我们承认:托马斯·布迪曼博士在TSE Systems GmbH公司提供的技术专长和设备定制。约翰·弗莱（巴特尔公司）和鲁迪博士耶格（CH Technologies公司）为他们的有益的讨论。吴天，山姆Mboggo，四月米勒和肖恩·戴维斯（Amgen公司），用于实验的帮助。

在这些研究中使用的小鼠按照指南实验动物的管理和使用，第八版24人照顾。小鼠关在协会的评估和实验动物的认证（AAALAC）的国际认证的工厂在玉米芯床上用品消毒通风的微型隔离房集团。当测量的支气管收缩，将小鼠用100mg / kg的戊巴比妥腹膜和麻醉深度麻醉因缺乏脚趾捏反射的监测，并通过腹膜内麻醉必要维持。在实验结束时，小鼠经断颈过量巴比妥酸盐后安乐死。安乐死的验证因缺乏呼吸的证实。没有生存的手术都对小鼠进行。所有研究方案得到机构动物护理和使用委员会（IACUC）的批准。 1.的Phar的产生配方和设备选择maceutical气溶胶注:配方和设备选择依赖于单独药物的物理化学性质被雾化，因此一般方案见下文和读取器是由曾25和26奥赖尔登提到的评论。 <ol><li> 干粉气雾剂 <ol><li>微粉化药物在球磨机，喷射磨或类似的装置27的干燥粉末，并确保所述微粉化的颗粒尺寸分布（PSD）包含可吸入（0.5-5微米质量中值空气动力学直径，MMAD）的颗粒尺寸的颗粒。共混物需要稀释，微粉化乳糖有效的测试化合物。 注意:如果没有足够的微粉以确定与级联冲击的PSD中，微粉化粉末的一个小的（子毫克）样品的PSD可以通过光散射进行测量，以确认其含有小/可吸入颗粒。 </li><li>生成使用莱特灰尘进料干粉气溶胶发生器干粉气雾剂。使用手动液压机在大约1000磅每平方英寸（psi），以产生由所述粉尘莱特饲料气溶胶发生器17用作输入粉末的压实的糕点包装微粉化药物/乳糖粉末到圆柱形储器。 </li><li>螺杆圆柱形贮存到尘埃莱特进料推进贮存直至刮刀与所述药饼接触。 </li><li>莱特灰尘进料至一个旋风分离器和所述入口的出口连接到设置为15升/分钟的气流速率（最大压力90 psi）的压缩空气源。 </li><li>设定进给速度控制，以每分钟（rpm）0.7革命和打开莱特灰尘进料气溶胶发生器。 注:在0.7rpm使用小赖特灰尘进料圆筒贮存时相当于1克/小时的试验品滤饼进料速率。赖特灰尘进料刮擦压实PO的薄层通过旋转贮存器wder时，将测试物品蛋糕。空气中携带尘出莱特灰尘进料的，通过用于解附聚声速喷嘴，并进入旋流器以除去不可吸入颗粒和附聚物。 </li><li>旋流器的出口连接到所述吸入部（ 图1）的中心气雾剂增压室。 注:化合物可以由300在赖特灰尘进料的储存器被压缩到1500psi的。目的是足够的压缩颗粒，它们将在贮存器倒立时被保留，但没有这么多，赖特灰尘进料不能刮去用于重新气雾化的薄层。必须记住的是在手动液压机压力表以磅读取和对小的灰尘进料储存器中的柱塞具有约0.25平方英寸的表面积。因此，250磅的0.25平方英寸的压缩力是等效至1000psi。 </li></ol></li><li> 雾化液体的气溶胶 <ol><li>溶于100mL水或生理食盐水的药物。 </li><li>加载100毫升的注射器与药物溶液和注射器放入设定在1毫升/分钟的流速注射泵。 </li><li>连接注射器泵到喷射喷雾器，并从进料管线通向喷雾器清除空气。 </li><li>加压空气源连接到喷射喷雾器，并设置在空气流量计10升/分钟。 </li><li>插入喷射喷雾器进入预分离器。预分离器连接到喷雾器吸入单元的中心气室气溶胶（ 图1）。 注意:许多药物化合物具有有限的水溶解度和被更好地配制为干粉气雾剂。如果稳定的悬浮液可以从微粉化（MMAD &lt;5μM）化合物来制备，它可与喷射喷雾器一起使用。注意应作为暂停可能堵塞喷雾器。从1毫克/毫升为有效化合物（如支气管扩张剂喷雾器进料浓度异丙托铵），以40mg / mL的悬浮液（对于像硫酸沙丁胺醇较少有效的化合物）已被使用。注射泵进料速度设定为1毫升/分钟为一个实际的原因;以允许气溶胶浓度平衡时间和45分钟长的暴露，而无需重新加载注射器完成。 </li></ol></li></ol> 2.气溶胶暴露实验设置<ol><li> 测量药物浓度（通过在绝对过滤收集气溶胶）和粒度分布（通过使用级联冲击收集气溶胶）进入所述吸入部后预分离器/旋流器的气溶胶的。使用与动物的每分钟通气量，体重，以及曝光时间沿着这些参数来估算沉积于肺部药物的剂量。 <ol><li>称量绝对过滤，记录过滤器的重量。放置过滤器进入过滤器保持器并组装过滤器保持器。连接的绝对过滤器保持器的入口到中央气雾剂增压室样品端口和所述出口设置以1L / min的流速在实验的持续时间来采样气溶胶的真空源。 