TWI565949B - Method for assessing asthma and lung function - Google Patents

Description

Method for assessing asthma and lung function

The present invention relates to a method for assessing asthma and lung function, and more particularly to a method for assessing asthma and lung function in a betel nut.

Areca is a common species estimated by about 600 million people around the world, especially India, the South Pacific Islands and Southeast Asia. Areca is also widely used by Asian immigrants in the United States, the United Kingdom, and Europe.

The alkaloids contained in betel nut are mainly arecoline and arecoline, and arecoline, which is a choline alkaloid, constitutes the dry weight of 0.15-0.67% betel nut chew. Arecoline and arecoline have been used as biomarkers for chewing betel nut habits, and chewing betel nut is recognized as an independent interference factor for oral cancer.

Asthma is a chronic inflammatory disease mediated by some chemokines. Eotaxin-1 belongs to the family of CC chemokines. When the respiratory tract is stimulated by allergens, it can effectively mobilize eosinophils into the respiratory tract, which is an important role in the cause of asthma. . Physicians often diagnose asthma with clinical diagnosis and pulmonary function tests, and there are currently many drugs available for the treatment of asthma. According to the guidelines for asthma diagnosis and treatment published by the Global Initiative For Asthma (GINA), it is currently based on the control of the patient's asthma state. However, due to the complexity of the pathogenesis of asthma, it involves both genetic factors and environmental factors. In the course of treatment, it is often found that the same dose of the drug is given to different patients, some patients are not very good at the drug, and some can achieve good control. As a result, approximately one in four patients still do not achieve the control status defined in the treatment guidelines after treatment.

It is currently known that asthmatic patients who continue to chew on betel nut will aggravate asthma symptoms, but few studies have investigated the relationship between chewing betel nut and asthma. If the specific substance test can be used to infer the risk of asthma in the betel nut chew or the state of lung function, and even by knowing the relationship between betel nut and asthma, specific drugs can be used to control the panting state of the betel nut. This is actually Betel nuts chew the gospel of the eater.

For the sake of the job, the applicant has carefully designed and researched the above-mentioned techniques, and has carefully designed the case to "evaluate the method of asthma and lung function". A brief description.

The present invention is the first to propose that eotaxin-1 is involved in the correlation between arecoline and asthma, and proposes that eotaxin-1 blood protein and arecoline can be used to assess the risk of asthma and the deterioration of lung function in betel nut users.

One of the purposes of the present invention is to provide a method for assessing whether a betel nut chew suffers from asthma, the method comprising: obtaining a sample from a betel nut chew; and measuring an eosinophilic chemokine-1 (eotaxin) in vitro. -1) or a concentration of arecoline in the sample; and comparing the concentration to a standard concentration, if the concentration is higher than the standard concentration, indicating that the betel nut chew suffers from asthma.

Another object of the present invention is to provide a method for assessing whether a betel nut chew has a high risk of suffering from asthma, the method comprising: obtaining a sample from a betel nut chew; and measuring an eosinophil activating chemokine in vitro - a concentration of 1 (eotaxin-1) or arecoline in the sample; and comparing the concentration to a standard concentration, if A concentration above the standard concentration indicates that the betel nut chew has a high risk of suffering from asthma.

Another object of the present invention is to provide a use of eotaxin-1 for assessing whether a betel nut chew suffers from asthma by the aforementioned method.

A further object of the present invention is to provide a method of assessing a lung function of a betel nut chew that suffers from asthma. The method comprises the steps of: obtaining an integral sample from the betel nut chew; measuring a concentration of a arecoline in the in vivo sample in vitro; and determining the lung function based on the concentration.

A further object of the present invention is to provide a use of arecoline for assessing a lung function of a betel nut chew that suffers from asthma by the aforementioned method.

A further object of the present invention is to provide an use of an eotaxin-1 inhibitor which is useful as a medicament for a betel nut chew that suffers from asthma.

