l -Type amino acid transporter 1 (lat1)-mediated targeted delivery of perforin inhibitors

International Journal of Pharmaceutics 498 (2016) 205–216

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

L-Type

amino acid transporter 1 (lat1)-mediated targeted delivery of perforin inhibitors Kristiina M. Huttunena,* , Johanna Huttunena , Imke Aufderhaara , Mikko Gynthera , William A. Dennyb , Julie A. Spicerb a b

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 November 2015 Received in revised form 9 December 2015 Accepted 12 December 2015 Available online 15 December 2015

Perforin is a cytolytic pore-forming glycoprotein secreted by cytotoxic effector cells. It is a key component of the immune response against virus-infected and transformed cells and has been implicated in a number of human diseases. Perforin activity can be inhibited by small-molecular-weight compounds, although less is known about their delivery to the site of action. Therefore, in the present study, it was explored if perforin inhibitors could be efficiently and site-selectively delivered firstly into the cytotoxic effector cells and secondly into lytic granules, in which perforin is stored. This was accomplished by designing and synthesizing four prodrugs of perforin inhibitors that could utilize L-type amino acid transporter (LAT1), since activated immune cells are known to over-express LAT1. The results demonstrate that cellular uptake of perforin inhibitors can be increased by LAT1-utilizing prodrugs (into human breast adenocarcinoma cells (MCF-7)). Furthermore, these prodrugs were also able to deliver perforin inhibitors into the cell organelles having lower pH (rat liver lysosomes). Therefore, by using these prodrugs, intracellular mechanisms of perforin inhibitory activity can be studied more thoroughly in future. Moreover, this prodrug approach can be applied for other drugs that would benefit from targeted delivery into cells expressing LAT1, such as cancer. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Prodrug Perforin inhibitor L-type amino acid Transporter 1 (LAT1) Targeted drug delivery Transporter-mediated drug delivery

1. Introduction Perforin is a cytolytic pore-forming glycoprotein secreted by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (Lichtenheld et al., 1988; Podack, 1992; Trapani and Smyth, 2002). It is a key component of the immune response against virus-infected and transformed cells. Perforin has also been implicated in a number of human immunopathologies and therapy-induced conditions, such as cerebral malaria, autoimmune neuroinflammation, insulin-dependent diabetes, allograft rejection and graft-versus-host disease (Kagi et al., 1997; Liesz et al., 2011; Potter et al., 2006; Veale et al., 2006). The mechanism of perforin function is complex. CTLs and NK cells contain secretory vesicles (granules) that store perforin along with other cytotoxic

Abbreviations: BBB, blood–brain barrier; CTLs, cytotoxic T lymphocytes; HPLC, high-performance liquid chromatography; LAT1, L-type amino acid transporter; MCF-7, human breast adenocarcinoma cell line; MCTs, monocarboxylate transporters; NK cells, natural killer cells; OATPs, organic anion transporting polypeptides; PSA, polar surface area. * Corresponding author. E-mail address: Kristiina.Huttunen@uef.fi (K.M. Huttunen). http://dx.doi.org/10.1016/j.ijpharm.2015.12.034 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

proteins, including granzymes (pro-apoptic serine proteases) (Brennan et al., 2011; Thiery et al., 2011). After killer cell conjugation to the target cell, exocytic delivery of the granule contents into the calcium-rich immune synapse results in calcium binding by perforin, dramatically changing its conformation, followed by the formation of trans-membrane pores composed of highly ordered perforin oligomers (Baran et al., 2009; Law et al., 2010). Formation of perforin pores facilitates entry of granzymes into the target cell, which in turn activates various apoptotic mechanisms. Perforin-mediated cytotoxicity can been inhibited by small-molecular-weight compounds both in vitro and in vivo (Lena et al., 2008; Spicer et al., 2013), but its is not yet clear at what point in the process these inhibitors interact with perforin; whether extracellularly or if they also have intracellular function(s). To study if perforin inhibitors could be efficiently and selectively delivered into the cytotoxic effector cells, we utilized a L-type amino acid transporter (LAT1) as a (pro) drug carrier. LAT1 is a sodium-independent heterodimeric trans-membrane protein that is mainly expressed in the blood–brain barrier, but also in placenta, tumors (Kanai et al., 1998) and activated immune cells (Hayashi et al., 2013; Nii et al., 2001; Sinclair et al., 2013). LAT1 transports not only large and neutral amino acids, but also

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several clinically used drugs and prodrugs, such as levodopa, gabapentin and melphalan (Boado et al., 1999; Uchino et al., 2002). The relatively high and selective expression of LAT1 at the blood– brain barrier (BBB), in activated immune cells and several types of cancer cells (Fuchs and Bode, 2005; Yanagida et al., 2001), makes it an important means of targeted drug delivery, especially in the case of infectious and inflammatory brain diseases as well as some forms of cancer. In the present study, we prepared four LAT1-utilizing prodrugs of selected perforin inhibitors and studied their ability to bind to LAT1 and to be delivered into LAT1-expressing cells (human breast adenocarcinoma cells; MCF-7). Moreover, the ability of these prodrugs and their parent compounds to be accumulated into cell organelles that have lower pH (rat liver lysosomes), was also evaluated, since such lysosomes closely resemble the secretory vesicles of NK cells and CTLs (Burkhardt et al., 1990; Dell’Angelica et al., 2000). 2. Materials and methods 2.1. General synthetic procedures All reactions were performed with reagents obtained from Sigma–Aldrich (St. Louis, MO, USA), Acros Organics (Waltham, MA, USA) or Merck (Darmstadt, Germany). 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) eluting with CH2Cl2/MeOH solution. 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. ESI–MS spectra were recorded by a Finnigan LCQ quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, CA, USA) equipped with an electrospray ionization source. Over 95% purities were obtained for the compounds 1–18 by elemental analysis (C, H, N) with a PerkinElmer 2400 Series II CHNS/O organic elemental analyzer (PerkinElmer Inc., Waltham, MA, USA). 2.2. Synthesis of the prodrugs 1–8 The compounds 1–4 were prepared as previously described by using 9-BBN to protect the amino acid group (Peura et al., 2013), coupling the amino acid and the perforin inhibitor by the aid of EDC and deprotecting the final compounds by ethylenediamine. More detailed procedures can be found from the supporting data. The compounds 5–8 were prepared by refluxing compound 13 or 14 with either hydroxylamine hydrochloride (compounds 5 and 7, respectively) or with hydrazine hydrate (compounds 6 and 8, respectively) in the presence of pyridine in EtOH or EtOH/DMSO mixture overnight. 2.2.1. (R)-2-Amino-3-(3-(((4-(5-(1-oxo-1,3-dihydroisobenzofuran-5yl) thiophen-2-yl)benzyl)oxy)carbonyl)phenyl)propanoic acid (1) Yellow solid (41%). 1H NMR ((CD3)2SO): d ppm 8.00 (s, 1H), 7.93– 7.84 (m, 4H), 7.79 (d, J = 3.8 Hz, 1H), 7.77 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 3.8 Hz, 1H), 7.58–7.54 (m, 3H), 7.44 (t, J = 7.7 Hz, 1H), 5.45 (s, 2 H), 5.39 (s, 2H), 3.62–3.58 (m, 1H), 3.20-3.09 (m, 1H), 2.96–2.85 (m, 1H); 13C NMR ((CD3)2SO): d ppm 172.33, 169.90, 165.60, 148.40, 144.17, 140.96, 139.15, 138.76, 136.07, 134.30, 132.80, 130.08, 129.38, 128.64 (2C), 128.40, 127.22, 127.04, 125.86, 125.52, 125.41 (2C), 125.25, 123.65, 118.83, 69.52, 65.51, 55.60, 37.77. MS (ESI
) for C29H22NO6S (M-H)
: calcd 512.55, found 512.10.

