Jacques Dupuy

Enhancement of moxidectin bioavailability in lamb by a natural flavonoid: quercetin

Moxidectin is an antiparasitic drug widely used in cattle, sheep and companion animals. Due to the involvement of P-glycoprotein (P-gp) and cytochrome P450 3A in the metabolism of moxidectin, we studied the influence of various P-gp interfering agents (ivermectin, quercetin and ketoconazole) on the metabolism of 14C moxidectin in cultured rat hepatocytes over 72 h. This in vitro study allowed selection of compounds which are able to increase the moxidectin bioavailability in lambs. From this, the modulation of moxidectin pharmacokinetics in plasma of lambs was studied after co-administration of 0.2 mg kg(-1) moxidectin (subcutaneously (SC)) and 0.2 mg kg(-1) ivermectin (SC), or 10 mg kg(-1) quercetin (SC), or 10 mg kg(-1) ketoconazole (orally). Ivermectin and quercetin increased significantly the quantity of 14C moxidectin in the rat hepatocytes. Ketoconazole co-administration led to a higher concentration of moxidectin in the rat hepatocytes. In vivo, only quercetin was able to modify the pharmacokinetics of moxidectin in plasma of lambs by increasing significantly the area under the plasma concentration-time curve. This study allowed the use of a natural agent, quercetin, to improve the bioavailability of moxidectin.

Eprinomectin in dairy goats : dose influence on plasma levels and excretion in milk

Abstract The plasma levels and milk excretion of eprinomectin were determined in goats following topical application at doses of 0.5 mg kg−1 and 1.0 mg kg−1. The area under the concentration–time curve (AUC) was 2 times lower for 0.5 mg kg−1 (8.24 ± 3.50 ng day−1 ml−1) than for 1.0 mg kg−1 (15.68 ± 8.84 ng day−1 ml−1), suggesting that the pharmacokinetics of eprinomectin in goats is dose independent. The bioavailability of eprinomectin in lactating compared with non-lactating goats is low. This is probably due to the physiological status of dairy animals, which present a marked decrease in body fat. Comparison of the eprinomectin concentrations in the milk and plasma demonstrated a parallel disposition of the drug with a milk-to-plasma ratio of 0.10–0.25. The amount of drug recovered in the milk was 0.3–0.5% of the total administered dose. In all cases, the maximum level of residue in milk remained below the maximum acceptable level of 30 ng ml−1 permitted in lactating cattle.

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.

Food-grade TiO 2 impairs intestinal and systemic immune homeostasis, initiates preneoplastic lesions and promotes aberrant crypt development in the rat colon

Food-grade titanium dioxide (TiO2) containing a nanoscale particle fraction (TiO2-NPs) is approved as a white pigment (E171 in Europe) in common foodstuffs, including confectionary. There are growing concerns that daily oral TiO2-NP intake is associated with an increased risk of chronic intestinal inflammation and carcinogenesis. In rats orally exposed for one week to E171 at human relevant levels, titanium was detected in the immune cells of Peyer’s patches (PP) as observed with the TiO2-NP model NM-105. Dendritic cell frequency increased in PP regardless of the TiO2 treatment, while regulatory T cells involved in dampening inflammatory responses decreased with E171 only, an effect still observed after 100 days of treatment. In all TiO2-treated rats, stimulation of immune cells isolated from PP showed a decrease in Thelper (Th)-1 IFN-γ secretion, while splenic Th1/Th17 inflammatory responses sharply increased. E171 or NM-105 for one week did not initiate intestinal inflammation, while a 100-day E171 treatment promoted colon microinflammation and initiated preneoplastic lesions while also fostering the growth of aberrant crypt foci in a chemically induced carcinogenesis model. These data should be considered for risk assessments of the susceptibility to Th17-driven autoimmune diseases and to colorectal cancer in humans exposed to TiO2 from dietary sources.

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.

