A Preclinical Study Comparing the Activity and Potency of OnabotulinumtoxinA and PrabotulinumtoxinA.

Objective: The goal of this study was to compare the unit-to-unit biological activity of the vacuum-dried formulation of prabotulinumtoxinA (prabotA) and onabotulinumtoxinA (onabotA) in preclinical assays. Methods: Reconstituted 100 U vials of prabotA and onabotA were tested in 3 distinct assays: plate-capture light chain activity (PC-LCA), measuringlight chain enzymatic activity after recovery of toxin from reconstituted product using a proprietary toxin capture step; cell-based potency assay (CBPA), measuring the intoxication steps of binding, translocation, and light chain activity (synaptosomal-associated protein 25 [SNAP25] cleavage); and mouse Digit Abduction Score (DAS), evaluating muscle paresis. Each assay tested 3 separate prabotA and onabotA lots on several independent test dates. Results: Multiple orthogonal assays established that when assessed on a unit-to-unit basis, the biological activity of prabotA is lower than that of onabotA. In the PC-LCA and CBPA assays, onabotA displayed 1.51 ± 0.14-fold higher (mean ± SD) and 1.33 ± 0.07-fold higher (mean of pooled lots ± SEM) activity than prabotA, respectively. Similarly, the mouse DAS data showed that onabotA had 1.4 ± 0.1-fold higher (mean ± SEM) potency than prabotA. Results of all 3 assays demonstrated differences in potency, efficacy, and duration of action between onabotA and prabotA on a unit-to-unit basis. Conclusion: Preclinical assays established differences in the biological activity of onabotA and prabotA, supporting that the units of biological activity are not interchangeable.

3D Proximal Tubule Tissues Recapitulate Key Aspects of Renal Physiology to Enable Nephrotoxicity Testing.

Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current pre-clinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Using Organovo's proprietary 3D bioprinting platform, we have developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFß as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies.

Methionine sulfoxide reductase B (MsrB) of Mycobacterium smegmatis plays a limited role in resisting oxidative stress.

Pathogenic mycobacteria including Mycobacterium tuberculosis resists phagocyte generated reactive oxygen intermediates (ROI) and this constitutes an important virulence mechanism. We have previously reported, using Mycobacterium smegmatis as a model to identify the bacterial components that resist intracellular ROI, that an antioxidant methionine sulfoxide reductase A (MsrA) plays a critical role in this process. In this study, we report the role of methionine sulfoxide reductase B (MsrB) in resistance to ROI by constructing a msrB mutant (MSDeltamsrB) and MsrA/B double mutant (MSDeltamsrA/B) strains of M. smegmatis and testing their survival in unactivated and interferon gamma activated mouse macrophages. WhilemsrB mutant exhibited significantly lower intracellular survival than its wild type counterpart, the survival rate seemed to be much higher than msrA mutant (MSDeltamsrA) strain. Further, the msrB mutant showed no sensitivity to oxidants in vitro. The msrA/B double mutant (MSDeltamsrA/B), on the other hand, exhibited a phenotype similar to that of msrA mutant in terms of both intracellular survival and sensitivity to oxidants. We conclude, therefore, that MsrB of M. smegmatis plays only a limited role in resisting intracellular and in vitro ROI.