This annex provides information for an in vivo stratum corneum sampling (or tape stripping (TS)) study for semi-solid formulations as a permeation kinetic method to show equivalence, in lieu of a therapeutic equivalence study.

本附录提供了半固体制剂的体内角质层取样（或胶带剥离（TS））研究的信息，作为渗透动力学方法，以显示等效性，而不是治疗等效性研究。

The S.C. sampling study is a minimally invasive technique that involves sequential removal of the outermost skin layer (i.e., the stratum corneum (S.C.)) using adhesive tapes after application of a drug-containing formulation. The amount of drug in the S.C. depends on three main processes: drug partitioning from the formulation into the SC, drug diffusion across the S.C., and drug partitioning out of the S.C. into the viable tissues. A major advantage of TS is that the experiment is conducted in vivo with a fully functioning cutaneous microcirculation so that drug clearance from the skin is unimpeded.

S.C.取样研究是一种微创技术，涉及在施用含药物制剂后使用胶带依次去除最外层皮肤（即角质层（S.C.））。S.C.中的药物量取决于三个主要过程：药物从制剂分配到SC中，药物在S.C.中扩散，药物从S.C.分配到活组织中。TS的一个主要优点是，该实验在体内进行，具有完全功能的皮肤微循环，因此药物从皮肤的清除是不受阻碍的。

TS data provide direct measurements and information on the local bioavailability of semi-solid drug products that act on or in the S.C. e.g. antifungal products. In cases when the target sites of action are beyond the S.C., TS data may provide a suitable surrogate to characterise the rate and extent of drug absorption to the underlying tissues.

TS数据提供了作用于S.C.或在S.C.中的半固体药物产品（例如抗真菌产品）的局部生物利用度的直接测量和信息。在目标作用位点超出S.C.的情况下，TS数据可以提供一个合适的替代物，以表征药物吸收到底层组织的速率和程度。

In vivo TS studies are only applicable for products where drug diffusion into and through the SC takes place. Thus, TS should not be used for testing of drug products to be applied on significantly damaged skin (e.g. open wounds, burns) or skin of premature new-born. In addition, any products that contain volatile drugs or target primarily the cutaneous appendages (e.g. hair follicles, sebaceous glands) are also not suitable.

体内TS研究仅适用于药物扩散进入和通过SC的产品。因此，TS不应用于测试应用于严重受损皮肤（如开放性伤口、烧伤）或早产新生儿皮肤的药物产品。此外，任何含有挥发性药物或主要针对皮肤附件（如毛囊、皮脂腺）的产品也不适用。

A TS study is not an automated process and careful consideration of the experimental design is vital. The experimental conditions of the pivotal study should be assessed individually for the concerned products and should be established by performing a pilot TS study. A summary of the development and optimisation of the TS method should be provided.

TS研究不是一个自动化的过程，仔细考虑实验设计至关重要。关键研究的实验条件应针对相关产品进行单独评估，并应通过进行TS试验研究来确定。应提供TS方法开发和优化的总结。

The following experimental conditions should be established and verified during the pilot study:

在试点研究期间，应建立并验证以下实验条件：

●TS study should be conducted on healthy, normal forearm (volar) skin areas with avdequate skin barrier function. The inclusion/exclusion criteria for skin conditions should be defined. Skin with tattoos, any signs of dermatological abnormality or exhibiting a significant density of terminal haivr should be excluded. The preparation and cleaning procedures prior to the experiment should be established and further, that the treatment sites are not damaged by these processes.

TS研究应在具有足够皮肤屏障功能的健康、正常前臂（掌侧）皮肤区域进行。应确定皮肤状况的纳入/排除标准。任何带有纹身、有皮肤病异常迹象或末端毛发密度明显的皮肤，应排除在外。制定实验前的准备和清洁程序，并进一步确保这些过程不会损坏处理部位。

●Skin integrity should be determined before and after the experiment. This is normally performed by the measurement of Transepidermal Water Loss (TEWL), although other techniques may be applicable if appropriate. The acceptance criteria should be fully discussed and justified.

