KR101099615B1 - Diagnostic Markers and Methods for Efficacy and Liver Toxicity of Statin - Google Patents

Abstract

The present invention relates to markers and methods for measuring the efficacy and stabilization of statins, and specifically, provides markers and methods for measuring the efficacy and liver toxicity of statins using significant differences in specific amino acid concentrations. The marker and measuring method has an advantage that the drug efficacy and toxicity of statins can be easily measured from urine samples that are easily collected.

Hyperlipidemia, statins, atorvastatin, efficacy, toxicity, diagnostic markers, electrophoresis, mass spectrometry, amino acids, urine

Description

Diagnostic Markers and Methods for Efficacy and Liver Toxicity of Statin}

The present invention relates to markers and methods for measuring drug efficacy and liver toxicity for statins.

Hyperlipidemia treatment is currently one of the most frequently used drugs in clinical treatment, and the administration period of the drug is a very long-term drug for life after the diagnosis of hyperlipidemia. The most widely used drug for the treatment of hyperlipidemia is the statin family, and its typical toxicity is known as liver toxicity.

To diagnose hepatotoxicity, it is common to measure ALT (aspartate aminotransferase) and ALT (astine aminotransferase), which are indicators of hepatotoxicity in the blood, but blood should be collected for analysis. Therefore, a simpler and more accurate method of analysis is required.

An object of one embodiment of the present invention is to provide a marker for measuring the efficacy and liver toxicity of statins.

It is an object of another embodiment of the present invention to provide a method for measuring the efficacy and liver toxicity of statins.

The present invention relates to markers and methods for measuring the drug efficacy and / or hepatotoxicity of statins, and more specifically, utilizing the concentration of specific amino acids in urine.

Markers and methods for measuring the efficacy and / or liver toxicity of statins according to the present invention have the advantage that the efficacy and toxicity of statins can be easily measured from urine samples that are easily collected.

The marker for measuring the efficacy of statin according to one embodiment of the present invention includes at least one selected from the group consisting of isoleucine, glutamine, glutamic acid and arginine. The marker can be, for example, amino acids in urine.

In another embodiment, the marker for measuring liver toxicity of statin is a marker for measuring liver toxicity of statin, and includes one or more from the group consisting of proline, 3-ureidopropionic acid and histidine. The marker can be, for example, amino acids in urine.

The marker is for maximizing the efficacy and reducing the toxicity by early detection of the efficacy and / or liver toxicity of statins. Among the many amino acids present in the human body, there is provided a marker for measuring efficacy and / or toxicity, including amino acids in which concentration changes occur specifically with respect to administration of statins and increased toxicity.

In one embodiment, the marker is characterized in that it comprises a specific amino acid in urine. Since the marker uses urine, which is easy to collect, there is an advantage that the marker is easy to analyze. In addition, one analysis can monitor the efficacy and toxicity of statins simultaneously or separately.

In one embodiment, the marker may determine that the efficacy of statins is effective when one or more concentrations selected from the group consisting of isoleucine, glutamine, glutamic acid and arginine are 70% or less of the onset of hyperlipidemia. More specifically, the marker can determine that the efficacy of statin is effective when the concentration of glutamic acid is 40% or less of the hyperlipidemic group.

In another embodiment, the marker is recognized that liver toxicity by statins is at least one concentration selected from the group consisting of proline, 3-ureidopropionic acid and histidine is 150% or more of the hyperlipidemic group Can be determined. More specifically, the marker can be determined that when the concentration of histidine is 250% or more of the hyperlipidemic group, hepatic toxicity by statins is recognized.

The statin can be applied without limitation, as long as it is a statin drug. In one embodiment, the statin is simvastatin, atorvastatin, atorvastatin, cerivastatin lovastatin, pitavastatin, rosuvastatin, prabastatin, prabastatin It may be at least one selected from the group consisting of fluvastatin and mevastatin. More specifically, the statin may be atorvastatin.

The present invention also provides a method for determining the efficacy and / or liver toxicity of statins by analyzing the concentration of specific amino acids.

In one embodiment, the measuring method comprises the step of analyzing the concentration of one or more amino acids selected from the group consisting of isoleucine, glutamine, glutamic acid and arginine, the concentration of the analyzed amino acid is less than 70% of the onset of hyperlipidemia In this case, it is characterized by determining that the efficacy of the statin is effective. More specifically, when the concentration of glutamic acid is 40% or less of the hyperlipidemic group, it can be judged that the efficacy of statin is effective.

