WO2019010482A1 - Compositions et méthodes pour moduler la maturation du col - Google Patents

Compositions et méthodes pour moduler la maturation du col Download PDF

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Publication number
WO2019010482A1
WO2019010482A1 PCT/US2018/041255 US2018041255W WO2019010482A1 WO 2019010482 A1 WO2019010482 A1 WO 2019010482A1 US 2018041255 W US2018041255 W US 2018041255W WO 2019010482 A1 WO2019010482 A1 WO 2019010482A1
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Prior art keywords
alkyl
aryl
pgdh
group
pge2
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PCT/US2018/041255
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English (en)
Inventor
Ruth Ann WORD
Hari Kishore Annavarapu
Bruce POSNER
Joseph READY
Sanford Markowitz
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Case Western Reserve University
University Of Texas Southwestern Medical
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Publication of WO2019010482A1 publication Critical patent/WO2019010482A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha

Definitions

  • Labor can be induced in a number of ways.
  • 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).
  • 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.
  • 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.
  • PGE2 prostaglandin E2
  • 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.
  • 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.
  • 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.
  • 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.
  • the labor inducing agent comprises at least one of dioprostone (PGE2) or misoprostol (PGE1).
  • the 15-PGDH inhibitor can include a compound having the following formula (V):
  • n 0-2
  • X 6 is independently is N or CR C
  • R 6 , R 7 , 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, C 3 -C2 0 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-C 6 alkyl), NC(0)(Ci-C 6 alkyl), O, and S), C 6 -C24 alkaryl, C 6 -C24 aralkyl, halo, -Si(Ci-C 3 alkyf , hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2 0 aryloxy, acyl (including C2-C24 alkylcarbonyl
  • alkylcarbonato (-O-(CO)-O-alkyl), C 6 -C2 0 arylcarbonato (-O-(CO)-O-aryl), carboxy
  • R 6 and R 7 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;
  • R 6 and R 7 can each independently be one of the following:
  • R 65 R 66 R 67 , R 68 , R 69 , R 7o R 7i R 72 R 73 a R 74 ⁇ ⁇ same or diff eren t and are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C2 0 aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C 6 alkyl), NC(0)(Ci- C 6 alkyl), O, and S), heteroaryl or heterocyclyl containing from 5-14 ring atoms, (wherein from 1-6 of the ring
  • the 15-PGDH inhibitor can inhibit the enzymatic activity of recombinant 15-PGDH at an IC 50 of less than 1 ⁇ , or preferably at an IC 50 of less than 250 nM, or more preferably at an IC 50 of less than 50 nM, or more preferably at an IC 50 of less than 10 nM, or more preferably at an IC 50 of less than 5 nM at a recombinant 15-PGDH concentration of about 5 nM to about 10 nM.
  • 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.
  • 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.
  • preterm e.g., thirty seven week
  • 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.
  • 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.
  • Figs. 2(A-E) illustrate a table and graphs showing PGE2-mediated gene regulation is Ca 2+ -dependent.
  • Figs. 3(A-E) illustrate graphs, an immunoblot, and plot showing PGE2 results in deacetylation of chromatin associated with the 15-PGDH gene promoter.
  • IP 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.
  • N 3.
  • FIGs. 4(AD) illustrate graphs, an immunoblot, and plot showing PGE2 mediated 15-PGDH repression is mediated by HDAC4.
  • C HDAC4 protein levels in hCSCs pretreated with DMSO or PF-04418948 (2 ⁇ ) followed by DMSO or PGE2. ⁇ -actin, loading control D.
  • HDAC4 in hCSCs in media + Ca 2+ pre-treated with DMSO or BAPTA-AM (1 ⁇ ) for 1 h, then with DMSO or PGE2 (25 nM) for 15 h.
  • FIGs. 5(A-G) illustrate images, graphs, a plot, and immunblot showing PGE2 mediated dephosphory/arfon of HDAC4 and nuclear Ocalization.
  • PGE2 acts through EP2 receptors to increase Ca 2+ -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.
