Oral administration of a novel RORγt antagonist attenuates psoriasis-like skin lesion of two independent mouse models through neutralization of IL-17.

BACKGROUND: Targeting the IL-17 pathway represents a highly effective strategy for the treatment of psoriasis, using antibodies against IL-17A and IL-17 receptor, suggesting that Th17 cells essentially contribute to development of psoriasis. Th17 differentiation depends on the key transcription factor, RORγt. OBJECTIVE: To develop a novel RORγt antagonist which is effective on psoriasis via oral administration. METHODS: A chemical library was screened using cell-based high-throughput methods, luciferase reporter assay, competitive binding assay, and T cell differentiation assay. To evaluate in vivo effects of a novel RORγt antagonist, A213, we orally administrated it to two independent mouse models of psoriasis; IL-23-injection model and K5.Stat3C transgenic mouse. RESULTS: Oral administration of A213 resulted in attenuation of skin inflammation in the both mouse models. At the same time, increased levels of IL-17A expression were significantly reduced in the skin lesions and skin-draining lymph nodes. CONCLUSION: These results implicate a new therapeutic application of RORγt antagonist for the treatment of psoriasis.

Evaluation of species difference in peripheral lymphocyte reduction effect of CS-0777, a sphingosine 1-phosphate receptor modulator, based on a pharmacokinetic/pharmacodynamic model analysis.

Pharmacokinetic (PK) and pharmacodynamic (PD) modeling was conducted for the reduction of peripheral lymphocytes after oral administration of CS-0777 to healthy rats, monkeys and experimental autoimmune encephalomyelitis (EAE) induced rats. The phosphorylated active metabolite of CS-0777, M1, is a selective sphingosine 1-phosphate receptor-1 modulator. A linear one- and two-compartment model with a reversible metabolism process characterized the time courses of CS-0777 and M1 concentrations in rats and monkeys, respectively. The relationship between lymphocyte counts and M1 concentrations in blood was well described by an indirect response model in all animals examined. An Imax of 0.815 and an IC50 of 6.58 nM in healthy rats, an Imax of 0.807 and an IC50 of 5.09 nM in the EAE rats, an Imax of 0.789 and an IC50 of 0.484 nM in monkeys were estimated by the indirect PD model. Since the IC50 values calculated in terms of the unbound plasma concentration in rats and monkeys were within a similar range, after correction of the IC50 in blood described above with the blood to plasma concentration ratio and the plasma free fraction of M1, it was revealed that there is no species difference in the essential activity of M1 against lymphocyte reduction. The sensitivity of the lymphocytes to M1 was not affected by the EAE status. Comparison of the simulated lymphocyte reduction in EAE rats after multiple dosing with CS-0777 and the actual EAE clinical scores implies that the significant suppressive effect on EAE did not require the elimination of all lymphocytes from the blood. Copyright © 2016 John Wiley & Sons, Ltd.

Component of Caramel Food Coloring, THI, Causes Lymphopenia Indirectly via a Key Metabolic Intermediate.

Caramel color is widely used in the food industry, and its many variations are generally considered to be safe. It has been known for a long time that THI (2-acetyl-4-(tetrahydroxybutyl)imidazole), a component of caramel color III, causes lymphopenia in animals through sphingosine 1-phosphate (S1P) lyase (S1PL) inhibition. However, this mechanism of action has not been fully validated because THI does not inhibit S1PL in vitro. To reconcile this situation, we examined molecular details of THI mechanism of action using "smaller" THI derivatives. We identified a bioactive derivative, A6770, which has the same lymphopenic effect as THI via S1PL inhibition. In the case of A6770 we observe this effect both in vitro and in vivo, and demonstrate that A6770 is phosphorylated and inhibits S1PL in the same way as 4-deoxypyridoxine. In addition, A6770 was detected in rat plasma following oral administration of THI, suggesting that A6770 is a key metabolic intermediate of THI.

Targeting cell surface TLR7 for therapeutic intervention in autoimmune diseases.

Toll-like receptor 7 (TLR7) senses microbial-derived RNA but can also potentially respond to self-derived RNA. To prevent autoimmune responses, TLR7 is thought to localize in endolysosomes. Contrary to this view, we show here that TLR7 is present on the cell surface of immune cells and that TLR7 responses can be inhibited by an anti-TLR7 antibody. The anti-TLR7 antibody is internalized with TLR7 and accumulates in endolysosomes as an immune complex. TLR7 responses in dendritic cells, macrophages and B cells are all inhibited by the anti-TLR7 antibody. Furthermore, the anti-TLR7 antibody inhibits in vivo cytokine production induced by a TLR7 ligand. Spontaneous TLR7 activation in Unc93b1(D34A/D34A) mice causes lethal inflammation. Progressive inflammation such as splenomegaly, thrombocytopenia and chronic active hepatitis are ameliorated by anti-TLR7 antibody treatment. These results demonstrate that cell surface TLR7 is a promising target for therapeutic intervention in autoimmune diseases.

