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Although opioids are widely used to treat moderate to severe pain, opioid addiction and the opioid overdose epidemic are becoming more serious. Although opioid receptor antagonists/partial agonists, such as naltrexone and buprenorphine, have relatively low selectivity for the μ-opioid receptor (MOP), they have been used for the management of opioid use disorder. The utility of highly selective MOP antagonists remains to be evaluated. Here, we biologically and pharmacologically evaluated a novel nonpeptide ligand, UD-030, as a selective MOP antagonist. UD-030 had more than 100-fold higher binding affinity for the human MOP (Ki = 3.1 nM) than for δ-opioid, κ-opioid, and nociceptin receptors (Ki = 1800, 460, and 1800 nM, respectively) in competitive binding assays. The [35S]-GTPγS binding assay showed that UD-030 acts as a selective MOP full antagonist. The oral administration of UD-030 dose-dependently suppressed the acquisition and expression of morphine-induced conditioned place preference in C57BL/6J mice, and its effects were comparable to naltrexone. These results indicate the UD-030 may be a new candidate for the treatment of opioid use disorder, with characteristics that differ from traditional medications that are in clinical use.

Background: Lysophosphatidic acid (LPA) is known as a fibrotic lipid mediator. Mice lacking LPA1 receptor or treated with a LPA1 antagonist are protected from bleomycin (BLM)-induced pulmonary fibrosis. However, the therapeutic efficacy has not yet been reported. Therefore we evaluated therapeutic effect of LPA antagonism and a role of LPA in the later fibrotic phase of BLM model using a novel LPA1 antagonist, UD-009. Methods: Potency and selectivity of UD-009 were assessed with recombinant LPA receptors and in normal human lung fibroblasts. In a BLM model, animals were administered with BLM i.v. for 5 days from day 0, and treated with UD-009 (30 mg/kg, p.o., b.i.d.) either from day 0 to 28 for prophylactic assay or from day 14 to 28 for therapeutic assay. The differentiation of fibroblasts was assessed by using primary cultured fibroblasts isolated from the lungs of BLM-injured mice by staining with anti-α-SMA antibody. Results: UD-009 is a potent and selective LPA1 antagonist. Prophylactic treatment of UD-009 significantly reduced collagen accumulation in the BLM model. Interestingly, UD-009 showed inhibition predominantly in the later phase of the model. Primary cultured fibroblasts isolated from the lungs of the BLM-injured mice receiving UD-009 decreased the expression of α-SMA compared to the control. Moreover, therapeutic treatment of UD-009 caused almost same effects as those of the prophylactic treatment. Conclusions: UD-009 is a newly identified LPA1 antagonist and shows clear therapeutic efficacy by restraining the fibrotic phase when the fibroblasts are activated and differentiated. LPA may therefore control progression of fibrotic disorders in IPF.

Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for anticancer drugs due to its dual roles, cell cycle regulation and gene transcription/RNA processing. We synthesized UD-017, a small-molecule, highly selective, orally active CDK7 inhibitor with a novel chemotype. In this study, we characterized anticancer effects on blood cancer cells and in vivo anticancer activity in combination with chemo- or immuno-oncology agents. Methods: We first evaluated antiproliferative activities of UD-017 on blood cancer cell lines and investigated the mechanism of activity of UD-017 using NCI-H929 cells (multiple myeloma). Apoptosis was assayed by FACS analysis of Annexin V-positive and PI negative. In vivo anticancer effect was evaluated in the mouse xenograft model with NCI-H929. We further evaluated the anticancer efficacy of UD-017 in combination with chemotherapeutics in an HCT-116 xenograft mouse model and with anti PD-1 antibody in B16F10 mouse allograft model. Results: UD-017 strongly inhibited the proliferation of blood cancer cell lines (myeloma) with IC50s of 10-100 nM range, more broadly than other representative CDK inhibitors. In the similar assay, UD-017 showed no cytotoxicity to human peripheral blood mononuclear cells up to 10 μM. In the mechanism study, phosphorylation of RNA polymerase II c-terminal domain and expression of c-Myc were both inhibited concomitant with the antiproliferative activity in NCI-H929 cells. Significant apoptosis was induced at around 100 nM. In vivo, UD-017 almost completely inhibited the cancer growth at 50 mg/kg, q.d. for 14-day treatment in a NCI-H929 xenograft model in mice. In the combination assay in vivo, UD-017 showed a clear synergistic effect at 50 mg/kg with 5-fluorouracil (5-FU, 15 mg/kg, i.p.) without further affecting the side effects of 5-FU in HCT-116 (colorectal carcinoma) xenografted mice. Also, UD-017 (100 mg/kg) in combination with anti PD-1 antibody (250 μg/body, i.p.) showed add-on anticancer effect in B16F10-allografted mice. All of the mice in the combination group survived during the dosing period without decreasing the number of blood cells. Conclusions: We propose UD-017 as a novel type of anticancer drug that shows complete anticancer responses in xenograft models of myeloma cancer cells, and also shows in vivo synergy in combination with chemotherapy and anti PD-1 antibody. These data support the rationale for further advancing towards clinical development. Citation Format: Yasuhiro Aga, Sayaka Ogi, Kazuhiro Onuma, Hidetoshi Sunamoto, Takashi Matsushita, Ayumi Ogawa, toru Hasegawa, Shigeyuki Kono, Noriaki Iwase, Shigeru Ushiyama. Evaluation of anticancer activities of UD-017, a novel selective and orally available CDK7 inhibitor, in blood cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4837.

Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for anticancer drugs due to its dual roles, cell cycle regulation and gene transcription/RNA processing. We synthesized UD-017, a small-molecule, highly selective, orally active CDK7 inhibitor with a novel chemotype. In this study, we characterized anticancer activities of UD-017 in vitro and in vivo using patient-derived (PD) cancer cells. Methods: We first evaluated an antiproliferative activity of UD-017 broadly in a panel of cancer cell assay including patient-derived 3-dimensional tumor clonogenic assay. To verify the potential of c-Myc expression as a biomarker, we investigated the correlation between c-Myc expression levels and anticancer activity in vitro and in vivo using a colorectal cancer cell. In the main part, anticancer efficacy of UD-017 was investigated in vivo in PD-xenograft (PDX) models using representative PD cancer cells. Results: In antiproliferative panel assays using over 100 cancer cell lines, UD-017 broadly inhibited a wide range of cancer cells from colon, breast, lung, kidney, blood, pancreas, osteosarcoma, sarcoma and urinary bladder cancers. Especially, UD-017 showed high sensitivity in solid tumor such as non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric, osteosarcoma and sarcoma cells with IC50s of 10-200 nM. UD-017 showed a good correlation of c-Myc expression levels with antiproliferative activity using colorectal cancer cells in vitro, and UD-017 reduced the intratumoral MYC mRNA levels by an administration at 100 mg/kg in a HCT-116 xenograft model in mice. In PDX models, UD-017 showed strong inhibition and even regression of tumor growth, and the tumors were almost disappeared on day 14 without body weight loss in non-small cell lung cancer (LXFL1121). In a pleuramesothelioma (PXF541) PDX model, UD-017 also showed regressive effect, and in gastric cancer (GXA3067) and sarcoma (SXFS117) PDX models, UD-017 completely inhibited the tumor growth. Conclusions: We propose UD-017 as a novel type of anticancer drug that shows complete antitumor responses in patient-derived xenograft models of diverse cancer types. c-Myc expression in cancer cells may be a biomarker for the antitumor effect of UD-017. These data support the rationale for further advancing towards clinical development. Citation Format: Takashi Matsushita, Sayaka Ogi, Kazuhiro Onuma, Hidetoshi Sunamoto, Ayumi Ogawa, Toru Hasegawa, Yasunori Tokunaga, Yasuhiro Aga, Shigeru Ushiyama. UD-017, a novel highly selective and orally active CDK7 inhibitor, shows a significant anticancer activity in patient-derived cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4835.

