技术文章

In Vitro Permeation Test Studies for Topical Drug Products Submitted in ANDAs Guidance for Industry

工业指南中ANDAs申请提交的外用制剂的体外渗透试验研究

U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) October 2022 Generic Drugs

美国卫生与公众服务部食品和药物管理局药物评估与研究中心（CDER）2022年10月仿制药

IINTRODUCTION

This guidance is intended to assist applicants who are submitting abbreviated new drug applications (ANDAs) for liquid-based and/or other semisolid products applied to the skin, including integumentary and mucosal (e.g., vaginal) membranes, which are hereinafter called “topical products.” Because of the complex route of delivery associated with these products, which are typically locally acting, and the potential complexity of certain formulations, topical products (other than topical solutions) are classified as complex products. This guidance provides recommendations for in vitro permeation test (IVPT) studies comparing a proposed generic (test) topical product and its reference standard (RS) for the purpose of supporting a demonstration of bioequivalence (BE) to the reference listed drug (RLD). The reference standard ordinarily is the RLD

本指南旨在帮助申请人提交用于皮肤的液体和/或其他半固体产品申请ANDA，包括皮肤和粘膜（如阴道），以下称为“局部产品”。由于这些产品复杂的递送途径，通常是局部起效的，以及某些制剂的潜在复杂性，外用制剂（外用溶液除外）被归类为复杂制剂。本指南为体外渗透试验（IVPT）研究提供了建议，该研究比较了拟申报仿制制剂及其参考制剂（RS），以支持证明与参比制剂的（RLD）生物等效性（BE）。参比制剂通常为RLD
 

This guidance does not address drug products that are administered via ophthalmic, otic, nasal, inhalation, oral, or injection-based routes, or that are transdermal or topical delivery systems (including products known as patches, topical patches, or extended release films).

本指南不适用于通过眼、耳、鼻、吸入、口服或注射途径给药的药物，也不适用于透皮或局部给药系统（包括贴片、局部贴片或缓释膜）。

It is beyond the scope of this guidance to discuss specific topical products to which this guidance applies. FDA recommends that applicants consult this guidance and any relevant product-specific guidances (PSGs) and any other relevant guidances for industry, when considering the design and conduct of IVPT studies that, in conjunction with other studies, as deemed necessary, may be appropriate to support a demonstration that a proposed generic topical product and its RLD are bioequivalent. FDA also recommends that applicants routinely refer to FDA’s guidance web pages, because additional guidances may become available that could assist in the development of a generic topical product.

本指南是不讨论适用该指南的特定外用制剂的指南。FDA建议申请人在考虑IVPT研究的设计和实施时，参考本指南和任何相关的特定产品指南（PSG）以及任何其他相关的行业指南，如有必要，结合其他研究，IVPT研究可能适合支持建议的通用外用制剂及其RLD具有生物等效性的证明。FDA还建议申请人定期查阅FDA的指导网页，因为可能会有更多的指导，有助于仿制药的开发。

In general, FDA’s guidance documents do not establish legally enforceable responsibilities. Instead, guidances describe the Agency’s current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in Agency guidance means that something is suggested or recommended, but not required.

一般来说，FDA的指导文件并没有确立法律上可强制执行的责任。相反，指南描述了机构目前对某一主题的想法，并且应仅视为建议，除非引用了具体的监管或法定要求。在机构指南中使用“应该”一词意味着有人建议或建议，但不是必须的。

II. BACKGROUND

This guidance has been developed as part of FDA’s “Drug Competition Action Plan,” which, in coordination with the Generic Drug User Fee Amendments (GDUFA) program and other FDA activities, is intended to increase competition in the market place for prescription drugs, facilitate the entry of high-quality and affordable generic drugs, and improve public health.

本指南是作为FDA“药品竞争行动计划”的一部分制定的，该计划配合GDUFA计划和其他FDA举措，旨在促进处方药市场的竞争，促进高质量和实惠的药品的进入市场，并改善公众医疗。

The Federal Food, Drug, and Cosmetic Act (FD&C Act) generally requires an ANDA to contain, among other things, information to show that the proposed generic drug product 1) is the same as the RLD with respect to the active ingredient(s), conditions of use, route of administration, dosage form, strength, and labeling (with certain permissible differences) and 2) is bioequivalent to the RLD. Thus, an ANDA will not be approved if the information submitted in the ANDA is insufficient to show that the test product is bioequivalent to the RLD.

《联邦食品、药品和化妆品法案》（FD&C法案）通常要求ANDA包含合适的信息，说明：仿制药与RLD相比1）具有相同活性成分、使用条件、给药途径、剂型、规格和标签（允许存在某些差异），和2）与RLD具有生物等效性。因此，如果ANDA中提交的信息不足以证明自制制剂与RLD具有生物等效性，则ANDA将不予批准。

An IVPT study may be used to assess the rate and extent to which a drug (i.e., an active ingredient) from a topical product becomes available at or near a site of action in the skin, and may be used to characterize and compare the rate and extent of bioavailability for a drug from a test topical product and RS. The IVPT flux profiles resemble pharmacokinetic profiles and can be analyzed using unique IVPT endpoints that are somewhat analogous to the pharmacokinetic endpoints of maximum concentration (Cmax) and the area under the concentration-time curve(AUC). Yet, IVPT studies characterize the rate and extent of absorption, not the distribution, metabolism and excretion that occurs in vivo. Therefore, while it is relevant to characterize the kinetics of topical drug bioavailability monitored by IVPT studies, the use in this guidance of the term “cutaneous pharmacokinetics” should not be construed to embody all aspects of pharmacokinetics—only those related to the absorption component that directly controls the rate and extent to which a topically applied drug becomes available locally at the site of action. This guidance focuses on general considerations and recommendations for the method development, method validation, and conduct of IVPT studies that are submitted in ANDAs and intended to support a demonstration of BE.

IVPT研究可用于评估局部产品中的药物（即活性成分）在皮肤作用部位或附近可用的速率和程度，并可用于表征和比较测试局部产品和RS中药物的生物利用率和程度。IVPT通量曲线类似于药代动力学曲线，可以使用*的IVPT终点进行分析，这些终点与最大浓度（Cmax）的药代动力学终点和浓度-时间曲线下的面积（AUC）有些相似。然而，IVPT研究表征的是吸收的速率和程度，而不是体内的分布、代谢和排泄。因此，虽然表征IVPT研究监测的局部药物生物利用度的动力学是相关的，本指南中使用的术语“皮肤药代动力学”不应被解释为仅体现与吸收成分相关的药代动力学的所有方面，吸收成分直接控制局部应用药物在作用部位局部可用的速率和程度。本指南侧重于在ANDA中提交的用于支持BE演示的方法开发、方法验证和IVPT研究的一般考虑和建议。

III. IVPT METHOD DEVELOPMENT

The development of an IVPT method that is suitable to support a demonstration of BE for a specific topical product routinely involves a systematic series of exploratory studies. Inappropriate or insufficient efforts to develop an IVPT method that is suitable for its intended purpose increases the likelihood that the subsequent IVPT validation, pilot, and pivotal studies will ultimately be inadequate to support a demonstration of BE. By contrast, appropriate and systematic IVPT method development studies help to identify IVPT study designs and protocol (method) parameters which reliably produce flux profiles that can facilitate a comparison of the cutaneous pharmacokinetics of a drug delivered topically to the skin from test topical products and RSs.

IVPT方法的开发适用于支持特定局部产品的BE演示，通常涉及一系列系统的探索性研究。开发适合其预期目的的IVPT方法的努力不当或不足，增加了后续IVPT验证、试点和关键研究最终不足以支持BE演示的可能性。相比之下，适当和系统的IVPT方法开发研究有助于确定IVPT研究设计和方案（方法）参数，这些设计和方案能够可靠地产生通量曲线，从而有助于比较从测试局部产品和RS局部递送至皮肤的药物的皮肤药代动力学。

A detailed and well-organized IVPT method development report should be submitted in an ANDA to show how the IVPT method was optimized, and to support a demonstration that the method parameters selected for the IVPT are appropriate or necessary, particularly in situations where the method parameters are different from the methods recommended in this guidance). The Agency’s interest in reviewing the method development report is to understand why specific IVPT method parameters were selected and whether the resulting IVPT method is suitably sensitive and reproducible. This method development report should clearly indicate/distinguish the method parameters used for each set of data, illustrate the efforts made to optimize the IVPT method, and demonstrate that the method parameters selected for the IVPT are appropriate.

应在ANDA中提交一份详细且组织良好的IVPT方法开发报告，以说明IVPT方法是如何优化的，并支持为IVPT选择的方法参数是适当或必要的，特别是在方法参数与本指南中推荐的方法不同的情况下）。机构审查方法开发报告的目的是了解为什么选择了特定的IVPT方法参数，以及所得IVPT方法是否具有适当的敏感性和重现性。该方法开发报告应明确指出/区分每组数据使用的方法参数，说明为优化IVPT方法所做的努力，并证明为IVPT选择的方法参数是合适的。

Applicants are encouraged to use the recommendations in this guidance, and if an applicant elects to use methods that are different from those recommended in this guidance, the IVPT method development report should demonstrate why it is scientifically justified to use an alternative approach than what is recommended in this guidance to optimize the IVPT method. Some examples of recommended procedures are described in subsequent sections, to help applicants identify circumstances when information should be submitted in the ANDA to explain why a different procedure was utilized.

鼓励申请人使用本指南中的建议，如果申请人选择使用与本指南中建议的方法不同的方法，IVPT方法开发报告应说明为什么使用本指南推荐的替代方法来优化IVPT方法在科学上是合理的。建议程序的一些示例在随后的章节中描述，以帮助申请人确定应在ANDA中提交信息的情况，以解释为什么使用不同的程序。

A. IVPT Method Parameters

All relevant parameters of the final IVPT method should be summarized (e.g., in a table) and submitted in the ANDA. Also, information should be provided to briefly explain the choice of the final IVPT method parameters like the equipment (e.g., a vertical diffusion cell (VDC)), skin source (e.g., cadaver), skin type (e.g., posterior torso), skin preparation (e.g., dermatomed), skin barrier integrity test (e.g., trans-epidermal water loss (TEWL) measurement), skin barrier integrity test acceptance criteria (e.g., < 15 grams/meter2 /hour (g/m2 /hr)), topical product dose amount (e.g., 15 milligrams/centimeter2 (mg/cm2 )), dose duration (e.g., 6 hours), study duration (e.g., 24 hours, 48 hours, etc.), receptor solution sampling times (e.g., 1, 2, 4, 6, 8, 12, 16, 20, and 24 hours), etc.

应总结最终IVPT方法的所有相关参数（例如，在表格中），并在ANDA中提交。此外，应提供信息，以简要解释最终IVPT方法参数的选择，如设备（例如，垂直扩散池（VDC））、皮肤源（例如，尸体）、皮肤类型（例如，后躯干）、皮肤准备（例如，皮肤科）、皮肤屏障完整性测试（例如，经表皮水分损失（TEWL）测量）、，皮肤屏障完整性测试验收标准（例如，<15克/米2/小时（g/m2/小时））、局部产品剂量（例如，15毫克/厘米2（mg/cm2））、剂量持续时间（例如，6小时）、研究持续时间（如，24小时、48小时等）、受体溶液取样时间（例如1、2、4、6、8、12、16、20和24小时）等。《IVPT测试中的皮肤研究》

B. IVPT Method Considerations

The choice of some IVPT method parameters like the equipment, skin source, skin type, skin preparation, and skin barrier integrity test procedures may be based upon investigator experience or convenience, like the availability of specific equipment or instrumentation in a laboratory, established tissue supply agreements, or other logistical considerations. However, if the chosen IVPT method parameters do not appear to be well-suited for a specific IVPT method, it is the applicant’s responsibility to systematically evaluate alternative method parameters, and ultimately, to validate that the IVPT method parameters chosen are suitable for the intended purpose. The recommended procedures for IVPT method validation are detailed in section IV of this guidance.

一些IVPT方法参数的选择，如设备、皮肤来源、皮肤类型、皮肤准备和皮肤屏障完整性测试程序，可能基于研究人员的经验或便利性，如实验室中特定设备或仪器的可用性、既定的组织供应协议或其他后勤考虑。然而，如果选定的IVPT方法参数似乎不适合特定的IVPT法，则申请人有责任系统地评估备选方法参数，并最终验证选定的IVPT方法参数是否适合预期用途。IVPT方法验证的推荐程序详见本指南第四节。

The choice of other IVPT method parameters like the topical product dose amount, dose duration, study duration (which may be longer than the dose duration), sampling schedule, sampling procedures, receptor solution composition, and sample analytical method may be different for each IVPT method, and such parameters of IVPT methods should be systematically developed, optimized, and/or validated for the relevant topical product, as appropriate. The IVPT method development studies should characterize how differences in these method parameters influence the resulting IVPT flux profile so that optimal study conditions can be objectively selected from among those evaluated.

其他IVPT方法参数的选择，如局部产品剂量、剂量持续时间、研究持续时间（可能长于剂量持续时间）、取样计划、取样程序、受体溶液组成和样品分析方法，对于每种IVPT方法可能有所不同，应系统开发、优化IVPT方法的此类参数，和/或对相关局部产品进行验证。IVPT方法开发研究应描述这些方法参数的差异如何影响最终的IVPT通量分布，以便从评估的条件中客观选择最佳研究条件。

The selection of the dose amount used in the study should be assessed for each IVPT method based upon studies performed during IVPT method development. Different dose amounts may be compared in parallel on replicate skin sections from the same set of donors to optimize the dose amount for the IVPT study. Considerations for selecting an optimal dose amount may include (1) the consistency with which the dose can be applied (potentially using different dispensing and/or spreading techniques), (2) the reproducibility of the flux profiles, (3) the influence of dose amount and dose duration on the shape of the flux profile, and (4) the approximate range of drug concentrations in receptor solution samples at different time points (relative to the sample analytical method limits of quantification).

应根据IVPT方法开发期间进行的研究，评估每种IVPT方法在研究中使用的剂量选择。可以在来自同一组供体的复制皮肤切片上平行比较不同剂量，以优化IVPT研究的剂量。选择最佳剂量的考虑因素可以包括（1）可以施加剂量的一致性（可能使用不同的分配和/或散布技术），（2）通量分布的再现性，（3）剂量和剂量持续时间对通量分布形状的影响，和（4）不同时间点受体溶液样品中药物浓度的近似范围（相对于样品分析方法的定量限值）。

The selected sampling schedule and study duration should be sufficient to characterize the cutaneous pharmacokinetics of the drug, which ideally includes a sufficiently complete flux profile to identify the maximum (peak) flux and a decline in the flux thereafter across multiple subsequent time points. A dose that remains on the skin for the duration of the study may continue to deliver the drug for a sustained period and may not necessarily exhibit a suitable decline in the flux at later time points. In such instances, it may be appropriate to develop an IVPT method that involves wiping off the applied dose after a suitable duration on the skin and continuing to monitor the receptor solution for an extended period thereafter, during which the decline in the flux profile can be characterized. The sampling frequency should be selected to provide a suitable resolution for the flux profile, and a minimum of eight non-zero sampling time points is recommended across the study duration (e.g., 48 hours).

