Principales aspectos de la producción científica y editorial de MEDISAN en el quinquenio 2017-2021 / Main aspects of MEDISAN's scientific and editorial production in the five- year period 2017-2021 i:en

Introducción: En el mundo académico, la publicación científica se ha convertido en un problema constante, tanto en sus elementos cuantitativos como en los cualitativos. Objetivo: Caracterizar el nivel de publicación de MEDISAN según algunos aspectos de su producción científica y editorial. Métodos: Se realizó una investigación observacional, descriptiva y transversal, del tipo bibliométrico, de la producción científica en la revista MEDISAN de Santiago de Cuba durante el quinquenio 2017-2021. La población de estudio quedó conformada por todos los artículos científicos publicados en ese periodo según los registros de las bases de datos SciELO Citation Index y Google Académico, así como los de la propia revista en la plataforma Open Journal Systems. Resultados: Durante el periodo analizado se publicaron 38 números, que incluyeron 679 artículos y 2367 autores. Se destacaron el año 2017 con mayor cantidad de publicaciones (31,8 %) y el año 2019 con una menor cifra (14,3 %). Los autores cubanos presentaron una contribución superior (629 artículos), sobre todo los de la provincia de Santiago de Cuba, donde sobresalieron los profesionales de la Universidad de Ciencias Médicas, con 50,1 % de toda la producción. El índice h5 reveló una tendencia al aumento de las citas que recibió la revista, dado que en el año 2021 su resultado fue 23 y en el 2022 se incrementó a 27. Conclusiones: El análisis de la producción científica y editorial reveló las fortalezas y debilidades de la revista, lo que permite trazar políticas para aumentar su calidad y, con ello, su visibilidad en bases de datos.

Introduction: In the academic world, scientific publication has become a constant problem, both in its quantitative and qualitative elements. Objective: To characterize MEDISAN's publication rate according to some aspects of its scientific and editorial production. Methods: An observational, descriptive and transversal research, of bibliometric type, was carried out on the scientific production of MEDISAN journal from Santiago de Cuba during the five-year period 2017-2021. The study population was made up of all scientific articles published in that period according to the records of SciELO Citation Index and Google Scholar databases, as well as those of the journal itself on the Open Journal Systems. Results: During the analyzed period, 38 issues were published, which included 679 articles and 2,367 authors. The year 2017 stood out with the highest number of publications (31.8%) and the year 2019 with a lower number (14.3%). Cuban authors had the greatest contribution (629 articles), especially those from Santiago de Cuba province, where professionals from University of Medical Sciences stood out, with 50.1% of all production. The h5 index revealed a trend of increasing citations received by the journal, since in 2021 it was 23 and in 2022 it increased to 27. Conclusions: The analysis of scientific and editorial production revealed the strengths and weaknesses of the journal, which makes it possible to draw up policies to increase its quality and, with it, its visibility in databases.

Desarrollo de la Clínica Virtual Docente en la universidad médica de Santiago de Cuba / Development of the Teaching Virtual Clinic in the medical university of Santiago de Cuba

Entre los entornos virtuales de enseñanza-aprendizaje figura la Clínica Virtual Docente, que es un espacio educativo de gran trascendencia en el contexto universitario de las ciencias médicas, el cual contiene situaciones de salud agrupadas en diferentes especialidades y secciones con características específicas en cuanto a objetivos y organización, lo que favorece la integración de actividades docentes, presenciales o no, y el intercambio social entre personas que comparten problemas e intereses afines, con la perspectiva de la construcción permanente de saberes, a partir de principios rectores bien definidos, como crear, compartir y colaborar.

Among the teaching-learning virtual environments figures the Teaching Virtual Clinic that is an educational space of great transcendency in the university context of medical sciences, which contains health situations grouped in different specialties and sections with specific characteristics as for objectives and organization, what favors the integration of teaching activities with or without physical presence, and the social exchange between people that share common problems and interests, with the perspective of the permanent construction of knowledge, starting from very well defined rectors principles, as creating, sharing and collaborating.

