1-Methyl-L-tryptophan

Liver fibrosis, a precursor to cirrhosis, is the result of the deposition of extracellular matrix (ECM) proteins and is mediated primarily by activated hepatic stellate cells (HSCs). In this study, we investigated the anti-fibrotic effects of interferon (IFN)-γ in activated HSCs in vitro and whether cell viability would be decreased by the inhibition of indoleamine 2,3-dioxygemase (IDO), which is responsible for cell cycle arrest. Following treatment with IFN-γ, cell signaling pathways and DNA content were analyzed to assess the inactivation of HSCs or the decrease in HSC proliferation. The IDO inhibitor, 1-methyl-L-tryptophan (1-MT), was used to determine whether IDO plays a key role in the regulation of activated HSCs, as IFN-γ increases the expression of IDO. IFN-γ significantly inhibited the growth of HSCs and downregulated the expression of α-smooth muscle actin (α-SMA) in the HSCs. IDO expression was markedly increased by IFN-γ through signal transducer and activator of transcription 1 (STAT1) activation and resulted in the depletion of tryptophan. This depletion induced G1 cell cycle arrest. When the cells were released from IFN-γ-mediated G1 cell cycle arrest by treatment with 1-MT, the apoptosis of the HSCs was markedly increased through the induction of IFN-γRβ, interferon regulatory factor (IRF-1) and FAS. Our results thus suggest that the inhibition of IDO enhances the suppression of activated HSCs, and therefore co-treatment with IFN-γ and 1-MT may be applied to ameliorate liver fibrosis.

Increased activity of the tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is associated with immunological and neurological disorders, and inhibition of its enzyme activity could be a therapeutic approach for treatment of these disorders. The aim of the present study was to establish a large animal model to study the accumulation of the potential IDO inhibitor 1-methyltryptophan (1-MT) in blood and different organs of domestic pigs (Sus scrofa domestica). Because 1-MT has not been previously evaluated in pigs, the pharmacokinetics of a single subcutaneous 1-MT application was investigated. Based on this kinetic study, a profile for repeated 1-MT applications over a period of five days was simulated and tested. The results show that a single administration of 1-MT increases its concentrations in blood, with the maximum concentration being obtained at 12 h. Repeated daily injections of 1‑MT generated increasing plasma concentrations followed by a steady-state after two days. Twelve hours after the final application, accumulation of 1-MT was observed in the brain and other organs, with a substantial variability among various tissues. The concentrations of 1-MT measured in plasma and tissues were similar to, or even higher, than those of tryptophan. Our data indicate that repeated subcutaneous injections of 1-MT provide a suitable model for accumulation of 1-MT in plasma and tissues of domestic pigs. These findings provide a basis for further research on the immunoregulatory functions of IDO in a large animal model.

Indoleamine 2,3-dioxygenase (IDO) is an immunomodulating enzyme that is overexpressed in many cancers with poor prognosis. IDO suppresses T cell immunity by catabolizing tryptophan into kynurenine (KYN), which induces apoptosis in T effector cells and enhances T regulatory cells, providing a powerful immunosuppressive mechanism in tumors. Thus, major efforts for developing IDO inhibitors have been undertaken. Among them, 1-Methyl-l-Tryptophan (MLT) and 1-Methyl-d-Tryptophan (MDT) effectively inhibit IDO in preclinical tumor models and the latter is under clinical evaluation. However, both MLT and MDT present poor pharmacokinetics, with the maximum serum concentration being below their 50% inhibitory concentration value. Herein, we have developed polymeric IDO inhibitors based on MLT, which can release active MLT after enzymatic degradation, toward establishing superior antitumor immunotherapies. These polymers were prepared by ring opening polymerization of an N-phenyl carbamate (NPC) derivative of MLT that was synthesized by carbamylation with diphenyl carbonate. By using ω-amino-poly(ethylene glycol) (PEG-NH2) as the macroinitiator, we prepared amphiphilic PEG-poly(MLT) block copolymers, which self-assembled into polymeric micelles in aqueous conditions. The PEG-poly(MLT) block copolymers could be readily degraded by chymotrypsin and the micelles were able to reduce the levels of KYN in activated macrophages. These results provide a strong rationale for pursuing MLT-based polymeric micelles as tumor-targeted prodrug systems.