WO2020141509A1 - Compositions et procédés d'inhibition de l'ovulation - Google Patents

Compositions et procédés d'inhibition de l'ovulation Download PDF

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WO2020141509A1
WO2020141509A1 PCT/IL2019/051429 IL2019051429W WO2020141509A1 WO 2020141509 A1 WO2020141509 A1 WO 2020141509A1 IL 2019051429 W IL2019051429 W IL 2019051429W WO 2020141509 A1 WO2020141509 A1 WO 2020141509A1
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inhibitor
abcc4
ptgs
pharmaceutical composition
ovulation
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PCT/IL2019/051429
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English (en)
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Ariel HOURVITZ
Yuval YUNG
Gil YERUSHALMI
Bat-El SHURAKI
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Hourvitz Ariel
Yung Yuval
Yerushalmi Gil
Shuraki Bat El
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Publication of WO2020141509A1 publication Critical patent/WO2020141509A1/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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • 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
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives

Definitions

  • the present invention relates to inhibition of ovulation using inhibitors of ABCC4, a transporter of prostaglandin E2, alone or in combination with inhibitors of prostaglandin synthesis.
  • Prostaglandins are a family of local mediators of a variety of physiological and pathological processes. Long associated with female reproduction, PGs have been implicated in fertilization, luteolysis, implantation, and parturition. PGs also play a key role in the ovulatory cascade. PG biosynthesis is under the control of prostaglandin synthase (PTGS) enzymes, also commonly known as cyclooxygenase (COX) enzymes. The inducible form PTGS2 (COX2) is critical for ovulation and expressed in the ovary in a cell type-specific manner. Prostaglandin E2 (PGE2) is the major PG product that mediates the ovulation process.
  • PTGS prostaglandin synthase
  • COX2 cyclooxygenase
  • PTGS inhibitors In humans, inhibition of ovulation by PTGS inhibitors was reported by several clinical studies and case reports in woman. For example, patients afflicted with arthritis taking NSAIDS (non-steroidal anti-inflammatory drugs, which are PTGS inhibitors) such as indomethacin or diclofenac on a daily basis were unable to conceive until they stopped taking the drugs (Akil et ak, Br J Rheumatol 35:76-78 (1996); and Mendonca et ak, Rheumatology (Oxford) 39:880-882 (2000)).
  • NSAIDS non-steroidal anti-inflammatory drugs, which are PTGS inhibitors
  • indomethacin or diclofenac on a daily basis were unable to conceive until they stopped taking the drugs (Akil et ak, Br J Rheumatol 35:76-78 (1996); and Mendonca et ak, Rheumatology (Oxford) 39:88
  • indomethacin and azapropazone were found to induce the luteinized unruptured follicles (LUF) syndrome (Killick et al., Fertil Steril 47:773-777 (1987)). Indomethacin was also shown to delay follicle rupture in 5 of 6 of indomethacin-treated patients (Athanasiou et al., Fertil Steril 65:556-560 (1996)).
  • PGs are charged anions that diffuse poorly through plasma membranes despite their lipid nature.
  • a carrier-mediated transport mechanism is therefore required for the translocation of PGs across biological membranes.
  • PG transporter also known as the solute carrier organic anion transporter family member 2A1 (SLC02A1)
  • SLC02A1 solute carrier organic anion transporter family member 2A1
  • PGT inhibitors were suggested as non-hormonal contraceptives: see for example Yerushalmi et al., supra, and WO 2016/113734, to the assignee of the present invention, which discloses methods and compositions for inhibiting ovulation in a female subject, including a subject in need of contraceptives and a subject undergoing fertility treatments, comprising administering to the female subject at least one PGT inhibitor.
  • ABCC4 ATP Binding Cassette subfamily C member 4
  • MRP4 Multidrug Resistance Protein 4
  • ABCC4 is an energy-dependent, transmembrane transporter with wide substrate selectivity: it transports several endogenous substrates, such as cyclic nucleotides, PGs, leukotrienes, folates as well as glutathione-conjugated molecules like bile acids and steroid hormones.
  • ABCC4 is also known as a mediator of drug resistance in several tumor types. ABCC4 is known as a PGE2 efflux transporter (Reid et al. Proc Natl Acad Sci U S A 100, 9244-9249 (2003)).
  • Yerushalmi et al. ( Mol Hum Reprod 20, 719-735 (2014)) characterized the human cumulus cell transcriptome during final follicular maturation and ovulation. They used global transcriptome sequencing to characterize the final stages of follicular maturation and ovulation pathways in humans. The aim of the study was to systematically identify ovulation-associated genes.
  • compositions and methods that inhibit ovulation, particularly non-hormonal inhibition of ovulation which may be useful, for example, as non-hormonal contraceptives, during fertility treatments, and more.
  • compositions, methods and kits for inhibiting ovulation in a female subject.
  • the compositions, methods and kits utilize inhibitors of ATP Binding Cassette subfamily C member 4 (ABCC4), a transporter of prostaglandin E2, alone or in combination with inhibitors of prostaglandin synthases (PTGS).
  • ABCC4 ATP Binding Cassette subfamily C member 4
  • PTGS inhibitors of prostaglandin synthases
  • the present invention discloses a novel target for non-hormonal ovulation inhibition, ABCC4, which was found to be remarkably effective on its own and also in combination with inhibition of prostaglandin synthesis.
  • ABCC4 is expressed in the human ovary and serves as an efflux transporter of prostaglandin E2 (PGE2) in the ovarian follicle.
  • PGE2 prostaglandin E2
  • the inventors have found that ABCC4 is upregulated in the ovary immediately before ovulation.
  • inhibitors of ABCC4 effectively inhibit ovulation.
  • Effective inhibition of ovulation was also achieved when an ABCC4 inhibitor was combined with a PTGS inhibitor.
  • the combination showed a synergistic effect, where effective inhibition of ovulation was achieved using doses in which each inhibitor alone was ineffective or showed only a limited effect.
  • the combination advantageously allows using low doses of the inhibitors, for effective and safer ovulation inhibition.
  • compositions, methods and kits for inhibiting ovulation are useful as non- hormonal contraceptives.
  • Most contraceptive options for women are progesterone- and estrogen-based hormonal contraceptives, with the drawbacks associated with hormonal treatments.
  • Selective inhibition of ovulation is an important target for the development of new contraceptives. Ovulation inhibition without alteration of ovarian steroid hormone synthesis and without alteration of, or interference with, the menstrual cycle is particularly valuable. This approach minimizes or even eliminates side effects experienced by many women who use current hormonal contraceptives, but still provides protection against undesired pregnancy.
  • some women are prohibited from taking hormonal contraceptives due to increased risk in developing serious medical illnesses, such as heart attack, migraines and blood clots. Thus, providing non-hormonal effective and safe contraceptives for women is of great importance.
  • compositions, methods and kits for inhibiting ovulation are further useful, for example, for controlling ovulation during fertility treatments.
