JP6446065B2 - Niacinamide for inducing the formation of antimicrobial peptides - Google Patents

Description

  The present invention relates to a new use of niacinamide for triggering the production of AMP (antimicrobial peptide) in the skin. This is applied to improve skin, scalp and oral immunity against microbial attack.

  The skin is the primary defense line that protects the human body from invading pathogens such as viruses and bacteria. As a primary defense organ, skin tissue remains in constant contact with the environment at all times and must therefore be countered and resolved against threats and loads from invading pathogens. The exposed skin surface is not only loaded by pathogenic foreign bacteria, but also interacts in contact with resident commensal bacteria. Despite all these loads from alien and commensal microorganisms, healthy skin remains uninfected and the number of resident microflora also remains constant. This equilibrium in the interaction between skin tissue and microorganisms is maintained because the skin has an elaborate defense strategy and antimicrobial peptides (AMP) form an important part of it.

  AMP forms an integral part of the skin's own defense system. AMPs were first discovered in insects and animals, and since then, those first discovered AMPs are considered promising antimicrobial agents. AMP is ubiquitous in nature and typically exhibits broad activity against invading bacteria, fungi, enveloped viruses and parasites (Braf and Gallo, 2006. AMP is generally a short peptide, human Are reported to have about 90 different AMPs, which generally have two main physical characteristics: a) a cationic charge and b) a significant proportion of hydrophobic residues. It is. The cationic charge of AMP promotes the selectivity of negatively charged microorganisms to the cytoplasmic membrane, whereas hydrophobicity facilitates the interaction of microbial species with the cell membrane.

  We continue to work to provide hygiene benefits to consumers through routes that enhance AMP levels in the skin. We want to provide this through the use of natural molecules that are perceived by the skin as being more skin-friendly and thus less unpleasant. In order to achieve this goal, various actives were tested and after extensive research came to the use of niacinamide or vitamin B3 to enhance AMP levels in the skin.

  Niacinamide or vitamin B3 is a well-known skin lightening active used in several products for skin care. Niacinamide has also been reported to have various other properties such as cellulite prevention, treating dermatitis, and improving water absorption into the skin (to name a few, Europe (Reported in Japanese Patent No. 1063963, European Patent No. 1063994, and European Patent No. 1063965). Niacinamide is also used in the treatment of many inflammatory skin conditions such as acne vulgaris, psoriasis and atopic dermatitis (Niren, NM (2006) Pharmacologic doses of nicotinamide treatment of inflammatory). skin conditions: a review.Cutis 77: 11-16.).

  WO 11333692 (Cedar's Sinai Medical Center) discloses the important role of C / EBPe in the innate immune response against pathogens. Specifically, in the absence of functional C / EBPe, mice have been shown to be severely impaired in their ability to clear S. aureus infection. Neutrophils are particularly affected and their susceptibility to S. aureus can be corrected by treatment with interferon gamma (IFN- →). Importantly, increased C / EBPe activity by inducing overexpression of C / EBPE or by application of nicotinamide or analogs, derivatives or salts thereof dramatically enhanced immune killing of S. aureus. Lead to remission of infection.

European Patent No. 1063963 European Patent No. 1063994 Specification European Patent No. 1063965 Specification International Publication No. 111333692 Pamphlet

Braff and Gallo, 2006 Niren, N.M. M.M. (2006) Pharmacological doses of nicotinamide in the treatment of inflammatory skin conditions: a review. Cutis 77: 11-16.

  We are an important step in improving skin immunity against microbial attack upon application of niacinamide to the skin (which can be the skin itself or the scalp or oral cavity). It has been found that the active induces the production of AMP, which is known to be. Thus, the benefit that consumers obtain is that the application of a composition comprising niacinamide protects the skin against pathogens that may attack in the future.

  The present invention provides the use of niacinamide to induce secretion of antimicrobial peptides (AMP) when applied to the external surface of the human body.

  Thus, the present invention also provides niacinamide for prophylactic induction of antimicrobial peptide (AMP) secretion when applied to the external surface of the human body.

