Genotype-dependent effects of inhibitors of the organic cation transporter, OCT1: predictions of metformin interactions.

Common genetic variants of the liver-specific human organic cation transporter 1 (OCT1; SLC22A1) have reduced transport capacity for substrates such as the antidiabetic drug metformin. The effect of the reduced OCT1 function on drug interactions associated with OCT1 has not been investigated and was, therefore, the focus of the study presented here. HEK293 cells expressing human OCT1-reference or the variants R61C, V408M, M420del and G465R were first used to study the kinetics and inhibition pattern of the OCT1 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). In the second part OCT1-mediated (14)C-metformin uptake was studied in the presence of drugs administered concomitantly with metformin. Transport studies using ASP(+) showed that the function of the variants decreased in the following order: OCT1-reference=V408M=M420del >R61C >>G465R. Variants M420del and R61C were more sensitive to drug inhibition, with IC(50) values up to 23 times lower than those of the OCT1-reference. Uptake studies using (14)C-metformin were in qualitative agreement with those using ASP(+), with the exception that a larger reduction in transport capacity was observed for M420del. Concomitantly administered drugs, such as verapamil and amitriptyline, revealed potential drug-drug interactions at clinical plasma concentrations of metformin for OCT1-M420del.

Ketobemidone is a substrate for cytochrome P4502C9 and 3A4, but not for P-glycoprotein.

The role of the major drug-metabolizing cytochrome P450 (CYP) enzymes as well as P-glycoprotein (PGP) was investigated in the disposition of ketobemidone in vitro. Formation of norketobemidone from ketobemidone was studied and compared with the activities of 11 major CYP enzymes in human liver microsomes. The formation of norketobemidone from ketobemidone (1 microM) correlated best with CYP2C9 activity, measured as losartan oxidation (rs = 0.82, n = 19, p < 0.001), but there was also a strong correlation with CYP3A4 activity. Additionally, a good correlation was observed with CYP2C19, CYP2C8 and CYP2B6 at a ketobemidone concentration of 50 microM. Inhibition studies confirmed the involvement of CYP2C9 and CTP3A4 in the formation of norketobemidone. The formation rate of norketobemidone was three times higher in the CYP2C9*1*1 genotype group compared with the CYP2C9*1*2, CYP2C9*1*3 and CYP2C9*3*3 genotypes (p < 0.01). Treatment with verapamil as a PGP inhibitor did not affect the transport of ketobemidone in Caco-2 cells, indicating that PGP is not involved. The data suggest that CYP2C9 and CYP3A4 play a major role in the formation of norketobemidone at clinically relevant concentrations.

Novel lipoamino acid- and liposaccharide-based system for peptide delivery: application for oral administration of tumor-selective somatostatin analogues.

Lipoamino acid and liposaccharide conjugates of somatostatin analogue TT-232 were synthesized to modify the physicochemical properties of the parent peptide. The relative position, the number, and the nature of the lipid and/or saccharide moieties were varied. Experiments in vitro clearly showed that many compounds modified at the N- and/or C-terminus with lipid or sugar moieties retained the biological activity of the parent compound. An interesting construct was synthesized containing lipid and sugar units at opposite ends of the somatostatin analogue, so that the entire molecule could be considered as an amphipathic surfactant.

Absorption enhancement in intestinal epithelial Caco-2 monolayers by sodium caprate: assessment of molecular weight dependence and demonstration of transport routes.