应在实验前后确定皮肤完整性。这通常是通过测量经表皮失水（TEWL）来进行的，但如果合适的话，其他技术也可能适用。应充分讨论并证明验收标准的合理性。

●Due to inter-subject variability, the products to be compared should be applied on the same subject. Additionally, a negative control that is non-equivalent to the comparator product should also be included to demonstrate the discriminatory power of the method. It is recommended to blind the investigator responsible for formulation application and tape stripping to minimise risk of bias.

由于受试者之间的差异，应将待比较的产品应用于同一受试者。此外，还应包括与对照品不等同的阴性对照，以证明该方法的歧视性。建议让负责配方应用和胶带剥离的研究人员盲试验，以最大限度地减少偏倚风险。

●The dosing amount should be determined based on the SmPC. During the pilot study, the dosage and area of dose application should be verified to achieve a quantifiable mass of active substance in the SC. The dosing technique, blinding and randomisation procedures should also be established.

剂量应根据SmPC确定。在试点研究期间，应验证剂量和剂量应用面积，以在SC中获得可量化的活性物质质量。还应建立剂量技术、盲法和随机化程序。

●A single dose approach should be followed, i.e. skin stripping is performed after a single application of the test and comparator products.

应采用单剂量方法，即在单次应用测试和对照产品后进行皮肤剥离。

●It is necessary that the products are compared at two time points (one uptake, one clearance) for each subject. The optimal uptake and clearance times depend on the characteristics of the drugs and products and should be determined during the pilot study. Ideally and when relevant, the uptake time should be sufficiently long for the drug to have attained the diffusional steady state. This can be established by testing at multiple uptake times and from which time the mass of drug recovered from the SC remains constant. The clearance time should be long enough to allow measurable transfer of drug from the SC into the viable skin (and beyond) but should not exceed 48 hours to avoid any skin desquamation effect. The clearance time providing at least a 25% decrease in the mass of drug recovered from the SC with respect to that at the uptake phase is preferred. In all cases, the sampling times should be carefully  considered and justified.

有必要在两个时间点（一次吸收，一次清除）对每个受试者的产品进行比较。最佳吸收和清除时间取决于药物和产品的特性，应在试点研究期间确定。在相关的理想情况下，吸收时间应足够长，以使药物达到扩散稳定状态。这可以通过在多次摄取时间进行测试来确定，从该时间起，从SC中回收的药物质量保持恒定。清除时间应足够长，以允许可测量的药物从SC转移到活的皮肤中（或更久），但不应超过48小时，以避免任何皮肤脱屑影响。相对于摄取阶段，从SC回收的药物质量提供至少25%减少的清除时间是优选的。在所有情况下，应仔细考虑并证明取样时间的合理性。

●The drug product should be removed from the skin surface after the specified uptake time. The cleaning procedure should be established to ensure that the residual formulation is efficiently removed from the treatment sites before stripping.

药物产品应在规定的摄取时间后从皮肤表面移除。应制定清洁程序，以确保在剥离前有效地从处理部位去除残留制剂。

●The adhesive tape chosen should meet the following requirements: a) does not lose mass when applied and rubbed against the skin surface; b) minimal weight loss and gain during storage; c) the drug is readily extracted from the SC adhered to the tape; d) the adhesive or other components of the tape do not interfere with the analytical quantification of the drug; and e) the adhesive power should be such that the majority of the SC is removed with a sufficiently low number of tapes (e.g. not more than 30 tapes).

选择的胶带应满足以下要求：a）在应用和摩擦皮肤表面时不会失去质量；b） 储存期间的最小化重量损失和增加；c） 药物容易从粘附在胶带上的SC中提取；d） 胶带的粘合剂或其他成分不干扰药物的分析定量；以及e）粘合力应使得大部分SC用足够低数量的胶带（例如不超过30条胶带）去除。

●The TS procedure followed must ensure that most of the SC (≥75%) is sampled for each skin site. The minimum and maximum number of tapes should be established based on the TEWL (or other relevant) criteria, e.g. eight-fold increment over baseline value, safety stop value.

