Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
21917~3
09-'i33773 ~ PCT~l$95!07153
LYSOZYME-DEGRADABLE BIO~ATERIALS
The present invention is related to a vehicle for
5 delivery of bioactive agents, and in particular, for
the delivery of bioactive ophthalmic agents to the
eye.
BACKGROUND OF THE INVENTION
Corneal shields made of collagen are known for use as
10 eye bandages and as a means of delivering drugs.
They provide sustained ocular drug delivery, an
advantage over frequent dosing with eye drops, and
the advantage of lower dose delivery, and therefore
fewer side effects, than systemic delivery. The rate
15 of drug release from shields is in part determined by
the rate at which the shield dissolves, and for
collagen shields this is determined by the level of
activity of the enzyme collagenase, which can vary
greatly from individual to individual. This leads to
20 the problem with collagen shields of widely varying
dissolution times and therefore uncertain dosing.
The enzyme lysozyme is known to ~e present in tears
at a relatively high and constant level, but collagen
is not lysozyme-degradable. Chitin, a natural
25 polymer, is a lysozyme substrate, but it is not
readily soluble in body fluids. However, the present
invention provides delivery materials comprising
SUBSTllUTE SHEET (RULE 26~
W095/33773 21 9 1 7 5 3 PCT/1!5'151~7153 -
chitin derivatives which are soluble an~ lyso~y~e-
degradable.
Accordingly, the present invention provides drug
delivery vehicles, such as corneal shields, made of
composite materials containing chitin derivatives
which are degraded by lysozyme. The invention also
provides vehicle materials comprising a mixture of
chitin derivatives and collagen which are dissolved
by lysozyme n yitro. Since lysozyme is a compon~nt
of normal tears, a shield susceptible to its
hydrolytic activity has the advantage of a constant
and reproducible dissolution rate compared to a
shield which can only be dissolved by collagenase.
The primary r--hRn;~m of drug delivery accordinq to
the invention is by sustained release through
enzymatic hydrolysis oE the vehicle. The key enzyme,
lysozyme, is present in increasing amounts in areas
of inflammation; thus it i8 an advantaqe to
incorporate an inflammation fiqhting drug in the
vehicle to be released at greater rates in areas of
qreater need.
It is thus an object of the present invention to
provide lysozyme-degradable delivery compositions for
controlled release of bioactive aqents into body
fluids or tissues.
This and other objects of the invention will be
apparent from the following description and Rppr~n~ed
claims, and from practice of the invention.
~ ~095~33773 21917 S 3 PCT1US95107153
--3--
SUMMARY OF THE IN~ENTION
The present invention provides a method for preparing
a lysozyme-degradable delivery vehicle for controlled
release of a bioactive agent. The method comprises
the steps of cross-linXing a vehicle composition
comprising water-soluble, chitih derivatives;
optionally, forming the cross-linked vehicle
composition into a desired shape; then contacting the
cross-linked vehicle composi~ion with a bioactive
agent to reversibly bind th bioactive agent to the
vehicle composition. Cros~ inking is preferably
effected by heating in a p;-eial vacuum, by W
irradiation or by the use of a cross-linking agent,
such as ethyl dimethylaminopropylcarbodiimide.
Alternatively, the bioactive agent is bound to the
vehicle composition before cross-linking.
As used herein, the term "binding affinity" is
the ratio of the amount of bound drug tthe bioactive
agent) to the amount of free drug, wherein
[Bound drug] = [the total amount of
drug found in a
biopolymer sample which
is contacted with a
solution of drug and
allowed to equilibrate]
~inus [the volume of
solution absorbed by
the biopolymer times
the concentration of
drug in the remaininq
unabsorbed solution]
[Free drug~ = [the total amount of
drug found in the
9 1
~09s~33773 ~ 1~11,J~ PC1~S95107153
biopolymer sample]
minus [Bound drug].
Thus,
Binding affinity = rBound druq~ = rBound druql
[Free drug] ~Total drug]-tBound drug]
Particularly preferred compositions have binding
affinities over 0.5, preferably l.0 and higher. Uscful
compositions have a binding affinity for the drug in the
range of l.0 to 5Ø
lO A high binding affinity allows the drug to be
incorporated after the fabrication of the delivery
devlce, since the material will bind substantial amounts
of drug from solution. Without this characteristic, drug
will need to be entrapped into the device during
15 fabrication to provide sustained release properties,
complicating the fabrication of a sterile product.
