WO2013155406A1 - Contragestion and treating inflammation by modulating sodium channel activity in the epithelium - Google Patents

Abstract

This invention is based on the discovery that activation of the endometrial epithelial sodium channel (ENaC) triggers PGE2 release and production, which is required for embryo implantation. This invention provides a system for developing pharmaceuticals that inhibit ENaC activity or decrease ENaC expression (for example, using small molecule drugs or siRNA). These pharmaceuticals can be used for birth control or contragestion, or to treat inflammatory conditions mediated by PGE2. This invention also provides a system for developing pharmaceuticals that activate endometrial ENaC or increasing its expression. These pharmaceuticals can be used for promoting embryo implantation.

Description

CONTRAGESTION AND TREATING INFLAMMATION BY MODULATING SODIUM CHANNEL ACTIVITY IN THE EPITHELIUM

RELATED APPLICATION

[0001] This International Application claims the priority benefit of U.S. patent application 61/623,520, filed April 12, 2012. The priority application is hereby incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

[0002] The invention relates generally to the fields of fertility, birth control, pregnancy, in vitro fertilization, parturition, and inflammatory disease. More specifically, it relates to modulation of epithelial sodium channel (ENaC) activity as a means for achieving contragestion, maintaining pregnancy, or treating an inflammatory condition.

BACKGROUND

[0003] The world population is projected to continue its grow until at least 2050, with the population reaching 9 billion in 2040. Some predictions put the population in 2050 as high as 11 billion. Almost all growth will take place in the less developed regions, where today's 5.3 billion population of underdeveloped countries is expected to increase to 7.8 billion in 2050.

Overpopulation strains resources that are fundamental to human existence, including fresh water, food, housing, energy, and the environment.

[0004] Accordingly, there is a need for new technologies that makes birth control more effective and more readily accessible throughout the world.

SUMMARY OF THE INVENTION

[0005] The inventors have discovered that activation of the epithelial sodium channel (ENaC) triggers PGE2 release and production. ENaC activity in the endometrium is required for embryo implantation. This invention provides a system for developing pharmaceuticals that inhibit ENaC activity or decrease ENaC expression (for example, using small molecule drugs or siRNA). These pharmaceuticals can be used for birth control or contragestion, or to treat inflammatory conditions mediated by PGE2. This invention also provides a system for developing pharmaceuticals that activate endometrial ENaC or increase its expression. These pharmaceuticals can be used for promoting embryo implantation. [0006] One embodiment of this invention is a method of identifying a pharmaceutical product as an agent for birth control or contragestion. The method typically comprises the steps of obtaining a compound that inhibits transport of sodium ion through an endometrial epithelium sodium channel; administering said compound to a test subject when an embryo is present in the uterus of the subject following fertilization; and then determining whether the compound affects implantation of said embryo in the uterus of the subject. This leads to a method for producing a pharmaceutical composition for birth control, the method comprising obtaining a compound that inhibits ENaC activity or expression, and formulating an effective amount of the compound with a

pharmaceutically compatible excipient for administration to the uterus of a human subject.

Compounds that inhibit transport of sodium ion through ENaC can be identified by combining the compound with cells or a culture systems that express functional ENaC, and measuring the effect of the compound on the flux of sodium ion. Other assays for the identification, screening, and validation of potential therapeutic agents are provided in the description that follows.

[0007] Another embodiment of the invention is a pharmaceutical composition formulated and labeled for use in birth control in a human subject, comprising an effective amount of a compound that inhibits an endometrial epithelium sodium channel. The compound can be a small molecule drug exemplified by but not limited to amiloride, Ac-LHPHLQRL-amide, and an

epoxyeicosatrienoic acid (EET). Alternatively, it can be antisense RNA, interfering RNA, or siRNA that inhibits expression of said endometrial epithelium sodium channel; or it can be an antibody that specifically binds and thereby blocks said endometrial epithelium sodium channel.

[0008] The invention also provides a method of birth control, contragestion, inducing labor, by inhibiting COX-2 activity, or by inhibiting production of prostaglandins such as PGE2 in the uterus of a subject. The method comprises administering to the subject an effective dose of an inhibitor of an endometrial epithelium sodium channel. The inhibitor may be administered into the uterus of the subject: either by way of injection, or by way of topical application.

[0009] Another embodiment of the invention is a method for improving the probability of implantation in a subject of an in vitro fertilized (IVF) embryo. The method comprises increasing the expression or activity of an epithelium sodium channel (ENaC) in the endometrium in the subject: for example, by administering into the uterus of a subject an effective amount of a serine protease.

[0010] A further embodiment of the invention is a method of identifying a pharmaceutical product as an agent for treating an inflammatory condition. The method generally comprises the steps of obtaining a compound that inhibits transport of sodium ion through an epithelium sodium channel, as already described. The compound is then administered to a test subject who has an inflammatory condition; and the ability of the compound to reduce inflammation is determined. This leads to a method for producing a product for treating an inflammatory condition in which an effective amount of an ENaC inhibitor is formulated with a pharmaceutically compatible excipient for administration to a human subject: either systemically, or locally at or around the site of inflammation.

[0011] Another embodiment of the invention is a pharmaceutical composition formulated and labeled for use in treating an inflammatory condition in a human subject, comprising an effective amount of a compound that inhibits an epithelium sodium channel. The compound can be a small molecule drug exemplified by but not limited to amiloride, Ac-LHPHLQRL-amide, and an epoxyeicosatrienoic acid (EET). Alternatively, it can be antisense RNA, interfering RNA, or siRNA that inhibits expression of said epithelium sodium channel; or it can be an antibody that specifically binds and thereby blocks the epithelium sodium channel.

[0012] The invention also provides method of treating an inflammatory condition in a subject, comprising administering to the subject an effective dose of an inhibitor of an epithelium sodium channel. The method comprises administering to the subject an effective dose of an inhibitor of an epithelium sodium channel at or near the site of the inflammation.

[0013] Other embodiments of the invention will be apparent from the description that follows.

DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 illustrates ENaC -mediated signal transduction events and PGE2 release from mouse endometrial epithelial cells. Figure 1(A), Trypsin-induced membrane depolarization assessed by voltage-sensitive fluorometric measurement. Figure 1(B), Changes in intracellular Ca²⁺ levels in endometrial epithelial cells (EECs) in response to trypsin in the presence of amiloride or nifedipin. Figure 1(C), Trypsin-induced PGE2 release from 3-day culture of EEC. Figure 1(D), Quantitative PCR analysis of COX-2 mRNA levels in EECs. Figure 1(E), Western blot analysis of protein levels of phosphorylated form of CREB (P-CREB). Figure 1(F), Fluorescence labeling of P-CREB in EECs with DAPI labeled nucleus. Figure 1(G), qPCR analysis of miR199a and miRlOl levels in EECs.

[0015] Figure 2 shows involvement of ENaC in trypsin-induced decidualization in endometrial epithelial/stromal cell co-culture. Figure 2(A), are bright-field photographs of cocultured stromal cells before and after treatment with trypsin. Figure 2(B), shows trypsin- induced intracellular cAMP increase in the co-culture, which could be inhibited by amiloride.

[0016] Figure 3 shows the requirement of ENaC activity for embryo implantation in vivo. Figure 3(A), Effect of intrauterine injection of amiloride, EIPA or aprotinin on implantation rate in mice. Figure 3(B), Western blotting data showing the effectiveness of in vivo knockdown of ENaCa (the alpha chain of ENaC) by intrauterine injection of siRNA. Figure 3(C), Effect of ENaCa knockdown on implantation rate in mice with a photograph showing implantation sites (arrowed) in the control uterine horn (siRNANC) as compared with treatment using siRNAENaCa. Figure 3(D), Hematoxylin and eosin stains samples showing the effect of ENaCa knockdown on decidualization in mice on day 5, with less decidual cells observed as compared with the control. Figure 3(E), Effect of ENaCa knockdown on uterine miRNA (micro RNA) levels, measured by qPCR, on day 4 and day 7. Figure 3(F), Western blot analysis showing the effect of ENaCa knockdown on the uterine expression of COX-2.

[0017] Figure 4 provides a Western blot analysis that compares endometrial expression of ENaCa between women undergoing IVF treatment with successful and failed pregnancy.

[0018] Figure 5 is a sketch depicting the role of ENaC in initiating decidualization.

[0019] Figure 6(A) shows L-type Ca2+ channel expression in mouse endometrial epithelial cells (EECs). Figure 6(B) is a patch-clamp recording of Ca2+ channel activities (n=7) before (left) and after (right) addition of nifidepine, a small-molecule calcium channel blocking drug.

[0020] Figure 7(A) shows the effect of ENaCa knockdown on decidualization in mice. The number of cells with morphology of decidual cells was significantly reduced in the siRNA-treated uteri, compared with the control. This illustrates the involvement of ENaC activity in embryo implantation. Figure 7(B) shows that interference with ENaC expression with siRNA substantially reduced the expression of implantation markers HoxAlO, IgF2 and LIF.

[0021] Figure 8 provides clinical features of a group of patients undergoing IVF (in vitro fertilization).

[0022] Figure 9 is a compilation of endometrial expression of ENaC amongst women in this study. The endometrial samples from women with failed pregnancy after IVF-ET had significantly lower ENaCa subunit expression.

[0023] Figures 10(A) and 10(B) are Western blots showing phosphorylation of CREB by COX-2. Figure 10(A) shows results of treating cells with the ENaC activator trypsin. Figure 10(B) shows results of actiatinv cells by mechanical stimulation. Inhibition of ENaC expression or function can have an anti-inflammatory effect when administered either lcoally or systemically.

DETAILED DESCRIPTION

[0024] The amiloride-sensitive epithelial sodium channel(ENaC), coded by SCNNl genes within the degenerin/ENaC superfamily, is localized in the apical membrane of a wide variety of epithelia including endometrial epithelium. It is known for its essential role in salt and water homeostasis. (Amiloride-sensitive sodium channel subunit beta [Homo sapiens] : Accession:

P51168.2 Gl: 8928561 ; Amiloride-sensitive sodium channel subunit alpha: Accession: P37088.1 Gl: 585966; Amiloride-sensitive sodium channel subunit gamma: Accession: P51170.4 Gl: 108885072; Amiloride-sensitive sodium channel subunit delta: Accession: P51172.2 Gl: 116242784; SEQ. ID NOs:7 to 12).

[0025] The data presented in this disclosure show that activation of an epithelial ion channel can trigger signaling cascade leading to increased release and production of PGE2. It has been discovered that interfering with ENaC in the uterus (either by channel inhibitors or specific siRNA) can suppress COX-2 and PG release, resulting in implantation failure in mice (Example 3).

Furthermore, abnormal ENaC expression is found in IVF patients with implantation failure

(Example 4).

[0026] Figure 5 provides a working model for how ENaC mediates decidualization. ENaC activation by serine proteases from the embryo causes epithelial cell membrane depolarization that triggers calcium influx leading to: (1) release of PGE2; (2) phosphorylation of CREB that may up- regulate the expression of COX-2; and (3) alteration in COX-2 -targeting micro-RNA. The endometrial epithelial released PGE2 in turn activates cAMP -related pathways in stromal cells, leading to stromal decidualization. This model is provided to assist the reader in understanding possible mechanisms that underlie aspects of the invention, without conveying a limitation on how the invention is practiced.

[0027] Thus, inhibiting ENaC activity and/or reducing its expression in the uterus is a modality for contragestion. Conversely, increasing ENaC activity and/or enhancing its expression in the uterus is a modality for improving the probability of implantation in a subject of an in vitro fertilized (IVF) embryo. Given the wide distribution of the ion channel and the importance of COX-2 and prostaglandins in inflammation, inhibiting ENaC activity and/or reducing its expression is also a new modality for treating inflammatory conditions throughout the body.

