KR20130102777A - Methods for predicting drug reaction of omeprazole using genotype of cyp2c19 gene - Google Patents

Methods for predicting drug reaction of omeprazole using genotype of cyp2c19 gene Download PDF

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KR20130102777A
KR20130102777A KR1020120023879A KR20120023879A KR20130102777A KR 20130102777 A KR20130102777 A KR 20130102777A KR 1020120023879 A KR1020120023879 A KR 1020120023879A KR 20120023879 A KR20120023879 A KR 20120023879A KR 20130102777 A KR20130102777 A KR 20130102777A
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omeprazole
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김승희
한순영
정면우
나한성
김영훈
신희정
하지혜
박현주
김해든
서두원
이석용
배정우
최창익
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대한민국 (식품의약품안전처장)
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Abstract

The present invention is a method for predicting the drug response of omeprazole, and after analyzing the genotype of the gene encoding cytochrome P450 2C19 (CYP2C19), to determine the appropriate dosage of omeprazole according to the analyzed genotype, The present invention relates to a method of predicting drug response of omeprazole by effectively measuring blood drug concentration. More specifically, the present invention provides a method for analyzing the genotype of the CYP2C19 gene of Koreans in order to predict the drug response of omeprazole. According to the present invention, it is possible to provide a method for analyzing the genotyping of the CYP2C19 gene for predicting drug response of omeprazole, and further, to calculate the appropriate drug dose for each genotype and to perform a verification study for this, finally allowing the drug approval considering the Korean drug genotype. It can be used to suggest a reflection of the matter.

Description

METHODS FOR PREDICTING DRUG REACTION OF OMEPRAZOLE USING GENOTYPE OF CYP2C19 GENE}

The present invention is a method for predicting the drug response of omeprazole using the genotype of the CYP2C19 gene, and after analyzing the genotype of the gene encoding cytochrome P450 2C19 (CYP2C19), an appropriate amount of omeprazole according to the analyzed genotype The present invention relates to a method of predicting drug response of omeprazole by effectively measuring blood drug concentration of omeprazole.

It is well known that mutations and frequencies of genes related to drug reactions between races and ethnic groups appear differently, and therefore, drug reactions between races and ethnic groups appear differently. Developed countries such as the United States (FDA), Europe (EMEA), and Japan (Ministry of Health, Labor and Welfare) provide safe drug use information by applying drug genetic information to drug approvals. It is very important to identify the difference in drug response.

Cytochrome P450 (CYP) enzyme is an enzyme that affects the pharmacokinetics of the drug and plays an important role in showing the efficacy and toxicity of the drug. CYP has several heterologous enzymes, and the main enzymes involved in drug metabolism include CYP2C9, CYP2C19, CYP2D6, and CYP3A4.

Substrate drugs of CYP2C19 include antifungal agents (Voriconazole), antiulcers (Omeprazole, Lansoprazole, etc.), anticonvulsants (S-mephenytoin, etc.), diabetes treatment (Tolbutamide, etc.) and sedatives (Diazepam, etc.).

Omeprazole is an anti-ulcer agent that inhibits the secretion of gastric acid by blocking the activity of H + / K + ATPase (proton pump) in gastric wall cells (Gonzalez et al., 2003). The pH in the stomach showed a significant change according to the concentration of omeprazole in blood (Furuta et al., 1999). CYP2C19 is known to show significant pharmacokinetic and pharmacodynamic differences (Chang et al., 1995; Chang et al. 1995; Tybring et al., 1997; Furuta et al., 1999; Sakai et al., 2001; Kita et al., 2002; Desta et al., 2002). Klotz, 2006; Qiao et al., 2006; Shirai et al., 2001; Sugimoto et al., 2006; Shimatani et al. 2005), probe drugs of CYP2C19. The gastric acid secretion effect of proton pump inhibitors (PPIs), including omeprazole, is directly proportional to AUC (area under the plasma concentration curve), with a significant correlation between pharmacokinetics and pharmacodynamics (Klotz et al., 2004).

