WO2023187197A1 - Conjugates of antimicrobial agents with a tocopheryl or tocotrienyl group - Google Patents

Conjugates of antimicrobial agents with a tocopheryl or tocotrienyl group Download PDF

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Publication number
WO2023187197A1
WO2023187197A1 PCT/EP2023/058567 EP2023058567W WO2023187197A1 WO 2023187197 A1 WO2023187197 A1 WO 2023187197A1 EP 2023058567 W EP2023058567 W EP 2023058567W WO 2023187197 A1 WO2023187197 A1 WO 2023187197A1
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conjugate
typically
group
moiety
formula
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PCT/EP2023/058567
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French (fr)
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Laeticia PINTO
Maria LAPTEVA
Verena SANTER
Yogeshvar KALIA
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Université De Genève
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions

Definitions

  • the present disclosure relates to prodrugs of antimicrobial agents, and particularly to conjugates of antimicrobial agents and tocopheryl groups.
  • the disclosure also relates to pharmaceutical formulations of such conjugates, to pharmaceutical compositions comprising such conjugates, and to such conjugates, formulations and compositions for use in medicine.
  • the conjugates, formulations and compositions find particular use in treating conditions such as acne, bacterial infection and fungal infections, for example by topical administration to a subject in need thereof.
  • Microbial infestation is a common cause of disease.
  • Pathogens such as bacteria and fungi are associated with many disease conditions and there is a pressing and ongoing need for new and/or improved therapies for treating such conditions.
  • Pathogens can infect many organs of the body. One such organ is the skin.
  • a wide number of skin disorders caused by microbial infection are known and present therapeutic targets for addressing with suitable agents.
  • Acne is the most common skin disease worldwide. Acne affects the pilosebaceous units of the skin, which consist of the sebaceous glands, sebaceous ducts, and their attached hair follicles. Acne (vulgaris) is a disease characterized by a clogging of the skin’s pilosebaceous units by the sebum produced by the sebaceous glands. Whilst there is still no clear explanation of the pathogenesis, acne has been associated with microbial infection e.g. by bacteria such as Cutibacterium acnes (previously known as Propionibacterium acnes). Accordingly, some treatments are known to be efficient such as antibiotics. Another condition associated with acne is rosacea. Like acne, the exact pathology of rosacea is unclear, but bacterial infection by bacteria such as P. acnes and others, including H. pylori, have been associated with rosacea.
  • Other skin disorders associated with microbial infection include cellulitis, erysipelas, microbial folliculitis, so-called “hot tub” folliculitis, furuncles, carbuncles, impetigo, erythrasma, tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, onychomycosis, etc.
  • Cellulitis affects the dermis and subcutaneous tissue and is associated with bacterial infection by bacteria such as Streptococcus and Staphylococcus, e.g Staphylococcus aureus. Cellulitis can be severe and lead to extreme discomfort. In extreme cases infections associated with cellulitis can spread to lymph nodes and the bloodstream and can be life-threatening.
  • Erysipelas affects the upper layers of the skin and leads to intense burning sensations. Erysipelas is associated with infection by bacteria such as Streptococcus and can particularly occur following a break in the skin or via secondary infection from bacteria associated with a primary nose or throat infection.
  • Microbial folliculitis may be caused by fungal or bacterial infection of the hair follicles in the skin. Severe cases of microbial folliculitis may lead to hair loss. “Hot tub” folliculitis is associated with infection by Pseudomonas bacteria, typically arising through contact with contaminated water sources such as whirlpools, hot tubs and jacuzzis. This condition may give rise to a painful rash which can in some cases be difficult to treat.
  • Furuncles are infections around a hair follicle. Unlike folliculitis, furuncles typically involve infection of entire pilosebaceous units. Furuncles can develop into abscesses which can be extremely severe. Carbuncles are clusters of furuncles and are often associated with severe bacterial infection. Such infection is typically associated with infection by bacteria such as Staphylococcus and may be associated with fever, weakness and exhaustion.
  • Impetigo is a bacterial infection of the top layer of the epidermis and is associated with bacterial infection by bacteria such as Streptococcus and Staphylococcus, e.g. Staphylococcus aureus. Impetigo can lead to severe sores and is highly contagious.
  • Erythrasma is a skin infection associated with bacterial infection by bacteria such as Corynebacterium minutissimum. It can lead to a painful rash.
  • Fungal skin infections are also common. Fungal skin infections include tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, and onychomycosis. Such conditions can be painful and in some cases severe. Given the prevalence of skin disorders associated with microbial infection, there is a pressing need for treatments.
  • WO 2012/177986 discloses nanoparticle based pro-drugs of antifungal and antibacterial conjugates with various carriers.
  • antimicrobial agents for example, in the form of a cream which can be rubbed into the skin.
  • antimicrobial agents comprised in such compositions are typically poorly selective and cause many side effects.
  • benzoyl peroxide is an anti-infective used topically in cases of mild and moderate acne for its antibacterial, keratolytic and comedolytic properties.
  • it is photosensitive and discolours the skin as well as hair and clothing of patients, as well as causing side effects such as irritation, skin dryness, and stinging.
  • Topical retinoids have been used, but have side effects such as skin irritation, pruritus, erythema and increased photosensitivity requiring limitation of sun exposure.
  • the side effects associated with conventional treatments for skin disorders associated with microbial infection are particularly problematic given that relatively high concentrations of such agents are typically required in order to deliver a suitable concentration into the site of infection, such as (for example) into the pilosebaceous units.
  • the non-specific administration of antimicrobial agents can give rise to development of microorganisms that are resistant to the administered agent. This can reduce the efficacy of future administrations of a particular agent in a given subject, and may in time contribute to the build-up of resistant microbes in a population.
  • the present inventors have recognised that some or all of the issues outlined above could be addressed, at least in part, by the improved delivery of antimicrobial agents to specific sites of infection.
  • the treatment of skin disorders including (but not limited to) acne could be improved by means to concentrate antimicrobial agents in the pilosebaceous units.
  • the inventors have surprisingly found that conjugating an antimicrobial agent to a tocopheryl or tocotrienyl group can allow the agent to concentrate in the pilosibaceous units.
  • this concentration may lead to multiple benefits.
  • the localised concentration of the agent in the pilosebaceous units means that the overall amount of the agent which needs to be administered to a subject in order to treat a given infection may be reduced relative to prior formulations of the agent. Side effects caused by the agent acting on areas other than the site of infection may consequently be mitigated.
  • by targeting the antimicrobial agent to the site of the infection development of bacterial resistance caused by repeated administration to other areas of the skin may also be reduced.
  • D is an antimicrobial agent
  • L is a linker
  • P is a polymeric spacer
  • X is a group selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, -S-, -S(O)-, -SO2-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, -NR 10 C(O)NR n -, -NR 10 C(O)O-, -OC(O)NR 10 , and a covalent bond;
  • Z is selected from Ci-12 alkylene, C2-12 alkenylene, and C2-12 alkynylene, wherein Z is unsubstituted or is substituted with 1, 2 or 3 substituents independently selected from halogen, -OR 10 , and -NR 10 R n ;
  • T is a tocopheryl or tocotrienyl group; and each R 10 and R 11 is independently selected from H and C1-2 alkyl.
  • D is an antibiotic agent, an antifungal agent, or a comedolytic retinoid, typically an antibiotic or antifungal agent.
  • D is an antibiotic or antifungal macrolide.
  • D is selected from erythromycin, adapalene, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline; and pharmaceutically acceptable salts thereof.
  • D is an antibiotic or antifungal macrolide.
  • D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline; and pharmaceutically acceptable salts thereof.
  • D is erythromycin or a pharmaceutically acceptable salt thereof or adapalene or a pharmaceutically acceptable salt thereof.
  • D is erythromycin or a pharmaceutically acceptable salt thereof.
  • P is a poly(ethylene glycol) (PEG), a poly(lactic acid) (PLA), a poly(lactic-co-glycolic acid) (PLGA), a polycaprolacton (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), a polyacrylamide (PAM), hyaluronic acid, heparin, polysialic acid, or a polypeptide.
  • P is poly(ethylene glycol) (PEG).
  • P has an average molecular weight of from about 100 to about 10,000 Da, more typically from about 500 to about 5000 Da, still more typically 1,000 Da.
  • X is -OC(O)-, -C(O)O- or -C(O)-.
  • T is vitamin E or a derivative thereof. More often, T is wherein the wavy line indicates the point of attachment to the moiety -OC(O)-Z-X-
  • L is -C(O)-L 1 -C(O)-; and antimicrobial agent D comprises a hydroxyl group which is esterified with the linker (L) at one of the -C(O)- moieties such that the moiety D-L- is a group of formula (Q) wherein the wavy line indicates the point of attachment to the moiety -P-X-Z-C(O)O-T; and L 1 is selected from Ci-i8 alkylene, C2-I8 alkenylene, and C2-I8 alkynylene, wherein L 1 is unsubstituted or is substituted with 1, 2 or 3 substituents selected from halogen, -OR 10 , and -NR 10 R n .
  • the conjugate is of Formula (II) wherein n is an integer from 1 to about 50; wherein often D is erythromycin and the conjugate is of Formula (III) In some embodiments, n is an integer from about 10 to about 30.
  • L 1 is unsubstituted Ci-12 alkylene. More typically L 1 is unsubstituted C2-7 alkylene.
  • Z is unsubstituted Ci-12 alkylene. More typically, Z is unsubstituted C1-4 alkylene.
  • L is -O-l - O)-; and antimicrobial agent D comprises a carboxylic acid group which is esterified with the linker (L) such that the moiety D-L- is a group of formula (QI) wherein - the wavy line indicates the point of attachment to the moiety -P-X-Z-C(O)O-T; and
  • L 1 is selected from Ci-18 alkylene, C2-I8 alkenylene, and C2-I8 alkynylene, wherein L 1 is unsubstituted or is substituted with 1, 2 or 3 substituents selected from halogen, -OR 10 , and -NR 10 R n .
  • the moiety X-Z forms one or more -O-CH2CH2 (PEG) units.
  • the conjugate comprises a moiety of Formula (VI)
  • D is adapalene
  • a pharmaceutical formulation for topical administration comprising a plurality of micelles in a vehicle, wherein said micelles each comprise a plurality of conjugates as described herein.
  • said micelles each comprise (i) a core comprising the tocopheryl or tocotrienyl groups of the conjugates and (ii) a solvent-accessible surface; and wherein the antimicrobial agent of the conjugates extends into the solvent from the solvent-accessible surface.
  • compositions comprising a conjugate as described herein and one or more pharmaceutically acceptable excipient, carrier and/or diluent.
  • the pharmaceutical composition is for topical administration.
  • conjugate as described herein, a pharmaceutical formulation as described herein, or a pharmaceutical composition as described herein, for use in treating a microbial skin infection in a subject in need thereof.
  • said conjugate, pharmaceutical formulation or pharmaceutical composition is for use in treating acne.
  • the microbial skin infection is a bacterial infection caused by bacteria of genus Cutibacterium (e.g. Cutibacterium acnes), Streptococcus (e.g. Streptococcus pyogenes), Staphylococcus (e.g. Staphylococcus aureus), Pseudomonas (e.g. Pseudomonas aeruginosa) or Corynebacterium (e.g. Corynebacterium minutissimum).
  • Cutibacterium e.g. Cutibacterium acnes
  • Streptococcus e.g. Streptococcus pyogenes
  • Staphylococcus e.g. Staphylococcus aureus
  • Pseudomonas e.g. Pseudomonas aeruginosa
  • Corynebacterium e.g. Corynebacterium minutissimum
  • said conjugate, pharmaceutical formulation or pharmaceutical composition is for use by topically administering said conjugate, pharmaceutical formulation or pharmaceutical composition to said subject.
  • Figure 7 Size distribution by number of the micelles of TPGS (A) and the micelles of compound 2 (B) by Zetasizer. Results described in the examples.
  • Figure 9 TEM images of (A) TPGS micelles at a magnification of 50’000; and the prodrug (compound 2) micelles at a magnification of 50'000 (B) and 100'000 (C).
  • the scale bar is 100 nm in (A) and 50 nm in (B) and (C). Results described in the examples.
  • Figure 17 Summary of the stability of compound 2 in contact with porcine liver esterase and skin extract. Results described in the examples.
  • FIG. 19 TEM images (50,000 x magnification) of (A) TPGS micelles; and the prodrug (compound 3) micelles (B; and zoomed in (C)).
  • the scale bar is 50 nm in (A) and (B) and 10 nm (C). Results described in the examples.
  • Figure 20 Release of erythromycin (A) and azelaic acid (B) from compound 3 administered to porcine skin samples comprising and excluding PSUs.
  • the left hand bar shows results for skin samples comprising PSUs and the right hand bar shows results for skin samples excluding PSUs. Results described in the examples.
  • FIG 21 Thin-layer chromatography (TLC) monitoring of the reaction progress of Example 3 (formation of an adapalene-copolymer conjugate), TLC taken after 20 hours.
  • Figure 22 1H NMR spectrum of adapalene (ADA) in CDCL3.
  • Figure 24 1H NMR spectrum of ADA-mPEG-PLGA conjugate in DMSO.
  • Figure 25 P31 NMR spectrum of ADA-mPEG-PLGA conjugate in DMSO.
  • Figure 26 UHPLC-PDA chromatogram showing ADA retention time.
  • Figure 27 UHPLC-PDA chromatogram showing generation of ADA and ADA-copolymer conjugate (new product) after saponification reaction.
  • Figure 28 Characterization of the micelle formulation for size distribution using DLS (size in nm).
  • a polynucleotide includes two or more polynucleotides
  • reference to “a polynucleotide-handling protein” includes two or more such proteins
  • reference to “a helicase” includes two or more helicases
  • reference to “a monomer” refers to two or more monomers
  • reference to “a pore” includes two or more pores and the like.
  • a Ci-20 alkyl group is a linear or branched alkyl group containing from 1 to 20 carbon atoms.
  • a Ci-20 alkyl group is often a Ci-18 alkyl group, or a C10-20 alkyl group.
  • a C10-20 alkyl group is often a C14-I8 alkyl group such as a Ci6 alkyl group.
  • a Ci-18 alkyl group is often a Ci-12 alkyl group such as a C1-9 alkyl group, e.g. a C1-7 alkyl group.
  • a C1-7 alkyl group may be C1-6 alkyl group such as a C1-4 alkyl group.
  • a C1-2 alkyl group is methyl or ethyl, typically methyl.
  • the alkyl groups may be the same or different.
  • a C2-20 alkenyl group is a linear or branched alkenyl group containing from 2 to 20 carbon atoms and having one or more, e.g. one or two or three, typically one double bonds.
  • a C2-20 alkenyl group is often a C2-I8 alkenyl group, or a C10-20 alkenyl group.
  • a C10-20 alkenyl group is often a C14-I8 alkenyl group such as a Ci6 alkenyl group.
  • a C2-I8 alkenyl group is often a C2-12 alkenyl group such as a C2-9 alkenyl group, e.g. a C2-7 alkenyl group.
  • a C2-7 alkenyl group may be C2-6 alkenyl group such as a C2-4 alkenyl group. Examples include ethenyl, propenyl and butenyl. For the avoidance of doubt, where two alkenyl groups are present, the alkenyl groups may be the same or different.
  • a C2-20 alkynyl group is a linear or branched alkynyl group containing from 2 to 20 carbon atoms and having one or more, e.g. one or two or three, typically one triple bonds.
  • a C2-20 alkynyl group is often a C2-I8 alkynyl group, or a C10-20 alkynyl group.
  • a C10-20 alkynyl group is often a C14-I8 alkynyl group such as a Ci6 alkynyl group.
  • a C2-I8 alkynyl group is often a C2-12 alkynyl group such as a C2-9 alkynyl group, e.g.
  • a C2-7 alkynyl group may be C2-6 alkynyl group such as a C2-4 alkynyl group. Examples include ethynyl, propynyl and butynyl. For the avoidance of doubt, where two alkynyl groups are present, the alkynyl groups may be the same or different.
  • a Ci-20 alkylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from a Ci-20 alkane. The two hydrogen atoms may be removed from the same carbon atom or from different carbon atoms.
  • a C1-20 alkylene group is a Ci-18 alkylene group, such as a Ci-12 alkylene group e.g. a C1-9 alkylene group.
  • a C1-9 alkylene group is a C1-7 or C1-6 alkylene group. Examples include methylene, ethylene, n-propylene, iso-propylene, n-butylene, sec -butylene and tertbutylene.
  • the alkylene groups may be the same or different.
  • a C2-20 alkenylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from a C2-20 alkene. The two hydrogen atoms may be removed from the same carbon atom or from different carbon atoms.
  • a C2-20 alkenylene group is a C2-I8 alkenylene group, such as a C2-12 alkenylene group e.g. a C2-9 alkenylene group.
  • a C2-9 alkenylene group is a C2-7 or C2-6 alkenylene group.
  • alkenylene groups examples include ethenylene, n-propenylene, iso-propenylene, n-butenylene, sec- butenylene and tert-butenylene.
  • alkenylene groups may be the same or different.
  • a C2-20 alkynylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from a C2-20 alkene. The two hydrogen atoms may be removed from the same carbon atom or from different carbon atoms.
  • a C2-20 alkynylene group is a C2-I8 alkynylene group, such as a C2-12 alkynylene group e.g. a C2-9 alkynylene group.
  • a C2-9 alkynylene group is a C2-7 or C2-6 alkynylene group.
  • alkynylene groups examples include ethynylene, n-propynylene, iso-prop ynylene, n-butynylene, sec- butynylene and tert-butynylene.
  • the alkynylene groups may be the same or different.
  • alkyl, alkenyl, alkynyl, alkylene, alkenylene or alkynylene group as used herein may be unsubstituted or substituted.
  • substituted alkyl, alkenyl or alkynyl groups typically carry one or more, e.g. 1, 2, 3 or 4, such as one, two or three e.g. one, or two, e.g. one substituent selected from halogen, -OR 10 , and -NR 10 R n , wherein R 10 and R 11 are as defined herein.
  • the substituents on a substituted alkyl, alkenyl or alkynyl group are typically themselves unsubstituted unless otherwise stated. Where more than one substituent is present, these may be the same or different.
  • a halogen is typically chlorine, fluorine, bromine or iodine and is typically chlorine, bromine or fluorine, especially chorine or fluorine, especially fluorine.
  • a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base.
  • Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as oxalic, citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p- toluenesulphonic acid.
  • Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g.
  • hydrochloride salts and acetate salts are preferred, in particular hydrochloride salts.
  • the stereochemistry is not limited.
  • compounds of Formula (I) containing one or more chiral centre may be used in enantiomerically or diastereoisomerically pure form, or in the form of a mixture of isomers. Further, for the avoidance of doubt, the compounds of the invention may be used in any tautomeric form.
  • the agent or substance described herein contains at least 50%, typically at least 60, 75%, 90% or 95% of a compound according to Formula (I) which is enantiomerically or diasteriomerically pure.
  • a compound of the invention comprises by weight at least 60%, such as at least 75%, 90%, or 95% of a single enantiomer or diastereomer.
  • the compound is substantially optically pure.
  • D is an antimicrobial agent.
  • D is a hydrophilic agent.
  • D is an antibiotic agent, an antifungal agent, or a comedolytic retinoid.
  • D is an antibiotic or antifungal agent.
  • D is an antibiotic or antifungal macrolide.
  • macrocylic lactone ring e.g. a 14-, 15- or 16-membered ring, to which one or more deoxy sugars, such as cladinose or desosamine, may be attached.
  • D is not limited to such compounds and other antimicrobial agents may be used.
  • D is an antibiotic agent.
  • D is selected from erythromycin, adapalene and derivatives thereof, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline; and pharmaceutically acceptable salts thereof.
  • D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline; and pharmaceutically acceptable salts thereof.
  • the structures of these compounds are shown below.
  • D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; and pharmaceutically acceptable salts thereof.
  • D is selected from erythromycin, clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, minocycline, and sarecycline and pharmaceutically acceptable salts thereof.
  • D is erythromycin or a pharmaceutically acceptable salt thereof, typically erythromycin.
  • D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline, and pharmaceutically acceptable salts thereof, D is typically attached to the moiety -L-P-X-Z-C(O)O-T of formula (I) by a covalent bond to the L moiety.
  • D comprises a compound of formula (IV) or formula (V) as described herein, or pharmaceutically acceptable salts thereof
  • D is typically attached to the moiety -L-P-X-Z-C(O)O-T of formula (I) by a covalent bond to the L moiety.
  • the bond between D and L is typically formed by removing a hydrogen atom from a suitable position on D and forming a covalent bond to a suitable atom on L.
  • the hydrogen atom that is removed may be a labile hydrogen atom e.g. at a side chain.
  • a hydrogen atom may be removed from an -OH group of D such that D is attached to L by an O-L bond thereby forming a moiety of form D-O-L-.
  • a hydrogen atom may be removed from an -NH2 group of D such that D is attached to L by an NH-L bond thereby forming a moiety of form D-NH-L-.
  • a hydrogen atom may be removed from a - C(0)-CH3 group of D such that D is attached to L by an CH2-L bond thereby forming a moiety of form D-C(0)-CH2-L-.
  • a hydrogen atom may be removed from a -C(O)-OH group of D such that D is attached to L by an O-L bond thereby forming a moiety of form D-C(O)-O-L-.
  • D is a macrolide comprising a macrocylic ring and one or more sugar rings and is attached to L by a bond to an -OH group on D
  • the -OH group is typically on a sugar ring.
  • D is erythromycin
  • D is typically attached to L by a bond to an -OH group on D at one of the positions indicated by an asterisk (*) in the structure below:
  • D is a comedolytic retinoid.
  • D may be adapalene or a derivative thereof.
  • D is or comprises a compound of formula (IV) or a pharmaceutically acceptable salt thereof: wherein
  • R 1 is selected from H, C1-4 alkyl which is unsubstituted or is substituted with one or more substituents selected from -OH, -CN, -NH2 or halogen; and C2-4 alkenyl;
  • R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 13 may be the same or different and are each independently selected from H, C1-4 alkyl which is unsubstituted or is substituted with one or more substituents selected from -OH, -CN, -NH2 or halogen; halogen, and -OR 12 ; and - R 14 is selected from H; C1-4 alkyl which is unsubstituted or is substituted with one or more substituents selected from -OH, -CN, -NH2 and halogen; C2-4 alkenyl, and -OR 12 ; each R 12 is independently selected from H and methyl.
  • R 1 is selected from H, methyl or ethyl, most typically H or methyl, and more preferably methyl.
  • R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 13 are each independently selected from H, methyl, -OMe, and -OH.
  • R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 13 are each independently selected from methyl, -OMe, and -OH, and the remainder of R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 13 are H.
  • all of R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 13 are H.
  • R 14 is selected from H, unsubstituted C1-4 alkyl, and -OR 12 ; wherein R 12 is selected from H and methyl. Often R 14 is -OR 12 ; wherein R 12 is selected from H and methyl. Preferably, R 14 is -OH.
  • R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 13 are each H;
  • R 14 is -OR 12 wherein R 12 is H.
  • D may thus be a compound of formula (V) or a pharmaceutically acceptable salt thereof:
  • a comedolytic retinoid as described herein, is typically a compound of formula (IV) or (V) and pharmaceutically acceptable salts thereof.
  • Formula (V) represents the structure of adapalene.
  • D is a compound of formula (IV)
  • D is often attached to the moiety L by a covalent bond to an oxygen atom at position R 14 .
  • L is a linker between D and P.
  • L comprises or consists of a polymeric linker, typically comprising a reactive functional group at each end of the linker for reaction with D and P, respectively.
  • the reactive functional group for reaction with D may be the same or different to the reactive functional group for reaction with P.
  • the or each reactive functional group may comprise -C(O)-, -C(O)O-, -O-, etc.
  • the or each reactive functional group may comprise -C(O)-, -C(O)O-, etc.
  • L is a group of form -C(O)-L 1 -C(O)-; and D comprises a hydroxyl group which is esterified with the linker (L) at one of the -C(O)- moieties such that the moiety D-L- is a group of formula (Q) wherein the wavy line indicates the point of attachment to the moiety -P-X-Z-C(O)O-T.
  • L is a group of form -O-L 1 -C(O)-
  • D comprises a carboxylic acid group (e.g. a group -C(O)-R 14 wherein R 14 is OH) and the carboxylic acid group is esterified with the linker (L) such that the moiety D-L- is a group of formula (QI) wherein the wavy line indicates the point of attachment to the moiety -P-X-Z-C(O)O-T.
  • the moiety X-Z forms one or more -O-CH2CH2 (PEG) units.
  • the conjugate provided herein comprises a moiety of Formula (VI) wherein PLGA is a polymeric spacer such as poly(lactic-co-glycolic acid).
  • the (PLGA) n (OCH2CH2) moiety represents a PLGA-PEG conjugate having an average molecular mass of about 2 to about 5 kDa, such as about 3 to about 4 kDa.
  • conjugate when a conjugate provided herein comprises a moiety of formula (Q) or (QI), the conjugate preferably comprises a moiety of formula (Q).
  • L 1 is selected from Ci-18 alkylene, C2-I8 alkenylene, and C2-I8 alkynylene, wherein L 1 is unsubstituted or is substituted with 1, 2 or 3 substituents selected from halogen, -OR 10 , and -NR 10 R n .
  • L 1 is selected from Ci-18 alkylene, C2-I8 alkenylene, and C2-I8 alkynylene
  • the alkylene, alkenylene or alkynylene group may be linear or branched, typically the alkylene, alkenylene or alkynylene group is linear.
  • L 1 is selected from Ci-12 alkylene, and C2-12 alkenylene, wherein L 1 is unsubstituted or is substituted with 1 or 2 substituents selected from -OR 10 , and - NR 10 R n . More typically, L 1 is selected from C1-9 alkylene and C2-9 alkenylene, wherein L 1 is unsubstituted or is substituted with 1 substituent selected from -OR 10 , and -NR 10 R n .
