阅读说明：本技术 聚赖氨酸树枝状聚合物在预防和管理易出现痤疮的皮肤和痤疮皮肤中的应用 (Use of polylysine dendrimers for the prevention and management of acne-prone skin and acne skin ) 是由 J·阿提亚-维格诺 E·卢茵 M·波莱尔 于 2020-02-21 设计创作，主要内容包括：本发明涉及聚赖氨酸树枝状聚合物或其盐用于管理易出现瑕疵或易出现痤疮的皮肤或用于治疗或预防寻常痤疮的用途。聚赖氨酸树枝状聚合物作为活性物用于恢复或重新平衡皮肤微生物群。聚赖氨酸树枝状聚合物还作为活性物用于改善或恢复待治疗的皮肤的外观和感觉。(The present invention relates to the use of polylysine dendrimers or salts thereof for the management of blemish-prone or acne-prone skin or for the treatment or prevention of acne vulgaris. Polylysine dendrimers are used as actives to restore or rebalance skin microbiota. Polylysine dendrimers are also used as actives to improve or restore the look and feel of the skin to be treated.)

1. Cosmetic, non-therapeutic use of polylysine dendrimers or salts thereof as active ingredient in the management of acne-prone skin and blemished skin.

2. A polylysine dendrimer or a salt thereof for use as an active ingredient in the treatment of acne vulgaris.

3. The cosmetic use according to claim 1 or polylysine dendrimer or salt thereof according to claim 2, wherein the polylysine dendrimer or salt thereof is used as an active ingredient to restore or rebalance skin microbiota, preferably to restore diversity of propionibacterium acnes (c.

4. The cosmetic use according to claim 1 or 3 or a polylysine dendrimer or salt thereof according to claim 2 or 3, wherein the polylysine dendrimer or salt thereof is used as an active ingredient to reduce the abundance of a Propionibacterium acnes strain on the skin, preferably a Propionibacterium acnes strain belonging to phylogenetic IA1, IA2, IB and IC, more preferably to phylogenetic IA 1.

5. The cosmetic use according to any one of claims 1 and 3 to 4 or a polylysine dendrimer or a salt thereof according to any one of claims 2 to 4, wherein the polylysine dendrimer or a salt thereof is used to increase a non-acne Propionibacterium acnes strain, preferably a Propionibacterium acnes strain belonging to phylogenetic II or III, preferably phylogenetic II, on the skin.

6. The cosmetic use according to any one of claims 1 and 3-5 or polylysine dendrimer or salt thereof according to any one of claims 2-5, wherein the polylysine dendrimer or salt thereof is used as an active ingredient for:

-improving skin appearance and feel, and/or

Restoring skin comfort and/or

-preventing, ameliorating or treating blemishes, and/or

Soothing the skin, and/or

-reducing or preventing skin redness or skin inflammation, and/or

-reduction of sebum secretion from the skin, and/or

Reducing visible lesions, such as comedones, pustules and papules, and/or

-improving skin desquamation.

7. The cosmetic use according to any one of claims 1 and 3-6 or polylysine dendrimer or salt thereof according to any one of claims 2-6, wherein the polylysine dendrimer is a second generation unbound poly-L-lysine dendrimer with a DPn of from 40 to 60, preferably from 46 to 50, for example 48.

8. The cosmetic use according to any one of claims 1 and 3-7 or a polylysine dendrimer or salt thereof according to any one of claims 2-7, wherein the polylysine dendrimer is a poly-L-lysine dendron having a linear poly-L-lysine core, wherein the epsilon amino groups are each substituted with a moiety of poly-L-lysine.

9. The cosmetic use according to claim 8 or polylysine dendrimer or salt thereof according to claim 8, wherein the linear poly-L-lysine core is from 6 to 10, preferably 8 lysine residues in length and/or the portion of poly-L-lysine grafted on the epsilon-amino group is from 2 to 6, preferably 5 lysine residues in length.

10. The cosmetic use according to any one of claims 1 and 3 to9 or a polylysine dendrimer or a salt thereof according to any one of claims 2 to9, wherein the polylysine dendrimer is in the form of a polycationic dendrimer, wherein the counter anion is acetate.

11. The cosmetic use according to any one of claims 1 and 3 to 10 or the polylysine dendrimer or salt thereof according to any one of claims 2 to 10, wherein the polylysine dendrimer is present in a non-pharmaceutical, cosmetic or dermocosmetic composition.

12. Cosmetic use according to any one of claims 1 and 3 to 11 or a polylysine dendrimer or salt thereof according to any one of claims 2 to 11, wherein the polylysine dendrimer is present as active ingredient in a cosmetic composition selected from the group consisting of soothing creams, exfoliating products, day creams or gel products, cleansing lotions or lotions, cleansing cleansers, blemish correction creams or sticks, and concealer sticks or creams, for use on acne skin, blemish prone skin or skin prone to acne.

13. The cosmetic use according to any one of claims 1 and 3 to 12 or a polylysine dendrimer or a salt thereof according to any one of claims 2 to 12, wherein the polylysine dendrimer is used in combination with an active ingredient selected from vitamin a, tretinoins such as adapalene, tretinoin or tazarotene; bactericides such as benzoyl peroxide and decanediol, azelaic acid, salicylic acid; anti-inflammatory agents such as dapsone or Melaleuca alternifolia (tea tree) oil; an anti-redness agent; a soothing agent; and combinations thereof.

14. A topical composition for managing acne skin, acne-prone skin or blemish-prone skin comprising as an active ingredient a polylysine dendrimer and one or more excipients.

15. The topical composition of claim 14, comprising:

-0.1 to 100ppm, preferably 0.1 to 10ppm, of a polylysine dendrimer, preferably a polylysine dendrimer according to any one of claims 7 to 10,

-0% to 20% of one or more additional active agents, and

-70% to 99.9999% of one or more excipients,

the percentages are expressed by weight relative to the total weight of the composition.

16. A method of treating or preventing acne in a subject, said method comprising the step of topically administering to said subject an effective amount of polylysine dendrimer or a composition thereof, preferably according to any one of claims 7 to 10.

17. A method of restoring or rebalancing the microbiota in acne skin, blemish-prone skin, or acne-prone skin comprising the step of topically applying to the subject an effective amount of a polylysine dendrimer, or a composition thereof, preferably according to any one of claims 7-10.

18. A method of improving the appearance and feel of skin, and/or restoring skin comfort and/or preventing, improving or treating blemishes and/or acne lesions and/or soothing the skin, and/or reducing the visibility of pores (and thus smoothing the skin), and/or reducing or preventing redness of the skin, and/or inflammation of the skin, and/or reducing sebum secretion from the skin and/or imparting a less shiny and/or oily appearance to the skin, and/or improving the skin, in a subject having acne, blemish prone skin or acne prone skin, comprising the step of topically applying to the subject an effective amount of a polylysine dendrimer or composition thereof, preferably according to any one of claims 7-10.

19. Use of a polylysine dendrimer in the manufacture of a topical composition, preferably according to any one of claims 1 to 18, for the management or treatment of acne-prone skin, blemish-prone skin or acne skin.

Background

Acne vulgaris (Acne vulgaris), also referred to simply as Acne (ace), is a chronic inflammatory disease common to sebaceous producing glands (pilosebaceous glands). Acne causes the formation of inflammatory bumps (papules, pustules, nodules, and cysts), comedones (blackheads and whiteheads), and scars on the skin. Acne is one of the most common conditions treated by dermatologists and other healthcare providers. Over 90% of the world's population is affected by acne at some point in their life. The pathogenesis of acne is multifactorial, involving four key factors with interrelated mechanisms: increased sebum production, hyperkeratosis, skin inflammation and propionibacterium acnes (Cutibacterium acnes) proliferation.

Propionibacterium acnes (formerly known as Propionibacterium acnes) are ubiquitous in the pilosebaceous glands of human skin, predominantly in the face and back. Propionibacterium acnes is a major member of the normal skin microbiota and plays an important role in maintaining the normal skin microbiota by inhibiting the development of some pathogenic bacteria, such as Staphylococcus aureus (staphyloccus aureus), and maintaining acidic pH acids in the pilosebaceous follicles, although propionibacterium acnes is a symbiont that is believed to be involved in the pathogenesis of acne by promoting inflammation. In fact, many studies have shown that, in the skin, propionibacterium acnes activate innate immunity through Toll-like receptors expressed by monocytes and keratinocytes and increase the secretion of interleukins. Furthermore, propionibacterium acnes has been shown to be associated with opportunistic infections, especially in the case of indwelling medical devices. Its dual role in skin microflora balance, acne inflammatory conditions and opportunistic infections leads to the following hypothesis: certain strains of propionibacterium acnes can be characterized by an increased pathogenic and inflammatory potential.

Phylogenetic studies provide valuable insight into the genetic population structure of propionibacterium acnes. Propionibacterium acnes have been shown to have an overall clonal structure, and isolates thereof can be divided into a number of statistically significant clades or phylogenetic groups designated IA1, IA2, IB, IC, II and III. The population of systems has been shown to differ in their association with a particular type of infection and differ in the putative virulence determinants, inflammatory potential, antibiotic resistance, aggregation properties and morphological features that they produce. The phylogenetic forms IA1, IA2, IB, IC of propionibacterium acnes have been identified as acne strains, while phylogenetic forms II and III are classified as non-acne strains. Phylogenetic IA1 has been shown to be the most abundant strain in the acne strain, while phylogenetic forms II and III are mainly present in healthy, non-acne skin.

In fact, many independent epidemiological studies have shown a correlation between clonal complexes of the phylum type IA1 and moderate to severe acne (Scholz et al, Ploss, 2014, e104199, Barnard et al Journal of Clinical Microbiology,2015,53, 1149-.

More recently, Dagnelie et al (Acta Derm Venoreol, 2018; 98: 262-. They showed that healthy patients had phylogenetic profiles IA1 (39.1%) and II (43.4%), whereas IA1 (84.4%) predominated in the acne group, especially on the back (95.6%): type IA1SLST is clearly associated with severe acne. Notably, Dagnelie et al reported that inflammatory severe acne in both the face and back was associated with significant loss of the diverse population of propionibacterium acnes. Since the microflora modulates the innate immunity of the skin, it is believed that this loss of diversity may trigger inflammation and promote inflammatory acne lesions. In a recent publication, evidence was provided by Dagnelie et al that loss of diversity in the Propionibacterium acnes subgroup did trigger activation of the innate immune system, triggering the development of skin inflammation (Dagnelie et al, JEADV, 2019; 33: 2340-.

