European Journal of Pharmaceutical Sciences 66 (2015) 36–40
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Amino acid ester prodrugs conjugated to the a-carboxylic acid group do not display affinity for the L-type amino acid transporter 1 (LAT1) Jarkko Rautio ⇑, Jussi Kärkkäinen, Kristiina Huttunen 1, Mikko Gynther 1 School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland
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Article history: Received 12 August 2014 Received in revised form 30 September 2014 Accepted 30 September 2014 Available online 8 October 2014 Keywords: LAT1 L-type amino acid transporter 1 Prodrug Quinidine Isoleucine
a b s t r a c t L-type amino acid transporter (LAT1) is an intriguing target for carrier-mediated transport of drugs as it is highly expressed in the blood–brain barrier and also in various types of cancer. Several studies have proposed that in order for compounds to act as LAT1 substrates they should possess both negatively charged a-carboxyl and positively charged a-amino groups. However, in some reports, such as in two recent publications describing an isoleucine–quinidine ester prodrug (1), compounds having no free a-carboxyl group have been reported to exhibit high affinity for LAT1 in vitro. In the present study, 1 was synthesized and its affinity for LAT1 was evaluated both with an in situ rat brain perfusion technique and in the human breast cancer cell line MCF-7 in vitro. 1 showed no affinity for LAT1 in either model nor did it show any affinity for LAT2 in an in vitro study. Our results confirm the earlier reported requirements for LAT1 substrates. Thus drugs or prodrugs with substituted a-carboxyl group cannot bind to LAT with high affinity. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction L-type amino acid transporter 1 (LAT1) is a sodium-independent heterodimeric transmembrane protein found in brain, testis and placenta. It is also known that the levels of functional LAT1 are also significantly up-regulated in the surface of several human tumor cells highlighting its essential role in the growth and proliferation of cells. LAT1 is responsible for transporting large neutral amino acids such as L-leucine, L-tryptophan, L-isoleucine and Lphenylalanine into cells (Kanai et al., 1998) but it can also carry amino acid-derived drugs such as levodopa, gabapentin, melphalan and baclofen (Cornford et al., 1992; Kageyama et al., 2000; van Bree et al., 1988; Wang and Welty, 1996). Furthermore, LAT1 has been demonstrated to be able to transport amino acid prodrugs, where the amino acids have been conjugated with drug molecules which are not LAT1 substrates as such (Gynther et al., 2008; Killian et al., 2007; Walker et al., 1994). In previous studies it has seemed that in order to achieve efficient binding to LAT1, then the substrates should have an unsubstituted carboxylic acid or alternatively a negatively charged group as well as an unsubstituted amine functionality (Geier et al., 2013; Gynther et al., 2008; Uchino et al., 2002; Ylikangas et al., 2013). ⇑ Corresponding author. Tel.: +358 40 353 2791. 1
E-mail address: jarkko.rautio@uef.fi (J. Rautio). Authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejps.2014.09.025 0928-0987/Ó 2014 Elsevier B.V. All rights reserved.
In these studies removal or the presence of substituents on either carboxylic acid or amine functionalities exerted a dramatic effect on the binding ability of these compounds resulting in only weak affinity or a complete inability to bind to LAT1. In addition, amino acid derivatives in which the carboxyl and primary amine groups were further away than 3 Å possessed only a modest affinity for LAT1 which was comparable to those compounds lacking either the negatively charged group or the amine group (Ylikangas et al., 2013). However, some studies have indicated possible involvement of amino acid transport systems, including LAT1, in the transport of compounds lacking the free carboxyl group across cell membranes (Jain et al., 2004; Patel et al., 2013, 2014). For example, in two recent articles, an isoleucine–quinidine ester prodrug (1 in Fig. 1) has been reported to exhibit high affinity for LAT1 both in human derived prostate cancer cells (PC-3)(Patel et al., 2013) and in the Madin–Darby canine kidney (MDCK–MDR1) cell line (Patel et al., 2014). In addition, affinity for LAT2 was reported (Patel et al., 2014). In this prodrug the isoleucine promoiety had been conjugated with quinidine from the carboxylic acid functionality which, as far as we are aware and according to the prevailing consensus, this modification should significantly reduce the affinity for LAT1. Since these results are at odds with the current knowledge, in the present study we synthesized the prodrug described by Patel et al. (2013, 2014) and investigated its affinity for LAT1 and LAT2. The LAT1 affinity experiments were carried out with two
J. Rautio et al. / European Journal of Pharmaceutical Sciences 66 (2015) 36–40
