Title Page Transport-metabolism interplay of atazanavir in...

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Title Page

Transport-metabolism interplay of atazanavir in rat hepatocytes

Johan Nicolaï, Tom De Bruyn, Louise Thevelin, Patrick Augustijns, Pieter Annaert

Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological

Sciences, O&N2, Herestraat 49 - box 921, 3000 Leuven, Belgium. (J.N., T.D.B., L.T., P.A., P.A.)

This study was partially supported by the Agency for Innovation by Science and Technology [IWT, Flanders, Belgium, project number 111193]; by the Scientific Research Network of the Research Foundation [FWO, Flanders, Belgium, grant number G.0662.09N] and by internal funds of the Lab for Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on December 28, 2015 as DOI: 10.1124/dmd.115.068114

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Running Title Page

Running Title: Transport-metabolism interplay of atazanavir

Address correspondence to:

Pieter P. Annaert

Drug Delivery and Disposition,

O&N2, Herestraat 49 - box 921

B-3000 Leuven, Belgium

E-mail address: [email protected]

Tel: +32 16 3 30303 or +32 16 3 30300

Fax: +32 16 3 30305

Number of text pages: 39

Number of tables: 4

Number of figures: 9

Number of references: 28

Number of words in the Abstract: 165

Number of words in the Introduction: 512

Number of words in the Discussion: 1160

Abbreviations: ATV, atazanavir; ABT, 1-aminobenzotriazole; HPGL, hepatocytes per gram

liver; IDV, indinavir; IPRL isolated perfused rat liver; Kpu,u, ratio of the intracellular to

extracellular unbound concentration; LOS, losartan; MPPGL, microsomal protein per gram liver;

MPPMC, microsomal protein per million cells; RLM, rat liver microsomes; SF, Scaling factors;

SRH, suspended rat hepatocytes


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Abstract

The aim of this study was to explore the mechanisms governing the intra- to extracellular

unbound concentration ratio (Kpu,u) for the HIV protease inhibitor atazanavir (ATV) in rat

hepatocytes. We previously proposed a new method to determine Kpu,u by using the unbound

Km values from metabolism studies with suspended rat hepatocytes and rat liver microsomes.

Based on this method, ATV Kpu,u valued 0.32, indicating that ATV hepatocellular clearance is

uptake rate-limited. This hypothesis was supported by the linear correlation between Kpu,u and

active uptake clearance (p = 0.04; R2=0.82) in the presence of increasing concentrations of the

uptake transport inhibitor losartan. Moreover, in contrast to an expected increase of Kpu,u upon

inhibition of ATV metabolism, a decrease of Kpu,u was observed, suggesting an increased impact

of sinusoidal efflux. In summary, involvement of active uptake transport does not guarantee high

intracellular accumulation; however it has a key role in regulating intracellular drug

concentrations and drug metabolism. These findings will help improve future IVIVE and likewise

PBPK-models.


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Introduction

Liver microsomes have been an established in vitro model to determine P450-mediated drug

elimination for over 50 years. (Rane et al., 1977) Currently, this relatively simple in vitro

technique still stands as an invaluable preclinical high-throughput tool in drug discovery settings.

However, absence of a plasma membrane with drug transporting proteins (e.g. SLC-transporters,

ABC-transporters) can cause liver microsomal-based clearance predictions to deviate from the in

vivo situation. (Lam and Benet, 2004; Lu et al., 2006) Apart from drug transporters (uptake and

efflux), intracellular metabolism and intracellular binding can also cause intracellular

concentrations to differ greatly from medium concentrations. (Parker and Houston, 2008) To

overcome this problem, suspended hepatocytes which possess a cell membrane with drug

transporting proteins as well as both phase I and phase II metabolizing enzymes, are becoming a

more preferred in vitro drug metabolism tool. (Di et al., 2012) In suspended hepatocytes, the

exposure of intracellular enzymes to unbound drug concentrations will more closely resemble in

vivo conditions. However, when drug elimination data from these in vitro tools are used, unbound

medium concentrations are often considered to equal intracellular unbound concentrations, which

is rarely the case. The ratio between unbound intra- to extracellular concentrations (Kpu,u) can

either be higher than 1 (metabolism/efflux rate-limited), equal to 1 (active/passive uptake ~

metabolism/efflux) or lower than 1 (uptake rate-limited) depending on the impact of each

eliminating pathway. (Pfeifer et al., 2013a) Not only clearance predictions, but also predictions

regarding drug toxicity, drug efficacy and drug-drug interactions could benefit from this

information. (Chu et al., 2013) Therefore, hepatic drug disposition models have been proposed to

calculate intracellular drug exposure by combining uptake/efflux and metabolism data. (Iwatsubo

et al., 1999; Shitara et al., 2005; Webborn et al., 2007; Umehara and Camenisch, 2012) Still, the


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development of methods to accurately measure intracellular protein binding or intracellular drug

distribution remains a great challenge. Several experimental techniques involving cell-

homogenization have been proposed, which disregard the dynamic interplay between drug

transporters and drug metabolizing enzymes. (Pfeifer et al., 2013a; Mateus et al., 2013) In

contrast, indirect methods relying on differences in kinetic parameters such as the Ki-method

presented by Brown et al. and the Km-method proposed by our group, will take this dynamic

interplay into consideration when calculating Kpu,u. (Brown et al., 2010; Nicolaï et al., 2015) The

Ki-method uses the ratio between the unbound Ki values of a drug metabolism inhibitor obtained

in microsomes and hepatocytes, to get an idea of the intracellular inhibitor accumulation.

However, these calculations depend on the distribution of both substrate and inhibitor into the

hepatocytes and are therefore potentially more challenging. On the other hand, the Km-method is

based on the ratio between a compound’s unbound metabolic Km in liver microsomes

(intracellular concentration) and it’s apparent Km in suspended rat hepatocytes (extracellular

concentration), resulting in a direct value for Kpu,u. Inherent to this method, Kpu,u will reflect the

impact of the processes controlling intracellular drug exposure. The current study aims to extend

application of the Km-method, from calculating the Kpu,u during our previous experiments with

verapamil (passive diffusion; Cyp3a1/2), to atazanavir (active uptake/efflux; Cyp3a1/2). (Nicolaï

et al., 2015) Thus, the HIV protease inhibitor atazanavir (ATV) was selected as a model

compound since its elimination involves P450-mediated drug metabolism (hCYP3A4/5;

rCyp3a1/2) and drug transport by sinusoidal ATP-binding cassette transporters (ABCC1; MRP1)

as well as SLC-transporters (SLCO1B1/3). (Swainston Harrison and Scott, 2005; Kis et al., 2010;

Wempe and Anderson, 2011; De Bruyn et al., 2015b) This will enable exploration of the

interplay between active uptake/efflux transport and intracellular metabolism which determine

the intracellular unbound drug exposure.


