Biomass-Based, Dual Enzyme-Responsive Nanopesticides: Eco-friendly and Efficient Control of Pine Wood Nematode Disease.

Pine wood nematode (PWN) disease is a globally devastating forest disease caused by infestation with PWN, Bursaphelenchus xylophilus, which mainly occurs through the vector insect Japanese pine sawyer (JPS), Monochamus alternatus. PWN disease is notoriously difficult to manage effectively and is known as the "cancer of pine trees." In this study, dual enzyme-responsive nanopesticides (AVM@EC@Pectin) were prepared using nanocoating avermectin (AVM) after modification with natural polymers. The proposed treatment can respond to the cell wall-degrading enzymes secreted by PWNs and vector insects during pine tree infestation to intelligently release pesticides to cut off the transmission and infestation pathways and realize the integrated control of PWN disease. The LC50 value of AVM@EC@Pectin was 11.19 mg/L for PWN and 26.31 mg/L for JPS. The insecticidal activity of AVM@EC@Pectin was higher than that of the commercial emulsifiable concentrate (AVM-EC), and the photostability, adhesion, and target penetration were improved. The half-life (t1/2) of AVM@EC@Pectin was 133.7 min, which is approximately twice that of AVM-EC (68.2 min). Sprayed and injected applications showed that nanopesticides had superior bidirectional transportation, with five-times higher AVM contents detected in the roots relative to those of AVM-EC when sprayed at the top. The safety experiment showed that the proposed treatment had lower toxicity and higher safety for nontarget organisms in the application environment and human cells. This study presents a green, safe, and effective strategy for the integrated management of PWN disease.

Functional Analysis of Insecticide Inhibition and Metabolism of Six Glutathione S-Transferases in the Rice Stem Borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are important detoxifying enzymes in insects. Our previous studies found that the susceptibility of Chilo suppressalis to abamectin was significantly increased when the CsGST activity was inhibited by glutathione (GSH) depletory. In this study, the potential detoxification mechanisms of CsGSTs to abamectin were explored. Six CsGSTs of C. suppressalis were expressed in vitro. Enzymatic kinetic parameters including Km and Vmax of recombinant CsGSTs were determined, and results showed that all of the six CsGSTs were catalytically active and displaying glutathione transferase activity. Insecticide inhibitions revealed that a low concentration of abamectin could effectively inhibit the activities of CsGSTs including CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1. However, the in vitro metabolism assay found that the six CsGSTs could not metabolize abamectin directly. Additionally, the glutathione transferase activity of CsGSTs in C. suppressalis was significantly increased post-treatment with abamectin. Comprehensive analysis of the results in present and our previous studies demonstrated that CsGSTs play an important role in detoxification of abamectin by catalyzing the conjugation of GSH to abamectin in C. suppressalis, and the high binding affinities of CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1 with abamectin might also suggest the involvement of CsGSTs in detoxification of abamectin via the noncatalytic passive binding and sequestration instead of direct metabolism. These studies are helpful to better understand the detoxification mechanisms of GSTs in insects.

Uptake and Biotransformation of Guvermectin in Three Crops after In Vivo and In Vitro Exposure.

Guvermectin, as a novel nucleoside-like biopesticide, could increase the rice yield excellently, but the potential environmental behaviors remain unclear, which pose potential health risks. Therefore, the uptake and biotransformation of guvermectin in three types of crops (rice, lettuce, and carrot) were first evaluated with a hydroponic system. Guvermectin could be rapidly absorbed and reached equilibrium in roots (12-36 h) and shoots (24-60 h) in three plants, and guvermectin was also vulnerable to dissipation in roots (t1/2 1.02-3.65 h) and shoots (t1/2 9.30-17.91 h). In addition, 8 phase I and 2 phase II metabolites, transformed from guvermectin degradation in vivo and in vitro exposure, were identified, and one was confirmed as psicofuranine, which had antibacterial and antitumor properties; other metabolites were nucleoside-like chemicals. Molecular simulation and quantitative polymerase chain reaction further demonstrated that guvermectin was metabolized by the catabolism pathway of an endogenous nucleotide. Guvermectin had similar metabolites in three plants, but the biotransformation ability had a strong species dependence. In addition, all the metabolites exhibit neglectable toxicities (bioconcentration factor <2000 L/kg b.w., LC50,rat > 5000 mg/kg b.w.) by prediction. The study provided valuable evidence for the application of guvermectin and a better understanding of the biological behavior of nucleoside-like pesticides.

