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Molecular characterization of chronic cutaneous wounds reveals subregion- and wound type-specific differential gene expression.
No full textA limited understanding of the pathology underlying chronic wounds has hindered the development of effective diagnostic markers and pharmaceutical interventions. This study aimed to elucidate the molecular composition of various common chronic ulcer types to facilitate drug discovery strategies. We conducted a comprehensive analysis of leg ulcers (LUs), encompassing venous and arterial ulcers, foot ulcers (FUs), pressure ulcers (PUs), and compared them with surgical wound healing complications (WHCs). To explore the pathophysiological mechanisms and identify similarities or differences within wounds, we dissected wounds into distinct subregions, including the wound bed, border, and peri-wound areas, and compared them against intact skin. By correlating histopathology, RNA sequencing (RNA-Seq), and immunohistochemistry (IHC), we identified unique genes, pathways, and cell type abundance patterns in each wound type and subregion. These correlations aim to aid clinicians in selecting targeted treatment options and informing the design of future preclinical and clinical studies in wound healing. Notably, specific genes, such as PITX1 and UPP1, exhibited exclusive upregulation in LUs and FUs, potentially offering significant benefits to specialists in limb preservation and clinical treatment decisions. In contrast, comparisons between different wound subregions, regardless of wound type, revealed distinct expression profiles. The pleiotropic chemokine-like ligand GPR15L (C10orf99) and transmembrane serine proteases TMPRSS11A/D were significantly upregulated in wound border subregions. Interestingly, WHCs exhibited a nearly identical transcriptome to PUs, indicating clinical relevance. Histological examination revealed blood vessel occlusions with impaired angiogenesis in chronic wounds, alongside elevated expression of genes and immunoreactive markers related to blood vessel and lymphatic epithelial cells in wound bed subregions. Additionally, inflammatory and epithelial markers indicated heightened inflammatory responses in wound bed and border subregions and reduced wound bed epithelialization. In summary, chronic wounds from diverse anatomical sites share common aspects of wound pathophysiology but also exhibit distinct molecular differences. These unique molecular characteristics present promising opportunities for drug discovery and treatment, particularly for patients suffering from chronic wounds. The identified diagnostic markers hold the potential to enhance preclinical and clinical trials in the field of wound healing
Investigating the human and nonobese diabetic mouse MHC class II immunopeptidome using protein language modeling.
No full textIdentifying peptides associated with the major histocompability complex class II (MHCII) is a central task in the evaluation of the immunoregulatory function of therapeutics and drug prototypes. MHCII-peptide presentation prediction has multiple biopharmaceutical applications, including the safety assessment of biologics and engineered derivatives in silico, or the fast progression of antigen-specific immunomodulatory drug discovery programs in immune disease and cancer. This has resulted in the collection of large-scale datasets on adaptive immune receptor antigenic responses and MHC-associated peptide proteomics. In parallel, recent deep learning algorithmic advances in protein language modeling have shown potential in leveraging large collections of sequence data and improve MHC presentation prediction.Here, we train a compact transformer model (AEGIS) on human and mouse MHCII immunopeptidome data, including a preclinical murine model, and evaluate its performance on the peptide presentation prediction task. We show that the transformer performs on par with existing deep learning algorithms and that combining datasets from multiple organisms increases model performance. We trained variants of the model with and without MHCII information. In both alternatives, the inclusion of peptides presented by the I-Ag7 MHC class II molecule expressed by nonobese diabetic mice enabled for the first time the accurate in silico prediction of presented peptides in a preclinical type 1 diabetes model organism, which has promising therapeutic applications.The source code is available at https://github.com/Novartis/AEGIS
SHP2 INHIBITOR TNO155 SYNERGIZES WITH ALK INHIBITORS IN ALK-DRIVEN NEUROBLASTOMA MODELS
No full textPurpose: Survival rates among patients with high-risk neuroblastoma remain low and novel therapies for recurrent neuroblastomas are required. ALK is commonly mutated in primary and relapsed neuroblastoma tumors and ALK tyrosine kinase inhibitors (TKIs) are promising treatments for ALK-driven neuroblastoma; however, innate or adaptive resistance to single agent ALK-TKIs remain a clinical challenge. Recently, SHP2 inhibitors have been shown to overcome ALK-TKI resistance in lung tumors harboring ALK rearrangements.
