Comprehensive Evaluation of the Binding of Lipocalin-Type

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Comprehensive Evaluation of the Binding of Lipocalin-Type Prostaglandin D Synthase to Poorly Water-Soluble Drugs Yoshiaki Teraoka, Satoshi Kume, Yuxi Lin, Shogo Atsuji, and Takashi Inui Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00590 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 24, 2017

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Molecular Pharmaceutics

Comprehensive Evaluation of the Binding of Lipocalin-Type Prostaglandin D Synthase to Poorly Water-Soluble Drugs Yoshiaki Teraoka,†,‡,# Satoshi Kume,†,§,¶,# Yuxi Lin,§ Shogo Atsuji,† and Takashi Inui*,† †

Department of Applied Life Sciences, Graduate School of Life and Environmental Sciences,

Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan ‡

Research Fellow of the Japan Society for the Promotion of Science

§

Cellular Function Imaging Team, Division of Bio-function Dynamics Imaging, RIKEN Center

for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 6500047, Japan ¶

Health Metrics Development Team, Integrated Research Group, RIKEN Compass to Healthy

Life Research Complex Program, RIKEN Cluster for Science and Technology Hub, Kobe, Japan KEYWORDS: drug delivery system, pharmaceutical solubilizer, poorly water-soluble drug, lipocalin-type prostaglandin D synthase, structure-based docking, isothermal titration calorimetry

ACS Paragon Plus Environment

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ABSTRACT: Low water solubility of candidate drug compounds is a major problem in pharmaceutical research and development. We developed a novel drug delivery system (DDS) for poorly water-soluble drugs using lipocalin-type prostaglandin D synthase (L-PGDS), which belongs to the lipocalin superfamily and binds a large variety of hydrophobic molecules. In this study, we comprehensively evaluated the capability of L-PGDS to bind and solubilize various poorly-water soluble drugs using structure-based docking. Docking simulations of 2892 commercially available approved drugs indicated that L-PGDS shows higher binding affinities for various drugs compared with 2-hydroxypropyl-β-cyclodextrin. Five drugs selected from the top 100 with the highest binding affinities for L-PGDS exhibited very low solubility in PBS (pH 7.4). However, in the presence of 1 mM L-PGDS, the apparent solubility of all drugs improved markedly, from 19.5- to 166-fold. Calorimetric experiments on two drugs, telmisartan and imatinib, revealed that L-PGDS forms a 1:2 complex with each drug, with dissociation constants of 0.4–40.0 µM. Kinetic simulations of drug dissolution with L-PGDS indicated that the difference in free energy change (∆∆G) between the insoluble state and the L-PGDS–bound state are within the range from −10 to +5 kJ mol−1. The ∆∆G value is a critical factor in evaluating whether a poorly water-soluble drug can be solubilized by L-PGDS. Collectively, these results demonstrate that in silico docking is a promising approach for identifying drug molecules suitable for the L-PGDS-based DDS.


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INTRODUCTION In pharmaceutical research and development, insolubility or low solubility in water is a major problem for new drug candidates.1 Poor solubility of drugs can lead not only to inaccurate results in in vitro enzyme and cell assays but also low oral bioavailability and difficulty with intravenous administration in in vivo assays.1,2 Nevertheless, poorly water-soluble compounds are often identified as drug candidates in currently used high-throughput screening approaches.3 Recent pharmaceutical studies demonstrated that approximately 65% of new drug candidates belong to classes II and IV of the biopharmaceutical classification system (BCS), exhibiting “low solubility and high permeability” or “low solubility and low permeability”, respectively.4,5 Although chemical modification and the prodrug approaches employed during the lead compound optimization process often enhance the aqueous solubility of drug candidates, drug potency nevertheless decreases in many cases. Thus, in order to make available hydrophobic drugs, drug delivery systems (DDSs) in which drug molecules are encapsulated and solubilized using delivery vehicles such as cyclodextrins,6 liposomes,7 polymer micelles,8 and dendrimers9 have been extensively examined. We recently developed a novel DDS using lipocalin-type prostaglandin D synthase (L-PGDS), a natural transporter protein and a non-toxic and nonimmunogenic molecule.10 We hypothesized that this DDS would facilitate the pharmaceutical development and clinical application of a variety of poorly water-soluble compounds. L-PGDS is a unique multi-functional protein that acts as a PGD2 synthase,11,12 a reactive oxygen species scavenger,13 a scavenger of biliverdin in the cerebrospinal fluid of subarachnoid hemorrhage patients,14 and an extracellular transporter for several small lipophilic molecules.15–17 L-PGDS has a classical lipocalin fold that consists of a long α-helix and an eight-stranded anti-