independent methods; in situ rat brain perfusion technique and in human breast cancer cell line MCF-7 in vitro, both of which have demonstrated their versatility in LAT1 affinity studies, (Gynther et al., 2008; Killian et al., 2007; Shennan et al., 2004) while LAT2 affinity was determined in MCF-7 cells. 2. Materials and methods 2.1. General synthetic methods All the reactions were performed with reagents of commercial high purity quality without further purification. Reactions were monitored by thin-layer chromatography using aluminum sheets coated with silica gel 60 F245 (0.24 mm) with suitable visualization. Purifications by flash chromatography were performed on silica gel 60 (0.063–0.200 mm mesh). 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 500 spectrometer (Bruker Biospin, Fällanden, Switzerland) operating at 500.13 MHz and 125.75, respectively, using tetramethylsilane as an internal standard. pH-dependent NH-protons of the compounds were not observed. The final products were also characterized by mass spectroscopy with a Finnigan LCQ quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, CA, USA) equipped with an electrospray ionization source and by elemental analysis (C, H, N) with a Perkin Elmer 2400 Series II CHNS/O organic elemental analyzer (Perkin Elmer Inc., Waltham, MA, USA). 2.2. Synthesis of isoleucine–quinidine (1) The compound 1 was synthesized as previously described (Patel et al., 2013). 1H NMR ((CD3)2SO): d ppm 8.76 (d, 3JHH = 4.5 Hz, 1H), 8.02 (d, 3JHH = 9.0 Hz, 1H), 7.64 (d, 3JHH = 4.2 Hz, 1H), 7.56–7.49 (m, 2H), 6.23–6.12 (m, 1H), 5.27–5.18 (m, 2H), 4.48–4.41 (m, 1H), 3.40 (s, 3H), 3.97–3.90 (m, 1H), 3.64–3.545(m, 1H), 3.51–3.41 (m, 1H), 3.31–3.20 (m, 2H), 2.78–2.69 (m, 1H), 2.45–2.38 (m, 1H), 2.19– 2.10 (m, 1H), 2.05–2.00 (m, 1H), 1.87–1.79 (m, 2H), 1.59–1.50 (m, 1H), 1.46–1.37 (m, 1H), 1.32–1.21 (m, 2H), 1.04 (d, 3JHH = 5.7 Hz, 3H), 0.88 (t, 3JHH = 7.3 Hz, 3H) ; 13C NMR ((CD3)2SO): d ppm 168.13, 158.16, 147.26, 143.77, 139.72, 137.32, 131.39, 125.53, 122.31, 118.39, 117.25, 101.40, 71.68, 57.36, 56.02, 48.39, 47.79, 36.58, 35.31, 26.35, 23.49, 22.19, 19.27, 14.82, 11.29. MS (ESI+) for C26H36N3O3 (M + H)+: Calcd 438.58, Found 438.19. Anal. Calcd for (C26H35N3O3 ⁄ 1.3 TFA): C, 58.64; H, 6.25; N, 7.17; Found: C, 58.60; H, 5.85; N, 7.09. 2.3. In vitro uptake experiments in MCF-7 cells MCF-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with L-glutamine (2 mM), heat-inactivated fetal bovine serum (10%), penicillin (50 IU/ml) and
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streptomycin (50 Ag/ml). MCF-7 cells were seeded at the density of 1 105 cells/well onto collagen-coated 24-well plates. The cells were used for the uptake experiments one day after seeding. After removal of the culture medium, the cells were carefully washed with pre-warmed Na+-free HBSS (Hank’s balance salt solution) containing 125 mM chlorine chloride, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.3 mM CaCl2, 5.6 mM glucose, and 25 mM HEPES (pH 7.4) and then pre-incubated in 500 lL of pre-warmed Na+-free HBSS at 37 °C for 10 min before adding substrates for the uptake experiment. The cells were then incubated at 37 °C for 5 min in 250 lL of uptake medium: Na+-free HBSS containing [14C]-L-leucine or [14C]-L-alanine. Subsequently, the cells were washed three times with ice-cold Na+-free HBSS. The cells were then lysed with 500 lL of 0.1 M NaOH and the lysate was mixed with 3.5 mL of Emulsifier safe cocktail (PerkinElmer, Waltham, MA, USA). The radioactivity was measured by liquid scintillation counting (Wallac 1450 MicroBeta; Wallac Oy, Finland). In our studies, Km and Vmax values were determined for [14C]-L-leucine and [14C]-L-alanine uptake under Na+-free conditions. The Km and Vmax values were 141.0 ± 13.6 lM and 9.5 ± 0.28 nmol/mg protein/min for [14C]-Lleucine, which are in agreement with those by Shennan et al. (2004) and >2 mM and 1.4 ± 0.6 nmol/mg protein/min for [14C]-Lalanine, respectively. For the inhibition experiments the uptake of 0.157 lM [14C]-L-leucine or 10.0 lM [14C]-L-alanine was examined in the presence or absence of test compounds. 2.4. In situ rat brain perfusion technique The ability of 1 to bind to rat LAT1 at the rat BBB was studied with the in situ rat brain perfusion technique, which has been described and validated earlier (Gynther et al., 2008). The 100% permeability-surface area (PA) product of [14C]-L-leucine, a known substrate of LAT1, was determined with 30 s perfusion of 0.2 lCi/ mL [14C]-L-leucine (0.64 lM). The perfusion fluid was infused through the common carotid artery at the rate of 10 mL/min for 30 s using a Harvard PHD 22/2000 syringe pump (Harvard Apparatus Inc., Holliston, MA). In order to determine the interaction of 1 with LAT1 at the BBB, a 100 lM concentration of 1 was coperfused for 30 s with [14C]-L-leucine. 1 was dissolved in the perfusion medium right before the perfusion, in order to avoid the release of isoleucine from the prodrug before the experiment. The PA product of [14C]-L-leucine after coperfusion was compared with the 100% PA product of [14C]-L-leucine. In the coperfusion studies the LAT1binding of the investigated compound is shown by decreased PA product of [14C]-L-leucine caused by a competitive binding to LAT1. All samples were analyzed for radioactivity by liquid scintillation counting (Wallac 1450 MicroBeta; Wallac Oy, Finland). Brain samples were dissolved in 0.5 mL of Solvable (PerkinElmer, Boston) overnight at 50 °C, and liquid scintillation cocktail (Ultima Gold, PerkinElmer, Boston) was added before the samples were analyzed. The perfusion medium consisted of a pH 7.4 bicarbonate-buffered physiological saline (128 mM NaCl, 24 mM NaHCO3, 4.2 mM KCl, 2.4 mM NaH2PO4, 1.5 mM CaCl2, 0.9 mM MgCl2 and 9 mM D-glucose). The solution was filtered, heated to 37 °C, and gassed with 95% O2, 5% CO2 to attain steady-state gas levels within the solution. 2.5. Animals
O
N
O
O
1
N
Fig. 1. Chemical structure of isoleucine–quinidine ester (1).
Male Wister rats 200–250 g, 7–8 weeks of age obtained from the National Laboratory Animal Centre (Kuopio, Finland) were used for in situ rat brain perfusions. Food and water was available ad libitum on a 12/12 h light/dark cycle with lights on at 6 am. Experiments were performed between 9:00 am and 16.00 pm in temperature and humidity-regulated rooms (22–24 °C, relative humidity: 60–70%). Rats were anesthetized with intraperitoneal injections of ketamine (50 mg/kg) and xylazine (5 mg/mL).