  • L 1 is unsubstituted C1-7 alkylene.
  • L 1 may be unsubstituted methylene, ethylene (C2 alkylene), C3 alkylene, C4 alkylene, C5 alkylene, Cf> alkylene, or C7 alkylene.
  • ethylene (C2 alkylene) L thus corresponds to malonate.
  • L 1 is unsubstituted ethylene (C2 alkylene) L thus corresponds to succinate.
  • L 1 is unsubstituted C3 alkylene L thus corresponds to glutarate.
  • L 1 is unsubstituted C4 alkylene L thus corresponds to adipate.
  • L 1 When L 1 is unsubstituted C5 alkylene L thus corresponds to pimelate. When L 1 is unsubstituted Cf> alkylene L thus corresponds to suberiate. When L 1 is unsubstituted C7 alkylene L thus corresponds to azelate. When L 1 is unsubstituted Cs alkylene L thus corresponds to sebacate. In preferred embodiments L 1 may be unsubstituted C2 to C7 alkylene, e.g. typically succinate or azelate.
  • T is a tocopheryl or tocotrienyl group.
  • Tocopheryl groups are derived from tocopherol compounds.
  • Tocotrienyl groups are derived from tocotrienol compounds.
  • Tocopherol and tocotrienyl groups comprise a cyclic substituted chromane ring with a hydroxyl group comprising the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I); and a hydrophobic side chain.
  • the hydroxyl group of the chromane ring may be esterified with the C(O)O moiety of the Formula (I) moiety thereby forming the conjugate.
  • Tocopheryl and corresponding tocotrienyl groups are typically structurally related, with tocophenyl groups typically comprising an unsaturated alkyl side chain and tocotrienyl groups differing from the corresponding tocopheryl groups by the presence of three double bonds in the side chain which thus comprises a alkenyl side chain.
  • T is vitamin E or a derivative thereof.
  • T is a moiety of formula (W): wherein: each R a , R b , R c and R d are independently H or unsubstituted C1-3 alkyl; and
  • R e is C1-20 alkyl, C2-20 alkenyl or C2-20 alkynyl, wherein R e is unsubstituted or is substituted by 1, 2 or 3 substituents independently selected from halogen, -OR 10 , and -NR 10 R n ; and the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I).
  • R a is H or methyl, typically methyl
  • R b is H or methyl, typically methyl
  • R c is H or methyl, typically methyl
  • R d is H or methyl, typically methyl
  • R e is C10-20 alkyl or C10-20 alkenyl; typically C10-20 alkyl; more typically C14-I8 alkyl.
  • the dashed bond is absent so that the indicated bond is a single C-C bond.
  • R a is methyl; R b is methyl; R c is methyl; R d is methyl; R e is C 10-20 alkyl or C 10-20 alkenyl; and the dashed bond is absent so that the indicated bond is a single C-C bond; or ii) R a is methyl; R b is H; R c is methyl; R d is methyl; R e is C 10-20 alkyl or C 10-20 alkenyl; and the dashed bond is absent so that the indicated bond is a single C-C bond; or iii) R a is H; R b is methyl; R c is methyl; R d is methyl; R e is C 10-20 alkyl or C 10-20 alkenyl; and the dashed bond is absent so that the indicated bond is a single C-C bond; or iv) R a is H; R b is H; R c is methyl; R d
  • R e is C10-20 alkyl; more typically C14-I8 alkyl.
  • R e is selected from:
  • the moiety of formula (W) is a moiety of formula (W 1): wherein R a , R b , R c , R d , R e , the dashed bond and the wavy line are as defined for formula (W).
  • R a is methyl; R b is methyl; R c is methyl; R d is methyl; and R e is such that formula (W) is a-tocopheryl; or ii) R a is methyl; R b is H; R c is methyl; R d is methyl; R e such that formula (W) is P-tocopheryl; or iii) R a is H; R b is methyl; R c is methyl; R d is methyl; R e is such that formula (W) is y-tocopheryl; or iv) R a is H; R b is H; R c is methyl; R d is methyl; R e is such that formula (W) is 6-tocopheryl; and the dashed bond is absent so that the indicated bond is a single C-C bond.
  • R a is methyl; R b is methyl; R c is methyl; R d is methyl; and R e is such that formula (W) is a- tocopheryl.
  • T is: wherein the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I).
  • D is selected from erythromycin, adapalene and derivatives thereof, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; and is attached to the - C(O)-L 1 -C(O)-P-X-Z-C(O)O-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and
  • L 1 is selected from Ci-12 alkylene, and C2-12 alkenylene, wherein L 1 is unsubstituted or is substituted with 1 or 2 substituents selected from -OR 10 , and -NR 10 R n ;
  • T is a moiety of formula (W): wherein: each R a , R b , R c and R d are independently H or unsubstituted C1-3 alkyl; and
  • R e is C1-20 alkyl, C2-20 alkenyl or C2-20 alkynyl, wherein R e is unsubstituted or is substituted by 1, 2 or 3 substituents independently selected from halogen, - OR 10 , and -NR 10 R n ; and the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I); and P, X, and Z are as defined herein.
  • D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; and is attached to the -C(0)-L 1 -C(0)-P-X-Z-C(0)0-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and L 1 is selected from C 1-12 alkylene, and C2-12 alkenylene, wherein L 1 is unsubstituted or is substituted with 1 or 2 substituents selected from -OR 10 , and -
  • T is a moiety of formula (W): wherein: each R a , R b , R c and R d are independently H or unsubstituted C1-3 alkyl; and
  • R e is C1-20 alkyl, C2-20 alkenyl or C2-20 alkynyl, wherein R e is unsubstituted or is substituted by 1, 2 or 3 substituents independently selected from halogen, -
  • the conjugate is of formula (la) or (lb), wherein:
  • D is selected from erythromycin, adapalene, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; and is attached to the X-Z-C(O)O-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and
  • L 1 is unsubstituted C1-7 alkylene
  • T is a moiety of formula (W): wherein:
  • R a is H or methyl, typically methyl
  • R b is H or methyl, typically methyl
  • R c is H or methyl, typically methyl
  • R d is H or methyl, typically methyl
  • R e is Cio-20 alkyl or C10-20 alkenyl; typically C10-20 alkyl; more typically C14-I8 alkyl; and the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I); and P, X, and Z are as defined herein.
  • conjugate is of formula (la), wherein:
  • D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; and is attached to the -QOj-L'-QOj-P-X-Z-QOjO-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and L 1 is unsubstituted C1-7 alkylene;
  • T is a moiety of formula (W): wherein:
  • R a is H or methyl, typically methyl
  • R b is H or methyl, typically methyl
  • R c is H or methyl, typically methyl
  • R d is H or methyl, typically methyl
  • R e is C10-20 alkyl or C10-20 alkenyl; typically C10-20 alkyl; more typically C14-I8 alkyl; and the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I); and P, X, and Z are as defined herein.
  • P is a polymeric spacer
  • P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), a polycaprolactone (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), a polyacrylamide (PAM), hyaluronic acid, heparin, polysialic acid, or a polypeptide.
  • PEG poly(ethylene glycol)
  • PLA polylactic acid
  • PLA poly(lactic-co-glycolic acid)
  • PCL polycaprolactone
  • PX poly(glycerol)
  • POX poly(oxazoline)
  • PVP poly(vinylpyrrolidone)
  • PAM polyacrylamide
  • P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), a polycaprolactone (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), or a polyacrylamide (PAM).
  • P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), or a polycaprolactone (PCL). More typically P is a poly(ethylene glycol) (PEG).
  • P has an average molecular weight of from about 100 Da to about 10,000 Da (10 kDa). More typically, P has an average molecular weight of from about 200 Da to about 5 kDa. Still more typically P has an average molecular weight of from about 300 Da to about 3 kDa, such as from about 500 Da to about 2 kDa, e.g. from about 700 to about 1500 Da, such as from about 800 to about 1200 Da, such as from about 900 to about 1100 Da, e.g. about 1 kDa.
  • the molecular weight of P is typically the number average molecular weight, as determined e.g. by MALDI mass spectroscopy.
  • P comprises a polymer comprising from about 5 to about 50 monomer units, such as from about 10 to about 40 monomer units, e.g. from about 15 to about 30 monomer units, e.g. from about 18 to about 28 monomer units, e.g. from about 20 to about 25 monomer units.
  • P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), a polycaprolactone (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), or a polyacrylamide (PAM) having an average molecular weight of from about 100 Da to about 10 kDa.
  • PEG poly(ethylene glycol)
  • PLA polylactic acid
  • PLA poly(lactic-co-glycolic acid)
  • PCL polycaprolactone
  • PX poly(oxazoline)
  • POX poly(vinylpyrrolidone)
  • PAM polyacrylamide
  • P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co- glycolic acid) (PLGA), or a polycaprolactone (PCL) having an average molecular weight of from about 200 Da to about 5 kDa. More typically P is a poly(ethylene glycol) (PEG) having an average molecular weight of from about 500 Da to about 2 kDa, e.g. from aobut 700 to about 1500 Da. Most typically P is poly(ethylene glycol) (PEG) having an average molecular weight of about 1 kDa.
  • a PEG having an average molecular weight of about 1 kDa typically comprises about 23 monomer ethylene glycol units.
  • P is PLGA having an average molecular weight of about 4 kDa.
  • X is a group selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, -S-, -S(O)-, -SO2-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, -NR 10 C(O)NR n -, -NR 10 C(O)O-, -OC(O)NR 10 , and a covalent bond.
  • X is selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, -NR 10 C(O)NR n -, -NR 10 C(O)O-, and -OC(O)NR 10 . More typically, X is selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, NR 10 C(O)-, and -C(O)NR 10 -.
  • X is selected from -OC(O)-, -C(O)O-, -C(O)-, and -O-. Yet more typically X is selected from -OC(O)-, -C(O)O- and -C(O)-. Most typically, X is -OC(O)-.
  • Z is selected from Ci-12 alkylene, C2-12 alkenylene, and C2-12 alkynylene, wherein Z is unsubstituted or is substituted with 1, 2 or 3 substituents independently selected from halogen, -OR 10 , and -NR 10 R n . More typically, Z is selected from C1-6 alkylene, and C2-6 alkenylene, wherein Z is unsubstituted or is substituted with 1 or 2 substituents independently selected from -OR 10 , and -NR 10 R n .
  • Z is selected from C1-4 alkylene, and C2-4 alkenylene, wherein Z is unsubstituted or is substituted with 1 substituent selected from -OR 10 , and -NR 10 R n . Yet more typically, Z is unsubstituted C1-4 alkylene, typically methylene or ethylene, most typically ethylene (C2 alkylene).
  • P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co- glycolic acid) (PLGA), a polycaprolactone (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), or a polyacrylamide (PAM) having an average molecular weight of from about 100 Da to about 10 kDa;
  • - X is selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, -NR 10 C(O)NR n -, -NR 10 C(O)O-, and -OC(O)NR 10 ; and Z is selected from C1-6 alkylene, and C2-6 alkenylene, wherein Z is unsubstituted or is substituted with 1 or 2 substituents independently selected from -OR 10 , and -NR 10 R n . More typically, in formula (I):
  • P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co- glycolic acid) (PLGA), or a polycaprolactone (PCL) having an average molecular weight of from about 200 Da to about 5 kDa;
  • PEG poly(ethylene glycol)
  • PLA polylactic acid
  • PLGA poly(lactic-co- glycolic acid)
  • PCL polycaprolactone
  • - X is selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, NR 10 C(O)-, and -C(O)NR 10 -;
  • Z is selected from C1-4 alkylene, and C2-4 alkenylene, wherein Z is unsubstituted or is substituted with 1 substituent selected from -OR 10 , and -NR 10 R n .
  • each R 10 and R 11 is independently selected from H and C1-2 alkyl. More typically, each R 10 and R 11 is independently selected from H and methyl. Most typically, each R 10 and R 11 is H.
  • the conjugate of formula (I) is of formula (la) or (lb): wherein:
  • D is selected from erythromycin, adapalene and derivatives thereof, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; typically erythromycin or a pharmaceutically acceptable salt thereof; and D is attached to the -C(O)-L 1 -C(O)-P-X- Z-C(O)O-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and L 1 is selected from unsubstituted C1-9 alkylene and unsubstit
  • T is a moiety of formula (W): wherein: o R a is H or methyl; o R b is H or methyl; o R c is H or methyl; o R d is H or methyl; and
  • P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co- glycolic acid) (PLGA), or a polycaprolactone (PCL) having an average molecular weight of from about 200 Da to about 5 kDa;
  • PEG poly(ethylene glycol)
  • PLA polylactic acid
  • PLGA poly(lactic-co- glycolic acid)
  • PCL polycaprolactone
  • X is selected from -OC(O)-, -C(O)O-, -C(O)-, and -O-;
  • Z is selected from unsubstituted Ci-6 alkylene and unsubstituted C2-6 alkenylene.
  • the conjugate of formula (I) is of formula (la): wherein:
  • D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; typically erythromycin or a pharmaceutically acceptable salt thereof; and D is attached to the -C(O)-L 1 -C(O)-P-X-Z-C(O)O-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and
  • L 1 is selected from unsubstituted C1-9 alkylene and unsubstituted C2-9 alkenylene;
  • T is a moiety of formula (W): wherein: o R a is H or methyl; o R b is H or methyl; o R c is H or methyl; o R d is H or methyl; and
  • P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co- glycolic acid) (PLGA), or a polycaprolactone (PCL) having an average molecular weight of from about 200 Da to about 5 kDa;
  • PEG poly(ethylene glycol)
  • PLA polylactic acid
  • PLGA poly(lactic-co- glycolic acid)
  • PCL polycaprolactone
  • X is selected from -OC(O)-, -C(O)O-, -C(O)-, and -O-;
  • Z is selected from unsubstituted C1-6 alkylene and unsubstituted C2-6 alkenylene.
  • conjugate of formula (I) is of formula (la) or
  • D is selected from erythromycin, adapalene, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; typically erythromycin or a pharmaceutically acceptable salt thereof; and D is attached to the -C(O)-L 1 -C(O)-P-X-Z-C(O)O-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and
  • L 1 is unsubstituted C1-7 alkylene
  • P is a poly(ethylene glycol) (PEG) having an average molecular weight of from about 500 Da to about 2 kDa;
  • X is selected from -OC(O)-, -C(O)O- and -C(O)-; and Z is unsubstituted C1-4 alkylene.
  • conjugate of formula (I) is of formula (la): wherein:
  • D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; typically erythromycin or a pharmaceutically acceptable salt thereof; and D is attached to the -C(0)-L 1 -C(0)-P-X-Z-C(0)0-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and
  • L 1 is unsubstituted C1-7 alkylene
  • T is P is a poly(ethylene glycol) (PEG) having an average molecular weight of from about 500 Da to about 2 kDa;
  • X is selected from -OC(O)-, -C(O)O- and -C(O)-; and Z is unsubstituted C1-4 alkylene.
  • the conjugate of formula (I) is a conjugate of formula (II): wherein: n is an integer from 1 to about 50, typically from about 10 to about 30, more typically from about 15 to about 30, more typically from about 18 to about 28, more typically from about 20 to about 25;
  • D is as defined herein; typically D is erythromycin;
  • L 1 is as defined herein; typically L 1 is unsubstituted C1-7 alkylene; and
  • Z is as defined herein, typically Z is unsubstituted C1-4 alkylene. wherein: n is an integer from about 10 to about 30, typically from about 15 to about 30, more typically from about 18 to about 28, more typically from about 20 to about 25;
  • L 1 is as defined herein; typically L 1 is unsubstituted C1-7 alkylene; and
  • Z is as defined herein, typically Z is ethylene.
  • a preferred conjugate of formula (I) is: wherein n is from about 15 to about 30, more typically from about 18 to about 28, more typically from 20 to about 25, typically 20, 21, 22, 23, 24, or 25. wherein n is from about 15 to about 30, more typically from about 18 to about 28, more typically from 20 to about 25, typically 20, 21, 22, 23, 24, or 25.
  • Another preferred conjugate provided herein is:
  • each n is the same or different and is each independently selected from about 1 to about 50, such as from about 10 to about 30 e.g. about 20 to about 25, typically 20, 21, 22, 23, 24, or 25. In some embodiments each n is the same or different and is each independently selected from about 1 to about 10, such as from about 2 to about 5.
  • the conjugates provided herein may form micelles which preferentially localise in hair follicles of the skin.
  • the drug moiety (D) is typically hydrophilic and is believed to localise at the surface of the micelles thereby produced.
  • the delivery of a drug (D) via the conjugates provided herein, e.g. when formulated as micelles is materially different to the delivery of hydrophobic drug molecules which may be concentrated in the hydrophobic core of a micelle e.g. formed by a lipid.
  • the dosage of the drug to the skin is determined by the number of conjugate monomers used to form the micelles which may be administered as described herein. This contrasts with methods in which a hydrophobic drug is contained in the hydrophobic core of a micelle in which the drug delivery depends on the loading of the micelle.
  • Indiscriminate delivery of drugs to the skin e.g. via simple prior formulations of such drugs leads to high concentrations of the drug contacting bacteria which are not directly responsible for an ongoing infection at the point of administration, e.g. those colonising uninfected areas of the skin. Repeated low-level administration in this way may lead to the build-up of antibiotic resistance in such bacteria.
  • the localised delivery of the drug comprised in the provided conjugates means that this build-up of resistance is decreased.
  • the localisation of the drug moiety (D) on the surface of the micelle formed by the conjugates provided herein may lead to beneficial effects compared to micelles which encapsulate their drug loads, e.g. those comprising a hydrophobic drug encapsulated in a hydrophobic core.
  • the bioavailability of the drug is typically improved when surface accessible as provided herein.
  • Side effects associated with the delivery of the conventional micelles to the skin e.g. from contact of the lipid shell of such micelles to uninfected areas of the skin may also be reduced as such contact is “shielded” by the drug molecules comprised in the provided conjugates.
  • the conjugates provided herein are believed to break down in the environment of the skin and in particular in the localised environment of hair follicles.
  • the breakdown of the conjugates is believed to be beneficially catalysed by esterases expressed by pathogenic microbes which may colonise pilosebaceous units.
  • Breakdown, e.g. by hydrolysis, of the conjugates provided herein releases the drug moiety (D) of the conjugate which exerts a localised therapeutic effect.
  • the breakdown e.g. hydrolysis
  • the breakdown is optimal in the vicinity of pilosebaceous units due to the localised concentration of esterase-expressing pathogenic microbes therein (e.g. under disease conditions such as those described herein) meaning that improved and targeted delivery of the drug moiety (D) is achieved.
  • the inventors believe that the conjugates provided herein have beneficial transdermal permeation properties. IN particular, the inventors have found that the rate of transdermal penetration of the disclosed conjugates may be advantageously slowed (e.g. compared to the equivalent drug in unconjugated form) thereby improving the efficacy of the drug in treating a microbial skin infection and decreasing potentially harmful absorption into the body.
  • the inventors also believe that the therapeutic effect exerted by the conjugates provided herein may be enhanced by the co-release of the linker moiety L.
  • L comprises a group of form -C(O)-L 1 -C(O)- and L 1 is a C7 alkylene group the moiety L corresponds to azelaic acid.
  • carboxylic acids are believed to exert a further therapeutic effect in targeting microbial skin infestations such as acne.
  • the therapeutic effect exerted by the conjugates provided herein is typically greater than that provided by prior formulations of the drug molecule (e.g. erythromycin).
  • the therapeutic effect exerted by the conjugate may be at least additive, e.g.
  • the therapeutic effect exerted by the conjugate may be synergistic, e.g. relative to the effect exerted by formulations of the drug moiety (D) and linking group (L) separately.
  • conjugates provided herein are believed to form micelles when contacted with a suitable vehicle.
  • the term “micelle” relates to a supramolecular assembly of conjugate molecules.
  • conjugate molecules in a micelle typically assemble into a three dimensional structure comprising a hydrophobic core and a hydrophilic shell.
  • the reverse structure may be adopted in the presence of a hydrophobic solvent, e.g. when the conjugate molecules are contacted with a non-aqueous or substantially non-aqueous hydrophobic vehicle.
  • the conjugates provided herein form micelles each comprising (i) a core comprising the tocopheryl or tocotrienyl groups of the conjugates and (ii) a solvent- accessible surface; wherein the antimicrobial agent of the conjugates extends into the solvent from the solvent-accessible surface.
  • the solvent-accessible surface is an aqueous-solvent-accessible surface.
  • the disclosed conjugates form micelles in which the drug moiety (D) is typically wholly or substantially grafted to the shell of the micelle, and extends into the solvent (e.g. an aqueous solvent).
  • the solvent e.g. an aqueous solvent
  • this differs from many drug-containing micelles described in the art in which a drug molecule (typically a hydrophobic drug molecule) is generally localised in the core of the micelle.
  • the micellar structures formed by the conjugates provided herein are advantageous as described herein.
  • the production of micelles by the conjugates provided herein can be confirmed by tests conventional in the art.
  • the successful formation of a conjugate can be confirmed using Nile Red assays and/or foaming assays.
  • Nile Red assays can detect micelle formation as Nile Red is lipophilic and concentrates in the lipophilic core of micelles formed in aqueous solution.
  • Foaming assays e.g. using TPGS as a control can be used to determine micelle formation.
  • the conjugates provided herein form substantially spherical micelles.
  • the invention is not limited to such and micelles having other shapes such as ellipsoids, cylinders, and bilayers, are within the scope of the invention.
  • some micelles formed by conjugates provided herein adopt “flower” shapes known in the art. The shape may be determined e.g. by TEM imaging.
  • the conjugates provided herein have a critical micelle concentration in water (e.g. at 25 °C) of below 100 mg/mL, such as from about 10 ng/mL to about 100 mg/mL, e.g. from about 1 pg/mL to about 10 mg/mL, e.g. from about 10 pg/mL to about 1 mg/mL e.g. about 100 pg/mL.
  • CMC can be determined by techniques that are routine in the art, such as by fitting observed parameters measured across a range of conjugate concentrations. Fit functions for properties such as electrical conductivity, surface tension, NMR chemical shifts, absorption, self-diffusion coefficients, fluorescence intensity and mean translational diffusion coefficients are known in the art.
  • the conjugates provided herein form micelles in aqueous solution (e.g. when assessed at concentrations of about 1 mg/mL) having an average size (e.g. a mean diameter as determined by TEM, or z-average diameter as assessed by DLS) of from about 2 to about 50 nm, e.g. from about 5 to about 25 nm such as from about 8 to about 15 nm, e.g. about 9 or 10 nm.
  • an average size e.g. a mean diameter as determined by TEM, or z-average diameter as assessed by DLS
  • the conjugates provided herein form micelles in aqueous solution (e.g. when assessed at concentrations of about 1 mg/mL) having a zeta potential of from about - 100 mV to about +100 mV, e.g. from about 0 mV to about +80 mV, such as from about +20 mV to about +60 mV, e.g. from about +30 mV to about + 50 mV.
  • the micelles formed by the conjugates provided herein have a positive potential (zeta potential > 0).
  • the conjugates provided herein form micelles in aqueous solution (e.g. when assessed at concentrations of about 1 mg/mL) having a polydispersity index of less than about 10, e.g. less than about 5, such as less than about 2, e.g. less than about 1.
  • the micelles formed by the conjugates provided herein are substantially monodisperse.
  • the vehicle in which the conjugates provided herein form micelles is a pharmaceutically acceptable vehicle.
  • a pharmaceutically acceptable vehicle Such vehicles are described in more detail herein in the context of pharmaceutical formulations.
  • the vehicle is sterile and pyrogen free.
  • conjugates provided herein can be prepared by any suitable method. Detailed general synthetic routes for representative compounds of the invention are set out in the Examples.
  • conjugates provided herein can typically be prepared by functionalising the drug moiety (D) at an appropriate reactive functional group such as an alcohol group, e.g. an OH group, e.g. an OH group comprised in a sugar ring attached to a macrolide macrocycle, with a moiety comprising the linker L and having a further reactive functional group for downstream reaction.
  • an appropriate reactive functional group such as an alcohol group, e.g. an OH group, e.g. an OH group comprised in a sugar ring attached to a macrolide macrocycle
  • a suitable moiety comprising linker L may comprise a carboxylic acid or anhydride equivalent thereof for reaction with the reactive functional group on drug moiety (D) thereby forming an ester bond to linking moiety L.
  • Linker L may further comprise a second reactive functional group such as a carboxylic acid or anhydride equivalent thereof.
  • a suitable moiety comprising linker L may be a moiety of form OH-C(O)-L 1 -C(O)-OH wherein L 1 is as described herein.
  • Reaction of the second reactive functional group or an activated derivative thereof with the moiety P-X-Z-C(O)O-T (e.g. at a reactive functional group comprised in moiety P) may then yield the final conjugate.
  • the moieties D, L and P-X-Z-C(O)O-T are typically commercially available or can be synthesized by standard techniques using methods disclosed in e.g. March’s Advanced Organic Chemistry, 8 th edition (Wiley, 2020).
  • a pharmaceutical formulation comprising a plurality of micelles in a vehicle, wherein said micelles each comprise a plurality of conjugates as described herein.
  • said micelles are as described herein.
  • the vehicle is a pharmaceutically acceptable vehicle such as a pharmaceutically acceptable solvent.
  • the vehicle is aqueous or substantially aqueous.
  • the vehicle comprises a buffered aqueous solution, e.g. buffered from pH 4 to pH 9 such as from pH 5 to pH 8 e.g. pH 6 to pH 7.
  • compositions comprising a conjugate as provided herein.
  • the composition comprises a pharmaceutically acceptable vehicle such as a pharmaceutically acceptable solvent.
