结核性胸膜炎患者胸腔积液中利福平、异烟肼和乙胺丁醇的药代动力学的研究

发布时间：2018-04-23 08:48

本文选题：利福平 + 异烟肼　；参考：《南方医科大学》2012年硕士论文

【摘要】：背景和目的结核性胸膜炎(Tuberculous pleuritis)是最常见的肺外结核(IEPTB)之一,近年来由于艾滋病的流行和结核分枝杆菌合并感染增加,使其发病率呈上升趋势,全球有将近三分之一的人感染结核,大约3-25%的结核病人会发生结核性胸膜炎,据报道统计,呼吸科住院病人中10%伴有胸腔积液,其中结核性胸腔积液占46.7%。结核性胸腔积液是结核性渗出性胸膜炎的表现,是干性胸膜炎的进一步发展形成,由近胸膜的原发病灶直接侵入胸膜腔,或经淋巴管血行播散至胸膜而引起的渗出性炎症。多急性发病且好发于青年人,临床上表现为单侧胸腔少至中等量的积液,常见的症状是胸痛、干咳、乏力等,大部分病人会发热,但是其中也有15%没有发热症状,如果积液量大,病人会呼吸困难。若得不到早期科学合理治疗,容易引起胸膜肥厚、粘连,形成包裹性积液,甚至脓胸,从而严重影响肺的呼吸功能。高达50%的病人会出现胸膜增厚,胸膜增厚导致肺限制性通气功能障碍,并可产生后遗症如继发性支气管扩张、不可逆性压缩性肺不张等使病情加重。胸膜腔位于肺和胸壁之间的一个的潜在腔,作为肺和胸壁之间的连接系统而成为呼吸系统结构的重要部分。过去认为,结核性胸腔积液的发病机制是宿主对结核菌或其代谢产物发生了迟发变态反应(DTH),结核杆菌不直接侵犯胸膜和胸腔。主要依据是大部分结核性胸膜炎病例胸腔积液结核菌培养阴性。目前临床实践发现胸膜活检有50%-80%的病例胸膜上有典型结核结节形成,故认为胸膜的病理损伤是结核性胸膜炎乃至结核性胸腔积液发生的主要机制,DTH在其中起一定作用。这说明在胸腔积液中或者胸膜上存在有结核菌。因此,能否有效杀死病灶部位的致病菌是治疗的一个根本措施。抗菌药物是治疗胸膜炎的主要药物,通常认为抗生素在胸腔积液中的浓度和血清中相似,所以,目前临床结核性胸膜炎的药物治疗方法一直沿用肺结核的化疗方案为主：短疗程异烟肼(INH)、利福平(RFP)、吡嗪酰胺(PZA)和盐酸乙胺丁醇(EMB)治疗2个月,再INH和RFP口服4个月,根据病情可延长疗程达12个月。但是,缺乏相关的实际数据支持这种经验用药方案,由于胸膜腔密闭结构的特殊性以及药物透过胸膜的差异性,导致胸膜腔内各种抗结核药物浓度透过程度和药动学过程不同。国内外的少量研究数据表明：在结核性脓肿中,INH浓度可能会降低,但高于最低抑菌浓度或杀菌浓度,RFP在病灶中浓度可能会低于最低抑菌或杀菌浓度。但是抗结核治疗只有各个抗结核药物分别达到各自的有效治疗浓度才会发挥协同作用,从而降低耐药的发生率。因此,这就需要增加对病灶部位抗结核药物的药动学和抗菌作用的认识,从而设计更好的治疗方案,改善治疗效果。文献检索结果表明,目前国内外尚无对抗结核药物在胸腔积液中完整的药代动力学及疗效分析的报道,因此,研究单次和多次给药后药物在胸腔积液中的动态过程以及药物的胸膜渗透是有较高的学术价值和重要的临床指导意义。本课题研究初治的结核性胸膜炎患者服药第1、2、3天后药物在结核性胸腔积液中的药代动力学,对各抗结核药物浓度进行检测,更直接地评价临床疗效,为临床药物治疗方案的调整提供重要的理论依据,尤其是对于治疗失败的病例,可以推断是否药物渗透受影响或药物耐药的情况发生,从而根据检测结果及时改变给药方案和用药剂量,以改善疗效,缩短疗程,减少耐药性的产生。内容和结果选取南方医院呼吸内科2010年6月-2012年4月23例结核性胸膜炎患者,纳入标准是：1.中等量或者大量渗出性胸腔积液患者；2.符合渗出性结核性胸膜炎的诊断标准：①结核中毒症状：发热,畏寒,胸痛,气短,出汗,乏力。②体征：局部叩诊浊音,呼吸音减弱。③经过细菌学、组织学或者超声波和CT胸片检查确诊。排除标准：复治结核,恶性胸腔积液患者,合并心血管、肝、肾或者造血系统等严重原发性疾病患者,精神病患者,具有慢性疾病或者其他器质性疾病患者,需同时应用影响抗结核药物药动学的患者,近期参加其他临床试验的患者。采样分为两部分：1.引流13名患者胸腔置管后初次服药的第1、2、3天每天空腹晨服药药后2、4、6、8、12、24h的胸腔积液,同时采集其中5名患者第3天服药前和服药后2、4、8、24h的血样；2.收集20名患者胸腔置管后初次服药后2h的胸腔积液,以及第3天服药后2h的胸腔积液和血样。所有样品经过前处理提取后,采用高效液相色谱方法检测样品中INH和RFP的浓度,EMB的浓度采用液相质谱联用仪测量。 RFP的色谱条件：KromasiL C18色谱柱(150mm×4.60mm,5micron);流动相：甲醇-10mmol/l磷酸二氢钾缓冲盐(磷酸调节pH4.5)(v/v62:38)；波长：340nm；流速：1.0ml/min；柱温：30℃,进样量：20μl。