{"paper_id":"045e4b84-8122-49cb-8301-a062ba15fd64","body_text":"Cyclooxygenase-2 (COX-2) and microsomal prostaglandin-E- synthase-1 (mPGES-1)\nplay a key role in several acute and chronic peripheral disease conditions. For\nexample, COX-2 and mPGES-1 are induced in the synovium of rheumatoid arthritis (RA)\npatients. 1 ,  2  Mice deficient in either COX-2 or mPGES-1\nshowed a reduced arthritis score in the collagen-induced arthritis (CIA)\nmodel. 3 ,  4  Prostaglandin-E 2  (PGE 2 )\nis synthesized by COX-2 and mPGES-1, and is found in synovial fluid of RA patients\nsupporting its role in inflammatory pathology. 5  PGE 2  exacerbates CIA in mice through modulation\nof the inflammatory cytokine IL-23 / IL-17 axis. 6\nPGE 2  exerts downstream signaling through activation of four\nmembrane bound G protein-coupled receptors, EP1, EP2, EP3 and EP4. EP2 and EP4\nreceptors promote cAMP signaling, EP1 mediates Ca +2  mediated signaling,\nand EP3 typically inhibits cAMP signaling. Two other prostanoid receptors, DP1 and\nIP, also promote cAMP mediated signaling, like EP2. 7\nCOX-2 selective inhibitors have been developed to reduce pain and\ninflammation in arthritis patients. However chronic use of COX-2 drugs (e.g.\nRofecoxib and Valdecoxib) has resulted in cardiovascular complications in patients,\nas a result they were withdrawn from the USA market. 8  The less selective COX-2 drug celecoxib\n(Celebrex) remains on the market with a black-box warning for cardiovascular\nthrombotic events. The adverse effects of COX-2 drugs have been attributed to\ninhibition of PGI2 synthesis, thus the IP receptor, which plays a cardioprotective\nrole. 9 ,  10  Future anti-inflammatory strategies could\ninvolve inhibition of specific prostanoid receptors or prostanoid synthases, rather\nthan generic block of the entire COX-2 cascade. We found that the EP2 receptor\nmediates a majority of COX-2 pro-inflammatory effects in the brain. 11 – 13  Although the EP4 receptor seems to exert anti-inflammatory\nactions in CNS and other diseases, 14  it promotes inflammation and exacerbates osteoarthritis\ndisease. 15 ,  16  Therefore, targeting EP2 or EP4 receptors\nshould avoid adverse effects mediated by COX-2 inhibition. 8  Recently, an EP4 receptor antagonist,\nGrapiprant, has been approved to treat dogs to reduce the pain and inflammation\nassociated with osteoarthritis, supporting the notion that prostanoid receptors are\npotential targets for development of anti-inflammatory therapy. 17 ,  18\nRecently Pfizer, 19 \nAmgen, 20  and we 21 ,  22  identified distinct classes of EP2 antagonists with good\npotency. However, these compounds displayed sub-optimal selectivity or in vivo\nplasma half-life to use in preclinical models of chronic diseases. We initially\nidentified a cinnamic amide class of EP2 antagonists by high-throughput\nscreening 23  and conducted\nstructure activity relationship studies to generate a second generation lead EP2\nantagonist TG8–15 ( 3a ,  Fig\n1 ), which displayed about 600–1000 fold selectivity for EP2 over\nEP4 and IP receptors, but only had 29-fold selectivity to DP1 receptors. 21  Moreover, compound TG8–15\ndisplayed plasma half-life of only 0.3 h after intraperitoneal administration in\nmice. Therefore, improvement in plasma half-life while maintaining EP2 potency,\nselectivity against prostanoid receptors and improved aqueous solubility were the\nmain goals for the present study before performing a preclinical evaluation of EP2\nantagonist in an animal model of chronic inflammatory disease such as arthritis. In\nthis report, we present lead optimization studies that led to discovery of\nTG8–69 ( Fig 1 ), which displays high\npotency, selectivity, aqueous solubility, and plasma half-life. Furthermore, we\ndemonstrate that TG8–69 dampens the EP2 mediated induction of\npro-inflammatory genes in a novel BV2 microglial cell line overexpressing the human\nEP2 receptor.