Biomarker for evaluating drug effect of olanzapine, method and application
阅读说明：本技术 奥氮平药效评估的生物标志物、方法及应用 (Biomarker for evaluating drug effect of olanzapine, method and application ) 是由 刘丽宏 安卓玲 贺玖明 万子睿 李鹏飞 丛菱 于 2020-11-16 设计创作，主要内容包括：本发明公开了奥氮平药效评估的生物标志物,包括溶血磷脂先乙醇胺(0：0/18:3)、溶血磷脂先乙醇胺(0：0/22:5)和十八碳三烯酸。本发明还公开了引物对在寻找奥氮平药物代谢动力学差异的关键药物基因中的应用。本发明还公开了奥氮平药物代谢动力学差异的关键药物基因的寻找方法。本发明还公开了γ-氨基丁酸转氨酶基因多态性于奥氮平药物代谢动力学研究及药效评估中的应用。本发明还公开了包含能够检测生物标志物的试剂的试剂盒。本发明还公开了生物标志物在评估奥氮平药效、奥氮平检测试剂盒及科学研究中的应用。本发明的生物标志物具有预测药物浓度的能力,为临床个体化用药提供重要依据。(The invention discloses biomarkers for olanzapine efficacy evaluation, which comprise lysophospholipid monoethanolamine (0:0/18:3), lysophospholipid monoethanolamine (0:0/22:5) and octadecatrienoic acid. The invention also discloses application of the primer pair in searching key drug genes of olanzapine pharmacokinetic difference. The invention also discloses a method for searching key drug genes of olanzapine pharmacokinetic difference. The invention also discloses application of the gamma-aminobutyric acid transaminase gene polymorphism in olanzapine pharmacokinetic research and pharmacodynamic evaluation. Also disclosed are kits comprising reagents capable of detecting biomarkers. The invention also discloses application of the biomarker in evaluation of olanzapine efficacy, olanzapine detection kit and scientific research. The biomarker has the capability of predicting the concentration of the drug, and provides an important basis for clinical personalized medicine.)
1. Biomarkers for olanzapine efficacy evaluation, comprising lysophospholipid monoethanolamine (0:0/18:3), lysophospholipid monoethanolamine (0:0/22:5) and octadecatrienoic acid.
2. The biomarker for olanzapine efficacy assessment according to claim 1, further comprising: lysophosphatidylethanolamine (0:0/20:3), lysophosphatidylethanolamine (18:1/0:0), galactoglycerol and 5-hydroxytryptamine.
3. Application of a primer pair in searching key drug genes of olanzapine pharmacokinetic difference, which is characterized in that the primer pair comprises a primer pair shown as SEQ ID NO: 43 and SEQ ID NO: 44, or a primer set shown in the specification.
4. The use of claim 3, wherein the primer pair further comprises the primer set as set forth in SEQ ID NO: 45 and as shown in SEQ ID NO: 46, or a pharmaceutically acceptable salt thereof.
5. The use of claim 3, wherein the primer pair further comprises the primer set as set forth in SEQ ID NO: 1 and as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3 and as shown in SEQ ID NO: 4, and the primer pair shown as SEQ ID NO: 5 and as shown in SEQ ID NO: 6, and the primer pair shown as SEQ ID NO: 7 and as shown in SEQ ID NO: 8, and the primer pair shown as SEQ ID NO: 9 and as shown in SEQ ID NO: 10, and the primer pair shown in SEQ ID NO: 11 and as shown in SEQ ID NO: 12, and the primer pair shown in SEQ ID NO: 13 and as shown in SEQ ID NO: 14, and the primer pair shown as SEQ ID NO: 15 and as shown in SEQ ID NO: 16, and the primer pair shown as SEQ ID NO: 17 and as shown in SEQ ID NO: 18, and the primer pair shown as SEQ ID NO: 19 and as shown in SEQ ID NO: 20, and the primer pair shown in SEQ ID NO: 21 and as shown in SEQ ID NO: 22, and the primer pair shown in SEQ ID NO: 23 and as shown in SEQ ID NO: 24, and the primer pair shown in SEQ ID NO: 25 and as shown in SEQ ID NO: 26, and the primer pair shown in SEQ ID NO: 27 and as set forth in SEQ ID NO: 28, and the primer pair shown in SEQ ID NO: 29 and as set forth in SEQ ID NO: 30, and the primer pair shown in SEQ ID NO: 31 and as set forth in SEQ ID NO: 32, and the primer pair shown as SEQ ID NO: 33 and as set forth in SEQ ID NO: 34, and the primer pair shown in SEQ ID NO: 35 and as shown in SEQ ID NO: 36, and the primer pair shown in SEQ ID NO: 37 and the nucleotide sequence as set forth in SEQ ID NO: 38, and the primer pair shown in SEQ ID NO: 39 and as shown in SEQ ID NO: 40, and the primer pair shown as SEQ ID NO: 41 and as shown in SEQ ID NO: 42, and the primer pair shown as SEQ ID NO: 47 and as shown in SEQ ID NO: 48, and the primer pair shown in SEQ ID NO: 49 and as shown in SEQ ID NO: 50, and the primer pair shown as SEQ ID NO: 51 and as set forth in SEQ ID NO: 52, and the primer pair shown as SEQ ID NO: 53 and as shown in SEQ ID NO: 54, and the primer pair shown as SEQ ID NO: 55 and as shown in SEQ ID NO: 56, and/or the primer set shown as SEQ ID NO: 57 and as shown in SEQ ID NO: 58, or a primer set.
