ABSTRACT:Midazolam (MDZ) is one of the most commonly used in vivo and in vitro CYP3A4 probe substrates for drug-drug interactions (DDI) studies. The major metabolic pathway of MDZ in humans consists of the CYP3A4-mediated 1-hydroxylation followed by urinary excretion as 1-O-glucuronide derivative. In the present study, following incubation of MDZ with human liver microsomes supplemented with UDP-glucuronic acid, two major high-performance liquid chromatography (HPLC) peaks were isolated. HPLC and liquid chromatography/tandem mass spectrometry analyses identified these two metabolites as quaternary direct N-glucuronides of MDZ, thus revealing an additional metabolic pathway for MDZ. Because a large number of currently available drugs and future drugs will be metabolized by the members of the CYP3A subfamily, the potential for drug-drug interaction (DDI) is substantial. DDIs involving the inhibition and induction of CYP3A4 are of great scientific and clinical relevance. Indeed, drugs with potent CYP3A4 inhibitory properties have been implicated in significant CYP3A4-mediated DDIs. Interactions of the benzodiazepines with the azole antifungal agents and especially the inhibition of CYP3A-mediated midazolam (MDZ) metabolism by ketoconazole have been widely studied. Concomitant administration of both drugs results in large, variable, and highly significant increases (5-16-fold) in MDZ exposure, depending on the dose regimen of ketoconazole used. Table 1 summarizes available clinical data on the effects of ketoconazole on MDZ plasma levels following p.o. or i.v. administration of MDZ.MDZ is a short-acting water-soluble imidazobenzodiazepine (Fig. 1) extensively used in clinical practice mainly for induction and maintenance of anesthesia, sedation for diagnostic and therapeutic procedures, and also as an oral hypnotic agent (Reves et al., 1985). MDZ is a well known CYP3A substrate because its metabolism has been the focus of many in vitro investigations (Fabre et al., 1988b;Kronbach et al., 1989;Wrighton and Ring, 1994;Ghosal et al., 1996; Maenpaa et al., 1998;Hosea et al., 2000;Wang et al., 2000). MDZ biotransformation is mediated by at least three different CYP3A isoenzymes: CYP3A4, CYP3A5, and CYP3A7 (Gorski et al., 1994;Kuehl et al., 2001). Because CYP3A7 is principally expressed in fetal tissues, CYP3A4 and CYP3A5 represent the main cytochrome P450 (P450) isoforms in adult liver and intestine (Guengerich, 1995).MDZ biotransformation yields two primary hydroxylated metabolites: 1Ј-hydroxy-MDZ (1Ј-OH-MDZ) and 4-hydroxy-MDZ. 1Ј-OH-MDZ represents the main metabolite because it accounts for 95% of net intrinsic clearance of MDZ in human liver microsomes (von Article, publication date, and citation information can be found at

Colonic mucosal protection is provided by mucous gel, mainly composed of secreted (Muc2) and membrane-bound (Muc1, Muc3, Muc4) mucins. Our aim was to determine the expression profile of secreted and membrane-bound mucins in experimental dextran sulfate sodium (DSS)-induced colitis. Acute colitis was induced in Balb/C mice by oral administration of 1.0% DSS (5 days) and chronic colitis was maintained by subsequent 0.15% DSS treatment (28 days). Clinical symptoms (mortality, weight gain), stool scores, and MPO activity confirmed the inflammatory state in the two phases of colitis. Muc2 gene expression was not modified by colitis, whereas Muc3 gene expression was increased (x2) only in the cecum and the distal colon of mice after acute colitis. Muc1 and Muc4 mRNA levels were more significantly increased in the cecum (x8-10) than in colonic segments (x4) after acute colitis. TFF3 involved in mucosal repair was up-regulated during colitis induction. These results indicate that Muc and TFF3 genes are regulated early in inflammation and suggest that their mRNA levels could be used as early markers of inflammation.

Colonic mucosal protection is provided by the mucus gel, mainly composed of mucins. Several factors can modulate the formation and the secretion of mucins, and among them butyrate, an endproduct of carbohydrate fermentation. However, the specific effect of butyrate on the various colonic mucins, and the consequences in terms of the mucus layer thickness are not known. Our aim was to determine whether butyrate modulates colonic MUC genes expression in vivo and whether this results in changes in mucus synthesis and mucus layer thickness. Mice received daily for 7 days rectal enemas of butyrate (100 mM) versus saline. We demonstrated that butyrate stimulated the gene expression of both secreted (Muc2) and membrane-linked (Muc1, Muc3, Muc4) mucins. Butyrate especially induced a 6-fold increase in Muc2 gene expression in proximal colon. However, butyrate enemas did not modify the number of epithelial cells containing the protein Muc2, and caused a 2-fold decrease in the thickness of adherent mucus layer. Further studies should help understanding whether this last phenomenon, i.e. the decrease in adherent mucus gel thickness, results in a diminished protective function or not.

Intrauterine growth restriction (IUGR) is a leading cause of perinatal mortality and morbidity and increases the risk for necrotizing enterocolitis. We hypothesized that colonic barrier disruption could be responsible for intestinal frailty in infants and adults born with IUGR. Mucins and trefoil factor family 3 (TFF3) actively contribute to epithelium protection and healing. Our aim was to determine whether IUGR affects colonic mucosa maturation. IUGR was induced by dietary protein restriction in pregnant dams. Mucins and Tff3 expression and morphologic maturation of the colonic mucosa were followed during postnatal development of the offspring. Before weaning, mucin 2 and Tff3 protein levels were reduced in colonic mucosa of rats with IUGR compared with controls. After weaning, expression of mucin 2 (mRNA and protein) and mucin 4 (mRNA) were lower in colonic mucosa of rats with IUGR. At the same time, IUGR was associated with a reduction of crypt depth and a higher percentage of crypts in fission. We conclude that IUGR impairs mucus barrier development and is associated with long-term alterations of mucin expression. The lack of an efficient colonic barrier induced by IUGR may predispose to colonic injury not only in neonatal life but also in later life.