U.S. patent application number 10/029551 was filed with the patent office on 2002-06-27 for treatment for diabetes.
Invention is credited to Brand, Stephen J., Lane, Anne, Nardi, Ronald V., Parikh, Indu.
Application Number | 20020081285 10/029551 |
Document ID | / |
Family ID | 22909247 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020081285 |
Kind Code |
A1 |
Parikh, Indu ; et
al. |
June 27, 2002 |
Treatment for diabetes
Abstract
Methods and compositions for treating diabetes mellitus in a
patient in need thereof are provided. The methods include
administering to a patient a composition providing a gastrin/CCK
receptor ligand, e.g., a gastrin, and/or an epidermal growth factor
(EGF) receptor ligand, e.g., TGF-.alpha., in an amount sufficient
to effect differentiation of pancreatic islet precursor cells to
mature insulin-secreting cells. The composition can be administered
systemically or expressed in situ by cells transgenically
supplemented with one or both of a gastrin/CCK receptor ligand
gene, e.g., a preprogastrin peptide precursor gene and an EGF
receptor ligand gene, e.g., a TGF-.alpha. gene. The methods also
include transplanting into a patient cultured pancreatic islets in
which mature insulin-secreting beta cells are proliferated by
exposure to a gastrin/CCK receptor ligand and an EGF receptor
ligand.
Inventors: |
Parikh, Indu; (Chapel Hill,
NC) ; Lane, Anne; (Westmount, CA) ; Nardi,
Ronald V.; (Nahwah, NJ) ; Brand, Stephen J.;
(Lincoln, MA) |
Correspondence
Address: |
Rae-Venter Law Group, P.C.
P.O. Box 60039
Palo Alto
CA
94306
US
|
Family ID: |
22909247 |
Appl. No.: |
10/029551 |
Filed: |
December 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10029551 |
Dec 20, 2001 |
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09241100 |
Jan 29, 1999 |
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09241100 |
Jan 29, 1999 |
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09127028 |
Jul 30, 1998 |
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09127028 |
Jul 30, 1998 |
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07992255 |
Dec 14, 1992 |
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Current U.S.
Class: |
424/93.21 ;
514/12.3; 514/12.6; 514/5.9; 514/8.9; 514/9.6 |
Current CPC
Class: |
Y10S 514/866 20130101;
A61P 5/48 20180101; A61P 3/10 20180101; A61P 7/00 20180101; A61K
48/00 20130101; C07K 14/595 20130101; A61P 43/00 20180101; A61K
38/2207 20130101; C07K 14/495 20130101; A61K 38/2207 20130101; A61K
38/1808 20130101; A61K 38/2207 20130101; A61K 38/1841 20130101;
A61K 38/2207 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/93.21 ;
514/12 |
International
Class: |
A61K 048/00; A61K
038/17 |
Claims
What is claimed is:
1. A method for treating diabetes mellitus in an individual in need
thereof, said method comprising: administering to said individual a
composition providing at least one receptor ligand selected from
the group consisting of a gastrin/CCK receptor ligand and an EGF
receptor ligand in an amount sufficient to effect differentiation
of pancreatic islet precursor cells to mature insulin-secreting
cells.
2. The method according to claim 1, wherein said at least one
receptor ligand is an EGF receptor ligand selected from the group
consisting of EGF1-53, EGF1-48, or its EGF1-47 or EGF1-49
congener.
3. The method according to claim 2, wherein said EGF1-53, EGF1-48,
or its EGF1-47 or EGF1-49 congener is human EGF1-53, EGF1-48, or
its EGF1-47 or EGF1-49 or its congener.
4. A method for providing a patient with diabetes in need thereof
with a population of mature insulin-secreting beta cells, said
method comprising: transplanting into said patient cultured
pancreatic islets which have been provided with a sufficient amount
of at least one receptor ligand selected from the group consisting
of a gastrin/CCK receptor ligand and an epidermal growth factor
receptor ligand to induce proliferation of mature insulin-secreting
beta cells of said islets prior to said transplanting.
5. The method according to claim 4, wherein said diabetes is Type 2
diabetes.
6. The method according to claim 4, wherein said gastrin/CCK
receptor ligand is a gastrin.
7. The method according to claim 4, wherein said epidermal growth
receptor ligand is TGF-.alpha. or an EGF selected from the group
consisting of EGF1-53, EGF1-48, or its EGF1-47 or EGF1-49
congener.
8. A method for expanding a population of pancreatic beta cells,
said method comprising: providing said pancreatic beta cells with a
sufficient amount of a gastrin/CCK receptor ligand and an epidermal
growth factor receptor ligand to induce proliferation of said
pancreatic beta cells, whereby an expanded population of pancreatic
beta cells is obtained.
9. A composition comprising: pancreatic .beta. cells, wherein said
culture is obtained by providing pancreatic islets with a
sufficient amount of a gastrin receptor agonist and an epidermal
growth factor receptor agonist to induce proliferation of said
pancreatic .beta. cells.
10. A method for treating diabetes in an individual in need
thereof, said method comprising: administering to said individual a
composition comprising at least one receptor ligand selected from
the group consisting of a proteinaceous gastrin/CCK receptor ligand
and a proteinaceous EGF receptor ligand in an amount sufficient to
effect differentiation of pancreatic islet precursor cells to
mature insulin-secreting cells, wherein said composition is
administered systemically.
11. The method according to claim 10, wherein said proteinaceous
gastrin/CCK receptor ligand is a gastrin.
12 The method according to claim 10, wherein said proteinaceous EGF
receptor ligand is a TGF-.alpha..
13. The method according to claim 10, wherein said diabetes is type
2 diabetes.
14. A method for stimulating pancreatic islet cell neogenesis in an
individual in need thereof, said method comprising: administering
to said individual a composition comprising at least one receptor
ligand selected from the group consisting of a gastrin/CCK receptor
ligand and an EGF receptor ligand in an amount sufficient to effect
differentiation of pancreatic islet precursor cells to mature
insulin-secreting islet cells, wherein said composition is
administered systemically.
15. The method according to claim 14, wherein said individual.
16. The method according to claim 14, wherein both said gastrin/CCK
receptor ligand and said EGF receptor ligand are administered.
17. The method according to claim 16, wherein at least one of said
gastrin/CCK receptor ligand and said EGF receptor ligand is a
proteinaceous receptor ligand.
18. A method for treating diabetes mellitus in an individual in
need thereof which comprises administering to the individual a
composition providing a gastrin/CCK receptor ligand and an EGF
receptor ligand in an amount sufficient to effect differentiation
of pancreatic islet precursor cells to mature insulin-secreting
cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/127,028, filed Jul. 30, 1998, which is a continuation U.S. Ser.
No. of 07/992,255, filed Dec. 14, 1992, which issued Mar. 23, 1999,
as U.S. Pat. No. .sub.______, which disclosures are incorporated
herein by reference.
INTRODUCTION
[0002] 1. Field of Invention
[0003] This invention relates to a method for treating diabetes
mellitus in an individual in need thereof by administering to the
individual a composition comprising a gastrin/CCK receptor ligand
and/or an EGF receptor ligand which effectively promotes
differentiation of pancreatic islet precursor cells to mature
insulin-secreting cells. The method is exemplified by
administration of gastrin and transforming growth factor alpha
(TGF-.alpha.) either alone or in combination to normal
streptozotocin (STZ) induced diabetic and genetically predisposed
diabetic Zucker rats.
[0004] 2. Background
[0005] Diabetes is one of the most common endocrine diseases across
all age groups and populations. In addition to the clinical
morbidity and mortality, the economic cost of diabetes is huge,
exceeding $90 billion per year in the U.S. alone, and the
prevalence of diabetes is expected to increase more than two-fold
by the year 2010.
[0006] There are two major forms of diabetes mellitus:
insulin-dependent (Type 1) diabetes mellitus (IDDM) which accounts
for 5 to 10% of all cases, and non-insulin-dependent (Type 2)
diabetes mellitus NDDM) which comprises roughly 90% of cases. Type
2 diabetes is associated with increasing age however there is a
trend of increasing numbers of young people diagnosed with NIDDM,
so-called maturity onset diabetes of the young (MODY). In both Type
1 and Type 2 cases, there is a loss of insulin secretion, either
through destruction of the .beta.-cells in the pancreas or
defective secretion or production of insulin. In NIDDM, patients
typically begin therapy by following a regimen of an optimal diet,
weight reduction and exercise. Drug therapy is initiated when these
measures no longer provide adequate metabolic control. Initial drug
therapy includes sulfonylureas that stimulate .beta.-cell insulin
secretion, but also can include biguanides, .alpha.-glucosidase
inhibitors, thiazolidenediones and combination therapy. It is
noteworthy however that the progressive nature of the disease
mechanisms operating in type 2 diabetes are difficult to control.
Over 50% of all drug-treated diabetics demonstrate poor glycemic
control within six years, irrespective of the drug administered.
