CA3165406C - Anti-inflammatory dendrimer formulation for the treatment of psoriasis - Google Patents

Abstract

The present invention relates to sugar-derived catanionic surfactant vesicles comprising anti-inflammatory dendrimers and to their use as a medicament, more particularly in the treatment of psoriasis.

Description

1 Description Title: Formulation of an anti-inflammatory dendrimer for the treatment of psoriasis Technical field

[0001] The present invention relates to vesicles of sugar-derived catanionic surfactants comprising anti-inflammatory dendrimers and their use as a medicinal product, more particularly in the treatment of psoriasis. Prior art

[0002] Psoriasis is a chronic inflammatory skin disease resulting from accelerated epidermal cell turnover fueled by inflammation. It manifests as red plaques covered with white scales that flake off the skin. This disease affects more than 125 million people worldwide and has a significant impact on the quality of life of those affected. Currently, there is no cure for psoriasis, and its causes are numerous and still poorly understood.

[0003] Treatments to alleviate symptoms have been developed, such as topical application of corticosteroids or vitamin D. These treatments relieve mild symptoms of psoriasis but are only marginally effective for treating the most severe forms. The most severe forms are treated with oral immunosuppressants such as cyclosporine, but these have significant side effects. Monoclonal antibodies and soluble receptors targeting pro-inflammatory mediators such as TNF, IL-12, IL-23, and IL-17 have been proposed. These biological drugs are expensive and their effectiveness decreases over time. They are also contraindicated in the case of certain comorbidities associated with psoriasis. 5 10 15 20 25 30 CA 03165406 2022-06-20 2

[0004] There therefore remains a need to develop new treatments for the treatment of psoriasis for topical application, thus limiting side effects.

[0005] Dendrimers are macromolecules composed of monomers combined to form a three-dimensional, multi-branched architecture with a defined structure. Dendrimers have a perfectly defined size and structure thanks to their iterative synthesis. Their multivalence makes them very attractive nano-objects for numerous applications, particularly in biology and medicine.

[0006] In particular, azabisphosphonate surface phosphorus dendrimers (ABP dendrimers) are capable of activating monocytes and inducing an anti-inflammatory response (WO2010/013086, Portevin D. et al. J Transl Med. 2009; 7:82). More specifically, the anti-inflammatory properties have been validated in animal models of rheumatoid arthritis in which monocytes are known to play a key role in inflammation and osteoclastogenesis (Hayder M. et al. 2011; Sci Trans Med. 3(81)). The anti-inflammatory effect of the ABP dendrimer has also been validated in models of uveitis (Fruchon et al. 2013. Molecules;18(8):9305-16) and multiple sclerosis (Hayder M. Biomacromolecules 2015; 16, 3425-3433). However, the potential effect of these dendrimers in the treatment of psoriasis has only been suggested in International Application WO2010/013086. Summary

[0007] The treatment of a dermatosis linked to keratinocyte hyperproliferation and/or an inflammatory context, such as psoriasis, via the cutaneous route requires penetration of the stratum corneum, which alone ensures the skin's barrier function, making its penetration by active ingredients very complex. Thus, to treat psoriasis via the cutaneous route, it is necessary to improve the penetration of active substances into the skin.

[0008] To allow for better skin penetration of the dendrimers, the inventors formulated the aza-bisphosphonate dendrimers with sugar-derived catanionic surfactants. The sugar-derived catanionic surfactants 5 10 15 20 25 CA 03165406 2022-06-20 3 spontaneously associate in the form of vesicles and can encapsulate hydrophobic active ingredients in their membrane bilayer.

[0009] While hydrophilic ABP dendrimers exhibit a low encapsulation rate with sugar-derived surfactants and a solid-to-fluid phase transition temperature of the membrane bilayer of 37°C, the inventors surprisingly discovered that the complex formed by combining the phosphonic acid form of the ABP dendrimer (ABP-OH) with a sugar-derived surfactant results in a decrease in the complex's transition temperature to 33°C. Thus, upon contact with the skin, this complex becomes fluid and is therefore able to diffuse more easily into the deeper layers of the skin, also enhancing its anti-inflammatory action. Furthermore, the encapsulation rate of these phosphonic acid forms is higher than that of hydrophilic ABP dendrimers, and the complexes are stable at a pH close to that of the skin.

[0010] In a first aspect, the invention relates to a vesicle comprising: - a catanionic surfactant of formula (I) or a mixture of catanionic surfactants of general formula (I): in which ( ® J is a sugar, R1 is selected from H and a linear or branched, saturated or unsaturated hydrocarbon chain of 1 to 20 chains, R2 is a linear or branched, saturated or unsaturated hydrocarbon chain of 1 to 20 chains, R3 and R4 are independently of each other a linear or branched, saturated or unsaturated hydrocarbon chain of 1 to 19 chains; and - a dendrimer of formula (II): [Chem. 2] 5 (II) in which the following formulas are selected from the pentoses, hexoses and groups of 10 : , , and, Z is chosen from -CH2- and -CH=N-, R is chosen from H and C1-C12-alkyl, A is chosen from and , where X is chosen from S and O, and 15 n is an integer between 3 and 8.

[0011] According to another aspect, the invention relates to a pharmaceutical composition comprising at least one vesicle according to the invention and a pharmaceutically acceptable excipient. 5 10 15 20 25 Date Recue/Date Received 2024-04-12 5

[0012] According to another aspect, the invention relates to a vesicle according to the invention or a pharmaceutical composition according to the invention for its use in the treatment of psoriasis.

[0013] Finally, the invention relates to a method for preparing a vesicle according to the invention. Brief description of the drawings Fig. 1 [0014] [Fig. 1] represents a diagram illustrating the obtaining of the bioactive formulation. Fig. 2 [0015] [Fig. 2] shows the phase transition temperature for the TriCat 12/ABP formulation. Fig. 3 [0016] [Fig. 3] shows the fluorescence quantification of the quantity of ABP-NIR dendrimer (ABP dendrimer in its fluorescent form), formulated or not with TriCat 12, which penetrated pig ear skin (Franz cells) after 24 h at 40°C. Fig. 4 [0017] [Fig. 4] shows in A, the size distribution measured by DLS of the vesicles formed by the formulation TriCat 12/G1-A (1/0.5), and in B, the scanning electron microscopy image of the vesicles formed by the formulation TriCat 12/G1-A (1/0.5). Fig. 5 [0018] [Fig. 5] shows the phase transition temperature of the formulation TriCat 12/G1-A. Fig. 6 [0019] [Fig. 6] shows the measurement of the mean hydrodynamic diameter and the intensity by DLS of the formulation TriCat 12/G1-A over two months at 4°C. 5 10 15 20 25 CA 03165406 2022-06-20 6 Fig. 7 [0020] [Fig. Fig. 7] shows the measurement of the mean hydrodynamic diameter and the intensity in DLS of the TriCat 12/G1-A formulation over two months at +4°C and -20°C. Fig. 8 [0021] [Fig. 8] shows the fluorescence quantification of the quantity of G1A-NIR dendrimer (G1A dendrimer in its fluorescent form) formulated or not with TriCat 12, which penetrated the skin of pig ear cells (Franz cells) after 24h at 35°C. Fig. 9 [0022] [Fig. Fig. 10 [0023] [Fig. 10] shows the observation by confocal microscopy of the fluorescence (in white) of the unformulated G1-A-NIR dendrimer (left image) and the formulated dendrimer with TriCat 12 (right image) which penetrated pig ear skin (Franz cells) after 24h at 35°C. Fig. 11 [0024] [Fig. 11] shows the flow cytometry analysis of the morphology (size and granulosity) of monocytes (on the graphs, each point is a cell). The top graph shows inactive control monocytes. The bottom left graph shows monocytes cultured in the presence of TriCat 12 vesicles alone (no activation). The bottom right graph shows monocytes cultured with TriCat 12 vesicles loaded with the G1-A dendrimer. Active monocytes are in the ellipse. Fig. 125 10 15 20 25 Date Received 2024-04-12 7 [0025] [Fig. 12] shows the therapeutic efficacy of the G1-A dendrimer and TriCat12 vesicles loaded with the G1-A dendrimer in the murine model of imiquimod-induced psoriasis (IMQ). Clinical scores (top graph) are given as a function of time expressed in days. Histological scores are given on the bottom graph. The "control" mice are the animals that were not treated. Fig. 13 [0026] [Fig. 13] shows the antiproliferative effect of compound G1-A, as a function of treatment time, on the N-TERT keratinocyte line (top graphs) and on primary human keratinocytes (bottom graphs). Fig. 14 [0027] [Fig. 14] shows in A, the size distribution measured by DLS of the vesicles formed by the formulation TriCat 12/G1-B (1/0.5), and in B, the scanning electron microscopy image of the vesicles formed by the formulation TriCat 12/G1-B (1/0.5). Fig. 15 [0028] [Fig. Fig. 15] shows the phase transition temperature of the TriCat 12/G1-B formulation. Fig. 16 [0029] [Fig. 16] shows the measurement of the mean hydrodynamic diameter and the DLS intensity of the TriCat 12/G1-B formulation over two months at 4°C. Fig. 17 [0030] [Fig. 17] shows the NMR 31P-{1H} spectrum in the presence of D2O and after pH adjustment to pH = 7 of compound G1-B (B) and the MAS NMR 31P-{1H} spectrum in the presence of D2O and after pH adjustment to pH = 7 of TriCat 12/G1-B vesicles in a xanthan gel (A) after 3 months of storage at pH=4 at 25°C in water protected from light. 185 10 15 20 25 CA 03165406 2022-06-20 8 [0031] [Fig. 18] shows the evolution of light transmission, after 1 day in water at room temperature, through a sample of TriCat 12/G1-B incorporated (bottom) or not (top) in a xanthan gel (physical stabilization). Fig. 19 [Fig. 19] shows the fluorescence quantification of the amount of dendrimer GI-B-NIR (dendrimer G1-B in its fluorescent form) formulated or not with TriCat 12, which penetrated pig ear skin (Franz cells) after 24 h at 35°C. Fig. 20 [0032] [Fig. Fig. 21 [0033] [Fig. 21] shows the observation by confocal microscopy of the fluorescence (in white) of the G1-B-NIR dendrimer (G1-B dendrimer in its fluorescent form) unformulated (A) and formulated with TriCat 12 (B) which penetrated the skin of a pig's ear (Franz cells) after 24h at 35°C. Fig. 22 [0034] [Fig. Figure 22 shows the flow cytometry analysis of monocyte morphology (size and granulosite) (on the graphs, each point represents a cell). The top graph shows inactive control monocytes. The bottom graph shows monocytes cultured in the presence of TriCat12 vesicles loaded with the G1-B dendrimer. Active monocytes are within the ellipse. Figure 23 [0035] [Figure 23] shows the therapeutic efficacy of the G1-B dendrimer and TriCat12 vesicles loaded with the G1-B dendrimer in the imiquimod-induced murine model of psoriasis. Clinical scores (top graph) are given as a function of time, expressed in days. Histological scores are given on the bottom graph. The "control" mice are the animals that were not treated. Fig. 24 [0036] [Fig. 24] shows the antiproliferative effect of compound G1-B, as a function of treatment time, on the N-TERT keratinocyte line (top graphs) and on primary human keratinocytes (bottom graphs). Fig. 25 [0037] [Fig. 25] shows the size distribution measured by DLS of vesicles formed by the TriCat 12/G1-C formulation (1/0.5). Fig. 26 [0038] [Fig. 26] shows the phase transition temperature of the TriCat 12/G1-C formulation. Fig. 27 [0039] [Fig. Figure 27 shows the measurement of the mean hydrodynamic diameter measured by DLS of the TriCat 12/G1-C formulation over one month at 4°C. Figure 28 [0040] shows the fluorescence quantification of the amount of G1C-NIR dendrimer (G1C dendrimer in its fluorescent form) formulated with TriCat 12, which penetrated pig ear skin (Franz cells) after 24 hours at 35°C. Figure 29 [0041] shows the flow cytometry analysis of the morphology (size and granulosity) of monocytes (on the graphs, each point represents a cell). The top graph shows inactive control monocytes. The graphs at the bottom show monocytes cultured with the G1-C dendrimer at 3 concentrations: 0.2 pM, 2 pM and 20 pM. Active monocytes are in the ellipse. 5 10 15 20 CA 03165406 2022-06-20 10 Fig. 30 [0042] [Fig. 30] shows the size distribution measured by DLS of vesicles formed by the formulation TriCat 8/G1-B (1/0.5) Fig. 31 [0043] [Fig. 31] shows the size distribution measured by DLS of vesicles formed by the formulation TriCat 16/G1-B (1/0.5) Fig- 32 [Fig. Figure 32 shows the measurement of the mean hydrodynamic diameter measured by DLS of the TriCat 16/G1-B formulation over 10 days at 4°C. Description of the implementation methods

[0044] The inventors have shown that the formulation of azabisphosphonate dendrimers in the form of phosphonic acid with sugar-derived catanionic surfactants promotes the penetration of the dendrimer into the skin, thus improving its anti-inflammatory effect for the treatment of psoriasis.

[0045] The present invention thus relates to a vesicle comprising: - a catanionic surfactant of general formula (I) or a mixture of catanionic surfactants of general formula (I): [Chern. 1] in which $ ) is a sugar, in particular is chosen from monosaccharides, disaccharides, polysaccharides and polyols, more particularly 5 10 15 20 CA 03165406 2022-06-20 11 is 1-deoxylactitol, R1 is chosen from H and a linear or branched, saturated or unsaturated hydrocarbon chain of 1 to 20 groups, in particular R1 is chosen from H and C1-C20-alkyl, more particularly, R1 is chosen from H and O4-O18-alkyl, more particularly still R1 is H, and R2 is a linear or branched, saturated or unsaturated hydrocarbon chain of 1 to 20 groups, in particular R2 is C1-C20-alkyl, more particularly R2 is C4-C18-alkyl, more particularly R2 is C8-C16-alkyl, even more particularly R2 is dodecyl, R3 and R4 are independently of each other a linear or branched hydrocarbon chain, saturated or unsaturated, of 1 to 19 chains, in particular R3 and R4 are independently of each other C1-C19-alkyl, more particularly R3 and R4 are C3-C17-alkyl, more particularly R3 and R4 are C7-C15-alkyl, even more particularly R3 and R4 are undecyl; and - a dendrimer of formula (II): [Chem.2] S / — is a disaccharide or a polyol, more particularly (II) in which C c is chosen from the pentoses, hexoses and the following formula groups: 12 , , , and in particular is chosen from the following formula groups: , , , and , more particularly is chosen from the following formula groups: , and , even more particularly is ; Z is chosen from -CH2- and -CH=N-, R is chosen from H and C1-C12-alkyl, in particular R is C1-C12-alkyl, more particularly R is C1-C8-alkyl, more particularly R is C1-C4-alkyl, still more particularly R is chosen from methyl, n-hexyl and noctyl; A is chosen from and , where X is chosen from S and O; in 5 Z X </ *9 9. / \ / £ \ 1 IO o \ rj \/ . >XV ( K v j r 7 -f 7 7'-< / Pi , yy z_ u /_ >\ ,2 .-2- X / V X i=v \/ % zv Xz=4- 7 ' \ z^=a.<^^ Z*\ XX Io | ^1/ |o| j/ U ii ii u 1CD 1 ch2 1 Yx' h2c^5 10 15 20 25 CA 03165406 2022-06-20 13 particular A is , or X is chosen from S and O ; more particularly X is S, and n is an integer between 3 and 8, in particular n is equal to 6.

[0046] For the purposes of this invention, "alkyl" means a hydrocarbon radical of the formula CnH2n+1, where n is an integer greater than or equal to 1. Alkyl radicals may be linear or branched, preferably linear. Particular alkyl radicals of the invention have from 1 to 20 carbon atoms, more particularly from 1 to 12 carbon atoms.

[0047] For the purposes of this invention, "sugar" means monosaccharides, disaccharides, polysaccharides, polyols, and their derivatives. Sugar derivatives include, in particular, sugar-type radicals in which a hydroxyl group has been removed. A particularly preferred sugar of the invention is 1-deoxylactitol.

