Note: Descriptions are shown in the official language in which they were submitted.
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Title: Compositions and Methods Comprising Gastrin Compounds
FIELD OF THE INVENTION
The invention relates generally to compositions and methods comprising a
gastrin compound, and
uses thereof.
BACKGROUND OF THE INVENTION
Gastrin and other growth factors have been implicated in the development of
fetal pancreas ( Brand
and Fuller, J. Biol. Chem. 263:5341-5347). Gastrin is transiently expressed in
the fetus in the pancreatic
islets and its expression is confined to the period when protodifferentiated
islet precursors form differentiated
islets. While the significance of pancreatic gastrin expression in islets is
unknown, elevated pancreatic
gastrin has been observed with nesidioblastosis. In particular, an abnormal
persistence of pancreatic gastrin
has been documented in a case of infantile nesidioblastosis (Hollande et al,
Gastroenterology, 71:251-262,
1976) and hypergastrinemia caused by gastrin-expressing islet cell tumors.
Atrophic gastritis has also been
associated with nesidioblastosis similar to that seen in differentiating fetal
islets (Sacchi et al, Virchows
Archiv B, 48:261-276, 1985). However, in neither observation was a casual
relationship established between
the nesidiobastosis and gastrin stimulation.
The citation of any reference herein is not an admission that such reference
is available as prior art
to the instant invention.
SUMMARY OF THE INVENTION
The invention provides a composition, in particular a pharmaceutical
composition, comprising one
or more gastrin compound that provides beneficial effects in the treatment of
diabetes and its complications.
In an aspect the invention provides a pharmaceutical composition, comprising
one or more gastrin
compound that provides beneficial effects, in particular sustained beneficial
effects, following treatment. The
beneficial effects provided by a composition of the invention can include
increased absorption, distribution,
metabolism and/or elimination of a gastrin compound. A composition can have
increased bioavailability
(absorbed more rapidly and to a higher degree) or provide enhanced therapeutic
effects, in particular
sustained beneficial effects.
The invention also provides a pharmaceutical composition intended for
administration to a patient
to provide beneficial effects, in particular sustained beneficial effects,
comprising a gastrin compound,
optionally together with pharmaceutically acceptable carriers, excipients, or
vehicles.
The invention also provides a pharmaceutical composition for the treatment of
a disease or
condition comprising a therapeutically effective amount of a gastrin compound
to provide a sustained
beneficial effect in a pharmaceutically acceptable carrier, excipient, or
vehicle.
In an embodiment, a pharmaceutical composition comprising a gastrin compound
is provided which
has been adapted for administration to a subject to provide sustained
beneficial effects to treat a condition or
disease. In a preferred embodiment, the composition is in a form such that
administration to a subject results
in blood glucose levels that are about normal that persist in the subject for
a sustained period of time after
cessation of treatment.
In another embodiment, the invention relates to a liquid drug formulation
comprising a gastrin
compound or pharmaceutically acceptable salts thereof, and to lyophilized drug
formulations that can be
reconstituted to provide suspensions that are stable and suitable for
parenteral administration.
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In a particular embodiment, the invention relates to an aqueous composition
comprising a gastrin
compound adapted to provide sustained beneftcial effects. The invention also
provides a drug comprising an
aqueous formulation of a gastrin compound that provides sustained beneficial
effects, or pharmaceutically
acceptable salts thereof with at least one solubilizer.
The present invention is directed to compositions comprising a gastrin
compound that provides
beneficial effects, in particular sustained beneficial effects, in the
treatment of a condition or disease in
particular, diabetes.
In another aspect, the invention features a composition comprising a gastrin
compound in a dosage
effective for inducing proliferation of islet precursor cells into an
increased amount of mature insulin
secreting cells, in particular for a sustained period following administration
of the gastrin compound.
Proliferation of islet precursor cells may be induced ex vivo or ira vivo. The
composition can be 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, excipeint, or
vehicle.
The invention additionally provides a method of preparing a stable
pharmaceutical composition
comprising one or more gastrin compound adapted to provide beneficial effects,
preferably sustained
beneficial effects, following treatment. A method can comprise mixing one or
more gastrin compound, and a
pharmaceutically acceptable carrier, excipient, or vehicle, in particular, a
pharmaceutically acceptable
carrier, excipient, or vehicle effective to physically stabilize the gastrin
compound(s). After compositions
have been prepared, they can be placed in an appropriate container and
labelled for treatment of an indicated
condition. For administration of a composition of the invention, such
labelling would include amount,
frequency, and method of administration.
The invention also contemplates the use of a composition comprising at least
one gastrin compound
for the preparation of medicaments for preventing and/or treating conditions
and/or diseases. The invention
additionally provides uses of a pharmaceutical composition of the invention in
the preparation of
2,5 medicaments for the prevention and/or treatment of conditions and/or
diseases. The medicaments provide
beneficial effects, preferably sustained beneficial effects following
treatment.
The invention provides a method for treating and/or preventing a condition
and/or disease in a
subject comprising administering to the subject a therapeutically effective
amount of one or more gastrin
compound to provide beneficial effects. In an aspect the invention provides a
treatment which results in
sustained beneficial effects following treatment.
The invention has particular applications in preventing and/or treating
diabetes. Thus, the invention
relates to a method of treatment comprising administering a therapeutically
effective amount of one or more
gastrin compound which upon administration to a subject with symptoms of
diabetes produces beneficial
effects, preferably sustained beneficial effects. In an embodiment, sustained
beneficial effects are evidenced
by one or more of the following: (a) an increase in C-peptide production, (b)
an increase in pancreatic insulin
production, and/or (c) about normal blood glucose levels.
In an embodiment, the invention provides a method for preventing and/or
treating Type I or Type II
diabetes comprising administering a therapeutically effective amount of a
composition of the invention.
In a further embodiment, the invention provides a method for amelioriating
progression of a
condition and/or disease or obtaining a less severe stage of a condition
and/or disease in a person suffering
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from Type I or Type II diabetes comprising administering a therapeutically
effective amount of a
composition of the invention. .
The invention relates to a method of delaying the progression of impaired
glucose tolerance or non-
insulin requiring Type II diabetes to insulin requiring Type II diabetes
comprising administering a
therapeutically effective amount of a composition of the invention.
The invention also relates to a method of increasing the insulin synthesis
capability of a subject
comprising administering a therapeutically effective amount of a composition
of the invention.
In embodiments of methods of the invention the subject is not treated with
insulin.
The invention provides a kit comprising one or more gastrin compound or a
pharmaceutical
composition of the invention. In an aspect, the invention provides a kit for
preventing and/or treating
diabetes, containing a composition comprising one or more gastrin compound, a
container, and instructions
for use. The composition of the kit can further comprise a pharmaceutically
acceptable carrier, excipient, or
vehicle.
These and other aspects, features, and advantages of the present invention
should be apparent to
those skilled in the art from the following drawing and detailed description.
DESCRIPTION OF THE DRAWINGS
The invention will be better understood with reference to the drawings in
which:
Figure 1 is a bar graph showing the results of treating NOD mice with recent-
onset diabetes with
either E1 (1 wg/Kg/day LP. for 14 days) or Gl (3 ~g/Kg/day LP. for 14 days).
Fasting blood glucose levels
(mM) at day 0 and day 35 after diabetes onset (FBG > 6.6 mM) are shown.
Figure 2 is a bar graph showing the results of treating NOD mice with recent-
onset diabetes with
either E1 or Gl as described for Figure 1. Pancreatic insulin content (fig
/pancreas) is shown for vehicle
alone at onset of diabetes and at day 35. Treatment stopped after day 14.
Figure 3 is a line graph showing the results of monitoring NOD mice with
recent-onset diabetes for
eight weeks, including an initial 18 day period of treatment with gastrin.
Fasting glucose levels (mM) at
week 0 and each week thereafter are shown.
Figure 4 is a graph showing treatment with Gl decreases fasting blood glucose
levels in
chronically diabetic insulin-dependent NOD mice and prevents death 14 days
after cessation of insulin
therapy.
DETAILED DESCRIPTION OF EMBODIMENTS
Glossary
Numerical ranges recited herein by endpoints include all numbers and fractions
subsumed within
that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is
also to be understood that all numbers
and fractions thereof are presumed to be modified by the term "about." The
term "about" means plus or
minus 0.1 to 50%, 5-50%, or 10-40%, preferably 10-20%, more preferably 10% or
15%, of the number to
which reference is being made. Further, it is to be understood that "a," "an,"
and "the" include plural
referents unless the content clearly dictates otherwise. Thus, for example,
reference to a composition
containing "a compound" includes a mixture of two or more compounds.
Compounds described herein can contain one or more asymmetric centers and may
give rise to
enantiomers, diasteriomers, and other stereoisomeric forms which may be
defined in terms of absolute
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stereochemistry as (R)- or (S)-. Therefore, the invention includes all such
possible diasteriomers and
enantiomers as well as their racemic and optically pure forms. Optically
active (R)- and (S)-isomers may be
prepared using chiral synthons or chiral reagents, or resolved using
conventional techniques. When the
compounds described herein contain centers of geometric asymmetry, and unless
specified otherwise, it is
intended that the compounds include both E and A geometric isomers. All
tautomeric forms are intended to
be included within the scope of the invention.
The terms "subject", "individual", "recipient" or "patient" refer to an animal
including a warm-
blooded animal such as a mammal, which is afflicted with or suspected of
having or being pre-disposed to a
disease or a condition described herein. Mammal includes without limitation
any members of the Mammalia.
In general, the terms refer to a human. The terms also include domestic
animals bred for food or as pets,
including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo
animals, goats, apes (e.g. gorilla or
chimpanzee), and rodents such as rats and mice. The methods herein for use on
subjectslindividuals/patients
contemplate prophylactic as well as curative use. Typical subjects for
treatment include persons susceptible
to, suffering from or that have suffered a condition or disease described
herein. In embodiments of the
invention a subject is diabetic.
The term "pharmaceutically acceptable carrier, excipient, or vehicle" refers
to a medium which does
not interfere with the effectiveness or activity of an active ingredient and
which is not toxic to the hosts to
which it is administered. A carrier, excipient, or vehicle includes diluents,
binders, adhesives, lubricants,
disintegrates, bulking agents, wetting or emulsifying agents, pH buffering
agents, and miscellaneous
materials such as absorbants that may be needed in order to prepare a
particular composition. Examples of
carriers etc include but are not limited to saline, buffered saline, dextrose,
water, glycerol, ethanol, and
combinations thereof. The use of such media and agents for an active substance
is well known in the art.
"Pharmaceutically acceptable salt(s)," includes salts of acidic or basic
groups which may be present
in the compounds suitable for use in the present invention. Examples of
pharmaceutically acceptable salts
include sodium, calcium, ammonium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamine, 2
ethylamino, ethanol, histidine, procai'ine, and potassium salts of carboxylic
acid groups and hydrochloride
salts of amino groups. Other pharmaceutically acceptable salts of amino groups
are hydrobromide, sulfate,
hydrogen sulfate, phosphate, acetate, oxalic, hydrogen phosphate, dihydrogen
phosphate, acetate, succinate,
citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-
toluenesulfonate (tosylate) salts.
The term "preventing and/or treating" refers to the administration to a
subject of a composition of
the invention either before or after onset of a condition or disease. A
treatment may be either performed in an
acute or chronic way.
A "beneficial effect" refers to an effect of a gastrin compound or composition
thereof including
favorable pharmacological and/or therapeutic effects, and improved
pharmacokinetic properties and
biological activity. In embodiments of the invention, beneficial effects
include but are not limited to the
following: reduced or absent islet inflammation, decreased or prevention of
disease progression, increased
survival, or treatment or reversal of a disease or condition.
In an embodiment, the beneficial effects can be evidenced in diabetes by one
or more of the
following: (a) a reduction in fasting blood glucose levels, in particular when
blood glucose levels are greater
than 7-10 mM; (b) reduction in glycosylated haemoglobin; (c) increase in serum
insulin concentration; (d) an
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increase in pancreatic insulin production or content; and/or (e) prevention of
disease progression. In a
particular embodiment, the beneficial effects comprise (a), (b) and (c), or
(a), (c), and (d).
In a preferred embodiment, the beneficial effect is a "sustained beneficial
effect" where the
beneficial effect is sustained for a prolonged period of time after
termination of treatment. A beneficial effect
may be sustained for a prolonged period of at least about 2 to 4 weeks, 2 to 5
weeks, 3 to 5 weeks, 2 to 6
weeks, 2 to 8 weeks, 2 to 10 weeks, 2 to 12 weeks, 2 to 14 weeks, 2 to 16
weeks, 2 to 20 weeks, 2 to 24
weeks, 2 weeks to 12 months, or 2 weeks to 18 months following treatment. The
period of time a beneficial
effect is sustained may correlate with the duration and timing of the
treatment. A subject may be treated
continuously for about or at least about 2 to 4 weeks, 2 to 6 weeks, 2 to 8
weeks, 2 to 10 weeks, 2 to 12
weeks, 2 to 14 weeks, 2 to 16 weeks, 2 weeks to 6 months, 2 weeks to 12
months, 2 weeks to 18 months,
periodically or continuously. A sustained beneficial effect may manifest as
one or more of increased C-
peptide production, increased pancreatic insulin production or concentration,
and about normal or low blood
glucose levels for a prolonged period following treatment.
The beneficial effect may be a statistically significant effect in terms of
statistical analysis of an
effect of a gastrin compound versus the effects without the compound.
"Statistically significant" or
"significantly different" effects or levels may represent levels that are
higher or lower than a standard. In
embodiments of the invention, the difference may be 1.5, 2, 3, 4, 5, or 6
times higher or lower compared
with the effect obtained without a gastrin compound.
"Therapeutically effective amount" relates to the amount or dose of an active
compound (e.g.
gastrin compound) or composition of the invention that will lead to one or
more desired beneficial effects, in
particular, one or more sustained beneficial effects. A therapeutically
effective amount of a substance can
vary according to factors such as the disease state, age, sex, and weight of
the individual, and the ability of
the substance to elicit a desired response in the individual. Dosage regima
may be adjusted to provide the
optimum therapeutic response (e.g. sustained beneficial effects). For example,
several divided doses may be
administered daily or the dose may be proportionally reduced as indicated by
the exigencies of the
therapeutic situation.
A "native-sequence polypeptide" or " a native polypeptide" comprises a
polypeptide having the
same amino acid sequence of a polypeptide derived from nature. Such native-
sequence polypeptides can be
isolated from nature or can be produced by recombinant or synthetic means. The
term specifically
encompasses naturally occurring truncated or secreted forms of a polypeptide,
polypeptide variants including
naturally occurring variant forms (e.g. alternatively spliced forms or splice
variants), and naturally occurring
allelic variants.
The term "polypeptide variant" means a polypeptide having at least about 70-
80%, preferably at
least about 85%, more preferably at least about 90%, most preferably at least
about 95% amino acid
sequence identity with a native-sequence polypeptide, in particular having at
least 70-80%, 85%, 90%, 95%,
98%, or 99% amino acid sequence identity to the sequences identified in any of
SEQ ID NOs. 1 through 5.
Such variants include, for example, polypeptides wherein one or more amino
acid residues are added to, or
deleted from, the N- or C-terminus of the full-length or mature sequences of
SEQ ID NOs: 1 through 5
including variants from other species, but excludes a native-sequence
polypeptide.
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Percent identity of two amino, acid sequences, or of two nucleic acid
sequences is defined as the
percentage of amino acid residues or nucleotides in a candidate sequence that
are identical with the amino
acid residues in a polypeptide or nucleic acid sequence, after aligning the
sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and not
considering any conservative
substitutions as part of the sequence identity. Alignment for purposes of
determining percent amino acid or
nucleic acid sequence identity can be achieved in various conventional ways,
for instance, using publicly
available computer software including the GCG program package (Devereux J. et
al., Nucleic Acids
Research 12(1): 387, 1984); BLASTP, BLASTN, and FASTA (Atschul, S.F. et al. J.
Molec. Biol. 215: 403-
410, 1990). The BLAST programs are publicly available from NCBI and other
sources (BLAST Manual,
Altschul, S. et al. NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al. J.
Mol. Biol. 215: 403-410,
1990). Skilled artisans can determine appropriate parameters for measuring
alignment, including any
algorithms needed to achieve maximal alignment over the full length of the
sequences being compared.
Methods to determine identity and similarity are codified in publicly
available computer programs.
An "analog" refers to a polypeptide wherein one or more amino acid residues of
a parent
polypeptide have been substituted by another amino acid residue, one or more
amino acid residues of a
parent polypeptide have been inverted, one or more amino acid residues of the
parent polypeptide have been
deleted, and/or one or more amino acid residues have been added to the parent
peptide. Such an addition,
substitution, deletion, and/or inversion may be at either of the N-terminal or
C-terminal end or within the
parent polypeptide, or a combination thereof.
Mutations may be introduced into a polypeptide by standard methods, such as
site-directed
mutagenesis and PCR-mediated mutagenesis. Conservative substitutions can be
made at one or more
predicted non-essential amino acid residues. A "conservative amino acid
substitution" is one in which an
amino acid residue is replaced with an amino acid residue with a similar side
chain. Amino acids with
similar side chains are known in the art and include amino acids with basic
side chains (e.g. Lys, Arg, His),
acidic side chains (e.g. Asp, Glu), uncharged polar side chains (e.g. Gly,
Asp, Glu, Ser, Thr, Tyr and Cys),
nonpolar side chains (e.g. Ala, Val, Leu, Iso, Pro, Trp), beta-branched side
chains (e.g. Thr, Val, Iso), and
aromatic side chains (e.g. Tyr, Phe, Trp, His). Mutations can also be
introduced randomly along part or all of
the native sequence, for example, by saturation mutagenesis. Following
mutagenesis the variant polypeptide
can be recombinantly expressed.
A "derivative" refers to a polypeptide in which one or more of the amino acid
residues of a parent
polypeptide have been chemically modified. A chemical modification includes
adding chemical moieties,
creating new bonds, and removing chemical moieties. A polypeptide may be
chemically modified, for
example, by alkylation, acylation, glycosylation, pegylation, ester formation,
deamidation, or amide
formation.
A "chimeric polypeptide" comprises all or part (preferably biologically
active) of a selected
polypeptide operably linked to a heterologous polypeptide (i.e. a polypeptide
other than the selected
polypeptide). Within the fusion protein, the term "operably linked" is
intended to indicate that a selected
polypeptide and the heterologous polypeptide are fused in-frame to each other.
The heterologous polypeptide
can be fused to the N-terminus or C-terminus of a selected polypeptide.
Chimeric and fusion proteins can be
produced by standard recombinant DNA techniques.
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A "gastrin compound" is understood to refer to any compound, including
peptides and non-peptide
compounds, which fully or partially, directly or indirectly, potentiate,
induce, mimic, or otherwise enhance
the activity of a gastrin or a gastrin/CCK receptor. In particular, a gastrin
compound can be used which fully
or partially associates andlor activates a gastrin/CCK receptor. A gastrin/CCK
receptor includes receptors
that associate with a gastrin.
In some applications of the invention, a gastrin compound may be a ligand that
associates, binds to,
interacts with or stimulates a gastrin/CCK receptor. A gastrin compound may be
selected that is a peptide or
non-peptide small molecule that has a suitable ICSO, for example an
ICS° of about ~ 0.7 nM, as measured by
methods known in the art (see Singh et al (1995) J. Biol. Chem. 270: 8429-
8438, and Kopin et al (1995) J.
Biol. Chem. 270: 5019-5023 describing ira vitro cell growth assays, and
receptor binding assays as described
in Singh et al (1995) J. Biol. Chem. 270: 8429-8438, and Kopin et al (1995) J.
Biol. Chem. 270: 5019-5023).
A "gastrin compound" can include native-sequence or synthetic gastrin
polypeptides, fragments,
analogs (e.g. muteins), derivatives, isoforms, variants, chimeric
polypeptides, polypeptides with sequence
identity, peptidomimetics, and pharmaceutically acceptable salts thereof, and
active metabolites and
prodrugs. In particular the term includes the various forms of gastrin,
preprogastrin, progastrin, such as
gastrin 34 (big gastrin), gastrin 17 (little gastrin), gastrin 8 (mini
gastrin), pentagastrin, tetragastrin and
fragments, analogs, and derivatives thereof.
Sequences for gastrins including big gastrin-34 (Bonato et al , 1986, Life
Science 39:959) and small
gastrin-17 (Bentley et al (1966) Nature 209:583) are shown in SEQ ID NOs. 1-5.
Big gastrin-34 is
essentially an extension of an amino acid sequence at the N-terminal end of
small gastrin-17. Big gastrin is
cleaved ira vivo to release gastrin-17. Glp at the N-terminal end is
pyroglutamate, which is a naturally
cyclized form of glutamate. In various embodiments, where cysteine or lysine
are added to a terminus of
gastrin having a pyroglutamate, the pyroglutamate is replaced with a
glutamate, or the pyroglutamate is
deleted. Further, each of a gastrin 34 and gastrin-17 can be used having a
methionine or a leucine at position
15, as shown in SEQ ID NOs: 1-4 herein. A gastrin compound can be sulfated or
nonsulfated. [See J.H.
Walsh, "Gastrin" in Gut Peptides: Biochemistry and Physiology, ed. J.H. Walsh
and G.J. Doclcray, Raven
Press Ltd., New York, 1994 for a review of Gastrin.]
A gastrin compound also includes active analogs, fragments and other
modifications, which for
example share amino acid sequence identity with an endogenous mammalian
gastrin or native-sequence
gastrin, for example, share 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence
identity.
Examples of gastrin compounds that may be used in the present invention
include the compounds
disclosed in U.S. Patent No. 6,288,301. In some applications of the invention,
a gastrin compound may be
selected that is a peptide or non-peptide agonist or partial agonist of the
gastrin receptor such as A71378 (Lin
et al., Am. J. Physiol. 258 (4 Pt 1): 6648, 1990). In other applications of
the invention, a gastrin compound
may be a gastrin/CCK receptor ligand including but not limited to gastrin
compounds described herein, or a
cholecystokinin (CCK) such as CCK 58, CCK 33, CCK 22, CCK 12 and CCK 8; and
the like.
Gastrin compounds also include substances that increase the secretion of
endogenous gastrins,
cholecystolcinins or similarly active peptides from sites of tissue storage.
Examples of these are the gastric
releasing peptide, omeprazole which inhibits gastric acid secretion and
increases plasma gastrin levels, Soya
bean trypsin inhibitor which increases CCK stimulation, and gastrin releasing
peptide, which stimulates
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gastrin secretion without binding to gastrin receptors.
Gastrin compounds may be prepared using conventional processes. For example,
small forms of
gastrin such as gastrin 17 are economically prepared by peptide synthesis, and
the synthetic peptides are
commercially available. In particular, gastrin compounds may be synthesized by
chemical synthesis using
techniques well known in the chemistry of proteins such as solid phase
synthesis (Merrifield, 1964, J. Am.
Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl,
1987, Methods of
Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart). The
synthesis may be performed
using manual procedures or by automation. Automated synthesis may be carried
out, for example, using an
Applied Biosystems 431A peptide synthesizer (Perkin Elmer).
Gastrin compounds can be prepared by recombinant methods well known to those
skilled in the art.
Thus, the invention contemplates the use of a nucleotide sequence encoding a
gastrin compound and
optionally a regulatory element, and a host cell comprising the nucleotide
sequence for the preparation of a
gastrin compound.
Gastrin compounds may also be obtained from commercial sources. For example,
synthetic human
gastrin 17 with methionine or leucihe at position 15 are available from Bachem
AG, Bubendorf,
(Switzerland), and from Research Plus Imc (New Jersey, USA).
"Host cells" comprising a nucleotide sequence of a gastrin compound include a
wide variety of
prokaryotic and eukaryotic host cells. For example, the polypeptides may be
expressed in bacterial cells such
as E. coli, Bacillus, or Streptonryces, insect cells (using baculovirus),
yeast cells, or mammalian cells. Other
suitable host cells can be found in Goeddel, Gene Expression Technology:
Methods in Erizymology 185,
Academic Press, San Diego, CA (1991). A host cell may also be chosen which
modulates the expression of
an inserted nucletotide sequence, or modifies (e.g. glycosylation or
phosphorylation) and processes (e.g.,
cleaves) the polypeptide in a desired fashion. Host systems or cell lines may
be selected which have specific
and characteristic mechanisms for post-translational processing and
modification of proteins. For long-term
high-yield stable expression of the protein, cell lines and host systems which
stably express the gene product
may be engineered.
"Regulatory element" refers to a genetic element or elements having a
regulatory role in gene
expression, for example, promoters or enhancers, Large numbers of suitable
vectors and promoters are
known to those of skill in the art and are commercially available for
generating recombinant constructs
encoding a gastrin compound. The following vectors are provided by way of
example. Bacterial: pBs,
phagescript, PsiX174, pBluescript SK, pBs KS, pNHBa, pNHl6a, pNHl8a, pNH46a
(Stratagene); pTrc99A,
pKK223-3, pKK233-3, pDR540, pRITS (Pharmacia). Eukaryotic: pWLneo, pSV2cat,
pOG44, PXTL pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). As defined herein "operably
linked" means that an
isolated polynucleotide and a regulatory element are situated within a vector
or cell in such a way that the
polypeptide is expressed by a host cell which has been transformed
(transfected) with the ligated
polynucleotide/regulatory element sequence. A regulatory element can be a
constitutive or induced
transcriptional regulatory region, for example, a transcriptional regulatory
region from an insulin gene that is
induced by increasing intracellular glucose concentrations.
""Conditions and/or diseases", "condition(s)" and "disease(s)" include
diabetes and its
complications. The term "diabetes" as used herein means any manifested
symptoms of diabetes in any
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mammal including experimental animal models, and including human forms such as
type I and type II
diabetes, early stage diabetes, and a pre-diabetic condition characterized by
mildly decreased insulin or
mildly elevated blood glucose levels. A "pre-diabetic condition" describes a
subject demonstrating a
symptom in terms of insulin or glucose level, and/or demonstrating a
susceptibilty to diabetes or a related
condition due to family history, genetic predisposition, or obesity in the
case of type II diabetes, and includes
a subject who has previously had diabetes or a related condition and is
subject to risk of recurrence.
"Insulinotropic activity" refers to an ability of a substance to stimulate
insulin secretion in response
to elevated glucose levels, to produce or increase glucose uptake by cells,
and decreased serum glucose or
blood glucose levels. Methods known in the art can be employed to assay for
insulinotropic activity. For
example, in vitro and in vivo methods may be used that measure gastrin
receptor binding activity, receptor
activation (see the methods described in EP 619,322 to Gelfand et al and US
Patent No. 5,120,712), and
insulin or C-peptide levels. Compounds or compositions described herein have
insulinotropic activity if islet
cells secrete insulin in the presence of the compounds or compositions above
background levels or levels in
the absence of the gastrin compounds or compositions.
"Islet neogenesis" means formation of new pluripotent pancreatic precursor
cells, pancreatic islet
precursor cells, or beta cells by proliferation and differentiation, which may
or may not have the
characteristics of stem cells which have the ability to reproduce in an
unlimited manner.
Compositions and Methods
The invention is related to compositions and methods that utilize one or more
gastrin compound to
provide beneficial effects, in particular enhanced beneficial effects, more
particularly sustained beneficial
effects.
In an embodiment, where the' disease or condition is diabetes, sustained
beneficial effects of a
composition or treatment of the invention can manifest as one or more of the
following:
a) An increase in pancreatic insulin levels relative to the levels measured in
the absence of a
gastrin compound after administration to a subject with symptoms of diabetes.
Preferably the
compounds induce at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%,
20%, 30%,
33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% increase in
pancreatic insulin
levels in a subject.
b) A reduction or an absence of symptoms of islet inflammation after
administration to a
subject with symptoms of diabetes.
c) A decrease in blood glucose levels relative to the levels measured in the
absence of a gastrin
compound in subjects with symptoms of diabetes. Preferably, the compounds
induce at least
about a 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease
in blood
glucose levels. Most preferably, the compounds yield blood glucose levels
about or close to the
levels common in a normal subject.
d) An improvement in glucose tolerance. In particular, at least about a 5-95%,
10-90%, 10-
80%, 10-70%, 10-60%, improvement in glucose tolerance.
e) An increase in C-peptide levels relative to the levels measured in the
absence of gastrin
compounds in subjects with symptoms of diabetes. Preferably, the compounds
induce at least
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about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%,
50%,
GO%, 70%, 80%, 90%, 95%, or 99% increase in C-peptide levels.
f) Maintenance of blood glucose levels at about normal for a prolonged period
of time, in
particular for at least 1 week, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks,
10 weeks, 12
weeks, 14 weeks, 16 weeks, 20 weeks, 24 weeks, 30 weeks, 40 weeks, 52 weeks,
or 78 weeks,
more particularly, 2 to 4 weeks, 2 to 5 weeks, 3 to 5 weeks, 2 to G weeks, 2
to 8 weeks, 2 to 10
weeks, 2 to 12 weeks, 2 to 1G weeks, 2 to 20 weeks, 2 to 24 weeks, 2 weeks to
12 months, or 2
weeks to 18 months.
g) A reduction, prevention, or slowing of the rate of disease progression in a
subject with
diabetes.
h) A reduction or prevention of the development of severe hyperglycemia and
ketoacidosis
with symptoms of diabetes.
i) An increase in survival in a subject with symptoms of diabetes.
j) A decrease in requirement for insulin injection/intake by at least 10-90%,
10-80%, 10-70%,
10-60%, 10-50%, 10-40%~ 10-30%, or 10-20%.
One or more of these beneficial effects can be demonstrated in a diabetic
subject or disease model,
for example, a non-obese (NOD) mouse with symptoms of diabetes.
A gastrin compound may be selected for particular applications in the present
invention based on
one or more of the following characteristics: ability to bind to a gastrin
receptor, ability to initiate a signal
transduction pathway resulting in proliferation and/or differentiation of beta
cells or insulinotropic activity;
ability to reduce glucose levels, insulinotropic activity; stimulation of beta
cell proliferation/differentiation;
and/or, an ifa vivo half life of at least about 5 minutes to 24 hours,
preferably 2 to 10 hours or 2 to 8 hours in
humans using conventional methods.
Pharmaceutical compositions and methods of the invention can be selected that
have sustained
beneficial effects, preferably statistically significant sustained beneficial
effects. In an embodiment, a
pharmaceutical composition with statistically significant sustained beneficial
effects is provided comprising
a gastrin compound selected from the group consisting of gastrin 17 and
analogs and derivatives thereof,
preferably synthetic human gastrin I having 17 amino acid residues with a Leu
residue at amino acid position
15. In a particular embodiment, a pharmaceutical composition with
statistically significant beneficial effects
is provided comprising gastrin-17(leu).
The invention contemplates the use of a composition of the invention for
preventing, and/or
ameliorating disease severity, disease symptoms, and/or periodicity of
recurrence of a condition and/or
disease. The invention also contemplates preventing and/or treating, in
mammals, conditions and/or diseases
using the compositions or treatments of the invention. In particular, the
present invention provides improved
methods and compositions for use of a gastrin compound for sustained treatment
of diabetes. The present
invention in an embodiment provides a composition comprising a gastrin
compound that achieves greater
efficacy, potency, and utility. The greater efficacy can be shown by improving
glucose tolerance in severe
diabetes with treatment resulting in sustained improvement of blood glucose
after ceasing treatment and also
in recent onset diabetes. An improvement in glucose tolerance may also be
observed with the compositions
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described herein using lower doses of gastrin, i.e. doses below 1-50 ~g/kg
body weight, in particular, 1-30
pg/kg body weight.
Greater efficacy and potency of a gastrin treatment of the invention improves
the therapeutic ratio
of treatment, reducing untoward side effects and toxicity. The methods of the
invention also enhance utility
improving long-standing diabetes even when treatment is begun long after the
completion of (3 cell
destruction.
While not wishing to be bound by a particular mechanism, improvement in
glucose tolerance after
treatment with gastrin may result from (3 cell regeneration and concomitant
increased (3 cell mass.
Histological analysis can show treatment with gastrin stimulates (3 cell
regeneration with an increase in the (3
cell mass as measured morphometrically. This can be confirmed biochemically by
an increase in pancreatic
insulin content. The increased (3 cell mass can also result in increased
secretion of insulin into the
bloodstream which can be shown by increased circulating C peptide in plasma.
Prolonged efficacious islet cell neogenesis can be achieved in accordance with
the invention
following administration of a gastrin compound or composition of the
invention. The gastrin compound or
composition can be administered iiz vivo to provide for proliferation and/or
differentiation of islet cells in a
subject or it can be administered ex vivo to cells for transplantation. A
gastrin compound can be introduced
to cells using methods known to a person skilled in the art including
recombinant techniques. For example, a
chimeric insulin promoter-gastrin fusion gene may be introduced ira vivo or ex
vivo to pancreatic cells to
express one or more gastrin compound.
The invention relates to a method for expanding and differentiating stem cells
or progenitor cells
into insulin secreting cells comprising contacting the stem cells or
progenitor cells with a therapeutically
effective amount of a gastrin compound or a composition of the invention or
sufficient amounts of a gastrin
compound or a composition to expand and differentiate stem cells or progenitor
cells. The stem cells may be
obtained from pancreatic islets, umbilical cords, embryos, or stem cell lines.
The amount and' duration of
expansion and differentiation is significantly different compared with that
achieved in the absence of the
gastrin compounds or composition. In an embodiment, the stem cells or
progenitor cells are contacted with
the gastrin compounds or composition in culture. In another embodiment, the
stem cells or progenitor cells
are contacted with the gastrin compounds or composition in a subject. The
gastrin compounds or
composition may be administered to a subject before, during, or after
implantation of stem cells in the
subject to expand and differentiate the stem cells in the subject for a
prolonged period. The method may
additionally comprise administering an immunosuppressive agent.
In an aspect, the invention provides a method for treating diabetes mellitus
by providing a
composition comprising a gastrin compound in an amount sufficient to effect
differentiation of pancreatic
islet precursor cells to mature insulin-secreting cells for a prolonged period
following administration. A
gastrin compound in the composition can be administered systemically, in
particular by injection, preferably
intravenously, in a physiologically acceptable carrier. Alternatively, a
gastrin compound can be expressed ira
situ, and pancreatic islet precursor cells are transformed either ex vivo or
in vivo with one or more nucleic
acid encoding a gastrin compound in an expression construct vector that
provides for expression of the
compound in the cells. A nucleic acid encoding a gastrin compound can be
contained in an expression
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construct that may include a preprogastrin peptide precursor coding sequence,
which following expression is
processed to gastrin. The expression construct can include regulatory
elements.
The invention also relates to inducing islet neogenesis in a subject
comprising contacting islet
precursor cells with a gastrin compound, or composition of the invention in a
sufficient amount to increase
and prolong proliferation of islet precursor cells in the subject thereby
inducing islet neogenesis. In an
aspect, the invention provides a method for stimulating prolonged beta cell
proliferation in a subject
comprising administering a therapeutically effective amount of a gastrin
compound or composition of the
invention. In an embodiment, the invention provides a method for increasing
the number andlor size of beta
cells in a subject for a prolonged period comprising administering a
therapeutically effective amount of a
gastrin compound or a composition of the invention.
Regenerative differentiation of pluripotent pancreatic precursor cells, for
example, pancreatic ductal
cells, into mature insulin-secreting cells for a prolonged period can be
obtained with the gastrin compounds,
compositions and methods described herein for treatment of diabetes mellitus,
particularly juvenile onset
diabetes, and by therapeutic administration of gastrin compounds or
compositions which are provided for
systemic administration, or for in situ expression within the pancreas.
The invention provides methods for treating diabetes mellitus in a patient in
need thereof by
administering a composition comprising a gastrin compound in an amount
sufficient to effect prolonged
differentiation of the patient's pancreatic islet precursor cells to mature
insulin-secreting cells and/or to
stimulate insulin synthesis in existing islet 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 compound, together
with transcriptional and
translational regulatory elements functional in pancreatic islet precursor
cells.
In an aspect, the invention provides a method for treating diabetes mellitus
in a patient in need
thereof which includes administering to the individual a composition that
provides a gastrin compound in a
dose sufficient to effect prolonged differentiation of pancreatic islet
precursor cells to mature insulin-
secreting cells. In another aspect, the invention provides a method for
treating insulin dependent diabetes,
especially Type I or juvenile diabetes mellitus, comprising administering,
preferably systemically, a
differentiation regenerative amount of a gastrin compound to a diabetic
mammal, to stimulate islet
neogenesis resulting in an increase in the number of functional glucose
responsive insulin secreting a cells in
the pancreas for a prolonged period following administration.
The invention contemplates a method of expanding a functional beta cell mass
of pancreatic islet
transplants in a diabetic patient for a prolonged period, the method
comprising administering to the patient a
therapeutically effective amount of a gastrin compound, or a composition of
the invention.
The invention in an embodiment provides a method for preventing and/or
treating diabetes, the
method comprising administering to a mammal in need thereof a composition
comprising a gastrin
compound in an amount sufficient to increase the number of pancreatic insulin
secreting (3 cells in the
mammal for a prolonged period following administration, thereby preventing
and/or treating the diabetes.
The composition is administered systemically. The mammal is a diabetic mammal,
for example, the
mammal has been diabetic for an extent of 1% of the lifespan of the mammal.
The gastrin compound is
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provided in an amount sufficient to induce differentiation of the pancreatic
islet precursor cells into glucose
responsive insulin secreting islet cells for a prolonged period.
Another embodiment of the invention provides a method for preventing andlor
treating diabetes, the
method comprising administering to a mammal in need thereof a composition
comprising a gastrin
compound in an amount sufficient to increase the amount and duration of
proliferation of islet precursor cells
in pancreatic tissue for a prolonged period following administration, thereby
preventing and/or treating the
diabetes.
In another aspect, the invention provides a method for preventing and/or
treating diabetes, the
method comprising administering to a mammal in need thereof a composition
comprising a gastrin
compound in an amount sufficient to increase the number of pancreatic insulin
secreting (3 cells in the
mammal for a prolonged period following administration; and determining the
amount of islet neogenesis,
thereby preventing and/or treating the diabetes. The amount of islet
neogenesis may be measured by one or
more of the following parameters: blood glucose, serum glucose, blood
glycosylated hemoglobin, pancreatic
(3 cell mass, serum insulin, and pancreatic insulin content. Administering the
composition reduces blood
glucose compared to blood glucose assayed prior to administering the
composition. Glycosylated
hemoglobin concentration is reduced for a prolonged period compared to
glycosylated hemoglobin
concentration in the mammal assayed prior to administering the composition.
Serum insulin concentration is
increased for a prolonged period compared to serum insulin concentration in
the mammal assayed prior to
administering the composition. Pancreatic insulin concentration is increased
for a prolonged period
compared to pancreatic insulin concentration in the mammal assayed prior to
administering the composition.
In a further aspect, the invention provides a method for inducing pancreatic
islet neogenesis in a
mammal, the method comprising administering to the mammal a composition
comprising a gastrin
compound, in an amount sufficient to increase the amount and duration of
proliferation of islet precursor
cells in pancreatic tissue for a prolonged period following administration,
thereby inducing pancreatic islet
neogenesis. The plurality of cells can be multicellular. The plurality of
cells are delivered systemically to
the mammal.
In a still further aspect, the invention provides a method for inducing islet
neogenesis therapy in a
cell of an animal for a prolonged period, comprising contacting the cell with
a nucleic acid sequence
encoding a gastrin compound operably linked to a regulatory element, for
example, an insulin promoter
receptor ligand, for example, a metallothionein promoter. For example, the
cell is a germ cell, or the cell is
an autologous cell cultured ex vivo.
The invention contemplates cell based treatment methods using a gastrin
compound of the
invention, or compositions of the invention. Thus, the invention contemplates
methods comprising treating
cells, or treating explanted pancreatic tissue of a mammal with a gastrin
compound or composition of the
invention and introducing the treated cells or pancreatic tissue to the mammal
to provide beneficial effects,
in particular sustained beneficial effects. See PCT/CA03/33595 for a
description of general culture and cell
based treatment methods.
A method for treating a subject with a condition or disease described herein
comprises contacting ex
vivo a plurality of cells with a gastrin compound, or a composition of the
invention, optionally culturing the
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cells, and administering the cells to the subject in need thereof to provide
beneficial effects, in particular
sustained beneficial effects.
In embodiments of cell based therapeutic methods the cells are pancreatic
ductal cells and the
amount of compounds or compositions used in the methods are generally
effective to increase the amount of
insulin secreting cells in the subject for a prolonged period. The cells may
be autologous (i.e. from the same
subject), or may be from another individual of the same species, or from a
different species.
The invention also contemplates a method for treating diabetes in a subject
comprising
transplanting a pancreatic islet preparation into the subject and
administering a therapeutically effective
amount of a gastrin compound, or a composition of the invention to provide
beneficial effects, in particular
sustained beneficial effects.
The invention also relates to a method for sustaining islet cells or precursor
cells in culture
comprising culturing the cells in the presence of a gastrin compound or a
composition of the invention in an
amount sufficient to sustain the cells in culture. The cells may be sustained
in culture for a significantly
longer period of time compared with cells cultured in the absence of the
compounds or composition.
Culturing cells in the presence of a gastrin compound or a composition of the
invention will be particularly
useful in preparing and maintaining cells intended for transplantation.
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 compound to increase the number of pancreatic beta cells
in the islets for a prolonged
period; optionally the population of pancreatic beta cells can be grown in
culture for a time sufficient to
expand the population of (3-cells prior to transplantation.
Another embodiment of the invention provides a method for treating diabetes,
the method
comprising: contacting ex vivo a plurality of cells with a composition
comprising a gastrin compound in an
amount sufficient to increase proliferation of islet precursor cells and the
amount of insulin secreting islet
cells; and administering the contacted plurality of cells to a mammal in need
thereof to produce a beneficial
effect, in particular a sustained beneficial effect. The cells can be
autologous. The composition is provided
in an amount sufficient to effect differentiation of stem cells, for example,
to effect differentiation of
pancreatic islet precursor cells in pancreatic tissue into mature insulin
secreting islet cells. The composition
is provided in an amount sufficient to increase proliferation of pancreatic
islet stem cells, for example, of
pancreatic islet precursor cells for a prolonged period. Stem cells can be
obtained either from a pancreatic
tissue or from a non-pancreatic tissue, such as liver or bone marrow.
The invention provides a method of treating a condition or disease comprising
administering a
gastrin compound or composition of the invention with a plurality of cells to
a subject in need thereof to
thereby produce a beneficial effect, preferably a. sustained beneficial
effect. In an aspect, the invention
provides a method for expanding and differentiating stem cells, in a diabetic
recipient of the cells, into
insulin secreting cells, the method comprising implanting the cells in the
recipient, and administering a
composition containing an effective dose of a gastrin compound to produce a
beneficial effect, in particular a
sustained beneficial effect. For example, the implanted cells are obtained
from a human, for example, are
obtained from human pancreatic islets, human liver, human bone marrow, human
umbilical cord, or human
embryos. Implanting the cells into the recipient may be by a route such as
injecting directly into an organ, for
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example, into the pancreas, the kidney, or the liver. Alternatively,
implanting the cells may be administering
by intravenous injection, for example, into the portal vein or into the
hepatic vein. In certain embodiments,
prior to implanting the cells are treated ex vivo with a composition
comprising a gastrin compound.
The present invention also includes methods of using the compositions of the
invention in
combination with one or more additional therapeutic agents including without
limitation immunosuppressive
agents (e.g. rapamycin, cyclosporine, ISAtx247, and FK506), antiobesity
agents, antidiabetic agents, appetite
regulating drugs, antihypertensive agents, agents for the treatment andlor
prevention of complications
resulting from or associated with a condition or disease, in particular
diabetes and obesity, anti-nausea, anti
headache medications, and general medications that treat or prevent side
effects
The invention also contemplates the use of a composition comprising at least
one gastrin compound
for the preparation of a medicament providing beneficial effects, preferably
sustained beneficial effects in
treating a condition or disease.
In an embodiment, the invention relates to the use of a therapeutically
effective amount of at least
one gastrin compound for preparation of a medicament for providing beneficial
effects, preferably sustained
beneficial effects, in treating a condition or disease.
In an embodiment the invention provides the use of a gastrin compound for the
preparation of a
medicament for increase (preferably prolonged increase) of the number and/or
size of beta cells in a subject
after treatment.
In another embodiment the invention provides the use of a gastrin compound for
the preparation of
a medicament for stimulation (preferably prolonged stimulation) of beta cell
proliferation after treatment.
In a still further embodiment the invention provides the use of a gastrin
compound for the
preparation of a medicament for prolonged or sustained treatment of Type I or
Type II diabetes.
The invention additionally provides uses of a gastrin compound or a
pharmaceutical composition of
the invention in the preparation of medicaments for beneficial effects,
preferably sustained beneficial effects,
in the treatment of diseases and conditions.
Therapeutic efficacy and toxicity of compositions and methods or the invention
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals such as by calculating a
statistical parameter such as the EDso ( the dose that is therapeutically
effective in 50% of the population) or
LDso (the dose lethal to 50% of the population) statistics. The therapeutic
index is the dose ratio of
therapeutic to toxic effects and it can be expressed as the EDSo/LDso ratio.
Pharmaceutical compositions
which exhibit large therapeutic indices are preferred.
The methods of the invention may further comprise measuring one or more of the
following
markers: blood glucose, serum glucose, blood glycosylated haemoglobin,
pancreatic beta cell mass, serum
insulin, pancreatic insulin levels, morphometrically determined beta cell
mass, amount of insulin secreting
cells, and glucose responsiveness of insdlin secreting cells.
Administration
A gastrin compound and compositions of the present invention can be
administered by any means
that produce contact of the active agents) with the agent's sites of action in
the body of a subject or patient
to produce a beneficial effect, in particular a sustained beneficial effect.
The active ingredients can be
administered simultaneously or sequentially and in any order at different
points in time, to provide the
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desired beneficial effects, in particular sustained beneficial effects. A
gastrin compound and composition of
the invention can be formulated for sustained release, for delivery locally or
systemically. It lies within the
capability of a skilled physician or veterinarian to select a form and route
of administration that optimizes the
effects of the compositions and treatments of the present invention to provide
beneficial effects, in particular
sustained beneficial effects.
Modes of parenteral administration include, but are not limited to,
transdermal, 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 and intestinal mucosa, etc.),
and may be administered
together with other biologically active agents. A preferred route of
administration is systemic, for example,
by subcutaneous injection. For parenteral administration, the compounds and
compositions described herein
may be combined with saline, PBS, or other suitable buffer, at an appropriate
pH. A sustained release
formulation can also be used for either or both therapeutic agents.
The compositions may be administered in oral dosage forms such as tablets,
capsules (each of
which includes sustained release or timed release formulations), pills,
powders, granules, elixirs, tinctures,
suspensions, syrups, and emulsions. They may also be administered in
intravenous (bolus or infusion),
intraperitoneal, subcutaneous, or intramuscular forms, all utilizing dosage
forms well known to those of
ordinary skill in the pharmaceutical arts. The compositions of the invention
may be administered by
intranasal route via topical use of suitable intranasal vehicles, or via a
transdermal route, for example using
conventional transdermal skin patches. A dosage protocol for administration
using a transdermal delivery
system may be continuous rather than intermittent throughout the dosage
regimen.
The dosage regimen of the invention will vary depending upon known factors
such as the
pharmacodynamic characteristics of the agents and their mode and route of
administration; the species, age,
sex, health, medical condition, and weight of the patient, the nature and
extent of the symptoms, the kind of
concurrent treatment, the frequency of treatment, the route of administration,
the renal and hepatic function
of the patient, and the desired effect.
An amount of a therapeutic of the invention which will be effective in the
treatment of a particular
condition or disorder to provide effects; in particular sustained beneficial
effects, will depend on the nature
of the condition or disorder, and can be' determined by standard clinical
techniques. The precise dose to be
employed in the formulation will also depend on the route of administration,
and the seriousness of the
condition or disorder, and should be decided according to the judgement of the
practitioner and each
patient's circumstances. Routine determinations of blood levels of insulin or
C peptide, and of fasting levels
of glucose or glucose challenges, are determined by one of ordinary skill in
the art.
Suitable dosage ranges for administration are particularly selected to provide
beneficial effects, in
particular sustained beneficial effects. The dosage ranges are generally about
0.01 micrograms to about 500
micrograms of a gastrin compound per kilogram body weight per day, for
example, about 0.01 micrograms
to about 1 micrograms/kg, about 0.1 micrograms/kg to about 10 micrograms/kg,
or about 1 microgram/kg to
about 50 micrograms/kg.
In another aspect the invention provides a pharmaceutical composition
comprising 0.1 to 30, 0.1 to
40, 0.1 to 50, and 0.1 to 60 micrograms/kg/day gastrin compound.
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In particular embodiments of the invention providing sustained beneficial
effects, the dosage range
for administration of a gastrin compound is 1-30 micrograms/kg body weight, in
particular 3-30
micrograms/kg body weight, more particularly 5-20 micrograms/kg body weight.
A composition or treatment of the invention may comprise a unit dosage of at
least one gastrin
compound to provide beneficial effects, in particular sustained beneficial
effects. A "unit dosage" refers to a
unitary i.e. a single dose which is capable of being administered to a
patient, and which may be readily
handled and packed, remaining as a physically and chemically stable unit dose
comprising either the active
agents as such or a mixture with one or more solid or liquid pharmaceutical
excipients, carriers, or vehicles.
In an aspect, a pharmaceutical composition is provided comprising a
therapeutically effective
suboptimal dosage of a gastrin compound that is effective at decreasing or
reducing glucose levels for a
sustained period or increasing beta cell proliferation or differentiation
following treatment.
In another aspect, an improved pharmaceutical composition is provided
comprising therapeutically
effective suboptimal amounts of a gastrin compound in a form for chronic or
acute therapy of a disease or
condition, in particular diabetes.
In an aspect the invention provides a pharmaceutical composition comprising 30-
3000, 100-3000,
100-6000, 1000-6000, 2000-6000, and 3000-6000 micrograms gastrin compound per
single unit.
A composition or formulation of the invention may be administered to a subject
for about or at least
about 2 weeks to 4 weeks, 2 weeks to 6 weeks, 2 weeks to 8 weeks, 2 weeks to
10 weeles, 2 weeks to 12
weeks, 2 weeks to 14 weeks, 2 weelcs to 16 weeks, 2 weeks to 6 months, 2 weeks
to 12 months, or 2 weeks
to 18 months, periodically or continuously. A composition of the invention may
be administered one or
more times per day, in particular 1 or 2 times per day.
The compositions of the present invention or fractions thereof typically
comprise suitable
pharmaceutically acceptable carriers, excipients, and vehicles selected based
on the intended form of '
administration, and consistent with conventional pharmaceutical practices.
Suitable pharmaceutical carriers, excipients, and vehicles are described in
the standard text,
Remington's Pharmaceutical Sciences, Mack Publishing Company. By way of
example for oral
administration in the form of a capsule or tablet, the active components can
be combined with an oral, non-
toxic pharmaceutically acceptable inert carrier such as lactose, starch,
sucrose, methyl cellulose, magnesium
stearate, glucose, calcium sulfate, dicalcium phosphate, mannitol, sorbital,
and the like. For oral
administration in a liquid form, the drug components may be combined with any
oral, non-toxic,
pharmaceutically acceptable inert carrier such as ethanol, glycerol, water,
and the like. Suitable binders (e.g.
gelatin, starch, corn sweeteners, natural sugars including glucose; natural
and synthetic gums, and waxes),
lubricants (e.g. sodium oleate, sodium stearate, magnesium stearate, sodium
benzoate, sodium acetate, and
sodium chloride), disintegrating agents (e.g. starch, methyl cellulose, agar,
bentonite, and xanthan gum),
flavoring agents, and coloring agents may also be combined in the compositions
or components thereof.
Compositions as described herein can 'further comprise wetting or emulsifying
agents, or pH buffering
agents.
The composition can be a liquid solution, suspension, emulsion, tablet, pill,
capsule, sustained
release formulation, or powder. The compositions can be formulated as a
suppository, with traditional
binders and carriers such as triglycerides. Oral formulations can include
standard carriers such as
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pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose,
magnesium carbonate, etc. Various delivery systems are known and can be used
to administer a composition
of the invention, e.g. encapsulation in liposomes, microparticles,
microcapsules, and the like.
In an aspect of the invention a pharmaceutical composition has a pH from about
7 to 10.
Formulations for parenteral administration of a composition of the invention
may include aqueous
solutions, syrups, aqueous or oil suspensions and emulsions with edible oil
such as cottonseed oil, coconut
oil or peanut oil. Dispersing or suspending agents that can be used for
aqueous suspensions include synthetic
or natural gums, such as tragacanth, alginate, acacia, dextran, sodium
carboxymethylcellulose, gelatin,
methylcellulose, and polyvinylpyrrolidone.
Compositions for parenteral administration may include sterile aqueous or non-
aqueous solvents,
such as water, isotonic saline, isotonic glucose solution, buffer solution, or
other solvents conveniently used
for parenteral administration of therapeutically active agents. A composition
intended for parenteral
administration may also include conventional additives such as stabilizers,
buffers, or preservatives, e.g.
antioxidants such as methylhydroxybenzoate or similar additives.
In an embodiment, a composition herein is formulated in accordance with
routine procedures as a
pharmaceutical composition adapted for subcutaneous or intravenous
administration to human beings to
provide a beneficial effect, in particular a sustained beneficial effect.
Typically, compositions for
subcutaneous or intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary,
the composition may also include a solubilizing agent and a local anesthetic
to ameliorate pain at the site of
the injection. Generally, the ingredients are 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, for example, indicating the quantity of active
agent. Where the composition is to
be administered by infusion, an ampoule of sterile water or saline for
injection can be provided so that the
ingredients may be mixed prior to administration.
Compositions of the invention can be formulated as neutral or salt forms.
Pharmaceutically
acceptable salts include those formed with free amino groups such as those
derived from hydrochloric,
phosphoric, acetic, oxalic, 'tartaric acids etc., and those formed with free
carboxyl groups such as those
derived from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-
ethylamino ethanol, histidine, procaine, etc.
In an embodiment, a solid form pharmaceutical composition is provided (e.g.
tablets, capsules,
powdered, or pulverized form) comprising a crystalline or amorphous gastrin
compound.
In another embodiment, a solid form pharmaceutical composition is provided
(e.g. tablets,
capsules, powdered, or pulverized form) comprising a crystalline or amorphous
gastrin compound.
In another embodiment, the invention relates to a liquid drug formulation
comprising
pharmaceutically acceptable salts of a gastrin compound, and to lyophilized
drug formulations that can be
reconstituted to provide suspensions that are stable and suitable for
parenteral administration.
In a particular embodiment, the invention relates to an aqueous composition
comprising
pharmaceutically acceptable salts of a gastrin compound, and a solvent system
which effects solubilization.
The invention also provides a drug comprising an aqueous formulation of
pharmaceutically acceptable salts
of a gastrin compound with at least one solubilizer.
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A composition of the invention may be sterilized by, for example, filh~ation
through a bacteria
retaining filter, addition of sterilizing agents to the composition,
irradiation of the composition, or heating the
composition. Alternatively, the compounds or compositions of the present
invention may be provided as
sterile solid preparations e.g. lyophilized powder, which are readily
dissolved in sterile solvent immediately
prior to use.
In addition to the formulations described herein, the compositions can also be
formulated as a depot
preparation. Such long acting formulations may be administered by implantation
(for example,
subcutaneously or intramuscularly) or by intramuscular injection. Thus, for
example, the fractions may be
formulated with suitable polymeric or hydrophobic materials (for example, as
an emulsion in an acceptable
oil), or ion exchange resins, or as sparingly soluble derivatives, for
example, as a sparingly soluble salt.
The compositions of the invention and components thereof may comprise soluble
polymers as
targetable drug carriers.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate
container and labelled for treatment of an indicated condition. For
administration of a composition of the
invention, such labelling would include amount, frequency, and method of
administration.
In embodiments of the invention, a pharmaceutical pack or kit is provided
comprising one or more
containers filled with one or more of the ingredients of a pharmaceutical
composition of the invention to
provide a beneficial effect, in particular a sustained beneficial effect.
Associated with such containers) can
be various written materials such as instructions for use, or a notice in the
form prescribed by a governmental
agency regulating the manufacture, use or sale of pharmaceuticals or
biological products, which notice
reflects approval by the agency of manufacture, use, or sale for human
administration.
According to another aspect of the invention, a kit is provided. In an aspect,
the kit comprises a
gastrin compound or a pharmaceutical composition. The kit is a package which
houses a container which
contains a composition of the invention and also houses instructions for
administering the composition to a
subject.
The invention will be described in greater detail by way of specific examples.
The following
examples are offered for illustrative purposes, and are not intended to limit
the invention in any manner.
Those of skill in the art will readily recognize a variety of noncritical
parameters which can be changed or
modified to yield essentially the same results.
Examules
Example 1
Effects of Gastrin or EGF on Fasting Blood Glucose and Pancreatic Insulin
Content in NOD
Mice with Recent Onset Diabetes
The purpose of this experiment was to determine whether gasttin (G1) or an EGF
(E1) alone can
improve diabetic conditions in NOD mice with recent onset diabetes.
Non-obese diabetic (NOD) female mice with chronic insulin-dependent diabetes
were purchased
from Taconic (Germanton, NY). The mice were housed and fed under pathogen-free
conditions and were
cared for according to the Canadian Council on Animal Care guidelines. The NOD
mice were monitored for
diabetes development (fasting blood glucose, FBG >6.6 mmolll). After the onset
of diabetes, the mice were
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treated with (i) vehicle (V) - saline phosphate buffer (n=4), (ii) E1 (E) 1
~g/kglday, given i.p. once daily
(n=5) for 14 days, (iii) Gl (G) 3 pg/kg/day, given i.p. once daily (n=5) for
14 days. The mice did not receive
insulin-replacement treatment. Fasting blood glucose levels and pancreatic
insulin levels were monitored. E1
is a 51 amino acid EGF analog; G1 is a gastrin analog that is the same length
as the native gastrin but
contains a single amino acid change at position 15.
In the vehicle-treated control animals, fasting blood glucose (FBG) levels
were doubled after 35
days. FBG levels of animals treated with either E1 or Gl remained close to
values recorded at diabetes onset
(day 0), in spite of disease progression (e.g. ongoing destruction of islet
cells) in this animal model. See
Figure 1. Islet cell neogenesis stimulated by EGF or gastrin compensates for
the destruction of these cells. In
addition, pancreatic insulin levels were also measured in all animals.
Pancreatic insulin levels for vehicle
treated controls decreased at day 35 due to destruction of [3-cells, whereas
animals treated with El or Gl
exhibited significantly elevated levels of pancreatic insulin levels in
comparison to the pretreatment values.
See Figure 2. The results of this study demonstrate that a short course (14
days) of treatment with either E1
or Gl after recent onset of diabetes in NOD mice can prevent progression of
diabetic conditions as well as
improve pancreatic insulin content for at least 3 weeks after therapy is
stopped.
Example 2
Prolonged Effect of Gastrin on Fasting Blood Glucose in NOD Mice With Recent
Onset
Diabetes
The purpose of this experiment was to determine whether treatment with gastrin
produced long term
improvement of diabetic conditions in NOD mice with recent onset diabetes. NOD
mice were purchased
from Taconic (Germanton, NY). The mice were housed and fed under pathogen-free
conditions and were
cared for according to the Canadian Council on Animal Care guidelines
Following an initial 18 day period of treatment with 3 ~ g/kg/day of gastrin,
non-obese diabetic
(NOD) mice were monitored for fasting blood glucose (FBG) levels for up to 8
weeks from the initiation of
therapy. A control group of mice treated with vehicle was also monitored.
In the vehicle-treated control animals, fasting blood glucose (FBG) levels
increased over time. FBG
levels of animals treated with gastrin remained decreased relative to the
control animals for at least 5 weeks
after cessation of treatment. See Figure 3. The results of this study
demonstrate that treatment with gastrin
reduces fasting blood glucose levels in NOD mice for at least 5 weeks after
therapy is stopped.
Example 3
Dose-Dependent Effects of Gastrin on Fasting Blood Glucose in NOD Mice with
Chronic Insulin-
Dependent Diabetes
Objective:
To determine whether gastrin,(Gl) can prevent development of severe
hyperglycemia and
3 5 death in NOD mice with chronic insulin-dependent diabetes.
Method:
NOD mice with chronic insulin-dependent diabetes and maintained on insulin
therapy were
distributed into different treatment groups treated with: (i) vehicle (n = 4);
(ii) Gl 1 pg/kg/day, given i.p.
twice daily (n = 4) for 28 days, (iii) Gl 5 pglkg/day, given i.p. twice daily
(n = 4) for 28 days, (iv) Gl 10
pg/kg/day, given i.p. twice daily (n = 4) for 28 days. Insulin therapy was
stopped 14 days after
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commencement of treatment with Gl. G1 is a 17 as gastrin analog that is the
same length as the native
gastrin molecule but contains a single amino acid change at position 15.
Results and Conclusions:
From day 0 to day 14, where the animals were maintained on insulin therapy,
fasting blood
glucose (FBG) levels for all treatment groups remained close to levels
recorded at day 0 except for the
group treated with 10 ~g/kg/day of Gl which exhibited a decrease in FBG. At
day 28, 14 days after the
cessation of insulin therapy, all animals in the vehicle group died from
diabetic ketoacidosis (DKA)
since these mice were completely dependent on insulin injections. However all
mice treated with G1
survived without insulin treatment for 2 weeks. Fasting blood glucose levels
for mice treated with 1
~g/kg/day of Gl remained elevated but there was a corresponding decrease of
fasting blood glucose
levels with increasing dose of G1 (5 and 10 pg/kg/day, respectively). See
Figure 4. These data suggest
that treatment with gastrin has the ability to significantly improve glucose
control without the use of
insulin therapy in chronically diabetic insulin-dependent NOD mice.
The present invention is not to be limited in scope by the specific
embodiments described herein,
since such embodiments are intended as but single illustrations of one aspect
of the invention and any
functionally equivalent embodiments are within the scope of this invention.
Indeed, various modifications of
the invention in addition to those shov~m and described herein will become
apparent to those skilled in the art
from the foregoing description and accompanying drawings. Such modifications
are intended to fall within
the scope of the appended claims.
All publications, patents and patent applications referred to herein are
incorporated by reference in
their entirety to the same extent as if each individual publication, patent or
patent application was
specifically and individually indicated to be incorporated by reference in its
entirety. The citation of any
reference herein is not an admission that such reference is available as prior
art to the instant invention.
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Seuuence Listins
SEQ ID NO. 1
N-terminal Glp-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu-Val-Ala-Asp-Pro-Ser-Lys-Lys-
Gln-Gly-Pro-Trp-
Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe
SEQ ID NO. 2
N-terminal Glp-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu-Val-Ala-Asp-Pro-Ser-Lys-Lys-
Gln-Gly-Pro-Trp-
Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe
SEQ ID NO. 3
N-terminal Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe
.
SEQ ID NO. 4
N-terminal Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe
SEQ ID NO. 5
mqrlcvyvli falalaafse aswkprsqqp daplgtganr dlelpwleqq gpashhrrql
gpqgpphlva dpskkqgpwl eeeeeaygwm dfgrrsaede n