  Furthermore, the present invention provides a method for providing protection to the scalp by inducing secretion of an antimicrobial peptide (AMP), comprising applying niacinamide to a person's hair / scalp. I will provide a.

  These and other aspects, features and advantages will become apparent to those skilled in the art from a reading of the following detailed description and appended claims. To avoid misunderstanding, any feature of one aspect of the invention may be utilized in any other aspect of the invention. The word “comprising” means “including” but is not intended to necessarily mean “consisting of” or “consisting of”. In other words, the listed steps or options need not be exhaustive. It should be noted that the examples set forth in the following description are intended to clarify the invention and are not intended to limit the invention to the examples themselves. Similarly, all percentages are weight / weight percentages unless otherwise indicated. All numbers in this description that indicate material amounts or reaction conditions, material physical properties and / or usage, unless otherwise stated in the working examples and comparative examples, are indicated by the word “about”. Should be understood as being modified by Numerical ranges expressed in the format “from x to y” are understood to include x and y. It should be understood that for a particular feature, where multiple preferred ranges are described in the form “from x to y”, all ranges with different endpoints are also contemplated.

  “Skin”, as used herein, is intended to include the external surface of mammals, particularly humans, and includes skin, scalp, hair and oral cavity. Use may be by incorporating niacinamide into a product that is not washed away or rinsed away, primarily to whiten the skin, but may also improve appearance, cleansing, malodor control or overall aesthetics. , Including any product applied to the human body. Use is preferably by incorporation into a composition that does not wash away. The composition may be in the form of a liquid, lotion, cream, foam, scrub, gel, bar soap or toner, or may be applied using an instrument or via a face mask, pad or patch. Non-limiting examples of such compositions include non-washing skin lotions and creams, shampoos, conditioners, shower gels, solid cosmetic soaps, antiperspirants, deodorants, hair removers, lipsticks, foundations, mascaras, sun restaners and Includes sunscreen lotion.

Niacinamide has a structure as described below.

  Niacinamide for use in the present invention preferably induces the secretion of AMP from keratinocytes. AMP secreted in this way provides improved immunity of the external surface of the body. The external surface includes the skin, scalp or oral cavity.

  In accordance with the present invention, it has been found that niacinamide activates keratinocytes, the main cells in the skin epidermis, to provide the benefits of the present invention, i.e., induce secretion of antimicrobial peptides (AMP). Thereby, niacinamide enhances the protective shield against pathogens. Thus, niacinamide provides the body with protection against infection by increasing the body's own defenses. In other words, the actives prepare the body surface in preparation for pathogen protection. The advantage of this is that it provides long-term protection against pathogens, for example up to 24 hours of protection.

  Compositions in which niacinamide is used as in the present invention may include the following preferred features. Niacinamide is preferably 0.1 to 5%, more preferably 0.5 to 5%, even more preferably 0.5 to 3%, optimally 1.0 to 3% by weight of the composition. Present at 0.0 weight percent. The composition preferably comprises a cosmetically acceptable base. The cosmetically acceptable base is preferably a cream, lotion, gel or emulsion.

Personal care compositions can be prepared using different cosmetically acceptable emulsifying or non-emulsifying systems and vehicles. Preferred cosmetically acceptable bases contain 1 to 25% fatty acids. A further preferred embodiment provides inclusion of 0.1 to 10% soap. A very suitable base is a cream. Vanishing cream is particularly preferred. Vanishing cream base generally comprises 5 to 25% w / w fatty acid and 0.1 to 10% w / w soap. Vanishing cream base gives the skin a matte feel that is highly appreciated. Especially preferably the _{C 20} fatty acid is vanishing cream base from C _12, further more preferred _is a _{C 18} fatty acids from _{C 14.} The most preferred fatty acid is stearic acid. The fatty acid may be a mixture of palmitic acid and stearic acid. The fatty acid in the composition is more preferably present in an amount in the range of 5 to 20% w / w of the composition. The soap in the vanishing cream base contains an alkali metal salt of a fatty acid such as sodium or potassium salt, most preferably potassium stearate. The soap in the vanishing cream base is generally present in an amount in the range of 0.1 to 10%, more preferably 0.1 to 3% w / w of the composition. When formulated as a vanishing cream, the personal care composition preferably comprises 60 to 85%, more preferably 65 to 80% w / w water. Water is generally present in many compositions prepared as in the present invention and is generally present at 40 to 85% by weight of the composition.