[Ibero-latinamerican clinical practical guidelines on pediatric caustic esophagitis: Physiopathology and clinical-endoscopic diagnosis (1st Part)]. / Guía de práctica clínica Ibero-Latinoamericana sobre la esofagitis cáustica en Pediatría: Fisiopatología y diagnóstico clínico-endoscópico (1a Parte).

Caustic ingestion represents a serious social-medical problem due to the devastating and irreversible consequences it can produce in the upper digestive tract. In Ibero-America, there are no published reliable data on the incidence or prevalence of caustic-induced injuries, and most of the available information on clinical presentation, diagnosis, treatment, and prognosis is based on retrospective clinical series and, indeed, its clinical management is often based primarily on expert opinion. Re cently as an initiative of the Latin American Society for Pediatric Gastroenterology, Hepatology and Nutrition (LASPGHAN) and with the cooperation of the Spanish Society for Pediatric Gastroente rology, Hepatology and Nutrition (SEGHNP), we have designed a Clinical Practice Guideline that include a series of statements and recommendations aimed at optimizing patient medical care which is based on the systematic review of evidence. Two (2) successive papers focused on the evaluation of physiopathological and clinical-endoscopic diagnostic features of caustic esophagitis in children (1st. Paper) and, on the other hand, the most relevant therapeutic considerations (2nd. Paper). We expect this guideline to become a useful tool for the physician in the difficult decision-making process when assessing patients after caustic ingestion.

[Ibero-Latinamerican Clinical Practical Guidelines on pediatric caustic esophagitis: Therapeutical aspects (Part 2)]. / Guía de práctica clínica Ibero-Latinoamericana sobre la esofagitis cáustica en Pediatría: Aspectos terapéuticos (2a. Parte).

Caustic ingestion represents a serious social-medical problem due to the devastating and irreversible consequences it can produce in the upper digestive tract. In Ibero-America, there are no published reliable data on the incidence or prevalence of caustic-induced injuries, and most of the available information on clinical presentation, diagnosis, treatment, and prognosis is based on retrospective clinical series and, indeed, its clinical management is often based primarily on expert opinion. Re cently as an initiative of the Latin American Society for Pediatric Gastroenterology, Hepatology and Nutrition (LASPGHAN) and with the cooperation of the Spanish Society for Pediatric Gastroente rology, Hepatology and Nutrition (SEGHNP), we have designed a Clinical Practice Guideline that include a series of statements and recommendations aimed at optimizing patient medical care which is based on the systematic review of evidence. Two (2) separate papers focused on the evaluation of physiopathological and clinical-endoscopic diagnostic features of caustic esophagitis in children (1st. Paper) and, on the other hand, the most relevant therapeutic considerations (2nd. Paper). We expect this guideline to become a useful tool for the physician in the difficult decision-making process when assessing patients after caustic ingestion.

Guía de práctica clínica Ibero-Latinoamericana sobre la esofagitis cáustica en Pediatría: aspectos terapéuticos (2a. Parte) / Ibero-Latinamerican Clinical Practical Guidelines on pediatric caustic esophagitis: therapeutical aspects (Part 2)

