WO2019010482A1 - Compositions and methods for modulating cervical ripening - Google Patents

Abstract

Compositions and methods of modulating cervical ripening including initiating cervical ripening and/or inducing labor as well inhibiting preterm cervical ripening and/or preterm birth in a female in need thereof can include at least one of 15-PGDH inhibitors, which can initiate, or amplify, PGE2-mediated cervical ripening, as well as EP2 receptor antagonists, HDAC4 inhibitors, and/or 15-PGDH activators, which can prevent preterm cervical ripening and preterm birth.

Description

COMPOSITIONS AND METHODS FOR MODULATING CERVICAL RIPENING

RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application

No. 62/529,762, filed July 7, 2017, the subject matter of which is incorporated herein by reference in its entirety.

GOVERNMENT FUNDING

[0002] This invention was made with government support under Grant Nos. HD080776 and HD082502 awarded by The National Institutes of Child Health and Human

Development. The United States government has certain rights to the invention.

BACKGROUND

[0003] It is a common clinical situation in obstetrics that labor needs to be induced due to an extended pregnancy, for example beyond the 41-42 weeks gestation time, or due to numerous medical complications, exemplified by pre-eclampsia, diabetes, essential hypertonia and Intra Uterine Growth Retardation (IUGR).

[0004] Labor can be induced in a number of ways. Non-limiting examples of methods to induce labor are physical stimulation processes; administration of oxytocin, prostaglandin E or derivatives thereof, such as misoprostol and dinoproston; rupturing the amniotic sac; expanding the cervix, administrating an intracervical balloon and use of intra cervical Foley catheter (providing an endogenous release of prostaglandin from decidua and cervix). Also combinations of these labor inducing processes can be used. Even if it is common practice to administer these agents or processes to induce labor, females subjected to labor induction suffer from frequent incidences of labor dystocia, including labor arrest, prolonged latent phase of labor and slow progress of labor (protracted labor). It is also estimated that 15-20% of the interventions to induce labor in females with unfavorable cervices fail following local application of prostaglandin E2.

[0005] Mechanisms of prostaglandin-mediated cervical ripening and labor induction are largely unknown. Likewise, it is not understood why some females at term respond to vaginal prostaglandins for cervical ripening whereas others do not. Although failure rates vary depending on preparation, method of administration, definition of failure, dose, and dosing interval, in general, the overall risk of the cervix remaining unchanged or unfavorable for induction of labor 12 to 24 hours after vaginal prostaglandins is 21.6%. Further, prostaglandin (PGE2) treatment leads to uterine hyperstimulation in 1-5.8% of which 31% are associated with abnormalities in fetal heart rhythm. Both problems require emergency treatment and, in many cases, urgent operative delivery.

SUMMARY

[0006] Embodiments described herein relate to compositions and methods of modulating cervical ripening, and particularly relate to compositions and methods of initiating cervical ripening and/or inducing labor as well inhibiting preterm cervical ripening and/or preterm birth in a female in need thereof. It was found that prostaglandin E2 (PGE2), a cervical ripening agent, mediates unique EP2-receptor- signaling pathways in human cervical stromal cells targeting its own synthesis by increasing COX-2 and PTGES expression and decreasing its metabolism by loss of its degradative enzyme 15-PGDH. Downregulation of 15-PGDH was also found to be crucial for PGE2-induced cervical ripening and preterm birth. It was further found that 15-PGDH inhibitors described herein can initiate, or amplify PGE2-mediated cervical ripening, and EP2 receptor antagonists, HDAC4 inhibitors, and/or 15-PGDH activators can prevent preterm cervical ripening and preterm birth. Accordingly, in some embodiments, compositions and methods of modulating 15-PDGH activity can be used to modulate cervical ripening, and induce or prevent preterm labor.

[0007] In some embodiments, a method of inducing cervical ripening and labor in a female in need thereof can include administering to the female a 15-PGDH inhibitor alone or in combination with another labor inducing agent.

[0008] In some embodiments, the labor inducing agent can include a prostaglandin or derivative thereof. The prostaglandin or derivative thereof can be selected from the group consisting of dioprostone, latanoprost, travoprost, fluprostenol, unoprostone, bimatoprost, cloprostenol, viprostol, butaprost, misoprostol, their salts, and their esters. In a preferred embodiment, the labor inducing agent comprises at least one of dioprostone (PGE2) or misoprostol (PGE1).

[0009] In some embodiments, the 15-PGDH inhibitor can include a compound having the following formula (V):

wherein n is 0-2

X⁶ is independently is N or CR^C

R⁶, R⁷, and R^c are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C₃-C2₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6 ring atoms (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(0)(Ci-C₆ alkyl), O, and S), C₆-C24 alkaryl, C₆-C24 aralkyl, halo, -Si(Ci-C₃ alkyf , hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (— CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C₂-C₂₄

alkylcarbonato (-O-(CO)-O-alkyl), C₆-C2₀ arylcarbonato (-O-(CO)-O-aryl), carboxy

(-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl

(-(CO)-NH(Ci-C₂₄ alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C ), cyanato (-0-CN), isocyanato (-0-N⁺=C), isothiocyanato (-S-CN), azido (-N=N⁺=N ), formyl (-(CO)-H), thioformyl (~(CS)~ H), amino (~NH₂), C1-C24 alkyl amino, C5-C2₀ aryl amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), imino (-CR=NH where R is hydrogen, C1-C24 alkyl, C5-C2₀ aryl, C₆-C24 alkaryl, C₆-C24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO₂-OH), sulfonato (-SO₂-O ), C1-C24 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), Ci-C₂₄ alkylsulfinyl (-(SO)-alkyl), C₅-C₂₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), sulfonamide (-SO₂-NH2, -SO₂NY₂ (wherein Y is independently H, aryl or alkyl), phosphono

(-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-P0₂), phosphino (— PH₂), poly alky lethers, phosphates, phosphate esters, groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, combinations thereof, and wherein R⁶ and R⁷ may be linked to form a cyclic or polycyclic ring, wherein the ring is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, and a substituted or unsubstituted heterocyclyl;

U¹ is N, C-R², or C-NR³R⁴, wherein R² is selected from the group consisting of a H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, 0-CH₂- CH₂X, CH₂-CH₂-CH₂X, 0-CH₂-CH₂X, X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, 0(CO)R', COOR' (wherein R' is H or a lower alkyl group), and wherein R¹ and R² may be linked to form a cyclic or polycyclic ring, wherein R³ and R⁴ are the same or different and are each selected from the group consisting of H, a lower alkyl group, O, (CH₂)_nlOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, CH₂-CH₂-CH₂X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, COOR' (wherein R' is H or a lower alkyl group), and R³ or R⁴ may be absent;

and pharmaceutically acceptable salts thereof.

[0010] In some embodiments, R¹ is selected from the group consisting of branched or linear alkyl including -(CH₂)n_!CH₃ (n^O-7), ² wherein n₂=0-6 and X is any of the following: CF_yH_z (y + z = 3), CCl_yH_z (y + z = 3), OH, OAc, OMe, R⁷¹, OR⁷², CN, N(R⁷³)₂,

"³ (n₃=0-5, m=l-5), and "⁴ (n₄=0-5).

[0011] In other embodiments, R⁶ and R⁷ can each independently be one of the following:

each R⁸, R⁹, R¹⁰ R¹¹ _, R¹² R¹³ _, R¹⁴ R¹⁵, R¹⁶ R¹⁷, R¹⁸, R¹⁹, R²⁰ R²¹, R²² R²³, R²⁴ R^:

_R26 _R27 _R28 _R29 _R30 _R31 _R32 _R33 _R34 _R35 _R36 _R37 _R38 _R39 _R40 _R41 _R42 _R43 _R44 _R45 _R46 _R47

R⁴⁸ R⁴⁹ _, R⁵⁰ R⁵¹ R⁵² R⁵³ R⁵⁴ R⁵⁵ R⁵⁶ R⁵⁷ _, R⁵⁸ R⁵⁹ R⁶⁰ R⁶¹ _, R⁶² R⁶ R⁶⁴. R⁶⁵ R⁶⁶ R⁶⁷ _, R⁶⁸ _, R⁶⁹ _{, R}7o _R7i _R72 _R73 _{a R}74 ^ ^ _{same or} diff_erent and are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C2₀ aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(0)(Ci- C₆ alkyl), O, and S), heteroaryl or heterocyclyl containing from 5-14 ring atoms, (wherein from 1-6 of the ring atoms is independently selected from N, NH, N(Ci-C₃ alkyl), O, and S), C₆-C24 alkaryl, C₆-C24 aralkyl, halo, silyl, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (— CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C₂-C₂₄ alkylcarbonato (-O-(CO)-O- alkyl), C₆-C₂₀ arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl (-(CO)-NH(Ci-C₂₄ alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C ), cyanato (-0-CN), isocyanato (-0-N⁺=C^~), isothiocyanato (-S-CN), azido (-N=N⁺=N ), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (~NH₂), Ci-C₂₄ alkyl amino, C5-C2₀ aryl amino, C₂-C₂₄ alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), sulfanamide (-S0₂N(R)2 where R is independently H, alkyl, aryl or heteroaryl), imino (-CR=NH where R is hydrogen, C1-C24 alkyl, C5-C2₀ aryl, C₆-C24 alkaryl, C₆-C24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-S0₂-0^~), Ci-C₂₄ alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), C C₂4 alkylsulfinyl (-(SO)-alkyl), C5-C2₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (wherein Y is independently H, aryl or alkyl), phosphono (-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-PO2), phosphino (— PH2), polyalkyl ethers (-[(CH2)_nO]_m), phosphates, phosphate esters [-OP(0)(OR)2 where R = H, methyl or other alkyl], groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, and combinations thereof, and pharmaceutically acceptable salts thereof.

[0012] In some embodiments, the 15-PGDH inhibitor can inhibit the enzymatic activity of recombinant 15-PGDH at an IC₅₀ of less than 1 μΜ, or preferably at an IC₅₀ of less than 250 nM, or more preferably at an IC₅₀ of less than 50 nM, or more preferably at an IC₅₀ of less than 10 nM, or more preferably at an IC₅₀ of less than 5 nM at a recombinant 15-PGDH concentration of about 5 nM to about 10 nM. [0013] Other embodiments described herein relate to a method of inhibiting cervical ripening and preterm labor in a female in need thereof. The method can include

administering to the female at least one of an EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator.

[0014] The administration of the EP2 receptor antagonist, an HDAC4 inhibitor, and/or a 15-PGDH activator can prevent and/or stop cervical shortening, premature cervical ripening, and/or preterm labor in a female subject in need thereof. Since at risk females are hard to prognoses, the EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator can be used, for example, in preterm (e.g., thirty seven week) gestation by prophylactic administration of the compounds into the cervix of pregnant females and/or in the serum or following a preterm labor and/or membrane rupture test.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figs. l(A-D) illustrate graphs showing PGE2 regulates its own metabolism via EP2 receptors. A. Relative mRNA levels of 15-PGDH (24 h), PTGES (24 h) and COX-2 (6 h) after treatment with DMSO or 100 nM of PGE2, misoprostol, PGF2D, sulprostone or PGD2. B. Feedback mechanism of PGE2 metabolism. PGE2 increases COX-2 and PTGES, simultaneously decreasing 15-PGDH thereby positively regulating its own metabolism C. Prostanoid receptor profile in human cervical stromal cells. Data represent Fragments Per Kilobase of exon per Million fragments mapped (FPKM) from RNA-Seq datasets. D. 15- PGDH mRNA levels in hCSCs pretreated with DMSO or EP2 selective antagonists (2 μΜ) for 1 h followed by treatment with either DMSO or PGE2 (100 nM) for 24 h. A&D. Data represent relative mean + SD of triplicates normalized to GAPDH from at least 3 different cell preps. *P < 0.05 compared with DMSO treatment. RFE (AU), Relative Fold Expression (Arbitrary Units).

[0016] Figs. 2(A-E) illustrate a table and graphs showing PGE2-mediated gene regulation is Ca²⁺-dependent. A. Pathway analysis of RNA-seq data (24h). B. Heatmap represents hierarchical clustering of known Ca²⁺ responsive genes and genes involved in calcium signaling differentially expressed (log2 fold change > 1.5) between control and PGE2 at 1 and 24 h. C. 15-PGDH mRNA in hCSCs after treatment with Ca²⁺ ionophore A23187 (10 nM) for 24 h. D, E. Cells maintained in serum-free DMEM and then incubated in DMEM, Ca²⁺-free DMEM, or Ca²⁺-free medium + CaCb (200 mg/L) for 6 h, pre- treated with either DMSO or BAPTA-AM (1 μΜ) for 1 h. Thereafter, cells were treated with DMSO or PGE2 (25 nM) x 15 h. Data represent 15-PGDH mRNA as mean + SD of triplicates normalized to GAPDH. *P < 0.01 compared with DMSO, ANOVA followed by Tukey's posthoc testing. RFE (AU), Relative Fold Expression (Arbitrary Units)

[0017] Figs. 3(A-E) illustrate graphs, an immunoblot, and plot showing PGE2 results in deacetylation of chromatin associated with the 15-PGDH gene promoter. A. Chromatin immunoprecipitation (IP) of acetylated histone H3 (AcH3) is compared with IP of IgG (negative control) in cells treated with DMSO or PGE2 (100 nM) x 24 h. Data represent average fold enrichment. *P < 0.05 compared with DMSO, Student's t test. N = 3. B. Representative immunoblot and quantitation of 15-PGDH levels in hCSCs treated with DMSO or HDAC inhibitor SAHA (2.5 μΜ), TSA (1 μΜ), or HDAC-42 (1 μΜ) for 24 h. Acetylated histone H3(AcH3) -positive control, β-actin -loading control. *P < 0.05 compared with DMSO, Student's t test. N = 3. C. Dose (24 h) - and time-dependent (SAHA (2.5 μΜ), TSA (1 μΜ), or HDAC-42 (1 μΜ)) increases in 15-PGDH after treatment with HDACi. *P < 0.05 compared with DMSO (C) or 0 time point (D), ANOVA, Dunn's post hoc testing. N = 3. E. Data represent Fragments Per Kilobase of exon per Million fragments mapped (FPKM) of Class I, IIA, IIB and IV HDACs expressed in hCSCs treated with DMSO or PGE2 (100 nM) for 1 or 24 h mined from RNA-Seq dataset. GAPDH and RPLP0 are shown as controls (1/100* FPKM values). *P < 0.05 compared with corresponding DMSO control.

[0018] Figs. 4(AD) illustrate graphs, an immunoblot, and plot showing PGE2 mediated 15-PGDH repression is mediated by HDAC4. A. HDAC4 and 15- PGDH mRNA after treatment with increasing concentrations of PGE2 for 24 h. B. HDAC4 mRNA in hCSCs pretreated with DMSO or EP2 selective antagonists (2 μΜ) for 1 h followed by either DMSO or PGE2 (100 nM) for 24 h. C. HDAC4 protein levels in hCSCs pretreated with DMSO or PF-04418948 (2 μΜ) followed by DMSO or PGE2. β-actin, loading control D. HDAC4 in hCSCs in media + Ca²⁺, pre-treated with DMSO or BAPTA-AM (1 μΜ) for 1 h, then with DMSO or PGE2 (25 nM) for 15 h. E. 15-PGDH and HDAC4 expression levels after siRNA- mediated knockdown with control negative siRNA or siHDAC4 for 48 h followed by treatment with DMSO or PGE2 (100 nM) for 24 h. F. 15-PGDH mRNA quantified in hCSCs treated with DMSO or PGE2 (25 nM) in combination with LMK235 (500 nM). Immunoblot of 15-PGDH protein levels in whole cell protein extracts prepared from hCSCs treated with DMSO or LMK- 235 (1 μΜ) for 24 h. β-actin- gel loading control. G, H. HDAC4 (G) and 15-PGDH (H) mRNA levels in hCSCs transfected with no siRNA, control siNeg, or si- HDAC4 with or without the HDAC4/5 enzyme inhibitor LMK-235. Bars represent relative mean + SD of triplicates. *P < 0.01, ANOVA followed by Dunnett's test using vehicle or DMSO/si-Neg as control. Experiment repeated in 3 cell preps with identical results.

[0019] Figs. 5(A-G) illustrate images, graphs, a plot, and immunblot showing PGE2 mediated dephosphory/arfon of HDAC4 and nuclear Ocalization. A. Immunocytostaining of hCSCs with HDAC4 antibody after treatment with DMSO or PG£"2 (100 nM) for 24 h.

Microscopic images were captured using 63X objective under similar settings. B. Data represent relative fluorescent signal intensity +SEM, quantified from confocal images. *P < 0.05 compared with DMSO, Student's t test. C, D. Representative immunoblot and quantitation of HDAC4 protein in nuclear and cytoplasmic fractions of hCSCs treated with PGE2 (100 nM) for indicated times. Histone H3 and β-actin, gel loading controls. *P < 0.05 compared with DMSO, Student's t test. N=3. E, F. Phosphorylation profile of HDAC4 (Ser246) and HDAC4 protein levels in response to PGE2 treatment in hCSCs as a function of time, β-actin, gel loading control. *P < 0.05 compared with DMSO, Student's t test. N=3. G. PGE2 acts through EP2 receptors to increase Ca²⁺-dependent dephosphorylation of cytoplasmic HDAC4. Dephosphorylation of HDAC4 relieves binding to its cytoplasmic chaperone 14-3-3 resulting in nuclear translocation and deacetylation of chromatin associated with the 15-PGDH gene promoter. Nuclear CaMKII restores nuclear HDAC4 levels by phosphorylation and export to the cytoplasm. EP2 receptor antagonists and HDAC inhibitors prevent PGE2-mediated 15-PGDH gene repression.

[0020] Figs. 6(A-B) illustrate plots and images showing HDAC4 mRNA and protein levels in human cervical stromal tissues at different stages of pregnancy. A. HDAC4 and 15- PGDH mRNA levels quantified in human cervical stromal tissues obtained from nonpregnant (NP, N = 7), pregnant early gestation (E. gest, N = 4), pregnant with cervix not ripe and not in labor (near term, Not Ripe, N = 7), term pregnant with cervix ripe before labor (Ripe, N = 8) and term pregnant in labor (IL, N = 7). Data represent relative mRNA levels normalized to GAPDH mRNA levels. P <0.05. ANOVA followed by Tukey's multiple comparisons test AU- Arbitrary Units. B Immunolocalization of HDAC4 in human cervical stroma of nonpregnant (NP) and pregnant females in early gestation (10-14 weeks), at term before cervical ripening (Not Ripe), term after cervical ripening but before labor (Ripe), and in labor (In Labor). Results were consistent representing 3-4 tissues in each group. Bar = 20 μιη.

[0021] Figs. 7(A-F) illustrate images and graphs showing PGE2 plus 15-PGDH inhibitor treatment induces preterm cervical ripening and labor in mice. A. Time (h) of delivery from the initiation of different treatments as indicated. Gestation day (days post coitum -dpc) are shown on right. *P < 0.01 ANOVA with Tukey's posthoc testing. B.

Preterm pups born after treatment with PGE2+SW033291 intact with fetal membranes and placenta. Bar = 2.5 mm. C. PGE2+SW033291 -induced delivery of litter mates born on dl7 (left) and early dl9 (right). D. Female reproductive tract dissected on dl7 for assessment of fetal health and gross morphological changes in cervix and uterus. Arrows indicate delivery of cervical pups in animals treated with PGE2+SW033291. B, bladder; V, vagina. E.

