WO2010126528A1 - Compositions et procédés pour réduire la probabilité d'un échec d'implantation ou d'une fausse couche chez des receveuses d'une insémination artificielle - Google Patents

Compositions et procédés pour réduire la probabilité d'un échec d'implantation ou d'une fausse couche chez des receveuses d'une insémination artificielle Download PDF

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Publication number
WO2010126528A1
WO2010126528A1 PCT/US2009/042481 US2009042481W WO2010126528A1 WO 2010126528 A1 WO2010126528 A1 WO 2010126528A1 US 2009042481 W US2009042481 W US 2009042481W WO 2010126528 A1 WO2010126528 A1 WO 2010126528A1
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csf
administered
day
mcg
insemination
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PCT/US2009/042481
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WO2010126528A9 (fr
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Darry L. Carter
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Nora Therapeutics, Inc.
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Priority to PCT/US2009/042481 priority Critical patent/WO2010126528A1/fr
Publication of WO2010126528A1 publication Critical patent/WO2010126528A1/fr
Publication of WO2010126528A9 publication Critical patent/WO2010126528A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention generally relates to methods of preventing implantation failure or miscarriage during assisted reproduction and, in particular, to methods for reducing the likelihood of or preventing implantation failure or miscarriage in recipients of intravaginal insemination, intracervical insemination, intratubal insemination, and intrauterine insemination.
  • assisted reproductive techniques such as artificial insemination, in vitro fertilization (IVF) and the like.
  • Artificial insemination (AI) is the process by which sperm is placed into the reproductive tract of a female for the purpose of impregnating the female by using means other than sexual intercourse. In humans, it is used as assisted reproductive technology, primarily to treat infertility but is also increasingly used to enable women without a male partner to produce children by using sperm provided by a sperm donor.
  • artificial insemination freshly ejaculated sperm, or sperm which has been frozen and thawed, is placed in the cervix (intracervical insemination or ICI), in the female's uterus (intrauterine insemination or IUI), in the vagina (intravaginal insemination or IVI), or in the fallopian tubes (intratubal insemination or ITI) by artificial means.
  • rate of artificial insemination may be as high as 15 percent each cycle. Although success rates are higher for other forms of fertility treatments, artificial insemination is often one of the first methods used. It is less invasive and less expensive than more complex procedures, such as in vitro fertilization.
  • pregnancy rates following six cycles of AI or one cycle of IVF can be as high as 60%, some recipients fail repeatedly.
  • Various uterine pathologies, such as thin endometrium, altered expression of adhesive molecules and immunological factors, may be the causes for repeated failure.
  • Spontaneous abortion occurs in 15% - 50% of diagnosed pregnancies in women between fifteen and forty-five years of age.
  • the formal definition of recurrent spontaneous abortion is three or more spontaneous abortions.
  • the American College of Obstetrics and Gynecology recommends that in women over the age of 35, a thorough workup should be undertaken after two spontaneous abortions.
  • Approximately 3- 4% of women are estimated to fit the formal definition of recurrent spontaneous abortion.
  • the risk of pregnancy loss increases from 15-20% in the first pregnancy to 40% after one spontaneous abortion.
  • Threatened abortion generally presents as cramping and bleeding for which treatment is bed rest. This conservative treatment provides palliative care for the mother but does little to alter the outcome.
  • the use of hormones is generally contraindicated due to the risk of congenital anomalies, including malformation of the vessels of the heart of the embryo and possible genital abnormalities in female offspring.
  • Preeclampsia and other hypertensive disorders of pregnancy are a leading global cause of maternal and infant illness and death.
  • Symptoms of preeclampsia include hypertension, edema and proteinuria with sudden weight gain, headaches and changes in vision.
  • Preeclampsia can prevent the placenta from getting enough blood which can cause low birth weight and other problems for the baby.
  • most women with preeclampsia still deliver healthy babies, some develop eclampsia, a serious condition that threatens the life of the mother and the fetus.
  • preeclampsia The risk of preeclampsia is higher in women carrying multiple babies, in teenage mothers and in women older than age 40. Typically, preeclampsia occurs in the late 2nd or 3rd trimesters (middle to late pregnancy) though occasionally it occurs earlier. Preeclampsia affects about 5% of all pregnancies. monitoring of blood pressure. Progression of the disorder is treated with fluids, antihypertensives and magnesium sulfate but delivery of the fetus provides the only remedy.
  • One objective of the present invention is to provide methods, compositions and kits comprising a granulocyte colony stimulating factor (G-CSF) in an amount effective to prevent miscarriage or implantation failure in an artificial insemination procedure.
  • G-CSF granulocyte colony stimulating factor
  • One aspect of the present invention relates to a method for preventing or reducing the likelihood of implantation failure or miscarriage in a recipient of artificial insemination.
  • the method comprises administering to the recipient of artificial insemination an effective amount of a composition comprising G-CSF. intravaginal insemination, intracervical insemination, or intratubal insemination.
  • composition is administered parenterally, administered enterally, or topically.
  • composition is administered by inhalation.
  • composition is administered prior to the artificial insemination.
  • the insemination is preceded by a controlled ovarian hyper stimulation procedure, and the composition is administered before, during, or after the time of controlled ovarian hyperstimulation.
  • composition is administered daily for one to thirty-five consecutive days.
  • composition is administered daily until the end of the first trimester.
  • composition is administered daily until the recipient presents a normal ThI response or a normal Th2 response or both.
  • the G-CSF is administered at a dose of between 0.1 mcg/kg/day to 600 mcg/kg/day.
  • the G-CSF is administered at a dose of between 0.5 mcg/kg/day to 300mcg/kg/day.
  • the G-CSF is administered at a dose of between 1 mcg/kg/day to 100 mcg/kg/day.
  • the G-CSF is administered at a dose of between 1 mcg/kg/day to 50 mcg/kg/day. mcg/kg/day to 10 mcg/kg/day.
  • the G-CSF is administered at a dose of between 1 mcg/kg/day to 2 mcg/kg/day.
  • the G-CSF is administered in the form of a nucleotide sequence encoding G-CSF.
  • the composition further comprises an additive selected from the group consisting of cytokines that suppress ThI immune response, cytokines that enhance Th2 immune response, cytokines that support successful pregnancy through non-immunologic mechanisms, anti-inflammatory agents, and inhibitors of proinflammatory cytokines.
  • the additive is selected from the group consisting of interferon alpha, interferon beta, macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), interleukin-1 (IL-I), IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-13, IL- 14, IL-15, IL-19, IL-20, IL-21, IL-22, IL-24, IL-25, IL-26, IL- 27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, and IL-35.
  • M-CSF macrophage colony stimulating factor
  • GM-CSF granulocyte macrophage colony stimulating factor
  • LIF leukemia inhibitory factor
  • TGF-beta transforming growth factor beta
  • composition further comprises a pharmaceutically acceptable carrier.
