WO2022255782A1 - Biomarkers for predicting response to treatment of rheumatoid arthritis - Google Patents
Biomarkers for predicting response to treatment of rheumatoid arthritis Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to lipid biomarkers for predicting drug treatment responsiveness in rheumatoid arthritis.
- Lipidomics is a field of study that focuses on qualitatively and quantifying the overall phenomena and changes that occur in the metabolic process of various lipidomes that occur in vivo to find out their biological and biochemical significance. It is also aimed at research to find disease-related biomarkers. In particular, with the recent rapid development of electrospray ionization-tandem mass spectrometry (ESI-MS-MS), structural analysis of lipid molecules has become possible, and the field of shotgun lipidomics can also be used for analysis of biological tissues.
- ESI-MS-MS electrospray ionization-tandem mass spectrometry
- rheumatoid arthritis is a representative autoimmune disease, caused by inflammation of a tissue called the synovium surrounding the joint, and is known to develop in about 1-2% of the total population (Alamanosa and Drosos, Autoimmun. Rev., 4:130-136 (2005)). It is known that the onset of rheumatoid arthritis is affected by genetic-environmental factors, and that genetic factors contribute about 60%.
- rheumatoid arthritis initially affects the smaller joints, such as those of the wrists, hands, ankles, and feet, and as the disease progresses, may also affect the joints of the shoulders, elbows, knees, hips, jaw, and neck; If disease activity is not controlled, irreversible joint deformity and disability occur over time. Also, unlike other arthritic conditions that only affect areas within or around the joints, rheumatoid arthritis is a systemic disease that can cause inflammation in tissues outside the joints throughout the body, including the skin, blood vessels, heart, lungs and muscles. .
- a drug treatment method in which analgesic anti-inflammatory drugs are administered in combination with various anti-rheumatic drugs is generally used to minimize joint damage, prevent functional loss, and reduce pain, and recently, biological treatments have been developed. It is used as a combination treatment along with anti-rheumatic drugs, and surgical therapy is performed when the severity of the disease is severe.
- Biologic disease-modifying anti-rheumatic drugs (bDMARDs), which have recently emerged as biologic treatments, include treatments that are indispensable for the treatment of rheumatoid arthritis, but conventional markers (especially CRP) are not effective in treating rheumatoid arthritis. Research results have been reported that the value is lowered regardless of whether or not it is mitigated. Therefore, it is essential to develop a new biomarker capable of accurately diagnosing the response (disease activity of arthritis) after treatment of rheumatoid arthritis.
- An object of the present invention is to provide a biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis.
- Another object of the present invention is to provide a kit for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis.
- Another object of the present invention is to provide a method for evaluating responsiveness to a therapeutic agent for rheumatoid arthritis.
- an object of the present invention is to provide an information providing method for predicting the prognosis of rheumatoid arthritis.
- the present invention provides acyl carnitine, ceramide sphingosine, lysophosphatidylcholine, ether-bridged LPC, phosphatidylcholine, ether-bridged PC, phosphatidylethanolamine, ether-bridged phosphatidylethanolamine, sphingomyelin and triacylglycerol. It provides a biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis, comprising one or more lipids selected from the group consisting of.
- the present invention provides a kit for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis.
- the present invention provides a method for evaluating responsiveness to a therapeutic agent for rheumatoid arthritis.
- the present invention provides an information providing method for predicting the prognosis of rheumatoid arthritis.
- Lipid biomarkers selected through lipidomics analysis in the present invention have specific expression levels that change according to reactivity after drug treatment, so they are useful for evaluating responsiveness to rheumatoid arthritis drugs and predicting the prognosis of rheumatoid arthritis There is an effect that can be used wisely.
- Figure 1 is a heatmap showing the expression of 37 significant lipids identified after comparison of samples obtained before and after treatment (samples compared using t-test days).
- Figure 2 is a diagram estimating the difference in lipid expression between good treatment responders / bad treatment responders using RHT (Regularized Hotelling's T 2 ) test.
- lipids are marked according to the system name and common name (IUPAC-IUBMB) according to world standards for applied chemistry, biochemistry, and molecular biology in Lipidomics.
- system name and common name IUPAC-IUBMB
- the present invention provides acyl carnitine (CAR), ceramide sphingosine (Cer-NS), lysophosphatidylcholine (LPC), ether-linked LPC (ether-linked LPC, LPC O), Phosphatidylcholine (PC), ether-linked phosphatidylcholine (PC O), phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (PE P), sphingomyelin ( It relates to a biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis, comprising at least one lipid selected from the group consisting of sphingomyelin (SM) and triacylglycerol (TG).
- SM sphingomyelin
- TG triacylglycerol
- the acyl carnitine is 12:0, 14:1 and 16:1, the ceramide sphingosine is 42:1 and the lysophosphatidylcholine is 16:0, 16:1, 18:1, 18:2, 18 :3, 20:1, 20:2, 20:3, 20:4, 20:5, 22:5, 22:6 or 24:0, and the ether-bridged LPC is 16:1, 18:0 or 24 :1, and phosphatidylcholine is 32:3, 34:2, 34:5, 36:1, 36:5 (1), 36:6 (1), 40:1, 40:4, 42:10 or 42: 6, the ether-bridged PC is 36:3, the phosphatidylethanolamine is 36:1 or 40:7, the ether-bridged phosphatidylethanolamine is 34:2, 36:3 or 40:6, and the sphingomyelin is 38:1 (1) or 38:1 (2), and triacylglycerols may be 48:5 (2), 50:6
- the treatment for rheumatoid arthritis may be disease modifying anti-rheumatic drugs (DMARDs) or may be biological DMARDs (bDMARDs).
- DMARDs disease modifying anti-rheumatic drugs
- bDMARDs biological DMARDs
- biomarker used in the present invention refers to an index that can detect changes in the body using proteins, DNA, RNA, metabolites, etc. The degree of response to the drug can be objectively measured. Biomarkers are being used to diagnose various incurable diseases such as cancer, stroke, and dementia, and in the present invention, they can be used to determine responsiveness after treatment of rheumatoid arthritis or to determine the prognosis of rheumatoid arthritis after treatment. In addition, biomarkers can be used in the new drug development process, and through this, the safety of the new drug can be secured and the cost required in the new drug development process can be reduced.
- the present invention relates to a kit for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis, comprising a lipid biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis.
- the kit may further include tools and/or reagents for collecting a biological sample from a subject or patient as well as tools and/or reagents for isolating lipids from the sample.
- the term "kit for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis” refers to a kit containing a lipid biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis of the present invention. Therefore, the expression “kit” can be used interchangeably or interchangeably with “composition”.
- diagnosis refers to determining a subject's susceptibility to a specific disease or disorder, determining whether a subject currently has a specific disease or disorder, or suffering from a specific disease or disorder Determining a subject's prognosis (eg, determining the responsiveness of rheumatoid arthritis to treatment), or therametrics (eg, monitoring a subject's condition to provide information about treatment efficacy) includes
- the term "reactive diagnostic biomarker, diagnostic biomarker, or diagnostic marker” is a substance capable of diagnosing reactivity after treatment of rheumatoid arthritis, and a lipid whose expression is increased or decreased compared to before treatment.
- biomarkers for diagnosing responsiveness to therapeutic agents for rheumatoid arthritis include CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 ( 1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P -34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) and TG
- the present invention provides (a) acyl carnitine, ceramide sphingosine, lysophosphatidylcholine ether-bridged LPC, phosphatidylcholine, ether-bridged PC, phosphatidylethanolamine, ether-bridged phosphatidylethanolamine in a biological sample isolated from a test subject, Measuring the expression level of one or more lipids selected from the group consisting of sphingomyelin and triacylglycerol; And (b) it relates to a method for evaluating the responsiveness to a therapeutic agent for rheumatoid arthritis, comprising determining the responsiveness to the therapeutic agent for rheumatoid arthritis.
- Cer-NS 42:1, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3 in a sample from a subject LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32: 3, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PE 36 :1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 ( 2)
- the concentrations of TG 54:9 and TG 54:9 were upregulated compared to the expression level in the sample before treatment, and the concentrations of CAR 12:0, CAR 14:1, and CAR 16:1 were decreased compared to the expression level in the sample before treatment.
- the term "disease activity score with 28-joint assessment (DAS28)" is a rheumatoid arthritis activity evaluation using 28 joints, and is an evaluation method that is currently widely used.
- the new criteria based on DAS28, which reflects disease activity, are in line with the treatment guidelines of developed countries in Europe and America, and are expected to contribute to the appropriate drug use of patients with rheumatoid arthritis who require the administration of anti-TNF drugs.
- the joints included in the DAS28 are the PIP joint (10), the MCP joint (10), the arm joint (2), the elbow joint (2), the shoulder joint (2), and the knee joint (2).
- the biological sample may be blood or serum.
- the expression level (concentration) of a lipid biomarker in a sample can be measured using any method known to those skilled in the art.
- Methods for measuring lipids include, but are not limited to, mass spectrometry, spectroscopic methods including soft ionization techniques for mass spectrometry, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). It doesn't work.
