purified polypeptide, topical formulation, isolated polynucleotide, vector, recombinant microorganis

专利摘要:
  The invention provides a purified polypeptide that inhibits (i) protease production and / or keratinocyte activity, (ii) inhibits IL-6 production and / or keratinocyte activity, (iii) inhibits phenol modulin production soluble alpha 3 of Staphylococcus aureus and / or (iv) inhibits agr production and / or activity by S. aureus. A topical formulation comprising the polypeptide is additionally provided. A recombinant microorganism is provided that comprises a vector or polynucleotide that encodes the polypeptide. An additional probiotic composition is provided which comprises the recombinant microorganism. The invention also provides kits and articles of manufacture comprising the polypeptide and / or the recombinant microorganism.
公开号:BR112020003508A2
申请号:R112020003508-4
申请日:2018-08-31
公开日:2020-09-01
发明作者:Richard L. Gallo；Michael Williams
申请人:The Regents Of The University Of California；
IPC主号:

专利说明:

[001] [001] This invention was made with the support of the Government under Contracts Nos AI117673, AR067547, AR062496 and AR064781, granted by the National Institutes of Health. The Government has certain rights in the invention. Cross-reference to related patent applications
[002] [002] This patent application claims its priority, under the terms of 35 U.S.C. §119, to Provisional Patent Application No. Serial 62 / 553,025, filed on August 31, 2017, the content of which is incorporated by reference. Technical description field
[003] [003] The description refers to compositions and methods for treating dermatological diseases and disorders and compositions that modulate the permeability of the skin barrier.
[004] [004] Microorganisms exemplary of the description (Staphylococcus epidermidis A11, Staphylococcus hominis C5, Staphylococcus hominis A9 and Staphylococcus warneri G2) were deposited on August 28, 2018, with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110- 2209, under ATCC Number _______ (S. epidermidis A11 81618 strain name, deposited on August 28, 2018),
[005] [005] The epidermis is the first line of defense of the immune system and protects and regulates interactions between microbes and the host organism. The control of this interaction is important because bacteria not only reside on the surface where they influence superficial keratinocytes, but also penetrate below the stratum corneum and into the dermis, where some species of bacteria have been shown to influence immune function. For example, Staphylococcus epidermidis (S. epidermidis) interacts with keratinocytes of the epidermis to prevent inflammation mediated by the toll-type 3 receptor (toll-like receptor 3), recruits mast cells and T cells and increases the tight junctions and production of antimicrobial peptides. Unlike the most common commensal bacteria, S. epidermidis, Staphylococcus aureus (S. aureus) is often pathogenic and has a negative influence on skin function. It is especially evident in skin diseases, such as atopic dermatitis (AD), where S. aureus promotes this disease.
[006] [006] The microbiome that inhabits the skin of individuals with AD revealed less global microbial diversity and greater abundance of S. aureus. Increased colonization by S. aureus was linked to greater disease severity for patients with AD. In mechanistic terms, it is not clear how S. aureus worsens the disease. Several S. aureus products have been shown to damage the barrier and / or trigger inflammation. These products include toxin A, superantigens, toxin 1 of toxic shock syndrome, enterotoxins, protein A, Panton-Valentine leukocidin, exfoliative toxins and serine Protease V8. Because of the potential pathogenic effects of these molecules, understanding the skin's response to colonization by S. aureus in the absence of clear clinical signs of infection is critical to understanding the pathogenesis of and for the development of future therapies. summary
[007] [007] The description provides a purified polypeptide comprising a sequence that is at least 98% identical to SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17 and which inhibits (i) the production of proteases and / or keratinocyte activity, (ii) inhibits IL-6 production and / or keratinocyte activity, (iii) inhibits the production of soluble phenol alpha 3 modulin from Staphylococcus aureus (S. aureus) and / or (iv ) inhibits agr production and / or activity by S. aureus. In one embodiment, the polypeptide is at least 98% identical to SEQ ID NO: 2. In another embodiment, the polypeptide comprises SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17. In yet another embodiment, the polypeptide consists of SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17. In another or more embodiments of any of the above, the polypeptide comprises one or more D-amino acids. In yet another or more embodiments, the polypeptide comprises a compound of Formula I, IA or IB (see below).
[008] [008] The description also provides a topical formulation comprising a polypeptide of the description or a compound of Formula I, IA
[009] [009] The description also provides an isolated polynucleotide that encodes a polypeptide of the description. In one embodiment, the polynucleotide comprises a sequence that hybridizes under stringent conditions to a polynucleotide that consists of SEQ ID NO: 1 or 3 and encodes a polypeptide comprising SEQ ID NO: 4. In another embodiment, the polynucleotide comprises SEQ ID NO: 1 or 3.
[0010] [0010] The description also provides vectors that comprise a polynucleotide of the description. The vector can be any vector suitable for expression in a cell or a microbial host.
[0011] [0011] The description also provides a recombinant microorganism that comprises a vector or polynucleotide of the description. In some embodiments, the microorganism does not naturally express a polypeptide from the description, but, through recombinant engineering, is modified to express a polynucleotide from the description. In yet another modality, the microorganism is attenuated by the fact that it has become non-pathogenic or has reduced pathogenicity compared to a wild-type organism of the same species. In yet another embodiment, p recombinant microorganism is a microorganism normally found (eg, commensal) on the skin of the mammal (eg, a human).
[0012] [0012] The description also provides a probiotic composition that comprises a recombinant microorganism from the description.
[0013] [0013] The description also provides a probiotic composition comprising a microorganism that expresses a polypeptide of the description (e.g., SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 and / or 17). In one embodiment, the microorganism is S. hominis, S. epidermidis, S. warneri or any combination thereof. In an additional embodiment, the microorganism is S. hominis C5, S. hominis A9, S. epidermidis A11 and / or S. warneri G2. In yet another or more modalities, the composition comprises
[0014] [0014] The description also provides a method of treating a dermatological disorder, which comprises administering an effective amount of Staphylococcus sp. coagulase-negative (CoNS), or an effective amount of a CoNS fermentation extract sufficient to inhibit protease activity in the skin, in which CoNS produces a polypeptide comprising a sequence that is at least 98% identical to SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17 and that inhibits protease production. In one embodiment, the dermatological disorder is selected from the group consisting of Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus. In another embodiment, administration is by topical application. In another or additional modality, CoNS is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphyloccus
[0015] [0015] The description also provides a method of treating a skin disease or disorder, which comprises measuring the protease activity of an individual's skin culture or the individual's skin; compare protease activity with that of a normal control; administering a composition of commensal skin bacteria and / or coagulase-negative Staphylococci fermentation extract, wherein the composition of commensal skin bacteria or fermentation extract comprises a polypeptide that is at least 98% identical to SEQ ID NO: 4 , 11, 12, 13, 14, 15, 16 or 17, and / or comprises a compound of Formula I, IA or IB, in which the composition is formulated in cream, ointment or a pharmaceutical composition that maintains the capacity of commensal bacteria of the skin to grow and replicate. In one embodiment, the coagulase-negative Staphylococcus is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus devisoci, Staphylococcus, Staphylococyl, Staphylococcus, hamster and Staphylococcus lugdunensis.
[0016] [0016] The description also provides a method of treating a skin disease or disorder, which comprises administering a purified polypeptide of the description or a probiotic composition comprising a bacterium that produces a polypeptide that is at least 98% identical to SEQ
[0017] [0017] The description also provides a method of treating a skin disease or disorder, which comprises administering a composition that inhibits the expression of soluble phenol modulin, wherein the composition comprises a purified polypeptide of the description or a compound of Formula I , IA or IB. In one embodiment, administration is topical. In another embodiment, the composition is a coagulase-negative Staphylococci fermentation extract.
[0018] [0018] The description also provides a topical probiotic composition comprising commensal probiotic bacteria from the skin selected from the group consisting of S. epidermidis A11, S. hominis C4, S. hominis C5, S. hominis A9, S. warneri G2 and any combination of them. In one embodiment, the composition is formulated into a lotion, shake-type lotion, cream, ointment, gel, foam, powder, solid, paste or tincture.
[0019] [0019] The description also provides a drug composition comprising a drug and a fermentation extract of S. aureus or probiotic S. aureus comprising a soluble phenol alpha 3 modulin. The description also provides the use of the composition for delivering a drug through the skin of an individual.
[0020] [0020] The description provides commensal / good bacteria and / or their products to prevent the increased activity of proteases on the skin. This is important in many disease states including atopic dermatitis, Netherton's syndrome and other skin conditions that suffer from abnormally increased protease activity and barrier breakdown.
[0021] [0021] This description also provides a factor and compositions to induce protease activity and, therefore, assist with proteolytic remodeling of the skin in the treatment of disorders related to wound repair, aging, sun damage, pigmentary abnormalities and
[0022] [0022] The description provides a method of treating a dermatological disorder, which comprises administering an effective amount of Staphylococcus sp. coagulase-negative (CoNS), or an effective amount of a CoNS fermentation extract sufficient to inhibit protease activity in the skin. In one embodiment, the dermatological disorder is selected from the group consisting of Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus. In another embodiment, administration is by topical application. In another modality, CoNS is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylocococcus, staphylococcus devi, Staphylococcus devi . In a specific modality, CoNS is S. epidermidis.
[0023] [0023] The description also provides a method of treating a skin disease or disorder, which comprises measuring the protease activity of an individual's skin culture or the individual's skin; compare protease activity with that of a normal control; administering a composition of commensal skin bacteria and / or fermentation extract of a coagulase-negative Staphylococcus, wherein the composition of commensal skin bacteria comprises at least one commensal bacterium which reduces the serine protease activity of the culture or skin, in which at least one of the commensal bacteria is formulated in cream, ointment or a pharmaceutical composition that maintains the ability of commensal bacteria in the skin to grow and replicate. In one embodiment, the coagulase-negative Staphylococcus is selected from the group consisting of Staphylococcus epidermidis,
[0024] [0024] The description also provides a method of treating a skin disease or disorder, which comprises administering an agent that inhibits the expression of kallikreins. The description also provides a method of treating a skin disease or disorder, which comprises administering an agent that inhibits the expression of soluble phenol modulin. In any of the foregoing modalities, administration is topical. In another embodiment, the agent is a fermentation extract of a coagulase-negative Staphylococcus In another embodiment, the coagulase-negative Staphylococcus is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolytici, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus devriesei, Staphylococcus hominis, Staphylococcus jettensis, Staphylococcus petrasii and Staphylococcus lugdunensis.
[0025] [0025] The description also provides a topical composition that comprises a plurality of skin bacteria. In one embodiment, the commensal probiotic bacteria on the skin is a species of coagulase-negative Staphylococcus. In another distinct modality, the commensal probiotic bacterium of the skin comprises Staphylococcus aureus. In an embodiment of any of the previous embodiments, the bacterium is formulated in a cream, lotion, tincture, gel or other topical formulation in which the bacterium remains viable.
[0026] [0026] The description also provides a topical probiotic composition which comprises a fermentation extract of commensal probiotic bacteria
[0027] [0027] In any of the described modalities, a topical probiotic composition is formulated in lotion, shake-type lotion, cream, ointment, gel, foam, powder, solid, paste or tincture.
[0028] [0028] The description provides a drug composition comprising a drug and an extract of S. aureus fermentation or a biotic composition of S. aureus.
[0029] [0029] The description provides a method for delivering drugs through the skin, which comprises placing a composition comprising a drug and a fermentation extract of S. aureus or biotic composition of S. aureus in contact with the skin. In one embodiment, the drug is a topical drug to be absorbed or adsorbed through the skin.
[0030] [0030] The description also provides a method of releasing a topical drug, wherein the method comprises placing a composition comprising a S. aureus or a S. aureus fermentation extract in contact with an individual's skin for a time, at a dose and under conditions that increase the permeability of the skin and, place the drug to be released in contact with the skin.