  The compositions of the present invention may include other optional ingredients such as skin lightening agents and one or more UV sunscreens. The composition according to the invention may comprise other diluents. Diluents act as dispersants or carriers for other materials present in the composition so as to facilitate their distribution when the composition is applied to the skin. Diluents other than water may include liquid or solid emollients, solvents, humectants, thickeners and powders.

  The composition may be delivered as a deodorant. Deodorant means personal deodorant benefits, eg in stick-type, roll-on or propellant media used for application in the axillary region, which may or may not contain antiperspirant actives It is a product.

  Deodorant compositions may generally be in the form of firm solids, soft solids, gels, creams and liquids and dispensed using an applicator appropriate to the physical characteristics of the composition.

  The composition may include a wide variety of other optional ingredients. The CTFA Cosmetic Ingredient Handbook, Second Edition, 1992, which is incorporated herein by reference in its entirety, is a wide variety of non-limiting commonly used in the skin care industry suitable for use in the compositions of the present invention. Of basic cosmetic and pharmaceutical ingredients. Examples are antioxidants, binders, biological additives, buffers, colorants, thickeners, polymers, astringents, fragrances, wetting agents, opacifiers, conditioners, exfoliants, pH adjusters, storage Contains agents, natural extracts, essential oils, skin sensates, skin sedatives and skin healing agents.

  The composition is formulated in any known form such as shampoo, non-washing cream, lotion, tonic or serum, the more preferred form being shampoo, cream or lotion.

  Another aspect of the present invention is a method for providing protection to the scalp by inducing secretion of an antimicrobial peptide (AMP), comprising applying niacinamide to a person's hair / scalp. Including methods. Niacinamide is preferably applied in a therapeutic amount to induce secretion of the antimicrobial peptide.

  Protection to the scalp by secretion of AMP preferably leads to extended or longer protection to the scalp against scalp related problems such as dandruff. The use of niacinamide provides an excellent dandruff control by enhancing AMP secretion as an alternative. Furthermore, through this mechanism, the scalp is given excellent dandruff control and, in certain cases, resistance to recurrent dandruff.

  Thus, enhanced secretion of AMP by application of niacinamide provides enhanced protection / defense, resistance to dandruff and advanced nutritional benefits. The benefits of the present invention can be perceived by people suffering from dandruff or scalp dryness and itching.

  According to a preferred embodiment, the use of niacinamide according to the invention is preferably non-therapeutic.

  The invention will now be further described by the following non-limiting examples.

[Example]
[Examples 1 to 6]
Examples to demonstrate that niacinamide induces LL-37 gene expression but does not alter psoriacin expression The following samples, as shown in Table 1, show their effect on psoriacin expression and expressed psoriasin Prepared to measure AMP (LL-37).

  The material used for each gene expression and the procedure used to measure it are described below.

Materials Normal human newborn epidermis primary keratinocytes (NHEK), keratinocyte growth medium (KGM) and growth supplements, antibiotics (penicillin and streptomycin), E. coli strain (ATCC strain 10536), lipo from E. coli 055: B5 strain Polysaccharide (LPS), 1 × phosphate buffered saline, 10 mM sodium phosphate buffer, niacinamide, sterile cup (both ends are open barrel-shaped, open end area is 2.27 cm ² , rod is hard Teflon (Appendix 1), Vaseline raw material (Appendix 2), Psoriasin (S100A7) (Cyclex) and LL-37 ELISA kit (Hicult) round tip, McConkey agar medium, TSA medium (Unilever research report number : BL110086) .

