Phytoecdysone compositions for intranasal administration

Abstract

The invention relates to a composition comprising at least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone and at least one cyclodextrin in the form of at least one inclusion complex, for use in the treatment or prevention of a pathology in mammals by intranasal route. Figure for abstract: Fig. 4

Description

Title of the invention: Phytoecdysone compositions for their intranasal administration Technical field of the invention

[0001] The invention relates to drug compositions. It falls within the field of drugs and their administration by intranasal route. The invention relates more particularly to phytoecdysone compositions intended for the prevention and / or treatment of pathologies. Prior art

[0002] Cyclodextrins (CDs) are cyclic oligosaccharides generally composed of 6 to 8 glucopyranose members linked by (a-1,4) bonds. Cyclodextrins with 6, 7 or 8 members are called respectively a-CD, [3-CD or y-CD. CDs have a truncated cone structure, delimiting a cavity in their center. This cavity is all the larger as the number of glucopyranose members composing the CD is large. The central cavity has an apolar and rather hydrophobic carbon environment capable of accommodating molecules with low water solubility, while the exterior has numerous hydroxyl groups, leading to good solubility in water.

[0003] Due to their apolar cavity, cyclodextrins are capable of forming inclusion complexes in aqueous media with a wide variety of hydrophobic guest molecules. This complexation allows the solubilization of hydrophobic molecules, which are very insoluble in an aqueous phase (Merkus et al., 1999).

[0004] The hydroxyl groups of the glucopyranose links can be substituted by different groups, which in particular has the consequence of influencing the solubility in water of the CDs obtained and the hydrophobicity of their cavity.

[0005] This property of CDs is used to solubilize drugs for their administration in aqueous phase by different routes. CDs are particularly indicated for molecules of biopharmaceutical class II (Biopharmaceutics Classification System (BCS) class II) which correspond to poorly soluble / permeable molecules. CDs are of little or no interest for BCS class III and IV molecules, respectively soluble / poorly permeable and poorly soluble / poorly permeable (Rassu et al. 2021). Indeed, class III and IV molecules retain low permeability although their solubility is good from the outset, or improved thanks to complexation with a CD. By permeable, we mean the capacity of a molecule to cross the intestinal mucosa and thus reach the general circulation.

[0006] Molecules solubilized in the presence of CDs are in dynamic equilibrium between their free and complexed states. Only free molecules are able to penetrate biological membranes, which implies that guest molecules must be released from the molecule / CD complex before they can be absorbed through the intestinal mucosa into the general circulation.

[0007] The intranasal (IN) route is a route of administration of drugs directly into the nasal cavity of a patient. It is used for local effects on the nasal mucosa (such as decongestants), but also for systemic action (anti-migraine, vaccines, calcitonin, etc.).

[0008] The IN route uses the very large surface area of ​​the capillaries located on the surface of the nasal mucosa, thus allowing easy access to the vascular system (via irrigation of the respiratory epithelium) but also to the central nervous system (CNS) (presence of the trigeminal nerves and the olfactory nerves in contact with the nasal cavity).

[0009] Given the very close proximity between the nasal mucosa and the CNS, a drug administered by the IN route has the opportunity to easily reach the brain. Indeed, when a substance reaches the olfactory mucosa, it is absorbed immediately and diffuses at the level of the brain and quickly reaches the cerebrospinal fluid, thus avoiding the barrier of the blood-brain barrier.

[0010] Thanks to direct absorption into the systemic circulation, the IN route, unlike the oral route, makes it possible to avoid the first hepatic and enteric passage and thus to avoid a first metabolization, which can increase the quantity of the directly effective drug.

[0011] Optimal IN administration requires dissolution of an effective dose of the drug in a very small volume of aqueous solution. CDs are therefore particularly indicated for improving the solubility of class II (poorly soluble / permeable) lipophilic (hydrophobic) molecules in small volumes for IN administration.

[0012] Phytoecdysones represent an important family of polyhydroxylated phytosterols structurally related to insect molting hormones. These molecules are produced by many plant species and participate in their defense against insect pests. The major phytoecdysone is 20-hydroxyecdysone (20E). 20E is pharmacologically active in mammals. It activates the Mas receptor, a receptor of the protective arm of the Renin Angiotensin System (Lafont et al., 2021) which induces a number of beneficial effects that have been described in preclinical studies in physiological and pathological contexts.

[0013] BIO101 is an oral preparation of 20E with a purity greater than or equal to 97%. Its preparation process is disclosed in international patent application WO2018197731 (Lafont et al., 2018). BIO101 is a new drug candidate clinically developed in the treatment of sarcopenia, in Duchenne muscular dystrophy and in COVID-19. These last two therapeutic applications are the subject of international patent applications WO2018197708 (Dilda et al., 2018) and WO2021198588 (Dilda et al., 2021). Semisynthetic derivatives of 20E have also been developed, as disclosed in international patent application WO2015177469 (Lafont et al., 2015), and are used for such therapeutic applications.

[0014] 20E has very limited oral bioavailability in mammals (Dinan et al. 2021). This property implies that administration of 20E as part of an oral pharmacological treatment would be unsuitable. Indeed, regardless of the mammal considered, more than 90% of the ingested product remains in the digestive tract and is eliminated in the feces (Dinan et al., 2021; Dioh et al., 2023).

[0015] Following oral administration, the prolonged presence of significant quantities (at least 90% of the administered dose) of 20E in the digestive tract in contact with the intestinal microbiota is responsible for the production of metabolites (Kumpun et al., 2011; Dinan et al., 2021; Dioh et al., 2023). These metabolites are absorbed by the intestinal mucosa and are found in the plasma. In the context of pharmaceutical development of 20E, circulating metabolites of 20E, produced only by the intestinal microbiota, must be identified and characterized when they reach certain plasma levels. These are then referred to as major metabolites. The identification and regulated characterization of these major metabolites have a significant impact on the duration and cost of drug development.

[0016] It therefore appears essential to develop new 20E administration strategies compatible with improved bioavailability and metabolic control. Presentation of the invention

[0017] The present invention aims to overcome the aforementioned drawbacks by proposing a solution allowing good bioavailability and better control of metabolism for phytoecdysones and 20-hydroxyecdysone (20E) derivatives.

[0018] Thus, according to a first aspect, the present invention relates to a composition comprising at least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone and at least one cyclodextrin in the form of at least one inclusion complex, for its use in the treatment or prevention of a pathology in mammals by intranasal route.

[0019] The inventors demonstrated that 20E belongs to biopharmaceutical class III (BCS Class III, soluble and poorly permeable, Rincon-Lopez et al., 2021).

[0020] The inventors have shown that 20E forms inclusion complexes with different cyclodextrins (CD). However, and unexpectedly, only certain CDs make it possible to significantly improve the water solubility of 20E. In accordance with the biopharmaceutical class III of 20E, the inventors have shown in an experimental model, in rats, that 20E-CD inclusion complexes do not provide any advantage in terms of oral bioavailability of 20E compared to administration of free 20E (solubilized in pure water). However, and unexpectedly, the inventors have shown that certain 20E-CD inclusion complexes allow particularly efficient passage of 20E through the nasal mucosa. The bioavailability of 20E administered in the form of inclusion complexes with certain CDs is then very largely improved compared to oral administration.