Resumen: La ingestión de cáusticos representa un grave problema médico-social por las consecuencias devastadoras e irreversibles que puede producir en el tracto digestivo superior. En Iberoamérica no se han publicado datos fidedignos sobre la incidencia o la prevalencia de lesiones inducidas por cáusticos. La información disponible sobre la presentación clínica, diagnóstico, tratamiento y pronóstico se basa en series retrospectivas de casos y, de hecho, su manejo clínico se sustenta en muchos casos fundamentalmente en la opinión de expertos. Recientemente como una iniciativa de la Sociedad Latinoamericana de Gastroenterología, Hepatología y Nutrición Pediátrica (SLAGHNP) y con la co laboración de colegas de la Sociedad Española de Gastroenterología, Hepatología y Nutrición Pediá trica (SEGHNP), hemos diseñado una Guía de Práctica Clínica (GPC) la cual incluye una serie de enunciados y recomendaciones dirigidos a optimizar la atención a los pacientes y que se basan en la revisión sistemática de la evidencia. En dos (2) manuscritos sucesivos nos hemos enfocado primero, en los aspectos fisiopatológicos y de diagnóstico clínico-endoscópico de la esofagitis cáustica en niños (1a. Parte) y en segundo lugar, en los aspectos más relevantes del tratamiento (2a. Parte). Esperamos esta guía se convierta en una herramienta útil para el clínico en el difícil proceso de toma de decisio nes a la hora de evaluar un paciente posterior a la ingesta de una sustancia cáustica.

Abstract: Caustic ingestion represents a serious social-medical problem due to the devastating and irreversible consequences it can produce in the upper digestive tract. In Ibero-America, there are no published reliable data on the incidence or prevalence of caustic-induced injuries, and most of the available information on clinical presentation, diagnosis, treatment, and prognosis is based on retrospective clinical series and, indeed, its clinical management is often based primarily on expert opinion. Re cently as an initiative of the Latin American Society for Pediatric Gastroenterology, Hepatology and Nutrition (LASPGHAN) and with the cooperation of the Spanish Society for Pediatric Gastroente rology, Hepatology and Nutrition (SEGHNP), we have designed a Clinical Practice Guideline that include a series of statements and recommendations aimed at optimizing patient medical care which is based on the systematic review of evidence. Two (2) separate papers focused on the evaluation of physiopathological and clinical-endoscopic diagnostic features of caustic esophagitis in children (1st. Paper) and, on the other hand, the most relevant therapeutic considerations (2nd. Paper). We expect this guideline to become a useful tool for the physician in the difficult decision-making process when assessing patients after caustic ingestion.

Intrinsic disorder controls two functionally distinct dimers of the master transcription factor PU.1.

Transcription factors comprise a major reservoir of conformational disorder in the eukaryotic proteome. The hematopoietic master regulator PU.1 presents a well-defined model of the most common configuration of intrinsically disordered regions (IDRs) in transcription factors. We report that the structured DNA binding domain (DBD) of PU.1 regulates gene expression via antagonistic dimeric states that are reciprocally controlled by cognate DNA on the one hand and by its proximal anionic IDR on the other. The two conformers are mediated by distinct regions of the DBD without structured contributions from the tethered IDRs. Unlike DNA-bound complexes, the unbound dimer is markedly destabilized. Dimerization without DNA is promoted by progressive phosphomimetic substitutions of IDR residues that are phosphorylated in immune activation and stimulated by anionic crowding agents. These results suggest a previously unidentified, nonstructural role for charged IDRs in conformational control by mitigating electrostatic penalties that would mask the interactions of highly cationic DBDs.

Mirtha Yris Cedeño Rodríguez: una bibliotecaria médica de excelencia / Mirtha Yris Cedeño Rodríguez: a medical librarian of excellence i: en

Con este artículo se quiso realzar las cualidades humanas y el quehacer científico de Mirtha Yris Cedeño Rodríguez, bibliotecaria médica santiaguera, quien durante su etapa laboral se caracterizó por una actitud digna en la atención a numerosos profesionales de diversas especialidades biomédicas, mediante asesorías, docencia, búsquedas bibliográficas, entre otros servicios bibliotecarios, con lo cual contribuyó, desde su modesta tribuna, a la formación de ellos y a la mejoría de la salud pública en Cuba. Actualmente está felizmente jubilada.

With this work we wanted to enhance the human qualities and scientific daily tasks of Mirtha Yris Cedeño Rodríguez, medical librarian from Santiago de Cuba who was characterized by a worthy attitude in the service to many professionals of diverse biomedical specialties during her working years, by means of consultancies, teaching, literature searching, among other librarians services, with which contributed, from her modest tribune, to their training and improvement of the public health in Cuba. At the moment she is happily retired.

