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2019
L-Type Amino Acid Transporter 1 (LAT1)-Utilizing Prodrugs Are
Carrier-Selective Despite Having Low Affinity for Organic Anion Transporting Polypeptides (OATPs)
Huttunen, Johanna
Elsevier BV
Tieteelliset aikakauslehtiartikkelit
© Elsevier B.V.
CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
http://dx.doi.org/10.1016/j.ijpharm.2019.118714
https://erepo.uef.fi/handle/123456789/7830
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L-Type Amino Acid Transporter 1 (LAT1)-Utilizing Prodrugs Are Carrier- Selective Despite Having Low Affinity for Organic Anion Transporting Poly- peptides (OATPs)
Johanna Huttunen, Mikko Gynther, Kati-Sisko Vellonen, Kristiina M.
Huttunen
PII: S0378-5173(19)30759-8
DOI: https://doi.org/10.1016/j.ijpharm.2019.118714
Reference: IJP 118714
To appear in: International Journal of Pharmaceutics Received Date: 24 May 2019
Revised Date: 22 August 2019 Accepted Date: 16 September 2019
Please cite this article as: J. Huttunen, M. Gynther, K-S. Vellonen, K.M. Huttunen, L-Type Amino Acid Transporter 1 (LAT1)-Utilizing Prodrugs Are Carrier-Selective Despite Having Low Affinity for Organic Anion Transporting Polypeptides (OATPs), International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/
j.ijpharm.2019.118714
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© 2019 Elsevier B.V. All rights reserved.
L
-Type Amino Acid Transporter 1 (LAT1)-Utilizing Prodrugs Are Carrier-Selective Despite Having Low Affinity for Organic Anion
Transporting Polypeptides (OATPs)
Johanna Huttunen,a Mikko Gynther,a Kati-Sisko Vellonen,a Kristiina M. Huttunen a,*
a School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland,
P.O. Box 1627, FI-70211 Kuopio, Finland
* Corresponding author, E-mail: kristiina.huttunen@uef.fi
Abstract
L-Type amino acid transporter 1 (LAT1) –utilizing prodrugs has been designed to improve drug delivery and targeting into the brain or cancer cells, since LAT1 is highly and selectively expressed on the blood-brain barrier as well as over-expressed in several types of cancer. However, less is known about the affinity of these compounds for the secondary transport mechanisms. The aim of this study was to evaluate interactions of nine LAT1-utilizing prodrugs with organic anion transporting polypeptides (OATPs). These results showed that LAT1-utilizing prodrugs can indeed, interact with OATPs, although it was considered to be minor compared to LAT1; the Km values of these compounds for LAT1 were 1-7 µM while the ones for OATPs were 73-406 µM. Moreover, utilization of LAT1 was 2-12-times more effective (compared as intrinsic clearance) than of OATPs, whose utilization seemed to be less significant at therapeutically used concentrations. According to these results, affinity for OATPs raised from the structural features of the parent drug moiety regardless of the structure of the promoiety. In conclusion, the present study shows that it is important to evaluate secondary transport mechanisms carefully, since they may have a role in pharmacokinetics of LAT1-utilizing prodrugs if LAT1 becomes saturated or un-functional.
Keywords
Affinity, Intrinsic clearance (CLint), L-Type amino acid transporter 1 (LAT1), Organic anion transporting polypeptides (OATPs), Prodrugs, Targeted drug delivery, Transport capacity
Abbreviations
BACE1, β-secretase 1; BBB, blood-brain barrier; COX, cyclo-oxygenase; 3D-QSAR, 3-Dimensional Quantitative Structure Activity; LAT1, L-type amino acid transporter 1; MCF-7, human breast cancer cell line; OATP, organic anion transporting polypeptide; PRB, probenecid; SLC, solute carriers; T4,
1. Introduction
Endogenous solute carriers (SLCs) are membrane-bound proteins that facilitate the transport of essential substances, such as amino acids, sugars, vitamins, electrolytes, nucleosides, bile acids, even macromolecules like proteins, across the biological membranes.1-3 Therefore, they regulate the supply of cell nutrients. It has also been proposed that transporters are major determinants for drug action as well as toxicity. For example, SLCs are able to carry not only nutrients, but also several drugs and toxins across the blood-brain barrier (BBB) into the brain parenchyma and therefore, these carriers play a crucial role in drug/toxin exposure within the brain.4,5 Thus, SLC transporters, consisting of nearly 500 proteins, are promising drug targets for future drug research and development; not only as a targeted drug carriers, but also as drug targets to modulate nutrient homeostasis, which may be impaired in brain diseases, or as diagnostic targets when identifying pathological changes associated with brain diseases.2,6 However, SLC transporters are still poorly characterized and utilized in rational drug research and development today.1 If the necessary structural features of the substrates for the selected transporter protein are known, novel compounds; substrates or inhibitors, that can interact with the transporter, can be developed.
