抗结核药物缓释微球在兔椎体中释放及分布规律的研究
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摘要
研究背景
据世界卫生组织的统计到目前为止结核病仍然是一种严重危害人类健康的慢性传染病,目前全球有约1/3的人口被感染结核分枝杆菌。目前全世界结核病患者逾2000万,每年新增近800万,1993年WHO宣布了“全球进入结核病紧急状态”。据文献报道在HIV阴性的患者约有3%-5%的患者在感染肺结核后出现脊柱结核,而HIV阳性患者这一数字则达到了60%,这向广大医务工作者和骨科工作者敲响了警钟和提出了新的要求。我国1979年全国第一次结核病流行病学调查结果显示,活动性肺结核患病率717/10万,全国约有活动性肺结核病人700万,据专家估计骨与关节结核占3%-5%,即有骨关节结核病人21万-35万。2000年全国第四次结核病流行病学调查结果,活动性肺结核患病率367/10万。估计全国有活动性肺结核病人500万,如按3%-5%计算,现有骨关节结核病人15-25万。外科手术可以直接切除脊柱结核病灶,但不能完全清除结核菌。而手术放置内固定则有可能成为术区残留结核菌粘附的金属床,加上骨骼特殊的成分和生理活性,以及抗结核药物自身半衰期较短和组织渗透性差的特点,治疗药物按一般给药途径难以有效转运到作用部位,成为了脊柱结核术后复发的一个重要原因。在脊柱结核外科手术结束时往往会在术区局部放置抗结核药物,但是局部的药物会随血液循环和自身的代谢迅速消失。聚乳酸(PLA)已经广泛应用于载药释放系统的实验研究[17][18],其中外消旋聚乳酸(D,L-polylactic-acid, PDLLA)是经FDA批准可用作医用手术防粘连膜和注射用微胶囊、微球及埋植剂等缓释制剂的材料。羟基磷灰石(hydroxyapatite,简称HA),分子式为Ca10(PO4)6(OH)2,其组成和结构均与人体硬组织(骨骼,牙齿)中的无机成份相似,具有优良的生物相容性和生物活性,植入人体后能与原骨组织形成生理结合。因此本题提出了设计了两种组织相容性好,缓释效果理想的抗结核药缓释载体,通过在术区埋置抗结核药物缓释剂,使骨骼中的抗结核药物浓度长期保持在有效杀菌药物浓度,并对缓释剂的离体释放和在动物椎骨中释放进行浓度检测。从而有效地提高脊柱结核的疗效,减少脊柱结核术后的复发,为骨结核的治疗提供理论依据和治疗手段。
本文对实验动物脊柱骨内埋置抗结核药物缓释剂后外周血液持续给予五联抗痨药物,采用液相-质谱联用技术(HPLC-MS)又叫液相色谱-质谱联用技术对药物浓度进行检测(它以液相色谱作为分离系统,质谱为检测系统)。对实验动物椎骨及外周血液中异烟肼(INH),利福平(RFP)和吡嗪酰胺(PZA),链霉素(MS)及乙胺丁醇(EMB)五种一线抗结核药物的浓度进行了全面检测。探讨正规化疗及埋置缓释剂后抗结核药物在实验动物脊柱骨中的分布情况,为探索进一步提高脊柱结核病灶清除术的患者术区抗结核药物浓度提供理论依据。
目的：
1.寻找一种能够在脊柱结核病灶术区内持续长时间释放抗结核药物的缓释载体,保障术区保持长期的有效杀菌浓度。
2.寻找出一种一次性进样检测全部一线五种抗结核药物浓度的检测方法,并确保该方法检测结果符合生物样本浓度检测的要求。
3.对实验动物进行造模干预后模拟人体抗结核治疗的过程,检测其病灶内的药物浓度并与人体抗痨治疗后药物浓度作比较,将异烟肼缓释微球放入实验动物病灶中进行浓度检测了解其代谢和分布规律。方法:
1.寻找出一种能够在脊柱结核病灶术区内持续长时间释放抗结核药物的缓释载体
(1)采用复乳-溶媒挥发法制备异烟肼聚乳酸微球：
①分别称量聚乙烯醇4g和1g加入超纯水各100ml,加热至100C°后充分搅拌溶解聚乙烯醇为4%和1%PVC液各100ml备用。
②称量INH标准品20mg,溶于4%PVC液1ml中；称量PLGA25mg溶于2ml二氯甲烷中。
③将含INH的4%PVC液体倒入二氯甲烷溶液中,冰水冲浴下磁力搅拌,分散1min,形成W/O液(water/oil)。
④倒入1%PVC液10ml,搅拌10min,形成W/O/W。将W/O/W倒入1%PVC20ml中搅拌12-18h,扩散有机相。
⑤将全部液体高速离心4000转/min离心10min得漂浮白色颗粒,将白色颗粒用BPS液体冲洗后再次离心,收集离心后的BPS液体测试紫外,取顶部白色漂浮物再次BPS液冲洗,离心,循环三次后用称量纸包裹白色颗粒晾晒干燥后备用。
(2)采用压力渗透技术备制羟基磷灰石异烟肼微球：
①称取质量比为10：10：80的Li2CO3, CaCO3, H3BO3粉料充分混合
②混合物置于硅碳棒高温炉中,在1100℃下加热熔融15min后冰水急速淬冷。
③将混合物干燥破碎后筛取100-140目(每平方英时筛网上的孔眼数目)之间的颗粒,再至于800℃管式炉中球化两次。
④将球化后的颗粒置于K2HPO4(0.25mol/L, pH=9)溶液中,静置于37℃恒温箱中一周后取出。
⑤用去离子水充分洗涤后再90℃恒温干燥24h,然后将微球置于800℃马弗炉中加热2h,冷却后即得到多孔HA微球。
⑥称量异烟肼标准品23.4mg加入超纯水1ml,超声10min后加温至90C°充分溶解药物备用。
⑦称量制备好的羟基磷灰石0.100g放入50ml烧杯,再放入真空干燥箱中保持真空度<0.058MPa放置12h,以排除微球孔隙中的气体增加载药量,异烟肼药液倒入微球超声15min,放置24h。
⑧将全部液体高速离心4000转/min离心10min将上清液收集测试紫外。取底部白色沉淀再次BPS液冲洗,离心,循环三次后用称量纸包裹白色颗粒晾晒干燥后备用。
2.寻找出能一次性进样检测全部一线五种抗结核药物浓度的检测方法,并确保该方法检测结果符合生物样本浓度检测的要求。
(1)确定出检测的HPLC-MS条件：
①色谱柱：亲水性高效液相色谱(HILIC)柱(150mm×2.1mmI.D.,3μm(Thermo, USA)。柱前连接有一个RRLC在线过滤器(Agilent, Germany)。柱温：40C°。
②流动相：由甲醇和含0.1%甲酸的5mM乙酸铵溶液(65：35,v/v)组成。流速：0.5mL/min。进样量：5μL。
③质谱离子化方式：正离子电喷雾离子化(ESI+)。采用多级反应离子监测(MRM)模式进行定量,相应的MRM离子对和质谱参数为。ESI+条件：喷雾电压5000V；离子源温度700℃；气帘气15psi,碰撞活化参数：M；雾化气：70psi；辅助气：65psi。
(2)样品提取过程：
①样品提取溶液：含5mM乙酸铵、0.1%甲酸和10%水的甲醇溶液。
②100μl标准曲线系列样品或质控样品加50μl内标工作液和300μl样品提取溶剂。待测血浆样品100μl,加50μL内标工作液和300μl样品提取溶剂。
③样品经充分涡旋2min后,于13000rpm离心10min。上清液转移至自动进样瓶中,进样5μl。
(3)建立标准曲线：
得出9个浓度低度的色谱图,再以五种药物的峰面积为X轴、三种药物的浓度为Y轴作线性性分析。RPZA=0.9982,RINH=0.9973,RSM=0.9963,REMB=0.9921,RRFP=0.9973Y PZA=0.000377x+0.00577Y INH=0.000654x+0.00384Y SM=8.7e-005x-0.000435Y EMB=0.104x+0.0596YRFP=0.00137x+0.00199
(4)样品处理：
①取样品血浆100μl,加内标液50μl,加提取液300μl(流动相/甲醇=65：35),涡旋15min后离心10min取上清液进样。
②骨组织样品则加入200μl骨组织提取液,加内标液50μl,加提取液300μl(流动相／甲醇=65：35),涡旋15min后离心10min取上清液进样。
③得5种抗结核药物和2中内标药物质谱图,带入标准曲线求出药物浓度。
3.对实验动物进行造模干预,模拟人体结核抗痨治疗的过程,为脊柱结核的基础研究搭建实验动物平台。
(1)实验动物的准备
①新西兰白兔(成年2-3岁)共38只(清洁级),雌雄不限。饲养条件为：23℃左右,相对湿度：70%左右,标准兔饲料饲养2周(基本热量需求)。
②提前一个月对新西兰兔予以地塞米松注射液(Dexamethasone,DSMS)0.32mg iv QOD(按照0.16mg/公斤折算),一周后改为片剂喂饲给药0.06mg poQD(按照0.03mg/公斤折算),抑制其体液免疫和细胞免疫。
(2)菌株备制过程
①将牛型结核分枝杆菌减毒菌株BCG在改良Sauton液体培养基中培养扩增2-3周。
②分装于离心管,离心后用10%小牛血清培养液稀释成均匀的5mg/ml混悬液,常温放置待用。使用时再次稀释并镜下调整浓度为1×105/ml。
(3)菌株接种过程。
①麻醉满意后常规碘酒消毒,酒精脱碘,铺无菌巾,术前器械高温消毒,取双侧第12肋末端连线的中点向下至双侧髂嵴连线的中点作约4cm纵向切口的后正中入路,显露腰4、5椎体棘突及棘上韧带。
②于腰5椎体左侧近椎间盘处紧贴椎体皮质骨部由上往下剥离,暴露腰5椎体的左半部。用电动球磨钻机在腰5椎体上钻一骨孔,深度约为0.4cm、孔径约0.25cm,骨孔道止血后填入医用明胶海绵,在明胶海绵上用注射器缓慢注射BCG菌株悬液0.1m1。
③1一0号丝线间断关闭切口,关闭深筋膜后皮下涂洒注射用青霉素粉剂(30万U/只),缝合皮肤后再次酒精消毒,无菌纱布覆盖切口上,并固定好。
④将动物松绑后送入3P级饲养房内饲养。
结果：
1.制作一种能够在脊柱结核病灶术区内持续长时间释放抗结核药物的缓释载体,保障术区保持长期的有效杀菌浓度。
①聚乳酸异烟肼微球孔隙率为63.68%,羟基磷灰石异烟肼微球孔隙率为82.18%。
②聚乳酸异烟肼微球载药率为26.62%,包封率为28.35%。羟基磷灰石异烟肼微球载药率为10.45%,包封率39.87%。
③两种缓释微球均能在第52天释放出有效杀菌浓度的异烟肼。
2.寻找出一种一次性进样检测全部一线五种抗结核药物浓度的检测方法,并确保该方法检测结果符合生物样本浓度检测的要求。
①本题所采用的质谱条件下可以一次性进样品检测出全部五种一线抗结核药物浓度,检测时间控制在3分钟以内,效率比单纯的液相色谱技术进行检测大大提高。
