U.S. patent application number 11/504217 was filed with the patent office on 2008-05-29 for specific antagonists for glucose-dependent insulinotropic polypeptide (gip).
Invention is credited to Linda Neville, Chi-Chuan Tseng, M. Michael Wolfe.
Application Number | 20080125371 11/504217 |
Document ID | / |
Family ID | 26708269 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080125371 |
Kind Code |
A1 |
Wolfe; M. Michael ; et
al. |
May 29, 2008 |
Specific antagonists for glucose-dependent insulinotropic
polypeptide (GIP)
Abstract
In one embodiment, this invention provides an antagonist of
glucose-dependent insulinotropic polypeptide (GIP) consisting
essentially of a 24 amino acid polypeptide corresponding to
positions 7-30 of the sequence of GIP. In another embodiment, this
invention provides a method of preventing and treating obesity and
non-insulin dependent diabetes mellitus (Type II) in a patient
comprising administering to the patient an antagonist of
glucose-dependent insulinotropic polypeptide (GIP). In yet another
embodiment, this invention provides a method of improving glucose
tolerance in a mammal comprising administering to the mammal an
antagonist of glucose-dependent insulinotropic polypeptide
(GIP).
Inventors: |
Wolfe; M. Michael; (Newton,
MA) ; Tseng; Chi-Chuan; (Newton, MA) ;
Neville; Linda; (Hull, MA) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Family ID: |
26708269 |
Appl. No.: |
11/504217 |
Filed: |
August 15, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08984476 |
Dec 3, 1997 |
7091183 |
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11504217 |
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60032329 |
Dec 3, 1996 |
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Current U.S.
Class: |
514/6.7 ;
514/21.4; 514/6.8; 530/325 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/605 20130101; A61P 3/10 20180101 |
Class at
Publication: |
514/13 ; 514/14;
514/15; 514/18; 530/325 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 38/06 20060101 A61K038/06; A61K 38/08 20060101
A61K038/08; C07K 14/00 20060101 C07K014/00; A61P 3/10 20060101
A61P003/10; A61K 38/10 20060101 A61K038/10 |
Goverment Interests
[0001] The work leading to this invention was supported in part by
Grant Nos. DK08753 and RO1DK48042 from the National Institutes of
Health. The U.S. Government may have certain rights to this
invention.
Claims
1. An antagonist of glucose-dependent insulinotropic polypeptide
(GIP) consisting essentially of a 24 amino acid polypeptide
corresponding to positions 7-30 of the sequence of GIP.
2. An isolated polypeptide comprising an amino acid sequence which
specifically interferes with the biological activity of
glucose-dependent insulinotropic polypeptide (GIP) when said
polypeptide is administered to an animal in an amount effective to
reduce intestinal uptake of glucose.
3. A polypeptide according to claim 2, wherein said polypeptide
comprises an amino acid identical to SEQ ID NO:2.
4. A polypeptide according to claim 2, wherein the polypeptide
comprises an amino acid sequence at least 95% identical to SEQ ID
NO:2.
5. A polypeptide according to claim 2, wherein said polypeptide
comprises an amino acid sequence at least 95% identical to SEQ ID
NO:8.
6. A polypeptide according to claim 2, wherein the polypeptide
comprises an amino acid sequence identical to SEQ ID NO:8.
7. A polypeptide according to claim 2, wherein said polypeptide
comprises an amino acid sequence at least 95% identical to SEQ ID
NO:3.
8. A polypeptide according to claim 2, wherein the polypeptide
comprises an amino acid sequence at least 95% identical to SEQ ID
NO:3.
9. A polypeptide according to claim 2, wherein said polypeptide
comprises an amino acid sequence identical to SEQ ID NO:9.
10. A polypeptide according to claim 2, wherein the polypeptide
comprises an amino acid sequence at least 95% identical to SEQ ID
NO:9.
11. A polypeptide according to claim 2, wherein said polypeptide
comprises an amino acid sequence identical to SEQ ID NO:5.
12. A polypeptide according to claim 2, wherein the polypeptide
comprises an amino acid sequence at least 95% identical to SEQ ID
NO:5.
13. A polypeptide according to claim 2, wherein said polypeptide
comprises an amino acid sequence identical to SEQ. ID NO 10.
14. A polypeptide according to claim 2, wherein the polypeptide
comprises an amino acid sequence at least 95% identical to SEQ ID
NO:10.
15. A polypeptide according to claim 2, wherein said polypeptide
comprises an amino acid sequence identical to SEQ ID NO:13.
15. A polypeptide according to claim 2, wherein the polypeptide
comprises an amino acid sequence at least 95% identical to SEQ ID
NO:13.
17. A polypeptide according to claim 2, wherein said polypeptide
comprises the amino acid sequence of SEQ ID NO:13.
18. A polypeptide according to claim 9, wherein the polypeptide
comprises an amino acid sequence at least 95% identical to SEQ ID
NO:13.
19. A polypeptide having an amino acid sequence which specifically
interferes with the biological activity of glucose-dependent
insulinotropic polypeptide (GIP) when said polypeptide is
administered to a mammal in an amount effective to reduce
absorption of glucose from the mammalian gut, said polypeptide
comprising the amino acid sequence of SEQ ID NO 6.
20. A polypeptide according to claim 39, wherein the polypeptide
comprises an amino acid sequence at least 95% identical to SEQ ID
NO:2.
21. An isolated polypeptide antagonist of glucose-dependent
insulinotropic polypeptide (GIP) receptor effective to reduce
glucose uptake from a mammalian intestine and reduce serum insulin
levels.
Description
FIELD OF THE INVENTION
[0002] This invention is directed to specific antagonists of
glucose-dependent insulinotropic polypeptide (GIP). This invention
is also directed to treatment of non-insulin dependent diabetes
through increasing glucose tolerance without requirement for
increased serum insulin, the treatment of obesity by the
administration of a GIP antagonist, the development of nonpeptide
GIP antagonist compounds, and compositions.
BACKGROUND
[0003] Insulin release induced by the ingestion of glucose and
other nutrients is due part to both hormonal and neural factors
(Creutzfeldt, et al., 1985, Diabetologia 28:565-573). Several
gastrointestinal regulatory peptides have been proposed as
incretins, the substance(s) believed to mediate the enteroinsular
axis and that may play a physiological role in maintaining glucose
homeostasis (Unger, et al., 1969, Arch. Intern. Med, 123:261-266;
Ebert R., et al. 1987, Diab. Metab. Rev., 3:1-16; Dupre J., 1991,
"The Endocrine Pancreas." Raven Press, New York, p 253). Among
these candidates, only glucose-dependent insulinotropic polypeptide
(GIP) and glucagon like peptide-1 (7-36)(GLP-1) appear to fulfill
the requirements to be considered physiological stimulants of
postprandial insulin release (Dupre, et al. 1973, J. Clin.
