U.S. patent application number 12/843411 was filed with the patent office on 2011-02-10 for gastrin compositions and formulations, and methods of use and preparation.
This patent application is currently assigned to Waratah Pharmaceuticals, Inc.. Invention is credited to Antonio Cruz.
Application Number | 20110034379 12/843411 |
Document ID | / |
Family ID | 32330051 |
Filed Date | 2011-02-10 |
United States Patent
Application |
20110034379 |
Kind Code |
A1 |
Cruz; Antonio |
February 10, 2011 |
Gastrin Compositions And Formulations, And Methods Of Use And
Preparation
Abstract
An embodiment of the invention provided herein is a
pharmaceutical composition comprising a gastrin compound having an
extended activity upon administration to a subject in comparison
with native gastrin. Methods are provided of conjugating portions
of the amino acid sequence of gastrin having functional ability to
bind to the gastrin/CCK receptor, to various carrier moieties,
including the use of amino acid spacer regions, and use of
bifunctional cross-linking reagents. Methods of treating a diabetes
patient with the compositions are provided.
Inventors: |
Cruz; Antonio; (Toronto,
CA) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Assignee: |
Waratah Pharmaceuticals,
Inc.
Toronto
CA
|
Family ID: |
32330051 |
Appl. No.: |
12/843411 |
Filed: |
July 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11701196 |
Jan 31, 2007 |
7803766 |
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12843411 |
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10535745 |
Nov 17, 2005 |
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PCT/CA03/01778 |
Nov 21, 2003 |
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11701196 |
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60428100 |
Nov 21, 2002 |
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60428562 |
Nov 22, 2002 |
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60430590 |
Dec 3, 2002 |
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60519933 |
Nov 14, 2003 |
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Current U.S.
Class: |
514/6.9 ;
530/399 |
Current CPC
Class: |
A61K 47/60 20170801;
A61K 31/436 20130101; A61K 38/1808 20130101; A61K 38/2207 20130101;
A61K 38/1808 20130101; A61P 3/10 20180101; A61K 38/26 20130101;
A61K 47/543 20170801; A61K 2300/00 20130101; A61K 38/26 20130101;
A61P 37/06 20180101; A61P 43/00 20180101; A61K 31/436 20130101;
A61K 38/2207 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/6.9 ;
530/399 |
International
Class: |
A61K 38/22 20060101
A61K038/22; C07K 14/595 20060101 C07K014/595; A61P 3/10 20060101
A61P003/10 |
Claims
1-28. (canceled)
29. A gastrin compound which comprises an amino acid sequence
comprising from the amino acid terminus
Z-Y.sub.m-X.sub.n-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6,
wherein Z is polyethylene glycol (PEG); X.sub.n is
Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala (SEQ ID NO:9);
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6 is
Tyr-Gly-Trp-Leu-Asp-Phe (SEQ ID NO:6) or Tyr-Gly-Trp-Met-Asp-Phe
(SEQ ID NO:5), Y.sub.m is an optional spacer region comprising m
amino acid residues of a small neutral amino acid, and wherein
there is a cysteine residue at the amino terminus of Y wherein m is
1 or greater, or at the amino terminus of X when m is 0, providing
that the gastrin compound binds a gastrin/CCK.sub.B receptor.
30. A gastrin compound according to claim 29 which comprises
Y.sub.m wherein m is 5 and Y is (Gly-Ala).
31. A gastrin compound according to claim 29 wherein m is 0.
32. A pharmaceutical composition for use in the treatment of
diabetes comprising a gastrin compound according to claim 29 and a
pharmaceutically acceptable carrier or excipient.
33. A pharmaceutical composition for use in the treatment of
diabetes comprising a gastrin compound according to claim 30 and a
pharmaceutically acceptable carrier or excipient.
34. A pharmaceutical composition for use in the treatment of
diabetes comprising a gastrin compound according to claim 31 and a
pharmaceutically acceptable carrier or excipient.
35. A gastrin compound according to claim 28 wherein
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6 is
Tyr-Gly-Trp-Leu-Asp-Phe (SEQ ID NO:6).
36. A gastrin compound according to claim 28 wherein
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6 is
Tyr-Gly-Trp-Met-Asp-Phe (SEQ ID NO:5).
37. A gastrin compound according to claim 28 wherein said gastrin
has extended activity upon administration to a subject in
comparison with native gastrin.
38. The gastrin compound of claim 28 wherein Y.sub.m is a spacer
region comprising m amino acid residues of a small neutral amino
acid.
39. The gastrin compound of claim 30 wherein Y.sub.m is alternately
glycine and alanine amino acids.
40. A pharmaceutical composition according to claim 30 wherein said
gastrin has extended activity upon administration to a subject in
comparison with native gastrin.
41. A pharmaceutical composition according to claim 30 wherein said
gastrin prevents severe hypoglycemia or increases pancreatic
insulin content upon administration to a subject.
42. A pharmaceutical composition comprising a gastrin compound
according to claim 35 and a pharmaceutically acceptable carrier or
excipient.
43. A pharmaceutical composition comprising a gastrin compound
according to claim 36 and a pharmaceutically acceptable carrier or
excipient.
Description
FIELD OF THE INVENTION
[0001] The invention in various embodiments provides gastrin
compositions having longer active function in vivo than gastrin
peptides, and methods of making and using the gastrin compositions
for treatment of diabetes.
BACKGROUND OF INVENTION
[0002] Therapeutic agents such as peptides and low molecular weight
proteins used in the D treatment of diseases suffer from
significant limitations. These agents are often eliminated by the
kidneys within a short period of time or are destroyed by proteases
therefore limiting their bioavailability, resulting in short plasma
half-life and lower drug concentrations than are required to be
efficacious. A high clearance of a therapeutic agent is not optimal
in cases where it is desired to maintain a high serum level over an
extended period of time to obtain maximal efficacy. Increased doses
or increased frequency of administration often result in a higher
therapeutic efficacy but a higher risk of side effects as well,
limiting the dose or frequency that can be administered.
[0003] Many peptide hormones have extremely short half-lives in the
bloodstream, resulting in the loss of biological activity not long
after administration. Gastrin is a peptide hormone that has been
shown in combination with other growth factors to be efficacious in
the treatment of diabetes. However, when gastrin is administered
alone, there is only limited efficacy. In addition, it has been
found that gastrin has a relatively short half life. Gastrin-17 for
instance has a circulating half-life of about 5-9 mins while
gastrin-34 has a circulating half-life of about 35 mins.
[0004] There is a need for compositions containing gastrin
compounds that have a protracted or long-acting action.
SUMMARY
[0005] A featured embodiment of the invention provides a
pharmaceutical composition comprising a gastrin compound having an
extended activity upon administration to a subject in comparison
with native gastrin. The gastrin component in this embodiment
contains at least amino acids selected from the group of: positions
29-34 of SEQ ID NO: 1; positions 29-34 of SEQ ID NO:2; positions
12-17 of SEQ ID NO: 3; and positions 12-17 of SEQ ID NO: 4, and the
gastrin is further associated with a protein, a polymer, a lipid or
a carbohydrate.
[0006] An alternative featured embodiment provides a gastrin
compound comprising:
Z-Y.sub.m-X.sub.n-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6,
wherein AA.sub.1 is Tyr or Phe, AA.sub.2 is Gly, Ala, or Ser,
AA.sub.3 is Trp, Val, or Ile, AA.sub.4 is Met or Leu, AA.sub.5 is
Asp or Glu, and AA.sub.6 is Phe or Tyr the AA.sub.6 being amidated;
wherein Z is a polymer which when the polymer is a protein, Z is
the amino acid sequence of the protein; Y.sub.m is an optional
spacer region comprising m amino acid residues of a small neutral
amino acid, and X is selected from any consecutive portions of:
residues 1-28 of SEQ ID NO: 1, residues 1-28 of SEQ ID NO: 2,
residues 1-11 of SEQ ID NO: 3, and residues 1-11 of SEQ ID NO: 4,
providing that the gastrin compound binds a gastrin/CCK receptor.
The AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6 is, for
example, Tyr-Gly-Trp-Met-Asp-Phe. Alternatively, the
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6 is
Tyr-Gly-Trp-Leu-Asp-Phe. In this formula, Z can be a protein, for
example, Z is human serum albumin.
[0007] Y.sub.m can be an amino acid sequence comprising m residues
having glycine alternating with alanine, for example,
[Gly-Ala].sub.5. or having random sequence of glycine and alanine.
The gastrin compound further can have a cysteine residue at the
amino terminus of Y, when m is greater than 1, or at the amino
terminus of X, when m is 0. The gastrin compound can further
comprise of a bifunctional crosslinking agent for linkage to Z. In
general, m is 0 to about 20 residues. In certain embodiments, if m
is 0, wherein
X.sub.n-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6
further comprises a bifunctional cross-linking agent for linkage to
Z.
[0008] The X in certain embodiments is selected from the group of
sequences: position 1 to position 11 of SEQ ID NO: 3; position 1 to
position 11 of SEQ ID NO: 4; position 2 to position 11 of SEQ ID
NO: 3; and position 2 to position 11 of SEQ ID NO: 4. The gastrin
compound in which Z is a protein can be recombinantly produced.
[0009] Another embodiment provided herein is a nucleotide sequence
encoding the gastrin compound in which the Z is a protein. Further
provided is a cell carrying this nucleotide sequence. The cell is a
bacterial or a yeast cell. When the cell is a bacterial cell it can
be, for example, a cell of a species of an Escherichia, a Bacillus,
or a Streptomyces. When the cell is a yeast cell, it can be a cell
of a species of a Saccharomyces, a Kluyveromyces, a
Schizosaccharomyces or a Pichia.
[0010] The gastrin compound generally contains a minimal gastrin
component which is at least amino acids at positions 29-34 of SEQ
ID NO:2 or positions 12-17 of SEQ ID NO:4. These are located at the
carboxy terminus of gastrins that occur in circulation, and in
various embodiments additional amino acids for example from gastrin
can be present.
[0011] The polymer component need not be limited to a protein, but
may be a synthetic chemical polymer such as a polyethylene glycol
(PEG) or a dextran. When the polymer is a protein, in various
embodiments it can be a serum protein, for example, a serum
albumin, for example, human serum albumin.
[0012] Another embodiment of a gastrin compound provided herein has
a structure C--Y.sub.m--X, wherein C is Cys or Lys, Y.sub.m is an
optional spacer region comprising m amino acid residues of a small
neutral amino acid, and X is at least six amino acid residues
comprising sequences selected from at least positions 12-17 of
gastrin-17 (SEQ ID NO: 3 and 4) and at least positions 29-34 of
gastrin-34 (SEQ ID NO: 1 and 2). The gastrin compound further is
conjugated to a polymer, for example, is conjugated to a
polyethylene glycol (PEG) or a dextran. The gastrin compound
further is alternatively conjugated to a protein. In certain
embodiments, the gastrin compound further includes a bifunctional
cross-linking agent wherein a first reactive end of the
cross-linking agent is covalently linked to C. A second reactive
end of the cross-linking agent is covalently linked to a polymer or
protein. The C--Y.sub.m--X can be produced recombinantly or it can
be synthesized by peptide synthesis.
[0013] Any of the gastrin compounds provided herein are, in certain
embodiments, provided in an effective dose. The gastrin compounds
provided herein can further include an agent for immune
suppression. The gastrin compounds provided herein can further
include a hypoglycemic agent. The gastrin compounds provided herein
can further include a pharmaceutically acceptable carrier. The
gastrin compounds provided herein can further include a growth
factor. For example, in certain embodiments the growth factor is a
glucagon-like peptide 1 receptor ligand. Alternatively, in certain
embodiments the growth factor is an EGF receptor ligand.
[0014] Also provided herein are embodiments of the invention which
include a method of manufacture of a medicament for treating a
subject having diabetes, comprising formulating a gastrin compound
according to any of those described herein, and administering the
gastrin compound to the subject. In certain embodiments of the
method, frequency of administering the gastrin compound is less
than frequency of administration of a native gastrin. The method
may further include measuring a physiological indicator of islet
neogenesis, for example, measuring fasting blood glucose (FBG). The
method may include decreasing insulin dependency.
[0015] Yet another embodiment of the invention provides a method of
making a gastrin compound, the method being associating an amino
acid sequence of a gastrin with a carrier composition. Accordingly,
prior to associating the gastrin with the carrier, the gastrin is
modified to comprise a cysteine substitution or an additional
cysteine residue. The cysteine substitution is a replacement of
pyroglutamate. The gastrin amino acid sequence comprises at least
positions selected from the group of: residues 29-34 of amino acid
sequence. SEQ ID NO: 1; residues 29-34 of amino acid sequence SEQ
ID NO: 2; residues 12-17 of amino acid sequence SEQ ID NO: 3; and
residues 12-17 of amino acid sequence SEQ ID NO: 4. In certain
embodiments, the cysteine is at the amino terminus of the gastrin.
Alternatively, prior to associating the gastrin with the carrier,
the method includes modifying the gastrin to further include a
bifunctional cross-linking agent.