  • kits for preventing or reducing the likelihood of implantation failure and miscarriage in a recipient of artificial insemination includes an effective amount of G-CSF; and a label with instructions for using the G-CSF to prevent or reduce the likelihood of implantation failure and miscarriage.
  • spontaneous abortion by administering an effective amount of G-CSF to the subject.
  • the methods can be administered to any female subject at risk for spontaneous abortion. Subjects at risk can be identified according to the methods described herein or according to methods known to practitioners in the art.
  • the subject is in the first or second trimester of pregnancy. In certain embodiments, the subject is in the first 20 weeks of pregnancy. In certain embodiments, the subject is in the first or second months of pregnancy. In certain embodiments, the methods will be administered before pregnancy is achieved.
  • Another aspect of the present invention provides methods of treating or preventing preeclampsia and preterm labor by administering to a subject in need thereof an effective amount of GCSF or an effective amount of mobilized peripheral blood stem cells.
  • the methods can be administered to any female subject at risk for preeclampsia or preterm labor.
  • Subjects at risk can be identified according to the methods described herein or according to methods known to practitioners in the art. Typically, the subject is in the second or third trimester of pregnancy.
  • the following terms shall have the following meanings: of alleviating or abrogating a disorder and/or its attendant symptoms.
  • the terms “prevent”, “preventing” or “prevention”, as used herein, refer to a method of barring a subject from acquiring a disorder and/or its attendant symptoms. In certain embodiments, the terms “prevent”, “preventing” or “prevention” refer to a method of reducing the risk of acquiring a disorder and/or its attendant symptoms.
  • the term "artificial insemination” refers to an assisted reproduction procedure where a donor's sperm is deposited into the vagina (intravaginal insemination), cervical canal (intracervical insemination), uterine cavity (intrauterine insemination), or fallopian tubes (intratubal insemination) of the recipient.
  • Intrauterine insemination is a subset of artificial insemination in which sperm that have been washed of seminal fluid are placed directly into the uterus to bypass the vagina and cervix. Artificial insemination can be performed without any fertility drugs on the day of ovulation or can be performed using fertility drugs to stimulate superovulation (i.e., release of more than one oocyte per cycle).
  • spontaneous abortion refers to delivery or loss of the product of conception before the 20th week of pregnancy.
  • spontaneous abortion includes but is not limited to miscarriage, threatened abortion, inevitable spontaneous abortion, incomplete spontaneous abortion, habitual or recurrent spontaneous abortion or missed abortion.
  • the term "threatened spontaneous abortion” refers to any bleeding or cramping of the uterus in the first 20 weeks of pregnancy.
  • missed abortion refers to delay in expulsion of a dead fetus.
  • assisted reproduction refers to clinical and laboratory techniques used to enhance fertility in humans and animals, including, but not limited to, in vitro fertilization, artificial insemination and the like.
  • in vitro fertilization refers to the procedure involving ovarian hyperstimulation (optionally), oocyte retrieval from the mother-to-be or a donor, fertilization outside the subject's body, embryo culture and embryo transfer.
  • embryo transfer refers to the procedure involving transfer to a subject's uterus, of the developing or cleaving embryos or pre-embryos, also termed preimplantation embryos.
  • implantation failure refers to the failure of an embryo produced by assisted reproduction or through artificial insemination to implant or to implant normally in the uterus of a recipient subject.
  • the tenia "preeclampsia” refers the development of hypertension with albuminuria or edema typically between the 20th week of pregnancy and the end of the first week postpartum. Any pregnant subject who develops a blood pressure of 140/90 mm Hg or higher, edema of the face or hands, albuminuria of >1+ or whose blood pressure rises by 30 mm Hg systolic or 15 mm Hg diastolic (even if less than 140/90 mm Hg) is considered preeclamptic. promotes and contributes to the maturity of cells, such as, hematopoietic and blood cells. Examples of CSF molecules include, but are not limited to, erythropoietin, G-CSF, GM-CSF, macrophage CSF, interleukin (IL)-3, IL-6 and stem cell factor.
  • IL interleukin
  • G-CSF granulocyte-colony stimulating factor
  • G-CSF granulocyte-colony stimulating factor
  • G-CSF includes derivatives, mimetics, variants and chemically modified compounds or hybrids thereof as described in U.S. Patent Nos. 5,399,345; 5,416,195; 5,981,551; 6,166,183 and 6,261,550, the contents of which are incorporated by reference in entireties.
  • G-CSF is commercially available under the names Neupogen® (Amgen), Tevagrastim® (Teva), Biograstim® (CT Arzneistoff), Ratiograstim® (Ratiopharm GmbH), Zarzio® (Sandoz GmbH), Filgrastim Hexal® (Hexal AG), Neulasta® (Amgen) , Granocyte® and Neutrogin® (Chugai), and Neu-up® (Kyowa Hakko).
  • granulocyte refers to a white blood cell containing granules, especially a leukocyte (white blood cell or corpuscle) containing neutrophil, basophil or eosinophil granules in its cytoplasm.
  • GM-CSF granulocyte/macrophage colony stimulating factor
  • GM-CSF granulocyte/macrophage colony stimulating factor
  • GM-CSF includes derivatives, mimetics, variants and chemically modified compounds or hybrids thereof as described in, for example, U.S. Patent Nos. 5,895,646; 5,891,429 and 5,908,763; the contents of which are incorporated by reference in entireties.
  • GM-CSF is commercially available under the trade names Leukine®, Berlex® and Leucomax® (Wyeth).
  • CSF-I refers to compounds or factors that stimulate proliferation or promote survival of monocytes and macrophages in an animal, including a human subject.
  • M-CSF includes derivatives, mimetics, variants and chemically modified compounds or hybrids thereof as described in, for example, U.S. Patent Nos. 5,837,230 and 5,888,495; the contents of which are incorporated by reference in entireties.
  • M-CSF is commercially available under the trade name Leukoprol® (Kyowa).
  • macrophage relates to a mononuclear, phagocytic white blood cell that can exit the circulation and enter tissue spaces.
  • terapéuticaally effective amount refers to that amount of an active agent being administered sufficient to prevent development of or alleviate to some extent one or more of the symptoms of the condition or disorder being treated.
  • premature labor also known as premature labor, refers to the beginning of regular contractions that cause the cervix to begin dilation and effacement before the 37th week of pregnancy.
  • an effective amount refers to that amount of an active agent being administered sufficient to prevent the disorder or prevent one or more symptoms of the disorder being treated. In certain embodiments, the term “effective amount” refers to that amount of an active agent being administered sufficient to reduce the risk of the disorder or one or more symptoms of the disorder.
