Pharmaceutical composition containing ATRA for use in the treatment of fungal infections caused by c. auris, c. glabrata, c. tropicalis and c. krusei

Abstract

Composition containing ATRA for the treatment of fungal infections caused by non-albicans Candida and in particular by one or more of C. krusei, C. tropicalis, C. auris, C. glabrata.

Description

PHARMACEUTICAL COMPOSITION CONTAINING ATRA FOR USE IN THE TREATMENT OF FUNGAL INFECTIONS CAUSED BY C. AURIS, C. GLABRATA, C.TROPICALIS AND C. KRUSEIFIELD OF THE INVENTION

[0001] The present invention relates to a pharmaceutical composition containing all- trans retinoic acid (ATRA) for the treatment of fungal infections caused by Candida spp., and in particular fungal infections caused by C. auris, C. glabrata, C. tropicalis and C. krusei.STATE OF THE ART

[0002] Over the last 20 years, the incidence of invasive fungal infections (IFIs) has increased significantly. In the global perspective, Candida spp. and Aspergillus spp. are the most frequent fungal aetiological agents, often responsible for invasive fungal infections in severely immunocompromised patients, burdened with a high morbidity and mortality rate. In recent years, fungal infections by environmental mycetes, previously considered non-pathogenic, have also been spreading. A wide range of pathogens belonging to the Mucorales order (e.g. Rhizopus spp), such as hyalohyphomycetes (e.g. Fusarium and Scedosporium spp) or pheohyphomycetes (e.g. Alternaria spp and Cladophialophora bantiana) together with Cryptococcus neoformans, constitute a major cause of infectious morbidity and mortality, with an estimated 600,000 deaths per year.

[0003] Mycoses caused by these organisms, especially the rhino-cerebral forms, are often difficult to treat and require specialist consultations (1 ). The increase of invasive fungal infections (IFIs), but also of non-invasive fungal infections, has been related to the increased prevalence of immunocompromised individuals, especially due to iatrogenic causes, such as oncological chemotherapies, or immunosuppressive therapies in autoimmune diseases, and to the increase of AIDS patients in countries with difficult access to treatment. In addition, improvements in surgical management, the introduction of selective immunosuppressive drugs, and the increase in the number of organ, solid or bone marrow transplant recipients experiencing neutropenia are further risk factors for the development of fungal infections. Similarly, the increased use of intravenous devices, prolonged stay in intensive care units and the administration of antibacterial therapies that alter the normal human microbiota are further predisposing factors for the occurrence of invasive fungal infections.

[0004] The GAFFI (Global Action Fund for Fungal Infections) has estimated that at least 150 patients die every hour worldwide from invasive fungal infections (IFIs), equalto 1 ,350,000 per year. Some neutropenic oncology patients need to undergo antifungal therapy in 1 / 3 of the cases when they do not respond to broad-spectrum antibiotic therapies because they are predisposed to developing fungal infections (2).

[0005] Among invasive fungal infections, invasive pulmonary aspergillosis (IPA) and systemic infections sustained by fungi belonging to the Candida genus are those most frequently encountered in the clinical setting. In particular, IPA represents the most common fungal infection among the invasive ones in the world, which is difficult to diagnose early. The responsible fungus, Aspergillus fumigatus, a ubiquitous fungus widespread in the environment, can cause a broad spectrum of diseases such as bronchopneumonia, allergic diseases, sinusitis, aspergilloma and pneumonia. The lung represents the hotbed from which the infection can spread to many other organs in susceptible patients, turning into a rapidly progressive systemic disease with often fatal outcome. During the recent international congress of the European Respiratory Diseases Society, it was stated that the aspergillosis problem is underestimated, despite the fact that there are about 300000 cases per year and 240000 in Europe alone. This is a major threat to the health of patients at risk, thus, with a mortality rate, if left untreated, of over 90%. However, even when invasive pulmonary aspergillosis (IPA) is treated, the mortality rate is 50% in immunocompromised patients. Guidelines for the treatment of IPA call for the use of antifungal drugs of the azole class. However, cases of resistance to the drugs used to occur.

[0006] Furthermore, during the pandemic triggered by Sars-CoV-2, there was an exponential increase in Mucormycosis infections, infections caused by various fungal organisms belonging to the Mucorales order, which are mainly widespread in India. This suggests that infectious diseases that are considered rare, may instead emerge especially in immunocompromised individuals, leading to a worsened prognosis.

[0007] In addition to aspergillosis, an increased occurrence of fungal infections caused by fungi belonging to the Candida genus has been observed in recent years, especially in immunocompromised patients.

[0008] Infections caused by microorganisms of the Candida genus can manifest themselves in various ways, for example, as localised diseases of the skin or nails, diseases affecting the mucous surfaces of the mouth, such as thrush, or of the vagina, or even as life-threatening systemic candidiases that spread through the blood stream, involving many organs such as the central nervous system, heart, kidneys, liver, bones, muscles, joints, spleen, and eyes.

[0009] The species most frequently responsible for candidiasis include Candida albicans, C. tropicalis, Candida kefyr, C. glabrata, C. krusei and Candida parapsilosis (3).

[0010] Although they all belong to the Candida genus, the species Candida albicans, C. glabrata (Nakaseomyces glabratus), C. auris, C. tropicalis and C. krusei (Pichia kudriavzevii) show marked differences in genotype, phenotype and drug-resistance profiles that justify separate clinical management for each.

[0011] C. albicans represents the most studied and virulent species, due to its remarkable morphological plasticity (dimorphism with blastospore — hypha transition), production of the cytolytic peptide toxin “candidalysin” (encoded by the ECE1 gene), and formation of highly structured mature biofilms. Its resistance to azoles is mainly mediated by mutations in the ERG11 gene (encoding 14a-lanosterol demethylase), alterations in the ERG3 and ERG5 genes, and over-regulation of ABC / MFS transporters such as CDR1 , CDR2, MDR1 , regulated by transcription factors such as TAC1 , MRR1 , UPC2.

[0012] In contrast, C. glabrata is a non-dimorphic, phylogenetically distant haploid yeast that mainly exploits the transcriptional regulation of PDR1 to confer overexpression of efflux pumps (SNQ2, CDR1 ), while resistance to echinocandins emerges through mutations in the hotspot codons F659, S663 of FKS2 and S629 of FKS1 , which alter the catalytic subunit of 1 ,3-[3-D-glucan synthase. Furthermore, mutations in mismatch repair genes (MSH2) can lead to mutated phenotypes that accelerate the acquisition of multiple resistances. Such mechanisms are, on the contrary, absent or rare in C. albicans. This explains the greater antifungal tolerance and intracellular persistence of C. glabrata with respect to Candida albicans.

[0013] C. auris, which has recently emerged as a critical nosocomial pathogen, is characterised by high thermotolerance (>42°C), environmental persistence and multi- fungal resistance. Point mutations in the ERG11 genes (Y132F, K143R), together with chromosome duplications and activating mutations in TAC1 b, contribute to extensive resistance to azoles; variants in the S639F / Y / P hotspot codons of FKS1 confer resistance to echinocandins, while resistance to amphotericin B is polygenic and variable. C. auris lacks the typical virulence factors of C. albicans, but its transmissibility, ability to colonise skin and hospital environments, and diagnostic difficulty make it a unique pathogen.

[0014] C. tropicalis shares some characteristics with C. albicans, such as partial dimorphism and biofilm formation, but has distinct virulence patterns, especially in neutropenic patients, and a high tendency to develop resistance through mutations in ERG11 , upregulation of MDR1 / CDR1 , and modifications in FKS1 , which are responsible for reduced sensitivity to echinocandins.

[0015] Finally, C. krusei exhibits intrinsic resistance to fluconazole, attributed to the low affinity of its sterol 14-demethylase for the drug, and high basal expression of efflux pumps. Unlike the preceding species, C. krusei is incapable of forming germinating tubes and does not exhibit dimorphism, but may persistently colonise the host, especially in the context of prolonged antifungal prophylaxis.

[0016] The evolutionary heterogeneity, confirmed by phylogenetic data, justifies the profound differences in virulence profiles and response to antifungals between the various species of C. albicans and C. non albicans. Indeed, each species represents a distinct biological entity with its own evolutionary trajectories, cellular structures, immune evasion strategies and pharmacological adaptation mechanisms. The phylogenetic tree shows a clear separation between two main clades: the CTG clade, comprising C. albicans and C. tropicalis, and the glabrata-\ike clade, which includes C. glabrata, C. krusei and C. auris. This genotypic distinction reflects profound differences in the mechanisms of pathogenicity, antifungal resistance and cell biology. In addition, as pointed out above, there are also non-trivial differences between C. albicans and C. tropicalis that cause, among other things, different responses to known drugs.

[0017] The treatment of such candidiasis is currently complicated by the fact that some strains, particularly C. glabrata and C. krusei have recently developed resistance to commonly used antifungal drugs. These drugs are usually azole-based. In particular, C. auris is proving to be a real global health threat, as this fungus is responsible for the most frequent nosocomial infections acquired by individuals weakened by other diseases, undergoing surgery or immunocompromised, and is proving resistant to the most common antifungal drugs currently available, such as azoles, echinocandins and amphotericin B.

[0018] This level of resistance had never been seen in fungal infections caused by other Candida species and is particularly worrying as it severely limits the treatment options available to patients with invasive infections, especially those caused by C. auris. The treatment of candidiasis from C. auris is also complicated by the fact that this species is not easily recognised by standard laboratory diagnostic tests, confusing it inparticular with other species such as C. haemulonii, C. lusitaniae, C. guillermondii and C. parapsilosis and this further compromises the efficacy of treatments for C. auris infections.

[0019] In addition, the drugs currently available for the treatment of fungal infections have high toxicity that risks further compromising the general health condition of patients who are treated with these drugs, especially if they are patients with a severely compromised clinical picture or who do not have a good general health condition. These problems are most evident when long-term treatment with currently known antifungal drugs is required.

[0020] The development of drug-resistant fungal strains and the high toxicity of traditional antifungals, especially in the case of prolonged and massive use, has increasingly reinforced the need to find new and more effective therapeutic strategies for fungal infections.

