CaMKII (calmodulin-dependent kinase type II) (Ki = 370 nM)

Upon two days of treatment with KN-93 hydrochloride, 95 percent of the cells were in the G1 phase. G1 arrest is reversible, and cells peak into the S and G2-M phases one day after KN-93 hydrochloride is released. KN-93 hydrochloride also inhibits the proliferation of NIH 3T3 fibroblasts in response to basic fibroblast growth factor, platelet-derived growth factor-BB, and epidermal growth factor [1]. While KN-93 hydrochloride strongly dissipates the proton gradient produced in stomach membrane vesicles and decreases cavity volume, it inhibits the action of H+ and K+-ATPase [2]. KN-93 hydrochloride (0.5 μM) inhibits the development of stress elevations in LV during early afterdepolarization and action potential lengthening. Early afterdepolarization is characterized by a rise in Ca2+-independent CaM kinase activity, which is inhibited by KN-93 hydrochloride [3].

KN-93 (5 μg) ameliorates levodopa-induced dyskinesia by lowering the expression of pGluR1S845 in a rat model of Parkinson’s disease. In MRL/lpr Foxp3-GFP mice, KN-93 results in a significant induction of Treg cells in the spleen, peripheral lymph nodes and peripheral blood, and decreases skin and kidney damage.

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KN-93 (1 mg/kg/day, ip) decreases phosphorylation of CaMKII and NF-κB in diabetic retina and inhibits retinal vascular leakage caused by diabetes[4].

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The multifunctional Ca++/calmodulin-dependent protein kinase II (CaM kinase) mediates Ca++-induced augmentation of L-type Ca++ current (ICa); therefore it may act as a proarrhythmic signaling molecule during early afterdepolarizations (EADs) due to ICa. To investigate the hypothesis that ICa-dependent EADs are favored by CaM kinase activation EADs were induced with clofilium in isolated rabbit hearts. All EADs were rapidly terminated with ICa antagonists. Hearts were pretreated with the CaM kinase inhibitor KN-93 or the inactive analog KN-92 (0.5 microM) for 10 min before clofilium exposure. EADs were significantly suppressed by KN-93 (EADs present in 4/10 hearts) compared to KN-92 (EADs present in 10/11 hearts) (P =.024). There were no significant differences in parameters favoring EADs such as monophasic action potential duration or heart rate in KN-93- or KN-92-treated hearts. CaM kinase activity in situ increased 37% in hearts with EADs compared to hearts without EADs (P =.015). This increase in CaM kinase activity was prevented by pretreatment with KN-93. In vitro, KN-93 potently inhibited rabbit myocardial CaM kinase activity (calculated Ki 100 microM). The actions of KN-93 and KN-92 on ICa and other repolarizing K+ currents did not explain preferential EAD suppression by KN-93. These data show a novel association between CaM kinase activation and EADs and are consistent with the hypothesis that the ICa and CaM kinase activation both contribute to EADs in this model. [3]

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Curcumin and KN-93 inhibit retinal vascular leakage induced by diabetes [4]
Evans blue was used in retinal flat mounts to evaluate the effect of curcumin on retinal blood vessel leakage. In control retinas, Evans blue fluorescence was located within blood vessels (Fig. 2A). In STZ-treated rats, focal leakage of the dye from capillaries and larger vessels was noted (Fig. 2B, arrows), in agreement with other reports. This leakage was not seen in STZ-treated rats which were administered curcumin (Fig. 2C) or KN93 (Fig. 2D). Evans blue in the retina was measured to assess BRB permeability (Fig. 2E). Evans blue levels were elevated in the retinas of STZ-treated diabetic rats (2.89±0.47 μg Evans blue/g wet wt retina) as compared to control animals (0.82±0.11 μg Evans blue/g wet wt retina). In agreement with the reduced vascular leakage indicating by whole mount imaging, this elevation was significantly reduced in STZ-treated rats administered curcumin (1.24±0.21 μg) or KN93 (1.37±0.35 μg).

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Curcumin and KN-93 reduce VEGF, iNOS and ICAM-1expession in the diabetic retina [4]
Vscular leakage and adhesion of leukocytes to retinal vessels is mediated by pro-inflammatory cytokines. Therefore, the effect of curcumin on the expression levels of VEGF, iNOS and ICAM-1 was measured. In comparison to non-diabetic controls, mRNA (Fig. 3A) and protein (Fig. 3B, C) measures were significantly elevated in the retina of STZ-treated diabetic rats. These increases were significantly reduced by administration of curcumin or KN93.