Twenty-eight alleles have been reported (http://www.cypalleles.ki.se/cyp2c19.htm) so far associated with decreased or absent enzyme activity of CYP2C19. Among them, CYP2C19 * 2 (75-85%) and * 3 alleles are the majority of PM (poor metabolizer), and Asians have 2C19 * 2 and * 3 alleles represent more than 99% of PM (de Morais et al., 1994a, b; Roh et al., 1996; Goldstein et al., 1997). In particular, CYP2C19 * 3 is an allele that is specific to Asians (Roh et al., 1996; Goldstein et al., 1997). For Westerners, the CYP2C19 * 2, * 4, * 5, * 6, * 7 and * 8 alleles represent 99.74% of PM alleles (Wedlund, 2000). CYP2C19 * 9 to * 15 are reported to occur only in Westerners and Africans (Blaisdell et al., 2002). CYP2C19 * 17 has increased enzyme activity (Sim et al., 2006), and CYP2C19 * 18 and CYP2C19 * 19 have been reported in Japan (Fukushima-Uesaka et al., 2005). Recently, CYP2C19 * 22, CYP2C19 * 27 and CYP2C19 * 28 were found in Africans (Matimba et al., 2009; Dr et al., 2010), and * 23 ~ * 26 were found in Chinese and Koreans (Zhou et al., 2009; Lee et al., 2009). ).

In many studies, gastric acid secretion was more significantly inhibited in the CYP2C19PM group than in the EM group when omeprazole was administered according to the genotype of CYP2C19 (Shirai et al., 2001; Sugimoto et al., 2006; Furuta et al., 1999; Shimatani et al., 2005). In the case of lansoprazole, the treatment effect of gastroesophageal reflux disease was more significant in the CYP2C19PM group (Furuta et al., 2002; Kawamura et al., 2003). Compared with Westerners, AUC is approximately fourfold higher in Asians (20 mg single dose) and dose control should be considered in Asians (Omeprazole prescribing information).

As is well known for the CYP2C19 gene, common SNPs include 681G> A (splicing defect, CYP2C19 * 2 allele) and 636G> A (premature stop codon, CYP2C19 * 3 allele). CYP2C19 * 2 alleles have similar frequencies in Asians and Westerners, but CYP2C19 * 3 alleles have been reported mainly in Asians and rarely in Westerners. Said genetic polymorphism produces inactive enzyme proteins, which are reported to account for more than 99% of the PM allele, especially in Japanese. Recently, more extensive research is being conducted, including the inclusion of transcriptional regulatory regions of the CYP2C19 gene in the analysis.

As such, the use of omeprazole as a probe drug affected by the CYP2C19 enzyme with a large difference in genetic polymorphism between races, the crosslinking test and the use of imported new drugs by examining the correlation between pharmacokinetic characteristics and genetic variation in Koreans There is a need to ensure safety and effectiveness.

An object of the present invention is to provide a method for analyzing the genotype of the CYP2C19 gene of Koreans to predict the drug response of omeprazole.

It is another object of the present invention to provide a method for predicting omeprazole drug response by determining an appropriate omeprazole dosage according to the genotype of the CYP2C19 gene of Koreans.

Another object of the present invention to provide a method for measuring the blood drug concentration of omeprazole for predicting the drug response of omeprazole.

According to an aspect of the present invention,

(a) obtaining exon 4 and exon 5 of a gene encoding cytochrome P450 2C19 (CYP2C19) from genomic DNA to determine nucleotide sequences;

(b) detecting the presence of the following single nucleotide polymorphism (SNP) in the determined base sequence;

1.CYP2C19 * 2: 39th G of SEQ ID NO: 1 (Exon 5) is replaced by A

2. CYP2C19 * 3: 155th G of SEQ ID NO: 2 (Exon 4) is replaced by A

(C) determining which group belongs to CYP2C19EM (Extensive Metabolizer), CYP2C19IM (Intermediate Metabolizer) and CYP2C19PM (Poor Metabolizer) according to the following genotypes using the detected single nucleotide polymorphism (SNP)

Figure pat00001

It provides a method of genotyping the CYP2C19 gene for predicting the drug response of omeprazole.

In addition, the present invention is a method for determining the base sequence in the step (a) is a PCR method using a primer capable of amplifying exon 4 and exon 5 of the gene encoding CYP2C19, the primer is SEQ ID NO: 3 to 6 The oligonucleotide of the present invention provides a method for genotyping the CYP2C19 gene for predicting drug response of omeprazole.

In addition, the present invention is oral administration of 20 to 80 mg of omeprazole to the CYP2C19 genotype EM group, IM group and PM group analyzed by the above method, and then genotypes by comparing the AUC (area under the plasma concentration curve) for each genotype Provided is a method for predicting drug response of omeprazole according to the CYP2C19 genotype, characterized by calculating the omeprazole drug dosage.

In addition, in the method for predicting drug response of CYP2C19 genotype omeprazole, the drug dosage is 75mg when CYP2C19 genotype is EM group, 60mg when IM group, and 20mg when PM group, CYP2C9 genotype drug response Provide a prediction method.