  • the present invention provides a method for inhibiting ovulation in a female subject, comprising administering to the female subject a pharmaceutical composition comprising at least one ATP Binding Cassette subfamily C member 4 (ABCC4) inhibitor.
  • ABCC4 ATP Binding Cassette subfamily C member 4
  • the method further comprises administering to the female subject at least one prostaglandin synthase (PTGS) inhibitor.
  • PGS prostaglandin synthase
  • the ABCC4 inhibitor is probenecid.
  • the PTGS inhibitor is selected from the group consisting of indomethacin, meloxicam, celecoxib, ibuprofen, azapropazone and rofecoxib. Each possibility represents a separate embodiment of the present invention.
  • the PTGS inhibitor is indomethacin. In additional particular embodiments, the PTGS inhibitor is meloxicam.
  • the ABCC4 inhibitor is probenecid and the PTGS inhibitor is indomethacin. In additional embodiments, the ABCC4 inhibitor is probenecid and the PTGS inhibitor is meloxicam.
  • the ABCC4 inhibitor and the PTGS inhibitor are administered in a single pharmaceutical composition. In other embodiments, the ABCC4 inhibitor and the PTGS inhibitor are administered each in a separate pharmaceutical composition. In some embodiments, the ABCC4 inhibitor and the PTGS inhibitor are administered simultaneously. In other embodiments, the ABCC4 inhibitor and the PTGS inhibitor are administered sequentially in either order.
  • the ABCC4 inhibitor and the PTGS inhibitor are administered orally.
  • the ABCC4 inhibitor alone or in combination with the PTGS inhibitor, is administered daily from one to six days prior the mid-cycle.
  • the ABCC4 inhibitor alone or in combination with the PTGS inhibitor, is administered daily during mid-cycle and at least one day thereafter.
  • the ABCC4 inhibitor, alone or in combination with the PTGS inhibitor are administered daily from day 9 of the menstrual cycle to day 14 of the menstrual cycle.
  • the method further comprises measuring LH levels in the blood or urine of the female subject.
  • the ABCC4 inhibitor alone or in combination with the PTGS inhibitor, is administered daily from the day that the level of LH surges until, and including, the day that the level of LH drops to baseline.
  • the method further comprises measuring estradiol levels in the blood or urine of the female subject.
  • the ABCC4 inhibitor alone or in combination with the PTGS inhibitor, is administered daily, at the day that the level of estradiol surges and at least four consecutive days thereafter.
  • the method further comprises measuring the levels of estradiol and LH in the blood or urine of the female subject, and administering the ABCC4 inhibitor, alone or in combination with the PTGS inhibitor, daily from the day that the level of estradiol surges and until the day that the level of LH drops to baseline.
  • the ABCC4 inhibitor alone or in combination with the PTGS inhibitor, is administered for emergency contraception.
  • the method comprises a single administration of the ABCC4 inhibitor, alone or in combination with the PTGS inhibitor.
  • the method comprises a plurality of administrations of the ABCC4 inhibitor, alone or in combination with the PTGS inhibitor, within several hours up to several days following the sexual intercourse.
  • the present invention provides a pharmaceutical composition effective in inhibiting ovulation in a female subject, the pharmaceutical composition comprising an ABCC4 inhibitor and a PTGS inhibitor, and further comprising pharmaceutically acceptable excipients or carriers.
  • the present invention provides a combined non- hormonal contraceptive comprising an ABCC4 inhibitor and a PTGS inhibitor in a single dosage form.
  • the contraceptive is an emergency contraceptive.
  • the present invention provides a pharmaceutical composition comprising at least one ABCC4 inhibitor, for use in inhibiting ovulation.
  • the pharmaceutical composition is for use together with a pharmaceutical composition comprising at least one PTGS inhibitor.
  • a pharmaceutical composition comprising an ABCC4 inhibitor and a pharmaceutical composition comprising a PTGS inhibitor, for combined use in inhibiting ovulation.
  • the present invention provides a kit for inhibiting ovulation in a female subject, the kit comprising: (i) a container containing a pharmaceutical composition comprising at least one ABCC4 inhibitor; and (ii) instructions for use of the pharmaceutical composition in inhibiting ovulation.
  • the kit further comprises a container containing a pharmaceutical composition comprising at least one PTGS inhibitor
  • the written instructions are written instructions for the combined use of the pharmaceutical composition comprising the at least one ABCC4 inhibitor and the pharmaceutical composition comprising the at least one PTGS inhibitor in inhibiting ovulation.
  • ABCC4 in cumulus granulosa cells in vivo.
  • A RNASeq expression (reads) of ABCC4 in compact cumulus granulosa cells (“CCGV”, surrounding immature germinal vesicle oocytes), and in expanded cumulus granulosa cells (“CCM2”, surrounding mature metaphase II oocytes).
  • B ABCC4 expression in vivo in cumulus granulosa cells versus mural granulosa cells aspirated from preovulatory follicles (>17 mm) during IVF procedures. ABCC4 was quantified by qPCR and normalized to b-actin expression. Data represent the mean ⁇ SEM of three independent experiments.
  • (C) In vitro expression of ABCC4 in mural granulosa cells. The cells were aspirated during IVF procedures and initially cultured for 4 days with daily medium replacement and then exposed to hCG (human chorionic gonadotropin) for the indicated time. ABCC4 was quantified by qPCR and normalized to b-actin expression. Data represent the mean ⁇ SEM of three independent experiments.
  • the image is a representative of three independent experiments. Protein levels of ABCC4 were analyzed by image studio lite (LI-COR Biosciences) and calculated relative to the b-actin level in the same sample. Results are expressed as mean ⁇ SEM of three independent experiments.
  • E Cultured mural granulosa cells were treated with forskolin (FSK, IOmM), phorbol 12-myristate 13-acetate (PMA, 20 nM) or hCG (10U), or pre-treated with H89 (PKA inhibitor) (IOmM) following by FSK treatment for 9 hours. ABCC4 was quantified by qPCR and normalized to b-actin expression. Data represent the mean ⁇ SEM of three independent experiments.
  • FIG. 1 The role of ABCC4 in the regulation of extra- and intra-cellular PGE2 levels in MGCs - ABCC4 inhibitors studies.
  • MGCs aspirated from preovulatory follicles (>17 mm) during IVF procedures were cultured for 3-4 days.
  • the cells were either stimulated with lU/ml hCG in the presence or absence of the ABCC4 inhibitors MK-571 (50mM) or probenecid (500mM) for 24 hours, or treated with each inhibitor without treatment of hCG, or remained untreated (control).
  • PGE2 levels in the culture media (A, B) and intracellular (C, D) were measured by PGE2 EIA kit.
  • Each sample included pooled cells from 2 to 4 women. The results are expressed as mean ⁇ SEM of four independent experiments.
  • FIG. 3 The role of ABCC4 in the regulation of extra- and intra-cellular PGE2 levels in MGCs - siRNA studies.