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2.6. Hydrolysis of 1 in aqueous solution at pH 7.4 The rate of chemical hydrolysis of 1 was studied in aqueous phosphate buffer solution of pH 7.4 (0.16 M, ionic strength 0.5) at 37 °C. Pseudo-first-order half-time (t1/2) for the hydrolysis of the compound was calculated from the slope of the plotted logarithm of remaining compound versus time. 2.7. HPLC assay Concentration of 1 in the samples was analyzed by Agilent 1100 HPLC system with UV-detector (Agilent Technologies Inc., Waldbronn, Karlsruhe, Germany). The detector wavelength was set at 235 nm. A mixture of acetonitrile and water containing 0.1% of formic acid at a flow rate of 0.8 mL/min was used as a mobile phase. The amount of acetonitrile in the mobile phase was 78% and 15%. Reversed-phase HPLC was conducted with a Zorbax RP-18 column (150 mm 4.6 mm, 5 lm, Agilent Technologies, Little Falls Wilmington, DE). 2.8. Data analysis Statistical differences between groups were tested using oneway ANOVA, followed by a two-tailed Dunnett’s t test (Figs. 2 and 4). In Fig. 2, a two-tailed independent samples t test was used. P < 0.05 was considered as statistically significant. All statistical analyses were performed using GraphPad Prism 5.03 for Windows.
protein/min at 7.5 lM of isoleucine to 2.99 ± 0.29 pmol/mg of protein/min at 100 lM of isoleucine). 3.2. Ability of 1 to bind to LAT1 in situ The ability of 1 to bind to LAT1was studied also with the in situ rat brain perfusion technique. The brain capillary permeability surface area (PA) of 0.2 lCi/mL of [14C]-L-leucine (i.e. 0.64 lM) after 30 s brain perfusion was determined to be 0.02059 ± 0.00323 mL/ s/g. In the presence of 100 lM 1, the [14C]-L-leucine PA product was not reduced (0.02082 ± 0.0005 mL/s/g (mean ± sd, n = 3)), (Fig. 3), thus demonstrating that 1 did not exhibit any affinity for LAT1 at the rat BBB. 3.3. Ability of 1 to bind to LAT2 in vitro Uptake inhibition of 1.5 lCi/mL of [14C]-L-alanine (i.e. 10.0 lM), a known LAT2 substrate, was measured in the presence of 50 lM and 100 lM 1 in MCF-7 cells in vitro. In addition, the ability of isoleucine to bind to LAT2 was determined at 50 lM and 100 lM concentrations. The results shown in Fig. 4 clearly demonstrated that 1 did not inhibit the uptake of [14C]-L-alanine, whereas isoleucine displayed statistically significant 36.9% uptake inhibition at 50 lM concentration and 56.1% uptake inhibition at 100 lM concentration.
3. Results 3.1. Ability of compounds to bind to LAT1 in vitro The ability of 1 to bind to LAT1 was investigated in competitive inhibition studies in MCF-7 cells at 50 lM and 100 lM concentrations with a known LAT1 substrate, L-leucine. The expression and function of LAT1 in MCF-7 cells corresponded with previously reported values (Shennan et al., 2004). In addition, as 1 was rapidly cleaved into quinidine and isoleucine in aqueous solutions at pH 7.4 (t1/2 = 3.69 ± 0.29 h, mean ± sd, n = 3), the ability of isoleucine to bind to LAT1 was determined at four concentrations ranging from 7.5 lM to 100 lM. The results shown in Fig. 2 clearly demonstrate that 1 did not inhibit the uptake of [14C]-L-leucine significantly whereas isoleucine exerted a concentration dependent uptake inhibition of [14C]-L-leucine (from 6.80 ± 0.49 pmol/mg of
Fig. 2. The ability of 1 to bind to LAT1 in MCF-7 cells. The uptake of [14C]-L-leucine was not significantly inhibited at 50 lM or 100 lM of 1. The uptake inhibition of [14C]-L-leucine was statistically significant at 15 lM, 50 lM and 100 lM concentrations of isoleucine. The asterisks denote a statistically significant difference from the respective control (⁄⁄⁄P < 0.001, one-way ANOVA, followed by Dunnett’s t test).
Fig. 3. The ability of 1 to bind to LAT1 present at the rat BBB. The PA-product of [14C]-L-leucine was not decreased in the presence of 100 lM 1.
Fig. 4. The ability of 1 to inhibit the uptake of [14C]-L-alanine in MCF-7 cells. 1 did not have any statistically significant effect on the uptake of [14C]-L-alanine at 50 lM or 100 lM concentrations. The uptake inhibition of [14C]-L-alanine was statistically significant at 100 lM concentration of isoleucine. The asterisk denotes a statistically significant difference from the respective control (P < 0.05, one-way ANOVA, followed by Dunnett’s t test).
J. Rautio et al. / European Journal of Pharmaceutical Sciences 66 (2015) 36–40
4. Discussion Several studies have investigated the structural features of LAT1 substrates. These have been proposed to include both a negatively charged a-carboxyl and positively charged a-amino groups (Gynther et al., 2008; Smith, 2005; Uchino et al., 2002). More recently, we generated a 3D pharmacophore for a set of LAT1 substrates whose binding affinities for LAT1 were determined using the in situ rat brain perfusion technique. The pharmacophore identified the following structural features as being responsible for efficient LAT1 binding; (1) negatively charged center and hydrogen bond donor (HBD) at 3 Å distance from each other, (2) aromatic feature following HBD and a negative charge, (3) hydrogen bond acceptor (HBA) next to the aromatic region, at 3.8 Å distance from the aromatic feature (Ylikangas et al., 2013). Recently reported compound 1 (Fig. 1) has been reported to exhibit high affinity for LAT1 both in human derived prostate cancer cells (PC-3) (Patel et al., 2013) and in MDCK–MDR1 cell line (Patel et al., 2014). In our previous in situ study, structurally similar ketoprofen prodrugs in which the amino acids phenylalanine and leucine were conjugated with ketoprofen from the carboxylic group did not show any affinity for LAT1 (Gynther et al., 2008). Because 1 does not fulfill the structural requirements for high affinity LAT1 substrates either, we wanted to prepare this prodrug and study its affinity for LAT1 in our methods to understand the differences between the laboratories. Firstly, we predicted the affinity of 1 to bind to the LAT1 by a CoMFA model generated recently in our laboratory (Ylikangas et al., 2014). Because 1 lacks the free carboxylic group, the model predicted negligible LAT1 binding affinity for the compound. Secondly, we synthesized compound 1 according to the previously described procedure (Patel et al., 2013) and evaluated its LAT1 affinity with two independent methods i.e. in situ rat brain perfusion technique and a human breast cancer cell line MCF-7 in vitro. Both are well established methods used in LAT1 affinity studies (Gynther et al., 2008; Killian et al., 2007; Shennan et al., 2004). In our experiments, 1 exerted no affinity for human LAT1 or LAT2 in vitro in MCF-7 cells. This was demonstrated in inhibition studies where 1 achieved