Saturable transport of H2‐antagonists ranitidine and famotidine across Caco‐2 cell monolayers

doi:10.1021/js980474k

Journal of Pharmaceutical Sciences

Volume 88, Issue 7, July 1999, Pages 680-687

https://doi.org/10.1021/js980474k Get rights and content

Abstract

The purpose of this study was to investigate the mechanism by which the H₂‐antagonists ranitidine and famotidine interacted with the paracellular space during their transport across Caco‐2 cell monolayers. Transport experiments with ranitidine and famotidine across Caco‐2 cell monolayers were performed to determine the apical‐to‐basolateral flux at various concentrations. Kinetic analysis of the transport data showed that ranitidine and famotidine were transported by both saturable and nonsaturable processes. Na⁺,K⁺‐ATPase inhibitor ouabain and metabolic inhibitors sodium azide +2‐deoxy‐D‐glucose did not affect ranitidine transport, suggesting that the active transport was not involved. Famotidine and some other guanidine‐containing compounds, e.g., guanethidine, Arg‐Gly, L‐arginine methyl ester, and L‐argininamide, inhibited the transport of ranitidine, whereas other guanidine‐containing compounds with an additional negative charge, e.g., L‐arginine, did not. 2,4,6‐Triaminopyrimidine (TAP), an inhibitor of paracelluar cationic conductance, also inhibited the transport of both ranitidine and famotidine. On the basis of these results, it is proposed that the saturable transport of ranitidine and famotidine across Caco‐2 cell monolayers appears to be via a facilitated diffusion process mediated by the paracellular anionic sites. This mechanism is consistent with the observation that ranitidine and famotidine caused a concentration‐dependent increase in transepithelial electrical resistance (TEER) across Caco‐2 cell monolayers, presumably by blocking the paracellular anionic sites and thus inhibiting the flux of cations (e.g., Na⁺).

References and Notes (30)

K.L. Lutz et al.
Molecular Structure of the Apical Junction Complex and its Contribution to the Paracellular Barrier
J. Pharm. Sci.
(1997)
M. Hu et al.
Transport of a Large Neutral Amino Acid in a Human Intestinal Epithelial Cell Line (Caco‐2): Uptake and Efflux of Phenylalanine
Biochim. Biophys. Acta
(1992)
D.T. Thwaites et al.
H⁺‐Coupled Dipeptide (Glycylsarcosine) Transport across Apical and Basal Borders of Human Intestinal Caco‐2 Cell Monolayers Displays Distinctive Characteristics
Biochim. Biophys. Acta
(1993)
A.H. Dantzig et al.
Cefaclor Uptake by the Proton‐Dependent Dipeptide Transport Carrier of Human Intestinal Caco‐2 Cells and Comparison to Cephalexin Uptake
Biochim. Biophys. Acta
(1992)
G.T. Knipp et al.
Paracellular Diffusion in Caco‐2 Cell Monolayers: Effect of Perturbation on the Transport of Hydrophilic Compounds that Vary in Charge and Size
J. Pharm. Sci.
(1997)
W.L. Hayton
Rate‐Limiting Barriers to Intestinal Drug Absorption: a Review
J. Pharmacokinet. Biopharm.
(1980)
Drug Transport across Gastrointestinal Epithelia
A. Tsuji et al.
Carrier‐Mediated Intestinal Transport of Drugs
Pharm. Res.
(1996)
B. Gumbiner
Structure, Biochemistry, and Assembly of Epithelial Tight Junctions
Am. J. Physiol.
(1987)
S.T. Ballard et al.
Regulation of Tight‐Junction Permeability during Nutrient Absorption across the Intestinal Epithelium
Annu. Rev. Nutr.
(1995)

E.E. Schneeberger et al.

Tight Junctions. Their Structure, Composition, and Function

Circ. Res.

(1984)

L.‐S. Gan et al.

Mechanism of Intestinal Absorption of Ranitidine and On‐dansetron Transport across Caco‐2 cell Monolayers

Pharm. Res.

(1993)

J.H. Lin

Pharmacokinetic and Pharmacodynamic Properties of Histamine H₂‐Receptor Antagonists

Clin. Pharmacokinet.

(1991)

L.‐S. Gan et al.

Modulation of the Tight Junctions of the Caco‐2 Cell Monolayers by H2‐Antagonists

Pharm. Res.

(1998)

J.H. Graf et al.

Vitamin D Increases Tight‐Junction Conductance and Paracellular Ca₂₊ Transport in Caco‐2 Cell Culture

Am. J. Physiol.

