The development of the dog heartworm is highly sensitive to sterols which activate the orthologue of the nuclear receptor DAF-12.

Prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections.

The transcription factor NHR-8: A new target to increase ivermectin efficacy in nematodes.

Resistance to the anthelmintic macrocyclic lactone ivermectin (IVM) has a great impact on the control of parasitic nematodes. The mechanisms by which nematodes adapt to IVM remain to be deciphered. We have identified NHR-8, a nuclear hormone receptor involved in the xenobiotic response in Caenorhabditis elegans, as a new regulator of tolerance to IVM. Loss-of-function nhr-8(ok186) C. elegans mutants subjected to larval development assays and electropharyngeogram measurements, displayed hypersensitivity to IVM, and silencing of nhr-8 in IVM-resistant worms increased IVM efficacy. In addition, compared to wild-type worms, nhr-8 mutants under IVM selection pressure failed to acquire tolerance to the drug. In addition, IVM-hypersensitive nhr-8(ok186) worms displayed low transcript levels of several genes from the xenobiotic detoxification network and a concomitant low Pgp-mediated drug efflux activity. Interestingly, some pgp and cyp genes known to impact IVM tolerance in many nematode species, were down regulated in nhr-8 mutants and inversely upregulated in IVM-resistant worms. Moreover, pgp-6 overexpression in nhr-8(ok186) C. elegans increased tolerance to IVM. Importantly, NHR-8 function was rescued in nhr-8(ok186) C. elegans with the homolog of the parasitic nematode Haemonchus contortus, and silencing of Hco-nhr-8 by RNAi on L2 H. contortus larvae increased IVM susceptibility in both susceptible and resistant H. contortus isolates. Thus, our data show that NHR-8 controls the tolerance and development of resistance to IVM in C. elegans and the molecular basis for this relates to the NHR-8-mediated upregulation of IVM detoxification genes. Since our results show that Hco-nhr-8 functions similarly to Cel-nhr-8, this study helps to better understand mechanisms underlying failure in drug efficacy and open perspectives in finding new compounds with NHR-8 antagonist activity to potentiate IVM efficacy.

Acquired Tolerance to Ivermectin and Moxidectin after Drug Selection Pressure in the Nematode Caenorhabditis elegans.

Ivermectin and moxidectin are the most widely administered anthelmintic macrocyclic lactones (MLs) to treat human and animal nematode infections. Their widespread and frequent use has led to a high level of resistance to these drugs. Although they have the same mode of action, differences in terms of selection for drug resistance have been reported. Our objective was to study and compare changes occurring upon ivermectin or moxidectin selection in the model nematode Caenorhabditis elegans C. elegans worms were submitted to stepwise exposure to increasing doses of moxidectin. The sensitivity of moxidectin-selected worms to MLs was determined in a larval development assay and compared with those of wild-type and ivermectin-selected strains. Selection with either ivermectin or moxidectin led to acquired tolerance to ivermectin, moxidectin, and eprinomectin. Importantly, moxidectin was the most potent ML in both ivermectin- and moxidectin-selected strains. Interestingly, this order of potency was also observed in a resistant Haemonchus contortus isolate. In addition, ivermectin- and moxidectin-selected strains displayed constitutive overexpression of several genes involved in xenobiotic metabolism and transport. Moreover, verapamil potentiated sensitivity to ivermectin and moxidectin, demonstrating that ABC transporters play a role in ML sensitivity in ML-selected C. elegans strains. Finally, both ivermectin- and moxidectin-selected strains displayed a dye-filling-defective phenotype. Overall, this work demonstrated that selection with ivermectin or moxidectin led to cross-resistance to several MLs in nematodes and that the induction of detoxification systems and defects in the integrity of amphidial neurons are two mechanisms that appear to affect the responsiveness of worms to both ivermectin and moxidectin.

Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions.

