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The aim of this study was to evaluate the suitability of a non-disruptive Raman spectroscopic method to quantify drug concentrations below 5 w% within a polymer matrix produced by hot-melt extrusion (HME). For calibration, praziquantel (PZQ)-polyvinylpyrrolidone-vinylacetat-copolymer (PVP-VA) mixtures were extruded. By focusing the laser light of the Raman probe to a diameter of 1 mm and implementing a self-constructed filament holder, the signal-to-noise (S/N) ratio could be reduced considerably. The obtained Raman spectra show quite high fluorescence, which is likely to be caused by dissolved pharmaceutical active ingredient (API) in the polymer matrix. For content determination, HPLC analysis was conducted as a reference method using the same filament segments. A partial least squares (PLS) model, regressing the PZQ concentrations from HPLC method analysis versus the off-line collected Raman spectra, was developed. The linear correlation for a suitable extrusion run for the production of low-dosed filaments (extrusion 1, two kneading zones) is acceptable (R2 = 0.9915) while the correlation for a extrusion set-up with low miscibility (extrusion 2; without kneading zone) is unacceptable (R2 = 0.5349). The predictive performance of the calibration model from extrusion 1 is rated by the root mean square error of estimation (RMSEE), which was 0.08%. This calibration can now be used to validate the content of low-dosed filaments during HME.
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Povidona , Espectrometría Raman , Espectrometría Raman/métodos , Povidona/química , Polímeros/química , Tecnología de Extrusión de Fusión en Caliente , Composición de Medicamentos/métodos , CalorRESUMEN
The online coupling of size exclusion chromatography (SEC) to capillary enhanced Raman spectroscopy (CERS) based on a liquid core waveguide (LCW) flow cell was applied for the first time to assess the higher-order structure of different proteins. This setup allows recording of Raman spectra of the monomeric protein within complex mixtures, since SEC enables the separation of the monomeric protein from matrix components such as excipients of a biopharmaceutical product and higher molecular weight species (e.g., aggregates). The acquired Raman spectra were used for structural elucidation of well characterized proteins such as bovine serum albumin, hen egg white lysozyme, and ß-lactoglobulin and of the monoclonal antibody rituximab in a medicinal product. Additionally, the CERS detection of the disaccharide sucrose, which is used as a stabilizing excipient, was quantified to achieve a limit of detection (LOD) of 120 µg and a limit of quantification (LOQ) of 363 µg injected on the column.
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Productos Biológicos , Espectrometría Raman , Cromatografía en Gel , Excipientes/análisis , Albúmina Sérica BovinaRESUMEN
Amorphous-Amorphous phase separation (AAPS) is an important phenomenon that can impede the performance of amorphous solid dispersions (ASDs). The purpose of this study was to develop a sensitive approach relying on dielectric spectroscopy (DS) to characterize AAPS in ASDs. This includes detecting AAPS, determining the size of the active ingredient (AI) discrete domains in the phase-separated systems, and accessing the molecular mobility in each phase. Using a model system consisting of the insecticide imidacloprid (IMI) and the polymer polystyrene (PS), the dielectric results were further confirmed by confocal fluorescence microscopy (CFM). The detection of AAPS by DS was accomplished by identifying the decoupled structural (α-)dynamics of the AI and the polymer phase. The α-relaxation times corresponding to each phase correlated reasonably well with those of the pure components, implying nearly complete macroscopic phase separation. Congruent with the DS results, the occurrence of the AAPS was detected by means of CFM, making use of the autofluorescent property of IMI. Oscillatory shear rheology and differential scanning calorimetry (DSC) detected the glass transition of the polymer phase but not that of the AI phase. Furthermore, the otherwise undesired effects of interfacial and electrode polarization, which can appear in DS, were exploited to determine the effective domain size of the discrete AI phase in this work. Here, stereological analysis of CFM images probing the mean diameter of the phase-separated IMI domains directly stayed in reasonably good agreement with the DS-based estimates. The size of phase-separated microclusters showed little variation with AI loading, implying that the ASDs have presumably undergone AAPS upon manufacturing. DSC provided further support to the immiscibility of IMI and PS, as no discernible melting point depression of the corresponding physical mixtures was detected. Moreover, no signatures of strong attractive AI-polymer interactions could be detected by mid-infrared spectroscopy within this ASD system. Finally, dielectric cold crystallization experiments of the pure AI and the 60 wt % dispersion revealed comparable crystallization onset times, hinting at a poor inhibition of the AI crystallization within the ASD. These observations are in harmony with the occurrence of AAPS. In conclusion, our multifaceted experimental approach opens new venues for rationalizing the mechanisms and kinetics of phase separation in amorphous solid dispersions.
