A phytic acid analogue INS-3001 prevents ectopic calcification in an Abcc6-/- mouse model of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic calcification disorders, affects the skin, eyes and the cardiovascular system due to inactivating mutations in the ABCC6 gene. There is no effective treatment for the systemic manifestations of PXE. In this study, the efficacy of INS-3001, an analogue of phytic acid, was tested for inhibition of ectopic calcification in an Abcc6-/- mouse model of PXE. In prevention study, Abcc6-/- mice, at 6 weeks of age, the time of onset of ectopic calcification, were treated with INS-3001 with 0.16, 0.8, 4, 20 or 100 mg/kg/day administered by subcutaneous implantation of osmotic pumps, as well as 4 mg/kg/day by subcutaneous injection thrice weekly or 14, 4 and 0.8 mg/kg/day once weekly subcutaneous injection. Mice were necropsied at 12 weeks of age. Histologic examination and quantitative calcium assay revealed that mice receiving 6 weeks of continuous INS-3001 administration via osmotic pumps showed dose-dependent inhibition of muzzle skin calcification with complete response at 4 mg/kg/day and a minimum effective dose at 0.8 mg/kg/day. INS-3001 plasma concentrations were dose-dependent and largely consistent during treatment for each dose. thrice weekly and once weekly subcutaneous injections of INS-3001 also prevented calcification. In established disease study, 12-week-old Abcc6-/- mice with extensive calcification were continuously administered INS-3001 at 4 mg/kg/day for a follow-up of 12 weeks. INS-3001 treatment was found to stabilize existing calcification that had developed at start of treatment. These results suggest that INS-3001 may provide a promising preventive treatment strategy for PXE, a currently intractable ectopic calcification disorder.

Development of a Kidney Calcification Inhibitor Employing Image-Based Profiling: A Proof-of-Concept Study.

Kidney calcification increases the risk of chronic kidney disease. However, to date, renal calcium phosphate crystallization, a main initiating and driving factor of kidney calcification, has not been explored as a drug target. Pre-clinical drug development is hampered by limited knowledge on the broad range of kidney calcification disorders, characterized by a multifactorial process of disease progression. In this work, we first established an in vitro calcification profiling platform to accelerate pre-clinical drug discovery. The image-based profiling assay allowed the rapid testing of several ionic stimuli and/or inhibitory molecules. We then leveraged a previously established library of inositol hexakisphosphate analogues to identify a renal calcium phosphate inhibitor. A lead compound showed in vitro and in vivo efficacy to prevent calcium phosphate-induced kidney damage. In conclusion, this work reports a renal calcium phosphate inhibitor that could efficiently reduce kidney damage and emphasizes the utility and translational value of the in vitro calcification platform.

Investigational Therapies for Primary Hyperoxaluria.

Recent years have brought exciting new insights in the field of primary hyperoxaluria (PH), both on a basic research level as well as through the progress of novel therapeutics in clinical development. To date, very few supportive measures are available for patients suffering from PH, which, together with the severity of the disorder, make disease management challenging. Basic and clinical research and development efforts range from correcting the underlying gene mutations, preventing calcium oxalate crystal-induced kidney damage, to the administration of probiotics favoring the intestinal secretion of excess oxalate. In this review, current advances in the development of those strategies are presented and discussed.

Inhibitors of Calcium Oxalate Crystallization for the Treatment of Oxalate Nephropathies.

Calcium oxalate (CaOx) crystal-induced nephropathies comprise a range of kidney disorders, for which there are no efficient pharmacological treatments. Although CaOx crystallization inhibitors have been suggested as a therapeutic modality already decades ago, limited progress has been made in the discovery of potent molecules with efficacy in animal disease models. Herein, an image-based machine learning approach to systematically screen chemically modified myo-inositol hexakisphosphate (IP6) analogues is utilized, which enables the identification of a highly active divalent inositol phosphate molecule. To date, this is the first molecule shown to completely inhibit the crystallization process in the nanomolar range, reduce crystal-cell interactions, thereby preventing CaOx-induced transcriptomic changes, and decrease renal CaOx deposition and kidney injury in a mouse model of hyperoxaluria. In conclusion, IP6 analogues based on such a scaffold may represent a new treatment option for CaOx nephropathies.

