FR3092490A1 - Squalamine-like aminosteroid compound for use in the treatment and / or prevention of dermatophytosis - Google Patents

Abstract

The present invention relates to a squalamine-like aminosteroid compound corresponding to general formula (I) comprising two amino chains -NHR, or one of its pharmaceutically acceptable salts, for its use by topical application in humans or animals in the treatment and / or prevention of dermatophytosis caused by at least one fungus and / or of bacterial superinfection of said dermatophytosis . It also relates to a pharmaceutical composition comprising an aminosteroid compound analogous to squalamine corresponding to the general formula (I), or one of its pharmaceutically acceptable salts. Figure for the abstract: none

Description

Squalamine analog aminosteroidal compound for use in the treatment and/or prevention of dermatophytosis

The present application relates to the therapeutic field. More specifically, the present application relates to an aminosteroidal compound similar to squalamine comprising two -NHR amino chains corresponding to the general formula (I) defined below, or one of its pharmaceutically acceptable salts, for its use by topical application in the treatment and/or the prevention of a dermatophytosis caused by at least one fungus, and/or of a bacterial cutaneous superinfection of said dermatophytosis, . It also relates to a pharmaceutical composition comprising an aminosteroidal compound similar to squalamine corresponding to the general formula (I), or one of its pharmaceutically acceptable salts.

Dermatophytosis, also called dermatophytosis or dermatomycosis, is superficial mycoses and more specifically infections of the skin or appendages due to filamentous microscopic fungi also called dermatophytes, which belong to the following three genera: Trichophyton, Microsporum and Epidermophyton . Dermatophytes are divided into three groups, anthropophilic species, Microsporum audouinii, Trichophyton rubrum, Trichophyton interdigitale, Trichophyton violaceum, Trichophyton schoenleini, Epidermophyton floccosum, anthropo-zoophilic species in which we find Microsporum canis transmitted by cats, Trichophyton mentagrophytes transmitted by horses and small rodents, Trichophyton verrucosum, Trichopyton ochraceums transmitted by cattle and Trichophyton tonsurans or even geophilic or telluric species such as Microsporum gypseum. These filamentous microscopic fungi characterized by the production of spores such as micronidia, macronidia, arthrospores and chlamydospores, are pathogenic and keratinophilic but respect the mucous membranes. They feed on the keratin that makes up the stratum corneum of the skin, body hair, hair and nails.

Transmission occurs through contact with contaminated hair or dander, with the presence of any kind of epidermal alteration as a contributing factor. This transmission can be of human-to-human origin via anthropophilic species through human contact with contaminated hair or dander or through contact with a contaminated environment such as collective showers, municipal swimming pool floors, sports or school locker rooms. This transmission can also take place from a mammalian animal to humans via zoophilic species or even from the soil to humans via geophilic species.

Epidemiological studies have shown that dermatophytosis are among the most prevalent skin pathologies in the world [1].

Different clinical forms of dermatophytosis exist, we thus distinguish the dermatophytosis of the folds, caused mainly by three dermatophytes, by human-to-human transmission, which are: theTrichophyton rubrum (70-80% of cases), theTrichophyton interdigitalis(15-20% of cases),Epidermophyton floccosum(5% of cases). Among these dermatophytosis of the folds also called intertrigo of the folds, we can mention the intertrigo inter-toes also called athlete's foot, which presents in the form of a simple dry or oozing desquamation, which can be associated with cracks or cracks. Blister bubbles on the inner surface of the toes with variable pruritus, or even involvement of the large folds, also called inguinal intertrigo, which presents with an itchy erythematous-scaly patch, with circinated contours.

We also distinguish dermatophytosis of glabrous skin most often caused by anthropophilic or anthropozoophilic dermatophytes from an infected animal, such as the so-called typical form of herpes circinata, the lesions of which form rounded or polycyclic plaques. with erythemato-vesiculo-squamous borders, which may be accompanied by intense pruritus or even the so-called inflammatory form of kerions which are of animal origin and whose appearance of the lesions is nodular and pustular.

Ringworms of the scalp are also distinguished, such as microsporic ringworms characterized by rounded patches of alopecia a few centimeters in diameter, or such as trichophytic ringworms presenting small scattered, scaly and possibly pustular lesions; or such as ringworms characterized by small inflammatory alopecic patches or such as inflammatory ringworms called kerions translated by an excessive immune reaction to dermatophytes, but also beard ringworms resulting in acute suppurative folliculitis accompanied by inflammatory papular areas . Finally, we also distinguish nail dermatophytosis at the level of the toenails in 80% of cases, caused essentially by T.rubrum (80%) and T.mentagrophytes (20%), almost always associated with dermatophytosis of the interdigital spaces and beginning at the level of the distal and lateral part of the nail or even dermatophytosis of the nails at the level of the nails of the fingers, also mainly caused by T. rubrum .

Among skin fold dermatophytosis, athlete's foot pathology, also called intertrigo inter-toes or intertrigo interdigito plantar or tinea pedis , is one of the most common infections and is found in approximately 15% of the general population. Indeed, dermatophytes proliferate easily in warm and humid areas such as the inter-toe space which is often an area neglected during drying and whose humidity associated with the heat of the foot and the confinement in the shoes make it a place conducive to the proliferation of microscopic filamentous fungi which are the cause.

The primary attack begins with a slight desquamation accompanied by scratching, not painful, in particular at the level of the ^4th and 5th ^toes becoming progressively itchy until the final stage corresponding to the cracking of the interdigital space and the bottom of the fold, which is painful and can lead to secondary bacterial infection. The focus of infection frequently spreads to the plantar level, it is often the cause of onychomycosis and scratching the infected area can also induce contamination of other parts of the body. This is why foot dermatophytosis is often associated with the presence of onychomycosis, caused largely by the same types of dermatophytes including Tricophyton rubrum and T. mentagrophytes . [2]

Dermatophytosis can lead to secondary bacterial infections that can have quite harmful consequences. For example, dermatophytosis of the foot, such as athlete's foot pathology, can lead to serious complications such as lymphangitis, erysipelas in particular of the lower limb, sepsis or necrosis of the toes, arteritis or even chronic or postoperative venous insufficiency [3].

Indeed, the inter-toe cracks that may be present in the pathology of athlete's foot, constitute an entry point for bacteria, thus leading to bacterial superinfections such as erysipelas which is an acute bacterial dermo-hypodermatitis not necrotizing, 85% caused by group A beta-hemolytic streptococci, most often affecting the lower limbs and manifested by circumscribed erythema accompanied by local edema and a febrile state. This secondary infection can sometimes turn into an abscess, fasciitis or even bacteremia. Only systemic antibiotic therapy can then put an end to the superinfection, taking into account in particular the risks of bacterial resistance [4].

Of the more than 200 million patients worldwide with diabetes, 60 to 70% of them suffer from diabetic neuropathy, which results in a decrease in the sensitivity of the contact nerves preventing the perception of small injuries or abnormalities of the foot that can grow and become infected. This is why these people have a very high susceptibility to infections by dermatophytes in the foot as well as a high susceptibility to bacterial superinfections that may accompany them. As indicated above, these bacterial superinfections can have quite serious consequences such as bacteremia. It is therefore very important to be able to treat and/or prevent dermatophytosis and its bacterial superinfection in patients with diabetes [5].

Among the existing treatments, there are local antifungals when the infection is isolated and / or whose extent is limited, such as imidazole type compounds such as bifonazole, econazole, pyridone type compounds such as ciclopiroxolamine or also compounds of the allylamine type such as terbinafine, said antifungals being administered in a galenic form adapted to the clinical appearance with an administration frequency ranging from 1 to 2 times per day for 1 to 8 weeks depending on the antifungal chosen . There are also systemic antifungals for dermatophytosis that is more pronounced or presents a certain risk of complications, such as griseofulvin by oral administration which is fungistatic on dermatophytes at a dose of 1g/day in adults and 10-20 mg/day in children but inducing photosensitivity and presenting numerous drug interactions, or even terbinafine which is currently the most effective fungicide against dermatophytes (250mg/day) but for which no galenic form suitable for children exists and presenting numerous undesirable effects such as digestive disorders and changes in taste, or else ketoconazole which presents numerous undesirable effects including drug-induced hepatitis and which is less effective on dermatophytes than on yeasts of the Candida genus. Another disadvantage of existing treatments, in particular antifungals and antibacterials, lies in the fact that these treatments can induce resistance in the fungi to be treated [6].