所选择的采样时间表和研究持续时间应足以表征药物的皮肤药代动力学，理想情况下，包括足够完整的通量曲线，以确定最大（峰值）通量以及随后在多个后续时间点的通量下降。在研究持续时间内留在皮肤上的剂量可能会持续给药一段时间，并且不一定会在以后的时间点出现适当的流量下降。在这种情况下，开发IVPT方法可能是合适的，该方法包括在皮肤上适当的持续时间后擦去施用的剂量，并在此后的较长时间内继续监测受体溶液，在此期间可以表征通量分布的下降。应选择采样频率，以便为通量分布提供合适的分辨率，并且建议在整个研究持续时间内（例如，48小时）至少八个非零采样时间点。

C. IVPT Method Procedures and Controls

Suitable technical procedures and control parameters should be established during method development. These may include procedures for preparing and mounting the skin on the diffusion cell in a consistent manner, determining the instrument settings that regulate the skin surface temperature within the specified range, performing the barrier integrity test appropriately, controlling the accuracy and precision of the dose amount dispensed on each skin section.

在方法开发过程中，应建立适当的技术程序和控制参数。这些可以包括以一致的方式制备皮肤并将其安装在扩散池上的程序，确定将皮肤表面温度调节在规定范围内的仪器设置，适当地进行屏障完整性测试，控制分配到每个皮肤部分的剂量的准确性和精密度。

For example, a dosing procedure may be developed that uses a positive displacement pipette to dispense a volumetrically controlled amount of a topical product, targeting the deposition on the skin of a certain mass (e.g., 15 mg/cm2 ) of topical product. If the inner diameter of the orifice in the dosing compartment of the diffusion cell is 15 millimeters (mm), and the effective dose area is ~1.77 cm2 , this would indicate a target dose of ~26.5 mg of topical product per diffusion cell. Experiments during method development may establish that, based upon the density of the topical product, a specific volumetric setting on a specific model of positive displacement pipette with a specific pipette tip repeatedly dispenses ~27.5 mg of topical product (e.g., characterized by multiple replicate pipette dispensations into a weigh boat on a fine balance). This pipette setting may be optimal for a dosing procedure where the dose spreading instrument, like the flat bottom of a high performance liquid chromatography (HPLC) glass vial, or the rounded end of a glass rod or capillary tube, is subsequently used to spread the dispensed dose evenly upon the skin section mounted in the diffusion cell, and where repeatedly weighing the dose-spreading instrument before and after the dose spreading indicates that the residual topical product remaining on the bottom of the glass vial after the dose spreading reproducibly amounts to ~1.0 mg of topical product (indicating that ~26.5 mg of the topical product would reproducibly be dosed to each skin section). Such characterizations of the technical procedures and control parameters for the IVPT method, like the reproducibility of the dosing procedure, should be established during method development and may not need to be demonstrated thereafter each time the same procedure is used.

例如，可以开发一种剂量程序，其使用正位移移液管来分配体积控制量的局部产品，靶向一定质量（例如，15mg/cm2）的局部产品在皮肤上的沉积。如果扩散池给药室的孔口内径为15毫米（mm），有效剂量面积约为1.77 cm2，则表明每个扩散池的目标剂量为约26.5 mg局部产品。方法开发过程中的实验可以确定，基于外用产品的密度，具有特定移液管的特定型号正移液管的特定体积设置可重复分配约27.5 mg外用产品（例如，以多次重复移液管分配为特征，将其分配到精密天平上的称量舟中）。这种移液管设置对于剂量分配过程可能是最佳的，其中剂量分配仪器，如高效液相色谱（HPLC）玻璃瓶的平底，或玻璃棒或毛细管的圆形端，随后用于将分配的剂量均匀地分配到安装在扩散池中的皮肤部分上，并且其中在剂量散布之前和之后重复称量剂量散布仪器表明，在剂量散布之后残留在玻璃瓶底部的残留局部产品可重复地达到约1.0mg局部产品（表明将可重复地将约26.5mg局部产品施用到每个皮肤部分）。IVPT方法的技术程序和控制参数的此类特征，如给药程序的再现性，应在方法开发过程中确定，此后可能不需要在每次使用相同程序时进行证明。‍

‍D. ‍‍IVPT Skin Barrier Integrity Testing: Common Methods

The technical procedures for the skin barrier integrity test should be established during IVPT method development. Three types of barrier integrity tests are common, however, there are currently no applicable compendial standard protocols or acceptance criteria for any of these three types of human skin barrier integrity tests. Nonetheless, recommended parameters for the three common types of barrier integrity tests are discussed below.

应在IVPT方法开发期间制定皮肤屏障完整性测试的技术程序。三种类型的屏障完整性测试是常见的，然而，目前没有适用于这三种类型人体皮肤屏障完整性检测的药典标准协议或验收标准。尽管如此，下面讨论了三种常见类型的屏障完整性测试的推荐参数。IVPT测试中的皮肤研究

1. Trans-Epidermal Water Loss Skin Barrier Integrity Test

A TEWL skin barrier integrity test involves a measurement near the outer surface of the skin of the rate at which water (vapor) is fluxing through the skin barrier from the underside of the skin section. For the test, the skin section is mounted in a diffusion cell (e.g., clamped in place between the donor and receptor compartments), with the underside of the skin in contact with the receptor solution in the receptor compartment (e.g., phosphate buffered saline, pH 7.4), and equilibrated to a skin surface temperature of 32°C ± 1°C. If skin sections are cut large enough to cover the flange of the diffusion cell in which they are mounted, then after they have equilibrated for several hours at a skin surface temperature of 32°C ± 1°C, it may be feasible to gently remove the donor compartment without disrupting a skin section’s adherence to the lower flange of the diffusion cell, thereby allowing the TEWL probe to be placed directly on the skin surface, instead of being placed atop the donor compartment. Typically, a minimum of three replicate measurements are made on each skin section, which are recorded after the measurements have stabilized.

TEWL皮肤屏障完整性测试包括在皮肤外表面附近测量水（蒸汽）从皮肤部分下侧流过皮肤屏障的速率。对于试验，将皮肤部分安装在扩散池中（例如，夹在供体和受体室之间的适当位置），皮肤下侧与受体室中的受体溶液（例如，磷酸盐缓冲盐水，pH 7.4）接触，并平衡至32°C±1°C的皮肤表面温度。如果皮肤部分被切割得足够大，足以覆盖其所安装的扩散池的法兰，那么在皮肤表面温度为32°C±1°C的条件下平衡数小时后，可以在不破坏皮肤部分与扩散池下法兰的粘附性的情况下轻轻移除供体隔室，从而允许TEWL探针直接放置在皮肤表面上而不是放置在供体隔室的顶部。通常，在每个皮肤部分上至少进行三次重复测量，并在测量稳定后进行记录。（下图，皮肤测试用经皮水分流失测量仪）

Commercially available devices to measure TEWL may differ in design and operational principles. The TEWL measured by devices with certain designs (e.g., an open chamber versus a closed chamber) may be relatively more susceptible to the influence of environmental conditions. Therefore, environmental temperature and humidity are typically controlled as precisely as possible (e.g., a temperature range of 21°C ± 2°C and a humidity range of 50% ± 20% relative humidity are ideal, if feasible). More precise control of the relative humidity (e.g., in the range of 40% – 50%) may reduce the variability of TEWL measurements for devices with certain designs. Certain designs of measurement probes and adapters for in vitro use are available by the manufacturers of TEWL devices, and may be appropriate to use. Inconsistency in the diameters for the measurement probe chamber, the measurement probe orifice, the in vitro adapters, and the skin area being measured, as well as variation in the distance of the probe sensor(s) from the skin surface, potentially because of the (variable) height of donor compartments (when applicable), could increase the variability of TEWL measurements. Inconsistent control of the alignment of the TEWL measurement device in relation to the donor compartment and/or the skin section may also increase the variability of TEWL measurements. Also, the TEWL measured by devices with certain designs may be relatively more susceptible to the influence of heat transfer from the hand that holds the probe. Applicants should follow relevant instructions in the manufacturer’s user manual for the specific TEWL measurement device used.

用于测量TEWL的市售设备在设计和操作原理上可能有所不同。由具有特定设计（例如，开放室与封闭室）的装置测量的TEWL可能相对更容易受到环境条件的影响。因此，通常尽可能精确地控制环境温度和湿度（例如，如果可行，理想的温度范围为21°C±2°C，湿度范围为50%±20%相对湿度）。更精确地控制相对湿度（例如，在40%–50%的范围内）可以减少具有特定设计的设备的TEWL测量的可变性。TEWL装置的制造商可提供用于体外使用的测量探针和适配器的某些设计，并且可能适合使用。测量探头室、测量探头孔口、体外适配器和被测皮肤面积的直径不一致，以及探头传感器与皮肤表面的距离变化，可能是由于供体隔室的（可变）高度（如适用），可能会增加TEWL测量的变异性。TEWL测量装置相对于供体隔室和/或皮肤部分的对准的不一致控制也可能增加TEWL测定的可变性。此外，由具有特定设计的设备测量的TEWL可能相对更容易受到握住探头的手的热传递的影响。申请人应遵循制造商用户手册中关于所用特定TEWL测量设备的相关说明。

No more than approximately 15 grams of water per square meter per hour (i.e., ≤ 15 g/m2 /hr) could be a reasonable skin barrier integrity acceptance (cutoff) criterion for a TEWL barrier integrity test on human torso or thigh skin; if this was selected as the cutoff criterion, skin sections with a TEWL > 15 g/m2 /hr would fail the test. Skin sections that fail a barrier integrity test should not be dosed, but may serve as non-dosed control skin sections. A higher cutoff (e.g., ≤ 20 g/m2 /hr) may also be reasonable if justified by experimental data demonstrating that the selected acceptance criterion appropriately discriminates skin sections with a compromised barrier integrity from those with a competent barrier integrity.

每平方米每小时不超过约15克水（例如，≤ 15g/m2/hr）可能是人体躯干或大腿皮肤上TEWL屏障完整性测试的合理皮肤屏障完整性验收（截止）标准；如果将此作为截止标准，TEWL>15 g/m2/hr的皮肤切片将无法通过测试。未通过屏障完整性测试的皮肤部分不应给药，但可作为未给药的对照皮肤部分。较高的截止（例如，≤ 20g/m2/hr）也可能是合理的，如果实验数据证明所选验收标准适当地区分屏障完整性受损的皮肤部分和屏障完整性合格的皮肤部分。

However, TEWL measurements for skin sections with a competent barrier integrity can vary depending upon the TEWL measurement device, the manner in which it is operated, and the environmental conditions (e.g., higher ambient humidity or greater distance from the skin surface may decrease the value of the TEWL measurement). Precise control of environmental and device/operational factors can minimize variability in TEWL measurements. Therefore, the technical procedures for measuring TEWL should be optimized during IVPT method development (or based upon prior optimization in the laboratory performing the test). Also, the TEWL measurement device should be appropriately calibrated (by the manufacturer, and for some devices, also before each set of tests). Applicants may provide information about the relevant calibration procedures specified by the manufacturer for the specific TEWL device used; this can be submitted in the ANDA along with the IVPT method development report, to support the appropriateness of the technical procedures established by the laboratory for TEWL measurements. When a TEWL barrier integrity test is used in any study phase (IVPT method development, pilot study, validation, and/or pivotal study) the ambient laboratory temperature and humidity during the TEWL barrier integrity test should be monitored and reported.

然而，对于具有合格屏障完整性的皮肤部分的TEWL测量可以根据TEWL测试设备、其操作方式和环境条件而变化（例如，较高的环境湿度或离皮肤表面更大的距离可能会降低TEWL的测量值）。精确控制环境和设备/操作因素可以最大限度地减少TEWL测量的变化。因此，应在IVPT方法开发过程中优化TEWL测量的技术程序（或基于实验室进行试验的事先优化）。此外，TEWL测量设备应进行适当校准（由制造商进行校准，对于某些设备，也应在每组测试之前进行校准）。申请人可提供制造商为所用特定TEWL装置规定的相关校准程序的信息；这可以与IVPT方法开发报告一起提交在ANDA中，以支持实验室为TEWL测量建立的技术程序的适当性。当TEWL屏障完整性测试用于任何研究阶段（IVPT方法开发、试点研究、验证和/或关键研究）时，应监测和报告TEWL阻隔完整性测试期间的实验室环境温度和湿度。

2.Tritiated Water Skin Barrier Integrity Test

An example of a recommended approach to a tritiated water skin barrier integrity test would be to mount the skin in a diffusion cell (e.g., clamped in place between the donor and receptor compartments) and allow it to equilibrate to a skin surface temperature of 32°C ± 1°C with the stratum corneum exposed to the air in the donor compartment and the underside of the skin in contact with the receptor solution (e.g., phosphate buffered saline, pH 7.4).

氚化水-皮肤屏障完整性测试的推荐方法的一个例子是将皮肤安装在扩散池中（例如，夹在供体和受体室之间的适当位置），并使其平衡至32°C±1°C的皮肤表面温度，使角质层暴露在供体室中的空气中，皮肤下侧与受体溶液（例如磷酸盐缓冲盐水，pH 7.4）。

A small amount of tritiated water (sufficient to cover the entire surface of the skin section) would be briefly dosed on the stratum corneum. This dose of tritiated water would be left on the surface for a precisely controlled and relatively brief period (e.g., 5 minutes) after which it would be removed from the skin surface (e.g., using a pipette to remove the bulk volume and then an absorbent low lint laboratory tissue to gently blot dry). The receptor solution would then be sampled at a precise duration after the removal of the tritiated water from the skin surface (e.g., 30 minutes after the removal of the 5-minute dose of tritiated water from the skin surface).

将少量的氚水（足以覆盖皮肤部分的整个表面）短暂地施加于角质层。该剂量的氚水将在表面上停留一段精确控制且相对较短的时间（例如，5分钟），之后将其从皮肤表面去除（例如，使用移液管去除大量体积，然后使用实验室吸水纸轻轻吸干）。然后在从皮肤表面去除氚水之后的精确持续时间（例如，从皮肤表面除去5分钟剂量的氚水后30分钟）对受体溶液进行取样。

While the entire volume of the receptor compartment may be removed and replenished, typically only an aliquot of the receptor solution (e.g., phosphate buffered saline, pH 7.4) is transferred to a suitable volume of scintillation fluid for counting. The volume of the aliquot typically depends upon the type of scintillation fluid used and the maximum amount of aqueous fluid that is suitable to mix with the scintillation fluid. A scintillation counter is then used to quantify the amount of radioactivity in the aliquot sampled, which can be used to calculate the amount of tritiated water that permeated into the larger (entire) volume of receptor solution; the calculation is performed using the specific activity of the tritiated water to equate a given amount of radioactivity to the equivalent volume of tritiated water that permeated per square centimeter of skin surface area.