In another embodiment, the measuring method comprises the step of analyzing the concentration of one or more amino acids from the group consisting of proline, 3-ureidopropionic acid and histidine, wherein the concentration of the analyzed amino acid, In the case of 150% or more, it is determined that hepatic toxicity by statins is recognized. More specifically, when the histidine concentration is 250% or more of the hyperlipidemic group, it can be determined that hepatic toxicity by statins is recognized.

The present invention also provides a method for simultaneously measuring the efficacy and liver toxicity of statins.

In one embodiment, the measuring method comprises the step of analyzing the amino acid concentration, wherein the amino acid,

At least one selected from the group consisting of isoleucine, glutamine, glutamic acid and arginine; And

At least one from the group consisting of proline, 3-ureidopropionic acid and histidine,

If at least one concentration selected from the group consisting of isoleucine, glutamine, glutamic acid and arginine is less than or equal to 70% of the onset of hyperlipidemia, the efficacy of statins is determined to be effective,

If one or more concentrations from the group consisting of proline, 3-ureidopropionic acid and histidine are 150% or more of the hyperlipidemic group, it is determined that liver toxicity by statins is recognized.

In one embodiment, the amino acid used in the above mentioned statin drug and / or liver toxicity measurement method may be an amino acid in urine. Since the urine sample is measured, it is easy to collect the sample and has the advantage of monitoring the efficacy and / or liver toxicity of statins through a single analysis.

In one embodiment, the process of analyzing the amino acid concentration may be by electrophoresis and mass spectrometry. In addition, prior to analyzing the amino acid concentration, the pretreatment process to remove the suspended matter and protein in the urine sample may be further subjected. In the pretreatment process, for example, the suspended solids may be removed by centrifugation and a high molecular weight protein may be removed using a molecular weight cut off filter.

Specifically, after administration of statins to a patient with hyperlipidemia, a urine sample is taken. The concentration of specific amino acids in the collected urine samples will be analyzed. A method for measuring the concentration of amino acids is not particularly limited, but may be measured by, for example, Capillary Electrophoresis-Mass Spectrometry (CE-MS). Based on the measured results, compared with the amino acid concentration of the hyperlipidemic group without the administration of statins, there is a significant difference in concentration. Prior to determination of amino acid concentrations, the collected urine samples are subjected to centrifugation and molecular weight cut off filters to remove suspended solids and proteins.

Detailed experiments on the selection and measurement method of the marker according to the present invention are as follows.

Hyperlipidemia-induced rat atorvastatin, a typical statin-based drug, was administered at a dose of 250 mg / kg / day for 7 days, followed by serum and histological examination for liver toxicity, and urine samples were collected. If necessary, the collected urine samples were subjected to a process of removing suspended solids and proteins using centrifugation and a molecular weight cut off filter. From the collected urine samples, amino acids whose concentrations change with drug administration and increased liver toxicity were detected using a Capillary Electrophoresis-Mass Spectrometry (CE-MS).

As a result of measuring the change in the concentration of the amino acid, the amino acid that increased or decreased in the hyperlipidemia-induced group and then returned to the normal group (hyperlipidemic-induced group) level by drug administration was determined as a factor of drug effect. In addition, in the normal group (non-hyperlipidemic group) and hyperlipidemia group, the concentration showed no significant difference or increased, and the amino acid whose concentration increased further after drug administration was determined as a factor of toxicity.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example 1 Induction of Hyperlipidemia and Drug Administration

First, hyperlipidemia was induced by intraperitoneal injection twice daily for three days at a dose of 1 g / kg of poloxamer-407 in a normal rat receiving normal diet. Hyperlipidemia-induced rat atorvastatin (atorvastatin; Lipitor, Pfizer Co., Korea) was orally administered at 250 mg / kg / day for 7 days (drug group). To maintain hyperlipidemia even during oral administration of atorvastatin, poloxamer-407 was intraperitoneally injected every three days.

The hyperlipidemia control group was induced with hyperlipidemia by intraperitoneal injection of poloxamer-407 at a dose of 1 g / kg once every three days for the same period as the experimental period of the drug administration group without administration of a statin drug. The normal control group was intraperitoneally injected with physiological saline instead of poloxamer-407 every three days for the same period as the experimental period of the drug administration group.

