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CN113015904B - 用于筛选个人护理产品的方法 - Google Patents

用于筛选个人护理产品的方法 Download PDF

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CN113015904B
CN113015904B CN201980074656.9A CN201980074656A CN113015904B CN 113015904 B CN113015904 B CN 113015904B CN 201980074656 A CN201980074656 A CN 201980074656A CN 113015904 B CN113015904 B CN 113015904B
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K·S·韦
T·迪可兰德里亚
D·R·芬利
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Abstract

筛选洗去型个人护理组合物的方法可包括结合水分和/或细胞增殖的测量使用外植体皮肤。

Description

用于筛选个人护理产品的方法
技术领域
本公开一般涉及利用皮肤外植体来筛选个人护理产品的方法。
背景技术
随着时间的推移，皮肤的清洁已成为个人卫生方案的一部分。由于该方案已被更广泛地采用，公司和个人已开发出用于清洁皮肤的产品。然而，皮肤是复杂的器官。有时，已开发用于清洁皮肤的产品通过过度干燥皮肤或损害皮肤的屏障性能而对皮肤有害。虽然已开发出一些测试方法来帮助筛选产品并了解它们对皮肤的影响，但这些方法常常依赖于不是真正预测性的和/或不是消费者相关的体外方法。此外，目前的体内临床研究昂贵、耗时并且通量有限。因此，需要用于个人护理产品的附加筛选方法。
发明内容
例如，一种筛选洗去型个人护理组合物的方法，包括：a.从第一培养基中选择第一皮肤外植体；b.向所述第一皮肤外植体施用对照产品；c.加入水并用对照产品洗涤所述第一皮肤外植体；d.用水从所述第一皮肤外植体冲洗掉所述对照产品并干燥所述第一皮肤外植体；e.将所述第一皮肤外植体于所述第一培养基中放置约24小时；f.从第二培养基中选择第二皮肤外植体；g.向所述第二皮肤外植体施用洗去型个人护理组合物；h.加入水并用所述洗去型个人护理组合物洗涤所述第二皮肤外植体；i.从所述第二皮肤外植体冲洗掉所述洗去型个人护理组合物并干燥所述第二皮肤外植体；j.将所述第二皮肤外植体于所述第二培养基中放置约24小时；k.针对所述对照产品，重复(b)至(e)持续至少两天；l.针对所述洗去型个人护理组合物，重复(g)至(j)持续至少两天；m.将所述第一皮肤外植体的状况和所述第二皮肤外植体的状况进行比较，以评估所述对照产品相对于所述洗去型个人护理组合物的功效。
下文将更全面地描述该示例和其他示例。
附图说明
图1为示出用皮肤外植体评价个人护理组合物的示例性时间线的图；
图2为如图1所示的示例性每日洗涤程序的图示；
图3为示出如通过笔式水分计(moisture pen)所测量，在第7天和第14天用水、个人护理产品(测试A)、个人护理产品(测试B)处理的皮肤外植体的皮肤水合的图；
图4为示出如通过MTT测定所测量，在第7天和第14天用水、个人护理产品(测试A)、个人护理产品(测试B)处理的皮肤外植体的MTT皮肤活力的图；
图5为示出如通过Ki67染色方法所测量，在第7天和第14天用水、个人护理产品(测试A)、个人护理产品(测试B)处理的皮肤外植体中Ki67活性水平的图；并且