Thus, most dermatophytosis treatments existing to date essentially act on the fungi known to be the main cause of these dermatophytosis. However, without wanting to be bound by any theory, it seems that cutaneous bacterial proliferation is not only involved in the superinfection of these dermatophytosis but that it is also involved in the development of the pathology itself, the presence of specific bacteria thus forming with the fungi, a particular microflora on the surface of the skin favoring the development of dermatophytosis [7].

There is therefore a need for a therapeutic solution for treating and/or preventing a dermatophytosis caused by at least one fungus and a bacterial superinfection of said dermatophytosis which is effective, has little or no adverse effects and which is capable of acting on the fungi and / or bacteria involved in dermatophytosis and / or bacterial superinfection of dermatophytosis without inducing resistance of said microorganisms.

The present invention therefore aims to provide an effective solution for treating and / or preventing dermatophytosis caused by at least one fungus, as well as a bacterial superinfection of a dermatophytosis which is effective, without adverse effects, acting on the mycotic population and bacterial, not inducing resistance of microorganisms and having a low cost.

To do this, the Applicant provides analog aminosteroidal compounds of squalamine corresponding to a general formula (I) comprising two amino chains, and/or their pharmaceutically acceptable salts, for their use by topical application in humans or animals for treating and/or preventing a dermatophytosis caused by at least one fungus, and a bacterial superinfection of said dermatophytosis. In particular, the dermatophytosis considered in the context of the present invention is dermatophytosis of the folds, also called intertrigo of the folds, it is in particular a dermatophytosis of the foot, also called pathology of athlete's foot, intertrigo inter- toes or plantar intertrigo interdigito and/or nail dermatophytosis.

Squalamine (formula Ia), which is a natural steroid, mainly isolated from the tissues of a small shark Squalus acanthias , has been shown to be a very active substance with antiangiogenic activity against cells but also antiviral and antibacterial activity. Formula Ia

Chemically, squalamine is an original molecule with an amphiphilic character. It thus comprises an apolar central part (a cholestane type skeleton) and two polar ends (a polyamine chain and a sulphate group). Initially, this water-soluble polyaminosterol aroused interest for its antiangiogenic and antimicrobial properties on a variety of Gram-positive ( Staphylococcus aureus, Enterococcus faecalis) and Gram-negative ( Escherichia coli, Pseudomonas aeruginosa ) bacteria, fungi ( Candida albicans, Candida tropicalis ) and protozoa.

It has been described in WO 20011/067501 that squalamine and its aminosteroidal analogues exhibit antibiotic activity when applied topically in the form of a lipophilic formulation, in particular an ointment, making it possible to reduce the concentration of Staphylococcus aureus .

Squalamine and some of the analogous aminosteroid derivatives of squalamine have been described in patent application EP2802214 A1, in particular the derivatives having an amino chain –NHR such as the compounds ASD1 and ASD2, for their disinfecting activity of inert objects, with an ability to kill pathogenic bacteria S. aureus and P. aeruginosa and pathogenic yeasts C. albicans and A. niger in vitro , at a concentration of at least 3 mM. It is worth noting that neither C.albicans nor A.niger are implicated in dermatophytosis and in particular in the pathology of athlete's foot.

Coulibaly et al [8], demonstrated the in vitro activity of squalamine and the aminosteroidal compound similar to squalamine ASD1, bearing a single amino chain, against various dermatophytes such as Trichophyton rubrum, Trichophyton mentagrophtes, Trichophyton soudanense, Microsporum canis, Microsporum audouinii, Microsporum persicolor, Microsporum cookie and Microsporum gypseum with a minimum inhibition concentration (MIC) between 1 and 4 mg/L for squalamine and 2 to 8 mg/L for ASD1. However, for Trichophyton rubrum , which is one of the most common fungi in dermatophytosis, the antifungal activity of these compounds has proved to be weak. Furthermore, neither squalamine nor the compound ASD1 has been tested against fungi of the species Epidermophyton floccosum which are strongly involved in dermatophytosis and in particular in the pathology of athlete's foot.

Another publication by Coulibaly et al [9] concerns a 3-week clinical study aimed at studying the effectiveness of a composition comprising 1% squalamine topically for the treatment of scalp ringworm called tineas capitis on 5 patients. . This study did not reveal a treatment for the pathology but only a partial clinical improvement with a better hair growth score in the group treated with squalamine compared to that treated with the placebo and with an MIC of about 12 mg/L against M. audouinii .

Thus, no document describes or suggests that the aminosteroidal compounds analogous to squalamine comprising two amino chains, corresponding to a general formula (I) according to the present invention explained below, and/or their pharmaceutically acceptable salts, can be useful. for their use by topical application in humans or animals for treating and/or preventing dermatophytosis caused by at least one fungus, as well as bacterial superinfection of said dermatophytosis with similar or better efficacy to that of squalamine while being easier and less expensive to synthesize.

In particular, none of the cited documents describes or suggests that the aminosteroidal compounds analogous to squalamine comprising two amino chains, corresponding to a general formula (I) according to the present invention explained below, and/or their pharmaceutically acceptable salts, for their use by topical application in humans or animals to treat and/or prevent dermatophytosis caused by at least one fungus and bacterial superinfection of said dermatophytosis would exhibit superior antifungal activity to other analogous aminosteroidal compounds of squalamine has two amino chains.

A first object of the invention relates to an aminosteroidal compound analogous to squalamine corresponding to the general formula (I) below: (I)

in which R and R' are chosen independently from the groups –(CR ₁ R ₂ ) _n -[X _k -(CR ₃ R ₄ ) _m ] _p -NR ₅ R ₆ in which:

- n and m are integers, identical or different ranging from 1 to 40,

- p is an integer ranging from 0 to 4,

- k= 0 or 1

- X is an oxygen atom or a -NR ₇ - group,

- the R ₁ to R ₄ groups, which are identical or different, are chosen from: a hydrogen atom; a C ₁ -C ₁₂ alkyl group; a C ₆ -C ₁₂ aryl group,

- the R ₇ group is chosen, independently, from a hydrogen atom, a C ₁ -C ₆ alkyl group or a –(CH ₂ )y-NHR ₈ group with y an integer ranging from 1 to 5 and R ₈ a hydrogen atom, a C ₁ -C ₁₂ alkyl group or a C ₆ -C ₁₂ aryl group, and

- the R ₅ and R ₆ groups, which are identical or different, are chosen from: a hydrogen atom; a C ₁ -C ₁₂ alkyl group or R ₅ and R ₆ together form an optionally substituted heterocyclic group comprising from 5 to 20 atoms,

m, X, R ₃ , R ₄ and R ₇ being chosen independently for each unit - [X-(CR ₃ R ₄ )m]-

the other carbons of the compound of formula (I) comprising an identical or different R0 radical chosen from H, NH ₂ , SH, or R ₁ , preferably R0 being H or OH but with a single R0 being OH

or one of its pharmaceutically acceptable salts, for its use by topical application in humans or animals in the treatment and/or prevention of dermatophytosis caused by at least one fungus.

A second object of the invention relates to a composition for use by topical application in humans or animals in the treatment and / or prevention of dermatophytosis caused by at least one fungus, comprising a compound of formula (I ) according to the invention, or one of its pharmaceutically acceptable salts and a pharmaceutically acceptable excipient.

The objects of the present invention are described more precisely below.

Figure 1 shows the chemical structure of compound ASD-1.