We found that a novel compound, R-130823 {2-(4-fluorophenyl)-4-(1-phenethyl-1,2,3,6-tetrahydropyridin-4-yl)-3-(pyridin-4-yl)-1 H -pyrrole}, had highly selective inhibition against mitogen-activated protein kinase p38α (IC 50 =22 nM). The release of tumor necrosis factor-α, interleukin-1β, -6 and -8 was inhibited in lipopolysaccharide-stimulated human blood pretreated by R-130823, with IC 50 values of 0.089, 0.066, 0.95 and 0.16 μM, respectively. R-130823 reduced the established hind paw swelling in rat adjuvant-induced arthritis, while methotrexate showed no suppression. In the same model, R-130823 ameliorated adjuvant-induced hyperalgesia with rapid onset and long duration comparable to a cyclooxygenase-2 inhibitor, celecoxib. In murine collagen-induced arthritis, R-130823 blocked the progress of arthritis when administered just after the onset of the arthritis. Histological analysis of the knee joints showed that proliferation of fibroblasts and synoviocytes and infiltration of neutrophils were ameliorated. In conclusion, R-130823 is expected to be an efficacious treatment for rheumatoid arthritis by blocking the p38 pathway.

Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for cancer drugs due to its dual roles, cell cycle regulation & gene transcription/RNA procession. Recent studies show that transcription of some oncogene is highly sensitive to inhibition of transcription such as inhibition by CDK7. The dependence of cancer cells on transcription can be therefore advantageous to CDK7 inhibitors as effective anti-cancer drugs. We synthesized a novel small molecule highly selective reversible CDK7 inhibitor, UD-017. In this study, we evaluated anti-cancer activity of UD-017 in the colorectal cancer cells, HCT-116, xenograft models in mice & investigated the rationale for CDK7 inhibition as anti-cancer drugs. Experimental procedures: We first examined the pharmacokinetics profile of UD-017 after an oral administration in mice. We then evaluated the anti-tumor efficacy of UD-017 in HCT-116 xenograft models in mice. We further investigated the pharmacodynamics of UD-017 by detecting the phosphorylation levels of the retinoblastoma (cell cycle) & RNA polymerase II (transcription) & expression levels of c-myc in the tumor tissues in the same model after oral administration of UD-017. Results: UD-017 was identified as an orally available inhibitor of CDK7. The bioavailability was approximately 50% in BALB/c mice. In the HCT-116 xenograft model in mice, UD-017 inhibited the cancer growth by 33%, 64% & 88% at 25, 50 & 100 mg/kg, respectively, with clear dose-dependence by once daily administration for 14 days. Throughout the experiment, UD-017 was well tolerated with no reduction in body weights & no myelosuppression. In order to evaluate the effect of UD-017 in combination with chemotherapeutics, UD-017 was administrated with 5-fluorouracil (5-FU) to HCT-116 xenografted mice with either 50 mg/kg of UD-017 alone, or 15 mg/kg ip of 5-FU alone, or in combination of UD-017 plus 5-FU. UD-017 showed clear combination effect with 5-FU without further affecting the side effects of 5-FU. Phosphorylation of the retinoblastoma was inhibited after oral administration of UD-017 time-dependently in the tumor tissues. Phosphorylation of RNA polymerase II COOH-terminal domain (CTD) was also inhibited & expression of c-myc was decreased. PPAP cleavage was subsequently induced, causing apoptotic death of the cancer cells. Conclusion: We propose UD-017 as a novel type of anti-cancer drug that shows favorable oral pharmacokinetics profile & complete anti-tumor responses in xenograft models of colorectal cancer cells, & also shows in vivo synergy in combination with chemotherapy. These data support the rationale for further advancing towards clinical development. IND-enabling studies are in progress. Citation Format: Yasuhiro Aga, Sayaka Ogi, Yasunori Tokunaga, Ayumi Ogawa, Kazuhiro Onuma, Takashi Matsushita, Hidetoshi Sunamoto, Toru Hasegawa, Shigeru Ushiyama. In vivo anti-tumor efficacy of UD-017, a novel highly selective & orally available CDK7 inhibitor, in colorectal cancer cells xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-297. doi:10.1158/1538-7445.AM2017-LB-297

