Variability of permeability estimation from different protocols of subculture and transport experiments in cell monolayers

Oltra-Noguera, Davinia; Mangas‐Sanjuán, Víctor; Centelles-Sangüesa, Amparo; González‐García, Ignacio; Sanchez-Castaño, Gloria; González‐Álvarez, Marta; Casabó, V.G.; Merino, Virginia; González‐Álvarez, Isabel; Bermejo, Marival

doi:10.1016/j.vascn.2014.11.004

Cited by 33 publications

(31 citation statements)

References 34 publications

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“…The permeability value was calculated depending on sink (low permeable compounds) or non‐sink (high permeable compounds) conditions on the acceptor side …”

Section: Methodsmentioning

confidence: 99%

“…Under sink conditions (negligible drug concentration in acceptor vs donor concentration or acceptor concentration <10% of donor concentration, no change of donor concentration during the assay, under a linear approximation of the appearance rates), the apparent permeability coefficients ( P app ) were calculated using the following formula:

P_{normalapp} = \frac{d Q}{d t} \times \frac{1}{A \times C_{0}}

where d Q /d t (mmol/s or mg/s) is the total amount of investigated drug substance transported to the acceptor side per unit time, A is the exposed surface area of the cell monolayer (0.7 cm 2 ), C 0 ( m or mg/ml) is the average initial concentration of drug substance in donor wells.…”

Section: Methodsmentioning

confidence: 99%

“…Under non‐sink conditions (continuous change of the donor and acceptor concentrations), the apparent permeability coefficient was calculated using the following equation:

C_{acc, t} = \frac{Q_{total}}{V_{normalacc} + V_{normaldon}} + ((C_{acc, t - 1} \times f) - \frac{Q_{normaltotal}}{V_{acc} + V_{don}}) \times e^{- P_{normalapp} \times A \times (\frac{1}{V_{normalacc}} + \frac{1}{V_{normaldon}}) \times Δ t,}

where C acc, t is the drug concentration in the acceptor chamber at time t , Q total is the total amount of drug in both chambers, V acc and V don are the volumes in acceptor and donor chamber, C acc ,t −1 is the drug concentration in acceptor chamber at previous time, f is the sample replacement dilution factor, A is the surface area, Δ t is the time interval and P app is the permeability coefficient.…”

Section: Methodsmentioning

confidence: 99%

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Demonstrating suitability of the Caco-2 cell model for BCS-based biowaiver according to the recent FDA and ICH harmonised guidelines

Jarc

Novak

Hevir³

et al. 2019

Journal of Pharmacy and Pharmacology

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Objective According to the regulatory guidelines, one of the critical steps in using in‐vitro permeability methods for permeability classification is to demonstrate the suitability of the method. Here, suitability of the permeability method by using a monolayer of cultured epithelial cells was verified with different criteria. Methods Imaging with a transmission electron microscope was used for characterisation of the cells. Monolayer integrity was confirmed by transepithelial electrical resistance measurements and permeability of zero permeability marker compounds. Real‐time polymerase chain reaction was employed to evaluate expression levels of 84 known transporters. Samples for bidirectional permeability determination were quantified by ultra‐performance liquid chromatography. Key findings The Caco‐2 cells grow in an intact monolayer and morphologically resemble enterocytes. Genes of 84 known transporters were expressed at different levels; furthermore, expression was time depended. Functional expression of efflux transporter P‐glycoprotein was confirmed. We established a correlation between permeability coefficients of 21 tested drug substances ranging from low, moderate and high absorption with human fraction absorbed literature data (R2 = 0.84). Conclusions Assay standardisation assures the consistency of experimental data. Only such fully characterised model has the ability to accurately predict drug's intestinal permeability at the early stage of research or for the BCS‐based biowaiver application.

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“…The permeability value was calculated depending on sink (low permeable compounds) or non‐sink (high permeable compounds) conditions on the acceptor side …”

Section: Methodsmentioning

confidence: 99%

P_{normalapp} = \frac{d Q}{d t} \times \frac{1}{A \times C_{0}}

Section: Methodsmentioning

confidence: 99%

“…Under non‐sink conditions (continuous change of the donor and acceptor concentrations), the apparent permeability coefficient was calculated using the following equation:

C_{acc, t} = \frac{Q_{total}}{V_{normalacc} + V_{normaldon}} + ((C_{acc, t - 1} \times f) - \frac{Q_{normaltotal}}{V_{acc} + V_{don}}) \times e^{- P_{normalapp} \times A \times (\frac{1}{V_{normalacc}} + \frac{1}{V_{normaldon}}) \times Δ t,}