[0048] In one embodiment, the vesicle according to the invention is characterized in that it comprises a single catanionic surfactant of formula (I).

[0049] In another embodiment, the vesicle according to the invention is characterized in that it comprises a mixture of catanionic surfactants of formula (I), in particular a mixture of two different catanionic surfactants of formula (I).

[0050] According to this embodiment, the molar ratio between the two different catanionic surfactants of formula (I) can be between 99/1 and 1/99.

[0051] In a particular embodiment, the vesicle according to the invention is characterized in that, in the catanionic surfactant of formula (I), C s ') is 1-deoxylactilol.

[0052] Thus, according to this embodiment, the catanionic surfactant present in the formulation is that of formula (1a): [Chern. 3]14 (Ia) in which R1, R2, R3 and R4 are as defined in formula (I). 5 [0053] In one embodiment, the vesicle according to the invention is characterized in that, in the dendrimer of formula (II), is .

[0054] Thus, according to this embodiment, the dendrimer present in the vesicle is that of formula (IIa): [Chem. 4] 10 (IIa) in which Z, R, X and n are such as defined in formula (II).

[0055] According to one variant of this embodiment, the dendrimer of formula (IIa) present in the vesicle is that in which Z is -CH=N-. 15 [0056] According to another variant of this embodiment, the dendrimer of formula (IIa) present in the vesicle is that in which Z is -CH2-. 5 10 15 20 25 CA 03165406 2022-06-20 15

[0057] According to another variant of this embodiment, the dendrimer of formula (Ila) present in the vesicle is that in which A is * or X is chosen from S and O.

[0058] According to another variant of this embodiment, the dendrimer of formula (Ila) present in the vesicle is that in which A is.

[0059] Advantageously, the molar ratio of catanionic surfactant/dendrimer in the vesicle according to the invention is between 50/1 and 1/1, in particular between 30/1 and 1/1, more particularly between 20/1 and 1/1, even more particularly between 10/1 and 1/1, and still more particularly between 5/1 and 1/1. In a very particular way, the molar ratio of catanionic surfactant/dendrimer in the vesicle according to the invention is about 2/1.

[0060] The vesicle according to the invention allows the dendrimer to be encapsulated in the catanionic surfactant.

[0061] Advantageously, the encapsulation rate of the dendrimer of formula (I) in the catanionic surfactant of formula (II) is greater than 50%. In particular, the encapsulation rate of the dendrimer of formula (I) in the catanionic surfactant of formula (II) is between 50% and 95%, more particularly between 60% and 85%, and even more particularly between 70% and 80%.

[0062] For the purposes of this invention, "encapsulation rate" means the ratio of the number of moles of dendrimer stabilized in the catanionic surfactant to the number of moles of dendrimer initially used for vesicle preparation. The encapsulation rate can be determined by any method known to those skilled in the art, in particular by UV-visible spectrophotometry or fluorescence spectroscopy.

[0063] Advantageously, the average diameter of the vesicles according to the invention is between 50 and 500 nm, in particular between 100 and 350 nm. 5 10 15 20 CA 03165406 2022-06-20

[0064] The average diameter can be determined by all methods known to a person skilled in the art, in particular by dynamic light scattering (DLS), notably by means of a He-Ne laser emitting monochromatic light of a wavelength of 633 nm.

[0065] Advantageously, the phase transition temperature of the vesicles according to the invention is between 30 and 35°C, in particular is 33°C.

[0066] Preparation process

[0067] The present application also relates to a method for preparing vesicles as described above.

[0068] The catanionic surfactant of formula (I) present in the vesicle according to the invention is formed by the association of an N-alkylaminosugar of formula (I'): [Chern. 5] S N (r) in which \ —"" is a sugar, R1 is chosen from H and a linear or branched hydrocarbon chain, saturated or unsaturated, of 1 to 20 chantons, in particular R1 is chosen from H and C1-C20-alkyl, R2 is a linear or branched hydrocarbon chain, saturated or unsaturated, of 1 to 20 chantons, in particular R2 is C1-C20-alkyl; and of a phosphinic acid of formula (I”): [Chern. 6] HO in which 5 10 15 20 25 CA 03165406 2022-06-20 17 R3 and R4 are independently of each other a linear or branched, saturated or unsaturated hydrocarbon chain of 1 to 19 chains, in particular R3 and R4 are independently of each other C1-C19-alkyl.

[0069] Thus, the method for preparing a vesicle according to the invention comprises successively the following steps: (a) mixing of one or more N-alkylaminosugars of formula (I’), in particular an N-alkylaminosugar of formula (I’), a phosphinic acid of formula (I”) and the dendrimer of formula (II) as defined above; and (b) optionally, separation of the vesicle obtained from the unencapsulated dendrimer.

[0070] Advantageously, the mixing step is carried out in an aqueous solution, in particular in water.

[0071] Advantageously, the N-alkylaminosugar, phosphinic acid and the dendrimer are mixed with a molar ratio between 100/50/1 and 1/1/1. In particular, the mixing step is carried out with an N-alkylaminosugar/phosphinic acid/dendrimer molar ratio of 2/2/1.

[0072] The mixing can be carried out using all techniques known to a person skilled in the art, in particular by magnetic stirring, by vortex stirring and/or by ultrasonic sonication, more particularly by vortex stirring followed by magnetic stirring or by ultrasonic sonication.

[0073] The mixing step can be carried out at room temperature or by heating to a temperature between room temperature and 100°C, in particular between 25°C and 75°C, for a period of between 5 minutes and 72 hours, in particular between 10 minutes and 48 hours.

[0074] Once the mixing step has been carried out, the vesicle obtained can then be separated from the non-encapsulated dendrimer.

[0075] The separation step can be carried out using all techniques known to a person skilled in the art, in particular by filtration.

[0076] Pharmaceutical composition 5 10 15 20 25 30 CA 03165406 2022-06-20 18

[0077] The present patent application also relates to a pharmaceutical composition comprising the vesicle as described above and a pharmaceutically acceptable excipient.

[0078] The term “pharmaceutically acceptable” refers only to ingredients in a pharmaceutical composition that are compatible with each other and not harmful to the patient. In one embodiment, a pharmaceutically acceptable excipient does not produce any side effects, allergic reactions, or other undesirable reactions when administered to an animal, preferably a human. For human administration, preparations must meet the standard criteria for sterility, pyrogenicity, general safety, and purity as required by regulatory agencies, such as the FDA or the EMA.

[0079] In particular, the pharmaceutical composition as described above shall be intended for topical or transcutaneous application.

[0080] The pharmaceutical composition as described above may thus include one or more excipients that are pharmaceutically acceptable for a formulation suitable for topical administration.

[0081] Pharmaceutically acceptable excipients may include any excipient known to those skilled in the art for the purpose of obtaining a composition for topical application in the form of a milk, cream, balm, oil, lotion, gel, foaming gel, ointment, or spray, preferably in gel form.

[0082] In particular, the pharmaceutical excipients may be gelling agents selected from: carbomers, polysaccharides, such as xanthan gum, guar gum, chitosans, carrageenans, cellulose and its derivatives such as hydroxypropylmethylcellulose in particular or hydroxyethylcellulose available under the name Natrosol, or the family of aluminum and magnesium silicates, the family of acrylic polymers, the family of modified starches, and gelling agents of the polyacrylamide family. Preferably, the pharmaceutical excipient comprises hydroxyethylcellulose, chitosan, or xanthan gum, preferably xanthan gum. 5 10 15 20 25 CA 03165406 2022-06-20 19 Therapeutic Use

[0083] In the present application, the inventors have shown that the dendrimers according to the invention have, in addition to an anti-inflammatory effect, an anti-proliferative effect on keratinocytes. The formulation of the dendrimers in vesicles according to the invention allows the active ingredient to cross the stratum corneum and reach the keratinocyte proliferation zone in the deep epidermis.

[0084] The present patent application relates to the vesicle or pharmaceutical composition of the invention as described above for its use in the treatment or prevention of a dermatosis linked to hyperproliferation of keratinocytes and/or an inflammatory context, preferably psoriasis, dermatitis such as atopic dermatitis, contact dermatitis, seborrheic dermatitis or ichthyoses, such as erythematous ichthyoses (rare diseases) like Peeling Skin Syndrome (PSS) type 1 or 6 or congenital ichthyosiform erythroderma, squamous cell carcinomas, basal cell or squamous cell carcinomas or acne.

[0085] In particular, the present patent application relates to the vesicle or pharmaceutical composition of the invention as described above for its use in the treatment or prevention of a dermatosis linked to hyperproliferation of keratinocytes and an inflammatory context, preferably dermatitis such as atopic dermatitis, contact dermatitis, seborrheic dermatitis or ichthyoses, such as erythematous ichthyoses (rare diseases) like Peeling Skin Syndrome (PSS) type 1 or 6 or congenital ichthyosiform erythroderma.

[0086] Dermatitis is an irritation of the skin. Dermatitis is a common condition and presents in many forms. It generally manifests as itching, dry skin, or a rash on swollen, reddened skin. It can also cause blisters, oozing, scabs, or peeling. 5 10 15 20 25 30 CA 03165406 2022-06-20 20

[0087] Preferably, the vesicle or pharmaceutical composition of the invention as described above is particularly useful for treating atopic dermatitis, contact dermatitis and seborrheic dermatitis.

[0088] Atopic dermatitis, also called atopic eczema, is a chronic inflammatory skin disease. It develops primarily in infants and children, but can persist or even appear in adolescents and adults. It is characterized by dry skin associated with eczema-like lesions (redness and itching, blisters, oozing, and crusting) that occur in flare-ups. Atopic dermatitis is a chronic inflammatory skin disease.

[0089] Contact dermatitis refers to a skin reaction resulting from exposure to allergenic (allergic contact dermatitis) or irritating (irritant contact dermatitis) substances. The skin reaction generally develops a few minutes to a few hours after exposure to the substance and can last from two to four weeks.

[0090] Seborrheic dermatitis generally affects the scalp and causes scaly patches, red skin, and persistent dandruff. It can also affect oily areas of the body, such as the face, upper chest, and back. Seborrheic dermatitis can be a long-term condition, with periods of improvement followed by seasonal flare-ups. In infants, this condition is called "cradle cap."

[0091] The vesicle or pharmaceutical composition of the invention as described above is particularly useful also for treating ichthyosis, preferably erythematous ichthyoses such as Peeling Skin Syndrome (PSS) of type 1 or 6 or congenital ichthyosiform erythroderma.

[0092] Ichthyosis is a congenital skin disease characterized by extremely dry, rough skin and an excessive amount of fine, free-edged scales, sometimes arranged like fish scales, which continually flake off. Erythematous ichthyosis is a dermatological lesion characterized by congestive redness of the skin, diffuse or localized, which blanchs with vitopressure. 5 10 15 20 25 30 CA 03165406 2022-06-20 21

[0093] Peeling skin syndrome type 1 or 6 are inflammatory forms of ichthyosis characterized by diffuse and superficial desquamation of the entire skin surface with underlying erythroderma, pruritus and atopy.

[0094] Congenital ichthyosiform erythroderma is characterized by the presence at birth of fine, whitish-grey scales of varying sizes, associated with erythroderma. Some newborns are enveloped in a soft collodion film (taut, shiny, and translucent) and develop scaly erythroderma after its desquamation.

[0095] The present patent application also relates to the vesicle or pharmaceutical composition as described above for its use in the treatment or prevention of a dermatosis linked to hyperproliferation of keratinocytes, such as basal or squamous cell carcinomas.

[0096] Basal cell carcinoma is a type of skin cancer that begins in the basal cells. Basal cell carcinoma often appears as a slightly translucent bump on the skin, although it can take other forms. Basal cell carcinoma most often appears on areas of skin that are exposed to the sun, such as the head and neck.

[0097] Squamous cell carcinoma is a common form of skin cancer that develops in the epidermoid cells that make up the middle and outer layers of the skin. Squamous cell carcinoma most often occurs on sun-exposed skin, such as the scalp, the backs of the hands, the ears, or the lips.

[0098] The present application also relates to the vesicle or pharmaceutical composition of the invention as described above for its use in the treatment or prevention of a dermatosis linked to an inflammatory context such as acne.

[0099] Acne is a common and chronic dermatological disease of the pilosebaceous system (which includes the hair follicle, the hair shaft and the sebaceous gland secreting sebum, at the root of the hair). It generally occurs in adolescence and is linked to the hypersecretion of sebum (hyperseborrhea) and to keratinization abnormalities leading to the obstruction of the excretory canal of the pilosebaceous follicle, and to the formation of comedones.

[0100] Preferably, the present patent application also relates to the vesicle or pharmaceutical composition as described above for its use in the treatment of psoriasis in a subject who needs it.

[0101] Psoriasis is a dermatosis linked to the overproduction of keratinocytes, cells that make up 90% of the epidermis, and to a chronic inflammatory context. In this pathology, keratinocytes play a role alongside skin immune cells and infiltrating immune cells. The overproduction of keratinocytes is accompanied by a defect in differentiation when they reach the outermost layer of the epidermis, the stratum corneum, hence the characteristic scaling plaques of the disease. In psoriasis, the renewal of the different layers of the epidermis occurs in 3 to 4 days, compared to 3 weeks for normal skin. In order to control the overproduction and the inflammatory context, it is therefore necessary to target these cellular players in the disease by allowing the active ingredient to cross the stratum corneum, which constitutes the skin's impermeable barrier. The formulation of dendrimers in vesicles according to the invention allows the active ingredient to penetrate the stratum corneum and reach the site of keratinocyte hyperproliferation in the deep epidermis. Thus, the vesicle or pharmaceutical composition as required is particularly useful for the treatment of psoriasis.

[0102] The term “psoriasis” refers to a chronic inflammatory skin disease inducing abnormal skin turnover, resulting in thick, red, scaly plaques. Psoriasis encompasses all forms of psoriasis, including plaque psoriasis, guttate psoriasis, infantile psoriasis, pustular psoriasis, erythrodermic psoriasis, inverse psoriasis, facial psoriasis, scalp psoriasis, nail psoriasis, and mucous membrane psoriasis. Plaque psoriasis, or psoriasis vulgaris, is the most common form, affecting more than 80% of patients, and is characterized by raised, red plaques with white scales on the surface. Guttate psoriasis, affecting approximately 10% of patients, is characterized by multiple teardrop-shaped lesions with few scales. Pustular psoriasis is the most severe form of psoriasis, in which plaques are covered with non-infectious white pustules. Erythrodermic psoriasis is a generalized inflammation with scales, affecting 90 to 100% of the skin. Inverse psoriasis is characterized by the presence of non-scaly plaques inside joints and skin folds.

[0103] The term "subject" refers to an animal, more particularly a mammal. Preferably, the subject is a human being. For the purposes of the present invention, a subject may be a patient, namely a person receiving medical care, undergoing or having undergone medical treatment, or being monitored in the context of the development of a disease.

[0104] The term “treat” or “treatment” refers both to therapeutic treatment and to prophylactic or preventive measures, in which the objective is to prevent or slow down (reduce) the targeted pathological condition, in particular keratinocyte proliferation and skin inflammation. Preferably, in the case of psoriasis treatment, the objective is to reduce the surface area of affected skin, decrease the degree of redness of the lesions, their thickness, and the intensity of scaling.

[0105] In particular, the vesicle or composition as described above for use as a medicinal product, in particular for the treatment or prevention of dermatosis related to hyperproliferation of keratinocytes and/or an inflammatory context, preferably for the treatment of psoriasis, are characterized in that they are formulated for topical application.

[0106] The present invention, according to another aspect, relates to a method for treating or preventing a dermatosis linked to keratinocyte hyperproliferation and/or an inflammatory context in a subject in need, comprising administering to a subject a therapeutically effective dose of a vesicle or a pharmaceutical composition according to the invention. Preferably, the subject is an animal, preferably a mammal, more preferably a human suffering from a dermatosis linked to keratinocyte hyperproliferation and/or an inflammatory context. 5 10 15 20 25 30 CA 03165406 2022-06-20 24

[0107] The present invention also relates to the use of a vesicle or a pharmaceutical composition as described above for the manufacture of a drug for the treatment or prevention of a dermatosis linked to hyperproliferation of keratinocytes and/or an inflammatory context.

