WO2016135037A1 - Dianhydrohexitol codrug - Google Patents

Abstract

The present invention relates to a codrug of a dianhydrohexitol having one of the following structures: Formula (1a) Formula (1b) Formula (1c) wherein D1 and D2 are drug moieties that are the same or different and are covalently linked to the dianhydrohexitol via a physiologically hydrolyzable bond; R₁ and R₂ are selected from the group consisting of hydrogen, -SO₃M and a physiologically hydrolyzable chemical group, wherein M is a cation, and the physiologically hydrolyzable chemical group is selected from the group consisting of alkyl, alkenyl, alkylcarbonyl, alkenylearbonyl arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylether, alkenylether, arylether, and heteroarylether groups wherein the alkyl group or moiety consists of unsubstituted or substituted, straight-chain or branched-chain or cyclic alkyl groups having 1-22 carbon, atoms, wherein the alkenyl moiety consists of unsubstituted or substituted, straight-chain or branched-chain or cyclic alkenyl groups having 2-22 carbon atoms, wherein the aryl moiety consists of unsubstituted or substituted, phenyl, or phenalkyl groups wherein the alkyl moiety contains 1-3 carbon atoms and the phenyl moiety is unsubstituted or substituted, and the heteroaryl moiety is an aromatic 5- or 6-membered heterocyclic ring containing one or two heteroatoms selected from, the group consisting of nitrogen, oxygen, and sulphur. The invention also relates to the treatment of wounds and biofilm-associated inventions using these compounds,

Description

Dianhydrohexito) codrug

1. FIELD OF THE INVENTION

Dianhydrohexitol compounds according to Formula (1) are suitable for use in the treatment o wounds, for example to reduce scarring, and can be used to treat biofdm associated infections. This invention relates to codrugs of these compounds. 2. BACKGROUND OF THE INVENTION

Wound healing is a complex process that, when properly orchestrated, leads to reestablishment of tissue integrity with minimal residual scarring. Normal wound healing includes a transition from a proliferative phase, during which extracellular matrix (ECM) proteins are elaborated, to a remodeling phase, during which the wound is strengthened through stromal organization. Abnormal wound healing may result in pathologic scarring, which represents a diverse spectrum of disorders that range from unsightly scars, to keloids, to life-threatening systemic diseases such as scleroderma. One example of pathologic dermal scarring is hypertrophic scars that are an unfavorable outcome of burns, trauma, or surgery. Although much has been learned about the pathophysiology of pathologic dermal scarring, most treatment modalities lack a defined mechanism, of action, are nonspecific, and have limited efficacy. There is therefore a need for new compounds that can effectively treat wounds, particularly reduce scarring.

Oftentimes, healing in a wound will be delayed or prevented by the presence of a biofdm. A biofilm is a community of sessile, stably attached microorganisms, especially bacteria, enmeshed in an extracellular polymer matrix, generally a polysaccharide matrix, exhibiting growth properties that are distinguished from those of planktonic, free-living microorganisms. Antibiotic resistance of bacteria in biofilms has been extensively documented and bacterial bio films play a role in a number of disease settings, including the exacerbation of cystic fibrosis, chronic urinary tract infections, chronic sinus infections, ear infections, acne, dental caries, periodontitis, nosocomial infections, chronic wounds, infections due to medical devices such as catheters, ventilators, prosthetic heart valves, contact lenses, intrauterine devices and dental plaque (see, e.g., Potera (1999) Science 283(19)1837-1839; Costerton et al. (1999) Science 284(5418) 13 18- 22; Bjamsholt (2013) APMIS 121 (s 1 6)1 -58). Bacterial biofilms in chronic wounds are generally not resolved by the host's immune system, and these biofilms have an increased resistance to systemic and topical antimicrobial/antibiotic agents and so are very difficult to eliminate. There is a need for new compounds that can effectively treat biofilm-associated infections.

The inventors have discovered that certain dianhydrohexitol compounds are suitable for treating wounds, and that these compounds reduce scarring in a healing wound. The dianhydrohexitol compounds also inhibit or eradicate biofilms and are therefore also suitable for use in methods to treat biofilm-associated infections. These compounds are particularly suitable for treating wounds containing a bioiilm. This invention relates to codings of those dianhydrohexitol compounds.

3. SUMMARY OF THE INVENTION

The invention provides codrugs represented by any one of the Formulae (la), (lb) and

Formula (1 a) Formula ( lb) Formula ( lc) wherein

Di and Da are drug moieties that are the same or different and are covalently linked to the dianhydrohexitol via a physiologically hydrolyzable bond;

R_t and R₂ are selected from the group consisting of hydrogen, -SO₃M and a physiologically hydrolyzable chemical group,

wherein M is a cation, and the physiologically hydrolyzable chemical group is selected from the group consisting of alkyl, alkenyl, alkylcarbonyl. alkenylcarbonyl arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylether, alkenylether, arylether, and heteroarylether groups wherein the alkyl group or moiety consists of unsubstituted or substituted, straight-chain or branched-chain or cyclic alkyl groups having 1 -22 carbon atoms, wherein the alkenyl moiety consists of unsubstituted or substituted, straight-chain or branched-chain or cyclic alkenyl groups having 2-22 carbon atoms, wherein the aryl moiety consists of unsubstituted or substituted phenyl, or phenalkyl groups wherein the alkyl moiety contains 1-3 carbon atoms and the phenyl moiety is unsubstituted or substituted, and the heteroaryl moiety is an aromatic 5- or 6-membered heterocyclic ring containing one or two heteroatoras selected from the group consisting of nitrogen, oxygen, and sulphur. The invention also provides topical formulations and medical devices comprising these codrags. The invention, also relates to the use of the codrags or topical formulations in methods of treating wounds (e.g. reduce scarring) and/or biofilm-associated infections.

4. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic representation of an example of the procedure in the animal model, for scar analysis.

Figure 2 shows an example of a H&E image and the corresponding Sirius red image illustrating the measurements that are used for scar size analysis.

Figure 3 shows the results of the quantitative scar index analysis. Isosorbide is significantly different from Control and Vehicle (p<0.05). Figure 4 shows a graph demonstrating the scar prevention properties after ^'treatment of a wound with. 30 Μ isosorbide sodium disulphate as compare to the vehicle control.

Figure 5 shows the effects of the isomers isoidide and isomannide on the scar in fall thickness excisional wounds in mice.

Figure 6 shows that compound A competes for esterase activity in a dose-dependent manner. Figure 7 shows that compound B competes for esterase activity in a dose-dependent manner.

Figure 8 shows that wounds treated with Compound A (300 μΜ) showed a 40% reduction in. scar formation as compared to wounds treated with a glycerol control.

5, DETAILED DESCRIPTION OF THE INVENTION

5.1 General remarks As used, herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. That said, the terms "comprises", "comprising", "includes", "including", "has", "having" or any other variation thereof can also be understood to cover the disclosed embodiment having no further additional components (i.e. consisting of those components).

Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be non-restrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

Further, when an aspect of the invention is described as being 'preferred', it should be understood that this preferred aspect of the invention can be combined with other preferred aspects of the invention.

5.2 Dianhydroieiitol component of the cod rug

The inventors have discovered that dianhydrohexitol compounds according to Formula (1) are suitable for treating wounds and biofilm associated infections:

Formula (1 ) wherein R] and R_? may be the same or different and are selected from, the group consisting of hydrogen, -SO3M and a physiologically hydrolyzable chemical group, wherein M is a cation, and the physiologically hydrolyzable chemical group is selected from the group consisting of alkyl, alkenyl, alkylcarbonyl, alkenylcarbonyl arylearbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkyl ether, alkenyiether, arylether, and heteroarylether groups wherein the alkyl group or moiety consists of unsubstituted or substituted, straight-chain or branched-chain or cyclic alkyl groups having 1-22 carbon atoms, wherein the alkenyl moiety consists of unsubstituted or substituted, straight-chain or branched-chain or cyclic alkenyl groups having 2-22 carbon atoms, wherein the aryl moiety consists of unsubstituted or substituted phenyl, or phenalkyl groups wherein the alkyl moiety contains 1 -3 carbon atoms and the phenyl moiety is unsubstituted or substituted, and the heteroaryl moiety is an aromatic 5- or 6-membered heterocyclic ring containing one or two heteroatoms selected from the group consisting of nitrogen, oxygen, and sulphur. Evidence for the wound healing and biofilm inhibition and eradication is provided in the examples below. Reference is also made to the data in PCT/EP2014/06801 1 which is incorporated herein by reference.

The present invention is directed to codrugs of the compounds of Formula ( 1). A codmg is the association in a unique molecule of at least two drugs, preferably synergistic, attached to each other, one drug being the canier for the other and vice versa. The concept of a coding is known in the art. For instance, sulfasalazine is the classic example of a colon-specific codrag of 5-aminosalicylic acid and sulfapyridine. Other known codrugs include benorylate, which is an esterified product of paracetamol and acetylsalicylic acid, and sultamicillin, which is an ester of ampicillin and sulbactam. The coding of the present invention includes a dianhydrohexitol as a component thereof. The codrug of the present invention also includes one or more drags as a component thereof. The drug may be connected to the dianhydrohexitol by a physiologically hydrolysable bond (e.g. ether, ester, carbonate, carbamate) or it can be connected via a physiologically hydrolysable linking group L. That is, if the drug that is to form a component of the codrug of the invention contains a functional group (e.g. -COOH ) which together with the hydroxy! group on the dianhydrohexito] can form a physiologically hydrolysable bond (e.g. -C(=0)0-), then the drug may be connected directly to the dianhydrohexitol via that bond (in this example the drug is connected to the dianhydrohexitol via an ester bond). If the drag does not contain such a functional group then a physiologically hydrolysable linking group may be used to connect the drug to the dianhydrohexitol.

