Discovery of novel antigiardiasis drug candidates.

Giardiasis is a severe intestinal parasitic disease caused by Giardia lamblia, which inflicts many people in poor regions and is the most common parasitic infection in the United States. Current standard care drugs are associated with undesirable side effects, treatment failures, and an increasing incidence of drug resistance. As follow-up to a high-throughput screening of an approved drug library, which identified compounds lethal to G. lamblia trophozoites, we have determined the minimum lethal concentrations of 28 drugs and advanced 10 of them to in vivo studies in mice. The results were compared to treatment with the standard care drug, metronidazole, in order to identify drugs with equal or better anti-Giardia activities. Three drugs, fumagillin, carbadox, and tioxidazole, were identified. These compounds were also potent against metronidazole-resistant human G. lamblia isolates (assemblages A and B), as determined in in vitro assays. Of these three compounds, fumagillin is currently an orphan drug used within the European Union to treat microsporidiosis in immunocompromised individuals, whereas carbadox and tioxidazole are used in veterinary medicine. A dose-dependent study of fumagillin in a giardiasis mouse model revealed that the effective dose of fumagillin was ∼ 100-fold lower than the metronidazole dose. Therefore, fumagillin may be advanced to further studies as an alternative treatment for giardiasis when metronidazole fails.

Determination of carbadox and olaquindox metabolites in swine muscle by liquid chromatography/mass spectrometry.

This paper presents LC-MS/MS method that was developed for the simultaneous determination and confirmation metabolites of carbadox (desoxycarbadox, quinoxaline-2-carboxylic) and olaquindox (3-methylquinoxaline-2-carboxylic acid) residues in pig muscle tissues at concentrations ≤3.0µgkg(-1). Pig muscle tissues were deproteinated with meta-phosphoric acid in methanol and then were extracted with ethyl acetate:dichloromethane (50:50, v/v). The whole extracts were evaporated to dryness in rotary evaporator at 45°C, and dry residues were re-dissolved in 0.5% isopropanol in 1% acetic acid. The LC separation was performed on a C8 column with a gradient system consisting of isopropanol/water/acetic acid and methanol as the mobile phase. Additionally SelexION™ technology to reduce matrix effect was used. The decision limit (CCα) ranged from 1.04µgkg(-1) to 2.11µgkg(-1) and the detection capability (CCß) ranged from 1.46µgkg(-1) to 2.89µgkg(-1). The total recoveries were from 99.8% to 101.2%. The results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC.

Complexation facilitated reduction of aromatic N-oxides by aqueous Fe(II)-tiron complex: reaction kinetics and mechanisms.

Rapid reduction of carbadox (CDX), olaquindox and several other aromatic N-oxides were investigated in aqueous solution containing Fe(II) and tiron. Consistent with previous work, the 1:2 Fe(II)-tiron complex, FeL2(6-), is the dominant reactive species as its concentration linearly correlates with the observed rate constant kobs under various conditions. The N-oxides without any side chains were much less reactive, suggesting direct reduction of the N-oxides is slow. UV-vis spectra suggest FeL2(6-) likely forms 5- or 7-membered rings with CDX and olaquindox through the N and O atoms on the side chain. The formed inner-sphere complexes significantly facilitated electron transfer from FeL2(6-) to the N-oxides. Reduction products of the N-oxides were identified by HPLC/QToF-MS to be the deoxygenated analogs. QSAR analysis indicated neither the first electron transfer nor N-O bond cleavage is the rate-limiting step. Calculations of the atomic spin densities of the anionic N-oxides confirmed the extensive delocalization between the aromatic ring and the side chain, suggesting complex formation can significantly affect the reduction kinetics. Our results suggest the complexation facilitated N-oxide reduction by Fe(II)-tiron involves a free radical mechanism, and the subsequent deoxygenation might also benefit from the weak complexation of Fe(II) with the N-oxide O atom.

The metabolism of carbadox, olaquindox, mequindox, quinocetone and cyadox: an overview.