Insulin therapy is regarded by many as the last resort in the
treatment of Type 2 diabetes, and there is patient resistance to
the use of insulin.
[0007] Pancreatic islets develop from endodermal stem cells that
lie in the fetal ductular pancreatic endothelium, which also
contains pluripotent stem cells that develop into the exocrine
pancreas. Teitelman and Lee, Developmental Biology, 121:454-466
(1987); Pictet and Rutter, Development of the embryonic encocrine
pancreas, in Endocrinology, Handbook of Physiology, ed. R. O. Greep
and E. B. Astwood (1972), American Physiological Society:
Washington, D.C., p.25-66. Islet development proceeds through
discrete developmental stages during fetal gestation which are
punctuated by dramatic transitions. The initial period is a
protodifferentiated state which is characterized by the commitment
of the pluripotent stem cells to the islet cell lineage, as
manifested by the expression of insulin and glucagon by the
protodifferentiated cells. These protodifferentiated cells comprise
a population of committed islet precursor cells which express only
low levels of islet specific gene products and lack the
cytodifferentiation of mature islet cells. Pictet and Rutter,
supra. Around day 16 in mouse gestation, the protodifferentiated
pancreas begins a phase of rapid growth and differentiation
characterized by cytodifferentiation of islet cells and a several
hundred fold increase in islet specific gene expression.
Histologically, islet formation (neogenesis) becomes apparent as
proliferating islets bud from the pancreatic ducts
(nesidioblastosis). Just before birth the rate of islet growth
slows, and islet neogenesis and nesidioblastosis becomes much less
apparent. Concomitant with this, the islets attain a fully
differentiated state with maximal levels of insulin gene
expression. Therefore, similar to many organs, the completion of
cellular differentiation is associated with reduced regenerative
potential; the differentiated adult pancreas does not have either
the same regenerative potential or proliferative capacity as the
developing pancreas.
[0008] Since differentiation of protodifferentiated precursors
occurs during late fetal development of the pancreas, the factors
regulating islet differentiation are likely to be expressed in the
pancreas during this period. One of the genes expressed during
islet development encodes the gastrointestinal peptide, gastrin.
Although gastrin acts in the adult as a gastric hormone regulating
acid secretion, the major site of gastrin expression in the fetus
is the pancreatic islets. Brand and Fuller, J Biol Chem.,
263:5341-5347 (1988). Expression of gastrin in the pancreatic
islets is transient. It is confined to the period when
protodifferentiated islet precursors form differentiated islets.
Although the significance of pancreatic gastrin in islet
development is unknown, some clinical observations suggest a rule
for gastrin in this islet development as follows. For example,
hypergastrinemia caused by gastrin-expressing islet cell tumors and
atrophic gastritis is associated with nesidioblastosis similar to
that seen in differentiating fetal islets. Sacchi, et al., Virchows
Archiv B, 48:261-276 (1985); and Heitz et al., Diabetes, 26:632-642
(1977). Further, an abnormal persistence of pancreatic gastrin has
been documented in a case of infantile nesidioblastosis. Hollande,
et al., Gastroenterology, 71:251-262 (1976). However, in neither
observation was a causal relationship established between the
nesidioblastosis and gastrin stimulation.
[0009] It is therefore of interest to identify agents that
stimulate islet cell regeneration which could be of value in the
treatment of early IDDM and in the prevention of .beta.-cell
deficiency in NIDDM.
[0010] Citations of a reference herein shall not be construed as an
admission that such reference is prior art to the present
invention.
RELEVANT LITERATURE
[0011] Three growth factors are implicated in the development of
the fetal pancreas, gastrin, transforming growth factor .alpha.
(TGR-.alpha.) and epidermal growth factor (EGF) (Brand and Fuller,
J. Biol. Chem. 263:5341-5347). Transgenic mice over expressing
TGF-.alpha. or gastrin alone did not demonstrate active islet cell
growth, however mice expressing both transgenes displayed
significantly increased islet cell mass (Wang et al, (1993) J Clin
Invest 92:1349-1356). Bouwens and Pipeleers (1998) Diabetoligia
41:629-633 report that there is a high proportion of budding
.beta.-cells in the normal adult human pancreas and 15% of all
.beta.-cells were found as single units. Single .beta.-cell foci
are not commonly seen in adult (unstimulated) rat pancreas; Wang et
al ((1995) Diabetologia 38:1405-1411) report a frequency of
approximately 1% of total .beta.-cell number.
[0012] Insulin independence in a Type 1 diabetic patient after
encapsulated islet transplantation is described in Soon-Shiong et
al (1994) Lancet 343:950-51. Also see Sasaki et al (Jun. 15, 1998)
Transplantation 65(11):1510-1512; Zhou et al (May 1998) Am J
Physiol 274(5 Pt 1):C1356-1362; Soon-Shiong et al (June 1990)
Postgrad Med 87(8):133-134; Kendall et al (June 1996) Diabetes
Metab 22(3):157-163; Sandler et al (June 1997) Transplantation
63(12):1712-1718; Suzuki et al (January 1998 ) Cell Transplant
7(1):47-52; Soon-Shiong et al (June 1993) Proc Natl Acad Sci USA
90(12):5843-5847; Soon-Shiong et al (November 1992) Transplantation
54(5):769-774; Soon-Shiong et al (October 1992) ASAIO J
38(4):851-854; Benhamou et al (June 1998) Diabetes Metab
24(3):215-224; Christiansen et al (December 1994) J Clin Endocrinol
Metab 79(6):1561-1569; Fraga et al (April 1998) Transplantation
65(8):1060-1066; Korsgren et al (1993) Ups J Med Sci 98(1):39-52;
Newgard et al (July 1997) Diabetologiz 40 Suppl 2:S42-S47.
SUMMARY OF THE INVENTION
[0013] The invention provides methods for treating diabetes
mellitus in a patient in need thereof by administering a
composition providing a gastrin/CCK receptor ligand, an EGF
receptor ligand, or a combination of both in an amount sufficient
to effect differentiation of the patient's pancreatic islet
precursor cells to mature insulin-secreting cells. The composition
can be administered systemically or expressed in situ by host cells
containing a nucleic acid construct in an expression vector wherein
the nucleic acid construct comprises a coding sequence for a
gastrin CCK receptor ligand or a coding sequence for an EGF
receptor ligand, together with transcriptional and translational
regulatory regions functional in pancreatic islet precursor cells.
Also provided are methods and compositions for treating diabetes in
a patient in need thereof by implanting into a diabetic patient
pancreatic islet cells that have been exposed in culture to a
sufficient amount of a gastrin/CCK receptor ligand and an EGF
receptor ligand to increase the number of pancreatic beta cells in
the islets; optionally the population of pancreatic beta cells can
be grown in culture for a time sufficient to expand the population
of .beta.-cells prior to transplantation. The methods and
compositions find use in treating patients with diabetes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A is an image that shows numerous insulin staining
cells in the metaplastic ducts from the TGF-.alpha. transgenic
pancreas upon immunoperoxidase staining. FIG. 1B is an image that
shows that most ductular cells stained less intensely for insulin,
while occasional ductular cells did stain with the same intensity
of insulin staining as the adjacent islets.
[0015] FIG. 2A schematically shows the structure of the chimeric
insulin promoter-gastrin (INSGAS) transgene. FIG. 2B illustrates
that the radioimmunassay of pancreatic extracts from INSGAS
transgenic mice shows high levels of gastrin immunoreactivity that
exceed the gastrin content in the gastric antrum expressed from the
endogenous murine gene. The INSGAS transgenic mice had high
expression of gastrin in the postnatal pancreas.
[0016] FIG. 3A is an image of the pancreatic histology of an
INSGAS/TGF-.alpha. mouse used in the study reported by Example 3.
The INSGAS/TGF-.alpha. pancreas had some areas of increased
ductular complexes and slightly increased interstitial cellularity.
The field shown here had the most severely abnormal histology in
the five animals used. FIG. 3B is an image of the pancreatic
histology of a control mouse from Example 3. FIG. 3C is an image of
the pancreatic histology of a TGF-.alpha. mouse from Example 3.
This field of a TGF-.alpha. mouse pancreas from the study reported
in Example 3 was typical and showed the interstitial cellularity
and fibrosis combined with florid ductular metaplasia that has been
described by Jhappan, et al, supra.
[0017] FIG. 4A is a histogram graphically illustrating
point=counting morphometric data which confirmed that at 17 weeks
the pancreas of the INSGAS/TGF-.alpha. mice had lower duct mass
than the pancreas of the TGF-.alpha. mice based on the study
reported in Example 3. FIG. 4B is a histogram which graphically
illustrates point=counting morphometric data which show that
co-expression of gastrin and TGF-.alpha. in the INSGAS/TGF-.alpha.
pancreas significantly increased the islet mass compared to the
islet mass of the corresponding non-transgenic control mice.
Further, TGF-.alpha. expression alone does not increase islet mass.
These data are based on the studies illustrated in Example 3.