注:在过滤器上的药物采样45分钟后的质量可在亚微克范围和/或与乳糖，氯化钠盐，或其它车辆进行混合。读取到0.1微克微量天平是必要的。为了获得沉积在过滤器的药物的准确的重量，必须将过滤器平衡并在湿度受控环境中称重。药物的过滤器上的重量只能在剂量计算中使用，如果有在制剂中没有车辆或车辆是水。当在除水以外的所述制剂的车辆中，物质的过滤器上的重量只给出了通过高效液相色谱（HPLC）的药物含量进一步分析的估计的起点。 </li><li>称重并记录7级联冲击级滤波器的权重和一个最后的"尾片";过滤。放置在每个级联冲击的七个阶段的过滤器和组装默瑟级联冲击28。级联冲击器的入口连接到一个中央气雾剂增压室样品端口和所述出口设置为样品在流速级联冲击在（通常为0.5或1升/分钟）校准气雾剂的持续时间的真空源实验。 </li><li>监测吸入单元与实时气溶胶监视器（见材料和​​试剂表）气溶胶内容，以确认该气溶胶发生器的功能，并产生在整个实验稳定气雾剂。实时气溶胶显示器的入口连接到一个中央气雾剂增压室样品端口和所述出口设置以1L / min的流速在实验的持续时间来采样气溶胶的真空源。 注:来自实时气溶胶监视器以μg/ L报告的信号，但被校准用于道路灰尘和必须重新校准为每个单独的药物气雾剂，得到正确的气溶胶浓度值。无需校准使用显示器来确认存在或不存在和气溶胶浓度的经时稳定性。 </li><li>设置过程控制参数（空气流量，真空，压力，气溶胶发生器功率）需要依赖于动物的数量将被连接到吸入单元的值。吸入部和气雾剂发生器被连续地控制/通过由制造商提供的计算机化的过程控制/数据采集系统（DACO）监测（见材料和​​试剂表）。空气流速向所述吸入单元应最低限度地在吸入单元中的所有动物的约2倍的总分钟通气率以避免CO 2的累积。 </li></ol></li><li> 负载的小鼠成鼻式暴露限位器在仅鼻吸入单元气溶胶之前。此外载荷约束应力控制动物到限制器呼吸室内空气。 注:剂量/反应实验由暴露于气溶胶​​不同的时间量的小鼠的多个组的。曝光时间被用于控制剂量/反应实验期间各组接受的剂量。 <ol><li>角度朝向天花板的限制筒了，而试图加载的动物，因为他们往往向上运行试图逃跑。指着管下来，同时装载鼓励转身逸出管的后面。确保鼠标的鼻子被定向到管的尖端并固定位置可变的柱塞进入所述限制器的后端。 注:柱塞被构造以允许小鼠的尾部为从保持件，其允许鼠标调节其体温而在限制器突出。 </li><li>调整柱塞以允许小鼠旋转，但不转头尾;确保气溶胶吸入。 </li><li>连续监测小鼠而在约束管中。定位在柱塞之后，小老鼠（&lt;20克），往往试图在管转动头对尾和采用的U位置，他们有呼吸困难。这种转向行为是最普遍的在第5分钟的克制，在这之后的小鼠很少尝试把头部到尾部。 </li></ol></li></ol> 3.气溶胶传递<ol><li>插入止动件，以堵塞吸入单元的输送口，并从过程控制软件内激活气溶胶发生器，压缩空气流量控制器，和吸入单元真空泵。 </li><li>一旦从实时气溶胶显示器读数表明气溶胶浓度必须达到平衡（〜30分钟， 图2），开始除去塞子和将含有小鼠到吸入单元鼻仅抑制管中。重复，直到暴露于药物的所有小鼠都连接到吸入单元。 注:在示例实验中，通过气溶胶发生器和稀释空气供给到吸入单元的总空气流量被设定在使用中提供一个0.5升/分钟的流速给每个动物暴露端口。例如，一个15升/分钟总空气流量足够在吸入单元，以供给每30个端口。这比所要求的小鼠的分钟通气得多的空气流动，但较大的空气流需要提供的能量（穿过气溶胶发生器压降）以产生（雾化/解附聚物）的气雾剂。 </li><li>一旦所有的动物被装载到曝光单元，打开连接到使用过程控制软件的绝对过滤器和级联冲击真空采样泵。 </li><li>当所有敞口完毕，关闭气溶胶发生器和从吸入部除去残留的小鼠。 注:动物会改变站头巾或戴口罩的人员进行处理。气溶胶传递的结束后，老鼠都是R从管emoved和管每次使用后消毒。 </li></ol> 4.计算所沉积的剂量的<ol><li>为了以μg计算所沉积的剂量29 / kg的（等式1）乘以药物在气雾剂X吸入分数x肺沉积分数的浓度（μg/ L）X分钟通气量（L /分）的曝光（分）×持续时间并且由体重（公斤）除以。 </li><li>通过乘以采样时间（分钟）的过滤器（L /分钟）除以药物的质量上由空气流量的绝对过滤器（微克）计算气溶胶浓度（μg/ L）。基于体重30异速生长公式估算鼠标的每分钟通气量。可吸入分数为1作为气溶胶通过预分离器，以便除去非可吸入颗粒，和肺沉积馏分从药物气雾剂MMAD确定使用（ 图3）由单分散气溶胶试验的校正曲线（ 图4）31。 </li></ol><img alt="方程" src="/files/ftp_upload/55454/55454eq1.jpg" />