 Example 2 Collection of Serum, Tissue and Urine Samples and Detection of Liver Toxicity

After the administration of the drug, urine samples were taken for 24 hours. After the urine sample was collected, blood was collected by cannulation in the femoral artery of the rat, and the collected blood was centrifuged to obtain serum. Liver, kidney, heart, muscle, and brain tissues were collected after blood collection was complete.

Urine samples were tested for amino acid concentrations, serum biochemistry, and liver tissue histological examination. Hyperlipidemia was determined by measuring the concentration of cholesterol in the blood. Hepatotoxicity was determined by the concentration of biochemical markers on serum and histological examination of each tissue.

Specific test results are shown in Table 1 below.

Experimental group
parameter Normal control Hyperlipidemia control Drug administration group
(250 mg / kg / day) AST (aspartate amino trasnferase) (IU / L) 70.4 ± 0.4 56.0 ± 15.8 88.2 ± 9.0 ≠, ** ALT (alanine amino transferase) (IU / L) 36.8 ± 2.7 21.8 ± 4.8 30.8 ± 29.1 ALP (alkline phosphatase) (IU / L) 446.7 ± 23.2 179.4 ± 41.3 250.0 ± 134.7 BUN (blood urea Nitrogen)
(mg / dL) 23.2 ± 3.6 21.7 ± 3.0 26.5 ± 4.6 Creatinine (mg / dL) 0.5 ± 0.1 0.3 ± 0.1 0.4 ± 0.1 Glucose (mg / dL) 202.1 ± 4.4 187.2 ± 23.0 177.2 ± 30.3 Total cholesterol
Total cholesterol (mg / dL) 81.0 ± 2.8 1871.5 ± 429.8 1229.0 ± 248.8 Total protein
(Total protein) (g / dL) 5.9 ± 0.1 4.8 ± 0.4 5.1 ± 0.4 Creatinine phosphatase
(CPK; Creatinine phosphokinase) (IU / L) 159.5 ± 14.9 164.0 ± 76.7 177.3 ± 76.0 Albumin (g / dL) 3.1 ± 0.0 2.7 ± 0.2 2.7 ± 0.3 Total bilirubin
(Total Bilirubin) (mg / dL) 0.1 ± 0.0 0.3 ± 0.2 0.5 ± 0.2 Albumin globulin ratio 1.1 ± 0.0 1.5 ± 0.6 1.5 ± 0.7 Triglyceride (mg / dL) 111.0 ± 52.3 7240.0 ± 1389.3 5265.0 ± 2340.1

≠: P <0.1: significance in comparison between normal group and drug group

**: P <0.05: significance in comparison between hyperlipidemic control group and drug administration group

Referring to Table 1, only the concentration of aspartate amino transferase (AST) indicating liver toxicity among the indicators of liver, kidney and muscle toxicity increased after drug administration. In addition, as shown in Figure 1, inflammation in the hepatic portal vein thickening and gallbladder thickening was observed in the drug administration group, through which it was confirmed that hepatotoxicity through administration of atorvastatin. After drug administration, no changes in other tissues were observed.

In the serum biochemical test, the concentration of total cholesterol and triglyceride (triglyceride) in the poloxamer-407 administration group increased, indicating that hyperlipidemia was induced. In addition, they showed a tendency to decrease their average concentration after drug administration, it was confirmed that the drug appeared.

Example 3 Pretreatment and Quantitative Analysis of a Urine Sample

After confirming liver toxicity by administration of atorvastatin, the concentration of amino acid in urine was quantified using a Capillary Electrophoresis-Mass Spectrometry (CE-MS).

In order to remove protein in the sample, the urine sample was centrifuged to remove the suspended solids in order to remove the protein in the sample, and then 200 µl of the supernatant was purified using a ultrafree-MC filter (0.1 µl, Durapore PVDF, molecular). weight cut off: 3000, Millipore Corp. Bedford, Mass., USA). The filtered solution was analyzed by injection into a Capillary Electrophoresis-Mass Spectrometry (CE-MS).

