Method for predicting cause-specific therapeutic effect of sensorineural hearing loss and diagnostic kit used therefor

Abstract

The present disclosure relates to a kit and information providing method capable of predicting or diagnosing the effect of a cause-specific treatment for sensorineural hearing loss, and more specifically, a kit and method according to one embodiment of the present invention enable the effect of a cause-specific treatment for sensorineural hearing loss to be diagnosed early and an optimal treatment method to be selected.

Description

Technical Field

[0001] This specification discloses a method for providing information for predicting or diagnosing a causative-specific therapeutic effect of sensorineural hearing loss and a diagnostic kit used therefor.

[0002] [National Research and Development Project that Supported the Present Invention] Problem Specific Number: 1711114924 Problem Number: 2018R1A2B2001054 [Department Name] Ministry of Science and ICT, Republic of Korea [Name of the Agency Responsible for Problem Management (Specialty)] National Research Foundation of Korea [Name of Research Project] Individual Basic Research (Ministry of Science and ICT) (R&D) [Name of Research Problem] Establishment of a spectrum of self-inflammatory hereditary hearing loss treatable with drugs: Discovery of genetic biomarkers and exploration of therapeutic effective substances [Contribution Rate] 1 / 1 [Name of the Agency Implementing the Problem] Pyeongtaek Seoul National University Hospital [Research Period] March 1, 2018 ~ February 28, 2021

Background Art

[0003] Gradually progressing sensorineural hearing loss (SNHL) is a widespread sensory defect. From sudden sensorineural hearing loss that deteriorates within 2 to 3 days after onset to hearing loss that deteriorates subacutely over several months longer than that period, there are various etiologies, and it can be said to be a syndrome rather than a single disease.

[0004] Since it is difficult to identify the cause of such sensorineural hearing loss, steroids are generally used systemically or locally as a common treatment. However, steroid treatment that is not cause-specific often does not achieve sufficient effects and has limitations such as showing resistance to steroid treatment itself.

[0005] Against such a background, the present inventors examined various etiologies of sensorineural hearing loss, studied potential biomarkers in the progression of sensorineural hearing loss and predictors for efficient treatment, and completed the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0006] In one aspect, an object of the present invention is to provide a method for providing information for predicting or diagnosing a cause-specific treatment effect of sensorineural hearing loss.

[0007] In another aspect, an object of the present invention is to provide a kit for predicting or diagnosing a cause-specific treatment effect of sensorineural hearing loss.

Means for Solving the Problems

[0008] In one aspect, the present invention provides a method for providing information for predicting or diagnosing a cause-specific treatment effect of sensorineural hearing loss, the method including: (a) inducing the secretion of IL-1β from a sample isolated from an individual; (b) measuring the secretion pattern of IL-1β induced in step (a); and (c) comparing the secretion pattern of IL-1β measured in step (b) with a normal individual sample.

[0009] In another aspect, the present invention provides a kit for predicting or diagnosing a cause-specific treatment effect of sensorineural hearing loss, the kit including an agent for inducing the secretion of IL-1β and an agent for measuring the secretion pattern of IL-1β.

Effects of the Invention

[0010] In one aspect, the method or kit according to an embodiment of the present invention is excellent in the effect of predicting or diagnosing a cause-specific treatment effect of sensorineural hearing loss.

[0011] In one aspect, the method or kit according to an embodiment of the present invention can be clinically used to determine treatment by diagnosing the cause-specific treatment effect of sensorineural hearing loss at an early stage and selecting an optimal treatment method.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0013] Hereinafter, the present invention will be described in detail.

[0014] In one aspect, the present invention provides a method for providing information for predicting or diagnosing a cause-specific therapeutic effect of sensorineural hearing loss, comprising: (a) inducing the secretion of IL-1β from a sample isolated from an individual; (b) measuring the secretion pattern of IL-1β induced in step (a); and (c) comparing the secretion pattern of IL-1β measured in step (b) with a normal individual sample. In one aspect of the present invention, "predicting or diagnosing a cause-specific therapeutic effect of sensorineural hearing loss" may mean predicting or diagnosing what the causative factor of sensorineural hearing loss is for a subject, or whether the subject's sensorineural hearing loss is effective for treatment with an antagonist of IL-1β. In one aspect of the present invention, the method or kit can be clinically used to determine treatment by diagnosing the cause-specific therapeutic effect of sensorineural hearing loss at an early stage and selecting an optimal treatment method.