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parallel β-barrel that forms a cap-shaped binding pocket for hydrophobic compounds.18–22 We demonstrated that human L-PGDS binds to a large variety of intravital lipophilic molecules in mammals, including heme metabolites, retinoids, thyroid hormones, and steroids.17,23 By exploiting this characteristic of L-PGDS, we demonstrated that the protein forms complexes with and enhances the apparent solubility of various poorly water-soluble drugs, including diazepam,10 6-nitro-7-sulfamoylbenzo[f] quinoxaline-2,3-dione,10 telmisartan,24 dipyridamole,25 and 7-ethyl-10-hydroxy-camptothecin (SN-38).26 In addition, using in vivo assays, we demonstrated that L-PGDS is suitable as a potent drug-delivery carrier for administration both orally10,24,25 and intravenously.10,26 These characteristics of L-PGDS led us to survey poorly water-soluble drugs to determine which are applicable to the L-PGDS-based DDS. To accomplish this, it was necessary to determine the effect of common physico-chemical properties such as molecular size, hydrophobicity, and chemical structure of drugs on solubilization by LPGDS. In this study, we comprehensively evaluated the capability of human L-PGDS to bind and solubilize various poorly water-soluble drugs using structure-based docking. First, the binding affinity for L-PGDS was predicted by in silico docking using AutoDock Vina for 2892 commercially available drugs approved for human use. From these predictions, five highly hydrophobic drugs exhibiting high binding affinity for L-PGDS were selected. Improvement in the solubility of these drugs in the presence of L-PGDS was determined experimentally, and the thermodynamic parameters for the binding of these drugs to L-PGDS were characterized using isothermal titration calorimetry (ITC). Finally, we performed kinetic simulations of drug dissolution with L-PGDS to evaluate the differences in free energy change (∆∆G) between the insoluble state and the L-PGDS–bound state. These computational and experimental results


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revealed the common physico-chemical properties associated with solubilization of drugs by LPGDS.

MATERIALS AND METHODS Docking simulations. The crystal structure of human C65A-substituted L-PGDS bound to polyethylene glycol has been determined (Protein Data Bank [PDB] code: 4ORR).22 Its coordinates were obtained from the PDB and used as the source of the L-PGDS structure for in silico docking. The model structure of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) was constructed based on the structure of β-cyclodextrin (β-CD) extracted from the crystal structure of the β-CD/β-amylase complex (PDB code: 1BFN).27 The bound compound and water molecules were removed from the PDB files. All hydrogen atoms were added to L-PGDS using the MolProbity server.28 Steric structures of drug molecules were obtained from the Zdd subset of the ZINC database, which includes commercially available drugs approved for use in humans.29 Gasteiger-Marsili charges30 were added to L-PGDS, HP-β-CD, and drug molecules using AutoDockTools31 and the Raccoon program.32 Non-polar hydrogen atoms in these molecules were not used in docking simulations. High-throughput docking of 2892 drug molecules to L-PGDS and HP-β-CD was performed using a molecular docking program, AutoDock Vina, version 1.1.2.33 Although L-PGDS may bind to multiple drug molecules, we targeted the primary binding site which is considered to be mainly involved in solubilization of drugs by L-PGDS. The cavity volumes of L-PGDS and HP-β-CD were estimated to be 1245 and 156 Å3, respectively, using the 3V website.34 The docking boxes for L-PGDS and HP-β-CD were set to grids of 28 × 26 × 32 Å and 22 × 22 × 22 Å, respectively, and the boxes were centered on