Endocrinol. Metab., 37:826-828; Nauck, et al., 1989; J. Clin.
Endocrinol. Metab., 69:6540662; Kreymann, et al. 1987, Lancet,
2:1300-1304; Mojsov, et al., 1987, J. Clin. Invest.,
79:616-619).
[0004] Following oral glucose administration, serum GIP levels
increase several fold (see Cleator, et al., 1975, Am. J. Surg.,
130:128-135; Nauck, et al. 1986, J. Clin. Endocrinol. Metab.,
63:492-498; Nauck, et al., 1986, Diabetologia, 29:46-52; Salera, et
al., 1983, Metabolism, 32:21-24; Kreymann, et al., 1987, Lancet,
2:1300-1304), and although the increment in plasma GLP-1
concentration in response to glucose is also significant, it is far
smaller in magnitude (Kreymann, et al., 1987, Lancet, 2:1300-1304;
Orskov, et al., 1987, Scand. J. Clin. Lab. Invest., 47:165-174;
Orskov, et al., 1991, J. Clin. Invest., 87:415-423; Shuster, et
al., 1988, Mayo Clin. Proc., 63:794-800). In human volunteers,
Nauck et al. (1993, J. Clin. Endocrinol. Metab., 76:912-917) showed
that GIP was a major contributor in the incretin effect after oral
glucose, whereas GLP-1 appeared to play a major role. Shuster et
al. (1988) also suggested that GIP was the most important, but not
the sole, mediator of the incretin effect in humans.
[0005] Some studies have demonstrated that GIP and GLP-1 are
equally potent in their capacity to stimulate insulin release
(Schmid, et al., 1990, Z. Gastroenterol., 28:280-284; Suzuki, et
al., 1990, Diabetes, 39:1320-1325), whereas others have suggested
that GLP-1 possesses greater insulinotropic properties (Siegel, et
al. 1992, Eur. J. Clin. Invest. 22:154-157; Shima, et al. 1988,
Regul. Pept., 22:245-252). Recently, using a putative specific
antagonist to the GLP-1 receptor, exendin (9-39), Wang et al. have
demonstrated that exenden reduced postprandial insulin release by
48% and thus concluded that GLP-1 might contribute substantially to
postprandial stimulation of insulin secretion (Wang, et al. 1995,
J. Clin. Invest., 95:417-421). More recent studies, however, have
shown that exendin might also displace GIP binding from its
receptor and thereby reduce GIP-stimulated cyclic adenosine
monophosphate (cAMP) generation (Wheeler, et al. 1995,
Endocrinology, 136:4629-4639; Gremlich, et al. 1995, Diabetes,
44:1202-1208). Therefore, the antagonist properties of exendin
(9-39) might not be limited to GLP-1.
[0006] The availability of a GIP-specific receptor antagonist would
be invaluable for determining the precise roles of these peptides
in mediating postprandial insulin secretion.
SUMMARY OF THE INVENTION
[0007] It is an object of this invention to provide specific
antagonists of glucose-dependent insulinotropic polypeptide
(GIP).
[0008] It is another object of this invention to provide
alternative methods for treatment of non-insulin dependent diabetes
which increase glucose tolerance without requirement for increased
serum insulin, for treatment of obesity with a GIP antagonist which
inhibits, blocks or reduces glucose absorption from the intestine
of an animal, and for development of nonpeptide GIP antagonist
compounds.
[0009] In one embodiment, this invention provides an antagonist of
glucose-dependent insulinotropic polypeptide (GIP) consisting
essentially of a 24-amino acid polypeptide corresponding to
positions 7-30 of the sequence of GIP.
[0010] In another embodiment, this invention provides a method of
treating non-insulin dependent diabetes mellitus in a patient
comprising administering to the patient an antagonist of
glucose-dependent insulinotropic polypeptide (GIP).
[0011] In yet another embodiment, this invention provides a method
of improving glucose tolerance in a mammal comprising administering
to the mammal an antagonist of glucose-dependent insulinotropic
polypeptide (GIP).
[0012] Using a reporter L-cell line stably transfected with rat GIP
receptor cDNA (LGIPR2), the inventors have identified a fragment of
GIP [GIP (7-30)-NH.sub.2] as a specific GIP receptor antagonist.
This antagonist (referred to as ANTGIP) inhibited GIP-stimulated
intracellular cAMP production in vitro, and ANTGIP competed with
GIP for binding to cellular receptors, but did not complete with
GLP-1. ANTGIP inhibited the GIP-dependent release of insulin in
vivo, but ANTGIP had no effect on glucose-, GLP-1-, GIP-, and
arginine-induced insulin release in anesthetized rats. In conscious
rats, ANTGIP inhibited postprandial insulin release, without
significantly affecting the serum glucose concentration. However,
despite its inhibiting effect on insulin release, ANTGIP has been
discovered to enhance glucose tolerance in an oral glucose
tolerance test.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A and 1B show cAMP-dependent .beta.-galactosidase
production by LGIPR2 cells in the presence of GIP or various GIP
fragments.
[0014] FIG. 2 shows dose-dependent inhibition of ANTGIP on
GIP-included cAMP-dependent .beta.-galactosidase production in
LGIPR2 cells.
[0015] FIG. 3 shows competition of .sup.125I-GIP and .sup.125I
GLP-1 (inset) binding by GIP, GLP-1 and ANTGIP.
[0016] FIG. 4 shows plasma insulin concentrations (.+-.SE) in
fasted anesthetized rats after 30 min of GIP, ANTGIP, or 0.9 NaCl
infusion.
[0017] FIG. 5 shows plasma insulin concentrations (.+-.SE) in
fasted anesthetized rats after a 30-min infusion of GLP-1 (0.4
nmol/kg), glucose (0.8 g/kg), or arginine (375 mg/kg) with (open
bars) or without (solid bars) ANTGIP (100 nmol/kg) (n=6 for each
group).
[0018] FIG. 6 shows postprandial plasma insulin and serum glucose
levels (.+-.SE) in conscious trained rats.
[0019] FIG. 7 shows plasma insulin level following oral glucose
administration to rats with or without ANTGIP injection.
[0020] FIG. 8 shows plasma glucose level following oral glucose
administration to rats with and without ANTGIP injection.