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Nitrocompuestos , Polímeros , Cristalización/métodos , Polímeros/química , Neonicotinoides , Solubilidad , Rastreo Diferencial de CalorimetríaRESUMEN
Label-free identification of tumor cells using spectroscopic assays has emerged as a technological innovation with a proven ability for rapid implementation in clinical care. Machine learning facilitates the optimization of processing and interpretation of extensive data, such as various spectroscopy data obtained from surgical samples. The here-described preclinical work investigates the potential of machine learning algorithms combining confocal Raman spectroscopy to distinguish non-differentiated glioblastoma cells and their respective isogenic differentiated phenotype by means of confocal ultra-rapid measurements. For this purpose, we measured and correlated modalities of 1146 intracellular single-point measurements and sustainingly clustered cell components to predict tumor stem cell existence. By further narrowing a few selected peaks, we found indicative evidence that using our computational imaging technology is a powerful approach to detect tumor stem cells in vitro with an accuracy of 91.7% in distinct cell compartments, mainly because of greater lipid content and putative different protein structures. We also demonstrate that the presented technology can overcome intra- and intertumoral cellular heterogeneity of our disease models, verifying the elevated physiological relevance of our applied disease modeling technology despite intracellular noise limitations for future translational evaluation.
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Glioblastoma , Espectrometría Raman , Humanos , Diferenciación Celular , Algoritmos , Aprendizaje AutomáticoRESUMEN
Glycosylphophatidylinositol (GPI)-anchored proteins play important roles in many biological processes, and mutations affecting proteins involved in the synthesis of the GPI anchor are reported to cause a wide spectrum of intellectual disabilities (IDs) with characteristic additional phenotypic features. Here, we describe a total of five individuals (from three unrelated families) in whom we identified mutations in PGAP3, encoding a protein that is involved in GPI-anchor maturation. Three siblings in a consanguineous Pakistani family presented with profound developmental delay, severe ID, no speech, psychomotor delay, and postnatal microcephaly. A combination of autozygosity mapping and exome sequencing identified a 13.8 Mb region harboring a homozygous c.275G>A (p.Gly92Asp) variant in PGAP3 region 17q11.2-q21.32. Subsequent testing showed elevated serum alkaline phosphatase (ALP), a GPI-anchored enzyme, in all three affected children. In two unrelated individuals in a cohort with developmental delay, ID, and elevated ALP, we identified compound-heterozygous variants c.439dupC (p.Leu147Profs(∗)16) and c.914A>G (p.Asp305Gly) and homozygous variant c.314C>G (p.Pro105Arg). The 1 bp duplication causes a frameshift and nonsense-mediated decay. Further evidence supporting pathogenicity of the missense mutations c.275G>A, c.314C>G, and c.914A>G was provided by the absence of the variants from ethnically matched controls, phylogenetic conservation, and functional studies on Chinese hamster ovary cell lines. Taken together with recent data on PGAP2, these results confirm the importance of the later GPI-anchor remodelling steps for normal neuronal development. Impairment of PGAP3 causes a subtype of hyperphosphatasia with ID, a congenital disorder of glycosylation that is also referred to as Mabry syndrome.
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Anomalías Múltiples/genética , Discapacidad Intelectual/genética , Mutación Missense , Trastornos del Metabolismo del Fósforo/genética , Receptores de Superficie Celular/genética , Anomalías Múltiples/patología , Fosfatasa Alcalina/sangre , Secuencia de Aminoácidos , Animales , Pueblo Asiatico/genética , Células CHO , Hidrolasas de Éster Carboxílico , Niño , Preescolar , Mapeo Cromosómico , Consanguinidad , Cricetinae , Cricetulus , Exoma , Femenino , Homocigoto , Humanos , Discapacidad Intelectual/patología , Datos de Secuencia Molecular , Pakistán , Linaje , Trastornos del Metabolismo del Fósforo/patología , Filogenia , Polimorfismo de Nucleótido Simple , Receptores de Superficie Celular/metabolismo , Arabia Saudita , Estados Unidos , Población Blanca/genéticaRESUMEN
We report that hypofunctional alleles of WNT1 cause autosomal-recessive osteogenesis imperfecta, a congenital disorder characterized by reduced bone mass and recurrent fractures. In consanguineous families, we identified five homozygous mutations in WNT1: one frameshift mutation, two missense mutations, one splice-site mutation, and one nonsense mutation. In addition, in a family affected by dominantly inherited early-onset osteoporosis, a heterozygous WNT1 missense mutation was identified in affected individuals. Initial functional analysis revealed that altered WNT1 proteins fail to activate canonical LRP5-mediated WNT-regulated ß-catenin signaling. Furthermore, osteoblasts cultured in vitro showed enhanced Wnt1 expression with advancing differentiation, indicating a role of WNT1 in osteoblast function and bone development. Our finding that homozygous and heterozygous variants in WNT1 predispose to low-bone-mass phenotypes might advance the development of more effective therapeutic strategies for congenital forms of bone fragility, as well as for common forms of age-related osteoporosis.
