Physiology and therapeutics of the free fatty acid receptor GPR40.

The G protein-coupled receptor 40 (GPR40) was deorphanized in 2003 as a receptor of medium- and long-chain free fatty acids (FFAs), now also called FFA receptor 1 (FFAR1). Studies have shown that GPR40 not only directly mediates FFA amplification of glucose-stimulated insulin secretion but also indirectly enhances insulin secretion by stimulating incretin release. Therefore, GPR40 has attracted considerable attention as a therapeutic drug target of type 2 diabetes mellitus, and numerous GPR40 ligands have been developed and investigated for their antidiabetic actions. Recently, one of these ligands, TAK-875, has been successfully tested in phase II clinical trials with reduced risk of hypoglycemia. This chapter will summarize studies on GPR40, including its molecular cloning and tissue distribution, physiology, pharmacology, and pathophysiology.

Genotoxicity evaluation of Mequindox in different short-term tests.

Quinoxaline-1,4-dioxides (QdNOs) are the potent heterocyclic N-oxides with interesting biological properties such as antibacterial, anticandida, antitubercular, anticancer and antiprotozoal activities. Here, we tested and compared the mequindox (MEQ) for mutagenic abilities in a battery of different short term tests according to OECD guidelines. When compared with the controls, a strong mutagenicity of MEQ and carbadox (CBX) was observed with an approximate concentration-effect relationship in Salmonella reverse mutation test, chromosome aberration test, unscheduled DNA synthesis assay and HGPRT gene mutation test, in the absence and presence of S(9)-mix. In in vivo micronucleus test, CBX produced significant increase in the proportion of micronucleus formation than MEQ in mice bone marrow cells. From these results, we can conclude that MEQ had a strong genotoxic potential to mammalian cells in vitro as well as in vivo and its mutagenicity is slightly higher than CBX. Our results, for the 1st time, discuss the genotoxic potential of MEQ. These results not only confirm the earlier findings about CBX but also extend the knowledge and awareness about the genotoxic risk of QdNO derivatives.

Two generation reproduction and teratogenicity studies of feeding quinocetone fed to Wistar rats.

To investigate the reproductive toxicity and teratogenic potential of quinocetone, a growth promoting agent, Wistar rats were fed different diets containing 0, 50, 300 and 1800 mg/kg quinocetone or 300 mg/kg olaquindox. Groups of 15 males and 30 females (F(0)) were fed through a 10-week prebreed period as well as during mating, gestation, parturition and lactation. At weaning, 12 males and 24 females of F(1) generation weanlings per group were selected randomly as parents for F(2) generation. Selected F(1) weanlings were exposed to the same diet and treatment as their parents. At the highest quinocetone group, body weights in F(0) and F(1) rats, fetal body weight on day 21 after birth and number of viable fetuses in F(0) and F(1) generation significantly decreased. In teratogenicity study, groups of 12 males and 24 females were fed with the same diets through a 12-week prebreed period and matting periods. Pregnant rats were subjected to cesarean section on GD 20 for teratogenic examination. At the highest quinocetone group, body weights and feed efficiency, fetal body lengths, tail lengths, litter weights and number of viable fetuses significantly decreased. The NOAEL for reproduction/development of quinocetone for rats was estimated to be 300 mg/kg diet.

Population pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in chicken based on retrospective data, incorporating first-pass metabolism.

A population pharmacokinetic (PPK) model for enrofloxacin and its metabolite ciprofloxacin in chicken based on retrospective data was developed. Plasma concentrations of enrofloxacin and its metabolite ciprofloxacin were determined in blood samples from chicken administered either enrofloxacin via oral and intravenous routes or ciprofloxacin via intravenous injection. The disposition of enrofloxacin and ciprofloxacin was described simultaneously by an integrated mathematic model. Two compartments were used to describe the enrofloxacin and ciprofloxacin disposition profiles. The formation of ciprofloxacin was through the central compartment of enrofloxacin. The integrated model was estimated with nonlinear mixed effects model (NONMEM). The total clearance of enrofloxacin (CLEN) and ciprofloxacin (CLCP) was 0.613 L/h and 1.15 L/h, respectively. Correlation between CLEN, the central compartment volume of distribution for enrofloxacin (V2) and CLCP was estimated. After intravenous administration of enrofloxacin, the transformation rate of enrofloxacin to ciprofloxacin was 0.429 L/h. The bioavailability factor after oral administration was 0.926, and 12.6% of enrofloxacin after oral administration was transformed to ciprofloxacin via first-pass effect. Pharmacodynamic (PD) evaluation was performed using area under concentration time curve of active moiety from 0 to 24 h and MIC collected from literature. This study is the first one to use PPK method to investigate parent drug and its metabolite disposition and PDs using an integrated model in veterinary medicine.

The metabolism and N-oxide reduction of olaquindox in liver preparations of rats, pigs and chicken.