[0021] FIG. 9 shows the effects of the GIP receptor antagonist,
ANTGIIP, on the absorption of free D-glucose from the lumen of the
jejunal test segment.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Glucose-dependent insulinotropic polypeptide (GIP) is
42-amino acid hormone that was originally described as a inhibitor
of acid secretion. More recently, however, it has been shown to be
potent stimulant for the release of insulin from the endocrine
pancreas.
[0023] The inventors have confirmed previous studies (Rossowski, et
al., 1992, Regul. Pep., 39:9-17) indicating that truncated GIP [GIP
(1-30)-NH.sub.2] might be one of the biologically active forms of
mature GIP. As shown in FIG. 1, GIP (1-30)-NH.sub.2 was nearly
equipotent to GIP (1-42) in stimulating cAMP dependent
.beta.-galactosidase production in LGIPR2 cells. These findings are
consistent with the observations of Wheeler, et al. (1995),
reported that both GIP(1-42) and GIP(1-30) exhibited similar
stimulatory properties for cAMP production in COS-7 cell
transiently expressing GIP receptor cDNA. Moreover, Kieffer et al.
(1993, Can. J. Physiol. Pharmacol., 71:917-922) found that GIP
(1-30) competitively inhibited binding of GIP (1-42) to the GIP
receptor in .beta.TC3 cells. These data suggest the possibility of
cellular processing of GIP (1-42) to yield biologically-active
.alpha.-amidated GIP (1-30).
Physiological Effects of GIP Antagonists
[0024] Insulin release induced by the ingestion of glucose and
other nutrients is due in part to both hormonal and neural factors
(see, e.g., Creutzfeldt, et al., 1985). Although a number of
gastrointestinal regulatory peptides have been proposed as putative
incretins; GIP and GLP-1 are the most likely physiological
insulinotropic peptides. Although both GIP and GLP-1 possess
significant insulinotropic properties, controversy exists regarding
their relative physiological roles in stimulating insulin
release.
[0025] Using a GLP-1 receptor antagonist exendin (9-39), Wang et
al. (1995) detected a 50% decrease in postprandial insulin
secretion in exendin-treated rats. Administration of exendin also
reduced 70% of insulin release following intraduodenal glucose
infusion (Kolligs, et al., 1995, Diabetes, 44:16-19). Recent
studies, however, have demonstrated that exendin also displaced GIP
binding from its receptor, and inhibits cAMP generation in response
to GIP stimulation (Wheeler, et al. 1995; Gremlich, et al. 1995).
Therefore, the antagonist properties of exendin do not appear to be
GLP-1 specific.
[0026] Successful synthesis by the present inventors of a specific
GIP receptor antagonist greatly facilitates investigation of the
relative contribution of GIP in mediating the enteroinsular axis.
The GIP fragment ANTGIP [GIP (7-30)-NH.sub.2] specifically inhibits
various GIP-dependent effects. In LGIPR2 cells, ANTGIP inhibited
the cAMP response to GIP in a concentration-dependent manner (see
FIG. 2), and in .beta.TC3 cells, the antagonist displaced GIP
binding from its receptor (see FIG. 3). Furthermore, ANTGIP
completely abolished the insulinotropic properties of GIP in fasted
anesthetized rats, while not affecting GLP-1, glucose-, or
arginine-stimulated insulin release indicating that this antagonist
is GIP-specific. ANTGIP alone demonstrated no stimulatory effect on
insulin release or cAMP generation in either intact rats or LGIPR2
cells, indicating the absence of any agonist properties. Studies
demonstrated that even at a concentration as high as 10.sup.-4 M,
ANTGIP did not stimulate a detectable increase in cAMP-dependent
.beta.-galactosidase level in LGIPR2 cells.
[0027] The inventors have observed a 72% decrease in postprandial
insulin release in response to the administration of ANTGIP to
rats. ANTGIP did not affect GLP-1 binding to its receptor, and the
insulinotropic effect of GLP-1 is preserved in vivo in the presence
of ANTGIP. Furthermore, postprandial GLP-1 levels were not affected
by ANTGIP. These findings are consistent with a dominant role for
GLP in mediating the enteroinsular axis.
[0028] Wang et al. demonstrated an approximate 50% reduction in
postprandial insulin levels in exendin-treated rats, whereas plasma
glucose levels increased minimally from 7.5 to 8.7 mmol/l. The
physiological significance of this minor increment in glucose level
was not clear to Wang, et al. The inventors found that serum
glucose concentrations remained largely unchanged despite a marked
decrease in serum insulin levels in ANTGIP-treated rats. The
results of the present study are consistent with the notion that
insulin is not the sole mediator of glucose homeostasis, but that
glucose maintenance is dependent on numerous neurohumoral factors.
These factors include hormones, such as pancreatic glucagon,
cortisol, and growth hormone, and physiological events, including
peripheral and hepatic glucose uptake.
[0029] The results of the present studies demonstrate that GIP
(7-30)-NH.sub.2 is a specific receptor antagonist of naturally
occurring GIP. GIP (7-30)-NH.sub.2 inhibits GIP-induced cAMP
generation and insulin release, but does not affect the
insulinotropic effects of other secretagogues such as glucose,
arginine, and GLP-1. Furthermore, circulating insulin levels
decreased by 72% in response to the concomitant administration of
GIP (7-30)-NH.sub.2 to chow-fed rats, indicating that GIP plays a
dominant role in mediating postprandial insulin secretion.
[0030] Strikingly, although GIP (7-30)-NH.sub.2 reverses the
insulin stimulatory properties of the parent compound, when the GIP
antagonist was administered to rats (injected intraperitoneally),
oral glucose tolerance was improved: a significant decrease in
serum glucose levels was detected at all time points in all rats.
In addition, plasma insulin levels were also diminished in these
same rats. These results are surprising--with the decrease in
insulin release, one would expect an increase in serum glucose.
However, GIP has several other peripheral effects which may include
an affect of GIP on peripheral glucose utilization, and the
decrease in serum glucose levels seen with GIP might be due to such
an effect.
[0031] The effect of GIP antagonists on serum glucose levels in the
absence of increased serum insulin suggests their use in patients
with noninsulin dependent diabetes mellitus (NIDDM). With the aging
of the United States population, an increase in the number of cases
of NIDDM has been predicted. In the past forty years, very few new
forms of therapy for this most prevalent disease have been
developed. GIP antagonists enhance tolerance to oral glucose, as
demonstrated herein, and therefore treatment of NIDDM patients with
these compounds is indicated.