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Densidad Ósea/genética , Huesos/patología , Mutación/genética , Osteogénesis Imperfecta/genética , Osteoporosis/genética , Proteína Wnt1/genética , Animales , Secuencia de Bases , Células Cultivadas , Niño , Preescolar , Femenino , Heterocigoto , Humanos , Recién Nacido , Proteínas Relacionadas con Receptor de LDL/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Osteoblastos/metabolismo , Osteoblastos/patología , Osteogénesis Imperfecta/patología , Osteoporosis/patología , Linaje , Fenotipo , EmbarazoRESUMEN
The use of biocidal compounds in polymers is steadily increasing because it is one solution to the need for safety and hygiene. It is possible to incorporate an antimicrobial moiety to a polymer. These polymers are referred to as intrinsic antimicrobial. The biocidal action results from contact of the polymer to the microorganisms, with no release of active molecules. This is particularly important in critical fields like food technology, medicine and ventilation technology, where migration or leaching is crucial and undesirable. The isomers N-(1,1-dimethylethyl)-4-ethenyl-benzenamine and N-(1,1-dimethyl-ethyl)-3-ethenyl-benzenamine (TBAMS) are novel (Co-)Monomers for intrinsic anti-microbial polymers. The secondary amines were prepared and polymerized to the corresponding water insoluble polymer. The antimicrobial activity was analyzed by the test method JIS Z 2801:2000. Investigations revealed a high antimicrobial activity against Staphylococcus aureus and Escherichia coli with a reduction level of >4.5 log10 units. Furthermore, scanning electron microscopy (SEM) of E. coli. in contact with the polymer indicates a bactericidal action which is caused by disruption of the bacteria cell membranes, leading to lysis of the cells.
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Antibacterianos/síntesis química , Antibacterianos/farmacología , Polímeros/química , Antibacterianos/química , Membrana Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacosRESUMEN
Autosomal recessive cutis laxa (ARCL) type 2 constitutes a heterogeneous group of diseases mainly characterized by lax and wrinkled skin, skeletal anomalies, and a variable degree of intellectual disability. ALDH18A1-related ARCL is the most severe form within this disease spectrum. Here we report on the clinical and molecular findings of two affected individuals from two unrelated families. The patients presented with typical features of de Barsy syndrome and an overall progeroid appearance. However, the phenotype was highly variable including cardiovascular involvement in the more severe case. Investigation of a skin biopsy of one patient revealed not only the typical alterations of elastic fibers, but also an altered structure of mitochondria in cutaneous fibroblasts. Using conventional sequencing and copy number analysis we identified a frameshift deletion of one nucleotide and a microdeletion affecting the ALDH18A1 gene, respectively, in a homozygous state in both patients. Expression analysis in dermal fibroblasts from the patient carrying the microdeletion showed an almost complete absence of the ALDH18A1 mRNA resulting in an absence of the ALDH18A1 protein. So far, only 13 affected individuals from seven unrelated families suffering from ALDH18A1-related cutis laxa have been described in literature. Our findings provide new insights into the clinical spectrum and show that beside point mutations microdeletions are a possible cause of ALDH18A1-ARCL.
