U.S. patent application number 10/173805 was filed with the patent office on 2003-09-11 for composition of an endogenous insulin-like growth factor-ii derivative.
This patent application is currently assigned to DAIICHI PHARMACEUTICAL CO., LTD.. Invention is credited to Hashimoto, Ryuji, Higashihashi, Nobuyuki, Sakano, Katsuichi.
Application Number | 20030170240 10/173805 |
Document ID | / |
Family ID | 18407338 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170240 |
Kind Code |
A1 |
Sakano, Katsuichi ; et
al. |
September 11, 2003 |
Composition of an endogenous insulin-like growth factor-II
derivative
Abstract
The effects of endogenous insulin-like growth factor can be
appreciated by administering compounds capable of increasing free
IGF in living bodies. Compounds are described which can elevate the
concentration of unbound IGF by converting endogenous IGF
(insulin-like growth factor) into free, biologically active IGF or
elevating the concentration of the complex of IGF and IGFBP
(insulin-like growth factor binding protein) in living bodies.
Medicaments can be prepared containing these compounds or these
compounds may be used in methods for the prevention and or
treatment of IGF-responsive diseases such as diabetes, amyotrophic
lateral sclerosis, or osteoporosis.
Inventors: |
Sakano, Katsuichi; (Tokyo,
JP) ; Higashihashi, Nobuyuki; (Tokyo, JP) ;
Hashimoto, Ryuji; (Tokyo, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 Pennsylvania Avenue, NW
Washington
DC
20037-3213
US
|
Assignee: |
DAIICHI PHARMACEUTICAL CO.,
LTD.
|
Family ID: |
18407338 |
Appl. No.: |
10/173805 |
Filed: |
June 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10173805 |
Jun 19, 2002 |
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09331851 |
Jun 28, 1999 |
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6428781 |
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09331851 |
Jun 28, 1999 |
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PCT/JP97/04881 |
Dec 26, 1997 |
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Current U.S.
Class: |
424/145.1 ;
514/16.9; 514/6.9; 514/8.5; 514/8.6; 514/8.7 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/4743 20130101; C07K 14/65 20130101 |
Class at
Publication: |
424/145.1 ;
514/12 |
International
Class: |
A61K 039/395; A61K
038/30 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 1996 |
JP |
8-349968 |
Claims
1. A method for elevating the concentration of a free insulin-like
growth factor, which comprises converting a binary complex composed
of an insulin-like growth factor and an insulin-like growth factor
binding protein in living bodies into the insulin-like growth
factor.
2. A method for elevating the concentration of a free insulin-like
growth factor, which comprises releasing, from a binary complex
composed of an insulin-like growth factor and an insulin-like
growth factor binding protein in living bodies, the insulin-like
growth factor.
3. A method for elevating the concentration of a free insulin-like
growth factor, which comprises inhibiting the binding of an
insulin-like growth factor and an insulin-like growth factor
binding protein in living bodies.
4. A method for elevating the concentration of a binary complex
composed of an insulin-like growth factor and an insulin-like
growth factor binding protein, which comprises converting a ternary
complex composed of the insulin-like growth factor, the
insulin-like growth factor binding protein and an acid labile
subunit in living bodies into the binary complex composed of the
insulin-like growth factor and insulin-like growth factor binding
protein.
5. A method for elevating the concentration of a binary complex
composed of an insulin-like growth factor and an insulin-like
growth factor binding protein, which comprises releasing, from a
ternary complex composed of the insulin-like growth factor, the
insulin-like growth factor binding protein and an acid labile
subunit in living bodies, the binary complex composed of the
insulin-like growth factor and insulin-like growth factor binding
protein.
6. A method for elevating the concentration of a binary complex
composed of an insulin-like growth factor and an insulin-like
growth factor binding protein, which comprises inhibiting the
binding of the binary complex of the insulin-like growth factor and
the insulin-like growth factor binding protein in living bodies to
an acid labile subunit.
7. A method for elevating the concentration of a free insulin-like
growth factor, which comprises converting a ternary complex
composed of an insulin-like growth factor, insulin-like growth
factor binding protein and an acid labile subunit in living bodies
into the insulin-like growth factor.
8. A method for elevating the concentration of a free insulin-like
growth factor, which comprises releasing, from a ternary complex
composed of an insulin-like growth factor, insulin-like growth
factor binding protein and an acid labile subunit in living bodies,
the insulin-like growth factor.
9. A method for elevating the concentration of a free insulin-like
growth factor, which comprises inhibiting the binding of an
insulin-like growth factor, insulin-like growth factor binding
protein and an acid labile subunit in living bodies.
10. A compound which converts a binary complex composed of an
insulin-like growth factor and an insulin-like growth factor
binding protein in living bodies into a free insulin-like growth
factor.
11. A compound which releases a free insulin-like growth factor
from a binary complex composed of an insulin-like growth factor and
an insulin-like growth factor binding protein in living bodies.
12. A compound which inhibits the binding of an insulin-like growth
factor and an insulin-like growth factor binding protein in living
bodies.
13. A compound which converts a ternary complex composed of an
insulin-like growth factor, an insulin-like growth factor binding
protein and an acid labile subunit in living bodies into a binary
complex composed of the insulin-like growth factor and insulin-like
growth factor binding protein.
14. A compound which releases, from a ternary complex composed of
an insulin-like growth factor, an insulin-like growth factor
binding protein and an acid labile subunit in living bodies, a
binary complex composed of the insulin-like growth factor and
insulin-like growth factor binding protein.
15. A compound which inhibits the binding of a complex of an
insulin-like growth factor and an insulin-like growth factor
binding protein in living bodies to an acid labile subunit.
16. A compound which converts a ternary complex composed of an
insulin-like growth factor, an insulin-like growth factor binding
protein and an acid labile subunit in living bodies into a free
insulin-like growth factor.
17. A compound which releases, from a ternary complex composed of
an insulin-like growth factor, insulin-like growth factor binding
protein and an acid labile subunit in living bodies, a free
insulin-like growth factor.
18. A compound which releases a free insulin-like growth factor by
inhibiting the binding of an insulin-like growth factor, an
insulin-like growth factor binding protein and an acid labile
subunit in living bodies.
19. A compound which substantially binds neither to an insulin-like
growth factor receptor nor to an insulin receptor but binds to an
insulin-like growth factor binding protein.
20. An insulin-like growth factor derivative, which substantially
binds neither to an insulin-like growth factor receptor nor to an
insulin receptor but binds to an insulin-like growth factor binding
protein.
21. An insulin-like growth factor derivative, which substantially
binds neither to an insulin-like growth factor receptor nor to an
insulin receptor but binds to an insulin-like growth factor binding
protein, and has an amino acid sequence similar to an insulin like
growth factor except for the addition, depletion or substitution of
one or more than one amino acid residue.
22. An insulin-like growth factor derivative, which substantially
binds neither to an insulin-like growth factor receptor nor to an
insulin receptor but binds to an insulin-like growth factor binding
protein, and has an amino acid sequence similar to human
insulin-like growth factor-II except that the 27-th tyrosine
residue and 43-rd valine residue each has been substituted with a
leucine residue.
23. An anti-insulin-like growth factor binding protein antibody,
which substantially binds neither to an insulin-like growth factor
receptor nor to an insulin receptor but binds to an insulin-like
growth factor binding protein.
24. An anti-insulin-like growth factor binding protein antibody,
which substantially binds neither to an insulin-like growth factor
receptor nor to an insulin receptor but binds to an insulin-like
growth factor binding protein-3.
25. A medicament, which comprises a compound as recited in any one
of claims 10 to 19.
26. A medicament, which comprises an insulin-like growth factor
derivative as recited in any one of claims 20 to 22.
27. A medicament, which comprises an anti-insulin-like growth
factor binding protein antibody as recited in claim 23 or 24.
28. A screening method of a compound as recited in any one of
claims 10 to 19, which comprises labeling any one of the
insulin-like growth factor, insulin-like growth factor binding
protein and acid labile subunit so as to be directly or indirectly
detectable.
Description
TECHNICAL FIELD
[0001] The present invention relates to compounds and methods for
elevating endogenous insulin-like growth factors and their
activities in living bodies.
BACKGROUND ART
[0002] Insulin-like growth factors (hereinafter "IGF") are found in
two distinct molecular forms called IGF-I and IGF-II, respectively.
Human IGF-I and IGF-II are 70 and 67 amino acids in length,
respectively. Compared to IGF-II, IGF-I has three more amino acids
at the site corresponding to the C peptide, which is a partial
structure of insulin. The amino acid sequence homology between
IGF-I and IGF-II is about 60%, while that between IGF-I and insulin
is about 40%. Although the liver and kidney are the major sites of
production for IGF-I in living bodies, northern blot analysis of
mRNA has revealed that IGF-I is produced by almost all tissues in
the body (D'Ercole, A. J., et al., Proc. Natl. Acad. Sci. USA. 81,
935 (1984); Humbel, R. E., et al., Eur. J. Biochem., 190, 445
(1990)). IGF-I is considered to act not only as an endocrine factor
but also as paracrine or autocrine factor.
[0003] IGF-I and IGF-II bind to distinct and specific receptors; an
IGF-I receptor and an IGF-II/cation-independent mannose-6-phosphate
receptor, respectively. However, since IGF-II has also been shown
to bind to the IGF-I receptor, the various biological activities
associated with IGF-II are thought to occur mainly through the
IGF-I receptor located on cell surface (Casella, S. J., et al., J.
Biol. Chem., 261, 9268 (1986); Sakano, K., et al., J. Biol. Chem.,
266, 20626 (1991)).
[0004] The IGF-I receptor shares a high degree of amino acid
sequence homology with the insulin receptor and the two molecules
resemble each other in their intracellular signal transduction
mechanism (Shemer, J., et al., J. Biol. Chem., 262, 15476 (1987);
Myers, M. G. Jr., et al., Endocrinology, 132, 1421 (1993)). IGFs
regulate glucose metabolism predominantly in the peripheral tissue,
which is different from insulin, as shown in animal model studies.
The receptors for IGFs and insulin are differentially localized in
tissues, and this may explain why the biological effect of IGFs in
the body is distinguishable from insulin's effect (Laager, R., et
al., J. Clin. Invest., 92, 1903 (1993)).
[0005] The blood of an average adult human contains about 100 nM of
IGF and about 100 pM of insulin (Baxter, R. C., in Modern Concepts
of Insulin-Like Growth Factors (Spencer, E. M., ed) pp.371,
Elsevier Science Publishing Co., New York-Amsterdam (1991)). Most
IGFs found in living bodies form complexes with an IGF-binding
protein (hereinafter "IGFBP"). It appears that a specific binding
protein exists for each IGF. The hypoglycemic effect of free IGF or
unbound IGF is about 5 to 10% of that of insulin (Guler, H. P. et
al., New Engl. J. Med., 317, 137 (1987)), indicating that
insulin-like growth factors are at concentrations of about 50 to
100-fold greater than insulin (Baxter, R. C., in Modern Concepts of
Insulin-Like Growth Factors (Spencer, E. M., ed) pp.371, Elsevier
Science Publishing Co., New York-Amsterdam (1991)).