Synthesis and SAR studies of benzyl ether derivatives as potent orally active S1P1 agonists.

We report herein the synthesis and structure-activity relationships (SAR) of a series of benzyl ether compounds as an S1P1 receptor modulator. From our SAR studies, the installation of substituents onto the central benzene ring of 2a was revealed to potently influence the S1P1 and S1P3 agonistic activities, in particular, an ethyl group on the 2-position afforded satisfactory S1P1/S1P3 selectivity. These changes of the S1P1 and S1P3 agonistic activities caused by the alteration of substituents on the 2-position were reasonably explained by a docking study using an S1P1 X-ray crystal structure and S1P3 homology modeling. We found that compounds 2b and 2e had a potent in vivo immunosuppressive efficacy along with acceptable S1P1/S1P3 selectivity, and confirmed that these compounds had less in vivo bradycardia risk through the evaluation of heart rate change after oral administration of the compounds (30 mg/kg, p.o.) in rats.

Synthesis and SAR of 1,3-thiazolyl thiophene and pyridine derivatives as potent, orally active and S1P3-sparing S1P1 agonists.

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P(3)-sparing S1P(1) agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC(50) value of 3.4 nM for human S1P(1), and over 5800-fold selectivity against S1P(3). In rat on host versus graft reaction (HvGR), the ID(50) value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.

Synthesis and evaluation of CS-2100, a potent, orally active and S1P(3)- sparing S1P(1) agonist.

Modulators of sphingosine phosphate receptor-1 (S1P(1)) have recently been focused as a suppressant of autoimmunity. We have discovered a 4-ethylthiophene-based S1P(1) agonist 1-({4-Ethyl-5-[5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl]-2-thienyl}methyl)azetidine-3-carboxylic acid (CS-2100, 8) showing potent S1P(1) agonist activity against S1P(3) and an excellent in vivo potency. We report herein the synthesis of CS-2100 (8) and pharmacological effects such as S1P(1) and S1P(3) agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effects on adjuvant-induced arthritis and experimental autoimmune encephalomyelitis (EAE) in animal models. The pharmacokinetic data were also reported. CS-2100 (8) had >5000-fold greater agonist activity for human S1P(1) (EC(50); 4.0 nM) relative to S1P(3) (EC(50); >20,000 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-2100 (8) in rats, lymphocyte counts decreased significantly, with a nadir at 8 and/or 12 h post-dose and recovery to vehicle control levels by 24-48 h post-dose. CS-2100 (8) is efficacious in the adjuvant-induced arthritis model in rats (ID(50); 0.44 mg/kg). In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for 0.3 and 1 mg/kg CS-2100 (8) groups in mice. While CS-2100 (8) showed potent efficacy in various animal disease models, it was also revealed that the central 1,2,4-oxadiazole ring of CS-2100 (8) was decomposed by enterobacteria in intestine of rats and monkeys, implicating the latent concern about an external susceptibility in its metabolic process in the upcoming clinical studies.

Discovery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist.

S1P(3)-sparing S1P(1) agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC(50) value of 4.0 nM for human S1P(1) and over 5000-fold selectivity against S1P(3). The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID(50) value was determined at 0.407mg/kg. The docking studies of CS-2100 with the homology model of S1P(1) and S1P(3) showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P(3), not in the case of Leu276 in S1P(1). This observation gives an explanation for the excellent S1P(3)-sparing characteristic of CS-2100.

Discovery of CS-0777: A Potent, Selective, and Orally Active S1P1 Agonist.

CS-0777 (3) is phosphorylated in vivo, and the phosphate of CS-0777 (CS-0777-P) (4) acts as a selective S1P receptor-1 (S1P1) modulator. We report herein the synthesis of CS-0777 and CS-0777-P, pharmacological effects such as S1P1 and S1P3 agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effect on experimental autoimmune encephalomyelitis (EAE), and also the pharmacokinetics in rats. CS-0777-P had ∼320-fold greater agonist activity for human S1P1 (EC50; 1.1 nM) relative to S1P3 (EC50; 350 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-0777 in rats, lymphocyte counts decreased significantly, with a nadir at 12 h postdose and recovery to vehicle control levels by 5 days postdose. In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for the 0.1 and 1 mg/kg CS-0777 groups in rats. CS-0777 is currently in clinical trials for the treatment of multiple sclerosis (MS).

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: part 3.

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.

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: part 2.

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).

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: part 1.

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).

Use of an exposure-response model to aid early drug development of an oral sphingosine 1-phosphate receptor modulator.