The case can be further explained by the following detailed explanation:

The first panel shows the effect of different concentrations of arecoline on eotaxin-1 in cultured dermal fibroblasts in the presence/absence of TNF-[alpha] and IL-4. *p=0.01. The experimental group indicated that fibroblasts were treated with 50 ng/ml of IL-4 and 100 ng/ml of TNF-α for 72 hours; the basal group showed that fibroblasts were not induced by IL-4 and TNF-α, only at different concentrations of areca nut. Alkali treatment.

The second panel shows the effect of different concentrations of arecoline on eotaxin-1 in the presence/absence of TNF-α and IL-4 in cultured gingival fibroblasts. *p=o.03. The experimental group indicated that fibroblasts were treated with 50 ng/ml of IL-4 and 100 ng/ml of TNF-α for 72 hours; the basal group showed that fibroblasts were not induced by IL-4 and TNF-α, only at different concentrations of areca nut. Alkali treatment.

The method of the present invention will be fully understood from the following description of the embodiments and can be accomplished by those skilled in the art. However, the implementation of this case is not limited to the following examples.

Statistical analysis of the present invention

The two groups were compared using χ2 and t-tests to assess statistical significance. Due to the skewed distribution of serum-specific immunoglobulin E (IgE), high-sensitivity C-reactive protein (HS-CRP), arecoline, arecoline, and eotaxin-1, the logarithm of the amount of expression was taken. To control the impact of potential interference factors, logistic regression was used for each interference factor to calculate the corrected odds ratio (OR) and 95% confidence interval. Four groups of experimental groups (with asthma) and control groups (without asthma) who chewed betel nut or did not chew betel nut were compared using one-way variance analysis and least significant difference (LSD) multiple comparison test. Release of eotaxin-1 between cells treated with different doses of arecoline. In this case, the Pearson correlation coefficient (r value) was calculated to assess the association between arecoline, arecoline and lung function. The attributive component of the present invention is estimated by the following formula: the frequency of chewing betel nut in the test group × [(winning ratio - 1) / odds ratio], wherein the odds ratio for the effect of chewing betel nut on asthma is based on multiple logistic regression models. To evaluate. In addition, the present invention also uses the software package SPSS 14.0 for statistical analysis.

Please refer to the first table for a comparison of features between a control group consisting of 600 asthmatic subjects and a control group consisting of 1200 non-asthmatic subjects. Prior to statistical analysis, the total IgE volume was logarithmically transformed to meet the assumption of a normal distribution. Immunoglobulin IgE has been shown to be a target for diseases causing asthma and allergies. As can be seen from the first table, the experimental group did significantly have a higher IgE amount than the control group. There was no significant difference in alcohol intake between the two groups, but smoking and chewing betel nuts The proportion of sputum was higher in the experimental group than in the control group, and there were also significant differences between the two groups in terms of BMI.

Please refer to the second table, which is a logistic regression analysis of the corrected correlation between betel nut chewing and asthma. The concentration detection of eotaxin-1 in the table can be achieved by a kit well known to those skilled in the art that can perform protein quantification, packaging products, reagents, and the like. The potential interference factors corrected in the second table included: age, gender, BMI, and smoking. After correction, relative to the control group who had never consumed betel nut, The risk of pre- and current betel nut chews suffering from asthma was 1.60 and 2.05 times that of the control group, respectively. The attributable risk of asthma caused by arecoline can be calculated as 0.435 (0.77/1.77). The proportion of the attributive component for all adults was 4.9% [(68/600) x 0.45], 11.7% for men and 1.2% for women. If eotaxin-1 is included in multiple logistic regression analysis, the association between current betel nut chews and asthma is significantly reduced. The Sobel-Goodman test is used to verify whether the relationship between the variables has a mediating effect. The present invention also uses this test to test whether eotaxin-1 mediates the association between arecoline and asthma and obtains 0.08 (p < 0.001). Sobel-Goodman coefficient. Regression models of asthma and betel nut chews (p < 0.001) and log-eotaxin (mediator variables) and betel nut chew regression models (p < 0.001) were statistically significant. Furthermore, in log-eotaxin (mediation variable) and betel nut chews in multiple regression of asthma, only log-eotaxin was significant for asthma (p < 0.001), while betel nut chews were less pronounced for asthma. (p<0.045). The total effect of betel nut chewing on the pathogenesis of asthma, about 50.5% is the mediating effect via eotaxin-1, and reached statistical significance. Taken together, the eotaxin-1 is an important role in mediating the association between arecoline and asthma.

