Position statement on use of pharmacological combinations in a single pill for treatment of hypertension by Argentine Federation of Cardiology (FAC) and Argentine Society of Hypertension (SAHA).

The present document provides scientific evidence reviewed and analysed by a group of specialist clinicians in hypertension that aims to give an insight into a pharmacological strategy to improve blood pressure control. Evidence shows that most hypertensive patients will need at least two drugs to achieve blood pressure goals. There is ample evidence showing that treatment adherence is inversely related to the number of drugs taken. Observational studies show that use of drug combinations to initiate treatment reduces the time to reach the treatment goal and reduces CVD, especially with single pill combinations (SPCs). This work, based on recommendations of the Argentine Federation of Cardiology and Argentine Society of Hypertension as a reference, aims to review the more recent evidence on SPC, and to serve as guidelines for health professionals in their clinical practice and to the wider use of SPCs for the treatment of hypertension. Evidence from clinical trials on the effectiveness and adverse effects of using SPCs are provided. An analysis is also made of the main contributions of SPCs in special populations, e.g., elderly and diabetic patients, and its use in high risk and resistant hypertension. The effects of SPCs on hypertensive-mediated organ damage is also examined. Finally, we provide some aspects to consider when choosing treatments in the economic context of Latin-America for promoting the most efficient use of resources in a scarce environment and to provide quality information to decision makers to formulate safe, cost-effective, and patient-centered health policies. Finally, future perspectives and limitations in clinical practice are also discussed.

Optimization of a Pyrimidinone Series for Selective Inhibition of Ca2+/Calmodulin-Stimulated Adenylyl Cyclase 1 Activity for the Treatment of Chronic Pain.

Adenylyl cyclase type 1 (AC1) is involved in signaling for chronic pain sensitization in the central nervous system and is an emerging target for the treatment of chronic pain. AC1 and a closely related isoform AC8 are also implicated to have roles in learning and memory signaling processes. Our team has carried out cellular screening for inhibitors of AC1 yielding a pyrazolyl-pyrimidinone scaffold with low micromolar potency against AC1 and selectivity versus AC8. Structure-activity relationship (SAR) studies led to analogues with cellular IC50 values as low as 0.25 µM, selectivity versus AC8 and other AC isoforms as well as other common neurological targets. A representative analogue displayed modest antiallodynic effects in a mouse model of inflammatory pain. This series represents the most potent and selective inhibitors of Ca2+/calmodulin-stimulated AC1 activity to date with improved drug-like physicochemical properties making them potential lead compounds for the treatment of inflammatory pain.

Acyclic nucleoside phosphonates with 2-aminothiazole base as inhibitors of bacterial and mammalian adenylate cyclases.

A series of novel acyclic nucleoside phosphonates (ANPs) was synthesized as potential adenylate cyclase inhibitors, where the adenine nucleobase of adefovir (PMEA) was replaced with a 5-substituted 2-aminothiazole moiety. The design was based on the structure of MB05032, a potent and selective inhibitor of fructose 1,6-bisphosphatase and a good mimic of adenosine monophosphate (AMP). From the series of eighteen novel ANPs, which were prepared as phosphoroamidate prodrugs, fourteen compounds were potent (single digit micromolar or submicromolar) inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT), mostly without observed cytotoxicity in J774A.1 macrophage cells. Selected phosphono diphosphates (nucleoside triphosphate analogues) were potent inhibitors of ACT (IC50 as low as 37 nM) and B. anthracis edema factor (IC50 as low as 235 nM) in enzymatic assays. Furthermore, several ANPs were found to be selective mammalian AC1 inhibitors in HEK293 cell-based assays (although with some associated cytotoxicity) and one compound exhibited selective inhibition of mammalian AC2 (only 12% of remaining adenylate cyclase activity) but no observed cytotoxicity. The mammalian AC1 inhibitors may represent potential leads in development of agents for treatment of human inflammatory and neuropathic pain.

24/7 Physical Therapy Intervention With Adult Patients in a Chilean Intensive Care Unit: A Cost-Benefit Analysis in a Developing Country.