[0021] In particular, there is a strong need to identify new compounds with antifungal activity. There is also a particular need to identify new antifungal compounds that are both effective in treating fungal infections and selective so as to reduce undesirable effects for the treated patient.

[0022] There is also a particular need to identify antifungal compounds that are both effective in treating fungal infections and have reduced cytotoxicity in order to avoid side effects for the patient.

[0023] There is also a particular need to identify antifungal compounds that are both effective in treating fungal infections and less likely to generate drug resistance.

[0024] There is also a particular need to identify compositions that can effectively treat fungal infections caused by C. krusei, C. tropicalis, C. auris, C. glabrata.

[0025] There is therefore a need to identify new compositions for use in the treatment of fungal infections that are effective and have limited side effects, and preferably are easy to access and clinically manage.

[0026] A study conducted by researchers from the Department of Haematology at the Sapienza University of Rome, entitled “Infectious complications in patients with acute promyelocytic leukaemia treated with the AIDA regimen” published on 15 May 2003 in the journal Leukemia, showed that patients with promyelocytic leukaemia treated with all-trans retinoic acid (ATRA) were less likely to develop fungal infections by Candida albicans and Aspergillus spp. (4). Based on this observation, experiments in vitro and subsequently in vivo were conducted to test the use of ATRA for the prevention of fungalinfections. Tests were conducted in vitro in a preclinical model of invasive pulmonary aspergillosis, which showed that ATRA exerts a fungistatic action in vitro against Aspergillus fumigatus and Candida albicans, blocking the germination and replication of these fungi. These in vitro experiments have also shown that the use of ATRA in combination with conventional antifungals such as Amphotericin B or Posaconazole exerts a synergistic effect in contrasting the germination of aspergillus conidia (5). This synergistic effect makes it possible to reduce the dosage of antifungal drugs in the treatment of infections by Aspergillus fumigatus, and consequently to limit toxic effects related to the prolonged and massive use of these drugs. A preclinical model of invasive pulmonary aspergillosis in the rat was developed in order to evaluate the beneficial antifungal effects of ATRA in vivo as well. The results showed that the administration of ATRA statistically significantly increased the survival of the animals, compared to the untreated control group (60% vs 20% 12 days after infection). Although these studies could infer efficacy of ATRA in treating fungal infections caused by Aspergillus fumigatus and Candida albicans, there was no indication that this molecule could also be effective in treating non-albicans Candida species, as these mycetes are extremely different from Candida albicans, despite belonging to the same fungal genus.

[0027] Of the several hundred known species of the Candida genus, only a few are reported to be capable of causing human infections, and these species differ significantly, however, in both virulence and adaptation strategies. Therefore, these Candida species are located at divergent points in a continuous spectrum of types of interactions between the host species and the microorganism. It appears, in fact, that the various species of Candida have developed distinct biochemical, metabolic and physiological adaptations over the course of evolution, which they use to adapt to commensal niches and achieve full virulence. The different species of Candida therefore differ from each other both in structure and genetic characteristics, but above all in different virulence factors, such as: isotype switching, drug resistance, biofilm formation and quorum sensing.

[0028] In fact, each species of Candida has peculiar features both genotypically and phenotypically and virulence characteristics that differ from the other species and that characterise and distinguish one species of Candida from another.

[0029] These differentiation factors between species of Candida include various hostrecognition biomolecules, e.g. adhesin receptors, i.e. proteins on the surface of cells that recognise fungal pathogens, morphogenesis, i.e. the reversible transition betweenunicellular yeast cells and filamentous growth forms. In particular, Candida species are in yeast form at room temperature, when exposed to temperatures of 37°C or higher they become a filamentous fungus and produce structures called hyphae, which can lead to infection in humans. Further differences can be found in the aspartic proteases secreted by the fungi of the different Candida species and phospholipases, i.e. molecules that destroy human tissue and allow Candida species to cause systemic or local infections in humans. By destroying tissue with these proteases, hyphae make their way into the skin or mucous membranes and can reach blood vessels, causing systemic infections.

[0030] Among the different virulence factors possessed by the various Candida species, “phenotypic switching”, i.e. the transition from yeast to filamentous form, is accompanied by changes in antigen expression, colony morphology and tissue affinities in all Candida species.

[0031] Moreover, each Candida species has different susceptibility to antifungals, so that, at the clinical level, each Candida infection is unique and requires specific therapy based on susceptibility testing, and no certain response can be predicted in the management of such infections. Each species of Candida in particular may only be sensitive to some of the existing antifungals, so to combat infections by each Candida species, a precise therapeutic strategy must be devised, both in terms of the compounds used and the methods of administration.

[0032] Candida is a saprophytic fungus normally present in several districts of the body (including the vagina and the gastrointestinal tract) in a latent form (as blastospora) and is usually controlled by the immune system and the local bacterial flora (the lactobacilli). Several types of Candida have been isolated, which are usually distinguished into Candida albicans and non-albicans Candida. Candida albicans is usually identifiable in about 80% of cases, other species, grouped and identified as non- albicans Candida, such as C. glabrata, Candida parapsilosis, C. krusei and C. tropicalis, responsible for about the remaining 20% of candidiasis cases. In recent years we are witnessing an increase in infections caused by non-albicans Candida species that are resistant to common treatments.

[0033] These species of non-albicans Candida, which used to cause infections only in immunocompromised individuals (such as HIV-positive individuals or diabetics), now also affect apparently healthy individuals, and their presence should be suspectedespecially in the presence of an infection with less pronounced symptoms but a greater tendency to become chronic.

[0034] It is well known that the treatment of Candida infections must be appropriate and differentiated according to the type of Candida underlying the infection: in particular, while Candida albicans responds well and quickly to common “azole” antimycotics, nonalbicans Candida strains have proven resistant to conventional treatments. On the other hand, as mentioned, such "non-albicans" species are becoming increasingly widespread and can cause severe infections especially in clinically compromised patients.

[0035] In particular, Candida albicans differs from non-albicans species such as C. krusei, C. glabrata, C. parapsilosis and C. auris in that it is the only species capable of growing in hyphal form during the virulence phase. Isotype switching from the yeast to the hyphal form plays a key role in the pathogenesis of C. albicans. Hyphae, in fact, not only promote the penetration of the fungus into the tissues and blood vessels of the host, causing systemic infections, but in the hyphal form C. albicans also expresses the gene ECE1, absent, however, in yeast cells, which codes for a toxin known as “candidalysin”. This toxin exerts a cytolytic action against epithelial cells, thus destroying the epithelial barrier and further promoting the spread of Candida at the tissue level. Candidalysin also induces the recruitment of neutrophils, and activates the TH17 lymphocyte response, triggering immune-mediated tissue damage.

[0036] Fungal hyphae also contribute to the production of biofilms as they act as a support, promoting the adhesion of the Candida yeast cells. Biofilms can be produced on both biotic and abiotic surfaces (catheters, valve prostheses, joints) and are more structured in Candida albicans than biofilms produced by non-albicans species.

[0037] Non-albicans Candida species, on the other hand, can grow in pseudohyphal form during virulence but are not capable of producing true hyphae, with the exception of C. krusei, which can grow in hyphal form under certain conditions, similar to Candida albicans. C. glabrata, on the other hand, exists in nature, both as a commensal and as a pathogenic fungus, only in the yeast form.

[0038] Therefore, Candida albicans, represents the most virulent species even though, unlike the non-albicans species, Candida albicans has retained sensitivity to drugs traditionally used to treat both superficial and systemic candidiasis.

[0039] In contrast, C. glabrata, Candida parapsilosis and C. auris have developed resistance to these drugs, while C. krusei has innate resistance to fluconazole.

[0040] The emerging species C. auris has acquired resistance to azoles and also to echinocandins, another class of antifungal drugs commonly used for systemic infections. The phenomenon of multi-drug resistance makes the management of such infections extremely difficult. Furthermore, C. auris produces biofilms that have been shown to be resistant to common disinfectants even on the skin of apparently healthy individuals. This encourages the spread of C. auris infections both by direct contact between different individuals and by contact with contaminated devices. Therefore, while the treatment of such infections is extremely difficult, it is of paramount importance to find treatment methods that can be effective in treating them.

[0041] Therefore, there remains a need to identify molecules or compounds that may be effective in treating fungal infections sustained by non-albicans Candida strains.

[0042] Therefore, there remains a need to identify molecules or compounds that may be effective in treating fungal infections of C. krusei, C. tropicalis, C. auris and C. glabrata strains.

[0043] In particular, there is a need to identify molecules or compounds that have a non-negative impact on the patient's general health and whose use does not generate undesirable side effects.

[0044] There is a need to identify molecules or compounds that do not induce drug resistance.

[0045] There is therefore a need to identify molecules or compounds of natural origin that can be used to develop new antifungal therapeutic strategies.SUMMARY OF THE INVENTION

[0046] An object of the present invention is to provide a molecule or compound having antifungal activity against non-albicans Candida species, such as C. auris, C. glabrata, C. tropicalis and C. krusei.

[0047] A further object of the invention is to provide a pharmaceutical composition for the treatment of fungal infections from non-albicans Candida, such as C. auris, C. glabrata, C. tropicalis and C. krusei.

[0048] An object of the present invention is a pharmaceutical composition comprising all-trans retinoic acid (ATRA) for the treatment of infections sustained by non-albicans Candida species.

[0049] According to the invention, a pharmaceutical composition comprising all-trans retinoic acid (ATRA) is provided for the treatment of infections sustained by one or moreof the following species of non-albicans Candida and in particular by one or more of C. auris, C. tropicalis, C. glabrata and C. krusei.

[0050] Preferably the composition of the invention contains ATRA in a proportion between about 0.01 wt% and about 10wt%, preferably between about 0.05wt% and about 5wt%, more preferably between about 0.1 wt% and about 1wt%.

[0051] The invention provides a composition that is effective in treating fungal infections, particularly those caused by mycetes that have demonstrated resistance to traditional drugs. This makes it possible to treat with the composition of the invention fungal infections for which known drugs have proved ineffective.