RFP在胸腔积液和血浆的线性范围分别是0.046875～6.0μg/ml和0.09375～12.0μg/ml,线性良好；回归方程分别是：C=2.627e-5*A-3.443e-2(R2=0.994), C=2.021e-5*A+3.784e-2(R2=0.999)；在此条件下,胸腔积液中RFP的最低检测限为0.02μg/ml,样品取样回收率大于80%,提取后室温下放置24h及反复冻融3次稳定性良好。 INH的色谱条件：流动相甲醇-20mM磷酸二氢钾(70%高氯酸,20%三乙胺,调节pH3.2)(v/v:30-70),色谱柱：NucLeosiL CN-RP(250mm,4.61/mm×5μm),流速：1.0ml/min,波长：340nm,温度：30℃,时间：20min,进样量：20μl。INH在血浆和胸液中的线性范围分别是0.01～10.0μg/ml,0.01～5.0μg/ml,线性良好；回归方程分别是C=4.35×10-6A+2.05×l0-2(R2=0.9999), C=4.44×10-6A+3.84×10-3(R2=0.9999),检测限均为0.01μg/ml;提取回收率理想,日内和日间精密度小于10%,样品处理后放置12h及反复冻融3次稳定性良好。 EMB的色谱条件：Agilent ZORBAX SB-C18柱(2.1x150mm,3.5mm);流动相：甲醇-0.1%甲酸水溶液(v/v=15：85)；流速：0.2ml/min;柱温为25℃；进样量：2μ1。质谱条件：电喷雾离子源(ESI)正模式,以多重反应离子监测(MRM)进行检测,检测离子为m/z205.1→m/z116.0(EMB), m/z130.1→m/z60.2(二甲双胍,内标)。EMB胸腔积液中的最低定量限为31.25ng/ml,胸腔积液和血浆线性范围分别是为31.25-4000ng/ml和31.25-8000ng/ml,回归方程分别是Y=0.211X-0.094(R2=0.991), Y=0.332X+0.386(R2=0.998),回收率高于80%,日内及日间RSD均小于10%。根据检测结果计算渗透率和胸腔积液中抗结核药物的药代动力学参数Tmax， Cmax, AUC及渗透率,结果如下：第3天给药后2h胸腔积液和血浆药物浓度之比为： RFP：16.051±8.345； INH：65.701±39.682； EMB：71.621±71.503。第3天药物在胸腔积液中的Tmax(h)、Cmax(μg/ml)、AUC0-24(mg/l·h)分别是： RFP：6.67±2.449,2.368±0.848,33.374±8.147； INH：3.78±1.563,2.759+1.013,26.258±13.580； EMB:5.11+1.054,1.673+0.696,17.843±4.657。第3天药物在血浆中Tmax(h)、Cmax(μg/ml)、AUC0-24(mg/l·h)分别是： RFP：3.60±0.894,7.820±1.034,72.878±16.306； INH：2.400±0.894,4.614+1.213,29.283±13.125; EMB：2.40±0.894,1.943±0.780,12.216±4.048。第3天药物在血浆和胸腔积液中AUC0-24(mg/1·h)的比值分别是： RFP：37.338+11.005,49.683+9.731; INH：79.194+24.098,111.792±7.341; EMB：131.164±108.840,180.997+123.952。血浆和胸腔积液RFP的AUC/MIC125,对胸腔积液和血浆中RFP的浓度和AUC做相关性分析,结果表明均无相关性(r=0.411,P=0.072；r=0.613,P=0.271)。血浆和胸腔积液INH的AUC/MIC125,对胸腔积液和血浆中INH的浓度和AUC做相关性分析,结果表明均呈正相关(r=0.702,P=0.001；r=0.995,P=0.000)。血浆和胸腔积液EMB的AUC/MIC125,对胸腔积液和血浆中EMB的浓度和AUC分别做相关性分析,结果表明均无相关(r=0.411,P=0.072；r=-0.248,P=0.687)。结论与讨论试验结果表明：RFP血浆和胸腔积液浓度均超过了最低杀菌浓度,RFP从血浆进入胸膜腔的渗透率较低,其在胸腔积液中的AUC较高,可能与RFP蛋白结合率高,使其在胸膜腔中呈一定蓄积作用,随着给药次数的增加,胸腔积液中Tmax减小,Cmax接近血浆稳态浓度。对于治疗非耐药结核性胸膜炎有效并且能抑制结核杆菌耐药。 INH的胸腔积液浓度较高,达峰时间较血浆稍晚,胸腔积液浓度随血浆浓度增加而升高,说明INH透过胸膜的速度快,吸收程度高。第3天胸腔积液AUC和血浆AUC相当,随着给药次数增加,推测第3天血浆和胸腔积液可达到稳态浓度,对没有包裹性胸腔积液的患者不需要胸腔注射给药INH即可达到有效的治疗浓度。