\n\nThe rat C6 glioma (C6G) cells stably expressing human DP1, EP2, EP4, or\nIP receptors were created in the laboratory 22 ,  23  and grown in\nDulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) supplemented with\n10% (v/v) fetal bovine serum (FBS) (Invitrogen), 100 U/ml penicillin and 100\nμg/ml streptomycin (Invitrogen), and 0.8 μg/ml G418\n(Invitrogen).\nIntracellular cAMP was measured with a cell-based homogeneous\ntime-resolved fluorescence resonance energy transfer (TR-FRET) method (Cisbio\nBioassays), as previously described. 23  The assay is based on generation of a strong FRET\nsignal upon the interaction of two molecules, an anti-cAMP antibody coupled to a\nFRET donor (Cryptate) and cAMP coupled to a FRET acceptor (d2). Endogenous cAMP\nproduced by cells competes with labeled cAMP for binding to the cAMP antibody\nand thus reduces the FRET signal. Cells stably expressing human DP1, EP2, EP4,\nor IP receptors were seeded into 384-well plates in 40 μl complete medium\n(4,000 cells/well) and grown overnight. The medium was carefully withdrawn and\n10 μl Hanks’ Buffered Salt Solution (HBSS) (Hyclone) containing 20\nμM rolipram was added into the wells to block phosphodiesterases. The\ncells were incubated at room temperature for 0.5 h and then treated with 10\nμl vehicle or test compound for 10 or 50 min before addition of\nincreasing concentrations of appropriate agonist: BW245C for DP1,\nPGE 2  for EP2 and EP4, or iloprost for IP. The cells were\nincubated at room temperature for 40 min, then lysed in 10 μl lysis\nbuffer containing the FRET acceptor cAMP-d2 and 1 min later another 10 μl\nlysis buffer with anti-cAMP-Cryptate was added. After 60–90 min\nincubation at room temperature, the FRET signal was measured by an Envision 2103\nMultilabel Plate Reader (PerkinElmer Life Sciences) with a laser excitation at\n337 nm and dual emissions at 665 nm and 590 nm for d2 and Cryptate (50 μs\ndelay), respectively. The FRET signal was expressed as: F665/F590 ×\n10 4 .\n200,000 BV2-hEP2 cells/well (passage 16–20) were grown overnight\nin Poly-D-lysine (Sigma, USA) coated 12 well plates in duplicate in DMEM-F12\n(Gibco, USA) media with 5% FBS (Gibco, USA) and 800 μg/ml G418 (Sigma,\nUSA). The cells were treated with either 0.3 μM or 1 μM\nTG8–69 for 1 hr, followed by 30 nM ONO-AE1-259-01 (provided by ONO\nPharmaceutical Co, Osaka, Japan) for 1 hr and then 100 ng/ml LPS (Sigma, USA)\nfor 2 hrs. For vehicle treatment, a similar dilution of compound vehicle (DMSO)\nin complete media was used. After all treatments, the entire media was removed\nfrom all the wells and RNAs were extracted from the treated cells using RNA\nextraction kit (Zymo Research, USA) as per the manufacturer’s protocol.\nRNA samples were converted into cDNA using cDNA conversion kit (Quanta, USA) and\nfurther used for qPCR analysis. SYBR Green super mix (Quanta, USA) and primers\nfor GAPDH, COX-2, ILβ, IL6 and hEP2 genes (IDT, USA, see  SI Table-2  for the primer\nsequences) were used for PCR reaction carried out in CFX96 Real Time System (Bio\nRad, USA). PCR data was presented as mean fold changes of gene expression in all\nthe treated samples normalized to vehicle treated cells.\nFor the anti-inflammatory assay ΔΔCT values were used for\nstatistical analysis as they were normally distributed compared to their fold\nchanges. ANOVA-with Holm-Sidak multiple comparisons test for post-hoc analysis\nwas used. P values were considered significant at *<0.05.\n\nThe earlier lead compound TG8–15 ( 3a ) contains three\nstructural moieties - the 2-methyl-1 H -indol-1-yl moiety, a middle\nbenzamide ring and a third tetrazole ring ( Fig\n1 ). Previous structure activity relationship (SAR) studies that led to\nidentification of  3a  indicated that the right side tetrazole ring is\ngood for high EP2 potency, aqueous solubility and mouse liver microsomal\nstability. 