虽然可以移除并补充整个体积的受体室，但通常仅将一等分的受体溶液（例如，磷酸盐缓冲盐水，pH 7.4）转移到合适体积的闪烁液中进行计数。等分试样的体积通常取决于所使用的闪烁流体的类型以及适合与闪烁流体混合的水性流体的最大量。然后，使用闪烁计数器来定量取样的等分试样中的放射性量，可用于计算渗入较大（整个）体积的受体溶液中的氚水的量；使用氚水的比活度进行计算，以将给定的放射性量等同于每平方厘米皮肤表面积渗透的氚水当量体积。

Approximately 1.5 equivalent(eq.) microliter (µL) of tritiated water per cm2 (i.e., ~1.5 eq. µL/cm2 or ~1.5 eq. mg/cm2 ) would be a reasonable skin barrier integrity acceptance (cutoff) criterion for a tritiated water barrier integrity test that involves a 5-minute dose followed by a 30- minute sampling duration (i.e., sampling 30 minutes after dose removal) on human torso or thigh skin. Skin sections with a tritiated water test result of > 1.5 eq. mg/cm2 would fail the test and be excluded from the population of skin sections dosed with the topical product; skin sections that fail a barrier integrity test should not be dosed, but may serve as non-dosed control skin sections. Other acceptance criteria may also be reasonable if justified by experimental data demonstrating that the selected acceptance criterion appropriately discriminates skin sections with a compromised barrier integrity from those with a competent barrier integrity.

人体躯干或大腿皮肤上氚水屏障完整性测试的合理皮肤屏障完整性验收（截止）标准是每cm2约1.5当量（eq）微升（µL）的氚水（例如，约1.5 eq. µL/cm2或约1.5 eq. mg/cm2）。该测试涉及5分钟剂量，随后30分钟采样持续时间（即，去除剂量后30分钟采样）。氚水测试结果大于1.5 eq. mg/cm2的皮肤切片判定为未通过测试，并被排除在使用局部产品的皮肤切片人群之外；未通过屏障完整性测试的皮肤部分不应给药，但可作为未给药的对照皮肤部分。如果实验数据证明所选验收标准能够适当区分屏障完整性受损的皮肤部分和屏障完整性合格的皮肤部分，则其他验收标准也可能合理。（下图，放射性测试用闪点计数器）

When calculating the results for a tritiated water barrier integrity test, it may be important to account for the surface area dosed. For example, if using an acceptance criterion of 1.5 eq. mg/cm2 with a diffusion cell that has an orifice diameter of 15 mm and a skin surface area of 1.77 cm2 , the mass of tritiated water that would be calculated to have permeated into the receptor compartment would be ~2.7 eq. mg/cm2 of tritiated water.

当计算氚水屏障完整性测试的结果时，给药表面积是一个很重要的影响因素。例如，如果使用1.5 eq. mg/cm2的验收标准和孔直径为15 mm、皮肤表面积为1.77 cm2的扩散池，则计算出渗入受体室的氚水质量为约2.7 eq. mg/cm2的氚。

3.Electrical Based Skin Barrier Integrity Tests

There are several variations of electrical based skin barrier integrity tests that report the test result as a measure of the resistance, conductance, or a related electrical concept that characterizes the bulk flow of electrical current across the skin. Transepithelial electrical resistance tests involving the skin may be referred to more specifically as Trans-Epidermal Electrical Resistance (TEER) skin barrier integrity tests. The test results may be described in units of conductance, which is the reciprocal of resistance. Electrical based skin barrier integrity tests often use instruments that are designed to measure the inductance (L), capacitance (C), and resistance (R) of electronic circuits or electrical components; these instruments are commonly known as LCR meters and have different settings (test parameters) that can be adjusted.

基于电的皮肤屏障完整性测试有几种变体，将测试结果报告为电阻、电导或相关电概念的测量值，以表征穿过皮肤的整体电流。涉及皮肤的经上皮电阻测试可以更具体地称为经表皮电阻（TEER）皮肤屏障完整性测试。测试结果可用电导单位表示，电导是电阻的倒数。基于电气的皮肤屏障完整性测试通常使用设计用于测量电子电路或电气部件的电感（L）、电容（C）和电阻（R）的仪器；这些仪器通常被称为LCR仪表，并具有可调整的不同设置（测试参数）。

An example of a recommended approach to a TEER skin barrier integrity test would be to mount the skin in a diffusion cell (e.g., clamped in place between the donor and receptor compartments) and allow it to equilibrate to a skin surface temperature of 32°C ± 1°C with the stratum corneum exposed to the air in the donor compartment and the underside of the skin in contact with an ionic solution (e.g., phosphate buffered saline, pH 7.4).

TEER皮肤屏障完整性测试推荐方法的一个示例是将皮肤安装在扩散池中（例如，夹在供体和受体隔室之间的适当位置），并使其恒定在32°C±1°C的皮肤表面温度，角质层暴露于供体隔室中的空气中，皮肤下侧与离子溶液接触（例如，磷酸盐缓冲盐水，pH 7.4）。

A small amount of the ionic solution (sufficient to cover the entire surface of the skin section) would be briefly dosed on the stratum corneum. Then, one lead/electrode from an LCR meter would be placed in contact with the solution in the receptor compartment while the other lead/electrode would be placed in contact with the solution in the donor compartment. After measuring the resistance across the skin (e.g., in kΩ, normalized for area, noting that resistance is inversely proportional to area) the solution in the donor compartment would be removed and the skin surface would be gently blotted dry with an absorbent low lint laboratory tissue. The skin (still mounted in the diffusion cell) would then be allowed to equilibrate with the dry air above for a sufficient duration to normalize the hydration state of the stratum corneum before being dosed with the test topical product or RS.

将少量离子溶液（足以覆盖皮肤部分的整个表面）短暂地施加到角质层上。然后，LCR测量仪的一根导线/电极与受体室中的溶液接触，而另一根导线或电极与供体室中的液体接触。测量皮肤上的电阻（例如，kΩ，按面积归一化，注意电阻与面积成反比）后，除去供体室中的溶液，并用吸水性低皮棉实验室纸巾轻轻吸干皮肤表面。然后让皮肤（仍安装在扩散池中）与上面的干燥空气平衡足够长的时间，以使角质层的水合状态正常化。

The results for a TEER skin barrier integrity test can vary substantially depending on the LCR meter settings (e.g., frequency) and the technical procedures used for the test. The acceptance criterion for a specific electrical based skin barrier integrity test method may be justified by experimental data demonstrating that the selected acceptance criterion appropriately discriminates skin sections with a compromised barrier integrity from those with a competent barrier integrity.

TEER皮肤屏障完整性测试的结果可能会根据LCR仪表设置（例如频率）和测试使用的技术程序而产生较大差异。可以通过实验数据证明，特定基于电气的皮肤屏障完整性测试方法的验收标准可以适当区分屏障完整性受损的皮肤部分和屏障完整性合格的皮肤部分。

E.IVPT Skin Barrier Integrity Testing: General Considerations

There are three general considerations for the development or adoption of technical procedures for any skin barrier integrity test method during IVPT method development:

在IVPT方法开发过程中，开发或采用任何皮肤屏障完整性测试方法的技术程序有三个一般考虑因素：

i. The technical procedures should not irreversibly alter the skin barrier. It may be acceptable to temporarily alter the hydration state of the stratum corneum by briefly depositing an aqueous solution on the surface of the skin, as long as sufficient time is afforded for the hydration of the stratum corneum to normalize before dosing of the topical product. The procedure described above for a brief (e.g., 5-minute) exposure of the skin surface to tritiated water followed by a 30-minute duration during which the hydration state of the stratum corneum is re-equilibrating would likely be appropriate. By contrast, a 30-minute exposure of the skin surface to an aqueous solution for an electricalbased test method, followed within 5 minutes by dosing of the topical product, may not be appropriate without further characterization of the influence of the hydration state of the stratum corneum on the discrimination sensitivity of the skin to differences in topical bioavailability. Similarly, if a portable lamp were placed close to the skin to improve visibility while study procedures were being performed, the heat from the lamp may alter the local (micro)environment of the skin in a manner that is not representative of the ambient environmental conditions in the laboratory; this should be avoided.

i、 技术程序不应给皮肤屏障造成不可逆转地改变。只要有足够的时间使角质层的水合作用正常化，就可以通过在皮肤表面短暂沉积水溶液来暂时改变角质层的水合状态。上述将皮肤表面短暂（例如5分钟）暴露于氚水，然后持续30分钟，在此期间角质层的水合状态重新平衡的过程可能是合适的。相比之下，如果不进一步表征角质层的水合状态对皮肤对局部生物利用度差异的辨别敏感性的影响，将皮肤表面暴露于基于电的测试方法的水溶液中30分钟，然后在5分钟内给药局部产品可能是不合适的。类似地，如果在研究时将便携式灯靠近皮肤放置以提高能见度，灯的热量可能会改变皮肤的局部(微)环境，而这种方式不能代表实验室中的环境条件，应该避免。

ii. The acceptance criterion should be a cutoff value for the test result, at which a skin section fails the test. Skin sections that fail a barrier integrity test should not be dosed but may serve as non-dosed control skin sections. Skin sections with a passing barrier integrity test result may be considered to have a competent barrier integrity and may be dosed. This acceptance criterion should be selected based upon an understanding of the distribution of test results (among multiple replicate skin sections from multiple donors) for the specific barrier integrity test procedure performed with the specific type and preparation of skin under conditions relevant to the IVPT pivotal studies submitted in the ANDA. The intention of the barrier integrity test is to identify (and exclude) skin sections whose barrier integrity (intactness) is compromised. The intent is not to reduce the inherent variability in barrier function (permeability) in human skin that is representative of real variation in the human population. Also, the relative permeability of the skin to a drug from a topical product may not necessarily correlate with the permeability of the skin to water, and therefore, constraining the variability of the skin permeability to water (using a stricter acceptance criterion that excludes a larger number of skin sections) may not necessarily reduce the variability in the IVPT study results.

ii．验收标准应为皮肤部分未通过测试结果时的临界值。未通过屏障完整性测试的皮肤部分不应给药，但可作为未给药的对照皮肤部分。屏障完整性测试结果合格的皮肤部分可被视为具有合格的屏障完整性，并可给药。应基于对测试结果分布(来自多个供体的多个重复皮肤切片)的理解来选择此接受标准，该测试程序是在与ANDA中提交的IVPT关键研究相关的条件下，使用特定类型和制备的皮肤进行的。屏障完整性测试的目的是识别（并排除）屏障完整性（完整性）受损的皮肤部分。其目的并不是减少人体皮肤屏障功能（渗透性）的固有变异性，这这代表了人群的真实变化。此外，皮肤对局部产品的药物的相对渗透性可能不一定与皮肤对水的渗透性相关，因此，限制皮肤对水渗透性的可变性（使用更严格的接受标准，排除更多的皮肤部分）不一定会降低IVPT研究结果的可变性。

iii. The acceptance criterion should be able to discriminate skin sections with a compromised barrier integrity. This may be demonstrated by measuring the barrier integrity of skin sections mounted and equilibrated in a diffusion cell before and after deliberately compromising the skin barrier (e.g., by repeatedly using adhesive tape to strip away increasing amounts of the stratum corneum, piercing the skin several times with a 30 gauge needle, or using other physical or chemical insults to damage the skin barrier). Based upon the acceptance criterion selected, the test result for skin sections that pass the test before being damaged should fail the test after the damage.

iii.验收标准应能够区分屏障完整性受损的皮肤部分。这可以通过在故意破坏皮肤屏障之前和之后测量在扩散池中安装和平衡的皮肤部分的屏障完整性来证明（例如，通过反复使用胶带剥离越来越多的角质层，用30号针刺穿皮肤几次，或使用其他物理或化学侮辱来破坏皮肤屏障）。根据选定的接受标准，受损前测试结果合格的皮肤，破损后的测试结果应不合格。

F.Differences Between IVPT Method Development and Validation

1.Optimization of an IVPT Method Prior to Advancing to IVPT Method Validation

Different study designs and method parameters may be evaluated during the IVPT method development phase. For example, if the selected study parameters initially involve a dose duration of 48 hours and a study duration of 48 hours, and the flux profile is measurable, but it is not feasible to identify the maximum (peak) flux and a decline in the flux thereafter across multiple subsequent time points, then it may be appropriate to evaluate other study parameters as part of the IVPT method development. For example, a different target dose of the topical product and/or a longer sampling duration may be evaluated. An alternate study design may involve a shorter dose duration (e.g., 4–6 hours) after which the applied dose is removed from the skin, and the receptor solution continues to be sampled across a study duration that is sufficient to identify the maximum (peak) flux and a decline in the flux thereafter across multiple subsequent time points. While shorter dose durations can help to improve the shape of IVPT flux profiles, the removal of the topical product dose from the skin surface can be challenging and often requires its own method development and optimization. Also, the design of sensitivity studies for such an IVPT study design may require a more sophisticated understanding of IVPT studies. While reasonable efforts should be made to develop an IVPT method that produces a well-defined maximum (peak) flux and a decline in the flux thereafter across multiple subsequent time points, this may not be feasible for certain topical products even with study durations of 96 hours, or, at least, may not be feasible to produce reliably in all donors. In such circumstances, the IVPT method development report should detail the systematic efforts made to optimize the IVPT method.

在IVPT方法开发阶段，可以评估不同的研究设计和方法参数。例如，如果所选的研究参数最初涉及48小时的剂量持续时间和48小时的研究持续时间，并且通量分布是可测量的，但无法确定最大（峰值）通量和通量下降，则可以评估其他研究参数作为IVPT方法开发的一部分。例如，可以评估局部产品的不同目标剂量和/或更长的取样持续时间。另一种研究设计可能涉及更短的剂量持续时间（例如，4-6小时），之后去除皮肤的上样剂量，并且在研究持续时间内继续对受体溶液进行采样，该研究持续时间足以确定最大（峰值）通量以及随后在多个后续时间点的通量下降。虽然更短的剂量持续时间有助于改善IVPT通量分布曲线的形状，但从皮肤表面去除局部产品剂量可能具有挑战性，通常需要自己开发方法和优化。此外，此类IVPT研究设计的敏感性研究设计可能需要对IVPT研究有更复杂的理解。尽管应尽合理努力开发一种IVPT方法，该方法可在随后的多个时间点产生明确定义的最大（峰值）通量和通量下降，但这对于某些局部产品来说可能不可行，即使研究持续时间为96小时，或者至少不可能在所有供体中稳定地产生。在这种情况下，IVPT方法开发报告应详细说明为优化IVPT方法所做的系统努力。

The IVPT method development studies, being exploratory in nature, are often performed using a sample analytical method that is not validated (e.g., an HPLC or ultrahigh performance liquid chromatography (UPLC) method, often involving mass spectrometry (MS)); also, IVPT method development studies are often conducted in a manner that is not compatible with a quality management system which would otherwise make the evidence generated suitable to support valid conclusions. Such method development studies would not be suitable to demonstrate the validity of an IVPT method, or associated results. Therefore, although it may appear to be redundant, certain experiments performed during IVRT method development may need to be repeated during IVPT method validation, using appropriate controls, like a validated analytical method and procedures that are compatible with a suitable quality management system.

