Pegylated drug complexed with bioadhesive polymer suitable for drug delivery and methods relating thereto

PEGylated drugs complexed with bioadhesive polymers, wherein the PEGylated drugs comprise a polyethylene glycol covalently bonded to the drugs are disclosed. The PEGylated drug/bioadhesive polyner complex and compositions thereof may be topically administered to body fluids or mucosal tissues. Methods of administering the PEGylated drug/bioadhesive polymer complex and compositions thereof to an animal are also disclosed.

TECHNICAL FIELD 
This invention relates generally to a PEGylated drug complexed with 
bioadhesive polymer and, more specifically, to a PEGylated drug complexed 
with a bioadhesive polymer wherein the complex is suitable for drug 
delivery to a mucosal tissue. 
BACKGROUND OF THE INVENTION 
Polyethylene glycol (PEG) has been widely used in biomaterials, 
biotechnology and medicine primarily because PEG is a biocompatible, 
nontoxic, nonimmunogenic and water-soluble polymer (Zhao and Harris, ACS 
Symposium Series 680: 458-72, 1997). In the area of drug delivery, PEG 
derivatives have been widely used in covalent attachment (i.e., 
"PEGylation") to proteins to reduce immunogenicity, proteolysis and kidney 
clearance and to enhance solubility (Zalipsky, Adv. Drug Del. Rev. 
16:157-82, 1995). Similarly, PEG has been attached to low molecular 
weight, relatively hydrophobic drugs to enhance solubility, reduce 
toxicity and alter biodistribution. Typically, PEGylated drugs are 
injected as solutions. 
A closely related application is synthesis of crosslinked degradable PEG 
networks or formulations for use in drug delivery since much of the same 
chemistry used in design of degradable, soluble drug carriers can also be 
used in design of degradable gels (Sawhney et al., Macromolecules 26: 
581-87, 1993). It is also known that intermacromolecular complexes can be 
formed by mixing solutions of two complementary polymers. Such complexes 
are generally stabilized by electrostatic interactions 
(polyanion-polycation) and/or hydrogen bonds (polyacid-polybase) between 
the polymers involved, and/or by hydrophobic interactions between the 
polymers in an aqueous surrounding (Krupers et al., Eur. Polym J. 
32:785-790, 1996). For example, mixing solutions of polyacrylic acid 
(PAAc) and polyethylene oxide (PEO) under the proper conditions results in 
the formation of complexes based mostly on hydrogen bonding. Dissociation 
of these complexes at physiologic conditions has been used for delivery of 
free drugs (i.e., non-PEGylated). In addition, complexes of complementary 
polymers have been formed from both homopolymers and copolymers. 
While significant advances have been made in the field of PEGylated drug 
delivery, there is still a need in the art for novel and improved 
PEGylated drug delivery formulations, particularly those that are useful 
in the area of sustained drug delivery. The present invention fulfills 
these needs, and provides further related advantages. 
SUMMARY OF THE INVENTION 
In brief, the present invention is directed to a PEGylated drug complexed 
with a bioadhesive polymer, wherein the PEGylated drug comprises a 
polyethylene glycol covalently bonded to the drug. Accordingly, in one 
aspect of the present invention, a PEGylated drug complexed with a 
bioadhesive polymer is disclosed wherein the PEGylated drug comprises a 
polyethylene glycol covalently bonded to the drug and wherein the 
polyethylene glycol has a molecular weight ranging from about 3 kD to 
about 50 kD, and preferably from about 5 kD to about 30 kD. The drug of 
the PEGylated drug complexed with a bioadhesive polymer may be either a 
protein or a peptide, and preferably is a protein. In an alternative 
embodiment, the drug of the PEGylated drug complexed with a bioadhesive 
polymer may be a hydrophobic drug (e.g., taxol or paclitaxel). 
In another aspect of the present invention, the bioadhesive polymer of the 
PEGylated drug complexed with a bioadhesive polymer may be either 
polyacrylic acid, polymethylacrylic, polyethylacrylic acid or chitosan, 
and preferably is polyacrylic acid. Alternatively, the bioadhesive polymer 
of the PEGylated drug complexed with a bioadhesive polymer may be either a 
random block or graft copolymer of one or more of polyacrylic acid, 
polymethylacrylic or polyethylacrylic acid. In addition, in one embodiment 
the PEGylated drug complexed with the bioadhesive polymer is stable at or 
below pH 4, and in another embodiment the PEGylated drug complexed with 
the bioadhesive polymer is stable up to about pH 7 and degrades at or 
above about pH 7. 