[0021] It was also discovered that intranasal (IN) administration of 20E in the form of an inclusion complex with certain cyclodextrins made it possible to avoid the production of 20E metabolites, these being not detectable in the plasma of the treated animals.

[0022] Finally, while passage through the nasal mucosa is facilitated by the administration of an inclusion complex of 20E with certain cyclodextrins, the inventors discovered, quite unexpectedly, that this new composition of 20E administered by the IN route did not allow any passage to the central nervous system. Indeed, neither 20E nor its major metabolites are detected in the brain tissue and in the cerebrospinal fluid after a single or repeated (7 days) administration of 20E in the form of an inclusion complex with certain cyclodextrins by the IN route.

[0023] In the present description, the term "treatment" means the achievement of a desired pharmacological and physiological effect. The term "treatment", as used in the present description, includes the prevention or partial prevention of one or more of the symptoms of the disease and / or the partial or total cure of the disease and / or the total or partial disappearance of one or more of its symptoms.

[0024] In the present description, the term “at least one” means a single compound (a phytoecdysone or a semi-synthetic derivative of 20-hydroxyecdysone) or a mixture of several such compounds.

[0025] In particular embodiments, the invention further meets the following characteristics, implemented separately or in each of their technically operative combinations.

[0026] A phytoecdysone that can be used according to the invention is, for example, 20-hydroxyecdysone (20E).

[0027] Semi-synthetic derivatives of 20-hydroxyecdysone are understood to mean in particular the known compounds which are the subject of patent application WO 2015 / 177469 in which their production by semi-synthesis is described.

[0028] Phytoecdysones and semi-synthetic derivatives of 20-hydroxyecdysone are advantageously purified to pharmaceutical grade.

[0029] According to a particular embodiment, the composition which is the subject of the present invention comprises 20-hydroxyecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone.

[0030] The 20-hydroxyecdysone used is preferably in the form of an extract of plants rich in 20-hydroxyecdysone or of a composition comprising 20-hydroxyecdysone as active agent. Extracts of plants rich in 20-hydroxyecdysone are, for example, extracts of Stemmacantha carthamoides (also called Leuzea carthamoides or Rhaponticum carthamoides), Cyanotis arachnoidea, Cyanotis vaga, Pfaffia glomerata and Pfaffia paniculata.

[0031] The extracts obtained are preferably purified to pharmaceutical grade.

[0032] In one embodiment, 20-hydroxyecdysone is included in an extract of a plant or a part of a plant, said plant being chosen from plants containing at least 0.5% of 20-hydroxyecdysone by dry weight of said plant. Said extract preferably comprises at least 95%, and preferably at least 97%, of 20-hydroxyecdysone. Said extract thus represents 20-hydroxyecdysone purified to pharmaceutical grade.

[0033] Said extract or said 20-hydroxyecdysone thus purified are hereinafter called BIO101. BIO101 remarkably comprises impurities, such as minor compounds, in individual proportions of between 0 and 0.5%.

[0034] The plant from which BIO101 is produced is preferably chosen from Stemmacantha carthamoides (also called Leuzea carthamoides or Rhaponticum carthamoides), Cyanotis arachnoidea, Cyanotis vaga, Pfaffia paniculata and Pfaffia glomerata.

[0035] According to a particular embodiment of the present invention, said cyclodextrin is chosen from cyclodextrins comprising 7 or 8 glucopyranose links linked by (a-1,4) bonds. These cyclodextrins are called respectively [3-CD or y-CD and are respectively of formula (I) and (II) below: [Chem. 1] [Chem. 2]

[0036] According to a preferred embodiment, the cyclodextrin is the cyclodextrin of formula (II) composed of 8 glucopyranose links linked by (a-1,4) bonds, called y-CD.

[0037] According to a particular embodiment, the cyclodextrin is a cyclodextrin which is composed of 7 or 8 glucopyranose links of which at least one of the hydroxyls in positions 2, 3 and 6 is substituted.

[0038] Preferably the cyclodextrin is a cyclodextrin which is composed of 7 or 8 glucopyranose members of which at least one of the hydroxyls in positions 2, 3 and 6 is substituted by a methyl group or the cyclodextrin is a cyclodextrin which is composed of 7 or 8 glucopyranose members of which at least one of the hydroxyls in positions 2, 3 and 6 is substituted by a 2-hydroxypropyl group.

[0039] The composition which is the subject of the present invention is intended to be used in the treatment of a pathology in mammals by intranasal route. In the present description, the term "pathology in mammals" preferably refers to any disorder or disease for which 20-hydroxyecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone have demonstrated pharmacological activity, i.e. a pathology for which 20-hydroxyecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone is deemed to be an effective treatment. Preferably, the invention relates to the composition which is the subject of the present invention, for its use in the treatment or prevention of a pathology chosen from myopathies and / or neuromuscular diseases, muscular dystrophies, indications requiring an improvement in performance and / or muscular endurance, diseases involving a defect in muscular anabolism and / or muscular catabolism, metabolic diseases, diseases related to blood sugar control, fibrotic diseases, inflammatory diseases, dermatological diseases, cardiovascular diseases, liver diseases, respiratory diseases, kidney diseases, bone diseases, stomach diseases, diseases of the digestive system, diseases of the reproductive function, hematological diseases, diseases related to menopause, parasitic diseases, cancers and healing.

[0040] According to a particular embodiment, the invention relates to the composition which is the subject of the present invention, for its use in the treatment or prevention of a pathology chosen from sarcopenia, sarcopenic obesity, muscle necrosis, loss of muscle strength following immobilization, Duchenne muscular dystrophy, Becker muscular dystrophy, infantile spinal muscular atrophy, diabetes, prediabetes, hyperglycemia, hyperlipidemia, metabolic syndrome, obesity, atherosclerosis, cardiac arrhythmia, myocardial contractility defect, diabetic cardiomyopathy, diabetic hepatopathy, diabetic nephropathy, viral hepatitis, gastric ulcer, celiac disease, allergic bronchial hyperreactivity, asthma, viral pneumonitis, COVID-19, pulmonary inflammation, renal fibrosis, osteoporosis, cartilage degeneration, decreased libido, giardiasis, lambliasis and hymenolepiasis.

[0041] According to a particular embodiment, the invention relates to the composition which is the subject of the present invention, for its use in the treatment or prevention of a pathology chosen from sarcopenia, loss of muscle strength following immobilization, Duchenne muscular dystrophy, spinal muscular atrophy, alteration of respiratory function in mammals infected with SARS-CoV-2, asthma and exacerbated bronchial reactivity.

[0042] In a particular embodiment, the dose of phytoecdysones administered in the form of an inclusion complex with at least one cyclodextrin is between 0.1 and 2 milligrams per kilogram per day in humans. Phytoecdysone is understood here to mean both phytoecdysones in general, in particular 20-hydroxyecdysone and preferably in extract form, and semisynthetic derivatives of 20-hydroxyecdysone.