As used herein, the term "drug" means a therapeutically active ingredient and the term "linking group" refers to any means by which the drug can be covalently linked to the dianhydrohexitol, Essentially, the codrug of the present invention can be represented by any one of the

Formulae (l a), (lb) and ( lc):

Formula (1 a) Formula ( lb) Formula ( 1 c) wherein

Di and D₂ are drug moieties that are the same or different and are covalently linked to the dianhydrohexitol via a physiologically hydrolyzable bond;

R i and R₂ are selected from the group consisting of hydrogen, -SGjM and a physiologically hydrolyzable chemical group,

wherein M is a cation, and the physiologically hydrolyzable chemical group is selected from the group consisting of alkyl, alkenyl, alkylcarbonyl, alkenylcarbonyl arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxyearbonyl, heteroaryloxycarbonyl, alkylether, alkcnylether, arylether, and heteroarylether groups wherein the alkyl group or moiety consists of unsubstituted or substituted, straight-chain or branched-chain or cyclic alkyl groups having 1-22 carbon atoms, wherein the alkenyl moiety consists of unsubstituted or substituted, straight-chain or branched-chain or cyclic alkenyl groups having 2-22 carbon atoms, wherein the aryl moiety consists, of unsubstituted or substituted phenyl, or phenalkyl groups wherein the alkyl moiety contains 1 -3 carbon atoms and the phenyl moiety is unsubstituted or substituted, and the heteroaryl moiety is an aromatic 5- or 6-membered heterocyclic ring containing one or two heteroatoms selected from the group consisting of nitrogen, oxygen, and sulphur.

Exemplary alkyl groups for the alkyl, alkylcarbonyl, alkoxycarbonyl, and alkylether, of Formulae (1), (l a), (lb) and (l c) include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec- butyl, tert-butyl, hcxyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, as well as alkyl groups derived from fatty acids such valeric acid, isovaleric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecy!ic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, and arachidic acid. Exemplary alkenyl groups for the alkenyl, alkenylcarbonyl, and alkenylether of Formulae (1 ), ( l a), ( l b) and (lc) include ethylene, propylene, butylene, peiitylene, hexylene, and octylene, including those derived from fatty acids such as myristoleie acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, and arachidonic acid.

Suitable substituents, for all embodiments disclosed herein include: 3-12 member heterocycle. C6-C 10 aryl, 5-12 member heteroaryl. halogen (e.g. F, CI, Br), -N0₂; N¾, NR¾ -CN, -COR, - COOR, -CONR₂, -OH, -OR, -OCOR, -SR, -SOR, -S0₂R, -SONR_¾ -SG₂NR₂, wherein each R in the substituent is independently selected from H, CI -CIO alkyl, 3-10 member heterocycle, C6- C 10 aryl, 5- 12 member heteroaryl.

M may be any suitable cation including, but not limited to, a hydrogen atom, a metal atom, or an ammonium or an alkyl ammonium ion. M is preferably a metal cation such as a cation of an alkali metal or an alkaline earth metal. Preferred metal cations are those selected from the metals Ag, Zn. Al, Li, Na, K, Mg and Ca. Metal cations based on Ag or Z are useful because these metals can provide additional or complementary bactericidal or enzyme inhibiting properties. M may be a monovalent or polyvalent cation.

In preferred embodiments. R i and R; are selected from hydrogen, -SO₃M, or a hydrocarbon group selected from alkyl, alkenyl, alkylcarbonyl, and alkenylcarbonyl, wherein the alkyl group or moiety consists of nsubstituted or substituted, straight-chain or branched-ehain or cyclic aikyl groups having 1 -22 carbon atoms, and wherein the alkenyl moiety consists unsubstituted or substituted, straight-chain or branehed-chain or cyclic alkenyl groups having 2-22 carbon atoms. Preferred compounds according to Formulae (l a), (lb) and ( lc) are those where Ri and R₂ are selected from the group consisting of H, -SO3M, -R3 or -COR3, wherein R? is a C I to C22 hydrocarbon group. More preferred are those compounds where R₃ is a linear C I -CI 8 alkyl group, a branched C3-C18 alkyl group, a linear C2-C18 alkenyl group, or a branched C3-C 1 8 alkenyl group. Preferred embodiments of compounds according to Formulae (1 ), (la), (lb) and (lc) are those where Ri and R₂ are selected from H, ~CH¾ -C2H5, -COCH_3> -COC₂H_s, or -SO3M.

The compound according to Formulae (la), (lb) and (l c) may be any of the possible stereoisomers (e.g. cnantiomers or diastereoisomers). Thus, compounds according to Formulae (la), (lb) and (lc) having the same stereochemistry as isosorbide, isoidide or isomannide can be used in the present invention. Of these, compounds having the stereochemistry of isosorbide are most preferred. Thus, this disclosure should be taken to mean that, for all of the compounds according to Formulae (la), ( l b) and (l c), including all of the preferred embodiments described herein, the structure of the compound can have the same stereochemistry as isosorbide. It is to be understood that any disclosure made herein with respect to the compounds of Formulae (l a), (lb) and (lc), extends to the stereoisomers, tautomers, pharmaceutically acceptable salts, polymorphs, and. solvates tiiereof.

"Pharmaceutically acceptable salts", as used herein, are salts that retain the desired biological activity of the parent compound and do not impart undesired toxieological effects. Pharmaceutically acceptable salt forms include various crystalline polymorphs as well as the amorphous form of the different salts. The pharmaceutically acceptable salts can be formed with metal or organic counterfoils and include, but are not limited to, alkali metal salts such as sodium or potassium; alkaline earth metal salts such as magnesium or calcium; and ammonium or tetraalkyl ammonium salts. The salts can be organic or inorganic in nature. Representative salts include acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, 5 calcium edetate, camsylate, carbonate, chlori.de, clavulanate, citrate,

edetate, edisyiate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, ma!eate, mandelate, mesylate, methylbromide. methylnitrate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, Nil e) methyl glucamine, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, potassium, salicylate, sodium, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, trimethylammonium and valerate.

"Solvates", as used herein, are addition, complexes in which the compound is combined with an ! 5 acceptable co-solvent in some fixed proportion, Co-solvents include, but are not limited to, ethyl acetate, lauryl lactate, myristyl lactate, cetyl lactate, isopropyl myristate, methanol, ethanol, 1- propanol, isopropanol, 1 -butanol. isobutanol, tert-butanol, acetone, methyl ethyl ketone, acetonitrile, benzene, toluene, xylene(s), ethylene glycol, dichJoromethane, 1,2-dichloroethane, N-methylformamide, N,iV-dimethylformamide, N-methylacetamide, pyridine, dioxane, and 20 diethyl ether. The term 'hydrate' is employed when the co-solvent is water.

5.4 Drug D'

Drugs D] and D₂ (collectively referred to as drug D) may be the same or different and can be any 25 biologically active moiety that possesses a functional group capable of forming a physiologically hydrolysable covalent bond with a dianhydrohexitol moiety so as to provide a codnig according to Formulae (la), ( lb) or ( c). Exemplary functional groups on drug D include hydroxyl groups, amine groups, carboxylate groups (including carboxylic acids and esters), acid anhydride groups, thiol groups, sulfonyl halide groups, etc. Preferred functional groups are -OH, -N¾, -C(¾H, - 30 COX (where X is a halogen) and -CO2 groups where R is a C1-C6 alkyl. As used herein, a physiologically hydrolysable covalent bond is any covalent bond that hydrolyses in a physiologic environment. This includes bonds that are hydrolysed by enzymes that are present in the physiologic environment. Suitable physiologically hydrolysable covalent bonds include ether, ester, ketoester (i.e. a-ketoester, β-ketoester), carbamate, carbonate, phosphate esther, phosphate amine, phosphonic ester, phosphite ester, phosphonite. phosphinite, phosphorane thiocarbonate, dithiocarbonate, sulphite, sulfonate, sulfanate, sulfonate, silanol, silyl ether, hydroxylamaine, oxime, imino ether. Preferred physiologically hydrolysable covalent bonds include ether, ester, carbamate, and carbonate, When drug D contains two or more suitable functional groups then the skilled person can choose to link drag D to the dianhydrohexitol by any of the suitable groups. Which group is used may depend upon the lability of the resulting bond in the target physiological environments such as the precise pH and ionic strength, of the physiologic fluid, and the presence or absence of enzymes that may catalyze hydrolysis reactions in vivo. Thus, targeted release of the constituent drugs that make up the cod rug may be achieved by choosing a covalent bond that is selectively labile under the conditions of the target organ, e.g., a base-labile ester for release in a basic environment.

The covalent bond may be one that is cleaved enzymaticaliy. This is particularly useful for selectively delivering the component drugs of the coding to an area that contains such enzymes. For example, the environment of a healing wound tends to contain an increased amount of esterases. This physiological environment can be exploited by connecting drug D to the dianhydrohexitol via an ester bond. When delivered to a wound, the ester bond of the coding will be enzymaticaliy hydrolysed to selectively release the component drugs in the wound environment.

The biologically active nioiety that can be used for drug D is not limited provided it can be linked to the dianhydrohexitol by a physiologically hydrolysable covalent bond. Those skilled in the art can choose drug D according to the desired therapeutic indication to be treated and so will appreciate that an exhaustive list of all the biologically active moieties that can be chosen for Di and D₂ is not practical. Detailed lists of the biologically active moieties that can. be used as drug D can be found in, e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics (12th ed., McGraw-Hill Professional, 201 1 ; ISBN- 10: 0071624422), Remington: The Science and

Practice of Pharmacy (22nd ed., Pharmaceutical Press, 2012; ISBN-10: 0857110624), The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals (15th ed., Royal Society of Chemistry, 2013; ISBN-10: 1849736707), and other such volumes. Each of the aforementioned references is incorporated herein by reference. That is, rather than list each and every biologically active compound that is disclosed in the aforementioned references, it is to be understood that, for the present invention, drug D can be chosen from any of the biologically active compounds that are disclosed in the aforementioned references. Preferred compounds in these references are those having one or more functional groups selected from -OH, -NH₂, -C0₂H, -COX (where X is a halogen e.g. F, CI, Br) and -C0₂R where R is a C 1 -C6 alkyl. Also preferred are those compounds having a molecular weight in the range of from 100 to 10,000 preferably 150 to 1000. Also preferred are those compounds having from 6 to 1000 carbon atoms, preferably 9 to 100 carbon atoms. By way of example, Drag D may be chosen from the analgesic compounds; anti-inflammatory steroidal compounds (corticosteroids); non-steroidal anti-inflammatory compounds (NSAIDs); antibiotic compounds; anti-fiingal compounds; antiviral compounds; .antiproliferative compounds; immunomodulatory compounds; cell transport/mobility impeding compounds; cytokines; extracellular matrix modulating compounds, amino acids; peptides; and proteins.

