U.S. patent application number 17/490409 was filed with the patent office on 2022-01-20 for methods and agents to treat infertility in females.
The applicant listed for this patent is University Of Cincinnati. Invention is credited to Lori R. Bernstein, Tom Thompson.
Application Number | 20220016208 17/490409 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-20 |
United States Patent
Application |
20220016208 |
Kind Code |
A1 |
Bernstein; Lori R. ; et
al. |
January 20, 2022 |
METHODS AND AGENTS TO TREAT INFERTILITY IN FEMALES
Abstract
Described are novel female fertility therapies. A first aspect
of the invention is directed to therapies that include FSH lowering
methodologies to prevent and/or treat egg infertility. A second
aspect of the invention is directed to agents that bind to activin,
bind to receptors that bind activin, or that otherwise disrupt
activin signaling (collectively referred to herein as "activin
pathway modifier agents" or "APM agents") and methods of utilizing
these agents to prevent and/or treat egg infertility. A third
aspect of the invention is directed to methods of administering an
effective amount of an APM agent to a subject to increase oocyte
yield and/or ovarian reserve. All three aspects of the invention
may be used in humans and in animals. Additional aspects of the
invention include therapeutic drug kits for treatment of humans and
animals based on the methodologies described above.
Inventors: |
Bernstein; Lori R.;
(Montgomery Village, MD) ; Thompson; Tom;
(Loveland, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University Of Cincinnati |
Cincinnati |
OH |
US |
|
|
Appl. No.: |
17/490409 |
Filed: |
September 30, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16098221 |
Nov 1, 2018 |
11185569 |
|
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PCT/US2017/031054 |
May 4, 2017 |
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17490409 |
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62331658 |
May 4, 2016 |
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International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 47/68 20060101 A61K047/68; A61P 15/08 20060101
A61P015/08; A61K 31/57 20060101 A61K031/57; A61K 38/24 20060101
A61K038/24 |
Claims
1. A method of treating and/or reducing the incidence of
infertility, miscarriages, trisomic pregnancies, or birth defects
in a female subject, wherein said infertility, miscarriages,
trisomic pregnancies, or birth defects result from oocyte
aneuploidy, embryonic aneuploidy, and/or fetal aneuploidy, said
method comprising administering to a female subject at least one
FSH lowering agent or activin pathway modifying agent; wherein said
FSH lowering agent is chosen from LH; an LH derivative; hCG; an hCG
derivative; an antibody against FSH; inhibin A; inhibin B;
follistatin; follistatin derivatives; ActRIIB:Fc; ActRIIA: Fc;
3.alpha. hydroxy-4-pregnen-20-one (3.alpha.HP); 3.alpha.
hydroxy-4-pregnen-20-one-acetate (3.alpha.HPA); and anti-Mullerian
hormone; wherein said activin pathway modifying agent is chosen
from ActRIIB:Fc; ActRIIA: Fc; follistatin; and follistatin
derivatives; and wherein said at least one FSH lowering agent or
activin pathway modifying agent is administered in an amount
effective to treat and/or prevent infertility, miscarriages,
trisomic pregnancies, or birth defects.
2. The method of claim 1 wherein the at least one FSH lowering
agent or activin pathway modifying agent is ActRIIB:Fc.
3. The method of claim 2 wherein ActRIIB:Fc is given at a dose
sufficient to maintain serum levels of ActRIIB:Fc between 4 mg/kg
and 10 mg/kg.
4. A method of increasing oocyte production in a female subject
comprising administering to the subject at least one FSH lowering
agent or activin pathway modifying agent; wherein said FSH lowering
agent is chosen from LH; an LH derivative; hCG; an hCG derivative;
an antibody against FSH; inhibin A; inhibin B; follistatin;
follistatin derivatives; ActRIIB:Fc; ActRIIA: Fc; 3.alpha.
hydroxy-4-pregnen-20-one (3.alpha.HP); 3.alpha.
hydroxy-4-pregnen-20-one-acetate (3.alpha.HPA); and anti-Mullerian
hormone; wherein said activin pathway modifying agent is chosen
from ActRIIB:Fc; ActRIIA: Fc; follistatin; and follistatin
derivatives; and wherein the at least one FSH lowering agent or
activin pathway modifying agent is administered in an amount
effective to increase oocyte production in the subject.
5. The method of claim 4 wherein the at least one FSH lowering
agent or activin pathway modifying agent is ActRIIB:Fc.
6. The method of claim 5 wherein ActRIIB:Fc is given at a dose
sufficient to maintain serum levels of ActRIIB:Fc between 4 mg/kg
and 10 mg/kg.
7. The method of claim 1 further comprising administering the at
least one FSH lowering agent or activin pathway modifying agent for
at least a portion of one reproductive cycle.
8. The method of claim 1 further comprising administering the at
least one FSH lowering agent or activin pathway modifying agent for
at least a portion of two or more reproductive cycles.
9. The method of claim 8 further comprising harvesting and/or
attempting fertilization of an oocyte(s) after completion of the
second reproductive cycle.
10. The method of claim 8 further comprising harvesting and/or
attempting fertilization of an oocyte(s) after completion of at
least the third reproductive cycle.
11. The method of claim 1 wherein the subject is one of a human
subject or a non-human animal subject.
12. A kit for preventing or treating egg infertility in a subject
comprising at least one of a FSH lowering agent or an APM agent in
an amount effective to prevent or treat the egg fertility.
13. The kit of claim 12 further comprising adjunct agents as needed
to promote ovulation and/or provide endometrial support.
14. The kit of claim 13 wherein the adjunct agents are chosen from
human chorionic gonadotropin, progesterone medications and
combinations thereof.
15. A kit for increasing oocyte production in a subject comprising
at least one FSH lowering agent or an APM agent in an amount
effective to increase oocyte production.
16. The kit of claim 15 further comprising adjunct agents as needed
to promote ovulation and/or provide endometrial support.
17. The kit of claim 16 wherein the adjunct agents are chosen from
human chorionic gonadotropin, progesterone medications, and
combinations thereof.
18. The kit of claim 12 wherein the kit further includes a medical
implement for administering the at least one FSH lowering agent or
APM agent.
19. The kit of claim 15 wherein the kit further includes a medical
implement for administering the at least one FSH lowering agent or
APM agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/098,221, filed on Nov. 1, 2018 (and
published as U.S. Publication No. 2019/0160144 on May 30, 2019),
which is a U.S. national phase filing under 35 U.S.C. .sctn. 371 of
International Patent Application No. PCT/US2017/031054, filed May
4, 2017 (and published as International Publication No. WO
2017/192847 on Nov. 9, 2017), which claims priority to and the
benefit of the filing date of U.S. Patent Application No.
