U.S. patent application number 09/272945 was filed with the patent office on 2001-12-20 for method of treating reproductive disorders.
Invention is credited to MAACK, CHRISTOPHER A..
Application Number | 20010053762 09/272945 |
Document ID | / |
Family ID | 21872740 |
Filed Date | 2001-12-20 |
United States Patent
Application |
20010053762 |
Kind Code |
A1 |
MAACK, CHRISTOPHER A. |
December 20, 2001 |
METHOD OF TREATING REPRODUCTIVE DISORDERS
Abstract
This is a method for treating reproductive disorders associated
with a failure of gamete maturation in subjects by administering a
composition containing a complex of insulin-like growth factor
(IGF) and insulin-like growth factor binding protein-3
(IGFBP-3).
Inventors: |
MAACK, CHRISTOPHER A.; (EL
CERRITO, CA) |
Correspondence
Address: |
Beth A. Burrous
FOLEY & LARDNER
3000 K street, N.W.
suite 500
Washington
DC
20007
US
|
Family ID: |
21872740 |
Appl. No.: |
09/272945 |
Filed: |
March 18, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09272945 |
Mar 18, 1999 |
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09033836 |
Mar 3, 1998 |
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6110833 |
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09033836 |
Mar 3, 1998 |
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08854354 |
May 12, 1997 |
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08854354 |
May 12, 1997 |
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08468048 |
Jun 6, 1995 |
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08854354 |
May 12, 1997 |
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08315198 |
Sep 29, 1994 |
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08315198 |
Sep 29, 1994 |
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08101255 |
Aug 3, 1993 |
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Current U.S.
Class: |
514/9.9 ;
435/336; 514/10.1; 514/10.2; 514/8.6; 514/8.7; 514/8.9;
530/303 |
Current CPC
Class: |
A61K 38/30 20130101;
H01L 27/11521 20130101 |
Class at
Publication: |
514/3 ; 530/303;
435/336 |
International
Class: |
A61K 038/28; C12N
005/16 |
Claims
1. A method for treating a subject for a reproductive disorder
associated with a failure of gamete maturation, said method
comprising administering to said subject a complex comprising an
insulin-like growth factor (IGF) and insulin-like growth factor
binding protein-3 (IGFBP-3), said complex being administered in an
amount sufficient to alleviate said condition.
2. The method of claim 1 wherein the complex comprises equimolar
amounts of IGF and IGFBP-3.
3. The method of claim 1 wherein the IGF is IGF-I.
4. The method of claim 3 wherein the IGF is recombinant human
IGF-I.
5. The method of claim 1 wherein thee IGFBP-3 is recombinant human
IGFBP-3.
6. The method of claim 1 wherein the IGF is IGF-II.
7. The method of claim 6 wherein the IGF-II is recombinant human
IGF-II.
8. The method of claim 6 wherein the IGFBP-3 is recombinant human
IGFBP-3.
9. The method of claim 1 wherein the administration is
parenteral.
10. The method of claim 9 wherein the parenteral administration is
performed by subcutaneous injection.
11. The method of claim 1 wherein the sufficient amount is that
amount of complex that results in an increase in serum estrogen in
females or serum androgen in males.
12. The method of claim 1 wherein the amount of complex
administered is at least about 0.05 mg IGF/kg of body
weight/day.
13. The method of claim 1 wherein said subject is a mammal.
14. The method of claim 1 wherein the disorder is polycystic
ovarian disease.
15. The method of claim 1 wherein the disorder is anovulation.
16. The method of claim 1 wherein the disorder is treat with
standard gonadotropin stimulation, superovulation, in vitro
fertilization and embryo transfer therapies.
17. The method of claim 1 wherein the disorder is poor response to
standard gonadotropin stimulation of normal ovulation.
18. The method of claim 1 wherein the disorder is male
infertility.
19. The method of claim 18 wherein the disorder is hypogonadotropic
hypogonadism.
20. The method of claim 1 wherein the disorder is also associated
with failure to respond to standard gonadotropin therapy.
Description
FIELD OF THE INVENTION
[0001] This process relates generally to the field of medical
therapy and particularly to the treatment of reproductive disorders
by administering a therapeutic composition containing a complex of
an insulin-like growth factor (IGF) and an insulin-like growth
factor binding protein (IGFBP).
BACKGROUND ART
[0002] Growth factors are polypeptides which stimulate a wide
variety of biological responses (eg. DNA synthesis, cell-division,
expression of specific genes, etc.) in a defined population of
target cells. A variety of growth factors have been identified
including transforming growth factor-.beta.1 (TGF-.beta.1),
TGF-.beta.2, TGF-.beta.3, epidermal growth factor (EGF),
platelet-derived growth factor (PDGF), fibroblast growth factor
(FGF), insulin-like growth factor-I (IGF-I), and IGF-II.
[0003] IGF-I and IGF-II are related in sequence and structure, with
each polypeptide having a molecular weight of approximately 7500
daltons. IGF-I mediates the effects of growth hormone and thus is
the primary mediator of growth after birth. IGF-I has also been
implicated in the actions of various other growth factors, since
treatment of cells with such growth factors leads to increased
production of IGF-I. In contrast, IGF-II is believed to have a
major role in fetal growth. Both IGF-I and IGF-II have insulin-like
activities (hence the name) and are mitogenic (stimulate cell
division) for the cells in reproductive tissue, muscle, skeletal
tissue and a wide variety of other tissues.
[0004] Unlike most growth factors, the IGFs are present in
substantial quantity in the circulation, but only a very small
fraction of this IGF is found in the free form in the circulation
or in other body fluids. Most circulating IGF is bound to an IGF
binding protein called IGFBP-3. IGF-I may be measured in blood
serum in which it is found at abnormally low levels in abnormal
growth-related conditions, eg, pituitary gigantism, acromegaly,
dwarfism, various growth hormone deficiencies, etc. Although IGF-I
is produced in many tissues, most circulating IGF-I is believed to
be synthesized in the liver.
