WO2024236051A1 - Long-acting pain-relief formulation - Google Patents

Abstract

The present invention relates to a composition comprising a β-cyclodextrin polymer (a); and at least one compound (b) selected from mycolactone and derivatives thereof.

Description

DESCRIPTION

TITLE: Long-acting pain relief formulation

The subject of the present invention relates to a composition comprising a p-cyclodextrin polymer and at least one compound chosen from mycolactone and its derivatives, and its uses, in particular for the treatment of pain.

Worldwide, more than 100 million people suffer from chronic pain related to many pathologies (osteoarthritis, cancer, lower back pain, sciatica, shingles, neuropathic pain, burns). In particular, neuropathic pain, resulting from direct damage to the sensory neuron, is resistant to most treatments. Indeed, pain is one of the most difficult symptoms to treat and to bear for the patient. Currently, there are many treatments to relieve pain, particularly based on the use of morphine-based derivatives. These treatments have many limitations such as: a short-term action, a narrow therapeutic window, significant undesirable side effects (nausea, constipation, balance disorders, drowsiness, habituation) as well as an increased risk of causing a phenomenon of dependence in the patient. There is therefore a real need to find alternatives allowing effective and long-term treatment of pain.

The use of mycolactone and its derivatives as an analgesic appears to be a promising alternative. Indeed, application WO 2015/189342 describes the use of these molecules for the treatment of pain as well as their mechanism of action as an agonist of the angiotensin 2 receptor type 2 (AT2R), type I cyclooxygenases and TRAAK family potassium channels. This thus involves a hyperpolarization of neurons preventing the transmission of nerve information responsible for pain to the brain. Thus, this use has the advantage of not inducing dependence in the patient.

However, the use of this powerful analgesic is largely limited by the chemical nature of mycolactone derivatives, which makes them difficult to administer. Indeed, these polyketide derivatives are extremely sensitive to light and therefore degrade rapidly. Furthermore, their chemical nature makes them completely insoluble in aqueous media and requires the use of an organic solvent for their administration. Finally, mycolactone and its derivatives have low diffusion in the body once administered.

The use of nanoparticles or polymers to facilitate the administration of particularly poorly soluble pharmaceutical agents is currently an important point in medical research. Thus, Gouveia et al. Nature Scientific Reports 7, 5390 (2017) describes the use of p-cyclodextrin nanoparticles to encapsulate ethionamide (ETH) used in particular for the treatment of tuberculosis. The incorporation of the active ingredient also allows for preservation of its activity, better release of the agent in the lungs and therefore more effective treatment of tuberculosis. However, this formulation has only been tested for administration in the lungs.

There is therefore a need to provide new, more effective treatments to treat pain.

There is also a need to find an adequate composition allowing the progressive and prolonged release of mycolactone and its derivatives, while protecting them from degradation and avoiding their potentially toxic effects.

The present invention responds in particular to these needs.

Indeed, as demonstrated in the examples, the inventors have surprisingly discovered that the combination of a p-cyclodextrin polymer with a molecule selected from mycolactone or its derivatives makes it possible to solubilize mycolactone or its derivatives in an aqueous medium without the use of an organic solvent. In addition, it advantageously makes it possible to protect mycolactone from degradation under UV irradiation.

So, a first object of the present invention is a composition comprising:

- (a) a p-cyclodextrin polymer; and

- (b) at least one compound selected from mycolactone and its derivatives.

A second object of the present invention is a process for preparing said composition.

Another subject of the present invention is a pharmaceutical composition comprising at least the composition according to the invention and at least one pharmaceutically acceptable excipient.

Another object of the present invention is a composition for its use in the treatment of pain. The composition according to the invention comprises a compound (a) which is a polymer of P-cyclodextrin.

By “p-cyclodextrin polymer” is meant all compounds that can be obtained by polymerization of a compound chosen from the p-cyclodextrin family, or by grafting a compound chosen from the p-cyclodextrin family onto a (co)polymer.

The term “cyclodextrin” has a common meaning in the state of the art and refers to a family of cyclic oligosaccharides, consisting of macrocyclic compounds comprising several glucopyranose subunits linked together by a 1,4-glycosidic bond.

Extriated cyclodextrins are mainly produced by enzymatic degradation of amylose and its derivatives.

The cyclodextrins mainly used are divided into three families: a-, p- and y-cyclodextrins. These families are distinguished by the number of units that make up the cyclic structure of the cyclodextrins. “a-cyclodextrin” is composed of 6 glucopyranose groups in their cyclic structure, “p-cyclodextrin” is composed of 7 glucopyranose groups in their cyclic structure and “y-cyclodextrin” is composed of 8 glucopyranose groups in their cyclic structure.

The chemical structure of p-cyclodextrin is as shown in formula (A).

Les molécules de p-cyclodextrine possèdent une structure en cône tronqué, délimitant une cavité en leur centre. Cette cavité présente un environnement carboné apolaire et hydrophobe (squelette carboné et oxygène en liaison éther), capable d'accueillir des molécules peu hydrosolubles, tandis que l'extérieur du cône tronqué présente de nombreux groupements hydroxyle, conduisant à une bonne solubilité de la p- cyclodextrine en milieu aqueux. Ce caractère amphiphile permet en outre à la p- cyclodextrine d’inclure dans sa cavité des molécules hydrophobes (ou des parties de celles- ci) pour former des complexes d’inclusion en milieu aqueux. [Chem 1]

p-Cyclodextrin molecules have a truncated cone structure, delimiting a cavity in their center. This cavity has an apolar and hydrophobic carbon environment (carbon skeleton and oxygen in ether bond), capable of accommodating poorly water-soluble molecules, while the exterior of the truncated cone has numerous hydroxyl groups, leading to good solubility of p-cyclodextrin in aqueous medium. This amphiphilic character also allows p-cyclodextrin to include hydrophobic molecules (or parts thereof) in its cavity to form inclusion complexes in aqueous medium.

