U.S. patent application number 11/715795 was filed with the patent office on 2007-07-05 for protection of female reproductive system from natural and artifical insults.
This patent application is currently assigned to Massachusetts General Hospital, Partners HealthCare Research Ventures & Licensing. Invention is credited to Richard N. Kolesnick, Johnathan L. Tilly.
Application Number | 20070157331 11/715795 |
Document ID | / |
Family ID | 24003781 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070157331 |
Kind Code |
A1 |
Tilly; Johnathan L. ; et
al. |
July 5, 2007 |
Protection of female reproductive system from natural and artifical
insults
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.
Inventors: |
Tilly; Johnathan L.;
(Windham, NH) ; Kolesnick; Richard N.; (New York,
NY) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Massachusetts General Hospital,
Partners HealthCare Research Ventures & Licensing
Boston
MA
|
Family ID: |
24003781 |
Appl. No.: |
11/715795 |
Filed: |
March 7, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09503852 |
Feb 15, 2000 |
7195775 |
|
|
11715795 |
Mar 7, 2007 |
|
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Current U.S.
Class: |
800/21 ;
424/93.7 |
Current CPC
Class: |
A61K 31/685 20130101;
A61P 15/00 20180101; A61K 31/688 20130101; A61K 31/66 20130101;
A61K 31/00 20130101; A61P 15/08 20180101; A61K 31/661 20130101 |
Class at
Publication: |
800/021 ;
424/093.7 |
International
Class: |
A01K 67/027 20060101
A01K067/027 |
Claims
1-36. (canceled)
37. A method for in vitro fertilization of a mammal comprising. (a)
obtaining at least one oocyte from a mammal; (b) incubating said
oocyte in a medium containing a composition comprising
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; (d) culturing said fertilized oocyte to produce
an embryo; and (e) transferring at least one embryo to the uterus
of a mammal, wherein said at least one embryo develops to term in
said mammal.
38. The method of claim 37, wherein said at least one oocyte is
immature when obtained from said mammal and becomes mature in step
(b).
39. The method of claim 37, wherein said mammal is human.
40. The method of claim 38, wherein said immature oocyte is
cultured for about five to about seven days at step (b).
41. The method of claim 37, wherein prior to said step (b) said at
least one oocyte is cryopreserved in a cryopreservation medium
containing said composition.
42. The method of claim 37, wherein said composition is
additionally present in steps (c) and (d).
43. The method of claim 37, wherein said composition is added
continuously or periodically to said culture media.
44. The method of claim 37, wherein the mammal of step (a) is the
same or different from the mammal of step (de).
45. The method of claim 37, wherein said mammal is a woman.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for protecting
female reproductive system against natural or artificial insults by
administering a composition comprising an agent that antagonizes
one or more acid sphingomyelinase (ASMase) gene products. In
particular, this invention relates to a method of protecting
ovaries from cancer therapy regimens, chemotherapy and
radiotherapy, by administering to women a composition containing
sphingosine-1-phosphate, or an analog thereof, prior to the
therapy. Methods to enhance ovarian functions, ameliorate symptoms
of menopause, and improve the success of in vitro fertilization are
also disclosed.
I. BACKGROUND OF THE INVENTION
[0002] Female gonads house a finite number of meiotically-arrested
germ cells (oocytes) enclosed within primordial follicles that
serve as the stockpile of eggs released at ovulation at each
menstrual cycle for potential fertilization. Gougeon, Endocr Rev.
17, 121 (1996); Morita & Tilly, Dev. Biol. 213 (1999). Once
depleted, the ovarian germ cell pool cannot be replenished. Thus,
exposure of women to a wide spectrum of agents that damage the
ovary, such as chemotherapeutic agents and radiotherapy, generally
leads to premature menopause and irreversible sterility. Waxman,
Soc. Med. 76, 144 (1983); Familiari et al., Hum. Reprod. 8, 2080
(1993); Ried & Jaffe, Semin. Roentgenol. 29, 6 (1994); and
Reichman & Green, Monogr. Natl. Cancer Inst. 16, 125
(1994).
