JP7777321B2 - Agent and method for inhibiting or promoting the growth of specific acne bacteria - Google Patents

Description

The present invention relates to an acne prevention and improvement agent and an agent that inhibits or promotes the growth of specific acne bacteria, each containing erythritol as an active ingredient.

Acne, also known as "acne vulgaris," is a chronic skin disease that often manifests as bumps such as blackheads, whiteheads, pimples, and cysts on the face, chest, shoulders, and back, and sometimes abscesses. Acne occurs when deposits of dead skin cells, bacteria, and dried sebum clog hair follicles. Propionibacterium acnes (hereinafter referred to as "P. acnes") is known to be the causative bacterium of acne. P. acnes grows by breaking down triglycerides, a neutral fat found in sebum, with lipase, and also secretes components involved in the development of inflammation.

Propionibacterium acnes is classified into ribotypes (RT), RT1-10, based on sequence polymorphisms in its 16S ribosomal RNA gene (16S rDNA). Recent reports have shown that while the total amount of Propionibacterium acnes present on the skin is similar between acne patients and healthy individuals (those without acne symptoms), the predominant ribotype of Propionibacterium acnes differs. According to these studies, RT1, RT2, and RT3 are uniformly distributed between patients and healthy individuals, whereas RT6 is distributed almost exclusively in healthy individuals, and RT4, RT5, RT8, and RT10 are more prevalent in acne patients. It has also been suggested that RT2 and RT6 Propionibacterium acnes have mutations in the lipase gene region, potentially resulting in relatively low lipase activity (Non-Patent Documents 1 and 2). In other words, it has been suggested that certain types of Propionibacterium acnes that have mutations in the lipase gene region have low lipase activity and are therefore not involved in the development or worsening of acne, and as a result may be at a disadvantage in acne patients.

Joseph McLaughlin et al., Propionibacterium acnes and Acne Vulgaris: New Insights from the Integration of Population Genetic, Multi-Omic, Biochemical and Host-Microbe Studies, Microorganisms 2019, 7(5), 128; https://doi.org/10.3390/microorganisms7050128 Shuta Tomida et al., Pan-Genome and Comparative Genome Analyses of Propionibacterium acnes Reveal Its Genomic Diversity in the Healthy and Diseased Human Skin Microbiome, mBio, May/June 2013 Volume 4 Issue 3 e00003-13

Based on the above, it is believed that the specific types of Propionibacterium acnes that are involved in the onset and worsening of acne, such as RT4, RT5, and RT8, or specific types that do not have mutations in the lipase gene region, and that RT2, RT6, or types that have mutations in the lipase gene region are not involved. Therefore, it is believed that inhibiting the growth of these types of bacteria that are involved in the onset and worsening of acne can contribute to the prevention or improvement of acne.

In addition, since Propionibacterium acnes is a normal skin flora, and its total abundance is the same in acne patients and healthy individuals, it is thought that by promoting the growth of specific bacterial strains that are not involved in the onset or worsening of acne (such as RT2, RT6, or bacterial strains with mutations in the lipase gene region), it is possible to relatively suppress the growth of bacterial strains that are involved in the onset or worsening of acne, thereby contributing to the prevention or improvement of acne.

The present invention therefore aims to provide a technology for inhibiting the growth of specific types of Propionibacterium acnes that may be involved in the onset or worsening of acne. It also aims to provide a technology for promoting the growth of specific types of Propionibacterium acnes that are not involved in the onset or worsening of acne. It also aims to provide a technology that contributes to the prevention or improvement of acne.

As a result of extensive research, the inventors have discovered that erythritol can inhibit the growth of certain types of Propionibacterium acnes that may be involved in the onset or worsening of acne, while promoting the growth of certain types of Propionibacterium acnes that are not involved in the onset or worsening of acne. Based on these findings, the inventors have completed the following inventions.

(1) The acne prevention or amelioration agent of the present invention contains erythritol as an active ingredient.

(2) A first aspect of the acne bacteria growth inhibitor of the present invention is an agent that inhibits the growth of one or more acne bacteria selected from ribotypes 4 (RT4), 5 (RT5), and 8 (RT8), and contains erythritol as an active ingredient.

(3) A second aspect of the acne bacteria growth inhibitor of the present invention is an agent for inhibiting the growth of acne bacteria that do not have a mutation in the DNA region shown in SEQ ID NO: 11, and contains erythritol as an active ingredient.

(4) A first aspect of the acne bacteria proliferation promoter of the present invention is an agent for promoting the proliferation of ribotype 2 (RT2) and/or ribotype 6 (RT6) acne bacteria, and contains erythritol as an active ingredient.