Masson's trichrome stain of mid-cervical transverse sections of mice treated with vehicle, PGE₂, SW033291 or PGE₂+SW033291. Animals were treated bi-daily on dl5 and dl6 with tissue collection on late dl6 prior to delivery. Bar = 200 μιη. epi, epithelium; str, stroma. F. Baseline dilation, maximal force generation, and distensibility of cervices from animals treated with vehicle (N=3), PGE₂ (N= 5), SW033291 (N=5), or PGE₂ + SW033291 (N = 5) Data represent mean + SEM. *P < 0.01 ANOVA with Tukey's posthoc testing.

[0022] Figs. 8(A-C) illustrate RNA-Seq data analysis and validation in human CSCs. A. Heat map of data from cells treated with DMSO (0.1%) or PGE₂ (100 nM) for 1 or 24 h. Data represent hierarchical clustering of differentially expressed genes with False Discovery Rate (FDR) < 0.05, log2 fold change > 1.5, normalized to 1 h DMSO. B. Principal component analysis demonstrating Biological Coefficient of Variation (BCV) of different treatment groups. Volcano plots providing FDR values and fold change for all gene transcripts in PGE₂-treated hCSCs at 1 h (Left) or 24 h (Right). Differentially expressed genes with FDR< 0.05 are indicated in red. C. Validation of RNA-Seq data. Six different genes were selected by fold-change in different pathways significantly altered by PGE2. (A) Data from RNA-Seq analysis at 24 h expressed as Fragments Per Kilobase of exon per Million fragments mapped (FPKM). (B) Relative quantification of mRNA in hCSCs prepared from non-pregnant (qPCR NP-hCSC) or (C) pregnant (qPCR P- hCSC) cervices treated with either DMSO or PGE2 (100 nM) for 24. Data represent relative mean mRNA levels + SD of triplicates normalized to GAPDH mRNA levels. *P < 0.05 compared with DMSO treated controls. RFE (AU), Relative Fold Expression (Arbitrary Units).

[0023] Figs. 9(A-E) illustrate plots and a graph showing PGE2-mediated gene regulation is Ca²⁺-dependent. Genomic browser snapshots of PGE2-mediated upregulation of C-FOS (A) and DUSPl (B) from RNA-seq dataset. C, D. Relative expression of Ca²⁺- responsive genes C-FOS and DUSPl in hCSCs treated with PGE₂ (100 nM) for different times quantified by RT-qPCR. Data represent relative mean mRNA levels + SD of triplicates after normalizing to GAPDH. *P < 0.01 compared with 0 h time point. ANOVA, RFE (AU), Relative Fold Expression (Arbitrary Units). E. 15-PGDH mRNA levels in hCSCs after treatment with DMSO, PGE₂ (50 nM) or 8-Bromo-cAMP (2 μΜ), PGE₂ + adenylate cyclase inhibitor (ACi, 10 μΜ) or PKA inhibitor (RpCAMPS 5 μΜ), or PI3K inhibitors Wortmannin (1 μΜ) or LY94002 (1 μΜ). Data represent relative mean mRNA levels + SD of triplicates after normalizing to GAPDH repeated in 3 cell preps. *P < 0.01 compared to DMSO treated controls. Pathway inhibitors did not differ from PGE2 alone, ANOVA followed by Tukey's test for multiple comparisons. RFE (AU), Relative Fold Expression (Arbitrary Units).

[0024] Figs. lO(A-B) illustrate plots and a graph showing calcium ionophore A23187 mimics PGE₂ mediated effects in hCSCs. Relative levels of 15-PGDH, COX-2, DUSPl and C-FOS mRNA in hCSCs treated with (A) increasing concentrations of the Ca²⁺ ionophore A23187 for 24 h, or (B) A23187 (1 μΜ) as a function of time. Data represent relative mean mRNA levels + SD of triplicates after normalizing to GAPDH mRNA. *P < 0.01 compared with 0 h time point. RFE (AU), Relative Fold Expression (Arbitrary Units).

[0025] Figs. 1 l(A-D) illustrate graphs and plots showing HDACi treatment blocks PGE2-mediated 15-PGDH gene repression both before and after PGE2 treatment. 15-PGDH mRNA levels in hCSCs treated with DMSO or PGE₂ (A, 100 nM) or A23187 (C, 1 μΜ) for 24 h followed by treatment with HDACi [TSA (1 μΜ)/8ΑΗΑ (2.5 μΜ)/ΗΟΑΟ-42 (1 μΜ)] for 12 h (delayed treatment), or with DMSO or HDACi [TSA (1 μΜ)/8ΑΗΑ (2.5

μΜ)/ΗΟΑ042 (1 μΜ)] for 6 h followed by treatment with PGE₂ (B, 100 nM) or A23187 (D, 1 μΜ) for 12 h (primed treatment). Data represent relative mean mRNA levels + SD of triplicates after normalizing to GAPDH mRNA levels. *P < 0.05 ANOVA, RFE (AU), Relative Fold Expression (Arbitrary Units). [0026] Figs. 12(A-B) illustrate plots and graph showing reciprocal regulation of 15- PGDH and HDAC4 by PGE₂. A. HDAC4 and 15-PGDH mRNA after treatment with PGE₂ (100 nm) as a function of time in minutes or hours as indicated. B. Misoprostol (100 nM) and butaprost (100 nM, EP2-selective agonist) increase HDAC4 mRNA (24h). Data represent relative mean mRNA levels + SD of triplicates after normalizing to GAPDH mRNA levels in at least 3 cell preps. *P < 0.05 ANOVA, RFE (AU), Relative Fold

Expression (Arbitrary Units).

[0027] Fig. 13 illustrates a graph showing PGE2 increases HDAC4 levels via EP2 receptor. Densitometric quantitation of immunoblots of HDAC4 protein in hCSCs pretreated with DMSO or PF-04418948 (2 μΜ) followed by DMSO or PGE₂. *P < 0.05 compared with PGE₂ treatment alone, Student's t test. N = 3.

[0028] Figs. 14(A-C) illustrate graphs showing HDAC2 and Sirtuins (Class III HDACS) do not regulate 15-PGDH gene in hCSCs. A. mRNA levels of HDAC2 and 15- PGDH in hCSCs transfected with negative siRNA control or HDAC2- specific siRNA. B. Data represents Fragments Per Kilobase of exon per Million fragments mapped (FPKM) of sirtuins expressed in hCSCs treated with DMSO or PGE2 (100 nM) for 1 or 24 h mined from RNA-Seq dataset. GAPDH and RPLP0 are shown as controls (1/100* FPKM values). *P < 0.05 compared with corresponding DMSO control. C. 15-PGDH mRNA levels in hCSCs treated with indicated concentrations of sirtuin inhibitor Aristoforin for 24 h. Data represent relative mean mRNA levels + SD of triplicates after normalizing to GAPDH mRNA levels in at least 3 cell preps. Relative Fold Expression (Arbitrary Units).

[0029] Fig. 15 Illustrates a graph showing PGE2 does not alter HDAC4 gene expression in SK-MEL5 and MCF7 cells. HDAC4 mRNA levels quantified by RT-qPCR in SK-MEL5 melanoma cells or MCF7 breast cancer cells treated with increasing concentrations of PGE2 as indicated for 24 h. Data represent relative mean mRNA levels + SD of triplicates after normalizing to GAPDH mRNA levels. RFE(AU), Relative Fold Expression (Arbitrary Units).

[0030] Figs. 16(A-B) illustrate graphs showing 15-PGDH is an HDAC4 target gene in hCSCs. A. mRNA levels of HDAC4 and 15-PGDH and protein levels of HDAC4 in hCSCs transfected with control negative siRNA or two different HDAC4 siRNAs (^aAmbion; ^bSanta Cruz Biotechnologies) for 56 h. *P < 0.001 compared to si-Neg. Student's t test. N = 3. B. mRNA levels of HDAC4 and 15- PGDH and protein levels of HDAC4 in hCSCs infected with control adenovirus or HDAC4-expressing adenovirus for 48 h followed by incubation in serum- free medium for 24 h. Arrows indicate two immunoreactive proteins *P < 0.001 compared with control adenovirus infected cells. Student's t test. RFE (AU), Relative Fold Expression (Arbitrary Units).

[0031] Fig. 17 illustrates graphs showing HDAC5 does not regulate 15-PGDH gene expression in hCSCs. mRNA levels of HDAC5 and 15-PGDH in hCSCs transfected with negative siRNA control or HDAC5 specific siRNA. Data represent relative mean mRNA levels + SD of triplicates after normalizing to GAPDH mRNA levels. *P = 0.00002 compared with siNeg transfected cells. ANOVA, RFE(AU), Relative Fold Expression (Arbitrary Units).

[0032] Figs. 18(A-D) illustrate graphs showing HDAC4 mediated repression of 15- PGDH is de-phosphorylation dependent. A. Relative mRNA levels of 15-PGDH and HDAC4 in hCSCs treated with DMSO, KN-62 (5 μΜ), KN-93 (5 μΜ), or PGE₂ (100 nM) for 24 h. B. Levels of 15-PGDH (a) and HDAC4 mRNA in hCSCs treated with DMSO, C2 ceramide (50 μΜ) or PGE₂ (100 nM) for 24 h. C, D. 15-PGDH mRNA levels in hCSCs pretreated with DMSO or okadaic acid (OA, 100 nM) for 1 h followed by treatment with DMSO or KN-93 (5 μΜ) for 23 h. Data represent mean mRNA levels + SD of triplicates after normalizing to GAPDH mRNA levels. *P < 0.01 compared with DMSO treatment. Student's t test. N = 3 in at least 3 cell preps. RFE (AU), Relative Fold Expression (Arbitrary Units). OA, okadaic acid.

[0033] Figs. 19(A-B) illustrate plots and images showing 16,16-dimethyl PGE2 induces HDAC4 and represses 15-PGDH gene expression and has adverse fetal effects in pregnant mice. A. HDAC4 and 15-PGDH mRNA levels in hCSCs treated with increasing

concentrations of 16,16-dimethyl PGE2 as indicated for 24 h. *P < 0.01 compared with DMSO treatment. ANOVA followed by Dunnett's with time 0 as control, n = 3. B. Female reproductive tract dissected on dl5, 6 h after treatment for assessment of health of pups and gross morphological changes in cervix and uterus. Arrow indicates pale ischemic pups; V, vagina; B, bladder. DETAILED DESCRIPTION

[0034] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0035] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0036] The terms "comprise," "comprising," "include," "including," "have," and "having" are used in the inclusive, open sense, meaning that additional elements may be included. The terms "such as", "e.g. ", as used herein are non-limiting and are for illustrative purposes only. "Including" and "including but not limited to" are used interchangeably.

[0037] The term "or" as used herein should be understood to mean "and/or", unless the context clearly indicates otherwise.

[0038] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term "about" or "approximately" refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length + 15%, + 10%, + 9%, + 8%, + 7%, + 6%, + 5%, + 4%, + 3%, + 2%, or + 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0039] It will be noted that the structure of some of the compounds of the application include asymmetric (chiral) carbon or sulfur atoms. It is to be understood accordingly that the isomers arising from such asymmetry are included herein, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. The compounds of this application may exist in stereoisomeric form, therefore can be produced as individual stereoisomers or as mixtures.

[0040] The term "isomerism" means compounds that have identical molecular formulae but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of one another are termed "diastereoisomers", and stereoisomers that are non-superimposable mirror images are termed "enantiomers", or sometimes optical isomers. A carbon atom bonded to four nonidentical substituents is termed a "chiral center" whereas a sulfur bound to three or four different substitutents, e.g. sulfoxides or sulfinimides, is likewise termed a "chiral center".

[0041] The term "chiral isomer" means a compound with at least one chiral center. It has two enantiomeric forms of opposite chirality and may exist either as an individual enantiomer or as a mixture of enantiomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a "racemic mixture". A compound that has more than one chiral center has 2n-l enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as either an individual diastereomer or as a mixture of diastereomers, termed a "diastereomeric mixture". When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Alternatively, when one or more chiral centers are present, a stereoisomer may be characterized as (+) or (-). Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964, 41, 116).

[0042] The term "geometric Isomers" means the diastereomers that owe their existence to hindered rotation about double bonds. These configurations are differentiated in their names by the prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite side of the double bond in the molecule according to the Cahn-Ingold- Prelog rules. Further, the structures and other compounds discussed in this application include all atropic isomers thereof.

[0043] The term "atropic isomers" are a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as a mixture, however as a result of recent advances in

chromatography techniques, it has been possible to separate mixtures of two atropic isomers in select cases. [0044] The terms "crystal polymorphs" or "polymorphs" or "crystal forms" means crystal structures in which a compound (or salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions.

[0045] The term "derivative" refers to compounds that have a common core structure, and are substituted with various groups as described herein.

[0046] The term "bioisostere" refers to a compound resulting from the exchange of an atom or of a group of atoms with another, broadly similar, atom or group of atoms. The objective of a bioisosteric replacement is to create a new compound with similar biological properties to the parent compound. The bioisosteric replacement may be physicochemically or topologically based. Examples of carboxylic acid bioisosteres include acyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147- 3176 (1996).

[0047] The phrases "parenteral administration" and "administered parenterally" are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0048] The term "treating" is art-recognized and includes inhibiting a disease, disorder or condition in a subject, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected. For example, the term "treating" can refer to the administration of a short chain dehydrogenase inhibitor (e.g., 15- PGDH inhibitor) to slow or inhibit the progression of congestive heart failure during the treatment, relative to the disease progression that would occur in the absence of treatment, in a statistically significant manner. Well known indicia such as left ventricular ejection fraction, exercise performance, and other clinical tests as enumerated below, as well as survival rates and hospitalization rates may be used to assess disease progression. Whether or not a treatment slows or inhibits disease progression in a statistically significant manner may be determined by methods that are well known in the art.

[0049] The term "preventing" is art-recognized and includes stopping a disease, disorder or condition from occurring in a subject, which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it. Preventing a condition related to a disease includes stopping the condition from occurring after the disease has been diagnosed but before the condition has been diagnosed. For example, the term "preventing" can refer to minimizing or partially or completely inhibiting the development of congestive heart failure in a mammal at risk for developing congestive heart failure (as defined in "Consensus recommendations for the management of chronic heart failure." Am. J. Cardiol., 83(2A): lA-38-A, 1999).

[0050] The term "pharmaceutical composition" refers to a formulation containing the disclosed compounds in a form suitable for administration to a subject. In a preferred embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed compound or salts thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, inhalational, and the like. Dosage forms for the topical or transdermal administration of a compound described herein includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, nebulized compounds, and inhalants. In a preferred embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

[0051] The term "flash dose" refers to compound formulations that are rapidly dispersing dosage forms. [0052] The term "immediate release" is defined as a release of compound from a dosage form in a relatively brief period of time, generally up to about 60 minutes. The term "modified release" is defined to include delayed release, extended release, and pulsed release. The term "pulsed release" is defined as a series of releases of drug from a dosage form. The term "sustained release" or "extended release" is defined as continuous release of a compound from a dosage form over a prolonged period.

[0053] The phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, the term includes compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0054] The phrase "pharmaceutically acceptable carrier" is art-recognized, and includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient. In certain embodiments, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. [0055] The compounds of the application are capable of further forming salts. All of these forms are also contemplated herein.

[0056] "Pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. For example, the salt can be an acid addition salt. One embodiment of an acid addition salt is a hydrochloride salt. The pharmaceutically acceptable salts can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile being preferred. Lists of salts are found in Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0057] The compounds described herein can also be prepared as esters, for example pharmaceutically acceptable esters. For example, a carboxylic acid function group in a compound can be converted to its corresponding ester, e.g., a methyl, ethyl, or other ester. Also, an alcohol group in a compound can be converted to its corresponding ester, e.g., an acetate, propionate, or other ester.

[0058] The compounds described herein can also be prepared as prodrugs, for example pharmaceutically acceptable prodrugs. The terms "pro-drug" and "prodrug" are used interchangeably herein and refer to any compound, which releases an active parent drug in vivo. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.) the compounds can be delivered in prodrug form. Thus, the compounds described herein are intended to cover prodrugs of the presently claimed compounds, methods of delivering the same and compositions containing the same. "Prodrugs" are intended to include any covalently bonded carriers that release an active parent drug in vivo when such prodrug is administered to a subject. Prodrugs are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Prodrugs include compounds wherein a hydroxy, amino, sulfhydryl, carboxy, or carbonyl group is bonded to any group that may be cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl, free carboxy or free carbonyl group, respectively. Prodrugs can also include a precursor (forerunner) of a compound described herein that undergoes chemical conversion by metabolic processes before becoming an active or more active pharmacological agent or active compound described herein.

[0059] Examples of prodrugs include, but are not limited to, esters (e.g., acetate, dialkylaminoacetates, formates, phosphates, sulfates, and benzoate derivatives) and carbamates (e.g., Ν,Ν-dimethylaminocarbonyl) of hydroxy functional groups, ester groups (e.g., ethyl esters, morpholinoethanol esters) of carboxyl functional groups, N-acyl derivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases and enaminones of amino functional groups, oximes, acetals, ketals and enol esters of ketone and aldehyde functional groups in compounds, and the like, as well as sulfides that are oxidized to form sulfoxides or sulfones.

[0060] The term "protecting group" refers to a grouping of atoms that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in Green and Wuts, Protective Groups in Organic Chemistry, (Wiley, 2.sup.nd ed. 1991); Harrison and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996); and Kocienski, Protecting Groups, (Verlag, 3^rd ed. 2003).

[0061] The term "amine protecting group" is intended to mean a functional group that converts an amine, amide, or other nitrogen-containing moiety into a different chemical group that is substantially inert to the conditions of a particular chemical reaction. Amine protecting groups are preferably removed easily and selectively in good yield under conditions that do not affect other functional groups of the molecule. Examples of amine protecting groups include, but are not limited to, formyl, acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, t-butyloxycarbonyl (Boc), p-methoxybenzyl, methoxymethyl, tosyl, trifluoroacetyl, trimethylsilyl (TMS), fluorenyl-methyloxycarbonyl, 2-trimethylsilyl- ethyoxycarbonyl, 1 -methyl- l-(4-biphenylyl) ethoxycarbonyl, allyloxycarbonyl,

benzyloxycarbonyl (CBZ), 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC), and the like. Those of skill in the art can identify other suitable amine protecting groups.

[0062] Representative hydroxy protecting groups include those where the hydroxy group is either acylated or alkylated such as benzyl, and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers. [0063] Additionally, the salts of the compounds described herein, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Nonlimiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0064] The term "solvates" means solvent addition forms that contain either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate, when the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one of the substances in which the water retains its molecular state as H2O, such combination being able to form one or more hydrate.

[0065] The compounds, salts and prodrugs described herein can exist in several tautomeric forms, including the enol and imine form, and the keto and enamine form and geometric isomers and mixtures thereof. Tautomers exist as mixtures of a tautomeric set in solution. In solid form, usually one tautomer predominates. Even though one tautomer may be described, the present application includes all tautomers of the present compounds. A tautomer is one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. This reaction results in the formal migration of a hydrogen atom accompanied by a switch of adjacent conjugated double bonds. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that are interconvertable by tautomerizations is called tautomerism.

[0066] Of the various types of tautomerism that are possible, two are commonly observed. In keto-enol tautomerism a simultaneous shift of electrons and a hydrogen atom occurs.

[0067] Tautomerizations can be catalyzed by: Base: 1. deprotonation; 2. formation of a delocalized anion (e.g., an enolate); 3. protonation at a different position of the anion; Acid: 1. protonation; 2. formation of a delocalized cation; 3. deprotonation at a different position adjacent to the cation.

[0068] The term "analogue" refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analogue is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.

[0069] A "patient," "subject," or "host" to be treated by the subject method may mean either a human or non-human animal, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder.

