http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, Early Online: 1–11 ! 2015 Informa UK Ltd. DOI: 10.3109/00498254.2015.1021403

RESEARCH ARTICLE

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Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins Andrew Rowland1, David Hallifax2, Matthew R. Nussio3, Joseph G. Shapter3, Peter I. Mackenzie1, J. Brian Houston2, Kathleen M. Knights1, and John O. Miners1 1

Department of Clinical Pharmacology, Flinders University, Adelaide, Australia, 2Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, UK and 3School of Chemical and Physical Sciences, Flinders University, Adelaide, Australia Abstract

Keywords

1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu50.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for b-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.

Drug distribution, drug–protein binding, surface plasmon resonance

Introduction The reversible binding of drugs to intra- and extra- cellular proteins is an important factor in determining drug disposition and response for many drugs, particularly those that are highly protein bound with low hepatic clearance. While the extent of binding to extra-cellular (plasma) proteins is well established for many drugs (Yamasaki et al., 2013), the kinetics (kon and koff) and affinity (KD) of binding are infrequently characterized. Similarly, the extent, affinity and kinetics of binding to intra-cellular proteins remain poorly characterized for most drugs. Given the potential importance of drug–protein interactions, both within plasma and in other compartments, a more thorough understanding of these interactions that considers both the relative extent and

Address for correspondence: Dr Andrew Rowland, Department of Clinical Pharmacology, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia. Tel: +61 8 8204 7546. E-mail: [email protected]

History Received 14 December 2014 Revised 16 February 2015 Accepted 17 February 2015 Published online 24 March 2015

time-course (kinetics) of drug binding to intra- and extracellular proteins is warranted. When considering extra-cellular drug binding proteins, human serum albumin (HSA) is the most abundant and is also the best characterized in terms of structure and function (Hamilton, 2004). Apart from functioning as the primary plasma transporter of unesterified fatty acids, HSA also binds a myriad of endogenous compounds and xenobiotics, particularly drugs with acidic or electronegative features. Like HSA, fatty acid binding proteins (FABPs), which are among the most abundantly expressed intra-cellular proteins, bind saturated and unsaturated long-chain fatty acids with high affinity (Chmurzynska, 2006; Veerkamp & Maatman, 1995; Zimmerman & Veerkamp, 2002). FABPs may also bind endogenous and exogenous lipophilic organic acids and their derivatives. For example, recent studies in this laboratory identified that human intestinal FABP (IFABP) has the capacity to bind acidic drugs (Rowland et al., 2009), albeit to a lesser extent than albumin. Similarly, human liver FABP (LFABP) binds numerous endogenous hydrophobic compounds, including eicosanoids, heme, fatty acyl CoA esters, retinol and bilirubin (Maatman et al., 1994;

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Takikawa & Kaplowitz, 1986; Veerkamp et al., 1999). Chuang et al. (2008) further demonstrated that rat LFABP binds a number of drugs including non-steroidal antiinflammatories, fibrates and benzodiazepines. The latter observation is consistent with X-ray crystallographic data which indicates that rat LFABP, unlike other proteins of this family, is able to simultaneously bind two molecules of fatty acid via high and low affinity binding sites (Thompson et al., 1997, 1999). Importantly, however, the capacity of human LFABP to bind drugs has not been investigated in a systematic manner, and the affinity and the kinetics of these potential interactions remain unknown. Recent reviews have demonstrated that under normal physiological conditions, the concentration ranges for HSA (in plasma) and LFABP (in hepatocytes) are comparable, at 3–5% and 1–5%, respectively (Furuhashi & Hotamisligil, 2008; Hamilton, 2004). Surface plasmon resonance (SPR) is a technology that facilitates the monitoring of drug–protein complex formation and dissociation in real-time, yielding both equilibrium (KD) and kinetic (kon and koff) data for the interaction. In recent years, SPR has been used to assess the kinetics of the interactions of several drugs with HSA (Frostell-Karlsson et al., 2000; Rich et al., 2001). However, this approach has not been utilized to assess interactions of drugs with other binding proteins, or to compare the kinetics of drug binding to these proteins. Here, for the first time, we compare both the extent and the kinetics of drug binding to HSA and LFABP, and examine the effect of binding to these proteins on compartmental drug distribution. Specifically, experiments were performed to (i) compare the extent of binding of 13 drugs (five acids, three bases, and five neutrals; Figure 1) to HSA and LFABP, (ii) assess the binding affinities and dissociation kinetics for three representative acidic drugs, naproxen (NAP), sulfinpyrazone (SFP) and torsemide (TOR), to each protein, and (iii) model these interactions within the framework of the currently accepted rapid equilibrium model to examine the effect on compartmental distribution of low fup drugs in a preliminary manner.