(5) A second aspect of the acne bacteria proliferation promoter of the present invention is an agent for promoting the proliferation of acne bacteria having a mutation in the DNA region shown in SEQ ID NO: 11, and contains erythritol as an active ingredient.

(6) In a second aspect of the agent for promoting the proliferation of Propionibacterium acnes according to the present invention, the mutation in the DNA region shown in SEQ ID NO: 11 may include one or more mutations selected from the following (i) to (iii):
(i) deletion of 6 bases at positions 138 to 143 of SEQ ID NO: 11;
(ii) deletion of one base at position 288 of SEQ ID NO: 11;
(iii) The occurrence of a stop codon at positions 370 to 372 of SEQ ID NO: 11.

(7) The Propionibacterium acnes-selective antibacterial agent of the present invention has a selective antibacterial effect against Propionibacterium acnes, inhibiting the growth of one or more Propionibacterium acnes strains selected from the group consisting of HL007PA1 strain, HL038PA1 strain, HL043PA2 strain, HL072PA1 strain, HL086PA1 strain, and HL110PA1 strain, and exhibiting physical properties that do not inhibit or promote the growth of the HL110PA3 strain, and contains erythritol as an active ingredient.

(8) The agent of the present invention may be used as an external preparation.

The present invention can inhibit the growth of specific types of Propionibacterium acnes that may be involved in the onset or worsening of acne. The present invention can promote the growth of specific types of Propionibacterium acnes that are not involved in the onset or worsening of acne. Therefore, the present invention can contribute to the prevention or improvement of acne.

Furthermore, erythritol, the active ingredient of the present invention, is an extremely safe substance for humans and animals, as evidenced by its use as a food product. Therefore, according to the present invention, it is possible to inhibit the growth of specific bacterial strains that may be involved in the onset or worsening of acne, or to promote the growth of specific bacterial strains that are not involved in the onset or worsening of acne, without any concerns about skin irritation or safety.

1 is a bar graph showing the turbidity of culture solutions obtained by culturing various strains of Propionibacterium acnes in media containing 0%, 5 (w/v)% or 10 (w/v)% erythritol. Significant differences are indicated by *. FIG. 1 is a diagram showing a schematic diagram of the lipase gene region of Propionibacterium acnes. FIG. 1 shows the base sequences (1st to 320th bases) obtained by sequencing the lipase gene region of each P. acnes strain. FIG. 1 shows the base sequences (321st to 400th bases) obtained by sequencing the lipase gene region of each P. acnes strain.

The present invention is described in detail below. The present invention provides an agent for preventing or ameliorating acne, an agent for inhibiting the growth of Propionibacterium acnes (first and second embodiments), an agent for promoting the growth of Propionibacterium acnes (first and second embodiments), and an Propionibacterium acnes-type selective antibacterial agent. In this specification, these agents may be collectively referred to as the "agent of the present invention" or "the agent" or any one of them may be referred to as such.

In the present invention, "ribotype" refers to a subtype (bacterial type) of P. acnes classified based on sequence polymorphism of the 16S ribosomal RNA gene (16S rDNA). P. acnes having the wild-type sequence of 16S rDNA (SEQ ID NO: 1) can be identified as ribotype 1 (RT1), subtypes having the T854C mutation in the wild-type sequence as ribotype 2 (RT2), subtypes having the T1007C mutation as ribotype 3 (RT3), subtypes having G1058C and A1201C mutations as ribotype 4 (RT4), subtypes having G1058C mutation as ribotype 5 (RT5), subtypes having T854C and C1336T mutations as ribotype 6 (RT6), and subtypes having G1004A and T1007C mutations as ribotype 8 (RT8) (S. Fitz-Gibbon et al., The Skin Microbiome Associated with Acne, Journal of Investigative Dermatology 133, 2152-2160; doi:10.1038/jid.2013.21; published online 28 February 2013).

The "DNA region shown in SEQ ID NO: 11" is a partial region of the lipase gene region of Propionibacterium acnes. This lipase gene region consists of two lipase genes (lipase coding regions identified by loci HMPREF0675_4855 and HMPREF0675_4856 in the SK137 strain) arranged in tandem with an intergenic region between them. SEQ ID NO: 11 consists of the carboxyl-terminal region of the first lipase gene, the intergenic region, and the amino-terminal region of the second lipase gene in this lipase gene region.