- the present invention provides (a) acyl carnitine, ceramide sphingosine, lysophosphatidylcholine ether-bridged LPC, phosphatidylcholine, ether-bridged PC, phosphatidylethanolamine, ether-bridged phosphatidylethanolamine in a biological sample isolated from a test subject, Measuring the expression level of one or more lipids selected from the group consisting of sphingomyelin and triacylglycerol; and
- (b) it relates to a method of providing information for predicting the prognosis of rheumatoid arthritis, comprising the step of determining the prognosis of rheumatoid arthritis by comparing the expression level of lipids in a sample of a subject with the expression level of a normal sample.
- Cer-NS 42:1, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3 in a sample from a subject LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32: 3, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PE 36 :1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 ( 2)
- the concentrations of TG 54:9 and TG 54:9 were upregulated compared to the expression level in the sample before treatment, and the concentrations of CAR 12:0, CAR 14:1, and CAR 16:1 were decreased compared to the expression level in the sample before treatment.
- the CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18: 2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O- 16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P -36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) and TG 54:9 are therapeutics If there is no change in the expression level compared to the expression level in the sample before treatment, it can be judged
- the biological sample may be blood or serum.
- the expression level (concentration) of a lipid biomarker in a sample can be measured using any method known to those skilled in the art.
- Methods for measuring lipids include, but are not limited to, mass spectrometry, spectroscopic methods including soft ionization techniques for mass spectrometry, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). It doesn't work.
- prognosis refers to the degree of joint deformity (joint damage) after treatment for rheumatoid arthritis, or the rate of progression of joint destruction caused by rheumatoid arthritis, that is, the increase or decrease in the severity of rheumatoid arthritis.
- the term “detection” or “measurement” means to quantify the concentration of a detected or measured object, and to measure “or”measurement” is a qualitative or qualitative concentration level of a given substance in a sample. It means assessing the presence, absence, quantity or amount (which may be an effective amount) of such a substance, including the derivation of, or otherwise assessing the value or categorization of a subject's clinical parameter.
- determining the level of a biomarker can be performed on an untreated or unfractionated sample, and the level of a lipid biomarker in an untreated or unfractionated sample obtained from a subject can be determined.
- determining the level of a biomarker may be quantitative or semi-quantitative.
- quantitative determination may include determining the absolute amount or concentration (expression level) of one or more lipid metabolites.
- Quantitative determination can also include determining the relative amount or concentration of one or more lipid metabolites relative to one or more other metabolites.
- the term "subject” or "patient” refers to any single individual in need of treatment, including humans, apes, monkeys, cows, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects participating in clinical research trials who do not show any clinical signs of disease or subjects participating in epidemiological studies or subjects used as controls. In addition, in the present invention, it refers to a mammal, preferably a human.
- biological sample means a biological sample obtained from a subject or patient, and encompasses various types of samples obtained from organisms that can be used for diagnosis or monitoring assays.
- the term encompasses blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens, or tissue cultures or cells derived therefrom and from their progeny.
- the term specifically covers clinical samples, and further includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluids and tissue samples.
- a biological sample may be a sample of bodily fluid or body tissue of a subject.
- a biological sample can be a sample of blood, plasma, serum, saliva, bile, urine, feces, or cerebrospinal fluid from a subject, or a sample derived from a cell, tissue, or organ from a subject.
- a variety of techniques are available for obtaining biological samples.
- lipids were collected from the serum of patients with rheumatoid arthritis (Active RA) with high disease activity before treatment, serum before treatment, and serum after 6 months of treatment. Separated and analyzed lipid bodies changed before and after treatment. Specifically, study participants were enrolled in the Center for Integrative Rheumatoid Transcriptomics and Dynamics (CIRAD) cohort (2015), and RA patients were defined as meeting the 2010 ACR/EULAR RA classification criteria (6). These patients were followed until March 2021 for definitive RA development.
- CIRAD Integrative Rheumatoid Transcriptomics and Dynamics
- Serum was collected from each participant at the time of cohort enrollment, and to confirm the lipid body changes according to the treatment results, a group showing high disease activity 6 months after treatment with DMARDs (disease modifying anti-rheumatic drugs) (DAS28 ⁇ 3.2) and a group with low disease activity (DAS28 ⁇ 3.2), the former was classified as a non-responder, and the latter was classified as a good responder, and serum was obtained.
- DMARDs disease modifying anti-rheumatic drugs
- DAS28 ⁇ 3.2 disease modifying anti-rheumatic drugs
- Lipids were extracted from serum collected from each group of patients using MTBE (tert-methyl butyl ether) method, and mass spectrometry-based lipidomics analysis was performed. Specifically, lipids were separated on a UPLC system equipped with a C18 column (2.1 ⁇ 100 mm, 1.7 ⁇ m) coupled with a guard column (2.1 ⁇ 5 mm, 1.7 ⁇ m), and data were collected. For data preprocessing, the collected data was imported into MS-DIAL ver 4.38, and batch effects were removed with LOESS (locally estimated scatterplot smoothing) algorithm. After peak annotation, unreliable lipids were removed when the relative standard deviation (%) of the quality control (QC) sample was >30%.
- MTBE tert-methyl butyl ether
- Referenced lipids were putatively identified based on precursor ion m/z values and product ion patterns consistent with data from MS-DIAL's LipidBlast database.
- MSI metabolomics standards initiative
- the identified lipid list was confirmed as an in-house lipid library including retention time. All lipid features were normalized according to the median intensity of each sample, and a t-test was performed to obtain FDR, and a paired t-test was used to compare groups before and after treatment. To compare three or more groups, significant differences were identified using Tukey's HSD and one-way ANOVA.
Abstract
The present invention relates to lipid biomarkers for predicting the response to drug treatment of rheumatoid arthritis. The expression levels of the lipid biomarkers selected through lipidome analysis according to the present invention specifically change according to the response following drug treatment, and thus the lipid biomarkers have the effect of being able to be effectively utilized for evaluating the response to a rheumatoid arthritis therapeutic agent and predicting the prognosis of rheumatoid arthritis.
Description
본 발명은 류마티스 관절염의 약물 치료 반응성 예측을 위한 지질 바이오마커들에 관한 것이다.The present invention relates to lipid biomarkers for predicting drug treatment responsiveness in rheumatoid arthritis.
지질체학(Lipidomics)은 생체 내에서 발생하는 여러 지질체군(lipidome)들의 대사과정에서 생기는 총체적인 현상 및 변화를 정성 및 정량하여 그 생물학적, 생화학적 중요성을 알아내는 것에 초점을 맞춘 학문 분야로써, 각 종 질환관련 표지물질(biomarker)을 찾는 연구에도 그 목적을 두고 있다. 특히, 최근 전자분무이온화-탠덤질량 분석법(ESI-MS-MS)의 비약적인 발전으로, 지질 분자들의 구조적 분석이 가능하게 되었고, 샷건 지질학(shotgun lipidomics) 분야가 생물학적 조직의 분석에도 활용될 수 있다.Lipidomics is a field of study that focuses on qualitatively and quantifying the overall phenomena and changes that occur in the metabolic process of various lipidomes that occur in vivo to find out their biological and biochemical significance. It is also aimed at research to find disease-related biomarkers. In particular, with the recent rapid development of electrospray ionization-tandem mass spectrometry (ESI-MS-MS), structural analysis of lipid molecules has become possible, and the field of shotgun lipidomics can also be used for analysis of biological tissues.
한편, 류마티스 관절염(rheumatoid arthritis, 이하 RA)은 대표적인 자가면역질환으로, 관절 주위를 둘러싸고 있는 활막이라는 조직의 염증 때문에 일어나며, 전체 인구의 약 1~2% 가량에서 발병하는 것으로 알려진 질환이다 (Alamanosa and Drosos, Autoimmun. Rev., 4:130-136 (2005)). 류마티스 관절염의 발병은 유전적-환경적 인자가 작용하며, 유전적인 요인이 대략 60% 정도 기여하는 것으로 알려져 있다. 초기 류마티스 관절염은 처음에 더 작은 관절, 예컨대 손목, 손, 발목 및 발의 관절에 영향을 미치며 질환이 진행됨에 따라, 어깨, 팔꿈치, 무릎, 둔부, 턱 및 목의 관절들에도 영향을 나타낼 수 있으며, 질병활성도(disease activity)가 조절되지 않으면 시간 경과에 따라 비가역적 관절변형과 장애가 발생한다. 또한, 관절 내 부위 또는 관절 주변 부위에만 영향을 미치는 다른 관절염 상태들과는 달리, 류마티스 관절염은 피부, 혈관, 심장, 폐 및 근육을 포함하는 신체에 걸쳐 관절 외 조직에서 염증을 야기할 수 있는 전신성 질환이다.On the other hand, rheumatoid arthritis (RA) is a representative autoimmune disease, caused by inflammation of a tissue called the synovium surrounding the joint, and is known to develop in about 1-2% of the total population (Alamanosa and Drosos, Autoimmun. Rev., 4:130-136 (2005)). It is known that the onset of rheumatoid arthritis is affected by genetic-environmental factors, and that genetic factors contribute about 60%. Early rheumatoid arthritis initially affects the smaller joints, such as those of the wrists, hands, ankles, and feet, and as the disease progresses, may also affect the joints of the shoulders, elbows, knees, hips, jaw, and neck; If disease activity is not controlled, irreversible joint deformity and disability occur over time. Also, unlike other arthritic conditions that only affect areas within or around the joints, rheumatoid arthritis is a systemic disease that can cause inflammation in tissues outside the joints throughout the body, including the skin, blood vessels, heart, lungs and muscles. .
