Effects of verapamil on the pharmacokinetics and hepatobiliary disposition of fexofenadine in pigs

Accepted Manuscript Effects of verapamil on the pharmacokinetics and hepatobiliary disposition of fexofenadine in pigs Erik Sjögren, Mikael Hedeland, Ulf Bondesson, Hans Lennernäs PII: DOI: Reference:

S0928-0987(13)00368-0 http://dx.doi.org/10.1016/j.ejps.2013.09.014 PHASCI 2881

To appear in:

European Journal of Pharmaceutical Sciences

Received Date: Revised Date: Accepted Date:

23 April 2013 19 August 2013 18 September 2013

Please cite this article as: Sjögren, E., Hedeland, M., Bondesson, U., Lennernäs, H., Effects of verapamil on the pharmacokinetics and hepatobiliary disposition of fexofenadine in pigs, European Journal of Pharmaceutical Sciences (2013), doi: http://dx.doi.org/10.1016/j.ejps.2013.09.014

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Effects of verapamil on the pharmacokinetics and hepatobiliary disposition of fexofenadine in pigs

Erik Sjögrena, Mikael Hedelandb, c, Ulf Bondessonb, c, Hans Lennernäsa

a

Department of Pharmacy, Biopharmaceutic Research Group, Uppsala University,

Box 580, SE-751 23 Uppsala, Sweden b

Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, Uppsala

University, Box 573, SE-751 23 Uppsala, Sweden c

National Veterinary Institute (SVA), Department of Chemistry, Environment and Feed Hygiene, SE-

751 89 Uppsala, Sweden.

Running title: The pharmacokinetics of fexofenadine in pigs

Address for correspondence Erik Sjögren, PhD. Biopharmaceutical Research Group Department of Pharmacy Uppsala University Box 580 SE-751 23 Uppsala Sweden Telephone: +46 18 471 4154 Fax: +46 18 471 4223 E-mail: [email protected]

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Abstract The pharmacokinetics (PK) of fexofenadine (FEX) in pigs were investigated with the focus on exploring the interplay between hepatic transport and metabolism when administered intravenously (iv) alone or with verapamil. The in vivo pig model enabled simultaneous sampling from plasma (preliver, post-liver and peripheral), bile and urine. Each animal was administered FEX 35 mg iv alone or with verapamil 35 mg. Plasma, bile and urine were analyzed with liquid chromatography-tandem mass spectrometry. Non-compartmental analysis (NCA) was used to estimate traditional PK parameters. In addition, a physiologically based pharmacokinetic (PBPK) model consisting of 11 compartments (6 tissues + 5 sample sites) was applied for mechanistic elucidation and estimation of individual PK parameters. FEX had a terminal half-life of 1.7 h and a liver extraction of 3%. The fraction of the administered dose of unchanged FEX excreted into the bile was 25% and the bile exposure was more than 100 times higher than the portal vein total plasma exposure, indicating carrier-mediated (CM) disposition processes in the liver. 23% of the administered dose of FEX was excreted unchanged in the urine. An increase in FEX plasma exposure (+50%) and a decrease in renal clearance (-61%) were detected by NCA as a direct effect of concomitant administration of verapamil. However, analysis of the PBPK model also revealed that biliary clearance was significantly inhibited (-53%) by verapamil. In addition, PBPK analysis established that metabolism and CM uptake were important factors in the disposition of FEX in the liver. In conclusion, this study demonstrated that CM transport of FEX in both liver and kidneys was inhibited by a single dose of verapamil.

Key words: fexofenadine; carrier-mediated transport; hepatic disposition; physiologically based pharmacokinetic modeling; drug interaction

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Abbreviations: AUC, area under the concentration-time curve Abile, amount excreted in bile Aurine , amount excreted in urine Aprot, microsomal content Atot.I, total amount of fexofenadine excreted into the jejunal segment Atot.urine, total amount of fexofenadine excreted into the urine B:P, blood to plasma volume ratio CLbile, biliary clearance CLdif, bidirectional passive diffusion clearance CLeff, efflux clearance CLH, hepatic clearance CLI, intestinal clearance CLint, metabolic intrinsic clearance CLmet, metabolic clearance CLupt, membrane uptake clearance CLR, renal clearance CLtot, total clearance CM, carrier-mediated Cmax, maximum peak plasma concentration Cprot, microsomal protein concentration CVK, central vein catheter E, extraction ratio fe.int, fraction of dose excreted into the intestine fe.urine, fraction of dose excreted in urine fe.bile, fraction of dose excreted in bile FEX, fexofenadine fup, fraction of unbound drug in plasma fuh, fraction of unbound drug in hepatocytes HPGL, hepatocellularity HPLC, high-performance liquid chromatography iv, intravenous Jmax, maximum rate of uptake (Jmax.upt) and efflux (Jmax.eff) kAC, microsomal activity rate constant ke, terminal rate constant Km, Michaelis constant KP, partition coefficient constant LCC, liver cell compartment LC-ESI-MS/MS, liquid chromatography-electrospray-tandem mass spectrometry LVC, liver vascular compartment MAC, mixed arterial compartment NCA, non-compartmental analysis OATP, organic anion transporting polypeptide Peff, effective permeability Pdif, passive permeability P-gp, P-glycoprotein PBPK, physiologically based pharmacokinetic PK, pharmacokinetic Q, rate of blood flow SFmet, metabolic scaling factor t½, apparent terminal half-life tmax, time to reach Cmax v, rate of drug movement

