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.

The pharmacodynamics of the flukicidal salicylanilides, rafoxanide, closantel and oxyclosanide.

The pharmacokinetics of oxyclosanide, rafoxanide and closantel were investigated in sheep (n = 5). All three drugs were extensively (greater than 99%) bound to plasma proteins and the plasma concentration/time curve was best described by a tri-exponential equation. Closantel and rafoxanide had long terminal half-lives (mean 14.5 and 16.6 days, respectively) compared with oxyclosanide (mean 6.4 days). In a study of the efficacy of rafoxanide against Fasciola hepatica, a dose rate of 7.5 mg kg-1 against 6-week-old flukes appeared to be similarly effective to a dose rate of 2.5 mg kg-1 against 10-week-old flukes (86% and 88% efficacy, respectively), as assessed at autopsies carried out on all sheep when the flukes were 14 weeks old. Part of this putative efficacy against immature flukes may be due to rafoxanide persisting in the plasma and affecting the mature flukes when they reach the bile ducts.

Incidence of experimental fascioliasis on hepatic disposition of [3H]tetracycline and [14C]rafoxanide in rats.

[7-3H]Tetracycline and [carbonyl-14C]rafoxanide were injected intravenously into anesthetized controls and rats in which experimental fascioliasis had been induced by 20 Fasciola hepatica metacercariae. The biliary excretion (1 and 3 h, respectively) of the radioactivity consisted of approximately 4% of the administered dose. In 4-week infested rats, biliary excretion of [3H]tetracycline and hepatic levels of radioactivity were decreased, whereas bile flow did not vary and plasma clearance of the antibiotic was significantly decreased in comparison with control animals. These differences could be the result of the fascioliasis-induced decrease in the hepatic uptake of tetracycline and the limited active transport for its output into bile canaliculi. No change in [14C]rafoxanide disposition was shown in infested rats.

[Metabolism, residue and excretion of the anthelmintic 131 I-rafoxanide in blood, milk, meat and urine of lactating cows]. / Metabolismus, Rückstände und Ausscheidung des Anthelminithikums 131J-Rafoxanid in Blut, Milch, Fleisch und Urin am laktierenden Rind.

Rafoxanide, labelled by 131J, was applied orally in the formulation "Ursovermit" to 2 lactating cows, 5 mg/kg bodymass, resp. Residues and halflifes in blood, milk and meat and the excretion in urine are given, related to 131J in the chloroform extract. 3,5-diiodosalicyclic acid was found as metabolite in amounts of 1-2,5%, relative to rafoxanide, resp.

[Residue determinations in milk following application of the fasciolicides rafoxanide and hexachloro-p-xylene]. / Rückstandsuntersuchungen bei Milch nach Anwendung der Fasziolizide Rafoxanid und Hexachlor-p-xylol.

The control of Fasciola hepatica in dairy cattle may lead to considerable residue problems in dairying. In the GDR, a hexachloro-p-xylene-based fasciolicide has been approved till recently, and another, rafoxanide-based fasciolicide has been on trial. The investigations have shown that the two active principles are excreted in the milk for a prolonged period, rafoxamide being found more suitable. Their marked lipophil behaviour leads to accumulation in the fat phase and, thus, in high-fat milk products.

[Degradation and residue dynamics of the anthelminthic 131J-rafoxanide in cattle blood, milk, meat and fat after various dosages]. / Abbau und Rückstandsdynamik des Anthelminthikums 131J-Rafoxanid in Blut, Milch, Fleisch und Fett bei unterschiedlicher Dosierung am Rind.

Rafoxanide, labelled by 131J, was applied orally in oily solution to cattle, 2,4 and 12 mg/kg bodymass, resp. The halflifes of degradation in blood is depending on the dosage and is 7 days related to the therapeutical dosage of 5 mg/kg bodymass against Fasciola hepatica. Residues and halflifer in blood, milk, meat and fat are given, related to 131J in the chloroform extract. Rafoxanide does not show persistence following oral application to cattle.

[Degradation and elimination of the anthelminitic 131I-rafoxanid in cattle and sheep]. / Untersuchungen zum Abbau und zur Ausscheidung des Anthelminthikums 131J-Rafoxanid an Rind und Schaf

Rafoxanide, labelled by 131J, was applied orally in oily solution to cattle and sheep, 12 and 17 mg/kg bodymass, resp. The half time of 131J in blood in 12,5 and 10,5 d for sheep and cattle, resp., the compound is adsorbed to proteins in blood. In the milk a maximal level of 3-3,5 ppm (calculated as total 131J) after 4-6 d was measured. More detailed results regarding the metabolism of rafoxanide are hitherto not possible.