LSD1 (histone demethylase)/MAO-B
Vafidemstat targets lysine-specific histone demethylase 1 (LSD1, KDM1A) and monoamine oxidase B (MAO-B). LSD1 is a histone demethylase that removes methyl groups from histone H3 lysine 4 (H3K4) and lysine 9 (H3K9), thereby regulating gene expression. MAO-B is a mitochondrial enzyme that degrades monoamines such as dopamine and phenylethylamine. By inhibiting LSD1, Vafidemstat modulates epigenetic regulation of gene expression, potentially restoring normal gene expression patterns in neurodegenerative diseases. By inhibiting MAO-B, it increases dopamine levels in the brain, which may have neuroprotective effects. Vafidemstat has IC50 values of 105 nM for LSD1 and 58 nM for MAO-B.

Vafidemstat (ORY-2001) is a new epigenetic medication used to treat neurodegenerative illnesses. It is a dual LSD1/MAO-B inhibitor. One protein that is involved in transcriptional regulatory complexes, LSD1, whose modulation can be utilized to rectify neuroinflammation and cognitive deficiencies as well as transcriptional abnormalities in neurodegenerative illnesses [1]. Alzheimer's disease can be treated with vefidemstat (ORY-2001) [2].
In vitro, Vafidemstat is a potent inhibitor of LSD1 (IC50 = 105 nM) and MAO-B (IC50 = 58 nM). The compound's dual inhibitory activity has been characterized in enzymatic assays using purified LSD1 and MAO-B. Its selectivity for LSD1 and MAO-B over other histone demethylases and monoamine oxidases has been assessed. Vafidemstat is an irreversible inhibitor of LSD1 and MAO-B.

In this study, researchers investigated the effect of ORY-2001 on immune-mediated and virus-induced encephalomyelitis, two experimental models of multiple sclerosis and neuronal damage. Oral administration of ORY-2001 ameliorated clinical signs, reduced lymphocyte egress and infiltration of immune cells into the spinal cord, and prevented demyelination. Interestingly, ORY-2001 was more effective and/or faster acting than a sphingosine 1-phosphate receptor antagonist in the effector phase of the disease and reduced the inflammatory gene expression signature characteristic ofEAE in the CNS of mice more potently. In addition, ORY-2001 induced gene expression changes concordant with a potential neuroprotective function in the brain and spinal cord and reduced neuronal glutamate excitotoxicity-derived damage in explants. These results pointed to ORY-2001 as a promising CNS epigenetic drug able to target neuroinflammatory and neurodegenerative diseases and provided preclinical support for the subsequent design of early-stage clinical trials. https://pmc.ncbi.nlm.nih.gov/articles/PMC9323733/

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In this study, researchers explored potential therapeutic agents and demonstrate that vafidemstat (ORY-2001), a lysine-specific histone demethylase 1 (LSD1) inhibitor that was effective in other models of neuropsychiatric/neurodevelopmental disorders, ameliorates impairments in sociality but not working memory in adult Kmt2c+/fs mice. Intriguingly, the administration of vafidemstat was shown to alter the vast majority of DEGs in the direction to normalize the transcriptomic abnormalities in the mutant mice (94.3 and 82.5% of the significant upregulated and downregulated DEGs, respectively, P < 2.2 × 10-16, binomial test), which could be the molecular mechanism underlying the behavioral rescuing. In summary, our study expands the repertoire of ASD models with high etiological and face validity, elucidates the cell-type resolved molecular alterations due to Kmt2c haploinsufficiency, and demonstrates the efficacy of an LSD1 inhibitor that might be generalizable to multiple categories of psychiatric disorders along with a better understanding of its presumed mechanisms of action. https://pmc.ncbi.nlm.nih.gov/articles/PMC11420081/
In vivo, Vafidemstat has been evaluated in preclinical models of neurodegenerative diseases, including Alzheimer's disease and multiple sclerosis. Its brain penetration and oral bioavailability make it suitable for CNS applications. Vafidemstat has demonstrated efficacy in experimental models of neurodegeneration and neuroinflammation. Clinical trials have been conducted to evaluate its safety and efficacy in humans.