OBJECTIVES: Physiotherapy in an adult intensive care unit (ICU) affects health outcome. To justify the investment in ICU physical therapy, the cost savings associated with its benefits need to be established. The main objective of this study is to evaluate the potential cost savings of implementing 24-hour, 7-days-per-week physiotherapist (24/7-PT) in a Chilean public high-complex specialized ICU. METHODS: Using clinical data from a literature review and a micro-costing technique, we conducted a cost-benefit analysis in the National Institute of Thorax in Chile. Our example scenario involves 697 theoretical admissions of adult patients with cardiovascular or respiratory diseases, and the costs and benefits by reduction of length of stay in ICU, days of mechanical ventilation, and days with respiratory infections during the first year and 5 years of admissions. A sensitivity analysis was considered according to the variability in total costs, production income, and clinical benefits. RESULTS: Net cost savings generated in our example scenario demonstrate that the implementation of 24/7-PT produces a minimum saving for the institution of $16 242 during the first year and $69 351 over a 5-year interval considering individual income production. Out of the 30 scenarios included in the sensitivity analyses, 26 (87%) demonstrated net savings. CONCLUSIONS: A financial model, based on literature review and actual cost data, projects that 24/7-PT intervention is a cost-benefit alternative in adult ICU patients with cardiovascular or respiratory diseases in Chile. It is necessary a scenario of at least 3 sessions per day with insurance payment for individual treatments to support the long-term implementation of a 24/7-PT program.

Mechanisms of mitochondrial DNA escape and its relationship with different metabolic diseases.

It is well-known that mitochondrial DNA (mtDNA) can escape to intracellular or extracellular compartments under different stress conditions, yet understanding their escape mechanisms remains a challenge. Although Bax/Bak pores and VDAC oligomers are the strongest possibilities, other mechanisms may be involved. For example, mitochondria permeability transition, altered mitophagy, and mitochondrial dynamics are associated with intracellular mtDNA escape, while extracellular traps and extracellular vesicles can participate in extracellular mtDNA escape. The evidence suggests that mtDNA escape is a complex event with more than one mechanism involved. In addition, once the mtDNA is outside the mitochondria, the effects can be complex. Different danger signal sensors recognize the mtDNA as a damage-associated molecular pattern, triggering an innate immune inflammatory response that can be observed in multiple metabolic diseases characterized by chronic inflammation, including autoimmune diseases, diabetes, cancer, and cardiovascular disorders. For these reasons, we will review the most recent evidence regarding mtDNA escape mechanisms and their impact on different metabolic diseases.

Genome-Wide Small Interfering RNA Screening Reveals a Role for Cullin3-Really Interesting New Gene Ligase Signaling in Heterologous Sensitization of Adenylyl Cyclase.

Heterologous sensitization of adenylyl cyclase (AC) is revealed as enhanced or exaggerated AC/cAMP signaling that occurs following persistent activation of Gα i/o-coupled receptors. This paradoxical phenomenon was discovered more than 40 years ago and was proposed as a cellular mechanism to explain the adaptive changes that occur following chronic exposure to drugs of abuse. However, the underlying molecular mechanisms of heterologous sensitization of AC remain largely unknown. In the present study, we performed a genome-wide cell-based RNA interference screen as an unbiased approach to identify genes associated with heterologous sensitization of AC. Following a series of validation and confirmation assays, three genes that form an E3 ligase complex, cullin3 (CUL3), neural precursor-cell-expressed and developmentally downregulated 8 (NEDD8), and really interesting new gene (RING)-box protein 1 (RBX1), were identified as specific modulators of heterologous sensitization of AC. Furthermore, based on the downstream actions of these genes, we evaluated the activity of proteasome inhibitors as well as the specific NEDD8-activating enzyme inhibitor, MLN4924 (Pevonedistat), in AC sensitization. We demonstrate that MG-132 and bortezomib treatments could mimic the inhibitory effects observed with gene knockdown, and MLN4924 was potent and efficacious in blocking the development of heterologous sensitization of endogenous and recombinant AC isoforms, including AC1, AC2, AC5, and AC6. Together, by using genetic and pharmacological approaches, we identified, for the first time, cullin3-RING ligases and the protein degradation pathway as essential modulators for heterologous sensitization of AC. SIGNIFICANCE STATEMENT: Through a genome-wide cell-based RNA interference screening, we identified three genes that form an E3 ligase complex, cullin3, neural precursor-cell-expressed and developmentally downregulated 8 (NEDD8), and really interesting new gene-box protein 1, as specific modulators of heterologous sensitization of AC. The effect of cullin3, NEDD8, or really interesting new gene-box protein 1 small interfering RNAs on heterologous sensitization was recapitulated by proteasome inhibitors, MG132 and bortezomib, and the specific NEDD8-activating enzyme inhibitor, MLN4924. These results suggest a novel hypothesis in which protein degradation is involved in the sensitization of AC signaling that occurs following chronic activation of Gαi/o-coupled receptors.