The formation of these inclusion complexes advantageously allows the encapsulation of various molecules, in particular mycolactone and possibly other molecules of therapeutic interest in the p-cyclodextrin molecules. Advantageously, a cyclodextrin polymer comprises more than twenty p-cyclodextrin cages which are not all occupied by mycolactone. It thus becomes possible to co-incorporate a variety of active molecules of interest in the family of antibiotics, analgesics, anticancer drugs, etc.

By “inclusion complex” we mean a system composed of a host molecule (here p-cyclodextrin) capable of accommodating a chemical species (here mycolatone and its derivatives).

The term “encapsulate” indicates that the chemical species is included within the host molecule.

According to one embodiment, the polymer (a) of p-cyclodextrin is prepared by polymerization reaction between the modified or unmodified p-cyclodextrin and at least one other chemical compound selected from the group consisting of epichlorohydrin, tartaric acid, citric acid, acylated poly(ethylene glycol), adipoyl chloride, succinyl chloride, glutaraldehyde, diphenyl carbonate, 1,4-butanediol diglycidyl ether, toluene diisocyanate, naphthalene diisocyanate, succinic anhydride, 1,2,4,5 benzene tetracarboxylic anhydride, monochlorotriazine, trimethoxysilane derivatives, diethynyl benzene, tetrafluoro terephthalonitrile. Another type of cyclodextrin polymer is obtained from porous organic-inorganic particles (metalorganic frameworks, MOFs) based on cyclodextrin which can then be crosslinked using the agents mentioned earlier in this paragraph.

Alternatively, water-soluble copolymers can be grafted with cyclodextrins. These copolymers are among the group consisting of: alginates, poly(ethylene imine), poly(N-hydroxy ethyl acrylamide), chitosan, hyaluronic acid, phenylalanine, polyanhydrides, polyaspartamide, cellulose.

According to another embodiment, the polymer (a) is the product of the reaction between modified or unmodified P-cyclodextrin and epichlorohydrin.

According to another embodiment, the polymer (a) is the product of the reaction between modified P-cyclodextrin and epichlorohydrin.

By "modified p-cyclodextrin" is meant any p-cyclodextrin that has been modified by at least one chemical reaction without changing the intrinsic properties of the p-cyclodextrin.

By chemical reaction we mean in particular addition reactions, substitution reactions, or even polymer grafting reactions.

Generally speaking, the modified p-cyclodextrins used according to the invention are in particular p-cyclodextrins whose hydroxyl groups, preferably the secondary hydroxyls of each glucose unit forming it, have been modified by the addition of one or more identical or different substituents.

A “modified p-cyclodextrin” used in the composition according to the invention is a p-cyclodextrin which carries one or more identical or different substituents, chosen from functionalized or non-functionalized alkyl radicals, hydroxyalkyl radicals, carboxyl, carboxylate, nitro, amino, sulfonate, sulfate, phosphate, ether, polyether, ammonium radicals and radicals comprising an ester function. As alkyl radical, mention may be made of a linear or branched alkyl radical having from 1 to 20 carbon atoms, preferably from 1 to 5 carbon atoms, more particularly a methyl or ethyl radical. As hydroxyalkyl radical, mention may be made of a hydroxyalkyl group having from 1 to 20 carbon atoms, preferably 1 to 5 carbon atoms, more particularly a -CH2CH2OH or -CH2OH radical. As a radical containing an ester function, we can cite an ester group having from 1 to 20 carbon atoms, preferably 1 to 5 carbon atoms, more particularly the groups -O(CO)CH3, -O(CO)CH2CH3.

As an example, we can cite the methylated, ethylated, propylated, succinylated, carboxylated, acetylated, 2-hydroxypropylated, polyoxyethylated extrinated cyclodextrins.

Preferably, the polymer (a) is a crosslinked polymer, i.e. the polymer is in a three-dimensional, non-linear network formed by the creation of bonds between the macromolecular chains of a polymer during the polymerization reaction. This polymer can then be modified in order to add new functionalities such as negative or positive charges, or by grafting fluorescent molecules.

Preferably, the polymer (a) is the product of the reaction between p-cyclodextrin and epichlorohydrin.

According to this embodiment, this polymer is known and described by Grefet al. Journal of controlled release: official journal of the controlled release Society 2006, 111 (3), 316- 324 under the name of pCD.

Polymers (a) can be prepared by adaptation or application of the method described by Gref et al. Journal of controlled release: official journal of the controlled release Society 2006, 111 (3), 316-324, and Othman et al., J Colloid Interface Sci 2011, 354 (2), 517-27.

More specifically, a polymer (a) can be prepared by polymerization reaction between p-cyclodextrin and epichlorohydrin in an alkaline medium in the presence of NaOH. For example, p-cyclodextrin is dissolved under vigorous stirring in a solution comprising 33% by mass of NaOH, then epichlorohydrin is then added under stirring and the reaction is stopped by adding acetone before the gelation point.

According to this embodiment, the chemical structure of the polymer (a) is as represented by the general formula (B):

[Chem 2]

In which the truncated cones represent the p-cyclodextrin molecules and n represents a repeating unit corresponding to the reacted epichlorohydrin molecules separating two p-cyclodextrin molecules.

The composition according to the invention comprises a compound (b) chosen from mycolactone and its derivatives. Mycolactone and its derivatives are 12-membered polyketide derivatives originally produced by different bacterial strains of the Mycobacterium ulcerans (Mu) family and which are implicated in Buruli ulcers (BU).

Compounds (b) are accessible.

They can for example be obtained by extraction and purification from Mycobacterium ulcerans extract). This method is described in George et al; SCIENCE Volume 283, Issue 5403, 5 February 1999.

It is possible to use different strains of Mycobacterium ulcerans that produce different isomers. The production of isomers depends on the area where the bacterial strain comes from.

