U.S. patent application number 12/426902 was filed with the patent office on 2009-11-19 for pharmaceutical formulation for regulating the timed release of biologically active compounds based on a polymer matrix.
This patent application is currently assigned to Rutgers, The State University of New Jersey. Invention is credited to Joachim B. Kohn, Deborah M. Schachter.
Application Number | 20090285895 12/426902 |
Document ID | / |
Family ID | 22615557 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090285895 |
Kind Code |
A1 |
Kohn; Joachim B. ; et
al. |
November 19, 2009 |
Pharmaceutical Formulation for Regulating the Timed Release of
Biologically Active Compounds Based on a Polymer Matrix
Abstract
A formulation containing a biologically active compound having a
structure with hydrogen bonding sites is blended with a polymer
having a structure with complementary hydrogen bonding sites, the
polymer forming hydrolytic degradation products that promote the
release of the biologically active compound from the polymer.
Inventors: |
Kohn; Joachim B.;
(Piscataway, NJ) ; Schachter; Deborah M.; (Edison,
NJ) |
Correspondence
Address: |
FOX ROTHSCHILD LLP;PRINCETON PIKE CORPORATE CENTER
2000 Market Street, Tenth Floor
Philadelphia
PA
19103
US
|
Assignee: |
Rutgers, The State University of
New Jersey
New Brunswick
NJ
|
Family ID: |
22615557 |
Appl. No.: |
12/426902 |
Filed: |
April 20, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10169410 |
Dec 9, 2002 |
7521061 |
|
|
PCT/US01/00030 |
Jan 2, 2001 |
|
|
|
12426902 |
|
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60174137 |
Dec 31, 1999 |
|
|
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Current U.S.
Class: |
514/1.1 ;
514/1.3 |
Current CPC
Class: |
A61K 38/12 20130101;
A61K 47/14 20130101 |
Class at
Publication: |
424/486 ; 514/2;
514/9 |
International
Class: |
A61K 38/02 20060101
A61K038/02; A61K 9/14 20060101 A61K009/14 |
Claims
1. An implant comprising: a weight percentage of a peptide drug
having a chemical structure with hydrogen bonding sites; and a
hydrolytically degradable polycarbonate copolymer having a molar
percentage of tyrosine-derived diphenol monomer units with pendant
carboxylic acid groups and a molar percentage of tyrosine-derived
diphenol monomer units with pendant carboxylic acid ester groups,
wherein the peptide drug is dispersed within the copolymer.
2. The implant of claim 1 wherein the polycarbonate copolymer
degrades hydrolytically promoting release of the peptide drug from
the implant.
3. The implant of claim 1 wherein the molar percentage of
tyrosine-derived diphenol monomer units with pendant carboxylic
acid groups is between about 5 mole percent and about 15 mole
percent.
4. The implant of claim 1 wherein the weight percentage of the
peptide drug is between about 15 weight percent and about 30 weight
percent.
5. The implant of claim 1 wherein the molar percentage of
tyrosine-derived diphenol monomer units with pendant carboxylic
acid groups and the weight percentage of the peptide drug is
effective to provide reproducible release profiles of the peptide
drug from the implant without an initial burst effect.
6. The implant of claim 1 wherein the copolymer is a
desaminotyrosyltyrosine copolymer of poly(desaminotyrosyltyrosine
ethyl ester carbonate).
7. The implant of claim 1 wherein the peptide drug is a cyclic
peptide.
8. A formulation comprising: a weight percentage of a biologically
active compound having a chemical structure with hydrogen bonding
sites; and a hydrolytically degradable polycarbonate copolymer
having a molar percentage of tyrosine-derived diphenol monomer
units with pendant carboxylic acid groups and a molar percentage of
tyrosine-derived diphenol monomer units with pendant carboxylic
acid ester groups, wherein the polycarbonate copolymer degrades
hydrolytically promoting release of the biologically active
compound from the formulation.
9. The formulation of claim 8 wherein the molar percentage of
tyrosine-derived diphenol monomer units with pendant carboxylic
acid groups and the weight percentage of the biologically active
compound is effective to provide reproducible release profiles of
the biologically active compound from the formulation without an
initial burst effect.
10. The formulation of claim 8 wherein the biologically active
compound is a pharmaceutically active compound.
11. The formulation of claim 10 wherein the pharmaceutically active
compound is a peptide.
12. The formulation of claim 11 wherein the peptide is a cyclic
peptide.
13. The formulation of claim 8 wherein the copolymer is a
desaminotyrosyltyrosine copolymer of poly(desaminotyrosyltyrosine
ethyl ester carbonate).
14. The formulation of claim 8 wherein the molar percentage of
tyrosine-derived diphenol monomer units with pendant carboxylic
acid groups is between about 5 mole percent and about 15 mole
percent.
15. The formulation of claim 8 wherein the weight percentage of
biologically active compound is between about 15 weight percent and
about 30 weight percent.
16. A method for delayed delivery of a peptide drug to a patient in
need thereof comprising: providing a formulation including the
peptide drug and a copolymer, wherein the peptide drug has a
chemical structure with hydrogen bonding sites, and wherein the
copolymer is a hydrolytically degradable polycarbonate copolymer
having tyrosine-derived diphenol monomer units with pendant
carboxylic acid groups and tyrosine-derived diphenol monomer units
with pendant carboxylic acid ester groups; and administering the
formulation to the patient so that release of the peptide drug
occurs after a predetermined time without an initial burst
effect.
17. The method of claim 16 wherein a weight percentage of the
peptide drug in the formulation is between about 15 weight percent
and about 30 weight percent.
18. The method of claim 16 wherein a molar percentage of
tyrosine-derived diphenol monomer units with pendant carboxylic
acid groups is between about 5 mole percent and about 15 mole
percent.
19. The method of claim 16 wherein the peptide drug is a cyclic
peptide.
20. The method of claim 16 wherein the copolymer is a
desaminotyrosyltyrosine copolymer of poly(desaminotyrosyltyrosine
ethyl ester carbonate).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/169,410, filed Jul. 1, 2002, now U.S. Pat. No. 7,521,061,
which is a National Stage of International Application No.
PCT/US01/00030, filed Jan. 2, 2001, which claims priority under 35
U.S.C. .sctn. 119(e) to U.S. Provisional Application Ser. No.
60/174,137, filed Dec. 31, 1999, the disclosures of all three of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to a new approach to the
delayed or pulsed release of biologically active compounds having
pharmaceutical activity, particularly peptides such as
INTEGRILIN.TM., from a polymer matrix. In this system no
complicated barrier mechanism is required to prevent the release of
the peptide during the lag time, a high loading of the
water-soluble peptide is readily achieved, and the length of the
delay of the release of the peptide is easily controlled.
[0003] Previously, limited release of INTEGRILIN.TM. was reported
from poly(DTH adipate), a member of the tyrosine-derived
polyarylates, despite high loadings of the peptide (30% w/w).
Subsequent investigations indicated that interactions between the
peptide and the polymer were responsible for the minimal release
(.about.5% of the loaded peptide). Since hydrogen bonding was a
component of the interactions, the release of the peptide from
poly(DTH adipate) was demonstrated to be sensitive to the pH within
the polymer matrix.
[0004] The literature is replete with examples of the delayed or
pulsed release of active agents using polymeric materials. However,
it is possible to divide these systems into two basic categories;
those that depend on an environmental stimulus to induce release of
the active agent from the polymeric matrix and those that are
designed to release the drug after particular intervals of time
have elapsed. Examples of environmental stimuli that have been used
for this application are electrical impulses, pH or temperature
changes, application of magnetic fields, or ultrasound.
[0005] Those systems that are time-controlled can further be
divided into those that use a barrier technology that is placed
around the active agent that is designed to degrade or dissolve
after a certain time interval, and those that use the degradation
of the polymer itself to induce the release of the active
agent.