遵循的TS程序必须确保对每个皮肤部位的大部分SC（≥75%）进行采样。胶带的最小和最大数量应根据TEWL（或其他相关）标准确定，例如比基线值增加8倍，安全停止值。

●Most commonly, the drug is first extracted from the tapes then quantified in the extraction solvent(s). Alternative methods of extraction/quantification may be used if justified. Satisfactory efficiency should be demonstrated for the proposed extraction method.最常见的是，首先从胶带中提取药物，然后在提取溶剂中定量。如果合理，可使用其他提取/定量方法。应证明所提出的提取方法具有令人满意的效率。

Detailed standard operating procedures should be prepared for the conduct of TS studies to ensure precise control of dosing, cleaning, stripping, extraction, quantification and other study variables or potential sources of experimental bias. The inclusion/exclusion criteria should be pre-defined and clearly stated in the protocol.

在进行TS研究时，应制定详细的标准操作程序，以确保精确控制剂量、清洗、剥离、提取、定量和其他研究变量或实验偏差的潜在来源。纳入/排除标准应预先定义，并在方案中明确说明。

The following study design is recommended for TS studies. The final protocol developed for each specific case should be justified.

TS研究建议采用以下研究设计。应该针对每种具体情况制定合理的最终方案。

●Subjects – TS studies should be performed in healthy volunteers. The subjects should be screened for suitability in line with the principles of bioequivalence studies.

受试者–TS研究应在健康志愿者中进行。应根据生物等效性研究的原则筛选合适的受试者。

●Treatment area –healthy skin of the volar forearm areas sufficient to accommodate at least six application sites per forearm. Skin integrity should be verified e.g. by TEWL measurement. The same number of application sites should be assigned to each forearm;

治疗区域–前臂掌侧区域的健康皮肤，每个前臂至少有六个应用部位。应通过TEWL测量进行皮肤完整性验证。应为每个前臂分配相同数量的应用部位；

●Number of subjects – the choice of the number of subjects should be justified based on the variability estimated from the pilot studies and demonstrated to be statistically relevant. A minimum of 12 subjects should be used to demonstrate equivalence;

受试者数量——受试者的数量选择应根据试点研究估计的变异性进行证明，并证明其具有统计学意义。应使用至少12名受试者来证明等效性；

●Number of replicates – at least two application sites per product (test, comparator and a negative control) per forearm. One forearm should be used for uptake samples and the other for clearance;

重复次数–每个前臂每个产品至少有两个应用部位（测试、对照和阴性对照）。一侧前臂用于吸收样本，另一侧用于清除；

●The products should be applied at pre-determined doses (±5%) and spread evenly over the entire demarcated application sites. Blank samples should be collected from the adjacent areas to verify the absence of background levels of drug or other compounds that may interfere with the quantification of drug in the treated SC;

产品应以预定剂量（±5%）涂抹，并均匀分布在整个划定的涂抹部位。应从相邻区域采集空白样品，以验证是否存在可能干扰治疗SC中药物定量的药物或其他化合物的背景水平；

●The application sites should be randomised to avoid bias. The application time should be staggered to allow time for S.C. sampling;

用药位点应随机，以避免选择偏向性。应用时间应错开，以留出S.C.取样时间；

●Un-occluded conditions, unless occlusion is recommended in the product information, or otherwise justified e.g. to prevent inadvertent removal of formulation.

非封闭条件，除非产品信息中建议封闭，或以其他方式证明封闭是合理的，例如防止无意中移除制剂。

●The formulation should be removed from all treatment sites (uptake and clearance) at the end of the uptake phase. The total cleaning time should be minimised to avoid any artefacts due to further drug diffusion. Skin integrity of the treated area should be checked before stripping;

在吸收阶段结束时，应从所有治疗部位（吸收和清除）移除制剂。应尽量缩短总清洗时间，以避免因药物进一步扩散而产生任何假阳性。剥离前应检查处理区域的皮肤完整性；

●The ‘uptake’ sites should be tape-stripped immediately after formulation removal. The ‘clearance’ sites should be tape-stripped at the pre-defined clearance times;