图6为示出如通过TUNEL测定所测量，在第7天和第14天用水、个人护理产品(测试A)、个人护理产品(测试B)处理的皮肤外植体中TUNEL阳性细胞的图。
具体实施方式
定义
“个人护理组合物”是指旨在局部施用至皮肤或毛发的组合物。个人护理组合物可从包装件中挤出或分配。个人护理组合物可为例如液体、半液体霜膏、洗剂或凝胶的形式。个人护理组合物的示例可包括但不限于香波、调理香波、沐浴剂、保湿沐浴剂、沐浴凝胶、皮肤清洁剂、清洁乳、洗发剂和沐浴剂、沐浴保湿剂、宠物香波、剃刮制剂和与一次性清洁布一起使用的清洁组合物。
如本文所用，“洗去型”是指局部施用到皮肤和/或毛发上并且旨在随后在施用数秒至数分钟内用水从皮肤和/或毛发上冲洗掉的组合物。洗去型产品的示例为沐浴剂。还可使用基底将产品擦去。
个人护理产品如个人清洁剂可有助于维持皮肤。清洁剂可有助于从皮肤移除污垢、碎屑和其他物质。清洁剂还可影响皮肤的水分、屏障和其他特性。在公司决定在市场上投入新的清洁剂之前，他们可能希望测试产品以了解其对皮肤的影响或者了解其对特定属性的性能或在某些情况下的性能。
这类测试可以多种方式进行。例如，可将清洁剂置于管中并摇动，以便接近产品在使用期间将产生的泡沫。当涉及测量产品对皮肤的影响时，这可能更复杂。公司可利用志愿者进行临床研究，但这些测试可能非常昂贵，可能花费很长时间，并且常常在一次可测试的产品数量方面受到限制。同样，在皮肤模拟物或人造皮肤上进行的测试可给出难以关联回真实皮肤或仅不能很好地预测对真实皮肤的影响的结果，因为它们可能缺乏人类皮肤的互连性、渗透性和/或复杂性。
测试个人护理产品的一种有益方式是用外植体皮肤测试。外植体皮肤是在医疗手术(如脂膜切除术)期间已从个人移除的皮肤。使用实际皮肤具有其优点。其比其中对活体人类受试者进行测试的临床研究便宜，并且允许筛选更多的产品(即，更高的通量)。此外，结果还允许更深入地了解产品对皮肤的影响的机械性质。
使用外植体皮肤的一个缺点是其具有有限的活力。为了延长皮肤外植体的活力，可培养外植体。例如，可将外植体置于具有适当培养基的容器中。合适的培养基的一个示例是达尔伯克氏改良伊格尔培养基(Dulbecco’s Modified Eagle Medium)。该培养基向皮肤外植体提供营养物质。该培养基可为等渗透的或非渗透的。该培养基可包含附加的添加剂，如抗霉菌剂、抗细菌剂、氨基酸、维生素、或它们的组合。此外，可移除皮肤外植体的皮下层以改善培养期间培养基向外植体中的吸收。外植体皮肤准备的示例包括在下文的实施例1中。
作为培养程序的一部分，可使用多孔膜。此类膜通常放置在皮肤外植体的下面，使得膜处于培养基中，并且培养基通过毛细管作用穿过膜来喂送皮肤外植体。可能期望的是，基于皮肤样本的尺寸和/或类型来选择膜，该膜允许培养基以特定速率穿过它。合适的膜的一个示例是品牌。
优化皮肤外植体活力的另一种机制是通过其储存参数。这些参数可包括例如时间、温度、湿度、二氧化碳水平等。可将皮肤外植体培养例如约2天至约19天、约5天至约19天、约5天至约14天、约5天至约10天、或约6天至约8天。可为例如33℃至40℃、35℃至40℃、或甚至36℃至38℃的培养温度可为合适的，但基于目标属性，其他温度也可为可接受的。用于培养皮肤外植体的湿度水平可为例如小于100％湿度、40％至90％湿度、50％至70％湿度、或者在小于30％、或20％至30％湿度的低湿度范围内。二氧化碳水平可为例如5％。
此外，还可操纵储存参数以模拟皮肤状况。例如，可将储存条件设定为低湿度，如20％-30％，以模拟冬季条件和/或生成模拟干燥皮肤的皮肤外植体。
外植体皮肤可用于筛选个人护理组合物的功效。可进行该测试，以评估个人护理组合物相对于对照如水，或者将组合物彼此进行比较。个人护理组合物的功效可涉及皮肤外植体的水分含量。个人护理组合物可影响皮肤的水分含量。皮肤外植体的水分含量的变化可暗示产品可具有对皮肤的干燥影响(对于水分含量相对于水降低)，中性影响(意味着相对于水相对没有变化)，或者可为保湿的(对于水分含量相对于水提高)。同样，在将个人护理组合物与第二组合物进行比较的情况下，皮肤外植体的水分含量可暗示产品在其干燥、中性或保湿影响方面的差异或类似性。