Exemplary antiproliferative agents that may be used as drag D include anthracyclines, vincaalkaloids, purine analogs, pyrimidine analogs, inhibitors of pyrimidine biosynthesis, and/or alkylating agents. Antiproliferative compounds suitable as one or more constituent moieties in the present invention include: adriamycin, alitretinoin (9-cis-retinoic acid); amifostine; arabinosyl 5-azacytosine; arabinosyl cytosine; 6-azacytidine; 6-azauridine; azaribine; 6-azacytidine; 5-aza- 2'-deoxycytidine; bexarotene (4-[l -(5,6,7,8-tetraliydro-3,5,5,8,8-pentamethyl-2- naphthaIeny1)ethenyl]benzoic acid); bleomycin; capecitabine (5'-deoxy-5-fluoro-cytidine); chlorambucil; cladribine; cytarabine; cyclocytidine; daunombicin; 3 -deazaurirdine; 2'-deoxy-5- fluorouridine; 5'-deoxy-5-iluorouridine: docetaxel; doxorubicin; epirubicin; estramustine; etoposide; exemestane (6-methylenandrosta-l ,4-diene-3,17-dione); fludarabine; fludarabin phosphate; fluorocytosine; 5-fluorouracil (5FU); 5-fluorouridine; 5-{luoro-2'-deoxyuridine (FUDR); gemcitabine; hydroxyurea; idarubicin; irinotecan; melphalan; methotrexate; 6-

U mercaptopurine; mitoxantrone; paclitaxel; pentostatin; N-phosphonoacetyl-L-aspartic acid; prednimustine; pyrazoftirin; streptozocin; temozolomide; teniposide; 6-thioguaninc; tomudex; topotecan; 5-trifluoromethyl-2'-deoxyuridine; valrubicin (N-trifluoroacetyladriamycin-14- valerate); vinorelbine; other modified nucleotides and nucleosides, and salts of the foregoing. Preferred antiproliferative agents are paclitaxel, docetaxel, methotrexate, and SFU, etc., and salts and prodrugs thereof.

Suitable corticosteroids for use as drug D include: 21 -acetoxypregnenolone, aielometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone,clocortolone, cloprednoh eottieosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasoiie, diflorasone, diflucortolone, difuprednate, eno olone. fluazacort, flucloronide. flumethasone, flunisoMde, fluocinolone acetonide, fluocinonide, fraoeortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinoni.de, halobetasol propionate, halometasone, hydrocortisone, loteprednol etabonate, mazipredone, medrysorte, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylaminoacetate. prednisolone sodium, phosphate, prednisone, prednival, prednylidene, rimexolone, rofleponide, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, and triamcmolone hexacetonide, etc., and salts and prodrugs thereof.

Suitable antibiotics for use as drug D include, amoxicillin, ampicillin, amylpenicillin. apalcillin, azidocil!in, azlocillin, aztreonam, bacampicillin, benzylpenicilliriic acid, biapenem, cefaclor, cefadroxil, eefamandole, cefatrizine, cefazedone, cefazolin, cefbuperazone, cefeapene pivoxil, cefclidin, cefdinir, cefditoren, cefepime, cefetamet, cefixime, ceftnenoxime, cefrnetazole, cefminox, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefozopran, cefpimizole, cefpiramide, cefpirome, ccfpodoxime proxetil, cefprozil, cefroxadine, cefsolodin, ceftazidime, eefteram, ceftezole, ceftibvjten, ceftiofur, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephaeetrilic acid, cephalexin, cephaloglycin, cephaloridine, cephalosporin. C, cephalothin, cephamycins, cephapirinic acid, cephradine, ciprofloxacin, clometocillin, cloxacillin, cyclacillin, dicioxacillin, fenbenicillin, flomoxef, floxacillin, hetacillin, imipenem, lenampicillin, loracarbef. mcropeinem, metampicillin, moxalactam, nocardicins (e.g., norcardicin A), oxacillin, panipenem, penicillin, penicillin G, penicillin , penicillin O, penicillin. S, penicillin. V, phenethicillin, piperacillin, pivampicillin, pivcefalexin, propicillin, rifomycin, sulbenicillin, sulfamethoxazole, sultamicil!in, talainpicillin,. temocillin, ticarcillin, tigemonam, tobramycin, trimethoprim., xylitol, etc., and salts and prodrugs thereof.

NSAID compounds suitable as drug D include; acetaminophen, aspirin, choline magnesium tri salicylate, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketorolac, ketoprofen, meclofenamic acid, mefenamic acid, naproxen, nahumstone, nabumetone, oxaprozin, piroxicam, phenylbutazone, sulindac, and tolmetin, or prodrugs, salts, or active metabolites thereof. When the coding is a compound according to Formula (l c), then one aspect of the invention relates to compounds per se where D_t and D₂ are not aspirin, or salicylic acid.

Suitable analgesic compounds for use as drug D include: benzodiazepam, buprenorphine, butorphanol, codeine, desmorphine, dezocine, dihydromorphine, dimepbeptanol, eptazocine, ethylmorphine, fentanyl, glafenine, hyd.rom.orph.one, isoladoi, ketobenidone, p-Iactophetide, levorphanoL itdocaine. moptazinol, metazocin, meperidine, methadone, metopon, morphine, nalbuphine, nalmefene, nalorphine, naloxone, norlevorphanol, normorphine, oxycodone, oxymorphone, pentazocine, phenperidine, phenyl ramidol, propoxyphene, tramadol, and viminol, etc., and salts and prodrugs thereof.

Antiandrogen compounds suitable for use as drag D include luteinizing hormone-releasing hormone (LHRH) agonists or progestational agents, bicalutamide, bifluranol, cyproterone. flutamide, nil tamide, osaterone. oxendolone.

Anti-cholinergic compounds suitable for use as drug D include biperiden, procyclidin, trihexylphenidyl hydrochloride, atropine, ipratropium bromide, and oxitropium bromide, etc., and salts and prodrugs thereof. Adrenergic compounds suitable for use as drug D include acebutolol, atenolol, betaxolol, timolol, propanoic!, etc., and salts and prodrugs thereof. Local anesthetic compounds suitable as one of more constituent moieties in the present invention include ambucaine, benzocaine, butamben, procaine, oxybuprocaine, tetracaine, etc., and salts and prodrugs thereof. Exemplary extracellular matrix modulating compounds that may be used as drug D are inhibitors and regulators of MMP's, TIMP's, elastase, tryptase, chyraase, DPP IV, Gzm, plasmin, thrombin, MMP's, kallikreins, matriptase. cathepsins, aldehyde dehydrogenase, FAP. Also compounds like vitamin C can be considered here.

In a preferred embodiment of the invention, the codrug is a compound according to Formula (la) where D | is an antibiotic and R₂ is -SO3M or a compound according to Formula ( l b) where D₂ is an antibiotic and Rj is -SO₃M. Such a codrug, when hydrolysed, delivers an antibiotic drug and a dianhydrohexitol sulphate, both of which have antibiotic properties. If M is chosen to be Ag, then the codrag is capable of delivering three agents having bactericidal effect (the antibiotic; the dianhydrohexitol sulphate; and the Ag cation). Such an embodiment is particularly useful for treating wounds, including biofilm-associated infections in wounds, especially when the antibiotic drug is covalently linked to the dianhydrohexitol sulphate via an ester bond.

5.5 Preparation of the codrug

The codrugs of the invention may be synthesized by any manner known in the art for linking two drug molecules together. In general, the drug D moiety is condensed with the dianhydrohexitol moiety under conditions suitable for forming a linkage that is labile under physiologic conditions. In some cases it is necessary to block and/or protect some reactive groups on one, the other, or both of the moieties. If necessary, drug D can be fiinctionalized with a linker for attachment to the dianhydrohexitol. Where the drug D is to be covalently linked via a linker, it is advantageous to first condense either drag D or the dianhydrohexitol moiety with the linker. In some cases it is advantageous to perform the reaction in a suitable solvent, such as acetonitriie, in the presence of suitable catalysts, such as carbodiimides including EDCI ( 1 -ethyl-3-( 3-dimcthylaminopropyl)- carbodiimide) and DCC (DCC: dicyclohexylcarbodiimide), or under conditions suitable to drive off water of condensation or other reaction products (e.g., reflux), or a combination of two or more thereof. After the first pharmaceutically active moiety is condensed with the linker, the combined first moiety and linker may then be condensed with the second pharmaceutically active moiety, Again, in some cases it is advantageous to perform the reaction in a suitable solvent, such as acetonitrile, in the presence of suitable catalysts, such as carbodiimides including EDO and DCC, or under conditions suitable to drive off water of condensation or other reaction products (e.g., reflux), or a combination of two or more thereof. Where one or more active groups have been blocked, it may be advantageous to remove the blocking and/or protecting groups under selective conditions, however it may also be advantageous, where the hydrolysis product of the blocking and/or protecting group and. the blocked group is physiologically benign, to leave the active groups blocked. Such blocking and/or protecting of groups is well known in the art (see e.g. W, Green, P. G. M. Wuts, Protective Groups in Organic Synthesis, Wiley- interscience, New York, 1 99).

The person, having skill, in the art: will recognize that, while diacids, dialcohols, amino acids, etc., are described, as being suitable linkers, other linkers are contemplated as being within the present invention. For instance, while the hydrolysis product of a codrug according to the present invention may comprise a diacid, the actual reagent used to make the linkage may be, for example, an acylhalide such as succinyl chloride. The person having skill in the art will recognize that other possible acid, alcohol, amino, sulfato, and sulfamoyl derivatives may be used as reagents to make the corresponding linkage.

