EP0763063A1

EP0763063A1 - Lysozyme-degradable biomaterials

Info

Publication number: EP0763063A1
Application number: EP95921610A
Authority: EP
Inventors: Jeff J. Prior; Ronald K. Yamamoto; George L. Brode
Original assignee: Vitaphore Corp
Current assignee: Vitaphore Corp
Priority date: 1994-06-02
Filing date: 1995-06-02
Publication date: 1997-03-19
Also published as: WO1995033773A1; JPH10504277A; AU2662995A; EP0763063A4; CA2191753A1

Abstract

A method is provided for preparing a drug delivery composition and device comprising cross-linking water-soluble, lysozyme degradable chitin with a cross-linking agent and loading the cross-linked chitin with a bioactive agent. Preferred embodiments are disclosed wherein the drug delivery composition also comprises collagen or other collagenase-degradable collagen derivative.

Description

LYSOZYME-DEGRADABLE BIOMATERIALS The present invention is related to a vehicle for 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 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 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 the problem with collagen shields of widely varying dissolution times and therefore uncertain dosing.

The enzyme lysozyme is known to be present in tears at a relatively high and constant level, but collagen is not lysozyme-degradable. Chitin, a natural polymer, is a lysozyme substrate, but it is not readily soluble in body fluids. However, the present invention provides delivery materials comprising chitin derivatives which are soluble and lysozyme- 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 .in vitro. Since lysozyme is a component 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 mechanism of drug delivery according to the invention is by sustained release through enzymatic hydrolysis of the vehicle. The key enzyme, lysozyme, is present in increasing amounts in areas of inflammation; thus it is an advantage to incorporate an inflammation fighting drug in the vehicle to be released at greater rates in areas of greater need.

It is thus an object of the present invention to provide lysozyme-degradable delivery compositions for controlled release of bioactive agents into body fluids or tissues.

This and other objects of the invention will be apparent from the following description and appended claims, and from practice of the invention. SUMMARY OF THE INVENTION

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-linking a vehicle composition comprising water-soluble, chitin derivatives; optionally, forming the cross-linked vehicle composition into a desired shape; then contacting the cross-linked vehicle composition with a bioactive agent to reversibly bind the bioactive agent to the vehicle composition. Cross-linking is preferably effected by heating in a partial vacuum, by UV 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 (the 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] riinus [the volume of solution absorbed by the biopolymer times the concentration of drug in the remaining unabsorbed solution]

[Free drug] = [the total amount of drug found in the biopolymer sample] minus [Bound drug] .

Thus,

Binding affinity = [Bound drug] = [Bound drug] [Free drug] [Total drug]-[Bound drug]

Particularly preferred compositions have binding affinities over 0.5, preferably 1.0 and higher. Useful compositions have a binding affinity for the drug in the range of 1.0 to 5.0.

A high binding affinity allows the drug to be incorporated after the fabrication of the delivery device, since the material will bind substantial amounts of drug from solution. Without this characteristic, drug will need to be entrapped into the device during 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., 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 for the amount of drug "bound". Dividing the amount bound by total drug absorbed gives the fraction bound, F_b. If the device has an affinity for the drug, F_b will be greater than zero. As the affinity becomes greater, F_b will approach the value 1.0. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph of the drop in viscosity from the reaction of CM-chitin with lysozyme.

Figure 2 is a graph of the π 9 of enzyme hydrolysis of several CM-chitin-collagen corneal shields.

Figure 3 is a graph of the rate of cleavage of CM- chitin icrospheres by lysozyme.

Figure 4 is a graph showing the amount of binding of carbonic anhydrase inhibitor to CM-chitin microspheres.

Figure 5 is a graph showing the amount of binding of aprotinin to CM-chitin microspheres.

Figure 6 is a graph of release of cytochrome-C from CM-chitin microspheres.

DETAILED DESCRIPTION OF THE INVENTION

The lysozyme-degradable delivery vehicle for controlled release of a bioactive agent according to the present invention may be formed by treating the drug release materials comprising a lysozymic- degradable water-soluble, chitin derivative with a cross-linking 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 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 with collagen or other collagenase-degradable collagen derivative. The preferred chitin:collagen ratios are from 0.01:1 to 100:1, preferably from about 0.25:1 to 4:1.

The thickness of the delivery vehicle 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-linking agent to drug-release materials 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 from the teachings herein that the degree of cross-linking, thickness 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-linked biopolymer.