[0006] The World Health Organization has classified the disease,
Diabetes mellitus, into roughly three categories on the basis of
their distinct clinical patterns:
[0007] (1) Insulin-dependent diabetes mellitus (hereinafter
"IDDM")
[0008] (2) Non insulin dependent diabetes mellitus (hereinafter
"NIDDM")
[0009] (3) Other diabetes mellitus (derived from pancreato-pathy
diseases or endocrinopathy)
[0010] A method for treating IDDM involves insulin therapy, while
diet therapy, kinesitherapy, or treatment with an oral hypoglycemic
agent or with insulin is mainly used in the treatment of NIDDM. In
recent years, IGF-I therapy has been considered as an alternative
treatment for insulin-dependent diabetes mellitus in cases where
administration of insulin alone is not effective (Kuzuya, H., et
al. , Diabetes 42, 696 (1993)). Also for NIDDM, effects of IGF have
been under investigation (Zenobi, P. D., et al., J. Clin. Invest.,
.90,. 2234 (1992) ; Moses, A. C., et al. , Diabetes, 45,
91(1996)).
[0011] Guler et al. observed that the intravenous injection of
IGF-I into adult humans in an amount of 100 .mu.g/kg resulted in
the lowering of blood glucose levels with the lowest level
occurring after 20 minutes (Guler, H. P., et al., New Engl. J.
Med., 317, 137 (1987)).
[0012] Takano et al. observed that hypoglycemic activity was
observed in adult humans following the subcutaneous injection of
IGF-I in an amount of 60 to 120 .mu.g/kg, and that administration
of IGF-I every 6 days in an amount of 100 .mu.g/kg lowered the uric
acid and creatinine levels in blood (Takano, K., et al.,
Endocrinol. Jpn., 37, 309 (1990)).
[0013] In addition, there are reports on the lowering of free fatty
acid levels in blood (Turkalj. I., et al., J. Clin. Endocrinol.
Metab., 75, 1186 (1992)), the lowering of neutral fats such as
triglyceride (Turkalj. I., et al., J. Clin. Endocrinol. Metab. 75,
1186 (1992); Zenobi, P. D., et al., J. Clin. Invest., 90, 2234
(1992)), and the lowering in total cholesterol level (Zenobi, P.
D., et al., Diabetologia 36, 465 (1993)). Increases in renal blood
flow and glomerular filtration rate (Elahi, D., et al., in Modern
Concepts of Insulin-Like Growth Factors (Spencer, E. M., ed) pp219,
Elsevier Science Publishing Co., New York-Amsterdam (1991)), have
been reported for IGF-I.
[0014] There is also a report that the administration of IGF-II was
effective for intractable diabetes mellitus (Usara, A., et al.,
Diabetes, 44, Suppl. 1, 33A, 1995)). Results from animal model
studies suggest the effectiveness of IGF-I in the reduction of
conditions associated with stress including glucose metabolism at
the time of hemorrhagic shock, the alleviation of side effects
caused by sugar infusion (Unexamined Japanese Patent Publication
(KOKAI) No. Hei 7-242565).
[0015] Administering IGF to animals has helped to identify the
numerous biological activities of IGF including hypoglycemic
activity, induction of proliferation, cell differentiation, and
anobolic activity. Local administration of IGF-I to the injured
peripheral nervous system results in the proliferation of
non-neural cells while stimulating neurons. It is reported that
IGF-I receptors are present on spinal cells and that administration
of IGF-I decreases cell death of motor neurons. In addition, it is
recognized that the administration of IGF increases the muscular
end plate, promotes the functional recovery of a damaged sciatic
nerve and prevents peripheral motor paralysis observed during
chemotherapy (Sjoberg, J., et al., Brain Res. , 485, 102
(1989).
[0016] Based on these foregoing experimental observations in the
peripheral nervous sytem, clinical tests using IGF-I in the
treatment of amyotrophic lateral sclerosis and degenerative
diseases of the motor neuron have been conducted (Lewis, M. E., et
al., Exp. Neurol., 124, 73(1993)). Similarly, the use of IGF in
promoting the survival of neuronal cells is recognized as being
important in the treatment of Alzheimer's disease, apoplexy,
amyotrophic lateral sclerosis, Parkinson's disease and the like
(Unexamined Japanese Patent Publication (KOHYO) No. Hei 6-510305).
In addition, the effectiveness of IGF-I in the treatment of
muscular dystrophy has also been reported (Vlachopapadopoulou, E.,
et al., J. Clin. Endocrinol. Metab., 80, 3715 (1995)).
[0017] The effects of IGF on diabetic neuropathy have also been
studied. In an IDDM rat model (STZ-rat: streptozotocin-diabetic rat
model), alleviation of diabetic neuropathy was observed when IGF
was administered at concentrations that did not lower blood glucose
levels (Zhuang, H-X, et al., Exp. Neurol., 140, pp198-205 (1996)).
It has also been reported that administration of IGF in an NIDDM
rat model (diabetic obese Zucker (fa/fa) rat), reduced the level of
IGF-II mRNA in the sciatic nerve, spinal nerves and brain nerves,
and alleviated the diabetic neuropathy when used at a concentration
that did not result in lowering of blood glucose levels (Zhuang,
H-X, et al., J. Pharmacol. Exp. Ther., 283, pp366-374 (1997)).
These findings suggest that IGF is effective in the treatment of
diabetic neuropathy.
[0018] The effects of IGF have also been studied on cardiac
function. When doxorubicin is administered to rats, it causes
myocardiopathy, but administration of IGF-I improves myocardial
function (Ambler, G. R., et al., Cardiovasc. Res., 27, 1368
(1993)). Consequently, IGF-I is thought to be useful in the
prevention and treatment of myocardiopathies including myocarditis
and myocardial infarction, cardiac disease, and acute attack via
its ability to increase cardiac rate and improve cardiac output
(Unexamined Japanese Patent Publication (KOHYO) No. Hei
6-504286).
[0019] The effects of IGF-I on acute renal insufficiency caused by
ischemia have also been reported. On day 5 after an ischemic
attack, IGF-I was administered three times daily by subcutaneous
injection for three days. The result was that IGF improved renal
function, promoted formation of new renal tubules, inhibited
proteolysis and promoted protein synthesis, and decreased
catabolism (Ding, H., et al., J. Clin. Invest., 91, 2281
(1993)).
[0020] It has also reported that the local administration of IGF-I
to a skin injury, i.e., wounds, burn injuries or the like, reduces
the length of recovery. In a burn injury model, the administration
of rat IGF-I increased body weight, weight of the enteromucosa,
mucosal DNA and protein expression, and decreased the transfer of
enterobacterium to the intestinal lymph gland, thereby improving
intestinal function and life prognosis (Huang, K. F., et al., Arch.
Surg., 128, 47 (1993)).
[0021] Together with a platelet-derived growth factor (hereinafter
"PDGF"), IGF-I promotes mitosis and protein synthesis of cultured
mesenchymal cells, and although curing of skin disorders is not
promoted by the single use of PDGF or IGF-I, the combined use of
both factors promotes the growth of connective and epithelial
tissues (Stiles, C. D., et al., Proc. Natl. Acad. Sci. USA, 76,
1279 (1987)). In another report, however, the single application of
either one of these growth factors was shown to stimulate wound
healing (Tsuboi, R., et al., J. Exp. Med., 172, 245 (1990)).
Therefore, attempts have been made to use IGF for promoting wound
healing (Unexamined Japanese Patent Publication (KOKAI) No. Sho
63-233925, Unexamined Japanese Patent Publication (KOHYO) Nos. Hei
3-505870 and Hei 6-506191, and Unexamined Japanese Patent
Publication (KOKAI) No. Hei 7-316066).
[0022] In addition, IGF-I is effective at improving immune
function. IGF-I is produced in the thymus and sites of inflammation
and is considered to be important in the regulation of
proliferation and function for T lymphocytes expressing the IGF-I
receptor (Tapson, V. F., et al., J. Clin. Invest., 82, 950 (1988)).
It is reported that IGF-I promotes the proliferation of lymphocytes
at nanomolar concentrations (Schimpff, R. M., et al., Acta
Endocrinol. 102, 21 (1983)). Accordingly, the use of IGF-I for
treatment of immunodeficient patients including AIDS patients is
under investigation (Unexamined Japanese Patent Publication (KOHYO)
No. Hei 6-508830).
[0023] Moreover, IGF-I is considered to be effective in the
treatment of osteoporosis since increases in bone mass have been
associated with IGF (Bennett, A. E., et al., J. Clin. Endocrinol.
Metab., 59, 701 (1984); Brixen, K., et al., J. Bone. Miner. Res.,
5, 609 (1990); Johannsson, A. G., et al., J. Intern. Med., 234, 553
(1993); Johannsson, A. G., et al., Lancet, 339, 1619 (1992); Riggs,
B. L., Am. J. Med., 95, Suppl.5A, 62S, (1993); Unexamined Japanese
Patent Publication (KOKAI) No. Hei 4-235135 and U.S. Pat. No.
4,861,757).
[0024] However, it is recognized that in their naturally occurring
state, almost all of the IGFs form complexes with IGFBP in living
bodies, which effectively serves to regulate their physiological
activity in vivo (Rechler, M. M., Vitam. Horm., 47, 1 (1993);
Clemmons, D. R., Growth Regul. 2, 80 (1992)).
[0025] Six IGFBPs (designated "IGFBP-1 to IGFBP-6") have been
identified thus far, and each of them exhibits a high degree of
amino acid sequence identity or homology. The homology is markedly
similar in the N-terminal and C-terminal regions where many of the
cysteine residues are located, while the proteins are much less
homologous in their intermediate domains. For all six human IGFBPs,
the respective positions for sixteen (16) cysteine residues is
conserved (IGFBP-1 to IGFBP-5 have conserved 18 cysteine residues)
(Shimasaki, S., et al., Prog. Growth Factor Res., 3, 243
(1991)).
[0026] The concentrations of IGFBP-1, IGFBP-2 and IGFBP-3 in the
blood of an adult human are about 2 nM, 5 nM and 100 nM,
respectively, and IGFBP-3 is a major binding protein for IGF
(Baxter, R. C., in Modern Concepts of Insulin-Like Growth Factors
(Spencer, E. M., ed) pp371, Elsevier Science Publishing Co., New
York-Amsterdam (1991)). When normal human serum is fractionated by
gel filtration under neutral conditions, the IGFs elute in the
vicinity of 150 kDa and are found as a ternary complex (Baxter, R.
C., et al., Proc. Natl. Acad. Sci. USA, 86, 6898 (1989)). This
ternary complex is composed of IGF-I (or IGF-II) (m.w. of about 7.5
Kda), IGFBP-3 (m.w. of 53 Kda and inert to acid) and a subunit
protein (m.w. of 84 KDa and labile to acid (Acid Labile Subunit or
".alpha.-subunit"; hereinafter "ALS")). It is hypothesized, that
when IGF binds to IGFBP-3, the major binding protein in blood, ALS
binds to this binary complex to form a ternary complex having a
total m.w. of 150 KDa.