Ozonoterapia: una propuesta terapéutica para la prevención y el tratamiento de la infección por coronavirus SARS-COV-2 "COVID 19" / Ozone therapy: a therapeutic proposal for the prevention and treatment of the SARS-COV-2 "COVID 19" coronavirus infection

Enfrentamos actualmente una crisis que ha redefinido el comportamiento humano y está cambiando la historia de la humanidad. "La simple perspectiva de perder la vida por algo invisible pero omnipresente agobia la existencia de cada individuo social". La infección por los coronavirus en general ha tenido un comportamiento benigno y no había pasado de ser una "gripa" hasta que, en el 2002, 2012 y 2019 aparecen epidemia por estos coronavirus con la capacidad de producir infecciones mortales. De ellas, que respectivamente se han llamado SARS-CoV, MERS-CoV, SARS-CoV-2, siendo ésta última la responsable de la pandemia actual. La infección por SARS-CoV-2 en la actualidad ha afectado alrededor de 2,5 millones de personas y ocasionado alrededor de 160 mil muertes. Hasta hoy la única medida que se ha mostrado efectiva en el control de la infección es el aislamiento preventivo, buscando con éste evitar la aparición de enfermedad potencialmente mortal, cuyo tratamiento tiene costos enormes en equipos y personal hospitalario. Este aislamiento conduce a un freno de la economía y a una incapacidad del estado a mediano plazo para contener la crisis social creciente en varios componentes, ansiedad, depresión, desempleo, descuido en la atención de otras enfermedades potencialmente mortales, aplazamiento de cirugías electivas y hambre. Además de considerar el aislamiento como la piedra angular, es importante proponer alternativas de prevención y tratamiento de la enfermedad. A la fecha en términos de tratamiento son muchos los medicamentos probados, sin demostrarse todavía una eficacia que aliente el optimismo. Conocedores que somos de la importancia de brindar a los pacientes un medio de prevención y un método terapéutico efectivo de la infección, queremos proponer la ozonoterapia como parte del abanico de posibilidades que se le pueda ofrecer a los afectados por la infección, pero mejor aún, la enorme posibilidad que ésta podría brindar para evitar la misma y/o la progresión de la enfermedad hacia estadios graves. La ozonoterapia, mezcla de sangre venosa con ozono obtenido a partir de oxígeno medicinal, no es un procedimiento nuevo y poco probado, como si muchos de los medicamentos en curso de investigación para el manejo de la infección. Ésta existe desde 1935 y con ella se han manejado ya infecciones virales con éxito, incluso en la pandemia actual ya hay registros de su uso en Ibiza y en España con resultados que nos motivan a proponerla hoy en Colombia. Su aplicación puede abarcar la prevención y el tratamiento al poderse por este medio eliminar el virus circulante e inducir una respuesta antiinflamatoria que evite la agravación de la enfermedad y permita la recuperación más rápida de los pacientes. Queremos apoyar con este procedimiento a los pacientes afectados y a los contactos de ellos para abaratar los costos de manejo del paciente hospitalizado y de manera preventiva ayudar a enfrentar la crisis con miras a contener el desajuste económico que se avecina si no aparece un tratamiento diferente que sea efectivo.

Health promotion before the HIV/AIDS epidemic in primary care in Punta Arenas / Promoção da saúde frente à epidemia de HIV/AIDS na atenção primária em Punta Arenas / Promoción de la salud frente a epidemia del VIH/AIDS en atención primaria en Punta Arenas

ABSTRACT Objective: to identify the attitudes and actions of health professionals in health promotion before the HIV/AIDS epidemic in Primary Health Care (PHC), Punta Arenas, Magallanes and Chilean Antartica Chilena region, Chile. Method: a qualitative, descriptive, and exploratory study carried out at PHC facilities in the city of Punta Arenas, Chile. Sixteen professionals who work in the five Family Health Centers participated. Data collection was carried out between March and May 2019. Data was obtained through a semi-structured interview. To organize data, ATLAS ti® was used, which were analyzed according to thematic content analysis. Results: two categories have emerged. The first category, Health promotion and prevention actions before HIV/AIDS, described the actions carried out by nurses in their daily work, such as counseling on STI/AIDS prevention, sexual/reproductive health and education for professionals and the community. The second category, Nurse-midwives' attitudes towards caring for people living with HIV/AIDS, includes caring without prejudice, active behavior and empathy due to high workload. Conclusion: health promotion and prevention actions carried out by PHC professionals are mainly related to counseling and education for professionals and the community. The need to integrate prevention measures with other professionals and vulnerable groups in the community stands out.