6. A method for finding a key drug gene for pharmacokinetic differences of olanzapine, comprising the step of amplifying using the primer pair of claim 3.
7. The application of the gamma-aminobutyric acid transaminase gene polymorphism in olanzapine pharmacokinetic research and pharmacodynamic evaluation.
8. The use of claim 7, wherein said γ -aminobutyric transaminase gene polymorphism comprises a GABA-T rs1641031 gene polymorphism and a GABA-T rs1641022 gene polymorphism.
9. A kit for evaluating the efficacy of olanzapine comprising a reagent capable of detecting a biomarker according to claim 1.
10. The use of the biomarker of claim 1 for evaluating the efficacy of olanzapine, olanzapine detection kits and scientific research.
Technical Field
The invention belongs to the technical field of olanzapine efficacy evaluation, and relates to a biomarker for olanzapine efficacy evaluation, a method and application thereof.
Background
Schizophrenia is a common chronic psychosis, and patients with schizophrenia in 1/3-1/2 do not respond well to psychiatric drugs. Olanzapine is a second generation atypical antipsychotic and has good clinical efficacy, but the pharmacokinetics of olanzapine has significant individual difference and has the risk of weight increase, and has side effects of metabolic disorder, lethargy, prolactin increase and the like. Pharmacogenomics is used for studying the relationship between gene polymorphism and drugs, reveals different drug treatment effects caused by individual differences, and plays an important role in personalized medicine. Since different pathophysiological processes may produce similar phenotypes, pharmacogenomics presents difficulties in pharmacotherapeutic applications for psychiatric disorders. Pharmacokinetics (PK) studies the processes of absorption, distribution, metabolism and excretion of drugs into the human body. The pharmacokinetic parameters are the basis for adjusting the drug dosage, and can effectively avoid toxic and side effects or substandard curative effect caused by excessive or insufficient drugs. Individual differences in drug efficacy or toxicity due to individual differences in metabolic enzymes may lead to unpredictability of drug properties. Endogenous small molecule metabolites are metabolic end products of human physiological processes, and metabolomics illustrates the downstream output and environmental upstream input of genomics through changes in endogenous metabolites. Therefore, the drug genes, pharmacokinetic parameters and small molecule metabolic markers illustrate the whole process of the body for treating the drugs and reacting the drugs on the body from different layers.
Until now, no relevant explanation of olanzapine related drug genes, pharmacokinetic parameters and metabolic markers exists, and prospective medication guidance for drug selection and dose adjustment by combining the three correlated biomarkers cannot be provided.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
It is yet another object of the invention to provide biomarkers for olanzapine efficacy assessment.
Another object of the present invention is to provide the application of primer pairs in searching key drug genes of olanzapine pharmacokinetic differences.
It is another object of the present invention to provide a method for finding key drug genes for pharmacokinetic differences of olanzapine.
Another objective of the invention is to provide application of gamma-aminobutyric acid transaminase gene polymorphism in olanzapine pharmacokinetic study and pharmacodynamic evaluation.
It is another object of the present invention to provide a kit for evaluating the efficacy of olanzapine comprising reagents capable of detecting said biomarkers.
The invention further aims to provide application of the biomarker in evaluation of olanzapine efficacy, olanzapine detection kit and scientific research.
Therefore, the technical scheme provided by the invention is as follows:
biomarkers for olanzapine efficacy evaluation include lysophospholipid monoethanolamine (0:0/18:3), lysophospholipid monoethanolamine (0:0/22:5), and octadecatrienoic acid.
Preferably, the biomarker for evaluating the drug effect of olanzapine further comprises: lysophosphatidylethanolamine (0:0/20:3), lysophosphatidylethanolamine (18:1/0:0), galactoglycerol and 5-hydroxytryptamine.