IVPT方法开发研究本质上是探索性的，通常使用未经验证的样品分析方法（例如，HPLC或超高效液相色谱（UPLC）方法，通常涉及质谱（MS））；此外，IVPT方法开发研究通常与质量管理体系不兼容，否则会使生成的证据适合支持有效结论。此类方法开发研究不适合证明IVPT方法或相关结果的有效性。因此，尽管IVRT方法开发过程中进行的某些实验看起来可能是多余的，但在IVPT方法验证过程中可能需要重复进行，使用适当的控制，如经过验证的与合适的质量管理系统兼容的分析方法和程序。。

It is important to clearly segregate and consistently identify those experiments and results that were part of IVPT method development separately from those that were part of IVPT method validation. It is also important to consistently identify all relevant method parameters and experimental conditions/controls for each set of IVPT results. Information in the method development report should clearly identify/distinguish when the results for apparently similar sets of experiments may have been obtained using different method parameters. Method development reports should clarify which sets of diffusion cells were run in parallel or separately (e.g., on separate days). In addition, the sample analytical method parameters used to analyze the samples from each set of IVPT experiments should be specified, and the report should indicate whether or not the sample analytical method was validated (either at the time of sample analysis or subsequently).

重要的是要明确的将IVPT方法开发中的实验和结果与IVPT方法验证中的实验与结果分开，并加以一致识别。对于每组IVPT结果，一致识别所有相关的方法参数和实验条件/对照同样重要。方法开发报告中的信息应清楚地识别/区分什么时候明显相似的实验结果可能是使用不同的方法参数获得的。方法开发报告应阐明哪些扩散测试池组是平行或单独运行的（例如，在不同的日子）。此外，应规定用于分析每组IVPT实验样品的样品分析方法参数，报告应说明样品分析方法是否经过验证（在样品分析时或随后）。

IV.IVPT METHOD VALIDATION

When all the relevant parameters of the IVPT method have been established, a pilot study should be performed using the final IVPT method and using a validated sample analytical method. The purpose of the pilot study is to validate the suitability of the selected IVPT method parameters by demonstrating that the performance characteristics of the IVPT method are appropriate to compare the cutaneous pharmacokinetics of a drug delivered topically from a test product and RS. The results from the pilot study, thereby, support the systematic validation of the IVPT method, which proceeds as a distinct study phase following IVPT method development.

当IVPT方法的所有相关参数都已确定后，应使用最终IVPT方法和经验证的样品分析方法进行试点研究。试点研究的目的是通过证明IVPT方法的性能特征适用于比较在测试产品和RS中局部传递的药物的皮肤药代动力学，来验证所选IVPT方法参数的适用性。因此，试点研究结果支持IVPT方法系统验证，这在IVPT方法开发之后作为一个特殊的研究阶段进行。

The results from this IVPT pilot study can help to estimate the number of donors that may be needed to adequately power the IVPT pivotal study. In addition to the test topical product and RS evaluated in the pilot study, a parallel assessment should be performed with a third topical product or formulation that is known or designed to be different from the RS, to validate the selectivity of the IVPT method to discriminate differences in bioavailability. The IVPT pilot study results should be plotted with error bars, comparing the permeation profiles for the three treatment groups in the pilot study. Separate plots should be prepared for average flux results and average cumulative permeation results. These data can be used to support specific IVPT method validation parameters (e.g., permeation profile and range).

IVPT试点研究的结果有助于估计可能需要的供体数量，以充分支持IVPT关键研究。除了试点研究中评估的试验外用产品和RS外，还应使用已知或设计与RS不同的第三种外用产品或配方进行平行评估，以验证IVPT方法对区分生物利用度差异的选择性。IVPT试点研究结果应绘制误差条，比较中试研究中三个治疗组的渗透曲线。应为平均通量结果和平均累积渗透结果绘制单独的图。这些数据可用于支持特定的IVPT方法验证参数（例如渗透图谱和范围）。

A pilot IVPT study performed with multiple skin donors (e.g., 4–6 skin donors) and a minimum of four replicate skin sections per donor per treatment group is recommended. As skin from an increasing number of donors is evaluated in the pilot study, the accuracy of the estimated number of donors needed to adequately power the IVPT pivotal study may improve. While skin from the same donors evaluated in the pilot study may also be used in the IVPT pivotal study, the results from the pilot study should not be combined with the results from the IVPT pivotal study for the purpose of statistical analysis.

建议对多个皮肤供者（例如4-6个皮肤捐赠者）进行IVPT试验研究，每个治疗组每个供者至少四个重复皮肤切片。随着越来越多的供者的皮肤在试点研究中得到评估，为IVPT关键研究提供足够动力所需捐献者估计数量的准确性可能会提高。虽然在试点研究中评估的同一供者的皮肤也可用于IVPT关键研究，但试点研究的结果不应与IVPT关键性研究的结果结合起来进行统计分析。

The equipment, methodologies, and study conditions used in the IVPT pilot study (and the eventual IVPT pivotal study) should be appropriately validated or qualified. If an applicant elects to use equipment, methodologies, or study conditions that are different from those recommended in this guidance, the applicant should demonstrate why it was necessary and scientifically justified to do so. Detailed protocols and well-controlled study procedures are recommended to ensure the precise control of dosing, sampling, and other IVPT study parameters, as well as potential sources of experimental bias.

IVPT试点研究（以及最终的IVPT关键研究）中使用的设备、方法和研究条件应经过适当验证或鉴定。如果申请人选择使用与本指南中建议的不同的设备、方法或研究条件，申请人应证明这样做的必要性和科学合理性。建议采用详细的方案和控制良好的研究程序，以确保精确控制给药、取样和其他IVPT研究参数，以及实验偏差的潜在来源。

The validation of the IVPT method should incorporate specific qualifications and controls (described below), performed using a validated sample analytical method, as applicable. The qualification of an IVPT method parameter refers to the process of defining what attributes make it suitable to perform its function in the IVPT method. For example, when repeated measurements of the temperature at the surface of skin mounted in a diffusion cell demonstrate that an IVPT equipment can maintain the skin surface temperature in the range of 32°C ± 1°C, the results can support a demonstration that the equipment is qualified to perform its function in an IVPT method for which a method parameter is the control of skin surface temperature in the range of 32°C ± 1°C across the relevant study duration.

IVPT方法的验证应包括具体的限定条件和控制（如下所述），如适用，使用经验证的样品分析方法进行。IVPT方法参数的限定指的是定义哪些属性适合在IVPT方法中执行其功能的过程。例如，当对安装在扩散池中的皮肤表面温度重复测量的结果表明IVPT设备可以将皮肤表面温度保持在32°C±1°C的范围内时，结果可以证明该设备有资格在IVPT方法中执行其功能，其中方法参数是在相关研究持续时间内将皮肤表面温度控制在32°C±1°C范围内。

A.Equipment Qualification

Suitable equipment for the IVPT method includes various models of VDCs and flow-through diffusion cells. The operating principles and specific test procedures differ among the various equipment; relevant procedures from the manufacturer may be used for installation, operational, and performance qualifications. The laboratory qualification of each diffusion cell should, at minimum, include 1) measurements of the diffusional area of the orifices of the donor and receptor compartments between which the skin is mounted, 2) the empirically measured volume of the receptor solution compartment in each VDC or the empirically measured outflow tube length for each flow-through diffusion cell, 3) the stability of the temperature measured at the skin surface (e.g., 32°C ± 1°C) across a relevant duration (e.g., 48 hours), and 4) the rate of stirring or agitation in VDCs, or the flow rate for flow-through diffusion cells, as applicable.

适用于IVPT方法的设备包括各种型号的VDC和流通扩散池。不同设备的工作原理和具体测试程序不同；制造商的相关程序可用于安装、操作和性能鉴定。每个扩散池的实验室鉴定至少应包括：1）测量皮肤安装在供体和受体隔间的孔口扩散面积，2）每个VDC中受体溶液隔间的经验测量体积或每个流通扩散池的经验测量流出管长度，3）在相关持续时间（例如48小时）内在皮肤表面测量的温度（例如32°C±1°C）的稳定性，以及4）VDC中的搅拌或搅拌速率，或流通扩散池的流速（如适用）。

If information related to the diffusional area of the orifices and the volume of the receptor solution compartment for each diffusion cell is available from the manufacturer, that information should be provided for each relevant diffusion cell, in addition to the empirical measurements made by the laboratory performing the IVPT studies. The equipment should control the diffusion cell temperature so that the skin surface temperature is verified to be stable (e.g., 32°C ± 1°C) for each diffusion cell before dosing (e.g., measured by a calibrated infrared thermometer), and monitored periodically throughout the duration of the experiment by repeatedly measuring the skin surface temperature of a non-dosed control diffusion cell that is run in parallel with the other replicate dosed diffusion cells and connected to the same water bath or thermoregulation system.

如果制造商提供了与每个扩散池的孔口扩散面积和受体溶液隔室体积相关的信息，则除了实验室进行IVPT研究的经验测量之外，还应提供每个相关扩散池的信息。设备应控制扩散池温度，以便在给药前验证每个扩散池的皮肤表面温度稳定（例如，32°C±1°C）（例如，通过校准的红外温度计测量），并在整个实验期间通过重复测量非剂量控制扩散池的皮肤表面温度进行周期性监测，该扩散池与其他重复剂量给药扩散池并联运行并连接到相同的水浴或温度调节系统。

B.Membrane (Skin) Qualification

Excised human skin is recommended as the membrane for the IVPT study. The validity of each skin section dosed in the study should be qualified using an appropriate test procedure to evaluate the stratum corneum barrier integrity. Acceptable barrier integrity tests may be based upon tritiated water permeation, TEWL, or electrical impedance/conductance measured across the skin. The test parameters and acceptance criteria used for the skin barrier integrity test should be justified for the specific method and instrumentation that is used during the study. The skin from all donors whose skin is included in the study should be prepared in a consistent manner and dermatomed to a relatively consistent thickness, within limits specified in the study protocol. The skin thickness should be measured and reported for each skin section included in the study. The assignment of replicate skin sections from a donor to each treatment group should be randomized, as feasible. It is acceptable to balance the distribution of skin thicknesses in each treatment group (test topical product or RS) by a procedure specified in the study protocol.

建议将切除的人体皮肤作为IVPT研究的膜。通过适当的测试程序来评估角质层屏障的完整性，来鉴定研究中使用的每个皮肤切片的有效性。可接受的屏障完整性测试可基于氚水渗透、TEWL或皮肤上测量的电阻抗/电导。用于皮肤屏障完整性测试的测试参数和验收标准应针对研究期间使用的特定方法和仪器进行验证。研究中包括的所有捐献者的皮肤应以一致的方式制备，并在研究方案中规定的范围内保持相对一致的皮肤厚度。应测量并报告研究中每个皮肤切片的皮肤厚度。在可行的情况下，应将供体的复制皮肤切片随机分配给每个治疗组。也可以通过研究方案中规定的程序来平衡每个治疗组（测试局部产品或RS）的皮肤厚度分布。

C.Receptor Solution Qualification

The composition and pH of the receptor solution used for the IVPT study should be qualified in relation to its compatibility with the skin as well as the stability and solubility of the drug in that receptor solution. The stability of the drug in the receptor solution samples should be validated as part of the receptor sample analytical method validation. The solubility of the drug in the IVPT receptor solution should be empirically determined in triplicate, to illustrate that the solubility of the drug in the receptor solution exceeds the highest sample concentration in the IVPT pivotal study, ideally by an order of magnitude. The solubility of the drug in the IVPT receptor solution should be sufficient to characterize the higher amounts of drug permeating from the increased drug delivery condition evaluated in the IVPT sensitivity assessment during IVPT method validation.

用于IVPT研究的受体溶液的组成和pH应符合其与皮肤的相容性以及药物在该受体溶液中的稳定性和溶解度。受体溶液样品中药物的稳定性应作为受体样品分析方法验证的一部分进行验证。药物在IVPT受体溶液中的溶解度应根据经验确定三份，以说明药物在受体溶液中溶解度超过IVPT关键研究中的最高样品浓度，理想情况下为一个数量级。药物在IVPT受体溶液中的溶解度应足以表征IVPT方法验证期间IVPT敏感性评估中评估的药物递送条件增加导致的药物渗透量增加。

The inclusion of 0.1% polyoxyethylene[20]oleyl ether (also known as Oleth-20, Volpo-20, or Brij-20; CAS number 9004-98-2) is recommended to enhance the solubility of physiological buffer based (aqueous) receptor solutions for hydrophobic drugs. If additional solubility is needed, small increases in the concentration of polyoxyethylene[20]oleyl ether (e.g., from 0.1% or 0.2%, which is typically adequate for most hydrophobic drugs, to higher concentrations) are recommended, but should not exceed 6% polyoxyethylene[20]oleyl ether. Other strategies to improve the solubility of the drug in the receptor solution that may have the potential to alter the permeability of the skin (e.g., inclusion of organic solvents and alcohols in the receptor solution) are not recommended and may invalidate the IVPT method.

建议加入0.1%聚氧乙烯[20]油酸酯（也称为Oleth-20、Volpo-20或Brij-20；CAS号9004-98-2），以提高疏水性药物生理缓冲液基（水）受体溶液的溶解度。如果需要额外的溶解度，建议将聚氧乙烯[20]油酸酯的浓度小幅度增加（例如，从0.1%或0.2%，这对于大多数疏水性药物来说通常是足够的，到更高的浓度），但不应超过6%的聚氧乙烯[20]油酸酯。不建议采用其他可能改变皮肤渗透性的提高药物在受体溶液中溶解度的策略（例如，在受体溶液内加入有机溶剂和醇），可能使IVPT方法无效。

The inclusion of an anti-microbial agent in the receptor solution (e.g., ~0.1% sodium azide or ~ 0.01% gentamicin sulfate) is recommended to mitigate potential bacterial decomposition of the dermis and/or epidermis in the diffusion cell, regardless of the study duration. Other antimicrobial agents may also be acceptable, and if used, information should be included in the ANDA to explain the reason for their selection (and for the concentration at which they were used).

