Inactive analog of KN-93; negative control

LX-2 cell growth is inhibited by KN-93 (5-50 μM; 24 hours), but KN-92 (5-50 μM; 24 hours) is ineffective in preventing cell growth[2]. KN-93, not KN-92, is shown to decrease p53 and p21 expression when cell cycle regulator expression is analyzed[2].

Cell Viability Assay[2]
Cell Types: Human hepatic stellate cells (LX-2)
Tested Concentrations: 5-50 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Ineffective in blocking cell growth.

[1]. Inhibition of the inositol trisphosphate receptor of mouse eggs and A7r5 cells by KN-93 via a mechanism unrelated to Ca2+/calmodulin-dependent protein kinase II antagonism. J Biol Chem. 2002;277(38):35061-35070.

[2]. KN-93, a specific inhibitor of CaMKII inhibits human hepatic stellate cell proliferation in vitro. World J Gastroenterol. 2007;13(9):1445-1448.

KN-93 is a Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibitor that inhibits, in a concentration-dependent and reversible manner, intracellular calcium ([Ca²⁺]ᵢ) signaling mediated by the inositol 1,4,5-triphosphate receptor (IP₃R) in mouse oocytes and permeabilized A7r5 smooth muscle cells. Both cell types primarily express type 1 IP₃R (IP₃R-1). KN-92 is an inactive analogue with no inhibitory effect. The inhibitory effect of KN-93 on Ca²⁺ signaling is independent of its effect on IP₃ metabolism and the abundance of Ca²⁺ stores, suggesting that it acts on IP₃R. The inhibitory effect was independent of CaMKII, as other CaMKII inhibitors (KN-62, peptides 281-309, and autologous calmodulin-associated inhibitory peptides) were ineffective under the same conditions, and CaMKII activation was also blocked in permeabilized cells. Furthermore, KN-93 was most effective in the absence of Ca²⁺. Analysis of Ca²⁺ release in A7r5 cells, IP₃R-1 degradation in oocytes, and [³H]IP₃ binding in Sf9 microsomes at different [IP₃] concentrations all indicated that KN-93 did not affect IP₃ binding. The inhibitory effects of KN-93 and calmodulin (CaM), alone or in combination, on Ca²⁺ release and [³H]IP₃ binding were consistent with the specific interaction between KN-93 and the CaM binding site on IP₃R-1. This is consistent with the weak effect of KN-93 in permeabilized 16HBE14o(-) cells, which mainly express type 3 IP(3)R lacking a high-affinity CaM binding site. These findings suggest that KN-93 directly inhibits IP(3)R-1 and may therefore be a useful tool for studying the regulation of IP(3)R function. [1]

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Objective: To investigate the effects of the CaMKII selective inhibitor KN-93 on the proliferation of human hepatic stellate cells and the expression of p53 or p21 proteins. Methods: Human hepatic stellate cells (LX-2) were incubated with different concentrations (0-50 μmol/L) of KN-93 or its inactive derivative KN-92. Cell proliferation was detected by CCK-8 assay, and the expression of two cell cycle regulators, p53 and p21, was detected by SDS-PAGE and Western blotting. Results: KN-93 (5-50 μmol/L) reduced the proliferation of human hepatic stellate cells in a dose-dependent manner. After 24 hours of treatment, the proliferation rate decreased from 81.76% (81.76% ± 2.58% vs 96.63% ± 2.69%, P < 0.05) to 27.15% (27.15% ± 2.86% vs 96.59% ± 2.44%, P < 0.01). Incubation with 10 μmol/L KN-93 resulted in a time-dependent decrease in cell growth, from 78.27% at 8 hours to 11.48% at 48 hours. However, the inactive derivative of KN-93, KN-92, did not effectively inhibit cell proliferation. Furthermore, cell cycle regulator expression analysis showed that KN-93, but not KN-92, reduced the expression of p53 and p21. Conclusion: KN-93 significantly inhibited the proliferation of LX-2 cells by regulating the expression of two specific cell cycle regulators, p53 and p21. [2]

C24H25CLN2O3S

456.98

456.127

C, 63.08; H, 5.51; Cl, 7.76; N, 6.13; O, 10.50; S, 7.02

176708-42-2

KN-92 phosphate;1135280-28-2;KN-92 hydrochloride;1431698-47-3

5353702

Typically exists as solid at room temperature

1.3±0.1 g/cm3

602.1±65.0 °C at 760 mmHg

317.9±34.3 °C

0.0±1.7 mmHg at 25°C

1.630

5.97

1

5

9

31

650

0

O=S(C1=CC=C(OC)C=C1)(NC2=CC=CC=C2CN(C/C=C/C3=CC=C(Cl)C=C3)C)=O

RUAOVVIUGUOYHA-AATRIKPKSA-N

InChI=1S/C24H25ClN2O3S/c1-27(17-5-6-19-9-11-21(25)12-10-19)18-20-7-3-4-8-24(20)26-31(28,29)23-15-13-22(30-2)14-16-23/h3-16,26H,17-18H2,1-2H3/b6-5+

N-[2-[[[(E)-3-(4-chlorophenyl)prop-2-enyl]-methylamino]methyl]phenyl]-4-methoxybenzenesulfonamide

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)

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.)