[0043] Preferably, the phytoecdysones in the form of an inclusion complex with at least one cyclodextrin are administered at a dose of phytoecdysones of 5 to 200 mg / day, in one or more doses, in an adult human, and a dose of 0.2 to 100 mg / day, in one or more doses, in human children or infants. Phytoecdysone is understood here to mean both phytoecdysones in general, in particular 20-hydroxyecdysone and preferably in extract form, and semi-synthetic derivatives of 20-hydroxyecdysone.

[0044] In particular embodiments of the present invention, said at least one semi-synthetic derivative of 20-hydroxyecdysone is chosen from: - a compound of general formula (III): [Chem. 3] in which: • R 1 is chosen from: a (Ci-C6)W(Ci-C6) group; a (Ci -C6)W(C1-C6)W(C1-C6) group; a (C1-C6)W(C1-C6)CO2(C1-C6) group; a (Ci-C6)A group, A representing a heterocycle optionally substituted by a group of the OH, OMe, (Ci-C6), N(Ci-C6), CO2(Ci-C6) type; a CH2Br group; W being a heteroatom chosen from N, O and S, preferably O and even more preferably S; and, - a compound having the formula (IV): [Chem. 4] (IV)

[0045] In the context of the present invention, the term "(Ci-C6)" means any alkyl group of 1 to 6 carbon atoms, linear or branched, in particular, the methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl groups. Advantageously, it is a methyl, ethyl, iso-propyl or t-butyl group, in particular a methyl or ethyl group, more particularly a methyl group.

[0046] In the context of the present invention, the term heterocycle preferably means a cycle comprising 5 or 6 atoms including one or two heteroatoms (O, S or N), the remaining atoms being carbon atoms.

[0047] In a preferred embodiment of the present invention, in general formula (I): - R x is chosen from: a (Ci-C6)W(Ci-C6) group; a (Ci -C6)W(C1-C6)W(C1-C6) group; a (C1-C6)W(C1-C6)CO2(C1-C6) group; a (Ci-C6)A group, A representing a heterocycle optionally substituted by a group of the OH, OMe, (Ci-C6), N(Ci-C6), CO2(Ci-C6) type; W being a heteroatom chosen from N, O and S, preferably O and more preferably S.

[0048] In particular embodiments of the present invention, said at least one semi-synthetic derivative of 20-hydroxyecdysone is a compound chosen from the following compounds: No. 1: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-17-(2-morpholinoacetyl)-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]p henanthren-6-one, No. 2: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(3-hydroxypyrrolidin-l-yl)acetyl]-10,13-dimethyl-2,3,4,5,9,ll,12,15,16,17-decahydro-lH-cyclopenta[a]phenanthren-6-one; No. 3: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(4-hydroxy-l-piperidyl)acetyl]-10,13-dimethyl-2,3,4,5,9,11, 12,15,16,17-decahydro-1H-c y clopenta[a] phenanthren-6-one; No. 4: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-[4-(2- hydroxyethyl)-l-piperidyl]acetyl]-10,13-dimethyl-2,3,4,5,9,ll,12,15,16,17-decahydro-lH-cyclopenta[a]phenanthren-6-one; - No. 5: (2S,3R,5R,10R,13R,14S,17S)-17-[2-(3-dimethylaminopropyl (methyl)amino)acetyl]-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5, 9,11,12,15,16,17-decahydro- lH-cyclopenta[a]phenanthren-6-one; No. 6: 2-[2-oxo-2-[(2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-6-oxo-2,3,4,5,9,11,12,15,16,17-decalihydro-1H-cyclopenta[a]phenanthrene-17-yl]ethyl]sulfanylacetate; No. 7: (2S,3R,5R,10R,13R,14S,17S)-17-(2-ethylsulfanylacetyl)-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro- 1H-cyclopenta[a]phenanthren-6-one; No. 8: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(2- hydroxyethylsulfanyl)acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro- 1H cyclopenta[a]phenanthren-6-one.

[0049] In embodiments, the composition which is the subject of the present invention is a pharmaceutical composition containing, as active ingredient, said at least one phytoecdysone and / or said at least one semi-synthetic derivative of 20-hydroxyecdysone, in a pharmaceutically acceptable vehicle.

[0050] In the present description, the term "pharmaceutically acceptable vehicle" means any vehicle useful for the preparation of a pharmaceutical composition and which is generally safe, non-toxic and neither biologically nor otherwise undesirable for the subject to be treated, in particular for mammals and in particular humans.

[0051] The vehicle of the pharmaceutical composition according to the invention can be solid, semi-solid or liquid. It can be a diluent, an adjuvant or any other vehicle conventional in itself for the constitution of pharmaceutical compositions.

[0052] The pharmaceutical composition according to the invention is presented in a form suitable for administration by intranasal route but can also be presented in any galenic form, in particular in a form suitable for administration by parenteral, rectal, pulmonary, intrathecal, systemic or topical route.

[0053] Any conventional pharmaceutically acceptable salt of the compound of general formula (III) may be used according to the invention. Examples that may be mentioned are chlorides, bromides, formates, acetates, etc.

[0054] In the present description, the term "pharmaceutically acceptable salt" is understood to mean, in a conventional manner in itself, any salt of the compound of general formula (III) comprising, as counterion, a substance which does not produce any adverse, allergic or otherwise undesirable reaction when administered to a subject, in particular to a mammal.

[0055] The composition according to the invention, in its desired form, can be prepared by any conventional method in itself for the preparation of pharmaceutical compositions.

[0056] The composition according to the invention may contain one or more conventional excipients / additives in themselves for the constitution of pharmaceutical compositions, for example chosen from preservatives, sweetening agents, flavoring agents, fillers, disintegrants, wetting agents, emulsifiers, surfactants, dispersants, lubricants, stabilizers, buffers, antibacterials, antifungals, etc., or any of their mixtures; and / or any compound allowing rapid, prolonged or delayed, and / or targeted, release of the active ingredient after its administration to the subject.

[0057] According to a particular embodiment, the composition which is the subject of the present invention may further contain one or more compounds other than phytoecdysones or semi-synthetic derivatives of 20-hydroxyecdysone and cyclodextrins, these compounds having the capacity to improve the membrane permeability of 20-hydroxyecdysone and its residence time on the nasal mucosa. Such compounds may be chosen, for example, from polymers, surfactants, lectins, gelatin, alginates, starches, lecithins, penetratins, hyaluronic acid and chelating agents.According to a preferred embodiment, these compounds are chosen from chitosan, pluronic acid, poloxamer 407, poloxamer 188, poly-L-arginine, polyurethane, polyacrylic acids, carbopol 934, sodium deoxycholate, sodium glycodeoxycholate, sodium glycocholate, sodium taurocholate, dioctylsulfosuccinate, tweens, ethylenediaminetetraacetic acid, zonula occludens toxins (ZOT).

[0058] The composition which is the subject of the present invention is preferably formulated in the form of unit doses. Administration to the mammal can thus be carried out in unit doses.

