H₁ Receptor
SASH1 (SAM and SH3 domain containing 1) - The drug was identified through connectivity mapping to induce SASH1 mRNA expression (p = 0.000005, z-score 2.431). In vitro validation confirmed that Chloropyramine treatment increases SASH1 protein levels in breast cancer cell lines. [1]
H1 receptor - Chloropyramine is a first-generation reversible H1-receptor antagonist, clinically approved for managing allergic conditions. [1]

Chloropyramine (25, 50 μM; 24 h, 48 h) causes SASH1 expression and apoptosis in breast cancer cell lines[1].

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Induction of SASH1 Protein Expression: Treatment of breast cancer cell lines with 25 or 50 μM Chloropyramine for 24 hours resulted in a significant increase in SASH1 protein levels, as determined by immunoblotting. This effect was observed in seven out of eight cell lines tested (T47-D, BT-549, MDA-MB-231, Hs578T, SUM1315, MCF7, MDA-MB-361), with MDA-MB-468 being the exception. [1]
Inhibition of Cell Growth: Chloropyramine treatment (0-100 μM) for 96 hours induced a dose-dependent reduction in cell confluence in 7 out of 8 breast cancer cell lines (T47-D, BT-549, MDA-MB-231, Hs578T, SUM1315, MCF7, MDA-MB-361), as monitored by live-cell imaging. The Caspase-3 deficient MCF7 cells showed no response to SASH1 overexpression but did exhibit reduced growth upon Chloropyramine treatment. [1]
Induction of Apoptosis: In the three most sensitive cell lines (T47-D, MDA-MB-231, and BT-549), treatment with Chloropyramine led to an increase in Annexin V-positive cells measured by flow cytometry 48 hours post-treatment, indicating the induction of apoptosis. [1]
SASH1-Dependent Cell Death Mechanism: To determine if the cytotoxic effect was dependent on SASH1, T47-D, MDA-MB-231, and BT-549 cells were transfected with SASH1-targeting siRNA prior to Chloropyramine treatment. Depletion of SASH1 partially rescued the cells from Chloropyramine-induced cell death, suggesting that the drug's effect is at least in part mediated through SASH1. [1]
Lack of Effect in Apoptosis-Incompetent Cells: While ectopic SASH1 overexpression did not induce cell death in Caspase-3 deficient MCF7 cells, Chloropyramine treatment did reduce their growth, suggesting potential additional SASH1-independent mechanisms of action. [1]

Chloropyramine (i.p.; 10 mg/kg) abolishes hypothermia in rats[2].

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Cell Line: Breast cancer cell lines
Concentration: 25 or 50 μM
Incubation Time: 24 h
Result: Increased SASH1 expression in breast cancer cell lines.

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Cell Culture and Drug Treatment: Breast cancer cell lines (MDA-MB-231, MDA-MB-361, T47-D, BT-549, MCF7, MDA-MB-468, Hs578T, SUM1315) were cultured under standard conditions (37°C, 5% CO2) in appropriate media supplemented with FCS and, for some lines, insulin and EGF. For drug treatment, cells were seeded and allowed to adhere for 24 hours. Chloropyramine was then added at indicated concentrations (0-100 μM). [1]
Immunoblotting for SASH1 Expression: Cells treated with 25 or 50 μM Chloropyramine for 24 hours were lysed in a buffer containing Hepes, KCl, glycerol, MgCl2, EDTA, NP-40, and protease/phosphatase inhibitors. Lysates were cleared by centrifugation, and protein concentration was determined by Bradford assay. 50 μg of protein was resolved on 4-12% gradient gels, transferred to nitrocellulose membranes, and blocked. Membranes were incubated with anti-SASH1 primary antibody overnight at 4°C, followed by secondary antibodies. Bands were visualized using an infrared scanner, and fluorescence intensity was quantified relative to β-actin using Image J software. [1]
Cell Confluence Assay: Cells were seeded at 2,500 cells per well in 96-well plates and allowed to adhere for 24 hours. Chloropyramine was then added, and cells were imaged every 2 hours for 96 hours using an IncuCyte ZOOM live-cell imager to calculate confluence relative to untreated controls. [1]
Apoptosis Assay (Annexin V/PI): Adherent and floating cells were harvested 48 hours post-Chloropyramine treatment, washed in PBS, and stained according to the Annexin V-FITC apoptosis detection kit protocol. Annexin V-positive (apoptotic) cells were detected using a flow cytometer and quantified with Flow Jo software. [1]
siRNA Knockdown Experiment: Cells were transfected with SASH1-targeting esiRNAs or non-specific control oligos using RNAiMax, according to the manufacturer's instructions. Double transfections were performed 24 hours apart. 24 hours after the initial transfection, Chloropyramine was added. Cell confluence was monitored for 96 hours post-treatment using the IncuCyte ZOOM system. SASH1 depletion was confirmed by immunoblotting 72 hours after the initial transfection. [1]
Overexpression Study: Full-length SASH1 cDNA cloned into pCMV6-GFP vector, or pCMV6-GFP alone, was transfected into cells using Lipofectamine 2000. Cells were harvested 24-48 hours post-transfection for analysis. Cell death was assessed by staining with Hoechst 33342 and propidium iodide (PI) and imaged using an IN Cell Analyzer 2200. The proportion of GFP-positive, PI-positive cells was quantified. [1]

