骨载异烟肼缓释微球的制备及其在动物模型中的释放研究

目的：观察探讨骨载异烟肼聚乳酸微球的设计和制备方法及其在动物模型中的释放效果。方法：选择24只雄性新西兰大白兔作为实验动物模型,采用复乳法制备缓释微球,对微球进行形态学观察、粒径分布观察、体内释药和体外释药观察。结果：平均微球粒径（10.59±0.3）μm,平均包封率（44.9±0.9）%,平均载药率（13.0±0.6）%。在24h、72h、1w、3w微球组滑膜药物浓度明显高于口服组和原药组,P＜0.05。与口服组和原药组对比,微球组0～24h药物波动差值明显更低,P＜0.05。结论：本研究制备的骨载异烟肼缓释微球具有良好的缓释性,能够在抗结核治疗中置入骨结核病灶对术后病灶发挥持久的疗效。     

DNA微阵列芯片法在海南地区结核病诊断及耐药性检测中的应用

目的探讨DNA微阵列芯片法在海南地区结核病诊断及耐药性检测中的应用。方法采用抗酸杆菌涂片法、罗氏培养法、比例法药敏试验及DNA微阵列芯片法对海南地区的2069例疑似结核病患者痰标本进行检测,并对结核分枝杆菌检出率、耐药性及耐药基因突变特征进行分析。结果DNA微阵列芯片法检测结核分枝杆菌检出率为明显高于罗氏培养法和抗酸杆菌涂片法(P<0.05)。以比例法药敏试验为金标准,DNA微阵列芯片法与比例法药敏试验检测耐利福平、耐异烟肼结核分枝杆菌及耐多药结核分杆菌的效果比较,差异有统计学意义(P<0.05),但均有较好的一致性(Kappa≥0.75)。耐利福平基因突变类型主要为rpoB531(C→T),占56.3%;耐异烟肼基因突变类型主要为KatG315(G→C),占84.6%;耐多药结核分杆菌基因突变类型主要为rpoB531+KatG315占54.6%。结论DNA微阵列芯片法可快速、准确地对海南地区疑似结核病患者的痰标本进行结核分枝杆菌及其耐药性检测,从而指导临床用药。     

Impact of iron loading and iron chelation on murine tuberculosis

Objective: To demonstrate in a mouse model of tuberculosis that excess iron load enhances the growth of Mycobacterium tuberculosis and to assess whether or not iron chelation may abrogate the effect of iron loading on Mycobacterium tuberculosis growth in the mouse model.
Methods: In the first experiment, female BALB/C mice were infected intravenously with 5.4 × 10⁴ CPU of M. tuberculosis H37Rv per mouse. Before infection, half of them were treated for 2 weeks with 50 mg/kg polymaltose ferric hydroxide, a source of iron. In a second experiment, female BALB/C mice were infected intravenously with 9 × 10⁴ CPU per mouse and half of them were iron loaded for 2 weeks before infection. Both iron-loaded and non-iron-loaded mice were treated with desferrioxamine (DFO), an iron chelator, or isoniazid. At each sacrifice, mice and their spleens were weighed, lung lesions were noted, and the number of M. tuberculosis CFU determined by quantitative cultures of spleen and lungs.
Results: In the first experiment, the number of CFU was significantly higher in the spleen of iron-treated mice than in non-iron-loaded mice at days 14 and 28 after infection. In the second experiment, iron loading enhanced the multiplication of M. tuberculosis in the spleen but not in the lungs, DFO displayed a modest but significant effect on the multiplication of M. tuberculosis in iron-loaded mice, and isoniazid therapy was effective in both iron-loaded and non-iron-loaded mice.
Conclusions: Iron loading of BALB/C mice enhanced the multiplication of M. tuberculosis in the spleens but not in the lungs. DFO exhibited significant activity against M. tuberculosis in iron-loaded mice, and isoniazid therapy was strongly bactericidal in both iron-loaded and non-iron-loaded mice.     