  • vehicle is aqueous or substantially aqueous.
  • a pharmaceutical formulation or pharmaceutical composition provided herein may be for topical administration.
  • the pharmaceutical formulation or pharmaceutical composition may be intended for and/or suitable for topical administration.
  • the pharmaceutical formulation or pharmaceutical composition comprises a conjugate which is optically active
  • the conjugate is typically a substantially pure optical isomer.
  • the composition contains up to 85 wt% of a conjugate of the invention. More typically, it contains up to 50 wt% of a conjugate of the invention.
  • the composition may comprise up to 20 wt% of a conjugate of the invention, such as up to 10 wt% of a conjugate of the invention.
  • Preferred pharmaceutical formulations and compositions are sterile and pyrogen free.
  • a pharmaceutical formulation or pharmaceutical composition may be provided as a kit comprising instructions to enable the kit to be used in the methods described herein or details regarding which subjects the method may be used for.
  • a pharmaceutical formulation or pharmaceutical composition as provided herein may comprise an additional therapeutic agent.
  • the additional therapeutic agent may be for example selected from antibiotics, antiseptics, retinoids, hormones, etc.
  • the pharmaceutical formulation or pharmaceutical composition comprises one or more antibiotics.
  • the pharmaceutical formulation or pharmaceutical composition comprises one or more antibiotics selected from aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, antibiotic glycopeptides and lipoglycopeptides (such as vancomycin), macrolides, monobactams (such as aztreonam), oxazolidinones (such as linezolid and tedizolid), penicillins, antibiotic polypeptides, rifamycins, sulphonamides, streptogramins (such as quinupristin and dalfopristin) and/or tetracyclines.
  • antibiotics selected from aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, antibiotic glycopeptides and lipoglycopeptides (such as vancomycin), macrolides, monobactams (such as aztreonam), oxazolid
  • the one or more antibiotics are typically selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and/or sarecycline.
  • the pharmaceutical formulation comprises one or more antibiotics said antibiotic is a different antibiotic to the drug moiety (D) comprised in the conjugate.
  • the drug moiety (D) may be erythromycin or a pharmacueitcally acceptable salt thereof, and the conjugate may be formulated in a pharmaceutical formulation comprising azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and/or sarecycline.
  • the pharmaceutical formulation comprises a retinoid such as tretinoin, adapalene, isotretinoin, retinol, tazarotene, alitretinoin, and/or bexarotene.
  • a retinoid such as tretinoin, adapalene, isotretinoin, retinol, tazarotene, alitretinoin, and/or bexarotene.
  • the pharmaceutical formulation comprises an antiseptic agent such as benzoyl peroxide, chlorhexidine (e.g. chlorhexidine gluconate or acetate), povidone-iodine, chloroxylenol, ethyl alcohol, isopropyl alcohol, hexachlorophene, benzalkonium chloride, hydrogen peroxide, cetrimide, methylbenzethonium chloride, benzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, dofanium chloride, domiphen bromide, proflavine hemisulphate, triphenylmethane, brilliant green, crystal violet, gentian violet, potassium permanganate, chlorocresol, chloroxylenol, chlorophene, hexachlorophane/hexachlorophene, triclosan, hydroxyquinoline sulphate, potassium hydroxyquinoline sulphate, chlorquinaldol, dequalinium chlor
  • conjugate as described herein with one or more further therapeutic agents, typically selected from antibiotics, antiseptics, retinoids, hormones, e.g. those agents described herein.
  • the two agents may be provided in a single formulation as described herein, or they may be separately formulated. Where separately formulated, the two agents may be administered simultaneously, separately or sequentially. They may be provided in the form of a kit, optionally together with instructions for their administration.
  • the products may also be referred to herein as combinations or pharmaceutical combinations.
  • the conjugates provided herein are therapeutically useful.
  • the present invention therefore provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in medicine.
  • the present invention provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in treating the human or animal body.
  • a method of treating a subject in need thereof comprising administering an effective amount of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein to said subject.
  • the use of a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament are examples of a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament.
  • a conjugate of the invention may be administered in the form of a solvate.
  • the conjugates provided herein are useful in the treatment of skin disorders arising from microbial infection.
  • the present invention provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in treating a microbial infection, e.g. a microbial skin infection, in a subject in need thereof.
  • a method of treating a microbial infection, e.g. a microbial skin infection, in a subject in need thereof comprising administering an effective amount of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein to said subject.
  • a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament for treating a microbial infection, e.g. a microbial skin infection.
  • the microbial infection may be caused by a bacterial infection such as infection by bacteria from the families Propionibacteriaceae, Streptococcaceae, Staphylococcaceae, Pseudomonadaceae, and/or Corynebacteriaceae.
  • the bacterial infection may be caused by bacteria of genus Cutibacterium (e.g. Cutibacterium acnes), Streptococcus (e.g. Streptococcus pyogenes), Staphylococcus (e.g. Staphylococcus aureus), Pseudomonas (e.g. Pseudomonas aeruginosa) or Corynebacterium (e.g. Corynebacterium minutissimum).
  • the bacterial infection is caused by Cutibacterium acnes or Staphylococcus aureus.
  • the bacterial infection may be caused by an opportunistic pathogen.
  • the present invention provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in treating infection by Cutibacterium (e.g. Cutibacterium acnes), Streptococcus (e.g. Streptococcus pyogenes), Staphylococcus (e.g. Staphylococcus aureus), Pseudomonas (e.g. Pseudomonas aeruginosa) or Corynebacterium (e.g. Corynebacterium minutissimum), in a subject in need thereof.
  • a method of treating infection by Cutibacterium e.g. Cutibacterium acnes
  • Streptococcus e.g. Streptococcus pyogenes
  • Staphylococcus e.g. Staphylococcus aureus
  • Pseudomonas e.g. Pseudomonas aeruginosa
  • Corynebacterium e
  • Streptococcus pyogenes Streptococcus pyogenes
  • Staphylococcus e.g. Staphylococcus aureus
  • Pseudomonas e.g. Pseudomonas aeruginosa
  • Corynebacterium e.g. Corynebacterium minutissimum
  • a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament for treating infection by Cutibacterium (e.g. Cutibacterium acnes), Streptococcus (e.g.
  • Streptococcus pyogenes Streptococcus pyogenes
  • Staphylococcus e.g. Staphylococcus aureus
  • Pseudomonas e.g. Pseudomonas aeruginosa
  • Corynebacterium e.g. Corynebacterium minutissimum
  • the skin infection may be selected from acne, rosacea, cellulitis, erysipelas, microbial folliculitis, “hot tub” folliculitis, furuncles, carbuncles, impetigo, erythrasma, tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, and onychomycosis, typically acne.
  • the present invention provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in treating acne, rosacea, cellulitis, erysipelas, microbial folliculitis, “hot tub” folliculitis, furuncles, carbuncles, impetigo, erythrasma, tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, and onychomycosis, typically acne, in a subject in need thereof.
  • Also provided herein is a method of treating acne, rosacea, cellulitis, erysipelas, microbial folliculitis, “hot tub” folliculitis, furuncles, carbuncles, impetigo, erythrasma, tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, and onychomycosis, typically acne, in a subject in need thereof, comprising administering an effective amount of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein to said subject.
  • a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament for treating acne, rosacea, cellulitis, erysipelas, microbial folliculitis, “hot tub” folliculitis, furuncles, carbuncles, impetigo, erythrasma, tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, and onychomycosis, typically acne.
  • a conjugate, pharmaceutical formulation or pharmaceutical composition described herein in the targeted treatment of bacterial infection in hair follicles.
  • a conjugate, pharmaceutical formulation or pharmaceutical composition described herein in the targeted treatment of bacterial infection in pilosebaceous units are also provided herein.
  • the conjugates provided herein may be used in conjunction with one or more further therapeutic agents such as one or more antibiotics, antiseptics, retinoids, or hormones.
  • the present invention provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in treating a microbial infection, e.g. a microbial skin infection, in a subject in need thereof, wherein such use comprises co-administering the conjugate, pharmaceutical formulation or pharmaceutical composition of the invention with a further therapeutic agent e.g. a further therapeutic agent described herein.
  • a method of treating a microbial infection e.g.
  • a microbial skin infection in a subject in need thereof, comprising co-administering an effective amount of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein and a further therapeutic agent e.g. a further therapeutic agent described herein to said subject.
  • a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament for treating a microbial infection, e.g. a microbial skin infection by co-administering the conjugate, pharmaceutical formulation or pharmaceutical composition described herein and a further therapeutic agent e.g. a further therapeutic agent described herein to a subject in need thereof.
  • the subject is a mammal, in particular a human.
  • it may be non-human.
  • Preferred non-human animals include, but are not limited to, primates, such as marmosets or monkeys, commercially farmed animals, such as horses, cows, sheep or pigs, and pets, such as dogs, cats, mice, rats, guinea pigs, ferrets, gerbils or hamsters.
  • the subject can be any animal that is capable of being infected by a bacterium.
  • conjugates, compositions and formulations described herein are useful in the treatment of bacterial infection which occurs after a relapse following an antibiotic treatment.
  • the conjugates, compositions and formulations can therefore be used in the treatment of a patient who has previously received antibiotic treatment for the (same episode of) bacterial infection.
  • a conjugate, composition or formulation described herein can be administered to the subject in order to prevent the onset or reoccurrence of one or more symptoms of the bacterial infection.
  • This is prophylaxis.
  • the subject can be asymptomatic.
  • the subject is typically one that has been exposed to the bacterium.
  • a prophylactically effective amount of the agent or formulation is administered to such a subject.
  • a prophylactically effective amount is an amount which prevents the onset of one or more symptoms of the bacterial infection.
  • a conjugate, composition or formulation described herein can be administered to the subject in order to treat one or more symptoms of the bacterial infection.
  • the subject is typically symptomatic.
  • a therapeutically effective amount of the conjugate, composition or formulation is administered to such a subject.
  • a therapeutically effective amount is an amount effective to ameliorate one or more symptoms of the disorder.
  • the compound, composition or combination of the invention may be administered in a variety of dosage forms.
  • the conjugate, composition or formulation of the invention is administered in a topical dosage form.
  • a conjugate, pharmaceutical formulation or pharmaceutical composition for topical administration may be provided in the form of a lotion, gel, ointment, cream, emulsion, suspension, paste, solution, powder, shampoo, aerosol foam, spray, jelly, film, sponge, swab, patch, or the like.
  • the various topical dosage forms may also be formulated for immediate release, controlled release, sustained release, or the like.
  • the pharmaceutical formulation or pharmaceutical composition may comprise one or more pharmaceutically acceptable excipient, carrier, and/or diluent.
  • pharmaceutically acceptable excipient examples particularly suitable for topical formulations include liquid oils, viscosity-modifying agents, thickening agents, gelling agents, alcohols, surfactants, chelating agents, buffers, preservatives, humectants, emollients, stabilizers, diluents, dispersing agents, emulsifiers, wetting agents, stabilizers, pH adjusters, solvents, and cosolvents.
  • Gelling agents include carbomers (carboxy vinyl polymers / cross-linked polyacrylic acid), such as Carbopol® and Noveon® polycarbophil.
  • Thickening agents include acacia, alginic acid and salts thereof, hyaluronic acid and salts thereof, carboxymethylcellulose, ethylcellulose, gelatin, collagen, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, methylcellulose, poloxamers, polyvinylpyrrolidone, polyvinyl alcohol, tragacanth, xanthan gum, magnesium aluminum silicate, and bentonite.
  • Surfactants include octylphenoxy polyethoxyethanol such as Octoxynol 1, 3, 5, 8, 9, 10, 12, 13, 16, 30, 40, 70 (wherein the number indicates the number of repeating oxyethylene units), sorbitan esters such as sorbitan monooleate and sorbitan monostearate, polysorbates (such as polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate and polyoxyethylene sorbitan monolaurate, commonly known as Tween® 80, Tween® 60, and Tween® 20, poloxamers (block polymers of ethylene oxide and propylene oxide, such as Pluronic® Fl 27 and Pluronic® Fl 08, poloxamines (block polymers of ethylene oxide and propylene oxide attached to ethylene diamine, such as Tetronic® 1508 and Tetronic® 908), and long chain fatty alcohols (e.g., oleyl alcohol, stearyl alcohol, myristyl alcohol, docosahex
  • Emulsifiers include sodium or potassium oleate, triethanolamine stearate, sodium lauryl sulfate, sodium dioctyl sulfosuccinate, sodium docusate, quaternary ammonium salts, glyceryl monostearate, polyoxyethylene monooleate, polyoxyethylene monostearate, polyoxyethylene monolaurate, potassium oleate, sodium lauryl sulfate, sodium oleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate, triethanolamine oleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan trioleate.
  • Humectants include glycerin, sorbitol, hexylene glycol, propylene glycol, and urea.
  • the pharmaceutical formulation or pharmaceutical composition may comprise one or more chelating agents (such as EDTA) and/or antioxidants (such as butylated hydroxytoluene (BHT) butylated hydroxyanisole (BHA), sodium metabisulfite, propyl gallate, or cysteine).
  • chelating agents such as EDTA
  • antioxidants such as butylated hydroxytoluene (BHT) butylated hydroxyanisole (BHA), sodium metabisulfite, propyl gallate, or cysteine).
  • the conjugate, pharmaceutical composition or pharmaceutical formulation may also be administered in any other suitable manner.
  • the conjugate, composition or formulation can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules.
  • the conjugate, composition or formulation may be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrastemally, transdermally or by infusion techniques.
  • the conjugate, composition or formulation may be administered as a suppository.
  • the conjugate, composition or formulation may be administered via inhaled (aerosolised) or intravenous administration.
  • Solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, com starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g.
  • Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tableting, sugar coating, or film coating processes.
  • Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspension or solutions for intramuscular injections or inhalation may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for inhalation, injection or infusion may contain as carrier, for example, sterile water or typically they may be in the form of sterile, aqueous, isotonic saline solutions.
  • Pharmaceutical compositions suitable for delivery by needleless injection, for example, transdermally, may also be used.
  • the conjugate, composition or formulation may be formulated for inhaled (aerosolised) administration as a solution or suspension.
  • the conjugate, composition or formulation may be administered by a metered dose inhaler (MDI) or a nebulizer such as an electronic or jet nebulizer.
  • MDI metered dose inhaler
  • nebulizer such as an electronic or jet nebulizer.
  • the conjugate, composition or formulation may be formulated for inhaled administration as a powdered drug, such formulations may be administered from a dry powder inhaler (DPI).
  • DPI dry powder inhaler
  • the conjugate, composition or formulation When formulated for inhaled administration, the conjugate, composition or formulation may be delivered in the form of particles which have a mass median aerodynamic diameter (MMAD) of from 1 to 100 pm, typically from 1 to 50 pm, more typically from 1 to 20 pm such as from 3 to 10 pm, e.g. from 4 to 6 pm.
  • MMAD mass median aerodynamic diameter
  • the reference to particle diameters defines the MMAD of the droplets of the aerosol.
  • the MMAD can be measured by any suitable technique such as laser diffraction.
  • a therapeutically or prophylactically effective amount of the conjugate, composition or formulation is administered to a subject.
  • the dose may be determined according to various parameters, especially according to the compound used; the age, weight and condition of the subject to be treated; the route of administration; and the required regimen.
  • a physician will be able to determine the required route of administration and dosage for any particular subject.
  • a typical daily dose is from about 0.01 to 100 mg per kg, typically from about 0.1 mg/kg to 50 mg/kg, e.g. from about 1 to 10 mg/kg of body weight, according to the activity of the specific agent, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • a typical daily dose is from about 0.01 to 100 mg per m 2 , typically from about 0.1 mg/m 2 to 50 mg/m 2 , e.g. from about 1 to 10 mg/m 2 of body surface area, according to the activity of the specific agent, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • the dose of the other active agent can be determined as described above.
  • the dose may be determined according to various parameters, especially according to the agent used; the age, weight and condition of the subject to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular subject.
  • a typical daily dose is from about 0.01 to 100 mg per kg, typically from about 0.1 mg/kg to 50 mg/kg, e.g. from about 1 to 10 mg/kg of body weight, according to the activity of the specific agent, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • a typical daily dose is from about 0.01 to 100 mg per m 2 , typically from about 0.1 mg/m 2 to 50 mg/m 2 , e.g. from about 1 to 10 mg/m 2 of body surface area, according to the activity of the specific agent, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • This example describes the synthesis of an erythromycin prodrug by linking erythromycin and an amphiphilic polymer in order for the prodrug to form micelles with a preferential targeted delivery to the PSU.
  • Erythromycin (ERY) was chosen and D-a-Tocopherol polyethylene glycol 1000 succinate (TPGS) was selected as the polymer. Thanks to the targeted delivery, the concentration of the drug to the real localization of the disease would be increased which could decrease the risk of resistance emergence and limit the side effects.
  • TPGS D-a-Tocopherol polyethylene glycol 1000 succinate
  • ERY was purchased from Fagron BV, (Rotterdam, Netherlands). All the other products were purchased from Sigma-Aldrich (Buchs, Switzerland). The solvents were ordered from Fisher Scientific AG (Reinach, Switzerland). All chemicals were at least of analytical grade.
  • the first step of the production of the prodrug TPGS -Erythromycin- succinate was the addition of a succinic linker to the drug in order to form the intermediate Erythromycin-succinic acid conjugate (compound 1) and then the realization of the esterification in a second step with the TPGS.
  • Erythromycin (733.93 mg), succinic anhydride (SA) (200.14 mg) and 4- (Dimethylamino)pyridine (4-DMAP) (244.34 mg) were mixed in 3 ml anhydrous dichloromethane (DCM) for 24 hours at room temperature, under agitation based on the protocol of Cao and Feng [6].
  • SA succinic anhydride
  • the identity of the product was controlled by MS (Waters XEVO TQ-MS, Baden-Dattwil, Switzerland, in soft positive mode), X H-NMR and 13 C-NMR (Bruker Avance Neo 600 MHz, Fallanden, Switzerland, in CDC13 and at 600MHz).
  • the second step of the synthesis was the esterification between the compound 1 and TPGS.
  • the compound 1 100 mg
  • DCC dicyclohexylcarbodiimide
  • TPGS (277.92 mg) was added to 4-DMAP (1.47 mg). Finally, the two solutions were combined. The reaction was left under agitation, at 40°C for 24 hours, in anhydrous conditions.
  • UV detection 254 nm, 280 nm
  • the product was characterized by MS (in soft positive mode), X H-NMR (in CDC13 and DMSO at 600MHz) and UPLC-UV-ELSD (Waters Acquity UPLC®, Waters Acquity ELSD, Baden-Dattwil, Switzerland) using a C18 column maintained at 45°C (Waters Xbridge® BEH C18, 2.1x50 mm, 2.5 pm, Baden-Dattwil, Switzerland) in accordance with the following method:
  • a MALDI-TOF analysis was done in positive mode (Bruker Autoflex, Billerica, USA).
  • the sample was dissolved (10 mg/mL) in CH3CN/H2O 50/50, v/v, with 0.1% TFA.
  • CHCA MALDI matrix (10 mg/ml) solution was prepared in the same solutions than the samples.
  • the sample solution was mixed 1:1 with the matrix solution.
  • a volume of 1 pL of this samplematrix mixture was spotted onto the stainless steel MALDI target plate (MTP 384 ground steel BC, cat n°280784, Bruker, Germany).
  • Nile Red is a red dye known to be very lipophilic, having a good affinity for the hydrophobic core of micelles [7], 15 pl of a solution of Nile Red at 5mg/ml in acetone was introduced in the solution used previously. The solution appeared transparent in the absence of the hydrophobic core formed by the micelles and dark pink if micelles were formed.
  • the evidenced micelles were further characterized by dynamic light scattering (DLS) at 25°C at an angle of 173° with a Zetasizer Nano ZS (Malvern instruments, Malvern, UK).
  • the polydispersity index (P.I.), hydrodynamic diameter, volume and number weighted diameter (dv and dn) were measured. The values were obtained after three rounds of ten measurements each.
  • the following step was the characterization of the morphology of the micelles using TEM (Tecnai 20 D38O, FEI Company, Hillsboro, USA) after negative staining on copper grids.
  • the stability of the micelles formed by the prodrug was also determined in different conditions of pH: 5 (acetate buffer), 7 (phosphate buffered saline (PBS)) and 9 (NaOH). Solutions were prepared at 1 mg/ml and aliquots were taken every hour for 12 hours and then at day 1, 2, 3, 7, 14, 21 and 28. The samples were analyzed by UPLC-UV-ELSD with the same method presented previously. The stability in contact with porcine liver esterase was determined too. A solution of 2 mg/ml of prodrug was prepared with 10 units of esterase and placed at 37°C to simulate the temperature of the organism. Samples were taken every hour for 20 hours and then at day 1, 2, 3, 7, 14, 21, and 28 and were analyzed by UPLC-UV-ELSD.
  • the characterization of the final product was made by X H-NMR, UPLC-UV-ELSD and MALDI-TOF.
  • the MALDI-TOF results obtained showed that there was not a unique mass for the prodrug (Figure 3). However, the main peak at 2363.301 m/z corresponds to the compound 2 with the loss of a molecule of water and a sodium adduct (M+Na + -H20). The distribution of peaks was as expected with the difference of 44 m/z between the peaks corresponding to the weight of one unit of PEG.
  • the structure of the prodrug was also confirmed by X H-NMR ( Figure 4). It was not possible to identify all the protons of the molecule, but the presence of both erythromycin and TPGS were identified. 1 H-NMR results indicated that some free TPGS was not removed during the purification. This was confirmed by UPLC-UV-ELSD analysis ( Figure 5). Indeed, two peaks were present corresponding to the compound 2 and free TPGS. The ratio between the prodrug and the free polymer is estimated to 20:7. This result combined with a relatively low yield (13.52%) for the second reaction indicates that the reaction and the purification step may be further improved.
  • the next step was to assess the capacity of the new prodrug to form micelles.
  • Solutions of the prodrug at 1 mg/mL (estimated as a concentration above CMC) were prepared, to check the formation of foam, which is a sign of micelles formation.
  • Nile Red which has a different color if it is in hydrophilic or lipophilic environment, was also used to confirm the formation of micelle. Positive results of the two tests are presented in Figure 6. Compound 1 produced less foam than the positive control but gave similar results for the Nile Red test.
  • TEM images were taken at different magnification and the size and potential zeta of the micelle measured with a Zetasizer ( Figure 7 and Figure 8).
  • the size obtained for the micelles formed by the prodrug was 9.222 nm, which was smaller than the one obtained for the TPGS (14.20 nm).
  • the z-average obtained was of 22.29 for TPGS and 21.06 for the compound 2.
  • the value obtained for TPGS was 0.220, which showed a small variation of size between the particles.
  • the Pdl obtained for the prodrug was 0.301 which is higher but is still an acceptable value.
  • a zeta potential of 46 mV was obtained which indicates that the micelles of the prodrug can be considered as positive.
  • TEM images yielded interesting information about the morphology of the micelles. These images indicated that the shape of the micelle is not completely round. Indeed, TPGS alone forms micelles by placing the tails of PEG units in the outside and the tocopherol in the core of the micelles. In this case, the tocopherol and PEG units kept their position, However, without being bound by theory the erythromycin is believed to incorporate inside the micelle creating a “flower” shape [9] due to its polarity.
  • the prodrug In acidic conditions (pH 5, Figure 10), the prodrug is in similar pH condition that the one observed in the skin. The remaining fraction of the compound 2 was plotted as a function of time. Thanks to the linearization of the function (use of the natural logarithm), the k O bs can be estimated from the slope of the regression curve, which then can be used to determine the half-life.
  • stratum comeum of the skin has a pH around 5 but just underneath, in the first viable layer of the skin called stratum granulosum it increases and the pH becomes physiological (pH 7.4) within the next layers [12]. This means that if the micelles have the capacity to leave the skin surface, the prodrug will disassemble.
  • pH of the skin is increased up to values of 6,35 [12]. In that case, the prodrug will disassemble more easily.
  • This example describes the synthesis of an erythromycin prodrug by linking erythromycin and an amphiphilic polymer via azelaic acid in order for the prodrug to form micelles with a preferential targeted delivery to the PSU.
  • the product was characterized by 1H-NMR (Bruker Avance Neo 600 MHz, Fallanden, Switzerland). About Img of product was analyzed in 750pL of chloroform (CDC13) at 600MHz. MS analysis (Advion, Plate Express, Ithaca, NY) was performed as well as melting point measurement (Biichi, Melting Point B-540, Switzerland) (gradient of l°C/min) using melting point tubes of 80mm (Biichi). All the following 1H-NMR and MS analyzes were carried out under the same conditions.
  • erythromycin Fagron, NL, batch: CEPEB001E18
  • azelaic acid anhydride compound 1
  • 4- dimethylaminopyridine (Acros organics, USA, lot: A0421414)
  • the reaction takes place for 24 hours, under argon, at room temperature.
  • TLC thin layer chromatography
  • Ethanol (EtOH) sugar developer (Fisher Chemical, UK, lot: 2176183)/Sulfuric acid (H2SO4) (Carlo Erba, France, lot: 0D249269H)/Anisaldehyde (Acros Organics, China, lot: A0403824) 9:1:1 was used to highlight compounds such as erythromycin. To do this, the TLC plate is immersed in the sugar developer and then heated. The developer colors the plate brown and the compounds appear black
  • the DCU is filtered through a sintered glass.
  • a liquid-liquid extraction was carried out using lOmL of IM HC1 to extract the 4- DMAP into the aqueous phase while the compound will be in the organic phase. The extraction was repeated twice. The organic phase was recovered, dried with magnesium sulphate and lyophilised. Purification of the product was carried out using a puriflash on a C18 column (Interchim Puriflash C18HQ 250x4.6 mm, 15 pm, Monthujon France). Sample: Compound 3 dissolved in dichloromethane
  • UV detection 254 nm, 280 nm
  • the synthesized product was characterised by TLC, 1H-NMR, MS, UHPLC-ELSD and MALDI-TOF to confirm the synthesis.