[0059] The invention is also expressed in terms of a method for treating a pathology, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising at least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone and at least one cyclodextrin in the form of at least one inclusion complex. This method may meet one or more of the characteristics described above with reference to the therapeutic use of the composition comprising at least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone and at least one cyclodextrin in the form of at least one inclusion complex as a medicament.

[0060] In the present description, the term "a subject in need thereof" means a subject affected or likely to be affected by a pathology as described above with respect to the composition which is the subject of the present invention, in particular sarcopenia, loss of muscle strength following immobilization, Duchenne muscular dystrophy, spinal muscular atrophy, impaired respiratory function in patients infected with SARS-CoV-2, exacerbated bronchial reactivity and asthma, or a subject requiring the induction of bronchodilation. This subject may in particular be a mammal, and in particular a human.

[0061] In the present description, the term "therapeutically effective amount" means the amount of the composition administered which, when administered to a subject to treat the disease, is sufficient to ensure such treatment of the disease. The therapeutically effective amount of the composition used according to the invention depends on several factors, such as the disease and its severity, the age, weight, etc., of the subject to be treated, the particular compound(s) of the composition used, the route and form of administration, etc. The therapeutically effective amount of the composition used according to the invention will be determined by the physician for each individual case. The composition may, for example, be administered to the subject in need thereof once, twice, or three times a day, over a long period, at regular intervals, or in a targeted manner.

[0062] The invention is also expressed in terms of a use of a composition comprising at least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone and at least one cyclodextrin in the form of at least one inclusion complex for the manufacture of a medicament for the treatment of a pathology. This use may contain one or more of the characteristics presented above, implemented separately or in each of their technically effective combinations. Brief description of tables and figures

[0063] The invention will be better understood on reading the following description, given by way of non-limiting example, and made with reference to the figures which represent:

[0064] [Fig-1] [Fig.l] illustrates a table showing the limiting solubility values ​​of BIO101 (expressed in mg / mL and molar concentration) when it is in the form of inclusion complexes consisting of BIO101 and a cyclodextrin. The BIO101 / CD molar ratio of saturated solutions is also presented. The cyclodextrins (CD) used have 6, 7 or 8 links and are called a-CD, [3-CD or y-CD respectively. The CDs have glucopyranose links in positions 2, 3 and 6, either free or substituted hydroxyls. BIO101 is added in excess to aqueous solutions of cyclodextrins at different concentrations (2%, 5% or 10% w / v). Stirring for 1 h at room temperature, then centrifugation and determination of the concentration of BIO101 solubilized in the supernatant by LC / MS.

[0065] [Fig-2] [Fig.2] illustrates in the form of graphs the maximum solubilities values of BIO101 from the table in [Fig. 1]. These represent respectively the maximum solubility values ​​of BIO101 expressed in mg / mL (graph A) and in molar concentration (graph B) when it is in the form of an inclusion complex with different cyclodextrins at concentrations of 2%, 5% and 10% w / v.

[0066] [Fig.3] The values ​​of the complexation constants between BIO101 and different cyclodextrins at concentrations of 2%, 5% and 10% w / v are shown in graph A of [Fig.3]. The values ​​of the molar ratios, which correspond to the number of BIO101 molecules complexed per cyclodextrin molecule (BIO101 / CD), are shown in graph B of [Fig.3].

[0067] [Fig.4] [Fig.4] is a graph grouping the pharmacokinetic profiles of BIO101 in rat plasma following a single oral administration of BIO101 solubilized in pure water at a dose of 50 mg / kg, or intranasal administration of BIO101 solubilized in pure water at a dose of 1.5 mg / kg or in the form of an inclusion complex with HPBCD or RAMEB at a dose of 10 mg / kg. Plasma extraction was performed by protein precipitation with methanol, followed by determination of the BIO101 concentration by LC / MSMS with Cyasterone as internal standard. Description of the embodiments

[0068] In the present description, “n” corresponds to the sample size.

[0069] 1. Description of the solubility study of BIP 101 in aqueous solution at different pH.

[0070] As a reminder, BIO101 is a known preparation of 20-hydroxyecdysone (20E) with a purity greater than or equal to 97%. BIP 101 is currently known to be administered orally to mammals.

[0071] The solubility tests were carried out in water as follows: 100 mg of BIP101 were added to 2 ml of water, in n=2. Several stirring conditions were tested: • Magnetic stirring with magnetic bar, 1 hour at room temperature (25°C), • Magnetic stirring with magnetic bar, 24 hours at room temperature (25°C), • Magnetic stirring with magnetic bar, 1 hour on hotplate at 50°C, • Magnetic stirring with magnetic bar, 24 hours on hot plate at 50°C, • Ultrasonic bath, 1 hour at room temperature (25°C).

[0072] After stirring, the solutions were centrifuged for 10 minutes at 15,800 g and all presented a pellet, demonstrating saturation in BIO101. The supernatants were diluted to 1 / 10,000 in purified water. Two samples of each solution were assayed by LC / MSMS, with a calibration of BIO101 from 500 to 10,000 ng / mL and Cyasterone as internal standard. The results of maximum solubility of BIO101 according to the different dissolution conditions are presented in Table 1 below: [Tables 1] [B1O101] (mg / ml) Magnetic stirring, lh, TA ILS + 0.2 Magnetic stirring, Ih, 50®C 11.7+0.5 Magnetic stirring, 24h, TA 13.2 + 0.2 Magnetic stirring, 24b, 5TC 10.8 ± 0.3 Ultrasonic, 1b 1LÜ + 0.1

[0073] The tests with magnetic stirring for 1 hour at room temperature were repeated with adjustment of the pH of the solutions to pH 1.2 using a hydrochloric acid solution, and pH 4.5, 6.8 and 7.5, using a Britton-Robinson buffer and sodium hydroxide. The maximum solubility values ​​of BIO101 at the different pHs are presented in Table 2 below: [Tables2] 15101011 (mg / mL) pHL2 they ± ai pH 4.5 11.6+0.5 pH 6.8 10.5 ± 0.2 pH 7.5 13.5 ±3.8

[0074] In each of the conditions tested, the concentration of BIO101 (and therefore of 20E) is greater than 11 mg / mL. The concentrations of BIO101 (20E) measured in each of the conditions are compared to the maximum quantity of BIO101 administered daily to humans (700 mg, Dioh et al. 2023) in a volume of 250 mL, i.e. 2.8 mg / mL. This concentration is significantly higher than the maximum quantity of BIO101 administered daily to humans (700 mg) in a volume of 250 mL, i.e. 2.8 mg / mL. BIO101 (20E) is therefore considered to be very soluble in aqueous solutions with a pH between 1.2 and 7.5.

[0075] 2, Description of the study allowing experimental determination of the value of LogPdeBIOlOl (BIO101).