快速检测耐异烟肼结核分支杆菌方法的研究

目的 应用多重聚合酶链反应-单链构象多态性分析（multiple—Polymerase Chain Reaction—Single Strand Conformation Polymorphism,multi—PCR—SSCP）方法,快速、特异地同时检出耐异烟肼（isoniazid,INH）结核分支杆菌aphC启动子、inhA、katG基因的突变情况,并与直接测序（derictsequencing,DS）结果相比较,参照药敏试验,探讨该方法的可行性。 方法 根据结核分支杆菌的aphC启动子序列、inhA序列、katG序列,分别设计出3对特异性寡聚核苷酸引物,采用multi—PCR-SSCP技术,同时检测对结核分支杆菌耐INH起作用的这三个基因的突变情况;同时进行耐药株的测序分析。 结果 对H37Rv标准株、临床分离INH敏感株（23株）及INH耐药株（35株）进行multi—PCR—SSCP分析,突变检出率82．9％（29／35）;耐药株测序分析突变检出率为85．7（30／35）。两种方法的符合率为97．1％E（29＋5／）／35]。 结论 耐药基因检测指导治疗是一种新探索,multi—PCR—SSCP方法敏感、特异,能同时快速有效地检测结核分支杆菌aphC启动子、inhA、katG三个INH耐药基因的突变,提高检验效率,有望成为临床指导用药的好方法。     

应用寡核苷酸探针膜杂交方法快速检测耐异烟肼结核分支杆菌基因型

目的应用寡核苷酸探针膜反向斑点杂交技术快速检测结核分支杆菌对异烟肼(isoniazid,INH)耐药性.方法设计与合成用于检测结核分支杆菌耐INH基因katG、inhA的寡核苷酸探针,点于硝酸纤维素膜上,与结核分支杆菌临床分离株生物素标记的聚合酶链反应(PCR)产物进行反向斑点杂交,并与PCR-单链构象多态性(Polymerase chain reaction-Single stranded conformation polymorphism,PCR-SSCP)和PCR-直接测序(PCR-direct sequencing,PCR-DS)结果比较.结果 20株INH敏感株中,仅9株出现野生型探针K1阳性杂交,余11株中,10株K1b'杂交阳性,PCR-DS显示katG基因315位密码子AGC→ACC,1株K1c'杂交阳性,PCR-DS显示katG基因315位密码子AGC→AAC;15株出现野生型探针inh1阳性杂交,另5株Inha1探针杂交阳性,PCR-DS显示inhA基因-15位C→T突变.36株耐药株中,17株K1探针杂交阳性,18株K1b'杂交阳性,1株与所试探针不杂交,PCR-DS显示katG基因279位密码子GGC→GAC;11株与突变型Inha1探针阳性杂交,25株与野生型探针inh1杂交阳性.katG基因膜杂交突变检出率为 50 %,inhA基因膜杂交突变检出率为 30.56 %.结论寡核苷酸探针膜杂交技术可能成为检测部分结核分支杆菌耐异烟肼基因型简便、快速的方法.     

UPLC-MS/MS测定人痰液中异烟肼、吡嗪酰胺和左氧氟沙星的浓度

目的建立超高效液相色谱串联质谱法(UPLC-MS/MS)同时检测痰液中3种药物异烟肼(isoniazid,INH)、左氧氟沙星(levofloxacin,LFX)和吡嗪酰胺(pyrazinamide,PZA)浓度的方法,冒在为实现临床个体化用药提供帮助。方法痰液样品离心后经甲醇/乙腈蛋白沉淀法预处理后进样液质联用仪进行分析。实验采用的流动相A为含0.1%甲酸的水溶液,流动相B为含0.1%甲酸的乙腈;流速0.35mL·min^-1;采用ACQUITY UPLAC HSS T31.8μm column(2.1mm×100mm,Waters公司)分离;采用电喷零电离源,采用多反应监测(multiple reaction monitoring,MRM)模式对待测物进行正离子扫描检测。此外,本试验盲态收集5例患者的痰液,用该法进行定量分析。结果测得痰液中INH、PZA和LFX分别在48~6000 ng·mL^-1(r=0.9988)、480~60000 ng·mL^-1(r=0.9993)、120~15000 ng·mL^-1(r=0.9995)内表现出良好的线性关系。INH、PZA和LFX 3种药物的日内和日间精密度均低于15%,且3种药物的提取回收率均处于97.21%~107.80%之间。7例受试者体内均检测到药物INH,质量浓度分别为44.74、120.1、301.5、481、595.5、1220及1570 ng·mL^-1;PZA的质量浓度分别为104.2、6273.34和3185 ng·mL^-1;1例受试者检出LFX,质量浓度为199.86 ng·mL^-1。结论本试验建立了痰液中INH、PZA及LFX的检测方法,具有灵敏度高、测定结果准确、快速等诸多优点,可以应用于临床治疗药物的监测。     

