本文選題：氨磷汀(WR-2721) + WR-1065-局部給藥��； 參考：《復(fù)旦大學(xué)》2013年碩士論文

【摘要】：研究背景與目的 研究背景：氨磷汀全身給藥對(duì)放射損傷具有顯著保護(hù)作用已經(jīng)得到了證明,并已應(yīng)用至臨床。但隨著臨床的多年應(yīng)用,發(fā)現(xiàn)氨磷汀全身用藥給患者帶來(lái)了諸多不利,如需要專門技術(shù)人員進(jìn)行注射,且低血壓發(fā)生率高、嚴(yán)重以及還可帶來(lái)其他臨床上難以防治的全身不良反應(yīng),因而阻礙了它在臨床上的廣泛應(yīng)用。近來(lái),有許多研究報(bào)道氨磷汀直腸內(nèi)給藥對(duì)減輕放射性直腸損傷有明顯作用,并有相應(yīng)的臨床研究予以支持。而對(duì)于頭頸部惡性腫瘤患者來(lái)說(shuō),放射治療是非常重要的治療方法,但不可避免地會(huì)帶來(lái)放射性損傷,因而如果氨磷汀局部給藥是可行且有效的,那么將它用于臨床防治頭頸部癌癥患者因放療所致的放射性損傷,無(wú)疑是給患者和醫(yī)生帶來(lái)了新的治療手段。另外,由于目前文獻(xiàn)中報(bào)道的氨磷汀及其活性代謝產(chǎn)物WR-1065的檢測(cè)方法存在許多缺陷,如操作復(fù)雜、難以同時(shí)直接檢測(cè)此兩種物質(zhì)、靈敏度低和耗時(shí)較久等,因而本實(shí)驗(yàn)也旨在建立更為快速、方便、靈敏度更高的檢測(cè)方法,為今后的科學(xué)研究和臨床藥物監(jiān)測(cè)提供新的方法。 目的：1.建立高效液相色譜質(zhì)譜法(HPLC-MS/MS)檢測(cè)氨磷汀濃度的檢測(cè)方法；2.初步探索氨磷汀貼敷于豚鼠頰粘膜這一局部給藥方法的可行性；3.初步研究氨磷汀貼敷于豚鼠頰粘膜后對(duì)其急性放射性頰黏膜炎保護(hù)作用的有效性。 方法 1.檢測(cè)方法的建立：收集6位健康成年志愿者唾液,并以其為基質(zhì),石杉?jí)A甲(Huperzine-A, Hup A))為內(nèi)標(biāo),采用蛋白沉淀法,用HPLC-MS/MS測(cè)定唾液中的氨磷汀。色譜柱為ZIC-HILIC親水色譜分析柱((100×2.1mm,3.5μm)),質(zhì)譜分析采用多反應(yīng)監(jiān)測(cè)掃描模式(MRM),離子源為電噴霧離子源(ESI)。 2.可行性的初步研究：收集5位自愿者唾液作為標(biāo)本,并與氨磷汀配制成濃度為4.5×10-3g/1,9×10-3g/1and18×10-3g/1氨磷汀溶液,靜置5、、20、40、60、80、100分鐘后檢測(cè)；選擇24只健康成年豚鼠,并將其分成A、B、C三組,A、B組各有9只豚鼠,再分為三個(gè)亞組,每個(gè)亞組各3只,A、B組豚鼠分別于其每側(cè)頰粘膜貼敷含50mg、100mg的棉片,貼敷時(shí)間為30分鐘；C組6只豚鼠,為空白對(duì)照組；A、B組豚鼠在給藥后0分鐘、15分鐘和30分鐘后,應(yīng)用HPLC-MS/MS檢測(cè)其血清及頰黏膜組織中的氨磷汀及其活性代謝產(chǎn)物WR-1065的濃度。統(tǒng)計(jì)分析采用SPSS17.0軟件包。 3.