②INH,RFP, PZA, SM和EMB回收率：分别为98.1%,97.6%,98.3%,98.1%和97.9%。日间及夜间变异率：在5.6%-12.3%。INH,RFP,PZA,SM,和EMB。精密度：分别为0.212%,0.119%,0.103%,0.351%和0.209%。
3.对实验动物进行造模干预后模拟人体抗结核治疗的过程,检测其病灶内的药物浓度并与人体抗痨治疗后药物浓度作比较,将异烟肼缓释微球放入实验动物病灶中进行浓度检测。
①采用牛型结核分枝杆菌减毒菌株BCG造模的实验动物,病灶组织学改变同H37Rv菌株存在很大差异,但形态学改变上有一定的相似性。
②实验动物病灶内的抗结核药物浓度与人体脊柱结核病灶内药物浓度有一定差异。实验动物服药后抗结核药物原药的浓度在髂骨和病灶中较为相似,在血液中存在少许差异。(2小时FRFP血=8.544,PRFP血=0.015,3小时FRFP血=6.182PRFP血0.040)而其代谢产物的差异则更为明显。
③在实验动物体内埋置异烟肼缓释微球,能有效的长期保持局部抗结核药物的高浓度水平。
结论：
1.聚乳酸和羟基磷灰石异烟肼微球能在术区内缓慢释放,长期保持局部术区的抗结核药物浓度。
2.液相-质谱联用技术可一次性进样检测五种一线抗结核药物浓度,该检测方法稳定性好,可重复性强,灵敏度高。
3.采用BCG作为实验菌株接种实验动物可在一定程度上模拟人体脊柱结核的病理过程,并作为一种实验动物平台,为后续研究打下基础。
Background
According to the World Health Organization, tuberculosis (TB)-a contagious disease caused by Mycobacterium tuberculosis, is a chronic infectious disease with a serious hazard to human health [2],there were about1/3people in the worlds had been infected with this Mycobacterium tuberculosis [3]. At present the whole world TB patients are more than20million with an annual increase of nearly8million, WHO announced that TB was a global emergency in1993. According to some reports that between3%and5%of HIV-negative patients have musculoskeletal involvements after pulmonary infection, compared with that in60%of HIV-positive patients[4] Thus, this disease warrants considerable attention. The prevalence rate of pulmonary tuberculosis was717/100thousand based on the result of the first Chinese National Tuberculosis Epidemiological Survey in1979. There was approximately7million of patients with active pulmonary tuberculosis, experts estimate that bone and joint tuberculosis accounted from3%to5%, so bone and joint tuberculosis patients account to210-350thousand [6]. Based on the result of the fourth Chinese National Tuberculosis Epidemiological Survey in2000the prevalence rate of pulmonary tuberculosis was367/100thousand. The number of patients with active pulmonary tuberculosis was estimated at5000thousand. If the prevalence rate of bone and joint tuberculosis is3%-5%, there should be150-250thousand patients with this kind of disease.(TB) focus can be resected directly by surgery, but TB bacteria are unresectable. The internal fixation could be the metal bed for the adherence of TB in the postoperative residues focus. Along with bones special composition, half-life of anti-TB drug is shorter and the organization permeability of it is poor. It is difficult for anti-TB drugs to be efficively transported to the foci by the general route, which could be an important cause of the recurrence of the spinal TB [15][16]. At the end of surgery for spinal tuberculosis anti-TB drugs often were placed in the surgery sites, but local drug will quickly disappear because of the blood circulation and metabolism. Polylactic acid (PLA) has been widely used in experimental study on drug release system, among them D,L-polylactic-acid (PDLLA) is approved by FDA to be used as a medical material with microcapsule, microsphere and implants and sustained-release preparation and injection to prevent adhesion film material. The composition and structure of Hydroxyapatite (HA) is similar to inorganic composition in human hard tissue (bone, teeth). HA has excellent biological compatibility and biological activity and can combine physiologily with the original bone. Therefore this study aimed to design of two kinds of low-release agent with good compatibility for transporting anti-tuberculosis. By embedding these anti-tuberculosis drugs controlled-release agents in the surgery sites, high concentration of anti-tuberculosis drug above effective bactericidal concentration was keep in the long-term and the concentration of drugs from releasing in animal vertebrae was monitored in vitro, in order to improve the curative effect of spinal TB, reduce the postoperative recurrence of them and provide a theoretical basis and treatment measures for bone tuberculosis. This study was based on the embedding anti-tuberculosis drugs controlled-release agent in the surgery sites of animal and administering five anti-tuberculosis drugs continuously in peripheral blood, then the concentration of drugs were determinated by HPLC-MS, to map the concentration and distribution of five first line anti-tuberculosis drugs-Isoniazid (INH), Rifampicin (RFP), Pyrazinamidea (PZA), Ethambutol (EMB) and Streptomycin (SM). The concentration of anti-tuberculosis drugs in experimental animal vertebrae was exploered after regular chemotherapy and embedded with slow-release agent in order to construct theory basis on further enhancing concentration of anti-tuberculosis drugs in surgery sites of spinal tuberculosis patients
