A Biopolymer‐delivered MMP‐2 Inhibitory for Treatment of Renal Fibrosis

Abstract

Renal fibrosis, the buildup of extracellular matrix (ECM) components and scar tissue leading to organ failure, is the final common pathway of chronic kidney disease (CKD). There are currently no pharmacological treatments for CKD that specifically target the fibrosis process. Matrix metalloproteinases (MMPs) are involved in ECM homeostasis and degrade ECM components during tissue repair. Although an increase in MMPs can have anti-fibrotic effects, some MMPs can also process and activate chemokines and cytokines that are pro-inflammatory. Studies have shown that in the initial stages of CKD, there is an increase in the expression and activity of MMP-2, a gelatinase known to activate pro-TGF-β, TNF-α, and TNF- β, which are known to be pro-inflammatory and profibrotic. Furthermore, MMP-2 can promote epithelial to mesenchymal transition (EMT), a process in which epithelial cells lose their cell-to-cell adhesion and gain the ability to migrate, proliferate, and differentiate to profibrotic mesenchymal cells. Our goal is to develop an MMP-2 inhibitor that specifically targets the renal cells to reduce or reverse the effects of fibrosis. We created a chimeric fusion protein between an MMP-2 inhibitory peptide derived from beta amyloid precursor peptide (APP-IP) and an elastin like polypeptide (ELP) drug carrier. ELP is has been shown to accumulate in the kidney and has a long and tunable plasma half-life, which would allow for the therapeutic to have a sustained activity with minimal off target effects. An MMP-2 fluorometric assay revealed that the ELP – MPP-2 inhibitory peptide fusion protein (ELP-APP-IP) maintained potent MMP-2 inhibitory activity (IC50 = 1 mM, Figure panel A). Biodistribution of ELP-APP-IP or ELP control was determined in Sprague Dawley rats following bolus intravenous injection (30 mg/kg). Pharmacokinetic analysis revealed a terminal half-life approximately 90 minutes for both ELP and ELP-APP-IP. Ex vivo imaging of the organs showed that both ELP and ELP-APP-IP localized to the kidneys at levels approximately 16 times higher than in other major organs (brain, liver spleen, heart and lungs, Figure panel B,C). Histological analysis revealed that ELP and ELP-APP-IP accumulated primarily in the renal cortex (Figure panel D). A pilot efficacy study was conducted in Dahl Salt Sensitive Rats (S-Rats) on a high salt diet (8% NaCl) as a model of hypertension induced glomerulosclerosis. S-Rats (male, 12 weeks of age) on high salt diet were treated with ELP or ELP-APP-IP (n = 5 per group) for 3 weeks via osmotic minipumps (10 mg/kg/day). Urine was collected via metabolic caging 24 hours prior to initiation of treatment and high salt diet, then again weekly throughout the study. 21 days after initiation of treatment, the rats were euthanized, and blood and major organs were harvested for analysis. Rats on a high salt diet treated with ELP-APP-IP had similar urine production to rats treated with ELP. However, there was a trend for reduced onset of proteinuria in rats treated with ELP-APP-IP (two way ANOVA, treatment factor p = 0.088, Figure panel E), suggesting ELP-APP-IP has the potential to reduce kidney damage and slow the progression of renal disease.