Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl-/- mice by administration of soluble (non-targeted) chimeric alkaline phosphatase

Abstract

Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven effective in preventing skeletal and dental defects in TNAP knockout (Alpl(-/-)) mice, a model for life-threatening HPP. Here, we show that the administration of a soluble, intestinal-like chimeric alkaline phosphatase (ChimAP) improves the manifestations of HPP in Alpl(-/-) mice. Mice received daily subcutaneous injections of ChimAP at doses of 1, 8 or 16 mg/kg, from birth for up to 53 days. Lifespan and body weight of Alpl(-/-) mice were normalized, and vitamin B6-associated seizures were absent with 16 mg/kg/day of ChimAP. Radiographs, μCT and histological analyses documented improved mineralization in cortical and trabecular bone and secondary ossification centers in long bones of ChimAP16-treated mice. There was no evidence of craniosynostosis in the ChimAP16-treated mice and we did not detect ectopic calcification by radiography and histology in the aortas, stomachs, kidneys or lungs in any of the treatment groups. Molar tooth development and function improved with the highest ChimAP dose, including enamel, dentin, and tooth morphology. Cementum remained deficient and alveolar bone mineralization was reduced compared to controls, though ChimAP-treated Alpl(-/-) mice featured periodontal attachment and retained teeth. This study provides the first evidence for the pharmacological efficacy of ChimAP for use in the treatment of skeletal and dental manifestations of HPP.

Keywords: Craniosynostosis; Enzyme replacement; Hypophosphatasia; Osteomalacia; Rickets; Seizures.

Fig. 1

Pharmacokinetics and improvement of survival and body weight of Alpl^−/− mice by ChimAP treatment. A) Subcutaneous injections of 1 mg/kg, 8 mg/kg and 16 mg/kg ChimAP into Alpl^+/− heterozygous mice led to dose-dependent peak plasma AP levels after 4 h and trough plasma AP levels at 24 h that were slightly higher than endogenous TNAP activity levels in both heterozygous and WT mice. Dosing once a day was deemed adequate for the in vivo efficacy studies. B) Kaplan–Meier curves show that ChimAP16 mice survived to the end of the experiment. Average survival for Vehicle, ChimAP1, and ChimAP8 mice was p19, p22 and p42.5, respectively. C) Alpl^−/− vehicle treated mice are lighter than WT littermates; ChimAP1 treatment improves body weight close to WT at p18. Vehicle treated mice do not survive longer and ChimAP8 treated mice are still lighter than WT littermates at p53. Whereas ChimAP16 treated mice present a body weight not significantly different from their WT littermates at p53.

Fig. 2

ChimAP treatment improves the skeletal phenotype of Alpl^−/− mice. Radiographs of spine, forelimb, rib cage, hindlimb and paws from Alpl^−/− mice treated with vehicle, ChimAP1, ChimAP8, ChimAP16, and untreated WT controls (magnification 5×). A) Alpl^−/− mice display a severe hypomineralization (arrows) in vertebrae, long bones and rib cage. Secondary ossification centers are missing in Alpl^−/− mice (asterisk). Treatment with ChimAP1 slightly corrects that phenotype (arrowheads) compared to untreated WT at p18. B) Treatment with ChimAP8 and ChimAP16 clearly corrects the bone phenotype in vertebrae, long bones and rib cage. Specifically the distal most extremities are improved as the development of the secondary ossification centers in the metatarsal region demonstrates (arrowhead).

