Title of Invention

TREATMENT OF DIABETES

Abstract Gomposinons and methods are provided for islet meogenesis therepy comprising a member of a group of factors that cumplomont a gasirin/CCK receplot lighted, with formulation, devices and methods for sustaned release delivery and for local delivery to larged organs.
Full Text WO2004/037195 PCT/US2003/033595
TREATMENT OF DIABETES
Field of the Invention
The invention relates to methods and compositions for treating a diabetic subject with
5 islet neogenesis therapy and an agent for immunosuppression, with formulations and methods
for local delivery and for sustained release of the compositions.
Background of the Invention
The severe forms of the common disease Diabetes Mellitus result from an absence or
10 relative deficiency of insulin secretion from the pancreatic b-cell, Consequently, diabetics are
dependent on exogenous insulin injections to prevent life threatening complications of high
blood glucose (hyperglycemia). Unless patients adhere to a very demanding regime of
glucose testing and insulin treatment (intensive insulin treatment), insulin treatment does not
prevent chronic long-term complications of organ damage caused by hyperglycemia.
15 Intensive insulin, treatment increases risk of acute hypoglycemia due to insulin overdosing,
with acute and serious alterations of consciousness state that can be fatal.
About one million people in the United States population suffer from juvenile or type
I diabetes, and about 30,000 new cases arise each year. Further, an extremely large and
rapidly increasing number of patients have forms of type II diabetes (also called adult onset
20 or insulin-resistance diabetes), at a level of epidemic proportions, a disease that can cause
pancreatic exhaustion and insulin insufficiency.
The abnormally high blood glucose (hyperglycemia) that characterizes diabetes, if left
untreated, results in a variety of pathological conditions, for example, non-healing peripheral
vascular ulcers, retinal damage leading to blindness, and kidney failure. Diabetes of both
25 types I and II are treated with insulin injection in response to blood glucose levels determined
by patient glucose self-monitoring, although not all type II-patients progress to requiring
insulin therapy. Typically, multiple doses of insulin are delivered by the patient per day.
Severs pathological consequences of diabetes are correlated with less rigorous control of
blood glucose level.
30 A potential treatment for diabetes would be to restore b-cell function so that insulin
release is dynamically regulated in response to changes in blood glucose levels. This can be
achieved by pancreas transplantation, but this approach is typically limited to diabetics
requiring kidney transplants for renal failure. Also, pancreas transplantation can require life
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long immunosuppression to prevent allogeneic graft rejection and autoimmune destruction of
the transplanted pancreas.
Recently, transplants of isolated human islet preparations have successfully reversed
insulin diabetes in human subjects for prolonged periods. However, a large amount of donor
5 islet cell material is required for each recipient, and the supply of islet call material has not
been sufficient to meet the demand.
Summary
A feature of the invention is a method for treating a diabetic condition, the method
10 including administering to a mammal a therapeutically effective amount of a cemposition
comprising a gastrin /CCK receptor ligand and a factor for complementing gastrin for islet
neogenesis therapy (a FACGINT), provided that the FACGINT is not an EGF receptor
ligand,
As the term is used herein, the FACGINT is at least one selected from the group of: a
15 Glucagon-like peptide 1 receptor ligand; a Glucagon-like peptide 2 receptor ligand; a gastric
inhibitory polypeptide (GIP) receptor ligand; a keratinocyte growth factor (KGF) receptor
ligand; a dipeptidyl peptidas+e IV inhibitor; a KEG protein receptor ligand; a Growth
Hozmone receptor ligand; a Prolactin (PRL) receptor ligand ; an Insulin-like Growth Factor
(IGF) receptor ligand; PTH-related protein (PTHrP) receptor ligand; hepatocyte growth factor
20 (HGF) receptor ligand; a bone morphogenetic protein (BMP) receptor ligand, a transforming
growth factor- (TGF-) receptor ligand; a laminin receptor ligand; vasoactive intestinal
peptide (VIP) receptor ligand; a fibroblast growth factor (FGF) receptor ligand; a
keratinocyte growth factor receptor ligand; a nerve growth factor (NGF) receptor ligand; an
islet neogenesis associated protein (INGAP) receptor ligand; an Activin-A receptor Ligand; a
25 vascular endothelial growth factor (VEGF) receptor ligand; an erythropoietin (EPO) receptor
ligand; a pituitary adenylate cyclasa activating polypeptide (PACAP) receptor ligand; a
granulocyte colony stimulating factor (G-CSF) receptor ligand; a granulocyte-macrophage
colony stimulating factor (GM-CSF); a platelet-derived growth factor (PDGF) receptor ligand
and a Secretin receptor ligand.
30 As used herein, the term "diabetes" means any physiologic indication of a short age of
insulin, a production of antibodies against insulin, or an excess of blood sugar. Diabetes is
exemplified but not limited to diabetes I, diabeties II, gestational diabetes, and a pre-diabetic
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condition. As used herein, the term "mammal" has the usual meaning of any member of
Mammalia, and includes humans.
In related embodiments, the FACGINT is a Glucagon-like peptide 1 (GLP-1) receptor
ligand; a Glucagon-like peptide 2 (GLP-2) receptor ligand; or a member of a Growth
5 Hormone receptor ligand family, and can be a Growth Hormone, such as Human Growth
Hormone (HGH), a Placental lactogen (PL), or a Prolactin (PRL), or an exendin such as
exendin-4.
Another feature of the invention provides a method for treating diabetes, the method
including contacting ex vivo a plurality of cells with a composition having at least one of a
10 FACGINT and a gastrin/CCK receptor ligand, provided that the FACGINT is not an EGF
receptor ligand; and administering the cells to a mammal in need thereof, thereby treatingg
the diabetes. In one embodiment of the method the cells are autologous, i.e., from the
subject. Alternatively, the cells are from another individual in the same species, or even from
another species. In the method using cells ex vivo, the cells can be pancreatic ductal cells.
15 Pancreatic cells are a source of islet precursor cells. A further embodiment of this method
involves, prior to the implanting step, treating the cells ex vivo with the composition. A
related method further includes, prior to the contacting step, culturing the cells ex vivo.
Generally, the terms "administering" or "contacting" mean that the user of the method
is provided the composition in an amount effective to increase the amount of insulin secreting
20 cells in the mammal.
Generally in these methods, the composition is administered syatemically. Further,
the amount of the FACGINT in the composition is substantially less than the minimum
effective dose of the FACGINT required to reduce blood glucose in the diabetic mammal in
the absence of a gastrin/CCK receptor ligand. The method can further include measuring a
25 parameter selected from the group of: blood glucose, serum, glucose, blood glycosylated
hemoglobin, pancreatic b-cell mass, serum insulin, pancreatic insulin content, and
morphometrically determined b" cell mass. In general, one of skill in the art of diagnosis of
diabetes would measure a glucose level in blood or serum following a fast, i.e., a period of no
feeding of the subject or patient, of duration typical of such a diagnosis. The standard
30 measurement is an assay of fasting blood glucose, or FBG.
The in vivo methods herein can further include measuring a parameter selected from
the group of: amount of insulin secreting cells, glucose responsiveness of insulating secreting
cells, amount of proliferation of islet precursor cells, and amount of mature insulin secreting
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cells, these measurements being made in a mammal that is an experimental animal such as a
genetically diabetic mouse (NOD mouse) or a rodent in which diabetes has been induced
(with streptozotiein or STZ).
Another embodiment herein is a method for inducing pancreatic islet neogenesis in a
5 mammal which is administering to the mammal a composition comprising a combination of a
FACGINT and a gastrin /CCK receptor ligand provided that the FACGINT is not an EGF
receptor ligand, in an amount sufficient to increase proliferation of islet precursor cells in
pancreatic tissue, thereby inducing pancreatic islet neogenesis.
Yet another embodiment herein in a method for inducing pancreatic islet neogenesis
10 in a mammal, the method comprising administering a composition comprising a combination
of a FACGIKT and a gastrin /CCK receptor ligand wherein the FACGINT is not an EGF
receptor ligand, in an amount sufficient to increase the number of pancreatic insulin secreting
b-cells in the mammal,
Accordingly, an embodiment of the invention is a composition composing a
15 gastrin/CCK receptor ligand and a FACGINT, provided that the FACGINT is not an EGF
receptor ligand. The composition in some embodiments is provided in a dosage effective for
inducing proliferation of islet precursor cells into an increased amount of mature insulin
secreting cells. Similarly, the composition in some embodiments is provided in a dosage
effective for inducing differentiation of an islet precursor cell into a mature insulin secreting
20 cell.
The composition can be provided in a phannaceutically acceptable carrier.
Also provided herein is a kit for treatingg diabetes, containing a composition
comprising a gastrio/CCK receptor ligand and a FACGENT, a container, and instructions for
use, provided that the FACGINT is not an EGF receptor ligand. The bit composition can be
25 provided in one or more unit doses. The composition of the kit further includes a
pharmaceutically acceptable carrier.
Another feature of the invention herein is a method for expanding and differentiating
stem cells into insulin secreting cells in a diabetic recipient of implanted, cells, which includes
implanting the stem cells in the recipient, and administering to the recipient a composition
30 containing an effective dose of each of a gastrin/CCK receptor ligand and a FACGINT
provided that the FACGINT is net an EGF receptor ligmd. In this method, the cells can be
obtained for example from a human or a porcine. Further, the implanted cells are obtained
from pancreatic islets, umbilical chords, embryos, or stem cell lines. Alternatively, a method
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for expanding and differentiating stem cells in a diabetic recipient of the cells into insulin
secreting cells, is implanting the cells in the recipient; and administering a sustained release
composition comprising an effective dose of each of a gastrin/CCK receptor ligand, and a
FACGINT or an EGF receptor ligand
5 En general according to these methods, the gastrin/CCK receptor ligand is gastrin. In
related embodiments, the gastrin is gastrin-17, for example, the gastrin is human gastrin 1-
17Leul5.
Further, according to these methods, implanting the cells in the recipient is using a
route such as injecting directly into an organ, and administering intravenously. Injecting the
10 cells is delivering locally into an organ, for example, the pancreas, the kidney, and the liver.
Further, injecting the cells is delivering to the portal vein percuteneously or transhepatically.
In any of these methods, a catheter can be inserted into a vein or artery leading from or to the
organ, using an imaging technology, such as ultrasound or MRI during the procedure.
Delivering the cells locally can be chosen from several technologies, including
15 endoscopic retrograde cholangiopancreatography (ERCP); endoscopic ultrasound-guided fine
needle delivery (EUS-FNAD); injection into a pancreatic artery; injection into a portal vein;
intrapancreatie injection; and injection into an hepatic artery. In these technologies the user
can be guided by one of several imaging technologies including ultrasound or MRI during the
procedure.
20 Another feature of the invention provided is a method for reducing an amount of stem
cells needed for transplantation to treat human diabetes, the method including administering
to the recipient an effective dose of each of a gastrin/CCK receptor ligand and a FACGINT,
the amount of cells being reduced in comparison to Implanting cells in the absence of
administering the effective dose to an otherwise identical recipient, provided that the
25 FACGINT is not an EGF receptor ligand.
In a related embodiment of the invention, the invention provides a composition
comprising a gastrin/CCK receptor ligand and at least one FACGINT, provided that the
FACGINT is not an EGF receptor ligand. The composition is provided, in a dosage that is
effective for inducing differentiation of an islet precursor cell into a mature insulin secreting
30 cells. The composition can further be provided in a pharmaceutically acceptable carrier.
A kit is provided for treating diabetes, the kit containing a composition comprising at
least one FACGINT provided that the FACGINT is not an EGF receptor ligand, and a
gastrin/CCK receptor ligand, a container, and instructions for use.
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Any of the above methods con further include administering to the subject an agent
for suppressing an immune response. In any of the above methods, the components of the
combination can be delivered simultaneously, for example, within one hour or within one
day, or can be delivered sequentially, for example, at an interval of greater than one day,
5 greater than two or more days, greater than one week.
An exemplary agent for suppressing immune response is a drug, for example, is at
least one of the group consisting of a rapamycin; a corticosteroid; an azathioprine;
mycophenolate mofetil; a cyclosporine; a cyclophosphamide; a methotrexate; a 6-
mercaptopurine; FK5O6; 15-deoxyspergualin; an FTY 720; a mitoxantrone; a 2-amino-l ,3-
10 propanediol; a 2-amino-2[2-(4-octylphenyl)ethyl]propane-l,3-diol hydrochloride; a 6-(3-
dimethyl-aminopropionyl) forskolin; and a demethimmunomycin.
Alternatively, an exemplary agent for suppressing immune response is a protein, for
example, the protein comprises an amino acid sequence of an antibody. Accordingly, the
agent for suppressing immune response is at least one of: hul 124: BTI-322; allotrap-HLA-
15 B270; OKT4A; Enlimomab; ABX-CBL; OKT3; ATGAM; basiliximab; daclizumab;
thymoglobulin; ISAtx247; Medi-500; Medi-507; Alefacept; efalizumab; infliuximab; and an
interferon. The islet neogenesls therapy composition and the agent for suppressing immune
response are administered sequentially or can be administered simultaneously.
In certain embodiments, at least one of the islet neogenesis therapy composition and
20 the agent for suppressing immune response is administered systemically. For example, the
islet neogenesis therapy composition is administered as a bolus. Thus, at least one of the islet
neogenesis therapy composition and the agent for suppressing immune response is
administered by a roule that is intravenous, subcutaneous, intrapetitoneal, or intramuscular.
Further, in certain embodiments, the agent for suppressing immune response is administered
25 orally. In general, the agent for suppressing immune response is at least one of FK506,
rapamycin, and daclizumab. Further, according to embodiments of any of the methods herein
other than those calling for measurement of certain experimental parameters, the subject can
be a human.
Also featured herein is a kit for treatment of a diabetic subject, comprising a
30 container, an immunosuppressive agent, and an INT composition comprising a FACGINT
provided that the FACGINT is not an EGF receptor ligang.
Also provided here is a pharmaceutical composition for sustained release of an
I.N.T.™ therapeutic composition, the composition comprising: a gastrin receptor ligand; and
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an EGF receptor ligand or a FACGINT; wherein at least one of the gastrin receptor ligand, or
the EGF receptor ligand or FACGINT, is a sustained release formulation.
This composition can further comprising an agent for immune suppression. Exemplary
embodiments of the sustained release formulation are pegylation of at least one of the
5 components of the composition, and formulation of at least one component in a
multi vesicular lipid-based liposome. Examples of the EGF receptor ligand is selected from
the group consisting of an EGF and a TGFo. Examples of the FACGINT is at least one
selected from the group of: a Glucagon-like peptide 1 recepror ligand; a Glucagon-like
psptide 2 receptor ligand; a gastric inhibitory polypeptide (GIP) receptor ligand; a
10 keratinocyte growth factor (KGF) receptor ligand; a dipeptidyl peptidase IV inhibitor; a KEG
protein receptor ligand; a Growth Hormone receptor ligand; a Prolactin (PRL) receptor ligand
; an Insulin-like Growth Factor (IGF) receptor ligand; PTH-related protein (PTHrP) receptor
ligand; hepatocyte growth factor (HGF) receptor ligand; a bone morphogenetic protein
(BMP) receptor ligand, a transforming growth factor- (TGF- receptor ligand; a laminin
15 receptor ligand; vascactive intestinal peptide (VIP) receptor ligand; a fibroblast growth factor
(FGF) receptor ligand; a keratinocyte growth factor receptor ligand; a nerve growth factor
(NGF) receptor ligand; an islet neogenesis associated protein- (INGAP) receptor ligend; an
Activin-A receptor ligand; a vascular endothelial growth factor (VEGF) receptor ligand; an
erythropoietin (EPO) receptor ligand; a pituitary adenylate cyclase activating polypeptide
20 (PACAP) receptor ligand; a granulocyte colony stimulating factor (G-CSF) receptor ligand; a