因此,INH治疗结核性胸膜炎有效并能抑制细菌耐药。大部分患者EMB胸腔积液和血浆浓度较低于最小抑菌浓度,胸腔积液中Tmax明显延后,AUC/MIC125,治疗作用很小,应根据EMB监测浓度进行剂量调整,在达到有效治疗的同时,尽量避免其毒副作用的发生。因此,RFP和INH连续每天给药1次,胸腔积液中能达到有效治疗浓度,INH不需要胸腔注射给药,长期用药时需要监测RFP浓度,防止肝药酶诱导作用使其浓度降低,建议EMB根据监测浓度调整剂量。 RFP、INH、EMB均为浓度依赖型抗菌药物,比较三种药物在胸腔积液中的渗透率,可推测RFP与INH、EMB经胸膜进入胸腔积液中的渗透方式可能不同,这可能与药物的极性、蛋白结合率或者胸膜的间皮屏障作用有关,从而使不同药物胸膜渗透率具有显著差异,这些机制还有待进一步的研究。
[Abstract]:Background and purpose
Tuberculous pleurisy (Tuberculous pleuritis) is one of the most common extrapulmonary tuberculosis (IEPTB). In recent years, due to the prevalence of AIDS and the increase of Mycobacterium tuberculosis combined infection, the incidence of tuberculosis is on the rise. There are nearly 1/3 people around the world infected with tuberculosis, about 3-25% of tuberculosis patients will have tuberculous pleuritis, according to reports. In the Department of respiration, 10% of the patients in the Department of respiration were accompanied by pleural effusion, in which tuberculous pleural effusion accounted for 46.7%. tuberculous pleural effusion, the manifestation of tuberculous exudative pleuritis, and the further development of dry pleuritis, which was directly intruded into the pleural cavity, or disseminated to the pleura through the lymphatic vessel blood, and the exudative inflammation caused by the lymphatic vessels. Multiple acute onset and good hair in young people, clinical manifestations of the unilateral pleural less to moderate amount of fluid, the common symptoms are chest pain, dry cough, fatigue and so on, most patients will fever, but 15% of them have no fever, if the amount of fluid is large, the patient will breathe difficult. If no early scientific and reasonable treatment, it is easy to cause the pleura. Hypertrophy, adhesion, forming Encapsulated Effusion, even empyema, seriously affects the respiratory function of the lung. Up to 50% of the patients will have pleural thickening, pleura thickening and pulmonary restrictive ventilation dysfunction, and can produce sequelae such as secondary bronchiectasis, irreversible compressible pulmonary atelectasis.