Mutations in the DNA region shown in SEQ ID NO: 11 refer to deletions, insertions, substitutions, and/or additions of bases in SEQ ID NO: 11, and the associated appearance of stop codons in the coding region. The number of bases deleted, inserted, substituted, and/or added can be any number, for example, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 6. Specific examples of mutations include the following (i) to (iii):
(i) deletion of 6 bases at positions 138 to 143 of SEQ ID NO: 11;
(ii) deletion of one base at position 288 of SEQ ID NO: 11;
(iii) The occurrence of a stop codon at positions 370 to 372 of SEQ ID NO: 11 (associated with deletion, insertion, substitution and/or addition of bases).

Whether the growth of P. acnes has been inhibited or promoted can be determined by a culture test, as shown in the Examples below. Specifically, two identical media are prepared, one with the addition of this agent and the other without. P. acnes is inoculated into both media and cultured for a predetermined period, after which the bacterial mass in the media is measured. The bacterial mass can be measured simply by the turbidity method, but any known method, such as the dry bacterial weight method, wet bacterial weight method, or real-time PCR method, can also be used as appropriate. If the bacterial mass is lower in the sample with the agent added than in the sample without the agent, it can be determined that the agent has inhibited the growth of P. acnes. Conversely, if the bacterial mass is higher in the sample with the agent added than in the sample without the agent added, it can be determined that the agent has promoted the growth of P. acnes.

"An acne-type selective antibacterial agent" refers to an agent that has selective antibacterial activity against acne bacteria. Here, this selective antibacterial activity means that the agent inhibits the growth of acne bacteria types that are involved in the onset and worsening of acne, but does not inhibit or promote the growth of acne bacteria types that are not involved in the onset and worsening of acne. Whether or not an agent has this selective antibacterial activity can be determined, for example, by whether or not it has physical properties that inhibit the growth of the HL007PA1 strain (HM496, BEI Resources, RT4), HL038PA1 strain (HM512, BEI Resources, RT4), HL043PA2 strain (HM514, BEI Resources, RT5), HL072PA1 strain (HM531, BEI Resources, RT5), HL086PA1 strain (HM539, BEI Resources, RT8), or HL110PA1 strain (HM552, BEI Resources, RT8), but do not inhibit or promote the growth of the HL110PA3 strain (HM554, BEI Resources, RT6).

Erythritol is a sugar alcohol that is naturally found in fruits such as grapes and pears, as well as fermented foods such as miso, soy sauce, and sake. Its chemical name is 1,2,3,4-butaneterol, and it is a tetracarbonate monosaccharide alcohol. It is a reduced form of erythrose, and is obtained industrially through fermentation.

Erythritol may be produced according to methods known to those skilled in the art, or commercially available erythritol may be used for convenience. Erythritol may also be used in any form, including liquid, powder, or granules.

This agent exerts its effects by suppressing the growth of specific types of Propionibacterium acnes, a normal skin flora, that may be involved in the onset or worsening of acne, or by promoting the growth of specific types of bacteria that are not involved in the onset or worsening of acne, thereby creating a skin environment that is less susceptible to the onset or worsening of acne. Therefore, this agent can be used as a formulation that acts on the skin, i.e., as a topical agent.

Examples of topical preparations of the present invention include those that are directly applied, pasted, or sprayed onto the skin (cosmetics, quasi-drugs, and pharmaceuticals), as well as hygiene products such as skin cleansers, bath additives, disinfectants, and bactericides. Product forms include aerosols, roll-ons, sticks, creams, sheets, lotions, emulsions, gels, powders, and tablets. Products containing the present agent can be manufactured by adding erythritol to ingredients commonly used in such products (e.g., oils, surfactants, alcohols, preservatives, chelating agents, antioxidants, thickeners, fragrances, bactericides, and other ingredients).

The erythritol content can be set appropriately depending on the product form and application, but examples include 0.01 to 35% by mass, preferably 0.01 to 30% by mass, more preferably 0.5 to 25% by mass, and even more preferably 1 to 20% by mass.

The present invention will now be described based on examples. Note that the technical scope of the present invention is not limited to the features demonstrated in these examples.

In this example, commercially available erythritol (product name: Erythritol, white granules, Bussan Food Science Co., Ltd.) was used. Propionibacterium acnes strains listed in Table 1 were used as acne bacteria. Strains Nos. 6 to 10, 12, and 13 were obtained from BEI Resources (USA), and their catalog numbers are also shown in Table 1. Strains Nos. 1 to 5 and 11 were provided by Akihito Endo (Associate Professor, Department of Food and Cosmetic Chemistry, Faculty of Bioindustry, Tokyo University of Agriculture).

