Medical devices and materials comprising biodegradable polyesters

Abstract

The invention provides a formulation comprising a biodegradable polyester compounded with a compound of formula (I): AA-AA-AA-X-Y (I). The invention also provides methods of making these formulations, medical devices such as sutures comprising the formulations, and methods of making the devices.

Description

[0001] This invention relates to antimicrobial agents, particularly formulations comprising polymers compounded with small peptides or peptide-like molecules. These formulations are particularly intended for use in medical devices.

[0002] The use of medical devices such as catheters, orthopedic instruments and other implants, as well as surgical fasteners such as sutures, has been increasing. Despite improvements in device design and surgical procedures, infections associated with these devices remain a major problem. Conventional antibiotic treatment often fails due to low antibiotic levels at and around the actual site of infection. Biofilms present on introduced biomaterials / devices can impair the effectiveness of antibiotic treatment, as can the presence of drug-resistant strains.

[0003] Antibiotic-release coatings are known for use in medical devices such as sutures and catheters. However, patients may become infected with resistant bacteria, and the concentration gradient of antibiotics due to the local release pattern increases the risk of competitive selection by resistant bacteria. In particular, sutures impregnated with triclosan are marketed, given the recognition that this antibiotic has been overused and has led to the development of drug-resistant bacterial strains; furthermore, it is known to contribute to cross-resistance to other antibiotics. Therefore, health authorities face pressure to restrict its use. Although studies have shown that triclosan-coated sutures reduce the incidence of surgical site infections, data on sutures coated with other active agents, such as chlorhexidine, are limited (Onesti et al., 2018 European Review for Medical and Pharmacological Sciences 22: pages 5729-5739).

[0004] Postoperative wound infection is the third most common hospital-acquired infection in the United States. Surgical site infections cause severe patient discomfort, are potentially life-threatening events, and prolong hospital stays. Microbial adhesion to the surfaces of sutures and other surgical fasteners has been identified as one of the causes of surgical site infections.

[0005] Therefore, there is a clear need for alternative sutures and other medical devices that can provide controlled release of antimicrobial agents and limit the colonization of the device itself.

[0006] The inventors have discovered that they can combine certain small antimicrobial peptides with biodegradable polyesters to provide materials that can be used on their own (e.g., devices made by 3D printing) or as a coating for another medical device.

[0007] Antimicrobial peptides are promising candidates as novel antimicrobial agents because they are active against a broad spectrum of planktonic bacteria and biofilms, including antibiotic-resistant strains. Furthermore, bacteria are unlikely to develop resistance to these rapidly acting peptides due to their mode of action (including disruption of lipid membranes rather than targeting protein targets). However, formulating peptides into matrices that provide controlled release is not straightforward; peptides are generally poorly soluble compared to other types of drugs and typically degrade at the temperatures required to mix or blend them into the matrix.

[0008] The inventors have prepared materials that can be used to provide controlled release of active antimicrobial agents, i.e., antimicrobial agents that are leachable and inhibit bacterial growth in the surrounding environment. The antimicrobial agents are also used to control microbial growth within materials and / or on devices coated with blends of polymers and antimicrobial agents.

[0009] Therefore, in one aspect, the present invention provides a formulation comprising a biodegradable polyester, said biodegradable polyester being formulated with a compound of formula (I).

[0010] AA-AA-AA-XY (I)

[0011] Wherein, in any order, two of the AA (amino acid) moieties are cationic amino acids, preferably lysine or arginine, but may be histidine or any non-genetically encoded or modified amino acid carrying a positive charge at pH 7.0, and one of the AA moieties is an amino acid having a highly lipophilic R group having 14 to 27 non-hydrogen atoms and preferably containing two or more, for example two or three fused or linked cyclic groups, which typically contain five or six non-hydrogen atoms, preferably six (in the case of fused rings, non-hydrogen atoms can of course be shared);

[0012] X is an N atom, which can be branched or unbranched C1-C atoms. 10 Alkyl or aryl (e.g., methyl, ethyl, or phenyl) substitutions are preferred, but not branched or unbranched C1-C. 10 Alkyl or aryl (e.g., methyl, ethyl, or phenyl) substitution, and the group may contain up to two heteroatoms selected from N, O, and S; and

[0013] Y is selected from R1-R2-R3.

[0014] R1-R2-R2-R3, R2-R2-R1-R3, R1-R3 and R4

[0015] in:

[0016] R1 is C, O, S, or N, with C being preferred;

[0017] R2 is C;

[0018] R1 and R2 can each be substituted with or not substituted with C1-C4 alkyl groups. Preferably, Y is -R1-R2-R3 (where R1 is preferably C) and preferably the group is not substituted. However, when Y is -R1-R2-R2-R3 or R2-R2-R1-R3, it is preferred that one or more of R1 and R2 are substituted.

[0019] R3 is a group comprising one to three cyclic groups, each cyclic group having five or six non-hydrogen atoms (preferably all C atoms, but optionally also containing N, O, or S), two or more of the cyclic groups may be fused; one or more of the rings may be substituted and these substitutions may include (but typically do not include) polar groups, suitable substituents, including halogens, preferably bromine or fluorine, and C1-C4 alkyl groups; R3 contains up to 15 non-hydrogen atoms, preferably 5 to 12, most preferably phenyl; and

[0020] R4 is an aliphatic moiety having 2 to 20 non-hydrogen atoms, preferably carbon atoms, but may incorporate oxygen, nitrogen, or sulfur atoms. Preferably, R4 contains 3 to 10, most preferably 3 to 6, non-hydrogen atoms, and the moiety may be straight-chain, branched, or cyclic. If the R4 group contains a cyclic group, it is preferably directly attached to the nitrogen atom of X.

[0021] Preferred compounds incorporate a straight-chain or branched R4 group, particularly a straight-chain or branched alkyl group, including ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and its isomers, hexyl and its isomers, etc.; propyl, isopropyl, butyl and isobutyl are particularly preferred.

[0022] In some embodiments, R4 is an aliphatic moiety (preferably alkyl) having 6 to 16 non-hydrogen atoms, preferably carbon atoms, but may incorporate oxygen, nitrogen or sulfur atoms, and said moiety may be straight-chain, branched or cyclic.

[0023] In some preferred embodiments, R4 is isopropyl.

[0024] In the R4 group containing a cyclic group, it is preferred that R4 is a cyclohexyl or cyclopentyl molecule.

[0025] Suitable non-genetically encoded amino acids and modified amino acids that can provide cationic amino acids include analogs of lysine, arginine, and histidine, such as high-lysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid, and high-arginine, as well as trimethyllysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-methylamidine-piperidine-4-carboxylic acid, and 4-guanidinophenylalanine.

[0026] The highly lipophilic R group of AA may contain heteroatoms such as O, N, or S, and typically contains no more than one heteroatom, preferably nitrogen. The R group preferably has no more than two polar groups, more preferably no polar groups or one polar group, and most preferably no polar groups.

[0027] The preferred peptide compound preferably has formula (II).

[0028] AA1-AA2-AA1-XY (II)

[0029] in:

[0030] AA1 is a cationic amino acid, preferably lysine or arginine, but may be histidine or any non-genetically encoded or modified amino acid with a positive charge at pH 7.0;

[0031] AA2 is an amino acid with a highly lipophilic R group, said R group having 14 to 27 non-hydrogen atoms and preferably containing two or more, for example two or three fused or linked cyclic groups, which will typically contain five or six non-hydrogen atoms, preferably six; and

[0032] X and Y are defined as above.

[0033] Further preferred compounds include compounds of formulas (III) and (IV):

[0034] AA2-AA1-AA1-XY (III)

[0035] AA1-AA1-AA2-XY (IV)

[0036] AA1, AA2, X, and Y are as defined above. More preferably, the molecule of formula (II) is preferred.

[0037] Among the compounds described above, certain compounds are particularly preferred. In particular, the following compounds are most preferred: the amino acid having a large lipophilic R group (conveniently referred to herein as AA2) is tributyltryptophan (Tbt) or a biphenylalanine derivative, such as Phe(4-(2-naphthyl)) [also referred to herein as Bip(4-(2-naphthyl)], Phe(4-(1-naphthyl)) [also referred to herein as Bip(4-(1-naphthyl)], Bip(4-n-Bu), Bip(4-Ph) or Bip(4-T-Bu); Phe(4-(2-naphthyl)) and Tbt. In some preferred embodiments, the amino acid having a large lipophilic R group is tributyltryptophan (Tbt).

[0038] Another preferred group of compounds are those in which Y is defined as -R1-R2-R3 as above, preferably in which R1 and R2 are unsubstituted, and most preferably in which R1 and R2 are both carbon atoms.

[0039] A further preferred group of compounds are those in which -XY together form the group -NHCH2CH2Ph.

[0040] The compounds comprise all enantiomers, including D and L amino acids, as well as enantiomers generated by the chiral center within the R group and the C-terminal capping group "-XY" of an amino acid. β and γ amino acids, as well as α amino acids, are included in the term "amino acid," and N-substituted glycine is also included in the term "amino acid," where all N-substituted glycine can be considered as AA units. The molecules of the present invention comprise β-peptides and condensates.

[0041] The most preferred compounds are as follows:

[0042]

[0043] t-Bu represents tert-butyl. The second compound incorporating the amino acid 2,5,7-tri-tert-butyl-L-tryptophan is the most preferred compound used in this invention (also referred to herein as AMC-109). Analogs of this compound incorporating other cationic residues in place of arginine (especially lysine) are also highly preferred. Analogs incorporating alternative C-terminal capping groups as defined above are also highly preferred.

[0044] A further preferred group of compounds includes those in which -XY are selected from -NHCH(CH3)2, -NH(CH2)5CH3, -NH(CH2)3CH3, -NH(CH2)2CH3, -NHCH2CH(CH3)2, -NHcyclohexyl, and -NHcyclopentyl, with particular preference for compounds in which -XY is the group -NHCH(CH3)2 or -NH(CH2)5CH3. A particularly preferred group of compounds includes those in which -XY is NHCH(CH3)2.

[0045] The preferred compounds are those in which AA1 is arginine, AA2 is tributyltryptophan, and -XY together form NHCH(CH3)2.

[0046] The compounds used in this invention are preferably peptides.

