Analysis of the passage of the marine biotoxin okadaic acid through an in vitro human gut barrier

The marine biotoxin okadaic acid (OA), produced by dinoflagellates, can accumulate in various bivalve molluscs. In humans, oral consumption of shellfish contaminated with OA induces acute toxic effects like diarrhea, nausea, vomiting and abdominal pain. However, tumorigenic and embryotoxic effects of OA have been also described. Current toxicokinetic studies with mice were performed with high cytotoxic oral doses leading presumably to a paracellular passage of OA through the gastrointestinal barrier. There are no studies available analyzing the absorption at low concentrations, which represent a realistic dietary exposure, making a reliable risk assessment difficult. Therefore, we performed a low-dose study using the human intestinal Caco-2 cell model to simulate the intestinal barrier. Low level exposure of 20-200 nM OA to the cell monolayer allows an only limited passage from the "luminal" to the "blood side". Furthermore, we could detect a significant efflux of OA, which led to the suggestion that active transport mechanisms are involved in the elimination process of OA. In conclusion, our results indicate that besides the well known defense mechanisms of humans against this marine biotoxin--vomiting and diarrhea--further detoxification mechanisms are available to limit the absorption of toxic OA.     

Evaluation of Function and Features of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells for Analyzing Peptide Drug Intestinal Absorption Profiles

Caco-2 cell monolayers are widely employed as an in vitro model of the intestinal barrier, capable of accurately predicting the absorption of conventional small-molecule drugs. However, this model may not be applicable to all drugs, and the accuracy of absorption prediction is typically poor for high molecular weight drugs. Recently, human induced pluripotent stem (iPS) cell-derived small intestinal epithelial cells (hiPSC-SIECs), exhibiting properties similar to those of the small intestine when compared with Caco-2 cells, have been developed and are considered a novel candidate model for in vitro evaluation of intestinal drug permeability. Therefore, we evaluated the utility of human hiPSC-SIECs as a new in vitro model to predict the intestinal absorption of middle-molecular weight drugs and peptide drugs. Firstly, we showed that the hiPSC-SIEC monolayer allowed faster transport of peptide drugs (insulin and glucagon-like peptide-1) than the Caco- 2 cell monolayer. Second, we revealed that hiPSC-SIECs require divalent cations (Mg²⁺ and Ca²⁺) to maintain barrier integrity. Third, we demonstrated that experimental conditions established for Caco-2 cells are not persistently applicable to hiPSC-SICEs when analyzing absorption enhancers. Comprehensively clarifying the features of hiPSC-SICEs is essential to establish a new in vitro evaluation model.     

Active secretion and enterocytic drug metabolism barriers to drug absorption

Intestinal phase I metabolism and active extrusion of absorbed drug have only recently been recognized as major determinants of oral drug bioavailability. Both CYP3A4, the major phase I drug metabolizing enzyme in humans, and the multidrug efflux pump, P-glycoprotein (P-gp), are present at high levels in the villus enterocytes of the small intestine, the primary site of absorption for orally administered drugs. Moreover, these proteins are induced by many of the same compounds and demonstrate a broad overlap in substrate and inhibitor specificities, suggesting that they act as a concerted barrier to drug absorption. Clinical studies have demonstrated that inhibition of CYP3A4-mediated intestinal metabolism can significantly improve the oral bioavailability of a wide range of drugs. Intestinal P-gp is a major route of elimination for both orally and intravenously administered anticancer drugs in animal models, and experiments with the Caco-2 cell line have provided strong evidence that inhibition of intestinal P-gp is another means by which oral drug bioavailability could be enhanced.     

