Topical Formulation In Form Of A Patch, A Bandage Or A Plaster Comprising Probiotic Bacteria, And Use Thereof In A Method For Treating Or Preventing Skin Disorders

Abstract

A topical pharmaceutical or cosmetic formulation is disclosed, comprising two polymer membranes enclosing probiotic bacteria having antimicrobial activities against skin pathogens sandwiched therebetween, one of the membranes being semipermeable and having a pore size which does not allow penetration of the bacteria, and medical and cosmetic uses thereof. Preferably the probiotic bacteria are of the order Lactobacillales, most preferably of the strain L. plantarum 8P-A3 (DSM 32835).

Claims

1-13. (canceled)

14: A topical pharmaceutical or cosmetic formulation comprising two polymer membranes enclosing probiotic bacteria having antimicrobial activities against skin pathogens sandwiched therebetween, wherein a first of the two membranes is semipermeable and has a pore size that prevents penetration of the bacteria.

15: The formulation of claim 14, wherein a second of the two membranes is water-impermeable.

16: The formulation of claim 14, wherein the pore size of the first membrane is at least 0.1 μm.

17: The formulation of claim 14, wherein the semipermeable membrane comprises at least one selected from polycarbonate, polyethersulfone, polysulfone cellulose, cellulosic esters, silicones, polyamides, polyamide imide, polyamide urea, polyacryl nitrile, polyethylene, polypropylene, polytetrafluorethylene, polyvinylidene fluoride, polyvinyl chloride, polypiperazine amide, and combinations thereof.

18: The formulation of claim 14, further comprising an insert provided with the probiotic bacteria between the two membranes, the formulation containing from about 10.sup.8 to 10.sup.10 colony forming unit probiotic bacteria per cm.sup.2 of the insert surface.

19: The formulation of claim 18, wherein the insert comprises one selected from nonwovens comprising viscose or polypropylene, polyesters, polyamides, mixtures thereof, and polyurethane foams.

20: The formulation of claim 18, wherein the insert has a water-absorption ranging from 500 g/m.sup.2 to 2200 g/m.sup.2.

21: The formulation of claim 14, wherein the formulation is in the form of a patch or plaster.

22: The formulation of claim 14 wherein the probiotic bacteria are of the order Lactobacillales.

23: The formulation of claim 14, wherein the probiotic bacteria are selected from: Lactobacillus species, Streptococcus salivarius K12, Streptococcus salivarius M18 and Bifidobacterium species.

24: The formulation of claim 14, wherein the probiotic bacteria comprise two or more probiotics.

25: The formulation of claim 14 comprising at least one of: a biologically pure culture of Lactobacillus plantarum strain 8P-A3 deposited at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, under accession number DSM 32835; and a Lactobacillus strain having at least 97% sequence similarity with the sequence shown in SEQ ID NO: 1 in its 16S rRNA gene.

26: The formulation of claim 14, further comprising protectants to preserve long term viability of the enclosed probiotic bacteria.

27: A method of treating a skin disorder, the method comprising: applying the formulation of claim 14, wherein the skin disorder is a skin or wound infection.

28: The method of claim 27, wherein the skin disorder is selected from acne, superinfected acne lesions, infected burn wound, venous leg ulcer, and infected neurodermitis lesions.

29: The method of claim 27, wherein the skin disorder is caused at least in part by one selected from Cutibacterium (Propionibacterium) species, Staphylococcus species, Streptococcus species, Enterobacteriaceae species, and Pseudomonas species.

30: A method of treating infectious skin disorder, the method comprising: topically applying the formulation of claim 25 including the Lactobacillus plantarum strain 8P-A3 or a Lactobacillus strain having at least 97% sequence similarity with the sequence shown in SEQ ID NO: 1 in its 16S rRNA gene.

31: The method of claim 30, wherein the infectious skin disorder is selected from acne vulgaris, infected lesions of venous leg ulcers, infected burn wounds, and infected skin lesions of neurodermatitis patients.

