CN116782919A - 通过结构修饰、化学增强和微针增强新型肽的皮肤渗透 - Google Patents
通过结构修饰、化学增强和微针增强新型肽的皮肤渗透 Download PDFInfo
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- CN116782919A CN116782919A CN202180071564.2A CN202180071564A CN116782919A CN 116782919 A CN116782919 A CN 116782919A CN 202180071564 A CN202180071564 A CN 202180071564A CN 116782919 A CN116782919 A CN 116782919A
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- peptides
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- menthol
- permeation enhancer
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Classifications
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Abstract
色素沉着过度是常见的皮肤病，具有严重的社会心理后果。十肽‑12是新合成的肽，已被发现在减少黑色素含量方面比对苯二酚更安全，在每天两次治疗16周后，功效高达50％以上。然而，由于其亲水性和大分子量，该肽的经皮渗透有限。因此，十肽‑12通过添加棕榈酸酯链进行修饰，试图克服这一限制。我们还测试了化学渗透增强剂和微针通过皮肤输送两种肽的效果。使用体外皮肤渗透模型发现增强的人体皮肤渗透。此外，我们检查了不同配方的肽保留。我们的数据显示微针贴片中的棕榈肽是最有效的。
Description
相关申请的交叉引用
本非临时专利申请要求于2020年10月20日提交的美国专利申请63/094,242的优先权，该申请与本申请中引用的所有其他参考文献一起通过引用并入。
序列表
无
技术领域
本发明涉及新型生物制剂领域。
发明内容
色素沉着过度是具有严重社会心理后果的常见皮肤病的例子。十肽-12(decapeptide-12)是一种新合成的肽，已被发现在减少黑色素含量方面比对苯二酚更安全，在每天两次治疗16周后，功效高达50％以上。然而，由于其亲水性和大分子量，该肽的经皮渗透有限。因此，十肽-12通过添加棕榈酸酯链进行修饰，试图克服这一限制。我们还测试了化学渗透增强剂和微针通过皮肤输送两种肽的效果。使用体外皮肤渗透模型发现增强的人体皮肤渗透。此外，我们检查了不同配方的肽保留。我们的数据显示微针贴片中的棕榈肽是最有效的。
色素沉着过度和黄褐斑只是可以治疗的病症的两个例子。可以治疗的其他病症包括：神经病学、皮肤病学、肿瘤学、免疫学等。该肽可以治疗内部和皮肤状况，而不仅仅是黄褐斑或色素沉着过度。这种肽具有抗炎和抗衰老功能，影响身体的每个系统和身体的每个器官。因此，这种肽可以涵盖影响身体及其器官和系统的所有病症。
下面提到的器官和系统有两种方法。进一步的抗衰老不需要治疗被认为是疾病或异常的东西。我们也能够对待一些正常的人想要做得更好或改进的东西。
有包括外皮系统、骨骼系统、肌肉系统、淋巴系统、呼吸系统、消化系统、神经系统、内分泌系统、心血管系统、泌尿系统、生殖系统在内的十一个器官系统，该肽可以治疗这些系统或治疗这些系统的病症。
