INZ-701 Prevents Ectopic Tissue Calcification and Restores Bone Architecture and Growth in ENPP1-Deficient Mice

Abstract

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the major enzyme that cleaves extracellular adenosine triphosphate (ATP) to generate pyrophosphate (PPi), an inorganic metabolite with potent anticalcification activity. Loss‐of‐function mutations cause hypopyrophosphatemia and lead to a state of ENPP1 deficiency, which has an acute infantile phase known as generalized arterial calcification of infancy (GACI) and a pediatric to adult phase known as autosomal‐recessive hypophosphatemic rickets type 2 (ARHR2). ENPP1 deficiency manifests as ectopic calcification of multiple tissues, neointimal proliferation, premature mortality, impaired growth, and bone deformities. INZ‐701, a human ENPP1‐Fc protein, is in clinical development as an enzyme replacement therapy for the treatment of ENPP1 deficiency. The pharmacokinetic and pharmacodynamic profile and therapeutic effect of INZ‐701 were investigated in Enpp1asj/asj mice, a murine model of ENPP1 deficiency. Enpp1asj/asj mice have undetectable plasma PPi, lower plasma phosphate, and higher FGF23 levels compared with wild‐type (WT) mice. Enpp1asj/asj mice on the acceleration diet, containing high phosphate and low magnesium, quickly develop clinical signs, including dehydration, rough hair coat, pinned ears, stiffed legs, and hunched back. Enpp1asj/asj mice treated with vehicle had aforementioned clinical signs plus severe ectopic calcification in multiple tissues and bone defects, characteristics of the clinical phenotype observed in GACI and ARHR2 patients. Our results showed a durable PPi response for more than 3 days after a single dose of INZ‐701. Treatment of ENPP1‐deficient mice every other day with INZ‐701 for 8 weeks restored circulating levels of PPi, prevented pathological calcification in all the tested organs, restored growth parameters, corrected bone defects, improved clinical signs, and decreased mortality in Enpp1asj/asj mice, demonstrating the potential of INZ‐701 to treat ENPP1 deficiency. © 2021 American Society for Bone and Mineral Research (ASBMR).

Case Report and Review of Literature: Autosomal Recessive Hypophosphatemic Rickets Type 2 Caused by a Pathogenic Variant in ENPP1 Gene

“…However, the use of burosumab in ARHR2 patients is currently not recommended since it is not approved for this condition, and there exist concerns about worsening of ectopic calcification (18,19). ENPP1 replacement therapy, currently being developed in animal models, may be a future treatment for ARHR2 (13,20).…”

“…Although no evidence of arterial calcification was observed in this case, loss-of-function variants in ENPP1 are also known to be associated with GACI (OMIM 208000), a life-threatening disease characterized by calcification of medium and large arteries (2). ENPP1 regulates mineralization by suppressing hydroxyapatite crystal deposition via hydrolysis of adenosine triphosphate into adenosine monophosphate and PPi (20). Therefore, ENPP1deficiency results in pathological calcification and overmineralization in vessels and soft tissues, resulting in GACI.…”

INZ‐701, a recombinant ENPP1 enzyme, prevents ectopic calcification in an Abcc6−/− mouse model of pseudoxanthoma elasticum

“…The Abcc6 −/− mice began treatment at 6 weeks of age, the earliest stages of ectopic calcification in these mice 23 . To suppress the potential immune response to INZ‐701, all mice were administered intraperitoneal 40 μg GK‐1.5, an anti‐CD4 monoclonal antibody (Thermo Fisher Scientific), at Day 1 followed by a weekly intraperitoneal boost of 25 μg GK‐1.5, as described previously for Enpp1 asj/asj mice 20,21 . Mice were maintained on the standard rodent diet and sacrificed after 2 or 8 weeks of treatment.…”

“…INZ-701, a soluble recombinant ENPP1 enzyme, normalized plasma PPi levels by utilizing circulating ATP as a substrate, and subsequently prevented mortality, vascular calcification and defects of bone mineralization in the Enpp1 asj/asj mouse model of ENPP1 deficiency. 20,21 The efficacy of INZ-701 on ectopic calcification in PXE, a PPi deficiency disorder due to deficiency in ABCC6 which works upstream of ENPP1, has yet to be demonstrated. Normalization of plasma PPi levels in the Abcc6 −/− mice via genetic overexpression of human ENPP1 transgene significantly reduced ectopic calcification, 22 suggesting that INZ-701, the ENPP1 protein therapeutic, could be efficacious for PXE.…”

“…The deficiency of PPi is the underlying cause of systemic ectopic calcification in PXE, suggesting that pharmacologic approaches increasing plasma PPi concentrations are expected to counteract ectopic calcification leading to clinical stabilization and improvement of the disease. INZ‐701, a soluble recombinant ENPP1 enzyme, normalized plasma PPi levels by utilizing circulating ATP as a substrate, and subsequently prevented mortality, vascular calcification and defects of bone mineralization in the Enpp1 asj/asj mouse model of ENPP1 deficiency 20,21 . The efficacy of INZ‐701 on ectopic calcification in PXE, a PPi deficiency disorder due to deficiency in ABCC6 which works upstream of ENPP1, has yet to be demonstrated.…”