Materials and methods Materials 1-Anilino-8-naphthalene sulfonate (ANS), arachidonic acid (AA), caffeine (CAF), b-estradiol (b-EST), frusemide (FRU), human serum albumin (HAS, fatty acid free), lignocaine (LIG), S-naproxen (NAP), nortriptyline (NOR), phenytoin (PHY), propofol (PRO), propranolol (PRP), sulfinpyrazone (SFP) and zidovudine (AZT) were purchased from SigmaAldrich (Sydney, Australia). Diazepam (DIA) and torsemide (TOR) were obtained from Hoffmann-La Roche (Basel, Switzerland) and Boehringer Mannheim (Mannheim, Germany). Homo sapien fatty acid binding protein 1 (liver) TrueClone cDNA (reference sequence NM_0001443.1) was purchased from Origene (Rockville, MD) and the tetra HIS HPR-conjugate kit from Qiagen (Melbourne, Australia). Solvents and other reagents were of analytical reagent grade unless otherwise specified. SPR buffers and reagents were supplied with research-grade CM5 sensor chips and were of analytical reagent grade or higher (BIACore AB, Uppsala, Sweden).

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Expression of recombinant LFABP cDNA encoding human LFABP was PCR amplified from human LFABP Trueclone cDNA using the method descried previously for the expression of purified histidine tagged IFABP (Rowland et al., 2009). Purification of the LFABP protein by immobilized nickel affinity chromatography was facilitated by the addition of six histidine residues to the C-terminus of wild-type LFABP cDNA using the following sets of PCR primers: LFABP 50 -primer 50 -ATTAGGATCCTTCATGGCGTTTGACAGCACTTGG-30 . LFABP 30 -primer 50 -ATTAAAGCTTGGTTAGTGATGGTGATGGTGATGAA TTCTCTTGCTGATTTCTCTTGAAG-30 . Briefly, the LFABP insert was ligated into the pCWori(+) bacterial expression plasmid and transformed into DH5a Escherichia coli. Plasmid DNA was purified and the nucleotide sequence was confirmed. Overnight sub-cultures were used to inoculate 400 mL cultures, which were grown at 37  C with shaking for approximately 3 h, at which time, the temperature was reduced to 30  C and isopropyl-b-D-thiogalactopyranoside (IPTG; 1 mM) was added. Cultures were grown at 30  C with shaking for an additional 40 h. Cells were then harvested using a French press. LFABP was recovered in the soluble fraction after centrifugation at 45 000 g for 90 min and was purified by chromatography on a pre-equilibrated Ni2+ affinity resin column (Ni-NTA agarose). The concentration of purified LFABP protein was determined according to Lowry et al. (1951). Immunoblotting of LFABP Immunoblotting was performed to demonstrate the expression of LFABP using the protocol previously described for the detection of His-tagged IFABP (Rowland et al., 2009). Denatured purified protein (1 mg) was separated by SDSPAGE and then transferred onto nitrocellulose membranes. Membranes were washed in TBS, blocked in 1% (w/v) blocking reagent (Qiagen, Melbourne, Australia) then washed again in TBS, and probed with a commercial anti-His antibody (Tetra-HIS HPR-conjugate; 1:1000 dilution). BM chemiluminescence blotting substrate was used for immunodetection. The membranes were exposed to Kodak X-Omat films (Sigma-Aldrich, St. Louis, MO) and films were processed manually. Measurement of ANS binding and competitive displacement by arachidonic acid Activity of expressed LFABP was confirmed by measurement of ANS binding and displacement of ANS by arachidonic acid (0.2–1.6 mM). The binding of ANS to LFABP and the displacement of this compound by arachidonic acid was quantified by fluorescence measurement as described previously (Rowland et al., 2009). Measurement of drug binding to LFABP and HSA by ultrafiltration The binding of compounds to LFABP and HSA was measured by ultrafiltration using Microcon centrifugal filter devices

Intra- and extra-cellular drug–protein binding

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DOI: 10.3109/00498254.2015.1021403

Figure 1. Chemical structures of compounds used in LFABP and HSA binding studies.

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Table 1. Substrate and chromatography conditions for the measurement of drug binding by ultra-filtration.

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% Mobile phase Drug

Substrate concentrations

A

B

Detector wavelength (nm)

Analyte retention time (min)

Caffeine (CAF) Diazepam (DIA) b-Estradiol (b-EST) Frusemide (FRU) Lignocaine (LIG) s-Naproxen (NAP) Nortriptyline (NOR) Phenytoin (PHY) Propofol (PRO) Propranolol (PRP) Sulfinpyrazone (SFP) Torsemide (TOR) Zidovudine (AZT)

10, 50, 100, and 250 mM 50, 100, 200, and 1000 mM 5, 25, and 100 mM 100, 1000, and 5000 mM 100, 300, and 1000 mM 10, 100, and 500 mM 0.2, 1, 2.5, and 100 mM 5, 20, and 100 mM 25, 125, and 500 mM 5, 25, and 100 mM 10, 50, and 100 mM 5, 25, and 100 mM 100, 500, 1000, and 3000 mM