류마티스 관절염의 치료 방법으로는 관절의 손상을 최소화하고, 기능 손실을 방지하며 통증을 줄이기 위하여 일반적으로 진통소염제를 다양한 항류마티스제제와 병합 투여하는 약물치료 방법이 사용되고 있고, 최근에는 생물학적 치료제가 개발되어 항류마티스 약제와 함께 병용 치료로 이용되고 있으며, 질병의 중증도가 심각한 경우에는 수술요법이 시행되고 있다. 생물학적 치료제로 최근 대두되고 있는 질환조절항류마티스약제(biologic disease-modifying anti-rheumatic drugs, bDMARD)는 류마티스 관절염 치료에 없어서는 안될 치료제들이 포함되어 있으나, 종래의 마커 (특히 CRP)의 경우 류마티스 관절염의 실제 완화 여부와 관계 없이 그 값이 낮아진다는 연구 결과들이 보고되고 있다. 따라서, 류마티스 관절염의 치료 후 반응 (관절염의 질병활성도)을 정확하게 진단할 수 있는 새로운 바이오마커의 개발이 필수적이다.As a treatment method for rheumatoid arthritis, a drug treatment method in which analgesic anti-inflammatory drugs are administered in combination with various anti-rheumatic drugs is generally used to minimize joint damage, prevent functional loss, and reduce pain, and recently, biological treatments have been developed. It is used as a combination treatment along with anti-rheumatic drugs, and surgical therapy is performed when the severity of the disease is severe. Biologic disease-modifying anti-rheumatic drugs (bDMARDs), which have recently emerged as biologic treatments, include treatments that are indispensable for the treatment of rheumatoid arthritis, but conventional markers (especially CRP) are not effective in treating rheumatoid arthritis. Research results have been reported that the value is lowered regardless of whether or not it is mitigated. Therefore, it is essential to develop a new biomarker capable of accurately diagnosing the response (disease activity of arthritis) after treatment of rheumatoid arthritis.
본 발명의 목적은 류마티스 관절염 치료제에 대한 반응성 진단용 바이오마커 조성물을 제공하는 것이다.An object of the present invention is to provide a biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis.
또한, 본 발명의 목적은 류마티스 관절염 치료제에 대한 반응성 진단용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis.
또한, 본 발명의 목적은 류마티스 관절염 치료제에 대한 반응성을 평가하기 위한 방법을 제공하는 것이다.Another object of the present invention is to provide a method for evaluating responsiveness to a therapeutic agent for rheumatoid arthritis.
아울러, 본 발명의 목적은 류마티스 관절염의 예후 예측을 위한 정보제공방법을 제공하는 것이다.In addition, an object of the present invention is to provide an information providing method for predicting the prognosis of rheumatoid arthritis.
상기 목적의 달성을 위해, 본 발명은 아실 카르니틴, 세라마이드 스핑고신, 리소포스파티딜콜린, 에테르-가교 LPC, 포스파티딜콜린, 에테르-가교 PC, 포스파티딜에탄올아민, 에테르-가교 포스파티딜에탄올아민, 스핑고미엘린 및 트리아실글리세롤로 이루어진 군에서 선택되는 하나 이상의 지질을 포함하는, 류마티스 관절염 치료제에 대한 반응성 진단용 바이오마커 조성물을 제공한다.In order to achieve the above object, the present invention provides acyl carnitine, ceramide sphingosine, lysophosphatidylcholine, ether-bridged LPC, phosphatidylcholine, ether-bridged PC, phosphatidylethanolamine, ether-bridged phosphatidylethanolamine, sphingomyelin and triacylglycerol. It provides a biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis, comprising one or more lipids selected from the group consisting of.
또한, 본 발명은 류마티스 관절염 치료제에 대한 반응성 진단용 키트를 제공한다.In addition, the present invention provides a kit for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis.
또한, 본 발명은 류마티스 관절염 치료제에 대한 반응성을 평가하기 위한 방법을 제공한다.In addition, the present invention provides a method for evaluating responsiveness to a therapeutic agent for rheumatoid arthritis.
아울러, 본 발명은 류마티스 관절염의 예후 예측을 위한 정보제공방법을 제공한다.In addition, the present invention provides an information providing method for predicting the prognosis of rheumatoid arthritis.
본 발명에서 지질체(lipidomics) 분석을 통해 선발한 지질 바이오마커들은 약물 치료 후 반응성에 따라 특이적으로 발현 수준이 변화하므로, 이를 류마티스 관절염 치료제에 대한 반응성 평가 용도 및 류마티스 관절염의 예후 예측 용도로 유용하게 활용할 수 있는 효과가 있다.Lipid biomarkers selected through lipidomics analysis in the present invention have specific expression levels that change according to reactivity after drug treatment, so they are useful for evaluating responsiveness to rheumatoid arthritis drugs and predicting the prognosis of rheumatoid arthritis There is an effect that can be used wisely.
도 1은 치료 전/후에 얻은 시료의 비교 후 식별된 37개의 유의미한 지질의 발현을 나타낸 Heatmap이다 (t-검정일 이용하여 비교된 시료). Figure 1 is a heatmap showing the expression of 37 significant lipids identified after comparison of samples obtained before and after treatment (samples compared using t-test days).
도 2는 RHT(Regularized Hotelling's T2) 검정을 이용하여 좋은 치료반응군/나쁜 치료반응군 사이의 지질 발현 차이를 추정한 도이다.Figure 2 is a diagram estimating the difference in lipid expression between good treatment responders / bad treatment responders using RHT (Regularized Hotelling's T 2 ) test.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.
달리 정의되지 않는 한, 본원에서 사용된 모든 기술적 및 과학적 용어는 본 발명이 속하는 분야의 당업자가 통상적으로 이해하는 것과 동일한 의미를 갖는다. 본원에 기술된 것들과 유사하거나 등가인 임의의 방법 및 재료가 본 발명을 테스트하기 위한 실행에서 사용될 수 있지만, 바람직한 재료 및 방법이 본원에서 기술된다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention, the preferred materials and methods are described herein.
본 발명에서 지질 표기는 지질체학(Lipidomics)에서 세계 응용화학, 생화학, 분자생물학 표준에 따른 시스템 이름 및 공통 이름(IUPAC-IUBMB)에 따라 표기하였다.In the present invention, lipids are marked according to the system name and common name (IUPAC-IUBMB) according to world standards for applied chemistry, biochemistry, and molecular biology in Lipidomics.
일 측면에서, 본 발명은 아실 카르니틴(acyl carnitine, CAR), 세라마이드 스핑고신(Ceramide sphingosine, Cer-NS), 리소포스파티딜콜린(lysophosphatidylcholine, LPC), 에테르-가교 LPC(ether-linked LPC, LPC O), 포스파티딜콜린(phosphatidylcholine, PC), 에테르-가교 PC(ether-linked phosphatidylcholine, PC O), 포스파티딜에탄올아민(phosphatidylethanolamine, PE), 에테르-가교 포스파티딜에탄올아민(ether-linked phosphatidylethanolamine, PE P), 스핑고미엘린(sphingomyelin, SM) 및 트리아실글리세롤(triacylglycerol, TG)로 이루어진 군에서 선택되는 하나 이상의 지질을 포함하는, 류마티스 관절염 치료제에 대한 반응성 진단용 바이오마커 조성물에 관한 것이다.In one aspect, the present invention provides acyl carnitine (CAR), ceramide sphingosine (Cer-NS), lysophosphatidylcholine (LPC), ether-linked LPC (ether-linked LPC, LPC O), Phosphatidylcholine (PC), ether-linked phosphatidylcholine (PC O), phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (PE P), sphingomyelin ( It relates to a biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis, comprising at least one lipid selected from the group consisting of sphingomyelin (SM) and triacylglycerol (TG).