[0027] It is considered that free IGF or a binary complex of IGF
and IGFBP are able to pass through the capillary wall, while the
ternary complex cannot (Rechler, M. M., Vitam. Horm., 47, 1(1993)).
As regards the half-life of human IGF in blood, that of free IGF is
as short as about 10 minutes, that of the binary complex of IGF and
IGFBP is about 30 minutes and that of the ternary complex composed
of IGF, IGFBP-3 and ALS is about 15 hours (Zapf, J., et al., in
Modern Concepts of Insulin-Like Growth Factors (Spencer, E. M., et)
pp.591, Elsevier Science Publishing Co., New York-Amsterdam
(1991)).
[0028] As a result of the ternary complex formation, the half-life
of IGF in blood is extended and its physiological activity is
suppressed. Also, formation of a binary complex of IGF and IGFBP,
extends the half-life of IGF in blood and is involved in the
regulation of the physiological activity of IGF (Baxter, R. C., et
al., Prog. Growth Factor Res., 1, 49 (1989)).
[0029] Little or no difference is observed in ALS among species,
and homology of ALS between a human and a rat is 78% (Dai, J., et
al., Biochem. Biophys. Res. Commun., 188, 304 (1992)). Earlier, it
was reported that ALS alone does not bind to IGF or IGFBP-3, but
more recently ALS has been shown to exist as a complex with IGFBP-3
in the serum of rats (Lee, C. Y., Endocrinology, 136, 4982
(1995)).
[0030] IGF administration to a living body does not elevate the
concentration of free IGF in blood, but elevates the concentration
of IGFBP-2. It is now considered that an IGF-dependent mechanism
exists in the living body for regulating the expression of IGFBP
(Zapf, J., et al., in Modern Concepts of Insulin-Like Growth
Factors (Spencer, E. M., et) pp.591, Elsevier Science Publishing
Co., New York-Amsterdam (1991)).
[0031] One regulatory mechanism associated with the ternary complex
of IGF, IGFBP-3 and ALS is seen with non-islet cell tumor
hyperglycaemia (hereinafter "NICTH"). Non-islet cell tumors, which
produce IGF-II, are associated with hypoglycaemia or low blood
glucose levels. NICTH-derived IGF-II is glycosylated in the
E-domain of the precursor protein. Normally, IGF-II exists as a 7.5
kDa nonglycosylated protein.
[0032] Glycosylated IGF-II forms a complex with IGFBP-3, but cannot
form a ternary complex with IGFBP-3 and ALS (Baxter, R. C., in
Modern Concepts of Insulin-Like Growth Factors (Spencer, E. M., ed)
pp371, Elsevier Science Publishing Co., New York-Amsterdam (1991))
so the glycolsylated form is considered to exert blood glucose
lowering action because of its inability to form complexes with
ALS. Glycosylated IGF-II is complexed with IGFBP-3 in blood, and
this complex is thought to pass through the capillary vessel wall
(Rechler, M. M., Vitam. Horm., 47, 1 (1993)) and to reach the
target site. Administration of the IGF-I and IGFBP-3 complex to
hypophysectomized rats, demonstrated IGF-I activity, although
weaker than the administration of IGF-I alone (Zapf, J., et al., J.
Clin. Invest., 95, 179 (1995)). On the basis of these results, the
complex of IGF and IGFBP-3 found in blood, is presumed to be
transported to target tissues or organs where the activity of IGF
is demonstrated.
[0033] The IGF-IGFBP complex can also be regulated by a
IGFBP-specific protease. IGFBP-3 concentrations in the blood of a
gravida during the last stage of pregnancy are slightly increased
when measured by a radioimmunoassy (hereinafter "RIA") using an
anti-IGFBP-3 antibody. However, when blood samples from the same
patient are analyzed by a western blot method using .sup.125I-IGF,
a decrease in IGFBP-3 concentration is observed. This apparent
discrepency in results is explained by the presence of a protease
found in the blood of the gravida, which specifically degrades
IGFBP-3 (Hossenlopp, P., et al., J. Clin. Endocrinol. Metab., 71,
797 (1990); Giudice, L. C., et al., J. Clin. Endocrinol. Metab.,
71, 806 (1990)). As a result of this proteolytic activity on the
IGFBP-3 protein, the affinity between IGF and IGFBP-3 is lowered,
and a direct increase in IGF activity is observed.
[0034] These observations demonstrate that activity of IGF in the
living body is highly regulated by IGFBP and ALS. Exogenous IGF
when administered is rapidly metabolized or complexed with IGFBP or
ALS. Even if IGF or a compound having IGF-like activity is
administered exogenously, the IGF activity is controlled by IGFBP
and/or ALS in the living body.
[0035] The inventors have found that endogenous IGF can be
increased by administering compounds which release IGF from binary
(IGF-IGFBP) and ternary (IGF-IGFBP-ALS) complexes or which increase
binary complex formation having IGF-like activity.
[0036] The compounds of the present invention have at least one the
properties of: converting a binary IGF-IGFBP complex or a ternary
IGF-IGFBP-ALS complex into free IGF; converting the ternary complex
into the binary IGF-IGFBP complex; dissociating the ternary complex
into IGF or the binary IGF-IGFBP complex; or inhibiting the
formation of the binary IGF-IGFBP complex or the ternary
IGF-IGFBP-ALS complex.
[0037] An object of the present invention is to utilize the
abundant amount of endogenous IGF which is otherwise
physiologically regulated by complexing with IGFBP and or ALS.
[0038] Another object of the invention is a method for elevating
the concentration of IGF, comprising converting complexed IGF into
free IGF.
[0039] Another object of the present invention is a method for
elevating the concentration of the binary complex, which has lower
IGF activity than free IGF but higher IGF activity than the ternary
complex.
DISCLOSURE OF THE INVENTION
[0040] Biologically active, unbound IGF can be obtained by:
[0041] conversion of the IGF-IGFBP complex or IGF-IGFBP-ALS complex
into IGF;
[0042] dissociation of IGF from the IGF-IGFBP complex or
IGF-IGFBP-ALS complex; or
[0043] inhibition of the binding of IGF and IGFBP or binding of
IGF, IGFBP and ALS.
[0044] A biologically active complex of IGF-IGFBP can be obtained
by:
[0045] conversion of the ternary complex to the binary complex;
[0046] dissociation of the binary complex from the ternary complex;
or
[0047] inhibition of the binding of the binary complex to ALS.
[0048] The concentrations of free IGF or the binary complex can be
increased by:
[0049] 1) a compound which coverts the binary complex in the living
body into free IGF;
[0050] 2) a compound which dissociates free IGF from the binary
complex in the living body;
[0051] 3) a compound which inhibits the binding of IGF and IGFBP in
the living body;
[0052] 4) a compound which converts the ternary complex in the
living body into the binary complex;
[0053] 5) a compound which dissociates the binary complex from the
ternary complex in the living body;
[0054] 6) a compound which inhibits the binding of the binary
complex in the living body to ALS;
[0055] 7) a compound which converts the ternary complex in the
living body into free IGF;
[0056] 8) a compound which dissociates [dissociates] IGF from the
ternary complex in the living body;
[0057] 9) a compound which inhibits the binding of IGF, IGFBP and
ALS in the living body;
[0058] 10) a compound which binds to IGFBP but does not bind to an
IGF receptor or an insulin receptor;
[0059] 11) an IGF derivative which binds to IGFBP but does not bind
to an IGF receptor or an insulin receptor;
[0060] 12) an IGF derivative having the addition, deletion or
substitution of one or more amino acid residues, and which binds to
IGFBP but does not bind to an IGF receptor or an insulin
receptor;
[0061] 13) an IGF derivative having an amino acid sequence similar
to human IGF-II except that the tyrosine residue at amino acid
position 27 and the valine residue at amino acid position 43 have
been substituted with a leucine residue, and which binds to IGFBP
but does not bind to an IGF receptor or an insulin receptor;
[0062] 14) an anti-IGFBP antibody which binds to IGFBP but does not
bind to an IGF receptor or an insulin receptor;
[0063] 15) an anti-IGFBP antibody which binds to IGFBP-3 but does
not bind to an IGF-I receptor or an insulin receptor.
[0064] Another aspect of the present invention is a medicament
comprising a compound which can elevate the concentration of free
IGF or the binary complex.
[0065] Another aspect of the present invention is a screening
method for identifying a compound which can elevate the
concentration of free IGF or the binary complex.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] FIG. 1 illustrates the restriction map of plasmid BP-3
up/pUCl19/.DELTA. in which rat IGFBP-3 gene 5' end region is
cloned.
[0067] FIG. 2 illustrates the restriction map of plasmid BP-3
down/pTV119N in which rat IGFBP-3 gene 3' end region is cloned.
[0068] FIG. 3 illustrates the restriction map of plasmid
BP-3/pTV119N in which rat IGFBP-3 is cloned.
[0069] FIG. 4 illustrates the restriction map of plasmid
RSV-LTR/rat IGFBP-3/SV2-Term/SV40-Pro/neo/SV2-Term/+Amp which is a
rat IGFBP-3 expression vector in animal cells.
[0070] FIG. 5 illustrates the restriction map of the secretory
expression vector used in the preparation of an expression vector
for E. coli.
[0071] FIG. 6 illustrates the restriction map of a plasmid
containing a rat IGFBP-3 gene fragment from which the signal
sequence has been removed.
[0072] FIG. 7 illustrates the restriction map of a plasmid which is
a rat IGFBP-3 secretory expression vector in E. coli.
[0073] FIG. 8 illustrates the affinity of IGF-I and IGF-II for rat
IGFBP-3 (RBP-3 E. coli) and the affinity of RBP-3 E. Coli for
IGF-II (Example 10).
[0074] FIGS. 9A and 9B illustrate the inhibitory activity of
various compounds (Table 1) against the binding of IGF-I and
IGFBP-3 (Example 11).
[0075] FIG. 10 illustrates the binding of IGFBP-3 to immobilized
RALS in the presence of IGF-I or IGF-II, indicating the capacity of
each of IGF-I and IGF-II to form the complex of IGF, IGFBP-3 and
ALS.
[0076] FIG. 11 illustrates the affinity of ALS for the complex of
IGF and IGFBP-3 (Example 13).
[0077] FIG. 12 illustrates the affinity of ALS for IGFBP-3 (Example
14).
[0078] FIG. 13 illustrates the IGF-I receptor binding assay using
.sup.125I-IGF-I and the IGF-I receptor (Example 15).
[0079] FIG. 14 illustrates the results of the insulin receptor
binding assay using .sup.125I-insulin and insulin receptor (Example
15).
[0080] FIG. 15 illustrates the inhibition of the binding of IGF and
IGFBP-3 by a human IGF-II derivative, [Leu27, Leu43]rIGF-II
(Example 16).
[0081] FIG. 16 illustrates the inhibition of the binding of IGF and
IGFBP-3 by an anti-IGFBP-3 antibody (Example 17).
[0082] FIG. 17 illustrates the total triglyceride concentration in
the blood of SD rats, 6 and 24 hours after the administration of
IGF-I, IGF-II, IGF-II derivative and anti-IGFBP-3 antibody (Example
18-1).