Olaquindox, N-(2-hydroxyethyl)-3-methyl-2-quinoxalinecarboxamide-1,4-di-N-oxide, is one of the quinoxaline-dioxides used widely as an antimicrobial growth promoter in pig production. Its toxicities were reported to be closely related to the formation of N-oxide reductive metabolites. The present study presents the metabolism and N-oxide reduction of olaquindox incubated with liver microsomes and liver cytosol of rats, pigs and chicken. Metabolites were identified and characterized with a novel LC/MS-ITTOF. Thirteen metabolites were found in liver microsomes of rats, three of which were identified to be novel. Seven metabolites were found in liver microsomes of pigs and chicken. The N-oxide reduction was the major metabolic pathway of olaquindox in liver microsomes of the three species. The N1-reduction of olaquindox to metabolite O2 was found in not only liver microsomes but also cytosol of the three species in the presence of NAD(P)H under hypoxic conditions. The N1-reduction could be inhibited by air and carbon monoxide, and be significantly stimulated by riboflavin under various conditions. The N1-reduction in the liver cytosol of rats and pigs could be enhanced by menadione, but the reduction in liver cytosol of chicken could not be. The N1-reduction activities in all animals were not abolished when liver microsomes and cytosol were boiled. These findings suggested that the N1-reduction of olaquindox could be mediated by non-enzymatic and enzymatic conditions. This N1-reduction of olaquindox could also be catalyzed by a quinone-dependent reducing system in liver cytosol of rats and pigs. Moreover, liver cytosol of rats and pigs had an ability of N4-reduction that catalyzed olaquindox to metabolite O1 in the presence of benzaldehyde under hypoxic conditions, but the liver cytosol of chicken did not. The N4-reduction could be inhibited markedly in the cytosol rats and pigs by menadione, chlorpromazine and promethazine. In addition, 7-hydroxycoumarin was also found to inhibit the formation of O1 in the cytosol of rats. The inhibitory results suggested that the N4-reduction might be catalyzed by aldehyde oxidase in the cytosol of pigs, and by aldehyde oxidase and xanthine oxidase in the cytosol of rats. In conclusion, the N1-reduction and N4-reduction of olaquindox are mediated by multiple mechanisms and significant species differences are involved in both reductions. This work is a contribution to the understanding of toxicities and the relativities between toxicities and metabolism of olaquindox.

Application of electrospray ionization hybrid ion trap/time-of-flight mass spectrometry in the rapid characterization of quinocetone metabolites formed in vitro.

The application of electrospray ionization hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography (LC/MS-IT-TOF) in the rapid characterization of in vitro metabolites of quinocetone was developed. Metabolites formed in rat liver microsomes were separated using a VP-ODS column with gradient elution. Multiple scans of metabolites in MS and MS(2) modes and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only a 30-min analysis. Most measured mass errors were less than 10 ppm for both protonated molecules and fragment ions using external mass calibration. The elemental compositions of all fragment ions of quinocetone and its metabolites could be rapidly assigned based upon the known compositional elements of protonated molecules. The structure of metabolites were elucidated based on the combination of three techniques: agreement between their proposed structure, the accurate masses, and the elemental composition of ions in their mass spectra; comparison of their changes in accurate molecular masses and fragment ions with those of parent drug or metabolite; and the elemental compositions of lost mass numbers in proposed fragmentation pathways. Twenty-seven phase I metabolites were identified as 11 reduction metabolites, three direct hydroxylation metabolites, and 13 metabolites with a combination of reduction and hydroxylation. All metabolites except the N-oxide reduction metabolite M6 are new metabolites of quinocetone, which were not previously reported. The ability to conduct expected biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurement, all in a single experimental run, is one of the most attractive features of this methodology. The results demonstrate the use of LC/MS-IT-TOF approach appears to be rapid, efficient, and reliable in structural characterization of drug metabolites.

Long-term dose-dependent response of Mequindox on aldosterone, corticosterone and five steroidogenic enzyme mRNAs in the adrenal of male rats.

Mequindox (MEQ) is a synthetic quinoxaline 1,4-dioxides (QdNOs) derivative which can effectively improve growth and feed efficiency in animals. This study was to investigate the dose-dependent long-term toxicity in the adrenal of male rats exposed to 180 days of MEQ feed. Our data demonstrated that high doses of MEQ in the diet for 180 days led to adrenal damage and steroid hormone decrease, combined with sodium decrease and potassium increase in rat plasma. Significant changes of GSH and SOD in plasma were observed in the high doses (110, 275 mg/kg) groups. At the same doses, MEQ treatment down-regulated the mRNA levels of CYP11A1, CYP11B1 and CYP11B2 which located in mitochondria, but up-regulated mRNA levels of CYP21 and 3beta-HSD which located in endoplasmic reticulum. In conclusion, we reported the dose-dependent long-term toxicity of MEQ on adrenal gland in male rats, which raise awareness of its toxic effects to animals and consumers, and its mechanism may involve in oxidative stress and steroid hormone biosynthesis pathway.