RESUMO Objetivo: identificar as atitudes e ações dos profissionais de saúde na promoção da saúde frente à epidemia de HIV/Aids na Atenção Primária de Saúde, Punta Arenas, região de Magalhães e Antártica Chilena, Chile. Método: estudo de enfoque qualitativo, descritivo, exploratório, realizado em estabelecimentos de Atenção Primária de Saúde da cidade de Punta Arenas, Chile. Participaram 16 profissionais que atuam nos cinco Centros de Saúde da Família. A coleta de dados foi realizada entre março e maio de 2019. Os dados foram obtidos através de entrevista semiestruturada. Foi utilizado o ATLAS-ti® para a organização dos dados, e estes foram analisados segundo a análise de conteúdo temática. Resultados: resultou em duas categorias. A primeira categoria, Ações de promoção da saúde e prevenção frente ao HIV/Aids descreve as ações realizadas pelas enfermeiras em seu trabalho diário, como o aconselhamento em prevenção de IST/Aids, saúde sexual/reprodutiva e a educação para profissionais e a comunidade. A segunda categoria, Atitudes das enfermeiras e parteiras frente à atenção de pessoas vivendo com HIV/Aids, abarca cuidar sem preconceitos, com escuta ativa e empatia diante da alta carga de trabalho. Conclusão: as ações de promoção da saúde e prevenção realizadas pelos profissionais de Atenção Primária de Saúde se relacionam principalmente com o aconselhamento e educação para profissionais e comunidade. Destaca a necessidade de transversalizar as medidas de prevenção a outros profissionais e grupos vulneráveis da comunidade.

RESUMEN Objetivo: identificar las actitudes y acciones de los profesionales de la salud en promoción de la salud frente a epidemia del VIH/SIDA en Atención Primaria de Salud, Punta Arenas, región de Magallanes y Antártica Chilena, Chile. Método: estudio de enfoque cualitativo, descriptivo, exploratorio, realizado en establecimientos de Atención Primaria de Salud de la ciudad de Punta Arenas, Chile. Participaron 16 professionales que desempeñan en los cinco Centros de Salud Familiar. La recolección de datos se realizó entre marzo y mayo de 2019. Los datos fueron obtenidos a través de entrevista semiestructurada. Para la organización de los datos, se utilizó ATLAS ti®, los cuáles fueron analizados según a análisis de contenido temática. Resultados: se obtuvo como resultados dos categorías. La primera categoría, Acciones de promoción de la salud y prevención frente al VIH/SIDA, describiu las acciones que realizan las enfermeras en su trabajo diario, como la consejería en la prevención de ITS/SIDA, salud sexual/reproductiva y la educación para profesionales y la comunidad. La segunda categoría, Actitudes de las enfermeras y matronas frente a la atención de personas viviendo con VIH/SIDA, inclue atender sin preconceitos, com escuta activa e empatía diante da alta carga laboral. Conclusión: las acciones de promoción de la salud y prevención realizadas por los profesionales de Atención Primaria de Salud se relacionan principalmente con la consejería y educación para profesionales y comunidad. Destaca la necesidad de transversalizar las medidas de prevención a otros profissionales y grupos vulnerables de la comunidad.

Derecho a la salud en el trabajo: vulneración y fragmentación en su comprensión y materialización / Right to health at work: violation and fragmentation in its understanding and materialization / Dereito à saúde no trabalho: vulneração e fragmentação em sua comprensão e materialização

Objetivo: describir la problemática de hombres y mujeres trabajadores que al percibir vulnerado su derecho a la salud en el trabajo por sufrir presuntos o declarados ATEL, buscaron apoyo e interpusieron uno o más recursos constitucionales para lograr su restablecimiento efectivo. Materiales y métodos: estudio mixto. Se aplicó encuesta a 189 trabajadores que sufrieron un presunto o reconocido evento de ATEL y que buscaron apoyo en los servicios de extensión solidaria de la Universidad de Antioquia o en organizaciones de derechos humanos en Medellín. La encuesta captó datos sociodemográficos, de seguridad social, laborales, del SGSST, de los antecedentes, ocurrencia y consecuencias de los ATEL y los recursos interpuestos. Para el abordaje cualitativo se realizó: i) una entrevista en profundidad con un médico laboral y abogado independiente y ii) cuatro grupos focales con trabajadores que sufrieron ATEL, sindicalistas, abogados y médicos representantes de ARL, académicos. Resultados: los trabajadores más vulnerables fueron los más afectados en su derecho. Los agentes que integran el SGRL hacen un abordaje fragmentado de la salud y el trabajo que facilita la vulneración del derecho. Los trabajadores que sufren ATEL deben enfrentar barreras económicas, administrativas y de acceso a salud para restituir su derecho. Conclusiones: el trabajo decente y digno es un horizonte hacia el cual se deben apuntar los esfuerzos para que los trabajadores más vulnerados históricamente conquisten y sostengan una vida plena, digna y saludable. Los recursos como la acción de tutela contribuyen a lograr el cumplimiento del derecho a la salud en el trabajo.