95 50 60 65 80 50 45 60 25 65 50 65 65

5 50 40 35 20 50 55 40 75 35 50 35 35

270 290 280 254 205 254 240 214 214 214 254 290 267

2.8 2.4 3.4 2.3 2.8 2.1 2.3 2.8 3.6 2.6 2.0 2.9 2.6

(Millipore, Bedford, MA; Rowland et al., 2009). Briefly, incubation samples contained the compound of interest (Table 1 and Figure 1), phosphate buffer (0.1 M, pH 7.4) and protein (LFABP or HSA; 0.5% w/v). Samples were incubated at 37  C for 120 min, after which time, a 100 mL aliquot was added to the reservoir compartment of the filter device and centrifuged at 14 000 g for 2 min. Under these conditions, less than 15% of the sample passed from the reservoir compartment to the filtrate compartment. Aliquots (10 mL) were collected from the reservoir and filtrate compartments and protein was precipitated by the addition of ice cold methanol containing 4% acetic acid (20 mL). Samples were cooled on ice, centrifuged at 4000g for 10 min at 4  C, and aliquots of the supernatant fraction (5 mL) were analyzed by HPLC. Substrate concentrations investigated are shown in Table 1.

rate of 20 mL/min at 25  C. HSA or LFABP (50 mg/mL) in 10 mM sodium acetate buffer (pH 5.2 for HSA or pH 4.5 for LFABP; pH optimized for ligand association with the chip) was then injected over the surface at a flow rate of 20 mL/min for 7 min. The surface was blocked with a 7 min injection of 1 M ethanolamine (pH 8.5) at a flow rate of 10 mL/min, followed by three 30 s pulses of 50 mM sodium hydroxide to remove all non-covalently bound protein and to stabilize the baseline. This protocol resulted in final immobilization densities of between 8000 and 10 000 RU (resonance units) for each protein. The same protocol (without the injection of protein onto the surface) was used to prepare the reference surface (typically Fc1). To ensure the highest level of sensitivity, the SPR detector response was normalized prior to kinetic experiments by calibrating the detector at various light intensities under conditions of total internal reflection (an automated process).

Quantification of drug binding to LFABP and HSA Samples from the reservoir and filtrate compartments were collected as described above. The compound of interest was separated from the incubation matrix on a Waters NovaPak C18 analytical column (3.9  150 mm2, 4 mm, Waters, Sydney, Australia) using a mobile phase comprising 10 mM triethylamine (pH adjusted to 2.5 with perchloric acid; mobile phase A) and acetonitrile (mobile phase B), at a flow rate of 1 mL/min (see Table 1 for mobile phase proportions). Column eluent was monitored at the optimal wavelength (determined by spectroscopic analysis) for each compound (Table 1). The substrate concentration in ultrafiltration samples was determined by comparison of peak areas to those of authentic standards using calibration curves that spanned the concentration ranges employed in binding studies. Surface plasma resonance SPR analyses were performed using a BIACore 2000 optical biosensor equipped with research-grade CM5 sensor chips (BIACore AB, Uppsala, Sweden). Ligand surfaces were prepared by immobilising HSA or LFABP onto a gold dextran surface using amine-coupling chemistry. The ligand surface (typically flow cell (Fc) 2) was activated for 7 min using 50 mM N-hydroxysuccinimide (NHS) and 0.2 M 3-(N,Ndimethylamino)propyl-N-ethylcarbodiimide (EDC) at a flow

Measurement of NAP, SFP and TOR binding to ligand surfaces by SPR Analytes (i.e., NAP, SFP and TOR; 0–200 mM; in 67 mM isotonic phosphate buffer pH 7.4 (plus 1% v/v ethanol for SFP) were injected over the reference and ligand surfaces (Fc1 and Fc2, respectively) for 1 min at a flow rate of 30 mL/min. Each injection consisted of a 1 min wait period, injection of analyte over 1 min, a 5 min undisturbed dissociation phase, followed by a 1 min injection of running buffer (to ensure no carry-over of drug in the integrated fluidics cartridge (IFC)). Data obtained from the reference surface were subtracted from results obtained from the ligand surface. Responses from injections of drugs were recorded over 70 time points between 10 s prior to the start of injection and 1 min after the injection. These responses were used to determine both the affinity (Kd) and the dissociation kinetic (koff) parameters for analyte binding to each ligand. Kd values were determined by fitting a multiple analytebinding mode equation to experimental data obtained during the injection phase of SPR analysis: R¼

n X Rmax n  ½S

Kdn þ ½S

ð1Þ

Intra- and extra-cellular drug–protein binding

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where R is the corrected response, Rmaxn is the maximum response attributable to the binding of analyte to the ligand in the nth binding mode, Kdn is the dissociation constant for the nth binding mode, [S] is the total analyte concentration, and n is the number of distinct binding modes (n ¼ 2 or 3). koff values were determined by fitting experimental data obtained during the post injection phase of SPR analysis to a Langmurian dissociation model using BiaEvaluation 3.2 RC1 software (BIACore AB, Uppsala, Sweden). Figure 2. Scheme of dynamic model for passive uptake and distribution in the suspended hepatocyte involving extra- and intracellular protein binding.