2.2.2. (R)-2-Amino-3-(3-((4-(5-(1-oxo-1,3-dihydroisobenzofuran-5yl)thiophen-2-yl)phenoxy)carbonyl)phenyl)propanoic acid (2) Orange–yellow solid (81%). 1H NMR ((CD3)2SO): d ppm 7.97 (s, 1H), 7.92–7.85 (m, 5H), 7.82 (d, J = 7.8 Hz, 1H), 7.76–7.70 (m, 3H), 7.57–7.44 (m, 3H) 5.45 (s, 2 H), 3.96–3.89 (m, 1H), 3.13–3.05 (m, 1H), 2.98–2.90 (m, 1H); 13C NMR ((CD3)2SO): d ppm 175.92, 169.97, 165.60, 148.43, 146.82, 144.68, 140.10, 138.93, 138.11, 134.70, 134.66, 128.40, 128.35, 128.19, 127.24, 126.87, 125.76, 125.64 (2C), 125.53, 124.51, 123.49, 120.59 (2C), 118.69, 69.54, 55.30, 37.19. MS (ESI
) for C28H21N2O5S (M-H)
: calcd 497.54, found 497.14. 2.2.3. (R)-2-Amino-3-(3-((4-(5-(1-oxo-1,3-dihydroisobenzofuran-5yl)thiophen-2-yl)phenyl)carbamoyl)phenyl)propanoic acid (3) Yellow solid (86%). 1H NMR ((CD3)2SO): d ppm 8.07 (s, 1H), 8.02– 7.98 (m, 2H), 7.94 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 8.6 Hz, 2H), 7.79 (d, J = 3.9 Hz, 1H), 7.68–7.63 (m, 2H), 7.53 (t, J = 7.6 Hz, 1H), 7.37 (d, J = 8.5 Hz, 2H), 5.45 (s, 2H), 3.49–3.44 (m, 1H), 3.26–3.20 (m, 1H), 3.03–2.97 (m, 1H); 13C NMR ((CD3)2SO): d ppm 175.78, 170.25, 164.73, 150.53, 148.70, 143.88, 141.18, 138.96, 138.60, 135.34, 131.13, 131.03, 129.01, 128.84, 128.16, 127.56, 126.96, 126.71 (2C), 126.05, 125.78, 123.87, 122.81 (2C), 119.01, 69.81, 55.33, 36.63. MS (ESI
) for C28H20NO6S (M-H)
: calcd 498.53, found 498.19. 2.2.4. (R,(E,Z))-2-Amino-3-(4-((4-(5-((2-oxo-5-thioxoimidazolidin4-ylidene) methyl) thiophen-2-yl)phenoxy)carbonyl)phenyl) propanoic acid (4) Dark red solid (52%). 1H NMR ((CD3)2SO): d ppm 7.78 (s, 1H), 7.66 (d, J = 7.4 Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.38 (d, J = 7.5 Hz, 1H), 7.34 (d, J = 7.4 Hz, 1H), 7.19 (d, J = 3.8 Hz, 1H), 7.17 (d, J = 3.8 Hz, 1H), 7.78 (d, J = 8.6 Hz, 2H), 6.18 (s, 1 H), 3.65–3.62 (m, 1H), 3.21–3.15 (m, 1H), 2.82–2.75 (m, 1H); 13C NMR ((CD3)2SO): d ppm 191.27, 179.88, 172.38, 166.54, 157.03, 149.34, 138.67, 138.36, 134.64, 132.04, 129.00, 128.62, 128.08 (2C), 126.55, 126.33, 125.65, 125.24, 121.57, 115.85 (2C), 55.81, 37.81. MS (ESI
) for C14H9N2O2S2 (M-[C10H10NO3])
: calcd 301.01, found 300.96. 2.2.5. (E,Z)-4-((5-(4-((E,Z)-1-(Hydroxyimino)ethyl)phenyl)thiophen2-yl) methylene)-5-thioxoimidazolidin-2-one (5) Dark red solid (74%). 1H NMR ((CD3)2SO), E/Z isomers were observed together: d ppm 12.40 (s, 1H), 11.98 (s,1H), 11.30 (s, 1H), 7.84 (d, J = 4.0 Hz, 1H), 7.74–7.71 (m, 4H), 7.68 (d, J = 4.0 Hz, 1H), 6.64 (s, 1H), 2.17 (s, 3H); 13C NMR ((CD3)2SO): d ppm 178.40, 165.40, 152.36, 146.43, 139.23, 136.69, 135.60, 135.22, 133.05, 132.44, 126.29, 125.91, 125.89, 125.36, 104.18, 11.32. MS (ESI
) for C16H12N3O2S2 (M
H)
: calcd 342.41, found 342.04. 2.2.6. (E,Z)-4-((5-(4-((E,Z)-1-Hydrazonoethyl) phenyl) thiophen-2yl) methylene)-5-thioxoimidazolidin-2-one (6) Red-brown solid (77%). 1H NMR ((CD3)2SO), E/Z isomers were observed separately: d ppm 10.98 (s, 1H), 9.08 (s,1H), 8.00 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.63 (d, J = 3.7 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.47–7.36 (m, 1H), 6.57 (s, 0.5H), 6.55 (s, 0.5H), 4.71 (s, 2H), 2.54 (s, 3H); 13C NMR ((CD3)2SO): d ppm 197.42, 170.42, 145.21, 141.74, 139.27, 138.84, 135.60, 132.99, 130.60, 129.70, 127.94, 125.79, 125.23, 125.15, 123.98, 11.61. MS (ESI
) for C16H13N4OS2 (M
H)
: calcd 341.43, found 341.49. 2.2.7. (E,Z)-5-(1-Oxo-1,3-dihydroisobenzofuran-5-yl) thiophene-2carbaldehyde oxime (7) Dark red solid (74%). 1H NMR ((CD3)2SO), E/Z isomers were observed separately: d ppm 12.17 (s, 0.5H), 11.40 (s, 0.5H), 8.35 (s, 0.5H), 8.03 (s, 0.5H), 7.99–7.93 (m, 1H), 7.90 (s, 1H), 7.89–7.85 (m, 1H), 7.75 (d, J = 4.0 Hz, 0.5H), 7.71 (d, J = 3.8 Hz, 0.5H), 7.53 (d, J = 4.0 Hz, 0.5H), 7.36 (d, J = 3.8 Hz, 0.5H), 5.44 (s, 0.5H), 5.44