[0031] [0031] The description provides a composition comprising a fermentation extract of S. aureus or a lotion, shake-type lotion, cream, ointment, gel, foam, powder, solid, paste or tincture containing viable S. aureus.
[0032] [0032] The details of one or more embodiments of the invention are
[0033] [0033] Figure 1A-D shows (AC) NHEKs that were treated for 24 hours with the supernatants of S. aureus (SA; Newman, USA300, 113, SANGER252) and S. epidermidis (ATCC12228, ATCC1457), filtered on membrane sterile, and the conditioned medium of NHEK was analyzed with specific substrates for trypsin-like protease, elastase-like and MMP proteases. (D) Proteases secreted by S. aureus (Newman) were analyzed for their influence on trypsin activity. The data represent the mean ± SEM (n = 4) and are representative of at least three independent experiments. ANOVAs one way (unidirectional) (aec) and ANOVAs one way (bidirectional) (d) were used and the significance was indicated by * P <0.05, *** P <0.001, **** P <0.0001 . ANOVA, analysis of variance; MMP, matrix metalloproteinase; NHEK, normal human epidermal keratinocyte.
[0034] [0034] Figure 2A-C shows (A) the activity of total proteases (5 µg per ml of BODIPY FL casein), measured in the conditioned medium of NHEK after the treatment of the supernatant of S. aureus (SA, Newman) during 0 -48 hours, (B) while aprotinin, a serine protease inhibitor (800 µg / mL), was applied to the conditioned medium after treatment for 24 hours. (C) Strains of S. aureus (USA300 LAC) WT and without proteases were compared for effects on trypsin activity in the conditioned medium of NHEK (Boc-Val-Pro-Arg-AMC, 200 mM). Two way ANOVAs (A, B) and one way ANOVAs (C) were used and significance was indicated by * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ANOVA, analysis of variance; NHEK, normal human epidermal keratinocyte; WT, wild type.
[0035] [0035] Figure 3A-F shows increases in KLK expression by S.
[0036] [0036] Figure 4A-D shows multiple KLKs that are responsible for the serine protease activity induced by S. aureus in human keratinocytes. NHEKs were treated with KLK6, 13 or 14 (15 nM) siRNA before differentiation with CaCl2 and the addition of S. aureus supernatant (Newman). Controls 1 and 2 with scrambled siRNA (-) (scrambled) were used at 15 nM and 45 nM, respectively. (A) The conditioned medium was analyzed for changes in trypsin activity (Boc-Val-Pro-Arg-AMC, 200 µM). (B-D) The levels of KLK6, KLK13 and KLK14 transcripts were evaluated by qPCR and normalized to the housekeeping gene, GAPDH, in order to confirm the efficiency of siRNA knockdown (expression reduction). The data represent the mean ± SEM (n = 4) and are
[0037] [0037] Figure 5A-C shows that multiple KLKs regulate DSG-1 and FLG cleavage induced by S. aureus in human keratinocytes. NHEKs treated with S. aureus supernatant (Newman) for 24 hours were evaluated for changes in the cleavage of (A) desmoglein-1 (DSG-1) and (B) profilagrin (Pro-FLG) after knockdown with KLK6 siRNA , 13 and 14 (15 nM) by immunoblotting. The housekeeping gene, a-tubulin, was used as a loading control. DSG-1 (complete) and Pro-FLG are indicated by black arrows. (C) Densitometry analysis of DSG-1 (complete) and Pro-FLG represented by the average number of pixels normalized to a-tubulin (n = 1). Immunoblots are representative of at least three independent experiments. KLK, kallikrein; NHEK, normal human epidermal keratinocyte; siRNA, small interference RNA.
[0038] [0038] Figure 6 describes a method for preparing fermentation extracts and activity tests.
[0039] [0039] Figure 7 shows that S. aureus soluble phenol modulins (PSMs), under the control of the Agr quorum sensing system (chemical signaling) (accessory regulatory gene), are responsible for increased serine protease activity in keratinocytes.
[0040] [0040] Figure 8 shows that PSMs of S. aureus increase serine protease activity and damage to the skin barrier in mice.
[0041] [0041] Figure 9 shows that S. aureus isolates from the skin with atopic dermatitis (AD) lesion can induce serine protease activity
[0042] [0042] Figure 10 shows that the ATCC14490 strain of coagulase-negative Staphylococci (CoNS) (S. epidermidis) is capable of producing autoinducing peptide (AIP) to deactivate the agr activity of S. aureus.
[0043] [0043] Figure 11 shows the effect of S. aureus and commensal bacteria on serine protease activity in atopic dermatitis.
[0044] [0044] Figure 12 shows the effect of S. hominis C5 on the agr activity of S. aureus.
[0045] [0045] Figure 13 shows the effect of several CoNS on the agr activity of S. aureus.
[0046] [0046] Figure 14A-J shows that PSMα from S. aureus disturbs epithelial barrier homeostasis. Human keratinocytes (NHEKs) were stimulated with sterile filtered supernatant from wild type S. aureus (SA) strains (WT), with knockout (silencing) of PSMα (ΔPSMα) or PSMβ (ΔPSMβ) for 24 hours and (A) trypsin activity and (B) KLK6 mRNA, compared to those of the housekeeping GAPDH gene, were analyzed (n = 4). (C) Synthetic PSM peptides were added to NHEKs for up to 24 hours to analyze changes in trypsin activity. (D, E) Analysis of transcripts by RNA-Seq of genes that underwent a change ≥2 times after treatment with PSMα3 was performed followed by analysis of gene ontology (GO). 8-week-old male C57BL / 6 mice (n = 6) were treated for 72 hours with SA WT, SA ΔPSMα or a strain of SA with knockout of 10 secreted proteases (Δproteases) (1e7 CFU). (F, G) Representative photographs of murine skin (dashed lines indicate the treatment area) and changes in epidermal thickness after treatment (scale = 200μm). (H-K) Changes in murine dorsal skin with WT strains or SA mutants in transepidermal water loss (TEWL) and SA CFU / cm2 were also evaluated. All bars
[0047] [0047] Figure 15A-G shows the characterization of the Staphylococcus epidermidis agr type I autoinductive peptide and skin deficiency with AD. (A, B) Inhibition by the supernatant of S. epidermidis agr types I-III of S. aureus (SA) USA300 LAC agr type I activity after 24 hours (n = 4) and representation of the known structure of the autoinducing peptide (AIP ) of S. epidermidis agr type I. (C) Effect of the RP62A strain of Staphylococcus epidermidis (S. epi) agr type I wild type (WT) or with autoinductive peptide knockout (ΔAIP) on the agr activity of SA after 24 hours. (D) Growth in sterile filtered supernatant from SA, with or without the S. epi WT or ΔAIP supernatant being applied to NHEKs for another 24 hours, by measuring NHEK trypsin activity (n = 4). (E) Consensus on genomes of S. epidermidis agr types I-III found on the skin with AD. (F, G) Ratio between relative abundance of S. epidermidis agr type I and SA in regions with a crisis of 8 individuals with AD, from the AD score from “less severe” to “more severe” based on the objective SCORAD and combined global data for all individuals based on atopic dermatitis severity. All error bars are represented in the standard error of the mean (SEM) and one way ANOVAs (A, C, D) and unpaired test (nonparametric) and Mann-Whitney (F) were used to determine the statistical significance indicated by : p <0.05 *, p <0.01 **, p <0.001 ***, p <0.0001 ****.
[0048] [0048] Figure 16A-F shows that multiple clinically isolated coagulase-negative Staphylococci inhibit the agr activity of S. aureus. (A) Clinically isolated coagulase-negative Staphylococci supernatants (CoNS), filtered through a sterile membrane, were added to the reporter strain S. aureus (SA) USA300 LAC agr type I P3-YFP for 24
[0049] [0049] Figure 17A-H shows that the AD CoNS clinical isolate induced damage to the murine skin barrier. The reporter strain S. aureus (SA) USA300 LAC agr type I pAmi P3-Lux (1e7 CFU) with or without live S. hominis C5 (1e8 CFU) was applied to 8 week old female C57BL / 6 mice for 48 hours (n = 5). (A, B) The activity of agr de SA was evaluated on murine dorsal skin by changes in luminescence. (C) Representative images of murine skin after treatment with SA for 48 hours (dashed squares indicate the treatment area). (DH) SA CFU / cm2 was determined and damage to the murine skin barrier and inflammation was assessed by analyzing changes in IL-6 RNA expression, transepidermal water loss (TEWL), trypsin activity and mRNA expression in Klk6 normalized to the Gapdh housekeeping gene. All error bars are represented in the standard error of the mean (SEM) and one-way ANOVAs were used to determine the statistical significance indicated by: p <0.05 *, p <0.01 **, p <0.001 *** , p <0.0001 ****.
[0050] [0050] Figure 18A-H shows that S. aureus PSMα alters essential genes for barrier and cytokine expression in human keratinocytes. (A-D) Human keratinocytes treated with synthetic PSMα3 were evaluated for changes in trypsin activity and expression of KLK6 transcripts normalized to the housekeeping GAPDH gene in a dose and time dependent manner. (E) Analysis by GO terms of negatively regulated genes with variation ≥2 in relation to the control in human keratinocytes treated with PSMα3 for 24 hours. (F-H) Changes in protein expression of cytokines IL-6, TNF-α or IL-1α in human keratinocytes treated with the supernatant of SA WT, SA Δpsmα or SA Δpsmβ for 24 hours. All error bars are represented in the standard error of the mean (SEM) and one-way ANOVAs were used to determine the statistical significance indicated by: p <0.05 *, p <0.01 **, p <0.001 *** , p <0.0001 ****.
[0051] [0051] Figure 19A-H shows that PSMα and S. aureus proteases are responsible for damage to the barrier and induction of inflammation in murine skin. Strains of S. aureus (SA) (1e7 CFU) wild type (WT), with knockout of PSMα (Δpsmα) without proteases (Δproteases) were applied to the dorsal skin of male rodents for 72 hours (n = 6), and changes in (A, E) trypsin activity, (B, F) Klk6 mRNA expression, (C, G) Il6 and (D, H) IL17a / f normalized for the housekeeping Gapdh gene were measured. All error bars are represented in the standard error of the mean (SEM) and one-way ANOVAs were used to determine the statistical significance indicated by: p <0.05 *, p <0.01 **, p <0.001 *** , p <0.0001 ****.
[0052] [0052] Figure 20A-C shows that strains of CoNS do not affect the growth of SA. The coagulase-negative Staphylococci supernatant (CoNS) affects the growth of the reporter strain SA agr type I P3-YFP as assessed by OD600nm (n = 3-4), including (A) clinical isolates of CoNS, (B) S. epidermidis (S. epi) agr type I-III and (C) S. epidermidis (S. epi) type
[0053] [0053] Figure 21A-B shows that the supernatant of S. hominis C5 inhibits SA agr type I-III, but not of S. hominis C5 type IV, added to the reporter strains of agr agr types I-IV P3-YFP by 24 hours (n = 3). (A) Activity of the reporter strain of SA agr type I-IV and (B) measurement of growth by OD600 nm when cultivated in the presence of the supernatant of S. hominis C5. All error bars are represented in the standard error of the mean (SEM) and one-way ANOVAs were used to determine the statistical significance indicated by: p <0.05 *, p <0.01 **, p <0.001 *** , p <0.0001 ****.
[0054] [0054] Figure 22A-F shows that the S. hominis C5 supernatant inhibits SA-induced skin barrier damage. S. aureus (SA) (1e7 CFU) with or without S. hominis C5 <3kDa supernatant, 10 x concentrate, was applied to female dorsal murine skin for 48 hours (n = 3). (A-B) Representative images of the dorsum of rodents (dotted lines indicate the treatment area) and SA CFU / cm2 recovered from murine skin after treatment with SA. (C-F) SA-induced skin barrier damage markers, including Il6, transepidermal water loss (TEWL), trypsin activity and Klk6 mRNA expression compared to the Gapdh housekeeping gene. All error bars are represented in the standard error of the mean (SEM) and one-way ANOVAs were used to determine the statistical significance indicated by: p <0.05 *, p <0.01 **, p <0.001 *** , p <0.0001 ****. Detailed description of
[0055] [0055] In this specification and in the appended claims, the forms in the singular “one”, “one”, “o” and “a” include referents in the plural unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a plurality of such agents and a reference to “the microorganism” includes reference to one or more
[0056] [0056] In addition, the use of "or" means "and / or" unless otherwise stated. Likewise, "understand", "understand", "understanding", "include", "includes" and "including" are interchangeable and are not intended to be limiting.