Please refer to the third table, which shows the association of corrected interference factors between asthma, betel nut chews, plasma biomarkers, and lung function in four groups of subjects (n=147). The four groups of subjects were further divided into two groups, the experimental group (with asthma) and the control group (without asthma), which were further divided into two groups with or without chewing betel nut. The amount of arecoline and arecoline in plasma was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis. The amounts of arecoline, arecoline, IgE, hs-CRP, leptin, TGF-β1, and eotaxin-1 were taken before the statistical analysis to determine the logarithm of the normal distribution. Pulmonary function is measured by Spirometry. The lung function indicators measured by the present invention include Forced vital capacity (FVC) and forced expiratory volume in the first second exhaled volume (Forced expired volume in one second, FEV1). ), whose value is expressed as a percentage (%) relative to the predicted value. The normal value of FVC% is more than 80% of the expected value, and the expected value is a predicted value derived from age, gender, and height control.

As can be seen from the third table, the amount of arecoline, arecoline and eotaxin-1 in the experimental group were significantly higher in the group currently eating betel nut, while FEV1% and FVC% were currently eaten. There was a significant drop in the betel nut group. Furthermore, based on the aforementioned statistical analysis, it was confirmed that eotaxin-1 is an important role mediating the association between arecoline and asthma. Therefore, the present invention can utilize the concentration of eotaxin-1 to evaluate whether a betel nut chew suffers from asthma, and the method is performed by the following steps. First, a blood sample of the betel nut chew was obtained, and the concentration of eotaxin-1 in the sample was measured in vitro. Secondly, comparing the concentration with a standard concentration, if the concentration is higher than the standard concentration, it indicates that the betel nut chew suffers from asthma or indicates that it has a high risk of suffering from asthma. As can be seen from the third table, the concentration of the standard can be from any of the following three groups: non-betel nut chews with asthma, betel nut chews without asthma, and non-betel nut chews without asthma. That is, as long as a betel nut chews the blood in the eater If the eotaxin-1 concentration is higher than the average eotaxin-1 concentration in the blood of any of the above three groups, it can be judged that the betel nut chew suffers from asthma or has a high risk of suffering from asthma. Preferably, the data of 147 subjects can be further classified according to the interference factor (ie, age, BMI, and smoking) in the database, and the interference factor of the assessee is considered when selecting the concentration of the standard. Selecting data that is close to its interference factor as a standard concentration increases the accuracy of the assessment. This method is to assess whether the betel nut chew suffers from asthma by statistical analysis. If the following clinical symptom assessment can be added, it will be more certain whether the assessor suffers from asthma: (1) there is wheezing; (2) Have the following medical history: i) cough, especially severe at night, ii) repeated wheezing, iii) repeated breathing difficulties, iv) repeated chest tightness; (3) more symptoms occur at night or worsen, leading to sleep disruption; (4) symptoms occur Or deterioration is related to seasonal alternation; (5) family history of asthma and allergies; (6) frequent frequency of wheezing, more than once a month; (7) coughing or wheezing during exercise; (8) no respiratory infection Night cough; (9) wheezing episodes in any season; (10) symptoms persist after more than 3 years of age; and (11) symptoms may appear or worsen if the following conditions are encountered: inhaled allergens (eg hairy animals, dust mites, cockroaches, mold, pollen, smoke), chemicals, drugs (such as aspirin, beta blocker, exercise, strong mood changes and respiratory tract) Infection (especially viral infection, feeling The symptoms will be more than 10 days) and so on.