Exemplary reaction schemes according to the present invention are illustrated below for linking drug D with the dianhydrohexitol (Z). These schemes can be generalized by substituting other therapeutic agents for D, functionalised if necessary, or by using other synthetic coupling techniques.

Scheme 1 fester linkage)

D-COOH + HO-Z→ D-COO-Z In this case drug D (e.g. aspirin, lipoxin) is joined to the dianhydrohexitol by an ester linkage. For instance, referring to Formulae (l a), (lb) and (l c), D| and/or D₂ is an aspirin, moiety linked, to the dianhydrohexitol via an ester bond. Dianliydrohexitol esters are usually obtained by direct esterification of the dianhydrohexitol with the corresponding carboxylic acid in presence of a catalyst (US 8,258,325 B2, US 2012/01 16101 Al ; US 6,693,209 B2; WO 99/45060), Transesterification of dianhydrohexitol with the ester have also been reported (US 8,258,325 B2). Esterification and transesterification can. also be used to generate mono substituted dianhydrohexitol (US 2,983,228) as well as reaction starting from acid anhydride (Lavergne et al. RSC Adv. 2013, 3, 5997). Diester and monoester derivatives can also been, obtained by reaction of dianhydrohexitol with acid halide (Machut et al. Green Chem. 2010, 12, 772; Lavergne et al. RSC Adv. 2013, 3, 5997), and isosorbide diaspirinate can. be obtained by this synthetic route (Gilmer et al. Eur. J. Phar. Sci. 2002 16, 297). Sensitive carboxylic acid substrate that would degrade when subject to the reaction, condition, used in the protocols mentioned above can be attached to isosorbide under milder condition using, for instance carbodiitnide coupling chemistry. (Gella et al. Mol. Cryst. Liq. Cryst. 2014 591 , 34; EP 2 332 942 Al).

Scheme 2 (carbonate linkage)

D-O-CO-X + HO-Z→ D-G-COO-Z

In this case drug D (e.g. ascorbate; estradiol; tobramycin) is joined to the dianhydrohexitol by a carbonate linkage. For instance, referring to Formulae (l a), (lb) and (lc), Di and/or Dj is an ascorbate linked to the dianhydrohexitol via a carbonate bond. Dianhydrohexitol carbonate derivatives can be synthesized by reaction with lialoformate (Kircheldorf et. al. Macromol. 1996, 29, 8077; Saito et al. Macromol. 2005, 38, 6485; Yokoe et al. J. Pol. Sci. A 2003, 41, 2312).

Scheme 3 (ether linkage)

D-X + HO-Z→ D-O-Z

In this case drug D (e.g. kinin; borneol; genipin) is joined to the dianhydrohexitol by an ether linkage. For instance, referring to Formulae (l a), (lb) and (lc), D) and/'or D₂ is a kinin linked to the dianhydrohexitol via an ether bond. Dianhydrohexitol ether derivatives can be synthesized, for instance, via Williamson reaction (Achet et al., Biomass 1986, 9, 247; Chatti et al Tetrahedron 2000, 56, 5877; Zhu et al Green Chem., 2008, 10, 532; Zhu, et al, Langmuir 2009, 25, 13419), where in this case the alcohol moiety is first converted into and halide and then is it coupled to the dianhydrohexitol Scheme 4 (carbamate linkage)

D-NCO + HO-Z→ D-N-CO-O-Z

In this case drag D (e.g. ampicillin; tobramycin; trimethoprim; sulfamethoxazole) is joined to the dianhydrohexitol by a carbamate linkage. For instance, referring to Formulae (la), (lb) and (lc), Dj and/or D₂ is an ampicillin moiety linked to the dianhydrohexitol via a carbamate bond.

Dianhydrohexitol carbamate derivatives can be obtained, for example, by reaction with isocyanate (Dillon et al, Bioorg. Med. Chem. 2010, 18, 1045). Scheme 5 (phosphate linkage)

X-GP-(OD)₂ + HO-Z→ (DO)₂-PO-0-Z

In this case drug D (e.g. buciadesine) is joined to the dianhydrohexitol by a phosphate linkage. For instance, referring to Fomiulae (l a), (lb) and (lc), Dj and'or _? is buciadesine linked to the dianhydrohexitol via a phosphate bond, Dianhydrohexitol phosphate derivatives can be obtained by reaction with halophophorus (see e.g. US 2012/0252911 Al which describes synthesis of a variety of P bonds) or by nucleophilic substitution (EP 2 574 615 Al).

Scheme 6 (thiocarbonate linkage)

X-SC-OR + HO-Z→ ( O)₂-PO-0-Z

In this case drug D (e.g. boraeol; ascorbate) is joined to the dianhydrohexitol by a thiocarbonate linkage. For instance, referring to Formulae (la), (lb) and (l c), Di and/or Da is an alcohol moiety linked to the dianhydrohexitol via a thiocarbonate bond. Dianhydrohexitol thiocarbonate derivatives can be synthesized by reaction, with halothionoformate (Ghosh et ai. J. Med. Cliem. 1996, 39, 3278).

5.6 Preferred drugs and linkage

In a preferred embodiment of the invention, drug Di and/or drug D₂ in any one of Formulae la, lb, or l c is linked to the dianhydrohexitol via an ester linkage and is selected from the following list:

1 1 -hydroxy- 9(Z)-undecenoic acid; 14S,21 R-dihydroxydocosahexaenoic acid; azelaic acid monomethyl ester; Hyaluronic acid; Hyaluronic acid benzyl ester; amino acid;; Aspirinate; Butyrate; Caffeinate; caffeic acid phenethyl ester; 4,5-di-O-caffeoylquinic acid; Acteoside; Peru late; tetracosane- 1 ,24-diyl di[(Z)-ferulateJ; Ethyl ferulate; Hcmin: .lasmonic acid; Lineolate; Oleanolic acid; Oleate; osniarinate;; Salicylic acid; Salvianolate; 1 B Salvianolate; Tartarate; Tartaric acid diamide; Tranilast; Ursolic acid; Peptide coupled via the C terminal amino acid or via pendent COOH groups (Asp, Glu residue); Phycocyanin; Captopril; Enalapril; Lisinopril; PD123319; Valsartan; Cardesartan; Omapatrilat; Ketorolac; Eicosanoids; Eoxins; Leukotrienes; Lipoxins (e.g. Lipoxin A4); stabilized form of Lipoxin as disclosed by Colm Duffy, in his Doctoral Thesis at University College Dublin entitled "Hetcroaromatic Lipoxin A4 Analogues, Synthesis and Biological Evaluation"; Prostacyclins; Prostaglandines; Resolvins; Thromboxanes; Sivelestat (ONO-5046/ElaspoI); N-(o-(p-Pivaloyloxybenzenc)sulfony!aminobenzoyl)glycine, N- (2-(4-(2,2-Dimethylpropionyloxy)phenylsulfonylamino)benzoyl)aminoacetic acid; Diprotin A; Isosorbide; Isoidide; Isomaimide; Ac-IETD-CHO; AC-IEPD-CHO; Granzyme B Inhibitor II (CAS 1258003-96-1 ); -( u-Carbonyl-Arg-Val-Arg-al)-Phe; compounds from US 20140056964 and EP2648735 A2; 1,3,5 tri-substituted benzenes with COOH groups (Goswami et al, Bioorg Med Chem. 2014 Jun 15;22(12):3187-203); PG 1 1680 (O'Brien et al . J. Med. Cliem. 2000, 43, 156-166); Tanomastat; MMI-166; I-Arginine; L-NMMA; Retinoate; 13- cis retinoate; 13,14- Di hydro Retinoate; 18-hydroxy retinoate; 4 4-hydroxy retinoate; 5,6-epoxy-13-cis retinoate; 5,8- Epoxy- 13-cis-retinoate; 7 9-trans retinoate; Hirudin; Biva!irudin; Lepirudin; Argatroban; Melagatran; Dabigatran; 1 ,5-Dansyl-Glu-Gly-Arg Chloromethyl Ketone; GGACK; droxidopa; oritavancin; penicillin; ampicilline; ciprofloxacin; aztreonani; and vancomycin. In this embodiment, when the coding is a compound according to Formula l c, D)_. and D₂ are preferably the same.

In another preferred embodiment of the invention, drug Dj and/or drag D₂ in any one of Formulae la, l b, or lc is linked to the dianhydrohexitol via a carbamate linkage and is selected from the following list:

DALDA; Deferoxamine; peptide coupled via the N terminal amino acid or via pendent NH groups (Lys, Asp residues); 4-(2-Aminoethyl)benzenesulfonylfluoride; Lisinopril; Neomycin; Alogiiptin; Gemigliptin; Linagliptin; Saxagiiptin; Sitagliptin; Dipeptidylpeptidase IV Inhibitor; compounds from WO 2005/026123; Linagliptin; Acriflavin; 0-(3- carbamimidoylphenyl)-l-serine amides (Goswami et al, Bioorg Med Chem. Lett. 2015 Feb 1 ;25(3):616-20); ketothiazole inhibitors ( Han et al;, ACS Med Chem Lett. 2014 Oct 9;5(11): 1219-24); 1,3,5 tri-substituted benzenes with primary amine (Goswami et al, Bioorg Med Chem. 2014 Jim 15;22(12):3187-203); 1400W; l-Arginine; 1-NAME; L-NMMA; Hirudin; Bivalirudin; Lepiradin; Amiloride hydrochloride dehydrate; GGAC ; compounds from US 20120108494; ampicilline; tobramycin; trimethoprim; sulfamethoxazole; and oritavancin. In this embodiment, when the codrug is a compound according to Formula lc, Dj and D₂ are preferably the same. In another preferred embodiment of the invention, drag Di and/or drag D₂ in any one of

Formulae la, lb, or lc is linked to the dianhydrohexitol via a carbonate linkage and is selected from the following list:

Dapagliflozin; tedizolid; ascorbate; ascorbic acid-2-phosphate; ascorbic acid-2-sulphate,