The Caco-2 cell monolayer: usefulness and limitations

BACKGROUND: The Caco-2 monolayer has been used extensively for the high-throughput screening of drug permeability and identification of substrates, inhibitors, and inducers of intestinal transporters, especially P-glycoprotein (P-gp). Traditionally, the Caco-2 monolayer is viewed as a single barrier rather than a polarized cell monolayer consisting of metabolic enzymes that are sandwiched between two membrane barriers with distinctly different transporters. OBJECTIVE: This review addressed the usefulness and limitations of the Caco-2 cell monolayer in drug discovery and mechanistic studies. METHODS: This mini-review covered applications of the Caco-2 monolayer, clarified misconceptions, and critically addressed issues on data interpretation. CONCLUSION: The catenary model extends the usefulness of Caco-2 monolayer and provides proper mechanistic insight and data interpretation.     

Experimental Basis for the High Oral Toxicity of Dinophysistoxin 1: A Comparative Study of DSP

Okadaic acid (OA) and its analogues, dinophysistoxin 1 (DTX1) and dinophysistoxin 2 (DTX2), are lipophilic and heat-stable marine toxins produced by dinoflagellates, which can accumulate in filter-feeding bivalves. These toxins cause diarrheic shellfish poisoning (DSP) in humans shortly after the ingestion of contaminated seafood. Studies carried out in mice indicated that DSP poisonous are toxic towards experimental animals with a lethal oral dose 2–10 times higher than the intraperitoneal (i.p.) lethal dose. The focus of this work was to study the absorption of OA, DTX1 and DTX2 through the human gut barrier using differentiated Caco-2 cells. Furthermore, we compared cytotoxicity parameters. Our data revealed that cellular viability was not compromised by toxin concentrations up to 1 μM for 72 h. Okadaic acid and DTX2 induced no significant damage; nevertheless, DTX1 was able to disrupt the integrity of Caco-2 monolayers at concentrations above 50 nM. In addition, confocal microscopy imaging confirmed that the tight-junction protein, occludin, was affected by DTX1. Permeability assays revealed that only DTX1 was able to significantly cross the intestinal epithelium at concentrations above 100 nM. These data suggest a higher oral toxicity of DTX1 compared to OA and DTX2.     

Protective effects of lactoferrin against intestinal mucosal damage induced by lipopolysaccharide in human intestinal Caco-2 cells

Indirect evidence suggests that lactoferrin (Lf), a major iron-binding protein in human milk, induces enterocyte growth and proliferation, depending on its concentration and affects the function and permeability of the intestinal mucosa. The bacterial endotoxin (lipopolysaccharide, LPS) is known to cause mucosal hyperpermeability in vivo. However, protective effects of Lf against LPS-mediated intestinal mucosal damage and barrier function in epithelial cells are not yet fully clarified. The aim of this study was to investigate whether Lf can reduce the cellular injury and alter epithelial hyperpermeability caused by LPS in human intestinal Caco-2 cells. When cell viability was measured by a WST-1 assay (tetrazolium salt-based assay), the protective effects against LPS-induced damage to Caco-2 cells were observed at doses of 800 and 1000 microg/ml Lf. The barrier function of Caco-2 monolayer tight junctions was assessed by measuring transepithelial electrical resistance (TEER) and permeability of FITC-labeled dextran 4000 (FD-4). The treatment of Caco-2 cells with Lf at doses of 400 and 1000 microg/ml significantly increased TEER as compared to treatment with LPS alone for 2 h (p<0.05). Further, at doses of 400 and 1000 microg/ml, Lf inhibited the enhancement of LPS-mediated permeability in Caco-2 cell monolayer. The results of this study suggest that Lf may have protective effects against LPS-mediated intestinal mucosal damage and impairment of barrier function in intestinal epithelial cells.     

Astragaloside IV attenuates sepsis-induced intestinal barrier dysfunction via suppressing RhoA/NLRP3 inflammasome signaling