Ribotypes No. 6-10, 12, and 13 are as described in Table 1 of Non-Patent Document 2. Ribotypes No. 1-5 and 11 were determined by sequencing the 16S rDNA of each strain using standard methods and based on the sequences obtained. The 16S rDNA base sequences of No. 1-5 and 11 are shown in SEQ ID NOs: 2-7, respectively. Of the ribotypes in Table 1, RT1, RT2, and RT3 are reported to be present at similar levels in the skin of acne patients and healthy subjects (those without acne symptoms), while RT6 is reported to be present almost exclusively in the skin of healthy subjects. RT4, RT5, and RT8 are reported to be present almost exclusively in the skin of acne patients (Non-Patent Document 2).

Example 1: Effect on Propionibacterium acnes Bacterial Count Each strain in Table 1 was spread on a modified CDC anaerobic sheep blood agar medium (Nippon Becton Dickinson) and anaerobically cultured at 30°C for 4 days using Anaeropack Kenki (Mitsubishi Gas Chemical). These strains were picked up with a chip and suspended in 0.5 mL of modified GAM bouillon "Nissui" (Nissui Pharmaceutical) to prepare a seed suspension. Erythritol was added to modified GAM bouillon "Nissui" to a final concentration of 0%, 5%, or 10% by mass to prepare a main culture medium. The main culture medium was dispensed into a 96-deep well plate (AxyGen Scientific, Inc.), and 20 μL of the seed suspension was inoculated into the plate and anaerobically cultured (main culture) at 30°C for 48 hours.

The bacterial cell concentration of the culture medium after the main culture was measured by the turbidity method. Specifically, 180 μL of water was dispensed into a 96-well flat-bottom plate (4845-96F, Watson). 20 μL of the culture medium was then added, and the turbidity (OD ₆₆₀ ) was measured by detecting the transmitted light intensity at a wavelength of 660 nm using a microplate reader (SpectraMax® M2, Molecular Devices Japan). The OD ₆₆₀ value was multiplied by 10 to determine the turbidity, and the same test was performed four times to calculate the average value. A Mann-Whitney U test was used to test for statistical significance between two groups: a control group without erythritol added to the main culture medium, and a 5% or 10% erythritol added group (5% group, 10% group). A p<0.05 asymptotic significance level was considered significant. The results are shown in Figure 1. In Figure 1, significant differences were indicated by an *.

As shown in Figure 1, for the RT2 and RT6 strains, turbidity increased significantly when erythritol was added (5% group, 10% group) compared to when it was not added (0% group). In contrast, for the RT1, RT3, RT4, RT5, and RT8 strains, turbidity decreased significantly when erythritol was added (5% group, 10% group) compared to when it was not added (0% group).

In other words, the addition of erythritol significantly increased the number of RT2 and RT6 strains, while the addition of erythritol significantly decreased the number of RT1, RT3, RT4, RT5, and RT8 strains. These results demonstrate that erythritol promotes the growth of P. acnes RT2 (a strain present at similar levels in the skin of acne patients and healthy subjects) and RT6 (a strain predominant in healthy skin without acne symptoms), and inhibits the growth of P. acnes RT1 and RT3 (strains present at similar levels in the skin of acne patients and healthy subjects), as well as RT4, RT5, and RT8 (strains predominant in the skin of acne patients).

Example 2: Sequence analysis of the lipase gene region
P. acnes has 13 genes encoding proteins that are presumed to function as lipases. One of these genes consists of two lipase genes (the lipase genes identified by loci HMPREF0675_4855 and HMPREF0675_4856 in the SK137 strain) arranged in tandem with an intergenic region, as shown in Figure 2 (total length 2036 bp, SEQ ID NO: 8). It has been reported that P. acnes strains belonging to RT2, RT6, and RT8 have deletion mutations in the intergenic region and the second lipase gene (HMPREF0675_4856) (Non-Patent Document 2). Therefore, the strains listed in Table 1 were analyzed for the presence or absence of mutations in the lipase gene region.

Specifically, each strain listed in Table 1 was placed in a disruption tube containing 500 μL of sterile water and two 5 μm-diameter zirconia beads. The cells were disrupted using a Micro Smash® MS-100 cell disrupter (Tomy Seiko) at 4,000 rpm for 90 seconds to obtain genomic DNA. This DNA was used as template DNA for PCR using Quick Taq™ HS DyeMix (TOYOBO), which amplified DNA from a portion of the lipase gene region. The primers used were SEQ ID NOs: 6 and 7. The reaction conditions were 32 cycles of 95°C for 10 seconds, 63°C for 20 seconds, and 68°C for 60 seconds.
<Primers for amplifying lipase genes>
Forward: 5'-ACGAGCCTGGTGTCAAAAACATCCTCGTTCA-3' (SEQ ID NO: 9)
Reverse: 5'-GCTTCCACAAGCGGATGTGTTTCCGGGTTGTA-3' (SEQ ID NO: 10)

The amplified DNA fragments were purified using the FastGene® Gel/PCR Extraction Kit (Nippon Genetics) and then sequenced by FASMAC Corporation (http://fasmac.co.jp/). The results are shown in Figures 3 and 4. The nucleotide sequences of strains No. 1 to 13 shown in Figures 3 and 4 are shown in SEQ ID NOs: 11 to 23, respectively.