  • subject refers to animals such as mammals, including, but not limited to, primates (such as humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like.
  • subject is a human female. immediate container of an article, for example the written material displayed on a vial containing a pharmaceutically active agent.
  • labeling refers to all labels and other written, printed or graphic matter on any article or any of its containers or wrappers or accompanying such article, for example, a package insert or instructional videotapes or computer data storage devices, such as CDs and DVDs, accompanying or associated with a container of a pharmaceutically active agent.
  • the present invention is directed to methods of preventing spontaneous abortion and implantation failure, and methods of treating or preventing preeclampsia and preterm labor described in detail below.
  • the present invention provides methods of preventing or reducing the likelihood of embryo implantation failure and miscarriage in recipients of artificial insemination and particularly in recipients of intrauterine insemination during assisted reproduction by administrating to a subject in need thereof an effective amount of G- CSF.
  • AI Artificial insemination
  • IUI Intrauterine insemination
  • IUI can be performed without any fertility drugs on the day of ovulation or can be performed using fertility drugs to stimulate superovulation (i.e., release of more than one oocyte per cycle).
  • ovarian stimulation (COH) provides better pregnancy rates.
  • COH is most often achieved using clomiphene citrate and human chorionic gonadotropin (HCG) to trigger superovulation. Insemination is performed within 2 days of HCG administration.
  • HCG human chorionic gonadotropin
  • COH can be achieved using purified or recombinant follicle stimulation hormone with or without recombinant or purified luteinizing hormone. Again, this is followed by administration of HCG to trigger superovulation.
  • the recipient In a typical AI procedure, the recipient usually is stimulated with medication to stimulate multiple egg development and the insemination is timed to coincide with ovulat ⁇ on-release of the eggs from the follicles.
  • sperm processing On the day of the procedure, a semen specimen is harvested and "washed” in the laboratory (called sperm processing or sperm washing). By this process, the sperm is separated from the other components of the semen and concentrated in a much smaller volume.
  • sperm processing takes about 20-60 minutes, depending on the technique utilized.
  • a speculum is placed in the vagina and the cervical area is gently cleaned. Then the separated and washed specimen consisting of a purified fraction of highly motile sperm is placed either in the vagina (intravaginal insemination, IVI), cervix (intracervical insemination, ICI), higher in to the uterine cavity (intrauterine insemination, IUI), or fallopian tubes (intrafallopian insemination, IFI) using a sterile, thin and soft catheter, Intrauterine insemination has a better success rate than intracervical insemination. Therefore, it is the preferred method at some fertility specialist centers and some general obstetrical practices.
  • ThI T-helper 1
  • Th2 T-helper 2
  • the G-CSF is typically administered before the insemination procedure. The administration is continued until implantation of the embryo to the uterine wall is achieved, until the risk of failed implantation is reduced or eliminated, or according to the judgment of a practitioner of skill in the ait.
  • the administration is continued until pregnancy is confirmed.
  • G-CSF is administered daily for 1-35 consecutive days.
  • G-CSF is administered daily until the end of first trimester.
  • the administration is started before the time of controlled ovarian hyperstimulation or about the time of controlled ovarian hyperstimulation, and continued daily until about 3 days, about 5 days, about 7 days, about 10 days, about 12 days, about 14 days, about 30 days, about 45 days, or about 60 days after insemination into the subject's vagina, cervix, uterus, or fallopian tubes.
  • the administration is started about 14 days, 7 days, 5 days, 3 days, and 1 day before insemination into the subject's started about the time of controlled ovarian hyperstimulation and continued daily until about the end of the first trimester.
  • the dose is administered for five consecutive days about the time of insemination and continued daily until about 3 days, about 5 days, about 7 days, about 10 days, about 12 days, about 14 days, about 30 days, about 45 days, or about 60 days after insemination into the subject's vagina, cervix, uterus, or fallopian tubes.
  • the administration is continued daily until the subject presents a normal ThI immune response or a normal Th2 immune response or both, according to the judgment of a practitioner of skill in the art.
  • an effective amount of G-CSF is administered to a subject at risk of implantation failure or miscarriage.
  • a subject at risk is a subject that has failed one or more artificial insemination procedures.
  • a subject at risk is a subject undergoing her first AI procedure.
  • the subject can also be in any other population at risk for failed embryo implantation or miscarriage as determined by a practitioner of skill in the art.
  • the subject has had one or more previous spontaneous abortions.
  • Other subjects at risk include those with unusually high ThI immune responses or unusually low Th2 immune responses.
  • the subject can also be in any other population at risk for failed embryo implantation or miscarriage as determined by a practitioner of skill in the art.
  • the G-CSF is administered to the subject prior to insemination.
  • the G-CSF is administered to a subject that is planning or attempting to become pregnant via artificial insemination.
  • the G-CSF can be administered to the mother-to-be during the controlled ovarian hyperstimulation procedure or prior to the insemination if no controlled ovarian hyperstimulation procedure is used.
  • the G-CSF treatment may also be used to prevent or reduce the likelihood of implantation failure or miscarriage in recipients of other assistant reproductive procedures, such as in vitro fertilization (IVF).
  • IVF in vitro fertilization
  • In vitro fertilization is an assisted reproduction procedure to overcome fertility problems caused by, for example, tubal disease, endometriosis, oligospermia, sperm antibodies and unexplained infertility.
  • the procedure can include controlled ovarian hyperstimulation with "fertility drugs” such as ovarian stimulants like clomiphene citrate and gonadotropin-releasing hormones.
  • "fertility drugs” such as ovarian stimulants like clomiphene citrate and gonadotropin-releasing hormones.
  • Hyperstimulation of the ovaries can induce growth of the egg (oocyte) and its encasing cells, collectively also termed the ovarian follicles. After sufficient follicular growth, final follicular maturation is induced and oocytes are retrieved or harvested.
  • the oocytes are fertilized in vitro with sperm and the embryos cultured. A small number of embryos, generally 2-4, are then transferred to the uterus. Despite the transfer
  • the G-CSF is typically administered before the assisted reproduction procedures. The administration is continued until implantation of the embryo to the uterine wall is achieved, until the risk of failed implantation or miscarriage is reduced or eliminated, or according to the judgment of a practitioner of skill in the art.
  • the administration is started before eggs, sperm, or embryos are transferred into the recipient. In certain embodiments, the administration is continued until pregnancy is confirmed. In certain embodiments, the administration is started about the time of controlled ovarian hyperstimulation and continued until about 3 days, about 5 days, about 7 days, about 10 days, about 12 days, about 14 days, about 30 days, about 45 days, or about 60 days after the transfer of embryo to the subject's uterus. In certain hyperstimulation and continued until about the end of the first trimester. In another embodiment, the dose is administered for five consecutive days about the time of embryo/gamete transfer. In certain embodiments, the administration is continued until the subject presents a normal ThI immune response for a pregnant subject or a normal Th2 immune response for a pregnant subject or both, according to the judgment of a practitioner of skill in the art.
