Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections.

One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies.IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.

Identification of osalmid metabolic profile and active metabolites with anti-tumor activity in human hepatocellular carcinoma cells.

BACKGROUNDS: Ribonucleotide reductase (RR) catalyzes the essential step in the formation of all four deoxynucleotides. Upregulated activity of RR plays an active role in tumor progression. As the regulatory subunit of RR, ribonucleotide reductase subunit M2 (RRM2) is regarded as one of the effective therapeutic targets for DNA replication-dependent diseases, such as cancers. Recent studies have revealed that osalmid significantly inhibits the activity of RRM2, but the metabolic profile of osalmid remains unknown. OBJECTIVE: The aim of this study was to clarify the metabolic profile including metabolites, isoenzymes and metabolic pathways of osalmid. The anti-human hepatocellular carcinoma activity and mechanism of metabolites were further investigated. MATERIALS AND METHODS: Ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was used for identifying metabolites and for characterizing phase I and phase II metabolic pathways with recombinant enzymes or in human liver microsomes of osalmid. The eHiTS docking system was used for potential RRM2 inhibitor screening among metabolites. Cytotoxicity assays were performed for evaluating cell proliferation inhibitory activity of metabolites. Cell cycle assays and cell apoptosis assays were assessed by flow cytometry. Western blotting analysis of RRM2, cyclin D1, p21, p53, phosphorylated p53, Bcl-2 and Bax was performed to explore the anti-hepatocellular carcinoma mechanism of the active metabolites. RESULTS: Ten metabolites of osalmid were identified, and none of them have been reported previously. Hydroxylation, glucuronidation, sulfonation, acetylation and degradation were recognized as the main metabolic processes of osalmid. Isozymes of CYP1A2, CYP2C9, UGT1A1, UGT1A6, UGT1A9, UGT2B7 and UGT2B15 were involved in phase I and phase II metabolism of osalmid. Metabolites M7, M8 and M10 showed higher binding affinities with the RRM2 active site than osalmid. Metabolite M7 exhibited potent inhibitory activity to hepatocellular carcinoma cell lines by both competitive inhibition and down-regulation of RRM2. Moreover, M7 significantly induced cell cycle arrest and apoptosis by activating p53-related pathways. CONCLUSIONS: The metabolic profile of osalmid was identified. M7 significantly inhibited human hepatocellular carcinoma progression by inhibiting RRM2 activity. Furthermore, M7 induced cell cycle arrest and apoptosis by activating p53-related signaling pathways.

Discovery and Structure Relationships of Salicylanilide Derivatives as Potent, Non-acidic P2X1 Receptor Antagonists.

Antagonists for the ATP-gated ion channel receptor P2X1 have potential as antithrombotics and for treating hyperactive bladder and inflammation. In this study, salicylanilide derivatives were synthesized based on a screening hit. P2X1 antagonistic potency was assessed in 1321N1 astrocytoma cells stably transfected with the human P2X1 receptor by measuring inhibition of the ATP-induced calcium influx. Structure-activity relationships were analyzed, and selectivity versus other P2X receptor subtypes was assessed. The most potent compounds, N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (1, IC50 0.0192 µM) and N-[3,5-bis(trifluoromethyl)phenyl]-4-chloro-2-hydroxybenzamide (14, IC50 0.0231 µM), displayed >500-fold selectivity versus P2X2 and P2X3, and 10-fold selectivity versus P2X4 and P2X7 receptors, and inhibited collagen-induced platelet aggregation. They behaved as negative allosteric modulators, and molecular modeling studies suggested an extracellular binding site. Besides selective P2X1 antagonists, compounds with ancillary P2X4 and/or P2X7 receptor inhibition were discovered. These compounds represent the first potent, non-acidic, allosteric P2X1 receptor antagonists reported to date.

HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, natural products, and known bioactive molecules.

All living organisms contain a unique class of molecular chaperones called 60 kDa heat shock proteins (HSP60 - also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus - MRSA). Intriguingly, during our studies we found that three known antibiotics - suramin, closantel, and rafoxanide - were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.

Metabolism of drugs and other xenobiotics in giant liver fluke (Fascioloides magna).

