Kinetic modelling of passive transport and active efflux of a fluoroquinolone across Caco-2 cells using a compartmental approach in NONMEM

González‐Álvarez, Isabel; Fernández-Teruel, Carlos; Garrigues, Teresa María; Casabó, V.G.; Ruiz-Garcı́a, Ana; Bermejo, Marival

doi:10.1080/00498250500354469

Cited by 35 publications

(38 citation statements)

References 45 publications

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“…A catenary model similar to those used by others (Ito et al, 1999;Tam et al, 2003;Irie et al, 2004;González-Alvarez et al, 2005) was employed to appraise various aspects of the effective permeability, efflux ratio, and data interpretation on transporters and enzymes in Caco-2 monolayers. The model comprised the absorptive and ABC transporters of the apical membrane and influx and efflux barriers at the basolateral membrane as two sets of potential barriers.…”

Section: Methodsmentioning

confidence: 99%

“…We are of the view that the single-barrier model for the Caco-2 cell monolayer is inadequate and that a catenary model comprising basolateral (B), apical (A), and cellular compartments (Ito et al, 1999;Tam et al, 2003;Irie et al, 2004;González-Alvarez et al, 2005) is more appropriate than what routine analyses subscribe, especially under nonlinear conditions. Given the importance of the Caco-2 system in drug discovery and development, we explored properties of the catenary model to gain a mechanistically based understanding of the asymmetric observations arising from transporters, enzymes, and effective permeability under both linear and nonlinear conditions.…”

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confidence: 99%

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Permeability, Transport, and Metabolism of Solutes in Caco-2 Cell Monolayers: A Theoretical Study

Sun¹,

Pang²

2007

Drug Metab Dispos

103

105

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ABSTRACT:We explored the properties of a catenary model that includes the basolateral (B), apical (A), and cellular compartments via simulations under linear and nonlinear conditions to understand the asymmetric observations arising from transporters, enzymes, and permeability in Caco-2 cells. The efflux ratio (EfR; P app,B3A / P app,A3B ), obtained from the effective permeability from the A3B and B3A direction under linear conditions, was unity for passively permeable drugs whose transport does not involve transporters; the value was unaffected by cellular binding or metabolism, but increased with apical efflux. Metabolism was asymmetric, showing lesser metabolite accrual for the B3A than A3B direction because of inherent differences in the volumes for A and B. Moreover, the net flux (total ؊ passive permeation) due to saturable apical efflux, absorption, or metabolism showed nonconformity to simple The majority of drugs available on the market are in oral dosage forms. For the assessment of permeability and oral drug absorption, in silico models (Stenberg et al., 2001) and high throughput systems, such as the parallel artificial membrane permeability assay (Kansy et al., 1998) and cell-based systems (Hidalgo et al., 1989) exist to relate drug permeability to absorption, especially for compounds that do not undergo intestinal metabolism (Usansky and Sinko, 2005). The most popular high-throughput screening tool for drug permeability is human colon carcinoma (Caco-2) (Hidalgo et al., 1989) or transfected Madin-Darby canine kidney (Irvine et al., 1999) cells. Upon culture, Caco-2 cells differentiate and become confluent to form monolayers with tight junctions and polarized apical/mucosal (A side) and basolateral/serosal (B side) membranes that are structurally and functionally similar to those of enterocytes.ABC efflux transporters such as P-glycoprotein (Pgp) multidrug resistance-associated protein 2 (MRP2), and the breast cancer-resistant protein (BCRP) are expressed on the mucosal membrane of Caco-2 (Hunter et al., 1993;Hirohashi et al., 2000), as are the absorptive transporters, such as the proton-coupled oligopeptide transporter (PEPT1) (Guo et al., 1999) and the organic anion transporting polypeptide (OATP) (Kobayashi et al., 2003). Likewise, on the serosal membrane, basolateral efflux transporters such as MRP3 (Hirohashi et al., 2000) and organic solute transporters ␣ and ␤ (OST␣-OST␤) (Okuwaki et al., 2007) are expressed in Caco-2. In addition, multiple metabolic enzymes such as the sulfotransferases, UDPglucuronosyltransferases (H. Sun, L. Zhang, E. C. Chow, G. Lin, K. S. Pang, Z. Zuo, unpublished data), and the glutathione S-transferases (Peters and Roelofs, 1989) N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; A, apical; B, basolateral; C D,0 , initial loading concentration in donor side at t ϭ 0; P app , effective permeability; EfR, efflux ratio; MK571,vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propioni...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Permeability, Transport, and Metabolism of Solutes in Caco-2 Cell Monolayers: A Theoretical Study