62/331,658, filed May 4, 2016, the disclosures of which are
incorporated by reference herein in their entireties.
FIELD
[0002] The present invention is directed to new female fertility
therapies and associated kits.
BACKGROUND
[0003] Rates of infertility, miscarriage, and trisomic pregnancies
increase dramatically in women as they age, and currently there is
no cure. For many advanced maternal age women (AMA, >35 years
old) there is increased risk of oocyte, embryonic and fetal
aneuploidy; infertility due to advanced maternal age, diminished
ovarian reserve, recurrent implantation failure; premature ovarian
failure, elevated estradiol, prior miscarriages, and/or prior
aneuploid conceptuses including trisomic pregnancies and births
including Down syndrome, Patau syndrome, and Edward syndrome that
lead to profound disabilities and early death. These problems are
collectively referred to as "egg infertility." Egg infertility
increases exponentially with age. The root cause of egg infertility
is a dramatic increase in the rates of chromosome segregation
errors in the oocyte. Egg infertility is a significant public
health problem, with 1 in 5 US women now attempting her first
pregnancy after the age of 35. By age 42, up to 87% of embryos are
aneuploid and 40-50% of women experience miscarriages and
infertility.
[0004] Several hundred thousand women in the US and 1.2 million
women throughout the developed world suffer from egg infertility
each year and are unable to have a baby because they cannot
conceive or maintain a pregnancy. Others afflicted with egg
infertility have babies with devastating trisomic diseases such as
Down syndrome. A therapy for couples afflicted with egg infertility
would be profoundly useful by preventing infertility, miscarriages,
Down syndrome, and other trisomies. There is a need for therapies
that prevent or treat egg infertility.
[0005] Primates, prize farm animals, and racing horses with
declining fertility also undergo assisted reproductive
technologies, including intrauterine insemination ("IUI") and in
vitro fertilization ("IVF"). Some species experience egg
infertility with maternal age. Presently, there are no methods for
preventing and/or treating egg infertility in animals.
[0006] Each year well over a million women throughout the US and
the world undergo ovarian stimulation with FSH medications to
induce the growth of multiple eggs for ovulation. Aromatase
inhibitors to induce ovulation by increasing endogenous levels of
FSH are an alternative therapy used for some infertility patients.
Patients who undergo these stimulation regimens include women
undergoing JUT and women undergoing IVF. They also include women
seeking to have a baby with their own eggs and women serving as
donors of their eggs. There is a need for therapies that increase
oocyte yield and/or ovarian reserve to improve assisted
reproduction technologies.
[0007] New therapeutic alternatives to FSH administration to
increase oocyte yield and/or ovarian reserve in assisted
reproductive technologies would be generally useful for women
undergoing assisted reproduction. Such therapeutic alternatives
would be especially useful for women with a poor response to FSH
stimulation, which includes women with high FSH, high estradiol
(E2), and diminished ovarian reserve ("DOR"). These women generally
show a poor response to ovarian stimulation regimens that employ
high FSH. Therapies to increase oocyte yield and/or ovarian reserve
would also be useful in animals.
[0008] As women age, serum FSH becomes elevated throughout the
menstrual cycle. From the mid-30s to the early 40s, high FSH occurs
in the context of regular cycles and the diminution of fertility
that occurs prior to the premenopausal onset of irregular
cyclicity. Since FSH mediates the process by which oocytes prepare
for meiotic chromosome segregation, FSH may play a role in
regulating fidelity of chromosome segregation. However, for many
years, OB/Gyn physicians have regarded high FSH as an epiphenomenon
of ovarian aging that is not a cause of egg infertility. Instead,
ovarian aging processes inherent within the oocyte that cause
molecular damage to the chromosome segregation apparatus were
deemed as the root cause of egg infertility. Accordingly, most
physicians regard high FSH as associated with, but not causative of
ovarian aging.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The drawing referenced herein form a part of the
specification.
[0010] FIG. 1 is a graph of data demonstrating the FSH-lowering
effects of ActRIIB:Fc in ovariectomized SAMP8 female mice.
[0011] FIG. 2 is a graph of data demonstrating the FSH-lowering
effects of ActRIIB:Fc in midlife SAMP8 mice.
[0012] FIG. 3 is a graph of data demonstrating the enhancing effect
of ActRIIB:Fc on oocyte yield in midlife SAMP8 mice.
[0013] FIG. 4 is a series of photomicrographs of the effects of
ActRIIB:Fc on chromosomes and spindles in ovulated oocytes from
SAMP8 mice.
[0014] FIG. 5 is a graph of data demonstrating decrease in spindle
misalignments in oocytes from SAMP8 mice treated with
ActRIIB:Fc.
[0015] FIG. 6 is a graph of data demonstrating the effect of
ActRIIB:Fc on litter size in midlife SAMP8 mice.
SUMMARY
[0016] Described herein are novel female fertility therapies. A
first aspect of the invention is directed to therapies that include
FSH lowering methodologies to prevent and/or treat oocyte, embryo
and fetal aneuploidy, female infertility due to advanced maternal
age, diminished ovarian reserve, premature ovarian failure, and/or
recurrent implantation failure, miscarriages, and trisomic
pregnancies (collectively referred to herein as "egg
infertility."). A second aspect of the invention is directed to
agents that bind to activin, bind to receptors that bind activin,
or that otherwise disrupt activin signaling (collectively referred
to herein as "activin pathway modifier agents", "APM agents") and
methods utilizing these agents to prevent and/or treat egg
infertility. A third aspect of the invention is directed to methods
of administering an effective amount of an APM agent to a subject
to increase oocyte yield and/or ovarian reserve, which may be used
with assisted reproductive technologies to treat female infertility
with an APM agent. All three aspects of the invention may be used
in human subjects and in animal subjects. Additional aspects of the
invention include therapeutic drug kits for treatment of humans and
animals based on the methodologies described above.
