ABSTRACT
PURPOSE: Solute carrier SLC22A4 encodes the carnitine/organic cation transporter OCTN1 and is associated with inflammatory bowel disease, although little is known about how this gene is linked to pathogenesis. The aim of the present study was to identify endogenous substrates that are associated with gastrointestinal inflammation. METHODS: HEK293/OCTN1 and mock cells were incubated with colon extracts isolated from dextran sodium sulfate-induced colitis mice; the subsequent cell lysates were mixed with the amino group selective reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), to selectively label OCTN1 substrates. Precursor ion scanning against the fragment ion of APDS was then used to identify candidate OCTN1 substrates. RESULTS: Over 10,000 peaks were detected by precursor ion scanning; m/z 342 had a higher signal in HEK293/OCTN1 compared to mock cells. This peak was detected as a divalent ion that contained four APDS-derived fragments and was identified as spermine. Spermine concentration in peripheral blood mononuclear cells from octn1 gene knockout mice (octn1-/-) was significantly lower than in wild-type mice. Lipopolysaccharide-induced gene expression of inflammatory cytokines in peritoneal macrophages from octn1-/- mice was lower than in wild-type mice. CONCLUSIONS: The combination metabolomics approach can provide a novel tool to identify endogenous substrates of OCTN1.
Subject(s)
Organic Cation Transport Proteins/metabolism , Animals , Colitis/chemically induced , Colitis/metabolism , Colon/chemistry , Colon/metabolism , Cytokines/metabolism , HEK293 Cells , Humans , Inflammatory Bowel Diseases/metabolism , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Metabolomics , Mice , Mice, Inbred C57BL , Mice, Knockout , Organic Cation Transport Proteins/genetics , Spermine/metabolism , Symporters , Tissue Extracts/pharmacologyABSTRACT
The hepatic uptake transporter organic anion transporting polypeptide (OATP) 1B1 is inhibited by some uremic toxins; however, direct inhibition can only partially explain the delayed systemic elimination of substrate drugs in renal failure patients. This study aimed to examine the long-lasting inhibition of OATP1B1 by uremic toxins and their metabolites. Preincubation of HEK293/OATP1B1 cells with 21 uremic toxins resulted in almost no change in the uptake of a typical substrate [3H]estrone-3-sulfate (E1S), although some directly inhibited [3H]E1S uptake. In contrast, preincubation with an indole metabolite, 6-hydroxyindole, reduced [3H]E1S uptake, even after the inhibitor was washed out before [3H]E1S incubation. Such long-lasting inhibition by 6-hydroxyindole was time-dependent and recovered after a 3-h incubation without 6-hydroxyindole. Preincubation with 6-hydroxyindole increased the Km for [3H]E1S uptake with minimal change in Vmax. This was compatible with no change in the cell-surface expression of OATP1B1, as assessed by a biotinylation assay. Preincubation with 6-hydroxyindole reduced [3H]E1S uptake in human hepatocytes without changes in OATP1B1 mRNA. Plasma concentration of 6-hydroxyindole in renal failure patients increased as renal function decreased, but might be insufficient to exhibit potent OATP1B1 inhibition. In conclusion, 6-hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor with elevated plasma concentrations in renal failure patients.
Subject(s)
Hepatocytes/drug effects , Indoles/pharmacology , Liver-Specific Organic Anion Transporter 1/antagonists & inhibitors , Renal Insufficiency/blood , Uremia/blood , Biological Transport , Dose-Response Relationship, Drug , Estrone/analogs & derivatives , Estrone/metabolism , HEK293 Cells , Hepatocytes/metabolism , Humans , Indoles/blood , Kinetics , Liver-Specific Organic Anion Transporter 1/genetics , Liver-Specific Organic Anion Transporter 1/metabolism , Renal Insufficiency/diagnosis , Renal Insufficiency/physiopathology , Up-Regulation , Uremia/diagnosis , Uremia/physiopathologyABSTRACT
PURPOSE: Pazopanib is an orally active, multi-targeted tyrosine kinase inhibitor. A previous phase I study demonstrated that coadministration of pazopanib with irinotecan increases the area under the plasma concentration-time curve (AUC) for SN-38, an active metabolite of irinotecan. To clarify the possible mechanism underlying that drug-drug interaction, we investigated the potential for pazopanib to inhibit UDP-glucuronosyltransferase (UGT)1A1 and organic anion-transporting polypeptide (OATP)1B1, which are involved in detoxification and hepatic uptake of SN-38, respectively. METHODS: Human liver microsomes (HLMs) and recombinant human UGT1A1, and HEK293 cells stably transfected with OATP1B1 were used to evaluate the inhibitory effects of pazopanib against glucuronidation, and hepatic uptake of SN-38, respectively. Kinetic analysis was performed to estimate inhibition constants, which were corrected for non-specific binding to enzyme sources (Ki,u values). RESULTS: Concentration-dependent inhibition of SN-38 glucuronidation was observed in the HLMs and recombinant human UGT1A1 experiments: Pazopanib noncompetitively inhibited SN-38 glucuronidation by HLMs (Ki,u = 1.6 ± 0.05 µM) and recombinant human UGT1A1 (Ki,u = 0.69 ± 0.02 µM). Pazopanib-induced increases in SN-38 AUC estimated using Ki,u values were comparable to those observed in patients of the phase I study who received both irinotecan and pazopanib. Such results suggest that the drug-drug interaction is at least partially mediated by inhibition of UGT1A1. In contrast, pazopanib did not inhibit OATP1B1-mediated SN-38 uptake at concentrations up to 60 µM. CONCLUSIONS: Results showed that pazopanib inhibits UGT1A1-mediated SN-38 glucuronidation, but not OATP1B1-mediated SN-38 uptake.