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
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
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.
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
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
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,N-trimethylsphingosine-1-thiophosphate; or pharmaceutically acceptable
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 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
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
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
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
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
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
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
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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