[0006] One approach of this category has been to prepare a
polymeric hydrogel composed of derivitized dextran and to
incorporate into the hydrogel, a model protein, I.sub.gG, with an
enzyme, endo-dextranase, that degrades the hydrogel. It was
observed that without the enzyme the release of the protein was
very slow. However, when the enzyme was included in the
formulation, the release rate was dependent on the concentration of
the enzyme. At high concentrations, the release was fast and
complete. At low concentrations, the release was delayed.
[0007] A correlation was found between the delay time and the rate
of the degradation of the hydrogel. The interpretation of the data
was that the mesh size of the hydrogel was too small for efficient
diffusion of a large protein molecule such as I.sub.gG, but as the
enzyme degraded the polymer, the mesh size increased and diffusion
was unimpeded.
[0008] Delayed release in association with hydrolytic degradation
of the polymer has also been investigated. Heller's poly(ortho
esters) are viscous ointments at room temperature and when mixed
with a model protein, lysozyme, demonstrated a delayed release
profile. The length of the delay time was found to correlate with
polymer molecular weight and alkyl substituent of the polymer.
These experiments, however, are limited by the fact that all of the
drug release experiments were conducted at room temperature,
perhaps, because the polymers are viscous at room temperature, but
not at the physiological temperature of 37.degree. C.
[0009] Ivermectin, a water insoluble antiparasitic agent for
veterinary applications, was encapsulated in PLGA (50:50)
microspheres and the subsequent pulsed release of this agent, in
vivo, was shown to be dependant on the degradation rate of the
polymer matrix. Pulsed and delayed release of active agents from
PLGA microspheres was most intensely studied by Cleland et al. The
PLA or PLGA microspheres are processed using a high kinematic
viscosity of polymer solution and a high ratio of polymer to
aqueous solution. This produces dense microspheres, which require
sever bulk erosion to release the drug. These conditions yield
microspheres that have low loading (generally 1% w/w), moderate
bursts, and lag times during which significant leaching of drug
occurs.
SUMMARY OF THE INVENTION
[0010] The technology described in this disclosure represents a
departure from the prior art. In this system, bonding interactions
between a polymer and an active compound are used to inhibit the
release of the active compound, and the polymeric degradation
products are used to control the length of time preceding release
of the active compound. The bonding interactions are composed of
hydrogen bonding and hydrophobic forces and develop when a highly
functional polymer is employed.
[0011] Therefore, according to one aspect of the present invention,
a formulation containing a biologically active compound is
provided, having a structure with hydrogen bonding sites, blended
with a polymer having a structure with complementary hydrogen
bonding sites, the polymer forming hydrolytic degradation products
that promote the release of the biologically active compound from
the polymer.
[0012] The formulation thus consists of two components, a polymer
and an active compound blended together. The present invention thus
provides a formulation system that uses the degradation products of
selected polymers to trigger the release of the active compound
from the matrix of the polymer. Using this method, active compounds
can be very simply formulated with the polymer and be programmed to
be released at desired intervals, requiring no sophisticated
barriers to prevent the premature release of the active agent.
[0013] There are many drugs that are more effective when given to
the patient in a pulsatile manner as opposed to a continuous
release fashion. For example, an area of great interest, currently,
for this type of delivery system is single-shot immunity. Immunity
is best induced by a pulsatile delivery of the agent, hence the
need for booster shots. It has been suggested that it would be more
economical and effective, especially in third world countries, if a
tetanus toxoid or gp120 (under development for an AIDS vaccine)
could be implanted once into the patient and the boosters be
automatic and preprogrammed from the implanted or injected
device.
[0014] Therefore, the present invention also includes a method for
the pulsatile delivery of a biologically active compound to a
patient in need thereof comprising administering to the patient the
formulation of the present invention.
[0015] This type of drug delivery is not only important for amino
acid based drugs but also for hormonal based drug delivery.
Fertility and birth control drug therapy for both animals and
humans is not continuous, but rather cyclic in nature since these
therapies work synergistically with the menstrual cycle and the
corresponding hormonal flux. This is another direction in drug
delivery in which this type of delayed pulsed release of an active
agent would be applicable.
[0016] Agricultural applications which require the timed dosing of
fertilizers, weed-killers, and other active agents is another area
where this invention would be important.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 depicts the chemical structure of tyrosine-derived
polyarylates;
[0018] FIG. 2 depicts the amino acid sequence of
INTEGRILIN.TM.;
[0019] FIG. 3 depicts release from poly(DTH adipate) films
containing 30% (w/w) peptide;
[0020] FIG. 4 depicts release from D,L-PLA and
poly(.epsilon.-caprolactone) films containing 30% (w/w)
peptide;
[0021] FIG. 5 depicts percent mass retention of poly(DTH adipate)
samples containing 30% (w/w) peptide;
[0022] FIG. 6 depicts percent mass retention data for D,L-PLA
samples containing 30% (w/w) peptide;
[0023] FIG. 7 depicts percent water absorption by films of PCL and
PLA containing 30% (w/w) peptide;
[0024] FIG. 8 depicts percent water absorption by films of poly(DTH
adipate) both with and without peptide;
[0025] FIG. 9 depicts percent molecular weight retention of neat
poly(DTH adipate) samples to that of poly(DTH adipate) containing
30% (w/w) peptide, and to that of 10% PEG/90% poly(DTH
adipate);
[0026] FIG. 10 depicts the effect of ionic strength on the release
of 30% (w/w) INTEGRILIN.TM. from poly(DTH adipate) films;
[0027] FIG. 11 depicts release from poly(DTH adipate) films
containing 30% (w/w) peptide at pH 2.2 without added
electrolytes;
[0028] FIG. 12 depicts water uptake of poly(DTH adipate) films
containing 30% (w/w) peptide at pH 2.2 without added
electrolytes;
[0029] FIG. 13 depicts hydrogen bonding between a tyrosine-derived
polyarylate and a peptide;
[0030] FIG. 14 depicts the chemical structure of poly(DTH
dioxaoctanedioate);
[0031] FIG. 15 depicts release of peptide from poly(DTH
dioxaoctanedioate);
[0032] FIG. 16 depicts the chemical structure of poly(DTE
carbonate);
[0033] FIG. 17 depicts release of peptide from poly(DTE carbonate)
samples containing 15% (w/w) peptide;
[0034] FIG. 18 depicts the chemical structure of
desaminotyrosyltyrosine (DT);
[0035] FIG. 19 depicts percent molecular weight retention of neat
poly(DT-co-DTH adipate) films with 0, 5, 10, 15 mole percent of
DT;
[0036] FIG. 20 depicts percent water uptake of neat poly(DT-co-DTH
adipate) films with 0, 5, 10, and 15 mole percent DT;
[0037] FIG. 21 depicts release of peptide from poly(DT-co-DTH
adipate) matrices;
[0038] FIG. 22 depicts release of peptide from 30% (w/w)
poly(DT-co-DTH adipate) films;
[0039] FIG. 23 depicts pH measurements of buffer of samples of
poly(DT-co-DTH adipate) with 15% (w/w) peptide; and
[0040] FIG. 24 depicts percent molecular weight retention of
samples of poly(DTH adipate) containing various percentages of DT
incubated in PBS at 37.degree. C.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0041] In its broadest embodiment, polymers that are suitable for
use in the present invention are any polymer that contains
hydrogen-bonding sites as part of its structure and degrades to
form products that promote the release of a biologically active
compound from the polymer. Biocompatible polymers are required for
biomaterial end-use applications.
[0042] Preferred polymers are copolymers containing a hydrophilic
monomer and a hydrophobic monomer. In a more preferred embodiment,
the copolymer is selected from the tyrosine-derived polyarylate
libraries disclosed in WO 99/24107 and WO 99/52962, the disclosures
of both of which are incorporated herein by reference. The
copolymers of WO 99/24107 contain a hydrophilic monomer with a
pendant carboxylic acid group, desaminotyrosyltyrosine, which
degrades to form acidic degradation products. The other monomer, a
desaminotyrosyltyrosine ester, also contains hydrogen bonding sites
for retention of the active compound. A water soluble yet
hydrophobic dicarboxylate monomer forms polyarylate linkages
between the two diols.
