【作者】 徐磊；

【导师】 王海波；

【作者基本信息】 山东大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 第一部分壳聚糖-磷酸甘油-地塞米松凝胶内耳局部给药的研究研究目的：目前临床上,糖皮质激素已应用于多种内耳疾病的治疗,然而由于血-迷路屏障的存在,全身给药途径造成其在内耳中的吸收与分布受到限制,为达到内耳组织间隙中的有效药物浓度,需使用较大的治疗剂量,易引发药物对全身的毒副作用。因此学者们一直在寻求内耳局部给药的解决方案。经鼓膜注射的方法虽然显现出一定的优越性,但这种给药方法的治疗效果不确定,存在很大的个体差异。所以开发一种新的内耳局部给药系统,使其能够可控、持续且有效地释放治疗因子至内耳已成为耳鼻咽喉科领域的一项前沿课题。当前研究探索可生物降解的壳聚糖-磷酸甘油凝胶系统携带及释放地塞米松进入内耳的特性,及其治疗内耳疾病的可行性。实验方法：本研究包括体外实验与动物体内实验两部分。体外实验：探讨体外环境中,壳聚糖-磷酸甘油-地塞米松凝胶在7天的期间内释放地塞米松曲线及其与凝胶分解之间的关系。动物体内实验：地塞米松凝胶直接注射于小鼠圆窗龛,术后定期采集外淋巴液及血浆样本,并通过液相色谱-串联质谱法测定样本中的地塞米松含量,以评价药物的释放曲线。同时,我们还采用听觉脑干反应评估了该凝胶系统以及注射凝胶的手术过程是否会对模型小鼠的听觉功能产生影响。实验结果：该研究建立的壳聚糖-磷酸甘油-地塞米松凝胶在室温环境下配制后为可注射的液态,在37℃的体温环境下,经过15-20分钟后,即转变为不可流动的半固态,非常适宜于内耳局部给药。体外实验：地塞米松凝胶在7天中,释放了接近一半的地塞米松总药量。最初的24小时中,地塞米松的释放量较多,达到总药量的35.17%,在接下来的几天内,药物逐渐呈缓慢释放趋势,并且药物的释放与凝胶的降解保持一致。动物体内实验：地塞米松凝胶圆窗龛局部注射后,在外淋巴液中可以持续7天检测到高药物浓度水平,其浓度远大于血浆中的药物浓度,具有统计学意义。小鼠模型的听力在术后第2天,受到一定程度的影响,以低频为主；而在术后第10天时,听力即恢复正常,与术前的听力闽值相比没有统计学差异。研究结论：该研究建立的壳聚糖—磷酸甘油-地塞米松凝胶系统证实能够在圆窗龛单次注射后超过7天的期间内,持续可控地释放地塞米松至小鼠内耳,并形成显著的高外淋巴液药物浓度。地塞米松的释放与凝胶的分解具有直接的关系。圆窗龛的开放过程是安全的微创手术操作,对内耳功能没有显著影响。这些结果均强有力地说明壳聚糖-磷酸甘油凝胶系统适用于作为一种创新性的缓释可控内耳局部给药载体,提出了一种内耳局部给药的新思路,对改进传统给药方法,提高内耳疾病的治疗效果做出了有意义的探索。第二部分壳聚糖-磷酸甘油凝胶应用于庆大霉素内耳局部给药的试验研究研究目的：经鼓膜注射庆大霉素已在世界范围内应用于梅尼埃病的治疗,但是,这种方法最大的缺点是治疗效果及出现听力损失的几率存在巨大的个体差异。其原因在于药物经鼓膜直接注射至中耳后,不能准确地控制与圆窗膜接触的药量,从而导致能扩散入内耳的药物剂量在个体之间存在巨大的差异。为克服现有方法的局限,建立一种安全有效并且方便的给药模式,使其能够在单次应用后,即可持续缓慢地释放庆大霉素至内耳,我们拟探索壳聚糖-磷酸甘油凝胶系统应用于庆大霉素内耳局部给药的可能。当前研究通过与传统鼓室内注射的对照,探索壳聚糖-磷酸甘油-庆大霉素凝胶在小鼠圆窗龛局部注射后,缓释庆大霉素至内耳的能力,及其对耳蜗、前庭系统功能的影响。同时,我们对比分析了该凝胶系统在携带庆大霉素与不携带任何药物两种状态下的超微结构及元素组成,以期更好地理解其特性。实验方法：体外实验：采用环境扫描电子显微镜及能量色散X射线光谱的方法,分析壳聚糖-磷酸甘油凝胶系统在携带庆大霉素与不携带任何药物两种状态下的超微结构及元素组成特点。动物体内实验：庆大霉素采用壳聚糖-磷酸甘油凝胶系统携带或传统经鼓膜直接注射的方法应用于小鼠中耳,术后定期采集外淋巴液及血浆样本,并通过液相色谱-串联质谱法测定样本中的庆大霉素含量,以评价药物的释放曲线。同时,我们还采用听觉脑干反应及垂直转鼓试验评估术前术后的听觉及前庭系统功能,对比这两种不同的给药途径对小鼠模型内耳功能的影响。实验结果：体外实验：环境扫描电子显微镜及能量色散X射线光谱分析结果显示空白凝胶与携带庆大霉素的凝胶在超微结构与元素组成方面存在一定差异。动物体内实验：两种庆大霉素给药方法在小鼠外淋巴液中产生了完全不同的药物释放曲线。庆大霉素凝胶注射组的药动学曲线呈明显的缓释特性,在第一天到第三天达峰值,并呈平台期,此后4天,呈线性趋势逐渐下降。而经鼓膜注射组药动学曲线则在第一天达到峰值浓度之后,急剧下降。两组资料中,血浆中庆大霉素浓度均明显低于外淋巴液的药物浓度。空白凝胶的应用对内耳功能没有明显影响。在注射相同剂量的40μg庆大霉素之后,采用凝胶的方法可以造成显著的平衡功能异常,同时没有明显的听力损失；而采用经鼓膜注射的方法对前庭和听觉功能均没有造成任何显著的改变。研究结论：该研究建立的壳聚糖-磷酸甘油-庆大霉素凝胶系统证实可以持续、有效地释放庆大霉素至内耳,并且个体之间的一致性很好；注射药物的手术过程安全可靠,对内耳功能没有显著影响。庆大霉素凝胶系统相比传统经鼓膜注射,表现出其优秀的特性,为提高梅尼埃病的治疗效果提供了一种新的方法和思路,做出了有意义的探索。更多还原