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只有A鼻吸入系统及其操作提供药用气雾剂啮齿动物的肺部进行了描述。在鼻式保持器抑制动物是用于暴露动物空降材料通常使用的方法。进行实验证明了抗胆碱能支气管扩张药异丙托溴铵34能够有效地逆转醋甲胆碱诱导的支气管收缩时通过鼻式吸入用的沉积剂量为0.1μg/ kg的ED 50递送至小鼠。在异丙托的有效剂量大于10倍的增加被要求支气管扩张以下气管内递送（ED 50气管内= 1.3微克/千克，数据未示出）输送。这是由于在通过气管内给药3，5，10产生的肺部药物的非均匀沉积图案。 10倍和更高的有效剂量吸入气管和给药技术之间的差异对其他药物已被他人4先前观察到的。 在气管内给药所产生的肺药物的非均匀沉积图案也由35肺减慢药物吸收，减少在其中药物进入全身循环的速率和减小看到的全身性副作用的可能性。因此，为了优化安全性/功效（治疗指数）的新吸入药物36时，应使用鼻只吸入递送。气管内沉积将通过产生错误的高有效剂量在肺中和低剂量全身循环得到TI的不准确估计。 如吸入途径提供新的药物的开发，它从临床前药效学研究的有效药物剂量适当地转换为预测的EF是必不可少的ficacious人用剂量的临床试验。 Tusko的臭名昭著的死亡大象37文献中常常被引用来提醒我们使用异速生长尺度预测物种间的药物剂量和种间药物剂量不应线性外推的身体群众的一个简单比较的基础上。这是通常使用的异速生长方式与0.67的异速生长指数b。从临床前药效学研究38预测人类药物剂量。使用鼠标吸入的0.9微克/千克为异丙托铵，0.67的异速指数，0.03千克小鼠体重，和60公斤人体重ED 90; 0.07微克的估计人类ED 90 /公斤（（0.9 *（0.03 / 60）（1-0.67）= 0.07）。该预测值可以被计算为从我们的鼠标数据的预测的人类剂量沉积比得上实际人类的0.26微克/千克有效沉积剂量，这可以从所传送的执行来计算的40微克39由60公斤乘以16的0.4（（40/60）* 0.4 = 0.26）人口腔吸入肺部沉积分数人体质量除以本身。有效的人吸入沉积量的估计也有助于计划在临床试验需要40吸入毒理学研究中使用的递送剂量。 显影唯一鼻吸入技术当专门的设备和大量的用于鼻吸入仅药物递送所需的试验化合物（克数量）可以是显著限制。如果需要特定的剂量（通常是毒理学和不药效剂量/应答）的研究，并且该校准运行将需要更多的药物可用，而不存在动物甲校准运行是必要的。此校准运行是必要的，因为每一种药物/制剂的物理化学性质可以变化足以极大地影响所沉积的剂量在肺部药物。空气和药物的进料速率，以在校准运行期间气雾剂发生器的优化是必需的，以实现所需的特定剂量的气溶胶颗粒大小和浓度。而在所述方法中所建议的气溶胶发生器的空气和药物的进料速率是一个合理的起点，有潜在的对特定的药物配制剂的非可吸入（粒度&gt; 5mm）的气雾剂将生成。在临床前研究阶段，往往存在足够的可用药物做校准运行，不可能事先知道什么剂量（如果有的话）进入肺部。另外，静电在气雾剂产生过程中产生的并影响了气溶胶浓度达到平衡所需的时间。重要的是要正确接地设备，以尽量减少静电荷是很重要的。以最小化静电另一种选择是湿度增加供给到吸入单元的空气，以增加导电性和消散上的颗粒25的静电荷。加湿供给到气溶胶发生器的空气期间短（&lt;1个小时）给药会话不需要用于动物的舒适性，但如果使用较长的给药时间应该被考虑。 药物可以释放到动物的被动鼻式吸入或绕过鼻咽沉积直接气管内给药方法肺部。鼻式吸入递送在吸入毒理学41的领域中常用的，但微在药物发现过程的早期使用。是需要进行，由于鼻式吸入研究的多学科研究小组:需要大量的药物，制定，产生和表征气溶胶所需的专门知识;操作复杂的设备，并测量呼吸疾病的动物模型药物疗效。本文所描述的递送技术被用来开发SmaI位升分子吸入药物，但在将来可应用于开发吸入生物制剂42,43。但愿这份手稿记录的程序和技巧，将有利于新的临床前药物发现和毒理学研究人员获得必要通过雾化吸入，以药物输送到啮齿动物的技能。

