U.S. patent application number 09/764070 was filed with the patent office on 2001-09-13 for glucagon-like peptide-2 analogs.
Invention is credited to Crivici, Anna E., Drucker, Daniel J., Sumner-Smith, Martin.
Application Number | 20010021767 09/764070 |
Document ID | / |
Family ID | 27503705 |
Filed Date | 2001-09-13 |
United States Patent
Application |
20010021767 |
Kind Code |
A1 |
Drucker, Daniel J. ; et
al. |
September 13, 2001 |
Glucagon-like peptide-2 analogs
Abstract
Analogs of glucagon-like peptide 2, a product of glucagon gene
expression, have been identified as intestinal tissue growth
factors. Their formulation as pharmaceutical, and therapeutic use
in treating disorders of the small bowel, are described.
Inventors: |
Drucker, Daniel J.;
(Toronto, CA) ; Crivici, Anna E.; (San Diego,
CA) ; Sumner-Smith, Martin; (Bolton, CA) |
Correspondence
Address: |
Bernhard D. Saxe
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W. Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
27503705 |
Appl. No.: |
09/764070 |
Filed: |
January 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09764070 |
Jan 19, 2001 |
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08835538 |
Apr 8, 1997 |
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6184201 |
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08835538 |
Apr 8, 1997 |
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08631273 |
Apr 12, 1996 |
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08835538 |
Apr 8, 1997 |
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08632533 |
Apr 12, 1996 |
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08835538 |
Apr 8, 1997 |
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08422540 |
Apr 14, 1995 |
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5990077 |
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Current U.S.
Class: |
530/320 |
Current CPC
Class: |
G01N 33/4833 20130101;
A61K 38/00 20130101; A61K 38/26 20130101; C07K 14/605 20130101;
A61P 1/00 20180101 |
Class at
Publication: |
530/320 |
International
Class: |
A61K 038/18 |
Claims
What is claimed is:
1. A GLP-2 analog which is characterized by intestinotrophic
activity and which conforms to Formula 1:
R1-(Y1)m-X1-X2-X3-X4-Ser5-Phe6-Ser7-Asp8-(P1-
)-Leu14-Asp15-Asn16-Leu17-
Ala18-X19-X20-Asp21-Phe22-(P2)-Trp25-Leu26-Ile2-
7-Gln-28-Thr29-Lys30-(P3)-(Y2)n-R2, wherein X1 is His or Tyr X2 is
Ala or an Ala-replacement amino acid conferring on said analog
resistance to DPP-IV enzyme; X3 is Pro, HPro, Asp or Glu; X4 is Gly
or Ala; P1 is Glu-X10-Asn-Thr-Ile or Tyr-Ser-Lys-Tyr; X10 is Met or
an oxidatively stable Met-replacement amino acid; X19 is Ala or
Thr; X20 is Arg, Lys, His or Ala; P2 is Ile-Asn, Ile-Ala or
Val-Gln; P3 is a covalent bond, or is Ile, Ile-Thr or Ile-Thr-Asn;
R1 is H or an N-terminal blocking group; R2 is OH or a C-terminal
blocking group; Y1 is one or two basic amino acids selected from
the group Arg, Lys, and His; Y2 is one or two basic amino acids
selected from the group Arg, Lys, and His; and m and n,
independently, are 0 or 1; and wherein at least one of X1, X2, X3,
X4, P1, X10, X19, X20, P2 and P3 is other than a wild type,
mammalian GLP-2 residue.
2. The GLP-2 analog according to claim 1, wherein said analog
incorporates at least one amino acid substitution selected from: a)
incorporation at X2 of an amino acid which renders said
intestinotrophic GLP-2 analog resistant to cleavage by human DPP-IV
enzyme; b) incorporation at X10 of an oxidatively stable,
Met-replacement amino acid; and c) incorporation at X20 of a basic
amino acid selected from His or Lys.
3. The GLP-2 analog according to claim 2, wherein X2 is substituted
with an amino acid which confers on said analog resistance to
digestion by DPP-IV enzyme.
4. The GLP-2 analog according to claim 3, wherein X2 is selected
from the group consisting of D-hPr, D-Pro, D-Ala, Gly, Val, Glu,
Lys, Arg, Leu, and Ile.
5. The GLP-2 analog according to claim 3 which incorporates at
least one amino acid substitution at the following positions: X1,
X3, X4, X10, X19, X20 and P2.
6. The GLP-2 analog according to claim 2, wherein X10 is
substituted with an amino acid chosen from the group consisting of
Val, Ile, Asn, Glx, Tyr, Phe, Leu, Nle, Ala, Ser, and Gly.
7. The GLP-2 analog according to claim 1 selected from the group
consisting of: [Tyr.sup.1]rGLP-2; [Ala.sup.4]rGLP-2;
[Val.sup.23Gln.sup.24]hGLP-2 and [Asn.sup.33]hGLP-2(1-33).
8. A pharmaceutical composition comprising a therapeutically
effective amount of a GLP-2 analog according to claim 1 and a
pharmaceutically acceptable carrier.
9. A method for promoting growth of small bowel tissue in a patient
in need thereof, comprising the step of delivering to the patient
the pharmaceutical composition of claim 8.
10. A method for treating a gastrointestinal disease, wherein the
method comprises administering to a patient having the
gastrointestinal disease a therapeutically effective amount of a
GLP-2 analog according to claim 1, together with a pharmaceutically
acceptable carrier to reduce a pathological effect or symptom of
the gastrointestinal disease.
11. The method of claim 10, wherein the gastrointestinal disease is
selected from the group consisting of ulcers, digestion disorders,
malabsorption syndromes, short-gut syndrome, cul-de-sac syndrome,
inflammatory bowel disease, celiac sprue, tropical sprue,
hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's
disease), small intestinal damage due to toxic or other
chemotherapeutic agents, and short bowel syndrome.
12. An analog of a human GLP-2 peptide, wherein the GLP-2 analog
comprises at least one amino acid substitution selected from the
group consisting of: a) incorporation at position X2 and/or X3 of a
replacement amino acid which renders the analog resistant to
cleavage by DPP-IV enzyme; b) incorporation at position X10 of a
replacement amino acid which is oxidatively stable; and c)
incorporation at position X20 of a replacement amino acid other
than Arg.
13. The GLP-2 analog according to claim 12 which incorporates an
amino acid substitution at position X2, and wherein the amino acid
substituted at position X2 is chosen from the group consisting of
D-hPr, D-Pro, D-Ala, Gly, Val, Glu, Lys, Arg, Leu, and Ile.
14. The GLP-2 analog of claim 13, wherein the analog is selected
from the group consisting of: [Gly.sup.2]hGLP-2;
[D-Ala.sup.2Thr.sup.19]hGLP-2; [Gly.sup.2Thr.sup.19]hGLP-2;
[Ala.sup.1Gly.sup.2]hGLP-2; [Gly.sup.2Ala.sup.3]hGLP-2;
[Gly.sup.2Ala.sup.4]hGLP-2; [Gly.sup.2Ala.sup.5]hGLP-2;
[Gly.sup.2Ala.sup.6]hGLP-2; [Gly.sup.2Ala.sup.7]hGLP-2;
[Gly.sup.2Ala.sup.8]hGLP-2; [Gly.sup.2Ala.sup.9]hGLP-2;
[Gly.sup.2Ala.sup.10]hGLP-2; [Gly.sup.2Ala.sup.11]hGLP-2;
[Gly.sup.2Ala.sup.12]hGLP-2; [Gly.sup.2Ala.sup.13]hGLP-2;
[Gly.sup.2Ala.sup.16]hGLP-2; [Gly.sup.2Ala.sup.17]hGLP-2;
[Val.sup.2Thr.sup.19]hGLP-2; [Gly.sup.2Ala.sup.20]hGLP-2
[Gly.sup.2Ala.sup.12]hGLP-2; [Gly.sup.2Ala.sup.24]hGLP-2;
[Gly.sup.2Ala.sup.27]hGLP-2; [Gly.sup.2Ala.sup.28]hGLP-2; and
[Gly.sup.2Ala.sup.31]hGLP-2.
15. The GLP-2 analog according to claim 12 which incorporates an
amino acid substitution at position X10, wherein the amino acid
substituted at position X10 is chosen from the group consisting of
Val, Ile, Asn, Glx, Tyr, Phe, Leu, Nle, Ala, Ser, and Gly.
16. The GLP-2 analog according to claim 15, wherein the analog is
selected from the group consisting of [Ser.sup.10]hGLP-2(1-33);
[Nle.sup.10]hGLP-2(1-33); [Ala.sup.10]hGLP-2(1-33);
[Leu.sup.10]rGLP-2(1-33); [Nle.sup.10]ratGLP-2(1-33);
[Gly.sup.2Ala.sup.10]hGLP-2(1-33); [Met()).sup.10]ratGLP-2(1-33)
and [Tyr.sup.9Ser.sup.10Lys.sup.11Tyr.sup.12 (desIle.sup.13)
]hGLP-2(1-33).
17. The GLP-2 analog according to claim 12 wherein the nalog is
selected from the group consisting of [Pro.sup.3]hGLP-2;
[HPr.sup.3]hGLP-2; [Glu.sup.3Thr.sup.19]hGLP-2; and
[Thr.sup.19Lys.sup.20 ]hGLP-2.
18. The GLP-2 analog of claim 12, wherein the analog is elected
from the group consisting of: [Ser.sup.2,Gln.sup.3]hGLP-2(1-33);
[Gly.sup.2, Ala.sup.24]hGLP-2(1-33); [Gly.sup.2,
Ala.sup.26]hGLP-2(1-33); [Gly.sup.2, Ala.sup.14]hGLP-2(1-33);
[Gly.sup.2, Ala.sup.23]hGLP-2(1-33); [Gly.sup.2,
Ala.sup.30]hGLP-2(1-33); [Tyr.sup.1, Gly.sup.2]hGLP-2(1-33);
[Gly.sup.2, Arg.sup.34]hGLP-2(1-34); [Gly.sup.2,
Tyr.sup.34]hGLP-2(1-34); [tBuGly.sup.2]hGLP-2; [Asp.sup.2]hGLP-2
(1-33); [Glu.sup.2]hGLP-2 (1-33); [Phe.sup.2]hGLP-2(1-33);
[His.sup.2]hGLP-2(1-33) ; [Ile.sup.2]hGLP-2(1-33);
[Lys.sup.2]hGLP-2 (1-33); [Met.sup.2]hGLP-2(1-33);
[Asn.sup.2]hGLP-2(1-33); [Pro.sup.2]hGLP-2(1-33) [Gln.sup.2]hGLP-2
(1-33); [Ser.sup.2]hGLP-2 (1-33); [Thr.sup.2]hGLP-2(1-33);
[Val.sup.2]hGLP-2(1-33); [Tyr.sup.2]hGLP-2(1-33)- ;
[D-Ala.sup.2]hGLP-2 (1-33); [Pen.sup.2]hGLP-2(1-33);
[bAla.sup.2]hGLP-2(1-33); [aAbu.sup.2]hGLP-2 (1-33);
[Nval.sup.2]hGLP-2 (1-33); [PhGly.sup.2]hGLP-2(1-33); and
[Aib.sup.2]hGLP-2(1-33).