Intestinal barrier dysfunction is a trigger for sepsis progression. NLRP3 inflammasome and RhoA contribute to sepsis and intestinal inflammation. The current study aimed to explore the effects of Astragaloside IV (AS-IV), a bioactive compound from Astragalus membranaceus, on sepsis-caused intestinal barrier dysfunction and whether NLRP3 inflammasome and RhoA are involved. Septic mice modeled by cecal ligation and puncture (CLP) operation were administered with 3 mg/kg AS-IV intravenously. AS-IV decreased mortality, cytokines release, I-FABP secretion, intestinal histological score and barrier permeability, and increased tight junction (TJ) expression in intestine in CLP model. Also, in Caco-2 cells subjected to lipopolysaccharide (LPS), 200 μg/mL AS-IV co-incubation reduced cytokines levels and enhanced in vitro gut barrier function without cytotoxicity. Subsequently, NLRP3 inflammasome and RhoA were highly activated both in intestinal tissue in vivo and in Caco-2 cells in vitro, both of which were significantly suppressed by AS-IV treatment. In addition, the benefits of AS-IV on Caco-2 monolayer barrier were largely counteracted by RhoA agonist CN03 and NLRP3 gene overexpression, respectively. Furthermore, LPS-induced NLRP3 inflammasome activation was abrogated by RhoA inhibitor C3 exoenzyme. However, NLRP3 knockdown by siRNA hardly affected RhoA activation in Caco-2 cells. These data suggest that AS-IV protects intestinal epithelium from sepsis-induced barrier dysfunction via inhibiting RhoA/NLRP3 inflammasome signal pathway.     

DNA breaks and cell cycle arrest induced by okadaic acid in Caco-2 cells, a human colonic epithelial cell line

Okadaic acid (OA) is a shellfish toxin produced by dinoflagellates, in mussels. It is a potent tumour promoter and represents a potential threat to human health even at low concentrations. OA targets mainly the gastrointestinal tract in acute poisoning, causing diarrhoea. Therefore the present investigations were designed to study the ability of okadaic acid to induce cytotoxicity and DNA lesions in a human colonic cell line (Caco-2). Incubation of Caco-2 cells with OA (3.75-60 ng/ml, i.e. 4.6 x 10(-3)-7.5 x 10(-2) microM) causes a significant reduction in cell viability. Moreover, okadaic acid inhibits protein and DNA synthesis with, respectively, IC50 of 16 and 6.5 ng/ml after 24 h incubation. It also provokes cell cycle arrest, characterised by an increase in the number of S phase cells, correlated with a significant decrease in G0/G1 phase cells at high concentration. One of the main results obtained in these investigations is the apoptosis induced by OA in Caco-2 cells of intestinal origin, shown by DNA laddering in agarose gel electrophoresis (250-1000 base pairs). OA also induces clastogenic effects evaluated by DNA fragmentation analysis using the method of Higuchi and Aggarwal (52% for 60 ng/ml) and comet assay (increase of the frequency of comets and their tails length). Therefore, the cell death induced by OA seems clearly to be concentration-dependent after 24 h of incubation. The cytotoxic properties of okadaic acid and its ability to damage DNA result in cell death, mainly by apoptosis. Since consumption of shellfish contaminated with acceptable okadaic acid concentrations exposes colonic cells to harmful concentrations of this toxin, the possibility that OA would display its toxic effects on intestinal cells in vivo should be evaluated in human primary intestinal cells and human intestinal slices for cytotoxic effects, DNA fragmentation and apoptosis.     

3D organoids derived from the small intestine: An emerging tool for drug transport research

Small intestine in vitro models play a crucial role in drug transport research. Although conventional 2D cell culture models, such as Caco-2 monolayer, possess many advantages, they should be interpreted with caution because they have relatively poor physiologically reproducible phenotypes and functions. With the development of 3D culture technology, pluripotent stem cells (PSCs) and adult somatic stem cells (ASCs) show remarkable self-organization characteristics, which leads to the development of intestinal organoids. Based on previous studies, this paper reviews the application of intestinal 3D organoids in drug transport mediated by P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2). The advantages and limitations of this model are also discussed. Although there are still many challenges, intestinal 3D organoid model has the potential to be an excellent tool for drug transport research.     