  • MGCs aspirated from preovulatory follicles (>17 mm) during IVF procedures were cultured for 2-3 days.
  • the cells were transfected with either ABCC4 siRNA or a scrambled (control) siRNA. 48 hours post transfection, the two groups of transfected cells were stimulated with lU/ml hCG, whereas two not-transfected groups were stimulated either with lU/ml hCG or none.
  • A The cells were subjected to total RNA extraction and ABCC4 mRNA expression was assessed by qPCR and calculated relative to the b-actin level in the same sample.
  • the results are expressed as mean ⁇ SEM of three independent experiments.
  • PGE2 levels in the culture media (B) and intracellular (C) were measured by PGE2 EIA kit. 1: 1 or 1:200 dilution was used for the extracellular or intracellular results respectively.
  • the culture media PGE2 concentration was calculated relative to total protein levels, measured by Bradford method.
  • the results are expressed as fold change with respect to hCG that was set to 1.
  • the results are expressed as mean ⁇ SEM of three to seven independent experiments. Each culture included pooled cells from 2 to 4 women.
  • FIG. 4 (A) ABCC4 mRNA expression pattern in mice ovaries during the preovulatory period. 25-day old female mice were injected with saline or 10U PMSG to stimulate follicle growth, and 48 hours later with an ovulatory dose of 10U hCG to induce ovulation. The mice were sacrificed 48 hours after PMSG and 9 hours after hCG treatment. The ovaries were collected and the total ovarian RNA were extracted. ABCC4 mRNA levels were assessed by qPCR and expressed relative to b-actin mRNA in each sample. The results are expressed as mean ⁇ SEM of four independent experiments. (B-J) Ovarian histology of probenecid-treated and control mice.
  • E Higher magnification of one postovulatory follicle forming an early corpus luteum.
  • F 16h after the administration of hCG + probenecid.
  • G Higher magnification of the time point 16h after the administration of hCG + probenecid.
  • H 40h after the administration of hCG.
  • I Higher magnification of postovulatory follicular cells 40h after the administration of hCG.
  • J 40h after the administration of hCG + probenecid.
  • Figure 5 The effect of ABCC4 inhibition by probenecid on genes that regulate ovulation and corpus luteum function. Immature 25-day-old female mice were superovulated with 10U PMSG for 48h, followed by injection of 10U hCG with or without the 400 mg/kg probenecid. Animals were sacrificed 3 hours after hCG administration. The ovaries were collected and the RNA were extracted. mRNA levels of each gene were assessed by qPCR and expressed relative to b-actin mRNA in each sample. The results are expressed as mean ⁇ SEM of five to seven independent experiments.
  • Figure 6 Inhibition of ovulation in vivo by a combination of probenecid and indomethacin or probenecid and meloxicam and the rescue effect of PGE2.
  • 25-day old C57BL/6 female mice were injected with 10U of PMSG and 48 h later (A) with an ovulatory dose of 10U hCG w/wo Probenecid (PB, 25 mg/kg) w/wo Indomethacin (Indo, 5mg/kg); (B) with an ovulatory dose of 10U hCG with Probenecid (PB, 25 mg/kg) and Meloxicam (ML, lOmg/kg) and w/wo PGE2 (2pg/kg). Mice were sacrificed 16 hours after the administration of hCG and the number of oocytes within the ampullas of both oviducts was recorded.
  • the present invention is directed to ovulation inhibition using inhibitors of ABCC4, alone or in combination with inhibitors of prostaglandin synthases (PTGS).
  • PTGS prostaglandin synthases
  • ABCC4 is an important mediator of ovulation.
  • the invention is based, in part, on the finding that inhibition of ABCC4, alone or in combination with inhibition of prostaglandin synthesis, results in remarkably effective inhibition of ovulation.
  • the combination exhibited a synergistic effect, where doses in which each agent alone was ineffective or showed only a minor effect, were able to completely inhibit ovulation when the inhibitors were combined together.
  • the combined inhibition of ABCC4 and prostaglandin synthesis using low doses of the inhibitors achieved over 90% inhibition of ovulation in a super-ovulation protocol.
  • the inhibition of ovulation as disclosed herein is temporary and there is no negative effect on future ovulation.
  • a further advantage conferred by the combination disclosed herein is that it allows using lower concentrations of each inhibitor, concentrations in which each inhibitor on its own is ineffective, thereby lowering or avoiding possible side effects.
  • ABCC4 a transmembrane prostaglandin carrier protein, is upregulated in preovulatory human granulosa cells (GCs);
  • hCG human chorionic gonadotropin
  • ERK extracellular signal-regulated kinase
  • compositions, methods and kits of the present invention may be used, for example:
  • non-hormonal contraceptives long term use
  • hormonal contraceptives such as women with breast cancer, women with thrombophilia (hypercoagulability), APLA (antiphospholipid antibodies) and the like.
  • a method for inhibiting ovulation in a female subject comprising administering to the female subject a pharmaceutical composition comprising an ABCC4 inhibitor, alone or in combination with a PTGS inhibitor.
  • a pharmaceutical composition comprising an ABCC4 inhibitor, for use in inhibiting ovulation.
  • the pharmaceutical composition is for use in combination with a pharmaceutical composition comprising a PTGS inhibitor.
  • At least one ABCC4 inhibitor optionally in combination with at least one PTGS inhibitor, for inhibiting ovulation in a female subject.
  • the use of at least one ABCC4 inhibitor for the preparation of a medicament for inhibiting ovulation in a female subject.
  • the medicament is for use in combination with at least one PTGS inhibitor.
  • a method of emergency contraception in a female subject comprising administering to the female subject a pharmaceutical composition comprising at least one ABCC4 inhibitor, for inhibiting ovulation and preventing pregnancy in said female subject.
  • the method further comprises administering a pharmaceutical composition comprising at least one PTGS inhibitor.
  • the method of emergency contraception comprises a single administration of the pharmaceutical composition comprising at least one ABCC4 inhibitor, optionally in combination with the pharmaceutical composition comprising at least one PTGS inhibitor.
  • the method of emergency contraception comprises more than one administration of the pharmaceutical composition comprising at least one ABCC4 inhibitor, optionally in combination with the pharmaceutical composition comprising at least one PTGS inhibitor.
  • the inhibitor(s) may be administered two or three times, with an interval of 6, 12, 18, or 24 hours, in order to inhibit ovulation and prevent pregnancy in said female subject.
  • the administration is performed within 120 hours after sexual intercourse.
  • the administration is performed within 96, 72, 48, 24, 12 hours or less after sexual intercourse.
  • a method for contraception in a female subject comprising administering to the female subject a pharmaceutical composition comprising at least one ABCC4 inhibitor, alone or in combination with at least one PTGS inhibitor.
  • a pharmaceutical composition for use as a contraceptive comprising at least one ABCC4 inhibitor, alone or in combination with at least one PTGS inhibitor.