The pleural cavity is a potential cavity between the lung and the chest wall, which is an important part of the respiratory system as a connection between the lung and the chest wall. In the past, the pathogenesis of tuberculous pleural effusion was the late onset metamorphosis (DTH) of the host to the Mycobacterium tuberculosis or its metabolites, and the Mycobacterium tuberculosis did not directly infringe the pleura and chest. The main basis is that the tubercle bacillus culture of pleural effusion is negative in most cases of tuberculous pleurisy. At present, clinical practice has found typical tuberculosis nodules on the pleura of 50%-80% cases with pleural biopsy, so the pathological injury of the pleura is the main mechanism of tuberculous pleurisy and even tuberculous pleural effusion. DTH plays a certain role in it. This indicates that there are tuberculous bacteria in pleural effusion or pleura. Therefore, it is a fundamental measure to effectively kill pathogenic bacteria in the lesions.
Antibacterials are the main drugs for the treatment of pleurisy. It is generally believed that the concentration of antibiotics in the pleural effusion is similar to that in the serum. Therefore, the current drug therapy for tuberculous pleurisy always follows the chemotherapy regimen of pulmonary tuberculosis: short course isoniazid (INH), Fu Ping (RFP), PZA, and ethambutol hydrochloric acid (EMB) After 2 months of treatment, INH and RFP were taken orally for 4 months, and the duration of the treatment could be prolonged for 12 months. However, the lack of relevant practical data supported the experience of the drug regimen. The specificity of the pleural cavity and the difference in the pleura through the pleura resulted in the permeability of various antituberculous drugs in the pleural cavity and the pharmacokinetic process. A small number of research data at home and abroad show that the concentration of INH may be reduced in tuberculous abscess, but higher than the minimum or bactericidal concentration, the concentration of RFP in the focus may be lower than the minimum bacteriostasis or bactericidal concentration. The same effect, thus reducing the incidence of drug resistance, therefore, it is necessary to increase the understanding of the pharmacokinetics and antiseptic effect of anti tuberculosis drugs on the focus, so as to design a better treatment plan and improve the therapeutic effect. The results of literature retrieval show that there is no complete pharmacokinetics and treatment of anti tuberculosis drugs in the pleural effusion at home and abroad. Therefore, it is of high academic value and important clinical significance to study the dynamic process of the drug in the pleural effusion and the pleura permeation after a single and multiple drug delivery.
This topic is to study the pharmacokinetics of drug in tuberculous pleural effusion after 1,2,3 days after the first treatment of tuberculous pleurisy, to detect the drug concentration of various anti tuberculosis drugs, to evaluate the clinical efficacy more directly, and to provide an important theoretical basis for the adjustment of the clinical drug treatment scheme, especially for the cases of treatment failure. It is inferred whether drug penetration is affected or drug resistance occurs, thus changing the dosage regimen and dosage in time to improve the efficacy, shorten the course of treatment and reduce the production of drug resistance according to the results of the test.