granulocyte-macrophage colony stimulating factor (GM-CSF); a platelet derived growth
factor (PDGF) receptor ligand; and a Secretin receptor ligand. Alternatively, the EGF
receptor ligand is a low molecular weight drug. The composition can be formulated for
parenteral administration. Alternatively, the composition can be formulated for oral
25 administration.
The composition can be formulated for a route of administration selected from the group
consisting of subcutaneous, intraperitoneal, intravenous, and intramuscular injection.
In one embodiment, at least one of the gastrin receptor ligand, or the EGF receptor ligand or
the FACGINT, is formulated for systemic administration.
10 Further, the above compositions can be formulated for local delivery, for example, the
local delivery 13 targeted to the pancreas. Exemplary types of local delivery include
endoscopic retrograde cholangiopancreatography (ERCP); endoscopic ultrasound-guided fine
needle aspiration delivery (EUS-FNAD); injection into a pancreatic artery; injection into a
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portal vein; vntrapancreasic injection; and injection into an hepatic artery. These can be
combined with imaging technologies such as ultrasound and MRI, performed during the
procedure.
The composition in some embodiments is formulated for a route of administration
5 selected from due group consisting of transdermal and mucosal delivery. Any of these
compositions can be formulated for delivery by a mechanical device, in combination with
formulation for sustained release, or alternatively, use of the mechanical device to deliver the
formulation over a sustained period of time. The device is, for example, a degradable
implant; a transcutaneous patch; a catheter; an implainable pump; a percutaneous pump; an
10 infusion pump; and an iontophoresis device.
The above compositions and pharmaceutical compositions can be formulated for a
route of administration selected from the group consisting of: subcutaneous, intraperitoreal,
intravenous, and intrapancieatic. For example, the intravenous route is into a portal vein. A
device can be used, and the device can be a pump. With sustained release formulations as
15 with some of the above methods and compositions, the administration can be local. For
example, the local administration can be delivered by a route selected from the group of:
endoscopic retrograde cholangiopanereetography (ERCP); endoscopic ultrasound-guided fine
needle aspuration delivery (EUS-FNAD); injection into a pancreatic artery; injection into a
portal vein; intrapancreatic injection; and injection into an hepatic artery Alternatively with
20. the sustained release forulations and devices, the administration can be systemic. The
pharmaceutical composition can be provided in an effective dose. Also featured herein is a
kit comprising at least one dose of a composition of any of the above sustained release
formulations.
Also featured is a method of reducing frequency of treating a diabetic subject with an
25 I.N.T.™ composition, the method including preparing at least one component of the
composition as a sustained release formulation and administering the composition to the
subject according to a protocol having greater intervals between treatments than for the
composition not so formulated and otherwise identical. Administering can be delivering by a
route selected from: endoscopic retrograde cholangiopancreatography (ERCP); endoscopic
30 ultrasound-guided fine needle aspiration delivery (EUS-FNAD; injection into a pancreatic
artery; injection into a portal vein; intrapancreatic injection; or injection into an hepatic
artery. Imaging technology can be used to guide a catheter in place during these procedures.
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Also featured is method of enhancing efficacy of an I.N.T.™ composition in a
diabetic subject, the method including: administering to the subject an. I.N.T.™ composition
having at least one component of the composition formulated to produce a sustained release;
and comparing efficacy in treating the subject of an amount of the composition administered
5 to efficacy of a composition not having a component so formulated and otherwise identical,
such that the efficacy of the I.N.T.™ composition having a sustained release formulated
compositian, as measured by a decrease in an amount of the sustained release formulated
agent required to reduce or eliminate symptoms of diabetes in the subject, is enhanced. In an
embodiment of this methods, comparing efficacy is further analyzing toxicity of the
10 composition, such, that fewer or milder unwanted symptoms following administering the
composition indicates decreased toxicity in the composition having at least one component
formulated to produce a sustained release, compared to toxicity of the I.N.T.™ composition
not having a component so formulated and otherwise identical, In any of these methods,
exemplary sustained release formulations of the component are pegylation and a
15 multivesicular lipid-based liposome.
In any of these methods enhancing efficacy of an I,N.T.™ composition, the
component having the sustained release formulation, is an EGF receptor ligand selected from
the group consisting of an EGF and a TGF. Alternatively in embodiments of these methods,
the FACGINT is at least one selected from the group of: a Glucagon-like peptide 1 receptor
20 ligand; a Glucagon-like peptide 2 receptor ligand; a gastric inhibitory polypeptide (GIP)
receptor ligand; a keratinocyte growth factor (KGF) receptor ligand; a dipeptidyl peptidase
IV inhibitor; a REG protein receptor ligand; a Growth Hormone receptor ligand; a Prolactin
(PRL) receptor ligand ; an Insulin-like Growth Factor (IGF) receptor ligand; PTH-relatied
protein (PTHrP) receptor ligand; hepatocyte growth factor (HGF) receptor ligand; a bone
25 morphogenetic protein (BMP) receptor ligand, a transforming growth factor- (TGF-)
receptor ligand; a laminin receptor ligand; vasoactive intestinal peptide (VIP) receptor ligand;
a fibroblast growth factor (FGF) receptor ligand; a keratinocyte growth factor receptor ligand;
a nerve growth fector (NGF) receptor ligand; an islet neogenesis associated protein (TNGAP)
receptor ligand; an Activin-A receptor ligand; a vascular endothelial growth factor (VEGF)
30 receptor ligand; an erythropoietin (EPO) receptor ligand; a pituitary adenylate cyclase
activating polypeptide (PACAP) receptor ligand; a granulocyte colony stimulating factor (G-
CSF) receptor ligand; a granulocyte-macrophage colony stimulating factor (GM-CSF); a
platelet-derived growth factor (PDGF) receptor ligand; and a Secretin receptor ligand.
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Even further as an alternative, a component is a low molecular weight drug. In any of these
methods, administering is delivering by a route selected from the group consisting of
parenteral, oral, transdermal, subcutaneous, mucosal, untraperitoneal, intravenous,
intrapancreatic and intramuscular.
5 For example, administering produces local distribution. Further, the method includes
administering the composition in an effective dose. Even farther, the method includes, prior
to administering, the composition is formulated for using a sustained release device. For
example, the device selected from the group of: degradable implant; transoutaneous patch;
catheter; implantable pump; percutaneous pump; infusion pump; and iontophoresis device.
10 For example, the device is a pump. Administering in any of these methods may be by the
intravenous route is injecting into a portal vein.
Also featured is a method of reducing frequency of treating a diabetic subject, the
method comprising preparing a device for administering an I.N.T.™ composition to the
subject by continuous release for a prolonged period; providing the device to the subject; and
15 re-iterating treating the subject by replacing or refilling the device, for example, a device
which is a pump. The pump can be a percutaneous pump; a flurorcarbon propellant pump; an
osmotic pump; a mini-osmotic pump; an implantable pump; or an infusion pump.
Alternatively, the device is selected from the group consisting of: a degradable implant; a
non-degradable implant; a mucoadhesive implant; a transcutaneous patch; a catheter, and an
20 iontophoresis device. An exemplary non-degradable implant is Silastic. Other examples of
degradable implants can be at least one of the materials selected from the group of: starch;
vinylstarch; dipropyleneglycol diacrylate (DFGDA); tripropyleneglycol diacrylate (TPGDA);
pectin; cellulose acetate: cellulose propionate; cellulose acetate butyrate; cellulose acetate
propionate (CAP); hydroxypropyl cellulose (HPC); hydroxypropyl cellulose/cellulose acetate
25 propionate (HPC/CAP); methyl methacrylate (MMA); butyl methacrylate (BMA);
hydroxymethyl methacrylate (HEMA); ethyl hexyl acylate (EHA); octadecyl methacrylate
(ODMA); and ethyleneglycol dimemacrylate (EGDMA).
Also featured is a method for expanding and differentiating stem cells into insulin
secreting cells in a diabetic recipient of the cells, having steps of: implanting the cells in the
30 recippient; and administering a sustained release composition camprising an effective dosa of
each of: a gastrin/CCK receptor ligand; and a FACGINT or an EGF receptor ligand, wherein
the stem cells are expanded and differentiated into insulin secreting cells in the recipient,
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Also featured is a composition for treating diabetes comprising a Glucagon-like
peptide-1 (GLP-1) receptor ligand and a gastrin/CCK receptor ligand. For example, the
GLP-1 receptor ligand is GLP-1. Also featured is a composition for treating diabetes
comprising a Growth Hormone (GH) receptor ligand and a gastrin/CCK receptor ligand. For
5 example, the GH is human growth hormone (HGH). Also featured is a composition for
treating diabetes comprising a prolactin (PL) receptor ligand and a gastrin/CCK receptor
ligand. For example, the PL is human PL. In any of these Compositions, the exemplary
gastrin is gastrin 1 having 17 amino acids with a Leu residue at amino acid position 15. Any
of these compositions can further have an agent for immune suppression. Any of these
10 compositions can further be formulated for sustained release.
Also featured is a method of treating a diabetic subject, which is administering to the
subject a composition comprising a gastrin/CCK receptor ligand and a Glucagon-like
peptide-1 (GLP-1) receptor ligand. Also featured is a method of treating a diabetic subject
which is administering to the subject a composition comprising a gastrin /CCK receptor
15 ligand and a Growth Hormone (GH) receptor ligand. Also featured is a method of treating a
diabetic subject which is administering to the subject a composition comprising a gastrin
/CCK receptor ligand and a prolactin (PL) receptor ligand. Any of these methods can include
administering an agent for immune suppression. Any of these methods can further include
administering at least one of the receptor ligands or agents using a sustained release device.
20 Any of these methods can include further include formulating at least one of the receptor
ligands or agents for sustained release. Any of these methods can be used with the diabetic
subject who has type 1 diabetes or type II diabetes.
Also featured is a method for expanding a functional b-cell mass of pancreatic islet
transplants in a diabetic patient recipient of a transplant of purified islets, the method being
25 administering to the mammal an effective dose of a gastrinCCK receptor ligand and a
FACGINT.
Also featured is a method for method of treating human diabetes comprising
transplanting a pancreatic islet preparation into a diabetic patient; and administering to the
patient an effective dose of a gastrin/CCK receptor ligand and a FACGINT. Accordingly in
30 this method, the FACGINT comprises a prolactin receptor ligand which is prolactin
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Detailed Description of Specific Embodiments
Compositions for islet neogenesis therapy (I.N.T.™) comprise a gastrin/CCK receptor
ligand, and an EGF receptor ligand or a Factor Complementing Gastrin Islet Neogenesis
Therapy (FACGINT).
5 Treatment of diabetes by administration with a combination of a FACGINT and a
gastrin/CCK receptor ligand, for example, gastrin, gives a surprising level of enhancement of
potency, efficacy and utility over treatment with the any single component alone. For this
reason, the term FACGINT as used herein means "complementary" to administration of
gastrin. The term "complementary" is not necessarily meant to imply a synergism between
10 gastrin and the FACGINT, rather the term means that in comparison to administration of
gastrin and the FACGINT, administration of the combination is more efficacious for
remediating the diabetes at the doses used in the combination.
The term, "receptor ligend" as used herein in connection with a receptor for a
particular ligand shall mean any composition or compound that binds to, interacts with, or
15 stimulates that receptor.
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: a PTH-related protein
20 (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, I- 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)
25 receptor such as GIF; a transforming growth factor beta (TGF) receptor ligand such as
TGFb (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-lika peptide 1 receptor
30 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
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(GH) receptor ligand such a GH, a Prolactin (PRL) receptor ligand such as PRL and plaoental
lactogen (PL); an Insulin-like growth factor (Type 1 and 2) receptor ligands such as IGF1
and IGF-2; an Erythropoletin (EPO) receptor ligand such as EPO
(http://www.drinet,org/html/august_2002_.htm); a betacellulin (also considered to be a
5 member of the EGF family); an Activin-A receptor ligand such as Activin-A; a vascular
endotheliat growth factor (VEGF) receptor ligand such as VEGF; a bone morpnogenesis
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 cyclasa activating polypeptide (PACAF) cecapiat ligand such to
10 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.
For any of the growth factors, enzymes, enzyme inhibitors, peptide, protein and
15 hormone compounds herein that are indicated to be an exemplary FACGINT, all known
analogues, variants, 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,
20 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. Tne primary amino acid
sequence of the protein may be augmented by conjugates, as by glycosylatian, acylation, or
by addition of any other supplementary molecules, such as one or more of a lipid, a
15 phosphate, 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. An agonist, 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 ummunoglobulins and other
30 cytokincs. Variants of FACGINTs that are proteinaceous in nature can result from alternative
splicing of a primary transcript, from proteolytic cleavage or from other past-translational
modifications including dimerization, polymerization, phosphorylation, glycosylation,
sulfation and deamidation- Conjugates may include, for example, a- composition comprising
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WO 2004/037195 PCT/US2003/033595
the FACGINT coupled to a non-naturally occurring polymer comprising a polyalkylene
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. Further, agents that induce synthesis of the FACGINT or
5 mimic the action of the FACGINT are contemplated as equivalent compounds. The singular
form, "TACGINT", may mean any one or more compounds from the exemplary FACGINTs
shown herein.
In several uses of the term herein, the term FACGINT is specified not to include an
EGF receptor ligand, as is made plain in the context. However EGF receptor liganda such as
10 EGF and TGF are capable of complementing gastrin for remediation of diabetic conditions
and are therefore exemplary FACGINTs as defined herein, and are included as components in
other embodiments of compositions, methods and kits herein. Certain embodiments of I.N.T.
therapy comprising administration of an EGF receptor ligand in combination with a.
gastrin/CCK receptor ligand have been described previously (U.S.patent numbers 5,385,956
15 and 6,288,301), references that do not use these agents in certain combinations and
formulations as shown herein.
The term, "pancreatic- progenitor cell" or "beta cell progenitor" or "islet precursor
cell" as used herein is a precursor cell capable of differentiating into a pancreatic beta islet
cell, "which may or may not have the characteristic of a stem cell that is the ability to
20 reproduce itself in an unlimited manner. "Beta cell neogenesis" or "Islet neogenesis" means
formation of new beta cells by differentiation, which may or may not have the characteristic
of a stem cell that is the ability to reproduce itself in an unlimited manner. To secrete insulin
in a "glucose-responsive" manner means to secrete insulin according to the glucose
concentration in the blood. In a physiologically normal mammal, beta cells of the islets of
25 Langerhans secrete insulin as blood glucose level is elevated, i.e., are induced to socrete
insulin in response to blood glucose level.
A receptor "agonist" is any composition without limitation, for example, a
polypeptide growth factor or cytokine that is endogsnously found in the mammal, or a variant
or portion thereof, or an analog, or any peptidomimetie or low molecular weight drug, that
30 has the capability to bind to and activate the receptor of the endogenously found polypeptide
factor. For any of the growth factors or cytokines described herein, an equivalent is
considered one that is substantially identical in amino acid sequence, for example, shares
50% sequence identity, shares 60% sequence identity, shares 70% sequence identity, or
14