[0005] Almost all IGF circulates in a non-covalently associated
ternary complex composed of IGF-I or IGF-II, IGFBP-3, and a larger
protein termed the acid labile subunit (ALS). This complex is
composed of equimolar amounts of each of the three components. The
ALS has no direct IGF binding activity and appears to bind only a
preformed IGF/IGFBP-3 complex. The ternary complex of
IGF+IGFBP-3+ALS has a molecular weight of approximately 150,000
daltons. This ternary complex is alleged to function in the
circulation "as a reservoir and a buffer for IGF-I and IGF-II
preventing rapid changes of free IGF." See, Blum, W. F., et al.,
"Plasma IGFBP-3 Levels as Clinical Indicators", In Modern Concepts
in Insulin-Like Growth Factors, (E. M. Spencer, ed., Elsevier,
N.Y.) pages 381-393, 1991.
[0006] Nearly all of the IGF-I or- IGF-II and IGFBP-3 in the
circulation are in complexes, so very little free IGF or IGFBP-3 is
detectable. Moreover, a high level of free IGF in plasma is
undesirable. It would lead to serious hypoglycemia because IGF has
insulin-like effects on circulating glucose levels. In contrast to
the IGFs and IGFBP-3, there is a substantial pool of free ALS in
plasma which assures that IGF/IGFBP-3 complex entering the
circulation immediately forms the ternary complex.
[0007] IGFBP-3 is the most abundant IGF-binding protein in the
circulation, but at least five other distinct IGF-binding proteins
(IGFBPs) have been identified in various tissues and body fluids.
Although these proteins bind IGFs, they each originate from
separate genes and have distinct amino acid sequences. Thus, the
binding proteins are not merely analogs of a common precursor.
Unlike IGFBP-3, the other IGFBPs in the circulation are not
saturated with IGFs. None of the IGF binding proteins other than
IGFBP-3 can form the 150 kd circulating ternary complex.
[0008] IGF-I and IGFBP-3 may be purified from natural sources or
produced from recombinant sources. For instance, IGF-I has been
purified from human serum for a number of years. See, Rinderknecht,
E. W., et al., Proc Natl Acad Sci (USA) 73, 2365-2369 (1976).
Recombinant IGF-I processes are shown in EPA 0,128,733, published
in December of 1984. IGFBP-3 may be purified from natural sources
using processes such as those shown in Baxter et al., Biochem.
Biophys. Res. Comm. 139, 1256-1261 (1986). IGFBP-3 may be
synthetically produced from recombinant sources as discussed in
Sommer, A. S., et. al., In Modern Concepts of Insulin-Like-Growth
Factors (E. M. Spencer, ed., Elsevier, New York), pages 715-728
(1991). This recombinant IGFBP-3 binds IGF-I in a 1:1 molar ratio.
The topical administration of the IGF-I/IGFBP-3 complex to rat and
pig wounds was significantly more effective than IGF-I alone.
Sommer et al., ibid. Subcutaneous administration of the complex to
hypophysectomized rats "substantially prevents the hypoglycemic
effects of IGF-I" administered alone. Sommer et al., ibid.
[0009] U.S. Pat. No. 5,037,805 issued to Ling discloses a method
for using a follicle-stimulating hormone inhibiting protein for
treating endometriosis and for use as a contraceptive. This protein
is identical to IGFBP-3. There is no suggestion to use this protein
in conjunction with IGF.
[0010] Patent Cooperation Treaty Publication No. WO 91/14704,
published on Oct. 3, 1991, and applied for by Applied Research
Systems ARS Holding N.V. discloses a method for using estrogen to
increase endogenous IGFBP-1 as a treatment for polycystic ovarian
disease (PCOD).
[0011] The theory was advanced that because PCOD is associated with
higher levels of free IGF-1 and lower levels of IGFBP-1,
administering estrogen to increase IGFBP-1 would decrease levels of
free IGF and reduce follicular overstimulation and overproduction
of androgens. The use of exogenous IGFBP-1 was not proposed.
[0012] All of the important elements of the IGF system are found in
the female reproductive organs of humans and other mammals,
including IGF-I and -II and the IGF binding proteins and receptors.
IGF-I and IGF-II have been implicated in the growth and
differentiated function of several of the tissues of these organs.
The presence of IGF-I and IGF-II in rat, pig and human ovarian
follicular fluid has been well documented. The presence of IGF-I
and IGF-II mRNAs in ovarian granulosa and theca-interstitial cells
suggests that at least some of the IGF found in follicular fluid
arises from local production in the surrounding cells. In fact, of
all of the tissues, in the rat, the ovary has the third highest
level of IGF-I mRNA. In humans, IGF-I synthesis may be restricted,
to the theca-interstitial cells, while IGF-II production appears to
occur only in granulosa-cells. The expression of the IGF-I genes in
the ovary seems to be under complex hormonal control. In vitro
studies indicate that growth hormone, gonadotropins, estrogen, and
epidermal growth factor stimulate production of murine and porcine
granulosa cell IGF-I; combination treatment often results in
a-synergistic stimulation of IGF-I gene expression There are some
inter-species differences observed in the hormonal control of IGF-I
production.
[0013] Type I IGF receptors, which act to transduce the mitogenic
and differentiation signals provided by the IGFs, have also been
demonstrated in mouse, pig and human ovarian granulosa and
theca-interstitial cells. The gonadotropins, follicle stimulating
hormone (FSH) and luteinizing hormone (LH), are pivotal hormones in
controlling the progression of differentiation events that lead to
a mature fertilizable ovum. FSH and LH have been shown to stimulate
an increase in the Type I IGF receptors in granulosa cells.