[0047] Compounds of formulas (I) through (IV) can be peptide mimics, and peptide mimics of peptides described and defined herein also represent compounds used according to the present invention. Peptide mimics are generally characterized by retaining the polarity, three-dimensional size, and functionality (bioactivity) of their peptide equivalents, but in which the peptide bonds are typically replaced by more stable bonds. "Stable" means more resistant to enzymatic degradation by hydrolases. Typically, the bonds replacing the amide bonds (amide substitutes) retain many properties of the amide bond, such as conformation, spatial volume, electrostatic properties, and the possibility of hydrogen bonding. Chapter 14 of *Drug Design and Development* (Krogsgaard, Larsen, Liljefors, and Madsen, eds., 1996, Horwood Academic Press) provides a general discussion of the design and synthesis techniques for peptide mimics. In the current context where molecules react with membranes rather than with specific active sites of enzymes, some of the issues of precisely mimicking affinity and efficacy or substrate function are irrelevant, and peptide mimics can be readily prepared based on a given peptide structure or the motif of a desired functional group. Suitable amide bond substitutes include the following groups: N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46, 47), transamide (Chorev, M. and Goodman, M., Acc. Chem. Res., 1993, 26, 266), thioamide (Sherman, DB. and Spatola, AF. J Am. Chem. Soc., 1990, 112, 433), thioesters, phosphonates, ketomethylene (Hoffman, RV. and Kim, HO. J Org. Chem., 1995, 60, 5107), hydroxymethylene, fluorovinyl (Allmendinger, T. et al., Tetrahydron Lett., 1990, 31, 7297), vinyl, methylene amino (Sasaki, Y. and Abe, J. Chem. Pharm. Bull., 1997). 45,13), methylene thiols (Spatola, AF, Methods Neurosci, 1993, 13, 19), alkanes (Lavielle, S. et al., Int. J. Peptide Protein Res., 1993, 42, 270) and sulfonamides (Luisi, G. et al., Tetrahedron Lett. 1993, 34, 2391).

[0048] The peptide mimic compounds of the present invention typically have three identifiable subunits, which are approximately equivalent in size and function to amino acids (AA units). Therefore, the term "amino acid" may be conveniently used herein to refer to the equivalent subunit of the peptide mimic compound. Furthermore, peptide mimics may have groups equivalent to the R group of amino acids, and the discussion herein of suitable R groups and N- and C-terminal modifying groups is applicable to peptide mimic compounds by analogy.

[0049] As discussed in the textbook cited above, in addition to the substitution of amide bonds, peptide mimics may involve replacing larger structural portions with dipeptide or tripeptide mimic structures, and in this case, the mimic portion involving the peptide bond, such as pyrrole-derived mimics, can be used as the dipeptide substitution. However, peptide mimics are preferred, and therefore, peptide mimic backbones in which the amide bonds have been substituted as described above are preferred.

[0050] Suitable peptide mimics include reduced peptides, in which the amide bond is reduced to a methyleneamine by treatment with a reducing agent (e.g., borane or hydride reagents, such as lithium aluminum hydride). Such reductions have the added advantage of increasing the overall cationicity of the molecule.

[0051] Other peptide mimics include peptide-like compounds formed, for example, by stepwise synthesis of amide-functionalized polyglycine. The backbone of some peptide mimics will be readily obtained from their peptide precursors, such as peptides that have already been hypermethylated, a suitable method described by Ostresh, JM et al. in Proc. Natl. Acad. Sci. USA (1994) 91, 11138-11142. Strongly basic conditions will favor N-methylation rather than O-methylation and result in methylation of some or all of the nitrogen atoms in the peptide bond and the N-terminal nitrogen.

[0052] Preferred peptide mimic backbones include polyesters, polyamines and their derivatives, as well as substituted alkanes and olefins. The peptide mimics preferably have N- and C-termini, which can be modified as described herein.

[0053] The compounds used according to the invention (e.g., peptides) exhibit antimicrobial activity (generally antibacterial activity), particularly through a direct membrane-effect mechanism of cytotoxicity, and can be termed membrane-acting antimicrobials. These compounds lyse cell membranes, destabilize cell membranes, or even penetrate cell membranes. This provides a significant therapeutic advantage over agents that act on or interact with protein components of the target cell (e.g., cell surface receptors). Although mutations can produce novel forms of target proteins leading to antibiotic resistance, it is less likely that free radical alterations to the lipid membrane will occur to prevent cytotoxic effects. Lysis leads to extremely rapid cell death, thus having the advantage of killing bacteria before they have a chance to multiply. Furthermore, the molecules may possess other useful properties that kill or impair target microorganisms, such as the ability to inhibit protein synthesis, and therefore they may have multi-target activity.

[0054] The compounds used in this invention can be synthesized in any convenient manner. Typically, any reactive groups present (e.g., amino, thiol, and / or carboxyl groups) will be protected throughout the synthesis. Therefore, the final step in the synthesis will be the deprotection of the protected derivatives of this invention.

[0055] When constructing peptides, in principle, the process can begin at either the C-terminus or the N-terminus; however, a C-terminus-starting procedure is preferred.

[0056] Methods for peptide synthesis are well known in the art, but for the purposes of this invention, synthesis on a solid support, which is well known in the art, may be particularly convenient.

[0057] A wide range of amino acid protecting groups is known, and suitable amine protecting groups may include benzyl ester (also known as Z), tert-butoxycarbonyl (also known as Boc), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), and 9-fluorenylmethoxycarbonyl (also known as Fmoc). It should be understood that when constructing a peptide starting from the C-terminus, the amine protecting group will be present on the α-amino group of each newly added residue and needs to be selectively removed before the next coupling step.

[0058] For example, carboxyl protecting groups that can be used include easily cleaved ester groups, such as benzyl (Bzl), p-nitrobenzyl (ONb), pentachlorophenyl (OPClP), pentafluorophenyl (OPfp), or tert-butyl (OtBu), as well as coupling groups on a solid support, such as methyl groups linked to polystyrene.

[0059] Thiol protecting groups include p-methoxybenzyl (Mob), triphenylmethyl (Trt), and acetamylmethyl (Acm).

[0060] Several procedures exist for removing amine and carboxyl protecting groups. However, these must be consistent with the synthetic strategy employed. The side-chain protecting groups must be stable to the conditions used to remove the temporary α-amino protecting group prior to the next coupling step.

[0061] Acid treatment, such as with trifluoroacetic acid, can be used to remove both amine protecting groups (such as Boc) and carboxyl protecting groups (such as tBu). Oxidizing agents (such as iodine) can be used to selectively remove thiol protecting groups (such as Trt).

[0062] Compounding (“compounded”) is a term used to describe the mixing and / or blending of two polymers or a polymer with one or more additives. This can be conveniently achieved by blending in a melt state or by combining multiple components in solution. Both methods are described herein. The resulting mixture is ideally homogeneous or nearly homogeneous. To avoid ambiguity, compounding does not involve a covalent bond between the polyester and the compound of formula (I), but rather compounding results in a non-covalent, releasable association between the polyester and the compound of formula (I). Therefore, the compound can be considered to releasably associate with the polyester.

[0063] Therefore, the compounds of formula (I) can be released (or leached or diffused) from the formulations of the present invention. This is important in the context of the present invention because the compounds of formula (I) have antimicrobial activity, and it is desirable that, in use, the compounds can be released from the formulation to areas where antimicrobial activity is required, such as to prevent or treat infections of wounds, surgical sites, or implantation sites of medical devices.

[0064] Preferably, during use, the compound of formula (I) is released from the formulation in a controlled (i.e., sustained) manner. For example, a therapeutically effective amount of the compound may be released for at least 6, 8, 12, or 14 hours. Therapeutically effective amounts will preferably result in a concentration of the compound delivered to the local environment exceeding the minimum inhibitory concentration (MIC) of the compound against the target bacteria. The release time can be prolonged by applying a layer of biodegradable polyester, unmixed with the active compound, to the top of the formulation of the present invention (e.g., as an outer layer on a medical device).

[0065] The ability of an active compound to be released from the formulation of the present invention can be readily determined by any suitable method, and such methods are familiar to those skilled in the art. Suitable methods are described in the examples herein. For example, a suture coated with the formulation according to the present invention can be contacted with an agar plate inoculated with bacteria (e.g., Staphylococcus spp.), and after an appropriate incubation time, the presence of an "inhibition zone" (i.e., an area with no bacterial growth or reduced bacterial growth) around the suture can be examined. The presence of an "inhibition zone" (e.g., compared to a test with a control suture (without the antimicrobial compound)) indicates that the compound can be released from the formulation.

[0066] The formulations of the present invention can be considered as controlled-release formulations of compounds of formula (I) released in vivo. Compounds of formula (I) can be considered as dispersed (releasably dispersed) through (or at least partially dispersed through) polyester.

[0067] The compounds of formula (I) make the polyester resistant to microbial colonization and biofilm formation. Therefore, the coating and / or device effectively maintains its own cleanliness, which is highly advantageous since indwelling or other medical devices are often sites of microbial attachment and growth.

[0068] Liquid culture medium tests can be used to evaluate the release of active compounds and their anticolonization effects. Instruments coated with the polyester of this invention can be immersed in liquid bacterial broth. Leakage efficacy (release) is determined by counting the number of surviving bacteria in the broth, and anticolonization efficacy is determined by counting the number of surviving bacteria adhering to the instrument after exposure to the bacterial broth.

[0069] The biodegradable polymers used in this invention are classified as polyesters because they contain repeating ester groups, but may also contain other functional groups. They are typically ester-terminated, but free carboxylic acid groups or alkyl ester groups can be used as end groups. Polymers terminated with esters and those terminated with alkyl esters generally exhibit longer degradation times than those terminated with free carboxylic acid groups.

[0070] Particularly suitable polymers include polylactide (D or L form), polyglycolic acid, polydioxanone, and polycaprolactone. Copolymers (including block copolymers) incorporating these same monomers can also be used, such as copolymers of D-lactide and L-lactide (although L-lactide polymers are preferred), copolymers of lactide and glycolide, and copolymers of lactide and caprolactone. A 50:50 enantiomer mixture of poly(D,L-lactide-co-glycolic acid) is preferred. Polymers including copolymers (which contain trimethylene carbonate monomers) are also suitable. The monomer ratio in the copolymer can vary, but copolymers including lactide typically contain 50% or more, for example, at least 60% or 70% lactide monomer. Therefore, formulations can comprise a single polymer type, a single copolymer, or a mixture of one or more polymers and / or copolymers.

[0071] Biodegradable polymers are typically synthetic. The use of biodegradable polymers is well-known in the biomedical field. The biodegradable polymers used in this invention should be non-toxic and not perceived as foreign by the patient. The biodegradation products should also be non-cytotoxic and readily eliminated from the body.