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 bioactive agent or agents, which may be antimicrobial drugs or acromolecules such as growth factors, antibacterial agents, antispasmodic -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 physostigmine, neostigmine and the like, antispasmodic agenvs such as atropine, methantheline, papaverine and the like, tranquilizers and muscle relaxants such as fluphenazine, chlorpro azine, triflupromazine, mephenesin, meprobamate and the like, antidepressants like a itriptyline, nortriptyline, and the like, antihistamines such as diphenhydra ine, di enhydrinate, tripelennamine, perphenazine, chlorprophenazine, chlorprophenpyradimine and the like, hyptotensive agents such as rauwolfia, reserpine and the like, cardioactive agents such as bendroflumethiazide, flumethiazide, chlorothiazide, a inotrate, propranolol, nadolol, procainamide and the like, angiotensin converting enzyme inhibitors such as captopril and enalapril, bronchodialators such as theophylline, steroids such as testosterone, prednisolone, and the like, antibacterial agents, e.g. , sulfona ides such as sulfadiazine, sulfa erazine, sulfa ethazine, sulfisoxazole and the like, antimalarials such as chloroquine and the like, antibiotics such as the tetracyclines, nystatin, streptomycin, 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, e.g., acid addition salts, basic salts like alkali metal salts, etc. Preferred materials for ophthalmic use are antibiotics, steroid drugs and antiglaucoma 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. Typically, 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 10 minutes to 240 minutes. At ambient temperature (about 20-25°C), the material is then ready for use.

In another preferred method, the bioactive agent and drug release material are dissolved in an aqueous solvent before 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. The following examples are presented for the purpose of illustration and are not intended to limit the inve- ion in any way.

EXAMPLE 1 r.ϋACTION OF LYSOZYME WITH CHITIN DERIVATIVES

The following chitin derivatives were tested: carboxymethyl-chitin, carboxyrethyl-chit ran, chitosan-lactate, and chitosar.. To 7.2 c 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 lysozyme was assayed with CM-chitosan, chitosan-lactate, and chitosan, no change in viscosity was seen. As shown in FIG. l, with CM-chitin as substrate, lysozyme 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. Che . 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. EXAMPLE 2 FORMATION OF CHITIN-COLLAGEN SHIELD

CM-chitin (Maruben Corp. Tokyo) was dissolved at 1.25% in H₂0. Wetting agent (Pluronic L-92) was added to 0.006%, and the solution filtered through a

5 um syringe filter. 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% CM-chitin. Shield molds were filled to 5.75 mg per shield and air dried at RT. Dehydrother al cross-linking was performed in high vacuum for various times at temperatures from 115 to 135°C.

EXAMPLE 3 LYSOZYME DEGRADATION OF CORNEAL SHIELDS

Shield halves are shaken at 37°C in tear buffer 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 is 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

6 90%) were almost completely dissolved after over¬ night incubation at 37°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 collagenase, 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. Ideal conditions for formulating a 24 hr hybrid shield appear to be 80% CM-Chitin, 20% collagen, DHT 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 rp with a Beckman KA-21 rotor. Ten ml of 1.25% CM-chitin was mixed with 2.5 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 acetone was 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/ral 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 average of 2 experiments. The reactions appears to reach completion in approximately 3 days. Figure 3.

EXAMPLE 5 MICROSPHERE LOADING

Since lysozyme which degrades CM-chitin microspheres is present at high levels in tears, the microspheres were tested for binding to two drugs which could be used in diseases of the eye: carbonic anhydrase inhibitor for glaucoma, and aprotinin for corneal ulcers. CM-chitin microsphere - drug binding was investigated by Scatchard analysis. As seen in Fig. 4 & 5, CAI and aprotinin bind the microspheres with a K_d of 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 10K 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:

Drug Fraction Bound Total Cone

Carbonic Anhydrase Inhibitor 0.84 to 0.93 0.25% to 0.14% CAI

Aprotinin 0.79 to 0.95 50 V/μl to 29 O/μl Cytochrome C: 0.98 0.5 mg/ml EXAMPLE 6 PROTEIN RELEASE FROM CM-CHITIN 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. The 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, pH 7.6, with 200 ul of 1 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 br er system was chosen consistent with a possible e in periodontal disease. The major ions in 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. 100 ul of a 50:50 slurry of microspheres equilibrated in buffer were mixed with 100 ul of 1 mg/rol cytochrome C for 1.5 hr. The microspheres were then pelleted and 100 ul of supernate replaced with lysozyme so that the final concentration was mg/ml. At each time point the microspheres were again pelleted and 100 ul removed and assayed for cytochrome C, and 100 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 when microspheres were allowed to sit overnight and over the weekend, respectively, demonstrating that the slow release was not reaching equilibrium over the 1.5 hour time points. The control reaction treated with buffer 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 ability 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 compounds 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 of the invention is to be limited solely with respect to the appended claims and equivalents.