[0083] FIG. 18 illustrates the total cholesterol concentration in
the blood of SD rats, 6 and 24 hours after the administration of
IGF-I, IGF-II, IGF-II derivative and anti-IGFBP-3 antibody (Example
18-1).
[0084] FIG. 19 illustrates the total cholesterol concentration in
the blood of an insulin-resistant rat, 6 hours after the
administration of IGF-I and anti-IGFBP-3 antibody (Example
18-2).
[0085] FIG. 20 illustrates the total triglyceride concentration in
the blood of an insulin-resistant rat, 6 hours after the
administration of IGF-I and anti-IGFBP-3 antibody (Example
18-2).
[0086] FIG. 21 illustrates the free fatty acid concentration in the
blood of an insulin-resistant rat, 6 hours after the administration
of IGF-I and anti-IGFBP-3 antibody (Example 18-2).
[0087] FIG. 22 illustrates the free IGF-I concentration in the
blood of SD rats, 1 and 6 hours after the administration of
anti-IGFBP-3 antibody (Example 18-3).
BEST MODE FOR CARRYING OUT THE INVENTION
[0088] A method for elevating the concentration of free IGF and a
method for elevating a binary complex are described as follows.
[0089] The method for elevating the concentration of free IGF
according to the present invention is achieved by utilizing free
IGF or IGF existing in the form of a binary IGF-IGFBP complex
and/or ternary IGF-IGFBP-ALS complex, all three forms of which
occur endogenously in the living body.
[0090] The term "living body" as used herein means the blood,
tissues or organs such as the liver or kidney of a human or mammals
other than humans such as a cow, horse, sheep or pig.
[0091] The term "free IGF" as used herein means IGF which has been
converted from or dissociated from a binary or ternary complex, or
soluble IGF which is unbound or uncomplexed with IGFBP and/or
ALS.
[0092] In the present invention, the concentration of free IGF is
elevated by:
[0093] converting, in the binary IGF-IGFBP complex, inactive IGF to
active IGF,
[0094] dissociating IGF from the binary IGF-IGFBP complex,
[0095] inhibiting the binding of IGF and IGFBP,
[0096] converting, in the ternary IGF-IGFBP-ALS complex, inactive
IGF to active IGF,
[0097] dissociating IGF from the ternary IGF-IGFBP-ALS complex,
[0098] inhibiting the binding of IGF to IGFBP and ALS, or the
like.
[0099] It is also possible to elevate the concentration of free IGF
by:
[0100] converting the ternary IGF-IGFBP-ALS complex to the binary
IGF-IGFBP complex,
[0101] dissociating the binary IGF-IGFBP complex from the ternary
IGF-IGFBP-ALS complex,
[0102] inhibiting the binding of the binary IGF-IGFBP complex to
ALS, or by increasing the concentration of the binary IGF-IGFBP
complex followed by:
[0103] converting the binary IGF-IGFBP complex to IGF,
[0104] dissociating IGF from the binary IGF-IGFBP complex.
[0105] Since the method of the present invention increases the
concentration of endogenous, biologically active IGF, the inventors
envision the use of this free IGF in a method for the prevention
and/or treatment of those diseases that are responsive to the
action of IGF.
[0106] In the present invention, the concentration of the binary
IGF-IGFBP complex is elevated by:
[0107] converting the ternary IGF-IGFBP-ALS complex into the binary
IGF-IGFBP complex,
[0108] dissociating the binary IGF-IGFBP complex from the ternary
IGF-IGFBP-ALS complex,
[0109] inhibiting the binding of the binary IGF-IGFBP complex to
ALS, or the like.
[0110] The inventors have envisioned another method for the
prevention and/or treatment of those diseases which are responsive
to the IGF-like activity of the binary IGF-IGFBP complex.
[0111] The compounds of the present invention are described as
follows.
[0112] The compounds of the present invention do not exhibit
binding to the insulin receptor or the IGF receptor. The compounds
are different from IGF or other IGF-like molecules.
[0113] The compound of the present invention which converts the
binary IGF-IGFBP complex into IGF, acts on the binary complex,
thereby converting it into IGF.
[0114] The compound of the present invention which dissociates IGF
from the binary IGF-IGFBP complex, acts on the binary complex,
thereby dissociating IGF.
[0115] The compound of the present invention which inhibits the
binding of IGF and IGFBP, inhibits the formation of the binary
IGF-IGFBP complex.
[0116] The compound of the present invention which converts the
ternary IGF-IGFBP-ALS complex into the binary IGF-IGFBP complex,
acts on the ternary complex, thereby converting it into the binary
IGF-IGFBP complex.
[0117] The compound of the present invention which dissociates the
binary IGF-IGFBP complex from the ternary IGF-IGFBP-ALS complex,
acts on the ternary complex, thereby dissociating therefrom the
binary IGF-IGFBP complex. The compound of the present invention
which inhibits the binding of the binary IGF-IGFBP complex to ALS,
inhibits the formation of the ternary IGF-IGFBP-ALS complex.
[0118] The compound of the present invention which converts the
ternary IGF-IGFBP-ALS complex into IGF, acts on the complex,
thereby converting it into IGF.
[0119] The compound of the present invention which dissociates IGF
from the ternary IGF-IGFBP-ALS complex, acts on the ternary
complex, thereby dissociating IGF.
[0120] The compound of the present invention which inhibits the
binding of IGF, IGFBP and ALS, inhibits the formation of the
ternary IGF-IGFBP-ALS complex, thereby increasing free IGF.
[0121] In the present invention, the binary IGF-IGFBP complex and
ternary IGF-IGFBP-ALS complex are formed by static interaction,
hydrogen bonding, hydrophobic interaction or the like. They include
any kind of complex without particular limitation to the kind of
IGF or IGFBP. Examples include a binary complex of IGF-I and
IGFBP-3, a ternary complex of IGF-I, IGFBP-3 and ALS.
[0122] When the compound of the present invention is administered
in vivo, free IGF or a binary IGF-IGFBP complex increase in a
living body, resulting in biologically active IGF which may be used
as a medicament or used in a method for the prevention and/or
treatment of diseases which are responsive to IGF.
[0123] The diseases that may be prevented or treated with the
inventive compounds include at least one of diabetes mellitus,
diabetic neuropathy, amyotrophic lateral sclerosis, and
osteoporosis.
[0124] Selection of a compound for the prevention and/or treatment
of a particular disease may depend on the tissue specific
expression of IGFBP and/or tissue-specific effects of IGF. More
specifically, a compound having a selective effect(s) on a
particular organ such as muscle or bone, would be a preferred
compound for the prevention or treatment of a muscle or bone
disease responsive to IGF.
[0125] No particular limitations are placed on the compounds of the
present invention except that they possess the activities as
previously described. The compounds may be produced by organisms
such as miroorganisms, plants and animals, or by cultured cells or
tissues from plants or animals, or by extraction from organisms, or
chemical synthesis. The compounds may be used alone or in
combination.
[0126] The compounds of the present invention include insulin-like
growth factor derivatives and anti-insulin-like growth
factor-binding protein antibodies.
[0127] The term "insulin-like growth factor derivative" means a
derivative of an insulin-like growth factor obtained by subjecting
insulin-like growth factor to chemical modification or the like. A
derivative may have the addition, deletion or substitution of one
or more amino acid residues produced by the genetic engineering
methods. The addition, deletion or substitution of an amino acid
residue can be carried out by any one technique such as that
described in the following reference (Genetic Engineering, 3, 1
(1981); Nucleic Acid Research, 10, 6487 (1982)).
[0128] Preferred examples of the insulin-like growth factor
derivative of the present invention include a derivative obtained
by substituting the tyrosine residue at amino acid position 27 and
the valine residue at amino acid position 43 in the sequence for
human insulin-like growth factor-II with a leucine residue.
[0129] The compound of the present invention may be administered
either parenterally or orally, but oral administration is the
preferred route of administration. The compounds of this invention
can be formulated into various pharmaceutical compositions in a
manner commonly employed in the art. Pharmaceutical compositions
can contain additives such as excipients, disintegrators, binders,
lubricants, fluidity improvers, dispersants, suspending agents,
emulsifiers, antiseptics or stabilizers as needed.
[0130] Typical dosage forms for parenteral administration include
ointments, plasters, suppositories, injections, eye drops, nose
drops, ear drops, inhalants, spirits, cataplasms, liniments and
lotions. Examples of the dosage forms for oral administration
include elixirs, capsules, granules, fine granules, pills,
suspensions, emulsions, powders, tablets, syrups, troches, dry
syrups and lemonade.
[0131] The dosage of the pharmaceutical composition containing the
compound of the present invention may be determined on the basis of
the route of administration, the particular disease being be
treated, the condition of the patient and the like.
[0132] The present inventors have identified two compounds, which
elevate the level of free IGF-I in blood and increase IGF activity
in a normal rat and a diabetic rat model. The compounds include an
IGF-II derivative and anti-IGF-II antibodies, described
hereinafter.
[0133] A description of a method for screening a compound of the
present invention is the following.
[0134] A compound of the present invention can be identified by
labeling any one of IGF, IGFBP and ALS (i.e., a labeled factor) for
use in a direct or indirect method of detection, and determining
whether the binding of the labeled factor (e.g., IGF) to an
unlabeled factor (e.g., IGFBP) is inhibited by the compound or
whether the compound can dissociate the labeled factor from a
binary or ternary complex.
[0135] In a direct method, the amount of the labeled factor can be
detected by appropriate physical measurement. For example, a factor
labeled with a radioisotope can be detected directly by the
measurement of radioactivity. A Scintillation Proximity Assay
(Cook, N. D., Drug Discovery today, 1, 287 (1996)) or a similar
method may also be used. When labeling is carried out with a color
agent or fluorescent dye, the amount of the labeled factor can be
detected by optical measurement.
[0136] In the indirect method, the factor is labeled with a
nondetectable agent, which through a chemical reaction, forms a
directly detectable molecule (e.g., dye, fluorescence dye) in a
stoichiometric amount. For example, where a factor is conjugated to
an enzyme, an enzyme substrate is added to produce a detectable dye
following the reaction in the presence of a test compound.
[0137] Radioisotopic measurements can be carried out in accordance
with art-recognized liquid phase methods. For example, after
reaction of .sup.125I-labelled IGF (.sup.125I-IGF) with IGFBP,
complex-bound .sup.125I-IGF can be separated from unbound
.sup.125I-IGF by:
[0138] 1) activated charcoal (Moses, A. C., et al., Endocrinology,
104, 536 (1979); Binoux, M., et al., J. Clin. Endocrinol. Metab 59,
453 (1984); Scott, C. D., et al., Endocrinology, 116, 1094 (1985);
Szabo, L., et al., Biochem. Biophys. Res. Commun., 151, 207 (1988);
Gelato, M. C., et al., J. Clin. Endocrinol. Metab., 70, 879 (1990);
Oh, Y. , et al. , Biochem. J. , 278, 249 (1991), etc.),
[0139] 2) a lectin protein recognizing the sugar chain portion of
IGFBP (Martin, J. L., et al., J. Biol. Chem., 261, 8754
(1986)),
[0140] 3) immunoprecipitation using a primary and secondary
antibody (Martin, J. L., et al., J. Biol. Chem. 261, 8754 (1986);
Baxter, R. C., et al., J. Biol. Chem., 264, 11843 (1989); Baxter,
R. C., Biochem. J., 271, 773 (1990) or the like).
[0141] Using any one of the above-described methods with
.sup.125I-IGF, .sup.125I-IGFBP or .sup.125I-ALS and an appropriate
primary antibody, it is possible to evaluate the formation of the
binary IGF-IGFBP complex or ternary IGF-IGFBP-ALS complex in the
presence of a test compound.