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(s, 0.5H); 13C NMR ((CD3)2SO): d ppm 170.65 (0.5C), 170.60 (0.5C), 149.05 (0.5C), 148.81 (0.5C), 145.84 (0.5C), 145.17 (0.5C), 143.76 (0.5C), 142.45 (0.5C), 140.25 (0.5C), 139.44 (0.5C), 139.11 (0.5C), 138.35 (0.5C), 132.87 (0.5C), 132.31 (0.5C), 131.19 (0.5C), 128.91 (0.5C), 126.84 (0.5C), 126.73 (0.5C), 126.15 (0.5C), 126.07 (0.5C), 125.60 (0.5C), 124.56 (0.5C), 119.99 (0.5C), 119.82 (0.5C), 70.39 (0.5C), 70.24 (0.5C). MS (ESI+) for C13H10NO3S (M + H)+: calcd 260.29, found 260.10. 2.2.8. (E,Z)-5-(5-(Hydrazonomethyl)thiophen-2-yl)isobenzofuran-1 (3H)-one (8) Dark red solid (83%). 1H NMR ((CD3)2SO), E/Z isomers were observed separately: d ppm 7.91 (s, 1H), 7.87 (s, 1H), 7.84–7.83 (m, 2H), 7.63 (d, J = 3.8 Hz,1H), 7.10 (d, J = 3.8 Hz,1H), 7.01 (s, 2H), 5.43 (s, 1H); 13C NMR ((CD3)2SO): d ppm 169.94, 148.35, 143.41, 139.29, 139.10, 132.49, 126.14, 126.03, 125.73, 125.42, 123.32, 118.62, 69.49. MS (ESI+) for C13H11N2O2S (M + H)+: calcd 259.31, found 259.06. 2.3. Synthesis of the compounds 9–18 The compounds 9–11 were prepared as described recently (Miller et al., 2015), the compounds 12, 14–18 as previously described (Spicer et al., 2013) and the compound 13 as previously described (Spicer et al., 2012). 2.4. Physicochemical properties of the studied compounds Partition coefficient (log P) values and polar surface areas (PSA) were calculated by ChemBioDraw Ultra v. 13.0.2.3021 software (PerkinElmer, Inc., Waltham, MA, USA) and dissociation constant (pKa) values by ChemAxon Marvin Sketch v. 15.2.9.0 (ChemAxon Ltd., Budapest, Hungary). Aqueous solubilities of the prodrugs 1–4 and 7–8 their parent drugs 9–13 were determined at room temperature by shaking solid compound (1 mg) in water (1 mL) for 1 h. The solution was filtered through a 0.45 mm membrane filter (Millex1-HV, 13 mm, Merck Millipore Co., Merck KGaA, Darmstadt, Germany) and analyzed by the HPLC method described below. The possible absorption to membrane filter was excluded by centrifugating the sample without filtration and analyzing and the samples. No filter absorption was detected by these compounds. Aqueous solubilities of compounds 14–18 has been reported earlier (Spicer et al., 2013). Prodrugs 5–6 were too insoluble to be reliably detected by UV-HPLC method. 2.5. Other chemicals All reagents and solvents used in analytical studies were commercial and high purity of analytical grade or ultra-gradient HPLC-grade purchased from Sigma, St. Louis, MO, USA, J.T. Baker (Denventer, The Netherlands), Merck (Darmstadt, Germany) or Riedel-de Haën (Seelze, Germany). Water was purified using a Milli-Q Gradient system (Millipore, Milford, MA, USA). 2.6. High-performance liquid chromatography (HPLC) analyses The concentration of the prodrugs was determined by HPLC, consisting of a Agilent 1100 binary pump (Agilent Technologies Inc., Wilmington, DE, USA), a 1100 micro vacuum degasser, a HP 1050 Autosampler, a HP 1050 variable wavelength detector (operated at 365 nm). The chromatographic separations were achieved on a Agilent Zorbax SB-C18 analytical column (4.6 mm  250 mm, 5 mm) (Agilent Technologies Inc., Wilmington, DE, USA) by using isocratic elution by acetonitrile and 0.1% formic acid buffer (pH ca. 3.0) with a ratio of 45:55 (v/v) at the flow rate of 1.0 mL/min at room temperature. The lower limit of quantification

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for all compounds was 0.1–0.5 pmol/mg of protein. These HPLC methods were also selective (no interfering peaks), accurate (calculated C compared to nominal C; 95–105%) and precise (RDS% <6%) over the range 1–75 mM in LAT1-mediated uptake studies in MCF-7 cells and over the range 0.5–10 mM in lysosomal uptake studies. 2.7. Ability of compounds to bind to LAT1 in MCF-7 cells by [14C]-Lleucine competition Human breast cancer (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 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 HBSS (Hank’s balance salt solution) containing 125 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 mM HEPES (pH 7.4) and then pre-incubated in 500 mL of pre-warmed HBSS at 37  C for 10 min. The cells were then incubated at 37  C for 5 min in 250 mL of uptake medium containing 0.157 mM of [14C]-Lleucine in HBSS and 50 or 100 mM of the test compound (or DMSO as blank). Subsequently, the cells were washed three times with ice-cold HBSS. The cells were then lysed with 500 mL 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 Inc., Finland). 2.8. LAT1-Mediated uptake of prodrugs into MCF-7 cells The uptake of prodrugs 1–4 were studied by adding 100 mM prodrugs in pre-warmed HBSS buffer (250 mL) on the top of the cell layer and incubating at 37  C for 30 min. Subsequently, the cells were washed three times with ice-cold HBSS. The cells were then lysed with 500 mL of 0.1 M NaOH. The supernatants were analyzed by the HPLC method described above and the concentration of each experiment was calculated from the standard curve that was prepared by spiking known amounts of each prodrug to cell lysate. The protein concentrations on each plate were determined as mean of 3 samples by Bio-Rad Protein Assay (EnVision, PerkinElmer, Inc.). The amount of uptake at lower temperature, in which the carrier-mediated uptake is hindered, was studied as described above at 4  C (on the ice-bath). The competitive uptake in the presence of L-tryptophan was carried out as described above at 37  C by HBSS buffer solution that contained 50 or 100 mM of studied compound and 2 mM of L-tryptophan. The inhibition of probenecid-sensitive transporters was studied by pre-incubating the cells by HBSS buffer that contained 500 mM of probenecid for 5 min. The pre-incubation mixture was then removed and the experiment was carried out as described above by HBSS buffer solution containing 50 or 100 mM of studied compound and 500 mM of probenecid (and 2 mM of L-tryptophan) to identify the interface between LAT1 and probenecid-sensitive transport. The total uptake of prodrugs 1 and 3 into the MCF-7 cells was also determined between the concentration range of 6.25–400 mM. 2.9. Preparation of rat liver subcellular fractions Rat liver S9 fraction was prepared by centrifuging liver homogenate at 9,000  g for 20 min at 4  C and collecting the supernatant. The liver homogenate was prepared by homogenizing freshly collected rat liver with isotonic Tris-HCl buffer (pH 7.4) (1:4 w/v). Liver cytosolic fraction was prepared from rat liver S9