[0057] [0057] It should also be understood that when descriptions of various modalities use the term "comprises", the experts in the subject would understand that, in some specific cases, a modality can be described alternatively with the wording "consists essentially of" or "consists of ”.
[0058] [0058] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning commonly understood by anyone in the subject to which this description belongs. Any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions.
[0059] [0059] Atopic dermatitis (AD) is among the most common immune disorders, and causes a substantial burden on the patient's quality of life and finances, in addition to imposing a serious risk of comorbidities. Defects in the skin 's barrier function are important features of. Eczematous skin lesions in AD patients increased levels of Th2 cytokines such as IL4 and IL13. Th2 cytokines promote decreased skin barrier function by inhibiting the expression of filaggrin. These cytokines also suppress the expression of human antimicrobial peptides such as catelicidin and b-defensin-2, a defect in AD that can lead to dysbiosis of the bacterial skin community and increase colonization by S. aureus. Therapy directed at the IL4 alpha receptor results in significant improvement of the disease. The strong association between
[0060] [0060] The cutaneous barrier of patients with AD can be compromised by increased proteolytic activity, as it was found that they exhibit increased expression of kallikreins (KLK). KLKs are a family of 15 serine proteases, among which, several are found predominantly in the upper layers of the granular and horny stratum of the epidermis. In Netherton's syndrome, the increased activity of serine proteases is due to the decrease in the activity of the Kazal-5 serine protease inhibitor. The greater enzymatic activity increases the desquamation, alters the processing of antimicrobial peptides and philagrin (FLG) and leads to the activation of receptor 2 mediated by proteases and inflammation. The increased activity of proteases can also play an important role in the communication of the microbiome with the skin's immune system and, recently, it has been shown to directly influence the production of epidermal cytokines and inflammation by enhancing the penetration of bacteria through the epidermis.
[0061] [0061] Dysbiosis of the cutaneous microbiome and colonization of the skin by Staphylococcus aureus are associated with exacerbations of atopic dermatitis (AD). The present description demonstrates that S. aureus has the ability to induce the expression of specific KLKs by keratinocytes and increase the overall proteolytic activity in the skin. This illustrates a system by which bacteria on the skin communicate with the host and suggests a previously unknown, but probably important mechanism for how colonization by S. aureus can increase the severity of the disease in patients with AD.
[0062] [0062] S. aureus can secrete multiple proteases in the skin that alter the integrity of the skin barrier. Serine protease V8 and toxins
[0063] [0063] Increased digestion of barrier proteins was observed after keratinocytes were activated by S. aureus. It is known that FLG results from the cleavage of Pro-FLG (400 kDa) larger in a monomeric form (37 kDa) that plays an important role in the formation of the physical barrier of the stratum corneum with keratin. It has been shown that the accelerated cleavage of Pro-FLG could be linked to increased skin desquamation (Hewett et al., 2005). Interestingly, increased cleavage of Pro-FLG was observed in human keratinocytes treated with S. aureus supernatant. Pro-FLG cleavage was partially blocked when KLK6 or KLK13 were silenced, indicating that S. aureus can decrease the integrity of the skin barrier in a KLK-dependent manner by cleaving Pro-FLG.
[0064] [0064] DSG-1 is an important adhesion protein of corneodesmosomes that, when cleaved, leads to increased desquamation. Complete DSG-1 (160 kDa) in keratinocytes is rapidly cleaved by KLK activity stimulated by S. aureus. It has been reported that KLK5, 6, 7 and 14 can cleave DSG-1, while KLK13 cannot. This showed that up-regulation of KLK6 and KLK14 can reinforce complete DSG-1 cleavage while providing evidence contrary to the notion that KLK13 is not involved in DSG-1 cleavage. Thus, S. aureus can cause KLKs to alter the cleavage of FLG, but also increase the cleavage of DSG-1 as another way to decrease the integrity of the skin barrier
[0065] [0065] The description demonstrates that soluble factor (s) produced by S. aureus has (m) a potent and previously unsuspected capacity to alter the activity of endogenous proteases produced by the keratinocyte. This occurred with a dilution of S. aureus products after which the activity of bacterial proteases was undetectable. Thus, S. aureus can promote the epidermis to increase the expression of endogenous proteolytic activity, thereby dramatically altering the balance of total proteolytic activity in the epidermis.
[0066] [0066] Strains other than S. aureus (Newman, USA300, 113 and SANGER252) and S. epidermidis (ATCC12228 and ATCC1457) had different effects on the activity of proteases in human keratinocytes. Strains of S. aureus including Newman and USA300 increased trypsin activity, while other strains of S. aureus and S. epidermidis increased elastase or MMP activity. Thus, bacteria could alter the activity of proteases in the epidermis depending on the species and strain of the bacteria. It is possible that other bacterial species and strains of S. aureus may also have a unique influence on the enzymatic balance of human skin. Interestingly, preliminary data revealed that purified toll-like receptor ligands do not induce trypsin activity or KLK expression
[0067] [0067] Proteolytic activity is highly positively regulated in many skin diseases, causing damage to the skin barrier. This is associated with worsening illness in almost all cases. The description demonstrates, in one respect, that commensal microbes and their bacterial products are useful in preventing increased protease activity in the skin. In particular, the description demonstrates that coagulase-negative Staphylococci can prevent Staphylococcus aureus-induced serine protease activity in the skin by inhibiting the Agr quorum sensing system (intra- and interspecies communication of microorganisms). Staphylococcus aureus, a pathogenic bacterial strain, can induce serine protease activity in the skin. Increased proteolytic activity breaks the skin barrier and worsens disease states, including Netherton's syndrome and atopic dermatitis. The description demonstrates that this increased serine protease activity can be prevented through the use of commensal, or good, bacteria from the skin and factors derived from them.
[0068] [0068] The description presents an unexpected keratinocyte response to S. aureus. Because of the increased cleavage of DSG-1 and FLG, S. aureus produces one or more factors that decrease the integrity of the skin barrier in a KLK-dependent manner.
[0069] [0069] The description demonstrates that S. aureus not only secretes proteases, but can also specifically activate keratinocytes to increase the expression of endogenous proteases. The description demonstrates that water-soluble alpha modulin (PSMα) is secreted by S. aureus and that it triggers the self-digestion of the epidermis. For example, three members of the KLK family appear to play a role in this increased enzyme activity.
[0070] [0070] The description also identifies commensal bacteria, genes and polypeptides that inhibit the quorum sensing system accessory regulatory gene (agr) of S. aureus and disable PSMα thereby inhibiting the activity of
[0071] [0071] The description demonstrates that coagulase-negative Staphylococci (CoNS) species that normally reside on the skin, such as S. epidermidis and S. hominis, protect against this biological activity of S. aureus by producing autoinductive peptides (AIP) that inhibit the quorum sensing system accessory regulatory gene (agr) from S. aureus and deactivate PSMα secretion.
[0072] [0072] Virtually all toxins from S. aureus are under the control of the accessory regulatory gene (agr) of virulence. The agr system triggers changes in gene expression, in particular cell density, through a process called quorum sensing. In addition to toxins, agr is known to positively regulate a wide variety of virulence determinants, such as exoenzymes (proteases, lipases, nucleases), and to negatively regulate the expression of surface-binding proteins. This adaptation is believed to control the production of certain virulence determinants of an infection when they are needed (eg, binding proteins at the beginning, when cell density is low and adhesion to host tissue is important, and degradation toxins and exoenzymes when infection is established and nutrients need to be obtained from host tissues.
[0073] [0073] Multiple clinical isolates from different CoNS species inhibited the activation of proteases and prevent epithelial damage both in vitro and in vivo without altering the abundance of S. aureus (eg, inhibited the biological activity of protease / agr activity, without changing the density of S. aureus). Furthermore, the description shows that patients with active AD have shown a decrease in the relative abundance of these beneficial microbes (eg, CoNS) compared to S. aureus, thus overcoming the inhibition of quorum sensing and enabling the rupture of the barrier by S. aureus. Taken together, the
[0074] [0074] The description also identified polynucleotide sequences, polypeptide sequences and fragments thereof that provide products that inhibit the activity of quorum sensing agr. These polynucleotides and polypeptides can be used to provide non-pathogenic or attenuated therapeutic and recombinant skin bacteria for use in topical formulations in the treatment of S. aureus infections and / or atopic dermatitis.
[0075] [0075] For example, the description provides self-inducing peptides (AIPs) that negatively regulate the activity of agr. Polynucleotides encoding AIPs are also provided.
[0076] [0076] The description provides a link between increased colonization of S. aureus and increased serine protease activity in the skin with AD and provides new targets and therapies, including, among others, fermentation extracts for positive regulation of protease activity (p. fermentation extracts of S. aureus) or fermentation extracts of commensal bacteria that negatively regulate the activity of proteases on the skin (eg, containing one or more AIPs from the description). In addition, the description provides (i) topical formulations comprising such purified AIP extracts or peptides, (ii) topical formulations comprising probiotic commensal bacteria (eg, non-pathogenic or attenuated bacteria that have been transformed with an AIP coding sequence , or preparations of commensal bacteria purified in a topical formulation). Additional therapeutic targets may be antibodies against KLKs and / or therapy with DSG-1 and / or FLG (eg, greater expression or delivery of these factors to individuals with AD).
[0077] [0077] In one embodiment, an AIP polypeptide of the description has the consensus sequence of X1X2X3X4CX5X6X7X8 (SEQ ID NO: 10), where X1 is
[0078] [0078] The description provides a compound of Formula I Formula I in which X1 is formed by 1-6 amino acids; X2 is an amino acid selected from valine (V), proline (P), methionine (M) and
[0079] [0079] In one embodiment, the description provides a compound of Formula IA: Formula IA in which X1 is formed by 1-6 amino acids; X2 is an amino acid selected from valine (V), proline (P), methionine (M) and threonine (T); where R1 is selected from the group consisting of
[0080] [0080] The description provides a purified polypeptide (e.g., an AIP peptide) comprising a sequence that is at least 98% identical to SEQ ID NO: 4 and that inhibits (i) protease production and / or activity of keratinocyte proteases, (ii) inhibits the production of IL-6 and / or keratinocyte activity, (iii) inhibits the production of soluble phenol alpha 3 modulin from Staphylococcus aureus (S. aureus) and / or (iv) the production and / or agr activity by S. aureus. In another embodiment, the description provides a compound of Formula IB: Formula IB
[0081] [0081] In yet an additional embodiment, the description provides a purified polypeptide comprising or consisting of SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17. In an additional embodiment, the polypeptide forms a structure Formula I, IA or IB.
[0082] [0082] In one embodiment, an AIP peptide of the description can comprise one or more D-amino acids.
[0083] [0083] The description provides a topical formulation comprising an AIP peptide with a consensus sequence of SEQ ID NO: 10 or a peptide of SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17 or a compound Formula I, IA or IB.
[0084] [0084] "Substantially identical" means that an amino acid sequence is largely, but not entirely, the same, but retains a functional activity of the sequence to which it is related. The percentage of identity that a polypeptide sequence or polynucleotide sequences share is based on the alignment of the sequence. It is common in the art to use multiple programs to perform alignment and determine identity. In general, two polypeptides or domains are "substantially identical" if their sequences are at least 85%, 90%, 95%, 98% or 99% identical, or if there are conservative variations in the sequence. A computer program, such as the BLAST program (Altschul et al., 1990) can be used to compare sequence identity.