[0003] Apoptotic cell death plays a fundamental role in normal germ
cell endowment and follicular dynamics in the ovary. Tilly &
Ratts, Contemp. Obstet. Gynecol. 41, 59 (1996); Tilly, Rev. Reprod.
1, 162 (1996); and Tilly et al., Cell Death Differ. 4, 180 (1997).
Cell fate in the ovary is likely dependent on the actions of
several proteins recently identified as key determinants of cell
survival or death (Adams & Cory, Science 281, 1322 (1998);
Green, Cell 94, 695 (1998); Thornberry & Lazebnik, Science 281,
1312 (1998); Reed, Oncogene 17, 3225 (1998); Korsmeyer, Cancer Res.
59, 1693 (1999). Among these identified in the ovary are p53 (Tilly
et al., Endocrinology 136, 1394 (1995); Keren-Tal et al., Exp. Cell
Res. 218, 283 (1995); and Makrigiannakis et al., J. Clin.
Endocrinol. Metab. 85, 449 (2000)), members of the bcl-2 gene
family (Tilly et al., Endocrinology 136-232 (1995); Ratts et al.,
Endocrinology 136, 3665 (1995); Knudson et al., Science 270, 99
(1995); Perez et al., Nature Med. 3 1228 (1997); Kugu et al., Cell
Death Differ. 5, 67 (1998); Perez et al., Nature Genet. 21, 200
(1999), and members of the caspase gene family (Flaws et al.,
Endocrinology 136, 5042 (1995); Perez et al., Nature Med. 3, 1228
(1997); Maravei et al., Cell Death Differ. 4, 707 (1997); Kugu et
al., Cell Death Differ. 5, 67 (1998); Boone & Tsang, Biol.
Reprod. 58, 1533 (1998); Bergeron et al., Genes Dev. 13, 1304
(1998); and Perez et al., Mol. Hum. Reprod. 5, 414 (1999)).
[0004] In addition, ceramide, a recently identified lipid second
messenger associated with cell death signaling (Spiegel et al.,
Curr. Opin. Cell Biol. 8, 159 (1996); Hannun, Science 274, 1855
(1996); and Kolesnick & Kronke, Annu. Rev. Physiol. 60, 643
(1998)) has been implicated in the induction of apoptosis in the
ovary (Witty et al., Endocrinology 137, 5269 (1996); Kaipia et al.,
Endocrinology 137, 4864 (1996); and Martimbeau & Tilly, Clin.
Endocrinol. 46, 241 (1997)).
[0005] Since the initial discovery of the sphingomyelin pathway,
numerous studies have been published on the potential role of
ceramide in signaling cell death (Hannun, (1996) id.; and Kolesnick
& Kronke (1998) id. It is now known that ceramide can also be
metabolized via ceramidase to sphingosine, which is then
phosphorylated by sphingosine kinase to generate
sphingosine-1-phosphate (S1P) (Cuvillier et al., Nature 381, 800
(1996); Spiegel et al., Ann. N.Y. Acad. Sci. 845, 11 (1998); and
Spiegel, J. Leukoc. Biol. 65, 341 (1999)).
[0006] In some cell types, S1P can effectively counterbalance
stress-kinase activation and apoptosis induced by membrane-permeant
ceramide analogs or external stressors known to work through
elevations in intracellular ceramide levels. Therefore, a rheostat
model has been proposed in which cell fate is controlled by shifts
in the balance between ceramide and S1P levels. However, the
physiologic importance of ceramide, and that of sphingomyelin
hydrolysis as a whole, in activating developmental or homoeostatic
paradigms of apoptosis have recently been questioned by some
investigators (Hofinann & Dixit, Trends Biochem. Sci 23, 374
(1998); and Watts et al., Cell Death Differ. 6, 105 (1999)). In
particular, Hofmann et al., describe a lack of developmental
defects that should be the consequence of inpaired apoptosis in the
acid sphingomyelinase (ASMase) gene knockout mouse as substantive
evidence against a role for ASMase-catalyzed sphingomyelin
hydrolysis and ceramide in signaling cell death (Kolesnick &
Kronke (1998) id.)
[0007] Earlier studies using pharmacologic and genetic approaches
have shown that several other components of the programmed cell
death regulatory pathway in oocytes, including Bcl-2 family members
(Ratts et al., Endocrinology 136, 3665 (1995); Perez et al., Nat.