Guía de práctica clínica Ibero-Latinoamericana sobre la esofagitis cáustica en Pediatría: fisiopatología y diagnóstico clínico-endoscópico (1a Parte) / Ibero-latinamerican clinical practical guidelines on pediatric caustic esophagitis: physiopathology and clinical-endoscopic diagnosis (1st Part)

Resumen: La ingestión de cáusticos representa un grave problema médico-social por las consecuencias devastadoras e irreversibles que puede producir en el tracto digestivo superior. En Iberoamérica no se han publicado datos fidedignos sobre la incidencia o la prevalencia de lesiones inducidas por cáusticos. La información disponible sobre la presentación clínica, diagnóstico, tratamiento y pronóstico se basa en series retrospectivas de casos y, de hecho, su manejo clínico se sustenta en muchos casos fundamentalmente en la opinión de expertos. Recientemente como una iniciativa de la Sociedad Latinoamericana de Gastroenterología, Hepatología y Nutrición Pediátrica (SLAGHNP) y con la co laboración de colegas de la Sociedad Española de Gastroenterología, Hepatología y Nutrición Pediá trica (SEGHNP), hemos diseñado una Guía de Práctica Clínica (GPC) la cual incluye una serie de enunciados y recomendaciones dirigidos a optimizar la atención a los pacientes y que se basan en la revisión sistemática de la evidencia. En dos (2) manuscritos sucesivos nos hemos enfocado primero, en los aspectos fisiopatológicos y de diagnóstico clínico-endoscópico de la esofagitis cáustica en niños (1a. Parte) y en segundo lugar, en los aspectos más relevantes del tratamiento (2a. Parte). Esperamos esta guía se convierta en una herramienta útil para el clínico en el difícil proceso de toma de decisio nes a la hora de evaluar un paciente posterior a la ingesta de una sustancia cáustica.

Abstract: Caustic ingestion represents a serious social-medical problem due to the devastating and irreversible consequences it can produce in the upper digestive tract. In Ibero-America, there are no published reliable data on the incidence or prevalence of caustic-induced injuries, and most of the available information on clinical presentation, diagnosis, treatment, and prognosis is based on retrospective clinical series and, indeed, its clinical management is often based primarily on expert opinion. Re cently as an initiative of the Latin American Society for Pediatric Gastroenterology, Hepatology and Nutrition (LASPGHAN) and with the cooperation of the Spanish Society for Pediatric Gastroente rology, Hepatology and Nutrition (SEGHNP), we have designed a Clinical Practice Guideline that include a series of statements and recommendations aimed at optimizing patient medical care which is based on the systematic review of evidence. Two (2) successive papers focused on the evaluation of physiopathological and clinical-endoscopic diagnostic features of caustic esophagitis in children (1st. Paper) and, on the other hand, the most relevant therapeutic considerations (2nd. Paper). We expect this guideline to become a useful tool for the physician in the difficult decision-making process when assessing patients after caustic ingestion.

La Clínica Virtual Docente en el contexto educativo de las ciencias médicas / Teaching Virtual Clinic in the educational context of medical sciences

La Clínica Virtual Docente ha ocupado un lugar primordial como entorno virtual de enseñanza-aprendizaje, puesto que supone un cambio esencial en la didáctica universitaria de las ciencias médicas, la que demanda un proceso docente más dinámico, interactivo y activo en el tratamiento de situaciones de salud, basado en el intercambio social entre personas que comparten problemas e intereses afines, y con la perspectiva de la construcción permanente de saberes, a partir de principios rectores bien definidos, como son el de crear, compartir y colaborar.