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Curcumin and KN-93 suppress phosphorylation of CaMKII and NF-κB in diabetic retina [4]
Phosphorylation of the p65 subunit of NF-κB plays an important role in regulating the expression of many genes, including those that encode pro-inflammatory cytokines and adhesion molecules. In addition, phosphorylation of CaMKII is a critical factor in the development of retinal vascular damage in diabetic mice. To evaluate the role of curcumin in the regulation of the phosphorylation of CaMKII and NF-κB p65, we examined retinas by Western blot. As shown in Figure 5, levels of phosphorylated CaMKII (Thr286) and NF-kB p65 (Ser536) were significantly elevated in retinas of STZ-treated diabetic rats as compared to controls. This elevation was normalized in STZ-treated diabetic rats that were administered curcumin (100 mg/kg/day) or KN93 (1 mg/kg/day).

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Both 2 μg and 5 μg KN-93 treatment lowered AIMs scores in levodopa priming PD rats without affecting the antiparkinsonian effect of levodopa. In agreement with behavioral analysis, KN-93 treatment (2 μg) reduced pGluR1S845 levels in PD rats. Moreover, KN-93 treatment (2 μg) reduced the expression of Gad1 and Nur77 in PD rats. Conclusion: These data indicated that intrastriatal injections of KN-93 were beneficial in reducing the expression of LID by lowering the expression of pGluR1S845 via suppressing the activation of CaMKII in PD rats. Decreased expression of pGluR1S845 further reduced the expression of Gad1 and Nur77 in PD rats [5].

For primary culture studies, rat retinal Müller cells were obtained and identified as described previously. Briefly, Sprague-Dawley rats at postnatal (PN) day 5 to PN7 were sacrificed and the enucleated eyes were washed under sterile conditions, and the anterior portions were discarded. The retinas were isolated, chopped into 1×1 mm fragments, treated with 0.1% trypsin at 37°C for 20 min, and then passed through mesh to remove any large retinal pieces. The strained isolates were centrifuged at 800 rpm for 5 min, and the supernatant fluid was removed. The precipitated cells were resuspended and seeded onto plastic culture flasks containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mmol/L glutamine, 0.1% penicillin/streptomycin and 10% fetal calf serum. The cultures were maintained in 5% CO2 at 37°C. The medium was routinely replaced every 3-4 d. Müller cells were identified by their expression of glutamine synthetase (GS) and vimentin, as judged by immunocytochemical staining. Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). All experiments were conducted using 80%-85% confluent cells. Before each experiment, the plated cells were incubated with serum-free DMEM medium for 1 h. After this, the medium was replaced with serum-free DMEM and the cells were treated with normal D-glucose (5.5 mmol/L) or high glucose (HG; 30 mmol/l glucose) in the presence or absence of 10 μmol/L KN-93, 100 μmol/L PDTC (pyrrolidine dithiocarbamate, a NF-kB inhibitor), or curcumin at the indicated concentrations. [4]

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Evaluation of Cell Viability: Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Briefly, Müller cells were seeded at a density of 10×104 cells per well in 96-well plates and cultured until sub-confluence. Next, cells were treated with curcumin for 24 h before incubation with MTT (5 mg/mL) at 37°C in 5% CO2 atmosphere for 4 h. The culture medium was then removed, and the formazan formed in the reaction was dissolved in 150 μL DMSO (dimethyl sulphoxide). The optical density of the solution was measured at 490 nm using a multifunctional microplate reader. Cell viability in each well was presented as a percentage of the control (vehicle-treated group).

Rat model of diabetic retinopathy and drug treatment [4]
Male Sprague-Dawley rats (8 weeks of age) weighing 180-200 g were used in this study. Rats were housed in ventilated microisolator cages with free access to water and food. The rats were randomly assigned to receive either 60 mg/kg STZ intraperitoneally or citrate buffer alone. Rats were categorized as diabetic when blood glucose levels exceeded 16.7 mmol/L at 48 h after STZ treatment. Two weeks after the induction of diabetes, rats were divided randomly into three subgroups: STZ-diabetic rats (n=12), STZ-treated diabetic rats administered curcumin (n=12), or STZ-diabetic rats administered KN-93 (n=12) for a 12-week period. Curcumin was suspended in saline containing 0.5% carboxymethylcellulose at a concentration of 20 mg/ml and administered via oral gavage at a total dose of 100 mg/kg/day. KN-93 was administered by intraperitoneal injection at 1 mg/kg/day. Control STZ-treated diabetic rats and non-diabetic controls (n=12) were gavage administered saline containing 0.5% carboxymethylcellulose on a daily basis. Body weights and blood glucose levels were measured every 2 weeks. At the completion of the administration protocol, animals were deeply anesthetized with pentobarbital and subsequently sacrificed. The eyes were then enucleated for investigation.

