Characterization of intestinal and hepatic P450 enzymes in cynomolgus monkeys with typical substrates and inhibitors for human P450 enzymes.

Cynomolgus monkeys are widely used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage. Characterization of cynomolgus monkey P450s such as the mRNA expression level, substrate specificity, and inhibitor selectivity were conducted to provide helpful information in designing monkey in vivo studies and monkey-to-human extrapolation. The expression levels of 12 monkey P450 mRNAs, which are considered to be important P450 subfamilies in drug metabolism, were investigated in the liver, small intestine (duodenum, jejunum, and ileum), and colon of individual monkeys. 3. In vitro activities and intrinsic clearance values were determined in monkey intestinal and liver microsomes (MIM and MLM, respectively) using nine typical oxidative reactions for human P450s. Paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, and S-mephenytoin 4'-hydroxylation showed low activities in MIM and MLM. IC50 values of eight selective inhibitors of human P450s were determined in MIM and MLM. Inhibitory effects of furafylline and sulfaphenazole were weak in monkeys on phenacetin O-deethylation and diclofenac 4'-hydroxylation, respectively. These results show profiles of monkey P450s in both the intestine and liver in detail and contribute to a better understanding of the species difference in substrate specificity and inhibitor selectivity between cynomolgus monkeys and humans.

Evaluation of intestinal metabolism and absorption using the Ussing chamber system equipped with intestinal tissue from rats and dogs.

The purpose of this study was to evaluate the intestinal metabolism and absorption in a mini-Ussing chamber equipped with animal intestinal tissues, based on the transport index (TI). TI value was defined as the sum of drug amounts transported to the basal-side component (Xcorr) and drug amounts accumulated in the tissue (Tcorr), which are normalized by AUC of a drug in the apical compartment, as an index for drug absorption. Midazolam was used as a test compound for the evaluation of intestinal metabolism and absorption. The metabolite formulation of midazolam was observed in both rats and dogs. Ketoconazole inhibited the intestinal metabolism of midazolam in rats and improved its intestinal absorption to a statistically significant extent. Therefore, the mini-Ussing chamber, equipped with animal intestinal tissues, showed potential to use the evaluation of the intestinal metabolism and absorption, including the assessment of species differences.

Effect of G-1 column (Adacolumn) therapy in rats with adjuvant arthritis on the migration and immunoreactivity of peripheral and splenic leukocytes.

The G-1 column (Adacolumn), a novel extracorporeal adsorption device, is now available for the treatment of such chronic inflammatory diseases as ulcerative colitis and rheumatoid arthritis. G-1 column treatment sometimes results in a rapid decrease in clinical inflammatory parameters and/or has a delayed beneficial effect on disease activity. In order to identify the scientific basis for such clinical benefits, we studied rats with adjuvant arthritis induced by immunization with Mycobacterium butyricum antigen. The potential role of G-1 column treatment on the migratory properties and immunoreactivities of leukocytes was investigated. Treatment of arthritic rats for 60 min with an extracorporeal perfusion through the G-1 column led to the adsorption of a small proportion (20%) of circulating granulocytes and monocytes. However, after G-1 treatment, the migration of radiolabeled blood granulocytes and monocytes to sites of acute dermal inflammatory reactions decreased significantly, in the case of granulocytes, almost by half. The migration of granulocytes to the inflamed hindpaws of severely affected animals was diminished in the G-1 treated group. Granulocytes that have passed through the G-1 column may stay in the bloodstream because of their markedly diminished number of adhesion molecules. A slightly increased accumulation in the liver and a decreased localization in the lung was also observed. These results may be relevant to the rapid clinical anti-inflammatory effect observed in rheumatoid arthritis and possibly also in ulcerative colitis, without any pulmonary complications. In contrast, the adsorption rate by the G-1 column of T lymphocytes was very low, and their migration pattern to sites of dermal inflammatory reactions was not altered after treatment. However, the antigen (Mycobacterium purified protein derivative) reactivity of T lymphocytes in blood was almost completely abolished after G-1 column treatment of arthritic rats. This unexpected qualitative effect on T lymphocytes of G-1 treatment warrants further detailed study.

Differential roles of VLA-4(CD49d/CD29) and LFA-1(CD11a/CD18) integrins and E- and P-selectin during developing and established active or adoptively transferred adjuvant arthritis in the rat.

The role of the integrins VLA-4 and LFA-1 and of the selectin adhesion molecules in autoimmune arthritis was investigated. Adjuvant arthritis was induced in Lewis rats by active immunization (s.c.) with Mycobacterium butyricum or by adoptive transfer of immune T cells. With active adjuvant arthritis, Lewis rats develop maximal polyarticular joint inflammation and migration of radiolabelled (111In and 51Cr) blood neutrophils and monocytes to the joints 14 days post Mycobacterium butyricum immunization. Using blocking monoclonal antibodies we osbserved that at this stage monocyte recruitment was dependent (85%) on P-selectin plus VLA-4 (alpha4B1) and neutrophil recruitment depended (> 80%) on P-selectin plus LFA-1 (CD11a/CD18). E-selectin played a minimal role in inflammatory cell recruitment to the already inflamed joint. In contrast, during the development of active adjuvant arthritis, blockade of P-selectin beginning at day 5 post-immunization had no effect on subsequent arthritis. However, E-selectin blockade at this stage reduced arthritic scores by 70% (P < 0.01) and combined E-selectin plus VLA-4 blockade prevented development of arthritis. Either treatment nearly abolished neutrophil and monocyte recruitment to joints at day 14 and prevented cartilage damage. VLA-4 blockade alone was less effective. Adoptive T-cell transfer of adjuvant arthritis to naive rats employed spleen/lymph node lymphocytes from Mycobacterium butyricum immunized rats stimulated with Concanavalin A in vitro (48 h). E-selectin +/- P-selectin blockade had no effect on the development of adoptive arthritis. However, VLA-4 integrin blockade inhibited adoptive arthritis severity by 55% (P < 0.01). LFA-1 blockade had no effect. In adoptive adjuvant arthritis, inhibition of arthritis clinically and by histology was essentially complete (> 90%) when E- and P-selectin blockade was combined with VLA-4 blockade. Thus, in the development of actively induced arthritis E-selectin plays an important role, likely mediating early antigen reactive T-cell recruitment to joints. In contrast, VLA-4 and multiple selectin mechanisms are involved in arthritis induction by ex vivo restimulated arthritogenic T cells. Furthermore, in actively induced adjuvant arthritis, P- and E-selectin and VLA-4 are differently important in the initiation of arthritis, and at the time of fully developed joint inflammation.