[0018] FIG. 5 shows the effects of TGF-.alpha. and gastrin on
glucose tolerance in streptozotocin induced diabetic Wistar rats
treated with PBS (solid black diamonds) or a combination of
TGF-.alpha. and gastrin i.p. daily for 10 days (solid purple
squares).
[0019] FIG. 6 shows the effect of TGF-.alpha. and gastrin treatment
on .beta.-cell neogenesis in three groups of treated Zucker rats
together with the corresponding PBS controls (n=6 per group) as
described in Example 7. The light blue bar represents lean
TFG+gastrin, the magenta bar represents ob TGF+gastrin, the yellow
bar represents the ob PBS control, the dark blue bar represents pre
TFG+gastrin and the purple bar represents the lean PBS control.
TGF-.alpha. and gastrin significantly increased the relative
proportion of single .beta.-cell foci in all the groups studied as
compared to PBS-treated control animals. Groups 4 and 5 are
significantly different (p<0.0015) as are Groups 1 and 2
(p<0.0041).
[0020] FIG. 7 shows the effect of TGF-.alpha. and gastrin treatment
on .beta.-cell neogenesis in lean and obese Zucker rats.
.beta.-cell neogenesis is quantified by differential counting of
total .beta.-cells and newly generated single .beta.-cell foci and
is expressed as a percentage of total .beta.-cells counted. The
percentage of single .beta.-cell foci in lean Zucker rats treated
with the growth factor combination was 10.5.+-.0.9 compared to
3.9.+-.1.1 (p=0.004) in the corresponding PBS control (FIGS. 7A and
7B). In the obese Zucker rats, the percent single .beta.-cell foci
in the pretreatment group was 8.7.+-.1.3 vs. 4.2.+-.1.1 (p=0.0015)
in the corresponding control group (FIGS. 7C and 7D). FIG. 7E is a
400.times.magnification of the ductal region of FIG. 7C (indicated
by an arrow) and provides clear evidence of the budding of
insulin-containing .beta.-cells from the ductal epithelial cells
characteristic of .beta.-cell neogenesis.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0021] The invention provides methods for treating diabetes
mellitus in a patient in need thereof by administering a
composition providing a gastrin/CCK receptor ligand such as
gastrin, an EGF receptor ligand, such as TGF-.alpha., or a
combination of both in an amount sufficient to effect
differentiation of pancreatic islet precursor cells to mature
insulin-secreting cells. When the composition is administered
systemically, generally it is provided by injection, preferably
intravenously, in a physiologically acceptable carrier. When the
composition is expressed in situ, pancreatic islet precursor cells
are transformed either in ex vivo or in vivo with one or more
nucleic acid expression constructs in an expression vector which
provides for expression of the desired receptor ligand(s) in the
pancreatic islet precursor cells. As an example, the expression
construct includes a coding sequence for a CCK receptor ligand,
such as preprogastrin peptide precursor coding sequence which,
following expression, is processed to gastrin or a coding sequence
for an EGF receptor ligand such as TGF-.alpha., together with
transcriptional and translational regulatory regions which provide
for expression in the pancreatic islet precursor cells. The
transcriptional regulatory region can be constitutive or induced,
for example by increasing intracellular glucose concentrations,
such as a transcriptional regulatory region from an insulin gene.
Transformation is carried out using any suitable expression vector,
for example, an adenoviral expression vector. When the
transformation is carried out ex vivo, the transformed cells are
implanted in the diabetic patient, for example using a kidney
capsule. Alternatively, pancreatic islet cells are treated ex vivo
with a sufficient amount of a gastrin/CCK receptor ligand and/or an
EGF receptor ligand to increase the number of pancreatic .beta.
cells in the islets prior to implantation into the diabetic
patient. As required, the population of pancreatic .beta. cells is
expanded in culture prior to implantation by contacting them with
the same receptor ligand(s).
[0022] The subject invention offers advantages over existing
treatment regimens for diabetic patients. By providing a means to
stimulate the adult pancreas to regenerate, not only is the need
for traditional drug therapy (Type 2) or insulin therapy (Type 1
and Type 2) reduced or even eliminated, but the maintenance of
normal blood glucose levels also may reduce some of the more
debilitating complications of diabetes. Diabetic complications
include those affecting the small blood vessels in the retina,
kidney, and nerves, (microvascular complications), and those
affecting the large blood vessels supplying the heart, brain, and
lower limbs (mascrovascular complications). Diabetic microvascular
complications are the leading cause of new blindness in people
20-74 years old, and account for 35% of all new cases of end-stage
renal disease. Over 60% of diabetics are affected by neuropathy.
Diabetes accounts for 50% of all non-traumatic amputations in the
USA, primarily as a result of diabetic macrovascular disease, and
diabetics have a death rate from coronary artery disease that is
2.5 times that of non-diabetics. Hyperglycemia is believed to
initiate and accelerate progression of diabetic microvascular
complications. Use of the various current treatment regimens cannot
adequately control hyperglycemia and therefore does not prevent or
decrease progression of diabetic complications.
[0023] As used herein, the term "gastrin/CCK receptor ligand"
encompasses compounds that stimulate the gastrin/CCK receptor.
Examples of such gastrin/CCK receptor ligands include various forms
of gastrin such as gastrin 34 (big gastrin), gastrin 17 (little
gastrin), 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 ligands that either alone or in
combination with EGF receptor ligands can induce differentiation of
cells in mature pancreas to form insulin-secreting islet cells.
Also contemplated are active analogs, fragments and other
modifications of the above. Such ligands also include compounds
that increase the secretion of endogenous gastrins,
cholecystokinins or similarly active peptides from sites of tissue
storage. Examples of these are omeprazole which inhibits gastric
acid secretion and soy bean trypsin inhibitor which increases CCK
stimulation.
[0024] As used herein, the term "EGF receptor ligand" encompasses
compounds that stimulate the EGF receptor such that when
gastrin/CCK receptors in the same or adjacent tissues or in the
same individual also are stimulated, neogenesis of
insulin-producing pancreatic islet cells is induced. Examples of
such EGF receptor ligands include EGF1-53, and fragments and active
analogs thereof, including EGF1-48, EGF1-52, EGF1-49. See, for
example, U.S. Pat. No. 5,434,135. Other examples include
TGF-.alpha. receptor ligands (1-50) and fragments and active
analogs thereof, including 1-48, 1-47 and other EGF receptor
ligands such as amphiregulin and pox virus growth factor as well as
other EGF receptor ligands that demonstrate the same synergistic
activity with gastrin/CCK receptor ligands. These include active
analogs, fragments and modifications of the above. For further
background, see Carpenter and Wahl, Chapter 4 in Peptide Growth
Factors (Eds. Sporn and Roberts), Springer Verlag, (1990).
[0025] A principal aspect of the invention is a method for treating
diabetes mellitus in an individual in need thereof by administering
to the individual a composition including a gastrin/CCK receptor
ligand and/or an EGF receptor ligand in an amount sufficient to
effect differentiation of pancreatic islet precursor cells to
mature insulin-secreting cells. The cells so differentiated are
residual latent islet precursor cells in the pancreatic duct. One
embodiment comprises administering, preferably systemically, a
differentiation regenerative amount of a gastrin/CCK receptor
ligand and an EGF receptor ligand, preferably TGF-.alpha., either
alone or in combination to the individual.
[0026] Another embodiment comprises providing a gastrin/CCK
receptor ligand and/or an EGF receptor ligand to pancreatic islet
precursor cells of explanted pancreatic tissue of a mammal and
reintroducing the pancreatic tissue so stimulated to the
mammal.
[0027] In another, the invention comprises providing a gastrin/CCK
receptor ligand and/or an EGF receptor ligand to pancreatic islet
precursor cells of explanted pancreatic tissue from a mammal to
expand the population of .beta. cells.
[0028] In another embodiment gastrin/CCK receptor ligand
stimulation is effected by expression of a chimeric insulin
promoter-gastrin fusion gene construct transgenically introduced
into such precursor cells. In another embodiment EGF receptor
ligand stimulation is effected by expression of an EGF receptor
ligand gene transgenically introduced into the mammal. The sequence
of the EGF gene is provided in U.S. Pat. No. 5,434,135.
[0029] In another embodiment stimulation by a gastrin/CCK receptor
ligand and an EGF receptor ligand is effected by coexpression of
(i) a preprogastrin peptide precursor gene and (ii) an EGF receptor
ligand gene that have been stably introduced into the mammal.
[0030] In another aspect the invention relates to a method for
effecting the differentiation of pancreatic islet precursor cells
of a mammal by stimulating such cells with a combination of a
gastrin/CCK receptor ligand and an EGF receptor ligand. In a
preferred embodiment of this aspect, gastrin stimulation is
effected by expression of a preprogastrin peptide precursor gene
stably introduced into the mammal. The expression is under the
control of the insulin promoter. EGF receptor ligand, e.g.,
TGR-.alpha., stimulation is effected by expression of an EGF
receptor ligand gene transgenically introduced into the mammal. In
furtherance of the above, stimulation by a gastrin and a
TGF-.alpha. is preferably effected by co-expression of (i) a
preprogastrin peptide precursor gene and (ii) an EGF receptor
ligand introduced into the mammal. Appropriate promoters capable of
directing transcription of the genes include both viral promoters
and cellular promoters. Viral promoters include the immediate early
cytomegalovirus (CMV) promoter (Boshart et al (1985) Cell
41:521-530), the SV40 promoter (Subramani et al (1981) Mol. Cell.