Use of a primer pair for searching key drug genes for pharmacokinetic differences of olanzapine, said primer pair comprising the nucleotide sequence as set forth in SEQ ID NO: 43 and SEQ ID NO: 44, or a primer set shown in the specification.
Preferably, in the application, the primer pair further comprises a primer sequence shown as SEQ ID NO: 45 and as shown in SEQ ID NO: 46, or a pharmaceutically acceptable salt thereof.
Preferably, in the application, the primer pair further comprises a primer sequence shown as SEQ ID NO: 1 and as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3 and as shown in SEQ ID NO: 4, and the primer pair shown as SEQ ID NO: 5 and as shown in SEQ ID NO: 6, and the primer pair shown as SEQ ID NO: 7 and as shown in SEQ ID NO: 8, and the primer pair shown as SEQ ID NO: 9 and as shown in SEQ ID NO: 10, and the primer pair shown in SEQ ID NO: 11 and as shown in SEQ ID NO: 12, and the primer pair shown in SEQ ID NO: 13 and as shown in SEQ ID NO: 14, and the primer pair shown as SEQ ID NO: 15 and as shown in SEQ ID NO: 16, and the primer pair shown as SEQ ID NO: 17 and as shown in SEQ ID NO: 18, and the primer pair shown as SEQ ID NO: 19 and as shown in SEQ ID NO: 20, and the primer pair shown in SEQ ID NO: 21 and as shown in SEQ ID NO: 22, and the primer pair shown in SEQ ID NO: 23 and as shown in SEQ ID NO: 24, and the primer pair shown in SEQ ID NO: 25 and as shown in SEQ ID NO: 26, and the primer pair shown in SEQ ID NO: 27 and as set forth in SEQ ID NO: 28, and the primer pair shown in SEQ ID NO: 29 and as set forth in SEQ ID NO: 30, and the primer pair shown in SEQ ID NO: 31 and as set forth in SEQ ID NO: 32, and the primer pair shown as SEQ ID NO: 33 and as set forth in SEQ ID NO: 34, and the primer pair shown in SEQ ID NO: 35 and as shown in SEQ ID NO: 36, and the primer pair shown in SEQ ID NO: 37 and the nucleotide sequence as set forth in SEQ ID NO: 38, and the primer pair shown in SEQ ID NO: 39 and as shown in SEQ ID NO: 40, and the primer pair shown as SEQ ID NO: 41 and as shown in SEQ ID NO: 42, and the primer pair shown as SEQ ID NO: 47 and as shown in SEQ ID NO: 48, and the primer pair shown in SEQ ID NO: 49 and as shown in SEQ ID NO: 50, and the primer pair shown as SEQ ID NO: 51 and as set forth in SEQ ID NO: 52, and the primer pair shown as SEQ ID NO: 53 and as shown in SEQ ID NO: 54, and the primer pair shown as SEQ ID NO: 55 and as shown in SEQ ID NO: 56, and/or the primer set shown as SEQ ID NO: 57 and as shown in SEQ ID NO: 58, or a primer set.
A method for finding a key drug gene for pharmacokinetic differences in olanzapine comprising the step of performing amplification using a primer pair as described above.
The application of the gamma-aminobutyric acid transaminase gene polymorphism in olanzapine pharmacokinetic research and pharmacodynamic evaluation.
Preferably, in the application, the gamma-aminobutyric acid transaminase gene polymorphism comprises GABA-T rs1641031 gene polymorphism and GABA-T rs1641022 gene polymorphism.
A kit for evaluating the efficacy of olanzapine comprising a reagent capable of detecting said biomarker.
The biomarker is applied to evaluation of olanzapine efficacy, olanzapine detection kits and scientific research.
The invention at least comprises the following beneficial effects:
according to the invention, research on olanzapine in vivo metabolism is carried out for the first time through a comprehensive analysis method of endogenous small molecule metabolic markers, pharmacokinetic parameters and drug genes, GABA-T causes individual difference of olanzapine drug metabolism peak reaching time and clearance, GABA-T influences 5-hydroxytryptamine metabolic pathway, and the endogenous small molecule biomarkers related to the drug genes have the capability of predicting drug concentration, so that an important basis is provided for clinical individualized medication.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
In FIG. 1, 1A is a correlation graph of olanzapine peak blood concentration (Cmax) and GABA-T rs 1641033; 1B is a correlation graph of olanzapine Clearance (CL) and GABA-T rs 1641033; 1C is a correlation plot of olanzapine Clearance (CL) and GABA-T rs 1641022.