1. Giant liver fluke Fascioloides magna is a dangerous parasite, which infects herbivores. It was imported to Europe from North America and started to spread. Benzimidazoles like albendazole, mebendazole, triclabendazole and salicylanilides closantel and rafoxanide are the most used anthelmintics to control fascioloidosis. However their effect might be altered via drug-metabolizing enzymes of this parasite. 2. The aim of our study was to determine the activities of drug-metabolizing enzymes in F. magna and the metabolism of above mentioned anthelmintics. 3. Activities of several oxidative, reductive and conjugative enzymes towards various model xenobiotic substrates were found in F. magna subcellular fractions. 4. Subcellular fractions from F. magna oxidized albendazole to its sulphoxide metabolite and reduced mebendazole to hydroxyl-mebendazole. Under ex vivo conditions, only very-low concentrations of these compounds were detected using high-performance liquid chromatography/mass spectrometry. 5. The results indicate that the giant liver fluke possesses the active xenobiotic-metabolizing system. The overexpression of this system may play an important role in parasite resistance against these anthelmintics.

Closantel nano-encapsulated polyvinyl alcohol (PVA) solutions.

The influence of closantel on the rheological and physicochemical properties (particle size and by UV-Vis absorption spectroscopy) of PVA aqueous solutions is studied here. About 1% PVA aqueous solutions were prepared by varying the closantel content. The increase of closantel content led to a reduction in the particle size of final solutions. All the solutions were buffered at pH 7.4 and exhibited shear-thinning behavior. Furthermore, in oscillatory flow, a "solid-like" type behavior was observed for the sample containing 30 µg/mL closantel. Indicating a strong interaction between the dispersed and continuous phases and evidencing an interconnected network between the nanoparticle and PVA, this sample also showed the highest shear viscosity and higher shear thinning slope, indicating a more intrincate structure disrupted by shear. In conclusion, PVA interacts with closantel in aqueous solution and the critical concentration for closantel encapsulation by PVA was about 30 µg/mL; above this concentration, the average particle size decreased notoriously which was associated to closantel interacting with the surface of the PVA aggregates and thus avoiding to some extent direct polymer-polymer interaction.

Isolation and characterization of endochitinase and exochitinase of Setaria cervi.

Chitin metabolism has been shown to have a role in the development of parasitic nematodes including filarial parasites and the enzymes associated with chitin metabolism have been considered as potential vaccine and drug target. Chitinases are members of the enzyme superfamily of glycoside hydrolases, which are characterized by the ability to hydrolyze glycosidic bonds in chitin chain by either an endolytic or an exolytic mechanism. In the present study, we have demonstrated the chitinase (exochitinase and endochitinase) activity in different stages of Setaria cervi (bovine filarial parasite) and have also purified and characterized the endochitinase from microfilarial stage of the parasite. The chitinase activity has been detected in adult and microfilarial stages of S. cervi using the fluorescent substrates. The S. cervi adult stage was found to have high activity of exochitinase (28.72±0.25 nmol/min/mg) while microfilarial stage showed high activity of endochitinase (24.40±0.25 nmol/min/mg). Native polyacrylamide gel electrophoresis, followed by staining of enzyme activity with fluorescent substrates, revealed single isoenzymic form of exochitinase in adults and endochitinase in microfilariae of S. cervi. The endochitinase from S. cervi microfilariae was purified employing chitin affinity matrix and DEAE-Sephacel ion-exchange chromatography. The enzyme was purified about 55 fold with an enzyme recovery of 22.33%. The purified enzyme exhibited a doublet of protein bands on SDS-PAGE at 65-70 kDa. The closantel (chitinase inhibitor) strongly inhibited the enzyme activity of S. cervi microfilariae endochitinase with a Ki value of 4.3±0.18 µM.

Synthesis and evaluation of salicylanilide derivatives as potential epidermal growth factor receptor inhibitors.

Two series of novel salicylanilide were synthesized as potential epidermal growth factor receptor (EGFR) inhibitors. The enzyme inhibitory activity against EGFR of all compounds was carried out, and their antiproliferative activities against the A549 and A431 cell lines were also evaluated. Of the compounds studied, majority of them exhibited high antiproliferative activities compared with gefitinib; especially, 12a and 12b exhibited stronger inhibitory activity against EGFR with IC50 values of 10.4 ± 2.25 and 15.4 ± 2.33 nm, respectively, which were comparable to the positive control of gefitinib (IC50 = 12.1 ± 2.21 nm). Compound 12b also showed outstanding inhibitory activity against A431 and A549 cell lines with the IC50 values of 0.42 ± 0.43 µm and 0.57 ± 0.43 µm, which was better than the positive controls. In the molecular modeling study, compound 12b was bound into the active pocket of EGFR with two hydrogen bond and with minimum binding free energy ▵Gb = -25.1125 kcal/mol. The result also suggested that compound 12b could bind the EGFR kinase well.