L-Type amino acid transporter 1 (LAT1) is a transmembrane heterodimeric protein consisting of LAT1 light chain (SLC7A5) and 4F2 heavy chain (CD98; SLC3A2). LAT1 (referred thereafter to LAT1/4F2hc complex) is highly expressed at the luminal and abluminal sides of the BBB in relation to other normal tissues and it transports large, neutral, aromatic or branched amino acids (L-Leu, L- Ile, L-Phe, L-Trp, L-Tyr, L-Met, L-His and L-Val) from systemic blood stream into the brain.7 It can also transport several clinically used amino acid -mimetic drugs and prodrugs, such as L-dopa, gabapentin and melphalan.8 In addition, LAT1 is extensively over-expressed in tumors and their metastases,9 and therefore it has attained a growing interest also as a diagnostic target as well as potential drug target to detect and treat several types of cancer.10
We have previously shown with several drugs that are not LAT1-substrates as such, that attaching a cleavable amino acid promoiety to these parent drugs, and thus creating prodrugs, can significantly improve their cellular11-14 and brain uptake.15-20 However, we have also noticed that these prodrugs utilizing a high affinity – low capacity transporter LAT1, can also use another transporter mechanism with higher concentrations, i.e., when LAT1 is occupied. We have previously proposed that this secondary transport mechanism could be a low affinity – high capacity transporter, such as organic anion transporting polypeptide (OATP) or organic anion transporter (OAT), depending on the structure of the prodrug and the attached parent drug.12,13 These secondary transport mechanisms are very important to understand as they can have an effect on the pharmacokinetic profile and targeting efficacy of these prodrugs, e.g. across the BBB, into the brain parenchyma or cancer cells.
The aim of the present study was to evaluate the cellular uptake mechanisms of 8 recently synthesized prodrugs designed to utilize primarily LAT1 and explore if these prodrugs can also use OATPs for their cellular uptake in human breast cancer cell, MCF-7. This study will give insights which properties promote the affinity for OATPs, how much the affinity for OATPs affects the total cellular uptake at physiological conditions and what is the relevance of these secondary transport mechanisms e.g., in LAT1-utilizing targeted drug delivery.
2. Material and Methods
2.1 Materials
All reagents and solvents used in analytical studies were commercial and high purity of analytical grade or ultra-gradient HPLC-grade. Tris–HCl, ethylenediaminetetraacetic acid-Na2 2xH2O (EDTA), urea choline chloride, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), L-tryptophan (L-Trp), fluorescein, naringin, rifampicin, L-thyroxin (T4), probenecid, diclofenac and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA); KCl and NaOH from J.T. Baker (Denventer, The Netherlands), MgSO4, KH2PO4 and CaCl2 (2xH2O) from Merck (Darmstadt, Germany), and glucose, methanol, formic acid, chloroform, acetonitrile (ACN) and 2- (N-morpholino)ethanesulfonic acid (MES) from VWR International, LCC (Radnor, PA. USA). Water was purified using a Milli-Q Gradient system (Millipore, Milford, MA, USA). The studied LAT1- utilizing prodrugs have been synthesized in-house and their structural characterization (1H NMR, 13C NMR, LC-MS) and over 95% purity (elemental analysis) have been confirmed in our earlier publications; the studied prodrugs were made of perforin inhibitors (1-2)12, ketoprofen (3-7)19 and ferulic acid (8-9)20.
2.2 Expression of LAT1 and OATPs in MCF-7 Cells
MCF-7 human breast adenocarcinoma cells (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Total RNA was extracted from MCF-7 cells (3 parallel wells having 250 000 cell per well from cell passages of 15-17) by using RNeasy Micro Kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions. The extracted RNA was treated with DNase (DNA fee, AMbion, TX, USA), and the RNA concentration was quantified by using the NanoVue spectrophotometer (Biochrom, Harvard Bioscience, USA). RNA was converted into cDNA by using M-MuLV reverse transcriptase and random hexamers (Fermentas, Hanover, MD, USA).
Amplification of the cDNA corresponding to 11 ng of RNA was performed by employing Prism 7500
sequence detection system (Applied Biosystems, Inc., Foster City, CA, USA) and TaqMan Assays (Applied Biosystems, Inc., Foster City, CA, USA) hs01001183_m1 (LAT1, SLC7A5), hs00374243_m1 (4F2hc, SLC3A2), hs00245360_m1 (OATP1A2, SLCO1A2), hs00272374_m1 (OATP1B1, SLCO1B1), hs00251986_m1 (OATP1B3, SLCO1B3) and hs1030343_m1 (OATP2B1, SLCO2B1). Normalized gene expression was calculated using QGene application and cyclophilin A as the reference gene.21
The protein expression levels of LAT1, 4F2hc, OATP1A2, OATP1B1, OATP1B3 and OATP2B1 in crude and plasma membrane fractions of MCF-7 were quantified using multiplexed MRM analysis according to the protocol described by Uchida et al.22 First, the crude and plasma membrane fractions were isolated using Membrane Protein Extraction Kit, ab65400 (Cambridge, MA, USA) according to the manufacturer’s instructions. The aliquots (50 µg of protein) were solubilized in 7.0 M guanidine hydrochloride, 500 mM Tris–HCl (pH 8.5) and 10 mM EDTA. The proteins were reduced with dithiothreitol and S-carbamoylmethylated with iodoacetamide. Subsequently, proteins were precipitated with 600 µL methanol and 150 µL chloroform. The precipitates were dissolved by addition of 6.0 M urea in 0.1 M Tris–HCl (pH 8.5) followed by a 5-fold dilution with 0.1 M Tris–HCl (pH 8.5), which was spiked with a mixture of internal standard peptides. This step was followed by the addition of Lys-C (Promega, Madison, WI, USA) and Protease-Max (Promega, Madison, WI, USA), and incubation at room temperature for 3.0 h. Finally, tosylphenylalanyl chloromethyl ketone- treated trypsin (Promega, Madison, WI, USA) was added for tryptic digestion of the samples (enzyme/substrate ratio of 1:100), which were incubated at 37°C for 16.0 h. An aliquot of 45 µL of the sample was diluted and acidified with 75.0 µL of 1.5 % (v/v) formic acid in water, followed by centrifugation at 14 000 × g for 5 min at 4°C and supernatants were subjected to LC−MS/MS analysis.
