos de la Atención en Salud , Humanos , Hipertensión Arterial Pulmonar/epidemiología , España/epidemiologíaRESUMEN
OBJECTIVES: The aim of this study was to review the current evaluation and funding processes for new drugs in different developed countries, to provide a comparative framework with detailed, homogeneous, and up-to-date information. METHODS: Scientific publications, reports and websites were reviewed between July and December 2021 using PubMed, Google Scholar, and grey literature sources. The main items searched were actors and processes, including timelines, characteristics of clinical and economic evaluations, participation of stakeholders, elements of price and reimbursement decisions, cost-effectiveness thresholds and specific funds. The analysed 13 countries were Australia, Canada, England, France, Germany, Italy, Japan, the Netherlands, Portugal, Scotland, South Korea, Spain and Sweden. RESULTS: Eight countries perform the assessment process separated from the pricing decision. Countries measure each drug's added therapeutic value through multi-attribute value scales, algorithms, non-prescriptive lists of criteria, or quality-adjusted life years (QALYs). Health technology assessment (HTA) methodologies differ in their outcome measures, elicitation techniques, comparators, and perspectives. The criteria used for pricing and reimbursement include humanistic, clinical, and economic aspects. Only Scotland, England, the Netherlands, Canada and Portugal use explicit efficiency thresholds. Health care professionals participate in all assessment committees, and patients are becoming increasingly involved in most countries. The official time from marketing authorisation to the completion of the evaluation and pricing processes varied from 126 to 540 days. CONCLUSIONS: Most analysed countries show a trend towards value-based approaches that consider value for money to society, but also other economic, clinical, and humanistic criteria. Good practices included robustness, transparency, independence, and participation.
Asunto(s)
Organización para la Cooperación y el Desarrollo Económico , Evaluación de la Tecnología Biomédica , Humanos , Países Bajos , Alemania , Francia , Análisis Costo-BeneficioRESUMEN
BACKGROUND: Multi-criteria decision analysis (MCDA) was proposed to surmount arbitrary clinical decisions in the field of biological therapies for psoriatic patients. At the same time, MCDA may further highlight the potential of bimekizumab for the treatment of moderate-to-severe psoriasis, compared to placebo, adalimumab, ustekinumab, secukinumab, and even ixekizumab and risankizumab. RESEARCH DESIGN AND METHODS: The EVIDEM framework was adapted to reflect relevant criteria for the assessment. Estimated values were obtained by means of an additive linear model combining weights and scores assigned by a multidisciplinary committee of 12 experts. Consistency and replicability were evaluated through an alternative weighting method and a re-test. RESULTS: Bimekizumab was assessed by the committee as an intervention with a positive value contribution for the treatment of moderate-to-severe psoriasis in comparison to any of the alternatives. The drug provides a substantial therapeutical benefits and improves the health results reported by the patients, as it combines a higher level of clearance, rapidity, and persistence with a similar safety and tolerability profile. CONCLUSIONS: Under a methodology with increasing use in the health field, bimekizumab was evaluated as a drug with a high added value for the treatment of moderate-to-severe psoriasis when compared to six different alternatives.
Asunto(s)
Psoriasis , Adalimumab/efectos adversos , Anticuerpos Monoclonales Humanizados , Técnicas de Apoyo para la Decisión , Humanos , Psoriasis/tratamiento farmacológico , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
BACKGROUND: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). METHODS: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. RESULTS: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. CONCLUSION: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.
RESUMEN
Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an anti-diabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIOâ¯+â¯E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIOâ¯+â¯E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIOâ¯+â¯E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIOâ¯+â¯E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIOâ¯+â¯E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIOâ¯+â¯E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIOâ¯+â¯E animals, which did not differ from each other. Our results indicate that obesity induced by high-calorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.
