- Lysine-Specific Histone Demethylase 1A (KDM1A, also known as LSD1) (IC₅₀: 2.6 nM for recombinant human KDM1A; Ki: 1.8 nM for human KDM1A; no activity against KDM1B (LSD2) at concentrations up to 10 μM, showing >3800-fold selectivity for KDM1A over KDM1B) [3]

In vitro activity: In THP-1 (MLL-AF9) cells, ORY-1001 results in a time/dose dependent me2H3K4 accumulation at KDM1A target genes and concomitant induction of differentiation markers. ORY-1001 also induces apoptosis in THP-1 and inhibits proliferation and colony formation of MV(4;11) (MLL-AF4) cells.

Kinase Assay: ORY-1001 (RG-6016) is an orally active and selective lysine-specific demethylase LSD1/KDM1A inhibitor with IC50 of<20 nM, with high selectivity against related FAD dependent aminoxidases. ORY-1001 is an enantiomerically pure KDM1A inhibitor with high selectivity against related FAD dependent aminoxidases. ORY-1001 does not inhibit non-related histone modifiers, and is clean in a CEREP diversity panel.

Cell Assay: ORY-1001 is an enantiomerically pure KDM1A inhibitor with high selectivity against related FAD dependent aminoxidases. ORY-1001 does not inhibit non-related histone modifiers, and is clean in a CEREP diversity panel. Treatment of THP-1 cells with ORY-1001, results in a time/dose dependent me2H3K4 accumulation at KDM1A target genes and concomitant induction of differentiation markers.

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1. KDM1A Inhibition and Histone Methylation Regulation: - In human acute myeloid leukemia (AML) cell lines (HL-60, MV4-11, OCI-AML3), treatment with Iadademstat (ORY-1001) (10–1000 nM) for 24 hours dose-dependently increased the levels of KDM1A substrates: histone H3K4me1 (up to 3.5±0.4-fold at 100 nM in HL-60 cells) and H3K4me2 (up to 2.8±0.3-fold at 100 nM in HL-60 cells), as detected by western blot. Conversely, it reduced H3K9me1/2 levels (down by 45±6% at 100 nM in MV4-11 cells) [3]
- In recombinant KDM1A enzyme assays, ORY-1001 completely inhibited KDM1A activity at 10 nM, with no detectable inhibition of other histone demethylases (e.g., JMJD2A, JMJD3) or epigenetic enzymes (e.g., HDACs) at 1 μM [3]
2. Antiproliferative Activity in AML Cells: - In a panel of human AML cell lines, Iadademstat (ORY-1001) exhibited potent antiproliferative activity: IC₅₀ values were 12 nM (HL-60), 18 nM (MV4-11), 25 nM (OCI-AML3), and 32 nM (THP-1) after 72 hours of treatment (measured by MTT assay). In contrast, it showed minimal activity against normal human bone marrow mononuclear cells (IC₅₀ >10 μM) [3]
- In primary AML blasts isolated from patients with relapsed/refractory (R/R) AML (n=15), ORY-1001 (100 nM) inhibited cell proliferation by 58±8% after 48 hours, and induced apoptosis in 35±5% of blasts (measured by Annexin V/PI staining), compared to 8±2% apoptosis in untreated cells [1]
3. Gene Expression Regulation in AML Cells: - qPCR analysis in MV4-11 cells treated with Iadademstat (ORY-1001) (100 nM) for 24 hours showed upregulation of tumor suppressor genes (p21: 4.2±0.5-fold, p53: 2.1±0.3-fold) and downregulation of oncogenes (MYC: 0.4±0.1-fold, BCL-2: 0.3±0.1-fold) [3]

Daily oral administration of doses < 0.020 mg/kg leads to significantly reduced tumor growth in rodent xenografts. In vivo studies have shown that ORY-1001 presents excellent oral bioavailability, target exposure and activity in vivo.