In addition, in the method for predicting drug response of CYP2C19 genotype-specific omeprazole, the oral administration is characterized in that the single-dose, CYP2C19 genotype-specific omeprazole provides a method for predicting drug response.

In addition, the CYP2C19 genotype-specific omeprazole drug response prediction method, AUC is measured by a method comprising the following steps, CYP2C19 genotype-specific omeprazole provides a method for predicting drug response.

(a) leaving the frozen sample stored at room temperature to dissolve, shaking, and adding lansoprazole as an internal standard thereto;

(b) adding methyl t-butyl ether (MTBE) to the sample, mixing and centrifuging;

(c) taking the organic solvent layer after the centrifugation, evaporating and drying under a nitrogen stream, and resolving the residue into a mobile phase; And

(d) taking a portion of the sample solution and injecting into HPLC-MS / MS to quantify.

CYP2C19 * 2 and CYP2C19 * 3 alleles with no enzymatic activity and with clinically significant results were studied. Thus, genotypes for the CYP2C19 * 1, * 2 and * 3 alleles were searched.

In order to measure the concentration of omeprazole in plasma, we developed a quantitative method using HPLC-MS / MS and an extraction method from plasma samples. Based on this, we evaluated the specificity, precision, accuracy, sensitivity, recovery rate, and stability after preparation. Proceeded.

According to the present invention, it is possible to provide a method for analyzing the genotyping of the CYP2C19 gene for predicting drug response of omeprazole, and further, to calculate the appropriate drug dose for each genotype and to perform a verification study for this, finally allowing the drug approval considering the Korean drug genotype. It can be used to suggest a reflection of the matter.

1 is a graph showing the trend of omeprazole concentration (single dose) in mean plasma according to genotype of CYP2C19.
2 is a graph showing the omeprazole concentration (repeat dose) in average plasma according to the genotype of CYP2C19.

Example  1: subject's genome DNA  extraction

Forty-two subjects with blood samples for analyzing the drug genotype were recruited and DNA was collected from their blood. The collected blood was centrifuged at 2,500 rpm for 10 minutes to separate a buffy coat, and genomic DNA was extracted from 300 ㎕ of smoke using a DNA purification kit (Promega Ltd., USA). DNA concentrations were determined by measuring absorbance at 260 nm and 280 nm using a UV spectrophotometer.

Example  2: CYP2C19  Gene gene Variant  Search

* 2 and * 3 alleles were selected from the CYP2C19 genes without enzyme activity and with clinically significant results. Thus, genotypes for the CYP2C19 * 1, CYP2C19 * 2 and CYP2C19 * 3 alleles were searched. In this case, CYP2C19 * 1 means wild-type, CYP2C19 * 2 means that the G of the 39th position of SEQ ID NO: 1 (exon 5) is replaced with A, and CYP2C19 * 3 is SEQ ID NO: 2 (Exon 4) Position 155 is where G is substituted for A.

To this end, in order to amplify exon 4 and exon 5 sites of the CYP2C19 gene, the genomic DNA obtained in Example 1 was used as a template, and a SNP genotyping kit (SNaPshot ™ Assay Kit; TaqMan ™ Universal PCR Master Mix, Applied Biosystems, Inc., USA), and PCR was performed using primers specific for each site (SEQ ID NOs: 3 to 6), and then each of these sites of the CYP2C19 gene was amplified.

At this time, PCR conditions were 94 ℃ 5 minutes, (94 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 30 seconds) × 35 times for CYP2C19 * 2, 72 ℃, 94 ℃ 5 minutes for CYP2C19 * 3 , (94 ° C. 30 sec, 48 ° C. 1 min, 72 ° C. 30 sec) × 40 times, 72 ° C., and the base sequences of the primers are as follows.

Figure pat00002

The amplified PCR product was applied to an automatic sequencer using the primers (SEQ ID NOS: 3 to 6), and each sequence was analyzed. Subsequently, each analyzed nucleotide sequence was applied to a sequencing computer program to determine whether the single nucleotide polymorphism (SNP) exists in the base of each gene fragment.

Example  3: drug genome test

1) Preparation of plasma sample

CROS2C19 * 1 / * 1, * 1 / * 2, * 1 / * 3, * 2 / * 2, * 2 / * 3, * 3 / * 3 For subjects, Losecec capsule (omeprazole, 20-80 mg) Was dosed once or repeatedly with 240 mL of water. Blood collection was performed for 0-12 hours. The collected blood was centrifuged at 3,000 rpm for 10 minutes and then plasma was collected and transferred to the plasma separation tube and stored at -70 ° C until analysis.

