Rat Pharmacokinetics and In Vitro Metabolite Identification of KM-819, a Parkinson's Disease Candidate, Using LC-MS/MS and LC-HRMS.

FAF1 (FAS-associated factor 1) is involved in the activation of Fas cell surface death receptors and plays a role in apoptosis and necrosis. In patients with Parkinson's disease, FAF1 is overexpressed in dopaminergic neurons in the substantia nigra. KM-819, an FAF1 inhibitor, has shown potential for preventing dopaminergic neuronal cell death, promoting the degradation of α-synuclein and preventing its accumulation. This study aimed to develop and validate a quantitative analytical method for determining KM-819 levels in rat plasma using liquid chromatography-tandem mass spectrometry. This method was then applied to pharmacokinetic (PK) studies in rats. The metabolic stability of KM-819 was assessed in rat, dog, and human hepatocytes. In vitro metabolite identification and metabolic pathways were investigated in rat, dog, and human hepatocytes. The structural analog of KM-819, namely N-[1-(4-bromobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl]-2-(phenylsulfanyl) acetamide, served as the internal standard (IS). Proteins were precipitated from plasma samples using acetonitrile. Analysis was carried out using a reverse-phase C18 column with a mobile phase consisting of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile. The analytical method developed for KM-819 exhibited linearity within the concentration range of 0.002-10 µg/mL in rat plasma. The precision and accuracy of the intra- and inter-day measurements were <15% for the lower limit of quantification (LLOQ) and all quality control samples. KM-819 demonstrated stability under various sample storage conditions (6 h at room temperature (25 °C), four weeks at -20 °C, three freeze-thaw cycles, and pretreated samples in the autosampler). The matrix effect and dilution integrity met the criteria set by the Food and Drug Administration and the European Medicines Agency. This sensitive, rapid, and reliable analytical method was successfully applied in pharmacokinetic studies in rats. Pharmacokinetic analysis revealed the dose-independent kinetics of KM-819 at 0.5-5 mg/kg, with a moderate oral bioavailability of ~20% in rats. The metabolic stability of KM-819 was also found to be moderate in rat, dog, and human hepatocytes. Metabolite identification in rat, dog, and human hepatocytes resulted in the discovery of six, six, and eight metabolites, respectively. Glucuronidation and mono-oxidation have been proposed as the major metabolic pathways. Overall, these findings contribute to a better understanding of the pharmacokinetic characteristics of KM-819, thereby aiding future clinical studies.

Pharmacological Intervention Targeting FAF1 Restores Autophagic Flux for α-Synuclein Degradation in the Brain of a Parkinson's Disease Mouse Model.

α-Synuclein accumulation is implicated in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). Previously, we reported that Fas-associated factor 1 (FAF1), which plays a role in PD pathogenesis, potentiates α-synuclein accumulation through autophagy impairment in dopaminergic neurons. In this study, we show that KM-819, a FAF1-targeting compound, which has completed phase I clinical trials, interferes with α-synuclein accumulation in the mouse brain, as well as in human neuronal cells (SH-SY5Ys). KM-819 suppressed the accumulation of monomeric, oligomeric, and aggregated forms of α-synuclein in neuronal cells. Furthermore, KM-819 restored the turnover rate of α-synuclein in FAF1-overexpressing SH-SY5Y cells, implicating KM-819-mediated reconstitution of the α-synuclein degradative pathway. In addition, KM-819 reconstituted autophagic flux in FAF1-transfected SH-SY5Y cells, also suppressing α-synuclein-induced mitochondrial dysfunction. Moreover, oral administration of KM-819 also interfered with α-synuclein accumulation in the midbrain of mice overexpressing FAF1 via an adeno-associated virus system. Consistently, KM-819 reduced α-synuclein accumulation in both the hippocampus and the midbrain of human A53T α-synuclein transgenic mice. Collectively, these data imply that KM-819 may have therapeutic potential for patients with PD.

Neurorestorative Effects of a Novel Fas-Associated Factor 1 Inhibitor in the MPTP Model: An [18F]FE-PE2I Positron Emission Tomography Analysis Study.

Fas-associated factor 1 (FAF1), a Fas-binding protein, is implicated in neuronal cell death in Parkinson's disease (PD). We examined the effects of a novel FAF1 inhibitor, KM-819, in dopaminergic neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model using [18F]FE-PE2I positron emission tomography (PET). The MPTP model was generated with subacute MPTP treatment (20 mg/kg/day, i.p.) for 5 consecutive days in C57bl/6J mice. This study included three groups: the control group (treatment with saline only), the MPTP model group with KM-819 treatment (20 mg/kg/day p.o.) for 6 days, and the MPTP model group without KM-819 treatment. [18F]FE-PE2I PET studies were conducted in the same animals before and after MPTP with or without KM-819 treatment to monitor changes in striatal dopamine transporter activity indicated by non-displaceable binding potential (BPND) of [18F]FE-PE2I, and the expression levels of tyrosine hydroxylase were assessed using immunohistochemistry before and after KM-819 treatment. After MPTP injection, decreased striatal BPND was observed in the MPTP model group compared with the control group. Striatal BPND increased in the MPTP model group with KM-819 treatment, but not in the MPTP model group without KM-819 treatment. The tyrosine hydroxylase expression levels also significantly increased in the MPTP model group with KM-819 treatment compared with the control group. This study indicates that inhibition of the Fas-mediated cell death pathway by KM-819 has neurorestorative effects in striatal dopamine neurons in the MPTP model. Further studies would be needed to investigate the potential of KM-819 as a therapeutic drug for PD treatment.

A first-in-human study to investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of KM-819 (FAS-associated factor 1 inhibitor), a drug for Parkinson's disease, in healthy volunteers.

BACKGROUND: KM-819 is a novel FAS-associated factor 1 (FAF1) inhibitor, and a neuroprotective agent, under clinical development for the treatment of Parkinson's disease as a disease-modifying drug. METHODS: This first-in-human, single and multiple ascending dose study investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of KM-819 in healthy volunteers. Additionally, the effect of age on safety and pharmacokinetics were assessed. The starting dose was determined considering the no observed adverse effect level based on preclinical studies, and the dose escalations in subsequent cohorts were decided based on safety, tolerability, and pharmacokinetic data from previous dose cohorts. RESULTS: After a single dose, the KM-819 plasma exposure showed a less than dose-proportional increase across a dose range of 10-400 mg. After repeated dosing, KM-819 plasma exposure increased in an approximately dose-proportional manner across the evaluated dose range (30-400 mg once daily for 7 days). The mean elimination half-life was 1.8 to 4.8 h with the lower KM-819 doses (≤30 mg), which increased to around 9 h with the higher doses (100-400 mg). When administered to the elderly population, KM-819 plasma exposure increased to 102% after a 200 mg once-daily dosing for 7 days. No clear treatment-related effects on the estimated pharmacodynamic variables were observed. Single or multiple doses of KM-819 were generally well tolerated. CONCLUSION: The data from this study can be used to guide rational drug dosing and choose therapeutic regimens in subsequent clinical studies.