CA2396344C

CA2396344C - Polymer blends as biodegradable matrices for preparing biocomposites

Info

Publication number: CA2396344C
Application number: CA002396344A
Authority: CA
Inventors: Ramaz Katsarava; Zemphira Alavidze
Original assignee: Surmodics Inc
Current assignee: Surmodics Inc
Priority date: 2000-01-11
Filing date: 2001-01-11
Publication date: 2009-09-08
Anticipated expiration: 2021-01-11
Also published as: US20050175991A1; EP1421855A2; AU2001249050A1; AU2001230889A1; EP1421855A3; EP1250143A2; CN1230206C; WO2001050872A3; AU4313101A; US20120148505A1; KR20030009338A; DE60106727D1; US20130164374A1; WO2001050866A2; WO2001051027A3; US20040247569A1; US20040029250A1; CA2430498A1; US20090053144A1; DE60116052D1

Abstract

The present invention provides bioerodable constructs for controlled release of bioactive materials. In a preferred mode, the constructs may be utilized adjacent to a biological surface. The constructs are based on a blend of two or more poly(ester-amide) polymers (PEA). Such polymers may be prepared by polymerization of a diol (D), a dicarboxylic acid (C) and an alpha-amino acid (A) through ester and amide links in the form (DACA)n. An example of a(DACA)n polymer is shown below in formula II.
Suitable amino acids include any natural or synthetic alpha-amino acid, preferably neutral amino acids.

Description

POLYMER BLENDS AS BIODEGRADABLE MATRICES

FOR PRFP.aRING BIOCOMPOSITES
BACKGROUND OF THE INVENTION

Field of the Invention This invention is directed to polymeric matrices designed for controlled release of biologically active substances, such as therapeutic bacteriophage which can kill bacteria capable of eausing diseasc.

Review of Related Art Bioactive composites based on biodegradable (or more precisely, bioerodible) polymers as matrices, impregnated by bactericidal substances are promising for the treatment of superficial infected wounds. On the one liand, bactericidal substances clean the wound from bacteria and malce favorable conditions for wound llcaling, and prevent bacterial invasion th--ough the lioles made in wound coverings for exudate drainage, on the otlier hand, biodegradable polynier which is able to timely release enough degradation products (polynieric debris) can activate macrophages to produce the required growtli factors acrd, in that way, can accelerate wound liealing (Pratt, et al.
(1994, "Dimethyltitanocene-Induced Surface Chernical Degradation of Synthetic Bioabsorbable Polyesters", J. Poh=ni. Sci. Part .4: Pohwr. C'hent., 32(5):949;
Greisler, (1988), "Small Diameter Vascular Prostheses: Macrophage-Biomaterial Interactions with Bioresorbable Vascular Prostheses". Transactions of ASAIO, 34:105 1).
Mori, et al., U.S. Patent 3,867,520, discloses a delivery system for therapeutic agents using films made of polyamino acid polymers with oil-like or wax-like substances dispersed in the fih1i. Therapeutic agents are dissolved in the carrier, and when the film is applied to an intenzal or external surface of the body, the carrier migrates to the surface of the film where the agent is released. However, these films are not biodegraded during use.

WO 01/51027 CA 02396344 2002-07-08 pCT/USO1/00807

2 Sidman, U.S. Patent 4,351,337, discloses an implantable delivery device comprising a matrix formed of a poly-alpha-amino acid component having one or nlore drugs and/or diagnostic agents physically contained therein. The drug or diagnostic a-ent is released throu-h diffusion and/or biodegradation resultin- from the action on the polynieric nzatrix of enzymes present in the host into which the implant is placed.
Taniharak, et al., U.S. Patent 5,770,229, discloses a medical polymer gel made up of a cross-linked polysaccharide with a drug attacked to the polysaccharide via a linkage that is cleavable by an endo(lenous enzynie. This system provides for delayed release of the attached drug from the polymer, but the release rate is subject to individual variation in the amoiult of the endo2enous enzyme, and the polymer, while biocompatible, is not biodegradable.

Kuroyangi and coworkers (1992, J. .41)Pl. 13ioiuater., 3:153 -161) have developed a woLuid dressing for bum care that is a hydropllobic poly-L-lcucine spongy matrix impregnated with antibacterial silver sulfadiazinc supported by a fine nylon mesh. Ti1is wound dressing suppresses bacterial growth wliile controlling fluid loss.
However, the dressinu is not de-raded, but rather sticks to the wound until it separates spontaneously fronl the healed skin.