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Materials and Methods

Materials. Atazanavir sulfate (ATV) was obtained through NIH Aids Reagent Program.

Indinavir sulfate (IDV) was obtained from Hetero Drugs Limited (Hyderabad, India). 1-

aminobenzotriazole (ABT), benzbromarone, glucose 6-phosphate (G6P), collagenase type IV (C.

hystolyticum, ≥ 125 CDU), Leibovitz-15 (L-15) powder (with glutamine), mineral oil, silicon oil

and sodium taurocholate hydrate (TCA) were all purchased from Sigma-Aldrich (Schnelldorf,

Germany). Trypan blue stain (0.4%), L-glutamine (200 mM), 10X phosphate buffered saline

(PBS) and fetal bovine serum (FBS) were obtained from Lonza SPRL (Verviers, Belgium).

Dimethylsulfoxide (DMSO) and methanol (MeOH) were purchased from Acros Organics (Geel,

Belgium). Acetonitrile (ACN), Na-acetate and acetic acid were purchased from Analar-Normapur

(VWR) (Leicestershire, England). Na4-NADPH was obtained from Merck Millipore (MA, USA).

Ammonium acetate was obtained from Fisher Chemical (Landsmeer, Netherlands). HEPES (4-

(2-hydroxyethyl)-1-piperazineethanesulfonic acid) was purchased from MP Biochemical

(Illkirch, France). MK571 was obtained from Merck Sharp & Dohme research lab (Rahway,

USA).

Animals. Male Wistar rats were purchased from Janvier (Le Genest Saint Isle, France)

and housed according to Belgian and European laws, guidelines and policies for animal

experiments, animal housing, and animal care in the Central Animal Facility of the KU Leuven.

All experiments involving lab animals were approved by The Institutional Ethical Committee for

Animal Experimentation (license number: .LA 1210261).


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Determination of unbound fractions. Fumic, fuhep, fuIPRL and fuplasma were all determined

by equilibrium dialysis using a HTDialysis apparatus (CT, USA) fitted with membranes with a

molecular mass cutoff of 12-14 kDa. The HTDialysis apparatus was subjected to circular

agitation (175 rpm, 37°C) and samples were taken from both sides in each well at 4h and 6h.

Dialysis experiments were conducted in triplicate at an atazanavir (ATV) concentration of 1 µM

(0.2% v/v DMSO). Fuhep was determined with freshly-isolated and cryopreserved hepatocytes,

metabolically inactivated by heat (50 °C, 15 min) or by an incubation with 1-aminobenzotriazole

(ABT) (1 mM) and compared to wells without cells. Integrity and viability of heat inactivated

cells were determined using the Trypan blue (0.04%) exclusion method. Fuhep and Fumic were

determined in their respective incubation media. Binding of ATV to protein (20% blood) in the

IPRL perfusate was determined by equilibrium dialysis with Krebs-Henseleit buffer (KHB) in the

reference compartment (118 mM NaCl, 5.17 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 23.8 mM

NaHCO3, 12.5 mM HEPES, 5 mM D-glucose and 5 mM Na-pyruvate, pH 7.4). Fuplasma was

determined in plasma and compared to diffusion into a PBS containing compartment.

Metabolism studies in RLM. Rat liver microsomes (RLM) were prepared from male

Wistar rats (177-244 g after 24h fasting) using sequential (ultra)centrifugation as described

previously and stored at -80°C. (Nicolaï et al., 2015) ATV and 1-aminobenzotriazole (ABT)

solutions were prepared using microsomal incubation buffer (MIB) (3 mM MgCl2, 100 mM

sodium phosphate buffer, pH 7.4) to acquire four-fold concentrated solutions with a maximum

DMSO content of 0.2%. RLM were gently thawed and kept on ice. Subsequently, they were

diluted with MIB to obtain a four or two-fold concentrated solution of RLM (i.e. 1 or 0.5

mg/mL). Incubations were performed on a shaking incubator (350 rpm, 37°C) in 48-well plates

(Greiner-Bio-One, Wemmel, Belgium). ATV (100 µL) was preincubated with RLM (200 µL,


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0.25 mg/mL final concentration) for 10 minutes before adding 100 µL of four-fold concentrated

prewarmed (37°C) NADPH solution (1 mM final concentration) containing glucose 6-phosphate

(3 mM final concentration). During inhibition experiments, ABT (100 µL) was preincubated with

RLM (100 µL, 0.25 mg/mL final concentration) and prewarmed (37°C) NADPH solution (1 mM

final concentration) containing glucose 6-phosphate (3 mM final concentration) for 30 minutes,

after which ATV (100 µL) was added (5 - 0.1 µM). Control wells were incubated in the absence

of ABT or NADPH. Samples (75 µL) were taken at different time points and added to an equal

volume of cold acetonitrile (ACN) containing internal standard (0.1 µM IDV). Samples were

stored at -20°C for at least 1 hour prior to analysis. Before analysis, samples were thawed,

vortexed and centrifuged (10,500 g) for 10 min at 21°C, the supernatant was transferred into

micro-inserts for LC-MS/MS analysis. Linearity studies were performed with respect to ATV

concentration, microsomal protein concentration and incubation time. Subsequently, optimal

conditions for determining the in vitro half-life were selected. All incubations were terminated at

predetermined time points to ensure linear disappearance of ATV on an Ln(C)/time plot.

Metabolism studies in SRH. Suspended rat hepatocytes were isolated from male Wistar

rats (180-257 g; average weight = 215 g) according to the two-step collagenase perfusion method,

cryopreserved and thawed as described previously. (Nicolaï et al., 2015) They were re-suspended

in L-15* (90% L-15, 3.6 mM L-glutamine, 9.9 mM D-glucose, 9 mM HEPES, 3.6 mM NaHCO3,

pH 7.4) after which they were evaluated using the Trypan blue (0.04%) exclusion method to

determine viability and cell density. Viability of freshly-isolated SRH and cryopreserved SRH

ranged from 85% to 92% and from 75% to 85%, respectively. ATV and ABT solutions were

prepared using L-15* to acquire two or four-fold concentrated solutions with a maximum DMSO

content of 0.2%. Cells were two-fold concentrated and preincubated on a shaking incubator


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(37°C, 350 rpm) for 10 min in Advanced TCTM 24-well plates (Greiner-Bio-One, Wemmel,

Belgium). Incubations were initiated by adding an equal volume of two-fold concentrated ATV

solution (L-15*, 0.05% DMSO). When inhibition experiments were performed, cells were

concentrated four-fold and preincubated (37°C, 350 rpm) in the presence of ABT for 30 min.

