Biomarker composition for diagnosing psoriasis comprising clostridium reptum strain as active ingredient

Abstract

The present invention relates to a biomarker composition for diagnosing psoriasis, comprising a Clostridium leptum strain as an active ingredient. It has been confirmed that the relative intestinal abundance of the strain is significantly reduced in a psoriasis patient group in comparison to that of a normal group, and the strain is effective in alleviating psoriasis symptoms and improving intestinal permeability, and thus can be effectively used as a composition for diagnosing, preventing, treating, or alleviating psoriasis.

Description

Biomarker composition for diagnosing psoriasis containing Clostridium leptomeria strain as an active ingredient

[0001] The present invention relates to a biomarker composition for diagnosing psoriasis comprising a Clostridium leptum strain as an active ingredient.

[0002] Psoriasis is a chronic, intractable skin disease characterized by recurrent rashes consisting mainly of red papules (raised spots) and plaques covered with silvery-white scales. Although the immune response involving IL-23 and IL-17 is known to be the core of the pathophysiology of psoriasis, it remains an intractable disease that can only be controlled so far. At the same time, it is a disease with a high demand for treatment because it causes skin lesions on the entire body, including exposed areas, and has a significant prevalence of nearly 1% of the total population.

[0003] Recently, the incidence of psoriasis in Korea has been rapidly increasing. Psoriasis is prevalent during adolescence and in the 20s to 40s, reducing the labor force of young adults who should be most active, and causing economic burdens on individuals and society due to the cost of treatment. While research on psoriasis treatment is active and the development of biological agents is surging, the burden of medical expenses associated with the use of biological agents is high, treatment failure is frequent even after use, and a loss of response may occur over time. Recently, various studies have estimated the role of gut microbes in the pathogenesis of psoriasis. It has been reported that psoriasis worsens when patients consume a Western diet (WD), which is high in fat and sugar and can affect gut microbes, and that imiquimod (IMQ) does not effectively induce psoriatic lesions in mice lacking gut microbes. Accordingly, changes in the intestinal environment and imbalances in the gut microbiome are associated with the development of psoriasis, and while the role of the gut microbiome in the diagnosis, treatment, and prevention of psoriasis is anticipated, research is still lacking.

[0004] The object of the present invention is to provide a biomarker composition for diagnosing psoriasis comprising a Clostridium leptum strain as an active ingredient.

[0005] Another objective of the present invention is to provide a composition for diagnosing psoriasis comprising a preparation capable of detecting the strain.

[0006] Another objective of the present invention is to provide a psoriasis diagnostic kit comprising the above-mentioned psoriasis diagnostic composition as an active ingredient.

[0007] Another objective of the present invention is to provide a method for providing information for the diagnosis of psoriasis.

[0008] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of psoriasis comprising a Clostridium leptum strain as an active ingredient.

[0009] Another objective of the present invention is to provide a health functional food composition for preventing or improving psoriasis, comprising a strain of Clostridium leptum as an active ingredient.

[0010] To achieve the above objective, the present invention provides a biomarker composition for diagnosing psoriasis comprising a Clostridium leptum strain as an active ingredient.

[0011] In addition, the present invention provides a composition for diagnosing psoriasis comprising a preparation capable of detecting the strain.

[0012] In addition, the present invention provides a psoriasis diagnostic kit comprising the above-mentioned psoriasis diagnostic composition as an active ingredient.

[0013] In addition, the present invention provides a method for providing information for diagnosing psoriasis, comprising the step of measuring the relative abundance of a Clostridium leptum strain in a biological sample isolated from an individual (step 1); and the step of determining that there is a possibility of psoriasis development if the relative abundance of the strain in step 1 decreases when compared to a normal individual (step 2).

[0014] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of psoriasis comprising a Clostridium leptum strain as an active ingredient.

[0015] In addition, the present invention provides a health functional food composition for preventing or improving psoriasis comprising a Clostridium leptum strain as an active ingredient.

