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Brief Article

Affinity of ketamine to clinically relevant transporters Markus Keiser, Mahmoud Hasan, and Stefan Oswald Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00627 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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Molecular Pharmaceutics

Brief Article

Affinity of ketamine to clinically relevant transporters Markus Keiser, Mahmoud Hasan and Stefan Oswald Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine Greifswald, Greifswald, Germany

Corresponding author: Jun.-Prof. Dr. Stefan Oswald Department of Clinical Pharmacology Center of Drug Absorption and Transport (C_DAT) University Medicine Greifswald Felix-Hausdorff-Str. 3 D-17489 Greifswald Germany E-mail: [email protected] Phone: +49 (0)3834-865643 Fax: +49 (0)3834-865631

Keywords: ketamine, intestinal absorption, P-gp, organic cation transporters

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Graphical Abstract

Abstract Ketamine is a widely used intravenous anaesthetic 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 from 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 (Km ~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 exist as 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.


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Introduction Ketamine is widely used as intravenously administered anaesthetic. It acts as non-competitive N-methyl D-aspartate receptor (NMDR) antagonist.1-3 S(+)-ketamine is known to be more potent in terms of the hypnotic and analgesic effects than the R(-)-enantiomer.4 Ketamine was also shown to exert pronounced analgesic effects at sub-anaesthetic doses in both chronic and acute pain.5 Ketamine is metabolized through N-demethylation and hydroxylation mainly by CYP2B6, CYP3A4 and CYP2C9 to norketamine, dehydronorketamine and various hydroxyketamines.6 Under consideration that the expression of these enzymes is markedly different in the human intestine and liver7, it is expected that the route of ketamine administration may have a profound effect on the metabolic pattern of the drug. In this regard, oral administration of ketamine was shown to increase the norketamine to ketamine ratio (metabolite/parent compound) compared to intravenous drug administration.8 Due to its strong analgesic effect with a beneficial profile of side effects compared to established opioids and the very recently observed antidepressant activity, oral administration of ketamine may become of interest in the future as already demonstrated in some smaller clinical studies.9-12 In this regard, the pharmacokinetic properties of oral, sublingual and intravenous ketamine were compared13 and it was suggested that small oral doses of S-ketamine might be an alternative to repeated intravenous dosing in chronic pain.14 In contrast to this, the process of intestinal ketamine absorption is only poorly understood as it is so far almost exclusively administered parenterally. From the physicochemical point of view, ketamine is a lipophilic rather basic compound (logP = 2.9, pKa = 7.5) that mainly exists in its cationic form in the intestinal lumen (91% at pH= 6.5) which is not expected to cross biological membranes via passive diffusion. In line with these properties, ketamine is characterized by a low oral bioavailability (11.2 - 24.5%) most likely due to the poor intestinal absorption and the ACS Paragon Plus Environment

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extensive first-pass metabolism.15 Moreover, intravenously administered ketamine undergoes predominately renal excretion (91-97% of the dose). Considering these physicochemical and pharmacokinetic properties, transporters may play a role in ketamine absorption, distribution to its site of action (i.e. brain) and excretion.16 Assuming that ketamine is present in intestinal lumen as a positively charged moiety, cation transporters such as OCTs (organic cation transporters) may be involved in its absorption. In line with this hypothesis, ketamine was found to inhibit function of human and rodent OCTs. 17,18

Several OCT substrates such as morphine, ranitidine or trospium are known to be considered by the intestinal efflux pump P-glycoprotein (P-gp, ABCB1).19 However, it is so far unknown whether this is also the case for ketamine. Under consideration that ketamine is mainly excreted via the kidneys, transporter proteins may also play an important role in its renal elimination. As multidrug and toxin extrusion proteins 1 and 2K (MATE1/2K) are localized in the apical membrane of renal tubule cells which were shown to work in tandem with basally expressed OCT transporters for other cations including metformin, creatinine and amantadine, this mechanism might also be relevant for ketamine.20 However, for all aforementioned transporters there is no clear direct evidence to be involved in ketamine disposition. Thus, it was the aim of this study to investigate its affinity to human P-gp, OCT1-3 and MATE1/2K in order to get deeper insights into their potential clinical relevance in ketamine absorption, distribution and excretion.