有十四种疾病系统，包括：肌肉骨骼系统；特殊感觉器官(光学)；听觉；传染病、免疫紊乱和营养缺乏症；呼吸系统；心血管系统；消化系统；泌尿生殖系统；血液和淋巴系统；皮肤；内分泌系统；神经系统疾病；精神障碍，以及牙齿和口腔状况。该肽可用于治疗这些疾病和系统。
在考虑以下详细描述和附图后，本发明的其他目的、特征和优点将变得显而易见，在附图中，相同的参考标号代表相同的特征。
附图说明
图1a和图1b显示了肽的结构。两种肽的分子结构：天然肽(1a)及其类似物棕榈肽(1b)。棕榈肽的N端被棕榈酰化，C端被修饰为酰胺，6位酪氨酸由L-变为D-。
图2显示了肽的合成流程。
图3a和图3b显示了两种肽在PG中的三种增强剂中的溶解度。第1组：天然肽；第2组：天然肽+增强剂1；第3组：天然肽+增强剂2；第4组：天然肽+增强剂3；第5组：Palm-D-ISO-Amid；第6组：Palm-D-ISO-Amid+增强剂1；第7组：Palm-D-ISO-Amid+增强剂2；第8组：Palm-D-ISO-Amid+增强剂3。两种不同肽在不同药物递送系统中24小时内的累积渗透曲线。MN贴片中的天然肽和PG中的棕榈肽、含5％(重量/体积)油酸的PG、含5％(重量/体积)樟脑的PG、含5％(重量/体积)薄荷醇的PG和MN贴片：量(a)和百分比(b)。MN＝微针，PG＝丙二醇。
图4a和图4b以量(4a)和百分比(4b)显示了PG中三种增强剂中的两种肽在24小时内的累积皮肤渗透。
图5a和图5b以量(a)和百分比(b)显示了24小时后渗透皮肤的肽的累积量。
图6a和图6b以量(6a)和百分比(6b)显示了两种肽在24小时后的皮肤保留情况。未检测到MN贴片中天然肽的保留，以及5％(重量/体积)樟脑/薄荷醇中棕榈肽的保留。
具体实施方式
黑色素是黑色素生成的最终产物，在吸收细胞质内产生的自由基、保护宿主免受各种类型的电离辐射以及决定人体皮肤、头发和眼睛的颜色方面起着至关重要的作用。然而，黑色素的过量产生会导致皮肤色素沉着过度，这是一种常见且不会危及生命的疾病。黄褐斑是一种色素沉着过度的形式，会导致皮肤出现棕色或灰色斑块，主要发生在面部区域。对苯二酚和维甲酸与局部皮质类固醇相结合，是治疗黄褐斑和色素沉着过度的成熟治疗剂。Hantash和他的团队报告了一种新型专有合成肽，即十肽-12，与对苯二酚相比，它对蘑菇和人类酪氨酸酶的竞争性抑制作用更强。对人类的其他研究证明了Lumixyl(TM)的治疗效果，Lumixyl(TM)是一种含有十肽-12的局部制剂，用于治疗黄褐斑。
虽然十肽-12可以减少黑色素的产生，对治疗黄褐斑有很好的疗效，但十肽-12的渗透特性仍不清楚。如图1a所示，十肽-12是一种具有多个氨基和羟基的极性分子，这可能会阻碍其通过亲脂性角质层(SC)的透皮吸收。为此，有多种选项可供选择，例如分子修饰、使用化学渗透增强剂(CPE)或微针(MN)。
对于分子修饰，许多研究集中在提高用于口服、肺部和鼻腔给药的肽的生物稳定性和生物利用度。由于SC屏障的亲脂性，以两性离子形式存在的肽只能最低限度地渗透皮肤。为此，通过增加其亲脂性对肽进行分子修饰可以增强其皮肤渗透性。在之前的一项研究中，我们已经表明，可以通过修改六肽的结构以使其更具亲脂性来实现增强的皮肤渗透。
化学物质可以与SC内的细胞间脂质相互作用，以增强药物通过皮肤的渗透。许多CPE，例如亚砜、脂肪酸和萜烯，在促进药物通过皮肤的渗透方面发挥着重要作用。据报道，CPE的亲脂性与其增强作用成正比。