Georgian Patent No. 1090 describes a wound dressing containing 45-50 wt.`%, biodegradable poly(ester-amide) based on natural alpha-amino acids impregnated witli 50-55 wt.`%, dried bacteriopha-e. The poly(estcr-amide) is not characterized in detail, but the dressing also has 0.05-0.15 wt."/,, surface inimobilized alpha-chvmotr_ypsin. The impregnated poly(ester-amide) is fonned into a film, and the film is used to accelerate healing of superficial wounds, including burns.

Tsitlanadze, et al., in an abstract from Irnt. S),mn. Biodegrad. Mcttei-, October 7-9, 1996, Hamburg, Germany, describe alpha-cllymotrypsin-catalyzed hydrolysis of regular poly(ester-aniides) (PEAs) of general formula I:

3 -C-(CHZ)m C-N-CH-C-O-(CH2)k-O-C-CH-NH
R R
n wherek=2,3,4,or6 m=4or8,and R = CH(CH3)2, CH2,CH(CH3)2, CH(CH3)CH-2CH3, (CHI)3CH3, CH2C6H5, or (CH,)3SCH3.

It is reported that alpha-chyniotrypsin is spontaneously immobilized on the surface of the PEAs from aqueous solution, and erodes the polymer surface under physiologic conditions, with increasing lysis for more hydi-opliobic R groups and more hydropliobic polymer backbone. A biocomposite material based on a PEA polymer containing bacte--iophages, antibiotic or anesthetic was prepared for study as artificial skin for healing burns and festering wounds.

SUMMARY OF THE INVENTION

The present invention provides bioerodable constructs for controlled release of bioactive materials. In a preferred mode, the constructs mav be utilized adjacent to a biological surface. The constructs are based on a blend of two or more poly(ester-aniide) polvniers (PEA). Such polvmers may be prepared by polymerization of a diol (D), a dicarboxylic acid (C) and an alpha-amino acid (A) through ester and amide links in the form (DACA),,. An example of a(DACA)õ polymer is shown below in formula II. Suitable amino acids include anv natural or synthetic alpha-amino acid, preferably neutral amino acids.

Diols may be any aliphatic diol, including alkylene diols like HO-(CH_1)k-OH
(i.e. non-branched), branched diols (e.g., propylene (ilycol), cyclic diols (e.g.
dianhydrohexitols and cyclollexanediol), or oligomeric diols based on etliylene glycol (e.g., diethylene glycol, triethylene glycol, tetraethylene glycol, or poly(ethylene glycol)s). Aromatic diols (e.g. bis-phenols) are less useful for these purposes since they

4 are more toxic, and polymers based on them have rigid chains that are less likely to biodegrade.

Dicarboxylic acids may be any aliphatic dicarboxylic acid, such as a,oO-dicarboxylic acids (i.e., non-branched), branched dicarboxylic acids, cyclic dicarboxylic acids (e.g. cyclohexanedicarboxylic acid). Aromatic diacids (like phthalic acids, etc.) are less useful for these purposes since they are more toxic, and polymers based on them have rigid chain structure, exhibit poorer film-forming properties and have nluch lower tendency to biodegrade.
Preferred PEA polymers have the formula II:

0 ~1 H ~ ~ H O 11 -0-(CH2)k-O-C- i H-N-C-(CH2)m C-N- i H-C-R R n where k = 2-12, especially 2, 3, 4, or 6, m = 2-12, especially 4 or 8, and R = CH(CH3)2, CH7~CH(CH3)2, CH(CH3)CH2CH3, (CH-2)3CH3, CH,C6H5, or (CH2)3SCH3.

The constructs optionally contain bioactive inclusions, which are released upon degradation (bioerosion) of the consti-uct.
In a preferred embodiment, this invention provides biodegradable constructs which comprise a first PEA polynier in which A is L-phenvlalanine (Plie-PEA) and a second PEA polymer in which A is L-leucine (Leu-PEA). Preferably, the ratio of Phe-PEA to Leu-PEA is from 10:1 to 1:1; more preferably, the ratio of Phe-PEA to Leu-PEA is from 5:1 to 2.5:1. The construct may be formed as a deformable sheet adapted to conform to a biological surface.
In another embodiment, this invention provides bioerodable constructs comprising PEA polymers and further comprising a bioactive agent, which may be selected from the group consisting of antiseptics, anti-infectives, such as bacteriophages, antibiotics, antibacterials, antiprotozoal agents, and antiviral agents, analgesics, anti-inflammator, agents including steroids and non-steroidal anti-inflanimatory agents includin~ Cn`:-2 inhibitors, anti-neoplastic agents, contraceptives, CNS active druy~s, liormones, and vaccines.
In yet anotlier embodiment, the bioerodable construct of this invention