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fraction by standard differential ultracentrifugation at 100,000  g for 1 h at 4  C and collecting the supernatant. All biological material were stored at
80  C until used. Lysosomal fraction was isolated from freshly collected rat livers in 0.25 M sucrose. The livers were homogenized with 0.25 M sucrose (1:4 w/v) and centrifuging it at 1000  g for 10 min at 4  C. The floating fat layer was removed and the supernatant was incubated with 1 mM CaCl2 for 5 min at 37  C and centrifuged at 15,000  g for 20 min at 4  C. The resulting pellet was re-suspended with iso-osmotic Percoll (in 0.25 M sucrose; 1.075 g/mL; pH 7.4) and centrifuged at 60 000  g for 15 min at 4  C. The pooled lysosomal fractions were centrifuged at 100 000  g for 60 min at 4  C. The middle layer (lysosomes) was diluted with 0.25 M sucrose (1:2, v/v) and centrifuged at 10 000  g for 30 min at 4  C. The lysosomal pellet was washed to remove Percoll and re-suspended in 0.5 mL of 0.25 M sucrose per gram of original tissue and homogenized gently. Protein concentrations of all fractions were determined by Bio-Rad protein assay (EnVision, PerkinElmer, Inc.). The enriched lysosomal fraction was used immediately for the uptake studies.

for 5 min at 12000  g at room temperature. The supernatants were analyzed by the HPLC method described above and used as controls for lysosomal accumulation. The pellets from the first step were washed three times with isotonic Tris-HCl buffer (pH 7.4), suspended with 100 mL of 0.1 M NaOH and sonicated for 10 min at room temperature. 100 mL of MeOH was added and the mixtures were centrifuged for 5 min at 12000  g at room temperature. The supernatants were analyzed by the HPLC for the lysosomal accumulation of each compound.

2.10. Lysosomal uptake of compounds 1–13

The rates of bioactivation of the prodrugs 1–4 in rat and human liver S9 fractions as well as in rat liver cytosol fraction were determined at 37  C. The incubation mixtures were prepared by mixing liver S9 fraction or cytosol fraction (final protein concentration 1.0 mg/mL) with isotonic Tris–HCl buffer (pH 7.4) and 5 mM prodrug stock solution in DMSO (the final concentration of prodrugs was 100 mM and the DMSO concentration 2%). The mixture was incubated for 6 h and the samples (100 mL) were withdrawn at appropriate intervals. The enzymatic reaction was terminated by the addition of ice-cold acetonitrile (100 mL) and the samples were centrifuged for 5 min at 12000  g at room

The amount of prodrugs 1–4 accumulated into lysosomes was evaluated by mixing prewarmed lysosomal fraction and drug solution in isotonic Tris–HCl buffer (pH 7.4) containing 0.25 M sucrose (diluted from 1 mM stock solution in DMSO) in micro test tubes (final protein concentration was ca. 10 mg/mL, the compound concentration 50 mM and DMSO concentration 2%). Test tubes were incubated at 37  C for 30 min, after which they were centrifuged for 5 min at 12000  g at room temperature. 100 mL of supernatant was mixed with 100 mL of MeOH and centrifuged

2.11. Other biological material Pooled human liver S9 fraction (>20 mg of protein/mL) was purchased from Sigma–Aldrich (St. Louis, MO, USA). The pooled rat and human plasma was obtained from control rats or healthy human donors by collecting a whole blood aseptically and centrifuging at 12,000  g for 10 min. All biological material was stored at
80  C until used. 2.12. In vitro bioactivation of prodrugs 1–4

Scheme 1. Conversion of compounds 9–12 to their corresponding LAT1-utilizing prodrugs 1–4 and conversion of compounds 13 and 14 to their corresponding granuletargeted prodrugs 5–8. Detailed synthesis procedures for prodrugs 1–4 can be found from the supporting data. (a) NH2OH, pyridine, EtOH (DMSO), reflux, 95–99%; (b) NH2NH2, pyridine, EtOH (DMSO), reflux, 83–99%.

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temperature and kept on ice until the supernatants were analyzed by the HPLC method described above. In blank reactions, the S9 fractions were replaced with same volume of buffer. The pseudo-first-order half-lives (t1/2) for the rates of bioconversion of the prodrugs were calculated from the slope of the linear portion of the plotted logarithm of remaining prodrug concentration versus time. The rates of bioactivation of the prodrugs 1–4 in rat and human plasma were determined at 37  C as above by adding 1 mM stock solution of the prodrug to plasma in a ratio of 1:10. The rates of chemical pH-dependent hydrolysis of the prodrugs 7–8 were determined at 37  C in 50 mM (ionic strength 0.15) HCl buffer at pH 1.0 and 2.0, citric acid buffer at pH 3.0, 4.0, 5.0 and 6.0, and Tris–HCl buffer at pH 7.4. The incubation mixtures were prepared by dissolving 12 mM of prodrug 7 or 8 in EtOH in preheated buffer solutions. The EtOH concentration in the incubation mixtures was 1.5% and the prodrug concentration about 200 mM. The mixtures were incubated at 37  C and the samples were withdrawn at appropriate intervals. Acetonitrile (ACN) was added to the samples (1:1, v/v) to hinder further hydrolysis during the HPLC analyses. The pseudo-first-order halflives (t1/2) for the hydrolysis of the prodrug were calculated from the slope of the linear portion of the plotted logarithm of remaining prodrug versus time. 2.13. Data analysis All statistical 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 Dunnett’s or Tukey’s test. All data are presented as mean  SD. 3. Results and discussion