일 구현예에서, 아실 카르니틴은 12:0, 14:1 및 16:1이고, 세라마이드 스핑고신은 42:1이며, 리소포스파티딜콜린은 16:0, 16:1, 18:1, 18:2, 18:3, 20:1, 20:2, 20:3, 20:4, 20:5, 22:5, 22:6 또는 24:0이고, 에테르-가교 LPC는 16:1, 18:0 또는 24:1이며, 포스파티딜콜린은 32:3, 34:2, 34:5, 36:1, 36:5 (1), 36:6 (1), 40:1, 40:4, 42:10 또는 42:6이고, 에테르-가교는 PC 36:3이며, 포스파티딜에탄올아민은 36:1 또는 40:7이며, 에테르-가교 포스파티딜에탄올아민은 34:2, 36:3 또는 40:6이고, 스핑고미엘린은 38:1 (1) 또는 38:1 (2)이며, 및 트리아실글리세롤은 48:5 (2), 50:6 (4) 또는 54:9일 수 있다.In one embodiment, the acyl carnitine is 12:0, 14:1 and 16:1, the ceramide sphingosine is 42:1 and the lysophosphatidylcholine is 16:0, 16:1, 18:1, 18:2, 18 :3, 20:1, 20:2, 20:3, 20:4, 20:5, 22:5, 22:6 or 24:0, and the ether-bridged LPC is 16:1, 18:0 or 24 :1, and phosphatidylcholine is 32:3, 34:2, 34:5, 36:1, 36:5 (1), 36:6 (1), 40:1, 40:4, 42:10 or 42: 6, the ether-bridged PC is 36:3, the phosphatidylethanolamine is 36:1 or 40:7, the ether-bridged phosphatidylethanolamine is 34:2, 36:3 or 40:6, and the sphingomyelin is 38:1 (1) or 38:1 (2), and triacylglycerols may be 48:5 (2), 50:6 (4) or 54:9.
일 구현예에서, 류마티스 관절염 치료제는 DMARDs(disease modifying anti-rheumatic drugs)일 수 있으며, 생물학적 DMARD(bDMARD)일 수 있다.In one embodiment, the treatment for rheumatoid arthritis may be disease modifying anti-rheumatic drugs (DMARDs) or may be biological DMARDs (bDMARDs).
본 발명에서 사용된 용어 "바이오마커(biomarker)"는 단백질이나 DNA, RNA, 대사물질 등을 이용해 몸 안의 변화를 알아낼 수 있는 지표를 의미하고, 바이오마커를 활용하면 생명체의 정상 또는 병리적인 상태, 약물에 대한 반응 정도 등을 객관적으로 측정할 수 있다. 암을 비롯해 뇌졸증, 치매 등 각종 난치병을 진단하는데 바이오마커가 활용되고 있으며, 본 발명에 있어서는 류마티스 관절염의 치료 후 반응성 여부를 판단하거나, 치료 후 류마티스 관절염의 예후를 판단하는데 이용될 수 있다. 또한, 신약개발과정에서 바이오마커를 활용할 수 있고, 이를 통해서 신약의 안전성을 확보할 수 있고 신약 개발 과정에서 소요되는 비용을 절감할 수 있는 효과도 얻을 수 있다.The term "biomarker" used in the present invention refers to an index that can detect changes in the body using proteins, DNA, RNA, metabolites, etc. The degree of response to the drug can be objectively measured. Biomarkers are being used to diagnose various incurable diseases such as cancer, stroke, and dementia, and in the present invention, they can be used to determine responsiveness after treatment of rheumatoid arthritis or to determine the prognosis of rheumatoid arthritis after treatment. In addition, biomarkers can be used in the new drug development process, and through this, the safety of the new drug can be secured and the cost required in the new drug development process can be reduced.
일 측면에서, 본 발명은 류마티스 관절염 치료제에 대한 반응성 진단용 지질 바이오마커 조성물을 포함하는, 류마티스 관절염 치료제에 대한 반응성 진단용 키트에 관한 것이다.In one aspect, the present invention relates to a kit for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis, comprising a lipid biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis.
일 구현예에서, 상기 키트는 대상체 또는 환자로부터 생체 시료를 수집하기 위한 도구 및/또는 시약 뿐 아니라 그 시료로부터 지질을 분리하기 위한 도구 및/또는 시약을 더 포함할 수 있다. In one embodiment, the kit may further include tools and/or reagents for collecting a biological sample from a subject or patient as well as tools and/or reagents for isolating lipids from the sample.
본 발명에서 용어 "류마티스 관절염 치료제에 대한 반응성 진단용 키트"는 본 발명의 류마티스 관절염 치료제에 대한 반응성 진단용 지질 바이오마커 조성물이 포함된 키트를 의미한다. 따라서, 상기 표현 "키트"는 "조성물"과 서로 교차 또는 혼용하여 사용이 가능하다. 본 명세서에서 용어 "진단"은 특정 질병 또는 질환에 대한 한 객체의 감수성(susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는 지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 객체의 예후(prognosis)(예컨대, 치료에 대한 류마티스 관절염의 반응성 결정)를 판정하는 것, 또는 테라메트릭스(therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링 하는 것)을 포함한다.In the present invention, the term "kit for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis" refers to a kit containing a lipid biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis of the present invention. Therefore, the expression "kit" can be used interchangeably or interchangeably with "composition". As used herein, the term "diagnosis" refers to determining a subject's susceptibility to a specific disease or disorder, determining whether a subject currently has a specific disease or disorder, or suffering from a specific disease or disorder Determining a subject's prognosis (eg, determining the responsiveness of rheumatoid arthritis to treatment), or therametrics (eg, monitoring a subject's condition to provide information about treatment efficacy) includes
본 발명에서 용어 "반응성 진단용 바이오 마커, 진단하기 위한 바이오 마커 또는 진단 마커(diagnosis marker)"란 류마티스 관절염 치료 후 반응성을 진단할 수 있는 물질로, 치료 전에 비하여 발현이 증가 또는 감소하는 양상을 보이는 지질과 같은 유기 생체 분자 등을 포함한다. 본 발명의 목적상, 상기 류마티스 관절염 치료제에 대한 반응성 진단용 바이오 마커는 CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) 및 TG 54:9으로서, 류마티스 관절염에서 특이적으로 발현 정도가 변화하는 지질이다. 이러한 마커들은 이들 마커들이 둘 이상 포함된 복합 마커인 것이 바람직하다.In the present invention, the term "reactive diagnostic biomarker, diagnostic biomarker, or diagnostic marker" is a substance capable of diagnosing reactivity after treatment of rheumatoid arthritis, and a lipid whose expression is increased or decreased compared to before treatment. and organic biomolecules such as For the purpose of the present invention, biomarkers for diagnosing responsiveness to therapeutic agents for rheumatoid arthritis include CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 ( 1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P -34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) and TG 54:9, a lipid whose expression level specifically changes in rheumatoid arthritis. Preferably, these markers are composite markers in which two or more of these markers are included.
일 측면에서, 본 발명은 (a) 검사 대상체로부터 분리한 생물학적 시료에서 아실 카르니틴, 세라마이드 스핑고신, 리소포스파티딜콜린 에테르-가교 LPC, 포스파티딜콜린, 에테르-가교 PC, 포스파티딜에탄올아민, 에테르-가교 포스파티딜에탄올아민, 스핑고미엘린 및 트리아실글리세롤로 이루어진 군에서 선택되는 하나 이상의 지질의 발현 수준을 측정하는 단계; 및 (b) 류마티스 관절염 치료제에 대한 반응성을 판단하는 단계를 포함하는, 류마티스 관절염 치료제에 대한 반응성을 평가하기 위한 방법에 관한 것이다.In one aspect, the present invention provides (a) acyl carnitine, ceramide sphingosine, lysophosphatidylcholine ether-bridged LPC, phosphatidylcholine, ether-bridged PC, phosphatidylethanolamine, ether-bridged phosphatidylethanolamine in a biological sample isolated from a test subject, Measuring the expression level of one or more lipids selected from the group consisting of sphingomyelin and triacylglycerol; And (b) it relates to a method for evaluating the responsiveness to a therapeutic agent for rheumatoid arthritis, comprising determining the responsiveness to the therapeutic agent for rheumatoid arthritis.
일 구현예에서, 대상체의 시료에서 Cer-NS 42:1, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2) 및 TG 54:9의 농도가 치료제 처리 전 시료에서의 발현 수준 대비 상향 조절되고, CAR 12:0, CAR 14:1 및 CAR 16:1의 농도가 치료제 처리 전 시료에서의 발현 수준 대비 하향 조절되면 치료적으로 반응성인 것으로 판단할 수 있으며, 반응성은 치료제 처리 후 DAS28 <3.2인 것일 수 있다.In one embodiment, Cer-NS 42:1, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3 in a sample from a subject , LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32: 3, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PE 36 :1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 ( 2) The concentrations of TG 54:9 and TG 54:9 were upregulated compared to the expression level in the sample before treatment, and the concentrations of CAR 12:0, CAR 14:1, and CAR 16:1 were decreased compared to the expression level in the sample before treatment. If controlled, it can be judged to be therapeutically responsive, and the responsiveness can be DAS28 <3.2 after treatment with a therapeutic agent.