  • the pharmaceutical composition is for use as an emergency contraceptive.
  • the ABCC4 inhibitor and the PTGS inhibitor are in separate pharmaceutical compositions. In other embodiments, the ABCC4 inhibitor and the PTGS inhibitor are in a single pharmaceutical composition.
  • a combination pharmaceutical composition effective in inhibiting ovulation in a female subject comprising at least one ABCC4 inhibitor and at least one PTGS inhibitor in a single dosage form.
  • a combined non-hormonal contraceptive comprising at least one ABCC4 inhibitor and at least one PTGS inhibitor in a single dosage form.
  • the contraceptive is an emergency contraceptive.
  • the non-hormonal contraceptive disclosed herein provides a fixed dose of each inhibitor, for combined effective inhibition of ovulation.
  • the female subject according to the present invention is a mammalian female subject, typically a human female subject.
  • the human female subject is a human female subject undergoing fertility treatment(s).
  • the compositions of the invention may be administered to non-human mammals, for example to livestock, to synchronize ovulation.
  • the pharmaceutical compositions according to the present invention are for use daily from one to six days prior the mid-cycle. In some embodiments, the pharmaceutical compositions according to the present invention are for use daily during mid-cycle and at least one day thereafter. In some embodiments, the pharmaceutical compositions according to the present invention are for use daily from day 9 of the menstrual cycle to day 14 of the menstrual cycle.
  • the pharmaceutical compositions according to the present invention are for use daily, from the day that the level of LH surges until, and including, the day that LH level drops to baseline. In some embodiments, the pharmaceutical compositions according to the present invention are for use daily, at the day that the level of estradiol surges and at the at least four consecutive days thereafter.
  • the pharmaceutical compositions according to the present invention are for use daily at the day that the level of estradiol surges and until the day that the level of LH drops to baseline.
  • ABCC4 ATP Binding Cassette subfamily C member 4
  • MRP4 multidrug resistance-associated protein 4
  • MOAT-B multi-specific organic anion transporter B
  • ABCC4 acts as a regulator of intracellular cyclic nucleotide levels and as a mediator of cAMP-dependent signal transduction to the nucleus.
  • ABCC4 also transports prostaglandins, for example PGE2, out of the cell where they can bind receptors.
  • PGE2 prostaglandins
  • the term“ABCC4 inhibitor” refers to a chemical agent/entity that is capable of inhibiting ABCC4 activity. This term encompasses pharmaceutically acceptable salts of the ABCC4 inhibiting chemical agent/entity.
  • the inhibitors are specific inhibitors of ABCC4.
  • the phrases“inhibiting ABCC4” and“inhibiting ABCC4 activity” are used interchangeably and comprise any one or more of the following: attenuating, reducing or preventing cellular processes, pathways or phenotypes associated with ABCC4. Each possibility represents a separate embodiment of the invention.
  • inhibiting ABCC4 comprises reducing, inhibiting, preventing or neutralizing the functionality of ABCC4.
  • inhibiting ABCC4 comprises reducing or inhibiting ABCC4 expression.
  • inhibiting ABCC4 comprises any one or more of inhibiting oocyte nuclear maturation, inhibiting cumulus expansion, inhibiting ovulation, inhibiting follicular rupture, and inhibiting (reducing or even preventing) prostaglandin efflux.
  • an inhibitor of ABCC4 according to the present invention is capable of inhibiting PGE2 efflux.
  • An exemplary method for determining inhibition of PGE2 efflux is detailed in the Examples section below.
  • the ABCC4 inhibitor is capable of inhibiting at least one of: oocyte nuclear maturation, cumulus expansion, ovulation and follicular rupture. Each possibility represents a separate embodiment of the invention.
  • the ABCC4 inhibitor is a small molecule.
  • the ABCC4 inhibitor is selected from the group consisting of probenecid, MK-571, Ceefourin 1 and Ceefourin 2. Each possibility represents a separate embodiment of the present invention.
  • Other ABCC4 inhibitors according to the following drug types may be used: the phosphodiesterase inhibitors sildenafd, zaprinast and trequinsin, the cardiovascular drugs verapamil, losartan, telmisartan and candesartan, and the flavonoids quercetin and silymarin.
  • the ABCC4 inhibitor is selected from the group consisting of sildenafd, zaprinast and trequinsin.
  • the ABCC4 inhibitor is selected from the group consisting of verapamil, losartan, telmisartan and candesartan. In some embodiments, the ABCC4 inhibitor is selected from the group consisting of quercetin and silymarin.
  • the ABCC4 inhibitor is probenecid.
  • Probenecid is identified by CAS Registry number 57-66-9. Probenecid is approved for human use as a medication that increases uric acid excretion in the urine. It is primarily used in treating gout and hyperuricemia.
  • Prostaglandin synthases (EC 1.14.99.1), also known as cyclooxygenases (COX), are enzymes responsible for formation of prostanoids, including prostaglandins, from arachidonic acid.
  • PTGS1 (COX1) is a constitutively expressed enzyme in many tissues
  • PTGS2 (COX2) is an inducible enzyme predominantly expressed at sites of inflammation.
  • PTGS convert free arachidonate first to prostaglandin G2 (PGG2) by a cyclooxygenase reaction, and then -in a second enzymatic step- to prostaglandin H2 (PGEh) by a hydroperoxidase reaction.
  • PGEh is then converted to the various prostaglandins (PGE2, PGD2, PGF2 a , etc.), prostacyclin, (PGI2) or thromboxane by additional synthetases that use PGEE as substrate.
  • PTGS are also known by the alternative names prostaglandin-endoperoxide synthases, PG synthetases, and prostaglandin G/H synthases. It is to be understood that the term "PTGS" as used herein is interchangeable with any alternative name or synonym of this protein known in the art including the aforementioned names.
  • PTGS inhibitor refers to a chemical agent/entity that is capable of inhibiting PTGS activity.
  • the term encompasses pharmaceutically acceptable salts of the PTGS inhibiting chemical agent/entity.
  • the inhibitors are specific inhibitors of PTGS.
  • PTGS inhibitors are also known as non-steroidal anti inflammatory drugs (NSAIDs).
  • NSAIDs non-steroidal anti inflammatory drugs
  • PTGS inhibitors encompass non-selective PTGS inhibitors (also termed non-specific PTGS inhibitors), which inhibit both isoforms of PTGS, and selective PTGS2 inhibitors (also termed specific PTGS2 inhibitors), which selectively inhibit PTGS2.
  • the PTGS inhibitor utilized by the compositions, methods and kits of the present invention is a non-selective PTGS inhibitor. In other embodiments, the PTGS inhibitor utilized by the compositions, methods and kits of the present invention is a selective PTGS2 inhibitor.
  • inhibiting PTGS comprises reducing, inhibiting, preventing or neutralizing the functionality of PTGS.
  • inhibiting PTGS comprises reducing or inhibiting PTGS expression.