[0085] [0085] The description also provides a polynucleotide (i.e., an "AIP polynucleotide") that encodes an AIP polypeptide of the description. For example, the description provides a polynucleotide that encodes SEQ ID NO: 2 or 4. In one embodiment, the polynucleotide hybridizes under stringent conditions to a polynucleotide that consists of SEQ ID NO: 3 and encodes a polypeptide of SEQ ID NO: 3 : 4. The “rigor” of hybridization reactions is easily determined by any technician in the subject and, in general, is a
[0086] [0086] An AIP polynucleotide can be cloned into several vectors for use in the description. For example, an AIP polynucleotide can be cloned into an expression vector or plasmid for use in transformation and / or expression in a recombinant host cell. Vectors for use in bacterial transformations are known. The four main types of vectors are plasmids, viral vectors, cosmids and artificial chromosomes. Common to all the vectors developed is an origin of replication, a multicloning site and a selectable marker. Any of these are suitable for use in the present. An AIP polynucleotide can be inserted into a clone, vector, shuttle (bifunctional vector), plasmid, BAC, or it can also be integrated into the bacterial genome. If a plasmid is used, the copy number of the plasmid can be between 5-500 copy numbers per cell. Exemplary plasmids and expression vectors include, among others: p252, p256, p353-2 (Leer et al. 1992), p8014-2, pA1, pACYC, pAJ01, pAl-derivative (Vujcic & Topisirovic 1993), pall, pAM -beta-1,2,3,5,8 (Simon and Chopin 1988), pAR1411, pBG10, pBK, pBM02, pBR322, pBR328, pBS-slpGFP, pC194 (McKenzie et al. 1986, 1987; Horinouchi & Weisblum 1982b) , PC194 / PUB110, pC30il, pC30il (Skaugen 1989), pCD034-1, pCD034-2, pCD256, pC12000, pC1305, pC1528, pCIS3, pCL2.1, pCT1138, pD125, pE194, pE194 / PLS1, pEGFPP, PEGFP , pF8801, pFG2, pFK series, pGK series, pGK12, pGK13, pIA, pIAV1,5,6,7,9, pIL.CatT, pIL252 / 3, pIL253, pIL7, pISA (down to E. coli), pJW563, pKRV3, pLAB1000 (Josson et al. 1990), pLB4 (Bates & Gilbert 1989, pLBS, pLE16, pLEB124, pLEB590, pLEB591, pLEB600, pLEB604, pLEP24Mcop, pLJ1 (Takiguchi et al. 1989), pLM, pLKS, p1, pLKS, pLK (Bates & Gilbert, 1989; Skaugen, 1989; Leer et al., 1992; Vujcic & Topisirovic, 1993; Eguchi et al., 2000; Kaneko et al., 2000; Danielsen, 2002; Dami ng et al., 2003; de las Rivas et al., 2004; van Kranenburg et al., 2005), pLP1 / 18/30, pLP18, pLP317, pLP317cop, pLP3537, pLP3537xyl, pLP402,
[0087] [0087] In one embodiment, the description provides a topical composition comprising an AIP polypeptide or peptide of the description. For example, in one embodiment, the topical composition comprises a purified polypeptide (e.g., an AIP peptide) comprising a consensus sequence of SEQ ID NO: 10, or a sequence that is at least 98% identical to any of SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17 and which inhibits (i) protease production and / or keratinocyte protease activity, (ii) inhibits IL-6 production and / or keratinocyte activity, (iii) inhibits the production of soluble phenol alpha 3 modulin from Staphylococcus aureus (S. aureus) and / or (iv) inhibits the production and / or agr activity by S. aureus. In another embodiment, the topical composition comprises a compound of Formula I, IA or IB (as defined above).
[0088] [0088] In another embodiment, the topical composition may comprise a non-pathogenic microorganism (including an attenuated microorganism that has been modified to reduce or eliminate pathogenic activity), in which the microorganism has been modified to express an AIP polypeptide. The microorganism can be modified to contain a vector and / or
[0089] [0089] In one embodiment, the compositions and methods of the present use non-pathogenic bacteria that have been modified to produce a compound of Formula I, IA and / or IB, by transforming the bacteria with an AIP polynucleotide of the description. In one embodiment, the bacteria in the population are non-pathogenic and non-invasive microorganisms and may, in certain embodiments, be a food-grade Gram-positive bacterial strain. In another embodiment, populations of transformed bacteria are prepared from bacteria that occur naturally in the skin's microbiome.
[0090] [0090] In certain embodiments, the bacteria, which form the population of bacteria in the composition and which are transformed to express a compound of Formula I, IA and / or IB, can be a collection of the same bacteria or a mixture of different bacteria in distinct phylogenetic levels. Bacteria that reside on the skin of healthy humans include species of bacteria that reside on the face of humans, such as Actinobacteria, including bacteria from the genus Corynebacterium and the genus Propionibacterium. In other embodiments, bacteria residing on the skin of healthy humans include species of bacteria that typically reside on skin other than the face, including, for example, bacteria of the genus Bacteroidetes and Proteobacteria. Other bacteria in the skin's microbiome include those listed below.
[0091] [0091] In one embodiment, the bacteria are of the genus Propionibacterium, including, but not limited to, Propionibacterium acidifaciens, Propionibacterium acidipropionici, Propionibacterium acidipropionici cepa 4900, Propionibacterium acnes, Propionibacterium australiense, Propionibacterium avidum, Propionibacterium cyclohexanicium. Freudenreichii, P. freudenreichii
[0092] [0092] In yet another embodiment, the description provides a probiotic composition for topical release, which comprises a commensal cutaneous bacterium CoNS of the description. In one embodiment, the CoNS bacterium comprises a bacterium that produces an AIP polypeptide and / or a compound of Formula I. In an additional embodiment, the topical composition contains
[0093] [0093] A commensal bacterium of the description can be isolated from human skin and identified using the methods described here. For example, the description provides a method for obtaining, identifying and cultivating a commensal bacterium described herein by rubbing the surface of human skin using, e.g. eg a foam-tipped swab. The swabs were placed in a tryptic soy broth. The broth is diluted on mannitol salt agar (MSA) plates, supplemented with 3% egg yolk. Colonies of pink color without halo, representing strains of coagulase-negative Staphylococci (CoNS), are collected and cultured in tryptic soy broth (TSB) before adding the sterile filtered supernatant, 25% by volume, to a reporter strain of S. aureus agr type I YFP grown in fresh TSB (for measurement of inhibition of agr activity of S. aureus after 24 hours of incubation). The agronomic activity of the reporter strain of S. aureus is measured with a fluorimeter. Strains with strong inhibition of agr activity of S. aureus are distinguished more by isolation and gDNA sequencing. gDNA is isolated using a number of commercially available kits (eg, DNeasy UltraClean Microbial Kit, Qiagen). The gDNA can be sequenced using several two-cycle sequencing platforms (eg, MiSeq; Illumin Inc., San Diego, CA), which can generate 2x 250-bp end reads. Adapters are removed using the Cutadapt program (see, p.
[0094] [0094] In this specification, the term "probiotic composition" or "topical probiotic composition" or "probiotic composition for the skin" includes a composition with probiotic bacteria commensal from the skin, a fermentation extract of probiotic bacteria commensal from the skin, a microorganism attenuated or modified that expresses an AIP polypeptide and an agent that (i) inhibits protease activity or (ii) promotes protease activity, and a pharmaceutical vehicle that maintains the viability of commensal bacteria in the skin.
[0095] [0095] In this specification, the term “topic” may include administration to the skin externally, as well as shallow injection (eg, intradermal and intralesional) such that a probiotic composition
[0096] [0096] In this specification, the term "fermentation extract" means a product of the fermentation of a probiotic bacterium commensal on the skin in a culture and under appropriate fermentation conditions. For example, the culture of S. aureus can produce PSMα3 which is useful for increasing the permeability of the skin barrier. An extract of S. aureus contains PSMα3 that can be applied to the skin to improve permeability, induce skin remodeling or promote permeability of the skin barrier to release drugs. Likewise, a fermentation extract from a CoNS bacterium that produces an AIP of the description can be grown, and the extract from such a culture can be used to inhibit pathologies associated with S. aureus (e.g., protease activity, dermatitis, etc. .).
[0097] [0097] In this specification, the term “commensal skin bacterium” includes a microorganism from the skin microbiome. The commensal probiotic bacterium of the skin can include a bacterial composition that promotes protease activity (a "commensal probiotic bacterium of the skin that promotes protease"). A protease commensal skin bacterium that promotes protease is typically a skin bacterium that produces soluble phenol alpha 3 modulin (PSMα3). A composition of probiotic commensal bacteria on the skin that promotes protease (or fermentation extract thereof) is useful, e.g. eg to promote skin reshaping, wound repair, aging, sun damage, pigmentation abnormalities and scarring. In one embodiment, a protease-promoting skin commensal probiotic bacterium comprises one or more bacteria with serine protease activity and / or which induce serine protease activity in the skin. For example, a protease promoting commensal bacterium from the protease-promoting skin may include a strain of S. aureus that produces soluble phenol alpha 3 modulin (PSMα3).
[0098] [0098] In another modality, the commensal probiotic bacterium of the skin
[0099] [0099] The term "put in contact" refers to exposing the skin to a topical probiotic composition such that the probiotic composition for the skin can modulate the activity of proteases (eg, serine protease activity) on the skin.
[00100] [00100] The terms "inhibit" or "effective inhibitory amount" refer to the amount of probiotic skin composition consisting of one or more probiotic microorganisms and / or fermented medium or extract and / or by-products of fermentation and / or synthetic molecules which is sufficient to cause, for example, the inhibition of protease activity (eg serine protease activity) on the skin or in a skin culture. The term "inhibit" also includes preventing or ameliorating a sign or symptoms of a disorder (eg, a rash, bruise or the like).
[00101] [00101] The term "therapeutically effective amount", in this specification, for the treatment of an individual affected by a disease or disorder, means a quantity of a probiotic skin composition or extract thereof sufficient to improve a sign or symptom of the disease or disorder. For example, a therapeutically effective amount can be measured as the amount sufficient to decrease an individual's dermatitis or rash symptom by measuring the severity of skin lesions frequency. Typically, the individual is treated with an amount to reduce a symptom of a disease or disorder by at least 50%, 90% or 100%. In general, the optimal dose will depend on the disorder and factors such as the individual's weight, the type of bacteria, the sex of the individual and the degree of symptoms. However, adequate doses can be easily determined by the person skilled in the art.
[00102] [00102] The term "purified" and "substantially purified" in this specification refers to cultures or co-cultures of microorganisms or biological agents (eg fermentation media and extracts, fractional fermentation media, by-products of fermentation, an AIP peptide, polypeptide, gene, polynucleotide, compound of Formula I etc.) that is substantially free of other cells or components found in the natural environment with which an agent produced in vivo would be naturally associated. In some embodiments, a co-culture probiotic may comprise a plurality of commensal bacteria on the skin.
[00103] [00103] The description provides whole cell preparations comprising a substantially homogeneous preparation of S. epidermidis, S. hominis and / or S. warneri. Such a preparation can be used in the preparation of compositions for the treatment of inflammation and microbial infections. Whole cell preparations can comprise S. epidermidis, S. hominis and / or S. warneri or can comprise a non-pathogenic vector (e.g., attenuated microbe) as described below. The description also provides fractions derived from whole cells comprising agents that reduce the activity of proteases in the skin that result from the activity of S. aureus.
[00104] [00104] The ability of a first bacterial composition to inhibit the protease activity of a second bacterial composition can be determined by measuring the protease activity of the second bacterial composition before and after placing the second composition in contact with the first composition. The contact of an organism with a topical probiotic composition of the description can occur in vitro, for example, by adding the topical probiotic composition to a bacterial culture to test the protease inhibitory activity of the bacterium. Alternatively, contact can occur in vivo, for example, by placing the topical probiotic composition in contact with an individual affected by a disease or skin disorder.