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Experimental design [5]
As shown in Figure 1, 6-hydroxydopamine (OHDA) injections were used to produce a rat model of PD. After 3 weeks of injections, rats showing stable apomorphine-induced rotations (>7 turns/minutes) were selected as valid PD rats. Forty valid PD rats were treated with levodopa plus benserazide twice daily for 21 days. Abnormal involuntary movements (AIMs) scores were measured on days 1, 7, 14, and 21 of treatment. In addition, one group of PD rats (PD group, n = 10) and another group of sham-operated rats (sham group, n = 10) were treated with vehicle twice daily for 21 days. On day 22, 40 levodopa-treated PD rats were randomly divided into four groups: levodopa + KN-93 (1 μg) group, levodopa + KN-93 (2 μg) group, levodopa + KN-93 (5 μg) group, and levodopa + vehicle group. These rats were intrastriatally treated with different doses of KN-93 (1 μg, 2 μg, or 5 μg) or vehicle before levodopa treatment, respectively. As a control, the sham group and PD group rats received intrastriatal administration of vehicle before vehicle treatment. Apomorphine-induced rotations were measured to observe the antiparkinsonian effect of KN-93 on day 1 and day 22. AIMs scores were measured to observe the antidyskinetic effect of KN-93 in rats on days of 1, 7, 14, 21, and 22. By the end of the experiment, rats were sacrificed by deep anesthesia using 3% phenobarbital. Western blot was used to determine levels of GluR1 and pGluR1S845 in rats. Real-time polymerase chain reaction (PCR) was used to measure levels of Gad1 and Nurr77 in rats.

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Treatment [5]
Forty PD rats were treated with levodopa (25 mg/kg plus benserazide 12.5 mg/kg, subcutaneously), twice daily for 21 days. On day 22, 40 PD rats were randomly divided into four groups (n = 10) and intrastriatally administrated with KN-93 (1 μg, 2 μg, or 5 μg) or same volume of vehicle before levodopa administration. As controls, 10 sham-lesioned rats and 10 PD rats were treated with vehicle twice daily for 21 days. On day 22, these vehicle-treated rats were intrastriatally administrated with vehicle before vehicle treatment.

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Dissolved in 4 μL of 0.9% physiological saline containing 0.02% ascorbic acid; 5 μg; Intrastriatal administration

Sprague Dawley female rats

[1]. G1 cell cycle arrest and apoptosis are induced in NIH 3T3 cells by KN-93, an inhibitor of CaMK (the multifunctional Ca2+/CaM kinase). Cell Growth Differ. 1995 Sep;6(9):1063-70.

[2]. Inhibition of acid secretion in gastric parietal cells by the Ca2+/calmodulin-dependent protein kinase II inhibitorKN-93. Biochem Biophys Res Commun. 1993 Sep 15;195(2):608-15.

[3]. KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart. J Pharmacol Exp Ther. 1998 Dec;287(3):996-1006.

[4]. Curcumin Attenuates Retinal Vascular Leakage by Inhibiting Calcium/Calmodulin-Dependent Protein Kinase II Activity in Streptozotocin-Induced Diabetes. Cell Physiol Biochem. 2016;39(3):1196-208.

[5]. Intrastriatal injections of KN-93 ameliorates levodopa-induced dyskinesia in a rat model of Parkinson's disease. Neuropsychiatr Dis Treat. 2013:9:1213-20.

KN-93 is a sulfonamide resulting from the formal condensation of p-methoxybenzenesulfonic acid with the anilino nitrogen of 2-(aminomethyl)-N-(2-hydroxyethyl)aniline in which the hydrogens of the primary amino group have been replaced by methyl and p-chlorocinnamyl groups. KN-93 is a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II. It has a role as an EC 2.7.11.17 (Ca(2+)/calmodulin-dependent protein kinase) inhibitor and a geroprotector. It is a sulfonamide, a tertiary amino compound, a primary alcohol, a member of monochlorobenzenes and a monomethoxybenzene.