有许多优点的药物呼吸道疾病治疗吸入给药。吸入给药直接将治疗剂作用的部位，肺。在肺中药物的局部浓度高提供了减少剂量和全身暴露显著的优势，并最大限度地提高疗效。这是一个重要优点，即能大大提高治疗指数（TI，药物引起过度的药物剂量，提供功效的副作用的剂量比）的药物。吸入β2 -肾上腺素能激动剂，皮质类固醇和抗胆碱能药物已被证明在提高患者的哮喘和COPD肺功能有效，同时最小化的全身副作用（心动过速，免疫抑制，和便秘）当这些药物口服观察到。新的药物类（例如，PDE4抑制剂1和BTK抑制剂2）最近证明改善在临床前的动物疾病模型，但是，类似于β2 -激动剂，皮质甾类和抗胆碱能药物肺功能有效，遭受全身副作用，可能通过吸入输送最小化。由于吸入与口服药物开发所增加的成本，吸入制剂只应考虑呼吸迹象成功后口服/全身给药剂量揭示限制基于机制的全身性的副作用。 临床前，吸入化合物经优化以增加TI，这需要在体内效力和副作用的测量。最初，这些测量可以在分开的测定中，通常是一个局部递送功效测量和全身递送副作用测量，但要真正比较化合物制成，疗效及副作用应在吸入给药后的相同的动物进行测定。这需要剂量/响应的研究，交流hieve给予肺足够化合物以诱导可测量的副作用。的唯一方法目前大剂量药物的均匀地分配到多个小动物的肺同时仅鼻吸入3，4，5。的优势和不同的吸入暴露弱点的技术最近已经审查6，7，8。专门的设备和大量的测试化合物（克数量）的所需要的鼻吸入仅药物递送，但验证的概念研究可以通过其他手段是可能的。 当药物的量是有限的（毫克的量），直接施用方法是一种选择，但所有从非均匀沉积遭受与更多的药物沿着中央气道浓缩和较差表示在实质/肺泡区域3，4，5。直接滴入提供有效剂量是始终较高，不能直接进行比较，以吸入剂量4。直接滴入方法包括鼻内9，气管内液体10，11和喷雾滴注12，或干粉末吹入13，14可被用作筛选工具来确定购买仅鼻吸入研究的近似剂量范围内，或确定效力/毒性的排名的一系列结构相似的药物15。由于中心气道沉积图案，直接给药方法可能更有用，以确定化合物的，关于中央气道比I（支气管扩张药或肥大细胞抑制剂）作用的影响n中的外周肺（抗炎药）。 与人类不同，谁可以从吸入器在一个单一的深呼吸吸入气溶胶浓缩的相当大的剂量，连续产生可呼吸的（0.5-5微米质量中值空气动力学直径，MMAD）气雾剂，长达一个小时，则需要沉积一种有效的药物剂量成一个仅鼻吸入系统16自主呼吸啮齿动物的肺。气溶胶发生器（喷射雾化器或赖特灰尘进料17），其可以连续地产生用于仅鼻吸入研究所需的气溶胶颗粒大小和浓度是不是在产生高质量（吸入）气溶胶非常有效的。所述药物进料速率为有效的这些气溶胶发生器（IC 50 pM至nM的功能性的基于细胞的测定）的化合物在1〜10毫克/分范围通常是和药物气雾剂通常小于1％会向呼吸区动物（图1）。许多产生的粒子的过大（&gt; 5微米），以进入肺并通过气溶胶分类器（预分离器或旋风分离器具有5μm切口的点）被除去以避免在鼻子大剂量的药物。添加到仅鼻吸入系统的低效率是可吸入颗粒的小颗粒尺寸范围（0.5至5微米MMAD）。许多小于0.5微米的呼出（如香烟烟雾），而不是沉积于肺部18中的气溶胶粒子的。此外，许多"较大"的气溶胶颗粒的（〜5微米）在鼻子存款被吸收或通过粘膜纤毛清除朝向在那里它们被吞咽到胃19中的咽喉后部输送。当仅使用鼻吸入，敷在鼻剂量总是比沉积于肺部剂量和鼻剂量可以向全身暴露和副作用20大。本质上，吸入DRU克剂量的小（在微克范围）最小化由鼻，肺，或胃肠道组织吸收药物的任何全身副作用的潜力。甚至当提供给动物的气溶胶的颗粒大小是在吸入范围，平均只有4％的气溶胶颗粒，它使在肺部动物沉积物的呼吸区。更有效的气溶胶发生器是可用的，但喷射式喷雾器和Wright灰尘进料是无与伦比它们对来自不同液体和干粉制剂，分别产生连续气溶胶能力。 从预分离器吸入气雾剂传递成是基于流过鼻子设计22中的鼻式暴露吸入部21。吸入单元具有3层（仅两层在图1中示出），并且每个层包含对动物和气溶胶取样10个曝光端口。这些端口位于外围周围的CEntral气溶胶全会。有意识的小鼠被放置在玻璃抑制保持器（6英寸长英寸直径的1.2）和呼吸吸入单元的气溶胶成分。小鼠未驯化的约束装置23。先前的经验表明，小鼠耐受管克制不到一个小时的持续时间类似地，具有或不具有适配2。 吸入单元设计为直接递送药物气溶胶的动物的肺，同时避免暴露于运营商。这些药物的效力/毒性通常是未知和多个工程安全控制来避免暴露给运营商。运营商必须始终佩戴个人防护装备（手套，实验服，呼吸器，安全眼镜）。吸入单元的外气室是下在操作期间的任何时候都负压，从而允许安全移除的单个或动物组无覆盖UT切断气雾发生器。吸入单元也包含在通过排气口在天花板维持在负压力，以防止气雾剂的任何逃逸进入室内在故障的情况下的二次外壳。从吸入系统中的所有流出物的空气被释放到环境中之前，一个HEPA过滤器过滤。在这个手稿中使用的鼻式暴露系统是从单一供应商（参见材料的补充表 ）购得。