[0015] In one embodiment, step (a) may induce the secretion of IL-1β using at least one selected from the group consisting of LPS, ATP, and CaCl2.

[0016] In one embodiment, the sample in step (a) may be whole blood, peripheral blood mononuclear cells (PBMCs) extracted from whole blood, serum, or saliva.

[0017] In one embodiment, in step (b), the measurement of the secretion pattern of IL-1β may be performed using a rapid test kit such as an enzyme-linked immunosorbent assay (ELISA), RT-PCR, or a rapid antigen test kit. Here, the measurement of the secretion pattern of IL-1β includes qualitatively measuring the presence or absence of the secretion of IL-1β or quantitatively measuring the secretion level of IL-1β.

[0018] In another aspect, the present invention provides a kit for predicting or diagnosing a causative-specific therapeutic effect of sensorineural hearing loss, comprising an IL-1β secretion-inducing agent and an agent for measuring the secretion pattern of IL-1β.

[0019] In one embodiment, the IL-1β secretion-inducing agent may include at least one selected from the group consisting of LPS, ATP, and CaCl2.

[0020] In one embodiment, the agent for measuring the secretion pattern of IL-1β may include at least one selected from the group consisting of primers, probes, and antibodies.

[0021] As used herein, the term "primer" means a polynucleotide having bases of a sequence that can complementarily bind to the ends of a specific region of a gene, which is used to amplify a specific region corresponding to a target site of the gene using PCR, or a variant thereof. The primer does not necessarily have to be completely complementary to the end of the specific region, and may be used as long as it is complementary enough to hybridize to the end to form a double-stranded structure.

[0022] As used herein, the term "probe" means a polynucleotide having bases of a sequence that can complementarily bind to a target site of a gene, a variant thereof, or a polynucleotide and a labeling substance bound thereto.

[0023] As used herein, the term "hybridization" means that two single-stranded nucleic acids form a dimer structure by pairing of complementary base sequences. Hybridization can occur not only when the complementarity between the sequences of single-stranded nucleic acids is perfect, but also when there are some mismatched bases.

[0024] In one embodiment, the antibody may be at least one selected from the group consisting of polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and combinations thereof. Specifically, the antibody includes not only polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and the complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules, such as Fab, F(ab’), F(ab’)2, and Fv. Antibody production can be easily manufactured using techniques well known in the art to which the present invention pertains, and commercially available antibodies may also be used.

[0025] In one embodiment, the kit according to one aspect of the present invention may further include not only the IL-1β secretion-inducing agent and the agent for measuring the IL-1β secretion pattern, but also a label capable of quantitatively or qualitatively measuring the formation of an antigen-antibody complex, ordinary tools, reagents, etc. used in immunological analysis.

[0026] In one embodiment, examples of the label that enables qualitative or quantitative measurement of the formation of the antigen-antibody complex include, but are not necessarily limited to, enzymes, phosphors, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes. Enzymes that can be used as detection labels include β-glucuronidase, β-glucosidase, β-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, β-lactamase, etc., and are not limited thereto. Fluorescent substances include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, etc., and are not limited thereto. Examples of the ligand include, but are not limited to, biotin derivatives. Luminescent substances include acridinium ester, luciferin, luciferase, etc., and are not limited thereto. Microparticles include colloidal gold, colored latex, etc., and are not limited thereto. Redox molecules include ferrocene, ruthenium complex compounds, viologen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, hydroquinone, K4W(CN) 8 , [Os(bpy)3] 2+ , [RU(bpy)3] 2+ , [MO(CN)8] 4- , etc., and are not limited thereto. Radioisotopes include 3 H, 14 C,32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 There are, for example, Re and the like, but not limited thereto.

[0027] In one embodiment, examples of the tool or reagent include, but are not limited to, a suitable carrier, solvent, detergent, buffer, stabilizer, etc. When the labeling substance is an enzyme, it may include a substrate and a reaction terminator capable of measuring enzyme activity. The carrier includes a soluble carrier and an insoluble carrier. Examples of the soluble carrier include a physiologically acceptable buffer known in the art, such as PBS. Examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharide, and other papers, glass, metal, agarose, and combinations thereof.