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V.F., femoral vein V.H., hepatic vein V.P., portal vein Vmax, the theoretical maximum reaction rate Vss, Volume of distribution at steady state W, weight

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1. Introduction The potential involvement of carrier-mediated (CM) transport in the disposition of xenobiotics in tissues has been known for several decades (Hedman and Meijer, 1998; Meijer et al., 1970; Schinkel et al., 1994; Solomon and Schanker, 1963). However, this has been properly acknowledged only recently and the increased number of investigations in the area over the last decade has increased our knowledge rapidly (Funk, 2008; Giacomini et al., 2010; Shitara et al., 2006). Nonetheless, more relevant in vivo data are required to balance the massive output of in vitro data tofully elucidate the pharmacokinetic (PK) relevance of membrane transporter function in the intestine and the liver (Giacomini et al., 2010). CM distribution processes are known to be important disposition processes for several drug compounds, such as rosuvastatin, topotecan and fexofenadine (FEX) (Kruijtzer et al., 2002; Simonson et al., 2004; Tannergren et al., 2003b). FEX is a histamine H1 receptor antagonist, widely used for treatment of seasonal allergic rhinitis. Several in vivo and in vitro studies have shown FEX (molecular weight =502 g/mol, pKa=4.2 (acid) 7.8 (base), PSA=124 Å2, logD7.4=0.23) to be poorly permeable; it has been classified as a BCS III compound (high solubility, poor permeation) (Petri et al., 2004; Tannergren et al., 2003a). Studies have also indicated that FEX is metabolized to only a minor degree in rats and humans (Cvetkovic et al., 1999; Lippert et al., 1995; Poirier et al., 2009; Yamazaki et al., 2010). FEX is a substrate for several human membrane transporter proteins in vitro, in particular the efflux protein P-glycoprotein (P-gp) and various uptake transporters from the organic anion transporting polypeptide (OATP) family (Cvetkovic et al., 1999; Dresser et al., 2002; Kobayashi et al., 2003; Perloff et al., 2002; Petri et al., 2004). The in vivo implications of membrane transporter function in the human disposition of FEX have been investigated in clinical studies and the drug is currently listed by the FDA as a model substrate for P-gp (Bailey et al., 2007; FDA, 2011; Hamman et al., 2001; Niemi et al., 2005; Tannergren et al., 2003b). It has been suggested that inhibition of intestinal P-gp is the main mechanism behind the observed drug-drug interactions following oral administration of FEX, resulting in increased systemic exposure to FEX. However, concomitant jejunal administration of the p-gp inhibitor verapamil, using a human in vivo perfusion technique, did not increase the low effective jejunal permeability (Peff = 0.06 × 10-6) of FEX (Tannergren et al., 2003a). Instead, the bioavailability of FEX increased 4-fold, suggesting alterations

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to other distribution processes or first pass/systemic elimination pathways in the liver (Tannergren et al., 2003b). An animal study, using a multiple-sampling-site pig model, was then carried out to further investigate the entero-hepatic disposition of FEX (Petri et al., 2006). The Peff of FEX was increased when a low dose (35 mg) of verapamil was co-administered in the jejunal segment, but no such effect was seen at a high dose (200 mg). FEX plasma exposure was not affected, regardless of dose, when verapamil was co-administered (Petri et al., 2006). It was hypothesised that these observations were caused by the involvement of multiple membrane transporter proteins in the intestine, and that the verapamil concentration was too low to efficiently inhibit the processes involved in FEX disposition in the liver. Neither the human study nor the pig study offered any clear conclusions on the systemic processes involved using traditional non-compartmental PK analysis (Petri et al., 2006; Tannergren et al., 2003b). The multiple-sampling-site, in vivo pig model, which is suitable for in vivo mechanistic investigations of intestinal absorption, liver disposition and biliary excretion, has been used in our research group for several years (Bergman et al., 2009; Sjödin et al., 2008; Thörn et al., 2009). Recently, a physiologically based pharmacokinetic (PBPK) model was developed for this in vivo model to facilitate mechanistic interpretations involved in the hepatic drug disposition after intravenous (iv) drug administration (Sjögren et al., 2012). The aim of this study was to further explore the in vivo mechanisms of the hepatic disposition of FEX in the porcine model, and to investigate the local PK of FEX when administered iv alone or with verapamil.