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these binding pockets. During the docking simulations, the rotational bonds of the drug molecules and the hydroxypropyl groups of HP-β-CD were set to be flexible, and L-PGDS was treated as a rigid body with non-rotational bonds. In the calculations of binding free energy, the contributions of molecular interactions such as hydrogen bonding, hydrophobic interactions, and molecular repulsion were considered. Models of complex structures exhibiting the most stable binding free energy were adopted based on the calculations for each drug. Chemicals. Telmisartan was supplied by Boehringer Ingelheim GmbH & Co. KG (Germany). Nilotinib, lapatinib, imatinib, atovaquone, and HP-β-CD were purchased from Carbosynth (UK), ChemieTek (USA), Selleck Chemicals (USA), Tokyo Chemical Industry (Japan), and Wako (Japan), respectively. The concentrations of the compounds in PBS at pH 7.4 were determined spectroscopically with a molar absorption coefficient in PBS containing 5% (v/v) dimethyl sulfoxide (DMSO) of ε296 for telmisartan = 23,600 M−1 cm−1, in PBS containing 50% (v/v) DMSO of ε332 for lapatinib = 19,500 M−1 cm−1 and ε500 for atovaquone = 2530 M−1 cm−1, and in DMSO of ε340 for nilotinib = 3660 M−1 cm−1 and ε333 for imatinib = 4500 M−1 cm−1. Purification of recombinant human L-PGDS. We used a recombinant human C65A/C167A-substituted L-PGDS mutant (ε280 = 25,900 M−1 cm−1) in which Cys65 and Cys167 were substituted with Ala to get rid of the enzymatic activity and to prevent the formation of incorrect intra- and intermolecular disulfide bonds, respectively. Mutant L-PGDS was expressed as a glutathione-S-transferase fusion protein in Escherichia coli BL21 (DE3; TOYOBO, Japan).17 The fusion protein was bound to glutathione Sepharose 4B (GE Healthcare) and incubated overnight with thrombin to release L-PGDS. The recombinant protein was further purified by gel filtration chromatography using HiLoad 26/600 Superdex 75 (GE Healthcare) in


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5 mM Tris/HCl buffer (pH 8.0), followed by dialysis against PBS. The purified protein showed a single band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (Figure S1). Drug solubility determinations. PBS, PBS containing 19 mg mL−1 (1 mM) L-PGDS, and PBS containing 19 mg mL−1 (12.5 mM) HP-β-CD was saturated with termisartan, nilotinib, lapatinib, imatinib, and atovaquone. Each 1 mL of solution, containing approximately 5 µmol of drug, was rotationally suspended at 37°C for 1–24 h. The concentration of drug in the supernatant under each condition was calculated from absorption spectra with baseline corrections. The values for the apparent dissociation constant (Kd) for binding of L-PGDS and HP-β-CD to the drug molecules were determined using the following equilibrium equation:35 d

:

D P − D + D = (1) D − D

where [D]free represents the equilibrium solubility of the drug in PBS without L-PGDS or HP-βCD; [D]app represents the apparent concentration of dissolved drug in the supernatant; [P]t represents the total protein or HP-β-CD concentration; and n represents the binding stoichiometry of the drug per its binding partner. ITC measurements. ITC experiments were performed using a MicroCal VP-ITC instrument (GE Healthcare) at 25°C in PBS containing 5% (v/v) DMSO. From an injection syringe, L-PGDS (490 and 510 µM) was titrated into 59 µM telmisartan and 51 µM imatinib, respectively, and HP-β-CD (6 and 4 mM) was titrated into 60 µM telmisartan and 41 µM imatinib, respectively. Titration experiments consisted of 55–57 injections spaced at intervals of 300 s. The injection volume was 5 µL for each, and the cell was continuously stirred at 286 rpm. The corresponding heat of dilution of L-PGDS and HP-β-CD titrated into buffer was used to


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correct the data. The thermodynamic parameters for binding telmisartan were determined using the two-sets of independent binding sites model, and those for binding imatinib were determined using the one-set of independent binding sites model, as supplied with MicroCal Origin software (ver. 7.0). Kinetic simulations using simultaneous differential equations. The reaction schemes of drug dissolution with L-PGDS are shown in Figure S2A. Kinetic simulations of drug dissolution were performed by numerically solving the simultaneous differential equations (Figure S2B) using IGOR Pro software, version 6.37. Free energy changes associated with drug insolubility (∆Ginsol) and drug binding to L-PGDS (∆Gb) were calculated from the following thermodynamic relationships: ∆ ! = "# ln (2) ∆' = "# ln ( (3)

where R represents the gas constant and T represents the temperature (298.15 K). The terms Kfree and Kd represent the equilibrium constant for drug dissolution and the dissociation constant for L-PGDS–drug binding, respectively. The values k+1 and k−1 (Kfree = k+1/k−1) represent the apparent rate constants for drug dissolution and insolubility, respectively. The values k+2 and k−2 (Kd = k−2/k+2) represent the rate constants for association and dissociation in L-PGDS–drug binding, respectively. For ∆G values of −25.0, −30.0, and −35.0 kJ mol−1, the corresponding K values are 41.7, 5.55, and 0.738 µM. For k+1 and k−2 was 1.00, the k−1 and k+2 values were calculated as 0.0240, 0.180, and 1.36 according to the K values. Using these parameters and the initial concentrations of L-PGDS (1 mM) and insoluble drug (5 mM), the differential equations were solved using the IntegrateODE function of IGOR Pro.


