Novel treatment for PXE: Recombinant ENPP1 enzyme therapy

Abstract

Pseudoxanthoma elasticum (PXE) is a genetic multisystem ectopic calcification disorder caused by inactivating mutations in the ABCC6 gene encoding ABCC6, a hepatic efflux transporter. ABCC6-mediated ATP secretion by the liver is the main source of a potent endogenous calcification inhibitor, plasma inorganic pyrophosphate (PPi); the deficiency of plasma PPi underpins PXE. Recent studies demonstrated that INZ-701, a recombinant human ENPP1 that generates PPi and is now in clinical trials, restored plasma PPi levels and prevented ectopic calcification in the muzzle skin of Abcc6^-/-mice. This study examined the pharmacokinetics, pharmacodynamics, and potency of a new ENPP1-Fc isoform, BL-1118, in Abcc6^-/- mice. When Abcc6^-/- mice received a single subcutaneous injection of BL-1118 at 0.25, 0.5, or 1 mg/kg, they had dose-dependent elevations in plasma ENPP1 enzyme activity and PPi levels, with an enzyme half-life of approximately 100 h. When Abcc6^-/- mice were injected weekly from 5 to 15 weeks of age, BL-1118 dose-dependently increased steady-state plasma ENPP1 activity and PPi levels and significantly reduced ectopic calcification in the muzzle skin and kidneys. These results suggest that BL-1118 is a promising second generation enzyme therapy for PXE, the first generation of which is currently in clinical testing.

Keywords: ABCC6; ENPP1; ectopic calcification; enzyme therapy; pseudoxanthoma elasticum; pyrophosphate.

Graphical abstract

Figure 1

PK and PD effects of BL-1118 in Abcc6^−/− mice after a single injection Abcc6^−/− mice were injected subcutaneously with 0.25, 0.5, or 1 mg/kg of enzyme. Blood samples were collected at eight time points via retro-orbital bleeds. (A) ENPP1 enzyme activity vs. time plots to highlight the AUC of the enzyme. (B) The PK analysis of the recombinant ENPP1 enzyme. The fractional ENPP1 enzyme activity, corresponding to the fractional activity compared with peak plasma concentration (Cmax), was sampled in mice at eight time points over 239 h. (C) The PD effect after a single injection of 0.25, 0.5, or 1 mg/kg of enzyme, as measured by plasma PPi concentrations in Abcc6^−/− mice. The shaded areas represent the baseline levels of PPi in plasma of WT and KO mice. Results are mean (SD). n = 6 blood samples per time point.

Figure 2

Steady-state biomarkers in Abcc6^−/− mice after 10 week BL-1118 treatment (A) Plasma ENPP1 activity. (B) Plasma PPi concentrations. Vehicle-treated groups of WT mice and Abcc6^−/− mice serve as controls of baseline levels of ENPP1 activity and PPi concentration in plasma. Values were expressed as mean (SD); ∗p < 0.05 and ⁺p < 0.01 compared to WT group for the same time points. n = 10 mice per group (5 male and 5 female). WT, wild type. KO, knockout.

Figure 3

Assessment of ectopic calcification in Abcc6^−/− mice after 10 week BL-1118 treatment (A) μCT images of muzzle skin biopsies and quantification of calcification volume. n = 4 mice per group (2 male and 2 female). (B) von Kossa-stained muzzle biopsies and quantification of calcium content. n = 10 mice per group (5 male and 5 female). (C) μCT images of kidneys and quantification of calcification volume. n = 4 mice per group (2 male and 2 female). (D) von Kossa-stained kidneys and quantification of calcium content. n = 10 mice per group (5 male and 5 female). The Abcc6^−/− control mice developed extensive ectopic calcification in the muzzle skin and kidneys. The WT mice were entirely negative for calcification. The Abcc6^−/− mice administered BL-1118 demonstrated significantly reduced calcification compared to vehicle-treated Abcc6^−/− control mice. Values were expressed as mean (SD); WT, wild type. KO, knockout.