Pharmacokinetic (PK) and exposure-response modeling of a selective sphingosine 1-phosphate receptor-1 modulator (CS-0777) was conducted in an iterative process to guide early clinical development decisions. A model based on preclinical data from monkeys was extrapolated to humans to support a single ascending dose (SAD) study. The model was updated after each cohort, providing guidance on both maximal inhibition and time to recovery for lymphocyte counts. A 2-compartment PK model with first-order absorption and elimination was found to describe the monkey and human datasets. The relationship between lymphocyte counts and active metabolite (M-1) concentrations was modeled via an indirect response model, whereby M-1 inhibited the reentry of lymphocytes to the circulation. The indirect-response model based on SAD data had an Imax of approximately 85% and an IC50 of 0.24 ng/mL. Additionally, based on SAD data, similar models were developed for lymphocyte subsets, including CD4 cells. Subsequently, simulations were utilized to design a multiple ascending dose study with adaptive dosing regimens that would meet targeted pharmacodynamic (PD) response thresholds (eg, minimum 40% reduction in lymphocytes) while maintaining CD4 counts above a reasonable safety threshold. In conclusion, model-based development and use of adaptive designs for dose optimization can reduce the time and number of subjects needed in early clinical development.

Anti-type II collagen antibody accelerates arthritis via CXCR2-expressing cells in IL-1 receptor antagonist-deficient mice.

Arthritis can be induced in mice by the injection of anti-type II collagen (anti-CII) Ab and LPS. To elucidate the role of IL-1 receptor antagonist (IL-1ra) in Ab-induced arthritis, WT and IL-1ra(-/-) mice were administered anti-CII Ab and LPS. These IL-1ra(-/-) mice developed severe arthritis even at low doses of anti-CII Ab and LPS, while WT mice did not. The cells that invaded the arthritic joints were mainly Gr-1(+) neutrophils, and the number of the cells in the joints remained high over 4 weeks in the IL-1ra(-/-) mice. KC, a ligand for CXCR2, is found in higher levels in the arthritic paws of IL-1ra(-/-) mice compared with the WT, and most of the cells that infiltrated into the joints of the IL-1ra(-/-) mice were CXCR2-expressing neutrophils. Administration of anti-CXCR2 Ab completely inhibited arthritis development. The anti-CXCR2 Ab decreased the number of neutrophils in the blood and also inhibited the migration of neutrophils to KC. These results suggested that the high susceptibility of IL-1ra(-/-) mice to anti-CII Ab-induced arthritis was due to the higher expression of chemotactic factors like KC and the sustained infiltration of CXCR2-expressing neutrophils into the joints.

Essential role of neutrophils in anti-type II collagen antibody and lipopolysaccharide-induced arthritis.

In mice arthritis model induced by anti-type II collagen (CII) antibodies and lipopolysaccharide (LPS), most of cells that infiltrated into the joint space were neutrophils. To investigate the role of neutrophils in the pathogenesis of arthritis, we depleted the neutrophils in vivo by injection of the antibody against Gr-1 expressed mainly on neutrophils. The neutrophil depletion completely inhibited the arthritis development. Furthermore, neutrophil depletion in mice that had already developed arthritis ameliorated the disease. These results showed that neutrophils are indispensable not only for the development, but also for the maintenance of arthritis. Next, we tried to develop arthritis in C5-deficient mice to investigate the involvement of C5a, one of chemotactic factors for neutrophils. C5-deficient mice showed significant reduction in arthritis development in comparison with wild type mice. Injection of pertussis toxin (Ptx) into the mice, which inhibits the signals from the inhibitory G-protein coupled-receptors including the C5a receptor, suppressed the development of arthritis. Furthermore, Ptx also ameliorated the arthritis when injected into mice that had already developed the disease. These results suggest the important role of chemotactic factors involving C5a and inhibitory G-protein (Gi)-coupled receptors not only in the development, but also in the maintenance of arthritis.

Syntheses of glucose derivatives of E5564-related compounds and their LPS-antagonistic activities.

Glucose analogues 6, 12, 17b, 19a, and 19b of E5564 were synthesized, and their LPS-antagonistic activities were measured. The antagonistic activities (IC(50)) on LPS-induced TNFalpha production of these five compounds toward human whole blood were 72.8, 3.0, 0.9, 7.5, and 1.4nM, respectively. Inhibitory doses (ID(50)) of compounds 12, 17b, 19a, and 19b on TNFalpha production induced by co-injection of galactosamine and LPS in C3H/HeN mice in vivo were measured. The values of these compounds were 0.9, ND (not determined), 1.6, and 0.9mg/kg, respectively.

Syntheses of glucose analogues of E5564 as a highly potent anti-sepsis drug candidate.

