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

本附錄提供了半固體制劑的體內(nèi)角質(zhì)層取樣（或膠帶剝離（TS））研究的信息，作為滲透動力學(xué)方法，以顯示等效性，而不是治療等效性研究。

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

S.C.取樣研究是一種微創(chuàng)技術(shù)，涉及在施用含藥物制劑后使用膠帶依次去除最外層皮膚（即角質(zhì)層（S.C.））。S.C.中的藥物量取決于三個主要過程：藥物從制劑分配到SC中，藥物在S.C.中擴(kuò)散，藥物從S.C.分配到活組織中。TS的一個主要優(yōu)點(diǎn)是，該實(shí)驗(yàn)在體內(nèi)進(jìn)行，具有完全功能的皮膚微循環(huán)，因此藥物從皮膚的清除是不受阻礙的。

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

TS數(shù)據(jù)提供了作用于S.C.或在S.C.中的半固體藥物產(chǎn)品（例如抗真菌產(chǎn)品）的局部生物利用度的直接測量和信息。在目標(biāo)作用位點(diǎn)超出S.C.的情況下，TS數(shù)據(jù)可以提供一個合適的替代物，以表征藥物吸收到底層組織的速率和程度。

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

體內(nèi)TS研究僅適用于藥物擴(kuò)散進(jìn)入和通過SC的產(chǎn)品。因此，TS不應(yīng)用于測試應(yīng)用于嚴(yán)重受損皮膚（如開放性傷口、燒傷）或早產(chǎn)新生兒皮膚的藥物產(chǎn)品。此外，任何含有揮發(fā)性藥物或主要針對皮膚附件（如毛囊、皮脂腺）的產(chǎn)品也不適用。

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

TS研究不是一個自動化的過程，仔細(xì)考慮實(shí)驗(yàn)設(shè)計(jì)至關(guān)重要。關(guān)鍵研究的實(shí)驗(yàn)條件應(yīng)針對相關(guān)產(chǎn)品進(jìn)行單獨(dú)評估，并應(yīng)通過進(jìn)行TS試驗(yàn)研究來確定。應(yīng)提供TS方法開發(fā)和優(yōu)化的總結(jié)。

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

在試點(diǎn)研究期間，應(yīng)建立并驗(yàn)證以下實(shí)驗(yàn)條件：

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

TS研究應(yīng)在具有足夠皮膚屏障功能的健康、正常前臂（掌側(cè)）皮膚區(qū)域進(jìn)行。應(yīng)確定皮膚狀況的納入/排除標(biāo)準(zhǔn)。任何帶有紋身、有皮膚病異常跡象或末端毛發(fā)密度明顯的皮膚，應(yīng)排除在外。制定實(shí)驗(yàn)前的準(zhǔn)備和清潔程序，并進(jìn)一步確保這些過程不會損壞處理部位。

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

應(yīng)在實(shí)驗(yàn)前后確定皮膚完整性。這通常是通過測量經(jīng)表皮失水（TEWL）來進(jìn)行的，但如果合適的話，其他技術(shù)也可能適用。應(yīng)充分討論并證明驗(yàn)收標(biāo)準(zhǔn)的合理性。

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

由于受試者之間的差異，應(yīng)將待比較的產(chǎn)品應(yīng)用于同一受試者。此外，還應(yīng)包括與對照品不等同的陰性對照，以證明該方法的歧視性。建議讓負(fù)責(zé)配方應(yīng)用和膠帶剝離的研究人員盲試驗(yàn)，以最大限度地減少偏倚風(fēng)險(xiǎn)。

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

劑量應(yīng)根據(jù)SmPC確定。在試點(diǎn)研究期間，應(yīng)驗(yàn)證劑量和劑量應(yīng)用面積，以在SC中獲得可量化的活性物質(zhì)質(zhì)量。還應(yīng)建立劑量技術(shù)、盲法和隨機(jī)化程序。

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

應(yīng)采用單劑量方法，即在單次應(yīng)用測試和對照產(chǎn)品后進(jìn)行皮膚剝離。

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

有必要在兩個時間點(diǎn)（一次吸收，一次清除）對每個受試者的產(chǎn)品進(jìn)行比較。最佳吸收和清除時間取決于藥物和產(chǎn)品的特性，應(yīng)在試點(diǎn)研究期間確定。在相關(guān)的理想情況下，吸收時間應(yīng)足夠長，以使藥物達(dá)到擴(kuò)散穩(wěn)定狀態(tài)。這可以通過在多次攝取時間進(jìn)行測試來確定，從該時間起，從SC中回收的藥物質(zhì)量保持恒定。清除時間應(yīng)足夠長，以允許可測量的藥物從SC轉(zhuǎn)移到活的皮膚中（或更久），但不應(yīng)超過48小時，以避免任何皮膚脫屑影響。相對于攝取階段，從SC回收的藥物質(zhì)量提供至少25%減少的清除時間是優(yōu)選的。在所有情況下，應(yīng)仔細(xì)考慮并證明取樣時間的合理性。