[0052] The composition of the invention is suitable for use in the treatment of fungal infections in humans or even in veterinary medicine.

[0053] Furthermore, the invention provides a composition that has extremely limited or no side effects on the patient's health. Thus, the invention provides a composition that can be used to treat fungal infections of patients with a delicate or compromised general clinical picture without risking further worsening the patient’s clinical condition. The inventors found such a composition to be extremely effective in treating fungal infections caused by non-Albicans Candida species and in particular one or more of C. auris, C. tropicalis, C. glabrata and C. krusei. There is no commercially available formulation or composition based on ATRA alone or in combination with traditional antifungals.

[0054] The inventors found that ATRA has a unique mechanism of action different from all molecules used as antifungals. ATRA interacts with two key proteins in the life cycle of Candida spp, HSP90 and 14a-demethylase. While 14a-demethylase is the target of azoles, drugs used in common clinical practice for fungal infections, the inventors found that ATRA is the only molecule capable of binding HSP90. This allows ATRA to potentially be used in all cases of infections sustained by azole-resistant mycetes.

[0055] Moreover, to date there is no known resistance on the HSP90 protein, making ATRA the first molecule in its class to function as an antifungal.

[0056] In a further aspect of the invention, a pharmaceutical composition is provided for the treatment of fungal infections comprising at least one compound suitable for binding the HSP90 protein.

[0057] In a further aspect of the invention a pharmaceutical composition is provided for the treatment of fungal infections comprising at least one compound suitable forbinding the HSP90 protein of a fungus, wherein said fungal infections are fungal infections sustained by one or more of the following species: Candida albicans, C. auris, C. tropicalis, C. glabrata and C. krusei.

[0058] Indeed, C. auris is a fungus that is multi-resistant to therapies commonly used in clinical practice, with a mortality rate of over 50% in systemic forms of infection. Similarly, C. krusei is intrinsically resistant to fluconazole, the first-line drug for Candida albicans infections, but ineffective on C. auris and C. krusei and C. glabrata. Similarly, C. tropicalis and C. glabrata, species markedly different from Candida albicans in both morphology and genome, show increasing rates of resistance not only to azoles but also to other drug classes such as echinocandins. As these species differ not only in their response to antifungals compared to Candida albicans, but also in their genome and in the expression of different transmembrane proteins, an antifungal effect of ATRA against these species was not to be expected, although it has been demonstrated. Furthermore, while previous experiments have demonstrated a synergistic action between sub- optimal doses of ATRA (0.25 mM) and sub-optimal doses of amphotericin against Aspergillus fumigatus, no synergistic effects were shown when combining ATRA with amphotericin against C. krusei, C. auris, C. tropicalis and C. glabrata, underlining the non-obviousness of the interaction and how ATRA could not be expected to work also on the Candida non-albicans species and in particular of the Candida non-albicans under investigation.

[0059] Furthermore, the composition of the invention can be used effectively to treat fungal infections caused by a plurality of mycetes. In fact, as discussed above, ATRA has proven to be effective for various fungal populations. Whereas with known compositions in the case of infections caused by a plurality of mycetes it was necessary to use several antifungal agents, the composition of the invention is effective against several species. This further reduces possible side effects for the patient without compromising the effectiveness of the treatment, in fact guaranteeing good treatment effectiveness.

[0060] According to the invention compositions for topical use comprising a composition containing all-trans retinoic acid (ATRA) are provided for the treatment of infections sustained by one or more of the following species C. auris, C. tropicalis, C. glabrata and C. krusei.

[0061] According to the invention compositions for oral use comprising a composition containing all-trans retinoic acid (ATRA) are provided for the treatment of infectionssustained by one or more of the following species C. auris, C. tropicalis, C. glabrata and C. krusei.

[0062] According to the invention compositions are provided for intravenous or intramuscular administration comprising a composition containing all-trans retinoic acid (ATRA) for the treatment of infections sustained by one or more of the following species C. auris, C. tropicalis, C. glabrata and C. krusei.

[0063] Preferably, it is an object of the present invention to provide a composition containing all-trans retinoic acid (ATRA) for the treatment of mucosal infections caused by one or more of the following species C. auris, C. tropicalis, C. glabrata and C. krusei.

[0064] According to the invention, a pharmaceutical composition comprising all-trans retinoic acid (ATRA) is provided for the treatment of infections sustained by one or more of the following species of non-albicans Candida and in particular by one or more of C. auris, C. tropicalis, C. glabrata and C. krusei in which said infections are fungal infections of the mucous membranes.

[0065] According to the invention, a pharmaceutical composition comprising all-trans retinoic acid (ATRA) is provided for the treatment of infections sustained by one or more species of non-albicans Candida and in particular by one or more of C. auris, C. tropicalis, C. glabrata and C. krusei wherein said fungal infections are fungal infections of the vaginal, nasal, oral, anal mucosa.

[0066] According to the invention, a pharmaceutical composition comprising all-trans retinoic acid (ATRA) is provided for use in the treatment of oesophageal fungal infections sustained by one or more of the following species of Non-albicans Candida in particular infections of the folds, intertriginous, dermal or cutaneous adnexa infections, scalp infections, infections of hairy areas, nail infections, wherein such infections are sustained by one or more of the following species of non-albicans Candida and in particular one or more of C. auris, C. tropicalis, C. glabrata and C. krusei.

[0067] According to the invention, a pharmaceutical composition comprising all-trans retinoic acid (ATRA) is provided for use in the treatment of fungal infections of the nose, sinuses or ear, including otomycosis, sustained by one or more of the following species of non-albicans Candida and in particular by one or more of C. auris, C. tropicalis, C. glabrata and C. krusei.

[0068] According to the invention, a pharmaceutical composition comprising all-trans retinoic acid (ATRA) is provided for use in the treatment of intracranial or meningeal fungal infections sustained by one or more of the following species of non-albicansCandida and in particular by one or more of C. auris, C. tropicalis, C. glabrata and C. krusei.

[0069] According to the invention, a pharmaceutical composition comprising all-trans retinoic acid (ATRA) is provided for use in the treatment of peritoneal or intra-abdominal fungal infections, including peritonitis or catheter-associated fungal biofilms, sustained by one or more of the following species of non-albicans Candida and in particular by one or more of C. auris, C. tropicalis, C. glabrata and C. krusei.

[0070] According to the invention, a pharmaceutical composition comprising all-trans retinoic acid (ATRA) is provided for use in the treatment of systemic, blood, bone marrow or haematogenic fungal infections sustained by one or more of the following species of non-albicans Candida and in particular by one or more of C. auris, C. tropicalis, C. glabrata and C. krusei.

[0071] According to the invention a composition is provided for the treatment of fungal infections of non-albicans Candida and in particular by one or more of C. auris, C. tropicalis, C. glabrata and C. krusei in immunocompromised patients and in particular in patients with one or more of acute myeloid leukaemia (AML), human stem cell transplantation (HSCT) and graft versus host disease (GvHD).

[0072] According to the invention, a composition is provided for the treatment of fungal infections of non-albicans Candida and in particular from one or more of C. auris, C. tropicalis, C. glabrata and C. krusei wherein said fungal infections are epidermal fungal infections.

[0073] According to the invention, a composition is provided for the treatment of fungal infections of non-albicans Candida and in particular from one or more of C. auris, C. tropicalis, C. glabrata and C. krusei wherein said fungal infections are pulmonary fungal infections.

[0074] Preferably according to the invention, the composition comprises ATRA in powder form.

[0075] Preferably according to the invention, the composition comprises ATRA powder dissolved in a 0.9% physiological saline solution of sodium chloride.

[0076] Preferably according to the invention, the composition comprises ATRA powder dissolved in an injectable solvent.

[0077] According to the invention a kit is provided comprising a composition according to the invention and a physiological solution intended to dissolve said composition.

[0078] Preferably, the pharmaceutical composition further comprises an additional antifungal agent.

[0079] This increases the effectiveness of the composition of the invention.

[0080] Preferably the at least one additional antifungal agent is selected from the group consisting of azoles such as fluconazole, voriconazole, posaconazole itraconazole, ketoconazole, isavuconazole, thioconazole, opelconazole, miconazole.

[0081] Preferably the at least one additional antifungal agent is selected from the group consisting of polyenes such as amphotericin B, nystatin or liposomal and lipid forms.

[0082] Preferably the at least one additional antifungal agent is selected from the group consisting of purine or pyrimidine nucleotide inhibitors such as flucytosine.

[0083] Preferably the at least one additional antifungal agent is selected from the group consisting of polyoxins, such as nikkomycin, preferably nikkomycin Z or other chitin inhibitors.

[0084] Preferably, the at least one additional antifungal agent is selected from the group consisting of echinocandins such as micafungin and anidulafungin.

[0085] Preferably, the composition of the invention comprises a plurality of antifungal agents.

[0086] Preferably said additional fungal agent is present in said composition at a percentage between approximately 0.01 wt% and about 10 wt%, preferably between about 0.05wt% and about 5wt%, more preferably between about 0.2wt% and about 2wt%.

[0087] Preferably, the composition of the invention further comprises at least one compound selected from at least one excipient, at least one carrier agent, at least one adjuvant agent.

[0088] A preferred version of this composition is in the form of a liquid solution, and is preferably intended to be administered intravenously. Preferably, the composition is formulated for the administration of an amount of ATRA between 5 and 100 mg / m2 / day, preferably between 10-95 mg / m2 / day, more preferably between 20-70 mg / m2 / day, preferably between 30-60 mg / m2 / day, preferably between 50-60 mg / m2 / day. Preferably this composition is administered intravenously over a period of 1 to 20 days. Intravenous administration may be performed 1 to 3 times a day.

[0089] In one version, the composition is configured in such a way that an amount of ATRA between 10 and 30 mg / m2 / day is administered over a period of 7 to 15 days for between 1 and 3 administrations per day.

[0090] Each dose of the composition may comprise 10-50mg of ATRA.

[0091] In one version, the composition is configured so that each dose comprises 25 mg of ATRA. This composition can be administered once or twice a day.