[0016] Yet another embodiment of the invention provides a method of
manufacture of a medicament for treating a diabetes patient, the
method comprising formulating a modified gastrin compound capable
of covalently reacting with a serum protein, and administering to
the patient the modified gastrin. The modified gastrin includes a
sequence of a native gastrin capable of binding to the gastrin/CCK
receptor and an amino terminal cysteine or lysine. Accordingly, the
sequence of the native gastrin is selected from the group of:
residues 29-34 of amino acid sequence SEQ ID NO: 1; residues 29-34
of amino acid sequence SEQ ID NO: 2; residues 12-17 of amino acid
sequence SEQ ID NO: 3; and residues 12-17 of amino acid sequence
SEQ ID NO: 4.
[0017] Also provided herein is a method of manufacture of a
medicament for maintaining for an extended period of time an
increased gastrin serum level compared with the serum level of a
peptide having an amino acid sequence of a gastrin, the method
comprising formulating a gastrin compound as described above, and
administering the gastrin compound.
[0018] Also provided herein is a kit comprising at least one
effective dose of a gastrin compound as described herein.
BRIEF DESCRIPTIONS OF DRAWINGS
[0019] FIG. 1 shows the effect of unmodified gastrin on fasting
blood glucose levels of NOD mice with recent onset diabetes after a
14 day treatment.
[0020] FIG. 2 shows the effect of unmodified gastrin on pancreatic
insulin levels of NOD mice with recent onset diabetes after a 14
day treatment
DETAILED DESCRIPTION OF INVENTION
[0021] Use of long acting gastrin compounds can result in decreased
clearance of gastrin or reduced degradation of gastrin by enzymes
thereby maintaining higher concentrations of plasma gastrin for
extended period of time and/or increasing half-life of gastrin thus
resulting in increased efficacy. Dosing regimens can be improved by
either using lower doses and/or reducing the frequency of
administration of gastrin to diabetic patients. In addition, with
the conjugation of gastrin to a carrier, it is possible that some
of the carriers can also mask gastrin from the immune system,
thereby reducing or preventing gastrin provoking immune reaction,
as well as increasing the half life and/or maintaining higher
concentrations of serum gastrin.
[0022] This invention in general embodiments provides compositions
having gastrin-like activity herein referred to as gastrin
compounds The term "gastrin compounds" as used herein means agents
that bind to, interacts with or stimulates the gastrin/CCK
receptor. Gastrin compounds include gastrin derivatives and
conjugates as well as peptide homologs, that are capable of
interacting with the gastrin/CCK receptor. The terms "derivatives"
and "conjugates" as used herein are equivalent, and are used to
indicate compositions that are chemically related, and can be
prepared by synthetic, biological, recombinant or chemical
means.
[0023] In various embodiments, a "modified", gastrin can be
prepared and used to treat a patient having a diabetes. The term,
"diabetes" as used herein means any physiologic indication of a
shortage of insulin, a production of antibodies against insulin, or
an excess of blood sugar or any manifested symptoms of diabetes in
any mammal including experimental animal models, and including
human forms such as type I and type II diabetes, early stage
diabetes, and a pre-diabetic condition characterized by mildly
decreased insulin or mildly elevated blood glucose levels. As used
herein, the term "mammal" has the usual meaning of any member of
Mammalia, and includes humans.
[0024] The modified gastrin can be a gastrin derivative or analog
comprising a minimal sequence of 6 amino acids (from the C-terminal
end), and further having addition of a reactive group such as a
cysteine residue capable of undergoing an addition reaction (Refer
to SEQ ID 1-4) In various embodiments, the gastrin may extend up to
34 amino acids ("Big" Gastrin or Gastrin-34), wherein at least one
reactive amino acid such as a cysteine residue or a lysine residue
is added or substituted at the N-terminal end. The addition of the
reactive amino acid such as a cysteine can be at a terminal region,
and in related embodiments, a spacer region can optionally precede
the added reactive amino acid. For example, the spacer can be
synthesized biologically as part of, or can be chemically attached
to the gastrin amino acid sequence, forming a structure which has a
gastrin sequence-spacer-cysteine. For instance, the spacer region
can be a sequence of several amino acids such as alanine or
glycine. The sequence of amino acids can be alternating amino acids
(e.g. glycine/alanine) or can be non-alternating, i.e., can be a
random sequence or a particular sequence. The sequence can consist
of at least one amino acid.
[0025] In alternative embodiments, a bifunctional cross-linking
agent which is a reactive component is added to the modified
gastrin, particularly to the gastrin having an added reactive group
at the amino terminus (e.g., a cysteine), or to a modified gastrin
having a spacer, via a homobifunctional or heterobifunctional
portion of the crosslinker to generate an a modified gastrin having
a reactive group such as a thiol of an amino group at one end.
(e.g., to form, as listed from the carboxy terminus, a
gastrin-spacer-cys-cross-linker-carrier; a gastrin-cys-cross-linker
group-carrier; gastrin-spacer-cys-cross-linker with reactive group
exposed, and a gastrin-cys-cross-linker with reactive group
exposed.)
[0026] The modified gastrin is then either injected in this state
into a patient, or is further conjugated in vitro to one or more
plasma components such as whole or fractionated serum obtained from
the patient; one ore more purified serum protein(s) such as
albumin, transferrin or an immunoglobulin; lipids/lipophilic
moieties/hydrophobic moieties; or to polymeric carriers such as
dextran or PEG prior to injection. The term polymer as used herein
and in the claims includes polymers of amino acids, sugars,
nucleosides, synthetic polymers (such as PEG) and mixtures thereof.
The polymer can be activated, for instance with a bifunctional
crosslinker or via other chemical means prior to conjugation.
Administering the activated gastrin compound or the gastrin
conjugate results in increased serum half life and/or maintained
high plasma concentration of gastrin for an extended period of
time, compared with administering native gastrin.
[0027] The invention in general embodiments provides gastrin
compositions which have a moiety that is a gastrin compound and can
be associated with a larger molecule such as a polymer, either
non-covalently, or as a covalent conjugate, or as a fusion protein
to another peptidic compound having an amino acid sequence. The
gastrin compounds provided herein have a longer half-life in
circulation in a subject animal or patient, and/or maintain higher
concentrations in vivo of the gastrin compounds for an extended
period of time compared to the native forms of gastrin. In
addition, the invention in other embodiments provides compositions
and methods of making and of using the gastrin compounds, provided
to the patient either alone or in combination with at least one
growth factor, with a hypoglycemic agent, or with an
immunosuppressant, for the treatment of diabetes. Examples of
growth factors include but are not limited to a EGF receptor ligand
such as EGF, a GLP-1 receptor ligand such as GLP-1, prolactin
receptor ligand such as prolactin and growth hormone receptor
ligand such as growth hormone. Examples of immunosuppressants
include but are not limited to cyclosporine, FK506, rapamycin, and
daclizumab. Non-limiting examples of hypoglycemic agents include
sulfonylureas, meglitinides, biguanides, thiazolidinediones, and
alpha-glucosidase inhibitors.
[0028] In one embodiment, a gastrin compound can be bound to a
comparatively larger structure or a plurality of structures in the
blood and still retain the ability to bind target proteins, i.e., a
gastrin/CCK receptor. Generally, gastrin, which would be otherwise
rapidly degraded in the body, is attached to a carrier protein;
using this composition, a longer-term of drug efficacy can be
achieved. Alternatively a gastrin compound can be conjugated to a
polymeric carrier such as a polyethylene glycol (PEG) or a dextran
to achieve similar objectives
[0029] In certain embodiments, chemical modification of gastrin is
used to provide compounds that react covalently or non-covalently
to carrier proteins or polymeric carriers, either in vitro (ex
vivo) or in vivo. In a related embodiment, the non-covalent
interaction is electrostatic or hydrophobic. In related
embodiments, conjugation of modified gastrin to the carrier is
carried out prior to injection. In other embodiments, the gastrin
is modified in such a manner that when injected, will have an
enhanced affinity to the carrier in the bloodstream. In another
embodiment, long acting gastrin compounds are obtained via chemical
modification with no requirement for a carrier protein either in
vivo or ex vivo.
[0030] In certain embodiments, the carrier protein is a plasma
protein. In related embodiment, the plasma protein is an albumin or
an immunoglobulin or components of an immunoglobulin. The
immunoglobulin or components of the immunoglobulin can be modified
or portions deleted prior to conjugation. In certain embodiments,
the polymeric carrier is polyethylene glycol or dextran. For
instance, activated PEG can be attached to gastrin compound via an
amino group in the gastrin compound (Vernonese, F M. Biomaterials
22 (2001)-405-41.7).
[0031] In other embodiments, the gastrin compound which is a
sequence of amino acids, is genetically fused with the carrier
protein, which is also a sequence of amino acids, prior to
injection, using standard recombinant genetic techniques. Gastrin
can be fused recombinantly to a carrier protein with or without a
linker/spacer, for example, comprising a sequence of small neutral
uncharged amino acids. A nucleic acid encoding gastrin can be
recombinantly fused or synthesized directly as a fusion to portions
or the whole of the carrier protein and the nucleic acid construct
or fusion protein can encode or incorporate a number of additional
amino acids to act as a spacer between the two proteins.
Recombinant fusion proteins can be expressed in yeast
(Saccharomyces, Pichia) or in standard bacterial systems, or
mammalian or insect cell systems can be used. Following standard
procedures for expression and/or purification, the fusion protein
can be used therapeutically. Modifications to the sequence of
gastrin compound polypeptide can be introduced during construction
of the fusion protein if necessary.
[0032] In one embodiment, the gastrin compound is modified to
introduce a reactive group such as those present on an amino acid
such as a lysine or cysteine so that the reactive group upon
further contacting another compound such as a carrier protein or
carrier non-proteinaceous polymer, can form covalent interactions
with the carrier proteins or polymers. For instance, a reactive
thiol group can be added to the gastrin molecule through an amino
group on lysine, for example, using succinimidyl 3-2-pyridyldithio
propionate (SPDP) followed by reduction with DTT to release the
active thiol group (("Protein thiolation and reversible
protein-protein conjugation. N-Succinimidyl
3-(2-pyridyldithio)propionate, a new heterobifunctional reagent."
Carlsson J, Drevin H, Axen R. Biochem J 173, 723-737 (1978)).
Further, the bifunctional group can also be added after the
cysteine or lysine has been added, so that one reactive end of the
crosslinking agent will react with cysteine/lysine while the other
reactive end at the other end is left exposed or is conjugated to a
carrier.
[0033] Thiols can be also incorporated at carboxylic acid groups by
EDAC-mediated reaction with cystamine, followed by reduction of the
disulfide with DTT. ("Introduction of sulfhydryl groups into
proteins at carboxyl sites." Lin C M, Mihal K A, Krueger R J.
Biochim Biophys Acta 1038, 382-385 (1990). In a non-limiting
example, reaction of an amino group on the lysine residue in
gastrin with succinimidyl
trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate ("Conjugation
of glucose oxidase from Aspergillus niger and rabbit antibodies
using N-hydroxysuccinimide ester of
N-(4-carboxycyclohexylmethyl)-maleimide." Yoshitake S, Yamada Y,
Ishikawa E, Masseyeff R. Eur J Biochem 101, 395-399 (1979))
introduces a thiol reactive group at amino sites of gastrin that
can subsequently react with cysteine residues of the carrier
protein or free thiol group on the activated polymer.
[0034] A gastrin compound-carrier complex can include additional
modular components including a spacer arm or element or other
component that can facilitate preparation or isolation of the
gastrin compound-carrier complex or enhance or maintain the
functional activity of the gastrin compound. The spacer arm can be
one or more amino acids, peptide, a peptidomimetic, or a small
organic molecule, and can comprise homobifunctional or
heterobifunctional crosslinking agents or chitin oligomers or
polyethylene glycol or related polymers.
[0035] In another embodiment, the carrier and gastrin compound can
be covalently crosslinked with or without a spacer arm. Examples of
non-spacer arms (zero-length crosslinkers) include EDC.
Homobifunctional crosslinkers that generate a spacer arm can be for
instance disuccinimidyl suberate and heterobifunctional
crosslinkers that generate a spacer arm can be for instance
2-iminothiolane, succinimidyl 6-[3-(2-pyridyldithio)propionamido]
hexanoate (LC-SPDP) and 4-(N-maleimido
methyl)cyclohexane-1-carboxylate (SMCC).
[0036] In various embodiments, the gastrin compound is associated
with a larger carrier moiety such as a polymer, for example a
protein. As the association may be covalent or non-covalent, the
protein may be considered to be a carrier protein. Classes of
carrier proteins can possess the properties of being non-antigenic,
i.e., are native human proteins, and are being capable of sustained
maintenance in circulation. An ideal carrier protein is one
normally found in the human circulatory system.
[0037] As used herein, the term "gastrin/CCK receptor ligand"
encompasses any compound, that binds to, interacts with or
stimulates the gastrin/CCK receptor. Examples of such gastrin/CCK
receptor ligands are given in U.S. Pat. No. 6,288,301 issued Sep.