no inhibition of either L-leucine (LAT1 specific substrate) or L-alanine (LAT2 specific substrate) cellular uptake at concentrations up to 100 lM. To confirm our in vitro results, we determined the ability of 1 to bind to LAT1 with an in situ rat brain perfusion technique. In this in situ study, 1 showed no affinity for LAT1 as it was not able to inhibit the brain uptake of L-leucine even at a concentration as high as 100 lM. Is there any way to explain the discrepancies between the results reported here and those of Patel et al. (2013)? One explanation may be possible impurities of the previously tested compound. In the report by Patel et al. (2013), the full NMR spectrum of 1 was not unfortunately presented and there was no indication of the purity of the final product. On the other hand, 1H and 13C NMR interpretations of 1 synthesized in our laboratory with mass and elemental analysis reported in the experimental part demonstrated over 99% purity for the final compound. The presence of even a small amount of L-isoleucine as an impurity in the affinity experiments can significantly affect the results. In addition, any delay in performing the inhibition experiments would expose the compound to premature hydrolysis in the aqueous solution prior to conducting the experiments. Compound 1 was found to be susceptible to chemical degradation in aqueous solution with halflives of 5.0 ± 0.3 h in DPBS at pH 7.4 (Patel et al., 2014) and 3.69 ± 0.29 h in aqueous phosphate buffer solution of pH 7.4 (37 °C). We calculated that within 30 min at 37 °C and pH 7.4 approximately 7.5 lM and 15 lM concentration of isoleucine would be released from 50 lM and 100 lM solutions of 1,
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respectively. The uptake inhibition of [14C]-L-leucine was statistically significant at a 15 lM isoleucine concentration (Fig. 2). Therefore, the presence of even small amounts of released isoleucine could also account for the observed uptake inhibition. Thirdly, it has to be noted that the applied models used in the present research are different than the ones used by Patel et al. (2013, 2014). Therefore, we cannot fully exclude the possibility of a transporter expressed in the cells used by Patel et al., for which both large neutral amino acids and 1 have affinity, but which is expressed in neither MCF-7 cells nor in the rat BBB. The presence of impurities, the release of isoleucine or a yet unknown transporter in cell models used by Patel et al. could also explain the discrepancies between the LAT2 affinity results. 5. Conclusion In the present study, we have demonstrated with two independent methods that 1 has no affinity for either LAT1 or LAT2. Although no definitive explanation for discrepancy between results of the present study and those of Patel et al. (2013, 2014) could not be found, this study emphasizes, that experiments on prodrugs which consist of promoieties which can significantly affect the results (e.g. amino acids in affinity studies), should be carried out with caution. In order to have good affinity for LAT1, the substrates should fulfill the necessary structural features which include negatively charged group (e.g. carboxyl group) and hydrogen bond donor (e.g. amino group) and modification at either group is likely to result in total loss or at least a significant decline in affinity for LAT1. Acknowledgements The work was financially supported by the Academy of Finland (#256837 for KH and #132637 for JR), Päivikki and Sakari Sohlberg’s Foundation (KH), Orion Research Foundation (MG) and Finnish Cultural Foundation (MG). The authors also want to thank M.Sc. Henna Ylikangas for predicting LAT1 inhibition by QSAR. References Cornford, E.M., Young, D., Paxton, J.W., Finlay, G.J., Wilson, W.R., Pardridge, W.M., 1992. Melphalan penetration of the blood-brain barrier via the neutral amino acid transporter in tumor-bearing brain. Cancer Res. 52, 138–143. Geier, E.G., Schlessinger, A., Fan, H., Gable, J.E., Irwin, J.J., Sali, A., Giacomini, K.M., 2013. Structure-based ligand discovery for the large-neutral amino acid transporter 1, LAT-1. Proc. Natl. Acad. Sci. U. S. A. 110, 5480–5485. Gynther, M., Laine, K., Ropponen, J., Leppanen, J., Mannila, A., Nevalainen, T., Savolainen, J., Jarvinen, T., Rautio, J., 2008. Large neutral amino acid transporter enables brain drug delivery via prodrugs. J. Med. Chem. 51, 932–936. Jain, R., Majumdar, S., Nashed, Y., Pal, D., Mitra, A.K., 2004. Circumventing Pglycoprotein-mediated cellular efflux of quinidine by prodrug derivatization. Mol. Pharm. 1, 290–299. Kageyama, T., Nakamura, M., Matsuo, A., Yamasaki, Y., Takakura, Y., Hashida, M., Kanai, Y., Naito, M., Tsuruo, T., Minato, N., Shimohama, S., 2000. The 4F2hc/LAT1 complex transports L-DOPA across the blood–brain barrier. Brain Res. 879, 115– 121. Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., Endou, H., 1998. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J. Biol. Chem. 273, 23629–23632. Killian, D.M., Hermeling, S., Chikhale, P.J., 2007. Targeting the cerebrovascular large neutral amino acid transporter (LAT1) isoform using a novel disulfide-based brain drug delivery system. Drug Deliv. 14, 25–31. Patel, M., Dalvi, P., Gokulgandhi, M., Kesh, S., Kohli, T., Pal, D., Mitra, A.K., 2013. Functional characterization and molecular expression of large neutral amino acid transporter (LAT1) in human prostate cancer cells. Int. J. Pharm. 443, 245– 253. Patel, M., Mandava, N.K., Pal, D., Mitra, A.K., 2014. Amino acid prodrug of quinidine: an approach to circumvent P-glycoprotein mediated cellular efflux. Int. J. Pharm. 464, 196–204. Shennan, D.B., Thomson, J., Gow, I.F., Travers, M.T., Barber, M.C., 2004. L-leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231): kinetics,
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regulation by estrogen and molecular identity of the transporter. Biochim. Biophys. Acta 1664, 206–216. Smith, Q.R., 2005. Carrier-mediated transport to enhance drug delivery to brain. Int. Congr. Ser. 1277, 63–74. Uchino, H., Kanai, Y., Kim, D.K., Wempe, M.F., Chairoungdua, A., Morimoto, E., Anders, M.W., Endou, H., 2002. Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1): insights into the mechanisms of substrate recognition. Mol. Pharmacol. 61, 729–737. Walker, I., Nicholls, D., Irwin, W.J., Freeman, S., 1994. Drug delivery via active transport at the blood–brain barrier: affinity of a prodrug of phosphonoformate for the large amino acid transporter. Int. J. Pharm. 104, 157. van Bree, J.B., Audus, K.L., Borchardt, R.T., 1988. Carrier-mediated transport of baclofen across monolayers of bovine brain endothelial cells in primary culture. Pharm. Res. 5, 369–371.