  • the retrieved oocytes or embryos are maintained and cultured in medium containing G-CSF prior to their transfer to the uterus of the recipient.
  • Another aspect of the present invention provides methods of preventing or reducing the likelihood of spontaneous abortion by administering to a subject in need thereof an effective amount of a G-CSF.
  • the subject can be any mammalian subject at risk for a spontaneous abortion.
  • the subject is a human female.
  • the subject has previously had one or more spontaneous abortions.
  • the subject has previously had two or more spontaneous abortions.
  • the subject has had recurrent spontaneous abortions, i.e., three or more spontaneous abortions.
  • the subject can be any subject in a population at risk for spontaneous abortion.
  • the subject can be a human female in an age group at risk for spontaneous abortion.
  • the subject can be a human female greater than 35 years of age, greater than 40 years of age or greater than 45 years of age.
  • the subject can be a human female less than 20 years of age or less than 15 years of age.
  • essentially a woman of any age that presents with a reproductive infirmity, such as spontaneous abortion, preeclampsia and preterm labor is a candidate for obtaining the materials and methods of the instant invention.
  • the subject can also be in any other population at risk for spontaneous abortion as determined by a practitioner of skill in the art.
  • the subject is threatening abortion.
  • the subject is obese, morbidly obese, has overall poor health or comorbid conditions that indicate a risk of spontaneous abortion to the skilled practitioner.
  • these conditions can be incompetent cervix, uterine anomalies, hypothyroidism, diabetes mellitus, chronic nephritis, acute infection, use of illicit drugs (such as cocaine or crack), immunologic problems, severe emotional shock and viral infection (especially cytomegalovirus, herpes virus and rubella) (see Merck Manual 17th edition, 1999, Merck Research Laboratories, Whitehouse Station, New Jersey, p. 2053).
  • the subject has had an include those with unusually high TM immune responses or unusually low Th2 immune responses.
  • the subject can also be in any other population at risk for spontaneous abortion as determined by a practitioner of skill in the art.
  • the G-CSF is administered to the subject prior to pregnancy.
  • the G-CSF is administered to a subject that is planning or attempting to become pregnant.
  • the G-CSF is administered to a pregnant subject.
  • the G-CSF can be administered at any time during the first or second trimester of pregnancy.
  • the G-CSF is administered during the first 20 weeks of pregnancy.
  • the G-CSF is administered to a subject who is pregnant or attempting to become pregnant through artificial insemination or natural conception.
  • the present invention provides methods of treating or preventing preeclampsia or preterm labor by administering to a subject in need thereof an effective amount of granulocyte colony stimulating factor.
  • preeclampsia and preterm labor is caused or associated with inappropriate immune responses in a pregnant subject.
  • subjects at risk for preeclampsia or preterm labor present inappropriate immune cytokines associated with a T- helper 1 (ThI) immune response known to those of skill in the art.
  • subjects that have healthy pregnancies typically present immune cytokines associated with a T-helper 2 immune response.
  • administration of GCSF can reduce the inappropriate ThI response and/or increase a Th2 immune response in a subject. This invention is thus based, in part, on the discovery that administration of GCSF can shift a subject's immune preeclampsia or preterm labor.
  • GCSF is administered to a subject presenting one or more signs or symptoms of preeclampsia or preterm labor
  • the subject can be any subject that presents any of the signs or symptoms of preeclampsia during pregnancy such as hypertension, swelling or edema and excessive protein in the urine.
  • the subject can be any subject that develops hypertension with albuminuria or edema between the 20th week of pregnancy and the end of the 1st week postpartum.
  • Particular subjects include pregnant females who develop a blood pressure of 140/90 mm Hg, edema of the face or hands or albuminuria of > 1+ or whose blood pressure rises by 30 mm Hg systolic or 15 mm Hg diastolic (even if less than 140/190 mm Hg) between the 20th week of pregnancy and the end of the 1st week postpartum.
  • Particularly preferred subjects are human females.
  • the GCSF is typically administered until the signs or symptoms of preeclampsia or preterm labor are alleviated or reduced as long as the therapeutic benefit outweighs the risk of adverse events according to the judgment of a practitioner of skill in the art.
  • the dosing can continue as long as the subject displays no toxic effects of the administration according to the judgment of a practitioner of in the art.
  • the treatment is continued until the subject presents a normal ThI immune response for a pregnant subject or a normal Th2 response for a pregnant subject, or both, according to the judgment of a practitioner of skill in the art,
  • GCSF is administered to a subject at risk for developing preeclampsia or preterm labor.
  • the subject can be any mammalian subject at risk for preeclampsia or preterm labor.
  • Subjects at risk include subjects carrying multiple babies, subjects younger than age 20 and subjects older than age 40. Further subjects include those pregnant for the first time (primigravida), subjects with preexisting hypertension and subjects ThI immune responses or unusually low Th2 immune responses.
  • the subject is a human female.
  • GCSF is administered as long as the subject is at risk for preeclampsia and as long as the therapeutic benefit outweighs the risk of adverse events and also, so long as no toxicity is observed according to the judgment of a practitioner of skill in the art.
  • GCSF is administered for the duration of the pregnancy.
  • administration is provided in the 2nd and 3rd trimester of pregnancy.
  • administration is continued after delivery for about one, about two, about three, about four, about five, about six, about seven or about eight weeks post partum.
  • the treatment is continued until the subject presents a normal ThI immune response for a pregnant subject or a normal Th2 immune response for a pregnant subject, or both, according to the judgment of a practitioner of skill in the art.
  • the GCSF can be administered according to any method of administration known to those of skill in the art. Preferred methods of administration include subcutaneous administration. Other effective modes of administration are described in detail in the sections below. G-CSF AND FORMULATION
  • the present invention provides methods of administering an effective amount of granulocyte colony stimulating factor (G-CSF) to prevent or reduce the likelihood of spontaneous abortion, implantation failure and miscarriage during and following artificial insemination.
  • G-CSF granulocyte colony stimulating factor
  • a range of modifications can be made to the wild-type G-CSF molecules so long as the known immune system modulating activity of the G-CSF is maintained.
  • assays There are a number of assays that can be used to ensure that any one modified G-CSF retains the desired immune system modulating activity.
  • Plural types of G-CSF molecules can be administered in the practice of the instant invention.
  • the plural G-CSF molecules can be administered concurrently, consecutively, or sequentially.
  • the G-CSF can be any G-CSF or any derivative, variant, mimetic, chemically modified version or hybrid thereof, as described in U.S. Patent Nos. 5,399,345; 5,416,195; 5,981,551 ; 6,166,183 and 6,261,550, the contents of which are hereby incorporated by reference in their entireties.