11, 2001, and include various forms of gastrin, such as gastrin 34
(big gastrin), gastrin 17 (little gastrin or small gastrin),
gastrin 14, gastrin 13, gastrin-10, and gastrin 8, pentagastrin,
tetragastrin; various forms of cholecystokinin such as CCK 58, CCK
33, CCK 22, CCK 12 and CCK 8; and other gastrin/CCK receptor
ligands. In general, gastrin/CCK receptor ligands share a carboxy
terminal amino acid sequence Trp-Met-Asp-Phe-amide. The
aforementioned methionine (Met) can be replaced by a leucine. Also
contemplated are active analogs, fragments and other modifications
of the above, including both peptide and non-peptide agonists or
partial agonists of the gastrin/CCK receptor such as A71378 (Lin et
al., Am. J. Physiol. 258 (4 Pt 1): G648, 1990).
[0038] Small forms of gastrin such as gastrin 17 are economically
prepared by peptide synthesis, and synthetic peptides are
commercially available. Synthetic human gastrin 17 such as human
gastrin 17 having methionine or leucine at position 15 are also
available from Bachem AG, Bubendorf, Switzerland, and from
Researchplus. Gastrin peptides as found in nature are
carboxyl-terminally amidated peptides, and amidation of the
carboxyl terminus amino acid is within the scope of gastrin
compounds herein.
[0039] Gastrin/CCK receptor ligands include also active analogs,
fragments and other modifications of the above ligands, which for
example share amino acid sequence with an endogenous mammalian
gastrin, for example, share 60% sequence identity, or 70% identity,
or 80% identity. Such ligands also include compounds that increase
the secretion of endogenous gastrins, cholecystokinins or similarly
active peptides from sites of tissue storage. Examples of these are
the gastric releasing peptide, omeprazole which inhibits gastric
acid secretion, and soya bean trypsin inhibitor which increases CCK
stimulation
[0040] The sequence of big gastrin-34 and small gastrin-17 are
shown herein. Big gastrin-34 is essentially an extension form of
small gastrin-17 haying an additional amino acid sequence at the
N-terminal end. Big gastrin is cleaved in vivo to release
gastrin-17. The symbol "Glp" at the N-terminal end is a
pyroglutamate residue, which is a naturally cyclized form of
glutamate. In various embodiments, gastrins having an N-terminal
pyroglutamate residues are modified to contain N-terminal cysteine
or lysine residues by either replacing the pyroglutamate with a
glutamate or glutamine, or deleting the pyroglutamate. Further,
each of a gastrin 34 and gastrin-17 can be used in a modified form
that has a methionine or a leucine at position 32 as shown herein
in SEQ ID No: 1-2, respectively, or at position 15 as shown in SEQ
ID No: 3-4, respectively.
TABLE-US-00001 (SEQ ID NO: 1) N-terminal
Glp-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-
Leu-Val-Ala-Asp-Pro-Ser-Lys-Lys-Gln-Gly-Pro-Trp-
Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp- Phe-NH.sub.2. (SEQ
ID NO: 2) N-terminal Glp-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-
Leu-Val-Ala-Asp-Pro-Ser-Lys-Lys-Gln-Gly-Pro-Trp-
Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Leu-Asp- Phe-NH.sub.2. (SEQ
ID NO: 3) N-terminal Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-
Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH.sub.2. (SEQ ID NO: 4) N-terminal
Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-
Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe-NH.sub.2.
[0041] In certain embodiments, the gastrin compound which is a
fusion, for example, of a gastrin amino acid sequence with an
optional spacer of amino acids at the amino terminus of the gastrin
sequence, and having a protein carrier, can be provided
transgenically to a subject. In an embodiment of a nucleic acid
construct for transgenic expression of such a fusion protein, a
human preprogastrin peptide precursor gene is fused to a gene
encoding a carrier protein, with or without a spacer, by techniques
similar to those as shown in U.S. Pat. No. 5,885,956.
[0042] The method for treating diabetes mellitus in an individual,
in need thereof includes administering to the individual a
composition that provides a gastrin compound and a FACGINT such as
EGF, GLP-1, prolactin and growth hormone. Included are derivatives,
analogs, and conjugates of these FACGINT. As used herein, the term
"FACGINT" means a factor that complements gastrin for islet
neogenesis therapy. The phrase, "a FACGINT" as used herein can also
mean "one or more FACGINTs" or "at least one FACGINT".
[0043] The term "FACGINT" includes a large variety of growth
factors and growth hormones, agents that modify one or more of the
factors hormones, and ligands and effectors for one or more
receptors involved in binding of these growth hormones and growth
factors as these terms are generally understood, exemplified but
not limited to: EGF receptor ligand, a PTH-related protein (PTHrP)
receptor ligand such as PTHrP (PTHrP; Garcia-Ocana, A. et al.,
2001, J. clin. Endocrin. Metab. 86: 984-988); a hepatocyte growth
factor (HGF) receptor ligand such as HGF (HGF; Nielsen, J. et al.,
1999, J Mol Med 77: 62-66); a fibroblast growth factor (FGF) such
as FGF, a keratinocyte growth factor (KGF) receptor ligand such as
KGF; a nerve growth factor (NGF) receptor ligand such as NGF; a
gastric inhibitory polypeptide (GIP) receptor such as GIP; a
transforming growth factor beta (TGF.beta.) receptor ligand such as
TGF.beta. (U.S. patent application 2002/0072115 published Jun. 13,
2002), a laminin receptor ligand such as laminin-1; an islet
neogenesis associated protein (INGAP) receptor ligand such as
INGAP; a bone morphogenetic factor (BMP) receptor ligand such as
BMP-2; a vasoactive intestinal peptide (VIP) receptor ligand such
as VIP; a glucagon-like peptide 1 receptor ligand such as GLP-1 and
exendin-4, glucagon-like peptide 2 (GLP-2) receptor ligand such as
GLP-2, and dipeptidyl peptidase IV inhibitors which indirectly
affect the levels of GLP-1 (Hughes, T. et al., 2002, Am Diabetes
Assoc Abstract 272-or) by inhibiting an enzyme involved in its
integrity; a REG receptor ligand such as REG protein; a Growth
hormone (GH) receptor ligand such a GH, a Prolactin (PRL) receptor
ligand such as PRL and placental lactogen (PL); an Insulin-like
growth factor (Type 1 and 2) receptor ligands such as IGF1 and
IGF-2; an Erythropoietin (EPO) receptor ligand such as EPO
(http://www.drinet.org/html/august.sub.--2002_.htm); a betacellulin
(also considered to be a member of the EGF family); an Activin-A
receptor ligand such as Activin-A; a vascular endothelial growth
factor (VEGF) receptor ligand such as VEGF; a bone morphogenesis
factor (BMP) receptor ligand such as BMP-2; a vasoactive intestinal
peptide (VIP) receptor ligand such as VIP; a vascular endothelial
growth factor (VEGF) receptor ligand such as VEGF; a pituitary
adenylate cyclase activating polypeptide (PACAP) receptor ligand
such as PACAP; a granulocyte colony stimulating factor (G-CSF)
receptor ligand such as G-CSF; a granulocyte-macrophage colony
stimulating factor (GM-CSF) receptor ligand such as GM-CSH; a
platelet-derived growth factor (PDGF) receptor ligand such as PDGF;
and a Secretin receptor ligand such as secretin.
[0044] For any of the growth factors, enzymes, enzyme inhibitors,
peptide, protein and hormone compounds herein that are indicated to
be an exemplary FACGINT, all known analogues and derivatives,
whether naturally occurring or made by mutagenesis or designed and
synthesized shall be considered equivalent to that FACGINT. Also
considered among equivalents are conjugates, i.e., compositions
derived by addition of one or more of a chemical group, and
mixtures thereof. Encoding genes may be altered by, for example,
oligonucleotide directed mutagenesis to produce FACGINT analogues
thereof, such as the human recombinant analogues. Further, an
identity or location of one or more than one amino acid residue may
be changed by targeted mutagenesis. The primary amino acid sequence
of the protein may be augmented by conjugates, as by glycosylation,
acylation, or by addition of any other supplementary molecules,
such as one or more of a lipid, a phosphate, a sulphate and/or an
acetyl group. Further, individual amino acid residues in the chain
may be modified by oxidation, reduction, or other derivatization.
The FACGINT may be cleaved to obtain any fragments which retain
activity. A prodrug or a metabolite of a FACGINT is equivalent to
that FACGINT. The whole polypeptide or protein or any fragment can
be fused with any other peptide or protein such as immunoglobulins
and other cytokines. Conjugates may include, for example, a
composition comprising the FACGINT coupled to a non-naturally
occurring polymer comprising a polyethylene glycol moiety. The term
also encompasses derivatives obtained by chemically modifying one
or more amino acid residues of the parent peptide, for instance by
alkylation, acylation, ester formation or amide formation.
Alternatively, agents that induce synthesis of the FACGINT or mimic
the action of the FACGINT are contemplated as equivalent compounds.
The singular form, "FACGINT", may mean any one or more compounds
from the exemplary FACGINTs shown herein. The terms "derivatives"
and "conjugates" as used herein are equivalent, and are used to
indicate compositions that are chemically related, and can be
prepared by synthetic, biological, or recombinant or chemical
means.
[0045] The term, "receptor ligand" as used herein in connection
with a receptor for a particular ligand shall mean any compositions
that binds to, interacts with, or stimulates that receptor.
[0046] A receptor ligand includes within the scope of the
definition a receptor agonist, for the receptor for any particular
FACGINT, whether or not the agonist is structurally related to the
FACGINT.
[0047] The term, "prolactin" as used herein means any polypeptide
which shares substantial sequence similarity with an endogenous
mammalian prolactin as this term is known in the art of protein
factors, for example, human prolactin, and which possesses the
activity of a prolactin. Endogenous human prolactin is a 199 amino
acid polypeptide produced by the pituitary gland. The term
encompasses prolactin analogs which are deletions, insertions, or
substitution mutants of endogenous prolactin, and retain the
activity, and includes prolactins from other species and naturally
occurring variants. The prolactin function includes a composition
having agonist activity for the prolactin receptor, as disclosed in
U.S. Pat. Nos. 6,333,031 (activating amino acid sequence) and
6,413,952 (metal complexed receptor ligand agonist), and G120RhGH,
which is an analog of human growth hormone that acts as a prolactin
agonist (Mode et al., 1966, Endocrinol. 137(2): 447-454), and a
ligand for the prolactin receptor as described in U.S. Pat. Nos.
5,506,107 and 5,837,460.
[0048] PRL, GH and PL are members of a family of polypeptide
hormones that share a structural, immunological and biological
functions (reviewed in, "Pancreatic Growth and Regeneration", Ed.
N. Sarvetnick, Ch. 1. Brejie, T. et al., 1997), and therefore
referred to herein as the PRL/GH/PL family. PRL and GH are secreted
by the anterior pituitary of vertebrate animals. PRL is involved in
a broad range of biological functions that include osmoregulation,
reproduction, lactation, and immunomodulation. GH is associated
with physiological processes related to growth and morphogenesis.
The related receptor ligands are referred to as "PRL/GH/PL"
receptor ligands. The FACGINTs can be classified into various
groups based on structural similarity of the peptides and proteins,
functional similarity with respect to complementation of gastrin,
functional similarity with respect to binding of one or more
receptors, and these groups are each within the scope of various
embodiments of the invention.
[0049] Glucagon-like peptide-1 is synthesized in intestinal
endocrine cells in molecular forms GLP-1 (having residues
conventionally designated as positions 7-36) which is an amide, and
similarly as GLP-1 (7-37). Initial studies of GLP-1 biological
activity in utilized the full length N-terminal extended forms of
GLP-1 (1-37 and 1-36 which latter is an amide). The larger GLP-1
molecules were generally lacking biological activity. It was later
found that removal of the first six amino acids resulted in a
shorter version of the GLP-1 molecule having substantially enhanced
biological activity.