[0108] This patent application also relates to a method for treating psoriasis in a subject in need, comprising the administration of a therapeutically effective dose of the vesicle or composition as described above to said subject. In particular, this application relates to a method for treating psoriasis in a subject in need, comprising the topical administration of a therapeutically effective dose of the vesicle or pharmaceutical composition as described above to said subject.

[0109] The present invention also relates to the use of a vesicle or a pharmaceutical composition as described above for the manufacture of a drug for the treatment or prevention of psoriasis.

[0110] The term “therapeutically effective dose” refers to the dose of therapeutic agent necessary and sufficient to slow down or stop the progression, aggravation or deterioration of one or more symptoms of a dermatosis linked to hyperproliferation of keratinocytes and/or an inflammatory context as described above, preferably of psoriasis disease, for example reducing the surface area of affected skin, decreasing the degree of redness of the lesions, their thickness and the intensity of desquamation. [0111]According to one embodiment, the vesicle or a pharmaceutical composition as described above may be used as a medicament, in particular for the treatment or prevention of a dermatosis related to keratinocyte hyperproliferation and/or an inflammatory context as described above, preferably psoriasis, in a subject in combination with at least one other therapeutic agent. Such additional therapeutic agents include, but are not limited to, anti-inflammatory agents, anti-infective agents, or immunosuppressive agents. The vesicle and the additional therapeutic agent may be administered at the same time or at different times, simultaneously or separately.

[0112] Thus, the treatment methods and pharmaceutical compositions of the present invention can use the vesicle of the invention as monotherapy, but these methods and compositions can also be used as polytherapy in which the vesicles of the invention are co-administered in combination with one or more other therapeutic agents.

[0113] The present invention will be better understood in the light of the following non-limiting examples and figures. Examples I. Materials and Methods 1. Synthesis of the compounds used

[0114] N-dodecylamino-1-deoxylactitol was synthesized according to the protocol described in the thesis of Pauline Castagnos (Castagnos, Pauline (2011). Catanionic vesicles: design and mechanisms of active ingredient delivery). 8.75 mmol (3.15 g) of α-lactose are dissolved in 20 mL of ultrapure water. 14.88 mmol (2.76 g) of dodecylamine are dissolved in 25 mL of methanol. 5 wt% (relative to the total mass of reactants) of palladium supported on carbon (0.338 g) is suspended in 10 mL of methanol. The amine, palladium, and finally lactose are placed in a tightly screwed reactor. [0115] After three purges under 20 bar of hydrogen, the reactor is filled with hydrogen under 22 bar, then detached from the hydrogen cylinder and placed in a sand bath thermostatically controlled at 60°C, so as to obtain a temperature of approximately 50°C in the reactor. The reaction mixture is thus left under magnetic stirring for 3 days. After cooling the reactor, the reaction mixture is returned to atmospheric pressure. 5 10 15 20 25 30 CA 03165406 2022-06-20 26

[0116] To remove excess palladium, the reaction medium is filtered through a 5 porosity filter. After compacting the filter with ultrapure water, the reaction medium, heated to 50°C, is deposited on it and then washed with 200 mL of an ultrapure water/methanol (1:1) solution at 50°C. The filtrate is then dried in a rotary evaporator. When only water remains, it is removed by lyophilization. A hygroscopic white powder is recovered (m=1.62 g, R=36%).

[0117] 1H NMR (D2O, 300MHz): 8 (ppm): 0.81 (m, 3H, CH3); 1.48 (m, 20H, aliphatic CH2); 3.48 to 3.84 (m, 20H, OH, CH and CH2 of sugar); 4.42 (d, 1H, anomeric H)

[0118] MS: m/z: 512.4

[0119] FT-IR (KBr): vmax (cm-1): 3435 (N-H st (secondary amine), alcohols), 2924 (C-H elongation), 2853 (CH, CH2, CH3 elongation), 1638 (N-H elongation), 1466 (CH2 angular deformation), 1380 (C-H), 1079 (C-N st (secondary amine)), 720 (N-H 8, presence of an aliphatic chain greater than 4C)

[0120] Elementary analysis: C: 53.90%, H: 9.68%, N: 2.79%, Rd < 0.02% (Ctheo: 56.34%; Htheo: 9.65%; Ntheo: 2.74%)

[0121] Carbon purity: 95.7%

[0122] Bis-α-(α-hydroxyalkyl)phosphorus amphiphiles were synthesized according to the protocol described in the article (Brun, A.; Etemad-Moghadam, G. New double-chain and aromatic (α-hydroxyalkyl)phosphorus amphiphiles. Synthesis 2002, 10, 1385-1390.) [0123] 2.5 mmol of sodium hypophosphite monohydrate (i.e., 2.38 g) are stirred in the presence of 30 mL of 1,4-dioxane. 56 mmol of dodecylaldehyde (i.e., 10.3 g) are added, as well as 4 mL of 37% hydrochloric acid. The reaction mixture is heated under reflux (101°C) for 24 h. After cooling, the flask was passed through a rotary evaporator until a solid brown paste was obtained. This paste was then washed successively with 15 mL of ultrapure water, 15 mL of tetrahydrofuran (THF), 4 x 15 mL of acetone, 15 mL of ultrapure water, 15 mL of tetrahydrofuran, 4 x 15 mL of acetone, and 15 mL of tetrahydrofuran. All 27 washes were performed using a sinter with a porosity of 4. A compact white powder (m = 2.00 g, R = 20.5%) was obtained.

[0124] 1H NMR (CDCl3/CD3OD, CD3OD locked, 55°C, 300MHz): δ (ppm): 0.85 (t, 6H, CH3); 1.25 (m, 36H, aliphatic CH2); 1.61 (m, 4H, CH2 in α); 1.77 (m, 4H, 5CH2α); 3.60 (m, 2H from CHOH)

[0125] 13C NMR (CDCl3 / CD3OD, CD3OD locked, 55°C, 300MHz): δ (ppm): 13.6 (s, CH3); 22.4 to 31.7 (CH2), between 60 and 70 (CH)

[0126] 31P NMR (CDCl3/CD3OD, CD3OD-locked, 55°C, 300 MHz): δ (ppm): 45.43 and 46.52: peaks due to phosphorus pseudo-asymmetry. No peak around 30 ppm: no monosubstituted ion remains.

[0127] MS: 433.3 (M-H)-

[0128] FT-IR (KBr): νmax (cm-1): 3313 (C-OH); 2918 (C-H); 2848 (C-H); 2369 (P-OH); 1465 (δCH2); 1222 (P=O); 1141 (P=O); 1115 (PO-OH); 1067 (P-OH); 960 (P-OH); 941 (P-OH) 15 [0129] Elemental analysis: C: 65.67%, H: 9.18% (Ctheo: 66.32%; Htheo: 11.83%)

[0130] Carbon purity: 99.0% ABP dendrimer [0131] [Chem 7] 20

[0132] The ABP dendrimer was synthesized according to the protocol described in Poupot et al. FASEB J. 2006, 20(13)2339-51. 28 Dendrimer G1-A [0133] [Chem 8]

[0134] The G1-A dendrimer was synthesized according to the protocol described in Poupot 5 et al. FASEB J. 2006, 20(13)2339-51. G1-A-NIR Dendrimer

[0135] Synthesis scheme [scheme 1] 29 Synthesis of compound b

[0136] 4-Hydroxybenzenepropanoic acid (90 mg, 0.54 mmol), 1-Ethyl-3-(3-5-dimethylaminopropyl)carbodiimide (EDC) (90.18 mg, 0.47 mmol), and 1.1 equivalents of 4-dimethylaminopyridine (DMAP) (48.64 mg, 0.39 mmol) are added to a solution of ADIBO-amine a (100 mg, 0.36 mmol) in 5 mL of 30-N,N-dimethylformamide (DMF). The reaction mixture is stirred overnight at room temperature. The reaction mixture is lyophilized, the crude residue is dissolved in 50 mL of dichloromethane (DCM), and then washed with water (3 x 25 mL). The organic phase is dried and concentrated under reduced pressure. The 5th residue is purified on a silica column (eluent: hexane/AcOEt, 60/40) to give product b with a yield of 85% in the form of a transparent oil. [0137] [Chem 9] 10 [0138] 1H NMR (500 MHz, Chloroform-d) δ: 2.01–1.92 (m, 1H, CH2-CON), 2.30– 2.23 (m, 2H, C’04-CH2-), 2.50-2.50 (m, 2H, CH2-CON), 2.80–2.69 (m, 3H, -CH2- CO-NH-), 3.25–3.19 (m, 1H, -CH2-NH-CO), 3.39–3.32 (m, 1H, -CH2-NH-CO), 3.72 (d, 2JHH=13.9 Hz, 1H, r-CH2-N-CO-), 5.16 (d, 2JHH=13.9 Hz, 1H, r-CH2-NCO-), 6.14–6.08 (t, 3JHH = 6.0 Hz, 1H, CO-NH), 6.71 (d, 2JHH = 8.2 Hz, 4H, C’03H), 15 6.95 (d, 3JHH = 8.2 Hz, 4H, C’02H), 7.17 (br s, 1H, HAr), 7.23-7.27 (m, , 1H, HAr), 7.35 – 7.27 (m, 2H, HAr), 7.49 – 7.35 (m, 4H, HAr), 7.70 (d, 3JHH = 7.6 Hz, 2H, HAr), 8.04 (s, 1H, OH) ppm. [0139] 13C NMR (126 MHz, Chloroform-d) δ: 30.80 (-CH2-CO-NH-). 34.75(s, CH2- CON), 35.22(s, -CH2-NH-CO), 38.55 (s, C’04-CH2-), 55.63 (-CH2-N-CO), 20 107.74(s, C≡ C’), 114.79(s, C≡ C’), 115.38(s, C’02), 122.48 (s, Cf), 122.91 (s, Cf’), 125.66 (s, Ce’), 127.27 (s, CHAr), 127.93 (s, CHAr’), 128.37 (s, CHAr), 128.53 (s, CHAr), 128.69 (s, CHAr), 129.05 (s, C’03), 129.29 (s, CHAr), 131.96 (s, C’O4), 132.09 (s, CHAr), 147.91 (s, CAr), 150.88 (s, CAr), 154.81 (s, C’O1), 172.44 (s, CONH) ppm. 25 Synthesis of compound d 31

[0140] Compound c (465 mg, 0.6 mmol) is added to a solution of compound b (130 mg, 0.30 mmol) in THF (30 mL) with Cs2CO3 (199 mg, 0.61 mmol). After overnight stirring at room temperature, the mixture is centrifuged, filtered, and then concentrated under reduced pressure. The crude residue is purified on a silica column (eluent: DCM/AcOEt, 60/40) to give compound d in 88% yield as a clear oil. Compound c can be prepared according to Rolland, O. et al. (2008) Chemistry – A European Journal, 14: 4836-4850. [0141] [Chem 10] 10 [0142] 31P NMR (121 MHz, Chloroform-d) δ: 7.40 (s, P=N). [0143] 1H NMR (600 MHz, Acetone-d6) δ: 1.96 - 1.88 (m, 1H, CH2CON). 2.34-2.31 (m, 2H, CH2-C’04), 2.44-2.48 (m, 0.5H, CH2CON), 2.47 – 2.53 (m, 0.5H, CH2CON), 2.82 – 2.79 (m, 2H, CH2CONH), 3.17-3.12 (m, 1H, CH2NH-), 3.26- 15 3.20 (m, 1H, CH2NH-), 3.65 (d, 1JHH = 14.0 Hz, 1H, CH2NCO), 5.12 (d, 1JHH = 14.0 Hz, 1H, CH2NCO), 6.79 (t, 3JHH = 6.1 Hz, 1H, CONH), 7.01 – 6.97 (m, 2H, C’02H), 7.13 (d, 3JHH = 8.4 Hz, 2H, C’03H), 7.21 (d, 3JHH = 8.4 Hz, 4H, C02H), 7.26 – 7.23 (m, 1H, CHAr), 7.29 – 7.27 (m, 6H, C02H), 7.33 – 7.30 (m, 1H, HAr), 7.36 - 7.40 (m, 1H, CHAr), 7.49 – 7.45 (m, 2H, CHAr,), 7.54 – 7.51 (m, 1H, HAr), 7.67 (d, 20 3JHH = 7.4 Hz, 1H, CHAr), 7.85-7.84 (m, 2H, C03H), 7.86-7.85 (m, 5H, C03H), 7.87- 7.86 (m, 3H, C03H), 9.99 (s, 3H, CHO), 10.00 (s, 1H, CHO), 10.01 (s, 1H, CHO). [0144] 13C NMR (151 MHz, Acetone-d6) δ: 30.43 (s, CH2CONH), 34.52 (s, CH2CON), 35.29 (s, CH2NH-), 37.08 (s, CH2-C’04), 54.95 (s, -CH2NCO), 107.98 (s, C≡ C), 114.35 (s, C≡ C), 120.59 (s, C’02H), 121.27 (s, C02H), 121.35 (s, C02H), 32 122.15 (s, CAr), 123.05 (s, CAr), 125.17 (s, CHAr), 126.87 (s, CHAr), 127.60 (s, CHAr), 127.92 (s, CHAr), 128.10 (s, CHAr), 128.79 (s, CHAr), 129.46 (s, C’03H), 131.30 (s, C03H), 131.34 (s, C03H), 132.51 (s, CHAr), 133.98 (s, CAr), 134.05 (s, CHAr), 134.11(s, CHAr), 139.32 (br s, CAr, C’04), 148.11 (br s, CAr, C’01), 148.62(s, 5 CAr), 151.80(s, CAr), 154.46 (br s, Cq, C01), 154.65 (br s, Cq, C01), 170.64 (s, CON), 170.85 (s, CONH), 190.65 (s, CHO), 190.80 (s, CHO). Composed of

[0145] To a solution of compound d (300 mg, 0.25 mmol) in 10 mL of CHCl3, 7 equivalents of 0.2 M dichlorothiophospho(N-methyl)hydrazide (8.75 mL, 1.75 mmol) in chloroform are added. The reaction mixture is stirred for 2 hours at room temperature. At the end of the reaction (monitored by 1H NMR), the solvent is concentrated under reduced pressure. The resulting residue is dissolved in 2 mL of THF and then precipitated by addition to a large volume of pentane (100 mL). The solid formed is filtered and dried to obtain compound e in 83% yield as a white powder. [0146] [Chem 11] [0147] 31P NMR (243 MHz, Chloroform-d) δ: 8.30 (s, N=P), 62.42 (s, P=S), 62.47 (s, P=S), 62.57(s, P=S). 20 [0148] 1H NMR (600 MHz, Chloroform-d) δ: 1.96-1.91 (m, 1H, -CH2CON-), 2.25 (t, 3JHH = 7.8 Hz, 2H, -CH2-C’04), 2.44-2.39 (m, 1H, CH2CON-), 2.84- 2.76 (m, 2H, -CH2-CONH-), 3.22 – 3.14 (m, 1H, -CH2-NHCO), 3.37-3.33 (m, 1H, -CH2-NHCO), 5 10 15 20 25 CA 03165406 2022-06-20 33 -CH2-CONH-), 3.22-3.14 (m, 1H, -CH2-NHCO), 3.37-3.33 (m,1H, -CH2-NHCO), 3.50 (d, 3Jhp = 13.9, 15H, CH3-NPS), 3.69 (d, 1Jhh = 14.0 Hz, 1H, -CH2-NCO), 5.13 (d, 1Jhh = 14.0 Hz, 1H, -CH2-NCO), 6.01 (t, 3Jhh = 6.2 Hz, 1H, -CONH-), 6.92 (d, 3Jhh = 8.1 Hz, 2H, C’o2H), 7.02-6.98 (m, 4H, Co2H, 2H, C’o3H), 7.06 (d, 3Jhh = 8.2 Hz, 6H, Co2H), 7.21 (d, 3Jhh = 7.6 Hz, 1H, HAp), 7.29 (d, 3Jhh = 6.1 Hz, 3H, HAr), 7.44-7.35 (m, 3H, Hat), 7.61-7.58 (m, 3Jhh = 8.7 Hz, 10H, Co3H), 7.64 (s, 4H, -CH=N-), 7.66 (s, 1H, -CHAr), 7.68 (s, 2H, -CH=N-). [0149] 13C NMR (151 MHz, Chloroform-d) 6: 30.76 (s, CH2CONH), 31.96 (d, 2Jcp = 12.6 Hz, CH3-NPS), 34.72 (s, -CH2CON-), 35.21 (s, -CH2-NH), 37.95 (s, CH2- C’ o4), 55.53(s, -CH2NCO), 107.78 (s, C=C), 114.75 (s, C=C), 120.88 (s, C'o2H), 121.30 (s, C'o3H), 121.39(s, Co2H), 122.48 (s, Cap), 122.90 (s, Cat), 125.59 (s, CHap), 127.27 (s, CHap), 127.86 (s, CHap), 128.33 (s, CHap), 128.50 (s, CHAr), 128.60 (s, Co3), 128.66 (s, CHat), 129.04 (s, CHap), 129.35 (s, CHAr), 131.14 (s, CHap), 131.24 (s, CHap), 131.26 (s, Cap), 132.02 (s, CHap), 138.03, 140.59, 140.62 (d, 3Jcp — 9.3 Hz, C=N), 140.74 (d, 3Jcp = 9.4 Hz, C—N), 140.71 (s, Cap), 140.77 (s, Cap), 147.98 (s, Cap), 148.54 (m, C'o1), 150.94 (s, Cap), 151.74 (d, 2Jcp = 9.8 Hz, Co1), 151.89 (brd, Co1), 171.24(s, CONH), 172.22 (s, CON). Synthesis of compound f