[0043] Members of the tyrosine-derived polyarylate library all
share the same highly functional structural template but are
distinguished from one another by subtle structural changes. The
functional groups of the main template provide sites for
interactions. These are pi stacking of its aromatic rings with an
aromatic ring of a peptide, or hydrogen bonding of the
.alpha.-amino carboxylate region with a corresponding group in the
peptide. The small structural variations between members allow the
fine-tuning of these interactions to suit particular proteins or
peptides.
[0044] Also preferred are any of the copolymers that can be derived
from the tyrosine-derived diphenol compounds of U.S. Pat. No.
5,587,507 and the tyrosine-derived dihydroxy monomers of WO
98/36013, the disclosures of both of which are also incorporated
herein by reference, using the process of WO 99/24107 for forming
free carboxylic acid moieties. In addition to the above-referenced
polyarylates, examples include the polycarbonates of U.S. Pat. No.
5,099,060, the polyiminocarbonates of U.S. Pat. No. 4,980,449, the
polyphosphazenes and polyphosphates of U.S. Pat. No. 5,912,225,
polyurethanes, including the polyurethanes of U.S. Pat. No.
5,242,997, the random poly(alkylene oxide) block copolymers of U.S.
Pat. No. 5,658,995, and a wide range of other polymers that can be
derived from the above-referenced tyrosine-derived diphenol
compounds, the tyrosine-derived dihydroxy compounds and similar
peptides. All of the above-referenced patent publications are
incorporated herein by reference. Notably, corresponding polymers
of the tyrosine-derived dihydroxy compounds can be made by any of
the processes of any of the above-referenced patents disclosing
polymers of tyrosine-derived diphenol compounds.
[0045] A particularly preferred copolymer is the
desaminotyrosyltyrosine (DT) copolymer of
poly(desaminotyrosyltyrosine hexyl ester adipate) (Poly(DTH
adipate)), depicted in FIG. 1 (y=4; R=hexyl). Poly(DT-CO-DTH
adipates) having a weight-average molecular weight between about
80,000 and about 200,000 daltons is particularly preferred.
[0046] In a preferred embodiment, the present invention uses pH
sensitivity to control the release of an active compound. It was
discovered that the accumulation of acidic polymer degradation
residues in the matrix of a polymer/peptide blend weakened the
interactions between the peptide and the polymer so that the
peptide could be released. An inverse correlation was demonstrated
between the mole percent of acid moieties in a polymer and the
length of the lag time preceding release indicating that timed
release of a peptide, or any active agent with hydrogen bonding
sites, can be controlled by mole percent of acid moieties in a
polymer.
[0047] Any biologically active compound with hydrogen-bonding sites
that can be physically dispersed within the polymer can be used as
an active compound for release (e.g. FIG. 13). Examples of hydrogen
bonding sites include primary and secondary amines, hydroxyl
groups, carboxylic acid and carboxylate groups, carbonyl (carboxyl)
groups, and the like. While one can apply the current invention to
any active compound that has hydrogen bonding sites, including
natural and unnatural antibiotics, cytotoxic agents and
oligonucleotides, amino acid derived drugs such as peptides and
proteins seem to be most appropriate for this technology. The
compositions of the present invention overcome some of the
difficulties encountered in previous attempts to formulate
controlled release devices that show reproducible release profiles
without burst and/or lag effects. In its most preferred embodiment,
the active compound is a peptide that is stable under mildly acidic
conditions.
[0048] Peptide drugs suitable for formulation with the compositions
of the present invention include natural and unnatural peptides,
oligopeptides, cyclic peptides, library generated oligopeptides,
polypeptides and proteins, as well as peptide mimetics and
partly-peptides. Peptide drugs of particular interest include
platelet aggregation inhibiting (PAI) peptides, which are
antagonists of the cell surface glycoprotein Iib/IIIa, thus
preventing platelet aggregation, and ultimately clot formation.
Preferred PAI peptides include the PAI peptides disclosed by WO
90/15620, the disclosure of which is incorporated herein by
reference, particularly INTEGRILIN.TM. (FIG. 2), a medically useful
cyclic PAI heptapeptide.
[0049] The compositions of the present invention are suitable for
applications where localized drug delivery is desired, as well as
in situations where systemic delivery is desired. Therapeutically
effective dosages may be determined by either in vivo or in vitro
methods. For each particular compound of the present invention,
individual determinations may be made to determine the optimal
dosage required. The range of therapeutically effective dosages
will naturally be influenced by the route of administration, the
therapeutic objectives, and the condition of the patient. For the
various suitable routes of administration, the absorption
efficiency must be individually determined for each drug by methods
well known in pharmacology. Accordingly, it may be necessary for
the therapist to titer the dosage and modify the route of
administration as required to obtain the optimal therapeutic
effect. The determination of effective dosage levels, that is, the
dosage levels necessary to achieve the desired result, will be
within the ambit of one skilled in the art. Typically, applications
of compound are commenced at lower dosage levels, with dosage
levels being increased until the desired effect is achieved. The
release rate of the drug from the formulations of this invention
are also varied within the routine skill in the art to determine an
advantageous profile, depending on the therapeutic conditions to be
treated.
[0050] A typical dosage might range from about 0.001 mg/kg to about
1000 mg/kg, preferably from about 0.01 mg/kg to about 100 mg/kg,
and more preferably fro about 0.10 mg/kg to about 20 mg/kg.
Advantageously, the compounds of this invention may be administered
several times daily, and other dosage regimens may also be
useful.
[0051] The compositions may be administered subcutaneously,
intramuscularly, colonically, rectally, nasally, orally or
intraperitoneally, employing a variety of dosage forms such as
suppositories, implanted pellets or small cylinders, aerosols, oral
dosage formulations and topical formulations, such as ointments,
drops and transdermal patches. Liposomal delivery systems may also
be used, such as small unilamellar vesicles, large unilamellar
vesicles and multilamellar vesicles.
[0052] The following non-limiting examples set forth herein below
illustrate certain aspects of the invention. All parts and
percentages are by weight unless otherwise noted and all
temperatures are in degrees Celsius. The PAI peptide was obtained
from COR Therapeutics of South San Francisco, Calif., and used
without further purification. Solvents were of "HPLC grade" and
were obtained from Fisher Scientific of Pittsburgh, Pa.
EXAMPLES
[0053] INTEGRILIN.TM. (antithrombotic injection) was chosen as the
model peptide to explore the drug delivery applications of these
materials (FIG. 2). This compound is a synthetic cyclic readily
water-soluble heptapeptide which is a highly potent glycoprotein
IIb/IIIa antagonist. This compound has successfully demonstrated
antithrombogenic behavior in vivo and devices fabricated by the
formulation of this peptide into a polymer matrix with this
property would have many useful cardiovascular applications. In
addition, this polymer contains an RGD sequence and therefore a
device containing this peptide could possibly find applications as
a component in scaffolds for tissue regeneration.
[0054] The blend of INTEGRILIN.TM. and poly(DTH adipate) was
described in the U.S. Pat. No. 5,877,223, the disclosure of which
is incorporated herein by reference. There, it was mentioned that
formulating films from these components using the coprecipitation
melt-press technique resulted in specimens that released only trace
amounts of peptide when incubated in PBS at 37.degree. C. This was
unexpected because the peptide is readily water-soluble.