[0050] The majority of circulating biologically active GLP-1 is
found in the GLP-1 (7-36)amide form, with lesser amounts of the
bioactive GLP-1(7-37) form also detectable. See Orskov, C. et al.,
Diabetes 1994, 43: 335-339. Both peptides show about the same
amount of biological function. GLP-1 is secreted from gut endocrine
cells in response to nutrient ingestion and plays multiple roles in
metabolic homeostasis following nutrient absorption. Regulation of
GLP-1 occurs by N-terminal degradation of the peptide by Dipeptidyl
Peptidase (DPP-IV)-mediated cleavage at the position 2 alanine
residue. For an Overview, see DPP-IV. The biological activities of
GLP-1 include stimulation of glucose-dependent insulin secretion
and insulin biosynthesis, inhibition of glucagon secretion and
gastric emptying, and inhibition of food intake. GLP-1 appears to
have a number of additional effects in the GI tract and central
nervous system, as reviewed in Drucker, D., Endocrin 142: 521-527,
2001. Exemplary GLP-1 compositions include: BIM 51077 (GLP-1 analog
resistant to DPP-IV digestion, available from Beaufour Ipsen);
AC2592 (GLP-1, from Amylin, San Diego Calif.); ThGLP-1 (GLP-1,
modified amino acids and fatty acid attachment, from
Theratechnologies, Saint-Laurent, Quebec, Canada); DAC:GLP-1
(Conjuchem, Montreal, Quebec, Canada); CJC-1131 or DAC.TM.:GLP-1
(GLP-1 analog engineered for covalent coupling to albumin,
Conjuchem), LY315902 and sustained release LY315902 (DDP-IV
resistant GLP-1 analog from Eli Lilly, Indianapolis, Ind.); low
molecular weight GLP-1 mimetic, Albugon (albumin: GLP-1 fusion
peptide from Human Genome Sciences, Rockville, Md.); Liraglutide or
NN2211 (long acting GLP-1 derivative that is obtained by acylation
of the GLP-1 molecule, which upon entering the bloodstream, is
extensively bound to albumin which protects it from degradation by
DPPIV and reduces renal clearance; Elbrond et al., Diabetes Care
2002 Aug. 25(8): 1398-404).
[0051] Exendin-4 is a novel peptide from Heloderma suspectum(Gila
monster) venom, having 53% homology with GLP-1(7-36)amide. It
functions as a long-acting potent agonist of the glucagon-like
peptide 1 (GLP-1) receptor, as it is resistant to degradation by
DDP-IV. Exendin-4 has properties similar to GLP-1, and regulates
gastric emptying, insulin secretion, food intake, and glucagon
secretion. Examples of exendin-4 include exenatide (synthetic form
also known as AC2993, Amylin); exenatide LAR (long acting form);
ZP10 (modified exendin-4 having addition of six lysine residues,
Aventis/Zealand Pharma); and AP10 (long acting formulation,
Alkermes, Cambridge Mass.). Physiological studies indicate that
sustained expression of exendin-4 in transgenic mammals does not
perturb glucose homeostasis, cell mass or food intake (Biaggio, L.
et al. J Biol Chem 275: 34472-34477, 2000), so that the
physiological effects of exendin-4 are not completely
understood.
[0052] Dipeptidyl peptidase IV (DPP-IV) inhibitors refer to
compounds that inhibit activity of DPP-IV, a membrane-associated
peptidase of 766 amino acids that includes in its substrates GLP-1,
GLP-2 and GIP. DPP-IV-mediated inactivation of GLP-1 is a
determinant of GLP-1 bioactivity in vivo. Examples of DPP-IV
inhibitors include isoleucine thiazolidide, valine-purrolidide,
NVP-DPP738 (Novartis, Cambridge, Mass.), LAF237 (Novartis), P32/98
(Probiodrug AG, Halle, Germany), and P93/01 (Probiodrug).
[0053] As used herein, the term "EGF receptor ligand" encompasses
compounds that stimulate the EGF receptor such that when
gastrin/CCK receptors in the same or adjacent tissue or in the same
individual are also stimulated, neogenesis of insulin-producing
pancreatic islet cells is induced. Examples of such EGF receptor
ligands include full length EGF, which is EGF1-53, and further
include EGF1-48, EGF1-49, EGF1-52, and fragments and active analogs
thereof. Other examples of EGF receptor ligands are TGF.alpha.
forms that include 1-48, 1-47, 1-51, and amphiregulin and pox virus
growth factor as well as any EGF receptor ligands that demonstrate
the same synergistic activity with gastrin/CCK receptor ligands.
These include active analogs, fragments and modifications of the
above. See also, Carpenter and Wahl, Chapter 4, in Peptide Growth
Factors (Eds. Sporn and Roberts), Springer Verlag, 1990.
[0054] The group of compounds that comprises the EGF receptor
ligands further includes "modified EGF", which includes variants of
normal or wild type EGF. Modifications have been shown to affect
one or more biological activity such as the rate of clearance of
EGF. The term includes peptides having an amino acid sequence
substantially similar to that of human EGF, for example, with one
or a few amino acid substitutions at various residue positions.
[0055] Recombinant EGF forms have been genetically engineered to
have alterations in structure and activities, for example, EGF
having a methionine at position 21 replaced by a leucine residue
has been described (U.S. Pat. No. 4,760,023). Recombinant human EGF
(hEGF) having 51 residues, i.e., lacking the two C-terminal
residues at positions 52 and 53 of hEGF, and having a neutral amino
acid substitution at position 51, retain EGF activity and are more
resistant to protease degradation during a microbial production
process, and following administration to a subject. A series of
nucleic acid molecules have been described that encode a family of
proteins that have significant similarity to EGF and TGF.alpha. (WO
00/29438). EGF muteins (mutated EGF) having histidine at residue 16
replaced with a neutral or acidic amino acid have been described
(WO 93/03757), such forms retaining activity at low values of pH.
Chemical analogues and fragments of EGF and TGF.alpha. retain
ability to bind various members of the EGF receptor family (U.S.
Pat. No. 4,686,283). Various modifications of EGF or TGF.alpha.
confer advantageous properties affecting one or more of recombinant
protein production, in vitro and in vivo stability, and in vivo
activity. A exemplary recombinant modified EGF receptor ligand used
in the Examples herein is a C-terminus deleted form of human EGF of
51 amino acids in length, having asparagine at position 51
(referred to herein as EGF51N), which retains substantially full
I.N.T..TM. activity, and has in vivo and/or in vitro stability that
is mat is at least about as great or greater than normal or wild
type hEGF (S. Magil et al., published May 15, 2003 as
PCT/US02/33907, and incorporated by reference herein in its
entirety).
[0056] The term, "growth hormone" as used herein encompasses any
polypeptide that shares substantial amino acid sequence identity
with an endogenous mammalian growth hormone and possesses a
biological activity of a mammalian growth hormone. Human growth
hormone is a polypeptide containing 191 amino acids in a single
chain, and a molecular weight of about 22 kDal (Goeddel et al.,
1979, Nature 281: 544-548; Gray et al., 1985, Gene 39: 247-254).
The term encompasses analogs having deletions, insertions or
substitutions and growth hormones from other species and naturally
occurring variants. See Cunningham et al., 1989, Science 243:
1330-1336, and 1989, Science 244: 1081-1085; and WO 90/05185, and
U.S. Pat. No. 5,506,107.
[0057] The term "treating" or "ameliorating" as used herein means
reducing or eliminating one or more symptoms of diabetes. The term,
"diabetes" as used herein means any physiologic indication of a
shortage of insulin, a production of antibodies against insulin, or
an excess of blood sugar or any manifested symptoms of diabetes in
any mammal including experimental animal, models, and including
human forms such as type I and type II diabetes, early stage
diabetes, and a pre-diabetic condition characterized by mildly
decreased insulin or mildly elevated blood glucose levels. A
"pre-diabetic condition" describes a mammal suspected of having a
diabetic or related condition, for example, not formally diagnosed
with diabetes, but demonstrating a symptom in terms of insulin or
glucose level, and susceptibility to diabetes or a related
condition due to family history, genetic predisposition, or obesity
in the case of type II diabetes, or has previously had diabetes or
a related condition and is subject to risk of recurrence.
[0058] As used herein, the term "immunosuppressant" or "agent for
immune suppression" means any agent that suppresses immune
response. Exemplary immunosuppressant agents are shown in Table 1,
and any derivatives of those agents or functional equivalents are
considered appropriate for embodiments of the invention as
described herein and in the claims. Immunosuppressive agents in
Table 1 or other equivalent agents are administered as supplied by
the manufacturers, normalizing to body weight of the subject as is
known by one of skill in the pharmacological arts. For example,
Tacrolimus is generally administered by injection or orally, and
Sirolimus is generally administered orally
TABLE-US-00002 TABLE 1 Exemplary agents for immune suppression, and
commercial sources Names Company Nature 2-amino-1,3-propanediol
derivatives Novartis Used for preventing or treating chronic
rejection in a patient receiving an organ or tissue allo-or xeno-
transplant 2-amino-2[2-(4- Yoshitomi Immunosuppression, from
octylphenyl)ethyl]propane- Pharmaceutical accelerated lymphocyte
1,3-diol hydrochloride Industries, Ltd homing
40-O-(2-hydroxyethyl)- Novartis Sirolimus (rapamycin) rapamycin,
SDZ-RAD, Pharmaceuticals derivative, used for acute Everolimus
(Certican .RTM.) kidney rejection; reduces rejection and graft
vasculopathy following heart transplantation by inhibiting cell
proliferation 6-(3-dimethyl-aminopropionyl) forskolin Matsumori
Akia Immunosuppressing action Nippon Kayaju Co useful also for
treating Ltd autoimmune disease 6-mercaptopurine Glaxo SmithKline
Used to treat Crohn's (Purinethol .RTM., 6-MP) disease,
inflammatory, bowel disease and for organ transplant therapy
ABX-CBL (CBL-1) Abgenix Mouse monoclonal AB targeted against human
T- cell, B cells, NK cells and monocytes, fortreatment of
steroid-resistant graft vs host diseases, potential use in
treatment of inflammatory and. autoimmune disorders Alefacept
(human LFA-3 University of Utah- Knocks out causative IgG1 fusion
protein, Dermatology memory T-lymphocytes; AMEVIVE .RTM.)
Dept/BIOGEN Used to treat psoriasis, a T- cell mediated
inflammatory disorder HLA-B2702 peptide SangStat Medical Human
peptide, blocks (Allotrap .RTM.) action of NK cells and T- cell
mediated toxicities, used for prevention of first kidney allograft
rejection Antisense ICAM-1 ISIS-Boehringer Mouse monoclonal AB
inhibitor (ISIS 2302), Ingleheim blocks white blood cell
Enlimoinab, BIRR1, adhesion to T-cell surface Alicaforsen) molecule
(ICAM-1r); treatment of kidney transplant rejection Azathioprine
(Imuran .RTM., Generic, Glaxo Treatment of rheumatoid Azasan .RTM.)
SmithKline, arthritis and prevention of Prometheus kidney
transplant rejection, Laboratories, and other autoimmune or
aaiPharina inflammatory disorders such as inflammatory bowel
disease BTI-322 MedImmune Mouse derived monoclonal- AB targeted to
CD2 receptor; used for prevention of first-time kidney rejection,
and treatment of resistant rejection Cladribine (Leustatin .RTM.)
Boehringer Antimetabolite and Ingleheim immunosuppressive agent
that is relatively selective for lymphocytes; used to treat
lymphoid malignancies, e.g., hairy- cell leukemia. Cyclophosphamide
(CTX, Generic Immunosuppressant t for Neosar .RTM., Cytoxan .RTM.,
treatment of arthritis and Procytox .RTM.) other auto-immune
disorders and cancers Cyclosporine (cyclosporin Novartis 11 amino
acid cyclic A, cyclosporin) peptide; blocks helper T- (Sandimmune
.RTM., Neoral .RTM., cell, immunosuppressant SangCya .RTM.) used in
organ transplant therapy and other immune diseases
Demethimmunomycin'' (L- Merck & Co Treatment of autoimmune
683,742: also described as diseases, infectious diseases
31-desmethoxy-31- and/or prevention of organ hydroxy-L-683,590)
transplant rejections Dexamethasone Generic An adrenocorticoid,
(Decadron, Dexone, effective Dexasone) immunosuppressant in various
disorders Docosahexaenoic acid Not Immunosuppressant by that (DHA)
applicable lowers the proportion of T cells expressing CD4 or CD8,
blocks antigen recognition process; Taku et al., Journal of
Agricultural and Food Chemistry, 2000; 48(4):1047 FTY720 (oral
myriocin Novartis Alters lymphocyte derivative) Pharmaceuticals
infiltration into grafted tissues; used for prevention of organ
rejection in kidney transplants Glatiramer acetate Teva Synthetic
peptide (copolymer-1, Copaxone .RTM.) Pharmaceuticals copolymer,
decoy that mimics structure of myelin so immune cells bind Copaxone
instead of myelin; for multiple sclerosis Glial fibrillary acidic
CalBiochem; Synx Possesses protein (GFAP) Pharma immunosuppressive
activities in diabetic animal models; Winer et al., Nature Medicine
9: 198 (2003) Gusperimus,(15- Bristol Myers-Squibb Intravenous
deoxyspergualin immunosuppressant; (Spanidin .RTM.) suppresses
production of cytotoxic T-cells, neutrophils and macrophages hu1124
(anti-CD11a) XOMA Humanized monoclonal antibody; targets CD11a
receptor on surface of T cells to selectively inhibit immune system
rejection of transplanted organs Infliximab (Remicade .RTM.)
Centocor (affiliate of Monoclonal AB, binds and Johnson and
inactivates human TNF- Johnson) alpha and; used to treat Crohn's
disease and rheumatoid arthritis Interferon Various companies
Immunomodulatory including Serono, properties Biogen etc ISAtx247
Isotechnika Used to treat autoimmune diseases such as rheumatoid
arthritis and psoriasis isotretinoin Immunosuppressant, reduces
ability of T cells to proliferate in response to immune challenge.