[0070] The terms "prophylactic" and "therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0071] The terms "therapeutic agent", "drug", "medicament" and "bioactive substance" are art-recognized and include molecules and other agents that are biologically,

physiologically, or pharmacologically active substances that act locally or systemically in a patient or subject to treat a disease or condition. The terms include without limitation pharmaceutically acceptable salts thereof and prodrugs. Such agents may be acidic, basic, or salts; they may be neutral molecules, polar molecules, or molecular complexes capable of hydrogen bonding; they may be prodrugs in the form of ethers, esters, amides and the like that are biologically activated when administered into a patient or subject.

[0072] The terms "therapeutically effective amount" and "pharmaceutically effective amount" are an art-recognized term. In certain embodiments, the term refers to an amount of a therapeutic agent that produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate, reduce or maintain a target of a particular therapeutic regimen. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. In certain embodiments, a therapeutically effective amount of a therapeutic agent for in vivo use will likely depend on a number of factors, including: the rate of release of an agent from a polymer matrix, which will depend in part on the chemical and physical characteristics of the polymer; the identity of the agent; the mode and method of administration; and any other materials incorporated in the polymer matrix in addition to the agent.

[0073] The term "ED50" is art-recognized. In certain embodiments, ED50 means the dose of a drug, which produces 50% of its maximum response or effect, or alternatively, the dose, which produces a pre-determined response in 50% of test subjects or preparations. The term "LD50" is art-recognized. In certain embodiments, LD50 means the dose of a drug, which is lethal in 50% of test subjects. The term "therapeutic index" is an art-recognized term, which refers to the therapeutic index of a drug, defined as LD50/ED50.

[0074] The terms "IC₅₀," or "half maximal inhibitory concentration" is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc.

[0075] With respect to any chemical compounds, the present application is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include C-13 and C-14.

[0076] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent can be bonded to any atom in the ring. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent can be bonded via any atom in such substituent. Combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds. [0077] When an atom or a chemical moiety is followed by a subscripted numeric range (e.g., Ci-_δ), it is meant to encompass each number within the range as well as all intermediate ranges. For example, "Ci_6 alkyl" is meant to include alkyl groups with 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6 carbons.

[0078] The term "alkyl" is intended to include both branched (e.g., isopropyl, tert-butyl, isobutyl), straight-chain e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl), and cycloalkyl (e.g., alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. Such aliphatic hydrocarbon groups have a specified number of carbon atoms. For example, Ci_6 alkyl is intended to include Ci, C₂, C₃, C₄, C₅, and C₆ alkyl groups. As used herein, "lower alkyl" refers to alkyl groups having from 1 to 6 carbon atoms in the backbone of the carbon chain. "Alkyl" further includes alkyl groups that have oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more hydrocarbon backbone carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has six or fewer carbon atoms in its backbone (e.g., Ci-C₆ for straight chain, C₃-C₆ for branched chain), for example four or fewer. Likewise, certain cycloalkyls have from three to eight carbon atoms in their ring structure, such as five or six carbons in the ring structure.

[0079] The term "substituted alkyls" refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Cycloalkyls can be further substituted, e.g., with the substituents described above. An "alkylaryl" or an "aralkyl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). If not otherwise indicated, the terms "alkyl" and "lower alkyl" include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl or lower alkyl, respectively.

[0080] The term "alkenyl" refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, cyclopentenyl, cyclohexenyl, cyclooctenyl, and the like. Generally, although again not necessarily, alkenyl groups can contain 2 to about 18 carbon atoms, and more particularly 2 to 12 carbon atoms. The term "lower alkenyl" refers to an alkenyl group of 2 to 6 carbon atoms, and the specific term "cycloalkenyl" intends a cyclic alkenyl group, preferably having 5 to 8 carbon atoms. The term "substituted alkenyl" refers to alkenyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to alkenyl or heterocycloalkenyl (e.g., heterocylcohexenyl) in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkenyl" and "lower alkenyl" include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively.

[0081] The term "alkynyl" refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups can contain 2 to about 18 carbon atoms, and more particularly can contain 2 to 12 carbon atoms. The term "lower alkynyl" intends an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to alkynyl substituted with one or more substituent groups, and the terms

"heteroatom-containing alkynyl" and "heteroalkynyl" refer to alkynyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkynyl" and "lower alkynyl" include linear, branched, unsubstituted, substituted, and/or heteroatom- containing alkynyl and lower alkynyl, respectively.

[0082] The terms "alkyl", "alkenyl", and "alkynyl" are intended to include moieties which are diradicals, i.e., having two points of attachment. A nonlimiting example of such an alkyl moiety that is a diradical is— CH₂CH₂— , i.e., a C₂ alkyl group that is covalently bonded via each terminal carbon atom to the remainder of the molecule.

[0083] The term "alkoxy" refers to an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group may be represented as -O-alkyl where alkyl is as defined above. A "lower alkoxy" group intends an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. Preferred substituents identified as "C₁-C6 alkoxy" or "lower alkoxy" herein contain 1 to 3 carbon atoms, and particularly preferred such substituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

[0084] The term "aryl" refers to an aromatic substituent containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Aryl groups can contain 5 to 20 carbon atoms, and particularly preferred aryl groups can contain 5 to 14 carbon atoms. Examples of aryl groups include benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, the term "aryl" includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,

benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles",

"heterocycles," "heteroaryls" or "heteroaromatics". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diaryl amino, and al kylaryl amino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl). If not otherwise indicated, the term "aryl" includes unsubstituted, substituted, and/or heteroatom-containing aromatic substituents.

[0085] The term "alkaryl" refers to an aryl group with an alkyl substituent, and the term "aralkyl" refers to an alkyl group with an aryl substituent, wherein "aryl" and "alkyl" are as defined above. Exemplary aralkyl groups contain 6 to 24 carbon atoms, and particularly preferred aralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groups include, without limitation, benzyl, 2-phenyl-ethyl, 3 -phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl,

4-benzylcyclohexylmethyl, and the like. Alkaryl groups include, for example, p- methylphenyl, 2,4-dimethylphenyl, p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7- cyclooctylnaphthyl, 3-ethyl-cyclopenta-l,4-diene, and the like.

[0086] The terms "heterocyclyl" and "heterocyclic group" include closed ring structures, e.g., 3- to 10-, or 4- to 7-membered rings, which include one or more heteroatoms. "Heteroatom" includes atoms of any element other than carbon or hydrogen. Examples of heteroatoms include nitrogen, oxygen, sulfur and phosphorus.

[0087] Heterocyclyl groups can be saturated or unsaturated and include pyrrolidine, oxolane, thiolane, piperidine, piperazine, morpholine, lactones, lactams, such as azetidinones and pyrrolidinones, sultams, and sultones. Heterocyclic groups such as pyrrole and furan can have aromatic character. They include fused ring structures, such as quinoline and isoquinoline. Other examples of heterocyclic groups include pyridine and purine. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, or an aromatic or heteroaromatic moiety. Heterocyclic groups can also be substituted at one or more constituent atoms with, for example, a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl,— CF₃, or— CN, or the like.

[0088] The terms "halo" and "halogen" refers to fluoro, chloro, bromo, and iodo. "Counterion" is used to represent a small, negatively charged species such as fluoride, chloride, bromide, iodide, hydroxide, acetate, and sulfate. The term sulfoxide refers to a sulfur attached to 2 different carbon atoms and one oxygen and the S-0 bond can be graphically represented with a double bond (S=0), a single bond without charges (S-O) or a single bond with charges [S(+)-0(-)].

[0089] The term "substituted" as in "substituted alkyl", "substituted aryl", and the like, as alluded to in some of the aforementioned definitions, is meant that in the alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation: functional groups such as halo, hydroxyl, silyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C₂-C₂₄ alkylcarbonato

(-O-(CO)-O-alkyl), C₆-C₂₀ arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(CO)-NH₂), mono-(Ci-C₂₄ alkyl)-substituted carbamoyl (-(CO)- NH(Ci-C₂₄ alkyl)), di-(Ci-C₄ alkyl)-substituted carbamoyl (-(CO)~N(C₁-C₂₄ alkyl)₂), mono- substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C), cyanato (-0-CN), isocyanato (-ON⁺C^"), isothiocyanato (-S-CN), azido (-N=N⁺=N^~), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH₂), mono- and di-(Ci-C₂₄ alkyl)-substituted amino, mono- and di-(Cs-C₂o aryl)- substituted amino, C₂-C₂₄ alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), imino (-CR=NH where R=hydrogen, Ci-C₂₄ alkyl, Cs-C₂o aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino (— CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-S0₂ -OH), sulfonato (-S0₂-0^~), d-C₂₄ alkylsulfanyl (-S-alkyl; also termed

"alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), C C₂₄ alkylsulfinyl (-(SO)-alkyl), C₅-C₂₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂ -aryl), phosphono (-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-P0₂), and phosphino (-PH₂); and the hydrocarbyl moieties Ci-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, Cs-C₂o aryl, C₆-C₂₄ alkaryl, and C₆-C₂₄ aralkyl.

[0090] In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. Analogously, the above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated.

[0091] When the term "substituted" appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase "substituted alkyl, alkenyl, and aryl" is to be interpreted as "substituted alkyl, substituted alkenyl, and substituted aryl." Analogously, when the term "heteroatom- containing" appears prior to a list of possible heteroatom-containing groups, it is intended that the term apply to every member of that group. For example, the phrase "heteroatom- containing alkyl, alkenyl, and aryl" is to be interpreted as "heteroatom-containing alkyl, substituted alkenyl, and substituted aryl. [0092] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.

[0093] The terms "stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation, and as appropriate, purification from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0094] The term "free compound" is used herein to describe a compound in the unbound state.

[0095] Throughout the description, where compositions are described as having, including, or comprising, specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0096] The term "small molecule" is an art-recognized term. In certain embodiments, this term refers to a molecule, which has a molecular weight of less than about 2000 amu, or less than about 1000 amu, and even less than about 500 amu.

[0097] All percentages and ratios used herein, unless otherwise indicated, are by weight.

[0098] The terms "gene expression" and "protein expression" include any information pertaining to the amount of gene transcript or protein present in a sample, as well as information about the rate at which genes or proteins are produced or are accumulating or being degraded (e.g., reporter gene data, data from nuclear runoff experiments, pulse-chase data etc.). Certain kinds of data might be viewed as relating to both gene and protein expression. For example, protein levels in a cell are reflective of the level of protein as well as the level of transcription, and such data is intended to be included by the phrase "gene or protein expression information". Such information may be given in the form of amounts per cell, amounts relative to a control gene or protein, in unitless measures, etc.; the term

"information" is not to be limited to any particular means of representation and is intended to mean any representation that provides relevant information. The term "expression levels" refers to a quantity reflected in or derivable from the gene or protein expression data, whether the data is directed to gene transcript accumulation or protein accumulation or protein synthesis rates, etc.

[0099] The terms "healthy" and "normal" are used interchangeably herein to refer to a subject or particular cell or tissue that is devoid (at least to the limit of detection) of a disease condition.

[00100] The term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include analogues of either RNA or DNA made from nucleotide analogues, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double- stranded polynucleotides. In some embodiments, "nucleic acid" refers to inhibitory nucleic acids. Some categories of inhibitory nucleic acid compounds include antisense nucleic acids, RNAi constructs, and catalytic nucleic acid constructs. Such categories of nucleic acids are well-known in the art.

[00101] The term "labor induction" is generally defined as an intervention that directly or indirectly onsets labor from myometrial contractions of the uterus (uterine contractions) to accomplish a progress resulting in delivery and childbirth. The reasons for inducing labor include, but are not limited to, an extended pregnancy for example beyond the 41-42 weeks gestation time or medical complications, exemplified by pre-eclampsia, diabetes, essential hypertonia and Intra Uterine Growth Retardation (IUGR).

[00102] The term "inducing labor" relates to a therapy where a direct response effect is requested from the administration. In the context of labor it is requested that the

administration directly leads to at least one of initiation of cervical ripening or promotion or stimulation of uterine contractions. In other terms, the present invention is not directed to a prophylactic therapy, wherein females may receive a therapy to prevent from or counteract protracted labor, before being elected for labor induction.

[00103] The term "elected for labor induction" has the meaning that the pregnant female has been elected for a clinical reason, as outlined with "labor induction", or a humanitarian reason to enter into labor and that the labor shall be induced with a directly intervening administration therapy that directly after the administration initiates a process that directly or indirectly leads to the onset of labor. .

[00104] The terms "dystocia" or "labor dystocia", as used in the context of describing the present invention, are general terms covering several conditions including labor arrest, prolonged latent phase of labor and slow progress of labor (protracted labor). Dystocia is particularly common after labor induction and more frequent among nulliparous than multiparous females.

[00105] Embodiments described herein relate to compositions and methods of modulating cervical ripening, and particularly relate to compositions and methods of initiating cervical ripening and/or inducing labor as well inhibiting preterm cervical ripening and/or preterm birth in a female in need thereof. It was found that prostaglandin E2 (PGE2), a cervical ripening agent, mediates unique EP2-receptor- signaling pathways in human cervical stromal cells targeting its own synthesis by increasing COX-2 and PTGES expression and decreasing its metabolism by loss of its degradative enzyme 15-PGDH. Downregulation of 15-PGDH was also found to be crucial for PGE2-induced cervical ripening and preterm birth. It was further found that 15-PGDH inhibitors described herein can initiate, or amplify PGE2-mediated cervical ripening, and EP2 receptor antagonists, HDAC4 inhibitors, and/or 15-PGDH activators can prevent preterm cervical ripening and preterm birth. Accordingly, in some embodiments, compositions and methods of modulating 15-PDGH activity can be used to modulate cervical ripening, and induce or prevent preterm labor.

[00106] In some embodiments, a method of inducing cervical ripening and labor in a female in need thereof can include administering to the female a 15-PGDH inhibitor alone or in combination with another labor inducing agent. By inducing labor, a shortened delivery time and the number of labor complications, e.g., Caesarian sections can be significantly reduced. Protracted labor is also associated with other maternal complications, e.g., post partum haemorrhage, instrumental deliveries and endometritis as well as an increased risk of fetal asphyxia and infection.

[00107] In some embodiments, the females who are elected to be induced into labor belong to a patient group associated with risks for clinical complications for the female or the fetus/neonate, or the females can be elected for humanitarian reasons. Patient groups include females in an extended pregnancy beyond 41-42 weeks gestation time, females suffering from medical complications, such as pre-eclampsia, diabetes, essential hypertonia and Intra Uterine Growth Retardation (IUGR).

[00108] The state of cervix can be established by routine methods among obstetricians, such as Bishop's Score (cervix score). It is well established that females with a Bishop's Score of 5 or less have an unripe cervix. Conventional therapies to establish cervical ripeness with PGE2 include administration every 12 hours at the most four times. One commonly employed way estimating ripeness is to estimate cervical dilation. A dilation of 4 cm or more can be considered to manifest a ripe cervix.

[00109] In some embodiments, the other labor inducing agents can include at least one compound chosen from prostaglandins, in particular prostaglandin PGEi, PGE2, their salts, their esters, their analogues and their derivatives, in particular those described in WO 98/33497, WO 95/11003, JP 97-100091, JP 96-134242, in particular agonists of the prostaglandin receptors. The other labor inducing compound may include at least one compound ,such as the agonists (in acid form or in the form of a precursor, in particular in ester form) of the prostaglandin F201 receptor, such as for example latanoprost, fluprostenol, cloprostenol, bimatoprost, unoprostone, the agonists (and their precursors, in particular the esters such as travoprost) of the prostaglandin E2 receptors, such as 17-phenyl PGE2, dioproston, viprostol, butaprost, misoprostol, sulprostone, 16,16-dimethyl PGE2, 11-deoxy PGEi, 1-deoxy PGEi, the agonists and their precursors, in particular esters, of the prostacycline (IP) receptor such as cicaprost, iloprost, isocarbacycline, beraprost, eprostenol, treprostinil, the agonists and their precursors, in particular the esters, of the prostaglandin D2 receptor, such as BW245C ((4S)-(3-[(3R,S)-3-cyclohexyl-3-isopropyl]-2,5-dioxo)-4- imidazolidinehept- anoic acid), BW246C ((4R)-(3-[(3R,S)-3-cyclohexyl-3-isopropyl]-2,5- dioxo)-4-imidazolidinehept- anoic acid), the agonists and their precursors, in particular the esters, of the receptor for the thromboxanes A2 (TP) such as I-BOP ([lS-[la,2a(Z),

3b(lE,3S),4a]]-7-[3-[3-hydroxy-4-[4-(iodophenoxy)-l-butenyl]-7-oxabicyclo- [2.2.1]hept-2- yl]-5-heptenoic acid).

[00110] In other embodiments, the at least one prostaglandin or prostaglandin derivative can include prostaglandins, such as the prostaglandins of series 2 including in particular PGF2₀₁ and PGE2 in saline form or in the form of precursors, in particular of the esters (example isopropyl esters), their derivatives, such as 16,16-dimethyl PGE2, 17-phenyl PGE2 and 16,16-dimethyl PGF2₀₁ 17-phenyl PGF2₀₁, prostaglandins of series 1, such as 11- deoxyprostaglandin El, 1-deoxyprostaglandin El in saline or ester form, or their analogues, in particular latanoprost, travoprost, fluprostenol, unoprostone, bimatoprost, cloprostenol, viprostol, butaprost, misoprostol, their salts or their esters. Advantageously, administration of at least one 15-PGDH inhibitor as described herein and can increase the success of prostaglandin induced cervical ripening but also facilitate lower doses of prostaglandins and thereby decrease induction to delivery interval and mitigate fetal death.

[00111] 15-PGDH inhibitors can be identified using assays in which putative inhibitor compounds are applied to cells expressing 15-PGDH and then the functional effects on 15-PGDH activity are determined. Samples or assays comprising 15-PGDH that are treated with a potential inhibitor are compared to control samples without the inhibitor to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative 15-PGDH activity value of 100%. Inhibition of 15-PGDH is achieved when the 15-PGDH activity value relative to the control is about 80%, optionally 50% or 25%, 10%, 5% or 1 %.

[00112] Agents tested as 15-PGDH can be any small chemical molecule or compound. Typically, test compounds will be small chemical molecules, natural products, or peptides. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays).

[00113] In some embodiments, the 15-PGDH inhibitor can include a compound having the following formula (I):

wherein n is 0-2;

Y¹, Y², and R¹ are the same or different and are each selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C2₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6 ring atoms (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(O) (Ci-C₆ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, -Si(d-C₃ alkyl)₃, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C₆-C2₀ arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl

(-(CO)-NH(C C₂4 alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C^~), cyanato (-0-CN), isocyanato (-0-N⁺=C), isothiocyanato (-S-CN), azido (-N=N⁺=N^~), formyl (-(CO)-H), thioformyl (~(CS)~ H), amino (~NH₂), C1-C24 alkyl amino, C5-C2₀ aryl amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), imino (-CR=NH where R is hydrogen, C1-C24 alkyl, C5-C2₀ aryl, C₆-C24 alkaryl, C₆-C24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O ), C1-C24 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), Ci-C₂₄ alkylsulfinyl (-(SO)-alkyl), C₅-C₂₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (wherein Y is independently H, arlyl or alkyl), phosphono

(-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-P0₂), phosphino (— PH2), poly alky lethers, phosphates, phosphate esters, groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, combinations thereof, and wherein Y¹ and Y² may be linked to form a cyclic or polycyclic ring, wherein the ring is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, and a substituted or unsubstituted heterocyclyl;

U¹ is N, C-R², or C-NR³R⁴, wherein R² is selected from the group consisting of a H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, O-CH2-CH2X, CH2-CH2-CH2X, O-CH2-CH2X, X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, 0(CO)R', COOR' (wherein R' is H or a lower alkyl group), and wherein R¹ and R² may be linked to form a cyclic or polycyclic ring, wherein R³ and R⁴ are the same or different and are each selected from the group consisting of H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, CH₂-CH₂-CH₂X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, COOR' (wherein R' is H or a lower alkyl group), and R³ or R⁴ may be absent;

X¹ and X² are independently N or C, and wherein when X¹ and/or X² are N, Y¹ and/or Y², respectively, are absent;

Z¹ is O, S, CR^aR^b or NR^a, wherein R^a and R^b are independently H or a Ci_₈ alkyl, which is linear, branched, or cyclic, and which is unsubstituted or substituted;

and pharmaceutically acceptable salts thereof.