DETAILED DESCRIPTION
[0017] Described herein are novel female fertility therapies. A
first aspect of the invention is directed to therapies that include
follicle-stimulating hormone ("FSH") lowering methodologies to
prevent and/or treat oocyte and embryo aneuploidy, infertility,
miscarriages, and trisomic pregnancies (collectively referred to
herein as "egg infertility"). A second aspect of the invention is
directed to agents that bind to activin, bind to receptors that
bind activin, or that otherwise disrupt activin signaling
(collectively referred to herein as "activin pathway modifier
agents", "APM agents") which are utilized to prevent and/or treat
egg infertility. A third aspect of the invention is directed to
methods of administering an effective amount of an APM agent to a
subject to increase oocyte yield and/or ovarian reserve, which may
be used with assisted reproductive technologies to treat female
infertility. All three aspects of the invention may be used in
human subjects and in animal subjects. Additional aspects of the
invention include therapeutic kits comprised of FSH lowering
medications or comprised of APM agents, to treat subjects,
including human subjects and animal subjects. Embodiments of the
kits contain the medications in an easy to use format for
administration based on the methodologies described herein. These
medications include FSH lowering agents and/or APM agents and may
also include adjunct medications such as luteinizing hormone ("LH")
and/or human chorionic gonadotropin ("hCG") or a gonadotropin
releasing hormone analogs to trigger ovulation; aromatase
inhibitors; and progesterone, estrogen, and testosterone and their
pharmaceutically acceptable derivatives.
[0018] An embodiment of the invention is directed to methods of
treating or preventing egg infertility employing agents that lower
FSH. Another embodiment of the invention is directed to methods of
treating or preventing egg infertility employing APM agents. Some
agents useful in embodiments of the invention include both FSH
lowering and APM properties, although others either have FSH
lowering properties or APM properties. Methods employing
FSH-lowering agents and APM agents may also be considered
individually because modification of activin pathway activities by
APM agents may exert their therapeutic effects by means that are at
least in part separable from their FSH-lowering properties.
[0019] In embodiments of the invention, the FSH lowering agents,
APM agents, or both the FSH lowering agents and the APM agents may
be used to treat subjects (including human subjects and animal
subjects) to accomplish at least one of the following two
goals:
[0020] (1) Prevent oocyte, embryo, and fetal aneuploidy. This
includes oocyte, embryonic, and fetal aneuploidy in subjects with
diminished ovarian reserve, high FSH levels, elevated estradiol
levels, recurrent implantation failure, premature ovarian failure,
prior miscarriages, and prior aneuploid conceptuses including
trisomic pregnancies and births.
[0021] (2) Prevent and/or treat infertility and increase fertility.
This includes subjects with diminished ovarian reserve, high FSH
levels, elevated estradiol levels, recurrent implantation failure,
premature ovarian failure, prior miscarriages, and prior aneuploid
conceptuses including trisomic pregnancies and births.
[0022] Another embodiment of the invention is directed to the use
of an APM agent to increase oocyte yield and/or ovarian reserve,
which may be useful in facilitating assisted reproductive
technologies, such as IUI and IVF in humans and animals.
[0023] The methods described herein include the steps of
administering an effective amount of a FSH lowering agent and/or an
APM agent to a female subject for at least a portion of at least
one reproductive cycle, and in some embodiments, for at least one
complete reproductive cycle, prior to attempting conception.
[0024] Alternatively, the FSH-lowering agents and/or APM agents are
administered to the subject for two or more cycles or a portion of
two or more cycles, with the pregnancy attempt avoided until the
end of the final cycle of treatment. This is done so that poor
quality eggs are eliminated and the "ideal" egg that has been
nurtured to grow in a young microenvironment is the egg with which
pregnancy is attempted.
[0025] In other embodiments, the FSH-lowering agents and/or the APM
agents may also be administered to the subject for the period of
egg growth (several menstrual or estrous cycles prior to ovulation
in humans and animals, respectively), again with the subject
avoiding pregnancy until the final cycle of treatment. The
exemplary APM agent ActRIIB:Fc is effective in preventing egg
aneuploidy and increasing fertility when administered for the
duration of the window of egg growth. The basis for therapeutic
activity of APM agents is not necessarily based on FSH-lowering
properties that many of them may work, in theory, by an independent
mechanism.
[0026] The same methodologies (above) apply to both FSH lowering
agents and APM agents and that it is intended for the methods to
induce the growth of multiple follicles as well as for the
prevention of egg aneuploidy and infertility.
[0027] An alternative method of FSH lowering utilizes the
administration of a gonadotropin releasing hormone antagonist.
Exemplary gonadotropin releasing hormone antagonist(s) ("GnRHant")
include cetrorelix, ganirelix, abarelix, elagolix, and/or
degarelix. In embodiments of the invention, one or more GnRHants
are administered along with low dose LH activity (LH or hCG) and a
surge dose of LH activity in the peri-ovulatory period; but no FSH
is needed. This is because GnRHants partially lower FSH while
strongly suppressing LH. Administering GnRH analog with LH activity
will normalize FSH through the cycles of treatment. In embodiments
of this alternative, the treatment is conducted for at least two
cycles, in both the follicular and luteal phases, prior to the
pregnancy attempt, as described above.
[0028] In embodiments of the invention, the FSH lowering agent, the
APM agent, or combinations thereof, are administered at a dose
effective to provide sustained suppression of FSH for several weeks
of treatment while not suppressing FSH menstrual cycling in the
subject. In other words, the dose of the FSH lowering agent, the
APM agent, or combinations thereof is administered in an amount
effective to result in the desired outcome, i.e., at least one of
treating egg infertility, preventing egg infertility, or to
increase oocyte yield and/or ovarian reserve, while maintaining
cyclicity in the subject during the period of treatment. Depending
on the metabolic rate of the FSH lowering agent, the APM agent, or
combinations thereof in the subject, dose of the agent or agents
may need to be repeatedly administered so that the serum levels
necessary to obtained the desired result is maintained for the
desired duration.
[0029] Another aspect of the invention is directed to kits for
treating female infertility. An embodiment of a kit contains
medications that lower FSH in a formulation and combination to
prevent and treat egg infertility. Another embodiment of the kit
contains APM agents in a formulation and combination to prevent and
treat egg infertility. Another embodiment of the kit contains APM
agents in a formulation to induce the growth of multiple follicles
and increase oocyte production for use with assisted reproduction
methods. Some embodiments of the kits may be used to treat human
subjects and other embodiments may be used to treat animal
subjects. Some embodiments of these kits may also include adjunct
medications such as LH, hCG, estrogen, progesterone, and/or
testosterone or their derivatives, aromatase inhibitor, and/or a
GnRH analog to induce ovulation and support reproductive
function.
[0030] Patient compliance and therapeutic success of treatments
with FSH-lowering drugs and APM agents will be facilitated by drugs
provided in a kit format that simplifies the therapeutic regimen
for the human subject. Kits will also facilitate drug
administration for animal owners and caretakers in a setting such
as a farm or zoo location. It will be appreciated that kits with
different concentrations of medications and durations of
therapeutic treatment are formulated to be physiologically
applicable to the animal species for which the treatments are
intended.
