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Estimation of In Vivo Half-Life from In Vitro Metabolic Clearance, Protein and Cell Membrane Affinity Assays
No full textThe prediction of in vivo plasma half-life in rats from easily accessible in vitro assays is not possible yet. We surmised that the existing models, which consider protein binding and metabolic clearance, miss a term including the propensity to bind to membrane. We developed an improved proportionality equation taking all parameters into account. This article discusses when it can be applied and how reliable predictions are
Next generation Bruton's tyrosine kinase inhibitors – characterization of in vitro potency and selectivity
No full textBruton's tyrosine kinase (BTK) mediates B cell receptor and Fc receptor signaling and is a key regulator of autoimmunity and allergy. A series of novel BTK inhibitors (BTKi) are currently in development for non-oncologic indications with covalent-irreversible (remibrutinib, evobrutinib, tolebrutinib, orelabrutinib), covalent-reversible (rilzabrutinib), and non-covalent reversible (fenebrutinib) binding modes. This study characterizes their in vitro potency and selectivity profiles under the same conditions to minimize assay differences across the different binding modes.
The covalent BTKi showed human in vitro blood BTK binding in a time- and concentration-dependent manner with remibrutinib being the most potent and fastest in onset of action. Cellular BTK pathway inhibition was determined in human blood B cells and basophils, and for covalent BTKi correlated well with BTK binding. In contrast to the covalent-irreversible remibrutinib, the non-covalent reversible fenebrutinib showed rapid loss of cellular BTK inhibition after washout. Kinase selectivity was assessed in a binding screen across the human kinome and followed by quantification of binding affinities for a selection of kinases. The BTKi ranked in their selectivity (most selective to least): remibrutinib, fenebrutinib, evobrutinib, orelabrutinib, rilzabrutinib and tolebrutinib. These data suggest that next generation BTKi show important differences in their in vitro target binding and selectivity when compared under the same condition
Cryo-EM structures of CRAF/MEK1/14-3-3 complexes in autoinhibited and open-monomer states reveal features of RAF regulation.
No full textCRAF (RAF1) is one of three RAF-family kinases that initiate MAP kinase signaling in response to activated RAS and is essential for oncogenic signaling from mutant KRAS. Like BRAF, CRAF is regulated by 14-3-3 engagement and by intramolecular autoinhibitory interactions of its N-terminal regulatory region. Unlike BRAF, it is thought to require tyrosine phosphorylation in its N-terminal acidic (NtA) motif for full catalytic activation. Here we describe cryo-EM reconstructions of full-length CRAF in complex with MEK1 and a 14-3-3 dimer. These structures reveal a fully autoinhibited conformation analogous to that observed for BRAF and two "open monomer" states in which the inhibitory interactions of the CRD and 14-3-3 dimer are released or rearranged, but the kinase domain remains inactive. Structure-function studies of the NtA motif indicate that phosphorylation or acidic mutations in this segment increase catalytic activity by destabilizing the inactive conformation of the kinase domain. Collectively, these studies provide a structural foundation for understanding the shared and unique regulatory features of CRAF and will inform efforts to selectively block CRAF signaling in cancer
Sharing data for 5-Chloro-1H-pyrazol-3-amine (CAS RN 916211-79-5)
No full textSharing Ames test data for 5-Chloro-1H-pyrazol-3-amine (CAS RN 916211-79-5) into the Vitic database in connection with the Intermediates/Aromatic amine data sharing. It is a small chemical structure which is in public domain
Targeting the IL-17A pathway for therapy in early-stage tendinopathy
No full textObjectives: Tendinopathy is a frequent clinical problem and represents an extraordinary health economic and socioeconomic burden with high unmet medical needs. Recent clinical evidence suggests blockade of interleukin 17A (IL-17A) for tendinopathy therapy. The present preclinical study elucidates the biological mechanisms of IL-17A pathway stimulation and blockade in tendinopathy.
Methods: We explored whether IL-17A and other IL-17 family members are differentially expressed in biopsies of healthy, early-stage and late-stage tendinopathic human rotator cuff tendons using RT-qPCR. IL-17 pathway signature genes in healthy human tendon-derived cells were identified following IL-17A stimulation using AmpliSeq RNA. The molecular, structural and functional consequences of IL-17A pathway stimulation were explored in healthy human tendon-derived cells and in a rat tendon fascicle model ex vivo. The effects of IL-17A pathway blockade were investigated in a rat model of rotator cuff tendinopathy in vivo.
Results: We provide evidence of differential expression of IL-17A mRNA (IL17A) versus other IL-17 family members in human rotator cuff early-stage tendinopathy. In human tendon-derived cells, stimulation with IL-17A induced the expression of the selected IL-17A pathway signature genes NFKBIZ, ZC3H12A, CXCL1, IL6, MMP3. Expression was inhibited by IL-17A blockade. In the rat ex vivo and in vivo models, IL-17A blockade alleviated inflammatory immune effector release, tendon structural degeneration, tendon inflammation and impaired tendon function.
