KR101799985B1 - Method for Diagnosis of Severe Asthma Using S1P and Sphingosine - Google Patents

Abstract

The present invention relates to a method for diagnosing severe asthma using sphingosine and S1P. More specifically, the present invention relates to a method for diagnosing aspirin hypersensitive asthma by measuring a level of S1P in serum and sphingosine in urine in a patient with asthma. According to the present invention, the technology to diagnose aspirin hypersensitive asthma which is inherence of severe asthma using a biomarker of S1P in serum and sphingosine in urine can be useful for finding out target treatment agents while ensuring clinical advantages and economic feasibility.

Description

Methods for Diagnosis of Severe Asthma Using S1P and Sphingosine {

The present invention relates to a method for diagnosing severe asthma using Sphingosine and S1P, and a method for diagnosing aspirin-hypersensitive asthma by measuring S1P in a urine spiking hyperlipidemia and serum of an asthmatic patient.

Sphingolipids are ubiquitous cell membrane components that regulate various cellular processes such as cell division, differentiation, apoptosis and childhood. Sphingolipid metabolism is a complex pathway that produces S1P (sphingosine-1-phosphate) from various precursors using ceramide backbones. Ceramides are produced in either sphingomyelin or de novo spiking high-quality synthesis. These ceramides are deacetylated to produce sphingosine, which is then phosphorylated by sphingosine kinases 1 and 2 to produce S1P.

On the other hand, the diagnosis of asthma is based on the observation of the clinical symptoms of the asthmatic patients and the intuition of the physician. Therefore, the accuracy of the diagnosis may be decreased. To determine the correct treatment stage, the physician and the patient There is a problem in that a trial and error process must be performed for a considerable period of time.

Severe asthma is a heterogeneous inheritance of eosinophilic asthma, asthma, steroid-resistant asthma, and aspirin-exacerbated respiratory disease (AERD), depending on the asthma inducing substance, the degree of lung function control, the type of airway inflammation, It is a complex disease that appears as a type. There is an attempt to define the intrinsic type by means of sputum eosinophilia, pulmonary function test, and respiratory nitric oxide (NO) measurement. However, there are still limitations and introduction of target treatment according to the definition of intrinsic type of severe asthma and inherent type The need for Therefore, it is essential to use early diagnosis tests using biomarkers.

Biomarkers are defined as features that are objectively measured and evaluated as indicators of normal biological processes, pathological processes, or pharmacological responses to therapeutic intervention. The definition of a biomarker has recently been defined as a protein that further changes in expression are correlated with an increased risk of disease or progression or predicting the response of a disease to a given therapy.

In recent years, studies have been actively carried out to utilize biomarkers of human diseases for early diagnosis by using metabolomic techniques to measure metabolic changes that represent the pathophysiological changes of asthma. However, studies on metabolic studies of severe asthma Is very limited.

The present inventors have made extensive efforts to diagnose severe asthma using a biomarker. As a result, the present inventors have identified the sphingolipid metabolites of severe asthmatic patients and confirmed that S1P of urinary sphingosine and serum can be screened by the metabolism technique, Thereby completing the present invention.

The purpose of the present invention is to determine the concentration of sphingosine in urine of asthmatic patients and to determine the concentration of sphingosine 1-phosphate (S1P) in serum to diagnose aspirin hypersensitive asthma And a method for providing necessary information.

In order to accomplish the above object, the present invention provides a method for measuring the concentration of sphingosine in a sample of urine separated from a subject; And comparing the measured concentration of sphingosine with a control. The present invention also provides a method for providing information necessary for diagnosis of severe asthma.

The present invention also provides a diagnostic kit for severe asthma comprising quantitative substances of sphingosine contained in urine.

The diagnostic technique of aspirin hypersensitive asthma, which is an intrinsic type of severe asthma using S1P biomarkers in urine sphingosine and serum according to the present invention, is advantageous in terms of clinical and economic advantages and is useful for finding a therapeutic target.