[0072] Biodegradability is determined by the rate of degradation during use, i.e., within an animal (or in contact with it). Biodegradable polyesters are also referred to as bioabsorbable (i.e., decomposed and absorbed by the body). Degradation varies significantly between polymers, but typically ranges from 1 or 2 weeks to 4 years or longer. Preferably, in situ, the polyester will completely degrade within less than 6 months, for example, 2-4 months. Although in other preferred embodiments, the polymer may remain for up to 4 years or even longer. Those skilled in the art are aware of the degradation times of the different polymers of interest and can select polymers based on their intended use. For example, sutures may only need to last a week or several weeks, while some implants, such as orthopedic or cardiac implants, may remain in the body for years. Blending with compounds of formula (I) does not significantly affect the degradation rate of the polymer.

[0073] The intrinsic viscosity of the polymer (measured in dL / g) will also vary between polymers, typically from 0.1 or 0.2 to 6 or 8, preferably from 0.5 to 2.5, more preferably from 0.8 to 2.2, and most preferably from 0.8 to 1.2.

[0074] The molecular weight (weight average) will also depend on the polymer selected and the specific medical application of interest. Typical molecular weights are 3,000-30,000, for example, 5,000-25,000. Although in some cases molecular weights can be used up to 50,000, 80,000, 100,000 or higher. Preferred molecular weights are 3,000-20,000, for example, 3,000-10,000 or 15,000.

[0075] Gel permeation chromatography can be used to measure intrinsic viscosity and molecular weight.

[0076] Suitable suppliers of medical-grade biodegradable polyester include Evonik (whose...) (Scope). Suitable polymers are readily available commercially and can be conveniently synthesized through a number of reactions, including direct condensation of alcohols and acids, ring-opening polymerization, and metal-catalyzed polymerization.

[0077] Two different methods can be conveniently used to prepare the formulations of this invention.

[0078] First, the polyester and the compound of formula (I) can be blended by melting them together. The polymer can be melted first, and then the compound of formula (I) can be added, or the compound can be added before melting. The addition of the compound has no significant effect on the melting point of the polymer. Surprisingly, the compounds of formula (I), especially the peptides of formula (I), are able to withstand the heating necessary to blend them with the biodegradable polyester. These molecules exhibit unexpected thermal stability.

[0079] Therefore, in another aspect, the present invention provides a method for preparing a formulation comprising a biodegradable polyester compounded with a compound of formula (I), the method comprising melting the biodegradable polyester compounded with a compound of formula (I). Embodiments of other aspects of the invention described herein are applicable mutatis mutandis to this aspect of the invention. This melt compounding is preferably carried out using rapid heating, for example, to 120-230 degrees Celsius (depending on the melting point of the polyester). Within 5 minutes, for example, within 4 minutes, such as about 3 minutes. In this way, the polymer melts before the peptide decomposes. The compound of formula (I) is preferably exposed to high temperatures (e.g., above 100 degrees Celsius) for only 2 or 3 minutes (allowing heat transfer to the mixture). Once the polyester is melted and mixed with the compound of formula (I), the mixture is removed from the heat source. In this embodiment, a polyester with a melting point below about 230 degrees Celsius is preferred.

[0080] Alternatively, and preferably, the polymer and the compound having chemical formula (I) can be mixed using a solvent. In one embodiment, the biodegradable polymer is dissolved in a first solvent, and the compound of formula (I) is dissolved in a second solvent miscible with the first solvent, and then the two solutions are mixed. Both solvents are preferably organic solvents, and preferably have a certain degree of polarity.

[0081] Both solvents must be chosen to be miscible and capable of dissolving the polymer and the compound of formula (I) separately. When mixed, the solvents must also retain their ability to solubilize their components; even when mixed, a miscible solvent may cause one component to precipitate. Suitable solvents for the polymer include chloroform, ethyl acetate, acetone, and tetrahydrofuran (THF); polar aprotic solvents may be preferred. Suitable solvents for the compound of formula (I) vary further and include water, methanol, ethanol, chloroform, THF, and DMSO. Ethyl acetate is the preferred solvent for the polymer, and an alcohol, such as ethanol, is the preferred solvent for the compound of formula (I).

[0082] Alternatively, both the compound and the polymer of formula (I) can be dissolved in a single solvent before, during, or after mixing. Solvents suitable for this method include THF and chloroform. It may be necessary to combine the compound of formula (I) with a solvent (e.g., THF) for an extended period, such as at least 4 hours, preferably at least 12 hours, possibly 1 day or more, possibly up to 7 or 14 days. Surprisingly, compounds of formula (I) (such as AMC-109) are soluble in both THF and chloroform.

[0083] Therefore, in another aspect, the present invention provides a method for preparing an formulation comprising a biodegradable polyester containing a compound of formula (I), the method comprising: forming a compound of formula (I), a biodegradable polyester, and a mixture of one or more solvents, the solvents being capable of dissolving the compound and the polyester, and optionally drying the mixture.

[0084] In a preferred embodiment, the present invention provides a method for preparing a formulation comprising a biodegradable polyester compounded with a compound of formula (I), the method comprising: (i) providing a first solution comprising a compound of formula (I); (ii) providing a second solution comprising a biodegradable polyester, wherein the second solution is miscible with the first solution; and (iii) mixing the first solution and the second solution. Embodiments of other aspects of the invention described herein, with necessary modifications, are applicable to this aspect of the invention. The solvents used to prepare the first and second solutions may be the same or different. Preferred solvents are as described above.

[0085] After mixing, the resulting mixture can be dried, which can be an active or passive process. Passive drying processes may take several days, for example, 1-6 days.

[0086] Another method for compounding polyester with the compound of formula (I) is electrospinning. Electrospinning is a known voltage-driven method controlled by electrohydrodynamic phenomena, wherein fibers can be made from a polymer solution. To obtain the formulation according to the invention via electrospinning, a solution (or “spinning solution”) containing the polyester and the compound of formula (I) is electrospinned into fibers containing said compound. This solution can conveniently contain two solvents as described above, which are miscible and each capable of dissolving either the polymer or the compound of formula (I). Alternatively, a single solvent, such as THF, can be used. Suitable electrospinning methods are described in Scaffaro et al., European Polymer Journal 96 (2017), 266-277.

[0087] On a w / w basis, the amount of compound of formula (I) in the blended polymer formulation can be as low as 2% or 3% to as high as 25% or 30%, typically 4% to 15% or 20%.

[0088] The formulations of the present invention are preferably incorporated into medical devices as a coating, although some medical devices may consist of the formulations of the present invention, or may have parts or components made of the formulation, for example, by 3D printing. Therefore, another aspect of the present invention is a medical device coated with the formulations of the present invention as defined herein, including partially coated medical devices. Preferably, the entire outer surface of the device is coated with the formulation of the present invention. This coating creates a layer of formulation on the surface of the device (on any surface in contact with the body, including surfaces in contact with bodily fluids). The thickness of this layer is selected to provide the desired function, particularly to provide the desired in-situ controlled release profile.

[0089] In another aspect of the invention, formulations of the invention as defined herein are provided, which have been applied to medical devices.

[0090] Instruments include sutures, surgical fasteners, catheters, threads, and implants, including orthopedic implants (such as hip and knee implants) as well as dental implants, needles, stents, cardiac rhythm devices, and deep brain stimulation devices. Sutures are particularly preferred.

[0091] The device to be coated can be immersed (possibly several times, e.g., 3-10 times) in the molten formulation of the present invention or a mixture comprising a biodegradable polyester, a compound of formula (I), and one or more solvents, and then dried or allowed to dry. Suitable solvents and miscible solvent mixtures are discussed herein. The immersion method is particularly suitable for devices such as sutures. Alternatively, the formulation of the present invention can be applied to medical devices, such as implants, by brushing onto the surface of the device, for example by spraying. Medical devices containing the formulation of the present invention can also be produced by 3D printing.

[0092] Suitable sutures to which the formulations of this invention can be applied include absorbable, optionally braided sutures. Such sutures may be made of nylon, including Ethicon, Surgilon, and Nurolan sutures. It is preferred to use sutures that are free of or have no coating other than the formulations of this invention. For example, the sutures may be pretreated to remove the silicone coating.

[0093] Absorbable (biodegradable) sutures are preferred medical devices according to the invention and can be formed as follows. They are conveniently made from polyesters selected from monomers including lactic acid (in two enantiomeric forms, alone or in combination), glycolic acid, caprolactone, and dioxane. To tailor the properties of the suture, two (or more) of these monomers are typically copolymerized, or shorter chains of different homopolymers may be block-polymerized. The most common polymer in absorbable sutures is a copolymer of L-lactic acid and glycolic acid, PLGA, preferably made from about 90% glycolic acid and about 10% L-lactic acid. Polyglactin 910 is such a copolymer, exhibiting high tensile strength and conveniently used as filaments in absorbable sutures. The coating is preferably a 65 / 35 molar ratio lactide-glycolic acid copolymer (e.g., Polyglactin 370) applied in an amount of 2-10% by weight of the filament. For treatment performance, an equal amount of calcium stearate (i.e., equal to the coating polymer) can be added. The compound of formula (I) is preferably blended in the coating or in another coating applied thereon, but may also be blended in the filament.

[0094] The suture of the present invention may contain 0.5-10 mg of compound of formula (I) per meter of suture length, preferably 1-5 mg / m, for example 1-3 mg / m.

[0095] In another aspect, the present invention provides a method for producing the medical device of the present invention, the method comprising: (i) providing a formulation comprising a biodegradable polyester, said biodegradable polyester being compounded with a compound of formula (I); and (ii) applying said formulation to the medical device (e.g., by immersing the device in said formulation, or by brushing (e.g., spraying) said formulation onto the device, or by brushing (e.g., spraying) said formulation onto the core of the device (such as the filament portion of a suture). After the formulation is applied to the medical device, it may be actively dried (e.g., by moderate heating or by applying an airflow) or allowed to dry. Embodiments of other aspects of the invention described herein, with necessary modifications, are applicable to this aspect of the invention. If a solvent-containing formulation is applied, the solvent will evaporate during drying and leave a coating (or layer) on said medical device comprising or composed of the formulation of the present invention.

[0096] In a similar manner, the present invention provides a method for producing the medical device of the present invention, the method comprising: (i) providing a formulation of a biodegradable polyester, said biodegradable polyester being compounded with a compound of formula (I); and (ii) applying said formulation onto a backing sheet or other carrier. In this way, medical devices composed of or substantially composed of the formulations of the present invention can be prepared, for example, to form films, membranes, sheets, or adhesives.

[0097] On the other hand, formulations comprising biodegradable polyesters prepared by the method of the present invention are provided, said biodegradable polyesters being compounded with a compound of formula (I), or medical devices having such formulations applied thereto. Embodiments of other aspects of the invention described herein, with necessary modifications, are applicable to this aspect of the invention.