The mycolactone used in these examples comes from strain 1615 which corresponds to the cited bibliographic reference George et al; SCIENCE Volume 283, Issue 5403, 5 February 1999. It is also possible to extract mycolactone from strains obtained directly from patients.

Alternatively, the compounds can be synthesized. A synthetic method for preparing mycolactone and its derivatives has been developed and described in application EP 2 594 561.

According to one embodiment, compound (b) is a compound of formula (I):

- Ri, R2, R4 and R5 are the same or different and are independently selected from the group consisting of H, Re, C(O)R ₇ , C(S)R ₇ , C(O)NHR ₇ and C(S)NHR ₇ ,

- R3 is selected from the group consisting of H, OH, ORe, OC(O)R ₇ , OC(S)R ₇ , OC(O)NHR ₇ , OC(S)NHR ₇ OR OCH(OH)R ₇ , - Re is a group selected from the group consisting of C1-C6 alkyl, C6-C12 aryl, C6-C12 heteroaryl and sugar derivatives, and

- R? is selected from H, C1-C2 alkyl, C6-C12 heteroaryl, and C6-C12 aryl groups, where

- Ri and R2, 2 and R3, and/or R4 and R5 together form an acetal group, as well as its pharmaceutically acceptable salts.

By “Ci-Ce alkyl” group is meant a linear or branched aliphatic hydrocarbon group comprising, unless otherwise stated, from 1 to 6 carbon atoms in total, and which may comprise one or more unsaturations. Examples include methyl, ethyl, n-propyl, butyl, isobutyl, tert-butyl, pentyl or hexyl groups.

By “C6-C12 aryl” group is meant monocyclic, bicyclic or tricyclic aromatic hydrocarbon compounds, comprising, unless otherwise stated, from 6 to 12 carbon atoms in total. Examples include phenyl and naphthyl groups.

By “C6-C12 heteroaryl” group is meant one of the monocyclic, bicyclic or tricyclic aromatic compounds comprising from 6 to 12 carbon atoms in total, at least one atom of which is a heteroatom chosen from the group consisting of nitrogen, phosphorus, oxygen and sulfur. By way of example, mention may be made in particular of the pyrrole, furan, thiophene, imidazole, furazan, oxazole, oxadiazole, oxatriazole, isoxazole, thiazole, isothiazole, pyrazole, triazole or tetrazole groups. By “sugar derivative” is meant compounds comprising at least one sugar unit chosen from mono- and polysaccharides. By way of example, the monosaccharide unit may be glucose, galactose, fructose or pentose.

ainsi que ses sels pharmaceutiquement acceptables. According to another embodiment, compound b) is a compound of formula (II):

and its pharmaceutically acceptable salts.

This compound is a compound of formula (I) in which Ri, R2, R4, Rs are -H groups and R3 is an -OH group; it corresponds to mycolactone

According to one embodiment, the composition according to the invention comprises a compound (b) which is encapsulated in the polymer (a) of p-cyclodextrin. By encapsulated in the polymer (a) we understand the incorporation of the mycolactone by solubilization in an aqueous medium. In practice, this is achieved by adding an aqueous solution of polymer (a) of p-cyclodextrin to a container containing pure mycolactone. By contact with (a) it is solubilized, with or without mechanical stirring. Alternatively, to accelerate the process, sonication or ultraturrax can be used.

Indeed, without wishing to be bound by any theory, the molecules of mycolactone and/or its derivatives form inclusion complexes with the molecules of p-cyclodextrin composing the polymer (a). The formation of these inclusion complexes also makes it possible to encapsulate the compounds (b) within the molecules of p-cyclodextrin composing the polymer.

Advantageously, the formation of these inclusion complexes makes it possible to spontaneously solubilize compounds (b) without the use of organic solvent.

Advantageously, the encapsulation of the compounds (b) in the p-cyclodextrin polymer (a) also makes it possible to significantly stabilize the compounds (b) against degradation, in particular under UV irradiation.

Advantageously, the very strong affinity of mycolactone and its derivatives for p-cyclodextrin allows a prolonged release of the compound (b) in the body after administration of the composition.

In other words, the composition according to the invention is a composition comprising a p-cyclodextrin polymer in which compound (b) is encapsulated.

Preferably, the composition according to the invention is in powder form or may be in aqueous solution, even more preferably the composition is in aqueous solution.

In particular, the powder can be obtained by freeze-drying said composition according to the invention in aqueous solution. Advantageously, the encapsulation of the compounds (b) by the p-cyclodextrin polymer makes it possible to surprisingly obtain an aqueous composition having a high concentration of compound (b).

According to one embodiment, the composition is an aqueous solution in which the concentration of compound (b) is between 0.001 mg/mL and 10 mg/mL, preferably from 0.05 to 5 mg/mL.

Preferably, the composition according to the invention is an aqueous solution in which the concentration of compound (a) is between 25 mg/mL and 250 mg/mL, preferably from 50 mg/mL to 150 mg/mL.

Preferably, the composition according to the invention comprises a ratio of the amount of compound (b) as a percentage by weight to the amount of polymer (a) as a percentage by weight which can be from 0.1 to 10, preferably from 0.5 to 7, and even more preferably from 1 to 5, the percentages being expressed relative to the total weight of the composition.

According to another embodiment, the polymer (a) of p-cyclodextrin can typically encapsulate at least one other active ingredient having for example a complementary, potentiated or synergistic action with the compound (b). Generally, the active ingredients are encapsulated within the cavities of the free p-cyclodextrin molecules forming the polymer (a), i.e. not already being inclusion complexes with a compound (b).

According to one embodiment, the composition according to the invention may further comprise one or more active ingredients (c) chosen from anti-cancer agents, analgesic agents, anesthetics, active molecules with a healing effect, antibacterial agents, antibiotics, antiseptics, hemostatics, antifungals, antivirals, antithrombotics, anti-inflammatories, antipruritics, contrast products, hormones.