[00114] In other embodiments, the 15-PGDH inhibitor can include a compound having the following

wherein n is 0-2

X⁴, X⁵, X⁶, and X⁷ are independently N or CR^C;

R¹, R⁶, R⁷, and R^c are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C₃-C2₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6 ring atoms (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(0)(Ci-C₆ alkyl), O, and S), C₆-C24 alkaryl, C₆-C24 aralkyl, halo, -Si(Ci-C₃ alkyl)₃, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (— CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C₂-C₂₄

alkylcarbonato (-O-(CO)-O-alkyl), C₆-C2₀ arylcarbonato (-O-(CO)-O-aryl), carboxy

(-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl

(-(CO)-NH(Ci-C₂₄ alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C^~), cyanato (-0-CN), isocyanato (-0-N⁺=C), isothiocyanato (-S-CN), azido (-N=N⁺=N^~), formyl (-(CO)-H), thioformyl (~(CS)~ H), amino (~NH₂), C1-C24 alkyl amino, C5-C2₀ aryl amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), imino (-CR=NH where R is hydrogen, C1-C24 alkyl, C5-C2₀ aryl, C₆-C24 alkaryl, C₆-C24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O ), C1-C24 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), Ci-C₂₄ alkylsulfinyl (-(SO)-alkyl), C₅-C₂₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (wherein Y is independently H, aryl or alkyl), phosphono

(-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-P0₂), phosphino (— PH2), poly alky lethers, phosphates, phosphate esters, groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, combinations thereof, and wherein R⁶ and R⁷ may be linked to form a cyclic or polycyclic ring, wherein the ring is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, and a substituted or unsubstituted heterocyclyl;

U¹ is N, C-R², or C-NR³R⁴, wherein R² is selected from the group consisting of a H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, 0-CH₂- CH₂X, CH2-CH2-CH2X, O-CH2-CH2X, X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, 0(CO)R', COOR' (wherein R' is H or a lower alkyl group), and wherein R¹ and R² may be linked to form a cyclic or polycyclic ring, wherein R³ and R⁴ are the same or different and are each selected from the group consisting of H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, CH₂-CH₂-CH₂X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, COOR' (wherein R' is H or a lower alkyl group), and R³ or R⁴ may be absent;

Z¹ is O, S, CR^aR^b or NR^a, wherein R^a and R^b are independently H or a Ci_₈ alkyl, which is linear, branched, or cyclic, and which is unsubstituted or substituted;

and pharmaceutically acceptable salts thereof.

[00115] In yet other embodiments, the 15-PGDH inhibitor can include a compound having the following formula (III) or (IV):

wherein n is 0-2

X⁶ is independently is N or CR^C;

R¹, R⁶, R⁷, and R^c are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C₃-C2₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6 ring atoms (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(0)(Ci-C₆ alkyl), O, and S), C₆-C24 alkaryl, C₆-C24 aralkyl, halo, -Si(Ci-C₃ alkyf , hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (— CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C₂-C₂₄

alkylcarbonato (-O-(CO)-O-alkyl), C₆-C2₀ arylcarbonato (-O-(CO)-O-aryl), carboxy

(-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl

(-(CO)-NH(Ci-C₂₄ alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺ ), cyanato (-0-CN), isocyanato (-0-N⁺=C ), isothiocyanato (-S-CN), azido (-N=N⁺=N ), formyl (-(CO)-H), thioformyl (~(CS)~ H), amino (~NH₂), C1-C24 alkyl amino, C5-C2₀ aryl amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), imino (-CR=NH where R is hydrogen, C1-C24 alkyl, C5-C2₀ aryl, C₆-C24 alkaryl, C₆-C24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O ), C1-C24 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), Ci-C₂₄ alkylsulfinyl (-(SO)-alkyl), C₅-C₂₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (wherein Y is independently H, aryl or alkyl), phosphono

(-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-P0₂), phosphino (— PH₂), poly alky lethers, phosphates, phosphate esters, groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, combinations thereof, and wherein R⁶ and R⁷ may be linked to form a cyclic or polycyclic ring, wherein the ring is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, and a substituted or unsubstituted heterocyclyl;

U¹ is N, C-R², or C-NR³R⁴, wherein R² is selected from the group consisting of a H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, 0-CH₂- CH₂X, CH2-CH2-CH2X, O-CH2-CH2X, X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, 0(CO)R', COOR' (wherein R' is H or a lower alkyl group), and wherein R¹ and R² may be linked to form a cyclic or polycyclic ring, wherein R³ and R⁴ are the same or different and are each selected from the group consisting of H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, CH₂-CH₂-CH₂X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, COOR' (wherein R' is H or a lower alkyl group), and R³ or R⁴ may be absent;

Z¹ is O, S, CR^aR^b or NR^a, wherein R^a and R^b are independently H or a Ci_₈ alkyl, which is linear, branched, or cyclic, and which is unsubstituted or substituted;

and pharmaceutically acceptable salts thereof.

[00116] In some embodiments, R¹ is selected from the group consisting of branched or linear alkyl including -(CH₂)niCH₃ (ni=0-7), ² wherein n2=0-6 and X is any of the following: CF_yH_z (y + z = 3), CCl_yH_z (y + z = 3), OH, OAc, OMe, R⁷¹, OR⁷², CN, N(R⁷³)₂,

"³ (n₃=0-5, m=l-5), and "⁴ (n₄=0-5).

[00117] In other embodiments, R⁶ and R⁷ can each independently be one of the following:

R _, R _, R _, R _, and R are the same or different and are independently selected from the group consisting of hydrogen, substituted or unsubstituted C₁-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C₃-C2₀ aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(O) (Ci-C₆ alkyl), O, and S), heteroaryl or heterocyclyl containing from 5-14 ring atoms, (wherein from 1-6 of the ring atoms is independently selected from N, NH, N(Ci-C₃ alkyl), O, and S), C6-C24 alkaryl, C₆-C24 aralkyl, halo, silyl, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C₂-C₂₄ alkylcarbonato (-O-(CO)-O- alkyl), C₆-C₂₀ arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl (-(CO)-NH(Ci-C₂₄ alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C ), cyanato (-0-CN), isocyanato (-0-N⁺=C^~), isothiocyanato (-S-CN), azido (-N=N⁺=N ), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (~NH₂), Ci-C₂₄ alkyl amino, C5-C2₀ aryl amino, C₂-C₂₄ alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), sulfanamide (-S0₂N(R)2 where R is independently H, alkyl, aryl or heteroaryl), imino (-CR=NH where R is hydrogen, C1-C24 alkyl, C5-C2₀ aryl, C₆-C24 alkaryl, C₆-C24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-S0₂-0^~), Ci-C₂₄ alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), C C₂4 alkylsulfinyl (-(SO)-alkyl), C5-C2₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (wherein Y is independently H, aryl or alkyl), phosphono (-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-PO2), phosphino (— PH2), polyalkyl ethers (-[(CH2)_nO]_m), phosphates, phosphate esters [-OP(0)(OR)2 where R = H, methyl or other alkyl], groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, and combinations thereof, and pharmaceutically acceptable salts thereof.

[00118] In still other embodiments, R⁶ and R⁷ can independently be a group that improves aqueous solubility, for example, a phosphate ester (-OPO₃H2), a phenyl ring linked to a phosphate ester (-OPO₃H2), a phenyl ring substituted with one or more methoxyethoxy groups, or a morpholine, or an aryl or heteroaryl ring substituted with such a group. [00119] In other embodiments, the 15-PGDH inhibitor can include a compound having the following

wherein n is 0-2

X⁶ is independently is N or CR^C

R\ R⁶, R⁷, and R^c are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C₃-C2₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6 ring atoms (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(0)(Ci-C₆ alkyl), O, and S), C₆-C24 alkaryl, C₆-C24 aralkyl, halo, -Si(Ci-C₃ alkyf , hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (— CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C₂-C₂₄

alkylcarbonato (-O-(CO)-O-alkyl), C₆-C2₀ arylcarbonato (-O-(CO)-O-aryl), carboxy

(-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl

(-(CO)-NH(Ci-C₂₄ alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C ), cyanato (-0-CN), isocyanato (-0-N⁺=C), isothiocyanato (-S-CN), azido (-N=N⁺=N ), formyl (-(CO)-H), thioformyl (~(CS)~ H), amino (~NH₂), C1-C24 alkyl amino, C5-C2₀ aryl amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), imino (-CR=NH where R is hydrogen, C1-C24 alkyl, C5-C2₀ aryl, C₆-C24 alkaryl, C₆-C24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O ), C1-C24 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), Ci-C₂₄ alkylsulfinyl (-(SO)-alkyl), C₅-C₂₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (wherein Y is independently H, aryl or alkyl), phosphono (-P(0)(OH)₂), phosphonato (-P(0)(0^~)₂), phosphinato (-P(0)(0^~)), phospho (-P0₂), phosphino (— PH₂), poly alky lethers, phosphates, phosphate esters, groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, combinations thereof, and wherein R⁶ and R⁷ may be linked to form a cyclic or polycyclic ring, wherein the ring is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, and a substituted or unsubstituted heterocyclyl;

U¹ is N, C-R², or C-NR³R⁴, wherein R² is selected from the group consisting of a H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, 0-CH₂-CH₂X, CH₂-CH₂-CH₂X, 0-CH₂-CH₂X, X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, 0(CO)R' , COOR' (wherein R' is H or a lower alkyl group), and wherein R¹ and R² may be linked to form a cyclic or polycyclic ring, wherein R³ and R⁴ are the same or different and are each selected from the group consisting of H, a lower alkyl group, O, (CH₂)_nlOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, CH₂-CH₂-CH₂X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, COOR' (wherein R' is H or a lower alkyl group), and R³ or R⁴ may be absent;

and pharmaceutically acceptable salts thereof.

[00120] In some embodiments, R¹ is selected from the group consisting of branched or linear alkyl including -(CH₂)niCH₃ (ni=0-7), ² wherein n₂=0-6 and X is any of the following: CF_yH_z (y + z = 3), CCl_yH_z (y + z = 3), OH, OAc, OMe, R⁷¹, OR⁷², CN, N(R⁷³)₂,

"³ (n₃=0-5, m=l-5), and (n₄=0-5).

[00121] In other embodiments, R⁶ and R⁷ can each independently be one of the following:

R _, R _, R _, R , and R _, are the same or different and are independently selected from the group consisting of hydrogen, substituted or unsubstituted C₁-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C₃-C2₀ aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(0)(Ci- C₆ alkyl), O, and S), heteroaryl or heterocyclyl containing from 5-14 ring atoms, (wherein from 1-6 of the ring atoms is independently selected from N, NH, N(Ci-C₃ alkyl), O, and S), C₆-C24 alkaryl, C₆-C24 aralkyl, halo, silyl, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (— CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C₂-C₂₄ alkylcarbonato (-O-(CO)-O- alkyl), C₆-C₂₀ arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl (-(CO)-NH(Ci-C₂₄ alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C ), cyanato (-0-CN), isocyanato (-0-N⁺=C^~), isothiocyanato (-S-CN), azido (-N=N⁺=N ), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (~NH₂), Ci-C₂₄ alkyl amino, C5-C2₀ aryl amino, C₂-C₂₄ alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), sulfanamide (-S0₂N(R)2 where R is independently H, alkyl, aryl or heteroaryl), imino (-CR=NH where R is hydrogen, C1-C24 alkyl, C5-C2₀ aryl, C₆-C24 alkaryl, C₆-C24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-S0₂-0^~), Ci-C₂₄ alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), C C₂4 alkylsulfinyl (-(SO)-alkyl), C5-C2₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (wherein Y is independently H, aryl or alkyl), phosphono (-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-PO2), phosphino (— PH2), polyalkyl ethers (-[(CH2)_nO]_m), phosphates, phosphate esters [-OP(0)(OR)2 where R = H, methyl or other alkyl], groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, and combinations thereof, and pharmaceutically acceptable salts thereof.

[00122] In still other embodiments, R⁶ and R⁷ can independently be a group that improves aqueous solubility, for example, a phosphate ester (-OPO₃H2), a phenyl ring linked to a phosphate ester (-OPO₃H2), a phenyl ring substituted with one or more methoxyethoxy groups, or a morpholine, or an aryl or heteroaryl ring substituted with such a group. [00123] In other embodiments, the 15-PGDH inhibitor can include a compound having the following

(VI)

wherein n = 0-2;

X⁶ is N or CR^C;

R¹ is selected from the group consisting of branched or linear alkyl including■

X

(0¾)ηιΟ¾ (ni=0-7), ⁿ² wherein n2=0-6 and X is any of the following: CF_yH_z (y + z =

3), CCl_yH_z (y + z = 3), OH, OAc, OMe, R⁷¹, OR⁷², CN, N(R⁷³)₂,

m=l-5), and ⁿ⁴ (n₄=0-5).

R⁵ is selected from the group consisting of H, CI, F, Ν¾, and N(R⁷⁶)2_;

R⁶ and R⁷ can each independently be one of the following:

each R⁸ R⁹ R¹⁰ R¹¹ R¹² R¹³ R¹⁴ R¹⁵ R¹⁶ R¹⁷ R¹⁸ R¹⁹ R²⁰ R²¹ R²² R²³ R²⁴ R²⁵

26 _R27 R²⁸ R²⁹ _{, R}30

R³¹,

_R 4488 R_R ⁴4⁹9_, R_R ⁵5⁰0 _RR5511 _RR5522 R_R ⁵5³3 _R54 _R55 _R56 _R57 _R58 _R59 _R60 _R61 _R62 _R63 _R64 _R65 _R66 _R67 _R68 _R69 _R 7700 R_R ⁷7¹1 _RR7722 R_R ⁷7³3 R_R ⁷7⁴4 R_R ⁷7⁶6 _{and R}c are the same or different and are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C2₀ aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(O) (Ci-C₆ alkyl), O, and S), heteroaryl or heterocyclyl containing from 5-14 ring atoms, (wherein from 1-6 of the ring atoms is independently selected from N, NH, N(Ci-C₃ alkyl), O, and S), C₆-C24 alkaryl, C₆-C24 aralkyl, halo, silyl, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C₂-C₂₄ alkylcarbonato (-O-(CO)-O- alkyl), C₆-C₂₀ arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl (-(CO)-NH(Ci-C₂₄ alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C^~), cyanato (-0-CN), isocyanato (-0-N⁺=C^~), isothiocyanato (-S-CN), azido (-N=N⁺=N ), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (~NH₂), C C₂4 alkyl amino, C5-C2₀ aryl amino, C₂-C₂₄ alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), sulfanamide (-S02N(R)2 where R is independently H, alkyl, aryl or heteroaryl), imino (-CR=NH where R is hydrogen, Ci-C2₄ alkyl, C5-C2₀ aryl, C₆-C2₄ alkaryl, C₆-C2₄ aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-S0₂-0^~), C C₂₄ alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), Ci-C₂₄ alkylsulfinyl (-(SO)-alkyl), C5-C2₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (wherein Y is independently H, aryl or alkyl), phosphono (-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-PO2), phosphino (— PH2), polyalkyl ethers (-[(CH2)_nO]_m), phosphates, phosphate esters [-OP(0)(OR)2 where R = H, methyl or other alkyl], groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, and combinations thereof, and pharmaceutically acceptable salts thereof.

[00124] In other embodiments, the 15-PGDH inhibitor can include a compound having the following

wherein n = 0-2;

X⁶ is N or CR^C;

R¹ is selected from the group consisting of branched or linear alkyl including -(CH2)niCH₃ (ni=0-7), ² wherein n2=0-6 and X is any of the following: CF_yH_z (y + z =

3), CCl_yH_z (y + z = 3), OH, OAc, OMe, R⁷¹, OR⁷², CN, N(R⁷³)₂, ^ns (n₃=0-5, m=l-5), and ⁿ⁴ (n₄=0-5).

R⁵ is selected from the group consisting of H, CI, F, NH2, and N(R⁷⁶)2_; ⁷ can each independently be one of the following:

each R⁸. R⁹ R¹⁰ Rⁿ _, R¹² R¹³ R¹⁴ R¹⁵. R¹⁶ R¹⁷. R¹⁸ R¹⁹ R²⁰ R²¹ R²² R²³. R²⁴ R²⁵. R²⁶ R²⁷ _, R²⁸ R²⁹ R³⁰ R ¹ R³ R³³ _, R³⁴ _, R³⁵ R³⁶ R³⁷ _, R³⁸ _, R³⁹. R⁴⁰ R⁴¹ _, R⁴² _, R⁴³ _, R⁴⁴ _, R⁴⁵ _, R⁴⁶ R⁴⁷.

_R48 _R49 _R50 _R51 _R52 _R53 _R54 _R55 _R56 _R57 _R58 _R59 _R60 _R61 _R62 _R63 _R64 _R65 _R66 _R67 _R68 _R69

_R7o _R7i _R72 _R73 _R74 _R76 _an(j _Rc _arg ^ _{same or} different and are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C2₀ aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(O) (Ci-C₆ alkyl), O, and S), heteroaryl or heterocyclyl containing from 5-14 ring atoms, (wherein from 1-6 of the ring atoms is independently selected from N, NH, N(Ci-C₃ alkyl), O, and S), C6-C24 alkaryl, C₆-C24 aralkyl, halo, silyl, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C₂-C₂₄ alkylcarbonato (-O-(CO)-O- alkyl), C₆-C₂₀ arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl (-(CO)-NH(Ci-C₂₄ alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C ), cyanato (-0-CN), isocyanato (-0-N⁺=C^~), isothiocyanato (-S-CN), azido (-N=N⁺=N ), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (~NH₂), Ci-C₂₄ alkyl amino, C5-C2₀ aryl amino, C₂-C₂₄ alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), sulfanamide (-S02N(R)2 where R is independently H, alkyl, aryl or heteroaryl), imino (-CR=NH where R is hydrogen, C1-C24 alkyl, C5-C2₀ aryl, C₆-C24 alkaryl, C₆-C24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-S0₂-0^~), Ci-C₂₄ alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), C C₂4 alkylsulfinyl (-(SO)-alkyl), C5-C2₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S02-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (wherein Y is independently H, aryl or alkyl), phosphono (-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-PO2), phosphino (— PH2), polyalkyl ethers (-[(CH2)_nO]_m), phosphates, phosphate esters [-OP(0)(OR)2 where R = H, methyl or other alkyl], groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, and combinations thereof, and pharmaceutically acceptable salts thereof.