The analyzer is equipped with a Beckman coulter MDQ capillary electrophoresis system (Beckman instruments, Fullerton, CA, USA) and a thermo LCQ Deca mass spectrometry equipped with an electrospray ion source. ) Was used. In the analysis, a silica capillary column having an inner diameter of 50, an outer diameter of 52 and 80 cm was used, and the temperature of the capillary chamber was 20 ° C. using a liquid coolant. Maintained.

Before starting the assay, the capillary was conditioned with 0.1 M NaOH for 30 minutes and 1 M formic acid for 3 hours. 50% methanol was used as a sheath liquid, and electrospray ion was used at a rate of 10 μl / min using a Nanospace 3001 S1-2 (Shinoido Fine Chemicals, Tokyo, Japan) pump. It was injected into the sauce. Samples were injected for 1 minute at a pressure of 0.8 psi, and amino acid separation was performed by flowing 1 M formic acid into a running electrolyte at a pressure of 1 psi at 2.5 kV.

The detection of amino acids was analyzed by SRM (Selective Reaction Monitoring) mode in positive ionization of the mass spectrometer. The spray voltage and current were 5 kV and 80, respectively, and the sheath gas flow rate was 8 arb (arbitrary unit). The ion capillary temperature was maintained at 275 ° C.

In FIG. 2, parent ions and product ions and representative electropherograms for each amino acid used in the SRM analysis are shown.

Example 3 Concentration Correction and Statistical Processing for Quantified Samples

Analyzes include proline, asparagine, glutamic acid, glutamine, glutamine, valine, leucine, arginine, histidine, glycine, iso Isoleucine, hydroxylysine, 2,3-diaminopropionic acid, cystine, 3-ureidopropionic acid, DL 19 amino acids of DL-ornitine, amino adipic acid, 2-fluoyl glycine, 3-methyl histidine, and tryptophan Proceed against.

For the analysis of the concentration of amino acids in urine, a calibration curve was prepared and quantified in the concentration range of 0.01 ~ 5 / ml. For all amino acids, in the calibration curve concentration range, the analysis was verified to be within 10% of accuracy and within 15% of accuracy.

The urine amino acid concentration derived from the calibration curve was corrected by dividing by the creatinine level of each sample to calculate the final concentration. In addition, in each group, a student t-test was performed to verify the significant difference in the concentration of each amino acid.

As a result of analyzing the concentration of amino acids in each group, isoleucine, glutamine, glutamic acid, and arginine were found to increase in urine in hyperlipidemia and significantly decreased after administration of atorvastatin. . In addition, proline, 3-ureidopropionic acid, and histidine showed no significant change in concentration in hyperlipidemia, but increased after drug administration and detected as a factor of liver toxicity. The results are shown in Table 2.

Experimental group
amino acid Normal control Hyperlipidemia control Drug administration group
250 mg / kg / day Glycine 130.077 ± 91.13 160.42 ± 38.81 73.27 ± 43.49 2,3-diaminopropionic acid
(2,3-Diaminopropionic acid) 7.447 ± 2.8 15.06 ± 7.64 13.89 ± 5.81 Proline 24.807 ± 10.75 30.16 ± 20.39 54.03 ± 2.78 * Valine 7.837 ± 0.57 143.36 ± 53.00 † 49.36 ± 18.15 Cystine 13.397 ± 3.80 133.91 ± 19.33 †† 107.88 ± 7.65 Leucine 7.087 ± 1.51 41.98 ± 23.69 20.57 ± 10.90 Isoleucine 30.327 ± 1.30 38.02 ± 12.18 15.44 ± 8.49 * 3-ureidopropionic acid
(3-ureidopropionic acid) 23.947 ± 4.28 62.29 ± 3.95 †† 123.56 ± 29.05 * Asparagine 31.127 ± 16.59 87.77 ± 60.90 43.22 ± 18.19 DL-Ornithine 8.347 ± 4.53 21.05 ± 9.26 17.50 ± 4.34 Glutamine 92.487 ± 3.06 280.72 ± 134.89 96.64 ± 70.69 * Glutamic acid 165.647 ± 60.12 269.58 ± 188.47 62.13 ± 47.18 * Histidine 2.937 ± 0.56 7.33 ± 3.99 25.50 ± 5.18 ** L2-aminoadipic acid
(L2-Amino adipic acid) ND ND ND Hydroxylysine
(Hydroxylysine) ND ND ND 2-fluoroyl glycine
(2-Fluroyl glycine) 9.847 ± 4.18 3.78 ± 1.73 25.45 ± 18.17 3-methyl histidine
(3-Methyl histidine) 5.837 ± 0.55 26.64 ± 12.08 † 18.28 ± 3.14 Arginine 32.73 ± 23.85 59.69 ± 7.43 32.72 ± 1.05 * Tryptophan 13.31 ± 3.62 10.73 ± 3.63 12.62 ± 0.87