21  We also\nshowed that high activity can be achieved with an isomeric replacement of the left\nhand indole ring. 21  In this paper,\nwe expand on the SAR by reporting additional heterocyclic replacements for the\ntetrazole ring of TG8–15 and the application of the tetrazole ring to the\npreviously reported isomeric indole left hand part.\nThe novel derivatives with modification on the right side hetero aromatic\ntetrazole ring are synthesized by following  Scheme\n1  (see  supporting\ninformation (SI)  for synthesis procedures, and chemical characterization\ndata of the derivatives). As shown in  Table\n1 , we synthesized a N-methylated tetrazole derivative  3b \n(TG8–185), which displayed 25-fold less potency than  3a . The\npyrazole derivatives  3c  and  3d  (TG8–130 and\nTG8–168) are found to be > 3-fold more potent than  3a . A\ntriazole derivative  3e  was 2-fold less potent than  3a , but\nimidazole derivatives  3f  and  3g  (TG8–237 and\nTG8–239) displayed similar potency to  3a . However, pyrimidine\nderivatives  3h  &  3i  (TG8–186, and\nTG8–238), and a thiazole derivative  3j  (TG8–280) were\n1.5–2-fold less potent than  3a . Interestingly an isoquinoline\nderivative  3k  (TG8–258) displayed similar potency to\n 3a , but a benzimidazole derivative  31  (TG8–246)\ndisplayed 13-fold less potency than  3a . These SAR data reinforce that\nnotion that modification on the right side ring maintains the high EP2 potency and\naqueous-solubility ( Table 1 ). Several of\nthese novel derivatives displayed a modest <100-fold selectivity (see below\nfor a discussion on selectivity) to EP2 over the DP1 and IP prostanoid receptors,\nand two selective compounds  3e  and  3i  showed low stability\nin mouse liver microsomes ( Table 1 ).\nReplacement of the left hand indole of TG8–15 with an isomeric indole\nresulted in TG8–69 ( Fig 1  &  Scheme 1 ), which, gratifyingly, displayed\nsuitable pharmacology and pharmaceutical properties as described in detail\nbelow.\nUsing a cAMP mediated TR-FRET based functional EP2 assay, 27  we first demonstrated that\nTG8–69 inhibits PGE 2  induced EP2 receptor activation in a\nconcentration-dependent manner ( Fig 2A ) in a\nC6-glioma cell line overexpressing human EP2 receptors. In this assay, TG8–69\ndisplayed a competitive mechanism of antagonism of EP2 receptors as shown by Schild\nregression analysis with mean K B  48.5 nM and mean slope of 1.2 (n = 4)\n( Fig 2B ). Schild K B  values\nrepresent the concentration required to cause a 2-fold rightward shift of\nEC 50  of a full agonist. Ideally, a perfect competitive antagonist\nwill display a slope of unity. To compare the potency of TG8–69 with a known\nEP2 antagonist PF-04418948 (a Pfizer compound, purchased from Cayman chemical), we\ntested PF-04418948 in parallel ( Fig\n2C – D ). Previously the Pfizer\ncompound was reported to have a Schild K B  value of 1.8 nM in a cAMP\nmediated functional assay using Chinese hamster ovary (CHO) cells overexpressing\nhuman EP2 receptors. 19 \nSurprisingly, in our  h EP2 overexpressing C6-glioma cell line, the\nPfizer compound displayed 80-fold lower potency with mean K B  = 147 nM,\nand mean slope 1.5 (n = 3) ( Fig 2C – D ). To understand whether the reduced potency is\ndue to equilibrium not being obtained during the bioassay, we tested these two\ncompounds head-to-head with longer pre-incubation time (50 min, vs. 10 min we\nnormally use; thus the total incubation time is 90 vs 50 min). However, neither the\nSchild slope nor the K B  were much affected for either of the two\ncompounds by extending assay incubation time from 50 to 90 min (see  SI Figure 1 ). To determine whether the\ncell line plays a role in the relative potency of these two compounds, we tested\nboth in a novel microglia cell line, BV2-hEP2, which expresses human EP2 receptors\n(Rojas et al., submitted). As shown in the  SI Figure 2 , the Pfizer compound showed\nSchild K B  105 nM, which is 5-times less potency than TG8–69, and\n60-times lower potency than previously reported value. 