Figure 2 represents the chemical structure of the compound BSQ-1

Figure 3 represents the chemical structure of the compound BSQ-2

Figure 4 represents the chemical structure of the compound BSQ-3

Figure 5 shows the chemical structure of compound BSQ-4.

Figure 6 shows the chemical structure of compound III-1.

detailed description

The present invention provides a solution for effectively treating and/or preventing dermatophytosis caused by at least one fungus, in particular athlete's foot pathology and/or nail dermatophytosis. It also provides a solution to treat and/or prevent bacterial superinfection of dermatophytosis. The compounds according to the present invention being more effective than the known compounds of the prior art, acting on a plurality of microorganisms involved in dermatophytosis and bacterial superinfection of dermatophytosis and not inducing resistance of the microorganisms involved in said pathologies.

The Applicant has demonstrated that the compounds according to the invention exhibit antifungal activity against multiple fungi involved in dermatophytosis as well as against bacteria involved or likely to be involved in these pathologies as well as in bacterial superinfection of dermatophytosis .

Surprisingly, the Applicant has demonstrated that the aminosteroidal compounds analogous to squalamine corresponding to the general formula (I) comprising two amino chains have better efficacy against multiple microorganisms involved in dermatophytosis and bacterial superinfection of these dermatophytosis in comparison to squalamine, and to the analogous aminosteroid compound of squalamine ASD-1 (FIG. 1) comprising a single amino chain but also and above all in comparison to the analogous aminosteroidal compound of squalamine III-1 (FIG. 6) also comprising two amino chains .

These results are particularly surprising because nothing suggested that the compounds of formula (I) according to the invention would have the ability to effectively inhibit the growth of multiple microorganisms, not only fungi but also bacteria, involved in dermatophytosis with a improved efficacy compared to the aminosteroidal compound analogous to squalamine III-1 also comprising two amino chains, in particular with regard to the Trichophyton rubrum strain which is strongly involved in dermatophytosis and in particular in the pathology of athlete's foot.

These results are also particularly interesting because they make it possible to propose a solution for effectively treating and/or preventing a dermatophytosis caused by at least one fungus and a bacterial superinfection of said dermatophytosis with better efficacy than the known compounds of the prior art and without induce adverse drug or skin effects, or resistance of the microorganisms involved in said pathologies. Indeed, the compounds for their use according to the invention do not induce resistance of the microorganisms because they make it possible to reduce, to inhibit the fungal and bacterial growth by a physicochemical action, in particular by destroying the wall of the bacteria by a mechanism of detergent-type physico-chemical action, causing the bacteria wall to burst and destroy.

Definitions

The terms "squalamine analog aminosteroidal compound" in the context of the present invention refer to synthetic derivatives aminosteroidal analogs of squalamine. More specifically, the synthetic aminosteroidal analog derivatives of squalamine within the meaning of the present invention correspond to the formula below:

in which :

- either all the bonds lined with dotted lines between the carbons of positions 7-8, 5-6, 4-5, and 3-4 represent a single bond, the skeleton being that of a 10, 13, 17 trimethyl cholestane ,

- either one of the bonds lined with dotted lines between the carbons of positions 7-8, 5-6, 4-5, and 3-4 represents a double bond and the other bonds lined with dotted lines are single bonds , the skeleton being that of a 10, 13, 17 trimethyl cholestene. More specifically, in the context of the present invention, these synthetic aminosteroidal analogue derivatives of squalamine comprise a polyamino chain in position 3 and 20 of the cholestane or cholestene skeleton, preferably cholestene. Even more specifically in the context of the present invention, the synthetic aminosteroidal analog derivatives of squalamine correspond to the formula below: (I)

More precisely still, the analogous aminosteroidal compound of squalamine according to the present invention corresponds to the general formula comprising a skeleton of formula (I) below:

in which R and R' are chosen independently from the groups

–(CR ₁ R ₂ ) _n -[X _k -(CR ₃ R ₄ ) _m ] _p -NR ₅ R ₆ in which:

- n and m are integers, identical or different ranging from 1 to 40,

- p is an integer ranging from 0 to 4,

- k= 0 or 1

- X is an oxygen atom or a -NR ₇ - group,

- the R ₁ to R ₄ groups, which are identical or different, are chosen from: a hydrogen atom; a C ₁ -C ₁₂ alkyl group; a C ₆ -C ₁₂ aryl group,

- the R ₇ group is chosen, independently, from a hydrogen atom, a C ₁ -C ₆ alkyl group or a –(CH ₂ ) _y -NHR ₈ group

with y an integer ranging from 1 to 5 and R ₈ a hydrogen atom, a C ₁ -C ₁₂ alkyl group or a C ₆ -C ₁₂ aryl group, and

- the R ₅ and R ₆ groups, which are identical or different, are chosen from: a hydrogen atom; a C ₁ -C ₁₂ alkyl group or R ₅ and R ₆ together form an optionally substituted heterocyclic group comprising from 5 to 20 atoms,

m, X, R ₃ , R ₄ and R ₇ being chosen independently for each unit -[X-(CR ₃ R ₄ ) _m ]-

the other carbons of said skeleton of formula (I) comprising an identical or different R0 radical chosen from H, NH ₂ , SH or R ₁ , preferably R0 being H or OH but with a single R0 being OH.

The term “alkyl” is understood to mean a saturated, linear or branched aliphatic group, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms and even more preferably from 1 to 3 carbon atoms. By way of example of an alkyl group, reference may be made to the methyl, ethyl, isopropyl, butyl, tert-butyl, hexyl, nonyl and dodecyl groups. Preferably, the alkyl groups in the context of the present invention are chosen from methyl, ethyl and isopropyl groups. Alkyl groups of 3 to 6 carbon atoms can form a cycloaklyle by linking its two ends, to form for example a cyclopropyl, a cyclobutyl, a cyclopentyl or a cyclohexyl. This cycloalkyl group, comprising a heteroatom such as oxygen, nitrogen, phosphorus, or sulfur thus forms a heterocyclic.

The term “aryl” “Ar” means a mono- or bicycle having 6 to 12 carbon atoms of formula CnH(n-2), such as phenyl, biphenyl or naphthyl groups as well as heteroaryls, i.e. say aryls comprising at least one heteroatom such as an oxygen, sulfur, and nitrogen atom which can be substituted by different substituents.

The compounds according to the invention can also be used in the form of “pharmaceutically acceptable salts” or “cosmetically acceptable salts”, which correspond to the salts of acidic or basic groups present in the specified compounds. Among the pharmaceutically or cosmetically acceptable acid salts, mention may be made, by way of non-limiting example, of the hydrochloride, hydrobromide, perchlorate, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate salts. , tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, ptoluenesulfonate. Among the basic salts, mention may be made, by way of non-limiting example, of aluminum, calcium, lithium, magnesium, potassium, sodium, zinc and diethanolamine salts. In the context of the present invention, the term “pharmaceutically acceptable salts” or “cosmetically acceptable salts” preferably means the hydrochloride, hydrobromide, perchlorate, triflate, phosphate, sulfate, dichloroacetate, lactate, succinate, tartrate, citrate and more preferably to salts in hydrochloride form.

Within the meaning of the present invention, the term "physiologically acceptable medium" means a cosmetically or dermatologically acceptable medium, that is to say compatible with topical application, not inducing cutaneous toxicity and well tolerated by the skin in particular, not inducing tightness, redness or stinging of the skin.

The term “pharmaceutically acceptable excipient” or “cosmetically acceptable excipient” refers to an excipient suitable for the pharmaceutical or cosmetic form of the composition and compatible with the other ingredients of the composition in which it is found. In the context of the present invention, this also means that the excipient must be suitable for topical application. Finally, in the context of the present invention, the excipient is also compatible with the compounds of the material forming the packaging in which the composition is contained.

By topical application or topically, reference is made in the context of the present invention to the skin and appendages of a mammal, a human or an animal and preferably a human.