[0028] In one embodiment, the kit may include a test unit for inducing the secretion of IL-1β from a sample separated from an individual, a measurement unit for measuring the secretion pattern of the induced IL-1β, and an analysis unit for comparing the measured secretion pattern of IL-1β with a normal individual sample.

[0029] In one embodiment, the test unit may induce the secretion of IL-1β from the sample with an IL-1β secretion-inducing agent such as, for example, LPS, ATP, CaCl2. In that case, the sample may be whole blood, PBMC extracted from whole blood, serum, or saliva.

[0030] In one embodiment, the kit may further include a PBMC separation and extraction unit.

[0031] In one embodiment, the measurement unit may measure the secretion pattern of IL-1β, for example, by ELISA, RT-PCR, rapid antigen test, or the like.

[0032] In one embodiment, the kit may further include a display unit that shows the difference in the secretion pattern of IL-1β or the change in the secretion ratio before and after inducing the secretion of IL-1β.

[0033] In one embodiment, the kit may further include an autoimmune disease or FTA-ABS (fluorescent treponemal antibody absorption test) test unit. Examples of the autoimmune disease include, but are not limited to, Granulomatosis with polyangiitis, Cogan syndrome, polyarteritis nodosa, and the like.

[0034] Hereinafter, the configuration and effects of the present invention will be described more specifically with reference to examples. However, the following examples are provided to assist in the understanding of the present invention, and the scope and range of the present invention are not limited thereby.

Examples

[0035] Selection of Subjects Seventeen clinically diagnosed patients with cryopyrin-associated periodic syndrome (CAPS) and two patients classified as having autoinflammatory type hearing loss (AIHL) were enrolled as subjects in the study according to an embodiment of the present invention. The subjects were examined and their symptoms were characterized to evaluate whether they had chronic infantile, neurological, cutaneous and articular (CINCA) syndrome, Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), or DFNA34 (nonsyndromic SNHL). Furthermore, two "seemingly AIHL" patients without a family history were enrolled for comparison with CAPS patients.

[0036] Written consent was obtained from all subjects, and in the case of minors, written consent was obtained from their parents or guardians. All steps of this study were approved by the institutional review boards of Seoul National University Hospital and Bundang Seoul National University Hospital.

Example

[0037] Diagnosis of the causative treatment effect of sensorineural hearing loss in subjects Clinical data including the gender, age, medical history, physical examination, and audiometry results of the subjects selected in Example 1 were obtained. The audiometric threshold was calculated as the average of the thresholds at 0.5, 1, 2, and 4 kHz, and the audiometric levels were classified into the following four categories: mild (26 - 40 dB), moderate (41 - 55 dB), moderately severe (56 - 70 dB), severe (71 - 90 dB), and profound (> 90 dB). The specific method for obtaining the above clinical data is as follows.

[0038] Molecular genetic diagnosis Genomic DNA was extracted from the peripheral blood or buccal swabs of the subjects according to the manufacturer's protocol. Subsequently, the entire NLRP3 gene was screened to confirm the causative mutation. When potential mutation candidates were identified by NLRP3 screening, segregation studies were performed for genetic diagnosis. When NLRP3 mutations that were potentially pathogenic were not detected, exome analysis was performed to examine other possible candidate genes, followed by a filtering process by bioinformatics analysis.

[0039] Clinical evaluation: Examination of hearing and radiological data Clinical features were documented, and two experienced pediatric rheumatologists and two otolaryngologists performed physical examinations to diagnose CAPS. Audiological evaluations were performed according to test eligibility (varying by age): pure tone audiometry and / or auditory brainstem response, and / or auditory steady-state response. Inner ear canal protocol MRI including FLAIR sequence was performed to evaluate whether tumorous conditions or inflammation were present in the cerebellopontine angle, inner ear canal, or cochlea.

[0040] Statistical analysis Statistical analysis was performed using Prism v.8.0 software for Windows (GraphPad Software, Inc., San Diego, CA, USA) and Statistics v.24 (IBM, Armonk, NY, USA). Fisher's exact test was used to determine the association between the improvement of the cochlea on brain MRI and the auditory outcomes. The Kruskal-Wallis test was used to compare the secretion of IL-1β according to the medical conditions of each individual (normal control group, DFNA34, AIHL) for LPS and LPS+CaCl2 treatments, and Bonferroni adjustment was performed for post hoc testing. P<0.05 was considered statistically significant.