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Material and Methods Chemicals Ketamine, N-methyl-4-phenylpyridinium (MPP+), tetraethylammonium (TEA), quinidine, pyrimethamine, verapamil, rhodamine-123 (Rh123), adenosinmonophosphate (AMP), and adenosintriphosphate (ATP) were obtained from Sigma-Aldrich (Taufkirchen, Germany). PSC833 was kindly provided by Novartis (Basel, Switzerland). [3H]-BSP (10.2 Ci/mmol; 1 µCi/µl), [3H]-TEA (55 Ci/mmol; 1 µCi/µl), [3H]-MPP+ (80 Ci/mmol; 1 µCi/µl), and [3H]ketamine (80 Ci/mmol; 1 µCi/µl) were purchased from Hartmann Analytic (Braunschweig, Germany). Parallel artificial membrane permeability assays (PAMPA) Passive diffusion of ketamine was investigated using the 96-well Corning® Gentest™ Precoated PAMPA Plate System (Corning Life Sciences, Tewksbury, MA, USA). PAMPA was performed according to the manufacturer’s instructions. In brief, 30 nM of radiolabelled ketamine was dissolved in 300 µl incubation buffer (142 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l K2HPO4, 1.2 mmol/l MgSO4, 1. mmol/l CaCl2, 5 mmol/l glucose, and 12.5 mmol/l HEPES; pH 6.5 or 7.4) and added to the bottom (donor) compartment. After incubation for 2, 3 or 4 hours at 37°C, 100 µl of incubation puffer was taken from the top (receiver) compartment, mixed with 1 ml of scintillation cocktail (Rotiszint ecoplus; Roth, Karlsruhe, Germany) and measured using liquid scintillation counting. The apparent permeability (Papp) was calculated using the equation Papp = (dQ/dt) × (1/A × C0), where dQ/dt (mg/s) is the linear transport rate of the substrate to the receiver compartment over time, A (cm2) the exposed area of the membrane, and C0 (mg/mL) the initial compound concentration in the donor compartment. Cell Culture Madin-Darby canine kidney (MDCKII) were purchased from the European Collection of Cell Cultures (Salisbury, United Kingdom) and were grown in Dulbecco’s modified Eagle’s ACS Paragon Plus Environment

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medium supplemented with 10% fetal bovine serum, and 4 mM L-glutamine at 37 °C, 95% humidity, and 5% CO2. Stably transfected MDCKII cells overexpressing OCT1, OCT2 or OCT3, and the respective vector-transfected control cells (VC) were generated as previously described.21,22 MDCKII cells expressing MATE1 and MATE2k were purchased from Solvo Biotechnology (Szeged, Hungary). Affinity of ketamine to P-gp P-gp-mediated transport of ketamine was investigated at pH 6.5 and 7.4 in inside-out lipovesicles purchased from Thermo Fisher Scientific (Darmstadt, Germany) as described previously.19 In brief, for the ATP-dependent transport 30 µg of total vesicle protein was incubated for 10 minutes at 37°C with [3H]-ketamine, dissolved in tris-mannitol buffer (50 mmol/l tris, and 50 mmol/l mannitol, pH 6.5 or 7.4) with unlabelled ketamine to reach a final concentration of 10 µmol/L. Vesicles were permeabilized with scintillation cocktail (Rotiszint ecoplus; Roth, Karlsruhe, Germany) after rapid filtration using glas fiber filters (0.7 µM pore size; GE Healthcare, Freiburg, Germany), and uptake of ketamine in lipovesicles was measured by using a liquid scintillation beta counter (type 1409, LKB-Wallac, Turku, Finland). In control experiments, control vesicles (CV) which do not contain P-gp were used. Functionality of P-gp lipovesicles was verified by measuring Rh123 accumulation in the presence of AMP or ATP and in presence or absence of the P-gp inhibitor PSC833 using the Infinite M200 Plate Reader (Tecan, Maennedorf, Switzerland). Affinity to Uptake Transporters For uptake assays, MDCKII cells expressing OCT1, OCT2 or OCT3, cells were seeded in 24well plates and incubated in full growth medium at an initial density of 100,000 cells/well for 2 days until cells reached a confluence of 90%. Experiments were performed as described previously.19 In brief, cells were washed once with the aforementioned incubation buffer (37 °C, pH 6.5 or 7.4). After the respective experiment, cells were washed three times with icecold incubation buffer and lysed with 500 µl 0.5% Triton X-100 (Merck, Darmstadt, ACS Paragon Plus Environment

