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TITLE: Combination Therapy Using Transferrin Fusion Proteins Comprising GLP-1
INVENTORS: Homayoun Sadeghi, Christopher Prior, and David J. Ballance
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application
60/598,031,
filed August 3, 2004, which is herein incorporated by reference in its
entirety.
[0002] This application is related to a Continuation-in-Part of
PCT/US03/26818, filed
August 28, 2003, which is a Continuation-in-Part of U.S. Application No.
10/378,094, filed
March 4, 2003, both of which are herein incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The present invention is related to transferrin fusion proteins
comprising
insulinotropic peptides with extended effective therapeutic in vivo half life.
The present
invention also relates to combination therapies using DPP-IV inhibitors and/or
neural
endopeptidase inhibitors (NEP) and insulinotropic peptides.
BACKGROUND OF THE INVENTION
Proteases
[0004] Proteolytic enzymes play an important role in regulating physiological
processes
such as cell proliferation, differentiation, and signaling processes by
regulating protein
turnover and processing. Proteolytic enzyme controls the levels of important
structural
proteins, enzymes, and regulatory proteins through proteolytic degradation.
Uncontrolled
proteolytic enzyme activity, eitlier increased or decreased, has been
implicated in a variety
of disease conditions including inflammation, cancer, arteriosclerosis, and
degenerative
disorders.
[0005] The International Union of Biochemistry and Molecular Biology (IUBMB)
has
recommended the use of the term "peptidase" for the subset of peptide bond
hydrolases
(Subclass E.C 3.4.). The widely used term protease is synonymous with
peptidase.
Peptidases comprise two groups of enzymes: the endopeptidases and the
exopeptidases,
which cleave peptide bonds at points within the protein and reinove amino
acids
sequentially from either N or C-terminus respectively. The term proteinase is
synonymous
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with endopeptidase. Proteolytic enzymes are classified according to their
catalytic
mechanisms. Four mechanistic classes have been recognized by the IiJBMB: the
serine
proteases, cysteine proteases, aspartic proteases, and metalloproteases.
[0006] Serine proteases are a large family of proteolytic enzymes containing a
serine
residue in the active catalytic site for protein cleavage. They are ubiquitous
being found in
viruses, bacteria, and eukaryotes. Serine proteases have a wide range of
substrate
specificities and can be subdivided into subfamilies on the basis of these
specificities.
There are over 20 subfamilies of serine proteases which are grouped into six
clans (SA, SB,
SC, SE, SF, and SG).
[0007] Prolyl oligopeptidase is a serine protease grouped in the SC clan. It
hydrolyzes
proline containing peptides at the carboxyl side of proline residues.
Presumably, it is
involved in the maturation and degradation of peptide hormones and
neuropeptides (Wilk et
al. 1983 Life Sci. 33, 2149-2157). Examples of prolyl oligopeptidase include
dipeptidyl
peptidase IV (DPP-IV), dipeptidyl peptidase II (DPP-II), fibroblast activation
protein, and
prolyl oligopeptidase. These enzymes display distinct specificities.
[0008] Proline is present in numerous peptide hormones. It determines certain
structural
properties of these peptides, such as conformation and stability of these
peptides, preventing
degradation by non-specific proteases. A number of peptidases exist which
attack the
proline bonds. These peptidases are not only involved in the cleavage of X-Pro
or Pro-X
bonds, but also in the degradation of corresponding alanyl bonds, with reduced
activity.
Peptidases having highly specific actions on proline-containing sequences are
attractive
targets of medicinal chemistry because some of them have been linked to the
modulation of
the biological activity of natural peptide substrates. For example, DPP-IV is
linked to the
treatment of diabetes through regulating the level of glucagon-like peptide-1
(GLP-1).
DPP-IV activity is increased in various diseases such as rheumatoid arthritis,
multiple
sclerosis, Grave's disease, and Hashimoto's thyroiditis, sarcoidosis, and
cancer. DPP-IV
activity is also increased in AIDS, Down's syndrome, anorexia/bulimia,
pregnancy and
hypogammaglobulinemia.
Dipeptidyl Peptidases Including DPP-IV
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[0009] Dipeptidyl aminopeptidase activity is peptidase activity which
catalyzes the
removal of dipeptides from the N-terminus of peptides, polypeptides, and
proteins.
Generally, a dipeptidyl aminopeptidase is capable of cleaving the dipeptide XY
from the
unsubstituted N-terminal amino group of a peptide, polypeptide or protein,
wherein X and Y
represent any amino acid residue. Examples of dipeptidyl peptidases (DPPs)
include
dipeptidyl peptidase I(DPP-I), dipeptidyl peptidase II (DPP-II), dipeptidyl
peptidase III
(DPP-III), and dipeptidyl peptidase (DPP-IV).
[0010] DPP-I, also known as cathepsin C, is a lysosomal cysteine protease that
is
expressed in most tissues. DPP-I has been implicated in the processing of
granzymes,
which are neutral serine proteases expressed exclusively in the granules of
activated
cytotoxic lymphocytes. DPP-II is a serine protease found in lysosomes. Like
DPP-IV, it
cleaves proline containing peptide bonds. In fact, DPP-II has a similar
substrate specificity
to DPP-IV but is only active at acidic pH. Dipeptidyl peptidase III (DPP-III)
is a
metalloprotease.
[0011] DPP-IV is a serine protease comprising the serine protease motif GWSYG
and
having broad substrate specificity. It hydrolyzes a peptide in sequence from
the amino
terminus to release an amino acid. However, the hydrolysis is terminated when
an amino
acid residue followed by proline is reached. As a result, a peptide having a
bond of X-Pro-
Y- (X and Y are optional amino acids) will be cleaved to yield X-Pro and Y-.
DPP-IV will
also cleave dipeptides with alanine in the penultimate position, though less
effectively than
dipeptides with proline (Yaron et al., 1993 Crit. Rev. Biochem. Mol. Biol.
28:31-81). The
enzyme will also cleave other sequences, but with still lower efficiency.
[0012] DPP-IV has been shown to be highly specific in releasing dipeptides
from the N-
terminal end of biologically active peptides with proline or alanine in the
penultimate
position of the N-terminal sequence of the peptide substrate. A large number
of potential
peptide substrates for DPP-IV have been identified. DPP-IV substrates include
peptide
hormones and chemokines. Examples of some peptide hormones are endomorphin-2,
GLP-
1, GLP-2, gastric inhibitory peptide (GIP), neuropeptide Y, growth hormone
releasing
hormone (GHRH) and substance P, and examples of some chemokines are RANTES,
GCP-
2, SDF-la, SDF-20, MDC, MCP-1, MCP-2, and MCP-3. DPP-II possesses almost
identical substrate specificity to DPP-IV.
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DPP-IV and Diabetes
[0013] Insulin-dependent diabetes mellitus (IDDM, or type I diabetes) is
currently treated
through the administration of insulin to patients. Non-insulin-dependent
diabetes mellitus
(NIDDM, or type II diabetes) is treated by diet, administration of
sulphonylureas to
stimulate insulin secretion or with biguanides to increase glucose uptake.
Resistant
individuals may need insulin therapy. Standard therapy requires daily
intravenous injection
of insulin which will treat the acute symptoms, but prolonged therapy results
in vascular
disease and nerve damage. Modem methods such as transplantation are expensive
and
require risky surgical intervention. Thus, there is a need to develop a highly
effective, low
cost alternative to the treatment of diabetes.
[0014] In recent years, there has been a growing interest in DPP-IV as a
target for
lowering the level of blood glucose. The use of inhibitors to block DPP-IV
enzyme or
DPP-IV-like enzyme activity in the blood of subjects leads to reduced
degradation of
endogenous or exogenously administered insulinotropic peptides such as, GIP,
GLP-1 or
analogs thereof. GIP and GLP-1, hormones that stimulate glucose-induced
secretion of
insulin by the pancreas, are substrates of DPP-IV. Specifically, since DPP-IV
removes the
amino-terminal His-Ala dipeptide of GLP-1 to generate GLP- 1 -(9-3 6)-amide,
which is
unable to elicit glucose-dependent insulin secretion from the islets, the
inhibition of such
DPP-IV or DPP-IV-like enzyme activity in vivo would effectively suppress
undesired
enzyme activity in pathological conditions in mammalian organisms.
[0015] PCT/DE97/00820 discloses alanyl pyrrolidide and isoleucyl thiazolidide
as
inhibitors of DPP-IV or DPP-IV-like enzyme activity. DD 296075 discloses
pyrrolidide
and isoleucyl thiazolidide hydrochloride. U.S. Patent 6,548,481 discloses
inhibitors
analogous to dipeptide compounds formed from an amino acid and a thiazolidine
or
pyrrolidine group, and salts thereof. Although these are functional inhibitors
of DPP-IV
activities, the use of these inhibitors in certain patients or certain forms
of the disease may
be problematic since the enzyme is responsible for activation or inactivation
of such a wide
range of bioactive peptides, i.e. DPP-IV inhibitors lack specificity for the
desired targets
GIP and GLP-1.
Protection of Therapeutic Peptides by Modification
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[0016] An alternative way to prevent therapeutic proteins and peptides such as
GIP or
GLP-1 from being cleaved by proteolytic enzymes is to modify the proteins and
peptides
themselves to block their exposure to proteolytic enzymes. Protein
modifications have been
shown to increase therapeutic polypeptides' stability, circulation time, and
biological
activity. Some general methods of modifying amino acids and peptides are
disclosed in
Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins --A Survey
of Recent
Developments (Weinstein, B., ed., Marcel Dekker, Inc., publ., New York 1983)
which is
incorporated herein by reference. Also, the review article of Francis (1992
Focus on
Growth Factors 3:4-10, (Mediscript, London)) describes protein modification
and fusion
proteins, which is incorporated herein by reference.
[0017] With the advance of recombinant DNA technology and automated
techniques, one
may now easily prepare large quantities of modified polypeptides that are
short, medium or
long. A large number of modified small polypeptide hormones may be synthesized
using
automated peptide synthesizers, solid-state resin techniques, or recombinant
techniques.
For example, large quantities of modified substrates of dipeptidyl peptidase,
for example,
the substrates of DPP-IV such as GLP-1, GIP, neuropeptide Y, and bradykinin
can be
produced using an automated peptide synthesizer.
SUMMARY OF THE INVENTION
[0018] The present invention provides transferrin fusion proteins comprising
therapeutic
peptides or proteins that are susceptible to protease cleavage. The present
invention also
provides transferrin fusion proteins comprising therapeutic peptides or
proteins that are
sensitive, resistant or partially resistant to protease cleavage. The protease
may be DPP-IV
or neutral endopeptidase (NEP). Moreover, the present invention provides
compositions
comprising transferrin fusion proteins and a second agent such as, but not
limited to,
inhibitors of DPP-IV and/or NEP. Further, the compositions may be
pharmaceutical
compositions used in the treatment of various diseases.
[0019] The present invention provides combination therapies comprising
administering a
transferrin fusion protein and at least one second agent in the treatment of
various diseases.
The transferrin fusion protein may be administered concurrently with the one
or more
second agent. Alternatively the transferrin fusion protein is administered
sequentially,
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either prior to or after the administration of the second agent. Preferably,
the second agent
is an inhibitor of DPP-IV or NEP.
[0020] The GLP-1 peptide moieties of the present invention may be modified to
contain one
or more mutations so that they are partially or fully resistant to protease
cleavage, such as
DPP-IV cleavage. The GLP-1 peptide may be GLP-1(7-37) (SEQ ID NO: 32) or GLP-
1(7-
36) (amino acids 1-30 of SEQ ID NO: 2). For example, these peptides may be
modified by
mutating A8 to G and/or K34 A.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 shows the restriction enzyme map of pREX0094.
[0022] Figure 2 shows the restriction enzyme map of plasmid pREX0198.
[0023] Figure 3 shows the restriction enzyme map of pSAC35.
[0024] Figure 4 shows the restriction enzyme m'ap of plasmid pREX0240.
[0025] Figure 5 shows the restriction enzyme map of pREX0052.
[0026] Figure 6 shows the restriction enzyme map of pREX0367.
[0027] Figure 7 shows the restriction enzyme map of pREX0368.
[0028] Figure 8 shows time course of incubation of GLP-1 and H-GLP-1 and DPP-
IV.
The graph shows the amount of active, full length peptide remaining, as
measured by an
ELISA specific for active GLP-1.
DETAILED DESCRIPTION
1. General Description
[0029] This invention is based, in part, on the need to develop a more
effective, low cost
alternative for the treatment of diabetes. Insulinotropic peptides, such as
GLP-1, are
promising therapeutic agents for the treatment of type 2 non-insulin-dependent
diabetes
mellitus as well as related metabolic disorders, such as pre-diabetes,
metabolic syndromes,
and obesity. Other useful insulinotropic peptides include exendin 3 and
exendin 4.
However, these insulinotropic peptides have short plasma half-lives in vivo,
mainly due to
rapid serum clearance and proteolytic degradation. Extensive work has been
done to inhibit
DPP-IV, the enzyme responsible for the degradation of GLP-1 or to modify GLP-1
in such
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a way that its degradation is slowed down while still maintaining biological
activity.
Despite these extensive efforts, a long lasting, active GLP-1 has not been
produced. There
is thus a need to modify GLP-1, exendin 3, exendin 4 and other insulinotropic
peptides to
provide longer duration of action in vivo, while maintaining their low
toxicity and
therapeutic advantages.
2. Defuu' tions
[0030] As used herein, the term "derivative" refers to a modification of one
or more amino
acid residues of a peptide by chemical means, either with or without an
enzyme, e.g., by
alkylation, acylation, ester formation, or amide formation.
[0031] As used herein, the term "derived from" refers to obtaining a molecule
from a
specified source such as obtaining a molecule from a parent molecule.
[0032] As used herein, the term "dipeptidyl aminopeptidase activity" refers to
a peptidase
activity which cleaves dipeptides from the N-terminal end of a peptide,
polypeptide, or
protein sequence. Generally, the dipeptidyl aminopeptidase is capable of
cleaving the
dipeptide XY from the unsubstituted N-terminal amino group of a peptide,
polypeptide, or
protein, wherein X or Y may represent any amino acid residue selected from the
group
consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser,
Thr, Trp, Tyr, and Val, but at least Ala, Arg, Asp, and/or Gly. Preferably, Y
is Pro or Ala.
All of X and Y may be different or identical. Examples of dipeptidyl
aminopeptidase
include, but are not limited to DPP-I, DPP-II, DPP-III, and DPP-IV.
[0033] As used herein, the terms "Glucagon-Like Peptide-1 (GLP-1)" and "GLP-1
derivatives" refer to intestinal hormones which generally simulate insulin
secretion during
hyperglycemia, suppress glucagon secretion, stimulate (pro) insulin
biosynthesis and
decelerate gastric emptying and acid secretion. Some GLP-ls and GLP-1
derivatives
promote glucose uptake by cells but do not simulate insulin expression as
disclosed in U.S.
Pat. No. 5,574,008 which is hereby incorporated by reference.
[0034] As used herein, the term "insulinotropic peptides" refers to peptides
with
insulinotropic activity. Insulinotropic peptides stimulate, or cause the
stimulation of, the
synthesis or expression of the hormone insulin. Such peptides include
precursors,
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analogues, fragments of peptides such as Glucagon-like peptide 1, exendin 3
and exendin 4
and other peptides with insulinotropic activity.
[0035] As used herein, "pharmaceutically acceptable" refers to materials and
compositions
that are physiologically tolerable and do not typically produce an allergic or
similar
untoward reaction, such as gastric upset, dizziness and the like, when
administered to a
human. Typically, as used herein, the term "pharmaceutically acceptable" means
approved
by a regulatory agency of the Federal or a state government or listed in the
U.S.
Pharmacopeia or other generally recognized pharmacopeias for use in animals,
and more
particularly in humans.
[0036] As used herein, the term "pharmaceutical composition" refers to a
composition
comprising an agent together with a pharmaceutically acceptable carrier or
diluent when
needed. Pharmaceutically acceptable carriers and additives are chosen such
that side effects
from the pharmaceutical compound are minimized and the performance of the
compound is
not canceled or inhibited to such an extent that treatment is ineffective.
[0037] As used herein, "physiologically effective amount" is that amount
delivered to a
subject to give the desired palliative or curative effect. This amount is
specific for each
drug and its ultimate approved dosage level.
[0038] As used herein, "therapeutically effective amount" refers to that
amount of
modified therapeutic polypeptide or peptide which, when administered to a
subject in need
thereof, is sufficient to effect treatment. The amount of modified therapeutic
polypeptide or
peptide which constitutes a "therapeutically effective amount" will vary
depending on the
therapeutic protein used, the severity of the condition or disease, and the
age and body
weight of the subject to be treated, but can be determined routinely by one of
ordinary skill
in the art having regard to his/her own knowledge and to this disclosure.
[0039] As used herein, "therapeutic protein" refers to proteins, polypeptides,
antibodies,
peptide fragments or variants thereof, having one or more therapeutic and/or
biological
activities. Therapeutic proteins encompassed by the invention include but are
not limited to
proteins, polypeptides, peptides, antibodies and biologics. The terms
peptides, proteins, and
polypeptides are used interchangeably herein. Additionally, the term
"therapeutic protein"
may refer to the endogenous or naturally occurring correlate of a therapeutic
protein. By a
polypeptide or peptide displaying a"therapeutic activity" or a protein that is
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"therapeutically active" is meant a polypeptide, peptide or protein that
possesses one or
more known biological and/or therapeutic activities associated with a
therapeutic protein
such as one or more of the therapeutic proteins described herein or otherwise
known in the
art. As a non-limiting example, a "therapeutic protein" is a protein,
polypeptide, or peptide
that is useful to treat, prevent or ameliorate a disease, condition or
disorder. Such a disease,
condition or disorder may be in humans or in a non-human animal, e.g.,
veterinary use.
[0040] As used herein, the term "treatment" or "treating" refers to any
administration of a
compound of the present invention and includes: (1) preventing the disease
from occurring
in an animal which may be predisposed to the disease but does not yet
experience or display
the pathology or symptomatology of the disease; (2) inhibiting the disease in
an animal that
is experiencing or displaying the pathology or symptomatology of the diseased
(i.e.,
arresting further development of the pathology and/or symptomatology); or (3)
ameliorating
the disease in an animal that is experiencing or displaying the pathology or
symptomatology
of the diseased (i.e., reversing the pathology and/or symptomatology).
[0041] As used herein, the term "biological activity" refers to the ability to
mediate a
biological function. "Biological activity" includes functional activity as
well as structural
activity.
[0042] As used herein, the term "palliative" refers to the ability to relieve
or soothe the
symptoms of a disease or disorder without affecting a cure. For example, an
agent that
alleviates pain without curing the condition or disease is a palliative agent.
[0043] As used herein, the term "prophylactic" refers to the having protective
effect such
as acting to defend against or prevent something, especially disease or
condition.
[0044] As used herein, "purified" protein or nucleic acid refers a protein or
nucleic acid
that has been separated from a cellular component. "Purified" proteins or
nucleic acids
have been purified to a level of purity not found in nature.
[0045] As used herein, the term "substantially pure" protein or nucleic acid
refers to a
protein or nucleic acid preparation that is lacking in all other cellular
components.
[0046] As used herein, the term "therapeutic" refers to having a curative,
restorative, or
remedial effect. For example, a"therapeutic agent" or a "therapeutic
composition" has a
curative effect.
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3. Specific Embodiments
Dipeptidyl Peptidases
[0047] Dipeptidyl peptidases are hydrolases that remove dipeptides from the
unsubstituted
N-terminal amino group of a peptide, polypeptide, or protein. Examples of
dipeptidyl
peptidases include but are not limited to DPP-I, DPP-II, DPP-III, DPP-IV,
attractin, and
fibroblast activation protein (FAP). New enzymes of this family or with
similar function
but different structure are emerging.
[0048] Dipeptidyl peptidase I (DPP-I), also known as cathepsin C, is a
lysosomal cysteine
protease belonging to the papain family. DPP-I is capable of sequentially
removing
dipeptides from the free amino terminus of various peptide and protein
substrates, thus
acting in the exopeptidase (specifically dipeptidyl peptidase) mode. The
cleavage is
ineffective if the fragmented bond has on either side a proline residue, or
the N-terminal
residue is lysine or arginine.
[0049] DPP-II is a serine protease found in lysosomes with unknown function.
Like DPP-
IV, it cleaves predominantly proline containing peptide bonds. In fact, DPP-II
has a similar
substrate specificity to DPP-IV but is only active at acidic pH. Mammalian DPP-
II and
DPP-IV can be distinguished using the inhibitors puromycin and bacitracin;
puromycin will
inhibit DPP-II only while bacitracin inhibits DPP-IV only (1988 J. Biol. Chem.
263, 6613-
6618). Dipeptidyl peptidase III (DPP-III) is a metalloprotease. DPP-V releases
N-terminal
X-Ala, His-Ser, and Ser-Tyr dipeptides.
[0050] DPP-VII, also known as quiescent cell proline dipeptidase, is a proline-
specific
dipeptidase. It has been suggested that DPP-VII and DPP-II are identical
proteases based
on a sequence comparison of human DPP-VII and rat DPP-II (78% identity) (Araki
et al.
2001 J. Biochem. 129, 279-288).
[0051] DPP-VIII is a human postproline dipeptidyl aminopeptidase that is
homologous to
DPP-IV and FAP (Abbott, C.A. et al., 2000 European Journal of Biochemistry
267, 6140).
Similar to DPP-IV, DPP-VIII is ubiquitous. The full-length DPP-VIII cDNA codes
for an
882-amino-acid protein that has about 27% identity and 51% similarity to DPP-N
and FAP,
but no transmembrane domain and no N-linked or 0-linked glycosylation.
Purified
recoinbinant DPP-VIII hydrolyzed the DPP-IV substrates Ala-Pro, Arg-Pro and
Gly-Pro.
Thus recombinant DPP-VIII shares a postproline dipeptidyl aminopeptidase
activity with
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DPP-IV and FAP. DPP-VIII enzyme activity had a neutral pH optimum consistent
with it
being nonlysosomal. The similarities between DPP-VIII and DPP-IV in tissue
expression
pattern and substrates suggests a potential role for DPP-VIII in T-cell
activation and
immune function similar to DPP-IV.
[0052] Olsen C. et al. (2002 Gene 299, 185-93) report the identification and
characterization of a novel DPP-IV-like molecule, termed dipeptidyl peptidase-
like protein
DPP-IX. Like DPP-IV, DPP-IX comprises the serine protease motif GWSYG (SEQ ID
NO: 110). The presence of this motif and the conserved order and spacing of
the Ser, Asp,
and His residues that form the catalytic triad in DPP-IV, places DPP-IX in the
DPP-IV gene
family.
[0053] Attractin (DPPT-L) is a 175-kDa soluble glycoprotein reported to
hydrolyze Gly-
Pro. Attractin contains a kelch repeat domain and shares no significant
sequence homology
with DPP-IV or any other peptidase. Fibroblast activation protein (FAP) is a
cell surface-
bound protease of the prolyl oligopeptidase gene family expressed at sites of
tissue
remodelling.