19. A pharmaceutical composition comprising a therapeutically
effective amount of a GLP-2 analog according to claim 12, and a
pharmaceutically acceptable carrier.
20. A method for promoting growth of small bowel tissue in a
patient in need thereof, comprising the step of delivering to the
patient the pharmaceutical composition of claim 19.
21. A method for treating a gastrointestinal disease, wherein the
method comprises administering to a patient having the
gastrointestinal disease a therapeutically effective amount of a
GLP-2 analog according to claim 12, together with a
pharmaceutically acceptable carrier to reduce a pathological effect
or symptom of the gastrointestinal disease.
22. The method of claim 21, wherein the gastrointestinal disease is
selected from the group consisting of ulcers, digestion disorders,
malabsorption syndromes, short-gut syndrome, cul-de-sac syndrome,
inflammatory bowel disease, celiac sprue, tropical sprue,
hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's
disease), small intestinal damage due to toxic or other
chemotherapeutic agents, and short bowel syndrome.
23. A method of identifying intestinotrophic analogs of GLP-2,
comprising the steps of: a) obtaining a GLP-2 analog according to
claim 1; b) treating a mammal with said sing a regimen capable of
eliciting an intestinotrophic effect when utilized for rat GLP-2;
and c) determining the effect of said analog on small bowel weight
relative to a mock treated control mammal, whereby said
intestinotrophic analog of GLP-2 is identified as an analog which
elicits an increase in said weight.
24. An intestinotrophic GLP-2 analog selected from the group
consisting of: ratGLP-2(4-33) Ac-ratGLP-2(1-33); ratGLP-2(1-30);
ratGLP-2(1-25); ratGLP-2(1-33)amide;
[Arg-.sup.2,Arg-.sup.1]ratGLP-2 (2-33); [Pro.sup.1]hGLP-2 (1-33);
[Gln.sup.20]hGLP-2(1-33); [Asp.sup.1]hGLP-2 (1-33);
[Tyr.sup.34]hGLP-2(1-34); [desNH2Tyr.sup.1]hGLP-2 (1-33);
[Thr.sup.5]hGLP-2(1-33); [ser.sup.16,
Arg.sup.17,Arg.sup.18]hGLP-2(1-33); [Agm.sup.34]hGLP-2(1-34);
[Arg.sup.30]hGLP-2(1-33); [Ala.sup.5, Ala.sup.7]hGLP-2 (1-33);
[Glu.sup.33)hGLP-2(1-33); [Phe.sup.25]hGLP-2(1-33); and
[Tyr.sup.25]hGLP-2(1-33).
25. A pharmaceutical composition comprising a therapeutically
effective amount of a GLP-2 analog according to claim 24, and a
pharmaceutically acceptable carrier.
26. A method for promoting growth of small bowel tissue in a
patient in need thereof, comprising the step of delivering to the
patient the pharmaceutical composition of claim 25.
27. A method for treating a gastrointestinal disease, wherein the
method comprises administering to a patient having the
gastrointestinal disease a therapeutically effective amount of a
GLP-2 analog according to claim 24, together with a
pharmaceutically acceptable carrier to reduce a pathological effect
or symptom of the gastrointestinal disease.
28. The method of claim 27, wherein the gastrointestinal disease is
selected from the group consisting of ulcers, digestion disorders,
malabsorption syndromes, short-gut syndrome, cul-de-sac syndrome,
inflammatory bowel disease, celiac sprue, tropical sprue,
hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's
disease), small intestinal damage due to toxic or other
chemotherapeutic agents, and short bowel syndrome.
29. The peptide [Gly.sup.2]hGLP-2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 08/631,273, filed Apr. 12, 1996, and application Ser. No.
08/632,533, filed Apr. 12, 1996, and a continuation-in-part of
application Ser. No. 08/422,540, filed Apr. 14, 1995, the
disclosures of which are incorporated by reference herein.
FIELD OF THE INVENTION
[0002] This invention relates to glucagon-related peptides having
intestinal tissue growth promoting properties, and to their use
therapeutically to treat various medical conditions resulting from
the impaired growth or loss of such tissue.
BACKGROUND TO THE INVENTION
[0003] Expression of the glucagon gene yields a tissue-determined
variety of peptide products that are processed from the 160 residue
proglucagon product. The organization of these peptides within the
proglucagon precursor was elucidated by the molecular cloning of
preproglucagon cDNAs from the rat, hamster and bovine pancreas.
These analyses revealed that preproglucagon contains not only the
sequence of glucagon and glicentin, but also two additional
glucagon-like peptides (GLP-1 and GLP-2) separated from glucagon
and each other by two spacer or intervening peptides (IP-I and
IP-II). These peptides are flanked by pairs of basic amino acids,
characteristic of classic prohormone cleavage sites, suggesting
they might be liberated after posttranslational processing of
proglucagon (Drucker, Pancreas, V1990, 5(4):484).
[0004] Analysis of the peptides liberated from proglucagon in the
pancreatic islets of Langerhans, for instance, suggests the primary
pancreatic peptide liberated is the 29-mer glucagon, whereas
glicentin, oxyntomodulin, IP-II and the glucagon-like peptides are
more prevalent in the small and large intestines. This
demonstration that the glucagon-like peptides are found in the
bowel has prompted research into the precise structure and putative
function(s) of these newly discovered gut peptides. Most studies
have focussed on GLP-1, because several lines of evidence suggested
that GLP-1 may be an important new regulatory peptide. Indeed, it
has been determined that GLP-1 is one of the most potent known
peptidergic stimulus for insulin release, an action mediated in a
glucose-dependent manner through interaction with receptors on
pancreatic B cells. GLP-1 and its derivatives are in development
for use in the treatment of diabetics.
[0005] The physiological roles of glicentin and oxyntomodulin, the
so-called "enteroglucagons", are also under investigation,
particularly with respect to regulation of acid secretion and the
growth of intestinal cells. Oxyntomodulin is capable of inhibiting
pentagastrin-stimulated gastric acid secretion in a dose-dependent
manner. The role of glicentin in mediating the changes of
intestinal adaptation and growth of the intestinal mucosa has been
investigated, and the intestinotrophic effect of glicentin and its
therapeutic use have recently been reported by Matsuno et al in EP
612,531 published Aug. 31, 1994.
[0006] In contrast to GLP-1 and other glucagon-related peptides,
the physiological role of glucagon-like peptide GLP-2 remains
poorly understood despite the isolation and sequencing of various
GLP-2 homologues including human, rat, bovine, porcine, guinea pig
and hamster. Using GLP-2 antisera raised against synthetic versions
of GLP-2, various groups have determined that GLP-2 is present
primarily in intestinal rather than pancreatic extracts (see Mojsov
et al, J. Biol. Chem., 1986, 261(25):11880; Orskov et al in
Endocrinology, 1986, 119(4):1467 and in Diabetologia, 1987, 30:874
and in FEBS Letters, 1989, 247(2):193; George et al, FEBS Letters,
1985, 192(2):275). With respect to its biological role, Hoosein et
al report (FEBS Letters, 1984, 178(1):83) that GLP-2 neither
competes with glucagon for binding to rat liver and brain tissues,
nor stimulates adenylate cyclase production in liver plasma
membranes, but, enigmatically, can stimulate adenylate cyclase in
both rat hyopthalamic and pituitary tissue, at 30-5 OpM
concentrations. An elucidation of the physiological role of GLP-2
would clearly be desirable.
SUMMARY OF THE INVENTION
[0007] There have now been discovered analogs of GLP-2 which
promote growth of small bowel tissue. It is accordingly a general
object of the present invention to provide such GLP-2 analogs and
to provide for their use therapeutically and for related
purposes.
[0008] In one aspect of the invention, the GLP-2-analogs exhibit
intestinotrophic activity and conform to the structural Formula
1:
R1-(Y1)m-X1-X2-X3-X4-Ser5-Phe6-Ser7-Asp8- (P1)
-Leul4-Asp15-Asn16-Leu17-Al-
a18-X19-X20-Asp21-Phe22-(P2)-Trp25-Leu26-Ile27-Gln28-Thr29-Lys30-(P3)-(Y2)-
n-R2,
[0009] wherein
[0010] X1 is His or Tyr
[0011] X2 is Ala or an Ala-replacement amino acid conferring on
said analog resistance to DPP-IV enzyme;
[0012] X3 is Pro, HPro, Asp or Glu;
[0013] X4 is Gly or Ala;
[0014] P1 is Glu-X10-Asn-Thr-Ile or Tyr-Ser-Lys-Tyr;
[0015] X10 is Met or an oxidatively stable Met-replacement amino
acid;
[0016] X19 is Ala or Thr;
[0017] X20 is Arg, Lys, His or Ala;
[0018] P2 is Ile-Asn, Ile-Ala or Val-Gln;
[0019] P3 is a covalent bond, or is Ile, Ile-Thr or
Ile-Thr-Asn;
[0020] R1 is H or an N-terminal blocking group;
[0021] R2 is OH or a C-terminal blocking group;
[0022] Y1 is one or two basic amino acids selected from the group
Arg, Lys, and His;
[0023] Y2 is one or two basic amino acids selected from the group
Arg, Lys, and His; and
[0024] m and n, independently, are 0 or 1; and
[0025] wherein at least one of X1, X2, X3, X4, P1, X10, X19, X20,
P2 and P3 is other than a wild type, mammalian GLP-2 residue.