[0098] Another aspect of the present invention provides the use of the formulations and devices of the present invention for therapeutic purposes.

[0099] "Therapy" includes both treatment and prevention; that is, it encompasses both therapeutic and preventative uses.

[0100] In some embodiments, the present invention provides formulations or medical devices of the present invention for treating or preventing infection in a subject. In some preferred embodiments, the infection or potential infection is a surgical site infection or wound infection, such as a wound or other site requiring closure by sutures or other surgical fasteners. In other preferred embodiments, the infection or potential infection is an infection associated with the implant (as described above), including the formation of a biofilm on or around the device.

[0101] Preferably, the infection is a bacterial infection, such as one caused by Gram-positive bacteria (e.g., bacteria of the spp. Staphylococcus or Streptococcus). In some embodiments, the infection is a Staphylococcus aureus infection. In some embodiments, the infection is a Staphylococcus epidermidis infection.

[0102] The formulations, devices, uses, and methods of the present invention are preferably effective against broad-spectrum bacteria, particularly against Gram-positive and Gram-negative bacteria (by the release of active compounds that completely or partially inhibit bacterial growth in the surrounding environment and / or completely or partially inhibit clustering effects), for example, they are effective against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis.

[0103] Another aspect of the invention provides the use of the formulations or medical devices of the invention for inhibiting bacterial growth in a subject. Embodiments of other aspects of the invention described herein, with necessary modifications, are applicable to this aspect of the invention.

[0104] Another aspect of the invention provides the use of the formulations or medical devices of the invention for treatment, preferably for treating or preventing infection in a subject. Embodiments of other aspects of the invention described herein, with necessary modifications, are applicable to this aspect of the invention.

[0105] From another perspective, this invention provides a method for treating or preventing infection, comprising administering (or applying) the formulation or medical device of this invention to a subject in need. Embodiments of other aspects of the invention described herein, with necessary modifications, are applicable to this aspect of the invention.

[0106] From another perspective, this invention provides a method for treating or preventing infection, comprising administering (or applying) a therapeutically effective amount of the formulation or medical device of this invention to a subject in need. Embodiments of other aspects of the invention described herein, with necessary modifications, are applicable to this aspect of the invention.

[0107] For the selected compound of formula (I), the therapeutically effective dose will be determined based on clinical assessment and the MIC value against the target bacteria.

[0108] Another aspect of the invention provides the use of the compound of formula (I) for treatment, preferably for treating or preventing infection in a subject, wherein the compound is administered (or applied to) the subject as a formulation comprising a biodegradable polyester compounded with the compound or as a medical device to which such a formulation is applied. Embodiments of other aspects of the invention described herein, with necessary modifications, are applicable to this aspect of the invention.

[0109] As used herein, the terms "subject" or "patient" include any mammal, such as humans and any livestock, domesticated animals, or laboratory animals. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, cattle, and monkeys. However, preferably, the subject or patient is a human subject. Therefore, the subject or patient treated according to the present invention is preferably a human.

[0110] In some embodiments, the subjects or patients are those who have an infection, are suspected of having an infection, or are at risk of having (or contracting) an infection. Patients at risk are a preferred group and include those who require medical implants, must undergo surgery, or require surgical or other wound closure. For these patients, the devices of the present invention, such as sutures, other fasteners, or prosthetic implants, incorporating the formulation of the present invention, may be selected. Such devices release an antimicrobial compound of formula (I) to promote the formation of an infection-free local environment around the device within the patient, and the presence of the compound of formula (I) on / within the device inhibits bacterial colonization of the device itself.

[0111] The present invention also provides a kit comprising one or more medical devices of the present invention. Preferably, the kit is used for the treatment methods and uses described herein. Preferably, the kit includes instructions for use of the kit components. Preferably, the kit is used to treat or prevent infection, such as as described elsewhere herein, and optionally includes instructions for using the kit components to treat such infection.

[0112] As used throughout the application, the terms “a” and “an” are used in the sense that they refer to “at least one,” “at least first,” “one or more,” or “a plurality” of the components or steps referenced, except where the upper limit is specifically stated hereafter.

[0113] Additionally, when the terms “comprise”, “comprises”, “has” or “having” or other equivalent terms are used herein, in some more specific embodiments, these terms include the terms “composed of” or “substantially composed of” or other equivalent terms.

[0114] The invention will now be further described with reference to the following non-limiting embodiments and accompanying drawings, wherein:

[0115] Figure 1 This is a graph showing the effect of compound 2 on a one-day topical treatment against Staphylococcus aureus FDA486 in a mouse skin infection model. The number of colony-forming units (CFU) is shown on the Y-axis, and the type of topical treatment applied to the mice is shown on the X-axis. Compound 2 is also referred to herein as AMC-109.

[0116] Figure 2 This is a graph showing the effect of one day of topical treatment with compound 2 against Streptococcus pyogenes in a mouse skin infection model. The number of colony-forming units (CFU) is shown on the Y-axis, and the type of topical treatment applied to the mice is shown on the X-axis. Compound 2 is also referred to herein as AMC-109.

[0117] Figure 3 This graph shows the effect of one day of topical treatment against Staphylococcus aureus FDA486 in a mouse skin infection model. Each mouse was treated at 9:00 AM, 12:00 PM, and 3:00 PM. Skin biopsies were collected at 6:00 PM. The median is shown.

[0118] Figure 4 This graph shows the effect of one day of local treatment against Streptococcus pyogenes CS301 in a mouse skin infection model. Each mouse was treated at 7:00 AM, 10:00 AM, and 1:00 PM. Skin biopsies were collected at 4:00 PM. The median is shown.

[0119] Figure 5 This graph shows the effect of one day of topical treatment against Staphylococcus aureus FDA486 in a mouse skin infection model. Each mouse was treated at 9:00 AM, 12:00 PM, and 3:00 PM. Skin biopsies were collected at 6:00 PM. The median is shown.

[0120] Figure 6 This is a graph showing a quantitative analysis of the amount of AMC-109 released by extracting coated sutures. The first dataset comes from batch-01 of Experiment 2, and the subsequent three datasets are from three different sutures in batch-02 of Experiment 2.

[0121] Figure 7 This is a photograph showing the results of the Staphylococcus aureus growth inhibition test during the first use.

[0122] Figure 8 This is a graph showing the effect of AMC-coated sutures on bacterial growth in liquid culture medium, measured by CFU in the growth medium after exposure to the sutures.

[0123] Figure 9 It is a photograph showing the inhibition region as described in Example 9.

[0124] Figure 10 These are photographs of the suture holes containing TSB injection sites, where various sutures were placed. The numbered holes are as follows:

[0125] 1: Staphylococcus aureus Ethibond coated with AMC-109

[0126] 2: Staphylococcus aureus Ethibond. uncoated

[0127] 3: Staphylococcus aureus growth control

[0128] 4: Staphylococcus epidermidis Ethibond coated with AMC-109

[0129] 5: Staphylococcus epidermidis Ethibond. was not coated.

[0130] 6: Staphylococcus epidermidis growth control

[0131] Figure 11 This is a graph showing the leakage (as a percentage of total) of AMC-109 containing bioabsorbable polyesters RG502 and L206S over time (minutes).

[0132] Example 1

[0133] peptide synthesis

[0134] Chemical reagents

[0135] The protected amino acids Boc-Trp-OH, Boc-Arg-OH, Boc-4-phenyl-Phe, and Ac-Arg-OH were purchased from Bachem, while Boc-4-iodophenylalanine, Boc-3,3-diphenylalanine, and Boc-(9-anthrayl)alanine were purchased from Aldrich. The C-terminal compounds of the peptides, namely benzylamine, 2-phenylethylamine, 3-phenylpropanine, (R)-2-phenylpropanine, (S)-2-phenylpropanine, N,N-methylbenzylamine, N,N-ethylbenzylamine, and N,N-dibenzylamine, were purchased from Fluka, except for N-ethylbenzylamine, which was purchased from Acros. Diisopropylethylamine (DIPEA), 1-hydroxybenzotriazole (1-HOBt), trichloropyrrolidinyl hexafluorophosphate (PyCloP), and O-(benzotriazol-1-yl)-N,N,N',N' tetramethylurea hexafluorophosphate (HBTU) were purchased from Fluka. 4-n-butylphenylboronic acid, 4-tert-butylphenylboronic acid, 4-biphenylboronic acid, 2-naphthylboronic acid, tri-o-tolylphosphine, benzyl bromide, and palladium acetate were purchased from Aldrich. Solvents were purchased from Merck and Riedel-de. Or Aldrich.

[0136] Preparation of amino acids

[0137] Preparation of Boc-2,5,7-tri-tert-butyltryptophan-OH: A mixture of H₂N-Trp-OH (1.8 g, 8.8 mmol) and t-BuOH (4.7 g, 63.4 mmol) in trifluoroacetic acid (19 mL) was stirred at 70 °C for 3 h. The resulting brownish translucent solution was reduced in volume on a rotary evaporator at room temperature for 30 min, followed by grinding with the dropwise addition of 60 mL of 7% (by weight) NaHCO₃. The resulting gray / white granular solid was then recovered by vacuum filtration and vacuum dried at room temperature for 24 h. The product was separated by crystallization from a near-boiling mixture of 40% ethanol in water. The volume was typically about 20 mL per gram of crude product.

[0138] The first crystallization from the crude product yields a separated product with a purity of 80-83% (HPLC) relative to all other substances in the sample and approximately 94-95% relative to known TBT analogs. The yield of this stage is 60-65%.

[0139] Benzylation of Boc-4-iodophenylalanine. Boc-4-iodophenylalanine (1 equivalent) was dissolved in an aqueous solution of 90% methanol and neutralized to a weakly alkaline pH (determined by litmus paper) by adding cesium carbonate. The solvent was removed by rotary evaporation, and the remaining water in the cesium salt of Boc-4-iodophenylalanine was further reduced by repeated azeotropic distillation with toluene. The resulting dried salt was dissolved in dimethylformamide (DMF), benzyl bromide (1.2 equivalent) was added, and the mixture was stirred for 6–8 hours. At the end of the reaction, DMF was removed under reduced pressure to form an oil containing the title compound. This oil was dissolved in ethyl acetate, and the resulting solution was washed with equal volumes of citric acid solution (three times), sodium bicarbonate solution, and brine. The title compound was separated by rapid chromatography using dichloromethane:ethyl acetate (95:5) as the eluent, as a pale yellow oil in 85% yield. Crystalline benzyl Boc-4-iodophenylalanine can be obtained by recrystallization from n-heptane.