  • the micelles composed solely of TPGS have a relatively round shape and have a size of approximately 10 nm, which confirms the analysis carried out by DLS.
  • the stability of the prodrug was tested at pH 5 and pH 9 by UHPLC-ELSD under the conditions above. At pH 9, a half-life time of 256.26 h ⁇ 28.536 was observed. At pH 5 the prodrug has an average half-life of 61.62 h ⁇ 3.253). Stability at pH 7 could not be determined.
  • Distribution of the prodrug was characterized in samples of porcine skin comprising PSUs and in samples of porcine skin without PSUs present.
  • Pig's ears (Carre de Rolle slaughterhouse, Switzerland) were cleaned under running cold water.
  • an air dermatome (Zimmer, UK, batch: 64662570) thin layers of skin (outer ear) of 0.750 pm are cut and placed in lx DPBS.
  • Skin samples were assessed in Franz cells with a diameter of 2cm, with ImL of the prodrug (3mg/lmL in water) in the donor compartment; 3O-33°C for 24 hours with stirring. After 24 hours, the skin is collected and cleansed lightly with lx DPBS.
  • the PSUs as well as the controls (without PSU) are taken with a biopsy punch 1 mm in diameter (Kai medical, Japan, lot: 000006).
  • Samples were placed in 1.5mL eppendorfs containing 120 pL of 50% acetonitrile mixture (Fisher Chemical, UK, batch: 2169048)-0.1%AF / 50% H2O-0.1%AF.
  • the eppendorfs are then agitated overnight to extract the PSUs. After extraction, the eppendorfs are centrifuged at 14,000 rpm for 15 minutes. Finally, 100 pl of supernatant was taken and analyzed by UHPLC-MS/MS (Waters Acquity UPLC®, Waters XEVO TQ-MS, Baden Dattwil, Switzerland):
  • This example therefore confirms the synthesis of an erythromycin prodrug by linking erythromycin and an amphiphilic polymer via azelaic acid in order for the prodrug to form micelles with a preferential targeted delivery to the PSU and delivery of the drug (erythromycin) and azelaic acid to the PSU.
  • This example describes i) synthesis and characterisation of an adapalene-copolymer conjugate, and ii) self-assembling micelle formulation of the synthesized conjugate and preliminary characterization of said conjugate.
  • This example demonstrates i.a. that adapalene can be used as the D moiety in conjugates provided herein.
  • Acne vulgaris is a highly prevalent dermatological condition of the pilosebaceous unit (PSU). It affects a significant proportion of the population - although not life-threatening, if left untreated, it can have serious physical and psychological consequences.
  • retinoids are used as first-line therapy either alone or in combination with other antimicrobials [13].
  • their oral administration carries a risk of foetal malformation and appropriate measures must be taken to avoid pregnancy - therefore, it is clearly an advantage to reduce the systemic exposure of these drugs.
  • Topical treatment remains the most common and popular method for the management of acne.
  • Adapalene (ADA; 412.52 Da) is a highly potent third generation retinoid that binds to nuclear retinoid receptors. Since it can modulate cellular differentiation and keratinization, ADA is used in the topical treatment of mild to moderate Acne vulgaris. ADA is exceptionally lipophilic (log P 8.11) and practically insoluble in water meaning that its formulation is a complex and challenging task [15].
  • the drug-copolymer conjugate was also used to develop self-assembling micelles, which can be used to selectively deliver the therapeutic agent to the PSU, which is the target site for acne - and could provide a better option for acne treatment [16].
  • the drug - copolymer conjugate contains a hydrolysable ester bond that can be cleaved either enzymatically in the skin or in the acidic pH environment of the acne which could release the drug in more targeted manner.
  • novel formulation developed here benefits in several ways, as a PSU-targeting formulation will help to improve therapy and minimize side-effects associated with current marketed formulations and so improve patient compliance.
  • Targeted follicular delivery may decrease the drug concentration required in the formulation as compared to existing products [13, 14].
  • avoiding the use of organic solvents and simple formulation development approach would help to decrease the manufacturing cost of the product and hence could reduce the cost of the therapy.
  • a copolymer (mPEG-PLGA) that possesses a free hydroxyl group on the hydrophobic chain was selected for covalent bonding with ADA (which contains a free carboxylic acid group).
  • the conjugate synthesis was performed using the optimized Mitsunobu reaction and synthesized conjugate was purified and characterized by different analytical techniques.
  • adapalene ADA
  • mPEG-PLGA methoxypoly(ethyleneglycol)-b-poly(lactide-co-glycolide)
  • the ADA-copolymer conjugate was developed using Mitsunobu reaction under inert condition and reaction progress was monitored by thin layer chromatography. Isolation was performed using silica gel column chromatography with hexane: ethyl acetate (9:1) as a mobile phase. Isolated product was characterized by TLC and 1H NMR spectroscopy. Further conjugate analysis was performed by saponification reaction and UHPLC-PDA to confirm ester bond formation. The micelle formulation was developed by solvent evaporation method and size was determined using dynamic light scattering.
  • Adapalene (ADA) was purchased from Hangzhou Dayangchem Co. Ltd, (Hangzhou, P.R. China).
  • mPEG-PLGA was purchased from Akina Corp. (West Lafayate, USA).
  • Diisopropylazodicarboxylate, triphenylphosphine were purchased from Sigma- Aldrich, Buchs, Switzerland. All other chemicals were at least of analytical grade.
  • ADA (12.4 mg), mPEG-PLGA (100 mg) and 0.037 mmol of triphenylphosphine (PPhs) (98.25 mg) were weighed and dissolved in 2 mL of dry tetrahydrofuran (THF). This mixture was then cooled to 0°C in ice bath. 0.037 mmol of diisopropylazodicarboxylate (DIAD) (76 .L) dissolved in dry THF (0.5 mL) was then added to above mixture at 0°C under vigorous stirring.
  • DIAD diisopropylazodicarboxylate
  • TLC thin layer chromatography
  • R20 refers to reaction mixture after 20 h and the remaining spots on TLC are starting material or reagents.
  • reaction mixture was dissolved in a mixture of THF: dichloromethane (DCM) (1:1) or in DCM and loaded on silica gel and solvent was evaporated using rotary evaporator (Biichi RE 121 Rotavapor®; Flawil, Switzerland) and then dry powder was loaded in prefilled silica gel column. Column elution was started with hexane and gradually the concentration of EtOAc was increased. The desired product was eluted in hexane:EtOAc (9:1) i.e. around 10% EtOAc concentration in hexane. Pure fractions were evaporated using rotary evaporator and compound was dried under high vacuum for further analysis.
  • the NMR spectrum of the new compound displayed the characteristic aromatic protons corresponding to the naphthalene ring; along with protons in the aliphatic region (belonging to mPEG-PLGA and the adamantyl ring from ADA) thereby confirming that new compound was a conjugate of adapalene and the mPEG-PLGA copolymer.
  • the newly synthesized compound possesses a different Rf value than adapalene and shows fluorescence.
  • ADA and ADA-copolymer conjugate were analysed using Waters Acquity®UPLC®H-class system and a PDA detector (Baden-Dattwil, Switzerland) and a Xbridge®BEH RP-C18, 2.1 x 50 mm, 2.5 pm column. Detection wavelength was 321nm.
  • the gradient elution method was used comprising mobile phase: ACN (80%) + THF (20%) 0.1% FA, and Water + 0.1 %FA. The flow rate was 0.2 mL/min and the injection volume was 5 pL.
  • a saponification test under basic condition and UHPLC-PDA analysis showed that adapalene could be regenerated from the newly synthesized compound and so confirmed the presence of ester bond.
  • Self-assembling micelle nanocarriers of -140 nm size were obtained using adapalene-mPEG-PLGA conjugate.
  • Adapalene-mPEG-PLGA conjugate was developed and characterized.
  • the developed conjugate was able to self-assemble into nanometer size micelles which could be used for acne treatment by targeting the pilosebaceous unit.
  • Micelle formulation was prepared using the solvent evaporation method [5, 16].
  • ADA-copolymer conjugate 10 mg was dissolved in 4 mL of acetone. The mixture was added drop wise under sonication (Branson Digital Sonifier® S- 450D; Carouge, Switzerland) to 4 mL of ultrapure water. Subsequently, acetone was slowly evaporated at 40 °C using a rotary evaporator (Biichi RE 121 Rotavapor®; Flawil, Switzerland). After equilibration overnight, the micelle solution was centrifuged at 10 000 rpm for 15 min (Eppendorf Centrifuge 5804; Hamburg, Germany) to remove the impurity/precipitate from the formulation and the supernatant was carefully collected and used for analysis.
  • the self-assembled micelle formulations were characterized to determine their hydrodynamic diameter (Z av ), volume- and number-weighted diameters (d v and d n , respectively), and polydispersity index (P.L), using dynamic light scattering (DLS) with a Zetasizer Nano-ZS (Malvern Instruments Ltd; Malvern, UK). Measurements were performed in triplicate at an angle of 90° and at a temperature of 25 °C.
  • ADA-copolymer conjugate micelle formulations had hydrodynamic diameters (Z av ) of 140 nm and the polydispersity index was 0.06;
  • Figure 28 shows the size distribution by intensity of the ADA-copolymer conjugate micelle formulation.
  • Kandekar SG et al. Selective delivery of adapalene to the human hairfollicle under finite dose conditions using polymeric micelle nanocarriers Nanoscale. 2018; 10: 1099- 1110

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Abstract

Provided herein is a conjugate of formula (I) wherein D, L, P, X, Z and T are as defined herein. The conjugate may be provided in a formulation or composition as defined herein. The conjugate may be used in the treatment of microbial infection, e.g. in the treatment of microbial skin infection such as acne.

Description

CONJUGATES OF ANTIMICROBIAL AGENTS WITH A TOCOPHERYL OR TOCOTRIENYL GROUP
Field
The present disclosure relates to prodrugs of antimicrobial agents, and particularly to conjugates of antimicrobial agents and tocopheryl groups. The disclosure also relates to pharmaceutical formulations of such conjugates, to pharmaceutical compositions comprising such conjugates, and to such conjugates, formulations and compositions for use in medicine. In some embodiments the conjugates, formulations and compositions find particular use in treating conditions such as acne, bacterial infection and fungal infections, for example by topical administration to a subject in need thereof.
Figure imgf000002_0001
Microbial infestation is a common cause of disease. Pathogens such as bacteria and fungi are associated with many disease conditions and there is a pressing and ongoing need for new and/or improved therapies for treating such conditions. Pathogens can infect many organs of the body. One such organ is the skin.
Skin disease are very prevalent worldwide and are often underestimated. Even though many skin disease are not life-threatening, they can cause many consequences for the patient as discomfort, embarrassment, loss of self-confidence and isolation. In 2013, the Global Burden of Disease Study revealed that skin and subcutaneous diseases were the fourth cause of nonfatal disease burden worldwide. For these different reasons, the development of novel or improvement of existing therapies is essential.
A wide number of skin disorders caused by microbial infection are known and present therapeutic targets for addressing with suitable agents.
One such skin disorder is acne. Acne is the most common skin disease worldwide. Acne affects the pilosebaceous units of the skin, which consist of the sebaceous glands, sebaceous ducts, and their attached hair follicles. Acne (vulgaris) is a disease characterized by a clogging of the skin’s pilosebaceous units by the sebum produced by the sebaceous glands. Whilst there is still no clear explanation of the pathogenesis, acne has been associated with microbial infection e.g. by bacteria such as Cutibacterium acnes (previously known as Propionibacterium acnes). Accordingly, some treatments are known to be efficient such as antibiotics. Another condition associated with acne is rosacea. Like acne, the exact pathology of rosacea is unclear, but bacterial infection by bacteria such as P. acnes and others, including H. pylori, have been associated with rosacea.
Other skin disorders associated with microbial infection include cellulitis, erysipelas, microbial folliculitis, so-called “hot tub” folliculitis, furuncles, carbuncles, impetigo, erythrasma, tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, onychomycosis, etc.
Cellulitis affects the dermis and subcutaneous tissue and is associated with bacterial infection by bacteria such as Streptococcus and Staphylococcus, e.g Staphylococcus aureus. Cellulitis can be severe and lead to extreme discomfort. In extreme cases infections associated with cellulitis can spread to lymph nodes and the bloodstream and can be life-threatening.
Erysipelas affects the upper layers of the skin and leads to intense burning sensations. Erysipelas is associated with infection by bacteria such as Streptococcus and can particularly occur following a break in the skin or via secondary infection from bacteria associated with a primary nose or throat infection.
Microbial folliculitis may be caused by fungal or bacterial infection of the hair follicles in the skin. Severe cases of microbial folliculitis may lead to hair loss. “Hot tub” folliculitis is associated with infection by Pseudomonas bacteria, typically arising through contact with contaminated water sources such as whirlpools, hot tubs and jacuzzis. This condition may give rise to a painful rash which can in some cases be difficult to treat.
Furuncles are infections around a hair follicle. Unlike folliculitis, furuncles typically involve infection of entire pilosebaceous units. Furuncles can develop into abscesses which can be extremely severe. Carbuncles are clusters of furuncles and are often associated with severe bacterial infection. Such infection is typically associated with infection by bacteria such as Staphylococcus and may be associated with fever, weakness and exhaustion.
Impetigo is a bacterial infection of the top layer of the epidermis and is associated with bacterial infection by bacteria such as Streptococcus and Staphylococcus, e.g. Staphylococcus aureus. Impetigo can lead to severe sores and is highly contagious.
Erythrasma is a skin infection associated with bacterial infection by bacteria such as Corynebacterium minutissimum. It can lead to a painful rash.
Fungal skin infections are also common. Fungal skin infections include tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, and onychomycosis. Such conditions can be painful and in some cases severe. Given the prevalence of skin disorders associated with microbial infection, there is a pressing need for treatments.
WO 2012/177986 discloses nanoparticle based pro-drugs of antifungal and antibacterial conjugates with various carriers.
Many current treatments for infections such as those outlined above involve the topical administration of antimicrobial agents, for example, in the form of a cream which can be rubbed into the skin. However, the antimicrobial agents comprised in such compositions are typically poorly selective and cause many side effects. For example, benzoyl peroxide is an anti-infective used topically in cases of mild and moderate acne for its antibacterial, keratolytic and comedolytic properties. However, it is photosensitive and discolours the skin as well as hair and clothing of patients, as well as causing side effects such as irritation, skin dryness, and stinging. Topical retinoids have been used, but have side effects such as skin irritation, pruritus, erythema and increased photosensitivity requiring limitation of sun exposure.
The side effects associated with conventional treatments for skin disorders associated with microbial infection are particularly problematic given that relatively high concentrations of such agents are typically required in order to deliver a suitable concentration into the site of infection, such as (for example) into the pilosebaceous units. Furthermore, the non-specific administration of antimicrobial agents can give rise to development of microorganisms that are resistant to the administered agent. This can reduce the efficacy of future administrations of a particular agent in a given subject, and may in time contribute to the build-up of resistant microbes in a population.
Summary
The present inventors have recognised that some or all of the issues outlined above could be addressed, at least in part, by the improved delivery of antimicrobial agents to specific sites of infection. For example, the treatment of skin disorders including (but not limited to) acne could be improved by means to concentrate antimicrobial agents in the pilosebaceous units.
In seeking to address such problems, the inventors have surprisingly found that conjugating an antimicrobial agent to a tocopheryl or tocotrienyl group can allow the agent to concentrate in the pilosibaceous units. The inventors have further appreciated that this concentration may lead to multiple benefits. For example, the localised concentration of the agent in the pilosebaceous units means that the overall amount of the agent which needs to be administered to a subject in order to treat a given infection may be reduced relative to prior formulations of the agent. Side effects caused by the agent acting on areas other than the site of infection may consequently be mitigated. Furthermore, by targeting the antimicrobial agent to the site of the infection, development of bacterial resistance caused by repeated administration to other areas of the skin may also be reduced.
Accordingly, the present disclosure provides a conjugate of formula (I):
D — L — P — X — Z — C /?
O— T [I] wherein
D is an antimicrobial agent;
L is a linker;
P is a polymeric spacer;
X is a group selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, -S-, -S(O)-, -SO2-, -NR10-, -NR10C(O)-, -C(O)NR10-, -NR10C(O)NRn-, -NR10C(O)O-, -OC(O)NR10, and a covalent bond;
Z is selected from Ci-12 alkylene, C2-12 alkenylene, and C2-12 alkynylene, wherein Z is unsubstituted or is substituted with 1, 2 or 3 substituents independently selected from halogen, -OR10, and -NR10Rn;
T is a tocopheryl or tocotrienyl group; and each R10 and R11 is independently selected from H and C1-2 alkyl.
In some embodiments, D is an antibiotic agent, an antifungal agent, or a comedolytic retinoid, typically an antibiotic or antifungal agent.
In some embodiments, D is an antibiotic or antifungal macrolide. In some embodiments, D is selected from erythromycin, adapalene, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline; and pharmaceutically acceptable salts thereof. In some embodiments, D is an antibiotic or antifungal macrolide. In some embodiments, D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline; and pharmaceutically acceptable salts thereof. In some embodiments, D is erythromycin or a pharmaceutically acceptable salt thereof or adapalene or a pharmaceutically acceptable salt thereof. In some embodiments, D is erythromycin or a pharmaceutically acceptable salt thereof.
In some embodiments, P is a poly(ethylene glycol) (PEG), a poly(lactic acid) (PLA), a poly(lactic-co-glycolic acid) (PLGA), a polycaprolacton (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), a polyacrylamide (PAM), hyaluronic acid, heparin, polysialic acid, or a polypeptide. In some embodiments, P is poly(ethylene glycol) (PEG). In some embodiments, P has an average molecular weight of from about 100 to about 10,000 Da, more typically from about 500 to about 5000 Da, still more typically 1,000 Da.
In some embodiments, X is -OC(O)-, -C(O)O- or -C(O)-.
In some embodiments, T is vitamin E or a derivative thereof. More often, T is
Figure imgf000006_0001
wherein the wavy line indicates the point of attachment to the moiety -OC(O)-Z-X-
P-L-D.
In some embodiments, L is -C(O)-L1-C(O)-; and antimicrobial agent D comprises a hydroxyl group which is esterified with the linker (L) at one of the -C(O)- moieties such that the moiety D-L- is a group of formula (Q)
Figure imgf000006_0002
wherein the wavy line indicates the point of attachment to the moiety -P-X-Z-C(O)O-T; and L1 is selected from Ci-i8 alkylene, C2-I8 alkenylene, and C2-I8 alkynylene, wherein L1 is unsubstituted or is substituted with 1, 2 or 3 substituents selected from halogen, -OR10, and -NR10Rn.
In some embodiments, the conjugate is of Formula (II)
Figure imgf000007_0001
wherein n is an integer from 1 to about 50; wherein often D is erythromycin and the conjugate is of Formula (III)
Figure imgf000007_0002
In some embodiments, n is an integer from about 10 to about 30.
In some embodiments, L1 is unsubstituted Ci-12 alkylene. More typically L1 is unsubstituted C2-7 alkylene.
In some embodiments, Z is unsubstituted Ci-12 alkylene. More typically, Z is unsubstituted C1-4 alkylene. In some embodiments, L is -O-l - O)-; and antimicrobial agent D comprises a carboxylic acid group which is esterified with the linker (L) such that the moiety D-L- is a group of formula (QI)
Figure imgf000007_0003
wherein - the wavy line indicates the point of attachment to the moiety -P-X-Z-C(O)O-T; and
L1 is selected from Ci-18 alkylene, C2-I8 alkenylene, and C2-I8 alkynylene, wherein L1 is unsubstituted or is substituted with 1, 2 or 3 substituents selected from halogen, -OR10, and -NR10Rn. In some embodiments, the moiety X-Z forms one or more -O-CH2CH2 (PEG) units. In some embodiments, the conjugate comprises a moiety of Formula (VI)
Figure imgf000008_0001
In some embodiments, D is adapalene.
Also provided herein is a pharmaceutical formulation for topical administration, comprising a plurality of micelles in a vehicle, wherein said micelles each comprise a plurality of conjugates as described herein. In some embodiments, said micelles each comprise (i) a core comprising the tocopheryl or tocotrienyl groups of the conjugates and (ii) a solvent-accessible surface; and wherein the antimicrobial agent of the conjugates extends into the solvent from the solvent-accessible surface.
Also provided herein is a pharmaceutical composition comprising a conjugate as described herein and one or more pharmaceutically acceptable excipient, carrier and/or diluent. In some embodiments, the pharmaceutical composition is for topical administration.
Also provided herein is a conjugate as described herein, a pharmaceutical formulation as described herein, or a pharmaceutical composition as described herein, for use in treating a microbial skin infection in a subject in need thereof.
In some embodiments, said conjugate, pharmaceutical formulation or pharmaceutical composition is for use in treating acne.
In some embodiments, the microbial skin infection is a bacterial infection caused by bacteria of genus Cutibacterium (e.g. Cutibacterium acnes), Streptococcus (e.g. Streptococcus pyogenes), Staphylococcus (e.g. Staphylococcus aureus), Pseudomonas (e.g. Pseudomonas aeruginosa) or Corynebacterium (e.g. Corynebacterium minutissimum).
In some embodiments, said conjugate, pharmaceutical formulation or pharmaceutical composition is for use by topically administering said conjugate, pharmaceutical formulation or pharmaceutical composition to said subject.
Brief Description of the Figures
Figure 1. MS spectrum of the intermediate ES (compound 1) in positive mode - the expected mass was 835.00 (M+H+) and the result obtained is 816.60 (M-H20+H+). Results described in the examples. Figure 2. XH-NMR spectrum (A) and 13C-NMR spectrum (B) of compound 1 in CDCI3.
Results described in the examples.
Figure 3. MALDI-TOF spectrum of compound 2 - the expected mass was of 2359.47 and the result obtained is 2363.301. Results described in the examples.
Figure 4. XH-NMR spectrum of compound 2 - the main protons of TPGS and the compound 1 were identified which confirms the structure. Results described in the examples.
Figure 5. UPLC-UV-ELSD chromatogram of compound 2. After purification, some TPGS is still present. The ratio between the compound 2 and TPGS is 20:7. Results described in the examples.
Figure 6. Foam (A) and Nile Red (B) tests to control the ability of compound 2 to form micelles - Water is used as a negative control and TPGS as a positive control. Results described in the examples.
Figure 7. Size distribution by number of the micelles of TPGS (A) and the micelles of compound 2 (B) by Zetasizer. Results described in the examples.
Figure 8. Zeta potential measurement for compound 2. Results described in the examples.
Figure 9. TEM images of (A) TPGS micelles at a magnification of 50’000; and the prodrug (compound 2) micelles at a magnification of 50'000 (B) and 100'000 (C). The scale bar is 100 nm in (A) and 50 nm in (B) and (C). Results described in the examples.
Figure 10. Stability of compound 2 at pH 5. Results described in the examples.
Figure 11. Stability of compound 2 at pH 7. Results described in the examples.
Figure 12. Stability of compound 2 at pH 9. Results described in the examples. Figure 13. Summary of the stability of compound 2 at varying pH. Results described in the examples.
Figure 14. Stability of compound 2 in contact with porcine liver esterase. Results described in the examples.
Figure 15. UPLC-UV-ELSD analysis of the erythromycin standard in green (single peak at -2.93) and the product of degradation after esterase hydrolysis of TPGS-ES in purple (split peak). Results described in the examples.
Figure 16. Stability of compound 2 in contact with an extract of porcine skin. Results described in the examples.
Figure 17. Summary of the stability of compound 2 in contact with porcine liver esterase and skin extract. Results described in the examples.
Figure 18. Nile Red tests to control the ability of compound 3 to form micelles - Water is used as a negative control and TPGS as a positive control. Results described in the examples.
Figure 19. TEM images (50,000 x magnification) of (A) TPGS micelles; and the prodrug (compound 3) micelles (B; and zoomed in (C)). The scale bar is 50 nm in (A) and (B) and 10 nm (C). Results described in the examples.
Figure 20. Release of erythromycin (A) and azelaic acid (B) from compound 3 administered to porcine skin samples comprising and excluding PSUs. For each skin sample the left hand bar shows results for skin samples comprising PSUs and the right hand bar shows results for skin samples excluding PSUs. Results described in the examples.
Figure 21. Thin-layer chromatography (TLC) monitoring of the reaction progress of Example 3 (formation of an adapalene-copolymer conjugate), TLC taken after 20 hours. Figure 22. 1H NMR spectrum of adapalene (ADA) in CDCL3.
Figure 23. 1H NMR spectrum of mPEG-PLGA in CDCI3
Figure 24. 1H NMR spectrum of ADA-mPEG-PLGA conjugate in DMSO.
Figure 25. P31 NMR spectrum of ADA-mPEG-PLGA conjugate in DMSO.
Figure 26. UHPLC-PDA chromatogram showing ADA retention time.
Figure 27. UHPLC-PDA chromatogram showing generation of ADA and ADA-copolymer conjugate (new product) after saponification reaction.
Figure 28. Characterization of the micelle formulation for size distribution using DLS (size in nm).
Detailed Description
The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein.
The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment.
It should be appreciated that “embodiments” of the disclosure can be specifically combined together unless the context indicates otherwise. The specific combinations of all disclosed embodiments (unless implied otherwise by the context) are further disclosed embodiments of the claimed invention.