To date, there are multiple treatment lines to manage acne, depending on the severity of the acne. The treatment is based essentially on two types of active ingredients: topical retinoids which exert an anti-inflammatory effect, normalize skin desquamation and exert acne dissolving effects, and antibiotics and bactericides, such as benzoyl peroxide, for reducing the overall presence of propionibacterium acnes on the skin. Treatment of moderate and severe acne typically involves topical combination therapy in combination with benzyl peroxide and retinoids or antibiotics. Oral treatment with antibiotics and isotretinoin can also be prescribed. For women, oral contraceptives may also be prescribed. Unfortunately, these treatments also have some drawbacks, such as skin irritation, dermatitis, or photosensitivity. The use of antibiotics is also controversial due to problems with antibiotic resistance and may lead to an imbalance in the microbiota.

However, none of these treatments are directed to rebalancing the skin microbiota to promote propionibacterium acnes non-acne strains more than propionibacterium acnes acne strains.

There is therefore a need for alternative methods of managing acne skin as well as skin prone to acne and skin prone to blemishes.

Disclosure of Invention

The present invention relates to the cosmetic, non-therapeutic use of polylysine dendrimers or their salts as active ingredient in the management of acne-prone skin and blemish-prone skin. The invention also relates to the use of a polylysine dendrimer or a salt thereof as an active ingredient for the treatment or prevention of acne vulgaris.

In some embodiments, polylysine dendrimers or salts thereof are used as active ingredients to restore or rebalance skin microbiota, preferably to restore propionibacterium acnes diversity on the skin. For example, polylysine dendrimers or salts thereof may be used as active ingredients for reducing the abundance of a propionibacterium acnes strain on the skin, preferably a propionibacterium acnes strain belonging to phylogenetic IA1, IA2, IB and IC, more preferably to phylogenetic IA 1. Alternatively or additionally, polylysine dendrimers or salts thereof may be used to increase the abundance of non-acneiform strains of propionibacterium acnes, preferably of the phylogenetic group II or III, preferably phylogenetic group II, on the skin.

In some embodiments, in acne skin, acne-prone skin, or blemish-prone skin, a polylysine dendrimer or a salt thereof is used as an active ingredient to:

-improving skin appearance and feel, and/or

Restoring skin comfort, and/or

-preventing, ameliorating or treating blemishes, and/or

Soothing the skin, and/or

-reducing or preventing skin redness or skin inflammation, and/or

-reduction of sebum secretion from the skin, and/or

Reducing visible lesions, such as comedones, pustules and papules, and/or

-improving skin desquamation.

In some embodiments, the polylysine dendrimer is a second generation unbound poly-L-lysine dendrimer (dendrogrift) having a DPn of 40 to 60, preferably 46 to 50, e.g., 48.

In some other embodiments, the polylysine dendrimer is a poly-L-lysine dendrimer having a linear poly-L-lysine core, wherein the epsilon amino groups are substituted with poly-L-lysine moieties (moiety). For example, the linear poly-L-lysine core is 6 to 10, preferably 8 lysine residues in length and/or the poly-L-lysine moiety grafted onto the epsilon-amino group is 2 to 6, preferably 5 lysine residues in length.

In some further embodiments, the polylysine dendrimer is in the form of a polycationic dendrimer, wherein the counter anion is acetate. An example of such a dendrimer is given in figure 7.

The polylysine dendrimer may be present as an active ingredient in a non-pharmaceutical, cosmetic or dermocosmetic composition. Examples of target cosmetic compositions include soothing creams, exfoliating products, daily creams or gel products, cleansing lotions or lotions, cleansing cleansers, blemish correction creams or sticks, and concealer sticks or creams.

In some embodiments, the polylysine dendrimer may be used in combination with an active ingredient selected from the group consisting of: vitamin a, tretinoin such as adapalene, tretinoin or tazarotene; bactericides such as benzoyl peroxide and decanediol, azelaic acid, salicylic acid; anti-inflammatory agents such as dapsone or Melaleuca alternifolia (tea tree) oil; anti-redness agents (anti-reddness agents); a soothing agent; and combinations thereof.

The present invention also relates to a topical composition for managing acne skin, acne-prone skin or blemish-prone skin comprising as an active ingredient a polylysine dendrimer and one or more excipients. The topical composition may comprise:

-0.1 to 100ppm of polylysine dendrimer, preferably according to any one of claims 7 to 10,

-0% to 20% of one or more additional active agents, and

-70% to 99.9999% of one or more excipients,

the percentages are expressed by weight relative to the total weight of the composition.

Another object of the present invention is a method of treating or preventing acne in a subject, the method comprising the step of topically administering to said subject an effective amount of a polylysine dendrimer or a composition thereof. The present invention also relates to a method for restoring or rebalancing the microbiota in acne skin, blemish-prone skin or acne-prone skin comprising the step of topically applying to the subject an effective amount of a polylysine dendrimer or a composition thereof.

The present invention also relates to a method for improving the appearance and feel of skin, and/or restoring skin comfort and/or preventing, ameliorating or treating blemishes and/or acne lesions and/or soothing the skin, and/or reducing pore visibility (and thus smoothing the skin), and/or reducing or preventing redness of the skin, and/or inflammation of the skin, and/or reducing sebum secretion from the skin and/or imparting a less shiny and/or oily appearance to the skin, and/or improving the skin, in a subject suffering from acne, having blemish-prone skin or having acne-prone skin, comprising the step of topically applying to said subject an effective amount of a polylysine dendrimer or a composition thereof.

The present invention also relates to the use of a polylysine dendrimer, preferably a poly-L-lysine dendrimer, in the preparation of a topical composition as described above for the management or treatment of acne-prone skin, blemish-prone skin or acne skin.

Brief description of the drawings

Figure 1 shows the anti-inflammatory effect of G2 dendrimers according to the invention in normal human dermal fibroblasts. In hydrocortisone (0.05. mu.g/mL) or G2 dendrimer (10)^-9M、10^-8M、10^-7M or 10^-6M), cells were treated with 0.0003 ng/mLIL-. alpha.and IL-8 production was determined by IL-8ELISA assay. P<0.05，**p<0.01，***p<0.001。

Fig. 2 is a schematic diagram showing a timeline of product application in the isolated model of acne vulgaris used in example 5. At D0, D1, D4 and D6, the G2 dendrimer was applied at 2ppm or 5ppm followed (1 hour) by propionibacterium acnes.

Figure 3 shows the effect of G2 dendrimers on TRL2 expression in human skin explants challenged with propionibacterium acnes. Explants not treated with propionibacterium acnes or G2 were used as controls.

FIG. 4 shows the effect of G2 dendrimer on membrane fluidity of acne strains of Propionibacterium acnes (RT4 and RT5) and non-acne strains of Propionibacterium acnes (RT 6). The G2 dendrimer specifically and significantly increased the membrane fluidity of the acne strain.

Fig. 5A-5E show the results of a clinical study aimed at evaluating the effect of treatment with the test cream containing G2 dendrimer (G2) with placebo cream (placebo) on inflammatory lesions (i.e. pustules and papules) (fig. 5A), on the number of retained lesions (i.e. blackheads and whiteheads) (fig. 5B) and on the sebum secretion rate (fig. 5C) after 28 days of treatment. Fig. 5E and 5D show the propionibacterium acnes strain diversity using simpson and shannon index, respectively, after 28 days of treatment. The protocol and results of the clinical trial are detailed in example 8.

FIGS. 6A and 6B show the effect of dendrimers of the invention on biofilm formation by non-acne strain RT6 and by acne strain RT 5. The G2 dendrimer reduced the biofilm thickness of both strains. Notably, the G2 dendrimer significantly reduced the biomass density in the biofilm formed by the RT5 strain, while having no significant effect on the biomass density of the biofilm formed by the RT6 strain.

FIG. 7 shows an example of a polylysine dendrimer of the present invention.

Detailed Description

Applicants show that a "dendrimer" of lysine residues, referred to herein as a "G2 dendrimer", exhibits antibacterial activity against a strain of propionibacterium acnes belonging to phylogenetic IA1 with a MIC of 20 ppm. In addition, the G2 dendrimer moderates keratolytic effects and reduces the immune and inflammatory cascades known to be associated with p.acnes colonization. Applicants further show that this poly-L-lysine dendrimer is capable of specifically targeting the acne strain of propionibacterium acnes. Notably, example 6 shows that G2 dendrimer significantly increased membrane fluidity of the acneionoid form belonging to phylogenetic IA1(RT4 and RT5) without significant effect on the non-acneionoid form belonging to phylogenetic II RT 6. In other words, this result indicates that the G2 dendrimers are capable of specifically destabilizing the cell membranes of the acne strains and thus inhibiting their proliferation without damaging the non-acne propionibacterium acnes strains. Furthermore, example 7 shows that G2 dendrimers significantly reduced the biomass density in biofilms formed by acne strain RT5, while having no significant effect on the biomass density in biofilms formed by non-acne RT6 strains. Polylysine dendrimers are therefore expected to reduce the inflammatory response triggered by the propionibacterium acnes biofilm (whatever the strain) by reducing the thickness of the biofilm while exerting a selective and specific inhibitory effect on the acne strain by destabilizing its biofilm.

The efficacy of the G2 dendrimers of the present invention in managing blemishes and lesions in acne skin and improving skin microbiota was clinically demonstrated. The applicant showed that the treatment of 28 days with a cream containing a low concentration (2ppm) of G2 dendrimer significantly improved the appearance of the skin, in particular by reducing the number of acne blemishes and lesions such as papules, pustules, blackheads and remaining lesions compared to the treatment with placebo cream. The cream containing G2 was further able to reduce the sebum secretion rate of the skin. Notably, genomic analysis performed on facial samples indicated that the G2 dendrimer-containing composition improved skin microbiota by increasing the diversity of the propionibacterium acnes strains: in skin samples treated with the composition containing G2, the abundance of the phylogenetic IA1 strain (identified in the literature as a strain that promotes acne) was reduced, while the abundance of the non-acne phylogenetic II strain was increased. In other words, polylysine dendrimer has been shown to be a substance capable of rebalancing the diversity of propionibacterium acnes in acne skin.

Together, these results demonstrate that polylysine dendrimers are effective agents to control blemishes and restore skin microflora in acne and acne-prone skin as well as blemish-prone skin.