[0092] In one version, the composition is configured so that each dose comprises 50 mg of ATRA. This composition may preferably be administered once a day at 50 mg.

[0093] A preferred version of this composition is in the form of a liquid solution, and is preferably intended to be administered intramuscularly.

[0094] Preferably the composition is configured in such a way that an amount of ATRA is administered intramuscularly in a dose between 5 and 150 mg / day, preferably between 10 and 140 mg / day, preferably between 20 and 130, preferably between 30 and 110, preferably between 40 and 100, preferably between 50 and 90, preferably between 60 and 80 mg / day for an administration time between 1 and 5 days.

[0095] In one version, the composition is configured in such a way that an amount of ATRA between 25 and 50 mg / day is administered over a period of 7 to 28 days, preferably between 7 and 15 days for between 1 and 3 administrations per day.

[0096] Each dose of the composition may comprise 25-50mg of ATRA.

[0097] In one version, the composition is configured so that each dose comprises 25 mg of ATRA. This composition can be administered once or twice a day.

[0098] In one version, the composition is configured so that each dose comprises 50 mg of ATRA. This composition may preferably be administered once a day at 50 mg.

[0099] In a preferred embodiment, the composition contains an amount of ATRA between 0.01 wt% and 0.2wt%, preferably between 0.05wt% and 0.15wt%, wherein said composition is intended for intralesional administration for an administration time of 1 day to 15 weeks.

[0100] In a preferred embodiment, the composition is a composition for intralesional administration by intradermal injection of trans retinoic acid containing an amount of ATRA between 0.01 wt% and 0.2wt%, preferably between 0.05% and 0.15%, for an administration time of 1 day up to 15 weeks.

[0101] In a preferred version, said composition is formulated for oral administration.

[0102] In a preferred version the composition for oral administration contains an amount of ATRA between 1 and 100 mg, preferably between 5 and 95 mg, preferablybetween 10 and 80 mg, preferably between 15 and 75 mg, preferably between 20 and 70 mg, preferably between 25 and 65 mg, preferably between 30 and 60 mg, preferably between 35 and 55 mg, preferably between 40 and 50 mg.

[0103] Preferably the composition is administered orally twice a day.

[0104] Preferably the composition is administered orally twice a day for up to 90 days.

[0105] Preferably the composition for oral administration contains 10-30 mg of ATRA and is intended to be administered over a period of 30 to 60 days.

[0106] In an embodiment, said composition is for topical use.

[0107] In an embodiment, the composition is for systemic use. Systemic use refers to the introduction of ATRA into the body to achieve a general and / or targeted effect in one or more physiological districts, organs, apparatuses and systems. Specifically, any composition containing or comprising ATRA e.g. for intramuscular, parenteral, intravenous, intrathecal, subcutaneous use.

[0108] According to an embodiment of the present invention, the composition is used for topical use, the composition will therefore preferably be in the form of: cream, ointment, pomade, lotion, tonic, gel, paste, foam, hydrophilic gel, emulsion, suspension or liquid.

[0109] Depending on the type of formulation, the composition may further comprise one or more excipients and / or carriers.

[0110] For lipophilic ointments, excipients can be selected from: solid, semi-solid and / or liquid paraffins, vegetable oils, waxes, liquid silicones, which generally have occlusive or protective properties. In some versions, the composition further comprises surfactants such as sorbitan esters, lanolin alcohols, fatty alcohols, fatty acid sulphates, fatty acid esters and / or polysorbates to make an ointment that emulsifies in water.

[0111] In some versions, said composition is in the form of a hydrophilic ointment. In this case, the composition further comprises excipients such as mixtures of polyethylene glycols.

[0112] In some versions, said composition is in cream form. In this case, as the cream is a multiphase preparation consisting of a lipophilic and a hydrophilic phase, emulsifiers are added. If the composition is a hydrophobic cream, the addable emulsifiers are preferably selected from wool alcohols, sorbitan esters and / or monoglycerides. If the composition is a hydrophilic cream, the emulsifiers will be sodium soaps, polysorbates and / or sulphates of fatty alcohols.

[0113] In some versions, the composition is a hydrophilic gel. In this case, this composition further comprises hydrophilic macromolecules such as sodium alginate, agar-agar, gums, gelatin, starch, cellulose derivatives and / or carboxy-vinyl polymers.

[0114] In some versions, the composition is a hydrophobic gel. In this case, the composition further preferably comprises excipients such as micronised silica, hydrogenated castor oil, stearyl ammonium hectorite, zinc stearates, calcium stearates and / or aluminium stearates.

[0115] In other versions, the composition further comprises other excipients selected from moisturising substances such as collagen, elastin, various protein hydrolysates, nucleic acid hydrolysates, natural moisturising factor; or absorption promoters such as DMSO, fatty acids, urea-azone and / or menthol.

[0116] Preferably the formulation for topical use is in the form of an ointment, topical cream, topical gel, liniment, paste, film, solution, hydrogel, liposome, transferable vesicle, cream, lotion, dermal patch, transdermal patch, transdermal spray.

[0117] Preferably this composition is in the form of an ointment, pessary, vaginal ring, intrauterine device (IUD), extra-amniotic infusion, intravesical infusion, nasal spray, ear drops, ointment, insufflation.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0118] In the context of the present invention, mycoses or fungal infections are understood to be any form of infection by mycetes in humans.

[0119] In the context of the present invention, mycoses or fungal infections from Candida are understood to be any form of infections by mycetes in humans sustained by Candida albicans and / or by Candida non- albicans.

[0120] In the context of the present invention, the singular forms “a” and “an” are intended to designate both the singular and the plural, unless expressly indicated to designate only the singular.

[0121] Furthermore, as used herein, “and / or” refers to and comprises all possible combinations of one or more of the associated listed elements, as well as the lack of combinations when interpreted alternatively (“or”). Thus, fungal infection caused by Candida albicans and / or non-albicans species; Candida albicans infections; or nonalbicans Candida infections.

[0122] The terms “administering”, “administration” or “administer” as used in this document refer to (1 ) providing, dispensing, dosing and / or prescribing, e.g. by or underthe direction of a health care professional or his authorised agent, and (2) insertion, application, intake or consumption, e.g. by a health care professional or the subject. For example, administration may include, without limitation, the current route of administration (e.g. gel, ointment, cream, aerosol, etc.) and may be formulated, alone or together, in appropriate unit dosage formulations containing pharmaceutically acceptable non-toxic conventional carriers, adjuvants, excipients and vehicles appropriate for each route of administration. The invention is not limited by the route of administration, formulation or dosing schedule.

[0123] The term “treatment” as used herein includes the alleviation, mitigation or improvement of fungal infections caused by Candida albicans or C. non-albicans or of one or more symptoms and signs thereof, irrespective of whether the infection is considered resolved and whether or not all symptoms and signs are resolved. The terms also include reducing or preventing the progression of the disease, or one or more signs and symptoms thereof, by blocking or preventing an underlying mechanism of infection, and achieving any therapeutic and / or prophylactic benefit.

[0124] The term “carrier” refers to a diluent, adjuvant, excipient or carrier with which the therapeutic agent is administered.FiguresIn the graphs in Figures 1 -8 the following indications are adopted:CTRL indicates control, i.e. a full well without solvent or compound;ATRA indicates the histograms for the wells with different concentrations of ATRA: 1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM;AmB indicates the histograms for the wells with different concentrations of Amphociterin: 2 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.12 pg / mL;DMSO indicates the histograms for the wells with different concentrations of DMSO (without additional compound) 1 %v / v, 0.5%v / v, 0.25%v / v, 0.06 %v / v.

[0125] Figure 1. Figure 1 shows the inhibitory effect of ATRA on the biofilm biomass of C. krusei. The Candida cells were cultured for 24 hours in the absence or presence of ATRA at various concentrations. Amphotericin B (AmB) was used as a positive control drug. Biofilm growth was assessed in terms of total biomass (A) by staining with crystal violet (CV) and quantified by measuring optical density (OD) using a spectrophotometer at 595 nm. The results are the mean of the O.D. values ± SD of three independent experiments, each conducted in triplicate. *p<0.05, **p<0.01 ; ***p<0.001.

[0126] Figure 2. Figure 2 shows the inhibitory effect of ATRA on the metabolic activity of C. krusei biofilm. The Candida cells were cultured for 24 hours in the absence or presence of ATRA at various concentrations (1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM). AmB was used as a positive control drug. The metabolic activity of the biofilm was assessed by XTT tetrazolium salt reduction assay and quantified by measuring the optical density (O.D.) using a spectrophotometer at 490 nm. *p<0.05, **p<0.01 ; ***p<0.001.

[0127] Figure 3. Figure 3 shows the inhibitory effect of ATRA on the biofilm biomass of C. tropicalis. The Candida cells were cultured for 24 hours in the absence or presence of ATRA at various concentrations (1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM). AmB was used as a positive control drug. Biofilm growth was assessed as total biomass (A) by staining with crystal violet (CV) and quantified by measuring optical density (OD) using a spectrophotometer at 595 nm. The results are the mean of the O.D. values ± SD of three independent experiments, each conducted in triplicate. *p<0.05, **p<0.01 ; ***p<0.001.

[0128] Figure 4. Figure 4 shows the inhibitory effect of ATRA on the metabolic activity of C. tropicalis biofilm. The Candida cells were cultured for 24 hours in the absence or presence of ATRA at various concentrations (1 mM, 0.5 mM, 0.25, 0.12 mM, 0.06 mM). AmB was used as a positive control drug. The metabolic activity of the biofilm was assessed by XTT tetrazolium salt reduction assay and quantified by measuring the optical density (O.D.) using a spectrophotometer at 490 nm *p<0.05, **p<0.01 ; ***p<0.001.

[0129] Figure 5. Figure 5 shows the inhibitory effect of ATRA on the biofilm biomass of C. auris. The Candida cells were cultured for 24 hours in the absence or presence of ATRA at various concentrations (1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM). AmB was used as a positive control drug. Biofilm growth was assessed as total biomass (A) by staining with crystal violet (CV) and quantified by measuring optical density (OD) using a spectrophotometer at 595 nm. The results are the mean of the O.D. values ± SD of three independent experiments conducted in triplicate. *p<0.05, **p<0.01 ; ***p<0.001 .