(1998)

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Having demonstrated that Caco-2 morphology, apical actin accumulation, and claudin-2 expression could be controlled by changing the underlying biomaterial, we next set out to study the permeability of mature Caco-2 monolayers to several pharmaceutical drug molecules. We selected four compounds that are known to be transported predominantly via the paracellular pathway: histamine H2 receptor antagonists famotidine (Pepcid®) [62] and ranitidine (Zantac®) [62], and β-antagonists nadolol (Corgard®) [12] and atenolol (Tenormin®) [63]. We also selected two drug molecules that are known to be predominantly transported via transcellular routes: corticosteroid dexamethasone (Decadron®) [64] and non-steroidal anti-inflammatory drug naproxen (Aleve®) [65].
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Ranitidine is a hydrophilic weak base and an H₂-receptor antagonist which is commonly used for gastroesophageal reflux, including during pregnancy. It has limited placental permeation and can be used as a pre-anesthetic antacid to prevent aspiration of acidic stomach contents. Recent studies suggest that diabetes and hypertension may influence placental permeation of hydrophilic drugs. Thus, this study aimed to investigate the influence of diabetes and hypertension on ranitidine's placental permeation in pregnant women.
Forty one pregnant women all scheduled for elective cesarean section entered the study: healthy control (n = 15), with hypertension (n = 16) and with gestational diabetes (n = 10). All women received 50 mg of ranitidine intravenously. Three samples of maternal plasma (after ranitidine application, at delivery and after delivery), and two umbilical cord samples (arterial and venous blood) were collected and analyzed for ranitidine concentrations. Maternal pharmacokinetic parameter were calculated as well as feto:maternal and umbilical cord arterial to venous concentration ratios.
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The Ussing chambers model is almost exclusively used in the presence of plain aqueous phosphate buffers as solvent system. In an attempt to further elucidate the effect of luminal ingredients and postprandial conditions on intestinal permeability, pooled fasted and fed state human intestinal fluids (FaHIF_pool, FeHIF_pool) were used. In addition, simulated intestinal fluids of both nutritional states (FaSSIF, FeSSIF) were evaluated as possible surrogate media for HIF. The use of FaHIF_pool generated a broad range of P_app values for a series of 16 model drugs, ranging from 0.03 × 10⁻⁶ cm/s (carvedilol) to 33.8 × 10⁻⁶ cm/s (naproxen). A linear correlation was observed between P_app values using FaSSIF and FaHIF_pool as solvent system (R = 0.990), justifying the use of FaSSIF as surrogate medium for FaHIF in the Ussing chambers. In exclusion of the outlier carvedilol, a strong sigmoidal relationship was found between P_app and fa_human of 15 model drugs, illustrated by correlation coefficients of 0.961 and 0.936 for FaHIF_pool and FaSSIF, respectively. When addressing food effects on intestinal permeability, the use of FeHIF_pool resulted in a significantly lower P_app value for nine out of sixteen compounds compared to fasting conditions. FeSSIF as solvent system significantly overestimated P_app values in FeHIF_pool. To conclude, the optimized Ussing chambers model using biorelevant media as apical solvent system holds great potential to investigate food effects in a more integrative approach, taking into account drug solubilisation, supersaturation and formulation effects.
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Thus, the higher permeability for both compounds in FaHIF compared to FaSSIF might be due to an inhibitory effect of components solely present in FaHIF on the associated efflux transporters. Another hypothesis is suggested by work of (Lee and Thakker, 1999), stating that ranitidine transport in Caco-2 appears to be a facilitated diffusion process mediated by paracellular anionic sites. The modulation or saturation of these anionic sites by components present in FaSSIF or FaHIF can lead to the significantly different permeability for ranitidine in these media.
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Research ArticlesSaturable transport of H2‐antagonists ranitidine and famotidine across Caco‐2 cell monolayers

Abstract

J. Pharm. Sci.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Pharm. Sci.

Rate‐Limiting Barriers to Intestinal Drug Absorption: a Review

J. Pharmacokinet. Biopharm.

Drug Transport across Gastrointestinal Epithelia

Carrier‐Mediated Intestinal Transport of Drugs

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Structure, Biochemistry, and Assembly of Epithelial Tight Junctions

Am. J. Physiol.

Regulation of Tight‐Junction Permeability during Nutrient Absorption across the Intestinal Epithelium

Annu. Rev. Nutr.

Tight Junctions. Their Structure, Composition, and Function

Circ. Res.

Mechanism of Intestinal Absorption of Ranitidine and On‐dansetron Transport across Caco‐2 cell Monolayers

Pharm. Res.

Pharmacokinetic and Pharmacodynamic Properties of Histamine H2‐Receptor Antagonists

Clin. Pharmacokinet.

Modulation of the Tight Junctions of the Caco‐2 Cell Monolayers by H2‐Antagonists

Pharm. Res.

Vitamin D Increases Tight‐Junction Conductance and Paracellular Ca2+ Transport in Caco‐2 Cell Culture

Am. J. Physiol.

Research Articles
Saturable transport of H₂‐antagonists ranitidine and famotidine across Caco‐2 cell monolayers

Pharmacokinetic and Pharmacodynamic Properties of Histamine H₂‐Receptor Antagonists

Vitamin D Increases Tight‐Junction Conductance and Paracellular Ca₂₊ Transport in Caco‐2 Cell Culture