Polymer Synthesis and Specifications
[0055] Tyrosine derived polyarylates were synthesized as described
in U.S. Pat. No. 5,877,224 and in WO 99/24107. Poly(DTH adipate)
R=hexyl and y=4), was the polymer specifically chosen for this
study though many of the physical phenomena reported here for this
polymer have been observed with others in this polymer family. The
polymers used had molecular weights ranging between 80-120 kDa. The
particular polymers synthesized were
poly(DT.sub.0.05co-DTH.sub.0.95adipate,
Poly(DT.sub.0.10-co-DTH.sub.0.09adipate), and
poly(DT.sub.0.15-co-DTH.sub.0.85 adipate). The molecular weights of
the polymers used ranged from 60-80 kDa. D,L-PLA and
poly(.epsilon.-caprolactone) were purchased from Medisorb and
Aldrich, respectively. Both were of molecular weight 100 kDa and
fabricated into release devices in the same manner as the poly(DTH
adipate).
Fabrication of Release Devices
[0056] The peptide was obtained from COR therapeutics, Inc. The
peptide, as received, was 98-99% pure and used without further
purification. Compression molded films were fabricated from a
co-precipitate containing 30% peptide and 70% polymer by weight.
This co-precipitate was prepared by dissolving 0.15 g of peptide in
5 ml of methanol (HPLC grade) and 0.35 g polymer in 5 ml of
methylene chloride (HPLC grade) and mixing the two solutions
together to form a clear solution. This resultant solution was
added drop-wise into 100 ml of stirred ethyl ether maintained at
-78.degree. C. White spongy precipitates were formed, filtered
using a sintered glass filter, and dried under vacuum. After drying
the co-precipitate was compression molded at 90.degree. C. under a
pressure of 5,000 psi. Films with a thickness of 0.1 nm (.+-.0.02
mm) were obtained.
Device Characterization
[0057] Peptide loading was determined by dissolving 10.0 mg of a
film in THF (HPLC grade) (1.0 ml) in a 10 ml volumetric flask and
adding PBS (phosphate buffer saline) until the 10 ml line. The
mixture was stirred for a minimum of 6 hours followed by HPLC
analysis of the drug content in the aqueous medium. Methylene
chloride replaced THF when characterizing samples composed of PLA
or poly(.epsilon.-caprolactone) due to their insolubility in
THF.
Peptide Release Study
[0058] Films were cut into 0.5 cm.sup.2 squares. The mean mass of
the samples was 21 mg (.+-.5). Each specimen was individually
placed into 20 ml glass scintillation vials containing 10 ml of
phosphate buffered saline (pH 7.4, 37.degree. C.). The standard PBS
solution used was composed of 10 mM phosphate buffer saline, 138 mM
NaCl and 2.7 mM KCl. The buffer was changed at each time point and
analyzed by HPLC for release of the peptide. There was a minimum of
three samples per time point, each sample originating from a
different film. The HPLC method involved a 3 cm C-18 Perkin Elmer
cartridge column with a gradient mobile phase which began at 80%
water/20% acetonitrile and ended with 75% water during a period of
5 minutes at a flow rate of 1 ml/min. Both the acetonitrile and
water contained 0.1% (v/v) trifluoroacetic acid. The column was
calibrated with known concentrations of the peptide dissolved in
PBS to establish a calibration curve and the INTEGRILIN.TM.
contained in the buffer of each sample was quantified using this
curve. The HPLC pump used was a Perkin Elmer Series 410 LC pump and
the detector used was a PE LC-235 diode array UV-VIS detector set
at 280 nm. The data collected was analyzed using a PE Nelson 3000
Series Chromatography Data System.
[0059] At designated times, the samples were removed, rinsed with
deionized water, blotted with a Kimwipe tissue and either placed in
a vial for subsequent vacuum drying for mass retention and
molecular weight retention studies or used for thermal gravimetric
analysis (TGA) water uptake studies. Those devices that were not
needed for gel permeation chromatography (GPC) or TGS studies were
dissolved in organic solvent subsequent to drying and the peptide
content extracted to ensure that all loaded peptide was accounted
for.
[0060] Molecular Weight Determination of the Polymers Using GPC
[0061] The molecular weights of the poly(DTH adipate) samples were
calculated relative to a set of monodispersed polystyrene standards
(Polymer Laboratories, Ltd. Church Station, U.K.) without further
corrections. The GPC chromatographic system consisted of a Waters
510 HPLC pump, a Waters 410 differential refractometer detector,
and a Digital Venturi's 466 PC running Millenium (Waters Corp.)
software for data processing. Two PL-gel columns 30 cm in length
(pore sizes of 10.sup.3 and 10.sup.5 .ANG., Polymer Laboratories
Ltd.) operated in a series at a flow rate of 1 ml/min in THF.
[0062] Samples composed of PLA or PCL were dissolved in methylene
chloride instead of THF, but otherwise analyzed in the same way as
the poly(DTH adipate) samples.
Water Content Determination Using Thermogravimetric Analysis
(TGA)
[0063] A small sample (10 mg) was cut from a specimen and placed in
an aluminum TGA pan. The sample was heated under a nitrogen flow at
a rate of 10.degree. C./min from room temperature to 225.degree. C.
The water uptake was measured by the loss in weight of the sample
as it was heated from room temperature until 150.degree. C.
Water Content Determination Using the Microbalance
[0064] At pre-determined time points, the samples were removed from
the buffer, rinsed with deionized water and blotted dry. The
sample's wet weight (W.sub.w) was immediately taken using an
electronic balance. The dry weight (W.sub.d) was taken after the
sample was dried under vacuum for at least two weeks, by this time
constant weight was achieved. The amount of water uptake was
calculated from the following equation:
% Water uptake=[(W.sub.w-W.sub.d)/W.sub.d].times.100 Eq. (2.1)
[0065] Differential Scanning Calorimetry Analysis (DSC) to Measure
the Melting Point of the Peptide and Melt Transitions in the
Polymer Film
[0066] DSC was used to determine the melting point of the peptide.
A sample of approximately 2 mg of peptide was weighed out and
sealed in a crimped aluminum DSC pan. The sample was heated at
12.degree. C./min from room temperature to 200.degree. C., under
nitrogen flow. DSC was also used to determine whether there is a
melting transition associated with the polymer films that contain
30% (w/w) peptide. A sample size of 6 mg of film was sealed in a
crimpled aluminum DSC pan and heated at 12.degree. C./min until
200.degree. C., under nitrogen flow. Melting point of the sample
was determined by the temperature at which the sharp endotherm of
melting occurred. All data was analyzed using the first-run
thermogram. An empty aluminum pan was used as a reference in each
experiment. The instrument used was a DSC 910 (TA instruments). The
instrument was calibrated with indium (m.p.=156.61.degree. C.)
before use.
Percent Mass Retention Study
[0067] The percent mass retention of the samples was calculated in
the following manner. The sample was removed from the PBS
incubation medium, rinsed in deionized water, and blotted with a
Kimwipe tissue. It was placed in a fresh vial and dried under
vacuum for 2 weeks. Following this dessication period, it was
weighed (W.sub.d). The mass obtained following incubation and
drying was compared to the initial mass (W.sub.o). The formula for
calculating percent mass retention is the following:
% Mass loss=[(W.sub.o-W.sub.d)/W.sub.o].times.100 Eq. (2.2)
Fabrication and Incubation of Films Under Acidic Conditions
[0068] The same formulation protocol mentioned above was followed
for these films, with the exception that concentrated JCl (12
molar) was added drop-wise to the stirred peptide/methanol solution
until the pH, as measured by a pH meter dropped from 6.8 to 2.
[0069] The acidic media for the in vitro incubation studies
conducted at pH of 2 was prepared in the following manner. Standard
PBS solution was used and 12 M HCl was added drop-wise into the PBS
until the PBS until the pH meter indicated that the desired pH had
been obtained.
Incubation of Films Under Varying Ionic Strength Conditions
[0070] Three sets of films were prepared in the standard method
mentioned above, one set was incubated in HPLC water, used as is.
Another was incubated in standard PBS buffer. The last set was
incubated in PBS buffer that was twice the concentration of the
standard PBS solution.