  • the G-CSF can be administered in the form of a nucleotide sequence encoding G-CSF or expression vectors encoding G-CSF described in U.S, Patent No. 5,422,248, the content of which is hereby incorporated by reference in its entirety.
  • the G-CSF can be formulated according to any formulation for administration known to those of skill in the art.
  • the G-CSF is a commercially available G-CSF available as a pharmaceutical composition, suitable for administration to an animal, including a human.
  • Such commercially available pharmaceutical compositions can be, for example, filgrastim (Neupogen® (Amgen), Tevagrastim® (Teva), Biograstim® (CT Arzneistoff), Ratiograstim® (Ratiopharm GmbH), Zarzio® (Sandoz GmbH), Filgrastim Hexal® (Hexal AG), pegfilgrastim (Neulasta®, Amgen), nartograstim (Neu-Up®, Kyowwa) or lenograstim (Neutrogin®, Granocyte®, Chugai).
  • Filgrastim, nartograstim, and lenograstim are useful for promoting neutrophil proliferation and are generally administered to individuals in need to increased neutrophils, for example, patients undergoing chemotherapy.
  • Filgrastim, nartograstim, and lenograstim progenitor cell collection and severe chronic neutropenia.
  • Off label uses include treatment of neutropenia in AIDS patients, aplastic anemia, hairy cell leukemia, myelodysplasia, drug- induced and congenital agranulocytosis and alloimmune neonatalneutropenia.
  • the usual treatment of neutropenia associated with myelosuppression is 5 mcg/kg/day, once daily either by bolus subcutaneously or short (15-30 minute) intravenous infusion or by continuous subcutaneous or intravenous infusion. Administration is once daily starting no earlier than 24 hours after chemotherapy and continues for 14 days or until the individual's absolute neutrophil count is 10,000/mm3.
  • the usual dose is 10 mcg/kg/day administered as an intravenous infusion over 4-24 hours or as a continuous 24 hour subcutaneous infusion.
  • the first dose is generally administered at least 24 hours after chemotherapy and at least 24 hours after bone marrow infusion. During recovery, the dose is adjusted according to the patient's absolute neutrophil count.
  • Filgrastim dosing for peripheral blood progenitor cells generally begins at 10 ⁇ g/kg/day subcutaneously either as a bolus or continuous infusion. It is recommended that filgrastim be given for at least four days before leukapheresis and continued until the last leukapheresis procedure. Doses of filgrastim for congenital neutropenia are 5 mcg/kg subcutaneously twice daily while idiopathic or cyclic neutropenia is generally treated with a dose of 5 mcg/kg subcutaneously once daily.
  • Pegfilgrastim is a monomethoxypolyethylene glycol conjugate of filgrastim.
  • the pharmaceutical composition is commercially available as preservative free solutions of 10 rng/ml pegfilgratim in prefilled single-dose syringes.
  • Pegfilgrastim is indicated to decrease infections in patients with febrile neutropenia undergoing myelosuppressive chemotherapy.
  • Recommended dosing is a single 6 mg subcutaneous injection administered once per chemotherapy cycle.
  • amount i.e., an amount effective to reduce or eliminate the risk of implantation failure or spontaneous abortion. The amount can be determined by the skilled practitioner guided by the description herein and the knowledge in the art. In preferred embodiments, the amount can be any amount of G-CSF that reduces the ThI response of the subject.
  • the amount can be any amount sufficient to increase or initiate a Th2 response in the subject.
  • Assays to determine TM and Th2 responses in the subject are well known to those of skill in the art (See e.g., Schust and Hill, 1996, J. Soc. Gynecol Investig. 3:259-61, Xing et al., 2001, Chin. Med. J. 114:921-4, Raghupathy et al, 1999, Cell Immunol 196:122- 30, Mauri et al., 1996, J. Immunol 26:1511-8, Doncorli et al, 1997, Eur. J. Imm. 27:1451-8, Raziuddin, 1998, J.
  • G-CSF is administered at doses of about 0.1 mcg/kg/day to 600 mcg/kg/day, 0.5 mcg/kg/day to 300 mcg/kg/day, 1 mcg/kg/day to 100 mcg/kg/day, 1 mcg/kg/day to 50 mcg/kg/day, 1 mcg/kg/day to 20 mcg/kg/day, 1 mcg/kg/day to 10 mcg/kg/day, 1 mcg/kg/day to 2 mcg/kg/day; and about 1.67 mcg/kg/day.
  • At least 0.01 rng, at least 0.02 mg 5 at least 0.05 mg at least 0.1 mg, at least 0.2 mg, at least 0.5 mg, at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 25 mg, at least 50 mg, at least 75 mg, at least 100 mg, at least 125 mg, at least 150 mg, at least 175 mg, at least 200 mg ? at least 300 mg, at least 600 mg or more is administered daily.
  • the present invention provides methods of administering to a subject in need thereof an effective amount of G-CSF as monotherapy. In other embodiments, the present invention provides methods of administering to a subject an effective amount of G-CSF in combination with at least one additive.
  • Additives include cytokines that suppress ThI immune response, cytokines that enhance Th2 immune response, and non-myeloablative immunosuppressive agents.
  • Additives immunologic mechanisms such as stimulation of lrophoblast cell proliferation, inhibition of trophoblast cell apoptosis, stimulation of trophoblasat invasion, or stimulation of angiogenesis. Additives may also include anti-inflammatory agents, and inhibitors of proinflammatory cytokines,
  • additives include, but are not limited to interferon alpha, interferon beta, macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), interleukin-1 (IL-I), IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-11 , IL-13, IL- 14, IL-15, IL-19, IL-20, IL-21, IL-22, IL-24, IL-25, IL-26, IL-27 > IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, and IL-35.
  • M-CSF macrophage colony stimulating factor
  • GM-CSF granulocyte macrophage colony stimulating factor
  • LIF leukemia inhibitory factor
  • TGF-beta transforming growth factor beta
  • additives may include inhibitors of pro -inflammatory cytokines such as, but not limited to, anti-TNF-alpha, pentoxifyllin, a ⁇ ti- VEGF 5 ami-CD28, anti-CD80, anti-CD86, anti-CD40L, and the like.
  • pro -inflammatory cytokines such as, but not limited to, anti-TNF-alpha, pentoxifyllin, a ⁇ ti- VEGF 5 ami-CD28, anti-CD80, anti-CD86, anti-CD40L, and the like.
  • anti-inflammatory agents include anti-inflammatory agents.
  • the anti -inflammatory agent can be one that reduces leukocyte populations or inhibits leukocyte function.
  • Other anti-inflammatory agents can be used as well.
  • vitamin D3 l ⁇ S-dihydroxycholecalciferol
  • corticosteroids such as prednisone or methylprednisolone can be used.