Objective:
1. To make a sustained release carrier of anti-tuberculosis drugs in the focus of spinal tuberculosis, to maintain effective bactericidal concentration in surgery site in the long-term.
2. To find a method for determinating the concentration of all first-line anti-tuberculosis drugs in one-time sample, and to ensure that the results of this method accord with the biological sample concentration detection requirements.
3. To simulate human intervention on treatment of tuberculosis with animal modeled spinal tuberculosis. The concentration of anti-tuberculosis drugs in focus of animals was compared with patients. Finally isoniazid control-released agents were inoculated on the focus and the concentration of anti-tuberculosis drugs was determinate.
Methods:
1. To make a sustained release carrier of anti-tuberculosis drugs in the focus of spinal tuberculosis, to maintain long-term effective bactericidal concentration in surgery site.
(1) Isoniazid polylactic acid microspheres were prepared by double emulsion/solvent evaporation method.
①Polyving akohol was weighted for4g and1g respectively and100ml of ultrapure water was added, then was heated and agitated fully for100℃for4%and1%PVC water respectively.
②Isoniazid was weighted for20mg and dissolved in4%PVC water1ml. PLGA was weighted for25mg and dissolved in2ml.dichlormethane
③4%PVC water with isoniazid was added in2ml dichlormethane and stirred by magnetic force with ice water in1min to obtain components (water/oil).
④10ml1%PVC water was added and stirred by magnetic force in10min to obtain components (water/oil/water), which was added in1%PVC water20ml and stirred12-18h.
⑤The liduid was centrifugated with high speed at4000turn/min in10min and the white flotation particles were obtained, then was flushed with BPS water then was centrifugated again. Then the BPS water after centrifugalization was collected for ultraviolet determination. The top flotation particles were collected again for flushing and centrifugating in three times. Then the white particles was collected and stored with paper bundle.
(2) Isoniazid polylactic acid microspheres were prepared by pressure infiltration technology.
①The components with Li2CO3, CaCO3, H3BO3=10:10:80were stirred fully.
②The above components were placed in silicon carbide furnace and heated with1100℃in15min until to be melted completely then rapid quenched with ice water.
③After the components were quenched the parcels in100-140mesh, then were collected and nodulized in tubular furnace two times.
④The parcels after nodulizing were placed in K2HPO4solution (0.25mol/L, pH=9) and placed in constant temperature oven at37℃for one week.
⑤The parcels were flushed with deionized water and constant temperature dried at90℃, in24h. Then were placed in muffle furnace to heat at800℃in2h and obtained with Porous hydroxyapatite microspheres.
⑥Standard isoniazid was weighted for23.4mg and putted into ultrapure water1ml, then was treated with ultrasound10min and heated to90℃,for dissolving drug Fully.
⑦The hydroxyapatite was weighted for0.1g and putted into beaker, then was placed into vacuum drying oven to keep the vacuum (<0.058MPa) in12h in order to remove gas in pores for increasing drug loading of microspheres.
⑧The liquid was centrifugated with high speed at4000turn/min in10min and the supernatant was collected to test ultraviolet. The bottom sediment particles were collected again for flushing and centrifugating in three times. Then the white particles were collected and stored with paper bundle.
2. To find a method to determinate the concentration of all first-line anti-tuberculosis drugs in one-time sample, and to ensure that the results of this method accord with the biological sample concentration detection requirements.
(1) To determine the detection HPLC-MS conditions
①chromatographic column was Hydrophilic high performance liquid chromatography (HILIC)150mm×2.1mmI.D.3μm(Thermo, USA). An online RRLC filter was connected with pre-column (Agilent, Germany), the column temperature was room temperature.