[0076] A preliminary study has determined that the limiting solubility values ​​of BIO101 (20E) after stirring for 24 hours at 20°C in octanol pre-saturated with water and in water pre-saturated with octanol are respectively 8.5 and 9.5 mg / mL. The Log P value is determined experimentally (OECD 107 guidance) after magnetic stirring overnight at 20°C in a binary octanol / water system. For this, 3 different volume ratios of a BIO101 octanol solution (pre-saturated with water) and water (pre-saturated with octanol) are chosen. For each of the 6 samples (3 different volume ratios passed in duplicate, 1 / 1, 1 / 2, and 2 / 1), the separation of the two phases (octanol and aqueous medium) is carried out by centrifugation. Each isolated phase is then diluted in a solvent mixture allowing its injection into a chromatographic system. The concentration in solution of each separated and analyzed phase is determined by HPLC with external standardization.

[0077] Six Log P values ​​are calculated: 2 per octanol / water ratio (1 / 1, 1 / 2 and 2 / 1). The six values ​​obtained (-0.05; -0.03; -0.03; -0.05; -0.05 and -0.04) are consistent in terms of variability with the OECD 107 guidance. The measured Log P value of BIO101 (20E) is -0.04 ±0.01.

[0078] 3. Description of the in vitro permeability study of BIO101 on monolayer of intestinal epithelial cells (CaCo-2 TC7).

[0079] Caco-2 TC7 cells are commonly used as an in vitro model to predict the intestinal permeability of compounds, due to their ability to form tight junctions mimicking the intestinal barrier in vivo. In this study, the apparent permeability (Papp) of BIO101 (20E) across a Caco-2 TC7 cell monolayer in the apical to basolateral direction (absorption direction) and in the basolateral to apical direction (secretion direction) was evaluated using a 2-hour kinetic study. Before and after the transport experiment, transepithelial electrical resistance (TEER) values ​​were checked and cell layers with initial TEER values ​​lower than 600 Q were discarded. The concentrations of BIO101 (20E) in the different compartments (apical and basolateral) were determined by LC-MS / MS with external standardization.BIO101 (20E) demonstrates very low transepithelial flux values ​​(Table 3 below). The Papp xlO6 values ​​are 0.22 and 0.40 from the apical pole to the basolateral pole and from the basolateral pole to the apical pole, respectively. The values ​​of these fluxes are not significantly different from each other, which excludes an efflux phenomenon towards the intestinal lumen.

[0080] Metoprolol, which is a biopharmaceutical class I compound (BCS class I, good solubility / good permeability) was used as a control compound. metoprolol demonstrates flux values ​​approximately 50 times higher than those measured for BIOlOl (20E). [Tables 3] Apical to eral Basolat Basolateral to a pical Compound Ratio Papp x106 Papp x106 BA / AB BIO101 0.22 0.40 1.62 Metoprolol 11.3 21.6 1.90

[0081] In view of its good solubility and its favorable dissolution profile in aqueous solution (Tables 1 and 2), its Log P value, its very low oral bioavailability and its low permeability of the intestinal epithelium (Table 3), BIO101 (20E) belongs to biopharmaceutical class III (BCS class III).

[0082] 4. Description of the limit solubility study of BIO101 complexes with different cyclodextrins ([Fig. 1 ] )

[0083] Solutions of cyclodextrins in water are prepared at 2%, 5% and 10% w / v in 5 mL vials. Due to the low solubility of [3-CD in water (<20 g / L), the solutions for this cyclodextrin are prepared at 0.5%, 1% and 1.5% w / v. BIO101 is then added in excess to the different cyclodextrin solutions. The solutions are stirred using a magnetic bar for 1 hour at room temperature (25°C).

[0084] After stirring, the solutions are centrifuged for 10 min at 15,800 g. The supernatants are collected and the concentration of BIO101 in solution is determined by LC / MSMS.

[0085] [Fig.l] is a table showing the limiting solubility values ​​of BIO101 (expressed in mg / mL and molar concentration) when it is in the form of inclusion complexes consisting of BIO101 and a cyclodextrin. The BIO101 / CD molar ratio of the saturated solutions is also shown. The cyclodextrins (CD) used have 6, 7 or 8 links and are called α-CD, γ-CD or γ-CD respectively. The CDs have glucopyranose links in positions 2, 3 and 6, either free or substituted hydroxyls. BIO101 is added in excess to aqueous solutions of cyclodextrins at different concentrations (2, 5 or 10% w / v). In the table in [Fig.l], a-CD is an unsubstituted alpha cyclodextrin; RAMEA stands for random-methylated-a-cyclodextrin; HPACD is a hydroxypropyl-a-cyclodextrin; [3-CD is an unsubstituted beta cyclodextrin; RAMEB stands for random-methylated-[3-cyclodextrin; HPBCD is a hydroxypropyl-[3-cyclodextrin; SBECD is a sodium salt of sulfobutylated-[3-cyclodextrin; DIMEB is a Heptakis(2,6-di-O-methyl)-[3-cyclodextrin; y- . CD is an unsubstituted gamma cyclodextrin; RAMEG stands for random-methylated-y-cyclodextrin; HPGCD is a hydroxypropyl-y-cyclodextrin.

[0086] The limiting solubility values ​​of BIO101 in the presence of 2%, 5% and 10% w / v of α-CDs (composed of 6 glucopyranose members) are very modestly improved compared to the maximum solubility of BIO101 alone in water. The best limiting solubility of BIO101 obtained with an alpha-type cyclodextrin is 14.2 mg / mL in the presence of 5% HPACD. The same is true for unsubstituted [3-CD] whose intrinsic solubility does not allow [3-CD] concentrations greater than 1.5% w / v to be reached. In this case, the limiting solubility of BIO101 is 13.9 mg / mL in the presence of 1.5% w / v of [3-CD]. In contrast, the limiting solubility of BIO101 in the presence of substituted [3-CDs] increases very significantly compared to that obtained in pure water or in the presence of unsubstituted [3-CDs]. For 10% w / v solutions of SBECD, HPBCD, RAMEB and DIMEB, the limiting solubility values ​​of BIO101 are 21.4, 32.0, 39.6 and 42.8 mg / mL, respectively.

[0087] When cyclodextrins comprising 8 glucopyranose links (y-CD) are used, the solubilization of BIO101 is very significantly improved, whether the cyclodextrin is substituted (methyl or hydroxypropyl groups) or not (unsubstituted y-CD). Indeed, at 2%, 5% and 10% w / v of y-CD, RAMEG and HPGCD the limiting solubility values ​​of BIO101 are respectively 16.6, 15.8, 17.6 mg / mL; 25.0, 22.8, 25.2 mg / mL; 37.5, 35.8, 38.5 mg / mL.

[0088] The maximum solubility values ​​of BIO101 at different CD concentrations are shown in [Fig.2]. Graph A of [Fig.2] represents the maximum concentration of BIO101 (mg / mL) solubilized in the absence of CD (0%) as well as in 2, 5 and 10% w / v CD solutions.