Wang, Y., Welty, D.F., 1996. The simultaneous estimation of the influx and efflux blood-brain barrier permeabilities of gabapentin using a microdialysispharmacokinetic approach. Pharm. Res. 13, 398–403. Ylikangas, H., Malmioja, K., Peura, L., Gynther, M., Nwachukwu, E.O., Leppänen, J., Laine, K., Rautio, J., Lahtela-Kakkonen, M., Huttunen, K.M., Poso, A., 2014. Quantitative insight into the design of compounds recognized by the L-type amino acid transporter 1 (LAT1). ChemMedChem. http://dx.doi.org/10.1002/ cmdc.201402281 [Epub ahead of print]. Ylikangas, H., Peura, L., Malmioja, K., Leppanen, J., Laine, K., Poso, A., LahtelaKakkonen, M., Rautio, J., 2013. Structure–activity relationship study of compounds binding to large amino acid transporter 1 (LAT1) based on pharmacophore modeling and in situ rat brain perfusion. Eur. J. Pharm. Sci. 48, 523–531.
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Amino acid ester prodrugs conjugated to the a-carboxylic acid group do not display affinity for the L-type amino acid transporter 1 (LAT1) Jarkko Rautio ⇑, Jussi Kärkkäinen, Kristiina Huttunen 1, Mikko Gynther 1 School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland
a r t i c l e
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Article history: Received 12 August 2014 Received in revised form 30 September 2014 Accepted 30 September 2014 Available online 8 October 2014 Keywords: LAT1 L-type amino acid transporter 1 Prodrug Quinidine Isoleucine
a b s t r a c t L-type amino acid transporter (LAT1) is an intriguing target for carrier-mediated transport of drugs as it is highly expressed in the blood–brain barrier and also in various types of cancer. Several studies have proposed that in order for compounds to act as LAT1 substrates they should possess both negatively charged a-carboxyl and positively charged a-amino groups. However, in some reports, such as in two recent publications describing an isoleucine–quinidine ester prodrug (1), compounds having no free a-carboxyl group have been reported to exhibit high affinity for LAT1 in vitro. In the present study, 1 was synthesized and its affinity for LAT1 was evaluated both with an in situ rat brain perfusion technique and in the human breast cancer cell line MCF-7 in vitro. 1 showed no affinity for LAT1 in either model nor did it show any affinity for LAT2 in an in vitro study. Our results confirm the earlier reported requirements for LAT1 substrates. Thus drugs or prodrugs with substituted a-carboxyl group cannot bind to LAT with high affinity. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction L-type amino acid transporter 1 (LAT1) is a sodium-independent heterodimeric transmembrane protein found in brain, testis and placenta. It is also known that the levels of functional LAT1 are also significantly up-regulated in the surface of several human tumor cells highlighting its essential role in the growth and proliferation of cells. LAT1 is responsible for transporting large neutral amino acids such as L-leucine, L-tryptophan, L-isoleucine and Lphenylalanine into cells (Kanai et al., 1998) but it can also carry amino acid-derived drugs such as levodopa, gabapentin, melphalan and baclofen (Cornford et al., 1992; Kageyama et al., 2000; van Bree et al., 1988; Wang and Welty, 1996). Furthermore, LAT1 has been demonstrated to be able to transport amino acid prodrugs, where the amino acids have been conjugated with drug molecules which are not LAT1 substrates as such (Gynther et al., 2008; Killian et al., 2007; Walker et al., 1994). In previous studies it has seemed that in order to achieve efficient binding to LAT1, then the substrates should have an unsubstituted carboxylic acid or alternatively a negatively charged group as well as an unsubstituted amine functionality (Geier et al., 2013; Gynther et al., 2008; Uchino et al., 2002; Ylikangas et al., 2013). ⇑ Corresponding author. Tel.: +358 40 353 2791. 1
E-mail address: jarkko.rautio@uef.fi (J. Rautio). Authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejps.2014.09.025 0928-0987/Ó 2014 Elsevier B.V. All rights reserved.
In these studies removal or the presence of substituents on either carboxylic acid or amine functionalities exerted a dramatic effect on the binding ability of these compounds resulting in only weak affinity or a complete inability to bind to LAT1. In addition, amino acid derivatives in which the carboxyl and primary amine groups were further away than 3 Å possessed only a modest affinity for LAT1 which was comparable to those compounds lacking either the negatively charged group or the amine group (Ylikangas et al., 2013). However, some studies have indicated possible involvement of amino acid transport systems, including LAT1, in the transport of compounds lacking the free carboxyl group across cell membranes (Jain et al., 2004; Patel et al., 2013, 2014). For example, in two recent articles, an isoleucine–quinidine ester prodrug (1 in Fig. 1) has been reported to exhibit high affinity for LAT1 both in human derived prostate cancer cells (PC-3)(Patel et al., 2013) and in the Madin–Darby canine kidney (MDCK–MDR1) cell line (Patel et al., 2014). In addition, affinity for LAT2 was reported (Patel et al., 2014). In this prodrug the isoleucine promoiety had been conjugated with quinidine from the carboxylic acid functionality which, as far as we are aware and according to the prevailing consensus, this modification should significantly reduce the affinity for LAT1. Since these results are at odds with the current knowledge, in the present study we synthesized the prodrug described by Patel et al. (2013, 2014) and investigated its affinity for LAT1 and LAT2. The LAT1 affinity experiments were carried out with two
