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BB-Cl-Amidine is a peptidylarginine deminase (PAD) inhibitor with potential for Canine and Feline Mammary Cancer via Activation of the Endoplasmic Reticulum Stress Pathway. BB-Cl-Amidine is a modified version of Cl-amidine that retains the functional components but possesses a C-terminal benzimidazole group designed to limit proteolysis of the C-terminal amide. BB-CLA reduced viability and tumorigenicity of canine and feline mammary cancer cell lines in vitro. BB-CLA activates the endoplasmic reticulum stress pathway in these cells by downregulating 78 kDa Glucose-regulated Protein (GRP78), a potential target in breast cancer for molecular therapy, and upregulating the downstream target gene DNA Damage Inducible Transcript 3 (DDIT3).
| ln Vitro |
BB-Cl-amidine inhibits NET formation by mouse bone marrow neutrophils. Pretreatment of neutrophils with 12.5 µM BB-Cl-amidine significantly reduced NET formation induced by PMA. [1]
BB-Cl-amidine did not inhibit the production of hydrogen peroxide (H2O2) by mouse neutrophils stimulated with PMA, indicating that it does not block the NADPH oxidase pathway. [1] In a cell viability assay using human osteosarcoma (U2OS) cells, BB-Cl-amidine had an EC50 of 8.8 ± 0.6 µM. This cellular potency was over 20-fold higher than that of the related inhibitor Cl-amidine (EC50 > 200 µM). [1] BB-Cl-amidine induced apoptosis in freshly isolated mouse splenocytes in vitro. [1] |
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| ln Vivo |
In MRL/lpr animals, therapy with BB-Cl-amidine somewhat decreased splenomegaly, but treatment with PAD inhibitors tended to raise the amount of anti-NET antibodies in circulation. Nevertheless, body weight and total IgG levels were unaffected by any of the PAD inhibitors. Cl-amidine and BB-Cl-amidine therapy actually greatly enhanced endothelium-dependent vasodilation. Additionally, there was a clear tendency of IRG downregulation in the BB-Cl-amidine group. Cl-amidine or BB-Cl-amidine therapy can considerably lessen oral hair loss and, in many situations, stop it altogether [1].
In lupus-prone MRL/lpr mice, daily subcutaneous treatment with BB-Cl-amidine (1 mg/kg/day) from 8 to 14 weeks of age significantly reduced ex vivo spontaneous and PMA-stimulated NET formation by bone marrow neutrophils compared to vehicle-treated mice. [1] Treatment with BB-Cl-amidine significantly improved endothelium-dependent vasorelaxation (a measure of vascular function) in aortic rings isolated from MRL/lpr mice. [1] BB-Cl-amidine treatment downregulated the expression of several type I interferon-regulated genes (e.g., Mx1, Ifit1, Ifi44) in the bone marrow of MRL/lpr mice and reduced Mx1 protein expression in the kidneys. [1] Treatment with BB-Cl-amidine decreased immune complex deposition (IgG and C3) in the glomeruli of MRL/lpr mice, reduced interstitial inflammation, and significantly lowered the urine albumin-to-creatinine ratio (proteinuria). [1] BB-Cl-amidine treatment significantly improved skin disease in MRL/lpr mice, preventing or reducing muzzle alopecia (fur loss). This improvement correlated with a reduction in dermal NETs (MPO-DNA complexes) and Mx1-positive cells in the skin. [1] Treatment with BB-Cl-amidine subtly reduced spleen weight (splenomegaly) in MRL/lpr mice. [1] BB-Cl-amidine treatment did not significantly alter the number of circulating neutrophils, lymphocytes, platelets, hematocrit, total IgG levels, or body weight in MRL/lpr mice. There was a trend towards increased circulating anti-dsDNA and anti-CRAMP (a NET component) autoantibody levels with treatment. [1] |
| Enzyme Assay |
The potency and selectivity of BB-Cl-amidine for PAD enzymes (PADs 1-4) were evaluated. The inhibitory potency is expressed as kinact/KI, which is the best measure for an irreversible inhibitor. The kinact/KI values for BB-Cl-amidine against PADs 1-4 are presented graphically in the manuscript and are similar to those of Cl-amidine. [1]
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| Cell Assay |
A cell growth inhibition assay was performed to evaluate the cellular potency of BB-Cl-amidine. Human osteosarcoma (U2OS) cells were treated with various concentrations of the inhibitor for 72 hours. Cell viability was then measured using the XTT assay, a colorimetric method based on the reduction of a tetrazolium salt by metabolically active cells. The EC50 value was calculated from the dose-response curve. [1]
An apoptosis assay was performed on freshly isolated mouse splenocytes. Cells were treated with BB-Cl-amidine, and apoptosis induction was assessed. The specific method of apoptosis detection (e.g., Annexin V/PI staining) is not detailed in the main text. [1] |
| Animal Protocol |
Animal/Disease Models: MRL/lpr mouse[1].