Possible causes of excess sludge reduction adding metabolic uncoupler, 3,3',4',5-tetrachlorosalicylanilide (TCS), in sequence batch reactors.

Two parallel sequence batch reactors (SBRs) were operated, with and without TCS addition, to research the causes of sludge reduction by uncouplers. Three possible mechanisms of sludge reduction by TCS were studied: (1) occurrence of metabolic uncoupling, (2) consumption of more energy to resist the infection of TCS, (3) promotion of lysis-cryptic growth by TCS addition. Results showed the remarkable reduction of electronic transport system (ETS) activity and specific cellular ATP (SATP) in TCS reactor, which proved the occurrence of metabolic uncoupling. The increasing amounts of extracellular polymeric substances (EPS), as measured by chemical methods and excitation-emission matrix (EEM) fluorescence spectra, implied microorganisms consumed more energy to resist TCS. The similar DNA concentrations of the effluents in two reactors indicated sludge lysis was not intensified by TCS. Therefore, uncoupler might not only cause metabolic uncoupling but also induce more energy consumption in the production of some substances to resist uncoupler.

Dependence of structure stability and integrity of aerobic granules on ATP and cell communication.

Aerobic granules are dense and compact microbial aggregates with various bacterial species. Recently, aerobic granulation technology has been extensively explored for treatment of municipal and industrial wastewaters. However, little information is currently available with regard to their structure stability and integrity at levels of energy metabolism and cell communication. In the present study, a typical chemical uncoupler, 3,3',4',5-tetrachlorosalicylanilide with the power to dissipate proton motive force and subsequently inhibit adenosine triphosphate (ATP) generation, was used to investigate possible roles of ATP and cell communication in maintaining the structure stability and integrity of aerobic granules. It was found that inhibited ATP synthesis resulted in the reduced production of autoinducer-2 and N-acylhomoserine lactones essential for cell communication, while lowered extracellular polymeric substance (EPS) production was also observed. As a consequence, aerobic granules appeared to break up. This study showed that ATP-dependent quorum sensing and EPS were essential for sustaining the structure stability and integrity of aerobic granules.

Salicylanilide derivatives block Mycobacterium tuberculosis through inhibition of isocitrate lyase and methionine aminopeptidase.

The global burden of tuberculosis, its health and socio-economic impacts, the presence of drug-resistant forms and a potential threat of latent tuberculosis should serve as a strong impetus for the development of novel antituberculosis agents. We reported the in vitro activity of salicylanilide benzoates and pyrazine-2-carboxylates against Mycobacterium tuberculosis (minimum inhibitory concentrations as low as 0.5 µmol/L). Nineteen salicylanilide derivatives with mostly good antimycobacterial activity were evaluated for the inhibition of two essential mycobacterial enzymes, methionine aminopeptidase and isocitrate lyase, which are necessary for the maintenance of the latent tuberculosis infection. Salicylanilide derivatives act as moderate inhibitors of both mycobacterial and human methionine aminopeptidase and they also affect the function of mycobacterial isocitrate lyase. 4-Bromo-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl pyrazine-2-carboxylate was the most potent inhibitor of mycobacterial methionine aminopeptidase (41% inhibition at 10 µmol/L) and exhibited the highest selectivity. 5-Chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide and 4-chloro-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl pyrazine-2-carboxylate caused 59% inhibition of isocitrate lyase at 100 µmol/L concentration and (S)-4-bromo-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl 2-acetamido-3-phenylpropanoate produced 22% inhibition at 10 µmol/L; this rate is approximately comparable to 3-nitropropionic acid. Inhibition of those enzymes contributes at least in part to the antimicrobial activity of the compounds.

SMP production by activated sludge in the presence of a metabolic uncoupler, 3,3',4',5-tetrachlorosalicylanilide (TCS).