In yet another aspect of the present invention, the PEGylated drug 
complexed with a bioadhesive polymer may be in combination with free PEG, 
polyvinylpyrrolidone, polyacrylamide or N-alkyl derivatives thereof, or 
polyvinyl alcohol. The free PEG, polyvinylpyrrolidone, polyacrylamide or 
N-alkyl derivatives thereof, or polyvinyl alcohol have molecular weights 
ranging from about 10 kD to about 500 kD, preferably from about 10 kD to 
about 200 kD, and more preferably about 18.5 kD. 
Instill another aspect of this invention, a method of delivering a drug to 
a body fluid or mucosal tissue comprising contacting the body fluid or 
mucosal tissue with the PEGylated drug complexed with a bioadhesive 
polymer is disclosed. The body fluid or mucosal tissue may be fluid or 
tissue of the alimentary tract, respiratory tract, eye, nose, vagina, 
lung, mouth, or throat. Alternatively, the body fluid or mucosal tissue is 
fluid or tissue of an open wound.

DETAILED DESCRIPTION OF THE INVENTION 
As mentioned above, the present invention is generally directed to a 
PEGylated drug complexed with a bioadhesive polymer wherein the complex is 
suitable for sustained drug delivery to a biological fluid or mucosal 
tissue. More specifically, the PEGylated drug of the present invention 
comprises a polyethylene glycol (PEG) covalently bonded to a drug. 
Covalent attachment of the PEG to the drug (known as "PEGylation") may be 
accomplished by known chemical synthesis techniques. For example, in one 
exemplary embodiment of the present invention, the PEGylation of protein 
may be accomplished by reacting NHS-activated PEG with the protein under 
suitable reaction conditions as generally depicted by the following 
reaction scheme: 
##STR1## 
In an alternative embodiment, the PEGylated drug of the present invention 
comprises a polyethylene glycol (PEG) bonded to a drug via a degradable 
bond such that after release from the complex with the bioadhesive 
polymer, the PEGylated drug is subsequently dissociated into drug and PEG. 
In the context of covalent bonds, the covalent bonds may degrade in 
aqueous biologic environments by hydrolysis (e.g., an ester 
--COO--.fwdarw.--COOH+HO--, or an anhydride, 
--CO--O--CO--.fwdarw.--COOH+HOOC--, or an amide 
--CONH--.fwdarw.--COOH+H.sub.2 N--) or by enzymolysis wherein an enzyme 
may effect similar reactions. Enzymes may also cause oxidation, reduction 
or other chemical reactions which lead to bond scission. 
After PEGylation, the PEGylated drug is complexed with a suitable 
bioadhesive polymer. As used within the context of the present invention, 
suitable bioadhesive polymers include (but are not necessarily limited to) 
polyacrylic acid (PAAc), polymethacrylic acid (PMAAc), polyethylacrylic 
acid (PEAAc) including lightly cross-linked polymers thereof, and 
chitosan. More specifically, the first three bioadhesive polymers of the 
present invention may be generally characterized as carboxylic 
acid-containing polymers (chitosan, however, contains no carboxyl groups). 
The carboxylic acid moieties of such polymers are typically non-ionized at 
pHs below 5 and become ionized when contacted with, for example, a 
biological fluid and/or mucosal tissue at higher pH values. Note the pK of 
the carboxyl group will generally rise with the hydrophobicity of its 
microenvironment such that the degree of ionization of the carboxyl group 
at pH 7.4 will decrease in the order of PAAc, PMAAc, and PEAAc. Thus, the 
composition of the carboxylic polymer or copolymer will determine the rate 
and extent of dissociation of its complex with PEG at pH 7.4, since the 
strength of the complex will depend on the presence of a significant 
number of repeat units having --COOH groups (as opposed to --COO.sup.- 
groups). 
This feature also influences the bioadhesive properties of the carboxylic 
polymer via hydration and swelling upon contact with the biological fluid 
and/or mucosal tissue. For example, the carboxylic acid groups of polymers 
such as PAAc are ionized upon contact with biological fluids or mucosal 
tissues, and the uptake of cations (such as Na.sup.+ and K.sup.+) provides 
neutralized carboxylic acid moieties (e.g., COO.sup.- Na.sup.+). This 
ionization is accompanied by the uptake of water which, in turn, results 
in swelling and can cause the polymer to become "sticky" or bioadhesive. 