[00105] [00105] A preparation of probiotic bacterium commensal from the skin can be obtained in numerous ways. Any of a variety of methods known in the art can be used to administer topical probiotic compositions to an individual. For example, a skin probiotic composition or extract or synthetic preparation from the description can be formulated for topical administration (eg, in lotion, cream, spray, gel or ointment). Such topical formulations are useful in the treatment or inhibition of the presence or of microbial, fungal, viral infections or inflammation in the skin. Examples of formulations include topical lotions, creams, soaps, wipes and the like.
[00106] [00106] In yet another embodiment, a topical probiotic composition is provided that comprises a plurality of commensal probiotic bacteria of the skin. When used for the treatment of dermatitis or other skin diseases or disorders associated with increased protease activity (e.g., serine protease), the composition comprises one or more bacteria that inhibit protease activity in the skin. In such cases, the commensal probiotic bacterium of the skin is a Staphylococcus sp. coagulase-negative. In one embodiment, the commensal probiotic bacterium of the skin is selected from the group consisting of S. epidermidis strain, S. hominis strain, S. warneri strain and any combination thereof. When increased protease activity is desired (eg, for wound care, skin reshaping, etc.), the probiotic commensal bacterial composition contains bacteria with greater protease activity or which stimulate protease activity (eg. , serine protease activity). In this embodiment, an exemplary commensal bacterial composition will comprise an S. aureus bacterium or an attenuated S. aureus bacterium that produces PSMα3.
[00107] [00107] In another embodiment, the topical probiotic composition comprises an extract of the commensal probiotic bacterium of the skin that promotes
[00108] [00108] In yet another embodiment, a topical probiotic composition is provided which essentially consists of a fermentation extract of S. aureus alone or in combination with a S. aureus. According to an additional aspect, the topical probiotic composition above can be formulated into a lotion, shake-type lotion, cream, ointment, gel, foam, powder, solid, paste or tincture.
[00109] [00109] In another embodiment, the topical probiotic composition comprises a fermentation extract of commensal probiotic bacteria from the skin. In many ways, the bacteria from which the extract is produced comprise a coagulase-negative Staphylococcus species. In one embodiment, the Staphylococcus species is selected from the group consisting of S. epidermidis strain, S. hominis strain, S. warneri strain and any combination of them that produce an AIP that inhibits the quorum sensing agr system and / or the production of proteases in the skin or in the skin microbiome. In one embodiment, the AIP comprises a consensus sequence of SEQ ID NO: 10 or a sequence that is at least 98% identical to SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17 with modulation activity of the agr quorum and / or a compound of Formula I, IA or IB.
[00110] [00110] In yet another embodiment, a topical probiotic composition is provided which essentially consists of a fermentation extract of Staphylococcus sp. coagulase-negative or a fermentation extract of S. epidermidis alone or in combination with a Staphylococcus sp. coagulase-negative or a S. epidermidis. In another embodiment, the composition comprises one or more of the deposited strains of microorganisms described herein (e.g., S. epidermidis A11, S. hominis A9, S. hominis C5 and / or S. warneri G2).
[00111] [00111] According to an additional modality, the composition
[00112] [00112] In another embodiment, a fermentation extract is provided that can be obtained by fermenting a bacterium selected from the group consisting of S. epidermidis strain, S. hominis strain, S. warneri strain and any combination thereof under fermentation conditions. In many ways, such fermentation extracts can be used to inhibit serine protease activity in the skin. In another embodiment, the fermentation extract is obtained from any one or more of the deposited strains of microorganisms described here (eg, S. epidermidis A11, S. hominis A9, S. hominis C5 and / or S. warneri G2). According to an additional modality, the fermentation extract can be formulated into a lotion, shake-type lotion, cream, ointment, gel, foam, powder, solid, paste or tincture.
[00113] [00113] In another embodiment, a bandage or dressing is provided comprising the topical probiotic compositions described above, a fermentation extract of the commensal probiotic bacteria described above, a commensal probiotic bacterium of the skin described above and any combination thereof. In several respects, a bandage or dressing is provided whose main constituents include a matrix and a commensal probiotic bacterium of the skin that inhibits the protease activity in the skin. In several respects, a bandage or dressing is provided whose main constituents include a matrix and a fermentation extract of probiotic bacterium commensal from the skin that inhibits the activity of proteases in the skin.
[00114] [00114] In another embodiment, a bandage or dressing is provided which comprises the topical probiotic compositions described above, an extract of the commensal probiotic bacteria from the skin described above, a probiotic commensal skin bacteria described above and any combination thereof. In several respects, a bandage or dressing is provided whose main constituents include a matrix and a probiotic bacterium
[00115] [00115] The description also provides a method for treating a skin disease or disorder associated with protease (eg, serine protease activity). Examples of such a disease or disorder include Netherton's syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and itching. In one embodiment, the presence of the disease or disorder is first determined by measuring the protease activity of a sample (eg, skin or a culture of skin bacteria) from an individual suspected of having the disease or disorder. If the sample shows greater than normal protease activity (eg, serine protease activity), then the individual is treated with a protease inhibiting commensal bacterial preparation, placing the preparation in contact with the skin of the protease. individual. In another embodiment, a culture of the individual showing high protease activity and comprising bacteria is brought into contact with a preparation in vitro to determine the culture's susceptibility to the preparation and its effect on protease inhibition.
[00116] [00116] A preparation or extract of commensal bacteria that inhibits protease can be combined with one or more known serine protease inhibitors. There are a number of commercial and clinically relevant serine protease inhibitors that can be used in the methods and compositions of the description. For example, serine protease inhibitors such as those described in, for example, U.S. Patent No. 5,786,328, U.S. Patent No. 5,770,568 or U.S. Patent No. 5,464,820, the contents of which are incorporated by reference. Exemplary serine protease inhibiting agents include antibodies that bind to and inhibit a serine protease polypeptide or functional fragment of
[00117] [00117] A pharmaceutical composition comprising a probiotic skin composition described herein, comprising a commensal bacteria (e.g., S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5 and / or S. warneri G2), a developed form thereof (eg, attenuated or genetically modified) or an attenuated microorganism comprising a coding sequence for an AIP peptide, can be formulated in any dosage form that is suitable for topical administration of local effect or systemic, including emulsions, solutions, suspensions, creams, gels, hydrogels, foams, dusting powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, ointments, films, aerosols, irrigations, sprays, suppositories, bandages, skin adhesives . The topical formulation comprising a probiotic described herein can also comprise liposomes, micelles, microspheres, nanosystems and mixtures thereof.
[00118] [00118] In one embodiment, a bandage or dressing is provided comprising a probiotic skin composition described herein comprising a commensal bacterium (e.g., S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5 and / or S. warneri G2), a developed form thereof (e.g., attenuated or genetically modified) or an attenuated microorganism comprising a coding sequence for an AIP peptide described herein. In several respects, a bandage or dressing is provided whose main constituents include a matrix and a composition
[00119] [00119] A "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, antibacterial and antifungal agents (as needed as long as they are not harmful to the probiotic commensal bacteria), isotonic agents or absorption retardants and the like. The use of such means and agents for pharmaceutically active substances is well known in the art. Except to the extent incompatible with the pharmaceutical composition, the use of any conventional medium or agent in therapeutic compositions and treatment methods is contemplated. Supplementary active compounds can also be incorporated into the compositions.
[00120] [00120] Pharmaceutically acceptable vehicles and excipients suitable for use in the topical formulations disclosed herein include, but are not limited to, aqueous vehicles, water miscible vehicles, non-aqueous vehicles, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending agents and
[00121] [00121] A pharmaceutical composition comprising a probiotic can be formulated in the forms of ointments, creams, sprays and gels. Suitable vehicles for ointment include oil or hydrocarbon vehicles, including lard, benzene lard, olive oil, cottonseed oil and other oils, white petrolatum; emulsifiable or absorption vehicles, such as hydrophilic petrolatum, hydroxystearin sulfate, glycerol and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointment; water-soluble vehicles for ointment, including polyethylene glycols of variable molecular weight; emulsion vehicles, either water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions, including cetyl alcohol, glyceryl monostearate, lanolin and stearic acid (see, Remington: The Science and Practice of Pharmacy). These vehicles are emollients, but, in general, require the addition of antioxidants and preservatives.
[00122] [00122] A suitable base for creams can be oil in water or water in oil. The vehicles for creams can be washable with water, and contain an oil phase, an emulsifier and an aqueous phase. The oily phase is also called the "internal" phase, which is usually composed of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase normally, although not necessarily, exceeds the oil phase by volume and, in general, contains a humectant. The emulsifier in a cream formulation can be a nonionic, anionic, cationic or amphoteric surfactant.
[00123] [00123] Gels are semi-solid, suspension type systems. Single-phase gels contain material that is substantially and evenly distributed throughout the liquid vehicle. Suitable gelling agents include polymers of
[00124] [00124] In another embodiment, a pharmaceutical composition comprising a compound of Formula I and / or a commensal probiotic disclosed herein, a derivative or analog thereof, may be formulated either alone or in combination with one or more additional therapeutic agents, including, among others. others, chemotherapy, antibiotics (provided they do not destroy the benefits of probiotics), antifungal agents, antipruritic agents, analgesics, protease inhibitors and / or antiviral agents.
[00125] [00125] Topical administration in this specification includes (intra) dermal, conjunctival, intracorneal, intraocular, ophthalmic, auricular, transdermal, nasal, vaginal, ureteral, respiratory and rectal administration. Such topical formulations are useful in the treatment or inhibition of cancers of the eye, skin and mucous membranes (eg, mouth, vagina, rectum). Examples of formulations on the market include topical lotions, creams, soaps, wipes and the like.
[00126] [00126] Solutions or suspensions for use in a pressurized container, pump, spray, atomizer or nebulizer can be formulated to contain ethanol, aqueous ethanol or an alternative agent suitable for dispersing, solubilizing or prolonging the release of the active agent described here, a propellant as a solvent; and / or a surfactant, such as sorbitan trioleate, oleic acid or an oligolactic acid.
[00127] [00127] Materials useful for forming an erodible matrix include, among others, chitin, chitosan, dextran and pullulan; gum agar, gum arabic, caraia gum, locust bean gum, tragacanth gum, loadenins, ghatti gum, guar gum, xanthan and scleroglucan gum; starches, such as dextrin and maltodextrin; hydrophilic colloids, such as pectin; phosphatides, such as lecithin; alginates; propylene glycol alginate; gelatine; collagen; and cellulosics, such as ethyl cellulose (EC), methyl ethyl cellulose (MEC), carboxymethyl cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate (CA), cellulose propionate (CP), cellulose butyrate ( CB), cellulose butyrate acetate (CAB), CAP, CAT, hydroxypropylmethylcellulose (HPMC), HPMCP, HPMCAS, hydroxypropylmethylcellulose trimellitate (HPMCAT) and ethylhydroxyethylcellulose (EHEC); polyvinylpyrrolidone; polyvinyl alcohol; polyvinyl acetate; glycerol esters of fatty acids; polyacrylamide; polyacrylic acid; copolymers of ethacrylic acid or methacrylic acid (EUDRAGIT, Rohm America, Inc., Piscataway, N.J.); poly (2-hydroxyethylmethacrylate); polylactides; copolymers of L-glutamic acid and ethyl L-glutamate; degradable copolymers of lactic acid-glycolic acid; poly-D - (-) - 3-hydroxybutyric acid; and other acrylic acid derivatives, such as butyl methacrylate homopolymers and copolymers, methyl methacrylate, ethyl methacrylate, ethyl acrylate, (2-dimethylaminoethyl) methacrylate and (trimethylaminoethyl) methacrylate chloride.