In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes two or more polynucleotides, reference to “a polynucleotide-handling protein” includes two or more such proteins, reference to “a helicase” includes two or more helicases, reference to “a monomer” refers to two or more monomers, reference to “a pore” includes two or more pores and the like.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
Definitions
As used herein, a Ci-20 alkyl group is a linear or branched alkyl group containing from 1 to 20 carbon atoms. A Ci-20 alkyl group is often a Ci-18 alkyl group, or a C10-20 alkyl group. A C10-20 alkyl group is often a C14-I8 alkyl group such as a Ci6 alkyl group. A Ci-18 alkyl group is often a Ci-12 alkyl group such as a C1-9 alkyl group, e.g. a C1-7 alkyl group. A C1-7 alkyl group may be C1-6 alkyl group such as a C1-4 alkyl group. Examples include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl. A C1-2 alkyl group is methyl or ethyl, typically methyl. For the avoidance of doubt, where two alkyl groups are present, the alkyl groups may be the same or different. As used herein, a C2-20 alkenyl group is a linear or branched alkenyl group containing from 2 to 20 carbon atoms and having one or more, e.g. one or two or three, typically one double bonds. A C2-20 alkenyl group is often a C2-I8 alkenyl group, or a C10-20 alkenyl group. A C10-20 alkenyl group is often a C14-I8 alkenyl group such as a Ci6 alkenyl group. A C2-I8 alkenyl group is often a C2-12 alkenyl group such as a C2-9 alkenyl group, e.g. a C2-7 alkenyl group. A C2-7 alkenyl group may be C2-6 alkenyl group such as a C2-4 alkenyl group. Examples include ethenyl, propenyl and butenyl. For the avoidance of doubt, where two alkenyl groups are present, the alkenyl groups may be the same or different.
As used herein, a C2-20 alkynyl group is a linear or branched alkynyl group containing from 2 to 20 carbon atoms and having one or more, e.g. one or two or three, typically one triple bonds. A C2-20 alkynyl group is often a C2-I8 alkynyl group, or a C10-20 alkynyl group. A C10-20 alkynyl group is often a C14-I8 alkynyl group such as a Ci6 alkynyl group. A C2-I8 alkynyl group is often a C2-12 alkynyl group such as a C2-9 alkynyl group, e.g. a C2-7 alkynyl group. A C2-7 alkynyl group may be C2-6 alkynyl group such as a C2-4 alkynyl group. Examples include ethynyl, propynyl and butynyl. For the avoidance of doubt, where two alkynyl groups are present, the alkynyl groups may be the same or different.
As used herein, a Ci-20 alkylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from a Ci-20 alkane. The two hydrogen atoms may be removed from the same carbon atom or from different carbon atoms. Typically, a C1-20 alkylene group is a Ci-18 alkylene group, such as a Ci-12 alkylene group e.g. a C1-9 alkylene group. Typically a C1-9 alkylene group is a C1-7 or C1-6 alkylene group. Examples include methylene, ethylene, n-propylene, iso-propylene, n-butylene, sec -butylene and tertbutylene. For the avoidance of doubt, where two alkylene groups are present, the alkylene groups may be the same or different.
As used herein, a C2-20 alkenylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from a C2-20 alkene. The two hydrogen atoms may be removed from the same carbon atom or from different carbon atoms. Typically, a C2-20 alkenylene group is a C2-I8 alkenylene group, such as a C2-12 alkenylene group e.g. a C2-9 alkenylene group. Typically a C2-9 alkenylene group is a C2-7 or C2-6 alkenylene group. Examples include ethenylene, n-propenylene, iso-propenylene, n-butenylene, sec- butenylene and tert-butenylene. For the avoidance of doubt, where two alkenylene groups are present, the alkenylene groups may be the same or different.
As used herein, a C2-20 alkynylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from a C2-20 alkene. The two hydrogen atoms may be removed from the same carbon atom or from different carbon atoms. Typically, a C2-20 alkynylene group is a C2-I8 alkynylene group, such as a C2-12 alkynylene group e.g. a C2-9 alkynylene group. Typically a C2-9 alkynylene group is a C2-7 or C2-6 alkynylene group. Examples include ethynylene, n-propynylene, iso-prop ynylene, n-butynylene, sec- butynylene and tert-butynylene. For the avoidance of doubt, where two alkynylene groups are present, the alkynylene groups may be the same or different.
An alkyl, alkenyl, alkynyl, alkylene, alkenylene or alkynylene group as used herein may be unsubstituted or substituted. Unless otherwise stated, substituted alkyl, alkenyl or alkynyl groups typically carry one or more, e.g. 1, 2, 3 or 4, such as one, two or three e.g. one, or two, e.g. one substituent selected from halogen, -OR10, and -NR10Rn, wherein R10 and R11 are as defined herein. The substituents on a substituted alkyl, alkenyl or alkynyl group are typically themselves unsubstituted unless otherwise stated. Where more than one substituent is present, these may be the same or different.
As used herein, a halogen is typically chlorine, fluorine, bromine or iodine and is typically chlorine, bromine or fluorine, especially chorine or fluorine, especially fluorine.
As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as oxalic, citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p- toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines and heterocyclic amines. Hydrochloride salts and acetate salts are preferred, in particular hydrochloride salts. In Formula (I), the stereochemistry is not limited. In particular, compounds of Formula (I) containing one or more chiral centre may be used in enantiomerically or diastereoisomerically pure form, or in the form of a mixture of isomers. Further, for the avoidance of doubt, the compounds of the invention may be used in any tautomeric form. Typically, the agent or substance described herein contains at least 50%, typically at least 60, 75%, 90% or 95% of a compound according to Formula (I) which is enantiomerically or diasteriomerically pure. Typically, a compound of the invention comprises by weight at least 60%, such as at least 75%, 90%, or 95% of a single enantiomer or diastereomer. Typically, the compound is substantially optically pure.
Compounds of the Invention
Provided herein is a conjugate of formula (I):
D — L — P — X — Z — C /? O— T [I].
In formula (I), D is an antimicrobial agent. Typically, D is a hydrophilic agent. Often D is an antibiotic agent, an antifungal agent, or a comedolytic retinoid. Typically, D is an antibiotic or antifungal agent. More typically D is an antibiotic or antifungal macrolide. As used herein, the term “macrolide” relates to a compound comprising a macrocylic lactone ring, e.g. a 14-, 15- or 16-membered ring, to which one or more deoxy sugars, such as cladinose or desosamine, may be attached. However D is not limited to such compounds and other antimicrobial agents may be used. Most typically, D is an antibiotic agent.
Typically, D is selected from erythromycin, adapalene and derivatives thereof, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline; and pharmaceutically acceptable salts thereof. More typically, D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline; and pharmaceutically acceptable salts thereof. The structures of these compounds are shown below.
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Typically, in one aspect, D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; and pharmaceutically acceptable salts thereof. Typically, in another aspect, D is selected from erythromycin, clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, minocycline, and sarecycline and pharmaceutically acceptable salts thereof. Most typically, D is erythromycin or a pharmaceutically acceptable salt thereof, typically erythromycin.
When D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline, and pharmaceutically acceptable salts thereof, D is typically attached to the moiety -L-P-X-Z-C(O)O-T of formula (I) by a covalent bond to the L moiety. Similarly, when D comprises a compound of formula (IV) or formula (V) as described herein, or pharmaceutically acceptable salts thereof, D is typically attached to the moiety -L-P-X-Z-C(O)O-T of formula (I) by a covalent bond to the L moiety. The bond between D and L is typically formed by removing a hydrogen atom from a suitable position on D and forming a covalent bond to a suitable atom on L. The hydrogen atom that is removed may be a labile hydrogen atom e.g. at a side chain. For example, a hydrogen atom may be removed from an -OH group of D such that D is attached to L by an O-L bond thereby forming a moiety of form D-O-L-. A hydrogen atom may be removed from an -NH2 group of D such that D is attached to L by an NH-L bond thereby forming a moiety of form D-NH-L-. A hydrogen atom may be removed from a - C(0)-CH3 group of D such that D is attached to L by an CH2-L bond thereby forming a moiety of form D-C(0)-CH2-L-. A hydrogen atom may be removed from a -C(O)-OH group of D such that D is attached to L by an O-L bond thereby forming a moiety of form D-C(O)-O-L-.
Typically, when D is a macrolide comprising a macrocylic ring and one or more sugar rings and is attached to L by a bond to an -OH group on D, the -OH group is typically on a sugar ring. For example, when D is erythromycin, D is typically attached to L by a bond to an -OH group on D at one of the positions indicated by an asterisk (*) in the structure below:
Figure imgf000021_0001
In another aspect, D is a comedolytic retinoid. For example, D may be adapalene or a derivative thereof. In some embodiments, D is or comprises a compound of formula (IV) or a pharmaceutically acceptable salt thereof:
Figure imgf000021_0002
wherein
R1 is selected from H, C1-4 alkyl which is unsubstituted or is substituted with one or more substituents selected from -OH, -CN, -NH2 or halogen; and C2-4 alkenyl;
R2, R3, R4, R5, R6, R7, R8, R9 and R13 may be the same or different and are each independently selected from H, C1-4 alkyl which is unsubstituted or is substituted with one or more substituents selected from -OH, -CN, -NH2 or halogen; halogen, and -OR12; and - R14 is selected from H; C1-4 alkyl which is unsubstituted or is substituted with one or more substituents selected from -OH, -CN, -NH2 and halogen; C2-4 alkenyl, and -OR12; each R12 is independently selected from H and methyl.
Typically, in formula (IV), R1 is selected from H, methyl or ethyl, most typically H or methyl, and more preferably methyl.
Typically, in formula (IV), R2, R3, R4, R5, R6, R7, R8, R9 and R13 are each independently selected from H, methyl, -OMe, and -OH. Typically one or two of R2, R3, R4, R5, R6, R7, R8, R9 and R13 are each independently selected from methyl, -OMe, and -OH, and the remainder of R2, R3, R4, R5, R6, R7, R8, R9 and R13 are H. Typically all of R2, R3, R4, R5, R6, R7, R8, R9 and R13 are H.
Typically, in formula (IV), R14 is selected from H, unsubstituted C1-4 alkyl, and -OR12; wherein R12 is selected from H and methyl. Often R14 is -OR12; wherein R12 is selected from H and methyl. Preferably, R14 is -OH.
Therefore, preferably in formula (IV):
- R1 is methyl;
- R2, R3, R4, R5, R6, R7, R8, R9 and R13 are each H; and
- R14 is -OR12 wherein R12 is H.
D may thus be a compound of formula (V) or a pharmaceutically acceptable salt thereof:
Figure imgf000022_0001
A comedolytic retinoid, as described herein, is typically a compound of formula (IV) or (V) and pharmaceutically acceptable salts thereof. Formula (V) represents the structure of adapalene. When D is a compound of formula (IV), D is often attached to the moiety L by a covalent bond to an oxygen atom at position R14.
In formula (I), L is a linker between D and P. Typically, L comprises or consists of a polymeric linker, typically comprising a reactive functional group at each end of the linker for reaction with D and P, respectively. The reactive functional group for reaction with D may be the same or different to the reactive functional group for reaction with P. For example, the or each reactive functional group may comprise -C(O)-, -C(O)O-, -O-, etc. For example, the or each reactive functional group may comprise -C(O)-, -C(O)O-, etc.
Typically, in formula (I), L is a group of form -C(O)-L1-C(O)-; and D comprises a hydroxyl group which is esterified with the linker (L) at one of the -C(O)- moieties such that the moiety D-L- is a group of formula (Q)
Figure imgf000023_0001
wherein the wavy line indicates the point of attachment to the moiety -P-X-Z-C(O)O-T.
In another embodiment, L is a group of form -O-L1-C(O)-, and D comprises a carboxylic acid group (e.g. a group -C(O)-R14 wherein R14 is OH) and the carboxylic acid group is esterified with the linker (L) such that the moiety D-L- is a group of formula (QI)
Figure imgf000023_0002
wherein the wavy line indicates the point of attachment to the moiety -P-X-Z-C(O)O-T.
In some embodiments, the moiety X-Z forms one or more -O-CH2CH2 (PEG) units.
Accordingly, in some embodiments, the conjugate provided herein comprises a moiety of Formula (VI)
Figure imgf000023_0003
wherein PLGA is a polymeric spacer such as poly(lactic-co-glycolic acid). In some embodiments, the (PLGA)n(OCH2CH2) moiety represents a PLGA-PEG conjugate having an average molecular mass of about 2 to about 5 kDa, such as about 3 to about 4 kDa.
When a conjugate provided herein comprises a moiety of formula (Q) or (QI), the conjugate preferably comprises a moiety of formula (Q).
Typically, in formula (Q) or (QI) [preferably (Q)], L1 is selected from Ci-18 alkylene, C2-I8 alkenylene, and C2-I8 alkynylene, wherein L1 is unsubstituted or is substituted with 1, 2 or 3 substituents selected from halogen, -OR10, and -NR10Rn. When L1 is selected from Ci-18 alkylene, C2-I8 alkenylene, and C2-I8 alkynylene, the alkylene, alkenylene or alkynylene group may be linear or branched, typically the alkylene, alkenylene or alkynylene group is linear.
More typically, L1 is selected from Ci-12 alkylene, and C2-12 alkenylene, wherein L1 is unsubstituted or is substituted with 1 or 2 substituents selected from -OR10, and - NR10Rn. More typically, L1 is selected from C1-9 alkylene and C2-9 alkenylene, wherein L1 is unsubstituted or is substituted with 1 substituent selected from -OR10, and -NR10Rn.
Still more typically, L1 is unsubstituted C1-7 alkylene. For example, L1 may be unsubstituted methylene, ethylene (C2 alkylene), C3 alkylene, C4 alkylene, C5 alkylene, Cf> alkylene, or C7 alkylene. When L1 is unsubstituted methylene L thus corresponds to malonate. When L1 is unsubstituted ethylene (C2 alkylene) L thus corresponds to succinate. When L1 is unsubstituted C3 alkylene L thus corresponds to glutarate. When L1 is unsubstituted C4 alkylene L thus corresponds to adipate. When L1 is unsubstituted C5 alkylene L thus corresponds to pimelate. When L1 is unsubstituted Cf> alkylene L thus corresponds to suberiate. When L1 is unsubstituted C7 alkylene L thus corresponds to azelate. When L1 is unsubstituted Cs alkylene L thus corresponds to sebacate. In preferred embodiments L1 may be unsubstituted C2 to C7 alkylene, e.g. typically succinate or azelate.
In formula (I), T is a tocopheryl or tocotrienyl group. Tocopheryl groups are derived from tocopherol compounds. Tocotrienyl groups are derived from tocotrienol compounds. Tocopherol and tocotrienyl groups comprise a cyclic substituted chromane ring with a hydroxyl group comprising the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I); and a hydrophobic side chain. The hydroxyl group of the chromane ring may be esterified with the C(O)O moiety of the Formula (I) moiety thereby forming the conjugate. Tocopheryl and corresponding tocotrienyl groups are typically structurally related, with tocophenyl groups typically comprising an unsaturated alkyl side chain and tocotrienyl groups differing from the corresponding tocopheryl groups by the presence of three double bonds in the side chain which thus comprises a alkenyl side chain.
Typically, T is vitamin E or a derivative thereof.
Typically, T is a moiety of formula (W):
Figure imgf000025_0001
wherein: each Ra, Rb, Rc and Rd are independently H or unsubstituted C1-3 alkyl; and
Re is C1-20 alkyl, C2-20 alkenyl or C2-20 alkynyl, wherein Re is unsubstituted or is substituted by 1, 2 or 3 substituents independently selected from halogen, -OR10, and -NR10Rn; and the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I).
Typically, in formula (W):
Ra is H or methyl, typically methyl;
Rb is H or methyl, typically methyl;
Rc is H or methyl, typically methyl;
- Rd is H or methyl, typically methyl; and
Re is C10-20 alkyl or C10-20 alkenyl; typically C10-20 alkyl; more typically C14-I8 alkyl.
Typically, in formula (W), the dashed bond is absent so that the indicated bond is a single C-C bond.
Typically, in formula (W): i) Ra is methyl; Rb is methyl; Rc is methyl; Rd is methyl; Re is C 10-20 alkyl or C 10-20 alkenyl; and the dashed bond is absent so that the indicated bond is a single C-C bond; or ii) Ra is methyl; Rb is H; Rc is methyl; Rd is methyl; Re is C 10-20 alkyl or C 10-20 alkenyl; and the dashed bond is absent so that the indicated bond is a single C-C bond; or iii) Ra is H; Rb is methyl; Rc is methyl; Rd is methyl; Re is C 10-20 alkyl or C 10-20 alkenyl; and the dashed bond is absent so that the indicated bond is a single C-C bond; or iv) Ra is H; Rb is H; Rc is methyl; Rd is methyl; Re is C 10-20 alkyl or C 10-20 alkenyl; and the dashed bond is absent so that the indicated bond is a single C-C bond.
Typically, in formula (W), Re is C10-20 alkyl; more typically C14-I8 alkyl. Typically
Re is selected from:
Figure imgf000026_0001
Typically, the moiety of formula (W) is a moiety of formula (W 1):
Figure imgf000026_0002
wherein Ra, Rb, Rc, Rd, Re, the dashed bond and the wavy line are as defined for formula (W).
More typically, in formula (W): i) Ra is methyl; Rb is methyl; Rc is methyl; Rd is methyl; and Re is
Figure imgf000026_0003
such that formula (W) is a-tocopheryl; or ii) Ra is methyl; Rb is H; Rc is methyl; Rd is methyl; Re
Figure imgf000026_0004
such that formula (W) is P-tocopheryl; or iii) Ra is H; Rb is methyl; Rc is methyl; Rd is methyl; Re is
Figure imgf000026_0005
such that formula (W) is y-tocopheryl; or iv) Ra is H; Rb is H; Rc is methyl; Rd is methyl; Re is
Figure imgf000027_0001
such that formula (W) is 6-tocopheryl; and the dashed bond is absent so that the indicated bond is a single C-C bond.
Most typically, in formula (W) Ra is methyl; Rb is methyl; Rc is methyl; Rd is methyl; and Re is
Figure imgf000027_0002
such that formula (W) is a- tocopheryl.
Therefore, typically, T is:
Figure imgf000027_0003
wherein the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I).
Typically, therefore, the conjugate of formula (I) is of formula (la) or (lb):
Figure imgf000027_0004
wherein:
D is selected from erythromycin, adapalene and derivatives thereof, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; and is attached to the - C(O)-L1-C(O)-P-X-Z-C(O)O-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and
L1 is selected from Ci-12 alkylene, and C2-12 alkenylene, wherein L1 is unsubstituted or is substituted with 1 or 2 substituents selected from -OR10, and -NR10Rn;
T is a moiety of formula (W):
Figure imgf000028_0001
wherein: each Ra, Rb, Rc and Rd are independently H or unsubstituted C1-3 alkyl; and
Re is C1-20 alkyl, C2-20 alkenyl or C2-20 alkynyl, wherein Re is unsubstituted or is substituted by 1, 2 or 3 substituents independently selected from halogen, - OR10, and -NR10Rn; and the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I); and P, X, and Z are as defined herein.
Typically, therefore, the conjugate of formula (I) is of formula (la):
Figure imgf000028_0002
wherein:
D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; and is attached to the -C(0)-L1-C(0)-P-X-Z-C(0)0-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and L1 is selected from C 1-12 alkylene, and C2-12 alkenylene, wherein L1 is unsubstituted or is substituted with 1 or 2 substituents selected from -OR10, and -NR10Rn;
T is a moiety of formula (W):
Figure imgf000029_0001
wherein: each Ra, Rb, Rc and Rd are independently H or unsubstituted C1-3 alkyl; and
Re is C1-20 alkyl, C2-20 alkenyl or C2-20 alkynyl, wherein Re is unsubstituted or is substituted by 1, 2 or 3 substituents independently selected from halogen, -
OR10, and -NR10Rn; and the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I); and P, X, and Z are as defined herein.
More typically, the conjugate is of formula (la) or (lb), wherein:
D is selected from erythromycin, adapalene, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; and is attached to the
Figure imgf000029_0002
X-Z-C(O)O-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and
L1 is unsubstituted C1-7 alkylene;
T is a moiety of formula (W):
Figure imgf000029_0003
wherein:
Ra is H or methyl, typically methyl;
Rb is H or methyl, typically methyl;
Rc is H or methyl, typically methyl;
- Rd is H or methyl, typically methyl; and
Re is Cio-20 alkyl or C10-20 alkenyl; typically C10-20 alkyl; more typically C14-I8 alkyl; and the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I); and P, X, and Z are as defined herein.
More typically, the conjugate is of formula (la), wherein:
D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; and is attached to the -QOj-L'-QOj-P-X-Z-QOjO-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and L1 is unsubstituted C1-7 alkylene;
T is a moiety of formula (W):
Figure imgf000030_0001
wherein:
Ra is H or methyl, typically methyl;
Rb is H or methyl, typically methyl;
Rc is H or methyl, typically methyl;
- Rd is H or methyl, typically methyl; and
Re is C10-20 alkyl or C10-20 alkenyl; typically C10-20 alkyl; more typically C14-I8 alkyl; and the wavy line indicates the point of attachment to the D-L-P-X-Z-C(O)O- moiety of Formula (I); and P, X, and Z are as defined herein.
In formula (I), P is a polymeric spacer.
Typically, P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), a polycaprolactone (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), a polyacrylamide (PAM), hyaluronic acid, heparin, polysialic acid, or a polypeptide. More typically, P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), a polycaprolactone (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), or a polyacrylamide (PAM). Still more typically, P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), or a polycaprolactone (PCL). More typically P is a poly(ethylene glycol) (PEG).
Typically, P has an average molecular weight of from about 100 Da to about 10,000 Da (10 kDa). More typically, P has an average molecular weight of from about 200 Da to about 5 kDa. Still more typically P has an average molecular weight of from about 300 Da to about 3 kDa, such as from about 500 Da to about 2 kDa, e.g. from about 700 to about 1500 Da, such as from about 800 to about 1200 Da, such as from about 900 to about 1100 Da, e.g. about 1 kDa. The molecular weight of P is typically the number average molecular weight, as determined e.g. by MALDI mass spectroscopy.
Typically, P comprises a polymer comprising from about 5 to about 50 monomer units, such as from about 10 to about 40 monomer units, e.g. from about 15 to about 30 monomer units, e.g. from about 18 to about 28 monomer units, e.g. from about 20 to about 25 monomer units.
Typically, therefore, P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), a polycaprolactone (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), or a polyacrylamide (PAM) having an average molecular weight of from about 100 Da to about 10 kDa. More typically, P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co- glycolic acid) (PLGA), or a polycaprolactone (PCL) having an average molecular weight of from about 200 Da to about 5 kDa. More typically P is a poly(ethylene glycol) (PEG) having an average molecular weight of from about 500 Da to about 2 kDa, e.g. from aobut 700 to about 1500 Da. Most typically P is poly(ethylene glycol) (PEG) having an average molecular weight of about 1 kDa. As those skilled in the art will appreciate, a PEG having an average molecular weight of about 1 kDa typically comprises about 23 monomer ethylene glycol units. In some embodiments, P is PLGA having an average molecular weight of about 4 kDa.
In formula (I), X is a group selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, -S-, -S(O)-, -SO2-, -NR10-, -NR10C(O)-, -C(O)NR10-, -NR10C(O)NRn-, -NR10C(O)O-, -OC(O)NR10, and a covalent bond. Typically, X is selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, -NR10-, -NR10C(O)-, -C(O)NR10-, -NR10C(O)NRn-, -NR10C(O)O-, and -OC(O)NR10. More typically, X is selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, NR10C(O)-, and -C(O)NR10-. More typically, X is selected from -OC(O)-, -C(O)O-, -C(O)-, and -O-. Yet more typically X is selected from -OC(O)-, -C(O)O- and -C(O)-. Most typically, X is -OC(O)-.
In formula (I), Z is selected from Ci-12 alkylene, C2-12 alkenylene, and C2-12 alkynylene, wherein Z is unsubstituted or is substituted with 1, 2 or 3 substituents independently selected from halogen, -OR10, and -NR10Rn. More typically, Z is selected from C1-6 alkylene, and C2-6 alkenylene, wherein Z is unsubstituted or is substituted with 1 or 2 substituents independently selected from -OR10, and -NR10Rn. Still more typically, Z is selected from C1-4 alkylene, and C2-4 alkenylene, wherein Z is unsubstituted or is substituted with 1 substituent selected from -OR10, and -NR10Rn. Yet more typically, Z is unsubstituted C1-4 alkylene, typically methylene or ethylene, most typically ethylene (C2 alkylene).
Typically, therefore, in formula (I):
P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co- glycolic acid) (PLGA), a polycaprolactone (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), or a polyacrylamide (PAM) having an average molecular weight of from about 100 Da to about 10 kDa;
- X is selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, -NR10-, -NR10C(O)-, -C(O)NR10-, -NR10C(O)NRn-, -NR10C(O)O-, and -OC(O)NR10; and Z is selected from C1-6 alkylene, and C2-6 alkenylene, wherein Z is unsubstituted or is substituted with 1 or 2 substituents independently selected from -OR10, and -NR10Rn. More typically, in formula (I):
P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co- glycolic acid) (PLGA), or a polycaprolactone (PCL) having an average molecular weight of from about 200 Da to about 5 kDa;
- X is selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, NR10C(O)-, and -C(O)NR10-; and
Z is selected from C1-4 alkylene, and C2-4 alkenylene, wherein Z is unsubstituted or is substituted with 1 substituent selected from -OR10, and -NR10Rn.
In formula (I), each R10 and R11 is independently selected from H and C1-2 alkyl. More typically, each R10 and R11 is independently selected from H and methyl. Most typically, each R10 and R11 is H.
