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Title:  Protection of the female reproductive system from natural and artificial insults
United States Patent: 
7,195,775
Issued: 
March 27, 2007

Inventors: 
Tilly; Jonathan L. (Windham, NH), Kolesnick; Richard N. (New York, NY)
Assignee: 
The General Hospital Corporation (Boston, MA)
Appl. No.: 
09/503,852
Filed: 
February 15, 2000


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

Described are methods for protecting the female reproductive system against natural and artificial insults by administering to women a composition comprising an agent that antagonizes one or more acid sphingomyelinase (ASMase) gene products. Specifically, methods disclosed herein serve to protect women's germline from damage resulting from cancer therapy regimens including chemotherapy or radiotherapy. In one aspect, the method preserves, enhances, or revives ovarian function in women, by administering to women a composition containing sphingosine-1-phosphate, or an analog thereof. Also disclosed are methods to prevent or ameliorate menopausal syndromes and to improve in vitro fertilization techniques.

SUMMARY OF THE INVENTION

The present invention provides a method of protecting female reproductive system against a natural or an artificial insult comprising: administering a composition comprising an agent that antagonizes one or more acid sphingomyelinase (ASMase) gene product, in an amount sufficient to protect said female reproductive system from normal or pre-mature aging or destruction caused by said natural or artificial insult. The artificial insult comprises chemical insult, radiation insult, surgical insult, or a combination thereof. Natural insults to reproductive system occurs as a consequence of aging, genetic background, physiological factors, environmental factors, or other developmental and genetic factors.

According to an object of the invention, the artificial insult comprises chemical insults, including for example, cytotoxic factors, chemotherapeutic drugs, hormone deprivation, growth factor deprivation, cytokine deprivation, cell receptor antibodies, and the like. Chemotherapeutic drugs include 5FU, vinblastine, actinomycin D, etoposide, cisplatin, methotrexate, doxorubicin, among others.

In accordance with another object of the invention, the artificial insult comprises radiation insult, including ionization radiation, x-ray, infrared radiation, ultrasound radiation, heat, or a combination thereof. Radiation is administered to a patient through an invasive radiation therapy, a non-invasive radiation therapy, or both.

Protection of female's reproductive system is achieved in females in all age groups consisting of pre-reproductive age, reproductive age and post-reproductive age group.

One of the preferred agents of this invention is a small molecule compound comprising lysophospholipid. More preferably the lysophospholipid is a sphingolipid compound, or an analog thereof. The most preferred agent of the invention is the compound of sphingosine-1-phosphate, or an analog thereof. The agent is administered in vitro, ex vivo, or in vivo. Preferred routes of administration include, orally, intravascularly, intraperitoneally, intrauterine, intra-ovarian, subcutaneously, intramuscularly, rectally, topically, or a combination thereof. Intra-ovarian administration is achieved by methods, including, for example, by direct injection into the ovary. The injection is made to the ovary in vivo or ex vivo.

According to another object of the invention, a method of preserving, enhancing, or reviving ovarian function in female mammals is disclosed. This method comprises administering to female mammals an effective amount of a composition comprising sphingosine-1-phosphate, or an analog thereof. The ovarian functions include fertility and normal menstrual cyclicity.

Yet another object of the invention is a method to prevent or ameliorate menopausal syndromes. Menopausal syndromes within the scope of this invention include somatic disorders, cognitive disorders, emotional disorders, and the like. The agent of the invention is administered on a regular daily, weekly, biweekly, monthly or annual intervals in order to achieve the intended therapeutic objective.

According to another object of the invention, an in vitro fertilization method is disclosed that comprises (a) obtaining at least one oocyte from a mammal; (b) incubating said oocyte in a medium containing sphingosine-1-phosphate, or an analog thereof, in an amount sufficient to maintain viability of said oocyte in culture; (c) fertilizing in vitro said oocyte with sperm to produce at least one fertilized oocyte (zygote); (d) culturing said fertilized oocyte to produce an embryo; and (e) transferring at least one embryo to the uterus of said mammal, wherein said at least one embryo develops to term in said mammal.

DETAILED DESCRIPTION OF THE INVENTION

This invention, as described herein, relates that compositions containing a novel therapeutic agent, administered in vivo or used in vitro, which protects female reproductive system from stress signals or insults induced by natural or artificial factors.