Content and results
23 cases of tuberculous pleurisy in the Department of respiratory medicine of the southern hospital in June 2010 -2012 April were included in the standard: 1. of the patients with equal or massive exudative pleural effusion; 2. conformed to the diagnostic criteria for exudative tuberculous pleurisy: (1) symptoms of tuberculosis poisoning: fever, cold, chest pain, shortness of breath, sweat, fatigue. Voiced sounds and respiratory sounds were weakened. (3) confirmed by bacteriology, histology or ultrasound and CT chest radiography.
Exclusion criteria: retreated tuberculosis, patients with malignant pleural effusion, patients with severe primary diseases such as cardiovascular, liver, kidney or hematopoietic systems, patients with psychosis, chronic diseases or other organic diseases, should be used in patients with antituberculous pharmacokinetics and in other clinical trials in the near future.
The samples were divided into two parts: 1. drainage 13 patients after the first medicine after the thoracic cavity for the first 1,2,3 day after the first dose of 2,4,6,8,12,24h in the pleural effusion, and 5 patients before and after third days of medication and after the drug 2,4,8,24h blood samples; 20 patients after the first medicine after the chest catheterization of the 2H pleural effusion, and third days The pleural effusion and blood samples of 2h after taking the medicine were obtained. All samples were extracted by preprocessing, and the concentration of INH and RFP in the samples was detected by high performance liquid chromatography. The concentration of EMB was measured by liquid phase mass spectrometry.
The chromatographic conditions of RFP: KromasiL C18 column (150mm x 4.60mm, 5micron); mobile phase: methanol -10mmol/l potassium dihydrogen phosphate buffer salt (phosphoric acid regulated pH4.5) (v/v62:38); wavelength: 340nm; flow rate: 1.0ml/min; column temperature: 30 degrees C, sampling quantity: 20 mu l.RFP in pleural and plasma linear range of 0.046875 to 6 micron and 0.093 75 to 12 mu g/ml, linear good, regression equation: C=2.627e-5*A-3.443e-2 (R2=0.994), C=2.021e-5*A+3.784e-2 (R2=0.999). Under this condition, the minimum detection limit of RFP in pleural effusion is 0.02 mu g/ml, sample recovery rate is more than 80%, and 24h and repeated freezing and thawing at room temperature are good for 3 times.
INH chromatographic conditions: mobile phase methanol -20mM potassium dihydrogen phosphate (70% perchloric acid, 20% three ethylamine, regulating pH3.2) (v/v:30-70), chromatographic column: NucLeosiL CN-RP (250mm, 4.61/mm * 5 micron), flow rate: 1.0ml/min, wavelength: 340nm, temperature: 30 degrees, time: 20min, the linear range of 20 Mu in plasma and thoracic fluid is 0.01 to 10 um, respectively. G/ml, 0.01 ~ 5 mu g/ml, good linearity; the regression equation is C=4.35 x 10-6A+2.05 x l0-2 (R2=0.9999), C=4.44 x 10-6A+3.84 x 10-3 (R2=0.9999), and the detection limit is 0.01 mu g/ml; the recovery rate is ideal, the intra day and day precision is less than 10%, and the stability of 12h and repeated freezing and thawing after the sample treatment is good.
The chromatographic conditions of EMB: Agilent ZORBAX SB-C18 column (2.1x150mm, 3.5mm); mobile phase: methanol -0.1% formic acid water solution (v/v=15:85); flow rate: 0.2ml/min; the column temperature is 25 C; the sample volume: 2 mu 1. mass spectrometry conditions: electrospray ion source (ESI) positive mode, detection by multiple reactivity monitoring (MRM) MB), the minimum quantitative limit of m/z130.1 to m/z60.2 (metformin, internal standard).EMB pleural effusion is 31.25ng/ml, and the linear range of pleural effusion and plasma is 31.25-4000ng/ml and 31.25-8000ng/ml respectively. The regression equation is Y=0.211X-0.094 (R2=0.991), Y=0.332X+0.386 (R2=0.998), and the recovery rate is higher than 80%, both within and between day and day RSD are less than those
The pharmacokinetic parameters Tmax, Cmax, AUC and permeability of anti tuberculosis drugs in pleural effusion were calculated according to the test results. The results are as follows:
After third days of administration, the ratio of 2H pleural effusion and plasma drug concentration was:
RFP:16.051 + 8.345;
INH:65.701 + 39.682;
EMB:71.621 + 71.503.