19  These data suggest that potency does not\ndepend on the cell line, therefore, the observed difference in potency of the Pfizer\ncompound might be attributed to the DiscoveRX assay method used by Pfizer. 19  However, both of these compounds,\nwhile displaying slope values > 1.0, did not impact the maximum cAMP response\nelicited by the full agonist PGE2, suggesting they are competitive antagonists.\nNonetheless, TG8–69 is about 3-times more potent than the Pfizer compound in\nthe same assay. Furthermore, we also tested TG8–69 in a binding assay, in\nwhich it inhibited the binding of radiolabeled H 3 -PGE 2  to EP2\nreceptors with K i  of 135 nM ( Fig\n3A ).\nEP2, DPI, EP4 and IP are Gas coupled prostanoid receptors. Among these DPI\nhas the closest sequence homology to EP2, followed by IP, then EP4, although both\nEP2 and EP4 share a common endogenous ligand PGE 2  for their\nactivation. 28  To determine\nthe selectivity of several novel derivatives ( Table\n1 ) and TG8–69 for EP2 over other Gas-coupled prostanoid receptors,\nwe created C6-glioma cell lines overexpressing DP1, EP4 and IP receptors and used\nthem for counter screening. 22  The\nnovel compounds shown in  Table 1  showed low\nselectivity to either DP1 or IP receptors, except compound  3e \n(TG8–184) which showed > 200-fold selectivity against DP1 and IP\nreceptors. Three other compounds  3c  (TG8–130),  3g \n(TG8–239) and  3h  (TG8–186) displayed > 200-fold\nselectivity against DP1 receptor, but their selectivity against IP receptor was\n< 100-fold ( Table 1 ). However,\ncompound TG8–69 showed >300-fold selectivity against DP1 and EP4, and\n>1000-fold selectivity against IP receptors ( Fig 3B ), so it should be useful for  in vitro  and\n in vivo  proof of concept studies. To learn the selectivity\nagainst EP1 and EP3 receptors, we tested TG8–69 against radiolabeled\nH 3 -PGE 2  binding to EP1 and EP3 receptors at Cerep\nlaboratories (CRO). Interestingly TG8–69 did not show any significant\ninhibition of H 3 -PGE 2  binding to EP1 and EP3 at 3 μM,\nbut it inhibited 90% H3-PGE2 binding to EP2 receptor at 1 μM concentration\n(see  SI Fig 3 ). Additional\ndose-response studies are needed to establish the fold selectivity to EP2 against\nEP1 and EP3 receptors.\nTo determine ADME properties, we first tested several modestly selective\nnovel compounds  3c, 3e, 3h, 3i  ( Table\n1 ) for stability in mouse liver microsomes, but, these compounds showed\n< 10 minutes of half-life. These compounds were not tested in human liver\nmicrosomes for stability. However interestingly compound TG8–69 showed\n> 60 minutes half-life in mouse liver microsomes and subsequently in human\nliver microsomes, when incubated at 1 μM concentration ( Fig 4A ). We then tested TG8–69 against a panel of\nseven cytochrome-450 (CYP450) enzymes in binding assays. The assays were conducted\nas reported according to the methods reported in 24 , and the details are provided in SI. Interestingly when\ntested at 10 μM concentration, TG8–69 showed < 30% inhibition\nin six out of seven CYP450 tested, but CYP2C8 was inhibited about 70%. Additional\nstudies are needed to determine the IC 50  against this and other CYP450\nenzymes. CYP2C8 is an epoxygenase enzyme, associated mainly with metabolism of long\nchain fatty acids, and relatively less involved (in comparison with CYP3A4 and\nCYP2D6) in the metabolism of xenobiotics. Nonetheless, additional studies using a\nknown CYP2C8 metabolizing substrate are needed to confirm whether TG8–69 is a\nstrong inhibitor that would influence a drug-drug interaction. Furthermore,\nTG8–69 did not inhibit the binding of dofetilide to potassium channel\n h ERG at 10 μM concentration ( Fig 4B ). These data indicate that TG8–69 may not\nhave a potential cardiotoxicity liability.