[00125] Examples of compounds having formulas (I), (II), (III), (IV), (V), (VI), or (VII) are selected from are described in U.S. Patent Application Publication Nos. 2015/0072998, 2017/0165241, 2017/0173028, and 2018/0118756, all of which are incorporated by referenc in their entirety. [00126] In certain embodiments, the 15-PGDH inhibitor having formula (I), (II), (III), (IV), (V), (VI), and (VII) can be selected that can ia) at 2.5 μΜ concentration, stimulate a Vaco503 reporter cell line expressing a 15-PGDH luciferase fusion construct to a luciferase output level of greater than 70 (using a scale on which a value of 100 indicates a doubling of reporter output over baseline); iia) at 2.5 μΜ concentration stimulate a V9m reporter cell line expressing a 15-PGDH luciferase fusion construct to a luciferase output level of greater than 75; iiia) at 7.5 μΜ concentration stimulate a LS174T reporter cell line expressing a 15-PGDH luciferase fusion construct to a luciferase output level of greater than 70; and iva) at 7.5 μΜ concentration, does not activate a negative control V9m cell line expressing TK-renilla luciferase reporter to a level greater than 20; and va) inhibits the enzymatic activity of recombinant 15-PGDH protein at an IC₅₀ of less than 1 μΜ.

[00127] In other embodiments, the 15-PGDH inhibitor can ib) at 2.5 μΜ concentration, stimulate a Vaco503 reporter cell line expressing a 15-PGDH luciferase fusion construct to increase luciferase output; iib) at 2.5 μΜ concentration stimulate a V9m reporter cell line expressing a 15-PGDH luciferase fusion construct to increase luciferase output; iiib) at 7.5 μΜ concentration stimulate a LS174T reporter cell line expressing a 15-PGDH luciferase fusion construct to increase luciferase output; ivb) at 7.5 μΜ concentration, does not activate a negative control V9m cell line expressing TK-renilla luciferase reporter to a luciferase level greater than 20% above background; and vb) inhibits the enzymatic activity of recombinant 15-PGDH protein at an IC₅₀ of less than 1 μΜ.

[00128] In other embodiments, the 15-PGDH inhibitor can inhibit the enzymatic activity of recombinant 15-PGDH at an IC₅₀ of less than 1 μΜ, or preferably at an IC₅₀ of less than 250 nM, or more preferably at an IC₅₀ of less than 50 nM, or more preferably at an IC₅₀ of less than 10 nM, or more preferably at an IC₅₀ of less than 5 nM at a recombinant 15-PGDH concentration of about 5 nM to about 10 nM.

[00129] In other embodiments, the 15-PGDH inhibitor can increase the cellular levels of PGE-2 following stimulation of an A459 cell with an appropriate agent, for example IL1- beta.

[00130] In some embodiments, al5-PGDH inhibitor can include a compound having the following formula (VIII):

wherein n is 0-2;

R¹, R⁶, and R⁷ are the same or different and are each selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C2₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6 ring atoms (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C₆ alkyl), NC(O) (C C₆ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, -Si(C C₃ alkyl)₃, hydroxyl, sulfhydryl, Ci-C2₄ alkoxy, C2-C2₄ alkenyloxy, C2-C2₄ alkynyloxy, C5-C2₀ aryloxy, acyl (including C2-C2₄ alkylcarbonyl (-CO-alkyl) and C₆-C2₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C₂-C₂₄ alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), C2-C2₄ alkylcarbonato (-O-(CO)-O-alkyl), C₆-C2₀ arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO ), carbamoyl (-(CO)-NH₂), Ci-C₂₄ alkyl-carbamoyl

(-(CO)-NH(Ci-C₂₄ alkyl)), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (-NH-(CO)-NH₂), cyano(-CN), isocyano (-N⁺C ), cyanato (-0-CN), isocyanato (-0-N⁺=C), isothiocyanato (-S-CN), azido (-N=N⁺=N ), formyl (-(CO)-H), thioformyl (~(CS)~ H), amino (~NH₂), Ci-C2₄ alkyl amino, C5-C2₀ aryl amino, C2-C2₄ alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), imino (-CR=NH where R is hydrogen, Ci-C2₄ alkyl, C5-C2₀ aryl, C₆-C2₄ alkaryl, C₆-C2₄ aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O ), Ci-C2₄ alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), Ci-C₂₄ alkylsulfinyl (-(SO)-alkyl), C₅-C₂₀ arylsulfinyl (-(SO)-aryl), Ci-C₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (wherein Y is independently H, aryl or alkyl), phosphono

(-P(0)(OH)₂), phosphonato (-P(0)(0^")₂), phosphinato (-P(0)(0^~)), phospho (-P0₂), phosphino (— PH₂), poly alky lethers, phosphates, phosphate esters, groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, combinations thereof, and wherein R⁶ and R⁷ may be linked to form a cyclic or polycyclic ring, wherein the ring is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, and a substituted or unsubstituted heterocyclyl; and pharmaceutically acceptable salts thereof.

[00131] 1 -PGDH inhibitors having formula (VIII) can be synthesized as shown:

[00132] Any reaction solvent can be used in the above preparation process as long as it is not involved in the reaction. For example, the reaction solvent includes ethers such as diethyl ether, tetrahydrofuran and dioxane; halogenized hydrocarbons, such as dichloromethane and chloroform; amines such as pyridine, piperidine and triethylamine; alkylketones, such as acetone, methylethylketone and methylisobutyl; alcohols, such as methanol, ethanol and propanol; non-protonic polar solvent, such as Ν,Ν-dimethylformamide, N,N- dimethylacetamide, acetonitrile, dimethylsulfoxide and hexamethyl phosphoric acid triamide. Among non-reactive organic solvents that are ordinarily used in the organic synthesis, preferable solvents are those from which water generated in the reaction can be removed by a Dean-Stark trap. The examples of such solvents include, but are not limited to benzene, toluene, xylene and the like. The reaction product thus obtained may be isolated and purified by condensation, extraction and the like, which is ordinarily conducted in the field of the organic synthesis, if desired, by silica gel column chromatography. The individual enantiomers of PGDH inhibitors having the formula III can be separated by a preparative HPLC using chromatography columns containing chiral stationary phases.

[00133] Further, embodiments of this application include any modifications for the preparation method of the 15-PGDH inhibitors described above. In this connection, any intermediate product obtainable from any step of the preparation method can be used as a starting material in the other steps. Such starting material can be formed in situ under certain reaction conditions. Reaction reagents can also be used in the form of their salts or optical isomers.

[00134] Depending on the kinds of the substituents to be used in the preparation of the 15-PGDH inhibitors, and the intermediate product and the preparation method selected, novel 15-PGDH inhibitors can be in the form of any possible isomers such as substantially pure geometrical (cis or trans) isomers, optical isomers (enantiomers) and racemates.

[00135] In some embodiments, a 15-PGDH inhibitor having formula (VIII) can include a compound w

and pharmaceutically acceptable salts thereof.

[00136] Advantageously, the 15-PDGH inhibitor having formula (IX) was found to: i) inhibit recombinant 15-PGDH at 1 nM concentration; ii) inhibit 15-PGDH in cell lines at 100 nM concentration, iii) increase PGE2 production by cell lines; iv) is chemically stable in aqueous solutions over broad pH range; v) is chemically stable when incubated with hepatocyte extracts, vi) is chemically stable when incubated with hepatocyte cell lines; vii) shows 253 minutes plasma half-life when injected IP into mice; and viii) shows no immediate toxicity over 24 hours when injected IP into mice at 0.6 μιηοΙε/ρεΓ mouse and at

1.2 μιηοΙε/ρεΓ mouse and also no toxicity when injected IP into mice at 0.3 μιηοΙε/ρεΓ mouse twice daily for 21 days.

[00137] In other embodiments, a 15-PGDH inhibitor having formula (IX) can include a compound with the following formula (IXa):

and pharmaceutically acceptable salts thereof.

[00138] In still other embodiments, a 15-PGDH inhibitor having formula (IX) can include a comp

and pharmaceutically acceptable salts thereof.

[00139] In other embodiments, the 15-PDHG inhibitor can comprise a (+) or (-) optical isomer of a 15-PGDH inhibitor having formula (IX). In still other embodiments, the 15-PDHG inhibitor can comprise a mixture at least one of a (+) or (-) optical isomer of a 15-PGDH inhibitor having formula (IX). For example, the 15-PGDH inhibitor can comprise a mixture of: less than about 50% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (IX) and greater than about 50% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (IX), less than about 25% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (IX) and greater than about 75% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (IX), less than about 10% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (IX) and greater than about 90% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (IX), less than about 1% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (IX) and greater than about 99% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (IX), greater than about 50% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (IX) and less than about 50% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (IX), greater than about 75% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (IX) and less than about 25% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (IX), greater than about 90% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (IX) and less than about 10% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (IX), or greater than about 99% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (IX) and less than about 1 % by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (IX).

[00140] In a still further embodiment, the 15-PDGH inhibitor can consist essentially of or consist of the (+) optical isomer of a 15-PGDH inhibitor having formula (IX). In yet another embodiment, the PDGH inhibitor can consist essentially of or consist of the (-) optical isomer of a 15-PGDH inhibitor having formula (IX).

[00141] In other embodiments, a 15-PGDH inhibitor having formula (VIII) can include a compound wi

and pharmaceutically acceptable salts thereof.

[00142] Advantageously, the 15-PDGH inhibitor having formula (X) was found to: i) inhibit recombinant 15-PGDH at 3 nM concentration; ii) increase PGE2 production by cell lines at 20nM; iii) is chemically stable in aqueous solutions over broad pH range; iv) is chemically stable when incubated with mouse, rat and human liver extracts, v) shows 33 minutes plasma half-life when injected IP into mice; viii) shows no immediate toxicity over 24 hours when injected IP into mice at 50 mg/kg body weight, and ix) is soluble in water (pH=3) at 1 mg/mL.

[00143] In other embodiments, a 15-PGDH inhibitor having formula (X) can include a compound with the following formula (Xa):

and pharmaceutically acceptable salts thereof.

[00144] In still other embodiments, a 15-PGDH inhibitor having formula (X) can include a compound w

and pharmaceutically acceptable salts thereof.

[00145] In other embodiments, the 15-PDHG inhibitor can comprise a (+) or (-) optical isomer of a 15-PGDH inhibitor having formula (X). In still other embodiments, the

15-PDHG inhibitor can comprise a mixture at least one of a (+) or (-) optical isomer of a 15-PGDH inhibitor having formula (X). For example, the 15-PGDH inhibitor can comprise a mixture of: less than about 50% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (X) and greater than about 50% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (X), less than about 25% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (X) and greater than about 75% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (X), less than about 10% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (X) and greater than about 90% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (X), less than about 1% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (X) and greater than about 99% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (X), greater than about 50% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (X) and less than about 50% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (X), greater than about 75% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (X) and less than about 25% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (X), greater than about 90% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (X) and less than about 10% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (X), or greater than about 99% by weight of the (-) optical isomer of a 15-PGDH inhibitor having formula (X) and less than about 1% by weight of the (+) optical isomer of a 15-PGDH inhibitor having formula (X).

[00146] In a still further embodiment, the 15-PDGH inhibitor can consist essentially of or consist of the (+) optical isomer of a 15-PGDH inhibitor having formula (X). In yet another embodiment, the PDGH inhibitor can consist essentially of or consist of the (-) optical isomer of a 15-PGDH inhibitor having formula (X).

[00147] It will be appreciated that the other 15-PGDH inhibitors can be used in the methods described herein. These other 15-PGDH inhibitors can include known 15-PGDH inhibitors including, for example, tetrazole compounds of formulas (I) and (II),

2-alkylideneaminooxyacetamidecompounds of formula (I), heterocyclic compounds of formulas (VI) and (VII), and pyrazole compounds of formula (III) described in U.S. Patent Application Publication No. 2006/0034786 and U.S. Patent No. 7,705,041 ; benzylidene-1,3- thiazolidine compounds of formula (I) described in U.S. Patent Application Publication No. 2007/0071699; phenylfurylmethylthiazolidine-2,4-dione and

phenylthienylmethylthiazolidine-2,4-dione compounds described in U.S. Patent Application Publication No. 2007/0078175; thiazolidenedione derivatives described in U.S. Patent Application Publication No. 2011/0269954; phenylfuran, phenylthiophene, or

phenylpyrrazole compounds described in U.S. Patent No. 7,294,641, 5-(3,5-disubstituted phenylazo)-2-hydroxybenzene-acetic acids and salts and lactones described in U.S. Patent No. 4,725,676, and azo compounds described in U.S. Patent No. 4,889,846.

[00148] Still other examples are described in the following publications: Seo SY et al. Effect of 15-hydroxyprostaglandin dehydrogenase inhibitor on wound healing.

Prostaglandins Leukot Essent Fatty Acids. 2015;97:35-41. doi: 10.1016/j.plefa.2015.03.005. PubMed PMID: 25899574; Piao YL et al. Wound healing effects of new 15- hydroxyprostaglandin dehydrogenase inhibitors. Prostaglandins Leukot Essent Fatty Acids. 2014;91(6):325-32. doi: 10.1016/j.plefa.2014.09.011. PubMed PMID: 25458900; Choi D et al. Control of the intracellular levels of prostaglandin E(2) through inhibition of the 15- hydroxyprostaglandin dehydrogenase for wound healing. Bioorg Med Chem.

2013;21(15):4477-84. doi: 10.1016/j.bmc.2013.05.049. PubMed PMID: 23791868; Wu Y et al. Synthesis and biological evaluation of novel thiazolidinedione analogues as 15- hydroxyprostaglandin dehydrogenase inhibitors. J Med Chem. 2011 ;54(14):5260-4. Epub 2011/06/10. doi: 10.1021/jm200390u. PubMed PMID: 21650226; Duveau DY et al.

Structure-activity relationship studies and biological characterization of human NAD(+)- dependent 15-hydroxyprostaglandin dehydrogenase inhibitors. Bioorg Med Chem Lett. 2014;24(2):630-5. doi: 10.1016/j.bmcl.2013.11.081. PubMed PMID: 24360556; PMCID: PMC3970110; Duveau DY et al. Discovery of two small molecule inhibitors, ML387 and ML388, of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase. Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD)2010; Wu Y et al.

Synthesis and SAR of thiazolidinedione derivatives as 15-PGDH inhibitors. Bioorg Med Chem. 2010; 18(4): 1428-33. doi: 10.1016/j.bmc.2010.01.016. PubMed PMID: 20122835; Wu Y et al. Synthesis and biological evaluation of novel thiazolidinedione analogues as 15- hydroxyprostaglandin dehydrogenase inhibitors. J Med Chem. 2011 ;54(14):5260-4. Epub 2011/06/10. doi: 10.1021/jm200390u. PubMed PMID: 21650226; Jadhav A et al. Potent and selective inhibitors of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD). Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD)2010; Niesen FH et al. High-affinity inhibitors of human NAD-dependent 15-hydroxyprostaglandin dehydrogenase: mechanisms of inhibition and structure-activity relationships. PLoS One. 2010;5(l l):el3719. Epub 2010/11/13. doi: 10.1371/journal.pone.0013719. PubMed PMID: 21072165; PMCID: 2970562; Michelet, J. et al. Composition comprising at least one 15- PGDH inhibitor. US20080206320 Al, 2008; and Rozot, R et al. Care/makeup compositions comprising a 2-alkylideneaminooxyacetamide compound for stimulating the growth of the hair or eyelashes and/or slowing loss thereof. US7396525 B2, 2008.

[00149] The 15-PGDH inhibitors and, optionally, the other labor inducing agents described herein can be provided in a pharmaceutical composition. A pharmaceutical composition containing the 15-PGDH inhibitors and, optionally, the other labor inducing agents described herein as an active ingredient may be manufactured by mixing the derivative with a pharmaceutically acceptable carrier(s) or an excipient(s) or diluting the 15-PGDH and, optionally, the other labor inducing agents inhibitors with a diluent in accordance with conventional methods. The pharmaceutical composition may further contain fillers, anti- cohesives, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives and the like. The pharmaceutical composition may be formulated into a suitable formulation in accordance with the methods known to those skilled in the art so that it can provide an immediate, controlled or sustained release of the 15-PGDH inhibitors and, optionally, the other labor inducing agents after being administered into a mammal.

[00150] In some embodiments, the pharmaceutical composition may be formulated into a parenteral or oral dosage form. The solid dosage form for oral administration may be manufactured by adding excipient, if necessary, together with binder, disintegrants, lubricants, coloring agents, and/or flavoring agents, to the 15-PGDH inhibitors and, optionally, the other labor inducing agents and shaping the resulting mixture into the form of tablets, sugar-coated pills, granules, powder or capsules. The additives that can be added in the composition may be ordinary ones in the art. For example, examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicate and the like. Exemplary binders include water, ethanol, propanol, sweet syrup, sucrose solution, starch solution, gelatin solution,

carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphonate and polypyrrolidone. Examples of the disintegrant include dry starch, sodium arginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic monoglyceride and lactose. Further, purified talc, stearates, sodium borate, and polyethylene glycol may be used as a lubricant; and sucrose, bitter orange peel, citric acid, tartaric acid, may be used as a flavoring agent. In some embodiments, the pharmaceutical composition can be made into aerosol formulations (e.g., they can be nebulized) to be administered via inhalation.

[00151] The 15-PGDH inhibitors and, optionally, the other labor inducing agents described herein may be combined with flavoring agents, buffers, stabilizing agents, and the like and incorporated into oral liquid dosage forms such as solutions, syrups or elixirs in accordance with conventional methods. One example of the buffers may be sodium citrate. Examples of the stabilizing agents include tragacanth, acacia and gelatin.

[00152] In some embodiments, the 15-PGDH inhibitors and, optionally, the other labor inducing agents described herein may be incorporated into an injection dosage form, for example, for a subcutaneous, intramuscular or intravenous route by adding thereto pH adjusters, buffers, stabilizing agents, relaxants, topical anesthetics. Examples of the pH adjusters and the buffers include sodium citrate, sodium acetate and sodium phosphate. Examples of the stabilizing agents include sodium pyrosulfite, EDTA, thioglycolic acid and thiolactic acid. The topical anesthetics may be procaine HC1, lidocaine HC1 and the like. The relaxants may be sodium chloride, glucose and the like.

[00153] In other embodiments, the 15-PGDH inhibitors and, optionally, the other labor inducing agents described herein may be incorporated into suppositories in accordance with conventional methods by adding thereto pharmaceutically acceptable carriers that are known in the art, for example, polyethylene glycol, lanolin, cacao butter or fatty acid triglycerides, if necessary, together with surfactants such as Tween.

[00154] The pharmaceutical composition may be formulated into various dosage forms as discussed above and then administered through various routes including an oral, inhalational, transdermal, subcutaneous, intravenous or intramuscular route. The dosage can be a pharmaceutically or therapeutically effective amount.