Sun¹,

Pang²

2007

Drug Metab Dispos

103

105

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“…1) was constructed with Stella 8.1.4 software (isee systems, Lebanon, NH) to simulate mass flow in a permeability experiment. Three-compartment models have been used to simulate in vitro permeability experiments (Ito et al, 1999;González-Alvarez et al, 2005). We introduce an apparent cellular distribution coefficient (K ϭ M bound /M free ) to the model.…”

Section: Methodsmentioning

confidence: 99%

Decrease in Intracellular Concentration Causes the Shift in K_m Value of Efflux Pump Substrates

Korjamo

Kemiläinen²,

Heikkinen³

et al. 2007

Drug Metab Dispos

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ABSTRACT:Passive permeability and active efflux are parallel processes in transcellular flux. Therefore, the observed kinetics of a transporter substrate depends on both of these factors. The transporter expression has been shown to affect both the apparent K m and V max values. Kinetic parameters can be obtained from various experimental settings, but these do not necessarily reflect the situation in transcellular flux. Kinetic absorption models need reliable estimates of saturable kinetics when accurate in silico predictions are to be made. The effect of increasing P-glycoprotein expression on apparent transport kinetics was studied using quinidine and digoxin as model compounds. The intracellular concentrations of drugs during the transport process were also measured. A dynamic simulation model was constructed to study the observed data. The apparent K m and V max values increased as the P-glycoprotein expression increased. Simulations reproduced the shift in both kinetic parameters as a function of efflux pump expression. In addition, the apparent K m value showed a strong inverse relationship to the passive permeability. In contrast, the apparent V max value reached a maximum at intermediate passive permeability and declined above and below this passive permeability. The true V max and K m values were never reached. The shift in K m was assigned to a decrease in intracellular concentration at the Pglycoprotein interaction site with both experimental and simulation data. In conclusion, the apparent kinetic parameters in transcellular permeability assays depend on passive permeability and efflux pump activity. Therefore, parameters that are obtained from in vitro assays should be cautiously applied to in vivo predictions.

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“…The model describes mass transfer across membranes via passive diffusion and transporter mediation, protein binding, and metabolism. Data analyses with the catenary model (Ito et al, 1999;Gonzá lez-Alvarez et al, 2005;Bourdet et al, 2006) and other biophysical models (Ho et al, 2000;Tran et al, 2005) offer more mechanistic insight and accurate depiction of the kinetic process of interest. To demonstrate the robustness of the catenary model, we used the data set on Ba transport and metabolism to reveal substrate inhibition kinetics in metabolism.…”

Section: Discussionmentioning

confidence: 99%

A Catenary Model to Study Transport and Conjugation of Baicalein, a Bioactive Flavonoid, in the Caco-2 Cell Monolayer: Demonstration of Substrate Inhibition