5 comprises an enzyme capable of hydrolytically cleaving the PEA polymer, such as a-cliymotrypsin. In a preferred cmbodiment, the enzyme is adsorbed on the surface of the construct. In a particularly preferred embodiment, the construct contains bacteriophage which are released by action of the enzyme.
This invention also provides a niethod of treating a patient having an ulcerative wot.ii,d coinprising inserting into the wound or covering the wound with a bioerodable construct according to claim 1, wherein the bioerodable construct contains a bioactive agent, which may be bacteriophage, an antibiotic, an antiseptic, or an analgesic. The wound treated by tliis invention may be open or infected, and the construct may be in the form of a deformable slieet. In a preferred einbodiment, the construct used in treatment of the wound contains bacteriophage specific for bacteria found in the wound.
The construct may also comprise an enzyme capable of hydrolytically cleaving the PEA
polymer.

There is no currently available biodegradable polymer or polymeric blend composed entirely of naturally occurring and nontoxic building blocks showing high plasticitv (e.g., pliability when hydi-ated) together with high enzyme-catalyzed biodegradation rates, solubility in common organic solvents like chloroform, and suitable for either impregnation or the spontaneous surface immobilization (adsorption) of the enzymes like trypsin, a-chyinotrypsin, and lipase. The polymeric blends of this invention provide all of these properties, permitting their use as matrices for wound dressing/healing devices wllich are plastic and act to release bioactive substances in a sustained/controlled fashion.

6 BRIEF DESCRIPTION OF THE FIGURE

The Figure shows lipase catalyzed biodegradation of polymers in vivo over a six month period.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The use of a bacteriophage lysate in the treatment of suppurative lesions that are inflamed or infected requires multiple and frequent applications (e.g., 3-5 times a day) which increases consumption of both the bacteriophage preparation and the wound dressings. From this point of view the application of a bacteriophage reservoir, which provides for controlled release and prolonged action, is superior.
Bioresorbable (or bioerodable) polvmers ai-e the most appropriate matrices for preparing reservoirs of bacteriophages and/oi- other bioactive compounds.
Bioactive composites based on bioerodable polymers are known for controlled release of drugs to provide desirable concentrations of bioactive substances in surrounding tissues.
Compositites made of bioerodable polymers disappear over time in a biological environment as the substance of the composite is egraded or dissolved by action of the surrounding biologic milieu. This degradation may be facilitated by enzymes whch calalyze cleavage of covalent bonds in the polymer. (Such enzymes mav be present in the bilologic niileu or may be added exogenously, wliether as part of the construct or otherwise.) Controlled or sustained release of a biologically active substance from a bioerodable construct refers to a delay in the dispersion of the biologically active substance relative to simple diffusion from its point of introduction into the biological environment. Controlled release is generally due to some factor which interferes with normal diffusion of the substance, such as a diffusion barrier or limited solubility of the diffusion substance. The bioerodable constructs of this invention present a diffusion barrier which is removed progressively as the polymer degrades.
More recently, it has also been established that the rapid release of polymer degradation products in a sufficient amount into the surrounding tissues activates macrophages for the production of growth factor" which mav accelerate wound

7 healing. It is beneficial for polymeric degradation products to be either normal metabolic components or easily digestible by cells. Polymers used as matrices should be plastic enough to tightly cover wounds. It is also highly desirable for the polymeric matrix to be able either to immobilize enzymes (e.g. trypsin, alpha-chymotrypsin, lipase, etc.) on the surface by a simple method or incorporate them in the bulk matrix.
These enzymes can participate in the wound healing processes and can also erode polymers (e.g., by catalyzing the hydrolysis of ester bonds in the polymeric backbone) with a constant and desirable rate to provide for the release of bactericidal compounds as well as sufficient matrix degradation products in the surrounding tissue to stimulate macrophages.
The inventor has synthesized new biodegradable poly(ester-amide)s (PEAs) composed of naturally occurring alpha-amino acids, including essential ones like L-phenylalanine and L-leucine, and nontoxic compounds like aliphatic and dicarboxylic acids. Suitable synthetic methods are reported in Arabuli, et al. (1994), "Heterochain Polymers based on Natural Amino Acids. Synthesis and enzymatic hydrolysis of regular poly(ester-amide)s based on bis-(L-phenylalanine) alpha,omega-alkylene diesters and adipic acid," Macromol. Chem. Phys., 195(6):2279, and Katsarava, et al.
(1999) "Amino Acid Based Bioanalogous Polymers. Synthesis and study of regular poly(ester-amide)s based on bis-(a-amino acid) a,tz)-alkylene diesters and aliphatic dicarboxylic acids", J. Polym. Sci.: Part A: Chemistry, 37:391-407. These rapidly bioresorbable, biocompatible poly(ester-amide)s may be used to form a bioerodable polymer matrix.
The poly(ester-amide)s of this invention do not contain any toxic components.
Alpha-amino acids, such as the essential amino acids L-phenylalanine and L-leucine, are naturally-occurring products. These normal metabolic components, upon release through biodegradation, are digested by cells. Fatty acids and diols are well known nontoxic products commonly used in the food industry. They are also used as building blocks for other classes of biodegradable polymers like polyanhydrides and poly-(ortho-