Med. 3, 1228 (1997); Morita et al., Mol. Endocrinol. 13, 841
(1999); Perez et al., Nat. Genet. 21, 200 (1999)); and caspases
(Perez et al.,(1997) id.; Bergeron et al., Genes Dev. 12, 1304
(1998)), are required for oocyte survival or death. However, cell
lineage specificity will certainly be an important issue to
consider based on observations that p53, a classic signaling
molecule for cancer therapy-induced tumor cell destruction (Ko
& Prives, Genes Dev. 10, 1054 (1996); and Ding et al., Crit.
Rev. Oncog. 9, 83 (1998)), is completely dispensable for oocyte
death initiated by cancer therapy (Perez et al., (1997) id.)
[0008] Although the sensitivity of oocytes to cancer therapy, and
the potential role of ceramide in signaling cell death are
reported, as evidenced above, little is known regarding the
mechanisms responsible for female germ cell destruction. Recently,
it has been shown that female mouse oocytes undergo a type of cell
death, referred to as apoptosis, when exposed in vitro to a
prototypical anti-cancer drug (doxorubicin, 14-hydroxydaunorubicin,
Adriamycin.RTM.). Perez et al., (1997) id. Moreover, it is shown
that culture of mouse oocytes in vitro with sphingosine-1-phosphate
protected the oocytes from death induced by subsequent doxorubicin
exposure. However, the protection was only tested in vitro with
only a single drug, and thus in vivo application remained
questionable.
[0009] The present invention is the first to show that protection
of the ovaries from natural or artificial insults is achieved in
vivo, and that this protection is accomplished by administration of
a composition containing an agent that antagonizes activity or
expression of one or more acid sphingomyelinase (ASMase) gene
products. The invention demonstrates that such agents have
promising therapeutic effects in combating ovarian failure, thus,
preserving fertility and normal ovarian functions under various
adverse conditions.
II. SUMMARY OF THE INVENTION
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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,
intra-uterine, 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.
[0015] 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.
[0016] 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.
[0017] 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.
III. BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1. Postnatal oocyte hyperplasia results from ASMase
gene disruption. Number of non-atretic primordial, primary and
small preantral follicles in young adult (day 42 postpartum)
wild-type (hatched bars) and ASMase gene knockout (solid bars)
female mice (mean.+-.SEM, n=3 mice per genotype; P<0.05 versus
respective wild-type value).
[0019] FIG. 2. ASMase-deficiency or sphingosine-1-phosphate
treatment attenuates programmed cell death in female germline
during fetal gametogenesis. (A) Rate of programmed cell death in
germline of ovaries obtained from wild-type (+/+) or ASMase-mutant
(-/-) female fetuses following in vitro culture without hormonal
support. Each data point represents the mean (.+-.SEM) number of
non-apoptotic germline remaining per ovarian section, and the
results are the combined data from 6 fetal ovaries per genotype
(P<0.05 versus respective wild-type value). (B) Effects of
fumonisin-B1 (FB1) and S1P on germ cell survival in wild-type fetal
ovaries cultured for 72 hours without hormonal support
(mean.+-.SEM, n=6 fetal ovaries per group). Over one-half of the
starting population of germline (0 h or Time 0) is preserved after
72 hours of hormone deprivation by either ASMase gene disruption or
by S1P treatment.
[0020] FIG. 3. Cell autonomous nature of the germline programmed
cell death defect caused by ASMase gene disruption or S1P
treatment. Representative analysis of cellular morphology (A, B)
and of DNA integrity as assessed by the comet assay (C, D) in pools
of non-apoptotic oocytes (ASMase-deficient oocytes treated with
doxorubicin or DXR; A, C) and apoptotic oocytes (wild-type oocytes
treated with DXR; B. D). (E) Apoptotic cell death response in
wild-type (+/+) versus ASMase-deficient (-/-) oocytes cultured
without (control, CON) or with 200 nM DXR for 24 hours, or in
wild-type oocytes microinjected with human recombinant ASMase or
human recombinant Bax. Mean.+-.SEM from 3 or more independent
experiments with the total number of oocytes used per group
indicated over the respective bar, P<0.05 versus respective
wild-type value, N.D., none detected. For both ASMase and Bax
microinjection, a significant (P<0.05) increase in apoptosis was
observed versus those levels observed in comparable numbers of
vehicle-injected oocytes cultured in parallel (20.+-.5%;
mean.+-.SEM, n=3 or more independent experiments).
