Challenges in Permeability Assessment for Oral Drug Product Development.

Drug permeation across the intestinal epithelium is a prerequisite for successful oral drug delivery. The increased interest in oral administration of peptides, as well as poorly soluble and poorly permeable compounds such as drugs for targeted protein degradation, have made permeability a key parameter in oral drug product development. This review describes the various in vitro, in silico and in vivo methodologies that are applied to determine drug permeability in the human gastrointestinal tract and identifies how they are applied in the different stages of drug development. The various methods used to predict, estimate or measure permeability values, ranging from in silico and in vitro methods all the way to studies in animals and humans, are discussed with regard to their advantages, limitations and applications. A special focus is put on novel techniques such as computational approaches, gut-on-chip models and human tissue-based models, where significant progress has been made in the last few years. In addition, the impact of permeability estimations on PK predictions in PBPK modeling, the degree to which excipients can affect drug permeability in clinical studies and the requirements for colonic drug absorption are addressed.

Decellularization of Full Heart-Optimizing the Classical Sodium-Dodecyl-Sulfate-Based Decellularization Protocol.

Compared to cell therapy, where cells are injected into a defect region, the treatment of heart infarction with cells seeded in a vascularized scaffold bears advantages, such as an immediate nutrient supply or a controllable and persistent localization of cells. For this purpose, decellularized native tissues are a preferable choice as they provide an in vivo-like microenvironment. However, the quality of such scaffolds strongly depends on the decellularization process. Therefore, two protocols based on sodium dodecyl sulfate or sodium deoxycholate were tailored and optimized for the decellularization of a porcine heart. The obtained scaffolds were tested for their applicability to generate vascularized cardiac patches. Decellularization with sodium dodecyl sulfate was found to be more suitable and resulted in scaffolds with a low amount of DNA, a highly preserved extracellular matrix composition, and structure shown by GAG quantification and immunohistochemistry. After seeding human endothelial cells into the vasculature, a coagulation assay demonstrated the functionality of the endothelial cells to minimize the clotting of blood. Human-induced pluripotent-stem-cell-derived cardiomyocytes in co-culture with fibroblasts and mesenchymal stem cells transferred the scaffold into a vascularized cardiac patch spontaneously contracting with a frequency of 25.61 ± 5.99 beats/min for over 16 weeks. The customized decellularization protocol based on sodium dodecyl sulfate renders a step towards a preclinical evaluation of the scaffolds.

Fully Synthetic 3D Fibrous Scaffolds for Stromal Tissues-Replacement of Animal-Derived Scaffold Materials Demonstrated by Multilayered Skin.

The extracellular matrix (ECM) of soft tissues in vivo has remarkable biological and structural properties. Thereby, the ECM provides mechanical stability while it still can be rearranged via cellular remodeling during tissue maturation or healing processes. However, modern synthetic alternatives fail to provide these key features among basic properties. Synthetic matrices are usually completely degraded or are inert regarding cellular remodeling. Based on a refined electrospinning process, a method is developed to generate synthetic scaffolds with highly porous fibrous structures and enhanced fiber-to-fiber distances. Since this approach allows for cell migration, matrix remodeling, and ECM synthesis, the scaffold provides an ideal platform for the generation of soft tissue equivalents. Using this matrix, an electrospun-based multilayered skin equivalent composed of a stratified epidermis, a dermal compartment, and a subcutis is able to be generated without the use of animal matrix components. The extension of classical dense electrospun scaffolds with high porosities and motile fibers generates a fully synthetic and defined alternative to collagen-gel-based tissue models and is a promising system for the construction of tissue equivalents as in vitro models or in vivo implants.

Glucose transporters in pancreatic islets.

The fine-tuning of glucose uptake mechanisms is rendered by various glucose transporters with distinct transport characteristics. In the pancreatic islet, facilitative diffusion glucose transporters (GLUTs), and sodium-glucose cotransporters (SGLTs) contribute to glucose uptake and represent important components in the glucose-stimulated hormone release from endocrine cells, therefore playing a crucial role in blood glucose homeostasis. This review summarizes the current knowledge about cell type-specific expression profiles as well as proven and putative functions of distinct GLUT and SGLT family members in the human and rodent pancreatic islet and further discusses their possible involvement in onset and progression of diabetes mellitus. In context of GLUTs, we focus on GLUT2, characterizing the main glucose transporter in insulin-secreting ß-cells in rodents. In addition, we discuss recent data proposing that other GLUT family members, namely GLUT1 and GLUT3, render this task in humans. Finally, we summarize latest information about SGLT1 and SGLT2 as representatives of the SGLT family that have been reported to be expressed predominantly in the α-cell population with a suggested functional role in the regulation of glucagon release.

Matrix decoded - A pancreatic extracellular matrix with organ specific cues guiding human iPSC differentiation.