[0031] An embodiment of the kit includes doses of FSH-lowering
agents and/or APM agents necessary to implement the treatments
described herein. The kits may include LH, hCG, or GnRH agonist to
induce ovulation, and may optionally include the medical
instruments, such syringes, necessary to administer the doses as
well as instructions for administering the doses.
[0032] The use of FSH-lowering agents and of APM agents to prevent
oocyte and embryo aneuploidy to treat and prevent female
infertility, miscarriages and trisomic births due to aneuploidy,
and to increase oocyte and/or ovarian reserve, and embryo yield has
not been previously described in the literature.
[0033] In embodiments of the invention, the APM agent is
ActRIIB:Fc. ActRIIB is a transmembrane receptor for activin.
ActRIIB:Fc (aliases include ACE-031 and ACVR2B) is a cloned soluble
chimera of ActRIIB that has the extracellular domain, lacks the
transmembrane and cytoplasmic kinase domains, and is fused to an
IgG Fc sequence. ActRIIB:Fc is an activin decoy receptor that
sequesters activin and inactivates activin signaling. In
embodiments of the invention, ActRIIB:Fc, or derivatives thereof
which may include amino acid deletions or substitutions that do not
negatively affect activin binding, is administered at a dose
effective to provide sustained suppression of FSH for several weeks
of treatment while not suppressing FSH menstrual cycling in the
subject. In other words, the dose of ActRIIB:Fc is administered in
an amount effective to result in the desired outcome, i.e., at
least one of treating egg infertility, preventing egg infertility,
or to increase oocyte yield and/or ovarian reserve, while
maintaining cyclicity in the subject during the period of
treatment. In embodiments, the dosage is given at a dose to
maintain serum levels of ActRIIB:Fc between 4 mg/kg and 10 mg/kg,
such as 7 mg/kg, plus or minus 1 mg/kg. Accordingly, depending on
the metabolic rate of ActRIIB:Fc in the subject, ActRIIB:Fc may
need to be repeatedly administered so that the serum levels
necessary to obtain the desired result is maintained for the
desired duration.
[0034] Conventional paradigms in the field point away from FSH as a
cause of aneuploidy and instead implicate molecular damage to the
egg and the follicle accumulated over many years. The conventional
thought in the OB/Gyn field is that the egg itself becomes old due
to molecular damage that inherently occurs during the process of
aging. These degenerative processes include cohesin defects,
telomere shortening, mitochondrial damage, reactive oxygen species
(ROS), and spindle defects. The current thinking is that this
damage takes years to accumulate and cannot be reversed by a short
term clinical treatment of the patient. The notion that preventable
mechanisms contribute to oocyte aneuploidy has not been previously
appreciated by those in the field, and treatment of oocyte
aneuploidy was even considered to be impossible. Thus, therapies
employing FSH lowering agents and APM agents have not previously
been considered.
[0035] Examples of FSH-lowering agents and of APM agents to treat
egg infertility are listed in Table 1. The same APM agents listed
in the right column may also be used to increase oocyte and/or
ovarian reserve for assisted reproductive technologies.
TABLE-US-00001 FSH lowering agents and Activin pathway modifier
other FSH pathway inhibitors agents (APM agents) FSH beta chain
peptides ActRIIB:Fc (ACE-031) (e.g., aa90-95; 81-95; 33-53) and
derivative agents FSH receptor peptides ActRIIA:Fc (ACE-011;
sotatercept) (e.g., as 551-555; 533-555; and derivative agents;
TGFI3 650-653; 649-653) antagonists with altered ligand
specificities toward activin ligands (PRDC, gremlin, chordin,
noggin, etc.) Cetrorelix, Ganirelix, Abarelix, Follistatin,
including splice variants Elagolix, and/or Degarelix GnRH 288 and
315 and derivative agents antagonist to partially suppress FSH
(e.g., with or without fusion throughout treatment; plus low dose
Fc regions, domain modifications LH activity (LH or hCG); ovulation
or deletions) induction with LH or hCG, for two or more cycles of
treatment, where pregnancy is avoided until the last cycle of
treatment. Anti FSH antibodies FSTL3 and derivative agents Inhibins
A, B BYM338 and derivative agents; other inhibitors of Type II
activin receptors including anti-activin-receptor antibodies and
derivative agents; Activin receptor binding activin peptides and
related agents with dominant negative effect on activin receptor
signaling Follistatin, including splice variants ACE-083 and
derivative agents 288 and 315 and derivative agents (e.g., with or
without fusion Fc regions, domain modifications or deletions)
ActRIIB:Fc (ACE-031) Antibodies or other engineered agents (e.g.
camelids, adnectins) directed to bind and antagonize Activin A, B
and/or AB ActRIIA:Fc (ACE-011) ALK4/5/7 kinase inhibitors (e.g.,
SB431542, LY-2157299) 313-hydroxy-4-pregnen- Myostatin 20-one
(3HP), 313-hydroxy-4-pregnen-20- Dominant negative Activins or
Decoy one-3-acetate (3HPA) Ligands, including dominant negatives
and decoys of Activin A, Activin B, Myostatin, GDF11, Nodal agents
that cannot signal but still bind receptors. Activin peptide
antagonists. RNA interference towards Cerberus FSH, FSH receptor
Anti-MCillerian Hormone RNA interference directed towards
(AMH)-alias Mallerian- ActrIIA, ActrIIB, ALK4, Smad2 inhibiting
substance Smad 3, Activin A or Activin B Modified Activin
pro-domain
Example 1--Materials and Methods
[0036] SAM P8 mouse colony. Mice were housed and bred in-house at
the University of Maryland School of Medicine (UM) animal facility
in Baltimore and cared for according to the National Academy of
Sciences guidelines for compassionate use and care of laboratory
animals, using UM Institutional Animal Care and Use Committee
("IACUC")-approved animal use protocols as described. Prior to the
period before their use in experiments, female mice were housed at
a density of 3-5 mice/cage. The presence of male cages and the
housing of females in open cages accessible to male smell with a
low density of females per cage are needed to achieve regular
cyclicity of the SAMP8 female mice in the colony. Three or more
weeks before blood draws or oocyte retrievals, female mice were
housed in a room that contained open air cages containing 2 mice
per cage. Cages containing 3-5 intact males per cage (aged 2-9
months) were interspersed with cages containing female mice, in a
1:3 ratio of male to female cages. These conditions promote regular
cyclicity in the SAMP8 female mice.