Teaching Virtual Clinic has occupied an essential place as virtual environment of the teaching-learning process, since it supposes an essential change in the university didactics of medical sciences, that demands a more dynamic, interactive and active teaching process in the treatment of health situations, based on the social exchange between people that share similar problems and interests, and with the perspective of the permanent construction of knowledge, starting from well defined guiding principles, as creating, sharing and collaborating.

Allogeneic Human Umbilical Cord Mesenchymal Stem Cells for the Treatment of Autism Spectrum Disorder in Children: Safety Profile and Effect on Cytokine Levels.

Individuals with autism spectrum disorder (ASD) suffer from developmental disabilities that impact communication, behavior, and social interaction. Immune dysregulation and inflammation have been linked to children with ASD, the latter manifesting in serum levels of macrophage-derived chemokine (MDC) and thymus, and activation-regulated chemokine (TARC). Mesenchymal stem cells derived from umbilical cord tissue (UC-MSCs) have immune-modulatory and anti-inflammatory properties, and have been safely used to treat a variety of conditions. This study investigated the safety and efficacy of UC-MSCs administered to children diagnosed with ASD. Efficacy was evaluated with the Autism Treatment Evaluation Checklist (ATEC) and the Childhood Autism Rating Scale (CARS), and with measurements of MDC and TARC serum levels. Twenty subjects received a dose of 36 million intravenous UC-MSCs every 12 weeks (four times over a 9-month period), and were followed up at 3 and 12 months after treatment completion. Adverse events related to treatment were mild or moderate and short in duration. The CARS and ATEC scores of eight subjects decreased over the course of treatment, placing them in a lower ASD symptom category when compared with baseline. MDC and TARC inflammatory cytokine levels also decreased for five of these eight subjects. The mean MDC, TARC, ATEC, and CARS values attained their lowest levels 3 months after the last administration. UC-MSC administration in children with ASD was therefore determined to be safe. Although some signals of efficacy were observed in a small group of children, possible links between inflammation levels and ASD symptoms should be further investigated. Stem Cells Translational Medicine 2019;8:1008-1016.

Factores de riesgo asociados a estadía hospitalaria prolongada en pacientes adultos / Risk factors associated with prolonged hospital stay in adult patients

Se realizó una investigación de casos y controles de pacientes adultos ingresados en el Hospital Provincial Docente Dr Joaquín Castillo Duany de Santiago de Cuba, durante 5 meses del 2017, con el propósito de estimar el efecto de determinados factores de riesgo como modificadores de la estadía hospitalaria, así como la magnitud del impacto en la potencial reducción del grado de exposición a estos. El grupo de estudio fue conformado por 40 pacientes y el de control por 80. Predominaron las lesiones osteomioarticulares en ambos grupos (con un total de 23,3 por ciento); en tanto, para los casos resultó más frecuente una estadía hospitalaria de 13 días y para los controles fue igual o superior a los 7 días como promedio. Entre los factores de riesgo fueron definidos, con un nivel de confianza de 95 por ciento, la edad superior a los 65 años (OR: 4; IC 95 por ciento: 1,2-17), la ocurrencia de episodios adversos (OR: 26; IC 95 por ciento: 8,1-80,3) y los retrasos en las decisiones médicas (OR: 19; IC 95 por ciento: 4-89). Pudo concluirse que el diseño epidemiológico permitió establecer relaciones de causalidad en la prolongación de la estadía hospitalaria y cuantificar la magnitud de su reducción si se controlan o eliminan los riesgos

An investigation of cases and controls in adult patients admitted at Dr Joaquín Castillo Duany Provincial Hospital from Santiago de Cuba was carried out during 5 months in 2017, with the purpose of estimating the effect of certain risk factors as modifiers of the hospital stay, as well as the magnitude of the impact in the potential reduction of the exposure degree to those factors. The study group was formed by 40 patients and the control group by 80. The osteomioarticular lesions prevailed in both groups (with a total of 23.3 percent); as long as, for the cases it was more frequent a hospital stay of 13 days and for the controls, equal or longer than 7 days as average. Among the risk factors were defined, with a confidence level of 95 percent, the age older than 65 years (OR: 4; IC 95 percent: 1.2-17), the occurrence of adverse episodes (OR: 26; IC 95 percent: 8.1-80.3) and the delays in the medical decisions (OR: 19; IC 95 percent: 4-89). It could be concluded that the epidemiological design allowed to establish causality relationships in the continuity of the hospital stay and to quantify the magnitude of its reduction if risks are controlled or eliminated

