| Background and purposeTuberculous (TB) pleurisy, considered as one of the most common form of extrapulmonary tuberculosis (EPTB). In recent years, the interaction of HIV pandemic with Mycobacterium tuberculosis has lead to its increased incidence. Nearly one-third people infected TB in worldwide every year, Among them, tuberculous pleurisy occur approximately3～25%. It is reported that46.7%of patients in10%of respiratory inpatients with pleural effusions are tuberculous pleural effusions. Tuberculous pleural effusions present tuberculous exudative pleurisy, and it is the further development of dry pleurisy. Bacteria from near pleural primary lesions direct invade into the pleural cavity, or by the lymphatic hematogenous dissemination to the pleura, then caused exudative inflammation. Most TB pleural effusions manifest as an acute illness and have a high incidence in younger adults. The clinical symptoms are a little or moderate size effusions in unilateral pleural and the most frequent symptoms are chest pain, dry cough, fatigue and so on. Most patients are febrile but approximately15%will be afebrile. Patients with TB pleural effusions may be dyspneic if the effusions is large. If lack of scientific and rational treatment early, patients easily occur pleural thickening, adhesions, the formation of encapsulated effusions or empyema. Accordingly, it will badly affect the respiratory function of lung. Up to50%of patients are with pleural thickening, and it can cause lung restrictive ventilatory dysfunction and can also cause secondary sequelae such as bronchiectasis, irreversible compression atelectasis and so on, it will be more worse.The pleural cavity is a potential cavity between lung and chest wall cavity, and form an important part of the structure of the respiratory system as the connection between the lung and chest wall system. Due to the TB pleural effusions culture were negative for most tuberculous pleurisy cases, the tuberculous pleural effusions pathogenesis used to be thought that the host occured the delayed allergic reaction (DTH) to Mycobacterium tuberculosis or its metabolites, and mycobacterium tuberculosis even did not invade direct into pleura and chest. But current clinical practice found that typical tuberculous nodules formed on pleural in50%～80%cases checked by pleural biopsy, and it suggesting that the pathological damage of the pleura is the main mechanism of tuberculous pleurisy and even pleural effusions, and DTH played a role in the process. This showed that the Mycobacterium tuberculosis existed in the pleural effusions or pleural TB. Therefore, to kill pathogenic bacteria in the lesion site effectively is one of the fundamental measures for treatment.Patients with bacterial infections of the pleural space are treated with antibiotics. In general, it is thought that antibiotic levels in pleural effsions are similar to those in the serum. Therefore current tuberculous pleurisy pharmacotherapy is based on tuberculosis clinical chemotherapy:short course of isoniazide, rifampicin, pyrazinamide and ethambutol for2months, and then continue isoniazide and rifampicin for the next4months, and courses can be extended up to12months according to conditions. However, the experience in drug application is lack of real data support. Due to the pleural cavity closed structure as well as differences penetration in drug through the pleura, resulting in anti-TB drug concentration various in pleural cavity and different penetration degree and pharmacokinetics process. A little research data in domestic and foreign showed that:isoniazide concentration in tuberculous abscess may be reduced, but higher than the minimum inhibitory concentration or bactericidal concentrations, and rifampicin concentration in the lesions may be lower than the minimum inhibitory or bactericidal concentration. Only each anti-TB drugs achieve effective therapeutic concentrations will be synergies in anti-TB treatment. The literature search results showed that there is lack of information on anti-TB drugs pharmacokinetics and efficacy analysis of pleural effusions reports from domestic and abroad, Therefore, there is a high academic value and an important clinical guiding significance on studying the dynamic process of the drugs in the pleural effusions