Figure 1 shows the change in serum sphingomyelin associated with airway hyperresponsiveness by methacholine during ASA-BPT (lysine-asprin bronchial challenge test). (a) shows the change in serum sphingomyelin in accordance with the ASA-BPT in the asthma group (AERD) worse by aspirin, (b) is a PC ₂₀ - both for the change in serum sphingomyelin in the methacholine and the ground state . ≪ / RTI >
Figure 2 (a) compares serum S1P and urine sphingosin levels between the aspirin hypersensitivity (AERD) and aspirin resistance (ATA) groups, and the basal state of serum S1P and urinary sphingosine and (b) _One (%) Reduction and (c) correlation with serum ferritin levels.
Figure 3 shows the results of an analysis of receiver operating characteristics (ROC) for the basal state of serum S1P and urine sphingosine predicting aspirin hypersensitivity (AERD).
Figure 4 shows the correlation between sphingolipid metabolite levels. (a1) showed a positive correlation between serum S1P and urinary sphingosine in the basal state of ASA-BPT, and (a2) showed a positive correlation between serum S1P and serum sphingosine in symptom onset after ASA-BPT It shows. Serum sphingomyelin (d18: 0/16: 0), (b2) serum sphingomyelin (d18: 0/18), (b3) 1) and (b4) serum LysoSM in the basal state.
FIG. 5 (a) shows the relationship between HLA - DBP1 * 0301 and vasospasm in the basal state, and FIG. 5 (b) shows the relationship between the urinary spiking hypercholesterolemia and the CYSLTR1-634C> T polymorphism in the symptomatic state after ASA-BPT .

Unless otherwise defined, 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. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, the concentration of sphingosine and the concentration of sphingosine 1-phosphate (S1P) in serum were measured in urine of a severe asthma-type aspirin-hypersensitive asthmatic patient who had worsened asthma symptoms after taking aspirin , And it was confirmed that the concentration was significantly increased compared with the aspirin resistant asthmatic patients group, which is a normal control group and a disease control group.

Metabolic studies are used to predict changes in physiological function by tracking changes in metabolic concentrations, the product of various metabolic pathways essential for maintaining physiological function. The present invention can provide a specific metabolite profile according to pathological changes by performing metabolic analysis on blood or urine of patients with severe asthma. This metabolic profile can be used as a marker for early diagnosis of aspirin hypersensitive asthma, which is an inherent type of severe asthma.

Accordingly, the present invention provides a method for measuring the concentration of sphingosine in a sample of urine separated from a subject in one aspect, And comparing the measured concentration of sphingosine with a control. The present invention also relates to a method for providing information necessary for the diagnosis of severe asthma.

The present invention, in another aspect, relates to a diagnostic kit for severe asthma comprising quantitative substances of sphingosine contained in urine.

In the present invention, the step of comparing the concentration of sphingosine with a sample of the control sample may be characterized in that severe asthma is determined when sphingosine is higher than normal. The normal concentration of sphingosine is preferably less than 33.5 pg / ml, but is not limited thereto.

In the present invention, it is preferable to further include a step of measuring the concentration of sphingosine 1-phosphate (S1P) in blood or a serum sample, and more preferably, Or measuring the concentration of sphingosine 1-phosphate (S1P) in a sample of serum; And comparing the measured concentration of sphingosine 1-phosphate (S1P) with a sample of control blood or serum. However, the present invention is not limited thereto.

In the present invention, the normal concentration of sphingosine 1-phosphate is preferably less than 93.5 ng / ml, but is not limited thereto.

The sphingolipid metabolite of the present invention may be characterized by being sphingosine 1-phosphate (S1P) or sphingosine. The sphingolipid of the present invention plays an important role as a main component of the structural membrane in an organism, and at the same time, as a signal transmitter of bioactivity. It is a general term for lipid with sphingosine, which is a long chain base, and can be roughly classified into sphingoglycolipids and sphingolipids.