Sodium caprate (C10), a medium chain fatty acid, is used clinically to enhance rectal absorption of the low molecular weight (MW) drug ampicillin. The main aim of this study was to investigate whether C10 also enhances the permeability of high MW model drugs in a model of the intestinal epithelium. The second aim was to present visual evidence of the route of enhanced transport across the epithelial cell layer. The studies were performed in Caco-2 monolayers cultured on permeable supports. The effects of non-toxic concentrations (< or = 13 mM) of C10 on drug transport across the monolayers was studied using monodisperse 14C-polyethylene glycols (MW 238-502; 14C-PEGs), 125I-Arg5-vasopressin (MW 1,208), 125I-insulin (MW 6,000) and FITC-labelled dextrans (MW 4,400 and 19,600; FD4 and FD20 respectively) as model drugs. Electron and confocal laser scanning microscopy were used to demonstrate transport routes across the epithelium. 10 mM C10 increased the permeability of all 14C-PEGs to approximately the same extent. 13 mM C10 increased the permeability of 125I-Arg8-vasopressin 10-fold. Only small increases in FD4 and FD20 permeabilities were observed. After C10 exposure, both tight junctions with normal morphology and those with dilatations showed an increased permeability to ruthenium red, indicating that C10 enhanced the paracellular transport of molecules with a MW < 1,000. Confocal microscopy showed that C10 increased the transport of FD4 and FD20 by the paracellular route. In conclusion, non-toxic concentrations of C10 can be used to enhance the permeability of drugs of MW up to approximately 1,200. Enhancement of the absorption of molecules larger than 4,000 is quantitatively insignificant. The enhanced permeability occurred via the paracellular pathway.

Transport of an influenza virus vaccine formulation (iscom) in Caco-2 cells.

The influenza virus envelope glycoproteins hemagglutinin and neuraminidase were administered to the apical or basolateral sides of Caco-2 monolayers either as native protein micelles (mic-ag) or after incorporation into the orally active adjuvant formulation, immune stimulating complexes (iscoms) (isc-ag). Biotin-conjugated isc-ag were localized in intracellular vesicles as early as 2 min after administration to the apical side at 37 degrees C. Ten minutes after administration, both intracellular vesicles and intercellular spaces were labeled, and extracellular labeling was observed on the basolateral side of the cells, indicating that isc-ag were transported across the epithelium within 10 min of exposure. Transport of 125I-labeled isc-ag and mic-ag in the apical-to-basolateral and basolateral-to-apical directions across Caco-2 monolayers was comparable at 37 degrees C. Gel chromatography analysis revealed that only 0.55-3.1% of transported isc-ag and mic-ag had a molecular weight of > 5,000, while 21.0-42.3% was eluted at a position corresponding to peptides of approximately 10 amino acids. Although isc-ag and mic-ag were transported and degraded by Caco-2 monolayers in comparable amounts, only transported isc-ag induced a dose-dependent proliferative response in vitro of T cells primed with influenza virus antigen. High-performance gel chromatography and reverse-phase high-performance liquid chromatography indicated that transported antigenic isc-ag consisted of hydrophobic peptides with a molecular weight of < or = 3,000. These results indicate that antigens incorporated into the orally active adjuvant formulation iscom are degraded to antigenic peptides during transport across the intestinal epithelium.

Intestinal tissue distribution and epithelial transport of the oral immunogen LTB, the B subunit of E. coli heat-labile enterotoxin.

LTB provokes a systemic immune response and exerts adjuvant effects on mucosal immune responses to unrelated antigens. The binding and uptake of fluorescein-labelled LTB in the normal villus epithelium was compared to that in Peyer's patch dome epithelium in mouse intestine. LTB was bound by the GM1-receptor and taken up extensively by both tissues, indicating that not only the Peyer's patches but also the normal villus epithelium play a significant role in the transport of orally administered antigens. These results were supported by transport studies in the human intestinal epithelial cell line Caco-2 using 125I-LTB. After 2 h incubation, 5.1 +/- 0.1% and 5.9 +/- 0.1% of the added radioactivity was transported in the apical to basolateral and basolateral to apical direction, respectively. Less than 1% of the transported radioactivity was immunoprecipitated with anti-LTB antiserum indicating that LTB was extensively degraded during the transport. The results suggest that normal enterocytes play a significant role in the binding, uptake and transport of orally administered LTB.