[0054] Prolyl endopeptidase (PEP), also called proline oligopeptidase (PO),
was first
discovered by Walter and coworkers as an oxytocin-degrading enzyme in the
human uterus
(Walter et al., Science 173, 827-829 (1971)). The enzyme cleaves peptide bonds
at the
carboxy-side of proline in peptides containing the sequence X-Pro-Y, where X
is a peptide
or N-terminal substituted amino-acid and Y is a peptide, amino acid, amide or
alcohol
(Yoshimoto et al., J. Biol. Chem. 253, 3708-3716 (1979)). The enzyme has a
high
specificity for the trans-conformation of the peptide bond at the imino-side
of proline (Lin
& Brandts, Biochemistry 22, 4480-4485 (1983)).
[0055] Prolyl oligopeptidase hydrolyzes angiotensin I and angiotensin II which
results in
the release of angiotensin (1-7). Angiotensin (1-7) has vasodilator activity
and modulates
the release of vasopressin, which is able to influence the process of memory
as was shown
by injecting rats with specific PEP-inhibitors. The injection reverses the
scopolamine
induced amnesia. This experiment is not only an example which provides
evidence for a
possible physiologic function for the enzyme, but moreover it has led to the
hypothesis that
inhibitors for PEP can influence the memory process and counter dementia
(Yoshimoto et
al. 1987 J. Pharmacobio-Dyn. 10, 730-735).
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Dipeptidyl Peptidase (DPP-IV) and Substrates
[0056] DPP-IV is a ubiquitously expressed molecule that has been implicated in
the
degradation of several peptides and hormones. Various types of DPP-IV have
been purified
and the enzymological properties have been revealed. For example, DPP-IV has
been
isolated from rat liver (Hopsu-Havu V. K. et al., 1966 Histochem., 7:197-201),
swine
kidney (Barth A. et al., 1974 Biol. Med. Chem., 32:157-174), small intestine
(Svensson B.
1978 Eur. J. Biochem., 90:489-498), liver (Fukasawa K. M. et al. 1981 Biochim.
Biophys.
Acta, 657:179-189), human submaxillary gland (Oya H., et al., 1972 Biochim.
Biophys.
Acta, 258:591-599), sheep kidney (Yoshimoto T. et al., 1977 Biochim. Biophys.
Acta,
485:391-401; Yoshimoto T. et al., 1978 J. Biol. Chem., 253:3708-3716) or
microorganisms
(Fukusawa K. M. 1981 Biochem. Biophys., 210:230-237; Yoshimoto T. 1982 J.
Biochem.,
91:1899-1906 (1982)).
[0057] In the human immune system, DPP-IV is identical to the T-cell surface
antigen
CD26 which is expressed by activated lymphocytes (T-, B-, and natural killer
cells).
CD26/DPP-IV is a Type II membrane glycoprotein with intrinsic dipeptidyl
peptidase IV
activity and the ability to bind adenosine deaminase Type I(ADA-1). It is
expressed on
epithelial cells constitutively, but on T lymphocytes, it is expressed under
tight cellular
regulation, with expression upregulated upon cell activation. CD26/DPP-IV has
been
shown to have dipeptidyl peptidase IV activity in its extracellular domain
(Hegen et al.,
1990 J. Immunol 144:2908-2914; Ulmer et al., 1990 Scand. J. Immunol. 31:429-
435) and
the costimulatory activity appears to be partially dependent upon this enzyme
activity
(Tanaka et al., 1993 Proc. Natl. Acad. Sci. USA 90:4586-4590). DPP-IV is
involved in the
regulation of chemokine function and may play an important role in HIV
infection.
[0058] US Patent 6,265,551 discloses a circulating, soluble form of DPP-
IV/CD26
isolated from human serum. The serum form shares similar enzymatic and
antigenic
properties with the ubiquitous membrane form; however, in several biochemical
aspects
there are distinct differences. In particular, the circulating serum form has
a molecular
weight of 175 kDa, in contrast to the 105 kDa molecular weight of the membrane
form, and
it does not bind ADA-1. Nevertheless, the circulating form expresses
functional
dipeptidylpeptidase IV activity and retains the ability to costimulate the T
lymphocyte
response to recall antigen.
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[00591 The proteolytic activity of DPP-IV resides in a stretch of
approximately 200 amino
acids located at the C-terminal end of the protein. The catalytic residues
(Ser-629, Asp-708,
His-740) are arranged in a unique order which is different from the classical
serine
proteases such as chymotrypsin and subtilisin. Proline specific dipeptidyl
peptidase activity
alters the biological activity of a large number of bioactive proteins and
polypeptides
comprising, amongst others, GLP-1, the neurotransmitter substance P. human
growth
hormone-releasing factor, erythropoietin, interleukin 2 and many others.
Potential DPP-IV
substrates are listed in Tables 1, 2 and 3. Modulation of these polypeptides
to affect DPP-
IV cleavage may be useful in the treatment of clinical conditions including
but not limited
to diabetes, inflammation, vascular diseases, auto-immune disease, multiple
sclerosis, joint
diseases and diseases associated with benign and malign cell transformation.
TABLE 1: Human cytokines, growth factors, neuro- and vasoactive peptides with
a
penultimate proline, which are putative substrates for DPP-IV
Polypeptide SEQ ID NO: N-terminal sequence
Interleukin-l.beta. 1 Ala-Pro-Val-Arg-Ser-
Interleukin-2 2 Ala-Pro-Thr-Ser-Ser-
Interleukin-5 3 Ile-Pro-Thr-Glu-Ile-
Interleukin-6 4 Val-Pro-Pro-Gly-Glu-
Interleukin-10 5 Ser-Pro-Gly-Gln-Gly-
Interleukin-13 (recombinant) 6 Ser-Pro-Gly-Pro-Val-
Complement C4a 7 Lys-Pro-Arg-Leu-Leu-
Granulocyte chemotactic protein II 8 Gly-Pro-Val-Ser-Ala-
Granulocyte macrophage colony stimulating 9 Ala-Pro-Ala-Arg-Ser-
Factor
Granulocyte colony stiunulating factor 10 Thr-Pro-Leu-Gly-Pro-
Erythopoietin 11 Ala-Pro-Pro-Arg-Leu-
Gastrin releasing peptide growth hormone 12 Phe-Pro-Thr-Ile-Pro-
Interferon inducible peptide 10 (.gamma.IP10) 13 Val-Pro-Leu-Ser-Arg-
Interferon regulatory factor 1(IRF-1) 14 Met-Pro-Ile-Thr-Arg
Interferon regulatory factor 2 (IRF-2) 15 Met-Pro-Val-Glu-Arg
Insulin-like growth factor-1 16 Gly-Pro-Glu-Thr-Leu-
Melanoma growth stimulating activity 17 Ala-Pro-Leu-Ala-Thr-
Migration inhibition factor 18 Met-Pro-Met-Phe-Ile-
Monocyte chemotactic protein I 19 Glu-Pro-Asp-Ala-Ile-
Neuro eptide Y 20 Tyr-Pro-Ser-Lys-Pro-
Pancreatic polypeptide 21 Ala-Pro-Leu-Glu-Pro-
Peptide YY 22 Try-Pro-Ile-Lys-Pro-
Prolactin 23 Leu-Pro-Ile-Cys-Pro-
RANTES 24 Ser-Pro-Tyr-Ser-Ser-
Substance P 25 Arg-Pro-Lys-Pro-Gln-
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Polypeptide SEQ ID NO: N-terminal sequence
Thrombopoietin 26 Ser-Pro-Ala-Pro-Pro-
Transforming protein (N-myc) version 1 27 Met-Pro-Gly-Met-Ile-
Transforming protein (N-myc) version 2 28 Met-Pro-Ser-Cys-Ser-
Tumor necrosis factor beta. 29 Leu-Pro-Gly-Val-Leu-
Vascular endothelial growth factor 30 Ala-Pro-Met-Ala-Glu-
TABLE 2: Human peptides and proteins with a penultimate alanine that are
putative
substrates for DPP IV
adenosine deaminase
Annexins
breast basic conserved protein
Cofilin
natural killer cell enhancing factor b
precursors of a-interferon
precursors of interleukin 1, a and 1, 0 and interleukin 13
precursors of macrophage inflammatory protein-2-a and 2-0
precursor of melanocyte stimulating hormone
precursor of oxytocin-neurophysin 1
growth hormone releasing hormone
P amyloid protein (1-28)
anxiety peptide
joining peptide of pro-opiomelanocortin
[0060] The present invention may utilize modified substrates of DPP comprising
one or
more additional amino acids at the N-terminus of the substrates to protect the
substrates
from DPP activity. The preferred substrates for modification according to the
present
invention are disclosed in Table 3.
[0061]
Table 3: Substrates for DPP-IV (CD26) Cleavage
DPP-IV Substrate SEQ Seauence
ID NO:
GIP 31 YAEGTFISDY SIAMDKIHQQ DFVNWLLAQK
GKKNDWKHNI TQ
GLP-1 32 HAEGTFTSDV SSYLEGQAAK EFIAWLVKG
(Ainuzo
Acids 1-
29
GLP-2 33 HADGSFSDEM NTILDNLAAR DFINWLIQTK ITD
growth hormone 34 YADAIFTNSY RKVLGQLSAR KLLQDIMSRQ
releasing hormone QGESNQERGA RARL
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DPP-IV Substrate SEQ Seguence
ID NO:
Glucagon (slow 35 HSQGTFTSDY SKYLDSRRAQ DFVQWLMNT
inactivation, unlike GIP
and the GLPs)
peptide histidine- 36 HADGVFTSDF SKLLGQLSAK KYLESLM
methionine
IGF-1 37 G PETLCGAELV DALQFVCGDR GFYFNKPTGY GSSSRRAPQT
GIVDECCFRS CDLRRLEMYC APLKPAKSAR SVRAQRHTDM
PKAQKEVHLK NASRGSAGNK TY
Bradykinin 38 RPPGFSPFR
Substance P 39 RPKPQQFFGL M
CLIP 40 RPVKVYPNGA EDESAEAFPL EF
Neuropeptide Y 41 YPSKPDNPGE DAPAEDMARY YSALRHYINL ITRQRY
peptide YY (DPP-IV 42 YPIKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY
activates it)
Prolactin 43 LPICPGGAA RCQVTLRDLF DRAVVLSITYI HNLSSEMFSE
FDKRYTHGRG FITKAINSCH TSSLATPEDK EQAQQMNQKD
FLSLIVSILR SWNEPLYHLV TEVRGMQEAP EAILSKAVEI
EEQTKRLLEG MELIVSQVHP ETKENEIYPV WSGLPSLQMA
DEESRLSAYY NLLHCLRRDS HKIDNYLKLL KCRIIHNNNC
human chorionic 44 (alpha subuiut) APDVQDCPEC TLQEDPFFSQ PGAPILQCMG
gonadotropni (HCG) CCFSRAYPTP LRSKKTMLVQ KNVTSESTCC VAKSYNRVTV
MGGFKVEDHT ACHCSTCYYH KS
human chorionic 45 (beta subunit) SKEPLRPRCR PINATLAVEK EGCPVCITVN
gonadotropin (HCG) TTICAGYCPT MTRVLQGVLP ALPQVVCNYR NVRFESIRLP
GCPRGVNPVV SYAVALSCQC ALCRRSTTDC GGPKDHPLTC
DDPRFQDSSS SKAPPPSLPS PSRLPKPSDT PILPQ
enterostatin 46 APGPR
gastrin-releasing peptide 47 VPLPAGGGTV LTKMYPRGNH WAVGHLM
IL-2 48 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML
TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL
RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR
WITFCQSIIS TLT
IL-lb 49 APVR SLNCTLRDSQ QKSLVMSGPY ELKALHLQGQ
DMEQQVVFSM SFVQGEESND KIPVALGLKE KNLYLSCVLK
DDKPTLQLES VDPKNYPKKK MEKRFVFNKI EINNKLEFES
AQFPNWYIST SQAENMPVFL GGTKGGQDIT DFTMQFVSS
endomorphin-2 50 YPFF
tyr-melanostatin 51 YPLG
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DPP-IV Substrate SEQ Seguence
ID NO:
aprotinin 52 RPDFCLEPPY TGPCKARIIR YFYNAKAGLC QTFVYGGCRA
KRNNFKSAED CMRTCGGA
RANTES 53 SPYSSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV
FVTRKNRQVC ANPEKKWVRE YINSLEMS
trypsinogen 54 NPLLILTFV AAALAAPFDD DDKIVGGYNC EENSVPYQVS
LNSGYHFCGG SLINEQWVVS AGHCYKSRIQ VRLGEHNIEV
LEGNEQFINA AKIIRHPQYD RKTLNNDIML IKLSSR.AVIN
ARVSTISLPT APPATGTKCL ISGWGNTASS GADYPDELQC
LDAPVLSQAK CEASYPGKIT SNMFCVGFLE GGKDSCQGDS
GGPVVCNGQL QGVVSWGDGC AQKNKPGVYT
KVYNYVKWIK NTIAANS
alphal-microglobulin 55 G PVPTPPDNIQ VQENFNISRI YGKWYNLAIG STCPWLKKIM
DRMTVSTLVL GEGATEAEIS MTSTRWRKGV CEETSGAYEK
TDTDGKFLYH KSKWNITMES YVVHTNYDEY AIFLTKKFSR
HHGPTITAKL YGRAPQLRET LLQDFRVVAQ GVGIPEDSIF
TMADRGECVP GEQEPEPILI PRV
interferon-inducible 56 VPLSRTVRCT CISISNQPVN PRSLEKLEII PASQFCPRVE
protein 10 (IP10) IIATMKKKGE KRCLNPESKA IKNLLKAVSK ERSKRSP
Eotaxin 57 GPASVPTTCC FNLANRKIPL QRLESYRRIT SGKCPQKAVI
FLTKLAKDIC ADPKKKYVQD SMKYLDQKSP TPKP
Monocyte 58 QPDAINAPVT CCYNFTNRKI SVQRLASYRR ITSSKCPKEA
chemoattractant protein VIFKTIVAKE ICADPKQKWV QDSMDHLDKQ TQTP
1 MCP-1
Monocyte 59 QPDSVSIPIT CCFNVINRKI PIQRLESYTR ITNIQCPKEA
chemoattractant protein VIFKTKRGKE VCADPKERWV RDSMKHLDQI FQNLKP
2 (MCP-2)
Monocyte 60 QPVGINTSTT CCYRFINKKI PKQRLESYRR TTSSHCPREA
chemoattractant protein VIFKTKLDKE ICADPTQKWV QDFMKHLDKK TQTPKL
3 (MCP-3)
Granulocyte chemotactic 61 GPV SAVLTELRCT CLRVTLRVNP KTIGKLQVFP
protein-2 AGPQCSKVEVV ASLKNGKQVC LDPEAPFLKK
VIQKILDSGN KKN
SDF-la 62 KPVSLSYRCP CRFFESHVAR ANVKHLKILN TPNCALQIVA
RLKNNNRQVC IDPKLKWIQE YLEKALNK
SDF-lb 63 KPVSLSYRCP CRFFESHVAR ANVKHLKILN TPNCALQIVA
RLKNNNRQVC IDPKLKWIQE YLEKALNKRF KM
Macrophage-derived 64 GPYGANMEDS VCCRDYVRYR LPLRVVKHFY
chemokine WTSDSCPRPG VVLLTFRDKE ICADPRVPWV KMILNKLSQ
b-casomorphin 65 YPFVEPI
Procolipase 66 APG PRGIIINLEN GELCMNSAQC KSNCCQHSSA
LGLARCTSMA SENSECSVKT LYGIYYKCPC ERGLTCEGDK
TIVGSITNTN FGICHDAGRS KQ
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DPP-IV Substrate SEQ Seguence
ID NO:
Vasoactive Intestinal 67 HSDAVFTDNYTRLRKQMAVKKYLNSILN
Peptide (VIP)
Pituitary Adenylyl 68 HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK
Cyclase-Activating
Peptide 38 (PACAP38)
Oxyntomodulin 69 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA
Growth hormone (1-43) 70 FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYS
Secretin 71 HSDGTFTSELSRLREGARLQRLLQGLV
Brain-derived natriuretic SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH
peptide
[0062] The substrates for modification comprise X-ProY, X-Ala-Y, X-Ser-Y, or X-
Gly-Y
at the amino terminus. Preferably, the substrate for modification is GLP-1.
Modified Polypeptides Protected from DPP Activity
[0063] The present invention provides modified polypeptides, such as modified
polypeptide substrates of DPP, comprising one or more additional amino acids
at the N-
terminus to protect the polypeptide substrates from DPP activity. In one
embodiment, the
modified polypeptides have one additional amino acid at their N-terminus as
compared to
the wild-type polypeptides. In another embodiment, the modified polypeptides
have five
additional amino acids at their N-terminus. Alternatively, the modified
polypeptides have
between one and five additional amino acids at their N-terminus. Any one of
the 20 amino
acids may be added to the N-terminus of the polypeptide substrate or non-
natural amino
acids may be added.
[0064] It is expected that any pharmaceutical polypeptide having peptide bonds
which
would be subject to cleavage in the circulation or anywhere in vivo after
administration
would benefit from modification in accordance with the present invention
because of the
protection from DPP cleavage that is afforded by the present invention.
[0065] In accordance with this aspect of the invention, it is possible to
remove at least
about 30%, preferably at least about 50%, more preferably at least about 70%,
still more
preferably at least about 90%, and most preferably at least about 99% of the
dipeptidyl
peptidase activity. It is also possible to completely remove the dipeptidyl
aminopeptidase
activity using the methods of the present invention.
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[00661 Likewise, it is possible to reduce the substrate's dipeptidyl peptidase
sensitivity by
at least about 30%, preferably at least about 50%, more preferably at least
about 70%, still
more preferably at least about 90%, and most preferably at least about 99% of
the dipeptidyl
peptidase sensitivity. It is also possible to completely remove the dipeptidyl
aminopeptidase sensitivity using the methods of the present invention.
[0067] Although the modified polypeptide or peptide substrates of the present
invention
are partially or substantially protected from DPP activity, the modified
polypeptide
substrates have retained at least about 10%, preferably at least about 30%,
more preferably
at least about 50%, more preferably at least about 70%, and still more
preferably at least
about 90%, and most preferably at least about 99% of their functional activity
and potency.
In some instances, the modified polypeptide or peptide substrates with lowered
functional
activity or potency will be useful. For example, when the modified polypeptide
or peptide
is fused to another polypeptide, such as transferrin, to form a fusion protein
with increased
serum stability and in vivo circulatory half-life, a modified polypeptide
peptide substrate
with lowered functional activity or potency may be useful.
[0068] In other instances, the modified polypeptides or peptides may have
increased
potency as compared to the non-modified polypeptides or peptides.
[0069] Modified polypeptide molecules of the invention are substantially
protected from
dipeptidyl peptidase cleavage as compared to an unmodified version of the same
polypeptide. Qualification of this substantial protection may vary by the
assay used to
compare the modified versus unmodified polypeptide. In order to exhibit
substantial
protection, however, the modified polypeptide will exhibit a detectable level
of resistance to
dipeptidyl peptidase cleavage in the assay. Such assays include but are not
limited to those
disclosed in Doyle et al. (2002 Endocrinology 142, 4462-4468), O'Harte et al.
(1999
Diabetes 48, 758-765) and Siegel et al. (1999 Regulatory Peptides 79, 93-102).
[0070] DPP stabilized polypeptide substrates of the present invention are also
more stable
in the presence of DPP in vivo than a non-stabilized polypeptide substrates. A
DPP
stabilized therapeutic polypeptide substrate generally has an increased
activity half-life as
compared to a non-stabilized peptide of identical sequence. Peptidase
stability may be
determined by comparing the half-life of the unmodified polypeptide substrate
in serum or
blood to the half-life of a modified counterpart therapeutic peptide in serum
or blood. Half-
life may be determined by sampling the serum or blood after administration of
the modified
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and non-modified peptides and determining the activity of the peptide. In
addition to
determining the activity, the length of the polypeptide substrates may also be
measured by
HPLC or Mass Spectrometry.
[0071] The present invention also provides modified polypeptides or peptides
having an
altered amino terminus according to the invention to protect against DPP
cleavage and
having internal and/or C-terminus amino acid alterations that do not affect
the functional
activity or potency of the polypeptide. These modified polypeptides would have
minor
amino acid changes that are usually conservative amino acid substitutions,
although non-
conservative substitutions are also contemplated.
[0072] The modified polypeptides or peptides of the present invention may also
have
altered functional activity. For instance, a modified polypeptide or peptide
with increased
functional activity may be useful. Alternatively, a modified polypeptide or
peptide with
decreased functional activity may be used. Thus, the modified polypeptides or
peptides of
the present invention also contain amino acid changes that do affect
functional activity or
potency. For example, the analogs of GLP-1 with altered functional activity
may be
modified at its amino terminus to protect against DPP cleavage.
[0073] Examples of conservative amino acid substitutions are substitutions
made within
the same group such as within the group of basic amino acids (such as
arginine, lysine,
histidine), acidic amino acids (such as glutamic acid and aspartic acid),
polar amino acids
(such as glutamine and asparagine), hydrophobic amino acids (such as leucine,
isoleucine,
valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine)
and small amino
acids (such as glycine, alanine, serine, threonine, methionine).
[0074] Non-conservative substitutions encompass substitutions of amino acids
in one
group by amino acids in anotlier group. For example, a non-conservative
substitution would
include the substitution of a polar amino acid for a hydrophobic amino acid.
For a general
description of nucleotide substitution, see e.g. Ford et al. (1991), Prot.
Exp. Pur. 2: 95-107.
[0075] The present invention provides obvious variants of the amino acid
sequence of the
modified polypeptides and peptides, such as naturally occurring mature forms
of the
polypeptides or peptides, allelic/sequence variants of the polypeptides, non-
naturally
occurring recombinantly derived variants of the peptides, and orthologs and
paralogs of the
polypeptides or peptides. Such variants can readily be generated using art-
known
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techniques in the fields of recombinant nucleic acid technology and protein
biochemistry.
Such variants can readily be identified/made using molecular techniques and
the sequence
information. Further, such variants can readily be distinguished from other
peptides based
on sequence and/or structural homology to the modified polypeptides or
peptides of the
present invention.
[0076] Preferably, the modified peptides of the present invention are GLP-1
and analogs
thereof comprising one or more additional amino acids at their N-terminus.
[0077] In some instances, the DPP such as DPP-IV may activate a peptide
instead of
inactivating it through cleavage. In such instances, modification of the
peptide could
substantially reduce, delay, or prevent peptide activation.
