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

工業(yè)指南中ANDAs申請?zhí)峤坏耐庥弥苿┑捏w外滲透試驗研究

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

美國衛(wèi)生與公眾服務部食品和藥物管理局藥物評估與研究中心（CDER）2022年10月仿制藥

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

本指南旨在幫助申請人提交用于皮膚的液體和/或其他半固體產品申請ANDA，包括皮膚和粘膜（如陰道），以下稱為“局部產品”。由于這些產品復雜的遞送途徑，通常是局部起效的，以及某些制劑的潛在復雜性，外用制劑（外用溶液除外）被歸類為復雜制劑。本指南為體外滲透試驗（IVPT）研究提供了建議，該研究比較了擬申報仿制制劑及其參考制劑（RS），以支持證明與參比制劑的（RLD）生物等效性（BE）。參比制劑通常為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).

本指南不適用于通過眼、耳、鼻、吸入、口服或注射途徑給藥的藥物，也不適用于透皮或局部給藥系統(tǒng)（包括貼片、局部貼片或緩釋膜）。

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.

本指南是不討論適用該指南的特定外用制劑的指南。FDA建議申請人在考慮IVPT研究的設計和實施時，參考本指南和任何相關的特定產品指南（PSG）以及任何其他相關的行業(yè)指南，如有必要，結合其他研究，IVPT研究可能適合支持建議的通用外用制劑及其RLD具有生物等效性的證明。FDA還建議申請人定期查閱FDA的指導網頁，因為可能會有更多的指導，有助于仿制藥的開發(fā)。

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.

一般來說，F(xiàn)DA的指導文件并沒有確立法律上可強制執(zhí)行的責任。相反，指南描述了機構目前對某一主題的想法，并且應僅視為建議，除非引用了具體的監(jiān)管或法定要求。在機構指南中使用“應該”一詞意味著有人建議或建議，但不是必須的。

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.

本指南是作為FDA“藥品競爭行動計劃”的一部分制定的，該計劃配合GDUFA計劃和其他FDA舉措，旨在促進處方藥市場的競爭，促進高質量和實惠的藥品的進入市場，并改善公眾醫(yī)療。

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.

《聯(lián)邦食品、藥品和化妝品法案》（FD&C法案）通常要求ANDA包含合適的信息，說明：仿制藥與RLD相比1）具有相同活性成分、使用條件、給藥途徑、劑型、規(guī)格和標簽（允許存在某些差異），和2）與RLD具有生物等效性。因此，如果ANDA中提交的信息不足以證明自制制劑與RLD具有生物等效性，則ANDA將不予批準。

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研究可用于評估局部產品中的藥物（即活性成分）在皮膚作用部位或附近可用的速率和程度，并可用于表征和比較測試局部產品和RS中藥物的生物利用率和程度。IVPT通量曲線類似于藥代動力學曲線，可以使用*的IVPT終點進行分析，這些終點與最大濃度（Cmax）的藥代動力學終點和濃度-時間曲線下的面積（AUC）有些相似。然而，IVPT研究表征的是吸收的速率和程度，而不是體內的分布、代謝和排泄。因此，雖然表征IVPT研究監(jiān)測的局部藥物生物利用度的動力學是相關的，本指南中使用的術語“皮膚藥代動力學”不應被解釋為僅體現(xiàn)與吸收成分相關的藥代動力學的所有方面，吸收成分直接控制局部應用藥物在作用部位局部可用的速率和程度。本指南側重于在ANDA中提交的用于支持BE演示的方法開發(fā)、方法驗證和IVPT研究的一般考慮和建議。

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方法的開發(fā)適用于支持特定局部產品的BE演示，通常涉及一系列系統(tǒng)的探索性研究。開發(fā)適合其預期目的的IVPT方法的努力不當或不足，增加了后續(xù)IVPT驗證、試點和關鍵研究最終不足以支持BE演示的可能性。相比之下，適當和系統(tǒng)的IVPT方法開發(fā)研究有助于確定IVPT研究設計和方案（方法）參數，這些設計和方案能夠可靠地產生通量曲線，從而有助于比較從測試局部產品和RS局部遞送至皮膚的藥物的皮膚藥代動力學。

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.

應在ANDA中提交一份詳細且組織良好的IVPT方法開發(fā)報告，以說明IVPT方法是如何優(yōu)化的，并支持為IVPT選擇的方法參數是適當或必要的，特別是在方法參數與本指南中推薦的方法不同的情況下）。機構審查方法開發(fā)報告的目的是了解為什么選擇了特定的IVPT方法參數，以及所得IVPT方法是否具有適當的敏感性和重現(xiàn)性。該方法開發(fā)報告應明確指出/區(qū)分每組數據使用的方法參數，說明為優(yōu)化IVPT方法所做的努力，并證明為IVPT選擇的方法參數是合適的。

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.

鼓勵申請人使用本指南中的建議，如果申請人選擇使用與本指南中建議的方法不同的方法，IVPT方法開發(fā)報告應說明為什么使用本指南推薦的替代方法來優(yōu)化IVPT方法在科學上是合理的。建議程序的一些示例在隨后的章節(jié)中描述，以幫助申請人確定應在ANDA中提交信息的情況，以解釋為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.

應總結最終IVPT方法的所有相關參數（例如，在表格中），并在ANDA中提交。此外，應提供信息，以簡要解釋最終IVPT方法參數的選擇，如設備（例如，垂直擴散池（VDC））、皮膚源（例如，尸體）、皮膚類型（例如，后軀干）、皮膚準備（例如，皮膚科）、皮膚屏障完整性測試（例如，經表皮水分損失（TEWL）測量）、，皮膚屏障完整性測試驗收標準（例如，<15克/米2/小時（g/m2/小時））、局部產品劑量（例如，15毫克/厘米2（mg/cm2））、劑量持續(xù)時間（例如，6小時）、研究持續(xù)時間（如，24小時、48小時等）、受體溶液取樣時間（例如1、2、4、6、8、12、16、20和24小時）等。《IVPT測試中的皮膚研究》

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方法參數的選擇，如設備、皮膚來源、皮膚類型、皮膚準備和皮膚屏障完整性測試程序，可能基于研究人員的經驗或便利性，如實驗室中特定設備或儀器的可用性、既定的組織供應協(xié)議或其他后勤考慮。然而，如果選定的IVPT方法參數似乎不適合特定的IVPT法，則申請人有責任系統(tǒng)地評估備選方法參數，并最終驗證選定的IVPT方法參數是否適合預期用途。IVPT方法驗證的推薦程序詳見本指南第四節(jié)。

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方法參數的選擇，如局部產品劑量、劑量持續(xù)時間、研究持續(xù)時間（可能長于劑量持續(xù)時間）、取樣計劃、取樣程序、受體溶液組成和樣品分析方法，對于每種IVPT方法可能有所不同，應系統(tǒng)開發(fā)、優(yōu)化IVPT方法的此類參數，和/或對相關局部產品進行驗證。IVPT方法開發(fā)研究應描述這些方法參數的差異如何影響最終的IVPT通量分布，以便從評估的條件中客觀選擇最佳研究條件。

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).

應根據IVPT方法開發(fā)期間進行的研究，評估每種IVPT方法在研究中使用的劑量選擇?？梢栽趤碜酝唤M供體的復制皮膚切片上平行比較不同劑量，以優(yōu)化IVPT研究的劑量。選擇最佳劑量的考慮因素可以包括（1）可以施加劑量的一致性（可能使用不同的分配和/或散布技術），（2）通量分布的再現(xiàn)性，（3）劑量和劑量持續(xù)時間對通量分布形狀的影響，和（4）不同時間點受體溶液樣品中藥物濃度的近似范圍（相對于樣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).