皮肤外植体的水分含量可根据工业标准测试进行测量。用于测量皮肤外植体的水分含量的方法的一些示例包括表皮电导、表皮电容、生物阻抗、扩散、以及它们的组合。表皮电导可用笔式水分计来测量。可将笔式水分计的探头置于皮肤外植体上，在皮肤外植体中其可测量皮肤中的水分水平以及/或者在皮肤外植体超过预设水分水平的情况下给出通知。笔式水分计的一个示例是由DermaLab制造的。
表皮电容可用皮肤水分测试仪(corneometer)来测量。皮肤水分测试仪可用于通过将探头抵靠皮肤外植体放置来测量皮肤外植体的水分。皮肤水分测试仪使用电介质的电容测量来检测水合水平。皮肤水分测试仪的一个示例是由Courage-Khazaka Electronic制造的CM 825。
生物阻抗可利用Skicon仪器来测量。Skicon仪器可用于通过将探头置于皮肤外植体上来测量皮肤外植体的水分。可接受的Skicon仪器的一个示例是由IBS Company制造的
个人护理组合物的功效还可通过观察来自皮肤外植体的水分扩散来评估。个人护理组合物可影响皮肤屏障保持水分的能力。通过皮肤外植体的水分损失水平可暗示个人护理组合物是否将影响皮肤屏障。皮肤外植体的水分扩散水平可测量为经皮水损失。如果经皮水损失水平高于水对照或比较产品的经皮水损失水平，则测试产品可对皮肤屏障功能具有负面影响；如果经皮水损失水平相同或类似，则测试产品具有中性影响(相对于水对照或在功效上类似于比较产品)；或者如果水损失小于水对照或比较产品的水损失，则所述产品可对屏障功能没有影响或对屏障功能的影响小于对照或测试产品。经皮水损失可用仪器如由制造的AquaFluxTM或用由Delfin Company制造的VapoMeter来测量。经皮水损失通过将皮肤外植体置于具有测量来自皮肤外植体的水蒸气的分压的两个传感器的圆筒中来测量，并且两个传感器之间的梯度与蒸发速率成正比。
个人护理组合物的功效还可通过皮肤外植体中细胞代谢活性来检测。个人护理组合物可影响皮肤外植体中细胞的代谢活性。皮肤外植体的细胞中的代谢活性水平可暗示个人护理组合物是否将影响皮肤外植体的状况。如果测试产品的细胞代谢活性水平高于水对照或比较产品的细胞代谢活性水平，则测试产品更有效(即，对皮肤更好)；如果细胞代谢活性水平相同或类似，则测试产品具有中性影响(相对于水对照或在功效上类似于比较产品)；或者如果细胞代谢活性小于水对照或比较产品的细胞代谢活性，则测试产品不太有效(即，对皮肤更差)。
细胞代谢活性可通过MTT测定来评估。MTT测定使用NAD(P)H-依赖性细胞氧化还原酶以反映存在的活细胞的数量。这些酶能够将四唑染料MTT 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物还原为其不溶性甲臜(呈紫色)。快速分裂的细胞表现出高MTT还原速率。
个人护理组合物的功效还可通过测量与细胞增殖相关的蛋白质水平来评价。此类蛋白质的一个示例处于Ki67蛋白质中。个人护理组合物可影响皮肤外植体中细胞的Ki67蛋白质水平。皮肤外植体的细胞中的Ki67蛋白质水平可暗示个人护理组合物是否将影响皮肤外植体的潜在健康。如果Ki67蛋白质水平高于水对照或比较产品的此蛋白质水平，则测试产品对皮肤外植体的潜在状况更好；如果此蛋白质水平相同或类似，则测试产品具有中性影响(相对于水对照或在功效上类似于比较产品)；或者如果Ki67蛋白质水平小于水对照或比较产品的此蛋白质水平，则测试产品对潜在状况更差。Ki67蛋白质水平可通过Ki67染色测定来测量。