[0140] The general procedure for Suzuki coupling is as follows: Benzyl Boc-4-iodophenylalanine (1 equivalent), arylboronic acid (1.5 equivalent), sodium carbonate (2 equivalent), palladium acetate (0.05 equivalent), and tri-o-tolylphosphine (0.1 equivalent) are added to a degassed mixture of dimethoxyethane (6 ml / mmol benzyl Boc-4-iodophenylalanine) and water (1 ml / mmol benzyl Boc-4-iodophenylalanine). The reaction mixture is maintained under argon and heated to 80 °C for 4–6 h. After cooling to room temperature, the mixture is filtered through a silica gel and sodium carbonate short septum. The filter cake is further washed with ethyl acetate. The filtrates are combined, and the solvent is removed under reduced pressure. The product is separated by rapid chromatography using a mixture of ethyl acetate and n-hexane as the eluent.

[0141] Preparation of Boc-Bip(n-Bu)-OBn: The title compound was prepared from 4-n-butylphenylboronic acid in 53% yield using a general Suzuki coupling procedure. Boc-Bip(n-Bu)-OBn was separated using an 80:20 ethyl acetate:n-hexane eluent.

[0142] Preparation of Boc-Bip(t-Bu)-OBn: The title compound was prepared from 4-tert-butylphenylboronic acid in 79% yield using the general procedure of Suzuki coupling. Boc-Bip(t-Bu)-OBn was separated using an 80:20 ethyl acetate:n-hexane eluent.

[0143] Preparation of Boc-Bip(4-Ph)-OBn: The title compound was prepared from 4-biphenylboronic acid in 61% yield using the general procedure of Suzuki coupling. Boc-Bip(4-Ph)-OBn was isolated by recrystallization of the crude product from n-heptane.

[0144] Preparation of Boc-Bip(4-(2-naphthyl))-OBn: The title compound was prepared from 2-naphthylboronic acid in 68% yield using the general method of Suzuki coupling. Boc-Bip(4-(2-naphthyl))-OBn was isolated by recrystallization of the crude product from n-heptane.

[0145] Preparation of Boc-Bip(4-(1-naphthyl))-OBn: The title compound was prepared from 2-naphthylboronic acid using the general procedure of Suzuki coupling. Boc-Bip(4-(1-naphthyl))-OBn was isolated by recrystallization of the crude product from n-heptane.

[0146] The general procedure for the deesterification of benzyl esters is as follows: The benzyl ester is dissolved in DMF and hydrogenated for 2 days under ambient pressure using 10% carbon-supported Pd as a catalyst. At the end of the reaction, the catalyst is removed by filtration, and the solvent is removed under reduced pressure. The free acid is separated by recrystallization from diethyl ether.

[0147] Preparation of Boc-Bip(4-n-Bu)-OH: The title compound was prepared from Boc-Bip(n-Bu)-OBn in 61% yield using a general procedure for deesterification.

[0148] Preparation of Boc-Bip(4-t-Bu)-OH: The title compound was prepared from Boc-Bip(t-Bu)-OBn in 65% yield using a general procedure for deesterification.

[0149] Preparation of Boc-Bip(4-Ph)-OH: The title compound was prepared from Boc-Bip(4-Ph)-OBn in 61% yield using a general procedure for deesterification.

[0150] Preparation of Boc-Bip(4-(2-naphthyl))-OH: The title compound was prepared from Boc-Bip(4-(2-naphthyl))-OBn in 68% yield using a general procedure for deesterification.

[0151] Preparation of Boc-Bip(4-(2-naphthyl))-OH: The title compound was prepared from Boc-Bip(4-(2-naphthyl))-OBn in 68% yield using a general procedure for deesterification.

[0152] General procedure for solution-phase peptide synthesis using HBTU. The peptide is prepared in solution using a Boc protection strategy via stepwise amino acid coupling, according to the following general procedure. A C-terminal peptide moiety with a free amino group (1 equivalent), a Boc-protected amino acid (1.05 equivalent), and 1-hydroxybenzotriazole (1-HOBt) (1.8 equivalent) are dissolved in DMF (2-4 ml / mmol amino group), followed by the addition of diisopropylethylamine (DIPEA) (4.8 equivalent). The mixture is cooled on ice, and O-(benzotriazole-1-yl)-N,N,N',N' tetramethylurea hexafluorophosphate (HBTU) (1.2 equivalent) is added. The reaction mixture is shaken at ambient temperature for 1-2 h. The reaction mixture is diluted with ethyl acetate and washed with citric acid, sodium bicarbonate, and brine. The solvent is removed under vacuum, and the Boc protecting group of the resulting peptide is deprotected in the dark using 95% TFA or anhydrous methanol solution of acetyl chloride.

[0153] A solution-phase amide was formed using PyCloP. Synthesis of Boc-Arg-N(CH2Ph)2: A solution of Boc-Arg-OH (1 equivalent), NH(CH2Ph)2 (1.1 equivalent), and PyCloP (1 equivalent) was prepared in anhydrous DCM (filtered through alumina) (2 ml) and DMF (1 ml). The solution was cooled on ice and DIPEA (2 equivalents) was added with stirring. The solution was stirred at room temperature for 1 h. The reaction mixture was evaporated, reconstituted in ethyl acetate, and washed with citric acid, sodium bicarbonate, and brine. The solvent was removed under vacuum, and the Boc protecting group of the resulting peptide was deprotected in the dark using 95% TFA.

[0154] Peptide purification and analysis. Reversed-phase HPLC was performed at a Delta-Pak (Waters) C1000000 rpm. 18 column( The peptide was purified on a 15 μm, 25 × 100 mm plate using a mixture of water and acetonitrile (both containing 0.1% TFA) as eluent. The peptide was then purified by RP-HPLC using analytical Delta-Pak (Waters) C1000 ppm. 18 column( Peptides were analyzed by RP-HPLC on a VG Quattro quadrupole mass spectrometer (15 μm, 3.9 × 150 mm) and on a VG Quattro quadrupole mass spectrometer (VG Instruments, Altringham, UK).

[0155] Example 2

[0156] The in vitro activity of peptides defined in this article

[0157] Materials and methods

[0158] antimicrobial agents

[0159] The pre-weighed vials of Compound 1 and Compound 2 were supplied by Lytix Biopharma.

[0160]

[0161] bacterial isolates

[0162] The bacterial isolates used in this study were from various sources worldwide, stored at GR Micro, and maintained with minimal subculturing. They were then deep-frozen at -70°C as concentrated suspensions in a high-protein matrix of undiluted horse serum. The bacterial species used and their characteristics are listed in Table 1. These species included 54 Gram-positive bacteria, 33 Gram-negative bacteria, and 10 fungi.

[0163] Determination of minimum inhibitory concentration (MIC)

[0164] The MIC was determined using the following microbroth dilution method for antimicrobial susceptibility testing, as published by the Clinical and Laboratory Standards Institute (CLSI, formerly known as NCCLS):

[0165] M7-A6, Volume 23, Issue 2, January 2003: Methods for antimicrobial susceptibility testing of aerobic bacteria using dilution; Approved Standard – 6th Edition. M100-S15, Volume 25, Issue 1, January 2005: Performance standards for antimicrobial susceptibility testing; 15th Supplement. M11-A6, Volume 24, Issue 2: Methods for antimicrobial susceptibility testing of anaerobic bacteria; Approved Standard – 6th Edition. M27-A2, Volume 22, Issue 15: Reference method for antifungal susceptibility testing using yeast broth dilution method; Approved Standard – 2nd Edition. M38-A, Volume 22, Issue 16: Reference method for antifungal susceptibility testing of filamentous fungi using broth dilution method; Approved Standard.

[0166] MIC estimation was performed using wet plates containing antibacterial or antifungal agents prepared by GR Micro.

[0167] A cationic-regulated Mueller-Hinton broth (Oxoid (Basingstoke, UK) and Trek Diagnostic Systems (East Grinstead, UK)) (supplemented with 5% lysed horse blood for Streptococcus spp., Corynebacterium jejuni, and Listeria monocytogenes) was used for aerobic bacteria, with an initial inoculum of approximately 10... 5 Colony forming units (CFU) / mL.

[0168] Haemophilus influenzae test medium (Mueller-Hinton broth containing 0.5% yeast extract and Haemophilus influenzae test medium supplement containing 15 mg / L hemoglobin and 15 mg / L NAD, both obtained from Oxoid, Basingstoke, UK) was used for Haemophilus influenzae and inoculated with approximately 10 5 CFU / mL.

[0169] Supplemental broth (SBB) was used for anaerobic strains, with an inoculum of approximately 10... 6 CFU / ml. SBB is a broth composed of 1% peptone, 0.5% "Lab-lemco", 1% glucose and 0.5% sodium chloride, supplemented with 5 μg / L thiamine and 1 μg / L vitamin K (both from Sigma Aldrich).

[0170] MICs of yeast and filamentous fungi were performed in MOPS-buffered RPMI 1640 medium (MOPS buffer from Sigma-Aldrich, RPMI 1640 from Invitrogen (Paisley, Scotland)). The yeast inoculum was 7.5 × 10⁻⁶. 2 -4×10 3 CFU / mL, filamentous fungi: approximately 8 × 10⁻⁶ 3 -1×10 5 CFU / mL.

[0171] Following standard procedure, all plates containing Mueller-Hinton broth were prepared in advance, frozen at -70°C on the day of preparation, and thawed on the day of use. MIC determinations for fungi, Haemophilus influenzae, and anaerobic bacteria were performed on plates prepared on the same day.

[0172] To evaluate whether freezing affects peptide activity, several MIC measurements were repeated using plates containing freshly prepared Mueller-Hinton broth.

[0173] control strain

[0174] The test strain group included the following control (reference) strains.

[0175] Escherichia coli ATCC 25922

[0176] Staphylococcus aureus ATCC 29213

[0177] Enterococcus faecalis ATCC 29212

[0178] Streptococcus pneumoniae ATCC 49619

[0179] Pseudomonas aeruginosa ATCC 27853

[0180] Candida cruzie ATCC 6258

[0181] The following control strains were included outside the test strain group, and, where appropriate, to check whether the comparison substances were within the range.

[0182] Haemophilus influenzae ATCC 49247

[0183] Candida glabrata ATCC 2019

[0184] Bacteroides fragilis ATCC 25285

[0185] Egertella lataniae ATCC 43055

[0186] result

[0187] The results are presented in a single row and column in Table 1. Results for the replicate control strains are shown in Table 2. It can be seen that the control strain results are highly reproducible, including data from plates containing frozen or freshly used Mueller Hinton broth. Frozen plates had no effect on the MICs of other bacterial strains.