WO 2004/037195 PCT/US2003/033595
shares 80% sequence identity with a raturally occurring peptide or protein as described
herein. In other embodiments, the agonist compositions herein include an agonist inducing
agent, which, are envisioned to be substances that, when given to an animal or provided to a
cell, organ or tissue in culture, ia capable of increasing the amount of that agonist produced
5 by the animal, cell, organ or tissue. For example, a prolactin release peptide stimulates the
secretion of prolactin. 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.
The invention in one embodiment provides a method for treating diabetic conditions
10 such as diabetes mellitus by administerins a composition comprising both a gastrin/CCK
receptor ligand, e,g. gastrin, and a FACGINT, e.g. GLP-1, PKL or GH. Without being
limited by any particular mechanism, the gastrin/CCK receptor ligand and the FACGINT are
provided in an amount of each sufficient to effect differentiation of pancreatic islet precursor
cells to mature insulin-secreting cells. Each of the FACGINT and the gastrin/CCK receptor
15 ligand in the composition can be administered systemically or locally. Alternatively, one or
both of the FACGINT and the gastrin/CCK receptor ligand can be expressed in situ, by cells
that have been provided with a nucleic acid fusion construct in an expression vector. The
fusion construct typically includes a pieprogastrin peptide precursor coding sequence, and
also a coding sequence for a FACGINT,
20 Administration of a gastrin/CCK receptor ligand and an EGF receptor Iigand can
achieve prolonged efficacy of islet cell neogenesis, such that a therapeutic benefit is retalned
long after cessation of treatment. See PCT application PCT/US02/00685 (WO 02/055152),
published 18 July 2002. The duration of the therapeutic benefit is greater than the duration of
the protocol for administration of the composition.
15 Regenerative differentiation of residual pluripotent pancreatic ductal cells into mature
insulin-secreting cells is obtained with the provided compositions and methods for treatment
of diabetes mellitus, particularly juvenile onset diabetes, and by therapeutic administration of
this combination of factors or compositions which, are provided for systemic administration,
or for in situ expression within the pancreas.
30 An approach to b-cell replacement that eliminates a requirement for cell transplantation is
stimulation of b-cell regeneration. Although early studies suggested that a b-cell has limited
capacity for regeneration, it has been increasingly realized that the insulin secreting b-cells of
the pancreas comprise a dynamic cell population. The mass of b-cells can expand through
15

WO 2004/037195 PCT/US2003/33595
proliferation of existing b-cells (b ecll replication). During pregnancy, prolactin, growth
hormone (Holstad, M, et al., J. Endocrinol. 163:229-234), and placental lactogen (Nielsen,
J.H., et al., J. Mol. Med. 77:62-66, 1999) stimulate the proliferation of b-cells to increase 
cell mass. However, this expanded mass of cells depends on continuing hormonal
5 stimulation. After parturition, the expanded b-cell mass decreasea to non-pregnant levels, in
response to the decrease in prolactin and placental lactogen (Logothetopoulos, J.,
(Logothetopoulos, J. (1972) in Handbook of Physiology (Am. Thysol. Soc., Washington,
DC), Section 7, Chapter 3, pp67-76)
From this physiological information, an important aspect in evaluating b-cell
10 regeneration in response to administration of a gastrin receptor ligand and either an EGF
receptor ligand or a FACGINT is Whether an axpanded b-cell mass can persist for a
significant time alter the cessation of treatment with growth factors. Use of a sustained
release formulation, in combination with the I.N.T. composition, with or without an agent for
immune suppression, can avoid a requirement for frequent visits to a medical setting, or for
15 self-medication.
The neogenesis of b-cells is measured as an increase in both cell number and cell
miss, resulting in an increase in plasma insulin levels or pancreatic insulin content
Prolonged efficacy in treatment of diabetes is a desired outcome of I.H.T.
An embodiment of the present invention provides improved methods and
20 compositions fox use of a FACGINT administered with a gastrin/CCK ligand to treat
diabetes. The present invention in one embodiment provides a combination of gastrin with a
FACGINT to achieve greater efficacy, potency, and utility than achieved with a component
alone, resulting in an improved therapeutic ratio for the combination. Treatment with a
combination of gastrin and a FACGINT gave a reduction in blood glucose that was greater
25 than observed after treatment with a component alone, and the reduction in blood glucose
was sustained for a prolonged period after ceasing treatment. The phrase, "a FACGINT' as
used herein can also mean "one or more FACGINTs" or "at least one FACGINT".
The b-cell stimulant Exendin (a GLP-1 analog) improved glucose tolerance and
increased b-cell mass in a partial pancreatectomy model with mild diabetes (Xu, G. et al.,
30 Diabetes 48:2270-2276, 1999). However, a causal relationship between the improved
glucose tolerance and b-cell regeneration was not conclusively established. GLP-1, as shown
here to be an exemplary FACGINT, when administered alone suppresses appetite and
enhances the clearance of glucose by reducing insulin resistance, a process which can be
16