본 발명에서, 용어 "DAS28 (disease activity score with 28-joint assessment)"은, 28개의 관절을 이용한 류머티스 관절염 활성도 평가이고, 현재 많이 사용되는 평가 방법이다. 질병활성도를 반영하는 DAS28에 근거한 새로운 기준은구미 선진국의 진료지침과 부합하고, 항 TNF 제제의 투여가 필요한 류마티스관절염 환자의 적절한 약제사용에 기여할 것으로 예상된다. DAS28에 포함되는 관절은 PIP관절(10), MCP 관절(10), 완관절(2), 주관절(2), 견관절(2), 슬관절(2)이다. In the present invention, the term "disease activity score with 28-joint assessment (DAS28)" is a rheumatoid arthritis activity evaluation using 28 joints, and is an evaluation method that is currently widely used. The new criteria based on DAS28, which reflects disease activity, are in line with the treatment guidelines of developed countries in Europe and America, and are expected to contribute to the appropriate drug use of patients with rheumatoid arthritis who require the administration of anti-TNF drugs. The joints included in the DAS28 are the PIP joint (10), the MCP joint (10), the arm joint (2), the elbow joint (2), the shoulder joint (2), and the knee joint (2).
일 구현예에서, 대상체의 시료에서 CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) 및 TG 54:9가 치료제 처리 전 시료에서의 발현 수준 대비 발현 정도에 변화가 없으면 치료적으로 비-반응성일 것으로 판단할 수 있으며, 비-반응성은 치료제 처리 후 DAS28 ≥3.2인 것일 수 있다.In one embodiment, CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18:2 in a sample from a subject , LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16 :1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 ( 1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P- 36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) and TG 54:9 treat therapies If there is no change in the expression level compared to the expression level in all samples, it can be determined to be therapeutically non-responsive, and non-responsiveness may be DAS28 ≥ 3.2 after treatment with a therapeutic agent.
일 구현예에서, 생물학적 시료는 혈액 또는 혈청일 수 있다.In one embodiment, the biological sample may be blood or serum.
일 구현예에서, 지질 바이오마커의 시료 내 발현 수준 (농도)은 관련 기술분야의 통상의 기술자에게 공지된 임의의 방법을 사용하여 측정될 수 있다. 지질을 측정하는 방법은 질량분석법(mass spectrometry), 질량 분광측정법에 대한 연성 이온화 기술, 예컨대 전기분무 이온화 (ESI) 및 매트릭스-보조 레이저 탈착/이온화 (MALDI)를 포함한 분광측정 방법을 포함하나 이에 제한되지는 않는다.In one embodiment, the expression level (concentration) of a lipid biomarker in a sample can be measured using any method known to those skilled in the art. Methods for measuring lipids include, but are not limited to, mass spectrometry, spectroscopic methods including soft ionization techniques for mass spectrometry, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). It doesn't work.
일 측면에서, 본 발명은 (a) 검사 대상체로부터 분리한 생물학적 시료에서 아실 카르니틴, 세라마이드 스핑고신, 리소포스파티딜콜린 에테르-가교 LPC, 포스파티딜콜린, 에테르-가교 PC, 포스파티딜에탄올아민, 에테르-가교 포스파티딜에탄올아민, 스핑고미엘린 및 트리아실글리세롤로 이루어진 군에서 선택되는 하나 이상의 지질의 발현 수준을 측정하는 단계; 및 In one aspect, the present invention provides (a) acyl carnitine, ceramide sphingosine, lysophosphatidylcholine ether-bridged LPC, phosphatidylcholine, ether-bridged PC, phosphatidylethanolamine, ether-bridged phosphatidylethanolamine in a biological sample isolated from a test subject, Measuring the expression level of one or more lipids selected from the group consisting of sphingomyelin and triacylglycerol; and
(b) 대상체의 시료에서 지질의 발현 수준을 정상 시료에서의 발현 수준과 비교하여 류마티스관절염의 예후를 판단하는 단계를 포함하는, 류마티스 관절염의 예후 예측을 위한 정보제공방법에 관한 것이다.(b) it relates to a method of providing information for predicting the prognosis of rheumatoid arthritis, comprising the step of determining the prognosis of rheumatoid arthritis by comparing the expression level of lipids in a sample of a subject with the expression level of a normal sample.
일 구현예에서, 대상체의 시료에서 Cer-NS 42:1, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2) 및 TG 54:9의 농도가 치료제 처리 전 시료에서의 발현 수준 대비 상향 조절되고, CAR 12:0, CAR 14:1 및 CAR 16:1의 농도가 치료제 처리 전 시료에서의 발현 수준 대비 하향 조절되면 류마티스관절염 예후가 좋은 것으로 판단할 수 있으며, 예후가 좋은 것은 치료적으로 반응성인 것일 수 있으며, 치료적으로 반응성은 DAS28 <3.2인 것일 수 있다.In one embodiment, Cer-NS 42:1, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3 in a sample from a subject , LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32: 3, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PE 36 :1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 ( 2) The concentrations of TG 54:9 and TG 54:9 were upregulated compared to the expression level in the sample before treatment, and the concentrations of CAR 12:0, CAR 14:1, and CAR 16:1 were decreased compared to the expression level in the sample before treatment. If controlled, the prognosis of rheumatoid arthritis can be judged to be good, and a good prognosis may be a therapeutic response, and a therapeutic response may be a DAS28 <3.2.
일 구현예에서, 대상체의 시료에서 상기 CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) 및 TG 54:9가 치료제 처리 전 시료에서의 발현 수준 대비 발현 정도에 변화가 없으면 류마티스 관절염의 예후가 나쁜 것으로 판단할 수 있으며, 예후가 나쁜 것은 치료적으로 비-반응성인 것일 수 있고, 치료적으로 비-반응성은 DAS28 ≥3.2인 것일 수 있다.In one embodiment, the CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18: 2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O- 16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P -36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) and TG 54:9 are therapeutics If there is no change in the expression level compared to the expression level in the sample before treatment, it can be judged that the prognosis of rheumatoid arthritis is poor, and a poor prognosis may be therapeutically non-responsive, and therapeutically non-responsive is DAS28 ≥ It may be 3.2.
일 구현예에서, 생물학적 시료는 혈액 또는 혈청일 수 있다.In one embodiment, the biological sample may be blood or serum.
일 구현예에서, 지질 바이오마커의 시료 내 발현 수준 (농도)은 관련 기술분야의 통상의 기술자에게 공지된 임의의 방법을 사용하여 측정될 수 있다. 지질을 측정하는 방법은 질량분석법(mass spectrometry), 질량 분광측정법에 대한 연성 이온화 기술, 예컨대 전기분무 이온화 (ESI) 및 매트릭스-보조 레이저 탈착/이온화 (MALDI)를 포함한 분광측정 방법을 포함하나 이에 제한되지는 않는다.In one embodiment, the expression level (concentration) of a lipid biomarker in a sample can be measured using any method known to those skilled in the art. Methods for measuring lipids include, but are not limited to, mass spectrometry, spectroscopic methods including soft ionization techniques for mass spectrometry, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). It doesn't work.
용어 “예후 (prognosis)”는 류마티스관절염 치료 후 관절의 변형 정도 (관절의 손상), 또는 류마티스관절염에 의한 관절파괴의 진행속도, 즉, 류마티스 관절염의 중증도의 증가 및 감소를 나타낸다.The term "prognosis" refers to the degree of joint deformity (joint damage) after treatment for rheumatoid arthritis, or the rate of progression of joint destruction caused by rheumatoid arthritis, that is, the increase or decrease in the severity of rheumatoid arthritis.
본 발명에서 사용된 용어 "검출" 또는 "측정"은 검출 또는 측정된 대상의 농도를 정량하는 것을 의미하며, 측정하는 것" 또는 "측정"은 시료 내에서 주어진 물질의 정성적 또는 정성적 농도 수준의 도출을 포함하여, 이러한 물질의 존재, 부재, 수량 또는 양 (유효량일 수 있음)을 평가하는 것, 또는 달리 대상체의 임상 파라미터의 값 또는 카테고리화를 평가하는 것을 의미한다. As used herein, the term "detection" or "measurement" means to quantify the concentration of a detected or measured object, and to measure "or"measurement" is a qualitative or qualitative concentration level of a given substance in a sample. It means assessing the presence, absence, quantity or amount (which may be an effective amount) of such a substance, including the derivation of, or otherwise assessing the value or categorization of a subject's clinical parameter.
본 발명에서, 바이오마커의 수준을 결정하는 것은 전처리되지 않거나 또는 미분획된 시료 상에서 수행할 수 있으며, 대상체로부터 수득된 전처리하지 않거나 미분획된 시료 중에서의 지질 바이오마커의 수준을 결정할 수 있다.In the present invention, determining the level of a biomarker can be performed on an untreated or unfractionated sample, and the level of a lipid biomarker in an untreated or unfractionated sample obtained from a subject can be determined.
본 발명에서, 바이오마커의 수준을 결정하는 것은 정량적 또는 반정량적일 수 있다. 일부 실시예에서, 정량적 결정은 하나 이상의 지질 대사물의 절대적 양 또는 농도 (발현 수준)를 결정하는 것을 포함할 수 있다. 또한, 정량적 결정은 하나 이상의 다른 대사물을 기준으로 하여 하나 이상의 지질 대사물의 상대적 양 또는 농도를 결정하는 것을 포함할 수 있다.In the present invention, determining the level of a biomarker may be quantitative or semi-quantitative. In some embodiments, quantitative determination may include determining the absolute amount or concentration (expression level) of one or more lipid metabolites. Quantitative determination can also include determining the relative amount or concentration of one or more lipid metabolites relative to one or more other metabolites.