In one preferred aspect, therefore, the conjugate of formula (I) is of formula (la) or (lb):
Figure imgf000033_0001
wherein:
D is selected from erythromycin, adapalene and derivatives thereof, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; typically erythromycin or a pharmaceutically acceptable salt thereof; and D is attached to the -C(O)-L1-C(O)-P-X- Z-C(O)O-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and L1 is selected from unsubstituted C1-9 alkylene and unsubstituted C2-9 alkenylene;
T is a moiety of formula (W):
Figure imgf000034_0001
wherein: o Ra is H or methyl; o Rb is H or methyl; o Rc is H or methyl; o Rd is H or methyl; and
Figure imgf000034_0002
P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co- glycolic acid) (PLGA), or a polycaprolactone (PCL) having an average molecular weight of from about 200 Da to about 5 kDa;
X is selected from -OC(O)-, -C(O)O-, -C(O)-, and -O-; and
Z is selected from unsubstituted Ci-6 alkylene and unsubstituted C2-6 alkenylene.
In one preferred aspect, therefore, the conjugate of formula (I) is of formula (la):
Figure imgf000034_0003
wherein:
D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; typically erythromycin or a pharmaceutically acceptable salt thereof; and D is attached to the -C(O)-L1-C(O)-P-X-Z-C(O)O-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and
L1 is selected from unsubstituted C1-9 alkylene and unsubstituted C2-9 alkenylene;
T is a moiety of formula (W):
Figure imgf000035_0001
wherein: o Ra is H or methyl; o Rb is H or methyl; o Rc is H or methyl; o Rd is H or methyl; and
Figure imgf000035_0002
P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co- glycolic acid) (PLGA), or a polycaprolactone (PCL) having an average molecular weight of from about 200 Da to about 5 kDa;
X is selected from -OC(O)-, -C(O)O-, -C(O)-, and -O-; and
Z is selected from unsubstituted C1-6 alkylene and unsubstituted C2-6 alkenylene.
In another preferred aspect, therefore, the conjugate of formula (I) is of formula (la) or
(lb):
Figure imgf000035_0003
wherein:
D is selected from erythromycin, adapalene, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; typically erythromycin or a pharmaceutically acceptable salt thereof; and D is attached to the -C(O)-L1-C(O)-P-X-Z-C(O)O-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and
L1 is unsubstituted C1-7 alkylene;
T is
Figure imgf000036_0001
P is a poly(ethylene glycol) (PEG) having an average molecular weight of from about 500 Da to about 2 kDa;
X is selected from -OC(O)-, -C(O)O- and -C(O)-; and Z is unsubstituted C1-4 alkylene.
In another preferred aspect, therefore, the conjugate of formula (I) is of formula (la):
Figure imgf000036_0002
wherein:
D is selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, cethromycin; clindamycin, tetracycline, metronidazole, doxycycline, minocycline, and sarecycline; and pharmaceutically acceptable salts thereof; typically erythromycin or a pharmaceutically acceptable salt thereof; and D is attached to the -C(0)-L1-C(0)-P-X-Z-C(0)0-T moiety at an -OH group of D thereby forming the conjugate of formula (la); and
L1 is unsubstituted C1-7 alkylene;
T is
Figure imgf000036_0003
P is a poly(ethylene glycol) (PEG) having an average molecular weight of from about 500 Da to about 2 kDa;
X is selected from -OC(O)-, -C(O)O- and -C(O)-; and Z is unsubstituted C1-4 alkylene.
In a preferred aspect the conjugate of formula (I) is a conjugate of formula (II):
Figure imgf000037_0001
wherein: n is an integer from 1 to about 50, typically from about 10 to about 30, more typically from about 15 to about 30, more typically from about 18 to about 28, more typically from about 20 to about 25;
D is as defined herein; typically D is erythromycin;
L1 is as defined herein; typically L1 is unsubstituted C1-7 alkylene; and
Z is as defined herein, typically Z is unsubstituted C1-4 alkylene.
Figure imgf000037_0002
wherein: n is an integer from about 10 to about 30, typically from about 15 to about 30, more typically from about 18 to about 28, more typically from about 20 to about 25;
L1 is as defined herein; typically L1 is unsubstituted C1-7 alkylene; and
Z is as defined herein, typically Z is ethylene.
A preferred conjugate of formula (I) is:
Figure imgf000038_0001
wherein n is from about 15 to about 30, more typically from about 18 to about 28, more typically from 20 to about 25, typically 20, 21, 22, 23, 24, or 25.
Figure imgf000038_0002
wherein n is from about 15 to about 30, more typically from about 18 to about 28, more typically from 20 to about 25, typically 20, 21, 22, 23, 24, or 25.
Another preferred conjugate provided herein is:
Figure imgf000039_0001
wherein each n is the same or different and is each independently selected from about 1 to about 50, such as from about 10 to about 30 e.g. about 20 to about 25, typically 20, 21, 22, 23, 24, or 25. In some embodiments each n is the same or different and is each independently selected from about 1 to about 10, such as from about 2 to about 5.
Without being bound by theory, the inventors believe that the conjugates provided herein may form micelles which preferentially localise in hair follicles of the skin. The drug moiety (D) is typically hydrophilic and is believed to localise at the surface of the micelles thereby produced. Those skilled in the art will appreciate that the delivery of a drug (D) via the conjugates provided herein, e.g. when formulated as micelles, is materially different to the delivery of hydrophobic drug molecules which may be concentrated in the hydrophobic core of a micelle e.g. formed by a lipid. For example, in the present disclosure, the dosage of the drug to the skin is determined by the number of conjugate monomers used to form the micelles which may be administered as described herein. This contrasts with methods in which a hydrophobic drug is contained in the hydrophobic core of a micelle in which the drug delivery depends on the loading of the micelle.
Without being bound by theory, the inventors believe that the improved localisation of the drug moiety (D) delivered by the conjugates provided herein may lead to decreased antibiotic resistance. Indiscriminate delivery of drugs to the skin e.g. via simple prior formulations of such drugs leads to high concentrations of the drug contacting bacteria which are not directly responsible for an ongoing infection at the point of administration, e.g. those colonising uninfected areas of the skin. Repeated low-level administration in this way may lead to the build-up of antibiotic resistance in such bacteria. The localised delivery of the drug comprised in the provided conjugates means that this build-up of resistance is decreased.
Without being bound by theory, the localisation of the drug moiety (D) on the surface of the micelle formed by the conjugates provided herein may lead to beneficial effects compared to micelles which encapsulate their drug loads, e.g. those comprising a hydrophobic drug encapsulated in a hydrophobic core. For example, the bioavailability of the drug is typically improved when surface accessible as provided herein. Side effects associated with the delivery of the conventional micelles to the skin e.g. from contact of the lipid shell of such micelles to uninfected areas of the skin may also be reduced as such contact is “shielded” by the drug molecules comprised in the provided conjugates.
Further without being bound by theory, the conjugates provided herein are believed to break down in the environment of the skin and in particular in the localised environment of hair follicles. In particular, the breakdown of the conjugates is believed to be beneficially catalysed by esterases expressed by pathogenic microbes which may colonise pilosebaceous units. Breakdown, e.g. by hydrolysis, of the conjugates provided herein releases the drug moiety (D) of the conjugate which exerts a localised therapeutic effect. The breakdown (e.g. hydrolysis) is optimal in the vicinity of pilosebaceous units due to the localised concentration of esterase-expressing pathogenic microbes therein (e.g. under disease conditions such as those described herein) meaning that improved and targeted delivery of the drug moiety (D) is achieved.
Further without being bound by theory, the inventors believe that the conjugates provided herein have beneficial transdermal permeation properties. IN particular, the inventors have found that the rate of transdermal penetration of the disclosed conjugates may be advantageously slowed (e.g. compared to the equivalent drug in unconjugated form) thereby improving the efficacy of the drug in treating a microbial skin infection and decreasing potentially harmful absorption into the body.
Also without being bound by theory, the inventors also believe that the therapeutic effect exerted by the conjugates provided herein may be enhanced by the co-release of the linker moiety L. For example when L comprises a group of form -C(O)-L1-C(O)- and L1 is a C7 alkylene group the moiety L corresponds to azelaic acid. Such carboxylic acids are believed to exert a further therapeutic effect in targeting microbial skin infestations such as acne. Accordingly, the therapeutic effect exerted by the conjugates provided herein is typically greater than that provided by prior formulations of the drug molecule (e.g. erythromycin). The therapeutic effect exerted by the conjugate may be at least additive, e.g. relative to the effect exerted by formulations of the drug moiety (D) and linking group (L) separately. The therapeutic effect exerted by the conjugate may be synergistic, e.g. relative to the effect exerted by formulations of the drug moiety (D) and linking group (L) separately.
Micelles
Without being bound by theory, the conjugates provided herein are believed to form micelles when contacted with a suitable vehicle.
As used herein, the term “micelle” relates to a supramolecular assembly of conjugate molecules. In the presence of an aqueous or substantially aqueous vehicle, conjugate molecules in a micelle typically assemble into a three dimensional structure comprising a hydrophobic core and a hydrophilic shell. The reverse structure may be adopted in the presence of a hydrophobic solvent, e.g. when the conjugate molecules are contacted with a non-aqueous or substantially non-aqueous hydrophobic vehicle.
Typically, the conjugates provided herein form micelles each comprising (i) a core comprising the tocopheryl or tocotrienyl groups of the conjugates and (ii) a solvent- accessible surface; wherein the antimicrobial agent of the conjugates extends into the solvent from the solvent-accessible surface. Typically the solvent-accessible surface is an aqueous-solvent-accessible surface.
Without being bound by theory the inventors believe that the disclosed conjugates form micelles in which the drug moiety (D) is typically wholly or substantially grafted to the shell of the micelle, and extends into the solvent (e.g. an aqueous solvent). Those skilled in the art will appreciate that this differs from many drug-containing micelles described in the art in which a drug molecule (typically a hydrophobic drug molecule) is generally localised in the core of the micelle. The micellar structures formed by the conjugates provided herein are advantageous as described herein.
The production of micelles by the conjugates provided herein can be confirmed by tests conventional in the art. For example, as described in the example, the successful formation of a conjugate can be confirmed using Nile Red assays and/or foaming assays. Nile Red assays can detect micelle formation as Nile Red is lipophilic and concentrates in the lipophilic core of micelles formed in aqueous solution. Foaming assays e.g. using TPGS as a control can be used to determine micelle formation. Typically, the conjugates provided herein form substantially spherical micelles. However, the invention is not limited to such and micelles having other shapes such as ellipsoids, cylinders, and bilayers, are within the scope of the invention. In particular, some micelles formed by conjugates provided herein adopt “flower” shapes known in the art. The shape may be determined e.g. by TEM imaging.
Typically, the conjugates provided herein have a critical micelle concentration in water (e.g. at 25 °C) of below 100 mg/mL, such as from about 10 ng/mL to about 100 mg/mL, e.g. from about 1 pg/mL to about 10 mg/mL, e.g. from about 10 pg/mL to about 1 mg/mL e.g. about 100 pg/mL. CMC can be determined by techniques that are routine in the art, such as by fitting observed parameters measured across a range of conjugate concentrations. Fit functions for properties such as electrical conductivity, surface tension, NMR chemical shifts, absorption, self-diffusion coefficients, fluorescence intensity and mean translational diffusion coefficients are known in the art.
Typically, the conjugates provided herein form micelles in aqueous solution (e.g. when assessed at concentrations of about 1 mg/mL) having an average size (e.g. a mean diameter as determined by TEM, or z-average diameter as assessed by DLS) of from about 2 to about 50 nm, e.g. from about 5 to about 25 nm such as from about 8 to about 15 nm, e.g. about 9 or 10 nm.
Typically, the conjugates provided herein form micelles in aqueous solution (e.g. when assessed at concentrations of about 1 mg/mL) having a zeta potential of from about - 100 mV to about +100 mV, e.g. from about 0 mV to about +80 mV, such as from about +20 mV to about +60 mV, e.g. from about +30 mV to about + 50 mV. Typically, the micelles formed by the conjugates provided herein have a positive potential (zeta potential > 0).
Typically, the conjugates provided herein form micelles in aqueous solution (e.g. when assessed at concentrations of about 1 mg/mL) having a polydispersity index of less than about 10, e.g. less than about 5, such as less than about 2, e.g. less than about 1. Typically the micelles formed by the conjugates provided herein are substantially monodisperse.
Typically, the vehicle in which the conjugates provided herein form micelles is a pharmaceutically acceptable vehicle. Such vehicles are described in more detail herein in the context of pharmaceutical formulations. Typically the vehicle is sterile and pyrogen free. Synthesis
The conjugates provided herein can be prepared by any suitable method. Detailed general synthetic routes for representative compounds of the invention are set out in the Examples.
In summary, conjugates provided herein can typically be prepared by functionalising the drug moiety (D) at an appropriate reactive functional group such as an alcohol group, e.g. an OH group, e.g. an OH group comprised in a sugar ring attached to a macrolide macrocycle, with a moiety comprising the linker L and having a further reactive functional group for downstream reaction. For example, a suitable moiety comprising linker L may comprise a carboxylic acid or anhydride equivalent thereof for reaction with the reactive functional group on drug moiety (D) thereby forming an ester bond to linking moiety L. Linker L may further comprise a second reactive functional group such as a carboxylic acid or anhydride equivalent thereof. Thus a suitable moiety comprising linker L may be a moiety of form OH-C(O)-L1-C(O)-OH wherein L1 is as described herein. Reaction of the second reactive functional group or an activated derivative thereof with the moiety P-X-Z-C(O)O-T (e.g. at a reactive functional group comprised in moiety P) may then yield the final conjugate. The moieties D, L and P-X-Z-C(O)O-T are typically commercially available or can be synthesized by standard techniques using methods disclosed in e.g. March’s Advanced Organic Chemistry, 8th edition (Wiley, 2020).
Pharmaceutical compositions and formulations
Provided herein is a pharmaceutical formulation comprising a plurality of micelles in a vehicle, wherein said micelles each comprise a plurality of conjugates as described herein. Typically, said micelles are as described herein. Typically the vehicle is a pharmaceutically acceptable vehicle such as a pharmaceutically acceptable solvent. Typically the vehicle is aqueous or substantially aqueous. Typically the vehicle comprises a buffered aqueous solution, e.g. buffered from pH 4 to pH 9 such as from pH 5 to pH 8 e.g. pH 6 to pH 7.
Also provided herein is a pharmaceutical composition comprising a conjugate as provided herein. Typically the composition comprises a pharmaceutically acceptable vehicle such as a pharmaceutically acceptable solvent. Typically the vehicle is aqueous or substantially aqueous. Typically, a pharmaceutical formulation or pharmaceutical composition provided herein may be for topical administration. The pharmaceutical formulation or pharmaceutical composition may be intended for and/or suitable for topical administration.
When the pharmaceutical formulation or pharmaceutical composition comprises a conjugate which is optically active, the conjugate is typically a substantially pure optical isomer.
Typically, the composition contains up to 85 wt% of a conjugate of the invention. More typically, it contains up to 50 wt% of a conjugate of the invention. For example the composition may comprise up to 20 wt% of a conjugate of the invention, such as up to 10 wt% of a conjugate of the invention.
Preferred pharmaceutical formulations and compositions are sterile and pyrogen free.
A pharmaceutical formulation or pharmaceutical composition may be provided as a kit comprising instructions to enable the kit to be used in the methods described herein or details regarding which subjects the method may be used for.
A pharmaceutical formulation or pharmaceutical composition as provided herein may comprise an additional therapeutic agent. The additional therapeutic agent may be for example selected from antibiotics, antiseptics, retinoids, hormones, etc.
In some embodiments the pharmaceutical formulation or pharmaceutical composition comprises one or more antibiotics. In some embodiments the pharmaceutical formulation or pharmaceutical composition comprises one or more antibiotics selected from aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, antibiotic glycopeptides and lipoglycopeptides (such as vancomycin), macrolides, monobactams (such as aztreonam), oxazolidinones (such as linezolid and tedizolid), penicillins, antibiotic polypeptides, rifamycins, sulphonamides, streptogramins (such as quinupristin and dalfopristin) and/or tetracyclines.
In embodiments wherein the pharmaceutical formulation or pharmaceutical composition comprises one or more antibiotics, the one or more antibiotics are typically selected from erythromycin, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and/or sarecycline. Typically when the pharmaceutical formulation comprises one or more antibiotics said antibiotic is a different antibiotic to the drug moiety (D) comprised in the conjugate. For example, the drug moiety (D) may be erythromycin or a pharmacueitcally acceptable salt thereof, and the conjugate may be formulated in a pharmaceutical formulation comprising azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and/or sarecycline.
In some embodiments the pharmaceutical formulation comprises a retinoid such as tretinoin, adapalene, isotretinoin, retinol, tazarotene, alitretinoin, and/or bexarotene.
In some embodiments the pharmaceutical formulation comprises an antiseptic agent such as benzoyl peroxide, chlorhexidine (e.g. chlorhexidine gluconate or acetate), povidone-iodine, chloroxylenol, ethyl alcohol, isopropyl alcohol, hexachlorophene, benzalkonium chloride, hydrogen peroxide, cetrimide, methylbenzethonium chloride, benzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, dofanium chloride, domiphen bromide, proflavine hemisulphate, triphenylmethane, brilliant green, crystal violet, gentian violet, potassium permanganate, chlorocresol, chloroxylenol, chlorophene, hexachlorophane/hexachlorophene, triclosan, hydroxyquinoline sulphate, potassium hydroxyquinoline sulphate, chlorquinaldol, dequalinium chloride and/or diiodohydroxyquinoline .
Also provided herein is therefore a combination of a conjugate as described herein with one or more further therapeutic agents, typically selected from antibiotics, antiseptics, retinoids, hormones, e.g. those agents described herein.
The two agents may be provided in a single formulation as described herein, or they may be separately formulated. Where separately formulated, the two agents may be administered simultaneously, separately or sequentially. They may be provided in the form of a kit, optionally together with instructions for their administration. The products may also be referred to herein as combinations or pharmaceutical combinations.
The conjugates provided herein are therapeutically useful. The present invention therefore provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in medicine. The present invention provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in treating the human or animal body. Also provided herein is a method of treating a subject in need thereof, comprising administering an effective amount of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein to said subject. Also provided is the use of a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament.
For the avoidance of doubt, a conjugate of the invention may be administered in the form of a solvate.
As explained above, the conjugates provided herein are useful in the treatment of skin disorders arising from microbial infection. Accordingly, the present invention provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in treating a microbial infection, e.g. a microbial skin infection, in a subject in need thereof. Also provided herein is a method of treating a microbial infection, e.g. a microbial skin infection, in a subject in need thereof, comprising administering an effective amount of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein to said subject. Also provided is the use of a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament for treating a microbial infection, e.g. a microbial skin infection.
The microbial infection may be caused by a bacterial infection such as infection by bacteria from the families Propionibacteriaceae, Streptococcaceae, Staphylococcaceae, Pseudomonadaceae, and/or Corynebacteriaceae. The bacterial infection may be caused by bacteria of genus Cutibacterium (e.g. Cutibacterium acnes), Streptococcus (e.g. Streptococcus pyogenes), Staphylococcus (e.g. Staphylococcus aureus), Pseudomonas (e.g. Pseudomonas aeruginosa) or Corynebacterium (e.g. Corynebacterium minutissimum). Typically, the bacterial infection is caused by Cutibacterium acnes or Staphylococcus aureus. The bacterial infection may be caused by an opportunistic pathogen.
Accordingly, the present invention provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in treating infection by Cutibacterium (e.g. Cutibacterium acnes), Streptococcus (e.g. Streptococcus pyogenes), Staphylococcus (e.g. Staphylococcus aureus), Pseudomonas (e.g. Pseudomonas aeruginosa) or Corynebacterium (e.g. Corynebacterium minutissimum), in a subject in need thereof. Also provided herein is a method of treating infection by Cutibacterium (e.g. Cutibacterium acnes), Streptococcus (e.g. Streptococcus pyogenes), Staphylococcus (e.g. Staphylococcus aureus), Pseudomonas (e.g. Pseudomonas aeruginosa) or Corynebacterium (e.g. Corynebacterium minutissimum) in a subject in need thereof, comprising administering an effective amount of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein to said subject. Also provided is the use of a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament for treating infection by Cutibacterium (e.g. Cutibacterium acnes), Streptococcus (e.g. Streptococcus pyogenes), Staphylococcus (e.g. Staphylococcus aureus), Pseudomonas (e.g. Pseudomonas aeruginosa) or Corynebacterium (e.g. Corynebacterium minutissimum).
The skin infection may be selected from acne, rosacea, cellulitis, erysipelas, microbial folliculitis, “hot tub” folliculitis, furuncles, carbuncles, impetigo, erythrasma, tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, and onychomycosis, typically acne. Accordingly, the present invention provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in treating acne, rosacea, cellulitis, erysipelas, microbial folliculitis, “hot tub” folliculitis, furuncles, carbuncles, impetigo, erythrasma, tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, and onychomycosis, typically acne, in a subject in need thereof. Also provided herein is a method of treating acne, rosacea, cellulitis, erysipelas, microbial folliculitis, “hot tub” folliculitis, furuncles, carbuncles, impetigo, erythrasma, tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, and onychomycosis, typically acne, in a subject in need thereof, comprising administering an effective amount of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein to said subject. Also provided is the use of a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament for treating acne, rosacea, cellulitis, erysipelas, microbial folliculitis, “hot tub” folliculitis, furuncles, carbuncles, impetigo, erythrasma, tinea corporis, tinea pedis, tinea cruris, pityroasos versicolor, cutaneous candidiasis, and onychomycosis, typically acne. Also provided herein is the use of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein in the targeted treatment of bacterial infection in hair follicles. Further provided herein is the use of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein in the targeted treatment of bacterial infection in pilosebaceous units.
As further explained above, the conjugates provided herein may be used in conjunction with one or more further therapeutic agents such as one or more antibiotics, antiseptics, retinoids, or hormones. Accordingly, the present invention provides a conjugate, pharmaceutical formulation or pharmaceutical composition described herein for use in treating a microbial infection, e.g. a microbial skin infection, in a subject in need thereof, wherein such use comprises co-administering the conjugate, pharmaceutical formulation or pharmaceutical composition of the invention with a further therapeutic agent e.g. a further therapeutic agent described herein. Also provided herein is a method of treating a microbial infection, e.g. a microbial skin infection, in a subject in need thereof, comprising co-administering an effective amount of a conjugate, pharmaceutical formulation or pharmaceutical composition described herein and a further therapeutic agent e.g. a further therapeutic agent described herein to said subject. Also provided is the use of a conjugate, pharmaceutical formulation or pharmaceutical composition as described herein in the manufacture of a medicament for treating a microbial infection, e.g. a microbial skin infection by co-administering the conjugate, pharmaceutical formulation or pharmaceutical composition described herein and a further therapeutic agent e.g. a further therapeutic agent described herein to a subject in need thereof.
In one aspect, the subject is a mammal, in particular a human. However, it may be non-human. Preferred non-human animals include, but are not limited to, primates, such as marmosets or monkeys, commercially farmed animals, such as horses, cows, sheep or pigs, and pets, such as dogs, cats, mice, rats, guinea pigs, ferrets, gerbils or hamsters. The subject can be any animal that is capable of being infected by a bacterium.
The conjugates, compositions and formulations described herein are useful in the treatment of bacterial infection which occurs after a relapse following an antibiotic treatment. The conjugates, compositions and formulations can therefore be used in the treatment of a patient who has previously received antibiotic treatment for the (same episode of) bacterial infection.
A conjugate, composition or formulation described herein can be administered to the subject in order to prevent the onset or reoccurrence of one or more symptoms of the bacterial infection. This is prophylaxis. In this embodiment, the subject can be asymptomatic. The subject is typically one that has been exposed to the bacterium. A prophylactically effective amount of the agent or formulation is administered to such a subject. A prophylactically effective amount is an amount which prevents the onset of one or more symptoms of the bacterial infection.
A conjugate, composition or formulation described herein can be administered to the subject in order to treat one or more symptoms of the bacterial infection. In this embodiment, the subject is typically symptomatic. A therapeutically effective amount of the conjugate, composition or formulation is administered to such a subject. A therapeutically effective amount is an amount effective to ameliorate one or more symptoms of the disorder.
The compound, composition or combination of the invention may be administered in a variety of dosage forms. Typically the conjugate, composition or formulation of the invention is administered in a topical dosage form.
A conjugate, pharmaceutical formulation or pharmaceutical composition for topical administration may be provided in the form of a lotion, gel, ointment, cream, emulsion, suspension, paste, solution, powder, shampoo, aerosol foam, spray, jelly, film, sponge, swab, patch, or the like. The various topical dosage forms may also be formulated for immediate release, controlled release, sustained release, or the like.
The pharmaceutical formulation or pharmaceutical composition may comprise one or more pharmaceutically acceptable excipient, carrier, and/or diluent. Examples particularly suitable for topical formulations include liquid oils, viscosity-modifying agents, thickening agents, gelling agents, alcohols, surfactants, chelating agents, buffers, preservatives, humectants, emollients, stabilizers, diluents, dispersing agents, emulsifiers, wetting agents, stabilizers, pH adjusters, solvents, and cosolvents.
Gelling agents include carbomers (carboxy vinyl polymers / cross-linked polyacrylic acid), such as Carbopol® and Noveon® polycarbophil.
Thickening agents include acacia, alginic acid and salts thereof, hyaluronic acid and salts thereof, carboxymethylcellulose, ethylcellulose, gelatin, collagen, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, methylcellulose, poloxamers, polyvinylpyrrolidone, polyvinyl alcohol, tragacanth, xanthan gum, magnesium aluminum silicate, and bentonite.