†: ≤0.05 and ††: ≤0.01 Significance between normal and hyperlipidemic controls

*: ≤0.05 and **: ≤0.01 Significance between hyperlipidemic and drug-administered groups

1 shows inflammation of the hepatic portal vein and thickening of the gallbladder after oral administration of atorvastatin (7 days, dose; 250 mg / kg / day).

2 shows the electropherograms for each amino acid used in SRM analysis.

Claims (16)

As a marker measuring the efficacy of statins, At least one selected from the group consisting of isoleucine, glutamine, glutamic acid and arginine, A marker for measuring the efficacy of statins, wherein at least one concentration selected from the group consisting of isoleucine, glutamine, glutamic acid and arginine is effective when statin is effective when the concentration of hyperlipidemia is 70% or less. The method of claim 1, The marker is a marker for measuring the efficacy of statins, characterized in that the amino acid in the urine. delete The method of claim 1, A marker for measuring the efficacy of a statin, wherein the glutamic acid concentration is determined to be effective when the concentration of glutamic acid is 40% or less of the hyperlipidemic group. As a marker for measuring liver toxicity of statins, At least one selected from the group consisting of proline, 3-ureidopropionic acid and histidine, A marker for measuring hepatotoxicity of statins, wherein at least one concentration selected from the group consisting of proline, 3-ureidopropionic acid and histidine is recognized as hepatotoxic by statins when at least 150% of hyperlipidemic onset groups are recognized. The method of claim 5, The marker is a marker for measuring liver toxicity of statins, which is an amino acid in urine. delete The method of claim 5, A marker for measuring liver toxicity of statins, wherein the concentration of histidine is determined to be hepatotoxicity caused by statins when the concentration of hyperlipidemia is 250% or more of the onset group of hyperlipidemia. The method according to claim 1 or 5, The statin is simvastatin, atorvastatin, atorvastatin, cerivastatin, lovastatin, pitavastatin, rosuvastatin, prabastatin, fluvastatin, fluvastatin And mevastatin (mevastatin) is one or more markers selected from the group consisting of. The method of claim 9, The statin is a atorvastatin (atorvastatin), characterized in that the marker. Analyzing the concentration of one or more amino acids selected from the group consisting of isoleucine, glutamine, glutamic acid, and arginine, wherein if the analyzed amino acid concentration is 70% or less of the onset of hyperlipidemia, the efficacy of statin is determined to be effective. Method for measuring the efficacy of statins. Hepatotoxicity with statins, comprising analyzing the concentration of at least one amino acid selected from the group consisting of proline, 3-ureidopropionic acid and histidine, wherein the concentration of the analyzed amino acid is at least 150% of the onset of hyperlipidemia The method for measuring liver toxicity of statins which is determined to be recognized. Analyzing the amino acid concentration, wherein the amino acid, At least one selected from the group consisting of isoleucine, glutamine, glutamic acid and arginine; And At least one selected from the group consisting of proline, 3-ureidopropionic acid and histidine, If at least one concentration selected from the group consisting of isoleucine, glutamine, glutamic acid and arginine is less than or equal to 70% of the onset of hyperlipidemia, the efficacy of statins is determined to be effective, A method for determining that liver toxicity by statins is recognized when at least one concentration selected from the group consisting of proline, 3-ureidopropionic acid and histidine is 150% or more of the hyperlipidemic group. The method according to any one of claims 11 to 13, The amino acid is a measuring method, characterized in that the amino acid in the urine. The method according to any one of claims 11 to 13, The step of analyzing the amino acid concentration is a measuring method characterized by electrophoresis and mass spectrometry. The method of claim 15, Before analyzing the amino acid concentration, the measurement method characterized in that it further undergoes a pretreatment step of removing the suspended matter and protein in the urine sample.

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