HDAC2 activity was determined using a fluorimetric assay. Compounds were dissolved in DMSO, and serial dilutions were further diluted in HDAC assay buffer (BPS). These working solutions were incubated in duplicate at room temperature for 3 h in a mixture containing HDAC assay buffer, BSA, and recombinant HDAC2 (BPS). The enzymatic reactions were initiated by the addition of a fluorogenic, acetylated peptide substrate (BPS) and proceeded for 30 min at 37 °C. Then, HDAC assay developer (BPS) was added, and after further incubation at room temperature, fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 microplate reader. Trichostatin A (TSA) was used as a reference inhibitor. https://pmc.ncbi.nlm.nih.gov/articles/PMC9323733/
In vitro enzyme/receptor binding (non-cell) assays for Vafidemstat involve measuring its inhibitory activity against LSD1 and MAO-B using purified enzymes. LSD1 activity is measured using histone H3 peptide substrates and varying concentrations of Vafidemstat. MAO-B activity is measured using a monoamine substrate (e.g., kynuramine or benzylamine) and quantifying the production of the oxidized product. IC50 values are calculated from inhibition curves.

SH-SY5Y cells were treated 24 h after seeding with SAHA, Fingolimod (FTY720), Fingolimod Phosphate (FTY720-P) and ORY- 2001. After 2 and 6 h of treatment, cells were harvested and centrifuged at 250× g for 4 min at room temperature and cell pellets used for total histone extraction with EpiQuik Total Histone Extraction Kit following manufacturer’s instructions. https://pmc.ncbi.nlm.nih.gov/articles/PMC9323733/
In vitro cell-based experiments for Vafidemstat are conducted using neuronal cell lines or primary neurons. Cells are treated with Vafidemstat at varying concentrations, and histone methylation levels (e.g., H3K4me1, H3K4me2) are measured by Western blot to confirm LSD1 inhibition. MAO-B activity is measured in cell lysates. Gene expression changes are assessed by qRT-PCR or RNA-seq.

Vafidemstat preparation and administration [https://pmc.ncbi.nlm.nih.gov/articles/PMC11420081/] In the pharmacological experiment with an LSD1 inhibitor, we utilized vafidemstat (also known as ORY-2001). Vafidemstat was dissolved in dimethyl-sulfoxide (DMSO) to prepare the stock solution (50 mg/mL). The stock solution was stored at −80 °C until use. Vafidemstat was administered via drinking water according to a previous report [22]. Before the experiment, we measured that 5 mL per day of water was taken by a mouse on average. We calculated the final concentration in the drinking water assuming the weight of a mouse to be 35 g. To administer 0.96 mg/kg day of vafidemstat to a mouse via drinking water, the stock solution was diluted by water to 6.72 μg/mL. The drinking water was changed every week. Vafidemstat was administered to 15 male mice per condition for 4 weeks before the behavioral tests.

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Induction and Treatment of Experimental Autoimmune Encephalomyelitis (EAE) Model [https://pmc.ncbi.nlm.nih.gov/articles/PMC9323733/] Treatment consisted of the administration of ORY-2001 (1, 0.5 and 0.05 mg/kg), ORY-LSD1 (0.18, 0.09 and 0.06 mg/kg), FTY720 (1 mg/kg), or rasagiline (3 mg/kg) by oral gavage starting after the onset of the disease (day 12 post-immunization). The dose range used for ORY-2001 and ORY-LSD1 was a function of their relative in vivo potency of KDM1A inhibition of each compound. The dose chosen for in vivo treatment of mice with rasagiline is sufficient for full inhibition of MAO-B and confers full protection toward a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) insult in mice. MPTP is a prodrug which is converted to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) by MAO-B in vivo. The dose chosen for fingolimod (FTY720) was the highest dose used in previously reported efficacy studies and provided full efficacy in an EAE model in mice