GIP Antagonists
[0032] A GIP antagonist according to this invention is any
composition which interferes with biological action of GIP. Such
compositions include antibodies specific for either GIP or GIP
receptors, antisense RNA which hybridizes with mRNA encoding GIP or
GIP receptor, or other genetic controls which knock out expression
of GIP or GIP receptor. GIP antagonists also include peptides or
other small molecules which bind to the GIP receptor and block the
cAMP response to GIP. Suitable assays for antagonist activity are
exemplified in Examples 1 and 2 below:
[0033] As described herein (see Example 1 below), the inventors
have now discovered a polypeptide fragment of GIP that is a
specific GIP receptor antagonist. While the 30-amino acid
N-terminal fragment [GIP (1-30)-NH.sub.2] was as effective in
stimulating cAMP increase through GIP receptors as the parent
hormone, a fragment missing the most N-terminal six amino acids
[GIP (7-30)-NH.sub.2] did not stimulate cAMP release in the same
system. Thus, the N-terminal hexamer appears to be important for
functional GIP signaling. GIP fragments missing the N-terminal 15
amino acids (e.g., GIP (16-30)-NH.sub.2) did not mimic GIP, but
neither did they inhibit GIP-dependent effects. Thus, the segment
from amino acids 7-15 appears to be especially important in
signaling through the GIP receptor. Fragment GIP (10-30)-NH.sub.2
was less effective as an antagonist, but retained some ability to
affect GIP receptor activation, as indicated by partial agonist
activity. Thus, peptide antagonists would appear to require the
segment from amino acids 7-9 of the GIP sequence, and some or all
of the amino acids from 10-30 or effective alternative amino acids
thereto are likely to promote binding to the receptor. It should
therefore be understood by those of skill in this art that the
present invention contemplates any polypeptide sequence which
effectively prevents GIP activation of its native receptor, such as
the sequence containing amino acids in positions 7-30 of the
sequence of the GIP sequence and polypeptides based upon sequences
containing amino acids in positions 7-30 of the sequence of the GIP
that include additional, deleted or alternative amino acids to form
effective GIP polypeptide antagonist. Polypeptides based on this
sequence may be designed for use as GIP antagonists according to
this invention by the skilled artisan, who will routinely confirm
that the resultant peptides exhibit antagonist function by testing
the peptides in in vitro and in vivo assays such as those described
in Examples 1 and 3-5 below.
[0034] Immunologic components specific for GIP or GIP receptors can
be employed as GIP antagonists. Such antagonists include with
specific monoclonal antibodies (either naked or conjugated to
cytotoxic agents) or specific activated cytotoxic immune cells.
Such antibodies or immune cells may be generated as reagents
outside the body, or may be generated inside the body by vaccines
which target GIP or GIP receptors.
[0035] Antibodies which are specifically reactive with GIP or the
hormone binding domain of GIP receptor, or antigenic recombinant
peptide fragments of either of those proteins, may be obtained in a
number of ways which will be readily apparent to those skilled in
the art. The known sequences of GIP (see Takeda, et al. 1987, Proc.
Natl. Acad. Sci USA, B84:7005-7008, and Genbank Accession No.
M18185), and GIP receptor (see Bonner, T. I., and Usdin, T. B.,
1995, Genbank Accession No U39231) can be used in conjunction with
standard recombinant DNA technology to produce the desired
antigenic peptides in recombinant systems (see, e.g., Sambrook et
al.). Antigenic fragments of GIP or GIP receptor can be injected
into an animal as a immunogen to elicit polyclonal antibody
production. Purification of the antibodies can be accomplished by
selective binding from the serum, for instance by using cells
transformed with DNA sequence encoding the respective proteins. The
resultant polyclonal antisera may be used directly or may be
purified by, for example, affinity absorption using recombinantly
produced protein coupled to an insoluble support.
[0036] In another alternative, monoclonal antibodies specifically
immunoreactive with either GIP or the hormone binding domain of GIP
receptor may be prepared according to well known methods (See,
e.g., Kohler and Milstein, 1976, Eur. J. Immunol., 6:611), using
the proteins or antigenic fragments described above as
immunogen(s), using them for selection or using them for both
functions. These and other methods for preparing antibodies or
immune cells that are specifically immunoreactive with GIP or GIP
receptor are easily within the skill of the ordinary worker in the
art.
[0037] Immunogenic compositions according to this invention for use
in active immunotherapy include recombinant antigenic fragments of
GIP or GIP receptor prepared as described above and expression
vectors (particularly recombinant viral vectors) which express
antigenic fragments of GIP or GIP receptor. Such expression vectors
can be prepared as described in Baschang, et al., U.S. Pat. No.
4,446,128, incorporated herein by reference, or Axel, et al.,
Pastan, et al., or Davis, et al., using the known sequences of GIP
or GIP receptor.
[0038] Still another GIP antagonist according to this invention is
an expression vector containing an antisense sequence corresponding
to all or part of an mRNA sequence encoding GIP or GIP receptor,
inserted in opposite orientation into the vector after a promoter.
As a result, the inserted DNA will be transcribed to produce an RNA
which is complementary to and capable of binding or hybridizing to
the mRNA. Upon binding to the GIP or GIP receptor mRNA, translation
of the mRNA is prevented, and consequently the protein coded for by
the mRNA is not produced. Suitable antisense sequences can be
readily selected by the skilled artisan from the sequences of GIP
or GIP receptor cited above. Production and use of antisense
expression vectors is described in more detail in U.S. Pat. No.
5,107,065 and U.S. Pat. No. 5,190,931, both of which are
incorporated herein by reference.
[0039] Alternative materials within the contemplation of the
skilled artisan which function as antagonists of GIP in the
procedures described in Examples 1 and 3-5 below may also be used
in the therapeutic methods according to this invention.
Therapeutic Use of GIP Antagonists
[0040] GIP (7-30)-NH.sub.2 acts as a receptor antagonist of GIP,
but also improves glucose tolerance contrary to the expected
consequence of blocking GIP-dependent insulin secretion. In
addition, a GIP receptor antagonist in accordance with the present
invention inhibits, blocks or reduces glucose absorption from the
intestine of an animal. In accordance with this observation,
therapeutic compositions containing GIP antagonists may be used in
patients with noninsulin dependent diabetes mellitus (NIDDM) to
improve tolerance to oral glucose or in animals, such as humans, to
prevent, inhibit or reduce obesity by inhibiting, blocking or
reducing glucose absorption from the intestine of the animal, as
demonstrated herein.
[0041] Therapeutic compositions according to this invention are
preferably formulated in pharmaceutical compositions containing one
or more GIP antagonists and a pharmaceutically acceptable carrier.