[0142] These methods may not be suitable for the screening of
multiple test compounds because of the extreme caution that must be
taken in handling of radioisotopes having a short half-life and the
centrifugation steps required in the separation method.
[0143] For the rapid screening of multiple test compounds, the
Scintillation Proximity Assay may be more appropriate.
Alternatively, non-radioisotopic, solid phase methods using an
enzyme as a labeling agent can be used conveniently.
[0144] The screening of the compound of the present invention which
converts the binary IGF-IGFBP complex into free IGF or dissociates
free IGF from the binary IGF-IGFBP complex can be carried out, for
example, by adding an enzyme-labeled IGF to immobilized IGFBP in
the presence of a test compound, reacting, washing and then
measuring the amount of bound enzyme activity. Alternatively, it is
possible to immobilize IGF and label IGFBP with an enzyme.
[0145] The compound of the present invention, which inhibits the
binding of IGF and IGFBP can be screened by simultaneously adding
enzyme-labeled IGF and a test compound to an immobilized IGFBP,
reacting, washing and then measuring the amount of bound enzyme
activity. Alternatively, it is possible to immobilize IGF, label
IGFBP with an enzyme and measure the activity of the enzyme.
[0146] The compound which converts the ternary IGF-IGFBP-ALS
complex into the binary IGF-IGFBP complex, the compound which
dissociates the binary IGF-IGFBP complex from the ternary
IGF-IGFBP-ALS complex, the compound which converts the ternary
IGF-IGFBP-ALS complex into free IGF or the compound which
dissociates IGF from the ternary IGF-IGFBP-ALS complex, each
according to the present invention, can be screened, for example,
by adding IGF and enzyme-labeled IGFBP to immobilized ALS in the
presence of a test compound, reacting, washing and then measuring
the amount of bound enzyme activity. Alternatively, IGF can be
labeled with an enzyme.
[0147] The screening may be carried out by adding enzyme-labeled
ALS to an immobilized binary IGF-IGFBP complex to form the ternary
complex, adding a test compound, reacting, washing and then
measuring the amount of bound enzyme activity.
[0148] Screening may also be carried out by adding IGFBP and
enzyme-labeled ALS to immobilized IGF to form the ternary complex,
adding a test compound, reacting, washing and then measuring the
amount of bound enzyme activity.
[0149] To identify those compounds which: convert the ternary
IGF-IGFBP-ALS complex into the binary IGF-IGFBP complex; dissociate
the binary IGF-IGFBP complex from the ternary IGF-IGFBP-ALS
complex; convert the ternary IGF-IGFBP-ALS complex into free IGF;
or dissociate free IGF from the ternary IGF-IGFBP-ALS complex, it
is possible to employ the above-described screening method for the
compound which converts the binary IGF-IGFBP complex into free IGF
or the compound which dissociates free IGF from the binary
complex.
[0150] The compound which inhibits the binding of the binary
IGF-IGFBP complex to ALS or the compound which inhibits the binding
of IGF, IGFBP and ALS, each according to the present invention, can
be screened by simultaneously adding the binary complex (complex of
IGF and enzyme-labeled IGFBP) in the presence of a test compound to
immobilized ALS, reacting, washing and then measuring the amount of
bound enzyme activity. Alternatively, it is possible to label IGF
with an enzyme.
[0151] It is also possible to carry out screening by adding
enzyme-labeled ALS in the presence of a test compound to an
immobilized binary complex (IGF and IGFBP), reacting, washing and
then measuring the amount of bound enzyme activity.
[0152] It is also possible to carry out screening by adding IGFBP,
enzyme-labeled ALS and a test compound to immobilized IGF,
reacting, washing and then measuring the amount of bound enzyme
activity.
[0153] The screening methods used to identify those compounds which
inhibit the binding of IGF and IGFBP may be used to confirm the
identity of those compounds which inhibit the binding of the binary
IGF-IGFBP complex to ALS or which inhibit the binding of IGF, IGFBP
and ALS.
[0154] The immobilization of IGF, IGFBP or ALS may be achieved by
any suitable art-recognized method. In both the direct and indirect
solid-phase methods, immobilization may be achieved by any one of
the following including avidin-biotin, hapten-anti-hapten antibody
or the like. Examples of the solid phase material include glass,
plastics such as polystyrene, polyacrylamide and cellulose acetate.
The solid phase may be in the form of a test tube, bead, microtiter
plate, disc, chip and the like. For screening of multiple sample
specimens, a commercially available multi-well microtiter plate is
preferred.
[0155] The enzymatic activity may be measured by known methods,
which are dependent on conditions such as substrate, buffer, pH,
temperature, etc. Examples of the enzymatic methods include
colorimetry, fluorescence and luminescence.
[0156] IGF, IGFBP or ALS may be labeled with an enzyme by
art-recognized methods and examples include a maleimide method, a
periodic acid method and a glutaraldehyde method.
[0157] The selection of an enzyme used for labeling and an
appropriate substrate may be considered as needed. For example,
when .beta.-D-galactosidase is used as an enzyme, examples of the
substrate include 2-nitrophenyl-.beta.-D-galactoside,
4-methylumbelliferyl-.beta.-D- -galactoside and
5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside. When peroxidase is
used as an enzyme, examples of the substrate include
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid),
3,3',5,5'-tetramethylbenzidine and 1,2-phenylenediamine. When
alkaline phosphatase is used as an enzyme, examples of the
substrate include 4-methylumbelliferyl phosphate and N-nitrophenyl
phosphate.
[0158] Screening is preferably carried out in a buffer solution.
Any buffer generally recognized for its use in the measurement of
enzyme activity is appropriate. Examples include sodium phosphate
buffer, glycine-sodium hydroxide buffer and Tris-HCl buffer
solution. The pH of the buffer may be adjusted as appropriate, but
is preferably 6.0 to 7.4 since the solubility of IGFBP is
pH-dependent. Buffers may also be supplemented with a salt or
surfactant. For example, when sodium chloride is added, a
concentration up to 0.15 M is preferred.
[0159] Industrial Applicability
[0160] According to the method of the present invention, IGF
activity can be expressed or increased in vivo, by converting
endogenous IGF into free IGF or a binary IGF-IGFBP complex.
[0161] The in vivo administration of a compound of the present
invention elevates the concentration of free IGF thereby increasing
the biological activity of IGF. Accordingly, the compound of the
present invention may be useful in the prevention and/or treatment
of diseases such as diabetes mellitus, amyotrophic lateral
sclerosis, osteoporosis and the like, or those diseases which are
responsive to IGF.
[0162] The present invention is described in more detail by the
following Examples and is not in any way limited by the
Examples.
EXAMPLES
Example 1
[0163] Preparation of Human IGF-I and IGF-II
[0164] Human IGF-I was purchased from GroPep Pty. Ltd. Human IGF-II
was obtained in a similar manner to that of Sakano, et al. (Sakano,
K., et al., J. Biol. Chem., 266, 20626 (1991)). Specifically, human
IGF-II was expressed in Escherichia coli by a recombinant gene
technique. The human IGF-II protein was extracted from cells,
refolded and purified by chromatography on a reverse-phase HPLC
column.
Example 2
[0165] Cloning of Rat IGFBP-3 Gene
[0166] The rat IGFBP-3 gene was cloned in accordance with the
literature (Shimasaki, S., et al., Biochem. Biophys. Res. Commun.,
165, 907 (1989)). PCR primers were used to amplify the 5' and 3'
ends of the IGFBP-3 gene from a rat pancreas cDNA library.
[0167] The sequences of the 5' primers were as follows:
1 5' -CGCCATGCATCCCGCGCGCC - 3' (SEQ I.D. No. 1) and 5'
-ACGCCGCACGCGTCGCCTTC -3' (SEQ I.D. No. 2)
[0168] The sequences of the 3' primers were as follows:
2 (SEQ I.D. No. 3) 5'-GCGCGGGCCCCGTGGTGCGCTGCGAACCGT-3' and (SEQ
I.D. No. 4) 5'-TGCTGATCACGTTGTTGGC-3- '
[0169] The PCR fragments were blunt-ended, phosphorylated, and
inserted into a pUC19 vector (SalI/Blunting/BAP), followed by
subcloning of the 5' fragment (5' end BP-3 up/pUCl9) and the 3'
fragment (3' end: BP-3 down/pUCl9) into the vector.
[0170] The SphI site located upstream from the transcription
initiating codon (ATG) of the 5' BP-3 up/pUIC19 clone was
eliminated to generate 5' BP-3 up/pUC19/.DELTA.SphI (refer to FIG.
1).
[0171] After digestion of the 3' BP-3 down/pUIC19 clone with
BamHI-PstI, the fragment containing BP-3 down was recovered,
blunt-ended, and inserted into pTV119N (HincII/BAP) containing a
lacZ promoter to generate 3' BP-3 down/pTV119N (refer to FIG.
2).
[0172] The 5' end clone (BP-3 up/pUC19/.DELTA.SphI) was digested
with M1uI/HindIII, and a 250 bp fragment containing BP-3 up was
recovered. The 3' end clone (BP-3 down/pTV119N) was treated with
M1uI/HindIII/BAP followed by subcloning of the 250 bp fragment
containing BP-3 up in order to generate a rat IGFBP-3 clone
designated BP-3/pTV119N (refer to FIG. 3).
Example 3
[0173] Construction of Vector for the Expression of Rat IGFBP-3
Gene in Animal Cells
[0174] The rat IGFBP-3 clone was inserted into a plasmid containing
the RSV-LTP (Rous sarcoma virus long terminal repeat) (Nawa, K., et
al., Biochem. Biophys. Res. Commun., 171, 729 (1990)) for
expression of the protein in animal cells. The BP-3/pTV119N plasmid
was digested with XbaI/HindIII to release a 900 bp fragment. The
fragment was inserted into an expression plasmid
(SV2-Term/SV40-Pro/neo/SV2-Term/+Amp), followed by subcloning into
the HindIII site of an approximate 600 bp fragment of RSV-LTR
obtained by HindIII digestion (RSV-LTR/rat
IGFBP-3/SV2-Term/SV40-Pro/neo/SV2-Term/+Amp) (refer to FIG. 4).