Doramectin inhibits glioblastoma cell survival via regulation of autophagy in vitro and in vivo.

Glioblastoma (GBM) is one of the most widespread and lethal types of cancer. However, there are currently no drugs or therapeutic strategies that can completely cure GBM. Doramectin (DRM) has a broad range of activities against endoparasites and ectoparasites, and is extensively used in livestock. In the present study, the effect of DRM on the induction of autophagy in U87 and C6 GBM and glioma cell lines, as well as the mechanism of autophagy, were examined. First, transmission electron microscopy, plasmid transfection and western blot analysis demonstrated that DRM could induce autophagy in U87 and C6 cells in vitro. Next, MTT and colony formation assays revealed that DRM­induced autophagy prevented U87 and C6 cell viability and colony formation ratio. In addition, DRM­induced autophagy promoted U87 and C6 cell apoptosis, as indicated by DAPI analysis and flow cytometry. Furthermore, transcriptome analysis demonstrated that DRM modulated a number of genes and pathways involved in autophagy. In a nude mouse xenograft model, immunohistochemical staining and the TUNEL assay demonstrated that the effect of DRM on the tumor was consistent with that in vivo. These data indicated that DRM induced autophagy mainly by blocking the PI3K/AKT/mTOR signaling pathway in GBM cells. DRM­induced autophagy promoted the inhibition of GBM cell proliferation and apoptosis in vitro and in vivo. The present study suggested that DRM may be an effective drug for the treatment of GBM.

Metabolism and interactions of Ivermectin with human cytochrome P450 enzymes and drug transporters, possible adverse and toxic effects.

The review presents metabolic properties of Ivermectin (IVM) as substrate and inhibitor of human P450 (P450, CYP) enzymes and drug transporters. IVM is metabolized, both in vivo and in vitro, by C-hydroxylation and O-demethylation reactions catalyzed by P450 3A4 as the major enzyme, with a contribution of P450 3A5 and 2C9. In samples from both in vitro and in vivo metabolism, a number of metabolites were detected and as major identified metabolites were 3″-O-demethylated, C4-methyl hydroxylated, C25 isobutyl-/isopropyl-hydroxylated, and products of oxidation reactions. Ivermectin inhibited P450 2C9, 2C19, 2D6, and CYP3A4 with IC50 values ranging from 5.3 µM to no inhibition suggesting that it is no or weak inhibitor of the enzymes. It is suggested that P-gp (MDR1) transporter participate in IVM efflux at low drug concentration with a slow transport rate. At the higher, micromolar concentration range, which saturates MDR1 (P-gp), MRP1, and to a lesser extent, MRP2 and MRP3 participate in IVM transport across physiological barriers. IVM exerts a potent inhibition of P-gp (ABCB1), MRP1 (ABCC1), MRP2 (ABCC2), and BCRP1 (ABCG2), and medium to weak inhibition of OATP1B1 (SLC21A6) and OATP1B3 (SLCOB3) transport activity. The metabolic and transport properties of IVM indicate that when IVM is co-administered with other drugs/chemicals that are potent inhibitors/inducers P4503A4 enzyme and of MDR1 (P-gp), BCRP or MRP transporters, or when polymorphisms of the drug transporters and P450 3A4 exist, drug-drug or drug-toxic chemical interactions might result in suboptimal response to the therapy or to toxic effects.

Uptake, translocation and subcellular distribution of pesticides in Chinese cabbage (Brassica rapa var. chinensis).

The extensive application of pesticides in agricultural activities has raised increasing concerns on crop contamination by pesticide residues. Vegetables seem more susceptible to pesticide contamination given the high-intensive application of pesticides during their entire growth, while information about transfer and cell diffusion characteristics of pesticides in vegetables is currently insufficient. Here, we investigated the uptake, translocation and subcellular distribution behaviors of four commonly used pesticides in Chinese cabbage (Brassica rapa var. chinensis) under laboratory hydroponic conditions. Root uptake of pesticides followed the order of fenbuconazole > avermectin > thiamethoxam > spirotetramat. Thiamethoxam was more readily to be translocated from vegetable root to shoot, while spirotetramat, fenbuconazole and avermectin preferentially accumulated in vegetable root. Cell soluble components were the dominant storage compartment for thiamethoxam. The majority of spirotetramat, fenbuconazole and avermectin were partitioned into the cell walls. Hopefully, results of this study would extend the current knowledge of pesticide bioconcentration behavior in food-crops and assist in properly evaluating the threats of pesticide residues to human health via food chain.