Substantial quantities of the Type II IGF receptor are found in the
ovary but the function of this receptor is obscure.
[0014] The third element of the IGF system, the IGF binding
proteins, are also synthesized in the ovary and are found in
ovarian follicular fluid. IGFBP-2, -3, -4, -5 and -6 have been
identified in porcine follicular fluid, while IGFBP-1, -2, -3 and
-4 are found in fluid from luteinizing human follicles. In the rat,
mRNAs for IGFBP-2, -3, -4 and -5 are detectable in the adult ovary
but IGFBP-1 is not. However, mRNAs for IGFBP-1, -2, -3, -4 and -5
are present in particular cell types in the human ovary. IGFBP-2,
-4, and -5 mRNAs were found primarily in granulosa cells from
atretic (i.e. degenerating) follicles. IGFBP-1 and IGFBP-3 mRNAs
were found in granulosa cells of dominant (i.e. mature) follicles
and IGFBP-3 mRNA, additionally in theca-interstitial cells.
[0015] As with IGF-I, the hormonal regulation of the IGFBPs is
quite complex. In rats, ovarian IGFBP-2 and -3 mRNA was reduced
after hypophysectomy, but subsequent treatment with
diethylstilbestrol (DES) led to an increase in IGFBP-2 mRNA and a
decrease in IGFBP-3 mRNA. Growth hormone treatment increased
IGFBP-3 mRNA in hypophysectomized rats, an effect which was
antagonized by DES or FSH. The response of the rat ovary and of
cultured rat ovarian granulosa cells to FSH was also complex, both
in vivo and in vitro. Low doses of FSH led to an increase in the
secretion of total IGFBPs, while higher doses depressed IGFBP
secretion.
[0016] As with many other tissues, IGF treatment of the various
cell types of the female reproductive system leads to both
mitogenesis and the expression of tissue-specific differentiated
functions. Presumably acting through the Type I IGF receptors, both
IGF-I and IGF-II have been shown to stimulate granulosa cell DNA
synthesis and proliferation in rats and pigs. Growth factors and
hormones (such as epidermal growth factor, platelet derived growth
factor, FSH and relaxin) can act synergistically with IGF-I to
augment this mitogenic effect.
[0017] The IGFs, either alone or with FSH or LH, can stimulate sex
steroid production in certain reproductive cells, such as granulosa
and theca-interstitial cells. For example, IGF-I alone stimulates
aromatase activity, as well as estrogen and progesterone synthesis,
in granulosa cells in a variety of species. In synergy with FSH or
LH, IGF-I treatment of granulosa cells 1) promotes a higher rate of
estrogen and progesterone production than occurs with either
hormone alone and 2) stimulates proteoglycan and LH receptor
production. Similarly, IGF-I acts synergistically with LH to
increase androgen production in cultured theca-interstitial cells.
In summary, many gonadotropin and sex steroid effects on ovarian
function may be facilitated or potentiated by endogenous or
exogenous IGFs in developing follicles.
[0018] IGF and IGFBP actions-have been studied in cultured rat
granulosa cells. Adding excess IGFBP-1, -2, or -3 to these cells
blocks the synergistic effects of IGF-I and FSH treatment on
itogenesis and steroid production. This presumably occurs by
competition for IGFs between the free IGFBPs and the IGF Type I
receptor.
[0019] Polycystic-ovarian disease (PCOD) has been extensively
studied. PCOD is a type of infertility characterized by
hyperandrogenism which prevents small ovarian follicles from
becoming dominant and maturing to the point of release and
fertilization. Several studies have analyzed IGF and IGFBP levels
in ovarian follicles at different stages of maturation in normally
ovulating and PCOD patients. Follicular fluids contain IGF-I and
IGFBP-2, -3, and -4, as well as a 29 kd IGFBP. IGFBP-3 is the
predominant IGFBP in follicular fluid. There were no differences in
IGF-I or IGFBP-3 levels in the fluid from atretic (degenerating)
follicles obtained from normal or PCOD patients compared to fluid
from healthy follicles from normal patients. However, the levels of
IGFBP-2, -4, and 29 kd IGFBP were elevated in fluid from atretic
follicles obtained from normal or PCOD patients compared to fluid
from healthy follicles from normal patients. The total IGF binding
capacity in atretic follicles was elevated compared to healthy
follicles. Similar results were seen in fluid obtained from healthy
and atretic follicles obtained from sheep. Since atretic follicular
fluid is seriously deficient in estrogen and IGF-I stimulates
production of estrogen by granulosa cells, it has been proposed
that the abundant IGF-I in atretic follicular fluid is inactive
because it binds to the increased IGFBP-2, -4, and 29 kd IGFBP.
This apparently occurs without any change in IGFBP-3, the
predominant IGFBP in follicular fluid.
[0020] In anovulatory women undergoing treatment to induce
ovulation, growth hormone may sensitize ovarian tissues to the
effects of gonadotropins. The standard therapy for ovulation
induction is treatment with a gonadotropin releasing hormone (GnRH)
agonist to suppress pituitary function, followed by repeated
treatments with gonadotropins (LH, FSH and human chorionic
gonadotropin (hCG)). The dose and length of gonadotropin treatment
for each patient is guided by serum steroid hormone levels and
follicular maturation as assessed by ultrasound. Several studies
have used growth hormone in conjunction with standard gonadotropin
therapy for ovulation induction. In a number of placebo-controlled
studies of anovulatory patients who had previously undergone
standard gonadotropin therapy, treatment with growth hormone
reduced the daily dose of gonadotropin and shortened the course of
treatment (resulting in a smaller cumulative gonadotropin dose)
required to stimulate normal ovulation compared to that required by
patients receiving placebo plus gonadotropins. This effect was
accompanied by an increase in the level of IGF-I in serum and
follicular fluid from growth hormone-treated patients compared to
placebo-treated patients.