2) Measurement of blood drug concentration

① Manufacture of calibration curve sample and quality control sample

Dissolve the omeprazole standard in 90% methanol to make the concentration of omeprazole 0.1 mg / mL and store frozen (-20 ℃), and dilute this solution with the freeze-preserved plasma so that the concentration in plasma of 100 μL is as follows. A standard sample was prepared. For the calibration, plasma samples were prepared to be 2, 10, 50, 200, 500, 1000, 1500 and 2000 ng / mL. The quality control sample (QC sample) had a plasma concentration of 5, 100, 1750. ng / mL and dilution quality control sample (3000, 4000, 6000, 8000 ng / mL) in order to evaluate the dilution amount for quantifying the sample exceeding the highest limit. DQC sample).

② verification of plasma sample processing and analysis conditions

5 μL of lansoprazole (15 μg / mL) was added to 100 μL of each QC sample and analytical standard plasma sample, and the mixture was shaken. 2.5 mL of methyl t-butyl ether (MTBE) was added thereto, mixed, and centrifuged at 2,500 rpm for 10 minutes. The organic solvent layer was taken, transferred to a clean test tube, evaporated and dried under nitrogen stream at 40 ° C., the residue was redissolved into 400 μL of the mobile phase, and 2.5 μL of the final sample solution was taken and injected into HPLC-MS / MS. A calibration curve was prepared with the area ratio of omeprazole to the area ratio of the internal standard obtained here. The experiment was performed five times a day to obtain reproducibility within the day, and the experiment was continuously performed for five days to obtain daily reproducibility.

Plasma samples collected at each hour from the subject, stored at -70 ° C, were left at room temperature to dissolve, shaken for 20 seconds, and then 100 μL of this plasma was transferred to a test tube, and lansoprazole (15 μg / mL) was used as an internal standard. After the addition of μL and pretreatment in the same manner as the calibration curve preparation method was injected into HPLC-MS / MS for analysis.

At this time, the HPLC-MS / MS analysis conditions are as follows.

The device is an Agilent 1200 Series HPLC system, the MS / MS is API 3200 (Applied Biosystems / MDS SCIEX), the column is Luna C 18 5 μm (Phenomenex, 2.0 × 50 mm), and the data processing device is Analyst software version. 1.4.2 (Applied Biosystems / MDS SCIEX) was used. 10 mM ammonium formate with 90% methanol as mobile phase (pH 3.5) A mixed solution was used and quantified at a flow rate of 0.25 mL / min. MS / MS conditions were quantified under the following conditions using positive and MRM modes.

a) compound parameters

Figure pat00003

b) source / gas parameters

Figure pat00004

③ Calculation of blood concentration

The concentration of omeprazole in plasma was determined from the prepared calibration curve. For samples exceeding the highest quantitative limit concentration, the concentration was calculated by multiplying by diluting fold with 4 times dilution with co-plasma.

Example  4: Genotype Dosage Verification test  - Omeprazole CYP2C19  Genotype Dosages Verification test - One time  administration

Genotypes according to CYP2C19 of omeprazole, namely CYP2C19EM group (Extensive Metabolizer), IM group (Intermediate Metabolizer), PM group (Poor Metabolizer) in doses of 75 mg, 60 mg and 20 mg, respectively, 15 healthy Korean subjects CYP2C19EM group, IM group The mean plasma drug concentration-time curves tested for 15 patients and 12 PM patients are shown in FIG. 1 and pharmacokinetic parameters are shown in Table 1. In this case, the three types of allele groups, the EM group, the IM group, and the PM group, respectively, mean the following.

EM group: CYP2C19 * 1 / * 1

IM group: CYP2C19 * 1 / * 2, * 1 / * 3

PM group: CYP2C19 * 2 / * 2, * 2 / * 3, * 3 / * 3

Table 1.

Figure pat00005

{(Mean ± standard deviation), NS; Not important. Three genotyping groups were compared by one-way ANOVA and post hoc Bonferroni t-test or Kruskal-Wallis one-way analysis of variance and post hoc Mann-Whitney test. * P <0.05 compared to CYP2C19EM; ** P <0.01; *** P <0.001. ## P <0.01 compared to CYP2C19IM; ### P <0.001.}

Since gastric acid secretion inhibitors such as omeprazole have significantly increased drug effects in the PM group than the EM group, adjusting the drug dose to maintain the drug concentration in the CYP2C19PM group can increase the effect of the drug. To adjust the effective blood concentration of the PM group. In conclusion, the effective dose of omeprazole according to the genotype of CYP2C19 was 75mg in the CYP2C19EM group, 60mg in the IM group and 20mg in the PM group. It was confirmed that the appropriate dose of omeprazole was considered.