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Induction and Treatment of Theiler’s Murine Encephalomyelitis Virus (TMEV) Model[https://pmc.ncbi.nlm.nih.gov/articles/PMC9323733/] Susceptible SJL/J mice (6 weeks old), previously anesthetized with isoflurane, were inoculated in the cerebral parenchyma with 2 × 106 plaque forming units (pfu) of the Daniel’s strain of TMEV, diluted in 30 μL of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS). The injection takes place in the right hemisphere of the cerebral cortex using a Hamilton syringe coupled to a pipette tip in such a way that it allows for an exposure of the needle of about 2–3 mm; thus, the virus is always injected at the same depth. Sham animals are subjected to the same protocol, but they only received 30 µL of DMEM supplemented with 5% fetal bovine serum. Treatment consisted of the administration of ORY-2001 by oral gavage at the dose of 0.3 and 1.0 mg/kg, starting at the onset of the clinical signs (day 72 post-infection), once a day for five consecutive days from day 72 to day 76 and from day 79 to day 83 post-infection. Control mice were treated with vehicle [2% v/v Tween-80 + 98% HPβCD (13% w/v)] following the same administration regime (Figure 4a).
In vivo animal experiments for Vafidemstat are performed in animal models of Alzheimer's disease, multiple sclerosis, and other neurodegenerative disorders. Vafidemstat is administered orally, and cognitive function, motor function, neuroinflammation, and neurodegeneration are assessed. Clinical trials in humans have also been conducted.

Absorption: Vafidemstat is a compound with high oral bioavailability and is rapidly absorbed in the human body after oral administration. Exposure: After oral administration, exposure is approximately dose-proportional. After 5 consecutive days of daily administration, there is evidence of moderate systemic accumulation. Distribution: Vafidemstat is designed to cross the blood-brain barrier. A cerebrospinal fluid (CSF) sub-study confirms this, showing that the ratio of free drug in CSF to plasma indicates its ability to penetrate the central nervous system. Metabolism: Phase I studies characterized its pharmacokinetic profile, but the specific metabolic pathways or enzymes involved are not detailed in the provided abstract. Studies have confirmed that Vafidemstat binds to its target KDM1A in peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Excretion: No information on excretion pathways is provided in the search results.
Vafidemstat is orally bioavailable with good brain penetration. The compound has a molecular weight of approximately 400 Da and suitable lipophilicity for CNS penetration. Detailed pharmacokinetic parameters, including Cmax, Tmax, AUC, and half-life, are available from preclinical and clinical studies.

Overall Tolerability: Vafidemstat consistently demonstrated good safety and tolerability in clinical trials, both in healthy volunteers (Phase I) and patients with mental illness (Phase IIa). Maximum Recommended Starting Dose (MRSD): Based on the No Observed Adverse Effect Level (NOAEL) of 0.2 mg/kg in the most sensitive preclinical animal model (rat), the MRSD for human trials was calculated to be 0.2 mg. Adverse Events (AEs): In the Phase I trial, adverse events were reported in all dose groups, but these were dose-independent and showed no significant difference in frequency compared to the placebo group. No serious adverse events (SAEs) were reported during dose escalation. Laboratory Parameters: Biochemical parameters, urinalysis, vital signs, and electrocardiograms did not change significantly during dose escalation. The only notable finding was a transient decrease in platelet count, observed at additional dose levels specifically used to monitor the targeted effect of KDM1A inhibition. Phase IIa validation: In the REIMAGINE Phase IIa basket trial (1.2 mg/day, for 8 weeks), vafidemstat was again demonstrated to be safe and well-tolerated, with no clinically significant adverse events observed.
Preclinical and clinical toxicology studies have been conducted to evaluate the safety profile of Vafidemstat. The compound is generally well-tolerated at therapeutic doses. Clinical trials have evaluated the safety and tolerability of Vafidemstat in patients with neurodegenerative diseases. The safety profile supports its continued development for CNS indications.

[1]. ORYZON presents data on the Phase I trial with ORY-2001 at the 13th International Conference on Alzheimer’s and Parkinson’s diseases.

[2]. ORYZON Reports Financial Results and Corporate Update for the 1st Half Ended June 30, 2018.