Genistein, Rosmarinate, Methyl, rosmarinate, Troglitazone, Trehalose, 14S.21 R- dihydroxydocosahexaenoic acid; Alkannins; Cholesterolate; Curcumin; Eilagic acid; 3',4'- bis(0,0-methylene)ellagic acid; Emodin; Glycerol; Kinin; LFM-A13; M6P; Pimecrolimus;

Shikonins; Sucrose octasulphate; Peptide coupled via the OH pendent group (Ser, Thr, Tyr residues); Iridoids; Aucubin; Genipin; Isosorbide; Balafomycins; Metronidazole; Rapamycin;

Dipeptidylpeptidase IV Inhibitor III; Lupeol; Saxagiiptin; Vildagliptin; Elasnin; Lyngbyastatins

8-10 (PMID: 20098596); Sivelestat; N-Co-ip-

Pivaloyloxybenzene)sulfonylaminobenzoyl)glycine_t N-(2-(4-(2,2- dimethylpropionyloxy)phenylsulfonylammo)benzoyl)aminoacetic acid; compounds from WO 2005/026123 and WO 1988010258 Al ; Estradiol; Estriol; Estrone; Chemotin; Digoxin; Doxorubicin; bi-Flavonoids; Morelloflavone; Compounds from US 20140056964 Al ; Z-IETD- FMK; Z-Ile-Glu(0-ME)-Thr-Asp(0-Me); fluoromethy! ketone; Z-IE(OMe)TD(OMe)-FMK; Granzyme B Inhibitor ΪΙ (CAS 1258003-96-1 ): compounds from EP2648735 A2; AC-IEPD- CHO; Protopanaxatriol; a-Terpineol; Borneo!; Thymol; 1 ,3.5 tri-substituted benzenes with OH group (Goswami et al, Bioorg Med Chem. 2014 Jun 15;22(! 2):3187-203); ketothiazole inhibitors (Han et al;, ACS Med Chem Lett. 2014 Oct: 9;5(1 1 ): 1219-24 ); compounds from Whittaker et al., Chem. Rev. 1999, 99, 2735-2776; compounds from Brown, Medical Oncology 1997, 14, 1-10; Marimastat; doxyeyeline; compounds from WO 2008134712 A2; Rebimastat; 868368-30-3; rifomycin; tobramycin; xy!itol; and Metastat. In this embodiment, when the codrag is a compound according to Formula l c, D₍ and D₂ are preferably the same.

In another preferred embodiment of the invention, dmg Di and/or drug D₂ in any one of Formulae la, lb, or l c is linked to the dianhydrohexitol via a phosphate linkage and is selected from the following list: bucladesinc and M6P; 58231 1 -81 -7 (Steffcnsen et al, Crit Rev Oral Bio! Med. 2001 ;12(5);373-98). In. this embodiment, when the codrug is a compound according to Formula (lc), D] and D₂ are preferably the same. In another preferred embodiment of the invention, drug D_t and/or drug D₂ in any one of Formulae la, lb, or lc is linked to the dianhydrohexitol via a thiocarbonate or dithiocarbonate linkage and is selected from the following list: peptide coupled via pendent SH groups (Cys, Asp residues); Captopril; and Oiiiapatrilat In this embodiment, when the codrog is a compound according to Formula l c, Di and Da are preferably the same.

5.7 Uses of the codrug

The codings having Formulae (l a), ( lb) and (l c) are suitable for treating wounds and for reducing scarring in a healing wound since they hydrolyse under physiological conditions to provide a dianhydrohexitol suitable for treating wounds. The codrug compounds can also be used to inhibit or eradicate biofilms and are therefore also suitable for use in methods to treat biofilm- associated infections. The cod rugs are particularly suitable for treating wounds containing a biofilm, particularly chronic wounds.

The term "biofilm" is to be understood as having its ordinary meaning. A biofilm is a sessile community of microorganisms characterized by cells that are attached to a substratum or interface or to each other, that are embedded in a matrix of extracellular polymers (more specifically extracellular polymers that they have produced), and that exhibit an altered phenotype with respect to growth rate and gene transcription (for example as compared to their "non-biofilm" or free-floating or planktonic counterparts).

The biofilms that may be treated in accordance with the present invention are not limited in terms of the microorganisms that they contain. The biofilm may comprise any class, genus or speci es of microorganism, namely any microorganism that may form a biofilm. Such microorganisms typically include bacteria, including any genus or species of bacteria. Thus, the bacteria may be gram positive or gram negative, or gram test non-responsive. The bacteria may be aerobic or anaerobic. The bacteria may be pathogenic or non-pathogenic. It is particularly surprising that the molecules defined herein are able to kill bacteria in. biofilms and the treatment of such biofilms is particularly preferred. Biofilms comprising or consisting of Gram positive bacteria are preferred targets. Biofilms comprising Staphylococcus are preferred targets, with biofilms comprising S. haemolyticus, S. aureus, and/or S. epidermidis being especially preferred. Biofilms may also contain fungi, algae and other organisms such as parasitic protozoa in addition, to the bacteria. Such mixed colony biofilms are also treatable accordin to the methods described herein.

A "biofilm-associated infection" is a microbial infection of an object or a subject where it is known or suspected that the microbes are present as a biofilm. As used herein, a subject refers to a human or any non-human animal subject. Typically a biofi!m-assoeiated infection will be an infection where the existence of a biofilm is relevant to a particular clinical condition, e.g. to the diagnosis or prognosis, to the treatment regimen, to the severity of the infection, to the duration of the infection up to the point of treatment or anticipated in the future. A variety of bacterial infections associated with biofilm formation may be treated with methods and compounds of die invention, such as cystic fibrosis, otitis media, bacterial endocarditis, kidney stones, legionnaire's disease, gingivitis, urinary tract infections, pulmonary infections, dental plaque, dental caries and infections associated with surgical procedures or bums. For instance, the compounds of the present invention can be used to treat any of the biofilm-associated infections discussed by Bjarnsholt (2013) A.PM1S 121 (sl36)l-58, particularly Table 1 in Section 6.3 of that document and the references cited therein. Exemplary biofilm-associated infections that occur with the use of medical devices and that can also be treated with methods and compounds of the invention include catheter associated infections, contact lens associated infections, as well as biofilm infections associated with the use of other devices such as joint prostheses and heart valves.

The present invention is particularly suited, to treating biofilms in chronic wounds. These include, but are not limited to, venous ulcers, diabetic ulcers, and pressure ulcers. Venous ulcers, for example, occur mostly in the legs, as a result of poor circulation (e.g., ischemia), malfunctioning valves of veins, or repeated physical trauma (e.g., repetitive injury). Pressure ulcers may be present when local pressure that is exerted at or around a wound site is greater than blood pressure, for instance, such that poor circulation, paralysis, and/or bed sores may contribute to, or exacerbate, the chronic wound. Diabetic ulcers may occur in individuals with diabetes mellitus, for example, persons in whom uncontrolled high blood sugar can contribute to a loss of feeling in the extremities, leading to repetitive injuries and/or neglect on the part of the individual, to attend to injuries. Biofilms in chronic wounds that have resulted from acute wounds such as gunshot or shrapnel wounds, burns, punctures, or surgical wounds (e.g. postoperative wound infections) can also be targeted by compounds according to Formula (1). Biofilms can be treated in other wounds such as those arising from radiation poisoning, malignancies, dermal infections, gangrene, nonhealing surgical wounds, pyoderma gangrenosum, traumatic wounds, acute arterial insufficiency, necrotizing fasciitis, and osteomyelitis (bone infection).

"Treatment" of a biofilm includes prophylactic treatment and encompasses a reduction in size of the biofilm, a reduction in number of living microorganisms within the biofilm and prevention or reduction in the tendency of microorganisms within the biofilm to break free and form new biofilm colonies. The size, structure, integrity, and number of microbes in a biofilm can be analysed by any convenient method. For instance, scanning and transmission electronic microscopy is often, used to assess the size, Integrity and structure of a biofilm. When a subject is treated, treatment includes an improvement, observed by clinician or patient, in one or more of the symptoms associated with the infection. The biofilm to be treated may be present on a surface. The surface is not limited and includes any surface on which a microorganism may occur. The surface may be biotic or abiotic, and inanimate (or abiotic) surfaces include any such surface which may be exposed to microbial contact or contamination. When the surface is abiotic the linking group can be chosen to release the constituent drugs when acted upon by enzymes present in the biofilm. Thus particularly included are surfaces exposed to microbial contact or contamination include in particular any part of: food or drink processing, preparation, storage or dispensing machinery or equipment, air conditioning apparatus, industrial machinery, e.g. in chemical or biotechnological processing plants, storage tanks and medical or surgical equipment.

A biotic or animate surface may include any surface or interface in or on the human or animal body. It may be any internal or external body surface, including of any tissue, which may include haematological or haemotopoietic tissue (e.g. blood). Dead or dying (e.g. necrotic) or damaged (e.g. inflamed or disrupted or broken) tissue is particularly susceptible to biofilm growth and such tissue is encompassed by the term "animate" or "biotic". The surface may be a mucosal or non- mucosal surface. Representative biotic surfaces include, but are not limited to any surface in the oral cavity, e.g. teeth, gingiva, gingival crevice, periodontal pocket, reproductive tract (e.g.. cervix, litems, fallopian tubes), the peritoneum, middle ear, prostate, urinary tract, vascular intima, conjunctiva, corneal tissue, the respiratory tract, lung tissue (e.g. bronchial and alveolar), heart valves, gastrointestinal tract, skin, scalp, nails and the interior of wounds, particularly chronic wounds, which may be topical or internal wounds.

