Detection method for hunner-type interstitial cystitis diagnosis marker, hunner-type interstitial cystitis diagnosis kit, and hunner-type interstitial cystitis therapeutic agent

Abstract

To provide a detection method for Hunner-type interstitial cystitis diagnosis markers, enabling specific detection of Hunner-type interstitial cystitis, a Hunner-type interstitial cystitis diagnosis kit, and a Hunner-type interstitial cystitis therapeutic agent.SOLUTION: Provided is a detection method for Hunner-type interstitial cystitis diagnosis markers, the detection method involving detecting, from the urine of a subject, the presence level of at least one microorganism selected from the group consisting of bacteria of the genus Trabulsiella, bacteria of the genus Peptoniphilus, bacteria of the genus Anaerococcus, and bacteria of the genus Anaerobacillus.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting a diagnostic marker for Hanna type interstitial cystitis, a diagnostic kit for Hanna type interstitial cystitis, and a therapeutic agent for Hanna type interstitial cystitis. [Background technology]

[0002] Hanna type interstitial cystitis (HIC) is an intractable disease (designated intractable disease 226) in which inflammation of the bladder occurs for unknown reasons, resulting in painful symptoms such as frequent urination, discomfort and pain in the bladder and urethra. Pathological findings of HIC include lymphocytic infiltration in the interstitium, and exfoliation of the bladder epithelium due to inflammation of the interstitium.

[0003] However, these findings are non-specific inflammatory reactions that are also seen in ordinary cystitis. Local treatment of Hanna lesions alone does not result in a complete cure, so early detection and early treatment are necessary.

[0004] In response to such demand, a method for assisting in the detection of ulcerative (Hanna type) interstitial cystitis using gene markers including CXCL10 has been proposed (see Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5758122 Summary of the Invention [Problem to be solved by the invention]

[0006] However, since increased expression of CXCL10 is also observed in interstitial cystitis other than Hanna type interstitial cystitis, a diagnostic marker that increases or decreases more specifically in Hanna type interstitial cystitis is desired. Therefore, an object of the present invention is to provide a method for detecting a diagnostic marker for Hanna type interstitial cystitis, which can specifically detect Hanna type interstitial cystitis, a diagnostic kit for Hanna type interstitial cystitis, and a therapeutic agent for Hanna type interstitial cystitis. [Means for solving the problem]

[0007] The present invention includes the following aspects. [1] A method for detecting a diagnostic marker for Hanna type interstitial cystitis, comprising: A detection method for detecting the amount of at least one microorganism selected from the group consisting of bacteria of the genus Trabulsiella, bacteria of the genus Peptoniphilus, bacteria of the genus Anaerococcus, and bacteria of the genus Anaerobacillus in urine from a subject. [2] The detection method described in [1], which detects 16S ribosomal DNA or 16S ribosomal RNA of the microorganism. [3] The detection method according to [1] or [2], further comprising detecting CXCL10. [4] The detection method according to any one of [1] to [3], wherein a bladder pain score is used. [5] The detection method according to any one of [1] to [4], wherein the urine is from a female. [6] A Hanna-type interstitial cystitis diagnostic kit comprising a primer set for amplifying 16S ribosomal DNA or 16S ribosomal RNA of at least one microorganism selected from the group consisting of bacteria of the genus Trabulsiella, bacteria of the genus Peptoniphilus, bacteria of the genus Anaerococcus, and bacteria of the genus Anaerobacillus, and / or a probe that binds to the 16S ribosomal DNA or 16S ribosomal RNA, or an amplification product thereof. [7] A therapeutic agent for Hanna-type interstitial cystitis, comprising as an active ingredient an antibacterial agent against at least one microorganism selected from the group consisting of bacteria of the genus Trabulsiella, bacteria of the genus Peptoniphilus, bacteria of the genus Anaerococcus, and bacteria of the genus Anaerobacillus. Effect of the Invention

[0008] According to the present invention, Hanna type interstitial cystitis can be specifically detected. [Brief description of the drawings]

[0009] [Figure 1] 1 shows the results of a bacterial flora analysis in an example. [Diagram 2] (Top) Correlation coefficients between the abundance of Peptoniphilus, Anaerococcus, and Anaerobacillus in HIC patients and the abundance of Trabulsiella and Lactobacillus in HIC patients. (Bottom) ROC curve based on the value of Trabulsiella / (Peptoniphilus + Anaerococcus + Anaerobacillus) in HIC patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] <Method for detecting diagnostic markers for Hanna type interstitial cystitis> The method for detecting the Hanna type interstitial cystitis diagnostic marker of the present invention is a method for detecting the amount of at least one microorganism selected from the group consisting of bacteria of the genus Trabulsiella, bacteria of the genus Peptoniphilus, bacteria of the genus Anaerococcus, and bacteria of the genus Anaerobacillus in urine derived from a subject.