For purposes of the present invention, bioadhesive polymers and copolymers 
may be formed by known chemical synthesis techniques such as by 
polymerizing suitable monomers to yield the desired polymer or copolymer. 
Accordingly, a suitable polymer may be derived, for example, from 
polymerizable carboxylic acids, resulting in the desired synthetic 
carboxylic acid-containing polymer. 
In contrast, the bioadhesivity of chitosan, a poly(D-glucosamide) as 
generally shown below, is due to its strong acid-base and ionic 
interactions with the negatively charged mucosal surfaces. Furthermore, 
chitosan is not polymerized; rather, it is obtained as a polymer of 
poly(D-glucosamide) from natural sources (e.g., crustaceans). 
##STR2## 
As used in the context of the present invention, the term "drug" includes 
the definition set forth in 21 C.F.R .sctn. 201(g)(1), "Federal Food, 
Drug, and Cosmetic Act Requirements relating to Drugs for Human and Animal 
Use" (hereby incorporated by reference). Under this definition, a drug 
means (a) articles recognized in the official United States Pharmacopeia, 
official Homeopathic Pharmacopeia of the United States, or official 
National Formulary, or any supplement thereof; and (b) articles intended 
for use in the diagnosis, cure, mitigation, treatment, or prevention of 
disease in man or other animals; and (c) articles (other than food) 
intended to affect the structure of any function of the body of man or 
other animals; and (d) articles intended for uses as a component of any 
articles specified in clause (a), (b) or (c) above; but does not include 
devices or their components, parts or accessories. 
In general, the PEGylated drug complexed with a carboxylated bioadhesive 
polymer in accordance with the present invention is generally stable at 
about pH 4 and below and readily falls apart at higher pH's when a 
significant fraction of the --COOH groups are ionized. That is, when 
subjected to an acidic environment, the complex formed between the 
PEGylated drug and the bioadhesive polymer is stable in that it does not 
readily dissociate; whereas when it is subjected to a non-acidic or 
neutral environment, the complex will dissociate at rates dependent on the 
degree of ionization of the --COOH groups. However, the pH at which the 
PEGylated drug/bioadhesive complex is stable may be controlled, for 
example, by appropriate selection of the bioadhesive polymer. For example, 
the use of polymethylacrylic acid (PMAAc) or polyethylacrylic acid 
(PEAAc), as opposed to polyacrylic acid (PAAc), will generally result in a 
complex that is stable at a pH approaching 7, thus further slowing the 
rate of drug release when the complex is exposed to body fluid or mucosal 
tissue having a pH of the same value. 
Moreover, the addition of one or more of a free PEG (i.e., polyethylene 
glycol not covalently bonded to a drug), a polyvinylpyrrolidone, a 
polyacrylamide (including N-alkyl derivatives thereof such as 
--N(R.sub.1)(R.sub.2) derivatives where R.sub.1 and R.sub.2 are 
independently selected from hydrogen and C.sub.1-8 alkyl) or polyvinyl 
alcohol (PVA) to the PEGylated drug/bioadhesive polymer complex, may 
further retard the rate at which PEGylated drug dissociates from the 
complex. Stated somewhat differently, the addition of one or more of a 
free PEG, polyvinylpyrrolidone, polyacrylamide (including its derivatives) 
or PVA may reduce the rate at which PEGylated drug becomes dissociated 
from the bioadhesive polymer, especially when the PEGylated drug includes 
PEGs below 10 kD in MW. Without prescribing to any particularly theory, it 
is believed that the addition of free PEG or other suitable polymer 
further condenses the PEGylated drug/bioadhesive polymer complex, thereby 
inhibiting the release of PEGylated drug when formed in an acidic 
environment and subsequently subjected to a non-acidic or neutral 
environment. 
Accordingly, the PEGylated drug/bioadhesive polymer complexes of the 
present invention are particularly useful for the sustained and/or 
controlled release of a drug as that term has been defined above. In this 
embodiment, the PEGylated drug/bioadhesive polymer complex may be 
equilibrated in an acidic solution containing one or more agents, and then 
dried (e.g., in air, or lyophilized) to yield compositions in the form of 
particles and/or films. In the case of particles, such particles may 
generally have a diameter of less than 1 mm, and are more typically from 
about 0.1 mm to 0.5 mm in diameter. In some cases nanoparticles may form 
with a diameter of 50 nm or below. Depending upon its intended use, the 
larger particles may be further reduced in size by mechanical milling and 
grinding techniques. 