The LC−MS/MS analysis was conducted by using an Agilent 1290 Infinity LC (Agilent Technologies, Waldbronn, Germany) system connected to an Agilent 6495 Triple Quadrupole Mass Spectrometer equipped with an ESI source (Agilent Technologies, Palo Alto, CA, USA). The HPLC method used for separation and elution of peptides has been described previously.23 The eluted peptides were simultaneously detected using the positive ion MRM mode. The dwell time was 20 ms per transition. The source temperature was 210°C with drying gas at a flow rate of 16 L/min. The nebulizer pressure was 45 psi and MS capillary voltage was 3 kV. The quantitation of the target protein was based on one unique peptide selected according to the in silico peptide selection criteria and previous report.22 Three or four MRM transitions for each specific peptide related to high intensity fragment ions were selected for quantification of a stable isotope-labeled peptide and the unlabeled investigated peptide (Table S1 in supplementary file). Data were acquired using the Agilent MassHunter Workstation Acquisition software (Agilent Technologies, Data Acquisition for Triple Quad., version B.03.01) and processed with Skyline software (version 4.2).
2.3 Functionality of LAT1 and OATPs in MCF-7 Cells
MCF-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with L-glutamine (2.0 mM; EuroClone S.p.A., Pero (MI), Italy), heat-inactivated fetal bovine serum (10%; Gibco, ThermoFisher Scientific, Waltham, MA, USA), penicillin (50 U/mL)-streptomycin (50 µg/mL) solution (EuroClone S.p.A., Pero (MI), Italy). MCF-7 cells (passages 8-25) were seeded at the density of 1 × 105 cells/well onto 24-well plates. The cells were used for the uptake experiments one day after seeding. All the studies were carried out as three biological replicates from the same cell passage. The function of LAT1 and OATPs were followed between the used cell passages (8-25) with a LAT1 probe substrate, [14C]-L- leucine, and OATP probe substrate fluorescein and noticed to be un-altered.
After removal of the culture medium, the cells were carefully washed with pre-warmed HBSS (Hank’s balanced salt solution) containing 125.0 mM choline chloride, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.3 mM CaCl2, 5.6 mM glucose, and 25.0 mM HEPES (pH 7.4 adjusted with 1 M NaOH) after removal of the culture medium. Pre-incubation was done with 500 μL of pre-warmed HBSS at 37 °C for 10 min before adding substrates (250 μL in HBSS) for the uptake experiments.
The functionality of LAT1 in MCF-7 cells was studied with a known LAT1 substrate; the cells were incubated with [14C]-L-leucine at 37 °C for 5 min (uptake linear up to 10 min) in uptake buffer (HBSS, 250 μL) consisting of 0.16-300 μM L-leucine (containing of 0.05-0.25 mCi/ml [14C]-L-Leu;
PerkinElmer, Waltham, MA, USA). After incubation the reaction was stopped by adding 500 µL of ice-cold HBSS and the cells were washed two times with ice-cold HBSS (2 x 500 µL). The cells were then lysed with 500 μL of 0.1 M NaOH (60 min) and the lysate was mixed with 3.5 mL of Emulsifier safe cocktail (Ultima Gold, PerkinElmer, Waltham, MA, USA). The radioactivity in the cells was measured by liquid scintillation counting (MicroBeta2 counter, PerkinElmer Waltham, MA, USA).
The concentration dependent uptake was also carried out with a competitive manner, in the presence of another LAT1 substrate, L-tryptophan (2.0 mM), the incubation buffer containing the same amounts of L-leucine as described above. The functionality of LAT1 in different pH values was carried out as described above by using MES buffer, in which 25.0 mM HEPES was replaced by MES and adjusted to pH 4.5, 5.5 and 6.5 by 1.0 M NaOH or HBSS buffer adjusted by 1.0 M NaOH to pH 8.5 and using 0.76 μM (0.25 mCi/ml) of [14C]-L-leucine. The concentrations of [14C]-L-leucine were calculated from the spiked standard curve at each pH and normalized with the protein concentrations.
