基因多型性與sulfamethoxazole-trimethoprim藥品代謝物血中濃度與不良反應之關聯性研究

Abstract

背景 Co-trimoxazole (sulfamethoxazole-trimethoprim)為廣效性抗生素，可用於治療多種感染症，尤其在肺囊蟲肺炎（PJP）治療上為第一線用藥。Sulfamethoxazole（SMX）主要由肝臟代謝，其中約70%代謝途徑經由乙醯轉移酵素（NAT）代謝成沒有毒性的產物N4-acetyl sulfamethoxazole（N4-acetyl SMX），其餘則會由CYP2C9代謝，此類代謝物在人體可能導致毒性。NAT同時包含NAT1與NAT2兩種酵素，均具基因多型性。臺灣地區的NAT1基因多型性分布比例與其他亞洲國家相似，約有20%為慢代謝者（slow acetylator, SA），NAT2則約有30%屬於慢代謝者。不同基因型會影響藥品的代謝，致使藥品或代謝物濃度在個體間的變異性大，而增加藥物相關不良反應（adverse drug reaction, ADR）如肝毒性、皮膚過敏等發生的風險。目前尚未有研究探討臺灣病人使用co-trimoxazole時，不同的NAT基因型對於藥品濃度及ADR的影響，因此本研究利用前瞻性收案，研究不同NAT基因型與SMX及其代謝物濃度及ADR的相關性，同時也一併描述SMX與trimethoprim（TMP）的血中濃度分布情形。 研究目的 本研究包含兩部分分析，第一部分為血中濃度分布情形，第二部分為研究NAT1、NAT2基因多型性與SMX、N4-acetyl SMX血中濃度與ADR之間的關聯性。 研究方法 研究收案自2014年1月19日至2015年6月18日間，於臺大醫院前瞻性收納年紀≥20歲且使用co-trimoxazole治療劑量≥ 5 mg/kg/day（以TMP劑量計算）的住院病人，簽署受試者同意書後納入研究分析。排除沒有血中濃度、NAT基因型資料有缺漏、做過異體骨髓移植且無法取得口水檢體檢測基因型的病人。 待達到穩定血中濃度後，於使用藥品前量測血中最低濃度（trough concentration, Ctrough），在服藥後3小時（或靜脈輸注完畢1小時）檢測藥品最高濃度（peak concentration, Cpeak）。使用高效能液相層析儀以UV偵測吸收波長來測定藥品濃度，基因多型性則以聚合酶連鎖反應- 限制酶片段長度多型性分析。 研究記錄收案期間病人的基本資料、實驗室檢查值、藥品使用的劑量、頻次、起始日期與抽血時間，並在用藥期間觀察使否有發生藥品不良反應，不良反應之評估以Naranjo scale評估與co-trimoxazole的相關性。 統計方法使用Mann-Whitney U test、Chi-square test比較兩組之間的差異，使用Wilcoxon signed-rank test比較多組樣本間的差異。利用簡單線性回歸（simple linear regression）及多重線性迴歸分析（multiple linear regression）影響藥品濃度的重要因子。所有檢定皆為雙尾檢定（two sided），以p-value小於0.05視為統計上的顯著差異。 研究結果 本研究共收案87人，排除4人後共納入82人進行血中濃度療劑監測分析；納入病人之共病症主要以腫瘤疾病（64.6 %）及後天免疫缺乏症候群為主（43.9%）；co-trimoxazole主要用來治療肺囊蟲肺炎感染。第二部份分別納入具NAT1基因型結果的63人、NAT2基因型的68人進行分析。 第一部分分析的82人中，使用治療劑量雖低於治療指引建議的15~20 mg/kg/day，不過合計有65%落在文獻建議之SMX為100~200 µg/mL範圍中，而有70%落在文獻建議之TMP為3~8 µg/mL範圍中，顯示不需要用到較高劑量即可達到療劑範圍內。 第二部分NAT基因型與濃度分析，欲校正不同給藥劑量頻次而利用代謝物與原型藥比值做為結果分析。NAT1基因型63人中，有49位（77.8%）快代謝者、14位（22.2%）慢代謝者；NAT2基因型68人中，有23位（33.8%）快代謝者、有23位（33.8%）中速代謝者、22位（32.4%）慢代謝者。NAT1/NAT2基因型與血中濃度最低比值、血中濃度最高比值、AUC比值沒有相關性，但其中在血中濃度最高比值、AUC比值中，均有NAT1/NAT2快代謝者較高的趨勢。 基因型與濃度及ADR分析中，排除與SMX及其代謝物確定為不相關的ADR後，一共發生50件相關ADR。發生相關ADR比起沒有發生ADR者，不論在濃度最低比值、最高比值或AUC比值皆顯著較低（p-value<0.05）。細部以基因型分組後，NAT1基因型快代謝者，沒發生ADR者的最低濃度、最高濃度比值顯著較高（p-value<0.05）；NAT2基因型中速代謝者，沒發生ADR者則在最低濃度、最高濃度、AUC比值中顯著較有發生ADR者高（p-value<0.05）。 結論 本研究為第一個使用co-trimoxazole治療劑量的病人中，比較NAT基因型與藥物代謝物與原型藥比值及不良反應間的相關性研究。血中濃度分布之分析中，治療劑量雖低於指引之建議，但多數病人血中濃度可以達到文獻建議的治療範圍之內。基因型分析中，NAT基因型雖然只有影響代謝物與原型藥血中濃度比值的趨勢，但是藥物濃度的比值在不良反應的發生上有統計顯著差異，NAT1快代謝者、NAT2中速代謝者之藥物濃度的比值在不良反應的發生上有統計顯著差異。

