| BackgroundAccording 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 patientsObjective: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.dichlormethane③4%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 conditions①chromatographic 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 process①Sample 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 treatment①The 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 preparation①A 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. |
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