Advanced Systems for Skin Delivery of Cyanocobalamin: A Model Molecule for > 1 kDa Drugs

La optimización de la biodisponibilidad de los fármacos brinda una segunda oportunidad a los fármacos que, por diversas razones, no pueden ejercer un efecto terapéutico óptimo. La piel se ha utilizado como plataforma para administrar medicamentos desde la antigüedad y, en la actualidad, se sigue considerando como una de las principales alternativas a la vía oral. Sin embargo, como interfaz entre el organismo y el medio ambiente externo, la capa más externa de la piel (estrato córneo) impide la entrada de sustancias. Esta oposición es de gran importancia para las moléculas de gran tamaño, considerando 500 Da como el tamaño máximo para que una molécula difunda de forma natural a través de la piel. La reformulación de medicamentos en forma de sistemas avanzados de administración de medicamentos (SAAM) suele ser necesaria para eludir la barrera del estrato córneo y producir un efecto terapéutico optimizado. El objetivo principal de esta tesis fue desarrollar diferentes SAAM que permitieran la difusión de moléculas grandes a través de la piel. Como molécula modelo se seleccionó la cianocobalamina (vitamina B12), que debido a su alto peso molecular (1355 Da) e hidrofobicidad, presenta una baja absorción transdérmica. Para mejorar dicha absorción y aumentar su permeabilidad a través de la piel, se utilizaron como recursos tecnológicos las vesículas lipídicas y microagujas poliméricas. De entre todos los tipos de vesículas, se eligieron concretamente los liposomas, transfersomas y etosomas convencionales y se caracterizaron haciendo especial énfasis en aquellas propiedades predictivas de una difusión y absorción mejoradas. Específicamente, los transfersomas y los etosomas mostraron las mejores características, ya que produjeron poblaciones de vesículas homogéneas de pequeño tamaño (< 200 nm) y estructuras flexibles. La liofilización se empleó para resolver los problemas de estabilidad a largo plazo que suelen afectar a este tipo de formulaciones y como un paso intermedio que permite su incorporación en un sistema de administración alternativo, concretamente las microagujas solubles. Los estudios in vitro y ex vivo confirmaron el aumento en la absorción del fármaco cuando se incorporó cianocobalamina en los SAAM en comparación con una disolución acuosa estándar. Como consecuencia de su diseño, las microagujas pudieron administrar el fármaco por vía transdérmica después de un período de latencia moderado, lo que sugiere su idoneidad para usarse como una alternativa a la administración parenteral. Alternativamente, las vesículas de lipídicas, que mostraron tiempos de latencia más prolongados para lograr concentraciones de fármaco cuantificables en la dermis, pudieron localizar la administración del fármaco en las capas externas de la piel mejorando significativamente la penetración de cianocobalamina en comparación con la solución estándar. El comportamiento neutralizador del óxido nítrico que presenta la cianocobalamina hace que sea posible tratar los trastornos inflamatorios de la piel como la dermatitis atópica o el eccema cuando se aplica tópicamente. La idoneidad y efectividad de las vesículas lipídicas para tratamientos tópicos se evaluó mediante un modelo de hipersensibilidad de tipo retardado in vivo. Los transfersomas cargados con cianocobalamina redujeron significativamente el aumento del grosor de la oreja habitual en procesos inflamatorios cutáneos, en comparación con el grupo no tratado, demostrando su eficacia. Además, los hallazgos histológicos como la reducción de la hiperplasia epidérmica, engrosamiento de la dermis y la infiltración de leucocitos confirmaron el efecto restaurador que las vesículas lipídicas de cianocobalamina pueden ejercer en la piel afectada por las patologías de carácter inmuno-inflamatorio.The bioavailability optimization of drugs provides a second opportunity to drugs, which for several reasons, cannot exert an optimal therapeutic effect. The skin has been used as a platform to deliver drugs since ancient times. Nowadays, it is still considered as one of the main alternatives to the oral route. However, as an interface between the body and the environments, the outer skin layer (stratum corneum) prevents the entrance of substances. This opposition is of major importance for large molecules, and 500 Da is consider de maximum size diffuse through the skin structure naturally. Re-formulation of drugs in advanced drug delivery systems (ADDS) is commonly needed to bypass the stratum corneum barrier and produce an optimised therapeutic effect. The main objective of this thesis was to develop different ADDS that allowed the diffusion of large molecules through the skin. As a model molecule, cyanocobalamin (vitamin B12) was selected, due to its high molecular weight (1355 Da) and hydrophobicity, what hinders its transdermal absorption. For this, lipid vesicles and polymeric microneedles were used to encapsulate and increase cyanocobalamin permeability though the skin. Among all the lipid vesicles developed over the years, conventional liposomes, transfersomes and ethosomes were chosen and characterised towards those properties predictive of an enhanced diffusion. Specifically, transfersomes and ethosomes showed the best features, as they were homogeneous populations of small size (< 200 nm) and flexible structures. Freeze-drying was used to solve the long-term stability issues that usually affect lipid vesicles, and as an intermediate step that allows their incorporation in an alternative delivery system, the dissolving microneedles. In vitro and ex vivo studies confirmed the increase in drug absorption when cyanocobalamin was incorporated in the ADDS when compared to a standard aqueous solution. As consequence of their conception, microneedles were able to deliver transdermally the drug after a moderate period, suggesting their suitability to be used as an alternative to parenteral administration. Lipid vesicles, which showed longer latency times to achieve quantifiable dermal concentrations of drug, were able to localize the drug delivery in the outer skin layers improving the penetration in comparison to the standard solution. Cyanocobalamin nitric oxide scavenger behaviour makes possible to ameliorate skin inflammatory disorders such as atopic dermatitis or eczema when applied topically. The feasibility of lipid vesicles for topical treatments was assessed by in vivo delayed-type hypersensitivity model. Cyanocobalamin-loaded transfersomes significantly reduced the increase in ear thickness in comparison to the untreated group, proving their effectiveness. In addition, histological findings such as reduced epidermal hyperplasia, dermis thicken, and leukocyte infiltration confirmed the skin restoring effect of transfersomes

