Studies on the oral delivery of phosphocitrate

Phosphocitrate is a potent inhibitor of hydroxyapatite formation with demonstrated efficacy in preventing the progression of calcific pathologies in animal models. In the present studies, the oral bioavailability of phosphocitrate was investigated following administration of the compound by gavage in rats. Using a calcergic model, inhibitory action by phosphocitrate was evaluated. Accordingly, the formation of small subcutaneous calcific plaques was chemically induced. Parameters measured included plaque weight, calcium, and phosphorus content. The oral and intraperitoneal routes of administration were compared and dose-response relationships established. Phosphocitrate was found capable of reducing calcific plaque formation when delivered by either route. However reduced bioavailability was apparent following administration by gavage revealing the oral route to be approximately 45 times less effective on a dose-weight basis than the intraperitoneal route. The possibility that poor absorption from the small intestine was responsible for the observed differences was explored. Transport studies using everted sacs and in situ ligated loops confirmed phosphocitrate absorption. The data revealed concentration-dependance and the absence of in vitro intestinal transfer at the lowest concentration (1mM) trialled. Further studies were necessary to evaluate the extent of phosphocitrate absorption from the small intestine. Experiments with radiolabelled phosphocitrate and using an in situ perfusion technique with hepatic portal vein cannulation yielded additional information. The studies provided conclusive evidence that limited amounts of intact phosphocitrate were being absorbed. First pass metabolism to citric acid was observed but this metabolism was not commensurate with the poor rate of absorption. Studies utilising tricarballylate, a known inhibitor of tricarboxylic acid transport in the intestine, indicated that the disappearance of phosphocitrate from the intestinal lumen was not affected by the presence of this transport inhibitor. Consideration of this and everted sac data gave credence to the suggestion that phosphocitrate transport did not involve an established tricarboxylic acid transporter. A preliminary investigation was made into the possibility of improving the oral bioavailability of phosphocitrate with aliphatic ester prodrugs of phosphocitrate. Initially the approach was to investigate the synthesis and ultimate benefit of short (ethyl) and medium chain (octyl) esters. The triethyl ester of phosphocitrate was synthesised and trialled but the isolation and purification of the trioctyl ester was not possible within the time frame of these studies. Conclusions reached in this study were that phosphocitrate was absorbed following oral administration but that the extent of absorption was limited. Triethyl phosphocitrate was prepared in an attempt to improve the transport characteristics of phosphocitrate. Plaque calcification was not reduced following the administration of this ester. The prodnig approach was discussed as a possible avenue for improving the absorption of phosphocitrate