[0021] FIG. 4. Complete protection of the female germline from
radiation-induced death in vivo by S1P administration. Morphometric
analysis of the number of non-atretic oocyte-containing follicles
at the four indicated stages of development remaining in vehicle
(PET)- or S1P-treated ovaries 14 days after a single treatment with
0.1 Gy of ionizing radiation (mean.+-.SEM, n=3 mice; P<0.05
versus 0 .mu.M S1P receiving radiation treatment; N.S., not
significantly different). ##STR1##
IV. DETAILED DESCRIPTION OF THE INVENTION
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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)).
[0026] 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).
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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 acutemyelocytic 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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).
[0040] 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.
[0041] 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.
[0042] 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.
Catheter 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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 continues administration.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] The invention will be more fully understood by reference to
the following examples. These examples are not to be construed in
any way as limiting the scope of this invention. All literature
cited herein is specifically incorporated by reference.
V. EXAMPLES
Example 1
Histomorphometric Evaluation of Oocyte Endowment
[0063] Ovaries are fixed (0.34 N glacial acetic acid, 10% formalin,
28% ethanol), embedded in paraffin, and serially sectioned (8
.mu.M). The serial sections from each ovary are aligned in order on
glass microscope slides, stained with hematoxylin/picric methyl
blue, and analyzed for the number of healthy (non-atretic)
oocyte-containing primordial, primary and small preantral follicles
as described by Perez et al. Nat. Genet. (1999) id. incorporated by
reference herein in its entirety.
Example 2
Histomorphometric Evaluation of Wild Type and ASMase -/-
Ovaries
[0064] ASMase -/- mice are generated as described by Horinouchi et
al., Nat. Genet. 10, 288 (1995), incorporated herein by reference
in its entirety. The histomorphometric evaluation of the oocyte
endowment of wild type mice and ASMase -/- sisters shows that
sphingomyelin hydrolysis is a key event in generating death signals
in the developing female germline. Compared with their wild-type
sisters, ASMase -/- females possess over 1.1.times.10.sup.3 more
quiescent oocyte-containing primordial follicles per ovary, as well
as significant hyperplasia of the growing (primary and small
preantral) follicle populations. Results are presented in Table 1
and FIG. 1. TABLE-US-00001 TABLE 1 Postnatal Oocyte Hyperplasia
Results From ASMase Gene Disruption Follicles +/+ -/- P value
Primordial 19120 .+-. 602 30480 .+-. 2397 P < 0.01 Primary 707
.+-. 93 1573 .+-. 141 P < 0.01 Preantral 13 .+-. 13 160 .+-. 46
P < 0.05
[0065] Number of non-atretic oocyte-containing primordial follicles
endowed in the ovarian reserve, and numbers of growing (primary and
small preantral) follicles, in wild-type (+1+) and ASMase-mutant
(-/-) female mice at day 4 postpartum (mean.+-.SEM, n=3 mice per
genotype).
[0066] The ovarian oocyte reserve remains significantly elevated in
ASMase -/- female mice in young adult life (FIG. 1), well prior to
the onset of any organ abnormalities or Niemann-Pick disease-like
symptoms that occurs in ASMase -/- mice during postnatal life.
[0067] To determine the basis of the extensive oocyte hyperplasia
in ASMase-/- neonates, fetal ovaries are harvested from wild-type
and mutant mice at embryonic day 13.5 (el3.5) for in vitro culture
as a model to recapitulate the events surrounding germline death
that occurs as a normal component of female gametogenesis. A
time-dependent activation of programmed cell death is observed in
germline of wild-type fetal ovaries cultured without hormonal
support for up to 72 hours (FIG. 2A). By comparison, the rate of
germ cell apoptosis is significantly attenuated in ASMase-deficient
fetal ovaries cultured in parallel (FIG. 2A). These findings
indicate that there exists an ovarian-intrinsic cell death defect
in the ASMase-deficient mouse, and point to enhanced survival of
the developing germline during oogenesis as the mechanism
underlying the enlarged oocyte pool seen in mutant females at
birth.
