Background: Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme of de novo pyrimidine nucleotide synthesis. Most cells can salvage pyrimidine nucleotides from the environment, but rapidly dividing cells, such as activated lymphocytes, are reliant upon de novo synthesis. Therefore, the development of DHODH inhibitors is of interest to treat immune-mediated disease states driven by the abnormal proliferation of T- and B-cells. PTC844 is a novel, orally administered, small molecule that has demonstrated uniquely potent and selective inhibition of DHODH compared with in-class compounds both approved and under development to treat immune-mediated disease (teriflunomide, the teriflunomide prodrug leflunomide and vidofludimus). PTC844 is in development for the treatment of immune-mediated diseases and is under investigation in healthy volunteers (study PTC844-SMAD-101-HV; EU trial number: 2022-501986-38-00).

Objectives: To demonstrate in vitro cellular activity and in vivo activity of PTC844 and its analogues in preclinical animal models of autoimmune disease. Further, to evaluate the pharmacokinetics and pharmacodynamics of PTC844 and its analogues to allow for translation to the clinical setting.

Methods: The in vitro activity of PTC844, PTC analogues, and in-class compounds (teriflunomide and vidofludimus) was measured in MOLM13 cells. In vivo activity of PTC844 was measured in a rabbit model of arthritis and in a mouse model of xenogeneic graft-versus-host disease (xGvHD; immunocompromised mice injected with human peripheral blood mononuclear cells). In vivo activity of PTC844 analogues was tested in mouse models of multiple sclerosis (MS; C57BL/6 mice immunized with Myelin Oligodendrocyte Glycoprotein [MOG _35-55 ]) and lupus (MRL/lpr mice). Levels of test compound and the DHODH substrate, dihydroorotate (DHO), were measured in brain tissue and plasma in preclinical animal studies using liquid chromatography–tandem mass spectrometry.

Results: PTC844 inhibited DHODH-dependent proliferation of MOLM13 cells with a half-maximal inhibitory concentration (IC ₅₀ ) of 1 nM, whereas teriflunomide and vidofludimus had an IC ₅₀ > 10 000 nM and ~5000 nM, respectively. The ratio of DHODH-dependent inhibition of proliferation to off-target inhibition was greater for PTC844 than for teriflunomide and vidofludimus (~25 000-fold vs ~9-fold and ~12-fold, respectively). PTC844 and its analogues demonstrated substantial preclinical efficacy in models of T- and B-cell driven immune-mediated disease. In the xGvHD mouse model, the median survival time (MST) with PTC844 was 76 days compared with 36 days without treatment, demonstrating better survival than cyclosporine, Janus kinase inhibitors or prednisolone. PTC884 showed synergistic effects when used in combination with these agents. In a rabbit model of arthritis, PTC844 reduced swelling by 58% at Day 40 compared with the group without treatment, a similar reduction to that observed with methotrexate. In a mouse model of MS, PTC844 analogues led to a 100% reduction in clinical signs compared with the group without treatment. In a mouse model of lupus, the MST was 21 weeks without treatment, whereas treatment with PTC844 analogues resulted in 64% survival at 45 weeks. PTC844 was distributed in the central nervous system (CNS) of mouse, rat and dog models, whereas little vidofludimus or teriflunomide was found in the CNS after oral administration. Inhibition of DHODH in vivo resulted in elevations in DHO levels, which allowed identification of DHO levels associated with efficacy in preclinical studies.

Conclusions: Preclinical findings support further testing of PTC844 in immune-mediated disease.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: Marla Weetall Employee of PTC Therapeutics, Joesphine Sheedy Employee of PTC Therapeutics, Anna Mollin Employee of PTC Therapeutics, Nicole Risher Employee of PTC Therapeutics, Lucy Lee Employee of PTC Therapeutics, Jana Narasimhan Employee of PTC Therapeutics, Mark Ma Employee of PTC Therapeutics, Chris Delagrange Employee of PTC Therapeutics, John Karafilidis Employee of PTC Therapeutics, Aaron Tansy Employee of PTC Therapeutics, Galina Bernstein Consultant for PTC Therapeutics, Mayzie Johnston Employee of PTC Therapeutics.