无论研究持续时间如何，建议在受体溶液中加入抗菌剂（例如，约0.1%*氮化钠或约0.01%硫酸庆大霉素），以减轻扩散测试池中真皮和/或表皮的潜在细菌分解。其他抗菌剂也可以接受，如果使用，应在ANDA中包含相关信息，以解释选择的原因（以及使用的浓度）。

D.Receptor Solution Sampling Qualification

The accuracy and precision of receptor solution sample collection at each time point should be appropriately qualified. Evidence to qualify a sampling procedure should illustrate that the sampling technique can reliably collect a consistent volume of the sample from the well-mixed volume of the receptor compartment at each sampling event, and that no artifacts are likely to be created by the sampling technique. Information should be included describing the equipment manufacturer’s specification for the accuracy and precision of receptor solution sampling, when available.

应适当限定每个时间点受体溶液样品采集的准确度和精密度。鉴定采样程序的证据应表明，采样技术可以在每次采样中从一定体积、均匀混合的受体室中收集一致体积的样品，并且采样技术不会产生污染。如果可用，应包括描述设备制造商关于受体溶液取样准确性和精密度的规范的信息。

For IVPT studies using a flow-through diffusion cell, it may be appropriate to qualify the lengths of tubing, and their associated dead volumes, to accurately calculate the lag time before a sample elutes through the tubing and is collected. For IVPT studies using a VDC, removal of the entire receptor solution volume and full volume replacement of the receptor solution at each time point may provide optimal solubility sink conditions. The sampling of small aliquots of the receptor solution for an IVPT study may introduce anomalous measurements of apparently negative flux in certain regions of the IVPT study and produce flux profiles that are difficult to interpret.

对于使用流通扩散池的IVPT研究，可能需要确定管道长度及其相关死体积，以准确计算样品通过管道洗脱并收集之前的滞后时间。对于使用VDC的IVPT研究，在每个时间点去除整个受体溶液体积并全部置换受体溶液可以提供最佳溶解度漏槽条件。在IVPT研究中对受体溶液的小等份取样可能会在IVPT研究某些区域引入明显负通量的异常测量，并产生难以解释的通量分布。

E.Environmental Control

Ambient laboratory temperature and humidity during the study should be monitored and reported. An environmentally controlled temperature range of 21°C ± 2°C is recommended, and a humidity range of 50% ± 20% relative humidity is recommended, if feasible.

研究期间应监测和报告实验室环境温度和湿度。环境温度宜控制在21°C±2°C，相对湿度宜控制在为50%±20%。

F.Permeation Profile and Range

The flux profile and cumulative permeation profile for the IVPT pilot study should be plotted across a range of sampling times, which corresponds to the IVPT pivotal study duration. The calculation of flux and cumulative total permeation is discussed in more detail below. The results of the IVPT pilot study should validate that the selected study parameters are suitable to adequately characterize the permeation profile (the cutaneous pharmacokinetics) of the drug within the selected study duration (the range of sampling time points).

IVPT试点研究的通量分布和累积渗透分布应在一定取样时间范围内绘制，这与IVPT关键研究持续时间相对应。下面将更详细地讨论通量和累积总渗透的计算。IVPT先导研究的结果应验证所选研究参数适合于在所选研究时间(采样时间点范围)内充分表征药物的渗透特性（皮肤药代动力学）。

A sufficiently complete flux profile should be adequate to identify the maximum (peak) flux and a decline in the flux thereafter across multiple subsequent time points in the IVPT pilot study. The results of the IVPT pilot study should also validate that the sampling frequency provides suitable resolution to adequately characterize the permeation profile (particularly the flux profile).

在IVPT试点研究中，一个足够完整的通量分布应足以确定多个后续时间点的最大（峰值）通量和此后通量的下降。IVPT试点研究的结果还应验证采样频率提供了适当的分辨率，以充分表征渗透曲线（尤其是通量曲线）。

G.Precision and Reproducibility

The flux and cumulative permeation results from the IVPT pilot study (and the eventual IVPT pivotal study) should be calculated, tabulated, and reported for each diffusion cell at each time point, with summary statistics to describe the intra-donor average, standard deviation, and percent coefficient of variation (%CV) among replicates, as well as the inter-donor average, standard error, and %CV. Complete results for all data values used in the calculations should be reported in a clear and organized manner, to facilitate the reconstruction of the flux and cumulative permeation results. The design of the study should be detailed and clear, and data values should be clearly associated with specific donors, replicates, treatment groups, time points, etc

IVPT先导研究（以及最终的IVPT关键研究）的通量和累积渗透结果应在每个时间点为每个扩散池进行计算、制表和报告，并提供汇总统计数据，以描述供体内平均值、标准偏差和重复之间的变异系数百分比（%CV），以及供体间平均值和%CV。计算中使用的所有数据值的完整结果应以清晰和有组织的方式报告，以便于重建通量和累积渗透结果。研究的设计应详细、清晰，数据值应与特定捐献者、重复、治疗组、时间点等明确相关。

H.Dose Depletion

The recovery of permeated drug in the receptor solution should be characterized in each diffusion cell as the cumulative total permeation of the drug in the receptor solution over the IVPT duration. This may be expressed as a percentage of the nominal amount of drug in the applied dose (which may be estimated based upon the nominal strength of the drug in the topical product and the approximate mass of topical product dosed on the skin).

受体溶液中渗透药物的回收应为在每个扩散池中表征为药物在IVPT持续时间内在受体溶液中的累积总渗透。这可以表示为药物标称量在应用剂量中的百分比（可以基于局部产品中药物的标称强度和皮肤上施用的局部产品的近似质量来估计）。

For example, if 10 mg of a topical product containing 5% drug was dosed on the membrane, the amount of drug in the applied dose may be estimated to be 0.5 mg (or 500 µg). If a cumulative total of 10 µg of drug diffused into the receptor solution across a 48-hour duration of the IVPT, it would be possible to estimate that the 500 µg dose would have been depleted by approximately 10 µg, amounting to an approximately 2% dose depletion. The average percentage dose depletion may thereby be estimated (not accounting for skin content) and should be reported.

例如，如果将含有5%药物的10mg局部产品应用在膜上，则应用剂量中的药物量估计为0.5mg（或500µg）。如果在IVPT的48小时持续时间内，累计10µg药物扩散到受体溶液中，可以估计500µg剂量将消耗约10µg，相当于约2%的剂量消耗。由此可以估计平均剂量消耗百分比（不考虑皮肤含量），并应予以报告。

I.Discrimination Sensitivity and Selectivity

The discrimination ability of the IVPT method may be described using two concepts: sensitivity and selectivity. The IVPT sensitivity studies are necessarily performed during IVPT method development to establish IVPT method parameters like the dose amount, dose duration, study duration, etc. However, the analysis of the results from these studies is qualitative in nature, and they need not be repeated during the IVPT method validation phase.

IVPT方法的鉴别能力可以用两个概念来描述：灵敏度和选择性。IVPT方法开发期间必须进行IVPT灵敏性研究，以确定IVPT方法参数，如剂量、剂量持续时间、研究持续时间等。然而，这些研究结果的分析本质上是定性的，在IVPT方法验证阶段无需重复验证。

The IVPT sensitivity studies are typically performed toward the end of the IVPT method development phase, and a key purpose of these studies is to incorporate the final IVPT method parameters for the target dose and dose duration to be used in the pivotal study so that the IVPT sensitivity studies can support a demonstration of the validity of the final IVPT method. Therefore, IVPT sensitivity studies are described within this section of the guidance in the context of IVPT validation (rather than method development) to avoid dissociating the discussions of IVPT sensitivity (which is performed to establish the suitability of the final IVPT method parameters) and IVPT selectivity (which is performed once the final IVPT method parameters are established, and which is based upon the IVPT pilot study that is performed as part of the IVPT method validation). With the exception of the alternative dose amounts or dose durations used in the IVPT sensitivity study, it is important that the IVPT method parameters are consistent across the IVPT sensitivity, pilot, and pivotal studies (including the anatomical region specified in the study protocol (e.g., posterior torso), the skin source, and skin preparation).

IVPT灵敏度研究通常在IVPT方法开发阶段的末尾进行，这些研究的一个关键目的是将最终IVPT方法参数的目标剂量和剂量时间用于关键研究，IVPT灵敏性研究可以支持证明最终IVPT方法的有效性。因此本节指南在IVPT验证（而非方法开发）的背景下描述了IVPT灵敏度研究，以避免将IVPT灵敏度（用于确定最终IVPT方法参数的适用性）和IVPT选择性（一旦确定了最终IVPT方法参数，即进行该试验，并基于IVPT试验研究，该试验研究是IVPT方法验证的一部分）的讨论分开。除了IVPT敏感性研究中使用的替代剂量或剂量持续时间外，重要的是IVPT方法参数在IVPT灵敏度、先导性和关键性研究（包括研究方案中规定的解剖区域（如躯干后部）、皮肤来源和皮肤制备）中保持一致。

1.IVPT Sensitivity

IVPT sensitivity is the ability of the IVPT method to detect changes in the cutaneous pharmacokinetics of the drug as a function of differences in drug delivery. If the IVPT method consistently demonstrates higher and lower flux profiles (i.e., higher and lower values for IVPT endpoints) in response to increased and decreased drug delivery, respectively (or in response to other conditions expected to increase and decrease drug delivery, respectively), the IVPT method may be considered sensitive.

IVPT灵敏度是IVPT方法检测药物的皮肤药代动力学变化作为药物传递差异的功能的能力。如果IVPT方法始终显示出较高和较低的通量分布（即IVPT终点的高和低），分别对药物递送的增加和减少作出反应（或对预期分别增加和减少药物递送量的其他条件作出反应），则IVPT方法可能被认为是灵敏的。

There are a few potential approaches by which to produce the differences in drug delivery that can be differentiated by a suitably discriminating IVPT method. Regardless of the approach used, the differences in the IVPT permeation profiles are not necessarily expected to be specifically proportional to differences in the dose amount, dose duration, or product strength. For example, three-fold differences in the dose amount (even if outside the recommended target dose range) may provide distinct flux curves but may not result in three-fold differences in the IVPT endpoints because the skin barrier may be rate-limiting both in vitro and in vivo.

有几种潜在的方法可以产生药物递送的差异，这些差异可以通过适当鉴别的IVPT方法进行区分。无论采用何种方法，IVPT渗透曲线的差异不一定与剂量、剂量持续时间或产品强度的差异成正比。例如，剂量的三倍差异（即使在推荐的目标剂量范围之外）可以提供不同的通量曲线，但不会导致IVPT终点的三倍差别，因为皮肤屏障在体外和体内都可能是限制速率的因素。

In other words, if the target dose for the pivotal IVPT study was 10 mg/cm2 , a 3-fold lower dose would be ~3 mg/cm2 and a 3-fold higher dose would be 30 mg/cm2 ; thus, an IVPT sensitivity study might compare the flux profiles from 3, 10, and 30 mg/cm2 doses of the topical product. Similarly, if the target dose for the pivotal IVPT study was 15 mg/cm2 , a 3-fold lower dose would be 5 mg/cm2 and a 3-fold higher dose would be 45 mg/cm2 ; thus, an IVPT sensitivity study might compare the flux profiles from 5, 15, and 45 mg/cm2 doses of the topical product. An IVPT sensitivity study performed with multiple skin donors (e.g., 4–6 skin donors) and a minimum of four replicate skin sections per donor per treatment group is recommended.

换言之，如果关键IVPT研究的目标剂量为10 mg/cm2，则低3倍的剂量为~3 mg/cm2且高3倍的浓度为30 mg/cm2；因此，IVPT敏感性研究可以比较3、10和30 mg/cm2剂量的局部产品的通量分布。类似地，如果关键IVPT研究的目标剂量为15 mg/cm2，低3倍的剂量为5 mg/cm2而高3倍的浓度为45 mg/cm2；因此，IVPT敏感性研究可能会比较5、15和45 mg/cm2剂量的局部产品的通量分布。建议对多个皮肤捐献者（例如4-6个皮肤捐赠者）进行IVPT灵敏度研究，每个治疗组每个捐献者至少四个重复皮肤切片。

Modulation of Dose Amount: An IVPT method development study with different dose amounts may provide supportive evidence that the IVPT methodology is sensitive to differences in drug delivery.

剂量调节：不同剂量的IVPT方法开发研究可能提供支持性证据，证明IVPT方法对药物递送的差异敏感。

This approach is well suited to topical products that contain volatile components that evaporate from the formulation following dose application to the skin. Modulating the dose amount for such topical products effectively alters the thickness of the applied dose. The majority of volatile components from a thinner dose will tend to evaporate more rapidly (compared to a thicker dose), and a thinner dose will tend to deliver less drug into the skin (and/or for a shorter duration) compared to a thicker dose.

这种方法非常适合于含有挥发性成分的局部产品，这些挥发性成分在涂抹到皮肤后从配方中蒸发。调节这种局部产品的剂量有效地改变了应用剂量的厚度。较薄剂量的大部分挥发性成分往往会蒸发得更快（与较厚剂量相比），较薄剂量进入皮肤的药量较少（和/或持续时间更短）。

Modulating the dose amount can be an effective technique to modulate differences in drug delivery for formulations with volatile components, like gels, lotions, and many creams. However, modulating the dose amount may not necessarily produce perceptible differences in drug delivery for topical products like petrolatum-based ointments, or other types of topical products that do not evaporate on the skin, or that may not experience dose-dependent differences in metamorphosis that can alter the rate and extent of drug delivery.

调节剂量可以是一种有效的技术，可以调节含有挥发性成分的制剂（如凝胶、乳液和许多乳膏）的药物递送差异。然而，调节剂量并不一定会对局部产品（如基于凡士林的软膏）或其他类型的局部产品（不会在皮肤上蒸发）产生明显的药物递送差异，或者可能不会经历可改变药物递送速率和程度的剂量依赖性**差异。

Modulation of Dose Duration: For many topical products, it may be more effective to modulate the dose duration, instead of the dose amount, to produce differences in drug delivery and associated changes in the cutaneous pharmacokinetics of the drug.

剂量持续时间的调节：对于许多外用产品，调节剂量持续时间而不是剂量可能更有效，以产生药物递送的差异和药物的皮肤药代动力学的相关变化。

An IVPT method development study with a controlled dose amount (e.g., 15 mg/cm2 ) dosed for different durations (e.g., 2 hours, 6 hours, and 12 hours) may be well suited to provide supportive evidence that the IVPT methodology is sensitive to differences in drug delivery from many topical products. The scenario described in this example would support an IVPT study design where a topical product dose of 15 mg/cm2 is dosed for 6 hours (the target duration for the IVPT study) and then wiped off. The applied dose may be removed with a series of cotton-tipped swabs, one or more of which may be dry and one or more of which may be moistened (e.g., with a soap solution or water). The initial (dry) swab typically removes the bulk of the dose and subsequent swabs are used to remove the residual dose (i.e., the residue of the topical product which may otherwise continue to deliver drug into the skin) and/or to rinse the skin.