In the present invention, the severe asthma is preferably an inherent aspirin-hypersensitive asthma, but the present invention is not limited thereto. Aspirin-hypersensitive asthma, which has worsened its asthma symptoms after taking aspirin, has been reported to be an inherited form of severe asthma.

In the present invention, the control group is preferably a normal or aspirin resistant asthmatic patient, but is not limited thereto.

In the present invention, the term sample refers to a metabolite obtained from a liquid sample of blood or urine origin.

According to one embodiment of the present invention, whole blood can be pretreated to detect the sample of the present invention. For example, filtration, distillation, extraction, separation, concentration, inactivation of interfering components, addition of reagents, and the like. In addition, the sample may include a substance produced by metabolism and metabolism, a biological enzyme, and a substance generated by chemical metabolism by a molecule.

In the present invention, the measurement can be characterized in that liquid chromatography through mass spectrometry is performed by a chromatography / mass spectrometer quantification device.

Also, in the present invention, the quantitative substance may be an antibody or an antibody that specifically binds to sphingosine, an antibody that specifically binds to sphingosine 1-phosphate (S1P), or an antibody that binds specifically to sphingosine But it is not limited thereto.

In the present invention, when the concentration of sphingosine is 33.5 pg / ml or more, or when the concentration of sphingosine 1-phosphate is 93.5 ng / ml or more, it is preferable to judge to be severe asthma, but the present invention is not limited thereto.

The chromatography used in the present invention can be carried out by liquid-solid chromatography (LSC), paper chromatography (PC), thin-layer chromatography (TLC), gas- Gas-Solid Chromatography (GSC), Liquid-Liquid Chromatography (LLC), Foam Chromatography (FC), Emulsion Chromatography (EC) Including, but not limited to, gel filtration chromatography (GLC), ion chromatography (IC), Gel Filtration Chromatography (GFC), or Gel Permeation Chromatography All chromatographies for routine use can be used. Preferably, the chromatography used in the present invention is liquid-solid chromatography, more preferably ultra performance liquid chromatography (UPLC).

Meanwhile, the mass analyzer used in the present invention is MALDI-TOF MS, Q-TOF MS or UPLC-LTQ-Orbitrap MS.

The metabolites of the present invention are separated from each other in the UPLC according to different mobility, and confirm the constituent components of the elemental composition as well as accurate molecular weight information using the information obtained through the Q-TOF MS.

If the amount of S1P and sphingosine in the sphingolipid metabolite of the present invention is higher than that of aspirin-resistant asthmatic patients, it can be judged as an inherited form of aspirin-hypersensitive asthma. That is, it is preferable, but not limited, that the optimal reference concentration of S1P and high sour ascites for diagnosis of asthma aspirin is 93.5 ng / mL of S1P and 33.5 pg / mL of urinary sphingolipid.

According to the present invention, the sensitivity of diagnosis of aspirin-hypersensitive asthma by measuring the amount of S1P in the blood was 72.2% and the specificity was 71.1%. The sensitivity of diagnosis of aspirin-hypersensitive asthma by measuring the amount of ascorbic acid in urine was 77.8% And 78.9% respectively.

The term diagnosis is used herein to refer to determining the sensitivity and specificity of an object for a particular disease or disorder, determining whether an object currently has a particular disease or disorder, the prognosis of an object that is afflicted with a particular disease or disorder determining the prognosis, or therametrics (e.g., monitoring the status of the object to provide information about the therapeutic efficacy).

Based on the present invention, S1P and urinary SPP concentration measurement based on the present invention can be used for the early diagnosis of aspirin-hypersensitive asthma, which is an inherent type of severe asthma, and is an important technology for early detection of severe asthma It is possible to use as.

[ Example ]

Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the technical scope of the present invention is not limited to these embodiments.