  • inhibiting PTGS activity comprises inhibiting (namely, reducing or even preventing) prostaglandin synthesis.
  • each possibility represents a separate embodiment of the invention.
  • the PTGS inhibitor is a small molecule.
  • the PTGS inhibitor is selected from the group consisting of indomethacin, meloxicam, celecoxib ibuprofen, azapropazone and rofecoxib. Each possibility represents a separate embodiment of the present invention. Other/additional NSAIDs may also be used.
  • the PTGS inhibitor is indomethacin.
  • Indomethacin is identified by CAS registry number 53-86-1. Indomethacin is used to relieve pain, swelling, and joint stiffness caused by arthritis, gout, bursitis, and tendonitis. It is also used to relieve pain from various other conditions. Indomethacin is a non-selective PTGS inhibitor.
  • the PTGS inhibitor is celecoxib.
  • Celecoxib is identified by CAS Registry number 169590-42-5. It is used to treat the pain and inflammation of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, acute pain in adults, painful menstruation, and juvenile rheumatoid arthritis.
  • Celecoxib is a PTGS2 (COX2) selective inhibitor.
  • the PTGS inhibitor is meloxicam.
  • Meloxicam is identified by CAS Registry number 71125-38-7. It is an analgesic and anti-pyretic drug. Meloxicam is a PTGS2 (COX2) selective inhibitor.
  • the term“inhibiting ovulation” refers to, but is not limited to, any one or more of the following: abrogating, blocking, halting, attenuating or preventing the process, onset or event of ovulation. Each possibility represents a separate embodiment of the invention. In some embodiments, this term includes, and is not limited to, administration of an ABCC4 inhibitor, alone in combination with a PTGS inhibitor, to a female subject in order to prevent ovulation in said female subject.
  • menstrual cycle refers to the process responsible for the production of ova, and the preparation of the uterus for pregnancy.
  • menstrual cycle refers to the process responsible for the production of ova, and the preparation of the uterus for pregnancy.
  • the count of an individual menstrual cycle in days starts at the first day of menstrual bleeding. Stimulated by gradually increasing amounts of estrogen in the follicular phase, discharges of blood (menses) slow then stop, and the lining of the uterus thickens.
  • Follicles in the ovary begin developing under the influence of a complex interplay of hormones, and after several days one or occasionally two become dominant (non-dominant follicles atrophy and die).
  • the dominant follicle releases an ovum in an event called ovulation.
  • the ovum only lives for 24 hours or less without fertilization while the remains of the dominant follicle in the ovary become a corpus luteum; this body has a primary function of producing large amounts of progesterone.
  • progesterone the endometrium (uterine lining) changes to prepare for potential implantation of an embryo and establish pregnancy. If implantation does not occur within approximately two weeks, the corpus luteum will involute, causing sharp drops in levels of both progesterone and estrogen. The hormone drop causes the uterus to shed its lining and egg in a process termed menstruation.
  • hCG is an analogue of luteinizing hormone (LH) used to induce ovulation in the clinic and in animals. Ovulation typically occurs in the mid-cycle of women’s menstrual cycle and follows a surge of LH which is preceded by a gradual elevation of estradiol.
  • LH luteinizing hormone
  • the ABCC4 inhibitors of the invention are particularly useful when administered during or a few days prior to the mid-cycle.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to the mid-cycle. Each possibility represents a separate embodiment of the invention.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered during the middle of the menstrual cycle, also termed herein "mid cycle” or “mid-cycle period", which can be determined and followed, for example, by measuring the levels of estradiol, LH or both in the blood or urine of the female subject to be treated, as detailed below.
  • mid cycle or “mid-cycle period”
  • the mid-cycle period is typically between day 9 to day 14 of the mensural cycle.
  • administration of the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is synchronized with the levels of estradiol and LH.
  • administration of the ABCC4 inhibitor is synchronized with the level of E3G, the urinary metabolite of estradiol, and LH.
  • estradiol and “E3G” are interchangeable.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered as LH level surges. In some embodiments, the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered as LH levels surges and on the following day. In some embodiments, the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered daily, beginning when estradiol levels start to build up and continuing until the level of LH drops to threshold level. In some embodiments, the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered daily, for approximately six days, as follows: about two to three days prior to the surge of LH, 1-2 days during which the level of LH is high, following a day or two.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered following LH surge.
  • the methods of the invention further comprise a step of detecting LH, and, optionally, estradiol and/or E3G, with suitable detection methods or kits, for example kits available in the market, such as, ovulation kits.
  • the methods of the invention further comprise a step of detecting LH, and, optionally, E3G, in the urine of the female subject, prior to the administering step.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered daily from one to four days prior the mid-cycle. In some embodiments, the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered daily during mid-cycle and at least one day thereafter. In some embodiments, the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered daily from day 9 of the menstrual cycle to day 14 of the menstrual cycle.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor is administered once a month on a single occasion, every month, more precisely on a single occasion during each menstrual cycle. In some embodiments, the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is administered several times during the menstrual cycle, for example, on each day surrounding the, or prior to, and optionally following, the mid-cycle or the event of LH surge.
  • the subject in need thereof is a woman having a regular menstrual cycle, e.g. a 28, 29, 30, or 31-day cycle.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) may be administered daily between the 9th and the 14th days of the menstrual cycle.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) may be administered daily during the 10th and the 14th days, the 11th and the 14th days, the 12th and the 14th days, or the 13th and the 20 14th days of the menstrual cycle.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) may be administered on the 14th of the menstrual cycle.
  • the 1st day of the women menstrual cycle is the first day of bleeding.
  • compositions, methods and kits of the present invention are further useful for suppressing spontaneous ovulation during fertility treatments, including, but not limited to, in vitro fertilization (IVF).
  • IVF in vitro fertilization
  • the compositions, methods and kits may be applied in order to synchronize the menstrual cycle and/or as part of controlled ovarian hyperstimulation protocol utilized in fertility treatments.
  • Controlled ovarian hyperstimulation typically involves use of fertility agents (commonly analogues of follicle-stimulating hormone (FSH) and/or human Menopausal Gonadotrophins (hMG)) to induce ovulation by multiple ovarian follicles.
  • FSH follicle-stimulating hormone
  • hMG human Menopausal Gonadotrophins
  • These multiple follicles may be isolated by oocyte retrieval, e.g. for IVF, or be given time to ovulate, resulting in superovulation which is the ovulation of a larger-than-normal number of eggs, generally in the sense of at least two.
  • an assisted reproductive technique such as IVF
  • controlled ovarian hyperstimulation confers a need to suppress spontaneous ovulation.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) may be utilized for suppressing spontaneous ovulation.
  • ovulation suppression may be achieved by either Gonadotropin-releasing hormone (GnRH) agonist or by GnRH antagonist administration.
  • GnRH Gonadotropin-releasing hormone
  • Those treatments are hormonal based and may be associated with side effects.
