CaMK II

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

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Following two days of KN-93 treatment, 95% of cells exhibit G1 arrest. G1 arrest is reversible; a peak of cells had proceeded into S and G2-M one day after KN-93 release. In NIH 3T3 fibroblasts, KN-93 also inhibits the development of cells that are stimulated by basic fibroblast growth factor, platelet-derived growth factor-BB, and epidermal growth factor[1]. The H+, K+-ATPase activity is inhibited by KN-93, but the proton gradient created in the gastric membrane vesicles is strongly dissipated, and the luminal space volume is decreased[2]. Increased left ventricular developed pressure during action potential extension and early afterdepolarizations is prevented by KN-93 (0.5 μM). Early afterdepolarizations cause a rise in Ca2+-independent CaM kinase activity, which KN -93 blocks[3]. KN-93 (10 μM) dramatically suppresses the increased glucose-induced CaMKII/NF-κB signaling, which in turn reduces Müller cell production of VEGF, iNOS, and ICAM-1[4].

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CaMK-II (the type II multifunctional Ca2+/calmodulin kinase) is a ubiquitous serine/threonine protein kinase that is activated by Ca2+ and calmodulin (CaM) and has been implicated in cell cycle control. NIH 3T3 fibroblast cytosolic extracts contain CaMK-II enzymatic activity and two major Ca2+/CaM-dependent phosphoproteins of M(r) 55,000 and 115,000. Reverse transcription-PCR indicates that the gamma B and gamma C isozymes of CaMK-II are predominately expressed. KN-93, a novel membrane-permeant synthetic inhibitor of purified neuronal CaMK-II, inhibits serum-induced fibroblast cell growth in a comparable dose-dependent fashion to its inhibition of CaMK-II activity. After 2 days of KN-93 treatment, 95% of cells are arrested in G1. G1 arrest is reversible; 1 day after KN-93 release, a peak of cells had progressed into S and G2-M. KN-92, a similar but inactive compound, had no effect on CaMK-II activity or cell growth. KN-93 also blocked cell growth stimulated by basic fibroblast growth factor, platelet-derived growth factor-BB, epidermal growth factor, and insulin-like growth factor-1. After 3 days of KN-93-induced G1 arrest, cell size and viability decreased and DNA fragmented, indicating apoptosis. These data suggest that CaMK-II is necessary for cell cycle progression through G1 and operates at a site common to the transduction of signals from growth and/or survival factors [1].

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A novel Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) inhibitor, KN-93 potently inhibits gastric acid secretion from parietal cells. As previously reported (1), treatment of parietal cells with a selective inhibitor of CaM kinase II, KN-62 resulted in the inhibition of cholinergic-stimulated rabbit parietal cell secretion, whereas it failed to inhibit the histamine and forskolin response. In contrast effects of carbachol, histamine and forskolin were significantly inhibited by KN-93 with an IC50 of 0.15, 0.3 and 1 microM, respectively; these effects occurred without any changes in intracellular cyclic AMP and Ca2+ levels. In the present study we investigated the mechanism by which KN-93 acts upon the acid-secreting machinery of gastric parietal cells. Neither redistribution of the proton pump activity nor the morphological transformation were affected by KN-93. The drug only weakly inhibited the H+, K(+)-ATPase activity but strongly dissipated the proton gradient formed in the gastric membrane vesicles and reduced the volume of luminal space. Thus KN-93 acts at pH gradient formation whereas KN-62 acts only at CaM Kinase II [2].

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 formed by the condensation of p-methoxybenzenesulfonic acid with the aniline nitrogen of 2-(aminomethyl)-N-(2-hydroxyethyl)aniline, wherein the hydrogen on the primary amino group is replaced by a methyl group and a p-chlorocinnamyl group. KN-93 is a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II. It functions as an EC 2.7.11.17 (Ca²⁺/calmodulin-dependent protein kinase) inhibitor and an anti-aging agent. It is a sulfonamide, tertiary amine compound, primary alcohol, monochlorobenzene compound, and monomethoxybenzene compound.