3D-printed products for topical skin applications : from personalized dressings to drug delivery

3D printing has been widely used for the personalization of therapies and on-demand production of complex pharmaceutical forms. Recently, 3D printing has been explored as a tool for the development of topical dosage forms and wound dressings. Thus, this review aims to present advances related to the use of 3D printing for the development of pharmaceutical and biomedical products for topical skin applications, covering plain dressing and products for the delivery of active ingredients to the skin. Based on the data acquired, the important growth in the number of publications over the last years confirms its interest. The semisolid extrusion technique has been the most reported one, probably because it allows the use of a broad range of polymers, creating the most diverse therapeutic approaches. 3D printing has been an excellent field for customizing dressings, according to individual needs. Studies discussed here imply the use of metals, nanoparticles, drugs, natural compounds and proteins and peptides for the treatment of wound healing, acne, pain relief, and anti-wrinkle, among others. The confluence of 3D printing and topical applications has undeniable advantages, and we would like to encourage the research groups to explore this field to improve the patient’s life quality, adherence and treatment efficacy

Cyanocobalamin ultraflexible lipid vesicles: Characterization and in vitro evaluation of drug-skin depth profiles

Atopic dermatitis (AD) and psoriasis are the most common chronic inflammatory skin disorders, which importantly affect the quality of life of patients who suffer them. Among other causes, nitric oxide has been reported as part of the triggering factors in the pathogenesis of both conditions. Cyanocobalamin (vitamin B12) has shown efficacy as a nitric oxide scavenger and some clinical trials have given positive outcomes in its use for treating skin pathologies. Passive skin diffusion is possible only for drugs with low molecular weights and intermediate lipophilicity. Unfortunately, the molecular weight and hydrophilicity of vitamin B12 do not predict its effective diffusion through the skin. The aim of this work was to design new lipid vesicles to encapsulate the vitamin B12 to enhance its skin penetration. Nine prototypes of vesicles were generated and characterized in terms of size, polydispersity, surface charge, drug encapsulation, flexibility, and stability with positive results. Additionally, their ability to release the drug content in a controlled manner was demonstrated. Finally, we found that these lipid vesicle formulations facilitated the penetration of cyanocobalamin to the deeper layers of the skin. The present work shows a promising system to effectively administer vitamin B12 topically, which could be of interest in the treatment of skin diseases such as AD and psoriasis