除化学增强外，还有物理方法，例如电穿孔、离子电渗疗法、金刚石微晶换肤术、激光辐射和超声波。但成本高等缺点限制了其应用。最近，MN已成为增强各种生物分子(包括寡核苷酸、肽、蛋白质和疫苗)皮肤渗透的强大工具。MN可以应用于皮肤表面，以创建一系列微观通道，生物分子可以通过这些通道到达真皮。此前，我们报道了微针在利多卡因、铜肽和核酸透皮递送中的应用。
在这项研究中，我们使用CPE和MN两者研究了具有更高亲脂性的十肽-12(天然肽，图1a)及其类似物(棕榈肽，图1b)皮肤渗透性，以及它们通过皮肤渗透的能力。丙二醇(PG)是一种广泛使用的CPE，与其他增强剂一起使用时具有协同作用。油酸、樟脑和薄荷醇可以增加药物通过皮肤的渗透。使用人类尸体皮肤样本确定了不同肽递送系统的功效。
表1.基于生物信息学(silico)预测的肽的物理化学特性。
方法
材料
肽和棕榈肽由BIO BASIC(加拿大安大略省)合成。PG和PBS片购自美国VWR。油酸、青霉素-链霉素和三氟乙酸购自美国Sigma-Aldrich。樟脑粉购自澳大利亚NewDirections。薄荷醇晶体购自美国WFmed。磷酸溶液(85重量％的H2O)购自美国SAFC。乙腈从泰国RCI labscan获得。甲醇从美国Honeywell获得。人体表皮由美国Science Care提供。所有材料均按原样使用，无需进一步纯化。
多肽合成与表征
肽和类似物是通过传统的固相化学肽合成方法合成的，使用基于FMOC的合成方法在Rink-amide树脂上对每种化合物进行了特定的修饰(图2)。通过制备型高效液相色谱(HPLC)实现最终产物的纯化。
肽类似物物理化学特性
对两种肽的物理化学特性进行了生物信息学预测。
饱和肽溶液制备
在PG中测定各种肽类似物的溶解度以制备饱和肽溶液。将过量的肽添加到2毫升Eppendorf管内的每种溶剂中，该管在孵育轨道摇床(OM15C，Ratek，澳大利亚)中持续摇动48小时。整个装置保持在室温下。然后将样品以12,000转每分钟(rpm)的速度离心10分钟。随后，将上清液转移至琥珀色安瓿瓶中用于测定。
人皮膜制备
人类切皮皮肤获自Science Care(美国亚利桑那州)。皮肤组织是从两具死亡年龄分别为66岁和57岁的男性尸体的大腿上切除的。研究中使用的皮肤样本没有标识符，也免于伦理审查。在使用前使用目测检查尸体皮肤的完整性，以确保皮肤表面不存在毛孔或破裂。此外，通过观察在第一个采样时间点渗透皮肤的肽量的快速和大量增加，确定了皮肤完整性的任何损害。
微型模具的制造
使用先前报道的方法，通过以嵌入式3M微通道皮肤系统(embedded3MMicrochannel Skin System)作为主模板的聚二甲基硅氧烷(PDMS)的热固化来制造模具。简而言之，将弹性体和固化剂混合并在95千帕下抽真空10分钟至20分钟，以去除夹带的气泡。然后，将混合物缓慢倒入塑料培养皿中。之后，放入70摄氏度的热风烘箱中烘烤2小时。使用手术刀片从培养皿中轻轻取出固化的PDMS。将MN母模从固化的PDMS上轻轻剥离，以获得PDMS模具。
载有棕榈肽的基于薄荷醇的MN贴片的制备
PDMS模具在65摄氏度的热板上加热约10分钟。在加热PDMS模具时，使用加热块(Major Science)将棕榈肽在65摄氏度的比色皿中熔化，直到所有内容物熔化。将2％的棕榈肽加入薄荷醇中并充分混合。然后从热板上取下PDMS模具，将混合材料倒入型腔中。模具放置在室温下，随后在材料固化时脱模。
体外皮肤渗透试验