Apoptosis is a mechanism by which cells are programmed to die under a wide range of physiological, biochemical and developmental stimuli. Apoptosis is also an important cellular response to a large variety of stress signals, induced by natural or artificial factors. Acid sphingomyelinase (ASMase) gene disruption is shown to suppress normal apoptotic deletion of oocytes, leading to ovarian hyperplasia. Ex vivo, ASMase-/- oocytes or wild-type oocytes treated with an agent, capable of antagonizing one or more ASMase gene products, resist developmental and anticancer treatment-induced apoptosis, thereby confirming cell autonomy of the death defect.

The invention, as disclosed and described herein, provides for a germ cell-autonomous death defect caused by ASMase-deficiency. Cell autonomous death is reversed by inhibition of ASMase gene products, which inhibition causes a significant hyperplasia of the female germline during fetal ovarian development. These data, demonstrate that antagonizers of ASMase gene products confer significant protection against natural or artificial insults on oocytes in vivo, or in vitro and, therefore, offer a new route for rapid therapeutic development to combat premature ovarian failure, and to prolong ovarian function and fertility in women.

At present, how antagonizers of ASMase gene products exert their pro- and anti-apoptotic effects in a female reproductive system remains to be elucidated. Without being limited to any specific mechanism of action underlying the invention described herein, one possible mechanism is that a stepwise program of cell death is activated in germline by both physiologic and pathologic stimuli, with alterations in the sphingolipid rheostat serving as an initial signal transduction pathway. Indeed, S1P has been shown to prevent activation of downstream executioner caspases in Jurkat T-cells exposed to short-chain ceramide analogs (Cuvillier et al., J. Biol. Chem. 273,2910(1998)), and ceramide has recently been implicated as a facilitator of Bax-induced cytochrome c release from mitochondria (Pastorino et al., J. Biol. Chem. 274, 31734 (1999)).

The direct connection between ceramide and Bax is especially relevant to the present invention since Bax-deficient oocytes are, like ASMase-deficient oocytes, resistant to cancer therapy-induced apoptosis (Perez et al., Nature Med. (1997) id.) Furthermore, microinjection of human recombinant Bax protein into oocytes duplicates the pro-apoptotic effects of both human recombinant ASMase microinjection and anti-cancer drug treatment (FIG. 3E).

The ASMase antagonizers, or the "agent" according to this invention, include any compound, that suppresses or inhibits activity and/or expression of one or more acid sphingomylinase (ASMase) gene products in vitro, ex vivo, or in vivo. The agent comprises, for example, any lipid, lysophospholipid, sphingolipid, protein, peptide, polypeptide, nucleic acid molecule, including DNA, RNA, DNA/RNA hybrids or an antisense molecule, small molecules, antibiotics, and the like. The terms protein, peptide, and polypeptide are used interchangeably herein.

A preferred agent according to the invention is a small molecule. In a more preferred embodiment of the invention, the agent comprises lysophospholipids, and most preferably, the agent is sphingosine-1-phosphate (S1P), or an analog thereof. Examples of analogs of sphingosine-1-phosphate, include but are not limited to, N,N-dimethylsphingosine-1-phosphate; N,N,N-trimethylsphingosine-1-phosphate; N-acetylsphingosine-1-phosphate; N-acylsphingosine-1-phosphate; sphingosine-1,3-diphosphate; sphingosine-3-phosphate; sphingosine-1-thiophosphate; N,N-dimethylsphingosine-1-thiophosphate; N,N,N-trimethylsphingosine-1-thiophosphate; or pharmaceutically acceptable salts thereof.

Sphingosine-1-phosphate is shown to be completely safe and without side effects on the ovaries. In one general embodiment of the invention, as disclosed herein, in vivo administration of the agent of the invention prior to an artificial insult resulted in a significant preservation of the germ cell reserve with complete protection of the quiescent (primordial) and growing (primary, preantral) follicle populations in ovaries exposed to the insult.

According to one general embodiment of the invention, artificial insults are the consequence of a therapy against a disease or a disorder. The disease or disorder comprises, for example, cancer, rheumatoid arthritis, angioplasy, or restenosis. Cancer includes, for example, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chondroma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, acute lymphocytic leukemia and acute myelocytic leukemia, chronic leukemia and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, or immunoglobulin heavy chain diseases.