Cubic Microcontainers Improve In Situ Colonic Mucoadhesion and Absorption of Amoxicillin in Rats

An increased interest in colonic drug delivery has led to a higher focus on the design of delivery devices targeting this part of the gastrointestinal tract. Microcontainers have previously facilitated an increase in oral bioavailability of drugs. The surface texture and shape of microcontainers have proven to influence the mucoadhesion ex vivo. In the present work, these findings were further investigated using an in situ closed-loop perfusion technique in the rat colon, which allowed for simultaneous evaluation of mucoadhesion of the microcontainers as well as drug absorption. Cylindrical, triangular and cubic microcontainers, with the same exterior surface area, were evaluated based on in vitro release, in situ mucoadhesion and in situ absorption of amoxicillin. Additionally, the mucoadhesion of empty cylindrical microcontainers with and without pillars on the top surface was investigated. From the microscopy analysis of the colon sections after the in situ study, it was evident that a significantly higher percentage of cubic microcontainers than cylindrical microcontainers adhered to the intestinal mucus. Furthermore, the absorption rate constants and blood samples indicated that amoxicillin in cubic microcontainers was absorbed more readily than when cylindrical or triangular microcontainers were dosed. This could be due to a higher degree of mucoadhesion for these particular microcontainers

Clinically relevant transmitted drug resistance to first line antiretroviral drugs and implications for recommendations

Background The aim was to analyse trends in clinically relevant resistance to first-line antiretroviral drugs in Spain, applying the Stanford algorithm, and to compare these results with reported Transmitted Drug Resistance (TDR) defined by the 2009 update of the WHO SDRM list. Methods We analysed 2781 sequences from ARV naive patients of the CoRIS cohort (Spain) between 2007-2011. Using the Stanford algorithm "Low-level resistance", "Intermediate resistance" and "High-level resistance" categories were considered as "Resistant". Results 70% of the TDR found using the WHO list were relevant for first-line treatment according to the Stanford algorithm. A total of 188 patients showed clinically relevant resistance to first-line ARVs [6.8% (95%Confidence Interval: 5.8-7.7)], and 221 harbored TDR using the WHO list [7.9% (6.9-9.0)]. Differences were due to a lower prevalence in clinically relevant resistance for NRTIs [2.3% (1.8-2.9) vs. 3.6% (2.9-4.3) by the WHO list] and PIs [0.8% (0.4-1.1) vs. 1.7% (1.2-2.2)], while it was higher for NNRTIs [4.6% (3.8-5.3) vs. 3.7% (3.0-4.7)]. While TDR remained stable throughout the study period, clinically relevant resistance to first line drugs showed a significant trend to a decline (p = 0.02). Conclusions Prevalence of clinically relevant resistance to first line ARVs in Spain is decreasing, and lower than the one expected looking at TDR using the WHO list. Resistance to first-line PIs falls below 1%, so the recommendation of screening for TDR in the protease gene should be questioned in our setting. Cost-effectiveness studies need to be carried out to inform evidence-based recommendations