【Abstract】 PartⅠA Sustained Local Dexamethasone Delivery System for Inner Ear ApplicationsObjectives/Hypothesis:Glucocorticosteroid has been widely used for the treatment of inner ear diseases in the clinic. However, Systemic drug administration increases the likelihood of systemic toxicities and side effects and creates an inequality in drug concentration with higher circulating levels in the serum but lower local levels at the inner ear where the drug is needed. So much interest has been generated in developing a system for local drug delivery to the inner ear for the treatment of inner diseases, such as Meniere’s disease and sudden idiopathic or noise-induced hearing loss. Intratympanic injection has overcome some risks and limitations of systemic drug delivery, while there remains a considerable amount of variability in clinical outcomes among those patients treated with intratympanic injections. So there is a clear need for a versatile system which capable of safely and efficiently delivering a sustained dose of the drugs. This system would allow to treat patients by using a single application. The present study investigates the effectiveness of a controlled and sustained local Chitosan-Glycerophosphate hydrogel system on the delivery of dexamethasone into the inner ear and its influences on the inner ear functions.Methods:In vitro:dexamethasone release and Chitosan-Glycerophosphate-hydrogel matrix degradation were characterized. In vivo:dexamethasone laden hydrogel was placed directly in the round window niche of mice. By collecting perilymph and plasma at different time points and analyzing the samples with high performance liquid chromophotography and mass spectrometry, we characterized the drug release of this drug delivery system. The safety of hydrogel on auditory function was also evaluated.Results:We developed a Chitosan-Glycerophosphate-hydrogel which is prepared in a liquid state at room temperature. At body temperature, it undergoes a phase transition becoming solid. In vitro:dexamethasone laden hydrogel released almost half of the dexamethasone it contained over 7 consecutive days with concurrent degradation of the hydrogel matrix to 15.3% of its starting weight. In vivo:Elevated levels of dexamethasone were detected in perilymph for 7 days following a single hydrogel application. In post-operative day 2, there was a little increase in hearing thresholds, and by post-operative day 10, the hearing thresholds returned to baseline.Conclusions:Chitosan-Glycerophosphate-hydrogel provides sustained and controlled drug release of dexamethasone within the inner ear of mice. The surgical procedure and administration of CGP-hydrogel to mouse ears is safe. This novel system provides a unique approach for sustained local drug delivery for inner ear therapy. PartⅡA Controlled and Sustained Local Gentamicin Delivery System for Inner Ear ApplicationsObjectives/Hypothesis:Intratympanic gentamicin injection has gained popularity worldwide for the treatment of Meniere’s disease, However, this delivery system remains a considerable amount of variability in clinical outcomes among those patients treated with intratympanic gentamicin injection as unstable portions of the gentamicin contact with the round window membrane (RWM) and enter the fluids of the inner ear by diffusing across the RWM. The present study investigates the effectiveness of a controlled and sustained local hydrogel system on the delivery of gentamicin into the inner ear and its superiority for the treatment of Meniere’s disease.Methods:Ultrastructure of hydrogel loaded with/without gentamicin was explored in vivo. Gentamicin was applied to the ear of mice either through intratympanic gentamicin injection or in the hydrogel system. Kinetics curves, hearing and balance functions were examined to study how the hydrogel system affected the gentamicin delivery and inner ear functions.Results:The micrographs and spectra analyses showed different structures between the blank hydrogel and drug-laden hydrogel. The two gentamicin-delivery methods yielded different kinetics curves. The hydrogel system achieved sustained-release over a 7-day period with a flat plateau phase from day 1 to day 3 and slow decent in the subsequent days. The intratympanic gentamicin injection curve dramatically declined after the peak concentration at day 1 and almost eliminated by day 3. Hydrogel system yielded noticeable balance dysfunction with no significant hearing changes. In contrast, intratympanic gentamicin injection exhibited no significant influences on the inner ear functions after applying the same dosage of 40μg gentamicin.Conclusions:The hydrogel system established in this research has been proved to be much more sustained, consistent and efficient than traditional intratympanic gentamicin injection for the transportation of gentamicin into the inner ear and hence offers new outlook for Meniere’s disease’s therapy.更多还原