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Aldehído Deshidrogenasa/genética , Enfermedades Cardiovasculares/complicaciones , Enfermedades Cardiovasculares/genética , Cutis Laxo/congénito , Cutis Laxo/genética , Eliminación de Gen , Aldehído Deshidrogenasa/metabolismo , Aminoácidos/sangre , Secuencia de Bases , Enfermedades Cardiovasculares/sangre , Preescolar , Cutis Laxo/sangre , Cutis Laxo/complicaciones , Resultado Fatal , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Homocigoto , Humanos , Recién Nacido , Masculino , Datos de Secuencia Molecular , Linaje , ARN Mensajero/genética , ARN Mensajero/metabolismo , Piel/patología , Piel/ultraestructuraRESUMEN
Archaeological wood can be preserved in waterlogged conditions. Due to their degradation in the ground, these archaeological remains are endangered after their discovery, since they decay irretrievably during drying. Conservation measures are used to preserve waterlogged archaeological objects, maintaining their shape and character as much as possible. However, different methods have been developed leading to varying results. This study compares their effectiveness in order to clarify their mode of action. The methods including alcohol-ether resin, lactitol/trehalose, melamine formaldehyde, polyethylene glycol impregnation prior to freeze-drying, saccharose and silicone oil were assessed by analysing mass changes and volume stability using structured-light 3D scanning. The state of the conserved wood samples including the spatial distribution of the conservation agent was examined using synchrotron micro-computed tomography. Raman spectroscopy was used to observe the agent´s spatial distribution within the cells. The findings demonstrated that melamine formaldehyde stabilises the degraded cell walls. The lumens are void, as in the case with alcohol-ether resin, while polyethylene glycol, silicone oil, saccharose and lactitol/trehalose also occupy the lumens. It is assumed that the drying method has an effect on the distribution of the solidifying agent. The knowledge gained affords insights into the mechanism of conservation methods, which in turn accounts for the varied outcomes. It also allows conclusions to be drawn about the condition and stability of conserved museum objects and serves as a starting point for the further development of conservation methods.
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In this study, deep UV resonance Raman spectroscopy (DUV-RRS) was coupled with high performance liquid chromatography (HPLC) to be applied in the field of pharmaceutical analysis. Naproxen, Metformin and Epirubicin were employed as active pharmaceutical ingredients (APIs) covering different areas of the pharmacological spectrum. Raman signals were successfully generated and attributed to the test substances, even in the presence of the dominant solvent bands of the mobile phase. To increase sensitivity, a low-flow method was developed to extend the exposure time of the sample. This approach enabled the use of a deep UV pulse laser with a low average power of 0.5 mW. Compared to previous studies, where energy-intensive argon ion lasers were commonly used, we were able to achieve similar detection limits with our setup. Using affordable lasers with low operating costs may facilitate the transfer of the results of this study into practical applications.
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Espectrometría Raman , Espectrometría Raman/métodos , Cromatografía Líquida de Alta Presión/métodos , Preparaciones Farmacéuticas/análisis , Preparaciones Farmacéuticas/química , Naproxeno/análisis , Metformina/análisis , Metformina/química , Epirrubicina/análisis , Rayos Ultravioleta , Medicamentos a GranelRESUMEN
Autosomal recessive cutis laxa type 2B (ARCL2B; OMIM # 612940) is a segmental progeroid disorder caused by mutations in PYCR1 encoding pyrroline-5-carboxylate reductase 1, which is part of the conserved proline de novo synthesis pathway. Here we describe 33 patients with PYCR1-related ARCL from 27 families with initial diagnoses varying between wrinkly skin syndrome, gerodermia osteodysplastica, De Barsy syndrome or more severe progeria syndromes. Given the difficult differential diagnosis of ARCL syndromes we performed a systematic comparison of clinical features of PYCR1-related ARCL. Intrauterine growth retardation, a characteristic triangular facial gestalt, psychomotor retardation, and hypotonia were the most relevant distinctive hallmarks of ARCL due to proline de novo synthesis defects. Corneal clouding or cataracts, athetoid movements, and finger contractures were rather rare features, but had a high predictive value. In our cohort we identified 20 different PYCR1 mutations of which seven were novel. Most of the mutations accumulated in exons 4 to 6. Missense alterations of highly conserved residues were most frequent followed by splice site changes and a single nonsense mutation. Analysis of genotype-phenotype correlation revealed that patients with mutations in the first two exons had lower average clinical scores and absent or only mild intellectual disability. Structural analyses predicted interference with PYCR1 multimerization for a subset of missense mutations. These findings have implications for the clinics as well as the pathomechanism of PYCR1-related ARCL.
