氨苯砜斑贴试剂的研发与初步应用

发布时间：2018-05-26 16:56

本文选题：氨苯砜综合征 + 药物超敏反应　；参考：《青岛大学》2017年博士论文

【摘要】：研究背景:氨苯砜(dapsone,DDS)具有抗菌、抗炎双重效应,是治疗麻风病的基本药物并被广泛应用于疟疾、HIV感染所致的卡氏肺囊虫肺炎及多种慢性炎症性疾病的治疗。氨苯砜化学名称为4,4’-对位氨基双苯砜,其分子量为248.31。氨苯砜口服吸收后在肝脏进行代谢,经细胞色素P450酶的作用,发生羟基化和乙酰化两个过程,代谢产物为羟胺氨苯砜和乙酰氨苯砜。羟胺氨苯砜在药物的药理作用及不良反应的产生方面起关键作用。其代谢产物在皮肤、心脏、肝脏和肾脏等全身多器官广泛分布。氨苯砜经口服后通过小肠吸收,口服2-8小时后达到血浆峰浓度。单次剂量50-300mg氨苯砜可到达血药浓度为0.63-4.82mg/L。在100mg/d的常规用量下,血浆峰浓度可达3.26 mg/L。约有20%的代谢产物直接经尿液排出,70-85%的代谢产物与葡萄糖醛酸共价结合后经尿液排出,此外,氨苯砜也可经胆汁排出体外。氨苯砜最大的副作用在于部分患者服药后会发生一种严重的药物超敏反应,临床上表现为突然发生的丘疹或剥脱性皮疹,伴发热、淋巴结肿大、单核细胞增多及肝脏或造血系统功能受损,通常在服用氨苯砜4-6周后发生(故又称为“5周皮炎”),发生率为0.5%~3.6%,死亡率9.9%。该药物不良反应在1951年被正式命名为氨苯砜综合征。当前对于该综合征的诊断主要依据Richardus和Smith于1989年提出的诊断标准,即患者在服用氨苯砜后出现皮疹、发热、肝功能异常及淋巴结肿大中的2条或以上即可诊断。但上述诊断由于无有效的实验室诊断指标的支持,缺乏特异性,往往很难与疾病本身伴随的感染、炎症反应相鉴别,且对于疾病的治疗,患者往往会在同一时间段服用多种药物,这也导致致敏药物难以判定及准确停用,以致临床漏诊、误诊现象严重。氨苯砜综合征是由T淋巴细胞介导的一种药物超敏反应,在机体首次接触致敏药物后,体内T淋巴细胞被激活致敏。当机体再次接触该药物后,致敏的T淋巴细胞活化增殖,释放细胞因子IFN-γ等,吸引和激活巨噬细胞,在局部产生以T细胞和巨噬细胞浸润为主的炎症反应。斑贴试验作为测定机体变态反应的一种辅助诊断方法即是基于以上原理产生,迄今在皮肤科的应用已有120多年的历史。目前国际上诊断临床常见的28种致敏药物(卡马西平、布洛芬、阿莫西林、双氯芬酸钠等)已研发了具有独立知识产权的斑贴试剂盒,已获得美国FDA、中国c FDA以及欧盟认证,在临床上广泛应用,为药物所致不良反应的诊断及鉴别诊断提供了有效的工具,获得了良好的社会经济效益。研究目的:本研究旨在研发氨苯砜斑贴试剂,建立氨苯砜综合征免疫确证方法,为疾病的诊断提供有效技术手段,以推动该技术在临床上的应用。研究对象:1、氨苯砜综合征病例组:临床拟诊氨苯砜综合征并携带HLA-B*1301位点的麻风病患者24例。2、氨苯砜综合征对照组:服用过氨苯砜并携带HLA-B*1301位点但未出现氨苯砜综合征的麻风病患者25例。健康志愿者50例。纳入标准:病例组:1、2008.01-2016.12新发的麻风病患者且服用过氨苯砜进行治疗;2、患者服用氨苯砜治疗过程中出现发热、皮疹、肝功能异常、淋巴结肿大等疑似氨苯砜综合症,符合Richardus和Smith提出的该病诊断标准;3、携带HLA-B*1301等位基因;4、同意参与本研究并签署书面知情同意书。对照组1、2008.01-2016.12全国新发的麻风病患者且服用氨苯砜进行治疗,服药时间至少1个月;2、患者服用氨苯砜治疗过程中未发现发热、皮疹、肝功能异常、淋巴结肿大等临床症状;3、携带HLA-B*1301等位基因;4、同意参与本研究并签署书面知情同意书。排除标准:1.对磺胺类、呋塞米类、噻嗪类、磺酰脲类以及碳酸酐酶抑制药过敏的患者;2.无法提供正确信息的患者,如精神障碍等;3.并发严重疾病的患者,如心衰、恶性肿瘤等;研究内容:本研究分为三部分:研究I:研发可供临床应用的氨苯砜综合征斑贴试剂,探索稳定的试剂基质及浓度范围并明确其稳定性情况:以临床诊断和激发试验阳性的1例疑似氨苯砜综合征患者为实验对象,探索氨苯砜斑贴试剂合适的基质及浓度范围;研究II:对配置的斑贴试剂进行含量测定,明确试剂的稳定性情况;研究III:明确氨苯砜斑贴试剂临床应用的敏感度及特异度:对临床拟诊且携带HLA-B*1301位点的23例疑似氨苯砜综合征患者及25例服用氨苯砜且携带HLA-B*1301位点但未出现疑似氨苯砜综合征症状的25例麻风病患者采用建立的斑贴试剂进行检测。选择50例健康志愿者用于排除斑贴试剂本身所致的皮肤刺激反应。使用卡方检验用于确定各组间差异的统计学意义,确定研发的斑贴试剂临床应用的敏感度及特异度情况。研究方法:研究I:采用溶解法观察凡士林基质、聚乙二醇200基质、聚乙二醇200基质+二甲基亚砜、聚乙二醇基质+油酸、聚乙二醇基质+氮酮在氨苯砜中的溶解程度,并根据斑贴试验结果调整斑贴试剂的浓度;研究II:采用高效液相色谱仪对配制的凡士林基质、聚乙二醇200基质的斑贴试剂进行含量测定,同时对其初步的稳定性情况进行考察;研究III:对研究对象进行氨苯砜斑贴试验,方法如下:1.将氨苯砜斑贴试剂按0.1%、0.5%、1%、5%、10%、15%、20%、25%的浓度摆放好,把对照放好。2.将斑试器从带有三角撕口的一侧撕开,然后将氨苯砜斑贴试剂及空白对照按顺序加在斑试器上。注意记录斑贴试剂的浓度顺序。3.让患者充分暴露背部皮肤,选择无皮疹、无色素沉着的正常皮肤进行试验。4.用酒精棉球对皮肤进行充分消毒,然后将斑试器贴在患者后背皮肤上,轻压排净空气,将斑试器贴牢。5.用记号笔在斑试器的四角做好标记。并在实验记录纸上做好记录,特别注意记录斑贴试剂不同的浓度的位置。6.嘱咐患者在48小时后撕掉斑试器,试验期间不宜洗澡、不宜干农活、不宜剧烈运动、不宜饮酒等。试验期间若发生强烈反应需撕掉斑试器。7.72小时后,也就是撕掉斑试器24小时后观察实验结果。用酒精棉球擦拭清洁受试部位,再次用记号笔对斑试器的四个角进行标注,仔细观察受试部位有无红斑、丘疹、水泡出现(部分患者实验效果不明显,需仔细观察)。根据国际接触性皮炎研究组ICDRG(International Contact Dermatitis Research Group)指南判读结果。斑贴试验结果均由同一技术人员判定。统计学方法:研究结果分析在意向性分析(intention to treat,ITT)人群进行,ITT人群是指所有进入研究并至少有过一次用药措施的受试者。数据统计应用SPSS 16.0版软件,根据研究目的和资料的性质选用卡方检验,得出试验组与对照组的各项指标差异是否有统计学意义。所有统计检验均采用双侧检验,检验水准定义为α=0.05,P值小于0.05将被认为所检验的差别有统计学意义。研究结果:研究I:经过氨苯砜凡士林基质、聚乙二醇200基质、聚乙二醇200基质+二甲基亚砜、聚乙二醇基质+油酸、聚乙二醇基质+氮酮5种基质的比较,最终选择聚乙二醇200基质+二甲基亚砜做为最佳制剂。研究II:采用聚乙二醇200作为基质的斑贴,常温状态以及4℃低温放置6个月样品含量无明显下降趋势,且低温保存与常温保存对样品稳定性无差异,采用此基质可保证样品在一定时间内稳定,无需现用现配。研究III:在全国范围内对23例氨苯砜综合征患者做斑贴试验,其中10例斑贴试验阳性,13例斑贴试验阴性。服用过氨苯砜且未出现氨苯砜综合征的麻风病患者25例氨苯砜斑贴试验均阴性,健康志愿者50例氨苯砜斑贴试验均阴性。结论:本研究建立了基于斑贴实验的氨苯砜综合症免疫确证方法,为该病的精准诊断提供了有效的实验室技术手段,也将为其他药物所致药物超敏反应综合征的免疫确证方法的建立提供思路与借鉴,对于在临床实践中判定患者的致敏药物、明确疾病诊断具有重要意义。