A sufficient drug affinity means that the amount of drug
absorbed into the device will be greater than the amount
of drug present through simple fluid equilibration, i.e.,
20 the concentration of drug in the device is greater than
the concentration of drug in a surrounding fluid medium.
As described above, if the amount of "free" drug present
through fluid equilibration is subtracted from the total
amount of drug in the device, one can determine a value
25 for the amount of drug "bound". Dividing the amount
bound by total drug absorbed gives the fraction bound, Fb.
If the device has an affinity for the druq, Fb will be
greater than zero. As the affinity becomes greater, Fb
will approach the value lØ
SUBSTITUTE SHEET (RULE 26)
~ o~s133773 2 1 g 1 7 5 3 PCIIUS~510~153
BRIEF CESCRIPTION OF ~HE FIG~R65
Figure l iB a graph of the drcp in viscosity from the
reaction of CM-chitin with lysozyme.
Figure 2 is a graph of the rate of enzyme hydrolysis
of several CM-chitin-collagen corneal shields.
Figure 3 is a graph of the rate of cleavage of CM-
chitin microspheres by lysozyme.
Figure 4 is a graph showing the amount of binding of
carbonic anhydrase inhibitor to CM-chitin
nicrospheres.
Figure 5 is a graph showing the amount of binding of
aprotinin to CN-chitin microspheres.
Figure 6 is a graph of release of cytochrome-C from
CM-chitin microspheres.
DET~ILED DESCRIPTION OF THE I~VENTION
The lysozyme-degradable delivery vehicle for
controlled release of a bioactive agent according to
thc present invention may be formed by treating the
drug release materials comprising a lysozymic-
degradable water-soluble, chitin derivative with a
cross-l~nking agent. The drug-release materials may
be formed into microspheres, films, slabs or molded
into desired shapes.
The drug-release materials used according to the
Z5 present invention include, but are not limited to
chitin derivatives which are modified at the 6-
hydroxy group with a substituent which renders the
chitin water-soluble. Preferred materials are 6-0-
carboxymethyl chitin. The drug-release material may
also incorporate a mixture of the chitin derivative
219I753
wossf33773 PCT/~595~07153
--6--
with collagen or o~her collagenase-degradable
collagen derivative. The preferred chitln:collagen
ratios are from 0.01:l to l00:l, preferably from
about 0.25:1 to 4:1.
The thickness of the dclivery vehlcle may be varied
as desired, depending upon the desired pharmaceutical
dosage and duration of delivery. Ordinarily, a
suitable matrix thickness will be in a range of about
.02 mm to 1.0 centimeter. For a corneal shield, the
thickness is typically .04 to .06 mm before
hydration; with 4 to 9 fold increase upon hydration.
The ratio of cross-linkinq agent to drug-release
~aterials will depend in part on the particular
chitin. Generally, it will be useful to employ a
weight ratio of cross-linking agent to drug-release
materials of from about 20:1 to about 0.5:1.
It will be realized fror~ the teachings herein that
the degree of cross-linking, thic~n~se and/or shape
of the cross-linked drug-release materials are all
parameters which may be controlled to attain a
desired release profile of the bioactive agent from
the cross-linXed biopolyr.er.