Vergelli et al., Immunopharmacology, 1997, 31:191. Medi-500 (T10B9)
MedImmune Intravenous monoclonal AB that targets human T-cells;
treats acute kidney rejection and graft-vs-host disease Medi-507
MedImmune/Bio- Intravenous humanized AB Transplant directed.
against CD2 T-cell; used to treat corticosteroid- resistant graft
vs host disease and prevention of kidney rejection Methotrexate
Wyeth Lederle, Antimetabolite used to treat (Rheumatrex .RTM.,
Generic Crohn's disease, severe Amethopterin, Trexall .RTM.)
psoriasis, and adult rheumatoid arthritis (and as an anti-cancer
drug) Mitoxantrone Immunex Antiproliferative effect on (Novantrone
.RTM.) cellular immune system including T-cells, B-cells and
macrophages; used to treat hormone-refractory prostate cancer,
acute myelogenous leukemia and multiple sclerosis mycophenolate
mofetil Roche Proliferation of T and B (CellCept .RTM.) lymphocytes
by blocking the synthesis of purine nucleotides; used in organ
transplant therapy and inflammatory bowel disease OKT4A R. W.
Johnson Mouse monoclonal AB Pharmaceutical targeted against human
ReSearch Institute CD4 T cell; used for prevention of kidney
transplant rejection when used in combination with other
immunosuppressant drugs Muromonab-CD3 R. W. Johnson Monoclonal AB
that binds (Orthoclone OKT3 .RTM.)0 Pharmaceutical to receptor
sites on T-cells, Research Institute preventing activation by
transplanted tissue Prednisolone (Deltasone .RTM., Corticosteroid,
suppresses Oraone .RTM.) inflammation associated with transplant
rejection basiliximab (Simulect .RTM.) Novartis Monoclonal AB that
binds Pharmaceuticals to receptor sites on T-cells, preventing
activation by transplanted tissue (renal transplant) S100.beta.
glial protein Possesses immunosuppressive activities in diabetic
animal models Sirolimus, Rapamycin Wyeth-Ayerst Immunosuppressant
and (Rapamune .RTM.) Laboratories potent inhibitor of cytokine
(e.g. IL-2)-dependent T-cell proliferation (kidney transplant)
Tacrolimus (Prograf; FK-506) Fujisawa Interferes with IL-2 TCR
communication Antithymocyte SangStat Medical Anti-human thymocyte
immunoglobulin Corporation, immunoglobulin; used in ATGAM,
Pharmacia and reversal of acute kidney Thymoglobulin .RTM. Upjohn
transplant rejection and will likely be used off-label for
transplant induction therapy efalizumab (Xanelim .RTM.) XOMA T-cell
modulator that target T-cells through interactions with adhesion
molecules on endothelial cell surface, target migration of T-cells
into the skin and target activation of T-cells; Used to treat
Psoriasis Daclizumab (Zenapax .RTM.) , Protein Design Monoclonal AB
inhibits HAT (Humanized Anti- Laboratories/Roche binding of IL-2 to
IL-2 Tac), SMART anti-Tac, receptor by binding to IL-2 anti-CD25,
and humanized receptor; suppresses T cell anti -IL2-receptor
activity against allografts (renal transplant)
[0059] Hypoglycemic agents or drugs can be insulin response
enhancers, and are typically used as for glycemic control in
diabetic patients. These agents include but are not limited to
sulfonylureas (e.g. acetohexamide, chlorpropamide, tolazamide,
tolbutamide, glyburide, glipizide, glimepiride), meglitinides (e.g.
repaglinide, nateglinide), biguanides (e.g. metformin),
thiazolidinediones (e.g. pioglitazone, rosiglitazone),
alpha-glucosidase inhibitors (e.g. Miglitol), glucagons
antagonists, potassium channel openers, insulin sensitizers,
hepatic enzyme inhibitors, glucose uptake modulators, compounds
modifying lipid metabolism and compounds lowering food intake.
Combinations of these agents can be used with the gastrin compounds
provided herein.
[0060] As used herein the term mammal shall include without
limitation any members of the Mammalia, such as a human, an ape, a
rodent such as a mouse or rat, a dog, a cat, an agriculturally
important animal or a protein pig, a goat, a sheep, a horse, or an
ape such as a gorilla or a chimpanzee. An individual mammal may be
non-diabetic, pre-diabetic, or diabetic, as specified herein.
[0061] Modes of systemic administration include, but are not
limited to, transdermal, intrathecal, intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal, and oral
routes. The compounds may be administered by any convenient route,
for example, by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal,
vaginal, nasal, and intestinal mucosa, etc.), and may be
administered together with other biologically active agents. An
exemplary route of administration is systemic, for example, by
subcutaneous injection. The gastrin compound if administered with a
FACGINT or immunosuppressant or a hypoglycemic agent can be
administered in a single combined dose, or the components can be
administered separately in any order.
Methods of Preparation of Compositions
[0062] Synthesis of Gastrin Peptides
[0063] Gastrin peptides can be produced by any suitable means, such
as expression in a recombinant host cell or by chemical synthesis.
For the latter, gastrin peptides for instance can be synthesized by
using solid phase Fmoc peptide synthesis on a
polydimethylacrylamide gel resin. The polypeptide is then purified
by standard methods.
[0064] Conjugating Partners/Carriers
[0065] Conjugating partners/carriers include plasma components such
as serum obtained from the patient, purified serum protein(s) such
as albumin, transferrin or an immunoglobulin, red blood cell
proteins such as glycophorin A and AE-1, sugar binding proteins
such as lectin, inactivated enzymes, phosphate and sulfate binding
proteins, cholic acid, lipids binding proteins, lipids/lipophilic
moieties/hydrophobic moieties; polymeric carriers such as dextran
or polyethylene glycol. When gastrin is conjugated to the
lipids/lipophilic moieties, it is possible that these conjugates
once injected can interact either non-covalently or covalently with
serum proteins such albumin which is known to bind to fatty acids,
for instance or lipid binding proteins in the serum. For the
purposes of the compositions and methods herein, serum and plasma
may be used interchangeably.] In order to conjugate gastrin to a
carrier, the carrier may first need to be activated via the
introduction of a reactive group. In some cases, the carrier can
already contain at least one reactive group, for instance cysteine
34 on albumin. After activation of the carrier (if necessary), the
carrier is conjugated to gastrin compound, hi general, carriers can
be covalently attached to proteins via reactive groups in the
protein chain, such as thiol groups, alpha and epsilon amino
groups, carboxyl groups or aromatic rings, all of which may already
be present, or can be added by preliminary chemical modification of
the protein which can be either incorporated during chemical
synthesis or by chemically modifying existing gastrin peptides or
by modifying a protein's amino acid sequence, using known molecular
biology methods. For instance, carrier can be attached to the
protein's epsilon amino groups in lysine residues or the thiol
groups on cysteine residues located at the N-terminal end.
Alternatively, the carrier can be attached to the protein via a
heterobifunctional or homobifunctional crosslinking agent.
[0066] Plasma Proteins
[0067] Plasma protein binding can be an effective means of
improving the pharmacokinetic properties of otherwise short-lived
molecules such as gastrin. One aspect of plasma proteins is a
native molecular mass larger than the kidney filtration cutoff
(.about.45 kDa), and thus an extended residence time in plasma.
Plasma proteins include but are not limited to Albumin, Alpha-1
Acid Glycoprotein, Alpha-1-Antichymotrypsin, Alpha-1-Antitrypsin,
Alpha-2-Antiplasmin, Alpha-2-HS-Glycoprotein,
Alpha-2-Macroglobulin, Angiotensinogen, Antithrombin III,
Apolipoprotein AI (HDL), Apolipoprotein AII (HDL), Apolipoprotein B
(LDL), Apolipoprotein CI (VLDL), Apolipoprotein CII (VLDL),
Apolipoprotein CIII (VLDL), Apolipoprotein E (VLDL),
Apotransferrin,
C1 Esterase Inhibitor, Ceruloplasmin, Ferritin, Fibrinogen, GC
Globulin, Haptoglobin (mixed type), Hemoglobin, Immunoglobin A,
Immunoglobin A1, Immunoglobin A1, Immunoglobin A2, Immunoglobin D,
Immunoglobin E, Immunoglobin G, Fab Fragment, Immunoglobin G, Fc
Fragment, Immunoglobin G, Immunoglobin G1, Immunoglobin G2,
Immunoglobin G3, Immunoglobin G4, Immunoglobin M, .mu. Chain,
Immunoglobin M, Fcs.sub.5.mu., Immunoglobin M, Immunoglobulin heavy
chain (H) Immunoglobulin light chain (L)-K-light chains,
gamma-light chains, Fab fragment of an immunoglobulin Fc fragment
of an immunoglobulin, Lactoferrin, Lipoprotein a, [Lp(a)],
Lipoprotein (high density), Lipoprotein (Low Density), Lipoprotein
(Very Low Density), Prealbumin, Prothrombin, Prothymosin-.alpha.,
Rheumatoid Factor, Steroid binding proteins, Transcortin,
Thyroxine-binding globulin. Transferrin and .alpha.-fetoprotein. A
list of plasma proteins can be found in Anderson and Anderson,
Molecular and Cellular Proteomics 2002, 1.11: 845.
[0068] Human serum albumin has a molecular mass of .about.67 kDa, a
half-life of 19 days in circulation, and is the most abundant
protein in plasma human. Albumin consists of 585 amino acids
forming a single polypeptide chain. Albumin interacts with a large
number of compounds including physiological ligands such as long
chain fatty acids, certain therapeutic drugs such as Warfarin and
Valproate and inorganic ligands such as calcium.
[0069] The antibody/immunoglobulin can include a complete
immunoglobulin or fragment thereof, where immunoglobulins include
the various classes and isotypes, such as IgA, IgD, IgE, IgG1,
IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab,
Fv and F(ab').sub.2, Fab', and the like. In addition, fragments,
aggregates, polymers, or conjugates of immunoglobulins can be used
where appropriate. The antibody can be monoclonal or polyclonal and
can be prepared by commonly used techniques such as immunization of
a host and collection of sera (polyclonal) or by preparing
continuous hybrid cell lines and collecting the secreted protein
(monoclonal), or by cloning and expressing nucleotide sequences or
mutated versions thereof coding at least for the amino acid
sequences required for specific binding of natural antibodies.
[0070] Polymeric Carriers
[0071] The conjugating partners can also include polymers, which in
various embodiments can be naturally occurring or synthetic.
Examples of polymers include proteins, glycopeptides,
polysaccharides such as dextran such as aminodextran or
carboxymethyldextran, and bipolymer derivatives of dextran such as
dextran sulfate, hydroxyethylstarch, cellulose and cellulose
derivatives, including methylcellulose and carboxymethyl cellulose,
starch and starch derivatives, inulin, heparin, heparin fragments;
synthetic polymers such as polyalkyl glycols (PAG) such as PEG and
derivatives thereof, polyoxyethylated polyols (POP) such as
polyoxyethylated glycerol (POG), polytrimethylene glycol (PTG),
polypropylene glycol (PPG), polyhydroxyethyl methacrylate,
polyvinyl alcohol (PVA), polyacrylic acid, polyethyloxazoline,
polyacrylamide, polyvinylpyrrolidone (PVP), polyamino acids,
polyurethane and polyphosphazene, poly(lactic acid-co-ethylene
glycol PLA-PEG, poly(D,L-lactic-co-glycolic acid PLGA,
poly(orthoester), Poly(lactide-co-glycolide)-block-poly(ethylene
glycols, polyoxyethylated polyols, polyvinylpyrrolidone,
polyhydroxyethyl methacrylate, polyvinyl alcohols, and polyurethane
ol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA),
poly(N-isopropylacrylamide, polyoxyethylated polyols,
polyhydroxyethyl methacrylate, N-(2-hydroxypropyl)methacrylamide
(HPMA), synthetic copolymers of hydrazone, polyvinyl pyrrolidone,
polyvinyl alcohol, polyamino acids, divinylether maleic anhydride,
N-(2-Hydroxypropyl)-methacrylamide, polypropylene glycol,
polyalkylene glycol and derivatives thereof, copolymers of
polyalkylene glycols and derivatives thereof, polyvinyl ethyl
ethers, and .alpha.,.beta.-Poly[(2-hydroxyethyl)-DL-aspartamide,
poly(N-acryloylmorpholine (PacM). In general, the synthetic
polymers can also be linear or branched, substituted or
unsubstituted, homopolymeric, or co-polymers of two or more
different synthetic monomers.
[0072] Pegylation of peptides and proteins can result in enhanced
therapeutic properties with retention of biological function.
Conjugation with PEGs (or with other polymers, like dextrans), by
increasing molecular size, can reduce the processes of renal
filtration, immune response, and degradation by proteolytic
enzymes, all of which act to enhance stability in vivo. PEG
(polyethylene glycol) is a linear strand of repeating monomers,
which can be chemically activated at terminal free hydroxyl groups.
Such activation can result in reactivity to a number of functional
groups, including sulfhydryl and amino groups. To prevent
cross-linking, one of the two terminal hydroxyl groups may be
capped with a methoxy group (mPEG). PEGs with multiple forks or
branches can also be synthesized, allowing for multiple points of
attachment for peptides on an individual PEG molecule.
[0073] Peptides containing a free sulfhydryl group (as Cysteine,
abbreviated Cys using the three-letter amino acid designation) will
readily undergo an alkylation reaction with at least one of mPEG
modified with maleimide, forming a stable thioester bond. In the
case of gastrin, a synthesized gastrin compound with a Cysteine
residue introduced in a region of the peptide that is not required
for its biological activity (for example, at its N-terminal), can
be used.