Sun

Zhang²,

Chow³

et al. 2008

J Pharmacol Exp Ther

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The transport and metabolism of baicalein (Ba) was studied in vitro and in Caco-2 cells. Protein binding of Ba with Caco-2 lysate showed that Ba was bound to two classes of sites: a higher affinity, lower capacity site (K A1 ϭ 27.6 Ϯ 4.7 M Ϫ1 , n 1 ϭ 10.6 Ϯ 0.6 nmol/mg) and lower affinity, higher capacity site (K A2 ϭ 0.015 Ϯ 0.0013 M Ϫ1, n 2 ϭ 413 Ϯ 21 nmol/mg). Incubation studies of Ba with Caco-2 lysate showed substrate inhibition of both glucuronidation and sulfation, with K m values of 0.14 Ϯ 0.034 and 0.015 Ϯ 0.0053 M, and K I values of 6.75 Ϯ 1.70 and 0.37 Ϯ 0.16 M, respectively. In the Caco-2 monolayer, Ba (8 -47 M) displayed good apparent permeabilities (P app ) across the membrane; P app was found to be increased with elevated loading concentration in both the absorptive and secretory directions. However, the efflux ratio was less than unity, negating the involvement of apical efflux transporters. The concentration ratios of Ba sulfate (BS) and glucuronide (BG) decreased with increased loading Ba concentration, suggesting that BS and BG are apically excreted via transporters, likely breast cancer resistance protein and multidrug resistance-associated protein 2, respectively. Data fit to the catenary model, composed of basolateral, cellular, and apical compartments, showed a low cellular unbound fraction (0.0019 Ϯ 0.00018), a high passive diffusion clearance (0.012 Ϯ 0.00029 ml/min/mg), and substrate inhibition, with sulfation being more readily saturated and inhibited than glucuronidation, as evidenced by smaller K m value (0.35 Ϯ 0.078 versus 1.95 Ϯ 0.57 M) and K I value (0.58 Ϯ 0.20 versus 7.90 Ϯ 1.10 M); these patterns paralleled those observed in the lysate incubation studies. The results showed that the catenary model aptly predicts substrate inhibition kinetics and offers significant and mechanistic insight into the transport and atypical metabolism of drugs in the Caco-2 monolayer.Flavonoids are polyphenolic compounds that are widely distributed in both edible plant and derived foods (Yang et al., 2001). Flavonoids possess anticarcinogenic activity and other beneficial effects, including the prevention of oxidation of low-density lipoproteins and development of coronary heart disease. These activities have aroused increasing interest among scientists (Yang et al., 2001). However, several studies have demonstrated that the oral bioavailabilities of flavonoids are low and associated with extensive first-pass metabolism, including glucuronidation, sulfation, and methylation (Kroon et al., 2004;Manach and Donovan, 2004 ABBREVIATIONS: MRP, multidrug resistance-associated protein; Ba, baicalein; BG, baicalein-7-O-␤-glucoronide; UGT, UDP-glucuronosyltransferase; BS, baicalein sulfate; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline; A, apical; B, basolateral; P app , apparent permeability; EfR, efflux ratio; n 1 , n 2 , the number of binding sites in the first and second class of binding sites, respectively; CLЈ int,sec and CLЈ d2 {BG or BS}, net efflux clearance of...

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Kinetic modelling of passive transport and active efflux of a fluoroquinolone across Caco-2 cells using a compartmental approach in NONMEM

Cited by 35 publications

References 45 publications

Permeability, Transport, and Metabolism of Solutes in Caco-2 Cell Monolayers: A Theoretical Study

Permeability, Transport, and Metabolism of Solutes in Caco-2 Cell Monolayers: A Theoretical Study

Decrease in Intracellular Concentration Causes the Shift in K_m Value of Efflux Pump Substrates

A Catenary Model to Study Transport and Conjugation of Baicalein, a Bioactive Flavonoid, in the Caco-2 Cell Monolayer: Demonstration of Substrate Inhibition

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Kinetic modelling of passive transport and active efflux of a fluoroquinolone across Caco-2 cells using a compartmental approach in NONMEM

Cited by 35 publications

References 45 publications

Permeability, Transport, and Metabolism of Solutes in Caco-2 Cell Monolayers: A Theoretical Study

Permeability, Transport, and Metabolism of Solutes in Caco-2 Cell Monolayers: A Theoretical Study

Decrease in Intracellular Concentration Causes the Shift in Km Value of Efflux Pump Substrates

A Catenary Model to Study Transport and Conjugation of Baicalein, a Bioactive Flavonoid, in the Caco-2 Cell Monolayer: Demonstration of Substrate Inhibition

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Decrease in Intracellular Concentration Causes the Shift in K_m Value of Efflux Pump Substrates