The shape of the cross-linked drug-release materials
may be formed by molding or casting before cross-
linking or, after cross-linking, it may be formed
into a desired shape by cutting. The cross-linked
materials will then be loaded with the desired
bioactive agent(s~, which is believed to occur either
by ionic binding involving ionic sites on the chitin
and~or collagen or by hydrophobic binding, or both,
with the desired bloactive agent or agents, which may
be antimicrobial drugs or macromolecules such as
growth factors, antibacterial agents, antispasmodic
21917~
o~S/337~3 PCT~S9~/07153
--7--
agents, or any other active biological bioactive
agent, such as adrenergic agents such as ephedrine,
desoxyephedrine, phenylephrine, epinephrine and the
like, cholinergic agents such as ph sostigmine,
neostigmine and the like, antispasmodic agents such
as atropine, methantheline, papaverine and the like,
tranquilizers and muscle relaxants such as
fluphenazine, chlorpromazine, triflupromazine,
mephenesin, meprobamate and the like, antidepressants
like amitriptyline, nortriptyline, and the like,
antihistamines such as diphenhydramine,
dimenhydrinate, tripelennamine, perphenazine,
chlorprophenazine, chlorprophenpyradimine and the
like, hyptotensive agents such as rauwolfia,
reserpine and the like, cardioactive agents such as
bendroflumethiazide, flumethiazide, chlorothiazide,
aminotrate, propranolol, nadolol, procainamide and
the like, anqiotensin converting enzyme inhibitors
such as captopril and enalapril, bronchodialators
such as theophylline, steroids such as testosterone,
prednisolone, and the like, antibacterial agents,
e.g., sulfonamides such as sulfadiazine,
sulfamerazine, sulfamethazine, sulfisoxazole and the
like, antimalarials such as chloroquine and the like,
antibiotics such as the tetracyclines, nystatin,
streptomycLn, cephradine and other cephalosporins,
penicillin, semi-synthetic penicillins, griseofulvin
and the like, sedatives such as chloral hydrate,
phenobarbital and other barbiturates, glutethimide,
antitubercular agents such as isoniazid and the like,
analgesics such as aspirin, acetaminophen,
phenylbutazone, propoxyphene, methadone, meperidine
and the like, etc. These substances are frequently
employed either as the free compound or in a salt
form, _.~., acid addition salts, basic salts like
alkali metal salts, etc. Prcferred materials for
ophthalmic use are antibiotics, steroid drugs and
219~753
~0~3~33773 PCT~SsS~U71~3 -
-a-
antiqlaucoma agents. Other therapeutic agents having
the same or different physiological activity can also
be employed in the pharmaceutical preparations within
the scope of the present invention. ~ypically, the
bioactive agent or agents dissolved in a suitable
solvent will be contacted with the drug-release
material by immersion. The loading may be readily
determined based upon the uptake by the drug release
materials of the bioactive agent.
In a preferred method for forming the drug-release
material, the bioactive agent or agents is dissolved
in water at a suitable concentration, typically about
0.1-2~ by weight, and the drug release material is
immersed therein for a period of about lD minutes to
240 minutes. At ambient temperature ~about 20-25~CI,
the material is then ready for use.
In another preferred method, the bioactive agent and
drug release material are dissolved in an aqueous
solvent be~ore cross-linking. Typical agent:drug
release material weight ratios are in the range of
about 1:100 to 5:100 in solution. The drug release
material is then cross-linked by treatment with the
cross-linking agent.
It will be realized that the chitin or collagen
derivative may be modified, for example, so as to be
made more hydrophilic or hydrophobic to adjust for
suitable binding properties to the bioactive agent.
Such modification may be performed by, for example,
esterification of acid groups prior to cross-linking,
thus making the drug releasing material more
hydrophobic.
219~7~3
~ o~l337l3 PCI~IS95l07l~3
_g_
The following examples are presented for the purpose
of illustration and are not intended to limit the
invention in any way.
EXAMPLE 1
REACTION OF LYSOZYME WITH CHITIN DERIVATIVES
The following chitin derivatives were tested:
carboxymethyl-chitin, carboxymethyl-chitOSan~
chitosan-lactate, and chitosan. To 7.2 g of a 1%
solution of each chitin derivative was added 0.8 ml
of 100 mM sodium phosphate, pH 7.0, and the viscosity
was determined as a function of time with a
Brookfield model DVII viscometer. At time zero, 80
uL of 2% lysozyme, (or chitinase, as a positive
control), was added and the viscosity of the solution
monitored to detect any drop in viscosity resulting
from hydrolysis of the polymer. In all cases the
enzyme chitanase resulted in a rapid reduction in the
viscosity of a 1% polymer solution. When lysozy~e
was assayed with CM-chitosan, chitosan-lactate, and
chitosan, no change in viscosity was seen. As shown
in FIG. 1, with CM-chitin as substrate, lysozy e
caused a rapid drop in viscosity.
The CM-Chitin - lysozyme reaction was quantitated by
assaying the reducing sugar formed upon cleavage of
the glycosidic bond of CM-chitin by the ferricyanide
reducing sugar assay (Park, J. & Johnson, M., J.
Biol. Chem. 181: 149, 1949). The non-continuous
assay was determined to be linear with respect to
time and enzyme concentration to at least 0.25 mg/ml.
