Synthesis and evaluation of novel hepatoprotective L-cysteine prodrugs

Numerous deleterious physiological effects result from cellular depletion of glutathione (GSH). GSH is abundant in living systems and exerts several protective functions. Reduction of the normally high levels of GSH in cells results from a wide array of endogenous and exogenous factors. The liver is frequently vulnerable to GSH depletion by exposure to toxins. Developing novel agents that increase GSH in the liver has been the primary goal of this work. Since GSH cannot be taken up by cells and is quickly catabolized, prodrug forms of its constituent L-cysteine, which supports the biosynthesis of GSH, have proved a successful therapeutic alternative. Prodrug versions of L-cysteine are required to overcome the toxicity of L-cystelne itself. Drug targeting as a means of directing agents to particular cells or organs is well established in the case of hepatocyte targeting. The hepatic asialoglycoprotein receptor (ASGPR) recognizes molecules bearing galactose moieties, which is followed by cellular uptake. Three L-cysteine prodrugs possessing galactose were synthesized using the condensation of L-cysteine with aldoses to form a thiazolidine carboxylic acid (TCA). TCAs dissociate nonenzymatically over time and have previously demonstrated GSH-maintaining ability. The galactose-bearing prodrugs were developed with the aim of increasing liver L-cysteine prodrug through ASGPR targeting. Two related glucose-bearing prodrugs were synthesized as controls. Prodrug toxicity was not seen in mice and apparent in cells only at high concentrations after prolonged exposures. Biodistribution of prodrugs in mice and protection using hepatocytes with and without the ASGPR were used to indicate targeting. Protection studies used acetaminophen (APAP), which is well documented as a GSH-depleting hepatotoxin. All five prodrugs protected against APAP, yet prodrug targeting is ambiguous. Additional protection mechanisms involving Phase II enzyme induction were addressed, because thiol induction has been documented. Prodrugs did not cause induction at the doses given, with the possible exception of one, but this was complicated by APAP induction. Studies determining liver and kidney GSH imply that the most conspicuous protection mechanism is the prevention of GSH depletion by APAP. Although ASGPR targeting is not immediately obvious from these studies, the principal goal, the development of novel hepatoprotective GSH-maintaining agents, was achieved.