Background: Cyclin dependent kinase 7 (CDK7) is a member of the CDK family, & has a dual function relating to the cell cycle progression & gene transcription/ RNA processing. Recently CDK7 has been emerged as an attractive target for anticancer drugs due to its dual role. We synthesized UD-017, a small molecule, highly selective CDK7 inhibitor with a novel chemotype. In this study, we characterized in vitro antiproliferative profile of UD-017 & elucidated underlying mechanism how CDK7 inhibition contributes to the antiproliferation of cancer cells. Experimental procedures: We conducted a large panel of 9 CDKs & 313 other kinases assay of UD-017 to determine its selectivity & analyzed kinetics for CDK7 enzyme inhibition. We evaluated the antiproliferative activity of UD-017 in over 100 multiple types of cancer cell lines. We then investigated the mechanism of antiproliferative activity of UD-017 using human colorectal cancer cell line, HCT-116, & human multiple myeloma cell line, NCI-H929. We also explored biomarker candidates mainly using colorectal cancer cell lines. Results: UD-017 inhibited CDK7 enzyme with an IC50 value of 16 nM, which is at least 300-fold more selective against other CDKs, CDK1 through CDK9. Inhibition of UD-017 was reversible & ATP-competitive. In a panel of 313 kinases assay, the compound inhibited CDK7 almost mono-specifically. In antiproliferative panel assays using over 100 cancer cell lines, UD-017 broadly inhibited a wide range of cancer cells from colon, breast, lung, kidney, blood, pancreas & urinary bladder cancers. Specifically, it inhibited the growth of HCT-116 & NCI-H929 with GI50 values of 29 nM and 6.8 nM, respectively. In the mechanism study of the antiproliferative activity, UD-017 inhibited phosphorylation of CDK1, CDK2 & the retinoblastoma (RB) & arrested the cell-cycle. Phosphorylation of RNA polymerase II c-terminal domain was also inhibited & expression levels of c-Myc and XIAP decreased & the cleavage of PARP increased, which were followed by significant induction of apoptosis. UD-017 showed both cell cycle arrest & induction of apoptosis. In the biomarker study, c-Myc & cyclin H showed good correlation with antiproliferative activities in 6 colorectal cancer cell lines and will be candidate biomarkers of UD-017. Conclusion: UD-017 is a potent & highly selective CDK7 inhibitor, & significantly inhibits the proliferation of human cancer cells with concomitant cell cycle arrest & induction of apoptosis. In vivo anti-tumor efficacy for UD-017 is warranted. Citation Format: Takashi Matsushita, Kazuhiro Onuma, Hidetoshi Sunamoto, Ayumi Ogawa, Sayaka Ogi, Toru Hasegawa, Shigeyuki Kono, Noriaki Iwase, Yasuhiro Aga, Shigeru Ushiyama. In vitro anti-cancer activity of UD-017, a novel potent & highly selective CDK7 reversible inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-111. doi:10.1158/1538-7445.AM2017-LB-111

e14086 Background: Cyclin dependent kinase 7 (CDK7) modulates mRNA transcription and some oncogenes are reported to be sensitive to inhibition of transcription in certain cancer cells. CDK7 inhibitors have been considered as an intriguing approach to treat cancers that depend on transcriptional regulation of their oncogenes. We synthesized a novel highly selective CDK7 inhibitor, UD-017, and found that the compound showed antitumor potency in a variety of cancers in vitro and in vivo. We therefore explored underlying mechanisms especially focusing on an oncogenic driver, c-Myc. Methods: We examined CDK7 selectivity of UD-017 against the other CDKs and kinases. We evaluated an antiproliferative activity of UD-017 in over 200 multiple types of cancer cell lines including patients-derived cancer cells. We then investigated the correlation between c-Myc expression levels and an antiproliferative activity of UD-017 in cancer cells. Antitumor efficacy of UD-017 was assessed in multiple types of cancer xenograft models and patients-derived xenograft model. We determined whether an intratumoral c-Myc expression levels correlated with in vivo antitumor efficacy of UD-017 in xenograft models. Results: UD-017 inhibited CDK7 enzyme (IC50= 16 nM) highly selectively among the CDKs (over 300-fold) and almost mono-specifically in a panel of 313 kinases assay. In a cellular antiproliferative panel assay, UD-017 broadly inhibited the proliferation of a variety of cancer cells and c-Myc expression levels showed the good correlation with antiproliferative activity. UD-017 showed favorable PK profile and good oral absorbability and showed the potent antitumor activity in multiple types of cancer xenograft models in mice. In correlation with the PK, UD-017 reduced the intratumoral c-Myc mRNA levels time-dependently after dosing of UD-017 in the colorectal cancer xenograft model. Conclusions: We identified a highly selective and orally available CDK7 inhibitor that showed the broad in vitro and in vivo antitumor activity in a variety of cancers, modulating c-Myc as an oncogenic driver. These data support the rationale for further advancing towards clinical development.