[00155] Therapeutically effective dosage amounts of the 15-PGDH inhibitor and, optionally, the other labor inducing agents may be present in varying amounts in various embodiments. For example, in some embodiments, a therapeutically effective amount of the 15-PGDH inhibitor may be an amount ranging from about 10-1000 mg (e.g., about 20 mg- 1,000 mg, 30 mg- 1 ,000 mg, 40 mg- 1,000 mg, 50 mg- 1,000 mg, 60 mg- 1 ,000 mg, 70 mg- 1,000 mg, 80 mg-1 ,000 mg, 90 mg- 1,000 mg, about 10-900 mg, 10-800 mg, 10-700 mg, 10- 600 mg, 10-500 mg, 100- 1000 mg, 100-900 mg, 100-800 mg, 100-700 mg, 100-600 mg, 100- 500 mg, 100-400 mg, 100-300 mg, 200-1000 mg, 200-900 mg, 200-800 mg, 200-700 mg, 200-600 mg, 200-500 mg, 200-400 mg, 300- 1000 mg, 300-900 mg, 300-800 mg, 300-700 mg, 300-600 mg, 300-500 mg, 400 mg- 1 ,000 mg, 500 mg- 1,000 mg, 100 mg-900 mg, 200 mg-800 mg, 300 mg-700 mg, 400 mg-700 mg, and 500 mg-600 mg). In some embodiments, the 15-PGDH inhibitor is present in an amount of or greater than about 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg. In some embodiments, the 15-PGDH inhibitor is present in an amount of or less than about 1000 mg, 950 mg, 900 mg, 850 mg, 800 mg, 750 mg, 700 mg, 650 mg, 600 mg, 550 mg, 500 mg, 450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, or 100 mg. [00156] In other embodiments, a therapeutically effective dosage amount may be, for example, about 0.001 mg/kg weight to 500 mg/kg weight, e.g., from about 0.001 mg/kg weight to 400 mg/kg weight, from about 0.001 mg/kg weight to 300 mg/kg weight, from about 0.001 mg/kg weight to 200 mg/kg weight, from about 0.001 mg/kg weight to

100 mg/kg weight, from about 0.001 mg/kg weight to 90 mg/kg weight, from about

0.001 mg/kg weight to 80 mg/kg weight, from about 0.001 mg/kg weight to 70 mg/kg weight, from about 0.001 mg/kg weight to 60 mg/kg weight, from about 0.001 mg/kg weight to

50 mg/kg weight, from about 0.001 mg/kg weight to 40 mg/kg weight, from about

0.001 mg/kg weight to 30 mg/kg weight, from about 0.001 mg/kg weight to 25 mg/kg weight, from about 0.001 mg/kg weight to 20 mg/kg weight, from about 0.001 mg/kg weight to

15 mg/kg weight, from about 0.001 mg/kg weight to 10 mg/kg weight.

[00157] In still other embodiments, a therapeutically effective dosage amount may be, for example, about 0.0001 mg/kg weight to 0.1 mg/kg weight, e.g. from about 0.0001 mg/kg weight to 0.09 mg/kg weight, from about 0.0001 mg/kg weight to 0.08 mg/kg weight, from about 0.0001 mg/kg weight to 0.07 mg/kg weight, from about 0.0001 mg/kg weight to

0.06 mg/kg weight, from about 0.0001 mg/kg weight to 0.05 mg/kg weight, from about

0.0001 mg/kg weight to about 0.04 mg/kg weight, from about 0.0001 mg/kg weight to

0.03 mg/kg weight, from about 0.0001 mg/kg weight to 0.02 mg/kg weight, from about

0.0001 mg/kg weight to 0.019 mg/kg weight, from about 0.0001 mg/kg weight to

0.018 mg/kg weight, from about 0.0001 mg/kg weight to 0.017 mg/kg weight, from about

0.0001 mg/kg weight to 0.016 mg/kg weight, from about 0.0001 mg/kg weight to

0.015 mg/kg weight, from about 0.0001 mg/kg weight to 0.014 mg/kg weight, from about

0.0001 mg/kg weight to 0.013 mg/kg weight, from about 0.0001 mg/kg weight to

0.012 mg/kg weight, from about 0.0001 mg/kg weight to 0.011 mg/kg weight, from about

0.0001 mg/kg weight to 0.01 mg/kg weight, from about 0.0001 mg/kg weight to 0.009 mg/kg weight, from about 0.0001 mg/kg weight to 0.008 mg/kg weight, from about 0.0001 mg/kg weight to 0.007 mg/kg weight, from about 0.0001 mg/kg weight to 0.006 mg/kg weight, from about 0.0001 mg/kg weight to 0.005 mg/kg weight, from about 0.0001 mg/kg weight to

0.004 mg/kg weight, from about 0.0001 mg/kg weight to 0.003 mg/kg weight, from about

0.0001 mg/kg weight to 0.002 mg/kg weight. In some embodiments, the therapeutically effective dose may be 0.0001 mg/kg weight, 0.0002 mg/kg weight, 0.0003 mg/kg weight,

0.0004 mg/kg weight, 0.0005 mg/kg weight, 0.0006 mg/kg weight, 0.0007 mg/kg weight, 0.0008 mg/kg weight, 0.0009 mg/kg weight, 0.001 mg/kg weight, 0.002 mg/kg weight, 0.003 mg/kg weight, 0.004 mg/kg weight, 0.005 mg/kg weight, 0.006 mg/kg weight, 0.007 mg/kg weight, 0.008 mg/kg weight, 0.009 mg/kg weight, 0.01 mg/kg weight,

0.02 mg/kg weight, 0.03 mg/kg weight, 0.04 mg/kg weight, 0.05 mg/kg weight, 0.06 mg/kg weight, 0.07 mg/kg weight, 0.08 mg/kg weight, 0.09 mg/kg weight, or 0.1 mg/kg weight. The effective dose for a particular individual can be varied (e.g., increased or decreased) over time, depending on the needs of the individual.

[00158] In some embodiments, a therapeutically effective dosage may be a dosage of 10 μg/kg/day, 50 μg/kg/day, 100 μg/kg/day, 250 μg/kg/day, 500 μg/kg/day, 1000 μg/kg/day or more. In various embodiments, the amount of the 15-PGDH inhibitor or pharmaceutical salt thereof is sufficient to provide a dosage to a patient of between 0.01 μg/kg and 10 μg/kg; 0.1 μg/kg and 5 μg/kg; 0.1 μg/kg and 1000 μg/kg; 0.1 μg/kg and 900 μg/kg; 0.1 μg/kg and 900 μg/kg; 0.1 μg/kg and 800 μg/kg; 0.1 μg/kg and 700 μg/kg; 0.1 μg/kg and 600 μg/kg; 0.1 μg/kg and 500 μg/kg; or 0.1 μg/kg and 400 μg/kg.

[00159] Various embodiments may include differing dosing regimen. In some embodiments, the 15-PGDH inhibitor and, optionally, the other labor inducing agents can be administered via continuous infusion. In some embodiments, the continuous infusion is intravenous. In other embodiments, the continuous infusion is subcutaneous. The dosing regimen for a single subject need not be at a fixed interval, but can be varied over time, depending on the needs of the subject.

[00160] For topical application, the composition can be administered in the form of aqueous, alcoholic, aqueous-alcoholic or oily solutions or suspensions, or of a dispersion of the lotion or serum type, of emulsions that have a liquid or semi-liquid consistency or are pasty, obtained by dispersion of a fatty phase in an aqueous phase (O/W) or vice versa (W/O) or multiple emulsions, of a free or compacted powder to be used as it is or to be incorporated into a physiologically acceptable medium, or else of microcapsules or microparticles, or of vesicular dispersions of ionic and/or nonionic type. It may thus be in the form of a salve, a tincture, milks, a cream, an ointment, a powder, a patch, an impregnated pad, a solution, an emulsion or a vesicular dispersion, a lotion, aqueous or anhydrous gels, a spray, a suspension, a shampoo, an aerosol or a foam. It may be anhydrous or aqueous. It may also comprise solid preparations constituting soaps or cleansing cakes. [00161] Other embodiments described herein relate to a method of inhibiting cervical ripening and preterm labor in a female in need thereof. The method can include

administering to the female at least one of an EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator.

[00162] The administration of the EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator can prevent and/or stop premature cervical ripening and preterm labor in a female subject in need thereof. Since at risk females are hard to prognoses, the EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator can be used, for example, in preterm (e.g., thirty seven week) gestation by prophylactic administration of the compounds into the cervix of pregnant females and/or in the serum or following a preterm labor and membrane rapture test.

[00163] The prostaglandin EP2 receptor antagonist can include any suitable EP2 receptor antagonist. By "suitable" it is meant that the antagonist is one which may be administered to the patient. The receptor antagonists are molecules which bind to their respective receptors, compete with the natural ligand (PGE2) and inhibit the initiation of the specific receptor-mediated signal transduction pathways. The EP2 receptor antagonist can be selective to the particular receptor (e.g., is not a EP4 receptor antagonist) and typically has a higher binding affinity to the receptor than the natural ligand. Although antagonists with a higher affinity for the receptor than the natural ligand are preferred, antagonists with a lower affinity may also be used, but it may be necessary to use these at higher concentrations. Preferably, the antagonists bind reversibly to their cognate receptor. Typically, antagonists are selective for a particular receptor and do not affect the other receptor; thus, typically, an EP2 receptor antagonist binds the EP2 receptor but does not substantially bind the EP4 receptor. In some embodiments, the EP2 receptor antagonist is selective for the particular EP2 receptor subtype. By this is meant that the antagonist has a binding affinity for the particular EP2 receptor subtype which is at least ten-fold higher than for at least one of the other EP receptor subtypes.

[00164] In some embodiments, the EP2 receptor antagonist includes AH6809 (Pelletier et at (2001) Br. J Pharmacol. 132, 999- 1008). Other examples of EP2 receptor antagonist are described in DE 10 2009 049 662 Al , which is incorporated by reference in its entirety. DE 10 2009 049 662 Al describes 2-5-disubstituted 2H-indazoles which, with high binding affinity, selectively antagonize the EP2 receptor. [00165] Still other examples of EP2 receptor antagonists are described in U.S. Patent Application Publication No. 2016/0089364, which is incorporated herein by reference in its entirety. It was shown in this application and PCT/EP2012/073556 that the EP2 receptor antagonists according to formula I or la have an antagonistic action at the EP2 receptor (see biological examples; Table 1).

[00166] The histone deacetylase 4 (HDAC4) inhibitor can include any compound or pharmaceutically acceptable salt thereof that is capable of interacting with HDAC4 and inhibiting its enzymatic activity. The term "inhibiting HDAC4 enzymatic activity" is intended to mean reducing the ability of a HDAC4 to remove an acetyl group from a protein, such as but not limited to a histone or tubulin. The concentration of inhibitor which reduces the activity of a HDAC4 to 50% of that of the uninhibited enzyme is determined as the IC₅₀ value. In some embodiments, such reduction of HDAC4 activity is at least 50%, such as at least about 75%, for example, at least about 90%. In some embodiments, HDAC4 activity is reduced by at least 95%, such as by at least 99%.

[00167] In some embodiments, such inhibition is specific, i.e., the HDAC4 inhibitor reduces the ability of a histone deacetylase to remove an acetyl group from a protein at a concentration that is lower than the concentration of the inhibitor that is required to produce another, unrelated biological effect. In some embodiments, the concentration of the inhibitor required for HDAC4 inhibitory activity is at least 2-fold lower, such as at least 5 -fold lower, for example, at least 10-fold lower, such as at least 20-fold lower than the concentration required to produce an unrelated biological effect.

[00168] Examples of HDAC4 inhibitors are described in U.S. Patent Application Publication Nos. 2017/0042892, 2009/0181943, 2009/0087412, and 2003/0152557 as well as U.S. Patent Nos. 9,693,994, 9,056,843, and 7,737,175, all of which are incorporated by reference in their entirety. HDAC inhibitors, for example, vorinostat (SAHA, ZOLINZA) and romidepsin (FK228, ISTODAX), are known Class IIA inhibitors, which includes HDAC4. In fact, vorinostat and romidepsin are FDA-approved for the treatment of cutaneous and peripheral T-cell lymphoma.

[00169] The 15-PGDH activator can include any compound that can promote or stimulate the activity of 15-PGDH. In certain embodiments, the 15-PDGH activator can include a compound having the formulas (XII), (XIII), (XIV), or (XV) described, for example, in U.S. Patent No. 9,790,233, which is incorporated herein by reference in its entirety.

[00170] The EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein can be provided in a pharmaceutical composition that includes

pharmaceutically acceptable carrier. In some embodiments, EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein can be provided alone or in combination with other components. The EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein can also be provided alone or in combination with other components in aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Compositions including the EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part of a prepared food or drug.

[00171] The dose administered to a patient should be sufficient to induce a beneficial response in the subject over time. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the case of diabetes. It is recommended that the daily dosage of the EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein can be determined for each individual patient by those skilled in the art. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound in a particular subject.

[00172] In other embodiments, the EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein can be administered to the female reproductive system intravaginally using, for example, a gel or cream or vaginal ring or tampon. The EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein may also advantageously be administered by intrauterine delivery, for example using methods well known in the art such as an intrauterine device.

[00173] Typically, the gel or cream is one which is formulated for administration to the vagina. It may be oil based or water based. Typically, the EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein is present in the cream or gel in a sufficient concentration so that an effective amount is administered in a single (or in repeated) application.

[00174] Typically, the vaginal ring comprises a polymer which formed into a "doughnut" shape which fits within the vagina. The EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein is present within the polymer, typically as a core, which may dissipate through the polymer and into the vagina and/or cervix in a controlled fashion. Vaginal rings are known in the art.

[00175] Typically, the tampon is impregnated with the EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein and that a sufficient amount of the EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein is present in the tampon.

[00176] The individual to be treated may be any pregnant female individual who would benefit from such treatment. Typically and preferably the individual to be treated is a human female. However, the methods of the invention may be used to treat female mammals, such as the females of the following species: cows, horses, pigs, sheep, cats and dogs. Thus, the methods have uses in both human and veterinary medicine.

[00177] The invention is further illustrated by the following example, which is not intended to limit the scope of the claims.

Example

[00178] In this example, we show that PGE2 utilizes cell-specific EP2 receptor-mediated increases in Ca²⁺ to dephosphorylate and translocate HDAC4 to the nucleus for repression of 15-hydroxy prostaglandin dehydrogenase (15-PGDH). The crucial role of 15-PGDH in cervical ripening was confirmed in vivo. Although PGE2 or 15-PGDH inhibitor alone did not alter gestational length, treatment with 1 -PGDH inhibitor+PGE2 or metabolism-resistant dimethyl-PGE2 resulted in preterm cervical ripening and delivery in mice. The ability of PGE2 to selectively auto-amplify its own synthesis in stromal cells by signaling transcriptional repression of 15-PGDH elucidates long-sought-after molecular mechanisms that govern prostaglandin action in the cervix. This example details unique mechanisms of action in the cervix and serves as a catalyst for (i) the use of 15-PGDH inhibitors to initiate, or amplify low-dose PGE2-mediated cervical ripening, or (ii) EP2 receptor antagonists, HDAC4 inhibitors, and 15-PGDH activators to prevent preterm cervical ripening and preterm, birth.

[00179] This example addresses the molecular mechanism of PGE2 action in cervical stromal cells and the underlying pathways leading to cervical ripening. We show that PGE2 initiates specific cellular events in cervical stromal cells. Through physiological, genetic and biochemical analyses on candidate hCSC genes, we show the downstream molecular mechanism by which PGE2 down-regulates 15-PGDH in the human cervix. Our results confirm that cervical ripening agents PGE2 and misoprostol regulate metabolism of PGE2 by transcriptionally inducing COX-2 and PTGES and repressing 15-PGDH via an EP2-HDAC4- dependent, feed forward regulatory mechanism selective to hCSCs. Our results further show that 15-PGDH is crucial for maintaining cervical competency during pregnancy in vivo, and thus, can be targeted by pharmacologic agents to induce cervical ripening and labor.

Materials and Methods

Cell culture and treatments

[00180] Human cervical stromal cells (hCSCs) were cultured as described previously. Briefly, all experiments in cervical stromal cells were conducted in cells obtained from nonpregnant females undergoing hysterectomy for benign gynecological conditions with one exception. The one exception was that cervical stromal cells from pregnant females undergoing cesarean-hysterectomy for placenta previa at term prior to the onset of labor were used to validate RNA-Seq data. Dissected stromal tissues (from internal os to mid-cervix) were separated from the epithelium, washed with DMEM, and minced into tiny pieces followed by incubation in DMEM supplemented with 10% fetal bovine serum for 18 to 20 days until cells grow onto the culture plates (Passage 0). Cells were then trypsinized, subcultured once (passage 1) and used for various treatments. Cells were incubated in serum- free media for 24 hours before treatments to completely remove FBS-derived prostanoids. PGE2, PGF2oc, PGD2, butaprost, misoprostol, sulprostone, PF-04418948, 8-bromo-cAMP, Wortmannin, A23187, TSA, SAHA, HDAC-42, KN-62, KN-93, C2-ceramide, okadaic acid and 16, 16-dimethy] PGE₂ were obtained from Cayman Chemical. Ly 294002 and Aristoforin were obtained from Santa Cruz Biotechnology. BAPTA-AM was from ThermoFisher Scientific. LMK-235 was from Selleck Chemicals. TG4-155 and TG8-4 were synthesized as described previously. All reagents unless mentioned were dissolved in dimethyl sulfoxide (DMSO) and further diluted in DMSO for treatment of cells. Final concentration of DMSO was <0.2% for all treatments. All experiments were conducted in triplicates in at least three cell culture preparations from cervices obtained from different subjects.

Human cervical tissues

[00181] Cervical stromal tissues were obtained from non-pregnant females as described above and from pregnant females undergoing cesarean hysterectomy according to protocols approved by Institutional Review Board at the University of Texas Southwestern Medical Center. Gestational age and other details were recorded and cervical ripening was ascertained using a modified Bishop's score in pregnant females. In cases that precluded a clinical exam (i.e., placenta previa), cervical length was substituted for effacement (>4 cm = 0, 2- 3 cm = 1, and 0-2 cm = 2), station was presumed to be 0, and dilation of hysterectomy specimen were used to categorize cervical ripening. Cervical stroma was separated from endocervical epithelium from the internal os to mid-cervix. Ectocervix was not included. Clinical characteristics of pregnant females from whom samples were obtained are shown in Table 1. Only the stromal region of the cervix was dissected and stored in RNA later for RNA extraction. Specimens were without contamination from cervical epithelium as determined by absent expression of epithelial 17 HSD2. Another small section of cervix from the mid- cervix was formalin fixed immediately and processed for immunostaining. Tissues from females with infections, trophoblast invasion into the cervix, cervical dysplasia, or with steroid treatment were excluded. Table 1

Table 2

Control(10% EtOH +

II 4 none 200 μΐ dl9 a.m.

5% Cremaphor +D5W)

dl9 (2 animals 12

75 μg (in 200

10 none SW033291 h after controls, 6 μΐ)

animals dl9 a.m. ) dl5 (All 4

III 4 dmPGE2 d 15 - 50 μ_§ complete delivery from 8-12 h after iniprtinn")

[n progress - no

2 PGE2 - 50 μg BID

delivery

50 μg BID +

D16 (1 30h) dl7(l

2 PGE2 SW033291 SW033291

48 h (75 μ_§ ΒΙϋ)

RNA sequencing and analysis

[00182] Total RNA samples were processed with the TruSeq Stranded Total RNA LT Sample Prep Kit from Illumina. Total RNA was isolated from two biological replicates of hCSCs treated with vehicle or PGE?. for 1 or 24 h was processed for whole-genome polyadenylated RNA sequencing (polyA-i- RNA-Seq). Total RNA samples were subjected to enrichment of polyA^"1" RNA using Dynabeads 01igo(dT)25 (Invitrogen). Thereafter, strand- specific RNA-Seq libraries were prepared as described previously and sequenced using an Illumina HiSeq 2500 using SBS v3 reagents for lOObp paired-end reads. Reads were trimmed to remove adaptor sequences and low quality bases using fastq-mcf, followed by mapping to human genome (hgl9) using Tophat (v2.0.10, doi:10.1038/nprot.2012.016) with igenome annotations (https://ccb.jhu.edu/software/tophat/igenomes.shtml). Duplicate reads were marked but not removed. FeatureCounts (doi:10.10 3/bioinformatics/btt656) was used for read counting, and edgeR (doi: 10.1093/nar/gks042) for expression abundance estimation and differential expression test. Pathways enriched in differentially expressed genes were identified by DAVID (doi: 10.1038/nprot.2008.211, doi: 10.1093/nar/gkn923).