Typically, the active ingredient is an antiseptic, such as chlorhexidine or benzalkonium chloride, ethanol, hexamidine, betadine, chlorinated derivatives, triclocarban.

According to another embodiment, the active ingredient (c) is an analgesic agent, such as lidocaine, codeine, tramadol.

According to another embodiment, the active ingredient (c) is an antibiotic agent such as vancomycin (VCM), amikacin, gentamicin or amoxicillin. According to another embodiment, the active ingredient (c) is an anticancer agent such as doxorubicin, cyclophosphamide, cisplatin, docetaxel, gemcitabine, or oxaliplatin.

Compound (b) and said active ingredients may be present in the form of a hydrate and/or a pharmaceutically acceptable salt. Indeed, these compounds may be present in the form of corresponding salts of pharmaceutically acceptable organic or mineral acid or organic or mineral base.

The term "pharmaceutically acceptable salts" refers to the relatively nontoxic, inorganic and organic acid addition salts and base addition salts of the compounds of the present invention. These salts may be prepared in situ during the final isolation and purification of the compounds. In particular, the acid addition salts may be prepared by separately reacting the purified compound in its purified form with an organic or inorganic acid and isolating the salt thus formed. Examples of acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptanate, lactobionate, sulfamates, malonates, salicylates, propionates, methylenebis-b-hydroxynaphthoates, gentisic acid, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexyl sulfamates and quinatelaurylsulfonates, and the like. (See for example SM Berge et al. "Pharmaceutical Salts" J. Pharm. Sci, 66: p.1-19 (1977)). Acid addition salts can also be prepared by separately reacting the purified compound in its acid form with an organic or inorganic base and isolating the salt thus formed. Acid addition salts include amine and metal salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminum salts. Sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide. Suitable basic amine addition salts are prepared from amines which have sufficient alkalinity to form a stable salt, and preferably include amines which are often used in medicinal chemistry because of their low toxicity and acceptability for medicinal use: ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'- dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzyl- phenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetra-methylammonium, tetraethylammonium, methylamine, dimethylamine, trimethyl-amine, ethylamine, basic amino acids, for example lysine and arginine, and dicyclohexylamine, and the like.

According to another embodiment, the polymer (a) of p-cyclodextrin can advantageously be associated with other compounds such as lipid compounds (cholesterol, injectable oils such as Migliol), glycoproteins (such as laminins), nanoparticles (NPs) thus advantageously conferring complementary properties to the composition according to the invention such as antibacterial or antifungal properties, targeting properties or even the inclusion of other active ingredients in the composition.

This encapsulation is generally possible if the size of said nanoparticles allows their inclusion within the cavities of the free p-cyclodextrin molecules.

Preferably, the size of the nanoparticles (d) is between 50 nm and 400 nm, even more preferably between 50 nm and 250 nm. The lipid compounds can form microdroplets of a size ranging from 50 nm to 2-5 microns within the formulation.

According to another object, the present invention also relates to a pharmaceutical composition comprising the composition according to the invention and at least one pharmaceutically acceptable excipient.

The pharmaceutical composition of the invention may be presented in different forms intended for topical, parenteral or intrathecal administration. Preferably, said composition is administered to a patient in need thereof.

Topical administration means the administration of a product to a body surface such as the skin or mucous membranes. Parenteral administration means administration of a product by injection. Parenteral administration includes intramuscular (IM), intravenous (IV), subcutaneous (SC), intradermal, intra-arterial or intra-articular injections.

Intrathecal administration means an injection into the subarachoidal space to reach the cerebrospinal fluid, which will in turn diffuse the injected product.

Injectable pharmaceutical forms, in particular for parenteral or intrathecal use, are generally injectable solutions which can be obtained, for example, by the following process: the composition according to the invention is dissolved, suspended or emulsified either in an aqueous medium (for example distilled water, physiological saline or Ringer's solution), with a dispersant (for example Tween® 80, HCO® 60 (Nikko Chemicals), polyethylene glycol, carboxymethylcellulose or sodium alginate), a preservative (for example methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, benzyl alcohol, chlorobutanol or phenol), an isotonic agent (for example sodium chloride, glycerol, sorbitol or glucose) and optionally other additives, such as, if desired, a solubilizing agent (for example sodium salicylate or sodium acetate) or a stabilizer (for example human serum albumin).

The pharmaceutical forms for topical use can be obtained from a solid, semi-solid or liquid composition containing the composition of the invention. For example, to obtain a solid form, the composition according to the invention can be mixed with excipients (for example lactose, trehalose, sucrose, mannitol, starch, microcrystalline cellulose or saccharose) and a thickener (for example natural gums, cellulose derivatives or acrylic polymers) in order to transform them into powder. The liquid pharmaceutical compositions are prepared in substantially the same way as the injectable forms, as indicated above. The semi-solid pharmaceutical forms are preferably in the form of aqueous or oily gels or in the form of ointments. These compositions may optionally contain a pH regulator (e.g. carbonic acid, phosphoric acid, citric acid, hydrochloric acid or sodium hydroxide), an antioxidant compound (vitamin C, tocopherol, polyphenols) and/or a preservative (e.g. a p-hydroxybenzoic acid ester, chlorobutanol or benzalkonium chloride). According to one embodiment, the pharmaceutical composition according to the invention comprises a pharmaceutically acceptable excipient chosen from dextran and its derivatives, preferably hydrophobized dextran.

Advantageously, the use of hydrophobized dextran in the pharmaceutical composition according to the invention allows the formation of gels or nanoparticles. In this case, it is possible to envisage the incorporation of nanoparticles as described in example 13 or oil droplets.

Preferably, the size of the nanoparticles (d) is between 50 nm and 400 nm, even more preferably between 50 nm and 250 nm. The lipid compounds can form microdroplets of a size ranging from 50 nm to 2-5 microns within the formulation.