Objective: to describe the problems of working men and women who, when perceiving their right to health at work violated due to alleged or declared workplace accidents or illnesses (ATEL for its acronym in Spanish) sought support and filed one or more constitutional resources to achieve their effective restoration. Materials and methods: Mixed study. A survey was applied to 189 workers who suffered an alleged ATEL event and who sought support in the solidarity extension services at Univesidad de Antioquia or in human rights organizations in the city of Medellin. The survey that captured sociodemographic data, social security data, labor data, the Occupational Health and Safety Management System (SGSST for its acronym in Spanish) data of the background, occurrence and consequences of ATELs and legal resources interposed. For the qualitative approach, an in-depth interview was conducted with an occupational physician and an independent lawyer and four focus groups with workers who suffered ATELs, trade unionists, lawyers and doctors representing the Occupational Risk Administrator (ARL for its acronym in Spanish), and academicians. Results: the most vulnerable workers were the most affected in their right. The agents that make up the General System of Occupational Risks (SGRL by its acronym in Spanish) make a fragmented approach to health and work that facilitates the violation of the right. Workers suffering from ATEL must face economic, administrative and access to health barriers to restore their right. Conclusions: Decent and respectable work is a horizon towards which efforts should be aimed so that the most historically vulnerable workers conquer and sustain full, dignified and healthy life. Resources such as guardianship action contribute to the fulfillment of the right to health at work.

Objetivo: descrever a problemática de homens e mulheres trabalhadores que ao perceber vulnerado seu direito à saúde no trabalho por sofrer presuntos ou declarados ATEL, procuraram apoio e interpuseram um ou mais recursos constitucionais para lograr seu restabelecimento efetivo. Materiais e métodos: estudo misto. Aplicou se enquete a 189 trabalhadores que sofreram um presunto ou reconhecido evento de ATEL e que buscaram apoio nos serviços de extensão solidaria da Universidade de Antioquia ou em organizações de diretos humanos em Medellín. A enquete captou dados sócios demográficos, de segurança social, laborais, do SGSST, dos antecedentes, ocorrência e consequências dos ATEL e os recursos interpostos. Para a abordagem qualitativa se realizou: i) uma entrevista em profundidade com um médico laboral e advogado independente e ii) quatro grupos focais com trabalhadores que sofreram ATEL, sindicalistas, advogados e médicos representantes de ARL, acadêmicos. Resultados: os trabalhadores mais vulneráveis foram os mais afetados em seu direito. Os agentes que integram o SGRL fazem uma abordagem fragmentada da saúde e o trabalho que facilita a vulneração do direito. Os trabalhadores que sofrem ATEL devem enfrentar barreiras econômicas, administrativas e de aceso a saúde para restituir seu direito. Conclusões: o trabalho decente e digno é um horizonte para o qual se devem apontar os esforços para que os trabalhadores mais vulnerados historicamente conquistem e sustenham uma vida plena, digna e saudável. Os recursos como a ação de mandato de segurança que contribui a obter o cumprimento do direito à saúde no trabalho.

Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D1 and D2 Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening.

Adenylyl cyclase type 5 (AC5), as the principal isoform expressed in striatal medium spiny neurons (MSNs), is essential for the integration of both stimulatory and inhibitory midbrain signals that initiate from dopaminergic G protein-coupled receptor (GPCR) activation. The spatial and temporal control of cAMP signaling is dependent upon the composition of local regulatory protein networks. However, there is little understanding of how adenylyl cyclase protein interaction networks adapt to the multifarious pressures of integrating acute versus chronic and inhibitory vs. stimulatory receptor signaling in striatal MSNs. Here, we presented the development of a novel bimolecular fluorescence complementation (BiFC)-based protein-protein interaction screening methodology to further identify and characterize elements important for homeostatic control of dopamine-modulated AC5 signaling in a neuronal model cell line and striatal MSNs. We identified two novel AC5 modulators: the protein phosphatase 2A (PP2A) catalytic subunit (PPP2CB) and the intracellular trafficking associated protein-NSF (N-ethylmaleimide-sensitive factor) attachment protein alpha (NAPA). The effects of genetic knockdown (KD) of each gene were evaluated in several cellular models, including D1- and D2-dopamine receptor-expressing MSNs from CAMPER mice. The knockdown of PPP2CB was associated with a reduction in acute and sensitized adenylyl cyclase activity, implicating PP2A is an important and persistent regulator of adenylyl cyclase activity. In contrast, the effects of NAPA knockdown were more nuanced and appeared to involve an activity-dependent protein interaction network. Taken together, these data represent a novel screening method and workflow for the identification and validation of adenylyl cyclase protein-protein interaction networks under diverse cAMP signaling paradigms.