Molecular biology of the Amiloride Sensitive Sodium Channel

[0028] The amiloride-sensitive epithelial sodium channel (ENaC) is a constitutively active ion-channel located in various tissues of the body. ENaC is located in the apical membrane of polarized epithelial cells particularly in the kidney (primarily in the collecting tubules), the lung and the colon. ENaC consists of three different subunits: specifically, α, β, and γ, or alternatively δ, β, and γ. Each of the subunits consists of two transmembrane helices and an extracellular loop. ENaC is involved in the transepithelial Na+-ion transport, which it accomplishes together with the

Na+/K+-ATPase.

[0029] Kellenberger et al. have provided a review of the epithelial sodium channel/degenerin family of ion channels and their structure. Physiol Rev. 2002; 82(3):735-67. Chan et al. have investigated the distribution and regulation of ENaC in the murine female reproductive tract. J Membr Biol. 2002;185(2): 165-76. Yang et al., have investigated differential expression and localization of ENaC in mouse endometrium during preimplantation. Cell Biol Int 28, 433-9 (2004). Quadri et al. have reviewed ENaCs as therapeutic targets. Am J Physiol Cell Physiol. 2012 (e-pub). For an overview, the reader may also refer to the text AMILORIDE-SENSITIVE SODIUM CHANNELS: PHYSIOLOGY AND FUNCTIONAL DIVERSITY, D.J. BENOS, 1999. Inhibiting ENaC activity or decreasing ENaC expression as a method of birth control

[0030] As shown in Examples 1 and 2, activation of ENaC in mouse endometrial epithelial cells by an embryo-released serine protease triggers Ca²⁺ influx that leads to PGE2 release, phosphorylation of transcription factor CREB, upregulation of cyclooxygenase 2 (COX-2), the enzyme important for prostaglandin (PG) production and implantation, and alteration of two COX-2-targeting the micro-RNAs: specifically, miR-199a and miR-101. As shown in Example 3, blocking or knocking down expression of uterine ENaC results in implantation failure accompanied with altered levels of uterine COX-2, miR-199a and miR-101.

[0031] Figure 3(A) shows that intrauterine injection with amiloride (an ENaC inhibitor) in an animal model on day 3 of pregnancy can induce dose-dependent reduction in the number of implanted embryos. Figure 3(B) shows significantly reduced levels of ENaC a-chain in the uteri injected with siRNAENaCa compared with control Figure 3(C) shows a reduction in implantation rate that occurs from knocking down expression of the ENaC alpha chain using siRNA.

[0032] Accordingly, the invention provides a method of birth control, contragestion, inducing labor, or inhibiting PGE2 production in the uterus by administering to the subject an effective dose of an inhibitor of an endometrial epithelium sodium channel.

Activating ENaC or increasing its expression as a method of promoting embryo implantation

[0033] As shown in Example 4 and Figures 4, 8, and 9, ENaC expression in the uterus prior to implanting an in vitro fertilized (IVF) embryo is significantly lower in women with implantation failure as compared with those with successful pregnancy. These results demonstrate that endometrial ENaC has a role in regulating PGE2 production and release required for embryo implantation. Defects in ENaC function may be a cause of miscarriage and low successful rate in IVF.

[0034] Thus, the invention provides a method for improving the probability of implantation in a subject of an embryo or blastocyst that is transplanted into the uterus: including but not limited to an in vitro fertilized (IVF) embryo. The method comprises increasing the expression or activity of an endometrial epithelium sodium channel (ENaC) in the subject: for example, by administering into the uterus of a subject an effective amount of a serine protease.

Inhibiting ENaC activity or decreasing ENaC expression as a method of treating inflammation

[0035] ENaC is traditionally viewed to play a primarily role in electrolyte and fluid reabsorption, hyperactivity of which can lead to dehydration of lumens, particularly the airways, giving rise to bacterial infection, thus inflammation, such as in the case of cystic fibrosis. Many currently available anti-inflammation drugs target COX-2 directly. [0036] Figures 10(A) and 10(B) show that ENaC plays a role in regulating COX-2 and prostaglandin release outside the uterus. This provides a basis for inhibiting ENaC activity or decreasing its expression as a method of treating inflammatory conditions of different kinds.

[0037] Thus, the invention also provides method of treating an inflammatory condition. An effective dose of an inhibitor of an epithelium sodium channel at or near the site of the

inflammation, thereby inhibiting COX-2 activity and the production of prostaglandins like PGE2. The method comprises administering to the subject an effective dose of an inhibitor of an epithelium sodium channel at or near the site of the inflammation.

Agents for inhibiting ENaC activity or decreasing ENaC expression

[0038] In order to inhibit ENaC activity or decrease its expression according to this invention, different options are available. An overview is provided in the reference ION CHANNELS AND THEIR INHIBITORS, S.P. Gupta, 2011.

Small molecule drugs

[0039] Small molecule drugs are known and have been developed that interfere with ENaC function. Vallet et al., Nature 389, 607-10 (1997), described the use of amiloride to inhibit ENaC. Kashlan et al. , J. Biol. Chem. 285:35216-35223, 2010, provides Ac-LHPHLQRL-amide as a potential ENaC inhibitor.

[0040] Other small-molecule ENaC inhibitors include arachidonic acid and its metabolites 11,12-epoxyeicosatrienoic acid (11,12-EET), 8,9-EET, and 14,15-EET. Sun et al., J. Am. Assoc. Nephrol. 21 : 1667-1677, 2010; Pavlov et al., Am J Physiol Renal Physiol. 2011 Sep;301(3):F672-81. Other ENaC inhibitors include BAY39-9437 (CT Poll et al., Bayer Pharmaceuticals, Am J Physiol Lung Cell Mol Physiol 281 :L16-L23, 2001), as well as those disclosed in international patent applications WO 07/071,400 and WO 07/071,396.

[0041] To identify small molecule drugs suitable for inhibiting ENaC according to this invention, compounds can be screened using a cell line that expresses ENaC constitutively. To measure ligand binding, isotopically labeled compounds are combined with the cells, and the label associated with the cells is determined after washing. To measure the effect on ENaC activity, test compounds are combined with the cells, and transport or flux of sodium ion across the membrane of the cells is monitored. A test compound can be identified as having an ability to inhibit ENaC activity if the cells treated with the compound show less transport of sodium ion than the same cells before or without treatment, or other cells that have been treated with a compound known not to modulate ENaC function. To measure the effect of a compound on ENaC protein expression, the compound is combined with the cells, and then the level of ENaC protein on the cells is determined by immunoassay or Western blot, in comparison with untreated control cells. Antisense technology

[0042] Another approach is to inhibit ENaC expression by way of interfering RNA and other antisense technology. See generally the texts THERAPEUTIC OLIGONUCLEOTIDES: ANTISENSE, RNAI, TRIPLE-HELIX, GENE REPAIR, ENHANCER DECOYS, CPG, AND DNA CHIPS; Yoon S. Cho- Chung, A.M. Gewirtz et al, 2003; and ANTISENSE THERAPEUTICS, M.I. Phillips, 2010. siRNA and other types of antisense RNA can be created against any of the chains of human ENaC or the animal model being used (SEQ ID NOs:3, 5, 7, 9, and 1 1).

[0043] Small interfering RNA (siRNA) reduces ENaC expression by way of the RNA interference pathway. siRNAs typically have a short (-21 -nucleotide) double-strand RNA with 2-nucleotide 3 ' overhangs on either end. Standard RNA chemistry can be adapted to improve specificity, stability and/or biocompatibility. Exemplary is Stealth RNAi™ technology, developed at Sequitur Inc., and available commercially from Life Technologies, Grand Island, NY, U.S.A. By way of illustration, siRNA for inhibiting expression of the ENaC alpha chain is AAA GCA AAC UGC CAG UAC AUG C (SEQ ID NO: 1) and GCA UGA UGU ACU GGC AGU UUG CUU U (SEQ ID NO:2). To confirm the effect of siRNA on the expression of one or more subunits of ENaC, the siRNA is combined with cells that constitutively express ENaC, and then the level of the corresponding ENaC subunit protein on the cells is determined by immunoassay or Western blot, in comparison with untreated control cells.

[0044] For further reading on the preparation and use of siRNA, the reader is referred to RNA INTERFERENCE: FROM BIOLOGY TO CLINICAL APPLICATIONS, W.P. Min et al., 2010; RNA

INTERFERENCE: APPLICATION TO DRUG DISCOVERY AND CHALLENGES TO PHARMACEUTICAL DEVELOPMENT, P.H. Johnson et al., 201 1.

Antibodies

[0045] Antibodies specific for an endometrial ENaC can be prepared according to standard protocols of immunization, harvesting antibody-expressing cells, immortalizing the harvested cells (for example, using Epstein Barr virus), and selecting clones that produce antibody having the desired specificity. The immunogen can be any of the ENaC chains or a fragment thereof produced by recombinant expression, or a synthesized peptide comprising a portion of an ENaC chain amino acid sequence (SEQ ID NOs:4, 6, 8, 10 or 12). Specific single-chain variable regions can be produced by contacting a library of immunocompetent cells or viral particles with the target antigen, and growing out positively selected clones. Marks et al., New Eng. J. Med. 335:730, 1996, and patent publications WO 94/13804, WO 92/01047, and WO 90/02809.

[0046] Specific antibody can be also be custom ordered from commercial services such as Pacific Immunology, Ramona, CA, U.S.A.; Precision Antibody , Columbia, MD, U.S.A., and AbMax Biotechnology Co., Ltd., Beijing, China. Binding of antibody to cell-surface ENaC can be determined in cells constitutively expressing ENaC by combining the antibody with the cells, and then determining antibody bound by immunohistochemistry for immunoglobulin. The ability of antibody to affect the presentation of ENaC on the cell surface (for example, by promoting endocytosis) can be determined by combining the antibody with the cells, and then determining the level of ENaC expression by immunohistochemistry for ENaC antigen. The ability of antibody to block ENaC function can be determined by combining the antibody with the cells, and measuring the effect on the flux of sodium ion.

[0047] For further reading on the preparation and use of therapeutic antibodies and their equivalents, the reader is referred to THERAPEUTIC MONOCLONAL ANTIBODIES: FROM BENCH TO CLINIC, Z. An, 2009; THERAPEUTIC ANTIBODIES: METHODS AND PROTOCOLS, A.S. Dimitrov, 2009; and RECOMBINANT ANTIBODIES FOR IMMUNOTHERAPY, M. Little, 2009.

Agents for activating ENaC or increasing its expression

[0048] As described in more detail below, selected agents that activate ENaC or increase its expression can be used in the preparation of pharmaceuticals for promoting embryo implantation into the uterine wall, thereby improving the rate of pregnancy.

[0049] V, Vallet et al., Nature 389, 607-10 (1997), describes an epithelial serine protease that activates ENaC. A survey of the identification and use of various proteases that modulate ENaC activity is provided by Kleyman et al., J. Biol. Chem. 284:20447-20451, 2009. In principal, a compound suitable for use in this context may or may not act on ENaC directly, as long as contacting the cell with the compound has the effect of activating ENaC or increasing its expression without causing undesirable side effects.

[0050] To identify compounds suitable for increasing ENaC activity or expression according to this invention, the compounds can be screened using a cell line that constitutively expresses ENaC. Test compounds are combined with the cells, and transport or flux of sodium ion across the membrane of the cells is monitored. A test compound can be identified as having an ability to promote ENaC activity or increases ENaC expression if the cells treated with the compound show more transport of sodium ion than the same cells before or without treatment, or other cells that have been treated with a compound known not to modulate ENaC function.