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water (1.49–1.53 mg/mL) than their parent drug 13 (0.52 mg/mL). Other physicochemical properties (log P, pKa, and PSA) of all studied compounds (1–18) can be found from the supporting data. 3.2. Ability of prodrugs 1–4 and their parent drugs 9–12 to bind to LAT1 in MCF-7 cells by [14C]-L-Leucine competition The ability of prodrugs 1–4 to bind to LAT1 was evaluated at 37  C after 5 min incubation via a competitive inhibition assay of [14C]-L-leucine uptake in MCF-7 cell line, in which the LAT1 expression and function corresponded with previously reported values (Shennan et al., 2004). Prodrugs 1, 2 and 4 (50 mM) inhibited the cellular uptake of [14C]-L-leucine (83.22  0.66%, 54.16  5.02% and 66.61  3.01%, respectively), whereas prodrug 3 had only a little affinity for LAT1 (7.61  4.66% inhibition of [14C]-L-leucine uptake). Furthermore, none of the corresponding parent drugs (compounds 9–12, 50 mM) was able to bind to LAT1 (Fig. 1). Prodrugs 2 and 4 exerted a concentration-dependent uptake inhibition of [14C]-L-leucine when compared at 50 mM and 100 mM concentrations (Fig. 1). At 100 mM concentrations the inhibitions were as high as 79.22  2.33% and 86.21 1.68%, respectively. The ability of prodrug 3 to inhibit uptake of [14C]-L-leucine at 100 mM concentration was detectable but not significant (16.93  15.99% inhibition). Interestingly, no concentration dependency in uptake inhibition was seen with prodrug 1, although it inhibited the uptake of [14C]-L-leucine significantly (over 80%). This was most probably due to the fact that between 50 and 100 mM the prodrug 1 has already reached its maximum inhibition of [14C]-L-leucine uptake. In conclusion, all four prodrugs were able to bind to LAT1 at 100 mM concentration. The prodrugs 1, 2 and 4 showed good affinity for LAT1, while prodrug 3 seemed to be a poorer substrate for LAT1 (Killian and Chikhale, 2001). However, all the prodrugs were selected for further uptake studies

3.1. Synthesis and physicochemical properties of the studied compounds The compounds 9–12 were selected as parent drugs for LAT1selective prodrug derivatization and compounds 13 and 14 for granule-targeted prodrugs (Scheme 1) as they possess functional groups that can be readily used for prodrug derivatization. Prodrugs 1–4 were designed according to the 3-dimentional quantitative relationship (3D-QSAR) model of LAT1 binding site (Ylikangas et al., 2014), having free amino and acid groups in the promoiety which should guarantee efficient affinity for LAT1. The parent drugs were attached to this promoiety via enzymatically labile ester and amide bonds. Prodrugs 5–8 were designed to have an acid-labile hydroxyimine or hydrazone group in order to be bioactivated via a pH-sensitive manner in slightly acidic cell organelles (Kumpulainen et al., 2006; Zhou et al., 2011). Prodrugs 1–4 were prepared as previously described by employing 9-BBN to protect the amino acid group, coupling the amino acid and the perforin inhibitor (9–12) using EDC and deprotecting the final compounds with ethylenediamine (Peura et al., 2013). Detailed synthesis procedures for prodrugs 1–4 can be found from the supporting data. Compounds 13 and 14 were converted to their corresponding pH-sensitive hydroxyimines (5 and 7, respectively) and hydrazones (6 and 8, respectively) by reacting them with hydroxylamine or hydrazine in the presence of pyridine (Scheme 1). All perforin inhibitors (9–18) have shown inhibitory activity toward perforin-induced lysis of 51Cr-labelled Jurkat lymphoma cells (Miller et al., 2015; Spicer et al., 2012, 2013). Aqueous solubility of prodrugs 1–4 determined after 1 h dissolution time was approximately 4–15-times greater (1.70– 8.14 mg/mL) than that of their corresponding parent drugs 9–12 (0.17–1.98 mg/mL). Prodrugs 7–8 were also 3-times more soluble in

Fig. 1. The ability of prodrugs 1–4 and their corresponding parent drugs (9–12) to bind to LAT1 in MCF-7 cells at 37  C. The uptake of [14C]-L-leucine was not significantly inhibited at 50 mM of parent drugs (9–12) and the prodrug 3 whereas it was statistically significant at 50 mM concentrations of prodrugs 1,2 and 4. The uptake inhibition of [14C]-L-leucine was statistically significant and concentrationdependent (at 50 and 100 mM) with prodrugs 2–4 but concentration-independent with prodrug 1. An asterisk denotes a statistically significant difference from the respective control (*P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA, followed by Dunnett’s test).

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Fig. 2. The uptake of prodrugs 1–4 and their parent drugs 9–12 into MCF-7 cells at 100 mM concentration after 30 min incubation at 37  C. An asterisk denotes a statistically significant difference from the respective control (***P < 0.001, one-way ANOVA, followed by Tukey’s test).

3.3. LAT1-Mediated transport of prodrugs 1–4 into cells Uptake of prodrugs 1–4 into the MCF-7 cell was studied after 30 min incubation of 100 mM prodrugs at 37  C. As seen in Fig. 2, the uptake was more efficient with prodrugs 1–3 than with prodrug 4. The parent drug of prodrug 4 is more polar than the ones of other prodrugs. A recent 3D-QSAR model of LAT1 binding site shows that LAT1 has a lipophilic pocket in which the attached parent drug of the prodrug should fit in (Ylikangas et al., 2014). Therefore, it can be concluded that although prodrug 4 showed that it can be efficiently bound to LAT1 in the inhibition studies (86% inhibition of [14C]-L-leucine uptake at 100 mM), the capacity of LAT1 to transport prodrugs with more polar parent drug moiety is low (1.02  0.19 nmol/min/mg of protein at 100 mM). In contrast, prodrug 3 with more lipophilic parent drug moiety did not inhibit [14C]-L-leucine uptake as effectively (24% inhibition at 100 mM) as prodrug 4, but was more efficiently transported into the cells (2.94  0.50 nmol/min/mg of protein). Moreover, the structure of prodrug 3 closely resembles the structures of prodrugs 1 and 2, which both had good affinity (84% and 70% inhibition of [14C]-Lleucine uptake, respectively) as well as high transport capacity (2.76  0.34 and 2.74  0.11 nmol/min/mg of protein at 100 mM, respectively). However, when compared to the uptake of the parent drugs 9 and 11 to the one of their prodrugs 1 and 3, respectively, only very small amounts of compounds 9 (0.61  0.12 nmol/min/mg of protein) and 11 (0.22  0.08 nmol/min/mg of protein) were transported into the cells (less than 20% of the amount of prodrugs’ uptake) and thus, prodrugs 1 and 3 improved the cellular uptake of their parents (Fig. 2). During the uptake studies, prodrugs 2 and 4 were partly converted to their parent drugs, which may have an effect on total uptake of these prodrugs. Moreover, these prodrugs were not able to improve the cell uptake of their parent drugs (3.35  0.64 and 3.71 1.30 nmol/min/mg of protein, respectively). Therefore, only prodrugs 1 and 3 were selected for further studies to explore their uptake mechanism more thoroughly. First, the uptake of prodrugs 1 and 3 (100 mM) was studied at 4  C, since the lower temperature is known to decrease carriermediated transport of substances (Kageyama et al., 2000). With both prodrugs the uptake was decreased to less than 30% of their total uptake (0.69  0.15 nmol/min/mg of protein for prodrug 1, and 0.69  0.07 nmol/min/mg of protein for prodrug 3) (Fig. 3). This confirmed that the uptake at 37  C was mainly carrier-mediated. The uptake of prodrugs (100 mM) was also studied in the presence of 2 mM L-tryptophan (L-trp) at 37  C, to see whether the prodrugs could compete with natural LAT1 substrates (Fig. 3). The L-trp-

