*6.5. Targeting TNAP*

Alkaline phosphatases are a group of isoenzymes located at the cell surface catalyzing the hydrolysis of organic phosphate esters in a variety of phosphate-containing physiological compounds. They contribute to a range of physiological functions such as DNA synthesis, in addition to regulating calcification [183–186]. Alkaline phosphatases are classified as tissue-specific and tissue non-specific. The isoenzymes found in the intestine, placenta, and germinal tissue are tissue-specific, whereas the tissue non-specific (TNAP), often referred to as the liver/bone/kidney alkaline phosphatase, is found in these and other tissues. These alkaline phosphatases are encoded by four different genes and each has distinct functions.

The first evidence of a physiological connection between TNAP and a pathology closely related to PXE was reported in 2011 by St Hilaire et al. [187], even though a molecular connection was established a few years later [10,168]. This was followed by a more elaborate series of experiments using cell culture and several mouse models targeting the transporter/enzymes of the ABCC6 pathway (Figures 1 and 3) [20]. Using primary skin fibroblasts from PXE patients with confirmed *ABCC6* mutations, Ziegler et al. found that these cells had elevated *ALPL* gene expression and associated TNAP enzymatic activity as compared to controls. The authors also investigated the effects of a TNAP inhibitor, SBI-425 (30 mg/kg/day), administered in the food of *Abcc6*−/<sup>−</sup> mice vs. etidronate (240 mg/kg/day) or normal chow starting at 6 weeks of age for 14 weeks. MicroCT scans at 20 weeks revealed significant attenuation of calcification in both the mice treated with the TNAP inhibitor or etidronate. Furthermore, SBI-425 inhibited TNAP activity, whereas etidronate did not. More promising still as a potential therapeutic, *Abcc6*−/<sup>−</sup> mice aged to 20 weeks and treated with the TNAP inhibitor did not show the same progressive calcification as control animals, although the treatment did not reverse existing calcification in this experimental context. Remarkably, Ziegler and colleagues found that plasma PPi levels did not increase after SBI-425 treatment, despite decreased plasma TNAP activity. This is somewhat surprising as PPi concentration in the plasma of *Nt5e*−/<sup>−</sup> mice is significantly reduced [188]. Indeed, the lack of CD73 (NT5E) function leads to less adenosine and higher TNAP activity, resulting in higher rates of PPi hydrolysis. This apparent discrepancy is consistent with other circumstantial evidence [111,144], suggesting that plasma levels do not adequately reflect the steady state levels of PPi in connective tissues where it is most relevant.

A follow-up study used a slightly different approach to explore the role of TNAP in the calcification phenotype of PXE and GACI using *Abcc6*−/−*Alpl*+/<sup>−</sup> double-mutant mice and *Enpp1*−/<sup>−</sup> animals [127]. Mice heterozygous for *Alpl* in an *Abcc6*-deficient background showed both reduced plasma TNAP activity and mineralization as compared to controls,

and the administration of SBI-425 led to similar results. By contrast, SBI-425 treatment of *Enpp1*−/<sup>−</sup> mice did not produce any significant change in mineralization.

Overall, these studies demonstrated that inhibition of TNAP is a convincing treatment strategy for PXE (but maybe not for GACI). In fact, it is convincing enough that a pharmaceutical company is investing in this approach.