Example 3
Treatment With Ceramide Synthase Inhibitor
[0068] In order to show that sphingomyelin hydrolysis, as opposed
to ceramide synthesis, is important for generating ceramide as a
death signal, wild-type fetal ovaries are maintained in vitro for
72 hours and various concentrations (5-500 .mu.M) of a ceramide
synthase inhibitor, fumonisin-B1 (FB1) are applied to these
ovaries. The results show that this treatment does not alter
survival rates in female germline (FIG. 2B). Importantly, however,
and in support of the rheostat model, the reduced incidence of germ
cell apoptosis conveyed by ASMase-deficiency is recapitulated by
culturing wild-type fetal ovaries with increasing concentrations of
S1P (FIG. 2B). Equivalent levels of in vitro germ cell survival are
obtained by either ASMase gene knockout (FIG. 2A) or by S1P
treatment (FIG. 2B).
Example 4
Cell Autonomous Nature of Response
[0069] To demonstrate that germline survival is a cell autonomous
or a germline-intrinsic response, individual oocytes are isolated
from adult wild-type and ASMase -/- female mice, and are cultured
ex vivo with or without the anti-cancer drug, doxorubicin (DXR), to
induce apoptosis. In addition to assessments of cellular morphology
and caspase activation, some oocytes in each group are processed
for DNA cleavage analysis as an endpoint for cell death using the
Trevigen Comet Assay kit. The apoptotic event is elicited in
wild-type, but not ASMase-deficient, oocytes by DXR (FIG. 3E).
Example 5
Microinjection Experiment
[0070] Human recombinant acid sphingomyelinase is synthesized and
purified as described by He et al., Biochim. Biophys. Acta 1432,
251 (1999), incorporated herein by reference in its entirety. Six
picoliters of vehicle or of a 1 mg/ml stock of the enzyme are
microinjected into single oocytes using a Zeiss Axiovert 135
inverted microscope equipped with Narishige micromanipulators and a
PLI-100 pico-injector. Oocytes that survived the microinjection
procedure (>75%) are then cultured and assessed for the
occurrence of apoptosis. Furthermore, microinjection of human
recombinant Bax protein into single oocytes and assessments of
apoptosis are made as described by Perez, et al.(1997) id.
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). For
both ASMase and Bax microinjection, a significant (P<0.05)
increase in apoptosis is observed versus those levels observed in
comparable numbers of vehicle-injected oocytes cultured in parallel
(20.+-.5%; mean.+-.SEM, n=3 or more independent experiments).
Example 6
In Vitro Oocyte Cultures
[0071] Female mice (43 days of age post-partum; Charles River
Laboratories, Wilmington, Mass.) are superovulated with 10 IU of
equine chorionic gonadotropin (eCG or PMSG) followed by 10 IU of
human chorionic gonadotropin 48 h later. Mature oocytes are
collected from the oviducts 16 h after hCG injection. Cumulus
enclosed oocytes are denuded by a 1-min incubation in 80 IU/ml of
hyaluronidase, followed by three washes with culture medium. The
medium used for all culture experiments is human tubal fluid
(Irvine Scientific, Santa Ana, Calif.) supplemented with 0.5%
bovine serum albumin (BSA).
[0072] Oocytes are cultured in 0.1 ml drops of culture medium (8-10
oocytes/drop) under paraffin oil, an incubated with or without DXR
(200 nM) and/or fumonisin-B1, sphingosine-1-phosphate or
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-FMK) for 24
h at 37 C in a humidified atmosphere of 5% CO.sub.2-95% air. At the
end of the incubation period, oocytes are fixed, stained with
Hoechst 33342 and checked microscopically for morphological changes
characteristic of apoptosis (condensation, budding, cellular
fragmentation, and chromatin segregation into apoptotic bodies).
The percentage of oocytes that goes through apoptosis out of the
total number of oocytes cultured per drop in each experiment is
then determined, and all experiments are independently repeated
four to ten times with different mice.