个人护理组合物的功效可通过测量天然保湿因子(NMF)生物标志物的表达水平来评价。NMF生物标志物与皮肤外植体维持其水合水平的能力相关。个人护理组合物可影响皮肤外植体中NMF生物标志物的表达水平。皮肤外植体中NMF生物标志物的表达水平可暗示个人护理组合物是否将影响皮肤维持水合的能力。如果NMF生物标志物表达水平高于水对照或比较产品的此生物标志物表达水平，则测试产品在保持皮肤外植体维持水合的能力方面更好；如果此生物标志物表达水平相同或类似，则测试产品具有中性影响(相对于水对照或在功效上类似于比较产品)；或者如果NMF生物标志物表达水平小于水对照或比较产品的此生物标志物表达水平，则测试产品在保持皮肤维持水合的能力方面更差。NMF生物标志物的表达水平可通过标准分析仪器诸如质谱仪来测量。
个人护理组合物的功效还可通过测量凋亡DNA片段化的水平来评价。用于测量此的方式的一个示例是TUNEL测定。TUNEL测定可利用标记有标记物的末端脱氧核苷酸转移酶来确定凋亡水平。个人护理组合物可影响皮肤外植体中凋亡DNA片段化的水平。皮肤外植体的凋亡DNA片段化水平可暗示个人护理组合物是否将影响皮肤外植体的潜在健康。如果凋亡DNA片段化水平高于水对照或比较产品的凋亡DNA片段化水平，则测试产品对皮肤外植体的潜在状况更差；如果凋亡DNA片段化水平相同或类似，则测试产品具有中性影响(相对于水对照或在功效上类似于比较产品)；或者如果凋亡DNA片段化水平小于水对照或比较产品的凋亡DNA片段化水平，则测试产品对潜在状况更好。
如上所述，使用外植体皮肤来评价洗去型个人护理组合物的功效。采购外植体皮肤并为使用做准备。为使用做准备可包括例如洗涤和干燥、移除皮下层、将外植体皮肤切割成适当尺寸的片等。将皮肤外植体置于培养基中。在第1天，在产品施用之前，进行水分、MTT皮肤活力、Ki67阳性细胞和TUNEL阳性细胞的基线测量。在基线测量之后，向皮肤施用测试产品。在此情况下，向外植体皮肤的单独片施用测试产品(水、第一个人护理组合物(测试A)和第二个人护理组合物(测试B)。
可如图2所示施用个人护理组合物。将外植体皮肤从培养基培养物中取出并置于烧杯中。向烧杯中的一片外植体皮肤施用50μL的个人护理组合物。使用泡夫将个人护理组合物铺展在外植体皮肤上。然后向烧杯加入1.33mL的蒸馏水。然后使用泡夫将外植体皮肤的表皮侧洗涤30秒。将外植体皮肤留在烧杯中90秒。然后通过倒出液体来移除具有外植体皮肤的烧杯中的液体。然后将外植体皮肤冲洗两次，每次用1mL的蒸馏水冲洗。将外植体皮肤从冲洗水中取出并用无菌棉擦干。然后将外植体皮肤置于具有新鲜培养基的容器中。当测试产品为水时，其施用犹如其为上述个人护理组合物。测试产品可每天施用一次，持续14天，如图1所例示，其中测试在第1天、第7天和第14天发生。
对于水对照组、个人护理组合物测试A(A)和个人护理组合物测试B(B)的皮肤外植体的水分在基线、第7天和第14天测量，如图1和图3所示。用笔式水分计测量水分。将笔式水分计置于皮肤外植体的表面上，在此处笔式水分计给出水分读数。如由图3可见，用笔式水分计测量个人护理组合物测试A和测试B以及水对照。在7天和14天，其上放置有个人护理组合物A的皮肤外植体具有比水对照和个人护理组合物B两者显著更高的水分含量。这示出了测试A具有比水对照和测试B更好的皮肤保湿。
如在图4中可见，测试用水对照、个人护理组合物测试A和个人护理组合物测试B处理的皮肤外植体的MTT皮肤活力。在第7天，个人护理组合物测试A的MTT皮肤活力高于水对照和个人护理组合物测试B，并且在第14天的时间段高于个人护理组合物B。这示出了测试组合物A具有比水对照和组合物测试B更好的细胞活力和皮肤状况。