Surfactants include octylphenoxy polyethoxyethanol such as Octoxynol 1, 3, 5, 8, 9, 10, 12, 13, 16, 30, 40, 70 (wherein the number indicates the number of repeating oxyethylene units), sorbitan esters such as sorbitan monooleate and sorbitan monostearate, polysorbates (such as polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate and polyoxyethylene sorbitan monolaurate, commonly known as Tween® 80, Tween® 60, and Tween® 20, poloxamers (block polymers of ethylene oxide and propylene oxide, such as Pluronic® Fl 27 and Pluronic® Fl 08, poloxamines (block polymers of ethylene oxide and propylene oxide attached to ethylene diamine, such as Tetronic® 1508 and Tetronic® 908), and long chain fatty alcohols (e.g., oleyl alcohol, stearyl alcohol, myristyl alcohol, docosahexaenoyl alcohol, etc.). Emulsifiers include sodium or potassium oleate, triethanolamine stearate, sodium lauryl sulfate, sodium dioctyl sulfosuccinate, sodium docusate, quaternary ammonium salts, glyceryl monostearate, polyoxyethylene monooleate, polyoxyethylene monostearate, polyoxyethylene monolaurate, potassium oleate, sodium lauryl sulfate, sodium oleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate, triethanolamine oleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan trioleate.
Humectants include glycerin, sorbitol, hexylene glycol, propylene glycol, and urea.
The pharmaceutical formulation or pharmaceutical composition may comprise one or more chelating agents (such as EDTA) and/or antioxidants (such as butylated hydroxytoluene (BHT) butylated hydroxyanisole (BHA), sodium metabisulfite, propyl gallate, or cysteine).
Whilst topical administration is typically preferred, the conjugate, pharmaceutical composition or pharmaceutical formulation may also be administered in any other suitable manner.
Thus, the conjugate, composition or formulation can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The conjugate, composition or formulation may be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrastemally, transdermally or by infusion techniques. The conjugate, composition or formulation may be administered as a suppository. The conjugate, composition or formulation may be administered via inhaled (aerosolised) or intravenous administration.
Solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, com starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tableting, sugar coating, or film coating processes.
Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspension or solutions for intramuscular injections or inhalation may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for inhalation, injection or infusion may contain as carrier, for example, sterile water or typically they may be in the form of sterile, aqueous, isotonic saline solutions. Pharmaceutical compositions suitable for delivery by needleless injection, for example, transdermally, may also be used.
If administered by inhalation, the conjugate, composition or formulation may be formulated for inhaled (aerosolised) administration as a solution or suspension. The conjugate, composition or formulation may be administered by a metered dose inhaler (MDI) or a nebulizer such as an electronic or jet nebulizer. Alternatively, the conjugate, composition or formulation may be formulated for inhaled administration as a powdered drug, such formulations may be administered from a dry powder inhaler (DPI). When formulated for inhaled administration, the conjugate, composition or formulation may be delivered in the form of particles which have a mass median aerodynamic diameter (MMAD) of from 1 to 100 pm, typically from 1 to 50 pm, more typically from 1 to 20 pm such as from 3 to 10 pm, e.g. from 4 to 6 pm. When the conjugate, composition or formulation is delivered as a nebulized aerosol, the reference to particle diameters defines the MMAD of the droplets of the aerosol. The MMAD can be measured by any suitable technique such as laser diffraction.
In use, a therapeutically or prophylactically effective amount of the conjugate, composition or formulation is administered to a subject. The dose may be determined according to various parameters, especially according to the compound used; the age, weight and condition of the subject to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular subject. A typical daily dose is from about 0.01 to 100 mg per kg, typically from about 0.1 mg/kg to 50 mg/kg, e.g. from about 1 to 10 mg/kg of body weight, according to the activity of the specific agent, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. A typical daily dose is from about 0.01 to 100 mg per m2, typically from about 0.1 mg/m2 to 50 mg/m2, e.g. from about 1 to 10 mg/m2 of body surface area, according to the activity of the specific agent, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
When the conjugate, composition or formulation is administered to a subject in combination with another active agent, the dose of the other active agent can be determined as described above. The dose may be determined according to various parameters, especially according to the agent used; the age, weight and condition of the subject to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular subject. A typical daily dose is from about 0.01 to 100 mg per kg, typically from about 0.1 mg/kg to 50 mg/kg, e.g. from about 1 to 10 mg/kg of body weight, according to the activity of the specific agent, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. A typical daily dose is from about 0.01 to 100 mg per m2, typically from about 0.1 mg/m2 to 50 mg/m2, e.g. from about 1 to 10 mg/m2 of body surface area, according to the activity of the specific agent, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The application is limited only by the claims.
Thus, the following Examples illustrate the invention. They do not however, limit the invention in any way. In this regard, it is important to understand that the particular assays used in the Examples section is designed only to provide an indication of chemical properties and biological activity. There are many assays available to determine such properties, and a negative result in any one particular assay is therefore not determinative. EXAMPLES
Table of abbreviations
XH-NMR proton nuclear magnetic resonance
13C-NMR carbon nuclear magnetic resonance
4-DMAP 4-(dimethylamino)pyridine
CMC critical micellar concentration
DCC dicyclohexylcarbodiimide
DCM dichloromethane
DCU dicyclohexylurea
ELSD evaporative light scattering detector
ERY erythromycin
ES erythromycin-succinate
EtOH ethanol
FA formic acid
HC1 chlorhydric acid
LLE liquid-liquid extraction
MALDI matrix assisted laser desorption ionization
MeOH methanol
MS mass spectrometry
NaOH sodium hydroxide
PBS phosphate buffered saline
P.I. polydispersity index
PSU pilosebaceous unit
SA succinic acid tl/2 half-life
TEM transmission electron microscopy
TPGS D-a-Tocopherol polyethylene glycol 1000 succinate
TOF time of flight
UPLC ultra performance liquid chromatography
UV ultraviolet Example 1
This example describes the synthesis of an erythromycin prodrug by linking erythromycin and an amphiphilic polymer in order for the prodrug to form micelles with a preferential targeted delivery to the PSU.
Erythromycin (ERY) was chosen and D-a-Tocopherol polyethylene glycol 1000 succinate (TPGS) was selected as the polymer. Thanks to the targeted delivery, the concentration of the drug to the real localization of the disease would be increased which could decrease the risk of resistance emergence and limit the side effects.
Background
Skin disease are very prevalent worldwide and are often underestimated. Even though most of the skin disease are not life-threatening, they can cause many consequences for the patient as discomfort, embarrassment, loss of self-confidence and isolation [1]. In 2013, the Global Burden of Disease Study revealed that skin and subcutaneous diseases were the fourth cause of nonfatal disease burden worldwide [2, 3]. For these different reasons, the development of novel or improvement of existing therapies is essential.
Acne is the most common skin disease worldwide. Unfortunately, there is still no clear explanation of the pathogenesis [4] . However, some treatments are known to be efficient such as antibiotics. These treatments are poorly selective and cause many side effects. For these reasons, the improvement of the delivery to the pilosebaceous unit (PSU) would be of great interest, in order to increase the concentration in the site of the disease and limit the side effects. The production of a novel biodegradable prodrug for the treatment of acne is described herein.
Methods
Materials
ERY was purchased from Fagron BV, (Rotterdam, Netherlands). All the other products were purchased from Sigma-Aldrich (Buchs, Switzerland). The solvents were ordered from Fisher Scientific AG (Reinach, Switzerland). All chemicals were at least of analytical grade. Synthesis
The first step of the production of the prodrug TPGS -Erythromycin- succinate (compound 2) was the addition of a succinic linker to the drug in order to form the intermediate Erythromycin-succinic acid conjugate (compound 1) and then the realization of the esterification in a second step with the TPGS.
Erythromycin (733.93 mg), succinic anhydride (SA) (200.14 mg) and 4- (Dimethylamino)pyridine (4-DMAP) (244.34 mg) were mixed in 3 ml anhydrous dichloromethane (DCM) for 24 hours at room temperature, under agitation based on the protocol of Cao and Feng [6].
Figure imgf000055_0001
Once the reaction was finished, the result was controlled by TLC (eluent: DCM/methanol (MeOH)/formic acid (FA) (50: 50: 10)). The spots that were not visible by UV were visualized using iode sublimation technique. The spots containing erythromycin were visualized by sugar staining composed of ethanol (EtOH), sulfuric acid 96% and anisaldehyde (9 :1 :1). Then, a liquid-liquid extraction (LLE) was performed with chlorhydric acid (HC1) IM, in order to remove the 4-DMAP. The identity of the product was controlled by MS (Waters XEVO TQ-MS, Baden-Dattwil, Switzerland, in soft positive mode), XH-NMR and 13C-NMR (Bruker Avance Neo 600 MHz, Fallanden, Switzerland, in CDC13 and at 600MHz). The second step of the synthesis was the esterification between the compound 1 and TPGS. First, the compound 1 (100 mg) was mixed with dicyclohexylcarbodiimide (DCC) (37.14 mg). Secondly, TPGS (277.92 mg) was added to 4-DMAP (1.47 mg). Finally, the two solutions were combined. The reaction was left under agitation, at 40°C for 24 hours, in anhydrous conditions.
Figure imgf000056_0001
Once the reaction was finished, the result was confirmed by TLC (eluent: DCM/MeOH (9:1)). The spots were either visualized by UV, iode staining or the same sugar staining than used previously. Then, the balloon was placed on ice for 30 minutes to precipitate the DCC transformed during the reaction in dicyclohexylurea (DCU). Again, the 4-DMAP was removed by LLE with HC1 IM. Finally, the last step consisted in a purification to separate the prodrug of the residual TPGS and ERY by a purification on a C 18 column (Interchim Puriflash C18HQ 250x4.6 mm, 15 pm, Montlucon France) with a gradient as follows:
Sample: Prodrug TPGS -Erythromycin- succinate lOmg/ml in MeOH
Solvents: (A) Water + 0.1% FA
(B) MeOH + 0.1% FA
Column: Interchim Puriflash C18HQ 250x4.6 mm, 15 pm
UV detection: 254 nm, 280 nm
Time (min) % A % B Flow (ml/min)
0 98 2 1
15 40 60 1
50 3 97 1
70 3 97 1
75 0 100 1
80 1 99 1 Characterization
The product was characterized by MS (in soft positive mode), XH-NMR (in CDC13 and DMSO at 600MHz) and UPLC-UV-ELSD (Waters Acquity UPLC®, Waters Acquity ELSD, Baden-Dattwil, Switzerland) using a C18 column maintained at 45°C (Waters Xbridge® BEH C18, 2.1x50 mm, 2.5 pm, Baden-Dattwil, Switzerland) in accordance with the following method:
Sample: TPGS -Erythromycin- succinate 500pg/ml in Water
Injection volume: 15pl
Solvents: (A) ACN + 0.1% FA
(B) Water + 0.1% FA
Column: Xbridge BEH C18 2.5pm, 2,1x50 mm XP
Column temperature: 45°C
ELSD detection: Detector gain: 500
Nebulizer mode: cooling
Drift tube: 70°C
Gas pressure: 50.0 psi
Time (min) % A % B Flow (ml/min)
0.00 2 98 0.5
6.00 100 0 0.5
10.00 100 0 0.5
11.00 2 98 0.5
14.00 2 98 0.5
In order to complete the identification of the synthesized prodrug, a MALDI-TOF analysis was done in positive mode (Bruker Autoflex, Billerica, USA). The sample was dissolved (10 mg/mL) in CH3CN/H2O 50/50, v/v, with 0.1% TFA. CHCA MALDI matrix (10 mg/ml) solution was prepared in the same solutions than the samples. Next, the sample solution was mixed 1:1 with the matrix solution. Finally, a volume of 1 pL of this samplematrix mixture was spotted onto the stainless steel MALDI target plate (MTP 384 ground steel BC, cat n°280784, Bruker, Germany). External TOF calibration (PepMix 4, LaserBio Labs, France) was performed prior the sample spot analysis in TOF quadratic mode. MALDI-TOF acquisitions were performed from m/z 500 to 5’000 in reflectron positive ionization polarity. Once the identity of the final product was established, the capacity of the prodrug to form micelles above CMC was verified. A solution of compound 2 at Img/ml was prepared to identify the presence or the absence of foam, TPGS being a well-known surfactant. Then a Nile Red test was performed: Nile Red is a red dye known to be very lipophilic, having a good affinity for the hydrophobic core of micelles [7], 15 pl of a solution of Nile Red at 5mg/ml in acetone was introduced in the solution used previously. The solution appeared transparent in the absence of the hydrophobic core formed by the micelles and dark pink if micelles were formed.
The evidenced micelles were further characterized by dynamic light scattering (DLS) at 25°C at an angle of 173° with a Zetasizer Nano ZS (Malvern instruments, Malvern, UK). The polydispersity index (P.I.), hydrodynamic diameter, volume and number weighted diameter (dv and dn) were measured. The values were obtained after three rounds of ten measurements each. The following step was the characterization of the morphology of the micelles using TEM (Tecnai 20 D38O, FEI Company, Hillsboro, USA) after negative staining on copper grids.
The stability of the micelles formed by the prodrug was also determined in different conditions of pH: 5 (acetate buffer), 7 (phosphate buffered saline (PBS)) and 9 (NaOH). Solutions were prepared at 1 mg/ml and aliquots were taken every hour for 12 hours and then at day 1, 2, 3, 7, 14, 21 and 28. The samples were analyzed by UPLC-UV-ELSD with the same method presented previously. The stability in contact with porcine liver esterase was determined too. A solution of 2 mg/ml of prodrug was prepared with 10 units of esterase and placed at 37°C to simulate the temperature of the organism. Samples were taken every hour for 20 hours and then at day 1, 2, 3, 7, 14, 21, and 28 and were analyzed by UPLC-UV-ELSD. Finally, the stability of the prodrug in contact with an extract of the skin was realized. Fresh skin from porcine ears (Slaughterhouse of Loex, Switzerland) was removed using a dermatome( Zimmer Air Dermatome, Warsaw, USA). The slices have a thickness of 750 pm. Then, 300 mg of this sliced skin per ml are cut in very small section and placed in PBS with a stirrer. The skin is extracted overnight. The solution is then centrifuged at 5000 rpm for 10 minutes and filtered on Chromafil Xtra PVDF-20/13 filters (Macherey-Nagel, Duren, Germany). The prodrug was then put in contact with this solution at a concentration of 2 mg/ml at 37°C to simulate the temperature of the organism. Samples were taken each hour for 12 hours and then at day 1, 2, 3, 8, 14 and 21. Samples were analyzed by UPLC-UV-ELSD. For all stability studies, the natural logarithm of the remaining fraction of prodrug was plotted as a function of time. The first order rate constant (kObs) of hydrolysis was estimated from the slope of the function and the half-life (ti/2) was calculated (ti/2 = | In (1/2)/ kObs |).
Results
Compound 1 was obtained after the first synthesis. The characterization was done by MS (Figure 1), 1 H-NMR and 13C-NMR (Figure 2). The results obtained with MS and 1 H- NMR, 13C-NMR demonstrate that the intermediate was successfully synthesized. In Figure 1, the m/z value at 816.60 corresponds to the mass of the intermediate with a loss of a water molecule and the addition of a proton [M-H20+H+]. Another peak is visible at 916.60 which may correspond to the intermediate but with two succinic acid molecules linked.
13C-NMR (Figure 2) was used to confirm that the succinic acid was linked on the hydroxyl group in position 2’. This was confirmed by MS too. An open access tool for MS-spectra prediction was used in order to determine if the different possible structures would create different fragments that would help to identify the correct form of the molecule [8]. The simulated spectrum identified a particular fragment (m/z: 258.1335992) shown below that can only be present if the ester link is on position 2’. This fragment is easily identified in the MS spectrum in Figure 1 :
Figure imgf000059_0001
m/z: 258.1335992 Thanks to these different elements of characterization, it was possible to clearly establish that the intermediate was successfully synthetized and that it corresponds to the expectations. The yield for this first synthesis was 34.7%.
Prodrug of TPGS-Erythromycin-Succinate
After obtaining compound 1, a second reaction was carried out to finally synthesize the prodrug (compound 2).
The characterization of the final product was made by XH-NMR, UPLC-UV-ELSD and MALDI-TOF. The MALDI-TOF results obtained showed that there was not a unique mass for the prodrug (Figure 3). However, the main peak at 2363.301 m/z corresponds to the compound 2 with the loss of a molecule of water and a sodium adduct (M+Na+-H20). The distribution of peaks was as expected with the difference of 44 m/z between the peaks corresponding to the weight of one unit of PEG.
The structure of the prodrug was also confirmed by XH-NMR (Figure 4). It was not possible to identify all the protons of the molecule, but the presence of both erythromycin and TPGS were identified. 1 H-NMR results indicated that some free TPGS was not removed during the purification. This was confirmed by UPLC-UV-ELSD analysis (Figure 5). Indeed, two peaks were present corresponding to the compound 2 and free TPGS. The ratio between the prodrug and the free polymer is estimated to 20:7. This result combined with a relatively low yield (13.52%) for the second reaction indicates that the reaction and the purification step may be further improved.
The next step was to assess the capacity of the new prodrug to form micelles. Solutions of the prodrug at 1 mg/mL (estimated as a concentration above CMC) were prepared, to check the formation of foam, which is a sign of micelles formation. Nile Red, which has a different color if it is in hydrophilic or lipophilic environment, was also used to confirm the formation of micelle. Positive results of the two tests are presented in Figure 6. Compound 1 produced less foam than the positive control but gave similar results for the Nile Red test.
TEM images (Figure 9) were taken at different magnification and the size and potential zeta of the micelle measured with a Zetasizer (Figure 7 and Figure 8). The size obtained for the micelles formed by the prodrug was 9.222 nm, which was smaller than the one obtained for the TPGS (14.20 nm). The z-average obtained was of 22.29 for TPGS and 21.06 for the compound 2. In the case of the polydispersity index (Pdl), the value obtained for TPGS was 0.220, which showed a small variation of size between the particles. In comparison, the Pdl obtained for the prodrug was 0.301 which is higher but is still an acceptable value. A zeta potential of 46 mV was obtained which indicates that the micelles of the prodrug can be considered as positive.
The TEM images yielded interesting information about the morphology of the micelles. These images indicated that the shape of the micelle is not completely round. Indeed, TPGS alone forms micelles by placing the tails of PEG units in the outside and the tocopherol in the core of the micelles. In this case, the tocopherol and PEG units kept their position, However, without being bound by theory the erythromycin is believed to incorporate inside the micelle creating a “flower” shape [9] due to its polarity.
Once the ability of the prodrug to form micelles was established, the stability of compound 2 in different conditions was assessed. The prodrug was tested at different pH (5, 7 and 9).
In acidic conditions (pH 5, Figure 10), the prodrug is in similar pH condition that the one observed in the skin. The remaining fraction of the compound 2 was plotted as a function of time. Thanks to the linearization of the function (use of the natural logarithm), the kObs can be estimated from the slope of the regression curve, which then can be used to determine the half-life. The half-life (ti/2) is the time required to observe a decrease in the concentration of the product by a factor 2. It is very interesting to observe that in that case, the prodrug is stable for a long time (ti/2 = 451,13 h ± 42,74).
At pH 7 the half-life calculated is 7.86 h ± 0.09 (Figure 11). The prodrug is then less stable with the increase of pH, believed (without being bound by theory) to be due to increased hydrolysis of the links between erythromycin, succinate and TPGS.
Surprising stability was observed at pH 9 (Figure 12). Rather than the half-life being even shorter than at pH 7, the opposite was found with a half-life of 387,77 h ± 79,53. Without being bound by theory, this higher value is believed to be caused by the fact that erythromycin at this pH is no more charged. The pKa of erythromycin is 8.88 so at pH 9, the molecule becomes essentially neutral, with increased lipophilicity resulting in its localization closer to the center of the micelles (hydrophobic environment). The ester links will thus be more hidden inside the hydrophobic core of the micelle. This conformation gives to the prodrug a protection against hydrolysis. Results are summarized in Figure 13.
The stratum comeum of the skin has a pH around 5 but just underneath, in the first viable layer of the skin called stratum granulosum it increases and the pH becomes physiological (pH 7.4) within the next layers [12]. This means that if the micelles have the capacity to leave the skin surface, the prodrug will disassemble. Another aspect to keep in mind is that in the case of acneic skin, the pH of the skin is increased up to values of 6,35 [12]. In that case, the prodrug will disassemble more easily.
Previous work showed that micelles preferentially target the PSU [5]. The pH of PSU is acidic due to the presence of C. acnes which hydrolysis sebum triglycerides and secretes propionic acid. It can then be supposed that the prodrug will be very stable in this environment which would create a slow rate release over time. Meanwhile, the prodrug still needs to be hydrolyzed. To test the ability of esterases present on the skin to hydrolyze the ester links of the prodrug, the stability of the compound 2 in contact with porcine liver esterase was tested (Figure 14). The half-life obtained for this study was 3,99 h ± 0,13. Compared to the previous values obtained for the stability at different pH, this is the shortest half-life obtained. Once the product is applied on the skin, it is therefore highly probable that esterase will be the main path for conversion of the prodrug in erythromycin.
In this experiment, it was possible to observe the increase in erythromycin concentration while the amount of prodrug was decreasing. However, the shape of the supposed erythromycin peak is larger compared to the one of the standards (Figure 15). This indicates that some erythromycin even after 28 days in contact with esterase may still be linked to PEG units, which would be useful for a sustained release formulation.
To clearly identify whether the esterase present in the skin was able to break down the prodrug, an experiment was performed by first realizing an extraction of the proteins of the skin in PBS and then putting the prodrug in contact with this extract in order to estimate the stability of the prodrug in contact with the skin (Figure 16). The results obtained showed a half-life of 4.75 h ± 0.52. A comparison of the stability of the prodrug in contact with liver esterase and skin extract is shown in Figure 17. This experiment confirms that the skin has the ability to break down the prodrug.
Example 2
This example describes the synthesis of an erythromycin prodrug by linking erythromycin and an amphiphilic polymer via azelaic acid in order for the prodrug to form micelles with a preferential targeted delivery to the PSU.
Synthesis
Synthesis of azelaic acid anhydride (compound 1 )
Figure imgf000063_0001
15 g azelaic acid (Fluorochem, UK lot: FCB037216) was dissolved in 60 mL acetic anhydride (Fluka, Switzerland, lot: BCBC5087V). The mixture was heated at 150° C under reflux for 6 hours. The product was then dried in a rotavapor (Biichi Labortechnik AG, Switzerland). Once the majority of the acetic acid had been removed, several toluene strips (Fisher Chemical, UK, lot: 1153755) were carried out. Finally, to remove the last traces of solvents, the product was placed in a freeze-dryer (Christ, Alpha 2-4 LSCbasic, Germany). In order to verify the identity of the synthesized compound, the product was characterized by 1H-NMR (Bruker Avance Neo 600 MHz, Fallanden, Switzerland). About Img of product was analyzed in 750pL of chloroform (CDC13) at 600MHz. MS analysis (Advion, Plate Express, Ithaca, NY) was performed as well as melting point measurement (Biichi, Melting Point B-540, Switzerland) (gradient of l°C/min) using melting point tubes of 80mm (Biichi). All the following 1H-NMR and MS analyzes were carried out under the same conditions.
Synthesis of erythromycin azelate (compound 2)
Figure imgf000064_0001
Erythromycin©
Second, erythromycin (Fagron, NL, batch: CEPEB001E18), azelaic acid anhydride (compound 1) and 4- dimethylaminopyridine (Acros organics, USA, lot: A0421414) (Table 2) were dissolved in anhydrous dichloromethane 99.8% Extra Dry (Acros organics, UK, lot: 2056803). The reaction takes place for 24 hours, under argon, at room temperature. After the reaction is complete, thin layer chromatography (TLC) (Supelco, Germany, lot: HX16996154) was performed in an eluent mixture consisting of dichloromethane (DCM) (Fisher Chemical, UK, lot: 2183446)/ methanol (MeOH) (Fisher Chemical, UK, lot: 2182333)/ Formic acid (AF) (Acros Organics, ES, lot: A03462240) 50:50:10. The products are visible on the TLC under UV (254 nm) under sublimated iodine (iodine deposits on the TLC plate and attaches to double bonds, unsaturated parts of the molecule to color them). Ethanol (EtOH) sugar developer (Fisher Chemical, UK, lot: 2176183)/Sulfuric acid (H2SO4) (Carlo Erba, France, lot: 0D249269H)/Anisaldehyde (Acros Organics, China, lot: A0403824) 9:1:1 was used to highlight compounds such as erythromycin. To do this, the TLC plate is immersed in the sugar developer and then heated. The developer colors the plate brown and the compounds appear black
In order to remove the 4-DMAP which was added during the synthesis as a reaction catalyst, a liquid-liquid extraction was carried out. To do this, the solution was placed in a separatory funnel where lOmL of HC1 (Acros organics, Germany lot: A0430657) IM are added in order to extract the 4-DMAP in the aqueous phase while the compound will be in the organic phase. The extraction is repeated twice. The organic phase was recovered and dried with dry magnesium sulphate (Fisher Chemical, Germany, lot: 2064885). In order to eliminate the anhydrous DCM, the product is put in the rotavapor and then in the lyophilizer in order to obtain a dry product.