[0026] Particularly preferred analogs according to Formula 1 are
those which are rendered resistant to cleavage by human DPP-IV
enzyme by replacing the Ala at position X2 with an alternative
amino acid. Other analogs of the invention are those which replace
the oxidatively sensitive Met at position X10 with an amino acid
residue which is oxidatively stable. In this manner, the analog
peptides have increased stability compared to GLP-2 peptides with
the wild-type Met residue at this position. Yet another preferred
embodiment of the invention is the incorporation at position X20 of
a basic amino acid selected from His or Lys. This substitution is
advantageous when the GLP-2 analogs are chemically synthesized. The
Arg residue which normally occurs at this position tends to
strongly bind solvents used in peptide synthesis procedures.
Substitution of the Arg allows easier formulation of the
synthetically produced GLP-2 analogs into pharmaceutically
acceptable composistions.
[0027] More particularly, and according to one aspect of the
invention, there are provided analogs of a GLP-2 peptide selected
from a mammalian GLP-2 species and N- and/or C-terminally modified
forms thereof, the analogs having intestinotrophic activity and
incorporating, relative to said mammalian GLP-2 peptide, at least
one amino acid substitution at a position which is conserved in
mammalian GLP-2's. In a preferred aspect, the GLP-2 analogs
incorporate a substitution selected from:
[0028] 1) incorporation at position 2 or at position 3 a
replacement amino acid conferring on said analog resistance to
Dipeptidyl Peptidase-IV (hereinafter referred to as DPP-IV);
and
[0029] 2) incorporation at position 10 of an oxidatively stable
Met-replacement amino acid; and
[0030] 3) incorporation at X20 of a replacement amino acid other
than Arg.
[0031] In still another of its aspects, the invention provides
intestinotrophic analogs of a mammalian GLP-2, preferably human
GLP-2, in which either the N- and/or C-terminus is modified by a
blocking group, or additional amino acids, or the substitution of a
modified or alternative amino acid. In yet another aspect, there
are provided intestinotrophic analogs of mammalian GLP-2,
preferably human GLP-2, in which positions X1, X5, X7, X16-X18,
X25, X30, or X33 incorporate a replacement amino acid other than
that found in naturally occurring mammalian GLP-2's. Optionally, in
a further aspect, the invention provides intestinotrophic analogs
in which from 4 to 8 residues are deleted from the C-terminus.
[0032] In another of its aspects, the invention provides a
pharmaceutical composition comprising a GLP-2 analog of the present
invention in a therapeutically effective amount, and preferably in
an intestinotrophic amount, and a pharmaceutically acceptable
carrier.
[0033] In a further aspect, the invention provides a method for
promoting growth of small bowel tissue in a patient in need
thereof, comprising the step of delivering to the patient an
intestinotrophic amount of a GLP-2 analog of the present
invention.
[0034] Besides promoting bowel growth, in another of its aspects
the invention provides a method for treating a gastrointestinal
disease by administering to a patient suffering from
gastrointestinal disease a therapeutically effective amount of a
GLP-2 analog of the invention, together with a pharmaceutically
acceptable carrier, in order to reduce a pathological effect or
symptom of the gastrointestinal disease.
[0035] In still another aspect of the invention, there is provided
a method useful to identify intestinotrophic analogs of GLP-2,
comprising the steps of:
[0036] 1) obtaining a GLP-2 analog conforming to Formula 1
represented above;
[0037] 2) treating a mammal with said analog using a regimen
capable of eliciting an intestinotrophic effect when utilized for
rat GLP-2; and
[0038] 3) determining the effect of said analog on small bowel
weight relative to a mock treated control mammal, whereby said
intestinotrophic analog of GLP-2 is identified as an analog which
elicits an increase in said weight.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The invention relates to therapeutic and related uses of
GLP-2 analogs, particularly for promoting growth of tissue of the
small bowel. The effect on growth elicited by the present GLP-2
analogs manifests as an increase in small bowel weight, relative to
a mock-treated control. In particular, GLP-2 analogs are considered
to have "intestinotrophic" activity if, when assessed in the murine
model exemplified herein, the analog mediates an increase in small
bowel weight of at least 10% relative to a control animal receiving
vehicle alone. Particularly suitable for therapeutic use are those
analogs which mediate an increase of at least 20% in small bowel
weight; preferred for therapeutic use are those which mediate an
increase in small bowel weight of 50% or more. Intestinotrophic
activity is noted most significantly in relation to the jejunum,
including the distal jejunum and particularly the proximal jejunum,
and is also noted in the ileum.
[0040] In addition to exhibiting intestinotrophic activity as just
defined, the GLP-2 analogs of the present invention incorporate an
amino acid substitution at one or more sites within a GLP-2 peptide
"background", which is either a mammalian GLP-2 species per se, or
is a variant of a mammalian GLP-2 species in which the C-terminus
and/or the N-terminus has been altered by addition of one or two
basic residues, or has been modified to incorporate a blocking
group of the type used conventionally in the art of peptide
chemistry to protect peptide termini from undesired biochemical
attack and degradation in vivo. Thus, the present peptides
incorporate an amino acid substitution in the context of any
mammalian GLP-2 species, including but not limited to human GLP-2,
bovine GLP-2, rat GLP-2, degu GLP-2, ox GLP-2, porcine GLP-2,
guinea pig GLP-2 and hamster GLP-2, the sequences of which have
been reported by many authors, including Buhl et al, J. Biol.
Chem., 1988, 263(18):8621.
[0041] In one aspect of the invention, the intestinotrophic analogs
of GLP-2 conform to the sequence of Formula 1 as follows:
R1-(Y1)m-X1-X2-X3-X4-Ser5-Phe6-Ser7-Asp8-(P1)-Leu14-Asp15-Asn16-Leu17-Ala1-
8-X19-X20-Asp21-Phe22-(P2)-Trp25-Leu26-Ile27-Gln-28-Thr29-Lys30-(P3)-(Y2)n-
-R2,
[0042] wherein
[0043] X1 is His or Tyr
[0044] X2 is Ala or an Ala-replacement amino acid conferring on
said analog resistance to DPP-IV enzyme;
[0045] X3 is Pro, HPro, Asp or Glu;
[0046] X4 is Gly or Ala;
[0047] P1 is Glu-XlO-Asn-Thr-Ile or Tyr-Ser-Lys-Tyr;
[0048] X10 is Met or an oxidatively stable Met-replacement amino
acid;
[0049] X19 is Ala or Thr;
[0050] X20 is Arg, Lys, His or Ala;
[0051] P2 is Ile-Asn, Ile-Ala or Val-Gln;
[0052] P3 is a covalent bond, or is Ile, Ile-Thr or
Ile-Thr-Asn;
[0053] R1 is H or an N-terminal blocking group;
[0054] R2 is OH or a C-terminal blocking group;
[0055] Y1 is one or two basic amino acids selected from the group
Arg, Lys, and His;
[0056] Y2 is one or two basic amino acids selected from the group
Arg, Lys, and His; and
[0057] m and n, independently, are 0 or 1; and
[0058] wherein at least one of X1, X2, X3, X4, P1, X10, X19, X20,
P2 and P3 is other than a wild type, mammalian GLP-2 residue.
[0059] Wild-type mammalian GLP-2 residues which occur at a specific
position are determined by aligning the sequences of GLP-2's
isolated from different mammalian species and comparing the
sequence to the human sequence, reproduced below, for
convenience:.
[0060] His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-1 5 10
[0061] Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-15 20
[0062] Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp 25 30
[0063] The amino acid residues which, for purposes of this
application, are known to occur at specific positions in wild type
mammalian GLP-2's are the following: position X13 may be Ile or
Val; Position X16 may be Asn or Ser; position X19 may be Alanine or
Threonine; position X20 may be Arg or Lys; position X27 may be Ile
or Leu; and position X28 may be Gln or His.
[0064] The present GLP-2 analogs may incorporate desired amino acid
substitutions into a "background" which is an N-terminally or
C-terminally modified form of a mammalian GLP-2 peptide. Such
analogs are represented in Formula 1 as those in which R1
constitutes an N-terminal blocking group, and/or when m is 1 then
Y1 is one or two basic amino acids such as Arg or Lys; and/or R2 is
a C-terminal blocking group; and/or when n is 1 then Y2 is
independently, one or two basic amino acids such as Arg or Lys.
[0065] In preferred embodiments of the invention, the GLP-2 analog
is an analog of full length GLP-2, i.e., GLP-2(1-33), and P3 is
accordingly the sequence Ile-Thr-Asn. Alternatively, the GLP-2
analogs may be C-terminally truncated, to yield GLP-2(1-32) forms
in which P3 is Ile-Thr, or GLP-2(1-31) forms in which P3 is Ile, or
GLP-2(1-30) forms in which P3 is a covalent bond.
[0066] The "blocking groups" represented by R1 and R2 are chemical
groups that are routinely used in the art of peptide chemistry to
confer biochemical stability and resistance to digestion by
exopeptidase. Suitable N-terminal protecting groups include, for
example, C.sub.1-5alkanoyl groups such as acetyl. Also suitable as
N-terminal protecting groups are amino acid analogues lacking the
amino function, for example, desamino-Tyrl. Suitable C-terminal
protecting groups include groups which form ketones or amides at
the carbon atom of the C-terminal carboxyl, or groups which form
esters at the oxygen atom of the carboxyl. Ketone and ester-forming
groups include alkyl groups, particularly branched or unbranched
C.sub.1-5alkyl groups, e.g., methyl, ethyl and propyl groups, while
amide-forming groups include amino functions such as primary amine,
or alkylamino functions, e.g., mono-C.sub.1-5salkylamino and
di-C.sub.1-5alkylamino groups such as methylamino, ethylamino,
dimethylamino, diethylamino, methylethylamino and the like. Amino
acid analogues are also suitable for protecting the C-terminal end
of the present compounds, for example, decarboxylated amino acid
analogues such as agmatine.
[0067] Embodiments of the invention specifically include such
analogs in which m is 0 and R1 is a blocking group such as acetyl;
and analogs in which m is 0 and R2 is a C-terminal blocking group
such as an amide or an amine, e.g., -NH2.
[0068] In a preferred aspect of the invention, the GLP-2 analogs
are analogs of either human GLP-2 or of rat GLP-2. Human GLP-2 is
herein referred to interchangably as hGLP-2(1-33). Rat GLP-2 has
the amino acid sequence of human GLP-2, but incorporates at
position 19 a Thr residue instead of an Ala residue. Rat GLP-2 is
accordingly referenced herein either as rGLP-2(1-33) or as the
Thr.sup.19 analog of human GLP-2, i.e., as
[Thr.sup.19]hGLP-2(1-33).
