Hepatoprotection by L-cysteine-glutathione mixed disulfide, a sulfhydryl-modified prodrug of glutathione.

L-Cysteine-glutathione disulfide, a ubiquitous substance present in mammalian cells, was shown to be highly effective in protecting mice against acetaminophen-induced hepatotoxicity. Since the corresponding D-cysteine-glutathione disulfide was totally ineffective in this regard, an enzymatic mechanism that provides glutathione directly to cells is postulated.

Double-prodrugs of L-cysteine: differential protection against acetaminophen-induced hepatotoxicity in mice.

A series of double-prodrugs of L-cysteine, designed to release L-cysteine in vivo and stimulate the biosynthesis of glutathione (GSH), were synthesized. To evaluate the hepatoprotective effectiveness of these double-prodrugs, male Swiss-Webster mice were administered acetaminophen (ACP) (2.45 mmol/kg (360 mg/kg), intraperitoneally (i.p.)). Prodrug (2.50 mmol/kg, i.p. or 1.25 mmol/kg, i.p., depending on the protocol) was administered 1 h before ACP as a priming dose. A supplementary dose of prodrug (2.5 mmol/kg, i.p. or 1.25 mmol/kg, i.p. depending on the protocol) was administered 0.5 h after ACP. The plasma alanine amino transferase (ALT) values, 24 h after ACP administration were transformed to logs and the 95% and 99% confidence intervals of the log values were plotted and compared for each group. Hepatoprotection was assessed by the degree of attenuation of plasma ALT levels. With these multiple dose schedules, the use of 2% carboxymethylcellulose as vehicle for the prodrugs was found to be detrimental; therefore, the prodrugs were dissolved in dilute aqueous base and the pH adjusted for administration. When a priming dose was given 1 h before ACP followed by a supplementary dose 0.5 h after ACP, only N,S-bis-acetyl-L-cysteine, where both the sulfhydryl and amino groups of L-cysteine were functionalized with the acetyl group, was found to be effective in protecting mice against the hepatotoxic effects of ACP. This suggests that these acetyl groups were rapidly hydrolyzed in vivo to liberate L-cysteine. In contrast, N-acetylation of 2(R,S)-methylthiazolidine-4(R)-carboxylic acid (MTCA) and its 2-n-propyl analog (PTCA), or N-acetylation of 2-oxothiazolidine-4-carboxylic acid (OTCA), reduced the hepatoprotective effects relative to the parent MTCA, PTCA, and OTCA, indicating that the release of L-cysteine in vivo from these N-acetylated thiazolidine prodrugs was metabolically unfavorable. The carbethoxy group, whether functionalized on the sulfhydryl or on the amino group of L-cysteine, or on the secondary amino group of MTCA, appears to be a poor "pro-moiety," since these carbethoxylated double-prodrugs of L-cysteine did not protect mice from ACP-induced hepatotoxicity.

Acetaminophen-induced suppression of hepatic AdoMet synthetase activity is attenuated by prodrugs of L-cysteine.

Administration of acetaminophen (ACP, 400 mg/kg, i.p.) to fasted, male Swiss-Webster mice caused a rapid 90% decrease in total hepatic glutathione (GSH) and a 58% decrease in mitochondrial GSH by 2 h post ACP. This was followed by a time-dependent decrease (72%) in hepatic AdoMet synthetase activity and rise in plasma ALT levels (>10000 U/l) at 24 h post ACP treatment. AdoMet synthetase activity was maintained at 82, 78 and 60% of controls, respectively, by the cysteine prodrugs PTCA, CySSME and NAC. Total hepatic and mitochondrial GSH levels were also protected from severe ACP-induced depletion by CySSME and MTCA. These results suggest that the maintenance of GSH homeostasis by cysteine prodrugs can protect mouse hepatic AdoMet synthetase, a sulfhydryl enzyme whose integrity is dependent on GSH, as well as the liver itself from the consequences of oxidative stress elicited by toxic metabolites of xenobiotics.