Clinical feasibility of umbilical cord tissue-derived mesenchymal stem cells in the treatment of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a progressively debilitating neurological condition in which the immune system abnormally erodes the myelin sheath insulating the nerves. Mesenchymal stem cells (MSC) have been used in the last decade to safely treat certain immune and inflammatory conditions. METHODS: A safety and feasibility study was completed on the use of umbilical cord MSC (UCMSC) as a treatment for MS. In this 1-year study, consenting subjects received seven intravenous infusions of 20 × 106 UCMSC over 7 days. Efficacy was assessed at baseline, 1 month and 1 year after treatment, including magnetic resonance imaging (MRI) scans, Kurtzke Expanded Disability Status Scale (EDSS), Scripps Neurological Rating Scale, Nine-Hole Peg Test, 25-Foot Walk Test, and RAND Short Form-36 quality of life questionnaire. RESULTS: Twenty subjects were enrolled in this study. No serious adverse events were reported. Of the mild AEs denoted as possibly related to treatment, most were headache or fatigue. Symptom improvements were most notable 1 month after treatment. Improvements were seen in EDSS scores (p < 0.03), as well as in bladder, bowel, and sexual dysfunction (p < 0.01), in non-dominant hand average scores (p < 0.01), in walk times (p < 0.02) and general perspective of a positive health change and improved quality of life. MRI scans of the brain and the cervical spinal cord showed inactive lesions in 15/18 (83.3%) subjects after 1 year. CONCLUSIONS: Treatment with UCMSC intravenous infusions for subjects with MS is safe, and potential therapeutic benefits should be further investigated. Trial registration ClinicalTrials.gov NCT02034188. Registered Jan 13, 2014. https://clinicaltrials.gov/ct2/show/NCT02034188.

Two-step nanoprecipitation for the production of protein-loaded PLGA nanospheres.

One of the first methods to encapsulate drugs within polymer nanospheres was developed by Fessi and coworkers in 1989 and consisted of one-step nanoprecipitation based on solvent displacement. However, proteins are poorly encapsulated within polymer nanoparticles using this method because of their limited solubility in organic solvents. To overcome this limitation, we developed a two-step nanoprecipitation method and encapsulated various proteins with high efficiency into poly(lactic-co-glycolic)acid (PLGA) nanospheres (NP). In this method, a protein nanoprecipitation step is used first followed by a second polymer nanoprecipitation step. Two model enzymes, lysozyme and α-chymotrypsin, were used for the optimization of the method. We obtained encapsulation efficiencies of >70%, an amount of buffer-insoluble protein aggregates of typically <2%, and a high residual activity of typically >90%. The optimum conditions identified for lysozyme were used to successfully encapsulate cytochrome c(Cyt-c), an apoptosis-initiating basic protein of similar size, to verify reproducibility of the encapsulation procedure. The size of the Cyt-c loaded-PLGA nanospheres was around 300-400 nm indicating the potential of the delivery system to passively target tumors. Cell viability studies, using a human cervical cancer cell line (HeLa), demonstrate excellent biocompatibility of the PLGA nanoparticles. PLGA nanoparticles carrying encapsulated Cyt-c were not efficient in causing apoptosis presumably because PLGA nanoparticles are not efficiently taken up by the cells. Future systems will have to be optimized to ascertain efficient cellular uptake of the nanoparticles by, e.g., surface modification with receptor ligands.