Pharmaceutical compositions and their use

[0051] This invention provides pharmaceutical compositions, comprising one or more small molecule drugs, peptides, siRNA, antibodies, and other active agents of this invention that affect ENaC activity or expression. [0052] Development of pharmaceutical composition for use in birth control or to treat inflammation involves obtaining a compound that inhibits transport of sodium ion through an epithelium sodium channel or reduces ENaC expression, as described above. The compound is then tested in a suitable animal model for the target disease (e.g., Example 3). Testing an agent for use in birth control comprises administering an ENaC inhibitor to a test subject when an embryo is present in the uterus of the subject following fertilization; and then determining whether the compound affects implantation of said embryo in the uterus of the subject. Testing an agent for use in treating an inflammatory condition comprises administering an ENaC inhibitor to a suitable animal model for the target disease, and then determining whether there has been a reduction in COX-2 activity, reduced production of prostaglandins such as PGE2, or a resolution of any of the symptoms or signs of the inflammatory condition.

[0053] Development of pharmaceutical composition for use in increasing the probability of embryo implantation in the uterine wall, or for increasing fertility involves obtaining a compound that promotes transport of sodium ion through an epithelium sodium channel or increases ENaC expression, as described above. The compound is then tested in a suitable animal model for pregnancy or implantation, where the rate of implantation is compared between treated animals and untreated controls.

[0054] Once an ENaC modulator compound has been selected and tested, an effective amount for treating the condition for which it is being formulated is combined that are both physiologically compatible at the treatment site, and compatible with the active agent.

[0055] For administration systemically (e.g., for the treatment of disseminated inflammatory disease), the active agent can be formulated in the form of a tablet for oral ingestion. For injection in or near a uterine horn, or at or near a site of inflammation, the active agent can be formulated for injection in a compatible buffer, and optionally lyophilized for transport and storage.

[0056] For administration from within the uterus, the composition may be formulated as a salve, gel, or cream. Possible excipients include a buffering agent, a wetting agent such as ethyl alcohol, a levigating agents such as mineral oil or glycerin, and/or a suspension agent such as carboxymethylcellulose or tragacanth. Other excipients may include components that give the topical formulation its desired consistency: such as specialty esters, fatty alcohols, ethoxylates, hard fats, stearic acids, and mono- and triglycerides. The composition may include a preservative to increase the shelf-life of the composition at room temperature.

[0057] The composition may also contain one or more agents that promote penetration of the active agent(s) into or through the mucosa. These may fall into chemical classes such as terpenes, non-ionic surfactants, azone, oleyl surfactants, fatty alcohols, and fatty acid esters. See, for example, US 2007/0269379 Al ; US 2008/0152597 Al ; US 2005/ 0070440 Al ;

US 2011/0196040 Al; and US 2005/0239724 Al . [0058] For general formulation, preparation, storage, and use of pharmaceutical and cosmetic compositions according to this invention, the reader may refer to DRUG DELIVERY: PRINCIPLES AND APPLICATIONS (Wiley Series in Drug Discovery and Development), B Wang et al., 2005;

PERCUTANEOUS ABSORPTION: DRUGS, COSMETICS, MECHANISMS, METHODS, RL Bronaugh et al., 2005; and the current edition of Remington 's Pharmaceutical Sciences.

Glossary

[0059] The term "contragestion", is used in this disclosure to mean reducing the probability of pregnancy of a female following fusion of a female gamete with a male gamete in the uterus.

Contragestion according to this invention can occur but is not limited to impairment of implantation of a fertilized embryo into a uterine wall in the subject. It is a method of birth control.

[0060] An "embryo" as the term is used in this disclosure means a vertebrate at any stage of development following fertilization and prior to birth.

[0061] An "inflammatory condition" is a condition in a mammalian subject wherein at least some of the symptoms and/or signs experienced by the subject are mediated by an inflammatory process. This includes both antigen-specific and non-specific responses to external or internal stimuli. Antigen-specific reactions include but are not limited to Type I hypersensitivity (allergy).

[0062] A sodium channel or ENaC "inhibitor" is a compound (for example, a small molecule drug, an siRNA, or an antibody) that is shown in the cell-based assays such as those provided in this disclosure to inhibit expression of said channel, and/or inhibit the ability of the channel to transport sodium ion across the membrane of the cell in which it is expressed.

[0063] A sodium channel or ENaC "activator" is a compound (for example, a small molecule drug, an siRNA, or an antibody) that is shown in the cell-based assays such as those provided in this disclosure to promote expression of said channel, and/or increase the ability of the channel to transport sodium ion across the membrane of the cell in which it is expressed.

[0064] A sodium channel or ENaC "modulator" is either an ENaC inhibitor or an ENaC activator.

[0065] A "small molecule drug" is a compound with pharmaceutical activity that is no more than 5,000 Da in size.

[0066] The term "antisense RNA" indicates a compound that acts to modulate the translation or other normal operation of a target RNA molecule by way of Watson-Crick base pairing between the compound and the target RNA. The compound itself may be but is not required to be ribonucleic acid.

[0067] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a polypeptide, through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used in this disclosure, the term encompasses intact antibodies of any species including human, antibody fragments, chimeras, humanized antibodies, single-chain variable regions, and equivalent proteins that include at least one antigen combining site of the desired specificity.

[0068] A "pharmaceutical excipient" is a liquid, semi-liquid or solid medium in which a biologically active agent in a pharmaceutical composition is administered to the treatment subject and delivered to the intended site of administration. The excipient is compatible with the active agent (i.e., it does not adversely interfere with the activity or stability of the agent), and is physiologically compatible with the subject (i.e., it is physiologically inert, sterile, and compounded under conditions suitable for human administration). Ingredients of a pharmaceutical excipient may include solvents, creams, gels, buffers, thickeners, emulsifying agents, preservatives, stabilizers, coloring agents, solid matrixes, coatings, and releasing agents.

[0069] An "effective dose" or "effective amount" of an active agent in a composition of this invention is an amount of the agent which (when given in the appropriate manner) is effective in achieving a treatment goal as indicated by the particular context. The amount is effective in achieving the goal in a substantial proportion or majority of subjects in the same situation and phenotype, and having the same condition or treatment goal as the subject actually being treated.

EXAMPLES

[0070] The illustrations provided below show that activation of ENaC in mouse endometrial epithelial cells by an embryo-released serine protease triggers Ca²⁺ influx that leads to PGE2 release, phosphorylation of transcription factor CREB, upregulation of cyclooxygenase 2 (COX-2), the enzyme important for prostaglandin (PG) production and implantation, and alteration of two COX-2 -targeting microRNAs, miR-199a and miR-101.

[0071] Blocking or knocking down uterine ENaC in mice results in implantation failure accompanied with altered levels of uterine COX-2, miR-199a and miR-101. Uterine ENaC expression levels prior to in vitro fertilization (IVF) treatment were found to be significantly lower in women with implantation failure as compared with those with successful pregnancy.

[0072] These data demonstrate that ENaC plays a significant role in regulating PGE2 production and release required for embryo implantation. A defect in ENaC activity or expression may be a cause of miscarriage and low success rate in IVF. Example 1. ENaC activation triggers Ca²⁺-dependent and implantation-related signaling events

[0073] The immediate consequence of sodium ion influx on activation of ENaC is the influx of Na+ into the cell, resulting in membrane depolarization. This example uses a primary culture of mouse endometrial epithelial cells (EECs), to test whether a serine protease known to be released by invading embryo, trypsin, could induce membrane depolarization.

[0074] Figure 1 illustrates ENaC -mediated signal transduction events and PGE2 release from mouse endometrial epithelial cells. Figure 1(A), Trypsin-induced membrane depolarization assessed by voltage-sensitive fluorometric measurement in 1 -day primary culture of mouse endometrial epithelial cells (EECs). Fluorescence photographs of EECs before (left) and after (right) treated with trypsin (20 μg/mL). Color coding bar showing increasing fluorescence intensity indicating membrane potentials from strongly hyperpolarized (purple) to strongly depolarized (red to white). The insets show higher resolution images of the circled areas. Bottom left: Calibration curve with measured changes in fluorescence intensity of DiBAC4(3) (% of baseline) as a function of membrane potential changes (calculated from changes in K-gluconate concentrations in the presence of 2 μΜ valinomycin). Bottom right: Summary of membrane potential changes (AVm) elicited by trypsin, trypsin after pretreatment with amiloride or aprotinin. (n = 15-31, ***p<0.001 compared with the trypsin-treated group).

[0075] Figure 1(B), Representative time course changes in intracellular Ca²⁺ levels (indicated by 340/380 ratio of Fura-2) in EECs in response to trypsin (20 μg/mL) in the presence of amiloride (10 μΜ) or nifedipine (10 μΜ), with corresponding statistical analysis (n= 20- 81, ***p<0.001 compared with the trypsin-treated group). Figure 1(C), Summary of trypsin-induced PGE2 release from 3-day culture of EEC (n = 4-6, **p<0.01, ***p<0.001). Figure 1(D), Quantitative PCR (qPCR) analysis of COX-2 mRNA levels in EECs under different conditions (n=4, ***p<0.001). Figure 1(E), Western blot analysis of protein levels of phosphorylated form of CREB (P-CREB). Data are presented as the ratio of P-CREB level to the level of β-tubulin (n=4, *p<0.05, **p<0.01, ***p<0.001). Figure 1(F), Fluorescence labeling of P-CREB in EECs with DAPI labeled nuclei (Bars= 20 μιη). Figure 1(G), qPCR analysis of miR199a and miRlOl levels in EECs (n=4, **p<0.01, ***p<0.001). All qPCR data are presented as relative values normalized with the absolute value under control condition (Ctrl). T: trypsin (20 μg/mL); Ami: amiloride (10 μΜ); Apr: aprotinin (20 μg/mL); Nif: nifidipine (10 μΜ); B: BAPTA/AM (50 μΜ). Data are means ± SEM.

[0076] As shown in Figure 1(A), trypsin (20 μg/mL) was found to induce membrane depolarization (+22.24 ± 2.2 mV) in EECs, which was abrogated by either amiloride (10 μΜ), a specific blocker of ENaC, or aprotinin (20 μg/mL), a protease inhibitor. Given that Ca²⁺ mobilization, which is usually triggered by membrane depolarization, had been reported to facilitate COX-2- dependent production/release of PGE2, it was hypothesized that the serine protease-induced ENaC mediated membrane depolarization could result in Ca mobilization leading to PGE2 production/release. The effect of trypsin was tested on intracellular Ca²⁺ mobilization. As shown in Figure 1(B), addition of trypsin (20 μg/mL) induced sustained Ca²⁺ elevation in EECs, which was inhibited by amiloride (10 μΜ) and aprotinin (20 μg/mL), suggesting the involvement of ENaC in mediating the effect of trypsin. The trypsin-induced Ca²⁺ rise was largely reduced in Ca²⁺-free solutions suggesting Ca²⁺ influx through Ca²⁺ channels. Consistent with this notion, nifedipine (10 μΜ), a blocker of the voltage-dependent Ca²⁺ channel, abolished the trypsin-induced Ca²⁺ rise.

[0077] These results suggest that the ENaC-mediated membrane depolarization induced by the serine protease may activate voltage dependent Ca²⁺ channel leading to Ca²⁺ influx. To test this, L-type voltage-dependent Ca²⁺ channel mRNA expression and functional channel activities in EECs by the patch-clamp technique. Figure 6(A) shows PCR detection of L-type Ca²⁺ channel expression in mouse endometrial epithelial cells (EECs). Figure 6(B) is a patch-clamp recording of voltage- dependent Ca²⁺ channel activities (n=7). Left panel shows whole-cell currents elicited from -40 to 20 mV in a EEC before (Ctrl) and after addition of nifidepine (3μΜ). Right panel shows corresponding current-voltage curves showing voltage-dependent characteristic of L-type Ca²⁺ channel.

[0078] An ELISA kit was used to test whether trypsin could induce ENaC-dependent PGE2 release. It was found that trypsin (20 μg/mL) could induce a nearly 3-fold increase in PGE2 release from the epithelial culture, which could be attenuated by the pretreatment with either amiloride (10 μΜ) or aproptonin (20 μg/mL) (Figure 1(C)). Moreover, the trypsin induced PGE2 release from EECs was abolished by BAPTA/AM (50 μΜ), an intracellular calcium chelator, suggesting the involvement of intracellular Ca²⁺ in triggering PGE2 release. Taken together, the results suggest that the activation of ENaC by the serine protease may result in membrane depolarization, which in turn leads to Ca²⁺ influx, resulting in PGE2 release from EECs.

