The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.
Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson's disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.
Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.
In a randomized placebo-controlled trial of patients with NASH, we found 12-week administration of GS-0976 20 mg decreased hepatic steatosis, selected markers of fibrosis, and liver biochemistry. ClinicalTrials.gov ID NCT02856555.
In the drive to develop drugs with well-characterized and clinically monitorable safety profiles, there is incentive to expand the repertoire of safety biomarkers for toxicities without routine markers or premonitory detection. Biomarkers in blood are pursued because of specimen accessibility, opportunity for serial monitoring, quantitative measurement, and the availability of assay platforms. Cytokines, chemokines, and growth factors (here referred to collectively as cytokines) show robust modulation in proximal events of inflammation, immune response, and repair. These are key general processes in many toxicities; therefore, cytokines are commonly identified during biomarker discovery studies. In addition, multiplexed cytokine immunoassays are easily applied to biomarker discovery and routine toxicity studies to measure blood cytokines. However, cytokines pose several challenges as safety biomarkers because of a short serum half-life; low to undetectable baseline levels; lack of tissue-specific or toxicity-specific expression; complexities related to cytokine expression with multiorgan involvement; and species, strain, and interindividual differences. Additional challenges to their application are caused by analytical, methodological, and study design–related variables. A final consideration is the strength of the relationship between changes in cytokine levels and the development of phenotypic or functional manifestations of toxicity. These factors should inform the integrated judgment-based qualification of novel biomarkers in preclinical, and potentially clinical, risk assessment. The dearth of robust, predictive cytokine biomarkers for specific toxicities is an indication of the significant complexity of these challenges. This review will consider the current state of the science and recommendations for appropriate application of cytokines in preclinical safety assessment.
The tetraspanins are a family of integral membrane proteins with four transmembrane domains. These molecules form multimolecular networks on the surfaces of many different cell types. Gene-targeting studies have revealed a role for tetraspanins in B-and T-lymphocyte function. We have isolated and deleted a novel tetraspanin, Tssc6, which is expressed exclusively in hematopoietic and lymphoid organs. Using a genetrapping strategy, we generated an embryonic stem (ES) cell line with an insertion in the Tssc6 locus. Mice were derived from these ES cells and, using RNase protection and reverse transcription-PCR, we demonstrated that the insertion resulted in a null mutation of the Tssc6 allele. Mice homozygous for the gene trap insertion (Tssc6 gt/gt mice) were viable and fertile, with normal steady-state hematopoiesis. Furthermore, responses to hemolysis and granulocyte colony-stimulating factor-induced granulopoiesis were equivalent to those of wildtype mice. Lymphoid development was normal in Tssc6 gt/gt mice. Whereas Tssc6 gt/gt B cells responded normally to lipopolysaccharide, anti-CD40, and anti-immunoglobulin M stimulation, Tssc6 gt/gt T cells showed enhanced responses to concanavalin A, anti-CD3, and anti-CD28. This increased proliferation by Tssc6-deleted T lymphocytes was due to increased interleukin 2 production following T-cell receptor stimulation. These results demonstrate that Tssc6 is not required for normal development of the hematopoietic system but may play a role in the negative regulation of peripheral T-lymphocyte proliferation.
The cooperative nature of tetraspanin–tetraspanin interactions in membrane organization suggests functional overlap is likely to be important in tetraspanin biology. Previous functional studies of the tetraspanins CD37 and Tssc6 in the immune system found that both CD37 and Tssc6 regulate T cell proliferative responses in vitro. CD37−/− mice also displayed a hyper-stimulatory dendritic cell phenotype and dysregulated humoral responses. In this study, we characterize “double knockout” mice (CD37−/−Tssc6−/−) generated to investigate functional overlap between these tetraspanins. Strong evidence for a cooperative role for these two proteins was identified in cellular immunity, where both in vitro T cell proliferative responses and dendritic cell stimulation capacity are significantly exaggerated in CD37−/−Tssc6−/− mice when compared with single knockout counterparts. Despite these exaggerated cellular responses in vitro, CD37−/−Tssc6−/− mice are not more susceptible to autoimmune induction. However, in vivo responses to pathogens appear poor in CD37−/−Tssc6−/− mice, which showed a reduced ability to produce influenza-specific T cells and displayed a rapid onset hyper-parasitemia when infected with Plasmodium yoelii. Therefore, in the absence of both CD37 and Tssc6, immune function is further altered when compared with CD37−/− or Tssc6−/− mice, demonstrating a complementary role for these two molecules in cellular immunity.
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