6.7.4. Bisphosphonate Treatment for PXE and GACI

Bisphosphonates are non-hydrolyzable analogs of PPi with two phosphonate (PO3) groups covalently linked to a central carbon (instead of an oxygen) and two side chains that determine chemical characteristics. There are two categories of bisphosphonates based on the presence of a non-nitrogenous or nitrogenous side chain. Because bisphosphonates mimic the structure of PPi, they have similar properties and can inhibit enzymes that utilize pyrophosphate. Bisphosphonates have been used clinically for decades in the

treatment of osteoporosis and Paget's disease of bone, as well as other applications related to mineralization.

The bisphosphonate etidronate has been used to treat GACI patients and early treatments improved GACI outcome [145]. Retrospective analyses have confirmed that survival beyond infancy is markedly improved with etidronate therapy [55,146]. However, this treatment seems to induce undesirable side-effects as a recent case report highlighted significant inhibition of skeletal calcification with paradoxical joint calcifications in a 7-year-old GACI patient [145]. Furthermore, studies on uremic rats or *Enpp1*−*/*<sup>−</sup> mice suggested that this bisphosphonate is incapable of preventing *de novo* calcification without alteration to bone structures [203,204].

For PXE, a first study reported the anti-calcification properties of zoledronate (nitrogenous) in an in vitro series of experiments with primary fibroblasts [147]. However, more elaborate studies with mouse models showed that only administration of etidronate (non-nitrogenous), but not alendronate (nitrogenous), prevented ectopic mineralization. However, etidronate treatment did not reverse existing mineralization [18,144,148]. The effect of etidronate was accompanied by alterations in the trabecular bone microarchitecture similar to what was described for *Enpp1*−*/*<sup>−</sup> mice. Although the results suggested that biphosphonates could be used as potential treatments for PXE, the high dosages and/or the bone alterations are potentially detrimental in the long term.

However, these animal studies and the fact that etidronate is an approved drug already tested for off-label applications against calcification in GACI and basal ganglia calcifications [205] were sufficiently convincing that a clinical trial termed Treatment of Ectopic Mineralization in PXE or TEMP was initiated in the Netherlands [126]. This investigation primarily tested effectiveness and safety of etidronate administration in PXE. The 12-month long investigation used a cyclical treatment of 20 mg/kg for 2 weeks every 12 weeks in a randomized, placebo-controlled phase I/II with 74 participants. The primary outcome was ectopic calcification as determined by <sup>18</sup>fluoride positron emission tomography scans in femoral arterial tissues. Secondary outcomes were computed tomography arterial calcification and ophthalmological changes. Safety outcomes were bone density, serum calcium, and phosphate. The overall results were somewhat mixed, with PXE patients that received etidronate seeing reduced arterial calcification and subretinal neovascularization events. However, they did not experience lower femoral <sup>18</sup>fluoride positron emission tomography activity, suggesting that active mineralization was still ongoing. Overall, no adverse effects from the treatment were reported.

Of note, another phase I/II clinical trial with a French cohort is in preparation and is called PyROphosPHate supplementation to fight ECtopIc calcification in PXE (PROPHECI, registration: https://scanr.enseignementsup-recherche.gouv.fr/project/PHRCN-PHRC-19-0402, accessed on 2 March 2021). This trial will use oral disodium PPi, but etidronate will be used as a control instead of a placebo.