Medical or surgical equipment or devices represent a particular class of surface on which a biofilm may form. This may include any kind of line, including catheters (e.g. central venous and urinary catheters), prosthetic devices e.g., heart valves, vascular grafts, artificial joints, false teeth, dental crowns, dental caps and soft tissue implants (e.g. breast, buttock and lip implants), Any kind of implantable (or "in-dwelling") medical device is included (e.g. stents, intrauterine devices, pacemakers, intubation tubes, prostheses or prosthetic devices, lines or catheters). An "in-dwelling" medical device may include a device in which any part of it is contained within the body, i.e. the device may be wholly or partly in-dwelling. Medical or surgical equipment on which a biofilm has already formed can be treated in accordance with the invention. To prevent or hinder the formation of a biofilm in the first place, the medical or surgical equipment can comprise a compound according to Formula (1), for instance, on a part or the whole of a surface thereof. The present invention therefore also relates to medical devices comprising a compound according to Formulae (l a), ( l b) and ( 1 c). In addition to the surprising inhibitory and eradication effect that compounds according to

Formula (1) have on a biofilm, it has also been found that when compounds according to Formula (1) are applied to a wound, the amount of scar tissue formed after wound healing is significantly reduced. Thus, the present invention further relates to methods of treating wounds, methods of reducing scar formation, methods for regenerating normal tissue and methods for promoting the generation of normal tissue and/or skin structures. The present invention also relates to cosmetic methods of treating wounds and cosmetic methods of reducing scar formation.

A '"scar" in the sense of the present invention means normal scars, hypertrophic scars, keloid scars, contracture scars, atrophic scars and striae. Symptoms of scars include skin discolorations (including redness, changes in pigmentation, or other discolorations e.g. from blanching), erythema, dry, flaky, or itchy skin, raised area above the surrounding skin, keloid formation, hypertrophy, scar pain, decreased vascularization of the scar and/or surrounding tissue, reduced pliability, and poor aesthetic appearance (including quality and texture of the scar tissue). Thus, for the purpose of the present invention, the reduction of scarring can also be considered as the treatment (prevention or amelioration) of these symptoms.

Wounds to be treated may be acute or chronic. Acute wounds are wounds that proceed orderly through the three recognised stages of the healing process (i.e. the inflammatory stage, the proliferative stage and the remodelling phase) without a protracted time course. Chronic wounds, however, are those wounds that do not complete the ordered sequence of biochemical events because the wound has stalled in one of the healing stages. Viewed alternatively a chronic wound is a wound that has not healed within at least 30 days, preferably at least 40 days, more preferably at least 50 days, most preferably at least 60 days. The wound to be treated may be a breach in, or denudement of, the tissue for instance caused by surgical incision or trauma, e.g., mechanical, thermal, electrical, chemical or radiation trauma; a spontaneously forming lesion such as a skin ulcer (e.g. a venous, diabetic or pressure ulcer); a blister (e.g. a friction or thermal blister or a blister caused by pathogen infection such as chicken pox); an anal fissure or a mouth ulcer. For the purpose of treating wounds and reducing scar formation in accordance with the present invention, compounds according to Formulae (la), ( l b) and (l c) wherein Rt and R₂ are H or - SO3M are preferably used.

5,8 Formulations

Whether the codrag compounds according to Formulae (la), (lb) or (lc) are to be used for treating biofilms or wounds, the compounds can be used alone or as a formulation comprising the compound. For instance, when the codrug compounds according to Formulae (l a), (lb) or (lc) are used to treat wounds, such as to reduce scarring, or used to treat a biofilm on a biotic surface, the compounds can be formulated as a composition suitable for topical administration. "Topical administration" in the sense of the present invention means administration to a definite surface, e.g. directly to the biofilm or wound surface or the area around the biofilm or wound surface. Suitable topical administration forms include lotions, creams, gels, ointments, pastes, emulsions, foams, mousses, solutions, sprays, dispersions, aerosols, alginates, hydr gels, hydrocolloids, sticks, bars, and films. Reference is made to '"Remington: He Science and Practice of Pharmacy, 2 I^s1 Ed., Chapter 39 " and Ansel 's Pharmaceutical Dosage Forms and Drug Delivery Systems, 9^th Ed., Section IV -10 and Section Vf for further information regarding topical formulations. Preferred topical formulations are lotions, creams, gels, and ointments. A suitable dosage of the formulation can be determined by the skilled person depending on the constituent drugs and according to the desired treatment.

The topical formulations of the present invention typically contain 1 to 50,000 μΜ, preferably from 1000 to 10,000 μΜ of the codrug compound according to Formulae (l a), (lb) or (lc). Alternatively or additionally, the topical formulations of the invention contain from 0.0001 to 100 wt.%, preferably 0.0005 to 10 wt.%, more preferably 0.001 to 2 wt.%, even more preferably 0.01 to 0.1 wt.% of the codrag compound according to Formulae (l a), (lb) or (lc). If a mixture of two or more of the active compounds of Formulae (l a), (lb) or (lc) is present, the above ranges apply to the sum of the weight contents of those compounds. Exemplary, but non-limiting, dosages when applied to a surface (e.g. skin or wound surface) include an amount that allows effective administration of a codrug compound of Formulae (la), (l b) or (lc) in an amount of 0.1 to 1 00.000 rag/nr or preferably 10 to 75,000 mg/m², more preferably 100 to 60,000 mg/m² and even, more preferably 1000 to 50,000 mg/m². The compounds of Formulae (l a), (lb) or (lc) can also be administered to a biofilm or wound by injection and thus can be formulated as an injection solution.

The coding compounds of Formulae (la), (lb) or (lc) suitable for practising the invention can be used in simultaneous or sequential combination with one or more other active agents ('additional active, agent'). For example, when applied topically the cod rug compound can be applied before, together with, or after an additional active agent that is normally delivered onto or through a biotic surface for either a local or systemic effect. When applied simultaneously, the codrag compound of the invention according to Formulae (la), (lb) or (lc) and the additional active- agent may be present in the same topical formulation (e.g. in the form of a lotion, cream, gel, or ointment). The additional active agent includes antibiotics, analgesics, anesthetics, antiinflammatory agents (e.g., steroidal compounds such as dexamethasone, betamethasone, prednisone, prednisolone, triamcinolone, hydrocortisone, alclometasone, amcinonide, diflorasone, etc. as well as non-steroidal anti-inflammatories), anti-itch and irritation-reducing compounds (e.g., antihistamines such as diphenhydramine and psoriasis treatments), antimicrobial agents, antiseptic agents (e.g., povidone-iodine, methylbenzethonium chloride, etc.), immunomodulating agents, vitamins and the like.

The dosage may be delivered by a single administration or by multiple applications as needed to achieve the most effective results. Dosing can continue for as long as is medically indicated, which will depend on the severity of the biofilm or the wound. The dosage may also be delivered in a controlled-release maimer such as with an adhesive patch or suture.

It is preferable to treat a wound as quickly as possible in view of increased skin permeability immediately after wounding. Low molecular weight (< 500 Da) and moderately lipophilic agents (logP 1 -3) can surmount the horny barrier layer and gain access to viable epidermis, dermis and blood vessels to a low, yet relevant extent.

When treating acute wounds, it is preferable that the compounds according to Formulae ( l ), (lb) or (! c) (or a formulation comprising these compounds) be applied to the wound (or closed wound) within 1 day of the wound being formed. It is more preferable to apply the compounds within 6 hours of wound formation, even more preferable within 1 hour of wound formation.

For wounds created during surgery the compounds according to Formulae (la), (lb) or (lc) (or a formulation comprising these compounds) are preferably applied to the open wound and again on the closed wound on a regular basis for at least one week. An example of a regular basis is one, two or three separate applications per day. An alternative option is to apply the active agent by means of an adhesive patch or suture. The present invention further relates to an adhesive patch comprising at least one compound according to Formulae (l a), (lb) or (lc). Adhesive patches are a particularly effective means of delivering the active compounds to a biofilm or wound. The adhesive patch of the invention comprises a backing layer and an adhesive layer disposed on at least one side of the backing. The adhesive patch of the invention can be sterilised by appropriate methods such as but not limited to irradiation. When selecting a suitable sterilization method, it is advisable to test and confirm that the sterilization does not lead to degradation or other inactivation of the compound of Formulae (la), ( lb) or (lc).

The backing layer is preferably one which i substantially impermeable to components of the adhesive layer, such as a drug and additives, and prevents the components from passing through the backing and evaporating off from the back to cause a decrease in content. The backing layer may also provide odour control exudate control. Examples of such backings include films of polyurethanes, polyesters such as poly(ethylene terephthalate), nylons, Saran^{l M}, polyethylene, polypropylene, poly( vinyl chloride), ethylene/ethyl acrylate copolymers, polytetrafluoroethylene,

I^'M

Surlyn ^' , and metal foils and laminated films composed of two or more thereof. Moisture vapor permeability (MVTR) of the film is not particularly limited but may, if required, be adjusted to range from about 300 to 3000 g/m²/24h, preferably from about 500 to 2000 g/m²/24h, more preferably from about 700 to 1700 g/m²/24h as measured by the water method in ASTM E96. The adhesive layer is not particularly limited and examples of suitable adhesives include those based on acrylic polymers; rubber-based adhesives such as styrene/d i ene/styrene block copolymers (e.g., styrene/isoprene/styrene block copolymers and styrene butadi ene/styrene block

2? copolymers), pol isoprene. polyisobutylene, and polybutadiene; silicone type adhesives such as silicone rubbers, dimethylsiloxane-based polymers, and diphenylsiloxane-based polymers; vinyl ether type adhesives such as poly(vinyi methyl ether), polyvinyl ethyl ether), and poly(vinyl isobutyl ether); vinyl ester type adhesives such as vinyl acetate/ethylene copolymers; and polyester type adhesives produced from a carboxylic acid ingredient such as dimethyl terephthalate, dimethyl isophthalate, or dimethyl phthalate and a polyhydric alcohol ingredient such as ethylene glycol. The adhesive layer may be either a cross-linked adhesive layer or an uncross-linked adhesive layer. Preferably, the adhesive layer is a pressure-sensitive adhesive layer. From the standpoint of adhesion to the skin, pressure-sensitive adhesives are preferred and pressure-sensitive adhesive layers containing no water are preferred.

