Immunocytochemistry for vancomycin using a monoclonal antibody that reveals accumulation of the drug in rat kidney and liver.

We prepared monoclonal antibodies against N-(γ-maleimidobutyryloxy)succinimide-conjugated vancomycin (VM). The monoclonal antibody was specific for conjugated or free VM. The monoclonal antibody enabled us to develop an immunocytochemical method for detecting the uptake of VM in the rat kidney and liver. Three hours after a single intravenous (i.v.) injection of VM at the therapeutic dose, the immunocytochemistry revealed that VM accumulated in large amounts in both the S1 and S2 segments and in much smaller amounts in the S3 segment of the proximal tubules as well as in the distal tubules and collecting ducts. The drug was detected in the cytoplasm, cytoplasmic irregular granules, nuclei, and microvilli of the proximal tubule cells. The distal tubules and collecting ducts contained scattered swollen cells in which both the nuclei and cytoplasm were heavily immunostained. Twenty-four hours after injection, most of the swollen cells returned back to normal size and had somewhat decreased immunostaining. Also, significant amounts of VM remained accumulated for as long as 8 days postadministration. In the liver, similar drug accumulation was observed in the Kupffer cells and the endothelial cells of the hepatic sinusoids but not in the hepatocytes, suggesting that vancomycin cannot be eliminated via the liver. Immunoelectron microscopic studies demonstrated that in the collecting ducts, uptake of VM occurred exclusively in the lysosomes and cytoplasm of the principal cells and scarcely in the intercalated cells. Furthermore, double fluorescence staining using rats simultaneously administered with VM and gentamicin strongly suggests that both drugs colocalized in lysosomes in the proximal tubule cells of kidneys.

An in vivo role of Mrp2 in the rat hepatocytes by immunocytochemistry for amoxicillin using the transporter-deficient EHBR.

An in vivo role of the multidrug resistant-associated protein (Mrp2) in rat hepatocytes was examined by immunocytochemistry (ICC) for amoxicillin (AMPC) by the use of the transporter-deficient Eisai hyperbilirubinemic rats (EHBR). The ICC revealed that in the liver of EHBR at 3-h post-administration, amoxicillin accumulated in the cytoplasmic pools and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries. However, no amoxicillin was observed on the surface of the lumina ranging from the bile capillaries to the interlobular bile ducts. The drug persisted at least for 6-h after administration. In contrast, in the control rat liver at 3-h post-administration, AMPC-adsorption occurred on such luminal surface, while AMPC accumulated to a less level in both the cytoplasm and nuclei of the hepatocytes. The drug completely disappeared in the hepatocytes at 6-h post-administration. These results strongly suggest that AMPC taken up into the cytoplasm of the hepatocytes excretes via Mrp2 into the bile flow. Furthermore, electron microscopy demonstrated that the lower electron density areas in large sizes, corresponding to the cytoplasmic pools in ICC for AMPC, occurred in the cytoplasm peripheral to the nuclei of the hepatocytes in EHBR at 3-h post-administration, and then disappeared 24 h after administration.

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 ß-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.