Attenuating effects of caffeic acid phenethyl ester and betaine on abamectin-induced hepatotoxicity and nephrotoxicity.

Abamectin (ABM) is a widely utilized potent anthelmintic and insecticidal agent. In this study, we investigated the protective effects of caffeic acid phenethyl ester (CAPE) and betaine (BET) against ABM-induced hepatotoxicity and nephrotoxicity in rats. Forty rats were divided into five groups, receiving either oral saline solution (normal control), oral ABM at a dose of 2 mg/kg BW (1/5 LD50), CAPE (10 µmol/kg BW intraperitoneally) followed by ABM, or BET supplementation at a dose of 250 mg/kg BW followed by ABM administration, while group V rats received a combination of i.p. CAPE and oral BET in the same doses before receiving ABM. Biochemical analysis showed that ABM administration significantly (p < 0.05) increased serum levels of aminotransferases, alkaline phosphatase, lactate dehydrogenase, and cholesterol, as well as serum creatinine and urea. Compared to the control group, ABM-intoxicated rats had significantly (p < 0.05) higher tissue concentrations of nitric oxide and malondialdehyde, as well as lower tissue glutathione concentration, total antioxidant capacity, and antioxidant enzymatic activity (glutathione peroxidase, superoxide dismutase, and catalase). Histopathological examination of hepatic and renal tissues of ABM-intoxicated rats showed acute inflammatory and necrotic changes. Pretreatment with CAPE and/or BET reversed the biochemical and histopathological alterations of ABM on the liver and kidneys. Therefore, CAPE and BET (alone or in combination) could be promising protective agents against ABM-induced hepatotoxicity and nephrotoxicity. Future studies should confirm our findings and evaluate the other molecular effects are involved in the combination chemoprotection of CAPE and BET.

Structural model, functional modulation by ivermectin and tissue localization of Haemonchus contortus P-glycoprotein-13.

Haemonchus contortus, one of the most economically important parasites of small ruminants, has become resistant to the anthelmintic ivermectin. Deciphering the role of P-glycoproteins in ivermectin resistance is desirable for understanding and overcoming this resistance. In the model nematode, Caenorhabditis elegans, P-glycoprotein-13 is expressed in the amphids, important neuronal structures for ivermectin activity. We have focused on its ortholog in the parasite, Hco-Pgp-13. A 3D model of Hco-Pgp-13, presenting an open inward-facing conformation, has been constructed by homology with the Cel-Pgp-1 crystal structure. In silico docking calculations predicted high affinity binding of ivermectin and actinomycin D to the inner chamber of the protein. Following in vitro expression, we showed that ivermectin and actinomycin D modulated Hco-Pgp-13 ATPase activity with high affinity. Finally, we found in vivo Hco-Pgp-13 localization in epithelial, pharyngeal and neuronal tissues. Taken together, these data suggest a role for Hco-Pgp-13 in ivermectin transport, which could contribute to anthelmintic resistance.

Preclinical evaluation of avermectins as novel therapeutic agents for alcohol use disorders.