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

藥物產(chǎn)品應(yīng)在規(guī)定的攝取時間后從皮膚表面移除。應(yīng)制定清潔程序，以確保在剝離前有效地從處理部位去除殘留制劑。

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

選擇的膠帶應(yīng)滿足以下要求：a）在應(yīng)用和摩擦皮膚表面時不會失去質(zhì)量；b） 儲存期間的最小化重量損失和增加；c） 藥物容易從粘附在膠帶上的SC中提取；d） 膠帶的粘合劑或其他成分不干擾藥物的分析定量；以及e）粘合力應(yīng)使得大部分SC用足夠低數(shù)量的膠帶（例如不超過30條膠帶）去除。

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

遵循的TS程序必須確保對每個皮膚部位的大部分SC（≥75%）進(jìn)行采樣。膠帶的最小和最大數(shù)量應(yīng)根據(jù)TEWL（或其他相關(guān)）標(biāo)準(zhǔn)確定，例如比基線值增加8倍，安全停止值。

●Most commonly, the drug is first extracted from the tapes then quantified in the extraction solvent(s). Alternative methods of extraction/quantification may be used if justified. Satisfactory efficiency should be demonstrated for the proposed extraction method.最常見的是，首先從膠帶中提取藥物，然后在提取溶劑中定量。如果合理，可使用其他提取/定量方法。應(yīng)證明所提出的提取方法具有令人滿意的效率。

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

在進(jìn)行TS研究時，應(yīng)制定詳細(xì)的標(biāo)準(zhǔn)操作程序，以確保精確控制劑量、清洗、剝離、提取、定量和其他研究變量或?qū)嶒?yàn)偏差的潛在來源。納入/排除標(biāo)準(zhǔn)應(yīng)預(yù)先定義，并在方案中明確說明。

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

TS研究建議采用以下研究設(shè)計(jì)。應(yīng)該針對每種具體情況制定合理的最終方案。

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

受試者–TS研究應(yīng)在健康志愿者中進(jìn)行。應(yīng)根據(jù)生物等效性研究的原則篩選合適的受試者。

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

治療區(qū)域–前臂掌側(cè)區(qū)域的健康皮膚，每個前臂至少有六個應(yīng)用部位。應(yīng)通過TEWL測量進(jìn)行皮膚完整性驗(yàn)證。應(yīng)為每個前臂分配相同數(shù)量的應(yīng)用部位；

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

受試者數(shù)量——受試者的數(shù)量選擇應(yīng)根據(jù)試點(diǎn)研究估計(jì)的變異性進(jìn)行證明，并證明其具有統(tǒng)計(jì)學(xué)意義。應(yīng)使用至少12名受試者來證明等效性；

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

重復(fù)次數(shù)–每個前臂每個產(chǎn)品至少有兩個應(yīng)用部位（測試、對照和陰性對照）。一側(cè)前臂用于吸收樣本，另一側(cè)用于清除；

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

產(chǎn)品應(yīng)以預(yù)定劑量（±5%）涂抹，并均勻分布在整個劃定的涂抹部位。應(yīng)從相鄰區(qū)域采集空白樣品，以驗(yàn)證是否存在可能干擾治療SC中藥物定量的藥物或其他化合物的背景水平；

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

用藥位點(diǎn)應(yīng)隨機(jī)，以避免選擇偏向性。應(yīng)用時間應(yīng)錯開，以留出S.C.取樣時間；

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

非封閉條件，除非產(chǎn)品信息中建議封閉，或以其他方式證明封閉是合理的，例如防止無意中移除制劑。

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

在吸收階段結(jié)束時，應(yīng)從所有治療部位（吸收和清除）移除制劑。應(yīng)盡量縮短總清洗時間，以避免因藥物進(jìn)一步擴(kuò)散而產(chǎn)生任何假陽性。剝離前應(yīng)檢查處理區(qū)域的皮膚完整性；

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

“吸收”部位應(yīng)在移除制劑后立即用膠帶剝離。“清理”部位應(yīng)在預(yù)定義的清理時間進(jìn)行膠帶剝離；

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

所需膠帶的確切數(shù)量應(yīng)根據(jù)剝離區(qū)域的TEWL測量值和試點(diǎn)研究確定的停止標(biāo)準(zhǔn)確定；

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

每個膠帶上去除的SC的質(zhì)量應(yīng)通過在剝離前后稱重膠帶的重量來確定。如果有適當(dāng)描述和合理理由，可以使用替代性的SC量化方法；

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

應(yīng)分析從每個處理部位收集的所有剝離膠帶。為評估它們對回收藥物總量的貢獻(xiàn)，前兩個膠帶應(yīng)與剩余膠帶分開分析。為了增強(qiáng)分析檢測能力，可以將后續(xù)的膠帶進(jìn)行分組（例如，每組含有所需的最低含量的SC）提取。SC中藥物的總質(zhì)量應(yīng)計(jì)算為從所有膠帶樣本中提取的總和。應(yīng)確定每個治療部位的質(zhì)量平衡，包括通過清潔從表面去除的藥物含量。一般情況下，可接受的總回收率是90-110%；如果出現(xiàn)較大的偏差，應(yīng)充分解釋。