  • Figs. 6(A-B) illustrate plots and images showing HDAC4 mRNA and protein levels in human cervical stromal tissues at different stages of pregnancy.
  • Data represent relative mRNA levels normalized to GAPDH mRNA levels. P ⁇ 0.05.
  • Figs. 7(A-F) illustrate images and graphs showing PGE2 plus 15-PGDH inhibitor treatment induces preterm cervical ripening and labor in mice.
  • 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 PGE 2 (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 PGE 2 -treated hCSCs at 1 h (Left) or 24 h (Right).
  • 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
  • FIGs. 9(A-E) illustrate plots and a graph showing PGE2-mediated gene regulation is Ca 2+ -dependent.
  • C, D Relative expression of Ca 2+ - responsive genes C-FOS and DUSPl in hCSCs treated with PGE 2 (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.
  • E E.
  • Figs. lO(A-B) illustrate plots and a graph showing calcium ionophore A23187 mimics PGE 2 mediated effects in hCSCs.
  • 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
  • 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.
  • Figs. 12(A-B) illustrate plots and graph showing reciprocal regulation of 15- PGDH and HDAC4 by PGE 2 .
  • 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
  • Fig. 13 illustrates a graph showing PGE2 increases HDAC4 levels via EP2 receptor.
  • N 3.
  • 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).
  • FIG. 15 Illustrates a graph showing PGE2 does not alter HDAC4 gene expression in SK-MEL5 and MCF7 cells.
  • Data represent relative mean mRNA levels + SD of triplicates after normalizing to GAPDH mRNA levels.
  • Figs. 16(A-B) illustrate graphs showing 15-PGDH is an HDAC4 target gene in hCSCs.
  • FIG. 17 illustrates graphs showing HDAC5 does not regulate 15-PGDH gene expression in hCSCs.
  • Data represent relative mean mRNA levels + SD of triplicates after normalizing to GAPDH mRNA levels.
  • *P 0.00002 compared with siNeg transfected cells.
  • 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 ⁇ ), KN-93 (5 ⁇ ), or PGE 2 (100 nM) for 24 h.
  • B Levels of 15-PGDH (a) and HDAC4 mRNA in hCSCs treated with DMSO, C2 ceramide (50 ⁇ ) or PGE 2 (100 nM) for 24 h.
  • C D.
  • 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.
  • 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.
  • 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.
  • 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”.
  • 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".
  • a stereoisomer 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).
  • 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.
  • 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
  • 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.
  • derivative refers to compounds that have a common core structure, and are substituted with various groups as described herein.
  • 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).
  • 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.
  • 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.
  • 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.
  • a short chain dehydrogenase inhibitor e.g., 15- PGDH inhibitor
  • 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.
  • 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.
  • 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).
  • the term "pharmaceutical composition” refers to a formulation containing the disclosed compounds in a form suitable for administration to a subject.
  • 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.
  • active ingredient e.g., a formulation of the disclosed compound or salts thereof
  • the dosage will also depend on the route of administration.
  • routes 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.
  • the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
  • flash dose refers to compound formulations that are rapidly dispersing dosage forms.
  • 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.
  • modified release is defined to include delayed release, extended release, and pulsed release.
  • pulsed release is defined as a series of releases of drug from a dosage form.
  • 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.
  • phrases "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.
  • a pharmaceutically acceptable carrier is non-pyrogenic.
  • 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) alg
  • “Pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.
  • 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.
  • 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).
  • esters for example pharmaceutically acceptable esters.
  • a carboxylic acid function group in a compound can be converted to its corresponding ester, e.g., a methyl, ethyl, or other ester.
  • an alcohol group in a compound can be converted to its corresponding ester, e.g., an acetate, propionate, or other ester.
  • the compounds described herein can also be prepared as prodrugs, for example pharmaceutically acceptable prodrugs.
  • 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.
  • prodrugs include, but are not limited to, esters (e.g., acetate, dialkylaminoacetates, formates, phosphates, sulfates, and benzoate derivatives) and carbamates (e.g., ⁇ , ⁇ -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.