所選擇的采樣時間表和研究持續(xù)時間應足以表征藥物的皮膚藥代動力學，理想情況下，包括足夠完整的通量曲線，以確定最大（峰值）通量以及隨后在多個后續(xù)時間點的通量下降。在研究持續(xù)時間內留在皮膚上的劑量可能會持續(xù)給藥一段時間，并且不一定會在以后的時間點出現(xiàn)適當的流量下降。在這種情況下，開發(fā)IVPT方法可能是合適的，該方法包括在皮膚上適當的持續(xù)時間后擦去施用的劑量，并在此后的較長時間內繼續(xù)監(jiān)測受體溶液，在此期間可以表征通量分布的下降。應選擇采樣頻率，以便為通量分布提供合適的分辨率，并且建議在整個研究持續(xù)時間內（例如，48小時）至少八個非零采樣時間點。

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.

在方法開發(fā)過程中，應建立適當的技術程序和控制參數。這些可以包括以一致的方式制備皮膚并將其安裝在擴散池上的程序，確定將皮膚表面溫度調節(jié)在規(guī)定范圍內的儀器設置，適當地進行屏障完整性測試，控制分配到每個皮膚部分的劑量的準確性和精密度。

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，可以開發(fā)一種劑量程序，其使用正位移移液管來分配體積控制量的局部產品，靶向一定質量（例如，15mg/cm2）的局部產品在皮膚上的沉積。如果擴散池給藥室的孔口內徑為15毫米（mm），有效劑量面積約為1.77 cm2，則表明每個擴散池的目標劑量為約26.5 mg局部產品。方法開發(fā)過程中的實驗可以確定，基于外用產品的密度，具有特定移液管的特定型號正移液管的特定體積設置可重復分配約27.5 mg外用產品（例如，以多次重復移液管分配為特征，將其分配到精密天平上的稱量舟中）。這種移液管設置對于劑量分配過程可能是最佳的，其中劑量分配儀器，如高效液相色譜（HPLC）玻璃瓶的平底，或玻璃棒或毛細管的圓形端，隨后用于將分配的劑量均勻地分配到安裝在擴散池中的皮膚部分上，并且其中在劑量散布之前和之后重復稱量劑量散布儀器表明，在劑量散布之后殘留在玻璃瓶底部的殘留局部產品可重復地達到約1.0mg局部產品（表明將可重復地將約26.5mg局部產品施用到每個皮膚部分）。IVPT方法的技術程序和控制參數的此類特征，如給藥程序的再現(xiàn)性，應在方法開發(fā)過程中確定，此后可能不需要在每次使用相同程序時進行證明。‍

‍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.

應在IVPT方法開發(fā)期間制定皮膚屏障完整性測試的技術程序。三種類型的屏障完整性測試是常見的，然而，目前沒有適用于這三種類型人體皮膚屏障完整性檢測的藥典標準協(xié)議或驗收標準。盡管如此，下面討論了三種常見類型的屏障完整性測試的推薦參數。IVPT測試中的皮膚研究

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皮膚屏障完整性測試包括在皮膚外表面附近測量水（蒸汽）從皮膚部分下側流過皮膚屏障的速率。對于試驗，將皮膚部分安裝在擴散池中（例如，夾在供體和受體室之間的適當位置），皮膚下側與受體室中的受體溶液（例如，磷酸鹽緩沖鹽水，pH 7.4）接觸，并平衡至32°C±1°C的皮膚表面溫度。如果皮膚部分被切割得足夠大，足以覆蓋其所安裝的擴散池的法蘭，那么在皮膚表面溫度為32°C±1°C的條件下平衡數小時后，可以在不破壞皮膚部分與擴散池下法蘭的粘附性的情況下輕輕移除供體隔室，從而允許TEWL探針直接放置在皮膚表面上而不是放置在供體隔室的頂部。通常，在每個皮膚部分上至少進行三次重復測量，并在測量穩(wěn)定后進行記錄。（下圖，皮膚測試用經皮水分流失測量儀）

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.

用于測量TEWL的市售設備在設計和操作原理上可能有所不同。由具有特定設計（例如，開放室與封閉室）的裝置測量的TEWL可能相對更容易受到環(huán)境條件的影響。因此，通常盡可能精確地控制環(huán)境溫度和濕度（例如，如果可行，理想的溫度范圍為21°C±2°C，濕度范圍為50%±20%相對濕度）。更精確地控制相對濕度（例如，在40%–50%的范圍內）可以減少具有特定設計的設備的TEWL測量的可變性。TEWL裝置的制造商可提供用于體外使用的測量探針和適配器的某些設計，并且可能適合使用。測量探頭室、測量探頭孔口、體外適配器和被測皮膚面積的直徑不一致，以及探頭傳感器與皮膚表面的距離變化，可能是由于供體隔室的（可變）高度（如適用），可能會增加TEWL測量的變異性。TEWL測量裝置相對于供體隔室和/或皮膚部分的對準的不一致控制也可能增加TEWL測定的可變性。此外，由具有特定設計的設備測量的TEWL可能相對更容易受到握住探頭的手的熱傳遞的影響。申請人應遵循制造商用戶手冊中關于所用特定TEWL測量設備的相關說明。

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.

每平方米每小時不超過約15克水（例如，≤ 15g/m2/hr）可能是人體軀干或大腿皮膚上TEWL屏障完整性測試的合理皮膚屏障完整性驗收（截止）標準；如果將此作為截止標準，TEWL>15 g/m2/hr的皮膚切片將無法通過測試。未通過屏障完整性測試的皮膚部分不應給藥，但可作為未給藥的對照皮膚部分。較高的截止（例如，≤ 20g/m2/hr）也可能是合理的，如果實驗數據證明所選驗收標準適當地區(qū)分屏障完整性受損的皮膚部分和屏障完整性合格的皮膚部分。

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，對于具有合格屏障完整性的皮膚部分的TEWL測量可以根據TEWL測試設備、其操作方式和環(huán)境條件而變化（例如，較高的環(huán)境濕度或離皮膚表面更大的距離可能會降低TEWL的測量值）。精確控制環(huán)境和設備/操作因素可以最大限度地減少TEWL測量的變化。因此，應在IVPT方法開發(fā)過程中優(yōu)化TEWL測量的技術程序（或基于實驗室進行試驗的事先優(yōu)化）。此外，TEWL測量設備應進行適當校準（由制造商進行校準，對于某些設備，也應在每組測試之前進行校準）。申請人可提供制造商為所用特定TEWL裝置規(guī)定的相關校準程序的信息；這可以與IVPT方法開發(fā)報告一起提交在ANDA中，以支持實驗室為TEWL測量建立的技術程序的適當性。當TEWL屏障完整性測試用于任何研究階段（IVPT方法開發(fā)、試點研究、驗證和/或關鍵研究）時，應監(jiān)測和報告TEWL阻隔完整性測試期間的實驗室環(huán)境溫度和濕度。

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-皮膚屏障完整性測試的推薦方法的一個例子是將皮膚安裝在擴散池中（例如，夾在供體和受體室之間的適當位置），并使其平衡至32°C±1°C的皮膚表面溫度，使角質層暴露在供體室中的空氣中，皮膚下側與受體溶液（例如磷酸鹽緩沖鹽水，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).

將少量的氚水（足以覆蓋皮膚部分的整個表面）短暫地施加于角質層。該劑量的氚水將在表面上停留一段精確控制且相對較短的時間（例如，5分鐘），之后將其從皮膚表面去除（例如，使用移液管去除大量體積，然后使用實驗室吸水紙輕輕吸干）。然后在從皮膚表面去除氚水之后的精確持續(xù)時間（例如，從皮膚表面除去5分鐘劑量的氚水后30分鐘）對受體溶液進行取樣。

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.