Example 4
[0175] Construction of Vector for the Expression of Rat IGFBP-3
Gene in E. coli
[0176] For expression in Escherichia coli, a secretory expression
plasmid containing a PhoA signal sequence was used. The PhoA signal
sequence was prepared with a synthetic DNA oligomer, and inserted
into the NcoI/HindIII cut site of the expression plasmid, pTrc99A
(product of Pharmacia Biotech AB) (refer to FIG. 5). In order to
express the rat IGFBP-3 protein in E. coli, it was necessary to
delete the rat signal sequence from the clone by cutting the
BP-3/pTV119 plasmid with NaeI/XbaI (Example 2).
[0177] The resulting fragment was inserted into the plasmid
BP-3/pTV119 previously treated with NcoI/Klenow/XbaI/BAP, whereby
the plasmid shown in FIG. 6 was prepared. Since the plasmid had an
NcoI site, it was necessary to cut with NcoI/EcoRI and blunt-end
with Mung Bean Nuclease, to recover the rat IGFBP-3 gene fragment
from which the signal sequence had been eliminated. The resulting
fragment was inserted into the secretory expression vector
(HindIII/Klenow/BAP) of FIG. 5 to prepare the secretory expression
plasmid of rat IGFBP-3 (refer to FIG. 7).
Example 5
[0178] Expression of Rat IGFBP-3 in CHO-K1 cells and Purification
Thereof
[0179] Rat IGFBP-3 was expressed in CHO-K1 cells. The plasmid of
FIG. 4 was transfected into CHO-K1 cells by the calcium phosphate
method. Recombinant cells were cloned in DMEM/F-12 medium
containing 0.4 mg/ml G-418, selected and subsequently cultured in T
medium containing 1% ITES. Culture supernatants were collected and
an enzyme inhibitor (2 mM benzamide/1 mM PMSF/100 U/ml Trasylol/2
mM EDTA) was added. The mixture was sterile filtered ("CAPSULE
FILTER 0.2 .mu.m sterilized"; product of Gelman Science), and the
pH adjusted to 6.0 with a solution of 1 M sodium acetate. The
mixture was then applied to "SP-Sepharose F.F. column" (product of
Pharmacia Biotech AB) equilibrated with a 10 mM sodium acetate
buffer (pH 6.0) containing 0.15 M sodium chloride. After successive
washes with the same buffer and a buffer containing 0.5 M sodium
chloride, elution was carried out with a buffer containing 1 M
sodium chloride. The eluate was diluted two-fold and adjusted to a
final pH of 7.0 with a sodium phosphate solution.
[0180] A ligand affinity column ("HiTrap affinity column,
NHS-activated" (product of Pharmacia Biotech AB)), on which IGF-II
had been immobilized, was prepared in a conventional manner.
[0181] After the column was equilibrated with a 50 mM sodium
phosphate buffer (pH 7.0) containing 0.45 M sodium chloride, the
SP-Sepharose F.F. elution sample obtained above was applied. After
washing successively with the same buffer, a 10 mM sodium acetate
buffer (pH 7.0) containing 1 M sodium chloride and water, elution
was carried out with 0.5 M acetic acid. The eluate thus obtained
was recovered, lyophilized, dissolved in 0.1% trifluoroacetic acid
(hereinafter "TFA") and then subjected to reverse phase HPLC.
[0182] Rat IGFBP-3 (hereinafter "RBP-3CHO") was isolated on a
reverse phase HPLC column ("CAPCELLPAK C18 SG300", 250.times.4.6 mm
I.D., Shiseido Co., Ltd) using a linear gradient of acetonitrile at
a flow rate of 1 ml/min, followed by lyophilization and
storage.
Example 6
[0183] Expression of Rat IGFBP-3 in E. coli and Purification
Thereof
[0184] E. coli clones expressing rat IGFBP-3 were cultured in LB
medium at 37.degree. C. with shaking at 250 rpm. When an absorbance
of O.D.=3.45 was reached, IPTG was added to a final concentration
of 0.3 mM, to induce protein expression. The cells were collected,
and the periplasma was recovered by an osmotic shock method
(Nossal, N. G. et al., J. Biol. Chem., 241, 3055 (1966)), followed
by adjustment of the pH to 7.2 with a phosphate buffer.
[0185] The cell lysate was applied to an IGF-II affinity column to
isolate the rat IGFBP-3 protein according to the method described
in Example 5. The protein fraction was eluted with 0.5 M acetic
acid followed by lyophilization. The lyophilizate was dissolved in
10 ml of 0.1% TFA and rat IGFBP-3 was eluted with an acetonitrile
linear gradient at a flow rate of 1 ml/min on a reverse-phase HPLC
column ("YMC-PACK PROTEIN-RP", 250.times.4.6 mm I.D., product of
YMC Corporation. Rat IGFBP-3 (hereinafter "RBP-3 E. coli") obtained
in this manner was lyophilized and stored.
Example 7
[0186] Purification of Rat ALS from Rat Serum
[0187] Rat ALS was purified from rat serum by a similar method to
that of Baxter, et al. (Baxter, R. C., et al., Endocrinology, 134,
848 (1994)).
[0188] The above-described IGFBP-3 (RBP-3 E. coli) of Example 6 was
immobilized on the affinity column of Example 5, on which IGF-II
had been immobilized, to prepare an affinity column for isolation
of rat ALS.
[0189] The final purification step was performed on a "DEAE-5PW
column" (75.times.7.5 mm I. D., TOSOH CORPORATION) by equilibrating
the column with a 10 mM sodium phosphate buffer (pH 8.0) containing
50 mM sodium chloride. Rat ALS (hereinafter "RALS") was eluted with
a sodium chloride linear gradient in the same buffer, followed by
lyophilization and storage.
Example 8
[0190] Preparation of Human IGF-II Derivative ([Leu27, Leu43]
rIGF-II)
[0191] Human [Leu 27] rIGF-II and human [Leu 43] rIGF-II bind to
the IGF-II/cation-independent mannose-6-phosphate receptor but
exhibit almost no binding to the IGF-I receptor or the insulin
receptor. As a consequence of the single amino acid substitutions
in each of the derivatives, these molecules have a significantly
lowered effect on cell proliferation (Sakano, K., et al., J. Biol.
Chem., 266, 20626 (1991)). Furthermore, it is known that the
binding of these derivatives to IGFBP-3 is almost similar to the
wild type (Bach, L. A., et al., J. Biol. Chem., 268, 9246
(1993)).
[0192] In view of the foregoing, the present inventors developed a
derivative ([Leu27, Leu43]rIGF-II) having two leucine residue
substitutions (tyrosine at amino acid residue 27 and valine at
amino acid residue 43 were each replaced with leucine), which
exhibits little or no binding to the IGF-I receptor and the insulin
receptor but which has affinity for IGFBP-3.
[0193] The derivative ([Leu27, Leu43] rIGF-II) was prepared in
accordance with the Sakano method of preparation of human IGF-II
(Sakano, K., et al., J. Biol. Chem., 266, 20626 (1991)).
[0194] A plasmid encoding [Leu27, Leu43] rIGF-II was constructed by
a standard mutagenesis technique using human IGF-II DNA and two
synthetic oligonucleotides (5'-CTGGAAAAGAGGAAACCTCTG-3' (SEQ I.D.
No. 5), and 5'-TTCTTCGAGGATACCTC-3' (SEQ I.D. No. 6)). The mutant
IGF-II were expressed in E. coli and purified.
Example 9
[0195] Preparation of Anti-Rat IGFBP-3 Antibody
[0196] Antibodies to the RBP-3CHO antigen (rat IGFBP-3 obtained in
Example 5) were obtained by immunizing rabbits (Japanese white
house rabbit, male, 5 in number). A total of 5 injections were
administered at 2-week intervals. The anti-serum was diluted
two-fold with PBS, adjusted to pH 7.4 and applied to a Protein A
column ("PROSEP-A", product of Bioprocessing Co.) equilibrated with
PBS. After washing with PBS, elution was carried out with a 0.1 M
glycine-hydrochloric acid buffer (pH 3.0). The eluate was
concentrated by ultrafiltration and buffered with PBS. Altogether,
five lots of polyclonal antibodies (anti-RBP-3 pAb #35, #36, #88,
#89, #90) were obtained.
Example 10
[0197] Binding of IGF and IGFBP-3 in a Solid-Phase System
[0198] Materials:
[0199] A 96-well microtiter plate manufactured by Costar Corp.; 50
mM sodium phosphate buffer (pH 6.5) as a basic buffer for
immuobilization; basic buffer supplemented with 0.03% Tween 20,
basic buffer supplementee with 1% BSA and basic buffer supplemented
with 0.25% BSA an 0.03% Tween 20 were used in the washing, blocking
and binding steps, respectively. After each reaction, wells were
washed twice with 400 .mu.l/well of buffer. Horseradish
peroxidase-labeled IGF-II (hereinafter "HRP-IGF-II") was prepared
using a commercially available kit (PIERCE Chemichal Company).
[0200] The above-described materials are also used in Examples 11
to 17, unless otherwise specified.
[0201] Method:
[0202] To the microtiter plate, RBP-3 E. Coli (150 ng/ml) was added
in an amount of 50 .mu.l/well and allowed to stand overnight at
4.degree. C. for immobilization. After blocking, 50 .mu.l/well of
HRP-IGF-II (final concentration diluted 12,000-fold) was added at
25.degree. C. for 2 hours in the presence or absence of a test
compound. Test compounds at various concentrations were added to
each well in a total volume of 50 .mu.l.
[0203] In the final step, a solution of
2,2'-azino-bis(3-ethylbenzthiazoli- ne-6-sulfonic acid)
(hereinafter "ABTS"; Kirkegaard & Perry Laboratories) was added
to each well (100 .mu.l/well) and allowed to stand at room
temperature. The absorbance (O.D.) at 405 nm was measured on a
plate reader ("Vmax", Molecular Devices).
[0204] The absorbance in the absence of a test compound is
designated "B.sub.0", and in the presence of a test compound is
designated as "B". The absorbance in the absence of both a test
compound and using a non-immobilized well (a well not containing
RBP-3 E. coli) is designated as nonspecific bound (NSB). The
percent binding of labeled IGF-II to immobilized IGFBP-3 in the
presence of a test compound is calculated from the following
equation:
(B-NSB)/(B.sub.0-NSB).times.100
[0205] The binding reactions for each of the test compounds, was
performed triplicate. The average value is plotted and the SD value
is indicated with a bar (refer to FIG. 8)
[0206] The above-described binding assay makes it possible to
evaluate the affinity of various IGF and IGFBP derivatives for
IGFBP and to screen for competitive inhibitors of IGF/IGFBP
binding.
Example 11
[0207] Inhibition of IGF and IBFBP-3 Binding by Competitive
Inhibitors
[0208] A competitive binding assay was performed in accordance with
Example 10, using the test compounds set forth in Table 1. Each
compound was dissolved or suspended in basic buffer containing 5%
methanol at a final concentration of 1 mg/ml. The resulting
solution or suspension was diluted with the reaction buffer 5-fold
or to a final concentration of 200 .mu.g/ml. The final
concentration of each compound at the time of measurement was 100
.mu.g/ml.
[0209] The percent binding of IGF-II to IGFBP-3 (RBP-3 E. coli) in
the presence of a test compound was calculated using the following
formula:
"percent total bound (100(%))-percent actual bound (%)"=percent
inhibitory activity (%) of the test specimen.