Biol. 1:854-864) and the major late promoter from Adenovirus 2
(Kaufman and Sharp (1982) Mol. Cell. Biol. 2:1304-13199).
Preferably, expression of one or both of the gastrin/CCK receptor
ligand gene and the EGF receptor ligand gene is under the control
of an insulin promoter.
[0031] Another aspect of the invention is a nucleic acid construct.
This construct includes a nucleic acid sequence coding for a
preprogastrin peptide precursor and an insulin transcriptional
regulatory sequence, which is 5' to and effective to support
transcription of a sequence encoding the preprogastrin peptide
precursor. Preferably, the insulin transcriptional regulatory
sequence includes at least an insulin promoter. In a preferred
embodiment the nucleic acid sequence coding for the preprogastrin
peptide precursor comprises a polynucleotide sequence containing
exons 2 and 3 of a human gastrin gene and optionally also including
introns 1 and 2.
[0032] Another embodiment of the invention is an expression
cassette comprising (i) a nucleic acid sequence coding for a
mammalian EGF receptor ligand, e.g., TGF-.alpha. and a
transcriptional regulatory sequence thereof; and (ii) a nucleic
acid sequence coding for the preprogastrin peptide precursor and a
transcriptional regulatory sequence thereof. Preferably, the
transcriptional regulatory sequence for the EGF receptor ligand is
a strong non-tissue specific promoter, such as a metallothionein
promoter. Preferably, the transcriptional regulatory sequence for
the preprogastrin peptide precursor is an insulin promoter. A
preferred form of this embodiment is one wherein the nucleic acid
sequence coding for the preprogastrin peptide precursor comprises a
polynucleotide sequence containing introns 1 and 2 and exons 2 and
3 of the human gastrin gene.
[0033] Another aspect of the invention relates to a vector
including the expression cassette comprising the preprogastrin
peptide precursor coding sequence. This vector can be a plasmid
such as pGem1 or can be a phage which has a transcriptional
regulatory sequence including an insulin promoter.
[0034] Another aspect of this invention relates to a composition of
vectors including (1) having the nucleic acid sequence coding for a
mammalian EGF receptor ligand, e.g., TGF-.alpha., under control of
a strong non-tissue specific promoter, e.g., a metallothionein
promoter; and a preprogastrin peptide precursor coding sequence
under control of an insulin promoter. Each vector can be a plasmid,
such as plasmid pGem1 or a phage in this aspect. Alternatively, the
expression cassette or vector also can be inserted into a viral
vector with the appropriate tissue trophism. Examples of viral
vectors include adenovirus, Herpes simplex virus, adeno-associated
virus, retrovirus, lentivirus, and the like. See Blomer et al
(1996) Human Molecular Genetics 5 Spec. No:1397-404; and Robbins et
al (1998) Trends in Biotechnology 16:35-40. Adenovirus-mediated
gene therapy has been used successfully to transiently correct the
chloride transport defect in nasal epithelia of patients with
cystic fibrosis. See Zabner et a. (1993) Cell 75:207-216.
[0035] Another aspect of the invention is a non-human mammal or
mammalian tissue, including cells, thereof capable of expressing a
stably integrated gene which encodes preprogastrin. Another
embodiment of this aspect is a non-human mammal capable of
coexpressing (i) a preprogastrin peptide precursor gene; and/or
(ii) an EGF receptor ligand, e.g., a TGR-.alpha. gene that has been
stably integrated into the non-human mammal, mammalian tissue or
cells. The mammalian tissue or cells can be human tissue or
cells.
[0036] Therapeutic Administration and Compositions
[0037] Modes of administration include but are not limited to
transdermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, and oral routes. The compounds can 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 and intestinal mucosa, etc.) and
can be administered together with other biologically active agents.
Administration is preferably systemic.
[0038] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a therapeutic, and a pharmaceutically
acceptable carrier or excipient. Such a carrier includes but is not
limited to saline, buffered saline, dextrose, water, glycerol,
ethanol, and combinations thereof. The formulation should suit the
mode of administration. Pharmaceutically acceptable carriers and
formulations for use in the present invention are found in
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Philadelphia, Pa., 17.sup.th ed. (1985), which is incorporated
herein by reference. For a brief review of methods for drug
delivery, see Langer (1990) Science 249:1527-1533, which is
incorporated herein by reference.
[0039] In preparing pharmaceutical compositions of the present
invention, it may be desirable to modify the compositions of the
present invention to alter their pharmacokinetics and
biodistribution. For a general discussion of pharmacokinetics, see
Remingtons's Pharmaceutical Sciences, supra, Chapters 37-39. A
number of methods for altering pharmacokinetics and biodistribution
are known to one of ordinary skill in the art (See, e.g., Langer,
supra). Examples of such methods include protection of the agents
in vesicles composed of substances such as proteins, lipids (for
example, liposomes), carbohydrates, or synthetic polymers. For
example, the agents of the present invention can be incorporated
into liposomes in order to enhance their pharmacokinetics and
biodistribution characteristics. A variety of methods are available
for preparing liposomes, as described in, e.g., Szoka et al (1980)
Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871,
4,501,728 and 4,837,028, all of which are incorporated herein by
reference. Various other delivery systems are known and can be used
to administer a therapeutic of the invention, e.g., microparticles,
microcapsules and the like.
[0040] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, or powder. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulations can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc.
[0041] In a preferred embodiment, the composition is formulated in
accordance with routine procedures such as a pharmaceutical
composition adapted for intravenous administration to human beings.
Typically, compositions for intravenous administration are
solutions in sterile isotonic aqueous buffer. Where necessary, the
composition also can include a solubilizing agent and a local
anesthetic to ameliorate any pain at the site of the injection.
Generally, the ingredients are supplied 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 indicating the quality 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 or saline. Where the composition is
administered by injection, an ampoule of sterile water for
injection or saline can be provided so that the ingredients may be
mixed prior to administration.
[0042] The therapeutics of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium and other divalent cations,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0043] The amount of the therapeutic of the invention which is
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques. The precise dose to be
employed in the formulation also will 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. However, suitable dosage ranges for
intravenous administration are generally about 20-500 micrograms of
active compound per kilogram body weight. Suitable dosage ranges
for intranasal administration are generally about 0.01 pg/kg body
weight to 1 mg/kg body weight. Effective dosages can be
extrapolated from dose-response curves derived from in vitro or
animal model test systems. Suppositories generally contain active
ingredient in the range of 0.5% to 10% weight; oral formulations
preferably contain 10% to 95% active ingredient.
[0044] In the gene therapy methods of the invention, transfection
in vivo is obtained by introducing a therapeutic transcription or
expression vector into the mammalian host, either as naked DNA,
complexed to lipid carriers, particularly cationic lipid carriers,
or inserted into a viral vector, for example a recombinant
adenovirus. The introduction into the mammalian host can be by any
of several routes, including intravenous or intraperitoneal
injection, intratracheally, intrathecally, parenterally,
intraarticularly, intranasally, intramuscularly, topically,
transdermally, application to any mucous membrane surface, corneal
installation, etc. Of particular interest is the introduction of
the therapeutic expression vector into a circulating bodily fluid
or into a body orifice or cavity. Thus, intravenous administration
and intrathecal administration are of particular interest since the
vector may be widely disseminated following such routes of
administration, and aerosol administration finds use with
introduction into a body orifice or cavity. Particular cells and
tissues can be targeted, depending upon the route of administration
and the site of administration. For example, a tissue which is
closest to the site of injection in the direction of blood flow can
be transfected in the absence of any specific targeting. If lipid
carriers are used, they can be modified to direct the complexes to
particular types of cells using site-directing molecules. Thus,
antibodies or ligands for particular receptors or other cell
surface proteins may be employed, with a target cell associated
with a particular surface protein.
[0045] Any physiologically acceptable medium may be employed for
administering the DNA, recombinant viral vectors or lipid carriers,
such as deionized water, saline, phosphate-buffered saline, 5%
dextrose in water, and the like as described above for the
pharmaceutical composition, depending upon the route of
administration. Other components can be included in the formulation
such as buffers, stabilizers, biocides, etc. These components have
found extensive exemplification in the literature and need not be
described in particular here. Any diluent or components of diluents
that would cause aggregation of the complexes should be avoided,
including high salt, chelating agents, and the like.