e14085 Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for cancer drugs due to its dual roles, cell cycle regulation and gene transcription/RNA procession. We synthesized UD-017, a small molecule, highly selective CDK7 inhibitor with a novel chemotype. In this study, we characterize antitumor activity of UD-017 in vitro and in vivo, and try to elucidate underlying mechanisms by which CDK7 inhibition contributes to the antitumor efficacy in colorectal cancer cells. Methods: We examined CDK7 selectivity of UD-017 against the other CDKs and kinases. We evaluated the antitumor activity in a variety of human cancer cell lines including colorectal cancer cells. We investigated the mechanism of antitumor activity of UD-017 using a human colorectal cancer cell line, HCT-116. In vivo antitumor efficacy of UD-017 was assessed in HCT-116 xenograft models and patient derived xenograft (PDX) model. Results:UD-017 inhibited CDK7 enzyme (IC50 = 16 nM), which is at least 300-fold more selective against other CDKs. In a panel of 313 kinases assay, UD-017 inhibited CDK7 almost mono-specifically. UD-017 potently inhibited the growth of human cancer cells including the patient-derived colorectal cancer cells.In the mechanism study, UD-017 inhibited phosphorylation of CDK1, 2 and retinoblastoma, which was followed by cell-cycle arrest. Inhibition of both RNA polymerase II phosphorylation and c-Myc expression was observed followed by apoptosis induction. HCT-116 xenograft model and PDX model demonstrated the potent antitumor activity of UD-017 with dose-dependence by daily oral administration for 2 weeks. Furthermore, UD-017 showed the combination effect with 5-FU without further affecting the side effects of the chemotherapy. Conclusions:UD-017 is a potent and highly selective CDK7 inhibitor, and significantly inhibits the growth of human cancer cell lines with cell-cycle arrest and apoptosis induction. UD-017 showed potent and dose-dependent antitumor response in the HCT-116 xenograft model and PDX model. Further investigation towards clinical development is warranted.

We compared plasma 11-dhTXB2 and TXB2 for their ability to reflect in vivo TX formation in rabbits treated with AA and CS-518, a TX synthase inhibitor. The average plasma level of TXB2 in rabbits was much higher than that of 11-dhTXB2, probably because of artificial formation of TXB2 during blood sampling. CS-518 (1 mg/kg, p.o.) caused a long-lasting suppression of the 11-dhTXB2 level, and its inhibitory effect on 11-dhTXB2 was much more extensive than that on TXB2. AA-infusion for 5 min resulted in transient and remarkable increases of both TXs, and prevention of such increases by CS-518 pretreatment (1 mg/kg, i.v.) was shown: inhibitions of 11-dhTXB2 and TXB2 were 85% and 40%, respectively. The inhibitory effect caused by CS-518, which was more clearly observed on plasma 11-dhTXB2 than on TXB2, was due to not only the completely inhibited levels without artificial formation but also the durable high levels based on the long half-life of 11-dhTXB2 in AA-infused rabbits. CS-518 injection during sustained AA-infusion also resulted in a 2-fold faster disappearance of plasma 11-dhTXB2 than was seen without CS-518, despite its long half-life. Considering the absence of artificial formation, the long half-life, and the good response to change of TX formation, plasma 11-dhTXB2 is superior to TXB2 as an index for monitoring in vivo TX synthase activity and its pharmacological modification with AA and CS-518.