  • esters e.g., acetate, dialkylaminoacetates, formates, phosphates,
  • 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).
  • 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.
  • 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.
  • FMOC 9-fluorenylmethyloxycarbonyl
  • NVOC nitro-veratryloxycarbonyl
  • 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.
  • 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.
  • 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.
  • 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.
  • tautomerism 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.
  • keto-enol tautomerism a simultaneous shift of electrons and a hydrogen atom occurs.
  • 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.
  • 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.
  • 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).
  • an analogue is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.
  • 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.
  • 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.
  • the subject is a mammal.
  • a patient refers to a subject afflicted with a disease or disorder.
  • 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).
  • the unwanted condition e.g., disease or other unwanted state of the host animal
  • terapéuticaally effective amount and “pharmaceutically effective amount” are an art-recognized term.
  • 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.
  • 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.
  • 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.
  • ED50 is art-recognized.
  • 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.
  • LD50 is art-recognized.
  • LD50 means the dose of a drug, which is lethal in 50% of test subjects.
  • therapeutic index is an art-recognized term, which refers to the therapeutic index of a drug, defined as LD50/ED50.
  • IC 50 half maximal inhibitory concentration
  • concentration of a substance e.g., a compound or a drug
  • concentration of a biological process, or component of a process including a protein, subunit, organelle, ribonucleoprotein, etc.
  • isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium
  • isotopes of carbon include C-13 and C-14.
  • 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.
  • 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.
  • branched e.g., isopropyl, tert-butyl, isobutyl
  • straight-chain e.g., methyl, ethyl, propyl, butyl, pentyl, hexy
  • Ci_6 alkyl is intended to include Ci, C 2 , C 3 , C 4 , C 5 , and C 6 alkyl groups.
  • 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.
  • a straight chain or branched chain alkyl has six or fewer carbon atoms in its backbone (e.g., Ci-C 6 for straight chain, C 3 -C 6 for branched chain), for example four or fewer.
  • certain cycloalkyls have from three to eight carbon atoms in their ring structure, such as five or six carbons in the ring structure.
  • 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.
  • 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.
  • 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.
  • substituted alkenyl refers to alkenyl substituted with one or more substituent groups
  • 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.
  • alkenyl and “lower alkenyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively.
  • 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.
  • alkyl alkenyl
  • alkynyl moieties which are diradicals, i.e., having two points of attachment.
  • a nonlimiting example of such an alkyl moiety that is a diradical is— CH 2 CH 2 — , i.e., a C 2 alkyl group that is covalently bonded via each terminal carbon atom to the remainder of the molecule.
  • 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 “C 1 -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).
  • 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.
  • aryl groups include benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
  • aryl includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,
  • aryl heterocycles 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 alkyl,
  • 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.
  • alkaryl refers to an aryl group with an alkyl substituent
  • 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.
  • aralkyl groups include, without limitation, benzyl, 2-phenyl-ethyl, 3 -phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl,
  • 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.
  • 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.
  • 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, sulfamo
  • 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,— CF 3 , or— CN, or the like.
  • 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.
  • 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.
  • substituents include, without limitation: functional groups such as halo, hydroxyl, silyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2 0 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C2 0 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C 6 -C 20 aryloxycarbonyl (-(CO)-O-aryl), C 2 -C 24 alkylcarbonato
  • alkylthio arylsulfanyl (-S-aryl; also termed “arylthio", C C 24 alkylsulfinyl (-(SO)-alkyl), C 5 -C 20 arylsulfinyl (-(SO)-aryl), Ci-C 24 alkylsulfonyl (-S0 2 -alkyl), C 5 -C 20 arylsulfonyl (-S0 2 -aryl), phosphono (-P(0)(OH) 2 ), phosphonato (-P(0)(0 " ) 2 ), phosphinato (-P(0)(0 ⁇ )), phospho (-P0 2 ), and phosphino (-PH 2 ); and the hydrocarbyl moieties Ci-C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, Cs-C 2 o aryl, C 6 -C 24 alkyl
  • 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.
