Investigating the antifibrotic effect of the antiparasitic drug Praziquantel in in vitro and in vivo preclinical models.

Tissue fibrosis underlies the majority of human mortality to date with close to half of all reported deaths having a fibrotic etiology. The progression of fibrosis is very complex and reputed irreversible once established. Although some preventive options are being reported, therapeutic options are still scarce and in very high demand, given the rise of diseases linked to fibroproliferative disorders. Our work explored four platforms, complementarily, in order to screen preventive and therapeutic potentials of the antiparasitic drug Praziquantel as a possible antifibrotic. We applied the mouse CCl4-driven liver fibrosis model, the mouse chronic schistosomiasis liver fibrosis model, as well as novel 2D and 3D human cell-based co-culture of human hepatocytes, KCs (Kupffer cells), LECs (Liver Endothelial Cells), HSCs (Hepatic Stellate Cells) and/or myofibroblasts to mimic in vivo fibrotic responses and dynamics. Praziquantel showed some effect on fibrosis marker when preventively administered before severe establishment of fibrosis. However, it failed to potently reverse already established fibrosis. Together, we provided a novel sophisticated multi-assay screening platform to test preventive and therapeutic antifibrotic candidates. We further demonstrated a direct preventive potential of Praziquantel against the onset of fibrosis and the confirmation of its lack of therapeutic potential in reversing already established fibrosis.

Discovery of Affinity-Based Probes for Btk Occupancy Assays.

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.

Btk inhibition treats TLR7/IFN driven murine lupus.

Bruton's tyrosine kinase (Btk) is expressed in a variety of immune cells and previous work has demonstrated that blocking Btk is a promising strategy for treating autoimmune diseases. Herein, we utilized a tool Btk inhibitor, M7583, to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon. In BXSB-Yaa lupus mice, Btk inhibition reduced autoantibodies, nephritis, and mortality. In the pristane-induced DBA/1 lupus model, Btk inhibition suppressed arthritis, but autoantibodies and the IFN gene signature were not significantly affected; suggesting efficacy was mediated through inhibition of Fc receptors. In vitro studies using primary human macrophages revealed that Btk inhibition can block activation by immune complexes and TLR7 which contributes to tissue damage in SLE. Overall, our results provide translational insight into how Btk inhibition may provide benefit to a variety of SLE patients by affecting both BCR and FcR signaling.

Using ovality to predict nonmutagenic, orally efficacious pyridazine amides as cell specific spleen tyrosine kinase inhibitors.

Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.

Selective inhibition of spleen tyrosine kinase (SYK) with a novel orally bioavailable small molecule inhibitor, RO9021, impinges on various innate and adaptive immune responses: implications for SYK inhibitors in autoimmune disease therapy.

INTRODUCTION: Spleen tyrosine kinase (SYK) is a key integrator of intracellular signals triggered by activated immunoreceptors, including Bcell receptors (BCR) and Fc receptors, which are important for the development and function of lymphoid cells. Given the clinical efficacy of Bcell depletion in the treatment of rheumatoid arthritis and multiple sclerosis, pharmacological modulation of Bcells using orally active small molecules that selectively target SYK presents an attractive alternative therapeutic strategy. METHODS: A SYK inhibitor was developed and assayed in various in vitro systems and in the mouse model of collagen-induced arthritis (mCIA). RESULTS: A novel ATP-competitive inhibitor of SYK, 6-[(1R,2S)-2-Amino-cyclohexylamino]-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylic acid amide, designated RO9021, with an adequate kinase selectivity profile and oral bioavailability, was developed. In addition to suppression of BCR signaling in human peripheral blood mononuclear cells (PBMC) and whole blood, FcγR signaling in human monocytes, and FcϵR signaling in human mast cells, RO9021 blocked osteoclastogenesis from mouse bone marrow macrophages in vitro. Interestingly, Toll-like Receptor (TLR) 9 signaling in human Bcells was inhibited by RO9021, resulting in decreased levels of plasmablasts, immunoglobulin (Ig) M and IgG upon B-cell differentiation. RO9021 also potently inhibited type I interferon production by human plasmacytoid dendritic cells (pDC) upon TLR9 activation. This effect is specific to TLR9 as RO9021 did not inhibit TLR4- or JAK-STAT-mediated signaling. Finally, oral administration of RO9021 inhibited arthritis progression in the mCIA model, with observable pharmacokinetics (PK)-pharmacodynamic (PD) correlation. CONCLUSIONS: Inhibition of SYK kinase activity impinges on various innate and adaptive immune responses. RO9021 could serve as a starting point for the development of selective SYK inhibitors for the treatment of inflammation-related and autoimmune-related disorders.

Suppression of glomerulonephritis in lupus-prone NZB × NZW mice by RN486, a selective inhibitor of Bruton's tyrosine kinase.