The functionality of OATPs in MCF-7 cells was studied with a known OATP substrate, fluorescein (1-100 µM) in a MES buffer at pH 5.5 with a similar manner as described for the determination of
LAT1 functionality by using 30 min incubation time (uptake linear up to 40 min). After cell lysis, the supernatants were transferred into 96-well plates and the fluorescence was measured at 575Ex and 610Em by multiplate reader (EnVision, Perkin Elmer, Inc., Waltham, MA, USA). The concentration dependent uptake was also carried out with a competitive manner, in the presence of OATP inhibitors, naringin (NRG; 500 µM) or rifampicin (RFP; 500 µM), the incubation buffer (pH 5.5) containing the same concentration of fluorescein as described above (1-100 µM). The functionality of OATPs in different pH values was carried out as described above by using MES buffer adjusted to pH 4.5, 5.5 and 6.5 or HBSS buffer adjusted to pH 8.5 and with using 200 μM of fluorescein. The concentrations of fluorescein were calculated from the spiked standard curve at each pH and normalized with the protein concentrations.
2.4 Ability of Compounds to Bind to LAT1
For the following experiments the MCF-7 cells were cultured, seeded, washed with HBSS and preincubated as described above. The ability of compounds to inhibit the uptake of a known LAT1 substrate [14C]-L-leucine was then evaluated by incubating the cells at 37 °C for 5 min in uptake buffer pH 7.4 (250 μL) containing 0.16 μM (0.05 mCi/ml) of [14C]-L-leucine and 0.1-1000 μM of the studied compound (or HBSS as blank). The studied compounds were LAT1-utilizing prodrugs of investigational immunosuppressive perforin inhibitors (1-2),12 anti-inflammatory ketoprofen (3-7)19 and antioxidant ferulic acid (8-9)20, L-Trp, T4 and probenecid (PRB). The experiment was then carried out as described for the [14C]-L-leucine uptake above and the radioactivity was measured by liquid scintillation counting. Half of maximum inhibitory concentration (IC50) values were calculated by nonlinear regression analysis (fitting the curve to log (concentration) vs. remaining normalized viability).
2.5 Transporter-Mediated Uptake of Compounds into Cells
For the following experiments the MCF-7 cells were cultured, seeded, washed with HBSS and preincubated as described above. Cellular uptake of prodrugs 1-9, L-Trp, T4 and PRB were then studied by adding compounds at the concentration of 1-100 μM in pre-warmed HBSS buffer (250 μL) on the cell layer and incubating at 37 °C for 30 min (uptake was linear with all compounds up to
30 min). Subsequently, the cells were washed three times with ice-cold HBSS and lysed with 500 μl of 0.1 M NaOH (60 min). The supernatants were analyzed by the liquid chromatography mass spectrometry (LC-MS/MS) methods described earlier for compounds 1-2 (perforin inhibitor prodrugs),16,20 compounds 3-7 (ketoprofen prodrugs),19 and compounds 8-9 (ferulic acid prodrugs),20 with an Agilent 1200 Series Rapid Resolution LC System together with an Agilent 6410 Triple Quadrupole Mass Spectrometer equipped with an electro-spray ionization source using a Poroshell 120 EC-C-18 column (50 mm × 2.1 mm, 2.7 μm; Agilent Technologies, Santa Clara, CA) for the liquid chromatographic separation of the analytes. Detailed LC-MS/MS parameters for these methods can be found from the table S2 (in supplementary file). The lower limit of quantification (LLOQ) for the compounds 1 and 2 was 0.5 nM and for the compounds 3-9, L-Trp, T4 and PRB 0.05 nM. These LC-MS/MS methods were also selective, accurate (RSD < 15%) and precise (RSD < 15%) over the range 1.0-100 nM. The cell-associated concentrations of each compound normalized to protein concentration were calculated from the standard curve that was prepared by spiking known concentrations of compounds to ACN including the selected internal standard (diclofenac to all compounds). The protein concentrations on each plate were determined as a mean of three samples by Bio-Rad Protein Assay, based on the Bradford dye-binding method, using BSA as a standard protein and measuring the absorbance (595 nm) by multiplate reader (EnVision, Perkin Elmer, Inc., Waltham, MA, USA).
The competitive uptake in the presence of LAT1 inhibitor (KMH-233)24 was carried out as described above with HBSS buffer pH 7.4 or MES buffer pH 5.5 containing 25 µM of the studied compound.
The cells were pre-incubated with 100 µM LAT1 inhibitor for 10 min and the incubation mixture was removed before adding the studied prodrug and LAT1 inhibitor on the cells. The competitive uptake (30 min) with the inhibitor was then carried out as the normal uptake described above. The concentrations of studied compounds were analyzed by the LC-MS/MS method and calculated from the spiked standard curve and normalized with the protein concentrations.
The competitive uptake in the presence OATP1A2/2B1 inhibitor 500 µM naringin,25,26 or OATP1B1/1B3 substrate/inhibitor 500 µM rifampicin27 in the presence of LAT1 substrate L-Trp (2.0 mM) was carried out as described above by HBSS buffer pH 7.4 or MES buffer pH 5.5 containing 100 µM of the studied compound. The cells were pre-incubated with L-Trp and naringin or rifampicin for 10 min and the incubation mixture was removed before adding the studied prodrug, L-Trp and naringin or rifampicin on the cells. The competitive uptake (30 min) was then carried out as the non- competitive uptake described above. The concentrations of studied compounds were analyzed by the LC-MS/MS method described above and calculated from the spiked standard curve.