[0089] If the a-CDs (a-CD, RAMEA and HPACD) and the unsubstituted [3-CD (|3-CD) do not present any convincing advantage in terms of solubilization of BIO101, the implementation of the substituted [3-CDs (SBECD, RAMEB, HPBCD and DIMEB) as well as that of the substituted y-CDs (RAMEG, HPGCD) or not (y-CD) have an obvious interest. If we exclude SBECD which is the least effective of the substituted [3-CDs, concentrations of 10% w / v of the latter CDs make it possible to improve the limiting solubility of BIO101 by 2.9 to 3.9 times.

[0090] Graph B represents the maximum molar concentration (mol / L) of BIO101 solubilized in the absence of CD (0 mol / L) as well as in CD solutions whose concentrations are expressed in molar concentrations (mol / L) corresponding to CD solutions at 2%, 5% and 10% w / v.

[0091] 5. Description of the characteristics of the inclusion complexes formed between BIO101 and different cyclodextrins ([Fig.3]).

[0092] When a compound forms a complex with a cyclodextrin molecule, an AL type diagram (Higuchi & Connors 1965) is obtained with a linear relationship, and the solubility of the molecule can be expressed according to the following formula (Jambhekar & Breen 2016; Gazpio et al. 2005): [Math. 1] in which St represents the solubility of the compound, So its solubility in the absence of cyclodextrin, Ki i the complexation constant, and [CD] the cyclodextrin concentration.

[0093] The slope and the ordinate at the origin (oo) of the curve obtained correspond to the following formula (Jambhekar & Breen 2016; Gazpio et al. 2005): [Math. 2] oo = So

[0094] The complexation constant can thus be deduced according to the following formula (Jambhekar & Breen 2016; Gazpio et al. 2005): [Math. 3] Slope Ki -î--------------- oo (1 -slope)

[0095] The complexation constants of BIO101 with the different CDs used are represented in graph A of [Fig.3].

[0096] The values ​​of the complexation constants confirm on the one hand the absence of interest of the a-CDs (a-CD, RAMEA and HPACD) and on the other hand the advantage that certain substituted [3-CDs (RAMEB, DIMEB) and all the y-CDs tested, whether substituted or not (y-CD, RAMEG, HPGCD) constitute for the formation of inclusion complexes with BIO101.

[0097] The molar ratios, which correspond to the number of molecules of BIO101 complexed per molecule of cyclodextrin, are calculated according to the following equation: [Math. 4] [BlOWIHmcm Rat© mo atre - —' [CD] (mol / L) and are represented in graph B of [Fig.3].

[0098] The values ​​of the molar ratios (BIO101 / CD) confirm on the one hand the absence of interest of the a-CDs (a-CD, RAMEA and HPACD) and on the other hand the advantage that constitute some [substituted 3-CDs (RAMEB, DIMEB) and all tested γ-CDs, whether substituted or not (γ-CD, RAMEG, HPGCD) for the formation of inclusion complexes with BIO101.

[0099] 6. Description of the manufacture of inclusion complexes of BIP 101 with HPBCD and RAMEB for use in pharmacokinetic experiments.

[0100] BIO101 was complexed with HPBCD. An equimolar mixture of BIO101 and HPBCD was solubilized in 540 mL of methanol (MeOH). The mixture was stirred at room temperature for 15 minutes and then concentrated to dryness by evaporation. This solubilization then evaporation step is repeated 3 times and then the glaze on the wall of the flask is taken up in water for lyophilization to obtain 11.3 g of white solid. The cyclodextrin / BIO101 ratio is determined by LCMS (here: 250.5 mg of BIO101 in 1 g of complex).

[0101] BIP 101 was complexed with RAMEB. An equimolar mixture of BIP 101 and RAMEB was solubilized in 400 mL of methanol (MePH). The mixture was stirred at room temperature for 15 minutes and then concentrated to dryness by evaporation. This solubilization then evaporation step is repeated 3 times and then the glaze on the wall of the flask is taken up in water for lyophilization to obtain 12.5 g of white solid. The cyclodextrin / BIP101 ratio is determined by LCMS (here: 267.5 mg of BIP 101 in 1 g of complex).

[0102] 7. Description of the plasma exposure study of BIP 101 following a single oral administration of BIP101 solubilized in water or in the form of inclusion complexes with different cyclodextrins ([Fig.4] and Table 4).

[0103] The pharmacokinetic study of BIP101 following oral administration was performed using male Wistar rats (Janvier Labs, 53940 Le Genest Saint Isle, France). BIP101 was administered at a dose of 50 mg / kg body weight alone (BIP 101 alone) or complexed with HPBCD (BIP 101-HPBCD) or complexed with RAMEB (BID101-RAMEB). After administration, blood was collected from the tail at t = 0.25 h; 0.5 h; 1 h; 2 h; 4 h; 6 h and 8 h; 10 h; 12 h; 24 h. Blood samples were centrifuged and plasmas collected. The dosage of plasma samples allowed the determination of the AUC: the area under the curve which corresponds to plasma exposure.

[0104] For the quantification of BIP 101, a calibration curve is carried out in the plasma with 10 standards (10 to 10000 ng / mL) and Cyasterone as internal standard.

[0105] The LC-MSMS analysis is carried out with a 1260 Infinity HPLC chain (Agilent Technologies, Santa-Clara, USA), and a QQQ6420 mass spectrometer (Agilent Technologies Santa-Clara, USA). The injection volume is 5 pL. BIP101 is eluted on a C18 reverse phase column (2.1*50 mm, 3 pm particles; Ace-C18-Excel, AIT) with a gradient of acetonitrile and water (containing 0.1% acid formic acid) and a flow rate of 0.3 mL / min. The mass spectrometer analyzes in MRM Mode - Positive. [Tables 4] ; BiOl Ci sed Bl 01 û; -HPBCD 810101 -RAMEB AUÇ^-(ng*h / mL) 293 ±27 2S0±49 268 ±20

[0106] Complexation of BIO101 with HPBCD or RAMEB does not increase its bioavailability upon oral administration.

[0107] 8. Description of the plasma exposure study of BIP 101 following a single intranasal administration of BIO101 solubilized in water or in the form of inclusion complexes with different cyclodextrins ([Fig.4] and Table 5).

[0108] The pharmacokinetic study of BIO101 following its intranasal administration was carried out using male Wistar rats (Janvier Labs, 53940 Le Genest Saint Isle, France). BIP 101 was administered intranasally after isoflurane anesthesia (2% for 3 minutes), in 50 μL at a dose of 1.5 mg / kg body weight when solubilized in pure water and at 10 mg / kg body weight when in the form of an inclusion complex, either complexed with HPBCD (BIO101-HPBCD) or complexed with RAMEB (BIO101-RAMEB).

[0109] After administration, blood was collected from the tail at t = 0.25 h; 0.5 h; 1 h; 2 h; 4 h; 6 h; 8 h; 10 h; 12 h; and 24 h post administration. The blood samples were centrifuged and the plasmas collected (n = 10 for each collection time). The dosage of the plasma samples allowed the determination of the pharmacokinetic parameters, namely the Cmax, which corresponds to the maximum concentration observed after administration of the molecule, the Tmax which is the time required to reach the maximum concentration after administration of the molecule and the AUC: Fit under the curve which corresponds to the plasma exposure ([Fig.4] and Table 5).