Artificial insults, according to the invention described herein, include chemical, radiation, and surgical insults. Examples of chemical insults include, cytotoxic factors, chemotherapeutic drugs, hormone deprivation, growth factor deprivation, cytokine deprivation, cell receptor antibodies and the like. Further non-limiting examples include TNF-alpha, TNF-beta, IL-1, INF-gamma, IL-2, insulin-like growth factor, transforming growth factor B1, vascular endothelial growth factor, fibroblast growth factor, 5FU, vinblastine, actinomycin D, etoposide, cisplatin, methotrexate, doxorubicin, and the like.

In accordance with another embodiment of the invention, the insult is a radiation insult. It is shown that germline of female mammals exposed to radiation are seriously damaged and administration of the composition of the invention in vivo, in vitro, or ex vivo protects oocytes from destruction induced by a therapeutically-relevant dose of ionizing radiation.

Radiation insult, according to the invention disclosed herein, encompasses both non-invasive (external) and invasive (internal) radiation therapies. In an external radiation therapy, treatment is affected by radiation sources outside the body, whereas in an invasive radiation therapy treatment is affected by radiation sources planted inside the body. The representative diseases treated by non-invasive or invasive radiation therapy include, for example, cancer, rheumatoid arthritis, angioplasy, or restenosis.

Invasive radiation therapy encompasses, for example, selective internal radiation therapy (SIRT), incorporation of the radioactive materials into small particles, microspheres, seeds, wires and the like. These objects are directly implanted into the various tissue, organs, or their respective arterial blood supply within the body.

Various methods for introducing radiation into an area treated for stenosis are known. Some methods deliver radiation in a solid medium, while others utilize liquid sources. For example, a procedure in reducing the restenosis rate is the introduction of radiation energy into the interior of the vessel. This procedure, known as "intravascular radiation therapy" (IRT) has been shown to inhibit fibroblast and smooth muscle cell hyperplasia.

U.S. Pat. No. 5,059,166, issued to Fischell, discloses an IRT method that relies on a radioactive stent that is permanently implanted in the blood vessel after completion of the lumen opening procedure. U.S. Pat. No. 5,302,168, issued to Hess, teaches use of a radioactive source contained in a flexible catheter. U.S. Pat. No. 5,503,613, issued to Weinberger, uses a liquid filled balloon to guide a solid source wire to a treatment site. U.S. Pat. No. 5,616,114, issued to Thornton et al., describes an apparatus and method for delivering liquid radiation into a balloon-tipped catheter. Radiation therapies disclosed by aforementioned patents, are disclosed merely as examples of radiotherapeutic regimens used to treat patients and are non-limiting.

The use of radioactive material in connection with therapies, such as those disclosed above, creates a risk of harmful exposure, both to the medical personnel and to patients. Precautionary measures need to be taken to protect against the harm caused by the leakage of liquid radiation into the blood stream during these therapies. Sensitive organs, such as the ovaries, are inevitably damaged depending on the invasiveness of the procedure used. The invention disclosed herein protects ovaries of both patients and medical personnel from a risk of harm caused by exposure to radiation during such therapies.

Radiation is emitted from a variety of radionuclides. These radionuclides encompass, for example, beta-ray emitters, gamma-ray emitters, or a radionuclide that emits both beta-ray and gamma-ray. Further examples of radionuclides include, Strontium 90, Iridium 192, Phosphorous 32, Rhenium 186, Rhenium 188, .sup.198Au, .sup.169Er, .sup.166Ho, .sup.153Sm, and .sup.165Dy, which are chosen according to the purpose of treatment.

Other radiation sources include sources used in nuclear magnetic resonance diagnosis in which the central ion of the complex salt must be paramagnetic. In particular, the radiation sources use the divalent and trivalent ions of the elements of atomic numbers 21 29, 42, 44 and 58 70. Suitable ions are, for example, the chromium(III), manganese(II), iron(II), nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III), and iron(III).

According to another embodiment of the invention disclosed herein, radiation insult includes ultrasound radiation. Ultrasound radiation is administered to patients, either alone or in combination with other therapies, for example, hormonal therapy, chemotherapy, or surgery. The therapeutic regimen is applied either preoperatively, i.e., to the tumor in situ or postoperatively, in the region of the tumor after removal of the primary cancerous lesion. The ultrasound therapy comprises both the invasive and non-invasive ultrasound treatments. The dosage of ultrasonic energy applied is, for example, above 22.5 watt/sec, and has a frequency in the range of, for example, about 1 KHz to about 3 MHz.