2.6 Data analysis
All data analyses, including Michaelis-Menten and Eadie-Hofstee analyses were performed using GraphPad Prism v. 5.03 software (GraphPad Software, San Diego, CA, USA). Statistical differences between groups were tested using one-way ANOVA, followed by a two-tailed Tukey’s multiple comparison test and presented as mean ± SD, with statistically significant difference denoted by * P
< 0.05, ** P < 0.01, *** P < 0.001.
3. Results and Discussion
3.1 Expression and Function of LAT1 and OATPs
In the present study, human breast adenocarcinoma cell line, MCF-7, was used to evaluate the selectivity of LAT1-utilizing compounds for LAT1 over OATPs, since this cell line has been previously reported to express several different OATPs in addition to LAT1.28-30 Therefore, the gene and protein expression of LAT1 and selected OATPs was confirmed by RT-PCR and LC-MS/MS, and the function of LAT1 and OATPs by a known radio-labeled LAT1-substrate [14C]-L-leucine (L- Leu) or OATP-substrate fluorescein at varying pH values and in the presence of known transporters inhibitors. The mean normalized gene expression of human LAT1 (SLC7A5), its heavy chain 4F2hc (SLC3A2), OATP1A2 (SLCO1A2), OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3) and OATP2B1 (SLCO2B1) in MCF-7 cells are presented in Figure 1A. As shown, genes of LAT1 and 4F2hc were expressed at high levels in these cells, while the expression of most of the studied OATPs was at low level and the expression of OATP1B3 was not detected at all in MCF-7 cells.
The protein expression of LAT1 and its heavy chain 4F2hc as well as OATP1A2, OATP1B1, OATP1B3 and OATP2B1 were quantitated from the crude membrane (CM) and plasma membrane (PM) fractions of MCF-7 cells. Both proteins required for the functional LAT1 expression (LAT1 and 4F2hc) were quantitated on the MCF-7 membrane fractions supporting the mRNA expression data (Figure 1B). However, all investigated OATP-transporters were below detection limit providing evidence of a lower expression of these transporters in MCF-7 cells compared to LAT1. Similar pattern in LAT1 and OATP-transporter expression has also been reported in HepG2 cells earlier.31 Contrarily, in hepatocytes OATP-transporters were found to be highly expressed while LAT1 expression was in turn, below detection limit. Moreover, although the exact functions has remained unclear, it is known that OATPs are regulated at the post translational level by glycosylation and
function.32,33 This can have a major role to the detection of OATP proteins from crude and plasma membranes by LC-MS/MS, although they may be expressed in selected cells.
[14C]-L-Leu was uptaken into the MCF-7 at pH 7.4 with a concentration dependent manner (Figure 2A). The transport capacity of L-Leu uptake was relatively low having Vmax value of 23.2 ± 1.5 nmol/min/mg of protein. In turn, the affinity of L-Leu to LAT1 was relatively high having Km value of 59.0 ± 10.8 µM. However, both Vmax and Km values were in accordance to the previous literature values and corresponded a cellular uptake mediated via high affinity - low capacity transporter, namely LAT1.9,34,35 Moreover, another LAT1-substrate L-Trp (2.0 mM) was able to compete with the uptake of L-Leu and decreased it significantly (12% at 25 µM and 51% at 250 µM of L-Leu), showing that LAT1 protein was functional on the plasma membrane of MCF-7 cells (Figure 2A).
Concentration-dependent cellular uptake of fluorescein via OATPs was carried out at the pH 5.5 according to the literature, although in the following experiment it was found that the optimal pH value would have been actually a bit lower (pH 4.5, Figure 2B and D).36,37 The high transport capacity of fluorescein uptake (Vmax of 2332 ± 198 nmol/min/mg of protein) and low affinity for the transporter(s) (Km value of 226 ± 30 µM) corresponded to the literature values of OATP-mediated fluorescein uptake.38 A known OATP1A2/2B1 inhibitor naringin (500 µM)25,26 was able to block the fluorescein uptake only by ca. 21%, while an OATP1B1/1B3 inhibitor rifampicin (500 µM)27 had more effect on fluorescein uptake, inhibiting the uptake by ca. 43% (Figure 2B). However, it needs to kept in mind that although these inhibitors have been reported to be selective towards these transporter subforms among OATP family, they may have affinity for other influx or efflux carriers.
The uptake of [14C]-L-Leu was not dependent on pH (Figure 2C) unlike the uptake of fluorescein (Figure 2D), proving the known fact that LAT1-mediated uptake is pH-independent,39 while OATP- mediated transport is pH-dependent favoring slightly acidic conditions. However, although pH 7.4 is not optimal in vitro condition for OATPs, they are able to carry their substrates into the MCF-7 cells at pH 7.4, with a proportion comparable to LAT1 capacity (uptake of 200 µM fluorescein into MCF-7 cells at pH 7.4 is ca. 44.01 nmol/min/mg protein in Figure 2D while the uptake of 200 µM [14C]-L- Leu at same conditions is ca. 19.4 nmol/min/mg protein in Figure 2A).