\nThe high potency and selectivity, and good in vitro ADME characteristics,\nprompted us to explore in vivo pharmacokinetics for TG8–69. As shown in  Table 2  and  Figure 5 , when C57BL/6 male mice were dosed at 5 mg/kg intravenously\n(i.v.), or 10 mg/kg by oral gavage (p.o.), TG8–69 exhibited a terminal\nhalf-life 6.7 h and 10.5 h respectively. Using AUC inf , the calculated\noral bioavailability for TG8–69 is 14.5%. To evaluate brain-to-plasma ratio\n(B/P ratio) for TG8–69, we conducted a separate study with oral dosing (10\nmg/kg) and determined plasma and brain tissue concentrations at 0.5, 2 and 6 hrs.\nThe data indicated that B/P ratio is 0.01–0.02 (see  Table 2 ) suggesting it is largely peripherally\nrestricted and should be useful for investigating chronic peripheral disease\nmodels.\nTo determine whether we can formulate this compound in an aqueous solution\nfor in vivo use, we tested its solubility in PBS containing 1% DMSO at pH 7.4 using\na nephelometry assay. 26  In this\nassay, when visible light is passed through a solution, part of the incident radiant\nenergy will be scattered. The measurement of the intensity of the scattered light as\na function of the concentration of the dispersed phase is the basis of the\nnephelometric assay. The nephelometric assay can be employed to determine either the\npoint at which a solute begins to precipitate out of a true solution to form a\nsuspension or the concentration at which a suspension when diluted further becomes a\nsolution. The scattered light will remain at a constant intensity until\nprecipitation occurs, at which point it will increase sharply as shown in  SI Figure 4 . In this assay,\nTG8–69 showed solubility of 172 ± 15 μg/mL (500 ± 40\nμM) indicating it is highly aqueous soluble ( SI Fig 4 ).\nThe applications of an EP2 antagonist will be found in several chronic\ninflammatory diseases such as rheumatoid arthritis. EP2 receptor activation\nexacerbates symptoms of experimental inflammatory bowel disease (colitis) by\nincreasing IL-23 expression and reducing IL-12, together causing T-cells to\ndifferentiate to Th17 effectors. 29 \nMoreover, deleterious roles of EP2 are reported in other peripheral events such as\ntumor angiogenesis and preterm delivery. 30 – 35  Therefore,\na well characterized preclinical candidate will enable proof-of-concept tests in\nthese and similar indications. Having developed a lead candidate EP2 antagonist\nTG8–69, we next investigated anti-inflammatory properties in vitro. A routine\nisolation of primary macrophages from peritoneal region, or microglia from mouse\nbrain proved to be low throughput because the primary cells behave variably\ndepending on the animal. Thus, we created a BV2 cell line, which was previously\nderived from mouse microglia, overexpressing human EP2 receptors (Rojas et al., in\npreparation). Upon activation of this cell line with 100 ng/mL lipopolysaccharide\n(LPS), mRNA levels of several proinflammatory genes were induced including COX-2,\nIL-6 and IL-1β. An EP2 specific agonist 0N0-AE1-259-01 at 30 nM further\nexacerbated the induction of these inflammatory genes. Gratifyingly, TG8–69\n(0.3 μM and 1.0 μM) blunted the upregulation of these inflammatory\ngenes in a concentration dependent manner ( Fig\n6 ). EP2 mRNA expression was not affected by either the agonist or the\nantagonist. In a control experiment, incubation of BV2-EP2 cell line with\nTG8–69 (1 uM) in the presence or absence of LPS did not show a significant\neffect on induction of inflammatory mediators and EP2 ( Fig 7 ). These data support the use of this EP2 antagonist as\nanti-inflammatory agent in chronic disease models.\nIn summary, we have developed a novel, selective, aqueous-soluble\nperipherally restricted EP2 antagonist with suitable pharmaceutical properties and\nshowed that this compound dampens pro-inflammatory gene expression in an in vitro\ncell culture model indicating this compound will be useful to explore in vivo models\nwhere EP2 is suspected to play a deleterious role.","source_license":"public-domain-us","license_restricted":false}