[0046] The amount of therapeutic vector used will be an amount
sufficient to provide for a therapeutic level of expression in a
target tissue. A therapeutic level of expression is a sufficient
amount of expression to decrease blood glucose towards normal
levels. In addition, the dose of the nucleic acid vector used must
be sufficient to produce a desired level of transgene expression in
the affected tissues in vivo. Other DNA sequences, such as
adenovirus VA genes can be included in the administration medium
and be co-transfected with the gene of interest. The presence of
genes coding for the adenovirus VA gene product may significantly
enhance the translation of mRNA transcribed from the expression
cassette if this is desired.
[0047] A number of factors can affect the amount of expression in
transfected tissue and thus can be used to modify the level of
expression to fit a particular purpose. Where a high level of
expression is desired, all factors can be optimized, where less
expression is desired, one or more parameters can be altered so
that the desired level of expression is attained. For example, if
high expression would exceed the therapeutic window, then less than
optimum conditions can be used.
[0048] The level and tissues of expression of the recombinant gene
may be determined at the mRNA level as described above, and/or at
the level of polypeptide or protein. Gene product may be
quantitated by measuring its biological activity in tissues. For
example, protein activity can be measured by immunoassay as
described above, by biological assay such as blood glucose, or by
identifying the gene product in transfected cells by immunostaining
techniques such as probing with an antibody which specifically
recognizes the gene product or a reporter gene product present in
the expression cassette.
[0049] Typically, the therapeutic cassette is not integrated into
the patient's genome. If necessary, the treatment can be repeated
on an ad hoc basis depending upon the results achieved. If the
treatment is repeated, the patient can be monitored to ensure that
there is no adverse immune or other response to the treatment.
[0050] The invention also provides for methods for expanding a
population of pancreatic .beta.-cells in vitro. Upon isolation of
the pancreas from a suitable donor, cells are isolated and grown in
vitro. The cells which are employed are obtained from tissue
samples from mammalian donors including human cadavers, porcine
fetuses or another suitable source of pancreatic cells. If human
cells are used, when possible the cells are major
histocompatability matched with the recipient. Purification of the
cells can be accomplished by gradient separation after enzymatic
(e.g., collagenase) digestion of the isolated pancreas. The
purified cells are grown in media containing sufficient nutrients
to allow for survival of the cells as well as a sufficient amount
of a .beta.-cell proliferation inducing composition containing a
gastrin/CCK receptor ligand and EGF receptor ligand, to allow for
formation of insulin secreting pancreatic .beta. cells. According
to the invention, following stimulation the insulin secreting
pancreatic .beta. cells can be directly expanded in culture prior
to being transplanted into a patient in need thereof, or can be
transplanted directly following treatment with .beta.-cell
proliferation inducing composition.
[0051] Methods of transplantation include transplanting insulin
secreting pancreatic .beta.-cells obtained into a patient in need
thereof in combination with immunosuppressive agents, such as
cyclosporin. The insulin producing cells also can be encapsulated
in a semi-permeable membrane prior to transplantation. Such
membranes permit insulin secretion from the encapsulated cells
while protecting the cells from immune attack. The number of cells
to be transplanted is estimated to be between 10,000 and 20,000
insulin producing .beta. cells per kg of the patient. Repeated
transplants may be required as necessary to maintain an effective
therapeutic number of insulin secreting cells. The transplant
recipient can also, according to the invention, be provided with a
sufficient amount of a gastrin/CCK receptor ligand and an EGF
receptor ligand to induce proliferation of the transplanted insulin
secreting .beta. cells.
[0052] The effect of treatment of diabetes can be evaluated as
follows. Both the biological efficacy of the treatment modality as
well as the clinical efficacy are evaluated, if possible. For
example, disease manifests itself by increased blood sugar, the
biological efficacy of the treatment therefore can be evaluated,
for example, by observation of return of the evaluated blood
glucose towards normal. The clinical efficacy, i.e. whether
treatment of the underlying effect is effective in changing the
course of disease, can be more difficult to measure. While the
evaluation of the biological efficacy goes a long way as a
surrogate endpoint for the clinical efficacy, it is not definitive.
Thus, measuring a clinical endpoint which can give an indication of
.beta.-cell regeneration after, for example, a six-month period of
time, can give an indication of the clinical efficacy of the
treatment regimen.
[0053] The subject compositions can be provided as kits for use in
one or more procedures. Kits for genetic therapy usually will
include the therapeutic DNA construct either as naked DNA with or
without mitochondrial targeting sequence peptides, as a recombinant
viral vector or complexed to lipid carriers. Additionally, lipid
carriers can be provided in separate containers for complexing with
the provided DNA. The kits include a composition comprising an
effective agent either as concentrates (including lyophilized
compositions), which can be diluted further prior to use or they
can be provided at the concentration of use, where the vials may
include one or more dosages. Conveniently, in the kits single
dosages can be provided in sterile vials so that the physician can
employ the vials directly, where the vials will have the desired
amount and concentration of agents. When the vials contain the
formulation for direct use, usually there will be no need for other
reagents for use with the method. Associated with such kits can be
a notice in the form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals or biological
products, which notice reflects approval by the agency of
manufacture, use or sale for human administration.
[0054] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Materials and Methods
[0055] The following materials and methods were used in the studies
reported by the working examples set forth below except as
otherwise noted.
[0056] Animals. Mice, FVB and CD strain, were obtained from Taconic
Farms, Inc., Germantown, N.Y. The TGF-.alpha. transgenic line MT-42
used, which expresses high levels of TGF-.alpha. from a
metallothionein promoter, is described in Jhappan et al, Cell,
61:1137-1146 (1990). Normal Wistar and Zucker rats were allowed
normal chow ad libidum with free access to water and were
acclimatized for one week prior to initiation of each study.
Freshly prepared streptozotocin at a dose of 80 mg/kg body weight
was administered by I.V. five to seven days after induction of
diabetes, the rats were randomly allocated into groups for
subsequent treatment. Hormones, TGF-.alpha. and rat gastrin were
reconstituted in sterile normal saline containing 0.1% BSA.
According to the predetermined treatment schedule for different
studies, each animal received a single, daily i.p. injection of
either TGF-.alpha. or gastrin alone (4.0 .mu.g/kg body weight) or
as a 1:1 (w/w) combination (total 8.0 .mu.g/kg) or PBS for a period
of 10 days.
[0057] INSGAS Transgene Construct. A Pvull-Rsa1 fragment
encompassing nucleotides -370 to +38 of the rat insulin I gene
(Cordell, B. G. et al, Cell, 18:533-543 (1979)) was ligated into
pGem1 (Promega Corp., Madison, Wis.). A 4.4 kb BamHl-EcoRl fragment
containing 1.5 kb introns 1 and 2 and exons 2 and 3 of the human
gastrin gene which encodes the preprogastrin peptide precursor was
isolated and subcloned downstream of the rat insulin I fragment in
pGem1 (Promega). The fragment is described in Wiborg, O., Proc.,
Natl. Acad. Sci. USA, 81:1067-1069 (1984) and Ito, R., et al Proc.
Natl. Acada. Sci. (USA), 81:4662-4666 (1984). The insulin
promoter-preprogastrin INSGAS transgene construct was excised as a
4.8 kb Xbal-EcoR1 fragment.
[0058] Generation and Characterization of Transgenic Mice. The
fragment, made as described above was prepared for microinjection
as follows. It was isolated by agarose gel electrophoresis,
purified by C.sub.sC1 gradient purification, and dialyzed
extensively against injection buffer (5 mM NaCl; 01. MM EDTA; 5 mM
Tris-HCl pH 7.4). Fertilized oocytes from FVB inbred mice (Taconic
Farms, Inc., supra) at the single-cell stage were microinjected
using standard techniques. See Hogan, B., et al, Manipulating the
mouse embryo: A laboratory manual, Cold Spring Harbor, N.Y. (1986).
Surviving embryos were then implanted into the oviducts of CD1
(Charles River Laboratories, Inc., Wilmington, Mass.) foster
mothers according to procedures in Hogan et at. Transgenic founder
mice were identified by DNA blot techniques using DNA isolated from
individual mouse tails, and a human gastrin exon 2 probe labelled
with 32 dCTP by random priming. F1 mice and their siblings were
similarly identified.
[0059] Homozygous MT-42 mice containing the MT-TGF-.alpha.
transgene derived from a CD-1 mouse strain (Jhappan, supra) were
crossed with heterozygotic INSGAS mice. After weaning, the
offspring were placed on acidified 50 mM ZnCl.sub.2 as previously
described in order to induce the metallothionein promoter (Jhappan,
supra).
[0060] Northern Blot Hybridization Assay. For Northern analysis,
total RNA was extracted from tissues by the method of Cathala et
al, DNA 2:329-335 (1983). Samples of 20 .mu.g of total RNA were
resolved on a 1% agarose denaturing gel and transferred to
nitrocellulose. RNA blots were hybridized with .sup.32P labelled
TGF-.alpha. riboprobes or exon 2 of human gastrin that did not
cross-hybridize with endogenous mouse gastrin mRNA.