According to one embodiment, the composition according to the invention may further comprise nanoparticles (d) chosen from inorganic nanoparticles, organic-inorganic hybrid nanoparticles, or polymeric nanoparticles.

By “nanoparticle” (NP) is generally meant a particle of spherical shape, with an average diameter (in number) of between 1 nm and 1 pm. In the context of the invention, said NPs typically have an average diameter of less than 400 nm, in particular less than 250 nm, with a polydispersity index of less than 0.25.

The number-average diameter and polydispersity can be measured by dynamic light scattering (DLS) or transmission electron microscopy (TEM).

Examples of inorganic nanoparticles include magnetic nanoparticles, silver nanoparticles with an antibacterial effect, gold nanoparticles and copper nanoparticles.

Organic-inorganic hybrid nanoparticles are, for example, iron trimesate particles MIL-100(Fe), or other iron carboxylates, or even UIO-66.

Examples of polymeric nanoparticles include PLGA particles (poly(lactic-co-glycolic acid), poly(ethylene glycol) (PEG)-PLGA (PEG-PLGA) copolymer or polycaprolactone (PCL).

Advantageously, the nanoparticles (d) can themselves include an active ingredient. According to another object, the present invention also relates to a process for preparing the composition according to the invention, comprising a step of mixing compounds (a) and (b) in aqueous solution.

According to another embodiment, the method according to the invention may optionally comprise a step of adding an active ingredient (c) and/or nanoparticles (d) to the aqueous solution.

Preferably, the method according to the invention can also comprise a freeze-drying step allowing said composition to be obtained in powder form.

Another object of the present invention relates to the use of the composition according to the invention for the treatment of pain.

The term "pain" also refers to "painful disorders" and designates more or less severe suffering, produced by an injury, a burn, a lesion or any other cause, which manifests a disruption of well-being, of the balance of health, the loss or reduction of physical integrity.

Pain is generally classified as acute or chronic. “Acute” pain means a sudden, short-lived experience associated with a specific cause such as a specific injury resulting from, for example, surgery, dental work, or a sprain.

By “chronic pain” we mean long-term pain leading to significant psychological and emotional problems. This pain is notably linked to many pathologies such as osteoarthritis, cancers or even diabetes.

The composition according to the invention is suitable for the treatment of a wide range of painful disorders, including when these appear as symptoms of pathologies, in particular acute pain, chronic pain, neuropathic pain, inflammatory pain, iatrogenic pain, including cancer pain, infectious pain, including herpetic pain, visceral pain, central pain, dysfunctional pain, including fibromyalgia, nociceptive pain, including post-surgical pain, and mixed types of pain involving the viscera, the gastrointestinal tract, the cranial structures, the musculoskeletal system, the spine, the urogenital system, the cardiovascular system and the CNS, including cancer pain, back and orofacial pain. Generally speaking, painful disorders are considered symptoms of a pathology that must be treated independently of said pathology for the well-being of the patient.

By "symptom" we mean an abnormal manifestation caused by a pathology of which the patient complains.

The term "pain treatment" as used herein means the relief, inhibition of progression or elimination of painful disorders as described herein, without necessarily treating the pathology that may be potentially responsible for them. Thus, pain treatment may typically be symptomatic and may possibly be etiological.

Generally, pain or "pain disorders" in the patient are usually treated by the administration of short-acting analgesics or painkillers.

By "analgesic" we mean drugs used to suppress sensitivity to pain, such as opioid derivatives.

By "analgesic" we mean drugs used to reduce pain.

Preferably, the composition according to the invention is used as an analgesic in order to treat pain or painful disorders.

According to another embodiment, the composition according to the invention is used as an analgesic in order to treat pain and painful disorders.

Advantageously, the composition according to the invention can be used in a complementary manner in order to treat painful disorders caused by a pathology.

Figures

[Fig 1] Figure 1. A: Improvement of mycolactone (ML) encapsulation with increasing pCD amount. B: Successive extractions of ML from ML-pCD.

[Fig 2] Figure 2. Protective role of pCD against the degradation of mycolactone exposed to UV.

[Fig 3] Figure 3. A) No additional cytotoxic effects caused by pCD. B-D) pCD does not modify the immunomodulatory effects (production of IL-6, TNF-a and I L-1 p) of mycolactone. Black = free ML; gray = incorporated ML (pCD-ML)

[Fig 4] Figure 4. Analgesic effect of pCD-encapsulated mycolactone in mice (#: difference between ML and pCD-ML).

[Fig 5] Figure 5. Biodegradable extruded cyclodextrins reduce the analgesic effect of mycolactone compared to pCD.

[Fig 6] Figure 6. Analgesic effect of pCD-encapsulated mycolactone in a mouse plantar incisional model (surgical pain model) (#: difference between ML and pCD-ML).

[Fig 7] Figure 7. Analgesic effect of pCD-encapsulated mycolactone in a mouse plantar incisional model (surgical pain model) (#: difference between ML and pCD-ML).

Examples

Example 1 - Synthesis and characterization of a cyclodextrin polymer (pCD)

In a 500 mL flask, 50 g of -CD are introduced into 80 mL of 33% NaOH and left stirring overnight. The next day, the flask is heated to 30°C and 35 mL of epichlorohydrin (EP) are added with stirring (Scheme 1). The reaction mixture is kept stirring at 30°C for 1 h-1 h30 in order to homogenize it well. The reaction is stopped when the viscosity increases, just before the solidification point, by adding a volume of 80 mL of acetone.

Scheme 1. Reaction scheme for the synthesis of pCD from epichlorohydrin-crosslinked p-CD.

The bath temperature is raised to 50°C and left stirring overnight. The next day, neutralization is carried out with a 6 M HCl solution to obtain a pH of 7. The non-soluble residue is removed and the remainder of the polymer is purified by dialysis using a Spectra/por membrane (cut-off threshold 100,000 g/mole) and then lyophilized. The pCD polymer thus obtained is characterized by NMR and SEC to determine its p-CD content, which is around 70% (wt).