The term “dermatophytosis” refers in the context of the present invention to dermatophytosis or dermatomycoses which are superficial mycoses and more precisely infections of the skin or appendages caused by at least one fungus or dermatophyte. In particular caused by at least one microscopic filamentous fungus belonging to one of the following genera: Trichophyton, Microsporum and Epidermophyton . More specifically in the context of the present invention, the dermatophytosis considered can also be caused by at least one bacterium, in particular by at least one bacterium belonging to the genus Micrococcus spp, Proteus spp, Corynebacterium spp, Brevibacterium spp.

In particular in the context of the present invention, reference is made by “dermatophytosis” to dermatophytosis of the folds such as dermatophytosis of the foot, to dermatophytosis of glabrous skin, to ringworm of the scalp or else to dermatophytosis of the nails.

Preferably in the context of the present invention, the dermatophytosis considered is dermatophytosis of the folds, also called intertrigo of the folds, and particularly preferably the dermatophytosis considered is dermatophytosis of the foot.

By “dermatophytosis of the foot” in the context of the present invention, reference is made in an equivalent manner to dermatophytosis inter-toes, dermatophytosis intertrigo inter-toes or even to the “pathology of athlete’s foot”. These dermatophytosis are located in the cutaneous area of the foot, more precisely at the plantar level and/or at the level of the toes, in particular between the toes.

Preferably in the context of the present invention, the dermatophytosis under consideration is a nail dermatophytosis.

By “nail dermatophytosis” or “onychomycosis” or “ tinea unguium ” is meant in the context of the present invention a dermatophytosis located on the nails. In particular, in the context of the present invention, reference is made by “nail dermatophytosis” to dermatophytosis of the toenails, said dermatophytosis possibly corresponding to a complication of dermatophytosis of the foot. The nail dermatophytosis according to the invention is preferably caused by the presence of a dermatophytosis of the foot, also called pathology of athlete's foot, and its presence may or may not be concomitant with that of a dermatophytosis of the foot.

By “fungus” in the context of the present invention, reference is made to “dermatophytes” which are filamentous microscopic fungi proliferating on keratin materials such as the skin and/or the appendages.

In the context of the present invention, the term “dermatophytosis caused by a fungus” means a dermatophytosis for which “the main agent causing the dermatophytosis” is a fungus, that is to say which is at the origin of the dermatophytosis. More specifically in the context of the present invention, it is considered that "dermatophytosis is caused by a fungus" or that a fungus is "the main agent causing dermatophytosis" when this fungus represents between 50 and 70% of the fungal population. , preferably between 45 and 80% and more preferably between 40 and 100%.

In the context of the present invention, the term "dermatophytosis caused by at least one bacterium" means the fact that a bacterium is not the main agent causing dermatophytosis but makes it possible to create a favorable environment for the development and / or the survival of a fungus which is the main agent causing dermatophytosis. In other words, the term "dermatophytosis caused by at least one bacterium" means a dermatophytosis in which the presence of the bacterium makes it possible to create a pathogenic synergistic association with the fungi favoring the initiation and / or the maintenance of the dermatophytosis .

The “minimum inhibitory concentration” or “MIC” corresponds to the smallest concentration of a compound sufficient to inhibit, in vitro , the growth of a fungus, a yeast or a strain of bacteria. In the context of the present invention, the MIC corresponds to the smallest concentration of an aminosteroidal compound similar to squalamine corresponding to the formula (I) according to the present invention, sufficient to inhibit in vitro the growth of a fungus or a strain of bacteria.

By "dilution" is meant the process which consists in obtaining a final solution at a lower concentration than that of the starting solution, by taking a volume of the initial solution and supplementing it with solvent. In the context of the present invention, several concentrations of the molecules were tested, with dilutions carried out to half, and therefore concentration values divided by 2. In the present invention, when the difference between the MICs of two molecules is greater than or equal to two dilutions, or when the ratio between the two MIC values of two molecules has been greater than or equal to four, a superiority of one molecule relative to the other is considered.

The terms "treatment", "treating" designate in the context of the present invention an amelioration or prophylaxis, or the reversal or slowing of the progression or the inhibition or delay of the onset of a disease or a disorder or at least one specific symptom or at least one measurable physical parameter associated with the disease or disorder.

The terms "prevention", "prevent" designate in the context of the present invention a reduction in the risk of acquiring a disease or disorder, or a reduction, inhibition or slowing of the appearance of at least one specific symptom or at least one measurable physical parameter associated with the disease or disorder.

In the context of the present invention, the disease or disorder with which the specific symptom or the measurable physical parameter is associated is a dermatophytosis caused by at least one fungus, and a bacterial superinfection of said dermatophytosis. In particular, the dermatophytosis considered is a foot dermatophytosis and/or a nail dermatophytosis.

The specific symptom or measurable physical parameter associated with the aforementioned disease or disorder may, by way of non-limiting example, be chosen from pruritus, itching, redness, skin dryness, skin lesions, desquamation, alopecia, skin crusts, splitting, thickening or even a change in the color of the nails.

In the context of the present invention, the term “subject” and/or “patient” means any mammal, human or animal, preferably human beings such as men, women, children. According to one aspect, the preferred subjects are sports persons, persons frequenting swimming pools in particular municipal swimming pools and/or sports halls, and/or saunas, and/or wearing closed shoes and/or persons suffering from immune deficiency and/or people with diabetes.

Preferably, the "subjects" and/or "patient", in the context of the present invention, are human beings affected or likely to be affected by diabetes, more preferably those affected or likely to be affected by foot pathology. diabetic.

The term "effective amount", "sufficient amount" or "effective therapeutic amount" means the dose of a compound of formula (I) as defined above, making it possible to treat and/or prevent dermatophytosis caused by at least a fungus, in particular dermatophytosis of the foot, preferably inter-toe and/or plantar dermatophytosis, and bacterial superinfection of said dermatophytosis. More preferably, the term “effective amount”, “sufficient amount”, or “effective therapeutic amount”, means the dose of a compound of formula (I) as defined in the present invention, making it possible to treat and/or prevent a specific symptom or measurable physical parameter associated with dermatophytosis, in particular dermatophytosis of the foot, preferably intertoe and/or plantar dermatophytosis, and bacterial superinfection of said dermatophytosis.

A first object of the invention relates to an aminosteroidal compound analogous to squalamine corresponding to the general formula (I) below: (I)

in which R and R' are chosen independently from the groups –(CR ₁ R ₂ ) _n -[X _k -(CR ₃ R ₄ ) _m ] _p -NR ₅ R ₆ in which:

- n and m are integers, identical or different ranging from 1 to 40,

- p is an integer ranging from 0 to 4,

- k= 0 or 1

- X is an oxygen atom or a -NR ₇ - group,

- the R ₁ to R ₄ groups, which are identical or different, are chosen from: a hydrogen atom; a C ₁ -C ₁₂ alkyl group; a C ₆ -C ₁₂ aryl group,

- the R ₇ group is chosen, independently, from a hydrogen atom, a C ₁ -C ₆ alkyl group or a –(CH ₂ )y-NHR ₈ group with y an integer ranging from 1 to 5 and R ₈ a hydrogen atom, a C ₁ -C ₁₂ alkyl group or a C ₆ -C ₁₂ aryl group, and

- the R ₅ and R ₆ groups, which are identical or different, are chosen from: a hydrogen atom; a C ₁ -C ₁₂ alkyl group or R ₅ and R ₆ together form an optionally substituted heterocyclic group comprising from 5 to 20 atoms,

- m, X, R ₃ , R ₄ and R ₇ being chosen independently for each unit - [X-(CR ₃ R ₄ )m]-

the other carbons of the compound of formula (I) comprising an identical or different R0 radical chosen from H, NH ₂ , SH, or R ₁ , preferably R0 being H or OH but with a single R0 being OH

or one of its pharmaceutically acceptable salts, for its use by topical application in humans or animals in the treatment and/or prevention of dermatophytosis caused by at least one fungus.