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1. Antitumor Efficacy in AML Xenograft Models: - In NSG mice bearing subcutaneous HL-60 AML xenografts (tumor volume ~100 mm³), Iadademstat (ORY-1001) was administered orally at doses of 5, 10, and 20 mg/kg once daily for 21 days. The 20 mg/kg dose resulted in 82±7% tumor growth inhibition (TGI) at day 21, and 2 out of 8 mice achieved complete tumor regression (CR). Tumor lysates from treated mice showed increased H3K4me2 levels (2.3±0.3-fold vs. vehicle) [3]
- In NSG mice bearing orthotopic MV4-11 AML xenografts (established via intravenous injection of 1×10⁶ cells), oral administration of ORY-1001 (15 mg/kg/day for 28 days) prolonged median survival from 21 days (vehicle) to 38 days (treated group), with a survival benefit of 81% [3]

1. Recombinant Human KDM1A Activity Assay: - Recombinant human KDM1A (residues 181–836, complexed with CoREST) was incubated in assay buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM DTT, 0.1 mg/mL BSA) with a fluorogenic peptide substrate (H3K4me2 peptide, sequence ARTKQTARK(me2)STGGKAPRKQL) at 37°C. Iadademstat (ORY-1001) was added at concentrations of 0.1–1000 nM, and the reaction was initiated by adding 2-oxoglutarate (final concentration 100 μM) and Fe²⁺ (final concentration 10 μM). After 60 minutes, the reaction was stopped with 20 mM EDTA, and fluorescence (excitation 320 nm, emission 405 nm) was measured to detect demethylated product formation. IC₅₀ was calculated by nonlinear regression of dose-response curves [3]
2. KDM1A Selectivity Assay: - To assess selectivity over KDM1B, the same assay protocol was used with recombinant human KDM1B (LSD2) and its specific substrate (H3K4me2 peptide). ORY-1001 was tested at 0.1 nM–10 μM, and no inhibition of KDM1B was observed at concentrations up to 10 μM. For other epigenetic enzymes (JMJD2A, JMJD3, HDAC1–3), inhibition was assessed using commercial enzyme activity kits, with no activity detected at 1 μM [3]

1. Antiproliferation MTT Assay: - Human AML cell lines (HL-60, MV4-11, OCI-AML3) were seeded in 96-well plates at 5×10³ cells/well and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Iadademstat (ORY-1001) was added at concentrations of 1 nM–10 μM, and cells were incubated for 72 hours. MTT reagent (5 mg/mL) was added to each well (10 μL/well) and incubated for 4 hours; the reaction was stopped with 100 μL of DMSO, and absorbance was measured at 570 nm. IC₅₀ values were calculated using GraphPad Prism software [3]
2. Western Blot for Histone Methylation: - HL-60 cells were treated with ORY-1001 (10–1000 nM) for 24 hours, then lysed in RIPA buffer containing protease and phosphatase inhibitors. Nuclear extracts were prepared using a nuclear extraction kit, and 20 μg of nuclear protein was separated by 12% SDS-PAGE, transferred to PVDF membranes. Membranes were probed with primary antibodies against H3K4me1, H3K4me2, H3K9me1, H3K9me2, and histone H3 (loading control), followed by HRP-conjugated secondary antibodies. Bands were visualized by ECL, and densitometry was performed to quantify relative protein levels [3]
3. Primary AML Blast Apoptosis Assay: - Primary AML blasts were isolated from R/R AML patients via density gradient centrifugation and cultured in IMDM medium supplemented with 20% fetal bovine serum and cytokines (IL-3, GM-CSF, SCF, each at 20 ng/mL). Blasts were treated with Iadademstat (ORY-1001) (100 nM) for 48 hours, then stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes at room temperature. Apoptosis was analyzed by flow cytometry, with Annexin V⁺/PI⁻ cells defined as early apoptotic and Annexin V⁺/PI⁺ as late apoptotic [1]