Background Co-trimoxazole (sulfamethoxazole-trimethoprim) is a broad-spectrum antibiotic combination, which is used as the first line treatment for Pneumocystis jirovecii pneumonia (PJP) and several infections. Sulfamethoxazole (SMX) is mainly acetylated to N-acetyl sulfamethoxazole (N4-acetyl SMX) via N-acetyltransferase (NAT).The other pathway for SMX is CYP2C9-mediated bioactivation to a reactive metabolite that may result in toxicity. NAT has genetic polymorphism. There are 20~30% slow acetylators (SA) in Asian population. It is reported that SA with significantly higher concentration of SMX may contribute to more adverse drug reaction (ADR). However, there are limited data of metabolite concentration and ADR in relation to genetic polymorphism in Taiwan. We aim to describe the plasma concentrations of SMX and TMP, and to investigate NAT genetic polymorphism and its correlation to concentration of co-trimoxazole and N4-acetyl SMX and ADR. Objectives To evaluate the impact of NAT genetic polymorphism on concentration of co-trimoxazole, N4-acetyl SMX and ADRs, as well as to describe the distribution of co-trimoxazole concentration. Methods This prospective study was conducted at the National Taiwan University Hospital from January 19 2014 to June 18 2015. Inpatients, who were older than or equal to 20 years old, used therapeutic doses（≥ 5 mg/kg/day, based on TMP component）of co-trimoxazole and signed informed consent were enrolled. Patients without concentration or genetic information, who had allogeneic bone marrow transplantation and whose saliva samples were not collected were excluded. Blood samples of peak concentration were drawn 3 hours after oral administration or 1 hour after completion of intravenous infusion, while trough concentration were collected just before the next dose. The plasma concentration of co-trimoxazole and N4-acetyl SMX were assayed by high performance liquid chromatography. DNA was extracted from blood or saliva samples. SNP were determined by polymerase chain reaction amplification and restriction fragment-length polymorphism method. Patients’ characteristics, lab data, dosing regimen, and the duration of treatment were recorded. The Naranjo scale was applied to evaluate the causality of the ADRs. Chi-square test and Mann-Whitney U test were used to compare the differences between two groups. Wilcoxon signed-rank test was used to compare the differences among three groups. Simple linear regression and multiple linear regression analysis were used to determine the factors contributing to plasma concentration. In all of the statistical analyses, a p-value of <0.05 was considered statistically significant. Result During the study period, a total of 87 patients were enrolled, but only 82 patients were included in the analysis. Most of them had hemato-oncological disease (64.6%) or acquired immunodeficiency syndrome (43.9%). Co-trimoxazole was mainly used for the treatment of PJP. There were 63 and 68 patients included in the NAT1, NAT2 genetic polymorphism analysis, respectively. 82 patents were included in the analysis of correlation between dosage and concentration. Although the dose administered were lower than the recommended dose according to the treatment guideline (15~20 mg/kg/day). 65% of the peak SMX levels and 70% of the peak TMP levels were within the therapeutic ranges for treating PJP (target peak concentration 100~200 µg/mL for SMX and 3~8 µg/mL for TMP). In terms of NAT polymorphism in relation to serum concentration, the latter was expressed as a proportion of peak concentration of SMX compared to N4-acetyl SMX in order to standardize variation of dosing regimen. For the analysis of NAT1 genetic polymorphism, 49 patients (77.8%) were genotyped as rapid acetylators (RA) and 14 patients (22.2%) as SAs in the patient population. For the analysis of NAT2 genetic polymorphism, 23 patients (33.8%) were genotyped as RAs, 23 patients (33.8%) as intermediate acetylators (IA) and 22 patients (32.4%) as SAs in the patient population. There were no significant differences between the ratio of N4-acetyl SMX/SMX and the NAT polymorphisms, but higher levels were seen in RAs group. We excluded the less-associated ADRs of SMX (such as hyperkalemia) in the analysis of NAT polymorphism. A total of 50 ADRs was evaluated as SMX-associated. We found that the ratio of N4-acetyl SMX/SMX was lower in the group of SMX-related ADRs (p-value<0.05). Also, there were significant differences in the ratio of N4-acetyl SMX/SMX between RA group of NAT1 polymorphism, and the IA group of NAT2 polymorphism (p-value<0.05). Conclusion Although lower dosing regimen was administered, plasma concentrations of co-trimoxazole were within the optimal therapeutic range in the most of the patients. There were significant differences in the ratio of N4-acetyl SMX/SMX and adverse drug reaction. The same result were found in RA group of NAT1 and IA group of NAT2 polymorphism.