The effect of oxidized cholesterol on barrier functions and IL-10 mRNA expression in human intestinal epithelium co-cultured with dendritic cells in the transwell system

The intestinal epithelium is exposed to oxygenated cholesterol products present in foodstuffs. In vitro studies demonstrate the effect of oxysterols on cytokine release by intestinal cells cultured alone. However, physiologically, the response of the intestinal epithelium to external agents occurs in the presence of dendritic cells (DCs). The aim of the study was to analyze the effect of 7-ketocholesterol on the barrier functions and IL-10 mRNA expression of Caco-2 cells in the presence of DCs, and secondly, on IL-10 mRNA expression in DCs. Caco-2 cells were co-cultured with monocyte-derived dendritic cells and induced with 7-ketocholesterol in a transwell system. DCs did not affect the transepithelial electrical resistance (TER) of the Caco-2 cell monolayer, but increased IL-10 mRNA expression in Caco-2 cells. 7-ketocholesterol decreased the TER of Caco-2 cells co-cultured with DCs and diminished IL-10 mRNA expression in Caco-2 cells induced by the presence of DCs. IL-10 mRNA expression fell in DCs co-cultured with Caco-2 cells after treatment with 7-ketocholesterol. Oxidized cholesterols present in gut mucosa may contribute to the decrease of epithelial barrier functions and the inappropriate development of an inflammatory response to food compounds.     

The food contaminant deoxynivalenol, decreases intestinal barrier permeability and reduces claudin expression

'The gastrointestinal tract represents the first barrier against food contaminants as well as the first target for these toxicants. Deoxynivalenol (DON) is a mycotoxin that commonly contaminates cereals and causes various toxicological effects. Through consumption of contaminated cereals and cereal products, human and pigs are exposed to this mycotoxin. Using in vitro, ex vivo and in vivo approaches, we investigated the effects of DON on the intestinal epithelium. We demonstrated that, in intestinal epithelial cell lines from porcine (IPEC-1) or human (Caco-2) origin, DON decreases trans-epithelial electrical resistance (TEER) and increases in a time and dose-dependent manner the paracellular permeability to 4 kDa dextran and to pathogenic Escherichia coli across intestinal cell monolayers. In pig explants treated with DON, we also observed an increased permeability of intestinal tissue. These alterations of barrier function were associated with a specific reduction in the expression of claudins, which was also seen in vivo in the jejunum of piglets exposed to DON-contaminated feed. In conclusion, DON alters claudin expression and decreases the barrier function of the intestinal epithelium. Considering that high levels of DON may be present in food or feed, consumption of DON-contaminated food/feed may induce intestinal damage and has consequences for human and animal health.     

LC-MS测定雷公藤甲素及其在Caco-2细胞上的转运特征

目的：建立体外模拟体内肠道细胞的Caco-2细胞Transwell模型,以此研究雷公藤甲素在Caco-2细胞模型上的跨膜转运特征。方法：采用聚酯碳酸酯膜连续培养Caco-2细胞21天,形成致密的单层细胞模型。然后对影响雷公藤甲素在Caco-2细胞模型上转运特征的因素包括浓度、时间及跨膜转运蛋白（P-糖蛋白,多药耐药蛋白,乳腺癌耐药蛋白）进行考察;同时采用LC-MS对溶液中的雷公藤甲素的含量进行测定。结果：雷公藤甲素主要以主动转运的方式进行吸收,且随着时间和药物浓度的增加,转运量明显增加。结论：雷公藤甲素在Caco-2细胞上转运存在一定的浓度及时间依赖性,且P-gp介导雷公藤甲素在Caco-2细胞上转运。     

Effects of Pluronic P85 Unimers and Micelles on Drug Permeability in Polarized BBMEC and Caco-2 Cells