The pharmaceutical composition may contain other components so long
as the other components do not reduce the effectiveness of the GIP
antagonist according to this invention so much that the therapy is
negated. Examples of such components include sweetening, flavoring,
coloring, dispersing, disintegrating, binding, granulating,
suspending, wetting, preservative and demulcent agents and the
like. Pharmaceutically acceptable carriers are well known, and one
skilled in the pharmaceutical art can easily select carriers
suitable for particular routes for administration (Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,
1985).
[0042] Also in accordance with the present invention, the GIP
receptor antagonist of the present invention may be lyophilized
using standard techniques known to those in this art. The
lyophilized GIP receptor antagonists may then be reconstituted
with, for example, suitable diluents such as normal saline, sterile
water, glacial acetic acid, sodium acetate, combinations thereof
and the like. The reconstituted GIP receptor antagonists in
accordance with the present invention may be administered
parenterally or orally and may further include preservatives or
other acceptable inert components as mentioned hereinbefore.
[0043] The pharmaceutical compositions containing any of the GIP
antagonists according to this invention may be administered by
parenteral (subcutaneously, intramuscularly, intravenously,
intraperitoneally, intrapleurally, intravesicularly or
intrathecally, topical, oral, rectal, or nasal route, as
necessitated by choice of drug and disease. The dose used in a
particular formulation or application will be determined by the
requirements of the particular state of disease and the constraints
imposed by the characteristics of capacities of the carrier
materials. The concentrations of the active agent in
pharmaceutically acceptable carriers may range from 0.1 nM to 100
.mu.M. The compositions described above may be combined or used
together or in coordination with another therapeutic substance.
[0044] Dose will depend on a variety of factors, including the
therapeutic index of the drugs, disease type, patient age, patient
weight, and tolerance of toxicity. Dose will generally be chosen to
achieve serum concentrations from about 0.1 .mu.g/ml to about 100
.mu.g/ml. Preferably, initial dose levels will be selected based on
their ability to achieve ambient concentrations shown to be
effective in in-vitro models, such as that used to determine
therapeutic index, and in-vivo models and in clinical trials, up to
maximum tolerated levels. Standard clinical procedure prefers that
chemotherapy be tailored to the individual patient and the systemic
concentration of the chemotherapeutic agent be monitored regularly.
The dose of a particular patient can be determined by the skilled
clinician using standard pharmacological approaches in view of the
above factors. The response to treatment may be monitored by
analysis of blood or body fluid levels of the glucose or GIP or GIP
antagonist according to this invention, measurement of activity if
the antagonist or its levels in relevant tissues or monitoring
disease state of the patient. The skilled clinician will adjust the
dose based on the response to treatment revealed by these
measurements.
[0045] One approach to therapy of NIDDM is to introduce vector
expressing antisense sequences to block expression of GIP and/or
GIP receptor. In one embodiment of this invention, a method is
provided which comprises obtaining a DNA expression vector
containing a cDNA sequence having the sequence of human GIP or GIP
receptor mRNA which is operably linked to a promoter such that it
will be expressed in antisense orientation, and transforming cells
which express GIP or GIP receptor, respectively, with the DNA
vector. The expression vector material is generally produced by
culture of recombinant or transfected cells and formulated in a
pharmacologically acceptable solution or suspension, which is
usually a physiologically-compatible aqueous solution, or in coated
tablets, tablets, capsules, suppositories, inhalation aerosols, or
ampules, as described in the art, for example in U.S. Pat. No.
4,446,128, incorporated herein by reference.
[0046] The vector-containing composition is administered to a
mammal exhibiting NIDDM in an amount sufficient to transect a
substantial portion of the target cells of the mammal.
Administration may be any suitable route, including oral, rectal,
intranasal or by intravesicular (e.g. bladder) instillation or
injection where injection may be, for example, transdermal,
subcutaneous, intramuscular in intravenous. Preferably, the
expression vector is administered to the mammal so that the target
cells of the mammal are preferentially transfected. Determination
of the amount to be administered will involve consideration of
infectivity of the vector, transection efficiency in vitro, immune
response of the patient, etc. A typical initial dose for
administration would be 10-1000 micrograms when administered
intravenously, intramuscularly, subcutaneously, intravesicularly,
or in inhalation aerosol, 100 to 1000 micrograms by mouth, 10.sup.5
to 10.sup.10 plaque forming units of a recombinant vector, although
this amount may be adjusted by a clinician doing the administration
as commonly occurs in the administration of other pharmacological
agents. A single administration may usually be sufficient to
produce a therapeutic effect, but multiple administrations may be
necessary to assure continued response over a substantial period of
time.
[0047] Further description of suitable methods of formulation and
administration according to this invention may be found in U.S.
Pat. Nos. 4,592,002 and 4,920,209, which are incorporated herein by
reference in their entireties.
[0048] The present invention also contemplates the use of the GIP
antagonists and/or its properties to develop nonpeptide compounds
which exhibit antagonist properties similar to the GIP polypeptide
antagonists as herein described using techniques known those versed
in the pharmaceutical industry.
EXAMPLES
[0049] In order to facilitate a more complete understanding of the
invention, a number of Examples are provided below. However, the
scope of the invention is not limited to specific embodiments
disclosed in these Examples, which are for purposes of illustration
only.
Example 1
Effects of Various Peptide Fragments on cAMP Production
[0050] To define the biologically active region of GIP, the effects
of several peptide fragments of GIP on stimulating cAMP-dependent
.beta.-galactosidase production in LGIPR2 cells were examined.
LGIPR2 cells are stably transfected with a cAMP-dependent promoter
from the VIP gene fused to the bacterial lac Z gene. When
intracellular cAMP increases within these cells, lac Z gene
transcription is activated, resulting in the accumulation of its
product, .beta.-galactosidase. The measurement of
.beta.-galactosidase in this system provided a convenient,
inexpensive, and nonradioactive method for detecting changes in the
levels of intracellular cAMP.
[0051] LGIPR2 cells were grown in Dulbecco's Modified Eagle's
Medium (DMEM) containing 4.5 g/L of glucose and 10% fetal calf
serum. For each assay, 10.sup.5 cells/well were seeded onto 24-well
plates. After incubation overnight, peptides were added in various
concentrations to the wells in the absence of
3-isobutyl-methylxanthine (IBMX) for 4 h, at which time maximal
stimulation of .beta.-galactosidase was determined. The medium was
then removed and wells rinsed once with phosphate-buffered saline
(PBS). The plates were then blotted briefly and frozen overnight at
-70.degree. C., and, after the addition of chlorophenol
red-.beta.-D-galactopyranoside, accumulated .beta.-galactosidase
was detected using a colorimetric assay, as described previously
(Usdin, et al., 1993, Endocrinology, 133:2861-2870).