8 ester)s approved by the U.S. Food and Drug Administration (FDA) for clinical trials and other practical applications.
It is very important that the poly(ester-amide)s used in this invention are soluble in organic solvents that do not inactivate bioactive compounds such as bacteriophages.
These polymers are soluble in chloroform in which the enzymes like trypsin, a-chymotrypsin, lipase are sufficiently stable for enzyme activity to survive the process of preparing enzyme-containing polymer constructs.
Enzymes can be added to polymeric solutions in cllloroform in order to form enzynze-containing polymeric matricies when the solution is cast onto glass plates and the solvent is evaporated. For polynieric films impregnated by enzvnies according to this method, the enzymes catalyze the liydrolysis (erosion) of PEAs, which is impoi-tant for the release of bioactive substances into the surrounding tissues. The biodegradation rates of PEAs can vary over a wide range, spanning, e.g., 101-10 mg/cm2 h. The degradation rate is a function of the enzyme activity in the composite. These polymers may be designed to release sufficietit matrix degradation products (polynieric debris) over time to activate niacrophages.

Enzymes may be spontaneously immobilized onto the si.irface of PEAs based on L-phenylalanine through the simple immersion of the polynieric films in aqueous enzyme solution for varying lengths of time. (Immersion for, e.g., for 15-20 min is typical.) PEAs based on L-leucine do not readily adsorb enzymes using this simple metliod, and tluIs, PEAs based on L-phenylalanine are niore suitable for preparing biodegradable niatrices with surface-immobilized enzymes. However, PEAs based on L-phenylalanine do not possess sufficient plasticity for use as wound coverings. PEAs composed of L-leucine are pliable wlien hydrated (i.e., water acts as a plasticizer) and more suitable for biological applications sucli as wound coverings (dressings); however the films prepared from L-leucine PEAs are very sticky, adhering to themselves, and inconvenient to work with. In addition, L-leucine based PEAs inlniobilize enzymes poorly.

9 The present inventor has discovered that the detrimental characteristics inherent in each class of PEAs can be overcome by blending them. Polynieric blends prepared from approxinlately 70% of L-phenvlalanine based PEAs and 30% of L-leucine based PEAs sllowed:

= good plasticity (necessary to cover wounds tightly), = lack of self-adhesion, and = abilitv to in-imobilize enzyrnes.
As contemplated by the present invention, the polymer blend which is the basis for the invention has sufficient plasticity to permit a film made with the polymer blend to be manually deformed to fit tiglztly to an irregular biological surface (e.g., a concave wound surface). Additionally, films made with the poiymer blend are readily separable by gentle manual force, leaving each sheet of film intact upon separation.
Finally, the surface of an object nlade with the polymer blend of this invention will adsorb proteins, such that measurable enzyme activity can be detected adliered to the surface of the object after it is dipped into a solution of the enzynle.
This invention provides polymer blends comprising at least two PEAs of formula II. Preferably the blend contains one PEA in which R corresponds to the side chain of phenylalanine (Phe-PEA) and one PEA in which R corresponds to the side chain of leucine (Leu-PEA). The ratio of Phe-PEA to Leu-PEA may vary from 10:1 to 1:1, but is preferably from 5:1 to 2.5:1. Other PEAs (and indeed otlier polymers) may be included in the blend, so long as the resultant blend still exhibits the desired properties described above. The other polymers in the blend vrill, of course, be soluble in the solvent in which the blend is dispersed for preparing the constructs according to this invention. Leu-PEA and Phe-PEA are soluble in polar organic solvents including dimethyl-formamide (DMF), dimethylacetamide, dimethylsulfoxide (DMSO), trifluoroethanol, hexafluoroisopropanol and the like, or neutral organic solvents including chloroform and the like. Chloroform and similar solvents are preferred for preparation of bioerodable films containing bioactive components due to greater volatility (important for preparing films) and reduced tendency to inactivate enzymes (such as chymotrypsin or lipase), bacteriophages or other bioactive components.
In a preferred mode, the polymer blend of this invention is formed into a bioerodable film. The films of this invention may be a single layer or multiple layers, 5 such as a bilayer film having one layer of a PEA blend and an adjacent layer of poly(siloxane elastomer). However, alternative bioerodable constructs using the polyiner blend are easily within the skill of the art and within the contemplation of this invention. For example, the polvmer blend may be used to provide a bioerodable coating on a support nlaterial wliich niav or may not be biodegradable, such as a fibrous