[0130] Figure 6. Figure 6 shows the inhibitory effect of ATRA on the metabolic activity of C. auris biofilm. The Candida cells were cultured for 24 hours in the absence or presence of ATRA at various concentrations (1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM). AmB was used as a positive control drug. The metabolic activity of the biofilm was assessed by XTT tetrazolium salt reduction assay and quantified by measuring theoptical density (O.D.) using a spectrophotometer at 490 nm. *p<0.05, **p<0.01 ;***p<0.001.

[0131] Figure 7. Figure 7 shows the inhibitory effect of ATRA on the biofilm biomass of C. glabrata. The Candida cells were cultured for 24 hours in the absence or presence of ATRA at various concentrations (1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM). AmB was used as a positive control drug. Biofilm growth was assessed as total biomass (A) by staining with crystal violet (CV) and quantified by measuring optical density (OD) using a spectrophotometer at 595 nm. The results are the mean of the O.D. values ± SD of three independent experiments conducted in triplicate. *p<0.05, **p<0.01 ; ***p<0.001.

[0132] Figure 8. Figure 8 shows the inhibitory effect of ATRA on the metabolic activity of the biofilm of C. glabrata. The Candida cells were cultured for 24 hours in the absence or presence ofATRA at various concentrations (1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM). AmB was used as a positive control drug. The metabolic activity of the biofilm was assessed by XTT tetrazolium salt reduction assay and quantified by measuring the optical density (O.D.) using a spectrophotometer at 490 nm. *p<0.05, **p<0.01 ; ***p<0.001.

[0133] Figure 9. Figure 9 shows the effect of ATRA on C. krusei biofilm formation. The Candida cultures were incubated for 24 hours in the absence or presence of ATRA at various concentrations (1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM). The phenotypic transition from yeast to hyphal morphotype was analysed by light microscopy after staining with crystal violet. AmB was used as a positive control drug. The images were acquired by an optical microscope (Olympus, Carl Zeiss, UK) with 40* magnification objectives. A representative experiment of three is shown. The column on the left shows images of the wells with different concentrations of Trifarotene: 1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM; the middle column shows the images for the wells with different concentrations of Amphociterin: 2 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.12 pg / mL; the column on the right shows images of the wells with different concentrations of DMSO (without any additional compound) 1 %v / v, 0.5%v / v, 0.25%v / v, 0.06 %v / v, at the top a control image (CTRL) is shown, i.e. full well without solvent or compound.

[0134] Figure 10. Effect of ATRA on biofilm formation of C. tropicalis. The Candida cultures were incubated for 24 hours in the absence or presence of ATRA at various concentrations (1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM). The phenotypic transition from yeast to hyphal morphotype was analysed by light microscopy after staining withcrystal violet. AmB was used as a positive control drug. The images were acquired by an optical microscope (Olympus, Carl Zeiss, UK) with 40* magnification objectives. A representative experiment of three is shown. The column on the left shows images of the wells with different concentrations of Trifarotene: 1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM; the middle column shows the images for the wells with different concentrations of Amphociterin: 2 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.12 pg / mL; the column on the right shows images of the wells with different concentrations of DMSO (without any additional compound) 1 %v / v, 0.5%v / v, 0.25%v / v, 0.06 %v / v, at the top a control image (CTRL) is shown, i.e. full well without solvent or compound.

[0135] Figure 11. Effect of ATRA on C. auris biofilm formation. The Candida cultures were incubated for 24 hours in the absence or presence of ATRA at various concentrations (1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM). The phenotypic transition from yeast to hyphal morphotype was analysed by light microscopy after staining with crystal violet. AmB was used as a positive control drug. The images were acquired by an optical microscope (Olympus, Carl Zeiss, UK) with 40* magnification objectives. A representative experiment of three is shown. The column on the left shows images of the wells with different concentrations of Trifarotene: 1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM; the middle column shows the images for the wells with different concentrations of Amphociterin: 2 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.12 pg / mL; the column on the right shows images of the wells with different concentrations of DMSO (without any additional compound) 1 %v / v, 0.5%v / v, 0.25%v / v, 0.06 %v / v, at the top a control image (CTRL) is shown, i.e. full well without solvent or compound.

[0136] Figure 12. Effect of ATRA on C. glabrata biofilm formation. The Candida cultures were incubated for 24 hours in the absence or presence of ATRA at various concentrations (1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM). The phenotypic transition from yeast to hyphal morphotype was analysed by light microscopy after staining with crystal violet. AmB was used as a positive control drug. The images were acquired by an optical microscope (Olympus, Carl Zeiss, UK) with 40* magnification objectives. A representative experiment of three is shown. The column on the left shows images of the wells with different concentrations of Trifarotene: 1 mM, 0.5 mM, 0.25 mM, 0.12 mM, 0.06 mM; the middle column shows the images for the wells with different concentrations of Amphociterin: 2 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.12 pg / mL; the column on the right shows images of the wells with different concentrations of DMSO (without anyadditional compound) 1 %v / v, 0.5%v / v, 0.25%v / v, 0.06 %v / v, at the top a control image (CTRL) is shown, i.e. full well without solvent or compound.

[0137] Figure 13. Figure 13 shows a table of MIC values of non-albicans Candida subjected to nine different antifungal actives, commonly used in the clinic. The non- albicans Candida mycetes subjected to treatment included isolated strains of C. tropicalis, C. glabrata and C. krusei, as well as the reference strain of C. auris CDC B11903, using the Sensitrite YeastOne (SYO) system. The results shown in Figure 13 are the geometric averages of the MIC values (pg / mL). Resistance is defined as the following MIC in micrograms per millilitre: FLU > 8 for C. krusei and C. tropicalis', > 64 for C. glabrata and for C. auris > 32. AmB > 2 for C. auris', VOR > 2 for C. krusei and > 1 for C. tropicalis. MICA > 4 for C. auris; > 0.25 for C. glabrata and > 1 for C. tropicalis and C. krusei; CASPO and ANID > 0.5 for C. glabrata', > 1 for C. tropicalis and C. krusei [Clinical and Laboratory Standards Institute], Clsi.org. https: / / clsi.org / shop / standards / m27m44s / (accessed 24 / 09 / 2025).]. The data shown in Figure 13 demonstrate the full sensitivity of C. glabrata and C. auris to all antifungal agents tested. In contrast, C. krusei showed intrinsic resistance to fluconazole, as expected, while C. tropicalis showed reduced sensitivity to fluconazole, with both species exhibiting MIC values above 4 pg / mL, indicating potential resistance or tolerance.

[0138] According to the present invention, a composition comprising all-trans retinoic acid (ATRA) is provided for use in the treatment of fungal infections.

[0139] The composition of the invention is intended for use in the treatment of fungal infections caused by non-albicans Candida species.

[0140] Preferably, the compound of the invention is intended for use in the treatment of fungal infections caused by one or more of C. krusei, C. tropicalis, C. auris, C. glabrata.

[0141] All-trans retinoic acid (ATRA), the formula for which is provided below, is a metabolite of the retinoid family that intervenes in the functions of vitamin A (retinol).

[0142] Retinoids, through their related nuclear receptors, exert powerful effects on cell growth, differentiation and apoptosis and hold significant promise for cancer therapy and chemoprevention. Differentiation therapy with ATRA has marked a major breakthrough and has become the drug of first choice in the treatment of acute promyelocytic leukaemia (APL). Conversions of 13-cis-retinoic acid and 9-cis-retinoic acid to all-trans- retinoic acid are very rapid. Currently, two distinct families of retinoid-responsive nuclear receptors have been identified and characterised: retinoic acid receptors (RAR) and retinoid receptors (RXR), each of which comprises three isoforms, a, [3 and y.

[0143] For these reasons, all-trans retinoic acid is increasingly included in anti-cancer treatment regimens for various cancers such as Kaposi's sarcoma, head and neck squamous cell carcinoma, ovarian carcinoma, bladder cancer, neuroblastoma, and has shown anti-angiogenic effects in several systems, inhibiting proliferation in vascular smooth muscle cells (VSMCs) and anti-inflammatory in rheumatoid arthritis.

[0144] ATRA (commercial form Vesanoid) is currently used in clinical practice as an antineoplastic drug for the treatment of acute promyelocytic leukaemia (APL), as well as for several years, for the treatment of severe acne. Based on our scientific evidence, it could also be used in the control of invasive fungal infections (IFI) due to an innovative mechanism of action, ATRA may represent a new class of antifungal drugs with the potential to become a new therapeutic aid in the treatment of IFI.

[0145] In addition to its antitumour effect, ATRA exerts a fungistatic in vitro action against Aspergillus fumigatus and Candida albicans, blocking the germination and replication of these fungi; sub-optimal doses of ATRA and conventional antifungals such as Amphotericin B or Posaconazole synergize in vitro in counteracting the germination of aspergillus conidia. This synergistic effect makes it possible to reduce the dosage of antifungal drugs, limiting exposure to the toxic effects related to prolonged and massive use of such drugs. Furthermore, in a model of invasive pulmonary aspergillosis in the rat, ATRA administered prophylactically increased the survival of animals statistically significantly compared to the untreated control group (60% vs 20% 12 days after infection). The protective efficacy of ATRA was also found to be fully comparable to that obtained with Posaconazole, one of the antifungal drugs commonly used in the treatment of I PA.

[0146] Advantageously, the inventors demonstrated that ATRA, unexpectedly, also has an antifungal effect on non-albicans Candida species.

[0147] The inventors demonstrated that ATRA is effective in the treatment of fungal infections caused by fungal species of non-albicans Candida. The inventors have demonstrated that ATRA is effective in the treatment of fungal species such as C. auris, C. krusei, C. tropicalis and C. glabrata which have, on the contrary, proved resistant to conventional therapies commonly used in clinical practice. These species have been shown to be intrinsically resistant to fluconazole, or to other azoles that have been shown to be effective in treating Candida albicans infections. These non-albicans Candida species are resistant to the drugs currently commonly used in the treatment of Candida albicans infections.