Rats
10 mg/kg
Intraperitoneal

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[1]. SASH1 mediates sensitivity of breast cancer cells to chloropyramine and is associated with prognosis in breast cancer. Oncotarget. 2016 Nov 8;7(45):72807-72818.

N'-[(4-chlorophenyl)methyl]-N,N-dimethyl-N'-(2-pyridyl)ethane-1,2-diamine is an aminopyridine drug. Clopidogrel is a first-generation antihistamine used in Eastern European countries to treat bronchial asthma, allergic rhinitis, allergic conjunctivitis, and other allergic reactions. It is also indicated for the treatment of angioedema, insect bite allergies, food and drug allergies, and anaphylactic shock. Indications: Used to treat allergic conjunctivitis, allergic rhinitis, bronchial asthma, and other atopic (allergic) diseases. Mechanism of Action: Clopidogrel binds to histamine H1 receptors. This blocks the effects of endogenous histamine, thereby temporarily relieving the negative symptoms caused by histamine. Pharmacodynamics: Clopidogrel is a competitive, reversible H1 receptor antagonist. It inhibits vasodilation, increased vascular permeability, and tissue edema associated with histamine release. In addition, clopidogrel also has certain anticholinergic properties. These effects, combined with its ability to cross the blood-brain barrier, can lead to side effects such as drowsiness, weakness, dizziness, fatigue, dry mouth, and constipation. In rare cases, visual impairment and increased intraocular pressure may also occur.

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Chloropyramine is a first-generation antihistamine (H1-receptor antagonist) approved in several European countries for managing allergic conditions like conjunctivitis and bronchial asthma. It is sedating due to its ability to cross the blood-brain barrier. [1]
This study identified Chloropyramine through an in silico connectivity screen (using the cmap database) as a drug that could induce SASH1 expression, a proposed tumor suppressor often downregulated in cancers. The drug's potential to increase SASH1 levels was validated in breast cancer cells, linking its anti-tumor activity, at least in part, to this mechanism. [1]
Previous studies by other groups have shown that Chloropyramine has anti-tumor activity in melanoma, neuroblastoma, breast, and pancreatic cancers, involving inhibition of FAK and VEGFR3 signaling. This study connects its activity to the FAK-regulating protein SASH1. [1]
The study suggests that further preclinical and clinical investigation of Chloropyramine for breast cancer therapy is warranted, potentially as a repurposed drug. Given its tolerability and ability to cross the blood-brain barrier, it may have applications in molecular oncology. Phase 0 and phase I biomarker-driven trials could help confirm its pharmacodynamic effect on SASH1 induction. [1]

C16H20CLN3

289.8

289.135

C, 66.31; H, 6.96; Cl, 12.23; N, 14.50

59-32-5

Chloropyramine hydrochloride; 6170-42-9

25295

Solid powder

1.158g/cm3

413.5ºC at 760 mmHg

25°C

203.9ºC

1.6

3.303

0

3

6

20

264

0

CN(CCN(C1=CC=CC=N1)CC1C=CC(Cl)=CC=1)C

ICKFFNBDFNZJSX-UHFFFAOYSA-N

InChI=1S/C16H20ClN3/c1-19(2)11-12-20(16-5-3-4-10-18-16)13-14-6-8-15(17)9-7-14/h3-10H,11-13H2,1-2H3

N'-[(4-chlorophenyl)methyl]-N,N-dimethyl-N'-pyridin-2-ylethane-1,2-diamine

Avapena; Synopen; Suprastin; Anaphylline; Chlorneoantergan; Chloropyramine; Chlorpyramine; Halopyramine

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.)