Highly Versatile Polyelectrolyte Complexes for Improving the Enzyme Replacement Therapy of Lysosomal Storage Disorders

Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically cross-linked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and α-galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity, and hemocompatibility of RGD-targeted and untargeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freeze-drying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows the activity of the therapeutic protein to remain intact. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders

Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults

Background Underweight and obesity are associated with adverse health outcomes throughout the life course. We estimated the individual and combined prevalence of underweight or thinness and obesity, and their changes, from 1990 to 2022 for adults and school-aged children and adolescents in 200 countries and territories. Methods We used data from 3663 population-based studies with 222 million participants that measured height and weight in representative samples of the general population. We used a Bayesian hierarchical model to estimate trends in the prevalence of different BMI categories, separately for adults (age ≥20 years) and school-aged children and adolescents (age 5–19 years), from 1990 to 2022 for 200 countries and territories. For adults, we report the individual and combined prevalence of underweight (BMI <18·5 kg/m2) and obesity (BMI ≥30 kg/m2). For schoolaged children and adolescents, we report thinness (BMI <2 SD below the median of the WHO growth reference) and obesity (BMI >2 SD above the median). Findings From 1990 to 2022, the combined prevalence of underweight and obesity in adults decreased in 11 countries (6%) for women and 17 (9%) for men with a posterior probability of at least 0·80 that the observed changes were true decreases. The combined prevalence increased in 162 countries (81%) for women and 140 countries (70%) for men with a posterior probability of at least 0·80. In 2022, the combined prevalence of underweight and obesity was highest in island nations in the Caribbean and Polynesia and Micronesia, and countries in the Middle East and north Africa. Obesity prevalence was higher than underweight with posterior probability of at least 0·80 in 177 countries (89%) for women and 145 (73%) for men in 2022, whereas the converse was true in 16 countries (8%) for women, and 39 (20%) for men. From 1990 to 2022, the combined prevalence of thinness and obesity decreased among girls in five countries (3%) and among boys in 15 countries (8%) with a posterior probability of at least 0·80, and increased among girls in 140 countries (70%) and boys in 137 countries (69%) with a posterior probability of at least 0·80. The countries with highest combined prevalence of thinness and obesity in school-aged children and adolescents in 2022 were in Polynesia and Micronesia and the Caribbean for both sexes, and Chile and Qatar for boys. Combined prevalence was also high in some countries in south Asia, such as India and Pakistan, where thinness remained prevalent despite having declined. In 2022, obesity in school-aged children and adolescents was more prevalent than thinness with a posterior probability of at least 0·80 among girls in 133 countries (67%) and boys in 125 countries (63%), whereas the converse was true in 35 countries (18%) and 42 countries (21%), respectively. In almost all countries for both adults and school-aged children and adolescents, the increases in double burden were driven by increases in obesity, and decreases in double burden by declining underweight or thinness. Interpretation The combined burden of underweight and obesity has increased in most countries, driven by an increase in obesity, while underweight and thinness remain prevalent in south Asia and parts of Africa. A healthy nutrition transition that enhances access to nutritious foods is needed to address the remaining burden of underweight while curbing and reversing the increase in obesit

Sistemas peguilados para administración de fármacos. Aplicación a cáncer colorrectal y enfermedad de Fabry