3,3',4',5-Tetrachlorosalicylanilide (TCS) is an effective metabolic uncoupler utilized for microbial yield reduction. However, its potential impact, in particular on the soluble microbial products (SMP) formation, is unknown yet. Herein we study the effect of TCS on SMP production and analyze the related mechanism. The addition of TCS in activated sludge system led to an increased production of SMP, especially proteins. The SMP were produced in proportion to the substrate utilization at a low TCS concentration, while more non-substrate-associated SMP were released at a high TCS concentration. TCS simulated the production of extracellular polymeric substances (EPS) and enhanced cell lysis, which both contributed to SMP production. FTIR and EEM analyses show that the SMP, EPS, and cell lysis products have similar functional groups and fluorescence properties, indicating a similar origin of these substances. In addition, a dose of TCS increased the release of high molecular weight compounds due to cell lysis. This study might benefit for a better understanding of the response of activated sludge to metabolic uncouplers like TCS.

Synthesis and antiproliferative activities against Hep-G2 of salicylanide derivatives: potent inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase.

A series of salicylanilide derivatives (compounds 1-32) were synthesised by reacting substituted salicylic acids and anilines. The chemical structures of these compounds were determined by (1)H-NMR, electrospray ionisation mass spectrometry (ESI-MS) and elemental analysis. The compounds were assayed for their antiproliferative activities against the Hep-G2 cell line by the 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Among the compounds tested, 22 and 28 showed the most favouable antiproliferative activities with 50% inhibitory concentration (IC(50)) values of 1.7 and 1.3 µM, respectively, which were comparable to the positive control of 5-fluorouracil (IC(50)=1.8 µM). A solid-phase ELISA assay was also performed to evaluate the ability of compounds 1-32 to inhibit the autophosphorylation of the epidermal growth factor receptor tyrosine kinase (EGFR TK). Docking simulations of 22 and 28 were carried out to illustrate the binding mode of the molecule into the EGFR active site, and the result suggested that both compounds 22 and 28 could bind the EGFR kinase well.

Metabolic uncoupling of Shewanella oneidensis MR-1, under the influence of excess substrate and 3, 3', 4', 5-tetrachlorosalicylanilide (TCS).

The dissociation between catabolism and anabolism is generally termed as metabolic uncoupling. Experimentally, metabolic uncoupling is characterized by a reduction in the observed biomass yield. This condition can be brought about by: (a) excess-substrate (as measured by S(0)/X(0)), and (b) addition of chemical uncouplers such as 3, 3', 4', 5-Tetrachlorosalicylanilide (TCS). An empirical model is proposed to quantify the uncoupling effects of both excess-substrate and uncoupler addition on the microbial cultures. Metabolic uncoupling of Shewanella oneidensis MR-1, under the influence of excess pyruvate and TCS, has been modeled using the proposed expression. The degree of uncoupling was measured as a fractional reduction in theoretical maximum observed yield. Excess-substrate was observed to successively reduce biomass yield as substrate concentration was increased. In the presence of TCS, conflicting trends were obtained for number yield and protein yield. This could, in part, be attributed to the observed increase in cellular protein content upon addition of TCS. Excess-substrate conditions dominated uncoupling, as compared to uncoupler addition. However, these two approaches were found to have additive effects and could, in conjunction, be employed to control biomass growth during microbial processes such as subsurface bioremediation and activated sludge treatment.

The binding of closantel to ovine serum albumin, and homogenate fractions of Haemonchus contortus.

Closantel binds to the serum proteins of the host and affects blood sucking parasites when they ingest the blood of treated hosts. Closantel binds specifically to ovine serum albumin (K(a) of 9. 3x10(6)M(-1)) at site I, the warfarin/phenylbutazone binding site of albumin Closantel also binds to invertebrate haemocyanin and haemolymph. The strongest binding of closantel in homogenates of H. contortus is found in fractions containing soluble proteins. This binding is of low affinity and, because the site itself is not fully denaturable, it may not be proteinaceous. There is no detectable difference in binding affinity between homogenate fractions from closantel susceptible and resistant isolates of adult or larval worms suggesting that closantel resistance is not due to changes in the closantel receptor or carrier.

The effect of feed intake level on the pharmacokinetic disposition of closantel in sheep.