[0021] Women undergoing induction of superovulation for in vitro
fertilization/embryo transfer (IVF/ET) have benefitted from
combined growth hormone and gonadotropin therapy, especially in
cases where their response to gonadotropin therapy alone has been
suboptimal. For these patients, the clinical endpoint is the
induction of multiple mature oocytes which can be successfully
fertilized in vitro. As discussed above, several studies have shown
that growth hormone with standard gonadotropin therapy
significantly reduced the amount of gonadotropin required to induce
superovulation in these "poor responders". Furthermore,
co-administration of growth hormone and gonadotropins increased the
number of mature oocytes that could be isolated from "poor
responders", the growth rate of the harvested oocytes, and the
success rate of cocyte fertilization in vitro and of viable
pregnancies after embryo transfer. As described above, adding
growth hormone to the standard therapeutic regimen significantly
increased serum and follicular fluid IGF-I levels. Moreover,
significantly higher IGF-I levels were found in follicular fluid
from mature follicles that fertilized and cleaved than in
follicular fluid from mature follicles that did not fertilize.
[0022] In contrast, the results of studies in normally ovulatory
women undergoing superovulation therapy for IVF/ET have been mixed.
Some studies have reported a reduced need for gonadotropins when
they were co-administered with growth hormone, and other studies
have reported no such reduction.
[0023] Growth hormone therapy has the disadvantage of causing
hyperglycemia, hyperinsulinemia, sodium retention, edema, and
growth hormone insensitivity when used for prolonged periods.
[0024] Although androgens, LH and FSH play major roles in the
development and functioning of the male reproductive system, the
importance of the growth hormone/IGF axis in male fertility is
demonstrated by the striking delay in puberty and resistance to
human chorionic gonadotropin (hCG) therapy experienced by boys with
growth hormone resistance.
[0025] As with the female reproductive system, all three elements
of the IGF system are present in the male reproductive tract. IGF-I
is synthesized by both major cell types of the testis (Leydig and
Sertoli cells). This synthesis appears to be primarily under the
control of the gonadotropins rather than growth hormone. Similarly,
the Type I IGF receptor is also present in both cell types, as well
as in early spermatids and secondary spermatocytes. The IGF type I
receptor is up-regulated by treatment with hCG. Finally, IGFBPs
(predominantly IGFBP-2 and -3) are produced in several testicular
cell types. In Sertoli cells IGFBP-2 and -3 expression can be
regulated by IGF-I and FSH. IGF-I and IGFBP-2 and -3 are also
produced in cultured prostatic cells and are found in seminal
plasma.
[0026] IGF-I affects function of a number of testicular cells. In
culture, IGF-I is mitogenic for. Sertoli cells and stimulates
plasminogen activator synthesis. IGF-I treatment of Leydig cells
leads to increased hCG receptors and to synergy with hCG in
stimulating androgen production.
[0027] The effects of IGF-I are not restricted to isolated cultured
cells. In growth hormone deficient dwarf mice, IGF-I treatment
increases testicular weight, LH receptor numbers, and the
steroidogenic response to hCG. IGF-I is important in
spermatogenesis: IGF-I treatment of sections of seminiferous
tubules in organ culture stimulated premitotic DNA synthesis in the
germ cells.
[0028] Growth hormone also has been used in male sterility. Four
men with hypogonadotropic hypogonadism who had not responded to
gonadtropins alone were given combined growth hormone and
gonadotropin therapy. All four had an increase in serum IGF-I
levels, three rapidly increased testosterone secretion, two
produced adequate amounts of sperm, and one patient impregnated his
wife. (Shoham et al., Fertility & Sterility 57:1044, 1992)
[0029] IGF given alone can cause hypoglycemia, cardiac arrhythmias
and suppress growth hormone, insulin and ALS production.
[0030] As indicated above, many cases of female or male infertility
caused by failures of gamete maturation are refractory to standard
gonadotropin therapy. Such cases include, but are not limited to,
polycystic ovarian disease patients and "poor responders" to
gnadotropin-induced superovulation among females, and
hypogonadotropic hypogonadism among males and require different or
additional therapy.
DISCLOSURE OF THE INVENTION
[0031] In accordance with one embodiment of the present invention,
there is provided a method for treating a subject for a
reproductive disorder associated with failure of gamete maturation,
wherein the subject is administered standard gonadotropin therapy
in conjunction with a complex comprising an insulin-like growth
factor (IGF) and insulin-like growth factor binding protein-3
(IGFBP-3) in an amount sufficient to alleviate the reproductive
disorder.
[0032] In accordance with another embodiment of the present
invention, the IGF used in the complex is provided as IGF-I. In a
further embodiment, IGF and IGFBP are present in equimolar amounts.
In still another embodiment, both IGF and IGFBP-3 are human
proteins obtained from recombinant sources.
[0033] In accordance with another embodiment of the present
invention, the complex of IGF and IGFBP-3 is administered
parenterally. In a further embodiment, the complex is administered
by subcutaneous injection.
[0034] In another embodiment, the subject to whom the complex is
administered is a mammal.
[0035] In yet another embodiment, the method provides for
administration of the IGF/IGFBP-3 complex in an amount sufficient
to increase serum estrogen in females and serum androgen in males
and thus improve fertility. In a further embodiment, the amount of
IGF/IGFBP-3 complex administered is about 0.01 to 10 mg/kg/day.
[0036] In still other embodiments, the method provides a treatment
for polycystic ovarian disease, anovulation and male
infertility.