WO 2004/037195 PCT/US2003/033595
independent of b-cell stimulation. Thus the finding that plasma insulin levels were lower, not
higher, in an Exendin treated group, suggests that the observed improved glucose tolerance
was not a result of b cell stimulation. Furthermore, evaluation of the effect of Exendin on b
cell growth is complicated by the pancreatectomy model of diabetes that was, used in these
5 studies. An inflammation resulting from the surgical ablation, of the pancreas canses
expression of growth factors that act on islets such as gastrin and TGF a, which by
themselves stimulate islet regeneration. Indeed, enhanced b -cell regeneration has been
reported after pancreatectomy alone (Bonner-Weir, S., Diabetes 42: 1715-1720, 1993;
Sharma, A., et al., Diabetes 48:507-513, 1999). Thus, it has not previously been clear that
10 Exendin could stimulate enhanced b -cell regeneration in the absence of these
pancreatectomy-induced arowth fectors.
The term, "diabetes" as used herein means any manifested symptoms of diabetes in
any mammal including expeirmental animal models, and including human forms such as type
I and type II diabetes, early stage diabetes, and a pre-diabetic condition characterized by
15 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 susceptibilty 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
20 diabetes or a related condition and is subject to risk of recurrence.
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 liganda are given in U.S. patent 6,288,301 issued Sept. 11, 2001, and
include various forms of gastrin, such as gastrin 34 (big gastrin), gastrin 17 (little gastrin),
25 and gastrin 8 (mini gastrin); various forms of cholecystokinin such as CCK 58, CCK 33,
CCK 22, CCK 12 and CCK 8; and other gastrin/CCK receptor liganda. In general,
gastrin/CCK receptor ligands share a carboxy teminal amino acit sequence Trp-Met-Asp-
Phe-amide, Also contemplated are active analogs, fragments and other modifications of the
above, including both peptide and non-peptide agonists or partial agonists of the gastrio/CCK
30 receptor such as A71378 (Lun et al.,Am.J.Physiol.258(4 Pt 1):G648,1990).
Small forms of gastrin such as gastrin 17 ate economically prepared by peptide
synthesis:, and synthetic peptides are commercially available. Synthetic human gastrin 17
such as human gastia 17 having methionine or leucine at position 15 are also available from
17

WO 2004/037195 PCT/US2003/033595
Bachem AG, Bubendorf, Switzerland, and from Researchplus. 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
5 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.
The method for treating diabetes mellitus in an individual in need thereof includes
10 administering to the individual a composition that provides both a gastrin/CCK receptor
ligand and a FACGINT. Without being limited by any particular mechanism, the
gastrin/CCK receptor ligand and the FACGINT are provided in doses sufficient to effect
differentiation of pancreatic islet precursor cells to mature insulin-secreting cells.
The term "treating" or "amenorating" as used herein means reducing or eliminating
15 one or more symptoms of a diabetes. A method provided herein for treating diabetes
comprises administering, without being limited by the specific mechanism, a differentiation
regenerative amount of both a gastrin/CCK receptor ligand and a FACGINT, to a diabetic
mammal, to stimulate islet neogenesis to increase the number of functional glucose
responsive insulin secreting b-cells in the pancreas. The method is effective for diabetes
20 generally, including Type I or juvenile diabetes mellitus. The combination of gastrin and
FACGINT would result in a significant enhancement of the islet neogenesis response over
that observed with the individual components. An exemplary gastrin/CCK receptor ligand is
gastrin, and exemplary FACGINTs are GLP-1, PRL and GH.
Another embodiment of the invention herein is a method comprising treating
25 explanted pancreatic tissue of a mammal with a gastrin/CCK receptor ligand and FACGINT
ex vivo, and introducing the treated pancreatic tissue to the mammal. Again in this method,
an exemplary gastrin/CCK receptor ligand is gastrin, and exemplary FACGENTs art GLP-1,
PRL and GH.
In another embodiment, the invention provides a method fer gastrin/CCK, raceptor
30 ligand stimulation, the method comprising providing a chimeric insulin promoter-gastrin
fusion gene to pancreatic cell and expressing the gane. In yet another embodiment, a
method of FACGINT stimulation is provided, comprising expressing a FACGINT gene that
was trangenically introduced into a mammal, for example, a gene encoding a FACGINT, for
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WO 2004/037195 PCT/US2003/033595
example, GLP-1, PRL or GH. The gastrin/CCK receptor ligand gene can similarly be
provided trangenically, pieferably a human preprogastrin peptide precursor gene as shown in
U.S. patent number 5,885,956.
As used herein the term mammal shall include without limitation any members of the
5 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 of a chimpanzee. An individual mammal may be nom-diabetic, pre-diabetic, or
diabetic, as specified herein.
Modes of systemic administration include, but are not limited to, transdermal.
10 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
15 systemic, for example, by subcutaneous injection.
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
20 pituitary gland. The term encompasses prolaction 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
compositian having agonist activity for the prolactin receptor, as disclosed in U.S. patent
number 6,333,031 (activating amino acid sequence) and 6,413,952 (metal complexed
25 receptor ligand agonist), and Gl20RhGH, which is an analog of human growth hormone that
sets as a prolactin agonist (Mode et al., 1966, Endocrimal. 137(2): 447-454) and a ligand for
the prolactin receptor as described in U.S. patents 5,506,107 and 5,837,460. Also included
are prolactin-related protein, S179D, human prolactin and placental lactogens.
PRL, GH and PL are members of a family of polypeptide hormones that share a
30 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
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WO 2004/037195 PCT/US2003/033595
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. Classification of FACGINTs into various
groups based on structural similarity of the peptides and proteins, functional similarity with
5 respect to complementation of gastrin, functional similarity with respect to binding of one or
more receptors, are each within the scope of various embodiments of the invention. Prolactin
receptor ligands include PRL and PL, and growth hormone receptor ligands include GH.
As used herein, the term "GLP-1 receptor ligand" encompasses any compound that
binds to, interacts with or stimulates the GLP-1 receptor. Examples of GLP-1 receptor ligand
10 I nclude GLP-1 and exendin-4. 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 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
15 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.
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
20 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-TV) -mediated cleavage at the position 1 alanine residue. For an overview,
see DPP-IV. The biological activities of GLP-1 include stimulation of glucose-dependent
25 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., Endoorin, 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
30 Amylin, San Diego CA); ThGLP-1 (GLP-1, modified amino acids and fatty acid attachment,
from Theratechnologies, Saint-Laurent, Quebec, Canada); CIC-1131 also known as
DAC™:GLP-1 (GLP-1 analog engineered for covalent coupling to albumin, Conjuchem,
Montreal, Quebec, Canada), LY315902 and sustained release LY315902 (DDP-IV resistant
20