Fig. 3. The uptake mechanism of prodrugs 1 and 3 (100 mM) into MCF-7 cells. The uptake was determined in the presence of OATP-substrate, probenecid, and LAT1substrate, L-tryptophan (Trp), and both probenecid and L-tryptophan at 37  C. In addition, the uptake was determined at 4  C. An asterisk denotes a statistically significant difference from the respective control (***P < 0.001, one-way ANOVA, followed by Tukey’s test).

treatment increased the uptake of prodrugs 1 and 3 by 1.86–2.05fold (5.67  0.55 and 5.48  0.29 nmol/min/mg of protein, respectively). Therefore, we concluded that L-trp most probably drove these prodrugs to use some other amino acid carrying transporter (s) that are expressed in MCF-7 cell line, such as organic anions transporting polypeptides (OATPs) (Stute et al., 2012; Wlcek et al., 2008). OATPs comprise a family of trans-membrane proteins that are ubiquitously distributed throughout the body (Kalliokoski and Niemi, 2009; Roth et al., 2012). They transport mainly organic anions across the cell membranes, but they have quite broad substrate specificity and therefore, they can transport also neutral as well as even cationic compounds. As some of OATPs can transport thyroid hormones that have also affinity for LAT1 (Kinne et al., 2011; Visser et al., 2011), we hypothesized that these transporters may also be involved to the uptake of prodrugs 1 and 3 when LAT1-mediated transport is inhibited by L-trp. Thus, the uptake of prodrug 1 and 3 (50 and 100 mM) was studied in the presence of the known unselective competing OATP1/OATP2 substrate, probenecid (500 mM) (Gibbs and Thomas, 2002; Sugiyama et al., 2001; Tollner et al., 2015) at 37  C. At 100 mM prodrug concentration, probenecid did not have an effect on the uptake of prodrug 1 (2.74  0.19 nmol/min/mg of protein), which had better affinity for LAT1, whereas the uptake of prodrug 3 was slightly decreased (12%; 2.89  0.38 nmol/min/mg of protein) in the presence of probenecid (Fig. 3). Prodrug 3 already started to use probenecid-sensitive transporters at concentrations below 100 mM as it had lower affinity for LAT1, whereas prodrug 1 used mainly LAT1 up to 100 mM concentration. Prodrug concentrations below 50 mM probenecid did not have any effect on the uptake of these prodrugs, since both were primarily using LAT1 for their transport (data not shown). The uptake of the prodrugs (100 mM) in the presence of the competitive LAT1 substrate, L-trp (2 mM), hindered them to bind to LAT1 and forced them to use other transporters (Fig. 3). This increased uptake was inhibited by probenecid (2.74  0.19 and 2.59  0.20 nmol/min/mg of protein, respectively), which revealed that these transporter were probenecid-sensitive and thus, possibly OATP1/OATP2 (Fig. 3). The decrease was significant (ca. 50%) with both prodrugs at 100 mM concentrations, but only significant (ca. 40%) for prodrug 1 at 50 mM concentration, as it had higher affinity for LAT1 (data not

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shown). However, L-trp and probenecid together did not reduce the uptake to zero, since they are only competing substrates and it is possible that when LAT1 and probenecid-sensitive influx transporters are blocked, there will be a third transport system overexpressed in MCF-7 cells, to which the prodrugs have even lower affinity but which is able to carry these prodrugs into the cells. Furthermore, it is also possible that these prodrugs have some affinity for efflux transporters that probenecid is able to inhibit. Nevertheless, these results indicate that LAT1 may be primary transporter in vivo, since it is not likely that prodrug plasma concentrations would exceed the interface concentration (50– 100 mM) where probenecid-sensitive transporters start to participate to disposition of these prodrugs. From Fig. 4A it can been seen that the LAT1-mediated uptake of prodrug 1 saturates at concentrations below 100 mM. With higher concentrations another transporter starts to carry prodrug 1 inside the MCF-7 cells. Therefore, we carried out Eadie-Hosftee plot analysis for these uptakes. Prodrug 1 had higher affinity for LAT1 than for the other transporter(s) (Km values were 36.68  4.61 mM and 125.86  6.85 mM, respectively). However, at higher concentrations the other transporter had higher capacity to transport the prodrug 1 into the cells compared to LAT1 (Vmax values were 10.18  0.28 nmol/mg/min and 1.41  0.12 nmol/mg/min, respectively). The same trend was seen also with prodrug 3, but as it was not able to bind to LAT1 as effectively as prodrug 1, the second transport system started to participate to the total uptake at lower concentrations (Fig. 4B). The Km and Vmax values were 13.57  0.01 mM and 0.60  0.01 nmol/mg/min for LAT1-mediated transport of prodrug 3 and 203.51  28.09 mM and 11.14  1.00 nmol/mg/min for probenecid-sensitive transport of prodrug 3. According to these results the affinity of prodrug 3 for LAT1 seems to be higher than prodrug 1, but as the prodrug 1 was transported via LAT1 into the cells with higher capacity, it is highly likely that by the Eadie–Hofstee analysis we were not able to