As shown in Figure 3, the following mutations (i) to (iii) were confirmed in the nucleotide sequences (SEQ ID NOS: 13-14 and 20-21) of strains No. 3 and No. 4 (RT2) and strains No. 10 and No. 11 (RT6):
(i) a deletion of 6 bases at positions 108 to 114 (counting from the next base after the stop codon of the first lipase gene) in the intergenic region (corresponding to positions 996 to 1001 of SEQ ID NO: 8 and positions 138 to 143 of SEQ ID NO: 11);
(ii) a deletion of one base at position 124 of the second lipase gene (corresponding to position 1146 of SEQ ID NO: 8 and position 288 of SEQ ID NO: 11);
(iii) Appearance of a stop codon due to a frameshift associated with the one-base deletion described in (ii) above at positions 206 to 209 of the second lipase gene (corresponding to positions 1228 to 1230 of SEQ ID NO: 8 and positions 370 to 372 of SEQ ID NO: 11).
On the other hand, no mutations were observed in the sequences of strains No. 1-2, 5-9, and 12-13 (RT1, RT3, RT4, RT5, RT8), and SEQ ID NOs: 11-12, 15-19, and 22-23 were identical sequences.

That is, it was revealed that the RT2 and RT6 strains, whose growth was promoted by erythritol in Example 1, have a mutation in the DNA region shown in SEQ ID NO: 11 in the lipase gene region, whereas the RT1, RT3, RT4, RT5, and RT8 strains, whose growth was inhibited by erythritol in Example 1, do not have a mutation in the DNA region. These results demonstrate that erythritol can promote the growth of P. acnes that has a mutation in the DNA region shown in SEQ ID NO: 11 in the lipase gene region. It was also demonstrated that erythritol can inhibit the growth of P. acnes that does not have a mutation in the DNA region.

Claims (5)

An acne-type selective antibacterial agent comprising erythritol, which has an acne-type selective antibacterial effect of inhibiting the growth of the following (a) and/or (b), and not inhibiting or promoting the growth of (c) and/or (d):
(a) one or more Propionibacterium acnes selected from ribotypes 4 (RT4), 5 (RT5), and 8 (RT8);
(b) Propionibacterium acnes that does not have a mutation in the DNA region shown in SEQ ID NO: 11;
(c) ribotype 2 (RT2) and/or ribotype 6 (RT6) Propionibacterium acnes;
(d) Propionibacterium acnes having a mutation in the DNA region shown in SEQ ID NO: 11. The agent according to claim 1 , wherein the mutation includes one or more selected from the following (i) to (iii):
(i) deletion of 6 bases at positions 138 to 143 of SEQ ID NO: 11;
(ii) deletion of one base at position 288 of SEQ ID NO: 11;
(iii) The occurrence of a stop codon at positions 370 to 372 of SEQ ID NO: 11. 2. The agent according to claim 1, wherein the acne type-selective antibacterial activity has physical properties of inhibiting the growth of one or more acne strains selected from HL007PA1 strain, HL038PA1 strain, HL043PA2 strain, HL072PA1 strain, HL086PA1 strain, and HL110PA1 strain, and not inhibiting or promoting the growth of HL110PA3 strain. The agent according to any one of claims 1 to 3 , which is used as an external preparation. A method for selectively inhibiting the growth of Propionibacterium acnes (excluding methods for surgery, treatment, or diagnosis of humans), comprising the step of applying erythritol to the skin to inhibit the growth of the following (a) and/or (b) , and not inhibiting or promoting the growth of (c) and/or (d):
(a) one or more Propionibacterium acnes selected from ribotypes 4 (RT4), 5 (RT5), and 8 (RT8);
(b) Propionibacterium acnes that does not have a mutation in the DNA region shown in SEQ ID NO: 11;
(c) ribotype 2 (RT2) and/or ribotype 6 (RT6) Propionibacterium acnes;
(d) Propionibacterium acnes having a mutation in the DNA region shown in SEQ ID NO: 11.