Incubations were initiated by adding two-fold concentrated ATV solution (L-15*, 0.05%

DMSO). Linearity studies were performed with respect to ATV concentration, hepatocyte density

and incubation time. Subsequently, optimal conditions for determining the in vitro half-life were

selected (i.e. different hepatocyte densities were selected for different incubation concentrations

of ATV). All reactions were stopped by adding one volume of sample to two volumes of ice-cold

MeOH containing internal standard (0.1 µM IDV). Samples were stored at -20°C for the duration

of at least 1 hour prior to analysis. Finally, samples were thawed and centrifuged (20,816 g) for

10 min at 21°C and the supernatant was transferred into micro-inserts for LC-MS/MS analysis.

Uptake studies in SRH. Freshly isolated hepatocytes from male Wistar rats (180-220 g)

were re-suspended in Krebs-Henseleit buffer which had been sparged with carbogen (95%/5%

O2/CO2), after which they were evaluated using the Trypan blue (0.04%) exclusion method to

determine viability and cell density. Cells were diluted to a four-fold concentrated cell density (1

million cells/mL final density). Two-fold concentrated ATV solutions and four-fold concentrated

uptake inhibitor solutions were prepared in KHB (0.5% final DMSO content). Uptake studies

were performed using the oil-spin method. The hepatocytes (175 µL) were preincubated for 10

min (37°C) in the presence or absence of an uptake transporter inhibitor i.e. benzbromarone or

losartan (175 µL). Following preincubation, ATV (350 µL) was added and uptake was assessed.

After 30 sec, triplicate 200 µL aliquots were rapidly pipetted on top of an oil layer (82:18 silicon

oil:mineral oil, 1.051 g/mL) above a NaCl solution (8% w/v) in 1.5 mL test tubes. The test tubes


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were centrifuged immediately (16,162 g) for 3 min. Without delay, the tubes were snap frozen in

cooled ethanol (-80°C). After all tubes were frozen, the tube-pellets were collected in glass test

tubes after cutting the bottom part (~ 80 µL) with a tube cutter. Pellets were lysed with 300 µL

70:30 MeOH:H2O containing internal standard (0.01 µM IDV) and shaken for at least 30 min

(300 rpm, room temperature). Following lysis, the mixture was collected in test tubes and stored

at -20°C until analysis. Finally, samples were thawed and centrifuged (20,816 g) for 10 min at

21°C and the supernatant was transferred into micro-inserts for LC-MS/MS analysis. To

determine active uptake transporter kinetics, passive uptake of ATV was assessed at different

concentrations in the presence of benzbromarone (75 µM) which resulted in complete inhibition

of active uptake (data not shown) and subtracted from total uptake.

Monolayer cultured rat hepatocytes. On the day before seeding, Advanced TCTM 24-

well plates (Greiner Bio-One, Wemmel, Belgium) were coated with 50 µL of ice-cold (4°C)

neutralized rat-tail collagen (~ 1.5 mg/mL, pH 7.4). Plates were stored in a humidified incubator

(37°C, 5% CO2) for 2h before hydrating them with 500 µL/well of warm (37°C) sterile PBS and

storing them overnight in the humidified incubator (37°C, 5% CO2). In-house cryopreserved rat

hepatocytes from male Wistar rats (180-206 g) were thawed as reported previously and

suspended in day-0 cell culture medium (William’s E medium, 1 g/L glucose, 10% FBS, 2 mM

glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 µM dexamethasone, 4 µg/mL human

insulin). Just before seeding, viability and cell density were determined using the Trypan blue

(0.04%) exclusion method (viability 80-90%). Following aspiration of PBS from the collagen

coated plates, hepatocytes were seeded at a density of 400,000 cells/well. Cells were left to attach

to the collagen for 2h inside the humidified incubator (37°C, 5% CO2) before shaking the plates

vigorously to remove unattached cells and replacing the medium with 500 µL of warm (37°C)


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day-0 medium. Cultures were kept inside the humidified incubator for 2h until the start of the

experiment.

Efflux studies in monolayer cultured rat hepatocytes. 4h after seeding, monolayer

cultured rat hepatocytes were washed two times with warm (37°C) HBSS (10 mM HEPES, pH

7.4) before incubating them for 30 min in the presence of 250 µL of ABT (500 µM) in KHB

(10% FBS) to avoid interference of ATV metabolism. Subsequently, 250 µL of KHB (10% FBS)

containing 20 µM of ATV was added to each well and cells were loaded for 20 min. Following

loading, cells were washed three times with ice-cold (4°C) HBSS (10% FBS) after which ATV

efflux was commenced by adding 500 µL of prewarmed (37°C) KHB (10% FBS) containing

either 0.01% DMSO or 100 µM of MK571. Medium samples (100 µL) were taken at 3, 5, 10 and

15 min and added to 210 µL of MeOH containing internal standard (0.1 µM IDV). Collagen

coated wells without cells were incubated to correct for diffusion of ATV from the collagen.

Additional wells were lysed immediately after loading (70:30 MeOH:KHB (FBS 10%),

containing 0.07 µM IDV) to evaluate loading efficiency. Samples were centrifuged (20,816 g) for

10 min at 21°C and the supernatant was transferred into micro-inserts for LC-MS/MS analysis.

Bioanalysis was performed on the day of experiments.