[0052] Preliminary studies using LGIPR2 cells demonstrated that
GIP(1-42) stimulated .beta.-galactosidase production in a
concentration-dependent manner, with the maximum effect observed at
4 h with 10.sup.-8 M. Various peptide fragments of GIP, including
GIP(21-30)NH.sub.2, GIP (16-30)-NH.sub.2, GIP (7-30)-NH.sub.2, GIP
(1-30)-NH.sub.2, GIP (10-30)-NH.sub.2, and GIP (31-44), were
synthesized at the Biopolymer Laboratory, Harvard Medical School,
based on previously published rat GIP cDNA sequence (Tseng, et al.,
1993, Proc. Natl. Acad. Sci. USA, 90:1992-1996). LGIPR2 cells were
incubated in the presence of 10.sup.-M GIP or different GIP
fragments for 4 h, and .beta.-galactosidase was measured as
described herein and expressed in optical density (O.D.) units.
FIGS. 1A and 1B show cyclic AMP-dependent .beta.-galactosidase
generation in LGIPR2 cells in response to incubation with different
fragments of GIP. Values are expressed as the mean .+-.SE of
quadruplicate measurements (*p<0.01, compared to control).
[0053] As demonstrated in FIG. 1A, 10.sup.-8 M GIP (1-30)-NH.sub.2
stimulated .beta.-galactosidase production to a similar degree,
while none of the other peptide fragments tested, including GIP
(7-30)-NH.sub.2, GIP (16-30)-NH.sub.2, GIP (21-30)-NH.sub.2, and
GIP (31-44), stimulated .beta.-galactosidase generation above
control levels. Furthermore, no changes in
cAMP-dependent-.beta.-galactosidase levels were detected when
LGIPR2 cells were incubated in the presence of higher
concentrations of the smaller peptide fragments.
[0054] To examine whether any of these fragments might serve as an
antagonist to GIP, LGIPR2 cells were incubated with 10.sup.-8 M GIP
(1-42) and one of the peptide fragments at two different
concentrations (10.sup.-8 M or 10.sup.-6 M) for 4 h. LGIPR2 cells
were cultured in the presence of 10.sup.-1 M GIP and various
concentrations of ANTGIP, as depicted on the horizontal axis if
FIG. 2. Values are expressed as the mean .+-.SE of quadruplicate
measurements. Only GIP (7-30)-NH.sub.2 (ANTGIP) was found to
attenuate the cAMP stimulatory effects exhibited by GIP (1-42); the
inhibition was concentration-dependent, with half-maximal
inhibition occurring at 10.sup.-7 M (FIG. 2).
[0055] FIG. 1B shows that peptide GIP (10-30)-NH.sub.2 is an
antagonist, albeit a weak one, as demonstrated by the reduction in
GIP-stimulated .beta.-gal levels when GIP (10-30)-NH.sub.2 is
present with GIP (1-42) compared to GIP (1-42) alone. On the other
hand, GIP (10-30)-NH.sub.2 also has agonist properties, as
demonstrated by .beta.-gal level of 0.39 O.D. .+-.0.03 stimulated
by GIP (10-30)-NH.sub.2 alone, compared to 0.95.+-.0.04 for GIP
(1-42).
Example 2
Receptor Binding Studies
[0056] Binding studies were performed in either LGIPR2 or .beta.TC3
cells to determine the relative affinities of GIP, ANTGIP, and
GLP-1 for both GIP and GLP-1 receptors. GLP(7-37) and porcine GIP
(5 .mu.g each) were iodinated by the chloramine-T method and were
purified using C-18 cartridges (Sep-Pak.RTM., Millipore, Milford,
Mass.) using an acetonitrile gradient of 30-45%. The specific
activity of radiolabeled peptides was 10-50 .mu.Ci/mg (Hunter, et
al., 1962, Nature, 194:495-498; Kieffer, et al., 1993, Can. J.
Physiol. Pharmacol., 71:917-922). Aliquots were lyophilized and
reconstituted in assay buffer at 4.degree. C. to a concentration of
3.times.10.sup.5 cpm/100 .mu.l. Binding studies was performed in
desegrated LGIPR2 or .beta.TC2 cells, the latter a generous gift
from Dr. S. Efrat (Diabetes Center, Albert Einstein College of
Medicine, New York). The .beta.TC2 cell line originally arose in a
lineage of transgenic mice expressing an insulin promoted, SV40
T-antigen hybrid oncogene in pancreatic .beta.-cells (Efrat, et
al., 1988, Proc. Natl. Acad. Sci. U.S.A., 85:9037-9041) and has
previously been demonstrated to be responsive to both GIP and GLP
(Kieffer, et al., 1993, Can. J. Physiol. Pharmacol., 71:917-922).
The receptor binding buffer contained 138 mM NaCl, 5.6 mM KCl, 1.2
mM MgCl.sub.2, 2.6 mM CaCl.sub.2, 10 mM Hepes, 10 mM glucose, and
1% bovine serum albumin (BSA, fraction V, protease free, Sigma).
For binding assays, LGIPR2 (GIP binding) or .beta.TC3 (GLP-1
binding) cells were cultured in DMEM containing 4.5 g/L of glucose
and 10% fetal bovine serum until 70% confluent. Cells were washed
once with PBS and then harvested with PBS-EDTA solution. .beta.TC3
cells were then suspended in assay buffer at a density of
2.times.10.sup.6 cells/ml, and LGIPR2 cells were used at a density
of 2.5.times.10.sup.5 cells/ml. Binding was performed at room
temperature in the presence of 3.times.10.sup.5 cpm/ml of
[.sup.125I]-GIP and -GLP. Nonsaturable binding was determined by
the amount of radioactivity associated with cells when incubated in
the presence of unlabeled 10.sup.-6 M GIP, GLP, or 10.sup.-4 M
ANTGIP. Specific binding was defined as the difference between
counts in the absence and presence of unlabeled peptide. GIP
binding was examined using LGIRP2 cells, and GLP-1 binding was
assessed using .beta.TC3 cells, and the results are shown in FIG.
3. Values are expressed as a percentage of maximum specific binding
and are the mean .+-.SE, with assays performed in duplicate.
