| Nowadays, modern drug therapy has been more inclined to develop personalized target therapy. With the development of human genomics, genetics based pharmacogenomics has been developed rapidly. Pharmacogenomics is a subject to investigate the association between genetic factors and drug response, which provides genetic evidences for personalized target therapies. Traditional therapy designs personalized dosage regimen through measuring drug concentration and calculating pharmacokinetic parameters, which is applicable to the drugs with consistent efficacy and plasma concentration. With respect to patients who had similar diagnosis, general condition and plasma concentration but different efficacy and adverse reaction, traditional pharmacokinetic mechanism is failed to explain that clinical phenomenon. Since 1950s, some scholars have found that drug response is different with different individual genetic background. In 1959, the concept of pharmacogenomics was formally proposed for the first time. Pharmacogenomics provided theory for similar plasma concentration but different drug efficacy and adverse reaction to some extent. In 1990s, pharmacogenomics was developed rapidly with the development of human genomics. Single nucleotide polymorphisms (SNPs) were involved in the basic research. For the decisive impact of genetic diversity on the differences in individual plasma concentration, clinical efficacy and adverse reaction, pharmacogenomics requires that the application of drug should take the population allelic frequency into consideration and prescription also should be personalized corresponding to individual genetic characteristics. So far, hundreds of drugs were labeled as genetic characteristic drugs by the U.S. Food & Drug Administration (FDA). This study aimed to explore the "associations of theophylline clearance with CYP1A2, CYP2E1 and CYP3A4 genetic polymorphisms" was performed on the basis of pharmacogenomics.Theophylline, a common anti-asthmatic drug, has been applied clinically for more than 80 years. Theophylline can relieve asthma effectively and ameliorate the symptom of respiratory tract spasm. Recent studies showed that low concentration of theophylline (5-10 mg/L) has the functions of anti-inflammation and immune regulation. Hence, theophyllines have been the recommended drugs for the prevention of chronic obstructive pulmonary disease (COPD) and asthma, even as a primary drug for the treatment of asthma in some areas of our country. However, theophylline has a narrow therapeutic window and its therapeutic concentration is close to the toxic concentration. The effective range of plasm concentration is 5-20 mg/L. Adverse reaction may happen when plasm concentration is higher than 20 mg/L. The common adverse reactions of theophylline include arrhythmia, myocardial ischemia, insomnia, delirium and epilepsia, even death. So theophylline should be prescribed under the monitoring of plasm concentration to ensure the safety and efficacy of administration. It has been a main monitoring drug at home and abroad.About 90% of theophylline is metabolized as 1,3-dimethyluric acid,1-methyl xanthine and 3-methyl xanthine in vivo.1-methyl xanthine is further metabolized as 1-methyl uric acid for its instability. The remaining 10% is excreted by kidney. For the high affinity of liver cytochrome P4501A2 (CYP1A2) to theophylline, the metabolism of low concentration of theophylline in vivo is mainly mediated by CYP1A2. Cytochrome P4502E1 (CYP2E1) has high content to theophylline, thus CYP2E1 is involved in the metabolism of high concentration of theophylline. Cytochrome P450 3A4 (CYP3A4) is also contributed in the metabolism of theophylline but with less effect for its low affinity and low content.Cytochrome P450, a group of ferroheme enzymes, has oxidation function and plays an important role in the metabolism of many endogenous and exogenous compounds. The subfamily of human liver cytochrome P450mainly includes CYP1, CYP2 and CYP3. The variation of cytochrome P450 activity in individuals may be related to genetic and environmental factors, mainly be affected by genetic factors. CYP1A2 is accounting for 13%-15% of the weight of liver cytochrome P450 and is one of the important members of liver metabolic enzyme. Significant interindividual differences in CYP1A2 expression were observed. Studies reported that there is 15-40 times of individual differences in mRNA expression. There are four reported SNPs associated with the activity and inductivity of CYP1A2:G-3860A (CYP1A2*1C),-2467delT (CYP1A2*1D), C-729T (CYP1A2*1E) and C-164A (CYP1A2*1F). CYP2E1 accounts for 7% of human liver metabolic enzyme and is involved in regulating the metabolism of some endogenous and exogenous intoxicants. Most substrates are characterized by relatively low molecular weight (<100) and relatively high lipotropy. CYP2E1 is a key metabolic enzyme involved in the process of alcohol and analgesic-antipyretic producing hepatotoxicity to acetyl aminophenol and anti-tuberculosis drugs. Additionally, it is also regarded as a very important enzyme for the metabolisms and activities of all kinds of toxic substances, including nitrosamine, benzene and chlorethylene. The gene