The present invention therefore relates to the use of polylysine dendrimers, preferably poly-L-lysine dendrimers, as active agents for the management of acne skin, acne-prone skin and blemish-prone skin.

The invention also relates to the use of polylysine dendrimers as active agents for the prevention or treatment of acne vulgaris and/or for the management of blemishes and skin lesions associated with acne vulgaris.

The present invention also relates to the use of a polylysine dendrimer, preferably a poly-L-lysine dendrimer, as an active agent in a subject having a skin type selected from acne skin, skin prone to acne and blemished-acne skin, for:

-improving skin appearance and feel, and/or

Restoring skin comfort and/or

-preventing, ameliorating or treating blemishes, and/or

-preventing, ameliorating or treating acne lesions and/or

Soothing the skin, and/or

-reducing the visibility of pores, and/or

-reducing or preventing skin redness or skin inflammation, and/or

-reducing sebum secretion from the skin and/or giving the skin a less shiny and/or greasy appearance

-reduction of visible lesions: blackheads, pustules and papules, and/or

Improvement of skin desquamation (e.g. by exerting keratolytic effect).

The invention also relates to the use of a polylysine dendrimer, preferably a poly-L-lysine dendrimer, to restore or rebalance the microbiota in acne skin, skin prone to acne or skin prone to blemishes. More precisely, the polylysine dendrimers of the present invention are used as substances for reducing the abundance of propionibacterium acnes strains in the skin, preferably in the skin where acne is likely to occur. In another or further embodiment, polylysine dendrimers of the present invention are used to increase the abundance of non-acne propionibacterium acnes in skin, preferably acne or skin prone to acne. The invention also relates to the use of polylysine dendrimers in the prevention or reduction of skin colonization by propionibacterium acnes strains. The invention further relates to the use of polylysine dendrimers for increasing the diversity of propionibacterium acnes in acne skin or skin prone to acne, for example by reducing the abundance of propionibacterium acnes phylogenetic IA1, while being able to increase the abundance of propionibacterium acnes phylogenetic II or III.

In a more general aspect, the present invention relates to the use of polylysine dendrimers, preferably poly-L-lysine dendrimers, as active agents for restoring the balance of skin microbiota, in particular in acne or skin prone to acne. In another aspect, the present invention relates to the use of a polylysine dendrimer, preferably a poly-L-lysine dendrimer, in the prevention or treatment of skin dysbiosis associated with an imbalance in the propionibacterium acnes species.

The invention also relates to the use of a polylysine dendrimer, preferably a poly-L-lysine dendrimer, as an active agent for maintaining or restoring the homeostasis of skin microbiota, in particular in a subject having acne skin, acne-prone skin or blemish-prone skin.

Another aspect of the invention relates to a method for managing a skin type selected from the group consisting of acne skin, acne-prone skin and blemish-prone skin in a subject, the method comprising the step of topically applying to the subject an effective amount of a polylysine dendrimer. Such methods can improve the appearance of the skin of a subject, for example, by treating, reducing and/or delaying the onset of skin imperfections and lesions in the subject.

The present invention also relates to a method of treating or preventing acne in a subject, the method comprising the step of topically administering to said subject an effective amount of a polylysine dendrimer. Another object of the present invention is a method of improving the appearance and feel of skin, and/or restoring skin comfort and/or preventing, improving or treating blemishes and/or acne lesions and/or soothing the skin, and/or reducing the visibility of pores (and thus smoothing the skin), and/or reducing or preventing redness and/or inflammation of the skin, and/or reducing sebum secretion from the skin and/or imparting a less shiny and/or oily appearance to the skin, and/or improving the skin, in a subject having acne skin, acne-prone skin or blemished-prone skin, comprising the step of topically applying to said subject an effective amount of a polylysine dendrimer. The invention also relates to a method of restoring or rebalancing the microbiota in acne skin or skin prone to acne, the method comprising the step of topically applying to the subject an effective amount of a polylysine dendrimer.

The present invention also relates to the use of a polylysine dendrimer, preferably a poly-L-lysine dendrimer, in the preparation of a topical composition as described above for the management or treatment of acne-prone skin, blemish-prone skin or acne skin.

The present invention also provides topical compositions and methods for treating acne vulgaris, reducing the inflammatory response of the skin, and promoting desquamation of the skin using polylysine dendrimers (e.g., G2 dendrimer) and/or salts thereof. For example, the present invention relates to a method for reducing an inflammatory response of the skin comprising applying to the skin of a subject in need of treatment a composition comprising a polylysine dendrimer (e.g., unbound poly-L-lysine dendrimer) or a salt thereof, thereby reducing an inflammatory response of the skin of the subject, wherein the inflammatory response comprises IL-8 release or TLR2 overexpression in response to propionibacterium acnes. The present invention also relates to a method of promoting skin cell exfoliation, comprising applying to the skin of a subject in need of treatment a composition comprising a polylysine dendrimer (e.g., unbound poly-L-lysine dendrimer) or a salt thereof, thereby promoting exfoliation of skin cells of the subject.

The polylysine dendrimers according to the invention may be incorporated as active ingredients in topical compositions, typically pharmaceutical, cosmetic or dermocosmetic compositions.

Typically, the polylysine dendrimer is present in the topical composition at the following concentrations: 0.1ppm to 100ppm, preferably 0.1ppm to 20ppm, more preferably 0.5 to 10ppm, such as 0.5 to 7ppm, for example 0.5 to 6 ppm. The concentration of polylysine dendrimer in the topical composition and the frequency of application of the topical composition may depend on the skin to be treated and/or the stage of treatment. For example, a concentration of less than 4ppm (e.g., 2ppm) may be sufficient to treat acne-prone skin, blemish-prone skin, and skin with mild acne, applied once per day. For treatment of skin with moderate or severe acne, higher concentrations of polylysine dendrimer (e.g., 4 to 10ppm, such as 4 or 6ppm) may be used, applied twice daily during the initial treatment. Once the skin lesions associated with acne are managed, the dosage and frequency of application can be reduced and maintenance therapy can be administered for several weeks.

Polylysine dendrimers are commonly used by a topical route (for example, by application to the skin area to be treated, for example, an area of skin afflicted with acne or having acne-prone or blemish-prone properties. Generally, the area of skin to be treated may exhibit one or more skin imperfections and/or skin lesions and/or an oily appearance (e.g., due to excessive production of sebum) as described below. The targeted skin areas include, but are not limited to, face, neck, chest, shoulder, and back.

The frequency of treatment may vary and depends on the type of skin of the subject and/or the severity of acne (if present). Typically, polylysine dendrimers are applied to the skin for treatment once or twice a day. Treatment with polylysine dendrimers may continue for several consecutive days, for example 7 to 28 days, or several months (at least 2, 3, 4, 6,8 or 10 months).

Long-term treatment with the polylysine dendrimers of the present invention, especially at concentrations below 10ppm, is expected to be well tolerated in patients with any significant side effects such as skin irritation, at least because the polylysine dendrimers promote the rebalancing of the skin microbiota by specifically targeting the acne strain of propionibacterium acnes.

The effect exerted by the dendrimer may be therapeutic, prophylactic and/or cosmetic, depending on the type of skin and the mode of application.

For example, the use of the polylysine dendrimers of the present invention may be cosmetic, non-therapeutic use when applied to blemish-acne skin.

The subject may be any subject suffering from acne or having skin susceptible to acne or to blemishes.

In some embodiments, the subject is a juvenile from 10 to 18 years of age, preferably from 12 to 16 years of age.

In some embodiments, the subject has acne and is over the age of 23 years.

In another aspect, the subject is a female or male over the age of 23.

In some other embodiments, the subject has skin that is susceptible to acne.

In some further embodiments, the subject has mild acne, moderate acne, or severe acne, preferably moderate or severe acne.

In further embodiments, the subject has a skin that is prone to blemishes.

As used herein, "blemish-prone skin (blephish-bone skin)" refers to a type of skin in which the skin has a tendency to develop certain comedones and comedos, such as blackheads and whiteheads, but is free of inflammatory lesions, such as papules and pustules.

"blemish prone skin" can be characterized by an oily and shiny appearance, as the sebaceous glands produce more sebum than other skin types. This increased sebum production usually occurs during puberty when the production of a hormonal substance known as androgen is increased, which in turn stimulates the sebaceous glands to produce more sebum than is needed. Certain hormonal treatments can also trigger excessive production of sebum from the skin. This seborrhea itself can also interfere with normal skin desquamation and cause skin blemishes. In some embodiments, a subject with blemish prone skin has no history of personal acne, particularly severe or moderate acne.

As used herein, "Acne-bone skin" refers to a skin type in which the skin has a tendency to develop some comedones and pox and even some very few inflammatory lesions (usually papules). In some embodiments, a subject having skin susceptible to the occurrence of acne refers to a subject with a history of personal acne, i.e., a subject that has experienced at least one acne episode; or subjects with a history of familial acne, i.e., subjects with at least one family member (mother, father or sister/brother) who has experienced moderate or severe acne.

"acne-prone skin" or "blemish-prone skin" does not refer in a strict sense to skin that has a skin disorder but that may degenerate into acne if not managed properly.

As used herein, "acne skin (acneic skin)", also referred to herein as "acne skin (acneic skin)" refers to skin that has acne (also referred to as acne vulgaris). Acne is a non-contact infectious inflammatory skin disorder involving the pilosebaceous unit, characterized by pox caused by inflamed and infected sebaceous glands. This disease is most common in adolescents, but symptoms persist into adulthood, and some people, especially women, first develop symptoms after the age of 25. Persistent or delayed acne is known as delayed acne (ace tarda). Acne commonly occurs on the face, neck, shoulders, chest and back. The severity of Acne ranges from mild Acne (known as Acne vulgaris (Acne Comedonica)) to moderate Acne (Acne Papulopustulosa) to severe Acne (Acne Conglobata)). Mild acne is characterized by the presence of multiple blackheads, whiteheads, and sometimes a few papules. Moderate acne is characterized by the presence of comedones, papules and pustules. The skin may appear red and inflamed. Finally, severe acne is an unusual form of acne characterized by a number of inflamed blemishes, including papules and pustules, which may aggregate together and form nodules and cysts. The pathogenesis of acne is multifactorial, involving four key factors with interrelated mechanisms: increased sebum production, hyperkeratosis, skin inflammation and propionibacterium acnes proliferation.