Isolated perfused rat liver. Male Wistar rats (290-330 g) were anesthetized and the

portal vein was cannulated. The thoracic vena cava inferior was severed without cannulation and

the abdominal vena cava inferior was closed with a surgical clip. The bile duct was cannulated

with 10-15 cm of PE-10 tubing (0.28 mm x 0.61 mm id x od). To maintain bile flow, TCA was

infused continuously into the circulating system (30 µmol/h). (Paumgartner et al., 1974) During

excision, the liver was perfused (30 ml/min) with sparged (95%/5% O2/CO2) KHB (37°C, pH


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7.4). Following hepatectomy, the liver was placed on a collecting platform in a humidified and

temperature controlled (37°C) chamber. Perfusion with KHB was continued until 30 min after

cannulation. After organ acclimatization, the perfusion system was switched to KHB + 20%

heparinized rat blood (flow rate = 22.6 ± 2.1 ml/min), containing 0.4 µmol of ATV to reach an

initial perfusate concentration of 5 µM, which was oxygenated while passing through silastic Q7-

4750 semipermeable tubing (Dow Corning Europe SA, Belgium) inside an artificial lung

ventilated with carbogen (95%/5% O2/CO2). The perfusate was sampled (100 µL) just before and

directly after the liver at 2 min intervals until 10 min and subsequently every 5 min until 25 min

after dosing. Bile samples were collected every 5 min. Liver functionality was monitored by

measuring bile flow, portal vein pressure (water column ~ 10-15 cm water) and visual

appearance. Perfusate samples were processed immediately (see Bioanalysis) and stored at -20°C

until analysis. Adsorption studies were performed to assess adsorption to the materials of the

perfusion setup. Tubing materials consisted of glass, THV220/221GZ (Polyfluor Plastics, the

Netherlands), Tygon® chemical and Teflon®.

Bioanalysis. IPRL samples (30 µL) were diluted with water (120 µL) and protein was

precipitated with ACN (300 µL) containing internal standard (0.1 µM IDV). Following a vortex

step (2 x 30 sec), samples were centrifuged (20,816 g) for 10 min at 21°C. The supernatant was

transferred to micro-inserts and sample vials before being injected directly into the LC-MS/MS

system. The LC-MS/MS system (Thermo Scientific, San Jose, USA) consisted of an Accela®

autosampler, an Accela® pump and a TSQ Quantum Access® triple quadrupole mass

spectrometer equipped with an electrospray ionization (ESI) source. A kinetex® XB – C18

column (50 mm x 2.1 mm, 2.6 µm), protected by a securityGuard ULTRA precolumn

(Phenomenex, The Netherlands) was used for chromatographic separation. The mobile phase for


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ATV analysis consisted of a 0.5 mM ammonium acetate buffer (pH 3.5) (A) and MeOH (B).

Gradient elution at a constant flow (400 µL/min) was performed as follows: 95% A decreased

linearly to 5% in 2 min; was kept constant for 1 min; followed by a linear increase back to 95% A

in 10 sec and re-equilibration for 1.0 min with 95% A, before the next injection. The total run

time amounted to 4 min. The column oven and autosampler tray temperature were set at 30°C

and 15°C, respectively. The MS was operated in positive ionization mode. Spray voltage was

3500 V and argon was used as collision gas at a pressure of 1.5 mTorr. The MS was operated in a

3-channel selected reaction monitoring (SRM) mode with a scan time of 75 ms. Appearance and

disappearance of ATV mother compound, together with the internal standard (IDV), were

measured during analysis. Transitions monitored were 705.4 � 168.1 m/z and 614.5 � 421.3

m/z with retention times of 3.16 and 2.94 min for ATV and IDV, respectively. Other ionization

parameter settings were: capillary temperature (170°C), vaporizing temperature (300°C), sheet

gas pressure (50 arbitrary units), auxiliary gas pressure (0 arbitrary units), ion sweep gas pressure

(40 arbitrary units) and collision energy (43 arbitrary units). Intra- and intermediate precision of

quality control samples (0.01 µM and 0.1 µM) was below 10%.

Data Analysis.

Uptake in SRH: Net active uptake values of ATV in SRH were calculated by subtracting the

uptake in the presence of 75 µM of benzbromarone, representing passive uptake, from total

uptake at 37°C. Using Graphpad Prism® version 5.00 for windows (GraphPad Software, Inc.,

California, USA), the Michaelis-Menten equation (Equation 1) was fitted to the data and Vmax,

Km (± SE) were determined. Passive uptake clearance was calculated from the slope of passive

uptake rate over concentration curve. Active uptake clearance was calculated by dividing Vmax


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by Km or by dividing the active uptake rate (pmol/min/million cells) by the concentration of

ATV at which active uptake was measured.

� � ��

(Equation 1)

The inhibitory effect-Emax model (Equation 2) was used to describe the concentration dependent

inhibition of losartan on the uptake of ATV in SRH. Fits were obtained using non-linear

regression in Graphpad Prism® 5.00 software.

� ��

��

(Equation 2)

E represents the uptake of ATV in SRH, Emax the uptake of ATV in the absence of inhibitor, E0

the uptake of ATV at the maximal inhibitory effect, (Emax – E0) the maximal inhibitory effect, n

the Hill factor and IC50 the concentration at which the inhibitor exerts its half maximal inhibitory

effect. Subsequently, the Cheng-Prusoff equation (Equation 3) was applied to determine Ki from

IC50, where S is the ATV concentration at which IC50 was determined and Km the Michaelis-

Menten constant for active uptake of ATV in SRH.

�� 1 � ��

(Equation 3)

Metabolism in SRH and RLM: The slope of ATV (Ln(C)) disappearance over time was applied

using the in vitro half-life method to determine the rate of metabolism (pmol/min/million cells or

mg protein) for ATV metabolism in SRH and RLM as shown in Equation 4. (Obach, 1999)

� � �0.693��

� volume of incubate �μl million cells )* mg protein. � �/�/0/1/�23�


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(Equation 4)

The rate of metabolism was fitted as a function of ATV concentration to acquire Michaelis-

Menten parameters Vmax and Km (± SE). The unbound intrinsic metabolic clearance was

calculated according to Equation 5.

�4��, � �4��56 � ��/7�� 56

(Equation 5)

Following the methodology described in our previous study, the Kpu,u was determined by

dividing the unbound Km value of ATV metabolism in RLM (Kmmic,u) by the unbound Km value

of ATV metabolism in SRH (Kmhep,u) (Equation 6). (Nicolaï et al., 2015)

��, ��, � �8 ,

(Equation 6)

By applying Kpu,u in Equation 7, the unbound intracellular intrinsic metabolic clearance in SRH

was calculated (Clint,hep,u,intracellular).

�4��,��, ,�� 4��,��, � �4��,��, �8 , (Equation 7)

According to Webborn et al., the metabolic Clint,hep,u can be calculated by combining Clint,mic,u

with passive and active uptake clearance of ATV (Equation 8). (Webborn et al., 2007)

Subsequently, by combining Equation 8 and Equation 9, Kpu,u can be calculated from the

unbound metabolic clearance in RLM and the active/passive uptake and efflux clearance values

in SRH (Equation 10).