The Effect of the Peptide on the Glass Transition Temperature of
Poly(DTH Adipate)
[0071] The glass transition temperature of sets of films was
measured using Dynamic Mechanical Analysis (DMA). Measurements were
performed on a DMA 983 from TA Instruments in a flexural bending
deformation mode of strain. Each set of films contained a different
weight percentage of peptide ranging from 0%-30% (w/w) of peptide.
Samples of approximate size 5.times.10.times.1 mm were cut from the
films and mounted on the instrument using low mass clamps. The
samples were cooled using a liquid nitrogen cooling accessory to
-30.degree. C. The frequency was fixed at 1 Hz and the amplitude
was 1 mm. The glass transition was read from the maxima of the E''
peak.
Reduction of the Peptide Using Dithiothreitol (DTT)
[0072] INTEGRILIN.TM. (4.12 mg) was placed in a 25 ml roundbottom
flask. To this flask was added 50.12 mg of dithiothreitol. A
minimum of 20 moles of DTT was required per disulfide bridge (this
is 62 moles of DTT per disulfide bridge). Then 3 ml of water was
added and flask was stoppered. The contents of flask were stirred
with a magnetic stirrer. Every few hours, an aliquot of reaction
mixture was removed from the flask, diluted with HPLC water, and
analyzed with HPLC. As the reaction continued, the peak at 1.7
minutes corresponding to the intact peptide decreased and the peak
at 2.5 minutes corresponding to the peptide with the cleaved
disulfide bridge increased. Virtually all of the peptide had been
reduced after stirring overnight.
[0073] Following the conversion, the reaction mixture was
lyophilized overnight. In order to extract the reduced product, 5
ml of diethyl ether was added to dissolve the DTT and precipitate
the peptide. This mixture was stirred for 3 hours and the resulting
suspension was filtered using filter paper. The filtered material
was dried under vacuum overnight.
[0074] The presence of free SH groups was assayed in the following
manner. A saturated solution of lead acetate in ethanol was made
and a few milliliters of this solution were poured into the vial
containing the dried reduced peptide. A yellow color developed
indicating the presence of the lead sulfur complex: As a control,
equal volume of this lead solution was added to the peptide with
the intact disulfide bridge and no yellow color was detected.
Evidence for and Investigation of Interactions Between the Peptide
and Poly(DTH Adipate)
[0075] Formulation of INTEGRILIN.TM. with Poly(DTH Adipate)
[0076] Films composed of poly(DTH adipate) containing loadings of
5, 10, 15, 20, and 30% (w/w) peptide were prepared. Films
containing even the highest loading were clear and flexible. In
contrast, the films composed either of D,L-PLA or
poly(.epsilon.-caprolactone) (PCL) containing the same load of
peptide were opaque and brittle. The clarity of the
peptide/polyarylate films indicated that the phase separation in
the case of the peptide and poly(DTH adipate) was sufficiently
reduced that the separate polymer and peptide domains were too
small to scatter light. This suggested an enhanced compatibility of
peptide and tyrosine-derived polymer relative to the D,L-PLA or PCL
and peptide.
[0077] The flexibility of the polyarylate films that contained
peptide relative to those composed of the peptide and either of
aliphatic polyesters can be explained by the lower glass transition
temperature of the polyarylate (37.degree. C.) as compared to that
of PLA (52.degree. C.), and the amorphous nature of the polyarylate
as compared to PCL.
Release of Peptide from Films Incubated at 37.degree. C. and at
pH=7.4
[0078] In this experiment the in vitro release behavior of the
peptide, under simulated physiological conditions, from various
polymer matrices was observed. Both the aliphatic polymers released
and the peptide completely within three hours. In contrast, the
poly(DTH adipate) demonstrated only trace release, over a period of
77 days, under the identical conditions (FIGS. 3 $ 4).
Percent Mass Retention of Incubated Samples
[0079] Poly(DTH adipate) samples containing 30% (w/w) peptide lost
on average 5% mass during the 77 day incubation period (FIG. 5). In
contrast, the D,L-PLA samples that were formulated in the identical
fashion as the poly(DTH adipate) samples lost about 30% of their
mass within two hours (FIG. 6). The results of these experiments,
therefore, were consistent with the data obtained from the HPLC. In
the case of the poly(DTH adipate) films containing 30% (w/w)
peptide, the HPLC data indicate that these films released less than
10% of the loaded peptide (FIG. 3). This translates into a mass
loss for the entire sample of about 3% over the 77 day period. This
is in agreement with the average 5% mass loss observed for these
samples.
[0080] In contrast to the poly(DTH adipate) samples that
demonstrated minimal mass loss, the PLA samples showed extensive
mass loss. These film samples also contained 30% (w/w) peptide.
HPLC data indicated that these samples released all of the peptide
that they contained, this translates into a 30% mass loss over the
three hour incubation period. The resulting percent mass retention
data is about 70% for these samples is therefore in agreement with
the HPLC results. Furthermore, since the peptide was released so
rapidly by the PLA and PCL matrices, it can be concluded that the
peptide is small enough to readily diffuse through the polymer
chains and the development of pore structures and interconnecting
channels is not necessary to release the molecules of peptide that
are deep within the film. Therefore there should be minimal
impedance for release of the peptide from the polyarylate.
Measurement of Water Absorption by Polymer Films During
Incubation
[0081] Specimens of poly(DTH adipate) containing 30% (w/w) peptide
absorbed about 10% by weight water within the first day and
maintained that level of swelling throughout the entire incubation
period. Also the presence of the peptide increases the water
absorption of the polymer from about 3% by weight to 10% by weight
(FIG. 8). Samples of PLA and PCL containing identical loading of
peptide to the poly(DTH adipate) also absorbed with water within
that range during the 2-3 hours that they were incubated (FIG.
7).
The Effect of the Peptide on the Molecular Weight of the
Polymer
[0082] One of the amino acid residues on the peptide is an aspartic
acid. Aspartic acid is a moiety that introduces acidity into the
polymer when the polymer is blended with the peptide. Consequently,
an investigation of the molecular weight degradation of the polymer
was made and compared to rate of degradation for the neat poly(DTH
adipate) (FIG. 9).
[0083] As an additional control, samples composed of a blend of 10%
(w/w) PEG and 90% (w/w) poly(DTH adipate) were included in these
studies because these samples absorb 20% by weight water as
measured by the TGA. This represents more water than is absorbed by
the polymer samples containing 30% (w/w) peptide and functions as a
control for the effect of the added water on the molecular weight
degradation of the polymer. The results of these studies were that
the samples containing peptide did degrade at a faster rate than
the samples that did not contain peptide. After a period of over 2
months the poly(DTH adipate) samples containing 30% (w/w) peptide
had undergone 40% molecular weight degradation. In contrast, those
samples without peptide demonstrated almost no degradation during
this time period.
[0084] In addition, the increased amount of water in the polymer
matrix did not affect the rate of molecular degradation at all.
There did not appear to be any significant difference in the rate
of molecular weight degradation between the poly(DTH adipate)
samples containing PEG and the neat samples. It was the presence of
the peptide that had the catalytic effect on the degradation of the
polymer. However, this increase in degradation rate was not
significant enough to affect the release of the peptide.
[0085] The glass transition temperature of neat poly(DTH adipate)
was compared to those of poly(DTH adipate) containing 15, 20, of
30% (w/w) peptide. The results indicated that the peptide did not
reduce the glass transition temperature of the polymer. In fact, in
every case where peptide was present the glass transition
temperature was higher relative to the neat polymer samples. The
fact that there is an effect on the glass transition temperature
indicates that there is a mixing on the molecular scale between the
peptide and the polymer. The increase in T.sub.g with the addition
of the peptide confirms that there is hydrogen bonding between the
peptide and the polymer.
