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Open AccessArticle

Three-Dimensional Oral Mucosal Equivalents as Models for Transmucosal Drug Permeation Studies

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Pharmaceutics 2023, 15(5), 1513; https://doi.org/10.3390/pharmaceutics15051513 - 17 May 2023

Viewed by 605

Abstract

Oral transmucosal administration, where drugs are absorbed directly through the non-keratinized, lining mucosa of the mouth, represents a solution to drug delivery with several advantages. Oral mucosal equivalents (OME) developed as 3D in vitro models are of great interest since they express the [...] Read more.

Oral transmucosal administration, where drugs are absorbed directly through the non-keratinized, lining mucosa of the mouth, represents a solution to drug delivery with several advantages. Oral mucosal equivalents (OME) developed as 3D in vitro models are of great interest since they express the correct cell differentiation and tissue architecture, simulating the in vivo conditions better than monolayer cultures or animal tissues. The aim of this work was to develop OME to be used as a membrane for drug permeation studies. We developed both full-thickness (i.e., connective plus epithelial tissue) and split-thickness (i.e., only epithelial tissue) OME using non-tumor-derived human keratinocytes OKF6 TERT-2 obtained from the floor of the mouth. All the OME developed here presented similar transepithelial electrical resistance (TEER) values, comparable to the commercial EpiOral™. Using eletriptan hydrobromide as a model drug, we found that the full-thickness OME had similar drug flux to EpiOral™ (28.8 vs. 29.6 µg/cm²/h), suggesting that the model had the same permeation barrier properties. Furthermore, full-thickness OME showed an increase in ceramide content together with a decrease in phospholipids in comparison to the monolayer culture, indicating that lipid differentiation occurred due to the tissue-engineering protocols. The split-thickness mucosal model resulted in 4–5 cell layers with basal cells still undergoing mitosis. The optimum period at the air–liquid interface for this model was twenty-one days; after longer times, signs of apoptosis appeared. Following the 3R principles, we found that the addition of Ca²⁺, retinoic acid, linoleic acid, epidermal growth factor and bovine pituitary extract was important but not sufficient to fully replace the fetal bovine serum. Finally, the OME models presented here offer a longer shelf-life than the pre-existing models, which paves the way for the further investigation of broader pharmaceutical applications (i.e., long-term drug exposure, effect on the keratinocytes’ differentiation and inflammatory conditions, etc.). Full article

(This article belongs to the Special Issue Innovative In Vitro/In Silico Approaches to Drug Permeation and Absorption: The Highway to Implement the 3Rs Principles in Drug Development)

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Open AccessArticle

Permeability Assessment of a High-Throughput Mucosal Platform

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Cosmin Butnarasu

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Olga Valentina Garbero

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Paola Petrini

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Livia Visai

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Sonja Visentin

Pharmaceutics 2023, 15(2), 380; https://doi.org/10.3390/pharmaceutics15020380 - 22 Jan 2023

Viewed by 1140

Abstract

Permeability across cellular membranes is a key factor that influences absorption and distribution. Before absorption, many drugs must pass through the mucus barrier that covers all the wet surfaces of the human body. Cell-free in vitro tools currently used to evaluate permeability fail [...] Read more.

Permeability across cellular membranes is a key factor that influences absorption and distribution. Before absorption, many drugs must pass through the mucus barrier that covers all the wet surfaces of the human body. Cell-free in vitro tools currently used to evaluate permeability fail to effectively model the complexity of mucosal barriers. Here, we present an in vitro mucosal platform as a possible strategy for assessing permeability in a high-throughput setup. The PermeaPad 96-well plate was used as a permeability system and further coupled to a pathological, tridimensional mucus model. The physicochemical determinants predicting passive diffusion were determined by combining experimental and computational approaches. Drug solubility, size, and shape were found to be the critical properties governing permeability, while the charge of the drug was found to be influential on the interaction with mucus. Overall, the proposed mucosal platform could be a promising in vitro tool to model the complexity of mucosal tissues and could therefore be adopted for drug-permeability profiling. Full article

(This article belongs to the Special Issue Innovative In Vitro/In Silico Approaches to Drug Permeation and Absorption: The Highway to Implement the 3Rs Principles in Drug Development)