P-selectin is an adhesion receptor for leukocytes on thrombin-activated platelets and endothelial cells. It contains a NH2-terminal carbohydrate-recognition domain, an epidermal growth factor motif, nine consensus repeats, a transmembrane domain, and a cytoplasmic tail. We expressed two soluble forms of P-selectin, one truncated after the ninth repeat (tPS) and the other lacking the transmembrane domain due to alternative RNA splicing (asPS). When visualized by electron microscopy, each was a monomeric rod-like structure with a globular domain at one end, whereas membrane P-selectin (mPS) from platelets formed rosettes with the globular domains facing outward. Sedimentation velocity and equilibrium studies confirmed that tPS and asPS were asymmetric monomers, whereas mPS was oligomeric. HL-60 cells adhered to immobilized tPS and asPS, although less efficiently than to mPS. 125I-Labeled tPS and asPS bound to approximately 25,000 sites/neutrophil and approximately 36,000 sites/HL-60 cell with an apparent Kd of 70 nM. Treatment of HL-60 cells with O-sialoglycoprotease eliminated the binding sites for asPS. We conclude that 1) P-selectin is a rigid, asymmetric protein; 2) monomeric soluble P-selectin binds to high affinity ligands with sialylated O-linked oligosaccharides on leukocytes; and 3) oligomerization of mPS enhances its avidity for leukocytes.

In order to develop a new class of anti-rheumatic drugs which inhibit production of proinflammatory cytokines such as TNFα, IL-1β, IL-6, and IL-8, a series of 3-pyridylpyrrole derivatives possessing a bicyclic tetrahydropyridine moiety at the 4-position of the pyrrole ring are synthesized and their pharmacological activities are evaluated.

The synthesis and biological activity of N- and α-position substituted tetrahydropyridine derivatives is presented.

In order to develop a new class of anti-rheumatic drug which inhibits production of proinflammatory cytokines such as TNFalpha, IL-1beta, IL-6, and IL-8, a series of 3-pyridylpyrrole derivatives possessing a bicyclic tetrahydropyridine moiety at the 4-position of the pyrrole ring were synthesized and their pharmacological activities were evaluated. The derivatives were found to have potent inhibitory activities on the production of the cytokines both in vitro and in vivo. Among them, compound 4a, (S)-2-(4-fluorophenyl)-4-(1,2,3,5,6,8a-hexahydroindolizin-7-yl)-3-(pyridin-4-yl)-1H-pyrrole (R-132811), achieved the most promising results in various in vitro and in vivo tests including several rheumatoid arthritis models ((i) inhibition of p38alpha, p38beta, p38gamma, and p38delta MAP kinases: IC(50)=0.034, 0.572, >10, and >10 microM, respectively; (ii) inhibition of TNFalpha, IL-1beta, IL-6, and IL-8 production in human whole blood: IC(50)=0.026, 0.020, 0.88, and 0.016 microM, respectively; (iii) inhibition of LPS induced TNFalpha, IL-1beta and IL-6 production in mice: ID(50)=0.93, 8.63, and 0.11 mg/kg, p.o., respectively; (iv) inhibition of anti-collagen antibody-induced arthritis in mice: ID(50)=2.22 mg/kg, p.o.; (v) inhibition of collagen-induced arthritis in mice: ID(50)=2.38 mg/kg, p.o.; (vi) prophylactic effect on adjuvant-induced arthritis in rats: ID(50)=3.1 mg/kg, p.o.; (vii) therapeutic effect on adjuvant-induced arthritis in rats: ID(50)=4.9 mg/kg, p.o.; (viii) analgesic effect on adjuvant-induced arthritic pain in rats: ID(50)=2.9 mg/kg, p.o.). As a result, compound 4a was chosen as a candidate for further pre-clinical studies.

We previously reported a novel pyrrole derivative 1 which possesses a tetrahydropyridine group at the beta-position with a proinflammatory cytokine TNFalpha production inhibitor. Herein, we report the synthesis and biological activity of N- and alpha-position substituted tetrahydropyridine derivatives. In this series, we found that compound 3o showed good inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS)-induced TNFalpha production in human whole blood, IC(50)=0.44 microM) and compound 3i demonstrated potent inhibitory activity in vivo (inhibition of LPS-induced TNFalpha production in mice, ID(50)=1.42 mg/kg).

We investigated proinflammatory cytokine TNFalpha production inhibitors in order to develop novel anti-inflammatory agents. According to the results, we found that 17, a pyrrole derivative possessing a tetrahydropyridine group at the beta-position, showed potent inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS) induced TNFalpha production in human whole blood, IC(50)=1.86 microM) and in vivo (inhibition of LPS induced TNFalpha production in mice, ID(50)=5.98 mg/kg).