  • the above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated.
  • substituted appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group.
  • substituted alkyl, alkenyl, and aryl is to be interpreted as “substituted alkyl, substituted alkenyl, and substituted aryl.”
  • 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.
  • heteroatom- containing alkyl, alkenyl, and aryl is to be interpreted as “heteroatom-containing alkyl, substituted alkenyl, and substituted aryl.
  • "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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • 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.
  • 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.
  • 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).
  • inducing labor relates to a therapy where a direct response effect is requested from the administration.
  • a direct response effect is requested from the administration.
  • 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.
  • 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. .
  • 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.
  • 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.
  • PGE2 prostaglandin E2
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • IUGR Intra Uterine Growth Retardation
  • cervix score 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.
  • 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
  • the at least one prostaglandin or prostaglandin derivative can include prostaglandins, such as the prostaglandins of series 2 including in particular PGF2 01 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 PGF2 01 17-phenyl PGF2 01 , 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.
  • prostaglandins such as the prostaglandins of series
  • 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 %.
  • Agents tested as 15-PGDH can be any small chemical molecule or compound.
  • 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).
  • the 15-PGDH inhibitor can include a compound having the following formula (I):
  • n 0-2;
  • Y 1 , Y 2 , and R 1 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-C2 0 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-C 6 alkyl), NC(O) (Ci-C 6 alkyl), O, and S), C 6 -C 24 alkaryl, C 6 -C 24 aralkyl, halo, -Si(d-C 3 alkyl) 3 , hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2 0 aryloxy, acyl (including C2-
  • X 1 and X 2 are independently N or C, and wherein when X 1 and/or X 2 are N, Y 1 and/or Y 2 , respectively, are absent;
  • Z 1 is O, S, CR a R 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;
  • the 15-PGDH inhibitor can include a compound having the following
  • n 0-2
  • X 4 , X 5 , X 6 , and X 7 are independently N or CR C ;
  • R 1 , R 6 , R 7 , and R c are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C 3 -C2 0 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-C 6 alkyl), NC(0)(Ci-C 6 alkyl), O, and S), C 6 -C24 alkaryl, C 6 -C24 aralkyl, halo, -Si(Ci-C 3 alkyl) 3 , hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2 0 aryloxy, acyl (including C2-C24 al
  • alkylcarbonato (-O-(CO)-O-alkyl), C 6 -C2 0 arylcarbonato (-O-(CO)-O-aryl), carboxy
  • R 6 and R 7 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;
  • Z 1 is O, S, CR a R 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;
  • the 15-PGDH inhibitor can include a compound having the following formula (III) or (IV):
  • n 0-2
  • X 6 is independently is N or CR C ;
  • R 1 , R 6 , R 7 , and R c are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C 3 -C2 0 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-C 6 alkyl), NC(0)(Ci-C 6 alkyl), O, and S), C 6 -C24 alkaryl, C 6 -C24 aralkyl, halo, -Si(Ci-C 3 alkyf , hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2 0 aryloxy, acyl (including C2-C24 alkyl
  • alkylcarbonato (-O-(CO)-O-alkyl), C 6 -C2 0 arylcarbonato (-O-(CO)-O-aryl), carboxy
  • R 6 and R 7 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;
  • Z 1 is O, S, CR a R 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;
  • R 6 and R 7 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 C 1 -C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C 3 -C2 0 aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C 6 alkyl), NC(O) (Ci-C 6 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-C 3 alkyl), O, and S), C6-C24 alkaryl, C 6 -C24 aralkyl, halo, silyl, hydroxyl, sulfhydryl, C1-C24
  • R 6 and R 7 can independently be a group that improves aqueous solubility, for example, a phosphate ester (-OPO 3 H2), a phenyl ring linked to a phosphate ester (-OPO 3 H2), a phenyl ring substituted with one or more methoxyethoxy groups, or a morpholine, or an aryl or heteroaryl ring substituted with such a group.