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Background: Levodopa remains the most effective drug for treating Parkinson's disease (PD). However, long-term levodopa treatment is associated with the development of levodopa-induced motor dyskinesia (LID), which limits its use in the treatment of PD. The mechanism of LID is not yet fully elucidated. Previous studies have shown that the calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 can improve LID in rats. However, the specific mechanism by which KN-93 exerts its anti-movement disorder effect is not fully understood. Methods: In this study, a rat model of PD was established using 6-hydroxydopamine (OHDA) injection. Subsequently, before levodopa treatment, different doses of KN-93 (1 μg, 2 μg, or 5 μg) were injected into the striatum of successfully induced rats. Abnormal involuntary movements (AIMs) scores and apomorphine-induced rotational behavior were measured in the PD rats. The phosphorylation level of GluR1 serine 845 (pGluR1S845) was determined by Western blotting. The levels of Arc and Penk proteins were determined by real-time polymerase chain reaction (PCR). Results: We found that both 2 μg and 5 μg KN-93 treatments reduced the abnormal involuntary movements (AIMs) scores in levodopa-pretreated Parkinson's disease (PD) rats without affecting the anti-Parkinson's disease effect of levodopa. Consistent with behavioral analysis, 2 μg KN-93 treatment reduced pGluR1S845 levels in PD rats. Furthermore, 2 μg KN-93 treatment also reduced Gad1 and Nur77 expression in PD rats. Conclusion: These data indicate that intrastriatal injection of KN-93 can help reduce levodopa-induced motor dysfunction (LID) in PD rats by inhibiting CaMKII activation and reducing pGluR1S845 expression. Reduced pGluR1S845 expression further reduced Gad1 and Nur77 expression in PD rats. [5]
In summary, we found that intrastriatal injection of KN-93 (2 μg or 5 μg) reduced LID expression in levodopa-pretreated PD rats. Furthermore, KN-93 treatment reduced pGluR1S845 levels and the expression of Gad1 and Nur77. We hypothesize that KN-93 can improve LID expression by reducing Gad1 and Nur77 expression, thereby reducing pGluR1S845 levels in PD rats. [5]

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Background: Curcumin possesses various pharmacological properties, including anti-inflammatory effects. Although previous studies have shown that curcumin is beneficial for diabetic retinopathy, its mechanism of action remains unclear. To address this issue, we investigated the effects of curcumin on diabetic retinal vascular damage and its mechanism of action using high-glucose-stimulated cultured Müller cells.
Methods: We investigated the in vivo effects of curcumin in streptozotocin-induced diabetic rat retinas and its in vitro effects in high-glucose-stimulated Müller cells. We administered curcumin, the calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93, or saline to experimental animals daily for 12 weeks. Primary cultures of rat Müller cells were incubated in normal glucose or high glucose medium with or without curcumin, KN93, or the nuclear factor κB (NF-κB) transcription protein inhibitor pyrrolidine dithiocarbamate (PDTC). We measured the mRNA and protein levels of vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) using real-time quantitative PCR and Western blotting. CaMKII and NF-κB levels in the retina were also detected by Western blotting. Vascular leakage was assessed using Evans blue staining. Results: Curcumin and KN93 significantly inhibited the activation of the CaMKII/NF-κB signaling pathway induced by diabetes or hyperglycemia, thereby reducing the expression of VEGF, iNOS and ICAM-1. These changes were associated with a reduction in diabetes-induced retinal vascular leakage. Conclusion: Curcumin protects the retinas of diabetic rats from early retinal vascular damage by inhibiting CaMKII activity. Curcumin is currently used to treat a variety of clinical diseases and may be beneficial for the treatment of diabetic retinopathy. [4]

C26H32CLN2O8PS

599.032646179199

598.13

C, 52.13; H, 5.38; Cl, 5.92; N, 4.68; O, 21.37; P, 5.17; S, 5.35

1913269-12-1

KN-93;139298-40-1;KN-93 hydrochloride;1956426-56-4

16760530

White to off-white solid powder

4

10

11

39

763

0

OP(O)(O)=O.O=S(C1=CC=C(OC)C=C1)(N(C2=CC=CC=C2CN(C/C=C/C3=CC=C(Cl)C=C3)C)CCO)=O

NNKJTPOXLIILMB-IPZCTEOASA-N

InChI=1S/C26H29ClN2O4S.H3O4P/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;1-5(2,3)4/h3-16,30H,17-20H2,1-2H3;(H3,1,2,3,4)/b6-5+;

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

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)

DMSO: 100 mg/mL (166.94 mM)
H2O: 50 mg/mL (83.47 mM)

Solubility in Formulation 1: ≥ 10 mg/mL (16.69 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 100.0 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: ≥ 10 mg/mL (16.69 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 100.0 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: ≥ 10 mg/mL (16.69 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 100.0 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.)