[0069] In particularly preferred embodiments of the invention, with
respect to both the Formula 1 analogs and the more specific human
or rat GLP-2 analogs, the GLP-2 analogs incorporate an amino acid
substitution selected from:
[0070] 1) incorporation at X2 and/or at X3 of a replacement amino
acid which renders said analog resistant to cleavage by DPP-IV
enzyme;
[0071] 2) incorporation at X10 of an oxidatively stable
Met-replacement amino acid; and
[0072] 3) incorporation at X20 of a replacement amino acid other
than Arg.
[0073] In still another of its aspects, the invention provides
intestinotrophic analogs of a mammalian GLP-2, preferably human
GLP-2, in which either the N- and/or C-terminus is modified by a
blocking group, or additional amino acids, or the substitution of a
modified or alternative amino acid. In yet another aspect, there
are provided intestinotrophic analogs of mammalian GLP-2,
preferably human GLP-2, in which positions X1, X5, X7, X16-X18,
X25, X30, or X33 incorporate a replacement amino acid other than
that found in naturally occurring mammalian GLP-2's. Optionally, in
a further aspect, the invention provides intestinotrophic analogs
in which from 4 to 8 residues are deleted from the C-terminus.
[0074] The DPP-IV-resistant class of GLP-2 analogs possess
particularly advantageous properties. As is demonstrated herein,
mammalian GLP-2 species have been found to be sensitive to cleavage
by DPP-IV enzyme. It has also been found that this sensitivity to
DPP-IV is the result of the recognition sequence Ala.sup.2
Asp.sup.3 found in all mammalian forms of GLP-2. There are
accordingly provided by the present invention a class of GLP-2
analogs which incorporate at X2 and/or X3 a replacement amino acid
which confers on the GLP-2 analog relative resistance to DPP-IV
mediated cleavage, as determined by any convenient in vitro or in
vivo assessment technique that is able to detect the presence of
GLP-2 digestion products. A DPP-IV resistant GLP-2 analog is
revealed as that GLP-2 analog which is processed or degraded at a
rate that is measurably slower than the rate at which human GLP-2
is processed or degraded, under the same conditions.
[0075] An assay suitable for assessing DPP-IV sensitivity and
resistance is described below in Example 3, in the context of
results actually obtained.
[0076] The X2 class of GLP-2 analogs is preferred herein. These
Ala.sup.2-substituted GLP-2 analogs can incorporate at X2 a
structurally wide variety of Ala-replacement amino acids to achieve
relative resistance to DPP-IV digestion. A similarly wide variety
of Ala-replacement amino acids allow also for the retention by the
analog of intestinotrophic activity. For purposes of identifying
those DPP-IV-resistant X2 analogs that also retain intestinotrophic
activity, the X2 analogs showing DPP-IV resistance are screened in
the assay of intestinotrophic activity described below in Example
4.
[0077] In embodiments of the present invention, the Ala.sup.2
replacements include stereoisomers of amino acids that would
otherwise be substrates for DPP-IV, for example D-Ala, D-HPr,
.beta.Ala, t-butyl-Gly, L-penicillamin, .alpha.-aminobutyric acid,
aminoisobutyric acid, norvaline, L-phenyl-Gly, and D-Pro, and
naturally occurring amino acids, for example, Glu, Lys, Ile, Ser,
Asp, Phe, Lys, Met, Asn, Pro, Gln, Thr, Tyr, Gly and Val. In
specific embodiments of the invention, there are provided the
following Ala.sup.2-substituted GLP-2 analogs:
[D-Ala.sup.2]rGLP-2(1-33), [Gly.sup.2]rGLP-2(1-33),
[Val.sup.2]rGLP-2(1-33) [Gly.sup.2]hGLP-2(1-33),
[tBuGly.sup.2]hGLP-2, [ASP.sup.2]hGLP-2(1-33),
[Glu.sup.2]hGLP-2(1-33), [Phe.sup.2]hGLP-2(1-33)- ,
[His.sup.2]hGLP-2(1-33) , [Ile.sup.2]hGLP-2(1-33),
[Lys.sup.2]hGLP-2(1-33), [Met.sup.2]hGLP-2(1-33),
[Asn.sup.2]hGLP-2(1-33)- , [Pro.sup.2]hGLP-2(1-33),
[Gln.sup.2]hGLP-2(1-33), [Ser.sup.2]hGLP-2(1-33),
[Thr.sup.2]hGLP-2(1-33), [Val.sup.2]hGLP-2(1-33)- ,
[Tyr.sup.2]hGLP-2(1-33), [D-Ala.sup.2]hGLP-2(1-33),
[Pen.sup.2]hGLP-2(1-33), [bAla.sup.2]hGLP-2(1-33),
[aAbu.sup.2]hGLP-2(1-33), [Nval.sup.2]hGLP-2(1-33),
[PhGly.sup.2]hGLP-2(1-33), and [Aib.sup.2]hGLP-2(1-33).
[0078] The X2 GLP-2 analogs may incorporate amino acid replacements
at other positions. In embodiments of the invention, such analogs
include those carrying amino acid substitutions also at one or more
of positions X1, X3, X4, X10, X19, X20 and X24, and therefore
include those which, according to Formula 1, include at least one
of the following substitutions: X1 is Tyr; X3 is Glu; X4 is Ala; P1
is Glu-X10-Asn-Thr-Ile where X10 is other than Met or P1 is
Tyr-Ser-Lys-Tyr; X10 is an oxidatively stable Met-replacement amino
acid; X19 is Thr; X20 is Lys or Ala; P2 is Val-Gln and P3 is a
covalent bond, Ile, or Ile-Thr or Ile-Thr-Asn.
[0079] In embodiments of the present invention, the X2 analogs of
GLP-2 include those which also incorporate one of the following
substitutions: X1 is Ala; X3 is Ala; X4 is Ala; X10 is an
oxidatively stable Met-replacement amino acid such as Val, Ile,
Asn, Glx, Tyr, Phe, and preferably Leu, Nle, Ala, Ser, and Gly; and
P2 is Ile-Ala. In specific embodiments of the invention, there are
provided the following GLP-2 analogs:
[D-Ala.sup.2Thr.sup.19]hGLP-2(1-33); [Gly.sup.2Thr.sup.19]hGLP-2
(1-33); [Val.sup.2Thr.sup.19]hGLP-2 (1-33);
(Gly.sup.2Ala.sup.24]hGLP-2(1- -33); [Gly.sup.2Ala.sup.10]hGLP-2
(1-33); [Ala.sup.1Gly.sup.2]hGLP-2(1-33)- ;
(Gly.sup.2Ala.sup.3]hGLP-2(1-33) and
[Gly.sup.2Ala.sup.4]hGLP-2(1-33).
[0080] In a related embodiment, the X2 analogs include those which
include the following substitutions:
GLP-2[Gly.sup.2Ala.sup.8]hGLP-2; [Gly.sup.2Ala.sup.11]hGLP-2;
[Gly.sup.2Ala.sup.21]hGLP-2; [Gly.sup.2Ala.sup.9]hGLP-2;
[Gly.sup.2Ala.sup.16]hGLP-2; [Gly.sup.2Ala.sup.17]hGLP-2;
[Gly.sup.2Ala.sup.28]hGLP-2; [Gly.sup.2Ala.sup.5]hGLP-2;
[Gly.sup.2Ala.sup.31]hGLP-2; [Gly.sup.2Ala.sup.27]hGLP-2;
[Gly.sup.2Ala.sup.12]hGLP-2; [Gly.sup.2Ala.sup.13]hGLP-2;
[Gly.sup.2Ala.sup.7]hGLP-2; [Gly.sup.2Ala.sup.6]hGLP-2;
[Ser.sup.2,Gln.sup.3]hGLP-2; (1-33); [Gly.sup.2, Ala.sup.25]hGLP-2;
(1-33); [Gly.sup.2, Ala.sup.26]hGLP-2; (1-33); [Gly.sup.2,
Ala.sup.14]hGLP-2 (1-33); [Gly.sup.2, Ala.sup.23]hGLP-2 (1-33);
[Gly.sup.2, Ala.sup.30]hGLP-2(1-33); [Tyr.sup.1,
Gly.sup.26]hGLP-2(1-33); [Gly.sup.2, Ala.sup.34]hGLP-2(1-33); and
[Gly.sup.2, Tyr.sup.34]hGLP-2(1-34);
[0081] Alternatively, in the case where X2 is Ala, hPr or Pro,
DPP-IV resistance can be conferred by replacing Asp.sup.3 with an
Asp-replacement amino acid, X3 which is Pro or hPr.
[0082] The present invention also provides, as another class of
GLP-2 analogs, those analogs in which X10 represents an oxidatively
stable, Met-replacement amino acid. It has been found that the
intestinotrophic activity of mammalian GLP-2 is reduced when the
Met in position 10 is oxidized, and also that intestinotrophic
activity is retained when replacement amino acids insensitive to
oxidation are substituted for Met.sup.10 replacement. Such
Met.sup.10-substituted GLP-2 analogs are accordingly more stable
during synthesis, work-up and storage. In embodiments of the
present invention, such X10 analogs of GLP-2 are analogs of human
GLP-2. In a specific embodiments of the invention, such analogs
include: [Ser.sup.10]hGLP-2(1-33); [Nle.sup.10]hGLP-2(1-33);
[Ala.sup.10]hGLP-2(1-33); [Leu.sup.10]rGLP-2(1-33);
[Met(O).sup.10]ratGLP-2(1-33); and [Nle.sup.10]ratGLP-2 (1-33).
[0083] The X10 analogs may also incorporate amino acid
substitutions at one or more positions other than X10. As noted
above, such analogs include, for instance, those incorporating
substitutions at X2, and those in which P1 is is Tyr-Ser-Lys-Tyr,
as well as those in which any one of the X substituents or the P
substituents represented in Formula 1 are other than wild type
residues or sequences. In specific embodiments, the GLP-2 analogs
include [Gly.sup.2Ala.sup.10]hGLP-2(1-33) and
[Tyr.sup.9Ser.sup.10Lys.sup.11Tyr.sup.12desIle.sup.13]hGLP-2(1-33).
[0084] In other embodiments of the invention, the GLP-2 analogs
incorporate single amino acid substitutions in the context of the
mammalian GLP-2 peptide background in which they are introduced.
Such analogs include, for example, those mammalian GLP-2 analogs
and particularly the human GLP-2 analogs in which X1 is Tyr; X3 is
Glu; X4 is Ala; P1 is Tyr-Ser-Lys-Tyr-(desIle); P2 is Ile-Ala or
Val-Gln; and P3 is a covalent bond, or is Ile or Ile-Thr.