同样在第7天和第14天测试与个人护理组合物测试A、个人护理组合物测试B和水对照接触的外植体皮肤的Ki67活力。在第7天，个人护理组合物A的Ki67活性高于水对照和个人护理组合物B，并且在第14天的时间段高于个人护理组合物B(参见图5)。这示出了组合物测试A具有比水对照和组合物测试B更好的Ki67活力和皮肤状况。
图6示出了同样在第7天和第14天测试细胞中TUNEL表达的个人护理组合物测试A、个人护理组合物测试B和水对照的测试结果。个人护理组合物测试A在第7天类似于水对照组且小于个人护理组合物测试B，并且在第15天远小于个人护理组合物测试B。这表明产品A可具有与水对照类似的细胞毒性，而产品B与水对照和产品A相比在细胞毒性方面表现更差。
合起来看，与水对照和测试产品B相比，测试产品A在皮肤水合方面具有显著更好的性能，并且对潜在皮肤状况具有影响较小。
实施例
实施例1：皮肤外植体准备
用于准备的材料包括：达尔伯克氏改良伊格尔培养基加谷氨酰胺、抗生素和抗霉菌剂、1X磷酸盐缓冲溶液(PBS(-ca，-mg)、150mm×15mm无菌培养皿、无菌纱布4×4、一次性安全手术刀、4mm一次性活检穿孔器、6孔细胞培养插入物、消毒镊子、棉签、6孔培养板、任选地具有12×12直尺的直纹消毒切割垫、刮刀柄部、组织冻存培养基、活检盒、冷冻组织冻存管、10％福尔马林、用于固定组织的运输容器、以及生物危害/锐器容器。
皮肤准备可涉及在皮肤到达之前准备培养基(3X培养基，包含500ml DMEM加15ml的抗生素和抗霉菌剂；3X是指用于初始温育小时的抗生素/抗霉菌剂的3倍水平；1X培养基：500ml DMEM加5ml的抗生素和抗霉菌剂)。
到达时，将皮肤从包装中取出，浸泡在3X DMEM培养基中并于4℃保存1小时。1小时后，将皮肤从3X DMEM中取出并用无菌1X PBS冲洗。将皮肤置于含有用1X DMEM浸泡的纱布的干净培养皿中。可在到达当天将样本置于培养物中；然而如果需要，可将样本于4℃保存过夜。
在培养物准备中，可对皮肤组织进行如下操作。在切割垫上，皮肤组织被定位成使得表皮向下，并且暴露脂肪。通过用无菌锯齿状镊子小心地抓住皮肤的一角并用与皮肤保持45度角的刀片刮除脂肪来用一次性手术刀移除脂肪。在移除脂肪后，可使用刮刀作为直的边缘导向件，用手术刀切下外科边缘，并将皮肤保持在适当位置。使用刮刀作为导向件，可使用直尺(诸如任选地设置在切割垫上的直尺)将皮肤切割成期望的形状和尺寸(诸如1.25cm宽的条带)以测量宽度。将该条带在含有1X PBS的100mm培养皿中冲洗。将该条带切成1.25cm的正方形，并置于100mm培养皿中的1X DMEM浸泡的纱布上，直到它们在30分钟后放入培养物中(其中15分钟、30分钟、45分钟或一小时的时间点用于各种非限制性示例中)。
实施例2：个人护理组合物测试A是被称为OlayTM超级保湿沐浴剂的商业沐浴剂。关键成分是：水、凡士林、十三烷醇聚醚硫酸钠、氯化钠、椰油酰胺基丙基甜菜碱、十三烷醇聚醚-3、芳香剂、牛油果树果脂(乳木果油)、瓜尔羟丙基三甲基氯化铵、苯甲酸钠、黄原胶、油酸甘油酯、乙二胺四乙酸二钠、柠檬酸、氢氧化钠、丙烯酸酯/丙烯酸C10-30烷基酯交联聚合物等。该产品可利用标准工业方案制备。
实施例3：个人护理组合物测试B是被称为DoveTM深层保湿的商业沐浴剂。关键成分是：水、椰油酰胺基丙基甜菜碱、羟丙基淀粉磷酸钠、月桂酸、月桂酰甘氨酸钠、月桂酰羟乙基磺酸钠、氢化大豆油、野大豆(大豆)油或油葵(向日葵)籽油、氯化钠、甘油、芳香剂、瓜尔羟丙基三甲基氯化铵、DMDM乙内酰脲、硬脂酸、柠檬酸、BHT、乙二胺四乙酸四钠等。该产品可利用标准工业方案制备。
本文所公开的量纲和值不应理解为严格限于所引用的精确数值。相反，除非另外指明，否则每个此类量纲旨在表示所述值以及围绕该值功能上等同的范围。例如，公开为“40mm”的量纲旨在表示“约40mm”。