[0037] Experimental bleeds, serum preparation, and quantization of
serum FSH levels. Survival bleeds and terminal bleeds, preparation
of mouse sera, and quantization of serum concentrations of FSH with
a Milliplex MAP Rat Pituitary magnetic bead panel (RPTGMAG-86K;
Millipore) were performed as according to manufacturer's
instructions. Standard pooled sera from ovariectomized rats were
included alongside test samples to normalize measurements between
assays and to assess intra- and inter-assay variability. Duplicate
FSH measurements were performed for each mouse sample as
described.
[0038] Purification of ActRIIB:Fc. ActRIIB:Fc was expressed in
Chinese hamster ovary cells and purified from conditioned media as
described.
[0039] Ovariectomy of female SAMP8 mice, and treatment of
ovariectomized mice with ActRIIB:Fc with several ActRIIB:Fc
dosages. Eighteen SAMP8 female mice aged 7.4-7.6 months were
ovariectomized according to standard methods. Mice were permitted
to recover for 2.3 weeks before commencement of injections. Mice
aged 8.2-8.5 months were injected subcutaneously (sc) with 0.1 ml
of sterile PBS saline solution. Survival bleeds were then performed
for control FSH measurements prior to ActRIIB:Fc injection.
[0040] Mice were then divided into two test groups comprised of 9
mice per group. Mice were injected sc with sterile PBS containing
ActRIIB:Fc. One test group received 4 mg/kg ActRIIB:Fc, and the
other test group received 10 mg/kg ActRIIB:Fc. Survival bleeds were
performed for both test groups 24 hours and 96 hours after
ActRIIB:Fc injection. Blood sera were prepared and FSH levels were
analyzed as described above.
[0041] Identification of estrous cycle days and estrous cycle
lengths in cycling mice by vaginal cytological analyses. Vaginal
smearing was performed to obtain cells for staining and cytological
analyses. Smearing and histological analyses stained smears was
performed as described.
[0042] Treatment of cycling mice with control saline and ActRIIB:Fc
and collection of blood sera for FSH measurements. Mice at a mean
age of 5.92 months of age (ranging from 5.6 to 6.2 months) were
injected subcutaneously with PBS (vehicle control). Blood was
collected as survival bleeds on the next morning of estrus 1-4 days
after the PBS injection, and processed to generate serum according
to methods described.
[0043] ActRIIB:Fc in PBS was injected subcutaneously into regularly
cycling SAMP8 female mice ranging from 5.99 to 6.35 months at a
dosage of 7 mg/kg. Blood was drawn 1-4 days later and processed to
generate serum samples on the next morning of estrus.
[0044] ActRIIB:Fc boosters were continued every 3-4 days after the
initial injection. The dosage of ActRIIB:Fc in the booster shots
was designed to supplement the ActRIIB:Fc remaining in the
bloodstream to restore a serum ActRIIB:Fc concentration of 7 mg/kg.
The amount of ActRIIB:Fc remaining in the bloodstream several days
after the ActRIIB:Fc injection was calculated using the equation
for exponential decay,
A=A.sub.oe.sup.-t/T
where t is the time elapsed since the prior injection (3 days or 4
days); A=the final serum concentration after the elapsed time t
since the prior injection; A.sub.o=7 mg/kg ActRIIB:Fc (initial
serum concentration of ActRIIB:Fc at the time of the first
injection); and T=15.87, the constant for exponential decay for
ActRIIB:Fc. This constant was calculated based on assuming an 11
day half-life of ActRIIB:Fc in mice. The booster dose of ActRIIB:Fc
was computed as 7 mg/kg minus A, where A is the dose in mg/kg of
ActRIIB:Fc remaining after the decay.
[0045] ActRIIB:Fc treatments were continued every 3-4 days using
this method for calculating booster dosage to reconstitute the
initial administered dosage until approximately 3 weeks of
treatment had been given, after which a final terminal bleed was
taken on the morning of estrus, usually the morning that oocytes
were collected from the mice at approximately 6.4 months of age
(age range of 6.2-6.6 months). Terminal blood collections were
performed according to standard methods.
[0046] Gonadotropin measurements in cycling SAMP8 mice--Duplicate
FSH measurements were performed for each mouse serum sample. To
assess FSH suppression, FSH levels were compared on the same cycle
day of the estrus cycle between serum samples of mice that had been
administered vehicle vs. ActRIIB:Fc. Number of mice:vehicle
control: 10; ActRIIB:Fc treated 1-4 days: 20 mice; ActRIIB:Fc
treated for 19-24 days: 9 mice.
[0047] Oocyte collection, processing and analyses. Oocytes were
collected from cycling SAMP8 female mice, processed, analyzed for
morphology, fixed, and stained according to standard methods. Mice
were smeared to characterize their estrous cycles. Mice were
injected IP with 5 IU hCG on the afternoon of proestrus to induce
synchronous ovulation for the mice undergoing oocyte retrievals.
Oocytes were collected 14 to 16 hours after hCG administration, and
were denuded of granulosa cells utilizing standard methods. Scoring
of oocyte morphology, removal of the zona pellucida, fixation,
immunofluorescent staining, and scoring of chromosome misalignments
and spindle aberrations were performed.
[0048] Analyses of SAMP8 litter sizes after treatment with
ActRIIB:Fc. Intact female SAMP8 mice aged 5.5-6 months were
injected intraperitoneally (ip) with ActRIIB:Fc at a dose of 7
mg/kg. This was followed by booster injections of ActRIIB:Fc
administered every 3-4 days to maintain the initial administered
dose of ActRIIB:Fc for 21-22 days, as described above. On the day
that the last injection was administered, the female mice were
housed with intact male SAMP8 mice aged 4.75 months for the
duration of 11 days. By these means females had the opportunity to
experience two evenings of proestrus in two different cycles in
which they could get pregnant. Males were then removed from the
female cages, and daily checks were initiated 10 days later and
continued for an additional 14 days.
[0049] Females were approximately 6.2-6.7 months of age at the
initiation of pairing with the male, and were 6.9-7.5 months of age
at parturition. Pups were counted daily each morning by 10 am. Pups
lived for several days with the dam, during which pup viability was
assessed. Litter sizes were compared to litter sizes of untreated
SAMP8 female mice where the untreated females and the males were of
the same age range as the experimental mice.
