氨磷汀（WR-2721）贴敷对豚鼠急性放射性颊黏膜炎保护作用的初步研究

发布时间：2018-05-26 10:09

本文选题：氨磷汀(WR-2721) + WR-1065-局部给药　；参考：《复旦大学》2013年硕士论文

【摘要】：研究背景与目的研究背景：氨磷汀全身给药对放射损伤具有显著保护作用已经得到了证明,并已应用至临床。但随着临床的多年应用,发现氨磷汀全身用药给患者带来了诸多不利,如需要专门技术人员进行注射,且低血压发生率高、严重以及还可带来其他临床上难以防治的全身不良反应,因而阻碍了它在临床上的广泛应用。近来,有许多研究报道氨磷汀直肠内给药对减轻放射性直肠损伤有明显作用,并有相应的临床研究予以支持。而对于头颈部恶性肿瘤患者来说,放射治疗是非常重要的治疗方法,但不可避免地会带来放射性损伤,因而如果氨磷汀局部给药是可行且有效的,那么将它用于临床防治头颈部癌症患者因放疗所致的放射性损伤,无疑是给患者和医生带来了新的治疗手段。另外,由于目前文献中报道的氨磷汀及其活性代谢产物WR-1065的检测方法存在许多缺陷,如操作复杂、难以同时直接检测此两种物质、灵敏度低和耗时较久等,因而本实验也旨在建立更为快速、方便、灵敏度更高的检测方法,为今后的科学研究和临床药物监测提供新的方法。目的：1.建立高效液相色谱质谱法(HPLC-MS/MS)检测氨磷汀浓度的检测方法；2.初步探索氨磷汀贴敷于豚鼠颊粘膜这一局部给药方法的可行性；3.初步研究氨磷汀贴敷于豚鼠颊粘膜后对其急性放射性颊黏膜炎保护作用的有效性。方法 1.检测方法的建立：收集6位健康成年志愿者唾液,并以其为基质,石杉碱甲(Huperzine-A, Hup A))为内标,采用蛋白沉淀法,用HPLC-MS/MS测定唾液中的氨磷汀。色谱柱为ZIC-HILIC亲水色谱分析柱((100×2.1mm,3.5μm)),质谱分析采用多反应监测扫描模式(MRM),离子源为电喷雾离子源(ESI)。 2.可行性的初步研究：收集5位自愿者唾液作为标本,并与氨磷汀配制成浓度为4.5×10-3g/1,9×10-3g/1and18×10-3g/1氨磷汀溶液,静置5、、20、40、60、80、100分钟后检测；选择24只健康成年豚鼠,并将其分成A、B、C三组,A、B组各有9只豚鼠,再分为三个亚组,每个亚组各3只,A、B组豚鼠分别于其每侧颊粘膜贴敷含50mg、100mg的棉片,贴敷时间为30分钟；C组6只豚鼠,为空白对照组；A、B组豚鼠在给药后0分钟、15分钟和30分钟后,应用HPLC-MS/MS检测其血清及颊黏膜组织中的氨磷汀及其活性代谢产物WR-1065的浓度。统计分析采用SPSS17.0软件包。 3.有效性的初步研究：选择32只健康成年豚鼠随机分成4组(A、B、C、D),每组8只豚鼠,A、B组豚鼠分别给予氨磷汀50mg和100mg；C组为生理盐水组,A、B、C三组豚鼠给药后均予以单次照射X线30Gy,照射后8天进行肉眼Parkins及Sonis评分,评分后立即分离取出双侧颊粘膜,行HE染色光镜下观察研究；D组为空白对照组。统计分析采用SPSS17.0软件包。结果 1.氨磷汀在唾液中检测到的线性范围为0.938～30mg·L-1,线性关系良好,典型代表方程为：y=0.072x+-0.00526(r=0.9991)(n=6),定量下限(Lowest Limit Of Quantification, LLOQ)浓度为0.938mg·L-1,(S/N10)。唾液样品的低、中、高三个质控浓度(1.0、5.0和25mg·L-1)批内、批间精密度(RSD)均小于15%,其方法回收率均大于85%。 2.氨磷汀在人体外唾液中较为稳定,在检测时间内均能保持较高浓度,且各时间点无明显差异(P0.05),且并未检测到其分解产物WR-1065；在给药后15分钟、30分钟与0分钟相比,豚鼠颊黏膜组织的氨磷汀及WR-1065浓度均显著升高(P0.05),而给药后15分钟与30分钟相比则无明显差异(P0.05)。 3.B组豚鼠平均得分为2.4分,A组豚鼠平均得分为2.9分,C组豚鼠平均得分为4.4分,D组豚鼠颊粘膜未观察到急性放射性损伤,为正常黏膜,平均得分为0分。A、B两组豚鼠评分经SPSS17.0(SNK法)统计分析比较无统计学差异(P0.05)；与D组豚鼠相比,A、B、C三组豚鼠急性放射性颊黏膜损伤明显(P0.05)；A、B组豚鼠与C组相比,则放射性损伤明显减轻(P0.05)；光镜下观察A、B、C三组豚鼠,均可见炎症细胞浸润(主要是淋巴细胞),其中C组可见大量炎症细胞浸润、血管扩张甚至上皮剥脱、溃疡、坏死、上皮增生等病理变化,A组豚鼠可见部分炎症细胞浸润、毛细血管扩张、上皮结构破坏、脱落、增生等变化,而B组豚鼠较A组豚鼠炎症轻,细胞浸润少以及上皮破坏、增生也较轻。结论 1.本研究所建立的氨磷汀检测方法直接、快捷、简便、灵敏度高及定性好,适用于唾液中氨磷汀的测定及科学研究。因而可用HPLC-MS/MS检测生物标本中氨磷汀及其活性代谢产物WR-1065的浓度。 2.氨磷汀局部应用于口腔是可行的,且其全身吸收极少,甚或可以忽略；在研究时间点内(5分钟-100分钟),尽管与最初浓度相比有下降,但氨磷汀在人唾液中仍能长时间保持较高浓度； 3.局部应用一定剂量氨磷汀于口腔能显著减轻急性放射性口腔颊黏膜损伤,且增加一定剂量,尽管不明显,但组织放射保护作用仍有增强。
[Abstract]:Research background and purpose