The deleterious effects of alcohol use disorders (AUDs) on human health have been documented worldwide. The enormous socioeconomic burden coupled with lack of efficacious pharmacotherapies underlies the need for improved treatment strategies. At present, there is a growing body of preclinical evidence that demonstrates the potential of avermectins [ivermectin (IVM), selamectin (SEL), abamectin (ABM), and moxidectin (MOX)] in treatment of AUDs. Avermectins are derived by fermentation of soil micro-organism, Streptomyces avermitilis, and have been extensively used for treatment of parasitic infections. From the mechanistic standpoint, avermectins are positive modulators of purinergic P2X4 receptors (P2X4Rs). P2X4Rs belong to P2X superfamily of cation-permeable ion channels gated by adenosine 5'-triphosphate (ATP). Building evidence has implicated a role for P2X4Rs in regulation of ethanol intake and that ethanol can inhibit ATP-gated currents in P2X4Rs. Investigations using recombinant cell models and animal models of alcohol drinking have reported that IVM, ABM, and MOX, but not SEL, were able to antagonize the inhibitory effects of ethanol on P2X4Rs in vitro and reduce ethanol intake in vivo. Furthermore, IVM was shown to reduce ethanol consumption via P2X4R potentiation in vivo, supporting the involvement of P2X4Rs in IVM's anti-alcohol effects and that P2X4Rs can be used as a platform for developing novel anti-alcohol compounds. Taken together, these findings support the utility of avermectins as a novel class of drug candidates for treatment of AUDs.

Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models.

The P2X4 receptor (P2X4R) is a member of a family of purinergic channels activated by extracellular ATP through three orthosteric binding sites and allosterically regulated by ivermectin (IVM), a broad-spectrum antiparasitic agent. Treatment with IVM increases the efficacy of ATP to activate P2X4R, slows both receptor desensitization during sustained ATP application and receptor deactivation after ATP washout, and makes the receptor pore permeable to NMDG+, a large organic cation. Previously, we developed a Markov model based on the presence of one IVM binding site, which described some effects of IVM on rat P2X4R. Here we present two novel models, both with three IVM binding sites. The simpler one-layer model can reproduce many of the observed time series of evoked currents, but does not capture well the short time scales of activation, desensitization, and deactivation. A more complex two-layer model can reproduce the transient changes in desensitization observed upon IVM application, the significant increase in ATP-induced current amplitudes at low IVM concentrations, and the modest increase in the unitary conductance. In addition, the two-layer model suggests that this receptor can exist in a deeply inactivated state, not responsive to ATP, and that its desensitization rate can be altered by each of the three IVM binding sites. In summary, this study provides a detailed analysis of P2X4R kinetics and elucidates the orthosteric and allosteric mechanisms regulating its channel gating.

Drug-transporter mediated interactions between anthelminthic and antiretroviral drugs across the Caco-2 cell monolayers.

BACKGROUND: Drug interactions between antiretroviral drugs (ARVs) and anthelminthic drugs, ivermectin (IVM) and praziquantel (PZQ) were assessed by investigating their permeation through the Caco-2 cell monolayers in a transwell. The impact of anthelminthics on the transport of ARVs was determined by assessing the apical to basolateral (AP → BL) [passive] and basolateral to apical (BL → AP) [efflux] directions alone, and in presence of an anthelminthic. The reverse was conducted for the assessment of the influence of ARVs on anthelminthics. METHODS: Samples from the AP and BL compartments were taken at 60, 120, 180 and 240 min and quantified either by HPLC or radiolabeled assay using a liquid scintillating counter for the respective drugs. Transepithelial resistance (TEER) was used to assess the integrity of the monolayers. The amount of compound transported per second (apparent permeability, Papp) was calculated for both AP to BL (PappAtoB), and BL to AP (PappBtoA) movements. Samples collected after 60 min were used to determine the efflux ratio (ER), quotient of secretory permeability and absorptive permeability (PappBL-AP/PappAP-BL). The reverse, (PappAP-BL/PappBL-AP) constituted the uptake ratio. The impact of SQV, EFV and NVP on the transport of both IVM and PZQ were investigated. The effect of LPV on the transport of IVM was also determined. The influence of IVM on the transport of SQV, NVP, LPV and EFV; as well as the effect PZQ on the transport of SQV of was also investigated, and a two-tailed p value of <0.05 was considered significant. RESULTS: IVM significantly inhibited the efflux transport (BL → AP movement) of LPV (ER; 6.7 vs. 0.8, p = 0.0038) and SQV (ER; 3.1 vs. 1.2 p = 0.00328); and increased the efflux transport of EFV (ER; 0.7 vs. 0.9, p = 0.031) suggesting the possibility of drug transporter mediated interactions between the two drugs. NVP increased the efflux transport of IVM (ER; 0.8 vs. 1.8, p = 0.0094). CONCLUSIONS: The study provides in vitro evidence of potential interactions between IVM, an anthelminthic drug with antiretroviral drugs; LPV, SQV, NVP and EFV. Further investigations should be conducted to investigate the possibility of in vivo interactions.