<table><tbody><tr><td>Nose-only exposure inhalation unit</td><td>TSE systems</td><td>700100-KNES-040-ss</td><td>Custom configurations available</td></tr><tr><td>DACO data acquisition system</td><td>TSE systems</td><td>700400-PRO-C-D/1</td><td></td></tr><tr><td>MC One Jet Mill</td><td>Jetpharma</td><td>DEC MicroJet 10</td><td></td></tr><tr><td>Turbula Mixer</td><td>GlenMills Inc</td><td>T2F</td><td></td></tr><tr><td>Micronized Lactose</td><td>DFE Pharma</td><td>Lactohale 200</td><td></td></tr><tr><td>Hydraulic press</td><td>Specac</td><td>GS15011</td><td></td></tr><tr><td>Cascade impactor filters</td><td>Pall Life Sciences</td><td>7219</td><td>Emfab filter</td></tr><tr><td>Absolute filters</td><td>Whatman</td><td>10370302</td><td>5 cm diameter</td></tr><tr><td>Real time aerosol monitor&lt;br/&gt; Microdust Pro Monitor</td><td>Casella</td><td>CEL-712</td><td></td></tr><tr><td>Ipratropium bromide</td><td>Spectrum Chemical</td><td>I1178</td><td>pre-micronized</td></tr><tr><td>flexiVent FX1 system</td><td>scireq</td><td>FV-FXCS</td><td></td></tr></tbody></table>

dry powder and nebulized aerosol inhalation of pharmaceuticals delivered to mice using a nose-only exposure system