Purpose. Using polarized bovine brain microvessel endothelial cells (BBMEC) monolayers as in vitro model of the blood brain barrier and Caco-2 monolayers as a model of the intestinal epithelium, the present work investigates the effects of Pluronic P85 block copolymer (P85) on the transport of the P-gycoprotein (P-gp)- dependent probe, rhodamine 123 (R123). Methods. The permeability and cell efflux studies are performed with the confluent cell monolayers using Side-Bi-Side diffusion cells. Results. At concentrations below the critical micelle concentration, P85 inhibits P-gp efflux systems of the BBMEC and Caco-2 cell monolayers resulting in an increase in the apical to basolateral permeability of R123. In contrast, at high concentrations of P85 the drug incorporates into the micelles, enters the cells and is then recycled back out to the apical side resulting in decrease in Rl 23 transport across the cell monolayers. Apical to basolateral permeability of micelle-incorporated R123 in BBMEC monolayers was increased by prior conjugation of P85 with insulin, suggesting that modified micelles undergo receptor-mediated transcytosis. Conclusions. Pluronic block copolymers can increase membrane transport and transcellular permeability in brain microvessel endothelial cells and intestinal epithelium cells. This suggests that these block copolymers may be useful in designing formulations to increase brain and oral absorption of select drugs.     

Effect of benzo[a]pyrene on P-glycoprotein-mediated transport in Caco-2 cell monolayer

The main exposure pathway of benzo[a]pyrene (Bap) for humans is considered to be via the daily diet. The purpose of this study was to investigate the effect of BaP on the intestinal transport of chemicals mediated by P-glycoprotein (P-gp). The intestinal epithelial membrane transport of rhodamine-123 (Rho-123), a substrate of P-gp, was examined using a monolayer of the human Caco-2 cell line grown in transwells. In the monolayer exposed to Bap for 72 h before transport experiments, the ratio of the apparent permeability coefficients (P(app)) of Rho-123 efflux increased compared to that of the control. The permeability of rhodamine-B (Rho-B), not a substrate of P-gp, showed no difference between the monolayers. Treatment with quinidine or cyclosporine A, which are P-gp inhibitors, decreased the P(app) of Rho-123 to the same degree in both monolayers. The transport of Rho-123 was not influenced by the presence of Bap. Thus, Bap seemed not to act directly on the efflux activity of P-gp and be a binding site competitor of Rho-123. In the Caco-2 cells that enhanced the efflux of Rho-123 by the treatment with Bap, an increase in mRNA expression of MDR 1 (P-gp) was confirmed compared to that of control by RT-PCR. Furthermore, Western blot analysis using a monoclonal antibody, C219, demonstrated the increase of P-gp in Caco-2 cells exposed to Bap, compared with controls. It was inferred that Bap exposure induced the expression of P-gp, which led to the observed increase in efflux transport of Rho-123. The possibility was suggested that Bap might affect the disposition of medicines by increasing P-gp expression.     

L-1416, a novel MDR reversing agent with possible reduced calcium antagonism

BackgroundMultidrug efflux transporter P-glycoprotein (P-gp) is highly expressed on membrane of tumor cells and supposed to be implicated in the resistance to tumor chemotherapy. However, currently none of P-gp inhibitors has been approved by Food and Drug Administration not only due to toxicity but also lack of efficacy in clinical trials.MethodsTo solve the problem, our lab synthesized a novel compound named 1416 [1-(2,6-dimethylphenoxy)-3,4-dimethoxyphenylethylamino) propane hydrochloride] with the hope of high P-gp inhibition and low side effects. Caco-2 cell monolayer and tumor bearing mice were used to evaluate the P-gp inhibition of 1416 in vitro and in vivo, respectively. One of its potential side effects, calcium antagonism was also evaluated.ResultsResults showed that 1416 showed a similar P-gp inhibition as verapamil in Caco-2 cell monolayer. No significant difference was observed in antitumor enhancement when the optical isomers of 1416 (D-1416 and L-1416) were co-administered with vinblastine. In calcium antagonism, L-1416 showed less calcium inhibition than both D-1416 and verapamil.ConclusionThe novel compound 1416 could significantly increase the antitumor effects of cytotoxic drugs and one of its optical isomers, L-1416, might be more promising due to its potential low calcium antagonism.     

