Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR ORAL DELIVERY OF PROTEINS
Field of the Invention
The present invention relates to a composition comprising a swellable
hydrogel matrix and a protein contained therein, and the use of such a
composition for
oral delivery of bioactive compounds in an active form to the intestines of a
vertebrate.
Background of the Invention
Two major problems exist in developing oral delivery systems for
proteinaceous compounds such as insulin. T'he first problem is the
inactivation of
many proteins by digestive enzymes in the gastrointestinal (GI) system, mainly
in the
stomach. This can be overcome by designing carriers which would protect the
protein
from the harsh environments of the stomach before releasing the drug into more
favorable regions of the GI tract, specifically the lower regions of the
intestine.
Additionally, protease inhibitors can be used to retard the action of enzymes
present in
the GI system which could degrade orally administered proteins. The other
problem is
the slow transport of intact large peptides, across the lining of the
intestine into the
blood stream. Researchers have attempted to bypass this hurdle with the
addition of
absorption enhancers which aid the transport of macromolecules across
boundaries.
However, currently available delivery vehicles suffer from a lack of
effectiveness.
Accordingly, an oral delivery system is desired that is effective and can be
prepared at
relatively low cost.
Summary of the Invention
The present invention is direcaed to a composition comprising a
hydrogel matrix carrier and a bioactive compound, and the use of that
composition to
deliver the compound in an active form to the intestines. One preferred
hydrogel
matrix comprises a copolymer network of poly(methacrylic acid-g-ethylene
glycol)
crosslinked with tetraethylene glycol dimethacrylate, "P(MAA-g-EG) hydrogels",
that
exhibit pH dependent swelling behavior due to the presence of acidic pendant
groups
and the formation of interpolymer complexes between the etheric groups on the
graft
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chains and protonated pendant groups. In acidic media, such systems are
relatively
unswollen due to the formation of the interpolymer complexes. In basic
solutions, the
pendant groups ionize and the complexes dissociate. The pH dependent swelling
of
these hydrogels together with their bioadhesive properties make these
hydrogels ideal
for oral delivery of proteins.
Brief Description of the Drawines
Fig. 1 Reversible complexation in P(MAA-g-EG) hydrogels. C
represents the entrapped bioactive compound.
Fig. 2 Equilibrium polymer volume fraction as a function of pH for
samples containing PEG grafts of MW 1000 and a MAA:EG molar ratio of 1:1 at
37°C.
Fig. 3 Equilibrium mesh size as a function of pH for samples
containing PEG grafts of MW 1000 and a MAA:EG molar ratio of 1:1 at
37°C.
Fig. 4 Controlled release of proxyphylline in solutions of pH of 3.2 (~)
and 7.4 (~) and vitamin B12 at pH of 3.2 (0) and 7.4 (O) in buffered saline
solutions at
37°C.
Fig. Sa Pulsatile release of theophyllin in-vitro from P(MAA-g-EG)
hydrogels at 37 ° C.
Fig. Sb Pulsatile release of vancomycin in-vitro from P(MAA-g-EG)
hydrogels at 3 7 ° C.
Fig. Sc Pulsatile release of insulin in-vitro from P(MAA-g-EG)
hydrogels at 37°C.
Fig. 6 Adhesive behavior of P{MAA-g-EG) hydrogels containing a 1:1
MAA/EG ratio and graft PEG chains of molecular weight 1000 at pH values of 3.2
and 7.4 in contact with bovine submaxillary gland mucin.
Fig. 7 Blood glucose concentration in rats after oral administration of
insulin loaded P(MAA-g-EG) hydrogels at 25 U/kg (o) and 50 U/kg (~) and 50
U/kg
insulin solution (~) (N = 5).
Fig. 8 Blood glucose concentration in rats after oral administration of
insulin loaded P(MAA-g-EG) hydrogels at (25 U/kg dose) without aprotinin (o)
and
with aprotirun (~) and 50 U/kg insulin solution (~) (N = 5).
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Fig. 9 Blood glucose response in healthy (~) and diabetic (o) male
Wistar rats after the oral administration of P(MAA-g-EG) microspheres (25
IU/kg
body weight insulin doses) using gelatin capsules.
Fig. 10 Blood glucose response in diabetic male rats after oral
administration of P(MAA-g-EG) microsphe:res (25 IU/kg body weight) using
Eudragit
capsules.
Fig. 11 Blood glucose response in healthy dogs (25kg) after the oral
administration of P(MAA-g-EG) microsphe:res (10 ILT/kg body weight insulin
doses)
using gelatin capsules.
Fig. 12 Blood glucose response in diabetic dogs (25 kg) oral
administration of P(MAA-g-EG) microsphe:res ( 10 IIJ/kg body weight insulin
doses)
using gelatin capsules.