[0079] It has been suggested that persistent elevation of PGs is required for decidualization. ENaC may also play a role in PGs production during implantation, for example, by regulating the expression of COX-2, a key enzyme in PGs synthesis. As shown by the quantitative PCR result (Figure 1(D)), a significantly elevated mRNA level of COX-2 was found in the EECs after 15 min treatment with trypsin (20 μg/mL), which was abolished by pre-treating the cells with either amiloride (10 μΜ) or nifidipine (10 μΜ). These results suggest that up-regulation of COX-2 in EECs may be induced by ENaC activation-dependent Ca²⁺ influx. In this regard, transcription of COX-2 has been reported to be activated by the phosphorylation of cAMP/Ca²⁺ response element binding protein (CREB), a transcription factor known to regulate PGs production.

[0080] Although ENaC has never been implicated in the regulation of gene transcription, the observed Ca²⁺ influx upon protease-induced ENaC activation prompted us to test whether ENaC activation could lead to CREB phosphorylation, thereby regulating COX-2 expression and PGs production in EECs. Indeed, Western blot analysis showed that the phosphorylated form of CREB (P-CPvEB) was significantly increased in EECs after the treatment with trypsin (20 μg/mL), which was attenuated by the pretreatment with amiloride (10 μΜ) or nifedipine (10 μΜ) (Figure 1(E)). Immunofluorescence also showed an increased accumulation of P-CREB in the nuclei of cultured EECs after treatment with trypsin, which was blocked by amiloride (Figure 1(F)). These results suggested that ENaC activation could regulate transcriptional activities of COX-2.

[0081] It has also been suggested that COX-2 can be regulated at post-transcriptional level by microRNAs (miRNAs), which are small non-coding RNAs that may interfere with target protein translation. In the present study, two miRNAs, miR-199a and miR-101, which had been shown to suppress COX-2 and be critically involved in embryo implantation, were significantly reduced in the trypsin-treated EECs, and this reduction could be reversed by either amiloride or nifidipine

(Figure 1(G)), suggesting post-transcriptional regulation of COX-2 via miRNAs by ENaC activation. Taken together, the results indicate that the persistently high levels of PGs required for decidualization and implantation may be achieved by the activation of ENaC with subsequent Ca²⁺ influx, which may upregulate COX-2 expression either by CREB-mediated transcriptional activity or by miR-199a and miR-101 targeting COX-2.

Example 2. ENaC activation induces decidualization in vitro

[0082] This example tests whether the activation of endometrial ENaC by the serine protease could indeed lead to stromal cell decidualization.

[0083] Figure 2 shows involvement of ENaC in trypsin-induced decidualization in endometrial epithelial stromal cell co-culture. Figure 2(A), Bright-field photographs (bars=50 μιη) show the morphology of the cocultured stromal cells before (Ctrl) and after treatment with trypsin (T, 20 μg/mL). Fluorescence photographs (bars=20 μιη) show the anti-desmin signal on the co- cultured stromal cells before and after trypsin treatment, in the absence or presence of amiloride (Ami, 10 μΜ) or PGE2 (10 μΜ). Figure 2(B), Summary of trypsin-induced intracellular cAMP increase in the co-culture, which could be inhibited by amiloride (Ami, 10 μΜ) and EP4 receptor antagonist AH2384 (AH, 10 μΜ). n= 6, *p<0.05, ***p<0.001.

[0084] An epithelial-stromal co-culture was established. As shown in Figure 2(A), the treatment of the co-culture with trypsin (20 μg/mL, 4-6h) induced significant morphological changes of the stromal cells from elongated cells into giant, polygonal and multi-nuclei cells, as

characterized for decidual cells in vitro. In addition, the expression of desmin, a reported marker of decidual cells, was found up-regulated in the trypsin-treated co-culture, as indicated by the immunostaining results (Figure 2(A)). Pretreatment with amiloride (10 μΜ) attenuated the effect of trypsin both in inducing morphological change and desmin expression on the stromal cells, while the inhibitory effect of amiloride could be reversed by adding PGE2 (10 μΜ) to the co-culture

(Figure 2(A)). [0085] These results suggested that the serine protease could induce stromal decidualization in vitro by activating ENaC in the endometrial epithelial cells, an action that could be mimicked by PGE2. As a decidualizing molecule, PGE2 is believed to activate EP2/EP4 receptors on stromal cells, which in turn leads to the accumulation of intracellular cAMP ([cAMP]i) and thus decidualization.

[0086] The next experiment tested whether ENaC activation could lead to EP2/EP4 receptor- mediated elevation of cAMP in EECs. Trypsin (20 μg/mL, 20 min) was added to the EEC-stroma co-culture and found elevated [cAMP]i level, which could be attenuated by pretreatment with amiloride (10 μΜ) (Figure 2(B)). Consistent with the morphological result (Figure 2(A)), adding PGE2 could also reverse the inhibitory effect of amiloride on the trypsin-induced [cAMP]i in the co- culture. Moreover, EP4 receptor antagonist, AH2384 abolished the enhancing effect of trypsin on [cAMP]i in the co-culture, confirming the involvement of the PG receptor in mediating the effect of ENaC activation on intracellular cAMP production. Taken together, the results obtained in vitro have clearly demonstrated that the activation of ENaC by the serine protease could lead to stromal decidualization involving PGE2 and it receptor.

Example 3. Interfering with ENaC expression/function impairs embryo implantation in mice

[0087] This example tests whether interfering with ENaC function in vivo could impair implantation/ decidualization.

[0088] Figure 3 shows requirement of ENaC for embryo implantation in vivo. Figure 3(A), Effect of intrauterine injection (on day 3 post mating) of amiloride, EIPA or aprotinin on implantation rate in mice, indicated by the number of implanted embryos observed in each uterine horn on day 7. Ctrl: control; Amil: amiloride (0.01-5 mM); EIPA: ethylisopropyl-amiloride (0.01- 0.1 mM); Apr: aprotinin (200 μg/mL). (n=8-32, ***p<0.001 compared with Ctrl. Figure 3(B), Western blotting data showing the effectiveness of in vivo knockdown of ENaCa by intrauterine injection of siRNAENaCa (n=4, **p<0.01 compared with siRNANC). Figure 3(C), Effect of ENaCa knockdown on implantation rate in mice with a photograph showing implantation sites (arrowed) in the control uterine horn (siRNANC) as compared with the siRNAENaCa treated one. Figure 3(D), H-E pictures showing the effect of ENaCa knockdown on decidualization in mice on day 5, with less decidual cells observed as compared with the control. Figure 3(E), Effect of ENaCa knockdown on uterine miRNA levels, measured by qPCR, on day 4 and day 7 (n=4, **p<0.01 ). Figure 3(F), Western blot analysis showing the effect of ENaCa knockdown on the uterine expression of COX-2 on day 4 and day 7 (n=4, **p<0.01).

[0089] As shown in Figure 3, intrauterine injection with amiloride (100 μΜ-5 mM) on day 3 of pregnancy could induce dose-dependent reduction in the number of implanted embryos in the amiloride -treated uterine horns compared with that in the vehicle-treated control horns on day 7 of pregnancy (Figure 3(A)). To exclude possible non-specific effect of amiloride on sodium-proton exchanger (NHE), ethyl isopropyl amiloride (EIPA), a specific blocker of NHE was also applied. Unlike amiloride, injection with EIPA (100 μΜ) showed no significant effect on implantation rate in mice (Figure 3(A)).

[0090] To confirm that serine proteases (to which ENaC is sensitive) are required for implantation, aprotinin was applied to inhibit serine proteases in the pregnant mice. Similar to amiloride, aprotinin (200 μg/mL) was also found to significantly reduce the number of implanted embryos (Figure 3(A)). The impaired implantation resulted from interfering with ENaC activation, either by protease inhibitor or ENaC blocker, indicates the requirement of ENaC in the process of embryo implantation. To further confirm that ENaC is required for implantation, the expression of ENaC was manipulated in vivo using siRNAs targeting ENaCa (siRNAENaCa). Both

siRNAENaCa (20 pmole/horn) and non-silencing siRNAs for negative control (siRNANC, 20 pmole/horn) were applied on day 3 of pregnancy by intrauterine injection.

[0091] The expression of ENaCa protein in the uteri was checked on day 4 by Western blotting (Figure 3(B)), showing significantly reduced protein level of ENaCa in the uteri injected with siRNAENaCa compared with those injected with siRNANC. The implanted embryo number counted on day 7 of pregnancy in the ENaC -knockdown uterine horns was found to be significantly reduced compared with the control (Figure 3(C)).

[0092] Using hematoxylin-eosin (H-E) staining, the morphology of the uteri was evaluated. It was observed that on day 5, the number of cells with vacuolated cytoplasm and condensed chromatin (typical morphology of decidual cells) was significantly reduced in the siRNAENaCa- treated uteri as compared with the control uteri (Figure 3(D)), indicating impaired decidualization upon ENaC knockdown. Thus, the in vivo ENaC knockdown experiment confirms the results obtained using amiloride or aprotinin, demonstrating the requirement of ENaC in implantation and decidualization.

[0093] The morphology of the uteri was observed on day 5. Figure 7(A) (H-E staining) show the effect of ENaCa knockdown on decidualization in mice. The number of cells with typical morphology of decidual cells was significantly reduced in the siRNA_ENaCa-treated uteri, compared with the control uteri, indicating impaired decidualization upon ENaC knockdown. This confirms the results obtained using amiloride or aprotinin, demonstrating the requirement of ENaC activity for implantation.

[0094] A role of ENaC in implantation is also demonstrated by the effect of amiloride and ENaC knockdown on implantation markers, HoxAlO, IgF2 and LIF. Figure 7(B) show results of a qPCR assay. Interference with ENaC function/expression substantially reduced the expression of these markers. This is consistent with the hypothesis that ENaC activation is an upstream event that modulates implantation. [0095] Previous studies have demonstrated a dynamic uterine expression pattern of COX-2 in peri-implantation in mice. The uterine COX-2 has been found to be at a low to undetectable level on day 4 before embryo attachment, with increasing expression levels after the attachment up to day 6.

[0096] The impaired implantation in ENaC knockdown mice was investigated to determine if it was associated with a disrupted COX-2 expression levels. The expression levels of COX-2, miR- 101 and miR-199a was evaluated in the siRNA-treated uteri of day 4 (before implantation) and day 7 (after implantation). As shown in Figure 3(E), the expression of both miRlOl and miR199a were significantly reduced on day 4, while enhanced on day 7 in siRNAENaCa treated uteri as compared with the control. Since these miRNAs are known to suppress COX-2, the reduced and increased levels of the miRNAs on day 4 and 7 upon ENaC knockdown, respectively, were consistent with the observed COX-2 protein levels, which were increased on day 4 but significantly reduced on day 7 in siRNAENaCa treated uteri (Figure 3(F)).

[0097] The altered miRNAs/COX-2 expression in ENaC knockdown animals with implantation failure confirms the involvement of ENaC in regulating COX-2/PGE2 during implantation. The altered miRlOl and miR199a levels seen in siRNAENaCa treated uteri indicate the possible involvement of ENaC in epigenetic regulation of COX-2 expression and thus PGE2 production during decidualization/implantation. Thus, the findings also reveal a previously unsuspected role of ENaC in epigenetic regulation, especially where miRNAs are concerned, although the detailed mechanisms await further investigation.

Example 4. Reduced uterine ENaC expression in patients with IVF failure

[0098] Despite major improvement in assisted reproduction techniques (ART), the clinical pregnancy rate per embryo transfer (ET) in fresh ART cycles, or successful rate of IVF, remains at a low end of 31%. Since ENaC is required for embryo implantation as demonstrated presently in mice, alteration in ENaC expression or function may be a cause of implantation failure during IVF in humans.