A specific activity of 0.097 umol/hr/mg lysozyme was
obtained.
21917~3
~VO ~35,~33773 PC'I'~-IS~071S3
--10-
EXAMPLE 2
FORMATION OF CHITIN-COLLAG~J SHIELD
CM-chitin (Maruben Corp. Tokyo~ was dissolved at
1.25% in H2O. Wetting agent (Pluronic L-92) was
added to 0.00~%, and the solution fLltered through a
5 um syringe ~ilter. The solution was degassed in 50
ml tubes and mixed with 1.5 to 2.3~ collagen slurry
so that the resulting polymer solution was 40, 60, or
80% C~-chitin. Shield molds were filled to 5.75 mg
per shield and air dried at RT. Dehydrothermal
cross-linking was performed in high vacuum for
various times at temperatures from 115 to 135~C.
~XAMPLE 3
LYSOZYME DEGRAD~TION OF CORNEAL SHIELDS
Shield halves are shaken at 37~C in tear bu~fer with
0.1~ lysozyme with and without either 0.05 or 0.5
mg/ml collagenase. Shields were visually scored on a
scale from 5 to 0 where 5 is unchanged and 0 i5
completely dissolved. The ferricyanide reducing
sugar assay was used to demonstrate enzyme hydrolysis
of the CM-chitin polymer in the shield. Figure 2.
Shields with high ratios of CM-chitin to collagen (75
~ 90%) were almost completely dissolved after over-
night incubation at 3~~C. A 100% CM-chitin film
dissolved completely. A mixture of lysozyme and
collagenase dissolved shields made of any ratio of
CM-chitin to collagen. Additional experiments
determining dissolution times in the presence of
normal tear lysozyme levels, with and without
collaqenase, demonstrated the CM-chitin - collagen
hybrid shields can dissolve more reproducibly than
100~ collagen shields. CM-chitin - collagen hybrid
shields were tested at 40, 60, and 80~ CM-chitin.
~ og~/33773 2 1 9 1 7 5 3 PCT~S95,'071~3
--11--
Ideal conditions for formulating a 24 hr hybrid
shield appear to be 80% CM-Chitin, 20% collagen, DH~
cross-linked 15 hr at 130~C and EtO sterilized. For
a 12 hr shield, 80% CM-chitin cross linked 15 hr at
125~C is preferred.
EXAMPLE 4
FORMATION OF CHITIN MICROSPHERES
CM-Chitin ~Nova Chemical, Toronto, lot ~1348, low
viscosity) was formed into microspheres by dissolving
CM-chitin by stirring overnight at room temperature
and homogenizing 5 min with a Virtis polytron
homogenizer. The final concentration was 1.25%; in
some cases the solution was clarified by pelleting 30
min at 15,000 rpm with a Beckman KA-21 rotor. Ten ml
of 1.25% CM-chitin was mixed with 2.S ml Span-80 by
vortexing 2 min. With Virtis polytron homogenization
at setting 45, toluene was slowly added to a final
volume of 55 ml. This suspension was slowly added to
4 volume of acetone with stirring. EDC, pre-
dissolved in 1 ml water, then mixed with 4 ml acetonewas added to the stirred suspension at various ratios
of EDC to CM-chitin. After 2 hr, microspheres were
washed 3 times by pelleting and re-suspending with
acetone and 3 times with the desired buffer.
Microspheres cross-linked at ratios of 1.0, 0.75, and
0.5 were split in two and one half of each treated
with lysozyme at 0.1 mg/ml overnight. The
microspheres of ratio 0.5 and 0.75 dissolved; the 1.0
ratio microspheres swelled to about twice original
volume. In a separate experiment, the ferricyanide
- reducing sugar assay was used to quantitate the
cleavage of the glycosidic bond of the CM-chitin
~ microspheres. Microspheres were incubated in tear
buffer at 37~C in a shaking water bath. Reactions
contained 0.1 mg/ml lysozyme. Each point is the
W095/33773 2191~ 5 3 PCT~I~9~I07I53 -
-12-
average of 2 experiments. The reactions appears to
reach completion in approximately 3 days. Figure 3.