[0037] While not wishing to be bound by any particular theory the
Inventors propose that the administered complex of IGF and IGFBP-3
results in the gradual release of free IGF in moderately elevated
levels. This can occur either before or after the circulating
IGF/IGFBP-3 complex is taken up into the testes or ovaries. The
added IGF in the ovaries or testes sensitizes these tissues to the
actions of gonadotropins, stimulates sex steroid production,
stimulates germ cell division, and improves maturation and
production of fertile mature oocytes and sperm.
MODES FOR CARRYING OUT THE INVENTION
[0038] Definitions:
[0039] As used herein, "reproductive disorders" are defined as
conditions that lead to reduced fertility due to the partial or
total failure to produce fully mature oocytes or sperm which are
capable of combining to produce a fertilized ovum. Such conditions
include, but are not limited to, polycystic ovary disease (PCOD),
failure to respond to gonadotropin therapy to induce normal
ovulation, failure to respond to gonadotropin therapy to induce
superovulation prior to in vitro fertilization/embryo transfer,
anovulation, hypogonadotropic hypogonadism, and male
infertility.
[0040] "Standard gonadotropin therapy" is defined as current
clinically accepted standard treatments of infertile patients with
a combination of gonadotropins and releasing factors. Such
gonadotropins and releasing factors include, but are not limited
to, follicle stimulating hormone (FSH), luteinizing hormone (LH),
human menopausal gonadotropin (hMG), human chorionic gonadotropin
(hCG) and gonadotropin releasing hormone (GnRH) agonists.
[0041] "Subjects" are defined as humans and mammalian farm animals,
sport animals and pets. Farm animals include, but are not limited
to, cows, hogs and sheep. Sport animals include, but are not
limited to, dogs and horses. The category pets includes, but is not
limited to, cats and dogs.
[0042] "Insulin-like growth factor (IGF)" comprises a family of
factors, including, but not limited to, IGF-I and IGF-II. IGF is a
polypeptide having a molecular weight of about 7500 daltons. IGF
may be obtained from natural sources or prepared by recombinant
means.
[0043] "Insulin-like growth factor binding proteins (IGFBP)"
comprises a family of binding proteins, including but not limited
to IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6. IGFBP
may be obtained from natural sources or prepared by recombinant
means. At least one form of IGFBP (for example, IGFBP-3) complexes
with IGF and with a third molecule known as ALS.
[0044] A "therapeutic composition" as used herein is defined as
comprising IGF complexed with its binding protein IGFBP-3. The
therapeutic composition also contains other substances such as
water, minerals and carriers such as proteins.
[0045] "Alleviation of the condition" is said to occur when the
subject to whom the IGF/IGFBP-3 complex is administered produces
mature gametes capable of combining with a competent oocyte or
sperm to produce a viable fertilized ovum. Alleviation has occurred
when a male with a reproductive disorder produces normal quantities
of normal, active sperm, or at least enough normal, active sperm to
impregnate a competent oocyte (one capable of fertilization).
Alleviation has occurred when a female with a reproductive disorder
ovulates and produces normal, competent oocyte(s). There are other
useful indicators of "alleviation," including, but not limited to,
normal levels of estrogens or androgens.
DESCRIPTION OF THE INVENTION
[0046] The method of the present invention contemplates treating
and alleviating infertility caused by defective gamete maturation
in males and females by administering a complex of IGF and IGFBP-3
in conjunction with standard gonadotropin therapy.
[0047] Nearly all IGF-I or IGF-II complexes with IGFBP-3.
IGF/IGFBP-3 normally circulates in the form of a complex in humans
and other mammals. This complex associates with a third protein
(ALS), which is present in excess over the molar concentration of
IGF and IGFBP-3. Therefore, ALS is found both associated with the
IGF/IGFBP-3 complex and in the free form. The resultant ternary
complex has a size of about 150 kd.
[0048] Administration of IGF and IGFBP-3, either from natural or
recombinant sources, as a preformed complex results in the
formation of the ternary complex with the excess ALS. This type of
treatment produces a prolonged increase in the level of circulating
IGF, which is gradually released from the ternary complex. This
mode of administration avoids the detrimental side effects
associated with administration of free IGF-I, e.g., hypoglycemia,
suppression of growth hormone and ALS production, and sudden
increase in free endogenous IGF-II released from normally
circulating IGF-II/IGFBP-3 complexes by equilibrium replacement
with exogenous free IGF-I in complexes with IGFBP-3.
[0049] The formulation, method of administration and dosage will
depend upon the disorder to be treated and the medical history of
the patient. These factors are readily determined in the course of
therapy. Suitable patients with reproductive disorders can be
identified by medical history, physical findings and laboratory
tests. The medical history may reveal such facts as delayed
puberty, female androgynism, irregular menstrual cycles, and
repeated failure to conceive. The female patient may have
polycystic ovarian disease, reduced response of immature ovarian
follicles to standard gonadotropin therapy and anovulation; whereas
the male patient may have a low sperm count. Indicative laboratory
results include low serum growth hormone or IGF levels or reduced
levels of serum estrogen (in females) or androgen (in males) in
response to standard gonadotropin therapy.
[0050] In accordance with the method of the present invention, the
formulation comprises a complex of IGF and IGFBP-3. Preferably, the
IGF is IGF-I, although IGF-II also is useful. Because IGF and
IGFBP-3 naturally complex in a 1:1 molar ratio, a composition of
equimolar amounts of IGF and IGFBP-3 is preferred. Nevertheless,
the product can be formulated with IGF:IGFBP-3 molar ratios ranging
from about 0.5 to 1.5. More preferably, the molar ratio is about
0.9 to 1.3; and most preferably, the product is formulated with
approximately a 1:1 molar ratio.