[0150] To a suspension of aminobismethylenephosphonate compound derived from tyramine, prepared according to Rolland et al. (2008), A European Journal, 14: 4836-4850, (680 mg, 1.78 mmol) in THF (15 mL) and CS2CO3 (1.15 g, 3.56 mmol), compound e (350 mg, 0.18 mmol) is added. After overnight stirring at room temperature, the insolubles are removed by centrifugation and the solvent is removed by reduced pressure. The dendrimer f is obtained as a clear oil with a yield of 97%. The compound can be purified by column chromatography. [0151] [Chern 12]34 [0152] 31P NMR (243 MHz, Chloroform-d) δ: 8.33 (s, NP), 26.81 (s, POMe), 63.15 (s, PS), 6312 (s, PS), [0153] 1H NMR (600 MHz, Chloroform-d) δ: 1.86 – 1.90 (m, 1H, CH2CON), 2.18 5 (t, 3JHH = 7.8 Hz, 2H, CH2-C’04), 2.37 – 2.41 (m, 1H, CH2CON), 2.74 (t, 3JHH = 7.6 Hz, 20H, C14-CH2), 3.03 (t, 3JHH = 7.6 Hz, 20H, CH2-N), 3.17 (d, 2JHP = 9.5 Hz, 40H, CH2-PO ), 3.25 (d, 3JHH = 10.3 Hz, 4H, CH3-N-), 3.25 (d, 3JHP = 10.0 Hz, 6H, CH3-N), 3.30 (d, 3JHP = 10.0 Hz, 5H, CH3-N), 3.71 (d, 3JHP = 10.4 Hz, 120H, POMe), 5.08 (d, 2JHH = 14 Hz, 1H, CH2-Ca’), 6.24 (t, 3JHH = 6.1 Hz, 1H, 10 CONH), 6.91 (d, 3JHH = 8.4 Hz, 2H, C’02H), 6.96 (d, 3JHH = 8.6 Hz, 1H, C’03H), 7.00 (d, 3JHH = 8.2 Hz, 5H, C02H), 7.04 (d, 3JHH = 8.4 Hz, 3H, C02H), 7.10–7.08 (m, 20H, C12H), 7.15 (d, 3JHH = 7.9 Hz, 20H, C13H), 7.24 (m, 2H, CHAr), 7.35– 7.31 (m, 2H, CHAr), 7.60 (d, 3JHH = 8.9 Hz, 10H, C03H), 7.63 (d, 3JHH = 9.0 Hz, 15H, C03H, CH=N). 15 [0154] 13C NMR (151 MHz, Chloroform-d) δ: 31.91 (s, CH2), 32.92 (d, 2JCP = 5.9 Hz, CH3-NPS), 33.01 (br s, C14-CH2, CH3-NPS), 34.71 (s, CH2CON), 35.25 (s, CH2-NH), 37.74 (s, CH2-C’04), 49.44 (d, 1JCP = 157.5 Hz, CH2-PO), 49.49 (d, 1JCP = 157.6 Hz, -CH2-PO), 51.15–53.51 (m, POMe), 55.42 (s, CH2NCO), 58.12 (t, 3JCP = 7.6 Hz, CH2-N), 107.84 (s, C≡ C), 114.71 (s, C≡ C), 120.81 (s, C’02), 20 121.24–121.18 (m, C12, C02), 122.36 (s, CHAr), 122.93 (s, CHAr), 125.50 (s, CHAr), 127.17 (s, CHAr), 127.80 (s, CHAr), 128.25-128.22 (m, C03), 128.37 (s, CHAr), 128.64 (s, CHAr), 129.02 (s, CHAdibo), 129.35 (s, C’03), 129.91 (s, C13),132.14– 132.07 (m, CO4), 136.54 (s, C14), 136.58 (s, C14), 138.01 (s, CAr), 138.72 (d, 3JCP=13.8 Hz, C=N), 148.00 (s, CAr), 148.64 (s, CAr), 148.93 (s, C11), 148.98 (s, 25 C11), 151.00 (s, CAr), 151.25 (br s, CO1), 171.52 (s, CONH), 171.90 (s, CON). 35 Synthesis of the NIR compound

[0155] This compound is prepared according to the following scheme, adapting the procedure described in S. A. Klymchenko, V. G. Pivovarenko, O. Turan, and D. P. Alexander, New Journal of Chemistry, 27(9), 1336-1343, 2003. 5 [0156] Synthesis Scheme [Scheme 2] Synthesis of 1-(3-azidopropyl)-4-methylquinolinium chloride 10 [0157] A basic resin (DOWEX MARATHON MSA) (3 g) is added to a solution of 1-(3-azidopropyl)-4-methylquinolinium iodide (1 g, 2.8 mmol) in a 50/50 H2O/MeCN mixture. The reaction mixture is stirred for 3 days at room temperature. The mixture is then filtered and concentrated to dryness under reduced pressure. The residue is solubilized in DCM (100 mL), then washed twice with H2O (30 mL). The organic phase is recovered, dried with Na2SO4, filtered, and concentrated under reduced pressure. The crude residue is rapidly purified on a silica column (eluent: DCM/MeOH, 90/10) to give a black solid with a 70% yield. [0158] [Chem 13] (CH2)3N3 I36 [0159] 1H NMR (300 MHz, Chloroform-d) δ: 2.71-2.33 (m, 2H, CH2), 3.05 (s, 3H, Me), 3.82 (t, 3JHH= 6.5 Hz, 2H, CH2-N3), 5.65-5.39 (m, 2H, CH2-N), 8.08 – 7.98 (m, 2H, CHAr), 8.20-8.3 (m, 1H, CHAr), 8.40 (d, 3JHH = 8.5 Hz, 1H, CHAr), 8.56 (d, 5 3JHH = 9.0 Hz, 1H, CHAr), 10.25 (d, 3JHH = 6.0 Hz, 1H, CHAr). [0160] 13C NMR (75 MHz, Chloroform-d) δ: 20.70 (s, Me), 29.45 (s, CH2), 48.35 (s, CH2), 54.99 (s, CH2-N), 119.10 (s, CHAr), 123.32(s, CHAr), 126.94 (s, CHAr), 129.51 (s, CAr), 130.13 (s, CHAr), 135.86 (s, CHAr), 137.14 (s, CAr), 148.79 (s, CAr), 158.56 (s, CHAr). 10 Synthesis of the compound NIR chloride of (E)-1-(3-azidopropyl)-4-(2-(6-(diethylamino)benzofuran-2-yl)vinyl)quinolinium

[0161] To a solution of 1-(3-azidopropyl)-4-methylquinolinium chloride (500 mg, 1.9 mmol) in EtOH (30 mL), 6-Diethylaminobenzo[b]furan-2-carbaldehyde (1.2 g, 7.5 mmol) and a catalytic amount of piperidine (3 to 4 15 drops) are added. The solution is stirred under reflux for 5 hours. The mixture is evaporated to dryness under reduced pressure. The residue is purified by silica gel chromatography (eluent: DCM/MeOH 90/10) to give the NMR in powder form with a yield of 65%. [0162] [Chem 14] 20 [0163] 35Cl NMR (59 MHz, Methylene Chloride-d2) δ: 233.98 (s, Cl-). [0164] 1H NMR (300 MHz, Methylene Chloride-d2) δ: 1.25 (t, 3JHH = 7.1 Hz, 6H, CH3-CH2-N), 2.46 – 2.23 (m, 2H, CH2-CH2-N=), 3.46 (q, 3JHH = 7.1 Hz, 4H, CH3- 37 CH2-N), 3.70 (t, 3JHH = 6.3 Hz, 2H, CH2-N3), 5.17 – 4.99 (m, 2H, CH2-N=), 6.62 – 6.56 (m, 1H, CHAr ), 6.69 (d, 3JHH = 8.9 Hz, 1H, CHAr), 7.15 (s, 1H, CHfuran), 7.37 (d, 3JHH= 8.9 Hz, 1H, CHAr), 7.56 (d, 3JHH= 14.9 Hz, 1H, CHC=C), 7.83 (d, 3JHH= 8.4 Hz, 1H, CHAr), 7.90 (d, 3JHH=15.2 Hz, 1H, CHC=C), 8.09 – 8.00 (m, 1H, 5 CHAr), 8.19 (d, 3JHH= 8.9 Hz, 1H, CHAr), 8.23 (d, 3JHH= 6.7 Hz, 1H, CHAr), 8.46 (d, 3JHH= 8.6 Hz, 1H, CHAr), 9.48 (d, 3JHH= 6.6 Hz, 1H, CHAr). [0165] 13C NMR (75 MHz, Methylene Chloride-d2) δ: 12.46 (s, CH3-CH2-N), 29.02 (s, CH2-CH2-N3), 45.08 (s, CH3-CH2-N), 48.40 (s, CH2-N3), 54.09 (s, CH2-N=), 91.85 (s, CHAr), 110.75 (s, CHAr ), 114.00 (s, CHAr ), 115.00 (s, CHAr ), 117.06 (s, 10 CHAr ), 118.09 (s, CAr ), 118.23 (s, CHAr ), 123.05 (s, CHAr ), 125.73 (s, CHAr ), 126.39 (s, CAr), 128.71 (s, CHAr), 130.00 (s, CHAr), 134.92 (s, CHAr), 137.89 (s, CAr), 146.12 (s, CHAr), 149.31 (s, CAr), 151.11 (s, CAr), 152.20 (s, CAr), 159.46 (s, CAr). Synthesis of compound g 15 [0166] To a solution of dendrimer f (300, 0.055 mmol) in anhydrous acetonitrile (3 mL), near-infrared fluorescent azide (NIR) (25.5 mg, 0.11 mmol) is added. The reaction mixture is stirred for three days at room temperature. The solvent is then removed under reduced pressure to give the dendrimer g with a quantitative yield in the form of a blue oil 20 [0167] [Chem 15] [0168] 31P NMR (121 MHz, CD2Cl2) δ : 8.40 (s, N3P3), 29.64 (s, -PO3Me2), 63.26 (s, -P=S-). 5 10 15 20 25 CA 03165406 2022-06-20 38 [0169] 1H NMR (500 MHz, CD2CI2) 6: 1.19 (t, 3Jhh = 7.1 Hz, 6H, CH3-CH2-N), 2.63 - 2.19 (m, 4H, CH2), 2.74 (br s, 22H, Ci4-CH2, CH2), 3.04 (br s, 20H, Ci4-CH2- CH-), 3.17 (d, 2Jhp = 9.4 Hz, 41H, -CH2-P, CH2), 3.39-3.26 (m, 17H, CH3-N, CH2), 3.60-3.45 (m, 4H, Me-CH2-N), 3.64-3.57 (m, 120H, POCH3), 7.10-6.73 (m, 16H, C03H, C’02H, C’03H, CHAr,), 7.13 (d, 3Jhh = 7.2 Hz, 24H, Ci2H, HAr), 7.21 (d, 3Jhh = 7.3 Hz, 22H, Ci3H, HAr), 7.37-7.25 (m, 4H, HAr), 7.58-7.38 (m, 4H, HAr), 8.10- 7.57 (m, 19H, CH=N, Co3H, HAr). [0170] 13C NMR (126 MHz, CD2CI2) 5: 29.66 (s, CH3), 32.76 (s, CH2), 32.88 (s, CH3), 45.08 (s, CH2), 49.34 (d, 1Jcp= 157.4 Hz, N-CH2-P), 49.40 (d, 1Jcp= 157.4 Hz, N-CH2-P), 52.49 (br s, POCH3), 58.07 (s, CH2), 58.12 (s, CH2), 58.17 (s, CH2), 120.89 (s, CHAr), 121.13 (s, C12), 128.21 (s, Co3), 129.38 (s, CHAr), 129.93 (s, C13), 132.29 (br s, CO4), 136.98 (s, C14), 138.98 (s, CH=N-), 148.94 (br s, C11), 148.98 (br s, C11), 151.25 (br s, CO1). Synthesis of compound G1-A-NIR

[0171] To a solution of dendrimer g (300, 0.05 mmol) in anhydrous CH3CN (30 mL) at 0°C, BrTMS (0.33 mL, 2.55 mmol) is added dropwise. After overnight stirring, the mixture is evaporated to dryness under reduced pressure. The crude residue is resuspended in 10 mL of MeOH. After one hour of stirring at room temperature, the solid is recovered by filtration and then washed twice with MeOH (20 mL) and with Et2O (20 mL). The resulting solid is dried under reduced pressure to give the G1-A-NIR dendrimer as a green powder with a quantitative yield. The compound is then converted to sodium selenium for analysis. To a suspension of phosphonic acid compound in water under stirring, 20 equivalents of a 0.1 M aqueous NaOH solution are added. The resulting solution is filtered through a 0.4 pM microfilter and then lyophilized to give the expected compound as a blue powder with a yield of 85%. [0172] [Chern 16]39 [0173] 31P NMR (121 MHz, Deuterium Oxide) δ: 6.78 (s, POHONa), 6.83 (s, POHONa), 9.26 (s, P=N), 64.09 (s, PS), 64.35 (s, PS). 5 Dendrimer G1-B [0174] [Chem 17]

[0175] The G1-B dendrimer is synthesized according to the reaction scheme shown in Scheme 3: 40 [Scheme 3] - Synthesis of compound 1-G’0 [0176] [Chem 18] 5

[0177] To a solution of N3P3Cl6 (2000 mg, 5.75 mmol) in THF (200 mL) at room temperature, K2CO3 (14.3 g, 103.5 mmol) and 4-hydroxybenzaldehyde (4634 mg, 37.95 mmol) are successively added. After 72 hours of stirring at room temperature, the mixture is filtered and then concentrated to dryness. The crude residue 10 is washed 4 to 5 times with MeOH to give compound 1-G’0 as a white powder with a yield of 81%. [P=N]341

[0178] 1H NMR (300 MHz, Chloroform-d): δ (ppm) 9.83 (s, 6H, CHO), 7.74 (d, 3JHH = 8.6 Hz, 12H, CO3H), 7.12 (d, 3JHH = 8.5 Hz, 12H, CO2H).

[0179] 31P NMR (121 MHz, Chloroform-d): δ (ppm) 7.15 (s, P0).