[0061] Peptide radioimmunassay of Gastrin. Tissues were extracted
and assayed for gastrin immunoreactivity by radioimmunoassay as
described previously using antibody 2604 which is specific for
biologically active C terminally amidated gastrin in a gastrin
radioimmunoassay as described in Rehfeld, J. F., Scand. J. Clin.
Lab. Invest. 30:361-368 (1972). Tyrosine monoiodinated human
gastrin 17 tracer was used in all assays and synthetic human
gastrin 17 was used as a standard.
[0062] Peptide Radioimmunoassay of TGF-.alpha.: Tissues were frozen
in liquid nitrogen, ground to a powder with mortar and pestle, and
subjected to acid-ethanol extraction as described in Todaro, G. J.
et al, Proc. Natl. Acad. Sci. (USA), 77:5258-5262 (1980). Extracts
were reconstituted with water, and protein concentrations
determined with a Coomassie blue dye binding assay (Bio-Rad
Laboratories, Hercules, Calif.). Aliquots from the pancreata were
tested in duplicate in a TGF-.alpha. radioimmunoassay, which
measured competition with .sup.125I TGF-.alpha. for binding to a
solid-phase rabbit antibody raised against the C-terminus of rat
TGF-.alpha. (kit from BioTope, Seattle, Wash.).
[0063] Blood Glucose. Blood glucose was determined either after
overnight fasting or after IPGTT by glucose oxidase method.
[0064] Tissue Insulin Analysis. At the end of each study, the
animals were sacrificed and pancreas removed. Small biopsies were
taken from separate representative sites throughout the pancreas
and immediately snap-frozen in liquid nitrogen for
immunohistochemistry, protein, and insulin determinations.
Snap-frozen pancreatic samples (n=5) were rapidly thawed, disrupted
ultrasonically in deionized water and aliquots taken for protein
determination and the homogenate subjected to acid/ethanol
extraction prior to insulin determination by RIA.
[0065] Histological Analysis. The pancreata were removed, weighed,
similarly oriented in cassettes, fixed in Bouin's solution and
embedded in paraffin by conventional procedures.
[0066] Tissue Preparation and Immunohistochemistry. Freshly excised
pancreata were dissected, cleared of fat and lymph nodes, fixed in
Bouin's fixative, and then embedded in paraffin for sectioning.
Routine sections were stained with hematoxylin and eosin according
to standard methods. Pancreatic tissue from adult 17 week old
MT-TGF-.alpha. (MT-42) transgenic mice were immunostained for
insulin to examine the effect of TGF-.alpha. over-expression on
islet development. Insulin positive cells in TGF-.alpha.-induced
metaplastic ductules were identified using immunoperoxidase
staining guinea pig anti-human insulin sera (Linco, Eureka, Mo.); a
pre-immune guinea pig serum was used as a control.
Immunohistochemistry was performed on 5.mu. paraffin sections by
the peroxidase/antiperoxidase method of Sternberger using a
monoclonal rabbit antigastrin antibody. See, Sternberger, L. A.,
Immunocytochemistry, 2.sup.nd Ed. (1979) N.Y.: Wiley. 104-170.
[0067] Point-Counting Morphometrics. The relative volume of islets,
ducts, or interstitial cells was quantitated using the
point-counting method described in Weibel, E. R., Lab Investig,
12:131-155 (1963). At a magnification of 400.times., starting at a
random point at one corner of the section, every other field was
scored using a 25 point ocular grid. An unbiased but systematic
selection of fields was accomplished using the markings of the
stage micrometer. Intercepts over blood vessels, fat, ducts, lymph
nodes, or interlobular space were subtracted to give the total
pancreatic area. A minimum of 5000 points in 108 fields
(systematically chosen using the stage micrometer) were counted in
each block, with the relative islet volume being the number of
intercepts over islet tissue divided by the number over pancreatic
tissue. The absolute islet mass or islets was calculated as the
relative islet volume times pancreatic weight. See, Lee, H. C., et
al, Endocrinology, 124:1571-1575 (1989).
[0068] Statistical Analysis. Differences between means were
compared for significant differences using the Student's t test for
unpaired data.
Example 1
Assay For Insulin Production in TGF Transgenic Pancreas
[0069] Immunoperoxidase staining showed numerous insulin staining
cells in the metaplastic ducts from the TGF-.alpha. transgenic
pancreas (FIG. 1A), whereas insulin staining cells were virtually
absent from the non-transgenic ducts (less than 6.1%). When at
least 600 ductular cells/animal were scored at a final
magnification of 400.times., insulin positive cells were seen at a
frequency of 6.0+/-0.9% (n=5) in the metaplastic ductules of
TGF-.alpha. transgenic mice. Occasional ductular cells stained with
the same intensity of insulin staining as the adjacent islets, but
most had less intense staining (FIG. 1B). The low level of insulin
staining of the ductular cells resembles that of
protodifferentiated cells reported in the ducts of the developing
pancreas. Pictet, R. and W. J. Rutter, Development of the embryonic
endocrine pancreas, in Endocrinology, Handbook of Physiology, ed.
R. O. Greep and E. B. Astwood (1972) American Physiological
Society: Washington, D.C. 25-66; and Alpert, S. et al Cell,
53:295-308 (1988).
[0070] However, despite the increased number of insulin positive
cells in the metaplastic ducts, the islet mass of the TGF-.alpha.
transgenic mice was not increased. The islet mass as quantitated by
point counting morphometrics was 2.14 mg+/-0.84 (mean +/-se, n=5)
in the TGF-.alpha. transgenic pancreas compared to 1.93 mg+/-0.46
(n=6) non transgenic litter mates.
[0071] Thus, TGF-.alpha. over-expression alone did not effect
transition of these protodifferentiated duct cells into fully
differentiated islets. This implies that islet differentiation
requires other factors absent from the adult pancreas of
TGF-.alpha. transgenic mice. Since differentiation of
protodifferentiated islet precursors occurs during late fetal
development, factors regulating this transition would likely be
expressed in islets during this period. Among the factors expressed
in the developing islets are the gastrointestinal peptides, the
gastrins.
Example 2
Pancreatic Gastrin Expression from the INSGAS Transgene
[0072] To examine the possible role of gastrin in regulating islet
differentiation, transgenic mice were created that express a
chimeric insulin promoter-gastrin (INSGAS) transgene in which the
insulin promoter directs pancreas specific expression of the
gastrin transgene (FIG. 2A). Unlike the gastrin gene, insulin gene
expression is not switched off after birth. Thus, the INSGAS
transgene results in a persistence of gastrin expression in the
adult pancreas.
[0073] The INSGAS transgene comprised 370 bp of 5' flanking DNA and
the first non-coding exon of the rat insulin I gene. Cordell, B.,
et al, Cell 18:533-543 (1979). It was ligated to a BamH1-EcoR1
fragment containing 1.5 kb intron 1 and exons 2 and 3 of the human
gastrin gene which encodes the preprogastin peptide precursor.
Wiborg, O., et al, Proc. Natl. Acad. Sci. USA, 81:1067-1069 (1984);
and Ito et al Proc. Natl. Acad. Sci. USA, 81:4662-4666 (1984). A
4.8 kb INSGAS fragment was isolated and microinjected into inbred
FVB, one cell mouse embryos. Hogan, B. et al, Manipulating the
mouse embroy: laboratory manual, (1986) N.Y.:Cold Spring
Harbor.
[0074] Gastrin immunoreactivity in pancreatic and stomach extracts
from transgenic and non-transgenic mice was assayed by
radioimmunoassay using antisera 2604 (Rehfeld, J., et al, Scand J
Clin. Lab. Invest., 30:361-368 (1972)) specific for the bioactive
amidated C-terminus of gastrin.
[0075] Beta cell specific gastrin expression from the INSGAS
transgene was observed based on immunostaining of pancreatic
tissues with a gastrin monoclonal antibody.
[0076] Northern blots of RNA isolated from different tissues of 8
week old INSGAS transgenic mice were hybridized with a human
gastrin exon 2 probe. High levels of gastrin transgene mRNA were
seen in the pancreas but not in any other tissues. This probe is
specific for the human gastrin gene; no hybridization is seen in
antral RNA of INSGAS and non-transgenic FVB mice express high
levels of murine gastrin mRNA. Radioimmunoassay of pancreatic
extracts from INSGAS transgenic mice showed high levels of gastrin
immunoreactivity that exceed the gastrin content in the gastric
atrium expressed from the endogenous murine gene (FIG. 2B). No
gastrin immunoreactivity was detected in pancreatic extracts of
non-transgenic control mice (FIG. 2B). The gastrin radioimmunoassay
is specific for carboxy amidated precursors, indicating that the
gastrin peptide precursor is efficiently processed
post-translationally to the bioactive peptide. Immunohistochemistry
with a gastrin monoclonal antibody shows pancreatic beta islet cell
specific expression of gastrin.
[0077] Although the INSGAS transgenic mice had high expression of
gastrin in the postnatal pancreas (FIG. 2B), the INSGAS transgenic
mice had pancreatic histology identical to controls. Islet mass as
quantitated by point-counting morphometrics (Weibel, E. R., Lab
Investig. 12:131-155 (1963)) was identical in 5-6 week old INSGAS
mice (1.78+/-0.21 mg, n=11) and age matched non-transgenic controls
(1.74+/-0.18 mg, n=11). Thus, sustained expression of gastrin in
the postnatal pancreas alone does not stimulate islet cell
growth.
Example 3
Histological Examination of TGF-.alpha. and TGF-.alpha./INSGAS
Pancreas
[0078] Stimulation of islet growth by gastrin may require
stimulation by other growth factors to create a responsive
population of cells. Therefore, effects of gastrin stimulation were
studied in TGF-.alpha. transgenic mice which have metaplastic ducts
that contain insulin expressing cells resembling
protodifferentiated islet-precursors. To assess the interaction
between gastrin and TGF-.alpha., three groups of mice were bred
with equivalent FVB/CD1 strain genetic backgrounds: non-transgenic
control, TGF-.alpha. single transgenic and INSGAS/TGF-.alpha.
double transgenics. All three groups of mice were placed on 50 mM
ZnCl.sub.2 at 3 weeks of age. At 17 weeks of age, the animals were
sacrificed and the pancreas removed for histological evaluation.
The pancreas from TGF-.alpha. and INSGAS/TGF-.alpha. mice had
similar gross morphological appearances: resilient, firm and
compact in contrast to the soft diffuse control pancreas.
TGF-.alpha. expression was equivalent in TGF-.alpha. and
INSGAS/TGF-.alpha. groups when measured by Northern blot analysis
(data not shown) and by radioimmunoassay. The pancreatic
TGF-.alpha. immunoreactive peptide levels were 12.2+/-1 and
18.9+/-8 ng/mg protein (Mean+/-SD) in the TGF-.alpha. and
INSGAS/TGF-.alpha. mice, respectively.
[0079] Light micrographs of hematoxylin stained paraffin sections
of pancreas from the three groups of mice studied (A:
INSGAS/TGF-.alpha.; B: FVB/CD1 controls; and C: TGF-.alpha.) were
made. The INSGAS/TGF-.alpha. pancreas had some areas of increased
ductular complexes and slightly increased interstitial cellularity;
the field shown (FIG. 3A) had the most severely abnormal morphology
seen in the five animals; most of the pancreas was
indistinguishable from controls (FIG. 3B). In contrast, the field
of TGF-.alpha. pancreas (FIG. 3C) was typical and showed the
interstitial cellularity and fibrosis combined with florid ductular
metaplasia described by Jhappan et al, supra.
[0080] Pancreatic gastrin synergistically interacts with
TGF-.alpha. to increase islet mass and inhibit the ductular
metaplasia induced by TGF-.alpha. over-expression. Mating the
homozygous MT-TGF-.alpha. (MT-42) mice (TGF-.alpha.) with
heterozygotic INSGAS mice gave offspring that were either
heterozygotic TGF-.alpha. single transgenic or double transgenic
containing both INSGAS and TGF-.alpha. transgenes
(INSGAS/TGF-.alpha.). Since INSGAS were FVB strain and TGF-.alpha.
were CD1 strain, TGF-.alpha. homozygotes and CD1 controls (CON)
were both mated with FVB to produce FVB/CD1 strain background for
all three groups of mice. Mice were treated with 50 mM ZnCl.sub.2
from 3 weeks until sacrifice at age 17 weeks. The pancreas was
removed, weighed, similarly oriented in cassettes, fixed in Bouin's
solution and embedded in paraffin. One random section from each
animal was used to quantitate the relative volumes of ductules and
islets by point-counting morphometrics (Weibel, E. R., Lab
Investig., 12:131-155 (1963)). At least 2000 points over tissue
were counted as intercepts of a 50 point grid at 170.times.
magnification; the entire section was covered without overlap. The
mass of ductules or islets was calculated by multiplying the
relative volume and the animal's pancreatic weight. To normalize
different mean body weights, the mass was expressed as .mu.g/g body
weight. Results are mean and standard errors for 5-6 animals in
each group as determined by Student's t test (p<0.05).
[0081] Expression of gastrin from the INSGAS transgene reduced the
ductular metaplasia caused by TGF-.alpha. over-expression. At 17
weeks, the pancreatic histology of the INSGAS/TGF-.alpha. mice
(FIG. 3A) resembled that of the control pancreas (FIG. 3B) more
than that of the TGF-.alpha. mice (FIG. 3C).
[0082] This was confirmed by quantitating pancreatic ductular mass
in the TGF-.alpha. and INSGAS/TGF-.alpha. transgenic mice and the
FVB/CD1 controls by point-counting morphometrics (FIG. 4A).
Co-expression of gastrin and TGF-.alpha. in the INSGAS/TGF-.alpha.
pancreas also significantly increased the islet mass compared to
controls (FIG. 4B), whereas islet mass was not increased by
expression of the TGF-.alpha. or gastrin transgenes alone. The
blood glucose concentration was not significantly different among
the three groups of mice.
Example 4
Effects of TGF-.alpha. and Gastrin on Pancreatic Insulin Content in
Normal Rats
[0083] This experiment was designed to study the effects on
pancreatic insulin content in non-diabetic animals treated with
TGF-.alpha., a gastrin, or a combination of TGF-.alpha. and a
gastrin as compared to control animals (untreated). Groups (n=5) of
normal Wistar rats were assigned to one of the following four
treatment groups.
[0084] Group I: TGF-.alpha.: recombinant Human TGF-.alpha. was
reconstituted in sterile saline containing 0.1% BSA and was
administered i.p. at a dose of 0.8 .mu.g/day for 10 days.
[0085] Group II: Gastrin: synthetic Rat Gastrin I was dissolved in
very dilute ammonium hydroxide and reconstituted in sterile saline
containing 0.1% BSA. It was administered i.p. at a dose of 0.8
.mu.g/day for 10 days.
[0086] Group III: TGF-.alpha.+Gastrin: a combination of the above
preparations was administered i.p. at the dose levels given above
for 10 days.
[0087] Group IV: Control animals received an i.p. injection of
vehicle alone for 10 days.
[0088] At the end of the study period (10 days), all animals were
sacrificed and samples of pancreas taken as follows: five biopsy
specimens (1-2 mg) of pancreatic tissue were taken from separate
representative sites in each rat pancreas and immediately snap
frozen in liquid nitrogen for analysis of insulin content. For
analysis of pancreatic insulin content, the snap frozen pancreatic
samples were rapidly thawed, disrupted ultrasonically in distilled
water and aliquots taken for protein determination and acid/ethanol
extraction prior to insulin radioimmunoassay (Green et al, (1983)
Diabetes 32:685-690). Pancreatic insulin content values were
corrected according to protein content and finally expressed as
.mu.g insulin/mg pancreatic protein. All values calculated as mean
+/-SEM and statistical significance evaluated using Student's
2-sample t-test.
1TABLE 1 Treatment of Normal Rats with TGF-a and Gastrin Pancreatic
Insulin Content Treatment (.mu.g insulin/mg protein) Control 20.6
+/- 6.0 TGF-.alpha. 30.4 +/- 7.4* Gastrin 51.4 +/- 14.0**
TGF-.alpha. + Gastrin 60.6 +/- 8.7*** *TGF-.alpha. vs. control, p =
0.34; **gastrin vs. control, p = 0.11; ***combination of
TGF-.alpha. and gastrin, p = 0.007.
[0089] As shown in Table 1, above, pancreatic insulin content was
significantly increased (p=0.007) in the TGF-.alpha.+gastrin
treated animals as compared to control animals; there was an
approximately three-fold increase in pancreatic insulin content as
compared to control animals. These data support the hypothesis that
the combination of TFG-.alpha. and gastrin does produce an increase
in the functional islet .beta.-cell volume. This increase reflects
an overall condition of .beta.-cell hyperplasia (increase in
number) rather than .beta.-cell hypertrophy (increase in size of
individual .beta.-cells).
Example 5
Effect of Combination of TGF-.alpha. and Gastrin on Pancreatic
Insulin Content in Diabetic Animals
[0090] The second experiment was designed to determine whether the
combination of TGF-.alpha. and gastrin could increase pancreatic
insulin content in diabetic animals (streptozotocin (STZ) treated)
to levels comparable to those in normal (non-STZ treated)
animals.
[0091] Normal Wistar rats received a single iv injection of STZ at
a dose of 80 mg/Kg body weight. This dose of STZ was intended to
ensure that the study animals were rendered diabetic but that they
retained a functioning but reduced .beta.-cell mass. The STZ was
dissolved immediately before administration in ice-cold 10 mM
citric acid buffer. The animals were monitored daily; persistent
diabetes was indicated by glycosuria and confirmed by non-fasting
blood glucose determinations. One week after induction of diabetes,
rats were randomly allocated into two groups (n=6) as follows.