Section: Methodsmentioning

confidence: 99%

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Demonstrating suitability of the Caco-2 cell model for BCS-based biowaiver according to the recent FDA and ICH harmonised guidelines

Jarc

Novak

Hevir³

et al. 2019

Journal of Pharmacy and Pharmacology

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“…However, they have several limitations. Sub-cloned cell lines used at different laboratories often vary in genetic status and biological behavior, such as growth rate, transepithelial resistance, metabolic activity, and the extent of expression of transport proteins (2). They also differ in functional and transport characteristics as their passage number increases.…”

Section: Introductionmentioning

confidence: 99%

The Contributions of Human Mini-Intestines to the Study of Intestinal Physiology and Pathophysiology

Hasan

et al. 2017

Annu. Rev. Physiol.

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The lack of accessibility to normal and diseased human intestine and the inability to separate the different functional compartments of the intestine even when tissue could be obtained have held back the understanding of human intestinal physiology. Clevers and his associates identified intestinal stem cells and established conditions to grow “mini-intestines” ex vivo in differentiated and undifferentiated conditions. This pioneering work has made a new model of the human intestine available and has begun making contributions to the understanding of human intestinal transport in normal physiologic conditions and the pathophysiology of intestinal diseases. However, this model is reductionist and lacks many of the complexities of normal intestine. Consequently, it is not yet possible to predict how great the advances using this model will be for understanding human physiology and pathophysiology, nor how the model will be modified to include multiple other intestinal cell types and physical forces necessary to more closely approximate normal intestine. This review describes recent studies using mini-intestines, which have readdressed previously established models of normal intestinal transport physiology and newly examined intestinal pathophysiology. The emphasis is on studies with human enteroids grown either as three-dimensional spheroids or two-dimensional monolayers. In addition, comments are provided on mouse studies in cases when human studies have not yet been described.

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“…SOPs were described and validated previously in our laboratory. [7][8][9][10][11] Hank's balanced salt solution (HBSS) supplemented with HEPES was used to fill 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 the receiver chamber and to prepare the drug solution that was placed in the donor chamber (pH=7 in both chambers). Transport studies were conducted in an orbital environmental shaker at constant temperature (37ºC) and at an agitation rate of 50 rpm.…”

Section: Experimental Techniquesmentioning

confidence: 99%

Investigating the Discriminatory Power of BCS-Biowaiver in Vitro Methodology to Detect Bioavailability Differences between Immediate Release Products Containing a Class I Drug

et al. 2015

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The purpose of this work is to investigate the discriminatory power of the Biopharmaceutics Classification System (BCS)-biowaiver in vitro methodology, i.e., to investigate if a BCS-biowaiver approach would have detected the Cmax differences observed between two zolpidem tablets and to identify the cause of the in vivo difference. Several dissolution conditions were tested with three zolpidem formulations: the reference (Stilnox), a bioequivalent formulation (BE), and a nonbioequivalent formulation (N-BE). Zolpidem is highly soluble at pH 1.2, 4.5, and 6.8. Its permeability in Caco-2 cells is higher than that of metoprolol and its transport mechanism is passive diffusion. None of the excipients (alone or in combination) showed any effect on permeability. All formulations dissolved more than 85% in 15 min in the paddle apparatus at 50 rpm in all dissolution media. However, at 30 rpm the nonbioequivalent formulation exhibited a slower dissolution rate. A slower gastric emptying rate was also observed in rats for the nonbioequivalent formulation. A slower disintegration and dissolution or a delay in gastric emptying might explain the Cmax infra-bioavailability for a highly permeable drug with short half-life. The BCS-biowaiver approach would have declared bioequivalence, although the in vivo study was not conclusive but detected a 14% mean difference in Cmax that precluded the bioequivalence demonstration. Nonetheless, these findings suggest that a slower dissolution rate is more discriminatory and that rotation speeds higher than 50 rpm should not be used in BCS-biowaivers, even if a coning effect occurs.

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Variability of permeability estimation from different protocols of subculture and transport experiments in cell monolayers

Cited by 33 publications

References 34 publications

Demonstrating suitability of the Caco-2 cell model for BCS-based biowaiver according to the recent FDA and ICH harmonised guidelines

Demonstrating suitability of the Caco-2 cell model for BCS-based biowaiver according to the recent FDA and ICH harmonised guidelines

The Contributions of Human Mini-Intestines to the Study of Intestinal Physiology and Pathophysiology

Investigating the Discriminatory Power of BCS-Biowaiver in Vitro Methodology to Detect Bioavailability Differences between Immediate Release Products Containing a Class I Drug

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