Experimental Design: We have assessed the efficacy of the SHP2 inhibitor TNO155 alone and in combination with the ALK-TKIs crizotinib, ceritinib, or lorlatinib for the treatment of ALK-driven neuroblastoma using in vitro and in vivo models.
Results: In comparison to wild-type, ALK-mutant neuroblastoma cell lines were more sensitive to SHP2 inhibition with TNO155. Treatment with TNO155 and ALK-TKIs synergistically reduced cell growth and promoted inactivation of ALK and MAPK signaling in ALK mutant neuroblastoma cell lines. ALK mutant cells engrafted into larval zebrafish and treated with single agent or dual SHP2/ALK inhibitors showed reduced growth and invasion. In murine ALK mutant xenografts, tumor growth was likewise reduced or delayed, and survival was prolonged upon combinatorial treatment of TNO155 and lorlatinib. Notably, we show that neuroblastoma cells harboring ALK-F1174L mutations that have become resistant to lorlatinib can be re-sensitized to lorlatinib when combined with TNO155 in vitro and in vivo.
Conclusions: Our results suggest that combinatorial inhibition of ALK and SHP2 could be a novel approach to treating ALK-driven neuroblastoma, including those with innate or acquired resistance to ALK inhibitors
Interleukin-26 potentiates type 2 skin inflammation in presence of interleukin-1β
No full textAtopic dermatitis (AD) is a debilitating inflammatory skin disorder. Biologics targeting the IL-4/IL-13 axis are effective in AD, but there is still a large proportion of patients that do not respond to IL-4R blockade. Further exploration of potentially pathogenic T cell-derived cytokines in AD may lead to new effective treatments. This study aimed to investigate the downstream effects of IL-26 on skin in the context of type 2 skin inflammation. We found that IL-26 alone exhibited limited inflammatory activity in skin. However, in presence of IL-1β, IL-26 potentiated the secretion of TSLP, CXCL1 and CCL20 from human epidermis through JAK/STAT signaling. Moreover, in an in vivo AD-like skin inflammation model, IL-26 exacerbated skin pathology and locally increased type 2 cytokines, most notably of Il13 in skin T helper cells. Neutralization of IL-1β abrogated IL-26-mediated effects, indicating that the presence of IL-1β is required for full IL-26 downstream action in vivo. These findings suggest that the presence of IL-1β enables IL-26 to be a key amplifier of inflammation in the skin. As such, IL-26 may contribute to the development and pathogenesis of inflammatory skin disorders such as AD
Compliance and regulatory considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.
No full textMulti-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under the good manufacturing practice (GMP) regime, due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. This article is the second part of a two-tiered publication aiming at providing guidance for implementation of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) in a QC laboratory. The first part [1] focuses on technical considerations, while this second part provides considerations related to GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG)
International Workshops on Genotoxicity Testing (IWGT): Origins, Achievements and Future Ambitions
No full textOver the past thirty years, the IWGT became one of the leading bodies/entities/authorities in the field of regulatory genotoxicology, not only due to the heterogeneity of participants in terms of geography and professional affiliation, but also due to its unique setup as recurring every four years.
This article summarizes the history of the IWGT, specifies some major achievements, and provides an outlook into the future
Does the Red Shift in UV-Vis Spectra Really Provide a Sensing Option for Detection of N-nitrosamines Using Metalloporphyrins?