OBJECTIVE: Bruton's tyrosine kinase (BTK) plays a critical role in B cell development and function. We recently described a selective BTK inhibitor, RN486, that blocks B cell receptor (BCR) and Fcγ receptor signaling and is efficacious in animal models of arthritis. The aim of this study was to examine the potential efficacy of BTK in systemic lupus erythematosus (SLE), using an NZB × NZW mouse model of spontaneous SLE. METHODS: Mice received RN486 or its vehicle (administered in chow) at a final concentration of 30 mg/kg for 8 weeks, starting at 32 weeks of age. RESULTS: The administration of RN486 completely stopped disease progression, as determined by histologic and functional analyses of glomerular nephritis. The efficacy was associated with striking inhibition of B cell activation, as demonstrated by a significant reduction in CD69 expression in response to BCR crosslinking. RN486 markedly reduced the secretion of IgG anti-double-stranded DNA (anti-dsDNA) secretion, as determined by enzyme-linked immunosorbent and enzyme-linked immunospot assays. Flow cytometric analysis demonstrated depletion of CD138(high) B220(low) plasma cells in the spleen. RN486 inhibited secretion of IgG anti-dsDNA but not IgM anti-dsDNA, suggesting that pharmacologic blockade of BTK resembles the reported transgenic expression of low levels of endogenous BTK in B cells. In addition, RN486 may also impact the effector function of autoantibodies, as evidenced by a significant reduction in immune complex-mediated activation of human monocytes in vitro and down-regulation of the expression of macrophage-related and interferon-inducible genes in both the kidneys and spleens of treated mice. CONCLUSION: Collectively, our data suggest that BTK inhibitors may simultaneously target autoantibody-producing and effector cells in SLE, thus constituting a promising therapeutic alternative for this disease.

Pyrrolopyrazines as selective spleen tyrosine kinase inhibitors.

We describe the discovery of several pyrrolopyrazines as potent and selective Syk inhibitors and the efforts that eventually led to the desired improvements in physicochemical properties and human whole blood potencies. Ultimately, our mouse model revealed unexpected toxicity that precluded us from further advancing this series.

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo.

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments.

RN486, a selective Bruton's tyrosine kinase inhibitor, abrogates immune hypersensitivity responses and arthritis in rodents.

Genetic mutation and pharmacological inhibition of Bruton's tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fcε receptor cross-linking-induced degranulation in mast cells (IC(50) = 2.9 nM), Fcγ receptor engagement-mediated tumor necrosis factor α production in monocytes (IC(50) = 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC(50) = 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and bone-protective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

Dual, constrictor-to-dilator, response of the mouse ductus arteriosus to the microsomal prostaglandin E synthase-1 inhibitor, 2-(6-chloro-1H-phenanthro[9,10d]imidazole- 2-yl)isophthalonitrile.

BACKGROUND: Microsomal prostaglandin E synthase-1 (mPGES1) is critical for prostaglandin E(2) formation in ductus arteriosus (DA) and, accordingly, in its patency. We previously reported that mPGES1 deletion, unlike cyclo-oxygenase (COX) suppression, is not followed by upregulation of relaxant nitric oxide (NO). Consequently, we proposed that a mPGES1 inhibitor may be better than currently used COX inhibitors in managing premature infants with persistent DA (PDA). OBJECTIVE: To assess the effect of the mPGES1 inhibitor, 2-(6-chloro-1H-phenanthro[9,10d]imidazole-2-yl)isophthalonitrile (MF63) on DA ex vivo and in vivo (p.o. to the mother). METHODS: Experiments were carried out with mice bearing human mPGES1. We utilized isolated, wire-mounted DA for isometric recording and a whole-body freezing technique to assess the DA caliber as it occurs in vivo. RESULTS: MF63 (10 µM) contracted the isolated DA. DA constriction was also seen in vivo after a single 10-mg kg(-1) dose. Conversely, a 30-mg kg(-1) dose gave inconsistent results, combining constriction with no effect. DA dilatation followed instead a repeated lower dose (twice daily for 3 days), and postnatal closure of the vessel was also delayed. Chronic pretreatment had no effect on endothelial NO synthase mRNA expression in fetal DA, nor did it modify the contraction to NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (100 µM). CONCLUSIONS: MF63 has a dual action on DA, the constriction being associated with accessory dilatation. The latter effect should be explained before considering further a mPGES1 inhibitor for management of PDA.

Trisubstituted ureas as potent and selective mPGES-1 inhibitors.

A novel series of trisubstituted ureas has been identified as potent and selective mPGES-1 inhibitors. These compounds are selective over other prostanoid enzymes such as PGF synthase and TX synthase. This series of inhibitors was developed by lead optimization of a hit from an internal HTS campaign. Lead compound 42 is potent in A549 cell assay (IC(50) of 0.34 µM) and in human whole blood assay (IC(50) of 2.1 µM). An efficient and versatile one-pot strategy for the formation of ureas, involving a reductive amination, was developed to generate these inhibitors.