Example  1: Subjects and samples

The subjects were 45 aspirin - hypersensitive patients (AERD) and 45 aspirin - resistant patients (ATA). The subjects were 29 normal subjects. All patients underwent clinical history, chest radiography, and pulmonary function tests. Values for asthma and FEV ₁ ≥ 50% of the subjects were estimated according to the American Thoracic Society criteria.

After inhaling normal saline, the subjects inhaled a physiological saline solution containing 0.03-25.0 mg / mL 2-fold volume of methacholine. Methacholine is an inhibitor of FEV ₁ To a baseline of 20% or until the last dose was administered. Immediately after the last dose or FEV ₁ When the baseline ≥20% reduction was considered positive. For the ASA-BPT (lysine-asprin bronchogenic test), patients did not use any anti-asthma medications, including leukotriene modifiers, for at least 3 days. ASA-BPT was performed by increasing the dose of aspirin solution (up to 300 mg / mL) and was defined as positive when FEV ₁ % decreased by more than 15% after administration.

Serum and urine were collected at different times, and the baseline state was measured before ASA-BPT and FEV ₁ Were measured. Aspirin-resistant patients were subjected to a second sampling 4 hours after the last dose of lysine-aspirin. The total IgE of serum was measured using ImmunoCAP system (ThermoFisher Scientific, Sweden) and its range is 2-5000 kU / L.

All subjects received AJIRB-GEN-314 SMP-13-108 approval from the Ajou University Hospital Clinical Trials Review Board (IRB).

Table 1 shows the clinical characteristics of the subjects. Aspirin hypersensitivity patients (AERD) showed a significant decrease in FEV ₁ (%) after ASA-BPT (lysine-asprin bronchodilation test) compared to aspirin resistant patients (ATA) P < 0.001). Aspirin hypersensitivity patients were younger (P = 0.045) than aspirin-resistant patients, and aspirin-hypersensitive patients had predicted FEV ₁ (%) and PC ₂₀ values (P <0.001 and P = 0.004) after ASA-BPT (lysine- Compared with patients with aspirin resistance. Patients with aspirin hypersensitivity had a higher incidence of sinusitis and nasal polyps (86.7% vs. 61.9%, P = 0.013 and 37.8% vs 16.7%, P = 0.033), compared to patients with aspirin resistance. There was no significant difference between AERD and ATA in other basic characteristics.

Example  2: Sphing Metabolite  Quantitative analysis

Serum and urine samples were diluted 5-fold each in 80% methanol and water at 4 ° C. The samples were mixed well using a vortexer and then filtered / centrifuged at 4 ° C and 18,341 x g for 20 minutes and then the supernatant was collected and collected in analytical vials.

5 [mu] l of each assay sample was injected through a column heated to 40 [deg.] C. (0.1-0.1% formic acid in 0.1% formic acid) in 98% Solvent A (20 mM ammonium formate in H ₂ O) and 2% Solvent B (0.1% formic acid in methanol) 2% to 98%, followed by 98% of solvent B for 2 minutes, 2% of solvent B 98% for 15.1 minutes to 17 minutes). Performance liquid chromatography system equipped with an ACQUITY UPLC BEH C18, 2.1x50-mm, 1.7 μm (Waters, Milford, MA) column in an Agilent 1290 Infinity II UHPLC system (Agilent Technologies, Santa Clara, Calif.) And Agilent 6530 quadrupole time (Cho et al., Sci. Rep., 5: 8145, 2015) were used to conduct the separation and mass analysis of the metabolites in the sample by linking the mass spectrometer (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA) .

All chromatogram data were converted to a compound exchange file using Mass Hunter DA software (Agilent Technologies) B.04.00 and analyzed using a Mass Profiler Professional (MPP) software B.12.01 (Agilent Technologies) Retention time aligning, centering, scaling, and log 2 transforming were performed.

