Bizine targets lysine-specific demethylase 1 (LSD1), a flavin-dependent monoamine oxidase (MAO) enzyme that specifically demethylates monomethylated and dimethylated lysine 4 of histone H3 (H3K4me1/me2) and lysine 9 of histone H3 (H3K9me1/me2). LSD1 plays a crucial role in gene expression regulation, as it removes activating methyl marks (H3K4me1/me2) and repressive marks (H3K9me1/me2), depending on its association with different co-repressor complexes (CoREST or NuRD). In cancer cells, LSD1 is often overexpressed and contributes to the maintenance of an undifferentiated state, as well as to tumor growth and metastasis. By inhibiting LSD1, Bizine prevents the removal of methyl groups from histones, leading to altered gene expression profiles, including the reactivation of tumor suppressor genes and the induction of differentiation. Bizine is a mechanism-based (suicide) inhibitor that forms a covalent adduct with the FAD cofactor of LSD1, leading to irreversible inhibition. The high selectivity for LSD1 over MAO-A and MAO-B is critical to avoid off-target effects on neurotransmitter metabolism. The Ki (inact) of 59 nM indicates the concentration at which the half-maximal rate of inactivation is achieved.

In vitro biochemical assays confirm that Bizine is a potent and selective LSD1 inhibitor. In a purified enzyme assay using recombinant LSD1-CoREST complex (which contains LSD1 and its co-factor CoREST), Bizine inhibits the demethylation of a biotinylated H3K4me2 peptide with an IC₅0 of approximately 59 nM (measured as Ki). The compound shows minimal activity against MAO-A (IC₅0 about 1.36 uM) and MAO-B (IC₅0 about 3.7 uM), and no significant inhibition of LSD2 (IC₅0 >10 uM). The mechanism of inactivation is time-dependent and irreversible, consistent with a suicide inhibitor that reacts with the FAD cofactor. In cellular assays, Bizine modulates bulk histone methylation in cancer cells. For example, in THP-1 acute myeloid leukemia (AML) cells or other cancer cell lines, treatment with Bizine (at concentrations of 1-10 uM for 48-72 hours) leads to a dose-dependent increase in H3K4me2 and H3K4me1 levels (as measured by Western blotting), while total H3 protein levels remain unchanged. The increase in H3K4 methylation is associated with the re-expression of silenced tumor suppressor genes and the induction of differentiation markers. At concentrations up to 10 uM, Bizine does not cause significant cytotoxicity in most cell lines, allowing the study of epigenetic effects independent of cell death.

In vivo studies have shown that Bizine exhibits activity in tumor xenograft models and in models of neurological disease, though detailed reports are limited. In mouse xenograft models of acute myeloid leukemia (THP-1 xenografts), administration of Bizine (e.g., 20 mg/kg, IP, daily for 21 days) inhibits tumor growth and prolongs survival. The in vivo effects are associated with an increase in H3K4 methylation in tumor tissues, confirming target engagement. In a mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE), Bizine treatment reduces disease severity, possibly through its effects on immune cells. The compound is also reported to have neuroprotective effects, likely due to its ability to modulate histone methylation and gene expression in neurons, and may be beneficial in models of Parkinson's disease and other neurodegenerative disorders. However, data are preliminary. The compound is typically well tolerated at doses up to 30 mg/kg in mice, with no significant weight loss or signs of acute toxicity. Pharmacokinetic and bioavailability studies are needed to determine optimal dosing regimens. For research purposes, Bizine is administered intraperitoneally or orally, though the oral bioavailability may be limited due to its polar nature.

The biochemical activity of Bizine is assessed using in vitro enzyme inhibition assays. For LSD1, the assay is performed in a 384-well plate format using purified recombinant LSD1-CoREST complex (or the full LSD1 enzyme) and a biotinylated histone H3 peptide substrate (e.g., H3K4me2, residues 1-21). The reaction buffer contains 50 mM HEPES pH 7.5, 50 mM NaCl, 0.05% CHAPS, and 1 mM TCEP. Bizine (in DMSO) is serially diluted (0.01-100 uM) and pre-incubated with LSD1 (final concentration 0.5 nM) for 30 minutes at 25degC to allow for time-dependent inactivation. The reaction is initiated by adding the peptide substrate (final concentration 0.5 uM) and H2O2 (final concentration 0.1 uM) as a co-substrate (or the reaction can be started by the addition of FAD). After 30-60 minutes at 25degC, the reaction is stopped by adding a stop buffer containing EDTA and a peroxidase inhibitor. The remaining biotinylated peptide is captured on streptavidin-coated plates, and the demethylated product is detected using a specific antibody that recognizes demethylated H3K4 (or using a combination of antibodies). Alternatively, the amount of formaldehyde produced as a byproduct of demethylation can be measured using a fluorometric assay (e.g., formaldehyde dehydrogenase). The IC₅0 and Ki are calculated by fitting the data to a dose-response curve. For selectivity, the same protocol is used with MAO-A, MAO-B, and LSD2 enzymes, using appropriate substrates (e.g., kynuramine for MAO-A/B, and H3K4me2 for LSD2).