雖然可以移除并補充整個體積的受體室，但通常僅將一等分的受體溶液（例如，磷酸鹽緩沖鹽水，pH 7.4）轉移到合適體積的閃爍液中進行計數。等分試樣的體積通常取決于所使用的閃爍流體的類型以及適合與閃爍流體混合的水性流體的最大量。然后，使用閃爍計數器來定量取樣的等分試樣中的放射性量，可用于計算滲入較大（整個）體積的受體溶液中的氚水的量；使用氚水的比活度進行計算，以將給定的放射性量等同于每平方厘米皮膚表面積滲透的氚水當量體積。

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.

人體軀干或大腿皮膚上氚水屏障完整性測試的合理皮膚屏障完整性驗收（截止）標準是每cm2約1.5當量（eq）微升（µL）的氚水（例如，約1.5 eq. µL/cm2或約1.5 eq. mg/cm2）。該測試涉及5分鐘劑量，隨后30分鐘采樣持續(xù)時間（即，去除劑量后30分鐘采樣）。氚水測試結果大于1.5 eq. mg/cm2的皮膚切片判定為未通過測試，并被排除在使用局部產品的皮膚切片人群之外；未通過屏障完整性測試的皮膚部分不應給藥，但可作為未給藥的對照皮膚部分。如果實驗數據證明所選驗收標準能夠適當區(qū)分屏障完整性受損的皮膚部分和屏障完整性合格的皮膚部分，則其他驗收標準也可能合理。（下圖，放射性測試用閃點計數器）

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.

當計算氚水屏障完整性測試的結果時，給藥表面積是一個很重要的影響因素。例如，如果使用1.5 eq. mg/cm2的驗收標準和孔直徑為15 mm、皮膚表面積為1.77 cm2的擴散池，則計算出滲入受體室的氚水質量為約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.

基于電的皮膚屏障完整性測試有幾種變體，將測試結果報告為電阻、電導或相關電概念的測量值，以表征穿過皮膚的整體電流。涉及皮膚的經上皮電阻測試可以更具體地稱為經表皮電阻（TEER）皮膚屏障完整性測試。測試結果可用電導單位表示，電導是電阻的倒數?；陔姎獾钠つw屏障完整性測試通常使用設計用于測量電子電路或電氣部件的電感（L）、電容（C）和電阻（R）的儀器；這些儀器通常被稱為LCR儀表，并具有可調整的不同設置（測試參數）。

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皮膚屏障完整性測試推薦方法的一個示例是將皮膚安裝在擴散池中（例如，夾在供體和受體隔室之間的適當位置），并使其恒定在32°C±1°C的皮膚表面溫度，角質層暴露于供體隔室中的空氣中，皮膚下側與離子溶液接觸（例如，磷酸鹽緩沖鹽水，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.

將少量離子溶液（足以覆蓋皮膚部分的整個表面）短暫地施加到角質層上。然后，LCR測量儀的一根導線/電極與受體室中的溶液接觸，而另一根導線或電極與供體室中的液體接觸。測量皮膚上的電阻（例如，kΩ，按面積歸一化，注意電阻與面積成反比）后，除去供體室中的溶液，并用吸水性低皮棉實驗室紙巾輕輕吸干皮膚表面。然后讓皮膚（仍安裝在擴散池中）與上面的干燥空氣平衡足夠長的時間，以使角質層的水合狀態(tài)正?；?。

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皮膚屏障完整性測試的結果可能會根據LCR儀表設置（例如頻率）和測試使用的技術程序而產生較大差異?？梢酝ㄟ^實驗數據證明，特定基于電氣的皮膚屏障完整性測試方法的驗收標準可以適當區(qū)分屏障完整性受損的皮膚部分和屏障完整性合格的皮膚部分。

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方法開發(fā)過程中，開發(fā)或采用任何皮膚屏障完整性測試方法的技術程序有三個一般考慮因素：

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、 技術程序不應給皮膚屏障造成不可逆轉地改變。只要有足夠的時間使角質層的水合作用正?；?，就可以通過在皮膚表面短暫沉積水溶液來暫時改變角質層的水合狀態(tài)。上述將皮膚表面短暫（例如5分鐘）暴露于氚水，然后持續(xù)30分鐘，在此期間角質層的水合狀態(tài)重新平衡的過程可能是合適的。相比之下，如果不進一步表征角質層的水合狀態(tài)對皮膚對局部生物利用度差異的辨別敏感性的影響，將皮膚表面暴露于基于電的測試方法的水溶液中30分鐘，然后在5分鐘內給藥局部產品可能是不合適的。類似地，如果在研究時將便攜式燈靠近皮膚放置以提高能見度，燈的熱量可能會改變皮膚的局部(微)環(huán)境，而這種方式不能代表實驗室中的環(huán)境條件，應該避免。

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．驗收標準應為皮膚部分未通過測試結果時的臨界值。未通過屏障完整性測試的皮膚部分不應給藥，但可作為未給藥的對照皮膚部分。屏障完整性測試結果合格的皮膚部分可被視為具有合格的屏障完整性，并可給藥。應基于對測試結果分布(來自多個供體的多個重復皮膚切片)的理解來選擇此接受標準，該測試程序是在與ANDA中提交的IVPT關鍵研究相關的條件下，使用特定類型和制備的皮膚進行的。屏障完整性測試的目的是識別（并排除）屏障完整性（完整性）受損的皮膚部分。其目的并不是減少人體皮膚屏障功能（滲透性）的固有變異性，這這代表了人群的真實變化。此外，皮膚對局部產品的藥物的相對滲透性可能不一定與皮膚對水的滲透性相關，因此，限制皮膚對水滲透性的可變性（使用更嚴格的接受標準，排除更多的皮膚部分）不一定會降低IVPT研究結果的可變性。

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.驗收標準應能夠區(qū)分屏障完整性受損的皮膚部分。這可以通過在故意破壞皮膚屏障之前和之后測量在擴散池中安裝和平衡的皮膚部分的屏障完整性來證明（例如，通過反復使用膠帶剝離越來越多的角質層，用30號針刺穿皮膚幾次，或使用其他物理或化學侮辱來破壞皮膚屏障）。根據選定的接受標準，受損前測試結果合格的皮膚，破損后的測試結果應不合格。

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方法開發(fā)階段，可以評估不同的研究設計和方法參數。例如，如果所選的研究參數最初涉及48小時的劑量持續(xù)時間和48小時的研究持續(xù)時間，并且通量分布是可測量的，但無法確定最大（峰值）通量和通量下降，則可以評估其他研究參數作為IVPT方法開發(fā)的一部分。例如，可以評估局部產品的不同目標劑量和/或更長的取樣持續(xù)時間。另一種研究設計可能涉及更短的劑量持續(xù)時間（例如，4-6小時），之后去除皮膚的上樣劑量，并且在研究持續(xù)時間內繼續(xù)對受體溶液進行采樣，該研究持續(xù)時間足以確定最大（峰值）通量以及隨后在多個后續(xù)時間點的通量下降。雖然更短的劑量持續(xù)時間有助于改善IVPT通量分布曲線的形狀，但從皮膚表面去除局部產品劑量可能具有挑戰(zhàn)性，通常需要自己開發(fā)方法和優(yōu)化。此外，此類IVPT研究設計的敏感性研究設計可能需要對IVPT研究有更復雜的理解。盡管應盡合理努力開發(fā)一種IVPT方法，該方法可在隨后的多個時間點產生明確定義的最大（峰值）通量和通量下降，但這對于某些局部產品來說可能不可行，即使研究持續(xù)時間為96小時，或者至少不可能在所有供體中穩(wěn)定地產生。在這種情況下，IVPT方法開發(fā)報告應詳細說明為優(yōu)化IVPT方法所做的系統(tǒng)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方法開發(fā)研究本質上是探索性的，通常使用未經驗證的樣品分析方法（例如，HPLC或超高效液相色譜（UPLC）方法，通常涉及質譜（MS））；此外，IVPT方法開發(fā)研究通常與質量管理體系不兼容，否則會使生成的證據適合支持有效結論。此類方法開發(fā)研究不適合證明IVPT方法或相關結果的有效性。因此，盡管IVRT方法開發(fā)過程中進行的某些實驗看起來可能是多余的，但在IVPT方法驗證過程中可能需要重復進行，使用適當的控制，如經過驗證的與合適的質量管理系統(tǒng)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).