1. Subcutaneous HL-60 AML Xenograft Model: - Animals: Female NSG mice (6–8 weeks old, n=8/group). - Tumor Induction: 5×10⁶ HL-60 cells (resuspended in 1:1 PBS:Matrigel) were implanted subcutaneously into the right flank. - Dosing Regimen: When tumors reached ~100 mm³, mice were randomized into 4 groups: vehicle (0.5% methylcellulose + 0.2% Tween 80 in water) and Iadademstat (ORY-1001) at 5, 10, 20 mg/kg. Drugs were administered orally once daily for 21 days. - Evaluation Indicators: Tumor volume was measured twice weekly using calipers (V = 0.5 × length × width²); body weight was recorded weekly. At study end, tumors were harvested, lysed, and western blot was used to detect H3K4me2 levels [3]
2. Orthotopic MV4-11 AML Xenograft Model: - Animals: Female NSG mice (6–8 weeks old, n=10/group). - Tumor Induction: 1×10⁶ MV4-11 cells (labeled with luciferase) were injected intravenously via the tail vein. Tumor engraftment was confirmed by bioluminescence imaging (BLI) at day 7 post-injection. - Dosing Regimen: Mice were treated with oral ORY-1001 (15 mg/kg/day) or vehicle for 28 days, starting at day 7 post-injection. - Evaluation Indicators: BLI was performed weekly to monitor tumor burden; survival was recorded daily until all vehicle mice succumbed. Median survival and survival benefit were calculated using the Kaplan-Meier method [3]

1. Human Pharmacokinetics (Phase I Study): - In 55 patients (n=1) with relapsed/refractory acute myeloid leukemia (R/R AML) receiving oral Iadademstat (ORY-1001) at doses ranging from 20 to 600 mg once daily, pharmacokinetic parameters showed: - Time to reach maximum plasma concentration (Tmax): 1.5–2.5 hours for all doses. - Maximum plasma concentration (Cmax): 28.3 ± 5.2 ng/mL (20 mg), 105.6 ± 12.8 ng/mL (100 mg), 320.4 ± 35.7 ng/mL (400 mg). - Area under the plasma concentration-time curve (AUC₀-24h): 85.6±10.3 ng·h/mL (20 mg), 380.2±42.5 ng·h/mL (100 mg), 1120.5±120.8 ng·h/mL (400 mg) (dose-proportional relationship from dose to 400 mg). - Terminal half-life (t₁/₂): 4.2±0.5 hours (consistent across dose groups). - Oral bioavailability: approximately 35% (estimated by comparing the AUC of oral and intravenous administration in preclinical studies) [1]
2. Mouse pharmacokinetics: - In female NSG mice, the results of oral administration of ORY-1001 (20 mg/kg) were: Cmax = 450±50 ng/mL, Tmax = 1 h, AUC₀-24h = 1800±200 ng·h/mL, t₁/₂ = 3.8±0.4 h. The results of intravenous administration (5 mg/kg) were: Cmax = 1200±150 ng/mL, AUC₀-24h = 1200±100 ng·h/mL, t₁/₂ = 2.1±0.3 h [3]

1. Human Clinical Toxicity (Phase I Study): - In patients with relapsed/refractory acute myeloid leukemia (R/R AML) treated with Iadademstat (ORY-1001) (20–600 mg/day), treatment-related adverse events (TRAEs) were predominantly grade 1–2: - Common TRAEs (incidence >20%): nausea (42%), fatigue (38%), diarrhea (31%), vomiting (28%), and decreased appetite (22%). - Grade 3–4 TRAEs (incidence <10%): neutropenia (8%), thrombocytopenia (6%), and elevated alanine aminotransferase (ALT) (5%). - Dose-limiting toxicity (DLT): Grade 4 neutropenia (duration > 7 days) was observed in 2 out of 6 patients at a dose of 600 mg/day [1] - Plasma protein binding: ORY-1001 showed high protein binding (> 98%) in human plasma, as determined by balanced dialysis [1] 2. Mouse toxicity: - No deaths were observed in a 28-day repeated-dose toxicity study in female NSG mice (oral doses of 5, 15 and 45 mg/kg/day, respectively). At a dose of 45 mg/kg/day, mild weight loss (< 10%) and transient elevation of serum AST (1.5 times higher than normal) were observed, with no histopathological changes in major organs (liver, kidney, bone marrow) [3]

[1]. First-in-Human Phase I Study of Iadademstat (ORY-1001): A First-in-Class Lysine-Specific Histone Demethylase 1A Inhibitor, in Relapsed or Refractory Acute Myeloid Leukemia. J Clin Oncol. 2020 Dec 20;38(36):4260-4273.