FIG. 2 is an OPLS-DA model of differentiating subgroups in one embodiment of the present invention, each point representing one volunteer, the left point representing volunteers with a low Cmax, and the right point representing volunteers with a high Cmax.
FIG. 3 is a graph showing the correlation between the intensity of differential metabolites and Cmax in one embodiment of the present invention.
FIG. 4 is a graph of differential metabolite intensity versus GABA-Trs1641031 in one embodiment of the present invention.
FIG. 5 is a graph of the correlation analysis of differential metabolite concentrations with GABA-Trs1641022 in one embodiment of the present invention.
FIG. 6 is a schematic diagram of a biomarker screening process in one embodiment of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The invention provides biomarkers for olanzapine efficacy evaluation, which comprise lysophospholipid monoethanolamine (0:0/18:3), lysophospholipid monoethanolamine (0:0/22:5) and octadecatrienoic acid.
In the above aspect, preferably, the method further includes: lysophosphatidylethanolamine (0:0/20:3), lysophosphatidylethanolamine (18:1/0:0), galactoglycerol and 5-hydroxytryptamine.
The invention provides application of a primer pair in searching for a key drug gene of olanzapine pharmacokinetic difference, wherein the primer pair comprises a primer pair shown in SEQ ID NO: 43 and SEQ ID NO: 44, or a primer set shown in the specification.
In the above scheme, preferably, the primer pair further comprises a primer set as shown in SEQ ID NO: 45 and as shown in SEQ ID NO: 46, or a pharmaceutically acceptable salt thereof.
In one embodiment of the present invention, preferably, the primer pair further comprises a primer set as shown in SEQ ID NO: 1 and as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3 and as shown in SEQ ID NO: 4, and the primer pair shown as SEQ ID NO: 5 and as shown in SEQ ID NO: 6, and the primer pair shown as SEQ ID NO: 7 and as shown in SEQ ID NO: 8, and the primer pair shown as SEQ ID NO: 9 and as shown in SEQ ID NO: 10, and the primer pair shown in SEQ ID NO: 11 and as shown in SEQ ID NO: 12, and the primer pair shown in SEQ ID NO: 13 and as shown in SEQ ID NO: 14, and the primer pair shown as SEQ ID NO: 15 and as shown in SEQ ID NO: 16, and the primer pair shown as SEQ ID NO: 17 and as shown in SEQ ID NO: 18, and the primer pair shown as SEQ ID NO: 19 and as shown in SEQ ID NO: 20, and the primer pair shown in SEQ ID NO: 21 and as shown in SEQ ID NO: 22, and the primer pair shown in SEQ ID NO: 23 and as shown in SEQ ID NO: 24, and the primer pair shown in SEQ ID NO: 25 and as shown in SEQ ID NO: 26, and the primer pair shown in SEQ ID NO: 27 and as set forth in SEQ ID NO: 28, and the primer pair shown in SEQ ID NO: 29 and as set forth in SEQ ID NO: 30, and the primer pair shown in SEQ ID NO: 31 and as set forth in SEQ ID NO: 32, and the primer pair shown as SEQ ID NO: 33 and as set forth in SEQ ID NO: 34, and the primer pair shown in SEQ ID NO: 35 and as shown in SEQ ID NO: 36, and the primer pair shown in SEQ ID NO: 37 and the nucleotide sequence as set forth in SEQ ID NO: 38, and the primer pair shown in SEQ ID NO: 39 and as shown in SEQ ID NO: 40, and the primer pair shown as SEQ ID NO: 41 and as shown in SEQ ID NO: 42, and the primer pair shown as SEQ ID NO: 47 and as shown in SEQ ID NO: 48, and the primer pair shown in SEQ ID NO: 49 and as shown in SEQ ID NO: 50, and the primer pair shown as SEQ ID NO: 51 and as set forth in SEQ ID NO: 52, and the primer pair shown as SEQ ID NO: 53 and as shown in SEQ ID NO: 54, and the primer pair shown as SEQ ID NO: 55 and as shown in SEQ ID NO: 56, and/or the primer set shown as SEQ ID NO: 57 and as shown in SEQ ID NO: 58, or a primer set.
The invention also provides a method for searching key drug genes of olanzapine pharmacokinetic difference, which comprises the step of amplifying by using the primer pair.
The invention also provides application of the gamma-aminobutyric acid transaminase gene polymorphism in olanzapine pharmacokinetic research and pharmacodynamic evaluation.
In one embodiment of the present invention, preferably, the gamma-aminobutyric acid transaminase gene polymorphism comprises a GABA-T rs1641031 gene polymorphism and a GABA-T rs1641022 gene polymorphism.
The invention also provides a kit for evaluating the efficacy of olanzapine, comprising a reagent capable of detecting said biomarker.
