Immunocytochemistry for bestatin and its application to drug accumulation studies in rat intestine and kidney.

The in vivo role of transporters in drug disposition, in the context of other transporters, and metabolism has not been established. We prepared an anti-bestatin serum against bestatin conjugated to albumin with glutaraldehyde (GA). The antiserum was specific for GA-conjugated bestatin and weakly reacted with free bestatin, but no reaction occurred with structurally unrelated compounds according to both the inhibition and binding ELISAs. The antiserum allowed us to develop an immunocytochemical (ICC) method for detecting the uptake of bestatin in the rat intestine and kidney. Three hours after a single oral administration of bestatin, the ICC method revealed that the drug distributed in the microvilli, cytoplasm and nuclei of the absorptive epithelial cells at much larger amounts than in all other cell types in the small intestine. However, no drug was detected in the mucin goblets in the epithelium. In the kidney, the drug distributed to a greater extent in the S3 segment than in the S1 and S2 segments of the proximal tubule, and also was detected in the microvilli of the proximal tubule cells (S1, S2 and S3). These findings that bestatin accumulated in large amounts, especially in the cells and/or at the sites where the transporters PEPT1 and PEPT2 occur, corresponded well to those observed with β-lactam amoxicillin in the previous ICC studies. Thus, this may indicate a possibility that both the transporters might be involved, at least in part, in the distribution of bestatin in the small intestine and kidney under the conditions examined.