[00128] [00128] In yet a further embodiment, a composition (e.g., a probiotic composition or a composition comprising a peptide or compound of Formula I) provided herein can be combined with one or more steroidal drugs known in the art, including, among others, aldosterone, beclomethasone, betamethasone, deoxychorticosterone acetate, fludrocortisone acetate, hydrocortisone (cortisol), prednisolone, prednisone, methylprenisolone, dexamethasone and triamcinolone.
[00129] [00129] In yet an additional embodiment, a composition (e.g., a probiotic composition or a composition comprising a Formula I peptide or compound) provided herein may be combined with one or more antifungal agents, including, among others, amorolfine, amphotericin B, anidulafungin, bifonazole, butenafine, butoconazole, caspofungin, cyclopyrox, clotrimazole, econazole, fenticonazole, philippine, fluconazole, isoconazole, itraconazole, ketoconazole, micafungin, oxamine, nazolone, miconazolone, niacin , sertaconazole, sulconazole, terbinafine, terconazole, tioconazole and voriconazole.
[00130] [00130] For use in the therapeutic applications described here, kits and manufactured articles are also described. Such kits may comprise a vehicle, package or container that is compartmentalized to accommodate one or more containers such as vials, tubes and the like, each of the container (s) comprising one of the separate elements to be used in a method described here. Suitable containers include, for example, vials, vials, syringes and test tubes. Containers can be formed from a variety of materials such as glass or plastic.
[00131] [00131] For example, the container (s) may comprise one or more compositions (e.g., a probiotic composition or a composition comprising a Formula I peptide or compound) provided herein, optionally in combination with another agent as disclosed above. Such kits optionally comprise a composition disclosed herein with a description of identification or label or instructions relating to its use in the methods described herein.
[00132] [00132] The following examples are provided to illustrate in more detail, but not to limit the invention. Examples Example 1
[00133] [00133] Culture of primary human keratinocytes. Neonatal NHEKs (ThermoFisher Scientific, Waltham, MA) were grown in EpiLife medium (ThermoFisher Scientific) supplemented with 1x EpiLife Defined Growth Supplement (ThermoFisher Scientific), CaCl2b60 µM and 1x antibiotic-antimycotic (PSA; 100 U / mL penicillin, 100 U streptomycin. / mL, amphotericin B 250 ng / mL; ThermoFisher Scientific) at 37ºC, 5% CO2. For the experiments, NHEKs were grown to 70% confluence, followed by differentiation in EpiLife medium with a high calcium content (CaCl22 mM) for 48 hours before treatment with sterile filtered bacterial supernatant. The use of these commercial cellular products derived from humans does not require free and informed consent. For treatments with the bacterial supernatant, differentiated NHEKs were treated with the sterile filtered bacterial supernatant with 5% in volume in relation to the EpiLife medium. For the experiments, only NHEKs between 3 and 5 passages were used.
[00134] [00134] Bacterial culture. All bacteria were grown in 3% tryptic soy broth (TSB; Sigma, St. Louis, MO) at 37ºC with agitation at 300 rpm. The S. aureus Newman strains, USA300, 113, SANGER252 and the strains of S. epidermidis ATCC12228 and ATCC1457 were cultured for 24 hours until the stationary phase, followed by centrifugation (4,000 rpm, room temperature [RT], 10 minutes) and sterile filtration (0.22 µm) of the supernatants before adding to the NHEKs. Briefly, the strain without proteases was grown for 24 hours in TSB 3% containing lincomycin 25 µg / mL and erythromycin 5 µg / mL followed by subculture in TBS 3% only for an additional 24 hours. For S. aureus colonization assay in live rodents, 2 x 106 colony forming units of the bacteria were applied to 8 mm TSB agar discs and allowed to dry for 30 minutes at RT before adding to the murine dorsal skin.
[00135] [00135] Murine model with bacteria discs. Mice
[00136] [00136] Zimography in situ. Murine skin sections (10 µm thick) were rinsed 1x with 1% Tween-20 in water for 5 minutes. The sections were treated with 2 µg / mL of BODIPY FL casein substrate for total protease activity (Thermo-Fisher Scientific) for 4 hours at 37ºC in a humidified chamber to measure total protease activity. The serine protease inhibitor, AEBSF (50 mM; Sigma), was applied to the cuts 30 minutes before the addition of the BODIPY FL casein as well. The slides were rinsed 1x in saline with phosphate buffer, followed by the application of the ProLong Gold Antifade mounting medium without DAPI (ThermoFisher Scientific) and a cover slide. The fluorescent signal was measured using an Olympus BX51 fluorescent microscope (Tokyo, Japan).
[00137] [00137] Protease activity assays. 50 ml of conditioned NHEK medium was added to 96-well black bottom plates (Corning, Corning, NY), followed by the addition of 150 ml of casein substrate BODIPY FL 5 µg / ml, 2 µg / ml of elastin (substrate of the type elastase; ThermoFisher Scientific) or gelatin 4 µg / mL (substrate for MMP; ThermoFisher Scientific) according to the manufacturer's instructions. In addition, the 200 µM Boc-Val-Pro-Arg-AMC peptide (substrate of the type
[00138] [00138] Quantitative PCR in real time. RNA was isolated from NHEKs using Purelink columns for RNA isolation (ThermoFisher Scientific) according to the manufacturer's instructions. The RNA was quantified with a Nanodrop spectrophotometer (ThermoFisher Scientific), and 500 ng of RNA was subjected to reverse transcription using the iScript kit for cDNA synthesis (Bio-Rad, Irvine, CA). Real-time quantitative PCR reactions were performed on a CFX96 real-time detection system (Bio-Rad) using specific primers for TaqMan genes and probes (ThermoFisher Scientific).
[00139] [00139] Immunoblotting. For cell lysis, 1x cold buffer from the radioimmunoprecipitation assay (RIPA) (Sigma) containing 1x protease inhibitor cocktail (Cell Signaling Technology, Danvers, MA) was applied to NHEKs followed by scraping. Cell lysates were incubated for 30 minutes on ice and centrifuged (13,000 r.p.m., 15 minutes, 4ºC) to remove cell debris. Samples were prepared by determining the protein concentration with assays of bicinconinic acid (BCA) (Pierce, Rockford, IL), followed by the addition of 40 mg of sample to 4x Laemmli sample buffer (Bio-Rad) containing b-mercaptoethanol 1 % and heating for 7 minutes at 95ºC. The samples were run on TGX Precast gels with 4-20% tris-glycine (Bio-Rad), transferred to membranes
[00140] [00140] KLK gene silencing. NHEKs were treated for 24 hours with selected silencer siRNA specific for KLK or a scrambled siRNA (-) control (ThermoFisher Scientific), 15 nM or 45 nM, using RNAiMAX (ThermoFisher Scientific) and OptiMEM medium (ThermoFisher Scientific). NHEKs were differentiated in medium with high calcium content (CaCl22 mM) for 48 hours, followed by a 24 hour treatment with sterile filtered supernatant from S. aureus (Newman), before the analysis of NHEK lysates and conditioned medium. .
[00141] [00141] Statistical analysis. For statistical analysis, one-way analysis of variance and two-way analysis of variance with P <0.05 were used, which is significant. The GraphPad prism version 6.0 program (GraphPad, La Jolla, CA) was used for statistical analysis of the results.
[00142] [00142] Staphylococci affect the proteolytic activity of human keratinocytes. To assess whether different strains of bacteria found on human skin are capable of inducing proteolytic activity of keratinocytes, primary cultures of human epidermal keratinocytes
[00143] [00143] S. aureus increases the activity of serine protease in the epidermis. Because of the large increase in trypsin activity induced by certain strains of S. aureus (Newman and USA300), and the potential role that this activity could play in diseases mediated by S. aureus, the experiments were centered on this organism to better understand how the bacteria induce protease activity in NHEKs. To evaluate the kinetics of the protease response to S. aureus, keratinocytes were treated for 0, 8, 24 and 48
[00144] [00144] To further validate the action of S. aureus on the protease activity in the epidermis, live S. aureus (USA300) was applied to the dorsal skin of mice. The skin at the application site was then submitted to biopsy and sectioned for analysis of total proteolytic activity by in situ zymography in the presence or absence of the serine protease inhibitor, 4-benzenesulfonyl fluoride (AEBSF). Total protease activity in the epidermis was increased qualitatively in the epidermis after treatment with S. aureus compared to skin treated with agar disks only, and the greater activity detected by the increase in fluorescence was largely eliminated by inhibition of serine activity. protease with AEBSF. Background autofluorescence in hair follicles was observed in all sections, including the control without substrate. These observations further demonstrate that the
[00145] [00145] S. aureus increases the expression of KLK in keratinocytes. KLKs are a family of serine proteases abundant in the epidermis with trypsin-like or chymotrypsin-like activity. To determine whether S. aureus could alter the expression of KLKs mRNA in keratinocytes, NHEKs were treated for 24 hours with the supernatant of S. aureus (Newman) and the expression of KLK1-15 was measured by quantitative PCR in real time. KLK5 showed the highest relative mRNA abundance, while KLK6, 13 and 14 consistently exhibited the highest number of increases after exposure to S. aureus (Figure 3a-e). All other KLKs analyzed showed subtle increases in mRNA expression after exposure to S. aureus except KLK1, which showed decreased expression. The mRNA for KLK2, 3 and 15 was not detected.
[00146] [00146] Both the cell lysates and the conditioned medium of NHEK were then analyzed for changes in KLK protein expression after treatment with S. aureus supernatant (Newman). Immunoblotting for KLK6 and 14 exhibited increased expression of these KLK proteins after treatment with S. aureus supernatant in the cell lysate and in the conditioned medium, while KLK13 was increased only in the conditioned medium. KLK5 did not change expression after treatment with S. aureus supernatant (Figure 3f).
[00147] [00147] KLK6, 13 and 14 contribute to increase serine protease activity in keratinocytes. Because KLK6, 13 and 14 revealed the greatest increase in expression in NHEKs after exposure to S. aureus, experiments were carried out to examine whether these KLKs were responsible for the observed increased serine protease activity. To selectively silence their expression, the small interference RNA (siRNA) was used. siRNA for KLK6 and KLK13 significantly decreased trypsin activity induced by S. aureus, while
[00148] [00148] S. aureus promotes the degradation of desmoglein-1 and FLG by inducing KLKs. Desmoglein-1 (DSG-1) and FLG are both important for regulating the integrity of the epidermal skin barrier. The immunoblotting technique showed that exposure of NHEKs to the supernatant of S. aureus (Newman) promoted the cleavage of the complete DSG-1 (160 kDa), and that the cleavage of DSG-1 was blocked by silencing by KLK6, 13 or 14 (Figure 5a). The cleavage of profilagrin (Pro-FLG) in NHEKs, mediated by S. aureus, indicated by the> 250 kDa band in the immunoblot, was also partially blocked by silencing by KLK6 and KLK13 siRNA (Figure 5b). The densitometry analysis further illustrates the ability with which the KLK6, 13 and 14 knockdown prevents the cleavage of DSG-1 or Pro-FLG (Figure 5c). Overall, all of these observations demonstrate that the ability of S. aureus to increase the proteolytic activity of keratinocytes by inducing KLK6, 13 and 14 can lead to the digestion of molecules essential for maintaining a normal epidermal barrier. Example 2
[00149] [00149] Bacterial preparations. All the bacteria used in this study are listed in Table A. All strains of Staphylococci (S. aureus, S. epidermidis, S. hominis, S. warneri, S. capitis and S. lugdunensis) were grown until the stationary phase in broth of 3% triptych soy (TSB) for 24 hours at 250 RPM in an incubator at 37ºC with 4 mL volumes or
[00150] [00150] Culture of normal human keratinocytes. Neonatal normal human epidermal keratinocytes (NHEKs; Thermo Fisher Scientific) were cultured in Epilife medium containing 60 μM CaCl2 (Thermo Fisher Scientific) supplemented with 1x Epilife Defined Growth Supplement (EDGS; Thermo Fisher Scientific) and 1x antibiotic-antimycotic (PSA; penicillin 100 U / mL, streptomycin 100 U / mL, amphotericin B 250 ng / mL; Thermo Fisher Scientific) at 37ºC, CO2 5%. NHEKs were only used for experiments between 3-5 passages. For the experiments, NHEKS were grown to 70% confluence, followed by differentiation in EpiLife medium with a high calcium content (2 mM CaCl2) for 48 hours to simulate the upper layers of the epidermis. For bacterial supernatant treatments, differentiated NHEKs were treated with sterile filtered bacterial supernatant with 5% by volume in relation to Epilife medium for 24 hours. Likewise, for treatments with synthetic PSM, 5-50 μg-mL of peptide were added to the NHEKs for 24 hours in DMSO.