重要的是要明確的將IVPT方法開發(fā)中的實驗和結果與IVPT方法驗證中的實驗與結果分開，并加以一致識別。對于每組IVPT結果，一致識別所有相關的方法參數和實驗條件/對照同樣重要。方法開發(fā)報告中的信息應清楚地識別/區(qū)分什么時候明顯相似的實驗結果可能是使用不同的方法參數獲得的。方法開發(fā)報告應闡明哪些擴散測試池組是平行或單獨運行的（例如，在不同的日子）。此外，應規(guī)定用于分析每組IVPT實驗樣品的樣品分析方法參數，報告應說明樣品分析方法是否經過驗證（在樣品分析時或隨后）。

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.

當IVPT方法的所有相關參數都已確定后，應使用最終IVPT方法和經驗證的樣品分析方法進行試點研究。試點研究的目的是通過證明IVPT方法的性能特征適用于比較在測試產品和RS中局部傳遞的藥物的皮膚藥代動力學，來驗證所選IVPT方法參數的適用性。因此，試點研究結果支持IVPT方法系統(tǒng)驗證，這在IVPT方法開發(fā)之后作為一個特殊的研究階段進行。

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試點研究的結果有助于估計可能需要的供體數量，以充分支持IVPT關鍵研究。除了試點研究中評估的試驗外用產品和RS外，還應使用已知或設計與RS不同的第三種外用產品或配方進行平行評估，以驗證IVPT方法對區(qū)分生物利用度差異的選擇性。IVPT試點研究結果應繪制誤差條，比較中試研究中三個治療組的滲透曲線。應為平均通量結果和平均累積滲透結果繪制單獨的圖。這些數據可用于支持特定的IVPT方法驗證參數（例如滲透圖譜和范圍）。

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.

建議對多個皮膚供者（例如4-6個皮膚捐贈者）進行IVPT試驗研究，每個治療組每個供者至少四個重復皮膚切片。隨著越來越多的供者的皮膚在試點研究中得到評估，為IVPT關鍵研究提供足夠動力所需捐獻者估計數量的準確性可能會提高。雖然在試點研究中評估的同一供者的皮膚也可用于IVPT關鍵研究，但試點研究的結果不應與IVPT關鍵性研究的結果結合起來進行統(tǒng)計分析。

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試點研究（以及最終的IVPT關鍵研究）中使用的設備、方法和研究條件應經過適當驗證或鑒定。如果申請人選擇使用與本指南中建議的不同的設備、方法或研究條件，申請人應證明這樣做的必要性和科學合理性。建議采用詳細的方案和控制良好的研究程序，以確保精確控制給藥、取樣和其他IVPT研究參數，以及實驗偏差的潛在來源。

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方法的驗證應包括具體的限定條件和控制（如下所述），如適用，使用經驗證的樣品分析方法進行。IVPT方法參數的限定指的是定義哪些屬性適合在IVPT方法中執(zhí)行其功能的過程。例如，當對安裝在擴散池中的皮膚表面溫度重復測量的結果表明IVPT設備可以將皮膚表面溫度保持在32°C±1°C的范圍內時，結果可以證明該設備有資格在IVPT方法中執(zhí)行其功能，其中方法參數是在相關研究持續(xù)時間內將皮膚表面溫度控制在32°C±1°C范圍內。

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.

適用于IVPT方法的設備包括各種型號的VDC和流通擴散池。不同設備的工作原理和具體測試程序不同；制造商的相關程序可用于安裝、操作和性能鑒定。每個擴散池的實驗室鑒定至少應包括：1）測量皮膚安裝在供體和受體隔間的孔口擴散面積，2）每個VDC中受體溶液隔間的經驗測量體積或每個流通擴散池的經驗測量流出管長度，3）在相關持續(xù)時間（例如48小時）內在皮膚表面測量的溫度（例如32°C±1°C）的穩(wěn)定性，以及4）VDC中的攪拌或攪拌速率，或流通擴散池的流速（如適用）。

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.

如果制造商提供了與每個擴散池的孔口擴散面積和受體溶液隔室體積相關的信息，則除了實驗室進行IVPT研究的經驗測量之外，還應提供每個相關擴散池的信息。設備應控制擴散池溫度，以便在給藥前驗證每個擴散池的皮膚表面溫度穩(wěn)定（例如，32°C±1°C）（例如，通過校準的紅外溫度計測量），并在整個實驗期間通過重復測量非劑量控制擴散池的皮膚表面溫度進行周期性監(jiān)測，該擴散池與其他重復劑量給藥擴散池并聯(lián)運行并連接到相同的水浴或溫度調節(jié)系統(tǒng)。

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.

建議將切除的人體皮膚作為IVPT研究的膜。通過適當的測試程序來評估角質層屏障的完整性，來鑒定研究中使用的每個皮膚切片的有效性。可接受的屏障完整性測試可基于氚水滲透、TEWL或皮膚上測量的電阻抗/電導。用于皮膚屏障完整性測試的測試參數和驗收標準應針對研究期間使用的特定方法和儀器進行驗證。研究中包括的所有捐獻者的皮膚應以一致的方式制備，并在研究方案中規(guī)定的范圍內保持相對一致的皮膚厚度。應測量并報告研究中每個皮膚切片的皮膚厚度。在可行的情況下，應將供體的復制皮膚切片隨機分配給每個治療組。也可以通過研究方案中規(guī)定的程序來平衡每個治療組（測試局部產品或RS）的皮膚厚度分布。

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研究的受體溶液的組成和pH應符合其與皮膚的相容性以及藥物在該受體溶液中的穩(wěn)定性和溶解度。受體溶液樣品中藥物的穩(wěn)定性應作為受體樣品分析方法驗證的一部分進行驗證。藥物在IVPT受體溶液中的溶解度應根據經驗確定三份，以說明藥物在受體溶液中溶解度超過IVPT關鍵研究中的最高樣品濃度，理想情況下為一個數量級。藥物在IVPT受體溶液中的溶解度應足以表征IVPT方法驗證期間IVPT敏感性評估中評估的藥物遞送條件增加導致的藥物滲透量增加。

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.

建議加入0.1%聚氧乙烯[20]油酸酯（也稱為Oleth-20、Volpo-20或Brij-20；CAS號9004-98-2），以提高疏水性藥物生理緩沖液基（水）受體溶液的溶解度。如果需要額外的溶解度，建議將聚氧乙烯[20]油酸酯的濃度小幅度增加（例如，從0.1%或0.2%，這對于大多數疏水性藥物來說通常是足夠的，到更高的濃度），但不應超過6%的聚氧乙烯[20]油酸酯。不建議采用其他可能改變皮膚滲透性的提高藥物在受體溶液中溶解度的策略（例如，在受體溶液內加入有機溶劑和醇），可能使IVPT方法無效。

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).

無論研究持續(xù)時間如何，建議在受體溶液中加入抗菌劑（例如，約0.1%*氮化鈉或約0.01%硫酸慶大霉素），以減輕擴散測試池中真皮和/或表皮的潛在細菌分解。其他抗菌劑也可以接受，如果使用，應在ANDA中包含相關信息，以解釋選擇的原因（以及使用的濃度）。

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.

