KR101061562B1 - How to predict the side effects of nonsteroidal anti-inflammatory drugs - Google Patents

Abstract

The present invention relates to a method for predicting side effects of a nonsteroidal anti-inflammatory agent, comprising injecting a non-steroidal anti-inflammatory agent (NSAID) into a living body and measuring the concentration of a specific metabolite contained in the urine of the living body. When the method of predicting the side effects of NSAID of the present invention is used in the development of new drugs, unlike the method using the change pattern of the expression level of the conventional gene, it is not necessary to establish a separate database can save time and cost In addition, since the measured metabolite concentration does not change depending on the NSAID, stable data can be obtained, and thus it may be widely used for the development of more effective NSAID.

Nonsteroidal anti-inflammatory agents, metabolites, allantoin, taurine, acetate, benzoate

Description

How to predict side effects of nonsteroidal anti-inflammatory drugs {Method for Predicting the Side Effects of NSAIDs}

The present invention relates to a method for predicting side effects of nonsteroidal anti-inflammatory agents. More specifically, the present invention relates to a method for predicting side effects of a nonsteroidal anti-inflammatory agent comprising the step of injecting a nonsteroidal anti-inflammatory agent into a living body and measuring the concentration of a specific metabolite contained in the urine of the living body. will be.

Nonsteroidal anti-inflammatory drugs (hereinafter referred to as NSAIDs for convenience) are widely used not only for rheumatic diseases but also for diseases such as neurological diseases, tumors and Alzheimer's disease. It is known as one of the most commonly used therapeutics in. Since the NSAID family of drugs is prescribed for so many patients, many side effects have been reported. For example, many side effects have been reported for NSAID analgesics such as aspirin and ibuprofen, but many patients are aware of these side effects and abuse these drugs. It is known to occur.

NSAIDs have an anti-inflammatory effect mainly by inhibiting the activity of cyclooxygenase (COX) in the body and inhibiting the synthesis of prostaglandin (PG). COX, whose activity is inhibited by NSAIDs, has two types of insoforms: COX-1, which is involved in gastrointestinal blood flow, gastric mucus secretion, and platelet aggregation, and COX-2, which is involved in pain, elevated body temperature, and inflammatory reactions. (Feng, L., et al., Archives of Biochemistry and Biophysics, 307 (2), 361-36, 1993). Most NSAIDs known to date are known to be non-selective inhibitors that inhibit the activity of both COX-1 and COX-2, and only a few NSAIDs are known as selective inhibitors that exhibit efficacy by inhibiting only COX-2 activity. Selective inhibitors cause side effects by inhibiting not only COX-2 but also COX-1 activity. Examples of such side effects have been reported such as gastrointestinal dysfunction, peptic ulcer, heart or renal dysfunction (Vane, J. R., Nature: New Biology, 231 (25), 232-235, 1971). Therefore, efforts have been made to develop NSAIDs as selective inhibitors that inhibit only the activity of COX-2.

According to the 2006 World Population Estimation published by the United Nations, in 2005, the proportion of the elderly aged 65 and over in Korea was 9.1%, and it is expected to increase to 24.1% in 2030, which is expected to enter the aging society. . As such, as elderly people gradually increase in Korea, senile diseases are increasing, and as the number of patients with aging osteoarthritis or rheumatoid arthritis increases, the use of NSAIDs as a therapeutic agent thereof is increasing. Accordingly, various cases of side effects of NSAIDs have been reported, but fundamental research results to prevent them are insufficient (see Lee Sang-woo et al., Korean Journal of Gastroenterology, 44: 246-251, 2004).

Recently, as part of efforts to solve the problem of side effects associated with drugs such as NSAID, a method of measuring the expression level of the gene has attracted attention. In other words, it is known that the expression level of some genes changes rapidly when the drug is administered in the body, which is known to have side effects. Compared to the database, it is anticipated that the use of methods to predict adverse effects on drugs will enable early detection of the adverse effects of new drugs. However, since there is no database containing changes in the expression level of genes and the expression levels of genes vary greatly according to patients and drugs, the method of measuring the expression level of genes is not practical. There was a problem that can not.

Therefore, if there is no need to build an extensive database of drugs and develop a method that can simplify the pattern of drug-dependent changes, it is expected that NSAIDs can predict more effectively whether they have adverse effects. No results have been reported.