2 year outcome for 8 year old female managed with partial cystectomy for primary bladder clear cell carcinoma.

Bladder cancer is rare in the pediatric population, and clear cell carcinoma is extremely rare with one other pediatric case reported. Here we report the clinical outcome for a medically complicated pediatric patient with muscle invasive clear cell carcinoma treated with partial cystectomy without neoadjuvant or adjuvant therapy. Final pathology was stage T2bN0M0 with negative margins. At 2 years, there is no disease recurrence by cystoscopy, chest and abdominal imaging. Postoperative issues have been related to reduced bladder capacity and compliance and the patient is currently managed with continuous urinary diversion and will require future definitive lower tract reconstruction.

Determining the Operating Costs of a Medical Surveillance Program for Copper Miners Exposed to High Altitude-Induced Chronic Intermittent Hypoxia in Chile Using a Combination of Microcosting and Time-Driven Activity-Based Costing.

BACKGROUND: Copper mining installations in Chile use a large number of workers who do their jobs at high altitudes, exposing them to the conditions of chronic intermittent hypobaric hypoxia. The Chilean Safety Association implements the surveillance program. OBJECTIVE: This organization, under the sponsorship of the Chilean Superintendency of Social Security, was interested in determining the costs involved in this program to support its decision-making processes and to improve its performance. METHODS: Direct operating costs of the Hypoxia Medical Surveillance Program were determined through on-site surveys applied to the organization's local agencies in charge. The microcosting method was used, quantifying personnel costs, consumables, and equipment and overhead costs. Time-driven activity-based costing was partially adapted for the allocation of personnel and equipment costs. Costs concerning activities, groups of activities and items, and average cost per exposed worker were determined. RESULTS: The annual costs of the program were $127 299.58. The highest costs corresponded to the assessment activities, which were $89 192.13, representing 60.06% of the total. The labor factor costs were $77 568.50, which represents 60.93% of the total. The average cost per worker in the program is $21.17. CONCLUSIONS: The partial adaptation of the time-driven activity-based costing method in combination with the microcosting method provides a suitable solution to determine the total costs of running a healthcare program of this kind. The information generated by this study will aid in the decision-making and management processes of the Hypoxia Medical Surveillance Program.

Cost-Effectiveness of Exercise-Based Cardiac Rehabilitation in Chilean Patients Surviving Acute Coronary Syndrome.

PURPOSE: To assess the cost-effectiveness of 3 models of exercise-based cardiac rehabilitation (CR) compared with standard care in survivors of acute coronary syndrome (ACS) within the public health system in Chile. METHODS: A Markov model was designed using 5 health states: ACS survivor, second ACS, complications, general mortality, and cardiovascular mortality. The transition probabilities between health states for standard care and corresponding relative risk for CR were calculated from a systematic review. Health benefits were measured with the EuroQol 5-dimensional 3-level (EQ-5D-3L) survey. Costs for each health state were quantified using the national cost verification study. The CR cost was estimated with a microcosting methodology. The time horizon was a lifetime and the discount rate was 3% per year for costs and benefits. Deterministic and probabilistic analyses were performed. Structural uncertainty was managed by designing 3 scenarios: CR as currently delivered in a specific Chilean public health center, CR as recommended by South American guidelines, and CR as proposed for low-resource settings. RESULTS: Cardiac rehabilitation versus standard care showed an incremental cost-effectiveness ratio for the standard model of $722, for the South American model of $1247, and for the low-resource model of $666. The tornado diagram showed higher uncertainty in relative risk for the complications state and for the second ACS state. CONCLUSION: Considering a cost-effectiveness threshold of 1 unit of gross domestic product per capita (∼$19 000), CR is highly cost-effective for the public health system in Chile.

Optimization of a 1,3,4-oxadiazole series for inhibition of Ca2+/calmodulin-stimulated activity of adenylyl cyclases 1 and 8 for the treatment of chronic pain.