3.2 Selection of Studied Compounds
LAT1-Utilizing prodrugs 1-9 (see structures in Table 1) selected for this study have been previously designed according to the 3-Dimensional Quantitative Structure Activity (3D-QSAR) model of the rat Lat1 binding site and therefore, the syntheses of these compounds have been published elsewhere.12,19,20 These compounds include prodrugs of investigational immunosuppressive agent, perforin inhibitors (1-2), clinically used non-steroidal anti-inflammatory drug and unselective cyclo- oxygenase-1/-2 (COX-1/-2) inhibitor, ketoprofen (3-7) and naturally occurring polyphenol antioxidant and β-secretase 1 (BACE1) inhibitor ferulic acid (8-9). In addition to the prodrugs, a known OATP substrate probenecid (PRB),40 LAT1 substrate L-Trp39 and LAT1/OATP substrate T441
were used as reference compounds. All the prodrugs have been tested to be stable in studied in vitro conditions, but been able to release their parent drug in vivo.12,16,19,20 Therefore, it was expected that these prodrugs did not release their parent drugs during the cellular uptake studies, although they can deliver their parent drugs e.g., across the BBB after intravenous administration.16,19,20
3.3 Ability of Compounds to Bind to LAT1
The ability of compounds to bind to LAT1 was evaluated via a competitive inhibition assay with [14C]-L-Leu in the MCF-7 cell line. Compounds 1, 3, 5 and 8 were able to compete for LAT1 binding with L-Leu, having IC50 values less than 2 µM (Table 1). Compared to previously published LAT1 inhibitor (KMH-233; IC50 of 18 µM in MCF-7)24, these prodrugs had higher affinity for LAT1 than the LAT1-inhibitor. Only the compound 9 and T4 had higher and thus, comparable IC50 value to the LAT1-inhibitor, while some of the LAT1-utilizing prodrugs (2, 4, 6 and 7) and L-Trp as well as the negative control, PRB, were not able to compete with L-Leu. We have noticed earlier, that if the uptake of competing compound is fast, such as with natural amino acids, the inhibition percentages can be relatively low, even if the compounds have high affinity and ability to utilize LAT1 and thus, to compete with radiolabeled LAT1 substrate.12,14 Nevertheless, these result were in accordance to the values predicted by 3D-QSAR model (47-74% for all studied compounds); aromatic promoieties attached from their meta-position to the parent drugs having the highest ability to bind LAT1 while non-aromatic more flexible promoieties having lower affinity for LAT1 (Table 1).42
3.4 Transporter-Mediated Uptake of Compounds into Cells
LAT1-Mediated transport across the cell membrane was studied over a 1-100 µM concentration range after 30 min incubation from the cell lysates by liquid chromatography mass spectrometry (LC- MS/MS). We have previously shown that the direct cell uptake is a very accurate method to determine the exact concentrations of compounds inside the cell compared to the cis- or trans- inhibitions studies of known LAT1-substrates.14 Moreover, the concentration-dependent uptake (1-100 µM) and Michaelis-Menten kinetics can reveal, if the transport is saturable and thus, carrier-mediated. As seen from the figure S1 (supplementary file), the uptake of all LAT1-utilizing compounds was saturable.
It was also noticed from these uptake curves (left side columns in figure S1) that compounds 3, 4, 8, 9 and T4 utilized another transporter at higher concentrations (data points outside of the uptake curves). However, as the Eadie-Hofstee transformations from the concentration dependent uptake
was also performed in this study, they revealed that all the studied compounds except PRB utilized two different transport mechanisms (right side columns in figure S1).
According to the Michaelis-Menten kinetic parameters (Vmax and Km values) calculated from the Eadie-Hofstee plot analysis, LAT1-prodrugs of perforin inhibitors (1-2), L-Trp and T4 were transported into MCF-7 cells via LAT1 with higheset capacity (Vmax ranged from 70 to 530 pmol/min/mg protein in Table 2) of all studied compounds. However, all LAT1-utilizing compounds, also those that were transported with lower capacity into the cells, showed relatively high affinity to LAT1 (Km values below 15 µM). Surprisingly, although T4 is relatively large in size (Mw. 776.87 g/mol) and had the lowest affinity of all LAT1-compounds for LAT1 (Km of 13.5 µM), it was effectively carried inside the MCF-7 cells via LAT1 (Vmax value 74.1 pmol/min/mg protein). The transport efficiency or intrinsic clearance (CLint) of all studied compounds was estimated by dividing Vmax by Km, showing the same trend that was seen with transport capacity (Vmax values); L-Trp > 1 >
2 > T4 >> 4 > 5 > 3 >> 6 >> 9 (Table 2). Thus, there was no correlation between transport efficiency via LAT1 and IC50 values of [14C]-L-leucine uptake among the studied compounds. Interestingly, the compounds 7 and 8 showed inducible or autoactivation of LAT1 (Figure S1 in supplementary file), which has been also seen in our previous studies with other LAT1-utilizing compounds, due to yet unknown mechanism.14 Therefore, the Michaelis-Menten kinetic parameters of LAT1-mediated uptake were not calculated for these compounds.
