American Life Science Pharmaceuticals (United States)

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

There is currently no therapeutic drug treatment for traumatic brain injury (TBI) despite decades of experimental clinical trials. This may be because the mechanistic pathways for improving TBI outcomes have yet to be identified and exploited. As such, there remains a need to seek out new molecular targets and their drug candidates to find new treatments for TBI. This review presents supporting evidence for cathepsin B, a cysteine protease, as a potentially important drug target for TBI. Cathepsin B expression is greatly up-regulated in TBI animal models, as well as in trauma patients. Importantly, knockout of the cathepsin B gene in TBI mice results in substantial improvements of TBI-caused deficits in behavior, pathology, and biomarkers, as well as improvements in related injury models. During the process of TBI-induced injury, cathepsin B likely escapes the lysosome, its normal subcellular location, into the cytoplasm or extracellular matrix (ECM) where the unleashed proteolytic power causes destruction via necrotic, apoptotic, autophagic, and activated glia-induced cell death, together with ECM breakdown and inflammation. Significantly, chemical inhibitors of cathepsin B are effective for improving deficits in TBI and related injuries including ischemia, cerebral bleeding, cerebral aneurysm, edema, pain, infection, rheumatoid arthritis, epilepsy, Huntington’s disease, multiple sclerosis, and Alzheimer’s disease. The inhibitor E64d is unique among cathepsin B inhibitors in being the only compound to have demonstrated oral efficacy in a TBI model and prior safe use in man and as such it is an excellent tool compound for preclinical testing and clinical compound development. These data support the conclusion that drug development of cathepsin B inhibitors for TBI treatment should be accelerated.

Data suggest that the there are common pathological mechanisms in neurodegeneration caused by Alzheimer's disease (AD) and by traumatic brain injury (TBI). For example, both AD and TBI result in elevated brain activity of the cysteine potease, cathepsin B (CatB). To explore further the commonality of CatB activity in AD and TBI pathology, we extended our previous studies, which showed that CatB is an AD drug target and that cysteine protease inhibitors are effective AD treatments, by studying the effects of deleting the CatB gene or treatment with a cysteine protease inhibitor in a TBI animal model on behavior and pathology. An open skull, single traumatic injury model of TBI was used. Briefly, mice were anesthetized, the skin retracted, the skullcap removed without dura disruption and the cortex injured using the controlled cortical impact device. The skullcap was then replaced and the skin sutured together. CatB gene knockout mice were generated and treated and compared to sufficient CatB treated and sham animals. Treated animals received one dose of the cysteine protease inhibitor, E64d, by gavage immediately post treatment and compared to vehicle treated and sham animals. Motor skills were assessed over seven days using a Rotor-Rod system after which the animals were sacrificed and the CA3 hippocampal neuron density and lesion volume determined by histology and brain CatB activity determined using a fluorometric activity assay. The CatB gene knockout and E64d treated animals had significantly improved motor skills, increased neuron densities and reduced lesion volumes relative to CatB sufficient and vehicle treated animals, respectively. Brain CatB activity was significantly increased relative to sham animals in the CatB sufficient and vehicle treated animals and abolished in CatB deficient and significantly reduced in E64d treated animals. The results validate CatB as a drug target for TBI and demonstrate that a cysteine protease inhibitor is an effective TBI treatment. The data support the hypothesis that increased CatB activity is a common pathology in both AD and TBI.

This highlight article describes three Alzheimer's disease (AD) studies presented at the 5th General Meeting of the International Proteolysis Society that address enzymatic mechanisms for producing neurotoxic beta-amyloid (Abeta) peptides. One group described the poor kinetics of BACE 1 for cleaving the wild-type (WT) beta-secretase site of APP found in most AD patients. They showed that cathepsin D displays BACE 1-like specificity and cathepsin D is 280-fold more abundant in human brain than BACE 1. Nevertheless, as BACE 1 and cathepsin D show poor activity towards the WT beta-secretase site, they suggested continuing the search for additional beta-secretase(s). The second group reported cathepsin B as an alternative beta-secretase possessing excellent kinetic efficiency and specificity for the WT beta-secretase site. Significantly, inhibitors of cathepsin B improved memory, with reduced amyloid plaques and decreased Abeta(40/42) in brains of AD animal models expressing amyloid precursor protein containing the WT beta-secretase site. The third group addressed isoaspartate and pyroglutamate (pGlu) posttranslational modifications of Abeta. Results showed that cathepsin B, but not BACE 1, efficiently cleaves the WT beta-secretase isoaspartate site. Furthermore, cyclization of N-terminal Glu by glutaminyl cyclase generates highly amyloidogenic pGluAbeta(3-40/42). These presentations suggest cathepsin B and glutaminyl cyclase as potential new AD therapeutic targets.

The regulated secretory pathway of neurons is the major source of extracellular Aß that accumulates in Alzheimer's disease (AD). Beta-secretase is required to generate Aß from its APP precursor. In this study, the major ß-secretase activity in these secretory vesicles was identified as cathepsin B by tandem mass spectrometry. Immunoelectron microscopy demonstrated colocalization of cathepsin B with Aß in regulated secretory vesicles of neuronal chromaffin cells. Cathepsin B showed efficient kinetics for cleaving the wild-type ß-secretase site. Significantly, the selective cathepsin B inhibitor, CA074, blocked the production of Aß40 from endogenous APP in regulated secretory vesicles isolated from neuronal chromaffin cells. Importantly, in primary neuronal chromaffin cell cultures, CA074Me reduced the content of Aß40 in secretory vesicles, demonstrated by decreased secretion of Aß40 via the regulated secretory pathway upon induction by KCl. These results demonstrate a role for cathepsin B in production of Aß, as novel ß-secretase in the regulated secretory pathway of brain neurons, and that inhibitors of cathepsin B may be considered as therapeutic agents to reduce Aß in AD.