Asunto(s)
Exenatida/uso terapéutico , Hipocampo/efectos de los fármacos , Obesidad/tratamiento farmacológico , Receptores Muscarínicos/efectos de los fármacos , Animales , Carbacol/farmacología , Ingestión de Energía , Exenatida/farmacología , Receptor del Péptido 1 Similar al Glucagón/fisiología , Hipocampo/fisiología , Masculino , Ratas , Ratas Wistar , Receptores Muscarínicos/fisiologíaRESUMEN
Here, we investigate the effects of obesity induced by monosodium glutamate (MSG) on cognitive impairment and whether this model induces any alteration in the affinity, density, and subtypes of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Healthy rats were used as controls, and MSG-obese rats were selected via the Lee index > 0.300. The effects of MSG-induced obesity on hippocampal spatial learning and memory processes were evaluated by using the working memory versions of the Morris’ water maze task and the evaluation of mAChRs by binding assay and their subtypes by immunoprecipitation assays. [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ between control and MSG, indicating that affinity is not affected by obesity induced by MSG. The maximum number of binding sites (Bmax) obtained in MSG subjects was lower than that obtained from control rats, indicating a decrease in the expression of total mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 subtype of MSG when compared with control rats (M2 to M5 subtypes did not differ between control and MSG). We also observed that MSG promotes a disruption of the spatial working memory which was accompanied by a decrease in the M1 mAChR subtype in rat hippocampus, thus suggesting deleterious long-term effects besides the obesity. In conclusion, these findings provide new insights into how obesity can influence spatial learning and memory that is hippocampal-dependent. The data suggest that the M1 mAChR subtype protein expression is a potential therapeutic target.
RESUMEN
INTRODUCTION: Glucagon-like peptide-1 (GLP-1) receptor (R) agonists are a class of incretin mimetic drugs that have been used for the treatment of type 2 diabetes mellitus and also considered strong candidates for the treatment of obesity. The original prototypical drug in this class is the exenatide, a synthetic peptide with the same structure as the native molecule, exendin-4, found in the saliva of the Gila monster (Heloderma suspectum suspectum lizard). OBJECTIVES: To identify and compare the anti-obesogenic, antidyslipidemic and antidiabetogenic effects of agonism in GLP-1R by exenatide on two distinct models of obesity: induced by hypothalamic injury (MSG) or high-calorie diet (DIO). METHODS: To obtain MSG, neonatal rats were daily subcutaneously injected with 4 g monosodium glutamate/kg, for 10 consecutive days. To obtain DIO, 72-75 days old rats received hyperlipid food and 30% sucrose for drinking up to 142-145 days old. Untreated healthy rats with the same age were used as control. General biometric and metabolic parameters were measured. RESULTS: MSG was characterized by decreased naso-anal length, food and fluid intake, plasma protein and glucose decay rate per minute after insulin administration (KITT), as well as increased Lee index (body mass0.33/naso-anal length), mass of retroperitoneal and periepididymal fat pads, glycemia, triglycerides (TG), LDL and VLDL. Exenatide ameliorated KITT and food and fluid intake, and it also restored glycemia in MSG. DIO was characterized by glucose intolerance, increased body mass, Lee index, fluid intake, mass of retroperitoneal and periepididymal fat pads, glycemia, glycated hemoglobin (HbA1c), TG, VLDL and total cholesterol, as well as decreased food intake and KITT. Exenatide restored glycemia, HbA1c, TG, VLDL, total cholesterol and body mass, and it also ameliorated food and fluid intake, KITT and mass of retroperitoneal fat pad in DIO. CONCLUSIONS: The hypothalamic injury and the high-calorie diet induce dyslipidemia and glycemic dysregulation in addition to obesity in rats. The usual therapeutic dose of exenatide in humans is antidiabetogenic in both these obesity models, but is anti-obesogenic and hypolipidemic only in diet-induced obesity. Agonists of GLP-1R are promising anti-obesogenic and antidyslipidemic drugs in the early stages of the obesity, in which the integrity of the nervous system was unaffected.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Hipoglucemiantes/farmacología , Hipolipemiantes/farmacología , Lagartos , Péptidos/farmacología , Ponzoñas/farmacología , Animales , Glucemia , Dieta/veterinaria , Ingestión de Alimentos/efectos de los fármacos , Exenatida , Femenino , Hipotálamo/efectos de los fármacos , Masculino , Obesidad/inducido químicamente , Ratas Wistar , Saliva/química , Glutamato de Sodio/farmacologíaRESUMEN
CAR-T cell therapy represents a therapeutic revolution in the prognosis and treatment of patients with certain types of hematological cancer. However, they also pose new challenges in the healthcare, regulatory and financial fields. The aim of the RET-A project was to undertake a strategic reflection on the management of CAR-T therapies within the Spanish National Health System, to agree on recommendations that will help to better deal with the new context introduced by these cell therapies in the present and in the future. This think tank involved 40 key agents and opinion leaders. The experts identified three great challenges for implementing advanced therapies in Spain: therapeutic individualisation, with a multidisciplinary approach; acceleration of access times, by minimizing bureaucracy; and increase in the number of centers qualified to manage the CAR-T therapies in the NHS. The experts agreed on the ideal criteria for designating those qualified centers. They also agreed on a comprehensive CAR-T care pathway with the timings and roles which would ideally be involved in each part of the process.