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separate these two transport mechanisms clearly from each other, especially in the case of prodrug 3, whose transport by LAT1 and probenecid-sensitive transporter(s) overlap remarkably. Taking the [14C]-L-leucine uptake inhibition and compounds' uptake results together, it is obvious that LAT1-utilizing prodrugs are carried more efficiently into the cells expressing LAT1 than their parent drugs. However, although an efficient binding to LAT1 usually corresponds with the increased cellular uptake (prodrugs 1 and 2), a prodrug with lower inhibition of [14C]-Lleucine uptake can be transported efficiently via LAT1 into the cells (prodrug 3). Furthermore, a high inhibition of competing substrate uptake does not always guarantee increased cellular uptake of a prodrug, for example if the prodrug is slowly transported via LAT1 into the cells (prodrug 4). Therefore, these type of studies should never rely only on inhibition results of a known substrate. Instead, the absolute uptake of studied compounds should always be determined. Thyroid hormones, i.e., T3 (3,30 ,5-triiodo-L-thyronine) and T4 (3,30 ,5,50 -teteraiodo-L-thyronine) and their structurally smaller metabolites rT3 (3,30 ,50 -triiodo-L-thyronine) and T2 (3,30 -diiodo-L-thyronine) are known to be transported via LAT1 in a size-related order (Friesema et al., 2001). However, they also utilize OATPs in addition to monocarboxylate transporters 8 and 10 (MCT8 and MCT10) for their cellular uptake (Kinne et al., 2011; Visser et al., 2011). It has been suggested that OATP1C1 is primarily responsible for T4 uptake from the blood into the brain across the BBB, which implies that LAT1 may not be able to transport thyroid hormones efficiently and fast enough due to the structure restrictions, whereas OATP1C1, with broader substrate specificity, is able to compensate the brain uptake of thyroid hormones. Therefore, LAT1 selectivity over probenecid-sensitive transporters and participation of each subtype of probenecid-sensitive transporters as well as possible other tertiary transporters to the cellular uptake of LAT1-utilizing prodrugs of perforin inhibitors should be studied more thoroughly in future, as possible lack of selectivity for LAT1 may impair the targeting potency of these prodrugs. However, it should be remembered that even though these prodrugs may have affinity for other transporters than LAT1 in vitro, the concentrations that are relevant in vivo make the difference for the prodrug disposition. On the other hand, participation of probenecid-sensitive transporters may also open up new horizons for tissue-selective targeting, since for example OATPs are expressed in numerous epithelia throughout the body, and especially in tissues (e.g., liver, kidney, intestine and placenta) that are linked to abnormal perforin activity in human pathologies and therapy-induced conditions. 3.4. Lysosomal uptake of compounds 1–18 and bioconversion of the prodrugs 1–8

Fig. 4. The uptake of prodrugs 1 (A) and 3 (B) into MCF-7 cells at concentration range 6.25–400 mM and Eadie–Hofstee plots for LAT1-mediated and probenecidsensitive transports.

The ability of prodrugs 1–4 and their parent drugs 9–12 to be accumulated in cell organelles having lower pH, such as cytolytic granules of effector cells, were studied in rat liver lysosomes due to the similarity and the ease of preparation in large quantities (Dell’Angelica et al., 2000). Cytolytic granules have an acidic pH and they contain soluble and transmembrane lysosomal proteins in addition to their specific lytic proteins, such as perforin and granzymes. To understand the lysosomotropic behavior of this general class of perforin inhibitors, the accumulation of compounds 15–18 into rat liver lysosomes was also studied. In addition, we wanted to study, if acid-labile prodrug structures, hydroxyimines and hydrazones, could deliver perforin inhibitors more efficiently into the lysosomes, since these compounds can be converted into double prodrugs having a LAT1-targeted promoiety attached to hydroxyimine or hydrazone functionalities. Therefore, we prepared hydroxyimines 7 and 5 and hydrazones 8 and 6 from the compounds 13 and 14, respectively. None of the compounds

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Table 1 Structures of the compounds 1–4 and 7–18 and their relative lysosomal uptake (%) and absolute lysosomal uptake (nmol/mg of protein). Compound

Structure

Relative uptake (%)

Absolute uptake (nmol/mg of protein)

1

97.84  5.05

n.d.a

2

97.82  0.63

n.d.a

3

93.16  0.41

n.d.a

4

75.73  4.29

1.24  0.07

7

58.24  0.36

n.d.a

8

24.42  .016

n.d.a

9

89.99  1.11

n.d.a

10

97.57  2.02

n.d.a

11

94.98  9.31

n.d.a

12

82.06  1.63

0.67  0.13

13

31.88  0.14

n.d.a

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Table 1 (Continued) Compound

Relative uptake (%)

Absolute uptake (nmol/mg of protein)

14

82.80  2.99

0.40  0.20

15

56.02  6.64

0.66  0.08

16

89.90  2.17

0.91  0.02

17

89.48  19.58

0.55  0.12

18

79.32  6.54

0.42  0.03

a

Structure

Not determined due to metabolism.

studied were classical lysosomotropic compounds, i.e., weak bases that can readily cross the plasma membranes at physiological pH but are protonated in slightly acidic cellular compartments, such as lytic granules and lysosomes, and therefore trapped inside (de Duve et al., 1974; Ishizaki et al., 2000; Kaufmann and Krise, 2007). Compound 11 has an amine group that has pKa value of 3.67 and therefore is the most likely to behave lysosomotropically. Compounds 10–11 that had the highest relative lysosomal uptake (Table 1), consist of three aromatic non-polar rings and therefore they have higher calculated partition coefficient (log P) values (3.47–4.03) and smaller polar surface area (PSA; 46.53–52.32) (see supporting data). These compounds were also some of the smallest compounds of those studied. However, slightly bigger and more

polar compounds 16 and 17 were also relatively highly accumulated into lysosomes. Compound 15, the most polar of the studied compounds (PSA value of 84.22) distributed almost evenly to both sides of lysosomes, while other compounds in the thiohydantoin series were more efficiently accumulated into lysosomes. Lipophilicity has been thought to have a major impact on lysosomal accumulation, since the permeation across the cell membranes by passive diffusion increases along with increased lipophilicity. Furthermore, after pH-driven uptake lipophilic compounds are able to bind to membrane lipids, which increases the trapping effect (Ishizaki et al., 2000). In our study, lipophilicity seemed to have a crucial effect on relative uptake (Fig. 5A), although our compounds were not weak bases. Furthermore, all

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Fig. 5. Correlation between relative lysosomal uptake (%) and log P (A), molecular weight (C) and polar surface area (E) and between absolute lysosomal uptake (mmol/mg of protein) and log P (B), molecular weight (D) and polar surface area (F) of compounds 9–18.

our compounds were relatively lipophilic (log P 1–4). In contrast, molecular size (Fig. 5C) and molecular polar surface area (Fig. 5E) had less effect on the lysosomal uptake. When the same comparison was done for the compounds with absolute uptake value, i.e., these compounds were not metabolized inside the lysosomes, lipophilicity and polarity had more impact on the total uptake than the size of the compounds (Fig. 5B, D, F). Unfortunately, compounds that had a lactone ring (9–11 and 13) were metabolized in lysosomes, most probably the lactone ring was opened and therefore we were not able to quantify the total amount of absolute uptake. However, from the thiohydantoin series, compound 16 was most efficiently accumulated into lysosomes, i.e., 0.91  0.12 nmol/mg of protein (Table 1), and it was more efficiently accumulated than a known lysosomotropic compound, chloroquine (0.57  0.02 nmol/mg of protein). Due to their low aqueous solubility (<0.01 mg/mL), the lysosomal accumulation of prodrugs 5 and 6 was not studied.