  • the 15-PGDH inhibitor can include a compound having the following
  • n 0-2
  • X 6 is independently is N or CR C
  • R ⁇ R 6 , R 7 , 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, C 3 -C2 0 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-C 6 alkyl), NC(0)(Ci-C 6 alkyl), O, and S), C 6 -C24 alkaryl, C 6 -C24 aralkyl, halo, -Si(Ci-C 3 alkyf , hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2 0 aryloxy, acyl (including C2-C24 alkylcarbon
  • alkylcarbonato (-O-(CO)-O-alkyl), C 6 -C2 0 arylcarbonato (-O-(CO)-O-aryl), carboxy
  • R 6 and R 7 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 C 1 -C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C 3 -C2 0 aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C 6 alkyl), NC(0)(Ci- C 6 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-C 3 alkyl), O, and S), C 6 -C24 alkaryl, C 6 -C24 aralkyl, halo, silyl, hydroxyl, sulfhydryl, C1-C24
  • R 6 and R 7 can independently be a group that improves aqueous solubility, for example, a phosphate ester (-OPO 3 H2), a phenyl ring linked to a phosphate ester (-OPO 3 H2), a phenyl ring substituted with one or more methoxyethoxy groups, or a morpholine, or an aryl or heteroaryl ring substituted with such a group.
  • the 15-PGDH inhibitor can include a compound having the following
  • n 0-2;
  • X 6 is N or CR C ;
  • R 1 is selected from the group consisting of branched or linear alkyl including ⁇
  • R 5 is selected from the group consisting of H, CI, F, ⁇ 3 ⁇ 4, and N(R 76 )2 ;
  • R 6 and R 7 can each independently be one of the following:
  • R 4488 R R 4 4 9 9 , R R 5 5 0 0 RR 5511 RR 5522 R R 5 5 3 3 R 54 R 55 R 56 R 57 R 58 R 59 R 60 R 61 R 62 R 63 R 64 R 65 R 66 R 67 R 68 R 69 R 7700 R R 7 7 1 1 RR 7722 R R 7 7 3 3 R R 7 7 4 4 R R 7 7 6 6 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-C2 0 aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from 1-3 of the ring atoms is independently selected from N, NH, N(Ci-C 6 alkyl), NC(O) (Ci-C 6 alkyl), O, and S), heteroaryl or heterocyclyl
  • the 15-PGDH inhibitor can include a compound having the following
  • n 0-2;
  • X 6 is N or CR C ;
  • R 5 is selected from the group consisting of H, CI, F, NH2, and N(R 76 )2 ; 7 can each independently be one of the following:
  • R 26 R 27 , R 28 R 29 R 30 R 1 R 3 R 33 , R 34 , R 35 R 36 R 37 , R 38 , R 39 .
  • R 40 R 41 , R 42 , R 43 , R 44 , R 45 , R 46 R 47 .
  • the 15-PGDH inhibitor having formula (I), (II), (III), (IV), (V), (VI), and (VII) can be selected that can ia) at 2.5 ⁇ 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 ⁇ 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 ⁇ 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 ⁇ concentration, does not activate a negative control V9m cell line expressing TK
  • the 15-PGDH inhibitor can ib) at 2.5 ⁇ concentration, stimulate a Vaco503 reporter cell line expressing a 15-PGDH luciferase fusion construct to increase luciferase output; iib) at 2.5 ⁇ concentration stimulate a V9m reporter cell line expressing a 15-PGDH luciferase fusion construct to increase luciferase output; iiib) at 7.5 ⁇ concentration stimulate a LS174T reporter cell line expressing a 15-PGDH luciferase fusion construct to increase luciferase output; ivb) at 7.5 ⁇ 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 IC 50 of less than 1 ⁇ .
  • the 15-PGDH inhibitor can inhibit the enzymatic activity of recombinant 15-PGDH at an IC 50 of less than 1 ⁇ , or preferably at an IC 50 of less than 250 nM, or more preferably at an IC 50 of less than 50 nM, or more preferably at an IC 50 of less than 10 nM, or more preferably at an IC 50 of less than 5 nM at a recombinant 15-PGDH concentration of about 5 nM to about 10 nM.