[0041] Genotypic characteristics of autoinflammatory hearing loss patients The genotypic and phenotypic characteristics of all subjects with clinically diagnosed CAPS or DFNA34 are shown in Figure 1. Among 19 subjects, genetic diagnosis was performed in 18 subjects (94.7%), and three new mutations in NLRP3 were discovered, and one mutation that occurred twice was discovered from two genetically unrelated subjects (c.1217T>C, Case 5 and 9). Furthermore, another mutation, c.1709A>G, was discovered from two unrelated subjects (Case 2 and 6). In two other families, autosomal dominant inheritance of NLRP3 mutations from mother to child was observed (Case 15 and 17), one of which was CINCA syndrome (Case 15) and the other was non-syndromic (DFNA34) (Case 17).

[0042] Audiological phenotypes as potential biomarkers for predicting disease severity and treatment response Among the 19 subjects, 2 (Case1 (FCAS) and 4 (CINCA syndrome)) had never undergone a hearing test, and 7 (Cass5, 8, 11, 12, 13-1, 15 and 16) had overall normal hearing thresholds. However, 4 out of the 7 (Case5, 8, 13-1, 15) showed mild hearing loss restricted to high frequencies. Thus, it can be seen that in CAPS patients, the high-frequency hearing is the most impaired. Interestingly, 4 subjects (Case3, 5, 6 and 13-2) showed asymmetric hearing loss (>15 dB difference between the right and left ears). Thirteen subjects with available audiometric and laboratory data were analyzed for hearing phenotypes related to inflammatory markers (Case2~3, Case5~14). Changes in hearing thresholds and inflammatory markers, including ESR and CRP, were plotted in the time domain (clinic visits) focused on the use of anakinra, an antagonist of IL-1β, to examine the role of hearing thresholds as potential biomarkers for disease progression and response to anti-IL1 therapy (Figures 2A~2M). In contrast to the immediate and consistent response of inflammatory markers to treatment, hearing thresholds showed differential responses to anakinra. Specifically, 7 genetically confirmed NLRP3-related syndrome patients (Case5, 6, 8, 9, 11, 12 and 13-1) initially started with normal or mild hearing loss and showed a stable or slightly improved hearing status in response to anakinra therapy. Also, the MWS subject (case14) with initially severe SNHL showed a gradual improvement in hearing despite the delayed anakinra treatment. In summary, 3 subjects with NLRP3-related CAPS showed a clear improvement in their hearing status in response to anakinra, from mild hearing loss to normal (Case11), from moderate to mild hearing loss (Case6), and from severe to reduced severe hearing loss (Case14).

[0043] Conversely, the hearing thresholds of one patient with CINCA syndrome (Case 2) and one patient with MWS (Case 7), who initially belonged to the moderate hearing loss range, deteriorated to severe hearing loss despite anakinra treatment and ultimately required CI. Another patient (Case 3) who showed obvious manifestations of CINCA syndrome but had no clear pathogenic NLRP3 mutations initially showed mild hearing loss and then deteriorated to moderate hearing loss despite continued anakinra treatment. More interestingly, two monozygotic twins (Case 13-1 and 13-2) with CINCA syndrome due to the same NLRP3 mutation showed different auditory phenotypes and various responses to anakinra treatment. Specifically, unlike Case 13-1, whose auditory status remained stable over the follow-up period, Case 13-2 showed a gradual deterioration of unilateral hearing even after receiving anakinra treatment.

[0044] ELISA analysis of IL-1β in cultured PBMC PBMCs were collected from peripheral venous blood samples of four subjects (normal control group 1 (NC01), Case 17-1 and 17-2, AIHL2). Plastic adherent PBMCs were stored in a -80 °C freezer container and cultured in a 12-well culture plate (2×10 6 cells per well) with serum-free RPMI medium for 20 minutes. The medium was replaced with 1 mL of RPMI containing 10% FBS with or without LPS over 3 hours. Then, the medium was replaced with 500 μL of serum-free RPMI with or without 1 mM CaCl2 for 60 minutes. The sample supernatant was collected, and the absorbance was measured at 450 nm using an ELISA kit for IL-1β (BMS224-2, Invitrogen, Carlsbad, CA, USA) to analyze the samples. The analysis results were calculated as multiples normalized by the normal control group (NC01) and are shown in Figure 3.