Stimulation of human primary skin keratinocytes Human primary keratinocytes prepared from neonatal foreskin were obtained from (Lonza India) and cultured in a serum-free keratinocyte growth medium supplemented with a defined growth supplement (Invitrogen). Keratinocytes were cultured in 12 and 24 well tissue culture plates (BD Falcon) and cells were treated at 60-70% confluence. Keratinocyte stimulation was performed in KGM with growth supplements and all experiments were performed between passages 3-5.

RNA isolation and quantitative real-time polymerase chain reaction (PCR)
NHEKs were cultured at 6 × 10 ⁴ cells per well in 12 well plates (BD, Falcon). After 24 hours of incubation, 50% of the medium was replaced with fresh medium. On day 3, cells were treated with fresh media for 16-18 hours with different concentrations of niacinamide. Cells were harvested in RLT buffer (Qiagen) and samples were stored at -70 ° C until used for RNA isolation.

Total RNA was isolated using Qiagen RN Easy Mini Kit according to manufacturer's instructions. Purity and RNA concentration were confirmed using a nanodrop (thermo scientific) instrument. RNA samples were loaded on a 1% agarose gel and the quality of the extract was observed using a UV transilluminator (Bangalore Genie, India). RNA samples were aliquoted and stored at -70 ° C. CDNA was made using aliquots (250 to 500 ng) of total RNA by using the Iscript cDNA synthesis kit (Bio-Rad, USA). The cDNA synthesis program was at 65 ° C. for 5 minutes followed by 25 ° C. for 5 minutes. cDNA synthesis was performed at 42 ° C. for 30 minutes, and final denaturation was performed at 85 ° C. for 5 minutes, followed by removal at 4 ° C.
Real-time PCR was performed using the SYBR Green method using a thermally controlled Chromo 4 machine (Bio-Rad, USA) as per manufacturer's instructions (Bio-Rad, USA). 5 μl of a 1:10 diluted sample of cDNA and 1.5 pmole of specific primers were added in each reaction to a standardized PCR program (ie 94 ° C. for 5 minutes followed by 50 cycles of “94 ° C. for 15 seconds. The results were analyzed by using the 2- ^ΔΔCT method.

Psoriasin and LL-37 ELISA
Primary keratinocytes were seeded in 12-well plates (BD Falcon). At 60-70% confluency, cells were treated with different concentrations of niacinamide for 72 hours. After 72 hours of incubation, the cell culture supernatant was stored at -70 ° C until used for ELISA. All ELISA reagents and samples were brought to room temperature prior to assay. Cell culture supernatant samples were centrifuged at 5000 rpm for 10 minutes before use. 100 μl of an undiluted sample of cell culture supernatant was used to confirm psoriacin levels by using a Cyclex ELISA kit. Clinical samples were diluted 1:25 in dilution buffer and 100 μl was used from the diluted samples to confirm psoriacin levels by ELISA. In the case of LL-37, 100 μl of undiluted and clinical samples from in vitro were used to confirm LL-37 levels by ELISA (Hicult Biotech).

Psoriacin and LL-37AMP gene expression in keratinocytes treated with niacinamide for 18 hours. LPS (20 μg / ml) used as a positive control to trigger gene expression of psoriasin and LL-37AMP in primary keratinocytes after 18 hours was a study using the procedure described above. The results are shown in Table 2 below from the viewpoint of fold change.

  The data in Table 2 above show that niacinamide was unable to induce psoriasin gene expression even at very high concentrations (1.22 mg / ml), but surprisingly, niacinamide is an ELISA technique. Shows that the secretion of psoriasin AMP detected using can be induced.

[Examples 7 to 12]
In vivo experiments with skin lotions containing niacinamide to demonstrate the induction of psoriacin and LL-37 secretion in the skin To support the findings from in vitro experiments, the effects of niacinamide on healthy human volunteers were tested in vivo did. Volunteers were asked to apply lotions with and without niacinamide (3%) to their forearms twice daily. After 7 days of application, the site was extracted in PBS and an ELISA was performed for detection of psoriasin and LL-37AMP.