“吸收”部位应在移除制剂后立即用胶带剥离。“清理”部位应在预定义的清理时间进行胶带剥离；

●The exact number of tapes required should be determined based on TEWL measurements of the stripped area and the stopping criteria established from the pilot study;

所需胶带的确切数量应根据剥离区域的TEWL测量值和试点研究确定的停止标准确定；

●The mass of SC removed per tape should be determined using a gravimetric method by weighing the tapes strips before and after stripping. Alternative methods of quantification of the SC can be used if suitably described and justified;

每个胶带上去除的SC的质量应通过在剥离前后称重胶带的重量来确定。如果有适当描述和合理理由，可以使用替代性的SC量化方法；

●All stripped tapes collected from each treatment site should be analysed. The first two tapes should be analysed separately from the remaining tapes, so their contribution to the total amount of drug recovered can be eva1uated. To enhance analytical detectability, the subsequent tapes can be combined in groups (e.g. each group containing the required minimum content of SC) for extraction. The total mass of drug in the SC should be calculated as the sum extracted from all tape strip samples. The mass balance, including the drug content removed from the surface by cleaning should be determined for each treatment site. The overall recovery of 90-110% would be acceptable without justification; larger variation should be fully explained.

应分析从每个处理部位收集的所有剥离胶带。为评估它们对回收药物总量的贡献，前两个胶带应与剩余胶带分开分析。为了增强分析检测能力，可以将后续的胶带进行分组（例如，每组含有所需的最低含量的SC）提取。SC中药物的总质量应计算为从所有胶带样本中提取的总和。应确定每个治疗部位的质量平衡，包括通过清洁从表面去除的药物含量。一般情况下，可接受的总回收率是90-110%；如果出现较大的偏差，应充分解释。

Cleaning the skin surface at the end of the application period prior to tape-stripping is important and must be capable of removing excess formulation (i.e. unabsorbed drug) efficiently without inadvertently ‘driving’ the drug into the barrier. The cleaning procedure usually involves quickly and gently wiping the skin with dry/wet tissue, cotton swabs and/or fresh alcohol wipes. The cleaning components should be known not to influence drug diffusion into and through the SC. A careful eva1uation and validation of an efficient skin cleaning procedure should be performed prior to the pivotal study, e.g. by demonstrating satisfactory recovery (>90%) of the drug formulation removed from the skin surface and the negligible drug content (＜10%) recovered by stripping the cleaned skin immediately after application. Other ways of validation may be used if suitably justified.

在胶带剥离前，非常重要的是在应用期结束时进行皮肤表面的清洁，必须能够有效地去除多余的制剂（即未吸收的药物），而不会无意中“驱使”药物进入屏障。清洁程序通常包括用干/湿纸巾、棉签和/或新鲜酒精湿巾快速轻轻擦拭皮肤。应该知道清洁组件不会影响药物扩散进入和通过SC。在正式研究之前，应仔细评估和验证有效的皮肤清洁程序，例如，通过证明从皮肤表面去除的制剂有令人满意的回收率（＞90%）和通过在施用后立即剥离清洁的皮肤而回收的可忽略的药物含量（＜10%）。如果能证明合理性，可以使用其他验证方法。

The bioanalytical method employed for drug quantification in the tape strips should be validated. The efficiency of the extraction procedures (including extraction of tape strips in groups) should be established and demonstrated as consistent prior to the pivotal study.

应验证用于胶带中药物定量的生物分析方法。在正式研究之前，应确定提取程序的效率（包括成组提取胶带），并证明其一致性。

The discriminatory power of the TS method should be demonstrated for batches with different quality attributes (a negative control), such as a drug formulation with ±50% of the proposed product strength, that is shown to be statistically different and non-equivalent to the test and comparator products. The analytical methods for determining the content of active substance in the tape-stripped SC should be validated according to the Guideline on Bioanalytical Method Validation.