Anthelmintic resistance has a great impact on livestock production systems worldwide, is an emerging concern in companion animal medicine, and represents a threat to our ongoing ability to control human soil-transmitted helminths. The Consortium for Anthelmintic Resistance and Susceptibility (CARS) provides a forum for scientists to meet and discuss the latest developments in the search for molecular markers of anthelmintic resistance. Such markers are important for detecting drug resistant worm populations, and indicating the likely impact of the resistance on drug efficacy. The molecular basis of resistance is also important for understanding how anthelmintics work, and how drug resistant populations arise. Changes to target receptors, drug efflux and other biological processes can be involved. This paper reports on the CARS group meeting held in August 2013 in Perth, Australia. The latest knowledge on the development of molecular markers for resistance to each of the principal classes of anthelmintics is reviewed. The molecular basis of resistance is best understood for the benzimidazole group of compounds, and we examine recent work to translate this knowledge into useful diagnostics for field use. We examine recent candidate-gene and whole-genome approaches to understanding anthelmintic resistance and identify markers. We also look at drug transporters in terms of providing both useful markers for resistance, as well as opportunities to overcome resistance through the targeting of the transporters themselves with inhibitors. Finally, we describe the tools available for the application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance.

Ivermectin exposure leads to up-regulation of detoxification genes in vitro and in vivo in mice.

The biodisposition of the antiparasitic drug ivermectin in host and parasite is decisive for its efficacy and strongly depends on the efflux by ATP-Binding Cassette (ABC) transporters and on its biotransformation by cytochromes P450. The purpose of this study was to evaluate, in vitro and in vivo, the ivermectin ability in modulating the expression of the most important genes involved in drug detoxification. Gene expression of ABC transporters and cytochromes was evaluated by RT-qPCR in murine hepatic and intestinal cell lines exposed to increasing ivermectin doses, and in liver and intestine of mice orally administered with single or repeated therapeutic doses of ivermectin (0.2 mg/kg). Plasma, brain, liver and intestinal concentrations of ivermectin and its main metabolite were measured by HPLC in ivermectin-treated mice. In hepatocyte cell line, ivermectin up-regulated expression of Abcb1a, Abcb1b, Abcc2, Cyp1a1, Cyp1a2, Cyp2b10; while Abcb1a, Abcb1b, Abcg2, Cyp1a1, Cyp1a2, Cyp2b10 and Cyp3a11 levels were induced in intestinal cell line. In mice, repeated administration of ivermectin induced the expression of Abcb1a, Abcc2, Cyp1a1 and Cyp2b10 in intestine while only Cyp3a11 was induced in liver. Compared with single administration, repeated ivermectin administration lowered plasma, liver and intestine drug concentration, while increasing main metabolite content in plasma and intestine. These findings can be regarded as a warning that repeated ivermectin exposure is able to induce detoxification systems in mammals that may lead to subtherapeutic drug concentration. This may also be an important consideration in the assessment of drug-drug interaction and toxicity for other ABC transporters and CYP450s substrates.

Relative neurotoxicity of ivermectin and moxidectin in Mdr1ab (-/-) mice and effects on mammalian GABA(A) channel activity.

The anthelmintics ivermectin (IVM) and moxidectin (MOX) display differences in toxicity in several host species. Entrance into the brain is restricted by the P-glycoprotein (P-gp) efflux transporter, while toxicity is mediated through the brain GABA(A) receptors. This study compared the toxicity of IVM and MOX in vivo and their interaction with GABA(A) receptors in vitro. Drug toxicity was assessed in Mdr1ab(-/-) mice P-gp-deficient after subcutaneous administration of increasing doses (0.11-2.0 and 0.23-12.9 µmol/kg for IVM and MOX in P-gp-deficient mice and half lethal doses (LD(50)) in wild-type mice). Survival was evaluated over 14-days. In Mdr1ab(-/-) mice, LD(50) was 0.46 and 2.3 µmol/kg for IVM and MOX, respectively, demonstrating that MOX was less toxic than IVM. In P-gp-deficient mice, MOX had a lower brain-to-plasma concentration ratio and entered into the brain more slowly than IVM. The brain sublethal drug concentrations determined after administration of doses close to LD(50) were, in Mdr1ab(-/-) and wild-type mice, respectively, 270 and 210 pmol/g for IVM and 830 and 740-1380 pmol/g for MOX, indicating that higher brain concentrations are required for MOX toxicity than IVM. In rat α1ß2γ2 GABA channels expressed in Xenopus oocytes, IVM and MOX were both allosteric activators of the GABA-induced response. The Hill coefficient was 1.52±0.45 for IVM and 0.34±0.56 for MOX (p<0.001), while the maximum potentiation caused by IVM and MOX relative to GABA alone was 413.7±66.1 and 257.4±40.6%, respectively (p<0.05), showing that IVM causes a greater potentiation of GABA action on this receptor. Differences in the accumulation of IVM and MOX in the brain and in the interaction of IVM and MOX with GABA(A) receptors account for differences in neurotoxicity seen in intact and Mdr1-deficient animals. These differences in neurotoxicity of IVM and MOX are important in considering their use in humans.