Vafidemstat is being investigated in the clinical trial NCT03867253 (testing the safety and preliminary efficacy of the novel drug ORY-2001 in mild to moderate Alzheimer's disease). Mechanism of Action Vafidemstat is a small-molecule irreversible histone demethylase LSD1 inhibitor and a dual inhibitor of LSD1/MAO-B. Vafidemstat (ORY-2001) is a novel dual inhibitor of LSD1 and MAO-B, representing an epigenetic drug candidate for the treatment of neurodegenerative diseases. It is the company's second compound to enter clinical trials. Preclinical studies have shown that Vafidemstat has significant therapeutic activity, restoring memory and improving other indicators in SAMP-8 mice (an accelerated aging and Alzheimer's disease model) and other disease models. LSD1 is a key component of the transcriptional regulatory complex, and its regulation can be used to correct transcriptional imbalances, reduce neuroinflammation, and alleviate cognitive impairment associated with neurodegenerative diseases. This Phase I study, conducted at the Drug Research Center of San Pau Hospital in Barcelona, aimed to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of Vafidemstat. The planned single-dose and multiple-escalation portions of the study were successfully completed. Results showed that oral Vafidemstat was well tolerated, with no clinically significant changes in laboratory parameters, vital signs, electrocardiograms, physical examination results, or treatment-related adverse events. Since the maximum tolerated dose had not yet been reached, the company, with approval from the Spanish Agency for Medicines and Health Products, decided to add a cohort receiving higher doses and establish a separate cohort to measure drug concentrations in cerebrospinal fluid to confirm blood-brain barrier penetration in humans. These new cohorts are currently underway. Pharmacodynamic assessment of peripheral LSD1 target binding using the company's proprietary detection method showed time- and dose-dependent binding, which was highly consistent with pharmacokinetic data. The Phase I study has generated comprehensive data that can be used to build dose-response models between humans and preclinical animal models, and to support the development of safe dosing regimens for long-term Phase II efficacy studies in patients with neurodegenerative diseases and neuroinflammation. The company plans to submit the corresponding clinical trial application in the second half of 2017. Dr. César Molinero, Chief Medical Officer of Oryzon, commented, “The results are in line with our expectations and are crucial for determining the dosage in our upcoming Phase II trial. Despite species differences, we observed excellent correlation between the first-in-human data and the preclinical data, and we are excited to advance the clinical development of Vafidemstat.” Dr. Tamara Metz, Vice President and Chief Scientific Officer, added, “Neurodegenerative diseases have a significant epigenetic component, and Vafidemstat has shown great potential in a variety of preclinical models. The company’s recently announced capital increase also provides the necessary resources to initiate the Phase II clinical trial and positions the company as a leader in this field.”
Vafidemstat is also known as ORY-2001. It is a dual LSD1/MAO-B inhibitor being developed for the treatment of neurodegenerative diseases. Its dual mechanism of action—modulating epigenetic regulation and increasing dopamine levels—provides a unique approach to neuroprotection and disease modification. The compound is an investigational agent and is not approved for clinical use.

C19H20N4O2

336.387703895569

336.158

C, 67.84; H, 5.99; N, 16.66; O, 9.51

1357362-02-7

66714983

White to off-white solid powder

2.2

2

6

7

25

410

2

O1C(N)=NN=C1CN[C@@H]1C[C@H]1C1C=CC(=CC=1)OCC1C=CC=CC=1

XBBRLCXCBCZIOI-DLBZAZTESA-N

InChI=1S/C19H20N4O2/c20-19-23-22-18(25-19)11-21-17-10-16(17)14-6-8-15(9-7-14)24-12-13-4-2-1-3-5-13/h1-9,16-17,21H,10-12H2,(H2,20,23)/t16-,17+/m0/s1

5-((((1R,2S)-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)methyl)-1,3,4-oxadiazol-2-amine

ORY 2001; ORY-2001; Vafidemstat; 1357362-02-7; ORY-2001; Vafidemstat [INN]; LZ82JLT4UP; UNII-LZ82JLT4UP; 1,3,4-Oxadiazole-2-methanamine, 5-amino-N-((1R,2S)-2-(4-(phenylmethoxy)phenyl)cyclopropyl)-; 5-[[[(1R,2S)-2-(4-phenylmethoxyphenyl)cyclopropyl]amino]methyl]-1,3,4-oxadiazol-2-amine; ORY2001; Vafidemstat

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~148.64 mM)

Solubility in Formulation 1: 5 mg/mL (14.86 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 5 mg/mL (14.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 5 mg/mL (14.86 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)