Please refer to the fourth table, which shows the amount of arecoline and arecoline for the association of the amount of eotaxin-1 and lung function in a chewing betel nut with asthma. The amounts of arecoline, arecoline and eotaxin-1 were taken before the statistical analysis to determine the logarithm of the normal distribution. From the r coefficient in the table, there is a significant positive correlation between arecoline and eotaxin-1, and a significant negative correlation between FEV1% and FVC% for lung function predictors; and betel nut base for FEV1% and FVC%. Significant negative correlation. Therefore, the higher the concentration of arecoline or arecoline in asthmatic patients, the lower their lung function. Take β±sd=-1.009±0.222 in the table as an example, which means that it is relative to not eating. For the asthmatic patients of betel nut, the lung function (FEV1%) decreased by 1.009% for every 1 ng/ml of arecoline (ng/ml) of betel nut patients who consumed betel nut. In some of the subjects in this study, some betel nut patients who consumed betel nut had a blood concentration of 40 ng/ml or more, which means that lung function (FEV1%) decreased by 40.04%, which has a great impact on lung function damage. .

According to the data of the fourth table, the present invention proposes a method of evaluating the lung function of a betel nut chew person suffering from asthma, which is performed by the following steps. First, a blood sample of the betel nut chew was obtained, and the concentration of arecoline in the sample was measured in vitro. Secondly, comparing the concentration with a standard concentration, the FEV1% of the betel nut chew will decrease by 1.009% and the FVC% will decrease by 0.806% for each increase of 1 ng/ml of the measured concentration. The concentration of the standard is preferably the average arecoline concentration in the blood of a non-beech chewing person who has asthma. Preferably, when selecting the concentration of the standard, the data of the evaluator may be selected to be similar to the interference factor as the standard concentration, thereby improving the accuracy of the evaluation. This method is a statistical analysis to assess the deterioration of lung function in betel nut chews with asthma, if combined with clinical pulmonary function measurement methods (such as the use of spirometry and peak expiratory flow (PEF) variability, etc.) More reliable evaluation results will be available.

Since betel palm is currently known to constitute a dry weight of 0.15-0.67% betel nut chews, those skilled in the art can also estimate the amount of arecoline in the blood by the amount of betel nut that the patient eats every day through a simple experiment. Therefore, it is also possible to roughly estimate the damage of lung function by the amount of betel nut that the patient eats every day.

In order to study the functional correlation between the amounts of arecoline and eotaxin-1, the following tests were carried out. First, dermal and gingival fibroblasts are obtained from skin samples of normal human skin or oral cavity. Skin or gum tissue was taken, cut to 1-2 cubic millimeters, and then cultured for 7 days in a DMEM dish containing 10% FBS to harvest the fiber cells. The third generation of human fibroblasts were inoculated. Dermal fibroblasts were harvested and induced with IL-4 and TNF-[alpha], both of which are standard agents that induce the release of eotaxin-1 by fibroblasts. Fibroblasts were treated with 50 ng/ml IL-4 and 100 ng/ml TNF-α (both from Peprotech, Inc., Rock Hill, NJ) for 72 hours to induce release of eotaxin. Cells were also pretreated with arecolumine at different concentrations (0, 10, 25, 100 and 200 μg/ml) for the first 24 hours. Three replicate cells were evaluated for each treatment condition. Finally, the supernatant of the cells was taken for measurement of the amount of eotaxin-1 released (mean ± SD) by ELISA. The amount of eotaxin-1 (mean ± SD) was analyzed by One Way ANOVA and Bonferroni multiple correction test (P value adjusted by multiplying by 10). The results are shown in the first and second figures.