The compounds of general formula (I) can be obtained by those skilled in the art by referring to the synthetic methods described in patent application WO2011067501 A1 in particular in examples 1 and 2, pages 18-19. A person skilled in the art can refer to it without difficulty to produce the compounds of general formula (I) according to the invention.

According to a preferred aspect, the present invention also relates to the isomeric forms of the analogous aminosteroid compound of squalamine according to the present invention. Indeed, said compound may contain one or more asymmetric centers and may therefore be in the form of a racemate and a racemic mixture, a single enantiomer, a mixture of diastereomers and an individual diastereomer. Depending on the nature of the various substituents of the compounds of formula (I) according to the invention, asymmetric centers may be present. All possible optical isomers and diastereoisomers in mixtures and as pure or partially purified compounds fall within the scope of the present invention.

A person skilled in the art knows how to synthesize the isomeric forms of the analog aminosteroidal compound of squalamine according to the invention and how to determine their stereochemistry. Similarly, the person skilled in the art knows how to separate the enantiomers or the diastereoisomers from a racemic mixture.

Preferably, the squalamine analogue aminosteroidal compound is an isomer, preferably said compound is in the form of an enantiomer or a diastereoisomer.

More preferably, the squalamine analogue aminosteroidal compound according to the present invention is in the form of a mixture of diastereoisomers or of individual diastereomers. In a particular embodiment, the present invention also relates to a squalamine analogue aminosteroidal compound having the general formula (I) for its use in the treatment and/or prevention of (a) dermatophytosis caused by at least one fungus and (b) bacterial cutaneous superinfection of said dermatophytosis.

In particular in the context of the present invention, dermatophytosis is caused by at least one fungus belonging to one of the following genera Trichophyton, Epidermophyton , Microsporum.

More particularly in the context of the present invention, dermatophytosis is caused by at least one fungus belonging to one of the following species Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton interdigitale, Trichophyton tonsurans, Epidermophyton floccosum, Microsporum canis.

More particularly still in the context of the present invention, dermatophytosis is caused by at least one fungus belonging to one of the following species Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum, Trichophyton tonsurans , preferably belonging to the species Trichophyton rubrum.

Preferably in the context of the present invention, the dermatophytosis is caused by a fungus belonging to the species Trichophyton rubrum and/or Epidermophyton floccosum. More preferably in the context of the present invention, the dermatophytosis is caused by a fungus belonging to the species Trichophyton rubrum.

In the context of the present invention, dermatophytosis is not caused by a fungus belonging to the Candida genus and in particular to the Candida albicans species.

In the context of the present invention, the Applicant proposes a solution making it possible to treat and/or prevent dermatophytosis by acting on the cutaneous microbiome by inhibiting not only the growth of fungi but also that of cutaneous bacteria which can cause said dermatophytosis and/or a bacterial superinfection of said dermatophytosis. Thus, according to a particular embodiment of the invention, the dermatophytosis and/or the bacterial cutaneous superinfection of said dermatophytosis are caused by at least one bacterium belonging to the genus Micrococcus spp, Proteus spp, Corynebacterium spp, Brevibacterium spp.

According to a preferred aspect, the dermatophytosis is caused by at least one bacterium belonging to the genus Micrococcus spp, Proteus spp, Corynebacterium spp, Brevibacterium spp.

In the context of the present invention, dermatophytosis and/or bacterial superinfection of dermatophytosis is not caused by at least one bacterium belonging to the Staphylococcus genus and preferably not by a bacterium belonging to the Staphylococcus aureus species.

Preferably, according to the present invention, dermatophytosis and/or bacterial cutaneous superinfection of said dermatophytosis are caused by at least one bacterium belonging to one of the following species Micrococcus luteus, Micrococcus sedentarius, Proteus mirabilis, Brevibacterium epidermidis Corynebacterium minutissimum, Cornyebacterium striatum , preferably Micrococcus luteus, Micrococcus sedentarius, Brevibacterium epidermidis, Cornyebacterium striatum, Corynebacterium minutissimum.

More particularly according to the present invention, dermatophytosis and/or bacterial cutaneous superinfection of said dermatophytosis are caused by at least one bacterium belonging to one of the following species Micrococcus sedentarius , Brevibacterium epidermidis , Cornyebacterium minutissimum . Said bacteria being able to cause said dermatophytosis via the creation of a favorable environment for the development and/or the survival of the fungus.

Even more specifically in the context of the present invention, dermatophytosis is caused by at least one fungus belonging to one of the following speciesTrichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosumand the bacterial skin superinfection of the said dermatophytosis is caused by at least one bacterium belonging to one of the following speciesMicrococcus sedentarius, Brevibacterium epidermidis, Cornyebacterium minutissimum.

According to a particular embodiment of the present invention, the dermatophytosis is caused by at least one fungus belonging to the species Trichophyton rubrum and/or Epidermophyton floccosum and the bacterial superinfection is caused by at least one bacterium belonging to the species Brevibacterium epidermidis and/or Micrococcus sedentarius .

According to a particularly preferred embodiment of the present invention, dermatophytosis is caused by at least one fungus belonging to the speciesTrichophyton rubrumand the bacterial skin superinfection of the said dermatophytosis is caused by at least one bacterium belonging to the speciesBrevibacterium epidermidis.

According to another particular embodiment of the present invention, the dermatophytosis is caused by at least one fungus belonging to the species Trichophyton rubrum and the bacterial cutaneous superinfection of said dermatophytosis is caused by at least one bacterium belonging to the species Micrococcus sedentarius .

According to an even more particular embodiment of the present invention, dermatophytosis is caused by a fungus belonging to one of the following speciesTrichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosumand the bacterial superinfection of the skin of the said dermatophytosis is caused by a bacterium belonging to one of the following speciesMicrococcus sedentarius, Brevibacterium epidermidis, Cornyebacterium minutissimum. Preferably, dermatophytosis is caused by a fungus belonging to the speciesTrichophyton rubrumand orEpidermophyton floccosum, preferably byTrichophyton rubrumand the bacterial superinfection is caused by a bacterium belonging to the speciesBrevibacterium epidermidisand orMicrococcus sedentarius, preferablyBrevibacterium epidermidis.

According to a particular and preferred embodiment of the present invention, the dermatophytosis is a foot dermatophytosis and/or a nail dermatophytosis.

More specifically, dermatophytosis of the foot corresponds to the pathology of athlete's foot, also called intertrigo inter-toes or plantar intertrigo interdigito, and nail dermatophytosis corresponds to onychomycosis.

Preferably in the context of the present invention, nail dermatophytosis is a consequence of foot dermatophytosis and is present concomitantly with said foot dermatophytosis.

The compound of formula (I) for its use according to the first object of the invention is intended to be used in humans, more precisely in a man, a woman, or a child or an animal, preferably a mammal.

According to a particular embodiment, the compound of formula (I) for its use according to the first object of the invention is intended to be used in subjects suffering from diabetes and preferably from the pathology of the diabetic foot.

In particular, the compound for its use according to the present invention corresponds to formula (I), in which R and R' are identical with

- n and m are identical or different integers between 0 and 14,

- k= 0

- p is an integer from 0 to 4, and

- R ₁ to R ₆ are identical or different and chosen from a hydrogen atom, a C ₁ to C ₁₂ alkyl, an optionally substituted C ₆ -C ₁₂ aryl group and a —COOalk group, alk being an alkyl in _C1 to _C3 ,

the other carbons of the compound of formula (I) comprising a radical R0 = H

Preferably, the compound for its use according to the present invention corresponds to the formula (I) in which

- R ₁ to R ₄ are identical,

- R ₅ and R ₆ are identical or different and chosen from a hydrogen atom, a C1 to C12 alkyl, an optionally substituted C6-C12 aryl group and a —COOalk group, alk being a C1 to C3 alkyl.