Mycobacterium tuberculosis infection in psoriatic patients treated with biologics: Real-world data from 18 Japanese facilities

Although rare, tuberculosis has been reported with biologic treatment against psoriasis in Japan, a tuberculosis medium-burden country. Mycobacterial infection often develops after a long incubation period and might not have been adequately identified in clinical trials or post-marketing surveillance. To determine the real-world incidence of tuberculosis in psoriatic patients treated with biologics, we conducted a retrospective, multicenter, observational study in 18 facilities in Western Japan. Psoriatic patients who visited a participating facility between 2010 and March 2017 and received biologic reagents were enrolled. Information on sex, age at first biologic treatment, results of interferon-γ release assay (IGRA) for Mycobacterium tuberculosis, treatment history with isoniazid, and onset of active and/or latent tuberculosis was collected. A total of 1117 patients (830 men and 287 women) were enrolled. The mean duration of biologic treatment was 3.54 years. Sixty-five patients (5.8%) showed positive IGRA results at screening. Active tuberculosis developed in two patients after the administration of tumor necrosis factor inhibitors (both involved miliary tuberculosis). Latent tuberculosis was observed in two patients treated with anti-interleukin-12/23p40 antibody. The incidence rate of tuberculosis, including latent tuberculosis, in this survey was 0.36%. Although the incidence rate of tuberculosis was low considering the observation period of biologic treatment, active tuberculosis was found in both the screening-negative group and a screening-positive subject after isoniazid prophylaxis (both miliary tuberculosis), concluding that negative screening or isoniazid treatment does not always assure that an individual has no tuberculosis. Hence, dermatologists still need to pay careful attention to tuberculosis at every patient visit.     

Isonicotinic hydrazide causes seizures in scrapie-infected hamstesr with shorter latency than in control animals: A possible GABAergic defect

Isonicotinic hydrazide, a drug that decreases the level of GABA, when injected subcutaneously in control and scrapie-infected hamsters induced tonic-clonic seizures in scrapie hamsters significantly earlier (P<0.0001) than in control animals. This suggests depression of the GABAergic system in scrapie-infected hamsters. To determine whether this lesion is pre or postsynaptic we measured the level of GABA, glutamate, cGMP and cAMP and the GABA-benzodiazepine receptor complex.     

Modulation of CYP2EI activity by isoniazid in rapid and slow N-acetylators

Aims An investigation was undertaken to compare the effects of isoniazid pretreatment on the CYP2El -mediated 6-hydroxylation of chlorzoxazone in healthy subjects of known N-acetylator phenotype.
Methods CYP2E1 activity was estimated based on the 6-hydroxylation of chlorzoxazone following single dose (250 mg) oral administration to seven slow and eight rapid N-acetylators who were in good health. Separate studies were performed prior to and 14 days after the subjects received 300 mg isoniazid dady. Additional investigations were undertaken 2 and 16 days after discontinuing treatment with the antitubercular agent.
Results Concomitant administration of chlorzoxazone with the final dose of isoniazid resulted in reduced metabolism in both phenotypes; however, the extent of inhibition of 6-hydroxylation was greater in the slow N-acetylators—about 80% v s 60%. Two days after stopping isoniazid administration, chlorzoxazone's pharmacokinetic parameters had returned to their baseline values and remained constant for a further 14 days in the rapid acetylators. In contrast, chlorzoxazone's 6-hydroxylation in slow acetylators was increased by about 60% compared with baseline at 2 days after dicontinuing isoniazid but had returned to its initial value 14 days later. Conclusions The interphenotypic difference in the time-dependent interactions of isoniazid with CYP2E1 probably reflect a higher drug exposure in slow acetylators. Inhibition of CYP2E1 activity occurs in both N-acetylator phenotypes but is less extensive in fast acetylators, during the time that effective levels of isoniazid are present in the body. Increased CYP2E1 activity reflective of enzyme induction, on the other hand, is only observable following isoniazid's elimination and is more extensive in slow than rapid acetylators. Even then, however, such induction is relatively modest and of short duration.