Commercially Available Cell-Free Permeability Tests for Industrial Drug Development: Increased Sustainability through Reduction of In Vivo Studies

Abstract

Replacing in vivo with in vitro studies can increase sustainability in the development of medicines. This principle has already been applied in the biowaiver approach based on the biopharmaceutical classification system, BCS. A biowaiver is a regulatory process in which a drug is approved based on evidence of in vitro equivalence, i.e., a dissolution test, rather than on in vivo bioequivalence. Currently biowaivers can only be granted for highly water-soluble drugs, i.e., BCS class I/III drugs. When evaluating poorly soluble drugs, i.e., BCS class II/IV drugs, in vitro dissolution testing has proved to be inadequate for predicting in vivo drug performance due to the lack of permeability interpretation. The aim of this review was to provide solid proofs that at least two commercially available cell-free in vitro assays, namely, the parallel artificial membrane permeability assay, PAMPA, and the PermeaPad^® assay, PermeaPad, in different formats and set-ups, have the potential to reduce and replace in vivo testing to some extent, thus increasing sustainability in drug development. Based on the literature review presented here, we suggest that these assays should be implemented as alternatives to (1) more energy-intense in vitro methods, e.g., refining/replacing cell-based permeability assays, and (2) in vivo studies, e.g., reducing the number of pharmacokinetic studies conducted on animals and humans. For this to happen, a new and modern legislative framework for drug approval is required.

Keywords: PAMPA; PermeaPad®; biowaiver; dissolution-permeation; in vitro AUC; permeability; unstirred water layer.

Figure 1

Schematic representation of the interplay of solubility and permeability in oral drug absorption (a) and the Biopharmaceutics Classification System (BCS) introduced by Amidon et al. in 1995 [5] (b). Once a dosage form is administered orally, it undergoes dissolution and drug molecules are then absorbed through the gastrointestinal (GI) tract through the process of permeation. The fraction dose absorbed, %A, depends mainly on the drug’s aqueous solubility and its permeability through biomimetic barriers.

Figure 2

The numbers of animals used for research and testing in the EU in the period 2015–2018 [11].

Figure 3

Overview of drug products from the 10 largest pharmaceutical companies (Table 1). (A) Drug product portfolios, in which products are divided into non-oral and oral products. Oral products are divided into high-solubility (BCS class I/III) and low-solubility (BCS II/IV) drugs. (B) BCS classes of oral drugs from the 10 pharmaceutical companies by their year of approval. BCS class I/III—green; BCS class II/IV—red; BCS class not available (N/A)—blue.

Figure 4

Pathways for drug absorption across an epithelial monolayer (a), the expected correlation between in vitro apparent permeability (P_app) and in vivo human permeability (P_h) (b), and the expected correlation between P_app and the fraction absorbed (A%) (c). Drug absorption can occur via the transcellular route (A), the paracellular route (B), carrier-mediated transport (C), or transcytosis (D). Efflux transporters actively transport the drug in the opposite direction (basolateral–apical, (E)).

Figure 5

Schematic representation of set-ups for permeability assays. Permeability assays are based on two compartments, the donor and the acceptor, separated by a (biomimetic) barrier, i.e., the yellow segments in the figure. In general, permeability tests can be performed utilizing macroscopic diffusion cells, in which the diffusion of the drug takes place vertically, i.e., from top to bottom (or the reverse) (a), or horizontally, i.e., from side to side (c). In order to increase the potential of this method for high-throughput screening (HTS), several permeability assays are available in the multiwell-plate format (b), in which a (biomimetic) barrier is attached to the top plate horizontally (b-I) or at an angle (b-II) in order to reduce the risk of air bubble formation.

Figure 6

Representation of a conventional physical model used to interpret the passive transport of a compound through a barrier of thickness h_b. The blue lines represent the changes in the concentration gradient within the donor (c_d) and acceptor (c_a). In this model, there are no boundary regions, i.e., interfaces, within the water phases of the donor and acceptor media and the barrier (X = −L and X = L, respectively).

Figure 7

Plot of the amount of drug permeated vs time (a) and profile of the flux vs time (b). In general, permeability assays suffer from a lag phase, in which the flux is not constant. The interpolation interval of the linear regression ((a) red line) for measuring the mass flux should thus be carefully selected in order to avoid the misinterpretation of the data and achieve the best possible coefficient of determination (e.g., >0.9). The end-point approach for calculating P_app ((a) light-blue line) leads to lower absolute P_app values than in the multiple-point approach.

Figure 8

Advanced physical model to describe the permeation process in vitro. This model considers the existence of unstirred water layers in the donor and acceptor, namely, UWL_d and UWL_a, as additive thin layers that need to be crossed by the solute. Moreover, in this model, the different phases (water–lipid–water) are considered to be separated by boundary regions (interfaces, at X = −L and X = L).

Figure 9

Schematic representation of the PAMPA barrier structure. The PAMPA barrier is a porous filter (0.22–0.45 µm pore size) that is soaked with a lipid mixture (yellow section). The rate-limiting step, controlling permeability, in this case consists mainly of the solute partition/distribution coefficient (LogP/logD) and the diffusivity in the lipid barrier (D_B). In the case of electrolytes, the molecules need to be neutralized (light blue dots) and then re-gain their charge once in the acceptor.

Figure 10

Schematic representation of a hydrated PermeaPad. This barrier is constituted by a liposomal gel contained within two low-retention inert layers. The drug molecules can diffuse through the liposome bilayers, i.e., exploiting partitioning, and/or they can diffuse directly through the para-liposomal space.

Figure 11

Simplified permeability model for a nanocarrier-based enabling formulation (a) and the corresponding mass transfer profile (b) and the in vitro AUC (c). According to the free-fraction theory [93], the effective gradient for permeation should be given by the free drug fraction, c_f, i.e., the unloaded drug. The mass flux profile in this case is highly influenced by the equilibrium constant (K_eq) of the loaded (in the nanocarrier) to the unloaded (free) drugs.

Figure 12

Structural differences between a porous material (a) and a fibrous material (b).

Figure 13

Correlations of P_app values measured using regenerated cellulose membranes and molecular weights (original data form Di Cagno et al. [66]). A quite evident correlation of P_app ≈ 1/mw emerged.

Figure 14

Schematic representation of a dissolution-absorption apparatus (a), e.g., macroFLUX, bioFLUX, and Dissoflux. Solid dosage forms are placed in the donor compartment, i.e., a dissolution vessel, under paddle stirring. The drug concentration is monitored in the donor and acceptor compartments, allowing the user to create dissolution vs time profiles (b) and drug permeated vs time profiles (c), respectively. In such set-ups, high mechanical and chemical stability of the barrier over time are essential.

Figure 15

Example of dissolution/permeation results obtained with Dissoflux with PermeaPad cartridges. The in vitro AUC_{0–6 h} (gray zone) was used to evaluate the bioequivalence of two solid dosage forms (marketed product and new formulation, personal communication from Electrolab). The dissolution media consisted of 0.05 M sodium phosphate (pH 6.8) and 1% tween 80.