As used herein, "blemish" (blenish) refers to any skin defect, such as redness, acne, or comedones, for example, referring to skin defects observed in blemish-prone skin (or acne-prone skin), typically persistent lesions, such as blackheads, whiteheads, and other pox.

As used herein, "acne lesions" refers to inflammatory, inflammatory lesions associated with acne, such as papules, pustules, cysts, and nodules observed in moderate and severe acne.

As used herein, an acne strain (acne strain) refers to a strain of propionibacterium acnes that is statistically more abundant in acne skin than in healthy skin. Propionibacterium acnes have been subdivided into six major phylogenetic forms, namely IA1, IA2, IB, IC, II and III (Dagnelie et al, 2018, supra). In the skin, bacteria belonging to phylogenetic IA1, such as RT4 and RT5 ribotypes, predominate in acne skin. Acne strains encompass phylogenetic IA1, 1a2, 1B and IC, preferably IA1 and IA2, more preferably IA1, such as ribotype I (RT1), ribotype 4(RT4) and ribotype 5(RT 5).

Non-acne strains (Non-acne strains) refer to strains of propionibacterium acnes that are statistically more abundant in healthy skin than in acne skin. Such strains include phylogenetic forms II and III, such as ribotype RT 6.

For more detailed information on the phylogenetic, clonal complex and single locus sequences of Propionibacterium acnes, reference is made to Dagnelie et al (supra) and Scholz et al, 2016.

In some embodiments, treatment with polylysine dendrimers of the present invention reduces the abundance of the propionibacterium acnes phylogenetic I strain in the total population of propionibacterium acnes present on the skin of a subject. In some other embodiments, treatment with polylysine dendrimers of the present invention increases the abundance of a phylogenetic II or III strain of propionibacterium acnes, preferably a phylogenetic II strain, in the total population of propionibacterium acnes present on the skin of a subject.

As used herein, the term "skin microbiota", "skin microbiome" or "skin flora" refers to microorganisms that reside on the skin, typically human skin, and encompasses bacteria, mycobacteria, fungi and parasitic bacteria. Most are present in the superficial layers of the epidermis and in the upper part of the hair follicles. The skin flora is typically non-pathogenic and is symbiotic (commensal) or symbiotic (mutualistic). These microorganisms may provide benefits including preventing transient pathogenic organisms from colonizing the skin surface by competing for nutrients, secreting chemicals against the pathogenic organisms, or stimulating the immune system of the skin.

"restoring or rebalancing" the skin microbiota means restoring the diversity of microorganisms to achieve a balance that promotes the health of the skin and/or the microbial distribution corresponding to healthy skin. In the context of the present invention, "restoring or rebalancing a microbiome" or "restoring or rebalancing a propionibacterium acnes distribution" in blemish prone skin, acne prone skin or acne skin means reducing the abundance of propionibacterium acnes strains on the skin. In some embodiments, it also includes promoting non-acne strains on the skin, for example by increasing their abundance. In some further embodiments, "restoring or rebalancing the propionibacterium acnes profile" means that the polylysine dendrimers of the present invention are capable of promoting diversity of propionibacterium acnes while modulating the propionibacterium acnes biomass density on the skin area being treated, such that the resulting propionibacterium acnes population is close to or similar to the microbiome of healthy skin, e.g., of a healthy skin area of a subject. It may also include the following facts: the applied composition or active ingredient does not exert a significant lethal effect on non-pathogenic commensal bacterial strains, in particular non-pathogenic commensal strains of propionibacterium acnes.

The expression "reducing the abundance of an acne strain" means reducing the amount/proportion of bacteria belonging to the propionibacterium acnes strain compared to the total amount of propionibacterium acnes bacteria present on a defined area of the skin of the subject, for example the facial area of the subject.

The expression "increasing the abundance of a non-acne strain" means increasing the number/proportion of bacteria belonging to the non-acne strain of propionibacterium acnes compared to the total amount of propionibacterium acnes bacteria present on a defined area of the skin of the subject, for example the facial area of the subject.

A significant increase/decrease in the abundance/ratio of a strain refers to a change of at least 0.1%, e.g., at least 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 45% relative to the abundance of the strain determined prior to treatment.

The abundance of the propionibacterium acnes strain can be determined by metagenomic assays performed on swabs from the targeted skin area as shown in example 8.

As used herein, "increasing the diversity of the microbiome" means increasing the number of different bacterial strains present on the skin. More precisely, "increasing the diversity of propionibacterium acnes" means increasing the number and/or abundance of non-acne strains on the skin.

As used herein, "managing acne-prone skin or blemish-prone skin" means improving the visual appearance of acne-prone skin or blemish-prone skin, e.g., with the treatment of the present invention, the skin has a less oily appearance, and/or exhibits fewer or reduced blemishes and pox, and/or blemishes are less visible and/or the skin has a lesser tendency to exhibit blemishes.

The expression "preventing" is intended to mean delaying or preventing the onset of a target disorder, such as blemishes, acne lesions or acne.

The expression "treating a condition" is intended to mean reducing, alleviating, softening, correcting or slowing the development of the target condition or its associated manifestations (e.g. acne and associated blemishes or lesions).

As used herein, a "Dendrimer" (dendromer) is a macromolecular, highly branched compound formed by a repeating reaction sequence starting from an initial core molecule, with successive layers or stages being added in successive "generations" to build up a three-dimensional, highly ordered polymer compound. The preparation of dendrimers has been described in the art, see for example US4,289,872 and US4,410,688, which describe dendrimers based on layers of lysine units.

The dendrimers of the present invention are polylysine dendrimers, more preferably poly-L-lysine dendrimers, such as poly-L-lysine dendrimers.

As with conventional dendrimers, the preparation of graft dendrimers (also referred to herein as dendritic grafts) in a generation-based scheme involves protectionThe radical operation is combined with a polymerization step, i.e. by a cyclic (so-called "grafting from") method of protection, polymerization of monomers and deprotection&Rodriguez Hernandez (2002) Macromolecules 35:8718-23) or by using preformed polymers instead of molecular compounds in cycles of protection, condensation and deprotection as AB_nConstruction of modules (so-called "grafting onto" method; tertstra&Gauthier (2004) prog.Polym.Sci.29: 277-327). Because the G2 dendrimer is no longer a monomolecular compound, the dendritic graft polymer has poorer structure controllability than conventional dendrimers (e.g., as described in U.S. Pat. No. 4,289,872), but generally has a more regular structure than hyperbranched polymers (e.g., as described in Rodriguez-Hernandez et al (2003) Biomacromolecules 4:249-258 and Klok et al (2002) Macromolecules 35: 8718-8723). However, due to their synthetic procedure, the molecular weight of the grafted dendrimers increases more rapidly with each generation than the molecular weight of the dendrimers.

In some embodiments, polylysine dendrimers according to the present invention are second generation (G2) grafted polylysine dendrimers, also interchangeably referred to herein as "G2 poly-L-lysine dendrimers", "G2 dendrimers", "grafted polylysine dendrimers" or "grafted homo-polylysine dendrimers", "lysine dendrimers" or "DGL". It has been shown that G2 dendrimers are more effective than linear polylysine in inhibiting propionibacterium acnes proliferation and promoting anti-inflammatory effects.

The G2 dendrimers used in the process of the invention may be prepared as described herein (example 1) or as described, for example, in US 2008/0206183 or Collet et al (2010) chem.eur.j.16: 2309-16.

In certain embodiments, polylysine dendrimers, such as the G2 dendrimer, are homopolylysine dendrimers consisting of 30 to 70 lysine residues, or more preferably 40 to 60 lysine residues, or most preferably 48 lysine residues. In certain embodiments, the polylysine dendrimer is a G2 dendrimer, said G2 dendrimer being a homopolylysine dendrimer comprising, consisting of, or consisting essentially of 48 lysine residues. Preferably, as used herein, the number of lysine residues refers to the average degree of polymerization (DPn) of the dendrimer or dendrimer.

In certain embodiments of the invention, the dendrimers of the invention are unbound. By "unbound" polylysine dendrimers or dendrimers is meant polylysine dendrimers or dendrimers that have not been covalently linked or associated with another material, such as a pharmaceutical or agricultural material, typically, for example, a therapeutic moiety. In other words, the dendrimers of the present invention may be free of any therapeutic, pharmaceutical or cosmetic moiety other than lysine. In some embodiments, a lysine residue is the only building block present in the polylysine dendrimers of the present invention.

In some embodiments, polylysine dendrimers of the present invention are dendrimers comprising a linear polylysine core, in which the side chains NH of one or several (preferably all) lysine residues of the core₂The group (. epsilon. -amino group) is partially substituted by polylysine. For example, polylysine dendrimers of the present invention may comprise a linear poly-L-lysine core (i.e., α -poly-L-lysine), in which the ε -NH of each lysine residue₂The groups are substituted with poly-L-lysine moieties, preferably alpha-poly-L-lysine. The linear core may be 2 to 10, such as 6 to 10, e.g. 8, lysine residues in length and the poly-L-lysine substituent may be 2 to 10, such as 3 to 8, e.g. 5, lysine residues in length.

For example, polylysine dendrimers of the present invention may comprise compounds of formula (I):

wherein n is an integer from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8 or 9, preferably 6, 7, 8 or 9, more preferably 7.

[ Lys ] represents a lysine residue and n1 represents the number of lysine moieties linked together to form a polylysine moiety, preferably an alpha-polylysine moiety, each n1 being independently selected from an integer of 2 to 10, such as 2, 3, 4, 5, 6, 7 or 8, preferably 3, 4, 5, 6 or 7, even more preferably 4, 5 or 6. In some embodiments, all n1 are the same and are, for example, 5.

The dendritic grafts of the present invention have an average degree of polymerization (DPn) of from 42 to 58, preferably from 46 to 50, more preferably about 48. As used herein, the average degree of polymerization (DPn) represents the average number of lysine monomers per dendrimer molecule in the polylysine dendrimers according to the present invention.

In some or further embodiments, the weight average molar mass (M) of the dendritic grafts in accordance with the present invention_w) 7500 to 10000, preferably 8400 to 8800, e.g. 8600g.mol^-1。

Polylysine dendrimers, including the G2 dendrimer of the present invention, are polycationic polymers. Although the G2 dendrimers synthesized and illustrated herein include Trifluoroacetate (TFA) as the counter anion, the anionic component can be exchanged with various other counter anions, such as bicarbonate, dihydrogen phosphate, fluoride, chloride, bromide, iodide, citrate, or acetate.