In Vivo Study Procedure E. coli Culture Preparation A glycerol stock of E. coli (10536) was inoculated into 30 ml TSB medium and the culture was incubated overnight at 37 ° C. in a shaker incubator. Overnight, the culture was sub-cultured on TSA slant and incubated overnight at 37 ° C., then the slant was stored at 4 ° C. (slant was freshly prepared once every 15 days).

E. coli cultures from slant were subcultured on TSA plates and incubated overnight at 37 ° C. E. coli plate cultures grown overnight were suspended in 6 to 7 ml sodium phosphate buffer (10 mM). The density of the culture was adjusted to 1 OD ₆₂₀ using a spectrophotometer and used for clinical studies.

Extract Cup Scrub Method This technique is included in the ASTM method, E2752-10 (a standard guide for the evaluation of antimicrobial residual effects). The cup and the rod are made of Teflon, and the area of the cup is 2.84 cm ² . The cup was used to hold buffer on the skin and the rod was used for scrubbing for better recovery of skin AMP and / or residual bacteria from the skin. The scrubbing duration was 1 minute. The extracted sample was collected in a 1.5 ml Eppendorf tube for analysis (Appendix 1).

Estimation of psoriasin and LL-37AMP and counting of residual E. coli from human skin by cup scrub method 10 μl of 1OD ₆₂₀ culture of E. coli was applied to the marked area of the forearm at 2.27 cm ² area (cream, base Where no cream or niacinamide cream is applied). The culture was spread evenly at each location. After a contact time of 15 minutes, samples were extracted by the cup scrub method in 1 ml PBS. Samples were collected in sterile microfuge tubes.

  Of the extracted samples, 800 μl was used by standard microbiological methods (100 μl of undiluted, −1 and −2 diluted samples were plated on MacConkey agar plates by spread plate method). Residual E. coli was counted. The remaining 200 μl sample was used to analyze psoriacin and LL-37 levels by ELISA using a standardized kit. A pure sample was used to confirm the level of LL-37 in the sample. In the case of psoriacin, the sample was diluted 1:25 in dilution buffer (provided with the kit) and 100 μl of diluted sample was used for analysis.

In vivo study design Volunteers were asked to come to the research facility after taking a bath every morning.

2. A 6.25 cm ² area (2.5 × 2.5 cm) area of the inner forearm was marked using a marker pen and ruler. Except for the wrist (5 cm from the base of the palm), three areas of the forearm were marked. (Each volunteer was given a marker pen to mark the area when it disappeared or disappeared).

3. An aliquot of the 62.5 mg formulation was weighed into a sterile container (35 mm plastic round dish) and given to the volunteers.

4). Volunteers were asked to apply the formulation (62.5 mg / 6.25 cm ² area) where the forearm was outlined. (The order of application was the base formulation followed by the formulation containing the active). The formulation was spread over the area for about 30 seconds.

5. After application of each formulation, the fingers were wiped with sterile tissue paper.

6). A similar aliquot of the 62.5 mg formulation was weighed in a sterile 35 mm plastic petri dish, sealed with parafilm, and given to each volunteer for application at night (before going to bed).

7). In the 7 day study, steps 3-6 were repeated for 7 days. In the case of a one day study, the assessment was performed on the second day.

8). On the eighth day, volunteers were asked to come to the research facility in the morning without washing their forearms.

9. The volunteer's forearm was washed (by the investigator) with a non-antibacterial soap (Lax soap).

10. Excess water was removed by putting dry using sterile tissue paper.

11. Volunteers were asked to wait 6 hours without touching their forearms.

12 Using a template, mark a 2.27 cm ² circular area on each cream application site with 10 μl of E. coli 10536 culture corresponding to 1 × 10 ⁸ to 1 × 10 ⁹ cells / ml in each circular area. Added and spread evenly.