Ketoconazole increases the plasma levels of ivermectin in sheep

The parasiticide ivermectin and the antifungal drug ketoconazole are drugs that interact with P-glycoprotein. We have tested the ability of ketoconazole at a clinical dose to modify the pharmacokinetics of ivermectin in sheep. Lacaune lambs were administered with a single oral dose of ivermectin alone at 0.2 mg/kg (n=5) or in combination with a daily oral dose of ketoconazole (10 mg/kg) given for 3 days before and 2 days after the ivermectin (n=5). The plasma kinetics of ivermectin and its metabolite were followed over 15 days by HPLC analysis. Co-administration of ketoconazole induced higher plasma concentrations of ivermectin, leading to a substantial increase in the overall exposure of the animals to the drug. Ketoconazole did not reduce the production of the main ivermectin metabolite but it may rather act by inhibiting P-glycoprotein, and thus increasing the absorption of ivermectin. The use of a P-gp reversing agent such as ketoconazole could be useful tool to optimize antiparasitic therapy in the face of the worldwide development of anthelmintic resistance.

Enhanced absorption of pour-on ivermectin formulation in rats by co-administration of the multidrug-resistant-reversing agent verapamil.

Abstract The effect of verapamil, a multidrug-resistance (Mdr)-reversing agent on the absorption of a pour-on formulation of ivermectin was evaluated in rats. Absorption of ivermectin was effectively enhanced (40%) by the presence of verapamil, suggesting that absorption of ivermectin involves Mdr-P-glycoprotein and that verapamil should act as a competitive inhibitor for the transport and extrusion of ivermectin by P-glycoprotein. This hypothesis is consistent with other studies describing verapamil as a blocking agent of P-glycoprotein involved in the efflux of ivermectin in a resistant strain of Haemonchus contortus.

4-Hydroxy-2(E)-nonenal metabolism differs in Apc(+/+) cells and in Apc(Min/+) cells: it may explain colon cancer promotion by heme iron.

Animal and epidemiological studies suggest that dietary heme iron would promote colorectal cancer. Oxidative properties of heme could lead to the formation of cytotoxic and genotoxic secondary lipid oxidation products, such as 4-hydroxy-2(E)-nonenal (HNE). This compound is more cytotoxic to mouse wild-type colon cells than to isogenic cells with a mutation on the adenomatous polyposis coli (APC) gene. The latter thus have a selective advantage, possibly leading to cancer promotion. This mutation is an early and frequent event in human colorectal cancer. To explain this difference, the HNE biotransformation capacities of the two cell types have been studied using radiolabeled and stable isotope-labeled HNE. Apc-mutated cells showed better biotransformation capacities than nonmutated cells did. Thiol compound conjugation capacities were higher for mutated cells, with an important advantage for the extracellular conjugation to cysteine. Both cells types were able to reduce HNE to 4-hydroxynonanal, a biotransformation pathway that has not been reported for other intestinal cells. Mutated cells showed higher capacities to oxidize 4-hydroxynonanal into 4-hydroxynonanoic acid. The mRNA expression of different enzymes involved in HNE metabolism such as aldehyde dehydrogenase 1A1, 2 and 3A1, glutathione transferase A4-4, or cystine transporter xCT was upregulated in mutated cells compared with wild-type cells. In conclusion, this study suggests that Apc-mutated cells are more efficient than wild-type cells in metabolizing HNE into thiol conjugates and 4-hydroxynonanoic acid due to the higher expression of key biotransformation enzymes. These differential biotransformation capacities would explain the differences of susceptibility between normal and Apc-mutated cells regarding secondary lipid oxidation products.

Plasma and milk kinetic of eprinomectin and moxidectin in lactating water buffaloes (Bubalus bubalis).