Naphthalene/quinoline amides and sulfonylureas as potent and selective antagonists of the EP4 receptor.

Two new series of EP(4) antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP(4) receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.

A novel series of potent and selective EP(4) receptor ligands: facile modulation of agonism and antagonism.

A novel series of EP(4) ligands, based on a benzyl indoline scaffold, has been discovered. It was found that agonism and antagonism in this series can be easily modulated by minor modifications on the benzyl group. The pharmacokinetic, metabolic and pharmacological profiles of these compounds was explored. It was found that these compounds show good pharmacokinetics in rat and are efficacious in pre-clinical models of pain and inflammation.

Discovery of 4-[1-[([1-[4-(trifluoromethyl)benzyl]-1H-indol-7-yl]carbonyl)amino]cyclopropyl]benzoic acid (MF-766), a highly potent and selective EP4 antagonist for treating inflammatory pain.

The discovery of a highly potent and selective EP(4) antagonist MF-766 is discussed. This N-benzyl indole derivative exhibits good pharmacokinetic profile and unprecedented in vivo potency in the rat AIA model.

The discovery of 4-{1-[({2,5-dimethyl-4-[4-(trifluoromethyl)benzyl]-3-thienyl}carbonyl)amino]cyclopropyl}benzoic acid (MK-2894), a potent and selective prostaglandin E2 subtype 4 receptor antagonist.

The discovery of highly potent and selective second generation EP(4) antagonist MK-2894 (34d) is discussed. This compound exhibits favorable pharmacokinetic profile in a number of preclinical species and potent anti-inflammatory activity in several animal models of pain/inflammation. It also shows favorable GI tolerability profile in rats when compared to traditional NSAID indomethacin.

Discovery of disubstituted phenanthrene imidazoles as potent, selective and orally active mPGES-1 inhibitors.

Phenanthrene imidazoles 26 and 44 have been identified as novel potent, selective and orally active mPGES-1 inhibitors. These inhibitors are significantly more potent than the previously reported chlorophenanthrene imidazole 1 (MF63) with a human whole blood IC50 of 0.20 and 0.14 microM, respectively. It exhibited a significant analgesic effect in a guinea pig hyperalgesia model at oral doses as low as 14 mg/kg. Both active and selective mPGES-1 inhibitors (26 and 44) have a relatively distinct pharmacokinetic profile and are suitable for clinical development.

Development of a homogeneous immunoassay for the detection of angiotensin I in plasma using AlphaLISA acceptor beads technology.

Plasma renin activity (PRA) is a well-established biomarker for assessing the efficacy of various antihypertensive agents such as direct renin inhibitors, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors (ACEIs). PRA measurements are obtained through the detection and quantification of angiotensin I (Ang I) produced by the action of renin on its natural substrate angiotensinogen. The most accepted and reproducible method for PRA measurement uses an antibody capture Ang I methodology that employs specific antibodies that recognize and protect Ang I against angiotensinase activities contained in plasma. The amount of Ang I is then quantified by either radioimmunoassay (RIA) or enzyme immunoassay (EIA). In the current report, we describe the optimization of a novel homogeneous immunoassay based on the AlphaScreen technology for the detection and quantification of antibody-captured Ang I using AlphaLISA acceptor beads in buffer and in the plasma of various species (human, rat, and mouse). Ex vivo measurements of renin activity were performed using 10 microl or less of a reaction mixture, and concentrations as low as 1 nM Ang I were quantified. Titration curves obtained for the quantification of Ang I in buffer and plasma gave similar EC(50) values of 5.6 and 14.4 nM, respectively. Both matrices generated an equivalent dynamic range that varies from approximately 1 to 50 nM. Renin inhibitors have been successfully titrated and IC(50) values obtained correlated well with those obtained using EIA methodology (r(2)=0.80). This assay is sensitive, robust, fast, and less tedious than measurements performed using nonhomogeneous EIA. The AlphaLISA methodology is homogeneous, does not require wash steps prior to the addition of reagents, and does not generate radioactive waste.

MF63 [2-(6-chloro-1H-phenanthro[9,10-d]imidazol-2-yl)-isophthalonitrile], a selective microsomal prostaglandin E synthase-1 inhibitor, relieves pyresis and pain in preclinical models of inflammation.

Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal prostaglandin E(2) (PGE(2)) synthase in the cyclooxygenase pathway. Inhibitors of mPGES-1 may block PGE(2) production and relieve inflammatory symptoms. To test the hypothesis, we evaluated the antipyretic and analgesic properties of a novel and selective mPGES-1 inhibitor, MF63 [2-(6-chloro-1H-phenanthro-[9,10-d]imidazol-2-yl)isophthalonitrile], in animal models of inflammation. MF63 potently inhibited the human mPGES-1 enzyme (IC(50) = 1.3 nM), with a high degree (>1000-fold) of selectivity over other prostanoid synthases. In rodent species, MF63 strongly inhibited guinea pig mPGES-1 (IC(50) = 0.9 nM) but not the mouse or rat enzyme. When tested in the guinea pig and a knock-in (KI) mouse expressing human mPGES-1, the compound selectively suppressed the synthesis of PGE(2), but not other prostaglandins inhibitable by nonsteroidal anti-inflammatory drugs (NSAIDs), yet retained NSAID-like efficacy at inhibiting lipopolysaccharide-induced pyresis, hyperalgesia, and iodoacetate-induced osteoarthritic pain. In addition, MF63 did not cause NSAID-like gastrointestinal toxic effects, such as mucosal erosions or leakage in the KI mice or nonhuman primates, although it markedly inhibited PGE(2) synthesis in the KI mouse stomach. Our data demonstrate that mPGES-1 inhibition leads to effective relief of both pyresis and inflammatory pain in preclinical models of inflammation and may be a useful approach for treating inflammatory diseases.

Genetic deletion of mPGES-1 suppresses intestinal tumorigenesis.

Elevated levels of prostaglandin E(2) (PGE(2)) are often found in colorectal cancers. Thus, nonsteroidal anti-inflammatory drugs, including selective cyclooxygenase-2 (COX-2) inhibitors, are among the most promising chemopreventive agents for colorectal cancer. However, their long-term use is restricted by the occurrence of adverse events believed to be associated with a global reduction in prostaglandin production. In the present study, we evaluated the chemopreventive efficacy of targeting the terminal synthase microsomal PGE(2) synthase 1 (mPGES-1), which is responsible for generating PGE(2), in two murine models of intestinal cancer. We report for the first time that genetic deletion of mPGES-1 in Apc-mutant mice results in marked and persistent suppression of intestinal cancer growth by 66%, whereas suppression of large adenomas (>3 mm) was almost 95%. This effect occurred despite loss of Apc heterozygosity and beta-catenin activation. However, we found that mPGES-1 deficiency was associated with a disorganized vascular pattern within primary adenomas as determined by CD31 immunostaining. We also examined the effect of mPGES-1 deletion on carcinogen-induced colon cancer. The absence of mPGES-1 reduced the size and number of preneoplastic aberrant crypt foci (ACF). Importantly, mPGES-1 deletion also blocked the nuclear accumulation of beta-catenin in ACF, confirming that beta-catenin is a critical target of PGE(2) procarcinogenic signaling in the colon. Our data show the feasibility of targeting mPGES-1 for cancer chemoprevention with the potential for improved tolerability over traditional nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors.

Predominance of cyclooxygenase 1 over cyclooxygenase 2 in the generation of proinflammatory prostaglandins in autoantibody-driven K/BxN serum-transfer arthritis.

OBJECTIVE: Prostaglandins (PGs) are found in high levels in the synovial fluid of patients with rheumatoid arthritis, and nonsteroidal blockade of these bioactive lipids plays a role in patient care. The aim of this study was to explore the relative contribution of cyclooxygenase (COX) isoforms and PG species in the autoantibody-driven K/BxN serum-transfer arthritis. METHODS: The prostanoid content of arthritic ankles was assessed in ankle homogenates, and the importance of this pathway was confirmed with pharmacologic blockade. The presence of COX isoforms was assessed by Western blotting and their functional contribution was compared using COX-1-/- and COX-2-/- mice as well as isoform-specific inhibitors. The relative importance of PGE2 and PGI2 (prostacyclin) was determined using mice deficient in microsomal PGE synthase 1 (mPGES-1) and in the receptors for PGI2. RESULTS: High levels of PGE2 and 6-keto-PGF1alpha (a stable metabolite of PGI2) were detected in arthritic joint tissues, correlating strongly with the intensity of synovitis. Pharmacologic inhibition of PG synthesis prevented arthritis and ameliorated active disease. While both COX isoforms were found in inflamed joint tissues, only COX-1 contributed substantially to clinical disease; COX-1-/- mice were fully resistant to disease, whereas COX-2-/- mice remained susceptible. These findings were confirmed by isoform-specific pharmacologic inhibition. Mice lacking mPGES-1 (and therefore PGE2) developed arthritis normally, whereas mice incapable of responding to PGI2 exhibited a significantly attenuated arthritis course, confirming a role of PGI2 in this arthritis model. CONCLUSION: These findings challenge previous paradigms of distinct "housekeeping" versus inflammatory functions of the COX isoforms and highlight the potential pathogenic contribution of prostanoids synthesized via COX-1, in particular PGI2, to inflammatory arthritis.