RNA extraction and RTqPCR

[00183] RNA extraction from cells was performed using RNA extraction kit from Invitrogen (AM1914). RNA extraction from human cervical stromal tissues (Table 1), was performed using guanidine hydrochloride extraction method as described elsewhere. cDNA synthesis was performed using iScript™ Reverse Transcription Supermix from BIO-RAD according to supplier's protocol (170-8841). Quantitative PGR (qPCR) was done using iTaq SYBR Green PGR Master mix (4309155) or TaqMan Gene expression master mix (4369016) from Applied Biosystems in an ABI 7900HT Fast Real-Time PGR system.

Protein extraction and immunoblotting

[00184] Post treatment cells were washed with cold Phosphate Buffered Saline (PBS) twice and scraped into RIPA buffer (50 mM Tris.HCl; 150 mM NaCl; 0.1% SDS; 0.5% Sodium deoxycholate; 1% NP40; protease inhibitor cocktail), vortexed for 30 s and incubated on ice for 30 min. Lysates were then centrifuged at 10000 rpm for 5 min at 4° C. Clear supernatants were collected and protein amounts were quantified using BCA protein assay kit from ThermoScientific (23223, 23224). Cytoplasmic and nuclear protein fractions were prepared. 40 μg of whole cell/cytoplasmic or 20 μg of nuclear protein extracts were resolved using Mini-PROTEAN TGX gels from BIO-RAD (456-9033) and transferred to PVDF membrane (BIO-RAD, 162-0177). Membranes were blocked with 5% skim milk solution in TBS for 1 h at room temperature followed by incubation with specific antibodies against 15- PGDH (Cayman Chemical, 1606 15), HDAC4 (Cell Signaling, 7628), phospho- HDAC4(Ser246)/HDAC5 (Ser259)/HDAC7(Serl55) (Cell Signaling, 3443), Acetylated histone H3 (Millipore, 06-599) or β-actin (Cell Signaling, 4967) overnight at 4°C. Blots were then washed with PBS containing 0.1% Tween 20 and further incubated with HRP- conjugated secondary antibodies (Anti rabbit- 170-6515 or Anti mouse- 170-6516, BIO- RAD) followed by washing and developed using SuperSignal West Pico chemiluminescent kit (Thermo Scientific, 34080). Immunospecific signals were detected using FujiFilm LAS- 3000 imager. For re-probing with different antibodies, membranes were stripped using OneMinute Advance Western Blot stripping kit (GM Biosciences, GM6031). Densitometry was performed using Multi Guage V3.0 software.

Chromatin Immunoprecipitation

[00185] ChIP assays were performed as described elsewhere. Human CSCs were treated with either DMSO or PGE2 (100 nM) for 24 h followed by fixing with formaldehyde. Immunoprecipitations were performed with either IgG or Acetylated Histone H3 antibodies (Millipore, 06-599).

siRNA-mediated knock down

[00186] hCSCs were maintained in serum free Opti MEM (11058-021, Life

Technologies) over night and transfected with 30 nM of either control negative siRNA (4390846, Ambion), HDAC2 siRNA (4390824, Ambion), HDAC4 siRNA (sc-35540, Santa Cruz Biotechnologies and 4392420, Ambion) or HDAC5 siRNA (4390824, Ambion) using Lipofectamine RNAimax Transfection reagent (13778-075) according to supplier's protocol. Transfection medium was replaced with fresh DMEM with 10% FBS and incubated further for 24 h followed by change to serum free DMEM for 24 h. For quantifying basal levels of 15- PGDH, cells were further incubated for 12 h before processing for RT-qPCR. For different pharmacological treatments cells were treated with either DMSO, PGE2, or LMK- 235 alone or PGE2 + LMK-235 for 24 h in serum-free media.

Adenovirus-mediated overexpression

[00187] hCSCs grown in 6 well dishes were infected with 5 μΐ (1 x 10⁶ pfu/mL) of either β-galactosidase expressing control adenovirus (000197 A, Applied Biological Materials Inc.) or HDAC4 expressing adenovirus (000426A, Applied Biological Materials Inc.) for 36 h in complete growth medium with 10% FBS followed by incubation in serum free growth medium for 24 h before processing for RT-qPCR (no reverse transcriptase controls were included to confirm that RNA preparations were free from HDAC4 DNA from residual adenovirus in RNA preparations) and immunoblotting.

Immunocytochemistry

[00188] hCSCs were grown on tissue culture 8 chambered glass slides (4808, Lab-Tek) and treated with either DMSO or PGE2 (100 nM) for 24 h. ICC was performed as described previously with few modifications. Post treatment cells were fixed with freshly prepared 4% formaldehyde for 15 min at room temperature. Cells were then rinsed with PBS three times (five minutes each) to remove formaldehyde. Cells were then blocked using 10% normal goat serum (50062Z, ThermoFisher Scientific) in PBS with 0.3% Triton X-100 for 1 h at room temperature. After blocking cells were incubated in anti HDAC4 antibody (sc-11418, Santa Cruz Biotechnologies) diluted in PBS with 0.3% Triton X-100 over night (dilution- 1 :500) at 4°C. After washing three times with fresh PBS for 5 min each, cells were then incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (A11008, Molecular probes by Invitrogen) diluted in PBS with 0.3% Triton X-100 (1 :500) for 1 h at room temperature in dark. Cells were then washed three times with PBS (5 min each) and mounted with DAPI (P36395, Molecular Probes by Invitrogen), coverslipped and imaged using a Leica TCS SP5 confocal microscope. Treatments were performed on the same slide and images were captured with identical settings. Negative controls consisted of cells undergoing identical procedures with nonspecific IgG substituted for the primary antibody. Fluorescence was absent in these negative controls.

Immunohistochemistry

[00189] Formalin-fixed, paraffin-embedded tissues were sectioned at 5 μιη and mounted on slides. Tissue sections from positive and negative sections were mounted on the same slide, Sections were immunostained with antibodies against HDAC4 (1: 150, sc-11418, Santa Cruz Biotechnologies) in EDTA buffer using heated steam as the antigen retrieval method. Immunostaining was negative in controls in which tissues underwent all treatments except without primary antibody.

Animals and treatments

[00190] To precisely determine the duration of gestation, C57BL/6 mice (with a gestation duration of 19 days) were time mated for 4 h (9:00 AM to 1:00 PM) after which females were separated from the males (day 0) and randomly divided into 4 treatment groups-(l) Vehicle (88.33% D5W [5% Dextrose in water]; 6.66% Ethyl alcohol; 3.33% Kolliphor EL (#C5135, Sigma); 1.66% DMSO), (2). PGE2 Q.68 mg/kg), (3) SW033291 (2.5 mg/kg) and (4) PGE2 + SW033291. Reagents were freshly prepared each time just before treatment and 300 μΐ was injected intra peritoneally (i.p.) using a 28G needle, every 12 h (9:30 AM and 9:30 PM) starting on day 15 and observed for time of delivery using camera surveillance. Treatment was terminated on appearance of first pup and the time of delivery was recorded for each treated animal. Final concentrations of vehicle components were similar in all treatment groups.

[00191] Separate treatment groups were used for histology and biomechanics. Cervices were collected from different treatment groups on dl6 36 h post treatment. Histology

[00192] Tissues were harvested from 3 animals per treatment group on gestation dl6. The female reproductive tract containing the vagina, cervix, bifurcation of the uterus and two lower pups were fixed in neutral buffered formalin x 24 h. Thereafter, the buffer was changed to 50% ethanol and embedded in paraffin. Transverse serial sections from the external cervical os were obtained every 500 μιη and Masson's trichrome staining was performed.

Biomechanical testing

[00193] Each cervix was suspended between two stainless steel wire mounts inserted through the cervical os and attached to a steel rod apparatus with a calibrated mechanical drive and to a force transducer. Tissues were maintained in a physiologic salt solution in water baths at 37°C with 95% O2 and 5% CO2. After acclimation for 15 minutes, each ring was equilibrated to slack length (ring diameter at resting tone) as measured by the calibrated mechanical drive. Rings were distended in 1 mm increments with 2 minute intervals between each increment to allow stabilization of forces before subsequent distention. This process was continued until failure (breakage) or until plateau of force generation. Force in Newtons was plotted against deformation, producing a sigmoid- shaped curve. Distensibility was considered the inverse of tissue stiffness which was calculated from the slope of the linear portion of the curve.

Statistical analysis

[00194] RNA-Seq data were analyzed as described above. Otherwise, for multiple groups, ANOVA followed by Dunnett' s posthoc testing (Vehicle as control) was used except for gestational timing in which an ANOVA was used followed by Tukey's posthoc testing. Student's t test was used to compare two independent groups.

Results

PGE2 regulates the transcriptome of human cervical stromal cells in vitro through EP2- mediated increases in intracellular Ca²⁺

[00195] RNA-Seq data analysis and validation experiments identified PGE2 mediated changes in the transcriptome of cervical stromal cells at both early (1 h) and late (24 h) time points. Interestingly, PGE2 controlled its own metabolism by downregulating the major PGE2 catabolic enzyme 15-PGDH and upregulating expression of two genes involved in PGE2 synthesis (COX-2 and PTGES) (Figs. 1A, B and Fig. 8; NCBI GEO accession number: GSE99392). Importantly, validation experiments confirmed that results obtained in cervical stromal cells from the nonpregnant cervix were relevant to those in cells from pregnant females at term (Fig. 8). PGE2 and misoprostol, but not PGF201, sulprostone (EP3 agonist), or PGD2, regulated 15-PGDH, PTGES, and COX-2 (Fig. 1A). These results are consistent with the prostanoid receptor expression profile in these cells in which EP2 receptors are highly expressed relative to other prostanoid receptors (Fig. 1C) and confirm that cervical ripening agents PGE2 and misoprostol regulate metabolism of PGE2 by activating COX-2 and PTGES and repressing 15-PGDH in a feed forward regulatory mechanism. Three different EP2- selective antagonists (PF-04418948, TG4-155 and TG8-4) blocked PGE2- mediated 15- PGDH gene repression (Fig. ID). Unlike classical PGE2 signaling in other cells, PGE2-EP2 interactions resulted in Ca²⁺-dependent signaling (e.g., DUSP1, c-fos), not cAMP, PKA and PI3-kinase (Fig.9).

[00196] Pathway analysis of the RNA-Seq data indicated that the most significantly affected pathway by PGE2 was Ca²⁺ signaling (Fig. 2A). Expression levels of 71 genes either regulated by Ca²⁺ or involved in Ca²⁺ signaling pathways were significantly changed (FDR<0.05) by PGE2 at either 1 or 24 h or both time points (Fig. 2B). Similar to PGE2, treatment with Ca²⁺ ionophore (A23187) decreased 15-PGDH and increased COX-2 mRNA (Fig. 2C, Fig. 10). Ca²⁺-dependent PGE2-mediated 15-PGDH repression was confirmed using a cell permeable intracellular Ca²⁺ chelator BAPTA-AM (Fig. 2D). Pretreatment with BAPTA-AM had little effect on PGE2-mediated 15-PGDH repression in growth medium containing Ca²⁺. In Ca²⁺-free medium, however, BAPTA-AM blocked PGE2-mediated 15- PGDH repression (Fig. 2E). Addition of Ca²⁺ to Ca²⁺-free medium reversed this effect (Fig. 2E). Taken together, these results confirm that PGE2-mediated downregulation of 15-PGDH is transduced through EP2 receptors and is Ca²⁺-dependent inhCSCs.

PGE2 downregulates 15-PGDH gene expression in vitro by increasing HDAC4

[00197] Since intracellular Ca²⁺ results in activation of histone deacetylases (HDACs), and HDAC inhibitors (HDACi) have been shown to induce 15-PGDH gene expression in several mammalian cell types, we hypothesized that PGE2-mediated Ca²⁺ signaling activates HDACs which, in turn, regulate 15-PGDH. ChIP results indicated that acetylated histone H3 levels associated with 15-PGDH promoter decreased ~5-fold in response to PGE2 treatment (Fig. 3A). Immunoblotting indicated that three different non-selective HDACi increased 15- PGDH protein (Fig. 3B). As a positive control for HDACi action, acetylated histone H3 levels were probed, which also increased several fold in response to various HDACi (Fig. 3B). Further, treatment with three different HDACi increased mRNA levels of 15-PGDH significantly in a dose- and time-dependent manner (Fig. 3C, D). Interestingly, HDAC inhibitors induced 15- PGDH mRNA expression even in the presence of PGE2 or A23187 (Fig. 11), irrespective of order of treatment (delayed or primed). Thus, HDACs are mediators of PGE2- induced 15-PGDH gene repression.

[00198] To identify the specific HDAC involved in PGE2-mediated regulation of 15- PGDH in hCSCs, we first studied PGE2-mediated changes in expression of various HDAC genes using our RNA- Seq dataset. Among Class I and II HDACs, HDAC5 and 9 mRNA decreased in response to PGE2 (Fig. 3E). In contrast, HDAC4 mRNA increased ~4-fold (Fig. 3E). Experiments confirmed that PGE2 increased HDAC4 mRNA in a dose- and time- dependent manner, concomitantly, decreasing 15-PGDH in hCSCs (Fig. 4 A, Fig. 12A). Misoprostol and an EP2 receptor-selective agonist (butaprost) also increased HDAC4 gene expression (Fig. 12B), and EP2 receptor- selective antagonists blocked PGE2-mediated activation of HDAC4 expression (Fig. 4B). PGE2 also increased protein levels of HDAC4 which was blocked by EP2 receptor antagonist PF-04418948 (Fig. 4C, Fig. 13). In contrast with colorectal carcinomas in which marked increases in HDAC2 activity diminished levels of 15-PGDH, siRNA-mediated knock down of HDAC2 did not affect 15-PGDH gene expression in hCSCs (Fig. 14). Among class III HDACs (sirtuins), SIRT2 levels increased 2- fold within 1 h of treatment (Fig. 14B), but, unlike HDAC inhibitors, the SIRT inhibitor, aristoforin, did not alter 15-PGDH gene expression (Fig. 14C).

[00199] To determine if Ca²⁺ is involved in PGE2-mediated HDAC4 gene expression, experiments were conducted as described in Fig. 2D. Whereas absence of extracellular Ca^2"1" alone was ineffective, chelation of intracellular Ca^{2 ~} by BAPTA-AM resulted in decreased HDAC4 mRNA (Fig. 4D). Further, pretreatment of hCSCs with BAPTA-AM completely blocked PGE2-mediated increases in HDAC4 gene expression in all conditions (Fig. 4D). Thus, in hCSCs, PGE2 increases HDAC4 gene expression via EP2 receptors and HDAC4 and 15-PGDH genes are inversely regulated by PGE2 in a Ca²⁺-dependent manner. Interestingly, PGE2 treatment did not change HDAC4 gene expression in cell types with different EP receptor profiles (MCF7 and MEL5, Fig. 15).

[00200] Next, the crucial role of HDAC4 in mediating 15-PGDH gene expression in hCSCs was established. Specifically, siRNA-mediated knockdown of HDAC4 increased, whereas adenovirus-mediated over expression decreased, basal levels of 15-PGDH mRNA suggesting that 15-PGDH is an HDAC4 target gene (Fig. 4E, Fig. 16). Notably, knockdown of HDAC4 abrogated PGEi-mediated downregulation of 15-PGDH gene expression (Fig. 4E), suggesting that HDAC4 is necessary to mediate downregulation of this gene. The opposing actions of HDAC4 siRNA and PGE2 resulted in no change in HDAC4 expression levels (Fig. 4E). These findings are compatible, therefore, with the lack of dramatic HDAC4 knockdown effects on PGE2-mediated downregulation of 15-PGDH (Fig. 4E). LMK-235 (an HDAC4/5 selective enzyme inhibitor) treatment resulted in 5- to 6-fold increases in basal levels of 15- PGDH mRNA and protein (Fig. 4F). LMK-235 also blocked PGE₂-mediated downregulation of 15-PGDH gene expression (Fig. 4F). However, siRNA-mediated knockdown of HDAC5 did not alter 15-PGDH gene expression in these cells indicating that the effects of LMK-235 are mediated through inhibition of HDAC4, not HDAC5 (Fig. 17). Interestingly, siRNA-mediated knockdown of HDAC4 together with LMK-235 treatment, led to cumulative loss of HDAC4 mRNA and synergistic activation of 15-PGDH gene expression (Fig. 4G, H). Collectively, results indicate that PGE2-mediated increases in HDAC4 expression and its deacetylase activity are crucial for PGE2-mediated

downregulation of 15-PGDH inhCSCs.

PGE2 mediates HDAC4 nuclear import in vitro

[00201] Immunofluorescence studies show weak HDAC4 immunostaining distributed in both cytoplasmic and nuclear compartments in baseline hCSCs treated with vehicle (Fig. 5A, B). Treatment with PGE2 resulted in not only an overall increase in HDAC4

immunofluorescence but also increased nuclear localization (Fig. 5A, B). Immunoblotting clearly shows that nuclear HDAC4 levels increased within 1 min of treatment with PGE2 and steadily increased as a function of time (Fig. 5C, D). Cytoplasmic HDAC4 levels did not change until 6 h after treatment after which levels increased and remained elevated (Fig. 5C, D). These changes in nuclear localization of HDAC4 were accompanied by changes in the phosphorylation status of HDAC4 (Fig. 5E, F). HDAC4 (Ser246) was dephosphorylated in response to PGE2 treatment within 1 hour. Total HDAC4 protein levels did not change during this time period, but increased at later times (Fig. 5E, F). Although immunoblotting of phospho- HDAC4 alone indicates that HDAC4 phosphorylation seems to recover at later time points, normalization to increased protein levels of HDAC4 indicates that phosphorylation of HDAC4 actually decreased with time (Fig. 5F). In sum, PGE2 induces HDAC4

dephosphorylation and nuclear relocalization.

[00202] Phosphorylation of class II HDACs by serine/threonine kinases regulates their cellular localization, stability and ability to regulate targeted gene expression.

Phosphorylated HDAC4 binds 14-3-3 protein, a complex retained in the cytoplasm.

Dephosphorylation of cytoplasmic HDAC4, on the other hand, leads to release from the complex and nuclear import of HDAC4. Nuclear Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) binds and phosphorylates nuclear HDAC4 for export to the cytoplasm. CaMKII inhibitors KN62 and KN93 downregulated 15-PGDH 40 to 60% mimicking PGE2 (Fig. 18A). As expected, inhibition of CaMKII did not affect HDAC4 gene expression (Fig. 18A).

Likewise, treatment with a cytosolic serine/threonine phosphatase activator C2 ceramide repressed 15-PGDH but with no effect on HDAC4 (Fig. 18B). Further treatment with okadaic acid, which was previously shown to block HDAC4 de-phosphorylation by inhibiting protein phosphatase 2A, prevented PGE2-mediated down regulation of 15- PGDH (Fig. 18C). Okadaic acid, however, did not alter KN-93 -mediated down regulation of 15-PGDH gene expression (Fig. 18D). These results, therefore, are compatible with the overall pathway in which PGE2 signaling not only regulates expression of HDAC4, but also HDAC4 cellular localization, thereby bringing about targeted repression of 15-PGDH (Fig. 5D).

HDAC4 levels and localization during late gestation in human cervical stromal tissues in vivo

[00203] To determine if HDAC4 expression is regulated in human cervical stromal tissues during the course of cervical ripening, relative levels of HDAC4 mRNA and protein localization were determined in human cervical stromal tissues from nonpregnant and pregnant females (Table 1). HDAC4 mRNA levels were increased significantly in stroma from pregnant females in late gestation (35-42 weeks) compared with those from

nonpregnant or pregnant females in early gestation (Fig. 6A). 15-PGDH mRNA levels were decreased significantly in stroma of females in labor compared to nonpregnant or stromal tissues prior to cervical ripening (Fig. 6A). Immunostaining indicated that HDAC4 was present in the cytoplasm of some, but not all, cervical stromal fibroblasts from nonpregnant females (Fig. 6Ba). Prior to cervical ripening in late pregnancy, HDAC4 immunoreactivity was abundant in most cervical stromal cells and localized to the cytoplasm (Fig. 6Bb). In the ripe cervix at term, HDAC4 immunoreactivity was present in virtually all cervical stromal cells and distributed in both cytoplasmic and nuclear compartments (Fig. 6Bc). In contrast with the unripe cervix, HDAC4 protein staining was intense and predominantly localized in the nucleus during cervical dilation in labor (Fig. 6Bd).