[0057] GIP and ANTGIP displaced the binding of [.sup.125I]GIP to
LGIPR2 cells in a concentration-dependent manner (FIG. 3), with an
IC.sub.50 of 7 mM for GIP (n=5) and 200 for ANTGIP (n=4). Binding
of [.sup.125I]GLP-1 to its .beta.TC3 cell receptor was displaced
fully by GLP-1, but negligibly by ANTGIP, with an IC.sub.50 of 4 nM
and 80 .mu.M, respectively (n=7; FIG. 3).
Example 3
Intravenous Infusion of Peptides in Fasting Anesthetized Rats
[0058] Adult male Sprague-Dawley rats (250-350 g) were purchased
from Charles River Co. (Kingston, Mass.). For infusion studies,
rats were fasted overnight and then anesthetized using
intraperitoneal sodium pentobarbital. The right jugular vein was
cannulated with silicon polymer, tubing (0.025 in I.D., 0.047 in
O.D., Dow Corning Corporation, Midland, Mich.), as described by Xu
and Melethil (21). The tubing was then connected to an infusion
pump (Harvard Apparatus Co., Inc., Millis, Mass.), and freshly made
0.9% NaCl, 5% glucose, arginine, GIP, or GLP-1 (peptides and
arginine dissolved in 0.9% NaCl) was infused at a rate of 0.1
ml/min. Blood (0.5 ml each) was obtained at 0, 10, 20, and 30 min
by translumbar vena cava puncture, as described by Winsett et al.
(1985, Am. J. Physiol., 249:G145-146), and samples were centrifuged
at 2,000 g for 10 min. Serum samples were separated and stored at
-20.degree. C. until assayed for insulin using a radioimmunoassay
kit (ICN Biochemicals, Costa Mesa, Calif.), and glucose, using a
One Touch Ii.beta. glucose meter (Lifescan, INS., Milpitas,
Calif.).
[0059] To examine the insulinotropic effect of GIP in vivo, fasted
anesthetized rats were perfused continuously with three different
concentrations of GIP (0.5, 1.0, and 1.5 nmol/kg) at a rate of 0.1
ml/min for 30 min (10.sup.-8 M equivalent to 1 nmol/kg/30 min).
Significant increases in plasma insulin levels were first detected
at 15 min, and after completion of the GIP infusion, insulin levels
were elevated with all three GIP concentrations (43.5.+-.2.7,
61.6.+-.4.2, and 72.4.+-.3.5 .mu.IU/ml, respectively) compared to
control (32.2.+-.3.3 .mu.IU/m, p<0.05, FIG. 4). The concomitant
administration of ANTGIP (100 nmol/kg) completely abolished the
insulinotropic properties of GIP (1.5 nmol/kg), with plasma insulin
returning to control values (FIG. 4). GIP was infused at 0.5, 1.0,
and 1.5 nmol/kg, with the largest insulin stimulatory response seen
with 1.5 nmol/kg. ANTGIP (100 nmol/kg) administered concomitantly
with GIP 1.5 nmol/kg completely abolished its insulinotropic
effect, whereas ANTGIP and 0.9% NaCl infusion had no effect on
insulin secretion (n=6 for each group, *p<0.05, compared with
basal levels).
[0060] To examine whether ANTGIP exerted a nonspecific effect on
.beta.-cell function, GLP-1 (0.4 nmol/kg), glucose (0.8 g/kg), or
arginine (375 mg/kg) was infused, in the presence or absence of the
antagonist for 30 min, as described by Wang et al. (13). FIG. 5
shows plasma insulin concentrations (.+-.SE) in fasted anesthetized
rats after a 30-min infusion of GLP-1 (0.4 nmol/kg), glucose (0.8
g/kg), or arginine (375 mg/kg) with (open bars) or without (solid
bars) ANTGIP (100 nmol per kg) (n=6 for each group, *p<0.05,
compared with basal levels). GLP-1, glucose, and arginine alone
each significantly increased insulin levels after 15 min of
infusion, and by 30 min, the insulin levels in GLP-1-, glucose-,
and arginine-infused rats were 50.3.+-.3.7, 63.1.+-.2.5,
69.7.+-.5.8 .mu.IU/ml respectively (p<0.01, compared with
control rats, 29.1.+-.2.9 .mu.IU/ml, FIG. 5). No significant change
in the insulin response was detected when ANTGIP was administered
concomitantly (FIG. 5).
Example 4
Insulinotropic Effect of GIP in Trained Conscious Fed Rats
[0061] Postprandial plasma insulin and serum glucose levels were
studied in conscious trained rats. Previous reports have indicated
that the stress response to injection in untrained rats might alter
their feeding and subsequently glucose and insulin levels (13). To
avoid such a response, rats were trained for 10 d before
experimentation. They were fasted from 17:00 to 08:00, and 0.9%
NaCl (0.3 ml) was injected subcutaneously at 08:00 before feeding.
After the injection of 0.9% NaCl, animals were given rat chow for
30 min, after which it was removed. At the end often days, the rats
were accustomed to the injection and ate quickly (consuming 4-6 g
of rat chow within 30 min).
[0062] On the day of the experiment, after fasting from 17:00 the
night before, trained rats were injected subcutaneously at 08:00
with 0.3 ml of either 0.9% NaCl or ANTGIP (100 nmol/kg). This dose
was chosen to approximately the amount of peptide used in the
anesthetized animal studies of Example 3. After injection, six of
the fasted control rats were killed to obtain baseline serum
glucose and insulin levels. ANTGIP- or 0.9% NaCl-treated rats (n=6
in each group) were exposed to chow for 30 min, after which food
was withdrawn. Rats were then anesthetized by intraperitoneal
sodium pentobarbital, and blood was collected by translumber vena
cava puncture at 20 and 40 min for the subsequent measurement of
plasma insulin, glucose, and GLP-1.
[0063] FIG. 6 shows postprandial plasma insulin and serum glucose
levels (.+-.SE) in conscious trained rats (* p<0.01 compared to
ANTGIP injection). In response to consuming chow, serum glucose and
plasma insulin levels increased significantly, with insulin levels
of 38.7.+-.5.3 and 58.9.+-.3.7 .mu.IU/ml at 20 and 40 min,
respectively (p<0.05, FIG. 6A). These increases in plasma
insulin level were nearly abolished by ANTGIP pretreatment; at 20
and 40 min, the plasma insulin concentrations were 25.3.+-.4.7 and
27.1.+-.2.6 .mu.IU/ml, respectively (p<0.01). Postprandial serum
glucose concentrations were similar in both saline- and
ANTGIP-treated rats (FIG. 6B). To determine whether the effects of
the GIP receptor antagonist were mediated through changes in GLP-1
release into the circulation, postprandial serum GLP-1 levels were
measured in both control and ANTGIP-treated animals.