Inhibition of vascular calcification by inositol phosphates derivatized with ethylene glycol oligomers.

Myo-inositol hexakisphosphate (IP6) is a natural product known to inhibit vascular calcification (VC), but with limited potency and low plasma exposure following bolus administration. Here we report the design of a series of inositol phosphate analogs as crystallization inhibitors, among which 4,6-di-O-(methoxy-diethyleneglycol)-myo-inositol-1,2,3,5-tetrakis(phosphate), (OEG2)2-IP4, displays increased in vitro activity, as well as more favorable pharmacokinetic and safety profiles than IP6 after subcutaneous injection. (OEG2)2-IP4 potently stabilizes calciprotein particle (CPP) growth, consistently demonstrates low micromolar activity in different in vitro models of VC (i.e., human serum, primary cell cultures, and tissue explants), and largely abolishes the development of VC in rodent models, while not causing toxicity related to serum calcium chelation. The data suggest a mechanism of action independent of the etiology of VC, whereby (OEG2)2-IP4 disrupts the nucleation and growth of pathological calcification.

Small-Molecule Allosteric Triggers of Clostridium difficile Toxin B Auto-proteolysis as a Therapeutic Strategy.

Clostridium difficile causes increasing numbers of life-threatening intestinal infections. Symptoms associated with C. difficile infection (CDI) are mediated by secreted protein toxins, whose virulence is modulated by intracellular auto-proteolysis following allosteric activation of their protease domains by inositol hexakisphosphate (IP6). Here, we explore the possibility of inactivating the C. difficile toxin B (TcdB) by triggering its auto-proteolysis in the gut lumen prior to cell uptake using gain-of-function small molecules. We anticipated that high calcium concentrations typically found in the gut would strongly chelate IP6, precluding it from pre-emptively inducing toxin auto-proteolysis if administered exogenously. We therefore designed IP6 analogs with reduced susceptibility to complexation by calcium, which maintained allosteric activity at physiological calcium concentrations. We found that oral administration of IP6 analogs attenuated inflammation and promoted survival in mouse models of CDI. Our data provide impetus to further develop small-molecule allosteric triggers of toxin auto-proteolysis as a therapeutic strategy.

Investigational new treatments for Clostridium difficile infection.

Significant progress has been made by industry and academia in the past two years to address the medical threats posed by Clostridium difficile infection. These developments provide an excellent example of how patient need has driven a surge of innovation in drug discovery. Indeed, only two drugs were approved for the infection in the past 30 years but there are 13 treatment candidates in clinical trials today. What makes the latter number even more remarkable is the diversity in the strategies represented (antibiotics, microbiota supplements, vaccines, antibiotic quenchers and passive immunization). In this review, we provide a snapshot of the current stage of these breakthroughs and argue that there is still room for further innovation in treating C. difficile infection.

Targeting bacterial toxins.

Protein toxins constitute the main virulence factors of several species of bacteria and have proven to be attractive targets for drug development. Lead candidates that target bacterial toxins range from small molecules to polymeric binders, and act at each of the multiple steps in the process of toxin-mediated pathogenicity. Despite recent and significant advances in the field, a rationally designed drug that targets toxins has yet to reach the market. This Review presents the state of the art in bacterial toxin targeted drug development with a critical consideration of achieved breakthroughs and withstanding challenges. The discussion focuses on A-B-type protein toxins secreted by four species of bacteria, namely Clostridium difficile (toxins A and B), Vibrio cholerae (cholera toxin), enterohemorrhagic Escherichia coli (Shiga toxin), and Bacillus anthracis (anthrax toxin), which are the causative agents of diseases for which treatments need to be improved.