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independent methods; in situ rat brain perfusion technique and in human breast cancer cell line MCF-7 in vitro, both of which have demonstrated their versatility in LAT1 affinity studies, (Gynther et al., 2008; Killian et al., 2007; Shennan et al., 2004) while LAT2 affinity was determined in MCF-7 cells. 2. Materials and methods 2.1. General synthetic methods All the reactions were performed with reagents of commercial high purity quality without further purification. Reactions were monitored by thin-layer chromatography using aluminum sheets coated with silica gel 60 F245 (0.24 mm) with suitable visualization. Purifications by flash chromatography were performed on silica gel 60 (0.063–0.200 mm mesh). 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 500 spectrometer (Bruker Biospin, Fällanden, Switzerland) operating at 500.13 MHz and 125.75, respectively, using tetramethylsilane as an internal standard. pH-dependent NH-protons of the compounds were not observed. The final products were also characterized by mass spectroscopy with a Finnigan LCQ quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, CA, USA) equipped with an electrospray ionization source and by elemental analysis (C, H, N) with a Perkin Elmer 2400 Series II CHNS/O organic elemental analyzer (Perkin Elmer Inc., Waltham, MA, USA). 2.2. Synthesis of isoleucine–quinidine (1) The compound 1 was synthesized as previously described (Patel et al., 2013). 1H NMR ((CD3)2SO): d ppm 8.76 (d, 3JHH = 4.5 Hz, 1H), 8.02 (d, 3JHH = 9.0 Hz, 1H), 7.64 (d, 3JHH = 4.2 Hz, 1H), 7.56–7.49 (m, 2H), 6.23–6.12 (m, 1H), 5.27–5.18 (m, 2H), 4.48–4.41 (m, 1H), 3.40 (s, 3H), 3.97–3.90 (m, 1H), 3.64–3.545(m, 1H), 3.51–3.41 (m, 1H), 3.31–3.20 (m, 2H), 2.78–2.69 (m, 1H), 2.45–2.38 (m, 1H), 2.19– 2.10 (m, 1H), 2.05–2.00 (m, 1H), 1.87–1.79 (m, 2H), 1.59–1.50 (m, 1H), 1.46–1.37 (m, 1H), 1.32–1.21 (m, 2H), 1.04 (d, 3JHH = 5.7 Hz, 3H), 0.88 (t, 3JHH = 7.3 Hz, 3H) ; 13C NMR ((CD3)2SO): d ppm 168.13, 158.16, 147.26, 143.77, 139.72, 137.32, 131.39, 125.53, 122.31, 118.39, 117.25, 101.40, 71.68, 57.36, 56.02, 48.39, 47.79, 36.58, 35.31, 26.35, 23.49, 22.19, 19.27, 14.82, 11.29. MS (ESI+) for C26H36N3O3 (M + H)+: Calcd 438.58, Found 438.19. Anal. Calcd for (C26H35N3O3 ⁄ 1.3 TFA): C, 58.64; H, 6.25; N, 7.17; Found: C, 58.60; H, 5.85; N, 7.09. 2.3. In vitro uptake experiments in MCF-7 cells MCF-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with L-glutamine (2 mM), heat-inactivated fetal bovine serum (10%), penicillin (50 IU/ml) and
H2 N
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streptomycin (50 Ag/ml). MCF-7 cells were seeded at the density of 1 105 cells/well onto collagen-coated 24-well plates. The cells were used for the uptake experiments one day after seeding. After removal of the culture medium, the cells were carefully washed with pre-warmed Na+-free HBSS (Hank’s balance salt solution) containing 125 mM chlorine chloride, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.3 mM CaCl2, 5.6 mM glucose, and 25 mM HEPES (pH 7.4) and then pre-incubated in 500 lL of pre-warmed Na+-free HBSS at 37 °C for 10 min before adding substrates for the uptake experiment. The cells were then incubated at 37 °C for 5 min in 250 lL of uptake medium: Na+-free HBSS containing [14C]-L-leucine or [14C]-L-alanine. Subsequently, the cells were washed three times with ice-cold Na+-free HBSS. The cells were then lysed with 500 lL of 0.1 M NaOH and the lysate was mixed with 3.5 mL of Emulsifier safe cocktail (PerkinElmer, Waltham, MA, USA). The radioactivity was measured by liquid scintillation counting (Wallac 1450 MicroBeta; Wallac Oy, Finland). In our studies, Km and Vmax values were determined for [14C]-L-leucine and [14C]-L-alanine uptake under Na+-free conditions. The Km and Vmax values were 141.0 ± 13.6 lM and 9.5 ± 0.28 nmol/mg protein/min for [14C]-Lleucine, which are in agreement with those by Shennan et al. (2004) and >2 mM and 1.4 ± 0.6 nmol/mg protein/min for [14C]-Lalanine, respectively. For the inhibition experiments the uptake of 0.157 lM [14C]-L-leucine or 10.0 lM [14C]-L-alanine was examined in the presence or absence of test compounds. 2.4. In situ rat brain perfusion technique The ability of 1 to bind to rat LAT1 at the rat BBB was studied with the in situ rat brain perfusion technique, which has been described and validated earlier (Gynther et al., 2008). The 100% permeability-surface area (PA) product of [14C]-L-leucine, a known substrate of LAT1, was determined with 30 s perfusion of 0.2 lCi/ mL [14C]-L-leucine (0.64 lM). The perfusion fluid was infused through the common carotid artery at the rate of 10 mL/min for 30 s using a Harvard PHD 22/2000 syringe pump (Harvard Apparatus Inc., Holliston, MA). In order to determine the interaction of 1 with LAT1 at the BBB, a 100 lM concentration of 1 was coperfused for 30 s with [14C]-L-leucine. 1 was dissolved in the perfusion medium right before the perfusion, in order to avoid the release of isoleucine from the prodrug before the experiment. The PA product of [14C]-L-leucine after coperfusion was compared with the 100% PA product of [14C]-L-leucine. In the coperfusion studies the LAT1binding of the investigated compound is shown by decreased PA product of [14C]-L-leucine caused by a competitive binding to LAT1. All samples were analyzed for radioactivity by liquid scintillation counting (Wallac 1450 MicroBeta; Wallac Oy, Finland). Brain samples were dissolved in 0.5 mL of Solvable (PerkinElmer, Boston) overnight at 50 °C, and liquid scintillation cocktail (Ultima Gold, PerkinElmer, Boston) was added before the samples were analyzed. The perfusion medium consisted of a pH 7.4 bicarbonate-buffered physiological saline (128 mM NaCl, 24 mM NaHCO3, 4.2 mM KCl, 2.4 mM NaH2PO4, 1.5 mM CaCl2, 0.9 mM MgCl2 and 9 mM D-glucose). The solution was filtered, heated to 37 °C, and gassed with 95% O2, 5% CO2 to attain steady-state gas levels within the solution. 2.5. Animals
O
N
O
O
1
N
Fig. 1. Chemical structure of isoleucine–quinidine ester (1).
Male Wister rats 200–250 g, 7–8 weeks of age obtained from the National Laboratory Animal Centre (Kuopio, Finland) were used for in situ rat brain perfusions. Food and water was available ad libitum on a 12/12 h light/dark cycle with lights on at 6 am. Experiments were performed between 9:00 am and 16.00 pm in temperature and humidity-regulated rooms (22–24 °C, relative humidity: 60–70%). Rats were anesthetized with intraperitoneal injections of ketamine (50 mg/kg) and xylazine (5 mg/mL).