Accordingly, the present inventors have diligently researched to develop a method for more effectively predicting whether or not NSAIDs cause side effects. As a result, the present inventors have administered a desired NSAID to a living body, and measured the concentration of a specific metabolite in urine. In this case, it has been confirmed that the NSAID can predict whether or not it shows side effects, thereby completing the present invention.

After all, the main object of the present invention is to provide a method for predicting side effects of NSAIDs by measuring the concentration of certain metabolites in urine.

When the method of predicting the side effects of NSAID of the present invention is used in the development of new drugs, unlike the method using the change pattern of the expression level of the conventional gene, it is not necessary to establish a separate database can save time and cost In addition, since the measured metabolite concentration does not change depending on the NSAID, stable data can be obtained, and thus it may be widely used for the development of more effective NSAID.

In order to develop a method for predicting side effects of NSAIDs, the present inventors came to pay attention to metabolomics method while performing various studies. The metabolite method is a research methodology for analyzing and interpreting the composition and concentration of metabolites (metabolomes) that are changed in various genetic, physiological or environmental conditions, and can be used in various research fields. In the development of new drugs, it is known that the application of the metabolic method in the field related to the identification of side effects of drugs can predict the efficacy or side effects of drugs by identifying the cause of the change in life phenomena caused by the administered drugs.

We administered three NSAIDs (celecoxib, indomethacin, and ibuprofen) to rats after 2 to 5 hours, and collected urine to examine the metabolites in urine by NMR method. As a result of applying the results to the analysis program, the metabolites that were directly related to the occurrence of adverse effects of the NSAID were selected. As a result, 35 metabolites included in the urine affected by the administration of the three drugs were selected. It was. As a result, surprisingly, among the 35 metabolites, the metabolites affected during the administration of the non-selective inhibitors (indomethacin and ibuprofen) with side effects and the selective inhibitors without the side effects (celecoxib) were affected. The metabolites were distinguished from each other.

Subsequently, a PLS-DA analysis program included in a statistical program (SIMCA-P, Umetrics Inc., Kinnelon, NJ, USA) was performed on the selected 35 metabolites to have a variable importance plot (VIP) value of 1. As a result of reselecting the metabolites showing the abnormality, 11 metabolites could be selected again. The selected 11 metabolites are metabolites (group 1) that are all significant when three drugs are administered, and metabolites that are not all significant when all three drugs are administered (group 2). ), Both metabolites (group 3) and non-selective inhibitors of indomethacin and ibuprofen, which did not show significant values, were treated with the selective inhibitor celecoxib. The group belonging to group 3 by dividing into a metabolite (group 4) which shows a significant value only and a non-significant value and a metabolite which shows a non-significant value (group 5). Allantoin and taurine can be used as biomarkers for non-selective inhibitors, and acetate and benzoate belonging to group 4 It was found that could be used as biomarkers for the inhibitors.

Specifically, administration of NSAIDs belonging to selective inhibitors with no side effects to rats significantly increased the concentrations of acetate and benzoate in the urine of rats, whereas the concentrations of allantoin and taurine did not significantly increase, thus acetate and benzo It was found that the Eight could be used as a biomarker for selective inhibitors.

In addition, when administration of NSAIDs belonging to non-selective inhibitors showing side effects to rats, the concentrations of allantoin and taurine in rat urine increased significantly, whereas the concentrations of acetate and benzoate did not significantly increase. It was found that it can be used as a biomarker for non-selective inhibitors.

Therefore, when the target NSAID was administered to the rat and the concentration change of the four metabolites contained in the urine of the rat was observed, it was possible to predict whether the desired NSAID would show side effects.