  • G-CSF G-CSF
  • additives that may be used with a G-CSF are those currently used to treat recurrent spontaneous abortion, miscarriage, or implantation failure, such as intravenous Ig and heparin.
  • the additive can be another CSF, erythropoietin or stem cell factor.
  • the CSF can be G-CSF, GM-CSF or macrophage CSF. known to those of skill in the art.
  • Preferred methods of administration include subcutaneous administration, parenteral administration, enteral administration, topical administration.
  • G- CSF may also be administered by inhalation.
  • the G-CSF can be formulated in any manner known to those of skill in the art for formulating and administering effective amounts of G-CSF.
  • Filgrastim or non-glycosylated G-CSF is available as a preservative-free pharmaceutical composition
  • the composition can be administered subcutaneously without further admixture.
  • Intravenous preparations require dilution with proper diluent, such as 5% dextrose, diluted to a final concentration of filgrastim of 5 to 15 mcg/ml.
  • Saline is not recommended as a diluent due to product precipitation.
  • Mixture with albumin is recommended to prevent adsorption to plastic or glass materials during preparation and infusion.
  • the final concentration of human albumin should be 2 mg/ml. It is highly recommended that filgrastim be refrigerated at 2° to 8° C.
  • the presently available pharmaceutical composition contains a small amount of acetate, Tween 80, sodium, and sorbitol. These excipients are used to achieve and maintain characteristics that are physiologically acceptable to the body and pharmaceutically practical. Such characteristics include tonicity, osmoticity, osmolality, osmolality, viscosity and shelf life.
  • Aqueous pharmaceutical compositions of G-CSF with increased serum half life have been described, for example, in U.S. Patent No. 5,919,757, incorporated herein by reference in its entirety.
  • the pharmaceutical compositions can comprise the G-CSF in a salt form.
  • suitable acids which are capable of forming salts with the proteins of the present invention including, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycoHc acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, rnaleic acid, fumaric acid, cinnarnic acid, anthranilic acid, citric acid, naphthalene sulfonic acid, sulfanilic acid and the like.
  • Suitable bases capable of forming salts with the subject proteins can include, for example, inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di-and tri-alkyl amines (for example, triethyl amine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (for example, ethanolamine, diethanolamine and the like).
  • inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like
  • organic bases such as mono-, di-and tri-alkyl amines (for example, triethyl amine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (for example, ethanolamine, diethanolamine and the like).
  • G-CSF can be in a variety of forms suitable for any route of administration, including, but not limited to, parenteral, enteral, topical or inhalation.
  • Parenteral administration refers to any route of administration that is not through the alimentary canal, including, but not limited to, injectable administration, i.e., intravenous, intramuscular and the like as described below.
  • Enteral administration refers to any route of administration which is oral, including, but not limited to, tablets, capsules, oral solutions, suspensions, sprays and the like, as described below.
  • enteral administration also refers to rectal and vaginal routes of administration.
  • Topical administration refers to any route of administration through the skin, including, but not limited to, creams, ointments, gels and transdermal patches, as described below (see also, Pennsylvania, 1990).
  • Parenteral pharmaceutical compositions of the present invention can be administered by injection, for example, into a vein (intravenously), an artery (intraarterially), a muscle (intramuscularly) or under the skin (intradermal ⁇ or subcutaneously) or in a depot composition.
  • Injectable pharmaceutical compositions can be sterile suspensions, solutions or emulsions of the G-CSF in aqueous or oily vehicles.
  • the compositions can also comprise formulating agents or excipients, such as suspending, stabilizing and/or dispersing agents.
  • the formulations for injection can be presented in unit dosage form, in ampules or in multidose containers, and can comprise added preservatives.
  • the pharmaceutical compositions contain buffers such as citrate, acetate, phosphate, tris (hydroxymethyl) amino methane or THAM (tromethamine).
  • G-CSF may be contained in an inert matrix or device for slow release after implantation of the matrix or device.
  • injectable compositions can be pharmaceutically appropriate compositions for any route of injectable administration, including, but not limited to, intravenous, intrarterial, intracoronary, pericardial, perivascular, intramuscular, subdermal, subcutaneous and intraarticular.
  • the injectable pharmaceutical composition can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use.
  • a suitable vehicle including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc.
  • the G-CSF can be forms and reconstituted prior to use in vivo.
  • the pharmaceutical composition can be provided as a depot preparation, for administration by implantation; e.g., subcutaneous, intradermal, or intramuscular injection.
  • the pharmaceutical composition can be formulated with suitable polymeric or hydrophobic materials as an emulsion in an acceptable oil or ion exchange resins, or as sparingly soluble derivatives; as a sparingly soluble salt form of the G-CSF, or derivative, mimetic or variant thereof.
  • the G-CSF can be present in an inert matrix or device for implantation to achieve prolonged release.
  • transdermal delivery systems manufactured as an adhesive disc or patch that slowly releases the active ingredient for percutaneous absorption can. be used.
  • permeation enhancers can be used to facilitate penetration of the G-CSF.
  • a particular benefit may be achieved by incorporating the G-CSF into a transdermal patch.
  • the pharmaceutical formulations can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., piegelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methyl cellulose); fillers (e.g., lactose, microcrystalKne cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).
  • binding agents e.g., piegelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methyl cellulose
  • fillers e.g., lactose, microcrystalKne cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • Liquid pharmaceutical compositions for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid pharmaceutical compositions can suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils
  • compositions can also comprise buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Pharmaceutical compositions for oral administration can be suitably prepared to provide controlled release of the G-CSF.
  • Enteral pharmaceutical compositions can be suitable for buccal administration, for example, in the form of tablets, troches or lozenges.
  • the G-CSF can be prepared as solutions (e.g. for retention enemas), suppositories or ointments.
  • Enteral pharmaceutical compositions can be suitable for admixture in feeding mixtures, such as, for mixture with total parenteral nutrition (TPN) mixtures or for delivery by a feeding tube (see, Dudrick et al., 1998, Surg. Technol. Int. VII:174-184; Mohandas et al., 2003, Natl. Med. J.
  • the G-CSF can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafiuoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafiuoroethane, carbon dioxide or other suitable gas.
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated comprising a powder mix of the compound and a suitable powder base such as lactose or starch.
  • Inhaled pharmaceutical compositions can be those, for example, described in U.S. Patent Nos. 5,284,656 and 6,565,841, incorporated herein by reference in their entirety. that can comprise one or more unit dosage forms comprising the G-CSF.
  • the pack can, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device can be accompanied by instructions for administration.
  • the pharmaceutical compositions can be for a single, one time use or can contain antimicrobial excipients, rendering the composition suitable for multiple, extended use with greater shelf stability; for example, a multi-use bottle.