Adenylyl cyclases type 1 (AC1) and 8 (AC8) are group 1 transmembrane adenylyl cyclases (AC) that are stimulated by Ca2+/calmodulin. Studies have shown that mice depleted of AC1 have attenuated inflammatory pain response, while AC1/AC8 double-knockout mice display both attenuated pain response and opioid dependence. Thus, AC1 has emerged as a promising new target for treating chronic pain and opioid abuse. We discovered that the 1,3,4-oxadiazole scaffold inhibits Ca2+/calmodulin-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in cells stably expressing either AC1 or AC8. We then carried out structure-activity relationship studies, in which we designed and synthesized 65 analogs, to modulate potency and selectivity versus each AC isoform in cells. Furthermore, molecular docking of the analogs into an AC1 homology model suggests the molecules may bind at the ATP binding site. Finally, a prioritized analog was tested in a mouse model of inflammatory pain and exhibited modest analgesic properties. In summary, our data indicate the 1,3,4-oxadiazoles represent a novel scaffold for the cellular inhibition of Ca2+/calmodulin-stimulated AC1- and AC8 cAMP and warrant further exploration as potential lead compounds for the treatment of chronic inflammatory pain.

Nucleobase Modified Adefovir (PMEA) Analogues as Potent and Selective Inhibitors of Adenylate Cyclases from Bordetella pertussis and Bacillus anthracis.

A series of 13 acyclic nucleoside phosphonates (ANPs) as bisamidate prodrugs was prepared. Five compounds were found to be non-cytotoxic and selective inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT) in J774A.1 macrophage cell-based assays. The 8-aza-7-deazapurine derivative of adefovir (PMEA) was found to be the most potent ACT inhibitor in the series (IC50 =16 nm) with substantial selectivity over mammalian adenylate cyclases (mACs). AC inhibitory properties of the most potent analogues were confirmed by direct evaluation of the corresponding phosphonodiphosphates in cell-free assays and were found to be potent inhibitors of both ACT and edema factor (EF) from Bacillus anthracis (IC50 values ranging from 0.5 to 21 nm). Moreover, 7-halo-7-deazapurine analogues of PMEA were discovered to be potent and selective mammalian AC1 inhibitors (no inhibition of AC2 and AC5) with IC50 values ranging from 4.1 to 5.6 µm in HEK293 cell-based assays.

A Novel CRISPR/Cas9-Based Cellular Model to Explore Adenylyl Cyclase and cAMP Signaling.

Functional characterization of adenylyl cyclase (AC) isoforms has proven challenging in mammalian cells because of the endogenous expression of multiple AC isoforms and the high background cAMP levels induced by nonselective AC activators. To simplify the characterization of individual transmembrane AC (mAC) isoforms, we generated a human embryonic kidney cell line 293 (HEK293) with low cAMP levels by knocking out two highly expressed ACs, AC3 and AC6, using CRISPR/Cas9 technology. Stable HEK293 cell lines lacking either AC6 (HEK-ACΔ6) or both AC3 and AC6 (HEK-ACΔ3/6) were generated. Knockout was confirmed genetically and by comparing cAMP responses of the knockout cells to the parental cell line. HEK-ACΔ6 and HEK-ACΔ3/6 cells revealed an 85% and 95% reduction in the forskolin-stimulated cAMP response, respectively. Forskolin- and Gαs-coupled receptor-induced activation was examined for the nine recombinant mAC isoforms in the HEK-ACΔ3/6 cells. Forskolin-mediated cAMP accumulation for AC1-6 and AC8 revealed 10- to 250-fold increases over the basal cAMP levels. All nine mAC isoforms, except AC8, also exhibited significantly higher cAMP levels than the control cells after Gαs-coupled receptor activation. Isoform-specific AC regulation by protein kinases and Ca2+/calmodulin was also recapitulated in the knockout cells. Furthermore, the utility of the HEK-ACΔ3/6 cell line was demonstrated by characterizing the activity of novel AC1 forskolin binding-site mutants. Hence, we have developed a HEK293 cell line deficient of endogenous AC3 and AC6 with low cAMP background levels for studies of cAMP signaling and AC isoform regulation.

Identification of FDA-Approved Small Molecules Capable of Disrupting the Calmodulin-Adenylyl Cyclase 8 Interaction through Direct Binding to Calmodulin.