after single and multiple doses, as well as pleura penetration of drugs.This study research the drugs pharmacokinetics of untreated patients with tuberculous pleurisy on day1,2,3after the daily dose regimen. Detection of anti-TB drugs concentration in tuberculous pleural effusions is a more direct method for assessment clinical efficacy, and it can provide an important theoretical basis for the adjustment of the clinical drug treatment programs, especially for treatment failure cases. It also can be inferred whether the drugs penetration was affected or drug resistance generated. According to the measurement results, change the dosing regimen and dose in time would improve the efficacy, shorten the treatment course and reduce drug resistance emergence.Content and ResultsFrom June2010to April2012,23cases with tuberculous pleurisy patients were recruited in the study in Respiratory Medicine of Nanfang Hospital. Inclusion criteria:1. a moderate amount or a lot of exudative tuberculous pleural effusions.2. accordance with diagnostic criteria for exudative tuberculous pleurisy:â‘ tuberculosis toxic symptoms:fever, chills, chest pain, shortness of breath, sweating, fatigue.â‘¡signs:local dull percussion note, diminished respiration.â‘¢After the bacteriology, histology or ultrasound and CT chest X-ray examination confirmed.Exclusion criteria:patients with retreatment tuberculosis, malignant pleural effusions, cardiovascular, accompany liver, kidney or hematopoietic system severe primary disease; the mentally ill, chronic disease or other organic disease patients; patients take the medicine that will affect the efficacy of anti-TB drugs; patients recently participated in other clinical trials.Sampling was divided into two parts:1. pleural fluid was drainaged from13patients with chest tube on day1,2,3after2,4,6,8,12,24h administration fasting in the morning. At the same time, blood samples in5of13patients were collected before administration and after taking medicine at2,4,8,24h on day3:2. pleural fluid was collected in20patients2h after initial administration on day1, and plasma and pleural fluid were both collected2h after administration on day3. After the extraction, isoniazide and rifampicin in all samples were detected by high performance liquid chromatography method and the ethambutol concentration was detected by LC-MS/MS.Rifampicin chromatographic conditions:KromasiL C18column (150mm×4.60mm,5micron), mobile phase:methanol—10mmol/1potassium dihydrogen phosphate buffered saline (phosphate adjust pH4.5)(v/v:62—38), wavelength:340nm, flow rate:1.0ml/min, column temperature:30℃, injection volume:20μL. The results showed a good linear relationship in0.09375～6.0μg/ml and0.09375～12.0μg/ml in the pleural effusions and plasma, respectively. The regression equations: C=2.627e-5*A-3.443e-2(R2=0.994), C=2.021e-5*A+3.784e-2(R2=0.999). Detection limit was0.02μg/ml, Samples extract recovery were more than80%. They were stable placed under room temperature for24h and after freezing and thawing three times.Isoniazide chromatographic conditions:mobile phase methanol—20mM potassium dihydrogen phosphate (70%perchloric acid,20%triethylamine, adjust pH3.2)(v/v:30—70), column:NucLeosiL, CN-RP(250mm,4.6mm x5μm), flow rate:1.0ml/min, wavelength:340nm, temperature:30℃, time:20min, injection volume:20μL. Isoniazide in plasma and pleural fluid in the linear range of0.01-10.0μg/ml,0.01～5.0μg/ml; regression equation were:C=4.35×10-6A+2.05×10-2(R2=0.9999), C=4.44×10-6A+3.84x10"3(R2=0.999), Detection limits were0.01μg/ml. The extraction recovery and the method recovery were good. intraday and interday precision were<10%. Samples were stable placed under room temperature for12h and repeated freezing and thawing three times.The ethambutol chromatographic conditions:Agilent ZORBAX SB-C18column (2.1×150mm,3.5mm), mobile phase:methanol—0.1%formic acid (v/v:15—85), flow rate:0.2ml/min, Column temperature was25℃, injection volume:2μL. Mass spectrometry conditions:electrospray ionization source (ESI) mode with multiple reaction monitoring (MRM) detection; the detected ion:m/z205.1â†'m/z116.0(ethambutol), m/z130.1â†'m/z60.2(metformin, internal standard). The lowest limit quantification of ethambutol in pleural effusions and plasma were31.25ng/ml, linear range in pleural effusions