[0148] On the other hand, C. tropicalis and C. glabrata in particular, are species that differ considerably from Candida albicans in both morphology and genome and, therefore, have different mechanisms of proliferation but also of interaction with drugs compared to Candida albicans.

[0149] C. tropicalis and C. glabrata show increasing rates of resistance not only to azoles but also to other drug classes such as echinocandins. Since these species differ not only in their response to antifungals compared to Candida albicans, but also in their genome and the expression of different transmembrane proteins, an antifungal effect of ATRA against these species was not to be expected.

[0150] Contrary to expectations, ATRA proved effective against such species of non- albicans Candida.

[0151] Although the studies of the prior art could allow an inference of an efficacy of ATRA in the treatment of fungal infections caused by Aspergillus fumigates and Candida albicans, due to the differences indicated above between the species of Candida albicans and non-albicans Candida, both genetic and in the dynamics of infection propagation, did not allow one to imagine or predict an efficacy of ATRA also against infections caused by mycetes of the non-albicans Candida type. Such mycetes, in fact, are extremely different from Candida albicans despite being classified within the same genus, both in aetiology and genetically.

[0152] Another advantage related to the administration of ATRA is the zero risk of possible drug interactions with amphotericin, echinocandins, cyclopyroxolamine, and azoles, thus being an adequate and safe drug in combination with the antifungals used in common clinical practice.

[0153] The composition can be administered to a subject in need by conventional methods.

[0154] In an embodiment, said composition is in the form of a preparation for oral administration.

[0155] In an embodiment, said composition is in the form of a preparation for intravenous administration.

[0156] In an embodiment, said composition is in the form of a preparation for intramuscular administration.

[0157] In an embodiment said composition is in the form of a preparation for topical administration, preferably said composition being in the form of a gel, cream, lotion, solution for nail application, composition for transdermal application.

[0158] In an embodiment, said composition is in the form of a preparation for oral liposomal administration.

[0159] In an embodiment, said composition is in the form of an aerosol preparation.

[0160] In other embodiments the composition can be administered in other forms that are equally suitable for implementing the present invention.

[0161] A person skilled in the art may decide to administer the composition by any conventional pharmaceutical form. Reference can be made to Remington’s Pharmaceutical Sciences, latest edition.

[0162] A person skilled in the art will decide the effective timing of administration, depending on the patient’s condition, the level of seventy of the pathology, the patient's response, and any other clinical parameters included in the general knowledge of this topic.

[0163] The composition of the invention also contains, together with the active ingredients, at least one pharmaceutically acceptable carrier or excipient.

[0164] Such a carrier may be a macromolecule, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, an antibody, a lipid molecule, an inactive viral particle or any other carrier known in the pharmaceutical industry.

[0165] An extensive discussion of commercially available carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991 ).

[0166] The carriers may additionally contain liquids such as water, saline solution, glycerol and ethanol.

[0167] In addition, particularly useful formulation aids may be present, e.g. solubilising agents, dispersing agents, suspending agents and emulsifying agents. These components allow pharmaceutical compositions such as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like to be formulated for oral administration.

[0168] All these components are known in the industry and can easily be selected by a person skilled in the art based on their general knowledge of the pharmaceutical industry.

[0169] Once formulated, the composition can be administered directly to the subject. The subjects to be treated can be animals; in particular, they can be human subjects.

[0170] The composition of the invention may be administered by any route, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal or by transcutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means of application. A preferred route is the oral route.

[0171] The compositions for oral administration can take the form of liquid solutions or suspensions or powders.

[0172] Most commonly, the compositions are in a unit dosage form to facilitate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unit dosages for humans and other mammals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, in combination with a pharmaceutically acceptable excipient. Typical unit dosage forms comprise premeasured ampoules or syringes for liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. In such compositions, the compounds of the invention are generally a minor component (e.g. from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight), the remainder comprising various carriers and auxiliary substances useful in composing the desired dosage form. Treatment may involve single-dose or multiple-dose administration.

[0173] Pharmaceutical compositions according to the present invention may also contain one or more additional active ingredients. This additional active ingredient may, for example, be a compound known for use in diabetic nephropathy.

[0174] Average quantities of active compound may vary and should be based on the recommendations and prescription of a qualified physician. The administration regimen, dosage and posology will be decided by the doctor based on his or her experience, the disease to be treated and the patient's condition, and on general knowledge in the field.

[0175] For each compound, the therapeutically effective dose can be estimated initially in cell culture assays or in animal models, usually mice, rats, guinea pigs, dogsor pigs. The animal model can also be used to determine the appropriate concentration range and route of administration. This information can be used to determine useful doses and routes of administration for humans. When calculating the Human Equivalent Dose (HED), we recommend using the conversion table provided in the document Guidance for Industry and Reviewers (2002, U.S. Food and Drug Administration, Rockville, Maryland, USA).

[0176] An average dose for administration in humans may be established during clinical trials, as is standard practice in the industry.

[0177] The specific effective dose for a human subject will then depend on the severity of the condition, the subject's general health, age, weight and sex, diet, time and frequency of administration, any combination of drugs and tolerance / response to therapy. This amount can be determined through routine trials and is a matter for the doctor's assessment.

[0178] Depending on the chosen route of administration, the compositions will be in solid or liquid form, suitable for oral or parenteral administration or any other chosen route of administration.

[0179] The invention will now be further illustrated by the following examples.EXAMPLESEXPERIMENTAL TESTS

[0180] In vitro studies were conducted to evaluate the antifungal effect of all-trans retinoic acid (ATRA) at different concentrations (1 mM, 0.5 mM, 0.25mM, 0.12 mM, 0.06 mM) against opportunistic pathogenic mycetes of the non-albicans Candida species, in particular C. auris, C. krusei, C. glabrata, and C. tropicalis, according to standard protocols.

[0181] The results obtained showed that ATRA exerted fungistatic activity at a concentration of 0.5 mM against all non-albicans Candida species tested. This effect was maintained up to seven days, the last evaluated time point.

[0182] Strong fungicidal activity of ATRA was demonstrated at 1 mM on all non- albicans Candida species (C. auris, C. glabrata, C. tropicalis and C. krusei).

[0183] Studies were conducted in vitro to assess the antifungal sensitivity / susceptibility of opportunistic pathogenic mycetes of the species non-albicans Candida (NAC), in particular C. auris, C. krusei, C. glabrata, and C. tropicalis, following standard protocols against nine antifungal drugs, commonly used in the clinic, includingAmphotericin B, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Voriconazole, Micafungin, Anidulafungin, Caspofungin.MATERIALS AND METHODSCandida strains and growth conditions

[0184] Clinical strains of C. tropicalis, C. krusei, C. glabrata were isolated. For C. auris the reference strain C. auris CDC B11903 was used.

[0185] The same experimental tests were performed on all strains of Candida used in the experiment, so in the following generic reference will be made to Candida species and it is understood that this definition includes, within the scope of this discussion, strains of C. tropicalis, C. krusei, C. glabrata and C. auris.

[0186] Strains of each Candida species were grown on Sabouraud dextrose agar plates (Difco Laboratories, Detroit, Ml, USA), fortified with chloramphenicol, and incubated at 30°C for each Candida species. After 24 hours of incubation, the Candida cells were harvested by washing with sterile saline or distilled water with 0.05% (v / v) Tween 20. The Candida cells were counted using a Burker chamber, and diluted to the desired seeding density in RPMI 1640 medium with 10% fetal bovine serum (FCS) (catalogue 9014-81-7 Sigma-Aldrich, Milan, Italy).

[0187] With regard to sensitivity / susceptibility studies of non-albicans Candida mycetes to antifungal drugs, the same experimental tests were performed on all strains of non-albicans Candida including C. auris, C. tropicalis, C. krusei and C. glabrata. Tests were conducted with SENSITITRE™ YEASTONE™ panels (SYO®, TREK Diagnostics Systems, Thermo-Fisher, UK) according to the manufacturer's instructions with minor modifications. Briefly, the inoculum for SYO® was prepared by selecting four well- isolated colonies of at least 1 mm in diameter. The Candida cells were emulsified in sterile saline solution and prepared to a turbidity of 0.5 McFarland standard. Subsequently, 20 pL of Candida suspension was transferred into SYO® broth and the final density of the working solution was adjusted to 2x103CFU / mL using a Burker chamber, according to the Clinical and Laboratory Standards Institute (CLSI) guideline M27M44S. [Clinical and Laboratory Standards Institute. Clsi.org. https: / / clsi.org / shop / standards / m27m44s / (accessed 2025-09-24).]. Then, 100 pL of the broth suspension was transferred to each well of the SYO® panels and incubated without agitation at 35°C for 24 hours.

[0188] After incubation, the plates were read visually under normal laboratory lighting. Clear yeast growth was observed with the colour changing from blue (negative,indicating no growth) to pink (positive, indicating growth). Several antifungal drugs were evaluated, including fluconazole, voriconazole, itraconazole, isavuconazole, posaconazole, caspofungin, micafungin, anidulafungin and amphotericin B. the results were expressed as the geometric mean (GM) of two independent experiments performed in duplicate.Antimicrobial agents

[0189] All-trans retinoic acid (ATRA) was supplied in freeze-dried form by the company Sigma-Aldrich, Milan, Italy (catalogue no. R2625; Sigma-Aldrich, Milan, Italy). The powder was dissolved in 50% dimethylsulfoxide (DMSO; Sigma-Aldrich, Milan, Italy) and diluted with the appropriate RPMI 1640 culture medium at a final DMSO concentration of 2.5% (v / v). RPMI 1640 containing 2.5% DMSO was used for each experimental point in all tests. The following ATRA concentrations were used: 1 mM, 0.5 mM, 0.25 mM, 0.12 mM and 0.06 mM.Cell growth rate of C. tropicalis, C. krusei, C. glabrata and C. auris

[0190] To evaluate the antifungal potential of ATRA on the growth of C. tropicalis, C. krusei, C. glabrata and C. auris, 2x105cells of each strain of Candida were grown in 96- well plates (Thermo Scientific™ Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat -Bottom Microplate) in 100 pL of RPMI 1640 medium in 10% FCS (catalogue no. 9014-81 -7; Sigma-Aldrich, Milan, Italy) in the absence or presence of ATRA orAmB.