[Abstract]:Background: dapsone (DDS) is a basic drug for the treatment of leprosy, and is widely used in the treatment of Pneumocystis carinii pneumonia and a variety of chronic inflammatory diseases caused by HIV infection. The chemical name of sulfoxide is 4,4 '- para Diphenyl Sulfoxide, its molecular weight is 248.31. amamyl sulfoxide After absorption, it is metabolized in the liver. Through the action of cytochrome P450 enzyme, there are two processes of hydroxylation and acetylation. The metabolites are hydroxyaminophen sulfoxide and acetaminophen sulfoxide. Hydroxylaminyl sulfoxide plays a key role in the pharmacological action and adverse reactions of the drug. Its metabolic products are in the skin, heart, liver and kidney. It is widely distributed. After oral administration of sulfoxide, it is absorbed through the small intestine and reaches the plasma peak concentration after 2-8 hours of oral administration. A single dose of 50-300mg sulfoxide can reach the concentration of 0.63-4.82mg/L. at 100mg/d, and the plasma peak concentration is up to 3.26 mg/L., about 20% of the metabolites are directly connected to the urine, and the metabolites of 70-85% and grapes Aldonic acid is covalently combined and excreted in urine. In addition, sulfoxide can also be discharged through bile. The biggest side effect of sulfoxide is that some patients take a serious drug hypersensitivity after taking medicine, which are clinically manifested by sudden papules or exfoliative rashes, fever, lymph node enlargement, mononuclear cells and liver or formation. The function of the blood system is impaired, usually after taking 4-6 weeks after the use of sulfoxide (which is also called "5 weeks of dermatitis") with a rate of 0.5%~3.6% and the death rate of 9.9%., which was formally named the sulfoxide syndrome in 1951. The current diagnosis of the syndrome is based on the diagnostic criteria proposed by Richardus and Smith in 1989, that is, the patient. The diagnosis of rash, fever, abnormal liver function and 2 or more of Zhong Dazhong's lymph nodes can be diagnosed after taking Diphenyl Sulfoxide, but the above diagnosis is often difficult to identify with the infection of the disease itself, the inflammatory reaction and the treatment of the disease. Taking a variety of drugs for a period of time, this also causes the allergy drugs to be difficult to determine and disable, so that the clinical misdiagnosis and misdiagnosis are serious. The sulfoxide syndrome is a drug hypersensitivity reaction mediated by T lymphocytes. After the body is first exposed to the sensitized drug, the T lymphatic cells are activated and sensitized. When the body is again exposed to the drug, The sensitized T lymphocytes activate and proliferate, release cytokine IFN- gamma, etc., attract and activate macrophages, and produce an inflammatory reaction dominated by T cells and macrophages. Patch test as an auxiliary diagnostic method for the determination of the body's allergy is based on the above origin. So far, more than 120 applications in the Department of dermatology have been used. At present, 28 common clinical sensitizing drugs (C Masi Bing, ibuprofen, amoxicillin, diclofenac sodium, etc.) have been developed and have developed a patch kit with independent intellectual property rights. It has obtained FDA, C FDA and EU certification in the United States. It is widely used in clinical practice to diagnose and identify adverse drug reactions caused by drugs. The purpose of this study is to develop a good social and economic benefit. The purpose of this study is to develop the reagents for the DDF patch and establish an immune confirmatory method for the sulfoxide syndrome to provide an effective technique for the diagnosis of the disease and to promote the clinical application of this technique. 