WO 2004/037195 PCT/US2003/033595
GLP-1 analog from Eli Lilly, Indianapolis, IN); a low molecular weight GLP-1 mimetic;
Albugon (albumin: GLP-1 fusion peptide from Human Genome Sciences, Rockville, MD);
Liraglutide also known as NN2211 (long acting GLP-1 derivative that is obtained by
acylation of the GLP-1 molecule, which upon entering the bloodstream, is extensively bound
5 to albumin which protects it from degradation by DPP-IV and reduces renal clearance,
available from Novo Nordisk, Denmark; Elbrond et al., Diabetes Care 2002 Aug 25(8): 1398-
404).
Exendin-4, an example of an exendin, is a novel peptide from Heloderma suspectum
(Gila monster) venom, having 53% homology with GLP-1 (7-36)amide. It functions as a
10 long-acting potent agonist of the glucagon-like peptide 1 (GLP-1) receptor, as it is resistant to
degradation by DDP-IV. Exeudin-4 has properties similar to GLP-1, and regulates gastric
emptying, insulin secretion, food intake, and slucagon secretion. Examples of exendin-4
include excnatide (synthetic form also known as AC2993, Amylin); exenatide LAR
(longactinf form); ZP10 (modified exendin-4 haviag addition of six lysine residues,
15 Aventis/Zealand Pharma); and AP10 (long acting formulation, Alkennes, Cambridge MA).
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.
20 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-madiated inactivanon 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, MA), LAF237 (Novartis), P32/98
25 (Probiodrug AG, Halle, Germany) and P93/01 (Probiodrug).
As used heren the term "EGF receptor ligand" encompasses compounda that
stimulate the EGP receptor such, that when gastuin/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 BGF receptor ligands include full length
30 EGF, which is EGF1-53, and further include EGF1 -4S, EGF1-49, EGF 1-52, and fragments
and active analogs thereof. Other examples of EGF receptor ligands are TGF 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
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WO 2004/037195 PCT/US2003/033595
ligands. These include active analogs, fragments and modifications of the above. See also,
Carpenter and Wahl, Chapter 4, in Peptide Growth Factors (Eds, Spom and Roberts),
Springer Verlag, 1990,
The group of compounds comprises the EGF receptor ligands further includes
5 "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.
10 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. patent number 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 armino acid substitution at position 51, retain EGF activity
15 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 (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
20 activity at low values of pH. Chemical analogues and fragments of EGF and TGF retain
ability to bind various members of the EGF receptor family (U.S. patent number 4,686,233).
Various modifications of EGF or TGF 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-
25 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 fall I.N.T.™ activity,
and has in vivo and/or in vitro stability that is that 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
incorporatad by reference herein in its entirety).
30 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
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WO 2004/037195 PCT/US2003/033595
weight of about 22 kDal (Goeddel et al, 1979, Nature 581; 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. Sec Cunningham
et al., 1989, Science 243: 1330-1336, and 1989, Science 244: 1081-1045; and WO 90/05185,
5 and U.S. patent number 5,506,107.
The term, "erythnopoietin" (EPO) as used herein is any endogenous mammalian EPO
or variant thereof, or EPO receptor agonist, for example the EPO mimetic EMPI (Johnson et
al., 2000, Nepbr Dial Tranpl 15:1274-1277); or numerics described (Wrighton et al., 1996.
Science 273:458-164; U.S. patent number 5,773,569; Kaushansky, 2001, Ann NY Acad Sci
10 938;l3l-138);aii antibody having EPO receptor agonist activity(see for example, U.S.
patent number 5,885,574; WO 96/40231); and amino acid sequence disclosed in U.S. patent
number 6,333,031, and 6,413,952.
The tern, "PACAP" as used herein means an endogenously produced PACA or
analog or variant thereof that shares substantial ammo acid identity or similarity, or has
15 biological activity as a PACAP receptor agonist such as maxaditan (Moro et al., 1997, J Biol
Chem 272:966-970. Useful PACAP variants include without limitation, 38 amino acid and
27 amino acid variants as disclosed in U.S. patent numbers 5,128,242; 5,198,542; 5,208,320;
and 6,242,563).
Pharmaceutical Compositions
20 The present invention in various embodiments provides pharmaceutical compositions
comprising a theraptuticalty effective amount of a combination of a FACGINT, and a
gastrin/CCK receptor ligand. 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
25 pharmaceutically acceptable carrier or excipient can be added. Such a earrier 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 symptorm of diabetes.
30 The effective amount can be determined empirically by a skilled artisan according to
established methods of measurement of relevant parameters, as described herein.
The compositions herein can farther comprise wetting or emulsifying agents, or pH
buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill,
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WO 2004/037195 PCT/US2003/033595
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 manuitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Various
5 delivery systems are known and can be used to administer a composition of the invention,
e.g., encapsulation in liposomes, microparticles, miciocapsules and the like.
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
10 solutions in sterile isctanic 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
15 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
20 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.
25 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 hydroclorit, phosphoric, acetic, oxalic, tartaric adds, 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,
30 procaine, etc.
The amount of the therapeutic of the invention which will be effective in the treatment
of a particular disorder or condition will depend on me nature of the disorder or condition,
and can be determined by standard clinical techniques. The precise dose to be employed in
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WO 2004/037195 PCT/US2003/033595
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 chellenges, are determinied by one of ordinary skill
5 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.
Suitable dosage ranges for administration are generally about 0.01 micrograms to about
10,000 micrograms of each active I.N.T.™ compound per kilogram body weight per day, for
exampla, about 0.01 micrograms to about 1 microgram/kg, about 0,1 micrograms/kg to about
10 10 micrograms/Kg, about 1 microgram/kg to about 500 micrograms/kg, or about 10
miaograma/kg to about 10 mg/kg of body weight per day. Stritable dosage ranges for
administration are thus generally about 0.01 micrograms/kg body weight/day to about 10
mg/kg body weight/day.
The invention in other embodiments provides a pharmaceutical pack or kit comprising
15 one or more containers filled with one of more of the ingredients of the pharmaceutical
composition of the invention. In such a pack or kit can be found a containre baviag a unit
dosage of each or both of a gastrin/CCK receptor ligand and/or one or more of a FACGINT,
or an EGF receptor ligand, and one or more of an immunosuppressive agent One or more of
these components of the pack or kit can be formulated for sustained release or for insertion as
20 a refill into a sustained release device, or can be formulated for local delivery. Associated
with such container(s) 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 pharmacuticals of biological products, which notice reflects approval by the agenty
of manufacture, use or sale for human administration. In some embodiments the kit or pack
25 may be associated with software embedded in a computer readible format.
The invention in one aspect features islet neogenesis therapy (I.N.T.™) compositions
and methods, for example, a gastrin and a FACGINT, in combination with
immunosuppressive agents, to stimulate the growth of new b-cells in vivo, increasing islet
mass, and result in improved glucose tolerance in diabetic subjects, for example, in diabetic
30 humans and in animals.
The gastrirn/CCK receptor ligand and the EGF receptor ligand or FACGINT, can be
administered in a single combined dose, or administered separately in any order. An
"effective combined dose" of these compositions is a dose that produces a decrease in fasting
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WO 2004/037195 PCT/US2003/033595
blood glucse, or an increase in amount of insulin secreting cells, or an increase in insulin
blood level, or an increase in -cell mass. The gastrin/CCK receptor ligand is, in one
embodiment, human gastrin of length 17 amino acid residues, the residue at position 15 being
leucine (l-17Leul5, referred to herein as gastrinl71eul5); farther, the EGF receptor ligand is
5 human EGF51N (S. Magil et al., published May 12,2003 as FCT/US02/33907, and
incorporated by reference herein in its entirely). The effective dose can contain a ratio of
gastrin/CCK receptor ligand to EGF receptor ligand that is greater than 1, for example, the
effective dose contains a ratio of gastrin/CCK receptor ligand to EGF receptor ligand greater
than 10. A convenient route of administering the dose is with a systemic injection, for
10 example, a subcutaneous bolus.
In a further embodiment, the recipient subject is administered an agent that suppresses
the immune system. For example, the agent is a low molecular weight organic chemical, for
example, is at least one of Tacrolimus, Sirolimus, cyclosporine, and cortisone and other drugs
as shown in Table 1. In an alternative embodiment, the agent is an antibody, for example, the
15 antibody is anti-CD11a and other antibodies also shown in Table 1. In yet another
alternative, the irnmunosuppriessive agent can be an antibody that is elaborated by the subject
following an immunization schedule, for example, against GFAP or against S100. The
subject can be diabetic, for example, the subject is a non-obese diabetic mouse (the NOD
mouse) or a streptozoticin-treated mouse. The subject can be a human, for example a
20 diabetic patient having type I or type II diabetes, or having a pre-diabetic condition, or having
gestational diabetes, or having had diabetes in the past, for example, having had gestational
diabetes in a past pregnancy.
Further, evaluating the size and function of newly developed  insulin secreting cells
or islets is measuring a standard physiological or diagnostic parameter including: islet b-cell
25 mass, islet b-cell number, islet b-cell percent, blood glucose, serum glucose, blood
glycosylated hemoglobin, pancreatic b-cell mass, pancreatic b-cell number, fasting plasma C
peptide content, serum insulin, and pancreatic insulin content.
As diabetes is in certain cases an autoimmune disease, an embodiment of I.N.T.TM
is systemic administration of therapeutically effective doses of, for example, the ligands of
30 receptors for each of EGF and gastrin/CCK or each of a FACGINT and gastrin/CCK, such as
a combination of a gastrin and an EGF or a combination of a FACGINT and gastrin/CCK, to
subjects or patients who are also treated with one or more agents that suppress the immune
system, i.e., immunosuppressive agents.
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A number of different endpoints can be used to determine whether gastrin and EGF or
FACGINT treatment, or treatment with the combination of gastrin and EGF or FACGMT
and an immunosuppiessive agent, improves the diabetes, for example, increases the
functional mass of b-cells in the islet transplants. These include measurement of enhanced
5 plasma levels of circulating human C peptide and human insulin, after injecting mice with 
cell stimulants such as glucose or arginine; a response to gastrin/EGF treatment demonstrated
by increased human insulin immunoreactivity or mRNA levels extracted from the istet
transplants, and increased number of b-cells, determined by morphhometuic mrasusement of
islets in treated animals.
10 The term "transplanting" as used herein means introducing a cellular, tissue or organ
composition into the body of a mammal by any method established in the art, or as indicated
herein. The composition is a "transplant", and the mammal is the recipient. The transplant
and recipient may be syngeneic, allogenic, or xenogeneic. The term, "autologous" as used
herein means that the transplant is derived from the cells, tissues or prgans of the recipient.
15 Enhanced b-cell function of human islets can also be demonstrated by reversal of the
hyperglycemia in recipient mice with streptozotogin induced or genetic (using a strain of
mice known as non-obese diabetic or NOD) diabetes. Enhanced b-cell finction after
treatment of the diabetic recipient subject with gastrin, with EGF or FACGINT and with one
or more immunosuppressive agents is demonstrated by improved survival upon withdrawal
20 of insulin, and by correcting hyperglycemia as indicated by fasting blood glucose level.
Further, increases in both pancreatic insulin and plasma C-peptide are observed.
Table 1. Exemplary agents for immune suppression, and commercial sources

