All references, including patents and patent applications, are hereby incorporated by reference in their entireties.
Arachidonic acid (abbreviated as AA herein) is a ubiquitous polyunsaturated fatty acid (PUFA) that is found esterified to phospholipids at the secondary alcohol of glycerol in all mammalian cellular membranes. Enzymatic hydrolysis of esterified AA by calcium (Ca2+)-induced cytosolic phospholipase 2 (cPLA2) releases free AA, which may be further catalytically converted by the cyclooxygenase (COX) into the intermediate prostaglandin H2 followed by subsequent enzymatic isomerization into the naturally occurring prostaglandins (PGs) and thromboxanes. The five primary prostanoids include prostaglandin F2α (PGF2α), prostaglandin D2 (PGD2), prostaglandin I2 (PGI2), thromboxane A2 (TxA2), and prostaglandin E2 (PGE2), (Jahn, U. et al., Angew. Chem. Int. Ed. 2008, 47, 5894-5955; Wymann, M. P. et al., Nat. Rev. Mol. Cell. Biol. 2008, 9, 162-176; Samuelsson, B. et al., Ann. Rev. Biochem. 1978, 47, 997-1029). These five prostaglandins are lipid mediators that interact with nine specific members of a distinct prostanoid subfamily of G-protein-coupled receptors (GPCRs), designated FP, DP1-2, IP, TP, and EP1-4, respectively (Breyer, R. M. et al., Annu. Rev. Pharmacol. Toxicol. 2001, 41, 661-690). Prostaglandin and PG receptor pharmacology, signaling, and physiology have been studied and well documented (Hata, A. N. et al., Pharmacol. Ther. 2004, 103(2), 147-166; ElAttar, T. M. A., J. Oral Pathol. Med. 1978, 7(5), 239-252; Poyser, N. L., Clinics in Endocrinology and Metabolism 1973, 2(3), 393-410). Prostaglandins are short-lived local signaling molecules that are not stored in cells or tissues but are produced as needed by specific cells of virtually all body tissues. Their target cells reside in the immediate vicinity of their secretion sites. Well-known PG functions include regulation of cell stimulation, growth, and differentiation, immune response and inflammation, allergy, asthma, pain, vasomotor action, neuromodulation, intraocular pressure, and platelet aggregation, as well as mediation of fever, managing of renal blood flow, and induction of labor (Negishi, M. et al., Prog. Lipid Res. 1993, 32(4), 417-434).
As is the case for most prostaglandins, the biosynthesis of PGE2 commences with liberation of free AA from its esterified form in the cell membrane. One key enzyme involved in PGE2 biosynthesis is prostaglandin H synthase (PGHS). PGHS possesses both a COX and a peroxidase function. The COX activity promotes conversion of free AA to the unstable endoperoxide prostaglandin G2 (PGG2) via double oxygen insertion. One inserted oxygen molecule is subsequently reduced by the peroxidase activity of PGHS to provide the versatile biosynthetic cascade intermediate PGH2. The glutathione-dependent enzyme prostaglandin E synthase (PGES) promotes isomerization of PGH2 to PGE2 via peroxide ring opening of PGH2 to provide the highly functionalized hydroxypentanone scaffold of PGE2.

The physiology of PGE2 and the pharmacology of its four known complementary receptor subtypes designated EP1, EP2, EP3, and EP4 are among the most widely studied and published fields of PG research (Sugimoto, Y. et al., J. Biol. Chem. 2007, 282(16), 11613-11617; Suzuki, J. et al., Prostaglandins 2010, 127-133; Regan, J. et al., Life Sciences 2003, 74(2-3), 143-153; Bouayad, A. et al., Current Ther. Res. 2002, 63(10), 669-681; Breyer, M. et al., Kidney Int, Suppl. 1998, 67, S88-S94; Breyer, M. et al., Amer. J. Physiol. 2000, 279(1, Part 2), F12-F23; Negishi, M. et al., Recent Res. Dev. Endocrinol. 2000, 1(1), 133-143; Ma, W. et al., Prog. Inflamm. Res. 2006, 39-93; Mutoh, M. et al., Current Pharmaceutical Design 2006, 12(19), 2375-2382; Hebert, R. et al., Current Topics in Pharmacology 2002, 6, 129-137; Coleman, R. et al., Pharm. Rev. 1994, 46(2), 205-229). PGE2 binds to each of the four EP receptors with high affinity (Anderson, L. et al., Journal of Reproduction and Fertility, 1999, 116, 133-141). The prostaglandin PGE1 (saturated α-chain analog of PGE2), the major eicosanoid synthesized biologically from dihomo-γ-linolenic acid (DGLA) in response to various stimuli, also binds efficiently to all four EP receptor subtypes.

The EP4 receptor is expressed in a wide variety of tissues including those of the skeletal, muscular, central and peripheral nervous, immune, respiratory, cardiovascular, digestive, excretory, and reproductive tissues and is known to be involved in such processes and conditions as bone growth and remodeling, osteoporosis, relaxation of smooth muscle, neuroprotection, ocular inflammation, immune response, and cancer. Modulation of the EP4 receptor may also be involved in the neonatal development of the circulatory system (Fan, F. et al., Clinical and Experimental Pharmacology and Physiology, 2010, 37, 574-580; Bouayad, A. et al., Current Ther. Res. 2002, 63(10), 669-681; Bouayad, A. et al., Am. J. Physiol. Heart Circ. Physiol. 2001, 280, H2342-H2349). Activation of the EP4 receptor by PGE2 increases intracellular cAMP levels, leading to downstream effects associated with antiapoptotic activity and cytoprotection (Fujino, H. and Regan, J., Trends in Pharmacological Sciences, 2003, 24(7), 335-340; Hoshino, T. et al., J. Biol. Chem., 2003, 278(15), 12752-12758; Takahashi, S. et al., Biochem. Pharmacol., 1999, 58(12), 1997-2002; Quiroga, J. et al., Pharmacol. Ther., 1993, 58(1), 67-91).
EP4 receptor agonists are reported to be useful in lowering intraocular pressure and to have application in treating glaucoma. Prasanna, G. et al., Exp. Eye Res., 2009, 89 (5), 608-17; Luu, K. et al., J. Pharmacol. Exp. Ther. 2009, 331(2), 627-635; Saeki, T. et al, Invest. Ophthalmol. Vis. Sci., 2009, 50 (5) 2201-2208.
EP4 receptor agonists are also reported to induce bone remodeling and to have use in the treatment of osteoporosis. Iwaniec, U. et al., Osteoporosis International, 2007, 18 (3), 351-362; Aguirre, J. et al., J. Bone and Min. Res., 2007, 22(6), 877-888; Yoshida, K. et al., Proc. Natl. Acad. Sci. USA, 2002, 99 (7), 4580-4585. Hayashi, K. et al., J. Bone Joint Surg. Br., 2005, 87-B (8), 1150-6.
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