본 발명에서 용어, "대상체" 또는 "환자"는 인간, 유인원, 원숭이, 소, 개, 기니아 피그, 토끼, 닭, 곤충 등을 포함하여 치료가 요구되는 임의의 단일 개체를 의미한다. 또한, 임의의 질병 임상 소견을 보이지 않는 임상 연구 시험에 참여한 임의의 대상 또는 역학 연구에 참여한 대상 또는 대조군으로 사용된 대상이 대상에 포함된다. 아울러, 본 발명에서는 포유동물, 바람직하게는 인간을 지칭한다. As used herein, the term "subject" or "patient" refers to any single individual in need of treatment, including humans, apes, monkeys, cows, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects participating in clinical research trials who do not show any clinical signs of disease or subjects participating in epidemiological studies or subjects used as controls. In addition, in the present invention, it refers to a mammal, preferably a human.
본 발명에서 용어, "생물학적 시료(샘플)"는 대상 또는 환자로부터 얻은 생물학적 시료를 의미하며, 진단 또는 모니터링 검정에 사용될 수 있는 유기체로부터 수득된 각종 시료 유형을 포괄한다. 상기 용어는 생물학적 기원의 혈액 및 기타 액상 시료, 고형 조직 시료, 예컨대 생검 표본, 또는 그로부터 및 그의 자손으로부터 유래된 조직 배양물 또는 세포를 포괄한다. 상기 용어는 구체적으로, 임상 시료를 포괄하고, 세포 배양 중인 세포, 세포 상등액, 세포 용해물, 혈청, 혈장, 뇨, 양수, 생물학적 유체 및 조직 시료를 추가로 포함한다. 상기 용어는 또한, 시약으로의 처리와 같은 조달, 가용화, 또는 특정 성분에 대한 강화 후 어떠한 방식으로든 조작된 시료를 포괄한다. 본 발명의 일 실시예에서는 혈액 또는 혈청을 시료로 하였다.As used herein, the term "biological sample (sample)" means a biological sample obtained from a subject or patient, and encompasses various types of samples obtained from organisms that can be used for diagnosis or monitoring assays. The term encompasses blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens, or tissue cultures or cells derived therefrom and from their progeny. The term specifically covers clinical samples, and further includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluids and tissue samples. The term also encompasses samples that have been manipulated in any way after procurement, such as treatment with reagents, solubilization, or enrichment for a particular component. In one embodiment of the present invention, blood or serum was used as a sample.
생물학적 시료는 대상체의 체액 또는 신체 조직의 시료일 수 있다. 예를 들어, 생물학적 시료는 대상체로부터의 혈액, 혈장, 혈청, 타액, 담즙, 뇨, 대변 또는 뇌척수액의 시료, 또는 대상체로부터의 세포, 조직 또는 기관으로부터 유래된 시료일 수 있다. 많은 실시예에서, 혈액, 혈장 또는 혈청을 생물학적 시료로서 사용하는 것이 바람직할 수 있다. 생물학적 시료를 수득하기 위한 각종 기술이 이용 가능하다.A biological sample may be a sample of bodily fluid or body tissue of a subject. For example, a biological sample can be a sample of blood, plasma, serum, saliva, bile, urine, feces, or cerebrospinal fluid from a subject, or a sample derived from a cell, tissue, or organ from a subject. In many embodiments, it may be desirable to use blood, plasma or serum as the biological sample. A variety of techniques are available for obtaining biological samples.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다. The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.
실시예. 류마티스관절염 치료 반응에 따른 지질체 분석 및 지질 마커 선발Example. Lipid body analysis and lipid marker selection according to rheumatoid arthritis treatment response
혈청 내 지질체가 RA 치료 결과를 예측할 수 있는지 확인하기 위해, 질병활성도가 높은 상태의 치료 전 류마티스관절염 환자 (Active RA)의 혈청, 이 환자의 치료 전 혈청 및 치료 6개월 뒤 혈청을 수집하여 지질을 분리하여 치료 전 후로 변화한 지질체를 분석하였다. 구체적으로, 연구 참여자들은 CIRAD (Center for Integrative Rheumatoid Transcriptomics and Dynamics) 코호트에 등록되었으며 (2015 년), RA 환자는 2010 ACR / EULAR RA 분류 기준 (6)을 충족하는 것으로 정의되었다. 이 환자들은 2021년 3월까지 확실히 RA가 발생했는지 추적 관찰되었다. 코호트 등록시 각 참여자들로부터 혈청을 수집하였으며, 치료 결과에 따른 지질체 변화를 확인하기 위해, DMARDs(disease modifying anti-rheumatic drugs)를 처리하고 6개월 후 높은 질병 활성도(disease activity)를 보이는 군 (DAS28 ≥3.2)과 낮은 질병 활성도를 보이는 군 (DAS28 < 3.2)으로 나누어, 전자를 나쁜 치료반응군 (non responder)으로, 후자를 좋은 치료 반응군 (Good responder)으로 나누어 혈청을 확보하였다. 또한, 12개월 동안 3회 연속 측정시 DAS28 ≤ 2.6인 환자로 정의된, 지속적인 관해 상태에 있는 19명의 RA 환자를 선택하였다. MTBE(tert-methyl butyl ether) 방법을 이용하여 상기에서 각 군의 환자들로부터 수집한 혈청에서 지질을 추출하고 질량분석법(mass spectrometry)-기반 지질체(Lipidomics) 분석을 시행하였다. 구체적으로, 가드 컬럼 (2.1×5 mm, 1.7 μm)과 연결된 C18 컬럼 (2.1×100 mm, 1.7 μm)이 장착된 UPLC 시스템 상에서 지질을 분리하였으며, 데이터를 수집하였다. 데이터 전처리를 위해, 수집한 데이터를 MS-DIAL ver 4.38으로 가져왔으며, 배치 효과(Batch effects)는 LOESS(locally estimated scatterplot smoothing) 알고리즘으로 제거하였다. 피크 참조(peak annotation) 후, QC(quality control) 시료의 RSD(relative standard deviation, %)가 >30%일 때 신뢰할 수 없는 지질을 제거하였다. 참조된 지질을 MS-DIAL의 LipidBlast database의 데이터와 일치하는 전구체 이온 m/z값 및 프로덕트 이온 패턴에 기반하여 추정적으로 식별하였다. MSI(metabolomics standards initiative) 1으로서 지질 식별의 신뢰도를 높이기 위해, 식별한 지질 목록을 머무름 시간(retention time)을 포함하는 in-house 지질 라이브러리로 확인하였다. 모든 지질 특징은 각 시료의 중앙 강도에 따라 정규화되었으며, FDR을 획득하기 위해 t-test를 수행하고 치료 전 후 군을 비교하기 위해, paired t-test를 이용하였다. 세 개 이상의 군을 비교하기 위해서 Tukey's HSD와 one-way ANOVA를 이용하여 유의한 차이를 식별하였다. heatmap, PCA 및 Random Forest 알고리즘을 이용한 종합 해석을 바탕으로 의심되는 이상치를 제외하였으며, R의 Hmisc package를 이용하여 상관 분석을 수행하고, rcorr 함수를 이용하여 Pearson's 상관 관계를 결정하였다. 또한, 두 가지 다른 다변량 통계 분석 모델 (unsupervised 및 supervised)을 적용하여 군들을 구별하였다 (unsupervised, PCA; supervised, PLS-DA). PLS-DA 모델은 LOOCV(leave-one-out cross validation)을 이용하여 교차 검증하였고, Q2 값은 모텔의 overfitting 값을 추정하는데 사용되었다. 또한, 환자 군 간의 세부적인 차이를 더 확인하기 위해, OPLS-DA(orthogonal projections to latent structures-discriminant analysis) 분석을 수행하였다. 또한, 바이오마커 후보는 OPLS-DA 모델에서 |r| <0.5인 경우 및 t-검정에서 FDR 값이 <0.25인 경우 선발하였다.In order to confirm whether the lipid profile in serum can predict the RA treatment outcome, lipids were collected from the serum of patients with rheumatoid arthritis (Active RA) with high disease activity before treatment, serum before treatment, and serum after 6 months of treatment. Separated and analyzed lipid bodies changed before and after treatment. Specifically, study participants were enrolled in the Center for Integrative Rheumatoid Transcriptomics and Dynamics (CIRAD) cohort (2015), and RA patients were defined as meeting the 2010 ACR/EULAR RA classification criteria (6). These patients were followed until March 2021 for definitive RA development. Serum was collected from each participant at the time of cohort enrollment, and to confirm the lipid body changes according to the treatment results, a group showing high disease activity 6 months after treatment with DMARDs (disease modifying anti-rheumatic drugs) (DAS28 ≥3.2) and a group with low disease activity (DAS28 < 3.2), the former was classified as a non-responder, and the latter was classified as a good responder, and serum was obtained. In addition, 19 RA patients in sustained remission, defined as patients with a DAS28 ≤ 2.6 at 3 consecutive measurements for 12 months, were selected. Lipids were extracted from serum collected from each group of patients using MTBE (tert-methyl butyl ether) method, and mass spectrometry-based lipidomics analysis was performed. Specifically, lipids were separated on a UPLC system equipped with a C18 column (2.1 × 100 mm, 1.7 μm) coupled with a guard column (2.1 × 5 mm, 1.7 μm), and data were collected. For data preprocessing, the collected data was imported into MS-DIAL ver 4.38, and batch effects were removed with LOESS (locally estimated scatterplot smoothing) algorithm. After peak annotation, unreliable lipids were removed when the relative standard deviation (%) of the quality control (QC) sample was >30%. Referenced lipids were putatively identified based on precursor ion m/z values and product ion patterns consistent with data from MS-DIAL's LipidBlast database. In order to increase the reliability of lipid identification as MSI (metabolomics standards initiative) 1, the identified lipid list was confirmed as an in-house lipid library including retention time. All lipid features were normalized according to the median intensity of each sample, and a t-test was performed to obtain FDR, and a paired t-test was used to compare groups before and after treatment. To compare three or more groups, significant differences were identified using Tukey's HSD and one-way ANOVA. Suspected outliers were excluded based on comprehensive analysis using heatmap, PCA, and random forest algorithms, correlation analysis was performed using the Hmisc package of R, and Pearson's correlation was determined using the rcorr function. In addition, two different multivariate statistical analysis models (unsupervised and supervised) were applied to distinguish groups (unsupervised, PCA; supervised, PLS-DA). The PLS-DA model was cross-validated using leave-one-out cross validation (LOOCV), and the Q2 value was used to estimate the overfitting value of the model. In addition, in order to further confirm the detailed differences between the patient groups, OPLS-DA (orthogonal projections to latent structures-discriminant analysis) analysis was performed. In addition, biomarker candidates are | r | Selection was made when <0.5 and when the FDR value was <0.25 in the t-test.