[0016] According to the present invention, by confirming that the relative abundance of Clostridium leptum in the intestines of a psoriasis patient group is significantly reduced compared to a normal group and that the strain exhibits an effect of improving psoriasis symptoms and intestinal permeability, it can be usefully utilized as a composition for the diagnosis, prevention, treatment, or improvement of psoriasis.

[0017] Figure 1 shows the results of analyzing changes in psoriasis lesions according to the type of animal model and diet.

[0018] Figure 2 shows the results of analyzing the effect of psoriasis on changes in the gut microbiome using an animal model. **p<0.01, ***p<0.001

[0019] Figure 3 shows the results of analyzing the effect of psoriasis on changes in the gut microbiome using a human model (specimen). *p<0.05.

[0020] Figure 4 shows the results of analyzing the effects of Clostridium leptomeria strains on psoriasis and intestinal permeability.

[0021] The present invention will be described in more detail below.

[0022]

[0023] The present invention provides a biomarker composition for diagnosing psoriasis comprising a Clostridium leptum strain as an active ingredient.

[0024] The above strain may have a reduced relative abundance in the intestines of the psoriasis patient group compared to the normal group.

[0025] In this specification, “diagnosis” refers to identifying the existence or characteristics of a pathological condition, and for the purposes of the present invention, means identifying psoriasis, and includes determining the subject’s susceptibility to psoriasis or at least one of its symptoms, therametrics (e.g., monitoring the subject’s condition to provide information on therapeutic efficacy), etc. It also includes a primary diagnosis of a clinical condition or a diagnosis of a relapsed disease.

[0026] In this specification, “biomarker” refers to an indicator capable of detecting changes within the body, and is a substance capable of confirming the normal or pathological state of a living organism or whether there are changes thereof. It may include organic biomolecules such as polypeptides, nucleic acids, lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), and psoriasis can be diagnosed as in the present invention using such a substance.

[0027]

[0028] In addition, the present invention provides a composition for diagnosing psoriasis comprising a preparation capable of detecting Clostridium leptum strains.

[0029] The above preparation may be one or more selected from the group consisting of antibodies, peptides, aptamers, compounds, primers, probes, and antisense oligonucleotides that specifically bind to the strain, but are not limited thereto.

[0030] In this specification, "primer" refers to a short nucleic acid sequence having a short free 3' hydroxyl group, capable of forming base pairs with a complementary template, and functioning as a starting point for template strand replication. The primer can initiate DNA synthesis in the presence of a reagent for a polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleotide triphosphates at an appropriate buffer solution and temperature. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

[0031] In this specification, "probe" refers to a fragment of several to hundreds of nucleotide sequences capable of specifically binding to the strain, and is labeled so that the strain can be identified. Suitable probes and hybridization conditions may be appropriately selected according to techniques known in the art.

[0032]

[0033] In addition, the present invention provides a psoriasis diagnostic kit comprising the above-mentioned psoriasis diagnostic composition as an active ingredient.

[0034] The above kit can be used to diagnose whether psoriasis has developed by measuring the relative intestinal abundance of the strain in a sample isolated from an individual suspected of having psoriasis.

[0035] In addition, the above kit may include not only a preparation for measuring the relative abundance of the strain in the intestine, but also one or more other component compositions, solutions, or devices suitable for the analysis method.

[0036] For example, the kit according to the present invention may be a kit comprising genomic DNA derived from a sample to be analyzed for performing PCR, a primer set specific to the strain of the present invention, an appropriate amount of DNA polymerase, a dNTP mixture, a PCR buffer solution, and water. The PCR buffer solution may include KCl, Tris-HCl, and MgCl2. In addition, components necessary for performing electrophoresis to verify whether the PCR product has been amplified may be additionally included in the kit of the present invention.

[0037] In addition, the kit according to the present invention may be a kit containing essential elements necessary for performing RT-PCR. In addition to each primer pair specific to a marker gene, the RT-PCR kit may include a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC-water, sterile water, etc. Additionally, it may include a primer pair specific to a gene used as a quantitative control.