Effects of budesonide on P-glycoprotein expression in intestinal cell lines

BACKGROUND AND PURPOSE: P-glycoprotein (P-gp) is an important efflux transporter that supports the barrier function of the gut against invading antigens and against administered drugs. Since glucocorticoids, such as budesonide, are frequently used during inflammatory bowel disease we investigated how budesonide influences P-gp expression in different intestinal cell lines. EXPERIMENTAL APPROACH: LS180 and Caco-2 cells were incubated with budesonide and changes in P-gp expression were determined on mRNA, protein and functional level. The mRNA expression levels of glucocorticoid receptor (GR) and pregnane X receptor (PXR) were determined in these cell lines. PXR receptor was transiently transfected into Caco-2 cells. KEY RESULTS: Budesonide showed an induction of P-gp in LS180 cells and a down-regulation in Caco-2 cells. Expression levels of nuclear receptors revealed high expression of PXR only in LS180 cells and exclusive expression of GR in Caco-2 cells. Mifepristone, an anti-glucocorticoid, could not reverse the down-regulation of P-gp by budesonide in Caco-2 cells. In PXR-transfected Caco-2 cells the budesonide-mediated down-regulation of P-gp was abolished. Furthermore the expression of cytochrome P450 3A4 (CYP3A4), another PXR target gene, was induced in PXR-transfected Caco-2 cells after budesonide treatment. CONCLUSIONS AND IMPLICATIONS: Budesonide has the potential to influence MDR1 expression in vitro. In LS180 cells, the induction of MDR1 by budesonide probably is mediated via PXR. The mechanism of the down-regulation in Caco-2 cells still remains unclear, but GR does not seem to be involved. Further studies are required to evaluate how budesonide alters P-gp expression in vivo.     

In vitro metabolism and transport of the new dipeptidyl peptidase 4 inhibitors,KR66222 and KR66223

1. We investigated the in vitro metabolism and transport of KR66222 and KR66223, new inhibitors of dipeptidyl peptidase (DPP) 4, using human liver microsomes (HLMs) and a Caco-2 cell monolayer.

2. Human liver microsomal incubation of KR66222 in the presence of the NADPH-generating system resulted in the formation of two metabolites, identified as S-oxidation (KR68334) and hydrolysis (KR66223) products using liquid chromatography/tandem mass spectrometry. The formation of KR66223 via an esterase and the formation of KR68334 via CYP3A5 and CYP3A4 seem to be major factors in the in vitro metabolism of KR66222 using HLMs. Additionally, KR66222 had a significantly greater basal to apical transport rate (2.5-fold) than apical to basal transport in the Caco-2 cell monolayer, suggesting the involvement of an efflux transport system. Further studies using inhibitors of efflux transporters and P-glycoprotein (P-gp) overexpressed cells revealed that P-gp was involved in the basal to apical transport of KR66222. These findings suggest that KR66222 undergoes a significant first pass effect, which may serve to decrease the bioavailability of KR66222.

3. The active metabolite, KR66223, was stable for 1?h at 37°C in pooled HLMs (98.9?±?2.6% of control) and did not undergo P-gp-mediated efflux in Caco-2 cells. Apparent permeability of KR66223 (4.96?×?10^?6 cm/s) was comparable to that of KR66222 (4.08?×?10^?6 cm/s).

4. In conclusion, considering pharmacokinetic variability and the intestinal first-pass effect caused by the involvement of CYP3A and P-gp, KR66223 seems to have better in vitro metabolism and permeability characteristics than KR66222.

     

Drug Discovery and Regulatory Considerations for Improving In Silico and In Vitro Predictions that Use Caco-2 as a Surrogate for Human Intestinal Permeability Measurements

There is a growing need for highly accurate in silico and in vitro predictive models to facilitate drug discovery and development. Results from in vitro permeation studies across the Caco-2 cell monolayer are commonly used for drug permeability screening in industry and are also accepted as a surrogate for human intestinal permeability measurements by the US FDA to support new drug applications. Countless studies carried out in this cell line with published permeability measurements have enabled the development of many in silico prediction models. We identify several common cases that illustrate how using Caco-2 permeability measurements in these in silico and in vitro predictive models will not correlate with human intestinal permeability and will further lead to inaccuracies in these models. We provide guidelines and recommendations for improving these models to more accurately predict clinically relevant information, thereby enhancing the drug discovery, development, and regulatory approval processes.     