Nucleic Acids Encoding Modified Polypeptides
[0078] The present invention provides nucleic acid molecules encoding modified
polypeptides and peptides that are partially or substantially protected from
DPP cleavage
and have functional activity and potency. In one embodiment, nucleic acid
molecules
provided by the present invention encode modified polypeptides and peptides
having at
least one additional amino acid at its N-terminus as compared to their wild-
type unmodified
polypeptide. In another embodiment, the nucleic acid molecules encode modified
polypeptides and peptides having five additional amino acids at their N-
terminus.
Alternatively, the nucleic acid molecules encode modified polypeptides and
peptides having
between one and five additional amino acids at their N-terminus. Preferably,
the nucleic
acid molecules encoding modified GLP-1 comprise sequence encoding one or more
additional amino acids at its N-terminus.
[0079] The nucleic acid molecules of the invention include deoxyribonucleic
acids
(DNAs), both single- and double-stranded deoxyribonucleic acids. However, they
can also
be ribonucleic acids (RNAs), as well as hybrid RNA:DNA double-stranded
molecules.
Contemplated nucleic acid molecules also include genomic DNA, cDNA, mRNA, and
antisense molecules. The nucleic acids molecules of the present invention also
include
native or synthetic RNA, DNA, or cDNA that encode a modified polypeptide, or
the
complementary strand thereof.
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[0080] To construct modified polypeptides that are partially or substantially
protected
from DPP activity but having functional activity and/or potency compared to
wild-type
unmodified polypeptides, the nucleic acid encoding the wild-type unmodified
polypeptide
can be used as a starting point and modified to encode the desired modified
polypeptide.
Numerous methods are known to add sequences or to mutate nucleic acid
sequences that
encode a polypeptide and to confirm the function of the polypeptides encoded
by these
modified sequences.
[0081] The present invention also provides nucleic acids encoding polypeptides
and
peptides having a modified amino terminus for protection against DPP cleavage
and having
internal and C-terminus amino acid alterations that do not substantially
affect the functional
activity or potency of the polypeptide. These modified polypeptides would have
minor
amino acid changes that are usually conservative amino acid substitutions,
although non-
conservative substitutions are also conteniplated. Nucleotide substitutions
using techniques
for accomplishing site-specific mutagenesis are well-known in the art.
Preferably, the
nucleic acids encode GLP- 1 analogs having one or more additional amino acids
at their N-
terminus.
[0082] As known in the art "similarity" between two polynucleotides or
polypeptides is
determined by comparing the nucleotide or amino acid sequence and the
conserved
nucleotide or amino acid substitutes of one polynucleotide or polypeptide to
the sequence of
a second polynucleotide or polypeptide. Also known in the art is "identity"
which means
the degree of sequence relatedness between two polypeptide or two
polynucleotide
sequences as determined by the identity of the match between two strings of
such
sequences. Both identity and similarity can be readily calculated
(Computational Molecular
Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing:
Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York,
1993;
Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
G., eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von
Heinje, G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,
J., eds.,
M Stockton Press, New York, 1991).
[0083] While there exist a number of methods to measure identity and
similarity between
two polynucleotide or polypeptide sequences, the terms "identity" and
"similarity" are well
known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje,
G.,
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Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M
Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J.
Applied Math.,
48: 1073 (1988). Methods commonly employed to determine identity or similarity
between
two sequences include, but are not limited to those disclosed in Guide to Huge
Computers,
Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and
Lipman, D.,
SIAM J. Applied Math. 48:1073 (1988).
[0084] Preferred methods to determine identity are designed to give the
largest match
between the two sequences tested. Methods to determine identity and similarity
are
codified in computer programs. Preferred computer program methods to determine
identity
and similarity between two sequences include, but are not limited to, GCG
program package
(Devereux, et al., Nucleic Acids Research 12(l):387 (1984)), BLASTP, BLASTN,
FASTA
(Atschul, et al., J. Molec. Biol. 215:403 (1990)). The degree of similarity or
identity
referred to above is determined as the degree of identity between the two
sequences
indicating a derivation of the first sequence from the second. The degree of
identity
between two nucleic acid sequences may be determined by means of computer
programs
known in the art such as GAP provided in the GCG program package (Needleman
and
Wunsch (1970) Journal of Molecular Biology 48:443-453). For purposes of
determining
the degree of identity between two nucleic acid sequences for the present
invention, GAP is
used witll the following settings: GAP creation penalty of 5.0 and GAP
extension penalty of
0.3.
Codon Optimization
[0085] The degeneracy of the genetic code permits variations of the nucleotide
sequence
of polypeptides, while still producing a modified polypeptide comprising an
identical amino
acid sequence as the polypeptide encoded by a first DNA sequence. The
procedure, known
as "codon optimization" (described in U.S. Patent 5,547,871 which is
incorporated herein
by reference in its entirety) provides one with a means of designing such an
altered DNA
sequence. The design of codon optimized genes should take into account a
variety of
factors, including the frequency of codon usage in an organism, nearest
neighbor
frequencies, RNA stability, the potential for secondary structure formation,
the route of
synthesis and the intended future DNA manipulations of that gene. In
particular, available
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methods may be used to alter the codons encoding a given fusion protein with
those most
readily recognized by yeast when yeast expression systems are used.
[0086] The degeneracy of the genetic code permits the same amino acid sequence
to be
encoded and translated in many different ways. For example, leucine, serine
and arginine
are each encoded by six different codons, while valine, proline, threonine,
alanine and
glycine are each encoded by four different codons. However, the frequency of
use of such
synonymous codons varies from genome to genome among eukaryotes and
prokaryotes.
For example, synonymous codon-choice patterns among mammals are very similar,
while
evolutionarily distant organisms such as yeast (S. cerevisiae), bacteria (such
as E. coli) and
insects (such as D. melanogaster) reveal a clearly different pattern of
genomic codon use
frequencies (Grantham, R., et al., Nucl. Acids Res., 8, 49-62 (1980);
Grantham, R., et al.,
Nucl. Acids Res., 9, 43-74 (1981); Maroyama, T., et al., Nucl. Acids Res., 14,
151-197
(1986); Aota, S., et al., Nucl. Acids Res., 16, 315-402 (1988); Wada, K., et
al., Nucl. Acids
Res., 19 Supp., 1981-1985 (1991); Kurland, C. G., FEBS Letters, 285, 165-169
(1991)).
These differences in codon-choice patterns appear to contribute to the overall
expression
levels of individual genes by modulating peptide elongation rates. (Kurland,
C. G., FEBS
Letters, 285, 165-169 (1991); Pedersen, S., EMBO J., 3, 2895-2898 (1984);
Sorensen, M.
A., J. Mol. Biol., 207, 365-377 (1989); Randall, L. L., et al., Eur. J.
Biochem., 107, 375-379
(1980); Curran, J. F., and Yarus, M., J. Mol. Biol., 209, 65-77 (1989);
Varenne, S., et al., J.
Mol, Biol., 180, 549-576 (1984), Varenne, S., et al., J. Mol, Biol., 180, 549-
576 (1984);
Garesl, J.-P., J. Theor. Biol., 43, 211-225 (1974); Ikemura, T., J. Mol.
Biol., 146, 1-21
(1981); Ikemura, T., J. Mol. Biol., 151, 389-409 (1981)).
[0087] The preferred codon usage frequencies for a synthetic gene should
reflect the
codon usages of nuclear genes derived from the exact (or as closely related as
possible)
genome of the cell/organism that is intended to be used for recombinant
protein expression,
particularly that of yeast species. As discussed above, in one preferred
embodinlent the
modified polypeptide is codon optimized, before or after modification as
herein described
for yeast expression.
Vectors
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[0088] Expression units for use in the present invention will generally
comprise the
following elements, operably linked in a 5' to 3' orientation: a
transcriptional promoter, a
secretory signal sequence, a DNA sequence encoding a modified polypeptide and
a
transcriptional terminator. As discussed above, any arrangement of the
modified
polypeptide and peptide may be used in the vectors of the invention. The
selection of
suitable promoters, signal sequences and terminators will be determined by the
selected host
cell and will be evident to one skilled in the art and are discussed more
specifically below.
[0089] Suitable yeast vectors for use in the present invention are described
in U.S. Patent
6,291,212 and include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-
1039,
1978), YEp13 (Broach et al., Gene 8: 121-133, 1979), pJDB249 and pJDB219
(Beggs,
Nature 275:104-108, 1978), pPPC0005, pSeCHSA, pScNHSA, pC4 and derivatives
thereof.
Useful yeast plasmid vectors also include pRS403-406, pRS413-416 and the
Pichia vectors
available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids
pRS403,
pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and
incorporate the
yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-41.6 are
Yeast
Centromere plasmids (YCps).
[0090] Such vectors will generally include a selectable marker, which may be
one of any
number of genes that exhibit a dominant phenotype for which a phenotypic assay
exists to
enable transformants to be selected. Preferred selectable markers are those
that complement
host cell auxotrophy, provide antibiotic resistance or enable a cell to
utilize specific carbon
sources, and include LEU2 (Broach et al. ibid.), URA3 (Botstein et al., Gene
8: 17, 1979),
HIS3(Struhl et al., ibid.) or POT1 (Kawasaki and Bell, EP 171,142). Other
suitable
selectable markers include the CAT gene, which confers chloramphenicol
resistance on
yeast cells. Preferred promoters for use in yeast include promoters from yeast
glycolytic
genes (Hitzeman et al., J Biol. Chem. 225: 12073-12080, 1980; Alber and
Kawasaki, J.
Mol. Appl. Genet. 1: 419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or
alcohol
dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms
for
Chemicals, Hollaender et al., (eds.), p. 355, Plenum, N.Y., 1982; Ammerer,
Meth. Enzymol.
101: 192-201, 1983). In this regard, particularly preferred promoters are the
TPI1 promoter
(Kawasaki, U.S. Pat. No. 4,599,311) and the ADH2-4c (see U.S. Patent
6,291,212)
promoter (Russell et al., Nature 304: 652-654, 1983). The expression units may
also
include a transcriptional terminator. A preferred transcriptional terminator
is the TPI1
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terminator (Alber and Kawasaki, ibid.). More preferably, the promoter is the
PRBI
promoter disclosed in EP 431880 and the terminator is the ADH1 terminator
disclosed in EP
60057, which are herein incorporated by reference in their entirety.
[0091] In addition to yeast, modified polypeptides and peptides of the present
invention
can be expressed in filarnentous fungi, for example, species of the genus
Aspergillus.
Examples of useful promoters include those derived from Aspergillus nidulayas
glycolytic
genes, such as the ADH3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1985)
and the
tpiA promoter. An example of a suitable terminator is the ADH3 terminator
(McKnight et
al., ibid.). The expression units utilizing such components may be cloned into
vectors that
are capable of insertion into the chromosomal DNA of Aspergillus, for example.
[0092] Mammalian expression vectors for use in carrying out the present
invention will
include a promoter capable of directing the transcription of the modified
polypeptides and
peptides. Preferred promoters include viral promoters and cellular promoters.
Preferred
viral promoters include the major late promoter from adenovirus 2(Kaufrnan and
Sharp,
Mol. Cell. Biol. 2: 1304-13199, 1982) and the SV40 promoter (Subramani et al.,
Mol. Cell.
Biol. 1: 854-864, 1981). Preferred cellular promoters include the mouse
metallothionein-1
promoter (Palmiter et al., Science 222: 809-814, 1983) and a mouse Vx (see
U.S. Patent
6,291,212) promoter (Grant et al., Nuc. Acids Res. 15: 5496, 1987). A
particularly
preferred promoter is a mouse VH (see U.S. Patent 6,291,212) promoter. Such
expression
vectors may also contain a set of RNA splice sites located downstream from the
promoter
and upstream from the DNA sequence encoding the modified polypeptide or
peptide.
Preferred RNA splice sites may be obtained from adenovirus and/or
immunoglobulin genes.
[0093] Also contained in the expression vectors is a polyadenylation signal
located
downstream of the coding sequence of interest. Polyadenylation signals include
the early or
late polyadenylation signals from SV40 (Kaufinan and Sharp, ibid.), the
polyadenylation
signal from the adenovirus 5 E1B region and the human growth hormone gene
terminator
(DeNoto et al., Nuc. Acids Res. 9: 3719-3730, 1981). A particularly preferred
polyadenylation signal is the VH (see U.S. Patent 6,291,212) gene terminator.
The
expression vectors may include a noncoding viral leader sequence, such as the
adenovirus 2
tripartite leader, located between the promoter and the RNA splice sites.
Preferred vectors
may also include enhancer sequences, such as the SV40 enhancer and the mouse
(see U.S.
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Patent 6,291,212) enhancer (Gillies, Cell 33: 717-728, 1983). Expression
vectors may also
include sequences encoding the adenovirus VA RNAs.
[0094] The expression vectors are also used for expressing fusion proteins
comprising the
modified polypeptide or peptide of the present invention fused to a second
polypeptide or
peptide, for example transferrin, to enhance the half-life of the modified
polypeptide or
peptide, as described below. Also, the modified polypeptide or peptide may be
fused to a
tag and/or a cleavage site for expression and release of the modified
polypeptide or peptide.
Transformation
[0095] Techniques for transforming fungi are well known in the literature, and
have been
described, for instance, by Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad.
Sci. USA 75:
1929-1933, 1978), Yelton et al., (Proc. Natl. Acad. Sci. USA 81: 1740-1747,
1984), and
Russell (Nature 301: 167-169, 1983). The genotype of the host cell will
generally contain a
genetic defect that is complemented by the selectable marker present on the
expression
vector. Choice of a particular host and selectable marker is well within the
level of ordinary
skill in the art.
[0096] Cloned DNA sequences comprising modified polypeptides and peptides of
the
invention may be introduced into cultured mammalian cells by, for example,
calcium
phosphate-mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro
and Pearson,
Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456,
1973.)
Other techniques for introducing cloned DNA sequences into mammalian cells,
such as
electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), or lipofection may
also be
used. In order to identify cells that have integrated the cloned DNA, a
selectable marker is
generally introduced into the cells along with the gene or cDNA of interest.
Preferred
selectable markers for use in cultured mammalian cells include genes that
confer resistance
to drugs, such as neomycin, hygromycin, and methotrexate. The selectable
marker may be
an amplifiable selectable marker. A preferred amplifiable selectable marker is
the DHFR
gene. A particularly preferred amplifiable marker is the DHFRr (see U.S.
Patent 6,291,212)
cDNA (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: 2495-2499, 1983).
Selectable markers are reviewed by Thilly (Mammalian Cell Technology,
Butterworth
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Publishers, Stoneham, Mass.) and the choice of selectable markers is well
within the level
of ordinary skill in the art.
Host Cells
[0097] The present invention also includes a cell, preferably a yeast cell
transformed to
express a modified polypeptides or peptides of the invention. In addition to
the transformed
host cells tliemselves, the present invention also includes a culture of those
cells, preferably
a monoclonal (clonally homogeneous) culture, or a culture derived from a
monoclonal
culture, in a nutrient medium. If the polypeptide is secreted, the medium will
contain the
polypeptide, with the cells, or without the cells if they have been filtered
or centrifuged
away.
[0098] Host cells for use in practicing the present invention include
eukaryotic cells, and
in some cases prokaryotic cells, capable of being transformed or transfected
with exogenous
DNA and grown in culture, such as cultured mammalian, insect, fungal, plant
and bacterial
cells. A vector comprising a nucleic acid sequence of the present invention is
introduced
into a host cell so that the vector is maintained as a chromosomal integrant
or as a self-
replicating extra-chromosomal vector. Integration is generally considered to
be an
advantage as the nucleic acid sequence is more likely to be stably maintained
in the cell.
Integration of the vector into the host chromosome may occur by homologous or
non-
homologous recombination.
[0099] The choice of a host cell will to a large extent depend upon the gene
encoding the
polypeptide and its source. The host cell may be a unicellular microorganism,
e.g., a
prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Either
prokaryotes or
eukaryotes can be used. As prokaryotic host cells, generally used cells such
as Escherichia
coli or Bacillus subtilis can be used.
[00100] When prokaryotic cells are used as host cells, a vector replicable in
the host cells
may be used. An expression plasmid can be preferably used in which a promoter,
an SD
sequence (Shine-Dalgamo sequence), and an initiation codon (e.g. ATG) required
for
starting protein synthesis are provided in the vector upstream of the gene of
the present
invention to facilitate expression of the gene. Examples of the above vector
include
generally-used plasmids derived from E. coli such as pBR322, pBR325, pUC12,
pUC13
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and the like. However, applicable vectors are not limited to these examples
and various
known vectors can also be used. Examples of commercially available vectors
usable in
expression system using E. coli include pGEX-4T (Amersham Pharmacia Biotech),
pMAL-
C2, pMAI-P2 (New England Biolabs), pET21/lacq (Invitrogen), pBAD/His
(Invitrogen) and
the like.
[00101] Examples of eukaryotic host cells include yeast cells and the like.
Examples of
preferably used craniate cells include COS cell (cell from monkey) (1981 Cell,
23, 175),
Chinese Hamster Ovary cells and the dihydrofolate reductase defective strain
derived
therefrom (1980 Proc. Natl. Acad. Sci., USA., 77, 4216) and the like, and
examples of
preferably used yeast cells include SacchaYomyces cerevisiae or the like.
However, cells to
be used are not limited to these examples. Preferably, a yeast cell is used to
express the
modified polypeptide or peptide.
[00102] Fungal cells, including species of yeast (e.g., Sacchaf omyces spp.,
Schizosaccharonayces spp., Pichia spp.) may be used as host cells within the
present
invention. Examples of fungi including yeasts contemplated to be useful in the
practice, of
the present invention as hosts for expressing the modified polypeptide or
peptides of the
inventions are Pichia (including species formerly classified as Hansenula),
Saccharoinyces,
Kluyveroinyces, Aspergillus, Candida, Torulopsis, Torulaspora,
Schizosaccharoinyces,
Citeromyces, Pachysolen, Zygosaccharonayces, Debaromyces, Trichoderma,
Cephalospof ium, Humicola, Mucor, Neurospora, Yari=owia, Metschnikowia,
Rh.odosporidiuna, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis,
and the like.
Examples of Saccharoinyces spp. are S. cerevisiae, S. italicus and S. rouxii.
Examples of
Kluyveyomyces spp. are K fYagilis, K. lactis and K mar'xianus. A suitable
Torulaspora
species is T. delbrueckii. Examples of Pichia spp. are P. aiagusta (formerly
H.
polyniorpha), P. anomala (formerly H. anomala) and P. pastoris.
[00103] Particularly useful host cells to produce the modfied polypeptide or
peptide of the
invention are the methanoltrophic Pichia pastoris (Steinlein et al. (1995)
Protein Express.
Purif. 6:619-624). Pichia pastoris has been developed to be an outstanding
host for the
production of foreign proteins since its alcohol oxidase promoter was isolated
and cloned;
its transformation was first reported in 1985. P. pastoris can utilize
methanol as a carbon
source in the absence of glucose. The P. pastoris expression system can use
the methanol-
induced alcohol oxidase (AOX1) promoter, which controls the gene that codes
for the
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expression of alcohol oxidase, the enzyme which catalyzes the first step in
the metabolism
of methanol. This promoter has been characterized and incorporated into a
series of P.
pastoris expression vectors. Since the proteins produced in P. pastoris are
typically folded
correctly and secreted into the medium, the fermentation of genetically
engineered P.
pastof=is provides an excellent alternative to E. coli expression systems.
[00104] Strains of the yeast Saccharonzyces cerevisiae are another preferred
host. In a
preferred embodiment, a yeast cell, or more specifically, a Saccharonzyces
cerevisiae host
cell that contains a genetic deficiency in a gene required for asparagine-
linked glycosylation
of glycoproteins is used. S. cerevisiae host cells having such defects may be
prepared using
standard techniques of mutation and selection, although many available yeast
strains have
been modified to prevent or reduce glycosylation or hypermannosylation.
[00105] To optimize production of the heterologous proteins, it is also
preferred that the
host strain carry a mutation, such as the S. cerevisiaepep4 mutation (Jones,
Genetics 85:
23-33, 1977), wllich results in reduced proteolytic activity. It is
particularly advantageous
to use a host that carries a mutation in the gene encoding the aspartyl
protease yapsin
1 (YAP3) or the gene encoding yapsin 2(MKC7), or both (Copley et al. 1998
Biochein. J.
330, 1333-1340), such that the proteolytic activity directed to basic residues
is reduced or
eliminated. Host strains containing mutations in other protease encoding
regions are
particularly useful to produce large quantities of the modified therapeutic
polypeptides or
peptides of the invention.
[00106] Host cells containing DNA constructs of the present invention are
grown in an
appropriate growth medium. As used herein, the term "appropriate growth
medium" means
a medium containing nutrients required for the growth of cells. Nutrients
required for cell
growth may include a carbon source, a nitrogen source, essential amino acids,
vitamins,
minerals and growth factors. The growth medium will generally select for cells
containing
the DNA construct by, for example, drug selection or deficiency in an
essential nutrient
which is complemented by the selectable marker on the DNA construct or co-
transfected
with the DNA construct. Yeast cells, for example, are preferably grown in a
chemically
defined medium, comprising a non-amino acid nitrogen source, inorganic salts,
vitamins
and essential amino acid supplements. The pH of the medium is preferably
maintained at a
pH greater than 2 and less than 8, preferably at pH 5.5 to 6.5. Methods for
maintaining a
stable pH include buffering and constant pH control, preferably through the
addition of
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ammonia, ammonium hydroxide or sodium hydroxide. Preferred buffering agents
include
citric acid, phosphate, succinic acid and Bis-Tris (Sigma Chemical Co., St.
Louis, Mo.).
Yeast cells having a defect in a gene required for asparagine-linked
glycosylation are
preferably grown in a medium containing an osmotic stabilizer. A preferred
osmotic
stabilizer is sorbitol supplemented into the medium at a concentration between
0.1 M and
1.5 M., preferably at 0.5 M or 1.0 M.
[00107] Cultured mammalian cells are generally grown in commercially available
serum-
containing or serum-free medium. Selection of a medium appropriate for the
particular cell
line used is within the level of ordinary skill in the art. Transfected
mammalian cells are
allowed to grow for a period of time, typically 1-2 days, to begin expressing
the DNA
sequence(s) of interest. Drug selection is then applied to select for growth
of cells that are
expressing the selectable marker in a stable fashion. For cells that have been
transfected
with an amplifiable selectable marker the drug concentration may be increased
in a stepwise
manner to select for increased copy number of the cloned sequences, thereby
increasing
expression levels.
[00108] Baculovirus/insect cell expression systems may also be used to produce
the
modified therapeutic polypeptides or peptides of the invention. The BacPAKTM
Baculovirus Expression System (BD Biosciences (Clontech) expresses recombinant
proteins at high levels in insect host cells. The target gene is inserted into
a transfer vector,
which is cotransfected into insect host cells with the linearized BacPAK6
viral DNA. The
BacPAK6 DNA is missing an essential portion of the baculovirus genome. When
the DNA
recombines with the vector, the essential element is restored and the target
gene is
transferred to the baculovirus genome. Following recombination, a few viral
plaques are
picked and purified, and the recombinant phenotype is verified. The newly
isolated
recombinant virus can then be amplified and used to infect insect cell
cultures to produce
large amounts of the desired protein.
