Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
  • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
  • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
  • A61K9/0051—Ocular inserts, ocular implants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin

Definitions

• 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.
• 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.
• the present invention provides delivery materials comprising chitin derivatives which are soluble and lysozyme- degradable.
• 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.
• 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.
• the bioactive agent is bound to the vehicle composition before cross-linking.
• binding affinity is the ratio of the amount of bound drug (the bioactive agent) to the amount of free drug, wherein
• 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.
• 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.
• 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 ⁇ 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.
• 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.
• 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
• 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.
• 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.
• 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.
• 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.
• 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.
• modification may be performed by, for example, esterification of acid groups prior to cross-linking, thus making the drug releasing material more hydrophobic.
• 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.
• 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.
• 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 H 2 0. Wetting agent (Pluronic L-92) was added to 0.006%, and the solution filtered through a
• 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 The ferricyanide reducing sugar assay was used to demonstrate enzyme hydrolysis of the CM-chitin polymer in the shield.
• 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.
• 80% CM-chitin cross linked 15 hr at 125°C is preferred.
• 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.
• 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.
• 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.
• CM- chitin microspheres 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.

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• 1995
  • 1995-06-02 JP JP8501270A patent/JPH10504277A/ja active Pending
  • 1995-06-02 AU AU26629/95A patent/AU2662995A/en not_active Abandoned
  • 1995-06-02 CA CA 2191753 patent/CA2191753A1/en not_active Abandoned
  • 1995-06-02 EP EP95921610A patent/EP0763063A4/de not_active Withdrawn
  • 1995-06-02 WO PCT/US1995/007153 patent/WO1995033773A1/en not_active Application Discontinuation

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