使用Franz型扩散池。将人表皮安装在供体隔室和受体隔室之间，并修剪掉两侧多余的皮肤以尽量减少横向扩散。SC面向供体隔室，渗透的皮肤面积为1.327平方厘米。受体隔室充满5.5毫升1毫摩尔PBS溶液，其中含有1％(体积/体积)的抗菌抗真菌溶液。将受体溶液过滤两次以防止在表皮下形成气泡。将250微升溶液添加到供体隔室并用包裹物覆盖。首先将载有肽的MN贴片切成相同的适当大小。然后使用手指-拇指握法将贴片贴在皮肤上20秒。使用透明胶带将贴片固定在皮肤上。制备一式三份。供体隔室和采样口用包裹物覆盖以最小化凝胶污染和蒸发。在研究期间使用磁力搅拌器以每分钟180转的速度混合受体溶液。该装置保持在32摄氏度。在不同的时间间隔收集500微升受体流体用于HPLC分析，并用500微升含有1％(体积/体积)抗菌抗真菌溶液的新鲜PBS代替。在24小时内测量渗透。
HPLC药物分析
渗透的肽量使用具有安捷伦ODS C18柱(4.6毫米x 250毫米x 5微米，170埃)的Shimadzu CBM-20A HPLC系统测定。流动相由流动相A(0.1％体积/体积的三氟乙酸水溶液)和流动相B(0.1％体积/体积的三氟乙酸乙腈溶液)组成，采用梯度洗脱程序，溶剂A和B的比例如下：天然肽(15％到35％B持续0到20分钟，15％持续20.01分钟到30分钟)，棕榈肽(55％到45％B持续0到10分钟，55％持续10.01分钟到30分钟)。速率为每分钟1.0毫升。注射体积为50微升，紫外检测在280纳米进行。使用5微克/毫升至50微克/毫升的天然肽标准溶液和1微克/毫升至50微克/毫升的棕榈肽标准溶液建立校准曲线。
统计分析
所有数据均使用GraphPad Prism 8(GraphPad软件公司，加利福尼亚州，美国)整理和准备。使用ANOVA计算统计显着性，p小于(<)0.05被认为具有统计显着性。
结果
肽修饰及物理化学特性
棕榈肽从其母体化合物(即天然肽)进行修饰，以减少两性离子的形成并增加亲脂性(图1a和图1b)。棕榈肽的N端被棕榈酰化，C端被修饰为酰胺，6位酪氨酸由L-变为D-。C端酯化后，如在棕榈肽中所见，最初可电离的羧基基不再形成带电离子。棕榈肽显示出比天然肽更高的LogP，为-0.751。然而，这两种肽都具有高分子量，使其难以渗透皮肤。
肽的体外皮肤渗透
PG常用于化妆品中，以其共溶剂和增强渗透作用而闻名。因此，纯PG和含有5％(重量/体积)增强剂的PG也用于制备肽溶液。不同给药系统中MN贴片和棕榈肽中天然肽的含量如图3a所示。在含有5％(重量/体积)薄荷醇的PG中观察到棕榈肽的最高渗透。如图3b所示，根据剂量标准化后，与其他制剂相比，MN贴片中渗透皮肤的天然肽百分比明显更高(P小于(<)0.0001)。
24小时内的累积肽渗透
两种肽在24小时内从不同载体的累积渗透如图4a和图4b所示。在5％(重量/体积)的薄荷醇中发现棕榈肽的渗透率最高(图4a)。对于MN贴片，渗透皮肤的天然肽量明显高于棕榈肽(P小于(<)0.0001)。按剂量标准化后(图4b)，MN贴片中的天然肽渗透率显着高于其他贴片(P小于(<)0.01)。
人体皮肤肽保留
由于肽减少了表皮黑色素含量，我们测量了这两种肽在24小时后的皮肤保留情况(图5a和图5b)。在以下三种配方的皮肤样本中未检测到肽：MN贴片中的天然肽、5％樟脑中的棕榈肽和5％薄荷醇中的棕榈肽。对于其余的配方，它们之间没有发现显着差异(图5a)。剂量归一化后(图5b)，当使用MN贴片时，棕榈肽保留在皮肤中的百分比显着高于其他两种配方(P小于(<)0.01，P小于(<)0.001)。
讨论
肽的皮肤屏障和修饰