Thus, the present invention includes polylysine dendrimers such as the G2 dendrimer and salts thereof. Suitable counter anions are acetate, trifluoroacetate, chloride, citrate, phosphate, carbonate and combinations thereof, preferably acetate, chloride, carbonate and combinations thereof, more preferably acetate.

In certain embodiments, the counter anion of the dendrimer is acetate. Indeed, the applicant showed that in vitro, the G2 dendrimer with acetate counter anions had improved anti-inflammatory activity compared to the G2 dendrimer with chloride or carbonate counter anions: indeed, in dermal fibroblasts in vitro, G2 dendrimers with acetate as a counter anion more efficiently managed IL-8 production following IL 1-a stimulation.

To exchange the TFA counter anion for the acetate counter anion, a variety of methods can be used. One method included the use of a size exclusion support (G25) and ammonium bicarbonate elution buffer (0.1M) in chromatographic mode. After chromatography, the polymer is recovered in bicarbonate buffer and dried in a rotary evaporator, providing the polymer with bicarbonate counter anions. The counter anion can then be exchanged for an acetate anion by neutralization with acetic acid at ph 6.5. The product was then dried by lyophilization. Alternatively, the polymer solution was dialyzed against ammonium acetate buffer to exchange TFA counter anion. As a further alternative, the polymer is applied to an anion exchange resin previously conditioned in acetate. The performance of the exchange can be determined by¹⁹F NMR, percent analysis or by looking for TFA ions in LC/MS.

When used in the methods and uses of the present invention, polylysine dendrimers, such as the G2 dendrimer, and/or salts thereof, may be provided as cosmetic active ingredients present in cosmetic, pharmaceutical or dermocosmetic compositions.

For example, cosmetic compositions include, but are not limited to, anti-acne products, municipal care products, smoothing creams, exfoliating products, household care products, cleansing lotions or lotions, cleansing cleansers, blemish correction creams or sticks, concealer sticks, and the like. In some embodiments, the polylysine dendrimer is incorporated into a non-pharmaceutical cosmetic or dermocosmetic composition, for example, suitable for use on acne-prone skin or blemish-prone skin.

Thus, in certain embodiments, polylysine dendrimers, such as the G2 dendrimer, are components of compositions or formulations for external or topical application to the skin. In certain embodiments, the composition is in the form of a cleanser, toner, emulsion, solution, gel, cream/cream, ointment, or foam. In this regard, formulations or compositions containing polylysine dendrimers, such as the G2 dendrimer, are suitable or adapted for external or topical application.

Compositions comprising polylysine dendrimers according to the invention, such as the G2 dendrimer, may be viscous or semi-viscous fluids, or less viscous fluids, such as may be used in sprays or aerosols. It may take the form of a solution, suspension or emulsion. It may take a solid form, such as a powder or granules, which may be designed to be added to a liquid (e.g., water) prior to use. In some embodiments, the formulation is or can be applied to a carrier such as a sponge, swab, brush, pad, paper towel, cloth, wipe, skin patch, or dressing (which includes a bandage, plaster, skin adhesive, or other material designed for application to a tissue surface) to facilitate its application.

The compositions according to the invention may be intended for pharmaceutical (which includes veterinary but preferably human) use, and/or for cosmetic, dermocosmetic or other non-medical care purposes (e.g. to clean the skin, or to improve the appearance, feel or smell of the skin).

The compositions according to the invention may contain excipients and other additives known for use in topical formulations (topical formulations). Suitable excipients in formulations designed for topical or local application are well known to those skilled in the art. Those included will depend on the intended mode of application and site of the formulation. In the context of formulations for Topical application to the skin, examples can be found, for example, in Transdermal and Topical Drug Delivery (Pharmaceutical Press,2003) by Williams and other similar references. See also Date et al ((2006) Skin pharmacol. physiol.19(1):2-16) for a review of topical dosing strategies, as well as Skin Delivery Systems ((2006) John J Wille, Ed, Blackwell Publishing.

The excipients used may be suitable for targeting or controlling the release of the composition or components of the composition at the intended site of administration, e.g., to the desired site and/or time of delivery. Such excipients may, for example, target the composition to an area of the skin, such as the stratum corneum or the pilosebaceous glands (piloeback folliculules), or to hair follicles. They may delay or otherwise control the release of the composition over a specified period of time.

When the composition is intended for topical application to the skin, examples of suitable additives (also referred to as excipients) include diluents, fillers, carriers, emollients, humectants, fragrances, antioxidants, preservatives, stabilizers, gelling agents, and surfactants; and/or other substances commonly used in formulations in the cosmetic or pharmaceutical fields. Others can be found in Transdermal and Topical Drug Delivery by Williams (see above). However, for the treatment of acne, it may be preferred that the composition does not contain an emollient.

Polylysine dendrimers, such as the G2 dendrimer, and/or salts thereof, may be used alone or together, or may be used in combination with one or more additional active ingredients, such as antimicrobial (particularly antibacterial) or anti-inflammatory agents. The additional active ingredients may be combined with the polylysine dendrimer in a single composition, or administered separately, including by different routes (e.g., by oral routes).

In some embodiments, the additional active ingredient and the polylysine dendrimer are combined in the same composition.

For example, the formulation may contain one or more agents selected from anti-acne agents, keratolytics (keratolytics) such as lactic acid, comedolytics (comedolytics), agents capable of normalizing keratinocyte and/or sebaceous gland cell function, anti-inflammatory agents such as licochalcone a (licochalconone a) and Glycyrrhiza Inflata (Glycyrrhiza Inflata) extracts, antiproliferative agents, antibiotics, antiandrogens, sebum inhibitors (sebostatic/sebosuppressive agents), also known as sebum regulators (e.g., the natural extract of lemon balm (Backhousia citriodora), antipruritic agents, immunomodulators, anti-irritants or soothing agents (e.g., the extract of willow (Epilobium angustifolium)), agents promoting wound healing, sunscreens, skin lightening agents, anti-aging substances, and mixtures thereof.

For example, anti-acne agents include, but are not limited to, vitamin a; tretinoin such as adapalene, tretinoin or tazarotene; bactericides such as benzoyl peroxide and decanediol, azelaic acid, salicylic acid; anti-inflammatory agents such as dapsone or melaleuca alternifolia (tea tree) oil; an anti-redness agent; soothing agents and combinations thereof.

As examples of sebum regulators, secoisolariciresinol diglucoside, rice flour, zinc gluconate, sarcosine, cinnamon bark extract of Cinnamomum zeylanicum, avocado extract, lemon balm leaf extract of australia, red clover flower extract of Trifolium Pratense (clover) and combinations thereof may be cited.

As anti-redness agents, mention may be made of saponins, flavonoids, ruscogenins, esculosides and extracts containing them, such as extracts of the genus Ruscus (Ruscus), and certain essential oils, such as lavender or rosemary essential oils.

Examples of the soothing agent include allantoin, aloe extract, Calendula (california officinalis) extract, birch (e.g., white birch (Betula alba)) extract, willowherb (willow herb) extract, Tasmania pepper (e.g., Tasmania Lanceolata) extract, chestnut (e.g., castanea sativa) extract, cornflower (e.g., cornflower (Centaurea) cyanus) extract, Centella (e.g., Centella asiatica) extract, wild horsetail (e.g., Equisetum arvense) extract, fennel (e.g., fennel (Foeniculum vulgare)) extract, common Hamamelis (e.g., Hamamelis virginiana) extract, ivora (e.g., Hedera helix) extract, lily flower (e.g., Hedera helix) extract, roselle flower (e.g., lily flower) extract, common anemone (Lilium sibirica) extract, and common anemone (e.g., lily flower) extract, common lily flower (Lilium sativum) extract, common lily flower (e.g., lily flower) extract, common lily flower extract (common lily flower extract, common pepper (common pepper, common pepper (common pepper) extract, common pepper (common pepper ) extract, common pepper (common pepper) extract, common pepper (common pepper) extract, common pepper (common pepper) extract, common pepper (common pepper) extract, common pepper (common pepper) extract, common pepper (common pepper) extract, common pepper (common pepper) extract, common pepper (common pepper) extract, common pepper (common pepper) extract, common pepper (common pepper) extract, common pepper (common pepper) extract, common pepper (common, Melissa (e.g. Melissa officinalis (Melissa officinalis)) extract, Scutellaria (e.g. Scutellaria baicalensis (Scutellaria baica) extract, Mimosa (e.g. Mimosa tenuiflora) extract, Potentilla (e.g. Potentilla erecta (Potentilla erecta)) extract, exopolysaccharide extract from extreme microorganisms (e.g. Alteromonas ferment (Alteromonas ferment)), extract of one or more oligosaccharides, e.g. flax extract, peptides such as palmitoyl tripeptide-8, and combinations thereof.

For example, a polylysine dendrimer may be present in the composition. The cosmetic composition may comprise one or more excipients and optionally one or more additional active agents having a cosmetic or pharmaceutical effect.

In some embodiments, the additional active agent present in the composition is a cosmetic active ingredient.

The expression "cosmetic active agent" means a compound capable of exerting at least one cosmetic effect on the skin. The term "cosmetic effect" is intended to mean any non-therapeutic effect aimed at modifying and/or improving the appearance or feel of the skin, protecting the skin from external attacks (sun, wind, humidity, dryness, chemical products), or preventing and/or correcting the phenomena associated with aging or puberty.

Typically, the composition comprises:

from 0.1 to 100ppm of polylysine dendrimers according to the invention

-0% to 20% of one or more additional active agents, and

-70% to 99.9999% of one or more excipients,

the percentages are expressed by weight relative to the total weight of the composition.

Preferably, the composition is a cosmetic or dermocosmetic composition.

In some embodiments, polylysine dendrimers (e.g., G2 dendrimers) are used in synergistic combinations with antimicrobial and/or anti-inflammatory agents, allowing for a reduction in the dosage of one or both agents to achieve a similar or improved effect. This would allow smaller doses to be used, thus reducing the potential incidence of toxicity and reducing the cost of expensive antimicrobial agents.