A Non-Targeted Approach Unravels the Volatile Network in Peach Fruit

Volatile compounds represent an important part of the plant metabolome and are of particular agronomic and biological interest due to their contribution to fruit aroma and flavor and therefore to fruit quality. By using a non-targeted approach based on HS-SPME-GC-MS, the volatile-compound complement of peach fruit was described. A total of 110 volatile compounds (including alcohols, ketones, aldehydes, esters, lactones, carboxylic acids, phenolics and terpenoids) were identified and quantified in peach fruit samples from different genetic backgrounds, locations, maturity stages and physiological responses. By using a combination of hierarchical cluster analysis and metabolomic correlation network analysis we found that previously known peach fruit volatiles are clustered according to their chemical nature or known biosynthetic pathways. Moreover, novel volatiles that had not yet been described in peach were identified and assigned to co-regulated groups. In addition, our analyses showed that most of the co-regulated groups showed good intergroup correlations that are therefore consistent with the existence of a higher level of regulation orchestrating volatile production under different conditions and/or developmental stages. In addition, this volatile network of interactions provides the ground information for future biochemical studies as well as a useful route map for breeding or biotechnological purposes

Tracking SARS-CoV-2 introductions in Mozambique using pandemic-scale phylogenies: a retrospective observational study

9 hojas, 4 figuras, 1 tabla. Demographic data of the participants are available in the appendix (pp 40–42). Genome sequences are publicly available on GISAID. All scripts used for the analysis, and beta and delta subtree files are publicly available at https://gitlab.com/tbgenomicsunit/mozcovid. The study protocol (appendix pp 1–35) and clinical questionnaire (appendix pp 36–39) will be available with publicationBackground: From the start of the SARS-CoV-2 outbreak, global sequencing efforts have generated an unprecedented amount of genomic data. Nonetheless, unequal sampling between high-income and low-income countries hinders the implementation of genomic surveillance systems at the global and local level. Filling the knowledge gaps of genomic information and understanding pandemic dynamics in low-income countries is essential for public health decision making and to prepare for future pandemics. In this context, we aimed to discover the timing and origin of SARS-CoV-2 variant introductions in Mozambique, taking advantage of pandemic-scale phylogenies. Methods: We did a retrospective, observational study in southern Mozambique. Patients from Manhiça presenting with respiratory symptoms were recruited, and those enrolled in clinical trials were excluded. Data were included from three sources: (1) a prospective hospital-based surveillance study (MozCOVID), recruiting patients living in Manhiça, attending the Manhiça district hospital, and fulfilling the criteria of suspected COVID-19 case according to WHO; (2) symptomatic and asymptomatic individuals with SARS-CoV-2 infection recruited by the National Surveillance system; and (3) sequences from SARS-CoV-2-infected Mozambican cases deposited on the Global Initiative on Sharing Avian Influenza Data database. Positive samples amenable for sequencing were analysed. We used Ultrafast Sample placement on Existing tRees to understand the dynamics of beta and delta waves, using available genomic data. This tool can reconstruct a phylogeny with millions of sequences by efficient sample placement in a tree. We reconstructed a phylogeny (~7·6 million sequences) adding new and publicly available beta and delta sequences. Findings: A total of 5793 patients were recruited between Nov 1, 2020, and Aug 31, 2021. During this time, 133 328 COVID-19 cases were reported in Mozambique. 280 good quality new SARS-CoV-2 sequences were obtained after the inclusion criteria were applied and an additional 652 beta (B.1.351) and delta (B.1.617.2) public sequences were included from Mozambique. We evaluated 373 beta and 559 delta sequences. We identified 187 beta introductions (including 295 sequences), divided in 42 transmission groups and 145 unique introductions, mostly from South Africa, between August, 2020 and July, 2021. For delta, we identified 220 introductions (including 494 sequences), with 49 transmission groups and 171 unique introductions, mostly from the UK, India, and South Africa, between April and November, 2021. Interpretation: The timing and origin of introductions suggests that movement restrictions effectively avoided introductions from non-African countries, but not from surrounding countries. Our results raise questions about the imbalance between the consequences of restrictions and health benefits. This new understanding of pandemic dynamics in Mozambique can be used to inform public health interventions to control the spread of new variants. Funding: European and Developing Countries Clinical Trials, European Research Council, Bill & Melinda Gates Foundation, and Agència de Gestió d'Ajuts Universitaris i de Recerca.This publication was produced by MozCovid which is part of the EDCTP2 programme supported by the EU (grant number RIA2020EF-3005-MozCOVID). The COVID-19 testing was supported by Emory Global Health Institute, University of Emory, through the CHAMPS Program funded by the Bill & Melinda Gates Foundation (under the grant OPP1126780 to Robert Breiman, subcontract SC00003286). This work was also supported by the European Research Council under the EU’s Horizon 2020 Research and Innovation Program grant (101001038; TB-RECONNECT), the European Commission–NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global), the Departament d’Universitats i Recerca de la Generalitat de Catalunya (AGAUR; 2021 SGR 01517), and the Ministerio de Ciencia e Innovación (Spanish Government) Project PID2019–104477RB-I00. CISM is supported by the Government of Mozambique and the Spanish Agency for International Development. ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya (http://cerca.cat/en/suma/). We also acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the Centro de Excelencia Severo Ochoa 2019–2023 Program (CEX2018–000806-S).Peer reviewe