P-glycoproteins and other multidrug resistance transporters in the pharmacology of anthelmintics: Prospects for reversing transport-dependent anthelmintic resistance.

Parasitic helminths cause significant disease in animals and humans. In the absence of alternative treatments, anthelmintics remain the principal agents for their control. Resistance extends to the most important class of anthelmintics, the macrocyclic lactone endectocides (MLs), such as ivermectin, and presents serious problems for the livestock industries and threatens to severely limit current parasite control strategies in humans. Understanding drug resistance is important for optimizing and monitoring control, and reducing further selection for resistance. Multidrug resistance (MDR) ABC transporters have been implicated in ML resistance and contribute to resistance to a number of other anthelmintics. MDR transporters, such as P-glycoproteins, are essential for many cellular processes that require the transport of substrates across cell membranes. Being overexpressed in response to chemotherapy in tumour cells and to ML-based treatment in nematodes, they lead to therapy failure by decreasing drug concentration at the target. Several anthelmintics are inhibitors of these efflux pumps and appropriate combinations can result in higher treatment efficacy against parasites and reversal of resistance. However, this needs to be balanced against possible increased toxicity to the host, or the components of the combination selecting on the same genes involved in the resistance. Increased efficacy could result from modifying anthelmintic pharmacokinetics in the host or by blocking parasite transporters involved in resistance. Combination of anthelmintics can be beneficial for delaying selection for resistance. However, it should be based on knowledge of resistance mechanisms and not simply on mode of action classes, and is best started before resistance has been selected to any member of the combination. Increasing knowledge of the MDR transporters involved in anthelmintic resistance in helminths will play an important role in allowing for the identification of markers to monitor the spread of resistance and to evaluate new tools and management practices aimed at delaying its spread.

Moxidectin and the avermectins: Consanguinity but not identity.

The avermectins and milbemycins contain a common macrocyclic lactone (ML) ring, but are fermentation products of different organisms. The principal structural difference is that avermectins have sugar groups at C13 of the macrocyclic ring, whereas the milbemycins are protonated at C13. Moxidectin (MOX), belonging to the milbemycin family, has other differences, including a methoxime at C23. The avermectins and MOX have broad-spectrum activity against nematodes and arthropods. They have similar but not identical, spectral ranges of activity and some avermectins and MOX have diverse formulations for great user flexibility. The longer half-life of MOX and its safety profile, allow MOX to be used in long-acting formulations. Some important differences between MOX and avermectins in interaction with various invertebrate ligand-gated ion channels are known and could be the basis of different efficacy and safety profiles. Modelling of IVM interaction with glutamate-gated ion channels suggest different interactions will occur with MOX. Similarly, profound differences between MOX and the avermectins are seen in interactions with ABC transporters in mammals and nematodes. These differences are important for pharmacokinetics, toxicity in animals with defective transporter expression, and probable mechanisms of resistance. Resistance to the avermectins has become widespread in parasites of some hosts and MOX resistance also exists and is increasing. There is some degree of cross-resistance between the avermectins and MOX, but avermectin resistance and MOX resistance are not identical. In many cases when resistance to avermectins is noticed, MOX produces a higher efficacy and quite often is fully effective at recommended dose rates. These similarities and differences should be appreciated for optimal decisions about parasite control, delaying, managing or reversing resistances, and also for appropriate anthelmintic combination.

Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line.