[0099] To evaluate this, the expression levels of ENaC was determined in human endometrial samples obtained prior to IVF treatment and compared between women with successful (n=16) and failed (n=16) pregnancy. The demographic and clinical data of the patients showed no significant differences in age, ovarian hormonal profiles, endometrial thickness and oocyte quality between the two groups.

[0100] Figure 4 presents a comparison of endometrial expression of ENaCa between women undergoing IVF treatment with successful and failed pregnancy. Western blot analysis of protein levels of ENaCa from endometrial biopsy samples collected before the cycle of IVF-ET treatment. Data are presented as the ratio of ENaCa subunit levels to the level of β-actin (n=16 in each group *p<0.05)

[0101] As shown in Figure 4, the endometrial samples from women with failed pregnancy after IVF-ET showed a significantly lower averaged level in ENaCa subunit expression as compared with that from women with successful pregnancy. Five out of 16 patients with failed pregnancy showed a very low endometrial ENaCa expression which was well below the lowest expression level found in those with successful pregnancy.

[0102] Figure 8 provides a statistical analysis of patients' clinical parameters. There were no significant differences in age, ovarian hormonal profiles, endometrial thickness and oocyte quality between the two groups.

[0103] Figure 9 is a comparison of endometrial expression of ENaC between women undergoing IVF treatment with successful and failed pregnancy. Western blot analysis was done to determine protein levels of ENaCa from endometrial biopsy samples collected before the cycle of IVF-ET treatment. The data are presented as the ratio of the level of ENaC subunits α, β, and γ compared with the level of β-Actin. n=16 (successful pregnancy) and 17 (failed pregnancy).

**: P<0.01 ; ***: P<0.001 ; ns: P>0.05. The endometrial samples from women with failed pregnancy after IVF-ET showed a significantly lower average level of ENaCa and ENaCy subunit expression, compared with women with successful pregnancy.

[0104] These results, together with the results obtained from mice in vitro and in vivo, suggest that abnormal ENaC expression could lead to implantation failure in humans. Since ENaC expression is subject to regulation by ovarian hormones, its normal expression pattern may be altered during IVF with ovarian overstimulation, which may contribute to the low pregnancy rate in IVF. It is also noted that mutations of ENaC are well-documented, with abnormally enhanced ENaC function as observed in Liddle syndrome and abnormally reduced ENaC activity as observed in pseudohypoaldosteronism type 1 syndrome. Thus, a defect in ENaC, either expression or function due to its mutations, may be one of the underlying mechanisms for spontaneous miscarriage and implantation failure during IVF

Example 5. Interfering with ENaC expression/function is anti-inflammatory

[0105] This example was conducted using the human airway epithelial cell line, HBE as a model for inflammatory disease.

[0106] Figure 10(A) is a Western blot detecting phosphorylation of CREB. The cells were treated with the ENaC activator trypsin (20 μg/ml). The trypsin increased phosphorylation of transcription factor CREB in a time-dependent manner. [0107] Figure 10(B) shows results of another experiment in which ENaC was activated by mechanical stimulation (shaking for 30 minutes). Activating cells in this way also led to CREB phosphorylation. The phosphorylation was abolished by the ENaC inhibitor amiloride (10 μΜ).

[0108] cAMP response element-binding protein (CREB) is a cellular transcription factor that binds cAMP response elements (CRE). CREB activity depens on phosphorylation, and regulates expression of COX-2. Prostaglandin-endoperoxide synthase 2 (COX-2) is an enzyme implicated in the biosyntehsis of inflammatory mediators such as prostaglandins (PGD2, PGE2, PGF_2a), prostacyclin (PGI2), and thromboxane A2. Since CREB facilitates COX-2 dependent production and release of mediators such as PGE2, inhibiting ENaC activity would decrease the production of such mediators, thereby having an anti-inflammatory effect.

[0109] The results shown here are similar to what was observed with the uterine endometrial epithelial cells (Example 3). Clearly, ENaC is involved in regulation of the COX-2 pathway in tissues outside the uterus. The data confirm that inhibition of ENaC either locally or systemically can be used as a means for treating inflammatory conditions.

Example 6. Methods used in preceding examples

[0110] Mice and intra-uterine injection. Female ICR mice were purchased from the Laboratory Animal Service Centre of the Chinese University of Hong Kong. All animal experiments were conducted in accordance with the university guidelines on animal experimentation and approval by the Animal Ethic Committee of the Chinese University of Hong Kong was obtained for all related procedures. The day a plug was found after mating was designated as day 1 post mating. Intrauterine injection was done on day 3 around 18:00-20:00 pm. Uteri were exposed during abdominal surgery under general anesthesia. 20 μΕ PBS was injected into the lumen of each uterine horn close to the utero-tubal junction.

[0111] Patients and endometrial sample collection. Woman patients, who were diagnosed of infertility without hydrosalpinx syndrome and sought IVF treatment at Women's Hospital of School of Medicine of Zhejiang University, were recruited with written consents. The endometrial samples were collected during endometrium biopsy examination on day 21 (mid-secretory phase) of menstrual cycle before IVF-ET treatment. Western blotting assay was performed on 16 samples each randomly selected from the sample pools of successful pregnancy and failed pregnancy, according to the pregnancy outcome after IVF-ET.

[0112] Primary culture. Endometrial epithelial were isolated and cultured as previously described. Briefly, uteri were obtained from immature ICR mice, sliced longitudinally and treated with 6.5 mg/mL trypsin and 25 mg/mL pancreatin in PBS at 0°C for 60 min and at room

temperature for another 45 min. The uteri were transferred to fresh PBS and gently shaken by hands for 30 sec to release epithelial cells. After collecting the released epithelial cells, the remaining uterine tissue was washed with fresh PBS twice, incubated in 0.1% trypsin at 37 °C for further 15 min, transferred to fresh PBS and shaken for 30 sec to release stromal cells For the cocultures, the epithelial cells were mixed with the stromal cells at ratio of 1 : 1 and incubated in DMEM/F-12 with 10%) (v/v) fetal bovine serum, 1%> (v/v) nonessential amino acids, 100 IU/mL penicillin, and 10 μg/mL streptomycin at 37 °C.

[0113] Patch-clamp. A bath solution containing (mM): Na-gluconate 145, KCl 2.7, CaCh 1.8, MgCh 2, glucose 5.5 and Hepes 10 (pH 7.4) and a pipette solution containing (mM): K- gluconate 135, KCl 10, NaCl 6, MgCh 2 and Hepes 10 (pH 7.2) were used for ENaC currents. To record Ca²⁺ channel currents, cells were bathed in the solution containing (mM): NaCl 130, BaCb 10, CaCh 0.2, TEAC1 3, MgCh 0.6, NaHCOs 14, NaH₂P04 1, Hepes buffer 33, glucose 5.5 (pH 7.2) with pipettes filled with a solution containing (mM): NaCl 10, TEAC1 100, MgS042, EGTA 5.5, CaCh 0.5 and Hepes 10 (pH 7.2).

[0114] Membrane potential measurement. Epithelial cells on cover-slips were washed with a bath solution containing (mM): NaCl 135, KCl 5.8, MgC12 1.2, CaC12 2.5, Hepes 10 and glucose 5 (pH 7.4), and then loaded with the voltage-sensitive dye DiBAC4(3) (1 μΜ). Fluorescence

(495/520 nm excitation/emission) was monitored and calculated to membrane potential change by adding K+-gluconate (5-60 mM) in the presence ofvalinomycin (2 μΜ), as previously described.

[0115] Intracellular calcium measurement. Epithelial cells on cover-slips were loaded with fura2 (3 μΜ) at 37 °C for 30 min. Fluorescence excited at 340 and 380 nm was monitored

[0116] PGE2 measurement. Epithelial cells were grown on Transwell-Col membranes (0.4 μιη) for 3 days. FBS concentration in the culture medium was reduced to 1%> 12 hours before the experiment. Immediately before starting the experiment, the cells were changed into no-FBS media. After treatment, cell-free supernatant was collected and PGE2 content was measured using an EIA kit (Cayman Chemical).

[0117] cAMP measurement. After treatment, co-cultured cells were lysed in 0.1 mM HC1 and 0.1%) Triton-200 for 10 min. cAMP content was measured using an ELISA kit (Assay Design).

[0118] Immunostaining. Cells on cover-slips were fixed with 4%> PFA in PBS for 15 min permeablized with 1%> Triton- 200 in PBS for 5 min and incubated in PBS containing 5%> rabbit serum for 30 min for blocking. Primary antibodies against desmin (Abeam, 1 : 100), and P-CREB (Cell Signaling, 1 : 100) were applied on cells for overnight at 4°C.

[0119] In vivo RNAi. Stealth™ RNA duplex oligoribonucleotides (AAA GCA AAC UGC CAG UAC AUG C and GCA UGA UGU ACU GGC AGU UUG CUU U targeting ENaCa

(siRNAENaCa), Stealth™ RNAi Negative Control Lo GC Duplex (siRNANC), and

Lipofectamine™ 2000 were purchased from Invitrogen. The intrauterine injection surgery was as described above done on day 3 of pregnancy. 20 pmole siRNAENaCa or siRNANC combined with lipofectamine was injected to each uterine horn. siRNAENaCa and siRNANC were injected respectively to each uterine horn of the pair in the same mouse. [0120] Statistics. Data are presented as mean ± SEM (n is the number of tissue preparations, or cells, or experiment times). For two groups of data, two-tail Student's t test was used. For three or more groups, data were analyzed by one-way ANOVA with Dunnett's post hoc test. A value of p<0.05 was considered to be statistically significant.

SEQUENCES

AAA GCA AAC UGC CAG UAC AUG C (SEQ ID NO:l)

GCA UGA UGU ACU GGC AGU UUG CUU U (SEQ ID NO: 2)