�4��,��, � �4��,��, � �4��, �,�� 4��, �,��4��,��, � �4��,��


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(Equation 8)

�4��,��, � �4��,��, � �8 ,

(Equation 9)

�8 , � �4��, �,�� 4��, �,��4��,��, � �4��,�� (Equation 10)

Clint,up,act is the active uptake clearance, Clint,up,pass is the passive uptake clearance, Clint,mic,u is the

microsomal unbound clearance and Clint,eff is the total efflux clearance (passive and active).

Clint,eff was calculated assuming that at steady state the sum of the influx rates (concentration x

Clint) equals the sum of the efflux and metabolic elimination rates (Supplemental Equation 1).

Influx rates were measured for the extracellular Kmhep,u of ATV metabolism in SRH. The

microsomal rate of metabolism was calculated for the intracellular concentration attained at the

extracellular concentration of Kmhep,u, which is Kmmic,u (Supplemental Table 1).

IPRL: For the IPRL data, Cp0, kel, Vd and Clint,IPRL were calculated as reported previously by

using the outflow concentrations, reflecting the equilibrium between liver tissue and perfusate

concentration. Clint,IPRL is subsequently scaled to in vivo with SF for g liver per kg b.wt.

(determined for each individual IPRL experiment). (Nicolaï et al., 2015) Finally, Clint,IPRL was

divided by fuIPRL to correct for binding of ATV in the perfusate (Clint,IPRL,u). The previously

published blood concentration–time profile of ATV, obtained after intravenous administration to

rats (n = 3), was fitted to a two-compartmental model. (De Bruyn et al., 2015a) Intrinsic

clearance values of ATV in RLM, SRH and IPRL were used to simulate the in vivo data as

reported previously (Supplemental Figure 3). (Nicolaï et al., 2015) All data were scaled with

conventional SF for MPPMC (0.37 mg microsomal protein per million cells), MPPGL (61 mg


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microsomal protein/g liver) and HPGL (163 million cells/g liver). (Smith et al., 2008) Activity-

based scaling factors (SF) for MPPMC, MPPGL and HPGL were calculated as reported

previously (Equations 11-13). (Nicolaï et al., 2015)

9::9� � ��/7��/7��

[Equation 11]

;:<= � �4��,��, �4��,��,

[Equation 12]

9::<= � 9::9� � ;:<=

[Equation 13]

Statistics: ANOVA (α level of 0.05; Dunnett’s post hoc test) was applied to compare control

conditions to experiments including inhibitors, using Graphpad Prism® version 5.00 for windows

(GraphPad Software, Inc., California, USA).


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Results

Determination of unbound fractions. The unbound fractions of total ATV

concentrations in the presence of hepatocytes, microsomes and IPRL-buffer, were determined

using equilibrium dialysis. Values for fuhep (fresh and cryopreserved), fumic and fuIPRL were 0.96 ±

0.1, 0.77 ± 0.2, 0.76 ± 0.1 and 0.27 ± 0.02, respectively. Fu values were used to correct total

(bound + unbound) clearance values, rendering the unbound clearance values. Fuplasma and fublood

were determined previously by our group and valued 0.075 and 0.85, respectively. (De Bruyn et

al., 2015a)

Metabolism studies in RLM and SRH. The Michaelis-Menten equation was fitted to

observed values of ATV metabolism in RLM (Figure 1) and SRH (Figure 2). All metabolic

parameters are summarized in Table 1. There was no statistically significant difference (p = 0.68)

between the unbound Kmhep values of ATV in freshly-isolated (0.83 ± 0.14) and cryopreserved

(0.94 ± 0.24) SRH. Kpu,u values were 0.32 ± 0.07 and 0.28 ± 0.1 for ATV in freshly-isolated and

cryopreserved SRH, respectively. Using Kpu,u, Clint,hep,u was calculated to Clint,hep,u,intracellular

resulting in values of 514 ± 149 and 459 ± 185 µl/min/million cells for freshly-isolated and

cryopreserved SRH, respectively. Clint,hep,u,intracellular was comparable to the scaled Clint,mic,u.

Clint,mic,u (1910 ± 323 µl/min/mg protein) was scaled to the cellular level using the activity-based

scaling factor (0.30 mg microsomal protein/million cells) for MPPMC, which was determined

previously by our group, to attain a value of 554 ± 105 µl/min/million cells. (Nicolaï et al., 2015)


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Uptake studies in freshly-isolated SRH. The uptake of ATV in SRH in the presence and

absence of the Oatp inhibitor benzbromarone is shown in Figure 3A. The slope of the uptake rate

of ATV in the presence of benzbromarone, representing the passive uptake clearance (Clint,up,pass),

amounted to 134 ± 4 µl/min/million cells. The Michaelis-Menten equation was fitted to the

observed values, resulting in a Km of 4.0 ± 0.5 µM and Vmax of 399 ± 22 pmol/min/million cells

(Figure 3B, Table 2).

Effect of uptake inhibition on ATV Kpu,u. To measure the effect of uptake transport

inhibition on Kpu,u and likewise Clint,hep,u, the uptake transport inhibitor should not interfere with

ATV metabolism. The effect of losartan (LOS) on the metabolism of ATV in RLM is shown in

Figure 4A. Only at concentrations higher than 10 µM of LOS, a statistically significant difference

with the control condition was observed (p < 0.05). On the contrary, when the inhibitory effect of

LOS on ATV uptake in SRH was determined, the maximal inhibitory effect was already attained

at 10 µM (Ki = 0.63 µM) (Figure 4B). Therefore, concentrations of LOS lower than or equal to

10 µM (1, 5 and 10 µM) were selected to determine the Kpu,u of ATV in SRH in the presence of

LOS. A linear correlation was observed (p = 0.04; R2=0.82) between the decrease in uptake

clearance and the decrease in Kpu,u in the presence of different concentrations of LOS (Figure 5).

Effect of metabolism inhibition on ATV Kpu,u. Figure 6 shows the effect of 1-

aminobenzotriazole (ABT) on ATV metabolism in both RLM and SRH. A proportional decrease

in both Vmax and Km of ATV metabolism was observed when different concentrations of ABT

were co-incubated with RLM (Figure 6A) (all fits are shown in Supplemental Figures 4-7).

However, when ATV was co-incubated with ABT in SRH, the Km decreased relatively slower

than the Vmax as a function of the ABT concentration (Figure 6B). Hence, based on Equation 6,


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ATV Kpu,u decreased with increasing ABT concentrations. In other words, the unbound

intracellular ATV exposure decreases as a function of the ABT concentration (Figure 7A).