Closantel (CLS), containing a trace of [14C]CLS, was administered intraruminally to sheep whose feed intake was maintained at either 800 or 400 g day-1. The kinetic disposition of [14C]metabolites was determined in rumen and abomasal fluid and particulate digesta and of CLS per se in plasma. The slower digesta flow rate in the sheep on low, compared with high, feed intake resulted in the proportion of the dose passing through the abomasum being reduced from 60 to 45%. Increased absorption of CLS from the rumen of sheep on low feed intake resulted in both higher maximum CLS concentration and greater area under CLS plasma concentration versus time curve, although the elimination half-life was independent of feed intake. Not only are the higher plasma CLS concentrations likely to increase efficacy against Haemonchus contortus, the threshold concentrations that are considered to inhibit the establishment of ingested H. contortus larvae were extended by 10-14 days. The extended CLS presence after reduced feeding, when integrated with parasite treatment programmes, provides an opportunity to reduce the impact of H. contortus infection.

Haemonchus contortus: the uptake and metabolism of closantel.

Closantel is an anthelmintic which associates with plasma albumin and is useful for the control of sheep parasites, such as Haemonchus contortus, that ingest blood. However, the utility of closantel for parasite control has been threatened by the emergence of resistance. The mechanisms of resistance are unknown. A closantel-resistant and a closantel-susceptible isolate of H. contortus were compared with respect to the distribution and metabolism of closantel. Neither strain appeared to metabolise closantel in vitro or in vivo. Following treatment of infected sheep with radioactively labelled closantel, isotope levels in closantel-resistant adult H. contortus were significantly lower than in susceptible worms. This reduced accumulation of drug could contribute to closantel resistance by mechanisms such as reduced feeding, failure to dissociate the drug-albumin complex in the gut or increased efflux of closantel from resistant worms.

Studies on tetrachloroethene respiration in Dehalospirillum multivorans.

Tetrachloroethene (PCE) respiration was studied in the tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, with respect to localization of the catabolic enzymes, the electron carriers potentially involved in electron transport, and the response to ionophores and specific inhibitors. Hydrogenase and formate dehydrogenase were recovered in the periplasmic cell fraction and were membrane-associated. Electron-accepting tetrachloroethene dehalogenase was found in the cytoplasmic fraction. In the PCE dehalogenase assay, only artificial electron donors with a standard redox potential of D. multivorans (Eo' = -445 mV) could serve as electron donor for PCE reduction. However, the reaction rate with ferredoxin was only 1% of that with methyl viologen, whereas the pyruvate-ferredoxin oxidoreductase exhibited almost the same reaction rates with methyl viologen and ferredoxin as electron acceptors for pyruvate oxidation. Reduced menadione (2-methyl-1, 4-naphthoquinone) did not serve as electron donor in the PCE dehalogenase reaction. 2-Heptyl-4-hydroxyquinoline-N-oxide (HOQNO) had no significant effect on PCE dechlorination in cell suspensions and in crude extracts. Whole cells catalyzed the reductive dechlorination of PCE with H2 or formate as electron donors. The dechlorination in cell suspensions rather than in cell extracts was inhibited by the ionophores carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP) and tetrachlorosalicylanilide (TCS), indicating that a membrane potential and/or a pH gradient may be required for the reaction in vivo.

Simultaneous determination of five fasciolicides in milk by liquid chromatography with electrochemical detection.

A sensitive and specific method is described for determination of 5 fasciolicides in milk. The drugs are used to control liver flukes in cattle. The milk sample was homogenized with acetone and acetonitrile, sonicated, and centrifuged. The supernatant was extracted with dichloromethane. The extract was evaporated to dryness, dissolved in 1% sodium hydrogen carbonate, and purified on a C18 cartridge. The 5 drugs were separated from the matrix by reversed-phase liquid chromatography (LC) and determined by dual-electrode coulometric detection on a Kaseisorb LC ODS-300-5 column. The mobile phase was acetonitrile-0.05M potassium dihydrogen phosphate (55 + 45) at pH 3.0. The flow rate was 1 mL/min at 40 degrees C. The applied potentials of detectors 1 and 2 were set at 0.20 and 0.55 V, respectively. The average recovery of the drugs added to milk at 0.01 and 0.1 micrograms/mL was 89.6%, and the coefficient of variation was 4.7%. The detection limits of the drugs in milk were 4-20 ng/mL. The method is used to monitor commercial milk samples and to determine the residual levels of these drugs in milk from cows treated with a fasciolicide.

Closantel intoxication in a dog.

A case of overdosage with closantel, a salicynalide derivative, in a dog is described. The dog received 6 times the recommended dosage. Closantel induced optic neuritis, retinal degeneration, partial deafness, hepatotoxicosis and myopathy. Only the blindness was irreversible. The therapy included albumin administration to reduce the acute toxicity of closantel.