[0085] Still other GLP-2 analogs of the invention incorporate
alterations of the N- or C-terminus by the addition of amino acids,
by blocking groups, or by substituted amino acids. There are also
provided intestinotrophic analogs of mammalian GLP-2, preferably
human GLP-2, in which positions X1, X5, X7, X16-X18, X25, X30, or
X33 incorporate a replacement amino acid other than that found in
naturally occurring mammalian GLP-2's. optionally, the invention
provides intestinotrophic analogs in which from 4 to 8 residues are
deleted from the C-terminus. Specific embodiments of such
intestinotrophic analogs include: Ac-ratGLP-2(1-33);
ratGLP-2(1-30); ratGLP-2(1-25); ratGLP-2(1-33)amide; [Arg-.sup.2,
Arg-.sup.1]ratGLP-2(2-33) [Pro.sup.1]hGLP-2(1-33);
[Gln.sup.20]hGLP-2(1-33); [Asp.sup.1]hGLP-2(1-33);
[Tyr.sup.34]hGLP-2(1-34); [desNH2Tyr.sup.1]hGLP-2(1-33);
[Thr.sup.5]hGLP-2(1-33);
[Ser.sup.16,Arg.sup.17,Arg.sup.18]hGLp-2(1-33);
[Agm.sup.34]hGLP-2(1-34); [Arg.sup.30]hGLP-2(1-33); [Ala.sup.5,
Ala.sup.7]hGLP-2(1-33); [Glu.sup.33]hGLP-2(1-33);
(Phe.sup.25]hGLP-2(1-33- ); and [Tyr.sup.25]hGLP-2(1-33).
[0086] The present GLP-2 analogs can be synthesized using standard
techniques of peptide chemistry and can be assessed for
intestinotrophic activity, all according to the guidance provided
herein. With respect to synthesis, the selected GLP-2 analog can be
prepared by a variety of techniques well known for generating
peptide products. Those GLP-2 analogs that incorporate only L-amino
acids can be produced in commercial quantities by application of
recombinant DNA technology. For this purpose, DNA coding for the
desired GLP-2 analog is incorporated into an expression vector and
transformed into a microbial, e.g., yeast, or other cellular host,
which is then cultured under conditions appropriate for GLP-2
expression. A variety of gene expression systems have been adapted
for this purpose, and typically drive expression of the desired
gene from expression regulatory elements used naturally by the
chosen host. Because GLP-2 does not require post translational
glycosylation for its activity, its production may most
conveniently be achieved in bacterial hosts such as E. coli. For
such production, DNA coding for the selected GLP-2 peptide may
usefully be placed under expression controls of the lac, trp or PL
genes of E. coli. As an alternative to expression of DNA coding for
the GLP-2 per se, the host can be adapted to express GLP-2 peptide
as a fusion protein in which the GLP-2 is linked releasably to a
carrier protein that facilitates isolation and stability of the
expression product.
[0087] In an approach universally applicable to the production of a
selected GLP-2 analog, and one used necessarily to produce GLP-2
analogs that incorporate non-genetically encoded amino acids and N-
and C-terminally derivatized forms, the well established techniques
of automated peptide synthesis are employed, general descriptions
of which appear, for example, in J. M. Stewart and J. D. Young,
Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical
Company, Rockford, Ill.; and in M. Bodanszky and A. Bodanszky, The
Practice of Peptide Synthesis, 1984, Springer-Verlag, N.Y.; Applied
Biosystems 430A Users Manual, 1987, ABI Inc., Foster City, Calif.
In these techniques, GLP-2 analog is grown from its C-terminal,
resin-conjugated residue by the sequential addition of
appropriately protected amino acids, using either the Fmoc or tBoc
protocols, as described for instance by Orskov et al, 1989,
supra.
[0088] For the incorporation of N- and/or C- blocking groups,
protocols conventional to solid phase peptide synthesis methods can
also be applied. For incorporation of C-terminal blocking groups,
for example, synthesis of the desired peptide is typically
performed using, as solid phase, a supporting resin that has been
chemically modified so that cleavage from the resin results in a
GLP-2 peptide having the desired C-terminal blocking group. To
provide peptides in which the C-terminus bears a primary amino
blocking group, for instance, synthesis is performed using a
p-methylbenzhydrylamine (MBHA) resin so that, when peptide
synthesis is completed, treatment with hydrofluoric acid releases
the desired C-terminally amidated peptide. Similarly, incorporation
of an N-methylamine blocking group at the C-terminus is achieved
using N-methylaminoethyl-derivatized DVB resin, which upon HF
treatment releases peptide bearing an N-methylamidated C-terminus.
Protection of the C-terminus by esterification can also be achieved
using conventional procedures. This entails use of resin/blocking
group combination that permits release of side-chain protected
peptide from the resin, to allow for subsequent reaction with the
desired alcohol, to form the ester function. FMOC protecting
groups, in combination with DVB resin derivatized with
methoxyalkoxybenzyl alcohol or equivalent linker, can be used for
this purpose, with cleavage from the support being effected by TFA
in dichloromethane. Esterification of the suitably activated
carboxyl function, e.g., with DCC, can then proceed by addition of
the desired alcohol, followed by deprotection and isolation of the
esterified GLP-2 peptide.
[0089] Incorporation of N-terminal blocking groups can be achieved
while the synthesized GLP-2 peptide is still attached to the resin,
for instance by treatment with suitable anhydride and nitrile. To
incorporate an acetyl blocking group at the N-terminus, for
instance, the resin-coupled peptide can be treated with 20% acetic
anhydride in acetonitrile. The N-blocked GLP-2 analog can then be
cleaved from the resin, deprotected and subsequently isolated.
[0090] Once the desired GLP-2 analog has been synthesized, cleaved
from the resin and fully deprotected, the peptide is then purified
to ensure the recovery of a single oligopeptide having the selected
amino acid sequence. Purification can be achieved using any of the
standard approaches, which include reversed-phase high-pressure
liquid chromatography (RP-HPLC) on alkylated silica columns, e.g.,
C.sub.4, C.sub.8-, or C.sub.18- silica. Such column fractionation
is generally accomplished by running linear gradients, e.g.,
10-90%, of increasing % organic solvent, e.g., acetonitrile, in
aqueous buffer, usually containing a small amount (e.g., 0.1%) of
pairing agent such as TFA or TEA. Alternatively, ion-exchange HPLC
can be employed to separate peptide species on the basis of their
charge characteristics. Column fractions are collected, and those
containing peptide of the desired/required purity are optionally
pooled. In one embodiment of the invention, the GLP-2 analog is
then treated in the established manner to exchange the cleavage
acid (e.g., TFA) with a pharmaceutically acceptable acid, such as
acetic, hydrochloric, phosphoric, maleic, tartaric, succinic and
the like, to generate a pharmaceutically acceptable acid addition
salt of the peptide.
[0091] For administration to patients, the GLP-2 analog or its salt
is desirably provided in pharmaceutically acceptable form, e.g., as
a preparation that is sterile-filtered, e.g., through a 0.22 .mu.
filter, and substantially pyrogen-free. Desirably, the GLP-2 analog
to be formulated migrates as a single or individualized peak on
HPLC, exhibits uniform and authentic amino acid composition and
sequence upon analysis thereof, and otherwise meets standards set
by the various national authorities which regulate quality of
pharmaceutical products.
[0092] For therapeutic use, the chosen GLP-2 analog is formulated
with a carrier that is pharmaceutically acceptable and is
appropriate for delivering the peptide by the chosen route of
administration. Suitable pharmaceutically acceptable carriers are
those used conventionally with peptide-based drugs, such as
diluents, excipients and the like. Reference may be made to
"Remington's Pharmaceutical Sciences", 17th Ed., Mack Publishing
Company, Easton, Pa., 1985, for guidance on drug formulations
generally. In one embodiment of the invention, the compounds are
formulated for administration by infusion, e.g., when used as
liquid nutritional supplements for patients on total parenteral
nutrition therapy, or by injection, e.g., sub-cutaneously,
intramuscularly or intravenously, and are accordingly utilized as
aqueous solutions in sterile and pyrogen-free form and optionally
buffered to physiologically tolerable pH, e.g., a slightly acidic
or physiological pH. Thus, the compounds may be administered in a
vehicle such as distilled water or, more desirably, in saline,
phosphate buffered saline or 5% dextrose solution. Water solubility
of the GLP-2 analog may be enhanced, if desired, by incorporating a
solubility enhancer, such as acetic acid.
[0093] The aqueous carrier or vehicle can be supplemented for use
as injectables with an amount of gelatin that serves to depot the
GLP-2 analog at or near the site of injection, for its slow release
to the desired site of action. Concentrations of gelatin effective
to achieve the depot effect are expected to lie in the range from
10-20%. Alternative gelling agents, such as hyaluronic acid, may
also be useful as depoting agents.
[0094] The GLP-2 analogs of the invention may also be formulated as
a slow release implantation device for extended and sustained
administration of GLP-2 analog. Examples of such sustained release
formulations include composites of biocompatible polymers, such as
poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose,
hyaluronic acid, collagen, and the like. The structure, selection
and use of degradable polymers in drug delivery vehicles have been
reviewed in several publications, including, A. Domb et al.,
Polymers for Advanced Technologies 3:279-292 (1992). Additional
guidance in selecting and using polymers in pharmaceutical
formulations can be found in the text by M. Chasin and R. Langer
(eds.), "Biodegradable Polymers as Drug Delivery Systems, " Vol. 45
of "Drugs and the Pharmaceutical Sciences," M. Dekker, N.Y., 1990.
Liposomes may also be used to provide for the sustained release of
a GLP-2 analog. Details concerning how to use and make liposomal
formulations of drugs of interest can be found in, among other
places, U.S. Pat. No 4,944,948; U.S. Pat. No. 5,008,050; U.S. Pat.
No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,452,747;
U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,311,712; U.S. Pat. No.
4,370,349; U.S. Pat. No. 4,372,949; U.S. Pat. No. 4,529,561; U.S.
Pat. No. 5,009,956; U.S. Pat. No. 4,725,442; U.S. Pat. No.
4,737,323; U.S. Pat. No. 4,920,016. Sustained release formulations
are of particular interest when it is desirable to provide a high
local concentration of a GLP-2 analog.