A release liner may also be disposed on the pressure-sensitive adhesive layer to improve ease of handling such as, for example, protect the adhesive layer and provide additional support (e.g. stiffness) before the patch is applied to a wound. The release liner is usually discarded before the adhesive patch is applied to the skin. It is therefore preferred, from the standpoint of inhibiting the adhesive patch from, giving an uncomfortable feeling during wear on the skin, that the backing should be softer than the release liner. The release liner is not particularly limited. Examples of the material thereof include materials which are known in this field. Suitable release liner materials may, for instance, include bases having a release layer of a plastic film or paper which surface is treated with a release agent such as a silicone, long-chain alkyl, fluorine, or molybdenum sulphide release agent; low-adhesive bases comprising fluorine-containing polymers; and low adhesive bases comprising non-polar polymers including olefmic resins such as polyethylenes and polypropylenes to improve release property from an adhesive layer. Specific examples thereof include films of plastics such as polyesters including polyethylene terephthalate), poly(vinyl chloride),, poly(vinylidene chloride), various acrylic and methacrylic polymers, polystyrene, polycarbonates, polyimides, cellulose acetate (acetate), regenerated cellulose (cellophane), and. celluloid and laminated films composed of wood-free paper, glassine paper, or the like and a polyolefin. From the standpoints of safety, profitability, and drug migration prevention, it is preferred to use a. polyester film,. A preferred option is a fluorinated and/or siliconized material. The thickness of the release liner is generally 10-200 pm, preferably 25-100 pm. It is preferred to use a release liner having maximum, peel force of less than 0,5 N/25mm when measured at a 180° peeling angle. Drug-containing adhesive patches are well known and examples of how to prepare them can be found, for example, in WO 2013/005760 Al, EP 2332585 A l . BP 2570122 Al , EP 2702984 A l , WO 201.2/014589 A l . WO 2009/1.24763 A2. WO 98/21578 Al , and the further references cited therein. Compounds according to Formulae (la), (lb) or (lc) can be employed in the patch in the skin-contact adhesive or in one or more additional layers in the patch (e.g.. in a drug reservoir). Suitable drug-delivery patches include gelled, or liquid reservoirs, such as in US 4,834,979, so- called "reservoir" patches; patches containing matrix reservoirs attached to the skin by an adjacent adhesive layer, such as in. US 6,004,578, so-called "matrix" patches; and. patches containing PSA reservoirs, such as in US 6,365, 178, US 6,024,976, US 4,751 , 087 and. US 6,149,935, so-called "drug-in-adhesive" patches.

In some embodiments, the drug reservoir can be provided in the form of a matrix layer containing drug, the matrix layer being adhered to the skin-contact adhesive of the patch. Such a matrix can be an adhesive layer and can include any of the adhesives described above. Alternatively, the matrix layer can be non-adhesive or weakly adhesive and rely upon a surrounding rim of skin- contact adhesive to secure the patch in place and keep the drug reservoir in contact with the skin surface.

Another topical administration form according to the present invention is a dressing with, an absorbing patch in. the middle (standard plaster type) where the compound according to Formulae (l a), (lb) or (lc) is provided in the absorbing patch or on the surface in a resorbable matrix, in the first case the material will be into the absorbing material and be released by either exudate or skin moisture. If needed a secondary separating membrane could be used to ensure the correct water transport. In the last embodiment the compounds are embedded, in a resorbable material such as but not limited to PLA, alginate, or CMC that will release the compound upon contact with, moisture. This film can have several architectures such as but not limited to a film, a porous film, a mesh, a fabric but it should allow the proper liquid transport. Underneath this film another potential absorbing material can be layered in order to capture the liquid such as blood, exudate, or sweat.

In some embodiments, the drag reservoir can be provided within the skin-contact adhesive of the patch. The drug can be mixed with the skin-contact adhesive prior to forming the patch or it may be applied to the skin-contact adhesive of the patch in. a separate process step. Examples of suitable methods for applying drug to an adhesive layer may be found in US 2003/054025 and

US 5,688,523. Commercially available materials that can be modified to deliver compounds according to Formulae (l a), (lb) or (lc) in the form of an adhesive patch include hydrocolloid dressing

T]Vf materials (e.g. DuoActive (Convatec), Comphile (Coloplast). Tegasorb (3M), Absocure (Nitto Medical Corporation), alginate dressing materials (e.g. Cartstat (Convatec), Sorb an (Alcare Co., Ltd), Algodam (Medion Inc.), Kurabio AG (Kuraray Co., Ltd) ), a hydrogen dressing materials (e.g. Jelleypalm (Taketora), New Jail (Johnson & Johnson), Intrasite (Smith & Nephew), Granugel (Convatec), Clearsite (Nippon Sigmax Co., Ltd)), polyuretfaane dressing materials (e.g. Tegaderm (3M), Opsite Wound (Smith & Nephew), 1V3000 (Smith & Nephew), Bioclusive (Johnson & Johnson)), hydropolymer dressing materials (e.g. Tieral (Johnson & Johnson)), and hydro fiber dressing materials.

By administering the compounds according to Formulae (la), (lb) or (lc) by means of an adhesive patch, one can ensure a more uniform application of the compound as compared to alternative topical formulations. Furthermore, the adhesive patch serves to protect the wound while it is healing. Preferably, the active compounds of the invention are formulated in the adhesive patch in an amount of 0.1 to 100,000 mg/irf or preferably 10 to 75,000 mg/mr, more preferably 100 to 60,000 mg/m² and even more preferably 1000 to 50,000 mg m².

In another embodiment of the invention, the cod rug according to Formula (1 ) can be used for target delivery of the drugs that make up the codrug. For example, a judicious choice of the group Rj or R-2 in Formula (l a) or (lb) will allow the hydrophobic or hydrophilic properties of the codmg to be tailored so that the codrug can be effectively delivered to the site of interest. As an example, a hydrophobic fatty acid chain can be chosen as R] or R₂ to impart hydrophobic properties to the eodrug. Alternatively, a hydrophilic moiety such as PEG could be chosen. Solutions of such codrugs can be manipulated to form micelles. This can have the advantage since in its pure form certain bulky codings may be difficult to formulate or deliver to a site of interest because of steric hindrance.

The present invention is now illustrated in greater detail by way of the following Examples, but it should be understood that the present invention is not deemed to be limited thereto.

6. Examples

6,1 Wound healing effect for compounds according to Formula (1)

Mouse models have contributed significantly to an understanding of skin biology and disease and so are an appropriate model for testing compounds for their wound healing effects. The mouse model used here can be considered one of the simplest and standard murine models for

investigating scar formation and wound healing.

Mice were weighed and then anaesthetized with 5% isoflurane in air for induction and then administered 2.5-3% of isoflurane in air for the remainder of the procedure. Four full-thickness skin wounds (Figure 1 ) were performed by cutting the folded skin on the back of the mice with a 7.5 mm x 3 mm elliptical biopsy punch (Elliptical Excisional Instrument, Acuderm Inc.). The skin on the back was raised and folded at the level of the vertebral column by using the thumb and the index finger. The folded skin was then placed on a wood tongue depressor with the animal laying on one side and two wounds were performed through the folded skin by using an elliptical biopsy punch (Elliptical Excisional Instrument, Acuderm Inc.), resulting in four wounds on the back skin: two on the right of the vertebral column and two on the left. The epidermis, the dermis and the fat tissue in the wound was carefully removed without touching other tissues. The wounds were then treated with a topical application of an ointment comprising compounds according to Formula (1 ) or a vehicle control. The treatment position of the wounds was randomised according to a list. The wound site was then covered with OP-Film (Somed

International, the Netherlands) and the borders fixed with cyanoacrylate glue (Loctite). The treatment was repeated at the day 1, 2, 3 and 4 post-surgery for a total of 5 treatments in the first 5 days of healing. At each time point the mice were briefly anaesthetized with isoflurane and a new dose of ointment was injected through the film onto the wound, after which the animal was left to recover. At day 5 all films were removed. The extent of scarring was measured in accordance with the procedure set out by Khorasani et al (201 1). Dermal thickness was defined as the distance from the epidermal-dermal junction down to the panniculus carnosus visualized by H&E stain. Four dermal thickness measurements were taken per sample— two adjacent to the wound site at 50 pm on either side, and two at a farther distance of 700 pm on either side of the wound. Fibrotic scar tissue was outlined using the freeform outline tool in ZEN to produce a pixel-based area measurement that could then be converted to square micrometers. Like the dermal thickness measurements, scar area

measurements were performed extended to the panniculus carnosus. Scar size is determined b the Scar Index, which is calculated by dividing the scar area (A, in pm²) by the corresponding average dermal thickness (T_avg, in pm):

Scar Index = A/T_avg

Figure 2 shows an example of a H&E image and the corresponding Sirius red image illustrating the measurements that are used for scar size analysis.

Histological analysis was performed and the scar index measured, A statistically significant decrease compared ( I -way ANOVA with Dunnet's comparison to Vehicle-treated group, p<0.05) in the scar index was observ ed when wounds were treated with isosorbide 30 μΜ (Composition 2), a scar index of 517.33 pm ± 247.92 pin was observed. As expected, a severe scar formation was observed in untreated and in vehicle-treated wounds where a high scar index corresponding to 1 152 pm ± 621.85 pm and 819 pm ± 285.89 pm, respectively was observed (Table 1 ;

Figure 3).

Table 1

The following saline-based compositions were prepared:

Composition 6 Saline solution (to act as control)

Composition 7 0.1 wt.% benzalkonium chloride (to act as positive control)

Composition 8 0.44 wt.% isosorbide

Composition 9 0.1 1 wt.% isosorbide disulfate (Ri and R₂ are -SO^Na)

Composition 10: 1.1 wt.% isosorbide disulfate (Ri and R₂ are -S(¾Na)

Compositions 6 to 10 were tested in accordance with the in vitro model described by Brackman et al. (J. Appl. Microbiol., 2013 ; 1 14(6): 1833-42) for determining biofUm inhibitory and eradicating activity of wound care products.

Essentially, S. aureus (Mu50) was seeded onto an artificial matrix and 1 ml of compositions 1 to 5 was added immediately thereafter. After 24 h incubation, the medium was removed, the samples were rinsed with saline, washed and then vortexed. The wash fluid was plated and the amount of colony forming units (CPUs) was determined. The tests were blinded to the researchers. The results are shown in Table 4.