[00151] [00151] Model of epicutaneous infection by S. aureus in mouse. C57BL / 6 male or female mice of comparable sex and age (Jackson) at 8 weeks of age were used for all experiments (n = 3-6), as specified in the figure captions. All animal experiments have been approved by the Institutional Animal Care and Use Committee. The mice hair was removed by scraping and applying Nair
[00152] [00152] Preparation of synthetic soluble phenol modulin. All synthetic soluble phenol (PSM) modulins were produced by LifeTein (Hillsborough, NJ). The peptides were produced with 95% purity and N-terminal (f) formylation. The PSM sequences were as follows: PSMα1: f-MGIIAGIIKVIKSLIEQFTGK (SEQ ID NO: 5), PSMα2: f-MGIIAGIIKFIKGLIEKFTGK (SEQ ID NO: 6), PSMα3: f-MEFVAKLFKFFKDLLGKFLGNN: SEQ: ID -MAIVGTIIKIIKAIIDIFAK (SEQ ID NO: 8), PSMβ2: f-MTGLAEAIANTVQAAQQHDSVKLGTSIVDIVANG VGLLGKLFGF (SEQ ID NO: 9).
[00153] [00153] The peptides were resuspended in DMSO and concentrated by Speedvac in stocks of 500 mg powder, stored at -80 C before reconstitution in DMSO for the experiments.
[00154] [00154] Isolation of RNA and quantitative PCR in real time. All RNA was isolated using the Purelink RNA isolation kit according to the manufacturer's instructions (Thermo Fisher Scientific). For NHEKs, 350 μL of RNA lysis buffer (with 1% β-mercaptoethanol) was added directly to the cells. For the mouse tissue, the skin with a total thickness of 0.5 cm2 was broken and homogenized by the technique
[00155] [00155] Generation of competent RP62A cells and transformation. Electrocompetent RP62A cells were prepared. Briefly, an overnight culture of S. epidermidis RP62A was diluted to OD600nm of 0.5 in preheated Brain Heart Infusion (BHI) broth, incubated for an additional 30 minutes at 37ºC with shaking, transferred to centrifuge tubes and then , cooled on ice for 10 minutes. The cells were harvested by centrifugation (10 minutes, 4000 RPM, 4ºC), washed in series with 1 volume, 1/10 of the volume and then 1/25 of the volume of cold water submitted to the autoclave, followed by further sedimentation (pellet). at 4 C after each wash. After the final wash, the cells were resuspended in 1/200 of the volume of cold sterile 10% glycerol and divided into tubes with aliquots of 50
[00156] [00156] Allelic repositioning of the AIP of S. epidermidis RP62A. The allelic repositioning plasmid pMAD (50) was used to selectively generate an in frame deletion (in reading phase) of the AIP coding sequence of agrD in S. epidermidis RP62A. Briefly, fragments downstream and upstream with approximately 1000 bp of the RIP62A AIP sequence were amplified by PCR and joined by gene splicing with overlapping extension or "SOEing". The stitched fragments and the pMAD vector were digested with BamHI and SalI, linked to each other by T4 DNA ligase (New England Biolabs) and subsequently used to chemically transform the S. epidermidis strain of clonal complex 10 with artificial E modification coli per plasmid, DC10B-CC10. The transformants were plated in LB with Amp 100 μg / mL and Cm 30μg / mL at 37ºC. The correct transformants were validated by restriction digestion and sequencing. The verified construction was noted as pMAD :: ΔAIP. Electrocompetent RP62A was then transformed with ~ 5μg of pMAD :: ΔAIP derived from DC10B-CC10 and then plated on BHI agar with Cm 10 μg / mL and 50 μL of 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside (X-Gal), 40 mg / mL, at 30ºC. A single blue colony was
[00157] [00157] RNA sequencing. The RNA was sent to the Genomic Centered Laboratory at the University of California, San Diego (UCSD) for library preparation and sequencing. The TruSeq mRNA Library Prep kit (Illumina) was used to prepare the library, followed by high throughput sequencing on a HiSeq 2500 sequencer (Illumina). The data were analyzed with the Partek Flow and Partek Genomics Suite software and the gene ontology analysis was performed using the PANTHER classification system (http [: //] pantherdb.org).
[00158] [00158] Histology. Full-thickness murine skin (0.5 cm2) was collected, fixed in paraformaldehyde (4%) and washed in PBS before overnight incubations with 30% and 10% sucrose before freezing the tissue in OCT mounting medium with ice dry. Cuts obtained with Cryostat (10 mm) were mounted on Superfrost Plus glass slides
[00159] [00159] Determination of the level of cytokines. The conditioned medium of NHEKs (25 μL) was used to quantify the protein concentration of several cytokines. Milliplex test kits with magnetic beads (Millipore) for 3 human cytokines (IL-6, IL-8, TNFα) were used according to the manufacturer's instructions on a Magpix 200 system (Luminex). Human IL-1α and IL-36α were quantified by ELISA (R&D Systems).
[00160] [00160] Quantification of bacteria CFU. The colony-forming units (CFU) of S. aureus were quantified by plating serial dilutions (10 μL) from 10-1 to 10-5 on Baird-parker (BD) agar plates containing 3% egg yolk emulsion with tellurite for 24 hours in an incubator at 37ºC followed by CFU counting. The CFU value for all strains of Staphylococci was also approximated using a spectrophotometer and also measuring OD600 nm of cells diluted 1:20 in PBS.
[00161] [00161] Transsepidermal water loss measurements. To determine damage to the epidermal skin barrier, transepidermal water loss (TEWL) from murine skin treated for 48-72 hours with S. aureus was measured using a TEWAMETER TM300 (C & K).
[00162] [00162] Analysis of trypsin activity. NHEK conditioned medium was added, in 50 μL, to 96-well black bottom plates (Corning) followed by the addition of 150 μL of the Boc-Val-Pro-Arg-AMC peptide (trypsin-like substrate; BACHEM) to a final concentration of 200 μM in 1x digestion buffer (10 mM Tris-HCl, pH 7.8) and incubated at 37ºC for 24 hours. The relative fluorescent intensity (ex: 354 nm, in: 435
[00163] [00163] Agr activity of S. aureus. The reporter strains S. aureus USA300 LAC agr type I P3-YFP (AH1677) or S. aureus USA300 LAC agr type I pAmi P3-Lux (AH2759) were used to detect the activity of S. aureus agr. For in vitro experiments, 1e6 CFU of S. aureus USA300 LAC agr type I P3-YFP was added to 300μL of TSB 3% along with 100 μL of sterile filtered supernatant from the dinner (25% by volume) and stirred (250 RPM) during 24 hours at 37ºC. The bacteria was then diluted 1:20 in PBS (200 μL at the end) and YFP (ex: 495 nm, at: 530 nm) was detected using the fluorimeter as above, and the bacterial density was determined by reading OD600nm on a spectrophotometer . For murine experiments, the activity of S. aureus USA300 LAC agr type I pAmi P3-Lux was determined using an IVIS machine and evaluating the luminescent intensity, after 2 minutes of exposure, by measuring the photons emitted using the LiveImaging software (PerkinElmer) .
[00164] [00164] Genome sequencing and assembly. Genomic DNA
[00165] [00165] Data from the microbiome and comparative analysis of the genomes. Metagenomic data publicly available by the shotgun method for skin with atopic dermatitis were analyzed. The relative abundance of S. aureus and S. epidermidis strains was obtained from published supplementary material ([www.] Sciencetranslationalmedicine.org/cgi/content/full/9/397/eaal4651/DC 1). The agrD characterization analysis was restricted to eight patients (AD01, AD02, AD03, AD04, AD05, AD08, AD09, and AD11) with information on 7
[00166] [00166] Quantification and statistical analysis. The Mann-Whitney non-parametric test was used to analyze the statistical significance of metagenomic data from patients with AD. ANOVA one way or ANOVA two way was used for statistical analysis, as indicated in the descriptions of several figures. All statistical analysis was performed with GraphPad Prism Version 6.0 (GraphPad, La Jolla, CA). All data are presented as mean ± standard error of the mean (SEM) and a P ≤ 0.05 value considered significant.
[00167] [00167] PSMα and proteases produced by S. aureus induce damage to the epidermal barrier. A primary function of human skin is to establish a physical barrier against the external environment. Specific toxins produced by S. aureus, such as soluble phenol modulins (PSM), are capable of promoting epithelial inflammation and, as proposed, are fundamental to boost the disease in AD (19-22). Therefore, to understand how S. aureus on the skin surface could influence inflammatory activity in the epidermal barrier, normal human epidermal keratinocytes (NHEK) were assessed for their ability to express proteolytic activity when exposed to a strain of S. aureus USA300 LAC containing a targeted deletion either in the PSMα or PSMb operon. PSMα production was necessary for the induction of trypsin-like serine protease activity and increased levels of kallikrein 6 (KLK6) mRNA (Figure 14A-B). The PSMα and PSMβ operons in S. aureus contain distinct peptides including PSMα1- 4 and PSMβ1-2. Thus, synthetic PSMα1-4 and PSMβ2 peptides were tested in NHEKs and it was found that (Figure 14C) all
[00168] [00168] To validate the role of the PSMα operon in the epidermal barrier in vivo, mice were colonized for 72 hours on the skin surface with equivalent numbers of wild type S. aureus USA300 LAC or the PSMα mutant strain. Wild type S. aureus induced erythema, desquamation and epidermal thickening while no changes were observed in bacterial abundance in the absence of PSMα (Figure 14F). Despite the increased epidermal thickness, an increase in transepidermal water loss (TEWL), a well-established method for assessing damage to the skin barrier, was observed after exposure to wild-type S. aureus, but not when PSMα was absent (Figure 14G ). However, disruption of the skin barrier of a totally differentiated epidermis in vivo was also dependent on the expression of S. aureus proteases. With the use of a mutant strain of S. aureus USA300 LAC devoid of 10 major secreted proteases, including aureolysin, V8, staphopain A / B and SplA-F, the visible evidence of injury and increased TEWL has been diminished in a way that is deficient in proteases of S. aureus despite the fully intact expression of PSMα (Figure 14F, H). Coincidentally with the macroscopic and histological changes observed as associated with the expression of PSMα or bacterial proteases, an increase in the activity of
[00169] [00169] Self-inducing peptide from S. epidermidis inhibits agr activity of S. aureus. Interestingly, both the PSMα peptides and secreted proteases from S. aureus are under the regulation of the quorum sensing agr system. In addition, it was found that clinical isolates of S. aureus have four distinct types of agr, with type I being the most prominent in individuals with AD. Although the colonization of the skin by S. aureus increases in AD, other bacterial species, such as coagulase-negative Staphylococci (CoNS) strains, including the commensal organism abundant in the human skin S. epidermidis, are also present, making it essential the way these bacteria communicate. Laboratory isolates of S. epidermidis agr type I have been shown to produce a self-inducing peptide (AIP) that inhibits agr type I-III systems of S. aureus, but not type IV through a cross-communication mechanism (crosstalk), while little knows of other types II and III of S. epidermidis on the influence they have on the activity of agr of S. aureus. Conditioned culture supernatants of S. epidermidis strains with agr types I, II or III were added to a reporter strain of S. aureus USA300 LAC agr type I to explore whether the agr activity of S. epidermidis could influence the agr system of S aureus. This experiment confirmed that S. epidermidis agr I was the only potent inhibitor
[00170] [00170] Deficiency in the relative abundance of S. epidermidis agr type I on the skin with AD. Having established the potential of a S. epidermidis laboratory strain to influence the effects of S. aureus on the function of human keratinocytes, experiments were carried out to determine the abundance of these bacteria in a clinical setting. Metagenomic data available on the skin microbiome of 8 individuals with AD with different severity (based on the objective SCORAD), collected from 7 body sites, were analyzed for the relative abundance of S. epidermidis based on the type of agr. Sequence alignments identified S. epidermidis genomes, based on types IIII of agr, in patients with AD and determined that the most frequent type of S. epidermidis agr on the skin with AD is that of type I agr (Figure 15E). The comparison of S. epidermidis agr I with S. aureus revealed that S. epidermidis agr type I becomes relatively less abundant in individuals with AD with greater disease severity (Figure 15F-G). These observations confirmed the presence of
[00171] [00171] Several species and strains of Staphylococci inhibit the agr activity of S. aureus. In order to further establish the physiological significance of quorum sensing interactions between S. aureus and other members of the skin microbiome, different clinical isolates of CoNS that cause AD were tested for the ability of their culture supernatants to inhibit S. quorum sensing activity. aureus USA300 LAC agr type I. Several species, including S. epidermidis, S. hominis, S warneri and S capitis, showed potent inhibitory activity against S. aureus agr activity (Figure 16A). As with the laboratory isolates of S. epidermidis, the strains of CoNS inhibited the agr activity of S. aureus without inhibiting the growth rate (Figure 316S). In addition, an analysis of the genomic sequence of the agrD coding region for AIP of the strain S. hominis C5 revealed a new AIP sequence in the AIP coding region similar to the presence of the coding region of S. epidermidis agr type I and with an octamer predicted for AIP sequence of S. hominis C5 (Figure 16B; SEQ ID NO: 4). Biochemical techniques of the active S. hominis C5 supernatant revealed that inhibition of agr activity of S. aureus was dependent on a factor with <3kDa (small size), sensitive to pH 11 (thiolactone ring) that could be precipitated with sulfate 80% ammonium (peptide) (Figure 16C).