Adenylyl cyclases (AC) catalyze the formation of cyclic AMP (cAMP) from ATP and are involved in a number of disease states, making them attractive potential drug targets. AC8, in particular, has been implicated in several neurological disorders. While development of small molecule AC inhibitors has generated some chemical leads, the lack of inhibitor specificity among AC family members has limited the identification of successful drug candidates. Therefore, finding alternative novel methods to suppress AC activity are needed. Because only AC1 and AC8 are robustly stimulated by calmodulin (CaM), we set out to explore the mechanism of disrupting the AC/CaM interaction as a way to selectively inhibit AC8. Through the development and implementation of a novel biochemical high-throughput-screening paradigm, we identified six small molecules from an FDA-approved compound library that are capable of disrupting the AC8/CaM interaction. These compounds were also shown to be able disrupt formation of this complex in cells, ultimately leading to decreased AC8 activity. Interestingly, further mechanistic analysis determined that these compounds functioned by binding to CaM and blocking its interaction with AC8. While these particular compounds could inhibit CaM interaction with both AC1 and AC8, they provide significant proof of concept for inhibition of ACs through disruption of CaM binding. These compounds, as dual AC1/AC8 inhibitors, provide important tools for probing pathological conditions where AC1/AC8 activity are enhanced, such as chronic pain and ethanol consumption. Furthermore, unlike tools such as genetic deletion, these compounds can be used in a dose-dependent fashion to determine the role of AC/CaM interactions in these pathologies.

Synthesis of α-Branched Acyclic Nucleoside Phosphonates as Potential Inhibitors of Bacterial Adenylate Cyclases.

Inhibition of Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF), key virulence factors with adenylate cyclase activity, represents a potential method for treating or preventing toxemia related to whooping cough and anthrax, respectively. Novel α-branched acyclic nucleoside phosphonates (ANPs) having a hemiaminal ether moiety were synthesized as potential inhibitors of bacterial adenylate cyclases. ANPs prepared as bisamidates were not cytotoxic, but did not exhibit any profound activity (IC50 >10 µm) toward ACT in J774A.1 macrophages. The apparent lack of activity of the bisamidates is speculated to be due to the inefficient formation of the biologically active species (ANPpp) in the cells. Conversely, two 5-haloanthraniloyl-substituted ANPs in the form of diphosphates were shown to be potent ACT and EF inhibitors with IC50 values ranging from 55 to 362 nm.

Identification of a selective small-molecule inhibitor of type 1 adenylyl cyclase activity with analgesic properties.

Adenylyl cyclase 1 (AC1) belongs to a group of adenylyl cyclases (ACs) that are stimulated by calcium in a calmodulin-dependent manner. Studies with AC1 knockout mice suggest that inhibitors of AC1 may be useful for treating pain and opioid dependence. However, nonselective inhibition of AC isoforms could result in substantial adverse effects. We used chemical library screening to identify a selective AC1 inhibitor with a chromone core structure that may represent a new analgesic agent. After demonstrating that the compound (ST034307) inhibited Ca2+-stimulated adenosine 3',5'-monophosphate (cAMP) accumulation in human embryonic kidney (HEK) cells stably transfected with AC1 (HEK-AC1 cells), we confirmed selectivity for AC1 by testing against all isoforms of membrane-bound ACs. ST034307 also inhibited AC1 activity stimulated by forskolin- and Gαs-coupled receptors in HEK-AC1 cells and showed inhibitory activity in multiple AC1-containing membrane preparations and mouse hippocampal homogenates. ST034307 enhanced µ-opioid receptor (MOR)-mediated inhibition of AC1 in short-term inhibition assays in HEK-AC1 cells stably transfected with MOR; however, the compound blocked heterologous sensitization of AC1 caused by chronic MOR activation in these cells. ST034307 reduced pain responses in a mouse model of inflammatory pain. Our data indicate that ST034307 is a selective small-molecule inhibitor of AC1 and suggest that selective AC1 inhibitors may be useful for managing pain.

Molecular Modeling Evaluation of the Enantiomers of a Novel Adenylyl Cyclase 2 Inhibitor.

Adenylyl cyclase 2 (AC2) is one of nine membrane-bound isoforms of adenylyl cyclase that converts ATP into cyclic AMP (cAMP), an important second messenger molecule. Upregulation of AC2 is linked to cancers like pancreatic and small intestinal neuroendocrine tumors (NETs). The structures of the various isoforms of adenylyl cyclases are highly homologous, posing a significant challenge to drug discovery efforts for an effective, isoform-selective modulator of AC2. In a previous study, a screen identified a potential isoform-selective and noncompetitive inhibitor of AC2, SKF83566. In the present study, molecular modeling is used to explore the mode of inhibition of AC2 by SKF83566 and to investigate the active enantiomer of SKF83566. Homology models of hAC2 were built based on canine AC5-C1a and rat AC2-C2a templates. With these models, a combination of flexible docking, molecular dynamics simulations, and free energy calculations using the MM/GBSA methodology suggested an allosteric mechanism in which (S)-SKF83566 binds to an allosteric site near ATP and alters the protein conformation of the ATP binding site, potentially preventing the adenosine moiety of ATP from forming an archlike shape to form cAMP. The predicted binding preference for the (S)-SKF83566 enantiomer and the predicted free energy are consistent with the experimental data.