[0011] As described later in the Examples, the present inventors performed 16S rRNA bacterial flora analysis using urine from patients with Hanna type interstitial cystitis and found that the abundance of bacteria of the genera Trabulsiella, Peptoniphilus, Anaerococcus, and Anaerobacillus was significantly different from that of healthy individuals.

[0012] Methods for detecting these bacteria are not particularly limited, and include methods using specific antibodies and methods that analyze gene sequences specific to the bacteria. Methods for analyzing bacteria-specific gene sequences include methods for analyzing 16S ribosomal RNA or 16S ribosomal DNA. In particular, a fragment of 16S ribosomal DNA or 16S ribosomal RNA may be amplified by PCR using primers and the amplified product may be analyzed, or a probe complementary to the bacteria-specific 16S ribosomal DNA or 16S ribosomal RNA gene sequence may be used to analyze the gene sequence by hybridization. From the viewpoint of quantitative determination, it is preferable to amplify a fragment of 16S ribosomal DNA or 16S ribosomal RNA by PCR and analyze the amplified product. Specific quantitative determination methods include next-generation sequencing (NGS) and real-time PCR (RT-PCR).

[0013] In a next-generation sequencer, an analyzed DNA fragment is called a read, and the product of the number of reads and the number of bases (read length) determined per read is the output data. In this embodiment, it is preferable to amplify a region in the 16S ribosomal DNA of bacteria of the genus Trabulsiella, Peptoniphilus, Anaerococcus, and Anaerobacillus by singleplex PCR or multiplex PCR, analyze the base sequence of the amplified product by a next-generation sequencer (NGS), and quantify the region by counting the number of reads. Multiplex PCR is a method of simultaneously amplifying multiple gene regions by simultaneously using multiple primer pairs in one PCR reaction system. At least a part of the combination of primers that anneal to the genes of the above-mentioned bacteria may be used in one PCR reaction system.

[0014] In real-time PCR, the amount of amplified product can be monitored by detecting the fluorescence intensity using the intercalator method, the probe method, the cycling probe method, etc. In this embodiment, it is preferable to quantify the regions in the 16S ribosomal DNA of bacteria of the genus Trabulsiella, Peptoniphilus, Anaerococcus, and Anaerobacillus by singleplex or multiplex real-time PCR. In multiplex PCR, at least a part of the combinations of various primers that anneal to the genes of the above-mentioned bacteria may be used in one PCR reaction system.

[0015] In real-time PCR, it is preferable to use a fluorescent dye-labeled probe that is complementary to a bacteria-specific 16S ribosomal DNA gene sequence. Quantitative methods include absolute quantitation, which determines the actual copy number of the target, and comparative quantitation, which determines the relative value between samples. These methods are used depending on the quality of data to be obtained.

[0016] As described later in the Examples, a strong correlation was observed between the abundance of each of the genus Peptoniphilus and Anaerococcus bacteria and the abundance of the genus Trabulsiella bacteria, and therefore it is preferable to use the value of Trabulsiella bacteria / (Peptoniphilus bacteria + Anaerococcus bacteria + Anaerobacillus bacteria) in the diagnosis of Hanna-type interstitial cystitis.

[0017] Hanna type interstitial cystitis is more prevalent among women, so it is preferable that the urine from the subject is from a woman. Patients with Hanna type interstitial cystitis are found in all age groups, but are particularly prevalent in postmenopausal women. As described later in the Examples, the amount of bacteria present differs before and after menopause, so it is preferable to use urine samples separately for diagnosis before and after menopause.