[0188] The obtained MIC data are very encouraging, indicating that the peptide has a fairly broad spectrum of activity.

[0189] Table 1: A single-row list of the in vitro activities of two antimicrobial peptides and a comparative peptide against a group of Gram-positive bacteria, Gram-negative bacteria and fungi.

[0190]

[0191]

[0192]

[0193] Table 2: In vitro activity of two antimicrobial peptides and comparative agents against ATCC control strains

[0194] (Including ATCC control strains other than the test strain group)

[0195]

[0196]

[0197] MHB, Mueller Hinton Broth; HTM, Haemophilus Assay Medium; SBB, Supplemental Brucella Broth.

[0198] Example 3: Stability against trypsin degradation and antimicrobial activity

[0199] The trypsin resistance and antimicrobial activity of the compound AA1-AA2-AA1-NHCH2CH2Ph ​​were detected.

[0200] Measurement and calculation of peptide half-life

[0201] Each peptide was dissolved in 0.1M NH4HCO3 buffer (pH 6.5) to obtain a final peptide concentration of 1 mg / ml. Trypsin solutions were prepared by dissolving 1 mg of trypsin in 50 ml of 0.1M NH4HCO3 buffer (pH 8.2). For stability assays, 250 μl of freshly prepared trypsin solution and 250 μl of peptide solution were incubated on a shaker at 37°C in 2 ml of 0.1M NH4HCO3 buffer (pH 8.6). 0.5 ml aliquots were taken at different time intervals and diluted with 0.5 ml of water:acetonitrile containing 1% TFA (60:40 v / v): analyzed as described above by RP-HPLC. Samples without trypsin, collected at 37°C at 0 h and 20 h, served as negative controls. The integral of the peak area at 254 nm of samples collected during the first 5 h of the assay was used to generate τ. 1 / 2 Peptides that showed no degradation during the first 24 hours were classified as stable.

[0202] Antibacterial test

[0203] MIC determinations for Staphylococcus aureus strain ATCC 25923, methicillin-resistant Staphylococcus aureus (MRSA) strain ATCC 33591, and methicillin-resistant Staphylococcus epidermidis (MRSE) strain ATCC 27626 were performed by Toslab using standard methods. Amsterdam, D. (1996), Susceptibility testing of antimicrobial agents in liquid media, Antibiotics in Laboratory Medicine, 4th ed. (Lorian, V.), pp. 75-78, Williams & Wilkins, Baltimore.

[0204] Table 3. Stability of AA1-AA2-AA1-NHCH2CH2Ph ​​peptide to trypsin (measured as half-life (τ)) 1 / 2 And the antibacterial activity shown as MIC.

[0205]

[0206] a The half-life was calculated using a medical calculator from Cornell University.

[0207] b Minimum Inhibition Concentration

[0208] c Staphylococcus aureus strain ATCC 25923

[0209] d Methicillin-resistant Staphylococcus aureus (ATCC 33591)

[0210] e Methicillin-resistant Staphylococcus epidermidis ATCC 27626

[0211] f Compounds not included in the definition of this invention

[0212] Example 4: In vivo activity of compound 2

[0213] Mice were infected with Staphylococcus aureus or Streptococcus pyogenes skin, followed by a total of three treatments at 3-hour intervals. Three hours after the final treatment, skin biopsies were collected for examination, and the number of colony-forming units (CFUs) present in the skin samples was determined. Results are as follows: Figure 1 and Figure 2 As shown, this represents the number of colony-forming units per mouse.

[0214] In Experiment 1 ( Figure 1 In this study, compound 2 was applied as part of a cream or gel containing 2% (w / w) of compound 2 to the skin of mice. The same cream or gel without compound 2 served as a negative control (placebo). It was clearly observed that, compared to the negative control, the number of CFUs was reduced when the cream or gel containing compound 2 was applied to the skin of mice, indicating that compound 2 exerts an antimicrobial effect against Staphylococcus aureus. The properties of the carrier, cream, or gel were not significantly affected.

[0215] In Experiment 2 Figure 2 In this study, compound 2 was applied at two different concentrations, as 1% or 2% gels. A placebo gel and the known antibacterial agent "bactroban" were used as controls. It was observed that the gel containing compound 2 was more effective than the placebo gel or bactroban in reducing CFU counts. The gel containing 2% compound 2 was more effective than the gel containing only 1% compound 2.

[0216] Example 5

[0217] Preparation of the compounds used in this invention and their physical, antimicrobial and hemolytic properties.

[0218] peptide synthesis —Relevant information is also provided in Example 1.

[0219] Chemicals:

[0220] Protected amino acids Boc-Arg-OH and Boc-4-phenyl-Phe were purchased from Bachem, while Boc-4-iodophenylalanine was purchased from Aldrich. Isopropylamine, propylamine, hexylamine, butylamine, hexadecylamine, isobutylamine, cyclohexylamine, and cyclopentylamine, constituting the C-terminus of the peptide, were purchased from Fluka. Diisopropylethylamine (DIPEA), 1-hydroxybenzotriazole (1-HOBt), trichloropyrrolidinyl hexafluorophosphate (PyCloP), and O-(benzotriazol-1-yl)-N,N,N',N' tetramethylurea hexafluorophosphate (HBTU) were purchased from Fluka. 4-n-butylphenylboronic acid, 4-tert-butylphenylboronic acid, 4-biphenylboronic acid, 2-naphthylboronic acid, tri-o-tolylphosphine, benzyl bromide, and palladium acetate were purchased from Aldrich. Solvents were purchased from Merck and Riedel-de. Or Aldrich.

[0221] Preparation of Boc-Phe(4-4'-biphenyl)-OBn: The title compound was prepared from 4-biphenylboronic acid in 61% yield using the general procedure of Suzuki coupling. Boc-Phe(4-4'-biphenyl)-OBn was isolated by recrystallization of the crude product from n-heptane.

[0222] Preparation of Boc-Phe(4-(2'-naphthyl))-OBn: The title compound was prepared from 2-naphthylboronic acid in 68% yield using the general procedure of Suzuki coupling. Boc-Phe(4-(2'-naphthyl))-OBn was isolated by recrystallization of the crude product from n-heptane.

[0223] Preparation of Boc-Phe(4-4'-biphenyl)-OH: The title compound was prepared from Boc-Phe(4-4'-biphenyl)-OBn in 61% yield using a general procedure for deesterification.

[0224] Preparation of Boc-Phe(4-(2'-naphthyl))-OH: The title compound was prepared from Boc-Phe(4-(2-naphthyl))-OBn in 68% yield using a general procedure for deesterification.

[0225] The general procedure for solution-phase peptide synthesis using HBTU is described in Example 1.

[0226] Example 1 describes the use of PyCloP to form a solution phase amide.

[0227] The purification and analysis of the peptides were as described in Example 1.

[0228] Table 4

[0229] General formula of the compound: Arg-AA2-Arg-XY

[0230]

[0231] Antimicrobial test

[0232] The MICs for Staphylococcus aureus strain ATCC 25923, methicillin-resistant Staphylococcus aureus (MRSA) strain ATCC 33591, and methicillin-resistant Staphylococcus epidermidis (MRSE) strain ATCC 27626 were determined by Toslab using standard methods. Amsterdam, D. (1996), Susceptibility testing of antimicrobial agents in liquid media, Antibiotics in Laboratory Medicine, 4th ed. (Lorian, V.), pp. 75-78, Williams and Wilkins, Baltimore.

[0233] Table 5

[0234] The antimicrobial and toxic properties of the compounds used in this invention

[0235]

[0236] Example 6

[0237] Large-scale in vitro screening of selected compounds

[0238] Materials and methods

[0239] antimicrobial agents

[0240] The pre-weighed vials of Compound 7 and Compound 8 were supplied by Lytix Biopharma.

[0241]

[0242] bacterial isolates

[0243] The bacterial isolates used in this study are as described in Example 2.

[0244] Determine the minimum inhibitory concentration (MIC).

[0245] MIC was determined as described in Example 2.

[0246] result

[0247] The results are displayed as a single-row list in Table 6.

[0248] The obtained MIC data are very encouraging, indicating that the peptide has a fairly broad spectrum of activity.

[0249] Table 6: A single-row list of the in vitro activities of two antimicrobial peptides against a group of Gram-positive bacteria, Gram-negative bacteria and fungi.

[0250]

[0251]

[0252]

[0253] Example 7: In vivo activity of compounds 7 and 8

[0254] Mice were infected with Staphylococcus aureus or Streptococcus pyogenes skin, followed by a total of three treatments at 3-hour intervals. Three hours after the final treatment, skin biopsies were collected for examination, and the number of colony-forming units (CFUs) in the skin samples was determined. Results are as follows: Figure 3 , Figure 4 and Figure 5 As shown, this represents the number of colony-forming units per mouse.

[0255] In Experiment 1 ( Figure 3 In this study, compound 7 was applied to mouse skin as part of a cream or gel containing 2% (w / w) of compound 7. The same cream or gel without compound 7 served as a negative control (placebo). Bactroban 2% cream was used as a positive control. It was clearly observed that, compared to the negative control, a reduction in CFU counts was observed when the cream or gel containing compound 7 was applied to mouse skin, indicating that compound 7 exerts an antimicrobial effect against Staphylococcus aureus. The efficacy of standard clinical treatment with Bactroban 2% cream was not significantly affected by the treatment regimen. The properties of the carrier, cream, or gel were not significantly affected.

[0256] In Experiment 2 Figure 4 In this study, compound 7 was applied at two different concentrations, as 1% or 2% gels. A placebo gel and the known antibacterial agent Bactroban (mupirocin) were used as controls. It was observed that the gel containing compound 7 was more effective than the placebo gel or Bactroban in reducing the number of CFUs from Streptococcus pyogenes CS 301 infection. The gel containing 2% compound 7 was more effective than the gel containing only 1% compound 7.

[0257] In Experiment 3 Figure 5 In this study, compound 8 was administered as a 2% cream formulation to a mouse skin infection model of Staphylococcus aureus FDA 486 infection. A placebo cream and two known antibacterial agents, fusidic acid (fusidic acid) ointment 2% and mupirocin (bactroban) cream 2%, were used as controls. It was observed that the cream containing compound 8 was more effective than the placebo and either fusidic acid or mupirocin in reducing CFU counts.