The PEGylated drug/bioadhesive polymer complex and compositions formulated 
therefrom may be administered to the body of an animal (including man) in 
any suitable manner, such as by topical administration. Topical 
administration generally includes application to a mucosal tissue (such as 
the respiratory and alimentary tracts, rectum and vagina, eyes, nose, 
mouth and throat, lungs, gastro-intestinal tract, as well as open wounds) 
which contain sufficient water/ion content to hydrate the bioadhesive 
polymer. Moreover, the PEGylated drug/bioadhesive polymer complex and 
compositions thereof may be formulated using known formulation techniques 
in any manner suitable for its intended application. For example, the 
PEGylated drug -bioadhesive polymer complex may be suspended or emulsified 
within a solution containing an acceptable carrier or diluent, or combined 
with, for example, a solution, cream, gel, ointment or powder. Typically, 
suitable PEGylated drug/bioadhesive polymer complex concentrations in the 
formulations of these compositions range from 0.1% to 50% by weight, and 
preferably from 0.5% to 30% by weight. 
The PEGylated drug/bioadhesive polymer complex and compositions thereof may 
also be formulated as a tablet, capsule, suppository, or even an aqueous 
(low pH) suspension. To this end, suppository formulations may be 
particularly suited for rectal administration of the PEGylated 
drug/bioadhesive polymer complex, while tablet and capsule form may be 
suitable for oral administration. Similarly, suspensions may be suitable 
for application to mucosal surfaces and/or tissues, such as eye, nose, 
vagina, lungs, mouth and throat, etc. The PEGylated drug/bioadhesive 
polymer complex may also be formulated for nasal, pulmonary or buccal 
administration by known techniques. Furthermore, the PEGylated 
drug/bioadhesive polymer complex may be formulated such that it may be 
implanted in the body of an animal by, for example, subcutaneous or 
intramuscular implantation, or may be implanted into bone. 
In addition to use as vehicles for the sustained and controlled release of 
drugs, the PEGylated drug/bioadhesive polymer complex compositions of the 
present invention may have utility for a variety of other applications, 
including (but not limited to) uses relating to separation techniques, 
diagnostics, and bioreactions with immobilized ligands or reactant. 
The following examples are provided for purposes of illustration, not 
imitation. 
EXAMPLES 
Materials and Methods: 
Papain MW 23,000, pl 8.75 
(available from Sigma) 
PEG Succinimidyl Succinate PEG (SS-PEG), MW 5,000 
Succinimidyl propionic acid PEG (SPA-PEG), MW 20,000 
Succinimidyl Succinate branched PEG (PG2-NHS), MW 2.times.20,000 
(available from Shearwater Polymers) 
Ionexchange chromatography: 
BioCad.sup.R PerSeptive Biosystems, Inc. 
Column Strong Cation Exchange 
POROS HS/M 4.6 mmD/100L, 1.66 ml 
Elution 
buffer: 
20 mM MES pH6.0, NaCl 0 to 1000 mM 
Mass spectrometry: 
Matrix Assisted Laser Deposition/lonization-time of flight Mass 
Spectrometry. (MALDI-TOF-MS) 
Papain, a model protein, was PEGylated by using NHS- activated PEG having 
molecular weights of 5 kD, 20 kD and branched 2.times.20 kD. Solutions of 
PEGylated papain, PAAc (450 kD), and/or free PEG (.about.18.5 kD) were 
mixed at pH 3.0 and cast and dried on the porous surface (pore size 8 
.mu.m) of the upper chamber of a Transwell.sup.R (Corning Costar). Release 
of PEGylated papain were examined in PBS, pH 7.4. 
In the case of SS-PEG PEGylated to papain, for example, papain was 
dissolved in 10 ml of a borate buffer (100 mM, pH 8.5)(1.0 mg/ml, 
4.35.times.10.sup.-7 mol). A 10 times excess molar amount of SS-PEG 
(4.35.times.10.sup.-6 mol, 21.8 mg) was added into the papain solution. 