[00172] [00172] Next, S. aureus was cultured in the presence of the sterile filtered supernatant of S. hominis C5 and the subsequent culture supernatant was applied to NHEKs as in Figure 14. Similarly to S. epidermidis agr type I, S. hominis C5 inhibited trypsin activity, KLK6 transcript production and IL-6 protein expression, induced by S. aureus, in NHEKs (Figure 16D-F). In addition, S. hominis C5 was able to inhibit multiple S. aureus agr systems in addition to the more
[00173] [00173] Clinical CoNS isolate inhibits the agr activity of S. aureus and its ability to promote AD. To establish the physiological relevance of quorum sensing interactions between CoNS and S. aureus in vivo, the agr activity of S. aureus was evaluated by IVIS using a strain of S. aureus USA300 LAC agr type I with P3-Lux promoter (luminescence ). S. aureus on the dorsal skin showed abundant agr activity, but when in the presence of live S. hominis C5, agr activity of S. aureus was inhibited (Figure 17A-B). In addition, S. hominis C5 also protected against erythema and desquamation induced by S. aureus (Figure 17C) without altering the abundance of S. aureus (Figure 17D). This phenotype was associated with improved evidence for inflammation, barrier disruption and protease activity in the skin and Klk6 expression (Figure 17E-H). In addition, when S. aureus was applied to murine dorsal skin in the presence of a S. hominis C5 supernatant concentrated to <3kDa, similar reductions in barrier damage and inflammation were observed without changes to the abundance of S. aureus. (Figure 22). These data show that the microbial community of CoNS in the skin probably contains new AIPs that promote epithelial barrier homeostasis by interspecies quorum sensing activity.
[00174] [00174] Several description modalities have been described. However, it will be understood that several modifications can be made without deviating from the spirit and scope of the description. Accordingly, other modalities fall within the scope of the following claims.

权利要求:
Claims (37)
[1]
1. Purified polypeptide, characterized by the fact that it comprises a sequence that is at least 98% identical to SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17 and that inhibits (i) protease production and / or keratinocyte activity, (ii) inhibits the production of IL-6 and / or keratinocyte activity, (iii) inhibits the production of soluble phenol alpha 3 modulin from Staphylococcus aureus (S. aureus) and / or ( iv) inhibits the production of agr and / or activity by S. aureus.
[2]
2. Purified polypeptide according to claim 1, characterized in that the polypeptide is at least 98% identical to SEQ ID NO: 2.
[3]
Purified polypeptide according to claim 1, characterized in that the polypeptide comprises SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17.
[4]
Purified polypeptide according to claim 1, characterized in that the polypeptide consists of SEQ ID NO: 4, 11, 12, 13, 14, 15, 16 or 17.
[5]
Purified polypeptide according to any one of claims 1 to 4, characterized in that the polypeptide comprises one or more D-amino acids.
[6]
Purified polypeptide according to any one of claims 1 to 4, characterized in that the polypeptide comprises a compound of Formula I, IA or IB.
[7]
7. Topical formulation, characterized in that it comprises a polypeptide as defined in any one of claims 1 to 4.
[8]
8. Isolated polynucleotide, characterized by the fact that it encodes the polypeptide as defined in any one of claims 1 to 4.
[9]
An isolated polynucleotide according to claim 8, characterized in that the polynucleotide comprises a sequence that hybridizes under stringent conditions to a polynucleotide that consists of SEQ ID NO: 1 and encodes a polypeptide that comprises SEQ ID NO: 4.
[10]
An isolated polynucleotide according to claim 8, characterized in that the polynucleotide comprises SEQ ID NO: 1 or 3.
[11]
11. Vector, characterized by the fact that it comprises the polynucleotide as defined in claim 8.
[12]
12. Vector, characterized by the fact that it comprises the polynucleotide as defined in claim 9 or 10.
[13]
13. Recombinant microorganism, characterized by the fact that it comprises the polynucleotide as defined in claim 8.
[14]
14. Recombinant microorganism, characterized by the fact that it comprises the polynucleotide as defined in claim 9 or 10.
[15]
15. Recombinant microorganism according to claim 13, characterized by the fact that the microorganism is an attenuated microorganism.
[16]
The recombinant microorganism according to claim 13, wherein the microorganism is a commensal microorganism.
[17]
17. Topical probiotic composition, characterized by the fact that it comprises a recombinant microorganism as defined in claim 15 or 16.
[18]
18. Topical probiotic composition, characterized by the fact that it consists of a microorganism that expresses a polypeptide as defined in claim 1.
[19]
19. Topical probiotic composition according to claim 18, characterized by the fact that the microorganism is S. hominis, S. epidermidis, S. warneri or any combination thereof.
[20]
20. Topical probiotic composition according to claim 19, characterized by the fact that the microorganism is S. hominis C5, S. hominis A9, S. epidermidis A11 and / or S. warneri G2.
[21]
21. Topical probiotic composition according to claim 18, characterized by the fact that the composition comprises a microorganism selected from the group of microorganisms with ATCC Number _______ (strain name S. epidermidis A11 81618, deposited on August 28, 2018 ), ATCC Number _______ (designation of the S. hominis strain C5 81618, filed on August 28, 2018), ATCC Number _______ (designation of the S. hominis strain A9 81618, filed on August 28, 2018), ATCC Number _______ ( strain name S. warneri G2 81618, deposited on August 28, 2018) and any combination of the previous strains.
[22]
22. Use of an effective amount of Staphylococcus sp. negative coagulase (CoNS), or an effective amount of a CoNS fermentation extract sufficient to inhibit protease activity in the skin characterized by the fact that it is to prepare a drug for the treatment of a dermatological disorder, in which CoNS produces a polypeptide comprising a sequence that is at least 98% identical to SEQ ID NO: 4 and that inhibits protease production.
[23]
23. Use according to claim 22, characterized by the fact that dermatological disorders are selected from the group consisting of Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, inflammation epidermal, dermal inflammation and itching.
[24]
24. Use according to claim 22, characterized by the fact that the administration is by topical application.
[25]
25. Use according to claim 22, characterized by the fact that CoNS is selected from the group consisting of
Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus devriesei, Staphylococcus homries, Staphylococcus hominisocylococcus hominis, Staphylococcus hominis, Staphylococcus hominis.
[26]
26. Use according to claim 22, characterized in that the CoNS fermentation extract comprises a polypeptide sequence of SEQ ID NO: 4 and / or a compound of Formula I.
[27]
27. Use according to claim 22, characterized by the fact that CoNS is selected from the group consisting of S. epidermidis A11, S. hominis C4, S. hominis C5, S. hominis A9, S. warneri G2 and any combination thereof.
[28]
28. Use of a composition of commensal bacteria from the skin and / or a coagulase negative Staphylococci fermentation extract, characterized by the fact that it is to prepare a medicine to treat a disease or skin disorder, in which: the bacterial composition skin meal or fermentation extract comprises a polypeptide that is at least 98% identical to SEQ ID NO: 4 and / or comprises a compound of Formula I; the composition is formulated in a cream, ointment or pharmaceutical composition that maintains the ability of the commensal bacteria in the skin to grow and replicate; and [/ or] the use comprises: measuring the protease activity of an individual's skin culture or individual's skin culture; and compare protease activity with that of a normal control.
[29]
29. Use according to claim 28, characterized by the fact that the coagulase negative Staphylococci is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus, Staphylococcus, pasteur devriesei, Staphylococcus Hominis, Staphylococcus jettensis, Staphylococcus petrasii and Staphylococcus lugdunensis.
[30]
30. Use of a purified polypeptide as defined in claim 1 or a probiotic composition comprising a bacterium that produces a polypeptide that is at least 95% identical to SEQ ID NO: 4 that inhibits kallikrein expression, characterized by the fact that it is for preparing a medicine to treat a skin disease or disorder.
[31]
31. Use of a composition that inhibits the expression of phenol-soluble modulin, wherein the composition comprises a purified polypeptide as defined in claim 1 or a compound of Formula I, characterized in that it is to prepare a medicament for treating a skin disease or disorder.
[32]
32. Use according to claim 30 or 31, characterized by the fact that the administration is topical.
[33]
33. Use according to claim 30 or 31, characterized in that the composition is a fermentation extract of a coagulase negative Staphylococcus.
[34]
34. Topical probiotic composition, characterized by the fact that it comprises a plurality of commensal probiotic bacteria from the skin selected from the group consisting of S. epidermidis A11, S. hominis C4, S. hominis C5, S. hominis A9, S. warneri G2 and any combination thereof.
[35]
35. Topical probiotic composition according to claim 34, characterized in that it is formulated as a lotion, shaking lotion, cream, ointment, gel, ointment, powder, solid, paste or tincture.
[36]
36. Medicinal composition, characterized by the fact that it comprises a drug and a fermentation extract of S. aureus or biotic of S. aureus comprising a soluble phenol alpha 3 modulin.
[37]
37. Method for delivering drugs through the skin, characterized by the fact that it comprises contacting the skin with a composition as defined in claim 36.
Figure 1B Figure 1A
Petition 870200045837, of 4/13/2020, p. 93/117 Trypsin activity Elastase activity (ΔOD435 nm h-1) MMP activity (ΔOD 515 nm h-1) (ΔOD 515 nm h-1)
Control vehicle Control vehicle Control vehicle 1/23
Figure 1C
Figure 2B Figure 1D
Petition 870200045837, of 4/13/2020, p. 94/117 Trypsin activity (ΔOD 435 nm h-1)
Protease activity Total protease activity (ΔOD 530 nm h-1) Total (ΔOD 530 nm h-1) 2/23
Figure 2A
Time (h)

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法律状态:
2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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