As a result, the method of predicting the side effects of the nonsteroidal anti-inflammatory drug of the present invention is (i) 2-5 hours after the administration of nonsteroidal anti-inflammatory drug (NSAID) to the experimental group of mammals except humans. Collecting urine to measure the concentration of metabolites associated with the occurrence of adverse effects of the drug in urine; And (ii) comparing the concentration of the metabolite with the concentration of the metabolite contained in the urine of the control group not administered the drug to determine whether side effects occur. At this time, the side effects are not particularly limited to this, gastrointestinal dysfunction, peptic ulcer, heart or renal dysfunction, the metabolite is not particularly limited, it is preferred to use allantoin, taurine, acetate or benzoate. In addition, when the concentration of allantoin and taurine in the metabolite is significantly increased in the urine of the experimental group than the urine of the control group, the concentration of acetate and benzoate does not increase to a significant level, the nonsteroidal anti-inflammatory agent Has the properties of non-selective inhibitors, which can be expected to cause side effects. The concentrations of acetate and benzoate in the metabolites are increased to a significant level in the urine of the experimental group than the urine of the control group, allantoin and taurine If the concentration of does not increase to a significant level, the nonsteroidal anti-inflammatory agent can be expected to have the properties of selective inhibitors, thereby causing no adverse effects, the concentration of allantoin, taurine, acetate and benzoate is particularly Without limitation, NMR analysis It is measured by is preferred.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1 NMR Analysis of Metabolites in Urine of Experimental Animals Administered NSAIDs

NSAIDs as selective or non-selective inhibitors were administered to rats and metabolites with varying concentrations in the urine of the rats were intended to be detected by the NMR method:

Specifically, indomethacin (Sigma Chem. Co., USA) and ibuprofen (international drug) and selective inhibitors, which have a chemical structure of propionate, which are known as a representative gastrointestinal drug among NSAIDs. Phosphorous celecoxib (foreign drug) was orally administered to male Sprague-Dawley-based 5-week old rats at doses of 25 mg / 2 mL kg, 133 mg / 5 mL / kg and 800 mg / 5 mL / kg, respectively. Urine was then collected before and after oral administration of the drug, respectively, and stored at -70 ° C.

Each collected urine was centrifuged (10,000 g, 4 ° C., 10 minutes) to obtain a supernatant, 510 μl of the supernatant and 60 μl of DSS (internal standard solution) were mixed, and then 30 μl of NaN ₃ was added. The mixture was mixed vigorously for 30 seconds and then titrated to pH 6.80 ± 0.02 to obtain a test sample, and 500 μl of the test sample was analyzed using ¹ H-NMR. In this case, the DSS was prepared by dissolving 0.1092 g of DSS (Aldrich CAS: 2039-96-5) and 0.6808 g of imidazole in 100 mL of D ₂ O (Cambridge Isotope Laboratories, Inc., UK). (See FIGS. 1 to 4).

The instrument used in the analysis was as follows:

Analyzer name: Varian Unity Inova 600 MHz NMR, 768AS autosampler

-NMR analysis method: Noesypresat 1D

-spcetral width: 9615.4Hz (9615.4Hz / 600MHz = 16.0ppm, 0 ~ 16ppm)

data point: 76924

Fourier Transformation (FT) spectrum

pulse time: acquisition time 4 sec, relaxation delay 2 sec, mixing time 0.4 sec

number of transient: 64

pre-saturation: relaxation delay 1.5 seconds, saturation power 6, saturation frequency conversion

-shimming: gradient shimming, VnmrJ software

1 shows the NMR spectra of the urine obtained from the control group and rats administered with each nonsteroidal anti-inflammatory agent. Specifically, FIG. 1A shows NMR spectra of urine obtained from rats of the control group, FIG. 1B shows NMR spectra of urine obtained from rats administered indomethacin, a non-selective inhibitor, and FIG. 1C is non-selective. NMR spectra of urine obtained from rats administered with ibuprofen as an inhibitor are shown, and FIG. 1D shows NMR spectra of urine obtained from rats administered with the selective inhibitor celecoxib.

Example 2 Statistical Analysis of Metabolites in Urine of Experimental Animals Administered NSAIDs

The MNR data obtained in Example 1 was applied to a statistical analysis program to select metabolites whose concentrations in urine were significantly changed depending on the type of NSAID (selective inhibitor or non-selective inhibitor).

Each peak in each NMR spectrum of FIG. 1 is divided into 0.04 ppm units to quantify the area of each range peak, and each endogenous metabolite contained in urine using a library included in the analysis program (Chenomx). Qualitatively, the concentration of each metabolite was quantified at the same time. Then, the analyzed data was applied to the SIMCA-P (version 11.0.0.0) program to carry out multivariate statistical analysis, from which biomarker candidates were derived.