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Germany) and 0.5% sodium deoxycholate (Sigma-Aldrich, Steinheim, Germany). One hundred microliters of cell suspension was mixed with 1 ml of scintillation cocktail and measured using liquid scintillation counting. Protein concentration was determined to quantify cell density after the experiments using the BCA assay according to the manufacturer’s instructions (Pierce, Rockford, USA). Functionality of OCT1, OCT2, and OCT3 cells were studied using [3H]-MPP+ dissolved with unlabelled MPP+ to reach a final concentration of 10 µmol/L in the presence or absence of 100 µmol/L verapamil. Functionality of MATE1 and MATE2k expressing cells were verified using [3H]-TEA dissolved with unlabelled TEA to reach a final concentration of 5 µmol/l in the presence or absence of 100 µmol/L quinidine or 10 µmol/L pyrimethamine, respectively. Uptake of ketamine was first measured in time-dependent uptake assays for 10 – 300 seconds in OCT1, OCT2 and OCT3 expressing cells or 600 seconds in MATE1 and MATE2k transfected cells using [3H]-ketamine dissolved with unlabelled ketamine to reach a final concentration of 100 µmol/L (OCTs) or 15 µmol/L (MATE1/2K). The Michaelis−Menten constant (Km) and the maximal uptake rate (Vmax) values for OCT1, OCT2, and OCT3 were calculated using 0−500 µmol/L of ketamine and an incubation time of 1 min. Ketamine net uptake rate was corrected to the respective transporter protein abundance, determined by a validated LC-MS/MS method using the ProteoExtract® Native Membrane Protein Extraction Kit (Merck, Darmstadt, Germany) according to the manufacturer’s instructions.23 Kinetic and Statistical Evaluation The OCT-mediated net uptake was obtained by subtracting the uptake in vector-transfected cells from that in OCT1, OCT2, and OCT3-expressing cells. Km and Vmax were assessed using Prism 5.01 (GraphPad Software, San Diego, USA). Km and Vmax are presented as arithmetic means ± standard deviation (M ± SD). The intrinsic clearance (Clint) was calculated by Vmax/Km.


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Results and discussion Ketamine is a lipophilic basic drug (pKa = 7.5) whose ionization depends on the pH of the surrounding medium. Consequently, it is expected to exist mainly as a cationic moiety (91%) in the upper small intestine (pH = 6.5) which may markedly diminish its free diffusion across biological membranes such as the intestinal epithelia. In this regard, Papp-values of ketamine in the PAMPA were assessed to be 1.49 ± 0.04 x 10-6 cm/s at pH 6.5 and 3.64 ± 0.76 × 10-6 cm/s at pH of 7.4, indicating a low permeability of ketamine at pH 6.5 which was more than doubled at pH 7.4 (Figure S1). Therefore, transporter proteins may contribute to the intestinal absorption of ketamine which was investigated in our study. Uptake by OCT transporters As some OCT transporters were convincingly shown to be expressed in the human intestine24,25, we investigated the relevance of OCT1-3 in the cellular uptake of ketamine by using transfected MDCKII cells overexpressing the respective human proteins. The functionality of each cell line was verified using MPP+ as probe substrates in presence or absence of the OCT-inhibitor verapamil at pH = 6.5 and 7.4 (Figure S2). Ketamine was shown to be taken up in a concentration-dependent manner by OCT1-3 (Figure 1). This is in line with previously published data that ketamine is an inhibitor of the aforementioned transporters.17,18 As expected from the physicochemical properties of ketamine, the affinity to the OCT3 transporter at pH = 6.5 was clearly higher than at pH = 7.4, while in OCT1 and OCT2 transfected cells no affinity could be calculated. The respective Km and Vmax data of the cellular net uptake are shown in Table 1. Uptake studies were performed at pH 6.5 to mimic the situation in the intestinal lumen and at pH 7.4 to conclude on the situation in the systemic circulation. The so far clinically used oral doses of ketamine (~25 mg) are expected to result in intestinal concentrations of about 420 µmol/L when given with 240 ml (1 cup) water as recommended by the FDA for clinical trials (Guidance for Industry: Bioavailability and ACS Paragon Plus Environment