Table 4

Compositions 7 to 10 provided a statistically significant (p<0.05) inhibitory effect on the amount of S. aureus CPUs present, Composition 8 comprising isosorbide was as effective as the positive control comprising benzalkonium chloride (Composition 7). Compositions 9 and 10 comprising isosorbide disulfate exhibited a greater inhibitory effect, with composition 10 effectively eradicating the bacteria. 6,3 Biofilm eradicating effect

The following saline-based compositions were prepared:

Composition 6: Saline solution (to act as control)

Composition 1 1 : 1 .0 wt.% benzalkonium chloride (to act as positive control)

Composition 12: 1 1 wt.% isosorbide disuSfate (Ri and R are -SOjNa) Compositions 6, 1 1 and 12 were tested in accordance with the in vitro model described by

Brackman et al. (2013) for determining biofilm inhibitory and eradicating activity of wound care products. Compositions 1 1 and 12 are effectively a 10x concentration of Compositions 7 and 10, respectively. These compositions are diluted 10-fold when added to the culture medium... Essentially, S. aureus (Mu50) was seeded onto an artificial matrix, and allowed, to grow for 24 h to form a biofilm. After 24 h the medium was removed, the biofilm was washed with saline, and 900 μΐ new medium was added. To this, 100 μ) of Compositions 6, 1 1 and 12 was added. After 24 h incubation, the medium was removed, the samples were rinsed with saline, washed and then vortexed. For quantification by plating, the sessile cells were removed by three cycles of vortexing (30 s) and sonication ( 30 s) and the number o S. aureus CPUs per biofilm was determined by plating the resulting suspensions. These tests were also blinded to the researchers and the results are shown in Table .5.

Table 5.

Compositions 1 1 and 12 provided, a statistically significant (p<0.05) biofilm eradicating effect. Composition 12 comprising isosorbide disulfate had a greater biofilm eradicating effect than the positive control comprising benzalkonium chloride (Composition 1 1 ). 6,4 Preparation and enzyme h drol ses of cod rugs

In the following examples, esterase activity was determined photometrically in PBS with 4- nitrophenyl acetate (l OmM dissolved in dimethyl sulfoxide) as substrate. The amount of 4- nitrophenol released was routinely determined at 405 nm at room temperature. One unit of esterase activity was defined as the amount of enzyme releasing 1 pmol of 4-nitrophenol per second under the assay condition. When the coding is liydrolysed by the enzyme, a decrease in nitrophenyl turnover can be expected (Yang et al, 201 1).

Isosorbide diacetylsalicylate (Compound A)

Compound A solution

1 mmol of powdered compound A was added to vials with 1 ml of acetone. The solution was diluted 100 times in PBS then 1 OmM solution was obtained. The solution was diluted in PBS containing 1% acetone to 5, 2.5 and 1.25 mM.

Saturated compound A solution

Excess cpantities of powdered compound A were added to vials and 1.5 ml of phosphate buffer saline pH 7.4 (PBS) was added. The suspension was vortexed and shaken for 10 hr at 37°C. The suspension was sampled and filtered through a 0.45 μιη PTFE membrane filter.

Enzyme activity evaluation

The enzyme activity was evaluated with 0.8 mM of 4-nitrophenyl acetate as substrate and 6.25 ng ml of esterase from porcine liver (Sigma- Aldrich) as enzyme on a UV-plate reader (Multiskan FC; Thermo Scienti fic) at 405 nm. This allowed the monitoring of 4-nitrophenol which absorbs strongly at this wavelength. As shown in Figure 6, compound A competed for the esterase activity and this competition inhibition was dose dependent and shows that the enzyme cleaves the ester bond of compound A and the reaction of hydrolysis of 4-nitrophenol acetate was slowed down.

Enzyme activity determination

The enzyme activity was determined with LC-MS (UFLCXR & LCMS2020; Shimadzu) to detect isosorbide and salicylic acid. Compound A was incubated in PBS containing 45 ng/ml of esterase from porcine liver (218 U/mg; Sigma-Aldrieh) or porcine ear skin extracts (esterase activity is about 960 nmol ml/s) for 12 hr at 37°C. 500 μΐ of solution was sampled and 500 μΙ_ of acetonitrile was added. The mixture was vortexed and left at 4°C for 1 Ohr. After that, the solution was filtered through, a 0.25 μ^ηι PTFE membrane filter and injected into LCMS. The results are- shown in the table below. Cleaved compounds isosorbide and salicylic acid, were detected in the sample treated by both enzyme while no isosorbide nor salicylic acid were detected from the solutions without enzyme.

The result of enzyme activity (Compound A)

6.4.2 Isosorbide diretinoate (Compound B)

Compound B solution

0.1 mmol of powdered compound B was added to vials with 1 ml of acetone. The solution was diluted 20 times in PBS then 2mM solution, was obtained. The solution was diluted in PBS containing 1% acetone to 1 , 0.5 and 0,25 niM.

Enzyme activity evaluation

The enzyme activity was evaluated with same method as compound A. However compound B absorbs at 405 nm wavelength as well so absorbance results were corrected by subtracting background noise. As shown in Figure 7, compound B also competed for the esterase activity and this competition inhibition was dose dependent.

6.4.3 Scar reducing ability of Compound A In the above enzyme activity test it was demonstrated that the cleaved compounds from.

Compound A (that is, isosorbide and salicylic acid), were detected in a sample treated by enzyme while isosorbide and salicylic acid were not detected in the solutions that did not contain enzyme. With this validated proof of concept, compound A was tested on the wounds of mice as described previously for compounds according to Formula (1). The results are shown in Figure 8 (ISA is Compound 8; GLY is glycerol control). Wounds treated with Compound A (300 μΜ) showed a 40% reduction in scar formation as compared to wounds treated with vehicle control thus demonstrating that codrags according to the invention are hydrolysed in vivo and are suitable for treating wounds. 7. Industrial applicability

The present invention provides methods, compounds and formulations that are useful in the treatment of wounds, in particular the reduction of scarring, as well as compounds useful for inhibiting and eradicating biofilm formation. Because of their dual action of treating wounds and inhibiting eradicating biofilm formation, the compounds of the present invention are particularly useful in methods to treat biofilm-associated infections in wounds, especially chronic wounds. The compounds of the present invention are also particularly useful for delivering additional therapeutic agents to a wound or biofilm in addition to the dianhydrohexitol.

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Claims

1.. A coding of a dianhydrohexitol having one of the following structures:

Formula (1 a) Formula (lb) Formula (ic) wherein

Di and D₂ are drug moieties that are the same or different and are covalently linked to the dianhydrohexitol via a physiologically hydrolyzable bond;

R| and R₂ are selected from the 'group consisting of hydrogen, -SO3M and a physiologically hydrolyzable chemical group,

wherein M is a cation, and the physiologically hydrolyzable chemical group is selected from the group consisting of alkyl, alkenyl, alkylcarbonyl, alkenylcarbonyl arylcarbonyl, heteroarylcarbonyl, alkoxyearbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylether, alkenyletlier, arylether, and heteroarylether groups wherein the alkyl group or moiety consists of unsubstituted or substituted, straight-chain or branched-chain or cyclic alkyl groups having 1-22 carbon atoms, wherein the alkenyl moiety consists of uiisuhstituted or substituted, straight-chain or branched-chain or cyclic alkenyl groups having 2-22 carbon atoms, wherein the aryi moiety consists of unsubstituted or substituted phenyl, or pheiialkyl groups wherein the alkyl moiety contains 1 -3 carbon atoms and the phenyl moiety is unsubstituted or substituted, and the heteroaryl moiety is an aromatic 5- or 6-membered heterocyclic ring containing one or two heteroatoms selected from the group consisting of nitrogen, oxygen, and sulphur,

2. A codrug according to claim 1, wherein Rj and R₂ are selected from the group consisting of H, -SO3M, -Rj or -COR3, wherein R> is a CI to C22 hydrocarbon group.

3. A codrag according to claim 1 or 2, wherein ¾ and R₂ are selected from the group consisting of H, -SC¾M, -R₃ or -COR3, wherein R₃ is a linear CI -CI 8 alkyl group, a branched C3-C18 alkyl group, a linear C2-C18 alkenyl group, or a branched C3-C18 alkenyl group.

4. A cod rug according to any one of claims 1 to 3, wherein R) and R2 are selected from H. - CH_3j -C2H₅, -COCH3, -COC₂H₅, or -SO₃M.

5. A cod rug according to any one of claims I to 4, wherein drug D| and/or drug D2 is covalently bound to the dianhydrohexitol via an ether, ester, carbamate, carbonate, phosphate, thiocarbonate, or dithiocarbonatc bond.

6. A codrag according to any one of claims 1 to 5, wherein drug D| and/or drug D₂ are selected from analgesic compounds, anti-inflammatory steroidal compounds, non-steroidal anti- inflammatory compounds, antibiotic compounds, anti-fungal compounds, antiviral compounds, anti-proliferative compounds, immunomodulatory compounds, cell transport/mobility impeding compounds, cytokines, extracellular matrix modulating compounds, amino acids, peptides, and proteins.

7. A codrag according to any one of claims 1 to 6, wherein drug Di and drug D₂ are the same.

8. A codrag according to any one of claims 1 to 7, wherein drug Di and drug D₂ are selected from antibiotics, aspirin, lipoxins, lupeol and retinoic acid.

9. A topical formulation or medical device, preferably an adhesive patch, comprising a codrag according to any one of claims 1 to 8.

10. A codrag according to any one of claims I to 8, or a topical formulation comprising codrug according to claim 9, for use in a method of treating a wound.

1 1. The coding or topical formulation for use according to claim 10, for use in a method of reducing scarring in a wound,

12. A codrag according to any one of claims 1 to 8, or a topical formulation, comprising a codrag according to claim 9, for use in a method of treating a biofilm-associated infection.

13. The codrag or topical formulation for use according to any one of claims 10 to 12, for use in a method of treating a biofilm-associated infection in a wound.

14. The coding or topical formulation for use according to any one of claims 10 to 13, for use in a method of treating a biofilm-associated infection in a chronic wound.

15. The codrag or topical formulation for use according to any one of claims 10 to 14, for use in a method of treating venous ulcers, diabetic ulcers, or pressure ulcers.