32: The method of claim 30, wherein the infectious skin disorder is caused at least in part by one selected from: Cutibacterium (Propionibacterium) species, Staphylococcus species, Streptococcus species, Enterobacteriaceae species, and Pseudomonas species.

33: A method for improving the appearance of the human skin, reducing fine lines and/or wrinkles, hydrating the skin and/or achieving a luminous complexion, the method comprising: applying the formulation of claim 14.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0078] FIG. 1 shows a section of an example of a plaster according to the invention comprising a semipermeable membrane (5), a composition comprising the probiotic bacteria (7), a water-impermeable membrane (4), fixation means (1, 2) and a protective layer (3). Further, it shows a hydrogel or a material for absorption of wound exudate (6) placed between the plaster and the diseased skin.

[0079] It is emphasised that the specifics shown in the FIGURE are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. The drawing is presented in the cause of providing what is believed to be the most useful and ready description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The legend of the drawing discloses to those skilled in the art how the different forms of the invention may be embodied in practice.

EXAMPLES

[0080] The following examples are provided as a pure illustration of the present invention and are not intended to be considered as limiting the protection scope as it appears from the appended claims.

Example 1

Topical Application of the Composition (“Probiotic Patch”) for the Treatment of Acne Vulgaris and Other Skin Disorders (Co-)Caused by Pathogenic Bacteria

[0081] Two commercially available polycarbonate membranes, one water-impermeable (0.02 mm thick, Makrofol®, Covestro, Leverkusen, Germany) and one semipermeable (thickness: 5-10 μm, pore size: 0.2 μm, Nuclepore®, Whatman, received through Reichelt Chemietechnik GmbH+Co., Heidelberg, Germany), each approximately 4 cm by 4 cm, were heat-sealed at three sides at 170° C. using a commercially available programmable film sealing device (“Fermant 22”, joke renotec, Bergisch Gladbach, Germany).

[0082] An insert of a commercially available nonwoven material (2.0 cm by 2.0 cm; fibre composition: viscose, polypropylene; weight: 114 g/cm.sup.2; thickness: 1.4 mm; water absorption: 950 g/m.sup.2, M1556, Freudenberg New Technologies KG, Weinheim, Germany) was put into the obtained membrane pouch sealed at three sides. This formulation of the polycarbonate membranes and the nonwoven insert was heat-sterilized by autoclaving at 121° C. for 20 minutes in a standardized procedure (DIN EN ISO 17665-1:2006).

[0083] The Lactobacillus plantarum strain 8P-A3 (DSM 32835) was grown in MRS-broth to the early stationary phase, harvested by centrifugation and resuspended in 0.1 of the original volume in fresh MRS medium containing 5% trehalose so that the suspension contained approximately 3×10.sup.10 CFU of probiotic bacteria per ml.

[0084] After equilibration of the L. plantarum suspension at room temperature for 1 hour, 200 μl of the suspension was pipetted into the welded pouches, incubated for 15 min. at 4° C., and those were immediately dried in a vacuum drying chamber under the following conditions: drying time: 24 hours, chamber temperature: approximately 22-25° C., final chamber pressure: 100-1500 Pa (1-15 mbar).

[0085] Before the following in-vitro testing for antibacterial activity of the dried pouches—for some experiments—they were heat-sealed at the fourth side (not yet sealed before). Some experiments were carried out with patches which were left open at the fourth side. Both types of patches showed identical effects.

[0086] In-vitro effects of the dried pouches containing the probiotic L. plantarum strain DSM 32835: The antimicrobial activity of the sealed pouches was tested in-vitro against the following pathogens as described in the following:

TABLE-US-00001 Species Designation Strain Collection (Website) Cutibacterium acnes DSM-1897 DSMZ.de Pseudomonas aeruginosa DSM-1117 DSMZ.de Staphylococcus aureus DSM-799 DSMZ.de

[0087] Pouches containing MRS medium with 5% trehalose but no probiotic bacteria and vacuum-dried as above served as controls.