[0180] 13C NMR (75 MHz, Chloroform-d): δ (ppm) 190.47 (s, CHO), 154.44 (dd, 5 2JCP = 5.1 Hz, 4JCP = 2.5 Hz, CO1), 133.74 (s, CO4), 131.38 (s, CO3), 121.21 (d, 4JCP = 2.5 Hz, CO2). - Synthesis of compound 1-G’’0 [0181] [Chem 19] 10 [0182] A solution of compound 1-G’0 (500 mg, 0.580 mmol) in THF (50 mL) at room temperature is added an 8M methylamine solution in EtOH (1.3 mL, 10.45 mmol). After overnight stirring at room temperature, the mixture is concentrated to dryness to give the 1-G’’0 dendrimer as a white powder. 15 [0183] 1H NMR (300 MHz, Acetonitrile-d3): δ (ppm) 8.24 (d, 4JHH = 1.7 Hz, 6H, CH=N), 7.55 (d, 3JHH = 8.6 Hz, 12H, CO3H), 6.99 (d, 3JHH = 8.5 Hz, 12H, CO2H), 3.50 (s, 18H, N-CH3)

[0184] 31P NMR (121 MHz, Acetonitrile-d3): δ (ppm) 8.66 (s, P0).

[0185] 13C NMR (75 MHz, Acetonitrile-d3): δ (ppm) 160.80 (s, C=N), 151.57 (dd, 20 2JCP = 5.1 Hz, 4JCP = 2.5 Hz, C01), 133.94 (s, C04) 129.13 (C03), 120.66 (dd, 3JCP = 3.2 Hz, 5JCP = 1.8 Hz, C02), 47.29 (s, CH3). - Synthesis of the compound 1-G’’’0-HCl [0186] [Chem 20] [P=N]342

[0187] To a solution of 1-G’’0 dendrimer (500 mg, 0.53 mmol) in a THF/MeOH (25/5) mL mixture, NaBH4 (201 mg, 5.32 mmol) is added. After overnight stirring at room temperature, the mixture is concentrated to dryness. The crude residue is diluted in 200 mL of DCM and then washed once with 50 mL of distilled water. The organic phase is recovered, dried with Na2SO4, filtered, and concentrated under reduced pressure. The product is then dissolved in 30 mL of MeOH to which 9.5 mL of HCl (1 M in MeOH) is added. After 2 hours of stirring at room temperature, the solution is filtered and then concentrated under reduced pressure. The residue is then washed 3 times with 25 mL of CH2Cl2 to give the product 1-G’’’O-HCl (protonated form HCl) in the form of a white powder.

[0188] 1H NMR (400 MHz, Methanol-d4): δ (ppm) 7.54 (d, 3JHH = 8.5 Hz, 12H, C03H), 7.05 (d, 3JHH = 8.3 Hz, 12H, C02H), 4.27 (s, 12H, CH2NH), 2.75 (s, 18H, N-CH3).

[0189] 31P NMR (162 MHz, Methanol-d4): δ (ppm) 8.29 (s, P0). 15 [0190] 13C NMR (101 MHz, Methanol-d4): δ (ppm) 151.07 (dd, 2JCP = 5.1 Hz, 4JCP = 2.5 Hz, C01), 131.46 (s, C03), 128.71 (s, C04), 121.12 (dd, 3J = 3.2, 5J = 1.7 Hz, (s, C02), 51.30 (s, CH2NH), 31.88 (s, N-CH3 - Synthesis of the compound 1-G’’’0 [0191] [Chem 21] 20).

[0192] The compound 1-G’’’0-HCl (300 mg) is solubilized in 25 mL of distilled water to which 25 mL of a 2 M NaOH solution is added. The aqueous phase is then extracted 3 times with 200 mL of CH2Cl2. The organic phase is then dried with Na2SO4, filtered, and concentrated under vacuum to give the 25 1-G’’’0 dendrimer as a clear oil with a yield of 80%.

[0193] 1H NMR (400 MHz, Chloroform-d): δ (ppm) 7.12 (d, 3JHH = 8.5 Hz, 12H, C03H), 6.91 (d, 3JHH = 8.3 Hz, 12H, C02H), 3.69 (s, 12H, CH2NH), 2.43 (s, 18H, N-CH3). 43

[0194] 31P NMR (162 MHz, Chloroform-d): δ (ppm) 8.71 (s, P0).

[0195] 13C NMR (101 MHz, Chloroform-d): δ (ppm) 149.55 (dd, 2JCP = 5.1 Hz, 4JCP = 2.5 Hz, CO1), 136.70 (s, CO3), 128.99 (s, CO4), 120.82 (dd, 3J = 3.4 Hz, 5J = 1.7 Hz, CO2), 55.43 (s, CH2NH), 36.06 (s, N-CH3). 5 - Synthesis of compound 1-G1 [0196] [Chem 22]

[0197] To a solution of PSCl3 (3.109 mL, 30.60 mmol) in CH2Cl2 (100 mL) at room temperature, a solution of 1-10 G’’’0 dendrimer (800 mg, 0.85 mmol) in the presence of Et3N (0.740 mL, 5.31 mmol) in CH2Cl2 (25 mL) is added dropwise over a period of 30 min. After 3 h of stirring at room temperature, the mixture is concentrated to dryness under reduced pressure. The crude residue is then solubilized in 200 mL of CH2Cl2 and filtered through silica gel to give the 1-G1 dendrimer as a clear oil with a yield of 82%.

[0198] 1H NMR (300 MHz, Chloroform-d): δ (ppm) 7.22 (d, 3JHH = 8.5 Hz, 12H, CO3H), 6.99 (d, 3JHH = 8.3 Hz, 12H, CO2H), 4.60 (d, 3JHP = 15.1 Hz, 12H, CH2NH), 2.82 (d, 3JHP = 16.2 Hz, 18H, N-CH3).

[0199] 31P NMR (121 MHz, Chloroform-d): δ (ppm) 64.12 (s, P1), 8.35 (s, P0). 20 [0200] 13C NMR (75 MHz, Chloroform-d): δ (ppm) 150.26 (dd, 2JCP = 5.1 Hz, 4JCP = 2.5 Hz, C01), 132.73 (d, 3JCP = 6.5 Hz, C04), 129.17 (s, C03), 121.29 – 121.15 (m, C02), 54.18 (d, 2JCP = 5.5 Hz, CH2NH), 35.11 (d, 2JCP = 2.5 Hz, N-CH3). - Compound 3-G’1(OMe) [0201] [Chem 23] 25 44

[0202] To a previously prepared solution of compound 1-G1 (1 mmol) in THF (30 mL) with Cs2CO3 (24 mmol) is added phenol aza-bis-dimethylphosphonate (see WO 2005/052031 A1) (12 mmol). After overnight stirring at room temperature, the mixture is centrifuged, filtered, and then concentrated under reduced pressure. The crude residue is diluted in a minimum of THF and then washed with a large volume of diethyl ether. After drying under reduced pressure, the dendrimer 3-G’1(OMe) is obtained as a clear oil with a quantitative yield. [0203] 31P-{1H} (243 MHz, CDCl3) NMR δ = 8.40 (s, N=P), 26.85 (s, PO), 68.32 (s, 10 PS); [0204] 1H NMR (600 MHz, CDCl3) δ = 2.75 (d, 3JHH = 10.1 Hz, 24H, C14-CH2), 2.77 (d, 3JHP = 7.6 Hz, 18H, CH3), 3.06 (d, 3JHH = 7.7 Hz, 24H, C14-CH2-CH2), 3.19 (d, 2JHP = 9.4 Hz, 48H, CH2-P), 3.73 (d, 3JHP = 10.6 Hz, 144H, POMe), 6.94 (d, 3JHH = 8.2 Hz, 12H, C02H), 7.07 (d, 3JHH = 8.1 Hz, 24H, C12H), 7.18 (br d, 3JHH = 8.2 15 Hz, 36H, C03 and C13H); [0205] 13C-{1H} NMR (151 MHz, CDCl3) δ = 33.05 (s, CH2), 33.64 s, (CH3N), 49.46 (dd, 1JCP = 157.3, 3JCP =7.2 Hz, CH2-P), 53.56-51.75 (m, POCH3), 53.47 (d, 2JCP = 10.3 Hz, C14-CH2), 58.22 (t, 3JCP = 7.7 Hz, C14-CH2-CH2), 120.91 (br d, 3JCP = 4.4 Hz, C02 and C12), 129.30 (s, C03), 129.87 (s, C13), 134.10 (br d, 2JCP = 4.5 Hz, 20 C04), 136.22 (s, C14), 149.35 (d, 2JCP = 7.5 Hz, C11), 150.01 (br s, C01) ppm. - Compound G1-B [0206] [Chem 24]

[0207] To a solution of 3-G1(OMe) dendrimer (1 g, 0.17 mmol) in anhydrous CH3CN25 (30 mL) at 0°C, BrTMS (1.34, 10.2 mmol) is added dropwise. After overnight stirring, the mixture is concentrated to dryness under reduced pressure. The residue is stirred for one hour in MeOH (10 mL), then washed twice with MeOH (20 mL) and once with Et2O (20 mL). The resulting solid is dried under reduced pressure to give the G1-B dendrimer as a white powder with a quantitative yield.

[0208] Compound G1-B is not sufficiently soluble in water to be analyzed by NMR; it is converted into monosodium hydroxide according to the following procedure:

[0209] To a suspension of G1-B in water under stirring, 24 equivalents of a 0.1 M aqueous NaOH solution are added. The resulting solution is filtered on a 0.4 pM microfilter and then lyophilized to give the saline form of G1-B (24 sodium atoms) as a white powder with a yield of 90%. [0210] 31P-{1H} (162 MHz, D2O/CD3CN) NMR6 = 6.93 (s, PO), 9.59 (s, N=P), 68.77(s, PS); [0211] 1H NMR (600 MHz, D2O/CD3CN) 5 =2.76 (d, 3Jhp = 10.7 Hz, 18H, CH3), 3.13 (t, 3Jhh = 8.8 Hz, 24H, Ci4-CH2), 3.47 (d, 2Jhp =11.9 Hz, 48H, CH2-P), 3.72 (t, 3Jhh = 8.6 Hz, 24H, Ci4-CH2-CH2), 4.47 (d, 2Jhp = 13.5 Hz, 12H, Co4-CH2), 6.91 (d, 2Jhh = 8.1 Hz, 12H, Co2H), 7.15 (d, 2Jhh = 8.1 Hz, 24H, Ci2H), 7.29 (d, 2Jhh = 8.3 Hz, 12H, Co3H),7.38 (d, 2Jhh = 8.2 Hz, 24H, Ci3H); [0212] 13C-{1H} NMR (151 MHz, D2O/CD3CN) 5 =29.07 (s, Ci4-CH2). 33.58 (s, CH3), 52.91 (d, 1Jcp = 127.6 Hz, CH2-P), 52.94 (s, Co4-CH2), 57.81 (s, Ci4-CH2-CH2). 121.14(brs, Co2), 121.42 (d,3JCp = 4.4 Hz C12), 129.61 (s, Co3), 130.59 (s, C13), 133.95 (s, C14), 134.89 (s, Co4), 149.18 (s, Co1), 149.60 (d, 3Jcp = 7.6 Hz, C11) ppm. Dendrimere G1-B-NIR

[0213] Synthesis diagram: [Diagram 4]46 Compound m [0214] [Chem 25] 5 47

[0215] A solution of 4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde (Fu H. et al. Molecules, 2014,16:17715-17726) (1 g, 4.83 mmol) in THF (30 mL) is added to a solution of methylamine (8 M in EtOH) (1.8 mL, 14.49 mmol). After overnight stirring at room temperature, the mixture is concentrated at 5 sec to give product m as a colorless oil with a yield of 95%. [0216] 1H NMR (300 MHz, Methanol-d4) δ (ppm): 1.39 – 1.74 (m, 3H, -CH2-), 1.75 – 1.92 (m, 2H, -CH2-), 1.90 – 2.06 (m, 1H, -CH2-O), 3.55 – 3.62 (m, 1H, -CH2- O), 3.79 – 3.87 (m, 1H, -CH2-), 5.46 (t, 3JHH = 3.1 Hz, 1H, O-CH-O), 7.08 (d, 10 3JHH = 8.8 Hz, 2H, C02H), 7.63 (d, 3JHH = 8.8 Hz, 2H, C03H), 8.21 (q, 4JHH = 1.6 Hz, 1H, -CH=N-). [0217] 13C NMR (75 MHz, Methanol-d4) δ (ppm): 18.46 (s, -CH2-), 24.89 (s, -CH2-), 29.95 (s, -CH2-), 46.32 (s, CH3-), 61.73 (s, -CH2-O), 96.10 (s, -CH-O), 116.17 (s, C02), 129.22 (s, C04), 129.30 (s, C03), 159.41 (s, C04), 163.46 (s, C=N). 15 Compound n [0218] [Chem 26]

[0219] A solution of compound m (1 g, 4.5 mmol), 10% Pd/C (225 mg), and MeOH (40 mL) are introduced into a Fisher-Porter tube. The tube is slowly placed under vacuum to expel the air. The H2 pressure is then adjusted to 5 bar. The reaction mixture is stirred for 2 hours at room temperature. After slow depressurization, the reaction mixture is collected, filtered, and concentrated under reduced pressure. The residue is purified by silica gel chromatography (eluent: AcOEt) to obtain compound n 25 as a clear oil with a yield of 80%. [0220] 1H NMR (300 MHz, Chloroform-d) δ (ppm): 1.33 – 1.72 (m, 3H, -CH2-), 1.77 – 1.82 (m, 2H, -CH2-), 1.87 – 2.07 (m, 1H, -CH2-O), 2.36 (s, 3H, CH3), 3.50 – 3.57 (m, 1H, -CH2-O), 3.61 (s, 2H, -CH2-N), 3.76 – 3.99 (m, 1H, -CH2-), 5.34 (t, 3JHH = 3.3 Hz, 1H, O-CH-O), 6.96 (d, 3JHH = 8.6 Hz, 2H, C02H), 7.09 – 7.26 (m,2H, 30 C02H). 48 [0221] 13C NMR (75 MHz, Chloroform-d) δ (ppm): 18.78 (s, -CH2-), 25.16 (s, -CH2-), 30.33 (s, -CH2-), 35.63 (s, -CH3), 55.31 (s, -CH2-N), 61.96 (s, -CH2-O), 96.38 (s, O-CH-O), 116.36 (s, C02), 129.25 (s, C03), 132.78 (s, C03), 156.12 (s, C04). Compound o 5 [0222] [Chem 27]

[0223] A solution of compound n (1 g, 4.52 mmol) in the presence of Et3N (0.62 mL, 4.52 mmol) in DCM (40 mL) is added dropwise to a solution of PSCl3 (2.2 mL, 22.6 mmol) in DCM (100 mL) over a period of 30 min. After 4 h of stirring at room temperature, the reaction mixture is concentrated to dryness under reduced pressure. The crude residue is then solubilized in 200 mL of DCM and filtered through silica gel to give dendron o as a clear oil with a yield of 85%. [0224] 31P NMR (162 MHz, Chloroform-d) δ (ppm): 63.18 (s, PS). 15 Compound p [0225] [Chem 28]

[0226] To a solution of compound o (1 mmol) in THF (30 mL) with Cs2CO3 (4 mmol), phenol aminobismethylene phosphonate derived from 20-tyramine (Rolland et al. (2008), A European Journal, 14: 4836-4850) (2 mmol) is added. After overnight stirring at room temperature, the mixture is centrifuged, filtered, and then concentrated under reduced pressure. The crude residue is then purified by silica gel chromatography with a mixture of eluent (MeOH/EtOAc) to give, after solvent removal under reduced pressure, compound p as a clear oil with 85% yield. [0227] 31P NMR (162 MHz, Chloroform-d) δ (ppm): 26.95 (s, POMe), 68.48 (s, P=N). [0228] 1H NMR (400 MHz, Chloroform-d) δ (ppm): 1.58-1.72 (m, 3H, -CH2), 1.81-1.94 (m, 2H, CH2), 1.96-2.05 (m, 1H, CH2), 2.78-2.83 (m, 7H, C14-CH2, CH3N), 3.09 (t, 3JHH = 7.6 Hz, 4H, CH2N), 3.22 (d, 3JHH = 8.9 Hz, 8H, ), 3.55-3.67 (m, 1H, 5 CH2-O), 3.75 (d, 3JHP = 10.3 Hz, 24H, POMe), 3.89-3.95 (m, 1H, CH2-O), 4.44 (d, 3JHP = 12.0 Hz, 2H), 5.41 (t, 3JHH = 3.3 Hz, 1H, O-CH2-O), 6.99 (d, 3JHH = 8.6 Hz, 2H, C02H), 7.12 (d, 3JHH = 8.4, 4H, C12H), 7.16-7.25 (m, 6H, C03H, C13H). Compound q [0229] [Chem 29] 10