Fig. 3

Improved mineralization in ChimAP-treated Alpl^−/− mice. A) Histological analysis of femora of vehicle treated Alpl^−/− (p18), ChimAP8 (p51), ChimAP16 (p53), and untreated WT mice (p53). Von Kossa staining revealed better bone mineralization with increased dosages ChimAP8 and ChimAP16. Secondary ossification centers and the cortical bones are strikingly better (black) ChimAP-treated Alpl^−/− mice compared to vehicle-treated Alpl^−/− mice. There is less trabecular bone in the ChimAP8 and ChimAP16 mice compared to WT, but increased mineralization in the trabecular region as well as more osteoid (pink). B/C) Analysis of BV/TV and OV/BV for ChimAP8 and ChimAP16 demonstrated less bone volume and higher osteoid volume than age-matched controls. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Improvement of osteomalacia and plasma PP_i levels in ChimAP-treated Alpl^−/− mice. A) Histological analysis of femora of vehicle treated Alpl^−/− (p18) (n = 3), ChimAP8 (p51) (n = 3), ChimAP16 p53 (n = 3), and age-matched untreated sibling p53 WT control mice (n = 3). Goldner’s Trichrome staining of the femora sections show severe osteomalacia in Alpl^−/−, and improved mineralization (green) in the cortical area and in the secondary ossification with increased dosages ChimAP8 and ChimAP16. Presence of enlarged areas of osteoid (red), suggests deposition of bone, but not yet mineralized. B/C) PP_i concentrations in the plasma of Alpl^−/− mice receiving ChimAP1 (n = 5), ChimAP16 (n = 5), and untreated age-matched sibling WT (n = 5) control mice. ChimAP1 treatment leads to a significant reduction of the elevated PP_i levels in Alpl^−/− mice at p18. At p53 ChimAP16 treatment resulted in a correction of plasma PP_i levels comparable to WT controls. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Prevention of hypertrophic zone expansion in the growth plates of ChimAP-treated Alp^l−/− mice. Histological analysis using Goldner’s Trichrome staining of non-decalcified growth plate sections obtained at the knee joint of vehicle-treated Alpl^−/− (p20), ChimAP8 (p51), ChimAP16 (p53), and untreated age-matched sibling WT control mice (p53), shown in 10× (upper panels) and 20× magnification (lower panels). HZ: hypertrophic zone.

Fig. 6

Absence of craniofacial defects in ChimAP-treated Alpl^−/− mice. μCT isosurface images of WT (A, D), vehicle-treated Alpl^−/−(B, E) and ChimAP16 (C, F) mouse skulls at p21 and p53, respectively. Neither frontal nor parietal bones of treated Alpl^−/− mice were significantly different from those of WT mice. Adult skulls of ChimAP16-treated Alpl^−/− mice did not appear different from WT in terms of size and shape.

Fig. 7

ChimAP treatment prevents dentoalveolar pathology in Alpl^−/− mice. Compared to (A) radiography and (D) μCT analysis of wild-type controls at p25–26, (B, F) untreated Alpl^−/− mouse mandibles feature hypomineralized and reduced alveolar bone (AB), short molars (M1, M2, and M3) with thin dentin (DE) and wide pulp chambers, and defective (white asterisk) incisor (INC) dentin. Compared to (E) histology of control periodontal tissues, (G) Alpl^−/− mice exhibit no acellular cementum (AC), and alveolar bone osteoid invades the PDL space, creating bone-tooth ankylosis (asterisk). (C, H) ChimAP8-treated Alpl^−/− mice show improved radiographic appearance of molar height, dentin thickness, and bone mineralization, though the incisor teeth remain defective. (I) Histologically ChimAP does not restore acellular cementum to the root surface. Compared to (J, L) controls at p53, (K, M) ChimAP16-treated Alpl^−/− mice exhibit reduced alveolar bone mineralization around molar teeth. Molar form and mineralization appear relatively normal in treated Alpl^−/− mice, while incisor teeth remain severely affected on the root analogue (white asterisk). Compared to histology of (N) WT control molars, (P) ChimAP16-treated Alpl^−/− mice feature a mix of mineralized alveolar bone and osteoid (asterisks), and a reduced but maintained PDL space. Poor periodontal attachment is evidenced by lack of cementum, PDL disorganization and detachment, and down growth of the junctional epithelium. Small regions of PDL attachment (chevron) to the tooth were noted. Compared to the strong and parallel organization of PDL collagen fibers in (O) control tissues, indicated by picrosirius staining under polarized light, (Q) ChimAP16-treated Alpl^−/− mice feature a less organized PDL, though regions of organization and attachment exist adjacent to breakthrough areas of tooth root attachment (white chevron).