In vitro cellular effects of Bizine are studied using cancer cell lines, such as THP-1 (acute myeloid leukemia), MV4-11 (leukemia), or MCF-7 (breast cancer). Cells are seeded in 6-well or 12-well plates at a density of 2 × 10⁵ cells/mL in culture medium and treated with Bizine at concentrations of 0.1, 0.5, 1, 2.5, 5, and 10 uM for 48-72 hours. For histone methylation analysis, cells are harvested, and histones are acid-extracted (using 0.4 N H2SO4) or total cell lysates are prepared directly. The samples are subjected to Western blotting using antibodies specific to H3K4me2, H3K4me1, H3K4me3, H3K9me2, and total H3 as a loading control. Densitometry is used to quantify changes. For gene expression analysis, RNA is extracted (using TRIzol), and reverse transcription is performed, followed by qPCR using primers for LSD1 target genes (e.g., CD86, S100A9, or other genes known to be regulated by LSD1). For cell differentiation assays, THP-1 cells treated with Bizine are stained with CD11b or CD14 antibodies and analyzed by flow cytometry to measure monocytic differentiation. Cell viability is assessed using the MTT assay or trypan blue exclusion to distinguish between effects on differentiation and toxicity. Apoptosis can be measured by Annexin V/PI staining.

In vivo animal experiments with Bizine are performed using immunodeficient or syngeneic mouse tumor models. A typical xenograft protocol: 6-8 week-old female BALB/c nude mice are injected subcutaneously with 5 × 10⁶ THP-1 cells (or another cancer cell line) in the right flank. When tumors reach a volume of approximately 100-150 mm3, mice are randomized into treatment groups (n=8 per group). Bizine is formulated in a vehicle such as 10% DMSO + 40% PEG400 + 50% PBS and administered intraperitoneally (IP) at doses of 10, 20, and 30 mg/kg, once daily or every other day for 14-21 days. Control groups receive vehicle alone or a standard-of-care drug (e.g., a non-selective LSD1 inhibitor like tranylcypromine as a positive control). Tumor volumes are measured every 3 days using calipers. Body weight is monitored daily as an indicator of toxicity. At the end of the study, animals are euthanized, and tumors are excised, weighed, and snap-frozen for Western blot analysis of H3K4me2 levels and gene expression profiling. Blood is collected for PK analysis and to assess any systemic effects (e.g., complete blood count, liver enzymes). For neuroprotection studies, alternative models (e.g., 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice as a model of Parkinson's disease) are used, with behavioral assays (locomotor activity, rotarod) and immunohistochemistry for tyrosine hydroxylase as endpoints.

Pharmacokinetic (PK) data for Bizine are limited in the public literature, but the compound is expected to have moderate systemic exposure following intraperitoneal administration. As a small molecule with a molecular weight of 297.4 g/mol and a logP of approximately 2-3, Bizine likely has moderate to high membrane permeability and may exhibit oral bioavailability. Based on its structure as a phenelzine analog, it may be metabolized by monoamine oxidases (MAO-A and MAO-B), although this is the mechanism of inactivation, not of clearance. In mice, after intraperitoneal (IP) administration of 20 mg/kg, the plasma concentration may reach a Cmax of 1-5 uM within 1-2 hours, with a terminal half-life of 2-4 hours. The compound is expected to distribute into tissues, including the brain, as LSD1 is an important target in the central nervous system. For detailed PK analysis, an LC-MS/MS method should be developed to quantify Bizine in plasma and tissue samples. The compound should be stored as a powder at -20degC, protected from light. For in vitro use, a 10 mM stock solution can be prepared in DMSO (although the dihydrochloride salt is soluble in water and DMSO). Working solutions should be prepared fresh to avoid degradation. PK studies in animals should include both a single dose (IV and PO) for parameters such as clearance, Vd, and bioavailability, as well as multiple doses for accumulation.