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Cutis Laxo/diagnóstico , Cutis Laxo/genética , Estudios de Asociación Genética , Pirrolina Carboxilato Reductasas/genética , Alelos , Exones , Facies , Orden Génico , Genotipo , Humanos , Modelos Moleculares , Mutación , Fenotipo , Conformación Proteica , Pirrolina Carboxilato Reductasas/química , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
This study describes the synthesis, radiofluorination and purification of an anionic amphiphilic teroligomer developed as a stabilizer for siRNA-loaded calcium phosphate nanoparticles (CaP-NPs). As the stabilizing amphiphile accumulates on nanoparticle surfaces, the fluorine-18-labeled polymer should enable to track the distribution of the CaP-NPs in brain tumors by positron emission tomography after application by convection-enhanced delivery. At first, an unmodified teroligomer was synthesized with a number average molecular weight of 4550 ± 20 Da by free radical polymerization of a defined composition of methoxy-PEG-monomethacrylate, tetradecyl acrylate and maleic anhydride. Subsequent derivatization of anhydrides with azido-TEG-amine provided an azido-functionalized polymer precursor (o14PEGMA-N3) for radiofluorination. The 18F-labeling was accomplished through the copper-catalyzed cycloaddition of o14PEGMA-N3 with diethylene glycol-alkyne-substituted heteroaromatic prosthetic group [18F]2, which was synthesized with a radiochemical yield (RCY) of about 38% within 60 min using a radiosynthesis module. The 18F-labeled polymer [18F]fluoro-o14PEGMA was obtained after a short reaction time of 2-3 min by using CuSO4/sodium ascorbate at 90 °C. Purification was performed by solid-phase extraction on an anion-exchange cartridge followed by size-exclusion chromatography to obtain [18F]fluoro-o14PEGMA with a high radiochemical purity and an RCY of about 15%.
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Autosomal recessive cutis laxa (ARCL) syndromes are phenotypically overlapping, but genetically heterogeneous disorders. Mutations in the ATP6V0A2 gene were found to underlie both, autosomal recessive cutis laxa type 2 (ARCL2), Debré type, and wrinkly skin syndrome (WSS). The ATP6V0A2 gene encodes the a2 subunit of the V-type H(+)-ATPase, playing a role in proton translocation, and possibly also in membrane fusion. Here, we describe a highly variable phenotype in 13 patients with ARCL2, including the oldest affected individual described so far, who showed strikingly progressive dysmorphic features and heterotopic calcifications. In these individuals we identified 17 ATP6V0A2 mutations, 14 of which are novel. Furthermore, we demonstrate a localization of ATP6V0A2 at the Golgi-apparatus and a loss of the mutated ATP6V0A2 protein in patients' dermal fibroblasts. Investigation of brefeldin A-induced Golgi collapse in dermal fibroblasts as well as in HeLa cells deficient for ATP6V0A2 revealed a delay, which was absent in cells deficient for the ARCL-associated proteins GORAB or PYCR1. Furthermore, fibroblasts from patients with ATP6V0A2 mutations displayed elevated TGF-ß signalling and increased TGF-ß1 levels in the supernatant. Our current findings expand the genetic and phenotypic spectrum and suggest that, besides the known glycosylation defect, alterations in trafficking and signalling processes are potential key events in the pathogenesis of ATP6V0A2-related ARCL.
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Cutis Laxo/congénito , Mutación/genética , ATPasas de Translocación de Protón/genética , Factor de Crecimiento Transformador beta1/metabolismo , Adolescente , Adulto , Apoptosis , Western Blotting , Brefeldino A/farmacología , Células Cultivadas , Preescolar , Cutis Laxo/genética , Cutis Laxo/metabolismo , Cutis Laxo/patología , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Técnica del Anticuerpo Fluorescente , Glicosilación/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Humanos , Lactante , Masculino , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Adulto JovenRESUMEN
Pharmaceutical and medicinal printing technologies allow personalization and on-demand manufacturing of drug and medicinal products. Being able to manufacture patient tailored dosage forms or medical devices in a pharmacy, medical office, dental laboratory, or hospital at the point of care raises new demands on quality control procedures. For Fused Deposition Modeling, for example, it must be ensured that the starting materials, the (drug-loaded) filaments, are not accidentally exchanged by the operator. This study investigated the potential of colorimetric measurements for direct and indirect determination of the identity of extruded filaments consisting of polymer matrix, different API and/or coloring agents. Since reflection measurements were affected by surface properties of the filaments, a self-constructed filament holder was utilized with an optical fiber positioned in a 180° angle to a white light LED to perform transmission measurements. It was possible to distinguish filaments with different API concentrations by their color values, taking into account that transmission partially decreased by increased API concentration. Therefore, the intensity of the light source had to be adjusted depending on the transparency of the filament. It was shown that colorimetry can be used as a quality control tool to detect differences in drug-loading and is able to distinguish various extruded batches. Additionally, if differences in API/polymer concentrations do not lead to significant changes in L*a*b values, coloring agents were used as additives in low concentrations to color code filaments. In future studies, the setup must be supplemented with a standardized light source and obscuring filters for light intensity adjustments.