[0045] Serum cytokine measurement (ELISA analysis) Under the following three conditions, the secretion levels of IL-1β in cultured PBMCs were compared among the control group (NC-01), the non-syndromic autoinflammatory hearing loss group (DFNA34), and the AIHL subjects (AIHL 2): without stimulation, stimulation with LPS, or stimulation with LPS + CaCl2. In Case17-2, the levels of IL-1β when stimulated with LPS were significantly higher than those in NC01 and Case17-1 (P = 0.008 and P = 0.016, respectively). Similarly, the secretion of IL-1β in Case17-2 in response to LPS + CaCl2 was higher than that in NC01 (P = 0.031 by Kruskal-Wallis test and Bonferroni correction) (Figure 3).

[0046] Overall, according to an embodiment of the present invention, in the case of sensorineural hearing loss with over-secretion of IL-1β compared to the normal control group, the hearing ability was improved in response to the treatment with an antagonist of IL-1β. Thereby, it was found that by using the method or kit according to an embodiment of the present invention, it is possible to predict or diagnose whether the cause of sensorineural hearing loss is due to NLRP3 mutation or whether the sensorineural hearing loss of the subject shows an effect on the treatment with an antagonist of IL-1β.

Claims

1. (a) inducing the secretion of IL-1β from a sample isolated from an individual; (b) measuring the secretion pattern of IL-1β induced in step (a); (c) comparing the secretion pattern of IL-1β measured in step (b) with a normal individual sample, wherein the individual is a sensorineural hearing loss individual having excessive secretion of IL-1β compared to a normal individual, when the hearing level of the individual before administration of an IL-1β antagonist is 60 dB or less, it is predicted that the hearing level after administration of the IL-1β antagonist is better than 60 dB, A method for providing information for predicting or diagnosing a cause-specific therapeutic effect of sensorineural hearing loss.

2. Step (a) induces the secretion of IL-1β using at least one selected from the group consisting of LPS, ATP, and CaCl ２ The method according to claim 1, wherein the secretion of IL-1β is induced using at least one selected from the group consisting of LPS, ATP, and CaCl

3. The method according to claim 1, wherein the sample in step (a) is whole blood, PBMC extracted from whole blood, serum or saliva.

4. The method according to claim 1, wherein in step (b), the measurement of the secretion pattern of IL-1β is performed by ELISA, RT-PCR or rapid antigen test.

5. comprising an agent for inducing the secretion of IL-1β and an agent for measuring the secretion pattern of IL-1β, wherein the individual is a sensorineural hearing loss individual having excessive secretion of IL-1β compared to a normal individual, when the hearing level of the individual before administration of an IL-1β antagonist is 60 dB or less, it is predicted that the hearing level after administration of the IL-1β antagonist is better than 60 dB, A kit for predicting or diagnosing a cause-specific therapeutic effect of sensorineural hearing loss.

6. The IL-1β secretion-inducing agent contains at least one selected from the group consisting of LPS, ATP, and CaCl ２ The kit according to claim 5, which comprises at least one selected from the group consisting of ２ .

7. The kit according to claim 5, wherein the agent for measuring the secretion pattern of IL-1β comprises at least one selected from the group consisting of primers, probes and antibodies.

8. The kit comprises a test section for inducing the secretion of IL-1β from a sample isolated from an individual, a measurement section for measuring the secretion pattern of the induced IL-1β, and an analysis section for comparing the measured secretion pattern of IL-1β with a normal individual sample, for predicting or diagnosing a cause-specific therapeutic effect of sensorineural hearing loss according to claim 5.

9. The kit according to claim 8, further comprising a display section showing a change in the difference or ratio of the secretion pattern of IL-1β before and after inducing the secretion of IL-1β.

10. The kit according to claim 8, further comprising a PBMC separation and extraction section.

11. The kit according to claim 8, further comprising an autoimmune disease or an FTA-ABS test section.

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