[표 1][Table 1]
(CAR: acyl carnitine; Cer: ceramide; LPC: lysophosphatidylcholine; LPC O: ether-linked LPC; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PE P: ether-linked PE; 및 TG: triacylglycerol)(CAR: acyl carnitine; Cer: ceramide; LPC: lysophosphatidylcholine; LPC O: ether-linked LPC; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PE P: ether-linked PE; and TG: triacylglycerol)
그 결과, 좋은 치료반응군 (치료 후 DAS28 <3.2)에서는 41 개의 혈청 지질 (t-test로 확인 된 37 개의 지질 및 OPLS-DA에 의해 23 개의 지질, 표 1)에서 유의한 변화가 나타났으나, 나쁜 치료반응군, 즉, 치료 비반응군 (치료 후 DAS28 ≥3.2)에서는 지질체에서 차이가 나타나지 않았다 (Regularized Hotelling’s T2 [RHT] test (20): 좋은 치료 반응군 = 33.724, p-value = 0.035; 치료 비-반응군 = 6.294, p-value = 1.000) (도 1). 통계적 차이는 RHT test를 통하여 검정하였다 (도 2). 특히, 치료 비-반응군에서와 달리, 좋은 치료반응군에서 혈청 LPC, EtherLPC, PC, PE, EtherPE, 세라마이드 (Cer-NS) 42:1, SM 및 TG 서브 클래스는 증가하고 CAR 서브 클래스는 감소하였다. 이를 통해, 류마티스관절염의 치료 반응과 관련된 지질로서, CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) 및 TG 54:9를 선발하였으며 (표 2), 이들 지질을 류마티스관절염의 약물 치료 후 반응을 예측할 수 있는 바이오마커로 사용할 수 있는 것을 알 수 있다.As a result, significant changes were found in 41 serum lipids (37 lipids confirmed by t-test and 23 lipids by OPLS-DA, Table 1) in the good treatment responders (DAS28 <3.2 after treatment). , there was no difference in the lipid body in the poor treatment responders, that is, the treatment non-responders (DAS28 ≥3.2 after treatment) (Regularized Hotelling's T2 [RHT] test (20): good treatment responders = 33.724, p-value = 0.035; treatment non-responders = 6.294, p-value = 1.000) (FIG. 1). Statistical differences were tested through the RHT test (FIG. 2). In particular, unlike in the treatment non-responders, serum LPC, EtherLPC, PC, PE, EtherPE, ceramide (Cer-NS) 42:1, SM and TG subclasses increased and CAR subclasses decreased in the good treatment responders. did Through this, as lipids related to the therapeutic response of rheumatoid arthritis, CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6(1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2 , PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) and TG 54: 9 was selected (Table 2), and it can be seen that these lipids can be used as biomarkers that can predict the response after drug treatment of rheumatoid arthritis.
[표 2][Table 2]
Claims (14)
- 아실 카르니틴(acyl carnitine, CAR), 세라마이드 스핑고신(Ceramide sphingosine, Cer-NS), 리소포스파티딜콜린(lysophosphatidylcholine, LPC), 에테르-가교 LPC(ether-linked LPC, LPC O), 포스파티딜콜린(phosphatidylcholine, PC), 에테르-가교 PC(ether-linked phosphatidylcholine, PC O), 포스파티딜에탄올아민(phosphatidylethanolamine, PE), 에테르-가교 포스파티딜에탄올아민(ether-linked phosphatidylethanolamine, PE P), 스핑고미엘린(sphingomyelin, SM) 및 트리아실글리세롤(triacylglycerol, TG)로 이루어진 군에서 선택되는 하나 이상의 지질을 포함하는, 류마티스 관절염 치료제에 대한 반응성 진단용 바이오마커 조성물.Acyl carnitine (CAR), ceramide sphingosine (Cer-NS), lysophosphatidylcholine (LPC), ether-linked LPC (LPC O), phosphatidylcholine (PC), ether-linked phosphatidylcholine (PC O), phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (PE P), sphingomyelin (SM) and triacyl A biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis, comprising at least one lipid selected from the group consisting of glycerol (triacylglycerol, TG).
- 제 1항에 있어서, 아실 카르니틴은 12:0, 14:1 및 16:1이고, 세라마이드 스핑고신은 42:1이며, 리소포스파티딜콜린은 16:0, 16:1, 18:1, 18:2, 18:3, 20:1, 20:2, 20:3, 20:4, 20:5, 22:5, 22:6 또는 24:0이고, 에테르-가교 LPC는 16:1, 18:0 또는 24:1이며, 포스파티딜콜린은 32:3, 34:2, 34:5, 36:1, 36:5 (1), 36:6 (1), 40:1, 40:4, 42:10 또는 42:6이고, 에테르-가교는 PC 36:3이며, 포스파티딜에탄올아민은 36:1 또는 40:7이며, 에테르-가교 포스파티딜에탄올아민은 34:2, 36:3 또는 40:6이고, 스핑고미엘린은 38:1 (1) 또는 38:1 (2)이며, 및 트리아실글리세롤은 48:5 (2), 50:6 (4) 또는 54:9인, 류마티스 관절염 치료제에 대한 반응성 진단용 바이오마커 조성물.According to claim 1, acyl carnitine is 12:0, 14:1 and 16:1, ceramide sphingosine is 42:1, lysophosphatidylcholine is 16:0, 16:1, 18:1, 18:2, 18:3, 20:1, 20:2, 20:3, 20:4, 20:5, 22:5, 22:6 or 24:0, and the ether-bridged LPC is 16:1, 18:0 or 24:1, and phosphatidylcholine is 32:3, 34:2, 34:5, 36:1, 36:5 (1), 36:6 (1), 40:1, 40:4, 42:10 or 42 :6, ether-bridging PC is 36:3, phosphatidylethanolamine is 36:1 or 40:7, ether-bridging phosphatidylethanolamine is 34:2, 36:3 or 40:6, sphingomyelin is 38:1 (1) or 38:1 (2), and triacylglycerol is 48:5 (2), 50:6 (4) or 54:9, a biomarker composition for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis .
- 제 1항의 조성물을 포함하는, 류마티스 관절염 치료제에 대한 반응성 진단용 키트.A kit for diagnosing responsiveness to a therapeutic agent for rheumatoid arthritis, comprising the composition of claim 1.
- (a) 검사 대상체로부터 분리한 생물학적 시료에서 아실 카르니틴, 세라마이드 스핑고신, 리소포스파티딜콜린 에테르-가교 LPC, 포스파티딜콜린, 에테르-가교 PC, 포스파티딜에탄올아민, 에테르-가교 포스파티딜에탄올아민, 스핑고미엘린 및 트리아실글리세롤로 이루어진 군에서 선택되는 하나 이상의 지질의 발현 수준을 측정하는 단계; 및 (a) acyl carnitine, ceramide sphingosine, lysophosphatidylcholine ether-bridged LPC, phosphatidylcholine, ether-bridged PC, phosphatidylethanolamine, ether-bridged phosphatidylethanolamine, sphingomyelin and triacylglycerol in a biological sample isolated from a test subject; Measuring the expression level of one or more lipids selected from the group consisting of; and(b) 류마티스 관절염 치료제에 대한 반응성을 판단하는 단계를 포함하는, 류마티스 관절염 치료제에 대한 반응성을 평가하기 위한 방법.(b) a method for evaluating responsiveness to a therapeutic agent for rheumatoid arthritis, comprising determining the responsiveness to the therapeutic agent for rheumatoid arthritis.