Conclusion: Our data provide evidence that IL-17A is a key contributor to the pathogenesis of tendinopathy by promoting tendon inflammation and degeneration and that IL-17A blockade may represent a potential therapy in early-stage tendinopathy
Strain-release trifluoromethoxylation and pentafluorosulfanoxylation of [1.1.0]bicyclobutanes: expanded access to fluorinated cyclobutane hybrid bioisosteres.
No full textMethods for formal bromo-trifluoromethoxylation and bromo-pentafluorosulfanoxylation of [1.1.0]bicyclobutanes using AgOCF3 or AgOSF5 and 1,3-dibromo-5,5-dimethylhydantoin are disclosed. These represent complementary strategies to the syntheses of SF5- and CF3SF4-containing cyclobutanes previously reported from our laboratory, ultimately enabling comparative structural studies and in vitro ADME profiling for various fluorinated cyclobutanes
Eosinophil innate immune memory after bacterial skin infection promotes allergic lung inflammation.
No full textMicrobial exposure at barrier interfaces drives development and balance of the immune system, but the consequences of local infections for systemic immunity and secondary inflammation are unclear. Here, we show that skin exposure to the bacterium Staphylococcus aureus persistently shapes the immune system of mice with specific impact on progenitor and mature bone marrow neutrophil and eosinophil populations. The infection-imposed changes in eosinophils were long-lasting and associated with functional as well as imprinted epigenetic and metabolic changes. Bacterial exposure enhanced cutaneous allergic sensitization and resulted in exacerbated allergen-induced lung inflammation. Functional bone marrow eosinophil reprogramming and pulmonary allergen responses were driven by the alarmin interleukin-33 and the complement cleavage fragment C5a. Our study highlights the systemic impact of skin inflammation and reveals mechanisms of eosinophil innate immune memory and organ cross-talk that modulate systemic responses to allergens
Transplantation of vascularized cardiac microtissue derived from human iPS cells enhances the impaired electrical conduction capacity in a porcine model of myocardial injury.
No full textThe submission is a brief research report format. The content describes the application of vascularized cardiac microtissues in a porcine model of myocardial infarction. Efficient regenerative medicine using human pluripotent stem cell-derived cardiac tissue requires achieving electrical synchronization between the graft and the host heart and improving conduction disturbances in the impaired heart. This study aimed to demonstrate that transplantation of human induced pluripotent stem cell (hiPSC)-derived vascularized cardiac microtissue (VCM) can improve these conduction disturbances using a porcine myocardial injury (MI) model. Furthermore, we investigated whether simultaneous pacing of healthy and post-transplantation lesion areas could improve this conduction disturbance
A systemically delivered AAV-CFTR gene therapy approach for cystic fibrosis
No full textCystic fibrosis (CF) is the most common monogenic lung disease and results from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). There have been over 2000 variants identified in patients that can result in loss of function of the CFTR protein leading to systemic disease and respiratory failure in adolescence. While some variants encode proteins with residual activity that can be corrected or potentiated by CFTR modulators, at least 10% of CF individuals cannot tolerate the modulators or have nonsense mutations which fail to make any protein. For all people with CF, a mutation agnostic gene replacement strategy could provide a cure for CF lung disease. Here, we propose using a systemic route of administration to deliver a functional CFTR minigene cargo with a lung tropic AAV capsid. This would serve to reach multiple organs, most importantly the lung epithelium, and would provide a functional CFTR transgene that could be expressed in any cell type with a ubiquitous promoter. To achieve this, we generated the smallest CFTR minigene tested in an AAV delivery to date. We demonstrate it is expressed and functions following transfection in cell-based assays and restores function to primary CF airway cells after viral delivery. Furthermore, we identify an AAV capsid that can transduce alveolar and airway epithelium with systemic delivery in non-human primates. These data provide tools for delivering a functional CFTR minigene that fits within the packaging capacity of an AAV and demonstration of lung transduction following systemic delivery in a large animal model. This strategy would serve to reach target airway cells while circumventing the strong mucosal barrier in CF airways and has the potential to restore CFTR function in additional CF affected organs
JAS Case Studies:Weight of Evidence Approach with Different Feedback from Health Authorities
No full textCase studies for ICH S11 application is presented on two compounds: Compound A, an immunomodulator, and Compound B, an anti-cancer drug. Presented for each compound are the rationales and strategies submitted to the health authorities to support the inclusion of pediatric patients in clinical trials. The strategies for both compounds were similar by using a weight of evidence approach that included preclinical safety data to support not conducting a JAS. Feedback from the health authorities differed in the need for a JAS for both compounds and lowering the age group for Compound B. Additionally, significant modifications were made to the proposed JAS protocol by the Health Authority without explanation. These case studies together highlight differences in Health Authority feedback despite using a similar weight of evidence approach to request a waiver. Finally, sponsors should be prepared to run a JAS in the case of disparate Health Authority feedback