[0088] For these experiments the pathogens C. acnes and S. aureus were incorporated into Wilkins-Chalgren (WC) agar or Brain Heart Infusion (BHI) agar, respectively, with an approximate cell count of 10.sup.3 per ml. The dried L. plantarum-containing patches obtained as described above (after storage for four days at room temperature) were applied onto the surface of the agar plates containing the pathogens. Then these agar plates were incubated anaerobically at 33° C. for two days (S. aureus) and six days (C. acnes), respectively.

[0089] For P. aeruginosa as a target bacterium, a “deferred antagonism” test was performed as follows:

[0090] The dried patches were put onto BHI agar plates and those were incubated anaerobically at 33° C. for two days. After that the patches were removed and the indicator P. aeruginosa strain (˜10.sup.5 CFU/mL) was streaked in two perpendicular lines across the surface area where the patches had been placed before the incubation for two days and been removed after. These test plates were incubated aerobically at 33° C. for 18 hours.

[0091] Inhibitory activity of the L. plantarum probiotic-containing patches applied to agar plates:

[0092] The incubation of the probiotic-containing patches for two days on agar plates under the conditions described above led to a complete (under the whole surface of the patch) in-vitro inhibition of the pathogens C. acnes, S. aureus, and P. aeruginosa.

[0093] Analogically to the in-vitro testing of antibiotics, this indicates an in-vivo activity of the composition when applied to body surfaces, especially, but not exclusively, the human skin.

Exaxple 2

[0094] Topical Application of the Composition (“Probiotic Patch”) for the Treatment of Acne Vulgaris and Other Skin Disorders (Co-)Caused by Staphylococcus aureus

[0095] In the same manner as in Example 1, patches were obtained using Streptococcus salivarius strain K12 as probiotic.

[0096] The Streptococcus salivarius strain K12 (isolated from BLIS K12™, probiotic powder) was grown in mWC (modified Wilkins-Chalgren) broth to the early stationary phase, harvested by centrifugation and resuspended in 0.1 of the original volume in fresh mWC medium containing 5% trehalose so that the suspension contained approximately 10.sup.9 CFU of probiotic bacteria per ml.

[0097] The pouches were obtained and dried as in Example 1

[0098] In-vitro effects of the dried pouches containing the probiotic S. salivarius strain K12: The antimicrobial activity of the sealed pouches was tested in-vitro against the following pathogen as described in the following:

TABLE-US-00002 Species Designation Strain Collection (Website) Staphylococcus aureus DSM-799 DSMZ.de

[0099] Pouches containing mWC medium with 5% trehalose but no probiotic bacteria and vacuum-dried as above served as controls.

[0100] For these experiments the pathogen S. aureus was incorporated into Brain Heart Infusion (BHI) agar with an approximate cell count of 10.sup.3 per ml. The dried S. salivarius-containing patches obtained as described above (after storage for four days at room temperature) were applied onto the surface of the agar plates containing the pathogen. Then these agar plates were incubated anaerobically at 33° C. for two days.

[0101] Inhibitory activity of the S. salivarius K12 probiotic-containing patches applied to agar plates:

[0102] The incubation of the probiotic-containing patches for two days on agar plates under the conditions described above led to a complete (under the whole surface of the patch) in-vitro inhibition of the pathogen S. aureus.

[0103] This inhibition was also achieved after 3 months storage at room temperature of the dried S. salivarius-containing patches obtained as described above.

[0104] An inhibition of this kind and strength could also be observed when one drop of a sterile hydrogel (˜50 μl, “DracoHydrogel”, Dr. AusbUttel & Co. GmbH, Witten, Germany) was placed between the patches and the test agar plate containing S. aureus.

[0105] Analogically to the in-vitro testing of antibiotics, this indicates an in-vivo activity of the composition when applied to body surfaces, especially, but not exclusively, the human skin.