- 제 4항에 있어서, 대상체의 시료에서 Cer-NS 42:1, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2) 및 TG 54:9의 농도가 치료제 처리 전 시료에서의 발현 수준 대비 상향 조절되고, CAR 12:0, CAR 14:1 및 CAR 16:1의 농도가 치료제 처리 전 시료에서의 발현 수준 대비 하향 조절되면 치료적으로 반응성인 것으로 판단하는, 류마티스 관절염 치료제에 대한 반응성을 평가하기 위한 방법.The method of claim 4, wherein Cer-NS 42: 1, LPC 16: 1, LPC 18: 1, LPC 18: 2, LPC 18: 3, LPC 20: 1, LPC 20: 2, LPC 20: 3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32 :3, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2) The concentrations of TG 54:9 and TG 54:9 were upregulated compared to the expression level in the sample before treatment, and the concentrations of CAR 12:0, CAR 14:1 and CAR 16:1 were compared to the expression level in the sample before treatment A method for assessing responsiveness to a therapeutic agent for rheumatoid arthritis, which is judged to be therapeutically responsive when downregulated.
- 제 5항에 있어서, 반응성은 치료제 처리 후 DAS28 <3.2인 것인, 류마티스 관절염 치료제에 대한 반응성을 평가하기 위한 방법. The method for evaluating responsiveness to a therapeutic agent for rheumatoid arthritis according to claim 5, wherein the responsiveness is DAS28 <3.2 after treatment with the therapeutic agent.
- 제 4항에 있어서, 대상체의 시료에서 CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) 및 TG 54:9가 치료제 처리 전 시료에서의 발현 수준 대비 발현 정도에 변화가 없으면 치료적으로 비-반응성일 것으로 판단하는, 류마티스 관절염 치료제에 대한 반응성을 평가하기 위한 방법.5. The method of claim 4, wherein the CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18: 2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O- 16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P -36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) and TG 54:9 are therapeutics A method for evaluating responsiveness to a therapeutic agent for rheumatoid arthritis, which is determined to be therapeutically non-responsive if there is no change in expression level compared to expression level in a sample before treatment.
- 제 7항에 있어서, 비-반응성은 치료제 처리 후 DAS28 ≥3.2인 것인, 류마티스 관절염 치료제에 대한 반응성을 평가하기 위한 방법. The method for evaluating responsiveness to a therapeutic agent for rheumatoid arthritis according to claim 7, wherein non-responsiveness is DAS28 ≧3.2 after treatment with the therapeutic agent.
- 제 4항에 있어서, 생물학적 시료는 혈액 또는 혈청인, 류마티스 관절염 치료제에 대한 반응성을 평가하기 위한 방법. The method according to claim 4, wherein the biological sample is blood or serum.
- (a) 검사 대상체로부터 분리한 생물학적 시료에서 아실 카르니틴, 세라마이드 스핑고신, 리소포스파티딜콜린 에테르-가교 LPC, 포스파티딜콜린, 에테르-가교 PC, 포스파티딜에탄올아민, 에테르-가교 포스파티딜에탄올아민, 스핑고미엘린 및 트리아실글리세롤로 이루어진 군에서 선택되는 하나 이상의 지질의 발현 수준을 측정하는 단계; 및 (a) acyl carnitine, ceramide sphingosine, lysophosphatidylcholine ether-bridged LPC, phosphatidylcholine, ether-bridged PC, phosphatidylethanolamine, ether-bridged phosphatidylethanolamine, sphingomyelin and triacylglycerol in a biological sample isolated from a test subject; Measuring the expression level of one or more lipids selected from the group consisting of; and(b) 대상체의 시료에서 지질의 발현 수준을 정상 시료에서의 발현 수준과 비교하여 류마티스관절염의 예후를 판단하는 단계를 포함하는, 류마티스 관절염의 예후 예측을 위한 정보제공방법.(B) an information providing method for predicting the prognosis of rheumatoid arthritis, comprising the step of determining the prognosis of rheumatoid arthritis by comparing the expression level of the lipid in the sample of the subject with the expression level in the normal sample.
- 제 10항에 있어서, 대상체의 시료에서 Cer-NS 42:1, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2) 및 TG 54:9의 농도가 치료제 처리 전 시료에서의 발현 수준 대비 상향 조절되고, CAR 12:0, CAR 14:1 및 CAR 16:1의 농도가 치료제 처리 전 시료에서의 발현 수준 대비 하향 조절되면 류마티스관절염 예후가 좋은 것으로 판단하는, 류마티스 관절염의 예후 예측을 위한 정보제공방법.The method of claim 10, wherein Cer-NS 42: 1, LPC 16: 1, LPC 18: 1, LPC 18: 2, LPC 18: 3, LPC 20: 1, LPC 20: 2, LPC 20: 3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32 :3, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2) The concentrations of TG 54:9 and TG 54:9 were upregulated compared to the expression level in the sample before treatment, and the concentrations of CAR 12:0, CAR 14:1 and CAR 16:1 were compared to the expression level in the sample before treatment A method for providing information for predicting the prognosis of rheumatoid arthritis, in which the prognosis of rheumatoid arthritis is judged to be good when downregulated.
- 제 11항에 있어서, 예후가 좋은 것은 치료적으로 반응성인 것인, 류마티스 관절염의 예후 예측을 위한 정보제공방법.The method of providing information for predicting the prognosis of rheumatoid arthritis according to claim 11, wherein a good prognosis is therapeutically responsive.
- 제 10항에 있어서, 대상체의 시료에서 CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18:2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O-16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P-36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) 및 TG 54:9가 치료제 처리 전 시료에서의 발현 수준 대비 발현 정도에 변화가 없으면 류마티스관절염 예후가 나쁜 것으로 판단하는, 류마티스 관절염의 예후 예측을 위한 정보제공방법.11. The method of claim 10, wherein the CAR 12:0, CAR 14:1, CAR 16:1, Cer-NS 42:1, LPC 16:0, LPC 16:1, LPC 18:1, LPC 18: 2, LPC 18:3, LPC 20:1, LPC 20:2, LPC 20:3, LPC 20:4, LPC 20:5, LPC 22:5, LPC 22:6, LPC 24:0, LPC O- 16:1, LPC O-18:0, LPC O-24:1, PC 32:3, PC 34:2, PC 34:5, PC 36:1, PC 36:5 (1), PC 36:6 (1), PC 40:1, PC 40:4, PC 42:10, PC 42:6, PC O-36:3, PE 36:1, PE 40:7, PE P-34:2, PE P -36:3, PE P-40:6, SM 38:1 (1), SM 38:1 (2), TG 48:5 (2), TG 50:6 (4) and TG 54:9 are therapeutics A method for providing information for predicting the prognosis of rheumatoid arthritis, in which the prognosis of rheumatoid arthritis is judged to be bad if there is no change in the expression level compared to the expression level in the sample before treatment.
- 제 13항에 있어서, 예후가 나쁜 것은 치료적으로 비-반응성인 것인, 류마티스 관절염의 예후 예측을 위한 정보제공방법.The method for providing information for predicting the prognosis of rheumatoid arthritis according to claim 13, wherein the poor prognosis is therapeutically non-responsive.
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| CUPPEN BART V. J., FU JUNZENG, VAN WIETMARSCHEN HERMAN A., HARMS AMY C., KOVAL SLAVIK, MARIJNISSEN ANNE C. A., PEETERS JUDITH J. W: "Exploring the Inflammatory Metabolomic Profile to Predict Response to TNF-α Inhibitors in Rheumatoid Arthritis", PLOS ONE, vol. 11, no. 9, pages 1 - 18, XP093010542, DOI: 10.1371/journal.pone.0163087 * |
| FERREIRA HELENA BEATRIZ, MELO TÂNIA, PAIVA ARTUR, DOMINGUES MARIA DO ROSÁRIO: "Insights in the Role of Lipids, Oxidative Stress and Inflammation in Rheumatoid Arthritis Unveiled by New Trends in Lipidomic Investigations", ANTIOXIDANTS, vol. 10, no. 45, pages 1 - 21, XP093010545, DOI: 10.3390/antiox10010045 * |
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| MACIEJEWSKI MATEUSZ, SANDS CAROLINE, NAIR NISHA, LING STEPHANIE, VERSTAPPEN SUZANNE, HYRICH KIMME, BARTON ANNE, ZIEMEK DANIEL, LEW: "Prediction of response of methotrexate in patients with rheumatoid arthritis using serum lipidomics", SCIENTIFIC REPORTS, vol. 11, no. 7266, pages 1 - 6, XP093010547, DOI: 10.1038/s41598-021-86729-7 * |
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