[0210] Measurements for each of the test compounds were performed
twice, and the average inhibitory activity was determined. Results
are shown in FIGS. 9A and 9B. Compound 27 (Ellagic acid), Compound
29 (Aclacinomycin A) and Compound 31 (heparin) exhibited potent
inhibitory activity compared to any of the other test
compounds.
3TABLE 1 No. Compound 1 Samarosporin 2 Hydroxy aspergillic acid 3
Kidamycin 4 Siccanin 5 Comenic acid 6 Kinetin 7
2-Chloro-4,6-bisethylamino-5-triazine 8 Methyl hesperidine 9
Oxyperitin 10 Protionamide 11 Quercetin 12 Flavone 13 Glycyrrhizin
14 Naringenin 15 2-Hydroxychalcone 16 N-(Methylamino)-succinamide
17 D-(+)-Catechin 18 2-Carbethoxy-5,7-dihydroxy-4-methoxyisoflavone
19 (-)-Epicatechin 20 Betulin 21 .alpha.-naphtoflavone 22 Curcumin
23 Tamarixetin-7-rutinoside 24 Aescin 25 Ursolsaure 26 Fisetin 27
Ellagic acid 28 Oleanolsaure 29 Aclacinomycin A 30 Sulfonazo III 31
Heparin 32 Chondroitin sulfate 33 Vitamin B.sub.12 34 Vitamin
B.sub.6
Example 12
[0211] Binding Assay for IGF and IGFBP-3 to ALS in a Solid Phase
System (1)
[0212] In accordance with the conditions described in Example 10,
streptavidin (1 .mu.g/ml) was added to a microtiter plate in an
amount of 50 .mu.l/well and allowed to stand overnight at 4.degree.
C. for immobilization. After blocking with the blocking buffer,
biotinylated RALS (rat ALS of Example 7: 50 ng/ml; Amersham
International plc) was added in an amount of 50 .mu.l/well,
followed by incubation at 25.degree. C. for 2 hours. RBP-3 E. coli
(final concentration: 25 ng/ml) was added to the reaction mixture,
and as a test compound, IGF-I or IGF-II in various concentrations
were added simultaneously in a total amount of 50 .mu.l/well.
Plates were incubated overnight at 4.degree. C. to form a ternary
complex. The anti-RBP-3 pAb #35 (3 .mu.g/ml) obtained in Example 8,
was added in an amount of 50 .mu.l/well to each of the wells
containing the ternary complex, followed by incubation at
25.degree. C. for 2 hours. A labeled secondary antibody
(anti-rabbit IgG, horseradish peroxidase linked whole antibody,
diluted to 1000-fold, product of Amersham International plc) was
added in an amount of 50 .mu.l/well, and the mixture was incubated
at 25.degree. C. for 2 hours. Finally, an ABTS solution was added
in an amount of 100 .mu.l/well and the mixture was allowed to stand
at room temperature for 20 minutes. The absorbance (O.D.) at 405 nm
was then measured.
[0213] The value (absorbance) when IGF-I or IGF-II is added as a
test compound is designated as "B", while the value (absorbance)
when 300 pM of IGF-II is added as a test compound is designated as
total bound (100%) . The value (absorbance) when assayed without
the addition of biotinated RALS is designated as nonspecific bound
(NSB). The percent binding is calculated from the following
equation:
(B-NSB)/(total bound-NSB).times.100
[0214] The measurements for each condition were performed in
triplicate. The average percent binding is plotted, and the SD is
indicated by a bar (refer to FIG. 10).
[0215] The aforementioned binding assay allows one to evaluate the
capacity of, for example, IGF or an IGF derivative to form a
ternary complex with IGFBP and ALS. In addition, it is possible to
screen for compounds that inhibit the formation of the ternary
complex of IGF, IGFBP and ALS by adding the compound simultaneously
(or before and after the addition) of RBP-3 E. coli and IGF-I or
IGF-II.
Example 13
[0216] Binding Assay for IGF and IGFBP-3 to ALS in a Solid-Phase
System (2)
[0217] In accordance with the conditions described in Example 10,
streptavidin (1 .mu.g/ml) was added to a microtiter plate in an
amount of 50 .mu.l/well and allowed to stand overnight at 4.degree.
C. for immobilization. After blocking with the blocking buffer,
biotinated RALS (200 ng/ml) was added in an amount of 50
.mu.l/well, followed by incubation at 25.degree. C. for 2 hours.
HRP-IGF-II (final concentration: diluted to 2000-fold) and RBP-3 E.
coli (final concentration: 25 ng/ml) were added simultaneously in a
total amount of 50 .mu.l/well, and incubated at 25.degree. C. for 2
hours. In the final step, after washing, an ABTS solution was added
in an amount of 100 .mu.l/well and the mixture was allowed to stand
at room temperature for 20 minutes. The absorbance (O.D.) at 405 nm
was then measured.
[0218] In an actual competitive binding assay, biotinylated RALS,
HRP-IGF-II (final concentration: diluted to 1/2000), RBP-3 E. coli
(final concentration: 25 ng/ml), and various concentrations of
RALS, as a competitive inhibitor, were allowed to react.
[0219] The absorbance assayed without the addition of biotinated
RALS is designated as nonspecific bound (NSB). The absorbance
assayed without an inhibitory compound is designated as "B.sub.0"
and that assayed with the addition of an inhibitory compound is
designated as "B". The percent binding of IGFBP-3 and IGF-II to
immobilized RALS in the presence of the competitive inhibitor,
RALS, is calculated from the following equation:
(B-NSB)/(B.sub.0-NSB).times.100
[0220] The measurement for each concentration of RALS was performed
in triplicate. The average percent value is plotted and the SD
value is indicated by a bar (refer to FIG. 11).
[0221] The aforementioned binding assay allows one to evaluate the
affinity of, for example, ALS or various ALS derivatives for the
complex of IGF and IGFBP-3, in addition to screening for inhibitors
of the ternary complex of IGF, IGFBP and ALS, or the binary complex
of IGF and IGFBP-3 to ALS.
Example 14
[0222] Binding Assay for IGFBP-3 to ALS in a Solid Phase System
[0223] In accordance with the conditions described in Example 10,
streptavidin (1 .mu.g/ml) was added to a microtiter plate in an
amount of 50 .mu.l/well and allowed to stand overnight at 4.degree.
C. for immobilization. After blocking with a blocking buffer,
biotinated RALS (200 ng/ml) was added in an amount of 50
.mu.l/well, followed by incubation at 25.degree. C. for 2 hours.
RBP-3 E. coli (100 ng/ml) was added in an amount of 50 .mu.l/well,
and incubated overnight at 4.degree. C. The anti-RBP-3 pAb #35 (3
.mu.g/ml) obtained in Example 9, was added in an amount of 50
.mu.l/well, followed by incubation at 25.degree. C. for 2 hours. A
secondary antibody was added in an amount of 50 .mu.l/well,
followed by incubation at 25.degree.C. for 2 hours. In the final
step, an ABTS solution was added in an amount of 100 .mu.l/well,
and the mixture was allowed to stand at room temperature for 20
minutes. The absorbance (O.D) at 405 nm was then measured.
[0224] In an actual a competition assay, biotinylated RALS, RBP-3
E. coli (final concentration: 100 ng/ml) and, various
concentrations of RALS in a total amount of 50 .mu.l/well, were
allowed to react.
[0225] The absorbance value in the absence of biotinylated RALS is
designated as nonspecific bound (NSB). The absorbance value in the
absence of an inhibitor is designated as "B.sub.0" and the
absorbance value in the presence of an inhibitor is designated as
"B". The percent binding of RBP-3 E. coli to immobilized RALS in
the presence of the competitive inhibitor, RALS, is calculated from
the following equation:
(B-NSB)/(B.sub.0-NSB).times.100
[0226] The measurement for each concentration of inhibitor was
performed in triplicate. The average absorbance is plotted and the
SD value is indicated by a bar (refer to FIG. 12).
[0227] Based on the aforementioned binding assay, it is possible to
evaluate the affinity of, for example, ALS or an ALS derivative for
IGFBP-3, in addition to screening for compounds, which inhibit the
formation of the complex of IGFBP-3 and ALS.
Example 15
[0228] Characterization of Human IGF-II Derivative [Leu27,
Leu43]rIGF-II. in vitro (1)
[0229] The [Leu27, Leu43]rIGF-II derivative described in Example 8,
was characterized for its binding affinity to human placental IGF-I
receptor and human placental insulin receptor using a radioreceptor
assay (Le Bon, T. R., et al., J. Biol. Chem., 261, 7685 (1986);
Fujita-Yamaguchi, Y., et al., J. Biol. Chem., 258, 5045
(1983)).
[0230] In the first step, .sup.125I-IGF (or .sup.125I-insulin)
(Amersham International plc) in an amount of 2.times.10.sup.4 cpm
was incubated with various amounts of the IGF receptor or the
insulin receptor, to determine an amount of the receptor to which
50% of .sup.125I-IGF (or .sup.125I-insulin) binds.
[0231] [Leu27, Leu43]rIGF-II was added at various concentrations to
aliquots of the mixture containing the determined amount of the
receptor and .sup.125I-IGF (or .sup.125I-insulin) (2.times.10.sup.4
cpm). The final volume of the reaction mix was brought to 300 .mu.l
using a 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA and
0.075% Triton X-100, and allowed to incubate overnight at 4.degree.
C. After incubation, 75 .mu.l of human .gamma.-globulin (4 mg/ml)
and 375 .mu.l of 20% PEG 6000 (pH 7.0) were added simultaneously,
followed by incubation at 4.degree. C. for 1 hour. The reaction
mixture was centrifuged (3000 rpm.times.30 min, 4.degree. C.), the
supernatant decanted, and the cpm value for the pellet was measured
by gamma counting.
[0232] In a control assay, the IGF-I receptor assay was performed
using various concentrations of IGF-I or IGF-II instead of [Leu27,
Leu43]rIGF-II. For the insulin receptor assay, various
concentrations of insulin or IGF-II were added instead of [Leu27,
Leu43]rIGF-II.
[0233] The cpm value in the absence of a receptor is designated as
nonspecific bound (NSB). The cpm value in the absence of a test
compound is designated as "B.sub.0" and the cpm value assayed in
the presence of a test compound is designated as "B". The percent
binding to a receptor is calculated from the following
equation:
(B-NSB)/(B.sub.0-NSB).times.100
[0234] The measurement for each concentration of test compound is
performed in triplicate. The average cpm is plotted and the SD
value is indicated by a bar.
[0235] FIG. 13 illustrates the results of the IGF-I
receptor-binding assay using .sup.125I-IGF-I and the IGF-I
receptor. The affinity of IGF-II for an IGF-I receptor was similar
to that of IGF-I, but the affinity of [Leu27, Leu43]rIGF-II for the
IGF-I receptor decreased to about 1 to 2% of that for IGF-I.