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29

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30

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As used herein, the term "immunosuppiessant" 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
5 considered appropriate for embodiments of the invention as described herein and in the
claims.
As used herein, a dosing schedule refers to a protocol for administering any of the
compositions provided herein, for example, the components that make up the I.N.T.™
composition or the combination of a FACGLNT and a gastrtn/CCK receptor ligand, and one
10 or more of an immunosuppressive agent, each in an effective dose, administered
simultaneously or within a particular interval of each other, for example, within one day of
cachother, 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.
15 Most insulin dependent diabetic patients require insulin injection at least on a daily
basis. Multiple doses per day of insulin are required under certain circumstances of illness or
diet for management of diabetes, and the insulin administration is indicated by results of
frequent glucose monitoring, another activity which is required of a diabetes patient for
optinal management of the disease, which is performed for example as often as five times
20 daily.
Remission from diabetes due to successful islet neogenesis therapy in combination
with an immunosuppressive agent is indicated by a decreased fasting blood level of glucose,
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WO 2004/037195 PCT/US2003/033595
and by a decreased level and duration of elevated blood glucose in response to a dietary
challenge of sugar consumption. Upon achieving successful islet neogenesis, insulin
administration is reduced from, for example, five injections to two injections per day; from
two injections to one injection per day; and from one to none, as indicated by data obtained
5 from monitoring blood glucose levels. One of ordinary skill in the art of diabetology, when
treating a diabetic patient, is familiar with adjusting insulin dosage to levels of blood glucose
following festing and under other physiological conditions.
Dosages of the compositions to be administered to a subject are adjusted for known
variations from species to species in standard data encompassing criteria for absorption,
10 distribution, half-life kinetics in circulation, metabolism, excretion, and toxicology of the
receptor ligands of the embodiments herein, for example, for each primate and rodent species.
In general, dosages are adjusted to be about 6-fold to about 100-fold greater for
administration to a rodent species than to a primate species.
Immunosuppressive agents in Table 1 or other equivalent agents are administered as
15 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 Sirolixmus is generally administered orally.
Modes of administration of the receptor ligand compositions and immunosuppressive
agents include but are not limited to subcutaneous, transdermal, intraihecal, intramuscular,
20 intraperitoneat, intravenous, intranasal, and oral routes. The compounds may be administered
by any convenient route, for example, by infusion or bolus injection, by pump, by absorption
through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,
etc.). The receptor ligands herein may be administered in combination with one or a plurality
of other biologically active agents. For example, a recipient of the compositions and methods
25 herein may be administered one or more antibiotics if a bacterial infection is present, or
aspirin if a headache is present. Administration of the receptor ligands herein is preferably by
a systemic route.
The invention in one embodiment provides a method for treating diabetes mellitus by
administering a composition comprising both a gastrin/CCK receptor ligand, e.g. gastrin, and
30 an EGF receptor ligand, or a FACGINT, e.g. GLP-1, GH, or prolactin in a formulation for
sustained release, to effect differentiation of pancreatic islet precursor cells to mature imulin-
secreting cells. Both the FACGINT and the gastrin/CCK receptor ligand in the composition
can be administered systemically or locally.
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Without being limited by any particular mechanism, prolonged efficacious islet cell
neogenesis is achieved following administration of both a gastrin/CCK receptor ligand, such
as gastrin, and an EGF receptor ligand or one or more of a member of the FACGINT group
is defined herein, such as a GLP, a GH, or a prolactin, alone or with an agent for immune
5 suppresion, and any one or more of these receptor ligands or factors or agents being
formulated for sustained releasc as described herein or by equivalent methods known or one
of ordinary skill in the pharmacological arts.
The invention in a general embodiment provides a method for preventing or treating
diabetes, the method comprising administering to a mammal in need thereof a composition
10 comprising a sustained release formulation of at least one of an EGF receptor ligand or a
FACGINT, such as a GLP-1, an exending-4, a growth hormone, a prolactin, in combination
with a gastrin/CCK receptor ligand, each of the EGF receptor ligand or the FACGIMT and
the gastrin/CCK receptor ligand in an amount sufficient to increase the number of pancreatic
insulin secreting b-cells in the mammal, thereby treating or preventing the diabetes.
15 Compositions for treating diabetic subjects and patients with a sustained release formulation
of a FACGINT comprise : a PTH-related protein (PTHrP) Receptor ligand such as PTHrP
(PTHrP) Gaicia-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., 1599, J Mol Med
77: 62-66); a fibroblast growth factor (FGF) such as FGF, a keratinocyte growth factor
20 (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 (TGFb) receptor ligand such as TGF (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 TNGAP; a bone morphogenetic factor
25 (BMP) receptor ligand such as BMF-2; a vasoactive intestinal peptide (VIP) receptor ligand
suchas VIP; a glucagon-like peptide 1 receptor ligand such as GLP-1 and extendin-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 ai., 2002, Am Diabetes
Assoc Abstract 272-or) by inhibiting an enzyme involved in its integrity; a REG receptor
30 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_2002_.htm); a betacellulin (also
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WO 2004/037195 PCT/US2003/033595
considered to be a member of the EGF family); an Activin-A receptor ligand such as Activin-
A; a vascular Activin-A; a vascular endothelial growth factor (VEGF) receptor ligand such as
VEGF; a bone morphogenesis factor (BMP) receptor ligand such as SMP-2; a vasoactive
intestinal peptide (VIP) receptor ligand such as VIP; a vascular endothelial growth factor
5 (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 tigand such as secretn. These sustained release
10 formulations may also include an agent for immune suppression.
In one embodiment, the sustained release formulation of the composition is
administered systemically. Alternatively, the composition is administered locally, or with a
device or means for local delivery. The mammal is a diabetic mammal, for example, the
mammal has been diabetic for an extent of at least about 1% of the lifespan of the mammal.
15 In general, the amount of the sustained release formulation, of the gastrin/CCK receptor
ligand, the FACGINT or the EGF receptor ligand in the composition is substantially lower
than the minimum effective dose of any of these alone required to reduce blood glucose in the
diabetic mammal. The FACGINT or the EGF receptor ligand and the gastrin/CCK receptor
ligand are provided in amounts sufficient in combination to induce, differentiation of the
20 pancreatic islet precursor cells into glucose responsive insulin secreting islet cells for a
prolonged period of time.
Another embodiment of the invention provides a method, for preventing or treating
diabetes, the method comprising administering to a mammal in need thereof a sustained
release formulation of a composition comprising a combination of a FACGINT or an EGF
25 receptor ligand, and a gastrin/CCK receptor ligand, in an amount sufficient to increase
proliferation of islet precursor cells in pancreatic tissue, thereby treating or preventing the
diabetes.
In another aspect, the invention provides a method for preventing or treating diabetes,
the method comprising administering to a mammal in need thereof a sustained release
30 formulation of a composition comprising a combination of a FACGINT or an EGF receptor
ligand, and a gastrin/CCK receptor ligand, each in an amount sufficient to increase the
number of pancreatic insulin secreting b-cells in the mammal; and determining the amount of
islet neogenesis, thereby treating or preventing the diabetes. Administering the composition
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WO 2004/037195 PCT/US2003/033595
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 compered to glycosylated
5 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.
10 In another aspect, the invention provides a method for inducing pancreatic islet
neogenesis in a mammal, the method comprising administering to the mammal a sustained
release formulation of a composition comprising a combination of a FACGINT or an EGF
receptor ligand and a gastrin/CCK receptor ligand, each in an amount sufficient to increase
proliferation of islet precursor cells in pancreatic tissue, thereby inducing pancreatic islet
15 neogenesis.
In another aspect, the invention provides a method for inducing pancreatic islet
neogenesis in a mammal, the method comprising administering a composition comprising a
sustained release formulation of a combination of a FACGINT or an EGF receptor ligand and
a gastrin/CCK receptor ligand, each in an amount sufficient to increase the number of
20 pancreatic insulin secreting b-cells in the mammal.
In another aspect, the invention features a composition comprising a gastrin/CCK
receptor ligand, and a FACGNIT or an EGF receptor ligand, any of which agents are
formulated in sustained release formulation. The composition is in a dosage effective for
inducing proliferation of islet precursor cells into an increased amount of mature insulin
25 secreting cells. Further, the composition is in a dosage effective for inducing differentiation
of an islet precursor cell into a mature insulin secreting cell. The composition can be in a
pharmaceutically acceptable carrier. The composition con include an agent for suppression
of an immune response.
In another aspect, the invention provides a kit for treating or preventing diabetes,
30 containing a composition comprising a gastrin/CCK receptor ligand and a FACGINT or an
EGF receptor ligand, any of which are present in sustained release formulation, a container,
and instructions for use. The composition can include an agent for immune suppression. The
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WO 2004/037195 PCT/US2003/033595
composition of the kit can further comprise a pharmaceutically acceptable carrier. The
composition of the kit can be present in a unit dosage.
Another embodiment of the invention provided herein is a method for expanding end
differentiating stem cells in a diabetic recipient of the cells into insulin secreting cells,
5 comprising implanting the cells in the recipient, and administering a composition containing
an effective dose of each of a gastrin/CCK receptor ligand and a FACGENT or an EGF
receptor ligand, one or mare of which are present as a sustained release formulation. For
example, the implanted cells are obtained from a human. Further, the implanted cells are
obtained from pancreatic islets, umbilical chords, embryos, or stem cell lines. Generally, the
10 gastrin/CCK receptor ligand is human gastrin l-17Leul5. Generally, the EGF receptor
ligand is an EGF or a TGFa, or is a polypeptide that structurally is substantially identical to
EGF or TGFa, respectively, and has substantially the same biological functions of each of
EGF or TGFa In a related embodiment, cell, for exemple, stem cells are implaned by a
route selected from: direct injection into an organ, and by intravenous administration. For
15 example, the cells are injected into an organ selected from the pancreas, the kidney, and the
liver. Alternatively, the cells are administered to the portal vein using a percutaneous or
transhepatic route. In either case, prior to implanting, the cells can be treated ex vivo with the
composition.
Another embodiment of the invention provided herein is a method of treating human
20 diabetes by implanting a reduced amount of stem cells into a diabetic recipient, the method
comprising administering to the recipient an effective dose of each of a sustained release
formulation of a gastrin/CCK receptor ligand and a FACGINT or an EGF receptor ligand, the
amount of cells reduced in comparison to implanting cells into an otherwise identical
recipient in the absence of administering the effective dose. The recipient can further be
25 administered an agent that suppresses the immune system. For example, the agent is a
chemical selected from the group consisting of FK506, rapamycin, cyclosporin and cortisone.
Alternatively, the agent is an antibody, for example, the antibody is anti-CD4. In any of the
methods provided that involve stem cells, the cells prior to implanting can be obtained from a
family member and stored for later use.
30 An embodiment of the present invention provides improved methods and
compositions for use of a FACGINT or an EGF receptor ligand administered with a
gastrin/CCK ligand, any one or more of these agents being formulated for sustained release,
to treat diabetes. The present invention in one embodiment provides a susteintd release
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WO 2004/037195 PCT/US2003/033595
formulation of any of a gastrin, in combination which a FACGINT or an EGF receptor ligand,
to achieve greater efficacy, potency, and utility than achieved with a FACGINT or an EGF
receptor ligand alone, or with a combination of any of these agents administered to provide
immediate bioavailability of the entire formulation. The present invention provides an
5 improved therapeutic radio for the sustained release formulation compared to the immediately
bioavailable formulation.
Use of a sustained release formulation can extend the reduction in blood sugar for an
even greater period of time.
The combination of a gastrin/CCK receptor ligand and an EGF receptor ligand or
10 FACGINT in a sustained release formulation, with systemic administration, had greater
potency than when it was administered in a non-sustained release direct formulation. An
improvement in glucose tolerance and an increase in pancreatic insulin levels were observed
with sustained release formulation of the gastrin/FACGINT combination or gastrin/EGF
receptor ligand combination.
15 The method for treating diabetes mellitus m an individual in need thereof includes
administering to the individual a sustained release formulation of a composition that provides
both a gastrin/CCK receptor ligand and a FACGINT, or gastrin/CCK receptor ligand and an
EGF receptor ligand, the compositions in doses sufficient to effect differentiation of
pancreatic islet precursor cells to mature insulin-secreting cells. The cells differentiated are
20 residual latent islet piecuisor cells in the pancreatic duct. A method for treating insulin
dependent diabetes, especially Type I or juvenile diabetes mellitus, comprises administering,
preferably systemically, a differentiation regenfsrative amount of both a gastrin/CCK receptor
ligand and a FACGINT or a gastrin/CCK receptor ligand and an EGF receptor ligand, either
or both agents in a sustained release formulation, to a diabetic mammal, to stimulate islet
25 neogenesis to increase the number of functional glucose responsive insulin secreting b-cells
in the pancreas. The combination of a gastrin and either a FACGINT or an EGF receptor
ligand, any of which in a sustained release formulation, would result in a significant
enhancement of the islet neo genesis response over that observed with the same agents but in
a non-sustained release direct formulation. An exemplary gastrin/CCK receptor ligand is
30 gastrin or its synthetic gastrin derivative as described herein, and exemplary FACGINTs are
GLP-1, GH, and prolactin. An exemplary EGF receptor ligand is a recombinant human EGF,
for example, EGF51N, a 51 omino acid long human recombinant mutant EGF having a
deletion at the C-terminal and an asparagine residue at position 51.
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WO 2004/037195 PCT/US2003/033595
Alternatively, the cells are administered to the portal vein using a percutaneous or
transhepatic route. In either case, prior to implanting, the cells can be treated ex vivo with the
composition.
Formulations for Sustained Release and Local Delivery and Methods of Administration
5 Admioistration of at least one of the agents that are a gastrin/CCK receptor ligand, or
an EGF receptor ligand or a FACGINT, is formulated to occur by sustained or controlled
release. "Sustained release" as used here and in the claims refers to a combination of
materials, devices, formulation, and/or administration of at least one I.N.T.™ therapeutic
agent, that creates a continuous or discontinuous, such as cyclical, supply of an amount of the
10 combination or at least one I.N.T. agent, or immune suppressing agent, to the recipient as a
function of time. The period of time over which release can be sustained is minutes, hours,
days, weeks, or even months. The release of the active agent may be constant over a long
period, alternatively release may be cyclical over the prolonged period release. Release may
be independent of conditions, alternatively release may be triggered in response to a
15 composition in the environment or to other external events. For example, release may be
triggered by an endogenous composition such as insulin or glucose, or release may be
triggered by an externally supplied composition such as in response to administration of a
drug.
Sustained release is contrasted to a non-sustained release formulation that delivers the
20 whole of the therapeutic agent dose instantaneously or very rapidly, or is biotrvailsble in the
entirety of the composition in a very short period following administration, making a large
proportion of the agent available to the recipient over a very short time period. Examples of
substantially instantaneous bioavailability of a dose include aqueous solutions of agents that
are administered parenterally, for example by a bolus intraperitoneal injection, or orally, or
25 even administered by intravenous drip over a period of several hours. Thus, intravenous
administration of a cardiovascular imaging agent is circulated throughout the arterial system
within 15 seconds; intravenous drip administration of an anti-tumor agent is entirely
bioavailable within seconds of finishing the drip. In contrast, sustained release formulations
benefit the recipient patient both by providing long term bioavaility, and by protein the
30 patient by avoiding potential side effects that might result from initial high levels of the
agent. See Mathiowitz, E., Encyclopedia of Controlled Drug Delivery. 1999 New York:
John Wiley.
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WO 2004/037195 PCT/US2003/033595
Sustained release formulations have been developed to provide systemic as well as
local (or targeted) administration of agents. A variety of material, devices and routes of
administration reviewed herein is used with the I.N.T.™ agent such as the gastrin/CCK
receptor ligand, and the EGF receptor ligand or the FACGINT.
5 Oral sustained release systems have been available, for example, hard gelatin capsules
conrtaining a plurality of types of pellets, cach type having a different thickoess of coaling
such that some pellets are uncoated more quickly. See Banga, A., Bus- Brief.: Pharmatech
2002: 151-154. In oral osmotic systems, fluid entering a tablet through a semi-permeable
membrane generates an osmotic pressure of core components that is independent of variables
10 in the gastrointestinal tract. To protect agents that are macramolecules for example proteins
such as growth factors, methods include use of site-specific delivery, protease inhibitor,
carrier systems, or formulations such as hydrogels that contain polyacrylic acid backbones
and bioadhesivc properties.
Most protein agents are delivered pareoterally, and controlled release methods for
15 parenteral delivery of proteins include use of lactic acid-based polymers such, as poly(D, L-
lactide-co-glycolide; PLGA), which forms biodegradable microspheres with a core
containing the agent, and that release the agent over a course of time of about one month.
Further, proteins or liposomes (see below) containing protein can be pegyiated by covalent
addition of polyetheylene glycol (PEG).
20 Transdermal delivery can be used both for systemic and for local delivery.
Permeation enhancers as described infra can be used in combination with a transdermal patch.
to improve delivery, or in combination with a device for iontophoresis, phonophoresis,
microporation, or elecroporation with possible wearable electrical devices.
The term, "local delivery" as used herein refers to administering by a route and
25 formulation or device or both such that a particular target organ or tissue is substantially
treated while other organs and tissues are not so treated, or that the extent of treatment of the
target tissue or organ receives at least two-fold, at least five-fold, or at least 10-fold greater
dose than non-target tissues or organs. In general herein, a target organ is the pancreas. As
shown herein, cells for transplantation or multi-cellular transplants can be locally delivered to
30 the pancreas by injection into the portal vein; drugs can be delivered by injection into
pancreatic arteries, hepatic arteries, portal vein, or pancreatic duct. It is possible to employ a
pump that is not implanted, i.e., remains external and delivers the composition to a target
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WO 2004/037195 PCT/US2003/033595
organ such as the pancreas by a catheter connecting the pump to the organ. It is also possible
to employ an implantable pump to deliver the composition locally.
Other procedures for local delivery include without limitation endoscopic retrograde
cholangiopancreatography (ERCP): endoscopic ultrasound-guided fine needle aspiration
5 biopsy (EUS-FNAB) which is adapted for delivery of pharmaceutical compositions provided
herein, rather than used for sampling. See for example, Wang et al., Transpl. Int 1995, 8;
268-272, While these technologies are devised for diagnostic or prognostic purposes, they
can be adapted for delivery of compositions herein, which may I.N.T.™ compositions as
described herein, to be combined with immune suppressing agents, and or as sustained
10 release formulations. Set also, Yano et al, 1994, Transpl Int 7 Suppl 1 :S187-193; Ricordi et
al., 1994. Transpl Proc26: 3479; and Munoz-Acedo et at., 1995, J Endocrin 145; 227-234.
Pumps which can be implantable or non-implantable (external) pumps for drug
delivery are in use for treatment of several diseases, for example, for cancer and for diabetes.
A pump can be a peristaltic pump, a fluorocarbon propellant pump, or an osmotic pump
1 5 including a mini-osmotic pump (Blanchard, S., 1996 Biomedical Engineering Applications,
North Carolina State University). Peristaltic pumps deliver a set amount of drug with each
electric pulse that drives the pump bead. The pump, electronics and power source are located
in a titanium housing covered in Silastic. Drug reservoirs are silicone rubber pouches that
can withstand substantial pressure, for example, 60 psi. The reservoir can be refilled
20 percutaneously through a polypropylene port. Fluorocarbon pumps use a fluorocarbon liquid
to operate the pump. Osmotic pumps use osmotic pressure to release the drug at a constant
rate. An exemplary pump is the MiniMed MicroMed 407C pump (Medtronic, Iuc.,
Northridge, CA). Further, an intrathecal drug delivery system (Medronic) which includes
two implantable components, an infusion pump, and an intraspinai catheter, can be used. The
25 pump is inserted abdominally in a subcutaneous pocket, while the catheter is inserted into the
intrathecal space of the spine, tunneled under ihe skiru, and connected to the pump.
Medication is then delivered at a constant or variable flow rate. Further, an intraperitoneal
pump, for instance an implantable pump, con be used to deliver the compositions herein
locally, for example via a catheter, or systemically.
30 Mucosal delivery to epithelial areas that are maintained in a moist candition and
which have close underlying vasculature can be more efficient than transdermal delivery
across a tissue which is a dry epidermal surface.Mucosal surfaces include: nasal,
41