<110> KOREA FOOD & DRUG ADMINISTRATION <120> METHODS FOR PREDICTING DRUG REACTION OF OMEPRAZOLE USING GENOTYPE          OF CYP2C19 GENE <130> 0001 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 177 <212> DNA <213> Homo sapiens <220> <221> exon (222) (1) .. (177) <223> exon5 of CYP2C19 gene <400> 1 atatgcaata attttcccac tatcattgat tatttcccgg gaacccataa caaattactt 60 aaaaaccttg cttttatgga aagtgatatt ttggagaaag taaaagaaca ccaagaatcg 120 atggacatca acaaccctcg ggactttatt gattgcttcc tgatcaaaat ggagaag 177 <210> 2 <211> 161 <212> DNA <213> Homo sapiens <220> <221> exon (222) (1) .. (161) <223> exon4 of CYP2C19 gene <400> 2 cttcaccctg tgatcccact ttcatcctgg gctgtgctcc ctgcaatgtg atctgctcca 60 ttattttcca gaaacgtttc gattataaag atcagcaatt tcttaacttg atggaaaaat 120 tgaatgaaaa catcaggatt gtaagcaccc cctggatcca g 161 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ttaatgttgg tctcgaaccg 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 tatcactttc cataaaagca ag 22 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 ataataatag acaattgatt atact 25 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 acttcagggc ttggtcaata 20

Claims (6)

A method for genotyping a CYP2C19 gene for predicting drug response of omeprazole, comprising the following steps:
(a) obtaining exon 4 and exon 5 of a gene encoding cytochrome P450 2C19 (CYP2C19) from genomic DNA to determine nucleotide sequences;
(b) detecting the presence of the following single nucleotide polymorphism (SNP) in the determined base sequence;
1.CYP2C19 * 2: 39th G of SEQ ID NO: 1 (Exon 5) is replaced by A
2. CYP2C19 * 3: 155th G of SEQ ID NO: 2 (Exon 4) is replaced by A
(c) determining which group belongs to CYP2C19EM (Extensive Metabolizer), CYP2C19IM (Intermediate Metabolizer) and CYP2C19PM (Poor Metabolizer) according to the following genotypes using the detected single nucleotide polymorphism (SNP).
Figure pat00006
 According to claim 1, wherein the method for determining the base sequence in step (a) is a PCR method using a primer capable of amplifying exon 4 and exon 5 of the gene encoding CYP2C19, the primer is SEQ ID NO: 3 to It is an oligonucleotide of 6, genotyping method of the CYP2C19 gene for predicting the drug response of omeprazole. CYP2C19 genotype EM group, IM group, and PM group analyzed by the method of claim 1 were orally administered with 20 to 80 mg of omeprazole, respectively, and then genotyped omeprazole drug administration by comparing AUC (area under plasma concentration curve) for each genotype. A method for predicting drug response of omeprazole according to genotype of CYP2C19, characterized in that the dose is calculated. 4. The method of claim 3, wherein the drug dosage is 75 mg in the CYP2C19 genotype, 60 mg in the IM group, and 20 mg in the PM group. 4. The method according to claim 3, wherein the oral administration is a single administration. The method of claim 3, wherein the AUC is measured by a method comprising the following steps: CYP2C19 genotype drug response prediction method of omeprazole:
(a) leaving the frozen sample stored at room temperature to dissolve, shaking, and adding lansoprazole as an internal standard thereto;
(b) adding methyl t-butyl ether (MTBE) to the sample, mixing and centrifuging;
(c) taking the organic solvent layer after the centrifugation, evaporating and drying under a nitrogen stream, and resolving the residue into a mobile phase; And
(d) taking a portion of the sample solution and injecting into HPLC-MS / MS under the following conditions I) and II) to quantify.
I) Compound Parameters
Figure pat00007

II) Source / Gas Parameters
Figure pat00008
KR1020120023879A 2012-03-08 2012-03-08 Methods for predicting drug reaction of omeprazole using genotype of cyp2c19 gene KR20130102777A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106544415A (en) * 2016-10-13 2017-03-29 广东药科大学 A kind of method and test kit of detection CYP2C19*3 polymorphic site genotype

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106544415A (en) * 2016-10-13 2017-03-29 广东药科大学 A kind of method and test kit of detection CYP2C19*3 polymorphic site genotype

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