[0230] Pyridinium p-toluenesulfonate (1.5 mmol) is added to a solution of compound p (1 mmol) in MeOH (30 mL). The reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure, and the residue is purified by silica gel chromatography (eluent: 15 MeOH/AcOEt) to give, after solvent removal under reduced pressure, compound q as a clear oil with a yield of 80%. [0231] 31P NMR (162 MHz, Chloroform-d) δ (ppm): 26.76 (s, POMe), 68.38 (s, PS). Compound r

[0232] To a suspension of dendron q (1 g, 1.04 mmol) in THF (30 mL) with 20 of Cs2CO3 (794 mg, 2.08 mmol), hexachlorocyclotriphosphazene (70 mg, 0.20 mmol) is added. After overnight stirring at room temperature, the reaction mixture is centrifuged, filtered through Celite, and then concentrated under reduced pressure. Compound r is obtained as a clear oil with a yield of 90%. 25 [0233] [Chem 30] 50 [0234] 31P NMR (121 MHz, Chloroform-d) δ (ppm): 6.50 (d, 2JPP = 83.3 Hz, P=N), 21.50 (dd, 2JPP = 85.2 Hz, 2JPP = 81.6 Hz, P=N), 26.41 (s, POMe), 68.12 (s, PS). Compound s 5 [0235] To a suspension of compound b (130 mg, 0.30 mmol) in THF (30 mL) with Cs2CO3 (199 mg, 0.61 mmol) is added compound r (989 mg, 0.2 mmol). After overnight stirring at 45°C, the mixture is centrifuged, filtered, and then concentrated under reduced pressure. The residue is purified on a silica column with DCM/MeOH (60/40) to give the dendrimer t in 90% yield. The product can be purified by flash chromatography on a silica column. [0236] [Chem 31] [0237] 31P NMR (162 MHz, Chloroform-d) δ (ppm): 8.45 (s, P=N), 26.89 (s, POMe), 68.33 (s, PS). Compound t

[0238] To a solution of dendrimer s (500, 0.093 mmol) in anhydrous acetonitrile (3 mL), the azide compound NIR (43.4 mg, 0.18 mmol) is added. The reaction mixture is stirred for three days at room temperature. The solvent is then removed under reduced pressure to give dendrimer t with a quantitative yield of 20 in the form of a blue oil. [0239] [Chem 32] [0240] 31P NMR (162 MHz, Chloroform-d) δ (ppm): 8.40 (s, P=N), 26.86 (s, POMe), 68.38 (s, PS). 5 G1B-NIR

[0241] To a solution of dendrimer t (500, 0.086 mmol) in anhydrous CH3CN (50 mL) maintained at 0°C, BrTMS (0.56 mL, 4.38 mmol) is added dropwise. After overnight stirring, the mixture is concentrated to dryness under reduced pressure. The crude residue is resuspended in 10 mL of MeOH. After 1 hour 10 minutes of stirring at room temperature, the solid is recovered by filtration and then washed twice with MeOH (20 mL) and with Et2O (20 mL). The resulting solid is then dried under reduced pressure to give the G1B-NIR dendrimer as a green powder with a quantitative yield. [0242] [Chem 33] 15 52

[0243] The compound is then converted to a sodium salt for analysis. To a suspension of the phosphonic acid compound in water under stirring, 20 equivalents of a 0.1 M aqueous NaOH solution are added. The resulting solution is filtered through a 0.4 µM microfilter and then lyophilized to give the expected compound as a blue powder with a yield of 85%. [0244] [Chem 34] [0245] 31P NMR (162 MHz, Deuterium Oxide) δ (ppm): 6.66 (s, PO3HNa), 6.69 (s, PO3HNa), 6.74 (s, PO3HNa), 9.42 (s, P=N), 68.49 (s, PS), 68.69 (s, PS). 10 Dendrimer G1-C [Chem 35] P3N3 O N O N O P P OH OX OH OX 6 N N N O N O P P O OH OX OH OX O X= H or Na 15 Compound u [Chem 36] 5 10 15 20 CA 03165406 2022-06-20 53 o

[0246] To a solution of cyanuric chloride (1 g, 5.4 mmol) in dichloromethane (60 mL) at 0°C, phenol aminobismethylenephosphonate derived from tyramine (see WO 2005/052031 A1) (2.1 g, 5.4 mmol) and N,N-diisopropylethylamine (296 mg, 5.7 mmol) are added. After one hour of stirring at 0°C, a further addition of phenol aminobismethylenephosphonate derived from tyramine (2.1 g, 5.4 mmol) and N,N-diisopropylethylamine (696 mg, 5.4 mmol) is made at 0°C. The reaction mixture is then left to stir at room temperature overnight. After filtration and removal of solvents under reduced pressure, the residue is purified by silica gel chromatography (eluent: DCM/MeOH; 98/O2) to give compound u as a yellow oil with a yield of 80%. [0247] 31P-{1H} NMR (121 MHz, Chloroform-d) 5 (ppm): 26.55 (s, POMe). [0248] 1H NMR (300 MHz, Chloroform-d) 5 (ppm): 2.72 (t, J = 7.3 Hz, 4H), 3.02 (t, J = 7.4 Hz, 4H), 3.10 (d, J = 9.4 Hz, 8H), 3.62 (d, J = 10.5 Hz, 24H), 6.96 (d, J = 8.5 Hz, 4H), 7.20 (d, J = 8.6 Hz, 4H). [0249] 13C-{1H} NMR (75 MHz, Chloroform-d) 5 (ppm): 32.94 (CH2), 49.39 (dd, J = 157.9 Hz, J = 7.5 Hz, CH2), 51.65-53.36 (m, CH3), 57.81 (t, J = 7.7 Hz, CH2), 120.83 (CH), 130.07 (CH), 137.69 (C), 149.54 (C), 172.39 (C), 173.36 (C). Compose v [chem 37]5 10 15 20 CA 03165406 2022-06-20 54 '''OMe H^OMe OMe II 'OMe O ^^OMe rOOMe

[0250] To a solution of compound u (2 g, 2.28 mmol) in THF (40 mL), p-hydroxy-N-methylbenzylamine (324 mg, 4.56 mmol) and N,N-diisopropylethylamine (794 mg, 6.84 mmol) are added at room temperature. The reaction mixture is then stirred at 60°C overnight. After filtration and removal of solvents under reduced pressure, the crude residue is purified by silica gel chromatography (eluent: DCM/MeOH; 95/05) to give compound v as a clear oil with a yield of 70%. [0251] 31P-{1H} NMR (121 MHz, Chloroform-d) 6 (ppm): 26.73 (s, POMe), 26.94 (s, POMe). [0252] 1H NMR (300 MHz, Chloroform-d) 6 (ppm): 2.84 - 2.79 (m, 4H), 3.02-3.15 (m, 4H, CH2, 3H CH3), 3.21 (d, 2Jhp = 9.1 Hz, 8H, CH2), 3.79-3.72 (m, 24H, CH3), 4.33 (s, 2H, CH2), 6.70 (br d, 4H, CH2), 7.02 (d, 3Jhh = 8.5 Hz, 2H, CH2), 7.09 (d, 3Jhh = 8.5 Hz, 2H, CH2), 7.19-7.28 (m, 4H, CH2). [0253] 13C-{1H} NMR (75 MHz, Chloroform-d) 5 (ppm): 32.82 (s, CH2), 33.17 (s, CH2), 35.34 (s, CH3), 47.15-51.27 (m, CH2), 52.74-52.64 (m, CH3), 57.14-58.97 (m, CH2), 115.45 (s, CH), 121.59 (s, CH), 121.96 (s, CH), 127.41 (s, Cq), 129.47 (s, CH), 129.64 (s, CH), 136.12 (s, Cq), 136.37 (s, Cq), 150.49 (s, Cq), 150.99 (s, Cq), 156.65 (s, Cq), 167.11 (s, Cq), 172.21 (s, Cq), 172.29 (s, Cq). Compose w [Chern 38]55

[0254] Hexachlorocyclotrisphosphazene (0.14 mmol) is added at room temperature to a suspension of compound v (1.02 mmol) in THF (30 mL) with Cs2CO3 (2.04 mmol). After overnight stirring at 40 °C, the mixture is centrifuged, filtered, and then concentrated under reduced pressure. Compound w is obtained as a clear oil with a quantitative yield. [0255] 31P-{1H} NMR (121 MHz, Chloroform-d) δ (ppm): 8.11 (s, N=P), 27.06 (br s, POMe). [0256] 1H NMR (300 MHz, Chloroform-d) δ (ppm): 2.77 (m, 24H), 2.87-2.96 (m, 10 18H), 3.05 (t, J = 8.3 Hz, 24H), 3.12-3.30 (m, 48H), 3.60-3.88 (m, 144H), 4.64 (s, 12H), 6.89 (d, J = 8.6 Hz, 10H), 7.01 (m, 16H), 7.03-7.10 (m, 24H), 7.14-7.24 (m, 24H). Compound G1-C 15 A. A solution of compound w (1 g, 0.16 mmol) in anhydrous CH3CN (50 mL) at 0°C is dropwise added bromotrimethylsilane (1.4 g, 9.6 mmol). After overnight stirring, the reaction mixture is concentrated to dryness under reduced pressure. The residue is stirred for one hour in MeOH (10 mL), then washed three times with MeOH (20 mL) and once with Et2O (20 mL). The resulting solid 20 is dried under reduced pressure to give compound G1-C as a neutral phosphonic acid, a white powder with a yield of 80%.

[0257] The compound is then converted to a sodium salt for analysis. To a suspension of the phosphonic acid compound in water under stirring, 25 equivalents of a 0.1 M aqueous NaOH solution are added. The resulting solution is filtered through a 0.4 µM microfilter and then lyophilized to give the expected compound as a white powder with a yield of 90%. [0258] 31P-{1H} NMR (162 MHz, Deuterium Oxide) δ (ppm): 9.64 (s, N-P), 6.70 (br s, PO3HNa). 5 [0259] 1H NMR (400 MHz, Deuterium Oxide) δ (ppm): 2.96 (br s, 18H, CH3), 3.20 (br s, 24H, CH2), 3.37-3.41 (m, 48H), 3.79 (br s, 24H, CH2), 6.72-6.98 (m, 10H, CH), 7.00-7.29 (m, 38H, CH), 7.36-7.65 (m, 24H, CH). [0260] 13C-{1H} NMR (101 MHz, Deuterium Oxide) δ (ppm): 28.95 (s, CH3), 53.42 (br s, CH2), 54.66 (br s, CH2), 57.35 ((br s, CH2), 121.02 (s, CH), 121.76 (s, CH), 10 129.16(s, CH2), 130.46 (,CH), 134.52 (s,C), 150.54(s,C), 171.65(br s,C=N Compound G1-C-NIR [0261] [chem 39] 15 Compound x [0262] [chem 40] o57 P3N3 O N O N O P P OMe). OMe OMe OMe 5 Cl N N N O N O P P O OMe OMe OMe OMe O

[0263] Hexachlorocyclotrisphosphazene (70 mg, 0.20 mmol) is added to a solution of compound v (0.9 g, 1.0 mmol) in THF (30 mL) with Cs2CO3 (794 mg, 2.08 mmol). After overnight stirring at room temperature, the mixture is centrifuged, filtered, and then concentrated under reduced pressure. Compound x is obtained as a clear oil with a yield of 90%. [0264] 31P-{1H} NMR (121 MHz, Chloroform-d) δ (ppm): 6.35 (d, 2JPP = 82.2 Hz, P=N), 20.76-20.98 (m, P=N), 26.41 (s, POMe). Compound y 10 [0265] [Chem 41]

[0266] To a solution of compound b (75 mg, 0.17 mmol) in THF (30 mL) with Cs2CO3 (199 mg, 0.34 mmol) is added compound x (900 mg, 0.17 mmol). After overnight stirring at 45°C, the mixture is centrifuged, filtered, and then concentrated under reduced pressure to give compound y with a quantitative yield. [0267] 31P-{1H} NMR (162 MHz, Chloroform-d) δ (ppm): 8.45 (s, P=N), 26.89 (br s, POMe). 5 10 15 20 CA 03165406 2022-06-20 58 Compound z [0268] [Chem 42]

[0269] To a solution of compound y (500 mg, 0.092 mmol) in anhydrous acetonitrile (5 mL), compound NIR (as described in S. A. Klymchenko; V. G. Pivovarenko; O. Turan; D. P. Alexander, New Journal of Chemistry, 27(9), 1336-1343; 2003) (43.4 mg, 0.18 mmol) is added. The reaction mixture is stirred for three days at room temperature. The solvent is concentrated under reduced pressure to give the dendrimer z in quantitative yield as a blue oil. [0270] 31P-{1H} NMR (162 MHz, Chloroform-d) 5 (ppm): 8.51 (s, P=N), 26.72 (br s, POMe). Compose G1-C-NIR

[0271] To a solution of dendrimer z (500, 0.086 mmol) in anhydrous CH3CN (50 mL) at 0°C, bromotrimethylsilane (0.56 mL, 4.38 mmol) is added dropwise. After overnight stirring, the mixture is concentrated to dryness under reduced pressure. The residue is stirred for one hour in MeOH (10 mL), then washed twice with MeOH (20 mL) and once with Et2O (20 mL). The resulting solid is dried under reduced pressure to give the G1-C-NIR dendrimer as a green powder with a quantitative yield. The compound is then converted to sodium selenium for analysis. To a suspension of phosphonic acid compound in water under stirring, 20 equivalents of a 0.1 M aqueous NaOH solution are added. The resulting solution is filtered through a 0.4 pM microfilter and then lyophilized to give the expected compound as a blue powder with a yield of 80%. CA 03165406 2022-06-20 59 [0272] 31P-{1H} NMR (162 MHz, Deuterium Oxide) 6 (ppm): 7.79 (br s, PO3HNa), 9.42 (s, P=N). 2. Preparation of vesicles 5 [0273] The dendrimer formulation is obtained by mixing in water the catanionic surfactant TriCat-n and the acid dendrimer precursor G1-X [Fig. 1].