By the same methodology, a polymer of a-CD and a polymer of y-CD are synthesized.

pCD samples can be fractionated by successive dialysis (using for example SpectraPor membranes with cut-off thresholds of 20, 50, 100, 300 and 1000 KDa) in order to obtain samples with low polydispersities.

Example 2 - Synthesis and characterization of a malic acid-based cyclodextrin polymer

In a 25 mL flask, 0.2 mmol NaH2PO4 2H2O (catalyst), 0.09 mmol p-CD, and 0.45 mmol malic acid in 2 mL water are mixed. The mixture is then concentrated by evaporation at 140 °C for 10 min then heated at 140 °C for 25 min under reduced pressure (vacuum pump). The polymer thus formed is taken up with 10 mL of MilliQ water and dispersed by ultrasound. The insoluble fraction is removed by filtration. The soluble fraction is then purified by dialysis (Spectra/por membrane, cut-off threshold 20000 g /mole) then lyophilized. The polymer is then characterized by ¹ H NMR and SEC in order to determine its p-CD content which is around 70% (wt).

This polymer is called pCD.

A biodegradable polymer of p-CD is synthesized in a similar manner, replacing malic acid with citric acid.

Example 3 - Synthesis of dextran modified with alkyl links

To synthesize dextran grafted with hydrophobic lauryl chains (DM), 4 g of dextran (40000 g/mol) were solubilized in 100 mL of dimethylformamide containing 1 g of lithium chloride. Then, 0.62 mL of lauryl chloride and 0.031 mL of pyridine were added to the dextran solution. The reaction was carried out at 80 °C for 3 h. The obtained MD was isolated by precipitation in isopropyl alcohol. It was then solubilized in distilled water, purified by dialysis for 48 h and then lyophilized. The grafting rate of alkyl chains determined by ¹ H NMR is around 6%.

Example 4 - Production of mycolactone

Mycolactone is purified from M. ulcerans strain 1615 culture according to the protocol described by Georges 1999 (1). Briefly, total mycobacterial lipids are extracted according to the Folch method. Then, after precipitation of the phospholipids in cold acetone, the supernatant containing the mycolactone is deposited on a silica plate to perform thin-layer chromatography. After migration, the silica on which the mycolactone has adsorbed (rf of 0.23) is scraped off and the mycolactone is desorbed from the silica by filtration (in chloroform/methanol). Finally, the mycolactone is quantified by high-performance liquid chromatography (2). 1. George KM, Chatterjee D, Gunawardana G, Welty D, Hayman J, Lee R, Small PL. 1999. Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence. Science 283:854-857.

2. Marion E, Prado S, Cano C, Babonneau J, Ghamrawi S, Marsollier L. 2012. Photodegradation of the Mycobacterium ulcerans toxin, mycolactones: considerations for handling and storage. PLoS One 7:e33600.

Example 5 - Incorporation of mycolactone into pCD

In a 1.5 mL amber glass bottle, 0.5 mg of ML (mycolactone) solubilized in ethanol are introduced. The ethanol is evaporated using a vacuum concentrator (SpeedVac). 1 mL of pCD solution at a concentration of 100 mg/mL is then added. The preparation is stirred for 48 hours and then stored at 4°C. This time allows complete incorporation of the ML into the pCD. This solution is called pCD-ML.

The experiment is reproduced by replacing pCD with a polymer of p-CD and a polymer of p-CD. ML does not solubilize, even by extending the incubation time up to 72 h.

Example 6- Improved mycolactone encapsulation with increasing pCD amount

From a 100 mg/mL pCD solution, cascade dilutions are carried out in MilliQ water to obtain the following pCD concentrations: 1, 10, 50 and 100 mg/mL. In a 1.5 mL amber glass vial, 0.5 mg of ML solubilized in ethanol are introduced. The ethanol is evaporated using a vacuum concentrator (SpeedVac). 1 mL of pCD solution at concentrations 1, 10, 50 and 100 mg/mL is then added. The preparation is stirred for 48 hours.

Aliquots are collected and assayed by HPLC as in Example 5, in order to determine the amount of ML incorporated. It is found that at least 100 mg/mL of pCD is required to incorporate 0.5 mg of ML (Figure 1 A).

After incubation with shaking, 30 μL of each of the pCD-ML preparations are taken and then diluted ^1/10 in acetonitrile. After centrifugation (3000 g, 5 min), the supernatants are assayed by HPLC. All the pellets are taken up in MilliQ water and then diluted at 1/ ^10th in acetonitrile before being centrifuged and assayed again by HPLC. These steps are repeated 3 times in order to extract all the contents of ML encapsulated at the different concentrations of pCD tested.

The remainder of the preparation is taken up in ethanol then measured using HPLC after being diluted ^1/10 in acetonitrile and centrifuged (3000g, 5min).

In conclusion, the higher the ML content in pCD, the more difficult it becomes to extract ML from pCD. Thus, 4 successive extractions are necessary to extract all ML from pCD (100 mg/mL of pCD and 0.5 mg of incorporated ML). This demonstrates the high affinity of ML for pCD. It is very likely that at high concentrations, the highly hydrophobic ML can self-associate within pCD (Figure 1 B).

Example 7 - stability of pCD-ML during storage

Surprisingly, the ML incorporated in pCD no longer adsorbs on glass or plastic containers, making its handling possible. This was demonstrated by measuring the ML concentration by HPLC, in the case of a pCD-ML solution prepared as in Example 5. After 3 months, there was less than 4% concentration variation.

Example 8 - Protective role of pCD against the degradation of mycolactone exposed to UV

50 pL of pCD-ML solution prepared according to Example 5 (corresponding to 0.5 mg/mL of ML) are placed in transparent glass tubes. Then the tubes are exposed to UV irradiation (room temperature, wavelength 312 nm): 15 min, 1 h, 2 h, 6 h, 24 h. One tube is kept in the dark and serves as a control. All tubes are hermetically sealed to avoid evaporation problems.