應適當限定每個時間點受體溶液樣品采集的準確度和精密度。鑒定采樣程序的證據應表明，采樣技術可以在每次采樣中從一定體積、均勻混合的受體室中收集一致體積的樣品，并且采樣技術不會產生污染。如果可用，應包括描述設備制造商關于受體溶液取樣準確性和精密度的規(guī)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.

對于使用流通擴散池的IVPT研究，可能需要確定管道長度及其相關死體積，以準確計算樣品通過管道洗脫并收集之前的滯后時間。對于使用VDC的IVPT研究，在每個時間點去除整個受體溶液體積并全部置換受體溶液可以提供最佳溶解度漏槽條件。在IVPT研究中對受體溶液的小等份取樣可能會在IVPT研究某些區(qū)域引入明顯負通量的異常測量，并產生難以解釋的通量分布。

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.

研究期間應監(jiān)測和報告實驗室環(huán)境溫度和濕度。環(huán)境溫度宜控制在21°C±2°C，相對濕度宜控制在為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試點研究的通量分布和累積滲透分布應在一定取樣時間范圍內繪制，這與IVPT關鍵研究持續(xù)時間相對應。下面將更詳細地討論通量和累積總滲透的計算。IVPT先導研究的結果應驗證所選研究參數適合于在所選研究時間(采樣時間點范圍)內充分表征藥物的滲透特性（皮膚藥代動力學）。

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試點研究中，一個足夠完整的通量分布應足以確定多個后續(xù)時間點的最大（峰值）通量和此后通量的下降。IVPT試點研究的結果還應驗證采樣頻率提供了適當的分辨率，以充分表征滲透曲線（尤其是通量曲線）。

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先導研究（以及最終的IVPT關鍵研究）的通量和累積滲透結果應在每個時間點為每個擴散池進行計算、制表和報告，并提供匯總統(tǒng)計數據，以描述供體內平均值、標準偏差和重復之間的變異系數百分比（%CV），以及供體間平均值和%CV。計算中使用的所有數據值的完整結果應以清晰和有組織的方式報告，以便于重建通量和累積滲透結果。研究的設計應詳細、清晰，數據值應與特定捐獻者、重復、治療組、時間點等明確相關。

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).

受體溶液中滲透藥物的回收應為在每個擴散池中表征為藥物在IVPT持續(xù)時間內在受體溶液中的累積總滲透。這可以表示為藥物標稱量在應用劑量中的百分比（可以基于局部產品中藥物的標稱強度和皮膚上施用的局部產品的近似質量來估計）。

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，如果將含有5%藥物的10mg局部產品應用在膜上，則應用劑量中的藥物量估計為0.5mg（或500µg）。如果在IVPT的48小時持續(xù)時間內，累計10µg藥物擴散到受體溶液中，可以估計500µg劑量將消耗約10µg，相當于約2%的劑量消耗。由此可以估計平均劑量消耗百分比（不考慮皮膚含量），并應予以報告。

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方法的鑒別能力可以用兩個概念來描述：靈敏度和選擇性。IVPT方法開發(fā)期間必須進行IVPT靈敏性研究，以確定IVPT方法參數，如劑量、劑量持續(xù)時間、研究持續(xù)時間等。然而，這些研究結果的分析本質上是定性的，在IVPT方法驗證階段無需重復驗證。

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靈敏度研究通常在IVPT方法開發(fā)階段的末尾進行，這些研究的一個關鍵目的是將最終IVPT方法參數的目標劑量和劑量時間用于關鍵研究，IVPT靈敏性研究可以支持證明最終IVPT方法的有效性。因此本節(jié)指南在IVPT驗證（而非方法開發(fā)）的背景下描述了IVPT靈敏度研究，以避免將IVPT靈敏度（用于確定最終IVPT方法參數的適用性）和IVPT選擇性（一旦確定了最終IVPT方法參數，即進行該試驗，并基于IVPT試驗研究，該試驗研究是IVPT方法驗證的一部分）的討論分開。除了IVPT敏感性研究中使用的替代劑量或劑量持續(xù)時間外，重要的是IVPT方法參數在IVPT靈敏度、先導性和關鍵性研究（包括研究方案中規(guī)定的解剖區(qū)域（如軀干后部）、皮膚來源和皮膚制備）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靈敏度是IVPT方法檢測藥物的皮膚藥代動力學變化作為藥物傳遞差異的功能的能力。如果IVPT方法始終顯示出較高和較低的通量分布（即IVPT終點的高和低），分別對藥物遞送的增加和減少作出反應（或對預期分別增加和減少藥物遞送量的其他條件作出反應），則IVPT方法可能被認為是靈敏的。

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.

有幾種潛在的方法可以產生藥物遞送的差異，這些差異可以通過適當鑒別的IVPT方法進行區(qū)分。無論采用何種方法，IVPT滲透曲線的差異不一定與劑量、劑量持續(xù)時間或產品強度的差異成正比。例如，劑量的三倍差異（即使在推薦的目標劑量范圍之外）可以提供不同的通量曲線，但不會導致IVPT終點的三倍差別，因為皮膚屏障在體外和體內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.

換言之，如果關鍵IVPT研究的目標劑量為10 mg/cm2，則低3倍的劑量為~3 mg/cm2且高3倍的濃度為30 mg/cm2；因此，IVPT敏感性研究可以比較3、10和30 mg/cm2劑量的局部產品的通量分布。類似地，如果關鍵IVPT研究的目標劑量為15 mg/cm2，低3倍的劑量為5 mg/cm2而高3倍的濃度為45 mg/cm2；因此，IVPT敏感性研究可能會比較5、15和45 mg/cm2劑量的局部產品的通量分布。建議對多個皮膚捐獻者（例如4-6個皮膚捐贈者）進行IVPT靈敏度研究，每個治療組每個捐獻者至少四個重復皮膚切片。

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.

劑量調節(jié)：不同劑量的IVPT方法開發(fā)研究可能提供支持性證據，證明IVPT方法對藥物遞送的差異敏感。

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.

這種方法非常適合于含有揮發(fā)性成分的局部產品，這些揮發(fā)性成分在涂抹到皮膚后從配方中蒸發(fā)。調節(jié)這種局部產品的劑量有效地改變了應用劑量的厚度。較薄劑量的大部分揮發(fā)性成分往往會蒸發(fā)得更快（與較厚劑量相比），較薄劑量進入皮膚的藥量較少（和/或持續(xù)時間更短）。

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.

調節(jié)劑量可以是一種有效的技術，可以調節(jié)含有揮發(fā)性成分的制劑（如凝膠、乳液和許多乳膏）的藥物遞送差異。然而，調節(jié)劑量并不一定會對局部產品（如基于凡士林的軟膏）或其他類型的局部產品（不會在皮膚上蒸發(fā)）產生明顯的藥物遞送差異，或者可能不會經歷可改變藥物遞送速率和程度的劑量依賴性**差異。

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.

劑量持續(xù)時間的調節(jié)：對于許多外用產品，調節(jié)劑量持續(xù)時間而不是劑量可能更有效，以產生藥物遞送的差異和藥物的皮膚藥代動力學的相關變化。

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.

具有不同持續(xù)時間（例如，2小時、6小時和12小時）的受控劑量（例如，15 mg/cm2）的IVPT方法開發(fā)研究可能非常適合提供支持性證據，證明IVPT方法對許多局部產品的藥物遞送差異敏感。本例中描述的方案將支持IVPT研究設計，其中局部產品劑量為15 mg/cm2，持續(xù)6小時（IVPT研究的目標持續(xù)時間），然后擦掉。可使用一系列棉簽去除應用的劑量，其中一個或多個棉簽可能干燥，一個或更多棉簽可能濕潤（例如，用肥皂溶液或水）。初始（干）拭子通常去除大部分劑量，隨后的拭子用于去除剩余劑量（即，局部產品的殘留物，否則可能繼續(xù)將藥物輸送到皮膚中）和/或沖洗皮膚。

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.

