Application of In Vitro Permeation Test (IVPT) for the Development of Transdermal and Topical Drugs

Since 3000 BC, ancient Egyptians and Babylonians have used extracts derived from animals, minerals, or plants on the skin for therapeutic purposes.¹ In 1979, the US Food and Drug Administration (FDA) approved the first systemic transdermal drug delivery system (TDDS) , the scopolamine patch. This was a significant milestone for TDDS and initiated a new phase in its development.²

TDDS offers notable advantages compared to conventional methods of drug administration, as they can circumvent first-pass metabolism in the liver, enhance patient compliance through non-invasive delivery, and maintain stable drug concentrations in the bloodstream for long-term therapeutic effects.³ Currently, the methods used to evaluate transdermal drug delivery are broadly divided into in vivo and in vitro. Several experimental studies have demonstrated that in vitro studies under simulated in vivo conditions, specifically in vitro permeation test (IVPT), can provide data equally important to those obtained from conventional in vivo studies.⁴ In this article, we will discuss the applications, relevant guidance requirements, skin models, and commonly used test designs of IVPT, and demonstrate the use of IVPT for formulation screening through a validation study.

Applications of in vitro permeation test (IVPT)

Unlike traditional in vivo tests, IVPT measures the extent and the rate of a compound across the dermal barrier, and facilitates precise measurement of the distribution in various layers of the skin (Figure 1). It helps reveal the differences in skin permeation during the early screening phase as well as the late development and submission phases. IVPT reduces the need for live animals and facilitates repeated testing on the same donor, making it particularly suitable for screening tests to compare different formulations of transdermal administration drugs. IVPT studies also play an important role in assessing bioequivalence (BE) for several topical products. Several recent FDA product-specific guidance listed IVPT as a comparable method to replace clinical trials, with additional support in product quality (Q1) and quantity (Q2). In combination with the in vitro release test (IVRT) results, IVPT can provide crucial data for microstructural equivalence (Q3) in evaluating the BE of topical formulations.

Figure 1. Schematic diagram of the structure of human skin

Typical applications of IVPT experiments include:

• Early screening of compounds,
• Formulation development and optimization of topical dermatological products,
• Assessment of drug BE and bioavailability,
• Safety assessments of pharmaceutical or cosmetic products.

Relevant guidance requirements

The pertinent points of current global guidelines are listed below (Table 1).^5–8

• Skin type: In the FDA and European Medicines Agency (EMA) guidelines, only human-isolated skin is specified as a test model for IVPT, whereas the Japanese Pharmaceuticals and Medical Devices Agency (PMDA) allows the use of animal-isolated skin for the test.

• Duration of the test: In both the EMA and PMDA guidelines, the test duration is recommended to be no longer than 24 hours, whereas the FDA recommends that 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).

• Sampling frequency: The FDA recommends a minimum of eight non-zero sampling time points; the EMA recommends a minimum of six sampling time points; and the PMDA recommends more than five sampling time points, including the endpoint.

• The guidelines share consensus on the parameters, such as test device selection and skin temperature control.

When designing each individual studies to be compliant to various guidelines, the actual objectives and conditions of the product should be considered comprehensively.

Table 1. Comparison between global guidelines

FDA, Food and Drug Administration; EMA, European Medicines Agency; PMDA, Pharmaceuticals and Medical Devices Agency

How to choose and build suitable skin models

The choice of species is the first consideration when preparing skin materials. Although human-isolated skin is the gold standard for IVPT experiments, animal skin is often used as a substitute due to ethical and cultural concerns and availability challenges.

Pig skin bears the closest resemblance to human skin, including similar stratum corneum, epidermal thickness, and hair follicle density.^8,9 Skin characteristics of different animal species are compared in Table 2.

Table 2. Skin characteristics of different animal species^9,10

Rodent skin is one of the most readily available types of skin material. However, owing to structural differences such as fat composition and hair follicle density, the drug permeation parameters of rodent-isolated skin tend to be higher than those of human-isolated skin, leading to overestimations of drug absorption.¹¹ Therefore, the preferred skin for IVPT experiments is the isolated skin of Bama pigs, which is similar to human skin. Table 3 shows the skin models commonly used in IVPT experiments.