10 or non-fibrous three-dimensional construct or a woven support. Suitable forms for the three-dimensional constrcts of tliis invention are foams, which mav be formed by conventional means. For example, Phe-PEA/Leu-PEA blends can be prepared as foams as follows: a suspension of bacteriophages and other bioactive substanses (about I-) in the solution of Plie-PEA/Leu-PEA blend (1 g) in chloroform (10 mL) can be cast onto llydrophobic surface and 90-99% of chloroform evaporated at r.t. under atmospheric pressure. Afterwards a reduced pressure may be applied at room temperature to remove residual chloroform, and the resulting foamed filni dried fot- 12 h under reduced pressure. According to another procedure 1-10 % (of chloroform volume) of n-pentane mav be added to the suspension above. The mixture may be cast onto hydrophobic surface and allowed to dry at room temperatLn-e for 24 h, and the foamed 61m mav be subjected to a Gnal dryin" iuider reduced pressure for 12 lh. Foamed fihns may also be obtained using ultrasonic disintey~ration techniques.
Constructs prepared with the polymers of tllis invention nlav be part of devices including a support material to be used as, for example, bandages for wounds or burn dressings. Of course, the blends forming a coating on a woven support will preferablv retain the flexibility and/or elasticity of blends used for film-forming, but a blend for coating a rigid, three-dimensional construct may be less elastic. Such biends may have higher Phe-PEA content, and coatings in which Phe-PEA is the only PEA polymer are within the contemplation of this invention for such applications.

Il In another niode, this invention contemplates constructs consisting all or in part of a blend accordinLi to this invention which mav be surgicallv inlplanted.
Constructs according to this invention may also be fonned into devices for wound packing, such as gel foams, or mav be used as coniponents in surgical appliances, such as Penrose drains, indwelling catheters, catheters for peritoneal dialysis, and any other appliances that are in contact with body cavities, the blood circulation, or the lymphatic circulation and are either used to treat potential infections or are at risk of becoming infected.
. This invention also contemplates appliances for oral hygiene, including gum implants (e.g., for periodontal disease or dental caries). Such constructs will preferably contain bioactivc material i-eleased in a controlled manner upon erosion of the construct.
Suitable sclections of particular bioactive ll1CluslOnS will be readily apparent to the skilled artisan in vicw of thc intended sitc of implantation. For example, composites containin- bactericidal a"enst such as bactcrioplia-c may be implanted in the body to treat osteomvelitis, etc. Alternatively, biocrodable composites of this invention could be used for sustained/controlled release of anticancer and/or other drugs at a target site.
Bloactive materials mav be released in a controlled fasliion by diffiision froni within the construct, or by degradation of the construct, or by a combination of these processes.
Bioactive and/or inactive biocompatible niaterials may be included in the erodablc construct in amoiuIts up to 60`%o or niore by weight, so long as their inclusion does not dcstrov the desii-able properties of films according to tilis invention. Bloactive materials contemplated for inclusion in the bioerodable constructs of this invention include, but are not limited to, antiseptics, anti-infectives, such as bacteriophages, antibiotics, antibacterials, antiprotozoal agents, and antiviral agents, analgesics, anti-inflanunatory agents including steroids and non-steroidal anti-inflanimatory agents including COX-2 inhibitors, anti-neoplastic agents, contraceptives, CNS active drugs, liormones, and vaccines. In particular, constructs nlay include one or more of calcium gluconate and otlier phage stabilizing additives, hyaluronidase, fibrinolvsine and other fibrinolvtic enzymes, methyluracyl and otlier agents stimulating metabolic processes, sodium hydrocarbonate, L-arginine and other vasodilators, Benzocaine and other pain killers, mono- and disaccharides, polysaccharides and mucopolysaccharides, Metronidazol and other anti-protozoa drugs, Clotrimazolum and other anti-fungal drugs, thrombine and other hemostatics, vitamins, Prednizolone and other anti-inflammatory steroids, and VoltarenTM (Sodium diclofenac) and other anti-inflammatory non-steroid drugs. Of course the skilled artisan will in any case confirm that particular construct formulations retain the desired properties as discussed herein, and constructs which exhibit none of these properties are outside the contemplation of this invention.
In one preferred mode, this invention provides a novel approach to management of poorly healing and poorly vascularized wounds (which may include diabetic foot ulcers, pressure ulcers in patients with reduced mobility, and other ulcers and open skin lesions. In medicine, poorly healing wounds, such as those seen in diabetic patients with foot ulcers, and in bedridden patients with pressure sores, represent a major and very expensive management problem. Use of antibiotics in this setting is generally not efficacious. Because of poor vascularization, antibiotics seldom achieve therapeutic levels in affected areas sufficient to eradicate infection. Moreover, because of the recurrent courses of antibiotics that such patients have often received, the bacterial pathogens causing the infections are often antibiotic resistant. In this mode, as well as other wound treatment embodiments, the controlled-release character of the polymer constructs according to this invention avoid the necessity of constant re-application of bactericidal material, as well as the need for associated dressing changes.
Biocomposites mediating a sustained/controlled release of appropriate therapeutic agents have proven to be especially efficacious for healing infected wounds and cavities. Film materials, so called "artificial skin", prepared from these biocomposites have important therapeutic effects:
= Polymer material, when applied to the surface of such wounds, acts as a protector from external mechanical actions and bacterial invasion, and further prevents heat and moisture loss that occur as a result of uncontrolled water evaporation from the injured surface; and The slow-release properties of the biologically-active compound can be exploited to prom:)te appropriate, steady release of anti-bacterial agents at the site of infection.
Use of biocomposite "artificial skin" does not require patient inimobilization, and tliereby facilitates a return to daily life activities, an important consideration in this class of patients.