The pharmacokinetics and mammary excretion of moxidectin and eprinomectin were determined in water buffaloes (Bubalus bubalis) following topical administration of 0.5mgkg(-1). Following administration of moxidectin, plasma and milk concentrations of moxidectin increased to reach maximal concentrations (C(max)) of 5.46+/-3.50 and 23.76+/-16.63ngml(-1) at T(max) of 1.20+/-0.33 and 1.87+/-0.77 days in plasma and milk, respectively. The mean residence time (MRT) were similar for plasma and milk (5.27+/-0.45 and 5.87+/-0.80 days, respectively). The AUC value was 5-fold higher in milk (109.68+/-65.01ngdayml(-1)) than in plasma (23.66+/-12.26ngdayml(-1)). The ratio of AUC milk/plasma for moxidectin was 5.04+/-2.13. The moxidectin systemic availability (expressed as plasma AUC values) obtained in buffaloes was in the same range than those reported in cattle. The faster absorption and elimination processes of moxidectin were probably due to a lower storage in fat associated with the fact that animals were in lactation. Nevertheless, due to its high excretion in milk and its high detected maximum concentration in milk which is equivalent or higher to the Maximal Residue Level value (MRL) (40ngml(-1)), its use should be prohibited in lactating buffaloes. Concerning eprinomectin, the C(max) were of 2.74+/-0.89 and 3.40+/-1.68ngml(-1) at T(max) of 1.44+/-0.20 and 1.33+/-0.0.41 days in plasma and milk, respectively. The MRT and the AUC were similar for plasma (3.17+/-0.41 days and 11.43+/-4.01ngdayml(-1)) and milk (2.70+/-0.44 days and 8.49+/-3.33ngdayml(-1)). The ratio of AUC milk/plasma for eprinomectin was 0.76+/-0.16. The AUC value is 20 times lower than that reported in dairy cattle. The very low extent of mammary excretion and the milk levels reported lower than the MRL (20ngml(-1)) supports the permitted use of eprinomectin in lactating water buffaloes.

Comparative effects of cytokines on constitutive and inducible expression of the gene encoding for the cytochrome P450 3A6 isoenzyme in cultured rabbit hepatocytes: consequences on progesterone 6β-hydroxylation

Cultured rabbit hepatocytes were used to compare the relative activities of cytokines to inhibit the constitutive or rifampicin (RIF)-induced expression of the cytochrome P450 3A6 gene (CYP3A6). Human recombinant cytokines tested were interleukin-1beta (IL-1beta) (2 U/mL), interleukin-2 (IL-2) (5,000 U/mL) and interferon-gamma (IFN-gamma) (50 U/mL). Hepatocytes were cultured in the presence or absence of 25 microM RIF for 24 hr, with or without cytokines alone or in combination. All these cytokines inhibited RIF-induced P4503A6 expression without apparent cellular toxicity. By contrast, only IFN-gamma treatment provided a significant decrease (41%) in the constitutive P4503A6 protein level. Moreover, cytokines differed in their ability to repress RIF-dependent transcriptional induction of CYP3A6: IL-1beta and IL-2 were approximately equipotent, causing an almost 40-50% suppression of CYP3A6 mRNA and protein levels, whereas IFN-gamma exerted repressive effects only on P4503A6-related erythromycin N-demethylase activity and inducible protein expression. In fact, although strongly reducing P4503A6 protein content (an approximate 70% decrease), IFN-gamma did not exhibit any influence on CYP3A6 mRNAs with the exception of its association with interleukins. All these results suggest that IL-1beta and IL-2 mainly promote a transcriptional repression mechanism, given the absence of effect of these cytokines on the basal P4503A6 level, whereas IFN-gamma exerts a post-transcriptional suppressive action on both induced and constitutive P4503A6 expression. Consequently, P4503A6-dependent progesterone 6beta-hydroxylase activity also presented a cytokine-specific pattern of inhibition, with a much greater sensitivity than P4503A6 immunoreactive protein to IL-1beta and IL-2 + IFN-gamma treatments. Thus, this study underlines the significant impact of inflammation on steroid metabolism.