After this mixture was allowed to rotate for 30 minutes at room 
temperature (i.e., 25-26.degree. C.), unreacted papain and PEG were 
removed by cation exchange chromatography. A fraction of the PEGylated 
papain was collected with monitoring of absorbance at 280 nm. One or two 
PEG molecules were observed by the MALDI-TOF-MS. 
Activity of PEGylated Papain: 
Substrate N.alpha.-Benzoly- L-Arginine-7-Amido-4-MethylCoumarin(BAAMCA) 
Sigma 
50 .mu.l of Papain of PEGylated Papain, 100 .mu.g/ml, was added into the 3 
ml BAAMCA, of 50 .mu.M, in 50 mM Tris-HCL, pH7.5 containing 5 mM 
L-Cysteine, and 2 mM EDTA and 1% DMSO. After the mixture was allowed to 
incubate for 0 to 60 min, at the room temperature, 25.degree. C., 
fluorescent intensity at, .lambda.ex 380 and .lambda.em 440, were 
measured. 
Complexation: 
Transwell.sup.R Polycarbonate membrane, pore size 8.0 .mu.m 
surface area 0.33 cm.sup.2 
Corning Coster 
Elution buffer of PEGylated Papain was exchanged to citric buffer, pH3.0, 
10 mM, by using the Sephadex-G column(PD-10.sup.R). PAAc, MW 450 kD, or 
free PEG 18.5 kD was dissolved in distilled water and pH was adjusted to 
3.0 by addition of NaOH. 2.0 mg of PAAc and 0.2 mg of PEGylated Papain 
and/or 0. 0.2, 0.4, 0.8 and 1.6 mg of free PEG were mixed in the upper 
chamber of Transwell.sup.R, and dried at 37.degree. C. for 24 hours. 
Release: 
600 .mu.l or 100 .mu.l of PBS, pH 7.4 (10 mM PB, 2.7 mM KCl, 137 mM NaCl) 
was added into the lower or upper chamber of Transwell.sup.R respectively. 
The Transwell was allowed to shake at room temperature during the release 
experiments. After an appropriate interval, 400 .mu.l of release medium 
from the lower chamber was sampled and the absorbance at 280 nm was 
measured. 
In view of the foregoing materials and methods, the following more specific 
and illustrative examples are presented. 
Example 1 
RELEASE OF PEGYLATED PAPAIN FROM THE FORMULATIONS 
This example corresponds to FIG. 1 which illustrates the percent release 
versus time of papain, 5 kD PEG-papain, 20 kD PEG-papain, and 2.times.20 
kD PEG-papain, respectively, from their corresponding formulations (the 
formulations include polyacrylic acid (PAAc)), wherein: 
Formulations: 0.2 mg Papain 
5 kD, 20 kD and 2.times.20 kDPEG-Papain 
2.0 mg 450 kD PAAc 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example shows that the 5 kD PEGylated-papain is not slowed by the PAAc 
because the 5 kD PEG is too low in MW to complex with PAAc, while the 20 
kD PEG-papain and the 2.times.20 kD PEG-papain are both retarded, because 
the 10 and 20 kD PEGs are large enough to form complexes with PAAc. 
Example 2 
RELEASE OF PAPAIN AND PEGYLATED PAPAIN--EFFECT OF A CONJUGATION OF PEG AND 
ADDITION OF FREE PEG 
This example corresponds to FIG. 2 which illustrates the effect of the 
addition of 18.5 kD free PEG on the percent release versus time of papain 
and 5 kD PEG-papain, respectively, from their corresponding formulations 
(the formulations include polyacrylic acid (PAAc)), wherein: 
Formulations: 0.2 mg 5 kD PEG-Papain 
2.0 mg 450 kD PAAc 
0 and 0.8 mg 18.5 kD free PEG 
Mixed at pH 3.0 and cast 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example shows that the addition of free 18.5 kD PEG to the 5 kD 
PEG-papain conjugate significanly retards its release rate, while it has 
only a slight retarding effect on the release of free papain. 