The ability of secondary transport mechanism, namely OATPs, to transport these compounds varied a lot. Interestingly, in this study natural amino acid, L-Trp was able to utilize OATPs with relatively high capacity (Vmax of 3420 pmol/min/mg protein), but with a relatively low affinity (Km of 79 µM) (Table 2), which has not been reported earlier. Compound 1 was transported at higher concentrations with very high capacity by secondary transport mechanism (Vmax of 8500 pmol/min/mg protein), with
a capacity that was much higher than the one of the known OATP-substrate, PRB (Vmax of 60 pmol/min/mg protein). However, the affinity of compound 1 for this transporter was one of the lowest (Km of 406 µM) and 7-times lower than the one of PRB (Km of 56 µM) and therefore, the relevance of this transport mechanism in physiological conditions is most likely minor. Curiously, its structural analogue, compound 2, was transported by this secondary transport mechanism (Vmax of 1010 pmol/min/mg protein) with much less capacity, although its affinity for the carrier was relatively high (Km of 42 µM), indicating that the secondary transport mechanism for compound 2 may also be some transporter other than OATP. The similar trend was seen also among KPF prodrugs; compounds 3, 4 and 6 most likely utilized also OATPs, while compounds 5 and 6 were carried by some other transport mechanism than OATPs according to their Vmax (238-636 vs. 12-53 pmol/min/mg protein) and Km
values (73-181 vs. 23-67 µM). However, it needs to be kept in mind that in this study there was only a limited amount of data points (1-100 µM), particularly for the secondary transport type, which may misrepresent the results. For example, the ferulic acid prodrugs 8 and 9 and T4 did not have enough data points in order to evaluate their secondary transport mechanism (Figure S1, Table 2), although these results implied that at very high concentrations these compounds started to utilize another mechanisms. Overall, the affinities for LAT1 seemed to be only slightly lower than the affinities of the studied compounds for their target proteins, perforin, COX-1/-2 or BACE1 (< 5 µM).43-45 Therefore, it can be concluded that LAT1 is a more relevant carrier for these prodrugs with clinically used concentrations than to OATPs.
According to the intrinsic clearance the secondary transport mechanism was most efficient with L- Trp, and compounds 1-2, while the other compounds were less effectively transported by OATPs into the cells. Overall, when comparing CLint values of both transport types LAT1-utilizing prodrugs were 2-12-times more effectively transported via LAT1 than OATPs. From this set of compounds it cannot be univocally concluded which structural properties promoted the affinity for OATPs. When
comparing the compounds 3, 4, 5, 6 and 7, with same parent drug (ketoprofen) and varying promoieties, no clear differences was seen in their LAT1- and OATP-utilization. Contrarily, the compounds 1 and 2 as well as 3 and 8, but also 5 and 9, which had the same promoieties but varied with their parent drugs, showed some variation in OATP-mediated transport. Therefore, it can be concluded that the parent drug moiety has greater role than the promoiety in OATP-utilization, as it can have a greater variance in different functional groups that can favor binding and translocation across the plasma membranes via OATPs.
To understand the role and significance of OATPs in the total cellular uptake of LAT1-utilizing prodrugs, competitive uptake studies were performed at pH 7.4 and 5.5 by incubating prodrugs in the presence of LAT1 inhibitor (KMH-233)24 or OATP inhibitors, naringin25,26 or rifampicin27 together with competitive LAT1-substrate L-Trp. When comparing the uptake of 25 µM compounds at pH 7.4 and 5.5 (Table 3), it can be distinguished which of the studied compounds were able to utilize OATPs, as their uptake was increased significantly when the pH was reduced to more favorable for OATPs.
Such compounds were L-Trp, T4 and PRB, whose ionization at their amino and acid residues stayed same along with decreased pH (pKa of L-Trp 2.8 and 9.4; T4 2.4, 6.9 (phenol) and 10.1; of PRB 3.4), implying that their increased uptake at pH 5.5 was mediated via a transporter instead of increased passive permeation. This comparison also revealed the pure LAT1-utilizers; prodrugs of ferulic acid 8-9 and ketoprofen 5-6, which had almost identical uptake values in both pH values. Curiously, the uptake of compounds 1-4 and 7 were decreased at lower pH, which may imply that there is another transport mechanism, other than OATPs involved in their total cellular uptake at physiological pH.