[0258] Example 8: Preparation of absorbable braided sutures with a coating containing AMC-109

[0259] The aim was to investigate the solution coating of typical biodegradable poly(lactide-co-glycoliide) polymers containing AMC-109 onto braided sutures. The work was divided into four aspects:

[0260] 1. Prepare a solvent (or solvent mixture) that allows the poly(lactide-co-glycolic acid) polymer and AMC-109 to dissolve.

[0261] 2. Coat the "naked" woven absorbable suture material with the solution from item 1 above.

[0262] 3. Investigate AMC-109 leakage in coated sutures.

[0263] 4. Investigate the microbiological effectiveness of the coated sutures.

[0264] Materials and methods:

[0265] polymer

[0266] RG502, poly(D,L-lactide-co-glycolic acid) Sigma-Aldrich No. 719889, is a 50:50 mixture of lactide and glycolide, ester-terminated, M w It has a pH of 7000-17000 and is a biodegradable polymer. It is similar to biodegradable polymers commonly used to coat commercial absorbable sutures. Its chemical structure is shown below:

[0267]

[0268] peptides

[0269] AMC-109 is referred to herein as "compound 2" and has the formula Arg-Tbt-Arg-NHCH2CH2Ph.

[0270] sutures

[0271] Syneture Surgilon 4-0 suture, a silicone-coated nylon braided suture, is used for research.

[0272] Preparation procedure of coated sutures

[0273] Before coating, the sutures are washed with ethyl acetate to remove most of the pre-existing coating.

[0274] Will RG502 was dissolved in ethyl acetate, and AMC-109 was dissolved in ethanol. In the first test (Test-01), 50 mg of RG502 was dissolved in 300 μl of ethyl acetate and mixed with 10 mg of AMC-109 dissolved in 50 μl of ethanol. In the second test (Test-02), 50 mg of RG502 was dissolved in 400 μl of ethyl acetate and mixed with 10 mg of AMC-109 dissolved in 110 μl of ethanol. The resulting solvent mixture was homogeneous.

[0275] The suture substrate (after washing with ethyl acetate) was coated by repeated immersion in a coating solvent mixture.

[0276] Microbiology

[0277] • Bacterial strain: Staphylococcus aureus

[0278] • Sutures used for counting (colony-forming units) CFU:

[0279] Surgilon 4-0 sutures with RG502 polymer, control.

[0280] °Surgilon 4-0 sutures with RG502 polymer / AMC-109

[0281] ° Sutures: Surgilon 4-0 (original), control

[0282] Trypsin-treated Soy Broth (TSB)

[0283] Mueller Hinton (MH) agar plates

[0284] Colonies of Staphylococcus aureus were picked from blood agar plates and a 0.5 McFarland solution (1×10⁻⁶) was prepared. 8 CFU), this solution is used for:

[0285] 1. Inoculate with MH agar plates

[0286] 2. Dilute in TSB to obtain 1×10 5 CFU

[0287] The sutures were placed on agar plates to observe the inhibition zone and inoculated into TSB to examine the antibacterial effect of coated and uncoated sutures.

[0288] The sample was incubated at 37°C for 16 hours.

[0289] Cultures from tubes inoculated with sutures in TSB were serially diluted for CFU assay. To investigate the long-term effects of the coated sutures, the sutures were rinsed and re-inoculated with bacteria from TSB at 37°C for 16 hours.

[0290] Three parallel experiments were conducted.

[0291] CFU was determined after colony counting.

[0292] result

[0293] Preparation of coated sutures

[0294] Coated sutures can be readily produced by treating the sutures with a solution of Resomer RG-502 and AMC-109. The composition of the solvent mixture is crucial for its ability to dissolve and mix Resomer RG-502 and AMC-109 (and avoid phase separation or precipitation).

[0295] Quantitative analysis of AMC-109 release levels

[0296] The coated sutures were extracted with water. The coated sutures were placed in 0.4 ml of water and allowed to stand for 30 minutes. The sutures were then removed, dried, and a second extraction was performed for 22 hours. The amount of AMC-109 released from the extract was analyzed. The results are as follows: Figure 6 As shown.

[0297] Antimicrobial efficacy test of AMC-109 coated sutures

[0298] The antimicrobial efficacy of sutures coated with Resomer RG-502 and AMC-109 was evaluated using an agar growth inhibition test and a liquid broth test.

[0299] Day 1:

[0300] Compared to untreated control sutures and sutures coated with RG-502 (without AMC-109), an inhibition zone was observed around sutures coated with AMC-109. Figure 7 ).

[0301] On day 1, no significant growth was observed in TSB medium in tubes containing AMC-109-coated sutures. CFU count dilutions were prepared to obtain AMC-109-coated sutures with counts of 0,500 and 3 × 10⁻⁶ CFU. 3 The CFU count was determined. Growth was observed in the control tubes, yielding a CFU count of 5.8 × 10⁻⁶. 8 and 4.7×10 8 (The numbers are the average of multiple parallel values) Figure 8 ).

[0302] Day 2:

[0303] On day 2, growth was visible in all tubes, and there was no visible difference between the Surgilon RG502 / AMC tubes and the control.

[0304] in conclusion

[0305] The sutures can be easily coated with a solution of biodegradable polymer and AMC-109. The resulting AMC-coated sutures exhibit antibacterial activity for up to 16 hours. A final reactant layer without AMC-109 can be added to reduce immediate diffusion and provide a more durable effect.

[0306] Example 9 - Sutures with AMC-109 / polymer coating

[0307] Materials and methods

[0308] Bacterial strains: Staphylococcus aureus ATCC29213 and Staphylococcus epidermidis RP42A

[0309] Sutures: Covered with polycaprolactone + 5% AMC-109 coating Excel polymer sutures (Johnson & Johnson). The polymer (i.e., polycaprolactone, average molecular weight (M...)) is used. W Approximately 14,000) and peptides were melted by rapid heating (over approximately 3 minutes) to 120°C in a glass vial, and mixed while the polycaprolactone was melting. The sutures were then dipped into the molten mixture to coat them.

[0310] Control: Uncoated Excel sutures

[0311] In one experiment, bacterial colonies were diluted to 0.5 McFarland and spread on Mueller Hinton agar plates. In another experiment, colonies were diluted to 0.5 McFarland and diluted 1:100 in trypsin-soy broth (TSB).

[0312] In the first experiment, AMC-coated sutures and uncoated controls were placed on inoculated plates. The plates were incubated at 37°C for 16 hours. In the second experiment, AMC-coated sutures and uncoated controls were placed in inoculation medium and incubated with shaking at 37°C for 16 hours.

[0313] result

[0314] Compared with the control, both Staphylococcus epidermidis and Staphylococcus aureus showed significant inhibition on agar plates around the AMC-109 coated suture lines. Figure 9 ).

[0315] Significant inhibition of bacterial growth was observed in wells 1 and 4. Figure 10 (This part already contains Ethibond sutures coated with AMC.)

[0316] The sutures were further stained with Syto 9 and propidium iodide and studied using a fluorescence microscope. A clear difference was observed between the coated and uncoated sutures; abundant bacterial growth was observed on the uncoated sutures.

[0317] Example 10: Absorbable sutures coated with AMC-109

[0318] The described method is scalable and suitable for industrial development.

[0319] Materials and methods:

[0320] sutures

[0321] •Coviden (Medtronic) Polysorbate 3-0

[0322] • Ethicon Vicryl plus 3-0 (containing triclosan, positive control)

[0323] Both types of sutures consist of Polyglactin 910 inner braided filaments, covered by a soft, lubricating outer layer of a mixture of poly(D,L-lactide-co-glycolic acid) (lactide:glycolic acid 65:35) and calcium stearate. The outer layer of the Vicryl plus suture also contains triclosan as an active ingredient.

[0324] polymer

[0325] Resomer (Evonik) RG-502, biodegradable poly(D,L-lactide-co-glycolic acid) (lactide:glycolic acid 50:50) M w 7000-17000, degradation time <3 months.

[0326] peptides

[0327] AMC-109 is as described above.

[0328] Suture peeling

[0329] The outer layer of the polysorbate suture was removed by washing the suture with ethyl acetate for 10 minutes, followed by washing with water for 2 minutes (partially). The suture was dried before coating.

[0330] Suture coating mixture

[0331] The coating mixture was prepared by dissolving RG-502 in ethyl acetate in one vial and AMC-109 in ethanol in a second vial. Mixing the two solutions produced a slightly turbid solution, which became clear upon the addition of 0.5 ml of ethyl acetate.

[0332] suture coating

[0333] Soak the polysorbate sutures (cut into 6cm pieces) in the coating mixture for 10 minutes and dry them, then soak them a second time in the freshly prepared coating mixture for 2 minutes.

[0334] Two batches of sutures were prepared. One batch was used for chemical extraction analysis and inhibition zone testing (Experiment 1), and the second batch was used for efficacy testing in liquid culture medium (Experiment 2).

[0335] Table 7. Mass and volume used in coating.

[0336]

[0337]

[0338] extract

[0339] Water extraction was performed on suture samples.

[0340] First extraction

[0341] Place the suture in a vial and add 1 ml of water. Let the sample stand for 1 hour. Analyze the extracted sample by HPLC.

[0342] Second extraction

[0343] The suture sample from the first extraction was placed in a new vial, water (1 ml) was added, and extraction was carried out for 3.5 h. The second extraction sample was analyzed by HPLC.

[0344] Microbiological evaluation

[0345] Bacterial strains:

[0346] Staphylococcus aureus (8325)

[0347] • Pseudomonas aeruginosa (PAO1)

[0348] Escherichia coli

[0349] Enterococcus faecalis

[0350] 0.5 McFarland (1×10⁻⁶) was prepared using overnight colonies of different bacterial strains in 0.5% NaCl. 8The solution was prepared at CFU / ml and further diluted to 10 with 2 ml LB (Lysogeny broth) in a tube. 5 CFU / ml. Bacterial solutions were used for inoculating agar plates for the growth zone inhibition test and for directly inoculating suture test samples in LB medium. All samples were incubated at 37°C for 18 hours. CFU counting was performed by preparing a series of dilutions from i) the solution of the inoculated test samples (LB medium) or ii) the solution of the samples with rinsing and vortexing (20 s) (1 ml sodium chloride, 0.5%). A series of dilutions were prepared in 1 ml NaCl (10... -1 -10 -6 ), 100 μl of each of the different dilutions was spread onto blood agar plates and incubated at 37°C for 18 hours.

[0351] result:

[0352] Quantitative analysis of AMC-109 release

[0353] Table 8. Amount of AMC-109 found in aqueous suture extract (μg / ml).

[0354]

[0355] *NQ indicates that the quantity cannot be quantified.