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RESULTS Structure-based docking of drug molecules to L-PGDS. To estimate the binding affinity of human L-PGDS for various drug molecules, we docked 2892 approved drugs in the Zdd subset of the ZINC database to L-PGDS. This drug subset contains commercially available drugs approved for use in humans, with relative molecular mass (Mr) and hydrophobicity (LogP) values ranging from 59.0 to 821 and −5.97 to 9.84, respectively (Figure S3). In docking analyses using AutoDock Vina, the predicted binding free energy of drugs with L-PGDS (L-PGDS∆Gv) ranged from −50.6 to −10.0 kJ mol−1, with a mean of −30.5 kJ mol−1 (Figure 1A). We also performed docking simulations to analyze interactions between the drug molecules and HP-βCD, a widely used pharmaceutical solubilizer.35 The predicted binding free energy of drugs with HP-β-CD (HP-β-CD∆Gv) ranged from −43.9 to −8.8 kJ mol−1, with a mean of −25.3 kJ mol−1 (Figure 1B). The ratio of L-PGDS∆Gv to HP-β-CD∆Gv exceeded 1.0 for 91.8% of the docked drugs (Figure 1C), with an average ratio of approximately 1.2. These results indicated that L-PGDS has a higher potential than HP-β-CD for binding a variety of drug compounds. The L-PGDS∆Gv and HPβ-CD

∆Gv values were dependent on molecular size, each exhibiting an inverse bell-shaped profile

with a peak at Mr 500–600 (Figure 1D,E). The values of ∆∆Gv (L-PGDS∆Gv − HP-β-CD∆Gv) decreased with increasing Mr up to a value of 700 (Figure 1F). By contrast, the L-PGDS∆Gv values exhibited a dependence on hydrophobicity that showed a moderate increasing tendency with increasing LogP (Figure 1G), whereas the HP-β-CD∆Gv values were relatively independent of drug hydrophobicity (Figure 1H). The ∆∆Gv values decreased in the range of higher LogP values (Figure 1I). These results suggest that in comparison with HP-β-CD, L-PGDS tends to form a stable complex with more hydrophobic and higher-molecular-mass drug molecules.


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Next, to experimentally validate the in silico docking results, we examined the chemical structures and physico-chemical properties of the top 100 drugs (3.7%) with the highest LPGDS

∆Gv values ranging from −50.6 to −40.2 kJ mol−1 (Table S1). As a benchmark, we

performed a docking simulation of SN-38, an anti-cancer drug that can be bound to L-PGDS,26 and the L-PGDS∆Gv value was −36.8 kJ mol−1, suggesting that the top 100 drugs bind to L-PGDS with the sufficiently high affinities. Furthermore, among the top 100 drugs with Mr values ranging from 300 to 700 and LogP values >3, we selected five drugs (nilotinib, telmisartan, lapatinib, imatinib, and atovaquone; Figure 2A) for further analysis. The physico-chemical properties, L-PGDS∆Gv and HP-β-CD∆Gv values, and medical indications of these drugs are shown in Table 1. In structural models of drug/L-PGDS complexes, each drug molecule was clearly located in the binding pocket of L-PGDS (Figures 2B–G and S4). The intermolecular interactions of drug/L-PGDS complexes were predicted to be primarily hydrophobic interactions and formation of 1–6 hydrogen bonds (Figure S5). By contrast, the binding free energies of the drugs in complex with HP-β-CD were predicted to be lower than those of the drugs in complex with L-PGDS (Table 1), as the drugs were partially buried within the relatively small hydrophobic cavity of HP-β-CD (Figure S6). Enhancement of drug solubility by L-PGDS. In order to investigate the effect of L-PGDS on the solubility of the five selected drugs, we first assessed the concentration of each drug in PBS with and without L-PGDS and HP-β-CD (Figure 3A). By 24 h, nilotinib, telmisartan, and imatinib dissolved in PBS only up to 2.7, 7.4, and 34.9 µM, respectively, and the concentrations of lapatinib and atovaquone were lower than the corresponding detection limits (Figure 3B–F, Table 2). However, in the presence of 19 mg mL−1 (1 mM) L-PGDS, the apparent solubility of nilotinib, telmisartan, lapatinib, imatinib, and atovaquone increased markedly by 24