為了證明IVPT研究的靈敏度，應將IVPT研究目標劑量持續(xù)時間（例如6小時）產生的滲透曲線與預期明顯降低藥物遞送的更短劑量持續(xù)時間進行比較，并且還與預期顯著增加藥物遞送的更長劑量持續(xù)時間（例如12小時）相比較。因此，IVPT靈敏度研究中比較的三個劑量持續(xù)時間旨在根據藥物遞送差異產生藥物的皮膚藥代動力學的明顯變化，從而支持IVPT方法的靈敏度證明。

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.

具體劑量持續(xù)時間可以基于IVPT方法開發(fā)期間進行的初始探索性IVPT研究來選擇，該研究表征了當劑量在皮膚上停留更長時間（例如24或48小時）時的滲透曲線。這種IVPT研究結果的一個重要特征是當通量以相對較快的速率增加時，滲透曲線初始階段的持續(xù)時間。

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，如果這樣的探索性研究表明，通量以陡峭的斜率增加，直到大約12小時，然后繼續(xù)以逐漸增加的速率遞送藥物，這可能表明，超過12小時（例如24小時）的劑量持續(xù)時間的滲透曲線可能與12小時劑量持續(xù)時間沒有明顯差異，特別是在相對較少數量的供體和復制體（例如每個劑量持續(xù)時間具有四個復制體的四個供體）中進行比較時。這也可能表明，在IVPT敏感性研究中，12小時的劑量持續(xù)時間可能是三個劑量持續(xù)時間中最長的一個很好的選擇。

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).

目標劑量持續(xù)時間的選擇應基于以下考慮因素，如樣品分析方法的靈敏度、產生滲透曲線的能力，該滲透曲線可明顯區(qū)別于較長（12小時）劑量持續(xù)時間產生的滲透曲線，和/或局部產品的標記使用（這可能表明局部產品應每4-6小時重新給藥一次）。

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靈敏度研究中，三個劑量持續(xù)時間中最短的時間選擇，應根據樣品分析方法的靈敏度，以及可明顯區(qū)別于目標維持時間（6小時）所產生的滲透曲線的能力來選擇。

· 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.

•調節(jié)產品規(guī)格：為驗證IVRT方法的靈敏度、專屬性和選擇性，通常采用改變配方制劑規(guī)格的方法。雖然采用改變配方制劑的規(guī)格的方法來證明IVPT方法的靈敏度和選擇性似乎很方便，但這種方式可能不會產生預期的結果。在某些情況下，這種策略可能會產生明顯不同的滲透曲線，然而，在很多情況下，由于在對比研究中采用的供體組和重復數較少（如，每種外用制劑規(guī)格采用4個供體組，每組供體4個重復），并不能產生明顯不同的滲透曲線。通常，不建議通過調整外用制劑規(guī)格的方法以證明IVPT的靈敏度，因為它可能不會持續(xù)產生預期的增加或減少的藥物遞送；然而，在某些情況下，相較于外用制劑的目標規(guī)格，較高和較低規(guī)格的配方制劑（相對于對照制劑（RS）的規(guī)格）可以適當地增加和降低藥物遞送和皮膚藥代動力學。

2.IVPT Selectivity/IVPT選擇性

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選擇性是IVPT方法區(qū)分“對照制劑（RS）“和“與RS在藥物遞送方面存在差異的外用制劑或配方”在藥物皮膚藥代動力學差異的能力。在IVPT初步研究中，通過將RS、自研外用制劑和“已知或設計不同于RS”的第三種外用制劑或配方進行平行評估，提供支持性證據，說明IVPT方法對藥物遞送的差異具有選擇性。如適用，應在研究報告中遞交，IVPT方法開發(fā)、驗證和初步研究中用到的所有外用制劑的相關批信息，包括但不限于：批配方、生產日期、批量、變更的生產工藝（如適用），以及效價和含量均勻度（如有）?？苫跐B透曲線和/或IVPT終點的定性或定量比較進行不等效性評估。

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.

與耐用性測試相關的一個主要假設是，當所有系統(tǒng)變量（如：溫度、攪拌速率）處于標準設置時，測試系統(tǒng)的性能一致。耐用性測試意義在于，它可以驗證當特定變量略有改變時，系統(tǒng)是否能提供一致的輸出結果，從而確定這些變量的操作范圍。然而，無論是體外還是體內，人體皮膚滲透性固有的變異性可能與與測試系統(tǒng)一致性相關的主要初始的假設不兼容。

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.

盡管如此，如果相關，應報告IVPT方法開發(fā)期間得到的關于支持IVPT方法或系統(tǒng)耐用性的研究結果。例如，IVPT方法對于接收室攪拌速率的顯著變化具有耐用性。與其類似地，藥物進入并通過人體皮膚的滲透曲線，可能對外用制劑規(guī)格的改變表現(xiàn)出耐用性。所以，對于驗證IVPT方法參數的耐用性并不總是可行的，在IVPT研究中應盡可能精準地控制IVPT研究程序。

V. SAMPLE ANALYTICAL METHOD VALIDATION/樣品分析方法驗證

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

雖然在IVPT方法開發(fā)的探索性研究中，可采用未經驗證的樣品分析方法，但作為IVPT方法驗證的一部分，所進行的IVPT研究都必須采用經過驗證的樣本分析方法。經驗證的IVPT方法應使用已驗證的樣品分析方法（如：HPLC/MS或UPLC/MS）。因此，本指南中關于IVPT方法驗證的章節(jié)中包含了對IVPT方法樣品分析方法的討論。

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，與IVPT方法相關的研究方案和報告與用于量化IVPT接收液樣品定量的分析方法不同。樣品分析方法的驗證本身并不能證明IVPT方法的有效性。因此，應分別提交IVPT方法驗證報告和樣品分析方法（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).

如果在方法開發(fā)過程中，使用的樣品分析方法與最終驗證的樣品分析方法不同，那么與其相關的IVPT方法研究結果則不能用于支持論證IVPT方法的有效性。應在IVPT方法驗證報告中，提供參考的樣品分析方法驗證的信息，并明確表明IVPT方法驗證報告中的所有相關結果均采用經驗證的樣品分析方法（不是與經驗證的樣品分析法有不同參數的樣品分析放法）獲得的。

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).

接收液樣品的分析方法（如：通常為HPLC/MS或UPLC/MS系統(tǒng)），應使用具有審計追蹤的色譜軟件（如，色譜數據系統(tǒng)），并應包括具有適當質量控制樣品的多點（6～8個）校準曲線，并應符合FDA生物分析方法驗證行業(yè)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).

接收液中樣品分析方法的驗證應包括稀釋完整性確認（如適用），以及樣品在最高相關溫度的接收液中放置最長時間的穩(wěn)定性評估；最高相關溫度可略高于32°C，因為接收液的溫度通常高于皮膚表面的溫度。對于在每個取樣時間點替換整個接收液的情況，最長相關時間為各相鄰取樣時間點中，間隔最長時間；但對于接收液部分取樣的情況，時間可能更長（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.

如果樣品以特定的分析方法進行處理（如：通過將樣品干燥后重新以較小的體積溶劑配制以提高有效分析靈敏度，或通過稀釋接收液樣品，使其達到已驗證分析方法的曲線范圍內），則應確認其處理方法（如：在樣品分析方法驗證期間確認稀釋完整性）。接收液樣品中藥物的穩(wěn)定性，應在與IVPT正式研究相關的條件下，在擴散池中皮膚下側接觸的接收液基質中進行。

VI. IVPT PIVOTAL STUDY/IVPT正式研究

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

IVPT正式研究方案通常應包括與BE研究相關的考慮因素

A. Handling and Retention of Samples/樣品的處理與保存

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.