EXAMPLE S
MICROSPI~ERE LOA~ING
Since lysozyme which degrades CM-chitin microspheres
is present at high levels in tears, the mlcrospheres
were tested for binding to two druqs which could be
used in diseases of the eye: carbonic anhydrase
inhibitor for glaucoma, and aprotinin for corneal
ulcers. C~-chitin microsphere - druq binding was
investigated by Scatchard analysis. As seen in Fig.
4 & 5, CAr and aprotinin bind the microspheres with a
Kd ~~ 0.53 and 0.025 mM, respectively. The protein,
aprotinin, binds the polyanionic microspheres more
tightly than the CAI, presumably due to its cationic
nature, with an isoelectric point close to 10.5.
The fraction bound was determined using carboxymethyl
chitin microspheres by incubating preformed
microspheres and drug in a known volume for 60 min.
at room temperature, then pelleting the microspheres
at lOR x g for 5 min., and assaying the supernate for
free drug concentration by uv absorption. The amount
bound is calculated from the total as described
above.
The fraction bound for representative drugs are
presented below:
Ih~ug Fract}on Bou*d Totsl CODC
Carbonic Anhydr3sc l*h;bitor 0.84 to 093 0.~5% to û.14~v CAI
Aprotiain 0.79 t~ 0 g5 50 U/~LI to 29 U/,l~l
t~tochrome C. 098 0~ mg/ml
~; ~
~ 0~133773 21917 5 3 PCT~1S95J071S3
-13-
EXAMP~E 6
PROTEIN RELEASE FROM CM-C~ITIN MICROSPHERES
Sustained release through enzyme catalyzed cleavage
of polymer matrix is particularly applicable to
macromolecules since they would be less likely to be
released through simple diffusion, as might be the
case with small molecules. ~he sustained release was
tested with a model system using the easily
detectable protein, cytochrome C. The amount of
protein loading was calculated by mixing 200 ul of a
50:50 slurry of microspheres equilibrated in 20 mM
phosphate buffer, p~ 7.6, with 200 ul of l mg/ml
cytochrome C. After pelleting microspheres the
protein in the supernate was determined from a
calibration curve to account for 0.5~ of the total,
or 99.5~ was bound to the microspheres. A buffer
system was chosen consistent with a possible use in
periodontal disease. The major ions in saliva are
sodium and bicarbonate at approximately one-third
physiological strength, with Ph in the range of 6.2
to 7.4. Therefore, a 50 mM sodium bicarbonate
buffer, pH 7.0 was used. l00 ul of a 50:50 slurry of
microspheres equilibrated in buffer were mixed with
lCO ul of l mg/ml cytochrome C for 1.5 hr. The
microspheres were then pelleted and l00 ul of
supernate replaced with lysozyme so that the final
concentration was mg/ml. At each time point the
microspheres were again pelleted and lOO ul removed
and assayed for cytochrome C, and lOO ul fresh
lysozyme in buffer added to replace the amount
withdrawn. Controls without lysozyme were run in
parallel.
Figure 6 shows a slow release of protein from the
microspheres over a 32 hour period, in lysozyme
containing buffer. Peaks of protein were released
_ _ _ _ _ , .. . ... ...
21g~753
wosa~33~73 PCT~S~10~1~3 -
-14-
when microsphere9 were allowed to sit overnlght ~nd
over the weekend, respectively, demonstrating that
the slow release was not reaching equilibrium over
the l.5 hour time polnts. The control reactlon
treated with bu~fer did not release appreciable
amounts of protein.
After the reaction in Figure 6 was complete, lysozyme
was added to the control reaction and protein was
then slowly released, demonstrating the abillty of
the microspheres to retain protein until released by
lysozyme.
These results clearly demonstrate the ability of CM-
chitin microspheres to bind to drugs and protein.
In the periodontal environment, protein can remain
bound through extensive washing and be slowly
released in the presence of lysozyme. The cytochrome
C was used as a model protein since it is easily
detectable, has a net positive charge, and would be
expected to behave like a peptide drug or cytokine
having a net positive charge. This system will
provide sustained release of such comroun~c in the
case of periodontal disease, where infected areas of
the mouth would have increased lysozyme levels.
The foregoing description and examples have been set
forth merely to illustrate the invention and are not
intended to be limiting. Since modifications of the
described embodiments incorporating the spirit and
substance of the invention may occur to persons
skilled in the art, the scope o~ the invention is to
be limited solely with respect to the appended claims
and equivalents.