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h, to 53.2, 1230, 233, 813, and 207 µM, respectively, values which were 19.5- to 166-fold higher than in PBS (Figure 3B–F, Table 2). In particular, the concentrations of telmisartan and imatinib reached the maximum solubility within 1 h (Figure 3C,E). By contrast, in the presence of 19 mg mL−1 (12.5 mM) HP-β-CD, the apparent solubility of nilotinib, telmisartan, lapatinib, imatinib, and atovaquone increased only slightly by 24 h, to 6.6, 65.1, 4.9, 366, and 50.9 µM, values which were 2.1–45.0% of the values obtained in the presence of L-PGDS (Figure 3B–F, Table 2). These results show that the drugs selected based on the docking simulations are efficiently solubilized by L-PGDS. Based on the solubility measurements, we calculated the apparent Kd values for the binding of L-PGDS and HP-β-CD to the drug molecules (Table 2). It is known that L-PGDS can bind up to three hydrophobic molecules23,26 and that β-CD commonly forms a 1:1 complex with a drug molecule.36 Thus, we determined the apparent Kd values for the binding of L-PGDS to 1–3 drug molecules and for the formation of a 1:1 HP-β-CD:drug molecule complex (Table 2). The apparent Kd values for binding of the drugs to L-PGDS were substantially lower than those for HP-β-CD, indicating that the high binding affinity of L-PGDS enhances the solubility of the five drugs we examined. Thermodynamic characterization of interactions between L-PGDS and drug molecules. To elucidate the binding mode of poorly water-soluble drugs to L-PGDS in detail, we performed ITC analyses in PBS (Figure 4). Two of the five selected drugs were examined, telmisartan and imatinib, because the solubility of the other three drugs was insufficient for ITC experiments. As shown in the thermograms, titration of L-PGDS into telmisartan and imatinib produced negative peaks, indicating that the binding reactions were exothermic (Figure 4A,B, upper panels). After integrating the peak areas, the integrated heat obtained for binding to the drug was plotted against the molar ratio ([L-PGDS]/[drug]) (Figure 4A,B, lower panels). The


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binding isotherms for telmisartan and imatinib were fitted using the two-sets of independent binding sites model and the one-set of independent binding sites model, respectively (Figure 4A,B). Although we also assessed intermolecular interactions between HP-β-CD and telmisartan and imatinib using ITC, no binding isotherm was detected for either drug due to their weak binding affinities (Figure 4C,D). The thermodynamic parameters obtained for complex formation between L-PGDS and the drug molecules are summarized in Table 3. The ITC results showed that L-PGDS forms a 1:2 complex with telmisartan in high- and low-affinity sites, with Kd-ITC values of 0.4 and 1.1 µM, respectively, and L-PGDS forms a 1:2 complex with imatinib in an identical binding site, with a Kd-ITC value of 40.0 µM (Table 3). The values of standard enthalpy change for binding (∆Hb°) to telmisartan were −26.2 kJ mol−1 for the high-affinity site and −39.3 kJ mol−1 for the low-affinity site, and the ∆Hb° value for binding to imatinib was −28.0 kJ mol−1 (Table 3). These results indicate that negative ∆Hb° contributes favorably to the binding reactions of L-PGDS with the drugs examined. The values for the entropy term (–T∆Sb°) for the high- and low-affinity sites in binding to telmisartan were −10.3 kJ mol−1 and 5.3 kJ mol−1, respectively (Table 3), indicating that the entropic gains also contribute to binding to the high-affinity site. The –T∆Sb° value for imatinib was 2.9 kJ mol−1 (Table 3). These results demonstrate that L-PGDS has multiple binding sites beneficial for DDS, and drug–L-PGDS interactions are the specific binding associated with favorable enthalpy changes. We also performed ITC analysis of the binding of L-PGDS to temozolomide, which is a small, hydrophilic molecule (LogP: −1.9; Mr: 194) and had a low affinity for L-PGDS in the docking study (L-PGDS∆Gv: −26.8 kJ mol−1). The binding reaction between L-PGDS and