Secretory Signal Sequences
[00109] The terms "secretory signal sequence" or "signal sequence" or
"secretion leader
sequence" are used interchangeably and are described, for example in U.S. Pat.
6,291,212
and U.S. Pat 5,547,871, both of which are herein incorporated by reference in
their entirety.
Secretory signal sequences or signal sequences or secretion leader sequences
encode
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secretory peptides. A secretory peptide is an amino acid sequence that acts to
direct the
secretion of a mature polypeptide or protein from a cell. Secretory peptides
are generally
characterized by a core of hydrophobic amino acids and are typically (but not
exclusively)
found at the amino termini of newly synthesized proteins. Very often the
secretory peptide
is cleaved from the mature protein during secretion. Secretory peptides may
contain
processing sites that allow cleavage of the signal peptide from the mature
protein as it
passes through the secretory pathway. Processing sites may be encoded within
the signal
peptide or may be added to the signal peptide by, for example, in vitro
mutagenesis.
[00110] Secretory peptides may be used to direct the secretion of modified
polypeptides
and peptides of the invention. One such secretory peptide that may be used in
combination
with other secretory peptides is the third domain of the yeast Barrier
protein. Secretory
signal sequences or signal sequences or secretion leader sequences are
required for a
complex series of post-translational processing steps which result in
secretion of a protein.
If an intact signal sequence is present, the protein being expressed enters
the lumen of the
rough endoplasmic reticulum and is then transported through the Golgi
apparatus to
secretory vesicles and is finally transported out of the cell. Generally, the
signal sequence
immediately follows the initiation codon and encodes a signal peptide at the
amino-terminal
end of the protein to be secreted. In most cases, the signal sequence is
cleaved off by a
specific protease, called a signal peptidase. Preferred signal sequences
improve the
processing and export efficiency of recombinant protein expression using
viral, mammalian
or yeast expression vectors. A preferred signal sequence is a mammalian or
human
transferrin signal sequence. In some cases, the native substrate signal
sequence may be
used to express and secrete modified polypeptide or peptides of the invention.
In order to
ensure efficient removal of the signal sequence, in some cases it may be
preferable to
include a short pro-peptide sequence between the signal sequence and the
mature protein in
which the C-terminal portion of the pro-peptide comprises a recognition site
for a protease,
such as the yeast kex2p protease. Preferably, the pro-peptide sequence is
about 2-12 amino
acids in length, more preferably about 4-8 amino acids in length. Examples of
such pro-
peptides are Arg-Ser-Leu-Asp-Lys-Arg, Arg-Ser-Leu-Asp-Arg-Arg, Arg-Ser-Leu-Glu-
Lys-
Arg, and Arg-Ser-Leu-Glu-Arg-Arg (SEQ ID NOS: 111-114, respectively).
Production of Modified Polypeptide Substrates Protected from DPP Cleavage
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[001111 The modified polypeptides of this invention that are partially or
substantially
resistant to DPP activity, may be prepared by standard synthetic methods,
recombinant
DNA techniques, or any other methods of preparing peptides and fusion
proteins.
[00112] The solid phase peptide synthesis method is generally described in the
following
references: Merrifield, J. Am. Chem. Soc., 888:2149, 1963; Barany and
Merrifield, In the
Peptides, E. Gross and J. Meinenhofer, Eds., Academic Press, New York, 3:285
(1980); S.
B. H. Kent. Annu. Rev. Biochem., 57:957 (1988). By the solid phase peptide
synthesis
method, a peptide of a desired length and sequence can be produced through the
stepwise
addition of amino acids to a growing peptide chain which is covalently bound
to a solid
resin particle. Automated synthesis may be employed in this method.
[00113] As discussed above, the modified polypeptide of the present invention
may also be
obtained using molecular biology techniques, employing nucleic acid sequences
that encode
those polypeptides. Those sequences may be RNA or DNA and may be associated
with
control sequences and/or inserted into vectors. The latter are then
transfected into host
cells, for example bacteria. The preparation of the vectors and their
production or
expression in a host is carried out by conventional molecular biology and
genetic
engineering techniques.
[00114] Moreover, the modified polypeptides of the present invention can also
be made by
recombinant techniques using readily synthesized DNA sequences in commercially
available expression systems.
[00115] The modified polypeptides of the present invention may be obtained by
recombinant means comprising (a) cultivating a host cell under conditions
conducive to
production of the polypeptide; and (b) recovering the polypeptide. The cells
are cultivated
in a nutrient medium suitable for production of the polypeptide using methods
known in the
art. For example, the cell may be cultivated by shake flask cultivation, small-
scale or large-
scale fermentation (including continuous, batch, fed-batch, or solid state
fermentations) in
laboratory or industrial fermentors performed in a suitable medium and under
conditions
allowing the polypeptide to be expressed and/or isolated. The cultivation
takes place in a
suitable nutrient medium comprising carbon and nitrogen sources and inorganic
salts, using
procedures known in the art (see, e.g., references for bacteria and yeast;
Bennett, J. W. and
LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press,
California, 1991).
Suitable media are available from coniunercial suppliers or may be prepared
according to
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published compositions (e.g., in catalogues of the American Type Culture
Collection). If
the modified polypeptide is secreted into the nutrient medium, the polypeptide
can be
recovered directly from the medium. If the modified polypeptide is not
secreted, it can be
recovered from cell lysates.
[00116] As an example, the modified polypeptides or peptides of the present
invention
including the modified polypeptide or peptide fusion protein may be made by
the
fermentation methodology disclosed in WO 0044772, which is herein incorporated
by
reference in its entirety.
[00117] The modified polypeptides may be detected using methods known in the
art that
are specific for the polypeptides. These detection methods may include use of
specific
antibodies, formation of an enzyme product, or disappearance of an enzyme
substrate,
binding to a specific receptor, or by detection of activation of a specific
receptor in a cell-
based assay. For example, an enzyme assay may be used to determine the
activity of the
modified polypeptide. The resulting modified polypeptide may be recovered by
methods
known in the art. For example, the modified polypeptide may be recovered from
the
nutrient medium by conventional procedures including, but not limited to,
centrifugation,
filtration, extraction, spray-drying, evaporation, or precipitation.
[00118] The polypeptides of the present invention may be purified by a variety
of
procedures known in the art including, but not limited to, chromatography
(e.g., ion
exchange, affinity, hydrophobic, chromatofocusing, and size exclusion),
electrophoretic
procedures (e.g., preparative isoelectric focusing, differential solubility
(e.g., ammonium
sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein
Purification, J. -C. Janson
and Lars Ryden, editors, VCH Publishers, New York, 1989).
Fusion Proteins and Protein Conjugates.
[00119] The present invention provides modified polypeptides or peptides
attached to a
heterologous molecule via recombinant means or covalent attachment. The
attachment to a
heterologous molecule, for example a plasma protein, extends the activity of
the modified
polypeptides or peptides for days to weeks. In some instances, only one
administration of
such modified therapeutic polypeptide or peptide need be given during this
period of time.
Greater specificity can be achieved, since the active compound will be
primarily bound to
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large molecules, where it is less likely to be taken up intracellularly to
interfere with other
physiological processes.
[00120] In another embodiment, the modified polypeptides or peptides of the
present
invention can be attached to heterologous sequences to form chimeric or fusion
proteins via
recombinant means. Such chimeric or fusion proteins comprise a modified
polypeptide or
peptide, partially or substantially protected from DPP cleavage, operatively
linked to a
heterologous protein having an amino acid sequence not substantially
homologous to the
modified polypeptide or peptide. "Operatively linked" indicates that the
modified
polypeptide or peptide and the heterologous protein are fused in-frame. The
heterologous
protein can be fused to the N-terminus or C-terminus of the modified
polypeptide or
peptide.
[00121] In one embodiment, the fusion protein does not affect the activity of
the modified
polypeptide of the invention per se. For example, the fusion protein can
include, but is not
limited to, enzymatic fusion proteins, for example beta-galactosidase fusions,
yeast two-
hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions.
Such fusion
proteins, particularly poly-His fusions, can facilitate the purification of
recombinant
modified polypeptide. In a further example, the fusion protein comprises an
amino acid
sequence between the modified peptide of the invention and the other moiety,
said amino
acid sequence providing a recognition sequence that enables release of the
modified peptide
of the invention following chemical or enzymatic cleavage. In certain host
cells (e.g.,
mammalian host cells), expression and/or secretion of a protein can be
increased by using a
heterologous signal sequence. In another embodiment, the modified polypeptide
or peptide
is fused to a molecule that will extend its serum stability or serum half-
life, such as a plasma
protein. Preferably, the modified polypeptide or protein is fused to serum
albumin,
immunoglobulin, or a portion thereof such as the Fc domain. More preferably,
the modified
polypeptide or peptide is fused to transferrin, lactotrasferrin,
melanotransferrin, or hybrids
thereof. Methods for making such fusion proteins are provided by U.S.
Applications
10/231,494 and 10/378,094, and International Application PCT/US03/26818, which
are
herein incorporated by reference in their entirety.
[00122] As discussed in these applications, the transferrin to be attached to
the modified
polypeptide or peptide may be modified. It may exhibit reduced glycosylation.
The
modified transferrin polypeptide may be selected from the group consisting of
a single
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transferrin N domain, a single transferrin C domain, a transferrin N and C
domain, two
transferrin N domains, and two transferrin C domains.
[00123] When the C domain of Tf is part of the fusion protein, the two N-
linked
glycosylation sites, amino acid residues corresponding to N413 and N611 (SEQ
ID NO: 3
of PCT/LJS03/26818, which is incorporated by reference herein in its entirety)
may be
mutated for expression in a yeast system to prevent glycosylation or
hypermannosylationn
and extend the serum half-life of the fusion protein and/or therapeutic
protein ( to produce
asialo-, or in some instances, monosialo-Tf or disialo-Tf). In addition to Tf
amino acids
corresponding to N413 and N611, mutations may be to the adjacent residues
within the N-
X-S/T glycosylation site to prevent or substantially reduce glycosylation. See
U.S. Patent
5,986,067 of Funk et al. It has also been reported that the N domain of Tf
expressed in
Pichia pastof-is becomes 0-linked glycosylated witli a single hexose at S32
which also may
be mutated or modified to prevent such glycosylation.
[00124] Accordingly, in one embodiment of the invention, the transferrin
fusion protein
includes a modified transferrin molecule wherein the transferrin exhibits
reduced
glycosylation, including but not limited to asialo- monosialo- and disialo-
forms of Tf. In
another embodiment, the transferrin portion of the transferrin fusion protein
includes a
recombinant transferrin mutant that is mutated to prevent glycosylation. In
another
embodiment, the transferrin portion of the transferrin fusion protein includes
a recombinant
transferrin mutant that is fully glycosylated. In a further embodiment, the
transferrin
portion of the transferrin fusion protein includes a recombinant human serum
transferrin
mutant that is mutated to prevent glycosylation, wherein at least one of
Asn413 and Asn611
(SEQ ID NO: 3 of PCT/US03/26818, which is incorporated by reference herein in
its
entirety) are mutated to an amino acid which does not allow glycosylation. In
another
embodiment, the transferrin portion of the transferrin fusion protein includes
a recombinant
human serum transferrin mutant that is mutated to prevent or substantially
reduce
glycosylation, wherein mutations may be to the adjacent residues within the N-
X-S/T
glycosylation site. Moreover, glycosylation may be reduced or prevented by
mutating the
serine or threonine residue. Further, changing the X to proline is known to
inhibit
glycosylation.
[00125] A chimeric or fusion protein can be produced by standard recombinant
DNA
techniques. For example, DNA fragments coding for the different protein
sequences are
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ligated together in-frame in accordance with conventional techniques. In
another
embodiment, the fusion gene can be synthesized by conventional techniques
including
automated DNA synthesizers. Alternatively, PCR amplification of gene fragments
can be
carried out using anchor primers which give rise to complementary overhangs
between two
consecutive gene fragments which can subsequently be annealed and re-amplified
to
generate a chimeric gene sequence (see Ausubel et al. 1992 Current Protocols
in Molecular
Biology). Moreover, many expression vectors are commercially available that
already
encode a fusion moiety (e.g., a GST protein). A modified polypeptide or
peptide encoding
nucleic acid can be cloned into such an expression vector such that the fusion
moiety is
linked in-frame to the modified polypeptide or peptide.
[00126] In another embodiment, the modified therapeutic polypeptide or peptide
is
conjugated via a covalent bond to a heterologous molecule via a covalent bond
to increase
its stability and protection from DPP activity.
[00127] As an example, the modified polypeptide or peptide is conjugated to a
blood
component via a covalent bond formed between the reactive group of the
modified peptide
and a blood component, with or without a linking group. Blood components may
be either
fixed or mobile. Examples of fixed blood components are non-mobile blood
components
and include tissues, membrane receptors, interstitial proteins, fibrin
proteins, collagens,
platelets, endothelial cells, epithelial cells and their associated membrane
and membraneous
receptors, somatic body cells, skeletal and smooth muscle cells, neuronal
components,
osteocytes and osteoclasts and all body tissues especially those associated
with the
circulatory and lymphatic systems. Example of mobile blood components are
blood
components that do not have a fixed situs for any extended period of time,
generally not
exceeding 5, more usually one minute. These blood components are not membrane-
associated and are present in the blood for extended periods of time and are
present in a
minimum concentration of at least 0.1 g/ml. Mobile blood components include
serum
albumin, transferrin, immunoglobulins such as IgM and IgG, al protease
inhibitor,
antithrombin III and a2-antiplasmin. The half-life of mobile blood components
is typically
at least about 12 hours.
[00128] The formation of the covalent bond between the blood component and the
modified therapeutic polypeptide or peptide may occur in vivo or ex vivo. For
ex vivo
covalent bond formation, the modified polypeptide or peptide is added to
blood, serum or
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saline solution containing the blood component, e.g. human serum albumin or
IgG to permit
covalent bond formation between the modified polypeptide or peptide and the
blood
component. Also, the modified polypeptide peptide may be modified with
maleimide or a
similarly reactive chemical group and reacted with a blood component in saline
solution.
Once the modified therapeutic polypeptide or peptide is reacted with the blood
component
to form a modified polypeptide or peptide conjugate, the conjugate may be
administered to
the patient. Alternatively, the modified therapeutic polypeptide or peptide
may be
administered to the patient directly so that the covalent bond forms between
the modified
therapeutic polypeptide or peptide and the blood component in vivo. Also, the
same
reaction may be carried out with a recombinant protein, for example, albumin.
[00129] The various sites with which the chemically reactive groups of the non-
specific
modified therapeutic polypeptide or peptide may react in vivo include cells,
particularly red
blood cells (erythrocytes) and platelets, and proteins, such as
immunoglobulins, including
IgG and IgM, serum albumin, ferritin, steroid binding proteins, transferrin,
thyroxin binding
protein, a-2-macroglobulin, and the like.
[00130] The modified polypeptide or peptide may contain or may be chemically
modified
to contain a reactive group for binding to thiol. In one embodiment of the
invention the
modified polypeptide or peptide may be conjugated to polyethylene glycol.
Alternatively,
the modified polypeptide or peptide may be conjugated to a polyethylene glycol
modified
glycolipid or polyehtylene glycol modified fatty acid.
[00131] In one aspect, the modified polypeptide or peptide may be conjugated
to a fatty
acid or fatty acid derivative to improve its stability. Examples of fatty
acids include, but are
not limited to, lauric, palmitic, oleic, and stearic acids. Examples of fatty
acid derivatives
include ethyl esters, propyl esters, cholesteryl esters, coenzyme A esters,
nitrophenyl esters,
naphthyl esters, monoglycerides, diglycerides, and triglycerides, fatty
alcohols, fatty alcohol
acetates, and the like.
[00132] In another aspect, the modified polypeptide or peptide may be
engineered into a
drug affinity complex (DACTM). A drug affinity complex has three parts: a drug
component
which is responsible for biological activity; a connector attaching the drug
component to the
reactive chemistry group; and a reactive chemistry group, at the opposite end
of the
connector, which is responsible for the permanent bonding of the construct to
certain target
proteins in the body. For example, Kim et al. (2003, Diabetes 52(3):751)
disclose a GLP-1-
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albumin drug affinity complex. Kim et al. show that the albumin-conjugated
DAC:GLP-1
bound to the GLP-1 receptor (GLP-1R) and activated cAMP formation in
heterologous
fibroblasts expressing the receptor. The results suggest that the albumin-
conjugated
DAC:GLP-1 mimics the native GLP-1. Kim et al. provide a new approach for
prolonged
activation of GLP-1R signaling.
[00133] The modified polypeptide or peptide drug affinity complex is designed
to be
administered by subcutaneous injection and then rapidly and selectively bonds
in vivo to
albumin. The bioconjugate formed has the same therapeutic activity and similar
potency as
endogenous polypeptide or peptide but has a pharmacokinetic profile in animals
that is
closer to that of albumin.
Pharmaceutical Composition
[00134] The present invention provides pharmaceutical compositions comprising
modified
therapeutic polypeptides and peptides partially or substantially protected
from DPP
cleavage, but substantially retaining their functional activity and potency.
Such
pharmaceutical compositions may be administered orally, parenterally, such as
intravascularly (IV), intraarterially (IA), intramuscularly (IM),
subcutaneously (SC),
intraperitoneally, transdermally, or the like. Adniinistration may in
appropriate situations
be by transfusion. In some instances, administration may be oral, nasal,
rectal, transdermal
or aerosol, where the modified polypeptide allows for transfer to the vascular
system. For
example, fusion or conjugation of a modified polypeptide of the invention to a
transferrin
moiety allows for transport of the modified polypeptide to the vascular system
or across the
blood-brain barrier via binding to the transferrin receptor, as described in
International
Application PCT/US03/26778, which is herein incorporated by reference in its
entirety.
Usually a single injection will be employed although more than one injection
may be used,
if desired. The modified therapeutic polypeptides or peptides may be
administered by any
convenient means, including syringe, trocar, catheter, or the like. The
particular manner of
adininistration will vary depending upon the amount to be administered,
whether a single
bolus or continuous administration, or the like. Preferably, the
administration will be
intravascularly, where the site of introduction is not critical to this
invention, preferably at a
site where there is rapid blood flow, e.g., intravenously, peripheral or
central vein. More
preferably, the pharmaceutical compositions will be administered
subcutaneously. Other
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routes may find use where the administration is coupled with slow release
techniques or a
protective matrix. The intent is that the modified therapeutic peptides or
polypeptides be
effectively distributed, for example, in the blood, so as to be able to react
with the blood or
tissue components.
[00135] Generally, the invention encompasses pharmaceutical compositions
comprising
effective amounts of modified therapeutic polypeptide or peptide of the
invention together
with pharmaceutically acceptable diluents, preservatives, solubilizers,
emulsifiers, adjuvants
and/or carriers. Such compositions may include diluents of various buffer
content (e.g.,
Tris-HCI, acetate, phosphate), pH and ionic strength; additives such as
detergents and
solubilizing agents (e.g., Polysorbate 80), anti-oxidants (e.g., ascorbic
acid, sodium
metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking
substances (e.g.,
lactose, mannitol); incorporation of the material into particulate
preparations of polymeric
compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes.
Hyaluronic
acid may also be used, and this may have the effect of promoting sustained
duration in the
circulation. Such compositions may influence the physical state, stability,
rate of in vivo
release, and rate of in vivo clearance of the present proteins and
derivatives. See, e.g.,
Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co.,
Easton, Pa.
18042) pages 1435-1712 which are herein incorporated by reference.
[00136] For example, the modified therapeutic polypeptides or peptides may be
administered in a physiologically acceptable medium, e.g., deionized water,
phosphate
buffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma,
proteinaceous
solutions, mamiitol, aqueous glucose, alcohol, vegetable oil, or the like.
Other additives
which may be included include buffers, where the media are generally buffered
at a pH in
the range of about 5 to 10, where the buffer will generally range in
concentration from about
50 to 250 mM, salt, where the concentration of salt will generally range from
about 5 to 500
mM, physiologically acceptable stabilizers, and the like. Examples of
physiological buffers,
especially for injection, include Hank's solution and Ringer's solution.
Transdermal
formulations may contain penetrants such as bile salts or fusidates.
[00137] The pharmaceutical compositions may be prepared as tablets or dragees,
sublingual
tablets, sachets, paquets, soft gelatin capsules, suppositories, creams,
ointments, dermal
gels, transdermal devices, aerosols, drinkable and injectable ampoules. The
compositions
may also be prepared in liquid form, or may be in dried powder, such as
lyophilized form
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convenient for storage and transport. Implantable sustained release
formulations are also
contemplated.
Oral Dosage Fonns
[00138] In one embodiment, the present invention provides pharmaceutical
compositions
comprising the modified therapeutic polypeptides or peptides in oral solid
dosage forms,
which are described generally in Remington's Pharmaceutical Sciences (1990),
18th Ed.,
Mack Publishing Co. Easton Pa. 18042, which is herein incorporated by
reference. Solid
dosage forms include tablets, capsules, pills, troches or lozenges, cachets or
pellets. Also,
liposomal or proteinoid encapsulation may be used to formulate the present
compositions
(as, for example, proteinoid microspheres reported in U.S. Pat. No.
4,925,673). Liposomal
encapsulation may be used and the liposomes may be derivatized with various
polymers
(e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms
for the
therapeutic is given in Chapter 10 of Marshall, K., Modem Pharmaceutics
(1979), edited by
G. S. Banker and C. T. Rhodes, herein incorporated by reference. In general,
the
formulation will include the modified therapeutic polypeptide or peptide, and
inert
ingredients which allow for protection against the stomach environment, and
release of the
biologically active material in the intestine.
[00139] If necessary, the modified therapeutic polypeptide or peptide may be
chemically
modified so that oral delivery is efficacious. Generally, the chemical
modification
contemplated is the attachment of at least one moiety to the modified
therapeutic
polypeptide or peptide itself, where said moiety permits uptake into the blood
stream from
the stomach or intestine. Also desired is the increase in overall stability of
the compound
and increase in circulation time in the body. Moieties useful as covalently
attached vehicles
in this invention may also be used for this purpose. Examples of such moieties
include:
PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl
cellulose,
dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for
example,
Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981),
Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp 367-83;
Newmark,
et al. (1982), J. Appl. Biochem. 4:185-9. Other polymers that could be used
are poly-l,3-
dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as
indicated
above, are PEG moieties.
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[00140] Likewise, the modified therapeutic polypeptide or peptide may be
recombinantly
fused to another polypeptide to increase its overall stability or improve oral
delivery. For
example, the modified therapeutic polypeptide or peptide may be fused to
transferrin,
melanotransferrin, or lactoferrin. Methods for making such fusion proteins are
described in
U.S. Application 10/378,094, which is herein incorporated by reference in its
entirety.
