Development of an oral phosphocitrate prodrug to control calcinosis

Phosphocitrate (PC) has previously demonstrated inhibitory potential against calcium crystallite formation associated with disease states such as urolithiasis, atherosclerosis and joint crystal deposition. The compound has proven active when injected intraperitoneally (ip) and intramuscularly. However, when administered orally, it has limited bioavailability. The present study has investigated synthesis of an oral PC prodrug and, in conjunction with determining the metabolic fate of the parent compound, assessed in viva inhibitory activity of the new PC derivative. Basic pharmacokinetics of PC were determined by a single intravenous (iv) injection Uugular vein) of radiolabelled PC followed by measurement within 60 min of the compound in plasma, urine and selected tissues. The plasma concentration-time profile of the compound followed a two-compartment model, with an elimination half-life of 30 min. Uptake of the administered dose accounted for less than 0.1 % in tissues, with 10 % in urine within 1 h. A capillary electrophoretic system was optimised for PC identification and measurement in urine. Longer-term studies involving iv (caudal vein) and ip administrations of non-labelled PC indicated total excretion of compound occurred within 12 hand 24 h, respectively. A synthetic strategy was developed for generating a methylacyloxy derivative of PC. The mono-sodium PC salt was reacted with chloromethylacetate under alkali conditions, with formamide as the solvent. Partial characterisation of the isolated product indicated that the PC derivative was water soluble, suggesting that it may not have been fully esterified, although phosphate esterification did occur as indicated by thin-layer chromatography. As anticipated, the derivative proved unable to prevent in vitro the transformation of amorphous calcium phosphate to hydroxyapatite. Orally administered prodrug inhibited chemically-induced subcutaneous plaques in rats with a similar potency to that of equimolar doses of ip administered parent compound. Similar urinary excretion patterns of the parent compound were observed whether PC was given orally as PC prodrug or ip as the parent compound. Enzymatic studies in vitro demonstrated slow hydrolysis of prodrug to parent compound in the presence of plasma with a more rapid hydrolysis noted using a rat liver homogenate. For the first time, it has been demonstrated that PC exhibits improved bioavailability when administered as an oral prodrug derivative, thus offering an alternate route of administration for its future consideration as a therapeutic drug against calcinosis.