Glycosylation improves α-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic)acid microspheres.

Enhancing protein stability upon encapsulation and release from polymers is a key issue in sustained release applications. In addition, optimum drug dispersion in the polymer particles is critical for achieving release profiles with low unwanted initial "burst" release. Herein, we address both issues by formulating the model enzyme α-chymotrypsin (α-CT) as nanoparticles to improve drug dispersion and by covalently modifying it with glycans to afford improved stability during encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres. α-CT was chemically modified with activated lactose (500 Da) to achieve molar ratios of 4.5 and 7.1 lactose-to-protein. The bioconjugates were co-lyophilized with methyl-ß-cyclodextrin followed by suspension in ethyl acetate to afford nanoparticles. Nanoparticle formation did not significantly impact protein stability: less than 5% of the protein was aggregated and the residual activity remained above 90% for all formulations. Using a solid-in-oil-in-water (s/o/w) methodology developed in our laboratory for nanoparticles, we obtained a maximum encapsulation efficiency of 61%. Glycosylation completely prevented otherwise substantial protein aggregation and activity loss during encapsulation of the non-modified enzyme. Moreover, in vitro protein release was improved for glycosylated formulations. These results highlight the potential of chemical glycosylation to improve the stability of pharmaceutical proteins in sustained release applications.

Moisture-induced solid state instabilities in alpha-chymotrypsin and their reduction through chemical glycosylation.

BACKGROUND: Protein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state. RESULTS: First, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme alpha-chymotrypsin (alpha-CT) under controlled humidity conditions. Results showed that alpha-CT aggregates and inactivates as a function of increased relative humidity (RH). Furthermore, alpha-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that alpha-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution). To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa)). The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size. CONCLUSION: Water sorption leads to aggregation, inactivation, and structural changes of alpha-CT as has been similarly shown to occur for many other proteins. These instabilities correlate with an increase in protein structural dynamics as a result of moisture exposure. In this work, we present a novel methodology to stabilize proteins against structural perturbations in the solid-state since chemical glycosylation was effective in decreasing and/or preventing the traditionally observed moisture-induced aggregation and inactivation. It is suggested that the stabilization provided by these chemically attached glycans comes from the steric hindrance that the sugars conveys on the protein surface therefore preventing the interaction of the protein internal electrostatics with that of the water molecules and thus reducing the protein structural dynamics upon moisture exposure.

The relation between moisture-induced aggregation and structural changes in lyophilized insulin.

OBJECTIVES: Long-term stability is a critical factor in the successful development of protein pharmaceuticals. Due to the relative instability of proteins in aqueous solutions, they are formulated frequently and stored as lyophilized powders. Exposure of such powders to moisture constitutes a substantial storage problem leading to aggregation and inactivation. We have investigated the structural consequences of moisture sorption by lyophilized insulin under controlled humidity conditions by employing Fourier transform-infrared (FT-IR) microscopy. METHODS: Lyophilized insulin samples were stored in humidity chambers under controlled conditions at 50(o)C. Protein aggregation studies were carried out by redissolving the insulin samples and measuring the amount of both soluble protein and insoluble aggregates. Near-UV circular dichroism spectra were collected to assess the tertiary structure. FT-IR microscopy studies were carried out to investigate secondary structural changes in solid-state insulin after incubation at different relative humidities. KEY FINDINGS: It was found that sorption of moisture was accompanied by small structural changes in lyophilized insulin at low levels of relative humidity (i.e. 11%). At higher relative humidity levels, structural changes were becoming more pronounced and were characterized by a loss in the alpha-helix and increase in beta-sheet content. The magnitude of the structural changes in tendency paralleled the solid-state instability data (i.e. formation of buffer-insoluble aggregates and loss in tertiary structure upon reconstitution). CONCLUSIONS: The results support the hypothesis that water sorption by lyophilized proteins enables structural transitions which can lead to protein aggregation and other deleterious phenomena.