Detailed Description of the Invention
The present invention is directed to compositions for delivering
biologically active proteins and pharmaceuticals to vertebrates via oral
administration.
The term bioaetive compound as used herein refers to any compound that has an
effect
on living cells, for example a compound that induces a biochemical effect in a
cell. In
accordance with one embodiment the orally administered composition comprises a
swellable hydrogel matrix, and a labile protein contained within the swellable
hydrogel
matrix. A labile protein as used herein includes any protein whose biological
activity is
destroyed or diminished by exposure to low pH or exposure to enzymes present
in the
digestive tract of warm blooded species.
Hydrogels are water swellable, cross-linked polymer matrices that are
well known to those of ordinary skill in the .art. See, for example, Dresback,
U.S.
Patent No. 4,220,152, issued September 2, 1980, the disclosure of which is
expressly
incorporated herein by reference. Hydrogels have been found to be an effective
delivery vehicle for orally delivering proteins to vertebrate species. The
swellable
properties of hydrogels can be utilized, first to protect the hydrogel
contents from the
harsh environments of the stomach as the composition passes through the
digestive
tract, and then to release the hydrogel contents into the more favorable
regions of the
GI tract, specifically the lower regions of the intestine. The hydrogel
compositions of
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the present invention have been found to pass through the stomach without
substantial
swelling and become localized in the small intestine, where they swell and
concomitantly release their contents.
Hydrogels can be impregnated or loaded with a variety of bioactive
compounds, including but not limited to pharmaceuticals, growth hormones,
vaccine
compositions, vitamins, steroids and peptides, and used as a delivery vehicle
for orally
administering such bioactive compounds. Compounds loaded into the hydrogel are
released in a controlled manner as the hydrogel becomes hydrated within the
animal's
digestive system. In one embodiment, the present hydrogel matrix is in a
pelletized
form comprised of polymethacrylic acid, and the polymethacrylic acid polymers
are
grafted with an ionic long chain polymer such as polyethylene glycol (PEG).
The hydrogel pellets are preferably synthesized by polymerizing
methacrylic acid, in the presence of a crosslinking agent. The crosslinking
agent can
be selected from a wide variety of biocompatible crosslinking agents known to
those
skilled in the art including tetraethylene glycol dimethacrylate, ethylene
dimethacrylate,
diethylene dimethacrylate, triethylene dimethacrylate, tetraethylene
dimethacrylate,
pentaethylene dimethacrylate, the corresponding diacrylates, or a star polymer
comprising methacrylate, acrylate or methylene bis-acrylamido groups.
Polymerization
is initiated with a free radical initiator such as thermal initiators
including organic
peroxides or UV radical initiators known to those skilled in the art.
In one embodiment the hydrogel matrix comprises a co-polymer of
methacrylic acid and a poly(alkylene glycol) monomethacrylate (or
monoacrylate}
crosslinked with a biocompatible crosslinking agents. "Poly(alkylene glycol)
monomethacrylate" as used herein includes polyethylene glycol)
monomethacrylate,
polypropylene glycol) monomethacrylate and poly(ethylene/propylene glycol)
monomethacrylate, wherein poly(ethylene/propylene glycol) monomethacrylate is
the
polymer formed by hydroxy functional methacrylate initiated polymerization of
a
mixture of ethylene oxide and propylene oxide. The resulting pendant
poly(alkylene
glycol) groups have a molecular weight ranging from about 200 to about 4000,
more
typically about 200 to about 2000, and in one embodiment about 200 to about
1200.
The molar ratio of methacrylic acid and poly(alkylene glycol) monomethacrylate
(or
monoacrylate) monomers is about 4:1 to about 1:4.
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In one preferred embodiment the hydrogel matrix comprises a polymer
of methacrylic acid and polyethylene glycol) monomethacrylate crosslinked with
tetraethylene glycol dimethacrylate, "P(MAA-g-EG) hydrogels". In preparation
of that
polymer, polyethylene glycol) monomethacrylate having a molecular weight of
about
200 to about 2000, more typically about 200 to about 1200 is co-polymerized
with
methacryiic acid and tetraethylene glycol dirnethacrylate. The molar ratio of
methacrylic acid and polyethylene glycol) monomethacrylate monomers ranges
from
about 4:1 to about 1:4. In one embodiment,, the molar ratio of methacrylic
acid and
polyethylene glycol) monomethacrylate monomers is about 1:1. The crossIinking
agent is added in an amount of about 0.25 to about 10.00 mol%, more preferably
at
about 0.25 to about 1.00 mol% and in one embodiment, about 0.75 mol%.