A key element in the nianagement of cllronically infected wounds is the suppression of pathogenic bacteria] flora. With biocoinposite niaterials, this can be acliieved bv introducing bacteriocidal substances into the biocomposite structure.
Antibiotics mav be used in this setting, but tlicir efficacy is increasingly limited by the cievelopnient of antibiotic i-esistance. More recently, tliere has been interest in the introduction into biocomposites of such bactericidal substances as silver sulfadiazine (and related diazine derivatives of sulfanilamide), furagin (and pharmaceutically acceptable salts thereof) and chlorohexydine (and pharmaceutically acceptable salts thereof). However, utilization of such compounds nlay be limited by their inherent toxicity, particularly for patients with underlying kidney or liver disease.
Incorporation of bacteriophages into such biocomposite materials provides an alternative approach. Bacteriopliage are viruses that kill specific bacteria.
The lysis of microorganisms by viruses was discovered at the beginning of the 20t1i century. Any one pliave tends to be hi~hly specific for certain bacteria, requiring that therapy be caretlilly targeted (i.e., there is iio analogy to the broad-spectruni antibiotics which can "kill everything"). However, tllis also means that phage therapy can be used to kill specific pathogens without disturbing normal bactcrial flora.
Phages have been reported to be effective in treating skin infections caused by Pseuclonionas, Staphylococcus, Klebsrella, Proteus, E. coli, and other pathogenic species; success rates in these studies have ranged from 75 to 100%, depending on the pathogen. However, for these studies bacteriophages were introduced in a variety of vehicles: aqueous liquid preparations, aerosols and creams.

The polymeric blend composed of L-phenylalanine, L-leucine, adipic acid, and butane-diol-1,4 has been successfully used for preparing bioactive composites containing bactericidal substances. The wound dressings obtained based on this biocomposite material showed high wound healing properties.
Starting from the materials mentioned above it seems that bioactive composite based on bioresorbable (bioerodable) polymer and containing a complex of bacteriophages as a bactericidal substance will be an effective dressing material with accelerated wound healing ability. Selection of suitable bacteriophage is described in U.S. Patent Nos. 6,699,701 or 6,703,040.
EXAMPLE
A complex of polyvalent bacteriophages directed toward Staphylococcus species, Streptococcus species, E. coli, Proteus species, and Pseudomonas aeruginosa with a titer of 2x 106 - 2x 107 plaque-forming units, was prepared and used as bioactive substance for this study. Bacteriophage were prepared as a lyophilized dry powder as follows: bacteriophages suspended in an aqueous sucrose-gelatin mixture were lyophilized, resulting in a dry mass that was ground into fine powder. In this process, 50 mg of dry preparation corresponds to 1 ml of liquid bacteriophage with a titer of 2x 106 - 2x 107 . None of the individual components of bioactive composites (polymer, organic solvent, alpha-chymotrypsin, lipase) affected bacteriophages activity -100% of starting activity was retained in all cases.
A bioactive film was prepared as follows: A fine suspension of dry bacteriophage in a polymer solution with an appropriate solvent was cast on a glass surface and dried to constant weight. A composite was obtained in the form of a film with the following characteristics: mass I g, film area - 60-65 cm2, thickness - 0.2-0.3 mm. Afterwards alpha chvmotrypsin was immobilized on the surface of the film.
Optionally, the film was perforated. For particular applications, analgesics and/or antibiotics were added to the composite as well.
5 The activity of the resultant film in in vitro experiments was determined using a bacterial lawn on solid media. Activitv was estimated by measuring the width of the zone of lysis. The activity of the tilm coincides with the activity of dry bacteriophages used; pure polymeric film did not reveal any bactei-icidal activity.
The kinetics of bacteriophage release from 9 cm disks of the film was studied in 10 phosphate buffer Lmder physiological conditions (see Table 1). One can see that release of bactei-iophages during first 24 hours both from a-cllvmotrypsin-immobilized and a-chymotrypsin-free films was coniparable; for enzyme-immobilized film it was only 1.5-2 times liigher. This can be explained by extensive desorption of bacteriophages from the surface zone of enzyme free filni. However, wlien the filnis were transferred 15 to fresli buffer at 24 hours and 120 hours, the enzyme-catalyzed erosion niechanism becanie important at later stages for releasing bacteriophages froni the bulk of the film, and difference in release rate reaclied niore than one order in magnitude.
Clearly, alpha-chyinotrypsin promotes the release of bacteriophages from bioactive coinposite.