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Bioequivalence Studies for Orally Administered Drug Products — General Considerations, 2003), which makes an interaction with intestinal OCTs (Km ~35-75 µM) plausible. Thus, we would conclude that the OCT-mediated cellular uptake is expected to be only of relevance in the human intestine, where OCT1 / 3 could be verified to be stably expressed at the apical membrane24,25 and where ketamine is predominately present as cation (91%). This scenario indicates saturation of the respective intestinal uptake transporters (OCT1 / 3) suggesting that the OCT-mediated uptake might be the rate-determining step in the intestinal absorption of the drug. In contrast to this, OCT- and transporter-mediated uptake in general is not expected for ketamine at the systemic pH of 7.4 because of its low ionization (56%) and substantial passive diffusion of lipophilic ketamine (logP = 2.9) across biological membranes. Efflux transport by P-gp To study role of P-gp efflux, uptake studies into P-gp lipovesicles were performed at pH 6.5 and 7.4. The functionality of the P-gp vesicle assays was verified using a P-gp probe substrate (Rh123) and inhibitor (PSC833) (Figure S2). Our experiments demonstrated that accumulation of ketamine (10 µmol/L) for up to 30 min at pH 7.4 in P-gp lipovesicles was independent from ATP, which is the known driving force for this transporter (Figure 2A). This indicated that ketamine may not be a substrate for P-gp. This was also indirectly confirmed by an in vitro experiment in which ketamine showed no inhibition of P-gp in MDR1-transfected HEK293 cells as detected by flow cytometry.26 Therefore, P-gp is not expected to contribute significantly to tissue (brain) distribution or renal excretion of ketamine. As we performed our study at pH 7.4, which is the assumed intracellular and systemic pH, our finding maybe due to passive diffusion of ketamine into the vesicles considering the low ionization degree at this pH and the high lipophilicity of ketamine. This limitation of the lipovesicle assay for highly lipophilic compounds is already well established.27,28 ACS Paragon Plus Environment

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However, a recent mechanistic study revealed that P-gp substrates do not necessarily have to enter the cell and to bind to P-gp from the intracellular space but may penetrate into the membrane from the extracellular space and bind to an intramembrane domain of the transporter. The conformation switch of P-gp as initiated by intracellular binding and hydrolysis of ATP results in extrusion of the respective substrate out of the cell membrane.29 To acknowledge this hypothesis, we finally (re-)performed our vesicular uptake experiments at pH 6.5 (i.e. the expected pH in the upper small intestine) and found that ketamine is a low affinity substrate of P-gp with no saturation of the transporter for up to 2000 µmol/L (Figure 2B). As already discussed for the OCT transporters, the relevance of P-gp seems to be limited to the human intestine. In line with this assumption, a clinical drug-drug interaction study of oral ketamine with the P-gp and CYP3A4 inhibitor clarithromycin increased AUC and Cmax of ketamine about 3-fold while the terminal half-live was not significantly affected.30 In addition, the exposure of the CYP enzyme-derived major metabolite nor-ketamine was not changed suggesting that this interaction was at least partly caused by inhibition of intestinal Pgp. Transport by MATE1/2K The relevance of MATE1/2K in the transport of ketamine was studied with MATE1/2Ktransfected MDCKII-cells. The functionality of the cells was confirmed by using a probe compound (TEA) and well established inhibitors (quinidine for MATE1 and pyrimethamine for MATE2K, Figure S2). Uptake screening experiments with up to 15 µmol/L ketamine did not show a significant difference between the control and transporter-transfected cells (Figure 3). As ketamine is excreted predominately via the kidneys, we investigated MATE1/2K which works in many cases in concert with OCT transporters in order to facilitate a vectorial transport across the renal tubule cells. However, although ketamine was identified as an OCT substrate we could not verify a significant affinity to MATE1/2K. Taking also into account that under the systemic pH of about 7.4 an OCT-mediated transport of ketamine is not ACS Paragon Plus Environment

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expected (Km ~ 365 µmol/L for OCT3 vs. systemic concentrations of ~ 0.09 µmol/L13), we conclude that the renal elimination seems not to be transporter dependent but is rather a function of glomerular filtration and tubular reabsorption. This conclusion is also supported by clinical data of ketamine, i.e. its renal clearance. Considering that the renal clearance of ketamine in human is about 65 ml/ml and that the drug was described to be bound to plasma proteins by about 47%, unbound glomerular filtration rate can be estimated to be about 63.6 ml/min. This value is nearly identical to the observed clinical value and thereby dismisses the involvement of transporter proteins in the renal excretion of ketamine. Conclusion Ketamine is a substrate of OCT1, 2 & 3 and a week substrate for P-gp but it is not recognized by MATE1/2K. Clinically relevant transporter function of P-gp and OCTs (i.e. OCT1) is only expected in the human intestine which should be considered in of cases of oral administration of ketamine.