阻塞性呼吸系统疾病，如哮喘和慢性阻塞性肺疾病（COPD）目前由吸入抗炎和支气管扩张药治疗。尽管多次治疗的可用性，这两种疾病都是不断增长的公共健康问题。大部分哮喘患者都很好地控制电流吸入疗法但重症哮喘患者有相当数量都没有。哮喘影响全世界估计有3亿人，大约20％有疾病是一种严重的。相较于哮喘，很少有有效的治疗慢性阻塞性肺病。人口估计有10％的人COPD并同时减少其他主要疾病死亡率的趋势是COPD增加。虽然开发药物的吸入给药是具有挑战性的，只有鼻子吸入单元使啮齿动物的预临床疗效和安全性/毒理学研究肺直接交付的新型药物。吸入给药具有用于呼吸系统疾病，其中肺中的高浓度提高疗效和减少全身性副作用的多个优点。吸入糖皮质激素和支气管扩张从这些优势中获益，并吸入输送还可以适用于未来的生物疗法的潜力。本文所描述的吸入单元可以产生，样品表征和在啮齿类动物的肺中均匀地沉积的药物气雾剂。这使得临床前确定疗效并存放于啮齿动物肺部药物剂量的安全性，启动临床开发之前所需的关键数据。

支气管扩张异丙托溴铵以1毫克/毫升的浓度溶解于0.9％的生理盐水。 100毫升的注射器中填充的异丙托铵配制剂溶液和将注射器插入到以1mL / min的流速注射泵设定喂喷射喷雾器（ 图1）。激活控制系统，用于吸入单元发起10L /分钟的空气流的喷射喷雾器，5L /分钟的稀释空气流向稀释室，和15.5 L /真空气流的分钟来绘制药物气雾剂出的吸入单元一旦在它通过小鼠的鼻子。注射泵和实时监测气溶胶采样泵被激活。二十四只小鼠插入到吸入单元的实时气溶胶监视器确认在吸入部的气溶胶浓度已达到平衡（15-30分钟， 图2）之后。样品泵的绝对过滤器（1升/分钟）和级联冲击器（0.5L /分钟）被开启一次所有小鼠连接到吸入单元。 8只小鼠的第一组5分钟，15分钟之后的第二组，和45分钟后的第三组后从吸入装置中取出。支气管收缩32被测量为在呼吸系统阻力（Rrs）的增加对乙酰甲胆碱的单一雾化剂量（30毫克/毫升）使用啮齿动物呼吸器33给药后异丙托品输送2个小时。在Rrs的增加的百分比对于每个动物，作为最大的Rrs过雾化乙酰甲胆碱（Rmax）为后3分钟间隔计算减去基线测量的乙酰甲胆碱（RBASE）之前的Rrs值由RBASE（在Rrs的=％增加（RMAX划分-Rbase）/ RBASE），被用于定量的支气管收缩。 气溶胶暴露的45分钟期间沉积在级联冲击过滤器和绝对过滤气溶胶溶解在50％乙腈和异丙托铵的通过HPLC（表1）定量的质量。的异丙托铵气溶胶的MMAD的x / GSD经计算为1.7×/ 1.5微米（ 图3）和用于使用小鼠用于气雾剂为1.7微米（ 图4）的MMAD的0.037的沉积分数（DF）。可吸入分数（IF）为1作为气溶胶通过预分离器，以便除去不可吸入颗粒。乘以采样时间穿过过滤器异丙托铵在气溶胶（0.5微克/升）的平均浓度从异丙托铵的质量计算上通过空气的流率除以绝对过滤器（22微克）拉（1L /分钟） （45分钟）。的平均体重的小鼠的为0.019 kg和它们的预测分钟通气计算为0.021升/分钟。使用等式1，所沉积的剂量为5，15和45个分钟暴露组分别为0.1，0.3和0.9微克/千克;分别。 ="1"&gt;异丙托8周龄的C57BL / 6小鼠抑制雾化醋甲胆碱诱导的支气管收缩。乙酰甲胆碱雾化给药（ 图5，上图）的秒内增大的Rrs。对照组0.62±0.03 CMH 2 O * S / mL的基线值增加到1.66±0.12 CMH 2 O * S / mL的最大值的RR（暴露于室内空气代替异丙托气溶胶），较168±9％增加RRS，70秒乙酰甲胆碱气雾剂给药之后。在呼吸系统阻力的增加％是由吸入剂量异丙托铵以剂量依赖性方式抑制。在Rrs的增加的百分比由51±9％，79±14％，和89±2％在沉积吸入剂量0.1，0.3异丙托铵的分别抑制（ 图5，下图），和0.9微克/千克，。 克" /&gt; 图1:吸入单元与干粉气溶胶发生器附接。用来产生从液体制剂气雾剂喷雾器被示在右边。 1）莱特灰尘进料; 2）旋流器，3）注射泵，4）喷射式喷雾器插入预分离器，5）稀释空气混合器。当递送雾化气溶胶，尘埃进料和气旋由注射器泵，喷射式喷雾器/预分离器，和稀释空气混合器代替。放大图（从奥尔德姆21修改）示出了在由插入所述约束管向所述吸入单元创建围绕动物的鼻子呼吸区气溶胶流动路径。气溶胶填充吸入单元的中心气雾剂（灰色）气室，流出到动物的鼻子在那里由在外部（白色）气室并进入废物收集过滤器在轻微真空拉回（未示出）。 <a href="http://ecsource.jove.com/files/ftp_upload/55454/55454fig1large.jpg" target="_blank">PL</a>轻松点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55454/55454fig2.jpg" /> 图2:实时气溶胶监视器测量证实在吸入部的气溶胶浓度达到平衡〜接通气雾剂发生器在30分钟后。 <a href="http://ecsource.jove.com/files/ftp_upload/55454/55454fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55454/55454fig3.jpg" /> 图3:收集在级联冲击（蓝色条线图）的每个阶段异丙托质量覆盖有用来计算气溶胶颗粒大小分布的MMAD和GSD曲线拟合（黑色曲线）。 <a href="http://ecsource.jove.com/files/ftp_upload/55454/55454fig3large.jpg" target="_blank">请</a>点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55454/55454fig4.jpg" /> 图4:小鼠肺作为MMAD的用于通过鼻式吸入递送气雾剂的功能沉积分数（从谢31修改）。对于粒径小于0.5μm的大多数气雾剂的呼出和肺不会沉积（如香烟烟雾）。对于颗粒尺寸大于5μm，最气溶胶是由鼻子过滤掉。 <a href="http://ecsource.jove.com/files/ftp_upload/55454/55454fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55454/55454fig5.jpg" /> 图5:在呼吸系统阻力迅速增加通过雾化乙酰甲胆碱诱导administe红色到小鼠是通过鼻式吸入递送至小鼠（n = 8每组，上图）异丙托铵阻断。异丙托抑制在呼吸系统阻力醋甲胆碱诱导的增加为0.1微克/千克的ED 50剂量沉积（* P &lt;0.05vs。对照，下图）。 <a href="http://ecsource.jove.com/files/ftp_upload/55454/55454fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="3">级联冲击数据用来计算MMAD和GSD </td></tr><tr><td>级过滤器</td><td>异丙托溴铵的过滤器 通过HPLC（毫克） </td><td>各级馏份 直径（mm） </td></tr><tr><td> 1 </td><td> 0.002 </td><td> 6 </td></tr><tr><td> 2 </td><td> 0.19 </td><td> 4.5 </td></tr><tr><td> 3 </td><td> 1.43 </td><td> 2.5 </td></tr><tr><td> 4</td><td> 2.55 </td><td> 1.5 </td></tr><tr><td>五</td><td> 2.95 </td><td> 1.2 </td></tr><tr><td> 6 </td><td> 1.03 </td><td> 0.7 </td></tr><tr><td> 7 </td><td> 0.37 </td><td> 0.5 </td></tr></tbody></table> 表1:使用的级联冲击器的数据来计算MMAD和GSD

Watch this Scientific Journal Video about Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System at JoVE.com

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

本文所描述的吸入单元可以产生，样品表征和在啮齿类动物的肺中均匀地沉积的药物气雾剂。这使得临床前确定的有效性和沉积于肺部药物剂量的安全性的;关键数据，使临床吸入药物的发展。

干粉和药品的雾化吸入气雾剂递送小鼠使用鼻只曝光系统

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

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Introduction to Respiratory System Drugs

SCIENTISTS IN ACTION

40.6K 次观看.  Amgen. 本文所描述的吸入单元可以产生，样品表征和在啮齿类动物的肺中均匀地沉积的药物气雾剂。这使得临床前确定的有效性和沉积于肺部药物剂量的安全性的;关键数据，使临床吸入药物的发展。 