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Open AccessArticle

Combinational Inhibition of P-Glycoprotein-Mediated Etoposide Transport by Zosuquidar and Polysorbate 20

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Rasmus Blaaholm Nielsen

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Pharmaceutics 2023, 15(1), 283; https://doi.org/10.3390/pharmaceutics15010283 - 14 Jan 2023

Cited by 1 | Viewed by 853

Abstract

P-glycoprotein (P-gp) limits the oral absorption of drug substances. Potent small molecule P-gp inhibitors (e.g., zosuquidar) and nonionic surfactants (e.g., polysorbate 20) inhibit P-gp by proposedly different mechanisms. Therefore, it was hypothesised that a combination of zosuquidar and polysorbate 20 may potentiate inhibition [...] Read more.

P-glycoprotein (P-gp) limits the oral absorption of drug substances. Potent small molecule P-gp inhibitors (e.g., zosuquidar) and nonionic surfactants (e.g., polysorbate 20) inhibit P-gp by proposedly different mechanisms. Therefore, it was hypothesised that a combination of zosuquidar and polysorbate 20 may potentiate inhibition of P-gp-mediated efflux. P-gp inhibition by zosuquidar and polysorbate 20 in combination was assessed in a calcein-AM assay and in a transcellular etoposide permeability study in MDCKII-MDR1 and Caco-2 cells. Furthermore, solutions of etoposide, zosuquidar, and polysorbate 20 were orally administered to Sprague Dawley rats. Zosuquidar elicited a high level of nonspecific adsorption to various labware, which significantly affected the outcomes of the in vitro studies. Still, at certain zosuquidar and polysorbate 20 concentrations, additive P-gp inhibition was observed in vitro. In vivo, however, oral etoposide bioavailability decreased by coadministration of both zosuquidar and polysorbate 20 when compared to coadministration of etoposide with zosuquidar alone. For future formulation development, the present study provided important and novel knowledge about nonspecific zosuquidar adsorption, as well as insights into combinational P-gp inhibition by a third-generation P-gp inhibitor and a P-gp-inhibiting nonionic surfactant. Full article

(This article belongs to the Special Issue Innovative In Vitro/In Silico Approaches to Drug Permeation and Absorption: The Highway to Implement the 3Rs Principles in Drug Development)

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Open AccessArticle

Conformational Sampling Deciphers the Chameleonic Properties of a VHL-Based Degrader

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Giuseppe Ermondi

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Diego Garcia Jimenez

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Matteo Rossi Sebastiano

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Jan Kihlberg

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Giulia Caron

Pharmaceutics 2023, 15(1), 272; https://doi.org/10.3390/pharmaceutics15010272 - 12 Jan 2023

Viewed by 1374

Abstract

Chameleonicity (the capacity of a molecule to adapt its conformations to the environment) may help to identify orally bioavailable drugs in the beyond-Rule-of-5 chemical space. Computational methods to predict the chameleonic behaviour of degraders have not yet been reported and the identification of [...] Read more.

Chameleonicity (the capacity of a molecule to adapt its conformations to the environment) may help to identify orally bioavailable drugs in the beyond-Rule-of-5 chemical space. Computational methods to predict the chameleonic behaviour of degraders have not yet been reported and the identification of molecular chameleons still relies on experimental evidence. Therefore, there is a need to tune predictions with experimental data. Here, we employ PROTAC-1 (a passively cell-permeable degrader), for which NMR and physicochemical data prove the chameleonic behaviour, to benchmark the capacity of two conformational sampling algorithms and selection schemes. To characterize the conformational ensembles in both polar and nonpolar environments, we compute three molecular properties proven to be essential for cell permeability: conformer shape (radius of gyration), polarity (3D PSA), and the number of intramolecular hydrogen bonds. Energetic criteria were also considered. Infographics monitored the simultaneous variation of those properties in computed and NMR conformers. Overall, we provide key points for tuning conformational sampling tools to reproduce PROTAC-1 chameleonicity according to NMR evidence. This study is expected to improve the design of PROTAC drugs and the development of computational sustainable strategies to exploit the potential of new modalities in drug discovery. Full article

(This article belongs to the Special Issue Innovative In Vitro/In Silico Approaches to Drug Permeation and Absorption: The Highway to Implement the 3Rs Principles in Drug Development)

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Open AccessArticle

Advanced Online Monitoring of In Vitro Human 3D Full-Thickness Skin Equivalents

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Roland Schaller-Ammann

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Pharmaceutics 2022, 14(7), 1436; https://doi.org/10.3390/pharmaceutics14071436 - 08 Jul 2022

Cited by 1 | Viewed by 1090

Abstract

Skin equivalents and skin explants are widely used for dermal penetration studies in the pharmacological development of drugs. Environmental parameters, such as the incubation and culture conditions affect cellular responses and thus the relevance of the experimental outcome. However, available systems such as [...] Read more.

