The modern way we comprehend complex and multifaceted patient-drug mechanisms indicates the need in thorough and multifactorial analysis of pharmaceutical and biological factors that produce an impact on bioavailability, pharmacokinetics and pharmacodynamics of drugs. Such factors as drug composition, production technology and the state of biological barriers of patients, taken both separately and collectively, can significantly alter or distort the desired pharmacological outcome. This increases the volume, labor intensity, economic and time expenditures on research of novel drugs and generics. As a response to these challenges, the pharmacokinetic approach to the assessment of bioavailability and bioequivalence gains a greater popularity [1], whereas in vitro research is accompanied by the expanded scope and improved accuracy. It results in the essential cut of expenses and duration of preliminary studies of bioavailability and bioequivalence; in some cases (during registration of generics), it can be the basic or the only method to establish bioequivalence [2, 3]. In vitro permeability test (IVPT) belongs to an essential group of in vitro methods.

This mini-review aims at describing the current state of using IVPTs to evaluate transdermal permeability, explore the limitations of use, and determine the key ways of optimization and improvement of these systems.

In vitro permeability test (IVPT) is a sensitive and reliable model system that was integral to the detection of fundamental parameters associated with absorption of both drugs and industrial chemicals via various biological barriers [4].

IVPT with animal tissues or cellular cultures has been used as an efficient and effective tool to evaluate release and permeation of drugs during the last several decades. It allowed to develop formulations and perform quality control of peroral, transmucosal and transdermal delivery of drugs [5].

Various IVPTs are used to evaluate transdermal permeability. Human or animal (especially porcine) skin is frequently used as model membranes. Unlike the majority of clinical methods testing bioequivalence, IVPT performs simultaneous assessment of several replicated skin samples from the same donor (subject). The most commonly used IVPT methods include vertical diffusion cell, flow-through diffusion cell, Ussing chamber and Transwell systems [5].

High sensitivity of IVPT is its unquestionable advantage, which outperforms that of in vivo research. In particular, it has been shown that IVPT is more sensitive and less variable than the vasoconstrictive test [6].

It ensures a more differentiated assessment of similarities and differences between topical agents. The research that demonstrates the advantage of IVPT over bioequivalence assessment as compared to the clinical method has been described [7]

CURRENT STATE OF IVPT AND IVPT-BASED RESEARCH

Currently, IVPT is widely used as the key or one of the basic bioequivalence tests. It is included into the programs that develop local and systemic pharmaceutical formulations used for external application [8].

At present, research using various modifications of IVPT for a wide spectrum of topical drugs is presented in scientific literature. IVPT of human skin in Franz diffusion cells confirmed that effective permeation of an active ingredient in transdermal hormonal replacement therapy could manage menopausal symptoms [9–11]. In another research, IVPT was used to assess the extent of dermal absorption of insulin molecules within the patented phospholipid foundation intended for the delivery of highly molecular API [12]. The research of lorazepam transdermal permeation with human skin and Franz diffusion cells was also described [13]. With the help of IVPT, the mechanism of drug distribution into skin and its absorption type could be evaluated in a differentiated way, making it possible to select the optimal foundation for topical tramadol [14]. Detailed research is presented that uses the IVPT method to explore the permeation of UV-absorber in sunscreen [15].

Thus, IVPT is currently successfully used both for systemically and locally acting preparations. As the method is sensitive and reproducible, it was included into the regulations of FDA and EMA as a test to assess bioequivalence of local drugs [2, 5, 16].

LIMITATIONS AND PROBLEMS CONNECTED WITH THE USE OF IVPT

Meanwhile, some researchers mention that the test has limitations and that in some cases the outcomes should be interpreted with caution.

Therefore, any skin sample taken from the same donor can have an unusual or abnormal absorption profile. Such results are commonly called outlier values as their transdermal absorption kinetics significantly differ from those of other samples [4].