Table Sustained Release of Bacteriophages and Antibiotics from Medicated Wound Covering Film Release of bacteriophages from 9 cm dia. Phe-PEA film disks into IOmL of Phosphate buffer 0.2 M, pH 7.4, T=37 C. A 9 cni Phe-PEA/bacteriophage film disk contains a proximately 1800 x 104 bacteriophages.
Titer of bacteriophages in 1 mL solution Time in Composite bacteriophage/Phe- Composite bacteriophage/Phe-PEA
hours PEA film with a-chymotrypsin film without surface-immobilized a-ch otr sin 1 2.0 x 104 1 . 3 x 10 3 5.Ox 10 3.Ox 10 24 8.Ox 10 4.Ox 10 24 h later, after transfer to a new 1 OmL portion of the buffer 1 3.2 x 10 1.3 x 10 3 9.0x10 3.1x10 96 200.0 x 10 90.0 x 10 120 h later, after transfer to a new l OmL portion of the buffer Time in Composite Composite bacteriophage/Phe-PEA film hours bacteriophage/Phe-PEA film without surface-immobilized with a-chymotrypsin a-chvmotr sin 1 2.5x10 0.06x10 4 5.Ox 10 0.20x 10 It should be noted that surface immobilized a-chymotrypsin can play an additional role namely it can decompose both peptides and denaturated proteins. This enzymatic debridment, as it is known from literature, leads to the sanitation of a wound and accelerates healing.
The activity of films according to this invention was checked periodically for 1.5 years against both preexisting laboratory strains and newly received bacterial strains, and the film retained activity over this period. The surface immobilized enzyme was active for this period as well. The Figure shows lipase catalyzed biodegradation of polymers in vivo over a six month period. The in vivo data is summarized in Table 2.

WO 01/51027 CA 02396344 2002-07-08 pCT/US01/00807 a.
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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A bioerodable construct for controlled release of bioactive materials, said construct comprising a blend of at least two poly(ester-amide) polymers (PEA) prepared by polymerizing a diol (D) a dicarboxylic acid (C) and an alpha-amino acid (A) through ester and amide links in the form (DACA)n, wherein the blend comprises a first PEA
polymer in which A is phenylalanine (Phe-PEA) and a second PEA polymer in which A
is leucine (Leu-PEA) at a ratio of Phe-PEA to Leu-PEA of 10:1 to 1:1.

2. A bioerodable construct for controlled release of bioactive materials, said construct comprising a blend of at least two poly(ester-amide) polymers (PEA) prepared by polymerizing a diol (D), a dicarboxylic acid (C), and an alpha-amino acid (A) through ester and amide links in the form (DACA)n, wherein the alpha-amino acid (A) of each PEA polymer of the blend is an amino acid having an aliphatic side chain, an amino acid having a sulfur-containing side chain, or an amino acid having a side chain containing aromatic rings, and wherein the alpha-amino acid of at least one of the PEA
polymers is phenylalanine or leucine.

3. A bioerodable construct for controlled release of bioactive materials, said construct comprising a blend of at least two poly(ester-amide) polymers (PEA), wherein each PEA polymer has the formula:

wherein k = 2-12, m = 2-12, and R = CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3, (CH2)3CH3, CH2C6H5, or (CH2)2SCH3, wherein the blend comprises a PEA polymer wherein R = CH2CH(CH3)2 or a PEA
polymer wherein R = CH2C6H5.

4. A bioerodable construct for controlled release of bioactive materials, said construct comprising a blend of at least two poly(ester-amide) polymers (PEA), wherein each PEA polymer has the formula:

wherein k = 2-12, m = 2-12, wherein R represents an amino acid side chain, and the amino acid side chain is an aliphatic side chain, a sulfur-containing side chain, or a side chain containing an aromatic ring, and wherein the blend comprises a PEA polymer wherein R is an amino acid side chain containing an aromatic ring or a PEA polymer wherein R is an aliphatic amino acid side chain.