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ANS binding and competitive displacement data analyses The stoichiometry of ANS binding to LFABP was determined by Scatchard ([S]bound/[P] versus [S]bound/[S]total) analysis. Kd values for ANS and arachidonic acid binding to LFABP were determined using a single binding mode equation: DF ¼

C  ½S Kd þ ½S

ð2Þ

where DF is the absolute percent change in fluorescence upon addition of ANS (or competitor), C is the capacity, Kd is the dissociation constant, and [S] is the concentration of added substrate. Dynamic model of hepatocyte drug distribution The impact of intracellular binding of drugs such as NAP, SFP, and TOR to LFABP on their uptake kinetics was demonstrated with a dynamic cell distribution model that included a medium containing albumin (at plasma concentration). Although it is generally assumed that unbound drug within hepatocytes is in rapid equilibrium with external unbound drug with regard to diffusion and dissociation from albumin, passive uptake might be rate limited by the kinetics of binding to LFABP and hence equilibration of intracellular unbound drug might be dependent on this. A scheme illustrating the model is shown in Figure 2. In contrast to the model reported by Baker & Parton (2007), the model here includes intracellular binding (in addition to extracellular binding) and excludes elimination processes. The equations describing the concentrations of unbound and bound drug in the external medium (3 and 4) and the unbound and bound drug concentrations within the hepatocyte (5 and 6) are as follows: dCmu  Vm ¼ Vc  ðkdiff  Ccu Þ  Vm  ðkdiff  Cmu Þ dt þ Vm  ½ðCmb  koff, m Þ  ðCmu  kon, m  Pm Þ ð3Þ dCmb ¼ ðCmu  kon, m  Pm Þ  ðCmb  koff, m Þ dt dCcu  Vc ¼ Vm  ðkdiff  Cmu Þ  Vc  ðkdiff  Ccu Þ dt þ Vc  ½ðCcb  koff, c Þ  ðCcu  kon, c  Pc Þ dCcb ¼ ðCcu  kon, c  Pc Þ  ðCcb  koff, c Þ dt

ð4Þ

ð5Þ

rate constant; Ccu is the concentration of unbound drug in hepatocyte; Cmu is the concentration of unbound drug in medium; Cmb is the concentration of bound drug in medium; kon,m is the medium protein binding association rate constant; Pm is the concentration of medium protein; dCmb/dt is the rate of change of bound drug in medium; koff,m is the medium protein binding dissociation rate constant; dCcu/dt is the rate of change of unbound drug concentration in hepatocyte; Vc is the volume of hepatocyte; Ccb is the concentration of bound drug in hepatocyte; koff,c is the hepatocyte protein binding dissociation rate constant; kon,c is the hepatocyte proteinbinding association rate constant; Pc is the cell protein concentration; and dCcb/dt is the rate of change of bound drug in hepatocyte. The equations were compiled in ModelMaker 4.0 (ModelKinetix, Beaconsfield, UK). The behavior of the separate components (e.g., the individual binding compartments) were tested by simulation to accord with expected outcomes regarding koff, kon, P and fu. The volume of the hepatocyte compartment was 0.5% of the total volume. Simulations were started with an instantaneous (bolus) dose of pre-equilibrated (according to predicted fu in the medium) bound/unbound drug to the medium. koff values used were chosen to include the observed range in this study: scenario A: 1 s1 (HSA – medium) and 1 s1 (LFABP – cell); scenario B: 0.2 s1 (HSA – medium) and 0.6 s1 (LFABP – cell). kon values were calculated from observed Kd (kon ¼ koff/Kd). Protein concentration (Pm, Pc) values were set to give fu values expected at 5% protein (both HSA and LFABP). The concentration–time profile outputs for unbound and total drug both in the medium and within in the hepatocyte were recorded. Recent reviews have demonstrated that under normal conditions, the physiological concentration ranges for HSA (in plasma) and LFABP (in hepatocytes) are 3–5% and 1–5% (Hamilton, 2004; Furuhashi & Hotamisligil, 2008). As such, in order to account for the maximal possible effect of each of these binding proteins, simulations were performed at a protein concentration of 5% for both proteins. The diffusion rate (kdiff) was set to be non-rate limiting at 10-fold greater than the koff for HSA.