參考21 CFR 320.38，320.63和FDA行業(yè)指南《生物利用度（BA）和生物等效性（BE）研究中試驗樣品的處理和保存》（2004年5月）和《21CFR 320.38(c) BE樣品留存的數量及生物利用度》（2020年8月），關于保留研究藥物樣品的考慮以及21 CFR 320.36關于保存BE檢測記錄的要求（如適用）。在采用自研外用制劑和RS進行IVPT對比研究前，應從收到的藥品中隨機選擇樣品進行留樣。

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.

應對可能影響研究結果的研究程序進行適當控制。此外，應報告可能影響研究結果解釋的實驗觀察結果，以及任何方案或標準操作程序（SOP）發(fā)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.

與皮膚相關的程序的控制，包括相同的皮膚處理方法（如：皮膚的剝離）和安裝在擴散測試池上的皮膚切片的厚度的研究中的一致性，及皮膚儲存條件，例如皮膚冷凍的儲存時間和取出的凍融循環(huán)次數。所有供給皮膚均應采用相同解剖位置，并報告所有供體的人口統(tǒng)計信息（年齡、種族、性別）。此外，在IVPT靈敏度、初步實驗和正式研究，應采用同一解剖部位的皮膚；否則，如果在不同的研究階段使用不同位置解剖部位，IVPT靈敏度和初步試驗的結果可能不足以支持用于正式研究的IVPT方法的區(qū)分力的論證，因為方法參數在各個研究中不一致。同樣，如果在皮膚部分下方使用非速率限制性支撐膜（如：同類IVRT方法驗證研究中使用的濾膜），則與IVPT靈敏度、初步試驗和正式研究中一致。

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.

與劑量或上樣相關的控制程序，包括：對上樣面積、上樣量、上樣技術、劑量維持時間的控制，以及在研究中每個擴散池采用的盲法和隨機化方法的一致性。不同的上樣技術可能改變上樣到皮膚上劑型的形態(tài)，擴散池孔口擴散面積的不一致可能會顯著影響上樣面積，從而導致通量計算的偏差。

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.

與取樣相關的程序控制包括取樣時間精度、取樣技術、取樣和替換接收液的時間間隔、取樣體積或流速，以及樣品的處理和存儲。

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.

與正式研究相關的控制程序，應包括來自每個皮膚供體的非劑量對照皮膚部分，包括在所有時間點對接收液進行取樣，以確保接收液中監(jiān)測的藥物濃度與IVPT正式研究中應用的劑量相關，而不是研究期間由于皮膚的藥物污染滲透進入。建議從每個擴散池中采集“零”濃度樣品，用于識別每個皮膚切片和/或每個擴散池的潛在污染。

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，研究人員應在一定的質量管理體系條件下，進行IVPT驗證和正式研究，該體系包括但不限于：

·Study personnel identification, training, qualification, and responsibilities/ 研究人員的識別、培訓、資格和職責

·Study management and study management personnel responsibilities/研究管理和研究管理人員職責

· Quality control (QC) and QC personnel responsibilities/質量控制（QC）和QC人員職責

· Quality assurance (QA) and QA personnel responsibilities/質量保證（QA）和QA人員職責

·Use of SOPs /標準操作規(guī)程（SOP）的制定

· Use of study protocols/研究方案的制定

· Use of study reports/研究報告的制定

· Maintenance and control of the study facility environment and systems/研究設施環(huán)境和系統(tǒng)的維護和控制。

·Qualification and calibration of instruments and computerized systems/儀器和計算機系統(tǒng)的鑒定和校準。

·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/良好的文件規(guī)范，包括但不限于：研究程序的及時記錄、實驗觀察及相關SOPs中規(guī)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/維護有助于重建研究事件和程序的適當記錄，包括研究樣本處理和存儲記錄（如：樣本跟蹤日志）、樣本分析過程的審計跟蹤、研究物料和試劑的控制以及電子數據控制。

·Archival of study records研究記錄存檔

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.

研究方案和最終報告中應提供盲法程序的詳細說明。試驗外用制劑和RS的包裝應保持相似的外，以確保研究者和任何實驗操作人員的充分致盲。

D.Randomization/隨機化

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.

應在IVPT研究方案中描述隨機化方法并提供隨機化計劃表，推薦以.xpt格式的SAS數據集形式（使用SAS XPORT程序創(chuàng)建）。建議由獨立的第三方在整個研究過程中生成并保存隨機化代碼，以減少偏差。如果研究中未涉及自研外用制劑和RS的包裝和標簽，申請人可以自行生成隨機代碼。隨機方案的密封副本應保存在研究現(xiàn)場，并應在現(xiàn)場檢查時提供給FDA研究人員，以便確認每個皮膚切片的具體信息。

E.Dosing/上樣

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，對于每個供體組，可以在擴散池（皮膚切片）上以交替給藥方式依次放置自研外用制劑和RS。對于每個供體組，可采用下述兩個方式中的一個進行隨機放樣（如下所示）：

a. ABABAB…

b. BABABA…

F. Study Design/  研究設計

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，應采用皮膚屏障完整性良好的離體人類皮膚和經驗證的擴散池系統(tǒng)，對自研外用制劑和RS的皮膚藥代動力學進行對比研究。在自研外用制劑和RS的對比研究中，每個試驗組應采用相同的皮膚供體、相同的皮膚切片重復數，并采用相同的IVPT方法參數。

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.

應盡可能精確地控制IVPT正式研究設計、方法和與擴散池設備取樣精度。例如，在連續(xù)的擴散池上，錯開上樣時間點，根據上樣時間的差異，從而同步取樣時間，以確保所有擴散池給藥和取樣之間的時間間隔一致。

G. Inclusion Criteria/納入標準

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，以下入選標準應適用：來自至少18歲的健康、正常、屏障功能皮膚，雄性和/或雌性供體。應在研究方案中規(guī)定符合納入標準的人口統(tǒng)計信息（如，年齡、種族、性別），并報告每個供體的個人信息。申請人可以增加其它納入標準。

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.

皮膚可以在外科手術的患者或尸體上獲取。所有使用的皮膚應有一致的來源。研究方案中規(guī)定的解剖區(qū)域（如軀干后部）應與研究中包括皮膚的所有供體一致。

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.

研究方案應根據屏障完整性測試結果規(guī)定皮膚部分的納入（接受）標準。

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.

研究方案應規(guī)定與皮膚儲存的溫度和儲存時間以及凍融循環(huán)次數納入相關的標準。

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.

研究方案應規(guī)定與IVPT研究中使用的皮膚采集/處理程序和皮膚厚度（例如，500μm±250μm厚度的皮膚）。

H. Exclusion Criteria/排除標準

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，采用以下排除標準?；加幸阎つw?。ú∈罚┑墓w皮膚；有紋身、妊娠紋或任何明顯異常跡象的皮膚應；皮膚表現(xiàn)出明顯的末梢毛發(fā)密度等。申請人可增加其他標準。

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.

雖然可以采用溫和地清洗或漂洗皮膚表面，但將皮膚浸泡在水溶液中幾分鐘以上可能會破壞皮膚屏障，應避免該方式。另外，不建議采用用刀片刮過的皮膚；磨料拋光；膠帶剝離；或使用酒精、溶劑或其他可能破壞皮膚屏障的強極性溶液進行清潔。

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.

來自供體的皮膚具有顯著的藥物背景水平或其他可能干擾受體溶液樣品中藥物定量的化合物應排除在研究之外。

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.

