LSD1 (histone demethylase)/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 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/

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/

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/

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).

[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 under investigation in clinical trial NCT03867253 (Testing the Safety and Preliminary Efficacy of the New Drug ORY-2001 in Mild to Moderate Alzheimer's Disease).

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Mechanism of Action
Vafidemstat is a small molecule irreversible inhibitor of histone demethylase LSD1 and also a dual inhibitor of LSD1/MAO-B.

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.)