[0274] The formula for TriCat12 is as follows: [0275] [Chern 42]

[0276] The TriCat8 formula is as follows: [0277] [Chern 43]

[0278] The TriCat16 formula is as follows: 15 [0279] [Chern 44]5 10 15 20 25 CA 03165406 2022-06-20 60 Counterexample: Preparation of a vesicle comprising the ABP dendrimer

[0280] 5.82 mg of ABP dendrimer, 1.02 mg of N-dodecylamino-1-deoxylactitol (L-Hyd 12), and 0.87 mg of bis-α-(hydroxydodecyl)phosphinic acid are added at room temperature to 2 mL of ultrapure water. The mixture is vortexed for 5 min and then sonically aspirated using an ultrasonic probe for 15 min at power level 3 with 30% active cycles, controlling the temperature at 45°C. Preparation of a vesicle comprising the G1-A dendrimer (TriCat12/G1-A)

[0281] 5.29 mg of G1-A dendrimer, 1.02 mg of N-dodecylamino-1-deoxylactitol (L-Hyd 12), and 0.87 mg of bis-α-(hydroxydodecyl)phosphinic acid are added at room temperature to 2 mL of ultrapure water. The mixture is vortexed for 5 min and then sonically aspirated using an ultrasonic probe for 15 min at power level 3 with 30% active cycles, controlling the temperature at 45°C. Preparation of a vesicle comprising the G1-X-NIR dendrimer (TriCat12/G1-X-NIR)

[0282] 5.31 mg of G1-X-NIR dendrimer, 1.02 mg of N-dodecylamino-1-deoxylactitol (L-Hyd 12), and 0.87 mg of bis-α-(hydroxydodecyl)phosphinic acid are added at room temperature to 2 mL of ultrapure water. The mixture is vortexed for 5 min and then sonically aspirated using an ultrasonic probe for 15 min at power level 3 with 30% active cycles, controlling the temperature at 45°C. Preparation of a vesicle comprising the G1-B dendrimer (TriCat12/G1-B)

[0283] 5.21 mg of G1-B dendrimer, 1.02 mg of N-dodecylamino-1-deoxylactitol (L-Hyd 12), and 0.87 mg of bis-α-(hydroxydodecyl)phosphinic acid are added at room temperature to 2 mL of ultrapure water. The mixture is vortexed for 5 min and then left under magnetic stirring (500 rpm) for 24 h at room temperature. Preparation of a vesicle comprising G1-C dendrimer (TriCat12/G1-C)

[0284] 5.3 mg of G1-C dendrimer, 1.02 mg of N-dodecylamino-1-deoxylactitol (L-Hyd 12), and 0.86 mg of bis-α-(hydroxydodecyl)phosphinic acid are added at room temperature to 2 mL of ultrapure water. The mixture is vortexed for 5 min and then sonically aspirated using an ultrasonic probe for 15 min at power level 3 with 30% active cycles, controlling the temperature at 45°C. Preparation of a vesicle comprising G1-B dendrimer and TriCat8 (TriCat8/G1-B)

[0285] 5.21 mg of G1-B dendrimer, 0.94 mg of N-octylamino-1-deoxylactitol (L-Hyd 8), and 0.86 mg of bis-α-(hydroxydodecyl)phosphinic acid are added at room temperature to 2 mL of ultrapure water. The mixture is vortexed for 5 min and then left under magnetic stirring (500 rpm) at 40°C for 15 minutes and then for 24 h at room temperature. Preparation of a vesicle comprising G1-B dendrimer and TriCat 16 (TriCat16/G1-B)

[0286] 5.21 mg of G1-B dendrimer, 1.16 mg of N-hexadecylamino-1-deoxylactitol (L-Hyd 16), and 0.86 mg of bis-α-(hydroxydodecyl)phosphinic acid are added at room temperature to 2 mL of ultrapure water. The mixture is vortexed for 5 min and then left under magnetic stirring (500 rpm) at 40°C for 15 minutes and then for 24 h at room temperature. 3. Measurement by DLS

[0287] The size and size distribution of the vesicles were measured by DLS (dynamic light scattering). Each analysis was performed on the samples, filtered at 1.2 µm with a Minisart cellulose acetate filter, and placed in plastic cuvettes (semi-micro polystyrene cuvettes, Brand). The measurements were performed on a Malvern Instruments Nano S or Nano ZS instrument, both equipped with a He-Ne laser that emits monochromatic light with a wavelength of 633 nm. The scattered intensity was measured at 25.0°C ± 0.1°C at an angle of 173°. The result is an average of 4 measurements and was processed using the Contin algorithm. 4. Measurement of the transition temperature

[0288] The samples are prepared saturated at 1.10⁻² M in TriCat12. Equimolar amounts of L-Hyd12 and phosphonic acid are dispersed at a concentration of 1.10⁻² M and of the dendrimer at a concentration of 5.10⁻⁴ M in 2 mL of water. The mixture is stirred at 300 rpm for 2 days. Two methods are performed: the first consists of lyophilizing the medium and then dispersing it in 100 µL of water followed by the classic vesicle formation protocol. The second method consists of sonicating 100 µL of the medium immediately without the lyophilization step. Identical results are obtained by both methods.

[0289] Another method consists of sonicating the mixture immediately following the classic protocol without going through the stirring stage; similar results are obtained, but with this method there is more background noise.

[0290] The measurements are then carried out on a DSC 1 STARe System instrument with a MultiSTAR® HSS8 sensor. In this device, the sample crucible and the reference crucible are placed in the same furnace. To maximize the intensity of the product signal, the reference crucible is filled with 75 pL of the solvent, while 75 pL of the solution to be analyzed are placed in the sample crucible. The developed method consists of heating the sample from 25 to 5°C at 5°C/min and then performing four cycles from 5 to 60°C at 2°C/min. 5. Measurement of the encapsulation rate

[0291] In order to evaluate the encapsulation efficiency of the dendrimer in TriCat12/dendrimer vesicles, a spectrofluorimetric assay was developed using the fluorescent analogue of the dendrimer, G1-X-NIR.

[0292] The total quantity of G1-X-NIR (encapsulated or not) was determined by measuring the fluorescence intensity (IF) of a sample in which the vesicular structures were disrupted by adding methanol to the solution (1 mL of methanol per 20 pL of vesicular solution).5 10 15 20 25 CA 03165406 2022-06-20 63

[0293] In parallel, the unencapsulated G1-X-NIR was separated from the vesicles by 1.2 µm filtration (Minisart Cellulose Acetate filter, Sartorius). A controlled solution of G1-X-NIR made it possible to verify the removal of 70% (SD = 3%) of G1-X-NIR by this technique (to be taken into account in the calculation). After dilution of the filtrate in methanol (1 mL methanol per 20 pL of filtrate), the measurement of the fluorescence intensity (IF) made it possible to quantify the predominantly encapsulated G1-X-NIR. [Math. 1] % elimination = [1- ((IF dendrimer© encap$uie)/(iF dendrimere totals)}] x lOOx 1.43 [Math. 2] EE (%) = 100- % elimination 6. Observation by cryoMEB

[0294] The observation of the vesicles was carried out by cryofracture scanning electron microscopy (cryoSEM). The instrument used for the analysis is an ESEM Quanta FEG250 from the company FEI (Platerforme TRI-Genotoul), operating at 5 kV and approximately 1 x 10⁴ Pa. The solutions are deposited on the sample holder and then solidified in a pasty nitrogen solution (-210°C). They are then fractured in a chamber at -140°C. The sample then undergoes a sublimation step at -90°C for 5 minutes. The sample then undergoes platinum plasma metallization for 60 seconds. It is transferred for observation to the cryogenic chamber of the SEM, maintained at all times at -140°C by a flow of gaseous nitrogen. 7. Measurement of the stability of the formulation at 4°C

[0295] TriCat12/dendrimer solutions are stored at 4°C in DLS tanks. At the time of size measurement, the solution is brought to room temperature for 5 min, then the size is measured at 25°C in DLS. 8. Measurement of formulation stability after freezing 5 10 15 20 25 30 Date Re9ue/Date Received 2024-04-12 64

[0296] To evaluate the freezing stability of the formulation, aliquots of the TriCat12/dendrimer formulation are frozen at -20°C. After a time t (days), the solution is thawed in an ultrasonic bath at 25°C for 10 min, filtered at 1.2 µm, and then the size is measured in DLS at 25°C. 9. Preparation of a pharmaceutical formulation of xanthan gum comprising TriCat12/G1-X vesicles

[0297] A 2% wt Xanthan gum solution in water is prepared: 20 mg of Xanthan gum is added by sprinkling under stirring at 500 rpm into 1 mL of 0.2 µm filtered water. The mixture is stirred for a minimum of 1 hour. Then, an equivolume mixture of Tricat12/G1-X vesicles and 2% wt Xanthan gum gel is prepared. This mixture is left under stirring at 500 rpm for a minimum of 4 hours. 10. Physical stability measurement

[0298] The study of the physical stability of TriCat12/G1-B vesicles and the pharmaceutical formulation of TriCat12/G1-B in xanthan gum is measured using a Turbiscan Lab™ Expert (Formulation). The Turbiscan™ detects variations in the intensity of transmitted light at different heights (z) of the sample over time, thus allowing direct monitoring of local physical heterogeneities. Measurements are performed according to a protocol of 145 scans/hour, then 145 scans/24h at 25°C. 11. Stability measurement by NMR

[0299] The G1-X compounds were solubilized in water, then the pH was adjusted to pH 4, and the solutions were stored at 25°C protected from light. Just before NMR analysis, the solution was lyophilized, and the resulting powder (20 to 50 mg) was diluted in 0.5 to 0.7 mL of D2O in the presence of 6 drops of CD3CN. The TriCat 12/G1-B preformulations immobilized in xanthan gel were prepared according to the invention; if necessary, the pH was adjusted to pH 4, and the solutions were stored at 25°C protected from light. The NMR spectra were recorded at 25°C after adjusting the pH to pH 7 and then lyophilizing. The resulting powder is moistened with a few drops of D2O before analysis by solid-state NMR (MAS NMR). 5 10 15 20 25 30 CA 03165406 2022-06-20 65 12. Evaluation of in vitro cutaneous penetration on Franz cells. Quantification of the fluorescent dendrimer (G1X-NIR) in the skin

[0300] The Franz continuous flow cell diffusion method (PermGear®, Standard Franz Cells) is used to study in vitro the cutaneous penetration of the formulation over time without disrupting the system. These experiments were performed on pig ear skin, which represents a predictive model of human cutaneous penetration, having histological and physiological properties similar to human skin. Pig ear skin samples were obtained from the slaughterhouse (Arcadie Sud-Ouest, Montauban) so that the skin tissue was intact. After dissecting the skin from the underlying tissue, the skin was cleaned with water, degreased, depilated, and then cut into squares of approximately 2 cm. These skin patches were frozen at -80°C for subsequent use.

[0301] During the experiment, the skins were thawed at room temperature and placed on the Franz cells in the donor compartment so that the side bathed in the receiving liquid corresponds to the inner face of the skin while the side in contact with the air is that of the stratum corneum.

[0302] A fluorescent dendrimer analog with near-infrared (G1-X-NIR) emission is used for in vitro skin penetration and retention studies. A dose (300 µl) of PBS (blank), G1-X-NIR alone, or formulated in TriCat12/G1-X-NIR vesicles was deposited onto the surface of the stratum corneum using a micropipette to cover the diffusion area of 1.77 cm² for 24 h. The outer surface of the cell was covered with parafilm to prevent evaporation and volume changes. The receptor compartment in contact with the inner skin surface was filled with 12 mL of a phosphate buffer solution (PBS; pH=7.45), thermostated at 37°C and maintained under constant stirring (300 rpm) to ensure constant volume renewal, which approximates physiological conditions.

[0303] After 24 h, the skin is rinsed with PBS to remove the remaining deposit and dried with absorbent paper. The treated area is then cut into small pieces in a pillbox with 3 mL of PBS and homogenized using a disperser (Ultra-Turrax Ika®- Werke, T 25 Basic) for 5 min at 21500 rpm. The dispersion is then filtered at 0.45 µm (Minisart Cellulose Acetate filter, Sartorius) and the dendrimer in the filtrate is quantified by spectrofluorometry (excitation wavelength = 632 nm and emission wavelength = 650 to 850 nm). Observation of cutaneous penetration by confocal microscopy

[0304] Confocal microscopy is chosen to evaluate the penetration depth of G1-X-NIR into the different layers of the skin (stratum corneum, viable epidermis and dermis). The first step is the diffusion of G1-X-NIR onto Franz cells, according to the protocol described above.

[0305] After 24 hours of application, the skin is gently rinsed with PBS and dried. The treated area is cut into three equal pieces to be representative of the entire skin. These three pieces are frozen, embedded in OCT (TissueTek®) in a plastic mold (Tissue-Tek® Cryomold®), in isopentane cooled to -80°C. 10 µm thick sections obtained using a cryostat (CM1950, Leica) are then observed using a Leica SP8 confocal microscope with an inverted objective (63X), using oil immersion. The parameters used during observation on the LAS X software are: excitation wavelength = 635 nm and emission wavelength = 650 to 800 nm. Skin treated with PBS is used as a blank to evaluate skin autofluorescence. The resulting images are then processed using Fiji software with the Bio-formats plugin. 13. Monocyte purification and FACS analysis

[0306] Human blood from healthy donors is collected from the French Blood Establishment (EFS, Toulouse, France). Peripheral blood mononuclear cells (PBMCs) are then separated from the blood using a density gradient with Pancoll's solution (PANBiotech GmbH) by centrifugation at 1200 rpm for 20 min at 20°C. From the PBMCs, monocytes are isolated by negative selection with antibodies directed against all blood cells (T cells, B cells, NK cells, dendritic cells, erythrocytes and granulocytes) except monocytes using the Dynabeads® Untouched™ Human Monocytes kit (Invitrogen). The purity of the monocytes was verified, by flow cytometry, 5 10 15 20 25 30 CA 03165406 2022-06-20 67 greater than 90% for each donor with an anti-CD14-APC-Cy7 antibody (Miltenyi Biotec).

[0307] Freshly purified monocytes were resuspended in a 48-well plate at 1 million per ml in RPMI 1640 + GLUTAMAX, penicillin, and streptomycin at 100 U/mL and 10% fetal bovine serum (FBS). All molecules were added at the beginning of the cultures at a concentration of 20 pM of G1-X except for TriCat12 alone, which was added at a concentration of 40 pM. After 5 days of culture at 37°C, monocyte morphology was analyzed by flow cytometry using a MACSQUANT Q10 cytometer (Miltenyi Biotec). All cytometry data were analyzed using Flowlogic™ software (Miltenyi Biotec). 14. Evaluation of in vivo anti-psoriatic activity in IMQ mice

[0308] Eight-week-old female Balb/c mice, weighing approximately 20 g, were used in these experiments. The experimental procedures were evaluated and approved by the Animal Experimentation Ethics Committee. The procedures were performed under gas anesthesia, administered in an induction chamber using 4% isoflurane inhalation followed by 3% isoflurane via mask. After one week of acclimatization, the mice's backs were shaved on day -1 (D-1) over an area of approximately 4 cm² using clippers, and residual hairs were removed with a depilatory cream. At day 7 (D7), 80 mg of Aldara™ 5% lmiquimod cream (IMQ) were applied and massaged onto the backs of mice daily for 7 consecutive days in the late afternoon. The following morning, the backs of the animals treated with IMQ were washed with a cotton swab soaked in water. The following treatments were then administered in a volume of 100 µL: water (control), G1-A or G1-B dendrimers dissolved in water (amount corresponding to a final dose of 13 mg/kg), and G1-A or G1-B dendrimers formulated in TriCat12 vesicles (amount corresponding to a final dendrimer dose of 13 mg/kg). On day 7, 5 hours after the last treatment, the mice were euthanized. The severity of skin inflammation on the backs of mice was assessed using a clinical score based on the PASI (Psoriasis Area Severity Index). Skin inflammation severity was determined by daily evaluation of erythema, scaling, and back thickness using a scale of 0 to 5 (0 = normal; 1 = mild; 2 = moderate; 3 = marked; 4 = very marked). Back thickness was measured using calipers. A total clinical score combining these three clinical criteria was then calculated (scale of 0 to 12).