[0110] For the quantification of BIO101, a calibration curve is carried out in the plasma with 10 standards (10 to 10000 ng / mL) and Cyasterone as internal standard.

[0111] The LC-MSMS analysis is performed with a 1260 Infinity HPLC chain (Agilent Technologies, Santa-Clara, USA), and a QQQ6420 mass spectrometer (Agilent Technologies Santa-Clara, USA). The injection volume is 5 pL. BIO101 is eluted on a C18 reverse phase column (2.1*50 mm, 3 pm particles; Ace-C18-Excel, AIT) with a gradient of acetonitrile and water (containing 0.1% formic acid) and a flow rate of 0.3 mL / min. The mass spectrometer analyzes in MRM Mode - Positive.

[0112] When BIO101 is administered alone (1.5 mg / kg solubilized in pure water) intranasally, a Cmax = 53 ng / mL, a Tmax = 0.5 h and an AUC() 8h = 119 ng.h / mL are observed. Intranasal administration of BIO101 complexed with HPBCD (BIO101-HPBCD) allows a Cmax = 152 ng / mL, a Tmax = 0.5 h and an AUC0_8h = 370 ng.h / mL to be achieved. Finally, during intranasal administration of BIO101 complexed with RAMEB (BIO101-RAMEB) we observe a Cmax = 349 ng / mL, a Tmax = 0.5 h and an AUCo-8h = 902 ng.h / mL ([Fig.4] and Table 5).

[0113] Thus, for the same dose of BIO101 (10 mg / kg), the complexation of BIO101 with RAMEB allows, at the plasma level, to expose 2.4 times more the animals treated than those which were treated with BIO101 complexed with HPBCD.

[0114] Furthermore, and remarkably, when comparing the plasma exposures of animals having received BIO101 orally at 50 mg / kg with those of animals having received BIO101 at 10 mg / kg complexed with RAMEB, it is observed that by reducing the dose of BIO101 administered by a factor of 5 (from 50 mg / kg orally to 10 mg / kg IN), the plasma exposure is multiplied by 3.1 times.

[0115] 9. Description of the central nervous system exposure study (brain tissues and cerebrospinal fluid) to BIO101 following a single intranasal administration of BIO101 solubilized in water or in the form of inclusion complexes with different cyclodextrins ([Fig.4] and Table 5).

[0116] When BIO101 was administered intranasally as a BIO101-RAMEB inclusion complex (10 mg BlOlOl / kg), the brain and cerebrospinal fluid (CSF) were also collected to assess potential passage of BIO101 to the central nervous system. CSF was collected 0.5 h, 1 h, 2 h, 6 h and 24 h after administration of BIO101-RAMEB in pentobarbital-anesthetized animals. Following CSF collection, the brain of each animal (n=10) was dissected after decapitation.

[0117] CSFs were prepared for analysis by half-dilution with methanol. Brains were lyophilized and then extracted by grinding them in a 50 / 50 methanol / water mixture.

[0118] For the quantification of BIO101, a calibration curve is carried out in water with 10 standards (10 to 10000 ng / mL) and Cyasterone as internal standard.

[0119] The LC-MSMS analysis is performed with a 1260 Infinity HPLC chain (Agilent Technologies, Santa-Clara, USA), and a QQQ6420 mass spectrometer (Agilent Technologies Santa-Clara, USA). The injection volume is 5 pL. BIO101 is eluted on a C18 reverse phase column (2.1*50 mm, 3 pm particles; Ace-C18-Excel, AIT) with a gradient of acetonitrile and water (containing 0.1% formic acid) and a flow rate of 0.3 mL / min. The mass spectrometer analyzes in MRM Mode - Positive. [Tables 5] BÎOWT Sëb! ■?,5 BIOWLHPBCD BSQWLRAMEB Ï4J .'KÿA'g Plasma 119 370 902" AUCtm (nçfhtmL) Cerebral tissue ND ND < LLOQ CSF ND ND < LLOQ *Mean plasma exposure value obtained during two separate experiments carried out under the same conditions. ND: not determined. < LLOQ: value lower than the lowest concentration in the calibration range: < 10 ng / mL for CSF and < 150 pg / mg for brain tissue.

[0120] Interestingly, the quantification of BIO101 in brain tissue and at the CSF level is below the limit of quantification (Table 5), indicating that complexation with RAMEB does not induce passage of BIO101 to the CNS.

[0121] 10. Description of the plasma and central nervous system exposure study (brain tissue and CSF) to BIO101 and two major metabolites of BIO101 following single or repeated intranasal administration of BIO101 solubilized in water or as an inclusion complex with a cyclodextrin (Table 6).

[0122] Plasma exposure, as well as brain tissue and CSF exposure, to BIO101 and two major metabolites of BIO101 (14d20E and 14dPost) following single or repeated (once daily for 7 days) intranasal administration of BIO101 in the form of the inclusion complex BIO101-RAMEB at 10 mg BlOlOl / kg was determined by integration of the pharmacokinetic profiles using Graphpad Prism software.

[0123] A slight increase in plasma exposure to BIO101 was observed when BIO101-RAMEB was administered intranasally repeatedly (once a day for 7 days) compared to a single administration (1300 ng*h / mL versus 902 ng*h / mL; Table 6). Under both conditions, neither of the two metabolites investigated was quantifiable ( <LLOQ).

[0124] At the level of brain tissue and in the CSF, neither BIO101 nor the two metabolites sought (14d20E and 14dPost) are found after chronic administration of 7 days (Table 6).

[0125] These results demonstrate that repeated administration of BIO101 complexed with RAMEB does not expose the CNS of animals that have been chronically treated and that the major metabolites of BIO101 are not formed. [Tableauxô] Pissma Tissa cerebral LCR 1300 < LLOQ C LLOQ 14Œ < LLOQ < LLOQ < LLOQ. < LLOQ < LLOQ < LLOQ Pfes???® and LŒ / Tfsea cé / efo-a? : pgWàg < LLOQ: No AUC calculated, measured concentrations are below the lowest concentration in the calibration range. Plasma: < 10 ng / mL for 14d20E and < 25 ng / mL for 14dPost. CSF: < 10 ng / mL for all analytes. Brain tissue: < 150 pg / mg for all analytes.

[0126] Bibliographic references

[0127] Dinan L, Balducci C, Guibout L, Foucault AS, Bakrim A, Kumpun S, Girault JP, Tourette C, Dioh W, Dilda PJ, Veillet S, Lafont R. Ecdysteroid metabolism in mammals: The fate of ingested 20-hydroxyecdysone in mice and rats. J Steroid Biochem Mol Biol. 2021 Sep;212:105896. Doi: 10.1016 / j.jsbmb.2021.105896. Epub 2021 Apr 2. PMID: 33819630.

[0128] Dioh W, Tourette C, Del Signore S, Daudigny L, Dupont P, Balducci C, Dilda PJ, Lafont R, Veillet S. A Phase 1 study for safety and pharmacokinetics of BIO101 (20-hydroxyecdysone) in healthy young and older adults. J Cachexia Sarcopenia Muscle. 2023 Jun; 14(3): 1259-1273. Doi: 10.1002 / jcsm.l3195. Epub 2023 Apr 13. PMID: 37057316; PMCID: PMC10235879.