Herein, the additional antimicrobial agent may be selected from the group consisting of biocides, disinfectants, antiseptics, antibiotics, bacteriophages, enzymes, anti-adherents, immunoglobulins, antimicrobially active antioxidants, and mixtures thereof; it can act as a bactericide, especially against propionibacterium. In certain embodiments, the antimicrobial agent is an antibiotic, such as a penicillin, a cephalosporin, a carbacephem, a carbapenem, a monobactam, an aminoglycoside, a glycopeptide, a quinolone, a tetracycline, a macrolide, or a fluoroquinolone. Examples of specific antibiotic agents include, but are not limited to, penicillin G, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin, piperacillin, imipenem, aztreonam, cephalothin, cefazolin, cefaclor, cefamandole sodium, cefoxitin, cefuroxime, cefonicid, cefmetazole (cefetazole), cefotetan, cefprozil, chlorocepham, ceftamex, cefoperazone, cefotaxime, ceftizoxime (ceffizoxim), ceftriaxone, ceftazidime, cefepime, cefpodoxime, sulfadiazine, fleroxacin, nalidixic acid, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, doxycycline, minocycline, tetracycline, amikacin, lomefloxacin, gentamicin, kanamycin, netilmicin, tobramycin, streptomycin, azithromycin, clarithromycin, erythromycin estolate, erythromycin ethylsuccinate, erythromycin glucoheptonate, erythromycin lactobionate, erythromycin stearate, vancomycin, teicoplanin, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin, metronidazole, cephalexin, roxithromycin, amoxicillin complex (Co-amoxicilliavanate), piperacillin, and tazobactam, as well as various salts, acids, bases, and other derivatives thereof, and combinations thereof.

Polylysine dendrimers (e.g., G2 dendrimer) and/or salts thereof may also be used in combination with antifungal agents. Exemplary antifungal agents include, but are not limited to, terbinafine hydrochloride, nystatin, amphotericin B, griseofulvin, ketoconazole, miconazole nitrate, flucytosine, fluconazole, itraconazole, clotrimazole, benzoic acid, salicylic acid, and selenium sulfide.

Polylysine dendrimers (e.g., the G2 dendrimer) and/or salts thereof may also be used in combination with antiviral agents. Exemplary antiviral agents include, but are not limited to, amantadine hydrochloride, rimantadine, acyclovir, famciclovir, foscarnet, ganciclovir sodium, idoxuridine, ribavirin, solivudine, trifluorothymidine (trifloridine), valacyclovir, vidarabine, didanosine, stavudine, zalcitabine, zidovudine, interferon alpha, and edexuridine.

Polylysine dendrimers (e.g., the G2 dendrimer) and/or salts thereof may also be used in combination with antiparasitic agents. Exemplary antiparasitic agents include, but are not limited to, pyrethrins/synergistic ethers (piperonyl butoxides), permethrin, diiodoquinoline, metronidazole, diethylcarbamazine citrate, piperazine, thienopyrimidine (pyramate), mebendazole, thiabendazole, praziquantel, albendazole, proguanil, quinidine gluconate injection, quinine sulfate, chloroquine phosphate, mefloquine hydrochloride, primaquine phosphate, atovaquone, co-trimoxazole (sulfamethoxazole/trimethoprim), and pentamidine isethionate.

As noted above, polylysine dendrimers (e.g., G2 dendrimer) and/or salts thereof may be used in synergistic combinations with other known anti-inflammatory agents. Anti-inflammatory agents include, but are not limited to, corticosteroids (e.g., hydrocortisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., nabumetone, indomethacin, naproxen, ibuprofen), anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-13), cytokine antagonists (e.g., IL-1 receptor antagonists, TNF- α monoclonal antibodies, soluble TNF receptors, platelet factor 4), and the like. See also, for example, US 6,190,691; US 5,776,892; US4,816,449; and US RE37,263.

The formulations used in the methods of the invention may be incorporated into and thus applied in the form of: a cosmetic; skin care formulations (e.g., skin cleansers, lotions, or moisturizers); a deodorant or antiperspirant; cleansing formulations (e.g., facial cleanser); pharmaceutical (including veterinary) formulations; cosmeceutical formulations; toilet products (e.g. bath or shower additives or soaps). The formulation may be or be incorporated into a water-washable skin treatment product such as a skin cleanser, or in particular a leave-on skin treatment product.

As described herein, polylysine dendrimers (e.g., G2 dendrimer) and/or salts thereof exhibit antibacterial activity and reduce adhesion of certain strains of propionibacterium acnes to skin cells. Accordingly, one aspect of the present invention is a method of treating acne vulgaris by applying to the skin of a subject in need of treatment an antibacterially effective amount of a composition containing unbound poly-L-lysine dendrimers and/or salts thereof.

As used herein, "antibacterial active" refers to an active that is capable of preventing or reducing the growth and proliferation of propionibacterium acnes, preferably a propionibacterium acnes strain.

As mentioned above, "acne (ace)" or "acne vulgaris (ace vulgaris)" are multifactorial diseases of the pilosebaceous glands of the face and upper torso, characterized by various inflammatory and non-inflammatory lesions, such as papules, pustules, nodules and open and closed acne. Thus, its treatment may include treatment (which includes prevention or alleviation) of any of these symptoms, and reference to use as an anti-acne agent may be construed accordingly. In particular, treatment of acne includes treatment (including prevention) of lesions associated with acne. It also includes inhibiting propionibacterium activity that may cause or be associated with acne or symptoms thereof. In the context of the present invention, it may be particularly useful for the treatment of inflamed acne lesions.

Treatment of acne includes both therapeutic and prophylactic treatment of humans or animals, but especially humans. It may involve completely or partially eradicating the condition, removing or ameliorating the associated symptoms, arresting the subsequent development of the condition, and/or preventing or reducing the risk of the subsequent occurrence of the condition. As described herein, it will be particularly directed to the use of unbound G2 dendrimer and/or salts thereof. In certain embodiments, the method comprises treating acne vulgaris associated with propionibacterium acnes present on facial skin.

Another aspect of the invention relates to a method of reducing the inflammatory response of the skin by applying to the skin of a subject in need of treatment a composition containing unbound poly-L-lysine dendrons and/or salts thereof. In certain embodiments, the inflammatory response comprises IL-8 release or TLR2 overexpression in response to propionibacterium acnes. Measurable reductions in IL-8 release or TLR2 overexpression may be assessed using any conventional technique or those exemplified herein. Preferably, the polylysine dendrimer (e.g., unbound poly-L-lysine dendrimer) and/or salt thereof provides at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% reduction in IL-8 protein production and/or TLR2 overexpression compared to skin not contacted with the unbound poly-L-lysine dendrimer and/or salt thereof.

Yet another aspect of the invention is a method of promoting skin cell exfoliation by applying to the skin of a subject in need of treatment a composition containing a polylysine dendrimer (e.g., unbound poly-L-lysine dendrimer) and/or a salt thereof. Exfoliation is a natural phenomenon associated with the fact that the epidermis, which forms the upper layer of the skin, is constantly regenerating. During the normal process of exfoliation, the uppermost keratinocytes detach from the epidermal surface. According to the present method, polylysine dendrimers, such as unbound poly-L-lysine dendrimers, are applied to the skin surface to promote exfoliation and/or stimulate the epidermal renewal process.

The "Applying", "Applying" or "administration" of a composition containing a polylysine dendrimer, such as the unbound G2 dendrimer and/or salts thereof, to the skin of a subject includes external or topical skin treatment with the composition. In certain embodiments, a polylysine dendrimer, such as the G2 dendrimer and/or salts thereof, is applied to the facial skin of a subject.

A subject in need of treatment with a composition comprising a polylysine dendrimer, such as the G2 dendrimer, and/or salts thereof, refers to any subject at risk for, susceptible to, or having a condition in which the G2 dendrimer and/or salts thereof will provide a benefit, such as anti-acne activity, anti-inflammatory activity, or keratolytic activity. In some embodiments, the methods of the present invention are particularly useful for reducing acne problems in adolescents, reducing skin inflammation, and improving the appearance or feel of adult skin.

An effective amount of a composition comprising a polylysine dendrimer, such as the unbound G2 dendrimer and/or salts thereof, can be referred to as providing a measurable inhibition (whether complete or partial) of bacterial growth of one or more strains of propionibacterium acnes; a measurable reduction in inflammatory responses, such as IL-8 release or TLR2 overexpression in response to propionibacterium acnes; and/or a measurable amount of skin cell shedding. It may also refer to the amount of blemishes in acne skin or skin prone to acne that can be ameliorated or treated.

An effective amount of a composition containing a polylysine dendrimer, such as the unbound G2 dendrimer and/or salts thereof, includes from about 0.01ppm to about 100ppm, or more preferably from about 0.1ppm to 50ppm, or most preferably from about 1ppm to 10ppm of the polylysine dendrimer, such as the unbound G2 dendrimer and/or salts thereof.

In certain aspects, the compositions of the invention are used for non-therapeutic purposes. According to this aspect, the composition is used as an anti-acne/anti-inflammatory agent or in particular a skin care agent for non-therapeutic purposes, for example for cosmetic purposes, for example to improve the appearance or feel of the skin, in particular facial skin.

The invention is described in more detail by the following non-limiting examples.

Example 1: synthesis of DGLG2

The synthesis of the first generation poly (L-lysine) was performed by a three-step sequence (scheme 1).

i)NO,O₂,MeCN,0℃,1h；ii)NaHCO₃(0.2N),0℃,15h；

iii)NH₃,H₂O/MeOH,40℃,15h。

Scheme 1

N-carbamoylation of Ne-trifluoroacetyl-L-lysine affords C-Lys (Tfa). NO-mediated nitrosation of C-Lys (Tfa) in acetonitrile yields monomeric Lys (Tfa) -NCA, which is then reacted in aqueous sodium bicarbonate solution (pH6.5) to yield Ne-protected oligo (L-lysine) G1P. Both the nitrosation and polycondensation steps are carried out in one pot, without isolation of the NCA intermediate, the polymer G1P obtained precipitating spontaneously from the reaction medium. The polymer G1P was isolated by filtration and the epsilon amino group was deprotected by base treatment of G1P with ammonia in methanol/water. Deprotection gives oligo (L-lysine) G1 with a DPn of about 8 and an overall yield of 50-60% starting from C-Lys (Tfa).

The dendritic material was then prepared by carrying out the above reaction sequence (scheme 1) in a multi-generation sequence. Crude Lys (Tfa) -NCA was subjected to NaHCO in the presence of poly (L-lysine) G1 (30% w/w; monomer unit 0.36 eq.)₃After reaction in aqueous solution, polymer G2P precipitated spontaneously from the reaction medium, which after isolation was subjected to basic Ne deprotection to give poly (L-lysine) G2 with a DPn of about 48 and an overall yield of 36% starting from C-lys (tfa). Subsequently, ion exchange is performed to convert the trifluoroacetate salt to an acetate salt.