  • the compositions, methods and kits of the present invention are useful for suppressing spontaneous ovulation in an assisted reproduction technique in a safer manner than the current GnRH based treatments.
  • a“pharmaceutical composition” refers to a preparation of one or more of the ABCC4 inhibitor(s) or PTGS inhibitor(s) described herein, with other components, such as pharmaceutically inactive components, including, pharmaceutically acceptable carriers and excipients. Commonly, the purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the pharmaceutical active compound, namely, the ABCC4 inhibitor or PTGS inhibitor.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. Each possibility represents a separate embodiment of the invention.
  • pharmaceutically acceptable refers to a carrier, an excipient or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the pharmaceutical active compound.
  • carrier refers to any substance suitable as a vehicle for delivering of the ABCC4 inhibitor or PTGS inhibitor of the present invention to a suitable biological site or tissue. As such, carriers can act as a pharmaceutically acceptable excipient of the pharmaceutical composition of the present invention.
  • the ABCC4 inhibitor and PTGS inhibitor may be administered by any convenient route, including oral, buccal, sublingual, parenteral (e.g., intramuscular, transdermal), vaginal (for example in the form of a gel, or a foam), gingival, nasal, rectal, etc.
  • parenteral e.g., intramuscular, transdermal
  • vaginal for example in the form of a gel, or a foam
  • gingival for example in the form of a gel, or a foam
  • nasal, rectal etc.
  • the ABCC4 inhibitor and PTGS inhibitor may be administered in any suitable dosage form, including but not limited to, pills, patches, vaginal rings, foam, gel, injections and the alike. Each possibility represents a separate embodiment of the invention.
  • the pharmaceutical composition comprising the at least one ABCC4 inhibitor or PTGS inhibitor is an oral dosage form. In some embodiments, the pharmaceutical composition is administered per os.
  • the effective dose also termed herein“therapeutically effective amount” of the at least one ABCC4 inhibitor and PTGS inhibitor may be determined by a person of skill in the art while taking into consideration various conditions of the subject, for example, general health conditions of the subject, age and weight.
  • the frequency of administration depends on the properties of the ABCC4 inhibitor and PTGS inhibitor and the conditions of the subject and may be, for example, a plurality of times a day (i.e. 2, 3,4, 5, or more times per day), or once a day. Each possibility represents a separate embodiment of the invention. In some embodiments, the administration is once daily.
  • kits for inhibiting ovulation in a female subject comprising: a container containing a pharmaceutical composition comprising at least one ABCC4 inhibitor; and written instructions for use of the pharmaceutical composition in inhibiting ovulation.
  • the kit further comprises a container containing a pharmaceutical composition comprising at least one PTGS inhibitor, and the written instructions are written instructions for the combined use of the pharmaceutical composition comprising the at least one ABCC4 inhibitor and the pharmaceutical composition comprising the at least one PTGS inhibitor in inhibiting ovulation.
  • the kit further comprises means for collecting urine from the female subject. In some embodiments, the kit further comprises means for measuring LH levels. In some embodiments, the kit further comprises means for measuring estradiol levels.
  • the kit is for inhibiting spontaneous ovulation during fertility treatments.
  • the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) is for administration in proximity with LH surge.
  • the kit further comprises means for detecting LH surge. Exemplary means may include antibodies capable of detecting LH in the urine of a subject.
  • the kit comprises one or more vessels for collecting a urine sample of the subject.
  • the kit further comprises means for measuring LH level.
  • the instructions for use indicate to use the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) in proximity with LH surge.
  • the instructions for use indicate to measure LH level daily and to use the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) in proximity with LH surge and at least one day after. In some embodiments, the instructions for use indicate to measure LH level daily, starting from day 5 of the menstrual cycle and up to the decrease of LH level to baseline, and to use the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) in proximity with LH surge and at least one day after.
  • the baseline may be the level of LH in any one or more of the days from day 1 through day 8 and from day 15 to day 28, of the menstrual cycle. In some embodiments, the baseline level of LH is the average of the daily level of LH in two or more days during day 1 through day 8 of the menstrual cycle and day 15 to day 28 of the menstrual cycle.
  • the kit further comprises means for detecting estradiol level.
  • the instructions for use indicate to use the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) upon an increase in estradiol level and during the 6 days following the increase.
  • the instructions for use indicate to measure estradiol and LH level daily, starting from day 5 of the menstrual cycle and up to the decrease of LH level to baseline, and to use the ABCC4 inhibitor (alone or in combination with the PTGS inhibitor) in proximity with an increase in estradiol level and at least 5 days thereafter.
  • Yerushalmi et al. (Mol Hum Reprod 20, 719-735 (2014), supra) characterized the human cumulus cell transcriptome during final follicular maturation and ovulation. They used global transcriptome sequencing to characterize the final stages of follicular maturation and ovulation pathways in humans. The aim of the study was to systematically identify ovulation-associated genes. Review of the resultant transcriptome database revealed ABCC4 to exhibit a 3 -fold increase in mature cumulus cells (CCs) compared to immature CCs.
  • CCs cumulus cells
  • GnRH Gonadotropin-Releasing Hormone
  • Normo-ovulatory young women ( ⁇ 37 years of age) undergoing IVF because of male factor infertility or pre -implantation genetic diagnosis were selected for this study.
  • Ovarian stimulation was carried out as previously described (Elizur et al., Reprod Biomed Online 10, 645-649 (2005); and Hourvitz et al., Reprod Biomed Online 13, 504-509 (2006)).
  • a "short antagonist" protocol was used wherein controlled ovarian hyperstimulation with Human Menopausal Gonadotropins (HMG; Menopur®) or recombinant Follicle-Stimulating Hormone (rFSH, either Gonal-F®; Merck Serono or Puregon Pen®; Schering Plough) was initiated 3 days after the onset of menses.
  • the initial gonadotropin dose used was dependent upon age, body mass index, and previous IVF treatment history.
  • Ovarian suppression with a GnRH antagonist (0.25 mg/day, Cetrorelix, Cetrotide®; Serono International, SR) was initiated when the leading follicle was more than 12 mm in diameter.
  • hCG human Chorionic Gonadotropin
  • CGC Cumulus granulosa cell
  • CGCs of each oocyte were removed with the use of hyaluronidase (SAGE, Trumbull, CT, USA) and a glass denudation pipette (Swemed, Billdal, Sweden).
  • SAGE hyaluronidase
  • PBS Phosphate- Buffered Saline
  • Follicular fluid was aspirated from follicles > 17 mm in each subject.
  • the follicular fluid was centrifuged and the pelleted MGCs were re-suspended in PBS (Sigma-Aldrich- Aldrich, St Louis, MO, USA). After allowing the cells to settle by gravity for a few minutes, the top portion of the medium was aspirated and the cells were repeatedly re suspended until the medium proved clear. The cells were then centrifuged at 1000 rpm for 5 minutes at room temperature and the resulting pellets were stored at -80°C until RNA isolation. Total MGCs from 3 different subjects were pooled to generate a single replicate. Each experiment was performed at least three times.