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Background: Levodopa remains the most effective drug for the treatment of Parkinson's disease (PD). However, long-term levodopa treatment is associated with the emergence of levodopa-induced dyskinesia (LID), which has hampered its use for PD treatment. The mechanisms of LID are only partially understood. A previous study showed that KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, could be used to ameliorate LID in rats. However, the precise mechanisms by which KN-93 acts as an antidyskinetic are not fully understood.
Methods: In the present study, a rat model of PD was induced by 6-hydroxydopamine (OHDA) injections. Then, the successfully lesioned rats were intrastriatally administered with a different dose of KN-93 (1 μg, 2 μg, or 5 μg) prior to levodopa treatment. Abnormal involuntary movements (AIMs) scores and apomorphine-induced rotations were measured in PD rats. Phosphorylated levels of GluR1 at Serine-845 (pGluR1S845) levels were determined by western blot. Arc and Penk levels were measured by real-time polymerase chain reaction (PCR).
Results: We found that both 2 μg and 5 μg KN-93 treatment lowered AIMs scores in levodopa priming PD rats without affecting the antiparkinsonian effect of levodopa. In agreement with behavioral analysis, KN-93 treatment (2 μg) reduced pGluR1S845 levels in PD rats. Moreover, KN-93 treatment (2 μg) reduced the expression of Gad1 and Nur77 in PD rats.
Conclusion: These data indicated that intrastriatal injections of KN-93 were beneficial in reducing the expression of LID by lowering the expression of pGluR1S845 via suppressing the activation of CaMKII in PD rats. Decreased expression of pGluR1S845 further reduced the expression of Gad1 and Nur77 in PD rats.[5]
Taken together, we found that intrastriatal administration of KN-93 (2 μg or 5 μg) reduced the expression of LID in levodopa primed PD rats. In addition, KN-93 treatment reduced pGluR1S845 levels and the expression of Gad1 and Nur77. We assume that KN-93 can ameliorate LID expression by reducing the expression of Gad1 and Nur77 which subsequently lowers the levels of pGluR1S845 in PD rats.[5]

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ackground: Curcumin possesses many pharmacological properties including anti-inflammatory effects. Although prior studies indicate that curcumin has beneficial effects for diabetic retinopathy, the mechanism of action is not known. To address this issue, we investigated the effect of curcumin against diabetes-induced retinal vascular damage and its mechanism of action by using cultured retinal Müller cells stimulated with high glucose.
Methods: We studied the effects of curcumin in vivo in the retinas of rats rendered diabetic by streptozotocin and in vitro in Müller cells stimulated with high glucose. We administered curcumin, or KN93, an inhibitor of calcium/calmodulin dependent protein kinase II (CaMKII), or saline vehicle to experimental animals on a daily basis for 12 weeks. Primary cultures of rat Müller cells were incubated with normal glucose or high glucose, with or without curcumin, KN93, or pyrrolidine dithiocarbamate (PDTC), an inhibitor of the transcription protein nuclear factor κB (NF-κB). We examined mRNA and protein levels of vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) by real-time RT-PCR and Western blotting, respectively. Retinal levels of CaMKII and NF-κB were examined by Western blotting. Vascular leakage was evaluated using Evans blue.
Results: Curcumin and KN93 significantly inhibited the activation of CaMKII/NF-κB signaling induced by diabetes or elevated glucose, and subsequently decreased the expression of VEGF, iNOS and ICAM-1. These changes were associated with a decrease of diabetes-induced retinal vascular leakage.
Conclusion: Curcumin protects the diabetic rat retina against early retinal vascular damage, by inhibition of CaMKII activity. Curcumin is currently used to treat a number of clinical conditions, and may prove beneficial for the management of diabetic retinopathy.[4]

C26H30CL2N2O4S

537.5

536.13

C, 58.10; H, 5.63; Cl, 13.19; N, 5.21; O, 11.91; S, 5.96

1956426-56-4

KN-93 hydrochloride;1956426-56-4;KN-93 phosphate;1913269-12-1; 1188890-41-6 (phosphate); 139298-40-1

73425340

White to off-white solid powder

2

6

11

35

713

0

CN(C/C=C/C1=CC=C(C=C1)Cl)CC2=CC=CC=C2N(CCO)S(=O)(=O)C3=CC=C(C=C3)OC.Cl

ATHMCQDBXQEIOK-IPZCTEOASA-N

InChI=1S/C26H29ClN2O4S.ClH/c1-28(17-5-6-21-9-11-23(27)12-10-21)20-22-7-3-4-8-26(22)29(18-19-30)34(31,32)25-15-13-24(33-2)14-16-25;/h3-16,30H,17-20H2,1-2H3;1H/b6-5+

N-[2-[N-(4-Chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.65 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.65 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.65 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)