[00141] For oral delivery dosage forms, it is also possible to use a salt of a
modified
aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl] amino) caprylate
(SNAC),
as a carrier to enhance absorption of the therapeutic compounds of this
invention. The
clinical efficacy of a heparin formulation using SNAC has been demonstrated in
a Phase II
trial conducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, "Oral
drug
delivery composition and methods" which is herein incorporated by reference in
its entirety.
[00142] The modified therapeutic polypeptides or peptides of this invention
can be
included in the formulation as fine multiparticulates in the form of granules
or pellets of
particle size about 1 mm. The formulation of the material for capsule
administration could
also be as a powder, lightly compressed plugs or even as tablets. The
therapeutic could be
prepared by compression.
[00143] Colorants and flavoring agents may all be included. For example, the
modified
therapeutic polypeptide or peptide may be formulated (such as by liposome or
microsphere
encapsulation) and then further contained within an edible product, such as a
refrigerated
beverage containing colorants and flavoring agents.
[00144] One may dilute or increase the volume of the pharmaceutical
composition of the
invention with an inert material. These diluents could include carbohydrates,
especially
mannitol, cc-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans
and starch.
Certain inorganic salts may also be used as fillers including calcium
triphosphate,
magnesium carbonate and sodium chloride. Some commercially available diluents
are Fast-
Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
[00145] Disintegrants may be included in the formulation of the therapeutic
into a solid
dosage form. Materials used as disintegrants include but are not limited to
starch including
the commercial disintegrant based on starch, Explotab. Sodium starch
glycolate, Amberlite,
sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,
orange peel, acid
carboxymethyl cellulose, natural sponge and bentonite may all be used. Another
form of
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the disintegrants are the insoluble cationic exchange resins. Powdered gums
may be used as
disintegrants and as binders and these can include powdered gums such as agar,
Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
[00146] Binders may be used to hold the modified therapeutic polypeptide or
peptide
together to form a hard tablet and include materials from natural products
such as acacia,
tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl
cellulose (EC)
and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions
to
granulate the therapeutic.
[00147] An antifrictional agent may be included in the formulation of the
pharmaceutical
composition of the invention to prevent sticking during the formulation
process. Lubricants
may be used as a layer between the modified therapeutic polypeptide or peptide
and the die
wall, and these can include but are not limited to; stearic acid including its
magnesium and
calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils
and waxes.
Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium
lauryl
sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and
6000.
[00148] Glidants that might improve the flow properties of the modified
therapeutic
polypeptide or peptide during formulation and to aid rearrangement during
compression
might be added. The glidants may include starch, talc, pyrogenic silica and
hydrated
silicoaluminate.
[00149] To aid dissolution of the modified therapeutic polypeptide or peptide
of this
invention into the aqueous environment a surfactant might be added as a
wetting agent.
Surfactants may include anionic detergents such as sodium lauryl sulfate,
dioctyl sodium
sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used
and could
include benzalkonium chloride or benzethonium chloride. The list of potential
nonionic
detergents that could be included in the formulation as surfactants are
lauromacrogol 400,
polyoxy140 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol
monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl
cellulose and
carboxymethyl cellulose. These surfactants could be present in the formulation
of the
protein or derivative either alone or as a mixture in different ratios.
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[00150] Additives may also be included in the formulation to enhance uptake of
the
modified therapeutic polypeptide and peptide. Additives potentially having
this property
are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
[00151] Controlled release formulation also may be desirable. The modified
therapeutic
polypeptide or peptide of this invention could be incorporated into an inert
matrix which
permits release by either diffusion or leaching mechanisms e.g., gums. Slowly
degenerating
matrices may also be incorporated into the formulation, e.g., alginates,
polysaccharides.
Another form of a controlled release of the compounds of this invention is by
a method
based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed
in a
semipermeable membrane which allows water to enter and push drug out through a
single
small opening due to osmotic effects. Some enteric coatings also have a
delayed release
effect.
[00152] Other coatings may be used for the formulation. These include a
variety of sugars
which could be applied in a coating pan. The modified therapeutic polypeptide
or peptide
could also be given in a film coated tablet and the materials used in this
instance are divided
into 2 groups. The first are the nonenteric materials and include methyl
cellulose, ethyl
cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose,
hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and
the
polyethylene glycols. The second group consists of the enteric materials that
are commonly
esters of phthalic acid.
[00153] A mix of materials might be used to provide the optimum film coating.
Film
coating may be carried out in a pan coater or in a fluidized bed or by
compression coating.
Pulmon.ar,y Delivefy Forms
[00154] In another embodiment, the present invention also provides
pharmaceutical
compositions comprising the modified therapeutic polypeptides or peptides for
pulmonary
delivery. The pharmaceutical composition is delivered to the lungs of a mammal
while
inhaling and traverses across the lung epithelial lining to the blood stream.
[00155] The present invention provides the use of a wide range of mechanical
devices
designed for pulmonary delivery of therapeutic products, including but not
limited to
nebulizers, metered dose inhalers, and powder inhalers, all of which are
familiar to those
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skilled in the art. Some specific examples of commercially available devices
suitable for
the practice of this invention are the Ultravent nebulizer, manufactured by
Mallinckrodt,
Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical
Products,
Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo
Inc.,
Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured
by Fisons
Corp., Bedford, Mass.
[00156] All such devices require the use of formulations suitable for the
dispensing of the
modified therapeutic polypeptide and peptide. Typically, each formulation is
specific to the
type of device employed and may involve the use of an appropriate propellant
material, in
addition to diluents, adjuvants and/or carriers useful in therapy.
[00157] The modified therapeutic polypeptide or peptide should most
advantageously be
prepared in particulate form with an average particle size of less than 10 m,
most
preferably 0.5 to 5 m, for most effective delivery to the distal lung.
[00158] Pharmaceutically acceptable carriers include carbohydrates such as
trehalose,
mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use
in formulations
may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be
used. PEG may be used (even apart from its use in derivatizing the protein or
analog).
Dextrans, such as cyclodextran, may be used. Bile salts and other related
enhancers may be
used. Cellulose and cellulose derivatives may be used. Amino acids may be
used, such as
use in a buffer formulation.
[00159] Also, the use of liposomes, microcapsules or microspheres, inclusion
complexes,
or other types of carriers is contemplated.
[00160] Formulations suitable for use with a nebulizer, eitherjet or
ultrasonic, will
typically comprise the inventive compound dissolved in water at a
concentration of about
0.1 to 25 mg of biologically active protein per mL of solution. The
formulation may also
include a buffer and a simple sugar (e.g., for protein stabilization and
regulation of osmotic
pressure). The nebulizer formulation may also contain a surfactant, to reduce
or prevent
surface induced aggregation of the protein caused by atomization of the
solution in forming
the aerosol.
[00161] Formulations for use with a metered-dose inhaler device will generally
comprise a
finely divided powder containing the inventive compound suspended in a
propellant with
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the aid of a surfactant. The propellant may be any conventional material
employed for this
purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydrofluorocarbon, or
a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations
thereof. Suitable
surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also
be useful as a
surfactant.
[00162] Formulations for dispensing from a powder inhaler device will comprise
a finely
divided dry powder containing the inventive compound and may also include a
bulking
agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in
amounts which
facilitate dispersal of the powder from the device, e.g., about 50 to 90% by
weight of the
formulation.
Nasal Delivery Forms
[00163] Nasal delivery of the pharmaceutical composition of the modified
polypeptide or
peptide of the present invention is also contemplated. Nasal delivery allows
the passage of
the protein to the blood stream directly after administering the modified
therapeutic
polypeptide or peptide to the nose, without the necessity for deposition of
the product in the
lung. Formulations for nasal delivery include those with dextran or
cyclodextran. Delivery
via transport across other mucous membranes is also disclosed.
Dosages
[00164] The dosage regimen involved in a method for treating the above-
described
conditions will be determined by the attending physician, considering various
factors which
modify the action of drugs, e.g. the age, condition, body weight, sex and diet
of the patient,
the severity of any infection, time of administration and other clinical
factors. Generally,
the daily regimen should be in the range of 0.01-1000 micrograms of the
inventive
compound per kilogram of body weight, preferably 0.1-150 micrograms per
kilogram.
Treatment of Diseases with Modified Therapeutic Proteins
[00165] The present invention provides various transferrin fusion proteins
that could be
used in the treatment of a variety of diseases. For example, the
pharmaceutical
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compositions comprising the fusion polypeptides or peptides of the present
invention could
be used to treat diseases such as, but not limited, to insulin resistance,
hyperglycemia,
hyperinsulinemia, or elevated blood levels of free fatty acids or glycerol,
hyperlipidemia,
obesity, Syndrome X, dysmetabolic syndrome, inflammation, diabetic
complications,
impaired glucose homeostasis, impaired glucose tolerance, type II diabetes,
prediabetes,
hypertriglyceridemia atherosclerosis, nervous system disorders, congestive
heart failure,
dyspepsia, and irritable bowel syndrome. The modified polypeptides and
peptides could
also be used to induce an anxiolytic effect on the CNS, to activate the CNS or
for post
surgery treatment.
[00166] The modified therapeutic polypeptides and peptides of the present
invention are
more stable in vivo than the nonmodified therapeutic polypeptides and peptides
because
they are fused to transferrin or modified transferrin or are partially or
substantially protected
from DPP activity. Accordingly, smaller amounts of the molecule may be
administered for
effective treatment. A lower dosage amount may in some instances alleviate
side effects.
[00167] In one embodiment, the modified therapeutic polypeptides and peptides
of the
present invention may be used as a sedative. Accordingly, the present
invention provides a
method of sedating a mammalian subject with an abnormality resulting in
increased
activation of the central or peripheral nervous system using the modified
polypeptides or
peptides of the invention. The method comprises administering a modified
therapeutic
polypeptides or peptides to the subject in an amount sufficient to produce a
sedative or
anxiolytic effect on the subject. The modified therapeutic polypeptides or
peptides may be
administered intracerebroventriculary, orally, subcutaneously,
intramuscularly, or
intravenously. Such methods are useful to treat or ameliorate nervous system
conditions
such as anxiety, movement disorder, aggression, psychosis, seizures, panic
attacks, hysteria
and sleep disorders.
[00168] Moreover, the present invention encompasses a method of increasing the
activity
of a mammalian subject, comprising administering a modified therapeutic
polypeptides or
peptides to the subject in an amount sufficient to produce an activating
effect on the subject.
The subject has a condition resulting in decreased activation of the central
or peripheral
nervous system. The modified therapeutic polypeptides or peptides are useful
in the
treatment or amelioration of depression, schizoaffective disorders, sleep
apnea, attention
deficit syndromes with poor concentration, memory loss, forgetfulness, and
narcolepsy, to
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name just a few conditions in which arousal of the central nervous system may
be
advantageous.
[00169] Also, insulin resistance following a particular type of surgery,
elective abdominal
surgery, is most profound on the first post-operative day, lasts at least five
days, and may
take up to three weeks to normalize. Thus, the post-operative patient may be
in need of
administration of the modified insulinotropic peptides of the present
invention for a period
of time following the trauma of surgery. Accordingly, the modified therapeutic
polypeptides or peptides of the invention may be utilized for post surgery
treatments. A
patient is in need of the modified insulinotropic peptides of the present
invention for about
1-16 hours before surgery is performed on the patient, during surgery on the
patient, and
after the patient's surgery for a period of not more than about 5 days.
[00170] Moreover, the modified therapeutic polypeptides and peptides, such as
the
insulinotropic peptides, of the invention may be utilized to treat insulin
resistance
independently from their use in post surgery treatment. Insulin resistance may
be due to a
decrease in binding of insulin to cell-surface receptors, or to alterations in
intracellular
metabolism. The first type, characterized as a decrease in insulin
sensitivity, can typically
be overcome by increased insulin concentration. The second type, characterized
as a
decrease in insulin responsiveness, cannot be overcome by large quantities of
insulin.
Insulin resistance following trauma can be overcome by doses of insulin that
are
proportional to the degree of insulin resistance, and thus is apparently
caused by a decrease
in insulin sensitivity.
[00171] Preferably, the present invention provides modified insulinotropic
peptides to
normalize hyperglycemia through glucose-dependent, insulin-dependent and
insulin-
independent mechanisms. As such, the modified insulinotropic peptides are
useful as
primary agents for the treatment of diabetes, especially type II diabetes
mellitus. The
present invention is especially suited for the treatment of patients with
diabetes, both type I
and type II, in that the action of the peptide is dependent on the glucose
concentration of the
blood, and thus the risk of hypoglycemic side effects are greatly reduced over
the risks in
using current methods of treatment
[00172] The dose of modified insulinotropic peptides effective to normalize a
patient's
blood glucose level will depend on a number of factors, among which are
included, without
limitation, the patient's sex, weight and age, the severity of inability to
regulate blood
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glucose, the underlying causes of inability to regulate blood glucose, whether
glucose, or
another carbohydrate source, is simultaneously administered, the route of
administration
and bioavailability, the persistence in the body, the formulation, and the
potency.
[00173] Preferably, the modified therapeutic peptides such as the
insulinotropic peptides, of
the present invention are used for the treatment of impaired glucose
tolerance, glycosuria,
hyperlipidaemia, metabolic acidoses, diabetes mellitus, diabetic neuropathy,
and
nephropathy. More preferably, the modified peptides are modified GLP-l and
analogs
thereof for the treatment of type II diabetes.
Monitoring the Presence of Modified Therapeutic Polypeptides and Peptides
[00174] The modified therapeutic polypeptides and peptides may be monitored
using
assays for determining functional activity, HPLC-MS, or antibodies directed
against the
polypeptide or peptide. For example, the blood of the mammalian host may be
monitored
for the activity of the modified therapeutic polypeptide or peptide and/or
presence of the
modified therapeutic polypeptide or peptide. By taking a portion or sample of
the blood of
the host at different times, one may determine whether the modified
therapeutic polypeptide
or peptide has become bound to the long-lived blood components in sufficient
amount to be
therapeutically active and, thereafter, the level of modified therapeutic
polypeptide or
peptide in the blood. If desired, one may also determine to which of the blood
components
the modified therapeutic polypeptide or peptide, such as a modified
insulinotropic peptide,
is bound.
[00175] As an example, assays for insulinotropic activity may be used to
monitor the
modified insulinotropic peptides of the present invention. The modified
insulinotropic
peptides of the present invention have an insulinotropic activity that at
least equals the
insulinotropic activity of the non-modified insulinotropic peptides. The
insulinotropic
property of a modified insulinotropic peptide may be determined by providing
that modified
peptide to animal cells, or injecting that peptide into animals and monitoring
the release of
immunoreactive insulin into the media or circulatory system of the animal,
respectively.
The presence of immunoreactive insulin is detected through the use of a
radioimmunoassay
which can specifically detect insulin. Although any radioimmunoassay capable
of detecting
the presence of IRI may be employed, it is preferable to use a modification of
the assay
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method of Albano, J. D. M., et al., (1972 Acta Endocrinol. 70:487-509), which
is herein
incorporated by reference in its entirety.
[00176] The insulinotropic property of a modified therapeutic polypeptide or
peptide may
also be determined by pancreatic infusion (Penhos, J. C., et al. 1969 Diabetes
18:733-738,
which is hereby incorporated by reference). The manner in which perfusion is
performed,
modified, and analyzed preferably follows the methods of Weir, G. C., et al.,
(J. Clin.
Investigat. 54:1403-1412 (1974)), which is hereby incorporated by reference.
[00177] HPLC coupled with mass spectrometry (MS) can be utilized to assay for
the
presence of modified therapeutic polypeptide and peptides as is well known to
the skilled
artisan. Typically two mobile phases are utilized, such as 0.1 % TFA/water and
0.1 %
TFA/acetonitrile. Column temperatures can be varied as well as gradient
conditions.
[00178] Another method to monitor the presence of modified therapeutic
polypeptides and
peptides is to use antibodies specific to the modified therapeutic
polypeptides and peptides.
The use of antibodies, either monoclonal or polyclonal, having specificity for
particular
modified therapeutic polypeptides or peptides, can assist in mediating any
such problem.
The antibody may be generated or derived from a host immunized with the
particular
modified therapeutic polypeptide or peptide, or with an immunogenic fragment
of the agent,
or a synthesized immunogen corresponding to an antigenic determinant of the
agent.
Preferred antibodies will have high specificity and affinity for the modified
therapeutic
polypeptide or peptide. Such antibodies can also be labeled with enzymes,
fluorochromes,
or radiolabels.
[00179] The antibodies may be used to monitor the presence of modified
therapeutic
polypeptides and peptides in the blood stream. Blood and/or serum samples may
be
analyzed by SDS-PAGE and western blotting. Such techniques permit the analysis
of the
blood or serum to determine the bonding of the modified therapeutic
polypeptides or
peptides to blood components.
Glucagon-Like Peptide-1 (GLP-1)
[00180] Recombinant DNA technology has been used to create new molecules with
increased the stability and biological activity. These molecules are
combinations of
biologically active proteins and peptides fused to a stabilizing protein with
naturally long
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half-life such as immunoglobin Fc portion, albumin and transferrin. These
fusion molecules
retain the biological activity of the active moiety with much longer
pharmacokinetics than
their natural unfused protein or peptide counterparts. Increase in the
pharmacokinetics also
improves the biological activity, reduces unwanted side effects and improves
convenience
to the patients. There are many examples of such fusion proteins like
interferon-albumin,
interferon-Fc, BNP-albumin, GLP-1-albumin, GLP-1-Transferrin,
[00181] Although the fusion proteins are stable and resistant to degradation,
the underlying
protease mechanism that degrades the active moiety may result in a slow
inactivation of the
molecule. Specifically, many peptides such as GLP-1, dynorphin (Berman YL,
Juliano L,
Devi LA J Biol Chem. 1995 Oct 6;270:23845-50), enkephalin (Gu ZF, Menozzi D,
Okamoto A, Maton PN, Bunnett NW Exp Physiol. 1993 Jan;78:35-48), BNP, ANP,
angiotensin, bradykinin, and PYY are very susceptible to proteases such as
dipeptidyl-
peptidase IV, neutral endopeptidase. These proteases individually or in
combination cause
rapid inactivation of the peptides in the circulation. The fusion of peptides
to large proteins
such as albumin, Fc and transferrin confers a significant resistance to
proteases. ' However,
it may not totally eliminate the effect of the inactivation by proteases.
Therefore,
combination of the fusion proteins and protease inhibitors can have better PK
and PD than
the fusion protein alone.
[00182] The present invention provides transferrin fusion proteins comprising
therapeutic
peptides that are resistant to proteases. Preferably, the modified therapeutic
peptides of the
present invention are modified insulinotropic peptides partially or
substantially protected
from DPP activity. More preferably, the modified insulinotropic peptides are
modified
GLP-1 peptides and analogs and fragments thereof. The modified GLP-1 peptides
and
analogs and fragments thereof are useful for treating diabetes, specifically
type II diabetes.
The N-terminal sequence of wild-type GLP-1 is His-Ala-Glu; modified GLP-1
polypeptides
of the invention may comprise an N-terminal sequence selected from the group
consisting
of: His-His-Ala-Glu (SEQ ID NO: 115), Gly-His-Ala-Glu (SEQ ID NO: 116), His-
Gly-
Glu, His-Ser-Glu, His-Ala-Glu, His-Gly-Glu, His-Ser-Glu, His-His-Ala-Glu (SEQ
ID NO:
82), His-His-Gly-Glu (SEQ ID NO: 83), His-His-Ser-Glu (SEQ ID NO: 84), Gly-His-
Ala-
Glu (SEQ ID NO: 85), Gly-His-Gly-Glu (SEQ ID NO: 86), Gly-His-Ser-Glu (SEQ ID
NO:
87), His-X-Ala-Glu, His-X-Gly-Glu, and His-X-Ser-Glu, wherein X is any amino
acid. As
described below, other modifications may be made to decrease and prevent
protease
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degradation and these molecules may be used in the methods and compositions of
the
invention. Further, any GLP-l moiety or GLP-1 analogs, derivatives, or mimetic
may be
used (as a fusion protein) in the methods and compositions of the invention.
[00183] The addition of an amino acid to the N-terminus of GLP-1 may prevent
dipeptidyl
peptidase from cleaving at the second amino acid of GLP-1 due to steric
hindrance.
Therefore, GLP- 1 will remain functionally active. Any one of the 20 amino
acids or a non-
natural amino acid may be added to the N-terminus of GLP-1. Histidine is also
a preferred
amino acid. In some instances, an uncharged or positively charged amino acid
may be used
and preferably, a smaller amino acid such as Glycine is added. The modified
GLP-1 with
the extra amino acid can then be fused to transferrin to make a fusion
protein. In one
embodiment, the GLP-1 peptide is modified to contain at least one additional
amino acid at
its amino terminus. In another embodiment, the GLP-1 peptide is modified to
contain at
least five additional amino acids at its amino terminus. Alternatively, the
GLP-1 peptide is
modified to contain between one and five additional amino acids at its amino
terminus.
[00184] Glucagon-Like Peptide-1 (GLP-1) is a gastrointestinal hormone that
regulates
insulin secretion belonging to the so-called enteroinsular axis. The
enteroinsular axis
designates a group of hormones, released from the gastrointestinal mucosa in
response to
the presence and absorption of nutrients in the gut, which promote an early
and potentiated
release of insulin. The incretin effect which is the enhancing effect on
insulin secretion is
probably essential for a normal glucose tolerance. GLP-1 is a physiologically
important
insulinotropic hormone because it is responsible for the incretin effect.
[00185] GLP-1 is a product of the proglucagon gene (Bell, et al., Nature,
1983, 304: 368-
371). It is synthesized in intestinal endocrine cells in two principal major
molecular forms,
as GLP-1(7-36)amide and GLP-1(7-37). The peptide was first identified
following the
cloning of cDNAs and genes for proglucagon in the early 1980s.
[00186] Initial studies done on the fnll length peptide GLP-1(1-37 and 1-
36amide) concluded
that the larger GLP-1 molecules are devoid of biological activity. In 1987,
three
independent research groups demonstrated that removal of the first six amino
acids resulted
in a GLP-1 molecule with enhanced biological activity.
[00187] The amino acid sequence of GLP-1 is disclosed by Schmidt et al. (1985
Diabetologia 28 704-707). Human GLP-1 is a 37 amino acid residue peptide
originating
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from preproglucagon which is synthesized in the L-cells in the distal ileum,
in the pancreas,
and in the brain. Processing of preproglucagon to GLP-1(7-36)amide, GLP-1(7-
37) and
GLP-2 occurs mainly in the L-cells. The amino acid sequence of GLP-1(7-37) is
SEQ ID
NO: 32 (X = Gly):
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-
Lys-Glu-Phe-Ile-Ala-Trp-Leu-V al-Lys-Gly-Arg-Gly.
In GLP-1(7-36)amide, the terminal Gly is replaced by NH2 .