2. Materials and Methods 2.1 Enzyme kinetic assay in pig liver microsomes Liver tissue was collected from pigs used in the study programme in medicine at Uppsala University. These domestic pigs (Hampshire, Yorkshire and Swedish Landrace) were from the same breeder, were of the same gender and age, and were treated with the same anaesthetic drugs as the animals in the in vivo multiple-sampling-site PK study (see section 2.4, experimental procedures). Liver microsomes were prepared as previously described (Bergman et al., 2009). Microsomal incubations for investigation of the metabolic oxidation and conjugation of FEX were conducted with minor

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modifications as previously described (Fisher et al., 2000; Sjögren et al., 2009). The metabolic stability at a final protein concentration of 1.2 mg/ml was investigated at 0.2 and 20 µM FEX. Briefly, 50 µg of alamethicin/mg microsomes were mixed and placed on ice for 15 min. MgCl (1 mM in incubation), saccharolactone (5 mM in incubation), reduced β-nicotinamide adenine dinucleotide phosphate (1 mM in incubation) and uridine diphosphoglucuronic acid (5 mM in incubation) were added and preincubated for 5 min. The reaction was then started by adding FEX (final concentration 0.2 or 20 µM) dissolved in phosphate buffer. Samples (n=5 in triplicates) of 100 µl were consecutively removed at selected time points up to 90 min. The reactions were terminated by adding the sample to 100 µl of acetonitrile, including the internal standard at a concentration of 1 µM. After termination, the samples were centrifuged for 10 min at 10 000 g, and an aliquot of the supernatant was withdrawn and stored at -20°C until sample analysis. Binding to hepatic microsomes was determined by ultrafiltration as described previously (Sjögren et al., 2009).

2.2 Animals The parallel-group PK study involved two groups of five male pigs of mixed breed (Hampshire, Yorkshire and Swedish Landrace). The animals were between 10 and 12 weeks old and weighed 29.1 ± 6.9 kg (range 20.0–38.4 kg). The study was approved by the ethics committee for animal experiments in Uppsala (Dnr: C 55/4) and was performed at the Clinical Research Department No. 2, University Hospital of Uppsala, Sweden.

2.3 Investigational drugs and experimental design Two solutions of FEX (fexofenadine hydrochloride, Toronto Research Chemicals, Toronto, Canada) were prepared by dissolving 28 mg and 7 mg in 1 ml and 2.5 ml of 99.5% ethanol, respectively. These ethanol solutions were then filtered through a Minisart High-Flow filter (0.2 µm) directly into 20 ml and 47.5 ml of glucose solution (50 mg/ml), respectively. The 1.33 mg/ml FEX solution (21 ml) was administered as a bolus at time 0 and the 0.14 mg/ml FEX solution (50 ml) was administered as an infusion (25 ml/h) over 120 min from time 0, through a central vein. The total dose of FEX administered was thus 35 mg. One group (the treatment group) was also given verapamil 35 mg

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(Isoptin ® 2.5 mg/ml; Abbot, Solna, Sweden) as a 25.0 mg bolus dose at time 0, and a 10.0 mg infusion through the portal vein (V.P.). The infusion solution was prepared by adding 5 ml Isoptin 2.5 mg/ml to 45 ml NaCl (9 mg/l). This solution (0.25 mg/ml) was then infused (20 ml/h) over 120 min from time 0. The total dose of verapamil was higher than that required to reach the recommended clinical levels in humans (5–10 mg by iv injection) so as to optimize the possibility of inhibiting the disposition processes in the liver. Administration of verapamil through the V.P. also ensured maximum exposure of the liver while reducing the risk of reaching toxic levels in the heart. The solution used for intestinal perfusion consisted of potassium chloride 5.4 mM, sodium chloride 30 mM, mannitol 35 mm, D-glucose 10 mM and PEG 4000 1.0 g/l, all dissolved in a 70 mM phosphate buffer with pH 6.5 and an osmolality of 290 mOsm/kg. The perfusion buffer in the study has been used previously, and has been validated in several perfusion studies in both humans and animals (Petri et al., 2006; Tannergren et al., 2003b). The jejunal segment was perfused with a single-pass technique at a flow rate of 2 ml/min for both the control and treatment groups.