Another research was devoted to the selection and preliminary testing of human skin samples prior to IVPT. In FDA and OECD Guidelines that regulate IVPT it is stated that every skin sample must be checked before use for corneal layer integrity. Three methods are currently considered acceptable such as:
1) transepidermal water loss (TEWL),
2) electric resistance, an
3) tritium water absorption.

Gender, race and skin age are also important (skin samples taken from men, black population, and elderly people) [3].

It confirms that use of human skin as a membrane is hampered due to the limited access, high variability of results and restricted use for drugs with low skin permeability [17].

Correlation between in vitro and in vivo IVPT models was clearly established for systemic delivery. However, it is disputed whether the test is a reliable tool for detection of insignificant changes, which is critically important for assessment of topical drugs delivered to various parts of skin [18]. The results of combined treatment of biometric data in a published study have shown that in vitro equivalence both for the original drug and the generic product cannot be obtained in all skin areas. Moreover, authors doubt the use of a negative control offered in the EMA Guideline draft [2].

According to some authors, the effect of external factors during IVPT is studied insignificantly. However, they can produce a profound effect on the outcomes. Temperature is one of the most commonly mentioned factors. In particular, IVPT modification that takes place in higher temperatures and uses the clinical study design as the foundation is to a greater extent consistent with the results of clinical studies of buprenorphine transdermal system [19]. The research also describes the use of combined IVPT with diclofenac and skin of one miniature pig and three human donors at 32 ± 1 °C and 42 ± 1 °C to simulate normal and higher skin temperature respectively. According to the results, heat can change the extent of topical drug permeability. However, the rise of its concentration will not pose a systemic risk just as expected [20].

In another research of transdermal systems (TDSs) with nicotine and fentanyl, the effect of continuous and transient heat exposure was explored during IVPTs. In this case, heat was applied for the maximum recommended TDS wear duration or for short duration. The research demonstrated the significance of heat effects. However, subsequent clinical studies are required to interpret results correctly [21].

METHODS INCREASING ACCURACY AND RELIABILITY OF IVPT RESULTS

Apart from temperature, some other conditions influenced by experimental environment and utilized materials can result in incorrect outcomes, which are occasionally abnormal. Researchers have lately concentrated on the implementation of various methods to increase reliability and relevance of IVPT methodology and interpretation of its results.

IVPT combined with another surrogate in vitro method or in vivo pharmacodynamic assessment belong to one of the most commonly used approaches described in scientific literature. In vitro/in vivo correlation (IVIVC) is essential when biopharmaceutical products are developed, as model development and validation can be followed by in vitro method, which ensures effective assessment and prediction of drug profile in vivo. The published study has shown that heat had a more pronounced effect on the increased bioavailability of fentanyl in vivo than in vitro.

Thus, researchers have come to a conclusion that IVPT can successfully predict effectiveness of fentanyl TDS in vivo at normal temperature and when it is compared how different TDS formulations release the drug when subjected to heat. They also concluded that additional testing methods could improve the ability to predict in vivo heat effects with less prognosis bias [22]. In another research, it was necessary to add to and confirm the IVPT-based results with reverse iontophoresis. The vasoconstriction method was used to check the obtained data. A combination of methods allowed to show high reproducibility of results [23]. In another research, an attempt was made to relate in vivo plasma concentration-time profiles after topical administration of drugs to IVPT results using mechanistic diffusion and compartmental models. Ultimately, it was assumed that mechanistic and physiologically IVPT-based pharmacokinetic models can predict local in vivo behavior [24].

Supplementation of IVPT with the model of physiological pharmacokinetics in subcutaneous administration ensures a promising mechanistic approach, which allows for a more exact interpretation of IVPT results and in vitro to in vivo extrapolation [25].

Improved handling and interpretation of data obtained during the testing is another common approach that increases the accuracy and reliability of IVPT results. For instance, the novel statistical approach to assessment of acyclovir cream bioequivalence testing results has shown that IVPT is a sensitive and discriminatory test. It can detect the differences in the rate and extent of acyclovir skin bioavailability when creams with different formulations are used [26]. Published research offers the scaling model for IVPT data statistical processing, which is capable of finding significant differences between a prospective generic (test) product and a reference standard. It is also more sensitive than scaling-deprived tests. Moreover, only smaller samples can have an adequate statistical power [27].