Each metabolite was identified by comparing the mass spectra with the standard. D-erythro-Sphingosine C-17 (Cayman Chemical Company, Ann Arbor, Mich.) Was used as an internal standard for sphingosine and S1P. The urine concentration was corrected for creatinine and 1,3-dimethyl-2-imidazolidinone (Sigma-Aldrich, St. Louis, MO) was used as the internal standard for creatinine. Each metabolite was quantitated using a calibration curve (r ² ≥ 0.99).

Example  3: On methacoline  Induced hypersensitivity and serum With Sphingomyelin  correlation

Serum and urinary sphingolipid compositions of AERD and ATA patients during ASA-BPT are shown in Table 2.

The AERD group's sphingoid profile showed a unique pattern compared to the ATA group. In the ATA group after ASA-BPT, there was a distorted increase pattern of ceramide and sphingomyelin, but in ceramide and sphingomyelin in the AERD group.

In the AERD group exposed to aspirin, serum sphingomyelin (d18: 0/18: 0, d18: 0/18: 1, d18: 0/24: 0, (p> 0.05, Table 2), but not in the ATA group (P> 0.05, Table 2). Serum sphingomyelin (d18: 0/16: 0, d18: 0/18: 1, d18: 0/24: 0, Lyso SM) showed a positive correlation with methacholine-induced airway hyperreactivity (P = 0.051, P = 0.067, P = 0.033, P = 0.014, P124 = 0.071, P = 0.050, FIG. S1P was significantly increased in the serum and urine samples of both AERD and ATA groups after ASA-BPT (P <0.05, Table 2).

Example  4: AERD  And ATA  Distinguishable As a biomarker S1P  And Sphingosine

Serum S1P and urinary sphingolipid levels were significantly higher in the AERD group than in the ATA group (P <0.001, FIG. 2a) after baseline, ASA-BPT, aspirin-induced symptoms and after ASA-BPT.

To determine the relationship between sphingolipid metabolism and airway inflammatory status, we evaluated the correlation between the level of sphingolipid metabolites in the basal state of ASA-BPT and the biomarkers and clinical characteristics of patients with asthma (Table 3) . Serum S1P and urinary sphingosin levels in the ASA-BPT baseline state correlated with a large decrease in FEV ₁ (%) during ASA-BPT (r = 0.451, P <0.001; r = 0.553, P <0.001 2b).

AERD's new biomarker, Ferrier, has been investigated. The serum level of S1P was weakly correlated with serum ferritin levels in the basal state (r = 0.405, P = 0.001; r = = 0.233, P = 0.033, Fig. 2c).

Using the ROC (receiver operating characteristics) method, serum S1P and urinary sphingosin levels were measured with the AERD diagnostic biomarker (FIG. 3). Serum S1P> 93.5 ng / mL and urine sphingosine 33.5 pmol / mg creatinine (pmol / mg Cr) were selected from the cu 145 t-off value of the optimal ROC curve to generate the highest levels of sensitivity and specificity. Serum S1P 72.2% sensitivity, 71.1% specificity (AUC = 0.705, P = 0.002) and urinary sphingosine 77.8% sensitivity and 78.9% specificity (AUC = 0.816, P <0.001) were found to discriminate AERD from ATA I got burned.

In addition, the correlation between 15-HETE, known as AERD diagnostic biomarker, and basal sphingolipid metabolism was investigated. Serum 15-HETE was positively correlated with serum sphingosine (r = 0.459, P <0.001, Table 3), while serum sphingomyelin (d18: 0/16: 0, d18: (R = -0.338, P = 0.005, r = -0.299, P = 0.013, r = -0.263, P = 0.03, r = = -0.282, P = 0.020, Table 3).

As a result of observing changes in the concentrations of sphingolipid metabolites in severe asthma by LC-MS, the levels of S1P and urinary sphingosine in the aspirin-hypersensitive asthmatic patients were significantly higher than those of the normal control group and the aspirin-resistant asthmatics Compared to the control group.