2.4 Experimental procedures – in vivo study All the animals were given a pretransportation sedative consisting of xylazine (Rompun® vet 20 mg/ml; Bayer AG, Leverkusen, Germany) 50 mg. At the research clinic, all the animals were sedated by intramuscular administration of tiletamine and zolazepam (Zoletil; Virbac S.A., Carros, France) 6 mg/kg, xylazine (Rompun® vet 20 mg/ml; Bayer AG, Leverkusen, Germany) 2.2 mg/kg and atropine (Atropin NM Pharma 0.5 mg/ml; Merck NM AB, Stockholm, Sweden) 0.04 mg/kg while still in the transport box. Morphine (10 mg/ml; Meda AB, Solna, Sweden) 1 mg/kg was administered through a peripheral vein in the ear of each pig as preoperative analgesia. A breathing tube was introduced through an incision in the throat to ventilate the pig during anaesthesia. The animal was ventilated with a 30% oxygen-air mix. A central vein catheter (CVK) was introduced through the jugular vein to its final location in the superior caval vein. A mixture of ketamine (Ketaminol® vet 100 mg/ml; Intervet, Stockholm, Sweden) 20 mg/kg/h, morphine (10 mg/ml; Meda AB, Solna, Sweden) 0.5 mg/kg/h and pancuronium bromide (Pavulon 2mg/ml; Organon AB, Gothenburg, Sweden) 0.25 mg/kg/h was given to the animals through the CVK throughout the experiment to keep the animal

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pain-free and anaesthetized. Rehydrex (Fresenius Kabi, Uppsala, Sweden) 10 ml/kg/h and Ringer's acetate (9 mg/ml; Baxter, Kista, Sweden) 8 ml/kg/h were also administered throughout the experiment. Macrodex 60 mg/ml (Pharmalink AB, Upplands Väsby, Sweden) was given to retain colloidal osmotic pressure. A catheter was guided by fluoroscopy via the jugular vein to the hepatic vein (V.H.) for posthepatic venous blood sampling. A midline laparotomy was performed and a catheter was positioned in the V.P. for sampling of pre-hepatic venous blood. Finally, a catheter was placed in the right femoral vein (V.F.) for peripheral blood sampling. Drainage of urine from the bladder and gastric fluids from the ventricle was also provided. Cannulation of the bile duct made bile collection possible throughout the experiment and a ligature placed below the point of insertion of the catheter prevented the bile from passing the catheter to reach the intestine. The Loc-I-Gut® perfusion tube (Synetics Medical, Stockholm, Sweden) was put in place in the jejunum by inserting it through an incision in the duodenum and then sliding it down inside the intestine. This multichannel tube is 175 cm long and has an external diameter of 5.3 mm. It is made of polyvinylchloride, contains six channels and is provided with two elongated inflatable latex balloons. The balloons are placed 10 cm apart and are each connected to one of the smaller channels. The two wider channels are used for infusion and aspiration of perfusate solution and the two remaining channels, which were not used in this study, are for administration of a marker substance and drainage. The single-pass perfusion technique is described in detail elsewhere (Lennernas et al., 1992). Briefly, a 10 cm long segment was created by inflating the balloons with approximately 15 ml of air. The segment was then rinsed with isotonic sodium chloride solution (37 °C) for at least 30 min before starting the experiment, to allow the animal to stabilize after surgery. After the stabilization period, the experiment was started (time 0) and the perfusion solution (at 37 °C; without any drugs) was pumped into the segment at a flow rate of 2 ml/min using a calibrated syringe pump (Model 355; Sage Instruments, Orion Research Inc, Cambridge, USA) in order to examine direct intestinal secretions. In addition, at time 0, the bolus doses of FEX or FEX plus verapamil were given to the control and treatment groups, respectively, and the infusions of FEX or FEX plus verapamil were immediately started via syringe pump(s) (Graseby 3100 Syringe Pump; Graseby Medical Ltd., Watford, UK). FEX was administered into the vena cava superior, and the verapamil bolus was administered into the V.P. The perfusate leaving the intestinal jejunal segment

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via the single-pass perfusion was collected on ice throughout the experiment at 10 min intervals. Bile was continuously collected on ice and weighed. Venous blood samples (5 ml) were drawn from the V.H., V.P. and V.F. sites at 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 300 and 360 min after the start of FEX administration. The blood samples were collected in BD Vacutainer® tubes (light green cap) containing sodium heparin and gel and were centrifuged at 3000 rpm for 10 min at 4 °C. The plasma was divided into two aliquots consisting of at least 1 ml each before storage. All bile, plasma, urine and perfusate samples were stored at -20°C pending analysis. The status of the animal was monitored throughout the experiment by recording heart rate, blood pressure, oesophageal temperature and breathing patterns. The animals were euthanized by administration of 10 ml potassium chloride (20 mmol) into the CVK at the end of the study.