[0092] Group I: TGF-.alpha.+Gastrin: STZ diabetic rats were treated
with a single i.p. injection of a combination of recombinant human
TGF-.alpha. and synthetic rat Gastrin 1; both preparations were
administered at a dose of 0.8 .mu.g/day for 10 days.
[0093] Group II: Control: STZ diabetic rats received an i.p.
injection of vehicle alone for 10 days.
[0094] At the end of the study period, all animals were sacrificed
and samples of pancreas taken and analyzed as described in Example
4 and the results are given in Table 2.
2TABLE 2 Treatment of Streptozotocin Rats with TGF-.alpha. and
Gastrin Pancreatic Insulin Content Treatment (.mu.g Insulin/mg
protein) Control (STZ alone) 6.06 +/- 2.1 STZ plus TGF-.alpha. +
Gastrin 26.7 +/- 8.9
[0095] The induction of diabetes by STZ was successful and produced
a moderate but sustained degree of hyperglycemia. Total
insulinopaenia was not sought so as to ensure that the study
animals retained a functioning, but reduced .beta.-cell mass.
[0096] As shown in Table 2, above, the pancreatic insulin content
of the control streptozotocin treated animals was less than one
third that of normal rats (20.6.+-.6.0 mg insulin/mg protein, see
Table 1 above) as a result of destruction of .beta.-cells by the
STZ. In STZ animals treated with a combination of TGF-.alpha. and
gastrin, the pancreatic insulin content was more than four-fold
that of the animals which received STZ alone, and statistically the
same as that of normal rats.
[0097] Diabetes mellitus is a disease in which the underlying
physiological defect is a deficiency of .beta.-cells as a result
either of destruction of the .beta.-cells due to auto-immune
processes or of exhaustion of the potential for the .beta.-cells to
divide due to chronic stimulation from high circulating levels of
glucose. The latter eventually leads to a situation when the
process of .beta.-cell renewal and/or replacement is compromised to
the extent that there is an overall loss of .beta.-cells and a
concomitant decrease in the insulin content of the pancreas. The
above results demonstrate that a combination of TGF-.alpha. and
gastrin can be used to treat diabetes by stimulating the production
of mature .beta.-cells to restore the insulin content of the
pancreas to non-diabetic levels.
Example 6
Effects of TGF-.alpha. and Gastrin on IPGTT in STZ-Induced Diabetic
Animals
[0098] Two groups (average body weight 103 g) of STZ induced
diabetic Wistar rats (n=6/group) were treated for 10 days with a
daily i.p. injection of either a combination of TGF-.alpha. and
gastrin or PBS. Fasting blood glucose was determined for all rats
on days 0, 6, and 10. In order to establish that this insulin was
secreted and fictional, IPGTT tests were performed. At day 10,
intraperitoneal glucose tolerance tests (IPGTT) were performed
following an overnight fast. Blood samples were obtained from the
tail vein, before and 30, 60 and 120 minutes after administration
of an i.p. glucose injection at a dose of 2 g/kg body weight. Blood
glucose determinations were performed as above. The blood glucose
levels were similar in both study groups at time 0 but the
TFG.alpha. and gastrin treated rats demonstrated a 50% reduction in
blood glucose values (see FIG. 5), as compared to control rats at
30, 60, and 120 min. following the i.p. glucose load.
Example 7
Effects of TGF-.alpha. and Gastrin on Body Weight Gain and Insulin
Content in Diabetes Prone Animals
[0099] Zucker rats were obtained at 30 days of age approximately
10-15 days prior to development of obesity. Besides the diabetes
prone Zucker rats (genotype fa/fa, autosomal recessive mutation for
obesity and diabetes), lean non-diabetic littermates (genotype +/+)
also were included in the study as described below. The rats were
monitored daily for development of obesity and diabetes by
determining body weight and blood glucose. The onset of diabetes in
Zucker rats usually started between days 45-50 and was confirmed by
a significant increase in blood glucose levels, as compared to the
levels in age-matched lean controls.
[0100] The study included 5 groups of 5 rats each as described in
Table 3. Groups 1 and 2 (lean, non-diabetic) were treated with a
TGF-.alpha. and gastrin combination or PBS respectively from day 0
to day 10. Groups 3, 4 and 5 included obese, early diabetic Zucker
rats, genotype fa/fa. Group 3 received a combination pretreatment
for 15 days (day--15 to day 0) prior to onset of diabetes and
continuing post onset of diabetes for 10 additional days (day 0 to
day 10). Group 4 was treated with a combination of TGF-.alpha. and
gastrin for 10 days after onset of diabetes and Group 5 was treated
with PBS over the same time period. At the end of the study, the
rats were sacrificed and the pancreas removed. Small biopsies were
taken from separate representative sites for protein and insulin
determinations as described above.
[0101] The body weight gain in obese diabetic Zucker rats with
pretreatment, treatment only or with saline (groups 3, 4, and 5 in
Table 3) did not show any significant differences among the groups.
It is interesting to note that even prolonged treatment (25 days,
group 3) with TGF-.alpha.+gastrin was without effect on normal
weight gain. Within error limits body weight gain was identical in
all the groups.
[0102] The effect of TGF-.alpha.+gastrin treatment on fasting blood
glucose in the obese Zucker rats was compared to the corresponding
PBS controls. Fasting blood glucose was first significantly
increased by day 15 (4.0.+-.0.6 vs. 5.0.+-.0.2) and this time point
was chosen as the starting time for the 10-day treatment period
with TGF-.alpha.+gastrin or with PBS control. Fasting blood glucose
levels were not significantly altered by the TGF-.alpha.+gastrin
treatment or by PBS. Fasting blood glucose values were lower in
lean, as compared to obese animals whether or not they were treated
with the growth factors or with PBS.
3TABLE 3 Pretreatment .+-. Body Wt Group Geotype Condition
Treatment (days) PBS Control Gain (% .+-. SE) 1. +/+ lean,
non-diabetic None Yes 117 .+-. 2.1 2. +/+ lean, non-diabetic 0 + 10
No 119 .+-. 1.9 3. fa/fa obese, early diabetic -15 + 10 No 202 .+-.
15 4. fa/fa obese, early diabetic 0 + 10 No 119 + 1.0 5. fa/fa
obese, early diabetic None Yes 129 + 1.3
[0103] The results of treatment with TGF-.alpha. and gastrin in the
Zucker rat model of Type 2 diabetes showed no significant
differences in blood glucose levels between the treatment and
control groups, probably reflecting the transient hypoglycemic
effect following a prolonged period (18 hrs) of fasting. The
immunohistochemical studies revealed significant increases in the
number of single foci of insulin containing cells in the
TGF-.alpha. and gastrin treated animals, as compared to control
animals. These findings demonstrated an increase in single
.beta.-cells in adult rat pancreas following treatment with
TGF-.alpha. and gastrin. Interestingly, such single .beta.-cell
foci are not commonly seen in adult (unstimulated) rat pancreas.
These findings support a therapeutic role for TGF-.alpha. and
gastrin in Type 1 and Type 2 diabetes since treatment is targeted
at both .beta.-cell neogenesis and replication.
[0104] The present invention is based in part on studies which
demonstrated numerous insulin staining cells in the
TGF-.alpha.-induced metaplastic ductules. The low level of exocrine
and endocrine gene expression in the metaplastic ductal cells
resembled that of protodifferentiated ductal cells seen in the
early stage of fetal pancreatic development. Formation of islets
(neogenesis) results from proliferation and differentiation of
these protodifferentiated insulin expressing cells. Histologically
this is manifested as islets appearing to bud from the pancreatic
ducts (nesidioblastosis). In the MT-42 TGF-.alpha. transgenic mice,
ductular metaplasia was not seen in the immediate post-natal
period, but only at 4 weeks of age. This indicates that TGF-.alpha.
over-expression induced insulin expression in duct epithelia rather
than prolonging the persistence of islet precursors found in fetal
pancreatic ducts. Although the metaplastic ductules contained
numerous insulin positive cells, the islet mass of the TGF-.alpha.
transgenic mice was not increased over controls. The studies
reported above demonstrate that complete islet cell neogenesis is
reactivated in vivo in mammals in the ductular epithelium of the
adult pancreas by stimulation with a gastrin/CCK receptor ligand,
such as gastrin, and/or an EGF receptor ligand, such as
TGF-.alpha.. Studies are reported on the transgenic over-expression
of TGF-.alpha. and gastrin in the pancreas which elucidate the role
of pancreatic gastrin expression in islet development and indicate
that TGF-.alpha. and gastrin each play a role in regulating islet
development. Thus, regenerative differentiation of residual
pluripotent pancreatic ductal cells into mature insulin-secreting
cells is a viable method for the treatment of diabetes mellitus, by
therapeutic administration of this combination of factors or
compositions which provide for their in situ expression within the
pancreas.
[0105] The present invention is not limited by the specific
embodiments described herein. Modifications that become apparent
from the foregoing description and accompanying figures fall within
the scope of the claims.
[0106] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entirety.
* * * * *