  • MGCs were collected from aspirated follicular fluid of follicles size > 17 mm from one subject (unless specified otherwise) and re-suspended in PBS (Sigma-Aldrich-Aldrich, St Louis, MO, USA). After allowing the cells to settle by gravity for a few minutes, the top portion of the medium was aspirated and the cells were repeatedly re-suspended until the medium proved clear and then placed on a Percoll ® gradient and centrifuged at 3000 RPM for 15 min. The MGCs were collected and washed with PBS, counted and plated in 24-well plates at a density of 100,000 cells/well, and incubated at 37°C in a humidified atmosphere with 5% CO2 in air.
  • PBS Sigma-Aldrich-Aldrich, St Louis, MO, USA
  • MGCs were cultured as above and then incubated with hCG or Forskolin (FSK) or Phorbol-12- Myristate- 13 -Acetate (PMA) in the presence or absence of the protein kinase A inhibitor H89 (all from LC Laboratories, Woburn, MA, USA) for an additional 9 h. Each experiment was performed at least three times.
  • the real-time PCR mix contained lpl of cDNA, fast SYBR Green Master Mix (Applied Biosystems), and specific primers for ABCC4 or other gene of interest and b-actin (housekeeping gene) in a total volume of 10m1. Cycling parameters were: 1 cycle at 95 °C for 20 seconds, and 40 cycles each at 95 °C for 3 seconds and at 60°C for 30 seconds. A melting curve analysis was performed at the end of each run to ensure measurements were based on the amplification of the target gene. All samples were run in duplicate. Analysis of the qPCR results was carried out with StepOne software. Relative gene expression was calculated using the delta-delta Ct method. Details of the primers used are shown in Table A below.
  • RNA sequencing (RNAseq)
  • CCGV cells compact cumulus granulosa cells
  • CCMII or CCM2 cells expanded cumulus granulosa cells from mature metaphase II (Mil) oocytes.
  • the CCGV cells were obtained from two women during in vitro maturation (IVM).
  • IVM in vitro maturation
  • the CCM2 cells were obtained from three women during IVF. See Yerushalmi et al. , Mol Hum Reprod 20, 719-735 (2014).
  • a cDNA library was prepared according to Illumina recommendations (preparing samples for mRNA sequencing; Illumina). Cluster generation and single-end sequencing was carried out using the standard Illumina procedures for the HiSeq 2000 sequencer (Illumina). All sequenced reads were mapped and aligned to the human genome. The number of reads that overlap each of the annotated genes was counted and the differentially expressed transcripts were identified (see Yerushalmi et al., 2014, supra).
  • IVM cycles were carried out as previously described (Fadini et al., Reprod Biomed Online 19, 343-351 (2009)). Briefly, sonographic assessment of the antral follicle count and of endometrial thickness was carried out on day 3 of a spontaneous menstrual cycle. The serum concentrations of estradiol and progesterone were also determined. Treatment with 150 IU/day rFSH for 3 days followed suit. After a second sonographic assessment on Day 6, 10,000 IU hCG (Pregnyl; Organon) was administered when the endometrial thickness was >5 mm and the leading follicle was >12 mm. Oocyte retrieval was carried out 36 hours later.
  • hCG Pregnyl; Organon
  • mice were purchased from Envigo Inc. Huntingdon, United Kingdom). The mice were maintained under controlled lighting (12 hour light/ 12 hour dark) conditions with continuous access to food and water.
  • mice 25-day-old female mice were initially treated with 10 U of Pregnant Mare Serum Gonadotropin (PMSG, Chronogest, Intervet, Israel) to stimulate follicular growth.
  • PMSG Pregnant Mare Serum Gonadotropin
  • An ovulatory dose of hCG (10 U) (Ovitrelle®, Merck Serono, Darmstadt, Germany) was administered 48 hours later.
  • probenecid at different concentrations (200-450 mg/kg), was administrated intra-peritoneally in the same time as hCG.
  • mice were sacrificed 48 hours after the initiation of PMSG treatment as well as 9 or 16, hours after hCG administration. All mice were sacrificed by CO2 asphyxiation, and the ovaries were removed and either flash frozen in liquid nitrogen, paraformaldehyde -fixed, or punctured in order to collect entrapped oocytes. Blood samples were collected at the time of euthanasia for progesterone measurement and the number of oocytes within the ampullas of each oviduct was recorded. Western blot
  • Cells were harvested using 0.5 mL PBS and pelleted. Cell pellets were lysed in TNE buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 2 mM EDTA, 1% NP-40, Sigma Aldrich St Louis, MO, USA) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), vortexed, and incubated for 10 min on ice before removal of nuclei and debris by centrifugation. Aliquots of the clarified supernatants were used to determine protein concentration using the Bradford method (Protein Assay Dye Reagent, Bio-Rad, Hercules, CA, USA).
  • Equal amounts (50 pg) of protein were loaded and separated by SDS -polyacrylamide gel electrophoresis (10% acrylamide). Proteins were then transferred onto nitrocellulose membranes. Membranes were blocked in 5% bovine serum albumin (BSA) in TBST (100 mL TBS 10X, 900 mL H 2 0, 1 mL Tween 20, Sigma Aldrich St Louis, MO, USA) for one hour and afterwards incubated with a primary antibody against ABCC4 (M4I-10, Abeam, 1 :500) or b-actin (housekeeping gene) overnight at 4°C.
  • BSA bovine serum albumin
  • the membranes were then treated with a goat anti-rat IgG HRP -conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and developed using an enhanced chemiluminescence kit (Thermo Scientific, Waltham, MA, USA).
  • MGCs were plated in 24-well plates at a density of 200,000 cells/well and cultured as described above for 6 days. The MGCs were then treated for 24 hours with hCG (1U) in the absence or presence of MK-571 (50 mM) or probenecid (500 pM). The concentration of PGEi in conditioned media was assessed using an enzyme immunoassay kit (EIA) (Cayman Chemical).
  • EIA enzyme immunoassay kit
  • MGCs were plated in 6-well plates at a density of 250,000 cells/well and cultured as described above for 2-3 days.
  • the transfection mixture included: (A) 9 pi Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) in 150 pi OptiMEM (Gibco by Life Technologies, Paisley, UK), and (B) 8 pi ABCC4 siRNA (sc-40750 MRP4 siRNA (h) - a pool of 3 target-specific 20-25 nt siRNAs designed to knock down gene expression) or Scramble siRNA(sc -36869 - control siRNA (Fluorescein Conjugate)-A, a scrambled nonspecific 19-25 nt siRNA designed to measure transfection efficiency) (Santa Cruz Biotechnology, Dallas, TX, USA) in 150 m ⁇ OptiMEM medium (10 mM final concentration).