  • 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.
  • al5-PGDH inhibitor can include a compound having the following formula (VIII):
  • n 0-2;
  • R 1 , R 6 , and R 7 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-C2 0 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-C 6 alkyl), NC(O) (C C 6 alkyl), O, and S), C 6 -C 24 alkaryl, C 6 -C 24 aralkyl, halo, -Si(C C 3 alkyl) 3 , hydroxyl, sulfhydryl, Ci-C2 4 alkoxy, C2-C2 4 alkenyloxy, C2-C2 4 alkynyloxy, C5-C2 0 aryloxy, acyl (including C2-C2 4
  • R 6 and R 7 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.
  • 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 ⁇ , ⁇ -dimethylformamide, N,N- dimethylacetamide, acetonitrile, dimethylsulfoxide and hexamethyl phosphoric acid triamide.
  • 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
  • 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.
  • embodiments of this application include any modifications for the preparation method of the 15-PGDH inhibitors described above.
  • 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.
  • 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.
  • a 15-PGDH inhibitor having formula (VIII) can include a compound w
  • 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 ⁇ / ⁇ mouse and at
  • a 15-PGDH inhibitor having formula (IX) can include a compound with the following formula (IXa):
  • a 15-PGDH inhibitor having formula (IX) can include a comp
  • 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).
  • 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%
  • the 15-PDGH inhibitor can consist essentially of or consist of the (+) optical isomer of a 15-PGDH inhibitor having formula (IX).
  • the PDGH inhibitor can consist essentially of or consist of the (-) optical isomer of a 15-PGDH inhibitor having formula (IX).
  • a 15-PGDH inhibitor having formula (VIII) can include a compound wi
  • a 15-PGDH inhibitor having formula (X) can include a compound with the following formula (Xa):
  • a 15-PGDH inhibitor having formula (X) can include a compound w
  • the 15-PDHG inhibitor can comprise a (+) or (-) optical isomer of a 15-PGDH inhibitor having formula (X).
  • the 15-PDHG inhibitor can comprise a (+) or (-) optical isomer of a 15-PGDH inhibitor having formula (X).
  • the 15-PDHG inhibitor can comprise a mixture at least one of a (+) or (-) optical isomer of a 15-PGDH inhibitor having formula (X).
  • 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
  • the 15-PDGH inhibitor can consist essentially of or consist of the (+) optical isomer of a 15-PGDH inhibitor having formula (X).
  • the PDGH inhibitor can consist essentially of or consist of the (-) optical isomer of a 15-PGDH inhibitor having formula (X).
  • 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),
  • 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.
  • 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.
  • 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,
  • the pharmaceutical composition can be made into aerosol formulations (e.g., they can be nebulized) to be administered via inhalation.
  • 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.
  • 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.
  • pH adjusters and the buffers include sodium citrate, sodium acetate and sodium phosphate.
  • 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.
  • 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.
  • pharmaceutically acceptable carriers for example, polyethylene glycol, lanolin, cacao butter or fatty acid triglycerides, if necessary, together with surfactants such as Tween.
  • 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.
  • a 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.
  • 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,
  • 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.
  • 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
  • 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
  • 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
  • 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
  • 0.0001 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
  • 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,
  • the effective dose for a particular individual can be varied (e.g., increased or decreased) over time, depending on the needs of the individual.
  • a therapeutically effective dosage may be a dosage of 10 ⁇ g/kg/day, 50 ⁇ g/kg/day, 100 ⁇ g/kg/day, 250 ⁇ g/kg/day, 500 ⁇ g/kg/day, 1000 ⁇ g/kg/day or more.