As can be seen from the results of the first panel, treatment of dermal fibroblasts with arecoline at concentrations of 0, 10, 25, 100 and 200 μg/ml alone induced only a small amount of eotaxin-1 release (for either arecoline) The dose was measured, and the amount of eotaxin-1 was approximately in the range of 0 ± 0 pg / ml to 23 ± 2 pg / ml). However, under the stimulation of TNF-α and IL-4, 100 μg/ml of arecoline treatment significantly induced skin fibers relative to eotaxin-1 (1850 ± 142 pg/ml) produced by benithine treatment at 0 μg/ml. The mother cells produced eotaxin-1 at 2700 ± 98 pg/ml (p = 0.01, using Bonferroni multiple calibration test).

Since the chewed betel nut will act directly on the oral mucosa rather than the skin, the above experiment was also repeated with gingival fibroblasts to consolidate the causal relationship, and the results are shown in the second panel. As can be seen from the results of the second panel, the arecoline concentrations from 0 to 200 μg/ml did not cause eotaxin-1 release (the eotaxin-1 measured for any of the arecoline test doses was 0 ± 0 pg/ml). In the presence of TNF-α and IL-4, 100 μg/ml of arecoline treatment significantly induced gingival fibrils relative to eotaxin-1 (1044 ± 95 pg/ml) produced by betelsine treatment at 0 μg/ml The cells produced eotaxin-1 at 1489 ± 78 pg/ml (p = 0.03). The results of both the first and second graphs indicate that the pattern in the cells also confirmed that arecoline can increase the amount of eotaxin-1 expression.

The pathogenesis of asthma is very complicated, and no one has ever studied the pathogenic relationship between betel nut and asthma. Under the guidance of GINA, the principle of asthma treatment is based on complete control of asthma to restore and avoid the patient's asthma symptoms and airway obstruction. The drugs for the treatment of asthma can be divided into two categories according to their main functions: control drugs (also known as long-term preventive drugs) and relieve drugs (also known as rapid relief drugs). Controlled drugs are therapeutic drugs that must be used daily for a long period of time, including anti-inflammatory drugs and long-acting bronchodilators. Anti-inflammatory drugs can stop the inflammatory response of the airway, and have the effect of preventing and suppressing the inflammation of the airway. Therefore, the frequency of deterioration of asthma patients can be reduced, chronic symptoms can be reduced, lung function is improved, airway hyperreactivity is reduced, and quality of life is improved. Currently used anti-inflammatory drugs are mainly steroids, pharyngeal Yongle, nedocromil, sustained-release theophylline and leukotriene Modifiers, among which steroids have the best efficacy, and they have both preventive and therapeutic effects. . Relief drugs mainly include short-acting or fast-acting bronchodilator drugs that rapidly relieve bronchoconstriction in asthmatic patients and their accompanying acute symptoms such as cough, chest tightness, and wheezing. Bronchodilators include short-acting or fast-acting sympathomimetic agonists, parasympathetic antagonists, and short-acting theophylline, with beta -type sympathetic stimulants being the most commonly used and most effective bronchodilator.