[0074] Peptides containing free amines (as Lysine or N-terminal
amino groups) can react with PEGs modified with succinimidyl esters
to produce stable amide linkages. Due to the lack of specificity of
the chemical reaction, as in the case of gastrin-34, which contains
3 lysine residues, any one, or more, of the lysine residues could
react with the modified PEG, resulting in a chemically, and
possibly functionally, heterogeneous mixture of conjugated
peptides, purification of which may not be trivial. Site-specific
PEGylation of a particular lysine residue may be easily attained
during peptide synthesis, by introducing a lysine residue in which
a PEG is already linked to the .epsilon.-amino side chain (as
described by Felix (1997)). Felix, A. M. (1997) Site-Specific
Poly(ethylene glycol)ylation of Peptides. In "Poly(ethylene
glycol). Chemistry and Biological Applications." Harris, J. M.
& Zalipsky S. Eds.
[0075] The "activated PEG" (or "pegylating agent") is any PEG
derivative, which can be used as protein modifier, because it
contains a functional group capable of reacting with some
functional group in the protein/peptide to produce the
PEG-protein/peptide conjugates. Activated PEGs can include
alkylating PEGs, acylating PEGs and PEG with an amino acid arm. One
can also pegylate at cysteine residue by using PEG maleimide (e.g.
mPEG-maleimide-20,000 from Shearwater Corporation, Huntsville,
Ala.). Amino PEG can be used to pegylate carboxyl groups. Examples
of active PEGS include methoxypolyethylene glycol,
diaminomethoxy-polyethylene glycol, methoxypolyethylene
glycol-p-nitro-phenylcarbonate, methoxypolyethylene glycol
succinimidyl succinate, methoxypolyethylene glycol tresylate,
methoxy-polyoxyethylene amine (aminoPEG),
methoxypolyoxyethylene-carboxylic acid, monomethoxy-PEG
p-nitrophenyl carbonate, N-N'-Carbonyldiimidazole-activated PEG and
methoxypolyoxyethyleneimidazole-carbonyl. PEGs of different
molecular weights are available commercially. The average molecular
weight of the reactant PEG can range from between about 5,000 and
about 100,000 daltons. The method of attachment is not critical,
but preferably does not alter, or only minimally alters, the
activity of the biologically active molecule. A preferred method of
attachment is via N-terminal linkage to a polypeptide. See
Veronese, Biomaterials 22(5):405 (2001) for a review of the
different activation strategies for PEG. Alternatively PEG can be
conjugated to the peptide/protein via glycopegylation by first
derivatizing gastrin with sugars molecules prior to conjugation
(i.e. glycopegylation approach e.g. using Neose's GlycoPEGylation
technology).
[0076] Dextran is a naturally occurring polymer that consists of
mainly a linear polysaccharide of repeating units of D-glucose
linked together in glycosidic bonds. Branch points may be present
in a dextran polymer and the branch type and degree of branching
vary by species. Dextran can be used here to conjugate to gastrin
compounds. The average molecular weight of the soluble dextran can
range from between about 10,000 and about 500,000 daltons. Dextran
polymer contains adjacent hydroxyl groups on each glucose monomer
that can be oxidized by sodium periodate to give a functional
aldehyde group that can then react with an amino group.
Polyaldehyde dextran can conjugate to amine groups by Schiff base
formation followed by reductive amination to create stable linkage,
for instance to the epsilon amino group on lysine residue. Dextrans
can be carboxymethylated by reaction with monochloroacetic acid to
give carboxymethyldextran, which can form dextran-hydrazide on
condensation with hydrazine, which reacts with the carbonyl group
or aldehyde groups. Sulfhydryl reactive dextran derivatives can be
prepared through the use of heterobifunctional crosslinking agent
containing for instance pyridyldisulfide, maleimide or iodoacetyl
groups on one end to direct the conjugation reaction to sulfhydryl
groups.
[0077] Conjugation to Lipophilic Moieties
[0078] A lipophilic substituent can be attached to a reactive group
of an amino acid at the N-terminal end of the gastrin peptide, or
optionally to a reactive group generated via a heterobifunctional
or homobifunctional crosslinker group. For instance, the carboxyl
group of the lipophilic substituent can react with the amino group
on lysine. For instance, the lipophilic substituents are in
particular long-chain groups containing e.g. 8-40 carbon atoms. For
instance, the lipophilic substituents can be a straight chain or
branched alkyl group, an acyl group of a straight chain or branched
fatty acid, an acyl group of a straight chain or branched alkane
.alpha.,.omega.-dicarboxylic dicarboxylic acid. For example,
lipophilic derivatives of gastrin can be synthesized by chemical
attachment to an amino group, for instance, at the N-terminal with
various fatty acids including lauric acid (n-dodecanoic acid),
myristic acid (n-tetradecanoic acid), palmitic acid (n-hexadecanoic
acid), palmitoleic acid (n-hexadecenoic acid), stearic acid
(n-octadecanoic acid), oleic acid (n-octadecenoic acid), acetic
acid, linoleic acid and arachidonic acid.
[0079] Alternatively, a hydrophobic moiety that is conformationally
rigid (i.e. presence of a double bond, a triple bond, or a
saturated or unsaturated ring) can be attached to the gastrin
peptide. For instance, a long chain fatty acids comprising of a
carboxyl group can be attached to amino groups found for example on
lysine residue.
[0080] Bifunctional Crosslinking Agents
[0081] Examples of crosslinking agents include but are not limited
to the following: Amino group directed homobifunctional
cross-linking reagents include: Bisimidoesters (Bisimidates), e.g.
methyl acetimidate-HCl, dimethyl suberimidate-2HCl;
Bis-N-Succinimidyl Derivatives, e.g. bis(sulfosuccinimidyl-suberate
(BSSS), succinate bis-(N-hydroxy-succinimide ester); Bifunctional
Aryl Halides, e.g. 1,5-dichloro-2,4-dinitrobenzene; Bifunctional
Acylating Agents, such as diisocyanates and diisothiocyanates, e.g.
1,6-hexamethylene diisocyanate; bifunctional sulfonyl halides, e.g.
phenol-2,4-disulfonyl-chloride; bis-nitrophenol esters, e.g.
bis-(p-nitrophenyl ester) of carboxylic acids; and bifunctional
acylazides, e.g. tartryl diazide; Dialdehydes, e.g.,
glutaraldehyde; Diketones, e.g. 2.5-hexanedione; and others such as
benzoquinone, 2-iminothiolane, erythreitolbiscarbonate, mucobromic
acid, mucochloric acid, ethylchloroformate,
p-nitrophenylchloroformate, succinimidyl 6-hydrazinonicotinate
acetone hydrazone and succinimidyl 4-formylbenzoate (for instance
the first modifies an amine with a hydrazine linker and the second
modifies an amine to an aldehyde linker, which can then be
crosslinked together
[0082] Another group of bifunctional crosslinking agents are the
Sulfhydryl group directed homobifunctional crosslinkers, which
include: Mercurial Reagent, e.g. 1,4-bis(bromomercuri)butane;
Disulfide Forming Reagents, e.g.
polymethylenebis(methanthiosulfonate); Bismaleimides, e.g.
N,N'-methylenebismaleimide, Bis (N-maleimidomethyl)ether,
bis-maleimidoethane, 1,4-bis-maleimidobutane,
1,4-bis-maleimidyl-2,3-dihydroxybutane, bis-maleimidohexane,
1,8-bis-maleimidotriethyleneglycol. Yet another group are
Alkylating Agents such as Bio-haloacetyl derivatives, e.g.
1,3-dibromoacetone; Di-alkyl halides, e.g.
di(2-chloroethyl)sulfide; S-triazines, e.g.
2,4-dichloro-6-methoxy-s-triazine; Aziridines, e.g.
2,3,4-tri(ethyleneimino)-s-triazine; and Bis-epoxides, e.g.
1,2,3,4-diepoxybutane; and others e.g. divinyl sulfone.
[0083] Other group directed homobifunctional crosslinkers are known
in the art of chemical cross-linking, such as Carboxyl group
directed reagents, e.g. bisdiazohexane; Phenolate and Imidazolyl
group directed reagents, e.g. bis-benzidine; Arginine Reagent, e.g.
p-phenylenediglyoxal; and others e.g.
ds-dichlorodiaminoplatinum(II) (used to crosslink
alpha2-macroglobulin with one or more methionine residues at or
near the recognition site, may crosslink complementary strands of
DNA adipid acid dihydrazide as well as crosslink glycoproteins,
acid phosphatase and invertase),
N,N'-bis(b-aminoethyl)-tartramide.
[0084] Also know in the art are Group selective heterobifunctional
crosslinkers, which include: Amino and sulfhydryl group directed
bifunctional reagents, e.g., N-succinimidyl 3-(2-pyridyldithio
propionate) (SPDP) and its analogs (LC-SPDP, Sulfo-LC-SPDP),
Succinimidyloxycarbonyl-.alpha.-methyl-.alpha.-(2-pyridyldithio)toluene
(SMPT) and its analog
Sulfosuccinimidyl-6-{.alpha.-methyl-.alpha.-(2-pyridyldithio)toluamido]he-
xanoate (Sulfo-LC-SMPT),
Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)
and its analog, Sulfosuccinidimidyl
4-(N-maleimidomethyl-cyclohexane-1-carboxylate (Sulfo-SMCC),
m-Maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) and its analog
m-Maleimidobenzoyl-N-hydroxy-sulfosuccinimide ester (Sulfo-MBS),
N-succinimidyl(4-iodoacetyl)-aminobenzoate (SIAB) and its analog,
Sulfo-succinimidyl(4-iodoacetyl-aminobenzoate (sulfo-SIAB),
Succinimidyl-4-(p-maleimidophenyl)butyrate (SMBP) and its analog,
Sulfosuccinimidyl-4(p-maleimidophenylbutyrate (sulfo-SMPB),
N-.gamma.-Maleimidobutyryl-oxysuccinimide ester (GMBS) and its
analog (Sulfo-GMBS), succinimidyl 6-[(iodoacetyl0)-amino]hexanoate
(SIAX) and its analog sucinnimidyl
6-[6-(((iodoacetyl)amino)-hexanoyl)amino]hexanoate (SIAXX),
Succinimdl 4-(((iodoacetyl)amino)methyl)-cyclohexane-1-carboxylate
(SIAC) and its analog, succinimidyl
6-((((4-(iodoacetyl)amino)methyl)-cyclohexane-1-cabonyl)amino)hexanoate
(SIACX), N-succinimidyl 4-maleimidobutyrate; Carboxyl and either
sulfhydryl or amino group directed bifunctional reagents, e.g.,
p-nitrophenyl diazoacetate; Carbonyl and sulfhydryl group directed
bifunctional-reagents, e.g.
1-(aminooxy)-4-[(3-nitro-2-pyridyl)dithio)]butane;
4-(-4-N_maleimidophenyl)butyric add hydrazide hydrochloride (MPBH),
4-(N-Maleimidomethyl)cyclohexane-1-caboxyl-hydrazide
(M.sub.2C.sub.2H), 3-(2-Pyridyldithio)propionyl hydrazide (PDPH)
and others e.g.
2-methyl-N'-benzenesulfonyl-N.sup.4-bromoacetylqumonediimide,
N-hydroxysuccinimidyl-p-formylbenzoate,
methyl-4-(6-formyl-3-azido-phenoxy)butyrimidate HCl, acrolein.
[0085] Further included in embodiments of suitable cross-linking
agents are the group of Zero-length crosslinking agents, which
includes: Carboxyl group activating agents, such as carbodiimides
e.g., 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
(EDC) (activates a carboxyl group than can then crosslink with a
NH2 group); isoxazolium derivatives; chloroformates;
carbonyldiimidazole; and N-carbalkoxydihydroquinolines; Disulfide
forming reagent e.g. cupric di(1,10-phenanthroline); Enzymes e.g.
transglutaminase, peroxidase (between lysine residues), xanthine
oxidase (forms disulfide bonds); and others e.g.
pyrroloquinolinequinone (converts lysines to semi-aldehydes that
can then react with other lysine residues).
Pharmaceutical Compositions
[0086] The present invention in various embodiments provides
pharmaceutical compositions comprising a therapeutically effective
amount of a combination of the gastrin compound alone or the
combination of a FACGINT, or an hypoglycemic agent with a gastrin
compound. All of the pharmaceutical compositions described herein
can be formulated with or without an agent for immune suppression,
and with or without components or devices for sustained release,
for delivery locally or systemically. A pharmaceutically acceptable
carrier or excipient can be added. Such a carrier includes but is
not limited to saline, buffered saline, dextrose, water, glycerol,
ethanol, and combinations thereof. The formulation should suit the
mode of administration. An "effective amount" as the term is used
herein is an amount of a therapeutic agent or combination of agents
sufficient to achieve a recognized medical endpoint, in this case,
remediation of a symptom of diabetes. The effective amount can be
determined empirically by a skilled artisan according to
established methods of measurement of relevant parameters, as
described herein.