Meal-stimulated serum GLP-1 concentrations were not affected by
ANTGIP administration. Following the ingestion of rat chow, serum
GLP-1 levels at 20 min were 280.+-.20 and 290.+-.10 pg/ml in
control and ANTGIP-treated rats, respectively; at 40 min, serum
GLP-1 concentrations were 320.+-.10 and 330.+-.20 pg/mgl,
respectively.
Example 5
Effect of ANTGIP on Glucose Tolerance and Plasma Insulin Levels
[0064] Oral glucose tolerance tests were performed on rats injected
intraperitoneally with ANTGIP (300 ng/kg) or 0.9% saline solution.
After the intraperitoneal injection of 0.9% NaCl or ANTGIP, an oral
glucose tolerance test was performed. The test was done by
administering a 40% glucose solution by oral gavage at a dose of 1
g per kg. The volume administered to each rat was approximately 0.5
ml. Blood was obtained at various time points for subsequent
measurement of plasma insulin and glucose levels.
[0065] As expected in view of the experiment in Example 4, rats
treated with ANTGIP showed reduced the plasma insulin levels (FIG.
7). Surprisingly, plasma glucose was diminished at all time points
in rats treated with ANTGIP, compared to control rats (FIG. 8).
Thus, ANTGIP increases glucose tolerance, despite its negative
effect on the insulinotropic response to GIP shown in Examples 3
and 4.
Example 6
Effect of GIP Receptor Antagonist on Intestinal Glucose
Absorption
[0066] Male Sprague-Dawley rats weighing about 200-250 g are fasted
overnight and anesthetized using intraperitoneal urethane (about
1.25 g per kg body weight). After midline laparotomy, an about
30-cm segment of jejunum, starting at about 5 cm distal to the
ligament of Treitz, is isolated and flushed with approximately 20
ml of about 0.9% NaCl. The jejunal test segments are each perfused
twice, initially with control buffer and then once again with
control buffer or with the test solution. The test solution
consists of Krebs-Ringer-bicarbonate buffer containing about 5
mmol/L [.sup.14C]D-glucose, and .sup.3H-labeled polyethylene glycol
is included in the luminal perfusate to correct for fluid movement.
The test or control solution is perfused through the jejunal
segment without recirculation at a flow rate of about 1.6 ml/min,
using a Harvard PHD 2000 syringe pump (Harvard Apparatus, Millis,
Mass.). The effluent from the luminal segment is collected at about
5-min intervals for about 30 min. After the initial period of
perfusion, the luminal contents in the jejunum are flushed with
about 20 ml of about 0.9% NaCl prior to the initiation of the
second period of perfusion. In all experiments, animals are
administered either about 0.9% NaCl (control) or ANTGIP (10 nmol/kg
body weight) though the inferior vena cava by single injection at
about time 0 min.
[0067] The enclosed FIG. 9 depicts the effects of the GIP receptor
antagonist, ANTGIP, on the absorption of free D-glucose from the
lumen of the jejunal test segment. Data points are believed to
represent the rate of glucose disappearance from the luminal
perfusate corrected for fluid movement. Results are expressed as
the mean .+-.SE of five experiments. Statistical significance (*)
is assigned if P<0.05. As seen in the figure, a ANTGIP is
believed to significantly reduce the absorption of D-glucose from
the jejunal test segment throughout the entire 30-mini period of
perfusion. Thus, it is believed that one of the mechanisms by which
GIP receptor antagonism may improve glucose tolerance is by
decreasing intestinal glucose absorption.
[0068] For purposes of clarity of understanding, the foregoing
invention has been described in some detail by way of illustration
and example in conjunction with specific embodiments, although
other aspects, advantages and modifications will be apparent to
those skilled in the art to which the invention pertains. The
foregoing description and examples are intended to illustrate, but
not limit the scope of the invention. Modifications of the
above-described modes for carrying out the invention that are
apparent to persons of skill in medicine, molecular biology,
pharmacology, and/or related fields are intended to be within the
scope of the invention, which is limited only by the appended
claims.
[0069] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference in their
entireties to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference.
Sequence CWU 1
1
14130PRTHomo sapiens 1Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser
Ile Ala Met Asp Lys 1 5 10 15Ile His Gln Gln Asp Phe Val Asn Trp
Leu Leu Ala Gln Lys 20 25 30224PRTHomo sapiens 2Ile Ser Asp Tyr Ser
Ile Ala Met Asp Lys Ile His Gln Gln Asp Phe 1 5 10 15Val Asn Trp
Leu Leu Ala Gln Lys 20315PRTHomo sapiens 3Lys Ile His Gln Gln Asp
Phe Val Asn Trp Leu Leu Ala Gln Lys 1 5 10 1549PRTHomo sapiens or
Rattus norvegicus 4Ile Ser Asp Tyr Ser Ile Ala Met Asp 1
5521PRTHomo sapiens 5Tyr Ser Ile Ala Met Asp Lys Ile His Gln Gln
Asp Phe Val Asn Trp 1 5 10 15Leu Leu Ala Gln Lys 2063PRTHomo
sapiens or Rattus norvegicus 6Ile Ser Asp 1730PRTRattus norvegicus
7Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys 1
5 10 15Ile Arg Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20
25 30824PRTRattus norvegicus 8Ile Ser Asp Tyr Ser Ile Ala Met Asp
Lys Ile Arg Gln Gln Asp Phe 1 5 10 15Val Asn Trp Leu Leu Ala Gln
Lys 20915PRTRattus norvegicus 9Lys Ile Arg Gln Gln Asp Phe Val Asn
Trp Leu Leu Ala Gln Lys 1 5 10 151021PRTRattus norvegicus 10Tyr Ser
Ile Ala Met Asp Lys Ile Arg Gln Gln Asp Phe Val Asn Trp 1 5 10
15Leu Leu Ala Gln Lys 201142PRTHomo sapiens 11Tyr Ala Glu Gly Thr
Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys 1 5 10 15Ile His Gln
Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys 20 25 30Lys Asn
Asp Trp Lys His Asn Ile Thr Gln 35 401242PRTRattus norvegicus 12Tyr
Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys 1 5 10
15Ile Arg Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys
20 25 30Lys Asn Asp Trp Lys His Asn Ile Thr Gln 35 401310PRTHomo
sapiens or Rattus norvegicus 13Asp Phe Val Asn Trp Leu Leu Ala Gln
Lys 1 5 101414PRTRattus norvegicus 14Gly Lys Lys Asn Asp Trp Lys
His Asn Leu Thr Gln Arg Glu 1 5 10
* * * * *