[0038]

[0039] In addition, the present invention provides a method for providing information for diagnosing psoriasis, comprising the step of measuring the relative abundance of a Clostridium leptum strain in a biological sample isolated from an individual (step 1); and the step of determining that there is a possibility of psoriasis development if the relative abundance of the strain in step 1 decreases when compared to a normal individual (step 2).

[0040] The above biological sample may be derived from one or more selected from the group consisting of tissue, cell, whole blood, blood, serum, saliva, sputum, cerebrospinal fluid, feces, and urine, but is not limited thereto.

[0041]

[0042] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of psoriasis comprising a Clostridium leptum strain as an active ingredient.

[0043] A pharmaceutical composition characterized by the above strain reducing intestinal permeability increased by psoriasis.

[0044] In addition, the strain may exhibit an effect of improving one or more inflammatory skin diseases selected from the group consisting of psoriasis, contact dermatitis, irritant contact dermatitis, allergic contact dermatitis, phototoxic and photoallergic contact dermatitis, contact urticaria syndrome, autosensitization dermatitis, stasis dermatitis, acne, atopic dermatitis, allergic dermatitis, seborrheic dermatitis, papular urticaria, and eczema, but is not limited thereto.

[0045] The above psoriasis may be psoriatic dermatitis.

[0046] The pharmaceutical composition of the present invention may be manufactured in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person skilled in the art to which the invention belongs.

[0047] The above-mentioned pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0048] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0049] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, and cataplasms, but is not limited thereto.

[0050] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically affinities for the target. Examples of diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and / or dispersion media.

[0051] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated into tablets, troches, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated into injectable solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for topical application, oils, creams, etc.

[0052] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, gender, health status, dietary constitutional specificity, properties of the formulation, degree of disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate, and form of the drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0053] The above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutical effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, method of administration, time of administration, route of administration, etc., and a person skilled in the art can easily determine and prescribe a dosage effective for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0054]

[0055] In addition, the present invention provides a health functional food composition for preventing or improving psoriasis comprising a Clostridium leptum strain as an active ingredient.

[0056] The present invention can be generally used as a commonly used food.

[0057] The food composition of the present invention may be used as a health functional food. The term “health functional food” refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Health Functional Foods Act, and the term “functional properties” refers to consuming the food for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0058] The above-mentioned health functional food composition may include ordinary food additives, and unless otherwise specified, suitability as a “food additive” shall be determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety.

[0059] Examples of items listed in the above “Food Additives Codex” include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0060] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a conventional hard capsule with the composition according to the present invention and additives such as excipients, and soft capsules can be manufactured by mixing the composition according to the present invention with additives such as excipients and filling it into a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0061] The definitions of terms regarding the above excipients, binders, disintegrants, lubricants, flavoring agents, etc., are those described in literature known in the art and include those with identical or similar functions. There are no special restrictions on the types of food mentioned above, and they include all health functional foods in the conventional sense.

[0062] In the present invention, the term “prevention” refers to any act of suppressing or delaying psoriasis by administering a composition according to the present invention.

[0063] In the present invention, the term “treatment” refers to any act of improving or beneficially altering the symptoms of psoriasis through the administration of a composition according to the present invention.

[0064] In this invention, the term “improvement” refers to any act of improving a poor condition of psoriasis through the administration of a composition according to this invention.

[0065] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0066]

[0067] [Experimental Example 1] Analysis of the Effect of Psoriasis on Changes in Gut Microbiota

