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The sequential activity of gut microbial and host processes can exert a powerful modulatory influence on dietary components, as exemplified by the metabolism of the amino acids tyrosine and phenylalanine to p-cresol by gut microbes, and then to p-cresol glucuronide (pCG) by host enzymes. Although such glucuronide conjugates are classically thought to be biologically inert, there is accumulating evidence that this may not always be the case. We investigated the activity of pCG, studying its interactions with the cerebral vasculature and the brain in vitro and in vivo. Male C57Bl/6 J mice were used to assess blood–brain barrier (BBB) permeability and whole-brain transcriptomic changes in response to pCG treatment. Effects were then further explored using the human cerebromicrovascular endothelial cell line hCMEC/D3, assessing paracellular permeability, transendothelial electrical resistance and barrier protein expression. Mice exposed to pCG showed reduced BBB permeability and significant changes in whole-brain transcriptome expression. Surprisingly, treatment of hCMEC/D3 cells with pCG had no notable effects until co-administered with bacterial lipopolysaccharide, at which point it was able to prevent the permeabilizing effects of endotoxin. Further analysis suggested that pCG acts as an antagonist at the principal lipopolysaccharide receptor TLR4. The amino acid phase II metabolic product pCG is biologically active at the BBB, antagonizing the effects of constitutively circulating lipopolysaccharide. These data add to the growing literature showing glucuronide conjugates to be more than merely metabolic waste products and highlight the complexity of gut microbe to host communication pathways underlying the gut–brain axis. [ABSTRACT FROM AUTHOR]

We demonstrate here a new variant on a statistical spectroscopic method for recovering structural information on unstable intermediates formed in reaction mixtures. We exemplify this approach with respect to the internal acyl migration reactions of 1-[beta]-O-acyl glucuronides (AGs), which rearrange at neutral or slightly alkaline pH on a minute to hour time scale to yield a series of positional glucuronide ring isomers and [alpha]/[beta] anomers from the 1-[beta] (starting material), i.e. 2-[beta], 2-[alpha], l-[alpha], 3-[beta], 3-[alpha], and 4-[beta], 4-[alpha] isomers together with the aglycon and [alpha]- and [beta]-glucuronic acid hydrolysis products. Multiple sequential 800 MHz cryoprobe [sup.1]H NMR spectra (1D and 2D J-resolved, JRES) were collected on a 5.1 mM solution of a synthetic model drug glucuronide, 1-[beta]-O-acyl (S)-alpha]-methyl phenylacetyl glucuronide (MPG) in 0.1 M sodium phosphate buffer in [D.sub.2]O at pD 7.4 over 18 h to monitor the reaction which leads to the formation of the eight positional isomers and hydrolysis products. As the reaction proceeds and new isomers form, the NMR signal intensities vary accordingly allowing the application of a novel kinetic variant on statistical total correlation spectroscopy (K-STOCSY) method to recover the connectivities between proton signals on the same reacting molecule based on their intensity covariance through time. We performed K-STOCSY analysis on both the standard 1D NMR spectra and the skyline projected singlets of the [sup.1]H-[sup.1]H JRES NMR spectra through time, i.e. the K-JRES-STOCSY experimental variant, which increases the effective spectral dispersion and is ideally suited for the analysis of heavily overlapped spin systems. High statistical correlations were observed between mutarotated [alpha]-and [beta]-anomers of individual positional isomers, as well as directly acyl migrated products and anticorrelation observed between signals from compounds that were being depleted as others increased, e.g. between the 1-[beta] and 2-[alpha]/2-[beta] isomers. This statistical kinetic approach enabled the recovery of structural connectivity information on all isomers allowing unequivocal resonance assignment, and this approach to spectroscopic information recovery has wider potential uses in the study of reactions that occur on the second-to-minute time scale in conditions where multiple sequential NMR spectra can be collected. JRES-STOCSY is also of potential use as a method for recovering spectroscopic information in highly overlapped NMR signals and spin systems in other types of complex mixture analysis.

Carboxylic acid-containing drugs are often metabolized to 1-[beta]-O-acyl glucuronides (AGs). These can undergo an internal chemical rearrangement, and the resulting reactive positional isomers can bind to endogenous proteins, with clear potential for adverse effects. Additionally any 1-[beta]-O-acyl-glucuronidated phase I metabolite of the drug can also show this propensity, and investigation of the adverse effect potential of a drug also needs to consider such metabolites. Here the transacylation of the common drug ibuprofen and two of its metabolites is investigated in vitro. 1-[beta]-O-Acyl (S)-ibuprofen glucuronide was isolated from human urine and also synthesized by selective acylation. Urine was also used as a source of the (R)ibuprofen, (S)-2-hydroxyibuprofen, and (S,S)-carboxyibuprofen AGs. The degradation rates (a combination of transacylation and hydrolysis) were measured using [sup.1]H NMR spectroscopy, and the measured decrease in the 1-[beta] anomer over time was used to derive half-lives for the glucuronides. The biosynthetic and chemically synthesized (S)-ibuprofen AGs had half-lives of 3.68 and 3.76 h, respectively. (R)-Ibuprofen AG had a half-life of 1.79 h, a value approximately half that of the (S)-diastereoisomer, consistent with results from other 2-aryl propionic acid drug AGs. The 2-hydroxyibuprofen and carboxyibuprofen AGs gave half-lives of 5.03 and 4.80 h, considerably longer than that of either of the parent drug glucuronides. In addition, two (S)-ibuprofen glucuronides were synthesized with the glucuronide carboxyl function esterified with either ethyl or allyl groups. The (S)-ibuprofen AG ethyl ester and (S)-ibuprofen AG allyl esters were determined to have half-lives of 7.24 and 9.35 h, respectively. In order to construct useful structure-reactivity relationships, it is necessary to evaluate transacylation and hydrolysis separately, and here it is shown that the (R)- and (S)-ibuprofen AGs have different transacylation properties. The implications of these findings are discussed in terms of structure-activity relationships.

Drug metabolism, using body enzymes, and clinical and industrial implications are discussed. The cytochrome P450 monooxygenases (CYPs) are largely repsonsible for drug oxidation. The organic chemist can play a role in the synthesis of drug substrates, metabolites and inhibitors needed for experimental studies. Molecular modeling, paracetamol, terfenadine, dietary flavonoids, such as those found in grapefruit, and morphine-6-glucuronide are discussed. Future implications for medicinal chemists are considered.