Example 7
In Vitro Embryo Cultures
[0073] Female mice are superovulated with eCG followed hCG
treatment (see above) and placed with fertile males immediately
after hCG injection. Sixteen hours after mating, one-cell embryos
(confirmed by the presence of two polar bodies) are harvested from
the ampullae and denuded of cumulus cells by a 1-min hyaluronidase
treatment. Embryos are then maintained in vitro in HTF supplemented
with 0.5% BSA in absence or presence of 200 nM DXR. Under in vitro
conditions, one-cell embryos progress to the morula stage of
development within 72 h (see in vitro oocyte cultures above for
details of methodology and culture conditions). See, Perez et
al.(1997) id., incorporated by reference herein in its
entirety.
Example 8
Bax-Null Mice
[0074] In vitro experiments: mature oocytes are harvested from
wild-type and Bax-null adult female mice at approximately 6 weeks
of age using the gonadotropin superovulation regimen described
above. Following hyaluronidase removal of cumulus cells, oocytes
are incubated for 24 h without or with 200 nM DXR, after which the
occurrence of apoptosis is assessed and described under in vitro
oocyte cultures.
[0075] In vivo experiments: age-matched adult wild-type and
Bax-null female mice are given two intraperitoneal injections of
DXR (10 mg/kg of body weight) 1 week apart, starting at
approximately 8 weeks of age post partum. One week following the
second injection, ovaries are collected, fixed, embedded in
paraffin, serial-sectioned, and stained with hematoxylin/picric
methyl blue. Follicular morphology and numbers of immature
(primordial) follicles present in each ovary are then assessed as
detailed previously.
Example 9
p53-Null Mice
[0076] Mature oocytes are collected from adult wild-type and p53
null female mice by superovulation, and incubated with or without
200 nM DXR for 24 h. Following culture, the occurrence of apoptosis
is assessed as described above (see, Example 6: in vitro oocyte
cultures).
Example 10
S1P Protection Against Radiation
[0077] Young adult (postpartum day 40) wild-type female mice are
anesthetized, and dorsal incisions are made to retrieve and expose
the ovaries. Five .mu.l of vehicle (PET) are injected into the
bursa of one ovary of the pair while 5 .mu.l of a stock of either
0.5 or 2 mM S1P, prepared in PET, are injected into the bursa of
the contralateral ovary. Based on an estimated bursai cavity volume
of 50 .mu.l, the final concentrations of S1P in the bursal cavity
for ovarian exposure following administration of the 0.5 and 2 mM
stocks are approximately 50 and 200 .mu.M, respectively. The
ovaries are returned to the peritoneal cavity, the incisions are
sutured, and the mice are allowed to recover for a 2 hour
pretreatment period prior to a single exposure to 0.1 Gy of
abdominally-directed ionizing radiation. After two weeks, ovaries
are collected, coded, and processed for histomorphometric
evaluation of non-atretic oocyte-containing follicle numbers as
described above (see Example 1). In the absence of irradiation, the
number of follicles at any stage of development in S1P-treated
ovaries does not significantly differ from the number of
corresponding follicles in vehicle-treated ovaries.
[0078] Nearly complete destruction (LD.sub.80) of the
oocyte-containing primordial follicle pool is observed in
vehicle-treated ovaries of mice two weeks after a single exposure
to 0.1 Gy of ionizing radiation (FIG. 4). In contrast, in vivo
administration of S1P two hours prior to irradiation resulted in a
significant and dose-dependent preservation of the germ cell
reserve, with complete protection of the quiescent (primordial) and
growing (primary, preantral) follicle populations in ovaries
exposed to the highest dose of S1P prior to irradiation (FIG.
4).
[0079] Moreover, since oocyte viability, growth and function are
required for continued development of follicles from a quiescent to
mature state (see, Morita & Tilly (1999) id., incorporated
herein by reference in its entirety), the observation that ovaries
pretreated with the highest dose of S1P prior to irradiation
retained a completely normal distribution of oocyte-containing
follicles at all stages of development (i.e., identical to the
non-irradiated controls) at two weeks post-irradiation (FIG. 4)
suggests that the protected oocytes are indeed viable and
functional.
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