Affinity of Ketamine to Clinically Relevant Transporters

Ketamine is a widely used intravenous anesthetic drug that has also a pronounced analgesic effect. Moreover, one of its metabolites was very recently shown to possess antidepressant activity. Consequently, oral administration of ketamine may become of interest in the future. There is evidence from in vitro data, drug–drug interactions, and the physicochemical properties of the drug that ketamine may be a substrate of drug transporters. Thus, it was the aim of this study to investigate the affinity of ketamine to clinically relevant transporter proteins that are expected to affect its intestinal absorption, distribution, and excretion. Ketamine was shown to be significantly taken up in a time- and concentration-dependent manner by OCT1–3. The affinity to OCT transporters at pH 6.5 (<i>K</i>_m ≈ 35–75 μM) was clearly higher than that at pH 7.4. In addition, ketamine permeability was markedly lower at pH 6.5 than at pH 7.4 in a parallel artificial membrane permeability assay (PAMPA). Ketamine showed a low but significant affinity to P-gp at pH 6.5. In contrast to this, we could not detect any transport of ketamine by MATE1/2K. In conclusion, ketamine is a substrate for OCT1–3 and P-gp but is not recognized by MATE1/2K. Considering that ketamine is a lipophilic base that mainly exists as a cationic moiety (>90%) in the intestinal lumen, we conclude that the OCT-mediated cellular uptake as well as P-gp efflux is expected to be only of relevance in the human intestine (i.e., in the case of oral drug administration), where OCT1, OCT3, and P-gp are stably expressed at the apical membrane. On the other side, P-gp is not expected to contribute significantly to tissue (brain) distribution or renal excretion of ketamine

Clinically Relevant Multidrug Transporters Are Regulated by microRNAs along the Human Intestine

Intestinal drug transporters are crucial determinants for absorption and oral bioavailability of drugs. In healthy tissue donors, a recent study revealed profound discrepancies between mRNA expression and protein abundance as well as differences in the protein content between small and large intestine for clinically relevant multidrug transporters as the ATP binding cassette transporter subfamily B member 1 (ABCB1) and subfamily C member 3 (ABCC3) and the solute carrier family 15 member 1 (SLC15A1, PEPT1). As the mechanisms underlying these observations remained unclear, the aim of the present study was to elucidate the intestinal regiospecific microRNA profile under physiological conditions and identify specific microRNAs contributing to the post-transcriptional regulation of major drug transporters. For this purpose, tissue samples were collected from six intestinal sites obtained from six healthy tissue donors. The expression of 754 microRNAs was determined using qRT-PCR based low density arrays, and microRNA expression levels were correlated with transporter protein abundance quantified by targeted proteomics. A total of 241 microRNA–transporter pairs were identified, showing significant negative correlations to protein abundance (<i>p</i> < 0.05). Out of these, for nine pairs, the binding of the microRNA to the respective transporter 3′-UTR was predicted in silico. Besides the already known interactions of miR-27a-3p–<i>ABCB1</i> and miR-193a-3p–<i>PEPT1</i>, reporter gene assays confirmed binding of miR-192-5p to the <i>ABCC3</i> 3′-UTR (reduction of reporter gene activity by 31%; <i>p</i> = 0.0012), miR-409-3p to the <i>ABCB1</i> 3′-UTR (reduction by 38%; <i>p</i> = 0.0006), and miR-193b-3p as well as miR-27a-3p to <i>PEPT1</i> 3′-UTR (reduction by 49% (<i>p</i> = 0.0012) and 20% (<i>p</i> = 0.0043), respectively). These results suggest that mucosal microRNA expression contributes to the explanation of discrepancies between mRNA expression and protein abundance as well as site-dependent differences in protein content along the human intestine under physiological conditions, as exemplified for ABCB1, ABCC3, and PEPT1

Clinically Relevant Multidrug Transporters Are Regulated by microRNAs along the Human Intestine