②Mobile phase was5mM ammonium acetate with methanol and0.1%formic acid65:35, v/v, Flow rate was0.5mL/min. Sample size was5μ L
③Mass spectrometric method was positive ion electrospray ionization (ESI+) Using multiple reaction monitoring (MRM) mode for quantitative. ESI+condition is at spray voltage5000V, ion source temperature is at700℃, Air curtain gas is15psi, collisional activation parameter was M, atomization gas is70psi, and auxiliary gas is65PSI.
(2) Sample extraction process
①Sample extraction solution was methanol solution containing5mM ammonium acetate,0.1%formic acid and10%of water.
②100μl standard curves of series of samples or quality control samples were added with50μl internal standard working solution and300μ1sample extraction solvent. The measured plasma samples were100μL, plus50μL internal standard working solution and300μl sample extraction solvent.
③Samples were fully stirred after2min,13000RPM centrifugated for10min. Supernatant was transferred to an automatic sample bottle, sample was5μL.
(3) To establish the standard curve:
The chromatogram with9concentration was obtained, the following regression equation of five anti-TB drugs with peak area (Y) and concentration level (X) was defined: RPZA=0.9982, RINH=0.9973, RSM=0.9963, REMB=0.9921, RRFP=0.9973YPZA=0.000377x+0.00577YINH=0.000654x+0.00384YINHm=8.7e-005x-0.000435YEMB=0.104x+0.0596YRFP=0.00137x+0.00199(4) Sample treatment
①The samples of plasma of100μL, L,50μL and internal standard solution, and extract300μL (mobile phase/methanol=65:35) were centrifuged at10min after stirred15min, then the supernatant was obtained for sample.
②The samples of bone of200μL bone tissue extract and internal standard solution of50μL, and extract300μL (mobile phase/methanol=65:35) were centrifuged10min after stirred15min, finally the supernatant was obtained for sample.
③Five kinds of anti-TB drugs and2internal standard drug mass spectrum were obtained and taken into the standard curve of drug to count concentration.
3. To simulate human intervention on treatment of tuberculosis after modeled with spinal tuberculosis. The concentration of anti-tuberculosis drugs in focus of animals was compared with patients. Finally isoniazid control-released agents were inoculated on the focus and the concentration of anti-tuberculosis drugs was determinate.
(1) Experimental animal preparation
①A total of38New Zealand white rabbits (adult2-3years)(clean grade), male or female are permitted. Feeding condition is at23℃, relative humidity is70%, standard rabbit diet for2weeks (the basic heat demand).
②One month in advance dexamethasone (DSMS) was used for injecting in rabbits0.32mg IV QOD (0.16mg/kg), after one week tablet was used for feeding administration of0.06mg PO QD (0.03mg/kg), which was used to inhibit the humoral immunity and cellular immunity.
(2) The preparation process of strain.
①The type of Mycobacterium tuberculosis BCG strains in modified Sauton liquid cultured2-3weeks.
②Packed in centrifuge tubes, centrifuged for20minutes,1200R/min, in saline to make uniform5mg/ml suspension at room temperature, placed for ready in ordinary temperature.
(3) Strain inoculation process.
①After the rabbit was anesthetized satisfactorily, routine iodine disinfection, alcohol was used to remove iodine, sterile towel was placed, preoperative instrument was sterilized. The4cm longitudinal incision was made from the midpoint of the of bilateral twelfth rib line to the midpoint of bilateral iliac crest line by posterior median approach.
②L5body was stripped down from proximal lumbar disc close to the vertebral cortical bone and the left part of vertebral body was exposed. L5vertebra was drilled a hole by the electric ball milling machine, The depth was about0.4cm, pore size was about0.25cm, bone tunnel was stopped bleeding with medical gelatin sponge, which affiliated Mycobacterium BCG suspension liquid with a syringe injection0.1ml(1×105CFU/ml)
③The wound was closed discontinuously with No.1-0silk line, after the deep fascia closed penicillin powder was used at the subcutaneous tissue of wound.
④The animal was relieved and taken into3P feeding room to rearing.
Results:
1. To make a sustained release carrier of anti-tuberculosis drugs in the focus of spinal tuberculosis, to maintain effective bactericidal concentration in surgery site in the long-term.
⑤The porosity of Polylactic acid-isoniazid microspheres is63.68%, the porosity of Hydroxyapatite-isoniazid microspheres is82.18%
⑥The loading rate and encapsulation of Polylactic acid-isoniazid microspheres was26.62%and28.35%respectively. The loading rate and encapsulation of Hydroxyapatite-isoniazid microspheres was10.45%and39.87%respectively.
③Two kinds of microspheres can all release enough isoniazid above effective bactericidal concentration of it in fifty-second days in vitro.
2. To find a method to determinate the concentration of all first-line anti-tuberculosis drugs in one-time sample, and to ensure that the results of this method accord with the biological sample concentration detection requirements.
①With the mass spectrometric conditions in this study all the samples can be detected to the concentration of all five first-line anti-TB drugs in one-time. The detection time control in3minutes and the detection efficiency is higher than that of pure liquid chromatography technique.
②Recovery rate of INH, RFP, PZA, SM and EMB is98.1%,97.6%,98.3%,98.1%and97.9%respectively. Daytime and nighttime variation rate of INH, RFP, PZA, SM and EMB is from5.6%to12.3%, Precision rate of INH, RFP, PZA, SM and EMB is0.212%,0.119%,0.103%,0.351%and0.209%respectively.
3. To simulate human intervention on treatment of tuberculosis after modeled with spinal tuberculosis. The concentration of anti-tuberculosis drugs in focus of animals was compared with patients. Finally isoniazid control-released agents were inoculated on the focus and the concentration of anti-tuberculosis drugs was determinate.
①There are great differences in histological changes of focus in animal model inoculated with BCG and H37RV, But the morphological changes have some similarity.
②There are some differences of anti-TB drugs concentration between experimental animal and human spinal TB, the concentration of anti-TB drugs in ilium and lesions are similar between experimental animal and patients with spinal TB and there is little difference in the blood after oral administration (F=8.544, P=0.015for RFP in serum2h, F=6.182, P=0.040for RFP in serum3h). Furthermore the difference of metabolite in focus of animal models and patients with spinal TB is more obvious.
③In the experimental animal implanted with isoniazid controlled-release microspheres can effective maintain high concentration levels of antit-TB drugs in long-term locally.
Conclusion:
1. Polylactic acid-INH and hydroxyapatite-INH controlled-release microspheres can slowly release isoniazid in surgery site to keep the high concentration of anti-tuberculosis drugs locally in long-term.
2. The concentration of all first-line anti-tuberculosis drugs, could be determinate in one-time sample with HPLC-MS technique. The detection method has good stability, high repeatability and high sensitivity.
3. The pathological process of experimental animal inoculated with BCG can roughly simulate human spinal tuberculosis, which is an experimental animal platform to base the foundation for further study.