[00188] GLP-1 like molecules possesses anti-diabetic activity in human
subjects suffering
from Type II (non-insulin-dependent diabetes mellitus (NIDDM)) and, in some
cases, even
Type I diabetes. Treatment with GLP-1 elicits activity, such as increased
insulin secretion
and biosynthesis, reduced glucagon secretion, delayed gastric emptying, only
at elevated
glucose levels, and thus provides a potentially much safer therapy than
insulin or
sulfonylureas. Post-prandial and glucose levels in patients can be moved
toward normal
levels with proper GLP-1 therapy. There are also reports suggesting GLP-1-like
molecules
possess the ability to preserve and even restore pancreatic beta cell function
in Type-Il
patients.
[00189] Any GLP-1 sequence may be modified by adding one or more amino acids
at its
amino terminus, including GLP-1(7-34), GLP-1(7-35), GLP-1(7-36), and GLP-1(7-
37).
GLP-1 also has powerful actions on the gastrointestinal tract. Infused in
physiological
amounts, GLP-1 potently inhibits pentagastrin-induced as well as meal-induced
gastric acid
secretion (Schjoldager et al., Dig. Dis. Sci. 1989, 35:703-708; Wettergren et
al., Dig Dis Sci
1993; 38:665-673). It also inhibits gastric emptying rate and pancreatic
enzyme secretion
(Wettergren et al., Dig Dis Sci 1993; 38:665-673). Similar inhibitory effects
on gastric and
pancreatic secretion and motility may be elicited in humans upon perfusion of
the ileum
with carbohydrate- or lipid-containing solutions (Layer et al., Dig Dis Sci
1995, 40:1074-
1082; Layer et al., Digestion 1993, 54: 385-38). Concomitantly, GLP-1
secretion is greatly
stimulated, and it has been speculated that GLP-1 may be at least partly
responsible for this
so-called "ileal-brake" effect (Layer et al., Digestion 1993; 54: 385-38). In
fact, recent
studies suggest that, physiologically, the ileal-brake effects of GLP-1 may be
more
important than its effects on the pancreatic islets. Thus, in dose response
studies GLP-1
influences gastric emptying rate at infusion rates at least as low as those
required to
influence islet secretion (Nauck et al., Gut 1995; 37 (suppl. 2): A124).
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[001901 GLP-1 seems to have an effect on food intake. Intraventricular
administration of
GLP-1 profoundly inhibits food intake in rats (Schick et al. in Ditschuneit et
al. (eds.),
Obesity in Europe, John Libbey & Company ltd, 1994; pp. 363-367; Turton et
al., Nature
1996, 379: 69-72). This effect seems to be highly specific. Thus, N-terminally
extended
GLP-1(1-36amide) is inactive and appropriate doses of the GLP-1 antagonist,
exendin 9-39,
abolish the effects of GLP-1(Tang-Christensen et al., Am. J. Physiol., 1996,
271(4 Pt
2):R848-56). Acute, peripheral administration of GLP-1 does not inhibit food
intake
acutely in rats (Tang-Christensen et al., Am. J. Physiol., 1996, 271(4 Pt
2):R848-56; Turton
et al., Nature 1996, 379: 69-72). However, it remains possible that GLP-1
secreted from the
intestinal L-cells may also act as a satiety signal.
[00191] In diabetic patients, GLP-1's insulinotropic effects and the effects
of GLP-1 on the
gastrointestinal tract are preserved (Willms et al, Diabetologia 1994; 37,
suppl. 1: A118),
which may help curtail meal-induced glucose excursions, but, more importantly,
may also
influence food intake. Administered intravenously, continuously for one week,
GLP-1 at 4
ng/kg/min has been demonstrated to dramatically improve glycaemic control in
NIDDM
patients without significant side effects (Larsen et al., Diabetes 1996; 45,
suppl. 2: 233A.).
[00192] Modified GLP-1 partially or substantially protected from DPP activity
and
modified GLP-1 analogs are useful in the treatment of Type 1 and Type 2
diabetes and
obesity.
[00193] As used herein, the term "GLP-1 molecule" means GLP-1, a GLP-1 analog,
or
GLP-1 derivative.
[00194] As used herein, the term "GLP-1 analog" is defined as a molecule
having one or
more amino acid substitutions, deletions, inversions, or additions compared
with GLP-1.
Many GLP-1 analogs are known in the art and include, for example, GLP-1(7-34),
GLP-
1(7-35), GLP-1(7-36), Val$ -GLP-1(7-37), Gln9-GLP1(7-37), D-Gln9-GLP-1(7-37),
Thr16-
Lys18-GLP-1(7-37), and Lysl$-GLP-1(7-37) (SEQ ID NO: 72). U.S. Patent
5,118,666
discloses examples of GLP-1 analogs such as GLP-1(7-34) and GLP-1(7-35).
[00195] The term "GLP-1 derivative" is defined as a molecule having the amino
acid
sequence of GLP- 1 or a GLP- 1 analog, but additionally having chemical
modiflcation of
one or more of its amino acid side groups, a-carbon atoms, terminal amino
group, or
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terminal carboxylic acid group. A chemical modification includes, but is not
limited to,
adding chemical moieties, creating new bonds, and removing chemical moieties.
[00196] As used herein, the term "GLP-1 related compound" refers to any
compound
falling within the GLP-1, GLP-1 analog, or GLP-l derivative definition.
[00197] WO 91/11457 discloses analogs of the active GLP-1 peptides 7-34, 7-35,
7-36,
and 7-37 which can also be useful as GLP-1 moieties.
[00198] EP 0708179-A2 (Eli Lilly & Co.) discloses GLP-1 analogs and
derivatives that
include an N-terminal imidazole group and optionally an unbranched C6 -Clo
acyl group in
attached to the lysine residue in position 34.
[00199] EP 0699686-A2 (Eli Lilly & Co.) discloses certain N-terminal truncated
fragments
of GLP-1 that are reported to be biologically active.
[00200] U.S. Patent 5,545,618 discloses GLP-1 molecules consisting essentially
of GLP-
1(7-34), GLP1(7-35), GLP-1(7-36), or GLP-1(7-37), or the amide forms thereof,
and
pharmaceutically-acceptable salts thereof, having at least one modirication
selected from the
group consisting of: (a) substitution of glycine, serine, cysteine, threonine,
asparagine,
glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine,
phenylalanine,
arginine, or D-lysine for lysine at position 26 and/or position 34; or
substitution of glycine,
serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine,
isoleucine,
leucine, methionine, phenylalanine, lysine, or a D-arginine for arginine at
position 36 (SEQ
ID NO: 73); (b) substitution of an oxidation-resistant amino acid for
tryptophan at position
31 (SEQ ID NO: 74); (c) substitution of at least one of: tyrosine for valine
at position 16;
lysine for serine at position 18; aspartic acid for glutamic acid at position
21; serine for
glycine at position 22; arginine for glutamine at position 23; arginine for
alanine at position
24; and glutamine for lysine at position 26 (SEQ ID NO: 75); and (d)
substitution of at least
one of: glycine, serine, or cysteine for alanine at position 8; aspartic acid,
glycine, serine,
cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine,
isoleucine, leucine,
methionine, or phenylalanine for glutamic acid at position 9; serine,
cysteine, threonine,
asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine,
methionine, or
phenylalanine for glycine at position 10; and glutamic acid for aspartic acid
at position 15
(SEQ ID NO: 76); and (e) substitution of glycine, serine, cysteine, threonine,
asparagine,
glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or
phenylalanine, or the
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D- or N-acylated or alkylated form of histidine for histidine at position 7
(SEQ ID NO: 77);
wherein, in the substitutions is (a), (b), (d), and (e), the substituted amino
acids can
optionally be in the D-form and the amino acids substituted at position 7 can
optionally be
in the N-acylated or N-alkylated form.
[00201] U.S. Pat. No. 5,118,666 discloses a GLP-1 molecule having
insulinotropic activity.
Such molecule is selected from the group consisting of a peptide having the
amino acid
sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-
Ala-
Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys (GLP-1, 7-34, see SEQ ID NO: 32) or
His-Ala-
Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-
Phe-
Ile-Ala-Trp-Leu-Val-Lys-Gly (GLP-1, 7-35, see SEQ ID NO: 32); and a derivative
of said
peptide and wherein said peptide is selected from the group consisting of: a
pharmaceutically-acceptable acid addition salt of said peptide; a
pharmaceutically-
acceptable carboxylate salt of said peptide; a pharmaceutically-acceptable
lower alkylester
of said peptide; and a pharmaceutically-acceptable amide of said peptide
selected from the
group consisting of amide, lower alkyl amide, and lower dialkyl amide.
[00202] U.S. Patent 6,277,819 teaches a method of reducing mortality and
morbidity after
myocardial infarction comprising administering GLP-1, GLP-1 analogs, and GLP-1
derivatives to the patient. The GLP-1 analog being represented by the
following structural
formula (SEQ ID NO: **): RI-XI-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-
X2-
Gly-Gln-Ala-Ala-Lys- X3-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R2 (SEQ ID NO: 78)
and
pharmaceutically-acceptable salts thereof, wherein: Rl is selected from the
group consisting
of L-histidine, D-histidine, desamino-histidine, 2-ainino-histidine, .beta.-
hydroxy-histidine,
homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine; Xl is
selected
from the group consisting of Ala, Gly, Val, Thr, Ile, and alpha-methyl-Ala; X2
is selected
from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; X3 is selected
from the group
consisting of Glu, Gln, Ala, Thr, Ser, and Gly; R2 is selected from the group
consisting of
NH2, and GIy--OH; provided that the GLP-1 analog has an isoelectric point in
the range
from about 6.0 to about 9.0 and further providing that when Rl is His, Xl is
Ala, X2 is Glu,
and X3 is Glu, R2 must be NH2.
[00203] Ritzel et al. (Journal of Endocrinology, 1998, 159: 93-102) disclose a
GLP-1
analog, [SerB]GLP-1, in which the second N-terminal alanine is replaced with
serine. The
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modification did not impair the insulinotropic action of the peptide but
produced an analog
with increased plasma stability as compared to GLP-1.
[00204] U.S. Patent 6,429,197 teaches that GLP-1 treatment after acute stroke
or
hemorrhage, preferably intravenous administration, can be an ideal treatment
because it
provides a means for optimizing insulin secretion, increasing brain anabolism,
enhancing
insulin effectiveness by suppressing glucagon, and maintaining euglycemia or
mild
hypoglycemia with no risk of severe hypoglycemia or other adverse side
effects. The
present invention provides a method of treating the ischemic or reperfused
brain with GLP-
1 or its biologically active analogues after acute stroke or hemorrhage to
optimize insulin
secretion, to enhance insulin effectiveness by suppressing glucagon
antagonism, and to
maintain euglycemia or mild hypoglycemia with no risk of severe hypoglycemia.
[00205] U.S. Patent 6,277,819 provides a method of reducing mortality and
morbidity after
myocardial infarction, comprising administering to a patient in need thereof,
a compound
selected from the group consisting of GLP-1, GLP-1 analogs, GLP-1 derivatives
and
pharmaceutically-acceptable salts thereof, at a dose effective to normalize
blood glucose.
[00206] U.S. Patent 6,191,102 discloses a method of reducing body weight in a
subject in
need of body weight reduction by administering to the subject a composition
comprising a
glucagon-like peptide-1 (GLP-1), a glucagon-like peptide analog (GLP-1
analog), a
glucagon-like peptide derivative (GLP-1 derivative) or a pharmaceutically
acceptable salt
thereof in a dose sufficient to cause reduction in body weight for a period of
time effective
to produce weight loss, said time being at least 4 weeks.
[00207] GLP-1 is fully active after subcutaneous administration (Ritzel et
al., Diabetologia
1995; 38: 720-725), but is rapidly degraded mainly due to degradation by
dipeptidyl
peptidase N-like enzymes (Deacon et al., J Clin Endocrinol Metab 1995, 80: 952-
957;
Deacon et a1.,1995, Diabetes 44: 1126-1131). Thus, unfortunately, GLP- 1 and
many of its
analogues have a short plasma half-life in humans (Orskov et al., Diabetes
1993; 42:658-
661). Accordingly, it is an objective of the present invention to provide
modified GLP-1 or
analogues thereof which have a protracted profile of action relative to GLP-
1(7-37). It is a
further object of the invention to provide derivatives of GLP-1 and analogues
thereof which
have a lower clearance than GLP-1(7-37). Moreover, it is an object of the
invention to
provide pharmaceutical compositions comprising modified GLP-1 or GLP-1 analogs
with
improved stability. Additionally, the present invention includes the use of
modified GLP-1
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or GLP-1 analogs to treat diseases associated with GLP-1 such as but not
limited to those
described above.
[00208] In one aspect of the present invention, the pharmaceutical
compositions
comprising modified GLP-1 and GLP-1 analogs may be formulated by any of the
established methods of formulating pharmaceutical compositions, e.g. as
described in
Remington's Pharmaceutical Sciences, 1985. The composition may be in a form
suited for
systemic injection or infusion and may, as such, be formulated with a suitable
liquid vehicle
such as sterile water or an isotonic saline or glucose solution. The
compositions may be
sterilized by conventional sterilization techniques which are well known in
the art. The
resulting aqueous solutions may be packaged for use or filtered under aseptic
conditions and
lyophilized, the lyophilized preparation being combined with the sterile
aqueous solution
prior to administration. The composition may contain pharmaceutically
acceptable
auxiliary substances as required to approximate physiological conditions, such
as buffering
agents, tonicity adjusting agents and the like, for instance sodium acetate,
sodium lactate,
sodium chloride, potassium chloride, calcium chloride, etc.
[00209] The modified GLP-1 and GLP-1 analogs of the present invention may also
be
adapted for nasal, transdermal, pulmonal or rectal administration. The
pharmaceutically
acceptable carrier or diluent employed in the composition may be any
conventional solid
carrier. Examples of solid carriers are lactose, terra alba, sucrose, talc,
gelatin, agar, pectin,
acacia, magnesium stearate and stearic acid. Similarly, the carrier or diluent
may include
any sustained release material known in the art, such as glyceryl monostearate
or glyceryl
distearate, alone or mixed with a wax.
[00210] It may be of particular advantage to provide the composition of the
invention in
the form of a sustained release formulation. As such, the composition may be
formulated as
microcapsules or microparticles containing the modified GLP-1 or GLP-1 analogs
encapsulated by or dispersed in a suitable pharmaceutically acceptable
biodegradable
polymer such as polylactic acid, polyglycolic acid or a lactic acid/glycolic
acid copolymer.
[00211] For nasal administration, the preparation may contain modified GLP-1
or GLP-1
analogs dissolved or suspended in a liquid carrier, in particular an aqueous
carrier, for
aerosol application. The carrier may contain additives such as solubilizing
agents, e.g.
propylene glycol, surfactants, absorption enhancers such as lecithin
(phosphatidylcholine)
or cyclodextrin, or preservatives such as parabenes.
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[00212] Generally, the modified polypeptides or peptides of the present
invention are
dispensed in unit dosage form together with a pharmaceutically acceptable
carrier per unit
dosage.
[00213] Moreover, the present invention contemplates the use of the modified
GLP-1 and
GLP-l analogs for the manufacture of a medicinal product which can be used in
the
treatment of diseases associated with elevated glucose level (metabolic
disease), such as but
not to limited to those described above. Specifically, the present invention
contemplates the
use of modified GLP-1 and GLP-1 analogs for the treatment of diabetes
including type II
diabetes, obesity, severe bums, and heart failure, including congestive heart
failure and
acute coronary syndrome.
[00214] The present invention also provides modified Exendin-3 and Exendin-4
peptides
partially and substantially protected from DPP activity. Exendin-3 and Exendin-
4 are
insulinotropic peptides comprising 39 amino acids (differing at residues 2 and
3) which are
approximately 53% homologous to GLP-1. The Exendin-3 sequence is
HSDGTFTSDLSKQMEEEAVRLFIEWLKNGG PSSGAPPPS (SEQ ID NO: 79), and the
Exendin-4 sequence is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG PSSGAPPPS
(SEQ ID NO: 80). The invention also encompasses the modified exendin-4
fragments
comprising the amino acid sequences such as Exendin-4 (1-3 1)
HGEGTFTSDLSKQMEEAVR LFIEWLKNGGPY (SEQ ID NO: 81). Additionally, the
present invention includes modified analogs of Exendin-3 and Exendin-4
peptides.
Modified GLP-1 Fusion Protein or Conjugate for Treating Diabetes, Prediabetes,
or
Obesity
[00215] The modified GLP-1 may be fused to a heterologous molecule for
increased
overall stability in vivo. The modified GLP-1 may be fused to a heterologous
molecule by
recombinant means or covalently attached to a heterologous molecule by methods
well
known in the art. Modified GLP-1 may be fused or covalently attached, for
example to a
plasma protein such as serum albumin or transferrin, an immunoglobulin, or a
portion
thereof such as the Fc domain. More preferably, the modified polypeptide or
peptide is
fused to transferrin, lactotransferrin, or melanotransferrin. Methods for
making such fusion
proteins are provided by U.S. Application 10/378,094, which is herein
incorporated by
reference in its entirety.
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[002161 The GLP-1 molecule may be attached to the heterologous protein via a
linker of
variable length to provide greater physical separation and allow more spatial
mobility
between the fused proteins and thus maximize the accessibility of the
therapeutic protein,
for instance, for binding to its cognate receptor. The linker peptide may
consist of amino
acids that are flexible or more rigid. For example, a linker such as a poly-
glycine stretch
may be used. The linker can be less than about 50, 40, 30, 20, 10, or 5 amino
acid residues.
The linker can be covalently linked to and between the heterologous protein
and GLP-1.
Preferably, the linker may be one Ser residue, two Ser residues, the peptide
Ser-Ser-Gly, the
peptide PEAPTD, the peptide (PEAPTD)2, the peptide PEAPTD in combination with
IgG
hinge linker, and the peptide (PEAPTD)2 in combination with IgG hinge linker.
These
linkers may be used to link GLP-1 to transferrin.
[00217] The transferrin to be attached to the modified polypeptide or peptide
may be
modified. It may exhibit reduced glycosylation. The modified transferrin
polypeptide may
be selected from the group consisting of a single transferrin N domain, a
single transferrin C
domain, a transferrin N and C domain, two transferrin N domains, and two
transferrin C
domains.
[00218] As discussed above, GLP-1 activates and regulates important endocrine
hormone
systems in the body and plays a critical management role in the metabolism of
glucose.
Unlike all other diabetic treatments on the market GLP-1 has the potential to
be restorative
by acting as a growth factor for (3-cells thus improving the ability of the
pancreas to secrete
insulin and also, to make the existing insulin levels act more efficiently by
improving
sensitivity and better stabilizing glucose levels. This reduces the burden on
daily
monitoring of glucose levels and potentially offers a delay in the serious
long term side
effects caused by fluctuations in blood glucose due to diabetes. Furthermore,
GLP-1 can
reduce appetite and reduce weight. Obesity is an inherent consequence of poor
control of
glucose metabolism and this only serves to aggravate the diabetic condition.
[00219] Clinical application of natural GLP-1 is limited because it is rapidly
degraded in
the circulation (half-life is several minutes). To maintain therapeutic levels
in the
circulation requires constant administration of high doses using pumps or
patch devices
which adds to the cost of treatment. This is inconvenient for long term
chronic use
especially in conjunction with all the other medications for treating diabetes
and monitoring
of glucose levels. The modified GLP-1 fusion proteins retain the activity of
GLP-1 but
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have the long half-life (14-17 days), solubility, and biodistribution
properties of transferrin.
These properties could provide for a low cost, small volume, monthly s.c.
(subcutaneous)
injection and this type of product is absolutely needed for long term chronic
use.
[00220] The modified GLP-1 also may be covalently attached to a blood
component to
increase its stability. For example, the modified GLP-1 may be covalently
attached to
serum albumin, transferrin, immunoglobulin, or the Fc portion of the
immunoglobulin. In
one embodiment, the modified GLP-1 may be attached to a fatty acid or a fatty
acid
derivative. In another embodiment, the modified GLP-1 may be engineered into a
drug
affinity complex (DAC). As discussed earlier, Kim et al. (2003, Diabetes
52(3):751)
disclose a GLP-1-albumin drug affinity complex. Kim et al. show that the
albumin-
conjugated DAC:GLP-1 mimics the native GLP-1. Kim et al. provide a new
approach for
prolonged activation of GLP-1R signaling.
[00221] Upon subcutaneous administration, the DAC:modified GLP-1 rapidly and
selectively bonds in vivo to albumin. The bioconjugate formed has the same
therapeutic
activity and similar potency as endogenous GLP-1 but has a pharmacokinetic
profile that is
closer to albumin.
Modified GLP-1 and its Fusion Protein in Combination with Other Therapeutic
Agents
[00222] In one aspect of the invention, the modified GLP- 1 peptide and its
fusion protein,
for example, GLP- 1 -Tf fusion protein, of the present invention are used in
combination with
at least one second therapeutic molecule such as Glucophage (metformin
hydrochloride
tablets) or Glucophage XR (metformin hydrochloride extended-release tablets)
to treat
type II diabetes, obesity, and other diseases or conditions associated with
abnormal glucose
levels.
[00223] Glucophage and Glucophage XR are oral antihyperglycemic drugs for
the
management of type II diabetes. Glucophage XR is an extended release
formulation of
Glucophage. Accordingly, Glucophage XR may be taken once daily because the
drug is
released slowly from the dosage form. Glucophage helps the body produce less
glucose
from the liver. Accordingly, Glucophage is effective in controlling blood
sugar level in a
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patient. Glucophage rarely causes low blood glucose (hypoglycemia) because it
does not
cause the body to make more insulin.
[00224] Glucophage also helps lower the fatty blood components, triglycerides
and
cholesterol, that are often high in people with Type II diabetes. Metformin
has been shown
to decrease the appetite and help people lose a few pounds when they starting
taking the
medicine.
[00225] Metformin has been approved for treatment with sulfonylureas, or with
insulin, or
as monotherapy (by itself). Metformin has been suggested for use in treating
various
cardiovascular diseases such as hypertension in insulin resistant patients (WO
9112003-
Upjohn), for dissolving blood clots (in combination with a t-PA-derivative)
(WO 9108763,
WO 9108766, WO 9108767 and WO 9108765-Boehringer Mannheim), ischemia and
tissue
anoxia (EP 283369-Lipha), atherosclerosis (DE 1936274-Brunnengraber & Co., DE
2357875-Hurka, and U.S. Pat. No. 4,205,087-ICI). In addition, it has been
suggested to use
metformin in combination with prostaglandin-analogous cyclopentane derivatives
as
coronary dilators and for blood pressure lowering (U.S. Pat. No. 4,182,772-
Hoechst).
Metformin has also been suggested for use in cholesterol lowering when used in
combination with 2-hydroxy-3,3,3-trifluoropropionic acid derivatives (U.S.
Pat. No.