WO 2004/037195 PCT/US2003/033595
pulmonary, rectal, buccal, ocular, and genital. Exemplary mucosal surfaces are nasal and
pulmonary.
Materials used for microspheres for sustained release are primarily polymers, and
include PEG, also called polyethylene oxide (PEO), and PGLA as described. A polymer
5 vehicle cart be injected directly, allowing for gradual hydrolysis or degradation within the
subject to release the therapeutic agent. The molecular weight of the polymer, e.g., PEG, can
be varied to control the release rate. Pegylatfcm or covalent attachment of PEG to a protein
therapeutic agent shields the protein from receptors involved in clearance mechanisms such
as that of the reticulo-endothelial system (RES). Alternatively, polysaccharides can be used
10 so target an agent to the RES (U.S. patent 5, 554,386 issued Sept 10, 1996). Organs of the
RES include liver, spleen, and bone marrow.
Homopolymers or co-polymers, such as poly(lactic acid-co-ethylene glycol) or PLA-
PEG, can form a viscous liquid which is mixed with a therapeutic protein agent. Viscosity is
varied by the molecular weight of the PLA-PEG. Under certain conditions the therapeutic
15 agent co-precipitates with the polymer upon injection into the subject and loss of the solvent
by diffusion, such that a depot is formed having favorable release kinetics (Whitaker, M., et
al. Bus. Brief: Pharmatech 2002:1-5).
Microparticles are formed of polymeric microspheres that encapsulate the therapeutic
agent. Polymers for use in microspheres include poly(lactic acid) or PLA; poly(glycolic acid)
20 or PGA; and copolymer PLA-PGA. An amount of the therapeutic agent such as the proteins
of the present invention, is released in stages such as an initial burst of protein non.
specifically associated with the exterior of the particles, a later stage by diffusion, and a final
stage by erosion can be controlled by polymer composition, molecular weight, size of the
microparticles, and physiological conditions such as pH, Protein stability during manufacture
25 of microsphsres is enhanced if the protein is in solid farm, such as a lycophized powdsr,
which is emulsified with solvent or by a frozen-atomizatian process or a sonication process,
and the suspension is then frozen in liquid nitrogen for further solvent extraction.
Microspheres can be produced from a supercritical fluid, e.g., supercritical carbon dioxide
(scCO2).
30 Biodegradable block polymers that are suitable foo drug delivery and methods of
synthesis are described by Kumar, N. et al., Adv. Drug Deliv. Rsv. 53 (2001): 23-44.
Copolymers can be random, alternating, or block (di or tri type) and can be linear, or star or
graft (comb-shaped) in configuration. A polymer can form a hydrogel, which is a three-
42

WO 2004/037195 PCT/US2003/033595
dimensional, hydrophilic polymeric network that holds a large amount of aqueous fluid. The
polymer used in a hydrogel is rendered insoluble by cross-linking or other chemical adducts.
Biodegradable implants can be prepared from materials such as at least one of the
materials selected from the group of: starch; vinylstarch; dipropyleneglycol diacrylate
5 (DPGDA); tripropyleneglycol diacrylate (TPGDA); pectin; cellulose acetate; cellulose
propionate; cellulose acetate butyrate; cellulose acetate propionate (CAP); hydroxypropyl
cellulose (HPC); hydroxypropyl cellulose/cellulose acetate propionate (HPC/CAP); methyl
methacrylate (MMA); butyl methacrylate (BMA); hydroxymethyl merthacrylate (HEMA);
ethyl hexyl acrylate (EHA); octadecyl methacrylate (ODMA); and ethyleneglycol
10 dimethacrylate (EGDMA). See Gil, M. et al., Boletim de Biotecnologia 2002, 72:13 -19.
In addition to polymers, naturally occurring and synthetic lipids can be used for
sustained release formulations, DepoFoam™ (Skye Pharma, London, England) forms a
multivesicular lipid-based particle (lipasome) for encapsulating therapeutic agents (see U.S.
Patent 5,993,850; and Ye, Q. et al., 2000 J. Controlled Rel. 64:155-166). The lipids are
15 amphipathic with a net negative charge, sterols, or Zwitterionic lipids, and methods for
making the liposomes are non-acidic. Methods for incorporating an active therapeutic agent
into the liposomes are also provided. The therapeutic agent can be one or more of a
gastrin/CCK receptor ligand, an EGF receptor ligand, and a FACGTNT.
Other lipids for liposomes are within the scope of the invention. A. plant polar lipid,
20 for example a ceramide such as a wheat ceramide, is useful for forming a gel with a protein
such as a prolamine, into which one or more therapeutic agents can be placed for transdermal
or transmucosal delivery. See U.S. Patent 6,410,048, issued June 25, 2002. Exemplary
prolamines include wheat gliadin, and corn zein. Other naturally occurting polymers used in
sustained release drug formulations and devices include collagen (EP-A-O 621 044), chitin
25 (U.S. patent 4,393,373), and chitosan, a deacylated form of chitin,
A permeation enhancer, for example a glycolipid, a non-esterified. fatty acid, an
aliphatic alcohol, a fatty acid ester of an aliphatic alcohol, a cyclohexanol, a fatty acid, ester
of glycerol, a glycol, or an aliphatic alcohol ether or a glycol, are typical permeation
enhancers, other components such as a stabilizer, a solubilizer, a surfactant and a plasticizer
30 can be present in a transdenmal device. See U.S. patent application 20020127254, published
September 12,2002.
Lipids and a variety of types of polymers are used to form "nanoparticles" for drug
delivery, reviewed by M. Kumar, 2Q02, J, Pharm. Pharmaceut Sci.234-258. Drug loadiag
43

WO 2004/037195 PCT/US2003/033595
into these particles is found to be greatest with most lipophilic therapeutic agents. Long
lasting release has been found with liposomes comprising polylactic acid, lecithin, and
phosphatidylcholine or cholesterol.
Local (targeted) sustained release is obtained by methods described herein, for
5 example, use of liposomes comprised of a material designed to target the RES, or comprised
of a different material to avoid cells of the RES. Additional targeting methods include use of
antibodies or soluble recombinant receptors in conjunction with the outer surface of
liposomes or microspheies. Further, sustained release formulations as described herein may
be further adjusted for use with any of the device, described herein, such as a pump, for local
10 delivery to a particular target organ.
The present invention in another embodiment provides sustained release
pharmaceutical compositions comprising a therapeutically effective amount of a combination
of a FACGINT or an EGF receptor ligand, and a gastrin/CCK receptor ligand. A
pharmaceutically acceptable earner or excipient can be added. Such a carrier includes but is
15 not limited to saline, buffered saline, dextrose, water, glycerol, ethaoi, and combinations
thereof. The formulation should suit the mode of administration.
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
20 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 then entirety herein.
25 EXAMPLES
Example 1. Treatment with a GLP-1 receptor ligand glucagons-like peptide 1 (GLP-11) and
a gastrin/CCK receptor ligand gastrin, ptevents disease progression in NOD mice with
recent-onset diabetes.
Mice of the non-obese diabetic (NOD) strain have a phenotype that shares many
30 features of disease pathogenesis with human type I diabetes. NOD mice typically axhibit
destructive autoimmune pancreatic insulitis and -cell destruction as eaily as four weeks of
age. Diabetes onset usually occurs at age 10-13 weeks in these mice, with typical blood
glucose levels observed to be between about 7mM to about 10 mM (compared to a range of
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WO 2004/037195 PCT/US2003/033595
about 3.0-6.6 mM normal mice), and a pancreatic insulin level that is lower by more than
about 95% than that in normal mice. As disease progresses, NOD mice exhibit increasingly
severe signs of chronic diabetes, with blood glucose levels reaching between about 25 to
about 30 mM, and pancreatic insulin level declining to become virtually non-existent. At that
5 severe stage of the disease, greater than about 99% of -cells have been destroyed.
In this example, the effect of treatment by a combination of GLP-1 and gastric was
examined in NOD mice with recent onset diabetes, to determine whether administration of
both GLP-I and gastrin would prevent severe hyperglycemia, ketoacidosis and death as well
as increase pancreatic insulin content in NOD mice with recent-onset diabetes. The I.N.T.TM
10 composition used was gastrin as synthetic human gastrin I having 17 amino acid residues
with a Leu residue at amino acid position 15, 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).
15 Non-obese diabetic (NOD) female mice, ages 12-14 weeks, were monitored for
development of onset of diabetes (fasting blood glucose > 8.0 to 15mmol), and within 48
hours after onset of symptoms, two groups of mice were each treated as follows: one group
was treated with vehicle only; and the other group was administered 100 g/kg/day of GLP-
1, each treatment administered via the intraperitoneal route twice daily.
20 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 hyperglytemia persisted after termination of therapeutic treatment At
25 14 days treatment was stopped, and at 18 days the mice were sampled to obtain FBG levels
(as shown in Table 2).
Table 2. Glucagon-like peptide - 1 (GLP-1) and gastrin combination therapy treats recent
onset diabetes in NOD mice
30
Group GLP-1 Gastrin Number FBG (mMV) at day:
0 7 14 18