Intestinal drug transporters are crucial determinants for absorption and oral bioavailability of drugs. In healthy tissue donors, a recent study revealed profound discrepancies between mRNA expression and protein abundance as well as differences in the protein content between small and large intestine for clinically relevant multidrug transporters as the ATP binding cassette transporter subfamily B member 1 (ABCB1) and subfamily C member 3 (ABCC3) and the solute carrier family 15 member 1 (SLC15A1, PEPT1). As the mechanisms underlying these observations remained unclear, the aim of the present study was to elucidate the intestinal regiospecific microRNA profile under physiological conditions and identify specific microRNAs contributing to the post-transcriptional regulation of major drug transporters. For this purpose, tissue samples were collected from six intestinal sites obtained from six healthy tissue donors. The expression of 754 microRNAs was determined using qRT-PCR based low density arrays, and microRNA expression levels were correlated with transporter protein abundance quantified by targeted proteomics. A total of 241 microRNA–transporter pairs were identified, showing significant negative correlations to protein abundance (<i>p</i> < 0.05). Out of these, for nine pairs, the binding of the microRNA to the respective transporter 3′-UTR was predicted in silico. Besides the already known interactions of miR-27a-3p–<i>ABCB1</i> and miR-193a-3p–<i>PEPT1</i>, reporter gene assays confirmed binding of miR-192-5p to the <i>ABCC3</i> 3′-UTR (reduction of reporter gene activity by 31%; <i>p</i> = 0.0012), miR-409-3p to the <i>ABCB1</i> 3′-UTR (reduction by 38%; <i>p</i> = 0.0006), and miR-193b-3p as well as miR-27a-3p to <i>PEPT1</i> 3′-UTR (reduction by 49% (<i>p</i> = 0.0012) and 20% (<i>p</i> = 0.0043), respectively). These results suggest that mucosal microRNA expression contributes to the explanation of discrepancies between mRNA expression and protein abundance as well as site-dependent differences in protein content along the human intestine under physiological conditions, as exemplified for ABCB1, ABCC3, and PEPT1

Expression of Drug Transporters and Drug Metabolizing Enzymes in the Bladder Urothelium in Man and Affinity of the Bladder Spasmolytic Trospium Chloride to Transporters Likely Involved in Its Pharmacokinetics

The cationic, water-soluble quaternary trospium chloride (TC) is incompletely absorbed from the gut and undergoes wide distribution but does not pass the blood–brain barrier. It is secreted by the kidneys, liver, and intestine. To evaluate potential transport mechanisms for TC, we measured affinity of the drug to the human uptake and efflux transporters known to be of pharmacokinetic relevance. Affinity of TC to the uptake transporters OATP1A2, -1B1, -1B3, -2B1, OCT1, -2, -3, OCTN2, NTCP, and ASBT and the efflux carriers P-gp, MRP2 and MRP3 transfected in HEK293 and MDCK2 cells was measured. To identify relevant pharmacokinetic mechanisms in the bladder urothelium, mRNA expression of multidrug transporters, drug metabolizing enzymes, and nuclear receptors, and the uptake of TC into primary human bladder urothelium (HBU) cells were measured. TC was shown to be a substrate of OATP1A2 (<i>K</i>_m = 6.9 ± 1.3 μmol/L; <i>V</i>_max = 41.6 ± 1.8 pmol/mg·min), OCT1 (<i>K</i>_m = 106 ± 16 μmol/L; <i>V</i>_max = 269 ± 18 pmol/mg·min), and P-gp (<i>K</i>_m = 34.9 ± 7.5 μmol/L; <i>V</i>_max = 105 ± 9.1 pmol/mg·min, lipovesicle assay). The genetic OATP1A2 variants *2 and *3 were loss-of-function transporters for TC. The mRNA expression analysis identified the following transporter proteins in the human urothelium: <i>ABCB1</i> (P-gp), <i>ABCC1–5</i> (MRP1–5), <i>ABCG2</i> (BCRP), <i>SLCO2B1</i> (OATP2B1), <i>SLCO4A1</i> (OATP4A1), <i>SLC22A1</i> (OCT1), <i>SLC22A3</i> (OCT3), <i>SLC22A4</i> (OCTN1), <i>SLC22A5</i> (OCTN2), and <i>SLC47A1</i> (MATE1). Immuno-reactive P-gp and OATP1A2 were localized to the apical cell layers. Drug metabolizing enzymes <i>CYP3A5, </i>-<i>2B6</i>, -<i>2B7</i> -<i>2E1</i>, <i>SULT1A1–4, UGT1A1–10</i>, and <i>UGT2B15,</i> and nuclear receptors <i>NR1H3</i> and <i>NR1H4</i> were also expressed on mRNA level. TC was taken up into HBU cells (<i>K</i>_m = 18.5 ± 4.8 μmol/L; <i>V</i>_max = 106 ± 11.3 pmol/mg·min) by mechanisms that could be synergistically inhibited by naringin (IC₅₀ = 10.8 (8.4; 13.8) μmol/L) and verapamil (IC₅₀ = 4.6 (2.8; 7.5) μmol/L), inhibitors of OATP1A2 and OCT1, respectively. Affinity of TC to OCT1 and P-glycoprotein may be the reason for incomplete oral absorption, wide distribution into liver and kidneys, and substantial intestinal and renal secretions. Absence of brain distribution may result from affinity to P-gp and a low affinity to OATP1A2. The human urothelium expresses many drug transporters and drug metabolizing enzymes that may interact with TC and other drugs eliminated into the urine