The hydrogels can be loaded with the desired compounds using
standard techniques known to those skilled i.n the art. In one embodiment the
P(MAA-
g-EG) hydrogels are formed as microparticles ranging in size from about 50 pm
to
about S00 pm in diameter, more preferably about 100-200 pm in diameter. The
hydrogel microparticles are formed in accordance with one embodiment by
forming a
polymerized matrix and grinding the matrix to form a hydrogel particulate
having the
desired average particle size. The hydrogel microparticles can be loaded with
the
desired compound and packaged in standard tablet or capsule forms using
standard
techniques known to those skilled in the art. In one embodiment the hydrogel
particles
are packaged in a gelatin capsule.
In accordance with one embodiment the hydrogels are loaded with
bioactive compounds by equilibrium portioning. More particularly, the
hydrogels are
hydrated in a solution having a pH>5.8 and containing the composition to be
loaded.
The hydrogels are then recovered and washed with a solution having a pH of <
5.8 and
the loaded hydrogels are then dried and stored at 4°C. Another method
of loading the
hydrogels of the present invention comprises the steps of adding an aqueous
solution
of the desired compound to a solution of monomers and a cross-linker, and
initiating
polymerization of the mixture.
The ability of a compound to diffuse through a crosslinked polymer
network is dependent on the degree to which the gel swells and the size of
compound.
As a hydrogel swells, the polymer chains between crosslink points are
elongated and
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the network mesh size or correlation length, ~, is increased allowing for
greater solute
permeation in the material. (See Fig. 1) In equilibrium swollen complexation
networks, the degree of network swelling is dependent on the number of
chemical and
physical crosslinks present in the system. In P(MAA-g-EG) networks, the
equilibrium
swelling ratio is strongly dependent on the pH of the surrounding environment.
The P(MAA-g-EG) hydrogels of the present invention, form temporary
physical crosslinks when exposed to acidic conditions (typically pH<5.8) due
to
hydrogen bonding between the polymethacrylate groups and the pendant
poly(alkylene
glycol) groups. These physical crosslinks are reversible in nature and
dependent on the
pH and ionic strength of the environment. Thus, the degree of crosslinking and
network mesh size, a, are strongly dependent on the pH and ionic strength of
the
surrounding environment. In acidic media, such systems are relatively
unswollen due
to the formation of the intermacromolecular complexes. In basic solutions, the
pendant groups ionize and the complexes dissociate. The equilibrium swelling
of
IS P(MAA-g-EG) hydrogels is shown in Fig. 2. The data is presented as the
polymer
volume fraction of the hydrogels (PEG grafts of 1000MW and MAA:EG molar ratio
of 1:1 ) as a function of pH. For the case of equimolar amounts of MAA and EG
at
low pH values, the degree of complexation was high and the polymer volume
fraction
in the gel in the swollen state, via, was almost 0.70. However, as the pH of
the
swelling solution increased above pH = 4.6, the complexes began to dissociate
and the
backbone chains extended resulting in a significant decrease in the
equilibrium polymer
volume fraction in the gel. The highly swollen, non-complexed hydrogels
contained
less than S% polymer as more water was incorporated into the structure.
Because of the complexation/decomplexation phenomena in the
P(MAA:g-EG) gels, the mesh size of the networks will vary significantly over
the pH
range of interest. Additionally, the moduli of the hydrogels for small
deformations
(less than 10%) were obtained in solutions of differing pH. Using these data,
the mesh
sizes were calculated as a function of pH by determining the end-to-end
distance of the
polymer chains between the crosslinks, both covalent and physical. The average
network mesh size or correlation length was dramatically affected by the pH of
the
swelling solution (Fig. 3). In low pH solutions in which complexation will
occur, the
network mesh sizes for P(MAA-g-EG) hydrogels were as low as 70 A. However, as
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the pH was increased the physical crosslinks dissociated and the polymer
chains
elongated resulting in an increase in the network mesh size by a factor of 3,
to almost
210 ~. More importantly, assuming ideal neaworks, the available area for
diffusion is
equal to the square of the mesh size. Thus, there exists 9 times greater area
for
diffusion in the non-complexed hydrogels (plH greater than 5.2) than the
complexed
hydrogels (pH less than 5.2). Because of the; reversible nature of the
cornplexation
phenomena, the P(MAA-g-EG) hydrogels are ideal for the oscillatory release of
drugs.
Additionally, due to the large changes in the network structure over a small
pH
change, these materials function well as carriers for peptides and proteins.