sequence of CYP2E1 gene is relatively conservative. So far, there are 10 reported SNPs,4 SNPs have functional activity as reported in Asians:G1132A (CYP2E1*2), G10023A (CYP2E1*3), G4768A (CYP2E1*4) and G-1293C/C-1053T(CYP2E1*5B). CYP3A4 is the most abundant metabolic enzyme in human liver, regulating more than 50% drug metabolism. The genetic sequence of CYP3A4 is also relatively conservative and few SNPs were discovered to affect the activity of enzyme. The SNPs with functional activity in Asians mainly includes:A13871G (CYP3A4*4), C15702G (CYP3A4*5),17661del (CYP3A4*6), T 20070C (CYP3A4*18A), C 23237T (CYP3A4*19) and C15389T (CYP3A4*22).Few literatures reported that the three mentioned metabolic enzymes genetic polymorphisms were associated with the metabolism of theophylline in vivo, and the study results remains controversial. It is reported that G-3860A (CYP1A2*1C) and C-164A (CYP1A2*1F) may affect the metabolism of theophylline, while there is also contrary report. As for the effect of CYP2E1 genotype on the metabolism of theophylline, only Korean scholars found that the haplotype composed of 5 SNPs (-1566T>A,-1295 G>C,-1055 C>T,-1027 T>C and-807T>C) would cause the decrease of theophylline clearance. No studies reported that CYP3A4 gene polymorphism affected on the metabolism of theophylline. Hence, no clear conclusion was made about the influences of CYP1A2, CYP2E1 and CYP3A4 genetic polymorphisms on the metabolism of theophylline.In our previous study, we analyzed the correlation of CYP1A2 activity and its functional sites in 426 healthy Chinese, and have screened 5 label SNPs and established haplotypes with 15 homozygotes and heterozygote with mutation frequency over 3%. Through analyzing the influences of the 5 SNPs and their 15 haplotypes on CYP1A2 enzymatic activity, we found that the mutation of Gâ†'>A of G-3113A may decrease the CYP1A2 activity. Homozygous CYP1A2*1B and CYP1A2*1C also slightly decrease the CYP1A2 activity. No significant differences in CYP1A2 activity were found in different genotypes of T3594G and CYP1A2*1F. CYP1A2 activity was significant different among haplotypes. Haplotypes with-3113A/A showed low enzymatic activity and haplotyes with-3113G/G showed high enzymatic activity. Based on the research, we further explored the influences of the mutant homozygotes of the 4 functional sites on the metabolism of theophylline and found that no obvious differences of theophylline metabolism existed among the 4 functional sites. Only 3 samples was screened in-3113A/A. For the relatively small sample size, further statistic analysis has not been made, but the plasma concentration of theophylline was found increasing in-3113A/A genotype. Thus, our previous study suggested that the G-3113A mutation of CYP1A2 may lead to the decreased CYP1A2 activity. For the limitation of sample size, we did not find significant difference of theophylline metabolism in CYP1A2 different genotype.Therefore, the mechanism of individual difference in theophylline metabolism is still unclear. This study attempts to identify the genetic mechanism of individual difference in theophylline metabolism by analyzing the influences of CYP1A2, CYP2E1, CYP3A4 mutations on theophylline clearance in vivo.MethodsA total of 111 outpatients were initially selected between September 2013 and July 2014. Ten of them lost and 101 participants (63 males and 38 females; 18 smokers and 83 non-smokers) enrolled in this study finally. Blood samples were collected from the 101 participants. Subsequently, the participants took 100 mg theophylline, twice a day and received subsequent visit two weeks later. Peripheral blood samples (2 ml) were collected in EDTA tube under fasting condition and then kept at -80℃. The study was approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University. Informed consent was collected from each participant. The trial was registered on USA ClinicalTrials.gov with the register number NCT02001935. High efficiency liquid chromatography (HPLC) was used for the measurement of theophylline and its metabolin (1,3-dimethyluric acid). Theophylline clearance was calculated by the ratio 1,3-dimethyluric acid/theophylline (1,3-DU/TP). DNA in peripheral blood leucocyte was extracted by phenol-chloroform. Direct sequence assay was performed for amplified PCR products with gene sequencing machine. Five SNPs, G-3860A (CYP1A2*1C), C-164A (CYP1A2*1F), C5347T (CYP1A2*1B), G-3113A and G-3533A (CYP1A2*7), were detected. The four SNPs, G-3860A (CYP1A2*1C), C-164A (CYP1A2*1F), C5347T (CYP1A2*1B) and G-3113A were found there were polymorphism in Chinese population. CYP1A2 G-3533A (CYP1A2*7) was also included in detection which was reported to be functional in Caucasians. Four SNPs of CYP2E1 were detected, including G1132A (CYP2E1*), G10023A (CYP2E1*3), G 4768A (CYP2E1*4) and G-1293C/C-1053T (CYP2E1*5B), which were reported to be functional in Asians. Five SNPs of CYP3A, A13871G (CYP3A4*4), C15702G (CYP3A4*5),17661del (CYP3A4*6), T 20070C (CYP3A4*18A) and C23237T (CYP3A4*19), which were reported to be functional in Asians were also detected. Haplotypes were established composed of the SNPs detected with polymorphism to further analyze the associations of