皮肤的最外层亲脂层，称为SC，是经皮渗透的主要障碍。该亲脂层确保只有小的和中等亲脂性的分子才能以足够的量穿过皮肤以引起治疗相关的效果。具有大分子量和亲水性的肽不能以足够的量渗透SC。因此，我们对天然肽的末端进行了修饰，合成了一种新的肽，即棕榈肽。由于棕榈肽比天然肽具有更高的亲脂性，因此在PG溶液中24小时的渗透量和皮肤保留量相应增加(图4a和图4b以及图5a和图5b)。它表明化学修饰在使用PG溶液促进肽透皮递送方面的可行性。
肽的皮肤保留
由于这两种肽靶向黑素细胞发挥其治疗作用，而黑素细胞位于表皮的基底层，我们评估了肽在侧皮膜中的保留。与使用MN贴片递送时无法检测到的天然肽皮肤保留相比，在MN贴片递送时棕榈肽有明显的皮肤保留(图6a和图6b)。因此，天然肽的结构修饰可以通过增加其皮肤保留性来大大提高其局部功效。
表2.三种化学渗透增强剂的特性。
CPE对肽皮肤渗透的影响
数十年来，CPE一直被研究用于促进药物透皮给药，并且许多都改善了透皮给药。一种作用机制被描述为“拉-推”效应。CPE和药物之间的高溶解度参数差异会推动药物分子通过SC。根据该理论并考虑到两种肽之间的性质差异，我们选择了具有高亲脂性的油酸及亲水性的樟脑和薄荷醇(表2)，之前曾报道过它们可以增强透皮渗透。我们预计含有5％(重量/体积)油酸的PG将表现出最佳的皮肤渗透和皮肤保留改善，因为这两种肽的LogP差异最大。
然而，结果与油酸对皮肤渗透具有最佳增强作用的预期不一致。对于天然肽，三种增强剂不会改变皮肤渗透。对于棕榈肽，樟脑和油酸有抑制作用，而薄荷醇有促进皮肤渗透的作用。这可能是由于CPE和皮肤之间的相互作用导致棕榈肽的分配行为改变。
MN对肽皮肤渗透的影响
在这项研究中，在皮肤渗透方面，MN贴片中的两种肽都显示出比CPE更多的益处。此外，天然肽比棕榈肽表现出更好的皮肤渗透性。这可以归因于天然肽在水中的溶解度高于棕榈肽。MN通过在皮肤中形成微型通道来破坏皮肤屏障，因此MN贴片中的天然肽可以直接输送到受体室中。天然肽作为具有高极性的大分子，倾向于扩散到受体PBS溶液中，其中天然肽可混溶。
MN对肽皮肤保留的影响
对于皮肤保留，在MN贴片中观察到棕榈肽保留在皮肤内的百分比最高，与其他组明显不同，尽管棕榈肽在MN贴片保留在皮肤内的百分比仍然很低(小于(<)0.1％)。这可能是因为MN破坏了皮肤屏障，肽直接输送到真皮和表皮。此外，棕榈肽较高的亲脂性有利于经皮渗透。另一方面，24小时后在皮肤样本中未检测到天然肽。这可能是由于两个原因。首先，天然肽比棕榈肽更具极性，导致其扩散到受体隔室的水溶液中。其次，MN贴片内的肽量非常有限，导致尽管渗透性高，但皮肤中的天然肽保留最少。
皮肤内棕榈肽浓度的估计
棕榈肽在皮肤中的最高浓度达到9.0微克/克，接近细胞实验的有效浓度(约13.95微克/克，根据10微摩尔估算得出)；更重要的是，根据细胞毒试验，只有当浓度超过100微摩尔时，肽才会抑制黑色素细胞的活力和增殖，远远超过皮肤所能达到的浓度。然而，没有证据表明棕榈肽浓度在皮肤中的功效，因此，需要进行更详细的药效学实验。
供体隔室中的无限剂量与有限剂量
在实验过程中供体侧渗透物的耗尽通常会导致渗透率降低，并最终导致累积渗透曲线达到平稳状态。另一方面，如果渗透剂以无限剂量应用，则供体侧有足够的渗透剂，可以忽略供体浓度的任何变化。无限剂量实验是那些在供体隔室中典型剂量超过每平方厘米10毫克的实验。
在这项研究中，MN贴片被用作有限剂量条件，并表现出递减率和累积渗透曲线的平台(图3a和图3b)。然而，该理论不适用于棕榈肽溶液的渗透曲线，该溶液被用作无限剂量条件(大于(>)每平方厘米13毫克)，但也会导致速率降低。
用于透皮递送和肽修饰的理想LogP