In
particular, the P(MAA-g-EG) hydrogels of the present invention can serve as a
delivery vehicle for compounds having a mollecular weight ranging from about
1,000 to
about 100,000, more preferably ranging from about 1,000 to about 20,000
The important parameter in evaluating the potential of a gel to serve as
a carrier for a particular drug is the ratio of the effective molecular size
(hydrodynamic
diameter, d~ to the network mesh size. In order to study the size-exclusion
characteristics of these networks, the release of two solutes of differing
molecular size,
proxyphylline (molecular weight 238 and d,, - 4.3 fir) and vitamin B,2
(molecular
weight 1355 and d,, = 17 ~), from complexe~d and non-complexed hydrogels was
studied (Fig. 4). In solutions of pH = 3.2, tine hydrogel polymers were highly
complexed, and the transport of drug was sil;nificantly hindered. Less than
10% of the
vitamin B1z diffused out of the network in two hours. However, due to its
smaller size,
almost 30% of the proxyphylline was released from the gel in the same time
period.
When the hydrogels were contacted with a solution of pH = 7.4, the interchain
complexes of the hydrogel disassociated due to ionization of the pendant acid
groups.
As a result, the hydrogels swelled to a large degree allowing for substantial
diffusion of
vitamin B12 and proxyphylline from the polymers.
The release data were fit to the short time approximation for the
solution of the classical Fickian expression for planar systems and the
diffusion
coefficients were calculated for the diffusion of proxyphylline and vitamin
B,z through
complexed and non-complexed P(MAA-g-EG) hydrogels (Table 1). The transport of
the larger molecular weight solute, vitamin Et,2, was more significantly
affected by
complexation than proxyphylline due to the increased ratio of solute diameter
to the
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network mesh size. The diffusion coefl=icient for vitamin B,z from the non-
complexed
hydrogels was two orders of magnitude higher than that for the complexed
hydrogels,
while the proxyphylline diffusion coefficient was only one order of magnitude
higher
for the non-complexed hydrogels relative to the complexed hydrogels.
Table 1. Diffusion coefficients for proxyphylline and vitamin B,2 in compiexed
and
non-complexed P(MAA-g-EG) hydrogels.
Solute pH ~(t~) d,,/~ D3.,Zx108
(cm2/s)
proxyphylline 3.2 70.8 0.060 0.403
proxyphylline 7.4 194.4 0.022 9.38
vitamin B,z 3.2 70.8 0.240 0.0168
vitamin B,2 7.4 194.4 0.087 6.75
To further investigate the ability of P(MAA-g-EG) hydrogels to
function as oral delivery vehicles for vitamins, pharmaceuticals and other
bioactive
compounds, the pulsatile release of various compounds was determined under
simulated gastrointestinal conditions. In vitro release experiments were
performed
using theophylline (MW=180.2) vancomycin (MW=1485.7) and insulin (MW=5733.2}.
See Example I . Each of the compounds was loaded into P(MAA-g-EG) hydrogels by
equilibrium partitioning and then the loaded hydrogels were soaked in 200 ml
of pH =
1.2, simulated gastric fluid for 2 hours. The polymer microparticles were then
transferred to pH = 6.8 phosphate buffer solutions. The insulin concentration
released
into the surrounding solution was monitored using HPLC and the results for
theophylline, vancomycin and insulin are shown in Fig. Sa-Sc respectively.
Release of the compounds from the hydrogel matrix is reduced in the
acidic solution (see Fig. 2, first two hours of exposure). However, a rapid
release of
the compound is observed in the pH 6.8 buffer solution. This trend became more
pronounced as the drug molecular weight increased. For example the P(MAA-g-EG)
hydrogels are effective delivery vehicles for insulin (MW = 5733.2): Less than
10% of
the insulin was released from the polymer in the simulated gastric fluid (pH -
1.3)
during the first phase of the experiment. However, after the particles were
placed in
pH = 7.4 buffer solution, the hydrogels swelled rapidly allowing for a rapid
release of
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insulin. These results indicate that the graft copolymers P(MAA-g-EG) are
useful for
development of oral insulin delivery system. As used herein the term insulin
is
intended to include purified human and animal natural insulin as well as
derivatives
thereof, such as insulin lispro and recombinant forms of insulin, and mono or
divalent
salts of insulin or insulin derivatives.
Additionally, P(MAA-g-EG) hydrogels exhibit strong mucoadhesive
characteristics due to the presence of the grafted PEG chains, which serve as
adhesion
promoters. Furthermore, the mucoadhesive characteristics of P(MAA-g-EG)
hydrogels is strongly dependent on the pH o~f the environmental fluid (See
Fig. 6).
The adhesion between the gel and the mucous is significantly greater in
conditions
simulating the intestinal pH (pH = 7.4) relative to conditions simulating the
stomach
environment. However, to truly compare thc: mucoadhesive characteristics of
the gels,
the work of adhesion was normalized to account for the polymer gel fraction.