视频: Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

清醒小鼠滴鼻的CNS药物治疗管理

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

科研

医学

Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles

Using a numerical analysis based on computerized fluid dynamics, a nose-only inhalation toxicity chamber with four different exposure concentrations is designed and validated for flow field uniformity and cross-contamination among the exposure ports for each concentration. The designed flow field values are compared with the measured values from exposure ports located horizontally and vertically. For this purpose, nanoscale sodium chloride particles are generated as test particles and introduced to the inhalation chamber to evaluate the cross-contamination and concentration maintenance among the chambers, for each concentration group. The results indicate that the designed multiconcentration inhalation chamber can be used in animal inhalation toxicity testing without cross-contamination among concentration groups. Moreover, the designed multiconcentration inhalation toxicity chamber can also be converted to a single-concentration inhalation chamber. Further testing with gas, organic vapor, or non-nanoscale particles will ensure the use of the chamber in the inhalation testing of other test articles.

A nose-only inhalation toxicity chamber capable of testing inhalation toxicity at four different exposure concentrations was designed and validated for flow field uniformity and cross-contamination between the exposure ports for each concentration. Here, we present a protocol to confirm that the designed chamber is effective for inhalation toxicity testing.

提供纳米颗粒四种曝光浓度的单鼻子吸入毒性试验室的研制

Using a numerical analysis based on computerized fluid dynamics, a nose-only inhalation toxicity chamber with four different exposure concentrations is ...

工程学

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

一次性充填机对治疗药物的肺吹入小动物

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter &lt; 200 nm and a geometric standard deviation &sigma;g &lt; 2.5 5. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size 6, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria 5.
A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m3 whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm3) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m3). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m3/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis.
In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.

A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation.

全身纳米气溶胶吸入暴露

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle ...

生物学

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

吸入麻醉下鼻内给药评价鼻腔给脑药物的新方法

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

小鼠干粉配方的内经管理

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke.
After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 &#181;m) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 &#181;L of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19.
The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

This study describes a mouse model to study the synergistic effect of nicotine on the progression of pulmonary fibrosis in experimental silicosis mice. The dual-exposure mouse model simulates the pathological progression in the lung after simultaneous exposure to nicotine and silica. The methods described are simple and highly reproducible.

在二氧化硅暴露小鼠模型中使用尼古丁促进肺上皮-间充质转化

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role ...

可定制的一次性给药器，用于将干粉气雾剂药物输送给小鼠

Disposable Dosators Intended for Dry Powder Delivery to Mice

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus metering and dosing, and a propellant-free dispersal mechanism. To develop pharmaceutical dry powder aerosol products, robust in vivo testing is essential. Typically, initial studies involve using a murine model for preliminary evaluation before conducting formal studies in larger animal species. However, a significant limitation in this approach is the lack of suitable device technology to accurately and reproducibly deliver dry powders to small animals, hindering such models&#39; utility. To address these challenges, disposable syringe dosators were developed specifically for intrapulmonary delivery of dry powders in doses appropriate for mice. These dosators load and deliver a predetermined amount of powder obtained from a uniform bulk density powder bed. This discrete control is achieved by inserting a blunt needle to a fixed depth (tamping) into the powder bed, removing a fixed quantity each time. Notably, this dosing pattern has proven effective for a range of spray-dried powders. In experiments involving four different model spray-dried powders, the dosators demonstrated the ability to achieve doses within the range of 30 to 1100 &#181;g. The achieved dose was influenced by factors such as the number of tamps, the size of the dosator needle, and the specific formulation used. One of the key benefits of these dosators is their ease of manufacturing, making them accessible and cost-effective for delivering dry powders to mice during initial proof-of-concept studies. The disposable nature of the dosators facilitates use in animal procedure rooms, where cleaning and refilling reusable systems and weighing materials is inconvenient. Thus, developing disposable syringe dosators has addressed a significant hurdle in murine dry powder delivery for proof-of-concept studies, enabling researchers to conduct more accurate and reproducible preliminary studies in small animal models for pulmonary drug delivery.

作者聚焦：开发一次性给药剂，用于小动物模型中干粉吸入器的临床前 测试 

Pharmaceutical dry powder development necessitates reliable in vivo testing, often using a murine model. Device technology for accurately and reproducibly delivering dry powder aerosols to mice is restricted. This study presents disposable dosators for pulmonary drug delivery at mouse-relevant doses, aiding initial proof-of-concept research.

用于将干粉输送给小鼠的一次性给药器

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus ...

干粉和药品的雾化吸入气雾剂递送小鼠使用鼻只曝光系统

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