[0356] The sutures obtained after coating contained 0.4–0.5 mg of AMC-109. The concentrations of AMC-109 in the aqueous extracts are compiled in Table 8. The data show that, after a total extraction time of 4.5 hours, the high-load sutures released 25 μg of AMC-109 into the aqueous solution. Under similar conditions, the low-load sutures released 5 μg of AMC-109.

[0357] The AMC-109 levels indicate that at least 30% of the AMC-109 embedded in the high-load coating was released into the water within 4.5 hours. The release from the low-load suture appears to be slightly lower.

[0358] Microbiological assessment

[0359] Growth inhibition zones were consistently observed around polysorbate coated with 15% AMC-RG502. In contrast, Vicryl triclosan showed growth inhibition only against Staphylococcus aureus and Escherichia coli (Table 9).

[0360] Table 9: Inhibition Zones in Regional Inhibition Tests

[0361]

[0362] Direct inoculation of suture test material into bacterial suspension resulted in reduced bacterial growth of all strains of 15% AMC-109 coated sutures in LB medium. Triclosan-coated sutures inhibited only Staphylococcus aureus growth (Table 10).

[0363] Table 10. CFU of suture samples directly inoculated in LB medium.

[0364] Community Forming Unit (CFU)

[0365]

[0366]

[0367] in conclusion

[0368] AMC-109 can be incorporated into absorbable sutures using the aforementioned solution coating technique. The coating solution is turbid, indicating it is a supersaturated solution. This improves coating efficiency.

[0369] AMC-coated sutures release at least 30% of their AMC-109 content into an aqueous environment within 4.5 hours. The amount of AMC-109 released depends on the amount incorporated into the suture.

[0370] AMC-109 coated sutures provide anticolonization efficacy against Gram-positive and Gram-negative bacteria, including important pathogens ineffective against triclosan.

[0371] Example 11 Casting of a bioabsorbable film containing AMC-109

[0372] 11.1 Bioabsorbable polymers

[0373] Resomer L206S (poly(L-lactide)-terminated) (Sigma Aldrich 719854) is soluble in dichloromethane (DCM), and AMC-109 is soluble in chloroform.

[0374] Resomer RG502 (poly-D,L-lactide-co-glycolic acid) (Sigma Aldrich 719889) is soluble in tetrahydrofuran (THF), and AMC-109 is also soluble in THF.

[0375] 11.2 Preparation of bioabsorbable membranes

[0376] Casting solution

[0377] Resomer RG502 bioresorbable polymer material and AMC-109 were dissolved separately in THF in such a manner that the amount of AMC-109 was 5% compared to the amount of bioresorbable polymer in the final coating solution. The dissolution of AMC-109 in THF took several hours.

[0378] Resomer L206S bioabsorbable polymer material and AMC-109 were dissolved separately in dichloromethane and chloroform, respectively. The ratio of AMC-109 to L206S was the same as described above.

[0379] Casting process

[0380] Thin film samples are prepared by placing 8 ml of casting solution on an aluminum foil with shallow indentations, or by pouring the coating solution onto a petri dish. After drying (for several days), the cast film is mechanically detached from its surface.

[0381] 11.3 Determination of AMC-109 leakage in bioabsorbable membranes containing AMC-109

[0382] extract

[0383] Samples were cut from the cast film and accurately weighed (100-150 mg) to calculate the amount of AMC-109 in the sample. The sample was placed in a vial, water (2 ml) was added, and the vial was shaken. Six consecutive extractions were performed. Each extraction replaced the old extract with 2 ml of deionized water. Extraction was carried out with shaking periods of 10 s, 5 min, 30 min, 3 h, 22 h, and 48 h. The amount of AMC-109 in each extract was determined by UV spectrophotometry at 280 nm using a pre-prepared standard curve. The results are shown below. Figure 11 middle.

[0384] 11.4 Microbiological evaluation of bioresorbable membranes containing AMC-109

[0385] Bacterial strains:

[0386] Staphylococcus aureus 8325

[0387] Modified AATCC-100 method

[0388] Overnight colonies of Staphylococcus aureus were diluted to 0.5 McFarland in 0.9% NaCl to produce 1 × 10⁻⁶ cells / day. 8 The bacterial concentration of each bacterium was determined. The solution was further diluted in TSB to 1 × 10⁻⁶. 5 One bacterium.

[0389] Cut the L206S film material into slices approximately 0.4 × 0.4 cm. Then immerse the material in dH2O for 2 minutes and air dry before use. Use 50 μl of bacterial solution (1 × 10⁻⁶). 5 Inoculate the samples. Place the samples on a glass slide and incubate in a humidified chamber at 37°C for 24 hours. Prepare two biological copies of each experimental material.

[0390] After incubation, the film material was thoroughly washed for 2 minutes to remove AMC-109, which is readily extractable or present directly on the surface. It was then placed in 1000 μl of NaCl and vortexed for 45 seconds, followed by serial dilutions (0-10). -6 ) and spread 100 μl of the plate for CFU counting.

[0391] Microbiological efficacy

[0392] Settlement Formation Unit

[0393] The CFU count was below the detection limit for materials containing AMC. Compared with the control material, the CFU count decreased by 7 log, as shown in the table below.

[0394] Table. CFU values ​​of bioabsorbable membrane samples containing AMC-109.

[0395] TPU / polymer AMC-109 Comparison L206S 0 <![CDATA[8.7×10 7 ]]> RG502 * *

[0396] *The RG502 material disintegrates and adheres firmly to the slide surface during the 24-hour incubation period. The material cannot be recycled.

[0397] 11.5 Conclusion

[0398] Resomers can be dissolved in a number of solvents, with THF and dichloromethane being the most widely applicable solvents tested.

[0399] AMC-109 can be mixed into polymers dissolved in THF or chloroform. The solvent used to dissolve AMC-109 must be miscible with the solvent used to dissolve Resomers.

[0400] The resulting casting liquid can be applied to some surfaces or used to cast films.

[0401] • The cast film initially leaks AMC-109 rapidly, and based on the properties of the resomer, maintains a low concentration for at least two days.

[0402] The Resomer RG502 film exhibited leakage behavior of concern. The polymer disintegrated over time and continuously leaked AMC-109 over extended periods.

[0403] • Resomer L206S films do not disintegrate as rapidly as Resomer RG502 and can maintain activity for a longer period of time.

Claims

1. A formulation comprising a biodegradable polyester, said biodegradable polyester being formulated with a compound of formula (I). AA-AA-AA-XY (I) in, In any order, two of the AA (amino acid) moieties are cationic amino acids, and one of the AA moieties is an amino acid having a lipophilic R group having 14 to 27 non-hydrogen atoms; X is an N atom, which can be branched or unbranched C1-C atoms. 10 Alkyl or aryl group substitution, wherein the group may contain up to two heteroatoms selected from N, O, and S; and Y is selected from R1-R2-R3, R1-R2-R2-R3, R2-R2-R1-R3, R1-R3, and R4. in: R1 is C, O, S, or N. R2 is C; R1 and R2 can each be substituted with C1-C4 alkyl groups or not substituted; R3 is a group comprising one to three cyclic groups, each of which has five or six non-hydrogen atoms, two or more of which may be fused, and one or more of which may be substituted; R3 comprises a maximum of 15 non-hydrogen atoms; and R4 is an aliphatic moiety having 2 to 20 non-hydrogen atoms, said moiety being straight-chain, branched, or cyclic. The polyester is selected from polylactide, polyglycolic acid, polydioxanone, and polycaprolactone and their copolymers. Wherein, the compound of formula (I) is uniformly dispersed in the polyester, and Wherein, there is no covalent bond between the polyester and the compound of formula (I).

2. The formulation according to claim 1, wherein, The compound is a peptide.

3. The formulation according to claim 1 or 2, wherein, The cationic amino acid is arginine and / or lysine.

4. The formulation according to claim 1, wherein, The lipophilic R group comprises two or more fused or linked cyclic groups.

5. The formulation according to claim 1, wherein, The amino acid having a lipophilic R group is selected from tributyltryptophan (Tbt) or biphenylalanine derivatives, wherein the biphenylalanine derivative is selected from Phe(4-(2-naphthyl)), Phe(4-(1-naphthyl)), Bip(4-n-Bu), Bip(4-Ph) or Bip(4-T-Bu).

6. The formulation according to claim 1, wherein, The compound is a compound of formula (II). AA1-AA2-AA1-XY (II) in: AA1 is a cationic amino acid; AA2 is an amino acid having a lipophilic R group, said R group having 14 to 27 non-hydrogen atoms; and X and Y are as defined in claim 1.

7. The formulation according to claim 1, wherein, The compound has the following structural formula. 。 8. The formulation according to claim 1, wherein, The intrinsic viscosity of the polyester is 0.1 to 8 dL / g.

9. The formulation according to claim 1, wherein, The molecular weight of the polyester is between 3,000 and 30,000.

10. The formulation according to claim 1, wherein, The polyester is poly(D,L-lactide-co-glycoli), poly(L-lactide), or polycaprolactone.

11. A medical device comprising or consisting of the formulation of any one of claims 1 to 10.

12. The apparatus according to claim 11, wherein, The medical device includes the formulation as a coating thereon.

13. The apparatus according to claim 12, wherein, The medical device is a surgical fastener or implant.

14. The apparatus according to claim 13, wherein, The medical device in question is a suture.

15. A method for preparing an formulation according to any one of claims 1 to 10, the method comprising melting the biodegradable polyester mixed with a compound of formula (I).

16. A method for preparing an formulation according to any one of claims 1 to 10, the method comprising a compound of formula (I), a biodegradable polyester, and a mixture of one or more solvents, said solvents being capable of dissolving said compound and said polyester.

17. The method of claim 16, the method comprising: (i) providing a first solution comprising a compound of formula (I); (ii) providing a second solution comprising a biodegradable polyester, wherein the second solution is miscible with the first solution; and (iii) mixing the first solution and the second solution.

18. A method of producing a medical device according to any one of claims 11 to 14, the method comprising: (i) providing a formulation according to any one of claims 1 to 10; and (ii) applying the formulation to the medical device.

19. The method according to claim 18, wherein, The formulation is applied by immersing the device in the formulation or by coating the device with the formulation.

20. The method according to claim 18 or 19, wherein, The preparation is dried after being applied to the medical device.

21. Use of the formulation according to any one of claims 1 to 10 in the preparation of a medicament for therapeutic purposes.

22. The use according to claim 21, wherein the therapy is for treating or preventing infection in the subject.

23. The use according to claim 21, wherein the therapy comprises exposing a subject in need to a therapeutically effective amount of the preparation according to any one of claims 1 to 10.

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