[0087] The compositions herein can further comprise wetting or
emulsifying agents, or pH buffering agents. The composition can be
a liquid solution, suspension, emulsion, tablet, pill, capsule,
sustained release formulation, or powder. The compositions can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Various delivery systems are known and can be used
to administer a composition of the invention, e.g., encapsulation
in liposomes, microparticles, microcapsules and the like.
[0088] In an exemplary embodiment, a composition herein is
formulated in accordance with routine procedures as a
pharmaceutical composition adapted, for example, for subcutaneous
administration to human beings. Typically, compositions for
subcutaneous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic to ameliorate pain at the
site of the injection. Generally, the ingredients are provided
either separately or mixed together in unit dosage form, for
example, as a dry, lyophilized powder or water-free concentrate in
a hermetically sealed container such as an ampoule or sachette, for
example, indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed
with an infusion bottle containing sterile pharmaceutical grade
water, buffer, or saline. Where the composition is administered by
injection, an ampoule of sterile water or saline for injection can
be provided so that the ingredients may be mixed prior to
administration. The compositions herein can in various components
thereof be formulated as suppositories, which contain active
ingredient in the range of about 0.5% to about 10% by weight; oral
formulations preferably contain about 10% to about 95% active
ingredient by weight. A daily dose is administered as a single
dose, or is divided into a plurality of smaller fractional doses,
to be administered several times during the day.
[0089] As used herein, a dosing schedule refers to a protocol for
administering any of the compositions comprising for instance a
modified gastrin compound as provided herein, or the modified
gastrin in combination with a FACGINT or with a hypoglycemic agent
and/or with an immunosuppressant, each in an effective dose,
administered simultaneously or within a particular interval of each
other, for example, within one day of each other, or as a combined
preparation, or separately, and includes the amount of the
composition delivered per unit time such as per day, and the
duration or period of time over which each composition is
administered.
[0090] In one aspect, the invention provides a method for
preventing or treating diabetes, the method comprising
administering to a mammal in need thereof composition of gastrin
compound alone or in combination with a FACGINT a FACGINT or with a
hypoglycemic agent and/or with an immunosuppressant, each in an
amount sufficient to increase the number of pancreatic insulin
secreting .beta. cells in the mammal; and determining the amount of
islet neogenesis, thereby treating or preventing the diabetes.
Determining the amount of islet neogenesis is measuring a parameter
selected from the group of: blood glucose, serum glucose, blood
glycosylated hemoglobin, pancreatic .beta. cell mass, serum
insulin, and pancreatic insulin content. Administering the
composition described herein reduces blood glucose compared to
blood glucose assayed prior to administering the composition, for
example, administering the composition reduces blood glucose by
about 50%, or by about 70%, compared to blood glucose assayed prior
to administering the composition. Glycosylated hemoglobin
concentration is reduced compared to glycosylated hemoglobin
concentration in the mammal assayed prior to administering the
composition. Serum insulin concentration is increased compared to
serum insulin concentration in the mammal assayed prior to
administering the composition. Pancreatic insulin concentration is
increased compared to pancreatic insulin concentration in the
mammal assayed prior to administering the composition.
[0091] The compositions, of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0092] The amount of the therapeutic of the invention which will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques. The precise dose to be
employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Routine determinations of blood
levels of insulin or C peptide, and of fasting levels of glucose or
glucose challenges, are determined by one of ordinary skill in the
art. Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems, by one of
ordinary skill in the art of pharmacology. Dosages of the
compositions to be administered to a subject are adjusted for known
variations from species to species using standard data encompassing
criteria for absorption, distribution, half-life kinetics in
circulation, metabolism, excretion, and toxicology of the receptor
ligands of the embodiments herein. Suitable dosage ranges for
administration are generally about 0.01 micrograms to about 10,000
micrograms of each active compound per kilogram body weight per
day, for example, about 0.01 micrograms to about 1 microgram/kg,
about 0.1 micrograms/kg to about 10 micrograms/kg, about 1
microgram/kg to about 500 micrograms/kg, or about 10 micrograms/kg
to about 10 mg/kg of body weight per day. Suitable dosage ranges
for administration are thus generally about 0.01 micrograms/kg body
weight/day to about 10 mg/kg body weight/day.
[0093] The invention in other embodiments provides a pharmaceutical
pack or kit comprising one or more containers filled with one or
more of the ingredients of the pharmaceutical compositions of the
invention. In such a pack or kit can be found a container having a
unit dosage of the extended use gastrin compound. Associated with
such containers) can be various written materials such as
instructions for use; or a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0094] Unless otherwise defined, all technical and scientific terms
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains. Methods
and materials similar or equivalent to those described herein can
be used in the practice of the present invention. The invention in
various embodiments now having been fully described, additional
embodiments are exemplified by the following Examples and claims,
which are not intended to be construed as further limiting. The
contents of all cited references are hereby incorporated by
reference in their entirety herein.
EXAMPLES
Example 1
Pharmacokinetic Example of Unmodified Gastrin Following
Administration by Intravenous Injection to Male Cynomolgus
Monkeys
[0095] This example was conducted to assess the pharmacokinetic
(PK) profile of unmodified gastrin (referred to herein as compound
B; see Table 3) following administration by a single intravenous
injection to male cynomolgus monkeys. The 17 amino acid gastrin
analog used has a single amino acid change at position 15, where
methionine has been substituted by leucine. While compound B thus
is not identical to naturally occurring gastrin, all available
evidence indicates that it is functionally equivalent to
gastrin.
Terms associated with PK analysis terms are defined as follows:
C.sub.max--The maximum observed plasma concentration
t.sub.max.--The time to maximum concentration AUC--Area under the
curve, a measure of total exposure to a drug over a period of time
Plasmat.sub.1/2--A measure of how long a drug stays in the blood,
the time it takes for the plasma concentration to fall 50%
Administration of Gastrin
[0096] Male animals were used in the example. Each animal received
gastrin by intravenous administration (3 .mu.g/kg, 10 .mu.g/kg and
30 .mu.g/kg). The dosing solutions for intravenous injection were
administered as a single bolus dose via the saphenous vein at a
dose volume of 1 mL/kg. A 0.2 mL saline flush was administered
following dosing in order to ensure administration of the complete
dose volume. The actual volume administered to each animal was
based on the animal's most recent body weight 1
Blood Collection
[0097] Serial blood samples (approximately 0.5 ml per time point)
were collected via the brachial or femoral vein at the following
time points: 0 (pre-dose), 1, 3, 5, 10, 15, 30 minutes, 1, 2, and 4
hours post dose.
[0098] Each sample was collected into a tube containing EDTA and
kept on wet ice pending centrifugation. The samples were
centrifuged under refrigeration for a minimum of 10 minutes at
1,500 g (RCF) and the resultant plasma was transferred into
duplicate tubes and placed on dry ice. All samples were stored
frozen for PK analysis as described below.
Assay for Gastrin Levels
[0099] Gastrin in plasma samples was measured with a well
established competitive radioimmunoassay for the quantitative
determination of gastrin (Russell et al., Postgraduate Medical
Journal 52: 645, 1976). The antibody used in the assay was raised
in rabbits against synthetic human gastrin I conjugated with
carbodiimide to bovine serum albumin. The antigen was labeled with
.sup.125I and the presence of the antigent:antibody reactions were
detected using a gamma counter.
Pharmacokinetics Analysis
[0100] To calculate pharmacokinetic parameters, blood levels of
gastrin at each time point for every animal were entered in to
Graphpad Prism 3.0 (GraphPad Software, San Diego, Calif., USA,
www.graphpad.com).
TABLE-US-00003 TABLE 2 Summary of mean PK parameters for gastrin in
primates after intravenous injection Dose COMPOUND Cmax AUC B
(.mu.g/kg) t 1/2 (min) (pg/ml) Tmax (min) (ng.ml/min) 3 5 27000 2
251 10 4 127487 1 939 30 5 267625 1 2526
[0101] Data from table 2 demonstrated the relatively short plasma t
1/2 of COMPOUND B which averages around 4-5 minutes for each of the
three dose groups. COMPOUND B appeared to be rapidly cleared from
the bloodstream of primates after a single bolus administration.
With increasing intravenous dose of COMPOUND B, an increasing
values of Cmax and AUC were observed, however increasing the dose
did not appear to affect the value of t 1/2. These data show that
administering greater amounts of COMPOUND B does not enhance short
lifetime of this peptide in circulation. In summary these data
demonstrate that gastrin is cleared from the blood stream within
minutes. Thus, the presence of biologically active compound in the
serum is limited by the rapid clearance of gastrin from the
serum.
Example 2
Effect of Unmodified Gastrin on Fasting Blood Glucose Levels and
Pancreatic Insulin Content in NOD Mice with Recent Onset
Diabetes
[0102] Non-obese diabetic (NOD) female mice were monitored for
diabetes development as determined by a fasting blood glucose (FBG)
level of >6.6 mmol/l. After diabetes onset, mice were treated
with (i) vehicle (n=4); or, (ii) gastrin (compound B as listed in
Table 3) in the amount of 3 .mu.g/kg/day, given i.p. once daily
(n=5) for 14 days. Mice did not receive insulin-replacement
treatment. Fasting blood glucose levels and pancreatic insulin
content were assessed for the two treatment groups at both day 0
and day 35 (21 days after cessation of treatment).
[0103] FIG. 1 shows that in the vehicle-treated control animals,
fasting blood glucose levels (FBG) were doubled after 35 days. In
contrast, treatment with gastrin prevented some of the increase in
glucose levels from rising in the diabetic NOD mice, however, the
FBG levels remained significantly higher than that observed in
normal mice (.about.3-7 mM). These data show that, in order to
reduce blood glucose to normal levels, one may require to increase
the efficacy of gastrin.
[0104] Pancreatic insulin levels for vehicle-treated groups
decreased at day 35 due to destruction of beta-cells, whereas
animals treated with gastrin exhibited significantly elevated
levels of pancreatic insulin levels in comparison with pretreatment
values. See FIG. 2. However, the increase of pancreatic insulin
levels after treatment of COMPOUND B to about 0.6 ug of
insulin/pancreas is still significantly lower than that of normal
mice (.about.12 ug/pancreas). These data together with the
pharmacokinetics analysis in Example 1 suggest that the efficacy of
treating diabetic NOD mice using unmodified gastrin may be limited
by the relatively short half life of plasma gastrin (.about.5
mins). Thus, the use of a longer acting gastrin may be more
efficacious in stimulating islet cell neogenesis, increasing
pancreatic insulin and preventing diabetes progression in NOD
mice.
Example 3
Peptide Synthesis of Gastrin Peptides
[0105] Gastrin peptides may be readily synthesized by anyone with
ordinary skills in the art, using standard techniques for solid
phase peptide synthesis, for example as described by Steward, J. M.
and Young, J. D. (1984) in "Solid Phase Peptide Synthesis",
2.sup.nd ed., Pierce Chemical Company. Purification of gastrin
peptides may be performed using standard techniques, for example
using reverse phase HPLC with a volatile binary gradient system
consisting of 0.1% TFA in H.sub.2O and 0.1% TFA in acetonitrile.
Monitoring elution by UV absorbance allows for collection of
purified peptide, which is then lyophilized to dryness and
subsequently dissolved for administration and testing, or for
further conjugation reactions where required.
[0106] Gastrin synthetic peptides may be synthesized containing any
consecutive, portion of residues 1-28 in addition to residues 29-34
of SEQ ID NO: 1 or 2 or containing any consecutive portion of
residues 1-11 in addition to residues 12-17 of SEQ ID NO: 3 or 4.
Additionally, gastrin peptides may be synthesized with a spacer
region at the N-terminal end comprised of small neutral amino acid
residues such as Gly and Ala. Gastrin synthetic peptides, either
with or without a spacer region, may also be synthesized with an
N-terminal Cys residue.
[0107] A summary of some gastrin peptides which are synthesized for
use in gastrin compositions herein, are listed in Table 3
(compounds A through H), and include "Big" Gastrin-34 (A), "Little"
or "Small" Gastrin-17 (B), and Gastrin-13 (C).
TABLE-US-00004 TABLE 3 Summary of gastrin compositions and their
components N-terminal Gastrin Residues gum Compound Polymer Cys
residue Linker Peptide SEQ ID NO: A No No No 1-34 2 B No No No 1-17
4 C No No No 5-17 4 D No Yes No 2-34 2 E No Yes No 2-17 4 F No Yes
No 5-17 4 G No Yes (GA).sub.5 2-17 4 H No Yes (GA).sub.5 5-17 4 I
PEG Yes No 2-34 2 J PEG Yes No 2-17 4 K PEG Yes No 5-17 4 L PEG Yes
(GA).sub.5 2-17 4 M PEG Yes (GA).sub.5 5-17 4 N HSA Yes No 2-34 2 O
HSA Yes No 2-17 4 P HSA Yes No 5-17 4 Q HSA Yes (GA).sub.5 2-17 4 R
HSA Yes (GA).sub.5 5-17 4 PEG is poly(ethylene glycol)-20,000; HSA
is human serum albumin; (GA).sub.5 is
Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala spacer.