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temozolomide could not be detected by calorimetric titration (Figure S7). These results suggest that L-PGDS does not bind to low-molecular-mass, hydrophilic drugs. Kinetic simulations of drug dissolution in the presence of L-PGDS. In order to obtain further thermodynamic insights into the solubilization of poorly water-soluble drugs in the presence of L-PGDS, we performed kinetic simulations based on a simple equilibrium model (Figure S2A) under different free energy balances between the insoluble state of drugs and the complexed state of drugs with L-PGDS. By numerically solving the simultaneous differential equations (Figure S2B), the contributions of free energy change for insolubility (∆Ginsol) and binding to drug (∆Gb) were evaluated (Figure 5). The differences of ∆G between the insoluble state and the L-PGDS-bound state were expressed as ∆∆G. In Figure 5A, E, and I, the ∆∆G value was equal to zero kJ mol−1, and the simulated solubility of drug was around 500 µM. By absolutely leaning the equilibrium toward a more stable binding free energy corresponding to the ∆∆G values of –5 and –10 kJ mol−1 (Figure 5B,C,F), the simulated solubility of the drugs increased to 889–1020 µM. By contrast, by decreasing the ∆Ginsol values to –30 and –35 kJ mol−1, the apparent solubility of the drugs declined markedly (Figure 5D,G,H). However, even when the ∆∆G value increased to +5 kJ mol−1, the concentration of drug was still above 100 µM in the presence of L-PGDS (Figure 5D,H). A further increase in the ∆∆G value to +10 kJ mol−1 significantly diminished the ability of L-PGDS to enhance the solubility of the drugs (Figure 5G). In addition, a clear relationship between ∆∆G and the ability of L-PGDS to solubilize the drugs was identified by fitting of the theoretical sigmoid curve for the simulated solubility (Figure S8). These results demonstrate that the ∆∆G value is an important thermodynamic criterion governing the solubilization of poorly water-soluble drugs by L-PGDS.


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DISCUSSION The L-PGDS-based DDS primarily targets poorly-water soluble drugs in BCS classes II and IV. BCS class II compounds account for a large proportion (42%) of new drug candidates,4 and their low solubility limits their rate of absorption, despite their high membrane permeability.5 In a previous study, we demonstrated that L-PGDS can bind to and solubilize BCS class II drugs such as diazepam,10 telmisartan,24 dipyridamole,25 and SN-3826 (a BCS class IV drug) without any chemical modification of drug structures. Furthermore, the high solubility of SN-38 in the presence of L-PGDS allows for its intravenous administration, facilitating the high anti-tumor activity of the SN-38/L-PGDS complex.26 In this case, it is thought that free SN-38 molecules in equilibrium with the complex penetrate the lipid bilayer of cell membranes. By comparison, LPGDS was shown to enhance the intestinal absorption of diazepam and telmisartan in oral administration, resulting in excellent bioavailability of these drugs.10,24 Orally administered drug/L-PGDS complexes are thought to behave as enteric capsules that are stable under gastric conditions; the complexes release the drug molecules following intestinal digestion of L-PGDS, thus enhancing the rate of drug absorption.24 Using in silico docking, in the present study, we identified four BCS class II drugs (nilotinib, telmisartan, imatinib, and atovaquone) and a BCS class IV drug (lapatinib) from among 2892 compounds in the drug database for which the solubility could be enhanced at neutral pH with L-PGDS. Structure-based docking can therefore be regarded as a feasible high-throughput approach for identifying poorly water-soluble drugs suitable for application to the L-PGDS-based DDS. Comprehensive evaluations of the binding of L-PGDS to various drugs revealed that those drugs that bind to L-PGDS with high affinity possess several common physico-chemical


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properties. That is, L-PGDS tends to form stable complexes with relatively large (Mr: 500–600) and more hydrophobic drug molecules (Figure 1D,G). In addition, the top 100 drugs examined in the docking study contain many planar and rod-like structures composed of multiple aromatic rings (Table S1). These structural features often decrease solubility due to π–π interactions between drug molecules, as has been observed for inhibitors of protein kinases targeted in many oncology projects.1 Indeed, the top 100 list included five kinase inhibitors: nilotinib, lapatinib, gefitinib, sorafenib, and imatinib (Nos. 9, 16, 19, 27, and 71 in Table S1). We examined the solubility of nilotinib, lapatinib, and imatinib, and compared with HP-β-CD, L-PGDS enhanced the apparent solubility of all three drugs (Figure 3). We previously showed that L-PGDS is an effective delivery vehicle for the anti-cancer drug SN-38,26 which also exhibits a planar template with multiple aromatic rings. These results, taken together, suggest that the binding pocket of LPGDS is suitable for a variety of poorly water-soluble anti-cancer drugs exhibiting the common physico-chemical properties identified here, and thus, L-PGDS is useful solubility enhancer. Physico-chemical equilibrium of drug dissolution in the presence of L-PGDS is thermodynamically governed. Poorly water-soluble drug molecules exist essentially as insoluble precipitates in aqueous solution due to their low critical micelle concentration. Dissociation from the precipitate to release free drug molecules is a thermodynamically unfavorable process, requiring the disruption of strong intermolecular interactions (Figure 6A). However, when hydrophobic drug molecules are encapsulated into the hydrophobic cavity of L-PGDS, the complex can be regarded as an amphiphilic capsule drug, a state that is energetically stable in water (Figure 6B). For ∆∆G values between the insoluble aggregate and the soluble complex ranging from −10 to +5 kJ mol−1, L-PGDS improved the apparent solubility of poorly watersoluble drugs in our theoretical simulations (Figure 5). Indeed, in the presence of 1 mM L-


