Tsuneo Hamaguchi

Intestinal and Hepatic Expression of Cytochrome P450s and mdr1a in Rats with Indomethacin-Induced Small Intestinal Ulcers

Background: Non-steroidal anti-inflammatory drugs induce the serious side effect of small intestinal ulcerations (SIUs), but little information is available regarding the consequences to drug metabolism and absorption. Aim: We examined the existence of secondary hepatic inflammation in rats with indomethacin (INM)-induced SIUs and assessed its relationship to the cytochrome P450 (CYP) and P-glycoprotein (mdr1a), the major drug-metabolizing factors in the small intestine and the liver. Methods: Gene expression of the CYP family of enzymes and mdr1a was measured with quantitative real-time polymerase chain reaction (qPCR). Vancomycin (VCM), a poorly absorbed drug, was administered intraduodenally to rats with SIUs. Results: INM induced SIUs predominantly in the lower region of the small intestine with high expression of inflammatory markers. Liver dysfunction was also observed, which suggested a secondary inflammatory response in rats with SIUs. In the liver of rats with SIUs, the expression of CYP2C11, CYP2E1, and CYP3A1 was significantly decreased, and loss of CYP3A protein was observed. Although previous studies have shown a direct effect of INM on CYP3A activity, we could not confirm any change in hepatic CY3A4 expression (major isoform of human CYP3A) in vitro. The plasma VCM concentration was increased in rats with SIUs due to partial absorption from the mucosal injury, but not in normal mucosa. Conclusions: INM-induced SIUs had a subtle effect on intestinal CYP expression, but had an apparent action on hepatic CYP, which was influenced, at least in part, by the secondary inflammation. Furthermore, drug absorption was increased in rats with SIUs.

The Effect of Ethanol on the Hydrolysis of Ester-type Drugs by Human Serum Albumin

Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and hydrolyzes compounds at both sites. Although the hydrolytic interaction of ester-type drugs with other drugs by HSA has been reported, there are only a few studies concerning the effect of pharmaceutical excipients on the hydrolysis of ester-type drugs by HSA. In the present study, we investigated the effect of ethanol (2 vol%; 345 mM) on the hydrolysis of aspirin, p-nitrophenyl acetate, and olmesartan medoxomil, which are ester-type drugs, with 4 different lots of HSA preparations. The hydrolysis activities of HSA toward aspirin, p-nitrophenyl acetate, and olmesartan medoxomil were measured from the pseudo-first-order degradation rate constant (kobs) of salicylic acid, p-nitrophenol, and olmesartan, respectively, which are the HSA-hydrolyzed products. Ethanol inhibited hydrolysis of aspirin by HSA containing low levels of fatty acids, but not by fatty acid-free HSA. Ethanol inhibited hydrolysis of p-nitrophenyl acetate by both fatty acid-free HSA and HSA containing low levels of fatty acids. In contrast, the hydrolysis of olmesartan medoxomil by HSA was insignificantly inhibited by ethanol, but inhibited not only by warfarin and indomethacin but also by naproxen, which are site I binding drugs and a site II binding drug, respectively. These results suggest that the inhibitory action of ethanol on the hydrolysis of ester-type drugs by HSA differs between site I binding drugs and site II binding drugs.

Down‐regulation of hepatic CYP3A1 expression in a rat model of indomethacin‐induced small intestinal ulcers

The liver and the small intestine are closely related in the processes of drug absorption, metabolism and excretion via the enterohepatic circulation. Small intestinal ulcers are a serious adverse effect commonly occurring in patients taking nonsteroidal anti‐inflammatory drugs. However, the influence of small intestinal ulcers on drug metabolism has not been established. This study examined the expressional changes of cytochrome P450 (CYP) in the liver using an indomethacin‐induced small intestinal ulcer rat model and in cultured cells. After the administration of indomethacin to rats, ulcers were observed in the small intestine and expression of CYP3A1, the major isoform of hepatic CYP, was significantly down‐regulated in the liver, accompanied by increased expression of inducible nitric oxide synthase, tumor necrosis factor α, interleukin (IL)‐1β and IL‐6, in the small intestine and the liver. The indomethacin‐induced small intestinal ulceration, the increase in inflammatory mediators in the small intestine and the liver, and the down‐regulation of CYP3A1 expression in the liver were inhibited by co‐administration of ampicillin, an antibacterial agent. In the human hepatic HepG2 cell line, IL‐1β, IL‐6 and NOC‐18, an NO donor, caused down‐regulation of CYP3A4, the major isoform of human CYP3A. Thus, this study suggests that after indomethacin treatment small intestinal ulcers cause the down‐regulation of CYP3A1 in the rat liver through an increase in ulcer‐derived inflammatory mediators. Copyright