据世界卫生组织的统计到目前为止结核病仍然是一种严重危害人类健康的慢性传染病,目前全球有约1/3的人口被感染结核分枝杆菌。目前全世界结核病患者逾2000万,每年新增近800万,1993年WHO宣布了“全球进入结核病紧急状态”。据文献报道在HIV阴性的患者约有3%-5%的患者在感染肺结核后出现脊柱结核,而HIV阳性患者这一数字则达到了60%,这向广大医务工作者和骨科工作者敲响了警钟和提出了新的要求。我国1979年全国第一次结核病流行病学调查结果显示,活动性肺结核患病率717/10万,全国约有活动性肺结核病人700万,据专家估计骨与关节结核占3%-5%,即有骨关节结核病人21万-35万。2000年全国第四次结核病流行病学调查结果,活动性肺结核患病率367/10万。估计全国有活动性肺结核病人500万,如按3%-5%计算,现有骨关节结核病人15-25万。外科手术可以直接切除脊柱结核病灶,但不能完全清除结核菌。而手术放置内固定则有可能成为术区残留结核菌粘附的金属床,加上骨骼特殊的成分和生理活性,以及抗结核药物自身半衰期较短和组织渗透性差的特点,治疗药物按一般给药途径难以有效转运到作用部位,成为了脊柱结核术后复发的一个重要原因。在脊柱结核外科手术结束时往往会在术区局部放置抗结核药物,但是局部的药物会随血液循环和自身的代谢迅速消失。聚乳酸(PLA)已经广泛应用于载药释放系统的实验研究[17][18],其中外消旋聚乳酸(D,L-polylactic-acid, PDLLA)是经FDA批准可用作医用手术防粘连膜和注射用微胶囊、微球及埋植剂等缓释制剂的材料。羟基磷灰石(hydroxyapatite,简称HA),分子式为Ca10(PO4)6(OH)2,其组成和结构均与人体硬组织(骨骼,牙齿)中的无机成份相似,具有优良的生物相容性和生物活性,植入人体后能与原骨组织形成生理结合。因此本题提出了设计了两种组织相容性好,缓释效果理想的抗结核药缓释载体,通过在术区埋置抗结核药物缓释剂,使骨骼中的抗结核药物浓度长期保持在有效杀菌药物浓度,并对缓释剂的离体释放和在动物椎骨中释放进行浓度检测。从而有效地提高脊柱结核的疗效,减少脊柱结核术后的复发,为骨结核的治疗提供理论依据和治疗手段。
本文对实验动物脊柱骨内埋置抗结核药物缓释剂后外周血液持续给予五联抗痨药物,采用液相-质谱联用技术(HPLC-MS)又叫液相色谱-质谱联用技术对药物浓度进行检测(它以液相色谱作为分离系统,质谱为检测系统)。对实验动物椎骨及外周血液中异烟肼(INH),利福平(RFP)和吡嗪酰胺(PZA),链霉素(MS)及乙胺丁醇(EMB)五种一线抗结核药物的浓度进行了全面检测。探讨正规化疗及埋置缓释剂后抗结核药物在实验动物脊柱骨中的分布情况,为探索进一步提高脊柱结核病灶清除术的患者术区抗结核药物浓度提供理论依据。
目的：
1.寻找一种能够在脊柱结核病灶术区内持续长时间释放抗结核药物的缓释载体,保障术区保持长期的有效杀菌浓度。
2.寻找出一种一次性进样检测全部一线五种抗结核药物浓度的检测方法,并确保该方法检测结果符合生物样本浓度检测的要求。
3.对实验动物进行造模干预后模拟人体抗结核治疗的过程,检测其病灶内的药物浓度并与人体抗痨治疗后药物浓度作比较,将异烟肼缓释微球放入实验动物病灶中进行浓度检测了解其代谢和分布规律。方法:
1.寻找出一种能够在脊柱结核病灶术区内持续长时间释放抗结核药物的缓释载体
(1)采用复乳-溶媒挥发法制备异烟肼聚乳酸微球：
①分别称量聚乙烯醇4g和1g加入超纯水各100ml,加热至100C°后充分搅拌溶解聚乙烯醇为4%和1%PVC液各100ml备用。
②称量INH标准品20mg,溶于4%PVC液1ml中；称量PLGA25mg溶于2ml二氯甲烷中。
③将含INH的4%PVC液体倒入二氯甲烷溶液中,冰水冲浴下磁力搅拌,分散1min,形成W/O液(water/oil)。
④倒入1%PVC液10ml,搅拌10min,形成W/O/W。将W/O/W倒入1%PVC20ml中搅拌12-18h,扩散有机相。
⑤将全部液体高速离心4000转/min离心10min得漂浮白色颗粒,将白色颗粒用BPS液体冲洗后再次离心,收集离心后的BPS液体测试紫外,取顶部白色漂浮物再次BPS液冲洗,离心,循环三次后用称量纸包裹白色颗粒晾晒干燥后备用。
(2)采用压力渗透技术备制羟基磷灰石异烟肼微球：
①称取质量比为10：10：80的Li2CO3, CaCO3, H3BO3粉料充分混合
②混合物置于硅碳棒高温炉中,在1100℃下加热熔融15min后冰水急速淬冷。
③将混合物干燥破碎后筛取100-140目(每平方英时筛网上的孔眼数目)之间的颗粒,再至于800℃管式炉中球化两次。
④将球化后的颗粒置于K2HPO4(0.25mol/L, pH=9)溶液中,静置于37℃恒温箱中一周后取出。
⑤用去离子水充分洗涤后再90℃恒温干燥24h,然后将微球置于800℃马弗炉中加热2h,冷却后即得到多孔HA微球。
⑥称量异烟肼标准品23.4mg加入超纯水1ml,超声10min后加温至90C°充分溶解药物备用。
⑦称量制备好的羟基磷灰石0.100g放入50ml烧杯,再放入真空干燥箱中保持真空度<0.058MPa放置12h,以排除微球孔隙中的气体增加载药量,异烟肼药液倒入微球超声15min,放置24h。
⑧将全部液体高速离心4000转/min离心10min将上清液收集测试紫外。取底部白色沉淀再次BPS液冲洗,离心,循环三次后用称量纸包裹白色颗粒晾晒干燥后备用。
2.寻找出能一次性进样检测全部一线五种抗结核药物浓度的检测方法,并确保该方法检测结果符合生物样本浓度检测的要求。
(1)确定出检测的HPLC-MS条件：
①色谱柱：亲水性高效液相色谱(HILIC)柱(150mm×2.1mmI.D.,3μm(Thermo, USA)。柱前连接有一个RRLC在线过滤器(Agilent, Germany)。柱温：40C°。
②流动相：由甲醇和含0.1%甲酸的5mM乙酸铵溶液(65：35,v/v)组成。流速：0.5mL/min。进样量：5μL。
③质谱离子化方式：正离子电喷雾离子化(ESI+)。采用多级反应离子监测(MRM)模式进行定量,相应的MRM离子对和质谱参数为。ESI+条件：喷雾电压5000V；离子源温度700℃；气帘气15psi,碰撞活化参数：M；雾化气：70psi；辅助气：65psi。
(2)样品提取过程：
①样品提取溶液：含5mM乙酸铵、0.1%甲酸和10%水的甲醇溶液。
②100μl标准曲线系列样品或质控样品加50μl内标工作液和300μl样品提取溶剂。待测血浆样品100μl,加50μL内标工作液和300μl样品提取溶剂。
③样品经充分涡旋2min后,于13000rpm离心10min。上清液转移至自动进样瓶中,进样5μl。
(3)建立标准曲线：
得出9个浓度低度的色谱图,再以五种药物的峰面积为X轴、三种药物的浓度为Y轴作线性性分析。RPZA=0.9982,RINH=0.9973,RSM=0.9963,REMB=0.9921,RRFP=0.9973Y PZA=0.000377x+0.00577Y INH=0.000654x+0.00384Y SM=8.7e-005x-0.000435Y EMB=0.104x+0.0596YRFP=0.00137x+0.00199
(4)样品处理：
①取样品血浆100μl,加内标液50μl,加提取液300μl(流动相/甲醇=65：35),涡旋15min后离心10min取上清液进样。
②骨组织样品则加入200μl骨组织提取液,加内标液50μl,加提取液300μl(流动相／甲醇=65：35),涡旋15min后离心10min取上清液进样。
③得5种抗结核药物和2中内标药物质谱图,带入标准曲线求出药物浓度。
3.对实验动物进行造模干预,模拟人体结核抗痨治疗的过程,为脊柱结核的基础研究搭建实验动物平台。
(1)实验动物的准备
①新西兰白兔(成年2-3岁)共38只(清洁级),雌雄不限。饲养条件为：23℃左右,相对湿度：70%左右,标准兔饲料饲养2周(基本热量需求)。
②提前一个月对新西兰兔予以地塞米松注射液(Dexamethasone,DSMS)0.32mg iv QOD(按照0.16mg/公斤折算),一周后改为片剂喂饲给药0.06mg poQD(按照0.03mg/公斤折算),抑制其体液免疫和细胞免疫。
(2)菌株备制过程
①将牛型结核分枝杆菌减毒菌株BCG在改良Sauton液体培养基中培养扩增2-3周。
②分装于离心管,离心后用10%小牛血清培养液稀释成均匀的5mg/ml混悬液,常温放置待用。使用时再次稀释并镜下调整浓度为1×105/ml。
(3)菌株接种过程。
①麻醉满意后常规碘酒消毒,酒精脱碘,铺无菌巾,术前器械高温消毒,取双侧第12肋末端连线的中点向下至双侧髂嵴连线的中点作约4cm纵向切口的后正中入路,显露腰4、5椎体棘突及棘上韧带。
②于腰5椎体左侧近椎间盘处紧贴椎体皮质骨部由上往下剥离,暴露腰5椎体的左半部。用电动球磨钻机在腰5椎体上钻一骨孔,深度约为0.4cm、孔径约0.25cm,骨孔道止血后填入医用明胶海绵,在明胶海绵上用注射器缓慢注射BCG菌株悬液0.1m1。
③1一0号丝线间断关闭切口,关闭深筋膜后皮下涂洒注射用青霉素粉剂(30万U/只),缝合皮肤后再次酒精消毒,无菌纱布覆盖切口上,并固定好。
④将动物松绑后送入3P级饲养房内饲养。
结果：
1.制作一种能够在脊柱结核病灶术区内持续长时间释放抗结核药物的缓释载体,保障术区保持长期的有效杀菌浓度。
①聚乳酸异烟肼微球孔隙率为63.68%,羟基磷灰石异烟肼微球孔隙率为82.18%。
②聚乳酸异烟肼微球载药率为26.62%,包封率为28.35%。羟基磷灰石异烟肼微球载药率为10.45%,包封率39.87%。
③两种缓释微球均能在第52天释放出有效杀菌浓度的异烟肼。
2.寻找出一种一次性进样检测全部一线五种抗结核药物浓度的检测方法,并确保该方法检测结果符合生物样本浓度检测的要求。
①本题所采用的质谱条件下可以一次性进样品检测出全部五种一线抗结核药物浓度,检测时间控制在3分钟以内,效率比单纯的液相色谱技术进行检测大大提高。