Background: the significant protective effect of the systemic administration of ammoniacine on radiation damage has been proved and has been applied to the clinic. However, with many years of clinical application, it has been found that the systemic use of ammoniacine has brought many disadvantages to patients, such as the need for special technical personnel to be injected, and the incidence of hypotension is high, serious and can also be brought. Other clinically uncontrollable systemic adverse reactions impede its widespread clinical application. Recently, many studies have reported a significant effect on alleviating radionuclide injury by transrectal administration of ammoniacine, with corresponding clinical studies to support it. For patients with head and neck malignancies, radiation therapy is very important. An important treatment, but inevitably brings radioactive damage, so if the local administration of ammoniacine is feasible and effective, it is no doubt that it is a new treatment for patients and doctors because of radiation injury caused by radiotherapy in the head and neck cancer patients. The detection methods of ammoniacine and its active metabolite WR-1065 have many defects, such as complex operation, difficult to direct detection of these two substances at the same time, low sensitivity and time consuming. Therefore, this experiment also aims to establish a more rapid, convenient and more sensitive detection method, which provides new scientific research and clinical drug monitoring for future. Method.
Objective: 1. to establish a high performance liquid chromatography mass spectrometry (HPLC-MS/MS) method for detecting the concentration of ammoniacine, and 2. to explore the feasibility of applying the local administration of ammoniacine to the buccal mucosa of guinea pigs, and 3. to study the effectiveness of the application of ammoniacine to the buccal mucosa of guinea pig to protect the acute radiological buccal mucositis.
Method
1. the establishment of the detection method: the saliva of 6 healthy adult volunteers was collected, and the Huperzine-A (Hup A) was used as the internal standard. The protein precipitation method was used to determine the ammoniacine in saliva by HPLC-MS/MS. The chromatographic column was ZIC-HILIC hydrophilic chromatographic column (100 * 2.1mm, 3.5 m), and the mass spectrometry analysis adopted the multi reaction monitoring scanning mode. MRM), the ion source is an electrospray ion source (ESI).