The invention also provides application of the biomarker in evaluation of olanzapine efficacy, olanzapine detection kit and scientific research.
In order to make the technical solution of the present invention better understood by those skilled in the art, the following examples are now provided for illustration:
the invention carries out correlation analysis on the drug genes and pharmacokinetic parameters, and searches and determines key drug genes causing individual pharmacokinetic differences; performing correlation analysis on PK and metabolic markers, and searching for small molecule biomarkers capable of predicting pharmacokinetic parameters; the data of the drug genes and the metabolic markers are subjected to correlation analysis, the difference change of endogenous metabolites caused by different drug genes is searched, and biomarkers related to drug effect are explored and found from multiple layers of drug genes, treatment of drugs by organisms, and endogenous metabolites of the organisms.
1 materials and methods
1.1 clinical Subjects
19 Chinese adult volunteers were enrolled from the olanzapine orally disintegrating tablet bioequivalence test (SWDXX-201627) at the Kyoto rising Hospital, Beijing, university of medicine. The bioequivalence study (http:// www.chinadrugtrials.org.cn/, identidler: CTR20170069) was conducted in a single study center at phase I clinical trial center in the rising Hospital, Beijing, between 16 and 14 days 5 and 2018 in 2017. The study was performed in compliance with the declaration of helsinki and the guidelines of clinical practice. All study documents, including study protocol and consent, were reviewed and approved by the review Committee of the Hospital institute of Chaojingyang (ethical approval number: 2016-Drug-43) before the start of the clinical trial. Informed consent was obtained from all subjects in this study to ensure that subjects were compliant with the study restrictions promulgated by the protocol of the clinical trial center. The whole blood sample used in genomics is the remainder of the pharmacokinetic study, and complies with the principle of exemption from informed consent. The pharmacokinetic experimental data is selected from the bioequivalence experimental results, and the metabonomic data is derived from metabonomic experimental data developed in the earlier stage of the project.
1.2 Single Nucleotide Polymorphism (SNP) selection, DNA extraction and detection
Early olanzapine metabonomics research discovers neurotransmitter metabolites related to drug metabolism, and relates to a tryptophan metabolic pathway, a glutamic acid and aminobutyric acid metabolic pathway and a dopamine and norepinephrine metabolic pathway, and the research selects metabolic enzymes related to the three metabolic pathways: tryptophan metabolism-related glucuronyl transferase (UGT); tyrosine, Tyrosine Hydroxylase (TH) involved in the metabolism of dopamine and norepinephrine; glutamate and GABA metabolism-related gamma-aminobutyric acid transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). The sites and amplification primers are detailed in Table 1. Plasma samples were the remainder of the pharmacokinetic study, genomic DNA was extracted using QIAamp DNA blood mini kit (QIAGEN), and DNA concentration was determined using NanoDrop 2000(Thermo Scientific). SNPs were genotyped by Polymerase Chain Reaction (PCR) and Sanger sequencing.
TABLE 1 specific primers in dideoxy end-termination (Sanger) sequencing assays
1.3 data analysis
Comparison of the data between the two groups was performed using the IBM SPSS 21(Armonk, new york, usa) independent sample T test (data obeying normal distribution) or the Mann-Whitney U test (data not obeying normal distribution), with P <0.05 considered statistically significant. The variables with VIP values greater than 1 and independent sample T test or Mann-Whitney U test P <0.05 were considered as potential biomarkers using SIMAC-P14.1 (Umetrics AB, Umea, Sweden) for orthogonal partial least squares discriminant analysis (OPLS-DA).
2. Results
2.1 Association analysis of pharmacokinetic parameters with drug genes
2.1.1 pharmacokinetic parameters of olanzapine
Obtaining pharmacokinetic parameters of 19 healthy volunteers after taking olanzapine, wherein the main parameters comprise: peak concentration (C)max) Area under drug concentration-time Curve (AUC)0-t) Clearance (CL), time to peak (T)max) And elimination half-life (T)1/2). Highly individualized differences in olanzapine's drug metabolism were found by pharmacokinetic parameters. Peak concentration (C)max) The variation is maximal, the difference between the maximum and minimum values is more than 6 times, and the difference between the maximum and minimum values of Clearance (CL) is more than 3 times. Peak concentration (C)max) Has great significance for drugs with large individual variation difference and clearance rate(CL) is an important index reflecting the elimination of the drug from the body, and previous studies show that the drug effect of olanzapine is related to the plasma concentration of the drug, and that the clearance rate between olanzapine individuals is greatly different (Table 2). Therefore, the peak concentration (C) was selectedmax) And Clearance (CL) were correlated.