[0051] In accordance with the method of the present invention, the
IGF and IGFBP-3 are human proteins obtained from natural or
recombinant sources. Most preferably, IGF and IGFBP-3 are human
IGF-I and IGFBP-3 made by recombinant means and designated rhIGF-I
and rhIGFBP-3, respectively. rhIGFBP-3 may be in glycosylated or
non-glycosylated form. E. coli is a preferred source of
non-glycosylated IGFBP-3. Glycosylated IGFBP-3 is preferably
obtained from Chinese hamster ovary (CHO) cells.
[0052] The method of the present invention provides for formulating
the complex in modes which are readily apparent to those skilled in
the art. Preferably, the IGF and IGFBP-3 are complexed prior to
administration to the treated subject. Preferably, the complex is
formed by mixing approximately equimolar amounts of IGF-I and
IGFBP-3 dissolved in physiologically compatible carriers such as
normal saline solution or phosphate buffer saline solution. Most
preferably, a concentrated solution of rhIGF-I and a concentrated
solution of rhIGFBP-3 are mixed together for a sufficient time to
form an equimolar complex.
[0053] Depending on the mode of administration, compositions of the
complex may be in the form of solid, semi-solid or liquid dosage
preparations, such as for example, tablets, pills, powders,
capsules, liquids, suspensions or the like. Physiologically
compatible carriers include intravenous solutions, such as normal
saline, serum albumin, 5% dextrose, plasma preparations, and other
protein-containing solutions. The preferred carrier for parenteral
administration of the complex is a sterile, isotonic aqueous
solution, such as normal saline or 5% dextrose. Alternatively, a
solution of the complex may be placed into an implant, such as an
osmotic pump, for the slow release of the complex over an extended
period of time. Alternatively, the complex may be provided in
sustained release carrier formulations such as semipermeable
polymer-carriers in the form of suppositories or microcapsules.
See, for instance, U.S. Pat. No. 3,773,919 for Microcapsular
Sustained Release Matrices Including Polylactides; Sidmon et. al.,
Biopolymers 22 (13): 547-556 (1983) for copolymers of L-glutamic
acid and .gamma.-ethyl-L-glutamate; Langer et al., J Biomed Res 15:
167-277 (1981) for poly(2-hydroxyethylmethacrylate) or the
like.
[0054] The mode of administration delivers the complex to the
subject in a safe, physiologically effective manner. The complex
may be given by intranasal, subcutaneous, intravenous,
intramuscular, intraperitoneal, or other conventional routes of
administration. Preferably, the complex is injected subcutaneously,
intravenously or intramuscularly. Most preferably, the complex is
administered by subcutaneous injection. By subcutaneous injection,
the complex appears not to be toxic or mitogenic at the injection
site. Lack of mitogenic effect at the injection site is a distinct
improvement over scarring following the injection of IGF alone.
[0055] The IGF/IGFBP complex is administered in conjunction with
standard gonadotropin therapy, including gonadotropins and
releasing factors. Such gonadotropins and releasing factors
include, but are not limited to, follicle stimulating hormone
(FSH), luteinizing hormone (LH), human menopausal gonadotropin
(hMG), human chorionic gonadotropin (hCG) and gonadotropin
releasing hormone (GnRH) agonists. FSH is available as
Metrodin.RTM. urofollitropin (Serono Laboratories, Randolph,
Mass.). hMG is obtained from the urine of post-menopausal women and
includes equal activities of FSH and LH. hMG is available as
PERGONAL.RTM. menotropins (Serono Laboratories) and as HUMEGON.RTM.
menotropins (Organon Laboratories, Cambridge, U.K.). hCG is
commercially available as PROFASI.RTM. HCG for Injection (Serono
Laboratories), as well as under the brand names, CHORIGON.RTM.
(Ikafarm, Ramat-Gan, Israel) and PREGNYL.RTM. (Organon, West
Orange, N.J.). GnRH agonists include LUPRON.RTM. leuprolide acetate
(TAP Pharmaceuticals, Deerfield, Ill.) and DECAPEPTYL.RTM.
triptorelin (Wyeth-Ayerst Laboratories, Philadelphia, Pa.),
SUPREFACT buserelin acetate (Hoechst, Hounslow, U.K.). Prescribing
information is available in the current edition of the PHYSICIANS'
DESK REFERENCE and in the clinical literature.
[0056] For female subjects, this standard therapy typically
consists of ovarian suppression with a gonadotropin releasing
hormone agonist, followed by induction of oocyte maturation and
release with gonadotropin treatments. For example, the ovaries are
suppressed by daily subcutaneous injections of GnRH agonist for up
to 14 days starting in the early phase of the reproductive cycle.
For example, 200-500 .mu.g/d of SUPREFACT, 100-500 .mu.g/d
DECAPEPTYL or 500-1,000 .mu.g/d LUPRON can be administered. Ovarian
suppression is evaluated by measuring the serum estradiol level at
the end of this initial period. If the level is below about 30-50
.mu.g/ml, ovarian suppression has occurred and GnRH agonist
treatments continue as gonadotropin treatments are begun. If the
serum estradiol is above this level, GnRH treatment continues until
ovarian suppression is achieved and again continues during
gonadotropin treatment. Oocyte maturation is promoted by daily
intramuscular injections of human menopausal gonadotropin (e.g.
HUMEGON, PERGONAL, NEOPERGONAL, Serono, Levallois, France), a
mixture of LH and FSH usually containing 150-225 IU of each hormone
per ampule. Subjects are often give 2-3 ampules/day at first, with
the dose adjusted to ensure a steady rise in serum estradiol in
treated patients. Injections are given daily for at least 5-6 days
and continue until the oocytes are mature (serum estradiol above
400-500 .mu.g/ml and 1-3 follicles greater than 14-18 mm diameter).
