A class of imidazoisoindole (III) heme-binding indoleamine-2,3-dioxygenase (IDO1) inhibitors were optimized via structure-based drug design into a series of tryptophan-2,3-dioxygenase (TDO)-selective inhibitors. Kynurenine pathway modulation was demonstrated in vivo, which enabled evaluation of TDO as a potential cancer immunotherapy target. As means of mitigating the risk of drug-drug interactions arising from cytochrome P450 inhibition, a novel property-based drug design parameter, herein referred to as the CYP Index, was implemented for the design of inhibitors with appreciable selectivity for TDO over CYP3A4. We anticipate the CYP Index will be a valuable design parameter for optimizing CYP inhibition of any small molecule inhibitor containing a Lewis basic motif capable of binding heme.

A series of different prodrugs of indoximod, including estesrs and peptide amides were synthesized with the aim of improving its oral bioavailability in humans. The pharmacokinetics of prodrugs that were stable in buffers, plasma and simulated gastric and intestinal fluids was first assessed in rats after oral dosing in solution or in capsule formulation. Two prodrugs that produced the highest exposure to indoximod in rats were further tested in Cynomolgus monkeys, a species in which indoximod has oral bioavailability of 6–10% and an equivalent dose-dependent exposure profile as humans. NLG802 was selected as the clinical development candidate after increasing oral bioavailability (>5-fold), Cmax (6.1–3.6 fold) and AUC (2.9–5.2 fold) in monkeys, compared to equivalent molar oral doses of indoximod. NLG802 is extensively absorbed and rapidly metabolized to indoximod in all species tested and shows a safe toxicological profile at the anticipated therapeutic doses. NLG802 markedly enhanced the anti-tumor responses of tumor-specific pmel-1 T cells in a melanoma tumor model. In conclusion, NLG802 is a prodrug of indoximod expected to increase clinical drug exposure to indoximod above the current achievable levels, thus increasing the possibility of therapeutic effects in a larger fraction of the target patient population.

Abstract IDO1 catalyzes degradation of tryptophan (Trp) into kynurenines, which plays an important role in the regulation of immune responses by triggering anergy on reactive effector T cells and by modulating differentiation and activation of regulatory T cells (Treg). Indoximod has been demonstrated to relieve IDO-mediated immunosuppression in vitro and in vivo, by creation of an artificial Trp-sufficiency signal that bypasses activation of GCN2 and inhibition of mTOR in conditions of Trp deprivation. Inhibition of the IDO pathway by indoximod in combination with immune-stimulatory treatments leads to increased T cell proliferation, Treg reprogramming and antitumor effect. Indoximod has demonstrated an excellent safety profile in human clinical trials and is being dosed orally at 1200 mg bid. Increases in doses above this level do not generally result in increased plasma concentration or drug exposure due to limiting dose-dependent oral bioavailability. Animal models suggest that increased therapeutic benefit could be achieved at higher levels of exposure. For this reason, we synthesized and tested the pharmacokinetic profile of several indoximod prodrugs in mice, rat and monkeys in both liquid and capsule formulations. We selected clinical development drug candidate NLG802, which increases indoximod exposure and plasma concentration ~ 2-fold in rats and ~ 5-fold in monkeys dosed with capsules of comparable formulation to that being used in clinical trials. NLG802 is rapidly absorbed and metabolized in vivo to indoximod. NLG802 DMPK profile and GLP toxicology studies have been carried out in rats and monkeys suggesting a safe toxicological profile at predicted therapeutic doses. In a preclinical tumor model of mice bearing established B16F10 tumors, administration of NLG802 markedly enhanced the anti-tumor responses of naïve, resting pmel-1 cells to vaccination with cognate hgp100 peptide plus CpG-1826 in IFA. In this tumor model, NLG802 plus pmel-1/vaccine produced significant tumor size reduction within 4 days of vaccination. Moreover, the tumor response was achieved at lower doses than equivalent molar doses of indoximod. In conclusion, NLG802 is a prodrug of indoximod predicted to increase clinical drug exposure to indoximod above the current achievable levels and will soon enter Phase 1 safety testing in oncology clinical trials. Citation Format: Mario R. Mautino, Sanjeev Kumar, Hong Zhuang, Jesse Waldo, Firoz Jaipuri, Hima Potturi, Erik Brincks, James Adams, Agnieszka Marcinowicz, Clarissa Van Allen, Nicholas Vahanian, Charles J. Link. A novel prodrug of indoximod with enhanced pharmacokinetic properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4076. doi:10.1158/1538-7445.AM2017-4076

Abstract Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase is an central immune checkpoint mechanism that controls peripheral immune tolerance and tumor resistance to immune attack. The IDO pathway mediates immunosuppressive effects by expression of the IDO1 gene by tumor cells and/or host antigen presenting cells. Tryptophan depletion and generation of kynurenine generate downstream signals through GCN2, WARS, mTOR and AHR that ultimately lead to anergy of tumor-specific effector CD8+ T cells and activation of Tregs. High expression of IDO correlates negatively with clinical prognosis in patients with a variety of malignancies. Recently, it has been described that tumors can also achieve immunosuppressive effects mediated by tryptophan degradation through expression of TDO, an enzyme functionally and structurally related to IDO1. Therefore, development of small molecules that can prevent tryptophan degradation mediated by IDO1, TDO or both is a prime goal for immunotherapy of cancer. Here we report the discovery of a novel class of imidazoisoindole-based compounds that show potent and selective inhibition of TDO and IDO1, as well as compounds that show potent dual inhibition of both TDO and IDO1. We present structure-activity relationship studies that explore the molecular determinants of potency and specificity for TDO and IDO1. Compounds of these classes were tested for activity in in vitro assays using purified recombinant human and murine TDO and IDO1 as well as in cells expressing IDO1 or TDO. Many of these compounds are 10-200 fold more potent (IC50 < 50 nM) than previously published TDO-specific inhibitors. The pharmacokinetic properties of these compounds are being investigated and lead optimization efforts are in progress. Citation Format: Mario R. Mautino, Richard A. Metz, Firoz Jaipuri, Jesse Waldo, Sanjeev Kumar, Agnieszka Marcinowicz-Flick, Hima Potturi, James T. Adams, Clarissa Van Allen, Nicholas N. Vahanian, Charles J. Link. Novel specific- and dual- tryptophan-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO) inhibitors for tumor immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1633. doi:10.1158/1538-7445.AM2014-1633