13. After a contact time of 15 minutes, E. coli was recovered by the cup scrub method using 1 ml of 1 × PBS. The duration of extraction was 1 minute.

14 Samples were analyzed for bacterial count by the spread plate method on McConkey agar.

The residual logs of E. coli in the forearm region in various experiments are summarized in Tables 3 and 4 below:

  The data in Table 3 above shows that the composition containing niacinamide can reduce bacteria even after 1 day of use, while Table 4 shows that this effect is prolonged (applied for 7 days). Is maintained over time.

[Example 13]
Experiment to determine the type and amount of antimicrobial peptide (AMP) secreted by induction with niacinamide The following procedure was used to determine the AMP induced as a result of niacinamide in keratinocte cells did.

1. Primary keratinocytes were seeded in 12 well plates with 1 ml of KGM complete medium. (Seeded at 60,000 cells / well for the fourth passage).

2. Plates were incubated for 48 hours in a CO ₂ incubator.

3. After 48 hours of incubation, the cells were treated in duplicate wells with and without niacinamide 1 mg / ml concentration (fresh media was used for treatment).

4). After 72 hours of incubation, the cell culture supernatant was transferred to each 1.5 ml Eppendorf tube.

5. The sample was then passed through a 20 kDa blocking filter to remove high molecular weight proteins.

6). The eluted sample containing less than 20 kDa protein was transferred to a 15 ml sterile centrifuge tube.

7). Cold acetone (HPLC grade) was then added to the eluted sample (1 ml sample: 4 ml acetone).

8). The tube was transferred to −20 ° C. for 24 hours to precipitate the protein.

9. After 24 hours, the tubes were centrifuged at 14000 g for 40 minutes at 4 ° C.

10. The supernatant was discarded and the protein pellet was analyzed for protein using mass spectrometry.

Data for increased levels of secretion of various AMPs are listed in Table 5. Increased levels of AMP secretion are measured by PSM values (peptide spectrum match scoring function), which is a relative measure of the abundance of peptides in the secretome.

  The data in Table 5 shows that there is a significantly higher level of secretion of various AMPs as a result of the use of niacinamide.

[Examples 14 to 17]
Human volunteer study of the ability of the skin to protect itself against infection Experiments were performed using the following protocol:
1. Volunteers were given Lux ™ soap for use and asked not to use any other soap or moisturizer on the forearm for 7 days prior to the start of the study.

2. Volunteers were asked to apply Vaseline ™ Healthy White formulated with Vaseline ™ Healthy White Base Cream and Niacinamide (1%, 2% and 3%) to various locations on the forearm.

3. Volunteers were asked to repeat step 1 twice a day for 1 day after bathing in the morning and immediately before going to bed at night. Volunteers were asked not to wash their forearms after applying the evening formulation.

4). On the second day, the investigator will wash the forearm with non-antibacterial soap and ask the volunteers to wait 6 hours under controlled conditions (24 ± 2 ° C. and 45% ± 5% RH) It was.

5). After a 6 hour wait, approximately 10 ⁶ E. coli were applied separately to each forearm. The contact time of E. coli to the skin was 15 minutes.

6). After 15 minutes, the sample was extracted from the forearm using the cup-rod method.

7). E. coli was counted using standard microbial methods.

Data for the number of E. coli for various treatments are summarized in Table 6.

  The data in Table 6 shows that the use of niacinamide enhances skin genetic immunity against invading bacteria.

  Thus, the present invention provides a new use of niacinamide, which was previously unknown, for inducing the secretion of antimicrobial peptides (AMP) when applied to the external surface of the human body.

Claims (4)

0.1 to 5% by weight of niacin for protecting the outer surface of the human body from pathogenic bacteria by inducing secretion of antimicrobial peptide (AMP) from keratinocytes when applied to the outer surface of the human body A composition comprising an amide.   The composition of claim 1 for improving immunity of the external surface.   The composition according to claim 1 or 2, wherein the external surface comprises skin, scalp or oral cavity.   4. A composition according to any one of claims 1 to 3 for non-therapeutic use.