具有不同持续时间（例如，2小时、6小时和12小时）的受控剂量（例如，15 mg/cm2）的IVPT方法开发研究可能非常适合提供支持性证据，证明IVPT方法对许多局部产品的药物递送差异敏感。本例中描述的方案将支持IVPT研究设计，其中局部产品剂量为15 mg/cm2，持续6小时（IVPT研究的目标持续时间），然后擦掉。可使用一系列棉签去除应用的剂量，其中一个或多个棉签可能干燥，一个或更多棉签可能湿润（例如，用肥皂溶液或水）。初始（干）拭子通常去除大部分剂量，随后的拭子用于去除剩余剂量（即，局部产品的残留物，否则可能继续将药物输送到皮肤中）和/或冲洗皮肤。

To support a demonstration of the sensitivity of the IVPT study, the permeation profile produced by the target dose duration for the IVPT study (e.g., 6 hours) should be compared with a shorter dose duration (e.g., 2 hours) that is expected to perceptibly decrease the drug delivery, and also be compared with a longer dose duration (e.g., 12 hours) that is expected to perceptibly increase the drug delivery. Thereby, the three dose durations compared in the IVPT sensitivity study are designed to produce perceptible changes in the cutaneous pharmacokinetics of the drug as a function of differences in drug delivery, and thereby support a demonstration of the sensitivity of the IVPT method.

为了证明IVPT研究的灵敏度，应将IVPT研究目标剂量持续时间（例如6小时）产生的渗透曲线与预期明显降低药物递送的更短剂量持续时间进行比较，并且还与预期显著增加药物递送的更长剂量持续时间（例如12小时）相比较。因此，IVPT灵敏度研究中比较的三个剂量持续时间旨在根据药物递送差异产生药物的皮肤药代动力学的明显变化，从而支持IVPT方法的灵敏度证明。

The specific dose durations may be selected based upon an initial exploratory IVPT study performed during IVPT method development that characterizes the permeation profile when the dose is left on the skin for a longer duration (e.g., 24 or 48 hours). An important feature of the results from such an IVPT study is the duration of the initial phase of the permeation profile, when the flux is increasing at a relatively rapid rate.

具体剂量持续时间可以基于IVPT方法开发期间进行的初始探索性IVPT研究来选择，该研究表征了当剂量在皮肤上停留更长时间（例如24或48小时）时的渗透曲线。这种IVPT研究结果的一个重要特征是当通量以相对较快的速率增加时，渗透曲线初始阶段的持续时间。

For example, if such an exploratory study indicates that the flux increases on a steep slope until approximately 12 hours, and then continues to deliver the drug at a gradually increasing rate thereafter, it may suggest that the permeation profile for a dose duration of longer than 12 hours (e.g., 24 hours) may not be perceptibly different from that of the 12- hour dose duration, especially when compared in a relatively small number of donors and replicates (e.g., four donors with four replicates each per dose duration). It may also suggest that a 12-hour dose duration may be a good choice for the longest of the three dose durations in the IVPT sensitivity study.

例如，如果这样的探索性研究表明，通量以陡峭的斜率增加，直到大约12小时，然后继续以逐渐增加的速率递送药物，这可能表明，超过12小时（例如24小时）的剂量持续时间的渗透曲线可能与12小时剂量持续时间没有明显差异，特别是在相对较少数量的供体和复制体（例如每个剂量持续时间具有四个复制体的四个供体）中进行比较时。这也可能表明，在IVPT敏感性研究中，12小时的剂量持续时间可能是三个剂量持续时间中最长的一个很好的选择。

The target dose duration should be selected based upon considerations like the sensitivity of the sample analytical method, the ability to produce a permeation profile that can be perceptibly discriminated from that produced by the longer (12 hour) dose duration, and/or the labeled use of the topical product (which may indicate that the topical product should be reapplied every 4–6 hours).

目标剂量持续时间的选择应基于以下考虑因素，如样品分析方法的灵敏度、产生渗透曲线的能力，该渗透曲线可明显区别于较长（12小时）剂量持续时间产生的渗透曲线，和/或局部产品的标记使用（这可能表明局部产品应每4-6小时重新给药一次）。

The shortest of the three dose durations in the IVPT sensitivity study should be selected based upon the sensitivity of the sample analytical method and its ability to produce a permeation profile that can be perceptibly discriminated from that produced by the target (6 hour) dose duration.

在IVPT灵敏度研究中，三个剂量持续时间中最短的时间选择，应根据样品分析方法的灵敏度，以及可明显区别于目标维持时间（6小时）所产生的渗透曲线的能力来选择。

· Modulation of Product Strength: To validate the sensitivity, specificity, and selectivity of an in vitro release test (IVRT) method, altered strength formulations are routinely prepared. While it may seem convenient to use these altered strength formulations in an attempt to demonstrate the sensitivity and selectivity of an IVPT method, doing so may not produce the desired outcomes. There may be circumstances when this strategy may produce perceptibly different permeation profiles, however, in many instances, the resulting permeation profiles may not be perceptibly different when compared in a relatively small number of donors and replicates (e.g., four donors with four replicates each per topical product strength). In general, the modulation of topical product strength to support a demonstration of IVPT sensitivity is not recommended because it may not consistently produce the expected increase or decrease in drug delivery; however, in certain situations, higher and lower strength formulations (relative to the nominal strength of the RS) may suitably increase and decrease the drug delivery and cutaneous pharmacokinetics relative to that from the nominal strength topical product.

•调节产品规格：为验证IVRT方法的灵敏度、专属性和选择性，通常采用改变配方制剂规格的方法。虽然采用改变配方制剂的规格的方法来证明IVPT方法的灵敏度和选择性似乎很方便，但这种方式可能不会产生预期的结果。在某些情况下，这种策略可能会产生明显不同的渗透曲线，然而，在很多情况下，由于在对比研究中采用的供体组和重复数较少（如，每种外用制剂规格采用4个供体组，每组供体4个重复），并不能产生明显不同的渗透曲线。通常，不建议通过调整外用制剂规格的方法以证明IVPT的灵敏度，因为它可能不会持续产生预期的增加或减少的药物递送；然而，在某些情况下，相较于外用制剂的目标规格，较高和较低规格的配方制剂（相对于对照制剂（RS）的规格）可以适当地增加和降低药物递送和皮肤药代动力学。

2.IVPT Selectivity/IVPT选择性

IVPT selectivity is the ability of the IVPT method to discriminate the cutaneous pharmacokinetics of the drug between the RS and a topical product or formulation that exhibits differences in drug delivery relative to the RS. The IVPT pilot study with the parallel assessment of the RS, the test topical product, and a third topical product or formulation that is known or designed to be different from the RS may provide supportive evidence that the IVPT methodology is selective for differences in drug delivery. Topical product batch information for all topical product lots used in IVPT method development, validation and pilot studies, as applicable, should be submitted in the study reports. The topical product information should include, but not be limited to, information about the batch formula, manufacturing date, batch size, altered manufacturing processes (if applicable) and, if available, potency and content uniformity. The evaluation of inequivalence may be based upon a qualitative or quantitative comparison of the permeation profiles and/or the IVPT endpoints.

IVPT选择性是IVPT方法区分“对照制剂（RS）“和“与RS在药物递送方面存在差异的外用制剂或配方”在药物皮肤药代动力学差异的能力。在IVPT初步研究中，通过将RS、自研外用制剂和“已知或设计不同于RS”的第三种外用制剂或配方进行平行评估，提供支持性证据，说明IVPT方法对药物递送的差异具有选择性。如适用，应在研究报告中递交，IVPT方法开发、验证和初步研究中用到的所有外用制剂的相关批信息，包括但不限于：批配方、生产日期、批量、变更的生产工艺（如适用），以及效价和含量均匀度（如有）。可基于渗透曲线和/或IVPT终点的定性或定量比较进行不等效性评估。

J.Robustness/耐用性

A primary assumption related to robustness testing is that the test system performs consistently when all system variables (e.g., temperature, stirring rate) are at nominal settings. A value of robustness testing is that it can verify whether the system continues to provide a consistent output when specific variables are slightly altered, thereby qualifying operational ranges for those variables. However, the variability inherent in the permeability of human skin, whether in vitro or in vivo, may not be compatible with the primary assumption related to the consistency of the test system.

与耐用性测试相关的一个主要假设是，当所有系统变量（如：温度、搅拌速率）处于标准设置时，测试系统的性能一致。耐用性测试意义在于，它可以验证当特定变量略有改变时，系统是否能提供一致的输出结果，从而确定这些变量的操作范围。然而，无论是体外还是体内，人体皮肤渗透性固有的变异性可能与与测试系统一致性相关的主要初始的假设不兼容。

Nonetheless, results from studies during IVPT method development that appear to support the robustness of the IVPT method or system should be reported, if relevant. For example, an IVPT method may be robust to substantial variations in the stirring rate of the receptor compartment. Similarly, the permeation profile of a drug into and through human skin may appear to be robust to certain differences in the topical product strength. Ultimately, because it may not always be feasible to validate the robustness of IVPT method parameters, IVPT study procedures should be controlled as precisely as possible.

尽管如此，如果相关，应报告IVPT方法开发期间得到的关于支持IVPT方法或系统耐用性的研究结果。例如，IVPT方法对于接收室搅拌速率的显著变化具有耐用性。与其类似地，药物进入并通过人体皮肤的渗透曲线，可能对外用制剂规格的改变表现出耐用性。所以，对于验证IVPT方法参数的耐用性并不总是可行的，在IVPT研究中应尽可能精准地控制IVPT研究程序。

V. SAMPLE ANALYTICAL METHOD VALIDATION/样品分析方法验证

While exploratory studies performed during IVPT method development may use an unvalidated sample analytical method, it is essential that all studies conducted as part of the IVPT method validation use a validated sample analytical method. A validated IVPT method should use a validated sample analytical method (e.g., HPLC/MS or UPLC/MS). Therefore, a discussion of the sample analytical method for the IVPT method is included in this guidance under this section on IVPT method validation

虽然在IVPT方法开发的探索性研究中，可采用未经验证的样品分析方法，但作为IVPT方法验证的一部分，所进行的IVPT研究都必须采用经过验证的样本分析方法。经验证的IVPT方法应使用已验证的样品分析方法（如：HPLC/MS或UPLC/MS）。因此，本指南中关于IVPT方法验证的章节中包含了对IVPT方法样品分析方法的讨论。

However, the study protocols and reports related to the IVPT method are distinct from those for the sample analytical method that is used to quantify drug concentrations in IVPT receptor solution samples. The validation of a sample analytical method, in and of itself, does not demonstrate the validity of an IVPT method. Separate and specific reports should be submitted for the validation of the sample analytical method (e.g., HPLC/MS or UPLC/MS) and for the validation of the IVPT method.

然而，与IVPT方法相关的研究方案和报告与用于量化IVPT接收液样品定量的分析方法不同。样品分析方法的验证本身并不能证明IVPT方法的有效性。因此，应分别提交IVPT方法验证报告和样品分析方法（如：HPLC/MS或UPLC/MS）。

Any results from studies of the IVPT method that are performed during method development using a different sample analytical method than that which is ultimately validated, cannot support a demonstration of the validity of the IVPT method. Information should be provided in the IVPT method validation report referencing the (separate) sample analytical method validation, and clearly indicate that all relevant results in the IVPT method validation report were obtained using a validated sample analytical method (as opposed to a sample analytical method with different parameters than those which were validated).

如果在方法开发过程中，使用的样品分析方法与最终验证的样品分析方法不同，那么与其相关的IVPT方法研究结果则不能用于支持论证IVPT方法的有效性。应在IVPT方法验证报告中，提供参考的样品分析方法验证的信息，并明确表明IVPT方法验证报告中的所有相关结果均采用经验证的样品分析方法（不是与经验证的样品分析法有不同参数的样品分析放法）获得的。

The receptor sample analysis procedures (e.g., typically involving an HPLC/MS or UPLC/MS system) should be performed using chromatography software (e.g., a chromatography data system) with audit trails, and should include a multi-point (6–8 concentration) calibration curve with suitable quality control samples, and should be validated in a manner compatible with the FDA guidance for industry Bioanalytical Method Validation (May 2018).

接收液样品的分析方法（如：通常为HPLC/MS或UPLC/MS系统），应使用具有审计追踪的色谱软件（如，色谱数据系统），并应包括具有适当质量控制样品的多点（6～8个）校准曲线，并应符合FDA生物分析方法验证行业指南要求。

The validation of the receptor sample analytical method should include relevant qualifications of dilution integrity, if applicable, as well as stability assessments with the highest relevant temperature in the receptor solution for the longest relevant duration; the highest relevant temperature may be warmer than 32°C because the temperature of the receptor solution is often higher than the temperature at the surface of the skin, and the longest relevant duration may be the longest interval between sampling time points for methods in which the entire receptor solution is replaced at each sampling time point, or it could be longer in scenarios with only partial sampling of the receptor solution (e.g., 34°C for 48 hours).

接收液中样品分析方法的验证应包括稀释完整性确认（如适用），以及样品在最高相关温度的接收液中放置最长时间的稳定性评估；最高相关温度可略高于32°C，因为接收液的温度通常高于皮肤表面的温度。对于在每个取样时间点替换整个接收液的情况，最长相关时间为各相邻取样时间点中，间隔最长时间；但对于接收液部分取样的情况，时间可能更长（例如34°C放置 48h）。

If the samples are processed in specific ways for analysis (e.g., by drying and reconstituting the receptor samples in a smaller volume to concentrate the sample and increase the effective analytical sensitivity, or by dilution of receptor solution samples into the validated curve range of the sample analytical method) those procedures should be validated (e.g., by qualifying the dilution integrity during the sample analytical method validation). The stability of the drug in the receptor solution sample should be validated in a receptor solution matrix that has been exposed to the underside of the skin in a diffusion cell under conditions relevant to the IVPT pivotal study.

如果样品以特定的分析方法进行处理（如：通过将样品干燥后重新以较小的体积溶剂配制以提高有效分析灵敏度，或通过稀释接收液样品，使其达到已验证分析方法的曲线范围内），则应确认其处理方法（如：在样品分析方法验证期间确认稀释完整性）。接收液样品中药物的稳定性，应在与IVPT正式研究相关的条件下，在扩散池中皮肤下侧接触的接收液基质中进行。

VI. IVPT PIVOTAL STUDY/IVPT正式研究

The IVPT pivotal study protocol should incorporate considerations relevant to BE studies, in general

IVPT正式研究方案通常应包括与BE研究相关的考虑因素

A. Handling and Retention of Samples/样品的处理与保存

Refer to 21 CFR 320.38, 320.63 and the FDA guidances for industry Handling and Retention of BA and BE Testing Samples (May 2004) and Compliance Policy for the Quantity of Bioavailability and Bioequivalence Samples Retained Under 21 CFR 320.38(c) (August 2020), as applicable, regarding considerations for retention of study drug samples and to 21 CFR 320.36 for requirements for maintenance of records of BE testing. Retention samples should be randomly selected from the drug supplies received before allocating topical product units for use in an IVPT study in which the test topical product and RS are compared.