[0050] Results
[0051] ActRIIB:Fc suppresses FSH in ovariectomized SAMP8 mice. Mice
were injected with ActRIIB:Fc or with saline solution. FSH levels
were measured after 24 hours and 96 hours of ActRIIB:Fc treatment
for mice injected with ActRIIB:Fc dosages of 4 mg/kg and 10 mg/kg.
FSH levels in treated animals were compared to FSH levels in blood
sera drawn just before ActRIIB:Fc treatment was initiated. Mean FSH
levels prior to treatment were 275,717 pg/mI. FSH levels in mice
treated with 4 mg/kg ActRIIB:Fc declined to 94,861, a 65.5%
suppression (P<0.0001, two tailed Mann-Whitney Test; Table 2 and
FIG. 1).
[0052] Mean FSH levels were 156,570 after 96 hours of ActRIIB:Fc
treatment (P<0.0001), a 43.2% suppression. These data indicate
that ActRIIB:Fc causes FSH suppression that is sustained and also
somewhat attenuated such that 66% of the suppression that is
achieved at 24 hours was sustained at 96 hours
(100.times.(Untreated-96 hours treated)/(untreated-24 hours
treated)=66%). Mice given 10 mg/kg ActRIIB:Fc had 52,628 pg/ml FSH
after 24 hours of ActRIIB:Fc treatment, an 80.9% suppression of FSH
(P<0.0001 vs. untreated control). Suppression was compared to
control untreated values. Therefore FSH suppression by 10 mg/kg of
ActRIIB:Fc is 100% sustained for 96 hours of ActRIIB:Fc treatment.
P values comparing FSH levels for sera sustained at 96 hours, with
mean FSH of 52,144 pg/ml, an 81.1% suppression of FSH from mice
before vs. after ActRIIB:Fc treatment for 24 or 96 hours at 4 or 10
mg/kg of ActRIIB:Fc also showed statistically significant
differences, with P<0.0001 comparing untreated SAMP8 ovx mice to
treated mice at 4 or 10 mg/kg after either 24 or 96 hours of
treatment (Table 2). No significant suppression of luteinizing
hormone (LH) was detected in mice treated for 24 or 96 hours with 4
mg/kg or 10 mg/kg of ActRIIB:Fc.
TABLE-US-00002 ActRIIB:Fc ActRIIB:Fc Number of Mean P vs. P vs.
O.sub.k dosage, treatment mice tested FSH, SEM, un-treated
Untreated Suppression mg/kg time, hours Per group pg/ml pg/ml Mean
% CV (range) (unpaired) (paired test) vs. control 0 (control) NA 18
275,717 10,054 3.61 (0.06-16.9) NA NA NA 4 24 9 94,861 13,376 2.84
(2.1-6.5) <0.0001 <0.0001 65.6% 96 9 156,570 10,226 2.49
(0.70-5.7) <0.0001 0.0003 43.2% 10 24 9 52,628 2,886 3.43
(0.32-6.7) <0.0001 <0.0001 80.9% 96 9 52,144 9,223 4.69
(0-12.5) <0.0001 <0.0001 81.1%
[0053] In summary, Table 2 and FIG. 1 demonstrate that ActRIIB:Fc
suppresses serum FSH levels in ovariectomized SAMP8 female
mice.
[0054] ActRIIB:Fc suppresses FSH in cycling midlife female SAMP8
mice. Based on the data from ovariectomized mice it was predicted
that a dosage of ActRIIB:Fc that is between 4 and 10 mg/kg such as
7 mg/kg would provide sustained suppression of FSH for several
weeks of treatment in cycling mice while not suppressing FSH
excessively, so that cyclicity might be maintained in the mice
during the period of treatment. Midlife SAMP8 mice ranging from 5.9
to 6.4 months of age were given 7 mg/kg ActRIIB:Fc treatment.
ActRIIB:Fc treated mice exhibited a significant suppression of FSH
levels on the day of estrus, from a mean of 40,773 pg/ml to a mean
of 7,906 pg/ml after 1-4 days of ActRIIB:Fc treatment, a 73.6%
inhibition (Table 3 and FIG. 4; P<0.0001).
[0055] A test group of SAMP8 mice received booster shots of
ActRIIB:Fc every three days. By these means, ActRIIB:Fc levels were
maintained between approximately 5.8 to 7 mg/kg for a period of 19
to 24 days. Mice continued to cycle during ActRIIB:Fc treatment.
Terminal bleeds were performed on the morning of estrus ("three
week treatment test group," 19-24 days after initial ActRIIB:Fc
injection depending on when the morning of estrus occurred). SAMP8
mice appeared healthy and showed no signs of pathology in
necropsies performed immediately after terminal bleeds were
performed. Mice have been treated with 10/mg/kg of ActRIIB:Fc for
over a month without toxicity (Lee S J, unpublished data). FSH
levels in mice treated with ActRIIB:Fc for 19-24 days were
significantly lower than in vehicle-treated mice, with a mean of
40,773 pg/ml in vehicle treated mice, vs. 22,100 pg/mI in
ActRIIB:Fc treated mice (Table 3 and FIG. 2; P=0.0002). The
percentage suppression for mice treated with ActRIIB:Fc for 19-24
days was 47.3% relative to the vehicle treated mice, less than for
1-4 day treated test group. LH levels were not significantly
different between vehicle-treated mice, and mice treated with
ActRIIB:Fc.
TABLE-US-00003 ActRIIB:Fc Number Mean treatment of mice FSH % time
tested pg/ml SEM Mean % CV (range) P Suppression 0 (vehicle
treated) 10 40,773 4,469 5.3% (1.31-14.72%) NA NA 1-4 days 20 7,906
1,778 2.1% (0-4.8%) <0.0001 73.6 19-24 days 9 22,100 2,355 2.05%
(0-4.81%) 0.0002 47.3
[0056] In summary, Table 3 and FIG. 2 demonstrate that ActRIIB:Fc
suppresses FSH in midlife SAMP8 mice after 1-4 days of treatment
and after 19-24 days of treatment.
