Transport Kinetics of Iron Chelators and Their Chelates in Caco-2 Cells

Huang, Xi‐Ping; Spino, Michael; Thiessen, Jake J.

doi:10.1007/s11095-005-9258-5

Cited by 26 publications

(24 citation statements)

References 27 publications

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“…UGT substrates typically contain electrophilic acceptor groups such as aliphatic alcohols and phenols, amines, and sulfhydryl or carbonyl groups (Dutton, 1980;Shipkova and Wieland, 2005). UGT1A6 preferentially metabolizes phenols (Harding et al, 1988;Orzechowski et al, 1994;Tukey and Strassburg, 2000), favoring formation of the 3-O-glucuronide, which was identified and measured in earlier studies (Kontoghiorghes et al, 1990;Huang et al, 2006). The second deferiprone glucuronide form, detected in glucuronidation assays with specific UGTs (UGT1A6, 1A8, 1A9, and 1A10) and certain human tissues, has not been reported previously.…”

Section: Discussionmentioning

confidence: 99%

“…Although the long-term safety profile of deferiprone is well defined, the mechanism of and factors involved in its adverse effects, such as gastrointestinal disturbances, arthralgia, neutropenia, and agranulocytosis, are unclear (Agarwal et al, 1990;Taher et al, 2001;Ceci et al, 2002;Cohen et al, 2003;Franchini and Veneri, 2004), thus emphasizing the need to decipher the underlying mechanisms and factors involved in these adverse effects. Deferiprone has good bioavailability, but its clearance is accelerated by rapid biotransformation: approximately 85% of the drug is metabolized to a nonchelating (inactive) 3-O-glucuronide conjugate (Huang et al, 2006) by UDP-glucuronosyltransferases (UGTs). Of all of the known human UGTs, 12 were previously evaluated for deferiprone glucuronidation in a single study, which revealed that only UGT1A6 was capable of catalyzing the glucuronidation of deferiprone (Haverfield et al, 2005).…”

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confidence: 99%

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Deferiprone Glucuronidation by Human Tissues and Recombinant UDP Glucuronosyltransferase 1A6: An in Vitro Investigation of Genetic and Splice Variants

Benoit‐Biancamano

Connelly²,

Villeneuve³

et al. 2008

Drug Metab Dispos

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ABSTRACT:Tissue iron overload constitutes a major health problem for people who require regular blood transfusions, such as those with ␤-thalassemia major. Deferiprone is a hydroxypyridinone iron chelator used therapeutically to remove this excess iron and prevent tissue damage. Deferiprone is metabolized by UDP-glucuronosyltransferases (UGTs) into deferiprone 3-O-glucuronide (DG), but a systematic evaluation of the contribution of individual human UGTs and the impact of genetic variations of UGTs have not been conducted. Sixteen human UGT1A and UGT2B were studied for deferiprone glucuronidation, and their clearances were compared in human tissue samples. DG was measured by liquid chromatography coupled with mass spectrometry. DG was primarily produced in vitro by UGT1A6, and a second glucuronide metabolite was discovered. UGT1A6, as well as liver and kidney human microsomes, had similar kinetic profiles and clearance (Cl int ‫؍‬ 1.4-3.0 l/min/mg), but clearance by intestinal microsomes was much lower (0.04 l/min/mg). Binding of deferiprone to microsomal preparations was not significant. Genetic variants of UGT1A6 had K m values similar to the reference protein (UGT1A6*1), but their V max values were reduced by 25 to 70%. The UGT1A6 splice variant isoform 2, detected in the liver and kidney, had no transferase activity for deferiprone. When UGT1A6_i2 was coexpressed with the classic UGT1A6_i1 isoform, velocity was reduced for deferiprone but remained similar for 4-nitrophenol or serotonin glucuronidation. In conclusion, deferiprone glucuronidation seems to depend almost totally on UGT1A6, especially in the liver. Genetic variations and differences in the expression of splice variants represent a potential source of variation in deferiprone metabolism.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Deferiprone Glucuronidation by Human Tissues and Recombinant UDP Glucuronosyltransferase 1A6: An in Vitro Investigation of Genetic and Splice Variants

Benoit‐Biancamano

Connelly²,

Villeneuve³

et al. 2008

Drug Metab Dispos

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“…Huang and collaborators (Huang et al 2006), using the Caco-2 cell model cultured on permeable membrane, demonstrated that DFO, CP20 and ICL670 were passively transported across the enterocyte cell monolayer with a membrane permeability modulated by the lipophilicity and the charge of the chelators (CP20 [ ICL670). The iron chelates [Fe(ICL670) 2 ] and [Fe(DFO)] were not transported across Caco-2 monolayers, while the [Fe(CP20) 3 ] chelate was able to cross the monolayer in both directions.…”

Section: Effect Of Iron Chelators On Iron Metabolismmentioning

confidence: 99%

Effects of deferasirox and deferiprone on cellular iron load in the human hepatoma cell line HepaRG

et al. 2009

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Two oral chelators, CP20 (deferiprone) and ICL670 (deferasirox), have been synthesized for the purpose of treating iron overload diseases, especially thalassemias. Given their antiproliferative effects resulting from the essential role played by iron in cell processes, such compounds might also be useful as anticancer agents. In the present study, we tested the impact of these two iron chelators on iron metabolism, in the HepaRG cell line which allowed us to study proliferating and differentiated hepatocytes. ICL670 uptake was greater than the CP20 uptake. The iron depletion induced by ICL670 in differentiated cells increased soluble transferrin receptor expression, decreased intracellular ferritin expression, inhibited (55)Fe (III) uptake, and reduced the hepatocyte concentration of the labile iron pool. In contrast, CP20 induced an unexpected slight increase in intracellular ferritin, which was amplified by iron-treated chelator exposure. CP20 also promoted Fe(III) uptake in differentiated HepaRG cells, thus leading to an increase of both the labile pool and storage forms of iron evaluated by calcein fluorescence and Perls staining, respectively. In acellular conditions, compared to CP20, iron removing ability from the calcein-Fe(III) complex was 40 times higher for ICL670. On the whole, biological responses of HepaRG cells to ICL670 treatment were characteristic of expected iron depletion. In contrast, the effects of CP20 suggest the potential involvement of this compound in the iron uptake from the external medium into the hepatocytes from the HepaRG cell line, therefore acting like a siderophore in this cell model.

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“…Uptake and/or efflux information was appraised in parallel studies to obtain information on the individual kinetic process (Bourdet et al, 2006). In some cases, data may be obtained from identical Transwells that provide data in the various compartments at each time point (Huang et al, 2006). In other studies, repetitive sampling at various time points in the receiver compartment of the same Transwell with volume replacement was used.…”

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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Transport Kinetics of Iron Chelators and Their Chelates in Caco-2 Cells

Abstract: Caco-2 cells are useful in screening iron chelators and chelates and estimating bioavailabilities. Structure and distribution coefficients partially predict passive transport through Caco-2 monolayers.

Cited by 26 publications

References 27 publications

Deferiprone Glucuronidation by Human Tissues and Recombinant UDP Glucuronosyltransferase 1A6: An in Vitro Investigation of Genetic and Splice Variants

Deferiprone Glucuronidation by Human Tissues and Recombinant UDP Glucuronosyltransferase 1A6: An in Vitro Investigation of Genetic and Splice Variants

Effects of deferasirox and deferiprone on cellular iron load in the human hepatoma cell line HepaRG

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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