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Discontinued treatment and non-adherence are oftentimes weaknesses of common first-line drug therapy against bladder conditions due to their negative side-effects. To overcome these limitations and increase patients' quality of life, intravesical therapies are continuously being explored. 3D-printing offers the possibility of freely tailoring drug delivery systems to manufacture indwelling devices that may administer drugs locally over an extended time and avoiding frequently repeated administrations while minimizing systemic side-effects. In the present work, pressure-assisted micro syringe printing has been used to develop flexible drug-loaded inserts applicable via common urinary catheter that can remain up to several weeks inside the urinary bladder. Three APIs (lidocaine hydrochloride, trospium chloride (TrCl) and hydrochlorothiazide (HCT)) with different properties and solubilities were investigated for their applicability together with two different pharmaceutical polymers (biodegradable polycaprolactone (PCL) and non-degradable ethylene vinyl acetate copolymer (EVA)). The fastest release was thereby observed for the PCL-TrCl combination and the slowest for EVA-HCT depending on the API's solubility in the dissolution medium and formation of API clusters within the matrix. It was further demonstrated that the dissolution profile could be modified by adapting drug loads between 5 and 15 % or the geometry of the printed inserts indicating the possibility of tailoring release profiles.
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Tecnología Farmacéutica , Vejiga Urinaria , Preparaciones de Acción Retardada , Liberación de Fármacos , Humanos , Impresión Tridimensional , Calidad de VidaRESUMEN
Individual dosing of pharmaceutics and personalized medicine have become important with regard to therapeutic safety. Dose adjustments, biorelevant drug release and combination of multiple active substances in one dosage form for the reduction in polymedication are essential aspects that increase the safety and acceptance of the patient's pharmacotherapy. Therefore, not only innovative drug products but also new analytical methods are needed during the drug development phase and for quality control that can simultaneously determine different active ingredients and cover wide concentration ranges. We investigated a liquid-core waveguide UV absorbance flow cell detector coupled to an existing HPLC-UV system. A Teflon AF 2400 capillary tubing of 20 cm length was connected in series to the HPLC flow line and enabled a lower limit of quantification of 1 ng/mL pramipexole (increase in sensitivity by 20 compared to common 0.9 cm flow cells). This allowed the low-concentration of pramipexole and the higher concentrations of levodopa and benserazide occurring during drug release to be determined in a single chromatographic run within 22.5 min.
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Autosomal recessive cutis laxa type 2 (ARCL2), a syndrome of growth and developmental delay and redundant, inelastic skin, is caused by mutations in the a2 subunit of the vesicular ATPase H+-pump (ATP6V0A2). The goal of this study was to define the disease mechanisms that lead to connective tissue lesions in ARCL2. In a new cohort of 17 patients, DNA sequencing of ATP6V0A2 detected either homozygous or compound heterozygous mutations. Considerable allelic and phenotypic heterogeneity was observed, with a missense mutation of a moderately conserved residue p.P87L leading to unusually mild disease. Abnormal N- and/or mucin type O-glycosylation was observed in all patients tested. Premature stop codon mutations led to decreased ATP6V0A2 mRNA levels by destabilizing the mutant mRNA via the nonsense-mediated decay pathway. Loss of ATP6V0A2 either by siRNA knockdown or in ARCL2 cells resulted in distended Golgi cisternae, accumulation of abnormal lysosomes and multivesicular bodies. Immunostaining of ARCL2 cells showed the accumulation of tropoelastin (TE) in the Golgi and in large, abnormal intracellular and extracellular aggregates. Pulse-chase studies confirmed impaired secretion and increased intracellular retention of TE, and insoluble elastin assays showed significantly reduced extracellular deposition of mature elastin. Fibrillin-1 microfibril assembly and secreted lysyl oxidase activity were normal in ARCL2 cells. TUNEL staining demonstrated increased rates of apoptosis in ARCL2 cell cultures. We conclude that loss-of-function mutations in ATP6V0A2 lead to TE aggregation in the Golgi, impaired clearance of TE aggregates and increased apoptosis of elastogenic cells.