We report here the preclinical anti-inflammatory profile of CS-706 [2-(4-ethoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl)-1 H -pyrrole], a novel cyclooxygenase-2 (COX-2) selective inhibitor. CS-706 selectively inhibited COX-2 in a human whole blood assay with an IC 50 of 0.31 μM, compared with an IC 50 of 2.2 μM for COX-1. The selectivity ratio of CS-706 was higher than those of the conventional non-steroidal anti-inflammatory drugs naproxen, indomethacin, and Diclofenac–Na, whereas it was lower than those of rofecoxib, valdecoxib and etoricoxib. It was similar to that of celecoxib. The pharmacokinetic profile of CS-706 showed rapid absorption and dose-proportional exposure after oral administration to rats. CS-706 inhibited prostaglandin E 2 production in inflamed tissue induced by yeast-injection in rats with potency similar to that of indomethacin. However, it inhibited gastric mucosal prostaglandin E 2 production in normal rats weakly compared with indomethacin. CS-706 ameliorated both yeast-induced inflammatory acute pain (ED 50 = 0.0090 mg/kg) and adjuvant-induced chronic arthritic pain (ED 50 = 0.30 mg/kg) in rats. CS-706 showed more potent antinociceptive activity than celecoxib and rofecoxib in these models. In an adjuvant-induced arthritic model in rats, CS-706 suppressed foot swelling prophylactically with an ID 50 of 0.10 mg/kg/day, and decreased foot swelling in the established arthritis therapeutically in a dose range of 0.040 to 1.0 mg/kg/day. Single administration of up to 100 mg/kg of CS-706 induced no significant gastric lesions in rats. In conclusion, CS-706 is a COX-2-selective inhibitor with a potent antinociceptive and anti-inflammatory activity and a gastric safety profile.

Synovial hyperplasia is a hallmark of rheumatoid arthritis (RA) and is regarded as a major destructive element of articular bone and cartilage. This pathological process is accompanied by the production of proinflammatory cytokines, prostaglandin E 2 (PGE 2 ), and matrix metalloproteinases (MMPs) in synoviocytes. We studied the spontaneous production of these substances in RA synoviocytes in spheroid culture. Synovial sarcoma cell line SW 982 formed a single spheroid in non-adherent culture plates. It produced interleukin (IL)-1β, IL-6, IL-8, PGE 2 , MMP-2 and MMP-13. Neither the addition of integrin antagonizing oligopeptide (GRGDSP) nor that of vitronectin receptor inhibitor SB-265123 to the culture inhibited any production. Phosphorylation of p38 mitogen-activated protein (MAP) kinase was observed during the culture. A novel p38 MAP kinase inhibitor, R-130823, inhibited the release of IL-6, IL-8 and MMP-13 in a concentration-dependent manner, but not that of IL-1β or MMP-2. Real-time RT-PCR analysis demonstrated that IL-6, IL-8 and MMP-13 were inhibited at the transcriptional level. R-130823 did not affect the production of PGE 2 in spheroid culture, while the addition of R-130823 suppressed IL-1β-induced PGE 2 synthesis in monolayer culture of SW 982 cells. The results suggest that spheroid culture induced proinflammatory factors and MMPs in signaling pathways both dependent and independent of p38 MAP kinase.

Plaunotol [(2E,6Z,10E)-7-hydroxymethyl-3,11,15-trimethyl-2,6,10,14-hexadecatetraen-1-ol], a new anti-ulcer drug, was investigated for its effect on prostaglandin (PG) production. In cultured cells of 3T6 fibroblasts, plaunotol and its main metabolite (1-carboxylic plaunotol) at concentrations of 10–100 μM increased PGE 2 and PGI 2 production 2- to 4-fold. These compounds increased the release of radioactive arachidonic aud from [ 14 C]arachidonic acid prelabeled 3T6 fibroblast cells 2-fold at 30 μM, and this increase was inhibited by addition of mepacrine, a phospholipase inhibitor. Plaunotol and its main metabolite had no effect on PG cyclooxygenase activity. These results indicate that plaunotol and its main metabolite stimulate PG production by activating cellular phospholipase. In gastric-mucosa slices, PGE 2 and PGI 2 production was increased significantly either by oral administration of plaunotol to rats at a dose of 300mg/kg or by addition of the main metabolite to the incubation medium. All these results suggest that plaunotol increases the PG levels in gastric mucosa by stimulating the PG biosynthesis, particularly the cellular phospholipase activity. The increased levels of PG may participate in the anti-ulcer activity of plaunotol.