and plasma were31.25～4000ng/ml and31.25～8000ng/ml, respectively. the regression equations were Y=0.211*X—0.0938(R2=0.991), Y=0.332*X+0.386(R2=0.998), recoveries were higher than80%, intra-and inter-day RSD were less than10%.Penetration rate and pharmacokinetic parameters of anti-TB drugs in plasma and pleural effusions (Tmax, Cmax, AUC0-24) were as follows:Pleural effusions and plasma drug concentration ratios on day3after2h of the third administration:RFP:16.051±8.345;INH:65.701±39.682;EMB:71.621±71.503.Tmax (h), Cmax (μg/ml), AUC0-24(mg/1·h) and t1/2(h) of anti-TB drugs in the pleural effusions on day3:RFP:6.67±2.449,2.368±0.848,33.374±8.147,12.949±10.329;INH:3.78±1.563,2.759±1.013,26.258±13.580,8.789±5.462;EMB:5.11±1.054,1.673±0.696,17.843±4.657,14.675±11.317.Tmax (h), Cmax (μg/ml), AUC0-24(mg/1·h) and ti/2(h) of anti-TB drugs in the plasma on day3:RFP:3.60±0.894,7.820±1.034,72.878±16.306,5.654±2.301;INH:2.400±0.894,4.614±1.213,29.283±13.125,4.922±2.177;EMB:2.40±0.894,1.943±0.780,12.216±4.048,5.322±2.489.AUC and Cmax ratios of anti-TB drugs in pleural effusions and plasma after the administration on day3: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.RFP in plasma and pleural effusions:AUC/MIC>125,, the pleural effusion and plasma concentration of RFP and AUC correlation analysis showed no correlation (r=0.411, P=0.072; r=0.613, P=0.271). INH in plasma and pleural effusions:AUC/MIC>125, the pleural effusions and plasma concentration of INH and AUC correlation analysis showed a positive correlation (r=0.702, P=0.001; r=0.995, P=0). EMB in plasma and pleural effusions:AUC/MIC<125, the pleural effusions and plasma concentration of EMB and AUC correlation analysis showed no correlation(r=0.411, P=0.072; r=0.248, P=0.687).DiscussionAcoording to the results showed:the concentration of RFP in plasma and pleural effusions were more than the minimum bactericidal concentration, and the permeability of RFP from plasma into the pleural cavity was lower. High AUC in pleural effusions may due to high protein binding of rifampicin, and caused a certain accumulation in the pleural cavity. As the administration times increased, Cmax in pleural fluid was closed to plasma steady-state concentration, but Tmax is decreases. For no-drug resistant, it is effective for antibacterial and can also inhibit Mycobacterium tuberculosis drug resistance.INH concentration in pleural effusions was higher and Tmax was later, pleural effusions concentration was increased with plasma concentration, and pleural effusions AUC was closed to the plasma AUC on day3, it is showed that INH through the pleural fast, and had a high degree of absorption. As the administration times increased, it can be inferred that INH concentration in pleural effusions and plasma could reach a steady state concentration on day3. No parcel pleural effusion in patients without pleural injection INH can achieve effective treatment concentration. Therefore, isoniazid therapy is effective and can inhibit tuberculosis bacterial drug resistance.EMB concentration in pleural effusion and plasma was below the inhibitory concentration for most patients. and showed greater individual differences. Tmax in pleural effusion was significantly delayed, AUC/MIC<125, and therapeutic effect was little. Dose adjustments should be based on EMB blood concentration monitoring. Achieving effective treatment while trying to avoid the toxic and side effect.Thus, RFP and INH continuous daily dosing, pleural effusion can achieve effective treatment concentration. It is necessary to monitor the concentration of RFP during long-term medication to prevent the effect of hepatic enzyme induction reduced its concentration. Recommendation EMB dose adjustment according to monitoring concentration.RFP, INH, EMB were concentration-dependent antibacterial drugs, comparison of the permeability of three drugs in pleural effusions, it can be hypothesized that the way RFP, INH and EMB through pleural penetrate into pleural effusion may be different, which may be associated with the drug’s polarity, protein binding rate or pleural mesothelial barrier effect. Therefor different drugs pleural permeability are significantly different, the mechanism remains to be further studied. |
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