2.5 Sample analysis 2.5.1 In vitro samples – pig liver microsomes The concentration of FEX in the in vitro samples was quantified using a modified high-performance liquid chromatography (HPLC) method with fluorescence detection, as described elsewhere (Hamman et al., 2001). Briefly, the HPLC system consisted of a CMA/200 refrigerated microsampler (CMAmicrodialysis, Sweden), an LC-10AD pump (Shimadzu, Kyoto, Japan) and an FP-1520 Intelligent Fluorescence Detector (Jasco, Japan) for fluorometric detection. Data were acquired and evaluated using CSW32 integrating software (Data Apex Ltd, Prague, Czech Republic). The samples were separated on a Hypersil Gold C18 column (4.6 x 250 mm, 5 µm particle size; Thermo Scientific, UK) after a sample injection volume of 100 µl. The mobile phase consisted of CH3CN and 20 mM ammonium acetate buffer, pH 7.4, at a ratio of 55:45. All peaks were isocratically eluted within 5 min at a mobile phase flow rate of 1 ml/min and were detected using fluorometric detection at wavelengths of 230 nm for excitation and 280 nm for emission. A standard curve was constructed using known concentrations of FEX in incubation media with deactivated microsomes. Diphenhydramine was used as internal standard. The lower limit of quantification of FEX for the method was validated to 50 nM (CV<20%).

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2.5.2 In vivo samples – plasma, bile, urine and intestinal perfusate The concentrations of FEX and verapamil were simultaneously quantified in samples collected from the in vivo study. For the plasma analysis, 1.0 ml of each plasma sample was mixed with 1 ml of sodium acetate buffer pH 4.0 (0.2 M) and 100 µl of internal standard solution ([2H6]-fexofenadine 7.6 ng/ml and [2H3]-verapamil 7.8 ng/ml). All samples were vortex mixed and centrifuged for 10 min at 3500 rpm. The samples were applied to Isolute C18 solid phase extraction columns (3 ml, 500mg; International Sorbent Technology Ltd, Mid Glamorgan, UK), which had been preconditioned sequentially with 2.0 ml methanol, 2.0 ml water and 1.5 ml sodium acetate buffer at pH 4.0 (0.2 M). After sample application, the columns were washed with 2.0 ml of water and dried for 5 min with nitrogen gas. The analytes were subsequently eluted with 5.0 ml of methanol containing 50 mM triethylamine. All eluates were collected and evaporated to dryness under a gentle stream of nitrogen gas at 50 °C. The residue in each vial was reconstituted in 50 µl of methanol and 0.1 % acetic acid in water (1:1, v/v) prior to liquid chromatography-electrospray-tandem mass spectrometry (LC-ESIMS/MS) analysis. For the bile analysis, 200 µl of each bile sample was mixed with 100 µl of internal standard solution ([2H6]-fexofenadine 7.6 ng/ml and [2H3]-verapamil 7.8 ng/ml) and 800 µl of 0.1 % aqueous acetic acid/methanol (1:1 v/v). All samples were vortex mixed and then centrifuged for 10 min at 3500 rpm. The supernatants were transferred to vials for LC-ESI-MS/MS analysis. For the urine analysis, 100 µL of each urine sample was mixed with 100 µl of the internal standard solution ([2H6]-fexofenadine 7.6 ng/ml and [2H3]-verapamil 7.8 ng/ml) and 900 µl of 0.1 % acetic acid (aq)/methanol (1:1, v/v). This mixture was then analyzed with LC-ESI-MS/MS. For the perfusate analysis, 1000 µl of each perfusate sample was mixed with 100 µl of the internal standard solution ([2H6]-fexofenadine 7.6 ng/ml and [2H3]-verapamil 7.8 ng/ml). This mixture was then analyzed with LC-ESI-MS/MS. An HP1100 liquid chromatograph with binary pump (Hewlett-packard, Waldbronn, Germany) and a Zorbax Eclipse XDB-C18 chromatographic column (50 x 2.1 mm id, particle diameter 5 mm; Agilent Technologies, Palo Alto, CA, United Sates) were used for the LC-ESIMS/MS procedures. The samples were eluted using a mobile phase composed of 0.1 % acetic acid in