Ivermectin is widely used in human and veterinary medicine for the control of helminth infections. Ivermectin is known to interact with P-glycoprotein (P-gp/MDR1), being a good substrate and a potent inhibitor, however, the influence of ivermectin on the expression of the transporter has not been investigated. Expression of P-glycoprotein was investigated in cultured mouse hepatocytes acutely exposed to ivermectin. The two P-glycoprotein murine isoforms, Mdr1a and Mdr1b, mRNA levels were assessed by real-time RT-PCR. Ivermectin induced a clear time- and concentration-dependent up-regulation of Mdr1a and Mdr1b mRNA levels (as early as a 12-h exposure and up to 2.5-fold at 10µM). Moreover, ivermectin-treated cells displayed enhanced cellular efflux of the P-glycoprotein substrate calcein that was inhibited by the P-glycoprotein blocker valspodar, providing evidence that the ivermectin-induced P-glycoprotein was functional. The mechanisms underlying these effects were investigated. Ivermectin-mediated Mdr1 mRNA induction was independent of the two nuclear receptors CAR and PXR, which are known to be involved in drug transporters regulation. Moreover, by using reporter cell lines that detects specific ligand-activated transcription factors, we showed that ivermectin did not displayed CAR, PXR or AhR ligand activities. However, studies with actinomycin D revealed that the half-life of Mdr1a and Mdr1b mRNA were significantly prolonged by two-fold in ivermectin-treated cells suggesting a post-transcriptional mode of ivermectin regulation. This study demonstrates for the first time that ivermectin induces P-glycoprotein overexpression through post-transcriptional mRNA stabilization, thus offering insight into the mechanism of reduced therapeutic efficacy and development of ivermectin-resistant parasites.

P-glycoprotein dysfunction contributes to hepatic steatosis and obesity in mice.

Although the main role of P-glycoprotein (Pgp) is to extrude a broad range of xenochemicals and to protect the organism against xenotoxicity, it also transports a large range of endogenous lipids. Using mice lacking Pgp, we have investigated the possible involvement of Pgp in lipid homeostasis in vivo. In a long term study, we have followed the food intake, body status and lipid markers in plasma and liver of wild-type and mdr1ab(-/-) mice over 35 weeks. Pgp-deficient mice showed excess weight, hypertrophy of adipose mass, high insulin and glucose levels in plasma. Some of these metabolic disruptions appeared earlier in Pgp-deficient mice fed high-fat diet. Moreover, hepatosteatosis with increased expression of genes involved in liver detoxification and in de novo lipid synthesis occurred in Pgp-deficient mice. Overall, Pgp deficiency clearly induced obesity in FVB genetic background, which is known to be resistant to diet-induced obesity. These data reinforce the finding that Pgp gene could be a contributing factor and possibly a relevant marker for lipid disorder and obesity. Subsequent to Pgp deficiency, changes in body availabilities of lipids or any Pgp substrates may affect metabolic pathways that favour the occurrence of obesity. This is of special concern because people are often facing simultaneous exposition to many xenochemicals, which inhibits Pgp, and an excess in lipid dietary intake that may contribute to the high prevalence of obesity in our occidental societies.

Interaction of anthelmintic drugs with P-glycoprotein in recombinant LLC-PK1-mdr1a cells.

Given the widespread use of formulations combining anthelmintics which are possible P-glycoprotein interfering agents, the understanding of drug interactions with efflux ABC transporters is of concern for improving anthelmintic control. We determined the ability of 14 anthelmintics from different classes to interact with abcb1a (mdr1a, P-glycoprotein, Pgp) by following the intracellular accumulation of rhodamine 123 (Rho 123), a fluorescent Pgp substrate, in LLC-PK1 cells overexpressing Pgp. The cytotoxicity of the compounds that are able to interfere with Pgp activity was evaluated in cells overexpressing Pgp and compared with parental cells using the MTS viability assay. Among all the anthelmintics used, ivermectin (IVM), triclabendazole (TCZ), triclabendazole sulfoxide (TCZ-SO), closantel (CLOS) and rafoxanide (RAF) increased the intracellular Rho 123 in Pgp overexpressing cells, while triclabendazole sulfone, albendazole, mebendazole, oxfendazole, thiabendazole, nitroxynil, levamisole, praziquantel and clorsulon failed to have any effect. The concentration needed to reach the maximal Rho 123 accumulation (E(max)) was obtained with 10 microM for IVM, 80 microM for CLOS, 40 microM for TCZ and TCZ-SO, and 80 microM for RAF. We showed that for these five drugs parental cell line was more sensitive to drug toxicity compared with Pgp recombinant cell line. Such in vitro approach constitutes a powerful tool to predict Pgp-drug interactions when formulations combining several anthelmintics are administered and may contribute to the required optimization of efficacy of anthelmintics.