However, prodrugs 7 and 8 showed that relative lysosomal accumulation can be increased from 31.88% (compound 13) to 58.24% (compound 7) through conversion to hydroxyimine structure, but curiously not with a hydrazone structure (24.42% with the compound 8) (Table 1). Bioactivation of prodrugs 5–8 is expected to occur chemically in medium with lower pH values. Both hydroxyimine and hydrazone prodrugs (7 and 8) were bioconverted to their parent drugs in acidic environment, with half-lives at pH 3.0 127.97  21.20 h and 26.75  3.05 min, respectively, but at pH 5.0 1507.39  70.94 h and 15.16  5.11 h, respectively (see supporting data). Thus, the hydrazone prodrug was bioactivated in acidic conditions more efficiently, although the bioconversion rate of both of the prodrugs was relatively slow. Therefore, it was concluded that although hydroxyimine could be able to deliver more efficiently ketone or aldehyde parent drug into the lytic granules, its pH-sensitive bioactivation to its parent drug may not be selective and fast enough to release the active parent

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drug sufficiently enough in the target cell organelles. The detailed bioconversion rate constants of prodrugs 7 and 8 at different pH can be found in supporting data. Prodrugs 1–4 retained the high ability of compounds 9–12 to be accumulated into lysosomes (Table 1). Log P value seemed to have the greatest effect, since the less lipophilic prodrug 4 (log P 0.32; 75.73% uptake) was not taken up as much as the more lipophilic prodrugs 1–3 (log P 1.50–2.75; 93.16–97.84% uptake). However, prodrug 4, which was the only prodrug that did not metabolize in lysosomes, had the absolute uptake value of 1.24  0.07 nmol/mg of protein. Thus, it was able to be accumulated into lysosomes more efficiently than any other compound from the thiohydantoin series or the known lysosomotropic compound, chloroquine. Therefore, the LAT1-utilizing prodrugs may be able to deliver perforin inhibitors more efficiently into the lytic granules of cytotoxic effector cells. All prodrugs (1–4) were chemically stable in aqueous buffer solution (pH 7.4). Prodrugs 1, 3 and 4 were also stable in human and rat plasma during the 6 h incubation, whereas prodrug 2 released its parent drug (Table 2). In rat and human liver S9 fractions only prodrugs 1 and 2 released their parent drugs, whereas prodrugs 3 and 4 remained intact. Prodrug 1 (with a methylene group between the phenolic ring and the ester bond) seemed to be slightly more stable than prodrug 2 (in which the ester bond is directly attached to the phenolic ring) in all biological media studied. Since the bioactivation rate of prodrugs 1 and 2 in the rat liver cytosolic fraction equalled the rate in rat liver S9 fraction, which contains both cytosol and other smaller cell organelles, such as microsomes and lysosomes, it is highly likely that the prodrugs 1 and 2 will release their parent drugs in the cytosolic side if the uptake into the lytic granules is slow. However, as lysosomes also contain hydrolytic enzymes, it is possible that these prodrugs may also be able to be bioconverted in lysosomes. Curiously the prodrug 4 was not bioconverted in these conditions, although some bioconversion was seen in MCF-7 cells. This was most probably due to the fact that the bioconversion rate in lysosomes was so slow that it was not possible to detect during a 30 min incubation. Although the amide prodrugs 3 and 4 did not release their parent drugs in vitro during 6 h incubation (Table 2), they are most likely able to be quantitatively bioconverted in vivo, which is a common feature of amide prodrugs (Gynther et al., 2010; Gynther et al., 2015).

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needs to be studied more thoroughly in future. Moreover, it may give new insights for targeting prodrugs to other tissues. These results also demonstrate that LAT1-utilizing prodrugs have the potential to deliver perforin inhibitors efficiently into cell organelles that have lower pH, such as lytic granules of cytotoxic effector cells. Furthermore, the ester prodrugs were able to release their parent drugs enzymatically in rat and human liver S9 fraction. Although amide prodrugs were more stable in vitro, it is highly likely that amide prodrugs will release their parent drug in vivo. Taking together, this shows that these prodrugs have the potential to deliver perforin inhibitors into the activated target cells and affect the intracellular kinetics of perforin inhibitors. In future, whether these compounds can elicit their perforin-inhibitory activity intracellularly could therefore be investigated. Moreover, it may be feasible to apply this prodrug approach to other drugs that require targeting to cells over-expressing LAT1, such as several types of cancer cells. Authorship contribution Kristiina M. Huttunen: conception and design of the study, acquisition of data, analysis and interpretation of data, drafting the article and revising it critically, final approval of the version to be submitted. Johanna Huttunen: acquisition of data, analysis and interpretation of data, drafting the article, final approval of the version to be submitted. Imke Aufderhaar: acquisition of data, analysis and interpretation of data, drafting the article, final approval of the version to be submitted. Mikko Gynther: design of the study, analysis and interpretation of data, drafting the article and revising it critically, final approval of the version to be submitted. William A. Denny: design of the study, analysis and interpretation of data, revising the article critically for important intellectual content, final approval of the version to be submitted. Julie A. Spicer: design of the study, analysis and interpretation of data, revising the article critically for important intellectual content, final approval of the version to be submitted. Conflicts of interest The authors state no conflicts of interest.

4. Conclusions Acknowledgements In conclusion, all the designed and synthesized LAT1-utilizing prodrugs inhibited cellular uptake of [14C]-L-leucine, which indicates that these prodrugs have a reasonably good affinity for LAT1. Moreover, two of the prodrugs (1 and 3) were also transported via LAT1 into MCF-7 cells in significantly higher amounts than their parent drugs. Curiously, these prodrugs started to utilize a second probenecid-sensitive transport mechanism at higher prodrug concentrations or if LAT1 was saturated by the excess of L-tryptophan. As targeting of LAT1-utilizing prodrugs may be altered if a second major transporting system is involved, the role of each probenecid-sensitive and possible other transporters Table 2 Bioconversion rates of prodrugs 1–4 in rat and human liver S9 fractions and rat and human plasma at 37  C presented as half-lives (t1/2 (min); mean  SD, n = 3). Prodrug

Rat liver S9

Human liver S9

Rat plasma

Human plasma

1 2 3 4

54.73  1.41 40.93  4.08 –a –a

337.20  37.70 158.70  8.36 –a –a

–a 129.21  6.58 –a –a

–a 88.68  4.65 –a –a

a

No bioconversion was detected during the 6 h incubation.

The authors would like to thank Ms. Tiina Koivunen for skillful technical assistant with the syntheses and Ms. Helly Rissanen for invaluable technical assistance with in vitro bioconversion studies. The work was financially supported by the Academy of Finland (#256837), Orion-Farmos Research Foundation, The Finnish MS Foundation, Emil Aaltonen Foundation, University of Eastern Finland (postdoctoral project funding), Jenny and Antti Wihuri Foundation and Sigrid Juselius Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2015.12.034. References Baran, K., Dunstone, M., Chia, J., Ciccone, A., Browne, K.A., Clarke, C.J., Lukoyanova, N., Saibil, H., Whisstock, J.C., Voskoboinik, I., Trapani, J.A., 2009. The molecular

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