5. The construct of claim 1, wherein the ratio of Phe-PEA to Leu-PEA is 5:1 to 2.5:1.

6. The construct according to any one of claims 1, 2, 3 or 4, wherein the construct is a deformable sheet adapted to conform to a biological surface.

7. The construct according to any one of claims 1, 2, 3 or 4, further comprising a bioactive agent.

8. The construct of claim 7, wherein the bioactive agent is an antiseptic, an anti-infective, a bacteriophage, an antibiotic, an antibacterial, an antiprotozoal agent, an antiviral agent, an analgesic, an anti-inflammatory agent, an anti-neoplastic agent, a contraceptive, a CNS active drug, a hormone, or a vaccine.

9. The construct according to claim 7, wherein the construct comprises an enzyme capable of hydrolytically cleaving the PEA polymer.

10. The construct according to claim 9, wherein the enzyme is .alpha.-chymotrypsin.

11. The construct according to claim 9, wherein the enzyme is adsorbed on the surface of the construct.

12. The construct according to claim 9, wherein the construct contains a bacteriophage which is released by action of the enzyme.

13. The construct of claim 2, wherein the amino acid having an aliphatic side chain is valine, leucine, isoleucine, or norleucine.

14. The construct of claim 2, wherein the amino acid having a sulfur-containing side chain is methionine.

15. The construct of claim 2, wherein the amino acid having a side chain containing an aromatic ring is phenylalanine.

16. The construct of claim 2, wherein the blend comprises a first PEA
polymer in which A is phenylalanine and a second PEA polymer in which A is leucine.

17. The construct according to claim 3, wherein the blend comprises a first PEA polymer wherein R = CH2CH(CH3)2, and a second PEA polymer wherein R =
CH2C6H5.

18. The construct of claim 17, wherein the first PEA polymer and second PEA polymer are present in a ratio of 10:1 to 1:1.

19. The construct of claim 18, wherein the ratio of first PEA polymer to second PEA polymer is 5:1 to 2.5:1.

20. The construct of claim 3, wherein k=2, 3, 4 or 6 and m=4 or 8.

21. The construct of claim 4, wherein the aliphatic amino acid side chain is a valine, leucine, isoleucine, or norleucine side chain.

22. The construct of claim 4, wherein the sulfur-containing side chain is a methionine side chain.

23. The construct of claim 4, wherein the side chain containing an aromatic ring is a phenylalanine side chain.

24. The construct of claim 4, wherein the blend comprises a first PEA
polymer in which R is a phenylalanine side chain, and a second PEA polymer in which R is a leucine side chain.

25. The construct according to any one of claims 1, 2, 3 or 4, wherein the construct is a device for wound packing.

26. The construct according to claim 25, wherein the construct is a foam.

27. The construct according to claim 1 or 2, wherein the diol (D) is not bisphenol and wherein the dicarboxylic acid (C) is not phthalic acid.

28. The construct according to any one of claims 1, 2, 3 or 4, wherein the blend is formed into a bioerodable coating on a support material or is formed into a bioerodable film.

29. The construct according to claim 28, wherein the blend is formed into a bioerodable coating comprising bioactive material.

30. The construct according to any one of claims 1, 2, 3 or 4, wherein the construct is a device that may be surgically implanted.

31. The construct according to claim 30, wherein the implantable device is an indwelling catheter or appliance for oral hygiene.

32. A bioerodable construct according to any one of claims 1, 2, 3 or 4, wherein the bioerodable construct is a deformable sheet containing a bioactive agent for treating a patient having an ulcerative wound.

33. Use of a bioerodable construct according to any one of claims 1, 2, 3 or 4, wherein the bioerodable construct is a deformable sheet containing a bioactive agent for treating a patient having an ulcerative wound.

34. The use according to claim 33, wherein the bioactive agent comprises a bacteriophage, an antibiotic, an antiseptic, or an analgesic.

35. The use according to claim 33, wherein the wound is open or infected.

36. The use according to claim 33, wherein the bacteriophage are specific for bacteria found in the wound.

37. The use according to any one of claims 33 to 36, wherein the construct also comprises an enzyme capable of hydrolytically cleaving the PEA polymer.

38. The construct of claim 8, wherein the anti-inflammatory agent is a steroid.

39. The construct of claim 8, wherein the anti-inflammatory agent is a non-steroidal anti-inflammatory agent.

40. The construct of claim 39, wherein the non-steroidal anti-inflammatory agent is a COX-2 inhibitor.