After these different UV exposure times, the ML is measured by HPLC. A series of ML extractions is carried out as described in example 6. For this, 450 pL of acetonitrile is added to each tube containing 50 pL of pCD-ML. After 6 hours of exposure, 80% of the ML contained in ethanol has been degraded while over the same period only 50% of the ML associated with pCD has been degraded (Figure 2). Example 9- In vitro evaluation of the cytotoxic and immunomodulatory effects of pCD on murine macrophages

Two preparations are made: an ethanolic solution of ML and an aqueous solution of pCD-ML at 0.5 mg/mL. Cascade dilutions are made to obtain solutions with concentrations of 100 - 10 - 1 pg/mL then 60 - 20 - 2 ng/mL in ML. 100 pL of each dilution are distributed per well containing 100 pL of cells (100,000 cells/well), i.e. a final concentration of 30 - 10 - 1 ng/mL of ML. The cells are incubated for 24 hours (at 37°C, 5% CO2), then stimulated with 20 pL of a lipopolysaccharide (LPS) solution at 500 ng/mL, i.e. 50 ng/mL LPS in each well. After 24 hours of incubation, the supernatants are collected.

To determine the cytotoxic effect of ML, the Lonza™ ToxiLight™ test (Fisher Scientific) is used. For this, 5 pL of each supernatant is mixed with 25 pL of reagent.

To determine the immunomodulatory effect of ML, ELISA kits (IL-6, TNF-a, IL-1b) are used, after dilution of the supernatants to ^1/10 for the IL-6 and TNF-a kits, and to 1 for the IL-1b kit.

It appears that the incorporation of ML into pCD does not significantly modify its immunomodulatory effect (Figure 3. A-D).

Example 10 - Analgesic effect of pCD-encapsulated mycolactone in mice

In 3 different tubes, solutions of pCD in water (100 mg/mL), ML in ethanol (3 mg/mL) and pCD-ML (3 mg/mL) are prepared as previously described. Subsequently, pCD is diluted with physiological saline to a concentration of 8.33 mg/mL. ML is diluted in corn oil (1) to a concentration of 250 pg/mL. Finally, the pCD-ML preparation is diluted with physiological saline to a concentration of ML of 250 pg/mL.

1. Babonneau J, Bréard D, Reynaert ML, Marion E, Guilet D, Saint Andre JP, Croue A, Brodin P, Richomme P, Marsollier L. 2019. Mycolactone as Analgesic: Subcutaneous Bioavailability Parameters. Front Pharmacol 10:378. The experiments were carried out on female Balb/c mice aged 7 to 10 weeks. 15 μL of preparation were injected subcutaneously into the footpad. Then, the latency time of the mice to withdraw their paw following a thermal stimulus was measured. using the Hargreaves foot test apparatus. The value expressed corresponds to the ratio of the test group to the control group (pCD alone).

As shown in Figure 4, the analgesic effect induced by pCD-ML is superior to that of ML alone and this effect lasts longer. For statistical analyses, a two-way ANOVA followed by a Dunnett's multiple comparison test was used: * p<0.05, p**<0.01, **** p<0.0001))

Example 11 - Biodegradable cyclodextrins reduce the analgesic effect of mycolactone

From pCD and biodegradable cyclodextrins (named pCD-citrate), solutions at a concentration of 100 mg/mL are prepared. ML is encapsulated in each of these solutions at a concentration of 3 mg/mL. Then, these preparations are diluted in physiological saline at a concentration of 250 pg/mL. In parallel, control solutions without ML are prepared in the same way.

These experiments were carried out on Balb/c mice aged 7 to 10 weeks. 15 μL of preparation were injected subcutaneously into the pad. Then, the latency time of the mice to withdraw their paw following a thermal stimulus was measured using the plantar test apparatus by the Hargreaves method. The value expressed corresponds to the ratio of the tested group/control group (pCD alone). As shown in Figure 5, the analgesic effect induced by pCD-ML was greater than that of ML alone and this effect lasted longer. For statistical analyses, a two-factor ANOVA followed by a Dunnett multiple comparison test was used. * p<0.05, p**<0.01, **** p<0.0001). In our conditions, only the incorporated ML had a significant analgesic effect. This result shows the specificity of ML-pCD to induce an analgesic effect.

Example 12 - Analgesic effect of pCD-encapsulated mycolactone in a plantar incisional model in mice (surgical pain model) In 2 different tubes, solutions of pCD in water (100 mg/mL) and pCD-ML (3 mg/mL) are prepared as previously described. Then these solutions are diluted with physiological serum in order to obtain final preparations at 3.75 pg of ML, 1.875 pg of ML and 0.937 pg of ML for 15 pL of solution.

The value expressed corresponds to the ratio of the test group/control group (pCD alone). As shown in Figures 6 and 7, the analgesic effect induced by pCD-ML is greater than that of ML alone and this effect lasts longer. For statistical analyses, a two-way ANOVA followed by a Dunnett multiple comparison test was used: * p<0.05, p**<0.01, **** p<0.0001)). In our conditions, only ML-pCD has a significant analgesic effect. This result shows the specificity of ML-pCD to induce an analgesic effect.

These experiments were carried out on C57BI/6 mice aged 7 to 10 weeks. The incisions were made on the short flexor muscle of the toes. 15 μL of preparation were injected subcutaneously as close as possible to the incision. Then, the latency time of the mice to withdraw their paw following a thermal stimulus was measured using the Hargreaves plantar test device. The results obtained show that up to a dose of 0.9 pg ML in pCD (Figure 7), a significant analgesic effect was observed more than 30 h after application. The effect was more lasting for doses < 3.75 pg of ML (Figure 6. A). This result is surprising but can be explained by the fact that at low doses there is no longer any adverse effect (immunomodulatory effect) of ML that could reduce its analgesic effect.