Claims

WHAT IS CLAIMED IS:

1. A method for preparing a cross-linked lysozyme- degradable delivery vehicle for controlled-release of a bioactive agent comprising the steps of cross-linking a biopolymer comprising water- soluble, lysozyme-degradable chitin, wherein said chitin contains a modified 6-hydroxy group, to form a cross-linked chitin derivative; optionally, forming said cross-linked chitin derivative into a desired shape; contacting said cross-linked chitin derivative with a solution containing said bioactive agent or agents to reversibly bind at least a portion of said bioactive agent(s) to said cross-linked chitin derivative to form said polymeric delivery vehicle, wherein the binding affinity of said cross-linked chitin derivative with said bioactive agent(s) is above about 0.5.

2. A method for preparing a lysozyme-degradable delivery vehicle for controlled-release of a bioactive agent comprising the steps of contacting biopolymer comprising a water- soluble, lysozyme-degradable chitin containing a modified 6-hydroxy group with a solution containing a bioactive agent to reversibly bind at least a portion of said bioactive agent to said chitin; optionally, forming said chitin into a desired shape; and cross-linking said biopolymer to form said cross-linked, lysozyme-degradable delivery vehicle, wherein the binding affinity of said cross-linked, lysozyme-degradable delivery vehicle with said bioactive agent is above about 0.5.

3. A method according to Claim 1 or 2 wherein said binding affinity is 1.0 or higher.

4. A method according to Claim 3 wherein said binding affinity is in the range of 1.0 to 5.0.

5. A method according to Claim 1 or 2 wherein said chitin comprises 6-0-carboxymethyl.

6. A method according to Claim 1 or 2 wherein said delivery vehicle further comprises collagen or other collagenase-degradable collagen derivatives.

7. A method according to Claim 1 or 2 wherein said bioactive agent comprises an antibiotic.

8. A method according to Claim 1 or 2 wherein said bioactive agent comprises a steroid.

9. A method according to Claim 1 or 2 wherein said bioactive agent comprises an anti-glaucoma agent.

10. A composition for controlled release of a bioactive agent into body fluids comprising lysozyme- degradable vehicle having a binding affinity for said bioactive agent above about 0.5; and said bioactive agent which is reversibly bound to said composition so that the time-release profile of said bioactive agent from said composition is controlled by said binding affinity in the presence of said fluids.

11. A composition according to Claim 10 wherein said binding affinity is 1.0 or higher.

12. A composition according to Claim 11 whereas said binding affinity is the range of 1.0 to 5.0.

13. A composition according to Claim 10 wherein said chitin comprises 6-0-carboxymethyl.

14. A composition according to Claim 10 wherein said composition further comprises collagen or other collagenase-degradable collagen derivatives.

15. A composition according to Claim 10 wherein said bioactive agent comprises an antibiotic agent.

16. A composition according to Claim 10 wherein said bioactive agent comprises a steroid.

17. A composition according to Claim 10 wherein said bioactive agent comprises an antiglaucoma agent.

18. A composition according to Claim 10 which is configured as a corneal shield or as a microsphere.

19. A composition according to Claim 10 where the device remains substantially intact while a substantial amount of said agent is released into said body fluids, and thereafter said composition is biodegraded.

20. A composition according to Claim 14 which is configured as a corneal shield or as a microsphere.

21. composition according to Claim 14 wherein said chitin comprises carboxymethylchitin.

22. A composition according to Claim 14 wherein said chitin and collagen are in the ratio from about 0.01 to 1, to about 100 to 1.

23. A composition according to Claim 22 wherein said chitin and collagen are in the ratio from about 0.25 to 1, to about 4 to 1.

24. A composition according to Claim 14 containing at least one bioactive agent.

25. A method of delivering a drug into a body tissue or fluid comprising contacting said tissue or fluid with a composition according to any one of Claims 10 through 24.