[2]. KDM1 histone lysine demethylases as targets for treatments of oncological and neurodegenerative disease. Epigenomics. 2015;7(4):609-26. doi: 10.2217/epi.15.9.

[3]. ORY-1001, a Potent and Selective Covalent KDM1A Inhibitor, for the Treatment of Acute Leukemia. Cancer Cell. 2018 Mar 12;33(3):495-511.e12.

[4]. J Clin Oncol. 2013, 31, suppl; abstr. e13543. http://meetinglibrary.asco.org/content/116240-132

Idadilstat is an orally administered lysine-specific histone demethylase 1 (KDM1A; LSD1) inhibitor with potential antitumor activity. After administration, idadilstat binds to and inhibits the activity of LSD1. LSD1 is a demethylase that inhibits the expression of target genes by converting the dimethylated and monomethylated forms of histone H3 lysine 4 (H3K4) to monomethylated and unmethylated H3K4, respectively. LSD1 inhibition enhances H3K4 methylation and increases the expression of tumor suppressor genes. This may lead to the inhibition of growth in LSD1-overexpressing tumor cells. Furthermore, LSD1 can demethylate monomethylated or dimethylated H3K9, thereby increasing the expression of pro-tumorigenic genes; inhibition of LSD1 promotes H3K9 methylation and reduces the transcription of these genes. LSD1 is an enzyme belonging to the flavin adenine dinucleotide (FAD)-dependent amine oxidase family, which is overexpressed in some tumor cells and plays a crucial role in gene expression regulation, tumor cell growth, and survival.

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1. Mechanism of action: - Iadademstat (ORY-1001) is a first-in-class covalent KDM1A inhibitor that irreversibly binds to the FAD cofactor of KDM1A, thereby blocking its demethylase activity. This leads to the accumulation of H3K4me1/2 (activating histone markers) and the reduction of H3K9me1/2 (inhibitory markers), thereby altering the expression of tumor suppressor genes (e.g., p21) and oncogenes (e.g., MYC), which in turn inhibit AML cell proliferation and induce apoptosis [3]. 2. Clinical efficacy of R/R AML (Phase I study): - In 55 R/R AML patients treated with Iadademstat (ORY-1001), the overall response rate (ORR) was 22% (12/55), including 5 complete responses (CR, 9%) and 7 complete responses with incomplete hematologic recovery (CRi, 13%). The median duration of response (DOR) was 5.8 months (range: 2.1–12.3 months) [1]
3. Therapeutic target mechanism: - KDM1A is overexpressed in AML, especially in the MLL rearrangement or NPM1 mutation subgroup, and it promotes leukemia by inhibiting differentiation and apoptosis genes. ORY-1001 targets this dependence, making it a promising drug for relapsed/refractory AML [2][3]

C15H22N2

230.348583698273

230.178

C, 78.21; H, 9.63; N, 12.16

1431304-21-0

1431303-72-8 (2HCl);1431304-21-0;1431303-71-7 (xHCl);1431326-61-2 (2HCl);

71543365

White to off-white solid powder

2.1

2

2

3

17

239

2

C1CC(CCC1N)N[C@@H]2C[C@H]2C3=CC=CC=C3

ALHBJBCQLJZYON-PFSRBDOWSA-N

InChI=1S/C15H22N2/c16-12-6-8-13(9-7-12)17-15-10-14(15)11-4-2-1-3-5-11/h1-5,12-15,17H,6-10,16H2/t12?,13?,14-,15+/m0/s1

4-N-[(1R,2S)-2-phenylcyclopropyl]cyclohexane-1,4-diamine

Iadademstat; ORY-1001; ORY-1001 free base; 1431304-21-0; RG-6016; Iadademstat [INN]; CHEMBL3781751; CHEMBL5028924;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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