[0191] ATRA is supplied in powder form. 3 mg of ATRA is dissolved in 1 mL of DMSO to obtain a solution containing 10 mM ATRA called the stock solution, (solution A). From this stock solution, dilutions are made to obtain the desired concentration. From solution A, five further solutions were prepared with different degrees of dilution in order to obtain solutions with different ATRA concentrations. In particular, the following solutions were prepared: solution B: ATRA 1 mM; solution C: ATRA 0.5 mM; solution D: ATRA 0.25 mM; solution E: ATRA 0.12 mM; solution F: ATRA 0.06 mM.Specifically, solution B (= 1 mM) was obtained from solution A by taking 100 microlitres of solution A and adding 900 microlitres of RPMI 1640 (which is the culture medium in which the Candida species used for the experiments grow). From solution B, solution C was prepared by performing a 1 :1 dilution, i.e. 500 microlitres of solution B was takenand added to 500 microlitres of RPMI, resulting in solution C (0.5 mM). The same process was used to prepare solutions D, E and F.

[0192] Compositions containing ATRA and having different final concentrations of ATRA were prepared. Compositions were prepared with final concentrations ranging from 1 mM to 0.06 mM, corresponding to 300-18.75 pg / mL ATRA. Each concentration of ATRA (1 mM, 0.5mM, 0.25 mM, 0.12mM and 0.06 mM) was added in a volume of 100 pL / well. Each test was repeated in triplicate for each ATRA concentration.

[0193] Amphotericin B (AmB) was supplied in freeze-dried form by the company Sigma-Aldrich, Milan, Italy. The powder was dissolved in 50% dimethylsulfoxide (DMSO; Sigma-Aldrich, Milan, Italy) and diluted with the appropriate RPMI 1640 culture medium at a final DMSO concentration of 2.5% (v / v). RPMI 1640 containing 2.5% DMSO was used for each experimental point in all tests.

[0194] For Amphotericin B, the following concentrations were used: 2 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, and 0.12 pg / mL. For each strain of Candida (C. tropicalis, C. krusei, C. glabrata and C. auris), positive control wells were prepared by plating 2x105cells of Candida in 200 pL of culture medium (RPMI), and negative control wells by plating 200 pL of culture medium / well only or 100 pL of culture medium plus 100 pL of each solution prepared for the different concentrations of ATRA or AmB in the absence of Candida.

[0195] Each of the experiments was conducted including positive control wells and negative control wells; three wells were prepared for each experiment, i.e. for each concentration of ATRA.

[0196] The plate was left to incubate at 30°C for 24 hours. At the end of the incubation period, the cell growth rate was assessed by calculating the optical density using a spectrophotometer at 510 nm.

[0197] The results are presented as the mean OD ± SD of three independent experiments, conducted in triplicate and expressed as the percentage of growth inhibition compared to the untreated control.Hyphal growth inhibition assay

[0198] The impact of ATRA on the germination of various Candida species (C. tropicalis, C. krusei, C. glabrata and C. auris) and hyphal growth was also assessed. For this purpose, 2x105cells were cultured in 96-well plates in 200 pL of RPMI 1640 medium with 10% FCS and incubated at 37°C in the absence or presence of different concentrations of ATRA (1 mM, 0.5 mM, 0.25 mM, 0.12mM and 0.06 mM).

[0199] Experimental tests were conducted after 3 hours of incubation and after 24 hours of incubation. Germ tube formation and hyphal growth were assessed by microscopic examination using an optical microscope (Olympus, Carl Zeiss, UK), at 40x magnification, after 3 and 24 hours of incubation. The images were documented with the digital camera provided.Biofilm quantification using Crystal Violet and XTT tests

[0200] To evaluate the effects of ATRA on the biofilm of C. tropicalis, C. krusei, C. glabrata and C. auris, in terms of both biomass and metabolic activity, 2x105Candida cells were grown in 96-well plates in 200 pL of 10% FCS RPMI 1640 medium and incubated at 37°C for 24 h in the absence or presence of different concentrations of ATRA (1 mM, 0.5 mM, 0.25 mM, 0.12 mM and 0.06 mM).

[0201] Crystal violet (CV) staining and the 2,3-Bis-(2-methoxy-4-nitro-5- sulfophophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction test were used to assess the biomass and metabolic activity of the biofilm, respectively, by calculating OD by spectrophotometer. Three independent experiments were performed in triplicate and the data were expressed as the arithmetic mean of the absorbance values (OD) ± SD. In all experiments, the OD values correspond to the absorbances of the individual samples from which the ODs of the negative controls (wells not containing cells) were subtracted.ResultsBiofilm quantification using Crystal Violet and XTT tests

[0202] The production of biofilms of non-albicans Candida species, such as C. tropicalis, C. krusei, C. glabrata and C. auris, was assessed in terms of total biomass, by staining with crystal violet and metabolic activity, using the XTT reduction colorimetric test, as previously described.

[0203] The data in Figure 1 -2 show that ATRA at a concentration of 1 mM inhibits the production of biofilm as biomass of C. krusei, and at 1 mM and 0.5 mM inhibits metabolic activity. In fact, no significant differences were observed between the mean OD values of biomass after 24 hours of exposure to ATRA or Amphocytherin B and those of the cells corresponding to time 0, which corresponds to the time when the Candida cells were placed in the wells, suggesting no biomass formation.

[0204] The data in Figure 3-4 show that ATRA at concentrations of 1 mM and 0.5 mM inhibits both the production of biofilm as biomass and metabolic activity of C. tropicalis. In fact, no significant differences were observed between the mean OD values ofbiomass after 24 hours of exposure to ATRA or Amphocytherin B and those of the cells corresponding to time 0, which corresponds to the time when the Candida cells were placed in the wells, suggesting no biomass formation.

[0205] The data in Figure 5-6 show that ATRA at 1 mM inhibits the production of biofilm as biomass of C. auris, and at 1 mM and 0.5 mM inhibits metabolic activity. In fact, no significant differences were observed between the mean OD values of the biomass after 24 hours exposure to ATRA or AmB and those of the cells corresponding to time 0.

[0206] The data in Figure 7-8 show that ATRA at 1 and 0.5 mM inhibits both the production of biofilm as biomass and as metabolic activity of C. glabrata. Furthermore, ATRA 0.25 mM was still able to inhibit the metabolic activity of the biofilm. In fact, no significant differences were observed between the mean OD values of the biomass after 24 hours of exposure to ATRA or AmB and those of the cells corresponding to time 0, which corresponds to the time when the Candida cells were placed in the wells, suggesting no biomass formation.

[0207] Doses of ATRA below 0.5 mM (0.25-0.12 mM), although not totally suppressing biofilm formation, induced a significant dose-dependent reduction in biofilm development, similar to that induced by Amphocytherin B (0.5-0.12 pg / ml), while the metabolic activity of C. glabrata was still inhibited at 0.25 mM. At concentrations below 0.12 mM no significant differences were found between ATRA and the untreated control. Evaluation of the synergistic effect of ATRA and conventional antifungals

[0208] In order to study the possible synergistic antifungal activity of ATRA with traditionally used antifungal drugs, a sub-inhibitory concentration of a traditional antifungal such as Amphotericin B (AmB) was preliminarily determined.

[0209] It was found that 3.15 pg / mL of Amphotericin B was the minimum concentration of Amphotericin B capable of inhibiting the germination of Aspergillus conidia, whereas at a concentration of 1 .57 pg / mL, Amphotericin B did not interfere with germination and only delayed hyphal growth was observed.

[0210] In order to investigate the possible synergy of ATRA with drugs used in clinical practice in infections sustained by non-albicans Candida, such as C. tropicalis, C. krusei, C. glabrata and C. auris, growth studies were conducted as reported in the previous section “Cell growth rate of C. tropicalis, C. krusei, C. glabrata and C. auris”

[0211] The use of sub-optimal doses of ATRA (0.25mM and 0.5 mM) in combination with sub-optimal doses of Amphotericin B (0.125 and 0.12 pg / mL), did not demonstratea synergistic effect in inhibiting the growth of C. tropicalis, C. krusei, C. glabrata and C. auris. This further demonstrates that the effect of the same compound on various types of fungi cannot be predicted a priori due to the differences discussed above. In fact, while in the case of Aspergillus there is a synergistic effect between ATRA and traditional drugs, against Candida there is no synergistic effect at all, which also underlines the different response of fungi to different antifungal molecules.

[0212] The results obtained showed that ATRA exerted fungistatic activity at a concentration of 0.5 mM against all non-albicans Candida species tested. This effect was maintained up to seven days, the last evaluated time point.

[0213] Strong fungicidal activity of ATRA was demonstrated at 1 mM on all non- albicans Candida species: C. auris, C. glabrata, C. tropicalis and C. krusei.

[0214] EXAMPLESExample 1 : Composition for intravenous administration

[0215] ATRA is supplied in the form of freeze-dried sterile powder. This powder is placed in a 100cc vial and reconstituted with 0.9% sodium chloride physiological solution to produce a suspension with a final ATRA concentration of 0.1 -100 mg / mL. Procedures are performed under low light conditions and intravenous bags and lines are covered to avoid excessive light exposure during infusion.Example 2 Composition for intramuscular administration

[0216] ATRA is supplied in the form of freeze-dried sterile powder. This powder is placed in a bottle and diluted with appropriate solvents such as 0.5% chlorobutanol as a preservative; 12% polysorbate 80, 0.1 % citric acid and sodium hydroxide to adjust the pH. The resulting solution is then mixed with a 0.9% sodium chloride solution to produce a suspension with a final ATRA concentration of between 0.5 and 150 mg / mL.