Results ð6Þ

where dCmu/dt is the rate of change of unbound drug in medium; Vm is the volume of medium; kdiff is the diffusion

LFABP expression and binding of ANS and arachidonic acid The expression of LFABP was confirmed by immunoblotting of the purified protein and by activity measurement. A band

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corresponding to the molecular mass of His-tagged LFABP (approximately 16 kDa) was detected by chemiluminescence following immunoblotting with a tetra-His antibody (data not shown). No other bands were observed. Coomassie staining of the SDS-polyacrylamide gel prior to transfer of the protein onto nitrocellulose was also consistent with the presence of a single protein with an approximate molecular mass of 16 kDa. Scatchard analysis of ANS binding to LFABP (x-axis intercept 2.9; data not shown) indicated that each LFABP has the capacity to simultaneously bind three molecules of ANS. Despite the simultaneous binding of three molecules, the kinetics of ANS binding to LFABP was well described by a single binding mode equation, with a Kd value of 2.8 mM. This value is in agreement with a previous report for the binding of ANS to unmodified human LFABP (namely 3.4 mM; Norris & Spector, 2002). Addition of arachidonic acid to samples containing LFABP and ANS resulted in a decrease in ANS fluorescence, consistent with displacement of ANS by arachidonic acid. The displacement of ANS from LFABP by arachidonic acid was well described by a single binding

mode equation, which provided a Kd value for arachidonic acid binding to LFABP of 138 nM. This value is similar to the previously reported Kd value for binding of arachidonic acid to unmodified human LFABP (namely 105–120 nM; Norris & Spector, 2002). Comparative binding of drugs to LFABP and HSA Test compounds (Figure 1) were classified as acids (FRU, NAP, PHY, SFP, and TOR), bases (LIG, NOR, and PRP), or neutrals (CAF, DIA, b-EST, PRO, and AZT) on the basis of charge state at pH 7.4 (Rowland et al., 2009). Fraction unbound (fu) was independent of substrate concentration, but varied between compounds and, to a lesser extent, between proteins. FRU, NAP, SFP, and TOR bound extensively to HSA, while only TOR bound extensively to LFABP; fu values ranged from 0.01 to 0.16 (Table 2). PHY binding to both proteins and FRU, NAP, and SFP binding to LFABP was moderate with fu values ranging from 0.34 to 0.65 (Table 2). Binding of the basic compounds LIG and PRP to both

Table 2. Unbound fractions (fu) for the binding of drugs to LFABP and HSA (0.5%). fu in the presence of Drug

Classification

Frusemide (FRU)

Acid

s-Naproxen (NAP)

Acid

Phenytoin (PHY)

Acid

Sulfinpyrazone (SFP)

Acid

Torsemide (TOR)

Acid

Lignocaine (LIG)

Base

Nortriptyline (NOR)

Base

Propranolol (PRP)

Base

Caffeine (CAF)

Neutral

Diazepam (DIA)

Neutral

b-Estradiol (b-EST)

Neutral

Propofol (PRO)

Neutral

Zidovudine (AZT)

Neutral

Drug concentration (mM)

LFABP

HSA

100 1000 5000 10 100 500 5 20 100 10 50 100 5 25 100 100 300 1000 0.2 1 100 5 25 100 10 50 100 250 50 100 200 1000 5 25 100 25 100 500 100 1000 3000

0.61 0.62 0.63 0.57 0.64 0.61 0.65 0.62 0.64 0.34 0.34 0.37 0.12 0.09 0.16 0.88 0.92 0.90 0.64 0.68 0.65 0.81 0.80 0.83 0.81 0.83 0.83 0.82 0.55 0.50 0.45 0.54 0.12 0.11 0.18 0.94 0.94 0.97 0.98 0.90 0.99

0.09 0.12 0.10 0.08 0.11 0.11 0.55 0.50 0.54 0.02 0.03 0.04 0.02 0.01 0.04 0.86 0.90 0.91 0.46 0.57 0.49 0.78 0.79 0.82 0.80 0.83 0.83 0.82 0.34 0.32 0.23 0.37 0.04 0.04 0.03 0.46 0.53 0.50 1.00 0.93 1.00

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proteins was minor (520%). However, NOR bound appreciably to both proteins; mean fu values were 0.66 (LFABP) and 0.51 (HSA). As observed for the acidic compounds, binding of NOR to each protein was independent of substrate concentration. Differing patterns of binding were observed for the neutral compounds: b-EST bound extensively to both proteins, with fu values 50.2. DIA binding was moderate; mean fu values for LFABP and HSA were 0.51 and 0.32, respectively. CAF binding to both proteins was minor (approximately 20%), and AZT binding to both proteins was negligible (55%). While PRO bound appreciably to HSA (mean fu 0.5), binding to LFABP was negligible (55%).