Example 3

[0106] Topical Application of the Co Position (“Probiotic Patch”) for the Treat Ent of Skin Disorders Like Venous Leg Ulcers or Infected Burn Wounds Caused by Escherichia coli, P. aeruginosa or Staphylococcus aureus

[0107] In the same manner as in Example 2, patches were obtained using Streptococcus salivarius strain M18 as probiotic bacterium.

[0108] The Streptococcus salivarius strain M18 (isolated from PROBIO-DENT®, tablets, SYXYL GmbH & Co. KG, Cologne, Germany) was grown in mWC (modified Wilkins-Chalgren) broth to the early stationary phase, harvested by centrifugation and resuspended in 0.1 of the original volume in fresh mWC medium containing 5% trehalose so that the suspension contained approximately 10.sup.9 CFU of probiotic bacteria per ml.

[0109] The pouches were obtained and dried as in Examples 1 and 2.

[0110] In-vitro effects of the dried pouches containing the probiotic S. salivarius strain M18: The antimicrobial activity of the sealed pouches was tested in-vitro against the following pathogens as described in the following:

TABLE-US-00003 Species Designation Strain Collection (Website) Escherichia coli DSM-1103 DSMZ.de Pseudomonas aeruginosa DSM-1117 DSMZ.de Staphylococcus aureus DSM-799 DSMZ.de

[0111] Pouches containing mWC medium with 5% trehalose but no probiotic bacteria and vacuum-dried as above served as controls.

[0112] For the experiment with the pathogen S. aureus as target bacterium, this was incorporated into Brain Heart Infusion (BHI) agar with an approximate cell count of 10.sup.3 per ml. The dried S. salivarius-containing patches obtained as described above (after storage for four days at room temperature) were applied onto the surface of the agar plates containing the pathogen. Then these agar plates were incubated anaerobically at 33° C. for two days.

[0113] For E. coli DSM-1103 and P. aeruginosa DSM-1117 as target bacteria, a “deferred antagonism” test was performed as follows:

[0114] The dried patches were put onto BHI agar plates and those were incubated anaerobically at 33° C. for two days. After that the patches were removed and the indicator E. coli and P. aeruginosa strain, respectively (˜10.sup.5 CFU/mL), were streaked in two perpendicular lines across the surface area where the patches had been placed before the incubation for two days and been removed after. These test plates were incubated aerobically at 33° C. for 18 hours.

[0115] Inhibitory activity of the S. salivarius M18 probiotic-containing patches applied to agar plates:

[0116] The incubation of the probiotic-containing patches for two days on agar plates under the conditions described above led to a complete (under the whole surface of the patch) in-vitro inhibition of the pathogens E. coli DSM-1103, P. aeruginosa DSM-1117, and S. aureus DSM-799.

[0117] An inhibition of this kind and strength could also be observed when one drop of a sterile hydrogel (˜50 μl, “DracoHydrogel”, Dr. Ausbüttel & Co, GmbH, Witten, Germany) was placed between the patches and the test agar plate with the S. aureus strain incorporated (test not done in the deferred antagonism tests with E. coli or P. aeruginosa as target bacteria).

[0118] Analogically to the in-vitro testing of antibiotics, this indicates an in-vivo activity of the composition when applied to body surfaces, especially, but not exclusively, the human skin.

[0119] Reference to Deposited Biological Material

[0120] The Lactobacillus plantarum strain 8P-A3, used in particular embodiments of the invention, was deposited by the assignee under the terms of the Budapest Treaty with Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany, on 14 Jun. 2018 and assigned Accession No. DSM 32835.

LIST OF REFERENCE SIGNS

[0121] 1, 2 fixation means [0122] 3 protective layer [0123] 4 water-impermeable membrane [0124] 5 semipermeable membrane [0125] 6 hydrogel or material for absorption of wound exudate [0126] 7 enclosed probiotic bacteria