The experiments of the present invention confirmed that eotaxin-1 is an important role mediating the association between arecoline and asthma. Specifically, the total effect of betel nut chewing on the pathogenesis of asthma is about 50.5% via eotaxin-1. Mediation effect. For the study of pathogenesis, 50.5% is a fairly high mediation effect. For more complex pathogens, many of the factors involved only show mediation effects of 5%, for example. Therefore, in the pancreatic control of the betel nut chew, those skilled in the art will understand that the purpose of effectively controlling asthma can be achieved particularly by inhibiting the production of eotaxin-1. That is, an inhibitor of eotaxin-1 can be used as an agent for a betel nut chew that suffers from asthma. Although some eotaxin-1 inhibitors are currently known for the treatment of asthma (eg Salmeterol and Formoterol, which are long-acting β 2 sympathetic stimulants and have been widely used for inhalation therapy for asthma), due to asthma The variety of drugs and their choice depends on the degree of asthma control in patients, so clinical physicians may not choose eotaxin-1 inhibitors as the first choice when selecting drugs. In view of betel nut chews, the present invention provides a preferred choice of clinical asthma control drugs that provide better asthma control effects by inhibiting eotaxin-1 for a particular subject of betel nut chews. In the stepwise treatment of asthma, in the third order (moderate persistent asthma), low-dose inhaled steroids can be treated with long-acting type B sympathetic bronchodilators; in the fourth order, medium to high doses can be used. Steroids are combined with long-acting type B sympathetic bronchodilators for treatment. According to clinical studies, low-dose inhaled steroids combined with long-acting dilators are more effective than high-dose inhaled steroids alone, ie, combined therapy improves asthma rather than two drugs. use. Thus, the eotaxin-1 inhibitors of the invention may also be combined with inhaled steroids or other controlled drugs.

Other embodiments of the invention are described below.

1. A high wind that assesses whether a betel nut chew suffers from asthma or has asthma a method comprising the steps of: obtaining a sample from a betel nut chew; determining a concentration of eotaxin-1 in the sample in vitro; and comparing the concentration to a concentration The concentration of the standard, if the concentration is higher than the concentration of the standard, indicates that the betel nut chew suffers from a high risk of asthma or suffering from asthma.

2. A method of assessing a lung function of a betel nut chew that suffers from asthma, comprising: obtaining an integral sample from the betel nut chew; measuring a concentration of a arecoline in the in vivo sample in vitro; The lung function is determined based on the concentration.

3. The method of embodiment 2 wherein the in vivo sample is a blood sample.

4. The method of any of the preceding embodiments, further comprising comparing the measured concentration to a standard concentration.

5. The method according to any of the preceding embodiments, further comprising a first second exhalation (FEV1%) of the betel nut chew per 1 ng/ml of the measured concentration relative to the concentration of the standard. ) will drop by 1.009%.

6. The method of any of the preceding embodiments 2-5, further comprising, for each concentration increase of 1 ng/ml relative to the concentration of the standard, a forced vital capacity (FVC%) of the betel nut chew is decreased 0.806%.

7. The method of any of the preceding embodiments, wherein the standard concentration is a concentration of a arecoline measured from a non-beca chewing eater having asthma.

8. Use of arecoline for assessing a lung function of a betel nut chew that suffers from asthma by the aforementioned method.

9. Use of eotaxin-1 for assessing whether a betel nut chew suffers from asthma or has a high risk of suffering from asthma in the manner described above.

10. Use of an eotaxin-1 inhibitor for use as a medicament for a betel nut chew that suffers from asthma.

The above embodiments are merely illustrative of the exemplary embodiments of the present invention, The scope of the present invention is not limited to the specific embodiments described above, and the present invention is modified by those skilled in the art, and is not intended to be limited by the scope of the application.

Claims (4)

A method for assessing whether a betel nut chew suffers from asthma, comprising the steps of: obtaining a sample from a betel nut chew; in vitro measuring an eotaxin-1 in the sample a concentration; and comparing the concentration to a standard concentration, if the concentration is higher than the standard concentration, indicating that the betel nut chew suffers from asthma. A method for assessing whether a betel nut chew has a high risk of asthma, including the following steps: obtaining a sample from a betel nut chew; in vitro measuring an eotaxin-1 in eosin-1 A concentration in the sample; and comparing the concentration to a standard concentration, if the concentration is higher than the standard concentration, indicating that the betel nut chew has a high risk of suffering from asthma. A use of eotaxin-1 for assessing whether a betel nut chew suffers from asthma, as described in claim 1 of the patent application. An use of an eotaxin-1 inhibitor for use as a medicament for a betel nut chew that suffers from asthma.