6.7.5. Pyrophosphate (PPi)

The idea of using PPi as a therapeutic for PXE and GACI came quickly after the discovery that ABCC6 modulates PPi production [8,9], though the idea of using PPi to prevent ectopic calcification was not new [206,207]. Considering the short half-life of PPi in plasma, several experimental approaches were devised to counteract calcification in PXE and GACI mouse models. Two groups proceeded to test different methodologies, with either a single daily bolus injection [18] or continuous delivery via drinking water [120]. The first study found that daily injections achieved near complete suppression of ectopic mineralization in several tissues despite a relatively poor bioavailability of about 0.5%. Importantly, and similar to other forms of treatments tested thus far, established mineralization could not be reversed [18].

Because daily injections are not necessarily practical for the lifelong treatment needed for PXE or GACI patients, oral administration was also evaluated [120]. Adding sodium PPi to drinking water sustainably raised plasma PPi in both *Abcc6*−/<sup>−</sup> mice as well as in human volunteers, and significantly decreased calcification in both PXE and GACI mouse models. However, calcification inhibition was not complete. One of the remarkable discoveries of the latter study was that, contrary to previous beliefs [208], PPi is bioavailable when administered orally, although poorly (~0.1%), but is still effective against mineralization.

Furthermore, there has been a recent and inadvertent discovery that modulating PPi from dietary sources can also be effective at reducing calcification in *Abcc6*−/<sup>−</sup> mice [19]. These findings were prompted by a routine change in the supplier of institutional rodent diet. The new chow was enriched in PPi (a fact unknown to the manufacturer and the research laboratory), leading to a doubling of plasma PPi and a halving of calcification in *Abcc6*−/<sup>−</sup> mice. Dietary PPi is also readily absorbed in humans with a bioavailability comparable to that of drinking water (~0.1%), which suggests that dietary preference could contribute to the considerable and still unexplained phenotypic heterogeneity in PXE [19].

These recent studies unambiguously showed that PPi supplementation via oral delivery or even by injection are arguably the most promising and credible strategies for treating PXE (and GACI) patients. Indeed, PPi would be a simple and low-cost medication and it has a very safe profile. It is a nontoxic, physiological metabolite as per the World Health Organization (WHO). The US Food and Drug Administration (FDA) lists it as Generally Recognized As Safe and it is designated as food additive E450(a) in Europe. PPi is thus widely used in the food industry as a preservative in canned seafood, in baking soda, in cured meat, etc. . . . and is even present in toothpaste.

For these reasons, a phase II clinical trial using capsulized disodium PPi is currently being conducted (NCT04441671) while the PROPHECI clinical trial (see above) for PXE patients is currently in preparation and also proposes to test the oral delivery of encapsulated disodium PPi.

Of note, several pharmaceutical companies are now investing in the testing and manufacturing of new PPi formulations for the treatment of calcification in PXE, GACI, and other diseases.