除非明确排除或以其他方式限制，本文中引用的每一篇文献，包括任何交叉引用或相关专利或专利申请以及本申请对其要求优先权或其有益效果的任何专利申请或专利，均据此全文以引用方式并入本文。对任何文献的引用不是对其作为与本发明的任何所公开或本文受权利要求书保护的现有技术的认可，或不是对其自身或与任何一个或多个参考文献的组合提出、建议或公开任何此类发明的认可。此外，当本发明中术语的任何含义或定义与以引用方式并入的文献中相同术语的任何含义或定义矛盾时，应当服从在本发明中赋予该术语的含义或定义。
虽然已举例说明和描述了本发明的具体实施方案，但是对于本领域技术人员来说显而易见的是，在不脱离本发明的实质和范围的情况下可作出各种其他变化和修改。因此，本文旨在于所附权利要求中涵盖属于本发明范围内的所有此类变化和修改。

Claims (17)

1.一种筛选洗去型个人护理组合物对于干燥皮肤益处的方法，包括：
a.从第一培养基中选择第一皮肤外植体，其中所述第一皮肤外植体在20％至30％湿度的水平下培养以模拟干燥皮肤；
b.向所述第一皮肤外植体施用对照产品；
c.加入水并用对照产品洗涤所述第一皮肤外植体；
d.用水从所述第一皮肤外植体冲洗掉所述对照产品并干燥所述第一皮肤外植体；
e.将所述第一皮肤外植体于所述第一培养基中放置24小时；
f.从第二培养基中选择第二皮肤外植体，其中所述第二皮肤外植体在20％至30％湿度的水平下培养以模拟干燥皮肤；
g.向所述第二皮肤外植体施用洗去型个人护理组合物；
h.加入水并用所述洗去型个人护理组合物洗涤所述第二皮肤外植体；
i.从所述第二皮肤外植体冲洗掉所述洗去型个人护理组合物并干燥所述第二皮肤外植体；
j.将所述第二皮肤外植体于所述第二培养基中放置24小时；
k.针对所述对照产品，重复(b)至(e)持续至少两天；
l.针对所述洗去型个人护理组合物，重复(g)至(j)持续至少两天；
m.将所述第一皮肤外植体的状况和所述第二皮肤外植体的状况进行比较，以评估所述对照产品相对于所述洗去型个人护理组合物的功效。
2.根据权利要求1所述的方法，其中所述对照产品相对于所述洗去型个人护理组合物的所述功效通过水分含量来评估，其中更高的水分含量更有效。
3.根据权利要求2所述的方法，其中所述水分含量经由表皮电导、表皮电容、生物阻抗、扩散、或它们的组合来测量。
4.根据权利要求3所述的方法，其中所述表皮电导用笔式水分计来测量。
5.根据权利要求3所述的方法，其中所述表皮电容通过皮肤水分测试仪来测量。
6.根据权利要求3所述的方法，其中所述生物阻抗通过Skicon仪器来测量。
7.根据权利要求1所述的方法，其中所述对照产品相对于所述洗去型个人护理组合物的所述功效通过细胞代谢活性来评估，其中更高的细胞代谢活性更有效。
8.根据权利要求7所述的方法，其中细胞代谢活性通过MTT测定来评估。
9.根据权利要求1所述的方法，其中所述对照产品相对于所述洗去型个人护理组合物的所述功效通过测量保湿生物标志物的表达水平来评估。
10.根据权利要求9所述的方法，其中所述保湿生物标志物为NMF生物标志物，其中更高的NMF生物标志物的表达水平更有效。
11.根据权利要求10所述的方法，其中所述NMF生物标志物的水平通过质谱来评估。
12.根据权利要求1或2所述的方法，其中所述对照产品为水。
13.根据权利要求1或2所述的方法，其中所述对照产品为第二个人护理组合物。
14.根据权利要求1或2所述的方法，其中重复(k)和(l)每天一次，持续3天至28天。
15.根据权利要求1或2所述的方法，其中重复(k)和(l)每天一次，持续5天至21天。
16.根据权利要求1或2所述的方法，其中重复(k)和(l)每天一次，持续5天至14天。
17.根据权利要求1或2所述的方法，其中重复(k)和(l)每天一次，持续7天至10天。
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