Example 13 - Co-incorporation of magnetic particles

A DM solution and a pCD solution are prepared, each at 75 mg/mL. Mixing equal volumes of these solutions instantly produces the formation of a soft gel system that settles to the bottom of the container. This gel can be collected and passed through a syringe (20G needle).

The same operation is performed, except that in the DM (1 mL) 0.5 mg of iron oxide nanoparticles (Aldrich, < 5 micron) ref 310069 are incorporated and left to incubate for 4 hours. After mixing with 1 mL of pCD a dark colored gelled system is formed. It can be moved using a magnet because it contains magnetic particles. Almost all of the magnetic particles are incorporated into this gel. Furthermore, the system maintains its cohesion even during significant dilution (adding 3 L of water).

The same experiment is performed by incorporating the ML into the pCD as in Example 5 and then mixing with the DM solution containing the magnetic particles. The same type of gel is formed.

Example 14 - Development of nanoparticles

Identical volumes of DM (10 mg/mL) and pCD (10 mg/mL) are mixed with or without ML. Nanoparticles of approximately 120 nm are instantly formed, and a cloudy solution is observed.

Example 15: Freeze-drying

Aqueous solutions of pCD-ML are prepared as previously described in Example 5, in amber vials. The final concentrations of pCD are 10 and 100 mg/mL, and those of ML 0.3 and 0.5 mL, respectively. These solutions are directly frozen at -80°C and then lyophilized (typically, using an Alpha 1-2 LD Plus lyophilizer, -65°C, 0.018 mbar, 24 h).

At the end of lyophilization, a white solid is obtained. pCD-ML solutions are easily reconstituted by adding water to this lyophilizate.

The lyophilized form protects ML better from degradation in light than the solubilized form.

Advantageously, the lyophilisate can be stored for more than 8 months protected from light, without detectable degradation of the incorporated ML. The integrity of the ML incorporated in the pCD is determined by HPLC (Agilent, with a C18 Kinetex® column 5pm x 250mm x 4.6mm; 100 Â). Detection is done at 30°C, at a wavelength of 363nm, with a flow rate of 1 mL/min, and an injection volume of 20pL. The gradient is: 0min = 90/10 water/acetonitrile; 3min = 50/50 water/acetonitrile; 13min = 0/100 water/acetonitrile; 23min = 0/100 water/acetonitrile; 33min = 90/10 water/acetonitrile.

Claims

1. Composition comprising: - (a) a p-cyclodextrin polymer; and - (b) at least one compound selected from mycolactone and its derivatives. 2. Composition according to claim 1, in which the compound (b) corresponds to the following formula (I):

in which: - Ri, R2, 4 and R5, identical or different, are independently chosen from the group consisting of H, Re, C(O)R ₇ , C(S)R ₇ , C(O)NHR ₇ and C(S)NHR ₇ , - R3 is selected from the group consisting of H, OH, ORe, OC(O)R ₇ , OC(S)R ₇ , OC(O)NHR ₇ , OC(S)NHR ₇ OR OCH(OH)R ₇ , - Re is a group selected from the group consisting of C1-C6 alkyl, C6-C12 aryl, and sugar derivatives, and - R ₇ is chosen from H, C1-C6 alkyl and C6-C12 aryl groups, or - R1 and R2, R2 and R3, and/or R4 and R5 together form an acetal group, as well as its pharmaceutically acceptable salts. 3. Composition according to claim 1 or 2, in which the compound (b) is mycolactone corresponding to the formula (II):

and its pharmaceutically acceptable salts 4. Composition according to any one of claims 1 to 3, in which the compound (b) is encapsulated in the polymer (a) of p-cyclodextrin. 5. Composition according to any one of claims 1 to 4, as it is present in powder form or in aqueous solution. 6. Composition according to one of claims 1 to 5, such that the concentration of compound (b) is between 0.001 mg/mL and 10 mg/mL, preferably from 0.05 to 5 mg/mL. 7. Composition according to claim 1 to 6, in which the polymer (a) is the product of the polymerization reaction between the modified or unmodified p-cyclodextrin and at least one other chemical compound selected from the group consisting of epichlorohydrin, tartaric acid, citric acid, acylated poly(ethylene glycol), adipoyl chloride, succinyl chloride, glutaraldehyde, diphenyl carbonate, 1,4-butanediol diglycidyl ether, toluene diisocyanate, naphthalene diisocyanate, succinic anhydride, 1,2,4,5 benzene tetracarboxylic anhydride, monochlorotriazine, trimethoxysilane derivatives, diethynyl benzene and tetrafluoro terephthalonitrile. 8. Composition according to any one of the preceding claims, in which the polymer (a) is a polymer of p-cyclodextrin, modified or not and crosslinked with epichlorohydrin. 9. Composition according to any one of the preceding claims, further comprising an active ingredient (c) chosen from anti-cancer agents, analgesic agents, anesthetics, active molecules with a healing effect, antibacterial agents, antibiotics, antiseptics, hemostatics, antifungals, antivirals, antithrombotics, anti-inflammatories, antipruritics, contrast products, hormones. 10. Composition according to any one of the preceding claims, comprising a compound (d) chosen from inorganic nanoparticles, organic-inorganic hybrid nanoparticles or polymeric nanoparticles. 11. Pharmaceutical composition comprising the composition according to any one of claims 1 to 10, and at least one pharmaceutically acceptable excipient. 12. Pharmaceutical composition according to claim 11, in which the pharmaceutically acceptable excipient is chosen from dextran and its derivatives. 13. Pharmaceutical composition according to claim 11, in which the pharmaceutically acceptable excipient is hydrophobized dextran, leading to the formation of gels or nanoparticles. 14. Process for preparing the composition according to any one of claims 1 to 10, comprising a step of mixing compounds (a) and (b) in aqueous solution. 15. Composition according to any one of claims 1 to 10, for its use in the treatment of pain.