Crizotinib and Ceritinib Induce Apoptosis and Necrosis in Primary Rat Hepatocytes with Distinct Capacity

Drug development makes up a major portion of biomedical engineering research interests. The FDA oversees the introduction, experimentation, and implementation of all drugs before market approval is granted. Even after market approval is granted, the FDA continues to monitor the safety of all drugs. Crizotinib and ceritinib are two anaplastic lymphoma kinase (ALK) inhibitors recently approved by the FDA. Both drugs are indicated for treatment of non-small cell lung cancers (NSCLC) with abnormal ALK gene, and they are approved with a companion diagnostic test that determines ALK abnormality. Clinical trial data suggest that crizotinib and ceritinib can cause liver injury, and this information has been included in the “Warnings and Precautions” section of their labeling. The mechanism of ALK inhibitor induced hepatotoxicity is unknown. This study aimed to observe if crizotinib and ceritinib are directly toxic to liver cells. Primary cultured rat hepatocytes were treated with crizotinib and ceritinib at clinically relevant concentrations for 4, 8 and 24 h, and apoptosis and necrosis were measured. A ~125% to ~150% increase in caspase 3/7 activity was observed at 8 h for ceritinib treated hepatocytes, and significant necrosis (~40%) occurred at 24 h. Ceritinib treated hepatocytes also showed remarkable cytochrome c release at 4 h, the time point when no cell death was detectable. Crizotinib showed no toxicity at 10-fold the maximal blood concentration (Cmax), while ceritinib became toxic at 3-fold Cmax and caused ~40% cell death at 6-fold Cmax, indicating that ceritinib, the second-generation ALK inhibitor, is significantly more toxic than the first-generation drug crizotinib. These data provide novel insights into the mechanisms of ALK inhibitors associated hepatotoxicity

Identification of Penicillin Binding Protein 4 (PBP4) as a critical factor for Staphylococcus aureus bone invasion during osteomyelitis in mice.

Staphylococcus aureus infection of bone is challenging to treat because it colonizes the osteocyte lacuno-canalicular network (OLCN) of cortical bone. To elucidate factors involved in OLCN invasion and identify novel drug targets, we completed a hypothesis-driven screen of 24 S. aureus transposon insertion mutant strains for their ability to propagate through 0.5 μm-sized pores in the Microfluidic Silicon Membrane Canalicular Arrays (μSiM-CA), developed to model S. aureus invasion of the OLCN. This screen identified the uncanonical S. aureus transpeptidase, penicillin binding protein 4 (PBP4), as a necessary gene for S. aureus deformation and propagation through nanopores. In vivo studies revealed that Δpbp4 infected tibiae treated with vancomycin showed a significant 12-fold reduction in bacterial load compared to WT infected tibiae treated with vancomycin (p<0.05). Additionally, Δpbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Δpbp4 S. aureus failed to invade and colonize the OLCN despite high bacterial loads on the implant and in adjacent tissues. Together, these results demonstrate that PBP4 is required for S. aureus colonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN