p38α (IC50 = 1 μM); p38β (IC50 = 11 μM)

RWJ-67657 suppresses TNF-α release from human peripheral blood mononuclear cells treated with lipopolysaccharide (LPS) at an IC50 of 3 nM and superantigen staphylococcal enterotoxin B-treated peripheral blood mononuclear cells at an IC50 of 3 nM. 13 nM[2] is the value. In MCF-7 cells, RWJ67657 (10 μM; 24 hours) decreases colony formation [3].

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In 1999, Johnson Pharmaceutical Research Institute reported the synthesis of RWJ-67657 (compound 39, Table 4) which is selective p38α and p38β inhibitor (IC50 = 1 and 11 μM, respectively). RWJ 67657 displayed no activity at p38γ and p38δ, and was reported to exhibit cardio protective and anti-inflammatory activity in addition to being orally active. RWJ-67657 is approximately tenfold more potent than the literature standard of p38 kinase inhibitor SB 203580 (compound 37, Table 4) in all p38-dependent in vitro systems tested [1].

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Monocyte/macrophage production of TNF-alpha is dependent on the mitogen-activated protein kinase p38. RWJ-67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol -2-yl]-3-butyn-1-ol) inhibited the release of TNF-alpha by lipopolysaccharide (a monocyte stimulus)-treated human peripheral blood mononuclear cells with an IC(50) of 3 nM, as well as the release of TNF-alpha from peripheral blood mononuclear cells treated with the superantigen staphylococcal enterotoxin B (a T cell stimulus), with an IC(50) value of 13 nM. This compound was approximately 10-fold more potent than the literature standard p38 kinase inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ-67657 inhibited the enzymatic activity of recombinant p38alpha and beta, but not gamma or delta, in vitro and had no significant activity against a variety of other enzymes. In contrast, SB 203580 significantly inhibited the tyrosine kinases p56 lck and c-src (IC(50) = 5 microM). RWJ 67657 did not inhibit T cell production of interleukin-2 or interferon-gamma and did not inhibit T cell proliferation in response to mitogens. [2]

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In MCF-7 cells, this effect could be blocked by addition of a potent p38 inhibitor, RWJ-67657 (Fig. 2). Interestingly, RWJ67657 (10 μM) also inhibited vector transfected (VEC + Veh) cells, indicating that MCF-7 cells have basal p38 activity.
To test the biological impact of p38 signaling on estrogen-mediated signaling, MCF-7 cells were treated with vehicle or E2 in combination with vehicle or RWJ-67657 for 24 h. E2-treated cells grew almost 200 colonies per test group over the next 2 wk as compared with less than 115 colonies per group for cells treated with both E2 and RWJ67657 (Fig. 5). Cells treated with only RWJ67657 did decrease colony formation, consistent with our findings that MCF-7 cells have a basal level of p38 signaling. These data indicate p38 activity is required for normal growth in MCF-7 cells and inhibition of p38 activity leads to decreased MCF-7 cell proliferation. [3]

Following oral administration, lipopolysaccharide injections in mice (50 mg/kg inhibits 87%) and rats (25 mg/kg inhibits 91%) result in decreased production of TNF-α [2]. Strong anti-inflammatory properties are shown by RWJ-67657 (50 mg/kg; oral; once daily for 7 days). After a diabetic stroke, angiogenesis and neurogenesis are jointly promoted by endothelial progenitor cell (EPC) transplantation and RWJ-67657 administration, which both lower inflammation and enhance EPC function [4].

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RWJ-67657 inhibited TNF-alpha production in lipopolysaccharide-injected mice (87% inhibition at 50 mg/kg) and in rats (91% inhibition at 25 mg/kg) after oral administration. Based on these favorable biological properties, RWJ 67657 may have use as a treatment for inflammatory diseases.[2]

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Neither EPC transplantation nor RWJ administration alone significantly improved diabetic stroke outcome although RWJ displayed a potent anti-inflammatory effect. By both improving the functioning of EPCs and reducing inflammation, EPC transplantation plus RWJ administration in vivo synergistically promoted angiogenesis and neurogenesis after diabetic stroke. In addition, the white matter remodeling, behavioral scores, and expressions of vascular endothelial growth factor and brain-derived neurotrophic factor were significantly increased in diabetic mice treated with both EPCs and RWJ-67657. Conclusions: The combination of EPC transplantation and RWJ administration accelerated recovery from diabetic stroke, which might have been caused by increased levels of proangiogenic and neurotrophic factors.[4]

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Improved Functional Recovery of Diabetic Mice After EPC and RWJ-67657 Cotreatment [4]
Diabetic stroke mice cotreated with EPCs and RWJ-67657/RWJ showed significant reductions in neurological functional deficits compared with control mice or mice treated only with EPCs according to the modified neurological severity score (Figure 3A) and foot-fault tests on day 21 (Figure 3B). The infarct areas are shown as hyperintensity on T2-weighted images (Figure 3C). EPC transplantation plus RWJ administration significantly decreased the ischemia-induced loss of the ipsilesional territory on day 7, achieving an additive effect (Figure 3D).

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Increased Angiogenesis and Neurogenesis of Diabetic Mice After EPC and RWJ-67657/RWJ Cotreatment [4]
The cotreatment of diabetic mice with EPCs and RWJ after stroke significantly increased the microvessel density (Figure 4A and B) in the peri-infarct area on day 7. Furthermore, the combination of RWJ and EPCs significantly increased the number of cells that were positive for doublecortin, a marker of immature neurons, in the subventricular zone of the diabetic mice on day 7 (Figure 4C and 4D). There were significant interactions between combined treatments with EPC and RWJ for angiogenesis and neurogenesis, meaning that the effects of the combination were synergistic.

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Increased Axonal Remodeling of Diabetic Mice After EPC and RWJ-67657/RWJ Cotreatment [4]
In vivo DTI analysis revealed that the EPC plus RWJ treatment significantly increased fractional anisotropy (FA) in the ipsilesional internal capsule (IC) on day 14 compared with the other 3 groups (Figure 5A). The fiber counts in the ICs of the mice treated with both EPCs and RWJ were significantly increased (Figure 5B). myelin basic protein+ fiber coherence significantly increased within the ipsilesional IC in the mice treated with both EPCs and RWJ on day 14 (Figure 5C and 5D). Similar additive effects with infarct volume reduction were observed in the white matter remodeling of diabetic mice treated with the combination of EPCs and RWJ.

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Altered Expression of Cytokines in Diabetic Mice After EPC and RWJ-67657/RWJ Cotreatment [4]
Western blotting showed that the phosphorylation of p38 MAPK and the expression of proinflammatory cytokines (interleukin-1β and tumor necrosis factor-α) were significantly reduced in the mice treated with RWJ alone or with RWJ plus EPCs on day 7 (Figure 6A–6C). Moreover, the VEGF expression level was increased in the mice treated with both EPC transplantation and RWJ administration, which had an additive effect (Figure 6D). There was no effect of the 2 monotherapies on brain-derived neurotrophic factor expression. However, a significant interaction was observed in the combination group resulting in brain-derived neurotrophic factor higher expression than the expected additive effect of the 2 monotherapies.

In Vitro Kinase Assay [3]
Roughly 3–5 μg of eluted purified GST fusion protein or 200 ng of purified MAPK activated protein kinase-2 (MAPKAPK-2) was then incubated for 30 min at 30 C with shaking with 0.06 U of activated p38α in the presence of Magnesium/ATP cocktail containing [γ-32P] according to the manufacturer’s protocol. Reactions were stopped by the addition of 20 μl 2× sodium dodecyl sulfate (SDS) sample buffer containing 0.1 M phenylmethylsulfonyl fluoride, protease inhibitor cocktail, phosphatase inhibitor cocktail, and β-mercaptoethanol and boiling samples for 5 min. Samples were analyzed by 4–12% SDS-PAGE, stained with coomassie blue to monitor expression, and subjected to autoradiography.

Cell Proliferation Assay[3]
Cell Types: MCF-7 Breast Cancer Cells
Tested Concentrations: 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: diminished colony formation.

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Colony Assay [3]
MCF-7 cells were plated in six-well plates at 1000 cells per well in DMEM with 5% DCC-FBS. The next day, the cells were pretreated with DMSO or RWJ-67657 at 10 μM followed by either DMSO or E2 (1 nM). The cells were then monitored microscopically for colony growth. The cells were fixed by adding glutaraldehyde (2.5% final concentration) directly to the well. Fifteen to 30 min later, the plates were washed and stained with a 0.4% solution of crystal violet in 20% methanol for 15–30 min. The crystal violet solution was removed and the plates washed and dried. Colonies with a cell confluence of 50 or greater were counted.

Animal/Disease Models: db/db mice with EPCs (male, 8 weeks old) [4]
Doses: 50 mg/kg
Route of Administration: po (po (oral gavage)) one time/day for 7 days
Experimental Results: Diabetic mice after EPCs transplantation combined treatment Increased angiogenesis and neurogenesis.

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Ischemic Stroke Models [4]
Photothrombotic ischemic stroke was induced in db/db mice (male, 8 weeks old;).14 EPCs (1×106) were injected into the mice via the ipsilateral internal carotid artery at 24 hours after surgery. RWJ (50 mg/kg per day) was intragastrically administered once a day for 7 days, with the first dose given 30 minutes before ischemic stroke induction.

[1]. Research advances in kinase enzymes and inhibitors for cardiovascular disease treatment. Future Sci OA. 2017 Aug 8;3(4):FSO204.

[2]. RWJ 67657, a potent, orally active inhibitor of p38 mitogen-activated protein kinase. J Pharmacol Exp Ther. 1999 Nov;291(2):680-7.

[3]. p38 mitogen-activated protein kinase stimulates estrogen-mediated transcription and proliferation through the phosphorylation and potentiation of the p160 coactivator glucocorticoid receptor-interacting protein 1. Mol Endocrinol. 2006 Ma.

[4]. Synergistic Effects of Transplanted Endothelial Progenitor Cells and RWJ 67657 in Diabetic Ischemic Stroke Models. Stroke. 2015 Jul;46(7):1938-46.

The targeting of protein kinases has great future potential for the design of new drugs against cardiovascular diseases (CVDs). Enormous efforts have been made toward achieving this aim. Unfortunately, kinase inhibitors designed to treat CVDs have suffered from numerous limitations such as poor selectivity, bad permeability and toxicity. So, where are we now in terms of discovering effective kinase targeting drugs to treat CVDs? Various drug design techniques have been approached for this purpose since the discovery of the inhibitory activity of Staurosporine against protein kinase C in 1986. This review aims to provide context for the status of several emerging classes of direct kinase modulators to treat CVDs and discuss challenges that are preventing scientists from finding new kinase drugs to treat heart disease.[1]

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Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by activated monocytes/macrophages and T lymphocytes, has been implicated in several disease states, including rheumatoid arthritis, inflammatory bowel disease, septic shock, and osteoporosis. Monocyte/macrophage production of TNF-alpha is dependent on the mitogen-activated protein kinase p38. RWJ-67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol -2-yl]-3-butyn-1-ol) inhibited the release of TNF-alpha by lipopolysaccharide (a monocyte stimulus)-treated human peripheral blood mononuclear cells with an IC(50) of 3 nM, as well as the release of TNF-alpha from peripheral blood mononuclear cells treated with the superantigen staphylococcal enterotoxin B (a T cell stimulus), with an IC(50) value of 13 nM. This compound was approximately 10-fold more potent than the literature standard p38 kinase inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ 67657 inhibited the enzymatic activity of recombinant p38alpha and beta, but not gamma or delta, in vitro and had no significant activity against a variety of other enzymes. In contrast, SB 203580 significantly inhibited the tyrosine kinases p56 lck and c-src (IC(50) = 5 microM). RWJ 67657 did not inhibit T cell production of interleukin-2 or interferon-gamma and did not inhibit T cell proliferation in response to mitogens. RWJ 67657 inhibited TNF-alpha production in lipopolysaccharide-injected mice (87% inhibition at 50 mg/kg) and in rats (91% inhibition at 25 mg/kg) after oral administration. Based on these favorable biological properties, RWJ 67657 may have use as a treatment for inflammatory diseases. [2]