In conclusion, the optimal baseline concentration of sphingosine 1-phosphate (S1P) in patients with aspirin-hypersensitive asthma is over 93.5 ng / mL, and the optimal baseline concentration of urinary sphingosine is 33.5 pg / mL. The sensitivity of the diagnosis of aspirin hypersensitivity asthma was 72.2% and the specificity was 71.1%. The sensitivity of diagnosis of aspirin hypersensitive asthma was 77.8% and the specificity was 78.9% .

Example  5: Sphing Metabolism  Correlation between

Serum S1P was positively correlated with urinary sphingosine (r = 0.408, P <0.001, Fig. 4, a1) in the ASA-BPT baseline state. Serum S1P showed a weak correlation with urinary sphingosine (r = 0.282, P = 0.007, Fig. 4, a2) after presentation of response symptoms according to ASA-BPT.

The basal serum sphingosine level was negatively correlated with serum sphingomyelin (d18: 0/16: 0, d18: 0/18: 0, d18: 0/18: 1, Lyso SM) , b1-4, Table 4). In addition, urinary sphingomyelin levels were positively correlated with urinary ceramide (Table 5).

Example  6: urine Sphingosine  Level and HLA - DBP1 * 0301 And CYSLTR1 -634C> T  Correlation between polymorphisms

The association of HLA-DBP1 * 0301 and CYSLTR1-634C> T polymorphisms with S1P and sphingosine in 89 asthmatic patients, including 44 AERD and 45 ATA, was investigated (Table 6).

Blood samples were collected before ASA-BPT from 89 AERD and ATA patients. Genomic DNA was isolated from peripheral blood using a Puregene DNA purification kit (Gentra, Minneapolis, MN, USA). High-resolution genotyping of HLA - DBP1 * 0301 was directly sequenced using specific primers and the ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Genotying of CysLTR1-634C> T gene polymorphism (rs321029) was performed by primer extension method using SNaPshot ddNTP primer extension kit (Applied Biosystems).

As a result, patients with the HLA-DBP1 * 0301 allele showed increased levels of urinary spiking hypercholesterolemia after symptom onset according to ASA-BPT (P = 0.012, Fig. 5a). Also, in CYSLTR1 -634C> T polymorphism, patients with heterotypic CT or homozygous TT genotypes had higher levels of urinary sphingolipids in the basal state of ASA-BPT than patients with homozygous CC genotype (P = 0.042, 5b).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (13)

How to provide information for diagnosis of severe asthma including the following steps:
(a) measuring the concentration of sphingosine in a sample of urine separated from a subject; And
(b) comparing the measured sphingosine concentration to a control.
The method according to claim 1, wherein the step (b) is judged to be severe asthma when the concentration of sphingosine is higher than normal.
3. The method of claim 2, wherein the normal concentration of sphingosine is less than 33.5 pg / ml.
3. The method of claim 1, further comprising measuring the concentration of sphingosine 1-phosphate (S1P) in the blood or serum sample.
5. The method of claim 4, wherein the normal concentration of sphingosine 1-phosphate is less than 93.5 ng / ml.
2. The method of claim 1, wherein the severe asthma is an inherited aspirin-hypersensitive asthma.
A diagnostic kit for severe asthma, comprising a sphingosine quantification substance contained in urine.
8. The kit according to claim 7, wherein when the concentration of sphingosine is 33.5 pg / ml or more, it is judged to be severe asthma.
The kit according to claim 7, wherein the quantitative substance is an antibody or an umbrella that specifically binds to sphingosine.
The kit according to claim 7, further comprising a quantitative substance of sphingosine 1-phosphate (S1P) contained in blood or serum.
[Claim 11] The kit according to claim 10, wherein the sphingosine 1-phosphate concentration is 93.5 ng / ml or more.
[Claim 11] The kit according to claim 10, wherein the quantitative substance is an antibody or an umbrella that specifically binds to sphingosine 1-phosphate.
8. The kit of claim 7, wherein the severe asthma is an inherent aspirin-hypersensitive asthma.

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