Finally, ovulation is induced by a single intramuscular injection
of 5,000-10,000 IU of human chorionic gonadotropin (e.g. PROFASI,
PREGNYL or CHORIGON). Therapy with rhIGF-I/IGFBP-3 complex is
combined with standard gonadotropin therapy as discussed below.
[0057] Standard gonadotropin therapy for male subjects with
failures of spermatogenesis includes, for example, either pulsatile
dosing with luteinizing hormone releasing hormone (LH-RH) or
gonadotropin therapy in order to raise serum testosterone levels
and induce sperm maturation. LH-RH (e.g. FERTIRAL brand, available
from Hoechst, Hounslow, U.K.) can, for example, be given as 15
.mu.g subcutaneous pulses every 90 minutes with a self-driven pump.
For subjects who do not respond to LH-RH therapy, hMG can be
administered three times per week and hCG twice per week for 3-6
months by intramuscular injection. In other embodiments, these
regimens are combined with administration of the rhIGF-I/IGFBP-3
complex.
[0058] The appropriate dose of IGF/IGFBP complex can be readily
determined by those skilled in the art, based on the usual patient
symptoms and laboratory values discussed above. In addition,
patient estrogen and androgen levels may be helpful. Typically, the
peak serum estradiol levels in hyperstimulated female subjects
should rise to approximately 1,000-3,000 .mu.g/ml, or at least 200
.mu.g/ml/mature follicle. For treated male subjects, serum
testosterone levels should rise to approximately 300-1,000
.mu.g/dl; and the sperm count should increase toward the normal
20-250 million/ml, while motility should increase, preferably
toward 50%. Preferably, when the complex is administered to humans
daily, the dosage of complex is about 0.01 to 10 mg of IGF-I or
IGF-II/kg of body weight/day, complexed to an approximately
equimolar amount of IGFBP-3. More preferably, the daily dosage of
the complex for humans is about 0.05 to 7.5 mg IGF/kg/day,
complexed to an equimolar amount of IGFBP-3. Most preferably, the
daily dosage of the complex for humans is about 0.1 to 5 mg
IGF/kg/day, complexed to an equimolar amount of IGFBP-3. If daily
dosages in excess of about 0.5 mg IGF/kg must be given, the dosage
may be divided and injected subcutaneously at two or more
sites.
[0059] If, in conjunction with standard gonadotropin therapy, the
IGF/IGFBP-3 complex is administered to humans twice a week, each
dose of complex is preferably about 0.0 to 10 mg IGF/kg of body
weight, complexed to an equimolar amount of IGFBP-3. More
preferably, for twice weekly administration, the dose of the
complex is about 0.1 to 7.5 mg IGF/kg, complexed to an equimolar
amount of IGFBP-3. Most preferably, for twice weekly
administration, the dose of the complex is about 0.5 to 5 mg
IGF/kg, complexed to an equimolar amount of IGFBP-3. There is no
known upper limit of dosage; however, it is preferable that a
single dose not exceed 10 mg IGF/kg of body weight, when the IGF is
complexed to an equimolar amount of IGFBP-3. These doses of
IGF/IGFBP-3 complex are not expected to cause significant
hypoglycemia since the IGF-IGFBP-3 slowly releases IGF to cellular
insulin receptors.
[0060] Preferably, the infertile patient is started with a
relatively low dose of the complex, such as 0.05 mg IGF/kg of body
weight/day in conjunction with standard gonadotropin therapy. The
various factors given above should be monitored to determine if
there is improvement. Preferably, the patient produces sufficient
mature ovarian follicles or sufficient motile sperm following such
treatment. If the patient improves with the low dose, the low dose
preferably should be continued with standard gonadotropin therapy
until a successful conception by normal means or by in vitro
fertilization/embryo transfer is achieved. Such an outcome may
require several rounds of therapy through several reproductive
cycles.
[0061] If the patient does not respond to standard gonadotropin
therapy plus low dose IGF/IGFBP-3 complex with sufficient
production of mature oocytes or sperm, the dose of complex should
be increased gradually until such an outcome is achieved.
[0062] The invention has been disclosed by direct description.
Following are examples showing the efficacy of the IGF/IGFBP-3
complex in stimulating processes critical to the maturation of
oocytes and sperm and in stimulating fertility. The examples are
only examples and should not be taken in any way as limiting to the
scope of the process.
EXAMPLES
Example 1
[0063] This experiment shows the effect of the rhIGF-I/IGFBP-3
complex alone and in conjunction with follicle stimulating hormone
(FSH) on estradiol production in cultured ovarian granulosa cells
isolated from patients with polycystic ovarian disease (PCOD). The
level of estradiol, a crucial hormone in ovarian follicle
development, is low in the fluid from ovarian follicles isolated
from patients with PCOD. This is thought to cause the cessation in
follicular development that characterizes PCOD.
[0064] Granulosa cells are prepared from PCOD patients' ovaries
which have been removed during a total abdominal hysterectomy.
Fluid from individual follicles is collected by aspiration. Then
the follicles are dissected from the ovaries and cut open. From the
open follicles, granulosa cells are scraped and washed free from
the basal lamina. Cells from several follicles are pooled and
cultured in serum-free McCoy's 5a medium in multi-well culture
dishes for 2, 4, or 6 days. The estradiol precursor androstenedione
is added at 10.sup.-7 M and the rhIGF-I/IGFBP-3 complex at doses in
the range from 0 to 100 ng IGF-I/ml complexed to an equimolar
amount of IGFBP-3. FSH in the range from 0 to 100 ng/ml is added to
cultures with and without the rhIGF-I/IGFBP-3 complex. Estradiol
secreted into the culture medium is assayed by a standard
radioeimmunoassay (RIA).
