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Scientific and Regulatory Policy Committee Points to Consider*: Primary Digital Histopathology Evaluation and Peer Review for Good Laboratory Practice (GLP) Nonclinical Toxicology Studies
No full textThe Society of Toxicologic Pathology’s Scientific and Regulatory Policy Committee formed a working group to consider the present and future use of digital pathology in toxicologic pathology in general and specifically its use in primary evaluation and peer review in Good Laboratory Practice (GLP) environments. Digital histopathology systems can save costs by reducing travel, enhancing organizational flexibility, decreasing slide handling, improving collaboration, increasing access to historical images, and improving quality and efficiency through integration with laboratory information management systems. However, the resources to implement and operate a digital pathology system can be significant. Given the magnitude and risks involved in the decision to adopt digital histopathology, this working group used pertinent previously published survey results and its members’ expertise to create a Points-to-Consider article to assist organizations with building and implementing digital pathology workflows. With the aim of providing a comprehensive perspective, the current publication summarizes aspects of digital whole slide imaging relevant to nonclinical histopathology evaluations, and then presents points to consider applicable to both primary digital histopathology evaluation and digital peer review in GLP toxicology studies. The appendices provide additional tabulated resources
THE MISSING LINK: ENABLING LOADING OF 2-CHLOROTRITYL CHLORIDE RESIN IN N-BUTYLPYRROLIDINONE AS A GREEN SOLVENT
No full text2-Chlorotrityl chloride resin is one of the most frequently used and most versatile resins for the large-scale manufacture of peptides. As a part of our efforts in greening solid phase peptide synthesis, here we disclose an efficient procedure for the loading of the first amino acid onto 2-chlorotrityl chloride resin using the green solvent N-butylpyrrolidinone. The application of design of experiment models allowed us to identify key critical parameters in the loading process. Results obtained herein suggest that the conditions found can be generalized and applied to any Fmoc amino acid, or serve as a starting point for loading optimization
Biaryl bond formation through biocatalytic oxidative cross-coupling reactions
No full textBiocatalysis offers compelling advantages in synthesis, often becoming the method of choice
based on sustainability, safety, and selectivity considerations. Despite these advantages, enzymes in
synthesis are typically dedicated to functional group interconversions in linear synthetic sequences and
have not been broadly integrated into the retrosynthetic logic for carbon skeleton assembly. In this article,
we disclose a biocatalytic platform for fragment coupling to assemble target molecules convergently.
Specifically, we report a strategy for biocatalytic phenolic cross-coupling through oxidative C–C bond
formation. Using cytochrome P450 enzymes, we demonstrate the ability to catalyze cross-coupling
reactions on a panel of phenolic substrates and further demonstrate the ability to tune these catalysts to
possess the desired reactivity, site-, and atroposelectivity. This streamlined method for constructing
sterically-hindered biaryl bonds provides an engineerable platform for assembling molecules with
programmable catalyst-controlled reactivity and selectivity unprecedented with small molecule catalyst
Therapeutic potential of macrophage colony-stimulating factor (CSF1) in chronic liver disease.
No full textResident and recruited macrophages control the development and proliferation of the liver. We showed previously in multiple species that treatment with a macrophage colony stimulating factor (CSF1)-Fc fusion protein initiated hepatocyte proliferation and promoted repair in models of acute hepatic injury in mice. Here we investigated the impact of CSF1-Fc on resolution of advanced fibrosis and liver regeneration, utilizing a non-resolving toxin-induced model of chronic liver injury and fibrosis in C57BL/6J mice. Co-administration of CSF1-Fc with exposure to thioacetamide (TAA) exacerbated inflammation consistent with monocyte contributions to initiation of pathology. After removal of TAA, either acute or chronic CSF1-Fc treatment promoted liver growth, prevented progression and promoted resolution of fibrosis. Acute CSF1-Fc treatment was also anti-fibrotic and pro-regenerative in a model of partial hepatectomy in mice with established fibrosis. The beneficial impacts of CSF1-Fc treatment were associated with monocyte-macrophage recruitment and increased expression of remodeling enzymes and growth factors. These studies indicate that CSF1-dependent macrophages contribute to both initiation and resolution of fibrotic injury and that CSF1-Fc has therapeutic potential in human liver disease
Teleocidin-producing genotype of Streptomyces clavuligerus ATCC 27064.