First, metabolites with increased concentrations were selected from the metabolites contained in the urine of rats to which the non-selective inhibitor was administered and rats to which the selective inhibitor was administered. At this time, for the non-selective inhibitor, the mean value of the metabolite concentration in the urine of each rat administered indomethacin and ibuprofen was used, and for the selective inhibitor, the metabolite contained in the urine of the rat treated with celecoxib Concentration was used. Subsequently, the concentration of the selected metabolite in the urine of the rat administered the non-selective inhibitor indomethacin and the rat administered the selective inhibitor celecoxib was changed to the concentration of the same metabolite in the urine of the control rat. The degree of sensitization according to the administration of the non-selective and selective inhibitors was compared (see Table 1).

Changes in the concentration of metabolites depending on the inhibitors administered Metabolite Non-selective inhibitor Selective inhibitors  1-methylnicotinamide 0.98 0.70 **  2-hydroxyisobutyrate 0.96 0.65 **  2-oxocaproate 1.19 0.89  2-oxoglutarate 0.20 ** 0.26 **  2-oxoisocaproate 2.48 ** 1.58 **  2-oxobalate 2.34 ** 1.55 **  3-phosphate sulfate 1.15 0.86  3-phenyllactate 1.47 * 0.62 **  4-hydroxyphenyllactate 1.49 * 1.13  acetate 0.76 0.13 **  Alanine 1.78 * 0.72 **  Allantoin 1.44 ** 0.95  Benzoate 0.71 0.30 *  Betaine 1.46 ** 1.22  Citrate 0.21 ** 0.32 **  Dimethylamine 1.44 ** 0.98  Formate 0.32 * 0.13 *  Fumarate 0.44 ** 0.32 **  Hippurate 0.26 ** 0.44 **  Kainurenine 1.70 ** 0.87  Lactate 1.81 * 0.53 **  N, N-dimethylglycine 1.04 0.67  N-acetylglycine 0.87 0.79  Niacinamide 0.64 ** 0.29 **  Nicotinate 1.29 0.48 *  Pantothenate 0.89 0.69 *  Phenyl Acetate 4.09 * 0.89  Phenylacetylglycine 1.1 1.51  Succinate 0.48 ** 0.27 **  Taurine 0.31 ** 0.78  Trigonelin 0.64 ** 0.39 **  Trimethylamine 0.43 0.04  Trimethyl N-oxide 1.26 1.28  Uracil 1.16 0.57 **  cis-aconate 0.97 0.68 **

*: Significance (p <0.05)

**: significance (p <0.01)

As shown in Table 1, most metabolites showed relatively higher concentrations in urine when non-selective inhibitors were administered than when selective inhibitors were administered. That is, among various metabolites, 2-oxoglutarate, citrate, hypofurite, phenylacetylglycine, taurine, trimethyl N-oxide, and uracil are more urine than when non-selective inhibitors are administered. Although relatively high concentrations were observed in, most of the other metabolites showed relatively higher concentrations in urine when the non-selective inhibitor was administered than when the selective inhibitor was administered.

Next, the concentration of each of the selected metabolites was applied to the PLS-DA analysis program included in the statistical program (SIMCA-P, Umetrics Inc., Kinnelon, NJ, USA), and significant metabolites were selected again. That is, among the metabolites shown in Table 1, the concentrations of the respective metabolites in the urine of rats to which indomethacin, ibuprofen and celecoxib were applied were subjected to the PLS-DA analysis, and the three drugs were administered. Any of the metabolites showing a variable importance plot (VIP) value of 1 or greater were reselected (see Table 2).