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Characterization of drug protein binding by SPR The acidic drugs NAP, SFP, and TOR (‘analytes’) were selected for further analysis by SPR on the basis of the differential fu values observed for binding to LFABP (0.61– 0.12), and consistently low fu values observed for binding to HSA (50.1). Analytes spanning concentration ranges that allowed meaningful interpretation of affinity data (typically 0.1–200 mM) were injected over HSA (ligand), LFABP (ligand), and non-derivatized (reference) surfaces. At all concentrations, the responses reached equilibrium rapidly and each analyte fully dissociated from the respective surfaces within 25 s. Each injection of analyte onto the ligand surface generated equilibrium binding data that were clearly discernible above background noise. The lowest NAP, SFP, and TOR concentrations measured (0.1 mM) yielded responses of 0.5, 1.3, and 1.7 RU, respectively. No binding was detected for any analyte injected over a reference surface, response values were invariably 50.2 RU across the analyte concentration ranges analyzed. A sample SPR sensogram demonstrating TOR binding to HSA is shown in Figure 3.

Intra- and extra-cellular drug–protein binding

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In all cases, the binding of analyte to the ligand surface exhibited atypical binding characteristics, with clearly nonlinear binding plots (Figure 4). Accordingly, on the basis of statistical robustness of fit, the affinities of NAP, SFP, and TOR binding to HSA (Figure 4, Panel A) and LFABP (Figure 4, panel B) were quantified by fitting the two binding mode equation to experimental data, except for NAP and TOR binding to HSA, where a three binding mode equation was used. Binding affinities (Kd) for analyte binding to the respective ligands are shown in Table 3. The data for analyte binding to HSA are consistent with previous results obtained using SPR, which demonstrated that warfarin binds to two sites on HSA with binding affinities of 3.8 and 273 mM (Rich et al., 2001). The dissociation kinetics of the interactions of NAP, SFP, and TOR with HSA and with LFABP were assessed at four analyte concentrations spanning the Kd for the lower affinity component of the interaction. Mean (±SD) koff values for NAP, SFP, and TOR binding to HSA were 0.97 ± 0.19, 0.50 ± 0.02, and 0.22 ± 0.06 s1, respectively, while the mean (±SD) koff values for NAP, SFP, and TOR binding to LFABP were 1.15 ± 0.20, 1.12 ± 0.11, and 0.56 ± 0.01 s1, respectively. The koff values reported here for the binding of NAP, SFP, and TOR to HSA are comparable in magnitude to values reported for the binding of other drugs (e.g., warfarin) to HSA, both by SPR (Rich et al., 2001) and other analytical techniques (Fehske et al., 1982; Hage et al., 1995). The dissociation kinetics of the interaction of SFP with HSA and LFABP were assessed at both 4 and 25  C. At 4  C, the bound SFP dissociated more slowly from both HSA and LFABP. Mean (±SD) koff values for SFP binding to HSA and LFABP at 4  C were 0.36 ± 0.02 and 0.80 ± 0.09 s1, respectively. The difference in dissociation rate was statistically significant (p ¼ 0.001; paired t-test). Dynamic model of hepatocyte drug distribution

Figure 3. SPR sensorgrams obtained from duplicate injections of torsemide (TOR; 0.1–200 mM) over a surface of 9.5 kRU immobilized HSA.

Simulations using a dynamic model of hepatocyte distribution were undertaken to determine the time course of total and unbound drug concentrations, both internal and external to the hepatocyte for two kinetic scenarios representing the extremes of koff and kon ratios between the extracellular protein (HSA) and the intracellular protein (LFABP) observed for the three drugs studied by SPR. From the simulated concentration–time profiles obtained, it can be seen that application of a koff of 1 s1 for both proteins (as observed for NAP) with a range of different kon values (17-fold) between the proteins (scenario A) results in a very rapid equilibration of the intracellular unbound drug concentration with the extra-cellular unbound drug concentration (Figure 5A). The equilibration of intra-cellular total drug was measurably slower but still rapid in terms of typical in vitro timescales. Applying a lower koff of 0.2 s1 for HSA with a 3-fold greater koff for LFABP (such as observed for TOR) together with a 2-fold difference in kon (scenario B) also resulted in a very rapid equilibration of intra-cellular unbound drug, despite the greater extent of intra-cellular binding and equilibration time of intra-cellular total drug (Figure 5B). In both scenarios (Figure 5), the intracellular binding rate was intentionally not rate-limited by the applied diffusion rate across the hepatocyte membrane,

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Figure 4. Eadie–Hofstee plots for drug binding to LFABP and HSA. Points show experimentally determined values, whereas curves are from model fitting. Panels 1(A) and (B) show binding of naproxen (NAP) to HSA and LFABP, respectively; Panels 2(A) and (B) show binding of sulfinpyrazone (SFP) to HSA and LFABP, respectively; and Panels 3(A) and (B) show binding of torsemide (TOR) to HSA and LFABP, respectively.