No full textStreptomyces clavuligerus is an industrially important producer of clavulanic acid (CA), a β-lactamase inhibitor which is used together with amoxicillin in one of the most widely prescribed antibacterial medicines, the co-amoxiclav. In a mid-eighties ATCC vial of S. clavuligerus ATCC 27064 culture, we have found a new genotype, which was apparently lost from the subsequent ATCC collection stocks, and has remained obscure to the scientific community. Most importantly, this genotype harbors teleocidin (lyngbyatoxin) biosynthetic genes, which are located on an enigmatic 138 kb chromosomal region and support accumulation of significant amounts of these highly toxic, tumor-promoting secondary metabolites in cultures of S. clavuligerus. While this genomic region is completely absent from all published sequences for S. clavuligerus ATCC strain, at least one of the industrial strains for commercial production of CA, originating from ATCC 27064, retained the genetic potential for production of teleocidins. The origin of teleocidin biosynthetic cluster can now be traced back to early S. clavuligerus stocks at the ATCC. Our work provides a genome sequence and a deposited monoisolate of this genotype. Given the scale of industrial use of S. clavuligerus world-wide and toxicity of teleocidins, we also discuss the environmental and safety implications and provide a method of abolishing teleocidin production without affecting productivity of CA. KEY POINTS: • Early stocks of S. clavuligerus ATCC 27064 produce toxic teleocidins • Teleocidin biosynthetic genes were found within a distinct S. clavuligerus genotype • The genotype has been passed on to some industrial clavulanic acid producer strains
Paradoxical Increase of Permeability and Lipophilicity with the Increasing Topological Polar Surface Area within a Series of PRMT5 Inhibitors.
No full textAn imidazolone → triazolone replacement addressed the limited passive permeability of a series of protein arginine methyl transferase 5 (PRMT5) inhibitors. This increase in passive permeability was unexpected given the increase in the hydrogen bond acceptor (HBA) count and topological polar surface area (TPSA), two descriptors that are typically inversely correlated with permeability. Quantum mechanics (QM) calculations revealed that this unusual effect was due to an electronically driven disconnect between TPSA and 3D-PSA, which manifests in a reduction in overall HBA strength as indicated by the HBA moment descriptor from COSMO-RS (conductor-like screening model for real solvation). HBA moment was subsequently deployed as a design parameter leading to the discovery of inhibitors with not only improved passive permeability but also reduced P-glycoprotein (P-gp) transport. Our case study suggests that hidden polarity as quantified by TPSA-3DPSA can be rationally designed through QM calculations
Exposure-Efficacy Analysis of Asciminib in Philadelphia Chromosome-Positive Chronic Myeloid Leukemia in Chronic Phase.
No full textAsciminib (Scemblix) is a first-in-class BCR::ABL1 inhibitor that works by specifically targeting the ABL myristoyl pocket (STAMP) and has potent activity against the T315I mutation. This study aimed to characterize the effect of asciminib exposure on disease progression and to elucidate factors influencing efficacy. Our analysis included 303 patients with chronic myeloid leukemia in chronic phase recruited in a phase I study with dose ranging from 10 to 200 mg twice a day (b.i.d.) or 40 to 200 mg once a day (q.d.) (NCT02081378) and in the phase III ASCEMBL (Study of Efficacy of CML-CP Patients Treated With ABL001 Versus Bosutinib, Previously Treated With 2 or More TKIs) study receiving asciminib 40 mg b.i.d. (NCT03106779). A total of 67 patients harbored the T315I mutation. A longitudinal pharmacokinetic/pharmacodynamic model was developed to characterize the exposure-efficacy relationship, in which the efficacy was assessed through BCR::ABL1 transcript levels over time. Specifically, a three-compartment model representing quiescent leukemic stem cells, proliferating bone marrow cells, and resistant cells was developed. Drug killing of the proliferating cells by asciminib was characterized by a power model. A subgroup analysis was performed on the patients with the T315I mutation using a maximum drug effect model to characterize the drug effect. The model demonstrated the appropriateness of a total daily dose of asciminib 80 mg in patients without the T315I mutation and 200 mg b.i.d. in patients with the T315I mutation with further validation in light of safety data. This model captured key characteristics of patients' response to asciminib and helped inform dosing rationale for resistant and difficult-to-treat populations
Iptacopan monotherapy in patients with paroxysmal nocturnal hemoglobinuria: a 2-cohort open-label proof-of-concept study.