[0068] 1-1. Animal Models

[0069] To investigate the effects of psoriasis on changes in the gut microbiome, experiments were conducted using animal models. Seven-week-old wild-type C57BL / 6 mice (hereinafter referred to as normal mice); and seven-week-old IL-10 knockout mice (hereinafter referred to as IL-10(- / -) mice) were purchased from Orient Bio and used, and animal experiments were performed at the Preclinical Center of Seoul Metropolitan Boramae Hospital (Animal Experiment Approval Number: IACUC No. 2020-0051). IL-10(- / -) mice were selected as an animal model because they exhibited significantly more psoriatic lesions compared to normal mice and maintained a chronic condition. A total of four experimental groups were established as follows. After feeding the experimental group a normal diet (hereinafter referred to as ND) or a high-fat western diet (hereinafter referred to as WD) for 2 weeks, imiquimod (hereinafter referred to as IMQ) was applied to the back of the ears of the experimental group once a day, 5 times a week (Monday to Friday), for a total of 4 weeks to induce psoriasis (amount applied per application: 31.25 mg, total amount applied: 625 mg). During the application period, the existing diet was maintained. Subsequently, the thickness of the applied ear was measured, and photographs were taken weekly to confirm the psoriasis lesions. In addition, after the end of the application period, the mice were sacrificed, ear blocks were prepared to measure the epidermal thickness, and stool analysis was performed.

[0070] 1) Normal Mouse + ND + IMQ Processing

[0071] 2) Normal mouse + WD + IMQ processing

[0072] 3) IL-10(- / -) Mouse + ND + IMQ processing

[0073] 4) IL-10(- / -) Mouse + WD + IMQ Processing

[0074] As a result, as shown in Figure 1, psoriasis lesions were most significantly observed in the WD-fed IL-10(- / -) mouse group visually. Additionally, as shown in Figure 2, the results of the Alpha-diversity analysis to confirm bacterial diversity in the stool of the experimental group showed that bacterial diversity was significantly reduced in the WD-fed IL-10(- / -) mouse group compared to the ND-fed IL-10(- / -) mouse group, the relative abundance of Clostridium leptomeria strains was also reduced, and epidermal thickness was significantly increased in the WD-fed IL-10(- / -) mouse group compared to the other experimental groups.

[0075] From the above results, it was confirmed that psoriasis lesions significantly appeared in IL-10(- / -) mice compared to normal mice, WD exacerbated psoriasis lesions compared to ND, and the relative abundance of Clostridium leptomeria strains in the psoriasis model significantly decreased.

[0076]

[0077] 1-2. Human Model

[0078] To investigate the effects of psoriasis on changes in the gut microbiome, experiments were conducted using human specimens (models) (Experiment Approval Number: IRB No. 30-2021-138). Stool samples were obtained from healthy individuals (n=48) and psoriasis patients (n=31) and pretreated. The DNA extraction and pretreatment process for pathogen detection in stool was performed as follows. Glass beads and SLX-Mlus buffer were added to 200 mg of stool sample and homogenized for 10 minutes. Subsequently, DS buffer and Proteinase K were added and mixed, then heated at 70°C for 10 minutes. Afterward, SP2 buffer was added and the sample was placed on ice for 5 minutes, followed by centrifugation to transfer the supernatant to a new tube. cHTR reagent was added, and the sample was centrifuged again to transfer the supernatant to a new tube. A mixture of BL buffer and ethanol was placed in the HiBind® DNA column for washing. The column was washed twice with VHB buffer and DNA wash buffer, and dried. DNA was eluted with elution buffer and stored at -20°C. Subsequently, gut microbiota analysis was performed using 16S rRNA analysis, and downstream analysis was performed using qiime2 and MicrobiomeAnalyst.

[0079] As a result, as shown in Figure 3, in the Alpha-diversity analysis, strain diversity was significantly reduced in the psoriasis patient group compared to normal individuals, and the relative abundance of Clostridium leptomeria strains was significantly reduced.