1 24 cases of leprosy with HLA-B*1301 site.2, 24 cases of leprosy syndrome, and 25 cases of leprosy with HLA-B*1301 site but without sulfoxide syndrome. Healthy volunteers were included in the standard: case group: 12008.01-2016.12 new leprosy patients and taking ammonia 2, in the treatment of sulfoxide, patients were treated with fever, rash, liver dysfunction, lymph node enlargement and other suspected sssone syndrome, conforming to Richardus and Smith's diagnostic criteria for the disease; 3, carrying HLA-B*1301 alleles; 4, agreed to participate in the study and signed a written informed consent. 12008.01-2016.12 in the control group. The national new leprosy patients were treated with Diphenyl Sulfoxide for at least 1 months. 2, patients did not find fever, rash, liver dysfunction, lymph node enlargement and other clinical symptoms during the treatment of Daba; 3, carrying HLA-B*1301 allele; 4, agreed to participate in the study and signed written informed consent. 1. pairs of exclusion criteria: 1. Patients with sulfonamides, furasamides, thiazides, sulfonylureas, and carbonic anhydrase inhibitors; 2. patients who could not provide correct information, such as mental disorders, and 3. patients with severe disease, such as heart failure, malignant tumor, etc.; study: This study was divided into three points: the study of I: development for clinical application of the SF patch test To explore the stability of the reagent matrix and concentration range and to clarify its stability: 1 patients with suspected ssone syndrome with positive clinical diagnosis and excitation test were used as experimental subjects to explore the appropriate matrix and concentration range of the reagents for the reagents. The content of patch reagents was determined by II: and the stability of the reagents was determined. A study of the sensitivity and specificity of the clinical application of III: specific Diphenyl Sulfoxide patch reagents: 23 patients with suspected clinical diagnosis and carrying HLA-B*1301 loci and 25 cases of leprosy with HLA-B*1301 loci but no symptoms of dsone syndrome with 25 cases of dsone syndrome Test. Select 50 healthy volunteers to remove the skin irritation reaction caused by patch reagent itself. Use the chi square test to determine the statistical significance of the differences between each group, determine the sensitivity and specificity of the clinical application of the patch reagent. Research methods: I: the use of dissolving method to observe Vaseline matrix, poly (two) Alcohol 200 matrix, polyethylene glycol 200 matrix + two methyl sulfoxide, polyethylene glycol matrix + oleic acid, polyethylene glycol matrix + azone in sulfoxide, and adjust the concentration of patch reagents according to the patch test results; study II: using high performance liquid chromatograph for the preparation of Vaseline matrix, polyethylene glycol 200 matrix patch reagents containing Measurement and Study on the initial stability of the study. The study of III: on the research object was carried out by the patch test of sulfoxide. The methods were as follows: 1. the concentration of the reagents was put in the concentration of 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, and the control was ripped from one side with the triangular tearing, and then the sulfoxide spot was tested. The concentration sequence of the agent and the blank control was added in the spot test device. Note the concentration sequence of the patch reagents,.3. let the patient