However, when incubating these compounds together with specific LAT1-inhibitor in both buffers at pH 5.5 and 7.4 (Figure 3A and B), it can be concluded that almost all compounds switched their transport mechanism to another transporter, when LAT1 was saturated. Only one ketoprofen prodrug
(6), ferulic acid prodrugs 8-9 and L-Trp showed higher preference towards LAT1, which was in accordance to the concentration dependent uptake results (Table 2). In some cases, such as with perforin inhibitors 1 and 2, changing from high affinity – low capacity transport (LAT1) to low affinity – high capacity transporter (OATPs) even rose the total cellular uptake. This phenomenon has also been seen not only on the cell surface, but also at the BBB.12,16
To confirm that OATP inhibitors, naringin and rifampicin can inhibit the low affinity – high capacity transport, the concentration of compounds was increased to 100 µM to ensure maximal OATP- mediated transport and its inhibition. As seen from the figure 4B, all compounds except ketoprofen prodrugs 2-4 were inhibited by either naringin or rifampicin or both at pH 5.5, although in the case of compound 9, the inhibition was not statistically significant. Curiously, the uptake of T4 and prodrug 6 was significantly increased in the presence of rifampicin. Rifampicin is known to induce expression and/or function of efflux transporters, such P-gp,46 which is overexpressed in MCF-7.47 Therefore, this results confirms that T4 and compound 6 have interactions with efflux transporter(s) or its/their regulating transcription factors.48 The same phenomenon was also seen at pH 7.4 (Figure 4A), in which the uptake of T4 and compound 6 was increased in the presence of naringin, which in turn is a known P-gp modulator.49 Curiously, the effects of rifampicin and naringin were pH-dependent.
At pH 7.4 (Figure 4A), the inhibition of compounds by naringin or rifampicin was not statistically as significant as at pH 5.5 (Figure 4B), even with OATP substrate PRB. This may indicate that the role and participation of OATPs to the total cellular uptake of LAT1-utilizing compounds at physiological conditions is less relevant. As concluded by fluorescein uptake, OATPs are able to carry their substrates into the MCF-7 comparable to LAT1 capacity. Therefore, all these results together show that even though highly LAT1-selective prodrugs can be designed and synthesize, they most likely are able to utilize some other low affinity – high capacity transport mechanism, but only
at higher concentrations or when LAT1 is saturated or not functional. This may have an impact on pharmacokinetics of these prodrugs, e.g., in diseases where LAT1 expression or function is altered.
However, we have recently showed that Alzheimer’s Disease phenotype (APP and PSI gene mutations) or lipopolysaccharide (LPS) -induced neuroinflammation do not affect the LAT1 functionality at the BBB or on the astrocytes, and therefore LAT1-utilizing prodrug approach can be useful when targeting drugs into the brain parenchyma in neuroinflammated conditions.50 Furthermore, OATP1A2 and OATP2B1 are expressed at the human BBB,51 which may compensate the brain uptake of LAT1-utilizing compounds, if LAT1-mediated uptake is compromised. Moreover, since LAT1 is highly over-expressed in many types of cancer, LAT1-prodrugs may indeed have a greater opportunity to deliver selectively cytotoxic drugs into the cancer cells. In addition, many subforms of OATPs are over-expressed in different types of cancer,52 and therefore compensational utilization of specific OATPs may also have advantages in cancer-targeting if LAT1 is saturated.
Nevertheless, in this study only OATPs were considered to be participating in total cellular uptake of LAT1-utilizing prodrugs. It needs to be kept in mind that there can also be other transporters involved and in some cases the affinity for secondary transport mechanisms may also impair the targeting efficacy. Therefore, it is really important to address all the possible transport mechanisms when designing and developing transporter-utilizing (pro)drugs.
4. Conclusions
In conclusion, even though prodrugs can be designed to have high affinity for LAT1 by utilizing computer-aided drug design tools such as pharmacophores and 3D-QSAR models, these compounds can also have affinity for other amino acid transporters or transporter carrying amino acid-mimetic compounds like OATPs. The affinity of secondary transport mechanism is highly dependent on the prodrug structure and particularly, the parent drug moiety has the highest effect on the selectivity for LAT1 over OATPs. Importantly, the role of OATPs in total cellular uptake maybe less obvious when studying cellular uptake into a native cells, such as cancer cells instead of using transporter transfected cells. Then lowering the pH to slightly acidic may help to understand the role of OATPs for the overall transport of studied compounds. However, the results gained at non-physiological pH needs to be interpret very carefully, since the transporter capacity of OATPs drops when increasing the pH to 7.4. The present study also showed that even LAT1-utilizing prodrugs can also have interactions with OATPs at higher concentrations, their physiological role may be significant only when LAT1 is saturated or dysfunctional. However, despite the minor role of OATPs, the possibility of other transport mechanism involved into the total uptake of LAT1-utilizing prodrugs should be studied in more details in future.
Acknowledgement
The authors would like to thank Ms. Tiina Koivunen for technical assistance with uptake studies and Prof. Seppo Auriola for guidance with LC-MS/MS proteomic methods. The study was financially supported by the Academy of Finland (grants 294227, 294229, 307057, 311939), Sigrid Juselius Foundation (grants 2015, 2016, 2017, 2018), Finnish Cultural Foundation (North Savo Regional Fund), Emil Aaltonen Foundation and the Biocenter Kuopio.
Conflicts of Interests
The authors declare no competing interest.
Author Contributions
Participated in research design: KMH, MG, K-SV, JH Conducted experiments: JH
Contributed to new reagents or analytical tools: KMH, MG, JH Performed data analysis: KMH, MG, K-SV, JH
Wrote or contributed to the writing of the manuscript: KMH, K-SV, MG, JH
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