  • the transfection mixture was incubated at room temperature for 5 minutes before added to the cells (300 m ⁇ ) with additional 300 m ⁇ of OptiMEM medium.
  • the cells without transfection reagent were covered with 600 m ⁇ OptiMEM.
  • an additional 1.4ml growth medium (DMED/F12 with 10% FCS media) was added to each well. The medium was changed 24 h later. All the groups, except for control, were cultured for 48 h post transfection and then stimulated with hCG for 24 h. The cells were then harvested and the medium was collected from one duplicate of the group for protein quantification using the Bradford assay.
  • Extracellular PGE2 levels were assessed using a PGE2 Enzyme Immunoassay (EIA) A kit. PGE2 levels were analyzed relative to protein levels. The cells in the other duplicate were subjected to RNA lysis buffer, diluted 1:200 for intracellular PGE2 levels and assessed by PGE2 EIA kit and to qPCR to determine ABCC4 mRNA expression levels.
  • EIA Enzyme Immunoassay
  • mice were removed immediately after the mice were sacrificed, dissected from the surrounding fallopian tubes and fat tissue, and flash frozen in liquid nitrogen. Frozen ovaries were crushed by mortar and pestle and the RNA was purified using the Micro RNA Isolation I kit (Zymo Research, CA, USA) according to the manufacturer’s instructions.
  • granulosa cells produce two distinct lineages: mural granulosa cells (MGCs) that line the follicular wall and cumulus granulosa cells (CGCs) that surround the oocyte.
  • CGCs include compact CGCs, that surround immature germinal vesicle (GV) oocytes, and expanded CGCs, that surround mature metaphase II (Mil) oocytes.
  • GV germinal vesicle
  • Mo metaphase II
  • the expression of ABCC4 was measured in each of these cell types.
  • Illumina-based RNA-sequencing (RNAseq) was carried out on RNA extracted from compact CGCs, designated as CCGV cells, and expanded CGCs, designated as CCM2 or CCM2 cells.
  • MGCs and CGCs were obtained from large preovulatory follicles (>17mm) during the IVF procedure.
  • MGCs expressed significantly higher (2.6-fold) levels of ABCC4 mRNA relative to CGCs (p 0.0184).
  • LHCGR leukinizing hormone/choriogonadotropin receptor
  • the LHCGR activates multiple signaling pathways, including protein kinase A (PKA) and protein kinase C (PKC).
  • PKA protein kinase A
  • PKC protein kinase C
  • the intracellular levels of PGE2 following siRNA and hCG treatment did not differ significantly from the hCG-treated controls ( Figure 3C).
  • the percentage of mice that ovulated and the number of oocytes ovulated was decreased in probenecid- treated mice in a dose-dependent manner, with a significant decrease from the concentration of 350 mg/kg probenecid.
  • Concurrent injection of hCG and 350 or 400 mg/kg of probenecid prevented ovulation in about 72% to 76% of the mice, resulting in a mean ovulatory quota of 9.4 and 4.7 oocytes per ovulating mouse, respectively.
  • mice receiving hCG plus probenecid contained numerous unruptured preovulatory follicles replete with entrapped oocytes, resembling ovaries obtained from mice treated with PMSG alone.
  • ovaries from hCG-treated mice displayed many corpora lutea composed of luteinized GCs containing lipid droplets in their cytoplasm ( Figures 4H, 41).
  • ovaries collected from hCG and probenecid-treated mice still contained entrapped oocytes in preovulatory follicles (Figure 4J).
  • Oocytes were collected from control mice (treated with hCG and PMSG) as well as from mice treated with different doses of probenecid (300, 400 and 450 mg/kg), which were co-injected with hCG.
  • the ovaries were removed 16 hr after hCG injection.
  • the results show that oocytes from the hCG-treated group underwent germinal vesicle breakdown (GVBD) and progressed to the metaphase II stage of meiosis.
  • GVBD germinal vesicle breakdown
  • the number of oocytes is represented as the percentage of oocytes in the subgroup relative to the total group.
  • the number of oocytes is represented as the percentage of oocytes in the subgroup relative to the total group.
  • ABCC4 Inhibition of ABCC4 affects the levels of mRNA encoded by genes that regulate ovulation and corpus luteum function.
  • the genes in question included those involved in oocyte maturation (Areg, Ereg), progesterone receptor signaling (Pgr), cumulus expansion ( Tnfaip6 ), follicular rupture (Adamtsl, Ctsl), gonadotropin signaling ( Fshr , Lhcgr), and steroidogenesis (Star, Cypllal, and p450Scc (cholesterol side cleavage chain)).
  • the regulation of genes whose products encode proteins involved in PGE2 synthesis and signaling were also investigated (Figure 5B).
  • ABCC4 expression during the ovulatory interval was tested in vitro as well as in vivo.
  • the in vitro model comprised primary MGCs that were incubated for 4 days without treatment followed by hCG stimulation.
  • the stimulation of cultured MGCs with hCG caused a significant increase in ABCC4 mRNA and protein expression, peaking between 9 and 12 hours after hCG stimulation.
  • the mRNA levels of ABCC4 did not change in the ovaries of mice undergoing a superovulation protocol. This may be due to a dilutional effect of multiple cell types, because they included the whole ovary and not isolated GCs.
  • the LHCG receptor activates multiple signaling pathways, including those involved in the activation of c AMP-dependent protein kinase A (PKA) and protein kinase C (PKC).
  • PKA c AMP-dependent protein kinase A
  • PKA protein kinase C
  • Ptgs2 PGE2 synthesis
  • Pgt PGE2 influx
  • Hpgd PGE2 metabolism
  • Example 2 Inhibition of ovulation in vivo by a combination of the ABCC4 inhibitor probenecid and the PG synthesis inhibitors indomethacin and meloxicam
  • mice Twenty five-day old female mice (C57BL/6 mouse strain, Envigo inc.) were injected with 10U of Pregnant mare's serum gonadotropin (PMSG, Sigma, St. Louis, MO, USA) to stimulate follicle growth. An ovulatory dose of 10U hCG was administered 48 h later to induce ovulation.
  • probenecid 25 mg/kg
  • meloxicam 10 mg/kg
  • indomethacin 5 mg/kg
  • mice were sacrificed 16 hours after the administration of hCG and the number of oocytes within the ampullas of both oviducts was recorded.
  • each inhibitor alone had no significant effect or only a limited effect, while the combination resulted in almost complete blockade of ovulation.

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Abstract

L'invention concerne des compositions, des procédés et des kits pour inhiber l'ovulation chez un sujet féminin, qui utilisent des inhibiteurs du transporteur ABCC4 de la prostaglandine E2, seuls ou en combinaison avec des inhibiteurs de la synthèse de la prostaglandine.
PCT/IL2019/051429 2018-12-31 2019-12-30 Compositions et procédés d'inhibition de l'ovulation WO2020141509A1 (fr)

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Non-Patent Citations (3)

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