[0057] ActRIIB:Fc increases yield of oocytes and of viable oocytes,
and it increases ovarian reserve in midlife SAMP8 mice to exceed
the yields in young SAMP8 mice. Ovulated oocytes were retrieved
from the reproductive tracts of SAMP8 mice. Nearly all oocytes
recovered were mature eggs that had a polar body. All oocytes were
freshly ovulated, with 0/881 of all oocytes exhibiting
post-ovulatory aging. No apoptotic oocytes were found in either
test group. The yield and viability were compared between oocytes
retrieved from young SAMP8 mice aged 2-3 months and midlife SAMP8
mice aged 6.1-8.68 months. A statistically significant decline was
observed in mean total oocyte yield per mouse with age, from 12.05
per mouse aged 2-3 months to 11.25 per mouse aged 6-8 months, a
decrease of 1.41 oocytes per mouse (P=0.0155, Mann-Whitney test;
Table 3, FIG. 3). The mean number of viable oocytes from
cumulus-oocyte complexes ("COCs") declined commensurately, from
11.25 per mouse to 9.86 per mouse in the same age groups, a
decrease of 1.39 oocytes/mouse (P=0.0119). Almost all oocytes that
were not viable were dead, with just 5 of all oocytes recovered
that were abnormal due to denudation of cumulus at the time of
retrieval (5/881 oocytes). The yield of non-viable and abnormal
oocytes (dead or denuded) was 0.8000 non-viable oocytes per mouse
from young mice and 0.7727 per mouse for midlife mice (P=0.4174,
not significant (NS)). Moreover, 225/241 of oocytes from the young
mice were viable oocytes (93.4%), vs. 217/234 from the midlife mice
(92.7%; P=0.4648 (NS), Fisher Exact test). These data indicate that
the decrease in yield of viable oocytes that occurs with age is
attributable to declining yield of total oocytes rather than to
declining viability of the oocytes that are ovulated.
[0058] Midlife mice were treated with 7 mg/kg of ActRIIB:Fc for
approximately 3 weeks (19-24 days). ActRIIB:Fc significantly
increased the mean total yield of oocytes in the midlife mice, from
10.64/mouse in untreated midlife mice to 14.33/mouse in ActRIIB:Fc
treated midlife mice, an increase of 3.7 oocytes/mouse (34.7%
increase, P=0.0031). The yield of viable oocytes increased
commensurately, from 9.86/mouse in untreated midlife mice to 12.73
per mouse, an increase of 2.9 oocytes/mouse (29.1% increase,
P=0.0051). The yield of non-viable/abnormal oocytes/mouse was not
significantly different between ActRIIB:Fc treated midlife mice and
untreated mice (1.6 non-viable oocytes/mouse treated with
ActRIIB:Fc vs. 0.7727 non-viable oocytes/untreated mouse, a
difference of 0.83/mouse, P=0.2131 (NS)). 191/215 of oocytes from
the ActRIIB:Fc-treated midlife mice were viable oocytes (88.8%),
vs. 217/234 from the midlife mice (92.7%; P=0.1023 (NS)). These
data indicate that the increase in oocyte yield caused by
ActRIIB:Fc in midlife mice is predominantly attributable to an
increase in the yield of viable oocytes.
[0059] The yield of total oocytes in midlife mice treated with
ActRIIB:Fc exceeds the yields from untreated young mice by a
statistically significant margin, with 14.33 total oocytes per
mouse from ActRIIB:Fc treated midlife mice vs. 12.05 per mouse from
young mice (2.28 more oocytes per midlife mouse treated with
ActRIIB:Fc-treated mouse, P=0.0292). The yield of viable oocytes
from ActRIIB:Fc-treated midlife mice also significantly exceeds
that from young mice, with 12.73/mouse from ActRIIB:Fc treated mice
vs. 11.25/mouse from untreated young mice (1.48 more oocytes per
midlife ActRIIB:Fc treated mouse than per young mouse; P=0.0456).
The yield of non-viable eggs/mouse is not significantly different
between the test groups (1.6/mouse in ActRIIB:Fc treated mice vs.
0.8 in young mice, an increase of 0.80/mouse (P=0.1280, NS), nor is
the fraction of viable and non-viable eggs (191/215 (88.8%) viable
oocytes/total oocytes for ActRIIB:Fc treated midlife mice vs.
225/241 (83.4%) viable oocytes/total oocytes for young mice;
P=0.0619). These data indicate that ActRIIB:Fc administered to
midlife mice with declining oocyte yield and viability restores
yield of total oocytes and of viable oocytes to levels that are
statistically higher than those of young SAMP8 mice.
TABLE-US-00004 Change In number Midlife + of eggs Midlife + Test
Group Midlife ActRIIB:Fc P after Young Midlife P Young ActRIIB:Fc P
Total mice (N) 22 15 -- 20 22 20 15 Total eggs/mouse 10.64 14.333
0.0031 +3.7 12.05 10.64 0.0155 12.05 14.333 0.0292 Viable
eggs/mouse 9.86 12.733 0.0051 +2.9 11.25 9.86 0.0119 11.25 12.733
0.0292 Non-viable eggs/mouse 0.7727 1.60 0.2131 +0.96 0.8000 0.7727
0.4174 0.8000 1.60 0.1280 Fraction of viable 21 7/2 34 191/215
0.1023 -- 225/241 217/234 0.4648 225/241 191/215 0.0619 eggs (%)
(92.7%) (88.8%) (93.4%) (92.7%) (93.4%) (88.8%) Fraction of 17/234
24/215 0.1023 -- 16/241 17/234 0.4648 16/241 24/215 0.0619
non-viable eggs (%) (7.26%) (11.2%) (6.63%) (7.26%) (6.63%)
(11.2%)
[0060] Table 4. Illustrates that ActRIIB:Fc treatment of midlife
mice increases total yield of oocytes and of viable oocytes,
restoring the yield to levels that are significantly greater than
those of young SAMP8 mice. Yield of ovulated oocytes was compared
between untreated young SAMP8 mice (N=20), untreated midlife mice
(N=22), and midlife mice treated with 7 mg/kg dosage of ActRIIB:Fc
for approximately 3 weeks (N=15; 19-24 days), with termination of
treatment defined by the morning of oocyte recovery. P denotes P
values comparing median numbers of oocytes recovered in the various
test groups using a Mann-Whitney test. P values and changes in
number of eggs after ActRIIB:Fc treatment are shown in bold for
sample comparisons that display significant differences.
[0061] FIG. 3 demonstrates that treatment of midlife SAMP8 female
mice with ActRIIB:Fc enhances oocyte yield. Test groups comprised
of midlife mice, midlife mice treated with ActRIIB:Fc, and young
mice are labeled on the x-axis. Gray bar graphs: Viable oocytes.
Black bar graphs: non-viable/abnormal oocytes. ActRIIB:Fc restores
the yield of viable oocytes to midlife mice and exceeds the yields
of viable oocytes that are recovered from young mice. Error bars
are SEM for viable oocytes (Normal) and for non-viable oocytes
(Abnormal).