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

在膠帶剝離前，非常重要的是在應(yīng)用期結(jié)束時進(jìn)行皮膚表面的清潔，必須能夠有效地去除多余的制劑（即未吸收的藥物），而不會無意中“驅(qū)使”藥物進(jìn)入屏障。清潔程序通常包括用干/濕紙巾、棉簽和/或新鮮酒精濕巾快速輕輕擦拭皮膚。應(yīng)該知道清潔組件不會影響藥物擴(kuò)散進(jìn)入和通過SC。在正式研究之前，應(yīng)仔細(xì)評估和驗(yàn)證有效的皮膚清潔程序，例如，通過證明從皮膚表面去除的制劑有令人滿意的回收率（＞90%）和通過在施用后立即剝離清潔的皮膚而回收的可忽略的藥物含量（＜10%）。如果能證明合理性，可以使用其他驗(yàn)證方法。

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

應(yīng)驗(yàn)證用于膠帶中藥物定量的生物分析方法。在正式研究之前，應(yīng)確定提取程序的效率（包括成組提取膠帶），并證明其一致性。

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

對于具有不同質(zhì)量屬性的批次（陰性對照），應(yīng)證明TS方法的區(qū)分力，例如具有±50%擬議產(chǎn)品強(qiáng)度的藥物配方，其與試驗(yàn)和對照產(chǎn)品在統(tǒng)計(jì)學(xué)上不同且不等效。根據(jù)《生物分析方法驗(yàn)證指南》，測定膠帶剝離SC中活性物質(zhì)含量的分析方法應(yīng)進(jìn)行驗(yàn)證

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

應(yīng)報(bào)告所有受試者的數(shù)據(jù)，并討論結(jié)果的有效性和變異性。統(tǒng)計(jì)分析中應(yīng)包括所有受試者和應(yīng)用部位。必須在協(xié)議中預(yù)先規(guī)定允許的排除理由。不能僅基于統(tǒng)計(jì)分析或動力學(xué)原因排除數(shù)據(jù)。

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

對于每種產(chǎn)品，都應(yīng)報(bào)告去除的SC厚度、使用的膠帶數(shù)量以及在吸收和清除時間測量的最終TEWL值。應(yīng)在等效性方面對測試產(chǎn)品和對照產(chǎn)品之間這些參數(shù)的任何差異都進(jìn)行討論。

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

應(yīng)為每個應(yīng)用部位繪制SC中的藥物含量分布圖，例如每個SC膠帶條（單個或成組）的藥物含量與SC深度的關(guān)系。

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

在分析之前，應(yīng)對每個受試者中每個產(chǎn)品的重復(fù)測量值進(jìn)行平均（總體幾何平均值）。

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

對于產(chǎn)品的比較，應(yīng)根據(jù)《生物等效性研究指南》（CPMP/EWP/QWP/1401/98 Rev.1/Corr）對等效參數(shù)：從吸收（Muptake）和清除（Mclearance）部位回收的藥物質(zhì)量進(jìn)行統(tǒng)計(jì)比較。

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

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

除非有正當(dāng)理由，試驗(yàn)和對照產(chǎn)品平均值比率的90%置信區(qū)間應(yīng)包含在80.00-125.00%的驗(yàn)收區(qū)間內(nèi)。

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

如果在低強(qiáng)度和有限擴(kuò)散的藥物產(chǎn)品中觀察到高變異性，并且在臨床證明的情況下，可以接受更寬的90%置信區(qū)間限，最大值為69.84–143.19。應(yīng)遵循《生物等效性調(diào)查指南》第4.1.10節(jié)“高變異藥物或藥物產(chǎn)品”中的程序。

In addition, for the test to be valid:

此外，為了使測試有效

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

等效參數(shù)（Muptake）和（Mclearance）的驗(yàn)收標(biāo)準(zhǔn)

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

試驗(yàn)和陰性對照產(chǎn)品平均值比率的90%置信區(qū)間應(yīng)完全超出80.00-125.00%的區(qū)間。●The 90% confidence interval for the ratio of means of the comparator and negative control products should be entirely outside the interval of 80.00- 125.00%.對照產(chǎn)品和陰性對照品平均值之比的90%置信區(qū)間應(yīng)完全超出80.00-125.00%的區(qū)間。●The 90% confidence interval for the ratio of means of the test product clearance (Mclearance) and (Muptake) comparator products should be entirely below 1.0.試驗(yàn)產(chǎn)品清除率（Mclearance）與對照產(chǎn)品（Muptake）平均值之比的90%置信區(qū)間應(yīng)完全低于1.0。

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

對照產(chǎn)品清除率（Mclearance）與對照產(chǎn)品（Muptake）平均值之比的90%置信區(qū)間應(yīng)完全低于1.0。

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

應(yīng)提供研究的總體結(jié)論。這應(yīng)該得到對TS數(shù)據(jù)的合理科學(xué)討論和解釋的支持。