Glucose analogues 5 and 9 of E5564 were synthesized, and their LPS-antagonistic activities were measured. The inhibitory activities (IC50) on LPS-induced TNFalpha production of these two compounds towards human whole blood cells were 0.06 and 0.83 nM, respectively. Inhibitory doses (ID50) of compounds 5 and 9 on TNFalpha production induced by coinjection of galactosamine and LPS in C3H/HeN mice in vivo were measured and were 0.55 and <0.20 mg/kg, respectively. And also C3H/HeN mice preinjected with compounds 5 and 9 were protected from lethality induced by coinjection of galactosamine and LPS; out of eight mice preinjected with 1 mg/kg of the compounds, one-six and three of eight mice were protected, respectively.

R-130823, a novel inhibitor of p38 MAPK, ameliorates hyperalgesia and swelling in arthritis models.

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)-1H-pyrrole}, had highly selective inhibition against mitogen-activated protein kinase p38alpha (IC50=22 nM). The release of tumor necrosis factor-alpha, interleukin-1beta, -6 and -8 was inhibited in lipopolysaccharide-stimulated human blood pretreated by R-130823, with IC50 values of 0.089, 0.066, 0.95 and 0.16 microM, 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.

Essential role of Fc gamma receptors in anti-type II collagen antibody-induced arthritis.

Anti-type II collagen (anti-CII) Ab is a well-known autoantibody observed in patients with rheumatoid arthritis. Injection of anti-CII Ab and LPS induces arthritis in mice in which anti-CII Ab as well as inflammatory cytokines, IL-1beta and TNF-alpha, play critical roles. We investigated the involvement of IgG FcRs (FcgammaRs) in this arthritis model. BALB/c mice injected with the F(ab')(2) of anti-CII Ab showed no signs of arthritis. Arthritis development was not observed in FcRgamma(-/-) mice and was partially suppressed in FcgammaRIII(-/-) mice despite the binding of anti-CII Ab and C3 to cartilage surface. Surprisingly, BALB/c mice lacking FcgammaRIIB, which is known as an inhibitory FcgammaR, developed arthritis with no exacerbation in arthritis score compared with wild-type (WT) mice, and only slight exacerbation was observed in the histopathological analysis. In contrast, aged FcgammaRIIB(-/-) BALB/c mice developed arthritis without LPS injection, suggesting an augmented susceptibility to arthritis in aged FcgammaRIIB(-/-) mice. No significant difference was observed among BALB/c-WT, -FcRgamma(-/-), and -FcgammaRIIB(-/-) mice on cytokine production induced by anti-CII Ab and LPS injection. Severe arthritis developed in BALB/c-WT and -FcgammaRIIB(-/-) mice, but not in BALB/c-FcRgamma(-/-) mice, after the injection of anti-CII Ab and inflammatory cytokines. These results suggest that the reason behind the nondevelopment of arthritis in FcRgamma(-/-) BALB/c mice is not due to a disorder in transient cytokine production, but to an irregularity downstream of cytokine production.

The importance of IL-1 beta and TNF-alpha, and the noninvolvement of IL-6, in the development of monoclonal antibody-induced arthritis.

Injection of anti-type II collagen Ab and LPS induces arthritis in mice. The levels of IL-1 beta, IL-6, and chemokines (macrophage inflammatory protein (MIP)-1 alpha, MIP-2, and monocyte chemoattractant protein-1) in the hind paws increased with the onset of arthritis and correlated highly with arthritis scores. The level of TNF-alpha was also elevated, but only transiently. Quantitative real-time PCR analysis revealed increases in cytokine and chemokine mRNA. To elucidate the contribution of inflammatory cytokines and chemokines in arthritis development more directly, recombinant proteins, neutralizing Abs, and knockout mice were used. The injection of rIL-1 beta or TNF-alpha, but not IL-6 or chemokines, induced arthritis when mice were i.v. preinjected with anti-type II collagen Ab. However, a single injection of recombinant cytokines or chemokines into the hind paws did not induce swelling. Arthritis development was inhibited by neutralizing Ab against IL-1 beta, TNF-alpha, or MIP-1 alpha. In contrast, the inhibitory effect by anti-MIP-2 Ab was partial and, surprisingly, Abs to IL-6 and monocyte chemoattractant protein-1 showed no inhibitory effect. Furthermore, arthritis development in IL-1R(-/-) mice and TNFR(-/-) mice was not observed at all, but severe arthritis was developed in IL-6(-/-) mice. These results suggest that IL-1 beta and TNF-alpha play more crucial roles than IL-6 or chemokines in this model. Because arthritis was also developed in SCID mice, the development of arthritis in the Ab-induced mice model is due to a mechanism that does not involve T or B cells.