Metabolomics reveals a novel vitamin E metabolite and attenuated vitamin E metabolism upon PXR activation.

Pregnane X receptor (PXR) is an important nuclear receptor xenosensor that regulates the expression of metabolic enzymes and transporters involved in the metabolism of xenobiotics and endobiotics. In this study, ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS), revealed altered urinary metabolomes in both Pxr-null and wild-type mice treated with the mouse PXR activator pregnenolone 16alpha-carbonitrile (PCN). Multivariate data analysis revealed that PCN significantly attenuated the urinary vitamin E metabolite alpha-carboxyethyl hydroxychroman (CEHC) glucuronide together with a novel metabolite in wild-type but not Pxr-null mice. Deconjugation experiments with beta-glucuronidase and beta-glucosidase suggested that the novel urinary metabolite was gamma-CEHC beta-D-glucoside (Glc). The identity of gamma-CEHC Glc was confirmed by chemical synthesis and by comparing tandem mass fragmentation of the urinary metabolite with the authentic standard. The lower urinary CEHC was likely due to PXR-mediated repression of hepatic sterol carrier protein 2 involved in peroxisomal beta-oxidation of branched-chain fatty acids (BCFA). Using a combination of metabolomic analysis and a genetically modified mouse model, this study revealed that activation of PXR results in attenuated levels of the two vitamin E conjugates, and identification of a novel vitamin E metabolite, gamma-CEHC Glc. Activation of PXR results in attenuated levels of the two vitamin E conjugates that may be useful as biomarkers of PXR activation.

Strong inhibition of estrone-3-sulfatase activity by pregnenolone 16alpha-carbonitrile but not by several analogs lacking a 16alpha-nitrile group.

In recent years, development of potent inhibitors for estrogen sulfatases has become an actively pursued strategy for chemoprevention and/or chemotherapy of estrogen-dependent human breast cancers. We report here our findings that pregnenolone 16alpha-carbonitrile (PCN) is a potent inhibitor of estrone-3-sulfatase activity of rats and also humans. PCN inhibited in a concentration-dependent manner the desulfation of estrone-3-sulfate catalyzed by liver microsomal and nuclear fractions of female Sprague-Dawley rats. The inhibition of estrone-3-sulfatase activity in these two subcellular fractions showed a biphasic pattern, with a highly sensitive phase seen at 78 nM to 1.25 microm of PCN followed by a markedly less-sensitive phase at > 2.5 microm of PCN. Interestingly, several of PCN's structural analogs without a 16alpha-nitrile group showed little or no inhibitory effect on rat liver microsomal E(1)-3-sulfatase activity. Double-reciprocal analysis showed that the inhibition of rat liver microsomal E(1)-3-sulfatase activity by PCN was essentially competitive in nature. When microsomes from six human term placentas were tested for their E(1)-3-sulfatase activity, PCN showed a similar biphasic inhibition of placental E(1)-3-sulfatase. Likewise, several of its structural analogs showed little or no inhibitory effect on placental E(1)-3-sulfatase activity. Computational analysis of the D-ring structure of PCN and other structurally similar analogs used in the study suggests that the potent sulfatase-inhibiting activity of PCN may be partly due to its unique steric orientation and size of the 16alpha-nitrile group. This knowledge may be useful for the rational design of more potent steroidal inhibitors of E(1)-3-sulfatase by introducing an additional nitrile group to their C16alpha-position.

An approach to QSAR of 16-substituted pregnenolones as microsomal enzyme inducers.

In this study, we attempt to correlate quantitatively the structure of eight 16-substituted pregnenolones with microsomal enzyme inducing activity. We also performed some electrostatic potential calculations to get further insight into the properties of these substituents. It was found that pregnenolone-16 alpha-carbonitrile is the most active steroidal inducer among the pregnenolone derivatives tested. The receptor-inducer interaction is facilitated by a favourable electronic effect of the 16 alpha-substituents. The orientation of the electronegative area at position 16 seems to influence activity. Lipophilic and volume effects of the 16 alpha-substituents do not seem to be important for microsomal enzyme induction. However, substituent length has some influence on drug metabolising enzyme activity, probably interfering with receptor-inducer interactions.

Effect of the position of the cyano-group of cyanopregnenolones on their drug metabolic inducing activity.

The effect of the position of the cyano-group of several cyanopregnenolones on the body's resistance to drugs and on drug metabolism was investigated. Female rats were pretreated with 2 alpha-, 6-, 16 alpha-, 17 alpha-cyano- or 16 alpha-cyanomethyl-pregnenolone or with pregnenolone, and the (in vivo) resistance to zoxazolamine, digitoxin and indomethacin, as well as the in vitro drug metabolism (post mitochondrial fraction) of zoxazolamine and ethylmorphine were determined. It was found that the 16-derivative was the most active in this respect, the 2- and 17-cyanopregnenolones were less active but significantly potent compared to controls, while the 6-cyano, the 16-cyanomethyl derivatives and pregnenolone were essentially inactive. These differences were explained in terms of an effective or poor fit of the steroids to their receptor. The poor performance of pregnenolone-16 alpha-acetonitrile was attributed to electronic effects. A hypothesis of some structural features of the receptor site for its interaction with the cyanopregnenolone inducers was presented.