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Acknowledgement We acknowledge Jenny Hagemann and Diana Busch for her excellent technical assistance. The work was supported by the grant 03IPT612X (InnoProfile) of the German Federal Ministry for Education and Research.

Conflict of interest The authors declare that they have no conflict of interest.

Author contributions Participated in research design: Hasan, Keiser, Oswald Conducted experiments: Hasan, Keiser Performed data analysis: Hasan, Keiser Wrote or contributed to the writing of the manuscript: Hasan, Keiser, Oswald


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25. Han, T. K.; Everett, R. S.; Proctor, W. R.; Ng, C. M.; Costales, C. L.; Brouwer, K. L.; Thakker, D. R. Organic cation transporter 1 (OCT1/mOct1) is localized in the apical membrane of Caco-2 cell monolayers and enterocytes. Mol. Pharmacol. 2013, 84 (2), 182-189. 26. Tournier, N.; Chevillard, L.; Megarbane, B.; Pirnay, S.; Scherrmann, J. M.; Decleves, X. Interaction of drugs of abuse and maintenance treatments with human Pglycoprotein (ABCB1) and breast cancer resistance protein (ABCG2). Int. J. Neuropsychopharmacol. 2010, 13 (7), 905-915. 27. Xia, C. Q.; Milton, M. N.; Gan, L. S. Evaluation of drug-transporter interactions using in vitro and in vivo models. Curr. Drug Metab 2007, 8 (4), 341-363. 28. Polli, J. W.; Wring, S. A.; Humphreys, J. E.; Huang, L.; Morgan, J. B.; Webster, L. O.; Serabjit-Singh, C. S. Rational use of in vitro P-glycoprotein assays in drug discovery. J. Pharmacol. Exp. Ther. 2001, 299 (2), 620-628. 29. Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.; Harrell, P. M.; Trinh, Y. T.; Zhang, Q.; Urbatsch, I. L.; Chang, G. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 2009, 323 (5922), 1718-1722. 30. Hagelberg, N. M.; Peltoniemi, M. A.; Saari, T. I.; Kurkinen, K. J.; Laine, K.; Neuvonen, P. J.; Olkkola, K. T. Clarithromycin, a potent inhibitor of CYP3A, greatly increases exposure to oral S-ketamine. Eur. J. Pain 2010, 14 (6), 625-629.


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Legends for Figures

Figure 1:

Uptake of ketamine into stably transfected OCT1-, OCT2- and OCT3-MDCKII cells at pH = 6.5 (left figures) and 7.4 (right figures). Total uptake of ketamine is shown in nmol × mg total protein-1 × min-1 (normalized to total protein). Net uptake of ketamine (transporter transfected cells – vector transfected cells (VC)) is presented after normalization to respective transporter protein abundance as determined by targeted proteomics and is shown in nmol × fmol specific protein-1 × min-1. All experiments were performed three times in triplicate.

Figure 2:

ATP-dependent uptake of 10 µmol/l ketamine in P-gp vesicles at pH = 6.5 and 7.4 in the presence or absence of the P-gp inhibitor PSC833 (A) and concentration-dependent uptake of ketamine into P-gp vesicles or in control vesicles (CV) at pH = 6.5 in the presence of ATP (B). All experiments were performed two times in duplicate.

Figure 3:

Uptake of ketamine into MATE1- (A) and MATE2K-transfected (B) and wildtype MDCKII-cells. All experiments were performed two times in duplicate.


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Table 1:


Data on transport kinetics of ketamine for OCT1-3 at pH 6.5 (intestinal lumen) and pH 7.4 (systemic circulation). pH = 6.5

pH = 7.4

OCT1

OCT2

OCT3

OCT1

OCT2

OCT3

Km (µmol/L)

73.9 ± 15.2

33.5 ± 20.3

52.9 ± 15.0

-

-

365 ± 125

Vmax (fmol × fmol-1 × min-1)#

182.7 ± 12.6

49.1 ± 8.3

124.7 ± 10.9

-

-

99.2 ± 18.5

2.81 ± 0.96

2.75 ± 2.22

3.09 ± 0.63

-

-

0.27 ± 0.10*

Clint (nl × fmol-1 × min-1)# #

*p

Affinity of Ketamine to Clinically Relevant ... - ACS Publications

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