  • the amount of the 15-PGDH inhibitor or pharmaceutical salt thereof is sufficient to provide a dosage to a patient of between 0.01 ⁇ g/kg and 10 ⁇ g/kg; 0.1 ⁇ g/kg and 5 ⁇ g/kg; 0.1 ⁇ g/kg and 1000 ⁇ g/kg; 0.1 ⁇ g/kg and 900 ⁇ g/kg; 0.1 ⁇ g/kg and 900 ⁇ g/kg; 0.1 ⁇ g/kg and 800 ⁇ g/kg; 0.1 ⁇ g/kg and 700 ⁇ g/kg; 0.1 ⁇ g/kg and 600 ⁇ g/kg; 0.1 ⁇ g/kg and 500 ⁇ g/kg; or 0.1 ⁇ g/kg and 400 ⁇ g/kg.
  • Various embodiments may include differing dosing regimen.
  • the 15-PGDH inhibitor and, optionally, the other labor inducing agents can be administered via continuous infusion.
  • the continuous infusion is intravenous.
  • 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.
  • 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.
  • 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
  • administering to the female at least one of an EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator.
  • 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.
  • preterm e.g. thirty seven week
  • the prostaglandin EP2 receptor antagonist can include any suitable EP2 receptor antagonist.
  • 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.
  • PGE2 natural ligand
  • 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.
  • the antagonists bind reversibly to their cognate receptor.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 IC 50 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%.
  • 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.
  • 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.
  • 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.
  • vorinostat and romidepsin are FDA-approved for the treatment of cutaneous and peripheral T-cell lymphoma.
  • the 15-PGDH activator can include any compound that can promote or stimulate the activity of 15-PGDH.
  • 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.
  • EP2 receptor antagonist, HDAC4 inhibitor, and/or 15-PGDH activator described herein can be provided in a pharmaceutical composition that includes
  • 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.
  • 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.
  • 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.
  • the gel or cream is one which is formulated for administration to the vagina. It may be oil based or water based.
  • 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.
  • 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.
  • 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.
  • the individual to be treated may be any pregnant female individual who would benefit from such treatment.
  • the individual to be treated is a human female.
  • 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.
  • the methods have uses in both human and veterinary medicine.
  • PGE2 utilizes cell-specific EP2 receptor-mediated increases in Ca 2+ to dephosphorylate and translocate HDAC4 to the nucleus for repression of 15-hydroxy prostaglandin dehydrogenase (15-PGDH).
  • 15-PGDH 15-hydroxy prostaglandin dehydrogenase
  • This example addresses the molecular mechanism of PGE2 action in cervical stromal cells and the underlying pathways leading to cervical ripening.
  • PGE2 initiates specific cellular events in cervical stromal cells.
  • 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.
  • 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.
  • hCSCs Human cervical stromal cells
  • 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] PGE 2 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.
  • DMSO dimethyl sulfoxide
  • 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
  • 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.
  • 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 iScriptTM Reverse Transcription Supermix from BIO-RAD according to supplier's protocol (170-8841).
  • Quantitative PGR 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.
  • 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.
  • PBS cold Phosphate Buffered Saline
  • 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).
  • hCSCs were maintained in serum free Opti MEM (11058-021, Life
  • hCSCs grown in 6 well dishes were infected with 5 ⁇ (1 x 10 6 pfu/mL) of either ⁇ -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.
  • control adenovirus 000197 A, Applied Biological Materials Inc.
  • HDAC4 expressing adenovirus 000426A, Applied Biological Materials Inc.
  • 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.
  • 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 ⁇ 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.
  • 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 ⁇ and Masson's trichrome staining was performed.
  • 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.
  • 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.
  • PGE2 regulates the transcriptome of human cervical stromal cells in vitro through EP2- mediated increases in intracellular Ca 2+
  • 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.
  • 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).
  • 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-EP2 interactions resulted in Ca 2+ -dependent signaling (e.g., DUSP1, c-fos), not cAMP, PKA and PI3-kinase (Fig.9).
  • RNA-Seq data indicated that the most significantly affected pathway by PGE2 was Ca 2+ signaling (Fig. 2A). Expression levels of 71 genes either regulated by Ca 2+ or involved in Ca 2+ 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 Ca 2+ ionophore (A23187) decreased 15-PGDH and increased COX-2 mRNA (Fig. 2C, Fig. 10). Ca 2+ -dependent PGE2-mediated 15-PGDH repression was confirmed using a cell permeable intracellular Ca 2+ chelator BAPTA-AM (Fig. 2D).