TABLE 2 pharmacokinetic parameters for olanzapine administration in healthy subjects
2.1.2SNP genotyping and frequency
And (3) detecting SNP sites of UGT1A1, UGT1A4, GABA-T, TH and SSADH related genes, and displaying the SNP frequency results of all the tested single nucleotide polymorphisms in the table 2. The above gene polymorphisms were all subjected to Hardy-Weinberg equilibrium analysis (the Hardy-Weinberg equibrium analysis). Due to sample number limitations, genotypes are divided into two categories: wild homozygotes, mutant heterozygotes, and mutant homozygotes. UGT1A1 rs873478, UGT1A1 rs4148325, UGT1A4 rs8330, GABA-T rs1641025 and SSADH rs2744574 are grouped according to the drug genotype, so that the number of people in each group is less than three, and the statistical analysis is meaningless, so that the method is not included in the later analysis research. The UGT1A4 rs2011425 and TH rs6357 genotypes of the subjects are all wild homozygous; the TH rs11042962, TH rs11042978, TH rs6356 and TH rs7119275 genotypes of the subjects are all mutation heterozygote types or mutation homozygote types, so the genotypes are not included in the later analysis research.
TABLE 3 frequency of SNP sites of UGT1A1, UGT1A4, GABA-T, TH, SSADH-related genes in healthy subjects
2.1.3 Effect of SNP genotyping on the pharmacokinetics of olanzapine
Subjects were divided into two groups according to drug genotype: wild homozygote; wild mutant heterozygotes and mutant homozygotes. The parameters of pharmacokinetics between groups were compared by independent sample T-test or Mann-Whitney U-test (table 4). Compared with subjects with rs1641031 AA genotype, the peak reaching concentration (Cmax) of GABA-T rs1641031 AC and CC genotypes is averagely increased by 59.2 percent (P is 0.036), and the Clearance (CL) is averagely reduced by 25.33 percent (P is 0.010). GABA-T rs1641022CA, AA genotype subjects, and rs1641022CC genotype subjects had an average increase in Clearance (CL) of 33.0% (P ═ 0.041) (fig. 1).
TABLE 4 pharmacokinetic parameters of olanzapine administration to healthy subjects for different drug genotyping
Values are expressed as mean ± sd, with significant differences at p <0.05
2.2 Association analysis of pharmacokinetic parameters with differential metabolites
At the early stage, 41 endogenous differential metabolites related to drug metabolism are screened out through non-targeted metabonomics research of an olanzapine test group and an unadministered olanzapine control group. Subjects were assigned to CmaxThe median of (a) is: high CmaxGroup (N ═ 5), where CmaxGroup (N ═ 6), low CmaxGroup (N ═ 5); the median score according to CL is: high CL group (N ═ 5), medium CL group (N ═ 6), and low CL group (N ═ 5). Screening for high C Using OPLS-DA modelmaxGroup and Low CmaxDifferential metabolites between group, high CL group and low CL group. OPLS-DA model for CL: r2X＝0.335；R2Y＝0.939； Q2At 0.225, the CL model parameters are not satisfactory and therefore no subsequent analysis is performed. For CmaxOPLS-DA model of (A)2X＝0.539；R2Y＝0.889,Q20.554 as shown in fig. 2, CmaxBetter model parameters, high CmaxGroup and Low CmaxGroups are clearly distinguished. The interclass independent sample T test or Mann-Whitney U test (fig. 3) was performed on metabolites with VIP values greater than 1, and 7 metabolites were screened: cmaxHigh group and CmaxComparison in the lower group, lysophosphatidylethanolamine [ lysoPE (0:0/20:3)]The average intensity of the metabolism peak of 63.18% (P ═ 0.026); lysophosphatidylethanolamine [ lysoPE (0:0/18:3)]The intensity of the metabolism peak is increased by 99.58% on average (P is 0.013); the metabolic peak intensity of the galactoglycerol (Galactosylglycerol) is increased by 71.29 percent on average (P is 0.008); lysophosphatidylethanolamine [ lysoPE (0:0/22:5)]The average intensity of the metabolism peak of the medicine is increased by 169.10% (P is 0.008); the metabolic peak intensity of 5-hydroxytryptamine (5-hydroxytryptamine) is increased by 569.43% on average (P ═ 0.032); the metabolic peak intensity of the octadecatrienoic acid (Calendic acid) is increased by 76.15 percent on average (P is 0.032); lysophosphatidylethanolamine [ lysoPE (18:1/0:0)]The average increase in metabolic peak intensity was 150.44% (P ═ 0.013), see table 5 for details.