ATV efflux in monolayer cultured rat hepatocytes. To evaluate whether ATV was a

substrate for efflux transporters present in short term cultured rat hepatocytes, efflux of ATV was

assessed in day-0 monolayer cultured rat hepatocytes in the absence and presence of 100 µM

MK571. Figure 7B shows that 100 µM of MK571 was able to decrease ATV efflux by >80%,

confirming that ATV efflux is almost entirely reliant on an inhibitable efflux process.

Activity-based scaling factors. As reported in our previous work, activity-based scaling

factors (SF) for MPPMC, MPPGL and HPGL can be calculated based on data from different

model systems. (Nicolaï et al., 2015) To evaluate whether such SF are compound dependent, they

were calculated for ATV and compared to the SF obtained previously with verapamil. As

described in Equations 11-12, unbound intrinsic Cl of ATV in IPRL is needed to calculate

MPPGL and HPGL. Clint,IPRL,u amounted to 826 ± 149 ml/min/kg b.wt. (Figure 8, Table 3).

Activity-based SF are shown in Table 4. To evaluate the predictive value of preclinical data

obtained during the present study, the in vivo pharmacokinetic profile of ATV in rats was

simulated (Supplemental Figure 3). All preclinical models performed well in predicting the in

vivo elimination of ATV with RSS values of 0.92, 0.95, 0.96 and 0.98 for simulations with

cryopreserved SRH, IPRL, freshly isolated SRH and RLM, respectively.


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Discussion

Transport-metabolism interplay of hepatic atazanavir (ATV) clearance was investigated to

elucidate the mechanisms governing intracellular unbound ATV exposure. As a measure for

intracellular drug exposure, the hepatocellular Kpu,u (0.32-0.28; Table 1) was calculated with

unbound metabolic Km values retrieved from (in vitro) metabolism experiments with suspended

rat hepatocytes (SRH) and rat liver microsomes (RLM). This approach for Kpu,u determination

was recently introduced by our group with verapamil as a model compound. (Nicolaï et al., 2015)

Interestingly, the low (< 1) value of Kpu,u for ATV was an indication for uptake rate-limited

metabolic clearance (Figure 9A) while a total liver-to-plasma concentration ratio of 3 has been

reported. (Fukushima et al., 2009) Uptake experiments with SRH revealed that intracellular ATV

concentrations were controlled, at least to some extent, by a saturable uptake process (Figure 3;

Table 2). This is in line with previous work, which showed the role of OATP/Oatp for

hepatocellular uptake and the rate-limiting effect of transporters on hepatic clearance of several

HIV protease inhibitors. (De Bruyn et al., 2015a; Brown et al., 2010) In contrast to findings from

the present study, uptake transporter involvement is most often associated with high cell-to-

medium concentration ratio’s. (Parker and Houston, 2008; Brown et al., 2010) The hypothesis

concerning uptake rate-limited metabolism was challenged further by investigating the impact of

uptake transport inhibition on Kpu,u. Given the nature of our approach to calculate Kpu,u (based on

metabolic Km values), an uptake transport inhibitor not interacting with ATV metabolism was

required. Under these conditions, a shift of the metabolic Km of ATV in SRH was anticipated

through inhibition of active uptake transport. Losartan (LOS) was evaluated as an inhibitor for

ATV uptake in SRH (Ki = 0.62 µM; Figure 4B). Whereas active uptake was completely inhibited

at 10 µM, inhibition of metabolism at this concentration of LOS in RLM was excluded (p < 0.05)


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(Figure 4A). Co-incubation of ATV with LOS during metabolism experiments in freshly-isolated

SRH revealed a statistically significant linear correlation (p = 0.04; R2=0.82; Figure 5) between

ATV uptake clearance and Kpu,u, confirming importance of ATV uptake for hepatocellular ATV

metabolism (Figure 9B). Overall, the current approach can be classified under ‘estimation of

intracellular drug concentrations by modeling and simulation’, one of the main indirect

methodology types referred to in a recent review on intracellular drug concentration

determination. (Chu et al., 2013) However, rather than applying a modeling approach, this article

aims to quantitatively link hepatocellular drug disposition mechanisms to the hepatocellular

Kpu,u.

Intuitively, and based on Equation 10 (Figure 7A), inhibition of intracellular metabolism

was expected to increase Kpu,u. (Chang et al., 2014) Ideally, a non-competitive metabolic

inhibitor (no effect on Km) should be applied to investigate the effect of metabolism inhibition on

Kpu,u. Thus, the inhibition profile of the general P450 inhibitor 1-aminobenzotriazole (ABT), for

ATV metabolism was determined. Surprisingly, both Km and Vmax of ATV metabolism in RLM

decreased proportionally when ABT concentrations were increased (Figure 6A). This identified

ABT as an uncompetitive inhibitor for ATV metabolism (Ki = 2.34 ± 0.02). To our knowledge,

this is the first report of ABT being identified as an uncompetitive inhibitor. Notwithstanding the

direct effect of ABT on the Km of ATV metabolism, it was still a suitable inhibitor to determine

the effect of P450 inhibition on Kpu,u. When ATV was co-incubated with ABT in SRH, the Km

of ATV metabolism decreased at a relatively slower rate with respect to ABT concentrations as

compared to Vmax (Figure 6B). However, inhibition of metabolism was expected to increase the

intracellular unbound ATV accumulation, which would be reflected by a relatively faster

decrease of Km as compared to Vmax. The latter would be the result of an increase in Kpu,u with

increasing concentrations of ABT i.e. lower metabolic capacity (Figure 7A). Since interaction of


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ABT with ATV uptake was ruled out (Supplemental Figure 8) and ABT has never been shown to

interact with efflux transporters, we deemed the direct influence of ABT on ATV transport

unlikely. (Kimoto et al., 2012) Additionally, no deviation from linearity was observed for the

metabolism of ATV as a function of hepatocyte density curves (Supplemental Figure 9), ruling

out the hypothesis that ATV metabolites interact with ATV disposition in the SRH setup. Finally,

in line with previous reports on transport-metabolism interplay, inhibition of intracellular

metabolism could increase the susceptibility of ATV for efflux transporters present in SRH. (Shi

and Li, 2014)

Indeed, the current data already show a relatively high total efflux clearance (Clint,eff = 420