The aim of this article is to get an overview of the metabolism of quinoxaline 1,4-di-N-oxides (QdNOs) used in food animals. The derivatives of QdNOs (carbadox, olaquindox, mequindox, quinocetone, and cyadox) are the potent synthetic antimicrobial agents that are used for improving the feed efficiency and controlling dysentery in food-producing animals. Studies have demonstrated that the toxicity of QdNOs is closely associated with the production of their metabolism, especially with the production of their reduced metabolites. To the best of our knowledge, no one has systematically compiled the metabolism data of QdNOs. Therefore, the metabolism of QdNOs in animals has been discussed in the review for the first time. These drugs undergo extensive metabolism prior to excretion. N-oxide group reduction is the major metabolic pathway of QdNOs. Moreover, the N1- and N4-oxide reductions of QdNOs by different reducing mechanisms are also described. Obvious differences in metabolic pathways for QdNOs were observed owing to the differences on the side chain of these drugs. Therefore, understanding the metabolic pathways of QdNOs in animals will provide the guides for further studies of metabolism and toxicology of these drugs, and will also provide abundant information for the food safety assessment.

Reduction of carbadox mediated by reaction of Mn(III) with oxalic acid.

Manganese(III) geocomponents are commonly found in the soil environment, yet their roles in many biogeochemical processes remain unknown. In this study, we demonstrated that Mn(III) generated from the reaction of MnO(2) and oxalic acid caused rapid and extensive decompositions of a quinoxaline-di-N-oxide antibiotics, viz carbadox. The reaction occurred primarily at the quinoxaline-di-N-oxide moiety resulting in the removal of one -O from N1-oxide and formation of desoxycarbadox. The reaction rate was accelerated by increasing amounts of Mn(III), carbadox and oxalate. The critical step in the overall reaction was the formation of a quinoxaline-di-N-oxide/Mn(III)/oxalate ternary complex in which Mn(III) functioned as the central complexing cation and electron conduit in which the arrangement of ligands facilitated electron transfer from oxalate to carbadox. In the complex, the C-C bond in oxalate was cleaved to create CO(2)(-•) radicals, followed by electron transfer to carbadox through the Mn(III) center. This proposed reaction mechanism is supported by the reaction products formed, reaction kinetics, and quantum mechanical calculations. The results obtained from this study suggest that naturally occurring Mn(III)-oxalic acid complexes could reductively decompose certain organic compounds in the environment such as the antibiotic quinoxaline-di-N-oxide.

Identification of carbadox metabolites formed by liver microsomes from rats, pigs and chickens using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry.

Carbadox (methyl-3-(2-quinoxalinylmethylene)-carbazate-N(1),N(4)-dioxide) is a chemotherapeutic growth promoter added to feed for starter pigs. In this work, the metabolism of carbadox in rat, pig and chicken liver microsomes has been studied firstly. The incubation mixtures were then processed and analyzed for metabolites with a sensitive and reliable method based on high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). With the help of chromatographic behavior and accurate mass measurements, it is possible to rapidly and reliably characterize the metabolites of carbadox. The structural elucidations of these metabolites were performed by comparing the changes in the accurate molecular masses and fragment ions generated from precursor ions with those of parent drug. The present results showed that the metabolism of carbadox in liver microsomes had qualitative species-difference. A total of seven metabolites were identified in rat liver microsomes. Five metabolites (Cb1-Cb3, Cb5, Cb7) were observed in pig and chicken liver microsomes. In addition, metabolite Cb6 was also detected in chicken liver microsomes. The peak areas of the metabolites in the three species are different. For the formations of Cb1, Cb2, Cb5 and Cb6, the rank order was rat>chicken>pig; Cb3; pig~chicken>rat. Cb1, Cb2 and Cb3 have been previously reported, whereas the other four metabolites were novel. The N→O group reduction and hydroxylation followed by N→O group reduction were the main metabolic pathways for carbadox in the three species.

Reaction kinetics and transformation of carbadox and structurally related compounds with aqueous chlorine.