The
normalized work of adhesion was two-orders of magnitude greater for hydrogels
in the
non-complexed state. Accordingly, the mucoadhesive properties of the P(MAA-g-
EG)
hydrogels wilt be relatively low as they pass through the stomach and remain
in a
complexed state. After reaching the intestinca the interchain complexes will
dissociate,
thus enhancing the adhesion to the hydrogels; to the mucosa of the intestine
relative to
stomach mucosa. Therefore, the residence time of insulin carriers is much
greater in
regions where the insulin cauld be absorbed (i.e. in the intestine) after oral
administration to a vertebrate.
The differences in the adhesive characteristics of the hydrogels at
different pH values are due to mobility of the: PEG chains in each material.
In the
highly swollen, non-complexed state, the pendant PEG chains are free and
readily
penetrated the mucosa to serve as anchors for adhesion. In the complexed
state, the
pendant PEG chains in the P(MAA-g-EG) form complexes with the backbone chains
and are unavailable for interactions with mucosal surfaces.
In accordance with the present invention the hydrogel compositions can
be utilized to administer a therapeutically effl~ctive amount of a protein to
a vertebrate.
The method comprises the step of orally administering to a vertebrate, a
composition
comprising the protein contained within a hydrogel carrier. The composition
contained
within the hydrogel matrix may further comprise protease inhibitors,
pharmaceutically
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acceptable carriers, stabilizing agents and biocompatible fillers known to
those of
ordinary skill in the art.
One preferred hydrogel carrier is P(MA.A-g-EG), and in one
embodiment the P(MA.A-g-EG) hydrogel matrix contains a pharmaceutically
S acceptable composition comprising insulin. Furthermore, in accordance with
one
embodiment the insulin composition further comprises a protease inhibitor or
an
absorption enhancer. Compositions comprising insulin contained within a P(MAA-
g-
EG) hydrogel have been shown to be surprisingly effective in delivering
insulin to the
blood stream of animals (see Examples 3 and 4). The hydrogel matrix is
typically
prepared in particulate form and packaged within a suitable oral delivery
vehicle (i.e.
tablet, capsule, etc.) using techniques known to those skilled in the art.
In one embodiment, the delivery system consists of microparticles of
crosslinked copolymers of poly{methacrylic acid) and polyethylene glycol) and
contains insulin. This system is particularly effective because the structure
of the
copolymers exhibits pH sensitive swelling behavior that allows for protection
of the
insulin while the composition passes through the harsh environment of the
stomach.
The pendant PEG chains also serve as adhesive promoters to increase the
resident time
of the hydrogel carrier at the intended delivery site. As noted in Example 2
the
mucoadhesive properties of the hydrogels is strongly influenced by pH thus
favoring
adhesion to intestinal surfaces over the surface of the stomach. Additionally,
the
presence of the pendant PEG polymers serve as peptide stabilizers and help
maintain
the biological activity of bioactive compounds such as insulin.
The inter-chain complex formation in the hydrogel copolymers is
sensitive to the nature and pH of the surrounding fluid as well as the
copolymer
composition and graft chain length. In the acidic environment of the stomach,
the
hydrogels are in the complexed state due to the formation of interpolymer
complexes
stabilized by hydrogen bonding between the carboxylic acid protons and the
etheric
groups on the grafted chains. In these conditions compounds having a molecular
weight size of at least 1000 (insulin, for example) cannot readily diffuse
through the
membrane because of the small pore size, ~, and thus these compounds are
protected
from the harsh environment of the stomach. As the particles pass through the
stomach
and into the intestine, the environmental pH increases above the transition pH
of the
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gel. The complexes immediately dissociate and the network pore size rapidly
increases
leading to the release of compounds having a molecular weight size of less
than
100,000. Accordingly the P(MAA-g-EG) h;ydrogels can be used as an effective
oral
delivery vehicle for compounds having a molecular weight ranging from about
1,000 to
about 100,000.
Ezample 1
pH Dependent Release of P(MAA-g-EG) H,ydrogel Contents
The ability of P(MAA-g-EG) hydrogels to function as delivery vehicles
was investigated for three compounds of different sizes: theophylline (MW
180.2),
vancomycin (MW 1485.7) and insulin (MW 5733.2).
P(MAA-g-EG) hydrogels were prepared at 37° C by free-radical
solution polymerization of methacrylic acid .and polyethylene glycol)
monomethacrylate, and the oligomer chains were crosslinked with tetraethylene
glycol
dimethacrylate. The ensuing hydrogels werE: rinsed for a week in deionized
water to
remove unreacted monomer and non-crosslinked oligomer chains, dried under
vacuum
and ground into a powder having an averagE; particulate diameter ranging from
100-
150 pm.
Drug incorporation experiments were performed using theophyliine
(MW 180.2), vancomycin (MW 1485.7) and insulin (MW 5733.2). Each drug was
dissolved in a pH 7.4 phosphate bui~er solution and P(MAA-g-EG) hydrogels were
added to the drug solution to load the hydrogels by equilibrium partitioning.