Effect of Ionic Strength of the Medium on the Release of the
Peptide
[0086] Poly(DTH adipate) films containing 30% (w/w) peptide were
prepared in the standard manner. The pH of the incubation media
remained about 7, but the ionic strength of the release media was
varied. The in vitro release of the peptide in HPLC water, in the
standard PBS solution (10 mM phosphate buffer saline, 138 mM NaCl,
2.7 mM DCl), and in PBS buffer formulated at twice the
concentration (20 mM phosphate buffer saline, 276 mM NaCl, 5.4 mM
KCl) was measured and compared (FIG. 10). It was observed that the
rate of release of peptide was four times greater in HPLC water as
compared to the release rate in phosphate buffer. These results
suggest some hydrophobic inter-actions between the peptide and
polymer. Most likely the source of these hydrophobic forces is the
pi stacking of the tryptophan ring of the peptide with the phenolic
ring of the polymer.
Incubation of Poly(DTH Adipate) Films Containing 30% (w/w) Peptide
in Acidic and Low Ionic Strength Conditions
[0087] Samples containing 30% (w/w) peptide were prepared under
standard conditions and incubated in HPLC water containing 0.1%
(v/v) trifluoroacetic acid, the pH of this solution was 2.2. The
release rate of the peptide in this study, where both pH and ionic
strength of the incubation media are lowered was greater (FIG. 11)
than when just one factor ws lowered. When just the pH was lowered,
12% of the loaded peptide was released within three days. When just
the ionic strength was reduced 8% of the loaded peptide was
released within three days. When both parameters were lowered
simultaneously 20% of the loaded peptide was released within this
time period. Despite enhanced release in these conditions, the
peptide was not "dumped out" as in the case of D.L-PLA but there
was a continuous diffusion of the peptide from the poly(DTH
adipate) matrix.
[0088] However, what was unexpected was the absorption of water
under these conditions (FIG. 12). Within the first day of
incubation these samples swelled three times relative to the
samples incubated in the standard PBS solution, and by the seventh
day these samples swelled by seven times. From FIG. 8, it can be
determined that the neat polymer, by itself, does not increase its
absorption of water during this initial 7 day time period when
incubated in the standard PBS solution. Moreover, since this
polymer is relatively hydrophobic, as determined from contact angle
experiments, it would not be expected that the change in incubation
conditions would promote such an increase in the percentage of
water uptake by the neat polymer. Therefore, it can be inferred
that it is the peptide within the matrix that is the source of this
large water uptake.
[0089] Therefore, incubation in the standard PBS solution favors
the interaction of the peptide with poly(DTH adipate) rather then
with water, hence, there was no increase in swelling beyond the
initial 10% even over many weeks of incubation in these conditions.
However, in conditions where the peptide-polymer interactions were
weakened, as in this case, where both the pH and ionic strength of
the incubation media were lowered, there is more of a driving force
for the peptide to interact with water and consequently, there was
a steady increase in the swelling of the film as more peptide
molecules were exposed and interacted with water.
[0090] Under these conditions of increased acidity and lowered
ionic strength the film samples also turned opaque immediately.
This opaqueness, noted only under the circumstances where there was
enhanced release of the peptide from the poly(DTH adipate) films,
appears to be correlated with increased water absorption by the
film samples. The weakening of the intensity of the peptide-polymer
interactions result in an increase in water absorption and the
developing opacity, is caused by the water that occupies the free
volume within the polymer matrix.
[0091] The absorption of 10% by weight water was sufficient to
release the peptide to completion in the case of the aliphatic
polymers. However, samples whose matrix was composed of poly(DTH
adipate) instead of PLA, absorbed 70% by weight water and yet not
release the peptide in the same "dumping" manner that the PLA and
PCL matrices did at 10% by weight of water absorption.
[0092] The interaction of the peptide with the tyrosine-derived
polyarylate arises from the unique structure of the polymer in
which the amide bond of each repeat unit is in close proximity to
the pendent ester in the same unit. This entire region can be
considered as one functional group, the .alpha.-amidocarboxylate
group and can act as a pocket for the hydrogen bonding of various
groups on the peptide.
Peptide-Polymer Interactions with Other Tyrosine-Derived
Polymers
[0093] Several other polymers were screened for the diffusion of
the peptide. The loadings of peptide used in these screening
experiment were lower than those used with poly(DTH adipate) but
were sufficient to expect release of this readily water-soluble
peptide barring any interactions to impede it.
[0094] Poly(DTH dioxaoctanedioate) was the first alternate but
structurally related polymer that was investigated. This polymer,
contains the DTH repeat unit which makes it similar to poly(DTH
adipate). However, this polymer is synthesized by polymerizing DTH
with dioxaoctanedioic acid instead of adipic acid. The objective of
this experiment was to observe the effect of a more hydrophilic
tyrosine-derived polymer on the diffusion of the peptide. It would
be expected that this compound is more hydrophilic than adipic acid
because there are two oxygens in the backbone spacer.
[0095] No peptide was released from these films (FIG. 15)
indicating that increasing the hydrophilicity of the polymer does
not have an effect on the release of the peptide. The water uptake
of these films was also measured and found to be 5% by weight in
the case of those films that contained 10% (w/w) peptide and 10% by
weight in the case of those films containing 20% (w/w) peptide.
This indicates that although the loading of the peptide is lower in
these specimens there is the same amount of water in the bulk in
poly(DTH adipate) specimens containing 30% (w/w) peptide as in
poly(DTH dioxaoctanedioate) containing 20% (w/w) peptide. In
addition, the structure of this polymer differs from poly(DTH
adipate) only in the structure of the flexible backbone unit. Since
the release behavior of this polymer is similar to that of poly(DTH
adipate), and the structural differences between the two polymers
lie only in the structure of the backbone spacer, it can be
concluded that most likely it is the DTH unit that is most integral
to the peptide-polymer interactions.
[0096] To confirm this last conclusion, another polymer structure
was substituted for poly(DTH adipate). It was the
poly(DTE.sub.0.95-co-PEG.sub.(1000) 0.05 carbonate). This polymer
is a random copolymer of desaminotyrosyl tyrosine ethyl ester (DTE)
and poly(ethylene glycol) (PEG). This copolymer shares the basic
desaminotyrosyl tyrosine alkyl ester repeat unit with the poly(DTH
adipate), but contains carbonate linkages and not ester in the
backbone, and no diacid component. The absence of the diacid
component and the similarity in the tyrosine-derived repeat unit
should further confirm that it is the tyrosine-derived component
and not the diacid that is involved in these interactions should
the peptide fail to diffuse from this polymer, also. It was also
postulated that since PEG is a very flexible molecule that can
hydrogen bond and the length of the PEG is longer than the length
of the diacid in the polyarylates, the PEG itself might interrupt
the hydrogen bonds and allow release of the peptide. Films
containing 10% (w/w) peptide were prepared.
[0097] Peptide release from these polymers also was minimal. The
water uptake of these samples was also measured, during the period
of incubation the film samples absorbed 10% by weight water. Again,
this is the same amount of water absorbed by the PLA, PCL, and
poly(DTH adipate) samples. Although the peptide loading is lower in
these films it is not surprising that the water uptake is as much
as samples of these other polymer systems since the PEG increases
the hydrophilicity of these samples. It appears from this data and
the previous data illustrating the minimal release of the peptide
from polymers containing the DTR unit that the tyrosine-derived
repeat unit as suggested above is the structure responsible for the
absence of diffusion of the peptide from the polymer.
[0098] Poly(DTE carbonate) (FIG. 16) was also formulated with 15%
(w/w) peptide. This polymer structure contains only the
desaminotyrosyltyrosine ethyl ester with carbonate linkages and
does not contain any PEG. These films also showed the same behavior
as the tyrosine-derived polyarylates (FIG. 17). The water uptake of
these films was also measured and found to be 6% by weight over the
incubation period.
Pulsatile Delivery of a Model Water Soluble Peptide Using Novel
Synthetic Copolymers
[0099] A monomer containing a free acid group randomly replaced the
DTH monomer at particular molar concentrations. The particular
monomer containing the free acid group is desaminotyrosyltyrosine
(DT) (FIG. 18). Three sets of films from this terpolymer were
prepared each set with a different molar concentration of DT. The
first set was poly(DT.sub.0.05-co-DTH.sub.0.95 adipate). Since the
DT content is the parameter that controls the rate of development
of acidity within these polymers, the objective of these
experiments was to observe the effect of increasing DT content of
the polymer on the release of the peptide.