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2.6. Hydrolysis of 1 in aqueous solution at pH 7.4 The rate of chemical hydrolysis of 1 was studied in aqueous phosphate buffer solution of pH 7.4 (0.16 M, ionic strength 0.5) at 37 °C. Pseudo-first-order half-time (t1/2) for the hydrolysis of the compound was calculated from the slope of the plotted logarithm of remaining compound versus time. 2.7. HPLC assay Concentration of 1 in the samples was analyzed by Agilent 1100 HPLC system with UV-detector (Agilent Technologies Inc., Waldbronn, Karlsruhe, Germany). The detector wavelength was set at 235 nm. A mixture of acetonitrile and water containing 0.1% of formic acid at a flow rate of 0.8 mL/min was used as a mobile phase. The amount of acetonitrile in the mobile phase was 78% and 15%. Reversed-phase HPLC was conducted with a Zorbax RP-18 column (150 mm 4.6 mm, 5 lm, Agilent Technologies, Little Falls Wilmington, DE). 2.8. Data analysis Statistical differences between groups were tested using oneway ANOVA, followed by a two-tailed Dunnett’s t test (Figs. 2 and 4). In Fig. 2, a two-tailed independent samples t test was used. P < 0.05 was considered as statistically significant. All statistical analyses were performed using GraphPad Prism 5.03 for Windows.
protein/min at 7.5 lM of isoleucine to 2.99 ± 0.29 pmol/mg of protein/min at 100 lM of isoleucine). 3.2. Ability of 1 to bind to LAT1 in situ The ability of 1 to bind to LAT1was studied also with the in situ rat brain perfusion technique. The brain capillary permeability surface area (PA) of 0.2 lCi/mL of [14C]-L-leucine (i.e. 0.64 lM) after 30 s brain perfusion was determined to be 0.02059 ± 0.00323 mL/ s/g. In the presence of 100 lM 1, the [14C]-L-leucine PA product was not reduced (0.02082 ± 0.0005 mL/s/g (mean ± sd, n = 3)), (Fig. 3), thus demonstrating that 1 did not exhibit any affinity for LAT1 at the rat BBB. 3.3. Ability of 1 to bind to LAT2 in vitro Uptake inhibition of 1.5 lCi/mL of [14C]-L-alanine (i.e. 10.0 lM), a known LAT2 substrate, was measured in the presence of 50 lM and 100 lM 1 in MCF-7 cells in vitro. In addition, the ability of isoleucine to bind to LAT2 was determined at 50 lM and 100 lM concentrations. The results shown in Fig. 4 clearly demonstrated that 1 did not inhibit the uptake of [14C]-L-alanine, whereas isoleucine displayed statistically significant 36.9% uptake inhibition at 50 lM concentration and 56.1% uptake inhibition at 100 lM concentration.
3. Results 3.1. Ability of compounds to bind to LAT1 in vitro The ability of 1 to bind to LAT1 was investigated in competitive inhibition studies in MCF-7 cells at 50 lM and 100 lM concentrations with a known LAT1 substrate, L-leucine. The expression and function of LAT1 in MCF-7 cells corresponded with previously reported values (Shennan et al., 2004). In addition, as 1 was rapidly cleaved into quinidine and isoleucine in aqueous solutions at pH 7.4 (t1/2 = 3.69 ± 0.29 h, mean ± sd, n = 3), the ability of isoleucine to bind to LAT1 was determined at four concentrations ranging from 7.5 lM to 100 lM. The results shown in Fig. 2 clearly demonstrate that 1 did not inhibit the uptake of [14C]-L-leucine significantly whereas isoleucine exerted a concentration dependent uptake inhibition of [14C]-L-leucine (from 6.80 ± 0.49 pmol/mg of
Fig. 2. The ability of 1 to bind to LAT1 in MCF-7 cells. The uptake of [14C]-L-leucine was not significantly inhibited at 50 lM or 100 lM of 1. The uptake inhibition of [14C]-L-leucine was statistically significant at 15 lM, 50 lM and 100 lM concentrations of isoleucine. The asterisks denote a statistically significant difference from the respective control (⁄⁄⁄P < 0.001, one-way ANOVA, followed by Dunnett’s t test).
Fig. 3. The ability of 1 to bind to LAT1 present at the rat BBB. The PA-product of [14C]-L-leucine was not decreased in the presence of 100 lM 1.
Fig. 4. The ability of 1 to inhibit the uptake of [14C]-L-alanine in MCF-7 cells. 1 did not have any statistically significant effect on the uptake of [14C]-L-alanine at 50 lM or 100 lM concentrations. The uptake inhibition of [14C]-L-alanine was statistically significant at 100 lM concentration of isoleucine. The asterisk denotes a statistically significant difference from the respective control (P < 0.05, one-way ANOVA, followed by Dunnett’s t test).