4,107,329-ICI), 1,2-diarylethylene derivatives (U.S. Pat. No. 4,061,772-
Hoechst),
substituted aryloxy-3,3,3-trifluoro-2-propionic acids, esters and salts (U.S.
Pat. No.
4,055,595-ICI), substituted hydroxyphenyl-piperidones (U.S. Pat. No. 4,024,267-
Hoechst),
and partially hydrogenated 1H-indeno-[1,2B]-pyridine derivatives (U.S. Pat.
No. 3,980,656-
Hoechst).
[00226] Montanari et al. (Pharmacological Research, Vol. 25, No. 1, 1992)
disclose that
use of metformin in amounts of 500 mg twice a day (b.i.d.) increased post-
ischemia blood
flow in a manner similar to 850 mg metformin three times a day (t.i.d.).
Sirtori et al. (J.
Cardiovas. Pharm., 6:914-923, 1984), disclose that metformin in amounts of 850
mg three
times a day (t.i.d) increased arterial flow in patients with peripheral
vascular disease.
[00227] The present invention provides the treatment of various diseases
comprising
modified GLP-1 of the present invention or its fusion protein in combination
with one or
more therapeutic agents such as metformin. In one embodiment, the modified GLP-
1 or its
fusion protein in combination with metformin is used to treat diseases and
conditions
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associated with abnormal blood glucose level, such as diabetes. Preferably,
the GLP-1/mTf
fusion protein in combination with metformin is used to treat type II diabetes
or obesity.
[00228] Other therapeutic agents that may be used in combination with modified
GLP-1 of
the present invention and its fusion proteins include but are not limited to
sulfonylurea and
sulfonylurea-like agents, thiazolidinediones, Peroxisome Proliferator-
Activated Receptor
(PPAR) gamma modulators, PPAR alpha modulators, Protein Tyrosine Phosphatase-
1B
inhibitors, Insulin Receptor Tyrosine Kinase activators, 1lbeta-hydroxysteroid
dehydrogenase inhibitors, glycogen phosphorylase inhibitors, glucokinase
activators, beta-3
adrenergic agonists, and glucagon receptor agonists.
DPP-IV Inhibitors
[00229] Inhibitors of DPP-IV have been shown to be promising in treating
various
conditions mediated by DPP-IV. For example, inhibitors of DPP-IV are an
extremely
promising approach in the treatment of glucose intolerance and in disorders
associated with
hyperglycemia, such as type II diabetes or obesity. Moreover, DPP-IV has been
shown to
play a part in the immune response, such as transplant rejection
(Transplantation 1997,
63(10):1495-1500). Accordingly, DPP-IV may be useful in the prevention of
transplant
rejection. Also, inhibitors of DPP-IV may be useful in the treatment of cancer
and the
prevention of cancerous metastases, since the binding of endothelial DPP-IV of
the lung to
fibronectin of cancerous cells promotes metastasis of those cells (J. Biol.
Chem. 1998,
273(37:24207-24215). DPP-IV is likewise thought to play an important part in
the
pathogenesis of periodontitis (Infect. Immun. 2000, 68 (2), 716-724) and to be
responsible
for the inactivation of GLP-2, a factor facilitating recovery of the intestine
after major
resection (J. Surg. Res. 1999, 87 (1), 130-133). Accordingly, DPP-IV-
inhibitors also are
potentially useful in recovery of the intestine.
[00230] WO 95/15309 discloses certain peptide derivatives which are inhibitors
of DPP-IV
and, therefore, are useful in treating a number of DPP-IV mediated processes.
WO
95/13069 discloses certain cyclic amine compounds which are useful in
stimulating the
release of natural or endogenous growth hormone. European Patent 555,824
discloses
certain benzimidazolyl compounds which prolong thrombin time and inhibit
thrombin and
serine-related proteases. Archives of Biochemistry and Biophysics, Vol. 323,
No. 1, pgs.
148-154 (1995) discloses certain aminoacylpyrrolidine-2-nitriles which are
useful as DPP-
IV inhibitors. Journal of Neurochemistry, Vol. 66, pgs. 2105-2112 (1996)
discloses certain
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Fmoc-aminoacylpyrrolidine-2-nitriles which are useful in inhibiting prolyl
oligopeptidase.
Bulletin of the Chemical Society of Japan, Vol. 50, No. 7, pgs. 1827-1830
(1977) discloses
the synthesis of an aminohexapeptide, viz., Z-Val-Val-lmPro-Gly-Phe-Phe-OMe,
and its
related aminopeptides. In addition, the antimicrobial properties of said
compounds were
examined. WO 90/12005 discloses certain amino acid compounds which inhibit
prolylendopeptidase activity and, therefore, are useful in treating dementia
or amnesia.
Derwent Abstract 95: 302548 discloses certain N-(aryl(alkyl)carbonyl)
substituted
heterocyclic compounds which are cholinesterase activators with enhanced
peripheral
selectivity useful in treating conditions due to the lowering of
cholinesterase activity.
Chemical Abstracts 84: 177689 discloses certain 1-acyl-pyrrolidine-2-
carbonitrile
compounds which are useful as intermediates for proline compounds exhibiting
angiotensin
converting enzyme (ACE) inhibiting activity. Chemical Abstracts 96: 116353
discloses
certain 3-amino-2-mercapto-propyl-proline compounds which are Ras farnesyl-
transferase
inhibitors useful in treating various carcinomas or myeloid leukemias. WO
95/34538
discloses certain pyrrolidides, phosphonates, azetidines, peptides and
azaprolines which
inhibit DPP-IV and, therefore, are useful in treating conditions ameliorated
by DPP-IV
inhibition. WO 95/29190 discloses certain compounds characterized by a
plurality of KPR-
type repeat patterns carried by a peptide matrix enabling their multiple
presentation to, and
having an affinity for, the enzyme DPP-IV, which compounds exhibit the ability
to inhibit
the entry of HIV into cells. WO 91/16339 discloses certain tetrapeptide
boronic acids
which are DPP-IV inliibitors useful in treating autoimmune diseases and
conditions
mediated by IL-2 suppression. WO 93/08259 discloses certain polypeptide
boronic acids
which are DPP-IV inhibitors useful in treating autoimmune diseases and
conditions
mediated by IL-2 suppression. WO 95/11689 discloses certain tetrapeptide
boronic acids
which are DPP-IV inhibitors useful in blocking the entry of HIV into cells.
East German
Patent 158109 discloses certain N-protected peptidyl-hydroxamic acids and
nitrobenzoyloxamides which are useful as, inter alia, DPP-IV inhibitors. WO
95/29691
discloses, inter alia, certain dipeptide proline phosphonates which are DPP-IV
inhibitors
useful in the treatment of immune system disorders. German Patent DD 296075
discloses
certain amino acid amides which inhibit DPP-IV. Biochimica et Biophysica Acta,
Vol.
1293, pgs. 147-153 discloses the preparation of certain di- and tri-peptide p-
nitroanilides to
study the influence of side chain modifications on their DPP-IV and PEP-
catalyzed
hydrolysis. Bioorganic and Medicinal Chemistry Letters, Vol. 6, No. 10, pgs.
1163-1166
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(1996) discloses certain 2-cyanopyrrolidines which are inhibitors DPP-IV. J.
Med. Chem.,
Vol. 39, pgs. 2087-2094 (1996) discloses certain prolineboronic acid-
containing dipeptides
which are inhibitors of DPP-IV. Diabetes, Vol. 44, pgs. 1126-1131
(September'96) is
directed to a study which demonstrates that GLP-I amide is rapidly degraded
when
administered by subcutaneous or intravenous routes to diabetic and non-
diabetic subjects.
[00231] U.S. Patent 6,727,261 provides pyrido[2,1-a]isoquinoline derivatives
as novel
DPP-IV inhibitors useful for the treatment and/or prophylaxis of diseases
which are
associated with DPP-IV, such as diabetes, particularly non-insulin dependent
diabetes
mellitus, and impaired glucose tolerance. These compounds are also useful in
the treatment
and/or prophylaxis of Bowl disease, Colitis Ulcerosa, Morbus Crohn, obesity
and/or
metabolic syndrome.
[00232] U.S. Patent 6,716,843 provides alpha-amino acid sulphonyl compounds
useful as
inhibitors for DPP-IV.
[00233] U.S. Patent 6,645,995 discloses 2-substituted unsaturated heterocyclic
compounds
wherein a nitrogen atom in the heterocyclic ring is attached via an amide bond
or a peptide
bond to an amino acid or an amino acid derivative. These compounds are potent
and
selective inhibitors of DPP-IV, and are effective in treating conditions that
may be regulated
or normalized via inhibition of DPP-IV.
[00234] U.S. Patent 6,617,340 discloses N-(substituted glycyl)-pyrrolidines,
and the use of
said compounds in inhibiting dipeptidyl peptidase-IV. U.S. Patent 6124,305
discloses N-
(substituted glycyl)-2-cyanopyrrolidines which inhibit DPP-IV. These compounds
are
effective in treating conditions mediated by DPP-IV.
[00235] Administration of the Novartis compound 1-[[[2-[(5-cyanopyridin-2-
yl)amino]
ethyl]amino]acetyl]-2- cyano-(S)- pyrrolidine (NVP DPP728) over a 4 week
period to 93
patients with Type 2 diabetes (mean HbAlc of 7.4%) reduced levels of plasma
glucose,
insulin, and HbAI, over the 4 week study period (see Diabetes Care 2002,
25(5):869-875).
Combination Therapy Using Inhibitors of DPP-IV
[00236] In one aspect, the present invention provides the use of a transferrin
fusion protein
comprising a therapeutic protein, polypeptide, or peptide in combination with
one or more
inhibitors of DPP-IV for the treatment of various conditions. The present
invention
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provides pharmaceutical compositions comprising the transferrin fusion protein
and one or
more inhibitors of DPP-IV. As disclosed in U.S. Application No. 10/378,094,
which is
herein incorporated by reference in its entirety, the transferrin to be
attached to the
therapeutic protein, polypeptide, or peptide may be modified. It may exhibit
reduced
glycosylation. The modified transferrin polypeptide may be selected from the
group
consisting of a single transferrin N domain, a single transferrin C domain, a
transferrin N
and C domain, two transferrin N domains, and two transferrin C domains. The
therapeutic
protein or peptide to be attached to transferrin may be in its native or
modified form.
Preferably, the transferrin fusion protein comprises GLP-1 as the therapeutic
peptide, linked
to a modified transferrin molecule, as described in U.S. Application No.
10/378,094.
Moreover, the combination therapy of the present invention comprises GLP-1/mTf
fusion
protein, one or more inhibitors of DPP-IV, and another therapeutic molecule.
Such a
molecule may be Glucophage or Glucophage XR.
[00237] In another aspect, the present invention provides the use of a
modified protein or
peptide that is resistant to dipeptidyl protease cleavage or its fusion
protein in combination
with one or more DPP-IV inhibitors. The present invention discloses
pharmaceutical
compositions comprising the modified protein or peptide or its fusion protein
in
combination with one or more DPP-IV inhibitors. Preferably, the modified
peptide is
modified GLP-1 and the fusion protein is modified GLP-1/mTf protein. Further,
the
combination therapy of the present invention comprises modified GLP-1 or
modified GLP-
l/mTf protein, one or more inhibitors of DPP-IV, and another therapeutic
molecule such as
Glucophage or Glucophage XR.
[00238] DPP-IV inhibitors may be used in methods of the invention to treat any
relevant
disease. For instance, a GLP-1-transferrin fusion protein as herein described
can be
combined with a DPP-IV inhibitor, such as 1-[[[2-[(5-cyanopyridin-2-yl)amino]
ethyl]amino]acetyl]-2- cyano-(S)- pyrrolidine (NVP DPP728) to treat
prediabetes, diabetes,
obesity, or a diabetic symptom. The therapeutic agents may be administered
sequentially or
concurrently.
[00239] The transferrin fusion protein may comprise the GLP-1(7-37) peptide
(SEQ ID
NO: 32) or the GLP-1(7-36) peptide (amino acids 1-30 of SEQ ID NO: 32). More
preferably, the GLP-1(7-37) peptide or GLP-1(7-36) peptide comprises an A8 to
G and K34
to A mutation. The transferrin protein may also comprise a linker between GLP-
1(7-37)
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peptide or GLP-1(7-36) peptide and the transferrin molecule. Preferably, the
linker is the
(PEAPTD)2 peptide.
Enhancement of the Pharmacokinetics and Pharmacodynamics of Fusion Proteins
with Combination Therapy using Endopeptidase Inhibitors.
[00240] The present invention also provides combination therapy using neutral
endopeptidase (NEP) inhibitors and transferrin fusion proteins. Moreover, the
present
invention includes combination therapy using NEP and DPP-IV inhibitors and
transferrin
fusion proteins. The NEP and DPPIV inhibitors may be administered concurrently
or
sequentially. Furthermore, the inhibitors and the transferrin fusion proteins
may be
administered concurrently or sequentially. The inhibitors may be administered
prior to or
after administering the transferrin fusion proteins.
[00241] The transferrin fusion protein comprise the GLP-1(7-37) peptide (SEQ
ID NO: 32)
or the GLP-1(7-36) peptide (amino acids 1-30 of SEQ ID NO: 32). More
preferably, the
GLP-1(7-37) peptide or GLP-1(7-36) peptide comprises an A8 to G and K34 to A
mutation.
The transferrin protein may also comprise a linker between GLP-1(7-37) peptide
or GLP-
1(7-36) peptide and the transferrin molecule. Preferably, the linker is the
(PEAPTD)2
peptide.
[00242] Neutral endopeptidase (NEP), which is also known as enkephalinase,
neprilysin,
and atriopeptidase, is a membrane-bound zinc metalloendopeptidase found in
many tissues
including the brain, kidney, lungs, gastrointestinal tract, heart, and
peripheral vasculature.
NEP plays a major role in the clearance of natriuretic peptides by degrading
circulating
natriuretic peptides, thus preventing their effects on vasodilation, blood
pressure and
volume. NEP, by degrading and inactivating the natriuretic peptides, is
associated with,
hypertension, heart failure, and renal failure.
[00243] In addition to degrading circulating natriuretic peptides, NEP also
degrades other
vasodilating substances including circulating bradykinins; adrenomedullin,
renal
vasodilating and natriuretic-diuretic peptide; and/or urodilatin, a renal form
of ANP.
[00244] NEP is also involved in the degradation of endothelin isoform ET-1, a
vasoconstrictor, and may be involved in the formation of ET-1 (Erunner-La
Rocca et al.,
Cardiovascular Research 51 (2001) 510-520). NEP also degrades angiotensin II,
a potent
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vasoconstrictor, endomorphins, and a number of peptides involved in metabolism
such as
bradykinin, GLP-1 (Hupe-Sodmann, K., McGregor, G P., Bridenbaugh, R et al
Regulatory
Peptides 1995; 58: 149-56, Hupe-Sodmann, K, Goeke, R., Goeke, B et al Peptides
1997;
18: 625-32), PYY (Medeiros MD, Turner AJ., Endocrinology. 1994 May;134(5):2088-
94)
and glucagon (Trebbien R et al Am J Physiol Endocrinol Metab. 2004
Sep;287(3):E431-8).
[00245] A number of NEP inhibitors such as phosphoramidon or NEP/ACE
inhibitors
(including omapatrilat disclosed in U.S. Pat. No. 5,508,272, gempatrilat
disclosed in U.S.
Pat. No. 5,552,397, sampatrilat and MDL100240 disclosed in U.S. Pat. No.
5,430,145) have
been reported in the literature as useful for the monotherapeutic treatment
of, for example,
hypertension and heart failure. Nathisuwan et al., "A Review of Vasopeptidase
Inhibitors:
A New Modality in the Treatment of Hypertension and Chronic Heart Failure,"
Pharmacotherapy, Vol. 22(1), pp. 27-42 (2002). Candoxatril and ecadotril are
the two
highly specific inhibitors of NEP presently undergoing trials as future drugs
for heart
failure. Both compounds are prodrugs which are metabolized in the body to
active
congeners. Candoxatril is activated in the liver to candoxatrilat, while
ecadotril is converted
to its active congener, S-thiorphan.
[00246] Some examples of ACE/NEP inhibitors disclosed in U.S. Pat. Nos.
5,508,272,
5,362,727, 5,366,973, 5,225,401, 4,722,810, 5,223,516, 5,552,397, 4,749,688,
5,504,080,
5,612,359, and 5,525,723, and European Patent Applications 0481,522,
0534363A2,
534,396, and 534,492.
[00247] The present invention provides combination therapy comprising
transferrin fusion
proteins and DPP-IV inhibitors and or ACE/NEP inhibitors for the treatment of
various
diseases or conditions. Such diseases or conditions include but are not
limited to diabetes,
preferably type II diabetes, congestive heart failure, obesity, hypertension,
and irritable
bowel syndrome.
Transgenic Animals
[00248] The production of transgenic non-human animals that express a modified
polypeptide or peptide that is protected from DPP activity is contemplated in
one
embodiment of the present invention. In some embodiments, transgenic non-human
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animals expressing fusion proteins comprising a modified polypeptide or
peptide and
having increased stability is contemplated.
[00249] The successful production of transgenic, non-human animals has been
described in
a number of patents and publications, such as, for example U.S. Patent
6,291,740 (issued
September 18, 2001); U.S. Patent 6,281,408 (issued August 28, 2001); and U.S.
Patent
6,271,436 (issued August 7, 2001) the contents of which are hereby
incorporated by
reference in their entireties.
[00250] The ability to alter the genetic make-up of animals, such as
domesticated
mammals including cows, pigs, goats, horses, cattle, and sheep, allows a
number of
commercial applications. These applications include the production of animals
which
express large quantities of exogenous proteins in an easily harvested form
(e.g., expression
into the milk or blood), the production of animals with increased weight gain,
feed
efficiency, carcass composition, milk production or content, disease
resistance and
resistance to infection by specific microorganisms and the production of
animals having
enhanced growth rates or reproductive performance. Animals which contain
exogenous
DNA sequences in their genome are referred to as transgenic animals.
[00251] The most widely used method for the production of transgenic animals
is the
microinjection of DNA into the pronuclei of fertilized embryos (Wall et al.,
J. Cell.
Biochem. 49:113 [1992]). Other methods for the production of transgenic
animals include
the infection of embryos with retroviruses or with retroviral vectors.
Infection of both pre-
and post-implantation mouse embryos witli either wild-type or recombinant
retroviruses has
been reported (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]; Janenich
et al., Cell
24:519 [1981]; Stuhlmann et al., Proc. Natl. Acad. Sci. USA 81:7151 [1984];
Jahner et al.,
Proc. Natl. Acad Sci. USA 82:6927 [1985]; Van der Putten et al., Proc. Natl.
Acad Sci.
USA 82:6148-6152 [1985]; Stewart et al., EMBO J. 6:383-388 [1987]).
[00252] An alternative means for infecting embryos with retroviruses is the
injection of
virus or virus-producing cells into the blastocoele of mouse embryos (Jabner,
D. et al.,
Nature 298:623 [1982]). The introduction of transgenes into the germline of
mice has been
reported using intrauterine retroviral infection of the midgestation mouse
embryo (Jahner et
al., supra [1982]). Infection of bovine and ovine embryos with retroviruses or
retroviral
vectors to create transgenic animals has been reported. These protocols
involve the micro-
injection of retroviral particles or growth arrested (i.e., mitomycin C-
treated) cells which
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shed retroviral particles into the perivitelline space of fertilized eggs or
early embryos (PCT
International Application WO 90/08832 [1990]; and Haskell and Bowen, Mol.
Reprod.
Dev., 40:386 [1995]. PCT International Application WO 90/08832 describes the
injection
of wild-type feline leukemia virus B into the perivitelline space of sheep
embryos at the 2 to
8 cell stage. Fetuses derived from injected embryos were shown to contain
multiple sites of
integration.
[00253] U.S. Patent 6,291,740 (issued September 18, 2001) describes the
production of
transgenic animals by the introduction of exogenous DNA into pre-maturation
oocytes and
mature, unfertilized oocytes (i.e., pre-fertilization oocytes) using
retroviral vectors which
transduce dividing cells (e.g., vectors derived from murine leukemia virus
[MLV]). This
patent also describes methods and compositions for cytomegalovirus promoter-
driven, as
well as mouse mammary tumor LTR expression of various recombinant proteins.
[00254] U.S. Patent 6,281,408 (issued August 28, 2001) describes methods for
producing
transgenic animals using embryonic stem cells. Briefly, the embryonic stem
cells are used
in a mixed cell co-culture with a morula to generate transgenic animals.
Foreign genetic
material is introduced into the embryonic stem cells prior to co-culturing by,
for example,
electroporation, microinjection or retroviral delivery. ES cells transfected
in this manner
are selected for integrations of the gene via a selection marker such as
neomycin.
[00255] U.S. Patent 6,271,436 (issued August 7, 2001) describes the production
of
transgenic animals using methods including isolation of primordial germ cells,
culturing
these cells to produce primordial germ cell-derived cell lines, transforming
both the
primordial germ cells and the cultured cell lines, and using these transformed
cells and cell
lines to generate transgenic animals. The efficiency at which transgenic
animals are
generated is greatly increased, thereby allowing the use of homologous
recombination in
producing transgenic non-rodent animal species.
Gene Therapy
[00256] The use of modified polypeptide or peptide constructs of the present
invention for
gene therapy is contemplated in one embodiment of this invention. The
polypeptide or
peptide has been modified to protect it from DPP activity by the addition of
one or more
additional amino acids at its N-terminus. For example, the nucleic acid
construct encoding
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GLP-1 comprising an additional His residue at its N-terminus is provided for
gene therapy.
Also, the nucleic acid construct encoding modified GLP-1/transferrin fusion
protein is
provided for gene therapy. The modified GLP-1 constructs of the present
invention are
protected from DPP activity and are more stable; thus, they are ideally suited
to gene
therapy treatments.
[00257] Briefly, gene therapy via injection of an adenoviras vector containing
a gene
encoding a soluble fusion protein consisting of cytotoxic lymphocyte antigen 4
(CTLA4)
and the Fc portion of human immunoglobulin G1 was recently shown in Ijima et
al. (June
10, 2001) Human Gene Therapy (United States) 12/9:1063-77. In this application
of gene
therapy, a inurine model of type II collagen-induced arthritis was
successfully treated via
intraarticular injection of the vector.
[00258] Gene therapy is also described in a number of U.S. patents including
U.S. Pat.
6,225,290 (issued May 1, 2001); U.S. Pat. 6,187,305 (issued February 13,
2001); and U.S.
Pat. 6,140,111 (issued October 31, 2000).
[00259] U.S. Patent 6,225,290 provides methods and constructs whereby
intestinal
epithelial cells of a mammalian subject are genetically altered to operatively
incorporate a
gene which expresses a protein which has a desired therapeutic effect.