[0217] This solution for injection is intended to be administered in a single daily dose.Example 3 Composition for oral administration

[0218] Semi-solid capsules were prepared by adding the active substance, ATRA, to lipophilic compounds. ATRA is a powder and can be dissolved in peanut oil, yellow wax, soybean oil, hydrogenated soybean oil, partially hydrogenated soybean oil, rapeseed oil, palm oil, olive oil to obtain a liquid mixture. The liquid mixture is then filled into the hard gelatin capsule. ATRA is incorporated into formulations consisting of glyceryl macrogolglyceride associated with soybean oil or derivative, medium-chain triglyceride. The provision of an oily excipient also improves absorption of the lipophilic drug by increasing the solubility of the drug in the lipid phase, but the release of the activeingredient from the formulation may be slowed down due to the high affinity of the drug. The use of dispersed systems (emulsions or suspensions) instead of lipophilic or hydrophilic carriers alone can improve drug absorption as well as increase the gastrointestinal contact surface area and aid absorption.

Claims

Claims1. Pharmaceutical composition comprising ATRA for use in the treatment of fungal infections, wherein the fungal infection is caused by non-albicans Candida and in particular by one or more of C. krusei, C. tropicalis, C. auris, C. glabrata.

2. Pharmaceutical composition for use according to the preceding claim wherein said composition comprises ATRA in a percentage between about 0.01 wt% and about 10 wt%, preferably between about 0.05wt% and about 5wt%, more preferably between about 0.1 wt% and about 1wt%.

3. Pharmaceutical composition comprising ATRA for use in the treatment of fungal infections, wherein the fungal infection is caused by non-albicans Candida and in particular by one or more of C. krusei, C. tropicalis, C. auris, C. glabrata and wherein said composition comprises ATRA in a percentage between about 0.01 mM and about 2 mM, preferably between about 0.1 mM and about 1 .25 mM, more preferably between about 0.5 mM and about 0.75 mM.

4. Pharmaceutical composition for use according to one of the preceding claims and further comprising an additional antifungal agent.

5. Pharmaceutical composition for use according to claim 4, wherein the at least one additional antifungal agent is selected from the group consisting of azoles such as for example fluconazole, voriconazole, posaconazole, itraconazole, ketoconazole, isavuconazole, thioconazole, opelconazole, miconazole.

6. Pharmaceutical composition for use according to claim 4, or 5, wherein the at least one additional antifungal agent is selected from the group consisting of polyenes such as amphotericin B, nystatin or liposomal and lipid forms.

7. Pharmaceutical composition for use according to one of claims 4 to 6, wherein the at least one additional antifungal agent is selected from the group consisting of purine or pyrimidine nucleotide inhibitors such as flucytosine.

8. Pharmaceutical composition for use according to one of claims 4 to 7, wherein the at least one additional antifungal agent is selected from the group consisting of polyoxins, such as nikkomycins, preferably nikkomycin Z or other chitin inhibitors.

9. Pharmaceutical composition for use according to one of claims 4 to 8, wherein the at least one additional antifungal agent is selected from the group consisting of echinocandins such as micafungin and anidulafungin.

10. Pharmaceutical composition for use according to one of claims 4 to 9, wherein said additional antifungal agent is present in said composition in a percentage betweenabout 0.1 wt% and about 10wt%, preferably between about 0.5wt% and about 5wt%, more preferably between about 0.2wt% and about 2wt%.11 . Pharmaceutical composition for use according to one of the preceding claims and further comprising at least one compound selected from at least one excipient, at least one carrier, at least one adjuvant.

12. Pharmaceutical composition for use according to one of the preceding claims wherein said composition is in the form of a liquid solution, and is preferably intended for intravenous administration.

13. Pharmaceutical composition for use according to the preceding claim for the administration of an amount of ATRA between 5 and 100 mg / m2 / day, preferably between 10-95 mg / m2 / day, more preferably between 20-70 mg / m2 / day, preferably between 30-60 mg / m2 / day, preferably between 50-60 mg / m2 / day for an administration time between 1 and 20 days.

14. Pharmaceutical composition for use according to claim 12 or 13, formulated in such a way that an amount of ATRA between 10 and 30 mg / m2 / day is administered for an administration time between 7 to 15 days.

15. Pharmaceutical composition for use according to one of claims 12 to 14 and comprising 10-50 mg ATRA.

16. Pharmaceutical composition for use according to one of claims 1 -11 wherein said composition is in liquid form and is intended for intramuscular administration.

17. Pharmaceutical composition for use according to the preceding claim configured in such a way that an amount of ATRA is administered intramuscularly in a dose between 5 and 150 mg / day, preferably between 10 and 140 mg / day, preferably between 20 and 130, preferably between 30 and 110, preferably between 40 and 100, preferably between 50 and 90, preferably between 60 and 80 mg / day for an administration time between 1 and 5 days.

18. Pharmaceutical composition for use according to one of claims 16 to 17 formulated in such a way that an amount of ATRA is administered intramuscularly in a dose between 25 and 50 mg / day for an administration time of 2 weeks.

19. Pharmaceutical composition for use according to one of claims 16 to 18 formulated in such a way that an amount of ATRA is administered intramuscularly in a dose between 5 and 25 mg / day for an administration time of 4 weeks.

20. Pharmaceutical composition for use according to one of claims 1 to 11 , containing an amount of ATRA between 0.01 and 0.2%, preferably between 0.05% and0.15%, wherein said composition is intended for intralesional administration for an administration time of 1 day up to 15 weeks.21 . Pharmaceutical composition for use according to one of claims 1 to 11 for intralesional administration by intradermal injection of trans retinoic acid containing an amount of ATRA between 0.01 and 0.2%, preferably between 0.05% and 0.15%, for an administration time of 1 day up to 15 weeks.

22. Pharmaceutical composition for use according to one of claims 1 to 11 for oral administration containing an amount of ATRA between 1 and 100 mg, preferably between 5 and 95 mg, preferably between 10 and 80 mg, preferably between 15 and 75 mg, preferably between 20 and 70 mg, preferably between 25 and 65 mg, preferably between 30 and 60 mg, preferably between 35 and 55 mg, preferably between 40 and 50 mg, wherein said composition is administered for a number of times per day such that a maximum amount of ATRA of 100 mg per day is administered.

23. Pharmaceutical composition for use according to the preceding claim wherein said administration is provided twice a day.

24. Pharmaceutical composition for use according to claim 22 or 23 wherein administration is provided for a maximum administration time of 90 days.

25. Pharmaceutical composition for use according to one of claims 22 to 24 wherein said composition contains between 10-30 mg of ATRA and wherein administration is provided for an administration time of 30 to 60 days.

26. Pharmaceutical composition for use according to any one of claims 1 to 11 for topical use.

27. Pharmaceutical composition for use according to any one of claims 1 to 5 for systemic use.

28. Pharmaceutical composition for use according to any one of claims 1 to 11 , wherein said composition is in the form of an ointment, topical cream, topical gel, liniment, paste, film, solution, hydrogel, liposome, nanosome, mucosome, transferable vesicle, cream, lotion, dermal patch, transdermal patch, transdermal spray, powder, nail varnish, nail polish.

29. Pharmaceutical composition for use according to any one of claims 1 to 11 , for use in treating fungal infections caused by one or more of C. krusei, C. tropicalis, C. auris, C. glabrata wherein said fungal infections are cutaneous fungal infections.

30. Pharmaceutical composition for use according to any one of claims 1 to 11 , for use in the treatment of fungal infections caused by one or more of C. krusei, C. tropicalis, C. auris, C. glabrata wherein said fungal infections are mucosal fungal infections.31 . Pharmaceutical composition for use according to any one of claims 1 to 11 , for use in the treatment of fungal infections caused by one or more of C. krusei, C. tropicalis, C. auris, C. glabrata wherein said fungal infections are fungal infections of the vaginal, nasal, oral, anal mucosa.

32. Pharmaceutical composition for use according to one of claims 1 to 11 , for use in the treatment of fungal infections sustained by one or more of the following species of Non-albicans Candida and in particular by one or more of C. auris, C. tropicalis, C. glabrata and C. krusei wherein said infections are fungal infections selected from a group comprising oesophageal fungal infections, fungal infections cutaneous infections, infections of the skin folds, hairy areas, scalp infections, fungal infections of the nose, sinuses or ear, including otomycosis, intracranial or meningeal fungal infections, peritoneal or intra-abdominal fungal infections, including peritonitis or catheter-associated fungal biofilms, systemic, blood, bone marrow or haematogenous fungal infections.

33. Pharmaceutical composition for use according to one of the preceding claims for use in the treatment of fungal infections wherein said fungal infection is an epidermal infection.

34. Pharmaceutical composition for use according to one of the preceding claims for use in the treatment of fungal infections wherein said fungal infection is a pulmonary infection.

35. Pharmaceutical composition for use according to the preceding claim wherein said composition is in the form of an ointment, pessary, vaginal ring, intrauterine device (IUD), extra-amniotic infusion, intravesical infusion, nasal spray, ear drops, ointment, insufflation, mucoadhesive microdisk.

36. Pharmaceutical composition for use according to one of the preceding claims for the treatment of fungal infections in immunocompromised patients and in particular in patients suffering from one or more of acute myeloid leukaemia (AML), human stem cell transplantation (HSCT) and graft versus host disease (GvHD).

37. Pharmaceutical composition for use according to one of the preceding claims wherein said ATRA is in powder form, said composition further comprising physiological solution or other injectable solvent to dissolve said powder.

38. Pharmaceutical composition for use according to one of the preceding claims wherein said composition is in the form of a liquid suspension of ATRA in 0.9% sodium chloride.

39. Kit comprising a composition according to one of the preceding claims and a physiological solution intended to dissolve said composition.

40. Method for preparing an injectable composition according to one of the preceding claims comprising dissolving a composition according to claim 27 in an injectable solution, preferably a 0.9% aqueous solution of sodium chloride to obtain an injectable solution comprising the composition of the invention for the treatment of fungal infections caused by one or more of C. krusei, C. tropicalis, C. auris, C. glabrata.

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