Prisoners in Their Habitat? Generalist Dispersal by Habitat Specialists: A Case Study in Southern Water Vole (Arvicola sapidus)

Habitat specialists inhabiting scarce and scattered habitat patches pose interesting questions related to dispersal such as how specialized terrestrial mammals do to colonize distant patches crossing hostile matrices. We assess dispersal patterns of the southern water vole (Arvicola sapidus), a habitat specialist whose habitat patches are distributed through less than 2% of the study area (overall 600 km2) and whose populations form a dynamic metapopulational network. We predict that individuals will require a high ability to move through the inhospitable matrix in order to avoid genetic and demographic isolations. Genotypes (N = 142) for 10 microsatellites and sequences of the whole mitochondrial Control Region (N = 47) from seven localities revealed a weak but significant genetic structure partially explained by geographic distance. None of the landscape models had a significant effect on genetic structure over that of the Euclidean distance alone and no evidence for efficient barriers to dispersal was found. Contemporary gene flow was not severely limited for A. sapidus as shown by high migration rates estimates (>10%) between non-neighbouring areas. Sex-biased dispersal tests did not support differences in dispersal rates, as shown by similar average axial parent-offspring distances, in close agreement with capture-mark-recapture estimates. As predicted, our results do not support any preferences of the species for specific landscape attributes on their dispersal pathways. Here, we combine field and molecular data to illustrate how a habitat specialist mammal might disperse like a habitat generalist, acquiring specific long-distance dispersal strategies as an adaptation to patchy, naturally fragmented, heterogeneous and unstable habitats

Demographic History of Indigenous Populations in Mesoamerica Based on mtDNA Sequence Data

The genetic characterization of Native American groups provides insights into their history and demographic events. We sequenced the mitochondrial D-loop region (control region) of 520 samples from eight Mexican indigenous groups. In addition to an analysis of the genetic diversity, structure and genetic relationship between 28 Native American populations, we applied Bayesian skyline methodology for a deeper insight into the history of Mesoamerica. AMOVA tests applying cultural, linguistic and geographic criteria were performed. MDS plots showed a central cluster of Oaxaca and Maya populations, whereas those from the North and West were located on the periphery. Demographic reconstruction indicates higher values of the effective number of breeding females (Nef) in Central Mesoamerica during the Preclassic period, whereas this pattern moves toward the Classic period for groups in the North and West. Conversely, Nef minimum values are distributed either in the Lithic period (i.e. founder effects) or in recent periods (i.e. population declines). The Mesomerican regions showed differences in population fluctuation as indicated by the maximum Inter-Generational Rate (IGRmax): i) Center-South from the lithic period until the Preclassic; ii) West from the beginning of the Preclassic period until early Classic; iii) North characterized by a wide range of temporal variation from the Lithic to the Preclassic. Our findings are consistent with the genetic variations observed between central, South and Southeast Mesoamerica and the North-West region that are related to differences in genetic drift, structure, and temporal survival strategies (agriculture versus hunter-gathering, respectively). Interestingly, although the European contact had a major negative demographic impact, we detect a previous decline in Mesoamerica that had begun a few hundred years before