The extracellular matrix represents a dynamic microenvironment regulating essential cell functions in vivo. Tissue engineering approaches aim to recreate the native niche in vitro using biological scaffolds generated by organ decellularization. So far, the organ specific origin of such scaffolds was less considered and potential consequences for in vitro cell culture remain largely elusive. Here, we show that organ specific cues of biological scaffolds affect cellular behavior. In detail, we report on the generation of a well-preserved pancreatic bioscaffold and introduce a scoring system allowing standardized inter-study quality assessment. Using multiple analysis tools for in-depth-characterization of the biological scaffold, we reveal unique compositional, physico-structural, and biophysical properties. Finally, we prove the functional relevance of the biological origin by demonstrating a regulatory effect of the matrix on multi-lineage differentiation of human induced pluripotent stem cells emphasizing the significance of matrix specificity for cellular behavior in artificial microenvironments.

Automated real-time monitoring of human pluripotent stem cell aggregation in stirred tank reactors.

The culture of human induced pluripotent stem cells (hiPSCs) at large scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Innovative monitoring options and emerging automated process control strategies allow for the necessary highly defined culture conditions. Next to standard process characteristics such as oxygen consumption, pH, and metabolite turnover, a reproducible and steady formation of hiPSC aggregates is vital for process scalability. In this regard, we developed a hiPSC-specific suspension culture unit consisting of a fully monitored CSTR system integrated into a custom-designed and fully automated incubator. As a step towards cost-effective hiPSC suspension culture and to pave the way for flexibility at a large scale, we constructed and utilized tailored miniature CSTRs that are largely made from three-dimensional (3D) printed polylactic acid (PLA) filament, which is a low-cost material used in fused deposition modelling. Further, the monitoring tool for hiPSC suspension cultures utilizes in situ microscopic imaging to visualize hiPSC aggregation in real-time to a statistically significant degree while omitting the need for time-intensive sampling. Suitability of our culture unit, especially concerning the developed hiPSC-specific CSTR system, was proven by demonstrating pluripotency of CSTR-cultured hiPSCs at RNA (including PluriTest) and protein level.

Altered pancreatic islet morphology and function in SGLT1 knockout mice on a glucose-deficient, fat-enriched diet.

OBJECTIVES: Glycemic control by medical treatment represents one therapeutic strategy for diabetic patients. The Na+-d-glucose cotransporter 1 (SGLT1) is currently of high interest in this context. SGLT1 is known to mediate glucose absorption and incretin secretion in the small intestine. Recently, inhibition of SGLT1 function was shown to improve postprandial hyperglycemia. In view of the lately demonstrated SGLT1 expression in pancreatic islets, we investigated if loss of SGLT1 affects islet morphology and function. METHODS: Effects associated with the loss of SGLT1 on pancreatic islet (cyto) morphology and function were investigated by analyzing islets of a SGLT1 knockout mouse model, that were fed a glucose-deficient, fat-enriched diet (SGLT1-/--GDFE) to circumvent the glucose-galactose malabsorption syndrome. To distinguish diet- and Sglt1-/--dependent effects, wildtype mice on either standard chow (WT-SC) or the glucose-free, fat-enriched diet (WT-GDFE) were used as controls. Feeding a glucose-deficient, fat-enriched diet further required the analysis of intestinal SGLT1 expression and function under diet-conditions. RESULTS: Consistent with literature, our data provide evidence that small intestinal SGLT1 mRNA expression and function is regulated by nutrition. In contrast, pancreatic SGLT1 mRNA levels were not affected by the applied diet, suggesting different regulatory mechanisms for SGLT1 in diverse tissues. Morphological changes such as increased islet sizes and cell numbers associated with changes in proliferation and apoptosis and alterations of the ß- and α-cell population are specifically observed for pancreatic islets of SGLT1-/--GDFE mice. Glucose stimulation revealed no insulin response in SGLT1-/--GDFE mice while WT-GDFE mice displayed only a minor increase of blood insulin. Irregular glucagon responses were observed for both, SGLT1-/--GDFE and WT-GDFE mice. Further, both animal groups showed a sustained release of GLP-1 compared to WT-SC controls. CONCLUSION: Loss or impairment of SGLT1 results in abnormal pancreatic islet (cyto)morphology and disturbed islet function regarding the insulin or glucagon release capacity from ß- or α-cells, respectively. Consequently, our findings propose a new, additional role for SGLT1 maintaining proper islet structure and function.

sept8a and sept8b mRNA expression in the developing and adult zebrafish.

Septins are highly conserved GTP-binding proteins involved in numerous cellular processes. Despite a growing awareness of their roles in the cell biology, development and signal transmission in nervous systems, comparably little is known about precise septin expression. Here, we use the well-established model organism zebrafish (Danio rerio) to unravel the expression of sept8a and sept8b, with special focus on the CNS. We performed whole mount RNA in situ hybridization on zebrafish 1-4 dpf in combination with serial sectioning of epon-embedded samples as well as on brain sections of adult zebrafish to obtain precise histological mapping of gene expression. Our results show a common expression of both genes at embryonic stages, whereas sept8a is mainly restricted to the gill arches and sept8b to specific brain structures at later stages. Brains of adult zebrafish reveal a large spatial overlap of sept8a and sept8b expression with few regions uniquely expressing sept8a or sept8b. Our results indicate a neuronal expression of both genes, and additionally suggest expression of sept8b in glial cells. Altogether, this study provides a first detailed insight into the expression of sept8a and sept8b in zebrafish and contributes to a more comprehensive understanding of septin biology in vertebrate model systems.