Metabolites Rescreened Based on VIP Value Metabolite VIP value Celecoxib Indomethacin Ibuprofen 2-oxoglutarate
acetate
Allantoin
Benzoate
Citrate
Dimethylamine
Formate
Hippurate
Phenylacetylglycine
Succinate
Taurine 2.77 (0.26) **
1.90 (0.13) **
1.98 (0.95)
1.39 (0.30) *
2.71 (0.32) **
0.13 (0.98) *
1.08 (0.13) *
1.12 (0.44) **
1.31 (1.51)
1.40 (0.27) **
0.61 (0.78) 2.61 (0.22) **
1.55 (0.75)
2.49 (1.27) **
0.59 (0.95)
2.71 (0.25) **
1.06 (1.52) **
0.94 (0.15) *
1.27 (0.27) **
0.63 (1.11)
0.99 (0.65)
1.45 (0.29) ** 2.42 (0.19) **
1.10 (1.78)
3.60 (1.63) **
0.78 (0.45)
2.58 (0.17) **
0.61 (1.35) **
0.69 (0.52)
1.18 (0.25) **
0.43 (1.09)
1.24 (0.29) **
1.28 (0.35) **

*: Significance (p <0.05)

**: significance (p <0.01)

The value in () shown next to the VIP value in Table 2 indicates the degree of change in the concentration of the metabolite determined in Table 1. The metabolites in Table 2 are metabolites (group 1) that show significant values when all three drugs are administered, and metabolites that do not show all significant values when all three drugs are administered (group 2). , Metabolites (group 3) that do not show significant values when indomethacin and ibuprofen are administered, but not when the selective inhibitor is administered, and celecoxib, an optional inhibitor. If only a significant value and non-selective inhibitors are administered, it can be divided into a metabolite that does not show a significant value (Group 4) and a metabolite that shows a non-specific significant value (Group 5).

Of the above-mentioned metabolites, allantoin belonging to a metabolite (group 3) which shows a significant value when both non-selective inhibitors indomethacin and ibuprofen are administered, and does not show a significant value when a selective inhibitor is administered ( allantoin) and taurine could be used as biomarkers for non-selective inhibitors.

In addition, acetate and benzoate belonging to the metabolite (group 4) that do not show a significant value only when the selective inhibitor celecoxib was administered, and when a non-selective inhibitor was administered ) Can be used as a biomarker for selective inhibitors.

From the above results, the urine of the experimental animal administered the desired NSAID was collected, and the concentration of allantoin, taurine, acetate and benzoate contained in the urine was measured, and the administered NSAID acted as a selective inhibitor to show side effects. It was confirmed that it would be possible to predict whether or not to show side effects by acting as a non-selective inhibitor.

Specifically, in the four metabolites contained in the urine, the concentration of acetate and benzoate is significantly increased compared to that of the control, and if the concentration of allantoin and taurine does not increase significantly compared to the control, the administration It can be determined that the NSAIDs act as selective inhibitors and will not show side effects.

On the other hand, among the four metabolites contained in the urine, the concentrations of allantoin and taurine are significantly increased compared to those of the control group, and if the concentrations of acetate and benzoate are not significantly increased compared to the control group, It can be determined that NSAIDs will act as non-selective inhibitors and show side effects.

1 shows the NMR spectra of the urine obtained from the control group and rats administered with each nonsteroidal anti-inflammatory agent.

Claims (6)

(Iii) 2 to 5 hours after the administration of nonsteroidal anti-inflammatory drugs (NSAIDs) to the experimental groups of mammals except humans, urine is collected and associated with the occurrence of side effects of the drug contained in urine. Measuring a concentration of a metabolite selected from the group consisting of allantoin, taurine, acetate, and benzoate; And, (Ii) comparing the concentration of the metabolite with the concentration of the metabolite contained in the urine of the control group not administered the drug, the concentration of allantoin and taurine was increased to a significantly higher level in the urine of the experimental group than that of the control group. If the concentration of acetate and benzoate does not increase to a significant level, it is expected that the side effects of the nonsteroidal anti-inflammatory drugs will occur, and the concentration of acetate and benzoate will be significantly higher in the urine of the experimental group than that of the control group. Non-steroidal anti-inflammatory drugs comprising the step of determining whether the side effects occur by predicting that the side effects of the non-steroidal anti-inflammatory drugs will not occur if the concentration is increased and the concentration of allantoin and taurine does not increase to a significant level To predict the side effects of it. The method of claim 1, Said side effect is gastrointestinal dysfunction, peptic ulcer, heart or renal dysfunction How to predict the side effects of nonsteroidal anti-inflammatory drugs. delete delete delete The method of claim 1, The concentration of allantoin, taurine, acetate and benzoate is measured by NMR analysis. How to predict the side effects of nonsteroidal anti-inflammatory drugs.

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