Comparative metabolomics reveals the mechanism of avermectin production enhancement by S-adenosylmethionine.

It was found that S-adenosylmethionine (SAM) could effectively improve avermectin titer with 30-60 µg/mL addition to FH medium. To clearly elucidate the mechanism of SAM on intracellular metabolites of Streptomyces avermitilis, a GC-MS-based comparative metabolomics approach was carried out. First, 230 intracellular metabolites were identified and 14 of them remarkably influenced avermectin biosynthesis were discriminative biomarkers between non-SAM groups and SAM-treated groups by principal components analysis (PCA) and partial least squares (PLS). Based on further key metabolic pathway analyses, these biomarkers, such as glucose, oxaloacetic acid, fatty acids (in soybean oil), threonine, valine, and leucine, were identified as potentially beneficial precursors and added in medium. Compared with single-precursor feeding, the combined feeding of the precursors and SAM markedly increased the avermectin titer. The co-feeding approach not only directly verified our hypothesis on the mechanism of SAM by comparative metabolomics, but also provided a novel strategy to increase avermectin production.

[Avermectin, from winning the Nobel Prize to "innovation in China"].

The uprise of the superpower nations is always accompanied by the breakthrough and advances of technologies and innovations in the history. Natural products play very important role in human health, such as anticancer molecular taxol, anti-infection drug artemisinin that save a lot of lives, metabolic disease treatment, nutrition and health care. However, more has never been explored. With the 2015 Nobel Prize in Physiology or Medicine awarded to William C. Campbell, Satoshi Omura, and Youyou Tu for the discovery of avermectins and artemisinin respectively, the second "Golden age" in the development of natural product is dawning. China is a "world factory" and natural drugs-rich country, but how to upgrade and advance the industry and realize the China dream? Avermectins, produced by Streptomyces avermitilis, are pesticide with high efficiency and low levels of side effects. However, the low producer and expensive development pattern of high consumption, high contamination is not sustainable. Solving the problem, increasing the production and utilization of raw material, reducing the energy consumption and cost of production, decreasing environmental pollution are key to transform China into a power house. In this paper, we case-study avermectins to review the industry development driven by fundamental research. Institute of Microbiology, Chinese Academy.of Sciences increased the production of avermectin 1000 folds to 9 g/L, which out licensed to new Veyong biochemical Ltd and avermectin Coalitions. As a result, Merck Sharp and Dohme ceased the manufacture of avermectins. The success also shed lights on the improvement of other natural product drugs in China.

Direct interaction of avermectin with epidermal growth factor receptor mediates the penetration resistance in Drosophila larvae.

With the widespread use of avermectins (AVMs) for managing parasitic and agricultural pests, the resistance of worms and insects to AVMs has emerged as a serious threat to human health and agriculture worldwide. The reduced penetration of AVMs is one of the main reasons for the development of the resistance to the chemicals. However, the detailed molecular mechanisms remain elusive. Here, we use the larvae of Drosophila melanogaster as the model organism to explore the molecular mechanisms underlying the development of penetration resistance to AVMs. We clearly show that the chitin layer is thickened and the efflux transporter P-glycoprotein (P-gp) is overexpressed in the AVM-resistant larvae epidermis. We reveal that the activation of the transcription factor Relish by the over-activated epidermal growth factor receptor (EGFR)/AKT/ERK pathway induces the overexpression of the chitin synthases DmeCHS1/2 and P-gp in the resistant larvae. Interestingly, we discover for the first time, to the best of our knowledge, that AVM directly interacts with EGFR and leads to the activation of the EGFR/AKT/ERK pathway, which activates the transcription factor Relish and induces the overexpression of DmeCHS1/2 and P-gp. These findings provide new insights into the molecular mechanisms underlying the development of penetration resistance to drugs.

Characterization of multidrug transporter-mediated efflux of avermectins in human and mouse neuroblastoma cell lines.