Example 4
Conjugation of Gastrin with PEG-20,000
[0108] Gastrin peptides modified with Cys at the N-terminal
(compounds D, E, F, G, and H in Table 3) are incubated for about 30
minutes at near neutral conditions (buffered at pH 6.5-7.5) with a
molar excess of tris[2-carboxyethyl]phosphine hydrochloride (TCEP;
a reducing agent which has no reactivity with maleimide moieties)
to ensure that the Cys residue is in the reduced state and
available for reaction.
[0109] A molar excess of mPEG-maleimide-20,000 (average MW 20,000;
obtained from Shearwater Corporation, Huntsville Ala., USA) is
added gradually with mixing to allow dissolution, and when fully
dissolved, the solution is mixed for an additional 1 to 4 hours to
allow for completion of the conjugation reaction. Purification of
the conjugate may be performed using an anion-exchanger, for
example Q-Sepharose at neutral pH, to which both the conjugate and
any free unreacted gastrin binds tightly, since the theoretical
isoelectric point (pI) for gastrin is 3.4, whereas the neutral
unreacted mPEG-maleimide does not bind. For further purification,
the conjugate is readily separated from unreacted gastrin based on
the large difference in their molecular weights using size
exclusion chromatography, for example using Sephadex G-50, using a
buffer suitable for therapeutic use, such as PBS.
[0110] These reactions result in the production of compounds I, J,
K, L and M in Table 3. Success of conjugation reactions is verified
using the Biuret reaction for peptide content, a colorimetric
procedure for PEG content (Habeeb, A.F.S.A. (1966) Anal. Biochem.
14: 328-336), and electrospray mass spectrometry to determine the
total MW of the conjugate.
Example 5
Conjugation with Human Serum Albumin
[0111] Serum albumin contains a single accessible, reduced Cysteine
residue (Cys-34), which is the most reactive thiol group among
human plasma proteins (Pedersen et al (1980) Eur. J. Biochem. 106:
291-295). This property allows specific conjugation of ligands,
including peptides, to serum albumin.
[0112] Human serum albumin is incubated for about 30 minutes at
near neutral conditions (buffered at pH 6.5-7.5) with a molar
excess of tris[2-carboxyethyl]phosphine hydrochloride (TCEP; a
reducing agent which has no reactivity with maleimide moieties) to
ensure that Cys-34 residue is in the reduced state and available
for reaction. A molar excess of bis-maleimidoethane (a
homo-bifunctional cross-linking agent having a short spacer, and
reactivity towards sulfhydryl groups) is added to activate Cys-34.
In separate reactions, gastrin peptides modified with Cys at the
N-terminal (compounds D, E, F, G, and H in Table 3) are added and
the resulting solution is mixed for an additional 1 to 4 hours to
allow for completion of the conjugation reaction. The conjugate is
readily separated from unreacted gastrin (and any gastrin dimers
formed) based on the large difference in their molecular weights
using size exclusion chromatography, for example using a buffer
suitable for therapeutic use, such as PBS. Unreacted HSA was not
further separated from HSA conjugated with Gastrin peptides.
[0113] These reactions result in the production of compounds N, O,
P, Q and R in Table 3. Success of conjugation reactions is verified
using electrospray mass spectrometry to measure total MW of
conjugate.
Example 6
PHARMACOKINETIC Comparison Between Unmodified and Modified Gastrin
Compounds Following Administration by Intravenous Injection to
Wistar Rats
[0114] This example was conducted to assess the pharmacokinetic
profile of unmodified and modified gastrin derivatives/conjugates
following iv injection in Wistar rats.
Administration of Gastrin
[0115] Rats (groups of three animals) receive the various test
compounds as synthesized in Example 3 at the dose level of 10
.mu.g/kg of gastrin equivalent by iv administration.
The following test compounds are used are all of the compounds
listed in Table 3 above. Please refer to Table 3 for more
details.
Blood Collection
[0116] Serial blood samples (approximately 0.4 ml per time point)
were collected at the following time points: 5, 60, 180, 480
minutes after the injection all animals. At each time point, blood
was taken from at 3 animals per group.
[0117] Each sample was collected into a tube containing EDTA and
kept on wet ice pending centrifugation. The samples were
centrifuged under refrigeration for a minimum of 10 minutes at
1,500 g (RCF) and the resultant plasma transferred into duplicate
tubes and placed on dry ice. All samples were stored frozen for PK
analysis.
Assay for Gastrin Levels
[0118] Human Gastrin 1 (G-17) Immunoassay Kit from R&D systems,
catalog number DE3400 was used to measure plasma COMPOUND B levels
using the ELISA method. This assay is based on the competitive
binding technique in which Gastrin I present in a sample competes
with a fixed amount of alkaline phosphatase-labeled Gastrin I for
sites on a rabbit polyclonal antibody. During the incubation, the
antibody becomes bound to the goat anti-rabbit antibody coated onto
the microplate. Following a wash to remove excess conjugate and
unbound sample, a substrate solution is added to the wells to
determine the bound enzyme activity. Immediately following color
development, the absorbance is read at 405 nm. The intensity of the
color is inversely proportional to the concentration of Gastrin I
in the sample.
Pharmacokinetics Analysis
[0119] Plasma concentration of gastrin (COMPOUND B) and its
different derivatives/conjugates were analyzed using the PK
Functions for Microsoft.RTM. Excel (Usansky J I, Desai A, Tang-Liu
D, Department of Pharmacokinetics and Drug Metabolism, Irvine,
Calif.). To assess the PK profiles, various test compounds and the
following PK values were calculated: C.sub.max, t.sub.max, AUC and
t.sub.1/2.
[0120] Since gastrins (e.g. COMPOUND B) are naturally occurring at
detectable levels in blood, baseline values obtained before dosing
were subtracted from the plasma levels obtained after COMPOUND B
administration. Data are expressed as mean.+-.SD.
[0121] The data show that modified gastrin compounds with cysteine
as functional groups on the N-terminus), or modified gastrin
compounds conjugated to PEG or HSA on the N-terminus had
significantly longer half lives compared to native gastrin. The
modified gastrin compounds/conjugates are present in serum at
higher concentrations for longer periods of time compared to the
administration of native gastrin 17 or gastrin 34. These data also
demonstrate the AUCs also increase with the chemical modification
of the gastrin molecules. It is envisioned that the modified
gastrin derivatives/conjugates provide the highest concentrations
for the longest period of time will have the greatest potential for
the efficacy required for islet cell neogenesis and regulation of
glucose levels in diabetic animals.
Example 7
Comparison of Modified Gastrin Compounds and Unmodified Gastrin in
Preventing Diabetes Progression in NOD Mice with Recent Onset
Diabetes
[0122] In this example, the effect of treatment by unmodified
gastrin and modified gastrin compounds/conjugates were examined in
NOD mice with recent onset diabetes, to determine whether
administration of the various modified gastrin
derivatives/conjugates would be more effective in preventing severe
hyperglycemia as well as increase pancreatic insulin content in NOD
mice with recent-onset diabetes as compared to unmodified gastrin.
Modified gastrin compounds/conjugates used are as follows (Refer to
Table 3 for more details): Compound B, which is gastrin prepared as
synthetic human gastrin I having 17 amino acid residues with a Leu
residue at amino acid position 15, Compound E, Compound G, Compound
J, Compound L, Compound O and Compound Q.
[0123] Non-obese diabetic (NOD) female mice, ages 12-14 weeks, are
monitored for development of onset of diabetes (fasting blood
glucose >8.0 to 15 mmol/l), and within 48 hours after onset of
symptoms, the different groups of mice are each treated with 3 or
10 .mu.g/kg/day of Gastrin equivalent, each treatment administered
via the intraperitoneal route daily.
[0124] Therapy is administered for 14 days. Animals are monitored
weekly for fasting blood glucose (FBG) levels. FBG levels are
measured at about 12 hours after food had been withdrawn, and 24
hours after the last peptide or vehicle injection. Upon cessation
of therapy, all mice are monitored for FBG levels for the next 4
weeks (weeks 2-6) so as to determine whether prevention of
hyperglycemia persisted after termination of therapeutic treatment.
At 14 days treatment is stopped.
[0125] The protocol includes sampling of these mice for data again
at 6 weeks, and blood collected for assay of FBG and plasma
C-peptide, and the mice are sacrificed for pancreatic insulin
determinations and scoring of islet inflammation (insulins). From
the outset of treatment, mice receive neither insulin-replacement
treatment nor immunosuppression. The following parameters are
assessed: survival rates, pancreatic insulin levels, presence of
islet inflammation and fasting blood glucose levels.
[0126] The data demonstrate that modified gastrin
derivatives/conjugates with a longer half life and AUCs were more
effective in preventing hyperglycemia in diabetic NOD mice. In some
cases, the modified gastrin compounds/conjugates completely reverse
the glucose levels to normal levels, indicating the stimulation of
significant levels of islet neogenesis in this model.
Example 8
Comparison of Modified Gastrin Compounds/Conjugates and Unmodified
Gastrin in Combination with GLP-1 in Preventing Diabetes
Progression in NOD Mice with Recent Onset Diabetes
[0127] In this example, the effect of treatment by a combination of
GLP-1 and unmodified gastrin and GLP-1 and modified gastrin
compounds/conjugates were examined in NOD mice with recent onset
diabetes, to determine whether administration of both GLP-1 and
gastrin would prevent severe hyperglycemia as well as increase
pancreatic insulin content in NOD mice with recent-onset diabetes.
The GLP-1 used was GLP-1 which is the biologically active fragment
of human/mouse GLP-1 (having residues at positions 7-36 compared to
the precursor from which the fragment is processed; obtained from
Bachem H6795). Modified gastrin compounds/conjugates used are as
follows: Compound B-COMPOUND B-gastrin as synthetic human gastrin I
having 17 amino acid residues with a Leu residue at amino acid
position 15, Compound E, Compound Q.
[0128] Non-obese diabetic (NOD) female mice, ages 12-14 weeks, were
monitored for development of onset of diabetes (fasting blood
glucose >8.0 to 15 mmol/l), and within 48 hours after onset of
symptoms, four groups of mice were each treated as follows: one
group was treated with vehicle only, and the other group was
administered 100 .mu.g/kg/day of GLP-1, and the remaining groups
were treated with combination of GLP-1 (100 .mu.g/kg/day) and
gastrin compound (3 .mu.g/kg/day gastrin equivalent), each
treatment administered via the intraperitoneal route daily.
[0129] Therapy was administered for 14 days. Animals were monitored
weekly for fasting blood glucose (FBG) levels. FBG levels were
measured at about 12 hours after food had been withdrawn, and 24
hours after the last peptide or vehicle injection. Upon cessation
of therapy, all mice were monitored for FBG levels for the next 4
weeks (weeks 2-6) so as to determine whether prevention of
hyperglycemia persisted after termination of therapeutic treatment.
At 14 days treatment was stopped.
[0130] The protocol includes sampling of these mice for data again
at 6 weeks, and blood collected for assay of FBG and plasma
C-peptide, and the mice are sacrificed, for pancreatic insulin
determinations and scoring of islet inflammation (insulitis). From
the outset of treatment, mice received neither insulin-replacement
treatment nor immunosuppression. The following parameters are
assessed: survival rates, pancreatic insulin levels, presence of
islet inflammation and fasting blood glucose levels.
[0131] The data demonstrate that GLP-1 in combination of modified
gastrin compounds/conjugates (Compound E or Q) with longer half
lives were more effecting in reducing blood glucose levels in
diabetic animals compare to GLP-1 with native gastrin (Compound B).
These data support the use of longer lasting modified gastrin
compounds/conjugates with GLP-1 or other growth factors.
Sequence CWU 1
1
9134PRTArtificial SequenceChemically Synthesized 1Xaa Leu Gly Pro
Gln Gly Pro Pro His Leu Val Ala Asp Pro Ser Lys1 5 10 15Lys Gln Gly
Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met 20 25 30Asp
Phe234PRTArtificial SequenceChemically Synthesized 2Xaa Leu Gly Pro
Gln Gly Pro Pro His Leu Val Ala Asp Pro Ser Lys1 5 10 15Lys Gln Gly
Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Leu 20 25 30Asp
Phe317PRTArtificial SequenceChemically Synthesized 3Xaa Gly Pro Trp
Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp1 5 10
15Phe417PRTArtificial SequenceChemically Synthesized 4Xaa Gly Pro
Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Leu Asp1 5 10
15Phe56PRTArtificial SequenceChemically Synthesized 5Tyr Gly Trp
Met Asp Phe1 566PRTArtificial SequenceChemically Synthesized 6Tyr
Gly Trp Leu Asp Phe1 574PRTArtificial SequenceGeneral gastrin/CCK
receptor ligands carboxy terminal amino acid sequence 7Trp Met Asp
Phe1810PRTArtificial SequenceChemically Synthesized 8Gly Ala Gly
Ala Gly Ala Gly Ala Gly Ala1 5 10910PRTArtificial
SequenceChemically Synthesized 9Gly Pro Trp Leu Glu Glu Glu Glu Glu
Ala1 5 10
* * * * *
References