Intra- and extra-cellular drug–protein binding

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Table 3. Affinity constants (Kd) for the binding of drugs to HSA and LFABP. Ligand

Analyte

HSA

s-Naproxen Sulfinpyrazone Torsemide s-Naproxen Sulfinpyrazone Torsemide

LFABP

Kd1

Kd2

Kd3

0.597 1.14 0.084 0.064 0.105 0.227

10.4 129 6.36 2.83 8.18 12.3

477 – 254 – – –

Units: Kd, micromolar (mM).

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for HSA. When equilibrium was reached, the fraction unbound in the extra-cellular medium (where HSA was set at the approximate maximum physiological concentration, 5%) was approximately 0.01 and 0.002 for scenarios A and B, respectively, consistent with the extensive plasma binding observed for these acidic drugs. Within the hepatocyte, the fraction unbound was approximately 0.14 and 0.01 for scenarios A and B, respectively. The unbound fractions within the hepatocyte and the extra-cellular medium were confirmed by conducting simulations with each compartment individually, which served to verify the performance of the model.

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Discussion

Figure 5. Simulated concentration–time profiles for hypothetical examples of drugs bound to HSA (in medium) and LFABP (within hepatocytes) with either (A) the same koff values of 1 S1 (different kon values) or (B) koff values of 0.2 and 0.6 S1 for HSA and LFABP, respectively (different kon values). Representative lines are (- — -) total drug in the medium; (——) total drug in the cell; (- - - -) unbound drug in the cell; (. . .. . .) unbound drug in the medium (unbound drug in cell and medium are coincident).

although it is not known if any of these drugs would have such a permeability limitation. These simulated kinetic time profiles showed that the time to reach equilibration was dependent on LFABP kon values although, for the drugs studied here, these values were of a similar range to values

Until recently the in vitro characterization of interactions of drugs with binding proteins has been limited to assessment of binding affinity via determination of an equilibrium dissociation constant (Kd), because methods for assessing such interactions (e.g., 1-anilino-8-naphthalene sulfonate (ANS) displacement, ultrafiltration, and equilibrium dialysis) are unable to elucidate real-time information as the analytical determination occurs only once equilibrium has been attained (Wright et al., 1996). Indeed, this study is the first to compare the extent (fu), affinity (Kd), and kinetics (koff) of drug binding to both an extra-cellular binding protein, HSA, and an intracellular binding protein, LFABP. Screening experiments were undertaken to assess the comparative extent of drug binding (as fu) to LFABP and HSA (0.5%) at three or four substrate concentrations (always including 100 mM) that spanned the known Km for the principal route of metabolism of each compound. These experiments were undertaken with a panel of 13 drugs (six acids, three bases, and four neutrals) from a variety of therapeutic classes. With the exception of NOR, which bound moderately to both LFABP and HSA, binding of the basic drugs (LIG and PRP) to both proteins was minor (520%). Of the four neutral compounds, DIA (mean fu 0.51) and b-EST (mean fu 0.14) bound appreciably to LFABP. With the exception of CAF, for which binding to both proteins was minor (20%), binding of the neutral compounds to HSA was more extensive; DIA (mean fu 0.32), PRO (mean fu 0.49), and b-EST (mean fu 0.04) all bound appreciably. Binding of AZT to both proteins was negligible (55%). By contrast, binding of all acidic drugs to both LFABP and HSA was appreciable. As with the basic and neutral drugs, the extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP (Table 2). These data confirm that HSA plays an important role in the extracellular transport of many acidic and polar neutral compounds, with only a minor role in the transport of basic compounds. Similarly, the more extensive binding of acidic compounds to LFABP (compared with neutral and basic compounds) is consistent with this protein playing a more important role in the intracellular transport of acidic compounds (compared with basic and neutral compounds). The capacity of LFABP (and other FABPs) to bind drugs raises the important consideration of accounting for intracellular protein binding when undertaking in vitro drug metabolism studies with intact hepatocytes as the enzyme source. As this in vitro system does not typically include