amiloride-sensitive sodium channel subunit alpha [Mus tnuscul

ACCESSION NP_035454 XP_987354

tnRNA: SEQ ID NO : 3

Underl i ned sequence is siRNA

1 agtcacagcc cagccacacc tggagccggg agcaggaggc agctccggcc tcctgcaacc 61 cacggtcccc gaggcagaga aggaggtagc agggagctgg aggccagggc tagagagcct 121 agagaagagg acccaggagg agatagggaa ggcaggaaag gaagtgaggc aggatcagag 181 agcctggcac agagagggag acccaaagag aagcgggagt cagctgggcc aagagggcgt 241 gaaagccgga gagtcaaaca gtccgggagg aaaaaggggc aagagggaga ggcgctaagc 301 cagacagggt gcctgctgtg gagacccagg gaggcgctag cgggcaaacg aaggtggcct 361 tcgctgtgac cacttcgctc tgtggccact ccagtgaagc tccgtgctgc ctggttggcc 421 ccaactccag aaggtcagct ggctcctgga gaggtggagg agggtgggag gactgaggaa 481 gagggaactc agcgtgggat gcgggcaccg tcacggacgg ccccattctg cctccatact 541 aatgatgctg gaccacacca gagcccctga gctcaacctt gacctagacc ttgacgtctc 601 caactcaccg aagggatcca tgaagggcaa caatttcaag gagcaagacc tttgtcctcc 661 tctgcccatg caaggactgg gcaaggggga caagcgtgaa gaacaggcgc tgggcccgga 721 accctcagag ccccggcagc ccaccgagga ggaggaggca ctgatcgagt tccaccgctc 781 ctaccgggag ctcttccagt tcttctgcaa caataccacc atccacggtg ccatccgcct 841 ggtgtgctcc aagcacaacc gcatgaagac ggccttctgg gcggtgctct ggctctgcac 901 cttcggcatg atqtactqqc aqtttqcttt gctgttcgag gaatacttca gctaccccgt 961 gagtctcaac atcaacctca attcggacaa gctggtcttc cctgccgtca ctgtgtgcac 1021 ccttaatcct tacagataca ctgaaattaa agaggatctg gaagagctgg accgcatcac 1081 ggaacagacg ctttttgacc tgtacaaata caactcttcc tacactcgcc aggctggggg 1141 ccgccgccgc agcacccgcg acctccgggg tgctctccca caccccctgc agcgcctgcg 1201 cacaccacct ccgcccaatc ccgcccgctc ggcgcgcagc gcgtcctcca gtgtacgcga 1261 caacaatccc caagtggaca ggaaggactg gaaaatcggc ttccaactgt gcaaccagaa 1321 caaatcagac tgcttctacc agacatactc atccggggtg gatgccgtga gagaatggta 1381 ccgcttccat tacatcaaca ttctgtccag actgcccgac acctcgcctg ctctagagga 1441 agaagccctg ggcagcttca tctttacctg tcgtttcaac caggccccct gcaatcaggc 1501 gaattattct cagttccacc accccatgta tgggaactgc tacactttca acaacaagaa 1561 caactccaat ctctggatgt cttccatgcc tggagtcaac aatggtttgt ccctgacact 1621 gcgcacagag cagaatgact tcatccccct gctgtccaca gtgacggggg ccagggtgat 1681 ggtgcacggt caggatgagc ctgcttttat ggatgatggt ggcttcaacg tgaggcctgg 1741 tgtggagacc tccatcagta tgagaaagga agccctggac agcctcggag gcaactacgg 1801 agactgcact gagaatggca gtgatgtccc tgtcaagaac ctttacccct ccaagtacac 1861 acagcaggtg tgcattcact cctgcttcca ggagaacatg atcaagaagt gtggctgtgc 1921 ctacatcttc taccctaagc ccaagggtgt agagttctgt gactacctaa agcagagctc 1981 ctggggctac tgctactata aactgcaggc tgccttctcc ttggatagcc tgggctgctt 2041 ctccaagtgc aggaagccgt gcagtgtgac caactacaag ctctctgctg gctactcaag 2101 atggccgtct gtgaagtccc aggattggat cttcgagatg ctatccttgc agaataatta 2161 cacgatcaac aacaaaagaa acggagttgc taaactcaac atcttcttca aagagctgaa 2221 ctataaaact aattcggagt ctccctctgt cacgatggtc agcctcctgt ccaacctggg 2281 cagccagtgg agcctgtggt tcggctcatc tgtgctgtcc gtggtggaaa tggcggagct 2341 catcttcgac ctcctggtca tcacactcat catgttactg cacaggttcc ggagccggta 2401 ctggtctcca ggacgagggg ccaggggtgc cagggaggtg gcctctaccc cagcttcctc 2461 cttcccttcc cgtttctgtc cccaccctac atccccgcca ccttctttgc cccagcaggg 2521 cacgacccct cccctggccc tgacagcccc tccacctgcc tatgctaccc taggcccctc 2581 tgcctctcca ctggactcgg ctgtgcctgg ctcttctgcc tgtgctccgg ccatggcact 2641 ctgagagagg agagtgctcc tctcacccag gccagtgctc ctgtcacttc agcacatctt 2701 ccacagctgc ccagctgtct ttggtgtgtc ccggaggaac aggctaagca aggggcccag 2761 gaagttgtcc agaggacagg ggctaatgag ctgctcagag ctgccctgcc cctgcttctg 2821 aacactgctt tccacacaag cacgggcaag tcccctttac ccttggatca gccaagccag 2881 acttggagct ttgacaagga acgttcccgg gaaacgacca aacgaaccga acacatataa 2941 acaaggcaca gagaagtggc cacagccttc ccaccccacg accagagact ggcctggcct 3001 cactgctttc aaggacacag atgtctgcta cccctcttga acttgggtgg ggaaccccac 3061 ccaaaagccc ccttgttagc tctttggcaa ttctccttcc ctcactcctc agggtggggg 3121 ctagagtaag cctgacatcc tcctccattc tcaagactct ctctctttca tttggtaccc 3181 tgtaccccag tgcctctgcg tcgcctcctt cttgtgtgcc ttctgagctg tttcttcagc 3241 ctagaaactc cctgctcaaa ggcacctttg cttttgtgaa ctcgttcacc ctatcctgtc 3301 tcccccagga ttgcccccct ctcccctcac ccccacagca tgctgtatta gatgctcaca 3361 ttcttttgtg tccatctccc tgggtagact gaactgtgct cagggatgag ctttgctcat 3421 ttttgtatcc ttccgttcta gcccagtatc ccacttggac caggtaggca gatactcaat 3481 aaatgcttgt tccatcaaaa aaaaaaaaaa aaaaa

in: SEQ ID NO: 4

1 mmldhtrape Inldldldvs nspkgsmkgn n keqdl cpp Iptnqglgkgd kreeqalgpe 61 pseprqptee eealiefhrs yrel fqffcn nttihgai rl vcsknnrmkt afwavlwlct 121 fgmmywqfal I eeyfsypv si ni nl nsdk 1 v pavtvct 1 npyrytei k edl eeldrit 181 eqtlfdlyky nssytrqagg rrrstrdl rg alphplqrl r tppppnpars arsasssvrd 241 nnpqvdrkdw kigfqlcnqn ksdcfyqtys sgvdavrewy rfhyinilsr 1 pdtspal ee 301 ealgsfiftc rfnqapcnqa nysqfhhpmy gncytfnnkn nsniwmssmp gvnnglsltl 361 rteqndfipl Istvtgarvm vhgqdepafm ddgg nvrpg vetsismrke aldslggnyg 421 dctengsdvp vknlypskyt qqvcihscfq enrm kkcgca yi fypkpkgv efcdylkqss 481 wgycyyklqa afsldslgcf skcrkpcsvt nyklsagysr wpsvksqdwi femlslqnny 541 tinnkrngva klniffkeln yktnsespsv tmvsllsnlg sqwslwfgss vlsvvetnael 601 ifdllvitli tnllhrfrsry wspgrgarga revastpass psrfcphpt spppslpqqg 661 ttpplaltap ppayatlgps aspldsavpg ssacapamal

Atniloride-sensitive sodium channel subunit beta [Homo sapiens]

Epithelial Na(+) channel subunit beta

Beta-ENaC

640 aa protein

Accession: P51168.2 Gl : 8928561

mRNA : SEQ ID NO: 5

1 gtgcttcccc gcccctgaac ctgctccctc ccagtcggtc tcgccgcgct cgccgggtgt 61 cccagtgtca ccaacactcg gccgccgccg ccagcttggc gcgcaccgcc gcctccgcca 121 ccgccgacag cgcgcatcct ccgtgtcccc gctccgccgc ccgagcaggt gccactatgc 181 acgtgaagaa gtacctgctg aagggcctgc atcggctgca gaagggcccc ggctacacgt 241 acaaggagct gctggtgtgg tactgcgaca acaccaacac ccacggcccc aagcgcatca 301 tctgtgaggg gcccaagaag aaagccatgt ggttcctgct caccctgctc ttcgccgccc 361 tcgtctgctg gcagtggggc atcttcatca ggacctactt gagctgggag gtcagcgtct 421 ccctctccgt aggcttcaag accatggact tccctgccgt caccatctgc aatgctagcc 481 ccttcaagta ttccaaaatc aagcatttgc tgaaggacct ggatgagctg atggaagctg 541 tcctggagag aatcctggct cctgagctaa gccatgccaa tgccaccagg aacctgaact 601 tctccatctg gaaccacaca cccctggtcc ttattgatga acggaacccc caccacccca 661 tggtccttga tctctttgga gacaaccaca atggcttaac aagcagctca gcatcagaaa 721 agatctgtaa tgcccacggg tgcaaaatgg ccatgagact atgtagcctc aacaggaccc 781 agtgtacctt ccggaacttc accagtgcta cccaggcatt gacagagtgg tacatcctgc 841 aggccaccaa catctttgca caggtgccac agcaggagct agtagagatg agctaccccg 901 gcgagcagat gatcctggcc tgcctattcg gagctgagcc ctgcaactac cggaacttca 961 cgtccatctt ctaccctcac tatggcaact gttacatctt caactggggc atgacagaga 1021 aggcacttcc ttcggccaac cctggaactg aattcggcct gaagttgatc ctggacatag 1081 gccaggaaga ctacgtcccc ttccttgcgt ccacggccgg ggtcaggctg atgcttcacg 1141 agcagaggtc ataccccttc atcagagatg agggcatcta cgccatgtcg gggacagaga 1201 cgtccatcgg ggtactcgtg gacaagcttc agcgcatggg ggagccctac agcccgtgca 1261 ccgtgaatgg ttctgaggtc cccgtccaaa acttctacag tgactacaac acgacctact 1321 ccatccaggc ctgtcttcgc tcctgcttcc aagaccacat gatccgtaac tgcaactgtg 1381 gccactacct gtacccactg ccccgtgggg agaaatactg caacaaccgg gacttcccag 1441 actgggccca ttgctactca gatctacaga tgagcgtggc gcagagagag acctgcattg 1501 gcatgtgcaa ggagtcctgc aatgacaccc agtacaagat gaccatctcc atggctgact 1561 ggccttctga ggcctccgag gactggattt tccacgtctt gtctcaggag cgggaccaaa 1621 gcaccaatat caccctgagc aggaagggaa ttgtcaagct caacatctac ttccaagaat 1681 ttaactatcg caccattgaa gaatcagcag ccaataacat cgtctggctg ctctcgaatc 1741 tgggtggcca gtttggcttc tggatggggg gctctgtgct gtgcctcatc gagtttgggg 1801 agatcatcat cgactttgtg tggatcacca tcatcaagct ggtggccttg gccaagagcc 1861 tacggcagcg gcgagcccaa gccagctacg ctggcccacc gcccaccgtg gccgagctgg 1921 tggaggccca caccaacttt ggcttccagc ctgacacggc cccccgcagc cccaacactg 1981 ggccctaccc cagtgagcag gccctgccca tcccaggcac cccgcccccc aactatgact 2041 ccctgcgtct gcagccgctg gacgtcatcg agtctgacag tgagggtgat gccatctaac 2101 cctgcccctg cccaccccgg gcggctgaaa ctcactgagc agccaagact gttgcccgag 2161 gcctcactgt atggtgccct ctccaaaggg tcgggagggt agctctccag gccagagctt 2221 gtgtccttca acagagaggc cagcggcaac tggtccgtta ctggccaagg gctctgtaga 2281 atcacggtgc tggtacagga tgcaggaata aattgtatct tcacctggtt cctaccctcg 2341 tccctacctg tcctgatcct ggtcctgaag acccctcgga acaccctctc ctggtggcag

2401 gccacttccc tcccagtgcc agtctccatc caccccagag aggaacaggc gggtgggcca

2461 tgtggttttc tccttcctgg ccttggctgg cctctggggc aggggtggtg gagagatgga

2521 agggcatcag gtgtagggac cctgccaagt ggcacctgat ttactctaga aaataaaagt

2581 agaaaatact gagtcca

Protein: SEQ ID NO: 6

1 MHVKKYLLKG LHRLQKGPGY TYKELLVWYC DNTNTHGPKR IICEGPKKKA MWFLLTLLFA

61 ALVCWQWGIF IRTYLSWEVS VSLSVGFKTM DFPAVTICNA SPFKYSKIKH LLKDLDELME

121 AVLERILAPE LSHANATRNL NFSIWNHTPL VLIDERNPHH PMVLDLFGDN HNGLTSSSAS

181 EKICNAHGCK MAMRLCSLNR TQCTFRNFTS ATQALTEWYI LQATNIFAQV PQQELVEMSY

241 PGEQMILACL FGAEPCNYRN FTSIFYPHYG NCYIFNWGMT EKALPSANPG TEFGLKLILD

301 IGQEDYVPFL ASTAGVRLML HEQRSYPFIR DEGIYAMSGT ETSIGVLVDK LQRMGEPYSP

361 CTVNGSEVPV QNFYSDYNTT YSIQACLRSC FQDHMIRNCN CGHYLYPLPR GEKYCNNRDF

421 PDWAHCYSDL QMSVAQRETC IGMCKESCND TQYKMTISMA DWPSEASEDW IFHVLSQERD

481 QSTNITLSRK GIVKLNIYFQ EFNYRTIEES AANNIVWLLS NLGGQFGFWM GGSVLCLIEF

541 GEIIIDFVWI TIIKLVALAK SLRQRRAQAS YAGPPPTVAE LVEAHTNFGF QPDTAPRSPN

601 TGPYPSEQAL PIPGTPPPNY DSLRLQPLDV IESDSEGDAI

Ami Tori de-sensitive sodium channel subunit alpha [Homo sapiens]