[0018] In the detection method of the present invention, it is preferable to further detect CXCL10. CXCL is an inflammatory chemokine and a non-specific inflammation marker. If the urinary level of CXCL10 is 30 or more, it supports the diagnosis of Hanna type interstitial cystitis.

[0019] In the detection method of the present invention, it is preferable to further use a bladder pain score. The bladder pain score can be the severity scale created by the Japanese Interstitial Cystitis Study Group. Bladder pain is expressed by the NRS (Numerical Rating Scale) which is expressed on an 11-point scale from 0 to 10. In the severity scale created by the Japanese Interstitial Cystitis Study Group, if the bladder pain score is 4 or more out of 11 points from 0 to 10, it is supported that the patient has Hanna type interstitial cystitis.

[0020] Diagnostic kit The Hanna type interstitial cystitis diagnostic kit of the present invention comprises a primer set for amplifying 16S ribosomal DNA or 16S ribosomal RNA of at least one microorganism selected from the group consisting of bacteria of the genus Trabulsiella, bacteria of the genus Peptoniphilus, bacteria of the genus Anaerococcus, and bacteria of the genus Anaerobacillus, and / or a probe that binds to the 16S ribosomal DNA or 16S ribosomal RNA or an amplification product thereof. The diagnostic kit of the present invention is suitably used for the above-mentioned <<Method for detecting diagnostic markers for Hanna type interstitial cystitis>> and comprises the configuration described in <<Method for detecting diagnostic markers for Hanna type interstitial cystitis>>.

[0021] Furthermore, in addition to the above primer set and / or probe, a kit for extracting DNA or RNA from urine may be provided.

[0022] <Hanna type interstitial cystitis treatment> The therapeutic agent for Hanna type interstitial cystitis of the present invention contains, as an active ingredient, an antibacterial agent against at least one type of microorganism selected from the group consisting of bacteria of the genus Trabulsiella, bacteria of the genus Peptoniphilus, bacteria of the genus Anaerococcus, and bacteria of the genus Anaerobacillus.

[0023] Examples of the antibacterial agent include nitroimidazoles, carbapenems, macrolides, new quinolones, cephalosporins, penicillins, quinolones, and new quinolone antibacterial agents.Specific examples of the antibacterial agent include metronidazole, doripenem, azithromycin, sitafloxacin hydrate, cefoperazone, prulifloxacin, piperacillin, garenoxacin, fosfomycin, and pazufloxacin.

[0024] The therapeutic agent for Hanna type interstitial cystitis of the present invention can be administered orally in the form of, for example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., or parenterally in the form of injections, suppositories, topical skin preparations, etc.

[0025] As the pharmaceutically acceptable carrier, those used in ordinary preparations can be used without any particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, etc.; excipients such as starch, crystalline cellulose, etc.; swelling agents such as alginic acid, etc.; solvents for injections such as water, ethanol, glycerin, etc.; adhesives such as rubber-based adhesives, silicone-based adhesives, etc. The pharmaceutically acceptable carriers can be used alone or in combination of two or more.

[0026] The therapeutic agent for Hanna type interstitial cystitis of the present invention may further contain additives, such as lubricants such as calcium stearate and magnesium stearate, sweeteners such as sucrose, lactose, saccharin and maltitol, flavorings such as peppermint and safflower oil, stabilizers such as benzyl alcohol and phenol, buffers such as phosphates and sodium acetate, solubilizers such as benzyl benzoate and benzyl alcohol, antioxidants, and preservatives. The additives may be used alone or in combination of two or more.

[0027] (Administration Method) The method of administration of the Hanna type interstitial cystitis therapeutic agent of the present invention is not particularly limited, and may be appropriately determined according to the symptoms, body weight, age, sex, etc. of the patient. For example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc. are orally administered. Injections are administered intravenously alone or mixed with normal replacement fluids such as glucose and amino acids, and further administered intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally as necessary. Alternatively, they are administered locally to the bladder. Intravesical administration includes two types: intravesical injection therapy and intravesical wall injection therapy. The latter is a method of injecting a drug transurethrally using a special needle with an endoscope. Suppositories are administered rectally. Skin external preparations are applied, pasted, or sprayed on the affected area.