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water and methanol. The mobile phase gradient was changed over time as follows: 0–3 min 30 % methanol, 3–4 min 30–90 % methanol, and 4–10 min 90 % methanol. The volumetric flow rate was 0.2 ml/min and the injection volume was 20 µl. A Quattro LC (Micromass, Manchester, UK) quadrupole-hexapole-quadrupole mass spectrometer with an electrospray interface operating at positive potential was connected to the column outlet. The mass spectrometer was tuned manually for sensitivity during direct infusion of a standard solution of FEX. The interfacial parameters used during analysis were capillary 2.80 kV, cone 35 V and extractor 4 V. The chromatography was performed at ambient temperature. The mass spectrometer was run in the selected reaction monitoring (SRM) mode, switching between the m/z transitions 502.2 → 171.0 for FEX [M+H+] and 508.2 → 177.0 for [2H6]-fexofenadine [M+H+] with collision energy of 35 eV, and between the m/z transitions 455.6 → 165.0 for verapamil [M+H+] and 458.6 → 165.0 for [2H3]-verapamil [M+H+]. The limits of quantification in all the biological matrices were 0.2 ng/ml and 0.70 ng/ml for FEX and verapamil, respectively, in plasma, 0.49 ng/ml for both analytes in bile, 0.2 and 0.49 ng/ml, respectively, in urine and 0.2 ng/ml for both analytes in perfusate.

2.6 Data analysis 2.6.1 In vitro enzyme kinetics The enzyme kinetics in pig liver microsomes were determined using the multiple depletion curves method as previously described (Sjögren et al., 2009). In short, the models were simultaneously fitted to the disappearance of FEX for both starting concentrations. Correction for loss of enzymatic activity was included as mono-exponential decay. The reaction rate was tested for both linear kinetics (equation 1) and non-linear kinetics (equation 2). dC = CL int × C prot × C × fu inc . × e − k AC ×t dt

eq. 1

where C is the concentration of FEX in the incubation, CLint is the metabolic intrinsic clearance, Cprot is the microsomal protein concentration used in the incubation and kAC is the rate constant describing the potential reduction in microsomal activity.

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dC ⎛ Vmax × C prot × C × fuinc. ⎞ −k AC ×t ⎟×e =⎜ dt ⎝⎜ K m + (C × fuinc. ) ⎠⎟

eq. 2

where Vmax and Km represent the theoretical maximum reaction rate (amount/time/mg protein) and the Michaelis constant (amount/ volume), respectively. For non-linear kinetics, CLint (volume/time/mg protein) was calculated by CLint = Vmax/Km.

2.6.2 Pharmacokinetics – non-compartmental analysis (NCA) In the NCA, the area under the concentration-time curve (AUC) was calculated by adopting the linear trapezoidal method for ascending values and the log trapezoidal method for descending values. The AUC0-6 was determined from time 0 to the last measured concentration (Clast) at 6h. The AUC from time 0 to infinity (AUC 0-∞) was determined by adding the AUC from the last measured concentration to that at infinity to the AUC0-6. This was achieved via extrapolation; the last measured concentration was divided by the terminal rate constant (ke) obtained by log-linear regression analysis of the last three to five concentrations. The apparent terminal half-life (t½) was obtained from t½ = ln2/ke. The maximum peak plasma concentration (Cmax) and the time at which the maximum peak occurred (tmax) were derived directly from the plasma concentration-time curves. The liver extraction ratio (EH) for the compounds was calculated using equation 3:

EH =

AUCV . P. − AUCV . H . AUCV . P.

eq. 3

where AUCV.P. and AUCV.H. are the areas under the plasma concentration-time curves over 0-6 h, from the V.P. and V.H., respectively.

The fraction of the dose excreted into the intestine (fe.int) and the intestinal clearance (CLI) were calculated from the relationships in equation 4:

f e. int =

Atot.I × 50 CLI = Doseiv CL

eq. 4

where Atot.I is the total amount of FEX excreted into the jejunal segment during the single pass

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perfusion and Doseiv is the total iv dose of FEX given to the animal. CL is the total body clearance of FEX, calculated by CL = AUC 0-∞/Doseiv. Ae.int was scaled up by 50 times as the total intestinal length has been reported to be approximately 5 m, i.e. 50 times longer than the perfused 10 cm segment (Bergman et al., 2009).

The fraction of the dose excreted in urine (fe.urine) was calculated according to equation 5:

f e.urine =

Atot.urine CLR = Doseiv CL

eq. 5

where Atot.urine is the total amount of FEX excreted into the urine, collected during the experiment (0– 6 h) and CLR is the renal clearance.

The amount of the dose excreted into the bile (Atot.bile) was calculated by taking the concentration in each sample and multiplying it by the collected volume representing each time period. The fraction of the dose excreted in bile (fe.bile) was calculated by dividing Atot.bile by the administered dose (equation 6).

f e,bile =

Atot .bile Dose

eq. 6

The biliary clearance (CLbile) was calculated using equation 7:

CL bile =

Atot .bile AUC V . P .( 0 − 6 )

eq. 7

where AUCV.P.(0-6) is the area under the plasma concentration-time curve in the V.P. over the period 0– 6 h after dosing.