重復皮膚切片中屏障完整性測試失敗率高的供體皮膚可能被排除在研究之外，應采用替代供體皮膚。

I. IVPT Endpoints/  IVPT終點

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正式研究的終點，是基于表征藥物滲透和通過皮膚中，并進入接收液的速率和程度的參數。具體來說，藥物滲透速率以通量（J）進行表征，而藥物滲透程度采用整個研究期間滲透到接收液中的藥物的總累積量（AMT）進行表征。

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（藥物滲透速率）為縱坐標Y-軸，時間為橫坐標X-軸作圖；其中Y-軸用“J”標識，以質量/面積/時間（如，納克/平方厘米/小時）為單位。通量分布模型通常類似于血漿藥代動力學分布模型，盡管通量是一個速率單位，并不代表濃度。也應對藥物的滲透程度作圖，以藥物累積滲透量（單位為質量/面積，如納克/平方厘米）為縱坐標Y-軸，時間為橫坐標X-軸。

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:

流量的計算應基于：每個時間點的接收液中的樣品濃度（如：2.0 ng/mL）；精確、經驗性的測量擴散池的體積（如：6.0mL），每個擴散池體積或有不同；上樣區(qū)域面積（如：1cm2）；研究的持續(xù)時間。例如，如果這里例舉的樣品表示給藥后2小時內的情況，則J將基于上述值計算為：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).

應計算并報告各擴散池的所有相鄰采樣點之間的通量，并繪制整個研究期間所有擴散池的通量曲線。上面計算的速率可以在2小時的實際時間點進行繪制，或者在0到2小時（即1小時）之間的中點繪制。

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，應計算并報告每個擴散池的總累計滲透量（AMT）。AMT終點為藥物滲透的累計量（整個研究期間的滲透總量），而不是采用梯形法則計算的通量曲線下的面積。

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.

應將局部起效的自研制劑和RSs的最大通量（Jmax，藥物通量曲線的最高峰）和AMT進行對比。這類似于全身起效的自研制劑和RSs的Cmax和AUC的比較，因為這些終點可分別用于表征各劑型（局部起效或全身起效）藥物到達作用部位的速率和程度。

A confidence interval (CI) should be calculated for each IVPT endpoint: 應計算每個IVPT終點的置信區(qū)間（CI）：

a. the natural log-transformed maximum flux (Jmax) 自然對數換算后的最大通量（Jmax）

b. the natural log-transformed total cumulative amount (AMT) permeated自然對數換算后的總累積滲透量（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.

申請人有責任確定足以支持IVPT關鍵研究所需的供體數量，然而，建議每個試驗組（自研制劑或RS）每個供體至少4個重復劑量。

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研究結束時，如果自研外用制劑和RS試驗組中所有供體的皮膚切片重復數均一致，建議采用平衡設計法進行統(tǒng)計分析。如果皮膚切片或擴散測試池因實驗損失/問題而被排除在最終統(tǒng)計分析之外，且所得數據集不平衡，則建議對不平衡設計進行統(tǒng)計分析。

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，對正式研究的統(tǒng)計分析方法進行了描述。附錄I中提供了用于測定平衡數據集和非平衡數據集BE的SAS代碼示例。附錄II中提供了模擬數據集的具體示例。附錄III中提供了測定BE的R代碼示例。

VII. SUBMITTING INFORMATION ON IVPT STUDIES IN AN ANDA/ANDA中遞交的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.

對于在ANDA中提交的用于支持BE演示的外用制劑的IVPT研究，應提交詳細的研究方案、相關SOP和詳細的報告，用于IVPT方法驗證（包括IVPT初步研究）和IVPT關鍵研究。此外，還應提交一份詳細的IVPT方法開發(fā)報告。這些方案、SOPs和報告應在電子通用技術文件（eCTD）的模塊5.3.1.2中遞交，并對相關的試驗方法、研究控制、質量管理程序和數據分析進行描述。

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接收液中樣品濃度的定量分析方法（如，HPLC/MS或UPLC/MS方法）與IVPT方法相關的研究方案、SOPs和報告不同。樣品分析方法驗證應提交單獨的方案和標準操作規(guī)程。樣本分析方法開發(fā)和驗證報告、試點和關鍵IVPT研究樣本分析報告以及與人體皮膚IVPT研究的樣本分析相關的相關SOP和協(xié)議應在eCTD的模塊5.3.1.4中提交。

VIII.IVPT PIVOTAL STUDY STATISTICAL ANALYSIS/IVPT正式研究統(tǒng)計分析

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.

兩個治療組將對應于自研外用制劑（T）和RS（R）。進行統(tǒng)計分析應考慮以下樣本n 組供體，其中r^T復制第j個供者的皮膚切片(j = 1.? , n) 適用于T組和r^R復制第j個供體的皮膚切片(j = 1.? , n) 可用于R組。每個供體（j）的每個復制品（i）應隨機分配給每個產品。

Define the following quantities: 定義如下：

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個供體的第i個皮膚復制品的試驗外用產品給藥的自然對數轉換IVPT終點（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個供體第i次皮膚復制的RS給藥的自然對數轉換IVPT終點（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個供體用外用制劑（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個供體用RS( j=1, 2, ? , n)給藥的皮膚切片的重復數

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組皮膚切片總數

n = the number of donors

n = 供體組的數量

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

如果可用于最終統(tǒng)計分析的皮膚切片重復次數相同的n 組和T組和R組的供體中，所得數據集是平衡的。為方便標記，在平衡數據集中，一個供體對一個治療組的皮膚重復次數表示為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.

當有失敗的觀察記錄（如：在研究期間觀察到皮膚可見撕裂和泄漏）或方案的偏差（如：擴散測試池中的接收室在第一個取樣時間點被發(fā)現(xiàn)為空）時，擴散測試池可從數據集中的重復數中排除。在這種情況下，如果來自同一供體的足夠皮膚仍充足，并且沒有對該擴散池的樣品進行分析，則可以采用替代擴散池。若沒有可替代擴散池，則該結果數據集是不平衡的。

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.

下文描述了在平衡數據集和非平衡數據集情況下評估BE的統(tǒng)計分析方法。對于納入統(tǒng)計分析的供體，每個（T和R）治療組的供體應至少有3個重復皮膚切片。

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:

步驟1.確定S_wr,，每個自然對數變換IVPT終點Jmax和AMT的RS的估計供體內標準偏差：

(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，使用縮放平均BE（SABE）方法確定步驟2、3.1和4.1中單個IVPT終點的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，使用常規(guī)平均BE（ABE）方法，通過兩個單側測試（TOST）程序（Schuirmann，1987），在步驟2、3.2和4.2中確定單個IVPT終點的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∗).

確定T和R乘積平均差的點估計(I^{^}), 其標準誤差(se(I^{^}）），以及相應的自由度(df∗)

For a balanced data set, determine I^{^}, se(I^{^}) and df∗ by the following: 對于平衡數據集，確定I^{^}, se(I^{^})和df∗通過以下方式：

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.

對于不平衡數據集，近似I^{^}, se(I^{^})和df∗ 在SAS中使用PROC MIXED（或PROC GLM）。附錄I中提供了示例代碼。

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

In the SABE approach, the hypotheses to be tested are: 在SABE方法中，要測試的假設是：

Rejection of the null hypothesis supports the conclusion of equivalence of the two products. 否定零假設支持兩種產品等價的結論。

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常規(guī)平均BE（ABE）方法

In the ABE approach, the hypotheses to be tested are: 在ABE方法中，要測試的假設是：

Rejection of the null hypothesis supports the conclusion of equivalence of the two products. 否定零假設支持兩種產品等價的結論。

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方法確定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: 為使自制制劑與參比制劑具有生物等效性，每個IVPT試驗終點必須滿足以下兩個條件：

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法確定BE測定

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重點測試中，如果μ_T-μ_R的90%置信區(qū)間必須包含在每個測試IVPT終點原始量表中的限值[1/m，m]內，其中m=1.2500，則證明自研制劑與對照制劑具有生物等效