[0095] The GLP-2 analog can be utilized in the form of a
sterile-filled vial or ampoule, that contains an intestinotrophic
amount of the peptide, in either unit dose or multi-dose amounts.
The vial or ampoule may contain the GLP-2 analog and the desired
carrier, as an administration-ready formulation. Alternatively, the
vial or ampoule may contain the GLP-2 peptide in a form, such as a
lyophilized form, suitable for reconstitution in a suitable
carrier, such as phosphate-buffered saline.
[0096] As an alternative to injectable formulations, the GLP-2
analog may be formulated for administration by other routes. Oral
dosage forms, such as tablets, capsules and the like, can be
formulated in accordance with standard pharmaceutical practice.
According to the present invention, the GLP-2 analog is
administered to treat patients that would benefit from growth of
small bowel tissue. The effects of GLP-2 analog on this tissue, as
evidenced by the results exemplified herein, is dramatic and would
clearly benefit those patients suffering from diseases or
conditions marked by abnormalities in the small intestinal tract
mucosa, which include ulcers and inflammatory disorders; congenital
or acquired digestion and absorption disorders including
malabsorption syndromes; and diseases and conditions caused by loss
of small bowel mucosal function particularly in patients undergoing
extended parenteral feeding or who, as a result of surgery, have
undergone resection of the small bowel and suffer from short-gut
syndrome and cul-de-sac syndrome. Therapeutic treatment with GLP-2
analog is administered so as to reduce or eliminate the disease
symptoms and/or improve the nutritional status in these patients
associated with their reduced intestinal tract mucosal function.
For example, GLP-2 analog is administrated to a patient with an
inflammatory bowel condition in an amount sufficient to ameliorate
the intestinal discomfort and diarrhea caused by the condition.
Additionally, GLP-2 analog may be administered to patients with
malabsorption disorders so as to enhance the nutritional absorption
and thereby improve the nutritional status of such patients.
[0097] In general, patients who would benefit from increased small
intestinal mass and consequent increased small bowel mucosal
function are candidates for treatment with GLP-2 analog. Particular
conditions that may be treated with GLP-2 analog include the
various forms of sprue including celiac sprue which results from a
toxic reaction to .alpha.-gliadin from wheat, and is marked by a
tremendous loss of villae of the small bowel; tropical sprue which
results from infection and is marked by partial flattening of the
villae; hypogammaglobulinemic sprue which is observed commonly in
patients with common variable immunodeficiency or
hypogammaglobulinemia and is marked by significant decrease in
villus height. The therapeutic efficacy of the GLP-2 analog
treatment may be monitored by enteric biopsy to examine the villus
morphology, by biochemical assessment of nutrient absorption, by
patient weight gain, or by amelioration of the symptoms associated
with these conditions. Other conditions that may be treated with
GLP-2 analog, or for which GLP-2 analog may be useful
prophylactically, include radiation enteritis, infectious or
post-infectious enteritis, regional enteritis (Crohn's disease),
small intestinal damage due to toxic or other chemotherapeutic
agents, and patients with short bowel syndrome.
[0098] The therapeutic dosing and regimen most appropriate for
patient treatment will of course vary with the disease or condition
to be treated, and according to the patient's weight and other
parameters. The results presented hereinbelow demonstrate that a
dose of GLP-2 peptide equivalent to about 100 .mu.g/kg (or less)
administered twice daily over 10 days can generate very significant
increases in small bowel mass. It is expected that much smaller
doses, e.g., in the .mu.g/kg range, and shorter or longer duration
or frequency of treatment, will also produce therapeutically useful
results, i.e., a statistically significant increase particularly in
small bowel mass. Also, it is anticipated that the therapeutic
regimen will include the administration of maintenance doses
appropriate for reversing tissue regression that occurs following
cessation of initial treatment. The dosage sizes and dosing regimen
most appropriate for human use are guided by the results herein
presented, and can be confirmed in properly designed clinical
trials.
[0099] An effective dosage and treatment protocol may be determined
by conventional means, starting with a low dose in laboratory
animals and then increasing the dosage while monitoring the
effects, and systematically varying the dosage regimen as well.
Numerous factors may be taken into consideration by a clinician
when determining an optimal dosage for a given subject. Primary
among these is the amount of GLP-2 normally circulating in the
plasma, which is on the order of 151 pmol/ml in the resting state,
rising to 225 pmol/ml after nutrient ingestion for healthy adult
humans. Orskow, C. and Helst, J.J., 1987, Scand. J. Clin. Lav.
Invest. 47:165. Additional factors include the size of the patient,
the age of the patient, the general condition of the patient, the
particular disease being treated, the severity of the disease, the
presence of other drugs in the patient, the in vivo activity of the
GLP-2 analog and the like. The trial dosages would be chosen after
consideration of the results of animal studies and the clinical
literature. It will be appreciated by the person of ordinary skill
in the art that information such as binding constants and Ki
derived from in vitro GLP-2 binding competition assays may also be
used in calculating dosages, as well as the calculated half-life of
the GLP-2 analog in vivo.
[0100] A typical human dose of a GLP-2 peptide would be from about
10 .mu.g/kg body weight/day to about 10 mg/kg/day, preferably from
about 50 .mu.g/kg/day to about 5 mg/kg/day, and most preferably
about 100 .mu.g/kg/day to 1 mg/kg/day. As the GLP-2 analogs of the
invention can be up to 10 to even 100 times more potent than GLP-2,
a typical dose of such a GLP-2 analog may be lower, for example,
from about 100 ng/kg body weight/day to 1 mg/kg/day, preferably 1
.mu.g/kg/day to 500 .mu.g/kg/day, and even more preferably 1
.mu.g/kg/day to 100 .mu.g/kg/day.
[0101] Similarly, the intestinotrophic GLP-2 analogs of the
invention are also useful for both augmenting bowel growth and
treating inflammatory disorders in livestock and pets.
EXAMPLE 1
GLP-2 analog synthesis
[0102] Solid phase peptide synthesis (SPPS) is carried out manually
in a 300 milliliter (ml) vessel on a 3 millimole (mmole) scale
using 6 grams (g) of chloromethyl (Merrifield) resin (for
C-terminal free acid peptides) with a substitution of 0.5
milliequivalents (meq) per gram. Amino acids are protected at the
amino-terminus with the t-butyloxycarbonyl (tBoc) group. The
side-chains of trifunctional amino acids are protected with the
benzyl (Bz, for serine and threonine), benzyloxymethyl (BOM, for
histidine), 2-bromobenzyloxycarbonyl (2-BrZ, for tyrosine),
2-chlorobenzyloxycarbonyl (2-ClZ, for lysine), cyclohexyl (cHex,
for aspartic and glutamic acids), and tosyl (Tos, for arginine)
groups. The first amino acid is coupled to the chloromethyl resin
through esterification of the protected amino acid in the presence
of potassium fluoride (KF). C-terminal amide peptides are
synthesized on a 4-methylbenzhydrylamine (MBHA) resin on a 3 mmol
scale using 6 g of resin with a substitution of 0.5 meq/g. The
first amino acid is coupled to the MBHA resin according to the
procedure described for peptide elongation.
[0103] Amino-group deprotection is carried out using 50%
trifluoroacetic acid (TFA) in dichloromethane (CH.sub.2Cl.sub.2),
followed by neutralization using two washes of 10% triethylamine
(Et.sub.3N) in CH.sub.2Cl.sub.2. Peptide elongation is carried out
using N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole
(DCC/HOBt) activation in CH.sub.2Cl.sub.2/dimethylformamide (DMF).
The growing peptide chain is capped after each elongation step with
20% Ac.sub.2O in CH.sub.2Cl.sub.2. The peptide-resin is washed
after each elongation, capping and deprotection step with
isopropanol (iPrOH) and methanol (MeOH). The washes are repeated
once. N-terminal acetyl peptides are prepared by acetylation of the
terminal amino-group with 20% Ac.sub.2O in CH.sub.2Cl.sub.2 after
deprotection and neutralization as described. Resin-bound products
are routinely cleaved by a low-high procedure using hydrogen
fluoride (HF) containing dimethylsulfide (DMS) and p-cresol as
scavengers.
[0104] Crude peptides are purified by preparative high pressure
liquid chromatography (HPLC) using a Vydac C18, 15-20 .mu.m
wide-pore, 2 inch.times.12 inch, reverse-phase silica column using
gradient elution with 0.1% TFA in water modified with acetonitrile.
Elution is monitored at 220 nanometers (nm). Each fraction
collected is analyzed for purity by analytical HPLC using a Vydac
C18, 5 .mu.m, 4.6.times.254 millimeter (mm), reverse-phase silica
column by gradient elution using 0.1% TFA in water modified with
acetonitrile, and monitored at 215 nm. Fractions demonstrating
greater than 95% purity are combined and lyophilized. Acetate salts
of the peptides are prepared from the TFA salts by dissolution of
the lyophilized powder in water, with addition of acetonitrile to
aid in dissolution where necessary. The solution is passed through
a protonated Bio-Rex-70 cation exchange resin. The resin is washed
with 5 bed-volumes of water, and the resin-bound peptide is eluted
with 50% acetic acid in water. The eluent is diluted with water and
lyophilized.
[0105] The final lyophilized powder is analyzed for purity by two
analytical reverse-phase HPLC methods using a Vydac C18, 5 .mu.m,
4.6.times.254 mm reverse-phase silica column. The two solvent
systems used are a gradient of water adjusted to pH 2.25 with
triethylamine phosphate, modified with acetonitrile, and a gradient
of 0.1% TFA in water, modified with acetonitrile. The column eluent
is monitored at 215 nm. The identity of each product is confirmed
by amino acid analysis and by electrocopy-mass spectroscopy.
[0106] The GLP-2 analogs are next formulated as described below in
Example 2. Each of the GLP-2 analogs is fully soluble in water at
room temperature unless otherwise noted.
EXAMPLE 2
GLP-2 analog formulation
[0107] The GLP-2 analogs were formulated for injection either in
phosphate buffered saline or as a gelatin-containing depot
formulation. For the PBS-formulated GLP-2 analog preparations, a
10X stock PBS solution was first prepared, using 80 g NaCl (BDH ACS
783), 2 g KCl (BDH ACS 645), 11.5 g Na.sub.2HPO.sub.4 (Anachemia
AC-8460), and 2 g KH.sub.2PO.sub.4 (Malinckrodt AR7100), which was
brought to a total volume of one litre with sterile distilled
water. The final working solution was obtained by 10:1 dilution of
the stock solution with sterile distilled water and adjusted to pH
7.3-7.4 if necessary, using sufficient volumes of 10 N Na OH. The
working solution was then autoclaved for 30 minutes. In the final
working PBS solution, concentrations were 137 mM NaCl, 2.7 mM KCl,
4.3 mM Na.sub.2HPO.sub.4.7H20, and 1.4 mM KH.sub.2PO.sub.4.