  • the pharmaceutical composition of interest can be in unit dose or unit-of-use packages.
  • a unit dose is targeted for a single use.
  • the unit dose form can be in a vial, which can contain a solution or a desiccated form for reconstitution, a pre-filled syringe, a transdermal patch and the like.
  • a unit-of-use package is a convenient prescription size, patient ready unit labeled for distribution by health care providers.
  • the package contains as much active ingredient as necessary for a typical treatment regimen.
  • the pharmaceutical composition can be labeled and have accompanying labeling to identify the composition contained therein and other information useful to health care providers and end users.
  • the information can include instructions for use, dose, dosing interval, duration, indication, side effects and other contraindications, warnings, precautions, storage recommendations and the like.
  • parenteral administration can be in a single bolus or as a continuous infusion.
  • parenteral administration can be a single intravenous infusion given over 15-30 minutes.
  • parenteral administration can be a continuous infusion of G-CSF diluted in 5% dextrose.
  • the methods provide for administration of G-CSF for a therapeutically or prophylactically effective time.
  • the G-CSF is administered prior to the onset or observation of the disorder or symptoms accompanying the disorder.
  • the G-CSF is administered during the disorder or during the time period that symptoms accompanying the disorder are observed.
  • the G-CSF is administered for a time after the disorder had cleared.
  • the G-CSF can be administered about one day, about two days, about three days, about four days, about one week, about two weeks and up to about eight weeks, following resolution of threatened abortion or after confirmation of pregnancy during assisted reproduction.
  • diagnostic assays can be used to ascertain subjects presented with reproductive inefficiencies that are correlated with particular immunologic parameters.
  • patients with repeated spontaneous abortion, miscarriage, or implantation failure and the like present with particular profiles of their immune system status.
  • subjects with high ThI cell number or cell activity and/or reduced Th2 cell number or cell activity, or an aberrant ratio of the two may be candidates for obtaining the above-described treatment.
  • number or cell activity is enhanced.
  • Another assay of interest is one that determines whether Th2 cell number or activity is decreased.
  • Yet another assay of interest is one that determines a higher ratio of ThI cell number to Th2 cell number, or ThI cell activity to Th2 cell activity.
  • a number of known assays can be used to make such determinations.
  • ⁇ interferon, tumor necrosis factor alpha, tumor necrosis factor ⁇ , IL-2, IL- 12, and IL-18 are markers of ThI cells.
  • assays for one or more of such cell-specific markers can provide the basis to conclude a higher than normal ThI status.
  • Th2 IL-4, IL-5, IL-6, IL-IO and IL- 13 are known markers of that cell type.
  • assays for one or more of such cell-specific markers can provide the basis to conclude a higher than normal Th2 status.
  • Diagnostic assays can be also used to ascertain subjects presenting with reproductive inefficiencies that are correlated with particular pathopysiologic parameters.
  • Pathophysiologic markers could include markers of cell stress such as heat shock proteins, markers of oxidative stress such as nitric oxide and free radicals, markers of cell injury such as hepatic transaminases and creatine kinase, and markers of cell death including caspase 1 & 3.
  • a diagnostic kit can be used to ascertain subjects presenting with reproductive inefficiencies that are correlated with serum or ovarian follicular fluid G-CSF concentrations. Such a kit would serve as a theranostic complimenting G-CSF as a therapeutic.
  • kits for carrying out the methods of the invention provides kits for carrying out the methods of the invention.
  • the present invention provides kits for preventing or reducing the likelihood of spontaneous abortion or implantation failure during CSF along with a label or labeling with instructions on using the G-CSF to prevent or reduce the likelihood of spontaneous abortion or implantation failure during and following artificial insemination according to the methods of the invention.
  • the kits can comprise components useful for carrying out the methods such as devices for delivering the G-CSF.
  • the kit can comprise components useful for the safe disposal of devices for delivering the G-CSF, e.g., a sharps container for used syringes.
  • kits for preventing or reducing the likelihood of spontaneous abortion and implantation failure in recipients of IVF comprise one or more effective doses of G-CSF along with a label or labeling with instructions on using the G-CSF to prevent or reduce the likelihood of spontaneous abortion, miscarriage or implantation failure during and following IVF.
  • the kits can comprise components useful for carrying out the methods such as devices for delivering the G-CSF.
  • the kit can comprise components useful for the safe disposal of devices for delivering the G-CSF, e.g., a sharps container for used syringes.
  • the G-CSF in the kit is formulated for subcutaneous administration. In another embodiment, the G-CSF in the kit is formulated for intramuscular administration. In another embodiment, the G-CSF in the kit is formulated for intravascular administration.
  • the kit may further contain other active compounds, such as CSFs (e.g., G- CSF, GMCSF 5 and macrophage CSF), erythropoietin, stem cell factors, anti-inflammatory agents, interleukins,etc. for slow release after implantation of the device.
  • CSFs e.g., G- CSF, GMCSF 5 and macrophage CSF
  • erythropoietin erythropoietin
  • stem cell factors e.g., interleukins, etc.
  • anti-inflammatory agents e.g., interleukins, etc.
  • the present invention provides a transdermal patch comprising G-CSF as an active ingredient.
  • the present invention provides an implantable device comprising G-CSF as an active ingredient.
  • the present invention provides an implantable device G-CSF embedded in an inert matrix.
  • the present invention provides a vaginal ring comprising G-CSF as an active ingredient and in some cases with another complimentary agent as an additional active ingredient.
  • Example 1 G-CSF Prevents Embryotoxic Effects of Cells from Women with Recurrent Spontaneous Abortion In Vitro
  • G-CSF is effective in preventing the death of mouse embryos in an in vitro clinical assay for spontaneous abortion.
  • Mouse bioassays have widely been used to detect embryotoxic effects of sera from subjects having reproductive difficulty. (See, Cameo, et al., 1999, Human Reprod. 14(4):959-63, Oksenberg and Brautbar 1986, Am. J. Reprod Immunol. Microbiol 11(4):118-24, Roussev et al., 1995, Am. Reprod. Immunol. 33(2):171-175 and Thomason et al., 1995, Am. J. Reprod. Immunol. 34(6):338-41.). women suffering from recurrent spontaneous abortion. The leukocytes are cultured, and the culture medium is removed from the leukocytes. This culture medium is then contacted with murine embryos. Toxic factors in the culture medium typically kill the murine embryos in this assay.
  • the mononuclear leukocytes are incubated with G-CSF prior to removal of the culture medium.
  • the culture medium is then removed from the leukocytes and contacted with murine embryos. Survival of the murine embryos indicates the reduction of embryotoxic factors in the culture medium and thereby the effectiveness of G-CSF administration for prevention of spontaneous abortion in this in vitro model.
  • G-CSF effectively inhibits a well-known in vivo model for spontaneous abortion.