参考21 CFR 320.38，320.63和FDA行业指南《生物利用度（BA）和生物等效性（BE）研究中试验样品的处理和保存》（2004年5月）和《21CFR 320.38(c) BE样品留存的数量及生物利用度》（2020年8月），关于保留研究药物样品的考虑以及21 CFR 320.36关于保存BE检测记录的要求（如适用）。在采用自研外用制剂和RS进行IVPT对比研究前，应从收到的药品中随机选择样品进行留样。

B. Control of Study Procedures/研究程序的控制

Study procedures that have the potential to influence the results of the study should be appropriately controlled. Also, experimental observations that may have the potential to influence the interpretation of the study results, as well as any protocol or standard operating procedure (SOP) deviations, should be reported.

应对可能影响研究结果的研究程序进行适当控制。此外，应报告可能影响研究结果解释的实验观察结果，以及任何方案或标准操作程序（SOP）发生偏差。

Control of procedures related to the skin include the consistent control across the study of the skin preparation (e.g., dermatoming of skin sections) and the thickness of skin sections mounted on diffusion cells, as well as the skin storage conditions, including the duration for which the skin was frozen and the number of freeze-thaw cycles to which the skin was exposed. Skin from the same anatomical location should be used from all donors, and the demographics (age, race, sex) should be reported for all donors. Also, the IVPT sensitivity, pilot, and pivotal studies should use skin from the same anatomical site; otherwise, if skin from different anatomical sites is used across the different study phases, it may not be possible for the results of the IVPT sensitivity and pilot studies to support a demonstration of the discrimination ability of the IVPT method used for the pivotal study because the method parameters would not be aligned across the respective studies. Similarly, if a non-rate-limiting support membrane is used beneath the skin section (e.g., a filter membrane used in a validated IVRT method for the same topical product) then it should be used in a consistent manner for the IVPT sensitivity, pilot, and pivotal studies.

与皮肤相关的程序的控制，包括相同的皮肤处理方法（如：皮肤的剥离）和安装在扩散测试池上的皮肤切片的厚度的研究中的一致性，及皮肤储存条件，例如皮肤冷冻的储存时间和取出的冻融循环次数。所有供给皮肤均应采用相同解剖位置，并报告所有供体的人口统计信息（年龄、种族、性别）。此外，在IVPT灵敏度、初步实验和正式研究，应采用同一解剖部位的皮肤；否则，如果在不同的研究阶段使用不同位置解剖部位，IVPT灵敏度和初步试验的结果可能不足以支持用于正式研究的IVPT方法的区分力的论证，因为方法参数在各个研究中不一致。同样，如果在皮肤部分下方使用非速率限制性支撑膜（如：同类IVRT方法验证研究中使用的滤膜），则与IVPT灵敏度、初步试验和正式研究中一致。

Control of procedures related to the dose include the control of the area of dose application, the dose amount, the dosing technique, the dose duration, and the blinding and randomization procedures for dosing. The test topical product and RS should be dosed in an identical and consistent manner for all diffusion cells in the study. Differences in dosing technique may alter the metamorphosis of the dosage form on the skin, and inconsistencies in the diameter of the area dosed on each diffusion cell may significantly influence the dosed area and contribute to errors in the calculation of flux.

与剂量或上样相关的控制程序，包括：对上样面积、上样量、上样技术、剂量维持时间的控制，以及在研究中每个扩散池采用的盲法和随机化方法的一致性。不同的上样技术可能改变上样到皮肤上剂型的形态，扩散池孔口扩散面积的不一致可能会显著影响上样面积，从而导致通量计算的偏差。

Control of procedures related to sampling include the control of sampling time precision, the sampling technique, the duration of sampling and replacement of receptor solution, the sample volume or flow rate, and sample handling and storage.

与取样相关的程序控制包括取样时间精度、取样技术、取样和替换接收液的时间间隔、取样体积或流速，以及样品的处理和存储。

Control of procedures related to the pivotal study should include a non-dosed control skin section from each skin donor, which should be mounted in a diffusion cell and otherwise treated identically to the dosed skin sections, including sampling of the receptor solution at all time points to ensure that drug concentrations monitored in the receptor solution are associated with the dose applied in the IVPT pivotal study, and not drug contamination in the skin from that donor that might permeate into the receptor solution across the duration of the study. A pre-dose “zero” sample collected from each diffusion cell is also recommended, which may identify potential contamination associated with each skin section and/or each diffusion cell.

与正式研究相关的控制程序，应包括来自每个皮肤供体的非剂量对照皮肤部分，包括在所有时间点对接收液进行取样，以确保接收液中监测的药物浓度与IVPT正式研究中应用的剂量相关，而不是研究期间由于皮肤的药物污染渗透进入。建议从每个扩散池中采集“零”浓度样品，用于识别每个皮肤切片和/或每个扩散池的潜在污染。

In addition, investigators should perform the IVPT validation and pivotal studies within a quality management system that includes, but is not limited to, documented procedures for:

此外，研究人员应在一定的质量管理体系条件下，进行IVPT验证和正式研究，该体系包括但不限于：

·Study personnel identification, training, qualification, and responsibilities/ 研究人员的识别、培训、资格和职责

·Study management and study management personnel responsibilities/研究管理和研究管理人员职责

· Quality control (QC) and QC personnel responsibilities/质量控制（QC）和QC人员职责

· Quality assurance (QA) and QA personnel responsibilities/质量保证（QA）和QA人员职责

·Use of SOPs /标准操作规程（SOP）的制定

· Use of study protocols/研究方案的制定

· Use of study reports/研究报告的制定

· Maintenance and control of the study facility environment and systems/研究设施环境和系统的维护和控制。

·Qualification and calibration of instruments and computerized systems/仪器和计算机系统的鉴定和校准。

·Good documentation practices including, but not limited to, contemporaneous documentation of study procedures and recording of experimental observations or deviations from procedures specified in the study protocol or in relevant SOPs/良好的文件规范，包括但不限于：研究程序的及时记录、实验观察及相关SOPs中规定的程序的偏差。

· Maintenance of suitable records that facilitate the reconstruction of study events and procedures, including study sample handling and storage records (e.g., sample tracking logs), audit trails for sample analysis procedures, control of study materials and reagents, and electronic data control/维护有助于重建研究事件和程序的适当记录，包括研究样本处理和存储记录（如：样本跟踪日志）、样本分析过程的审计跟踪、研究物料和试剂的控制以及电子数据控制。

·Archival of study records研究记录存档

C. Blinding Procedure/盲法程序

A detailed description of the blinding procedure should be provided in the study protocol and final report. The packaging of the test topical product and RS should be similar in appearance to maintain adequate blinding of the investigator and any experimental operators.

研究方案和最终报告中应提供盲法程序的详细说明。试验外用制剂和RS的包装应保持相似的外，以确保研究者和任何实验操作人员的充分致盲。

D.Randomization/随机化

The method of randomization should be described in the protocol of the IVPT study and the randomization schedule provided, preferably in a SAS data set in .xpt format (created using the SAS XPORT procedure). It is recommended that an independent third party generate and hold the randomization code throughout the conduct of the study to minimize bias. The applicant may generate the randomization code if not involved in the packaging and labeling of the test topical product and RS dosed in the study. A sealed copy of the randomization scheme should be retained at the study site and should be available to FDA investigators at the time of site inspection to allow for verification of the treatment identity of each skin section.

应在IVPT研究方案中描述随机化方法并提供随机化计划表，推荐以.xpt格式的SAS数据集形式（使用SAS XPORT程序创建）。建议由独立的第三方在整个研究过程中生成并保存随机化代码，以减少偏差。如果研究中未涉及自研外用制剂和RS的包装和标签，申请人可以自行生成随机代码。随机方案的密封副本应保存在研究现场，并应在现场检查时提供给FDA研究人员，以便确认每个皮肤切片的具体信息。

E.Dosing/上样

In the IVPT pivotal study, the test topical product and RS should be dosed in an alternating pattern on successive diffusion cells (skin sections) from each donor. One of two dosing sequences (illustrated below) may be randomly assigned for each donor:

在IVPT正式研究中，对于每个供体组，可以在扩散池（皮肤切片）上以交替给药方式依次放置自研外用制剂和RS。对于每个供体组，可采用下述两个方式中的一个进行随机放样（如下所示）：

a. ABABAB…

b. BABABA…

F. Study Design/  研究设计

The IVPT pivotal study should compare the cutaneous pharmacokinetics of the drug from the test topical product versus that from the RS using excised human skin with a competent skin barrier mounted on a qualified diffusion cell system. The IVPT pivotal study should use a design that directly compares the test topical product and RS on skin from the same set of donors, each with the same number of replicate skin sections per donor per treatment group (dosed with either test topical product or RS topical), using the same IVPT method parameters.

在IVPT正式研究中，应采用皮肤屏障完整性良好的离体人类皮肤和经验证的扩散池系统，对自研外用制剂和RS的皮肤药代动力学进行对比研究。在自研外用制剂和RS的对比研究中，每个试验组应采用相同的皮肤供体、相同的皮肤切片重复数，并采用相同的IVPT方法参数。

The IVPT pivotal study design, methodology, and diffusion cell equipment considerations relating to sampling precision should be controlled as precisely as possible. For example, it may be appropriate to stagger the dose application on successive diffusion cells and to synchronize the sampling time points with the dosing time for that diffusion cell, to ensure consistent durations between dosing and sampling of all diffusion cells.

应尽可能精确地控制IVPT正式研究设计、方法和与扩散池设备取样精度。例如，在连续的扩散池上，错开上样时间点，根据上样时间的差异，从而同步取样时间，以确保所有扩散池给药和取样之间的时间间隔一致。

G. Inclusion Criteria/纳入标准

In general, the following inclusion criteria should apply: healthy, normal, barrier-competent skin from male and/or female donors of at least 18 years of age. Inclusion criteria related to donor demographics (e.g., age, race, sex) should be specified in the study protocol and demographic information should be reported for each donor. Additional criteria may be added by the applicant

通常，以下入选标准应适用：来自至少18岁的健康、正常、屏障功能皮肤，雄性和/或雌性供体。应在研究方案中规定符合纳入标准的人口统计信息（如，年龄、种族、性别），并报告每个供体的个人信息。申请人可以增加其它纳入标准。

The skin may be harvested following excision from patients undergoing a surgical procedure or excised from cadavers. A consistent source is recommended for all the skin used. The anatomical region specified in the study protocol (e.g., posterior torso) should be consistent for all donors whose skin is included in the study.

皮肤可以在外科手术的患者或尸体上获取。所有使用的皮肤应有一致的来源。研究方案中规定的解剖区域（如躯干后部）应与研究中包括皮肤的所有供体一致。

The study protocol should specify the inclusion (acceptance) criteria for skin sections based upon the barrier integrity test result, which should be reported for each skin section.

研究方案应根据屏障完整性测试结果规定皮肤部分的纳入（接受）标准。

The study protocol should specify inclusion criteria related to the temperature and duration of skin storage as well as the number of freeze-thaw cycles, all of which should be reported for each donor’s skin.

研究方案应规定与皮肤储存的温度和储存时间以及冻融循环次数纳入相关的标准。

The study protocol should specify the inclusion criteria related to the skin harvesting/processing procedures and skin thickness (e.g., dermatomed skin of 500 μm ± 250 μm thickness) used in the IVPT study.

研究方案应规定与IVPT研究中使用的皮肤采集/处理程序和皮肤厚度（例如，500μm±250μm厚度的皮肤）。

H. Exclusion Criteria/排除标准

In general, the following exclusion criteria should apply. Skin from subjects with a known (history of) dermatological disease should be excluded from the study. Skin with tattoos, stretch marks, or any visible sign of abnormality should be excluded from the study. Skin exhibiting a significant density of terminal hair is not recommended and should be excluded from the study. Additional criteria may be added by the applicant.

通常，采用以下排除标准。患有已知皮肤病（病史）的供体皮肤；有纹身、妊娠纹或任何明显异常迹象的皮肤应；皮肤表现出明显的末梢毛发密度等。申请人可增加其他标准。

While gentle washing or rinsing of the skin surface is appropriate, submerging the skin in an aqueous solution for more than a few minutes may damage the skin barrier and should be avoided; such skin sections should be excluded from the study. Also, skin that has been subjected to shaving with a blade; abrasive polishing; tape-stripping; or cleansing with alcohols, solvents, or other strong solutions that could damage the skin barrier should be excluded from the study.

虽然可以采用温和地清洗或漂洗皮肤表面，但将皮肤浸泡在水溶液中几分钟以上可能会破坏皮肤屏障，应避免该方式。另外，不建议采用用刀片刮过的皮肤；磨料抛光；胶带剥离；或使用酒精、溶剂或其他可能破坏皮肤屏障的强极性溶液进行清洁。

Skin from donors with significant background levels of the drug or other compounds that may interfere with the quantification of the drug in receptor solution samples should be excluded from the study.

来自供体的皮肤具有显著的药物背景水平或其他可能干扰受体溶液样品中药物定量的化合物应排除在研究之外。

Skin from donors exhibiting a high barrier integrity test failure rate among replicate skin sections may be excluded from the study, and skin from an alternative donor may be used instead.

重复皮肤切片中屏障完整性测试失败率高的供体皮肤可能被排除在研究之外，应采用替代供体皮肤。

I. IVPT Endpoints/  IVPT终点

The endpoints for the IVPT pivotal study are based upon parameters that characterize the rate and extent to which the drug permeates into and through the skin and becomes available in the receptor solution. Specifically, the rate of drug permeation is characterized by the flux (J) and the extent of drug permeation is characterized by the total cumulative amount (AMT) of drug permeated into the receptor solution across the study duration.

IVPT正式研究的终点，是基于表征药物渗透和通过皮肤中，并进入接收液的速率和程度的参数。具体来说，药物渗透速率以通量（J）进行表征，而药物渗透程度采用整个研究期间渗透到接收液中的药物的总累积量（AMT）进行表征。

The flux (rate of drug permeation) should be plotted as J on the Y-axis in units of mass/area/time (e.g., nanograms (ng)/cm2 /hr) versus time on the X-axis. Flux profiles commonly resemble plasma pharmacokinetic profiles, however, it is important to distinguish that the flux is a rate, rather than a concentration. The extent of drug permeation should also be plotted, as the total cumulative amount (AMT) of drug permeated on the Y-axis in units of mass/area (e.g., ng/cm2 ) versus time on the X-axis.