ABC transporters play an important role in the disposition of avermectins in several animal species. In this study the interactions of three key avermectins, abamectin, emamectin and ivermectin, with human and mouse homologues of MDR1 (ABCB1/Abcb1a) and MRP (ABCC/Abcc), transporters endogenously expressed by human SH-SY5Y and mouse N2a neuroblastoma cells were investigated. In both cell lines, retention of the fluorescent dye H33342 was found to be significantly increased in the presence of avermectins and cyclosporin A. These effects were shown to be unresponsive to the BCRP inhibitor Ko-143 and therefore MDR1/Mdr1-dependent. Avermectins inhibited MDR1/Mdr1a-mediated H33342 dye efflux, with apparent Ki values of 0.24±0.08 and 0.18±0.02µM (ivermectin); 0.60±0.07 and 0.56±0.02µM (emamectin) and 0.95±0.08 and 0.77±0.25µM (abamectin) in SH-SY5Y and N2a cells, respectively. There were some apparent affinity differences for MDR1 and Mdr1a within each cell line (affinity for ivermectin>emamectin≥abamectin, P<0.05 by One-Way ANOVA), but importantly, the Ki values for individual avermectins for human MDR1 or mouse Mdr1a were not significantly different. MK571-sensitive retention of GSMF confirmed the expression of MRP/Mrp efflux transporters in both cell lines. Avermectins inhibited MRP/Mrp-mediated dye efflux with IC50 values of 1.58±0.51 and 1.94±0.72µM (ivermectin); 1.87±0.57 and 2.74±1.01µM (emamectin) and 2.25±0.01 and 1.68±0.63µM (abamectin) in SH-SY5Y and N2a cells, respectively. There were no significant differences in IC50 values between individual avermectins or between human MRP and mouse Mrp. Kinetic data for endogenous human MDR1/MRP isoforms in SH-SY5Y cells and mouse Mdr1a/b/Mrp isoforms in N2a cells are comparable for the selected avermectins. All are effluxed at concentrations well above 0.05-0.1µM ivermectin detected in plasma (Ottesen and Campbell, 1994; Ottesen and Campbell, 1994) This is an important finding in the light of toxicity seen in the Mdr1-deficient animal models CF-1 mice, Mdr1ab (-/-) double knockout mice and Collie dogs. We also confirm MRP/Mrp-mediated avermectin transport in both N2a and SH-SY5Y cell lines.

X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors.

Cys-loop receptors are neurotransmitter-gated ion channels that are essential mediators of fast chemical neurotransmission and are associated with a large number of neurological diseases and disorders, as well as parasitic infections. Members of this ion channel superfamily mediate excitatory or inhibitory neurotransmission depending on their ligand and ion selectivity. Structural information for Cys-loop receptors comes from several sources including electron microscopic studies of the nicotinic acetylcholine receptor, high-resolution X-ray structures of extracellular domains and X-ray structures of bacterial orthologues. In 2011 our group published structures of the Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in complex with the allosteric partial agonist ivermectin, which provided insights into the structure of a possibly open state of a eukaryotic Cys-loop receptor, the basis for anion selectivity and channel block, and the mechanism by which ivermectin and related molecules stabilize the open state and potentiate neurotransmitter binding. However, there remain unanswered questions about the mechanism of channel opening and closing, the location and nature of the shut ion channel gate, the transitions between the closed/resting, open/activated and closed/desensitized states, and the mechanism by which conformational changes are coupled between the extracellular, orthosteric agonist binding domain and the transmembrane, ion channel domain. Here we present two conformationally distinct structures of C. elegans GluCl in the absence of ivermectin. Structural comparisons reveal a quaternary activation mechanism arising from rigid-body movements between the extracellular and transmembrane domains and a mechanism for modulation of the receptor by phospholipids.

Pharmacokinetics and metabolism of eprinomectin in cats when administered in a novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel.