Some authors developed mathematical models based on IVTP results that predict the rate and extent of drug absorption during several days irrespective of a daily dosage. Therefore, the authors suggest how to solve the pressing problem of IVPT and interpret the results obtained when the drug is tested multiple times [28]. The statistical hypothesis testing, in particular the mixed scaled average bioequivalence (MSABE), is a method that increases the significance of IVPT results. Inclusion of MSABE into the adaptive design, modifications of adaptive methods used in IVPT bioequivalence studies, such as Bonferroni adjustment and modal capacity function, show the advantages of these adaptive methods [29]. The published research also displays effectiveness of correlations between the IVPT and in vivo study results using deconvolution and convolution in data processing [30].

Some researchers endeavor to improve the IVPT and save resources by prematurely terminating unpromising experiments using an early decision-making algorithm [31]. In addition, it is proposed to simulate IVPT results with the GastroPlus software version 9.7 for predicting pharmacokinetics across the transdermal route [32].

Another example of IVPT simulation and predicting results is the study with bottom-up multiphysical modeling, which made it possible to evaluate the mechanism of percutaneous absorption kinetics of heterogeneous topical preparations [33]. Moreover, the researchers stress that it is essential to select the suitable bioanalytical methods and procedures [34] and suggest the options of sample optimization and automatization during IVPT [35].

In some cases, IVPT is upgraded and adapted when biological membranes are replaced with artificial ones. The artificial membranes have uniform, predictable properties and minimal variability, which is their absolute advantage. The reconstructed human epidermis models were validated as skin surrogates for safety testing and investigated for transdermal absorption testing using discriminant and modified in vitro permeability test (IVPT). The results showed a higher permeability of the reconstructed epidermis, which potentially allows for more differentiated comparisons of product penetration profiles [17]. In another published study, IVPT modes were tested using various synthetic membranes (hydrophilic polyamide nylon, Tuffrin polysulfone, and STRAT-M (SM) membrane) and various types of receiving medium (phosphate buffer containing different concentrations of sodium lauryl sulfate). In vitro-in vivo correlations were established using the GastroPlus software. The best IVPT method was chosen based on establishing a two-point correlation with in vivo data with minimal prediction errors (%PE) AUC0-24 and Cmax. The results showed that the IVPT method, which used a diffusion system with a Franz diffusion cell, a SM membrane, and a phosphate buffer without a surfactant, allowed us to construct the best IVIVR model with a correlation coefficient (R2) of 0.9966 and exponential function Y = (1.35)⁵ × X3.6 [36]. At the same time, it is necessary to take into account the fact that the use of synthetic membranes does not allow us to assess individual differences in the permeability of skin taken from different sites and from different donors. This information, if properly processed, is of interest and can be systematized to better understand real cases of abnormal permeability [4]. A promising area of IVPT adaptation is the development of a two-layer (stratum corneum with viable epidermis) diffusion model with appropriate mathematical and statistical tools for processing the results [37].

CONCLUSION

Thus, it can be asserted that IVPT is extensively and effectively applied in international practice today to assess the absorption of externally applied drugs. Like any model or method, IVPT carries its own flaws and limitations. However, the growing awareness of these limitations allows to take them into account when planning an experiment and interpreting the results. In addition, a large number of studies have recently been devoted to various ways of increasing effectiveness of the method. In general, main directions for modification and modernization of the IVPT method include as follows:
- optimization of methods for processing and interpretation of results, collection of additional information, use of new methods of data processing;
- optimization achieved due to the use of accumulated correlation data of IVIVT methods to create predictive mathematical models and data processing algorithms;
- technical optimization, i. e. physical modification of existing test systems, making them more complex, bringing the model closer to real skin conditions.

It can be assumed that additional ways to develop IVPT systems will include more complicated structures of test systems, introduction of additional testing modes, and functional combination of IVPT with a two-component model.