2. preliminary study of feasibility: collect 5 volunteers saliva as specimens, and make up a concentration of 4.5 x 10-3g/1,9 x 10-3g/1and18 x 10-3g/1 ammoniacine solution with ammoniacine, static 5, 20,40,60,80100 minutes after detection; select 24 healthy adult guinea pigs, and divide them into A, B, C three, A, and B group 9 Guinea pigs each, then divided into three subgroups, In each subgroup, 3 A and B guinea pigs were attached to each cheek mucosa with 50mg, 100mg and 30 minutes, and 6 guinea pigs in group C were blank control group. A, B group guinea pigs were used to detect the serum and the active metabolites WR-106 in the serum and buccal mucosa after 0, 15 and 30 minutes after the administration. The concentration of 5. The SPSS17.0 software package is used for statistical analysis.
3. preliminary study of effectiveness: 32 healthy adult guinea pigs were randomly divided into 4 groups (A, B, C, D), each group of 8 guinea pigs, A, B group were given amino acid 50mg and 100mg, and C group was the saline group, A, B, and C three guinea pigs were irradiated by single irradiation after the 8 days after the irradiation. The bilateral buccal mucosa was observed by HE staining under light microscope. D group was blank control group. SPSS17.0 software package was used for statistical analysis.
Result
1. the linear range of 1. ammoniacine in saliva is 0.938 ~ 30mg. L-1. The linear relationship is good. The typical representative equation is y=0.072x+-0.00526 (r=0.9991) (n=6). The quantitative lower limit (Lowest Limit Of Quantification, LLOQ) is 0.938mg L-1. The sperm density (RSD) is less than 15%, and the recovery rate is greater than 85%..
2. alpstine was stable in human saliva and kept high concentration in the detection time, and there was no significant difference at every time point (P0.05), and the decomposition product WR-1065 was not detected. The concentration of alptin and WR-1065 in the buccal mucosa of guinea pigs increased significantly (P0.05) after 15 minutes after administration and 30 minutes after the Administration (P0.05). There was no significant difference between 15 minutes and 30 minutes (P0.05).
The average score of guinea pigs in group 3.B was 2.4 points, the average score of guinea pigs in group A was 2.9, and the average score of guinea pigs in group C was 4.4. No acute radiation injury was observed in the buccal mucosa of group D. The average score was 0.A, and B two group of guinea pig scores were not statistically different (P0.05) by SPSS17.0 (SNK method). Compared with D group, A and B, B, B, B, B, B, B, B, B, B The acute radiation-induced buccal mucosa damage in the three groups of guinea pigs was obvious (P0.05); A, group B and C were significantly less radioactive (P0.05). The infiltration of inflammatory cells (mainly lymphocytes) was observed in groups of A, B, C in group C, and a large number of inflammatory cell infiltration, vascular dilatation and even epithelial exfoliation, ulcers and necrosis were found in group C. Pi Zengsheng and other pathological changes, A group of guinea pigs can see some inflammatory cells infiltration, capillary dilatation, epithelial structure destruction, shedding, hyperplasia and other changes, while group B guinea pigs are lighter than the A group of guinea pigs, small cell infiltration and epithelial destruction, hyperplasia is also lighter.
conclusion
1. the determination of ammoniacine in this study is direct, quick, simple, high sensitivity and good qualitative. It is suitable for the determination and scientific research of ammoniacine in saliva. Therefore, HPLC-MS/MS can be used to detect the concentration of ammoniacine and its active metabolite WR-1065 in biological specimens.
2. the local use of ammoniacine is feasible in the oral cavity, and the absorption of the whole body is very small or negligible; in the study time point (5 minutes -100 minutes), although it has decreased compared with the initial concentration, it still maintains a high concentration in human saliva for a long time.
3. a certain dose of a certain dose of ammoniacine can significantly reduce the injury of acute radiation-induced oral buccal mucosa, and increase a certain dose, although it is not obvious, but the radioprotective effect of tissue is still enhanced.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R818

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