Epithelial Na(+) channel subunit alpha

Alpha-ENac

669 aa protein

Accession: P37088.1 Gl : 585966

SEQ ID NO: 7

1 gtgcttcccc gcccctgaac ctgctccctc ccagtcggtc tcgccgcgct cgccgggtgt 61 cccagtgtca ccaacactcg gccgccgccg ccagcttggc gcgcaccgcc gcctccgcca 121 ccgccgacag cgcgcatcct ccgtgtcccc gctccgccgc ccgagcaggt gccactatgc 181 acgtgaagaa gtacctgctg aagggcctgc atcggctgca gaagggcccc ggctacacgt 241 acaaggagct gctggtgtgg tactgcgaca acaccaacac ccacggcccc aagcgcatca 301 tctgtgaggg gcccaagaag aaagccatgt ggttcctgct caccctgctc ttcgccgccc 361 tcgtctgctg gcagtggggc atcttcatca ggacctactt gagctgggag gtcagcgtct 421 ccctctccgt aggcttcaag accatggact tccctgccgt caccatctgc aatgctagcc 481 ccttcaagta ttccaaaatc aagcatttgc tgaaggacct ggatgagctg atggaagctg 541 tcctggagag aatcctggct cctgagctaa gccatgccaa tgccaccagg aacctgaact 601 tctccatctg gaaccacaca cccctggtcc ttattgatga acggaacccc caccacccca 661 tggtccttga tctctttgga gacaaccaca atggcttaac aagcagctca gcatcagaaa 721 agatctgtaa tgcccacggg tgcaaaatgg ccatgagact atgtagcctc aacaggaccc 781 agtgtacctt ccggaacttc accagtgcta cccaggcatt gacagagtgg tacatcctgc 841 aggccaccaa catctttgca caggtgccac agcaggagct agtagagatg agctaccccg 901 gcgagcagat gatcctggcc tgcctattcg gagctgagcc ctgcaactac cggaacttca 961 cgtccatctt ctaccctcac tatggcaact gttacatctt caactggggc atgacagaga 1021 aggcacttcc ttcggccaac cctggaactg aattcggcct gaagttgatc ctggacatag 1081 gccaggaaga ctacgtcccc ttccttgcgt ccacggccgg ggtcaggctg atgcttcacg 1141 agcagaggtc ataccccttc atcagagatg agggcatcta cgccatgtcg gggacagaga 1201 cgtccatcgg ggtactcgtg gacaagcttc agcgcatggg ggagccctac agcccgtgca 1261 ccgtgaatgg ttctgaggtc cccgtccaaa acttctacag tgactacaac acgacctact 1321 ccatccaggc ctgtcttcgc tcctgcttcc aagaccacat gatccgtaac tgcaactgtg 1381 gccactacct gtacccactg ccccgtgggg agaaatactg caacaaccgg gacttcccag 1441 actgggccca ttgctactca gatctacaga tgagcgtggc gcagagagag acctgcattg 1501 gcatgtgcaa ggagtcctgc aatgacaccc agtacaagat gaccatctcc atggctgact 1561 ggccttctga ggcctccgag gactggattt tccacgtctt gtctcaggag cgggaccaaa 1621 gcaccaatat caccctgagc aggaagggaa ttgtcaagct caacatctac ttccaagaat 1681 ttaactatcg caccattgaa gaatcagcag ccaataacat cgtctggctg ctctcgaatc 1741 tgggtggcca gtttggcttc tggatggggg gctctgtgct gtgcctcatc gagtttgggg 1801 agatcatcat cgactttgtg tggatcacca tcatcaagct ggtggccttg gccaagagcc 1861 tacggcagcg gcgagcccaa gccagctacg ctggcccacc gcccaccgtg gccgagctgg 1921 tggaggccca caccaacttt ggcttccagc ctgacacggc cccccgcagc cccaacactg 1981 ggccctaccc cagtgagcag gccctgccca tcccaggcac cccgcccccc aactatgact 2041 ccctgcgtct gcagccgctg gacgtcatcg agtctgacag tgagggtgat gccatctaac 2101 cctgcccctg cccaccccgg gcggctgaaa ctcactgagc agccaagact gttgcccgag 2161 gcctcactgt atggtgccct ctccaaaggg tcgggagggt agctctccag gccagagctt 2221 gtgtccttca acagagaggc cagcggcaac tggtccgtta ctggccaagg gctctgtaga 2281 atcacggtgc tggtacagga tgcaggaata aattgtatct tcacctggtt cctaccctcg 2341 tccctacctg tcctgatcct ggtcctgaag acccctcgga acaccctctc ctggtggcag 2401 gccacttccc tcccagtgcc agtctccatc caccccagag aggaacaggc gggtgggcca 2461 tgtggttttc tccttcctgg ccttggctgg cctctggggc aggggtggtg gagagatgga 2521 agggcatcag gtgtagggac cctgccaagt ggcacctgat ttactctaga aaataaaagt 2581 agaaaatact gagtcca

Protein: SEQ ID NO: 8

1 MEGNKLEEQD SSPPQSTPGL MKGNKREEQG LGPEPAAPQQ PTAEEEALIE FHRSYRELFE 61 FFCNNTTIHG AIRLVCSQHN RMKTAFWAVL WLCTFGMMYW QFGLLFGEYF SYPVSLNINL 121 NSDKLVFPAV TICTLNPYRY PEIKEELEEL DRITEQTLFD LYKYSSFTTL VAGSRSRRDL 181 RGTLPHPLQR LRVPPPPHGA RRARSVASSL RDNNPQVDWK DWKIGFQLCN QNKSDCFYQT 241 YSSGVDAVRE WYRFHYINIL SRLPETLPSL EEDTLGNFIF ACRFNQVSCN QANYSHFHHP 301 MYGNCYTFND KNNSNLWMSS MPGINNGLSL MLRAEQNDFI PLLSTVTGAR VMVHGQDEPA 361 FMDDGGFNLR PGVETSISMR KETLDRLGGD YGDCTKNGSD VPVENLYPSK YTQQVCIHSC 421 FQESMIKECG CAYIFYPRPQ NVEYCDYRKH SSWGYCYYKL QVDFSSDHLG CFTKCRKPCS 481 VTSYQLSAGY SRWPSVTSQE WVFQMLSRQN NYTVNNKRNG VAKVNIFFKE LNYKTNSESP 541 SVTMVTLLSN LGSQWSLWFG SSVLSVVEMA ELVFDLLVIM FLMLLRRFRS RYWSPGRGGR 601 GAQEVASTLA SSPPSHFCPH PMSLSLSQPG PAPSPALTAP PPAYATLGPR PSPGGSAGAS 661 SSTCPLGGP

Amiloride-sensitive sodium channel subunit gamma [Homo sapiens]

Epithelial Na(+) channel subunit gamma

Gamma-ENaC

649 aa protein

Accession: P51170.4 Gl : 108885072

mRNA : SEQ ID NO : 9

1 aagagcccgc ggtggcgctg ccaggggatg ctagcccgag agcgagcaga ggagcagcgc 61 acccgcacga gccttggacc ctttggaacc gaaagcacgc ccgtcctcag agtcccgtcc 121 tcaaagtccc atcctcgcca tggcacccgg agagaagatc aaagccaaaa tcaagaagaa 181 tctgcccgtg acgggccctc aggcgccgac cattaaagag ctgatgcggt ggtactgcct 241 caacaccaac acccatggct gtcgccgcat cgtggtgtcc cgcggccgtc tgcgccgcct 301 cctctggatc gggttcacac tgactgccgt ggccctcatc ctctggcagt gcgccctcct 361 cgtcttctcc ttctatactg tctcagtttc catcaaagtc cacttccgga agctggattt 421 tcctgcagtc accatctgca acatcaaccc ctacaagtac agcaccgttc gccaccttct 481 agctgacttg gaacaggaga ccagagaggc cctgaagtcc ctgtatggct ttccagagtc 541 ccggaagcgc cgagaggcgg agtcctggaa ctccgtctca gagggaaagc agcctagatt 601 ctcccaccgg attccgctgc tgatctttga tcaggatgag aagggcaagg ccagggactt 661 cttcacaggg aggaagcgga aagtcggcgg tagcatcatt cacaaggctt caaatgtcat 721 gcacatcgag tccaagcaag tggtgggatt ccaactgtgc tcaaatgaca cctccgactg 781 tgccacctac accttcagct cgggaatcaa tgccattcag gagtggtata agctacacta 841 catgaacatc atggcacagg tgcctctgga gaagaaaatc aacatgagct attctgctga 901 ggagctgctg gtgacctgct tctttgatgg agtgtcctgt gatgccagga atttcacgct 961 tttccaccac ccgatgcatg ggaattgcta tactttcaac aacagagaaa atgagaccat 1021 tctcagcacc tccatggggg gcagcgaata tgggctgcaa gtcattttgt acataaacga 1081 agaggaatac aacccattcc tcgtgtcctc cactggagct aaggtgatca tccatcggca 1141 ggatgagtat cccttcgtcg aagatgtggg aacagagatt gagacagcaa tggtcacctc 1201 tataggaatg cacctgacag agtccttcaa gctgagtgag ccctacagtc agtgcacgga 1261 ggacgggagt gacgtgccaa tcaggaacat ctacaacgct gcctactcgc tccagatctg 1321 ccttcattca tgcttccaga caaagatggt ggagaaatgt gggtgtgccc agtacagcca 1381 gcctctacct cctgcagcca actactgcaa ctaccagcag caccccaact ggatgtattg 1441 ttactaccaa ctgcatcgag cctttgtcca ggaagagctg ggctgccagt ctgtgtgcaa 1501 ggaagcctgc agctttaaag agtggacact aaccacaagc ctggcacaat ggccatctgt 1561 ggtttcggag aagtggttgc tgcctgttct cacttgggac caaggccggc aagtaaacaa 1621 aaagctcaac aagacagact tggccaaact cttgatattc tacaaagacc tgaaccagag 1681 atccatcatg gagagcccag ccaacagtat tgagatgctt ctgtccaact tcggtggcca 1741 gctgggcctg tggatgagct gctctgttgt ctgcgtcatc gagatcatcg aggtcttctt 1801 cattgacttc ttctctatca ttgcccgccg ccagtggcag aaagccaagg agtggtgggc 1861 ctggaaacag gctcccccat gtccagaagc tccccgtagc ccacagggcc aggacaatcc 1921 agccctggat atagacgatg acctacccac tttcaactct gctttgcacc tgcctccagc 1981 cctaggaacc caagtgcccg gcacaccgcc ccccaaatac aataccttgc gcttggagag 2041 ggccttttcc aaccagctca cagataccca gatgctggat gagctctgag gcagggttga 2101 gaagacagat ctagtcagga ccaccagcca tggtctaagg acatggatcg ggtgccccca 2161 gacgtgtgca caggggaccc tctgccccac tctgggcttt tcagatactc tgaccaaaaa 2221 gcctgcttta aaccgcaaga tggggcctgg gcatgcgcag gaggagccat cgggtactac 2281 gcagcaacac tcacaactgt ccaggctgag ataaatcccg ggacctgaac tattagcacg 2341 tcactagaga ctgggagccg aggcagtggt gctggcccaa gtgaaggcca gagtgaggac 2401 tgatgcagct ctttacgggt cttgagaggg aaggactctt ccaaagcccc aaagccgagg 2461 gtttcaccca cactgccagc ctgggttggg gcccaaggat gtgaccttga gtgtcaaggc 2521 tggacagcta ctgccagatg ccaaagatag gagaaagtgc cagccctgaa gctggagccg 2581 tttgtgaata aactgttctt catcattgac actggagaaa ggtgtcctcc atgccctcag 2641 gcagcagaga actggcccag agcccttgga gtgttggtgg agatcagagt gccgtggtgg 2701 aggtctggga ctatgtcaga gtgtcctcac tttggggcat gggtgggtcc aggagatgga 2761 tttagttatt caattttgtg gatgaataaa ttgaggcaca gaaagattaa gttaccggcc 2821 caaggtgaca cagtgaggag gtggcagagc taggatttga acccagacaa tctgacttca 2881 tgattttgca tccaattcgt gtctgtgcct tttaaagggt gaggtctgtg tcactttggg 2941 tggggagggg gagcatggtg ggccatgctc tgggcagctg ttccaagaca gagctgaccc 3001 ttccattacc aatggcctgt ccctcaccaa caagccaact gccacagatg acccacttca 3061 taccacattc acatctttcc acctgaaatg gctaacaggt attaaatcct tggttggtgt 3121 ttaaagccaa cccaagaaca gggtgttagg tactgtttta agcacctaga taggttagca 3181 taggggactg caaatggtgt tccgcaaagc aaatcctgtt tttgaccatg aactaagaat 3241 tttattttat tttatttttt attttttgta gaaataggat ctcactatgt tgcccaggct 3301 gttcttgaac tcctggcctc aaatgatctt cccacttcag cctcccaaag tgcagggatt 3361 acaggcacaa gccaccgtgc ccagccaaga atttttattt ttgcttttaa atagcaaaag 3421 aagaagattc tgtgactcat gaaaatgata tgagattcga attccagtgt ctataaataa 3481 agttttattg gtacacagca aaaaaaaaaa aaaaaa