The
hydrogel matrix was then contacted with an acid solution to induce the
formation of
interpolymer complexes, and thus reduce the pore size of the hydrogel matrix.
The
hydrogel microspheres were then collected by filtration, and dried under
vacuum.
Incorporation effic''rencies were calculated from the residual drug amount of
the
concentrations of the initial solutions and the filtrate obtained from the
washings of the
isolated hydrogels, as determined from HPL,C analysis.
Drug release experiments were performed following the Japanese
pharmacopoeia (JP) paddle method. The compositions were stirred with a paddle
at
100 rpm and 37° C in a first (pH 1.2) and a second (pH 6.8) fluid of
JP. After 2 hours
of treatment with the first fluid, the polymer samples were collected by
filtration and
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transferred to the second fluid of pH 6.8. The drug concentration was
monitored by
HPLC.
The mean insulin incorporation efficiency into the hydrogel matrix
reached 94% at 30 min after starting the experiment, thus the polymer is
thought to be
a suitable carrier for insulin. The results of the release experiments for
theophylline,
vancomycin and insulin from P(MAA-g-EG) hydrogels are shown in Fig. Sa, Sb and
Sc, respectively. Release of the compounds from the hydrogel matrix is reduced
in the
acidic solution {see first two hours of exposure). However, a rapid release of
the
compound is observed in pH 6.8 buffer solution. This trend became more
pronounced
as the drug molecular weight increased; less than 10% of the insulin was
released from
the polymer in the simulated gastric fluid (pH - 1.3) during the first phase
of the
experiment. However, after the particles were placed in pH = 7.4 buffer
solution, the
hydrogels swelled rapidly allowing for a rapid release of insulin. These
results indicate
that the graft copolymers P(MAA-g-EG) are useful for development of oral
insulin
delivery system.
Example 2
In vitro Mucoadhesion Studies
P(MAA-g-EG) hydrogels were prepared in thin films by a solution
polymerization technique. The hydrogels were swollen to equilibrium in DMGA
buffered saline solutions of pH = 3.2 and 7.4. The swollen hydrogels were cut
into
disks with diameters of 20 cm and placed in a tensile tester at 25 ° C
and 90% RH.
The polymer samples were adhered to the upper holder of the tester using
cyanacrylate
medical adhesive, whereas a sample of gelled bovine submaxillary mucin was
affixed
on the lower jaws using the adhesive. The two jaws were brought together for
15 min
and then separated at 1 mm/min. The detachment force was measured as a
function of
displacement. The work of fracture, equivalent to the work of bioadhesion was
calculated as the area under the curve.
P(MAA-g-EG) hydrogels function well as oral insulin devices because
they are able retard the action of protease inhibitors and also because they
adhere to
the mucosa of the intestinal wall, allowing for intimate contact and thus
aiding in
absorption of the drug.
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When the insulin containing hydrogels were placed in intestinal fluid,
the hydrogels swelled rapidly allowing insulin to be released. Insulin
containing,
P(MAA-g-EG) microparticles were swollen :for 1 hour phosphate buffered saline
solutions and then transferred into intestinal :Fluid. The proteolysis of the
insulin in the
intestinal fluid was monitored using an insulin EIA kit. Greater than 50% of
the
biological activity of the insulin was maintained for over 1 hour in the
presence of
proteolytic enzymes. In comparison, when insulin is dissolved in intestinal
fluid, the
biological activity is rapidly lost. P(MAA-g-EG) hydrogels serve to protect
the insulin
by binding calcium to the ionized pendant groups which in turn retards the
action of
proteolytic enzymes.
Additionally, P(MAA-g-EG) hydrogels exhibit mucoadhesive
characteristics due to the presence of the graft PEG chains which serve as
adhesion
promoters. The mucoadhesive characteristics of P(MAA-g-EG) hydrogels were
strongly dependent on the pH of the environmental fluid (Fig. 6). The area
under the
curves of Fig. 6 is equivalent to the adhesive: force between the gel and the
mucosa. In
conditions simulating the intestinal pH (pH == 7.4) the adhesion between the
gel and the
mucous was significantly greater. However, to truly compare the mucoadhesive
characteristics of the hydrogels, the work of adhesion was normalized to
account for
the polymer gel fraction (See Table 2). The normalized work of adhesion was
two-
orders of magnitude greater for hydrogels in the non-complexed state relative
to
complexed hydrogels. Accordingly, the hydrogels adhere to the mucosa of the
intestine to a much greater extent than the stomach. Therefore, the residence
time of
insulin carriers is much greater in regions where the insulin could be
absorbed.