µl/min/million cells; Table 2; Supplemental Equation1; Supplemental Table 1) as compared to

the passive uptake clearance (Clint,up,pass = 134 µl/min/million cells) of ATV. Additionally, the

non-specific Mrp inhibitor MK571 decreased ATV efflux in rat monolayer cultures by >80%

(Figure 7B). The intracellular gradient of unbound ATV, driven by intracellular metabolic ATV

depletion, may become less steep upon the inhibition of P450 and decrease overall flux of ATV

towards the endoplasmic reticulum. As a consequence, deep intracellular drug exposure at the site

of metabolism is decreased, thus increasing the likelihood of ATV binding to membrane-bound

efflux transporters (Figure 9C). This concept of inhomogeneous intracellular unbound drug

distribution is not improbable, since several small molecules have been shown to have specific

affinities for subcellular organelles, altering local intracellular drug concentrations. (Pfeifer et al.,

2013a; Matijašić et al., 2012; Fu et al., 2014) Likewise, the Km-method, which reflects

intracellular unbound ATV concentrations at the level of P450 enzymes, depends on ATV

concentrations in the vicinity of the endoplasmic reticulum. This inhomogeneous drug

distribution could imply that different substrate concentrations should be considered at the

intracellular locations of P450-enzymes and ABC-transporters, respectively.


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In the present study, uptake and metabolism parameters were determined in SRH and

RLM and combined to determine Kpu,u and Clint,eff for SRH (Equations 6 and 10). If Clint,eff would

be estimated from monolayer- or sandwich-cultured hepatocyte experiments, it should be

corrected for altered expression levels of efflux transporters in these systems. (De Bruyn et al.,

2013) Alteration of expression levels as a function of culture time or internalization of canalicular

efflux transporters upon isolation will affect intracellular ATV concentrations and likewise Kpu,u.

Therefore, Kpu,u determined for SRH should not be transferred to other systems with different

expression patterns of drug transporters and/or metabolic enzymes.

Consistent with our previous study, in which activity-based scaling factors (SF) were

calculated using verapamil (Nicolaï et al., 2015), SF were calculated using ATV metabolic

clearance in RLM, SRH and IPRL. The calculated SF valued 0.27-0.24 mg microsomal protein

per million cells (MPPMC), 40-46 mg protein per g liver (MPPGL) and 150-191 million cells/g

liver for freshly-isolated and cryopreserved SRH, respectively (Table 4). These values were

similar to the activity-based SF calculated with verapamil and the P450-content based SF

reported in literature. However, as discussed in our previous article, the activity-based SF for

MPPGL and HPGL in freshly-isolated SRH as determined with verapamil (80 mg protein/g liver;

269 million cells/g liver), deviated from all other SF. (Nicolaï et al., 2015) Confidence in current

calculations improved, since the activity-based SF values calculated during this study coincided

reasonably well with previously determined values, even though they were obtained with

different RLM, SRH, IPRL preparations and even another compound. Additionally, Clint,mic,u

equaled the intracellular unbound clearance of ATV in both freshly-isolated and cryopreserved

SRH. This confirmed the previously reported preserved intracellular metabolic capacity of P450

enzymes following cryopreservation of SRH. (Nicolaï et al., 2015)


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In summary, during the present study, intracellular unbound ATV concentrations were correlated

with the rate of active uptake transport, illustrating that total uptake is the rate-limiting process in

ATV hepatic clearance in the rat. Consistently, despite active uptake transport but in line with

sinusoidal efflux transport and significant intracellular metabolism, a low Kpu,u value was

obtained (0.32). Inhibition of ATV metabolism with ABT unexpectedly decreased rather than

increased the Kpu,u, pointing towards a possible mechanistic interplay between P450-mediated

ATV metabolism and hepatocellular efflux transporters. Simultaneously, ABT was identified as

an uncompetitive inhibitor of ATV metabolism. The current findings will help improve our

understanding of the link between mechanisms governing intracellular hepatic drug disposition,

aiming to ameliorate future PBPK modeling algorithms.


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Acknowledgements

Janna Mertens, for her understanding patience and loving care.


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Authorship Contributions

Participated in research design: Johan Nicolaï, Pieter Annaert

Conducted experiments: Johan Nicolaï, Louise Thevelin

Contributed new reagents or analytical tools: Johan Nicolaï, Tom De Bruyn

Performed data analysis: Johan Nicolaï, Pieter Annaert

Wrote or contributed to the writing of the manuscript: Johan Nicolaï, Pieter Annaert, Tom De

Bruyn, Patrick Augustijns


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Footnotes

Johan Nicolaï received a PhD scholarship from the Agency for Innovation by Science and

Technology [Agentschap voor innovatie door wetenschap en technologie (IWT), Flanders,

Belgium], project number 111193. This study was partially supported by FWO grant G.0662.09N

and by internal funds of the Lab for Drug Delivery and Disposition, KU Leuven Department of

Pharmaceutical and Pharmacological Sciences.


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Legends for Figures

Figure 1. Michaelis-Menten plot of atazanavir metabolism in RLM (A) and the corresponding

atazanavir intrinsic clearance in RLM (B) as a function of atazanavir concentrations. Points

represent mean values ± SD of four incubations performed in triplicate with two different batches

of RLM.

Figure 2. Michaelis-Menten plots of atazanavir metabolism in freshly-isolated (�) and

cryopreserved (�) SRH. (A) Corresponding atazanavir intrinsic metabolic clearance in SRH as a

function of atazanavir concentrations. (B) Points represent mean values ± SD of triplicate

incubations with two batches of SRH.

Figure 3. Total (�), passive (�) and active (�) uptake rates of atazanavir in SRH as a function

of the atazanavir concentration. (A) Passive uptake rates were measured in the presence of the

uptake transport inhibitor benzbromarone (75 µM) and active uptake rates were calculated from

the difference between total and passive uptake rates. Close up of the Michaelis-Menten plot of

atazanavir active uptake rates in SRH as a function of the atazanavir concentration. (B) Points

represent mean values ± SD of triplicate incubations with two batches of freshly-isolated SRH.

Figure 4. Metabolism (% of control) of atazanavir in RLM in the presence of different

concentrations of losartan. (A) Concentration-dependent inhibition of atazanavir uptake by

losartan in SRH. (B) Points represent means ± SD of triplicate incubations with a single batch of

RLM and two batches of SRH.


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Figure 5. Linear correlation (p = 0.04; R2=0.82) between the Kpu,u and atazanavir uptake

clearance, in the absence and presence of 1, 5 and 10 µM of losartan. Values represent means ±

SEM of Kpu,u values determined with triplicate incubations in four different batches of freshly-

isolated SRH, compared to uptake clearance (means ± SD) of atazanavir in SRH in the presence

of losartan (0 µM, 1 µM, 5 µM, 10 µM) in two batches of SRH.