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PGDS, the apparent solubility of telmisartan and imatinib was limited around 1 mM (Figure 3C,E, Table 2), although L-PGDS could bind to two molecules of these drugs in the ITC studies (Figure 4A,B, Table 3). Note that the ITC measurements were performed in the soluble condition with DMSO where the aggregation equilibrium may be neglected. These seemingly contradictory results imply that the solubility of drugs with L-PGDS was determined by the energetic balances between aggregation of drugs and formation of drug/L-PGDS complex. Thus, the ∆∆G is a critical factor governing the solubilization of poorly water-soluble drugs by LPGDS (Figure 6). We previously reported that the ability of human L-PGDS to bind intravital hydrophobic molecules is tuned by enthalpy–entropy compensation,23 suggesting that hydrophilic and hydrophobic interactions contribute to complex formation. In agreement with this hypothesis, docking models suggested that the five drugs selected in this study form hydrogen bonds and hydrophobic interactions with L-PGDS (Figure S5). To estimate the drug binding sites in LPGDS, we performed frequency analyses of contacting residues for models of complexes between the top 100 drugs and L-PGDS (Figure S9). Six residues (Lys59, Tyr107, Ser109, Trp112, Tyr116, and Ser133) located around the EF-loop and in the H2-helix and G-strand formed hydrogen bonds with high frequency (>15%) (Figure S9A,B). By contrast, nine residues (Trp54, Leu55, Met94, Tyr107, Trp112, Tyr116, Val118, Phe143, and Met145) located around the EF-loop and in the H2-helix, D-strand, and H-strand were found to be involved in hydrophobic interactions with high frequency (>40%) (Figure S9C,D). The binding sites estimated by in silico analyses are generally consistent with those determined by NMR titration experiments of mouse L-PGDS and drug molecules.10 These results suggest that both hydrogen bonding and hydrophobic interactions in the common regions contribute to the formation of


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thermodynamically favorable complexes between L-PGDS and drug molecules. Furthermore, the partially flexible docking where side chains of the 12 residues identified above are treated as flexible may enable a more accurate prediction of drug–L-PGDS binding without the excessive computational costs.

CONCLUSION In this study, we demonstrated that L-PGDS tends to bind to hydrophobic drugs with planar and/or rod-like shapes and can enhance the solubility of a variety of poorly water-soluble drugs through the formation of stable drug/L-PGDS complexes. Structure-based docking of compounds from a large dataset enabled us to correctly select drugs that can be solubilized by LPGDS. Thus, use of the L-PGDS-based DDS with in silico docking could rescue many poorly water-soluble drug candidates dropped from pharmaceutical development.


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Figure 1. Structure-based docking of drug molecules to L-PGDS and HP-β-CD. We examined 2892 approved drugs in the Zdd subset of the ZINC database. (A–C) Histograms of L-PGDS∆Gv (A), HP-β-CD∆Gv (B), and the ratio L-PGDS∆Gv/HP-β-CD∆Gv (C). (D–I) Relationship between ∆Gv values and physico-chemical properties of the drug molecules. Dependence of molecular size on


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L-PGDS

∆Gv (D), HP-β-CD∆Gv (E), and ∆∆Gv (F). Dependence of hydrophobicity on L-PGDS∆Gv (G),

HP-β-CD

∆Gv (H), and ∆∆Gv (I). Data represent mean ± SE.


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Figure 2. Structures of selected drugs and drug/L-PGDS complex models. (A) Chemical structures of nilotinib, telmisartan, lapatinib, imatinib, and atovaquone. (B–G) Structural models of drug/L-PGDS complexes obtained by the docking simulations. (B) Overlapped docking poses of nilotinib (magenta), telmisartan (orange), lapatinib (yellow), imatinib (green), and atovaquone (blue). L-PGDS and the drugs are represented as ribbon and stick models, respectively. (C–G)


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Individual docking poses of nilotinib (C), telmisartan (D), lapatinib (E), imatinib (F), and atovaquone (G). L-PGDS is represented as a cutaway surface model. The helix and β-strand regions are shown in pink and light blue, respectively. These graphical poses were prepared using CueMol (http://www.cuemol.org/en/).