Intestinal cell
transformation is accomplished by administration of a formulation composed
primarily of
naked DNA, and the DNA may be administered orally. Oral or other
intragastrointestinal
routes of administration provide a simple method of administration, while the
use of naked
nucleic acid avoids the complications associated with use of viral vectors to
accomplish
gene therapy. The expressed protein is secreted directly into the
gastrointestinal tract and/or
blood stream to obtain therapeutic blood levels of the protein thereby
treating the patient in
need of the protein. The transformed intestinal epithelial cells provide short
or long term
therapeutic cures for diseases associated with a deficiency in a particular
protein or which
are amenable to treatment by overexpression of a protein.
[00260] U.S. Pat. 6,187,305 provides methods of gene or DNA targeting in cells
of
vertebrate, particularly mammalian, origin. Briefly, DNA is introduced into
primary or
secondary cells of vertebrate origin through homologous recombination or
targeting of the
DNA, which is introduced into genomic DNA of the primary or secondary cells at
a
preselected site.
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[002611 U.S. Pat. 6,140,111 (issued October 31, 2000) describes retroviral
gene therapy
vectors. The disclosed retroviral vectors include an insertion site for genes
of interest and
are capable of expressing high levels of the protein derived from the genes of
interest in a
wide variety of transfected cell types. Also disclosed are retroviral vectors
lacking a
selectable marker, thus rendering them suitable for human gene therapy in the
treatment of a
variety of disease states without the co-expression of a marker product, such
as an
antibiotic. These retroviral vectors are especially suited for use in certain
packaging cell
lines. The ability of retroviral vectors to insert into the genome of
mammalian cells has
made them particularly promising candidates for use in the genetic therapy of
genetic
diseases in humans and animals. Genetic therapy typically involves (1) adding
new genetic
material to patient cells in vivo, or (2) removing patient cells from the
body, adding new
genetic material to the cells and reintroducing them into the body, i.e., in
vitro gene therapy.
Discussions of how to perform gene therapy in a variety of cells using
retroviral vectors can
be found, for example, in U.S. Pat. Nos. 4,868,116, issued Sep. 19, 1989, and
4,980,286,
issued Dec. 25, 1990 (epithelial cells), WO 89/07136 published Aug. 10, 1989
(hepatocyte
cells) , EP 378,576 published Jul. 25, 1990 (fibroblast cells), and WO
89/05345 published
Jun. 15, 1989 and WO/90/06997, published Jun. 28, 1990 (endothelial cells),
the disclosures
of which are incorporated herein by reference.
[00262] Without further description, it is believed that one of ordinary skill
in the art can,
using the preceding description and the following illustrative examples, make
and utilize the
claimed invention. The following working examples therefore, specifically
point out
preferred embodiments of the present invention, and are not to be construed as
limiting in
any way the remainder of the disclosure. All articles, publications, patents
and documents
referred to throughout this application are hereby incorporated by reference
in their entirety.
EXAMPLES
Example 1: Modified GLP-1 Having Dipeptidyl-Peptidase IV Protection
[00263] This Example describes modified GLP-1 peptides protected from DPP-IV
activity.
The following peptides were synthesized using standard solid phase Fmoc
chemistry and
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purified by reverse phase HPLC using a C18 column and quantitated by
absorbance at
220nm. The purified peptides were analyzed by mass spectrometry (MALDI-TOF):
GLP-1
NHZ-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-
Ala-
Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH (amino acids 1-30 of SEQ ID
NO:
32)
GLP-1 (A8G)
NHZ-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-
Ala-
Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH (SEQ ID NO: 90)
H-GLP-1
NH2-His-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-
Ala-
Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH (SEQ ID NO: 91)
H-GLPr1 (A8G)
NH2-His-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-
Ala-
Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH (SEQ ID NO: 92)
HH-GLP-1
NH2-His-His-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
Gln-
Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH (SEQ ID NO: 93)
G-GLP-1
NH2-Gly-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-
Ala-
Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH (SEQ ID NO: 94)
H-Exendin-4
NHZ-His-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-
Ala-
Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-
Pro-
Ser-COOH (SEQ ID NO: 95)
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Dipeptidylpeptidase-IV Treatment
[00264] Equimolar concentrations of each peptide (6 M) were treated with 2 g
of
recombinant human DPP-IV (1 g/ L, R&D Systems, Minneapolis, MN) in 25 mM Tris-
Cl
(pH 8.0). Control reactions excluding DPP-IV were set up in parallel for each
peptide. The
digests were incubated at room temperature for 2 hours, at which time the
reactions were
diluted 10-fold in Krebs-Ringer buffer (Biosource International, Camarillo,
CA)
supplemented with 1mM 3 -Isobutyl- 1 -methylxanthine (IBMX, Calbiochem, San
Diego,
CA). The peptides were then analyzed to determine residual GLP-1 receptor
activating
activity, as described below.
Cyclic AMP Stimulation Assay
[00265] Four 96-well tissue culture plates were seeded with CHO-GLPIR cells
(Montrose-
Rafizadeh, et al. 1997 J. Biol. Cliem. 272, 21201-21206) at a density of 2 x
104 cells/well in
RPMI/10% FBS medium one day prior to treatment. The next day the cells
appeared
uniformly distributed with an approximate confluency of 60-80 percent. One day
after
seeding the culture plates the cells were washed twice with Krebs-Ringer
buffer (KRB)
followed by incubation in KRB for lhr at 37 C to lower the intracellular
levels of cAMP.
This was followed by incubation for 10 minutes in KRB/IBMX to inhibit
intracellular
enzymes that break down cAMP. Dilutions of each test compound were prepared in
KRB/IBMX and triplicate wells of CHO-GLP1R cells were treated with 50 1 of
test
compound per well for exactly 20 minutes at 37 C. The treatment was halted by
washing
the cultures twice with ice-cold phosphate-buffered saline. Lysates were
prepared by the
addition of 0.1m1 lysis buffer 1B (Amersham Biosciences cAMP Biotrak EIA kit)
for 10
minutes at room temperature. The entire volume of each cell extract was then
assayed to
determine the cAMP concentration using the cAMP Biotrak Enzyme Immunoassay
System
(Amersham Biosciences Corporation, Piscataway, NJ, product code RPN225)
according to
kit instructions. Peptides of the invention were found to be more resistant to
DPP-IV than
the unmodified forms.
Active GLP-1 specific ELISA
[00266] Alternatively, DPP-IV degradation of GLP-1 and GLP-1 derivatives of
the
invention was assayed using an ELISA system (Glucagon-Like Peptide-1 [Active]
ELISA
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kit [Linco Research, Inc., St. Charles, MO]) that is specific for intact,
active GLP-1 and
does not recognize GLP-1 in which the N-terminal two amino acids have been
removed due
to the action of DPP-IV, i.e. GLP-l(9-36 or 9-37). Equimolar concentrations of
GLP-1 and
H-GLP-1 (1200pM) were treated with recombinant human DPP-IV (200ng/ L, R&D
Systems, Minneapolis, MN) in 25 mM Tris-Cl (pH 8.0) and the reaction stopped
by dilution
in the assay buffer supplied with the kit, which contains protease inhibitors.
[00267] The kit comprises a 96-well microtitre plate coated with anti-GLP-1
monoclonal
antibody. The plate was washed (25mM Borate-buffered Saline x4 in a plate
washer,
ThermoLabsystems Ultrawash Plus), then incubated with peptide samples (300pM
and 10-
fold serial dilutions down the plate) for 3 hours at room temperature. After
washing as
described above, the plate was incubated with Alkaline-Phosphatase-conjugated
anti-GLP
antibody (supplied as a ready-to-use component of the kit) for 2 hours at room
temperature.
After washing, 4-Methylumbelliferyl Phosphate (MUP) substrate (1:200 dilution
in 50mM
Borate pH 9.5) was applied to all wells, and incubated in the dark at room
temperature for
30 minutes. The plate was read at 355 excitation and 460nm emission
wavelengths on a
SpectraMax Gemini EM fluorescence plate reader. As H-GLP-1 bound less readily
to the
monoclonal antibody than GLP-1 itself, the concentration of active H-GLP-l
remaining
after DPP-IV treatment was determined using an H-GLP-1 standard curve. Figure
8 shows
that H-GLP-1 is substantially more resistant to the action of DPP-IV than GLP-
1.
Example 2: Modified GLP-1 Fusion Protein
[00268] This Example describes a fusion protein comprising a modified GLP-1
protected
from DPP-IV activity fused to a modified transferrin molecule.
[00269] In order to construct a sequence encoding the transferrin secretion
leader followed
by GLP-1 and the N-terminal part of transferrin, the following overlapping
primers were
designed:
P0236-
TTCCCATACAAACTTAAGAGTCCAATTAGCTTCATCGCCA (SEQ ID NO: 96)
P0237-
GGTTTAGCTTGTTTTTTTATTGGCGATGAAGCTAATTGGACTCTTAAGTTTGTAT
GGGAA (SEQ ID NO: 97)
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P0244-
ATAAAAAAACAAGCTAAACCTAATTCTAACAAGCAAAGATGAGGCTCGCCGTG
GGAGCCC (SEQ ID NO: 98)
P0245-
CAGGACGGCGCAGACCAGCAGGGCTCCCACGGCGAGCCTCATCTTTGCTTGTTA
GAATTA (SEQ ID NO: 99)
P0248-
TGCTGGTCTGCGCCGTCCTGGGGCTGTGTCTGGCGCATGCTGAAGGTACTTTTA
CTTCTGATGTTTCTTC (SEQ ID NO: 100)
P0249-
AATTCTTTAGCAGCTTGACCTTCCAAATAAGAAGAAACATCAGAAGTAAAAGT
ACCTTCAGCATGCGCCAGACACAGCCC (SEQ ID NO: 101)
P0250-
TTATTTGGAAGGTCAAGCTGCTAAAGAATTTATTGCTTGGTTGGTTAAAGGTAG
GGTACCTGATAAAACT (SEQ ID NO: 102)
P0251-
AGTTTTATCAGGTACCCTACCTTTAACCAACCAAGCAATA (SEQ ID NO: 103)
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The positions of these primers are shown below.
Af11I
-+----
...........P0236............
721 ccaatgttac gtcccgttat attggagttc ttcccataca aacttaagag tccaatta.g0
ggttacaatg cagggcaata taacctcaag aagcrgtatgt tt_g,aattctc agqttaatqq
........... P0237............
>>P0236.>> .....................P0244........................
~ , . ,._. . ... . .
781 ttc;atcgcca ataa3aaaac.aagctaaacc taattctaac aaqcaaagat ga,ggctcgcc
~agtagcggt tatttt_tttg ttcgatttg4 =ttaagattg ttcgtttcta ctecgagcgg
...........P0237............ ........... P0245 ............
...nL..... >
m r 1 a
>>P0244.>> >> .....................P0248........................
~. ..,..... _.
841 gt t~ggagccc tcrctaatctg cgccgtcctg Rggctatgtc Cq cgcatgC tga~ggtaCt
OaCCC;tcggg acgaccagac gcggC~,ggac cccgacacag accgCgt~acc~,dct'CcCatqg
...........P0245............ << ........... P0249 ............
> ...................... nL......................
v g a 1 1 v c a v 1 g 1 c 1 a
...GLP-1..... >
h a e g t
.....P0248....... ............... P0250 ...................
901 ittacttctg atgtttcttc ttatttgcraa ggtCaagC'tg et aaagaatt tattgcttgg
aaatc{aagac LaCaaa~aao aataaacctt coac{ttGaac qatttctta.a at~.acg_, aaCo
_ ~ ~ ..._... ,.. . .. . .. . _ ~ : __..
...................P0249.......................... << .P0251
.......................>
> ............................. GLP-1......
f t s d v s s y 1 e g q a a k e f i a w
Kpn2
-----+
.........P0250 ..............
961 ttggttaaact Utagggtaac tgataaaaat, gtgagatggt gtgcagtgtc ggagcatgag
aaccaatttc catcccatgg actattttg,~,cactctacca cacgtcacag cctcgtactc
. . .,_... _.
...........P0251............
>.... GLP-1....
1 v k g r
..................... mTf...................... >
v p d k t v r w c a v s e h e
(SEQ ID NO: 104 is the coding strand; SEQ ID NO: 105 is the encoded protein.)
The primers (8 L of 20pmol conc.) were combined and heated to 65 C for 5 min.
and then
the annealing reaction was allowed to cool slowly to room temperature.
[00270] After adding T4 DNA ligase to the annealing reaction and incubating
for a further
2hr at room temperature, 1 L of the reaction was removed and used in a PCR
reaction to
amplify the completed insert with the outer primers P0236 and P0251. The PCR
conditions
were as follows:
min at 94 C
25 cycles of : 30sec at 94 C
30sec at 50 C
1 min at 72 C
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7 min at 72 C
hold at 4 C
[00271] The resulting PCR product was digested AflII and KpnI and ligated into
pREX0094
(Figure 1) which had previously been digested with AflIl and KpnI. The
ligation was used
to transform E. coli. The DNA from the resultant clones was sequenced and a
clone correct
the length of the AfIII/KpnI insert was selected and designated pREX0198
(Figure 2). Next,
pREX0198 was digested with NotI and PvuI and inserted into pSAC3 5 (Figure 3)
to create
pREX0240 (Figure 4).
[00272] To create a plasmid encoding the natural transferrin secretion leader
followed by
H-GLP-1(7-36) fused to modified transferrin (mTf), overlapping primers P0424
and P0425
were designed to add the extra N-terminal histidine to the sequence encoded by
pREX0198.
P0424 5' to 3'
CTGTGTCTGGCGCATCATGCTGAAG (SEQ ID NO: 106)
P0425 5' to 3'
CTTCAGCATGATGCGCCAGACACAG (SEQ ID NO: 107)
[00273] pREX0198 was used as the template for the initial PCR reactions using
the two
overlapping mutagenic primers and two outer primers in separate reactions,
i.e. P0424 plus
P0012 and P0425 plus P0025. The products of these reactions were then used as
templates
in a second round of PCR with just the outer primers, i.e. P0012 plus P0025,
in order to join
them together. The reaction conditions for both rounds of PCR were 1 x 94 C
for 1 min, 20
x 94 C for 30 seconds, 50 C for 30 seconds, 72 C for 1 minute and 1 x 72 C for
7 minutes
to finish.
[00274] The PCR product from the final reaction was digested with AflII and
KpnI and
ligated into AfIII/KpnI digested pREX0052 (Figure 5) to create pREX0367
(Figure 6). The
construct was DNA sequenced to confirm the insertion of the codon for the
extra histidine.
[00275] pREX0367 was then digested with Notl and PvuI (the latter to destroy
the
ampicillin resistance gene) and ligated into pSAC35 previously digested with
NotI to create
pREX0368 (Figure 7).
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[00276] pREX0368 was transformed into the host Saccharonayces cerevisiae
strain by
electroporation and transformed colonies selected on the basis of leucine
prototrophy on
buffered minimal medium plates. After selection of single colonies, yeast
transformants
were stocked in 40% Trehalose and stored at -70 C. Expression was determined
by growth
in liquid minimal medium buffered to pH6.5 and analysis of supernatant by SDS-
PAGE,
western blot and ELISA.
[00277] The plasmids encoding GLP-1/mTf (pREX0100) and H-GLP-1/mTf were
constructed as described in U.S. Application 10/378,094, filed March 4, 2003,
which is
herein incorporated by reference in its entirety. To produce the GLP-1/mTf
fusion protein,
the amino acid sequence of GLP-1(7-36) and GLP-1(7-37) may be used.
haegtftsdvssylegqaakefiawlvkgr (amino acids 1-30 of SEQ ID NO: 32)
haegtftsdvssylegqaakefiawlvkgrg (SEQ ID NO: 32)
[00278] For example, the peptide sequence of GLP-1(7-36) may be back
translated into
DNA and codon optimized for yeast:
catgctgaaggtacttttacttctgatgtttcttcttatttggaaggtcaagctgctaaagaa
h a e g t f t s d v s s y 1 e g q a a k e
tttattgcttggttggttaaaggtaga (SEQ ID NO: 117)
f i a w 1 v k g r (amino acids 1-30 of SEQ ID NO: 32)
[00279] The primers were specifically designed to form 5' XbaI and 3' Kpnl
sticky ends
after annealing and to enable direct ligation into XbaI/KpnI cut pREX0052,
just 5' of the
end of the leader sequence and at the N-terminus of mTf. Alternatively, other
sticky ends
may be engineered for ligations into other vectors.
XbaI
-+-----
, ... ,. ., ,
. acttttactt ctgatgtttc'ttcttatttg
1 aggtct'ctag agaaaaggca tgctgaaggt .
tccagagatc tcttttccgt acgacttcca tgaaaatgaa qactacaaag aagaataaac
...... FL.......
r s 1 e k r
>> ........ .......... GLP-1.................... >
h a e g t f t s d v s s y 1
KpnI
------+
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61 .. .., ,., _
gaaggtcaag ctgctaaa9a,atttattact tggttggtta aaggtagggt acctgata
;ettccagttc gacgatttct t3aataacga accaaccaaz ttccatccca tggactat
> ...................... GLP-1......................
e g q a a k e f i a w 1 v k g r
..mTf..
v p d
SEQ ID NOs: 118 and 119
[00280] After annealing and ligation, the clones were sequenced to confirm
correct
insertion. This vector was designated pREX0094. The cassette was cut out of
pREX0094
with NotI and sub-cloned into Notl cut yeast vector, pSAC35, to make pREX0100.
[00281] This plasmid was then electroporated into the host Saccharornyces
yeast strains
and transformants selected for leucine prototrohy on minimal media plates.
Expression was
determined by growth in liquid minimal media and analysis of supernatant by
SDS-PAGE,
western blot, and ELISA.
[00282] GLP-1/mTf and H-GLP-1/mTf were expressed and purified from
fermentation
cultures, grown under standard conditions by cation exchange and anion
exchange
chromatography.
Dipeptidylpeptidase-IV Treatment
[00283] Equimolar concentrations of GLP-1/mTf and H-GLP/1-mTF (2 M) were
treated
with recombinant human DPP-N (1 g/ L, R&D Systems) in a solution of 25mM Tris-
Cl
(pH 8.0). Control reactions excluding DPP-IV were set-up in parallel for each
fusion
protein. The digests were incubated at room temperature for 2 hours, at which
time the
reactions were diluted 20-fold in Krebs-Ringer buffer (Biosource
International)
supplemented with 1 mM IBMX (Calbiochem).
Cyclic AMP Stimulation Assay
[00284] Tissue culture plates (24-well) were seeded with CHO-GLP1R cells at a
density of
1 x 105 cells per/well in RPMI/10% FBS medium one day prior to treatment. The
next day
the cells appeared uniformly distributed with an approximate confluency of 60-
80 percent.
One day after seeding the culture plates the cells were washed twice with
Krebs-Ringer
buffer (KRB) followed by incubation in KRB for lhr at 37 C to lower the
intracellular
levels of cAMP. This was followed by incubation for 10 minutes in KRB/IBMX to
inhibit
intracellular enzymes that break down cAMP. Dilutions of each test compound
were
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prepared in KRB/IBMX and triplicate wells of CHO-GLP1R cells were treated with
0.15m1
of test compound per well for exactly 50 minutes at 37 C. The treatment was
halted by
washing the cultures two times with ice-cold phosphate-buffered saline.
Lysates were
prepared by the addition of 0.2m1 lysis buffer 1B (Amersham Biosciences cAMP
Biotrak
EIA kit) for 10 minutes at room temperature, then 100 1 of each cell extract
was then
assayed to determine the cAMP concentration using the cAMP Biotrak Enzyme
Immunoassay System (Amersham Biosciences) according to kit instructions.
[00285] H-GLP-1/mTf was found to be more resistant to DPP-IV than GLP-l/mTf
Example 3: Modified GLP-1/mTf for the Treatment of Diabetes
[00286] In this Example, modified GLP-1/mTf of the present invention is used
as a
therapeutic agent to treat diabetes. Modified GLP-1/mTf is administered to
Zucker rats, a
standard aniinal model for type II diabetes. Zucker rats have abnormally high
blood glucose
levels. It has been shown that treatment of these animals with GLP-1 induces
insulin
secretion and reduces blood glucose.
[00287] Zucker rats are fasted overnight and then treated with H-GLP-1 or H-
GLP-1 fused
to transferrin (H-GLP-1/mTf). Thirty minutes after subcutaneous injection of H-
GLP-1 or
H-GLP-1/mTf, the animals are subjected to a Glucose Tolerance Test (GTT). For
this test,
fasted animals are fed glucose solution (1.5mg/g body weight), and the blood
glucose is
measured at appropriate time intervals. Soon after the glucose administration,
the blood
glucose level of the untreated animals rises and slowly drops towards the base
line while the
animals which are injected with H-GLP-1 or H-GLP-1/mTf show faster
normalization of
blood glucose level due to the insulinotropic effect of the GLP-1.
[00288] In a further experiment, modified H-GLP-1 or H-GLP-1/mTf is used to
normalize
the high fasting glucose of the Zucker rats without glucose administration.
While the blood
glucose levels remain high in the untreated animals, a significant drop is
seen in the H-GLP-
1 or modified H-GLP-1/mTf treated animals.
Example 4: Modified Glucagon Having Dipeptidyl-Peptidase IV Protection
[00289] This Example describes modified glucagon molecules protected from DPP-
IV
activity.
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[00290] The following peptides are synthesized using standard solid phase Fmoc
chemistry
and purified by reverse phase HPLC using a C18 column and quantitated by
absorbance at
220nm. The purified peptides are analyzed by mass spectrometry (MALDI-TOF):
Glucagon
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-
Ala-
Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH (SEQ ID NO: 35)
H-Glucagon
NH2-His-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Val-Leu-Asp-Ser-Arg-
Arg-
Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH (SEQ ID NO: 108)
[00291] The peptides are pre-treated with DPP-IV as described above and then
assayed for
the ability to activate the glucagon receptor using a recombinant cell line
expressing a
cloned glucagon receptor.
Example 5: Modified GIP Having Dipeptidyl-Peptidase IV Protection
[00292] This Example provides modified GIP molecules protected from DPP-IV
activity.
[00293] The following peptides are synthesized using standard solid phase Fmoc
chemistry
and purified by reverse phase HPLC using a C 18 column and quantitated by
absorbance at
220nm. The purified peptides are analysed by mass spectrometry (MALDI-TOF):
GIP
NHz-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-
Gln-
Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-
Asn-
Ile-Thr-Gln-COOH (SEQ ID NO: 31)
Y-GIP
NH2-Tyr-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-
His-Gln-
Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-
Asn-
Ile-Thr-Gln-COOH (SEQ ID NO: 109)
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[00294] The peptides are pre-treated with DPP-IV as described above and then
assayed for
the ability to activate the GIP receptor using a recombinant cell line
expressing a cloned
GIP receptor.
[00295] It should be understood that the foregoing discussion and examples
merely present
a detailed description of certain preferred embodiments. It therefore should
be apparent to
those of ordinary skill in the art that various modifications and equivalents
can be made
without departing from the spirit and scope of the invention. All journal
articles, other
references, patents, and patent applications that are identified in this
patent application are
incorporated by reference in their entirety.
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