Figure 6. Representative figure showing the Michaelis-Menten parameters (Vmax, Km)

describing atazanavir metabolism in RLM in the presence of different concentrations of ABT.

(A) Michaelis-Menten parameters (Vmax, Km) for atazanavir metabolism in SRH in the presence

of different concentrations of ABT. (B) In contrast to incubations with RLM, decrease of Km in

SRH is less pronounced as compared to Vmax. Bars represent means ± SE of triplicate

incubations in single batches of RLM and SRH. Repeated experiments confirm this disconnect

between RLM and SRH (Supplemental Figures 4-7).

Figure 7. Observed (�) and calculated ([ATV] = Cmax,plasma,u; �) change in atazanavir Kpu,u as a

function of ABT concentration. (A) The calculated change in Kpu,u as a function of ABT

concentration was obtained with Equation 10.(Webborn et al., 2007) Efflux of atazanavir in

monolayer cultured rat hepatocytes in the absence (full line) and presence (dotted line) of the

non-specific Mrp inhibitor MK571 (100 µM). (B) Points represent means ± SD of efflux

experiments performed in triplicate in three separate batches of rat hepatocytes.

Figure 8. Perfusate concentration (µM)-time profiles of atazanavir during IPRL experiments. Cin

and Cout represent perfusate concentrations immediately before and after the liver, respectively.

Points represent means ± SD of three separate liver perfusions.


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Figure 9. Schematic overview illustrating the impact of atazanavir (ATV) disposition pathways

on the hepatocellular Kpu,u in the absence of an inhibitor (A), presence of losartan (LOS) (B) or

presence of ABT (C). Size of arrows and ATV indicate the extent of atazanavir flux and local

atazanavir concentrations, respectively.


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Tables

Metabolism parameter Model system

Fresh SRH Cryo SRH RLM

Km (µM) 0.86 ± 0.13 1.22 ± 0.19 0.35 ± 0.04

Km x fu (µM) 0.83 ± 0.14 0.94 ± 0.24 0.26 ± 0.04

Kpu,u

(ratio of unbound intra- to extracellular concentration) 0.32 ± 0.07 0.28 ± 0.09 -

Vmax (pmol/min/million cellsa or mg proteinb) 136 ± 6a 122 ± 6a 506 ± 17b

Clint (µl/min/million cellsc or mg proteind) 158 ± 24c 100 ± 16c 1446 ± 175d

Clint / fu (µl/min/million cellsc or mg proteind) 165 ± 28c 129 ± 34c 1910 ± 323d

Clint / (fu x Kpu,u) (µl/min/million cells) 514 ± 149 459 ± 185 554 ± 105e

Table 1. Michaelis-Menten parameters (Km, Km x fu, Vmax) describing atazanavir metabolism

in RLM and SRH, together with corresponding total and unbound intrinsic clearance values (Clint

and Clint/fu). Km and Vmax values were obtained by fitting the Michaelis-Menten equation to the

observed rate of metabolic atazanavir disappearance as a function of atazanavir concentration

(Figure 2). Values are means ± SD of triplicate experiments with two batches of SRH and two

batches of RLM. a pmol/min/million cells; b pmol/min/mg protein; c µl/min/million cells; d

µl/min/mg protein; e Clint,mic,u was recalculated to match the unit of µl/min/million cells, using the

activity-based scaling factor (MPPMC, 0.30 mg microsomal protein/million cells) previously

determined with verapamil no Kpu,u-correction was applied here. (Nicolaï et al., 2015)


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Transport parameter Value

Km (µM) 4.0 ± 0.5

Vmax (pmol/min/million cells) 399 ± 22

Clint,up,act (µl/min/million cells) 100 ± 14

Clint,up,pass (µl/min/million cells) 134 ± 4

Clint,eff (µl/min/million cells) 420 ± 99a

% active uptake at Cmax,u 37 %

% passive uptake at Cmax,u 63 %

Table 2. Michaelis-Menten parameters for atazanavir uptake in SRH together with corresponding

passive and active uptake clearance values. Km and Vmax were obtained by fitting the

Michaelis-Menten equation to the observed rate of active atazanavir uptake in SRH as a function

of atazanavir concentration (Figure 3). Values are means ± SD of triplicate incubations in two

batches of freshly-isolated SRH. Cmax,u (1.048 µM) equals the unbound Cmax in human plasma.

aClint,eff was determined from uptake and elimination rates as reported in the supplemental data

(Supplemental Equation 1; Supplemental Table 1).


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PK Parameter IPRL

(5 µM)

t1/2 (h) 0.06 ± 0.01

Vdss (L) 0.38 ± 0.09

Cl (ml/min/kg b.wt.) 223 ± 36

Clint,u (ml/min/kg b.wt.) 826 ± 149

Table 3. Pharmacokinetic parameters describing atazanavir disposition in the IPRL system. T1/2

and Vdss were determined as reported under data-analysis. Values are means ± SD of IPRL

experiments with three different livers.


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Activity-baseda

Atazanavir

Activity-basedb

Verapamil

P450-content based

Scaling factor Fresh SRH Cryo SRH Fresh SRH Cryo SRH Conventional (rat)

MPPMC 0.27 ± 0.1 0.24 ± 0.1 0.30 ± 0.02 0.28 ± 0.03 0.37c

MPPGL 40 ± 16 46 ± 23 80 ± 15 43 ± 14 44.5b/ 45d / 61e

HPGL 150 ± 31 191 ± 49 269 ± 44 153 ± 27 120d/ 163e

Table 4. Activity-based rat scaling factors for MPPMC (mg protein/million cells), MPPGL (mg protein/g liver) and HPGL (million

cells/g liver). Scaling factors were calculated for atazanavir following the described methodology and compared to scaling factors

based on verapamil data and ‘conventional’ rat scaling factors as reported in literature. Values are means ± SD. a determined in the

present study using atazanavir, b determined in our previous study using verapamil (Nicolaï et al., 2015), c calculated from the

conventional MPPGL and HPGL stated by Smith et al. (Smith et al., 2008), d reported by Barter et al. (Barter et al., 2007), e reported

for the Wistar rat by Smith et al. (Smith et al., 2008)

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

ard. Published on Decem

ber 28, 2015 as DO

I: 10.1124/dmd.115.068114

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Figures


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