Example 2: inhibition of propionibacterium acnes by G2 dendrimers

The effectiveness of the G2 dendrimer in inhibiting the growth of propionibacterium acnes in culture was evaluated. G2 dendrimer with 10^-2M to 10^-5Final concentrations in the M range were used. Experiments were performed using the propionibacterium acnes strain IP53117T (see FR2844715a1), a strain known to cause acne. This strain belongs to phylogenetic form 1A1 ribotype 1(RT1) and is therefore an acne strain.

Bacteria (10)⁸CFU/ml) were seeded into 96-well plates containing a range of G2 dendrimer concentrations. After 24 to 48 hours of incubation at 37 ℃, the MIC (minimum inhibitory concentration) was visually defined as the lowest concentration at which no growth was visible. Based on the results of two independent experiments, this analysis showed that the MIC of the G2 dendrimer for Propionibacterium acnes strain IP53117T was 1.6-2.3X10^-5M (10-20 ppm). Thus, the G2 dendrimer was significantly inhibitedGrowth was made against the tested propionibacterium acnes strain and thus showed bactericidal effect at high doses (20 ppm).

Example 3: effect of G2 dendrimers on Propionibacterium acnes adhesion in keratinocyte cell cultures

The initial stage of acne vulgaris is the establishment, i.e., colonization, of bacteria on the skin surface. Colonization depends on bacterial adhesion. Thus, it was determined whether the G2 dendrimer could inhibit adhesion of the propionibacterium acnes strain to keratinocytes in culture.

Keratinocytes were isolated from the human epithelial cell line NCTC2544 (Neufahrt et al (1978) Arch.Dermaol.Res.256(3): 255-60). Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 1% antibiotic (penicillin/streptomycin) and 1% L-glutamine at 37 deg.C and 5% CO₂And maintaining the culture at 95% humidity.

To monitor bacterial adhesion, adenine of a strain of IP53117T bacteria (belonging to the acne phylogenetic IA1) was labeled with tritium and adhesion of the bacteria to the keratinocyte surface was determined by counting radioactivity.

Method 1. Bacteria to be labeled (2X 10)⁸microorganisms/mL) with varying amounts of G2 dendrimer (final concentration 10)^-5M to 6.25x10^-6M) are combined and deposited on the surface of a monolayer of keratinocytes. Incubate for 2 hours (37 ℃, 5% CO)₂) Thereafter, the cells were washed with Phosphate Buffered Saline (PBS) to remove non-adherent bacteria.

Method 2. Keratinocytes were pretreated with G2 dendrimer for 24 hours. The labelled bacteria (2X 10) were then used⁸Individual microorganisms/mL) was added to the cell monolayer for 2 hours without prior removal of the G2 dendrimer.

The results of this analysis show that after 2 hours, the dendrimers adhere at 10 compared to the adhesion of propionibacterium acnes in the absence of the G2 dendrimer^-5M、5X10^-6M and 2.5X10^-6Adhesion of propionibacterium acnes on the keratinocyte surface was inhibited by 50%, 40% and 29% at M, respectively. After 24 hours of pretreatment, withAdhesion of Propionibacterium acnes in the presence of the G2 dendrimer in contrast to the G2 dendrimer at 10^-5M、5X10^-6M and 2.5X10^-6Adhesion of propionibacterium acnes on the keratinocyte surface was inhibited by 50%, 51% and 35% at M, respectively. Based on the results of these two assays (with or without pretreatment), it was shown that the G2 dendrimer exhibited strong anti-adhesion activity. Thus, the G2 dendrimer may be used to block the initial stages of acne vulgaris, i.e. by preventing excessive bacterial adhesion or colonization by the acne strain.

Example 4: effect of G2 dendrimer on inflammation of Normal human dermal fibroblasts

There is evidence that propionibacterium acnes is involved in initiating inflammatory responses. In particular, it has been shown that stimulation of Toll-like receptor 2(TLR2) by Propionibacterium acnes leads to increased concentrations of interleukins 8 and 12(IL-8 and IL-12) (Kim et al (2002) J. Immunol.169(3): 1535-41). Thus, the effect of G2 dendrimer on inflammation of Normal Human Dermal Fibroblasts (NHDF) was evaluated. NHDF was isolated from human dermis at 37 ℃ with 5% CO₂And 95% humidity in DMEM containing 10% fetal bovine serum, 1% antibiotics (penicillin/streptomycin) and 1% L-glutamine.

IL-8 mediates the inflammatory response of the skin by recruiting neutrophils and other immune cells to invade injured or inflamed tissues. IL-8 secretion is induced in cultured human dermal fibroblasts following activation with the proinflammatory cytokine IL-1 α (Larsen et al (1989) Immunology 68(1): 31-6). Thus, in the presence or absence of G2 dendrimer (10)^{- 9}M、10^-8M、10^-7M or 10^-6M) or hydrocortisone (0.05. mu.g/mL) as a positive anti-inflammatory control, unconjugated NHDF cells were treated with IL 1-alpha (0.1 ng/mL). IL-8 release from fibroblasts was determined by enzyme-linked immunosorbent assay (ELISA).

The results of this assay (FIG. 1) show that hydrocortisone reduced IL-8 production by 83%. Under the same experimental conditions, the G2 dendrimer reduced IL1- α -induced IL-8 production by 66 to 81%. Thus, the G2 dendrimer attenuates the inflammatory cascade in human dermal fibroblasts known to be associated with colonization by propionibacterium acnes.

Example 5: anti-acne and keratolytic activity of G2 dendrimers in human skin explants

To demonstrate the anti-acne and keratolytic activity of the G2 dendrimer, human skin explants were exposed to lyophilized propionibacterium acnes (propionibacterium acnes strain IP 53117T-RT 1-phylogenetic IA1) to mimic acne in vivo in vitro.

And (4) treating the skin. 15 individual skin samples were obtained from patients undergoing plastic surgery (caucasian women, 26 years old). The skin explants were kept in culture for 8 days. Treatment with G2 dendrimer (2 and 5ppm) was performed for 1 hour on days 0, 1, 4 and 6 while applying lyophilized propionibacterium acnes (30 μ L) (fig. 2). On day 8, explants were fixed and embedded in paraffin. Sections 5 μm thick were stained with Masson trichrome or anti-TLR 2 antibody.

Evaluation of keratolytic Effect. Explants were analyzed for general morphology, in particular the stratum corneum. The images were taken with a leica orthoplan microscope equipped with a digital camera (tri-CCD) driven by the acquisition and archiving software leica im 1000.

Immunostaining for TLR 2. TLR2 is known to mediate the inflammatory response of skin to acne (Kim et al (2002) J.Immunol.169(3): 1535-41; McInturff & Kim (2005) Semin. Cutan. Med. Surg.24(2): 73-8). Thus, anti-TLR 2 (clone TL 2.1; Santa Cruz) was used to evaluate TLR2 protein in frozen sections. Sections were incubated with anti-TLR 2 antibody at 1:25 dilution overnight at 4 ℃ followed by detection of antibody binding with biotin/streptavidin-FITC conjugate. Nuclei were stained with propidium iodide.

Quantification of IL 1-alpha cytokine. The concentration of IL1- α in the medium was determined using an ELISA assay kit (Cayman, Ann Arbor, MI). Absorbance was measured using a microplate reader Tecan Infinite M200 Pro in combination with Magellan software.

And (6) obtaining the result. Topical application of 2ppm of G2 dendrimer resulted in moderate separation of the upper layers of the stratum corneum, with the lower layers remaining more stratified. In contrast, application of 5ppm of G2 dendrimer induced lower detachment. In view of this keratolytic activity, G2 dendrimers can be used to promote skin desquamation.

With respect to TLR2 expression, topical application of G2 dendrimer at 2ppm and 5ppm resulted in complete inhibition of propionibacterium acnes-induced TLR2 overexpression.

To assess the inflammatory response, the production of IL 1-a was quantified (fig. 3). At day 1 (D1), propionibacterium acnes did not induce a significant increase in IL1- α production compared to control conditions (i.e., untreated explants). Further, 2ppm and 5ppm of G2 dendrimer significantly reduced IL 1-a production by 49% and 42%, respectively, on day 1, compared to treatment with propionibacterium acnes. After 4 days of survival (D4), the release of IL 1-a was significantly reduced by 53% under control conditions. Furthermore, 2ppm and 5ppm of the G2 dendrimer provided a reduction in IL1- α production (28% and 27% reduction, respectively) at day 4 compared to treatment with propionibacterium acnes. Thus, the G2 dendrimer reduces the growth of propionibacterium acnes, attenuates the immune and inflammatory cascades to achieve optimal skin protection, and provides keratolytic action.

Example 6: the G2 dendrimer selectively increased membrane fluidity in a propionibacterium acnes strain belonging to propionibacterium acnes phylogenetic IA 1.

The effect of the G2 dendrimer on the membrane fluidity of three bacterial strains of propionibacterium acnes was tested. Two test strains of propionibacterium acnes were "acne" strains belonging to phylogenetic IA1(RT4 and RT5), while the last was a non-acne strain belonging to phylogenetic II (RT 6).

Several colonies of RT4, RT5 and RT6 (Propionibacterium acnes strain) were scraped and placed in fortified Clostridium medium (RMC medium). The tube was vortexed for a few seconds and homogenized, then completely filled with RMC to create an anaerobic state. The tubes were then incubated at 37 ℃ for at least 48 hours.

Bacterial cultures were collected and OD was taken at 580 nm. After inoculation, the bacteria were transferred to a microplate and treated with or without the G2 dendrimer. After 2 hours in the anaerobic chamber, the plates were transferred to a microplate reader for 72 hours (OD580nm, measured every 30 minutes).

Then, the bacterial production time of each propionibacterium acnes strain was analyzed. The G2 dendrimer was tested at the following concentrations: 6.10^-6M and 6.10^-7M。

-results of

The results are shown in fig. 4. After 72 hours incubation, G2 dendrimer (6.10) compared to control^-6M) significantly reduced the membrane stiffness of acne strains RT4 and RT 5. Notably, 6.10^-6The G2 dendrimer of M did not show any significant effect on RT6 strain. In other words, the G2 dendrimer showed a specific effect on the membrane fluidity of acne strains RT4 and RT5 compared to the non-acne strain (RT 6).