  • PGE2 downregulates 15-PGDH gene expression in vitro by increasing HDAC4
  • HDACs histone deacetylases
  • HDACi HDAC inhibitors
  • HDACi 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.
  • HDAC5 and 9 mRNA decreased in response to PGE2 (Fig. 3E).
  • HDAC4 mRNA increased ⁇ 4-fold (Fig. 3E).
  • PGE2 increased HDAC4 mRNA in a dose- and time- dependent manner, concomitantly, decreasing 15-PGDH in hCSCs (Fig. 4 A, Fig. 12A).
  • HDAC4 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).
  • 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).
  • Phosphorylated HDAC4 binds 14-3-3 protein, a complex retained in the cytoplasm.
  • CaMKII nuclear Ca 2+ /calmodulin-dependent protein kinase II
  • 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).
  • HDAC4 levels and localization during late gestation in human cervical stromal tissues in vivo
  • 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
  • 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).
  • Fig. 7A 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).
  • Fig. 7D we assessed the impact of treatment on histomorphology of the cervix.
  • 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.
  • 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.
  • 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.
  • RNA-Seq data confirmed that PGE2 activates Ca 2+ signaling pathways in hCSCs.
  • Phosphorylated HDAC4 binds 14-3-3 protein, a complex retained in the cytoplasm.
  • Dephosphorylation of cytoplasmic HDAC4 by protein phosphatase 2A leads to release from the complex and nuclear import of HDAC4.
  • Global inhibition of serine/threonine phosphatases with okadaic acid confirmed that
  • HDAC4 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.
  • RNA-Seq data did not reveal PGE2-mediated increased expression of proteases (or downregulation of protease inhibitors), hyaluronan synthases, or
  • progesterone receptors 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.
  • cytokine-cytokine receptor interactions e.g., CCL8, CXCL-1 and -2, IL1R1, and several members of the TNF/TNF receptor superfamily
  • 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.
  • EP2 receptor antagonists ii) HDAC inhibitors
  • activators of 15-PGDH activators of 15-PGDH
  • 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.
  • HDAC inhibitors target HDAC4 to increase basal levels of 15-PGDH that neutralizes active PGE2 and PGF2D.
  • 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.
  • 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.
  • development of EP2 antagonists and HDAC4 inhibitors may successfully interrupt the vicious cycle of preterm cervical ripening and preterm birth.

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Abstract

Des compositions et des procédés de modulation de la maturation du col comprenant l'initiation de la maturation du col et/ou l'induction du travail ainsi que l'inhibition de la maturation prématurée du col et/ou de la naissance avant terme chez une femme en ayant besoin peuvent comprendre au moins l'un des inhibiteurs de 15-PGDH, qui peut initier, ou amplifier, une maturation du col médiée par PGE2, ainsi que des antagonistes du récepteur EP2, des inhibiteurs de HDAC4 et/ou des activateurs de 15-PGDH, qui peuvent empêcher la maturation du col prématurée et la naissance avant terme.
PCT/US2018/041255 2017-07-07 2018-07-09 Compositions et méthodes pour moduler la maturation du col WO2019010482A1 (fr)

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US11345702B1 (en) 2020-01-23 2022-05-31 Myoforte Therapeutics, Inc. PGDH inhibitors and methods of making and using
WO2023032925A1 (fr) * 2021-08-30 2023-03-09 国立大学法人京都大学 Méthode et réactif de test
US11690847B2 (en) 2016-11-30 2023-07-04 Case Western Reserve University Combinations of 15-PGDH inhibitors with corticosteroids and/or TNF inhibitors and uses thereof
US11718589B2 (en) 2017-02-06 2023-08-08 Case Western Reserve University Compositions and methods of modulating short-chain dehydrogenase

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US11718589B2 (en) 2017-02-06 2023-08-08 Case Western Reserve University Compositions and methods of modulating short-chain dehydrogenase
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