According to a preferred embodiment, the compound for its use according to the present invention corresponds to formula (I) in which - R ₁ to R ₄ are hydrogen atoms,

- R ₅ and R ₆ are identical or different and chosen from a hydrogen atom, a C ₁ to C ₁₂ alkyl, an optionally substituted C ₆ -C ₁₂ aryl group and a —COOalk group, alk being an alkyl in _C1 to _C3 .

Preferably, the compound for its use according to the present invention corresponds to the formula (I) in which

- R ₁ to R ₄ are hydrogen atoms,

- R ₅ and R ₆ are identical and chosen from a hydrogen atom, a C ₁ to C ₁₂ alkyl, an optionally substituted C ₆ -C ₁₂ aryl group and a —COOalk group, alk being a C ₁ alkyl at _C3 .

According to an even preferred embodiment, the compound for its use according to the present invention corresponds to formula (I) in which R ₅ and R ₆ are identical and chosen from a hydrogen atom and a C ₁ to C ₃ alkyl .

In an even more preferred embodiment, the compound for its use according to the present invention corresponds to formula (I) in which R ₅ and R ₆ are identical and chosen from a hydrogen atom or a methyl, an ethyl, an isopropyl .

Even more preferably, the compound for its use according to the present invention corresponds to formula (I) in which n=12, k=0, p=0 and m=0.

According to an advantageous and preferred aspect, the compound for its use according to the present invention corresponds to formula (I) in which R and R′ are identical with

- n= 12, k= 0, p= 0

- R ₁ to R ₆ all correspond to a hydrogen atom, with the other carbons of the compound of formula (I) comprising a radical R0 = H.

According to another advantageous and preferred aspect, the compound for its use according to the present invention corresponds to the formula (I) in which R and R′ are identical with

- n= 12, k= 0, p= 0

- R ₁ to R ₄ are hydrogen atoms, R ₅ to R ₆ are methyls with the other carbons of the compound of formula (I) comprising a radical R0 = H.

Advantageously, the compound corresponding to formula (I) for its use according to the present invention is in the form of hydrochloride, hydrobromide, perchlorate, triflate, phosphate, sulphate, dichloroacetate, lactate, succinate, tartrate, citrate salts, preferably in the form of hydrochloride.

According to a preferred embodiment, the compound corresponding to formula (I) for its use according to the present invention is chosen from the compounds of the following formulas:

In the context of the present invention, the above compounds are successively called BSQ-1, BSQ-2, BSQ-3 and BSQ-4.

In an even more preferred embodiment, the compound corresponding to formula (I) for its use according to the present invention is the compound BSQ-1, of formula:

According to one particular aspect, the compound corresponding to formula (I) for its use according to the present invention, preferably compound BSQ-1 is in the form of a mixture of diastereoisomers or of individual diastereomers.

A second object of the invention relates to a composition for use by topical application in humans or animals in the treatment and / or prevention of dermatophytosis caused by at least one fungus, comprising a compound of formula (I ) according to the first object, or one of its pharmaceutically acceptable salts and a pharmaceutically acceptable excipient.

The composition according to the present invention is a composition comprising a compound of formula (I) as defined above, for its use by topical application, in particular a pharmaceutical composition intended for topical application. The composition according to the present invention is therefore a dermatological composition.

Preferably in the context of the present invention, the dermatophytosis is a foot dermatophytosis and/or a nail dermatophytosis.

According to a particular aspect, the invention relates to a composition comprising a compound of formula (I) according to the first object, or one of its pharmaceutically acceptable salts and a pharmaceutically acceptable excipient, for use by topical application in humans or animal in the treatment and/or prevention of a dermatophytosis caused by at least one fungus and of a bacterial superinfection in accordance with the first object of the invention.

In particular, the composition according to the present invention comprises the compound of formula (I) at a concentration ranging from 0.1 to 10%, preferably from 0.5 to 5%, more preferably at least 1% by weight relative to the total weight of the composition.

A person skilled in the art will ensure that the composition according to the invention comprises the compound of formula (I) or one of its pharmaceutically acceptable salts, in sufficient quantity to obtain the desired therapeutic or preventive effect and according to the dermatophytosis considered, in taking into account in particular the age, the sex, the weight of the subject, but also the therapeutic profile of the subject considered, for example if it is a diabetic subject suffering or potentially suffering from the pathology of the diabetic foot.

The composition according to the invention is in a form suitable for topical application to the skin, in particular in the form of a dermatological pharmaceutical composition. By way of example, the composition may be in the form of a solution, a lotion, an emulsion obtained, for example, by dispersing a fatty phase in an aqueous phase (O/W) or an aqueous phase in a fatty phase. (W/O). The topical composition according to the invention may be in anhydrous, aqueous or emulsion form, preferably in aqueous form. It can be in the form of a lotion, cream, ointment, gel, varnish, powder, shampoo, polymeric patch, hydrogel, washing base, spray, mousse, aerosol, ointment or other suitable form.

According to one particular embodiment, the composition is in the form of a lotion, cream, ointment, gel, varnish, powder, spray, mousse, aerosol or ointment. Preferably, the composition according to the present invention is in the form of a cream.

In a particular embodiment, the composition according to the present invention comprises the compound of formula (I) as defined above in the form of the hydrochloride, hydrobromide, perchlorate, triflate, phosphate, sulphate, dichloroacetate, lactate, succinate salt. , tartrate, citrate preferably in hydrochloride form.

In a preferred embodiment, the composition according to the present invention is integrated into a medical or cosmetic, preferably medical, device intended to be applied to the skin, for example a patch or a textile intended to be applied to the skin. In another particular embodiment, the composition according to the invention is integrated into a patch or a textile intended to be applied to the skin, preferably at the level of the skin area affected or likely to be affected by dermatophytosis.

According to a preferred aspect, the composition according to the invention is in the form of a polymeric patch capable of being applied to the skin at the level of a skin zone affected or likely to be affected by dermatophytosis, in particular dermatophytosis of the foot, of preferably inter-toe and/or plantar dermatophytosis, and bacterial superinfection of said dermatophytosis. The polymeric patch preferably comprises the compound of formula (I) within a polymeric layer in which the compound of formula (I) is in a composition of hydrogel form, said hydrogel being preferably capable of gradually releasing the compound of formula (I) included in the composition according to the invention.

According to a particular aspect, the present invention relates to a textile material in which the composition according to the invention is integrated. According to another preferred aspect, the composition according to the invention is integrated into a textile, for example socks, said textile being able to be applied to the skin at the level of a skin zone affected or likely to be affected by dermatophytosis. , in particular dermatophytosis of the foot, preferably inter-toe and/or plantar dermatophytosis, and bacterial superinfection of said dermatophytosis. The textile preferably being capable of gradually releasing the compound of formula (I) included in the composition according to the invention.

The pharmaceutical composition according to the present invention comprises at least one compound of formula (I) as defined above as the sole active ingredient, or in combination with one or more other active ingredients. According to a preferred aspect, the compound of formula (I) as defined above is the only active ingredient present in the pharmaceutical composition according to the present invention.

The composition according to the present invention may comprise one or more other inert additives such as, for example, emollients, moisturizing agents, stabilizing agents, pH regulating agents, emulsifying agents, antioxidants, solvents, preservatives.

A person skilled in the art will appropriately choose the additives and excipients constituting the composition according to the invention according to the desired galenic form. He will also be able to choose the galenic form most suited to the subject in question.

According to one embodiment, the composition according to the present invention is administered repeatedly, preferably 1 to 2 times per day, over a period ranging from 1 day to 6 months, renewable, preferably over a period of at least 4 weeks, more preferably over a period of at least 8 to 16 weeks. The administration of said composition can be carried out in a cycle with or without a rest period.

According to another embodiment, the composition according to the present invention is administered in a single manner.

The present invention also relates to a method of treatment using a compound of formula (I) as defined in the context of the present invention, preferably a pharmaceutical composition comprising said compound for its topical application, in an effective therapeutic amount in a subject affected or likely to be affected by a dermatophytosis caused by at least one fungus and/or a bacterial superinfection of said dermatophytosis. Said dermatophytosis preferably being foot dermatophytosis and/or nail dermatophytosis.