[0108] The GLP-2 analog, as a powdered peptide, is added to the
working PBS solution as required to generate formulations having
the desired peptide concentrations. For example, to generate a PBS
solution of GLP-2 analog at 130 mg/l, 5.2 mg of GLP-2 analog is
dissolved in 40 ml of PBS to yield a GLP-2 concentration of 130
.mu.g/ml, 0.5 ml is injected twice daily.
[0109] To generate the gelatin-based GLP-2 analog formulations, a
gelatin solution was first prepared by dissolving 12 grams of
gelatin (Sigma, G-8150 Lot #54H07241 Type A from Porcine skin
[9000-70-8] .about.300 Bloom) in 100 ml distilled water. The
gelatin solution was then autoclaved, warmed at 37.degree. C., and
the GLP-2 peptide previously dissolved in phosphate buffered saline
as described above was then added to achieve specific, desired
peptide concentrations. For instance, to generate a gelatin-based
PBS solution of the GLP-2 at a concentration of 13 mg/l, 10 ml of a
PBS solution prepared with 5.2 mg of GLP-2 was diluted with 30 ml
of the 20% working gelatin solution as first described above. The
solution was mixed by gentle pipeting, to yield a final solution of
130 mg/l GLP-2 in PBS/15% gelatin.
EXAMPLE 3
Assay for Resistance to DiPeptidyl Peptidase IV
[0110] The following peptides were tested for resistance to
dipeptidyl peptidase IV (DPP-IV): a control peptide, rGLP-2; the
[D-Ala.sup.2]rGLP-2 analog; and the [Gly.sup.2]rGLP-2 analog. To
perform the assay, 2.5 microliters (.mu.l) of a solution of human
placental DPP-IV (Calbiochem, La Jolla, CA, cat. # 317624)
containing 0.125 milliunits (mU) of enzyme in 50% glycerol, 10 mM
Tris, pH 7.8, EDTA and 0.02% NaN.sub.3 was added to 50 .mu.l of a
solution of the test peptide prepared at a concentration of 0.2
mg/ml in PBS at pH 7.4. The mixture was incubated at 37.degree. C.
in a circulating water bath for 24 hours. The incubation was
quenched by the addition of 50 .mu.l of a solution of diprotin A
prepared at a concentration of 4 mg/ml in PBS. Each peptide was
tested in duplicate.
[0111] Each sample was analyzed by reverse-phase (RP) HPLC as
follows: 90 .mu.l of the quenched incubation mixture was injected
onto a Rainin Dynamax 300 .ANG., C18, 5 micron, 4.6.times.250
millimeter column. The samples were eluted with 0.1%
trifluoroacetic acid (TFA) in water modified with 0.1% acetonitrile
using a linear gradient and a flow rate of 1 ml per minute. Sample
components were detected at 214 nanometers (nm). The extent of
cleavage was measured by relative integration of the peak
corresponding to the cleavage product compared to that of the
remaining undigested parent peptide. The cleavage product of the
control peptide, rGLP-2(1-33), which should be rGLP-2(3-33), was
confirmed to have result from cleavage between residues Ala.sup.2
and Asp.sup.3 by comparison of the retention time of this component
to that of a synthetic peptide standard, rGLP-2(3-33), and by
collection of the product from the HPLC and analysis by mass
spectrometry.
[0112] After the 24 hour incubation, 22% of the control peptide,
rGLP-2, was cleaved by DPP-IV. No cleavage products were detected
for the peptides [D-Ala.sup.2]rGLP-2 and [Gly.sup.2]rGLP-2 after 24
hours.
EXAMPLE 4
GLP-2 analog assessment
[0113] Recipients were CD1 mice obtained from Charles River
Laboratory (Ontario, Canada). The CD1 mice were aged-matched
females at time of injection (n=3-4 per group), 6 weeks of age,
unless otherwise specified. The animals were allowed a minimum of
24 hours to acclimatize to the laboratory facility before the
initiation of each experiment. Animals were identified by ear
punch. The mice were not restricted by diet or activity during the
experiments. The light/dark cycle was 12 hours, between 6 pm to 6
am. Controls were age-and sex-matched (n=3-4) animals. Mice were
injected subcutaneously, twice a day (b.i.d.), with 2.5 .mu.g
peptide in a total volume of 0.5 cc of PBS and were monitored daily
in the laboratory facility. Animals were sacrificed 10 or 14 days
after injection, and were fasted at least 20 hours before
sacrifice.
[0114] The mice were anaesthetised with C0.sub.2 and exsanguinated
by cardiac puncture. Blood was collected in 75 .mu.l of TED
(Trasysol; EDTA (5000 KIU/ml: 1.2 mg/ml; Diprotin-A), and the blood
was centrifuged at 14 k.times.g for 5 minutes and the plasma was
stored at -70 prior to analysis. The small bowel was removed from
the peritoneal cavity, from pylorus to cecum, cleaned weighed and
measured. For comparative purpose, sections from each animal were
obtained from the identical anatomical position. Fragments each
measuring 1.5-2.0 cm in length were obtained 8.+-.2 cm, 18.+-.2 cm,
32.+-.2 cm from pylorus for histomorphometry representing proximal
jejunum, distal jejunum and distal ileum. Each small bowel fragment
was opened longitudinally on its antimesenteric border in a tissue
block and then placed on 10% formalin (vol./vol.) overnight, then
transferred to 70% ETOH.
[0115] Percentage change in small bowel weight was calculated by
dividing the mean change in bowel weight of analog treated mice,
relative to mice treated with vehicle only, by the mean bowel
weight of mice treated with vehicle only, and multiplying this
figure by 100.
[0116] Results of intestinotrophic activity assessment are shown in
Table 1:
1TABLE 1 % Increase in GLP-2 analog small # (1-33 unless noted)
bowel weight 1 rGLP-2 45 2 [Tyr.sup.1]rGLP-2 37 3
[D-Ala.sup.2]rGLP-2 86 4 [Gly.sup.2]rGLP-2 70 5 [Val.sup.2]rGLP-2
65 6 [Gly.sup.2]hGLP-2 59 7 [Gly.sup.2Ala.sup.20]hGLP-2 46 8
[Gly.sup.2Ala.sup.10]hGLP-2 33 9 [Ala.sup.1Gly.sup.2]hGLP-2 23 10
[Gly.sup.2Ala.sup.3]hGLP-2 18 11 [Gly.sup.2Ala.sup.4]hGLP-2 36 12
[Glu.sup.3]rGLP-2 33 13 [Ala.sup.4]rGLP-2 30 14
[Tyr.sup.9Ser.sup.10Lys.sup.11Tyr.sup.12(desIle.sup.13)] 41 hGLP-2
15 [Leu.sup.10]rGLP-2 26 16 [Nleu.sup.10]rGLP-2 52 17 [Met
SO.sub.2.sup.10]rGLP-2 8 18 [Lys.sup.20]rGLP-2 62 19
[Val.sup.23Gln.sup.24]hGLP-2 36 20 Amidated C-term 23 21
[Gly.sup.2Ala.sup.24]hGLP-2 67 22 [Gly.sup.2Ala.sup.8]hGLP-2 33 23
[Gly.sup.2Ala.sup.11]hGLP-2 42 24 [Gly.sup.2Ala.sup.21]hGLP-2 35 25
[Gly.sup.2Ala.sup.9]hGLP-2 31 26 [Gly.sup.2Ala.sup.16]hGLP-2 36 27
[Gly.sup.2Ala.sup.17]hGLP- -2 36 28 [Gly.sup.2Ala.sup.28]hGLP-2 31
29 [Gly.sup.2Ala.sup.5]hGLP-2 38 30 [Gly.sup.2Ala.sup.31]hGLP-2 28
31 [Gly.sup.2Ala.sup.27]hGLP-2 22 32 [Gly.sup.2Ala.sup.12]hGLP- -2
18 33 [Gly.sup.2Ala.sup.13]hGLP-2 18 34 [Gly.sup.2Ala.sup.7]hGLP-2
22 35 [Gly.sup.2Ala.sup.6]hGLP-2 18
[0117] It can be readily seen from the above table that analogs of
mammalian GLP-2 molecules can have enhanced intestinotrophic
activity. Furthermore, it is clear that depending on the
substitution made, various levels of intestinotrophic activity are
manifest. For example, [Gly.sup.2]rGLP-2 has a greatly enhanced
intestinotrphic activity compared to the naturally occuring
molecule; [Gly.sup.2]hGLP-2 has a substantially increased
intestinotrophic activity compared with rGLP-2 and
(Leu.sup.10]rGLP-2 has less than a 50% increase in intestinotrophic
activity.
[0118] The following experiments were conducted to confirm that the
increase in bowel weight seen in experimental animals treated with
DPP-IV resistant analogs of GLP-2, compared with animals treated
with wild-type GLP-2, was attributable in part to the DPP-IV
resistant nature of the molecules. This experiment took advantage
of the availability of a DPP-IV deficient rat strain, Fisher 334
DPP-IV- rats. The effect of rGLP-2 injections on small bowel weight
in the Fisher DPP-IV deficient rats was compared to that in normal
Sprague-Dawley rats.
[0119] In the following experiments the Sprague-Dawley rats were
injected s.c. twice daily with rGLP-2 in the Gelatin formulation.
The Fisher rats were injected s.c. twice daily with GLP-2 peptides
(both rGLP-2, and rGLP-2 analogs) in PBS.
[0120] Normal Sprague-Dawley rats treated with rGLP-2, 2.5 .mu.g
b.i.d. or 25 .mu.g b.i.d., showed no % change in small bowel weight
compared to control animals given vehicle alone. However, Fisher
334 DPP-IV-rats (DPP-IV deficient animals) demonstrated
approximately a 40-50% increase in % change in small bowel weight
when treated with 20 .mu.g b.i.d. rGLP-2 compared to animals
treated with vehicle only. Moreover, DPP-IV deficient animals
treated with 20 .mu.g [Gly.sup.2]rGLP-2 showed a 50-60% increase in
% change in small bowel weight compared with animals given vector
alone. Further, Fisher 344 wild-type rats showed 75-85% increase in
small bowel weight, over control animals given vehicle alone, when
treated with 20 .mu.g b.i.d. of [Gly.sup.2]rGLP-2.