Example 3 
EFFECT OF FREE PEG ON TIE RELEASE OF PEGYLATED PAPAIN FROM THE FORMULATIONS 
This example corresponds to FIG. 3 which illustrates the effect of the 
addition of 18.5 kD free PEG on the percent release versus time of papain, 
5 kD PEG-papain, 20 kD PEG-papain, and 2.times.20 kD PEG-papain, 
respectively, from their corresponding formulations (the formulations 
include polyacrylic acid (PAAc)), wherein: 
Formulations: 0.2 mg Papain 
5 kD, 20 kD and 2.times.20 kD PEG-Papain 
20. mg 450 kD PAAc 
0.8 mg 18.5 kD PEG 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example shows that the addition of free 18.5 kD PEG does not 
significantly retard the release of 20 kD or 2.times.20 kD PEG-Papain, 
while the addition significantly retards the 5 kD PEG-Papain even below 
the 20 kD or 2.times.20 kD PEG-Papain. 
Example 4 
EFFECT OF FREE PEG ON THE RELEASE OF PEGYLATED PAPAIN FROM THE FORMULATIONS 
This example corresponds to FIG. 4 which illustrates the effect of the 
addition of different amounts of 18.5 kD free PEG on the percent release 
versus time of 5 kD PEG-papain from its corresponding formulations (the 
formulations includes polyacrylic acid (PAAc)), wherein: 
Formulations: 0.2 mg 5 kD PEG-Papain 
2.0 mg 450 kD PAAc 
0,0.2,0.4,0.8 and 1.6 mg 185 kD free PEG 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example shows that there may be an optimum amount of free 18.5 kD PEG 
in any formulation, and in this particular formulation it is approximately 
0.2-0.4 mg. 
Example 5 
EFFECT OF FREE PEG ON THE RELEASE OF PEGYLATED PAPAIN FROM THE FORMULATIONS 
This example corresponds to FIG. 5 which provides a bar chart that 
illustrates the effect of the addition of different amounts of 18.5 kD 
free PEG on the percent released in an 8 hour period of 5 kD PEG-papain 
from its corresponding formulations as shown in FIG. 4, wherein: 
Formulations: 0.2 mg 5 kD PEG-Papain 
2.0 mg 450 kD PAAc 
0,0.2,0.4,0.8 and 1.6 mg 18.5 kD free PEG 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example plots the data of Example 4 in an alternative bar chart 
format. 
Example 6 
EFFECT OF FREE PEG ON THE RELEASE OF PEGYLATED PAPAIN FROM THE FORMULATIONS 
This example corresponds to FIG. 6 which illustrates the effect of the 
addition of different amounts of 18.5 kD free PEG on the percent release 
versus time of 20 kD PEG-papain from its corresponding formulations (the 
formulations includes polyacrylic acid (PAAc)), wherein: 
Formulations: 0.2 mg 20kD PEG-Papain 
2.0 mg 45 kD PAAc 
0,0.2,0.4,0.8 and 1.6 mg 18.5 kD free PEG 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example shows that the addition of free 18.5 kD PEG may only enhance 
the release rate of a 20 kD PEG-papain conjugate. 
Example 7 
EFFECT OF FREE PEG ON THE RELEASE OF PEGYLATED PAPAIN FROM THE FORMULATIONS 
This example corresponds to FIG. 7 which provides a bar chart that 
illustrates the effect of the addition of different amounts of 18.5 kD 
free PEG on the percent released in an 8 hour period of 20 kD PEG-papain 
from its corresponding formulations as shown in FIG. 6, wherein: 
Formulations: 0.2 mg 20 kD PEG-Papain 
2.0 mg 450 kD PAAc 
0,0.2,0.4,0.8 and 1.6 mg 18.5 kD free PEG 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example plots the data of Example 6 in an alternative bar chart 
format. 
Example 8 
EFFECT OF FREE PEG ON THE RELEASE OF PEGYLATED PAPAIN FROM THE FORMULATIONS 
This example corresponds to FIG. 8 which illustrates the effect of the 
addition of 18.5 kD free PEG on the percent release versus time of papain 
and 5 kD PEG-papain, respectively, from their corresponding formulations 
(the formulations include polyacrylic acid (PAAc)), wherein: 
Formulations: 0.2 mg 2.times.20 kD PEG-Papain 
2.0 mg 450 kD PAAc 
0 and 0.8 mg 18.5 kD free PEG 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example shows that the addition of free 18.5 kD PEG does not lead to 
significantly retarded release of free papain, rather it speeds up the 
release of 2.times.20 kD PEG-papain. 