A new anti-inflammatory agent, sodium 2-[4-(2-oxocyclopentylmethyl)phenyl]propionate dihydrate (CS-600), was investigated for its inhibition of prostaglandin (PG) synthesis in vivo and in vitro. CS-600 caused a marked decrease in the level of urinary PGE2 and PGF2 alpha in rats. The dose of CS-600 which resulted in a 50% decrease of urinary PGE2 excretion was 1.9 mg/kg, p.o., and this value agreed well with the ID50 of the drug for carrageenin edema (1.2 mg/kg, p.o.). This suggests that CS-600 inhibits prostaglandin synthesis in vivo. However, CS-600 had only weak inhibitory activity against in vitro prostaglandin synthesis by bovine seminal vesicle microsomes (IC50:760 microM). A main plasma metabolite of CS-600, which was produced by stereospecific reduction of the cyclopentanone moiety to transhydroxy cyclopentane, exhibited potent inhibitory activity toward the prostaglandin synthetase of bovine seminal vesicle microsomes (IC50:11 microM). In cell cultures of 3T6 fibroblasts from mice, CS-600 inhibited production of PGE2 and PGF2 alpha by the cells at low concentrations (IC50 for PGE2:1.6 microM). The active metabolite exhibited more potent inhibition (IC50:0.29 microM), and conversion of CS-600 into the active metabolite occurred in the cell system. Inhibition of prostaglandin synthetase in the membrane fraction of the fibroblast cells was also investigated. Available evidence indicates that CS-600 is a pro-drug and exerts its pharmacological activities after conversion to the active metabolite.

15-Hydroxyprostaglandin dehydrogenase was partially purified from hog gastric mucosa by about 1000-fold with a 13.5 % yield. Its molecular weight was estimated to be 32,000 daltons by gel filtration. The enzyme was inhibited by some metabolites of plaunotol [(2E, 6Z, 10E)-7-hydroxymethyl-3,11,15-trimethyl-2, 6,10,14-hexadecatetraen-1-ol], a new anti-ulcer drug. The inhibition patterns for substrates, prostaglandin E 1 and NAD + were both uncompetitive with Ki values of 7.8 and 19.7 μM, respectively.

RS-5186, sodium 6-[2-[1-(1H)-imidazolyl]methyl-4,5-dihydrobenzo[b]thiophene]- carboxylate, inhibited platelet thromboxane A2 (TXA2) synthetase with IC50 values of 6 nM and 13 nM for human and rabbit microsomes, respectively. It had a selectivity for TXA2 synthetase 10(5)-fold greater than that for cyclooxygenase, PGI2 synthetase, 5-lipoxygenase and phospholipase A2. When administered orally or intravenously to dogs at 1 mg/kg, RS-5186 suppressed serum TXB2 levels almost completely with sustained duration of action: the suppression during 0.5 hr to 8 hr after dosing was more than 90%, and was 70-80% at 24 hr. Similar suppression of serum TXB2 levels was observed in rats and rabbits. Such suppression by RS-5186 was more potent than that by OKY-046 and CV-4151. Serial administration of RS-5186 (0.1 mg/kg/day p.o.) to dogs for 7 days decreased the serum TXB2 levels constantly during the medication, and no rebound phenomenon was observed after the medication was stopped. In a thrombotic model induced by sodium arachidonate injection in rabbits, RS-5186 at 1 mg/kg p.o. completely protected against sudden death (ED50 = 0.12 mg/kg, 1 hr after dosing) and this protective effect extended over 8 hr. All these results show that RS-5186 is a potent and highly selective TXA2 synthetase inhibitor with a long duration of action, and suggest that the compound could be useful in diseases where TXA2 is involved.