In order to verify the identity of the synthesized compound, the product was characterized by TLC, 1H-NMR and MS. In addition, UHPLC-ELSD detection (Waters Acquity UPLC®, Waters Acquity ELSD, Baden Dattwil, Switzerland) using a C18 column at 45°C (Waters Xbridge® BEH C18, 2.1x50 mm, 2.5 pm, Baden, Dattwil , Switzerland) was carried out. Sample: Compound 2: 1 mg/ml in Water
Injection volume: 45pl
Solvents: (A) ACN + 0.1% FA
(B) Water + 0.1% FA
Column: Xbridge BEH C18 2.5pm, 2.1x50 mm XP Column temperature: 45°C
ELSD detection: Detector gain: 500
Nebulizer mode: cooling
Drift tube: 70°C
Gas pressure: 50.0 psi
Time (min) % A % B Flow (ml/min)
0.00 2 98 0.5
6.00 100 0 0.5
10.00 100 0 0.5
11.00 2 98 0.5
14.00 2 98 0.5
Synthesis of erythromycin-azelate-TPGS (compound 3)
Figure imgf000066_0001
DCU
Compound 3
Third, 100 mg compound 2 was mixed with 45 mg dicyclohexylcarbodiimide (DCC) (Alfa Aesar, Germany, batch: 10216365) and anhydrous DCM. TPGS (256 mg) and 4-DMAP (45 mg) was mixed with anhydrous DCM. The two mixtures were combined for 24 hours under argon reflux at 40 °C. Once the reaction was complete, TLC was carried out in a 90:10 DCM/MeOH eluent mixture. Reaction progress was monitored by UV, iodine and sugar staining as above. At the end of the reaction, the flask containing the product was placed in ice for one hour in order to precipitate the transformed DCC into DCU during the synthesis. Once this step had been completed, the DCU is filtered through a sintered glass. In order to remove the 4-DMAP which was added during the synthesis as a reaction catalyst, a liquid-liquid extraction was carried out using lOmL of IM HC1 to extract the 4- DMAP into the aqueous phase while the compound will be in the organic phase. The extraction was repeated twice. The organic phase was recovered, dried with magnesium sulphate and lyophilised. Purification of the product was carried out using a puriflash on a C18 column (Interchim Puriflash C18HQ 250x4.6 mm, 15 pm, Monthujon France). Sample: Compound 3 dissolved in dichloromethane
Solvents: (A) Water + 0.1% FA
(B) MeOH + 0.1% FA
Column: Interchim Puriflash C18HQ 250x4.6 mm, 15 pm
UV detection: 254 nm, 280 nm
Time (min) % A % B Flow (ml/min)
0 0 100 1
35 0 100 1
65 0 100 1
30 70 1
70 30 70 1
The synthesized product was characterised by TLC, 1H-NMR, MS, UHPLC-ELSD and MALDI-TOF to confirm the synthesis.
Characterization
Formation of micelles was confirmed by Nile Red test (as described above). Results are shown in Figure 18. The dark pink color for the prodrug (compound 3) confirms the formation of micelles.
DLS analysis (25° at an angle of 173° with Zetasizer Nano ZS; Malvern instruments, Malvern, UK) indicated that the average size of the micelles formed by compound 3 was 16.61 nm. The polydisperion index (Pdl) was 0.260. The zeta potential was 29.8 mV indicating the micelles are positively charged.
In order to know more about the morphology of the micelles of compound 3, the latter was analyzed by TEM (Thermoscientific, USA, Talos L120C (120KeV, LaB6)). As shown in Figure 19, the micelles composed solely of TPGS have a relatively round shape and have a size of approximately 10 nm, which confirms the analysis carried out by DLS.
The stability of the prodrug was tested at pH 5 and pH 9 by UHPLC-ELSD under the conditions above. At pH 9, a half-life time of 256.26 h ± 28.536 was observed. At pH 5 the prodrug has an average half-life of 61.62 h ± 3.253). Stability at pH 7 could not be determined.
Distribution of the prodrug (compound 3) was characterized in samples of porcine skin comprising PSUs and in samples of porcine skin without PSUs present. Pig's ears (Carre de Rolle slaughterhouse, Switzerland) were cleaned under running cold water. Using an air dermatome (Zimmer, UK, batch: 64662570) thin layers of skin (outer ear) of 0.750 pm are cut and placed in lx DPBS. Skin samples were assessed in Franz cells with a diameter of 2cm, with ImL of the prodrug (3mg/lmL in water) in the donor compartment; 3O-33°C for 24 hours with stirring. After 24 hours, the skin is collected and cleansed lightly with lx DPBS. The PSUs as well as the controls (without PSU) are taken with a biopsy punch 1 mm in diameter (Kai medical, Japan, lot: 000006). Samples were placed in 1.5mL eppendorfs containing 120 pL of 50% acetonitrile mixture (Fisher Chemical, UK, batch: 2169048)-0.1%AF / 50% H2O-0.1%AF. The eppendorfs are then agitated overnight to extract the PSUs. After extraction, the eppendorfs are centrifuged at 14,000 rpm for 15 minutes. Finally, 100 pl of supernatant was taken and analyzed by UHPLC-MS/MS (Waters Acquity UPLC®, Waters XEVO TQ-MS, Baden Dattwil, Switzerland):
Injection volume: 5pl
Solvents: (A) ACN + 0.1% FA
(B) Water + 0.1% FA
Column: Xbridge BEH C18 2.5pm, 2.1x50 mm XP
Column temperature: 45°C
Figure imgf000068_0001
Time (min) % A % B Flow (ml/min)
0.00 2 98 0.5
6.00 100 0 0.5
10.00 100 0 0.5
11.00 2 98 0.5
14.00 2 98 0.5
MS/MS analysis confirmed that the peaks for the erythromycin and azelaic acid products from the prodrug were higher in skin samples comprising PSUs than in samples of skin absent PSUs (Figure 20).
This example therefore confirms the synthesis of an erythromycin prodrug by linking erythromycin and an amphiphilic polymer via azelaic acid in order for the prodrug to form micelles with a preferential targeted delivery to the PSU and delivery of the drug (erythromycin) and azelaic acid to the PSU.
Example 3
This example describes i) synthesis and characterisation of an adapalene-copolymer conjugate, and ii) self-assembling micelle formulation of the synthesized conjugate and preliminary characterization of said conjugate. This example demonstrates i.a. that adapalene can be used as the D moiety in conjugates provided herein.
Background
Acne vulgaris (acne) is a highly prevalent dermatological condition of the pilosebaceous unit (PSU). It affects a significant proportion of the population - although not life-threatening, if left untreated, it can have serious physical and psychological consequences. Among the different therapeutic agents indicated for the treatment of acne, retinoids are used as first-line therapy either alone or in combination with other antimicrobials [13]. However, their oral administration carries a risk of foetal malformation and appropriate measures must be taken to avoid pregnancy - therefore, it is clearly an advantage to reduce the systemic exposure of these drugs. Topical treatment remains the most common and popular method for the management of acne. However, this is frequently associated with local side-effects such as skin dryness, peeling, and skin irritation after application of conventional formulations that can affect patient compliance [13,14]. Adapalene (ADA; 412.52 Da) is a highly potent third generation retinoid that binds to nuclear retinoid receptors. Since it can modulate cellular differentiation and keratinization, ADA is used in the topical treatment of mild to moderate Acne vulgaris. ADA is exceptionally lipophilic (log P 8.11) and practically insoluble in water meaning that its formulation is a complex and challenging task [15].
Here a new chemical entity using adapalene and biocompatible and biodegradable copolymer (i.e. mPEG-PLGA) was developed and characterized. The drug-copolymer conjugate was also used to develop self-assembling micelles, which can be used to selectively deliver the therapeutic agent to the PSU, which is the target site for acne - and could provide a better option for acne treatment [16]. The drug - copolymer conjugate contains a hydrolysable ester bond that can be cleaved either enzymatically in the skin or in the acidic pH environment of the acne which could release the drug in more targeted manner.
The chemical conjugation approach solves the aqueous solubility problem of adapalene [15, 16].
The novel formulation developed here benefits in several ways, as a PSU-targeting formulation will help to improve therapy and minimize side-effects associated with current marketed formulations and so improve patient compliance. Targeted follicular delivery may decrease the drug concentration required in the formulation as compared to existing products [13, 14]. Moreover, avoiding the use of organic solvents and simple formulation development approach would help to decrease the manufacturing cost of the product and hence could reduce the cost of the therapy.
A copolymer (mPEG-PLGA) that possesses a free hydroxyl group on the hydrophobic chain was selected for covalent bonding with ADA (which contains a free carboxylic acid group). The conjugate synthesis was performed using the optimized Mitsunobu reaction and synthesized conjugate was purified and characterized by different analytical techniques.
The molecular structure of adapalene (ADA; MW 412 Da, pKa 8.0, log P 8.12), and methoxypoly(ethyleneglycol)-b-poly(lactide-co-glycolide) (mPEG-PLGA; MW-4000 Da), are shown below.
Figure imgf000071_0001
Methods
The ADA-copolymer conjugate was developed using Mitsunobu reaction under inert condition and reaction progress was monitored by thin layer chromatography. Isolation was performed using silica gel column chromatography with hexane: ethyl acetate (9:1) as a mobile phase. Isolated product was characterized by TLC and 1H NMR spectroscopy. Further conjugate analysis was performed by saponification reaction and UHPLC-PDA to confirm ester bond formation. The micelle formulation was developed by solvent evaporation method and size was determined using dynamic light scattering.
Materials
Adapalene (ADA) was purchased from Hangzhou Dayangchem Co. Ltd, (Hangzhou, P.R. China). mPEG-PLGA was purchased from Akina Corp. (West Lafayate, USA).
Diisopropylazodicarboxylate, triphenylphosphine were purchased from Sigma- Aldrich, Buchs, Switzerland. All other chemicals were at least of analytical grade.
Synthesis
ADA (12.4 mg), mPEG-PLGA (100 mg) and 0.037 mmol of triphenylphosphine (PPhs) (98.25 mg) were weighed and dissolved in 2 mL of dry tetrahydrofuran (THF). This mixture was then cooled to 0°C in ice bath. 0.037 mmol of diisopropylazodicarboxylate (DIAD) (76 .L) dissolved in dry THF (0.5 mL) was then added to above mixture at 0°C under vigorous stirring.
After this addition, the temperature was slowly increased to 25°C with continuous stirring. The reaction was then continued under an inert atmosphere at room temperature (25°C) for 20 h. The below scheme shows the chemical conjugation of mPEG-PLGA.
Figure imgf000072_0001
To monitor the reaction progress thin layer chromatography (TLC) analysis was performed. A small aliquot of reaction mix was dissolved in THF:methanol (1:1) solvent and a small spot was applied on TLC plate using capillary tube. Then spot was eluted in hexane:ethyl acetate (7:3) solvent system twice and visualized under UV/visible light.
Although multiple conditions were tried, the best conversion rate was observed using the protocol described herein and TLC for those conditions are shown in Figure 21. R20 refers to reaction mixture after 20 h and the remaining spots on TLC are starting material or reagents.
Purification of the major product formed after reaction completion was performed on silica gel chromatography. In short, reaction mixture was dissolved in a mixture of THF: dichloromethane (DCM) (1:1) or in DCM and loaded on silica gel and solvent was evaporated using rotary evaporator (Biichi RE 121 Rotavapor®; Flawil, Switzerland) and then dry powder was loaded in prefilled silica gel column. Column elution was started with hexane and gradually the concentration of EtOAc was increased. The desired product was eluted in hexane:EtOAc (9:1) i.e. around 10% EtOAc concentration in hexane. Pure fractions were evaporated using rotary evaporator and compound was dried under high vacuum for further analysis.
Characterisation
Characterization using 1H NMR
The characterization of starting material and isolated compound was performed using 1H NMR. In short, ~ 5 mg of compound was dissolved in CDCI3/DMSO and spectra were recorded using Bruker 400 Hz NMR machine and data was analysed using Mnova software (version 12.0). 1H NMR showed the presence of characteristic protons in aromatic region (6-9 ppm) as well as the disappearance of carboxyl group (-COOH) proton, which is the site of esterification.
1H NMR analysis of ADA, mPEG-PLGA copolymer and isolated new product confirmed the formation of ADA-copolymer conjugate. ADA 1H NMR spectrum (Figure
22) clearly shows the presence of one proton ~13 ppm which belong to acid group (- COOH) present in ADA. In the case of mPEG-PLGA copolymer NMR spectrum (Figure
23) all protons are present in the range of 0-5 ppm (aliphatic region) as there is no aromatic proton in the molecule. Since the conjugate was formed by ester bond formation between the acid group of adapalene and hydroxyl group of mPEG-PLGA, the carboxylic acid proton of ADA which appeared distinctly around 13 ppm was absent in the 1H NMR spectrum of the new compound (Figure 24). In addition, the NMR spectrum of the new compound displayed the characteristic aromatic protons corresponding to the naphthalene ring; along with protons in the aliphatic region (belonging to mPEG-PLGA and the adamantyl ring from ADA) thereby confirming that new compound was a conjugate of adapalene and the mPEG-PLGA copolymer.
However, 1H NMR spectrum of the new compound also showed some additional protons which could be due to impurity present in the isolated product. In the esterification reaction triphenylphosphine was used and hence there is the possibility of triphenylphosphine being present. This was tested by recording a 3 IP NMR (Figure 25); however, no triphenylphosphine was observed.
Characterization using UHPLC-PDA analysis
The newly synthesized compound possesses a different Rf value than adapalene and shows fluorescence.
ADA and ADA-copolymer conjugate were analysed using Waters Acquity®UPLC®H-class system and a PDA detector (Baden-Dattwil, Switzerland) and a Xbridge®BEH RP-C18, 2.1 x 50 mm, 2.5 pm column. Detection wavelength was 321nm. The gradient elution method was used comprising mobile phase: ACN (80%) + THF (20%) 0.1% FA, and Water + 0.1 %FA. The flow rate was 0.2 mL/min and the injection volume was 5 pL.
ADA eluted at 5.85 min using the UHPLC-PDA method (Figure 26). The conjugate eluted at 6.65 min (Figure 27). This indicated that the new product was more apolar in nature and this was expected as after conjugation of ADA with copolymer, the product should be more apolar due to the presence of the hydrophobic chain on the copolymer molecule.
Analysis by saponification reaction
To confirm the presence of an ester linkage in the product saponification reaction was performed. The compound was dissolved in mixture of THF:MeOH (1:1) and 100 pL of this was added to 900 pL of water and then saponified using 1 N NaOH solution to achieve pH of 9-10. Samples were vortexed for 5 min. Then this solution was mixed with THF:MeOH (1:1) solvent system in 1:1 ratio and samples were analysed by developed UHPLC-PDA method as described above.
A saponification test under basic condition and UHPLC-PDA analysis showed that adapalene could be regenerated from the newly synthesized compound and so confirmed the presence of ester bond. Self-assembling micelle nanocarriers of -140 nm size were obtained using adapalene-mPEG-PLGA conjugate.
In the saponification reaction the ester linkage underwent hydrolytic cleavage due to the basic pH conditions to yield the reactant acid (ADA) and alcohol (mPEG-PLGA) molecules. The new conjugate produced ADA after 5 min of reaction time which was observed at 5.85 min and ADA-copolymer conjugate was eluted at 6.65 min in the chromatogram. As the copolymer did not contain any chromophores it was not seen on the chromatogram.
Micelle formulation and size characterisation
Adapalene-mPEG-PLGA conjugate was developed and characterized. The developed conjugate was able to self-assemble into nanometer size micelles which could be used for acne treatment by targeting the pilosebaceous unit.
Micelle formulation was prepared using the solvent evaporation method [5, 16].
Briefly, a known amount of ADA-copolymer conjugate (10 mg) was dissolved in 4 mL of acetone. The mixture was added drop wise under sonication (Branson Digital Sonifier® S- 450D; Carouge, Switzerland) to 4 mL of ultrapure water. Subsequently, acetone was slowly evaporated at 40 °C using a rotary evaporator (Biichi RE 121 Rotavapor®; Flawil, Switzerland). After equilibration overnight, the micelle solution was centrifuged at 10 000 rpm for 15 min (Eppendorf Centrifuge 5804; Hamburg, Germany) to remove the impurity/precipitate from the formulation and the supernatant was carefully collected and used for analysis. The self-assembled micelle formulations were characterized to determine their hydrodynamic diameter (Zav), volume- and number-weighted diameters (dv and dn, respectively), and polydispersity index (P.L), using dynamic light scattering (DLS) with a Zetasizer Nano-ZS (Malvern Instruments Ltd; Malvern, UK). Measurements were performed in triplicate at an angle of 90° and at a temperature of 25 °C.
A clear micelle solution was obtained using ADA-copolymer conjugate. The conjugate underwent self-assembly to form nano-sized micelles. ADA-copolymer conjugate micelle formulations had hydrodynamic diameters (Zav) of 140 nm and the polydispersity index was 0.06; Figure 28 shows the size distribution by intensity of the ADA-copolymer conjugate micelle formulation.
Results
The results demonstrate that the adapalene-mPEG-PLGA conjugate was successfully synthesized and characterized. This conjugate could be formulated into aqueous micelles of nanometer size. This novel drug-copolymer conjugation approach solves the aqueous solubility problem of the poorly water soluble anti-acne agent adapalene and provides an option for targeted delivery to PSU which could enhance safety and efficacy in acne treatment.
References
1. Narayan, S., Dermatological history and examination. Medicine, 2017. 45(6): p. 352-358.
2. Seth, D., et al., Global Burden of Skin Disease: Inequities and Innovations. Curr Dermatol Rep, 2017. 6(3): p. 204-210.
3. Karimkhani, C., et al., Global Skin Disease Morbidity and Mortality: An Update From the Global Burden of Disease Study 2013. JAMA dermatology, 2017. 153(5): p. 406-412.
4. Shannon, J.F., Why do humans get acne? A hypothesis. Medical Hypotheses, 2020. 134: p. 109412.
5. Lapteva, M., et al., Self-assembled polymeric nanocarriers for the targeted delivery of retinoic acid to the hair follicle. Nanoscale, 2015. 7(44): p. 18651-62. 6. Cao, N. and S.S. Feng, Doxorubicin conjugated to D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS): conjugation chemistry, characterization, in vitro and in vivo evaluation. Biomaterials, 2008. 29(28): p. 3856-65.
7. Lapteva, M., et al., Self-assembled mPEG-hexPLA polymeric nanocarriers for the targeted cutaneous delivery of imiquimod. European Journal of Pharmaceutics and Biopharmaceutics, 2019. 142: p. 553-562.
8. Allen, F., et al., CFM-ID: a web server for annotation, spectrum prediction and metabolite identification from tandem mass spectra. Nucleic Acids Research, 2014. 42(W1): p. W94-W99.
9. Malo de Molina, P. and M. Gradzielski, Gels Obtained by Colloidal Self-Assembly of Amphiphilic Molecules. Gels, 2017. 3(3).
10. Paesen, J., et al., Study of the stability of erythromycin in neutral and alkaline solutions by liquid chromatography on poly(styrene-divinylbenzene). International Journal of Pharmaceutics, 1995. 113(2): p. 215-222.
11. Brisaert, M., M. Heylen, and J. Plaizier- Vercammen, Investigation on the chemical stability of erythromycin in solutions using an optimization system. Pharmacy World and Science, 1996. 18(5): p. 182-186.
12. Prakash, C., et al., Skin Surface pH in Acne Vulgaris: Insights from an Observational Study and Review of the Literature. The Journal of clinical and aesthetic dermatology, 2017. 10(7): p. 33-39.
13. Gollnick H, Cunliffe W, Berson D, et al. Management of acne: a report from a global alliance to improve outcomes in acne. J Am Acad Dermatol. 2003;49:Sl-S37
14. Castro GA, Ferreira LAM. Novel vesicular and particulate drug delivery systems for topical treatment of acne. Expert Opin Drug Deliv. 2008;5:665-679.
15. Bernard BA. Adapalene, a new chemical entity with retinoid activity. Skin Pharmacol. 1993;6:61-69.
16. Kandekar SG, et al. Selective delivery of adapalene to the human hairfollicle under finite dose conditions using polymeric micelle nanocarriers Nanoscale. 2018; 10: 1099- 1110

Claims

1. A conjugate of formula (I):
D — L — P — X — Z — C /?
O— T [I] wherein
D is an antimicrobial agent;
L is a linker;
P is a polymeric spacer;
X is a group selected from -OC(O)-, -C(O)O-, -C(O)-, -O-, -S-, -S(O)-, -SO2-, -NR10-, -NR10C(O)-, -C(O)NR10-, -NR10C(O)NRn-, -NR10C(O)O-, -OC(O)NR10, and a covalent bond;
Z is selected from Ci-12 alkylene, C2-12 alkenylene, and C2-12 alkynylene, wherein Z is unsubstituted or is substituted with 1, 2 or 3 substituents independently selected from halogen, -OR10, and -NR10Rn;
T is a tocopheryl or tocotrienyl group; and each R10 and R11 is independently selected from H and C1-2 alkyl.
2. A conjugate according to claim 1 wherein D is an antibiotic agent, an antifungal agent, or a comedolytic retinoid.
3. A conjugate according to claim 1 or 2, wherein D is an antibiotic or antifungal agent, preferably wherein D is an antibiotic or antifungal macrolide.
4. A conjugate according to any one of the preceding claims, wherein D is selected from erythromycin, adapalene, azithromycin, clarithromycin, fidaxomicin, carbomycin, josamycin, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, telithromycin, dirithromycin, and cethromycin; clindamycin, tetracycline, metronidazole, sulfacetamide, doxycycline, minocycline, dapsone, and sarecycline; and pharmaceutically acceptable salts thereof.
5. A conjugate according to any one of the preceding claims, wherein D is erythromycin or a pharmaceutically acceptable salt thereof or adapalene or a pharmaceutically acceptable salt thereof; preferably wherein D is erythromycin or a pharmaceutically acceptable salt thereof.
6. A conjugate according to any one of the preceding claims, wherein P is a poly(ethylene glycol) (PEG), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), a polycaprolactone (PCL), a poly(glycerol) (PG), a poly(oxazoline) (POX), a poly(vinylpyrrolidone) (PVP), a polyacrylamide (PAM), hyaluronic acid, heparin, polysialic acid, or a polypeptide; wherein preferably P is poly(ethylene glycol) (PEG); and wherein P preferably has an average molecular weight of from about 100 to about 10,000 Da, more preferably from about 500 to about 5000 Da, still more preferably about 1,000 Da.
7. A conjugate according to any one of the preceding claims, wherein X is -OC(O)-, -C(O)O- or -C(O)-.
8. A conjugate according to any one of the preceding claims, wherein T is vitamin E or a derivative thereof, preferably wherein T is
Figure imgf000078_0001
wherein the wavy line indicates the point of attachment to the moiety -OC(O)-Z-X-P-L-D.
9. A conjugate according to any one of the preceding claims, wherein L is -C(O)-L'- C(O)-; and wherein antimicrobial agent D comprises a hydroxyl group which is esterified with the linker (L) at one of the -C(O)- moieties such that the moiety D-L- is a group of formula (Q)
Figure imgf000078_0002
wherein the wavy line indicates the point of attachment to the moiety -P-X-Z-C(O)O-T; and L1 is selected from Ci-18 alkylene, C2-I8 alkenylene, and C2-I8 alkynylene, wherein L1 is unsubstituted or is substituted with 1, 2 or 3 substituents selected from halogen, -OR10, and -NR10Rn.
10. A conjugate according to any one of the preceding claims, wherein said conjugate
Figure imgf000079_0001
wherein n is an integer from 1 to about 50; wherein preferably D is erythromycin and the conjugate is of Formula (III)
Figure imgf000079_0002
wherein n is preferably an integer from about 10 to about 30.
11. A conjugate according to claim 9 or claim 10, wherein L1 is unsubstituted Ci-12 alkylene, wherein preferably L1 is unsubstituted C2-7 alkylene;
12. A conjugate according to any one of the preceding claims, wherein Z is unsubstituted Ci-12 alkylene, wherein preferably Z is unsubstituted C1-4 alkylene.
13. A conjugate according to any one of claims 1 to 8, wherein L is -O-l C O)-, and wherein antimicrobial agent D comprises a carboxylic acid group which is esterified with the linker (L) such that the moiety D-L- is a group of formula (QI)
Figure imgf000080_0001
wherein the wavy line indicates the point of attachment to the moiety -P-X-Z-C(O)O-T; and L1 is selected from Ci-i8 alkylene, C2-I8 alkenylene, and C2-I8 alkynylene, wherein L1 is unsubstituted or is substituted with 1, 2 or 3 substituents selected from halogen, -OR10, and -NR10Rn.
14. A conjugate according to any one of claims 1 to 6, 8, 9, 11 or 13, wherein the moiety X-Z forms one or more -O-CH2CH2 (PEG) units.
15. A conjugate according to any one of claims 1 to 8 or 13 to 14, wherein said conjugate comprises a moiety of Formula (VI)
Figure imgf000080_0002
wherein preferably D is adapalene.
16. A pharmaceutical formulation for topical administration, comprising a plurality of micelles in a vehicle, wherein said micelles each comprise a plurality of conjugates according to any one of claims 1 to 15.
17. A pharmaceutical formulation according to claim 16, wherein said micelles each comprise (i) a core comprising the tocopheryl or tocotrienyl groups of the conjugates and (ii) a solvent-accessible surface; and wherein the antimicrobial agent of the conjugates extends into the solvent from the solvent-accessible surface.
18. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 15 and one or more pharmaceutically acceptable excipient, carrier and/or diluent; wherein preferably said pharmaceutical composition is for topical administration.
19. A conjugate according to any one of claims 1 to 15, a pharmaceutical formulation according to claim 16 or claim 17 or a pharmaceutical composition according to claim 18, for use in treating a microbial skin infection in a subject in need thereof; wherein preferably i) said conjugate, pharmaceutical formulation or pharmaceutical composition is for use in treating acne; and/or ii) the microbial skin infection is a bacterial infection caused by bacteria of genus Cutibacterium (e.g. Cutibacterium acnes), Streptococcus (e.g. Streptococcus pyogenes), Staphylococcus (e.g. Staphylococcus aureus), Pseudomonas (e.g. Pseudomonas aeruginosa) or Corynebacterium (e.g. Corynebacterium minutissimum ) .
20. A conjugate, pharmaceutical formulation or pharmaceutical composition for use according to claim 19, wherein said use comprises topically administering said conjugate, pharmaceutical formulation or pharmaceutical composition to said subject.
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