The bile flow was calculated using the total volume of bile quantitatively collected during each time interval divided by the duration of the interval.

2.6.3 Pharmacokinetics – physiologically based pharmacokinetic (PBPK) analysis

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A previously developed PBPK model was used for mechanistic elucidation of the PK of iv FEX with and without co-administration of an iv dose of verapamil (Sjögren et al., 2012). The model structure and physiology parameters are described in detail elsewhere (Sjögren et al., 2012). In the absence of specific pig data, some parameters were adopted from rat data, assuming similarity. These cases are clearly highlighted. Briefly, all tissues except the liver were modelled as well stirred compartments with perfusion-limited distribution. The change in tissue concentration was described according to equation 8:

dCT = dt

∑v

⎛ C × QT × B : P ⎞ ⎟ − ⎜⎜ T ⎟ K P ,T ⎠ ⎝ VT

eq. 8

where T indicates tissue, and C is the concentration of FEX, Q is the rate of blood flow, B:P is the blood to plasma volume ratio, V is the volume and KP is the partition coefficient constant of the relevant tissue. The inlet rate is determined by the sum of the outlet rates (Σv) from the tissues (i.e., compartments) upstream in the blood circulation. KP,T values for the two lumped tissue compartments were estimated as previously described (Sjögren et al., 2012). Other KP,T values were collected from Poirier et al., based on the tissue composition models described in Rodgers et al. and originating from a rat model (Poirier et al., 2009; Rodgers et al., 2005; Rodgers and Rowland, 2006). The concentration of FEX in kidney tissue was accounted for by renal excretion according to equation 9:

dC kid dt

⎛ C kid ((QK × B : P ) + (CLren × fu p ))⎞ ⎟ v MACKID − ⎜⎜ ⎟ K P ,kid ⎝ ⎠ = Vkid

eq. 9

where Ckid is the concentration of FEX in the kidney, CLren is the renal clearance of unbound FEX, QK is the rate of kidney blood flow, fup is the fraction of unbound FEX in plasma in the rat (fup = 0.34 (Mahar Doan et al., 2004)) and vMACKID is the inlet rate of FEX from the mixed arterial compartment (MAC). The rate of appearance of unchanged FEX in the urine was modelled according to equation 10:

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eq. 10

dAurine = C kid × fu p × CLren dt where Aurine is the amount of FEX excreted in the urine.

The liver was modelled as two compartments: the liver vascular compartment (LVC), which is linked to the blood stream, and the liver cell compartment (LCC) (Sjögren et al., 2012). The LCC represents the intracellular compartment of the hepatocytes from which elimination was assumed to occur by metabolism and biliary excretion. The transport of drug to and from the LVC and LCC was modelled using permeability limited distribution as a bidirectional passive diffusion clearance (CLdif) and a unidirectional membrane uptake clearance (CLupt) to the LCC from the LVC. FEX is a poorly permeating compound and excretion to the bile was therefore considered to occur solely by CM transport as efflux clearance (CLeff). Reabsorption from the bile to the LCC was assumed not to occur. Passive membrane diffusion was considered as a possible distribution mechanism and quantification was based on in vitro measurements in rat hepatocytes, scaled according to equation 11:

CLdif = Pdif × HPGL × WT .liver

eq. 11

where Pdif is the passive permeability corrected for protein content in the hepatocyte assay (Pdif = 2.2 µl min-1 per 106 hepatocytes) (Poirier et al., 2009), HPGL is the hepatocellularity (120 × 106 hepatocytes per g liver) and WT.liver (31.6 g/kg body weight) is the liver weight.

The CM uptake and excretion were described by non-linear kinetics according to equations 12 and 13.

CLupt =

CLeff =

(K

J max .upt

m.upt

× K P. LVC ) + (C LVC × fu p ) J max .eff

eq. 12

eq. 13

K m.eff + (C LCC × fu h )

Michaelis constants for the total cellular CM uptake (Km.upt) and CM efflux (Km.eff) for FEX were adopted from previous measurements in the rat (Km.upt = 271 µM, Km.eff = 598 µM, (Poirier et al., 2009; Poirier et al., 2008). CLupt and CLeff were determined via estimation of the maximum rate of

16

uptake (Jmax.upt) and efflux (Jmax.eff). fuh is the fraction of unbound FEX in the hepatocytes (fuh=0.856 (Poirier et al., 2009)) and KP.LVC represents the instantaneous and at all times proportional partitioning of FEX to tissue components in the liver other than the hepatocytes (e.g. cellular components within cell types other than hepatocytes, interstitial water components, connective tissues, tissue fat components, etc.). To minimize the complexity of the model, the same level of nonspecific binding as was used for LVC and LCC was adopted (i.e. one level throughout the liver) even though it is acknowledged that differences may exist; i.e., KP.LVC = 1/fuh. CLVC and CLCC are the concentrations of FEX in the LVC and LCC, respectively.