No full textN-nitrosamines are widespread cancerogenic compounds in human environment, including water, tobacco products, food, and medicinal products. Their presence in pharmaceuticals has recently led to several recalls of important medi-cines from the market and now require strict controls and tight limits of N-nitrosamines. Analytical determination of N-nitrosamines is expensive, laborious, and time-inefficient making development of simpler and faster techniques for their detection crucial. Several reports published in the previous decade have demonstrated cobalt porphyrin-based chemosensors selectively bind N-nitrosamines, which produces a red shift of characteristic Soret band in UV-Vis spec-tra. In this manuscript thorough re-evaluation of metalloporphyrin:N-nitrosamine adducts was performed using vari-ous characterization methods. Herein, we demonstrate that while N-nitrosamines can interact directly with cobalt-based porphyrin complexes, the red shift in UV-Vis spectra is not selectively assured and might result also from the interaction between impurities in N-nitrosamines and porphyrin skeleton or interaction of other functional groups within the nitrosamine structure and the metal ion within the porphyrin. We show that pyridine nitrogen is the inter-acting atom in tobacco-specific nitrosamines (TSNAs), as pyridine itself is an active ligand and not the cyclic N-nitrosamine fragment. When using Co(II) porphyrins as chemosensors, acidic and basic impurities in dialkylnitrosa-mines (e.g. formic acid, dimethylamine) are also UV-Vis spectra red shift producing species. Treatment of these ni-trosamines with solid bases prevents the observed UV-Vis phenomena. These results imply that cobalt based metal-loporphyrins cannot be considered as selective chemosensors for UV-Vis detection of N-nitrosamine moiety contain-ing species. Therefore, special caution in interpretation of UV-Vis red shift for chemical sensors is suggested
Machine Learning for Small Molecule Drug Discovery in Academia and Industry
No full textAcademic and pharmaceutical industry research are both key for progresses in the field of molecular machine learning. Despite common open research questions and long-term goals, the nature and scope of investigations typ- ically differ between academia and industry. Herein, we highlight the op- portunities that machine learning models offer to accelerate and improve compound selection. All parts of the model life cycle are discussed, including data preparation, model building, validation, and deployment. Main chal- lenges in molecular machine learning as well as differences between academia and industry are highlighted. Furthermore, application aspects in the design- make-test-analyze cycle are discussed. We close with strategies to potentially improve collaboration between academic and industrial institutions
Engineering circuits of human iPSC-derived neurons and rat primary glia
No full textNovel in vitro platforms based on human neurons are needed to improve early drug testing and address the stalling drug discovery in neurological disorders. Topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons have the potential to become such a testing system. In this work, we build in vitro co-cultured circuits of human iPSC-derived neurons and rat primary glial cells using microfabricated polydimethylsiloxane (PDMS) structures on microelectrode arrays (MEAs). The circuits are achieved by seeding different neuron-to glia ratios either as dissociated cells or pre-aggregated spheroids. An antifouling coating is developed to prevent axonal overgrowth in undesired locations of the microstructure. We assess the electrophysiological properties of different types of circuits over more than 50 days, including their stimulation-induced neural activity. Finally, as a proof-of-concept for screening of activity altering compounds, we demonstrate the effect of magnesium chloride on the electrical activity of circuits
High-sensitivity ICP-MS is a valid method for the preclinical characterization of metal-conjugates to be developed as future radiopharmaceuticals
No full textThe preclinical characterization of radiopharmaceuticals requires specialized laboratories to enable the manipulation of open radioactive sources and is associated with a dose burden to personnel. The aim of the study was, therefore, to investigate the in vitro and in vivo predictivity of inductively coupled plasma mass spectrometry (ICP-MS) of non-radioactive analogous metal-conjugates for the development of future radiopharmaceuticals. For proof-of-concept, [175Lu]Lu-/[177Lu]Lu-PSMA-617 and [159Tb]Tb-/[161Tb]Tb-PSMA-617 have been employed. Sample preparation procedures for the quantification of minute amounts of lutetium-175 and terbium-159 in cell and organ samples of in vitro and in vivo experiments by ICP-MS were developed and validated for their robustness and accuracy. Quantification limits for the investigated lanthanides by ICP-MS were identified at concentrations in the range of parts per quadrillion (ppq) to parts per trillion (ppt), recovery of the lanthanides after sample preparation was 98%, and matrix effect was reduced so that it was negligible (<1% in cell digests and <5% in organ digests). ICP-MS technology and conventional γ-counting technique provided highly similar results with an absolute <5% difference regarding the total uptake and the internalization for the Lu-PSMA-617 and the Tb-PSMA-617 in PSMA-positive tumor cells, respectively. In vivo, equal results for both methods using Lu- and Tb-labeled PSMA-617 were obtained. Most prominent was the uptake in PSMA-positive PC-3 PIP tumor xenografts for both, the Lu-PSMA-617 (48 ± 9 % ID/g, and 50 ± 9% IA/g, respectively, at 1 h p.i.) and Tb-PSMA-617 (44 ± 5 % ID/g, and 45 ± 5% IA/g, respectively, at 1 h p.i.). This study demonstrated that high-sensitivity ICP-MS, using stable metal isotopes, allows the reliable and predictive quantification of analogous radiometal-labeled conjugates in vitro and in vivo. Hence, ICP-MS can be employed as a valid alternative to state-of-the-art radioactive assays. This allows the development of novel radiopharmaceuticals in conventional research laboratories and supports radiopharmaceutical research in academia and industry