[0028] (Dosage) The dosage of the Hanna type interstitial cystitis therapeutic agent of the present invention varies depending on the symptoms, body weight, age, sex, etc. of the patient and cannot be determined in general, but in the case of oral administration, for example, 1 μg to 10 g, for example, 0.01 to 2000 mg of active ingredient per day may be administered. In addition, in the case of injection, for example, 0.1 μg to 1 g, for example, 0.001 to 200 mg of active ingredient per day may be administered. In addition, in the case of suppository, for example, 1 μg to 10 g, for example, 0.01 to 2000 mg of active ingredient per day may be administered.

[0029] (Treatment method) The Hanna type interstitial cystitis treatment method of the present invention includes local treatment by transurethral Hanna lesion electric cauterization and treatment of areas other than the Hanna lesion by administration of the Hanna type interstitial cystitis treatment agent. Local treatment by electric cauterization alone does not completely cure the disease, so administration of the Hanna type interstitial cystitis treatment agent provides a more efficient therapeutic effect. EXAMPLES

[0030] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0031] Urine samples were collected from 20 premenopausal healthy subjects, 10 premenopausal HIC patients, 20 postmenopausal healthy subjects, and 20 postmenopausal HIC patients. Urine aliquots were pelleted by centrifugation, and total DNA was isolated from the pellet using the DNeasy Blood & Tissue kit (QIAGEN). The isolated DNA was sequenced using a next-generation sequencer after amplifying 16S rDNA sequences using a primer set specific to the V1-V2 hypervariable region. Data analysis was performed using statistical analysis software (Prism, GraphPad Software, San Diego, CA, USA). All microorganisms at the phylum level were compared, those with low relative abundance (<0.1%) at the genus level were filtered, and the remaining top 50 microorganisms were analyzed. The results are shown in Figure 1. As shown in Figure 1, premenopausal HIC patients had higher abundances of Peptoniphilus and Anaerococcus than premenopausal healthy controls, postmenopausal HIC patients had higher abundances of Anaerococcus than postmenopausal healthy controls, and postmenopausal HIC patients had lower abundances of Trabulsiella than postmenopausal healthy controls.

[0032] Based on the results of Figure 1, the correlation coefficients between the abundance of each of Peptoniphilus, Anaerococcus, and Anaerobacillus and the abundance of each of Trabulsiella and Lactobacillus in HIC patients were calculated. The results are shown in the upper part of Figure 2. A strong correlation was observed between the abundance of each of Peptoniphilus and Anaerococcus and the abundance of Trabulsiella.

[0033] Based on the results in Figure 1, the value of Trabulsiella / (Peptoniphilus+Anaerococcus+Anaerobacillus) in HIC patients was calculated, and an ROC curve was created. The results are shown in the lower part of Figure 2. The AUC was 0.77. [Industrial Applicability]

[0034] According to the present invention, Hanna type interstitial cystitis can be specifically detected.

Claims

1. A method for detecting a diagnostic marker for Hanna type interstitial cystitis, comprising: A detection method for detecting the amount of at least one microorganism selected from the group consisting of bacteria of the genus Trabulsiella, bacteria of the genus Peptoniphilus, bacteria of the genus Anaerococcus, and bacteria of the genus Anaerobacillus in urine derived from a subject.

2. The detection method according to claim 1 , wherein 16S ribosomal DNA or 16S ribosomal RNA of the microorganism is detected.

3. The method according to claim 1 or 2, further comprising detecting CXCL10.

4. The detection method according to any one of claims 1 to 3, which uses a bladder pain score.

5. The detection method according to any one of claims 1 to 4, wherein the urine is from a female.

6. A diagnostic kit for Hanna type interstitial cystitis, comprising a primer set for amplifying 16S ribosomal DNA or 16S ribosomal RNA of at least one microorganism selected from the group consisting of bacteria of the genus Trabulsiella, bacteria of the genus Peptoniphilus, bacteria of the genus Anaerococcus, and bacteria of the genus Anaerobacillus, and / or a probe that binds to the 16S ribosomal DNA or 16S ribosomal RNA, or an amplification product thereof.

7. A therapeutic agent for Hanna-type interstitial cystitis, comprising as an active ingredient an antibacterial agent against at least one microorganism selected from the group consisting of bacteria of the genus Trabulsiella, bacteria of the genus Peptoniphilus, bacteria of the genus Anaerococcus, and bacteria of the genus Anaerobacillus.

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