fully expose the back skin, choose no rash, the normal skin without the pigmentation, the.4. use the alcohol cotton ball to sterilize the skin fully, and then paste the spot test device on the back of the patient's back skin, gently press the net air and spot the spot. The test device is fastened to.5. with the mark pen in the four corner of the spot test device. And the record is made on the experiment paper. Special attention is paid to the location of the different concentration of the patch reagents..6. tells the patient to tear out the spot test device after 48 hours. After 24 hours of ripping off the spot test device, the test result was observed after 24 hours of ripping out the spot test device. Cleaning the tested parts with the alcohol cotton ball and marking the four corners of the spot test device again with the marker pen, and carefully observing whether there were erythema, papules, and bubble out of the subjects. The results were judged according to the ICDRG (International Contact Dermatitis Research Group) guide of the international contact dermatitis research group. The results of the patch test were determined by the same technician. Statistical method: the results of the study were analyzed in the intention analysis (intention to treat, ITT) population, and the ITT population was at least one in the study and at least one The subjects of the secondary drug use. Data statistics application of SPSS 16 software, according to the purpose of the study and the nature of the data of the selection of chi square test, the test group and the control group of the indicators of the difference is statistically significant. All statistical tests are both tested by bilateral test, water calibration sense is alpha =0.05, P value less than 0.05 will be considered to be tested The difference has statistical significance. The results of the study were to study the comparison of I: through the Vaseline matrix, polyethylene glycol 200 matrix, polyethylene glycol 200 matrix + two methyl sulfoxide, polyethylene glycol matrix + oleic acid, polyethylene glycol matrix + azone 5 substrates, and finally selected polyethylene glycol 200 base + two methyl sulfoxide as the best preparation. The content of ethylene glycol 200 as the matrix patch, the normal temperature state and the low temperature of 4 centigrade for 6 months did not decrease obviously, and there was no difference in the stability of the sample at low temperature and storage at normal temperature. The use of this matrix could guarantee the stability of the sample for a certain time and no need for present use. In the study of III:, 23 cases of sulfoxide syndrome were studied throughout the country. The spot patch test was used in 10 cases of plaque test positive and 13 cases of patch test negative. 25 cases of leprosy patients taking ddsone and 25 cases of leprosy patch test were negative and 50 cases of healthy volunteers were negative. Conclusion: This study established the immunization of sssone syndrome based on patch test. The method of proof provides an effective laboratory technique for the accurate diagnosis of the disease. It will also provide some ideas and reference for the establishment of the immune confirmation method of the drug hypersensitivity syndrome caused by other drugs. It is of great significance to determine the sensitizing drugs of the patients in clinical practice and to clear the diagnosis of the disease.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R595.3

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