有效性的初步研究：選擇32只健康成年豚鼠隨機(jī)分成4組(A、B、C、D),每組8只豚鼠,A、B組豚鼠分別給予氨磷汀50mg和100mg；C組為生理鹽水組,A、B、C三組豚鼠給藥后均予以單次照射X線30Gy,照射后8天進(jìn)行肉眼Parkins及Sonis評(píng)分,評(píng)分后立即分離取出雙側(cè)頰粘膜,行HE染色光鏡下觀察研究；D組為空白對(duì)照組。統(tǒng)計(jì)分析采用SPSS17.0軟件包。 結(jié)果 1.氨磷汀在唾液中檢測(cè)到的線性范圍為0.938～30mg·L-1,線性關(guān)系良好,典型代表方程為：y=0.072x+-0.00526(r=0.9991)(n=6),定量下限(Lowest Limit Of Quantification, LLOQ)濃度為0.938mg·L-1,(S/N10)。唾液樣品的低、中、高三個(gè)質(zhì)控濃度(1.0、5.0和25mg·L-1)批內(nèi)、批間精密度(RSD)均小于15%,其方法回收率均大于85%。 2.氨磷汀在人體外唾液中較為穩(wěn)定,在檢測(cè)時(shí)間內(nèi)均能保持較高濃度,且各時(shí)間點(diǎn)無(wú)明顯差異(P0.05),且并未檢測(cè)到其分解產(chǎn)物WR-1065；在給藥后15分鐘、30分鐘與0分鐘相比,豚鼠頰黏膜組織的氨磷汀及WR-1065濃度均顯著升高(P0.05),而給藥后15分鐘與30分鐘相比則無(wú)明顯差異(P0.05)。 3.B組豚鼠平均得分為2.4分,A組豚鼠平均得分為2.9分,C組豚鼠平均得分為4.4分,D組豚鼠頰粘膜未觀察到急性放射性損傷,為正常黏膜,平均得分為0分。A、B兩組豚鼠評(píng)分經(jīng)SPSS17.0(SNK法)統(tǒng)計(jì)分析比較無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)；與D組豚鼠相比,A、B、C三組豚鼠急性放射性頰黏膜損傷明顯(P0.05)；A、B組豚鼠與C組相比,則放射性損傷明顯減輕(P0.05)；光鏡下觀察A、B、C三組豚鼠,均可見(jiàn)炎癥細(xì)胞浸潤(rùn)(主要是淋巴細(xì)胞),其中C組可見(jiàn)大量炎癥細(xì)胞浸潤(rùn)、血管擴(kuò)張甚至上皮剝脫、潰瘍、壞死、上皮增生等病理變化,A組豚鼠可見(jiàn)部分炎癥細(xì)胞浸潤(rùn)、毛細(xì)血管擴(kuò)張、上皮結(jié)構(gòu)破壞、脫落、增生等變化,而B(niǎo)組豚鼠較A組豚鼠炎癥輕,細(xì)胞浸潤(rùn)少以及上皮破壞、增生也較輕。 結(jié)論 1.本研究所建立的氨磷汀檢測(cè)方法直接、快捷、簡(jiǎn)便、靈敏度高及定性好,適用于唾液中氨磷汀的測(cè)定及科學(xué)研究。因而可用HPLC-MS/MS檢測(cè)生物標(biāo)本中氨磷汀及其活性代謝產(chǎn)物WR-1065的濃度。 2.氨磷汀局部應(yīng)用于口腔是可行的,且其全身吸收極少,甚或可以忽略；在研究時(shí)間點(diǎn)內(nèi)(5分鐘-100分鐘),盡管與最初濃度相比有下降,但氨磷汀在人唾液中仍能長(zhǎng)時(shí)間保持較高濃度； 3.局部應(yīng)用一定劑量氨磷汀于口腔能顯著減輕急性放射性口腔頰黏膜損傷,且增加一定劑量,盡管不明顯,但組織放射保護(hù)作用仍有增強(qiáng)。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R818

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