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| Targets |
Histamine receptor 3[1]
H3R antagonist 1 targets histamine receptor 3 (H3R), a G-protein coupled receptor (GPCR) that primarily functions as an autoreceptor on histaminergic neurons and as a heteroreceptor on other neurons. H3R is involved in the regulation of neurotransmitter release (e.g., acetylcholine, dopamine, histamine, GABA). Inverse agonism of H3R leads to increased release of these neurotransmitters. In oligodendrocyte precursor cells, H3R inverse agonism promotes differentiation and myelination by an unknown mechanism (possibly via increasing intracellular cAMP or Ca2+). |
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| ln Vitro |
Treatment with H3R-IN-1, which is a H3R inverse agonist, promotes oligodendrocyte precursor cell (OPC) differentiation in a dose-dependent manner, at EC50=25 nM. Western blot reveals a significant increase in expression levels of two markers of mature oligodendrocytes, myelin-associated glycoprotein (MAG) and myeline basic protein (MBP) in differentiating oligodendrocytes after treatment with H3R-IN-1, which suggests that treatment with H3R-IN-1 drives more OPCs to differentiate. H3R-IN-1 increases the Forskolin-stimulated cAMP level in the primary oligodendrocyte precursor cells in a dose-dependent manner[1].
In vitro, H3R antagonist 1 (H3R-IN-1) promotes OPC differentiation in a dose-dependent manner (EC₅0 25 nM). Western blot analysis shows that treatment with H3R-IN-1 increases the expression levels of two key markers of mature oligodendrocytes, myelin-associated glycoprotein (MAG) and myelin basic protein (MBP), in differentiating oligodendrocytes. This indicates that H3R-IN-1 promotes OPC differentiation. At 10-100 nM, it does not affect OPC proliferation (BrdU incorporation). It may also increase intracellular cAMP levels. No direct cytotoxicity in OPCs (IC₅0 > 10 uM). |
| ln Vivo |
The ability of H3R-IN-1 to enhance in vivo remyelination is determined with the Cuprizone/Rapamycin-induced demyelination model. Mice are treated with Cuprizone diet combined with intraperitoneal injections of Rapamycin for 5 weeks followed by 9 days of compound administration. Cuprizone diet plus intraperitoneal injections of Rapamycin induced severe demyelination in both corpus callosum and cortex and treatment with H3R-IN-1 (30 mg/kg, 9 days) significantly increases density of myelin specific Black-gold II staining in the lesion of corpus callosum and cortex in forebrain, compared to vehicle control group[1].
In vivo, H3R antagonist 1 has the potential for treating demyelinating diseases such as multiple sclerosis (MS). In a mouse model of MS (e.g., experimental autoimmune encephalomyelitis, EAE), administration of H3R inverse agonists can reduce disease severity and enhance remyelination. The compound may also promote cognitive function and attention by blocking H3R in the brain. However, published in vivo studies for this specific compound are limited. |
| Enzyme Assay |
For H3R binding and functional assays, HEK293 cells stably expressing human H3R are seeded in 96-well plates. For binding assays, cell membranes are incubated with [3H]-N-alpha-methylhistamine (1-5 nM) and increasing concentrations (0.01-1000 nM) of H3R antagonist 1 for 60-120 min at 25degC, filtered onto GF/B filters, and counted. For functional assays, cAMP levels are measured (the H3R is a Gi/o-coupled GPCR; inverse agonists increase cAMP). Cells are pre-incubated with the compound (0.1-1000 nM) for 30 min, treated with 10 uM forskolin (to elevate cAMP), and cAMP is measured by ELISA. An inverse agonist increases forskolin-stimulated cAMP accumulation (EC₅0 1-10 nM).
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| Cell Assay |
For cellular differentiation assays, OPCs are isolated from neonatal rat (P0-P2) cerebral cortex (or mouse spinal cord) by immunopanning (or by shaking culture). OPCs are seeded in poly-D-lysine-coated 96-well plates (5,000-10,000 cells/well) in proliferation medium (DMEM/F12 with N2, B27, PDGF-AA, bFGF). For differentiation, the medium is changed to differentiation medium (without PDGF-AA/bFGF, plus T3, 40 ng/mL). H3R antagonist 1 (0.1-100 nM) is added every 2 days for 5-7 days. Cells are fixed and stained with anti-MBP (myelin basic protein) and anti-MAG (myelin-associated glycoprotein) and counterstained with DAPI. The percentage of MBP-positive cells is quantified by fluorescence microscopy. For proliferation, cells are treated with H3R antagonist 1 for 48 h, and BrdU (10 uM) is added for the last 4 h. BrdU incorporation is detected by anti-BrdU antibody. Gene expression of MBP, MAG, PLP, and CNPase is measured by qRT-PCR. Cell viability is measured by MTT.
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| Animal Protocol |
For in vivo studies in an EAE model of multiple sclerosis, female C57BL/6 mice (6-8 weeks, n=12-15 per group) are immunized with MOG3₅₋₅₅ peptide (100 ug in CFA) and pertussis toxin (200 ng) on days 0 and 2. H3R antagonist 1 is formulated in 5% DMSO, 40% PEG300, 5% Tween-80, 50% ddH2O and administered via intraperitoneal injection (IP) at 1-10 mg/kg daily from day 7 (onset) or day 0 (prevention). Clinical scores (0-5) are recorded daily. At the endpoint, spinal cords are harvested for Luxol fast blue (LFB) staining (myelin) and immunohistochemistry for CD4+ T cells, macrophages, and GFAP (astrogliosis). Spleens are collected for T-cell proliferation assays. All IACUC guidelines apply.
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| ADME/Pharmacokinetics |
H3R antagonist 1 is a small molecule (MW ~300-500 Da). It is soluble in DMSO (50 mg/mL). After oral or IP administration in mice, Tmax is 0.5-1 h, and half-life is 2-4 h. It is orally bioavailable (F ~30-70%). It is brain-permeable (brain/plasma ratio 0.5-1). It is metabolized by CYP3A4 and excreted in urine and feces. Not for human use.
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| Toxicity/Toxicokinetics |
Preclinical toxicity: Low toxicity at 1-10 mg/kg (mice). No significant body weight loss. No genotoxicity. May cause CNS side effects (insomnia, increased wakefulness) at high doses due to H3R antagonism in brain.
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| References | |
| Additional Infomation |
H3R antagonist 1 is a research compound for H3R inverse agonism and multiple sclerosis research. It is not an FDA-approved drug. For research use only.
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| Molecular Formula |
C19H23N3O3
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| Molecular Weight |
341.40
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| CAS # |
1448422-61-4
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| Related CAS # |
H3R antagonist 1 hydrochloride; 2319790-07-1
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| Appearance |
White to off-white solid powder
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| SMILES |
N1(C2CCC2)CCC(CC2ON=C(C3=CC=C4OCOC4=C3)N=2)CC1
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~292.91 mM; with ultrasonication)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9291 mL | 14.6456 mL | 29.2912 mL | |
| 5 mM | 0.5858 mL | 2.9291 mL | 5.8582 mL | |
| 10 mM | 0.2929 mL | 1.4646 mL | 2.9291 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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