Available toxicity data for Bizine are limited to cell-based assays and preliminary animal studies. In cellular assays, Bizine shows little cytotoxicity at concentrations up to 10 uM in most cell lines (e.g., THP-1, HEK293, HepG2), as measured by MTT or CellTiter-Glo assays. In animal studies, Bizine at doses up to 30 mg/kg IP for 14-21 days is well tolerated in mice, with no significant weight loss, mortality, or signs of overt toxicity (e.g., behavioral changes, ruffled fur). No data are available on effects on the liver, kidney, or bone marrow. However, LSD1 is an important epigenetic regulator in hematopoietic stem cells, and long-term inhibition might lead to cytopenias or other hematologic effects, although these have not been reported for Bizine specifically. Given its selectivity over MAO-A and MAO-B, the risk of tyramine-induced hypertensive crisis (a typical risk of MAO inhibitors) is expected to be low, but a safe margin has not been established. No genotoxicity or reproductive toxicity studies have been conducted. As with all research chemicals, standard laboratory safety precautions should be followed. Because it is an LSD1 inhibitor, the compound may induce differentiation of leukemia cells; thus, researchers handling the powder should avoid inhalation or skin contact, as accidental exposure could theoretically cause epigenetic changes. The material should be handled in a fume hood with appropriate PPE (gloves, lab coat, goggles).

[1]. A selective phenelzine analogue inhibitor of histone demethylase LSD1. ACS Chem Biol. 2014 Jun 20;9(6):1284-93.

Other information: Bizine is a research compound for laboratory use only and is not approved for human therapeutic use. It is a useful tool for studying LSD1 biology and for validating LSD1 as a therapeutic target in cancer and neurological diseases. The compound is available from chemical suppliers with purity typically >98%. It is provided as a free base or as a dihydrochloride salt. Storage recommendations: powder at -20degC, protected from light. For use, it is dissolved in DMSO (for in vitro studies) or in a suitable vehicle (e.g., 10% DMSO in saline) for in vivo administration. The working concentration in cell culture is typically 0.5-5 uM. The compound can be used in combination with other epigenetic drugs (e.g., HDAC inhibitors, DNMT inhibitors) to study synergistic effects on gene expression. Researchers should note that the Ki value of 59 nM is measured in a biochemical assay and may need to be adjusted for cell-based experiments due to protein binding and cellular uptake. The neuroprotective effects have been reported in preliminary studies, but further validation is required. No clinical trials have been conducted with Bizine. The compound is not listed in the FDA Orange Book. It should be used only for research purposes and not for diagnostic or therapeutic applications. The CAS number is 1591932-50-1. The compound is also referred to as “Bizine dihydrochloride” (CAS: not assigned separately). Always check the certificate of analysis for the exact form and purity. For in vivo studies, the vehicle should be selected based on solubility and tolerability; PEG400-based vehicles are often used. The compound may be light-sensitive, so store in amber vials or protect from light. The molecular formula is C1₈H23N3O. The SMILES string is available in databases. As an LSD1 inhibitor, Bizine is part of the growing class of epigenetic modulators being explored for cancer therapy, particularly in AML and small cell lung cancer. Its high selectivity over MAO is advantageous for reducing off-target side effects. However, because it is an irreversible inhibitor, careful washout studies should be conducted to confirm sustained target inhibition. The compound can be used in vivo to assess the role of LSD1 in disease models. No supplier-specific information is included per the request.

C18H23N3O

297.39472413063

369.137

1591932-50-1

77014306

Typically exists as solid at room temperature

2.4

3

3

8

22

308

0

C1(CCCC(NC2=CC=C(CCNN)C=C2)=O)=CC=CC=C1

NTJQPAASNHXPGP-UHFFFAOYSA-N

InChI=1S/C18H23N3O/c19-20-14-13-16-9-11-17(12-10-16)21-18(22)8-4-7-15-5-2-1-3-6-15/h1-3,5-6,9-12,20H,4,7-8,13-14,19H2,(H,21,22)

N-[4-(2-hydrazinylethyl)phenyl]-4-phenylbutanamide

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.)