Table 2. Work of adhesion for P(MAA-g-1_?G) hydrogels containing a 1:1 MAA/EG
ratio and graft PEG chains for molecular weight 1000.
pH Work of Adhesion Polymer Volume Normalized Work
W' 106 (J) Fraction, OZ,S of Adhesion
W/(O,-.l~' 106 (J)
3.2 I 5.38 ~ 0.693 ~ 62.1
7.4 1 9.34 I 0.049 I 6720
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The differences in the adhesive characteristics of the hydrogels at
different pH values were due to mobility of the PEG chains in each material.
In the
highly swollen, non-complexed state, the graft PEG chains were free and
readily
penetrated the mucosa to serve as anchors for adhesion. In the complexed
state, the
graft PEG chains in the P(MAA-g-EG) formed complexes with the backbone chains
and were unable to penetrate the gel/mucosa interface and form temporary
anchors.
Example 3
In vivo Administration of Insulin to Rats
The graft copolymers were prepared by free radical solution
polymerization of methacrylic acid and polyethylene glycol) monomethacrylate.
The
ensuing hydrogels were rinsed for 7 days in deionized water to remove
unreacted
monomer and uncrosslinked oligomer chains. The hydrogels were dried under
vacuum
and ground into powders. The powders were filtered to obtain particles with
diameters of 100 - 1 SO Vim. Crystalline porcine insulin (26.9 U/mg) was
loaded by
equilibrium partitioning. The drug loaded particles were filtered and washed
to
remove surface drug and dried under vacuum.
Male Wistar rats (200 g) were fasted for 24 hours. The rats were
restrained in the supine position and administered insulin loaded polymer
microparticles using a gelatin capsule, which dissolves instantly in the
stomach. Serum
glucose was monitored by collecting 0.2 ml aliquot blood samples from the
jugular
vein prior to the experiment and at 0.25, 0.5, 1, 2, 4, 6 and 8 hours after
dosing.
Serum was separated by centrifugation at 3000 rpm for 3 minutes and frozen
until
analysis. The serum insulin levels were determined by enzyme immunoassay using
an
insulin EIA kit. The serum glucose levels were determined by the glucose
oxidase
method using a glucose B-Test kit.
Fig. 7 summarizes the blood glucose response of rats receiving insulin
doses contained in P(MAA-g-EG) microparticles. Within 2 hours of receiving the
polymeric dosage form, a strong hypoglycemic effect {lowering of the blood
glucose
level) was observed. The reduction of the blood glucose levels depends
strongly on
the insulin dose. No response was observed in rats receiving the insulin
solutions.
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The effects of administering a composition comprising a P(MAA-g-EG)
hydrogel containing insulin and the protease; inhibitor, aprotinin, are shown
in Fig. 8.
Control groups of rats were administered hydrogels containing insulin without
a
protease (for comparison) and a group was. administered a SOU/Kg insulin
solution (to
serve as a control). The two groups receiving polymeric dosage forms of
insulin had a
large decrease in blood glucose concentration within two hours of
administration.
Those rats receiving a combination of insulin and the protease inhibitor,
aprotinin,
showed the greatest reduction in blood glucose levels. Aprotinin retards the
action of
the degradative enzymes in the intestine andl allows the insulin released
locally to
remain active longer. Thus, the amount of insulin transported into the
bloodstream is
highest in the rats receiving the hydrogel insulin and protease inhibitor
composition
(encapsulated within the P(MAA-g-EG) hydrogel), resulting in a greater
reduction in
the blood glucose concentration.
Example 4
In Yivo Studies in Diabetic Rats and Dogs
Diabetes was induced in healthy, male Wistar rats by administration of
streptozotocin. Healthy dogs were made diabetic by administration of alloxan.
P(MAA-g-EG) microspheres were prepared by a free-radical bulk, suspension
polymerization of methacrylic acid and polyethylene glycol) dimethacrylate
(PEG MW
= 1000). Tetraethylene glycol dimethacryla.te was added as the crosslinking
agent.
2,2'-Azobisisobutyronitrile (AIBN) was added in the amount of 0.5% of the
total
monomers as the thermal reaction initiator.
Drug loading was accomplished by equilibrium portioning of the insulin
into the P(MAA-g-EG) microparticles. Bovine pancreatic insulin was dissolved
in 200
Nl of 1 N NaOH. The insulin solution was .diluted with 20 ml of phosphate
buffer
solution (pH = 7.4) and normalized with 200 Nl of 0.1 N NaOH. Loading was
accomplished by swelling initially dry, P(M,AA-g-EG) for 24 hours in the
insulin
solution. The particles were then filtered and washed with 100 ml of 0.1 N HCl
solution to collapse the microparticles and "squeeze out" the remaining buffer
solution.
The drug loaded microspheres were dried under vacuum and stored at 4°
C. The
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degree of loading was determined from HPLC analysis of the insulin
concentrations of
the initial solutions and the filtrate from the washings.