Table 3. Common IVPT skin models

Commonly used apparatus and parameter configuration

Figure 2 shows devices commonly used for IVPT, which include static cells (SCs) and continuous flow cells (CFCs).¹² SCs have a fixed-volume receiving chamber, and the receiving fluid is continuously agitated during the test. Currently, SCs are the most commonly used type of diffusion cell. In CFCs, the receiving medium flows continuously to simulate blood flow in the body. However, due to the introduction of new parameters, more preliminary tests are needed to determine the relevant test parameters.

Figure 2. Schematic diagram of commonly used diffusion cells (left, SC; right, CFC)

When conducting experiments with diffusion cells, it is necessary to configure the parameters appropriately. Figure 3 describes a typical set of parameters for IVPT in a static cell.¹³ This design is generally consistent with the requirements outlined in the “Draft Guidance on Acyclovir” provided by the FDA, e.g., more than four donors per group, more than four replicates per donor, at least eight sampling time points during a test, and single-dose administration with a dosage range of 5–15 mg/cm². When selecting parameters for BE testing, it is imperative to perform method development and validation before critical tests are conducted.

Figure 3. Configuration of parameters for IVPT ¹² 

WuXi AppTec DMPK’s IVPT validation study

Compound A is a widely used nonsteroidal anti-inflammatory drug with rapid absorption and clearance and a short half-life when administered systemically. Using compound A as a model drug, the in vitro permeation of six different formulations was evaluated using the dorsal skin of Bama pigs mounted on vertical Franz diffusion cells (PermeGear, Inc., Hellertown, PA, USA). In the comparison, Group 1 was a clarified solution, Group 2 was a commercialized gel, and Groups 3–6 were independently screened Poloxamer formulations.

The dorsal skin of Bama pigs at 750 um was used as the test system. The prepared skin was placed between the donor and receptor chambers with the stratum corneum facing up, and only skin samples with qualified transepidermal water loss (TEWL) values were used in the actual comparison. A single dose of a commercial formulation of compound A (1.16%, w/w) was applied to the skin in the donor chamber. Normal skin temperature conditions were simulated during the test, with the skin temperature maintained at 32 ± 1 °C. Receiver fluid (200 μL) was collected at predetermined time points. After the test, the stratum corneum was cleaned and removed. Epidermis and dermis were subsequently separated and collected to determine the distribution of the compound in the skin. The results are shown in Figures 4 and 5.

Figure 4. Cumulative permeation (left) and permeation rate (right) of each group of formulations over time

Figure 5. Retention of compound A in the epidermis (left) and dermis (right) 24 h after drug administration

Figure 4 shows that the vertical Franz diffusion cell clearly distinguished the formulations in terms of their skin permeation characteristics. For example, compared to the clarified solution formulations in Group 1, the commercialized gel formulations in Group 2 had a significantly lower permeation rate and cumulative drug permeation rate. For the four Poloxamer formulations, the solvent ratio affected the transdermal permeability of the drug. These results will act as a solid foundation for subsequent formulation screening and optimization processes. Figure 5 shows the retention of compound A in the epidermis and dermis in each group of formulations 24 h after administration. Compound A's epidermal and intradermal retention was significantly lower in the Poloxamer-containing formulations (Groups 3 – 6).

Concluding remarks

There has been great interest in TDDS among researchers and pharmaceutical companies. The development of high-quality transdermal drug formulations remains challenging. Percutaneous absorption of drugs is a multifactorial and multi-step process. Various factors, including the type of animal, skin type, skin pretreatment steps, and physicochemical properties of the tested compound and delivery system, all have influence on IVPT outcome. Not only does IVPT serve as a highly valuable research tool for Q3 characterization during generic evaluations of topical formulations, but it also provides useful information for compound screening and the optimization of solvent formulations. As the advancement of permeation models progresses in the future, this valuable tool will facilitate the rapid development and optimization of complex formulations for topical and transdermal administration.

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References：

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5. Food and Drug Administration. Draft Guidance on Acyclovir, 2016.

6. European Medicines Agency. CHMP: Draft guideline on quality and equivalence of topical products, 2018.

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13. SANTOS L L, SWOFFORD N J, SANTIAGO B G. In vitro permeation test (IVPT) for pharmacokinetic assessment of topical dermatological formulations. Curr Prot Pharmacol, 2020, 91(1): e79Current Protocols in Pharmacology 91 (2020): e79.

Authors: Xuan Dong, Chunyun Xu, Cheng Tang

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