Doses: 1 mg/kg. Route of Administration: Administer subcutaneously (sc) (sc) daily from 8 to 14 weeks of age. Experimental Results: Endothelium-dependent vasodilation was Dramatically improved and demonstrated a strong trend of IRG downregulation. For in vivo efficacy studies in the MRL/lpr lupus model, BB-Cl-amidine was dissolved in a vehicle consisting of 25% DMSO in PBS. [1] Lupus-prone MRL/lpr mice (starting at 8 weeks of age) were treated daily with BB-Cl-amidine at a dose of 1 mg/kg via subcutaneous injection. Treatment continued for 6 weeks until the mice were euthanized at 14 weeks of age. Vehicle-treated mice received the 25% DMSO/PBS solution. [1] For pharmacokinetic studies, C57BL/6 mice were injected with BB-Cl-amidine at a dose of 1 mg/kg. Plasma was collected at various time points for analysis. [1] |
| ADME/Pharmacokinetics |
In C57BL/6 mice, the plasma half-life of BB-Cl-amidine (approximately 1.75 hours) was significantly longer than that of Cl-amidine (approximately 15 minutes) following a single injection. [1]
In mouse liver microsomal stability assays, the stability of BB-Cl-amidine was similar to that of Cl-amidine, indicating that both are comparable in their resistance to metabolic degradation by liver enzymes. [1] Compared to Cl-amidine, BB-Cl-amidine exhibited enhanced cellular activity, attributed to its higher cellular uptake, which is likely due to its higher hydrophobicity. [1] |
| Toxicity/Toxicokinetics |
In vitro experiments showed that BB-Cl-amidine could induce apoptosis in freshly isolated spleen cells. [1] In vivo experiments showed that after 6 weeks of treatment with BB-Cl-amidine in MRL/lpr mice, there were no significant changes in circulating blood cell counts (neutrophils, lymphocytes, platelets), hematocrit, or body weight. The number of splenic dendritic cells or T cells also did not change significantly. [1]
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| References | |
| Additional Infomation |
BB-Cl-amidine is a novel second-generation PAD inhibitor and a C-terminal bioisostere of Cl-amidine. Its structure introduces a benzimidazole group at the C-terminus to prevent proteolysis and a biphenyl group at the N-terminus to increase hydrophobicity and enhance cellular uptake. [1] Its main mechanism of action is to inhibit PAD4-mediated histone citrullination, which is essential for NET formation. By inhibiting NET, BB-Cl-amidine reduces the exposure of immunostimulatory molecules such as DNA and antimicrobial peptides, thereby reducing the production of type I interferon and alleviating subsequent inflammation and tissue damage. [1] This study shows that in the MRL/lpr lupus model, inhibition of PAD by BB-Cl-amidine has a protective effect and improves vascular, renal and skin disease. This contrasts with the exacerbation of lupus observed by NOX2 gene knockout inhibition of NET formation in the same model, highlighting the criticality of the NET inhibition strategy. [1] This study suggests that PAD inhibition may be safer than broadly disrupting NADPH oxidase activity by targeting downstream steps of NETosis (citrullination) while preserving upstream neutrophil functions (such as ROS production). [1]
|
| Molecular Formula |
C₂₆H₂₆CLN₅O
|
|---|---|
| Molecular Weight |
459.97
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| Exact Mass |
459.182
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| Elemental Analysis |
C, 67.89; H, 5.70; Cl, 7.71; N, 15.23; O, 3.48
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| CAS # |
1802637-39-3
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| Related CAS # |
BB-Cl-Amidine hydrochloride;2436747-41-8
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| PubChem CID |
129021946
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| Appearance |
Solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.655
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| LogP |
3.86
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
9
|
| Heavy Atom Count |
33
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| Complexity |
645
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| Defined Atom Stereocenter Count |
1
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| SMILES |
ClC/C(/N)=N\CCC[C@@H](C1=NC2C=CC=CC=2N1)NC(C1C=CC(C2C=CC=CC=2)=CC=1)=O
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| InChi Key |
YDOAWJHYHGBQFI-QHCPKHFHSA-N
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| InChi Code |
InChI=1S/C26H26ClN5O/c27-17-24(28)29-16-6-11-23(25-30-21-9-4-5-10-22(21)31-25)32-26(33)20-14-12-19(13-15-20)18-7-2-1-3-8-18/h1-5,7-10,12-15,23H,6,11,16-17H2,(H2,28,29)(H,30,31)(H,32,33)/t23-/m0/s1
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| Chemical Name |
N-[(1S)-1-(1H-benzimidazol-2-yl)-4-[(2-chloro-1-iminoethyl)amino]butyl]-[1,1'-biphenyl]-4-carboxamide
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| Synonyms |
BB-Cl-Amidine BB-CLA
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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 |
| 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 : ~125 mg/mL (~271.76 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1741 mL | 10.8703 mL | 21.7405 mL | |
| 5 mM | 0.4348 mL | 2.1741 mL | 4.3481 mL | |
| 10 mM | 0.2174 mL | 1.0870 mL | 2.1741 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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