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10

A. Rowland et al.

an extracellular protein source (such as albumin), protein binding is rarely considered. However, data presented here and in previous reports from this laboratory (Rowland et al., 2009) demonstrate that FABPs present in the hepatocyte may reduce the unbound concentration of drug available for metabolism, thereby resulting in an underestimation of enzyme binding affinity (overestimation of Km), and intrinsic clearance. Given the limited binding of basic (and most neutral) compounds to either HSA or LFABP, it is unlikely that binding would have significant implications for the determination of intracellular concentration of biologically active ‘‘free’’ compound. However, it is possible that the relative interactions of basic and neutral compounds with alternate extra- and intra- cellular binding proteins (such as a-globulins) may affect the intracellular ‘‘free’’ concentration of these compounds. As such, while interactions of basic and neutral compounds with HSA and LFABP were not selected for further analysis in the current study, investigation of the binding to alternate extra- and intra-cellular binding proteins is warranted. On the basis of the screening data, the affinity (Kd) and dissociation kinetics (koff) of NAP, SFP, and TOR binding to HSA and LFABP were assessed by SPR. Based on the fu values observed in the screening experiments, NAP binds ‘‘moderately’’ (35%) to LFABP, SFP binds ‘‘appreciably’’ (65%) to LFABP, and TOR binds ‘‘extensively’’ (>90%) to LFABP, whereas all three compounds bind ‘‘extensively’’ to HSA. Experimental data for NAP and TOR binding to HSA were best described by a three binding mode equation, while experimental data for SFP binding to HSA and the binding of all three drugs to LFABP were best described by a two binding mode equation. Despite the comparatively lower fu values observed for all three compounds in the presence of HSA compared with LFABP, the binding affinities for the interactions of the three compounds with the two proteins were generally comparable. Indeed, the interaction with the highest affinity (lowest Kd) was NAP binding to LFABP, which was also the interaction that exhibited the lowest extent of binding (highest fu). The relative order of binding affinities for the interaction of the three compounds with the two proteins was NAP (LFABP) > TOR (HSA) > SFP (LFABP) > TOR (LFABP) > NAP (HSA) > SFP (HSA). The observation of two modes for the binding of compounds to LFABP is consistent with X-ray crystallographic data, which demonstrates two distinct oleic acid binding sites (Thompson et al., 1997, 1999). In addition, a previous study demonstrated that rat LFABP has the capacity to bind a variety of drugs with differing affinities (Chuang et al., 2008). The ‘‘tightest’’ binding to rat LFABP was generally reported for compounds possessing a carboxylic acid group, although binding affinities varied by more than an order of magnitude. Due to limitations of instrument sensitivity and relatively slow sampling rate, it was not possible to quantify the dissociation rate (koff) experimentally at low substrate concentrations (very high-affinity component). However, the approach employed in the current study did facilitate quantification of koff value for the interactions of NAP, SFP, and TOR with HSA and LFABP at substrate concentrations corresponding to the low-affinity binding mode (kd2). As the

Xenobiotica, Early Online: 1–11

analyte concentrations associated with the kd2 binding mode most closely resemble the observed plasma concentrations for these drugs, this assessment was considered appropriate for modeling and the most relevant in terms of in vivo interactions. Mean koff values for the dissociation of compounds to both proteins were in the order of 0.2–1 s1. As differences in kon/koff values are only considered significant on a logarithmic scale, these data indicate similar dissociation kinetics for the two proteins. Again, no correlation was observed between the kinetics of dissociation (koff) and the extent of binding. Taken together, these data indicate that while HSA exhibits a greater capacity to bind (particularly acidic) compounds compared with LFABP, this is likely due to a greater number of potential binding sites on this protein, and hence a greater total binding capacity. Significantly, while the extent of binding of compounds to HSA is greater, the affinity and dissociation kinetics of the two proteins are similar, thus facilitating rapid equilibrium as described by the model of Berezhkovskiy (2004). The implication that the comparable kinetics for the binding of NAP, SFP, and TOR with HSA and LFABP would sustain rapid and hence effective equilibration between the hepatic cytosol and the extracellular medium was demonstrated dynamically using a hepatocyte simulation model. Simulations, which involved drug introduced into the extracellular medium pre-bound to the extracellular protein (HSA), showed rapid equilibration of unbound drug, with little change in unbound concentration, between the extracellular medium and the intracellular compartment and non-rate limiting trans-cellular diffusion. Although the koff values observed in this study were of a similar magnitude for both HSA and LFABP, the equilibration time for uptake of total drug was controlled by the association to LFABP but intracellular binding to LFABP was nevertheless shown to be rapid. In the absence of transport mediated uptake, the measurement of drug clearance by hepatocytes in vitro might reasonably be assumed to depend simply on the concentration of unbound drug in the incubation medium. These finding reenforce the need to experimentally assess unbound drug concentrations.

Conclusions HSA and LFABP have the capacity to bind acidic and polar neutral compounds. The extent of binding of moderate to strong acids and the polar neutral compound b-estradiol to HSA was higher than the binding to LFABP. SPR experiments demonstrated similar binding affinities and kinetics for the three representative acidic drugs NPA, SFP, and TOR. Mechanistic simulations of compartmental distribution, based on in vitro kinetics constants from SPR experiments, demonstrated rapid equilibration of unbound drug between the extra- and intra-cellular compartments. Hence, the unbound concentration of acidic drugs in hepatocytes is likely to be in equilibrium with the unbound plasma concentration.

Acknowledgements Technical assistance from Mr DJ Elliot and Dr BC Lewis is gratefully acknowledged.

DOI: 10.3109/00498254.2015.1021403

Declaration of interest The authors report that they have no conflicts of interest. This work was funded, in part, by a project grant from the National Health and Medical Research Council of Australia [Grant ID 595920].

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Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) us...
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