45


GLP- 1 (100 g/kg/day) and gastrin (3 g/kg/day) were administered by i.p. injection to NOD
femate mice, twice a day, to each group as shown above. Diabetic mice, aged 11-14 weeks,
were used within two days of diabetes onset (generally considered to be an FBG of more than
10 63 mM).
The protocol includes sampling of these mice for data again at 6 weeks, and blood
collected for assay of FBG and plasma C-peptsde, and the mice are sacrificed for pancreatic
insulin determinations and scoring of islet inflammation (insulitis). From the outset of
15 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.
Results show that in animals of the vehicle-treated control group (group 1), fasting
blood glucose (FBG) values increased progressively during the time course of this mock
20 treatment, from 11 mM glucose at day 0 to 24 mM at day 18.
In contrast, in mice in treated with GLP-1 and gastrin, FBG values (7,9 mM glucose)
were significantly less compared to the vehicle-tieated mice (24.4 mM), reduced to a level, in
fact, that is less than one third of the level that developed in the vehicle trated mice; Table 2).
Most significant and surprisingly, the combination of GLP-1 and gastrin was more effective
25 in decreasing FBG levels than either gastrin alone or GLP-1 alone (having FBG levies of 19.0
mM and 15.8 mM, respectively). Only in mice treated with the combination was the FBG
reduced to a level that is in a normal range. The improved control of blood glucose levels in
mice treated with GLP-1 and gastrin may be expected to be associated with a significantly
increased content of insulin in the pancreas of these mice.
30 in summary, the results in this study show that treatment with a short course of low
doses of GLP-1 and gastrin treatment in mice with recent onset of diabetes prevented disease
progression, and reversed the disease condition to yield a blood glucose level of about
normal. Further, this significant decreased blood glucose level in mice treated with gastrin
and GLP-1 persisted for an additional time period after cessation of treatment. It is expected
35 that in these animals, data will show improved pancreatic insulin content, and that these
effects will be shown to be sustained for a prolonged period of time after termination of
therapy.
46

WO 2004/037195 PCT/US2003/033595
Further groups of NOD female mice were treated with a prolactin receptor ligand,
prolactin (PRL), and a gastrin/CCK receptor ligand, and with a growth hormone receptor
ligaod, growth hormone (GH), and a gastrin/CCK receptor ligand, gastrin. It is anticipated
that data, vail indicate that these treatment yield effects on FBG and other parameters that are
5 similar to data obtained herein with GLP-1.
Example 1. Comparison of compositions with and without Pegylation
In order to determine whether a sustained release formulation would provide greater
efficacy than an otherwise identical formulation that is formulated far bolus or non-sustained
10 release delivery, a study is performd comparing a protocol of treating NOD mice with an
I.N.T.™ composition either in a pegylated or non-pegylated formulation. Pegylation of at
least one component of the I.N.T.™ composition can prolong the amount of time the
therapeutic agent is retained ina na active form in the body.
The study shows that administration of sustained release formulation of at least one
15 agent of an I.N.T.™ composition is able to improve the diabetic conditions of the NOD mice,
as compared to an I.N.T.™ composition formulated for direct, i.e., non-sustained release
administration.
Example 3. Comparison of frequency of dosages composition administration
20 In order to determine whether a sustained release formulation would provide greater
efficacy than a non-sustained release direct formulation, an experiment is devised having
comparing a protocol of three-time a day administration to the once-a-day administration of
an I.N.T,™ composition to NOD mice.
The results show that prolongation of bioavailability of the I.N.T.™ composition,
25 comparable to that obtained with a sustained release formulation of an I.N.T composition, can
improve efficacy, i.e., can better remidiate symptome of the diabetic condition of the NOD
mice, as compared to a direct or non-sustained release formulation of an I.N.T.TM composition,
and can reduce the frequency of dosages required for such remediation of symptoms.
30
47

Claims:
1. A composition, comprising a gastrin/CGK receptor ligand and a factor for
camplementing gastrin for islet neogenesis therapy (a FACGINT), provided that the
FACGINT is not an EGF receptor ligand, and a pharmaceutically acceptable carrier.
2. A composition according to claim 1 in a dosage effective for inducing differentiatian
of an islet precursor cell into a mature insulin secreting cell.
3. A composition according to claim 1 or 2, wherein the FACGINT is at least one factor
selected from the group consisting of a Glucagon-likie peptide 1 receptor ligand; a
Glucagon-like peptide 2 receptor ligand; a gastric inhibitory polypeptide (GOP) receptor
ligand; a keratinocyte growth factor (KGF) receptor ligand; a dipeptidyl peptidase IV
inhibitor: a REG protein receptor ligand; a Growth. Hormocne receptor ligand; a Prolactin
(PRL) receptor ligand; an Insulin-like Growth Factor (IGF) receptor ligand; PTH-related
protein (PTIHrP) receptor ligand; hepatocyte growth fector (HGF) receptor ligand; a. bone
morphogenetic protein (BMP) receptor ligand, a transforming growth factor- (TGF-)
receptor ligand; a laminin receptor ligand; vasoactive intestinal peptide (VIP) receptor
ligand; a fihroblast growth factor (FGF) receptor ligand; a nerve growth facton (NGF)
receptor ligand; an islet neogenesis associated protein (INGAP) receptor ligand; an
Activin-A receptor ligand; a vascular endothelial growth factor (VEGF) receptor ligand;
an erythropoietin (EPO) receptor ligand; a pituitary adenylate cyclase activating
polypeptide (PACAP) receptor ligand; a granulocyte colony stimulating factor (G-CSF)
receptor ligand; a granulocyte-macrophage colony stimulating factor (GM-CSF); a
platelet-derived growth factor (PDGF) receptor ligand; and a Secretin receptor ligand.
4. A composition according to claim 1, 2 or 3 wherein the amount of the FACGINT in
the composition is substantially less than the minimum effective dose of the FACGINT
required to reduce blood glucose in a diabetic mammal in the absence of a gastrin/CCK
48

receptor liganda.t
5, A composition according to claim 1, 2, 3, or 4 for treating diabetes comprising a
Glucagon-like peptide-1 (GLP-1) receptor ligand and a gastrin/CCK receptor ligand.
6. A composition according to claim 5 wherein the GLP-1 receptor ligand is GLP-1
7. A composition according to claim 3 wherein the FACGINT is a Growth Hormone
receptor ligand.
8. A composition according to claim 7 wherein the Growth Hormone receptor ligand is
human growth hormone.
9. A. composition according to claim 3 wherein the FACGINT is prolactin (PL) receptor
ligaind.
10. A composition according to claim 9 wherein the PL receptor ligand is human PL.
11. A composition according to any one of claims 1 to 10 wherein the gastrin is gastrin I
having 17 amino acids with a Leu residue at amino acid position 15
12. A composition according to any one of claims 1 to 11 further comprising an agent for
immune suppression.
13. A composition according to any one of claims 1 to 12 for sustained release wherein
at least one of the gastrin receptor ligand or FACHINT is a sustained release formulation.
AMENDED PAGE
49

14. A kit for treating or preventing diabetes, the kit comprising a composition according
to any one of claims 1 to 13, a container, and instructions for use.
15. Use of a composition according to any one of claims 1 to 14 far the preparation of a
medicament for treating diabetes.
16. Use of a composition according to any one of claims 1 to 14 for the preparation of a
medicament for contacting ex vivo a plurality of cells to treat diabetes.
17. Use according to claim 16 wherein the cells are pancreatic ductal cells.
18. Use of a composition according to any one of claims 1 or 13 for the preparation of a
medicament for inducing pancreatic islet neogenesis in a mammal by increasing
proliferation of islet precursor cells in pancreatic tissue and thereby inducing pancreatic
islet neogenesis, or by increasing the number of pancreatic insulin secreting b-cells in the
mammal,
19. Use of a composition according to any one of claims 1 to 13 for the preparation of a
medicament for incereasing proliferation of pancreatic islet precursor cells in pancreatic
tissue of a mammal.
20. Use of a composition according to any one of claims 1 to 13 for the preparation of a
medicament for expanding and differentiating stem cells into insulin secreting cells in a
diabetic recipient of implanted cells
21. Use of a composition according to any owe of claims 1 to 13 for the preparation of a
medicament for reducing an amount of stem cells needed for transplantation to treat
AMENDED PAGE
50

human diabetes, wherein the amount of cells is reduced in comparison to an amount of
cells needed in the absence of administering the composition to an otherwise identical
recipient.
22. A method for producing a preparation of insulin secreting tells comprising treating
stem cells capable of expanding and differentiating into insulin secreting cells in vitro
with an effective amount of a composition according to any one of claims 1 to 13 to
provide a preparation of insulin secreting cells
51


Gomposinons and methods are provided for islet meogenesis therepy comprising a member of a group of factors
that cumplomont a gasirin/CCK receplot lighted, with formulation, devices and methods for sustaned release delivery and for local
delivery to larged organs.

Documents:

00910-kolnp-2005-abstract.pdf

00910-kolnp-2005-claims.pdf

00910-kolnp-2005-description complete.pdf

00910-kolnp-2005-form 1.pdf

00910-kolnp-2005-form 3.pdf

00910-kolnp-2005-form 5.pdf

00910-kolnp-2005-international publication.pdf

910-KOLNP-2005-CORRESPONDENCE 1.1.pdf

910-KOLNP-2005-CORRESPONDENCE.pdf

910-KOLNP-2005-FORM 13.pdf

910-KOLNP-2005-FORM 3.1.pdf


Patent Number 244597
Indian Patent Application Number 910/KOLNP/2005
PG Journal Number 51/2010
Publication Date 17-Dec-2010
Grant Date 13-Dec-2010
Date of Filing 17-May-2005
Name of Patentee WARATAH PHARMACEUTICALS, INC.
Applicant Address 415 YONGE STREET, SUITE 1103, TORONTO, ONTARIO M5B 2E7, CANADA.
Inventors:
# Inventor's Name Inventor's Address
1 BRAND STEPHEN J 161 BEDFORD ROAD, LINCOLN, MA 01773 UNITED STATES OF AMERICA.
2 PASTRAK ALEKSANDRA 120 GLENVALE BOULEVARD, TORONTO, ONTARIO M4G 2V9, CANADA.
3 HEW YIN 6 JOANNA CRESCENT, THORNHILL, ONTARIO L4J 5E7, CANADA.
4 CRUZ ANTONIO 89 DUNLOE ROAD, TORONTO, ONTARIO M5P 2T7, CANADA.
PCT International Classification Number A61K
PCT International Application Number PCT/US2003/033595
PCT International Filing date 2003-10-22
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/428,562 2002-11-22 U.S.A.
2 60/428,100 2002-11-21 U.S.A.
3 60/420,187 2002-10-22 U.S.A.
4 60/420,399 2002-10-22 U.S.A.