Example 9 
RELEASE OF PEGYLATED PAPAIN AND TRYPSIN INHIBITOR 
This example corresponds to FIG. 9 which illustrates the percent release 
versus time of 5 kD PEG-papain and 5 kD PEG-trypsin inhibitor, 
respectively, from their corresponding formulations (the formulations 
include polyacrylic acid (PAAc)), wherein: 
Formulations: 0.2 mg 5 kD PEG-Papain or -Trypsin inhibitor 
2.0 mg 450 kD PAAc 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
In this example, trypsin inhibitor is negatively charged at pH 7.4, whereas 
papain is positively charged. Thus, this example shows that in the absence 
of free 18.5 kD PEG, both the 5 kD PEG-trypsin inhibitor and 5 kD 
PEG-papain are released at about the same rate. 
Example 10 
EFFECT OF FREE PEG ON THE RELEASE OF PEGYLATED PAPAIN AND TRYPSIN INHIBITOR 
This example corresponds to FIG. 10 illustrates the effect of the addition 
of 18.5 kD free PEG on the percent release versus time of 5 kD PEG-papain 
and 5 kD PEG-trypsin inhibitor, respectively, from their corresponding 
formulations (the formulations include polyacrylic acid (PAAc)), wherein: 
Formulations: 0.2 mg 5 kD PEG-Papain or -Trypsin inhibitor 
2.0 mg 450 kD PAAc 
0.8 mg 18.5 kD free PEG 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example shows that when a condensing polymer such as free 18.5 kD PEG 
is added, the charge on the protein is also important to the retardation; 
that is, this example shows that the retardation of the 5 kD PEG-Papain 
(cationic at pH 7.4) is much greater than for 5 kD PEG-trypsin inhibitor 
(anionic at pH 7.4). 
Example 11 
RELEASE OF PEGYLATED PAPAIN AND TRYPSIN INHIBITOR FROM FORMULATIONS WITH 
PMAAC 
This example corresponds to FIG. 11 which illustrates the percent release 
versus time of 5 kD PEG-papain and 5 kD PEG-trypsin inhibitor, 
respectively, from their corresponding formulations (the formulations 
include polymethacrylic acid (PMAAc)), wherein: 
Formulations: 0.2 mg 5 kD PEG-Papain or -Trypsin inhibitor 
2.0 mg 500 kD PMAAc 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example shows that when PMAAc is substituted for PAAc, the 
postively-charged protein is released much more slowly than the 
negatively-charged protein in the absence of any added free PEG (Compare 
to FIGS. 9 and 10). This also shows that both ionic interactions between 
the protein and the bioadhesive polymer as well as the hydrophobic 
character of the bioadhesive polymer are important. The latter will also 
affect the pK of the --COOH group and the strength of the complex at pH 
7.4. 
Example 12 
EFFECT OF PH ON THE RELEASE OF PEGYLATED PAPAIN FROM FORMULATIONS WITH 
PMAAC 
This example corresponds to FIG. 12 which illustrates the effect of casting 
the formulations at pH 3.0 and pH 6.0, respectively, on the release versus 
time of 5 kD PEG-papain; from the respective formulations (the 
formulations include polymethacrylic acid (PMAAc)), wherein: 
Formulations: 0.2 mg 5 kD PEG-Papain 
2.0 mg 5 kD PMAAc 
Mixed at pH 3.0 or 6.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example shows that the importance of casting the formulation at a low 
pH of 3.0 where the complex of PMAAc and PEG is strongly formed, as 
opposed to pH 6.0 where the complex may not be so strongly formed due to 
ionization of some of the --COOH groups. 
Example 13 
RELEASE OF PEGYLATED PAPAIN FROM THE PMAAC OR PMAAC GEL 
This example corresponds to FIG. 13 which illustrates the percent release 
versus time of 5 kD PEG-papain from formulations that include polyacrylic 
acid (PAAc) and polymethacrylic acid (PMAAc), respectively, wherein: 
Formulations: 0.2 mg 5 kD PEG-Papain 
2.0 mg 450 kD PAAc or 500 kD PMAAc 
Mixed at pH 3.0 and cast. 
Release medium: PBS pH7.4 (Salt concentration 0.15M) 
This example shows (similar to FIGS. 9 and 11) that increased hydrophobic 
character of the bioadhesive polymer may lead to a stronger --COOH:PEG 
complex. 
From the foregoing it will be appreciated that, although specific 
embodiments of the invention have been described herein for purposes of 
illustration, various modifications may be made without deviating from the 
spirit and scope of the invention. Accordingly, the invention is not 
limited except as by the appended claims.