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Humanos , Neoplasias Hematológicas , Inmunoterapia Adoptiva , Sistemas Nacionales de Salud , Consenso , EspañaRESUMEN
Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.
RESUMEN
Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an anti-diabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIO+E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIO+E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIO+E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIO+E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIO+E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIO+E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIO+E animals, which did not differ from each other. Our results indicate that obesity induced by high-calorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an anti-diabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIO+E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIO+E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIO+E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIO+E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIO+E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIO+E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIO+E animals, which did not differ from each other. Our results indicate that obesity induced by high-calorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.
RESUMEN
Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.
RESUMEN
Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.(AU)
Asunto(s)
Animales , Ratas , Serpientes , Venenos Elapídicos/efectos adversos , Fosfolipasas A2 , Fosfatos de Inositol , Acetilcolina , Receptores Muscarínicos/análisis , Análisis de Secuencia de ProteínaRESUMEN
The smoke of crack cocaine contains cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). AEME possesses greater neurotoxic potential than cocaine and an additive effect when they are combined. Since atropine prevented AEME-induced neurotoxicity, it has been suggested that its toxic effects may involve the muscarinic cholinergic receptors (mAChRs). Our aim is to understand the interaction between AEME and mAChRs and how it can lead to neuronal death. Using a rat primary hippocampal cell culture, AEME was shown to cause a concentration-dependent increase on both total [(3)H]inositol phosphate and intracellular calcium, and to induce DNA fragmentation after 24 hours of exposure, in line with the activation of caspase-3 previously shown. Additionally, we assessed AEME activity at rat mAChR subtypes 1-5 heterologously expressed in Chinese Hamster Ovary cells. l-[N-methyl-(3)H]scopolamine competition binding showed a preference of AEME for the M2 subtype; calcium mobilization tests revealed partial agonist effects at M1 and M3 and antagonist activity at the remaining subtypes. The selective M1 and M3 antagonists and the phospholipase C inhibitor, were able to prevent AEME-induced neurotoxicity, suggesting that the toxicity is due to the partial agonist effect at M1 and M3 mAChRs, leading to DNA fragmentation and neuronal death by apoptosis.
Asunto(s)
Cocaína/análogos & derivados , Hipocampo/metabolismo , Síndromes de Neurotoxicidad/metabolismo , Neurotoxinas/toxicidad , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M3/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células CHO , Cocaína/toxicidad , Cricetinae , Cricetulus , Fragmentación del ADN/efectos de los fármacos , Femenino , Hipocampo/patología , Síndromes de Neurotoxicidad/patología , Ratas , Factores de TiempoRESUMEN
We report the effect of acute estrogen treatment in the expression of muscarinic acetylcholine receptors (mAChRs) in myometrium. Strips were obtained from rats in estrus (control) and treated with estrogen, 24h before the experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed and m2, m3 and m5 mAChR mRNA subtypes were detected in myometrium from both groups. [(3)H]Quinuclidinyl benzilate [(3)HQNB] binding studies indicated that estrogen treatment did not change the affinity and density of mAChRs in myometrial membranes. Displacement curves of [(3)HQNB] with different mAChRs antagonists indicated a one-site fit for all antagonists tested. Comparison of pK(i) values indicated a significant correlation to M(2)-mAChR subtype. Functional studies, however, showed that estrogen treatment increased myometrium sensitivity to carbachol and the calculated apparent affinity values were significantly correlated to M(3)-mAChR. Furthermore, the pharmacological profile of the two populations of mAChR was not affected by estrogen. In conclusion, these results provide evidence for the presence of M(2)- and M(3)-mAChR, at the mRNA and protein level, in the rat myometrium and indicate that estrogen induces an increase in myometrial responsiveness to mAChR agonists.