TABLE 5 independent sample T-test or Mann-Whitney U-test for VIP > 1 metabolites
Values are expressed as mean ± sd, with significant differences at p <0.05
2.3 Association of differential metabolites with drug genes
Subjects were grouped according to the GABA-T rs1641031 gene polymorphism, GABA-T rs1641022, and the peak intensities of 41 endogenous metabolites before drug administration were compared between two groups (independent sample T test or Mann-Whitney U test), P <0.05, which metabolites were statistically different between groups (tables 6 and 7). The peak intensity of Cortisol (Cortisol) metabolism is increased by 60.0% on average in GABA-Trs1641031 AC, CC genotype subjects and rs1641031 AA genotype subjects (P ═ 0.009); the metabolic peak intensity of Tryptophan (Tryptophan) is increased by 29.68 percent on average (P is 0.015); the intensity of a metabolic peak of 5-hydroxytryptophan (5-hydroxytryptophan) is increased by 24.64 percent on average (P is 0.015); the intensity of the metabolic peak of lysophosphatidylethanolamine [ LysoPE (0:0/18:3) ] is increased by 54.08% on average (P ═ 0.033); the intensity of the metabolic peak of lysophosphatidylethanolamine [ LysoPE (0:0/22:5) ] is increased by 96.40% on average (P ═ 0.044); the metabolic peak intensity of octadecatrienoic acid (Calendic acid) increased by 73.46% on average (P ═ 0.042) (fig. 4). Compared with subjects with GABA-Trs1641022AC, CC genotype and rs1641031 AA genotype, the metabolism peak intensity of sphingosine (sphingosine) is reduced by 39.00% on average (P is 0.002); the intensity of the metabolic peak of 5-hydroxytryptophan (5-hydroxytryptophan) was reduced by 20.67% on average (P ═ 0.024) (fig. 5).
TABLE 6 differentiation of endogenous metabolites of olanzapine administration after grouping based on GABA-Trs1641031
Values are expressed as mean ± sd, with significant differences at p <0.05
TABLE 7 differences in metabolites of olanzapine after GABA-Trs1641022 grouping
Values are expressed as mean ± sd, with significant differences at p <0.05
3. Discussion of the related Art
3.1 drug Gene and pharmacokinetic parameters
The invention firstly discovers that the polymorphism of the GABA-T related gene has influence on the pharmacokinetics of olanzapine. Under the catalysis of GABA-T, GABA reacts with pyruvic acid to generate Succinic Semialdehyde (SSA) and alanine, and succinic semialdehyde dehydrogenase oxidizes SSA to form succinic acid which enters a tricarboxylic acid cycle, and the reaction forms the GABA branch of the tricarboxylic acid cycle. Previous studies have demonstrated that an increase or decrease in GABA-T activity results in a decrease or increase in GABA levels. Olanzapine has an activating effect on the GABA system, which leads to a long-term decrease in glutamate and GABA levels in the nucleus accumbens of adolescent rats. Research proves that olanzapine has influence on GABA neurotransmission related gene expression of human neuroblastoma cells, GABA system and olanzapine metabolism have correlation, and finds that olanzapine can promote GABA degradation by improving GABA-T activity. The invention discovers that the polymorphism of the GABA-T rs1641031 gene has significant influence on the peak reaching concentration and the clearance rate of olanzapine for the first time, and the GABA-T rs1641022 has significant influence on the clearance rate of olanzapine.
3.2 differential metabolite and pharmacokinetic parameters
The drug metabolism of olanzapine is associated with the metabolic pathways of various neurotransmitters. The invention firstly performs correlation analysis on the metabonomics and pharmacokinetic data of olanzapine. The research finds that: lysophosphatidylethanolamine (0:0/20:3) [ lysoPE (0:0/20:3)]Lysophosphatidylethanolamine (0:0/18:3) [ lysoPE (0:0/18:3)]Lysophosphatidylethanolamine (0:0/22:5) [ lysoPE (0:0/22:5)]Lysophosphatidylethanolamine (18:1/0:0) [ lysoPE (18:1/0:0)]Galactose glycerol (Galactosylglycerol), 5-hydroxytryptamine (5-hydroxytryptamine), and octadecenoic acid (Calendic acid)maxSubjects of high group and CmaxThe subjects in the lower group were clearly distinguished, i.e. the above biomarker was compared with olanzapine CmaxAre significantly related. Previous researches report that olanzapine has antagonism on 5-hydroxytryptamine receptors, and the drug concentration of olanzapine is increased possibly due to the fact that 5-hydroxytryptamine is too high in concentration. Additional studies have shown that in patients with 5-hydroxytryptamine deficiency, lipid metabolism is affected and lysophospholipids are also down-regulated. In the invention, the concentration of 5-hydroxytryptamine is obviously increased in a subject with high olanzapine peak concentration compared with that of olanzapine peak concentration, the concentration of various lysophospholipids is obviously increased, and the 5-hydroxytryptamine and lipid metabolic pathways are closely related to the drug concentration of olanzapine.