No full textIptacopan (LNP023) is a novel, oral selective inhibitor of complement factor B under clinical development for paroxysmal nocturnal hemoglobinuria (PNH). In this ongoing open-label phase 2 study, PNH patients with active hemolysis were randomized to receive single-agent iptacopan twice-daily, at a dose of either 25 mg for 4 weeks followed by 100 mg for up to 2 years (cohort 1) or 50 mg for 4 weeks followed by 200 mg for up to 2 years (cohort 2). At the time of interim analysis, of 13 PNH patients enrolled, all 12 evaluable for efficacy achieved the primary endpoint of reduction in serum lactate dehydrogenase (LDH) levels by at least 60% by week 12 as compared to baseline; mean LDH levels dropped rapidly and durably, namely by 77% and 85% at week 2 and by 86% and 86% at week 12 in cohorts 1 and 2, respectively. Most patients achieved a clinically meaningful improvement in hemoglobin levels and all but one patient remained transfusion-free up to week 12. Other markers of hemolysis, including bilirubin, reticulocytes and haptoglobin, showed consistent improvements. No thromboembolic events were reported, and iptacopan was well tolerated, with no severe or serious adverse events reported up until the data cutoff. In addition to the previously reported beneficial effect of iptacopan add-on therapy to eculizumab, this study showed that iptacopan monotherapy in treatment-naïve PNH patients resulted in normalization of hemolytic markers and rapid transfusion-free improvement of hemoglobin levels in most patients. Registered at www.clinicaltrials.gov as NCT03896152
Model-informed drug development for immuno-oncology agonistic anti-GITR antibody GWN323: MABEL-based dose selection, translational and clinical PK/PD modeling
No full textGWN323, an agonistic human anti-GITR (glucocorticoid-induced TNFR-related protein) IgG1 antibody, was studied clinically as an immuno-oncology therapeutic agent. A model-based minimum anticipated biological effect level (MABEL) approach integrating in vitro and in vivo data was used to inform dose selection for the first-in-human (FIH) study in patients with cancer. Data evaluated included the pharmacokinetics (PK), soluble GITR (sGITR) and tumor shrinkage in Colon26 syngeneic mice administered with DTA-1.mIgG2a (a mouse surrogate GITR antibody), cytokine release of GWN323 in human peripheral blood mononuclear cells, GITR binding affinity, and GWN323 PK exposure at the highest non-severe toxic dose in monkeys. A PK model was developed to describe DTA-1.mIgG2a PK and the relationship between DTA-1.mIgG2a exposure and sGITR was also modeled. Human GWN323 PK was predicted by allometric scaling of mouse PK. Based on the totality of PK/PD modeling and the in vitro and in vivo pharmacology and toxicology data, MABEL was estimated and informed the starting dose selection of the FIH study. Based on tumor kinetic PK/PD modelling of tumor inhibition by DTA-1.mIgG2a in Colon26 mice and the predicted human PK of GWN323, the biologically active dose of GWN323 was predicted and informed dose escalation of the FIH study. GWN323 PK from the FIH study was described by a population PK model and the relationship between ex vivo interleukin-2 release, a target-engagement marker, and GWN323 concentration was also modeled. The clinical PK/PD modeling data supported the biological active dose predicted from the translational PK/PD modeling in a “learn and confirm” paradigm of model-informed drug development of GWN323
A fluorescent biosensor for measuring Ras activity in living cells
No full textThe small GTPase Ras is a critical regulator of cell growth and proliferation. Its activity is
frequently dysregulated in cancers, prompting decades of work to pharmacologically target Ras.
Understanding Ras biology and developing effective Ras therapeutics both require probing Ras
activity in its native context, yet tools to measure its activities in cellulo are limited. Here we
developed a ratiometric Ras activity reporter (RasAR) that provides quantitative measurement of
Ras activity in living cells with high spatiotemporal resolution. We demonstrated that RasAR can
probe live-cell activities of all the primary isoforms of Ras. Given that the functional roles of
different isoforms of Ras are intimately linked to their subcellular distribution and regulation, we
interrogated the spatiotemporal regulation of Ras utilizing subcellularly targeted RasAR and
uncovered the role of Src kinase as an upstream regulator to inhibit HRas. Furthermore, we
showed that RasAR enables capture of KRasG12C inhibition dynamics in living cells upon
treatment with KRasG12C covalent inhibitors, including ARS1620, Sotorasib, and Adagrasib.
We found in living cells a residual Ras activity lingers for hours in the presence of these
inhibitors. Together, RasAR represents a powerful molecular tool to enable live-cell
interrogation of Ras activity and facilitate the development of Ras inhibitors