以通量（药物渗透速率）为纵坐标Y-轴，时间为横坐标X-轴作图；其中Y-轴用“J”标识，以质量/面积/时间（如，纳克/平方厘米/小时）为单位。通量分布模型通常类似于血浆药代动力学分布模型，尽管通量是一个速率单位，并不代表浓度。也应对药物的渗透程度作图，以药物累积渗透量（单位为质量/面积，如纳克/平方厘米）为纵坐标Y-轴，时间为横坐标X-轴。

The flux should be calculated based upon: the receptor sample concentration (e.g., 2.0 ng/mL) at each time point; the precise, empirically measured volume of that specific diffusion cell (e.g., 6.0 mL) which may vary between individual cells; the area of dose application (e.g., 1 cm2 ); and the duration for which the receptor volume was accepting the drug. For example, if the sample exemplified here represented a 2-hour period following dosing, then J would be calculated based upon the values above as:

流量的计算应基于：每个时间点的接收液中的样品浓度（如：2.0 ng/mL）；精确、经验性的测量扩散池的体积（如：6.0mL），每个扩散池体积或有不同；上样区域面积（如：1cm2）；研究的持续时间。例如，如果这里例举的样品表示给药后2小时内的情况，则J将基于上述值计算为：J = [(2.0 ng/mL) x (6.0 mL)]/(1 cm2 )/(2 hrs) = 6 ng/cm2 /hr

This flux should be calculated and reported for each diffusion cell for each sampling interval and plotted across the entire study duration to generate the flux profile for each diffusion cell. The rate calculated above may be plotted at the 2-hour time point, or at the midpoint between 0 and 2 hours (i.e., 1 hour).

应计算并报告各扩散池的所有相邻采样点之间的通量，并绘制整个研究期间所有扩散池的通量曲线。上面计算的速率可以在2小时的实际时间点进行绘制，或者在0到2小时（即1小时）之间的中点绘制。

In addition, the AMT should be calculated and reported for each diffusion cell. This cumulative amount of drug that has permeated (in total across the entire study) should be reported as the AMT endpoint, rather than using a trapezoid rule to calculate the area under the flux curve.

此外，应计算并报告每个扩散池的总累计渗透量（AMT）。AMT终点为药物渗透的累计量（整个研究期间的渗透总量），而不是采用梯形法则计算的通量曲线下的面积。

The maximum flux (Jmax) at the peak of the drug flux profile and the AMT should both be compared for locally-acting test topical products and RSs. This is somewhat analogous to the comparison of the Cmax and AUC for systemically-acting test products and RSs, inasmuch as the pair of endpoints in each case facilitates a comparison of the rate and extent to which the drug from each type of product (locally-acting or systemically-acting) becomes available at the site of action.

应将局部起效的自研制剂和RSs的最大通量（Jmax，药物通量曲线的最高峰）和AMT进行对比。这类似于全身起效的自研制剂和RSs的Cmax和AUC的比较，因为这些终点可分别用于表征各剂型（局部起效或全身起效）药物到达作用部位的速率和程度。

A confidence interval (CI) should be calculated for each IVPT endpoint: 应计算每个IVPT终点的置信区间（CI）：

a. the natural log-transformed maximum flux (Jmax) 自然对数换算后的最大通量（Jmax）

b. the natural log-transformed total cumulative amount (AMT) permeated自然对数换算后的总累积渗透量（AMT）

It is the responsibility of the applicant to determine the number of donors required to adequately power the IVPT pivotal study, however, a minimum of four dosed replicates per donor per treatment group (test product or RS) is recommended.

申请人有责任确定足以支持IVPT关键研究所需的供体数量，然而，建议每个试验组（自研制剂或RS）每个供体至少4个重复剂量。

At the completion of the study, if the number of skin replicates is the same for all donors in the test topical product and RS treatment groups in the IVPT study, a statistical analysis for a balanced design is recommended. If skin sections or diffusion cells are excluded from the final statistical analysis because of experimental loss/issues, and the resulting data set is unbalanced, a statistical analysis for an unbalanced design is recommended.

在IVPT研究结束时，如果自研外用制剂和RS试验组中所有供体的皮肤切片重复数均一致，建议采用平衡设计法进行统计分析。如果皮肤切片或扩散测试池因实验损失/问题而被排除在最终统计分析之外，且所得数据集不平衡，则建议对不平衡设计进行统计分析。

Approaches to statistical analysis of the pivotal study are described in section VIII of this guidance. Appendix I provides example SAS code for determining BE with both a balanced dataset and an unbalanced dataset. Appendix II provides numerical examples with simulated data sets. Appendix III provides example R code for determining BE.

在本指南的第VIII部分，对正式研究的统计分析方法进行了描述。附录I中提供了用于测定平衡数据集和非平衡数据集BE的SAS代码示例。附录II中提供了模拟数据集的具体示例。附录III中提供了测定BE的R代码示例。

VII. SUBMITTING INFORMATION ON IVPT STUDIES IN AN ANDA/ANDA中递交的IVPT研究信息

For IVPT studies with topical products submitted in ANDAs that are intended to support a demonstration of BE, detailed study protocols, relevant SOPs, and detailed reports should be submitted for the IVPT method validation (including the IVPT pilot study) and the IVPT pivotal study. In addition, a detailed report describing the IVPT method development should be submitted. These protocols, SOPs, and reports should be submitted in module 5.3.1.2 of the electronic Common Technical Document (eCTD) and should describe experimental procedures, study controls, quality management procedures, and data analyses.

对于在ANDA中提交的用于支持BE演示的外用制剂的IVPT研究，应提交详细的研究方案、相关SOP和详细的报告，用于IVPT方法验证（包括IVPT初步研究）和IVPT关键研究。此外，还应提交一份详细的IVPT方法开发报告。这些方案、SOPs和报告应在电子通用技术文件（eCTD）的模块5.3.1.2中递交，并对相关的试验方法、研究控制、质量管理程序和数据分析进行描述。

Note that the study protocols, SOPs, and reports related to the IVPT method are distinct from those for the sample analytical method that is used to quantify drug concentrations in IVPT receptor solution samples (e.g., an HPLC/MS or UPLC/MS method). Separate protocols and SOPs should be submitted for the sample analytical method validation. Sample analytical method development and validation reports, pilot and pivotal IVPT study sample analysis reports, as well as associated SOPs and protocols relevant to the sample analysis of an IVPT study with human skin should be submitted in Module 5.3.1.4 of the eCTD.

注意，用于IVPT接收液中样品浓度的定量分析方法（如，HPLC/MS或UPLC/MS方法）与IVPT方法相关的研究方案、SOPs和报告不同。样品分析方法验证应提交单独的方案和标准操作规程。样本分析方法开发和验证报告、试点和关键IVPT研究样本分析报告以及与人体皮肤IVPT研究的样本分析相关的相关SOP和协议应在eCTD的模块5.3.1.4中提交。

VIII.IVPT PIVOTAL STUDY STATISTICAL ANALYSIS/IVPT正式研究统计分析

The two treatment groups would correspond to the test topical product (T) and the RS (R). The statistical analysis should consider a sample of n donors, for which r^T replicate skin sections from the j ^thdonor (j = 1, ⋯ , n) are available for the T group and r^R replicate skin sections from the j ^th donor (j= 1, ⋯ , n) are available for the R group. Each replicate (i) from each donor (j) should have been randomly assigned to each product.

两个治疗组将对应于自研外用制剂（T）和RS（R）。进行统计分析应考虑以下样本n 组供体，其中r^T复制第j个供者的皮肤切片(j = 1.⋯ , n) 适用于T组和r^R复制第j个供体的皮肤切片(j = 1.⋯ , n) 可用于R组。每个供体（j）的每个复制品（i）应随机分配给每个产品。

Define the following quantities: 定义如下：

T_ij= the natural log-transformed IVPT endpoint (Jmax or AMT) dosed with the test topical product for the i^th skin replicate from the j^th donor (i = 1, 2, ⋯ ,r^T，j =1, 2, ⋯ , n)

T_ij=第j个供体的第i个皮肤复制品的试验外用产品给药的自然对数转换IVPT终点（Jmax或AMT）(i = 1, 2, ⋯ , r^T，j =1, 2, ⋯ , n)

R_ij = the natural log-transformed IVPT endpoint( Jmax or AMT) dosed with the RS for the i^th skin replicate from the j^th donor (i= 1, 2, ⋯ , r^R，

j = 1, 2, ⋯ , n)

R_ij=第j个供体第i次皮肤复制的RS给药的自然对数转换IVPT终点（Jmax或AMT）(i= 1, 2, ⋯ , r^R，j= 1, 2, ⋯ , n)

r^T_j= the number of skin replicates from the j th donor dosed with the test topical product ( j = 1, 2, ⋯ , n)

r^T_j=第j个供体用外用制剂（j = 1, 2, ⋯ , n)

r^Rj = the number of skin replicates from the j th donor dosed with the RS ( j=1, 2, ⋯ , n)

r^Rj=第j个供体用RS( j=1, 2, ⋯ , n)给药的皮肤切片的重复数

r^∗=r^R₁+r^R₂+……+r^R_n = the total number of skin replicates in the R group

r^∗ =r^R₁+r^R₂+……+r^R_n=R组皮肤切片总数

n = the number of donors

n = 供体组的数量

If the numbers of skin replicates available for the final statistical analysis are the same for the n donors for the T group and the R group, the resulting data set is balanced. For simplicity of notation, the common number of skin replicates for one donor for one treatment group in a balanced data set is denoted as r=r^T₁+r^T₂+……=r^T_n=r^R1+r^R2=……r^R_n

如果可用于最终统计分析的皮肤切片重复次数相同的n 组和T组和R组的供体中，所得数据集是平衡的。为方便标记，在平衡数据集中，一个供体对一个治疗组的皮肤重复次数表示为r=r^T₁+r^T₂+……=r^T_n=r^R1+r^R2=……r^R_n

A diffusion cell may be excluded from among the replicates in a data set when there is a documented observation of a failure (e.g., visual observation that a skin section tears and leaks during the study) or a protocol deviation (e.g., the receptor compartment in a diffusion cell is discovered to be empty at the first sampling time point). In such instances, if sufficient skin remains from the same donor, and no samples from that diffusion cell have been analyzed, a replacement diffusion cell can be set up and studied. Otherwise (if the diffusion cell cannot be replaced) the resulting data set becomes unbalanced.

当有失败的观察记录（如：在研究期间观察到皮肤可见撕裂和泄漏）或方案的偏差（如：扩散测试池中的接收室在第一个取样时间点被发现为空）时，扩散测试池可从数据集中的重复数中排除。在这种情况下，如果来自同一供体的足够皮肤仍充足，并且没有对该扩散池的样品进行分析，则可以采用替代扩散池。若没有可替代扩散池，则该结果数据集是不平衡的。

The statistical analysis methods for assessing BE in the cases of a balanced data set and an unbalanced data set are described below. For a donor to be included in the statistical analysis, there should be at least 3 replicate skin sections from the donor for each (T and R) treatment group.

下文描述了在平衡数据集和非平衡数据集情况下评估BE的统计分析方法。对于纳入统计分析的供体，每个（T和R）治疗组的供体应至少有3个重复皮肤切片。

Step 1. Determine S_wr,the estimated within-donor standard deviation of the RS, for each of the natural log-transformed IVPT endpoints Jmax and AMT:

步骤1.确定S_wr,，每个自然对数变换IVPT终点Jmax和AMT的RS的估计供体内标准偏差：

(a) If S_wr≥0.294, use the scaled average BE (SABE) approach to determine BE for the individual IVPT endpoint(s) in Steps 2, 3.1, and 4.1

（a） 如果S_wr ≥0.294，使用缩放平均BE（SABE）方法确定步骤2、3.1和4.1中单个IVPT终点的BE

(b) If S_wr＜0.294, use the regular average BE (ABE) approach through the two one-sided tests (TOST) procedure (Schuirmann, 1987) to determine BE fo the individual IVPT endpoint(s) in Steps 2, 3.2, and 4.2

（b） 如果S_wr＜0.294，使用常规平均BE（ABE）方法，通过两个单侧测试（TOST）程序（Schuirmann，1987），在步骤2、3.2和4.2中确定单个IVPT终点的BE

Step 2. Determine the point estimate for the mean difference of T and R products (I^{^}), its standard error (se(I^{^})), and the corresponding degrees of freedom (df∗).

确定T和R乘积平均差的点估计(I^{^}), 其标准误差(se(I^{^}）），以及相应的自由度(df∗)

For a balanced data set, determine I^{^}, se(I^{^}) and df∗ by the following: 对于平衡数据集，确定I^{^}, se(I^{^})和df∗通过以下方式：

For an unbalanced data set, approximate I^{^}, se(I^{^}) and df∗ by using PROC MIXED  (or PROC GLM) in SAS. The example code is provided in Appendix I.

对于不平衡数据集，近似I^{^}, se(I^{^})和df∗ 在SAS中使用PROC MIXED（或PROC GLM）。附录I中提供了示例代码。

Step 3.1. Scaled Average BE (SABE) Approach标度平均BE（SABE）方法

In the SABE approach, the hypotheses to be tested are: 在SABE方法中，要测试的假设是：

Rejection of the null hypothesis supports the conclusion of equivalence of the two products. 否定零假设支持两种产品等价的结论。

Determine (1-α)*100% upper confidence bound for (μT-μR)2-θσ²_WRbased on Howe’s Approximation (Howe, 1974) (α = 0.05):

Note that t（1-α）,df∗ is （1-α） ∗ 100^th percentile of the Student’s t-distribution with df∗ degrees of freedom and x²（1-α），（r*-n） is （1-α） ∗ 100th percentile of the Chi- square distribution with （r*-n）degrees of freedom

Step 3.2. Regular Average BE (ABE) Approach常规平均BE（ABE）方法

In the ABE approach, the hypotheses to be tested are: 在ABE方法中，要测试的假设是：

Rejection of the null hypothesis supports the conclusion of equivalence of the two products. 否定零假设支持两种产品等价的结论。

Determine the (1 − 2α)*100% confidence interval for μT-μR (α = 0.05):

where t（1-α）,df∗ is （1-α） ∗ 100th  percentile of the Student’s t-distribution with df∗ degrees of freedom.

Step 4.1. BE Determination with SABE Approach用SABE方法确定BE

For the test product to be bioequivalent to the reference standard, both of the following conditions must be satisfied for each IVPT endpoint tested: 为使自制制剂与参比制剂具有生物等效性，每个IVPT试验终点必须满足以下两个条件：

a.the 95% upper confidence bound for (μ_T-μ_R)²-θσ²_WR must be less than or equal to zero (numbers should be kept to a minimum of four significant figures for comparison).

b.the point estimate of the T/R geometric mean ratio must fall within the pre- specified limits [1/m，m], where m = 1.2500.

Step 4.2. BE Determination with ABE Approach 采用ABE法确定BE测定

For the test product to be bioequivalent to the reference standard, the 90% confidence interval for μ_T-μ_R must be contained within the limits [1/m，m] in the original scale for each IVPT endpoint tested, where m = 1.2500.

在IVPT重点测试中，如果μ_T-μ_R的90%置信区间必须包含在每个测试IVPT终点原始量表中的限值[1/m，m]内，其中m=1.2500，则证明自研制剂与对照制剂具有生物等效