theophylline clearance and the genotype.Statistic methodsAll data is expressed as mean ± SD. The participants were grouped with different genotypes. The comparison of theophylline clearance in different genotypes of single SNP was performed by one-way analysis of variation (one-way ANOVA). The comparisons between gender and two different genotypes were performed with independent-sample t test. Results were considered statistically significant with P< 0.05. All statistical analysis was conducted by applying SPSS 13.0.Results1. One sample was lost in the detection of theophylline concentration, so 100 participants’ data was calculated. With the dosage of theophylline 100 mg twice a day, the average stable concentration was 3.52±1.45 mg/L (mean±SD) with the maximum 8.31 mg/L and the minimum 1.13 mg/L. The average theophylline clearance rate (1,3-DMU/TP) was 0.058±0.033 with the maximum 0.259 and the minimum 0.041. Significant individual difference was existed in theophylline clearance.2. The mutation frequency of CYP1A2 G-3860A (CYP1A2*1C) of the 101 participants was 30.7%,30.0% for C-164A (CYP1A2*1F),15.8% for C5347T (CYP1A2*1B) and 9.9% for G-3113A. The results were consistent with our previous study while was significant different compared to the results in Caucasians. G-3533A (CYP1A2*7) was reported to exist polymorphism in Caucasians, but we did not find the mutation of CYP1A2*7 in Chinese. Further analysis of the difference of theophylline clearance in the four SNPs different genotypes found that there were no significant difference of theophylline clearance in different genotypes of CYP1A2 G-3860A, C-164A and C5347T (P>0.05). Only 2 samples were screened with the-3113 AA genotype, so AA genotype and GA genotype were combined to one group. There were significant difference between GG group and GA+AA group (t=2.450, P=0.016). Theophylline clearance was decreased with CYP1A2-3113 Gâ†'A.3. The mutation frequency of CYP2E1 G1132A (CYP2E1*2) was 0.5%, while the reported mutation frequency in Asians was 1.2%. No mutation was found in G10023A (CYP2E1*3) and G4768A (CYP2E1*4) in this study. The mutation frequency of G-1293C/C1053T (CYP2E1*5B) was 18.8%, close to the reported frequency of 19%-27% in Asians, was different from the reported 0.8%-1.0% in Caucasians. Further analysis showed that no significant difference of theophylline clearance was found in different genotypes of CYP2E1*5B (P=0.731).4. There was relatively low mutation frequency in 5 detected SNPs of CYP3A4. Only one heterozygote of A13871G (CYP3A4*4) was detected with the mutation frequency 0.5% and 4 heterozygote of T20070C (CYP3A4*18A) was screened with the mutation frequency 2%. No mutations were screened in C15702G (CYP3A4*5), 17661del (CYP3A4*6) and C 23237T (CYP3A4*19). Further analysis of wild-type homozygote and heterozygote, no difference of theophylline clearance was found between CYP3A4*18A genotypes.5. A total of 15 haplotypes were established composed of the 4 SNPs of CYP1A2 which were detected to be mutated. By further analysis the association of theophylline clearance between different haplotypes, we found that theophylline clearance increased with the haplotype of-3113G/G-164A/A-3860 G/G-5347T/C. There were no significant differences of theophylline among other haplotypes.Conclusions1. This study indicates that the average stable plasma concentration is 3.52mg/L with the theophylline dosage of 100 mg, twice a day, which is less than the common concentration for relieving asthma (10-20mg/L). Theophylline has the function of immune regulation and reversing glucocorticoids resistance with concentration of less than 10mg/L. We recommend low dose of theophylline applied combined with glucocorticoids to strengthen glucocorticoid sensitivity.2.14 SNPs of CYP1A2, CYP2E1 and CYP3A4 was detected in this study. Four SNPs of CYP1A2 and one SNP of CYP2E1 have relatively high mutation frequency: CYP1A2 G-3860A (CYP1A2*1C), C-164A (CYP1A2*1F), C5347T (CYP1A2*1B), CYP1A2 G-3113A, and G-1293C/C1053T (CYP2E1*5B). The mutation frequencies were close to reported frequencies in Asians, but significantly different from Caucasians. There were low mutation frequencies or no mutation in other SNPs, which was slightly different from the reported mutation frequency. This study also identified the ethnicity differences existed among CYP1A2, CYP2E1 and CYP3A genes.3. After analyzing the effects of different genotypes of the SNPs with relative high mutation frequency on theophylline clearance rate, we found that CYP1A2-3113Gâ†'A mutation may lead to theophylline clearance deceased. Other SNPs mutation had no effect on theophylline clearance. This is the first report that CYP1A2-3113Gâ†'A affects theophylline metabolism. The higher mutation frequency of CYP1A2 G-3113A in Chinese than Caucasians may account for the slower theophylline metabolism in Chinese than Caucasians.4. The comparison among haplotypes found that theophylline clearance increased with the haplotype of -3113G/G-164A/A-3860G/G-5347T/C. Thus, the study indicated that investigating the effects of gene polymorphism on theophylline metabolism should take haplotypes into consideration. |
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