[0062] ActRIIB:Fc therapeutically reduces the rates of chromosome
misalignments and spindle aberrations in ovulated oocytes from
midlife SAMP8 mice.
[0063] Midlife SAMP8 mice have elevated FSH compared to young SAMP8
mice. Chromosome misalignments and spindle aberrations are highly
predictive of impending aneuploidy. If chronic elevation of FSH
increases the rates of oocyte chromosome misalignments and spindle
aberrations in midlife mice, then the treatment of midlife mice for
the duration of oocyte growth with the FSH-lowering agent
ActRIIB:Fc (about 3 estrous cycles) would be expected to decrease
the rates of chromosome misalignments and spindle aberrations in
freshly ovulated oocytes. Freshly ovulated oocytes from SAMP8 mice
were stained to visualize chromosomes and spindles and examined in
immunofluorescence microscopy (FIG. 4). Each individual oocyte was
scored as having either well-aligned or misaligned meiotic
chromosomes and spindles, according to methods described. Treatment
of midlife mice with ActRIIB:Fc at a dose of 7 mg/kg for 19-24 days
significantly decreased the rate of chromosome misalignments, from
31/193 (16.1%) in the untreated mice, to 11/159 (6.9%) in the
ActRIIB:Fc-treated mice (P=0.0060), a 2.33-fold suppression (FIGS.
4 and 5). The fraction of oocytes from ActRIIB:Fc-treated mice that
had with misaligned chromosomes was also significantly lower than
that which was observed in the young mice, who had 26/210 (12.4%;
P=0.0200). These data demonstrate that ActRIIB:Fc lowers the rate
of oocyte chromosome misalignments below that of both midlife and
young SAMP8 mice.
[0064] The frequency of spindle aberrations in SAMP8 oocytes
significantly increases with age. 1/195 (0.51%) oocytes from young
SAMP8 exhibit spindle aberrations, vs. 14/188 (7.4%) of oocytes
from midlife SAMP8 mice, a 14.5-fold increase (P=0.0003). The rate
of spindle aberrations in oocytes from midlife SAMP8 mice is
significantly decreased by ActRIIB:Fc treatment, to 4/152 (2.63%),
a 2.8-fold decrease (P=0.0200). The rate of spindle aberrations in
the oocytes of midlife mice after ActRIIB:Fc treatment is
statistically indistinguishable from that of the young mice
(P=0.1179, NS). ActRIIB:Fc thus restores the rate of spindle
aberrations in midlife SAMP8 mice to that of young SAMP8 mice.
[0065] FIG. 4 demonstrates that treatment of midlife SAMP8 mice
with ActRIIB:Fc for 19-24 days restores organization of chromosomes
and spindles in ovulated oocytes. Regularly cycling mice were
injected with hCG on the afternoon of proestrus after 19-24 days of
ActRIIB:Fc treatment to trigger ovulation. Freshly ovulated oocytes
were recovered from ovarian ampullae on the morning of estrus.
Oocytes were fixed, stained, and examined in fluorescence
microscopy. A and B: Oocytes from midlife SAMP8 mice. C, D, E and
F: Oocytes from midlife SAMP8 mice treated with ActRIIB:Fc. G and
H: Oocytes from untreated young mice.
[0066] FIG. 5 demonstrates that treatment of midlife SAMP8 female
mice with ActRIIB:Fc significantly reduces the incidence of
chromosome misalignments in ovulated oocytes. Chromosome
misalignments were scored in individual oocytes examined in
fluorescence microscopy. Fraction and percentage of oocytes with
misaligned chromosomes is significantly lower in mice treated with
ActRIIB:Fc. P value was calculated with a Fisher exact test. In
midlife SAMP8 female mice 14 of 188 oocytes (7/4%) had spindle
aberrations. In SAMP8 mice treated with ActRIIB:Fc, only 4 of 152
oocytes had spindle aberrations. ActRIIB:Fc resulted in a
significant (P=0.02) 2.8 fold reduction in oocytes with spindle
aberrations.
[0067] The data further demonstrate that treatment of midlife SAMP8
female mice with ActRIIB:Fc significantly reduces the incidence of
spindle aberrations in ovulated oocytes.
[0068] ActRIIB:Fc improves fertility lost with age in SAMP8 female
mice. SAMP8 female mice display a significant decline in litter
sizes with age, from a mean of 8.22 pup per litter at the age of
3-4 months, to a mean of 5.06 pups/litter by the age of 5.8 months
(P<0.0001). The median number of pups per litter also decreased
from 9 per litter in young SAMP8 to 5 per litter in midlife SAMP8.
Regular estrous cyclicity is observed in both age groups.
[0069] Treating midlife mice with ActRIIB:Fc restores some of the
fertility that is lost with age. Three weeks of treatment of
midlife female SAMP8 mice with ActRIIB:Fc significantly increased
the fertility of midlife SAMP8 mice. Litter sizes increased to a
mean of 6.3 pups/litter and a median of 7 pups per litter, a 39-50%
recovery of litter size (P=0.0306).
[0070] FIG. 6 demonstrates that ActRIIB:Fc increases the fertility
of midlife SAMP8 mice. Midlife SAMP8 female mice were treated for 3
weeks with ActRIIB:Fc and then mated with young SAMP8 males.
Control young and midlife mice were untreated prior to mating with
young males. Number of pups per litter was counted in each test
group, Mating plugs in SAMP8 mice are difficult to discern, so the
mice that mated could not be definitively distinguished from those
that had not mated. Therefore, dams that had no litters were
excluded from the dataset. P value was calculated with a
Mann-Whitney test. Young SAMP8 mice (3-4 months) had an average
litter size of 8.22 pups (N=27 litters, Median 9 pups/litter).
Midlife untreated SAMP8 mice (5.8 months) had an average litter
size of 5.061 pups (N=33 litters, Median=5 pups/litter). Midlife
SAMP8 mice treated with ActRIIFB:Fc had an average litter size of
6.294 pups (N=17 litters, Median+7). The P valve for the midlife
untreated and the midlife treated was 0.0306.
[0071] While the present invention has been illustrated by the
description of one or more embodiments thereof, and while the
embodiments have been described in considerable detail, they are
not intended to restrict or in any way limit the scope of the
appended claims to such detail. Additional advantages and
modifications will readily appear to those skilled in the art. The
invention in its broader aspects is therefore not limited to the
specific details, representative apparatus and method and
illustrative examples shown and described. Accordingly, departures
may be from such details without departing from the scope or spirit
of the general inventive concept.
* * * * *