According to another embodiment of this invention, radiation insult includes, x-ray, infrared, and heat. Heat is used to selectively induce apoptosis in intended cells or tissues. Preferably heat is used to treat inflammation. The term inflammation includes inflamed atherosclerotic plaques, restenosis, and arteritis such as that found in systemic lupus, myocarditis of the autoimmune etiology, arteriovenous fistulea, dialysis grafts or other vascular prosthesis. The phrase "treating inflammation" also includes treating a region of a vein prior to or after balloon angioplasty, or related interventions that could result in inflammation and subsequent thrombosis, acute closure or restenosis.

Heat may be transferred to the target cells by a variety of methods. For example, heat is transferred into an inflamed plaque in a blood vessel by means of a catheter, stent, or liquid heat. Catherter or stents are heated electrically or with microwave or radio frequency radiation or other means. Heat is also generated from internal or external devices, such as radio frequency sources outside the body. The present invention protects ovaries from the risk of over-exposure to heat waves or liquid heat during heat therapy.

Natural insults, as defined herein, include damages resulting from physiological, biochemical or developmental processes occurring in a female body. A manifest natural, insult is apoptosis due to aging. Natural insults are influenced, for example, by genetic background of the female, environmental affects, or both. The functional life span of female gonads is defined by the size and rate of depletion of the endowment of oocytes enclosed within follicles in the ovaries at birth. This continuous loss of oocytes throughout life, referred to by many as the female biological clock, is driven by a genetic program of cell death that is controlled by physiological and biochemical pathways and players and is conserved from worms to humans (Morita & Tilly (1999) id.) This invention, as disclosed herein, demonstrates the effect of antagonizers of ASMase gene products in combating normal or pre-mature germ cell depletion in a female mammal.

Without being limited to any specific mechanism of action underlying the invention described herein, one possible mechanism for the effect of antagonizers of ASMAse gene products is through preventing apoptosis of granulosa cells as well as, or instead of, directly preventing apoptosis of oocytes. Granulosa cells support, nourish, and help to mature oocytes throughout postnatal life.

Examples of disease and disorders resulting from a natural insult include, disturbances in menstruation, abnormal uterine bleeding, abnormal ovulatory cycles, amenorrhea, pelvic pain, sexual dysfunction, in fertility, menstrual cyclicity, and pre-mature menopause among others.

Other insults include surgical insults wherein a woman's reproductive system, in part or in whole, is surgically removed. In particular, hormonal imbalance, resulting from the removal of one or both ovaries, is fully or partially restored by administration of the therapeutic agent of the invention.

Reproductive system includes any cell, tissue, organ, and tract that are involved in part or in whole in sexual reproduction. Cells include variety of somatic cells, for example, granulosa cells that nourish and mature oocytes, as well as germ cells.

Included withing the scope of this invention are methods to protect women's ovaries from natural and artificial insults, not only to keep them fertile, but also to preserve enough ovarian function to prevent menopause and its associated disorders. Women are subject to natural or artificial insult in any age group. These age groups are pre-reproductive, reproductive or post-reproductive age groups. Pre-mature menopausal syndromes are initiated by a wide variety of artificial or natural conditions. Menopausal disorders, include, for example, somatic disorders such as osteoporosis, cardiovascular disease, somatic sexual dysfunction, loss of libido; cognitive disorders, such as loss of memory; emotional disorders, such as depression, and the like.

The composition of the invention is administered on a continuous or semi-continuous, or temporary basis, depending on the type of insult and objectives of the therapy intended. For example, if protection of the reproductive system from long term natural insults is intended, administration of the composition of this invention on a continuous or semi-continuous basis is preferred. In a continuous administration, the composition is generally administered regularly, on a predetermined interval, for an indefinite period of time. Predetermined intervals comprise daily, weekly, biweekly, or monthly, or yearly intervals.

If protection from artificial insults are intended both short term and long term administration are suggested, depending on the type of insult and the objective of the therapy intended. An example of a short term administration is the administration to protect ovaries from radiation or chemical insults. In short term administration, the composition is administered, at least once, in a period of from about thirty days prior to immediately prior to exposure to the insult. More preferably the composition is administered from about fifteen days to about two days, and most preferably from about seven days to about two hours prior to exposure to the insult. The administration of the composition is terminated prior to ovarian exposure to the insult, or it is continued during exposure or after the exposure is terminated.