[0236] FIG. 14 illustrates the results of the insulin
receptor-binding assay using .sup.125I-insulin and the insulin
receptor. The affinity of IGF-II for an insulin receptor was
similar to that of insulin, but the affinity of [Leu27,
Leu43]rIGF-II for the insulin receptor decreased to about 1 to 2%
of that for insulin.
Example 16
[0237] Characterization of Human IGF-II Derivative ([Leu27,
Leu43]rIGF-II), in vitro (2)
[0238] In accordance with the procedure of Example 9, the inventors
determined that [Leu27, Leu43]rIGF-II inhibits the binding of IGF
and IGFBP-3.
[0239] [Leu27, Leu43]rIGF-II inhibited the binding of labeled
IGF-II to immobilized IGFBP-3 at concentrations similar to those
seen for IGF-II (refer to FIG. 15)
Example 17
[0240] Characterization of Anti-IGFBP-3 Antibody
[0241] Using the procedure of Example 10, the anti-IGFBP-3
antibodies (anti-RBP-3 pAb) of Example 9 were shown to inhibit the
binding of IGF and IGFBP-3.
[0242] The antibodies, #35 and #90, exhibited the strongest
inhibitory activity; 0.2632% as a molar ratio (2% as a weight
ratio) when the activity of RBP-3 E. coli (IGFBP-3) was set at
100%. The antibodies, #36 and #89, exhibited almost the same
inhibitory activity; 0.0875% as a molar ratio, while the antibody,
#88, exhibited slightly less activity; 0.0525% as a molar
ratio.
[0243] All of the inventive antibodies inhibited the binding of IGF
and IGFBP-3 (refer to FIG. 16).
Example 18
[0244] Characterization of Human IGF-II Derivative and Anti-IGFBP-3
Antibody, in vivo
[0245] To determine the effectiveness of the inventive method and
the inventive compound, in vivo, the following experiments were
conducted:
[0246] (1) the compound of the present invention was administered,
in vivo, to compare its effects to IGF, and
[0247] (2) the compound of the present invention was administered,
in vivo, to determine if it increased the concentration of free
IGF-I in the blood of a rat.
[0248] It is well known that administration of IGF, in vivo,
results in the lowering of blood lipid levels. Its recognized
specific effects are:
[0249] (a) lowering the level of free fatty acid (Turkalj. I., et
al., J. Clin. Endocrinol. Metab., 75, 1186 (1992)),
[0250] (b) lowering the level of triglyceride (Turkalj. I., et al.,
J. Clin. Endocrinol. Metab., 75, 1186 (1992); Zenobi, P. D., et
al., J. Clin. Invest 90, 2234 (1992)),
[0251] (c) lowering of total cholesterol vis--vis the lowering of
LDL-cholesterol (Kazumi, T., Metabolism, 35, 1024 (1986); Zenobi,
P. D., et al., Diabetologia 36, 465 (1993)), and the like.
[0252] The present inventors next investigated whether these same
effects are brought about by the administration of the inhibitory
compounds of Examples 8 and 9.
Example 18-1
[0253] Evaluation of Blood Lipid Level (1) [Leu27, Leu43]rIGF-II
(1,000 .mu.g/kg) was administered, subcutaneously, to a group of
nonfasting SD rats (n=3, 8 weeks old, male, average weight:300 g,
purchased from Charles River Japan).
[0254] Anti-RBP-3 pAb #35 (20 mg/head) was administered,
intraperitoneally, to another group of SD rats.
[0255] As a negative control, physiological saline (500 .mu.l/head)
was administered, subcutaneously, and as a positive control group,
IGF-I (50 .mu.g/kg, 200 .mu.g/kg) was administered,
subcutaneously.
[0256] About 500 .mu.l of blood was sequentially collected into
EDTA-coated glass tubes from the caudal vein. Thirty minutes after
collection, the blood was centrifuged (3,500 rpm.times.12 mm
25.degree. C.), and plasma supernatant was recovered and stored at
-80.degree. C. until assayed. The total cholesterol and total
triglyceride concentrations of the plasma were measured in
accordance with a "Hitachi 715 model" automatic analyzer using the
enzyme method of "Autocera CH02 and TG2" (each, manufactured by
Daiichi Pure Chemicals Co., Ltd.).
[0257] Compared to the negative control group, the total glyceride
concentration 6 hours after the administration of IGF-I (50
.mu.g/kg) and anti-RBP-3 pAb #35 was significantly decreased, and
[Leu27, Leu43]rIGF-II showed a tendency toward lowering total
glyceride levels as well (refer to FIG. 17). Compared to the
negative control group, the total cholesterol concentration 6 and
24 hours after the administration of IGF-I (200 .mu.g/kg) was
significantly decreased. A significant decrease 24 hours after the
administration of [Leu27, Leu43]rIGF-II was also observed. The
lowering tendency was also recognized even after the administration
of anti-RBP-3 pAb #35 (refer to FIG. 18).
[0258] The administration of the inventive inhibitory compounds
reduced lipid levels to that seen for IGF-I.
[0259] FIGS. 17 and 18 demonstrate the results obtained from the
inventive compounds: the percent concentrations of the total
triglyceride and total cholesterol are shown before and after
administration of the compounds.
Example 18-2
[0260] Evaluation of Blood Lipid Level (2)
[0261] Zucker fatty rats (insulin-resistant rat model, 6 to 11
weeks old, male, purchased from Tokyo Jikken Dobutsusha) were
fasted 20 hours before administration. The anti-RBP-3 pAb #36 (40
mg/head, n=5) was administered, subcutaneously, to a group of the
fasting rats.
[0262] As a negative control group, physiological saline (500
.mu.l/head, n=18) was administered, subcutaneously, and as a
positive control, IGF-I (300 .mu.g/kg, n=8) or IGF-II (1,200
.mu.g/kg, n=8, or 600 .mu.g/kg, n=8) was administered,
subcutaneously. From each group, blood was collected 6 hours after
administration. The total cholesterol and total triglyceride
concentrations in the plasma were measured in accordance with
Example 18-1. Based on the enzyme method of "NEFAC-test Wako"
(product of Wako Pure Chemicals), the concentration of free fatty
acid in plasma was measured in a 96-well microtiter plate (Costar)
with a 1/20 scale in the standard operating method, and then
measuring the absorbance (Abs 550 nm) by a plate reader ("Vmax",
product of Molecular Devices Ltd.).
[0263] Compared to the negative control group, the total
cholesterol concentration showed a significant decrease for all of
the treatment groups 6 hours after administration (refer to FIG.
19). As regards the total glyceride concentration, a significant
decrease was observed for all of the treatment groups 6 hours after
administration (refer to FIG. 20). A similar decrease in
concentration for free fatty acid was also observed for the
treatment groups (refer to FIG. 21).
[0264] The administration of an inhibitory antibody (i.e., anti
IGFBP-3 antibody which inhibits the binding of IGF and IGFBP-3)
mediates the lowering of lipid levels such as that seen with
IGF-I.
[0265] In FIGS. 19, 20 and 21, the percent concentrations for total
cholesterol and total triglyceride are shown before and after
administration of the inventive inhibitory compound.
Example 18-3
[0266] Evaluation of Free IGF-I Level in Blood
[0267] The anti-RBP-3 pAb #90 (40 mg/head) was administered,
intraperitoneally, to a group of non-fasting SD rats (n=7, 8 weeks
old, male, 10 average weight: 300 g, purchased from Charles River
Japan). As a negative control group, physiological saline (500
.mu.l/head) was administered, subcutaneously. The sequential blood
collection was carried out in accordance with Example 18-1.
[0268] Free IGF-I levels in plasma and total IGF-I levels in plasma
were analyzed by a reverse-phase cartridge ("Sep-Pak C18
cartridge") method (Hizuka, N., et al., Growth Regulation, 1, 51
(1991)) and the formic acid/acetone extraction method (Bowsher, R.
R., et al., Endocrinology, 128, 805 (1991), respectively.
[0269] The rat IGF-I radioimmunoassay was carried out under
conventional methods (Moses, A. C., et al., Eur. J. Biochem., 103,
401 (1980); Daughaday, W. H., Methods Enzymol., 146, 248 (1987)).
.sup.125I-IGF-I and somatomedin-C antiserum (anti IGF-I antibody),
each produced by Eiken Chemical Ltd., were used and rat IGF-I
produced by Gropep Pty Ltd. was used as standard. In the first
step, .sup.125I-IGF-I (7.77.times.10.sup.3 cpm/100 .mu.l),
approximately 50 .mu.l of antiserum, the IGF-I standard and test
compound were mixed. The mixture was adjusted to 300 .mu.l with an
assay buffer (25 mM sodium phosphate buffer (pH 7.5) containing
0.25% BSA, 0.05% Tween 20 and 0.1% NaN.sub.3) and incubated
overnight at 4.degree. C. To the mixture, 75 .mu.l of 4 mg/ml human
.gamma.-globulin and 375 .mu.l of 25% PEG 6000 were added, and the
mixture incubated at 4.degree. C. for 1 hour, followed by
centrifugation (3,000 rpm.times.20 mm, 4.degree. C.). The
supernatant was decanted and the cpm incorporation in the pellet
was measured by .gamma.-counting.
[0270] Compared to the negative control, the concentration or ratio
of free IGF-I to total IGF-I showed a significant increase 6 hours
after the administration of anti-RBP-3 pAb#90 (refer to FIG.
22).
[0271] In FIG. 22, the ratios of free IGF-I/total IGF-I) 1 and 6
hours after administration are shown.
Sequence CWU 1
1
6 1 20 DNA Rattus norvegicus STS (1)..(20) One of PCR primers for
cloning of the 5' end of IGFBP-3 gene from rat pancreas cDNA
library The Sequence is described at page 38, line 19 of the
specification. 1 cgccatgcat cccgcgcgcc 20 2 20 DNA Rattus
norvegicus STS (1)..(20) Another PCR primer for cloning of the 5'
end of IGFBP-3 gene from rat pancreas cDNA library The Sequence is
described at page 38, line 20 of the specification. 2 acgccgcacg
cgtcgccttc 20 3 30 DNA Rattus norvegicus STS (1)..(30) One of PCR
primers for cloning of the 3' end of IGFBP-3 gene from rat pancreas
cDNA library The Sequence is described at page 38, line 22 of the
specification. 3 gcgcgggccc cgtggtgcgc tgcgaaccgt 30 4 19 DNA
Rattus norvegicus STS (1)..(19) Another PCR primer for cloning of
the 5' end of IGFBP-3 gene from rat pancreas cDNA library The
Sequence is described at page 38, line 23 of the specification. 4
tgctgatcac gttgttggc 19 5 21 DNA Artificial Sequence misc_feature
(1)..(21) Description of Artificial Sequence Synthetic DNA oligomer
which was used in preparation of [Leu27]rIGF-II The Sequence is
described at page 45, line 24 of the specification. 5 ctggaaaaga
ggaaacctct g 21 6 17 DNA Artificial Sequence misc_feature (1)..(17)
Description of Artificial Sequence Synthetic DNA oligomer which was
used in preparation of [Leu43]rIGF-II The Sequence is described at
page 45, line 24 of the specification. 6 ttcttcgagg atacctc 17
* * * * *