Effect of structural modification of α-aminoxy peptides on their intestinal absorption and transport mechanism

A representative α-aminoxy peptide 1 has been demonstrated to have a potential for the treatment of human diseases associated with Cl(-) channel dysfunctions. However, its poor intestinal absorption was determined. The purpose of this study was to delineate the transport mechanism responsible for its poor absorption and also to prepare peptide analogues by structural modifications of 1 at its isobutyl side chains without changing the α-aminoxy core for retaining biological activity to improve the intestinal absorption. The poor intestinal absorption of 1 was proved to be due to the P-glycoprotein (P-gp) mediated efflux transport in Caco-2 cell monolayer, intestinal segments in Ussing chamber and rat single pass intestinal perfusion models. Four analogues with propionic acid (2), butanamine (3), methyl (4) and hydroxymethyl side chains (5) were synthesized and tested using the same models. Except for the permeability of 2, the absorbable permeability of the modified peptides in Caco-2 cell monolayer and their intestinal absorption in rats were significantly improved to 7-fold (3), 4-fold (4), 11-fold (5) and 36-fold (2), 42-fold (3), 55-fold (4), 102-fold (5), respectively, compared with 1 (P(app), 0.034 ± 0.003 × 10(-6) cm/s; P(blood), 1.61 ± 0.807 × 10(-6) cm/s). More interestingly, the structural modification remarkably altered transport mechanism of the peptides, leading to the conversion of the active transport via P-gp mediation (1, 2), to MRP mediation (3), MRP plus BCRP mediation (4) or a passive diffusion (5). Furthermore, P-gp mediated efflux transport of 1 and 2 was demonstrated to not alter the P-gp expression, while 1 but not 2 exhibited uncompetitive inhibitory effect on P-gp ATPase. The results demonstrated that intestinal absorption and transport mechanism of the α-aminoxy peptides varied significantly with different structures, and their absorption can be dramatically improved by structural modifications, which allow us to further design and prepare better α-aminoxy peptide candidates with appropriate pharmacokinetic fates, including intestinal absorption, for potential clinical use.     

Development of Caco-2 cells co-expressing CYP3A4 and NADPH-cytochrome P450 reductase using a human artificial chromosome for the prediction of intestinal extraction ratio of CYP3A4 substrates

The Caco-2 cells co-expressing cytochrome P450 (CYP) 3A4 and NADPH-cytochrome P450 reductase (CPR) were developed using a human artificial chromosome (HAC) vector. The CYP3A4 and CPR genes were cloned into the HAC vector in CHO cells using the Cre-loxP system, and the microcell-mediated chromosome transfer technique was used to transfer the CYP3A4-CPR-HAC vector to Caco-2 cells. After seeding onto semipermeable culture inserts, the CYP3A4-CPR-HAC/Caco-2 cells were found to form tight monolayers, similar to the parental cells, as demonstrated by the high transepithelial electrical resistance (TEER) value and comparable permeability of non-CYP3A4 substrates between parent and CYP3A4-CPR-HAC/Caco-2 cell monolayers. The metabolic activity of CYP3A4 (midazolam 1′-hydroxylase activity) in the CYP3A4-CPR-HAC/Caco-2 cells was constant from 22 to 35 passages, indicating that HAC vectors conferred sufficient and sustained CYP3A4 activity to CYP3A4-CPR-HAC/Caco-2 cells. The strong relationship between the metabolic extraction ratios (ER) obtained from the CYP3A4-CPR-HAC/Caco-2 cells and calculated intestinal extraction ratios in humans (Eg) from reported intestinal availability (Fg) was found for 17 substrates of CYP3A4 (r² = 0.84). The present study suggests that the CYP3A4-CPR-HAC/Caco-2 cell monolayer can serve as an in vitro tool that facilitates the prediction of intestinal extraction ratio (or availability) in humans.