El término "polímero terapéutico" fue acuñado para describir fármacos poliméricos, polímeros conjugados a proteínas, fármacos y aptámeros, micelas de co-polímeros y vectores no-virales multicomponentes los cuales contienen algún enlace covalente. El polietilenglicol (PEG) es uno de los polímeros más empleado, convirtiéndose en la década de los 50, en el primer polímero terapéutico aprobado para su uso con humanos. El principal objetivo de esta tesis doctoral es explorar el uso de distintos sistemas basados en PEG para generar nanoconjugados de moléculas pequeñas y de proteínas, como método de mejora de las propiedades farmacocinéticas y farmacodinámicas. El 5-Fluorouracilo (5FU) es empleado como tratamiento de primera línea en el cáncer colorectal, pero su rápida catabolización a metabolitos inactivos, su corto tiempo de circulación en plasma y su baja acumulación en el tumor, han hecho que se planteen un gran número de estrategias para conseguir una biomodulación más adecuada. La estrategia presentada en esta tesis se basa en la síntesis de nanoconjugados de 5FU con dendrones de OEG, desarrollados en nuestro grupo de investigación. Estos dendrones están compuestos compuestos por un núcleo de DTPA y ramas de oligoetilenglicol (OEG) de tamaño definido y tienen cuatro posiciones equivalentes sobre las cuales se conjuga el derivado del fármaco 5FU, y una posición diferenciada, que se denomina punto focal, sobre cuya rama se une un fluoróforo o se acetila. La unión del 5FU al dendrón ha presentando grandes problemas de estabilidad que generaban compuestos asociados a la pérdida temprana parcial o total de unidades de 5FU contenido en los conjugados. A pesar de todo esto, se pudieron llevar a cabo varios estudios biológicos, entre los que cabe destacar un estudio de biosdistribución en ratones. A pesar que los conjugados 5FU-OEG-dendrón no presentaron toxicidad, su acumulación en tumor fue muy baja, proporcionando pocas ventajas respecto al fármaco libre. En el segundo capítulo se estudiaron distintos conjugados basados en PEG con el antitumoral 7-Et11-10-hidroxi-camptotecina (SN38). El SN38 es un derivado de la camptotecina, que a pesar de presentar una alta actividad frente a un gran número de tumores, tiene una baja solubilidad en disolventes acuosos o mezclas apropiadas para su administración in vivo. Nuestro objetivo era aprovechar las características solubilizadoras del PEG para generar nanoconjugados que fueran más solubles y facilitara así su administración. Como primera aproximación se realizaron conjugados del SN38 a dendrones de morfología idéntica a los ya empleados, pero con ramas de OEG de mayor peso molecular, aumentando así la relación en peso de PEG/SN38 y asegurándonos así una buena solubilidad. Estos dendrones fueron sintetizados pero no se pudieron caracterizar apropiadamente. Así se optó por una segunda estrategia donde se conjugaba el fármaco SN38 a cadenas lineales de PEG, las cuales podían contener o no un péptido director que previamente se había sintetizado junto con otros y seleccionado. Los resultados de la biodistribución in vivo de estos nanoconjugados mostraron resultados prometedores. Se consiguieron nanoconjugdos con buena solubilidad en medios acuosos y un claro incremento de la acumulación del fármaco en el tejido tumoral en el caso del conjugado que contenía el péptido director. En el tercer capítulo de la tesis se presentan nanoconjugados basados en la PEGilación a una proteína, la α-Galactosidasa A, empleada en terapia sustitutiva contra la enfermedad de Fabry. Se realizaron nanoconjugados con dos proteínas α-Galactosidasa A, expresadas de diferentes maneras, obteniendo patrones de PEGilación similares en ambos casos y con PEGs que contenían o no distintos péptidos directores. Algunos de estos conjugados presentaron resultados in vitro prometedores, ya que la internalización y eficacia mostrada por ellos no difería en gran medida de la mostrada por la proteína que actualmente es administrada en pacientes. Finalmente, también se estudió el uso de un derivado colesterol-PEG-péptido director en la formación de vesículas unilamelares pequeñas para la encapsulación de la proteína antes mencionada. Se ha demostrado que la introducción de nuestro sistema colesterol-PEG-péptido director proporciona sistemas más unilamelares, homogéneos respecto a su tamaño y que presentan una mayor internalización, directamente relacionada con la presencia del péptido director.Polyethylenglycol (PEG) is one of the most employed therapeuthic polymers, being in the 50s, the first therapeuthic polymer to be approved for human use. The main objective of this PhD project is to explore diverse type of PEG and OEG-based conjugates as drug delivery systems of small drugs or proteins, with the aim to improve drug pharmacokinetics and pharmacodynamics properties. First, a drug delivery system based on a conjugate of the drug 5-Fluorouracil (5FU) with an OEG-based dendrimer was described. 5FU is a drug used in the treatment of colorectal cancer, but its quick catabolization in inactive metabolits, short half-live time in plasma and low accumulation in tumor makes necessary new strategies to improve its biomodulation. Here, the proposed PEGylated platform was a dendron structure composed by four oligoethylenglycol arms linked to a DTPA core, that contains 5FU linked through the functional group of each of the four equivalent arms. A biodistribution study demonstrated that despite OEG-dendron-5FU conjugate is not toxic, presented a low accumulation in tumor and a quick renal elimination. Secondly, we present a new conjugate based on the use of 7-Ethyl-10-hydroxy-camptothecin (SN38) as a drug. Our objective was to take advantage of the properties of PEG as solubilizer to improve the administration of SN38. The designed systems were highly soluble in aqueous solutions and biodistribution experiments in vivo showed differences between the conjugate that contain a targeting peptide (SN38-PEG- TCP-1) to those without (SN38-PEG). The results showed a clear effect in the drug accumulation in cancer tissues when the drug delivery system contains a specific targeting moiety. The third chapter of this work is an example of a protein PEGylation. The protein to be PEGylated was a-Galactosidase A, which is used as enzymatic replacement theraphy for the treatment of Fabry disease. Some of the conjugates showed promising results in vitro preliminary studies. In this last chapter were also presented the results obtained with liposomes that contained cholesterol-PEG-peptides on its composition and the use of these liposomes as drug delivery system for a-Galactosidase

Highly Versatile Polyelectrolyte Complexes for Improving the Enzyme Replacement Therapy of Lysosomal Storage Disorders

Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically cross-linked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and α-galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity, and hemocompatibility of RGD-targeted and untargeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freeze-drying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows the activity of the therapeutic protein to remain intact. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders

Highly Versatile Polyelectrolyte Complexes for Improving the Enzyme Replacement Therapy of Lysosomal Storage Disorders

Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically cross-linked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and α-galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity, and hemocompatibility of RGD-targeted and untargeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freeze-drying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows the activity of the therapeutic protein to remain intact. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders

Highly Versatile Polyelectrolyte Complexes for Improving the Enzyme Replacement Therapy of Lysosomal Storage Disorders

Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically cross-linked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and α-galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity, and hemocompatibility of RGD-targeted and untargeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freeze-drying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows the activity of the therapeutic protein to remain intact. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders