Note: Descriptions are shown in the official language in which they were submitted.
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GLUCAGON-LIKE PEPTIDE 1(GLP-1) PHARMACEUTICAL FORMULATIONS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Application Serial
No. 10/632,878,
filed July 22, 2003 and claims the benefit under 35 U.S.C. 119(e) to U.S.
Provisional
Application No. 60/744,882, filed on April 14, 2006. Each of the above-
mentioned priority
applications is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of pharmaceutical
formulations. The
present invention discloses dry powder formulations comprising
diketopiperazine (DKP)
particles in combination with glucagon-like peptide 1(GLP-1). The present
invention has utility
as a pharmaceutical formulation for treating diseases such as diabetes,
cancers, and obesity but is
not limited to such diseases. More particularly, the present invention has
utility as a
pharmaceutical formulation for pulmonary delivery.
BACKGROUND TO THE INVENTION
[0003] Glucagon-like peptide 1(GLP-1) as disclosed in the literature is a 30
or 31 amino
acid incretin, released from the intestinal endocrine L-cells in response to
fat, carbohydrate
ingestion, and protein from a meal. Secretion of this peptide hormone is found
to be impaired in
individuals with type 2 diabetes mellitus making it a potential candidate for
the treatment of this
and other related diseases.
[0004] In the non-disease state, GLP-1 is secreted from the intestinal L-cell
in response to
orally ingested nutrients, (particularly sugars), stimulating meal-induced
insulin release from the
pancreas, inhibiting glucagon release from the liver, as well as other effects
on the
gastrointestinal tract, and brain. GLP-1 effect in the pancreas is glucose
dependent, minimizing
the risk of hypoglycemia during exogenous peptide administration. GLP-1 also
promotes all
steps in insulin biosynthesis and directly stimulates B-cell growth and
survival as well as B-cell
differentiation. The combination of these effects results in increased B-cell
mass. Furthermore,
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GLP-1 receptor signaling results in a reduction of B-cell apoptosis, which
further contributes to
increased B-cell mass.
[0005] In the gastrointestinal tract, GLP-1 inhibits GI motility, increases
the secretion of
insulin in response to glucose, and decreases the secretion of glucagon,
thereby contributing to a
reduction of glucose excursion. Central administration of GLP-1 has been shown
to inhibit food
intake in rodents, suggesting that peripherally released GLP-1 may directly
affect the brain. This
is feasible since it has been shown that circulating GLP-1 can access GLP-1
receptors in certain
brain areas; namely the subfornical organ and the area postrema. These areas
of the brain are
known to be involved in the regulation of appetite and energy homeostasis.
Interestingly, gastric
distension activates GLP-1 containing neurons in the caudal nucleus of the
solitary tract,
predicting a role for centrally expressed GLP-1 as an appetite suppressant.
These hypotheses are
supported by studies employing the GLP-1 receptor antagonist, exendin (9-39)
where opposite
effects were seen. In humans, administered GLP-1 has a satiating effect
(Verdich et al., 2001),
and when given by continuous subcutaneous infusion over a 6 weeks regime,
diabetics exhibited
a reduction in appetite, which led to significant reductions in body weight
(Zander et al., 2002).
[0006] GLP-1 has also been shown to be effective in patients with type 2
diabetes,
increasing insulin secretion and normalizing both fasting and postprandial
blood glucose when
given as a continuous intravenous infusion (Nauck et al., 1993). In addition,
infusion of GLP-1
has been shown to lower glucose levels in patients previously treated with non-
insulin oral
medication and in patients requiring insulin therapy after failure on
sulfonylurea therapy (Nauck
et al., 1993). However, the effects of a single subcutaneous injection of GLP-
1 provided
disappointing results, as is noted in the art and discussed herein below.
Although high plasma
levels of immunoreactive GLP-1 were achieved, insulin secretion rapidly
returned to
pretreatment values and blood glucose concentrations were not normalized
(Nauck et al., 1996).
Only when repeated subcutaneous administrations were performed was the effect
on fasting
blood glucose comparable to intravenous administration (Nauck et al., 1996).
Continuous
subcutaneous administration for 6 weeks was shown to reduce fasting and
postprandial glucose
concentrations, and lower HbAlc levels (Zander et al., 2002). The short-lived
effectiveness of
single subcutaneous injections of GLP-1 was related to its circulatory
instability. It was shown
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that GLP-1 was metabolized by plasma in vitro and that the enzyme dipeptidyl
peptidase-IV
(DPP-IV) was responsible for this degradation (Mentlein et al., 1993).
[0007] With the physiological significance of GLP-1 in diabetes and the
demonstration that
exogenous GLP-1 is rapidly amino-terminally degraded in both healthy and type
2 diabetic
subjects, many studies have addressed the possibility of manipulating the in
vivo stability of
GLP-1 as a novel approach to an antidiabetic agent for the treatment of
diabetes (Deacon et al.,
2004). Two separate approaches have been pursued: 1) the development of
analogs of GLP-1
that are not susceptible to enzymatic degradation and 2) the use of selective
enzyme inhibitors to
prevent in vivo degradation and enhance levels of the intact, biologically
active peptides. Long-
acting GLP-1 analogs (e.g., Liraglutide (Novo Nordisk, Copenhagen, Denmark));
exenatide
(exendin-4; Byetta ) (Amylin Inc., San Diego, CA) and (exenatide-LAR, Eli
Lilly, Indianapolis,
IN)) that are resistant to degradation, called "incretin mimetics," have been
investigated in
clinical trials. Dipeptidyl peptidase IV inhibitors (e.g., Vildagliptin
(Galvus) developed by
Novartis, Basel, Switzerland) and Januvia (sitagliptin) developed by Merck,
Whitehouse Station,
New Jersey)~ that inhibit the enzyme responsible for incretin degradation are
also under study
(Deacon et al., 2004). Thus, the multiple modes of action of GLP-1 (e.g.,
increased insulin
release, delayed gastric emptying, and increased satiety) together with its
low propensity for
hypoglycemia appear to give it advantages over currently available therapies.
[0008] [However, despite these approaches/advances in GLP-1 therapy, none of
the drugs
currently available for diabetes are able to achieve therapeutic targets
(HbAlc, fasting blood
glucose, glucose excursions) in all patients and none of them are without side
effects such as
toxicity, hypoglycemia, weight gain, nausea and stress from vomiting.
Therefore, there is still a
need in the art for stable GLP-1 formulations having long term effectiveness
and optimal
absorption when administered as a pharmaceutical.
SUMMARY OF THE INVENTION
[0009] Stable, inhalable glucagon-like peptide 1(GLP-1) formulations for use
as
pharmaceutics are deficient in the art. In overcoming the deficiencies in the
art, the present
invention provides formulations of GLP-1 in combination with diketopiperazine
(DKP) particles
as a pharmaceutic.
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[0010] Therefore, in particular embodiments of the present invention, a dry
powder
composition comprising a GLP-1 molecule and a diketopiperazine or a
pharmaceutically
acceptable salt thereof is provided. In further embodiments, the dry powder
composition of the
present invention comprises a GLP-1 molecule selected from the group
consisting of a native
GLP-1, a GLP-1 metabolite, a GLP-1 analog, a GLP-1 derivative, a dipeptidyl-
peptidase-IV
(DPP-IV) protected GLP-1, a GLP-1 mimetic, an exendin, a GLP-1 peptide analog,
or a
biosynthetic GLP-1 analog. In still yet a further embodiment of the present
invention, the dry
powder composition comprises a diketopiperazine having the formula 2,5-diketo-
3,6-di(4-X-
aminobutyl)piperazine, wherein X is selected from the group consisting of
succinyl, glutaryl,
maleyl, and fumaryl. In another embodiment, the dry powder composition
comprises a
diketopiperazine salt. In still yet another embodiment of the present
invention, there is provided
a dry powder composition, wherein the diketopiperazine is 2,5-diketo-3,6-di(4-
fumaryl-
aminobutyl)piperazine.
[0011] The present invention further contemplates a dry powder composition
wherein the
GLP-1 molecule is native GLP-l, or an amidated GLP-1 molecule wherein the
amidated GLP-1
molecule is GLP-1 (7-36) amide.
[0012] In still yet another particular embodiment of the present invention,
there is provided
a process for preparing a particle comprising a GLP-1 molecule and a
diketopiperazine
comprising the steps of: providing a GLP-1 solution comprising a GLP-1
molecule; providing a
solution of a particle-forming diketopiperazine or a suspension of particles
of a diketopiperazine;
and combining the GLP-1 solution with the diketopiperazine solution or
suspension. In other
particular embodiments of the invention, the process for preparing a particle
comprising a GLP-1
molecule and a diketopiperazine further comprises removing solvent from the
solution or
suspension by lyophilization, filtration, or spray drying. In still yet a
further embodiment, the
particle of the invention is formed by removing solvent or is formed prior to
removing solvent.
[0013] In an embodiment of the invention, in the process for preparing a
particle having a
GLP-1 molecule and a diketopiperazine, there is provided a GLP-1 molecule
selected from the
group consisting of a native GLP-l, a GLP-1 analog, a GLP-1 derivative, a
dipeptidyl-peptidase-
IV (DPP-IV) protected GLP-1, a GLP-1 mimetic, an exendin, a GLP-1 peptide
analog, or a
biosynthetic GLP-1 analog. In another embodiment, the process for preparing a
particle having a
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GLP-1 molecule and a diketopiperazine comprises a diketopiperazine provided as
a suspension
of particles. In a further embodiment, the diketopiperazine is provided in
solution and the
process includes adjusting the pH of the solution to precipitate the
diketopiperazine and form
particles.
[0014] In other particular embodiments of the invention the GLP-1 solution is
at a
concentration of about 1 g/ml-50 mg/ml, more preferably about 0.lmg/ml-10
mg/ml. In yet
another particular embodiment of the invention, the GLP-1 solution is at a
concentration of about
0.25 mg/ml.
[0015] In another process for preparing a particle comprising a GLP-1 molecule
and a
diketopiperazine, the process further comprises adding an agent to the
solution, wherein the
agent is selected from salts, surfactants, ions, osmolytes, chaotropes and
lyotropes, acid, base,
and organic solvents. The agent promotes association between the GLP-1 and the
diketopiperazine particle and also improves the stability and/or
pharmacodynamics of the GLP-1
molecule. In some embodiments of the invention, the agent is a salt such as,
but not limited to,
sodium chloride. It is also contemplated the agent may be a surfactant such as
but not limited to,
Tween, Triton, pluronic acid, CHAPS, cetrimide, and Brij, H(CH2)7SO4Na. The
agent may be
an ion, for example, a cation or anion. The agent may be an osmolyte
(stabilizer), such as, but
not limited to Hexylene-Glycol (Hex-Gly), trehalose, glycine, polyethylene
glycol (PEG),
trimethylamine n-oxide (TMAO), mannitol, and proline. The agent may be a
chaotrope or
lyotrope, such as, but not limited to, cesium chloride, sodium citrate, and
sodium sulfate. The
agent may be an organic solvent for example, an alcohol selected from methanol
(MeOH),
ethanol (EtOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP).
[0016] In another particular embodiment of the present invention, there is
contemplated a
process for preparing a particle comprising a GLP-1 molecule and a
diketopiperazine, wherein
the process comprises adjusting the pH of the particle suspension to about 4
or greater. In
further embodiments of the invention, the process for preparing a particle
comprises a GLP-1
molecule and a diketopiperazine, wherein the GLP-1 molecule in the particle
has greater
stability.
[0017] Further contemplated in the present invention is a method of
administering an
effective amount of a GLP-1 molecule to a subject in need thereof, comprising
providing to the
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subject a GLP-1/diketopiperazine particle. The method of administering may be
intravenously,
subcutaneously, orally, nasally, buccally, rectally, or by pulmonary delivery
but is not limited to
such. In one embodiment, the method of administering is by pulmonary delivery.
In still yet
another embodiment of the invention, the method of administering comprises
treating a condition
or disease selected from the group consisting of diabetes, ischemia,
reperfused tissue injury,
dyslipidemia, diabetic cardiomyopathy, myocardial infarction, acute coronary
syndrome, obesity,
catabolic changes after surgery, hyperglycemia, irritable bowel syndrome,
stroke,
neurodegenerative disorders, memory and learning disorders, islet cell
transplant and
regenerative therapy.
[0018] In another embodiment of the invention, the method of administration of
the GLP-
1/diketopiperazine particle composition results in improved pharmacokinetic
half-life and
bioavailability of GLP-l.
[0019] In still yet a further particular embodiment of the present invention,
there is
provided a method of preparing a dry powder composition with an improved
pharmacokinetic
profile, comprising the steps of: providing a solution of a GLP-1 molecule;
providing a particle-
forming diketopiperazine; forming particles; and combining the GLP-1 and the
diketopiperazine;
and thereafter removing solvent by a method of drying to obtain a dry powder,
wherein the dry
powder has improved pharmacokinetic profile. The improved pharmacokinetic
profile
comprises increased half-life of GLP-1 and/or improved bioavailability of GLP-
l. The increased
half-life of GLP-1 is greater than or equal to 7.5 minutes.
[0020] In one embodiment of the present invention, a dry powder composition is
provided
comprising a GLP-1 molecule and a diketopiperazine or a pharmaceutically
acceptable salt
thereof. In another embodiment, the GLP-1 molecule is selected from the group
consisting of
native GLP-ls, GLP-1 metabolites, GLP-1 analogs, GLP-1 derivatives, dipeptidyl-
peptidase-IV
(DPP-IV) protected GLP-ls, GLP-1 mimetics, GLP-1 peptide analogs, or
biosynthetic GLP-1
analogs.
[0021] In an embodiment of the present invention, the diketopiperazine is a
diketopiperazine having the formula 2,5-diketo-3,6-di(4-X-
aminobutyl)piperazine, wherein X is
selected from the group consisting of succinyl, glutaryl, maleyl, and fumaryl.
In another
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embodiment, the diketopiperazine is a diketopiperazine salt. In another
embodiment, the
diketopiperazine is 2,5-diketo-3,6-di(4-fumaryl-aminobutyl)piperazine.
[0022] In an embodiment of the present invention, the GLP-1 molecule is native
GLP-1. In
another embodiment, the GLP-1 molecule is an amidated GLP-1 molecule. In
another
embodiment, the amidated GLP-1 molecule is GLP-1(7-36) amide.
[0023] In one embodiment of the present invention, a process is provided for
forming a
particle comprising a GLP-1 molecule and a diketopiperazine comprising the
steps of: providing
a GLP-1 molecule; providing a diketopiperazine in a form selected from
particle-forming
diketopiperazine, diketopiperazine particles, and combinations thereof; and
combining the GLP-
1 molecule and the diketopiperazine in the form of a co-solution, wherein the
particle comprising
the GLP-1 molecule and the diketopiperazine is formed.
[0024] In one embodiment of the present invention, the process further
comprises removing
a solvent from said co-solution by lyophilization, filtration, or spray
drying. In another
embodiment, the particle comprising said GLP-1 molecule and the
diketopiperazine is formed by
removing the solvent. In another embodiment, the particle comprising the GLP-1
molecule and
the diketopiperazine is formed prior to removing the solvent.
[0025] In another embodiment, the GLP-1 molecule is selected from the group
consisting
of a native GLP-l, a GLP-1 analog, a GLP-1 derivative, a dipeptidyl-peptidase-
IV (DPP-IV)
protected GLP- 1, a GLP-1 mimetic, a GLP-1 peptide analog, or a biosynthetic
GLP-1 analog. In
another embodiment, the GLP-1 molecule is provided in the form of a solution
comprising a
GLP-1 concentration of about 1 g/ml -50 mg/ml. In another embodiment, the GLP-
1 molecule
is provided in the form of a solution comprising a GLP-1 concentration of
about O.lmg/ml - 10
mg/ml. In another embodiment, the GLP-1 molecule is provided in the form of a
solution
comprising a GLP-1 concentration of about 0.25 mg/ml.
[0026] In another embodiment of the present invention, the diketopiperazine is
provided in
the form of a suspension of diketopiperazine particles. In another embodiment,
the
diketopiperazine is provided in the form of a solution comprising particle-
forming
diketopiperazine, the process further comprising adjusting the pH of the
solution to form
diketopiperazine particles. In another embodiment, the process further
comprises adding an
agent to said solution or suspension, wherein the agent is selected from the
group consisting of
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salts, surfactants, ions, osmolytes, chaotropes and lyotropes, acids, bases,
and organic solvents.
In another embodiment, the agent promotes association between the GLP-1
molecule and the
diketopiperazine particles or the particle-forming diketopiperazine. In
another embodiment, the
agent improves the stability or pharmacodynamics of the GLP-1 molecule. In
another
embodiment, the agent is sodium chloride.
[0027] In another embodiment of the present invention, the process further
comprises
adjusting the pH of the suspension or solution. In another embodiment, the pH
is adjusted to
about 4.0 or greater. In yet another embodiment, the GLP-1 molecule in the
particle has greater
stability than native GLP- 1.
[0028] In another embodiment, the co-solution comprises a GLP-1 concentration
of about 1
g/ml-50 mg/ml. In another embodiment, the co-solution comprises a GLP-1
concentration of
about 0.1 mg/ml-10 mg/ml. In another embodiment, the co-solution comprises a
GLP-1
concentration of about 0.25 mg/ml.
[0029] In still yet another embodiment of the present invention, the process
further
comprises adding an agent to the co-solution, wherein the agent is selected
from the group
consisting of salts, surfactants, ions, osmolytes, chaotropes and lyotropes,
acids, bases, and
organic solvents. In another embodiment, the agent promotes association
between the GLP-1
molecule and the diketopiperazine particles or the particle-forming
diketopiperazine. In another
embodiment, the agent improves the stability or pharmacodynamics of the GLP-1
molecule. In
another embodiment, the agent is sodium chloride.
[0030] In another embodiment, the process further comprises adjusting the pH
of the co-
solution. In another embodiment, the pH is adjusted to about 4.0 or greater.
[0031] In one embodiment of the present invention, a method is provided of
administering
an effective amount of a GLP-1 molecule to a subject in need thereof the
method comprising
providing to the subject a particle comprising GLP-1 and diketopiperazine. In
another
embodiment, the providing is carried out intravenously, subcutaneously,
orally, nasally,
buccally, rectally, or by pulmonary delivery. In another embodiment, the
providing is carried
out by pulmonary delivery.
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[0032] In another embodiment, the need comprises the treatment of a condition
or disease
selected from the group consisting of diabetes, ischemia, reperfused tissue
injury, dyslipidemia,
diabetic cardiomyopathy, myocardial infarction, acute coronary syndrome,
obesity, catabolic
changes after surgery, hyperglycemia, irritable bowel syndrome, stroke,
neurodegenerative
disorders, memory and learning disorders, islet cell transplant and
regenerative therapy.
[0033] In another embodiment, the provision of the particle results in
improved
pharmacokinetic half-life and bioavailability of GLP-1 as compared to native
GLP-1.
[0034] In one embodiment of the present invention, a method is provided of
forming a
powder composition with an improved GLP-1 pharmacokinetic profile, comprising
the steps of:
providing a GLP-1 molecule; providing a particle-forming diketopiperazine in a
solution;
forming diketopiperazine particles; combining the GLP-1 molecule and the
solution to form a
co-solution; and, removing solvent from the co-solution by spray-drying to
form a powder with
an improved GLP-1 pharmacokinetic profile.
[0035] In another embodiment, the improved GLP-1 pharmacokinetic profile
comprises an
increased GLP-1 half-life. In another embodiment, the increased GLP-1 half-
life is greater than
or equal to 7.5 minutes. In another embodiment, the improved GLP-1
pharmacokinetic profile
comprises improved bioavailability of GLP-1 as compared to native GLP-l.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The following drawings form part of the present specification and are
included to
further demonstrate certain aspects of the present invention. The invention
may be better
understood by reference to one or more of these drawings in combination with
the detailed
description of specific embodiments presented herein.
[0037] FIGs. lA-1D. Structural analysis of GLP-1 at various concentrations (pH
4, 20 C).
FIG. 1A - The far-UV circular dichroism (CD) of GLP-1 illustrates that as the
concentration
increases, the secondary structure of the peptide is transformed from a
predominantly
unstructured conformation to a helical conformation. FIG. 1B - The near-UV CD
illustrates that
the tertiary structure increases with increasing concentration of peptide
suggesting that GLP-1
self-associates. FIG. 1C - Fluorescence emission of GLP-1 at various
concentrations (pH 4,
20 C) resulting from tryptophan excitation at 280 nm. FIG. 1D - Transmission
FTIR of GLP-1
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at various concentrations (pH 4, 20 C). The amide I band at 1656 crri-i
indicates that GLP-1 has
a a-helical structure at concentrations _ 2 mg/mL.
[0038] FIGs. 2A-2D. Structural analysis of low concentration GLP-1 at varying
ionic
strength (pH 4, 20 C). FIG. 2A - The far-UV CD of 1.0 mg/mL GLP-1 illustrates
that
increasing the concentration of salt converts the unordered structure of GLP-1
into more ordered
a-helical structures. FIG. 2B - The near-UV CD of 1.0 mg/mL peptide
demonstrates that
increasing the NaC1 concentration also enhances the tertiary structure of GLP-
l. FIG. 2C -
Intrinsic fluorescence emission of 1.0 mg/mL GLP-1 at varying NaC1
concentrations (pH 4,
20 C) following tryptophan excitation at 280 nm. At high peptide
concentrations, the maxima
decreases in intensity and shifts to a lower wavelength, which is indicative
of a well-defined
tertiary structure. FIG. 2D - Tertiary structural analysis of 10 mg/mL GLP-1
at varying ionic
strength (pH 4, 20 C). The near-UV CD spectra demonstrate that increased ionic
strength
enhances the tertiary structure of self-associated GLP-l.
[0039] FIGs. 3A-3B. Structural analysis of 10 mg/mL GLP-1 at various
temperatures (pH
4). FIG. 3A - The near-UV CD illustrates that GLP-1 oligomers dissociate with
increasing
temperature. FIG. 3B - Structural analysis of 10 mg/mL GLP-1 at various
temperatures (pH 4).
FIG. 3C - Structural analysis of 0.05 mg/mL GLP-1 at various temperatures (pH
4). The far-UV
CD illustrates that the peptide is insensitive to temperature.
[0040] FIGs. 4A-4B. Structural analysis of GLP-1 at varying pH (20 C). FIG. 4A
- The
far-UV CD of 10 mg/mL GLP-1 at varying pH (20 C). As the pH is increased, self-
associated
GLP-1 precipitates between pH 6.3 and 7.6 but retains a helical structure at
pH 1.5 and 11.7.
FIG. 4B - Enlarging the spectrum at pH 7.6 reveals that the secondary
structure of GLP-1 is
unordered as a result of the concentration decrease.
[0041] FIG. 5. Resistance of 1 mg/mL GLP-1 to both deamidation and oxidation
as
demonstrated by HPLC. Deamidation conditions were achieved by incubating GLP-1
at pH 10.5
for 5 days at 40 C. Oxidative conditions were achieved by incubating GLP-1 in
0.1 % H202 for
2 hours at room temperature.
[0042] FIGs. 6A-6B. The effect of agitation on the tertiary structure of 1.5
and 9.4 mg/mL
GLP-1 (pH 4). The near-UV CD (FIG. 6A) and the fluorescence emission of GLP-
1(FIG. 6B)
both illustrate that the tertiary structure of GLP-1 peptide does not
significantly change due to
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agitation. Samples were agitated for both 30 and 90 min at room temperature
and the
fluorescence emission spectra were collected after tryptophan excitation at
280 nm.
[0043] FIGs. 7A-7C. The effect of 10 freeze-thaw cycles on the tertiary
structure of 1.6,
5.1 and 8.4 mg/mL GLP-1 (pH 4). Near-UV CD (FIG. 7A) and fluorescence emission
of GLP-1
(FIG. 7B) both show that the tertiary structure of the peptide does not
notably change due to
multiple freeze-thaw cycles. Samples were frozen at -20 C and were defrosted
at room
temperature. Fluorescence emission spectra were collected after tryptophan
excitation at 280
nm. Similar experiments showing the effect of 11 freeze-thaw cycles on the
secondary structure
of 10 mg/mL GLP-1 (pH 4) by far-UV CD were conducted (FIG. 7C).
[0044] FIGs. 8A-8B. Salt Studies. Loading curves for GLP-1/FDKP as a function
of pH
and NaC1 concentration (FIG. 8A). Loading was performed at 5 mg/mL FDKP and
0.25 mg/mL
GLP-1. NaC1 concentrations are expressed as mM. FIG. 8B - Depicts the amount
of GLP-1
detected in the reconstituted FDKP-free control samples as a function of pH
and NaC1
concentration.
[0045] FIGs. 9A-9B. Surfactant Studies. Loading curves for GLP-1/FDKP as a
function
of pH and surfactant (FIG. 9A). Loading was performed at 5 mg/mL FDKP and 0.25
mg/mL
GLP-1. FIG. 9B - Depicts the amount of GLP-1 detected in the reconstituted
FDKP-free
control samples as a function of pH and surfactant added.
[0046] FIGs. 10A-10D. Ion Studies. Loading curves for GLP-1/FDKP as a function
of
pH and ions. Loading was performed at 5 mg/mL FDKP and 0.25 mg/mL GLP-1 (FIGs.
10A
and 11C). Ion concentrations are indicated in the legend (mM)JRight-hand
curves depicts the
results for 1M NaC1. FIGs. lOB and 10D - Depict the amount of GLP-1 detected
in the
reconstituted FDKP-free control samples as a function of pH, ions and 1 M
NaC1.
[0047] FIGs. 11-11B. Osmolyte Studies. Loading curves for GLP-1/FDKP as a
function
of pH and in the presence of common stabilizers (osmolytes; FIG. 11A). Loading
was
performed at 5 mg/mL FDKP and 0.25 mg/mL GLP-1. FIG. 11B - Depicts the amount
of GLP-
1 detected in the reconstituted FDKP-free control samples as a function of pH
and osmolyte.
"N/A" indicates no osmolyte was present in the sample.
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[0048] FIGs. 12A-12B. Chaotrope/lyotrope Studies. Loading curves for GLP-
1/FDKP as a
function of chaotrope or lyotrope concentration at pH 3.0 (FIG. 12A) and pH
4.0 (FIG. 12C).
Loading was performed at 5 mg/mL FDKP and 0.25 mg/mL GLP-1. FIGs. 12B and 12D -
Depict the amount of GLP-1 detected in the reconstituted FDKP-free control
samples as a
function of pH in the presence of the various chaotropes or lyotropes. "N/A"
indicates no
chaotropes or lyotropes were present in the sample.
[0049] FIGs. 13A-13B. Alcohol Studies. Loading curves for GLP-1/FDKP as a
function
of pH and alcohols. Loading was performed at 5 mg/mL FDKP and 0.2 5mg/mL GLP-
1. Four
alcohol concentrations were evaluated for each alcohol 5%, 10%, 15%, and 20%
v/v (FIG.
13A). TFE=trifluoroethanol; HFIP=hexafluoroisopropanol. FIG. 13B - Depicts the
amount of
GLP-1 detected for reconstituted FDKP-free control samples as a function of pH
and alcohol
(20%).
[0050] FIGs. 14A-14B. Loading from GLP-1/FDKP concentration studies (FIG.
14A).
Loading was performed at 5 mg/mL FDKP and the GLP-1 concentration analyzed is
listed in the
X-axis. FIG. 14B - Scanning Electron Microscopy (SEM) images of multiple GLP-
1/FDKP
formulations (at 10000x magnification) depicts clusters of spherical and rod-
like GLP-1/FDKP
particle formulations. (Panel A) 0.5 mg/mL GLP-1 and 2.5 mg/mL FDKP; (Panel B)
0.5
mg/mL GLP-1 and 10 mg/mL FDKP; (Panel C) 0.5 mg/mL GLP-1 and 10 mg/mL FDKP in
20
mM sodium chloride, 20 mM potassium acetate and 20 mM potassium phosphate, pH
4.0; and
(Panel D) 10 mg/mL GLP-1 and 50 mg/mL FDKP in 20 mM sodium chloride, 20 mM
potassium
acetate and 20 mM potassium phosphate, pH 4Ø
[0051] FIG. 15. Depicts the effect of stress on multiple GLP-1/FDKP
formulations. The
legend indicates the mass-to-mass percentage of GLP-1 to FDKP particles and
the other
components that were present in solution, prior to lyophilization. The samples
were incubated
for 10 days at 40 C.
[0052] FIGs. 16A-16C. Structure of GLP-1. FIG. 16A - Depicts the glycine-
extended
form of GLP-1 (SEQ ID NO. 1) and the amidated form (SEQ ID NO. 2). FIG. 16B -
Inhibition
of DPPIV activity by aprotinin. FIG. 16C - Inhibition of DPPIV activity by
DPPIV inhibitor.
[0053] FIG. 17. Detection of GLP-1 after incubation in lung lavage fluid.
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[0054] FIGs. 18A-18B. Depicts the quantitation of GLP-1 in plasma. FIG. 18A
shows
quantitation in 1:2 dilution of plasma. FIG. 18B shows quantitation in 1:10
dilution of plasma.
[0055] FIGs. 19A-19B. Effect of GLP-1 and GLP-1 analogs on cell survival.
Effect of
GLP-1 on rat pancreatic epithelial (ARIP) cell death (FIG. 19A). Annexin V
staining depicting
inhibition of apoptosis in the presence of GLP-1 and staurosporine (Stau) as
single agents and in
combination (FIG. 19B). The concentration of GLP-1 is l5nM and the
concentration of
stauropsorine is 1 M
[0056] FIG. 20. Effect of the GLP-1 analog exendin-4 on cell viability. ARIP
cells were
treated with 0, 10, 20 and 40 nM exendin 4 for 16, 24 and 48 hours.
[0057] FIG. 21. The effect of the multiple GLP-1/FDKP formulations on
staurosporine-
induced cell death. ARIP cells pre-treated with GLP-1 samples were exposed to
5 M
staurosporine for 4 hours and were analyzed with Cell Titer-G1oTM to determine
cell viability.
Samples were stressed at 4 and 40 C for 4 weeks. Control samples, shown on
the right (Media,
GLP-1, STAU, GLP+STAU), illustrate the viability of cells in media (without
GLP-1 or
stauroporine), with GLP-1, with stauropsorine and with GLP-1 and staurosporine
(note: the
graph legend does not apply to the control samples). All of the results shown
are averages of
triplicate runs.
[0058] FIGs. 22A-22B. Pharmacokinetic studies depicting single intravenous
injection
(IV; FIG. 22A) and pulmonary insufffaltion (IS; FIG. 22B) in rats using
various concentrations
of GLP-1/FDKP formulations. The legends indicate the mass-to-mass percentage
of GLP-1 to
FDKP particles for the formulations analyzed.
[0059] FIGs. 23A-23B. Decrease in the cumulative food consumption in rats
dosed with
GLP-1/FDKP formulations at 2 hours (FIG. 23A) and 6 hours (FIG. 23B) post
dose.
[0060] FIG. 24. Pharmacodynamic study of GLP-1/FDKP administered via pulmonary
insufflation in male obese Zucker rats. The data depicts the glucose
measurements at 0, 15, 30,
45, 60 and 90 minutes for the control (air; group 1) and the GLP-1/FDKP
treated (group 2).
[0061] FIG. 25. Pharmacodynamic study of GLP-1/FDKP administered via pulmonary
insufflation in male obese Zucker rats. The data depicts the GLP-1
measurements at 0, 15, 30,
45, 60 and 90 minutes for the control (air; group 1) and the GLP-1/FDKP
treated (group 2).
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[0062] FIG. 26. Pharmacodynamic study of GLP-1/FDKP administered via pulmonary
insufflation in male obese Zucker rats. The data depicts the insulin
measurements at 0, 15, 30,
45, 60 and 90 minutes for the control (air; group 1) and the GLP-1/FDKP
treated (group 2).
[0063] FIG. 27. Pharmacokinetic study of GLP-1/FDKP with various GLP-1
concentrations administered via pulmonary insufflation in female rats. The
data depicts the
GLP-1 measurements at 0, 2, 5, 10, 20, 30, 40 and 60 minutes for the control
(air; group 1) and
GLP-1/FDKP treated groups 2, 3 and 4 administered 5%, 10% and 15% GLP-1
respectively.
[0064] FIG. 28. Pharmacokinetic study of GLP-1/FDKP with various GLP-1
concentrations administered via pulmonary insufflation in female rats. The
data depicts the
FDKP measurements at 0, 2, 5, 10, 20, 30, 40 and 60 minutes for the control
(air; group 1) and
GLP-1/FDKP treated groups 2, 3 and 4 administered 5%, 10% and 15% GLP-1
respectively.
[0065] FIG. 29. Pharmacodynamic study of GLP-1/FDKP in female rats
administered
GLP-1 /FDKP containing 15% GLP-1 (0.3 mg GLP-1) via a single daily pulmonary
insufflation
(n=10) for 4 consecutive days. The data depicts average food consumption
measured at predose,
1, 2, 4 and 6 hours post dose for 4 consecutive days.
[0066] FIG. 30. Pharmacodynamic study of GLP-1/FDKP in female rats
administered
GLP-1 /FDKP containing 15% GLP-1 (0.3 mg GLP-1) via a single daily pulmonary
insufflation
(n=10) for 4 consecutive days. The data depicts average body weight measured
at predose, 1, 2,
4 and 6 hours post dose for 4 consecutive days.
[0067] FIG. 31. Toxicokinetic study of GLP-1/FDKP in monkeys administered GLP-
1/FDKP via oronasal administration once daily (for 30 minutes a day) for 5
consecutive days.
The data depicts the peak plasma concentrations (Cm,,x) of GLP-1 in males and
females. Animals
received control (air; group 1), 2 mg/kg FDKP (group 2) or 0.3, 1.0, or 2.0
mg/kg GLP-1/FDKP
(groups 3, 4, and 5 respectively).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0068] Stable, inhalable glucagon-like peptide 1(GLP-1) formulations for use
as
pharmaceutics are deficient in the art. This is due to the instability of GLP-
1 peptide in vivo.
GLP-1 compounds tend to remain in solution under a number of conditions, and
have a relatively
short in vivo half-life when administered as a solution formulation. Further,
dipeptidyl-peptidase
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IV (DPP-IV,) which is found to be present in various biological fluids such as
the lung and
blood, greatly reduces the biological half-life of GLP-1 molecules. For
example, the biological
half-life of GLP-1(7-37) has been shown to be 3 to 5 minutes; see U.S. Patent
No. 5,118,666.
GLP-1 has also been shown to undergo rapid absorption in vivo following
parenteral
administration. Similarly, amide GLP-1(7-36) has a half-life of about 50
minutes when
administered subcutaneously; see also U. S. Patent No. 5,118,666.
[0069] The rapid clearance and short half-life of GLP-1 compositions in the
art present a
deficiency that the current invention overcomes. The present invention
overcomes the
deficiencies in the art by providing an optimized native GLP-1/FDKP (fumaryl
diketopiperazine)
formulation especially suited for pulmonary delivery. In other particular
aspects, the present
invention provides formulations of a native GLP-1 molecule that can elicit a
GLP-1 response in
vivo. Use of variants of native GLP-1 in such formulations is also
contemplated.
[0070] To overcome the deficiencies in the art, the present invention provides
formulations
of GLP-1 in combination with diketopiperazine (DKP) particles. In particular
embodiments of
the invention, the GLP-1/DKP formulations are provided for administration to a
subject. In
further particular embodiments, the GLP-1/DKP formulations comprise fumaryl
diketopiperazine (FDKP), but are not limited to such, and can include other
DKPs (asymmetrical
DKPs, xDKPs) such as 2,5-diketo-3,6-di(4-succinyl-aminobutyl)piperazine
(SDKP),
asymmetrical diketopiperazines including ones substituted at only one position
on the DKP ring
(for example "one armed" analogs of FDKP), and DKP salts. In other particular
embodiments of
the invention, administration of the GLP-1/FDKP formulation is by pulmonary
delivery.
[0071] In developing therapeutic formulations of GLP-1 molecules the
structural
characteristics of GLP-1 in solution were evaluated by employing various
biophysical and
analytical techniques which included far-ultraviolet circular dichroism (far-
UV CD), near-
ultraviolet circular dichroism (near-UV CD), intrinsic fluorescence, fourier
transform infrared
spectroscopy (FTIR), high pressure liquid chromatography (HPLC), and mass
spectroscopy
(MS). The technique of circular dichroism (CD) is a powerful tool used to
analyze the structural
changes of a protein under varying experimental conditions and is well known
in the art. The
experimental conditions under which these analyses were conducted included:
the effects of
concentration, ionic strength, temperature, pH, oxidative stress, agitation,
and multiple freeze-
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thaw cycles on the GLP-1 peptide. These analyses were designed to characterize
the major
routes of degradation as well as to establish conditions that manipulate the
structure of GLP-1
peptide in order to achieve preferred GLP-1/ DKP formulations having desirable
pharmacokinetic (PK) and pharmacodynamic (PD) characteristics.
[0072] It was observed that as the concentration of GLP-1 increased, the
secondary
structure of the peptide was transformed from a predominantly unstructured
conformation to a
more helical conformation. Increasing the ionic strength in solution caused
the structure of
GLP-1 to increase until it reversibly precipitated. The presence of NaC1
increased the tertiary
structure of GLP-1 as is evident by an increase in intensity of the nearCD
bands as depicted in
FIG. 2D. This occurs even for the low concentrations of the peptide where
there is no evidence
of self-association. Increased ionic strength readily converted unstructured
GLP-1 into the a-
helical form as depicted by the farCD minima shifts toward 208nm and 222nm,
(FIG. 2A) and
self-associated conformations as depicted by the tryptophan emission shifts to
lower wavelength
with increased salt and the nearCD patterns in FIGs. 2B and 2D. Temperature
and pH affected
the conformations of GLP-1 differently in that the unordered structure of GLP-
1 was not altered
by either of these parameters. On the other hand, the self-associated
conformation of GLP-1 was
found to be sensitive to thermal denaturation and its solubility sensitive to
pH as depicted in FIG.
4A and 4B which shows GLP-1 peptide reversibly precipitates between pH 6.3-7.6
at a peptide
concentration of l0mg/ml. The various conformations of GLP-1 were found to be
generally
stable to agitation and multiple freeze-thaw cycles. Neither deamidation nor
oxidation was
observed for GLP-1.
[0073] Adsorption of GLP-1 to FDKP particles was also observed under a variety
of
conditions which included variation in pH, GLP-1 concentration, and in the
concentration of
various surfactants, salts, ions, chaotropes and lyotropes, stabilizers, and
alcohols. The
absorption of GLP-1 to FDKP particles was found to be affected strongly by pH,
specifically,
binding occurred at about pH 4.0 or greater. Other excipients were found to
have a limited effect
on the absorption of GLP-1 to FDKP particles.
[0074] In developing the GLP-1/DKP formulations of the present invention, a
number of
parameters that would affect or impact its deliverability and absorption in
vivo were evaluated.
Such parameters included, for example, the structure of the GLP-1 peptide, the
surface charges
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on the molecule under certain formulation conditions, solubility and stability
as a formulation, as
well as susceptibility to serine protease degradation and in vivo stability;
all of which play a
critical role in generating a formulation that can be readily absorbed which
exhibits an extended
biological half-life.
[0075] The stability of GLP-1/FDKP formulations obtained was tested under a
variety of
conditions both in vitro and in vivo. The stability of GLP-1 was analyzed by
HPLC analysis and
cell-based assays. In addition, stability of GLP-1 was examined in lung lavage
fluid (which
contains DPP-IV). It was also found that the stability of native GLP-1 was
concentration
dependent in solution.
[0076] In vitro GLP-1 biological activity studies were also employed for
studies of GLP-
1/FDKP loading, and determining the effect in vivo. This strategy contributed
to further
identification of lead GLP-1/FDKP formulation methods. Further, based on the
fact that GLP-1
has been shown to play a role in increasing (3-cells mass by inhibiting
apoptosis, stimulating (3-
cell proliferation and islet neogenesis, the proliferative and anti-apoptotic
potential of the GLP-
1/FDKP formulations of the invention were examined through a cell-based assay.
[0077] Thus, the present invention provides optimized formulations comprising
native
human GLP-1 combined with fumaryl diketopiperazine (FDKP) that are stable and
resistant to
degradation.
[0078] II. GLP-1 Molecules
[0079] In particular embodiments of the present invention there are provided
optimized
formulations comprising native human glucagon-like peptide 1(GLP-1) combined
with a
diketopiperazine such as fumaryl diketopiperazine (FDKP). Such GLP-1/FDKP
formulations of
the present invention are stable and resistant to degradation.
[0080] Human GLP-1 is well known in the art and originates from the
preproglucagon
polypeptide synthesized in the L-cells in the distal ileum, in the pancreas
and in the brain. GLP-
1 is a 30-31 amino acid peptide that exists in two molecular forms, 7-36 and 7-
37, with the 7-36
form being dominant. Processing of preproglucagon to GLP-1(7-36) amide and GLP-
1(7-37)
extended form occurs mainly in the L-cells. It has been shown in the art that,
in the fasted state,
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plasma levels of GLP-1 are about 40 pg/ml. After a meal, GLP-1 plasma levels
rapidly increase
to about 50-165 pg/ml.
[0081] The term "GLP-l molecules" as used herein refers to GLP-1 proteins,
peptides,
polypeptides, analogs, mimetics, derivatives, isoforms, fragments and the
like. Such GLP-1
molecules may include naturally occurring GLP-1 polypeptides (GLP-1(7-37)OH,
GLP-1(7-
36)NH2) and GLP-1 metabolites such as GLP-1(9-37). Thus, in particular
embodiments of the
invention, GLP-1 molecules include: a native GLP-1, a GLP-1 analog, a GLP-1
derivative, a
dipeptidyl-peptidase-IV (DPP-IV) protected GLP-1, a GLP-1 mimetic, a GLP-1
peptide analog,
or a biosynthetic GLP-1 analog.
[0082] As used herein, an "analog" includes compounds having structural
similarity to
another compound. For example, the anti-viral compound acyclovir is a
nucleoside analogue
and is structurally similar to the nucleoside guanosine which is derived from
the base guanine.
Thus, acyclovir mimics guanosine (is biologically analogous with) and
interferes with DNA
synthesis by replacing (or competing with) guanosine residues in the viral
nucleic acid and
prevents translation/transcription. Thus, compounds having structural
similarity to another (a
parent compound) that mimic the biological or chemical activity of the parent
compound are
analogs. There are no minimum or maximum numbers of elemental or functional
group
substitutions required to qualify a compound as an analog provided the analog
is capable of
mimicking, in some relevant fashion, either identically, complementarily or
competitively, with
the biological or chemical properties of the parent compound. Analogs can be,
and often are,
derivatives of the parent compound (see "derivative" infra). Analogs of the
compounds
disclosed herein may have equal, lesser or greater activity than their parent
compounds.
[0083] As used herein, a "derivative" is a compound made from (or derived
from), either
naturally or synthetically, a parent compound. A derivative may be an analog
(see "analog"
supra) and thus may possess similar chemical or biological activity. However,
unlike an analog,
a derivative does not necessarily have to mimic the biological or chemical
activity of the parent
compound. There are no minimum or maximum numbers of elemental or functional
group
substitutions required to qualify a compound as a derivative. For example,
while the antiviral
compound ganclovir is a derivative of acyclovir, ganclovir has a different
spectrum of anti-viral
activity and different toxicological properties than acyclovir. Derivatives of
the compounds
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disclosed herein may have equal, less, greater or even no similar activity
when compared to their
parent compounds.
[0084] As used herein, a "metabolite" is any intermediate or product of
metabolism and
includes both large and small molecules. As used herein and where appropriate,
the
definition applies to both primary and secondary metabolites. A primary
metabolite is directly
involved in normal growth, development, and reproduction of living organisms.
A secondary
metabolite is not directly involved in those processes, but typically has
important ecological
function (e.g., an antibiotic).
[0085] As used herein, the term "biosynthetic" refers to any production of a
chemical
compound by a living organism.
[0086] As used herein, "particle-forming" refers to chemical, biosynthetic, or
biological entities or compounds that are capable of forming solid particles,
usually in a liquid
medium. The formation of particles typically occurs when a particle-forming
entity is exposed
to a certain condition(s) such as, for example, changes in pH, temperature,
moisture, and/or
osmolarity/osmolality. Exposure to the condition(s) may result in, for
example, binding,
coalescence, solidification and/or dehydration such that a particle is formed.
A precipitation
reaction is one example of a particle-forming event.
[0087] As used herein, "co-solution" is any medium comprised of at least two
chemical,
biological and/or biosynthetic entities. For example, a co-solution may be
formed by combining
a liquid comprising at least one chemical, biological and/or biosynthetic
entity with a solid
comprising a chemical, biological and/or biosynthetic entity. In another
example, a co-solution
may be formed by combining a liquid comprising at least one chemical,
biological and/or
biosynthetic entity with another liquid comprising a chemical, biological
and/or biosynthetic
entity. In a further example, a co-solution may be formed by adding at least
two solids, each
comprising at least one chemical, biological and/or biosynthetic entity, into
a single solution.
[0088] Native GLP-l, as contemplated in the present invention, is a
polypeptide having the
amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2. Native GLP-1 peptide
undergoes rapid
cleavage and inactivation within minutes in vivo.
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[0089] GLP-1 analogs of the present invention may include the exendins, which
are
peptides found to be GLP-1 receptor agonists; such analogs may further include
exendins 1 to 4.
Exendins are found in the venom of the Gila-monster and share about 53% amino
acid homology
with mammalian GLP-1. Exendins also have similar binding affinity for the GLP-
1 receptor.
Exendin-3 and exendin-4 were reported to stimulate cAMP production in, and
amylase release
from, pancreatic acinar cells (Malhotra et al., 1992; Raufman et al., 1992;
Singh et al., 1994).
The use of exendin-3 and exendin-4 as insulinotrophic agents for the treatment
of diabetes
mellitus and the prevention of hyperglycemia has been proposed (U.S. Patent
No. 5,424,286).
[0090] Carboxyl terminal fragments of exendin such as exendin[9-39], a
carboxyamidated
molecule, and fragments 3-39 through 9-39 have been reported to be potent and
selective
antagonists of GLP-1 (Goke et al., 1993; Raufman et al., 1991; Schepp et al.,
1994; Montrose-
Rafizadeh et al., 1996). The literature has also demonstrated that exendin[9-
39] blocks
endogenous GLP-1 in vivo, resulting in reduced insulin secretion (Wang et al.,
1995; D'Alessio
et al., 1996). Exendin-4 potently binds to GLP-1 receptors on insulin-
secreting (3-TCl cells, to
dispersed acinar cells from pancreas, and to parietal cells from stomach.
Exendin-4 peptide also
plays a role in stimulating somatostatin release and inhibiting gastrin
release in isolated stomachs
(Goke et al., 1993; Schepp et al., 1994; Eissele et al., 1994). In cells
transfected with the cloned
GLP-1 receptor, exendin-4 is reportedly an agonist, i.e., it increases cAMP,
while exendin[9-39]
is identified as an antagonist, i.e., it blocks the stimulatory actions of
exendin-4 and GLP-l.
exendin has also been found to be resistant to degradation.
[0091] Another embodiment the present invention contemplates the use of
peptide
mimetics. Peptide mimetics, as are know to the skilled artisan, are peptides
that biologically
mimic active determinants on hormones, cytokines, enzyme substrates, viruses
or other bio-
molecules, and may antagonize, stimulate, or otherwise modulate the
physiological activity of
the natural ligands. Peptide mimetics are especially useful in drug
development. See, for
example, Johnson et al., "Peptide Turn Mimetics" in BIOTECHNOLOGY AND
PHARMACY,
Pezzuto et al., Eds., Chapman and Hall, New York (1993). The underlying
rationale behind the
use of peptide mimetics is that the peptide backbone of proteins exists
chiefly to orient amino
acid side chains in such a way as to facilitate molecular interactions. A
peptide mimetic is
expected to permit molecular interactions similar to the natural molecule.
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[0092] In further embodiments it is contemplated that the GLP-1 molecules of
the
invention will have at least one biological activity of native GLP-1 such as
the ability to bind to
the GLP-1 receptor and initiate a signal transduction pathway resulting in
insulinotropic activity.
In further embodiments of the invention, a GLP-1 molecule may be a peptide,
polypeptide,
protein, analog, mimetic, derivative, isoform, fragment and the like, that
retains at least one
biological activity of a naturally-occurring GLP-l. GLP-1 molecules may also
include the
pharmaceutically acceptable salts and prodrugs, and salts of the prodrugs,
polymorphs, hydrates,
solvates, biologically-active fragments, biologically active variants and
stereoisomers of the
naturally-occurring human GLP-1 as well as agonist, mimetic, and antagonist
variants of the
naturally-occurring human GLP-l, the family of exendins including exendins 1
through 4, and
polypeptide fusions thereof. A GLP-1 molecule of the invention may also
include a dipeptidyl-
peptidase-IV (DPP-IV) protected GLP-1 that prevents or inhibits the
degradation of GLP-l.
[0093] GLP-1 molecules of the present invention include peptides,
polypeptides, proteins
and derivatives thereof that contain amino acid substitutions, improve
solubility, confer
resistance to oxidation, increase biological potency, or increase half-life in
circulation. Thus,
GLP-1 molecules as contemplated in the present invention comprise amino acid
substitutions,
deletions or additions wherein the amino acid is selected from those as are
well known in the art.
The N- or C- termini of the molecule may also be modified such as by
acylation, acetylation,
amidation, but is not limited to such. Thus, in the present invention, the
term "amino acid" refers
to naturally occurring and non-naturally occurring amino acids, as well as
amino acid analogs
and amino acid mimetics that function in a manner similar to naturally
occurring amino acids.
Naturally encoded amino acids are the 20 common amino acids (alanine,
arginine, asparagine,
aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,
isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
and valine) and
pyrolysine and selenocysteine. Amino acid analog refers to compounds that have
the same basic
chemical structure as a naturally occurring amino acid, i.e., an a carbon that
is bound to a
hydrogen, a carboxyl group, an amino group, and an R group, such as,
homoserine, norleucine,
norvaline, methionine sulfoxide, methionine methyl sulfonium, citrulline,
hydroxyl glutamic
acid, hydroxyproline, and praline. Such analogs have modified R groups (such
as norleucine),
but retain the same basic chemical structure as a naturally occurring amino
acid. Amino acids
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contemplated in the present invention also include (3-amino acids which are
similar to a-amino
acids in that they contain an amino terminus and a carboxyl terminus. However,
in (3-amino
acids two carbon atoms separate these functional termini. (3-amino acids, with
a specific side
chain, can exist as the R or S isomers at either the alpha (C2) carbon or the
beta (C3) carbon.
This results in a total of four possible diastereoisomers for any given side
chain.
[0094] GLP-1 molecules of the present invention may also include hybrid GLP-1
proteins,
fusion proteins, oligomers and multimers, homologues, glycosylation pattern
variants, and
muteins thereof, wherein the GL-P-1 molecule retains at least one biological
activity of the
native molecule, and further regardless of the method of synthesis or
manufacture thereof
including, but not limited to, recombinant (whether produced from cDNA,
genomic DNA,
synthetic DNA or other form of nucleic acid), synthetic, and gene activation
methods.
Recombinant DNA technology is well known to those of ordinary skill in the art
(see Russell,
D.W., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y., 2001.
[0095] III. Diketopiperazines
[0096] Diketopiperazines, are well known in the art for their ability to form
microparticles
that are useful for drug delivery and stabilization. In the present invention
diketopiperazines are
employed to facilitate the absorption of GLP-1 molecules thereby providing a
stable formulation
that is resistant to degradation.
[0097] Various methodologies may be employed wherein diketopiperazines can be
formed
into particles that incorporate GLP-1 molecules, or particles onto which GLP-1
molecules can be
adsorbed. This may involve mixing of the diketopiperazine solutions with
solutions or
suspensions of GLP-1 molecules followed by precipitation and subsequent
formation of particles
comprising diketopiperazine and GLP-l. Alternatively, the diketopiperazine can
be precipitated
to form particles and subsequently mixed with a solution of GLP-1 molecules.
Association
between the diketopiperazine particle and the GLP-1 molecule can be driven by
solvent removal
or a specific step, such as a pH adjustment, can be included prior to drying
in order to promote
the association.
[0098] In a preferred embodiment, diketopiperazines of the present invention
include but
are not limited 3,6-di(fumaryl-4 aminobutyl)-2,5-diketopiperazine also known
as (E)-3,6-bis[4-
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(N-carboxyl-2-propenyl)amidobutyl]-2,5-diketopiperazine (which may also be
referred to as
fumaryl diketopiperazine or FDKP).
[0099] Other diketopiperazines contemplated in the present invention include,
without
limitation, derivatives of 3,6-di(4-aminobutyl)-2,5-diketopiperazine such as:
3,6-di(succinyl-4-
aminobutyl)-2,5-diketopiperazine (also referred to herein as 3,6-bis(4-
carboxypropyl)amidobutyl-2,5-diketopiperazine; succinyl diketopiperazine or
SDKP); 3,6-
di(maleyl-4-aminobutyl)-2,5-diketopiperazine; 3,6-di(citraconyl-4-aminobutyl)-
2-5-
diketopiperazine; 3,6-di(glutaryl-4-aminobutyl)-2,5-diketopiperazine; 3,6-
di(malonyl-4-
aminobutyl)-2,5-diketopiperazine; 3,6-di(oxalyl-4-aminobutyl)-2,5-
diketopiperazine and
derivatives therefrom. In other embodiments, the present invention
contemplates the use of
diketopiperazine salts. Such salts may include, for example, any
pharmaceutically acceptable
salt such as the Na, K, Li, Mg, Ca, ammonium, or mono-, di- or tri-
alkylammonium (as derived
from triethylamine, butylamine, diethanolamine, triethanolamine, or pyridines,
and the like) salts
of diketopiperazine. The salt may be a mono-, di-, or mixed salt. Higher order
salts are also
contemplated for diketopiperazines in which the R groups contain more than one
acid group. In
other aspects of the invention, a basic form of the agent may be mixed with
the diketopiperazine
in order to form a drug salt of the diketopiperazine, such that the drug is
the counter cation of the
diketopiperazine. An example of a salt as contemplated herein, includes in a
non-limiting manner
FDKP diNa. Drug delivery using DKP salts is taught in U.S. Patent Application
No:
11/210,710, incorporated herein by reference for all it contains regarding DKP
salts.
[00100] As disclosed elsewhere herein, the present invention also employs
novel
asymmetrical analogs of FDKP, xDKPs such as: (E)-3-(4-(3,6-dioxopiperazin-2-
yl)butylcarbamoyl)-acrylic acid; (E)-3-(3-(3,6-dioxopiperazin-2-yl)propyl-
carbamoyl)acrylic
acid; and (E)-3-(4-(5-isopropyl-3,6-dioxopiperazin-2-yl)-
butylcarbamoyl)acrylic acid and
disclosed in U.S. Provisional Patent Application entitled "Asymmetrical FDKP
Analogs for Use
as Drug Delivery Agents" filed on even date herewith and incorporated herein
in its entirety
(Atty Docket No. 51300-00041)
[00101] Diketopiperazines can be formed by cyclodimerization of amino acid
ester
derivatives, as described by Katchalski, et al., (J. Amer. Chem. Soc. 68:879-
80; 1946), by
cyclization of dipeptide ester derivatives, or by thermal dehydration of amino
acid derivatives in
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high-boiling solvents, as described by Kopple, et al., (J. Org. Chem. 33:862-
64;1968), the
teachings of which are incorporated herein.
[00102] Methods for synthesis and preparation of diketopiperazines are well
known to one
of ordinary skill in the art and are disclosed in U.S. Patents 5,352,461;
5,503,852; 6,071,497;
6,331,318; 6,428,771 and U.S. Patent Application No. 20060040953. United
States Patent No.
6,444,226 and 6,652,885, describe preparing and providing microparticles of
diketopiperazines
in aqueous suspension to which a solution of active agent is added in order to
bind the active
agent to the particle. These patents further describes a method of removing a
liquid medium by
lyophilization to yield microparticles comprising an active agent, altering
the solvent conditions
of such suspension to promote binding of the active agent to the particle is
taught in U.S. Patent
Application Serial No: 60/717,524 and 11/532,063 both entitled "Method of Drug
Formulation
Based on Increasing the Affinity of Active Agents for Crystalline
Microparticle Surfaces"; and
11/532,065 entitled "Method of Drug Formulation Based on Increasing the
Affinity of Active
Agents for Crystalline Microparticle Surfaces." See also United States Patent
No. 6,440,463 and
U.S. Patent Application Serial No: 11/210,709 filed on August 23, 2005 and
U.S. Patent
Application No. 11/208,087). In some instances, it is contemplated that the
loaded
diketopiperazine particles of the present invention are dried by a method of
spraying drying as
disclosed in, for example, U.S. Patent Application Serial No. 11/678,046 filed
on February 22,
2006 and entitled "A Method For Improving the Pharmaceutic Properties of
Microparticles
Comprising Diketopiperazine and an Active Agent." Each of these patents and
patent
applications is incorporated by reference herein for all they contain
regarding diketopiperazines.
[00103] IV. Therapeutic Formulations of GLP-1/DKP Particles
[00104] The present invention further provides a GLP-1/FDKP formulation for
administration to a subject in need of treatment. A subject as contemplated in
the present
invention may be a household pet or human. In certain embodiments, the
treatment is for Type
II diabetes, obesity, cancer or any related diseases and/or conditions
therefrom. Humans are
particularly preferred subjects.
[00105] Other diseases or conditions contemplated in the present invention
include, but are
not limited to, irritable bowel syndrome, myocardial infarction, ischemia,
reperfused tissue
injury, dyslipidemia, diabetic cardiomyopathy, acute coronary syndrome,
metabolic syndrome,
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catabolic changes after surgery, neurodegenerative disorders, memory and
learning disorders,
islet cell transplant and regenerative therapy or stroke. Other diseases
and/or conditions
contemplated in the present invention are inclusive of any disease and/or
condition related to
those listed above that may be treated by administering a GLP-1/FDKP dry
powder formulation
to a subject in need thereof. The GLP-1/FDKP dry powder formulation of the
present invention
may also be employed in the treatment of induction of beta cell
differentiation in human cells of
type-II diabetes and hyperglycemia.
[00106] In still a further embodiment of the present invention, it is
contemplated that the
subject may be a household pet or animal, including rats, rabbits, hamsters,
guinea pigs, gerbils,
woodchucks, cats, dogs, sheep, goats, pigs, cows, horses, monkeys and apes
(including
chimpanzees, gibbons, and baboons).
[00107] It is further contemplated that the GLP-1/FDKP particle formulations
of the
invention can be administered by various routes of administration known to
persons of ordinary
skill in the art and for clinical or non-clinical purposes. The GLP-1/FDKP
compositions of the
invention may be administered to any targeted biological membrane, preferably
a mucosal
membrane of a subject. Administration can be by any route, including but not
limited to oral,
nasal, buccal, systemic intravenous injection, subcutaneous, regional
administration via blood or
lymph supply, directly to an affected site or even by topical means. In
preferred embodiments of
the present invention, administration of GLP-1/FDKP composition is by
pulmonary delivery.
[00108] Other alternative routes of administration that may be employed in the
present
invention may include: intradermal, intraarterial, intraperitoneal,
intralesional, intracranial,
intraarticular, intraprostatic, intrapleural, intratracheal, intravitreal,
intravaginal, rectal,
intratumoral, intramuscular, intravesicular, mucosally, intrapericardial,
bronchial administration
local, using aerosol, injection, infusion, continuous infusion, localized
perfusion bathing target
cells directly, via a catheter, via a lavage, in cremes, in lipid compositions
(e.g., liposomes), or
by other method or any combination of the foregoing as would be known to one
of ordinary skill
in the art (see, for example, Remington's Pharmaceutical Sciences, 1990,
incorporated herein by
reference for all it contains regarding methods of administration).
[00109] As a dry powder formulation, the GLP-1/DKP particles of the present
invention can
be delivered by inhalation to specific areas of the respiratory system,
depending on the particle
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size. Additionally, the GLP-1/DKP particles can be made small enough for
incorporation into an
intravenous suspension dosage form. For oral delivery, the particles can be
incorporated into a
suspension, tablets or capsules. The GLP-1/DKP composition may be delivered
from an
inhalation device, such as a nebulizer, a metered-dose inhaler, a dry powder
inhaler, and a
sprayer.
[00110] In further embodiments, administration of an "effective amount" of a
GLP-1/DKP
formulation to a patient in need thereof is contemplated. An "effective
amount" of a GLP-
1/DKP dry powder formulation as contemplated in the present invention refers
to that amount of
the GLP-1 compound, analog or peptide mimetic or the like, which will relieve
to some extent
one or more of the symptoms of the disease, condition or disorder being
treated. In one
embodiment, an "effective amount" of a GLP-1/DKP dry powder formulation would
be that
amount of the GLP-1 molecule for treating diabetes by increasing plasma
insulin levels, reducing
or lowering fasting blood glucose levels, and increasing pancreatic beta cell
mass by at least
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50%, or
greater, but not limited to
such. In another preferred embodiment the present invention contemplates
treating obesity by
administering to a subject in need of such treatment a pharmaceutically
effective amount of the
GLP-1 molecule. In such instances an "effective amount" of a GLP-1/DKP dry
powder
formulation would be that amount of the GLP-1 molecule for treating obesity by
reducing or
lowering body weight by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50%,
or greater, but is
not limited to such. The present invention also contemplates administering an
"effective
amount" of a GLP-1/DKP dry powder formulation for controlling satiety, by
administering to a
subject in need of such treatment a pharmaceutically effective amount of the
GLP-1 molecule.
In a non-limiting manner, the GLP-1 molecule can be an exendin molecule such
as exendin-1 or
-4. In such instances, an "effective amount" of a GLP-1/DKP dry powder
formulation would be
that amount of the GLP-1 molecule that reduces the perception of hunger and
food intake (as
measured by mass or caloric content, for example) by at least about 5, 6, 7,
8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,
45, 50%, or greater, but
not limited to such. An "effective amount" of a GLP-1/DKP dry powder
formulation may be
further defined as that amount sufficient to detectably and repeatedly
ameliorate, reduce,
minimize or limit the extent of the disease or condition or symptoms thereof.
Elimination,
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eradication or cure of the disease or condition may also be possible utilizing
an "effective
amount" of the inventive formulation..
[00111] In administering a GLP-1/FDKP composition of the present invention to
a subject in
need thereof, the actual dosage amount of the composition can be determined on
the basis of
physical and physiological factors such as body weight, severity of condition,
the type of disease
being treated, previous or concurrent therapeutic interventions, idiopathy of
the patient and the
route of administration. A skilled artisan would be able to determine actual
dosages based on
one or more of these factors.
[00112] The GLP-1/DKP formulation of the present invention can be administered
once or
more than once, depending the disease or condition to be treated.
Administration of the GLP-
1/DKP formulation can be provided to the subject at intervals ranging over
minutes, hours, days,
weeks or months. In some instances, timing of the therapeutic regimen may be
related to the
half-life of the GLP-1 molecule upon administration. In further embodiments,
in treating
particular or complex diseases or conditions such as cancer, for example, it
may be desirable to
administer a GLP-1/DKP formulation of the present invention with a
pharmaceutical excipient or
agent. In such cases, an administration regimen may be dictated by the
pharmaceutical excipient
or agent.
[00113] V. EXAMPLES
[00114] The following examples are included to demonstrate certain embodiments
of the
invention. It should be appreciated by those of skill in the art that the
techniques disclosed in the
examples, which elucidate representative techniques that function well in the
practice of the
present invention. However, those of skill in the art should, in light of the
present disclosure,
appreciate that many changes can be made in the specific embodiments that are
disclosed and
still obtain a like or similar result without departing from the spirit and
scope of the invention
Example 1
Biophysical and Analytical Analyses of the Structure of GLP-1
[00115] To analyze both the structure and behavior of GLP-1 a number of
biophysical and
analytical techniques were employed. These techniques included far-ultraviolet
circular
dichroism (far-UV CD), near-ultraviolet circular dichroism (near-UV CD),
intrinsic
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fluorescence, fourier transform infrared spectroscopy (FTIR), high pressure
liquid
chromatography (HPLC), and mass spectroscopy (MS); all of which are well know
to one of
ordinary skill in the art. A wide range of conditions were employed to
investigate the effects of
concentration, ionic strength, temperature, pH, oxidative stress, agitation,
and multiple freeze-
thaw cycles on the GLP-1 peptide; all of which are described in further detail
below. These
analyses were also employed to characterize the major routes of degradation
and to establish
conditions that manipulate peptide structure of GLP-1 in order to achieve
certain GLP-1/DKP
formulations.
[00116] Experimental Procedure
[00117] GLP-1 was purchased either from American Peptide (Sunnyvale, CA) or
AnaSpec
(San Jose, CA), or prepared in house (MannKind Corporation, Valencia, CA).
Aqueous GLP-1
samples, of varying concentration, were analyzed at pH 4.0 and 20 C (unless
otherwise noted).
Samples were generally prepared fresh and were mixed with the appropriate
additive (e.g., salt,
pH buffer, H202 etc., if any), prior to each experiment. Secondary structural
measurements of
GLP-1 under various conditions were collected with far-UV CD and transmission
fourier
transform infrared spectroscopy (FTIR). In addition, both near-UV CD and
intrinsic
fluorescence were employed to analyze the tertiary structure of GLP-1 by
monitoring the
environments surrounding its aromatic residues, namely tryptophan.
[00118] Concentration-dependent Structures of GLP-1
[00119] Circular dichroism (CD) spectra was used to analyze the a-helix,
random coil, (3-
pleated sheet, (3-turns and random coil that may be displayed by a molecule
such as a protein or
peptide. In particular, far-UV CD was used to determine the type of secondary
structure, for
example pure a-helix, (3-sheet, etc., in proteins and peptides. On the other
hand, near-UV CD
was used to analyze the tertiary structures of a molecule. Thus, in order to
examine the effect of
concentration on GLP-1 structures, far- and near- UV CD techniques were
employed.
[00120] The far-UV CD in FIG. lA demonstrated that GLP-1 forms two distinct
structures
which include a-helices and random coils, over a wide range of concentrations
(for example:
1.8, 4.2, 5.1, 6.1, 7.2 and 8.6 mg/mL). At low concentrations (< 2 mg/mL), GLP-
1 is primarily
unstructured, as determined by the large single minima at 205 nm. As the
concentration is
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increased, the peptide adopts an a-helical structure as determined by the two
minima at 208 nm
and 224 nm (FIG. lA).
[00121] Tertiary structural analysis suggests that the high concentration
structures of GLP-1
are self-associated conformations (i.e., oligomers). Both the near-UV CD and
fluorescence
emission data support this hypothesis. The positive bands between 250 - 300 nm
in the near-UV
CD (FIG. 1B) reveal that GLP-1 has a defined tertiary structure which
increases at higher
concentrations. More specifically, these bands indicate that the aromatic
residues of the peptide
are largely immobilized and exist in a well-defined environment.
[00122] Similarly, the fluorescence emission of GLP-1 at various
concentrations (pH 4.0,
20 C) showed that the aromatic residue tryptophan (which also displayed
intense bands in near-
UV CD spectra) exists in a well-defined tertiary structure; the data shown
resulted from
tryptophan excitation at 280 nm (FIG. 1C). The fluorescence maximum at 355 nm
for low
concentrations of GLP-1 indicated that the tryptophan is solvent exposed and
that there is no
significant tertiary structure. At high peptide concentrations, the maxima
decreased in intensity
and shifted to a lower wavelength, indicative of a more-defined tertiary
structure.
[00123] In order to further determine the underlying secondary structure of
GLP-1 self-
associated conformation, FTIR analysis was performed at various concentrations
(pH 4.0, 20 C).
The amide I band at 1656 crri i clearly indicates that GLP-1 has a a-helical
structure at
concentrations > 2 mg/mL (FIG. 1D). Therefore, GLP-1 does not form (3-sheet
structures;
instead it is more likely that the peptide generates a helix bundle at high
concentrations.
[00124] Additionally, it was experimentally shown that these various
structures of GLP-1
were not generated via sample handling. Dilutions from a concentrated stock
solution compared
to GLP-1 prepared by directly dissolving the peptide in buffer generated
similar far-UV CD,
near-UV CD, and fluorescence emission spectra.
[00125] The Effect of Ionic Strength on GLP-1
[00126] Studies were also conducted to determine the effect of ionic strength
on GLP-1
peptide. FIG. 2A (far-UV-CD) illustrates that increasing the concentration of
salt (from 100
mM to 1000 mM) converts the unordered structure of GLP-1 into a a-helical
conformation, as
revealed by the minimas at 208 and 224 nm. Upon raising the NaC1 concentration
to 1M, much
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of the peptide (at 1.0 mg/mL) precipitates out of solution (FIG. 2A).
Nevertheless, this type of
precipitate was shown to dissolve upon dilution with water, thus establishing
that at high ionic
strength GLP-1 can be reversibly precipitated.
[00127] Salt was also shown to generate and improve the tertiary structure of
GLP-1. This
is exemplified in FIG. 2B (near-UV-CD) where 1.0 mg/mL GLP-1 displays no
signal in the
absence of salt, but exhibits a clear tertiary structure that intensifies with
increasing ionic
strength. These results were confirmed with the fluorescence emission of 1.0
mg/mL GLP-1
(FIG. 2C) at varying NaC1 concentrations (pH 4.0, 20 C) following tryptophan
excitation at 280
nm. Increasing ionic strength caused the fluorescence maximum to shift to
lower wavelengths,
indicating that the tertiary structure of 1.0 mg/mL GLP-1 is both generated
and enhanced.
[00128] Additionally, tertiary structural analysis of 10 mg/mL GLP-1 at
varying ionic
strength (pH 4.0, 20 C) using near-UV CD spectra, demonstrated that the GLP-1
self-associated
conformation is also enhanced with increased ionic strength (FIG. 2D).
[00129] The data therefore suggests that ionic strength has a dramatic effect
on the structure
of GLP-l, causing the protein both to assume an a-helical conformation and
associate into
oligomers. Further, increasing the ionic strength in solution causes the
oligomerization of GLP-
1 to increase until it reversibly precipitates. This occurrence is evident at
low concentrations of
the peptide, where there is initially no tertiary structure, as well with high
concentrations of the
peptide that already display substantial secondary and tertiary structure.
Thus, increased ionic
strength readily converts unstructured GLP-1 into the a-helical and self-
associated
conformations. Moreover, the observed spectroscopic results are comparable to
the affects of
increased peptide concentration shown previously.
[00130] The Effect of Temperature and pH on GLP-1
[00131] Studies were also conducted to determine whether the self-associated
conformation
of GLP-1 is sensitive to changes in either temperature or pH. FIG. 3A (near-UV-
CD)
demonstrates that the tertiary structure of 10 mg/mL GLP-1 significantly
dissociates as
temperature increases. On the other hand, temperature does not have an affect
on low
concentrations (0.05 mg/mL) of GLP-1 at various temperatures and pH 4.0; see
FIG. 3B and 3C
(far-UV-CD). The far-UV CD illustrates that the peptide is insensitive to
temperature.
Therefore, increased molecular motion significantly hinders self-association
of GLP-1.
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[00132] Conversely, FIG. 4A (far-UV-CD) demonstrates that the solubility of
the a-helical
GLP-1 conformation is pH sensitive. Although the structure of 10 mg/mL GLP-1
is relatively
uniform (i.e., GLP-1 remains helical) at pH 4.4 and below, some precipitation
occurs when the
pH is raised to near or at neutral (between pH 6.3 and 7.6) and an unordered
spectrum is
generated. Samples where precipitation occurred have less intensity as a
result of less soluble
GLP-1 being present in the solution. This unordered structure is determined by
the single
minima observed at 208 nm in FIG. 4A (far-UV-CD), which is further depicted in
FIG. 4B
(near-UV-CD) and likely results from a decrease of GLP-1 in solution following
precipitation.
This precipitation may occur when the pH is raised above the pI of 5.5 for GLP-
1. However, as
the pH was raised from near neutral to 11.7, most of the precipitate re-
dissolved, indicating the
precipitation is reversible. The remaining un-dissolved precipitate for GLP-1
at pH 11.7 would
cause the amount of peptide in solution to decrease and hence reduce the
intensity of the far-UV
CD spectrum, as observed in FIG. 4A. It was also observed that GLP-1 is
extremely insoluble
when lyophilized GLP-1 powder is mixed with pH 9 buffer to a high
concentration of GLP-l.
[00133] Stability of GLP-1
[00134] The stability of GLP-1 peptide was examined by determining its
resistance to
deamidation and oxidation in addition to the effects of agitation and freeze-
thaw cycles.
[00135] GLP-1 (1 mg/mL) at pH 10.5, was incubated for 5 days at 40 C following
which
reverse-phase HPLC and electrospray mass spectrometry (MS) were performed for
deamidation
and oxidation analyses. Oxidation studies were also conducted on GLP-1 samples
(1 mg/mL)
incubated for 2 hours in the presence of 0.1% H202 using both HPLC and MS.
[00136] FIG. 5 depicts the stability of GLP-1 under conditions of deamidation
and
oxidation. The HPLC chromatograms illustrate that GLP-1 elutes at the same
retention time and
that no degradation peaks result for the destabilizing conditions analyzed.
Additionally, MS
analyses yielded a similar mass for all the samples, 3297 g/mol, indicating
that the mass is
unaltered. The data also illustrates that the peptide remains pure and intact
when incubated
under various conditions. Thus, deamidation of GLP-1 was not observed. GLP-1
was also
shown to be stable to oxidative stress as observed in the presence of 0.1 %
H202, where the purity
and mass of GLP-1 remained intact, as determined by HPLC and MS respectively.
Overall,
there were no changes in the retention times or the mass values and no
degradation peaks
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resulted, thereby demonstrating that GLP-1 peptide is resistance to both
deamidation and
oxidation.
[00137] The effects of agitation and consecutive freeze-thaw cycles on various
concentrations of GLP-1 were analyzed with near-UV CD, and intrinsic
fluorescence. Agitation
of 9.4 and 1.5 mg/mL GLP-1 produced no significant alterations in the peptide
as observed by
near-UV CD (FIG. 6A), and fluorescence emission (FIG. 6B). The samples were
agitated for
30 and 90 min at room temperature and the fluorescence emission spectra were
collected after
tryptophan excitation at 280 nm. In independent freeze-thaw studies, solutions
containing GLP-
1(pH 4.0) at 1.6, 5.1 and 8.4 mg/mL were frozen at -20 C and thawed at room
temperature. The
effect of 10 freeze-thaw cycles on GLP-1 was conducted and analyzed by near-UV
CD (FIG.
7A) and fluorescence emission (FIG. 7B). Fluorescence emission spectra were
collected after
tryptophan excitation at 280 nm. Both analyses show that the tertiary
structure of the peptide
does not notably change due to multiple freeze-thaw cycles. In similar
experiments, the effect of
11 freeze-thaw cycles on the secondary structure of 10 mg/mL GLP-1 (pH 4.0)
was analyzed
(FIG. 7C). The far-UV CD illustrates that the secondary structure of the
peptide does not
change significantly as a result of multiple freeze-thaw cycles.
[00138] Overall, the biophysical analyses obtained from the above experiments
showed that
the structure of the GLP-1 peptide is strongly influenced by its concentration
in solution. As the
concentration of GLP-1 was increased, a-helical structures became more
prominent. In addition,
increasing the ionic strength enhanced, and in some cases generated, tertiary
GLP-1 structures.
Example 2
GLP-1/FDKP Adsorption Studies
[00139] The interaction of GLP-1 with diketopiperazine (DKP) particles in
suspension was
evaluated by conducting adsorption studies. The variables in adsorption
studies explored the
effects of electrostatics, hydrogen bonding, water structure, protein
flexibility, and specific salt-
pairing interactions on the GLP-1/DKP interaction. In addition, several common
protein
stabilizers were tested for interference with GLP-1 adsorption to DKP
surfaces.
[00140] Using pre-formed DKP suspension particles (i.e., FDKP), conditions
where GLP-1
adsorbs to the surfaces of preformed DKP particles were studied. A FDKP
particle suspension,
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in which the FDKP particles are pre-formed, was combined with 3X pH buffer and
3X solution
of an additive or excipient. The final solution contained a FDKP concentration
of 5,mg/ml and a
GLP-1 concentration of 0.25,mg/ml (5% w/w). Unbound GLP-1 in the supematant
was filtered
off the suspension. The FDKP particles with the associated GLP-1 protein were
dissolved
(reconstituted) with 100,mM ammonium bicarbonate and filtered to separate out
any aggregated
GLP-1 protein. The amount of GLP-1 in both the supematant and reconstituted
fractions was
quantitated by HPLC. A series of experiments were conducted in which
conditions employed
included use of additives such as salts, surfactants, ions, osmolytes,
chaotropes, organics, and
various concentrations of GLP-l. The results from these studies are described
below.
[00141] Salt studies. - The effect of salt on the binding of GLP-1 to FDKP
particles was
observed by HPLC analysis. Loading of the GLP-1/FDKP particles was performed
at 5 mg/mL
FDKP and 0.25 mg/mL GLP-1 in the presence of 0, 25, 50, 100, 250, 500, 1000
and 1500 mM
NaC1(FIG. 8A). The amount of GLP-1 detected in reconstituted FDKP-free control
samples as
a function of pH and NaC1 concentration was also assessed (FIG. 8B). The pH in
both data sets
was controlled with a 20 mM phosphate/20 mM acetate mixture.
[00142] As observed in FIG. 8A, the optimal binding (adsorption) of GLP-1 to
FDKP
particles was strongly influenced by the pH of the suspension. At a pH of 4
and above, about
3.2% to about 4% binding of GLP-1 to FDKP particles was observed where the GLP-
1/FDKP
ratio in solution was 5% w/w. Essentially no adsorption of GLP-1 to FDKP
particles was
evident at pH 2.0 in the presence of 0 and 25 mM NaC1, but some apparent
loading was observed
with increased ionic strength. GLP-1 precipitation was observed in the FDKP-
free controls with
_1M NaC1 FIG. 8B. This apparent loading at _1M NaC1 is the result of
reversible precipitation
(salting out) of the GLP-1 peptide at high ionic strength. High-salt controls
of GLP-1 free of
FDKP particles also exhibited high GLP-1 levels in the reconstituted samples,
indicating that
GLP-1 had been trapped in the filters when the supematant had been collected.
Below 1M NaC1,
there was no evidence of GLP-1 precipitation in the absence of FDKP particles.
[00143] Surfactant studies. - The effect of surfactants on the binding of GLP-
1 to FDKP
particles was observed by HPLC analysis. Loading was performed at 5 mg/ml FDKP
and
0.25 mg/mL GLP-1 in the presence of a surfactant (FIG. 9A). The amount of GLP-
1 detected in
reconstituted FDKP-free control samples as a function of pH and surfactant
concentration was
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also assessed (FIG. 9B). The pH and the control sample conditions were as
described for the
above ionic strength study. Surfactants employed in this study included: Brij
78 at 0.09 mM,
Tween 80 at 0.01 mM, Triton X at 0.2 mM, Pluronic F68 at 0.12 mM, H(CH2)7SO4Na
at 0.9
mM, CHAPS at 0.9 mM, Cetrimide at 0.9 mM. Loading curves for GLP-1 in the
presence of
each surfactant are shown are for GLP-1/FDKP as a function of pH.
[00144] The data show that the pH-adsorption curves for GLP-1/FDKP particles
were not
influenced by the presence of surfactants near their critical micelle
concentration (CMC) - that
is, the small range of concentrations separating the limit below which
virtually no
aggregates/micelles are detected and the limit above which virtually all
additional surfactant
molecules form aggregates. Therefore, it is further suggested that any of
these surfactants could
be used to optimize stability and/or pharmacokinetics (PK) as discussed below.
As demonstrated
above for the salt study, interaction of GLP-1 with FDKP particles was
influenced by the pH of
the suspension.
[00145] Ion studies. For this experiment, two different ion studies were run
to determine
the effect of ions on the binding of GLP-1 to FDKP particles. In both studies,
Cl- was the
counterion for cations and Na+ was the counterion for anions. Loading of the
GLP-1/FDKP
particles was performed as described for the previous experiments. The pH was
controlled as
described supra. The samples were prepared with a pH buffer of either pH 3.0,
3.5, 4.0, or 5.0 in
the presence and absence of NaC1(which was used to better assess cases of high
ionic strength).
Additional ions were included in individual samples as follows: LiC1 at 20 or
250 mM, NH4C1 at
20 or 250 mM, NaF at 20 or 250 mM, and NaCH3COO at 20 or 250 mM.
[00146] The data from the first ion study, as depicted in FIG. 10A, shows the
loading curves
for GLP-1/FDKP, as a function of pH and ions. In the absence of NaC1, fluoride
at a
concentration of either 20 or 250 mM strongly influenced (enhanced) adsorption
at low pH with
the NaF at a concentration of 250 mM exhibiting maximal binding regardless of
pH. This
pattern was observed due to the fluoride in the solution, not the sodium,
because sodium
bicarbonate did not have the same effects at 20 and 250 mM. Furthermore these
effects were not
a result of the sodium in the sample because salt at similar concentration, as
shown in FIG. 8, did
not show this effect. In the presence of 1M NaC1, all of the ions gave a high
'apparent' load. The
'apparent' load for the 1M NaC1 samples resulted from the GLP-1 peptide
salting out of solution
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in the presence of high ionic strength. This is further illustrated in FIG.
lOB, which shows that
GLP-1 is present in the reconstituted FDKP-free control samples containing 1M
NaC1. The
amount of GLP-1 detected for these control samples increased for larger ion
concentrations,
because they added to the total ionic strength in the samples.
[00147] In the second ion experiment (FIG. 10C) the GLP-1/FDKP samples were
prepared
in the presence of KC1 at 20 or 250 mM, imidiazole at 20 or 250 mM, Nal at 20
or 250 mM, or
NaPO4 at 20 or 250 mM. The data shows that at 250 mM imidazole decreased
loading in the
presence of 1M NaC1 and both 250 mM phosphate and 250 mM gave a high
'apparent' load
(FIG. lOC). Based on the amount of GLP-1 detected in the reconstituted FDKP-
free control
samples at OM and 1M NaC1 concentrations (FIG. 10D), these affects resulted
from the
influence of the ions on the GLP-1 peptide itself and not on the interaction
of the peptide with
FDKP particles. Sodium phosphate and sodium iodide caused some salting-out of
GLP-1 in the
absence of NaC1. Additionally, imidazole helped to solublize the GLP-1 in the
1M NaC1
samples and so gave lower 'apparent' loading. Precipitation was also observed
in the OM NaC1
controls with 250 mM phosphate and iodide.
[00148] Osmolyte studies. The effect of osmolytes on the binding of GLP-1 to
FDKP
particles was also observed by HPLC analysis. FIG. 11A shows the loading
curves for GLP-
1/FDKP as a function of pH in the presence of common stabilizers (osmolytes).
Loading of the
GLP-1/FDKP particles was performed as described for the previous experiment.
Similarly, the
pH was controlled as described supra. The samples were prepared at pH 3.0 and
in the presence
of 20, 50, 100, 150, 200 or 300 mM of an osmolyte (stabilizer). The osmolytes
were Hexylene-
Glycol (Hex-Gly), trehalose, glycine, PEG, TMAO, mannitol or proline; N/A
indicates no
osmolyte. In a similar experiment, the concentration of the osmolyte
(stabilizer) in the samples
was held constant at 100 mM and the pH varied from 2.0 to 4Ø
[00149] None of the osmolytes (stabilizers) studied had a dramatic impact on
GLP-1
adsorption to FDKP surfaces either when the pH was held at pH 3.0 and the
concentrations of
the osmolytes were varied (FIG. 11A; left hand curves) or when the osmolyte
concentration was
held constant at 100 mM and pH was varied (FIG. 11A; right hand curves). No
precipitation of
GLP-1 was detected in the reconstituted FDKP-free control samples (FIG. 11B).
These
osmolytes may be utilized to optimize stability and/or pharmacokinetics.
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[00150] Chaotrope and Lyotrope studies. Ionic species that affect the
structure of water
and proteins (chaotropes and lyotropes) were studied to determine the role
that these factors play
in GLP-1 adsorption to FDKP. Loading of the GLP-1/FDKP particles was performed
as
described for the previous experiments. Similarly, the pH was controlled as
described supra.
The samples were prepared at pH 3.0 and in the presence of 0, 20, 50, 100,
150, 200 or 300 mM
of the following chaotropes or lyotropes: NaSCN, CsC1, NazSO4, (CH3)3N-HC1,
Na2NO3, Na
Citrate, and NaC1O4. In a similar experiment, the concentration of the
chaotrope or lyotrope in
the samples was held constant at 100mM and the pH varied from 2.0 to 4Ø
[00151] FIG. 12A shows the loading curves for GLP-1/FDKP as a function of pH
and
chaotrope and/or lyotrope. At low pH (< 3) significant variations in loading
occurred for the
different chaotropes analyzed, especially at higher chaotrope concentrations.
However at pH 4,
this variation was not observed (FIG. 12C). Thus, these agents appear to
promote binding of
GLP-1 to the FDKP particles at unfavorable lower pH, but have little impact at
the higher pH
conditions that are favorable to binding. The data from the reconstituted FDKP-
free control
samples suggests that the loading variations observed at pH 3.0 is due in part
to specific
chaotropes affecting the salting-out (precipitation) of GLP-1 peptide to
various degrees (FIG.
12B and 12D). This was noted for strong chaotropes such as NaSCN and NaC1O4.
[00152] Organic studies. Alcohols known to induce helical conformation in
unstructured
peptides by increasing hydrogen-bonding strength were evaluated to determine
the role that
helical confirmation plays in GLP-1 adsorption to FDKP. Loading of the GLP-
1/FDKP particles
was performed as described for the previous experiments. Similarly, the pH was
controlled as
described supra. The effects of each alcohol was observed at pH 2.0, 3.0, 4.0,
and 5Ø The
alcohols used were: methanol (MeOH), ethanol (EtOH), trifluoroethanol (TFE),
or
hexafluoroisopropanol (HFIP). Each alcohol was evaluated at a concentration of
5%, 10%, 15%,
and 20% v/v.
[00153] FIG. 13A shows the loading curves for GLP-1/FDKP as a function of pH
for each
alcohol at each concentration. At pH 3.0, low concentration of HFIP (5%)
results in a high
adsorption, as demonstrated by the mass ratio of GLP-1 to FDKP particles. Only
the strongest
H-bond strengthening (helix-forming) alcohol, HFIP, had an effect on
adsorption in the buffered
suspensions. At higher concentrations of HFIP (20%), GLP-1/FDKP adsorption was
inhibited.
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FIG. 13B shows that at 20% alcohol concentration, no significant precipitation
of GLP-1 was
noted in the reconstituted FDKP-free control samples.
[00154] This suggests that conformational flexibility of a drug (i.e., entropy
and the number
of FDKP-contacts that can be formed) may play a role in adsorption. The data
suggests that H-
bonding may play a role in GLP-1 interaction with FDKP surfaces under the
above conditions.
Based on the data, it is further speculated that if H-bonding served as a
dominant and a general
force in FDKP-GLP-1 interactions, more and stronger effects would have been
expected.
[00155] Concentration studies. - The adsorption of GLP-1 to FDKP particle
surfaces at
varying concentrations of GLP-1 was investigated. FIG. 14A shows loading
curves from GLP-
1/FDKP as a function of GLP-1 concentration at various pHs. GLP-1
concentrations were at
0.15, 0.25, 0.4, 0.5, 0.75, 1.0, 1.5, 2.0, 5.0, or 10 mg/mL. The pH of the
samples was at 2.5, 3.0,
3.5, 4.0, 4.5 or 5Ø
[00156] An increase in GLP-1 loading on FDKP particles was observed when the
FDKP
concentration was held constant at 5 mg/mL and the GLP-1 concentration
increased. Nearly
20% GLP-1 adsorption on FDKP particles was observed when the concentration of
GLP-1 was
mg/mL at pH 4. Surprisingly, no saturation of adsorption of GLP-1 loading on
FDKP
particles was observed at high concentrations of GLP-1. This observation is
probably
attributable to the self association of GLP-1 into a multi-layer.
[00157] Analysis of the morphology of GLP-1/FDKP formulations by scanning
electron
microscopy (SEM) shows that GLP-1/FDKP particles are present as crystalline or
plate like
structures which can form aggregates comprising of more than one GLP-1/FDKP
particles (FIG.
14B). These formulations were prepared by lyophilizing a solution containing:
(Panel A) 0.5
mg/mL GLP-1 and 2.5 mg/mL FDKP; (Panel B) 0.5 mg/mL GLP-1 and 10 mg/mL FDKP;
(Panel C) 0.5 mg/mL GLP-1 and 10 mg/mL FDKP in 20 mM sodium chloride, 20 mM
potassium acetate and 20 mM potassium phosphate, pH 4.0; and (Panel D) 10
mg/mL GLP-1 and
50 mg/mL FDKP in 20 mM sodium chloride, 20 mM potassium acetate and 20 mM
potassium
phosphate at pH 4Ø
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[00158] Summary of Results
[00159] Overall, the adsorption studies on the interaction of GLP-1 with FDKP
particles
showed that GLP-1 binds to the DKP particle surfaces in a pH-dependent manner,
with high
adsorption at pH 4 or above. The adsorption of GLP-1 to DKP particle surfaces
was found to be
most strongly affected by pH, with essentially no adsorption at pH 2.0 and
substantial interaction
at pH _ 4Ø As observed, sodium and fluoride ions enhanced adsorption at low
pH. Other
additives such surfactants, and common stabilizers had only a slight effect on
the adsorption of
GLP-1 to FDKP particle surfaces.
[00160] In addition, the properties of GLP-1 itself influenced the results of
these
experiments. The behavior of GLP-1 was found to be atypical and surprising in
that there was
no saturation of adsorption observed, which was attributed to GLP-1 self-
association at high
concentrations. The self-association of GLP-1 at high concentration, allows
for the possible
coating of DKP particles with multiple layers of the GLP-1 peptide thereby
promoting higher
percent load of the GLP-1 peptide. This surprising self-association quality
proves to be
beneficial in preparing stable GLP-1 administration forms. Further, the self-
associated
conformation of GLP-1 may be able to lessen or delay its degradation in blood.
However, it is
noted that care must be taken when working with associated GLP-1 since it is
sensitive to
temperature and high pH.
Example 3
Inte2rity Analysis of GLP-1/FDKP FormulationsBased on the results from the
experiments in
Examples 1 and 2, a series of GLP-1 formulations having the characteristics
described in Table 1
were selected for the cell viability assay as discussed herein. Most of the
formulations contained
GRAS ("generally recognized as safe") excipients, but some were selected to
allow the
relationship between stability and adsorption to be studied.
Table 1. Selected GLP-1/FDKP Formulations for Integrity Phase Analysis.
Modifier Amount (mM) No Buffer pH 3.0 pH 4.0 pH 5.0
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
None X X X X
NaC1 1000 X X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - ~
NaC1 20 X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - ~
-38-
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WO 2007/121411 PCT/US2007/066728
Tween 80 0.01% X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
HepSulf 0.90% X
Brij 78 0.09% X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - ~
F- 250 X
~ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
F- 20 X X
~ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
Li+ 20 X X X
Phosphate 250 X X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
Phosphate 20 X X X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
Imidizole 250 X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
Mannitol 20 X
Glycine 20 X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - ~
Me3N=HC1 50 X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
Citrate 50 X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
AmZSOq 50 X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
C104 50 X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - .
EtOH 20% X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - -
TFE 20% X
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
[00162] Further, based on the results obtained in Examples 1 and 2, a series
of formulations
were also selected for phase II integrity studies of GLP-1/FDKP. Table 2 below
shows the six
GLP-1 formulations chosen for phase II integrity. After the powders were
prepared, they were
blended with blank FDKP to yield similar masses of both the GLP-1 peptide and
FDKP in each
formulation.
Table 2. GLP-1/FDKP formulations chosen for phase II integrity. The
formulation made from
mg/ml GLP-1 in 20mM NaC1 and pH 4.0 buffer is desribed as the salt-associated
formulation.
GLP-1 Concentration Mass ratio Water 20mM NaC1 +
(GLP/FDKP) (no pH 4.0 buffer
buffer)
0.5 mg/mL 0.05 X X
3.0 mg/mL 0.10 X X
10 mg/mL 0.20 X X
[00163] The effect of stress on the GLP-1/FDKP formulations in Table 2 was
analyzed by
HPLC (FIG. 15). The samples containing 5%, 10% or 20% GLP-1/FDKP loaded in
H20; or
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5%, or 10% GLP-1/FDKP loaded in NaC1 + pH 4.0 buffer, were incubated for 10
days at 40 C.
The HPLC chromatograms demonstrate that the GLP-1 peptide elutes at the same
retention time
and that no degradation peaks are present. Furthermore, MS analyses yielded a
similar mass for
all the samples, 3297 g/mol, indicating that the mass is uniform for all the
samples analyzed.
The data show the mass-to-mass ratio of GLP-1 to FDKP particles and the other
components that
were present in solution, prior to lyophilization. Overall, the GLP-1/FDKP
formulations were
shown to be stable to stress.
Example 4
Stability of GLP-1 Incubated in Lun Lava2e Fluid
[00164] The stability of GLP-1 in biological fluids such as lung fluid and
blood was
analyzed given that dipeptidyl-peptidase IV (DPP-IV), found in biological
fluids, cleaves and
inactivates GLP-l.
[00165] Dipeptidyl-peptidase IV (DPP-IV) is an extracellular membrane-bound
serine
protease, expressed on the surface of several cell types, in particular CD4+ T-
cells. DPP-IV is
also found is blood and lung fluids. DPP-IV has been implicated in the control
of glucose
metabolism because its substrates include the insulinotropic hormone GLP-1
which is
inactivated by removal of its two N-terminal amino acids; see FIG. 16A. DPP-IV
cleaves the
Ala-Glu bond of the major circulating form of human GLP-1 (GLP-1 (7-36))
releasing the N-
terminal two residues. DPP-IV exerts a negative regulation of glucose disposal
by degrading
GLP-1 thus lowering the incretin effect on (3 cells of the pancreas.
[00166] Studies were conducted to determine inhibition of GLP-1 degradation in
rat blood
and lung fluid in the presence of aprotinin or DPP-IV inhibitor. Aprotinin, a
naturally occurring
serine protease inhibitor, which is known in the art to inhibit protein
degradation was added to
the samples post collection at 1, 2, 3, 4 and 5 TIU/ml. DPP-IV activity was
then measured by
detecting the cleavage of a luminescent substrate containing the DPP-IV
recognized Gly-Pro
sequence. Bronchial lung lavage fluid was incubated with proluminescent
substrate for 30 min
and cleavage product was detected by luminescence.
[00167] The data showed an increase in inhibition of DPP-IV activity, as
detected by the
inhibition of peptide degradation in various biological fluids (as discussed
herein) with
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increasing aprotinin concentration (FIG. 16B). Similar results were observed
with DPP-IV
inhibitor added to the samples post collection at 1.25, 2.5, 5, 10 and 20
l/ml (FIG. 16C).
Addition of inhibitors post-sample collection allowed for more accurate
evaluation of the
samples.
[00168] The stability of GLP-1 was also examined in lung lavage fluid using a
capture
ELISA mAb that recognizes GLP-1 amino acids 7-9. GLP-1 was incubated in lung
lavage fluid
(LLF) for 2, 5, 20 and 30 mins. The incubation conditions were: 1 or 10 g
(w/w) of LLF and 1
or 10 g (w/w) GLP-1 as depicted in FIG. 17. No GLP-1 was detected in LLF
alone. With the
combination of LLF and GLP-1 at various concentrations there was a high
detection of GLP-1
comparable to that of GLP-1 alone, indicating that GLP-1 is stable, over time,
in lung lavage
fluid (FIG. 17). Stability of GLP-1 in undiluted lung lavage fluid was
confirmed in similar
studies; at 20 minutes 70-72% GLP-1 integrity was noted (data not shown).
[00169] In addition, the stability of GLP-1 in rat plasma was examined. Plasma
was
obtained from various rats (as indicated by Plasma 1 and Plasma 2 in the
figure legend) and
diluted 1:2 or 1:10 (v/v). One microgram of GLP-1 was added to 10 l plasma or
PBS. Samples
were incubated at 37 C for 5, 10, 30 or 40 mins. The reaction was stopped on
ice, and 0.1U of
aprotinin was added. The data shows a high concentration of GLP-1 in plasma
dilutions 1:2 and
1:10 over all timepoints tested (FIGs. 18A and 18B). Overall, the data
indicate that GLP-1 is
surprisingly stable in both lung lavage fluid and plasma in which the serine
protease DPP-IV is
found.
Example 5
Effect of GLP-1 Molecules on Apoptosis and Cell Proliferation
[00170] To examine whether GLP-1 inhibits apoptosis a screening assay was
conducted to
determine the effect of GLP-1 on inhibition of (3-cell death. Rat pancreatic
epithelial (ARIP)
cells (used as a pancreatic (3-cell model; purchased from ATCC, Manassas, VA)
were pretreated
with GLP-1 at 0, 2, 5, 10, 15 or 20 nM concentration for 10 minutes. The cells
were then left
untreated or were treated with 5 M staurosporine (an apoptosis inducer) for
4.5 hours. Cell
viability was evaluated using Cell Titer-G1oTM (Promega, Madison, WI). A
decrease in the
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percent of cell death was noted with an increase in GLP-1 concentration of up
to 10 nM in the
staurosporine treated cells (FIG. 19A).
[00171] Further examination of the effect of GLP-1 on apoptosis was determined
by FACS
analysis using Annexin V staining. Annexin V staining is a useful tool in
detecting apoptotic
cells and is well known to those of skill in the art. Binding of Annexin V to
the cell membrane,
allows for the analysis of changes in phospholipids (PS) asymmetry before
morphological
changes associated with apoptosis occurred and before membrane integrity is
lost. Thus, the
effect of GLP-1 on apoptosis was determined in cells treated with 15 nm GLP-1,
l M
staurosporine for 4 hrs, l M staurosporine + 15 nm GLP-1 or neither
staurosporine nor GLP-1
(experimental control). The data shows that GLP-1 inhibited staurosporine
induced apoptosis by
about 40% (FIG. 19B).
[00172] Similar results of inhibition of apoptosis were observed using a GLP-1
analog,
exendin-4, which binds to the GLP-1 receptor in a similar manner to GLP-1.
ARIP cells were
treated with 5 M staurosporine in the presence of 0, 10, 20 or 40 nM exendin
for 16, 24, or 48
hours respectively. The data (FIG. 20) shows that at 10 nM, exendin was
completely ineffective
at inhibiting apoptosis as there was 100% cell death. At 20 and 40 nM exendin
inhibited
apoptosis to some degree with about 50% inhibition at 48 hours in the presence
of 40 nM of
exendin-4.
Example 6
Effect of Candidate GLP-1/ FDKP Formulations on Cell Death
[00173] Cell-based assays were conducted to assess the ability of GLP-1/FDKP
formulations, (as disclosed in Example 3, Table 1 above), to inhibit cell
death. These GLP-
1/FDKP particle formulations were either in a suspension or lyophilized. The
formulations were
analyzed for their ability to inhibit staurosporine-induced cell death in ARIP
cells. ARIP cells
pre-treated with GLP-1 samples were exposed to 5 M staurosporine for 4 hours
and were
analyzed with Cell Titer-G1oTM (Promega, Madison, WI) to determine cell
viability.
[00174] Samples of the various GLP-1/FDKP formulations were either left
unstressed or
were stressed at 4 or 40 C for 4 weeks. Each GLP-1/FDKP sample was used at 45
nM in a cell-
based assay to detemine their ability to inhibit stauorosporine induced cell
death. Control
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samples, shown on the right, illustrate the viability of cells in media alone,
with GLP-1 alone,
with staurosporine alone, or in the presence of both GLP-1 and staurosporine
(note: the graph
legend does not apply to the control samples. Each bar represents a separate
triplicate). All of
the results shown are averages of triplicate runs.
[00175] The data shows that all stressed GLP-1/FDKP lyophilized formulations
inhibited
staurosporine-induced cell death (FIG. 21). However, cell death was not
inhibited by many of
the GLP-1/FDKP suspension formulations.
Example 7
Pulmonary Insufflation of GLP-1/DKP Particles
[00176] To examine the pharmacokinetics of GLP-1/FDKP, plasma concentrations
of GLP-1
were evaluated in female Sprague Dawley rats administered with various
formulations of GLP-
1/FDKP via intravenous injections or pulmonary insufflation. In the
preliminary studies, GLP-1
at approximately 4% and 16% (w/w) of the GLP-1/FDKP particle formulations was
used. Rats
were randomized into 12 groups with groups 1, 4, 7 and 10 receiving GLP-1
solution
administered via pulmonary liquid instillation or IV injection. Groups 2, 5,
8, and 11 received
GLP-1/FDKP salt-associated formulation (as disclosed in Table 2), administered
via pulmonary
insufflation or IV injection. Groups 3, 6, 9, 12 received the GLP-1/FDKP salt-
associated
blended formulation administered via pulmonary insufflation or IV injection.
The GLP-1/DKP
formulation was a salt-associated formulation at approximately 16% load. To
achieve an
approximate 4% load, the 16% formulation was blended with DKP particles in a
3:1 mixture.
Pulmonary insufflation or intravenous injection was at 0.5 or 2.0 mg of
particles (16% or 4%
GLP-1 load, respectively) for a total GLP-1 dose of 0.08 mg.
[00177] In a separate group of animals (Groups 7-12), administration was
repeated on Day
2. Groups 1, 4, 7, and 10 were administered 80 g of a GLP-1 solution. Groups
2, 5, 8, and 11
were administered a GLP-1/DKP salt-associated formulation (-16% GLP-1 load).
Groups 3, 6,
9, 12 received the GLP-1/DKP salt-associated blended formulation (-4% GLP-1
load).
[00178] The experiment was performed twice using the same formulations, with
dosing and
blood collection on two consecutive days. Blood samples were taken on the day
of dosing for
each group at pre-dose (time 0), and at 2, 5, 10, 20, 30, 60 and 120 minutes
post dose. At each
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time-point, approximately 150 L whole blood was collected from the lateral
tail vein into a
cyro-vial tube containing approximately 3 U/mL aprotinin and 0.3% EDTA,
inverted and stored
on ice. Blood samples were centrifuged at 4000 rpm and 40 1 of plasma was
pipetted into 96-
well plates which were stored at -80 C until analyzed for GLP-1 levels by
ELISA following
manufactures' recommendations (Linco Research, St Charles, MO). It was
determined that the
optimal conditions were when the assay buffer was GLP-1 in the presence of
serum (5% FBS)
alone and no matrix.
[00179] Intravenous Administration: Groups 5, 6, 10, 11 and 12 received
various GLP-
1/FDKP formulations and GLP-1 solution intravenously (IV); (FIG. 22A). Groups
5 and 6 were
administered 15.8% GLP-1/FDKP and groups 11 and 12 were administered another
dose of
15.8% GLP-1/FDKP on a consecutive day; group 10 was administered GLP-1
solution as a
control. The concentration of GLP-1/FDKP was detected at time points of 0, 2,
5, 10, 20, 40, 60,
80, 100, and 120 mins. All groups showed a detectable increase in GLP-1 plasma
levels after
intravenous administration, with maximal concentrations observed at 2 minutes
post treatment.
Plasma levels of active GLP-1 returned to background levels by 20 minutes post
treatment for all
groups. No significant difference was observed in the kinetics of these
various formulations of
GLP-1/FDKP and GLP-1 solution when administered by intravenous injection. It
was noted that
plasma levels of GLP-1 returned to baseline levels at 10-20 minutes post dose
in rats treated via
intravenous injections suggesting physiological kinetics (i.e., about 95 % of
GLP-1 was
eliminated within 10 mins).
[00180] Single Insufflation Administration: Groups 1, 2, 3, 7, 8 and 9 12
received various
GLP-1/FDKP formulations or GLP-1 solution by pulmonary insufflation (FIG.
22B). Group 1
was administered 80 g of a GLP-1 control by pulmonary liquid instillation
(LIS); group 2 was
administered 15.8% GLP-1/FDKP by pulmonary insufflation (IS); group 3 was
administered
3.8% GLP-1/FDKP by pulmonary insufflation (IS); group 7 was administered 80 g
of a GLP-1
control by pulmonary liquid instillation (LIS); group 8 was administered 15.8%
GLP-1/FDKP by
pulmonary insufflation (IS); and group 9 was administered 3.8% GLP-1/FDKP by
pulmonary
insufflation (IS). The concentration of GLP-1/FDKP was measured at time points
of 0, 2, 5, 10,
20, 40, 60, 80, 100, and 120 mins.
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[00181] All groups showed a detectable increase in plasma GLP-1 concentration
following
pulmonary administration. Maximum plasma concentration of GLP-1 varied with
the
formulation/composition used. Groups 2 and 8 showed maximal plasma levels of
GLP-1 at 10-20
minutes post treatment as indicated by the AUC, while groups 3 and 9 showed
significant levels
of active GLP-1 at 5-10 minutes, and groups 1 and 7 showed a more rapid and
transient increase
in plasma levels of active GLP-l. Plasma levels of active GLP-1 returned to
background levels
by 60 minutes post treatment in groups 2, 3, 7 and 8, while groups 1 and 7
reached background
levels by 20 minutes post treatment.
[00182] Eight nanomolar GLP-1 appears to be efficacious in a diabetic rat
model; the GLP-1
dose was 80 g (3000-fold greater than the reported efficacious dose); plasma
GLP-1 levels were
10-fold greater with pulmonary delivery versus a 3 hr infusion (Chelikani et
al., 2005) at 30
minutes post dose; and the bioavailability of GLP-1/FDKP delivered via
pulmonary insufflation
was 71%. These results are further reported in Table 4 below. Plasma levels of
GLP-1 returned
to baseline levels at 30-60 minutes post dose in most rats treated via
pulmonary delivery. All
rats showed an increase in plasma concentrations of GLP-1 after intravenous
administration or
pulmonary insufflation of various GLP-1/FDKP formulations, except for 1 rat in
group 2.
[00183] Conclusion: A difference was observed in the pharmacokinetics profiles
of GLP-
1/FDKP formulations compared to GLP-1 solution. Plasma concentrations of GLP-1
were more
sustained in rats treated by pulmonary insufflation with GLP-1/FDKP
formulations relative to
those treated with GLP-1 solution. All animals showed a progressive decrease
in plasma
concentrations of GLP-1 between 20 and 60 minutes post dose. These results
showed relative
consistency in 2 experiments performed on 2 consecutive days.
Table 4. Bioavailability of GLP-1/FDKP Formulations
GLP-1 ,j,i/z Tmax Cmax 30 min AUC
Group Formulations Dose Route (min) (min) post dose *min/mL)
( g)/ M (pM) (-pM) (pM
1 GLP-1 80/24 LIS 1.0 5 1933 0 29350
2 FDKP-GLP-1 80/24 IS 9.9 10 3154 1000 145082
3 FDKP-GLP-1 * 80/24 IS 7.7 10 2776 400 60171
* Blended 3:1 with FDKP particles
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Example 8
GLP-1/FDKP Reduces Food Intake in Rats
[00184] GLP-1 is also known in the art to work in the brain to trigger a
feeling of satiety and
reduce food intake. Based on this role of GLP-1 in satiety and reduction of
food intake,
experiments were conducted to determine whether GLP-1/FDKP formulations of the
present
invention were effective as agents to reduce feeding and thereby have
potential for controlling
obesity.
[00185] Two groups of female Sprague Dawley rats were dosed with either a
control (air) or
15.8% GLP-1/FDKP formulation at a dosage of 2 mg/day (0.32 mg GLP-1/dose) by
pulmonary
insufflation. The control group consisted of five rats and the test group
consisted of ten rats.
Each rat was provided with a single dose for 5 consecutive days and the food
intake measured 2
and 6 hours following each dose. The body weight of each rat was collected
daily.
[00186] The preliminary data shows that at 2 and 6 hours post dose, there was
an overall
decrease in the cumulative food consumption in the rats dosed with GLP-1/FDKP
formulations
(FIGs. 23A and 23B). The decrease was more pronounced at day 4 at 2 hours post
dosing
(p=0.01). At 6 hours the decrease was more pronounced at days 1 and 2(p<0.02).
There was no
effect on food consumption at 24 hours post dose.
Example 9
Toxicity Studies
[00187] Repeat dose toxicity studies to evaluate the potential toxic effects
and toxicokinetic
profile of GLP-1/DKP after multiple administrations were conducted. Fourteen
day study in rats
and a twenty-eight day study in monkeys was performed. GLP-1/DKP will be dosed
daily, via
the inhalation route. In studies where animals were dosed for 28 days, a
proportion of the
animals will be sacrificed immediately after the dosing regimen while other
animals will be
allowed up to a one month recovery period prior to sacrifice. All animals will
be evaluated for
clinical signs, various physiological parameters including GLP-1, glucose,
insulin, organ
weights, and clinical pathology and histopathology of various organs.
[00188] A series of GLP mutagenicity studies were performed to evaluate the
mutagenic
potential of diketopiperazine particles. These studies included the in vitro
Ames and
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Chromosomal aberration assays, both which are well known to those of skill in
the art. In
addition, an in vivo mouse micronucleus assay, as is known to the skilled
artisan, was also
conducted. The genotoxicity data shows that there was no evidence of potential
for mutagenicity
or genetic toxicity with diketopiperazine particles.
[00189] Studies were also conducted to assess the effect of diketopiperazine
particles on
reproductive toxicity. These studies included fertility, embryo-fetal
development and postnatal
development studies in rats and rabbits. Diketopiperazine particles
administered via
subcuteanous injection does not impair fertility or implantation in rats and
there is no evidence
of teratogenicity in rats or rabbits. Diketopiperazine particles did not
adversely affect fertility
and early embryonic development, embryo fetal development, or prenatal or
postnatal
development.
[00190] Given that a number of pharmaceuticals have been removed from the
clinical
market due to their propensity to cause LQT syndrome (acquired LQTS or Long Q-
T syndrome
is an infrequent, hereditary disorder of the heart's electrical rhythm that
can occur in otherwise-
healthy people) an hERG assay was employed to examine the pharmacology of
diketopiperazine
particles. The hERG assay was utilized given that the vast majority of
pharmaceuticals that
cause acquired LQTS do so by blocking the human ether-a-go-go related gene
(hERG)
potassium channel that is responsible for the repolarization of the
ventricular cardiac action
potential. Results from the hERG assay indicated an IC50 >100 M for
diketopiperazine
particles. In addition, results from nonclinical studies with diketopiperazine
particles showed no
effect on the QTc interval (the heart rate-corrected QT interval) as
prolongation was not
observed in the dog (9-month or safety pharmacology cardiovascular studies).
There were no
effects of diketopiperazine particles, when administered intravenously, on CNS
or cardiovascular
systems evaluated in the safety pharmacology core battery.
Example 10
Effect of GLP-1 on R-cell Mass
[00191] GLP-1 is known to promote all steps in insulin biosynthesis and
directly stimulate
B-cell growth and survival as well as B-cell differentiation. The combination
of these effects
results in increased B-cell mass. Furthermore, GLP-1 receptor signaling
results in a reduction of
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B-cell apoptosis, which further contributes to increased B-cell mass. GLP-1 is
known to
modulate (3-cell mass by three potential pathways: enhancement of (3-cell
proliferation;
inhibition of apoptosis of (3-cells; and differentiation of putative stem
cells in the ductal
epithelium.
[00192] To demonstrate the effect of GLP-1 on (3-cell mass, cells were treated
at day 1, 3
and 5 with GLP-1/FDKP and compared to untreated cells. Administration of
active GLP-1
increased (3-cell mass by up to 2-fold as suggested in the literature (Sturis
et al., 2003). In
addition, examination of the effect of various GLP-1 receptor (GLP-1R)
agonists on diabetes
demonstrated that GLP-1R agonists prevent or delay occurrence or progression
of diabetes.
[00193] The effects of GLP-1/FDKP on (3 cell proliferation, insulin and
glucose were
assessed in male Zucker Diabetic Fatty/Obese (ZDF) rats (n=8/group). Animals
received either
control (air) or 2 mg GLP-1/FDKP containing 15% (0.3 mg) GLP-1 daily for 3
consecutive days.
An intraperitoneal (IP) glucose tolerance test was conducted and blood samples
were collected
for plasma GLP-1 and glucose analysis pre-dose, and at 15, 30, 45, 60 and 90
minutes post-dose.
Pancreatic tissues were collected for insulin secretion, (3 cell mass, and
apoptosis analysis via
immunohisto chemistry.
[00194] An IP glucose tolerance test (IPGTT, FIG. 24) was conducted on day 4
of dosing.
After an overnight fast, on day 3, animals received a glucose bolus via
intraperitoneal injection
followed immediately by control (air) or GLP-1/FDKP adminstration via
pulmonary insufflation.
Blood was collected prior to the glucose challenge and at various timepoints
out to 90 minutes
post dose. At 30 minutes post-dose, Group 1 showed a 47% increase in glucose
levels compared
to predose whereas Group 2(GLP-1/FDKP) showed a 17% increase in glucose levels
compared
to predose values. Glucose levels were significantly lower across all
timepoints following the
glucose tolerance test in the treated versus control animals (p<0.05).
[00195] GLP-1 levels were also measured on day 3 of dosing (FIG. 25). The
maximum
concentration of plasma GLP-1 levels in Group 2 was 10,643 pM at 15 minutes
post-dose.
[00196] In addition, insulin levels were measured at various timepoints on day
3 along with
glucose measurements followng the IP glucose tolerance test. Both control
(air) Group 1 and
Group 2 (GLP-/DKP) demonstrated an initial decrease in insulin concentration
from pre-dose
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levels, 46% and 30%, respectively, by 15 minutes post-dose (FIG. 26). However,
at 30 minutes
post-dose, insulin levels in Group 2 returned to baseline whereas insulin
levels in Group 1
continued to decrease to 64% of pre-dose values. In treated animals, insulin
levels at 45
minutes, 60 minutes, and 90 minutes were near pre-dose values with deviations
of less than
1.5%.
[00197] Slides were prepared for insulin immunostaining and microscopic
evaluation of
insulin expression. Based on quantitative assessment of insulin expression by
light microscopy,
there was a treatment-related increase in insulin expression within the
pancreas of male ZDF rats
that was dose-related, although statistical significance was not attained
(p=0.067); as determined
by the percentage of (3 islet cells expressing insulin.
[00198] Apoptosis analysis was also conducted on the pancreatic tissue of ZDF
rats.
Exocrine and endocrine pancreas cells were evaluated by the TUNEL assay
(Tornusciolo D.R. et
al., 1995). Approximately 10,000 cells in the pancreas (exocrine and
endocrine) were scored.
Most TUNEL-positive cells were exocrine. There were no differences in
apoptosis labeling
index in treated versus control groups.
[00199] In addition, (3 cell proliferation was evaluated in the pancreas of
Zucker Diabetic
obese rats dosed once daily for 3 days with control (air) or GLP-1/FDKP via
pulmonary
insulfflation. Slides were prepared for co-localization of insulin and Ki67 (a
proliferation
marker) using immunohistochemistry. Microscopic evaluation of cell
proliferation was
conducted within insulin-positive islets and in the exocrine pancreas in a
total of 17 ZDF rats.
Based on quantitative assessment of cell proliferation, there were no
treatment-related effects on
cell proliferation within the islet beta cells or exocrine cells of the
pancreas in male ZDF rats.
[00200] Overall, this study shows that GLP-1/FDKP administered at 2 mg or 0.3
mg GLP-1
via pulmonary insufflation lowered blood glucose levels in diabetic fatty rats
(model for Type 2
diabetes) following a glucose tolerance test and increased the number of
insulin secreting cells
per islet.
Example 11
Preparation of GLP-1/FDKP particle formulations
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[00201] An alternative methodology for preparing GLP-1/FDKP particle
formulations was
also employed. The formulations were prepared as follows: A 10 wt% GLP-1 stock
solution
was prepared by adding 1 part GLP-1 (by weight) to 9 parts deionized water and
adding a small
amount of glacial acetic acid to obtain a clear solution. A stock suspension
of FDKP particles
(approximately 10 wt% particles) was divided into three portions. An
appropriate amount of
GLP-1 stock solution was added to each suspension to provide target
compositions of 5 and 15
wt% GLP-1 in the dried powder. After addition of the protein solution, the pH
of the
suspensions was approximately 3.5. The suspensions were then adjusted to
approximately pH
4.4-4.5, after which the suspensions were pelletized in liquid nitrogen and
lyophilized to remove
the ice.
[00202] The aerodynamics of the powders is characterized in terms of
respirable fraction on
fill (RF Based on Fill), i.e., the percentage (%) of powder in the respirable
range normalized by
the quantity of powder in the cartridge, which was determined as follows: five
cartridges were
manually filled with 5 mg of powder and discharged through MannKind's MedTone
inhaler
(described in U.S. Patent Application No. 10/655,153).
[00203] This methodology produced a formulation with a good RF on fill. The
powder with
wt% GLP-1 was measured at 48.8 %RF/fill while the powder containing
approximately 15
wt% GLP-1 was 32.2 %RF/fill.
Example 12
Pharmacokinetics of GLP-1/FDKP with Various GLP-1 Concentrations
[00204] To assess the pharmacokinetic properties of GLP-1/FDKP with various
concentrations of GLP-1, eighteen female Sprague Dawley rats weighing between
192.3 grams
to 211.5 grams were divided into four treatment groups: Control GLP-1 (Group
1, n=3); GLP-
1/FDKP formulations (Groups 2-4, n=5/group). Animals received one of the
following test
articles: control (air) via pulmonary instillation; 2.42 mg GLP-1/FDKP
containing 5% GLP-1
(0.12 mg GLP-1); 1.85 mg GLP-1/FDKP containing 10% GLP-1 (0.19 mg GLP-1), or
2.46 mg
GLP-1/FDKP containing 15% GLP-1 (0.37 mg GLP-1) via pulmonary insufflation.
Blood
samples were collected and assayed for serum FDKP and plasma GLP-1 levels
predose and at
various timepoints (2, 5, 10, 20, 30, 40 and 60 minutes) post dose.
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[00205] The maximum plasma GLP-1 concentrations (Cma.X) following the
administration of
GLP-1/FDKP (5% formulation) were 2321 pM at a T. of 5 minutes post dose; 4,887
pM at a
Tmax of 10 minutes post dose (10% formulation); and 10,207 pM at a Tmax of 10
minutes post
dose (15% formulation). As depicted in FIG. 27 significant GLP-1 levels out to
30 minutes post
dose was observed. The area under the curve (AUC) levels for GLP-1 were 10622,
57101,
92606, 227873 pM*min for Groups 1-4, respectively. Estimated half-life of GLP-
1 was 10 min
for GLP-1/FDKP at 10% or 15% GLP-1 load.
[00206] As depicted in FIG. 28, maximum FDKP concentrations were determined to
be 8.5
g/mL (Group 2), 4.8 g/mL (Group 3) and 7.1 g/mL (Group 4) for the GLP-1/FDKP
formulations at 5%, 10% and 15% GLP-l, respectively. The time to maximum
concentrations
(Tmax) was 10 minutes. This data shows that, FDKP and GLP-1 exhibited similar
absorption
kinetics and similar amounts of FDKP were absorbed independent of the GLP-1
load on the
particles.
[00207] Overall, the study showed that plasma GLP-1 levels were detected at
significant
levels after single dose administration of GLP-1/FDKP via pulmonary
insufflation in Sprague
Dawley rats. Dose related increases in plasma GLP-1 levels were observed with
maximum
concentrations achieved at approximately 10 min post dose and with observable
GLP-1 levels at
40 minutes post dose. All animals survived until the completion of the study.
Example 13
Pharmacodynamic Properties of GLP-1/FDKP Administered via Pulmonary
Insufflation
[00208] To assess the pharmacodynamic properties of GLP-1/FDKP, female Sprague
Dawley rats were divided into 2 treatment groups. Animals received either
control (air; n=5) or
2 mg GLP-1/FDKP containing 15% GLP-1 (0.3 mg GLP-1) via a single daily
pulmonary
insufflation (n= 10) for 4 consecutive days.
[00209] Food consumption was measured during the dark cycle at predose, 1, 2,
4 and 6
hours post dose for 4 consecutive days (FIG. 29). Food consumption was
decreased on Days 1,
2 and 3 after daily single dose administration of GLP-1/FDKP via pulmonary
insufflation in the
treated animals compared to the control (air) group (p<0.05). There were
statistically significant
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decreases in food consumption for animals in the treated group versus control
(air) on Day 1 at
the 1 hour and 6 hour timepoint and on Day 2 at the 4 hour, 6 hour and at
predose on Day 3.
[00210] Body weights (FIG. 30) were measured daily at predose for 4
consecutive days.
Body weights at the initiation of dosing ranged from about 180 to 209 grams.
Although
statistical significance between treated and control (air) animals was not
reached, body weight
were lower in treated animals. All animals survived until scheduled sacrifice.
Examples 14 - 16
Toxicokinetics (TK) Studies
[00211] Examples 14 to 16 below disclose repeat-dose toxicity studies
performed in rats and
monkeys to evaluate the potential toxic effects and toxicokinetic profile of
GLP-1/FDKP
inhalation powder. The data indicates no apparent toxicity with GLP-1/FDKP
inhalation powder
at doses several fold higher than those proposed for clinical use.
Additionally, there appeared to
be no differences between the male and female animals within each species.
Example 14
Toxicokinetics of GLP-1/FDKP Administered for 5 days via Pulmonary
Insufflation in
Monkeys
[00212] Studies were conducted to determine the toxicity and toxicokinetic
profile of GLP-
1/FDKP via oronasal administration (the intended human therapeutic route of
administration) to
the cynomolgus monkey (Macaca fascicularis), once daily (for 30 minutes a day)
for 5
consecutive days. Oronasal administration involved the monkeys wearing a mask
over their
mouth and nose and breathing the test formulation for 30 min.
[00213] Fourteen days prior to the start of treatment, the animals were
acclimated to the
restraint and dosing procedures. At the start of treatment (Day 1), male
animals were between
30 months and 56 months old and ranged in weight from 2.3 to 4.0 kg; females
were between 31
months and 64 months and ranged in weight from 1.6 to 3.4 kg. Ten (5 male and
5 female) non-
naive cynomolgus monkeys were assigned to 5 groups (2 animals per group) as
depicted in
tables 5 and 6 below. The non-naive monkeys are colony animals who have
previously received
the formulations to be tested. However, these formulations have short half
lives and are not
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expected to be present or have any effect on the monkeys during the dosing
experiments
disclosed herein. Animals received control (air), 2 mg/kg FDKP or 0.3 (0.04 mg
GLP-1), 1.0
(0.13 mg GLP-1), or 2.0 (0.26 mg GLP-1) mg/kg GLP-1/FDKP.
Table 5: Targeted and estimated achieved mean dose levels (determined by
gravimetric
analysis*):
Group Group 3 Estimated Dose Level (mg/kg /da
Number Designation FDKP 2 GLP-1 2 GLP-1/FDKP 2
Target Achieved Target Achieved Target 1 Achieved
1 Air Control 0 0 0 0 0 0
2 Vehicle 2.00 2.10 0 0 2.0 2.10
Control
3 Low Dose 0.26 0.31 0.04 0.05 0.3 0.35
4 Mid Dose 0.87 0.81 0.13 0.14 1.0 0.93
High Dose 1.74 1.85 0.26 0.28 2.0 2.13
~Gravimetric analysis is performed by weighing the filter papers in the
inhalation chamber both before,
during and after dosing to calculate the aerosol concentration in the chamber
and to determine the
duration of dosing.
1 Based on an assumed body weight of 2.5 kg.
2 Based on the measured body weights (average for male and female).
3 The targeted and achieved dose levels quoted assume that the proportion of
GLP-1 in the generated
atmosphere is 13%. The estimation of total inhaled dose assumed 100%
deposition within the respiratory
tract.
Table 6: Targeted and achieved mean aerosol concentrations (determined by
gravimetric
analysis*):
Aerosol Concentration m /L
Group Group FDKP 1 GLP- 1 1 GLP-1/FDKP
Number Designation Target Achieved Target Achieved Target Achieved
1 Air Control 0 0 0 0 0 0
2 CVehicle ontrol 0.160 0.189 0 0 0.160 0.189
3 Low Dose 0.021 0.027 0.003 0.004 0.024 0.031
4 Mid Dose 0.070 0.073 0.010 0.011 0.080 0.084
5 High Dose 0.139 0.142 0.021 0.021 0.160 0.163
~Gravimetric analysis is performed by weighing the filter papers in the
inhalation chamber both before,
during and after dosing to calculate the aerosol concentration in the chamber
and to determine the
duration of dosing.
1 The targeted and achieved aerosol concentrations quoted assume that the
proportion of GLP-1 in the
generated atmosphere is 13%. The estimation of total inhaled dose assumed 100%
deposition within the
respiratory tract.
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[00214] Whole blood samples (1.4 mL/blood sample) were obtained on Day 5 at
the
following time points: Pre-dose, 10, 30, 45, 60, 90, 120 minutes and 4 hours
post-dose. Blood
was collected via venipuncture from the femoral vein. Blood samples were
divided into 2
aliquots; one for plasma GLP-1 analysis (0.8 mL) and the other (0.6 mL) for
serum FDKP
analysis. For plasma GLP-1 analysis, at each timepoint, the whole blood (0.8
mL) was collected
into 1.3 mL EDTA tubes (0.1% EDTA). DPP-IV inhibitor (Millipore - Billerica,
MA) was
added (10 L/mL of blood) to the tubes approximately 5-10 seconds after blood
collection
(yielding a concentration of DPP-IV of 100 M). Tubes were inverted several
times and
immediately placed onto wet ice. Whole blood samples were maintained on wet
ice until
centrifuged, (2 -8 C) at 4000 rpm for approximately 10 minutes, to produce
plasma. Plasma
samples were transferred into appropriate vials and maintained on dry ice
prior to storage in a
freezer at -70 (+10) C. Plasma concentrations (CmaX), TmaX, AUC, and Ti/z
were determined for
GLP-1.
[00215] After inhalation administration of GLP-1/FDKP for four consecutive
days,
detectable levels of GLP-1 were found in all pre-dose samples on Day 5. On Day
5, peak plasma
concentrations (CmaX) of GLP-1 were achieved within approximately 10 minutes
following dose
administration (FIG. 31).
[00216] Dose related increases in GLP-1 CmaX and AUCiast (area under the
concentration-
time curve from time zero to the time of the last quantifiable concentration)
as a function of the
dose were observed in both male and female monkeys on Day 5. Over the dose
range studied,
less than dose proportional increases in GLP-1 AUCiast were observed with
increasing doses in
both male and female monkeys, except for males at the 1 mg/kg/day dose level.
A 6.7 fold
increase in dose from 0.3 to 2.0 mg/kg/day only resulted in a 2.9 fold
increase in AUCiast in
males and 1.1 fold increase in AUCiast in females.
[00217] The peak concentration of GLP-1 averaged 17.2, 93.1 and 214 pg/mL in
males and
19.3, 67.9 and 82.8 pg/mL in females when administered GLP-1/FDKP at dose
levels of 0.3, 1.0
and 2.0 mg/kg/day respectively. Plasma levels of GLP-1 declined rapidly with
apparent
elimination half-lives ranging from 4 minutes to 24 minutes.
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[00218] The AUC values for GLP-1 were 21.6, 105 and 62.3 pg*h/mL in males and
33.4
23.7 and 35.4 pg*h/mL in females when administered GLP-1/FDKP at dose levels
of 0.3, 1.0
and 2.0 mg/kg/day respectively.
[00219] There were no apparent gender differences in TK parameters of GLP-1
observed at
the lowest dose level. However, male monkeys displayed consistently higher
AUCiast values
than female monkeys at the mid and high dose levels investigated. Some samples
from the
vehicle control and control (air) monkeys showed measurable levels of GLP-l.
This may have
been caused by the contamination of the air inhaled by the animals or may have
been a measure
of endogenous GLP-1 in those particular monkeys. It should be noted that
control animals were
exposed in different rooms to the GLP-1/FDKP treated animals.
[00220] Since the biological half-life of GLP-1 is less than 15 minutes, the
GLP-1 from the
administration of GLP-1/FDKP should be completely eliminated within 24 hours.
Therefore,
endogenous levels of GLP-1 were the likely explanation for consistently
quantifiable levels of
GLP-1 in time zero samples collected on Day 5 in all GLP-1/FDKP treated
animals. Subtracting
the time zero values from the observed concentrations of GLP-1 post dosing
should reflect the
change in GLP-1 due to the administration of GLP-1/FDKP.
[00221] For serum FDKP analysis, at each timepoint, the whole blood (0.6 mL)
was
collected into tubes containing no anticoagulant, allowed to clot at room
temperature for a
minimum of 30 minutes and separated by centrifugation to obtain serum. FDKP
analysis and
serum concentrations (Cm,,x), Tm,,x, AUC, and Ti/z) were determined. After
inhalation
administration of GLP-1/FDKP for four consecutive days, detectable levels of
FDKP were found
in all post-dose samples on Day 5. On Day 5, peak plasma concentrations (CmaX)
of FDKP were
achieved approximately 10 to 30 minutes following dose administration.
[00222] There was a dose related increase in FDKP AUCcc (area under the
concentration-
time curve from time zero extrapolated to the infinite time), as a function of
the dose, observed
in both male and female monkeys on Day 5. However, in females there was no
difference in
FDKP AUCcc, between 0.3 and 1.0 mg/kg/day but a dose related increase was
noted between 1
and 2 mg/kg/day. In all instances where an increase was observed, it was less
than dose
proportional. A 6.7 fold increase in dose from 0.3 to 2.0 mg/kg/day resulted
in a 2.7 fold
increase in AUCiast in males and 3.0 fold increase in AUCoo in females. The
peak concentration
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(CmaX) of FDKP averaged 200, 451 and 339 ng/mL in males and 134, 161 and 485
ng/mL in
females administered GLP-1F/DKP at dose levels 0.3, 1.0 and 2.0 mg/kg/day
respectively. The
AUCcc values for FDKP were 307, 578 and 817 ng.h/mL in males and 268, 235 and
810
ng.h/mL in females administered GLP-1/FDKP at dose levels of 0.3, 1.0 and 2.0
mg/kg/day
respectively. AUCoo and Cm~ levels in animals administered FDKP only at a dose
of 2.1
mg/kg/day (Group 2) were of the same order of magnitude as animals receiving
GLP-1/FDKP at
2.13 mg/kg/day, with the exception that the TmaX was slightly longer at 30 to
45 minutes
following dose administration.
[00223] Overall, GLP-1/FDKP was well tolerated with no clinical signs or
effects on body
weights, food consumption, clinical pathology parameters, macroscopic or
microscopic
evaluations. It is also noted that inhalation administration of GLP-1/FDKP to
cynomolgus
monkeys at estimated achieved doses of up to 2.13 mg/kg/day (corresponding to
a dose of 0.26
mg/kg/day GLP-1) administered for 30 minutes a day for 5 days is not
associated with any dose
limiting toxicity.
Example 15
Toxicokinetics of GLP-1/FDKP Administered for 14 days via Pulmonary
Insufflation in
Rats
[00224] This study evaluated the potential toxicity of GLP-1/FDKP after daily
administration via pulmonary insufflation for 14 consecutive days. Rats
received control (air),
FDKP particles at 10 mg/kg, or 1 (0.15 mg GLP-1), 3 (0.45 mg GLP-1) or 10 (1.5
mg GLP-1)
mg/kg GLP-1/FDKP as a daily pulmonary insufflation for 14 consecutive days
(n=24/sex/group). Animals were observed daily for clinical signs of toxicity;
body weight and
food consumption were also recorded.
[00225] On Days 1 and 14, GLP-1 Cm,,x was achieved within approximately 10 to
15 minutes
following dose administration in all dose groups. Peak concentrations of GLP-1
at 10 mg/kg/day
GLP-1/FDKP averaged 6714 and 6270 pg/mL on Day 1 and 2979 and 5834 pg/mL on
Day 14 in
males and females, respectively. Plasma levels of GLP-1 declined with apparent
elimination
half-lives ranging from 0.7 hours to 4.4 hours. Mean AUC levels of GLP-1 were
2187 pM*h in
males and 2703 pM*h in females at the highest dose of 10 mg/kg/day GLP-1/FDKP.
Minimal or
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no accumulation of GLP-1 was observed and there were no gender differences in
Cm~, half-life
and TmaX. AUC values of GLP-1 were slightly higher in female rats than in male
rats across all
doses. The No Observable Adverse Effect Level (NOAEL) in rats administered GLP-
1/FDKP
for 14 consecutive days via pulmonary insufflation was 10 mg/kg/day GLP-1/FDKP
(1.5
mg/kg/day GLP-1).
[00226] Approximately 24 hours after the final dose, animals (12/sex/group)
were sacrificed;
clinical pathology, macroscopic and microscopic evaluations were performed.
The toxicokinetic
(TK) satellite animals (12/sex/group) were sacrificed on Day 14 of dosing
after the final blood
collection. There were no deaths or clinical observations related to GLP-
1/DKP. There were no
differences in body weights or in food consumption between control and treated
animals. At 10
mg/kg GLP-1/FDKP in females only, liver weights and liver to body weight
ratios were
significantly lower compared to the control (air) group.
[00227] There were no clear differences noted from the results for hematology,
coagulation,
chemistry, urinalysis, or urine chemistry between rats administered vehicle
and air controls.
There were no gross or histopathological findings in tissues that were
determined to have
potential toxicity due to administration of GLP-1/FDKP.
Example 16
Toxicokinetics of GLP-1/FDKP Administered for 28 days via Pulmonary
Insufflation in
Monkeys
[00228] This study evaluated toxicity and toxicokinetics of GLP-1/FDKP
administered daily
via inhalation for at least 4 weeks. To assess the reversibility, persistence
or delayed occurrence
of any effects, there was a 4-week recovery period.
[00229] Animals received one of the following treatments: Group 1: control
(air); Group 2:
3.67 mg/kg/day FDKP particles; Group 3: 0.3 mg/kg/day GLP-1/FDKP
(0.045mg/kg/day GLP-
1); Group 4: 1 mg/kg/day GLP-1/FDKP (0.15mg/kg/day GLP-1) or Group 5: 2.6
mg/kg/day
GLP-1/FDKP (0.39 mg/kg/day GLP-1). Forty-two cynomolgus monkeys were divided
into 2
groups: main study (n = 3/sex/group) and recovery (n= 2/sex/group) in groups
1, 2, and 5. Group
1: air control Group 2: FDKP (-4 mg/kg/day); Group 3: 0.3 mg/kg/day GLP-1/FDKP
(low
dose); Group 4: .0 mg/kg/day GLP-1/FDKP (mid dose); Group 5: 2.6 mg/kg/day GLP-
1/FDKP
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(high dose). As is typically, in monkey studies only the high dose and
controls were evaluated at
recovery.
[00230] Animals were observed twice daily for mortality and morbidity and at
least once
daily, 30 minutes post-dose, for abnormalities and signs of toxicity. Body
weight data was
collected weekly and qualitative food consumption was assessed daily. Blood
was collected for
toxicokinetics on Days 1, 28, and 56. Three animals/sex/group were
anesthetized, weighed,
exsanguinated, and necropsied on Day 29. The remaining animals in Groups 1, 2
and 5
(n=2/sex/group) were anesthetized, weighed, exsanguinated, and necropsied on
Day 57. At
necropsy, selected organs were weighed and selected tissues were collected and
preserved. All
tissues from each animal were examined microscopically.
[00231] There were occasional fluctuations in body weight across all groups;
however, there
was no treatment related effect on body weight. Generally, all animals
maintained or gained
minor amounts of weight over the course of the study. Higher incidence and
frequency of loose
or liquid feces was observed at high doses. There were no significant changes
noted in any
clinical chemistry parameters that were considered to be treatment-related
with the exception of
a moderate increase in lactate dehydrogenase (LDH) and aspartate
aminotransferase (AST) in
high dose females at Day 29 (the end of treatment); see Table 7. The levels of
LDH were also
very slightly raised in males. These changes had resolved by the end of the
recovery period and
were not correlated to any microscopic findings in the liver. The change in
AST levels in the
high dose female group was primarily due to one out of the five animals.
Table 7: Mean % change in ALT, AST and LDH
% Change in Mean Value
Group ALT (u/L) AST (U/L) LDH (U/L)
Females
1. Control -2 52 -9
2. 3.67 mg/kg/day FKDP -13 -34 -53
3. 0.3 mg/kg/day GLP-1/FKDP -11 53 -14
4. 1.0 mg/kg/day GLP-1 /FKDP -15 9 -11
5. 2.6 mg/kg/day GLP-1/FKDP 32 422 117
Males
1. Control -16 -42 -62
2. 3.67 mg/kg/day FKDP 14 60 -6
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3. 0.3 mg/kg/day GLP-1/FKDP 24 168 69
4. 1.0 mg/kg/day GLP-1/FKDP 49 32 7
5. 2.6 mg/kg/day GLP-1/FKDP -16 30 6
[00232] There was no evidence of any treatment-related macroscopic or
histological changes
at dose levels up to 2.6 mg/kg/day GLP-1/FDKP. GLP-1/FDKP was well tolerated
with no
significant clinical signs or effects on body weights, food consumption,
hematology, urinalysis,
insulin analysis, ophthalmoscopy, ECG, macroscopic or microscopic changes
observed in doses
up to 2.6 mg/kg/day GLP-1/FDKP (0.39 mg/kg/day GLP-1). Inhalation
administration of FDKP
at an estimated achieved dose of up to 3.67 mg/kg/day for 28 days for up to 30
minutes a day
was also not associated with any toxicity.
[00233] Dose related increases in GLP-1 and FDKP Cmax and AUCiast as a
function of dose
were observed in both male and female monkeys on Day 1. Over the dose range
studied, less
than dose-proportional increases in GLP-1 Cmax but not AUCiast were observed
with increasing
doses in both male and female monkeys on Day 28. Peak concentrations of GLP-1
at 2.6
mg/kg/day GLP-1/FDKP averaged 259 pg/mL in males and 164 pg/mL in females.
Plasma levels
of GLP-1 declined with elimination half lives varying from 0.6 to 2.5 hours.
Mean AUC values
for GLP-1 were 103 pg*hr/mL in males and 104 pg*hr/mL in females at the high
dose. Female
monkeys displayed higher AUC and Cmax values at the low dose compared to
males. Peak
concentrations of FDKP at 2.6 mg/kg/day GLP-1/FDKP averaged 1800 ng/mL in
males and
1900 pg/mL in females.
[00234] In conclusion, inhalation administration of GLP-1/FDKP to cynomolgus
monkeys at
estimated achieved doses of up to 2.6 mg/kg/day GLP-1/FDKP or 0.39 mg/kg/day
GLP-1
administered for 28 days for up to 30 minutes a day was clinically well
tolerated. The NOAEL
was 2.6 mg/kg/day GLP-1/FDKP (0.39 mg/kg/day GLP-1). As described in Example
19 below,
the maximum human dose in the Phase I study will be 1.5 mg GLP-1/FDKP per day
or -0.021
mg/kg GLP-1 (assuming 70 Kg human). Additional studies will dose to 3.0 mg GLP-
1/FDKP per
day or -0.042 mg/kg GLP- 1.
Example 17
Preparation of Exendin/FDKP Formulations
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[00235] Exendin-4/FDKP was prepared by combining an acidic exendin-4 peptide
(SEQ ID
No. 3) solution with a FDKP particle suspension. The acidic peptide solution
was 10% (w/w) of
peptide dissolved in 2% acetic acid. The FDKP suspension contained
approximately 10% (w/w)
FDKP particles. The acidic exendin-4 peptide solution was added to the FDKP
particle
suspension as it gently mixed. The exendin-4/FDKP mixture was slowly titrated
with a 25%
ammonia solution to pH 4.50. The mixture was then pelleted into liquid
nitrogen and
lyophilized.
[00236] The % Respirable Fraction on Fill (%RF on Fill) contents for a 15%
Exendin-
4/FDKP powder was 36%, with a Percent Cartridge Emptying of 99%. A 15% GLP-
1/FDKP
powder produced at a similar scale showed a %RF on Fill contents of 34%, with
a Percent
Cartridge Emptying of 100%.
Example 18
Pharmacokinetics of Exendin/FDKP Administered via Pulmonary Insufflation
[00237] Repeat dose preliminary toxicity studies to examine the
pharmacodynamic and
pharmacokinetics profile of exendin-4 (a GLP-1 analogue) in an exendin-4/FDKP
formulation at
various concentrations, and after multiple administrations via the pulmonary
route are in
progress.
[00238] Twenty-eight day studies in rats and monkeys are performed.
Exendin/FDKP is
dosed daily, via the inhalation route. In studies where animals are dosed for
28 days, a
proportion of the animals are sacrificed immediately after the dosing regimen
while other
animals are allowed up to a one month recovery period prior to sacrifice. All
animals are
evaluated for clinical signs of toxicity; various physiological parameters
including blood levels
of Exendin-4, glucose, and insulin; organ weights, and clinical pathology and
histopathology of
various organs.
[00239] The intial study groups consisted of five animals per group with two
control groups:
air and Exendin administered intravenously. There were six pulmonary
insufflation groups
which received approximately 2.0 mg doses of Exendin/FDKP at 5%, 10%, 15%, 20%
and 25%,
and 30% Exendin load (w/w). Whole blood was collected for blood glucose and
Exendin
concentrations out to an 8 hour time point.
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[00240] The data (Cm,,,, Ti/z and TmaX), are collected, demonstrating that
Exendin/FDKP
formulations have comparable or better pharmacokinetics than GLP-1/FDKP.
Example 19
Pharmacokinetics of GLP-1/xDKP Administered via Pulmonary Insufflation in Rats
[00241] To determine whether different DKPs may influence the pharmacokinetic
profile of
GLP-1/FDKP formulations, various GLP-1/xDKP formulations were made as
disclosed in U.S.
Provisional Patent Application entitled "Asymmetrical FDKP Analogs for Use as
Drug Delivery
Agents" filed on even date herewith and incorporated herein in its entirety
(Atty Docket No.
51300-00041).
[00242] Studies were conducted in rats divided into 6 treatment groups
consisting of five
animals per group. The control group (n=3) received GLP-1 via liquid
instillation. GLP-1/FDKP
(0.3mg GLP-1), administered by pulmonary insufflation, was also used as a
second control.
Each of the GLP-1/xDKP treated groups received GLP-1/xDKP formulations via
pulmonary
insufflation at -2.0mg doses of xDKP loaded with GLP-1 at 10% and 15%. The
xDKPs used
were (E)-3-(4-(3,6-dioxopiperazin-2-yl)butylcarbamoyl)-acrylic acid), (3,6-
bis(4-
carboxypropyl)amidobutyl-2,5-diketopiperazine), and ((E)-3,6-bis(4-(Carboxy-2-
propenyl)amidobutyl)-2,5-diketopiperazine disodium salt) loads. Whole blood
was collected for
evaluation of GLP-1 concentrations at 5, 10, 20, 30, 45, 60 and up to 90
minutes post dose.
Example 20
A Phase la, Sin2le-Dose, Open-Label, Ascendin2 Dose, Controlled Safety and
Tolerability
Trial of GLP-1/FDKP Inhalation Powder in Healthy Adult Male Subiects
[00243] GLP-1 has been shown to control elevated blood glucose in humans when
given by
intravenous (iv) or subcutaneous (sc) infusions or by multiple subcutaneous
injections. Because
of the extremely short half-life of the hormone, continuous subcutaneous
infusion or multiple
daily subcutaneous injections would be required. Neither of these routes is
practical for
prolonged clinical use. Experiments in animals showed that when GLP-1 was
administered by
inhalation, therapeutic levels could be achieved.
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[00244] Several of the actions of GLP-l, including reduction in gastric
emptying, increased
satiety, and suppression of inappropriate glucagon secretion appear to be
linked to the burst of
GLP-1 released as meals begin. By supplementing this early surge in GLP-1 with
GLP-1/FDKP
inhalation powder a pharmacodynamic response in diabetic animals can be
elicited. In addition,
the late surge in native GLP-1 linked to increased insulin secretion can be
mimicked by post-
prandial administration of GLP-1/FDKP inhalation powder.
[00245] The Phase 1 a clinical trial of GLP-1/FDKP inhalation powder is
designed to test the
safety and tolerability of selected doses of a new inhaled glycemic control
therapeutic product
for the first time in human subjects. Administration makes use of the MedTone
Inhaler device,
previously tested. The primary intent of this clinical trial is to identify a
range of doses for GLP-
1/FDKP inhalation powder by pulmonary inhalation that are safe, tolerable and
can be used in
further clinical trials to establish evidence of efficacy and safety. The
doses selected for the
phase la clinical trial are based on animal safety results from non-clinical
trials of GLP-1/FDKP
inhalation powder described in above Examples, in rats and primates.
[00246] Twenty-six (26) subjects are enrolled into 5 cohorts achieve up to 4
evaluable
subjects in each of cohorts 1 and 2 and up to 6 evaluable subjects in each of
cohorts 3 to 5 who
meet eligibility criteria and complete the clinical trial. Each subject is
dosed once with
Glucagon-Like Peptide-1 (GLP-1) as GLP-1/FDKP Inhalation Powder at the
following dose
levels: cohort 1: 0.05 mg; cohort 2: 0.45 mg; cohort 3: 0.75 mg; cohort 4:
1.05 mg and cohort 5:
1.5 mg of GLP-1. Dropouts will not be replaced. These dosages assume a body
mass of 70 kg.
Persons of ordinary skill in the art can determine additional dosage levels
based on the studies
disclosed above.
[00247] The objectives of this trial are to determine the safety and
tolerability of ascending
doses of GLP-1/FDKP inhalation powder in healthy adult male subjects. The
tolerability of
ascending doses of GLP-1/FDKP inhalation powder as determined by monitoring
pharmacological or adverse effects on variables, including reported adverse
events (AE), vital
signs, physical examinations, clinical laboratory tests and electrocardiograms
(ECG) will be
evaluated.
[00248] The secondary objectives are to evaluate additional safety and
pharmacokinetic
parameters. These include additional safety parameters, as expressed by the
incidence of
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pulmonary and other AEs and changes in pulmonary function between Visit
1(Screening) and
Visit 3 (Follow-up); pharmacokinetic (PK) parameters of plasma GLP-1 and serum
fumaryl
diketopiperazine (FDKP) following dosing with GLP-1/FDKP inhalation powder, as
measured
via AUCo-120(min) plasma GLP-1 and AUCo-48o min serum FDKP; and additional PK
parameters of
plasma GLP-1 include: tm.plasma GLP-l; Cm.plasma GLP-l; and T1/2plasma GLP-l.
Additional
PK parameters of serum FDKP include: Tm. serum FDKP; Cm. serum FDKP; and T~/2
serum
FDKP.
[00249] Trial Endpoints are based on a comparison of the following
pharmacological and
safety parameters determined in the trial subject population. Primary
endpoints will include:
Safety endpoints will be assessed based on the incidence and severity of
reported AEs, including
cough and dyspnea, nausea and/or vomiting, as well as changes from screening
in vital signs,
clinical laboratory tests and physical examinations. Secondary endpoints will
include: PK
disposition of plasma GLP-1 and serum FDKP (AUCo-12o min plasma GLP-1 and AUCo-
48o min
serum FDKP); additional PK parameters of plasma GLP-1 (Tmax plasma GLP-l, Cmax
plasma
GLP-1 T~/2 plasma GLP-1; additional PK parameters of serum FDKP (Tmax serum
FDKP, Cmax
serum FDKP); and additional safety parameters (pulmonary function tests
(PFTs)) and ECG.
[00250] The Phase la, single-dose trial incorporates an open-label, ascending
dose structure
and design strategy that is consistent with 21 CFR 312, Good Clinical
Practice: Consolidated
Guidance (ICH-E6) and the Guidance on General Considerations for Clinical
Trials (ICH-E8) to
determine the safety and tolerability of the investigational medicinal product
(IMP).
[00251] The clinical trial will consist of 3 clinic visits: 1) One screening
visit (Visit 1); 2)
One treatment visit (Visit 2); and 3) One follow-up visit (Visit 3) 8-14 days
after Visit 2.
Administration of a single dose of GLP-1/FDKP inhalation powder will occur at
Visit 2.
[00252] This clinical trial will evaluate safety parameters in each cohort.
The cohort
scheduled to receive the next dose concentration will not be dosed until a
review of all safety and
tolerability data for the first or prior doses is conducted by the principal
investigator (PI). A half-
hour dosing lag time will be implemented between subjects in each cohort to
ensure subject
safety. The dose may be halted if 3 or more subjects within a cohort,
experience severe nausea
and/or vomiting or when the maximum dose is reached, or at the discretion of
the PI.
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[00253] Five doses of GLP-1/FDKP inhalation powder (0.05, 0.45, 0.75, 1.05 and
1.5 mg of
GLP-1) will be assessed. To accommodate all doses, formulated GLP-1/FDKP will
be mixed
with FDKP inhalation powder. Single-dose cartridges containing 10 mg dry
powder consisting of
GLP-1/FDKP inhalation powder (15% weight to weight GLP-1/FDKP) as is or mixed
with the
appropriate amount of FDKP inhalation powder will be used to obtain the
desired dose of GLP- 1
(0.05 mg, 0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg).: 1. The first 2 lowest dose
levels will be
evaluated in 2 cohorts of 4 subjects each and the 3 higher dose levels will be
evaluated in 3
cohorts of 6 subjects each. Each subject will receive only 1 dose at 1 of the
5 dose levels to be
assessed. In addition to blood draws for GLP-1 (active and total) and FDKP
measurements,
samples will be drawn for glucagon, glucose, insulin and C-peptide
determination.
[00254] Numerous references have been made to patents and printed publications
throughout this specification. Each of the above-cited references and printed
publications are
individually incorporated herein by reference in their entirety.
[00255] Unless otherwise indicated, all numbers expressing quantities of
ingredients,
properties such as molecular weight, reaction conditions, and so forth used in
the specification
and claims are to be understood as being modified in all instances by the term
"about."
Accordingly, unless indicated to the contrary, the numerical parameters set
forth in the following
specification and attached claims are approximations that may vary depending
upon the desired
properties sought to be obtained by the present invention. At the very least,
and not as an
attempt to limit the application of the doctrine of equivalents to the scope
of the claims, each
numerical parameter should at least be construed in light of the number of
reported significant
digits and by applying ordinary rounding techniques. Notwithstanding that the
numerical ranges
and parameters setting forth the broad scope of the invention are
approximations, the numerical
values set forth in the specific examples are reported as precisely as
possible. Any numerical
value, however, inherently contains certain errors necessarily resulting from
the standard
deviation found in their respective testing measurements.
[00256] It is readily apparent to one skilled in the art that various
embodiments and
modifications can be made to the invention disclosed herein, without departing
from the scope
and spirit of the invention.
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[00257] As used herein, the use of the word "a" or "an" when used in
conjunction with the
term "comprising" in the claims and/or the specification may mean "one," but
it is also consistent
with the meaning of "one or more," "at least one," and "one or more than one."
[00258] It is contemplated that any method or composition described herein can
be
implemented with respect to any other method or composition described herein.
[00259] The use of the term "or" in the claims is used to mean "and/or" unless
explicitly
indicated to refer to alternatives only or the alternatives are mutually
exclusive.
[00260] Throughout this application, the term "about" is used to indicate that
a value
includes the standard deviation of error for the device or method being
employed to determine
the value.
[00261] Other objects, features and advantages of the present invention will
become
apparent from the preceeding description and examples as well as the claims.
It should be
understood, however, that the detailed description and the specific examples,
while indicating
specific embodiments of the invention, are given by way of illustration only,
since various
changes and modifications within the spirit and scope of the invention will
become apparent to
those skilled in the art from this detailed description.
REFERENCES
[00262] The following references, to the extent that they provide exemplary
procedural or
other details supplementary to those set forth herein, are specifically
incorporated herein by
reference.
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[00268] Johnson JD et al: RyR2 and calpain-l0 delineate a novel apoptosis
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