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Cutis Laxo/metabolismo , Cutis Laxo/fisiopatología , Vesículas Citoplasmáticas/metabolismo , Mutación , ATPasas de Translocación de Protón/metabolismo , Tropoelastina/metabolismo , Secuencia de Aminoácidos , Apoptosis , Supervivencia Celular , Células Cultivadas , Preescolar , Estudios de Cohortes , Cutis Laxo/genética , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Aparato de Golgi/metabolismo , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Transporte de Proteínas , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/genéticaRESUMEN
Individualised medicine is continuously gaining attention in pharmaceutical research. New concepts and manufacturing technologies are required to realise this therapeutic approach. Off-label drugs used in paediatrics, such as metoprolol tartrate (MPT), are potential candidates for innovations in this context. Orodispersible films (ODFs) have been shown as an accepted alternative dosage form during the last years and inkjet printing is traded as seminal technology of precise deposition of active pharmaceutical ingredients (APIs). The objective of this study was to combine both technologies by developing imprinted ODFs based on hypromellose with therapeutically reasonable MPT single doses of 0.35 to 3.5 mg for paediatric use. After preselection, suitable ink compositions were analysed by confocal Raman microscopy regarding MPT distribution within the imprinted ODFs. Adjusted print settings, speed, print direction and angle, characterised the final ODF surface structure. The present investigations show that uniform dosages with acceptance values between 1 and 6 can be achieved. Nevertheless, changes in calibrated printed quantity due to nozzle aging have a significant effect on the final applied dose. At the lowest investigated quantity, the RSD was ±28% and at the highest, ±9%. This has to be considered for implementation of inkjet printing as a pharmaceutical production tool in the future.
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Orodispersible mini-tablets (ODMTs) are a promising dosage form for the pediatric use showing increasing interest from pharmaceutical industry. However, a scale-up process for ODMTs from a compaction simulator to a rotary tablet press following FDA and EMA guidelines has not been performed and investigated yet. Isomalt (galenIQ™721) and Ludiflash® both excipients with proven suitability for the development of ODMTs have been investigated in transfer and scale-up from a compaction simulator to a rotary tablet press. ODMTs with isomalt and Ludiflash® were produced on the rotary tablet press monitoring the product temperature over time and assessing the properties of the residual powder in the feed shoe. Critical quality attributes like tensile strength, mass and disintegration time were evaluated. The transfer from compaction simulator to rotary tablet press succeeded as for both excipients similar disintegration times, tabletability and compactibility profiles were obtained. However, during scale-up, disintegration time significantly increases over time for both excipients. Monitoring of the product temperature revealed that with increasing batch size the product temperature increases as well having a significant impact on disintegration time. The properties of ODMTs produced with the residual powder are comparable in tabletability and disintegration time compared with ODMTs produced from fresh powder.
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Excipientes , Niño , Composición de Medicamentos , Humanos , Polvos , Comprimidos , Resistencia a la TracciónRESUMEN
In recent years, the development of peptide drugs and alternative routes of administration, such as buccal and sublingual routes, has become increasingly important to the pharmaceutical industry. Performing experiments under physiologically relevant conditions is still a challenge that has not yet been fully mastered. The requirements associated with these alternative administration routes (e.g. permeation testing for buccal administration) push common analytical detection systems in pharmaceutical technology to their limits, especially with regard to large molecules and peptides. An HPLC-coupled coaxial liquid-core waveguide fluorescence detector has been developed and evaluated within this study to overcome these limits by achieving a more sensitive detection. Desmopressin acetate was selected as the peptide drug with the aim of investigating its permeation behavior during the clinically relevant application period of one hour. Based on the detector system, a complete validation according to the requirements of international guidelines was successfully performed. The results of the validation showed an increase in sensitivity resulting in a limit of detection of 4.7 ng/mL and a lower limit of quantification of 9.5 ng/mL. Moreover, it has been demonstrated that the permeation of desmopressin can be observed in clinically relevant dosages and time periods of up to one hour using this innovative detector system.