Role of P-glycoprotein in the disposition of macrocyclic lactones: A comparison between ivermectin, eprinomectin, and moxidectin in mice.

Macrocyclic lactones (MLs) are lipophilic anthelmintics and substrates for P-glycoprotein (P-gp), an ATP-binding cassette transporter involved in drug efflux out of both host and parasites. To evaluate the contribution of P-gp to the in vivo kinetic disposition of MLs, the plasma kinetics, brain concentration, and intestinal excretion of three structurally different MLs (ivermectin, eprinomectin, and moxidectin) were compared in wild-type and P-gp-deficient [mdr1ab(-/-)] mice. Each drug (0.2 mg/kg) was administered orally, intravenously, or subcutaneously to the mice. Plasma, brain, and intestinal tissue concentrations were measured by high-performance liquid chromatography. The intestinal excretion rate after intravenous administration was determined at different levels of the small intestine by using an in situ intestinal perfusion model. P-gp deficiency led to a significant increase in the area under the plasma concentration-time curve (AUC) of ivermectin (1.5-fold) and eprinomectin (3.3-fold), whereas the moxidectin AUC was unchanged. Ivermectin and to a greater extent eprinomectin were both excreted by the intestine via a P-gp-dependent pathway, whereas moxidectin excretion was weaker and mostly P-gp-independent. The three drugs accumulated in the brains of the mdr1ab(-/-) mice, but eprinomectin concentrations were significantly lower. We concluded that eprinomectin disposition in mice is controlled mainly by P-gp efflux, more so than that of ivermectin, whereas moxidectin disposition appears to be mostly P-gp-independent. Given that eprinomectin and ivermectin have higher affinity for P-gp than moxidectin, these findings demonstrated that the relative affinity of MLs for P-gp could be predictive of the in vivo kinetic behavior of these drugs.

Physicochemical characterization of molecular assemblies of miltefosine and amphotericin B.

This study describes the interactions between two amphiphilic molecules with antileishmanial activity, amphotericin B (AmB) and miltefosine [hexadecylphosphocholine (HePC)], the latter being effective by the oral route. The effect of HePC on the aggregation state of AmB in aqueous solution and the interactions between the two agents were monitored using absorption spectroscopy and circular dichroism. Structural characterization of the mixed aggregates formed in water by dynamic light scattering (DLS) and cryofracture electron microscopy was performed. At concentrations above its critical micelle concentration, HePC was shown to interact with AmB, leading to an increase in the proportion of AmB in its monomeric form as a result of a micellar solubilization mechanism with a capacity of 26 +/- 3 mmol of AmB solubilized/mol of HePC, that is, nearly 40 molecules of HePC per molecule of AmB in the mixed micelles. These were revealed as individual and spherical aggregates close to 10 nm in diameter by both electron microscopy and DLS. Such a micellar formulation provides a new AmB-based system which might be useful in delivering AmB orally for visceral leishmaniasis bitherapy.

Inward translocation of the phospholipid analogue miltefosine across Caco-2 cell membranes exhibits characteristics of a carrier-mediated process.

Miltefosine (hexadecylphosphocholine, HePC) is the first effective oral agent for the treatment of visceral leishmaniasis. The characteristics of HePC incorporation into the human intestinal epithelial cell line Caco-2 were investigated in order to understand its oral absorption mechanism. The results provide evidence for the involvement of a carrier-mediated mechanism, since the association of HePC at the apical pole of Caco-2 cells was (1) saturable as a function of time with a rapid initial incorporation over 5 min followed by a more gradual increase; (2) saturable as a function of concentration over the range studied (2-200 microM) with a saturable component which followed Michaelis-Menten kinetics (apparent K (m) 15.7 micromol/L, V (max) 39.2 nmol/mg protein/h) and a nonspecific diffusion component; (3) partially inhibited by low temperature and ATP depletion, indicating the temperature and energy-dependence of the uptake process. Moreover, we demonstrated, by an albumin back-extraction method, that HePC is internalized via translocation from the outer to the inner leaflet of the plasma membrane and that HePC may preferentially diffuse through intact raft microdomains. In conclusion, our results suggest that incorporation of HePC at the apical membrane of Caco-2 cells may occur through a passive diffusion followed by a translocation in the inner membrane leaflet through an active carrier-mediated mechanism.