[0129] Gazpio C, Sanchez M, Garcla-Zubiri IX, Vélaz I, Martinez-Ohârriz C, Martin C, Zornoza A. HPLC and solubility study of the interaction between pindolol and cyclodextrins. J Pharm Biomed Anal. 2005 Mar 9;37(3):487-92. Doi: 10.1016 / j.jpba.2004.11.008. Epub 2004 Dec 10. PMID: 15740908.

[0130] Higuchi T, Connors K. A., “Phase Solubility Techniques,” Advanced Analytical Chemistry of Instrumentation, Vol. 4, 1965, pp. 117-212.

[0131] Jambhekar SS, Breen P. Cyclodextrins in pharmaceutical formulations I: structure and physicochemical properties, formation of complexes, and types of complex. Drug Discov Today. 2016 Feb;21(2):356-62. Doi: 10.1016 / j.drudis.2015.11.017. Epub 2015 Dec 11. PMID: 26686054.

[0132] Kumpun S, Girault JP, Dinan L, Blais C, Maria A, Dauphin-Villemant C, Yingyongnarongkul B, Suksamrarn A, Lafont R. The metabolism of 20-hydroxyecdysone in mice: relevance to pharmacological effects and gene switch applications of ecdysteroids. J Steroid Biochem Mol Biol. 2011 Aug;126(l-2):l-9. Doi: 10.1016 / j.jsbmb.2011.03.016. Epub 2011 Mar 23. PMID: 21439380.

[0133] Merkus FW, Verhoef JC, Marttin E, Romeijn SG, van der Kuy PH, Hermens WA, Schipper NG. Cyclodextrins in nasal drug delivery. Adv Drug Deliv Rev. 1999 Mar l;36(l):41-57. Doi: 10.1016 / s0169-409x(98)00054-4. PMID: 10837708.

[0134] Ras su G, Sorrenti M, Catenacci L, Pavan B, Ferraro L, Gavini E, Bonferoni MC, Giunchedi P, Dalpiaz A. Versatile Nasal Application of Cyclodextrins: Excipients and / or Actives? Pharmaceutics. 2021 Jul 30 ;13(8) :1180. Doi: 10.3390 / pharmaceutics 13081180. PMID: 34452141; PMCID: PMC8401481.

Claims

Claims

1. Composition comprising at least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone and at least one cyclodextrin, in the form of at least one inclusion complex, for its use in a form suitable for intranasal administration in the treatment or prevention of a pathology in mammals.

2. Composition for use according to claim 1, wherein said cyclodextrin is chosen from cyclodextrins comprising 7 or 8 glucopyranose links linked by (a-1,4) bonds.

3. A composition for use according to claim 2, wherein the cyclodextrin is a cyclodextrin composed of 8 glucopyranose links linked by (a-1,4) bonds.

4. A composition for use according to claim 2 or 3, wherein at least one of the hydroxyls in positions 2, 3 and 6 of the glucopyranose links of the cyclodextrin is substituted.

5. A composition for use according to claim 4, wherein said at least one of the hydroxyls in positions 2, 3 and 6 is substituted by a methyl group or said at least one of the hydroxyls in positions 2, 3 and 6 is substituted by a 2-hydroxypropyl group.

6. A composition for use according to any one of claims 1 to 5, wherein the pathology is selected from sarcopenia, loss of muscle strength following immobilization, Duchenne muscular dystrophy, spinal muscular atrophy, impaired respiratory function due to SARS-CoV-2 infection, exacerbated bronchial reactivity and asthma.

7. Composition for use according to any one of claims 1 to 6, comprising 20-hydroxyecdysone.

8. Composition for use according to claim 7, wherein the 20-hydroxyecdysone is included in an extract of a plant or a part of a plant, said plant being chosen from plants containing at least 0.5% of 20-hydroxyecdysone by dry weight of said plant, said extract comprising at least 95%, and preferably at least 97%, of 20-hydroxyecdysone.

9.

10.

11. Composition for its use according to claim 8, remarkably comprising impurities in individual proportions of between 0 and 0.5% by dry weight of the extract. Composition for its use according to any one of claims 8 to 9, in which the plant is chosen from Stemmacantha carthamoides, Cyanotis arachnoidea, Cyanotis vaga, Pfaffia glomerata and Pfaffia paniculata. Composition for use according to any one of claims 1 to 10, in which said at least one semisynthetic derivative of 20-hydroxyecdysone is chosen from: - a compound of general formula (III): [Chem. 3] (NEITHER) in which: • R 1 is chosen from: a (Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-C6)W(Ci-C6) group; a (C i-C6)W(Ci-C6)CO2(Ci-C6) group; a (Ci-C6)A group, A representing a heterocycle optionally substituted by a group of the OH, OMe, (Ci-C6), N(Ci-C6), CO2(Ci-C6) type; a CH2Br group; W being a heteroatom chosen from N, O and S, preferably O and even more preferably S; and, - a compound having the formula (IV): (IV)

12. Composition for use according to claim 11, wherein in general formula (I): R x is chosen from: a (Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-C6)CO2(Ci-C6) group; a (Ci-C6)A group, A representing a heterocycle optionally substituted by a group of the OH, OMe, (CrC6), N(CrC6), CO2(CrC6) type; W being a heteroatom chosen from N, O and S, preferably O and more preferably S.

13. Composition for use according to any one of claims 11 to 12, in which the semi-synthetic derivative of 20-hydroxyecdysone is chosen from the following compounds: No. 1: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,1 3-dimethyl-17-(2-morpholinoacetyl)-2,3,4,5,9,11,12,15,16, 17-decahydro-1H-cyclopenta[a]phenanthren-6-one, No. 2: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(3-hydroxypyrrolidin-1-yl)acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; No. 3: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(4-hydroxy-l-piperidyl)acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1 H-cyclopenta[a]phenanthren-6-one; No. 4: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2- [4- (2-hy droxy ethy 1) -1 -piperidy 1] acety 1] -10,13 -dimethyl 1- 2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phena nthren-6-one; No. 5: (2S,3R,5R,10R,13R,14S,17S)-17-[2-(3-dimethylaminopropyl (methyl)amino)acetyl]-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; No. 6: 2-[2-oxo-2-[(2S,3R,5R,10R,13R,14S,17S)-2,3,14-tri hydroxy-10,13-dimethyl-6-oxo-2,3,4,5,9,11,12,15,16,17-de cahydro-lH-cyclopenta[a]phenanthren-17-yl]ethyl]sulfanyl ethyl acetate; No. 7: (2S,3R,5R,10R,13R,14S,17S)-17-(2-ethylsulfanylacetyl)-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,1 6,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; No. 8: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(2-hydroxyethylsulfanyl)acetyl]-10,13-dimethyl-2,3,4,5, 9,11,12,15,16,17-decahydro-1H cyclopenta[a]phenanthren-6-one.

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