②INH,RFP, PZA, SM和EMB回收率：分别为98.1%,97.6%,98.3%,98.1%和97.9%。日间及夜间变异率：在5.6%-12.3%。INH,RFP,PZA,SM,和EMB。精密度：分别为0.212%,0.119%,0.103%,0.351%和0.209%。
3.对实验动物进行造模干预后模拟人体抗结核治疗的过程,检测其病灶内的药物浓度并与人体抗痨治疗后药物浓度作比较,将异烟肼缓释微球放入实验动物病灶中进行浓度检测。
①采用牛型结核分枝杆菌减毒菌株BCG造模的实验动物,病灶组织学改变同H37Rv菌株存在很大差异,但形态学改变上有一定的相似性。
②实验动物病灶内的抗结核药物浓度与人体脊柱结核病灶内药物浓度有一定差异。实验动物服药后抗结核药物原药的浓度在髂骨和病灶中较为相似,在血液中存在少许差异。(2小时FRFP血=8.544,PRFP血=0.015,3小时FRFP血=6.182PRFP血0.040)而其代谢产物的差异则更为明显。
③在实验动物体内埋置异烟肼缓释微球,能有效的长期保持局部抗结核药物的高浓度水平。
结论：
1.聚乳酸和羟基磷灰石异烟肼微球能在术区内缓慢释放,长期保持局部术区的抗结核药物浓度。
2.液相-质谱联用技术可一次性进样检测五种一线抗结核药物浓度,该检测方法稳定性好,可重复性强,灵敏度高。
3.采用BCG作为实验菌株接种实验动物可在一定程度上模拟人体脊柱结核的病理过程,并作为一种实验动物平台,为后续研究打下基础。
Background
According to the World Health Organization, tuberculosis (TB)-a contagious disease caused by Mycobacterium tuberculosis, is a chronic infectious disease with a serious hazard to human health [2],there were about1/3people in the worlds had been infected with this Mycobacterium tuberculosis [3]. At present the whole world TB patients are more than20million with an annual increase of nearly8million, WHO announced that TB was a global emergency in1993. According to some reports that between3%and5%of HIV-negative patients have musculoskeletal involvements after pulmonary infection, compared with that in60%of HIV-positive patients[4] Thus, this disease warrants considerable attention. The prevalence rate of pulmonary tuberculosis was717/100thousand based on the result of the first Chinese National Tuberculosis Epidemiological Survey in1979. There was approximately7million of patients with active pulmonary tuberculosis, experts estimate that bone and joint tuberculosis accounted from3%to5%, so bone and joint tuberculosis patients account to210-350thousand [6]. Based on the result of the fourth Chinese National Tuberculosis Epidemiological Survey in2000the prevalence rate of pulmonary tuberculosis was367/100thousand. The number of patients with active pulmonary tuberculosis was estimated at5000thousand. If the prevalence rate of bone and joint tuberculosis is3%-5%, there should be150-250thousand patients with this kind of disease.(TB) focus can be resected directly by surgery, but TB bacteria are unresectable. The internal fixation could be the metal bed for the adherence of TB in the postoperative residues focus. Along with bones special composition, half-life of anti-TB drug is shorter and the organization permeability of it is poor. It is difficult for anti-TB drugs to be efficively transported to the foci by the general route, which could be an important cause of the recurrence of the spinal TB [15][16]. At the end of surgery for spinal tuberculosis anti-TB drugs often were placed in the surgery sites, but local drug will quickly disappear because of the blood circulation and metabolism. Polylactic acid (PLA) has been widely used in experimental study on drug release system, among them D,L-polylactic-acid (PDLLA) is approved by FDA to be used as a medical material with microcapsule, microsphere and implants and sustained-release preparation and injection to prevent adhesion film material. The composition and structure of Hydroxyapatite (HA) is similar to inorganic composition in human hard tissue (bone, teeth). HA has excellent biological compatibility and biological activity and can combine physiologily with the original bone. Therefore this study aimed to design of two kinds of low-release agent with good compatibility for transporting anti-tuberculosis. By embedding these anti-tuberculosis drugs controlled-release agents in the surgery sites, high concentration of anti-tuberculosis drug above effective bactericidal concentration was keep in the long-term and the concentration of drugs from releasing in animal vertebrae was monitored in vitro, in order to improve the curative effect of spinal TB, reduce the postoperative recurrence of them and provide a theoretical basis and treatment measures for bone tuberculosis. This study was based on the embedding anti-tuberculosis drugs controlled-release agent in the surgery sites of animal and administering five anti-tuberculosis drugs continuously in peripheral blood, then the concentration of drugs were determinated by HPLC-MS, to map the concentration and distribution of five first line anti-tuberculosis drugs-Isoniazid (INH), Rifampicin (RFP), Pyrazinamidea (PZA), Ethambutol (EMB) and Streptomycin (SM). The concentration of anti-tuberculosis drugs in experimental animal vertebrae was exploered after regular chemotherapy and embedded with slow-release agent in order to construct theory basis on further enhancing concentration of anti-tuberculosis drugs in surgery sites of spinal tuberculosis patients