Differences in Esterase Activity to Aspirin and p-Nitrophenyl Acetate among Human Serum Albumin Preparations

Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and also hydrolyzes some compounds at both sites. In the present study, we investigated differences in esterase activity among HSA preparations, and also the effects of warfarin, indomethacin, and naproxen on the hydrolytic activities of HSA to aspirin and p-nitrophenyl acetate. The esterase activities of HSA to aspirin or p-nitrophenyl acetate were measured from the pseudo-first-order formation rate constant (kobs) of salicylic acid or p-nitrophenol by HSA. Inter-lot variations were observed in the esterase activities of HSA to aspirin and p-nitrophenyl acetate; however, the esterase activity of HSA to aspirin did not correlate with that to p-nitrophenyl acetate. The inhibitory effects of warfarin and indomethacin on the esterase activity of HSA to aspirin were stronger than that of naproxen. In contrast, the inhibitory effect of naproxen on the esterase activity of HSA to p-nitrophenyl acetate was stronger than those of warfarin and indomethacin. These results suggest that the administration of different commercial HSA preparations and the co-administration with site I or II high-affinity binding drugs may change the pharmacokinetic profiles of drugs that are hydrolyzed by HSA.

Tu1232 Expressions of CYP3A and P-Glycoprotein Were Decreased in the Secondary Inflammatory Response of Dextran Sodium Sulfate-Induced Mice Colitis and Its Contribution to the Blood Concentration of Cyclosporine A

Background: Small intestinal lesions induced by NSAIDs are of great concern in clinical settings. Various hypotheses, mainly of which related to the inhibition of prostaglandin synthesis, have been proposed for the origin of these inflammatory responses, however, the precise mechanism is yet to be known. The cellular process of the lesions must involve upand down-regulations of a large number of proteins and complex interactions between them. To elucidate it, global and systematic identification of the proteins in intestinal cells affected by non-steroidal anti-inflammatory drugs (NSAIDs) is essential. Two-dimensional polyacrylamide gel electrophoresis allows the analysis of total proteins from both cells and tissues, so it can help to elucidate the complex interactions and mechanisms inside cells. Aims: To identify the proteins that play a critical role in NSAIDs toxicity in intestinal cells and to clarify the mechanism of intestinal mucosal injuries induced by NSAIDs. Materials and Methods: We performed 2-DE on IEC-6 rat normal intestinal cells that were treated with indomethacin (200 μΜ, 24 h) or a vehicle control. The differentially displayed proteins were identified through matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Results: We found 18 up-regulated and 8 down-regulated proteins that showed a statistically significant difference of p < 0.05 and a ≥1.5 average fold difference in spot volume as a result of indomethacin treatment. Among these, we found that collagen I and the proteins involved in collagen I synthesis and maturation were all down-regulated. Consistent with this, immunohistochemical analysis showed that the indomethacin-treated rat intestinal mucosal cells exhibits decreased collagen I expression on its apical surface. Furthermore, the cell-protective effect of collagen I on intestinal mucosal cells was demonstrated by the use of a collagen-synthesis inhibitor and cell cultivation on collagen-coated plates versus uncoated plates. Conclusions: For the first time in the literature, a proteomic approach was employed to identify the global proteome alterations of the intestinal mucosa using indomethacin-treated and untreated IEC-6 cells. These results show that collagen I may play an important role in cytoprotection against indomethacin-induced intestinal mucosal injury. Insights gained from the current study may facilitate the development of new therapeutic strategies against NSAID-induced intestinal injury.