Degradation Mechanism of the Poly(DT-co-DTH Adipate) Polymers
[0100] The degradation of the tyrosine-derived polyarylates
proceeds via an acid hydrolysis mechanism that is similar to the
hydrolysis of poly(DTE carbonate). The pendent ester groups in
contact with water would hydrolyze initially and the resulting acid
groups would begin the hydrolysis of the backbone ester, liberating
DTH and adipic acid. The adipic acid contributes to the acidity
within the matrix and further promotes the hydrolysis of both
backbone and pendent ester groups. However, it has been
demonstrated that this is a relatively slow process, only 40%
degradation occurs during a 2 month degradation period and the
degradation rate begins to plateau after reaching this extent of
degradation (FIG. 9).
[0101] The addition of DT to the polymer backbone accelerates the
degradation process. The degradation rate is hastened because the
hydrolyzed pendent ester is already present and randomly scattered
throughout the polymer prepared for the random scission for the
polymer chains. Moreover, since the degradation products of the
terpolymer are more acidic than those of poly(DTH adipate) due to
the increase in concentration of DT relative to DTH there is an
autocatalytic effect similar to what has been observed with PLA/PGA
derived polymers.
[0102] An investigation comparing the effect of increasing mole
percent of DT on the degradation rate of poly(DTH adipate) was made
(FIG. 19). It was observed that with the addition of anywhere
between 5 and 15 mole percent DT of sample films had chemically
degraded by 90-95% within 3 months. These data indicate that DT
does catalyze degradation, but it also indicates that there is a
maximum limit to the catalytic effect of the DT. This is seen in
the similarity in rate of degradation between the polymer
containing 10 mole percent of DT and that with 15 mole percent of
DT.
[0103] In addition, it can also be concluded from the percent
molecular weight retention data that the autocatalytic effect is
present in these polymers. This can be observed in the rate of
degradation of the polymer with 5 mole percent DT. The rate of
degradation begins much slower than the rate of degradation of the
polymers with higher percentages of DT. However after 120 days the
percent molecular weight retention is approximately the same as it
is for the samples with higher DT content. This suggests that
whatever initial acidity developed in the matrix of the polymer
containing 5 mole percent DT resulted in further increased
hydrolysis of the pendant chain of the DTH repeat unit, converting
the DTH repeat unit to the DT repeat unit. In this manner, the
number of DT repeat units increased from the original 5 mole
percent and consequently, the rate of degradation of this polymer
increased until it was able to "catch up" with the rate of
degradation of the polymers containing higher mole percentages of
DT.
Water Uptake of Poly(DT-co-DTH Adipate) Films
[0104] In spite of the increased hydrophilicity of DT versus DTH
there is no significant effect of mole percent of DT on amount of
water absorbed by the polymer when samples are initially incubated
(FIG. 20). Moreover significant water uptake does not appear to
cause degradation, rather it seems to be an effect of the
degradation. This can be observed by comparing the water uptake
during the first two weeks of incubation. During these two weeks
the rate of degradation is highest fro the polymers containing 10
or 15 mole percent DT, yet this is also the period of time that is
associated with the lowest percent water uptake. This is further
observed by noting that the polymers that do absorb more than 20%
water do not absorb this until they have degraded by 90%.
Visual Inspection of Poly(DT-co-DTH Adipate) Containing Peptide
[0105] The Three types of terpolymers were formulated in the same
technique as used for the copolymer, poly(DTH adipate), and two
loadings of peptide were explored. One loading was 15% (w/w)
peptide and the other 30% (w/w) peptide. Those films containing 15%
(w/w) peptide were completely transparent, there was not difference
between the neat copolymer films and films that contained 15% (w/w)
peptide. Samples from films that contained 30% (w/w) were slightly
hazy. All of the polymers were flexible and easy to handle.
Analysis of Miscibility of Terpolymer Using DSC
[0106] Thermograms of these terpolymers indicated only one glass
transition which was in the vicinity of the glass transition of
poly(DTH adipate). The appearance of only one glass transition
indicates there is a miscibility between the DTH adipate and the DT
adipate, not surprising since they share a very similar structure.
Moreover, the range of temperatures over which the glass transition
occurs is about 6.degree. C. This is about the same for poly(DTH
adipate) indicating that the polymer is quite homogeneous.
[0107] There was a trend of increasing T.sub.g with increasing mole
percent of DT, this is quite expected since an increase in the
amount of DT could result in an increase in hydrogen bonding
between the chains and thereby increasing the rigidity of the
polymer (Table 1). The homogeneity of the copolymer, in all
probability, contributes to the transparency and clarity of those
films that contain peptide.
TABLE-US-00001 TABLE 1 T.sub.g of
poly(DT.sub.x-co-DTH.sub.1-xadipate)x = 5, 10, 15 Mole Percent DT
in Polymer T.sub.g(.degree. C.) 0 37 5 37 10 43 15 46
Peptide Release from Terpolymer Films Containing 15% (w/w)
Loading
[0108] The incubation conditions were the same used in the above
experiments. The results of these experiments were a delayed
release, and the length of the delay time was a function of the
mole percent of DT. The set of films containing 15 mole percent DT
was characterized by a lag time of 20 days, after this lag time,
60% of the loaded peptide was released over a period of 40 days
(FIG. 21). Samples containing 10% DT were associated with a lag
time of close to 60 days. This delay period was followed by a
release phase where 60% of the loaded peptide was released within
30 days. Samples with 5% DT never released the peptide even after
110 days of incubation. The control in this experiment was poly(DTH
adipate) samples containing 15% (w/w) peptide which, also, did not
release the peptide. In all samples no burst was observed and no
leaching of the peptide occurred during the lag time.
[0109] The correlation of shorter lag time with increasing mole
percent of DT is not unexpected. The samples containing larger mole
percents of DT would be expected to accumulate acidic degradation
products faster creating a higher concentration of these products
in the bulk of the polymer resulting in the weakening of the
peptide-polymer interactions earlier than those polymers with lower
percentages of DT.
Peptide Release from Terpolymer Films Containing 30% (w/w)
Peptide.
[0110] In contrast to the terpolymer samples containing 15% (w/w)
peptide that were not characterized by a burst of peptide, these
samples which contained 30% (w/w) all demonstrated large bursts
(FIG. 22). The bursts were proportional to the mole percent of DT
in the polymer. The reason for the large burst observed with the
higher peptide loading could be the following: since the pK.sub.a
of the tyrosine acid proton is approximately 2, it would be
expected that when incubated initially, the majority of DT acid
protons would be lost to the medium. Therefore, any peptide
molecules that would be hydrogen bonded to this proton would no
longer be interacting with this group once the proton is lost, and
therefore these peptide molecules would be lost as a burst. In
addition, after the loss of the acid proton, the carboxyl-ate group
of the DT might actually compete with the peptide for interaction
sites on the DTH repeat unit resulting in the release from the
films of the peptide molecules that lost the competition.
Furthermore, the higher the DT content in the polymer the more
competition for the peptide and consequently, the size of the burst
is correlated with increasing mole percent of DT. However,
following the initial changes that occur when the specimens are
first incubated a new equilibrium is established of all the
components, and no further release of peptide occurs for many days.
This phenomenon is not observed with those terpolymer samples
containing the lower loading of peptide because there are many
fewer molecules of peptide and sufficient DTH sites for both the
interaction of the peptide and the DT.