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4. Discussion Several studies have investigated the structural features of LAT1 substrates. These have been proposed to include both a negatively charged a-carboxyl and positively charged a-amino groups (Gynther et al., 2008; Smith, 2005; Uchino et al., 2002). More recently, we generated a 3D pharmacophore for a set of LAT1 substrates whose binding affinities for LAT1 were determined using the in situ rat brain perfusion technique. The pharmacophore identified the following structural features as being responsible for efficient LAT1 binding; (1) negatively charged center and hydrogen bond donor (HBD) at 3 Å distance from each other, (2) aromatic feature following HBD and a negative charge, (3) hydrogen bond acceptor (HBA) next to the aromatic region, at 3.8 Å distance from the aromatic feature (Ylikangas et al., 2013). Recently reported compound 1 (Fig. 1) has been reported to exhibit high affinity for LAT1 both in human derived prostate cancer cells (PC-3) (Patel et al., 2013) and in MDCK–MDR1 cell line (Patel et al., 2014). In our previous in situ study, structurally similar ketoprofen prodrugs in which the amino acids phenylalanine and leucine were conjugated with ketoprofen from the carboxylic group did not show any affinity for LAT1 (Gynther et al., 2008). Because 1 does not fulfill the structural requirements for high affinity LAT1 substrates either, we wanted to prepare this prodrug and study its affinity for LAT1 in our methods to understand the differences between the laboratories. Firstly, we predicted the affinity of 1 to bind to the LAT1 by a CoMFA model generated recently in our laboratory (Ylikangas et al., 2014). Because 1 lacks the free carboxylic group, the model predicted negligible LAT1 binding affinity for the compound. Secondly, we synthesized compound 1 according to the previously described procedure (Patel et al., 2013) and evaluated its LAT1 affinity with two independent methods i.e. in situ rat brain perfusion technique and a human breast cancer cell line MCF-7 in vitro. Both are well established methods used in LAT1 affinity studies (Gynther et al., 2008; Killian et al., 2007; Shennan et al., 2004). In our experiments, 1 exerted no affinity for human LAT1 or LAT2 in vitro in MCF-7 cells. This was demonstrated in inhibition studies where 1 achieved no inhibition of either L-leucine (LAT1 specific substrate) or L-alanine (LAT2 specific substrate) cellular uptake at concentrations up to 100 lM. To confirm our in vitro results, we determined the ability of 1 to bind to LAT1 with an in situ rat brain perfusion technique. In this in situ study, 1 showed no affinity for LAT1 as it was not able to inhibit the brain uptake of L-leucine even at a concentration as high as 100 lM. Is there any way to explain the discrepancies between the results reported here and those of Patel et al. (2013)? One explanation may be possible impurities of the previously tested compound. In the report by Patel et al. (2013), the full NMR spectrum of 1 was not unfortunately presented and there was no indication of the purity of the final product. On the other hand, 1H and 13C NMR interpretations of 1 synthesized in our laboratory with mass and elemental analysis reported in the experimental part demonstrated over 99% purity for the final compound. The presence of even a small amount of L-isoleucine as an impurity in the affinity experiments can significantly affect the results. In addition, any delay in performing the inhibition experiments would expose the compound to premature hydrolysis in the aqueous solution prior to conducting the experiments. Compound 1 was found to be susceptible to chemical degradation in aqueous solution with halflives of 5.0 ± 0.3 h in DPBS at pH 7.4 (Patel et al., 2014) and 3.69 ± 0.29 h in aqueous phosphate buffer solution of pH 7.4 (37 °C). We calculated that within 30 min at 37 °C and pH 7.4 approximately 7.5 lM and 15 lM concentration of isoleucine would be released from 50 lM and 100 lM solutions of 1,
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respectively. The uptake inhibition of [14C]-L-leucine was statistically significant at a 15 lM isoleucine concentration (Fig. 2). Therefore, the presence of even small amounts of released isoleucine could also account for the observed uptake inhibition. Thirdly, it has to be noted that the applied models used in the present research are different than the ones used by Patel et al. (2013, 2014). Therefore, we cannot fully exclude the possibility of a transporter expressed in the cells used by Patel et al., for which both large neutral amino acids and 1 have affinity, but which is expressed in neither MCF-7 cells nor in the rat BBB. The presence of impurities, the release of isoleucine or a yet unknown transporter in cell models used by Patel et al. could also explain the discrepancies between the LAT2 affinity results. 5. Conclusion In the present study, we have demonstrated with two independent methods that 1 has no affinity for either LAT1 or LAT2. Although no definitive explanation for discrepancy between results of the present study and those of Patel et al. (2013, 2014) could not be found, this study emphasizes, that experiments on prodrugs which consist of promoieties which can significantly affect the results (e.g. amino acids in affinity studies), should be carried out with caution. In order to have good affinity for LAT1, the substrates should fulfill the necessary structural features which include negatively charged group (e.g. carboxyl group) and hydrogen bond donor (e.g. amino group) and modification at either group is likely to result in total loss or at least a significant decline in affinity for LAT1. Acknowledgements The work was financially supported by the Academy of Finland (#256837 for KH and #132637 for JR), Päivikki and Sakari Sohlberg’s Foundation (KH), Orion Research Foundation (MG) and Finnish Cultural Foundation (MG). The authors also want to thank M.Sc. Henna Ylikangas for predicting LAT1 inhibition by QSAR. References Cornford, E.M., Young, D., Paxton, J.W., Finlay, G.J., Wilson, W.R., Pardridge, W.M., 1992. Melphalan penetration of the blood-brain barrier via the neutral amino acid transporter in tumor-bearing brain. Cancer Res. 52, 138–143. Geier, E.G., Schlessinger, A., Fan, H., Gable, J.E., Irwin, J.J., Sali, A., Giacomini, K.M., 2013. Structure-based ligand discovery for the large-neutral amino acid transporter 1, LAT-1. Proc. Natl. Acad. Sci. U. S. A. 110, 5480–5485. Gynther, M., Laine, K., Ropponen, J., Leppanen, J., Mannila, A., Nevalainen, T., Savolainen, J., Jarvinen, T., Rautio, J., 2008. Large neutral amino acid transporter enables brain drug delivery via prodrugs. J. Med. Chem. 51, 932–936. Jain, R., Majumdar, S., Nashed, Y., Pal, D., Mitra, A.K., 2004. Circumventing Pglycoprotein-mediated cellular efflux of quinidine by prodrug derivatization. Mol. Pharm. 1, 290–299. Kageyama, T., Nakamura, M., Matsuo, A., Yamasaki, Y., Takakura, Y., Hashida, M., Kanai, Y., Naito, M., Tsuruo, T., Minato, N., Shimohama, S., 2000. The 4F2hc/LAT1 complex transports L-DOPA across the blood–brain barrier. Brain Res. 879, 115– 121. Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., Endou, H., 1998. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J. Biol. Chem. 273, 23629–23632. Killian, D.M., Hermeling, S., Chikhale, P.J., 2007. Targeting the cerebrovascular large neutral amino acid transporter (LAT1) isoform using a novel disulfide-based brain drug delivery system. Drug Deliv. 14, 25–31. Patel, M., Dalvi, P., Gokulgandhi, M., Kesh, S., Kohli, T., Pal, D., Mitra, A.K., 2013. Functional characterization and molecular expression of large neutral amino acid transporter (LAT1) in human prostate cancer cells. Int. J. Pharm. 443, 245– 253. Patel, M., Mandava, N.K., Pal, D., Mitra, A.K., 2014. Amino acid prodrug of quinidine: an approach to circumvent P-glycoprotein mediated cellular efflux. Int. J. Pharm. 464, 196–204. Shennan, D.B., Thomson, J., Gow, I.F., Travers, M.T., Barber, M.C., 2004. L-leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231): kinetics,
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