Prior to administration of the insulin loaded P(MAA-g-EG) hydrogels,
the male Wistar rats (250 g) were fasted for 24 hours. The rats were
restrained in the
supine position and administered the insulin loaded P{MAA-g-EG) microparticles
and
the control solutions via the mouth using gelatin capsules and capsules
prepared using
Eudragit L100. The gelatin capsules dissolved readily in the stomach while the
Eudragit capsules dissolved at significantly slower rate.
During the experiment, the rats were separated (4 animals per cage) and
allowed to drink water. A 0.2 ml aliquot of blood was collected from the
jugular vein
at 0.25, 0.5, 1, 2, 4, 6 and 8 hours following dosing. The blood serum was
separated
by centrifugation at 3000 rpm for 3 minutes. The serum glucose levels were
determined by the glucose oxidase method using a glucose B-test kit.
The diabetic dogs (25 kg) were fasted for 24 hours prior to
administration of the formulations. The polymer dosage forms were administered
orally using gelatin capsules. The dogs were fed at the time of
administration.
During the experiment, the dogs were caged and allowed to drink
water. Blood samples were taken from an in-dwelling catheter. Serum glucose
levels
were determined using a portable glucose analyzer.
While a number of systems have been effective in lowering the blood
glucose levels of healthy animals following oral administration of polymeric
carriers
containing insulin, similar results have not been observed in diabetic
animals. The
blood glucose response of diabetic and healthy rats after oral administration
of insulin
containing P(MAA-g-EG) microparticles using gelatin capsules (25 IU/kg doses)
is
shown in Fig. 9. The blood glucose levels of the diabetic rats were lowered by
up to
40% of the initial level. The reduction in blood glucose levels lasted for
greater than 8
hours, and the degree to which the glucose levels were suppressed was in fact
greater
for the diabetic animals than the healthy animals. Additionally, the strong
hypoglycemic effects were observed to last longer in the diabetic animals.
The blood glucose response of diabetic rats following oral
administration of Eudragit capsules containing insulin loaded P(MAA-g-EG)
microparticles (25 IU/kg doses) is shown in Fig. 10. The glucose levels of
rats
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receiving these dosages were reduced by greater than SO% for at least eight
hours
following a single administration. The microparticles encapsulated in Eudragit
were
more effective than gelatin capsules presumably because the microparticles
contained
in the Eudragit capsules were exposed to the harsh environment of the upper GI
tract
for shorter periods of time due to the slow diissolution of the Eudragit
capsules.
The blood glucose of healthy dogs was significantly lowered following
the oral administration of a single polymeric dosage from (10 IU/kg). At time
zero,
the dogs were fed and the normal response of the body would be to maintain the
basal
level. After feeding, the blood glucose level:; increased, however, within two
hours of
dosing, the blood glucose levels were reduced by greater than 20% due to
uptake of
insulin in the upper small intestine. Additionally, a second decrease occurred
around
the eight hour point, consistent to what was previously see in rats, probably
due to
colonic absorption of insulin. Additionally, the blood glucose levels steadily
decline
after eight hours, probably due to colonic absorption of the insulin.
The glucose response of diabetic dogs also verifies the uptake of insulin
following oral administration. The blood glucose levels of diabetic dogs was
controlled by the oral administration of insuliin containing P(MAA-g-EG)
microparticles using gelatin capsules (10 IUlkg doses). Following feeding and
administration of the polymer dosage form, the glucose levels of the dogs rose
rapidly,
initially. However, after one hour the glucose levels began to stabilize for
the next
three hours as the insulin was absorbed. The; blood glucose levels of the
diabetic dogs
which received the polymer dosage forms was 40% less than dogs that had not
received any insulin.
Oral insulin delivery systems must be able to protect the drug from the
harsh environment of the stomach and deliver the insulin in an biologically
active
conformation for extended period of time to more favorable regions for
absorption
along the GI tract such as the upper small inl:estine. Because of their
nature,
complexing P(MAA-g-EG) hydrogels are ideal for this application.
P(MAA-g-EG) hydrogels are able to effectively deliver biologically
active insulin via the oral route. These materials have been shown to reduce
the blood
glucose levels in diabetic rats and dogs and maintain the blood glucose at
near normal
levels for greater than eight hours. These materials function well because the
majority
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of the insulin contained in the hydrogels is not released until the materials
reach the
upper small intestine. While in the intestine, the hydrogels adhere strongly
to the
mucosa allowing for intimate contact between the carrier and the absorption
site.
Additionally, the polymers serve to retard the activity of proteolytic enzymes
in the
intestine allowing the insulin to remain active for longer periods of time
prior to
absorption. The inhibitory elect of the polymers on enzyme function is
believed to be
derived from the polymers' ability to form complexes with cations, such as
calcium,
necessary for enzymatic function.