Protein: SEQ ID NO: 10

1 MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT 61 LTAVALILWQ CALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE 121 TREALKSLYG FPESRKRREA ESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR 181 KVGGSIIHKA SNVMHIESKQ VVGFQLCSND TSDCATYTFS SGINAIQEWY KLHYMNIMAQ 241 VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMH GNCYTFNNRE NETILSTSMG 301 GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETA MVTSIGMHLT 361 ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAA 421 NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL 481 LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS 541 CSVVCVIEII EVFFIDFFSI IARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD 601 DLPTFNSALH LPPALGTQVP GTPPPKYNTL RLERAFSNQL TDTQMLDEL

Amiloride-sensitive sodium channel subunit delta [Homo sapiens]

Epithelial Na(+) channel subunit delta

Delta-ENac

638 aa protein

Accession: P51172.2 GI : 116242784

mRNA : SEQ ID NO: 11

1 atggctgagc accgaagcat ggacgggaga atggaagcag ccacacgggg gggctctcac

61 ctccaggctg cagcccagac gccccccagg ccggggccac catcagcacc accaccacca 121 cccaaggagg ggcaccagga ggggctggtg gagctgcccg cctcgttccg ggagctgctc 181 accttcttct gcaccaatgc caccatccac ggcgccatcc gcctggtctg ctcccgcggg 241 aaccgcctca agacgacgtc ctgggggctg ctgtccctgg gagccctggt cgcgctctgc 301 tggcagctgg ggctcctctt tgagcgtcac tggcaccgcc cggtcctcat ggccgtctct 361 gtgcactcgg agcgcaagct gctcccgctg gtcaccctgt gtgacgggaa cccacgtcgg 421 ccgagtccgg tcctccgcca tctggagctg ctggacgagt ttgccaggga gaacattgac 481 tccctgtaca acgtcaacct cagcaaaggc agagccgccc tctccgccac tgtcccccgc 541 cacgagcccc ccttccacct ggaccgggag atccgtctgc agaggctgag ccactcgggc 601 agccgggtca gagtggggtt cagactgtgc aacagcacgg gcggcgactg cttttaccga 661 ggctacacgt caggcgtggc ggctgtccag gactggtacc acttccacta tgtggatatc 721 ctggccctgc tgcccgcggc atgggaggac agccacggga gccaggacgg ccacttcgtc 781 ctctcctgca gttacgatgg cctggactgc caggcccgac agttccggac cttccaccac 841 cccacctacg gcagctgcta cacggtcgat ggcgtctgga cagctcagcg ccccggcatc 901 acccacggag tcggcctggt cctcagggtt gagcagcagc ctcacctccc tctgctgtcc 961 acgctggccg gcatcagggt catggttcac ggccgtaacc acacgccctt cctggggcac 1021 cacagcttca gcgtccggcc agggacggag gccaccatca gcatccgaga ggacgaggtg 1081 caccggctcg ggagccccta cggccactgc accgccggcg gggaaggcgt ggaggtggag 1141 ctgctacaca acacctccta caccaggcag gcctgcctgg tgtcctgctt ccagcagctg 1201 atggtggaga cctgctcctg tggctactac ctccaccctc tgccggcggg ggctgagtac 1261 tgcagctctg cccggcaccc tgcctgggga cactgcttct accgcctcta ccaggacctg 1321 gagacccacc ggctcccctg tacctcccgc tgccccaggc cctgcaggga gtctgcattc 1381 aagctctcca ctgggacctc caggtggcct tccgccaagt cagctggatg gactctggcc 1441 acgctaggtg aacaggggct gccgcatcag agccacagac agaggagcag cctggccaaa 1501 atcaacatcg tctaccagga gctcaactac cgctcagtgg aggaggcgcc cgtgtactcg 1561 gtgccgcagc tgctctcggc catgggcagc ctctgcagcc tgtggtttgg ggcctccgtc 1621 ctctccctcc tggagctcct ggagctgctg ctcgatgctt ctgccctcac cctggtgcta 1681 ggcggccgcc ggctccgcag ggcgtggttc tcctggccca gagccagccc tgcctcaggg 1741 gcgtccagca tcaagccaga ggccagtcag atgcccccgc ctgcaggcgg cacgtcagat 1801 gacccggagc ccagcgggcc tcatctccca cgggtgatgc ttccaggggt tctggcggga 1861 gtctcagccg aagagagctg ggctgggccc cagccccttg agactctgga cacctga

Protein: SEQ ID NO: 12

1 MAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL 61 TFFCTNATIH GAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS 121 VHSERKLLPL VTLCDGNPRR PSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR 181 HEPPFHLDRE IRLQRLSHSG SRVRVGFRLC NSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI 241 LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHH PTYGSCYTVD GVWTAQRPGI 301 THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTE ATISIREDEV 361 HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEY 421 CSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA 481 TLGEQGLPHQ SHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV 541 LSLLELLELL LDASALTLVL GGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD 601 DPEPSGPHLP RVMLPGVLAG VSAEESWAGP QPLETLDT [0121] For all purposes in the United States of America, each and every publication and patent document cited herein is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.

[0122] While the invention has been described with reference to the specific embodiments, changes can be made and equivalents can be substituted to adapt to a particular context or intended use, thereby achieving benefits of the invention without departing from the scope of what is claimed.

Claims

CLAIMS The invention claimed is:

1. A method of birth control, contragestion, inducing labor, or inhibiting COX-2 in the uterus of a subject, comprising administering to the subject an effective dose of an inhibitor of an endometrial epithelium sodium channel.

2. The method of claim 1, wherein the inhibitor is a small molecule drug selected from

amiloride, Ac-LHPHLQRL-amide, and an epoxyeicosatrienoic acid (EET).

3. The method of claim 1, wherein the inhibitor is an antisense RNA, interfering RNA, or siRNA that inhibits expression of said endometrial epithelium sodium channel.

4. The method of claim 1, wherein the inhibitor is an antibody that specifically binds and thereby blocks said endometrial epithelium sodium channel.

5. The method of any of claims 1 to 4, which is a method of birth control.

6. The method of aany of claims 1 to 4, which is a method for inducing labor.

7. The method of any of claims 1 to 6, which is a method for inhibiting uterine COX-2.

8. The method of any of claims 1 to 7, wherein the inhibitor is administered into the uterus of the subject.

9. A method of treating an inflammatory condition in a subject, comprising administering to the subject an effective dose of an inhibitor of an epithelium sodium channel at or near the site of inflammation.

10. The method of claim 9, wherein the inhibitor is a small molecule drug selected from

amiloride, Ac-LHPHLQRL-amide, and an epoxyeicosatrienoic acid (EET).

11. The method of claim 9, wherein the inhibitor is an antisense RNA, interfering RNA, or siRNA that inhibits expression of said epithelium sodium channel.

12. The method of claim 9, wherein the inhibitor is an antibody that specifically binds and thereby blocks said epithelium sodium channel.

13. The method of any of claims 9 to 12, which is a method for inhibiting COX-2.

14. The method of any of claims 9 to 13, wherein the inhibitor is administered at or around the site of the inflammation.

15. A method for improving the probability of implantation in a subject of an in vitro fertilized (rVF) embryo, comprising increasing the expression or activity of an endometrial epithelium sodium channel (ENaC) in the subject.

16. The method of claim 15, wherein the increasing the expression or activity of ENaC

comprises administering into the uterus of a subject an effective amount of a serine protease.

17. A method of identifying a pharmaceutical product as an agent for birth control or

contragestion, comprising:

a) obtaining a compound that reduces expression of an endometrial epithelium channel and/or inhibits transport of sodium ion through said channel;

b) administering said compound to a test subject at or around the time when an embryo is present in the uterus of the subject following fertilization; and then

c) determining whether the compound affects implantation of said embryo in the uterus of the subject.

18. A method for producing a pharmaceutical composition for birth control, the method

comprising obtaining a compound that was identified according to the method of claim 17, and then formulating an effective amount of the compound with a pharmaceutically compatible excipient for administration to the uterus of a human subject.

19. A pharmaceutical composition formulated and labeled for use in birth control in a human subject, comprising an effective amount of a compound that inhibits an endometrial epithelium sodium channel.

20. The pharmaceutical composition of claim 19, wherein the compound is a small molecule drug selected from amiloride, Ac-LHPHLQRL-amide, and an epoxyeicosatrienoic acid (EET).

21. The pharmaceutical composition of claim 19, wherein the compound is an antisense RNA, interfering RNA, or siRNA that inhibits expression of said endometrial epithelium sodium channel.

22. The pharmaceutical composition of claim 19, wherein the compound is an antibody that specifically binds and thereby blocks said endometrial epithelium sodium channel.

23. A method of identifying a pharmaceutical product as an agent for treating an inflammatory condition, comprising:

a) obtaining a compound that reduces expression of an epithelium channel and/or inhibits transport of sodium ion through said channel;

b) administering said compound to a test subject who has an inflammatory condition; and then

c) determining whether the compound reduces inflammation in the subject.

24. A method for producing a product for treating an inflammatory condition, comprising obtaining a compound that was identified according to the method of claim 23, and then formulating an effective amount of the compound with a pharmaceutically compatible excipient for administration to the uterus of a human subject.

25. A pharmaceutical composition formulated and labeled for use in treating an inflammatory condition in a human subject, comprising an effective amount of a compound that inhibits an epithelium sodium channel.

26. The pharmaceutical composition of claim 25, wherein the compound is a small molecule drug selected from amiloride, Ac-LHPHLQRL-amide, and an epoxyeicosatrienoic acid (EET).

27. The pharmaceutical composition of claim 25, wherein the compound is an antisense RNA, interfering RNA, or siRNA that inhibits expression of said epithelium sodium channel.

28. The pharmaceutical composition of claim 25, wherein the compound is an antibody that specifically binds and thereby blocks said epithelium sodium channel.