The potential release of carbadox (CDX), a commonly used antibacterial agent in swine husbandry, into water systems is of a concern due to its carcinogenic and genotoxic effects. Until this study, the reactivity of carbadox (possessing quinoxaline N,N'-dioxide and hydrazone moieties) toward aqueous chlorine has yetto be investigated in depth. Chemical reactivity, reaction kinetics, and transformation pathways of carbadox and structurally related compounds with free chlorine under typical water treatment conditions were determined. This study found that only CDX and desoxycarbadox (DCDX), a main metabolite of CDX with no ring N-oxide groups, react rapidly with free chlorine while other structurally related compounds including olaquindox, quindoxin, quinoxaline N-oxide, quinoxaline, and quinoline N-oxide do not. The reaction kinetics of CDX and DCDX with chlorine are highly pH dependent (e.g., the apparent second-order rate constant, kapp, for CDX ranges from 51.8 to 3.15 x 10(4) M(-1)s(-1) at pH 4-11). The high reactivity of CDX and DCDX to chlorine involves deprotonation of their hydrazone N-H moieties where initial chlorine attack results in a reactive intermediate that is further attacked by nucleophiles in the matrix to yield non-chlorinated, hydroxylated, and larger molecular weight byproducts. All of the CDX's byproducts retain their biologically active N-oxide groups, suggesting that they may remain as active antibacterial agents.

Oxidation of sulfonamides, macrolides, and carbadox with free chlorine and monochloramine.

The oxidation of 10 antibiotics-carbadox, erythromycin-H(2)O, roxithromycin, sulfadimethoxine, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxazole, sulfathiozole, and tylosin during chlorination and monochloramination in laboratory and surface waters was investigated to identify kinetics and treatment effectiveness. A kinetic model that incorporates pH-based speciation of both oxidant species and sulfonamide antibiotics was developed and validated. Specific rate constants for the individual ionic species were developed for the dominant reactant pairs. Liquid chromatography/mass spectrometry, preceded by solid phase extraction, was used to analyze antibiotics in kinetic experiments. With experimental conditions of 25 degrees C and reaction times of up to 2 h, an initial concentration of 1 mg/L of free chlorine removed an average of 88 percent of the antibiotics over a pH range of 6.1-9.1. Monochloramine was less effective at typical drinking water dosage concentrations of 3 mg/L, with average removals of 35, 10, and 0 percent at a pH of 6.1, 7.6, and 9.1, respectively.

Sorption and related properties of the swine antibiotic carbadox and associated N-oxide reduced metabolites.

Carbadox (CBX) (methyl 3-[2-quinoxalinylmethylene]-carbazate N1, N4 dioxide) is a chemotherapeutic growth promoter and antibacterial drug added to feed for starter pigs. Toxicity of CBX and at least one of its metabolites (bis-desoxycarbadox; DCBX) has resulted in a number of studies regarding its stability and residence time in edible swine tissue; however, little is known on its environmental fate pertinent to the application of antibiotic-laden manure to agricultural fields. We measured sorption of CBX and DCBX by soils, sediment, and homoionic clays from 10 mM KCl and 5 mM CaCl2 solutions, sorption of two N-oxide reduced metabolites (N4 and N1) by a subset of soils from 5 mM CaCl2, octanol-water partition coefficients (Kow) of CBX and all three metabolites, and CBX solubility in water and mixed solvents. Sorption appeared well-correlated to organic carbon (OC) for the soils (e.g., log (Koc, L/kg OC) = 3.96 +/- 0.18 for CBX). However, sorption was enhanced in the presence of K+, competitive sorption from the metabolites was observed, and sorption by clay minerals was large (approximately 10(5) L/kg for SWy(-1)). Sorption by clays was inversely correlated to surface charge density (e.g., sorption decreased from 10(5) to 10 L/kg as charge density increased from 1 to 2 micromolc/m2), similar to what has been observed for nitroaromatic compounds. In the absence of a clay surface, hydrophobic-type forces dominated with Kow values and reverse-phase chromatographic retention times increasing with the loss of oxygen from the aromatic nitrogens. Therefore, it is likely that both OC and clay contribute significantly to sorption of carbadox and related metabolites by soils with relative contributions most dependent on clay type.