According to one embodiment, the present invention relates to a method of treatment consisting in applying topically a pharmaceutical composition comprising a compound of formula (I) as defined in the context of the present invention, in an effective amount in a subject affected or likely to be affected by dermatophytosis caused by at least one fungus, preferably foot dermatophytosis, and/or bacterial superinfection associated with said dermatophytosis.

According to another embodiment, the present invention relates to a method of prevention consisting in applying topically a pharmaceutical composition comprising a compound of formula (I) as defined in the context of the present invention, in an amount effective in a subject likely to be affected by dermatophytosis, preferably foot dermatophytosis and/or bacterial superinfection associated with said dermatophytosis. By "subject likely to be affected" is meant, for example, people who have already been affected by dermatophytosis, and / or people with excessive sweating, and / or frequenting swimming pools, and / or sports halls, and/or saunas, and/or wearing closed shoes and/or people with immune deficiency, diabetes and/or diabetic foot disease.

The invention also relates to the use of a compound of general formula (I) as defined in the present application for the manufacture of a medicament or of a pharmaceutical composition for the treatment and/or prevention of dermatophytosis. caused by at least one fungus by topical application in humans or animals and bacterial superinfection of said dermatophytosis.

According to another aspect, the present invention relates to a kit comprising in the same packaging the composition according to the invention and a device in which the composition is capable of being integrated, preferably a textile.

The invention will be further illustrated by the following figures and examples. However, these examples and these figures should in no way be interpreted as limiting the scope of the invention.

Example 1 : Comparative test of the minimum inhibitory concentrations (MIC in μg/mL) of the aminosteroidal compounds analogous to squalamine BSQ-1 to BSQ-4 according to the invention, of squalamine, of the aminosteroidal compound analogous to squalamine ASD1, and of compound III-1 on fungi causing dermatophytosis and bacteria susceptible to cause dermatophytosis and bacterial superinfection of dermatophytosis.

Material:

The molecules squalamine, BSQ-1, BSQ-2, BSQ-3 and BSQ-4 were synthesized internally by BIOSQUAL. The bacterial strains were provided by the Institut Hospitalo-Universitaire – Méditerranée infection . The strains of dermatophytes were provided by the Parasitology laboratory of the IHU Méditerranée infection.

1. Pour la détermination de la CMI des molécules sur les champignons :

Method:

1. For determining the MIC of molecules on fungi:

The minimum inhibitory concentration of the molecules towards fungi was determined by the plate microdilution method.

The tests were carried out in the RPMI 1640 liquid medium prepared from the powder (Sigma Aldrich R6504 buffered with 3-morpholino-1-propanesulfonic acid at 0.165 mol/L (Sigma Aldrich M1254)

For squalamine and BSQ molecules 1 to 4, stock solutions at 5 mg/mL were prepared. A 1:50 dilution was made in RPMI-1640 medium (Sigma Aldrich R6504) in order to obtain a stock solution at 200 µg/mL. A volume of 200 µL of this solution was placed in the first well of the 96-well plate. The following wells were filled with 100 μL of RPMI and ½ dilutions were made by transferring 100 μL of the solution from the first well to the next well and so on.

For terbinafine and amphotericin B, a stock solution was prepared at 1600 µg/mL in dimethysulfoxide (DMSO). Successive dilutions were made in the RPMI medium in order to obtain solutions with final concentrations ranging from 32 µg/mL and less.

The antifungal activity of the molecules was tested using the microdilution method in 96-well plates prefilled with 100 µL of solutions containing the molecules.

Cultures of mushrooms from 7 to 14 days old on Sabouraud Dextrose agar were used for the preparation of the inoculum. The plates were scraped with a sterile loop and the conidia and hyphae fragments were placed in sterile water and left to sediment. The supernatant was adjusted to 1 McFarland and this suspension was diluted 1/10th with RPMI-1640 to obtain the final inoculum concentration. 100 μL of this suspension were added to the plates pre-filled with the molecules. Fungal growth control wells (without antifungal) were provided in the plates. The incubation was done at 30°C for 5 days and the MICs of the molecules were evaluated with the naked eye in comparison to the fungal control.

1. Pour la détermination de la CMI des molécules sur les bactéries :

An additional experiment was carried out following the same protocol as that described below to measure the efficacy of compound III-1 on 4 strains of dermatophytes: T. rubrum , T. interdigitale , T. tonsurans , E. floccosum .

1. For determining the MIC of molecules on bacteria:

The minimum inhibitory concentration of the molecules was determined by the plate microdilution method.

Stock solutions containing the molecules at 5 mg/mL were prepared. Successive dilutions were prepared in water to reach the concentration of 55.55 µg/mL. In 96-well plates, 90 µL of Müeller Hinton II liquid medium (MH II, cation adjusted) (Becton Dickinson, Le Pont de Claix, France) were placed in all the wells and 90 µL of the antibacterial solution at 55.55 µg /mL were added to the first well. ½ dilutions were made by transferring 90 µL of the first solution to the following wells containing the culture medium alone.

A bacterial suspension of the different bacteria was prepared and adjusted to 0.5 McFarland. This suspension was diluted 1:20 in MH II medium. Subsequently, 10 μL of this suspension were added to the solution containing the antibacterial agents in order to obtain a final volume of 100 μL with approximately 5×10 ⁵ cfu/mL of bacteria in each well. The concentrations tested in the plates were: 25 – 12.5 – 6.25 – 3.125 – 1.56 – 0.78 – 0.39 – 0.195 µg/mL.

A bacterial control column was provided in the plate as well as a culture medium control column. The plates were incubated at 37°C for 24 – 48 hours and the MICs were determined visually and after adding vital dye (iodonitrotetrazolium chloride, Sigma Aldrich).

The MICs indicated in Table 1 relating to the ASD-1 compound on certain fungi and bacteria are taken from the publication by Coulibaly et al (1). The equipment and method used in these studies are similar to those used to obtain MIC results for BSQ compounds and squalamine, which is why the data are comparable.

Results:

Table 1: MIC (µg/mL) of compounds BSQ-1 to BSQ-4 on fungi involved in dermatophytosis.

The MICs indicated below were determined according to the “Materials and methods” section indicated above:

These results demonstrate a superior activity of BSQ molecules compared to that of squalamine, compound ASD-1 and compound III-1 on fungi, with an MIC of between 1.56 and 6.25 µg/mL. In particular, the antifungal activity of these compounds against the T. rubrum strain (3.125 μg/mL) is greater than that of squalamine (12.5 μg/mL), than that of the ASD-1 compound (8 μg/mL) but especially to that of compound III-1 (12.5 μg/mL) with a difference of two dilutions. Similarly for the BSQ-1 compound, it is noted that the MIC obtained with this compound on the Epidermophyton floccosum strain is 1.56 μg/mL whereas it is 6.25 μg/mL for squalamine, and 3.25 μg/mL for the compound III-1.

The BSQ-1 to 4 compounds and in particular the BSQ-1 compound exhibit a superior activity against the fungi involved in dermatophytosis in comparison with the ASD-1 and III-1 compounds.

Table 2: MIC (µg/mL) of aminosteroidal compounds analogous to squalamine BSQ-1 to BSQ-4 and compound ASD-1 on bacteria likely to cause dermatophytosis and bacterial superinfection of dermatophytosis.

The MICs of the BSQ molecules on bacteria capable of inducing dermatophytosis and bacterial superinfection of dermatophytosis are between 0.78 and 3.125 μg/mL. These results highlight a superior activity of the BSQ compounds in terms of antibacterial activity compared to the ASD1 compound, with in particular a difference of two dilutions on the Brevibacterium epidermidis strain and of the BSQ-1 to 3 compounds on Micrococcus sedentarius compared to that of the compound ASD1.