The Tmax (H), Cmax (g/ml) and AUC0-24 (mg/l. H) of third day drugs in pleural effusion were:
RFP:6.67 + 2.449,2.368 + 0.848,33.374 + 8.147;
INH:3.78 + 1.563,2.759+1.013,26.258 + 13.580;
EMB:5.11+1.054,1.673+0.696,17.843 + 4.657.
Third days in the plasma, Tmax (H), Cmax (g/ml), AUC0-24 (mg/l. H) were:
RFP:3.60 + 0.894,7.820 + 1.034,72.878 + 16.306;
INH:2.400 + 0.894,4.614+1.213,29.283 + 13.125;
EMB:2.40 + 0.894,1.943 + 0.780,12.216 + 4.048.
The ratio of AUC0-24 (mg/1? H) in plasma and pleural effusion on the third day was:
RFP:37.338+11.005,49.683+9.731;
INH:79.194+24.098111.792 + 7.341;
EMB:131.164 + 108.840180.997+123.952.
The AUC/MIC125 of RFP in plasma and pleural effusion was correlated with the concentration of RFP in pleural effusion and plasma and AUC. The results showed no correlation (r=0.411, P=0.072; r=0.613, P=0.271). The AUC/MIC125 of INH in plasma and pleural effusion was correlated with INH concentration in pleural effusion and plasma and correlation analysis of AUC. 702, P=0.001; r=0.995, P=0.000). AUC/MIC125 of EMB in plasma and pleural effusion. The correlation of EMB concentration and AUC in pleural effusion and plasma was analyzed, and the results showed no correlation (r=0.411, P=0.072; r=-0.248, P=0.687).
Conclusion and discussion
The results showed that the concentration of plasma and pleural effusion in RFP was higher than the lowest bactericidal concentration. The permeability of RFP from plasma into the pleural cavity was low, and the AUC in the pleural effusion was higher. It might have a high binding rate with the RFP protein and made it accumulate in the pleural cavity. With the increase of the number of drug delivery, the Tmax decreased in the pleural effusion and Cmax connection. Near plasma steady state concentration is effective in treating non drug resistant tuberculous pleurisy and can inhibit the drug resistance of Mycobacterium tuberculosis.
The concentration of INH pleural effusion was higher than that of plasma, and the concentration of pleural effusion increased with the increase of plasma concentration, indicating that INH was faster and more absorbed by the pleura. The third day pleural effusion was equivalent to AUC and plasma AUC. With the increase of the number of drug delivery, it was speculated that the plasma and pleural effusion could reach steady state concentration, and there was no inclusion in the pleural effusion. Patients with pleural effusion do not need intrapleural administration of INH to achieve an effective therapeutic concentration. Therefore, INH is effective in treating tuberculous pleurisy and can inhibit bacterial resistance.
In most patients, the concentration of EMB pleural effusion and plasma is lower than the minimum inhibitory concentration, and the Tmax in the pleural effusion is obviously delayed, and the effect of AUC/MIC125 is very small. The dosage should be adjusted according to the monitoring concentration of EMB. While the effective treatment is achieved, the occurrence of its toxic and side effects is avoided as much as possible.
Therefore, RFP and INH are administered 1 times a day. The effective treatment concentration in the pleural effusion is achieved. INH does not need to be injected into the thoracic cavity. The concentration of RFP needs to be monitored in the long term, and the concentration of the liver is prevented from reducing the concentration of the liver drug enzyme induction. It is suggested that EMB adjust the dose according to the monitoring concentration.
RFP, INH and EMB are all concentration dependent antimicrobial agents. Comparing the permeability of three drugs in the pleural effusion, it is possible to speculate that the infiltration of RFP and INH in the pleural effusion may be different, which may be related to the polarity of the drug, the binding rate of protein, or the mesothelial barrier effect of the pleura, so that the permeability of the pleura of different drugs can be obtained. These mechanisms still need further study.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R521.7

【参考文献】