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Nuclear hormone receptors, such as the estrogen receptors (ERs), are regulated by specific kinase signaling pathways. Here, we demonstrate that the p38 MAPK stimulates both ERalpha- and ERbeta-mediated transcription in MCF-7 breast carcinoma, Ishikawa endometrial adenocarcinoma, and human embryonic kidney 293 cells. Inhibition of this potentiation using the p38 inhibitor, RWJ-67657, blocked estrogen-mediated transcription and proliferation. Activated ERs promote gene expression in part through the recruitment of the p160 class of coactivators. Because no direct p38 phosphorylation sites have been determined on either ERalpha or beta, we hypothesized that p38 could target the p160 class of coactivators. We show for the first time using pharmacological and molecular techniques that the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) is phosphorylated and potentiated by the p38 MAPK signaling cascade in vitro and in vivo. S736 was identified as a necessary site for p38 induction of GRIP1 transcriptional activation. The C terminus of GRIP1 was also demonstrated to contain a p38-responsive region. Taken together, these results indicate that p38 stimulates ER-mediated transcription by targeting the GRIP1 coactivator. [3]

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Background and purpose: An immature vascular phenotype in diabetes mellitus may cause more severe vascular damage and poorer functional outcomes after stroke, and it would be feasible to repair damaged functional vessels using endothelial progenitor cell (EPC) transplantation. However, high glucose induces p38 mitogen-activated protein kinase activation, which can accelerate the senescence and apoptosis of EPCs. The aim of this study was to investigate the combined effects of EPC transplantation and p38 mitogen-activated protein kinase inhibitor administration on diabetic stroke outcomes.
Methods: Bone marrow-derived EPCs were injected intra-arterially into db/db mice after ischemic stroke induction. RWJ-67657 (RWJ), a p38 mitogen-activated protein kinase inhibitor, was administered orally for 7 consecutive days, with the first dose given 30 minutes before stroke induction. Functional outcome was determined at days 0, 1, 7, 14, and 21. Angiogenesis, neurogenesis, infarct volume, and Western blotting assays were performed on day 7, and white matter remodeling was determined on day 14.
Results: Neither EPC transplantation nor RWJ-67657/RWJ administration alone significantly improved diabetic stroke outcome although RWJ displayed a potent anti-inflammatory effect. By both improving the functioning of EPCs and reducing inflammation, EPC transplantation plus RWJ administration in vivo synergistically promoted angiogenesis and neurogenesis after diabetic stroke. In addition, the white matter remodeling, behavioral scores, and expressions of vascular endothelial growth factor and brain-derived neurotrophic factor were significantly increased in diabetic mice treated with both EPCs and RWJ.
Conclusions: The combination of EPC transplantation and RWJ administration accelerated recovery from diabetic stroke, which might have been caused by increased levels of proangiogenic and neurotrophic factors.[4]

C27H24FN3O

425.49736

425.19

C, 76.21; H, 5.69; F, 4.46; N, 9.88; O, 3.76

215303-72-3

3008319

white solid powder

1.1±0.1 g/cm3

611.8±65.0 °C at 760 mmHg

124℃

323.8±34.3 °C

0.0±1.8 mmHg at 25°C

1.599

5.18

1

4

8

32

611

0

C1=CC=C(C=C1)CCCN2C(=NC(=C2C3=CC=NC=C3)C4=CC=C(C=C4)F)C#CCCO

QSUSKMBNZQHHPA-UHFFFAOYSA-N

InChI=1S/C27H24FN3O/c28-24-13-11-22(12-14-24)26-27(23-15-17-29-18-16-23)31(25(30-26)10-4-5-20-32)19-6-9-21-7-2-1-3-8-21/h1-3,7-8,11-18,32H,5-6,9,19-20H2

4-(4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(pyridin-4-yl)-1H-imidazol-2-yl)but-3-yn-1-ol

RWJ67657 RWJ-67657 RWJ 67657

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)

DMSO : ~125 mg/mL (~293.77 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.89 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.

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Solubility in Formulation 2: 2.08 mg/mL (4.89 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.89 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 20.8 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.)