Four studies were conducted to determine the pharmacokinetic characteristics and in vitro metabolism of eprinomectin, a semi-synthetic avermectin, in cats. Pharmacokinetic parameters including bioavailability of eprinomectin were determined in a parallel study design comprised of one group of eight cats which were treated once topically at 0.12 mL/kg bodyweight with BROADLINE(®), a novel combination product (fipronil 8.3% (w/v), (S)-methoprene 10% (w/v), eprinomectin 0.4% (w/v) and praziquantel 8.3% (w/v)), delivering a dose of 0.5mg eprinomectin per kg body weight, and a group of six cats which received 0.4% (w/v) eprinomectin at 0.4 mg/kg bodyweight once by intravenous injection. For cats treated by topical application, the average eprinomectin (B1a component) maximum plasma concentration (Cmax) was 20 ng/mL. The maximum concentrations were reached 24h after dosing in the majority of the animals (six of eight cats). The average terminal half-life was 114 h due to slow absorption ('flip-flop' kinetics). Following intravenous administration the average Cmax was 503 ng/mL at 5 min post-dose, and the mean elimination half-life was 23 h. Eprinomectin was widely distributed with a mean volume of distribution of 2,390 mL/kg, and the clearance rate was 81 mL/h/kg. Mean areas under the plasma concentration versus time curves extrapolated to infinity were 2,100 ngh/mL and 5,160 ngh/mL for the topical and intravenous doses, respectively. Topical eprinomectin was absorbed with an average absolute bioavailability of 31%. In a second parallel design study, the dose proportionality of eprinomectin after single topical administration of BROADLINE(®) was studied. Four groups of eight cats each were treated once topically with 0.5, 1, 2 or 5 times the minimum recommended dose of the combination, 0.12 mL/kg bodyweight. Based on comparison of areas under the plasma concentration versus time curves from the time of dosing to the last time point at which eprinomectin B1a was quantified, and Cmax, dose proportionality was established. In addition, the metabolic pathway of eprinomectin using cat liver microsomes, and plasma protein binding using cat, rat, and dog plasma were studied in vitro. Results of the analyses of eprinomectin B1a described here showed that it is metabolically stable and highly protein bound (>99%), and thus likely to be, as with other species, excreted mainly as unchanged parent drug in the feces of cats.

Phenotypic and phylogenetic characterization of an abamectin-degrading bacterial strain isolated from a citrus orchard.

Bacterial strain GB-01 was isolated from abamectin-contaminated soils by continuous enrichment culture. The preliminary identification of strain GB-01 as a Burkholderia species was based mainly on simple biochemical and substrate utilization tests; however, these tests alone cannot accurately differentiate all the species within the genus Burkholderia. The strain GB-01 was subjected to taxonomic analysis through a polyphasic approach, in which phenotypic, genotypic, and phylogenetic information was gathered to conclude the classification of this microbe. Phenotypic information comes from basic bacteriological tests and substrate utilization patterns using the Biolog GN2 MicroPlating system and automated miniature biochemical test kits, i.e. API 20 NE, ID 32 GN and API 50 CH, as well as analyzing the whole cell fatty acid profile. Genotypic information was gathered from whole genome DNA base composition (G+C mol%), and DNA-DNA hybridization with its closest species, while phylogenetic information was collected from the comparative analysis of 16S rRNA and recA gene sequences. The results of polyphasic analysis concluded that strain GB-01 is an atypical strain of the Burkholderia diffusa species.

Overexpression of metK shows different effects on avermectin production in various Streptomyces avermitilis strains.

The S-adenosylmethionine synthetase gene (metK) from Streptomyces avermitilis was cloned into multi-copy vector pIJ653 and integrative vector pSET152 yielding two metK expression plasmids pYJ02 and pYJ03, respectively. When wild-type strain ATCC31267 was transformed with these two plasmids, avermectin production was increased about 2.0-fold and 5.5-fold, respectively. The introduction of integrative expression plasmid pYJ03 into the engineered strain GB-165, which produces only avermectin B, promoted the production of avermectin approximately 2.0-fold. However, introduction of pYJ02 did not influence avermectin accumulation in GB-165. Moreover, transformation of the avermectin-overproducing industry strain 76-05 with these two plasmids did not stimulate avermectin production. These results showed that there were different effects of metK expression levels on avermectin production in various S. avermitilis strains. Additionally, the transcript levels of metK, aveR (the avermectin pathway-specific regulatory gene) and aveA1 (one avermectin biosynthesis gene) meet the expectation of fermentation levels of avermectin in wild-type strain and its recombinant strains. The gene expression levels of metK, aveR and aveA1 in GB-165 and 76-05 were much higher then those in wild-type strain, which probably limited the increasement of avermectin by overexpression of metK.

Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models.

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 µM, P = 0.017; 10 µM, P = 0.001; 25 µM, P = 0.008; and 50 µM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.