Asunto(s)
Estrógenos/farmacología , Miometrio/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Animales , Carbacol/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , ARN Mensajero/análisis , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Wistar , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M2/genética , Receptor Muscarínico M3/efectos de los fármacos , Receptor Muscarínico M3/genética , Receptores Muscarínicos/genéticaRESUMEN
The aim of the present study was to investigate the effects of different periods of ovariectomy and 17ß-estradiol (E2) replacement on the expression of Cytochrome C, apoptosis inducing factor (AIF) and Endonuclease-G (Endo-G) in mitochondrial and cytosolic fractions obtained from hippocampus of the adult female rats. In addition, the expression of phosphorylated CREB (phospho-CREB) was also analyzed in hippocampus. Ovariectomy or E2 treatment did not change the expression of Cytochrome C and AIF. Ovariectomy (15, 21 and 36 days) decreased the expression of Endo-G in the mitochondrial fractions and increased it in the cytosolic fractions obtained from hippocampus. The treatment with E2 after 15 days of ovariectomy for 7 days or 21 days, and throughout the post-ovariectomy period prevented the effects of ovariectomy on Endo-G expression. Our results suggest that ovariectomy-induced apoptotic cell death in hippocampal tissue could be mediated by Endo-G, but not by AIF, via a caspase-independent apoptotic pathway. Furthermore, ovariectomy decreased the expression of phospho-CREB and the treatment with E2 prevented these effects. In conclusion, E2 may help maintain long-term neuronal viability by regulating the expression of members of the Bcl-2 family. Regulation of Endo-G released from mitochondria, but not of Cytochrome C and AIF, is also involved in the neuroprotective actions of E2. Furthermore, CREB may be involved in the expression of Bcl-2. These data provide new understanding into the mechanisms involved in the neuroprotective role of estrogen.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Endodesoxirribonucleasas/metabolismo , Estradiol/farmacología , Terapia de Reemplazo de Estrógeno , Hipocampo/metabolismo , Ovariectomía , Animales , Factor Inductor de la Apoptosis/metabolismo , Citocromos c/metabolismo , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Fosforilación/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an anti-diabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIO+E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIO+E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIO+E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIO+E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIO+E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIO+E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIO+E animals, which did not differ from each other. Our results indicate that obesity induced by high-calorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an anti-diabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIO+E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIO+E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIO+E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIO+E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIO+E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIO+E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIO+E animals, which did not differ from each other. Our results indicate that obesity induced by high-calorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.
RESUMEN
The aim of the present study was to investigate the involvement of estrogen receptors in the activation of phospholipase C (PLC)-phosphoinositide hydrolysis in the hippocampus from rats in estrous and proestrous phases. 17ß-Estradiol (E2) and ESR1-selective agonist PPT, but not ESR2-selective agonist DPN, induced a rapid increase on total [³H]-inositol phosphate accumulation in the hippocampus from both rats. These effects are mediated by PLC activation, since the inhibition of this protein decreased the total [³H]-inositol phosphate accumulation. The pretreatment with ESR1 and ESR2 antagonist ICI 182,780, but not with GPER antagonist G-15, blocked the total [³H]-inositol phosphate accumulation induced by E2 and PPT, confirming that ESR1 is upstream component regulating this rapid effect. SRC family of protein tyrosine kinases inhibitor PP2 blocked the total [³H]-inositol phosphate accumulation induced by E2 and PPT in hippocampus, suggesting that ESR1 undergoes translocation from the nuclei to the plasma membrane region via SRC to activate rapid signaling pathways. Furthermore, the magnitude of the response to E2 and PPT was higher in hippocampus from rats in proestrous than in estrous. On the other hand, the expression of the ESR1 is higher in hippocampus from rats in estrous than in proestrous, indicating that the regulation of this receptor by estrous cycle does not play a role in the magnitude of the response to E2 and PPT in hippocampus. In conclusion, our results indicate that E2 activates SRC-mediated translocation of ESR1 to the plasma membrane, which results in the activation of PLC-inositol phosphate signaling pathway in rat hippocampus. Thus, these rapid estrogen actions in hippocampus might be a key step mediating cellular events important for learning and memory.