References

[1] O. Coulibaly “In vitro activity of aminosterols against dermatophytes”, Medical Mycology, 2013, 309-312

[2] F.Baudraz et al, “Diagnosis and treatment of onychomycosis”, Rev Med Switzerland, 2005, vol.1, 30323

[3] French Association of Teachers of Parasitology and Mycology (ANOFEL) “Dermatophytoses or Dermatophyties”, Source French-speaking Virtual Medical University, 2014

[4] D. Genné et al, Erysipelas: clinical manifestations and management, Rev Med Switzerland, 2013, vol.9, 1812-1815

[5] O. Vanhooteghem, “Chronic interdigital dermatophytic infection: A common lesion associated with potentially severe consequences”, Diabetes Research and clinical practice, 2011, 23-25

[6] Rossi et al, “ Antifungal resistance mechanisms in dermatophytes ” Mycopathologia, 2008, 166: 369-383

[7] A.W Kligman et al, “The Interaction of fungi and bacteria in the pathogenesis of Athlete’s foot”, Skin microbiology, 1981, pp 203-219

[8] O. Coulibaly et al, “In vitro activity of aminosterol against dermatophytes”, Medical Mycology, 2013, 51, 309-312

[9] O. Coulibaly et al, “A double-blind randomized placebo-controlled clinical trial of squalamine ointment for tinea capitis treatment”, Mycopathologia, 2015, 179:187-193

Claims (23)

Aminosteroidal compound analog of squalamine corresponding to the general formula (I) below:
(I)
in which R and R' are chosen independently from the groups –(CR ₁ R ₂ ) _n -[X _k -(CR ₃ R ₄ ) _m ] _p -NR ₅ R ₆ in which:
- n and m are integers, identical or different ranging from 1 to 40,
- p is an integer ranging from 0 to 4,
- k= 0 or 1
- X is an oxygen atom or a -NR ₇ - group,
- the R ₁ to R ₄ groups, which are identical or different, are chosen from: a hydrogen atom; a C ₁ -C ₁₂ alkyl group; a C ₆ -C ₁₂ aryl group,
- the R ₇ group is chosen, independently, from a hydrogen atom, a C ₁ -C ₆ alkyl group or a –(CH ₂ )y-NHR ₈ group with y an integer ranging from 1 to 5 and R ₈ a hydrogen atom, a C ₁ -C ₁₂ alkyl group or a C ₆ -C ₁₂ aryl group, and
- the R ₅ and R ₆ groups, which are identical or different, are chosen from: a hydrogen atom; a C ₁ -C ₁₂ alkyl group or R ₅ and R ₆ together form an optionally substituted heterocyclic group comprising from 5 to 20 atoms,
- m, X, R ₃ , R ₄ and R ₇ being chosen independently for each unit - [X-(CR ₃ R ₄ )m]-
the other carbons of the compound of formula (I) comprising an identical or different R0 radical chosen from H, NH ₂ , SH, or R ₁ , preferably R0 being H or OH but with a single R0 being OH
or one of its pharmaceutically acceptable salts, for its use by topical application in humans or animals in the treatment and/or prevention of dermatophytosis caused by at least one fungus. A compound for its use according to claim 1, in the treatment and/or prevention of (a) dermatophytosis caused by at least one fungus and (b) bacterial skin superinfection of said dermatophytosis. Compound for its use according to one of Claims 1 to 2, in which the dermatophytosis is caused by at least one fungus belonging to one of the following genera Trichophyton, Epidermophyton , Microsporum. Compound for its use according to one of Claims 1 to 3, in which the dermatophytosis is caused by at least one fungus belonging to one of the following species Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton interdigitale, Trichophyton tonsurans, Epidermophyton floccosum, Microsporum canis . Compound for its use according to one of Claims 1 to 4, in which the dermatophytosis is caused by at least one fungus belonging to one of the following species Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum, Trichophyton tonsurans , preferably belonging to the species Trichophyton rubrum. Compound for its use according to one of Claims 2 to 5, in which dermatophytosis and/or bacterial skin superinfection of said dermatophytosis, are caused by at least one bacterium belonging to the genusMicrococci spp, Proteus spp, Corynebacterium spp, Brevibacterium spp. Compound for its use according to claim 6, in which the dermatophytosis and/or the bacterial cutaneous superinfection of the said dermatophytosis, are caused by at least one bacterium belonging to one of the following species Micrococcus sedentarius, Brevibacterium epidermis, Cornyebacterium minutissimum. Compound for its use according to one of Claims 2 to 7, in which the dermatophytosis is caused by at least one fungus belonging to one of the following speciesTrichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosumand the bacterial skin superinfection of the said dermatophytosis is caused by at least one bacterium belonging to one of the following speciesMicrococcus sedentarius, Brevibacterium epidermidis, Cornyebacterium minutissimum. Compound for its use according to one of Claims 2 to 8, in which the dermatophytosis is caused by at least one fungus belonging to the speciesTrichophyton rubrumand the cutaneous superinfection of the said dermatophytosis is caused by at least one bacterium belonging to the speciesBrevibacterium epidermidis. Compound for its use according to one of Claims 1 to 9, in which the dermatophytosis is dermatophytosis of the foot and/or dermatophytosis of the nails. Compound for its use according to one of Claims 1 to 10, in subjects suffering from diabetes and preferably from the pathology of the diabetic foot. Compound for its use according to one of Claims 1 to 11, corresponding to formula (I) in which R and R' are identical with

• n and m are identical or different integers between 0 and 14,
• k= 0
• p is an integer from 0 to 4, and
• R ₁ to R ₆ are identical or different and chosen from a hydrogen atom, a C1 to C12 alkyl, an optionally substituted C6-C12 aryl group and a —COOalk group, alk being a C1 to C3 alkyl, the other carbons of the compound of formula (I) comprising a radical R0=H.

Compound for its use according to one of Claims 1 to 12, in which

• R ₁ to R ₄ are hydrogen atoms,
• R ₅ and R ₆ are identical or different and chosen from a hydrogen atom, a C ₁ to C ₁₂ alkyl, an optionally substituted C ₆ -C ₁₂ aryl group and a —COOalk group, alk being a C ₁ to C ₃ .

Compound for its use according to one of Claims 1 to 13, in which R ₅ and R ₆ are identical and chosen from a hydrogen atom and a C ₁ to C ₃ alkyl. Compound for its use according to one of Claims 1 to 14, in which R ₅ and R ₆ are identical and chosen from a hydrogen atom or a methyl, an ethyl or an isopropyl. Compound for its use according to one of Claims 1 to 15, in which n=12, k=0, p=0 and m=0. Compound for its use according to one of Claims 1 to 16, the said compound being in the form of hydrochloride, hydrobromide, perchlorate, triflate, phosphate, sulphate, dichloroacetate, lactate, succinate, tartrate, citrate salts, preferably in the hydrochloride form. Compound for its use according to one of Claims 1 to 17, the said compound being chosen from the compounds of the following formulae:



Composition for use by topical application in humans or animals in the treatment and/or prevention of dermatophytosis caused by at least one fungus, comprising a compound according to any one of claims 1 to 18, or a of its pharmaceutically acceptable salts and a pharmaceutically acceptable excipient. Composition according to Claim 19, in which the compound of formula (I) is present at a concentration ranging from 0.1 to 10%, preferably from 0.5 to 5%, more preferably at least 1% by weight relative to the total weight of the composition. Composition according to one of Claims 19 to 20, the said composition being in the form of a lotion, cream, ointment, gel, varnish, powder, spray, mousse, aerosol or ointment. Composition according to one of Claims 19 to 21, in which the compound is present in the form of a hydrochloride, hydrobromide, perchlorate, triflate, phosphate, sulphate, dichloroacetate, lactate, succinate, tartrate or citrate salt, preferably in the hydrochloride form . Composition according to one of Claims 19 to 22, the said composition being integrated into a patch or a textile intended to be applied to the skin, preferably at the level of the skin zone affected or likely to be affected by dermatophytosis.