3.3 drug genes and endogenous metabolites
Compared with the subjects with the GABA-T rs1641031 AC and CC genotypes and the subjects with the rs1641031 AA genotypes, the concentrations of Cortisol (Cortisol), Tryptophan (Tryptophan), octadecatrienoic acid (Calendic acid), 5-hydroxytryptophan (5-hydroxytryptophan), lysophosphatidylethanolamine (0:0/18:3) [ LysoPE (0:0/18:3) ] and lysophosphatidylethanolamine (0:0/22:5) [ LysoPE (0:0/22:5) ] are remarkably increased. Compared with GABA-Trs1641022AC, CC genotype subjects and rs1641031 AA genotype subjects, the concentrations of sphingosine (sphingosine) and 5-hydroxytryptophan (5-hydroxytryptophan) are obviously reduced. It is noted that compared with the rs1641031 AA genotype subjects, the GABA-T rs1641031 AC and CC genotype subjects have higher concentration of 5-hydroxytryptophan, and tryptophan is generated into 5-hydroxytryptophan by tryptophan hydroxylase and further decarboxylated to form 5-hydroxytryptophan. Compared with rs1641031 AC, CC genotype subjects and rs1641031 AA genotype subjects, the concentration of tryptophan which is the upstream metabolite of 5-hydroxytryptophan is obviously increased. The results of the invention show that the GABA-T related metabolic pathway of 5-hydroxytryptamine in the body is related to the in vivo metabolism of olanzapine.
3.4 pharmacokinetic parameters & drug genes & differential metabolites
Compared with a GABA-T rs1641031 AC and CC genotype subject and an rs1641031 AA genotype subject, the clearance rate of olanzapine drug metabolism is obviously reduced; the concentration of 5-hydroxytryptophan is obviously increased; the concentration of tryptophan, an upstream metabolite of 5-hydroxytryptophan, is significantly increased. Compared with a subject with a GABA-Trs1641022AC and CC genotype and a subject with an rs1641022 AA genotype, the clearance rate of olanzapine is obviously increased, and the concentration of 5-hydroxytryptophan is reduced. The result shows that the metabolic pathway of 5-hydroxytryptamine is closely related to GABA-T gene polymorphism, and 5-hydroxytryptamine concentration is inversely proportional to olanzapine clearance rate.
Compared with a GABA-T rs1641031 AC and CC genotype subject and an rs1641031 AA genotype subject, CmaxThe improvement is remarkable; compared with LysoPE (0:0/18:3), LysoPE (0:0/22:5) and Calendic acid, the GABA-T rs1641031 AC, CC genotype subjects and rs1641031 AA genotype subjects are obviously increased; cmaxHigher subjects and CmaxLysoPE (0:0/18:3), LysoPE (0:0/22:5), Calendic acid were also significantly elevated compared to lower subjects.
Thus, GABA-T rs1641031 correlates with peak concentration and clearance of olanzapine; GABA-T rs1641022 is associated with the clearance rate of olanzapine, GABA-T related gene polymorphism has an influence on a 5-hydroxytryptamine metabolic pathway, and lysophosphatidylethanolamine (0:0/18:3), lysophosphatidylethanolamine (0:0/22:5) and octadecatrienoic acid can distinguish subjects with high drug concentration and low drug concentration of olanzapine and are expected to be biomarkers for predicting the drug effect of olanzapine (figure 6).
4. Conclusion
According to the invention, research on olanzapine in vivo metabolism is carried out for the first time through a comprehensive analysis method of endogenous small molecule metabolic markers, pharmacokinetic parameters and drug genes, GABA-T causes individual difference of olanzapine drug metabolism peak reaching time and clearance, GABA-T influences 5-hydroxytryptamine metabolic pathway, and the endogenous small molecule biomarkers related to the drug genes have the capability of predicting drug concentration, so that an important basis is provided for clinical individualized medication. The invention has the problems of small sample number, lack of relevant data such as the medication information and the outcome of clinical patients and the like, and still needs to carry out the verification of large samples and clinical data in future research.
SEQUENCE LISTING
<110> Beijing Chaoyang Hospital affiliated to capital medical university
<120> biomarker for evaluating drug effect of olanzapine, method and application
<130> 2019
<160> 58
<170> PatentIn version 3.5
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