Downregulation of 15-PGDH is crucial for PGE2-induced preterm labor in mice

[00204] A precisely-timed pregnant mouse model was used to determine the importance of PGE2-mediated regulation of 15-PGDH for cervical ripening and labor. Intraperitoneal injection of pregnant mice with PGE2 did not alter duration of gestation with all mice delivering at the expected time on dl9 dpc (days post coitum) similar to vehicle-treated animals (Fig. 7A). We reasoned that high levels of 15-PGDH enzyme and rapid inactivation of PGE2 leads to failure of PGE2 to induce preterm labor. Interestingly, treatment with metabolism resistant 16, 16, -dimethyl PGE2 (dmPGE2) that also induced HDAC4 and decreased 15-PGDH mRNA levels in hCSCs (Fig. 19A) caused preterm birth on dl5 within 8 to 12 h. All pups were dead at the time of delivery with evidence of mechanical trauma. Sacrifice of pregnant animals 6 h after dmPGE2 treatment on dl5 revealed a tonically contracted uterus, intrauterine death, and pallid pups consistent with lack of oxygenation. Since dmPGE2 is a competitive inhibitor of 15-PGDH, we sought to determine if inhibition of 15-PGDH led to cervical ripening and preterm birth. However, treatment with a 15-PGDH inhibitor, SW033291, alone did not affect gestational length, and offspring were born healthy and full-term (Fig. 7A). Interestingly, treatment with a combination of PGE2 and SW033291 induced preterm labor within 12-48 h in 100% of animals (Fig. 7A). Combination treatment did not cause fetal death in utero and premature pups born on dl5 or dl6 delivered atraumatically with intact placentas and fetal membranes (Fig. 7B). Premature pups born on late dl6 or dl7 were alive but died shortly thereafter due to extreme prematurity (Fig. 7C). Perhaps, the most interesting aspect of this treatment (PGE2+SW033291) is that mice delivered only two pups closest to the cervix whereas the remaining pups were retained delivering at the expected time on day 19 alive and healthy suggesting that the predominant effect of PGE2+15-PGDH inhibitor was on the cervix and lower uterine horns (Fig. 7D). Thus, we assessed the impact of treatment on histomorphology of the cervix. In vehicle- treated animals, the endocervix was lined by a layer of 4-5 pseudostratified columnar epithelial cells that progressively differentiate from the basal epithelium to fully developed mucus-secreting cells toward the lumen (Fig. 7E). The collagenous stromal matrix was dense and well-organized. Epithelial and stromal morphology was similar in vehicle-, PGE2- or SW033291 alone-treated animals (Fig. 7E). In contrast, treatment with PGE2+SW033291 resulted in increased size and numbers of mucus-laden epithelial cells and dramatic remodeling of the collagenous matrix surrounding the stromal fibroblasts and smooth muscle cells (Fig. 7E). Biomechanical properties of cervices (on dl6) from treated animals were determined. Although similar in PGE2- and SW033291 -treated animals, baseline cervical dilation was increased in combination-treated animals compared with controls (Fig. 7F). This increase in baseline dilation was accompanied by significant increases in distensibility without compromise of maximal force (Fig. 7F). The results, together with the preterm delivery phenotype, indicate that 15-PGDH plays a crucial role in maintenance of cervical competency during pregnancy in the presence of increasing levels of PGE2 and that downregulation of cervical 15-PGDH is a prerequisite for PGE2 action at term for delivery.

[00205] The result described herein utilized cervical stromal cells in culture to elucidate the molecular pathways of PGE2 action in the human cervix. Human cervical tissues confirmed differential regulation of HDAC4 localization during human cervical ripening. Finally, studies in the mouse indicate that downregulation of 15-PGDH is a crucial component of successful PGE2-induced cervical ripening and labor. The experiments suggest that continuous doses of PGE2/1 may fail to induce cervical ripening and labor if endogenous levels of cervical 15-PGDH are high. 15- PGDH, therefore, represents an enzyme important for cervical competency during pregnancy.

[00206] It is well-known that COX-2-derived PGE2 plays a major role in cervical ripening during term and preterm birth. Inhibition of COX-2 during pregnancy, however, is relatively contraindicated because PGE2 interacting with EP4 receptors is crucial for patency of the fetal ductus arteriosus. The studies reported herein indicate that EP2, not EP4, receptors mediate the effects of PGE2 in cervical stromal cells. Activation of EP2 receptors led to decreased expression of 15-PGDH through detailed intracellular events unique from EP2 signaling in other cells. Specifically, activation of stromal cell EP2 receptors led to increases in intracellular Ca²⁺, Ca²⁺-dependent dephosphorylation of cytoplasmic HDAC4 which is then localized to the nucleus to modify the transcriptome of human cervical stromal cells. Further, we show that PGE2-mediated downregulation of its own catabolic enzyme, 15- PGDH, was required for PGE2-mediated cervical ripening.

[00207] RNA-Seq data confirmed that PGE2 activates Ca²⁺ signaling pathways in hCSCs. Early cellular events after EP2 activation included Ca²⁺-dependent dephosphorylation of HDAC4 and its nuclear import. Phosphorylation of class II HDACs by serine/threonine kinases regulates their cellular localization, stability and ability to regulate targeted gene expression. Phosphorylated HDAC4 binds 14-3-3 protein, a complex retained in the cytoplasm. Dephosphorylation of cytoplasmic HDAC4 by protein phosphatase 2A, on the other hand, leads to release from the complex and nuclear import of HDAC4. Global inhibition of serine/threonine phosphatases with okadaic acid confirmed that

dephosphorylation of HDAC4 was an important signaling event. After the initial

dephosphorylation of HDAC4 and changes in gene expression (including cfos), HDAC4 increased as a function of time and DUSPl mRNA remained increased for up to 24 h. These findings suggest that EP2 signaling results in an early increase in intracellular Ca^2÷ and dephosphorylation of HDAC4, but that later events continue to promote the signals.

[00208] In our mouse model, inhibition of 15-PGDH alone did not alter gestational length. Treatment of pregnant mice with a combination of PGE2 and 15-PGDH inhibitor SW033291 induced preterm cervical ripening and labor in all animals tested. More provocative is that delivery was always incomplete. Pups from the ovarian end of each horn were retained and delivered at term on dl9 with normal growth and development.

Maintenance of pups at the ovarian ends suggests that this ripening was not accompanied by strong uterine contractions or progesterone withdrawal in which the uterus empties completely.

[00209] Interestingly, RNA-Seq data did not reveal PGE2-mediated increased expression of proteases (or downregulation of protease inhibitors), hyaluronan synthases, or

progesterone receptors. Further, although PGE2 suppressed collagen type VI gene expression, the predominant fibrillar collagens were not affected. Decreases in COLA6A as well as differential regulation of integrin receptors may alter the matrix environment and matricellular signaling in the cervix. An important consideration, however, is PGE2-mediated increases in genes involved in cytokine-cytokine receptor interactions (e.g., CCL8, CXCL-1 and -2, IL1R1, and several members of the TNF/TNF receptor superfamily), suggesting that PGE2 may alter matrix remodeling of the cervix indirectly through stromal cell recruitment and activation of immune cell types within the cervix. Nonetheless, the key finding is the crucial role of 15-PGDH in regulating all aspects of PGE2 action in the cervix.

[00210] This example identifies (i) EP2 receptor antagonists, (ii) HDAC inhibitors, and (iii) activators of 15-PGDH as potential interventions to prevent preterm shortening of the cervix and preterm birth. With the discovery of several biologically active EP2 selective antagonists in the last 4 years, these can be considered as potential safer alternatives for COX-2 inhibitors in pregnant females with preterm shortening of the cervix or risk for preterm birth. Although differences in EP receptor profiles in the cervix of rats and mice compared with humans do not allow us to test EP2 selective antagonists in animals, the potential for these therapeutic targets to impact initiation, propagation, and rate of cervical ripening in pregnant females is supported by downregulation of 15-PGDH in cervical stromal tissues of pregnant females during cervical ripening, EP2 receptor predominance in cervical tissues obtained from pregnant females, increased HDAC4 mRNA in the pregnancy cervix, and progressive increases in nuclear localization of HDAC4 in stromal cells of the cervix from before ripening, during ripening before labor, to the dilated ripe cervix in labor.

Further, we confirmed previous studies in which the HDACi TSA delayed parturition in mice and found that extension of treatment time delayed parturition for up to 3 d without immediate adverse effects on the fetus. Taken together with our in vitro data, we suggest that HDAC inhibitors target HDAC4 to increase basal levels of 15-PGDH that neutralizes active PGE2 and PGF2D. With many HDACi considered as therapeutics for various diseases and well-established pharmacokinetics and toxicity profiles, identification of HDAC4 as a key regulatory intermediate for PGE2 action on the cervix may lead to new strategies to inhibit, or prevent, preterm cervical ripening and pretermbirth.

[00211] We propose that 15-PGDH inhibitors with a short half-life and safe

pharmacological profile may not only increase the success of PGE2-induced cervical ripening but also facilitate use of lower doses of PGE2/1 and thereby decrease the induction-to-delivery interval, an important consideration if induction of labor is conducted in an adverse perinatal environment. In contrast, development of EP2 antagonists and HDAC4 inhibitors may successfully interrupt the vicious cycle of preterm cervical ripening and preterm birth.

[00212] From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety.

Claims

Having described the invention, we claim:

1. A method inducing cervical ripening and/or labor in a female subject in need thereof, the method comprising:

administering to the subject a therapeutically effective amount of a 15-PGDH inhibitor.

2. The method of claim 1, wherein the 15-PGDH inhibitor is administered alone or in combination with a labor inducing agent.

3. The method of claim 2, wherein the labor inducing agent comprises a prostaglandin.

4. The method of claim 2, wherein the labor inducing agent comprises at least one of at least one of dioprostone (PGE2) or misoprostol (PGE1).

5. The method of claim 1, wherein the 15-PGDH inhibitor has the following formula (V):

wherein n is 0-2

X⁶ is independently is N or CR^C

R⁶, R⁷, and R^c are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C₃-C2₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6 ring atoms, C₆-C24 alkaryl, C₆-C24 aralkyl, halo, -Si(Ci-C₃ alkyl)3, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C₂- C24 alkynyloxy, C5-C2₀ aryloxy, acyl, acyloxy, C2-C24 alkoxycarbonyl, C₆-C2₀

aryloxycarbonyl, C2-C24 alkylcarbonato, C₆-C2₀ arylcarbonato, carboxy, carboxylato, carbamoyl, C1-C24 alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, foimyl, thioformyl, amino, C1-C24 alkyl amino, C5-C2₀ aryl amino, C2-C24 alkylamido, C6-C2₀ arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato, C1-C24 alkylsulfanyl, arylsulfanyl, C1-C24 alkylsulfinyl, C5-C2₀ arylsulfinyl, C1-C24 alkylsulfonyl, C5-C2₀ arylsulfonyl, sulfonamide, phosphono, phosphonato, phosphinato, phospho, phosphino, poly alky lethers, phosphates, phosphate esters, groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, combinations thereof, and wherein R⁶ and R⁷ may be linked to form a cyclic or polycyclic ring, wherein the ring is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, and a substituted or unsubstituted heterocyclyl;

U¹ is N, C-R², or C-NR³R⁴, wherein R² is selected from the group consisting of a H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, 0-CH₂- CH₂X, CH2-CH2-CH2X, O-CH2-CH2X, X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, 0(CO)R', COOR' (wherein R' is H or a lower alkyl group), and wherein R¹ and R² may be linked to form a cyclic or polycyclic ring, wherein R³ and R⁴ are the same or different and are each selected from the group consisting of H, a lower alkyl group, O, (CH₂)niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, CH₂-CH₂-CH₂X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, COOR' (wherein R' is H or a lower alkyl group), and R³ or R⁴ may be absent;

and pharmaceutically acceptable salts thereof.

6. The method of claim 1, wherein the 15-PGDH inhibitor has the following formula (VI):

wherein n = 0-2;

X⁶ is N or CR^C; selected from the group consisting of branched or linear alkyl including

X

(CH₂)n_!CH₃ (n₁₌0-7), "² wherein n₂=0-6 and X is any of the following: CF_yH_z (y + z =

3), CCl_yH_z (y + z = 3), OH, OAc, OMe, R , OR , CN, N(R ,

7'¾3, ,

m=l-5), and ⁿ⁴ (n₄=0-5).

R⁵ is selected from the group consisting of H, CI, F, Ν¾, and N(R⁷⁶)_2;

6 and ⁷ can each independently be one of the followin :

each R⁸ _, R⁹ _, R¹⁰ R¹¹ _, R¹² R¹³ _, R¹⁴ R¹⁵ _, R¹⁶ R¹⁷ _, R¹⁸ _, R¹⁹ _, R²⁰ R²¹ _, R²² R²³ _, R²⁴ R

_R26 _R27 _R28 _R29 _R30 _R31 _R32 _R33 _R34 _R35 _R36 _R37 _R38 _R39 _R40 _R41 _R42 _R43 _R44 _R45 _R46 _R47 _R48 _R49 _R50 _R51 _R52 _R53 _R54 _R55 _R56 _R57 _R58 _R59 _R60 _R61 _R62 _R63 _R64 _R65 _R66 _R67 _R68 _R69

_R70 _R71 _R72 _R73 _R74 _R76 _{and R}c

are the same or different and are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C₃-C2₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6 ring atoms, C₆-C24 alkaryl, C₆-C24 aralkyl, halo, -Si(Ci-C₃ alkyl)3, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl, acyloxy, C2-C24 alkoxycarbonyl, C₆-C2₀ aryloxycarbonyl, C2-C24 alkylcarbonato, C₆-C2₀ arylcarbonato, carboxy, carboxylato, carbamoyl, C1-C24 alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, C1-C24 alkyl amino, C5-C2₀ aryl amino, C2-C24 alkylamido, C₆-C2₀ arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato, C1-C24 alkylsulfanyl, arylsulfanyl, C1-C24 alkylsulfinyl, C5-C2₀ arylsulfinyl, C1-C24 alkylsulfonyl, C5-C2₀ arylsulfonyl, sulfonamide, phosphono, phosphonato, phosphinato, phospho, phosphino, poly alky lethers, phosphates, phosphate esters, groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, and combinations thereof, and

pharmaceutically acceptable salts thereof.

or pharmaceutically acceptable salts thereof.

8. A method initiating cervical ripening and/or inducing labor in a female subject in need thereof, the method comprising:

administering to the subject therapeutically effective amounts of a 15-PGDH inhibitor and a labor inducing agent.

9. The method of claim 8, wherein the labor inducing agent comprises a prostaglandin.

10. The method of claim 8, wherein the labor inducing agent comprises at least one of dioprostone (PGE2) or misoprostol (PGE1).

11. The method of claim 8, wherein the 15-PGDH inhibitor has the following formula (V):

wherein n is 0-2

X⁶ is independently is N or CR^C

R⁶, R⁷, and R^c are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C₃-C2₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6 ring atoms, C₆-C24 alkaryl, C₆-C24 aralkyl, halo, -Si(Ci-C₃ alkyl)3, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C₂- C24 alkynyloxy, C5-C2₀ aryloxy, acyl, acyloxy, C2-C24 alkoxycarbonyl, C₆-C2₀

aryloxycarbonyl, C2-C24 alkylcarbonato, C₆-C2₀ arylcarbonato, carboxy, carboxylato, carbamoyl, C1-C24 alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, C1-C24 alkyl amino, C5-C2₀ aryl amino, C2-C24 alkylamido, C₆-C2₀ arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato, C1-C24 alkylsulfanyl, arylsulfanyl, C1-C24 alkylsulfinyl, C5-C2₀ arylsulfinyl, C1-C24 alkylsulfonyl, C5-C2₀ arylsulfonyl, sulfonamide, phosphono, phosphonato, phosphinato, phospho, phosphino, poly alky lethers, phosphates, phosphate esters, groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, combinations thereof, and wherein R⁶ and R⁷ may be linked to form a cyclic or polycyclic ring, wherein the ring is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, and a substituted or unsubstituted heterocyclyl;

U¹ is N, C-R², or C-NR³R⁴, wherein R² is selected from the group consisting of a H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, 0-CH₂- CH₂X, CH2-CH2-CH2X, O-CH2-CH2X, X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, 0(CO)R', COOR' (wherein R' is H or a lower alkyl group), and wherein R¹ and R² may be linked to form a cyclic or polycyclic ring, wherein R³ and R⁴ are the same or different and are each selected from the group consisting of H, a lower alkyl group, O, (CH₂)_niOR' (wherein nl=l, 2, or 3), CF₃, CH₂-CH₂X, CH₂-CH₂-CH₂X, (wherein X=H, F, CI, Br, or I), CN, (C=0)-R\ (C=0)N(R')₂, COOR' (wherein R' is H or a lower alkyl group), and R³ or R⁴ may be absent;

and pharmaceutically acceptable salts thereof.

12. The method of claim 8, wherein the 15-PGDH inhibitor has the following formula (VI):

wherein n = 0-2;

X⁶ is N or CR^C;

R¹ is selected from the group consisting of branched or linear alkyl including■

X

(0¾)ηιΟ¾ (ni=0-7), "² wherein n2=0-6 and X is any of the following: CF_yH_z (y + z =

3), CCl_yH_z (y + z = 3), OH, OAc, OMe, R , OR , CN, N(

m=l-5), n₄

and (n₄=0-5).

R⁵ is selected from the group consisting of H, CI, F, Ν¾, and N(R⁷⁶)2_;

R⁶ and R⁷ can each independently be one of the following:

each R⁸ R⁹ R¹⁰ R¹¹ R¹² R¹³ R¹⁴ R¹⁵ R¹⁶ R¹⁷ R¹⁸ R¹⁹ R²⁰ R²¹ R²² R²³ R²⁴ R²⁵

48

R⁴⁹ _, R⁵⁰ R⁵¹ _, R⁵² _, R⁵³

70 _R72

R⁷¹ R⁷³ R⁷⁴ R⁷⁶

the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C₃-C2₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6 ring atoms, C₆-C24 alkaryl, C₆-C24 aralkyl, halo, -Si(Ci-C₃ alkyl)₃, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2₀ aryloxy, acyl, acyloxy, C2-C24 alkoxycarbonyl, C₆-C2₀ aryloxycarbonyl, C2-C24 alkylcarbonato, C₆-C2₀ arylcarbonato, carboxy, carboxylato, carbamoyl, C1-C24 alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, C1-C24 alkyl amino, C5-C2₀ aryl amino, C2-C24 alkylamido, C₆-C2₀ arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato, C1-C24 alkylsulfanyl, arylsulfanyl, C1-C24 alkylsulfinyl, C5-C2₀ arylsulfinyl, C1-C24 alkylsulfonyl, C5-C2₀ arylsulfonyl, sulfonamide, phosphono, phosphonato, phosphinato, phospho, phosphino, poly alky lethers, phosphates, phosphate esters, groups incorporating amino acids or other moieties expected to bear positive or negative charge at physiological pH, and combinations thereof, and

pharmaceutically acceptable salts thereof.