[0309] At the euthanasia stage, skin samples from the back were harvested, immediately fixed in 4% paraformaldehyde, and embedded in paraffin blocks. For histological evaluation, hematoxylin and eosin (H&E) staining was performed on 5 µm deparaffinized skin sections. Images were obtained using a slide scanner (Panoramic Slide Scanner 250, 40x objective). The severity of inflammation, at the histological level, was assessed based on the following histopathological characteristics of psoriasis: acanthosis, spongiosis, hyperkeratosis, parakeratosis, and immune infiltrate. For each of these five criteria, a score ranging from 0 (normal) to 4 (severe) was assigned, and the scores were then cumulative to give the total histological score (scale of 0 to 20). 15. Proliferation of keratinocytes

[0310] To study the effect of dendrimers on keratinocyte proliferation in vitro, the expression of the proliferation marker Ki67 (nuclear marker) was studied by immunohistochemistry after treatment or without (control) with 20 pM of G1-A or G1-B for 24, 48, or 72 h. Two types of keratinocytes were studied: human N-TERT cell line and primary human keratinocytes. Following treatment with the dendrimers, the cells were washed, fixed in para-formaldehyde (PFA), and permeabilized with Triton. Next, the fixed cells were incubated with a rabbit primary antibody against Ki67, then washed and incubated with a rabbit secondary antibody coupled to the fluorochrome Alexa Fluor 488. The cell nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI). The keratinocytes were then observed using a wide-field fluorescence microscope. For each observation area, two images were acquired: one with an ultraviolet laser to observe the DAPI labeling of the nuclei; and one with a green laser to observe the Ki67 labeling. These images were processed using Imaged software. On the image corresponding to the DAPI labeling, the nuclei were counted to obtain the total number of cells per image. On the image corresponding to 5 10 15 20 25 30 CA 03165406 2022-06-20 69 Ki67 labeling, the Ki67-positive nuclei are counted to obtain the number of proliferative cells and to calculate the percentage of proliferative cells. II. Results 1. Formulation TriCat12/ABP 1.1 Characterization of vesicates

[0311] The TriCat12/ABP formulation preparation process yields vesicles with an average diameter of approximately 250–300 nm and a bilayer transition temperature of 37°C [Fig. 2]. The TriCat12/ABP formulation preparation process is capable of encapsulating, on average, 25% of the dendrimer initially committed to the formulation. Separation of the unencapsulated dendrimer is difficult to achieve using techniques known to those skilled in the art. The zeta potential is -55 ± 4 mV and the pH of the solution is ~6.5. 7.2 Skin penetration of the formulation

[0312] Fluorescence quantification of the amount of ABP-NIR dendrimer (ABP dendrimer in its fluorescent form), formulated or not with TriCat 12, that penetrated pig ear skin (Franz cells) shows that after 24 h at 40°C, the ABP dendrimer, formulated or not, penetrates the skin only slightly [Fig. 3]. 2. TriCat12/G1-A Formulation 2.1 Characterization of Vesicles

[0313] The formulation preparation process using a precursor, the dendrimer in its phosphonic acid form, increases its insertion capacity into the hydrophobic portion of the vesicles, thus achieving an average encapsulation rate of 75% and allowing for easy separation of the unencapsulated active ingredient by filtration. Formulation of the G1-A dendrimer with TriCat-12 results in the formation of vesicles with an average diameter of approximately 100-350 nm [Fig. 4].5 10 15 20 25 30 CA 03165406 2022-06-20 70

[0314] The TriCat 12/G1-A formulation has a phase transition temperature of 33°C. Below 33°C, the vesicle membrane bilayer is rigid and stable. Above 33°C (skin temperature oscillating between 30 and 35°C), this membrane becomes fluid and its bio-targeting properties are increased (diffusion into tissues, fusion with cells, release of active ingredients) [Fig. 5].

[0315] The Zeta potential is -53 ± 2.5 mV and the pH of the solution ~ 2.8.

[0316] Similarly, the formulation of the G1-A-NIR dendrimer with TriCat-12 results in the formation of vesicles with an average diameter of approximately 100–350 nm. The pH of the solution is ~2.8. The TriCat 12/G1-A-NIR formulation is stable for at least 1 month when stored at 4°C in solution. 2.2 Stability of the TriCat12/G1-A formulation

[0317] The TriCat 12/G1-A formulation is stable for more than two months when stored at 4°C in solution [Fig. 6].

[0318] Experiments were carried out to verify whether the vesicles could be damaged by a freezing step. These experiments demonstrate that the formulations can be frozen and thawed without changes to their physicochemical properties [Fig. 7]. 2.3 Penetration of the formulation into the skin

[0319] The Franz continuous flow cell diffusion method is used to study in vitro the cutaneous penetration of the formulation over time. These experiments were performed on pig ear skin, whose histological and physiological properties are similar to those of human skin. These experiments show that after 24 hours, the amount of dendrimer that has penetrated the skin is significantly greater when it is formulated [Fig. 8].

[0320] Confocal microscopy was chosen to evaluate the depth of penetration of the dendrimer into the different layers of the skin (stratum corneum, viable epidermis and the dermis). Confocal microscopy experiments show that the dendrimer only penetrates the epidermis and the dermis5 10 15 20 25 30 CA 03165406 2022-06-20 71 when it is formulated in vesicles, otherwise it remains in the superficial layers of the skin (stratum corneum) [Fig. 9].

[0321] Experiments performed on human skin, obtained from human skin biopsies. These experiments show that after 24 hours, the amount of dendrimer that has penetrated the skin is significantly greater when it is formulated [Fig. 10]. 2.4 Monocyte Activation

[0322] A change in morphology (increased granulosite and size) reflects activation of primary human monocytes. The results show that TriCat12 vesicles alone do not modify the morphology of human monocytes, whereas TriCat12 vesicles loaded with the G1-A dendrimer lead to an increase in granulosite and size. Furthermore, it is noted that nearly half of the cells are within the ellipse of active monocytes [Fig. 11]. 2.5 In vivo activity in psoriatic mice

[0323] The results of monitoring the Clinical Score of the 3 groups of mice (control: untreated IMQ mice, IMQ mice treated with 13 mg/kg of free G1-A dendrimer, and mice treated with 13 mg/kg of G1-A dendrimer formulated in TriCat12 vesicles) show a decrease in the clinical score in the 2 treated groups. For untreated control mice, the mean clinical score is 9.5 at day 7. The clinical score is decreased to 6.5 and 5.7 for the free G1-A dendrimer and TriCat12/G1-A formula dendrimer groups, respectively [Fig. 12, top graph].

[0324] The mean histological score is also decreased from 16.5 for untreated control mice to 14.5 and 10.4 for the free G1-A dendrimer and TriCat12/G1-A formula dendrimer groups, respectively [Fig. 12, bottom graph]. 2.6 Keratinocyte Proliferation

[0325] Figure 13 shows the antiproliferative effect of compound G1-A, as a function of treatment time, on the N-TERT keratinocyte line (top graphs) and on primary human keratinocytes (bottom graphs). A decrease in the total cell number and the percentage of proliferative cells is observed [Fig. 13]. 3. TriCat 12/G1-B Formulation 3.1 Vesicle Characterization

[0326] Formulation of the G1-B dendrimer by TriCat-12 results in the formation of vesicles with an average diameter of approximately 200-300 nm [Fig. 14]. The average encapsulation rate is 70% in G1-B dendrimer.

[0327] The Zeta potential is -41 ± 2.6 mV and the pH of the solution is ~ 2.7

[0328] Below 33°C, the membrane bilayer of the formulations is rigid and very stable. Above 33°C (skin temperature fluctuating between 30 and 35°C), this membrane becomes fluid and its bio-targeting properties are increased (diffusion into tissues, fusion with cells, release of active ingredients) [Fig. 15]. 3.2 Formulation Stability

[0329] The formulations are stable (in terms of diameter and quantity (represented by the diffuse intensity DOR)) for more than two months when stored at 4°C in solution [Fig. 16]. 3.3 Pharmaceutical composition of TriCat 12/G1-B with Xanthan Gum

[0330] The chemical stability of the G1-B compounds in preformulations immobilized in a xanthan gel was evaluated by NMR in comparison with that of the G1-B compound alone at pH = 4. The results show that the stability of the G1-B compound in the TriCat 12/G1-X preformulation immobilized in a xanthan gel according to the invention is much superior to that of G1-B under the same conditions. 31P NMR spectroscopy shows a degradation of the G1-B compound after 3 months of storage at pH = 4 at 25°C (spectrum B of [Fig. 17], signal indicating acid hydrolysis at approximately 48 ppm symbolized by an arrow). Under the same conditions, compound G1-B in the TriCat 12/G1-X preformulation immobilized in a xanthan gel remains perfectly stable and no by-products are observed (spectrum A of [Fig. 17]).5 10 15 20 25 30 CA 03165406 2022-06-20 73

[0331] The formulation of TriCat 12/G1-B vesicles in a 1% xanthan gel is stable in terms of the physical stability of the sample even when stored at room temperature [Fig. 18]. For the formulation of TriCat 12/G1-B vesicles in a xanthan gel, no significant change in transmission through the sample is observed over the entire height of the sample after 24 hours, whereas for TriCat 12/G1-B vesicles stored in water, transmission increases at the top of the volume, showing a clarification of the solution due to slight sedimentation of the vesicles. The pharmaceutical formulation of the vesicles in xanthan gel thus allows for better storage at room temperature. 3.4 Penetration of the formulation into the skin

[0332] The Franz continuous flow cell diffusion method is used to study the in vitro skin penetration of the formulation over time. These experiments were performed on pig ear skin, whose histological and physiological properties are similar to those of human skin. These experiments show that after 24 hours, the amount of G1-B-NIR dendrimer that has penetrated the skin is significantly greater when it is formulated in TriCat 12/G1-B-NIR vesicles [Fig. 19].

[0333] Experiments on pigskin were also carried out to verify by confocal microscopy whether the dendrimer could penetrate the deep layers of the skin. These images show that the dendrimer penetrates the epidermis better when formulated in vesicles, otherwise it preferentially remains in the superficial layers of the skin (stratum corneum) [Fig. 20].

[0334] Experiments on pigskin were performed to verify by confocal microscopy whether the dendrimer formulated in vesicles and in 1% xanthan gel could penetrate the deep layers of the skin. These images show that the dendrimer penetrates deeply into the epidermis and dermis when formulated in vesicles and in xanthan gel; otherwise, it remains preferentially in the superficial layers of the skin (stratum corneum) [Fig. 21].5 10 15 20 25 CA 03165406 2022-06-20 74 3.5 Monocyte Activation

[0335] A change in morphology (increased granulosite and size) reflects activation of primary human monocytes. The results show that TriCat12 vesicles alone do not modify the morphology of human monocytes [Fig. 11], whereas TriCat12 vesicles loaded with the G1-B dendrimer lead to an increase in granulosite and size. Furthermore, it is noted that nearly 2/3 of the cells are within the ellipse of active monocytes [Fig. 22]. 3.6 In vivo activity in psoriatic mice

[0336] The results of monitoring the Clinical score of the 3 groups of mice (control: untreated IMQ mice, IMQ mice treated with 13 mg/kg of free G1-B dendrimer, and mice treated with 13 mg/kg of G1-B dendrimer formulated in TriCat12 vesicles) show a decrease in the Clinical score in the 2 treated groups. For untreated control mice, the mean Clinical score is 9.6 at day 7. The clinical score is decreased to 7.8 and 6.4 for the free G1-B dendrimer and TriCat12/G1-B formula dendrimer groups, respectively [Fig. 23, top graph].

[0337] The mean histological score also decreased from 14.8 for untreated control mice to 12.4 and 10.8 for the free G1-B dendrimer and TriCatl2/G1-B formula dendrimer groups, respectively [Fig. 23, bottom graph]. 3.7. Keratinocyte Proliferation

[0338] Figure 24 shows the antiproliferative effect of compound G1-B, as a function of treatment time, on the N-TERT keratinocyte line (top graphs) and on primary human keratinocytes (bottom graphs). A decrease in the total number of cells and the percentage of proliferative cells is observed. 4. TriCat 12/G1-C Formulation 4.1 Vesicle Characterization 5 10 15 20 25 CA 03165406 2022-06-20 75

[0339] The formulation of the G1-C dendrimer by TriCat-12 results in the formation of vesicles, the average diameter of which is around 300 nm [Fig. 25]. The average encapsulation rate is 80% in G1-C dendrimer.

[0340] Below 34°C, the membrane bilayer of the formulations is rigid and very stable. Above 34°C (skin temperature fluctuating between 30 and 35°C), this membrane becomes fluid and its bio-targeting properties are increased (diffusion into tissues, fusion with cells, release of active ingredients) [Fig. 26]. 4.2 Formulation Stability

[0341] The formulations are stable (in terms of diameter) for more than one month when stored at 4°C in solution [Fig. 27]. 4.3 Penetration of the formulation into the skin

[0342] Experiments on pigskin were performed to verify by confocal microscopy whether the dendrimer formulated in the vesicles could penetrate the deep layers of the skin. The images show that the dendrimer penetrates deeply into the epidermis and dermis when formulated in TriCat12/G1-C vesicles [Fig. 28]. 4.4 Monocyte Activation

[0343] A change in morphology (increased granulosite and size) reflects activation of primary human monocytes. The results show that the G1-C dendrimer at 20 pM leads to an increase in granulosite and size. Furthermore, it is noted that nearly half of the cells are within the ellipse of active monocytes [Fig. 29]. 5. TriCat 8/G1-B Formulation 5.1 Characterization of Vesicles

[0344] The formulation of the G1-B dendrimer by TriCat-8 gives rise to the formation of vesicles, the average diameter of which is around 300 nm [Fig. 30]. 6. TriCat 16/G1-B formulation 6.1 Characterization of vesiclesCA 03165406 2022-06-20 76

[0345] Formulation of the G1-B dendrimer by TriCat-16 results in the formation of vesicles with an average diameter of approximately 300-400 nm [Fig. 31]. 6.2 Formulation stability

[0346] TriCat-16/G1B formulations are stable (in terms of diameter) for 5 to 10 days when stored at 4°C in solution [Fig. 32].

Claims

77 ANNOTATED VERSION Claims [Claim 1] Vesicle comprising: - a catanionic surfactant of formula (I) or a mixture of catanionic surfactants of general formula (I): (I) in which is a sugar, R1 is selected from the group comprising: H, a linear hydrocarbon chain and a branched hydrocarbon chain, said hydrocarbon chain being saturated or unsaturated, of 1 to 20 carbon atoms, R2 is a linear or branched hydrocarbon chain, said hydrocarbon chain being saturated or unsaturated, of 1 to 20 carbon atoms, R3 and R4 are independently of each other a linear or branched hydrocarbon chain, said hydrocarbon chain being saturated or unsaturated, of 1 to 19 carbon atoms; and - a dendrimer of formula (II): (II) wherein 78 ANNOTATED VERSION is selected from the group comprising the pentoses, hexoses and the groups of formulas: , , , and , Z is -CH2- or -CH=N-, R is H or C1-C12-alkyl, A is or , where X is S or O, and n is an integer between 3 and 8. [Claim 2] Vesicle according to claim 1, characterized in that, in the sugar-derived catanionic surfactant of formula (I), is 1-deoxylactilol. [Claim 3] Vesicle according to claim 1 or 2, characterized in that, in the dendrimer of formula (II), is . [Claim 4] Vesicle according to any one of claims 1 to 3, characterized in that the molar ratio of catanionic surfactant to dendrimer is between 50/1 and 1/1. 79 ANNOTATED VERSION [Claim 5] Vesicle according to any one of claims 1 to 4, characterized in that the encapsulation rate of the dendrimer of formula (I) in the catanionic surfactant of formula (II) is greater than 50%. [Claim 6] Vesicle according to any one of claims 1 to 5, characterized in that the average diameter is between 50 and 500 nm. [Claim 7] Pharmaceutical composition comprising at least one vesicle according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient. [Claim 8] Vesicle according to claims 1 to 6 or pharmaceutical composition according to claim 7 for use as a medicinal product. [Claim 9] Use of vesicles according to claims 1 to 6 or of pharmaceutical compositions according to claim 7 in the manufacture of a medicinal product. [Claim 10] Vesicle according to claims 1 to 6 or pharmaceutical composition according to claim 7 for its use in the treatment of psoriasis. [Claim 11] Use of vesicle according to claims 1 to 6 or of pharmaceutical composition according to claim 7 in the manufacture of a medicinal product for the treatment of psoriasis. [Claim 12] Vesicle for its use in the treatment of psoriasis according to claim 10 by topical administration. [Claim 13] Use of vesicle in the manufacture of a medicinal product for the treatment of psoriasis according to claim 11 by topical administration. [Claim 14] A method for preparing a vesicle according to claims 1 to 6 comprising the step of: (a) mixing one or more N-alkylaminosugars of formula (I’): 80 ANNOTATED VERSION (I’) in which is a sugar, R1 is selected from the group comprising H, a linear hydrocarbon chain and a branched hydrocarbon chain, said hydrocarbon chain being saturated or unsaturated, of 1 to 20 carbon atoms, R2 is a linear or branched hydrocarbon chain, said hydrocarbon chain being saturated or unsaturated, of 1 to 20 carbon atoms; and a phosphinic acid of formula (I’’): (I’’) wherein R3 and R4 are independently of each other a linear or branched hydrocarbon chain, said hydrocarbon chain being saturated or unsaturated, of 1 to 19 carbon atoms, and the dendrimer of formula (II) as defined in claim 1. [Claim 15] A process according to claim 14, comprising, after step (a), a step of: (b) separating the vesicle obtained from the unencapsulated dendrimer. R2 HO HO