以往的研究表明，药物渗透的理想LogP约为2。这表明棕榈肽的LogP(-0.751)与理想值之间存在较大差异。
在这项研究中，我们修改了天然肽以增加其亲脂性。以前，我们报道了通过改变侧链的官能团来改善肽在皮肤上的渗透，这将亲脂性从LogP的-6.37增加到1.75。这可能提出了可行的解决方案。
结论
在研究中，我们使用化学修饰、CPES和MN贴片来改善肽的皮肤渗透和保留。CPE对皮肤渗透有积极影响，但不会促进棕榈肽在皮肤上的保留。MN贴片改善了棕榈肽的皮肤渗透和保留。对于靶向位于表皮基底层的黑素细胞的肽，结构修饰和MN贴片可以提高其功效。为了进一步改善肽的皮肤保留，可以测试修饰氨基侧链而不是末端的结构。
本发明的描述是为了说明和描述的目的而给出的。其并非旨在详尽无遗或将本发明限制为所描述的精确形式，并且根据上述教导可以进行许多修改和变化。选择和描述实施方案是为了最好地解释本发明的原理及其实际应用。该描述将使本领域的其他技术人员能够在各种实施方案中以及根据适合于特定用途的各种修改来最好地利用和实践本发明。本发明的范围由所附权利要求限定。
Claims (20)
1.通过执行细胞渗透来治疗受试者的方法，该方法包括向有此需要的受试者施用包含有效量的一种或多种肽的组合物，其中所述一种或多种肽包括：
(a)
2.根据权利要求1所述的方法，其中所述细胞是哺乳动物细胞。
3.根据权利要求2所述的方法，其中所述哺乳动物细胞是皮肤细胞。
4.根据权利要求3所述的方法，其中所述皮肤细胞是黑色素细胞。
5.根据权利要求1所述的方法，其中所述施用是通过微针进行的。
6.根据权利要求1所述的方法，其中所述施用是通过化学渗透增强剂进行的。
7.根据权利要求6所述的方法，其中所述化学渗透增强剂包括丙二醇、油酸、樟脑和薄荷醇中的至少一种。
8.根据权利要求7所述的方法，其中所述一种或多种肽以约5％(重量/体积)的浓度与薄荷醇一起存在。
9.根据权利要求1所述的方法，其中：
所述一种或多种肽由以下组成：
(a)并且所述施用是通过微针进行的。
10.根据权利要求1所述的方法，其中：
一种或多种肽由以下组成：
并且所述施用是通过化学渗透增强剂进行的。
11.肽，由以下组成：
12.用于治疗患有病症的受试者的治疗剂，其包含有效量的一种或多种肽，其中所述一种或多种肽包括：
(a)
13.根据权利要求12所述的治疗剂，其中所述病症包括以下中的至少一种：色素沉着过度、黄褐斑、神经病学、皮肤病学、肿瘤学、免疫学、抗炎功能、抗衰老功能、外皮系统、骨骼系统、肌肉系统、淋巴系统、呼吸系统、消化系统、神经系统、内分泌系统、心血管系统、泌尿系统、生殖系统、肌肉骨骼系统、特殊感觉器官(视觉)、听觉、传染性疾病、免疫失调、营养缺乏、呼吸系统、心血管系统、消化系统、泌尿生殖系统、血液和淋巴系统、皮肤、内分泌系统、神经系统病症、精神障碍或牙科和口腔。
14.根据权利要求12所述的治疗剂，还包含化学渗透增强剂。
15.根据权利要求14所述的治疗剂，其中所述化学渗透增强剂包括丙二醇、油酸、樟脑和薄荷醇中的至少一种。
16.根据权利要求14所述的治疗剂，其中：
所述化学渗透增强剂包括薄荷醇；并且
所述至少一种或多种肽由以下组成：
。
17.根据权利要求16所述的治疗剂，其中所述薄荷醇以约5重量％存在。
18.治疗病症的试剂盒，包括：
一种或多种肽，其中所述一种或多种肽包括：
(a)
19.根据权利要求18所述的试剂盒，还包括微针。
20.根据权利要求18所述的试剂盒，还包含化学渗透增强剂。
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