  • the murine mating pair CBA x DBA/2 results in a spontaneous abortion rate of approximately 40%.
  • female CBA mice are treated according to the methods of the invention. They are treated with G-CSF prior to mating, at the time of mating and immediately after mating. A reduction of the rate of spontaneous abortion in mice treated with G-CSF relative to control mice indicates that G-CSF effectively prevents spontaneous abortion in this in vivo model.
  • Example 3 G-CSF Prevents Habitual Abortion In Vivo
  • J. C. is a 36-year-old married white female with an obstetrical history of three uncomplicated vaginal deliveries at full term (all male children) followed by six consecutive first trimester miscarriages (each at 10-12 weeks). Conception was natural in each of the successful pregnancies and in each miscarriage. Each miscarried fetus was karyotyped, and all were normal. The couple then experienced three years of secondary infertility. At that point, she sought a consultation with a reproductive endocrinologist (RE).
  • RE reproductive endocrinologist
  • the RE performed a detailed workup to attempt to identify the cause of the couple's reproductive failures. No anatomic or endocrinologic etiology was identified. Both J. C. and her husband were found to be karyotypically normal. A standard andrology workup for the husband was negative.
  • J.C.'s past medical history was significant in that J.C. had a remote past history of seasonal allergies and ten years of allergy desensitization shots. Based on this medical history, a series of immunologic tests including measurement of ThI and Th2 cytokine production in vitro were ordered. As noted previously in this application, allergy is desensitization works by presenting the allergen in a manner that favors ThI cytokine production instead of Th2 cytokine production. In many individuals, this shift from Th2 to ThI dominance becomes more generalized and antigen non-specific. The series of tests ordered for J.C.
  • Thl/Th2 cytokines produced by the patient's peripheral blood mononuclear cells (PBMC) in response to the non-specific mitogen phytohemagglutinin (PHA).
  • PBMC peripheral blood mononuclear cells
  • PHA mitogen phytohemagglutinin
  • J.C.'s PBMC produced greater than 10,000 units per ml of the prototypic ThI cytokine gamma interferon in response to PHA.
  • Levels of the prototypic Th2 cytokine IL-4 and the counter regulatory Th2 cytokine IL-IO were undetectable.
  • the RE performed intrauterine insemination (IUI) using J.C.'s husband's sperm.
  • IUI intrauterine insemination
  • the first attempt at IUI resulted in a positive HCG at 7 days, The rhG-CSF administration was initiated the following day.
  • the regimen consisted of 100 meg/day of rhG-CSF (Neupogen) injected subcutaneously for a total of 30 days, a cumulative dose of 3000 meg.
  • the rhG-CSF regimen was carried out for the full 30 days and then discontinued.
  • the patient experienced no rhG-CSF-related side effects at any point during the regimen.
  • N. C. is a healthy 35-year-old married white female with an obstetrical history of primary infertility including three failed IUIs and one failed IVF.
  • N.C.'s first IUI resulted in monozygotic twins, one of which revealed no fetal heartbeat at 6 weeks and the other which had a confirmed weak fetal heartbeat at 6 weeks but no heartbeat by the 7 th week.
  • the second IUI resulted in a singleton pregnancy and fetal demise at 8 weeks. A heartbeat was seen at the 7 th week but was negative by the 8 th week.
  • Karyotyping was performed and revealed an abnormal karyotype (69 XXY).
  • N.C.'s third IUI resulted in a probable ectopic pregnancy treated with methotrexate.
  • N.C.'s last pregnancy attempt was a cycle of IVF.
  • N.C. was transferred from her RE's care to the high-risk obstetrical unit in a hospital where she delivered a healthy baby boy. Both mother and child are doing well.
  • N.C. opted to undergo another IVF cycle at a different clinic without the benefit of rhG-CSF therapy. This cycle failed and was classified as a biochemical pregnancy (positive beta HCG, no evidence of gestational sac or embryo).
  • N.C. contacted the inventor to request that he provide consultation regarding the use of rhG-CSF in her next IVF cycle.
  • the inventor agreed and a clinical plan identical to her previous IVF cycle using rhG-CSF was pursued.
  • N.C. began rhG-CSF (lOOmcg per day) five days prior to embryo transfer (i.e., on the day of oocyte retrieval) in a fresh IVF cycle. The pregnancy is ongoing and her RE has transferred her to the care of a general obstetrician. At her last examination (at 20 weeks), all measurements were normal for gestational age and fetal heartbeat was strong.
  • JJ. is a 33-year-old married white female with a history of primary subfertility and seven failed pregnancies. Over a period of three years, JJ. suffered three first-trimester miscarriages and three chemical pregnancies. Four of the pregnancies involved the use of fertility drugs and natural conception. Two of the pregnancies occurred through IUI. The [0166] J.J.'s RE perfo ⁇ ned a standard workup to attempt to determine cause for JJ. 's failures. The workup failed to identify a cause. Both members of the couple were found to be karyotypically normal. J.J. and her RE decided that she should consult with a Reproductive Immunologist.
  • JJ, and her RE sought a consultation with the inventor and decided to undergo another cycle of IVF with rhG-CSF treatment.
  • JJ. underwent another cycle of IVF with frozen embryos from her previous cycle. Although JJ. was scheduled to begin rhG-CSF at 100 meg per day five days prior to embryo transfer, JJ. was not able to begin rhG-CSF until three days before embryo transfer. The rhG-CSF was continued at 100 meg per day for 30 days after embryo transfer. The cumulative dose of rhG-CSF was 3300 meg. JJ. completed her course of rhG-CSF and experienced no rhG-CSF related side effects.
  • JJ. underwent another ultrasonic evaluation at 10 weeks gestation, and a strong heartbeat was identified and all measurements were exactly appropriate for dates. JJ. was transferred to the care of a general obstetrician and delivered a healthy baby girl. Both the mother and the child are healthy and doing well.
  • skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

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Abstract

L'invention porte sur des procédés et des trousses pour prévenir ou réduire la probabilité d'un échec d'implantation ou d'une fausse couche chez une receveuse d'une insémination artificielle. Les procédés comprennent l'administration chez la receveuse d'une insémination artificielle ayant besoin d'un tel traitement d'une quantité efficace de facteur de stimulation de colonie de granulocytes (G-CSF).
PCT/US2009/042481 2009-05-01 2009-05-01 Compositions et procédés pour réduire la probabilité d'un échec d'implantation ou d'une fausse couche chez des receveuses d'une insémination artificielle WO2010126528A1 (fr)

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ITMI20122063A1 (it) * 2012-12-03 2014-06-04 Marco Sbracia Gm-csf per l'utilizzo nella prevenzione dell'aborto spontaneo e del fallimento dell'impianto dell'embrione
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