Background and Aims Peripheral Artery disease (PAD) is a common vascular disease associated with functional impairment and increased risk of cardiovascular events in End Stage Kidney Disease (ESKD) patients undergoing dialysis. Poor limb salvage outcomes and high post-amputation mortality in hemodialysis (HD) patients highlight the need for earlier medical therapies. Cilostazol and pentoxifylline are approved for PAD. Their use in HD patients stays limited and cilostazol use requires caution in this population. Clinical studies demonstrate associations between arterial calcification and adverse outcomes in PAD patients. SNF472, a selective calcification inhibitor that interferes in the formation and growth of hydroxyapatite, is in Phase 3 for calciphylaxis treatment. This study aims to evaluate the effects of SNF472 on limb functional recovery and blood perfusion in a Vitamin D3 (VitD)-induced arterial calcification rat model. Method Arterial calcification was induced in 32 Sprague Dawley rats by 3 consecutive daily s.c. doses of 120 kIU/kg VitD. Rats were divided into four groups and treated during 12 days by: placebo s.c, placebo p.o, SNF472 (20 mg/kg/day, s.c.) or cilostazol (20 mg/kg/day, p.o.). An additional group of 8 rats without VitD received vehicle only (sham). Efficacy was evaluated at day 12 and 17 (5 days after treatment stop). Posterior limb blood perfusion was measured using Laser Doppler Imaging and limb walking ability was evaluated by measuring Maximum Walking Distance (MWD) and Maximum Walking Time (MWT) using a treadmill. Rats were sacrificed at day 26 (14 days after treatment stop), and aortas were collected for calcium analysis. Results VitD-induced arterial calcification was associated with decreased blood perfusion and impairment of limb walking ability (MWT and MWD) compared to sham. SNF472 reduced aorta calcification by 41% compared to placebo. No effects of cilostazol on vascular calcification were observed. The inhibition of calcification in SNF472-treated animals was associated with significant higher limb blood perfusion compared to placebo or Cilostazol (1.28 and 1.37-fold higher, respectively at day 12: p< 0.001) and it was translated into a significant improvement in walking ability compared to placebo (515±114 meters vs 334±187 meters, respectively: p<0.05). Conclusion SNF472 shows improvements in vascular calcification, blood perfusion and a functional parameter like walking distance in a PAD vascular calcification rat model. These results suggest that SNF472 may represent a new therapeutic approach for the treatment of PAD associated with high vascular calcification such as in renal disease.

Background and Aims Peripheral artery disease (PAD) is a common co-morbidity in end-stage kidney disease (ESKD) patients undergoing dialysis. The prominent vascular calcification in these patients is associated with severity of symptoms and adverse outcomes. Although there are medical therapies approved for PAD, none of them has been specifically approved por PAD-ESKD. For example, cilostazol is approved for PAD but its use is limited in PAD-ESKD patients. Surgical interventions are challenging in these patients due to their specific vascular calcification and show worse outcomes. Therefore, the aims of this study were to evaluate the effects of the investigational drug SNF472, a selective inhibitor of vascular calcification, on blood perfusion and limb functional recovery in a Vitamin D3 (VitD)-induced rat model of arterial calcification and limb ischemia. Method Three consecutives daily subcutaneous (s.c) doses of VitD were administered to 66 male Sprague Dawley rats to induce limb ischemia. Rats were randomized into four groups at day 5 after the start of VitD treatment (when all parameters were measured in a satellite group) and were treated for nine days with placebo s.c., placebo peroral (p.o.), SNF472 (40 mg/kg/day, s.c.) or cilostazol (40 mg/kg/day, p.o.). An additional control group did not receive VitD (sham) and was administered with placebo s.c. during these last nine days. Posterior limb blood perfusion was measured using laser Doppler imaging at baseline (before calcification induction), day 4 (before treatment start) and day 13 (end of treatment). Walking ability was evaluated by measuring Maximum Walking Distance (MWD) and Maximum Walking Time (MWT) using a rat treadmill at baseline, day 4 and day 11. Rats were sacrificed at day 13, and heart and femoral arteries were collected for calcium analysis. Results Administration of VitD induced heart and femoral artery calcification by day 5. This calcification was associated with decreased limb blood perfusion and impairment of walking ability (both MWT and MWD) compared to sham. Treatment with SNF472 inhibited calcification progression in femoral arteries by 41% and in heart by 56% compared to placebo. The inhibition of calcification progression by SNF472 led to a 29% increase in limb blood perfusion (p< 0.001) and a significant improvement in walking ability (49% in MWD and 43% in MWT; p< 0.05) compared to placebo. Calcification inhibition in femoral arteries was positively correlated with both MWD and MWT (p< 0.0001). No effects of cilostazol were observed in tissue calcification, limb blood perfusion or walking ability. Conclusion SNF472 attenuates the progression of vascular calcification and improves blood perfusion and the functional parameters MWT and MWD in a rat model of PAD vascular calcification. These results support investigation of SNF472 as a potential therapy for PAD-ESKD patients who have vascular dysfunction due to a high degree of arterial calcification.

Background and Aims Vascular calcification (VC) is a major contributor to increased morbidity and mortality in End Stage Kidney Disease (ESKD) patients undergoing dialysis. SNF472, a salt of inositol hexaphosphate (InsP6), is a selective calcification inhibitor that interferes in the formation and growth of ectopic hydroxyapatite (HAP). SNF472 is currently in Phase 3 clinical trials for the treatment of calciphylaxis in ESKD patients on dialysis. Inositol-1,2,3,5-tetraphosphate-4,6-bisPEG100 (InsP4bisPEG or INS3001) results from the PEGylation of inositol tetraphosphate (InsP4) with polyethylene glycol (PEG) 100. Our aim was to compare the relative bioavailability of SNF472 and InsP4bisPEG and their efficacy in the inhibition of calcification in silico, in vitro and in vivo. Method Subcutaneous (10 mg/kg) pharmacokinetics of InsP4bisPEG and SNF472 were assessed in Sprague Dawley (SD) rats. To evaluate the adsorption binding affinity (Eads) of SNF472, InsP4bisPEG and other inositol phosphates to the HAP crystal surface, computational studies were performed using Density Functional Theory calculations with DMOL3 (MS2016). The in vitro efficacy of the compounds was evaluated using a pharmacodynamic assay to measure the calcification potential of human plasma. An in vivo efficacy study (calcification induced by 3 consecutives daily s.c. administrations of 150 kIU/kg vitamin D3) was performed with SD rats receiving s.c. vehicle, or equimolar doses (36 µmol/kg) of SNF472 or InsP4bisPEG once daily. Results The PEGylation of inositol tetraphosphate in positions 4 and 6 increased the exposure and t1/2 of the compound when given subcutaneously compared to SNF472. Molecular modelling revealed that SNF472 binds to the HAP surface with higher affinity than InsP4bisPEG and INSP4 (ΔEads=-352 kcal/mol for SNF472, ΔEads=-177 kcal/mol for InsP4bisPEG and ΔEads=-146 Kcal/mol for InsP4, taking inositol as reference). These results were correlated with the inhibition of calcium phosphate crystallization in plasma in vitro. SNF472 treated animals presented significantly lower calcium levels in aorta, which were 38% and 55% lower than placebo and InsP4bisPEG treated animals, respectively. Conclusion The differential pharmacokinetic profile of InsP4bisPEG (INS3001) does not translate into higher, but lower, efficacy than SNF472 against vascular calcification when comparing equimolar doses.