对于具有不同质量属性的批次（阴性对照），应证明TS方法的区分力，例如具有±50%拟议产品强度的药物配方，其与试验和对照产品在统计学上不同且不等效。根据《生物分析方法验证指南》，测定胶带剥离SC中活性物质含量的分析方法应进行验证

Data from all subjects should be reported and the validity and variability of the results should be discussed. All treated subjects and application sites should be included in the statistical analysis. The permitted reasons for exclusion must be pre-specified in the protocol. Data exclusion based on statistical analysis or for kinetic reasons alone is not acceptable.

应报告所有受试者的数据，并讨论结果的有效性和变异性。统计分析中应包括所有受试者和应用部位。必须在协议中预先规定允许的排除理由。不能仅基于统计分析或动力学原因排除数据。

For each product, the thickness of SC removed, the number of tapes used and final TEWL value measured at both uptake and clearance times should be reported. Any differences in these parameters between the test and comparator products should be discussed with respect to equivalence.

对于每种产品，都应报告去除的SC厚度、使用的胶带数量以及在吸收和清除时间测量的最终TEWL值。应在等效性方面对测试产品和对照产品之间这些参数的任何差异都进行讨论。

A plot of drug content profile in the SC should be presented for each application site, e.g. the drug content of each SC tape strip (single or grouped) versus SC depth.

应为每个应用部位绘制SC中的药物含量分布图，例如每个SC胶带条（单个或成组）的药物含量与SC深度的关系。

The duplicated measurements for each product in each subject should be averaged (population geometric mean) prior to analysis.

在分析之前，应对每个受试者中每个产品的重复测量值进行平均（总体几何平均值）。

For the comparison of products, the equivalence parameters: mass of drug recovered from the uptake (Muptake) and clearance (Mclearance) sites, should be statistically compared, according to the Guideline on the Investigation of Bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1/ Corr ).

对于产品的比较，应根据《生物等效性研究指南》（CPMP/EWP/QWP/1401/98 Rev.1/Corr）对等效参数：从吸收（Muptake）和清除（Mclearance）部位回收的药物质量进行统计比较。

The acceptance criteria for equivalence parameters (Muptake) and (Mclearance) are:

●The 90% confidence interval for the ratio of means of the test and comparator products should be contained within the acceptance interval of 80.00- 125.00%, unless justified.

除非有正当理由，试验和对照产品平均值比率的90%置信区间应包含在80.00-125.00%的验收区间内。

●Wider 90% confidence interval limits, to a maximum of 69.84 – 143.19, may be accepted in the case of high variability observed with low strength and limited diffusion drug products, and if clinically justified. The procedure in the Guideline on Investigation of Bioequivalence,  “Section 4.1.10 Highly variable drugs or drug products” should be followed.

如果在低强度和有限扩散的药物产品中观察到高变异性，并且在临床证明的情况下，可以接受更宽的90%置信区间限，最大值为69.84–143.19。应遵循《生物等效性调查指南》第4.1.10节“高变异药物或药物产品”中的程序。

In addition, for the test to be valid:

此外，为了使测试有效

●The acceptance criteria for equivalence parameters (Muptake) and (Mclearance)

等效参数（Muptake）和（Mclearance）的验收标准

●The 90% confidence interval for the ratio of means of the test and negative control products should be entirely outside the interval of 80.00- 125.00%.

试验和阴性对照产品平均值比率的90%置信区间应完全超出80.00-125.00%的区间。●The 90% confidence interval for the ratio of means of the comparator and negative control products should be entirely outside the interval of 80.00- 125.00%.对照产品和阴性对照品平均值之比的90%置信区间应完全超出80.00-125.00%的区间。●The 90% confidence interval for the ratio of means of the test product clearance (Mclearance) and (Muptake) comparator products should be entirely below 1.0.试验产品清除率（Mclearance）与对照产品（Muptake）平均值之比的90%置信区间应完全低于1.0。

●The 90% confidence interval for the ratio of means of the comparator product clearance (Mclearance) and (Muptake) comparator products should be entirely below 1.0.

对照产品清除率（Mclearance）与对照产品（Muptake）平均值之比的90%置信区间应完全低于1.0。

The overall conclusions of the study should be provided. This should be supported by a sound scientific discussion and interpretation of the TS data.

应提供研究的总体结论。这应该得到对TS数据的合理科学讨论和解释的支持。