Skin equivalents and skin explants are widely used for dermal penetration studies in the pharmacological development of drugs. Environmental parameters, such as the incubation and culture conditions affect cellular responses and thus the relevance of the experimental outcome. However, available systems such as the Franz diffusion chamber, only measure in the receiving culture medium, rather than assessing the actual conditions for cells in the tissue. We developed a sampling design that combines open flow microperfusion (OFM) sampling technology for continuous concentration measurements directly in the tissue with microfluidic biosensors for online monitoring of culture parameters. We tested our design with real-time measurements of oxygen, glucose, lactate, and pH in full-thickness skin equivalent and skin explants. Furthermore, we compared dermal penetration for acyclovir, lidocaine, and diclofenac in skin equivalents and skin explants. We observed differences in oxygen, glucose, and drug concentrations in skin equivalents compared to the respective culture medium and to skin explants. Full article

(This article belongs to the Special Issue Innovative In Vitro/In Silico Approaches to Drug Permeation and Absorption: The Highway to Implement the 3Rs Principles in Drug Development)

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Open AccessArticle

Investigating the Mechanisms behind the Positive Food Effect of Abiraterone Acetate: In Vitro and Rat In Situ Studies

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Pharmaceutics 2022, 14(5), 952; https://doi.org/10.3390/pharmaceutics14050952 - 28 Apr 2022

Cited by 1 | Viewed by 1130

Abstract

The anticancer agent abiraterone suffers from an extensive positive food effect after oral intake of the prodrug abiraterone acetate (Zytiga). The underlying processes determining postprandial abiraterone absorption were investigated in this study. The impact of lipids and lipid digestion products on (i) the [...] Read more.

The anticancer agent abiraterone suffers from an extensive positive food effect after oral intake of the prodrug abiraterone acetate (Zytiga). The underlying processes determining postprandial abiraterone absorption were investigated in this study. The impact of lipids and lipid digestion products on (i) the solubility of abiraterone acetate and abiraterone, (ii) the conversion of abiraterone acetate to abiraterone, and (iii) the passive permeation of abiraterone was determined in vitro. The interaction of abiraterone acetate and abiraterone with vesicles and colloidal structures in the simulated fed state media containing undigested lipids and lipid digestion products enhanced the solubility of both compounds but limited the esterase-mediated hydrolysis of abiraterone acetate and the potential of abiraterone to permeate. Rat in situ intestinal perfusion experiments with a suspension of abiraterone acetate in static fed state simulated media identified abiraterone concentrations in the perfusate as the main driving force for absorption. However, experiments with ongoing lipolysis in the perfusate highlighted the importance of including lipid digestion as a dynamic process when studying postprandial abiraterone absorption. Future research may employ the in situ perfusion model to study postprandial drug absorption from a dynamic lipolysis-mediated intestinal environment to provide reference data for the optimisation of relevant in vitro models to evaluate food effects. Full article

(This article belongs to the Special Issue Innovative In Vitro/In Silico Approaches to Drug Permeation and Absorption: The Highway to Implement the 3Rs Principles in Drug Development)

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Open AccessArticle

Modulation of Paracellular-like Drug Transport across an Artificial Biomimetic Barrier by Osmotic Stress-Induced Liposome Shrinking

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Jonas Borregaard Eriksen

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Pharmaceutics 2022, 14(4), 721; https://doi.org/10.3390/pharmaceutics14040721 - 28 Mar 2022

Cited by 5 | Viewed by 1771

Abstract

Various types of artificial biomimetic barriers are widely utilized as in vitro tools to predict the passive “transcellular” transport of drug compounds. The current study investigated if the sandwich barrier PermeaPad^®, which is composed of tightly packed phospholipid vesicles enclosed between [...] Read more.