[0111] Samples containing 10% DT were also characterized by a
second release phase. This second release phase occurred at
approximately 60 days which is also when the release of the peptide
ws observed in samples of this polymer containing 15% (w/w)
peptide. This release is due to the weakening of the interactions
associated with the drop in pH of the polymer matrix. This polymer
is quite unique among this grouping because these specimens alone
can be considered a pulsatile release system. The films containing
15% DT also demonstrated a second release phase at about 40 days
but it is much smaller than the second release phase of the
specimens containing 10% DT. Samples with 5% DT, again as in the 5%
DT samples containing 15% (w/w) peptide, presumably, never reached
the critical pH necessary for release of the peptide, and,
therefore, following the burst no more peptide was released.
Analysis of the Buffer Media of the Poly(DT-co-DTH Adipate)
Containing 15% (w/w) Peptide for pH Changes
[0112] The buffer media were analyzed for pH changes at each buffer
change (FIG. 23). Since the release of the peptide depends on the
lowering of the pH of the matrix a detectable lowering of the pH
should coincide with the release of the peptide. As expected, those
films composed of the polymer system with 15 mole percent of DT
demonstrated a drop of the pH below 7.2, first. This reduction in
pH began at approximately 30 days which was 10 days after release
of the peptide commenced. The pH of the media remained around 7.0
for the remainder of the incubation.
[0113] Samples containing 10 mole percent of DT were characterized
by a drop in pH below 7.2 beginning around 60 days, which is
precisely when release of the peptide commenced. The pH of the
media of these samples remained approximately 7.0 for the remainder
of the incubation period.
[0114] Specimens containing 5 mole percent of DT behaved exactly
like the poly(DTH adipate) samples that did not contain any DT at
all. Both types of samples remained between 7.3 and 7.4 for the
first 100 days of the incubation. After this time both types of
samples dropped to and remained at 7.2 for the remainder of the
incubation period. The data indicate that there is a correlation
between release of the peptide and generation of acidic degradation
products. Specifically, only those samples that released peptide
were associated with a drop in pH below 7.2 and this drop coincided
with peptide release.
[0115] In addition, control samples of poly(DTH adipate) containing
15% (w/w) peptide were placed in buffer at pH of 7.0. This again,
was to observe whether environmental pH affects the release of the
peptide. Trace release of the peptide was seen from these control
samples. No difference in the behavior of these samples as compared
to samples incubated in buffer at 7.4 was observed.
Chemical Integrity of the Released Peptide from Poly(DT-co-DTH
Adiapte) Matrices
[0116] The only polymer matrix of the group of polymers
investigated in these experiments that released any of the peptide
with the cleaved disulfide bond was the
poly(DT.sub.0.15-co-DTH.sub.0.85 adipate) samples which contained
the lower loading of peptide. These samples began the release phase
after a lag time of 20 days and continued this steady release until
approximately 60 days of incubation. Intact peptide was released
within the first 20 days of the release phase. However from the
44.sup.th day of incubation and beyond, fully one third of the
peptide released was associated with a cleaved disulfide bond.
Again, peptide with a cleaved disulfide bond was not observed in
association with any other polymer system in these studies.
Percent Molecular Weight Retention During In Vitro Incubation of
Poly(DT-co-DTH Adipate) Samples Containing 15% (w/w) Peptide
[0117] The percent molecular weight retention data of the various
sets of films containing 15% (w/w) peptide were not significantly
different than samples without peptide (FIGS. 24 and 19). This
suggests that the catalytic degradation effect on the polymer of
the DT is more important than the catalytic effect of the aspartic
acid group of the peptide. In addition, although the polymer
containing 15% DT released the peptide far earlier than the polymer
with 10% DT the molecular weight degradation rate was the same. The
explanation for this observation is that there is a sufficient
number of DT repeat units in both polymer systems to reach the
maximum rate of hydrolysis. However due to the increased amount of
DT in the poly(DT.sub.0.15-co-DTH.sub.0.85 adipate) relative to the
polymer with 10 mole percent DT, the degradation products also
contain more DT and therefore critical concentration of acidic
products necessary for release of the peptide is reached earlier
with these samples than the polymers with 10 mole percent of
DT.
Comparison of the Mechanism of Degradation of the Poly(DT-co-DTH
Adipate) Samples with and without Peptide
[0118] The poly(DT-co-DTH adipate) polymers without peptide appear
to degrade through the same mechanism. The reates may be different
especially between those polymers that contain 5 mole percent of DT
and those that contain more DT but the end result appears similar.
After 16 weeks of incubation the polymers have all developed a
significant amount of low molecular weight fractions and there does
not appear to be a preference for the formation of one particular
fraction over another.
[0119] In contrast, the samples that were formulated with peptide
do not all degrade by the same mechanism. Samples with 5 mole
percent DT behave similarly to the neat samples; there is the
random scission of the chains forming a variety of oligomers and no
special preference for the formation of a specific degradation
product is observed in the GPC chromatograms of the degraded
samples. This observed behavior was consistent for this polymer
system whether it was loaded with 15% (w/w) peptide, 30% (w/w)
peptide or neat.
[0120] However, those films of poly(DT.sub.0.10-co-DTH.sub.0.90
adipate) containing peptide (it was the same for both loadings of
peptide) exhibited a distinct preference for the formation of
monomer during the degradation process. The monomer (DTH) has a
retention time of 18.6 minutes in the GPC and starting from the
5.sup.th week of incubation and beyond there is the presence of a
well defined peak at this retention time in the chromatograms of
these samples. This implies that the polymer degrades in the random
scission manner until it reaches about 20% molecular weight
retention at five weeks of incubation. After this point the polymer
begins to degrade in an unzipping process. This unzipping process
means that the degradation begins from the chain ends and moves in
along the chain. Consequently, each scission results in the
formation of monomer. The poly(DT.sub.0.15-co-DTH.sub.0.85 adipate)
samples that contain peptide exhibited this same behavior as
described for the poly(DT.sub.0.10-co-DTH.sub.0.90 adipate) samples
that contain peptide.
[0121] Physical Disintegration During In Vitro Incubation of
Samples of Poly(DT-co-DTH Adipate) Samples Containing Peptide
[0122] There were no significant physical changes in these samples
for the first two weeks of incubation. However, by the third week
all the samples have become opaque, and by the fourth week there
was significant shredding of the samples containing 15% DT. IN
fact, so much shredding has occurred that the buffer media has
turned opaque. No significant shredding of the samples containing
10% DT occurred before 70 days and the samples containing 5% DT
never shredded at all.
[0123] This shredding is most likely related to the dissolution of
the water soluble degradation products of the polymers. Other
polymers with the higher free acid content contain a significant
amount of water soluble degradation products (DT and adipic acid)
and therefore shredding is common to both of them. Since shredding
occurs in both of these polymer systems the resulting films
following incubation of 80 or more days appeared transparent with
only a "skin" of the material left. All of the bulk had
disappeared. Shredding never occurs in the polymer with the lower
free acid content since it never develops enough water soluble
components within the bulk and therefore, though, these samples
swelled and curled during the incubation period they remained
smooth and intact. The same phenomena was observed with the
poly(DT-co-DTH adipate) samples containing 30% (w/w) peptide.
[0124] The foregoing illustrates that polymers that form hydrolytic
degradation precuts promote the release of biologically active
compounds from the polymer matrix in comparison to polymers of
similar structure to do not hydrolytically degrade. Neither polymer
initially releases the biologically active compound. However, a
delayed pulsatile release is obtained from polymers that
hydrolytically degrade as the degradation precuts accumulate, while
significant quantities of biologically active compound are never
released from the polymers that do not hydrolytically degrade.
[0125] The foregoing examples and description of the preferred
embodiment should be taken as illustrating, rather than as
limiting, the present invention as defined by the claims. As will
be readily appreciated, numerous variations and combinations of the
features set forth above can be utilized without departing from the
present invention as set forth in the claims. Such variations are
not to be regarded as a departure from the spirit and scope of the
invention, and all such modifications are intended to be included
within the scope of the following claims.
Sequence CWU 1
1
117PRTHomo sapiensMOD_RES(1)..(1)desaminoCys 1Cys Arg Gly Asp Trp
Pro Cys1 5
* * * * *