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Figure 3. Assessment of the solubility of poorly water-soluble drugs in the presence of L-PGDS. (A) Experimental scheme of the solubility measurements. (B–F) Reaction time–dependent changes in the solubility of nilotinib (B), telmisartan (C), lapatinib (D), imatinib (E), and atovaquone (F) in PBS (open circles), PBS with HP-β-CD (blue closed circles), and PBS with LPGDS (red closed circles). The concentrations of lapatinib and atovaquone in PBS were lower than the detection limits.


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Figure 4. Calorimetric analysis of the binding of L-PGDS and HP-β-CD to telmisartan and imatinib. (A and B) L-PGDS in the injection syringe was titrated into 59 µM telmisartan (A) and 51 µM imatinib (B) in the cell. (C and D) HP-β-CD in the injection syringe was titrated into 60 µM telmisartan (C) and 41 µM imatinib (D) in the cell. Thermograms and binding isotherms are shown in the upper and lower panels, respectively. Lines in the lower panels (A and B) represent theoretical curves.


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Figure 5. Kinetic simulations of drug dissolution in the presence of L-PGDS at 25°C. In the simulations, the values of free energy changes associated with insolubility (∆Ginsol) and binding (∆Gb) were set to −25 and −25 kJ mol−1 (A), −25 and −30 kJ mol−1 (B), −25 and −35 kJ mol−1 (C), −30 and −25 kJ mol−1 (D), −30 and −30 kJ mol−1 (E), −30 and −35 kJ mol−1 (F), −35 and −25 kJ mol−1 (G), −35 and −30 kJ mol−1 (H), and −35 and −35 kJ mol−1 (I). The concentrations of free drug (black dashed lines), drug/L-PGDS complex (solid blue lines), and apparent


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dissolved drug (solid red lines) were plotted against relative time. The values of ∆∆G (∆Gb − ∆Ginsol) and the apparent solubility of the drug in equilibrium are shown in each panel.


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Figure 6. Thermodynamic aspects of the dissolution of poorly water-soluble drugs in the presence of L-PGDS. (A) In an aqueous solution such as PBS, hydrophobic compounds are often in an insoluble and/or precipitate state with a lower free energy than the soluble state. (B) In the presence of L-PGDS, the equilibrium shifts toward the drug/L-PGDS complex state. This energetically favorable state, with ∆∆G values ranging from –10 to +5 kJ mol−1, results in an increase in the apparent solubility of the drug.


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Table 1. Physico-chemical properties of the selected drugs and their free energy of binding to L-PGDS and HP-β-CD Popular name (ZINC ID)

Mra

LogPb

L-PGDS

∆Gvc (kJ mol−1)

∆Gvd (kJ mol−1)

Nilotinib (ZINC06716957)

530

4.99

−46.0

−32.2

Chronic myeloid leukemia

Telmisartan (ZINC01530886)

514

7.60

−44.4

−31.4

Hypertension

Lapatinib (ZINC01550477)

582

6.16

−44.4

−30.1

Breast cancer

Imatinib (ZINC19632618)

495

3.89

−41.0

−29.7

Chronic myeloid leukemia

Atovaquone (ZINC12504271)

366

4.96

−40.2

−29.3

Pneumocystis jirovecii pneumonia

HP-β-CD

Medical indicationse

a

Relative molecular mass obtained from ZINC database. bHydrophobicity obtained from ZINC database. cValues of free energy of binding to L-PGDS for each drug predicted using AutoDock Vina. dValues of free energy of binding to HP-β-CD for each drug predicted using AutoDock Vina. eRepresentative medical indications for each drug.


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Table 2. Apparent dissociation constants for L-PGDS and HP-β-CD bound to the selected drugsa + PBS Drug

Solubility (µM)

+ HP-β-CD Solubility (µM)

Kd1:1 (µM)

+ L-PGDS Solubility (µM)

Kd1:2 (µM)

Kd1:3 (µM)

51.3

17.0

10.6

Nilotinib

2.7

6.6

8930

Telmisartan

7.4

65.1

1590

1230

N. D.b

5.9

8.4

Lapatinib

Comprehensive Evaluation of the Binding of Lipocalin-Type

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