Various types of artificial biomimetic barriers are widely utilized as in vitro tools to predict the passive “transcellular” transport of drug compounds. The current study investigated if the sandwich barrier PermeaPad^®, which is composed of tightly packed phospholipid vesicles enclosed between two support sheets, contributes to a transport mechanism that is paracellular-like, representing one of the alternative pathways of passive transport in vivo, primarily of relevance for hydrophilic drug compounds. To this end, we pretreated the commercial PermeaPad^® barrier with NaCl solutions of either high or low osmolality prior to permeation experiments on reversed Franz cell setups with hydrophilic model compounds calcein and acyclovir and hydrophobic model compounds hydrocortisone and celecoxib. Our starting hypothesis was that the liposomes formed in the barrier during the hydration step should either shrink or swell upon contact with test media (drug solutions) due to osmotic pressure difference as compared to the pretreatment solutions. Apparent permeabilities for calcein and acyclovir across the PermeaPad^® barrier were found to increase approximately 2.0 and 1.7 fold, respectively, upon hypo-osmotic pretreatment (soaking in hypotonic medium, while the permeation of hydrocortisone and celecoxib remained unchanged. A control experiment with lipid-free barriers (support sheets) indicated that the permeation of all the compounds was virtually unchanged upon hypo-osmotic pretreatment. In conclusion, soaking PermeaPad^® in a medium of lower osmotic pressure than that used during the permeation study appears to induce the osmotic shrinking of the lipid vesicles in the barrier, leaving wider water channels between the vesicles and, thus, allowing hydrophilic compounds to pass the barrier in a paracellular-like manner. Full article

(This article belongs to the Special Issue Innovative In Vitro/In Silico Approaches to Drug Permeation and Absorption: The Highway to Implement the 3Rs Principles in Drug Development)

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Open AccessArticle

Human Lactobacillus Biosurfactants as Natural Excipients for Nasal Drug Delivery of Hydrocortisone

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Massimiliano Pio di Cagno

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Barbara Luppi

Pharmaceutics 2022, 14(3), 524; https://doi.org/10.3390/pharmaceutics14030524 - 26 Feb 2022

Cited by 6 | Viewed by 1527

Abstract

The inclusion of a chemical permeation enhancer in a dosage form is considered an effective approach to improve absorption across the nasal mucosa. Herein we evaluated the possibility of exploiting biosurfactants (BS) produced by Lactobacillus gasseri BC9 as innovative natural excipients to improve [...] Read more.

The inclusion of a chemical permeation enhancer in a dosage form is considered an effective approach to improve absorption across the nasal mucosa. Herein we evaluated the possibility of exploiting biosurfactants (BS) produced by Lactobacillus gasseri BC9 as innovative natural excipients to improve nasal delivery of hydrocortisone (HC). BC9-BS ability to improve HC solubility and the BS mucoadhesive potential were investigated using the surfactant at a concentration below and above the critical micelle concentration (CMC). In vitro diffusion studies through the biomimetic membrane PermeaPad^® and the same synthetic barrier functionalized with a mucin layer were assessed to determine BC9-BS absorption enhancing properties in the absence and presence of the mucus layer. Lastly, the diffusion study was performed across the sheep nasal mucosa using BC9-BS at a concentration below the CMC. Results showed that BC9-BS was able to interact with the main component of the nasal mucosa, and that it allowed for a greater solubilization and also permeation of the drug when it was employed at a low concentration. Overall, it seems that BC9-BS could be a promising alternative to chemical surfactants in the nasal drug delivery field. Full article

(This article belongs to the Special Issue Innovative In Vitro/In Silico Approaches to Drug Permeation and Absorption: The Highway to Implement the 3Rs Principles in Drug Development)

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Review

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Open AccessReview

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

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Ann-Christin Jacobsen

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Sonja Visentin

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Cosmin Butnarasu

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Paul C. Stein

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Massimiliano Pio di Cagno

Pharmaceutics 2023, 15(2), 592; https://doi.org/10.3390/pharmaceutics15020592 - 09 Feb 2023

Cited by 1 | Viewed by 1150

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Innovative In Vitro/In Silico Approaches to Drug Permeation and Absorption: The Highway to Implement the 3Rs Principles in Drug Development)

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