Effects of housing conditions on behaviors and biochemical parameters in juvenile cynomolgus monkeys (Macaca fascicularis).

To investigate the effects of environmental enrichment on laboratory monkeys, we studied behavioral and physiological differences following changes in housing conditions. Ten male and female juvenile cynomolgus monkeys were first housed in pairs for 8 weeks after quarantine/acclimatization (singly housed) and subsequently housed alone for the next 8 weeks. Monkeys were subjected to evaluations of body weight gain, stereotypic or affiliative behaviors, cortisol, 4-ethylphenyl sulfate (4EPS) and catecholamine concentrations in biological samples, and blood chemistry tests under both housing conditions. Under paired housing, the stereotypic behavioral score decreased in both sexes, and the affiliative behavioral score increased in males and showed an increasing trend in females. Under single housing, the stereotypic score increased in both sexes, and the affiliative score decreased in males. Paired housing decreased serum calcium and urine cortisol concentrations in both sexes and decreased plasma cortisol in males and plasma 4EPS concentrations in females. The stereotypic score was positively correlated with serum calcium, plasma and urine cortisol, and plasma 4EPS concentration and negatively correlated with the affiliative score. The feces painting score, affiliative score, and plasma cortisol and serum calcium concentrations showed sex differences, suggesting differences in responsiveness to environmental changes between males and females. In conclusion, paired housing improved behavioral abnormalities in juvenile cynomolgus monkeys, suggesting that it may be an effective environmental enrichment paradigm. Calcium, cortisol, and 4EPS concentrations in biological samples may be useful indices for evaluating the effects of environmental enrichment.

Coordinated roles of pregnane X receptor and constitutive androstane receptor in autoinduction of voriconazole metabolism in mice.

The antifungal efficacy of voriconazole (VRC) differs among host species, with potent efficacy in humans but less in rodents. We investigated the possible involvement of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in the species-specific efficacy of VRC through pharmacokinetic analyses using genetically modified mice and primary human hepatocytes. VRC (30 mg/kg) was orally administered to wild-type, Pxr-null, Car-null, and Pxr- and Car-null (Pxr/Car-null) mice for 7 days. Hepatic VRC metabolism was significantly increased by VRC administration, and the elimination rates of plasma VRC were much higher on day 7 than on day 1 in wild-type mice. This autoinduction was also observed in Pxr-null and Car-null mice but not in Pxr/Car-null mice, suggesting coordinated roles of PXR and CAR in the autoinduction of VRC metabolism in mice. Hepatic Cyp3a11 mRNA levels were increased by VRC administration, hepatic metabolic activities for VRC were correlated with CYP3A activities, and the induced VRC metabolism was inhibited by ketoconazole (a CYP3A inhibitor). In primary human hepatocytes, VRC barely increased mRNA levels of CYP3A4 and CYP2B6 (human PXR/CAR target genes) at its therapeutic concentrations. In conclusion, these results suggest that VRC is metabolized mainly by CYP3A11 in mouse livers and that PXR- and CAR-mediated CYP3A11 induction, namely, autoinduction of VRC metabolism, is a primary reason for the ineffectiveness of VRC in mice. A limited ability of VRC to activate human PXR/CAR at its clinical concentration might explain the VRC efficacy in humans. Therefore, the ability to activate PXR/CAR might determine the VRC efficacy in different mammalian species.

Strategy for structural elucidation of drugs and drug metabolites using (MS)n fragmentation in an electrospray ion trap.

Triple-stage quadrupole (TSQ) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and ion trap ESI-MS/MS can be used to cleave protonated molecules to produce carbocations and neutral molecules in the positive ion mode. Dissociation products which correspond to protonated forms of neutral fragment molecules can also be trapped and detected. These protonated molecules in turn can cleave via carbocation cleavage, ipso cleavage, onium cleavage or McLafferty or related rearrangements. One can elucidate the structures of metabolites from the differences in m/z ratios of the fragments arising from the original drug compound and its metabolite. This strategy for structural elucidation is further facilitated by estimates of the reactivity of drugs with oxygen diradicals involved in cytochrome P-450 cycles.

New SRM data dependent exclusion (MS)n measurement for structural determination of drug metabolites using LC/ESI/Ion trap MS.

New SRM (selected reaction monitoring) data dependent exclusion (MS)(n) measurement makes it possible to obtain MS(3) fragmentation data for all MS(2) fragments, useful for structural determination of drug metabolites using ESI ion trap. MS(2) fragments are produced by cleavage of all protonated molecules at the lone electron pairs of heteroatoms or the pi electrons of double and triple bonds, benzene rings and hetero-rings of drugs. Usually, data dependent MS(3) measurement cleaves only MS(2) fragment of highest intensity, that normally does not contain important metabolic sites. Fragmentation data from all parts of drug metabolites is required to determine structure. In addition to the usual basic measurement of protonated molecules and (MS)(n) fragmentation of drug metabolites, we demonstrate the use of SRM data dependent (MS)(n) measurement, plus new SRM data dependent exclusion (MS)(n) measurement for structural determination of metabolites.

Strategy for structure elucidation of drug metabolites derived from protonated molecules and (MS)n fragmentation of zotepine, tiaramide and their metabolites.

TSQ ESI MS/MS and ion trap ESI MS(2) cleave protonated molecules. MS(2) at m/z 332 of zotepine cleaved m/z 245 (10%), m/z 287 (5%) and m/z 315 (100%) fragment ions at protonated positions. MS(2) at m/z 356 of tiaramide cleaved m/z 338 (18%), 313 (10%), 226 (100%), 198 (78%) and 131 (60%) fragment ions at protonated positions. The ESI ion trap MS produced new internal protonated molecules in an ion trap, such as m/z 113 and m/z 88 from m/z 131 protonated piperazinonium, and m/z 245 protonated 8-chloro dibenzo[b,f]thiepin. ESI ion trap (MS)(n) (n>or=3) cleaved new internal protonated molecules. It also causes carbocation cleavage, alpha cleavage, onium cleavage and McLafferty cleavage. We can easily elucidate the structure of metabolites from the difference in m/z of corresponding fragments between unchanged compound and its metabolite. Reactive oxygen diradicals involved in cytochrome P-450 cycles react with electron rich groups and reactive C-H bonds of zotepine and tiaramide to produce metabolites of 2-hydroxyzotepine, 3-hydroxyzotepine, norzotepine, zotepine-N-oxide, zotepine-S-oxide, Tiaramide carboxylic acid, dehydroxyethyltiaramide and tiaramide-N-oxide. The strategy for structure elucidation of drug metabolites was established on the basis of the reactivity of unchanged drug with reactive oxygen diradicals involved in cytochrome P-450 cycles and theory associated with protonated molecules and (MS)(n) fragmentation of drug metabolites.