Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy

Abstract

Parkinson’s disease (PD) is a neurodegenerative aging disorder in which postmortem PD brain exhibits neuroinflammation, as well as synucleinopathy-associated protein phosphatase 2A (PP2A) enzymatic activity loss. Based on our translational research, we began evaluating the PD-repurposing-potential of an anti-inflammatory, neuroprotective, and PP2A stimulatory oral drug that is FDA-approved for multiple sclerosis, FTY720 (fingolimod, Gilenya®). We also designed two new FTY720 analogues, FTY720-C2 and FTY720-Mitoxy, with modifications that affect drug potency and mitochondrial localization, respectively. Herein, we describe the metabolic stability and metabolic profiling of FTY720-C2 and FTY720-Mitoxy in liver microsomes and hepatocytes. Using mouse, rat, dog, monkey, and human liver microsomes the intrinsic clearance of FTY720-C2 was 22.5, 79.5, 6.0, 20.2 and 18.3 μL/min/mg; and for FTY720-Mitoxy was 1.8, 7.8, 1.4, 135.0 and 17.5 μL/min/mg, respectively. In hepatocytes, both FTY720-C2 and FTY720-Mitoxy were metabolized from the octyl side chain, generating a series of carboxylic acids similar to the parent FTY720, but without phosphorylated metabolites. To assess absorption and distribution, we gave equivalent single intravenous (IV) or oral doses of FTY720-C2 or FTY720-Mitoxy to C57BL/6 mice, with two mice per time point evaluated. After IV delivery, both FTY720-C2 and FTY720-Mitoxy were rapidly detected in plasma and brain; and reached peak concentrations at the first sampling time points. After oral dosing, FTY720-C2 was present in plasma and brain, although FTY720-Mitoxy was not orally bioavailable. Brain-to-plasma ratio of both compounds increased time-dependently, suggesting a preferential partitioning to the brain. PP2A activity in mouse adrenal gland increased ~2-fold after FTY720-C2 or FTY720-Mitoxy, as compared to untreated controls. In summary, FTY720-C2 and FTY720-Mitoxy both (i) crossed the blood-brain-barrier; (ii) produced metabolites similar to FTY720, except without phosphorylated species that cause S1P1-mediated-immunosuppression; and (iii) stimulated in vivo PP2A activity, all of which encourage additional preclinical assessment.

Metabolic stability and its role in the discovery of new chemical entities

“…-During in vitro metabolic stability testing, systems based on different preclinical species are employed (i.e., human, mouse, rat, dog and monkey). As different model organisms are used in the experiments, interspecies variations in metabolic stability are typically observed (9,30,38,(94)(95)(96)(97)(98).…”

“…Enoru et al (97) assessed the preclinical metabolism of new blood-brain-barrier penetrant fingolimod analogues: FTY720-C2 and FTY720-Mitoxy. The in vitro intrinsic clearance of FTY720-C2 was low in dog, moderate in mouse, monkey and human, and high in rat, whereas in the case of the second analogue, FTY720-Mitoxy, the intrinsic clearance was found to be low in mouse, rat and dog, moderate in human and high in monkey.…”

A Pilot Microbiota Study in Parkinson’s Disease Patients versus Control Subjects, and Effects of FTY720 and FTY720-Mitoxy Therapies in Parkinsonian and Multiple System Atrophy Mouse Models

“…We showed that FTY720 improves GI physiology/function and reduces GI aSyn pathology in A53T Tg parkinsonian mice [22]. In CNP-aSyn MSA mice we have assessed FTY720-Mitoxy, our novel neuroprotective FTY720derivative, [27] that though not orally bioavailable, readily crosses the blood brain barrier [31], stimulates trophic factor expression in neurons and oligodendroglia [32,33], and is not immunosuppressive by decreasing circulating T cells [34]. Preliminary data confirm the restorative effects of FTY720-Mitoxy on motor, sudomotor, and GI function in Tg CNP-aSyn MSA mice [21].…”

Could α-Synuclein Modulation of Insulin and Dopamine Identify a Novel Link Between Parkinson’s Disease and Diabetes as Well as Potential Therapies?

“…In aging aSyn transgenic mice given long term oral FTY720, the mice had reduced aSyn pathology and increased levels of the protective molecule, brain derived neurotrophic factor (BDNF) (Vidal-Martinez et al, 2016). In collaboration with medicinal chemists, we made two non-immunosuppressive FTY720s that also enhance PP2Ac activity, and BDNF expression (Vargas-Medrano et al, 2014; Enoru et al, 2016; Segura-Ulate et al, 2017a). FTY720 and our novel FTY720-based-derivatives, may thus have therapeutic potential for both diabetes and PD.…”