The dosage of the therapeutic agent is adjusted according to, for example, the duration and the objective of the treatment intended. A lower dosage of the agent is required in a more prolonged and continuos administration.

The administration is achieved in vivo, in vitro or ex vivo. The in vivo administration encompasses orally, intravascularly, intraperitoneally, intra-uterine, intra-ovarian, subcutaneously, intramuscularly, rectally, topically, or a combination thereof. Intra-ovarian administration is achieved by several methods, including, for example, by direct injection into the ovary. The injection is made to the ovary in vivo or ex vivo.

According to another aspect of this invention, an in vitro fertilization method is described that uses the therapeutic agent of this invention to protect the viability of female germline at different stages of in vitro fertilization. These stages, include in vivo, ex vivo, and in vitro periods of fertilization and pregnancy. In vivo stages of fertilization and pregnancy include, for example, one or more of the following periods: the period prior to isolation of oocytes, the period after implantation of the embryo in the uterus, and the period during pregnancy. In vitro, and ex vivo stages include, for example, one or more of the following: cryopreservation of oocytes, culture or growth of oocytes prior to fertilization, fertilization stage, culture or growth of embryo post-fertilization.

Oocytes isolated from women are at different stages of development and are either mature or immature. Immature oocytes reach maturity in vitro or in vivo conditions. In vitro fertilization, according to the invention, is achieved by the use of a mammal's own oocytes or a different mammal's oocytes. After the embryo is implanted in the subject mammal, in vivo administration of the therapeutic agent is terminated, or it is continued for a time period thereafter to ensure continued viability and normal development of the embryo in vivo.

In vitro fertilization method, according to the invention disclosed and described herein, increases the chances of successful fertilization, pregnancy and normal development of the embryo in the uterus. Furthermore, it ensures availability of immature or mature oocytes for fertilization, and makes it possible to preserve fertility and increases availability of donor oocytes for women who do not have their own functional oocytes.

Also embraced within the scope of this invention are compositions comprising one or more agents of the invention in association with one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants (collectively referred to herein as "carrier" materials) and, if desired, other active ingredients.

According to an embodiment of the invention, the agent is combined with one or more adjuvants appropriate to the indicated route of administration. If administered per os, the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets may contain a controlled-release formulation as may be provided in a dispersion of active compound in hydroxypropylmethyl cellulose.

Formulations for parenteral administration are, for example, in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions are prepared, for example, from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cotton seed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art.

The compositions of the invention are adapted to be administered by any suitable route, and in a dose effective for the treatment intended. Therapeutically effective doses of the composition required to prevent or preserve the female reproductive system from insults are readily ascertained by one of ordinary skill in the art.

For oral administration, the composition is in the form of, for example, a tablet, capsule, suspension or liquid. The composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are tablets or capsules. Preferably, the oral units contain an amount of active ingredient from about 1 to 1000 mg, more preferably from about 25 to 500 mg, and most preferably from about 100 to 250 mg. A suitable daily dose may vary widely, however, a dose of from about 0.01 to 3000 mg/kg body weight, or from about 0.1 mg to about 100 mg/kg of body weight per day is preferred. A more preferred dosage will be a range from about 1 mg to about 100 mg/kg of body weight. Most preferred dosage is a dosage in a range from about 1 to about 50 mg/kg of body weight per day.

The dosage regimen of the agents and/or compositions of this invention is selected in accordance with a variety of factors and thus may vary widely. A main factor to consider is the objective of therapy, for example, protecting female germline from radiation or chemotherapy, prolonging fertility, preventing menopause, preserving normal menstrual cyclicity, ameliorating or preventing post-menopausal conditions, are among many therapeutic objectives that are intended and encompassed within the scope of the invention. Other factors include, for example, the age, weight, severity and type of the insult, the route of administration, and the type of therapeutic agent employed.
 


Claim 1 of 17 Claims

1. A method of treating a female reproductive system by administering to a female patient a composition comprising sphingosine-1-phosphate in an amount sufficient to inhibit apoptosis induced by an artificial insult, wherein said administration is in vivo or ex vivo, and wherein said artificial insult is a chemotherapeutic drug or radiation.
 

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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