[0121] The above results strongly indicate that GLP-2 is
inactivated in vivo in normal rats by cleavage of the two
N-terminal residues by a DPP-IV-like enzyme. Furthermore, these
results demonstrate that a GLP-2 analog modified so as to be
resistant to cleavage by DPP-IV caused a substantial increase in
bowel weight in normal rats, presumably as a result of increased
GPL-2 half-life in vivo.
[0122] As the DPP-IV cleavage site is conserved in all known
naturally occurring forms of GLP-2 it seems likely that in mammals
DPP-IV cleavage is an important, and probably the primary mechanism
whereby GLP-2 is inactivated in vivo. Importantly, GLP-2 analogs
resistant to DPP-IV cleavage have enhanced intestinotrophic
activity compared to native form.
EXAMPLE 5
[0123] Experiments assessing the small bowel inducing activity of
various analogs was repeated as described above for Example 4. In
these experiments, the small bowel inducing activity of each analog
in mice was calculated relative to that of native rat GLP-2(1-33)
(expressed as 100% of activity). Resistance of analogs to DPP-IV
cleavage was performed as described above in Example 3. Results are
tabulated below in Table 2.
2TABLE 2 % % Activity Cleaved Description of Sequence Rel. to by
Identification ALE-0109 rGLP-2 DPP-IV ratGLP-2(1-33) Native rat
sequence 100% 58% ratGLP-2(4-33) N-3; 3 N-terminal res. 6% removed
Ac-ratGLP-2(1-33) N-Acetyl 44% 0% anglerfish GLP-1 Native
anglerfish GLP-1 7% [Arg-1]ratGLP-2(-1-33) Arg added to N-terminus
11% 0% ratGLP-2(1-30) C-3; 3 C-terminal res. 23% removed
ratGLP-2(1-25) C-8; 8 C-terminal res. 8% removed
ratGLP-2(1-33)amide C-terminal amide 56% [Arg-2,Arg-1]ratGLP-2
Arg--Arg added to N- 43% 0% (2-33) terminus [DAla2]ratGLP-2(1-33)
Ala2->D-Ala2 210% 0% [Gly2]ratGLP-2(1-33) Ala2->Gly2 196% 0%
[Tyr1]ratGLP-2(1-33) His1->Tyr1 81% 100% [Ala4]ratGLP-2(1-33)
Gly4->Ala4 59% 72% [Glu3]ratGLP-2(1-33) Asp3->Glu3 65% 50%
[Leu10]ratGLP-2(1-33) Met10->Leu10 51% [Nle10]ratGLP-2(1-33)
Met10->Nle10 100% [Lys20]ratGLP-2(1-33) Arg20->Lys20 120%
[Ser2,Gln3]hGLP-2(1-33) Ala2->Ser2, 11% 0% Asp3->Glu3
[Val23,Gln24]hGLP-2 Ile23->Val23, 82% (1-33) Asn24->Gln24
[Val2]GLP-2(1-33) Ala2->Val2, TFA 145% 0% Degu GLP-2 Native degu
sequence 70% [Tyr9,Ser10,Lys11,Tyr12, Glu9->Tyr9, 94%
des-Ilel3]hGLP-2(1-33) Met10->Ser10, Asn11->Lys11,
Thr12->Tyr12, and deletion of Ile13 [Gly2,Ala8]hGLP-2(1-33)
Ala2->Gly2, 120% Asp8->Ala8 [Gly2,Ala11]hGLP-2(1-33)
Ala2->Gly2, 150% Asn11->Ala11 [Gly2,Ala21]hGLP-2(1-33)
Ala2->Gly2, 125% Asp21->Ala21 [Gly2,Ala24]hGLP-2(1-33)
Ala2->Gly2, 139% 0% Asn24->Ala24 [Gly2,Ala25]hGLP-2(1-33)
Ala2->Gly2, 18% Trp25->Ala25 [Gly2,Ala26]hGLP-2(1-33)
Ala2->Gly2, 28% Leu26->Ala26 [Gly2,Ala27]hGLP-2(1-33)
Ala2->Gly2, 82% Ile27->Ala27 [Gly2,Ala9]hGLP-2(1-33)
Ala2->Gly2, 112% Glu9->Ala9 [Gly2,Ala10]hGLP-2(1-33)
Ala2->Gly2, 82% Met10->Ala10 [Gly2,Ala12]hGLP-2(1-33)
Ala2->Gly2, 67% Thr12->Ala12 [Gly2,Ala13]hGLP-2(1-33)
Ala2->Gly2, 67% Ile13->Ala13 [Gly2,Ala14]hGLP-2(1-33)
Ala2->Gly2, 30% Leu14->Ala14 [Gly2,Ala16]hGLP-2(1-33)
Ala2->Gly2, 130% Asn16->Ala16 [Gly2,Ala17]hGLP-2(1-33)
Ala2->Gly2, 130% Leu17->Ala17 [Gly2,Ala20]hGLP-2(1-33)
Ala2->Gly2, 105% Arg20->Ala20 [Gly2,Ala23]hGLP-2(1-33)
Ala2->Gly2, 24% Ile23->Ala23 [Gly2,Ala28]hGLP-2(1-33)
Ala2->Gly2, 130% Gln28->Ala28 [Gly2,Ala30]hGLP-2(1-33)
Ala2->Gly2, 24% Lys30->Ala30 [Gly2,Ala31]hGLP-2(1-33)
Ala2->Gly2, 100% Ile31->Ala31 [Gly2]hGLP-2(1-33)
Ala2->Gly2 300% 0% [Ala1,Gly2]hGLP-2(1-33) His1->Ala1, 53% 0%
Ala2->Gly2 [Gly2,Ala3]hGLP-2(1-33) Ala2->Gly2, 44% 0%
Asp3->Ala3 [Gly2,Ala4]hGLP-2(1-33) Ala2->Gly2, 100% 0%
Gly4->Ala4 [Gly2,Ala5]hGLP-2(1-33) Ala2->Gly2, 136% 0%
Ser5->Ala5 [Gly2,Ala6]hGLP-2(1-33) Ala2->Gly2, 67%
Phe6->Ala6 [Gly2,Ala7]hGLP-2(1-33) Ala2->Gly2, 82%
Ser7->Ala7 [Pro1]hGLP-2(1-33) His1->Pro1 12% 0%
[Met(O)10]ratGLP-2(1-33) Met10 sulfoxide 18% [Gln20]hGLP-2(1-33)
Arg20->Gln20 80% [Asp1]hGLP-2(1-33) His1->Asp1 60% 0%
[tBuGly2]hGLP-2 Ala2->t-butyl-Gly2 123% 0% [Tyr34]hGLP-2(1-34)
Addition of C-terminal 135% Tyr [Asp2]hGLP-2(1-33) Ala2->Asp2
83% 0% [Glu2]hGLP-2(1-33) Ala2->Glu2 93% 0% [Phe2]hGLP-2(1-33)
Ala2->Phe2 50% 0% [His2]hGLP-2(1-33) Ala2->His2 0% 0%
[Ile2]hGLP-2(1-33) Ala2->Ile2 90% 0% [Lys2]hGLP-2(1-33)
Ala2->Lys2 90% 0% [Met2]hGLP-2(1-33) Ala2->Met2 170% 0%
[Asn2]hGLP-2(1-33) Ala2->Asn2 67% 0% [Pro2]hGLP-2(1-33)
Ala2->Pro2 50% 16% [Gln2]hGLP-2(1-33) Ala2->Gln2 100% 0%
[Ser2]hGLP-2(1-33) Ala2->Ser2 167% 0% [Thr2]hGLP-2(1-33)
Ala2->Thr2 117% 0% [Val2]hGLP-2(1-33) Ala2->Val2 180% 13%
[Tyr2]hGLP-2(1-33) Ala2->Tyr2 57% 0% [D-Ala2]hGLP-2(1-33)
Ala2->D-Ala2 130% 0% hGLP-2(1-33) Native human sequence 100% 60%
[desNH2Tyr1]hGLP-2 His1->desamino-Tyr1 125% 15% (1-33)
[Thr5]hGLP-2(1-33) Ser5->Thr5 73% [Ser16,Arg17,Arg18]
Asn16->Ser16, 59% hGLP-2(1-33) Leu17->Arg17, Ala18->Arg18
[Asn33]hGLP-2(1-33) Asp33->Asn33 91% [Pen2]hGLP-2(1-33)
Ala2->L-penicillamine2 58% 20% [bAla2]hGLP-2(1-33)
Ala2->beta-Ala2 118% 0% [aAbu2]hGLP-2(1-33) Ala2->alpha- 84%
49% aminobutyric acid2 [Nval2]hGLP-2(1-33) Ala2->Nval2
(norvaline) 111% 0% [PhGly2]hGLP-2(1-33) Ala2->L-phenyl-Gly2 37%
0% [Agm34]hGLP-2(1-34) Addition of C-terminal 97% agmatine (Agm is
des-COOH Arg) [Arg30]hGLP-2(1-33) Lys30->Arg30 103% 59%
[Pro3]hGLP-2(1-33) Asp3->Pro3 140% 0% [Ala5, Ala7]hGLP-2(1-33)
Ser5->Ala5, 108% 77% Ser7->Ala7 [Tyr1, Gly2]hGLP-2(1-33)
His1->Tyr1, 241% 0% Ala2->Gly2 [Glu33]hGLP-2(1-33)
Asp33->Glu33 121% 55% [Phe25]hGLP-2(1-33) Trp25->Phe25 114%
54% [Tyr25]hGLP-2(1-33) Trp25->Tyr25 64% 48% [Gly2,
Tyr34]hGLP-2(1-34) Ala2->Gly2 + C- 297% 0% terminal Tyr
[Aib2]hGLP-2(1-33) Ala2->aminoisobutyric 202% 0% acid2 [Gly2,
Arg34]hGLP-2 Ala2->Gly2 + C- .about.250% 0% (1-34) terminal
Arg
EQUIVALENTS
[0124] The foregoing written specification is sufficient to enable
one skilled in the art to practice the invention. Indeed, various
modifications of the above-described means for carrying out the
invention which are obvious to those skilled in the field of
molecular biology, protein chemistry, medicine or related fields
are intended to be within the scope of the following claims.
* * * * *