[0065] Untreated cultures produce detectable estradiol for only the
first two-day culture period and not thereafter. When either the
rhIGF-I/IGFBP-3 complex or FSH are added to the cultures on day 0,
the cells respond with a dose-related increase in the production
and secretion of estradiol into the culture medium. The effect of
the combination of FSH and the IGF-I/IGFBP-3 complex on estradiol
production is substantially greater than either factor alone. This
effect is observed throughout the course of the culture period.
Example 2
[0066] This experiment shows the effect of the rhIGF-I/IGFBP-3
complex on superovulation prior to in vitro fertilization/embryo
transfer (IVF/ET) in "poor responder" patients. The patients in
this study are PCOD patients who had a suboptimal response (ie.
fewer than 6 oocytes collected and fewer than 4 embryos developed).
with at least one IVF/ET cycle of ovarian stimulation using the
standard combined regimen of gonadotropin releasing hormone (GnRH)
agonist and human menopausal gonadotropin (hMG).
[0067] The patients are assigned to one of two groups: One group
receives placebo and one group receives rhIGF-I/IGFBP-3 complex, in
addition to standard gonadotropin treatment for IVF/ET. The placebo
and rhIGF-I/IGFBP-3 complex (0.05 to 0.5 rhIGF-I/kg/day complexed
to an equimolar amount of rhIGFBP-3) are administered by daily
subcutaneous injection throughout the period of hMG treatment until
hCG administration. The standard gonadotropin therapy given to both
groups is as follows: At the beginning of the study, ovarian
suppression, as measured by reduced plasma estradiol levels, is
accomplished by GnRH agonist treatment. This treatment is
maintained daily until hCG treatment. Following the establishment
of ovarian suppression, hMG is administered at a rate of at least 3
ampules per day for at least 6 days. Increases in the daily dosage
or days of treatment are determined by the follicular response of
each subject as assessed by ultrasound scans of the patients'
ovaries. hCG is administered when plasma estradiol levels are
sufficiently elevated and at least three large follicles can be
detected. Oocytes are recovered by transvaginal aspiration and
subjected to the standard IVF/ET culture and fertilization
procedures.
[0068] Treatment of these "poor-responding" PCOD patients with the
rhIGF-I/IGFBP-3 complex results in a significant reduction in the
number of ampules of gonadotropin required to stimulate
superovulation compared to those treated with placebo. Furthermore,
more follicles develop, and more oocytes are collected, fertilize
and cleave in rhIGF-I/IGFBP-3 complex treated patients than in
placebo-treated patients. These data support the utility of using
the IGF/IGFBP-3 complex in the treatment of infertility.
Example 3
[0069] This experiment demonstrates that the rhIGF-I/IGFBP-3
complex increases testicular growth and steroidogenic response in
growth hormone deficient mice. Immature Snell dwarf mice, which are
growth hormone deficient and exhibit delayed testicular maturation,
are used in the study. The animals receive either placebo or
rhIGF-I/IGFBP-3 complex (1.0 to 10 mg rhIGF-I/kg/day complexed to
an equimolar amount of rhIGFBP-3) in daily subcutaneous injections
for 7 to 14 days. At the end of the treatment period, an hCG
injection is administered and blood and various organs are
collected. Plasma testosterone and IGF-I levels are measured using
standard RIA procedures and testicular hCG binding sites are
measured by a radioreceptor assay.
[0070] Compared to placebo-treated animals, administration of the
rhIGF-I/IGFBP-3 complex substantially increases circulating IGF-I
and testicular weight in the Snell dwarf mice. Leydig cell function
in rhIGF-I/IGFBP-3 complex treated mice is also stimulated compared
to placebo treated animals, as evidenced by a significant increase
in plasma testosterone levels following the hCG challenge, and in
testicular hCG binding sites. These results demonstrate that the
IGF/IGFBP-3 complex promotes testicular maturation.
Example 4
[0071] This experiment shows that the rhIGF-I/IGFBP-3 complex
promotes spermatogenesis in patients who have failed to respond to
standard gonadotropin therapy alone. The patients included in the
study are those diagnosed as suffering from hypogonadotropic
hypogonadism on the basis of delayed puberty and reduced serum
gonadotropin and testosterone levels. In addition, the included
patients have failed to respond to standard gonadotropin therapy
with improved levels of serum testosterone or sperm production.
[0072] Patients are treated with standard gonadotropin therapy
which consists of 24 weeks of thrice weekly intramuscular
injections of hMG, FSH and LH and twice weekly injections of hCG.
This standard therapy is supplemented with daily subcutaneous
injections of either placebo or rhIGF-I/IGFBP-3 complex (0.05 to
0.5 mg IGF-I/kg/day complexed to an equimolar amount of rhIGFBP-3)
for the treatment period. Blood and semen samples are collected
during weeks 12 and 24. Testicular volume is measured by
ultrasonography at the end of the treatment period. Serum IGF-I and
testosterone levels are measured by standard RIAs.
[0073] The patients receiving the rhIGF-I/IGFBP-3 complex in
addition to standard gonadotropin therapy show a significant
increase in serum IGF-I and testosterone concentrations compared to
placebo-treated patients, especially at 24 weeks. Semen volume and
sperm density also significantly increase in the rhIGF-I/IGFBP-3
complex treated group, although testicular volume does not increase
in all complex-treated patients. Semen volume and sperm density
increases have been associated with successful improvement in
fertility and demonstrate the utility of combined gonadotropin and
IGF/IGFBP-3 complex therapy in treating male infertility.
[0074] This invention has been detailed both by example and by
direct description. It should be apparent that one having ordinary
skill in this art would be able to surmise equivalents to the
invention as described in the claims which follow but which would
be within the spirit of the description above. Those equivalents
are to be included within the scope of this invention.
* * * * *