Objective:
1. To make a sustained release carrier of anti-tuberculosis drugs in the focus of spinal tuberculosis, to maintain effective bactericidal concentration in surgery site in the long-term.
2. To find a method for determinating the concentration of all first-line anti-tuberculosis drugs in one-time sample, and to ensure that the results of this method accord with the biological sample concentration detection requirements.
3. To simulate human intervention on treatment of tuberculosis with animal modeled spinal tuberculosis. The concentration of anti-tuberculosis drugs in focus of animals was compared with patients. Finally isoniazid control-released agents were inoculated on the focus and the concentration of anti-tuberculosis drugs was determinate.
Methods:
1. To make a sustained release carrier of anti-tuberculosis drugs in the focus of spinal tuberculosis, to maintain long-term effective bactericidal concentration in surgery site.
(1) Isoniazid polylactic acid microspheres were prepared by double emulsion/solvent evaporation method.
①Polyving akohol was weighted for4g and1g respectively and100ml of ultrapure water was added, then was heated and agitated fully for100℃for4%and1%PVC water respectively.
②Isoniazid was weighted for20mg and dissolved in4%PVC water1ml. PLGA was weighted for25mg and dissolved in2ml.dichlormethane
③4%PVC water with isoniazid was added in2ml dichlormethane and stirred by magnetic force with ice water in1min to obtain components (water/oil).
④10ml1%PVC water was added and stirred by magnetic force in10min to obtain components (water/oil/water), which was added in1%PVC water20ml and stirred12-18h.
⑤The liduid was centrifugated with high speed at4000turn/min in10min and the white flotation particles were obtained, then was flushed with BPS water then was centrifugated again. Then the BPS water after centrifugalization was collected for ultraviolet determination. The top flotation particles were collected again for flushing and centrifugating in three times. Then the white particles was collected and stored with paper bundle.
(2) Isoniazid polylactic acid microspheres were prepared by pressure infiltration technology.
①The components with Li2CO3, CaCO3, H3BO3=10:10:80were stirred fully.
②The above components were placed in silicon carbide furnace and heated with1100℃in15min until to be melted completely then rapid quenched with ice water.
③After the components were quenched the parcels in100-140mesh, then were collected and nodulized in tubular furnace two times.
④The parcels after nodulizing were placed in K2HPO4solution (0.25mol/L, pH=9) and placed in constant temperature oven at37℃for one week.
⑤The parcels were flushed with deionized water and constant temperature dried at90℃, in24h. Then were placed in muffle furnace to heat at800℃in2h and obtained with Porous hydroxyapatite microspheres.
⑥Standard isoniazid was weighted for23.4mg and putted into ultrapure water1ml, then was treated with ultrasound10min and heated to90℃,for dissolving drug Fully.
⑦The hydroxyapatite was weighted for0.1g and putted into beaker, then was placed into vacuum drying oven to keep the vacuum (<0.058MPa) in12h in order to remove gas in pores for increasing drug loading of microspheres.
⑧The liquid was centrifugated with high speed at4000turn/min in10min and the supernatant was collected to test ultraviolet. The bottom sediment particles were collected again for flushing and centrifugating in three times. Then the white particles were collected and stored with paper bundle.
2. To find a method to determinate the concentration of all first-line anti-tuberculosis drugs in one-time sample, and to ensure that the results of this method accord with the biological sample concentration detection requirements.
(1) To determine the detection HPLC-MS conditions
①chromatographic column was Hydrophilic high performance liquid chromatography (HILIC)150mm×2.1mmI.D.3μm(Thermo, USA). An online RRLC filter was connected with pre-column (Agilent, Germany), the column temperature was room temperature.