Intestinal absorption of miltefosine: contribution of passive paracellular transport.

PURPOSE: This study aimed to characterize the transepithelial transport of miltefosine (HePC), the first orally effective drug against visceral leishmaniasis, across the intestinal barrier to further understand its oral absorption mechanism. MATERIALS AND METHODS: Caco-2 cell monolayers were used as an in vitro model of the human intestinal barrier. The roles of active and passive mechanisms in HePC intestinal transport were investigated and the relative contributions of the transcellular and paracellular routes were estimated. RESULTS: HePC transport was observed to be pH-independent, partially temperature-dependent, linear as a function of time and non-saturable as a function of concentration. The magnitude of HePC transport was quite similar to that of the paracellular marker mannitol, and EDTA treatment led to an increase in HePC transport. Furthermore, HePC transport was found to be similar in the apical-to-basolateral and basolateral-to-apical directions, strongly suggesting that HePC exhibits non-polarized transport and that no MDR-mediated efflux was involved. CONCLUSIONS: These results demonstrate that HePC crosses the intestinal epithelium by a non-specific passive pathway and provide evidence supporting a concentration-dependent paracellular transport mechanism, although some transcellular diffusion cannot be ruled out. Considering that HePC opens epithelial tight junctions, this study shows that HePC may promote its own permeation across the intestinal barrier.

Interaction between miltefosine and amphotericin B: consequences for their activities towards intestinal epithelial cells and Leishmania donovani promastigotes in vitro.

The aim of this study was to evaluate the potential of a combination of two antileishmanial drugs, miltefosine (HePC) and amphotericin B (AMB), when administered by the oral route. Caco-2 cell monolayers were used as a validated in vitro model of the intestinal barrier and Leishmania donovani promastigotes as a model for evaluating the effect of the drug combination. Spectroscopic measurements demonstrated that HePC and AMB associate, leading to the formation of mixed aggregates in which AMB is solubilized as monomers. The incubation of the association of HePC and AMB with Caco-2 cell monolayers, at a concentration higher than 5 microM, led to (i) a reduction of the HePC-induced paracellular permeability enhancement in Caco-2 cell monolayers, (ii) an inhibition of the uptake of both drugs, and (iii) a decrease in the transepithelial transport of both drugs, suggesting that a pharmacokinetic antagonism between HePC and AMB could occur after their oral administration. However, the combination did not exhibit any antagonism or synergy in its antileishmanial activity. These results demonstrated a strong physicochemical interaction between HePC and AMB, depending on the concentration of each, which could have important consequences for their biological activities, if they are administered together.

Modulation of intestinal barrier properties by miltefosine.

Miltefosine (hexadecylphosphocholine, HePC) is the first effective oral agent for the treatment of visceral leishmaniasis. This study aimed to determine whether this oral administration alters the integrity and transport capacities of the intestinal barrier. The objectives of this study were: (i) to evaluate the cytotoxicity of HePC, (ii) to investigate the effects of HePC on paracellular and transcellular transport and (iii) to investigate the influence of HePC on three major transporters of the intestinal barrier, namely, P-glycoprotein, the human intestinal peptide transporter (PepT-1) and the monocarboxylic acid transporter (MCT-1) in Caco-2 cell monolayers, used as an in vitro model of the human intestinal barrier. We show that HePC reduced the transepithelial electrical resistance and increased D-[14C]mannitol permeability in a dose-dependent manner but had no effect on [3H]testosterone permeability, demonstrating that HePC treatment enhances paracellular permeability via an opening of the tight junction complex without affecting the transcellular route. Morphological studies using confocal fluorescence microscopy showed no perturbation of the normal distribution of ZO-1, occludin or E-cadherin but revealed a redistribution of the tight junction-associated protein claudin-1 and the perijunctional actin after incubation with HePC. Finally, HePC was found to inhibit the intestinal P-glycoprotein in the Caco-2 cell model after a single short exposure. These results suggest that HePC could modify the oral bioavailability of other therapeutic compounds absorbed via the paracellular route or which are substrates of the intestinal P-glycoprotein.