[0080]

[0081] [Experimental Example 2] Analysis of the Effects of Clostridium leptomeria Strain on Psoriasis and Intestinal Permeability

[0082] To investigate the effects of the Clostridium leptomata strain (hereinafter referred to as CL) on psoriasis and intestinal permeability, experiments were conducted using an animal model. Male IL-10(- / -) mice aged 6–8 weeks were purchased from Orient Bio, and the animal experiments were performed at the Preclinical Center of Seoul Metropolitan Boramae Hospital (Animal Experiment Approval Number: IACUC No. 2020-0051). A total of two experimental groups were established as follows. Specifically, the experimental groups were fed WD-40 for 2 weeks, and during the WD-40 intake period (2 weeks), CL was administered once daily at a dose of 1 × 10⁶ 9 CFU (200 μL) was administered orally. PBS of the same volume as CL was administered to the control group in the same manner. After the administration of CL was completed, 5% IMQ cream was applied to the back of the mice's ears at a dose of 62.5 mg per day for a total of 3 days to induce psoriasis-like dermatitis.

[0083] 1) Control group (n=4): IL-10 (- / -) mice + WD + IMQ treatment + PBS

[0084] 2) CL administration group (n=5): IL-10(- / -) mice + WD + IMQ treatment + CL

[0085] Subsequently, body weight, ear thickness, colon length, and intestinal permeability of the experimental group were analyzed. Body weight was measured on days 0, 3, 7, 14, and 19 following IMQ application. Ear thickness was measured using a digital thickness gauge (±0.01 mm) on days 15 and 18 following IMQ application. Colon length was measured after sacrificing the mice following the completion of IMQ application, with the southern colon-rectum straight line measured using a vernier caliper. For intestinal permeability, FITC-dextran (4 kDa, 100 mg / mL, 0.6 mg / g body weight) was orally administered on the last day of IMQ application, serum fluorescence (Excitation 485 nm / Emission 528 nm) was measured 4 hours after administration, and the standard curve (r2 The concentration was converted to (= 0.998). Statistical analysis was performed using the unpaired t-test or Mann-Whitney U (when normality was not satisfied), and p<0.05 was considered a statistically significant difference.

[0086] As a result, as shown in Figure 4, there was no significant difference between the experimental groups regarding changes in body weight, confirming that LC does not exhibit significant toxicity such as inducing changes in body weight. Additionally, regarding ear thickness, on day 18 of IMQ application, ear thickness significantly decreased in the CL administration group compared to the control group (p=0.02). This confirmed that CL improves psoriasis (psoriatic dermatitis). Furthermore, regarding colon length, colon length increased in the CL administration group compared to the control group, and regarding intestinal permeability, blood FITC-dextran levels decreased in the CL administration group (3.5±0.4μg / mL) compared to the control group (4.7±0.6μg / mL) (p=0.11), confirming a tendency for improved intestinal permeability.

[0087]

[0088] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker composition for diagnosing psoriasis containing a Clostridium leptum strain as an active ingredient.

2. A biomarker composition according to claim 1, characterized in that the relative abundance of the strain in the intestines decreases in the psoriasis patient group compared to the normal group.

3. A composition for diagnosing psoriasis comprising a preparation capable of detecting Clostridium leptum strains.

4. A composition according to claim 3, wherein the preparation is one or more selected from the group consisting of antibodies, peptides, aptamers, compounds, primers, probes, and antisense oligonucleotides that specifically bind to the strain.

5. A psoriasis diagnostic kit comprising the psoriasis diagnostic composition of paragraph 3 as an active ingredient.

6. A step of measuring the relative abundance of Clostridium leptum strains in biological samples isolated from individuals (Step 1); and A method for providing information for diagnosing psoriasis, comprising a step (second step) of determining that there is a possibility of psoriasis development when the relative abundance of the strain of the first step decreases compared to a normal individual.

7. A method for providing information according to claim 6, characterized in that the biological sample is derived from one or more selected from the group consisting of tissue, cell, whole blood, blood, serum, saliva, sputum, cerebrospinal fluid, feces, and urine.

8. A pharmaceutical composition for the prevention or treatment of psoriasis comprising a strain of Clostridium leptum as an active ingredient.

9. A pharmaceutical composition according to claim 8, characterized in that the strain reduces intestinal permeability increased by psoriasis.

10. A health functional food composition for the prevention or improvement of psoriasis containing a strain of Clostridium leptum as an active ingredient.

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