②Mobile phase was5mM ammonium acetate with methanol and0.1%formic acid65:35, v/v, Flow rate was0.5mL/min. Sample size was5μ L
③Mass spectrometric method was positive ion electrospray ionization (ESI+) Using multiple reaction monitoring (MRM) mode for quantitative. ESI+condition is at spray voltage5000V, ion source temperature is at700℃, Air curtain gas is15psi, collisional activation parameter was M, atomization gas is70psi, and auxiliary gas is65PSI.
(2) Sample extraction process
①Sample extraction solution was methanol solution containing5mM ammonium acetate,0.1%formic acid and10%of water.
②100μl standard curves of series of samples or quality control samples were added with50μl internal standard working solution and300μ1sample extraction solvent. The measured plasma samples were100μL, plus50μL internal standard working solution and300μl sample extraction solvent.
③Samples were fully stirred after2min,13000RPM centrifugated for10min. Supernatant was transferred to an automatic sample bottle, sample was5μL.
(3) To establish the standard curve:
The chromatogram with9concentration was obtained, the following regression equation of five anti-TB drugs with peak area (Y) and concentration level (X) was defined: RPZA=0.9982, RINH=0.9973, RSM=0.9963, REMB=0.9921, RRFP=0.9973YPZA=0.000377x+0.00577YINH=0.000654x+0.00384YINHm=8.7e-005x-0.000435YEMB=0.104x+0.0596YRFP=0.00137x+0.00199(4) Sample treatment
①The samples of plasma of100μL, L,50μL and internal standard solution, and extract300μL (mobile phase/methanol=65:35) were centrifuged at10min after stirred15min, then the supernatant was obtained for sample.
②The samples of bone of200μL bone tissue extract and internal standard solution of50μL, and extract300μL (mobile phase/methanol=65:35) were centrifuged10min after stirred15min, finally the supernatant was obtained for sample.
③Five kinds of anti-TB drugs and2internal standard drug mass spectrum were obtained and taken into the standard curve of drug to count concentration.
3. To simulate human intervention on treatment of tuberculosis after modeled with spinal tuberculosis. The concentration of anti-tuberculosis drugs in focus of animals was compared with patients. Finally isoniazid control-released agents were inoculated on the focus and the concentration of anti-tuberculosis drugs was determinate.
(1) Experimental animal preparation
①A total of38New Zealand white rabbits (adult2-3years)(clean grade), male or female are permitted. Feeding condition is at23℃, relative humidity is70%, standard rabbit diet for2weeks (the basic heat demand).
②One month in advance dexamethasone (DSMS) was used for injecting in rabbits0.32mg IV QOD (0.16mg/kg), after one week tablet was used for feeding administration of0.06mg PO QD (0.03mg/kg), which was used to inhibit the humoral immunity and cellular immunity.
(2) The preparation process of strain.
①The type of Mycobacterium tuberculosis BCG strains in modified Sauton liquid cultured2-3weeks.
②Packed in centrifuge tubes, centrifuged for20minutes,1200R/min, in saline to make uniform5mg/ml suspension at room temperature, placed for ready in ordinary temperature.
(3) Strain inoculation process.
①After the rabbit was anesthetized satisfactorily, routine iodine disinfection, alcohol was used to remove iodine, sterile towel was placed, preoperative instrument was sterilized. The4cm longitudinal incision was made from the midpoint of the of bilateral twelfth rib line to the midpoint of bilateral iliac crest line by posterior median approach.
②L5body was stripped down from proximal lumbar disc close to the vertebral cortical bone and the left part of vertebral body was exposed. L5vertebra was drilled a hole by the electric ball milling machine, The depth was about0.4cm, pore size was about0.25cm, bone tunnel was stopped bleeding with medical gelatin sponge, which affiliated Mycobacterium BCG suspension liquid with a syringe injection0.1ml(1×105CFU/ml)
③The wound was closed discontinuously with No.1-0silk line, after the deep fascia closed penicillin powder was used at the subcutaneous tissue of wound.
④The animal was relieved and taken into3P feeding room to rearing.
Results:
1. To make a sustained release carrier of anti-tuberculosis drugs in the focus of spinal tuberculosis, to maintain effective bactericidal concentration in surgery site in the long-term.
⑤The porosity of Polylactic acid-isoniazid microspheres is63.68%, the porosity of Hydroxyapatite-isoniazid microspheres is82.18%
⑥The loading rate and encapsulation of Polylactic acid-isoniazid microspheres was26.62%and28.35%respectively. The loading rate and encapsulation of Hydroxyapatite-isoniazid microspheres was10.45%and39.87%respectively.
③Two kinds of microspheres can all release enough isoniazid above effective bactericidal concentration of it in fifty-second days in vitro.
2. To find a method to determinate the concentration of all first-line anti-tuberculosis drugs in one-time sample, and to ensure that the results of this method accord with the biological sample concentration detection requirements.
①With the mass spectrometric conditions in this study all the samples can be detected to the concentration of all five first-line anti-TB drugs in one-time. The detection time control in3minutes and the detection efficiency is higher than that of pure liquid chromatography technique.
②Recovery rate of INH, RFP, PZA, SM and EMB is98.1%,97.6%,98.3%,98.1%and97.9%respectively. Daytime and nighttime variation rate of INH, RFP, PZA, SM and EMB is from5.6%to12.3%, Precision rate of INH, RFP, PZA, SM and EMB is0.212%,0.119%,0.103%,0.351%and0.209%respectively.
3. To simulate human intervention on treatment of tuberculosis after modeled with spinal tuberculosis. The concentration of anti-tuberculosis drugs in focus of animals was compared with patients. Finally isoniazid control-released agents were inoculated on the focus and the concentration of anti-tuberculosis drugs was determinate.
①There are great differences in histological changes of focus in animal model inoculated with BCG and H37RV, But the morphological changes have some similarity.
②There are some differences of anti-TB drugs concentration between experimental animal and human spinal TB, the concentration of anti-TB drugs in ilium and lesions are similar between experimental animal and patients with spinal TB and there is little difference in the blood after oral administration (F=8.544, P=0.015for RFP in serum2h, F=6.182, P=0.040for RFP in serum3h). Furthermore the difference of metabolite in focus of animal models and patients with spinal TB is more obvious.
③In the experimental animal implanted with isoniazid controlled-release microspheres can effective maintain high concentration levels of antit-TB drugs in long-term locally.
Conclusion:
1. Polylactic acid-INH and hydroxyapatite-INH controlled-release microspheres can slowly release isoniazid in surgery site to keep the high concentration of anti-tuberculosis drugs locally in long-term.
2. The concentration of all first-line anti-tuberculosis drugs, could be determinate in one-time sample with HPLC-MS technique. The detection method has good stability, high repeatability and high sensitivity.
3. The pathological process of experimental animal inoculated with BCG can roughly simulate human spinal tuberculosis, which is an experimental animal platform to base the foundation for further study.
引文
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