This increase in membrane fluidity is expected to destabilize the bacterial membranes of the acne strain of acne phylogenetic IA1, limiting their growth, while maintaining or even promoting non-acne phylogenetic strains of phylogenetic type II.

Example 7: effect of G2 dendrimers on biofilm formation by acne strains

Scheme (iii)

Bacterial strains and bacterial cultures

Propionibacterium acnes ribotype 5(RT5) HL043PA2/HM-514 and Propionibacterium acnes non-acne strain ribotype 6(RT6) HL110PA3/HM-554 were obtained from BEIRESources American type culture Collection (Va, USA). These two strains belong to phylogenetic forms IA1 and II (McDowell,2017), respectively. Bacteria, stored at-80 ℃, RT6 strain was initially plated on brain heart infusion (BHI, BD) agar plates, RT5 strain was initially plated on enhanced clostridial media (RCM) agar plates and incubated using bdgaspack (tm) system at 37 ℃ under hypoxic conditions. Colonies were transferred to 15mL sterile conical tubes (Falcon) containing 12mL of RCM, vortexed and after homogenization, the Falcon tubes were completely filled with RCM medium and incubated at 37 ℃ for 72 hours.

Biofilm formation on glass slides

To evaluate the exposure to G2 dendrimerDuring this period, the Propionibacterium acnes biofilm formed using a 24-well plate with a flat glass bottom (sensory, Greinerbio-one, Germany). The 72 h bacterial culture (see above) was centrifuged at 7,500g for 10 min at room temperature. The bacterial pellet was resuspended in 5ml of Sterile Physiological Water (SPW). Finally, 300. mu.l of bacterial inoculum (OD)_580nm0.8) into eight central wells of a 24-well plate. The plates were incubated under anaerobic conditions for 3 hours to promote primary adhesion of the bacteria. After this incubation period, the physiological water in each well was carefully removed and 1ml of bacterial culture medium (RCM) supplemented or unsupplemented with G2-dendrimer was added. The plates were incubated in an anaerobic WhitleyA85 workstation for 72 hours at 37 ℃.

After 72 hours (biofilm formation), the RCM medium in each well was carefully removed to remove planktonic cells. Next, 300 μ Ι syto9 green fluorescent nucleic acid stain (thermolasher) was added to each well and incubated for 20 minutes in dark conditions. After this time, SYTO9 stain was removed and 300 μ Ι spw was added to each well. CLSM observations were performed immediately using Zeiss lsm710(Carl Zeiss microscopiy, Oberkochen, germany) using a 63x oil immersion objective. Syto9 was excited at 488nm and fluorescence emission was detected in the range 500 to 550 nm. Images were taken per micron throughout the depth of the biofilm. Visualization and processing of three-dimensional (3D) images was performed using zen2.1sp1 software (Carl Zeiss microscopi, Oberkochen, germany). Quantitative analysis of image stacks was performed using COMSTAT software (http:// www.imageanalysis.dk /) (Heydorn et al, 2000). The maximum and average biofilm thicknesses (μm) and their biomass volumes (μm) were determined³/μm²). Each study was repeated a minimum of 3 times.

-results of

The G2 dendrimer was shown to reduce the biofilm thickness of the RT5 (-48%) and RT6 (-32%) strains. Notably, the reduction in biofilm thickness was significantly more important for acne strain RT5 than for non-acne strain RT 6. Furthermore, as shown in fig. 6, G2 dendrimers significantly reduced the biomass density in RT5 (-50%) biofilms, while having no effect on the biomass density in RT6 biofilms.

In summary, polylysine dendrimers are expected to reduce the inflammatory response elicited by the propionibacterium acnes biofilm (whatever the strain) by reducing the thickness of the biofilm, while exerting a selective and specific inhibitory effect on the acne strain by destabilizing the biofilm of the acne strain. This destabilization was not observed for the non-acne strain RT6, as the biomass density remained unchanged.

Notably, such results are consistent with example 6, which shows that polylysine dendrimer increased membrane fluidity of the acne RT5 strain, while not increasing membrane fluidity of the non-acne RT6 strain.

Example 8: clinical research

Cream containing 2ppm g2 dendrimer (test cream) or placebo cream was applied twice daily on the half-face for 28 days on 20 caucasian subjects with greasy skin on the face and a tendency to develop acne (50% of subjects between 14 and 25 years, 50% of subjects between 26 and 40 years). The test formulation (G2 dendrimer formulation) is shown below:

the G2 dendrimer significantly improved the appearance of acne skin by reducing acne lesions and modulating sebum secretion rates.

The placebo formulation was similar to the test formulation except that the G2 dendrimer was replaced with water. After 28 days of use, the potential for acne development and the anti-blemish effect were evaluated in comparison with the number of lesions on the face before application (D0):

-clinical examination: after 28 days of use, the test creams were evaluated for their comedogenic potential and anti-blemish effect compared to the number of lesions on the face before application (D0). On days D0 and D28, the dermatologist examined the skin and counted the total inflammatory lesions as well as papules and pustules on each half of each subject enrolled. The dermatologist also determined the number of total remaining lesions and blackheads on each half of each patient. The results are shown in FIGS. 5A, 5B and 5C.

-Sebum level measurement: on day 28, courose and KHAZAKA SM 810 PC were usedThe amount of sebum secreted to the skin surface was quantitatively evaluated. The results are shown in FIG. 5D.

As a result:

as shown in fig. 5A-5C, the test cream containing the G2 dendrimer significantly reduced the number of blackheads, retained lesions, and inflammatory lesions after 28 days of treatment compared to the placebo cream. Moreover, the test cream also significantly reduced the sebum secretion rate of acne skin compared to the placebo cream (fig. 5D). All these data taken together indicate that the G2 dendrimer is an effective agent for managing acne conditions.

G2 dendrimers improve the microbiota of acne skin

After 28 days of treatment, a skin sample (swab) was taken from each half of the face of each selected volunteer in order to quantify the propionibacterium acnes strain by metagenomic analysis and to determine the effect of the test cream on the diversity of propionibacterium acnes compared to the placebo cream.

The principle of this metagenomic study is to amplify a region of dermatophytes acnes and sequence the amplicons. The amplicon sequences were compared to the SLST reference database (http:// medbac. dk/SLST/pacnes) which links the amplicon sequences to SLST types and clades and phylogenetic forms. The method is based on an improved method of Scholtz et al (Scholz,2014, supra). The abundance and diversity of SLST types and clades and phylogenetic types were calculated for each sample and compared to evaluate the therapeutic effect between the groups of volunteers treated with the active formula and volunteers treated with the placebo formula.

Scheme(s)

The swab was frozen and stored at-80 ℃ until DNA extraction. Extraction and purification of DNA was performed using Qiagen DNeasy PowerSoil Kit (Cat # 12888). Briefly, cells were lysed by a combination of mechanical (bead collision) and chemical (detergent) treatments. The extracted DNA was then purified on a silica membrane.

The original propionibacterium acnes SLST method (Scholz,2014, supra) amplified a 612 base pair region that was too large to be sequenced using Illumina technology. A new primer was designed to amplify a region or 497 base pairs with the forward primer located 74 nucleotides downstream of the original and the reverse 26 nucleotides upstream. . This modification would not allow for the differentiation of SLST types a1 and a6 or SLST types E3 and E7. In both cases, these variants belong to the same clade and phylogenetic type. DNA sequencing was performed on Illumina Miseq using paired-end technology. All sequence processing was performed using the QIIME2 suite (https:// qiime2.org /). After mass filtering, the clustered and chimeric sequences were removed by overlapping the pooled read pairs. The SLST type was assigned to the sequence against the reference database for the cutaneous bacterium acnes SLST (http:// medbac. dk/SLST/pacnes) using a 99% homology threshold.

And (6) analyzing the data.

Inputting data

The raw data were normalized using the sum-scaling (TSS) procedure, i.e., for a given sample, the abundance of each SLST divided by the sum of the abundances of all taxa.

The abundance data was further filtered by removing SLST types observed in only one sample before statistical comparisons between groups were made.

Sparse

The sparse curve was plotted using the software of Past3.20 (https:// palaeo-electronic. org/2001_1/past/issue1_01.htm), f (sequence number) ═ SLST number. These curves allow a graphical assessment of whether the sequencing depth is sufficient (when the curve reaches the plateau).

Alpha (α) -diversity.

Shannon and simpson diversity indices were calculated using the past3.20 software. These two α diversity metrics were used to characterize the categorical diversity of each sample: shannon entropy-like diversity and simpson (1-D) diversity. Both values of these indices depend on richness (number of different taxa) and population uniformity, with simpson's index being less affected by low abundance taxa. In both cases, the index increases with diversity. The number (abundance) of slslss is proposed, which can also be considered as a measure of diversity, with the bias of giving the same weight to high-abundance and low-abundance slss.

In all cases, calculations were performed using the SLST type normalized abundance data.

And (4) comparing the abundance.

Strain type abundances at each level were compared using parametric paired Student T test and nonparametric Wilcoxon test and symbolic test. Calculations were performed using SLST type normalization and filtered abundance data.

Results

This study led to the identification of 32 SLST types in the test samples, all of which are related to the phylogenetic type described in the literature.

Phylogenetic IA1 is the most representative one, with SLSTA1 being the predominant, followed by phylogenetic II, IB, IA2, IC and III. This redistribution is the same for samples treated with the test cream or placebo cream. The phylogenetic reassignment varies depending on the individual (age, sex) and the sampling area. Phylogenetic IA1 was more abundant for most of the back samples compared to the face samples. Our findings are consistent with the studies by Dagnelie et al (Dagnelie,2018, supra) which further showed that phylogenetic IA1 is more abundant in acne skin than in healthy skin, whereas phylogenetic II is less abundant on acne skin than healthy skin.

As shown in fig. 5E and 5F, treatment with the test cream increased strain diversity compared to the placebo cream after 28 days of treatment. We further noted that treatment with the test composition resulted in a decrease in the phylogenetic abundance of acne IA1 and an increase in non-acne phylogenetic II compared to placebo cream, especially in male subjects and subjects at least 23 years of age. The G2 dendrimer therefore exerts a beneficial effect on the propionibacterium acnes population in acne skin by limiting the acne strains while promoting non-acne strains. In other words, the G2 dendrimer can be used to rebalance the propionibacterium acnes population in acne skin and acne-prone skin.