The rate of metabolism, i.e. the metabolic clearance (CLmet), was described by non-linear or linear kinetics, depending on the results acquired from the in vitro experiment. CLint or Vmax from the microsome experiment were scaled up according to the liver microsomal content (Aprot.liv =1.14 g/kg body weight) (Sjögren et al., 2012). CLmet was estimated via estimation of a metabolic scaling factor (SFmet) according to equation 14 (linear) or equation 15 (non-linear).

CLmet = CLint × Aprot.liv × SFmet CLmet =

Vmax × Aprot .liv × SFmet

eq. 14 eq. 15

K m + (C LCC × fu h )

Km was considered to have the same value in vivo as that estimated in the in vitro experiments.

The concentration of FEX in the liver was described by combining equations 11 – 15 and integrating the physiological parameters for blood and tissue according to equations 16 (LVC) and 17 (LCC).

dCLVC dt

⎛ C ((Q × B : P ) + ((CLdif + CLupt )× fu p ))⎞ ⎟ vMACLVC + vLCCLVC − ⎜ LVC L ⎜ ⎟ K P, LVC ⎝ ⎠ = VLVC

eq. 16

where QL represents the rate of liver blood flow, VLVC is the LVC volume and vMACLVC and vLCCLVC represent the inlet rates of FEX from the MAC and the LCC, respectively.

17

dC LCC v LVCLCC − (C LCC × fu h × (CLmet + CLeff + CLdif )) = dt VLCC

eq. 17

where VLCC is the volume of the LCC and vLVCLCC represents the inlet rate (including both the passive and CM processes) of FEX from the LVC to the LCC. The rate of appearance of unchanged FEX in the biliary compartment was modelled according to equation 18:

eq. 18

dAbile = C LCC × fu h × CLbile dt where Abile is the amount of FEX excreted to bile.

Hepatic clearance (CLH), involving both transport and metabolism, was calculated according to equation 19 as previously described (Liu and Pang, 2005; Webborn et al., 2007):

CL H =

Q H × B : P × fu p × (CL met + CL bile ) × (CL upt + CL dif

)

Q H × B : P × (CL met + CL dif + CL bile ) + ((CL upt + CL dif )× (CL met + CL bile ) × fu p )

eq. 19

where QH is the rate of liver blood flow.

CLR was calculated using the well-stirred model (equation 20):

CLR =

QK × fu p × CLren

QK + (CLren × fu p B : P )

eq. 20

where QK is the rate of kidney blood flow. The total clearance (CLtot) was calculated as the sum of CLH and CLR, assuming negligible contribution from other elimination pathways.

The volume of distribution at steady state (Vss) was calculated using equation 21 (Poulin and Theil, 2002):

Vss = V pla + (Very × E : P) + ∑ K p ,i × VT ,i (1 − Ei )

18

eq. 21

where Vpla is the plasma volume, Very is the volume of erythrocytes, E:P is the erythrocyte/plasma concentration ratio (E:P = (B:P-(1-hematocrit))/hematocrit), VT,i is the physiological volume (weight) of the i-th tissue, and KP,i and Ei are the partition coefficient constant and extraction ratio, respectively. E was calculated as E = CL/(Q×B:P) where CL is the plasma clearance. A hematocrit of 0.27 was adopted (Lundahl et al., 2011).

In all, six parameters were estimated simultaneously through the PBPK analysis: CLmet, CLupt, CLeff, CLren, and KP.T for the two lumped tissue compartments. Akaike information criteria, visual examination of data, residual plots and the precisions of parameter estimation were used to evaluate the fit of the models. All analyses of kinetic data were performed (weighted 1/ŷ2) using WinNonlin Professional software V5.2 (Pharsight Corp., CA). Mean values were presented together with standard deviation (SD) or as otherwise stated.

3. Results 3.1 In vitro – enzyme kinetics The 90 min turnover of FEX in the hepatic microsomes was 4.4 ± 0.09 % and 2.1 ± 0.38 % (n=3) at a starting concentration of 0.2 µM and 20 µM, respectively. The low variability in the data enabled the adopted analysis methodology to determine CLint with high precision: CLint = 0.38 ± 0.12 µl/min/mg protein. By adopting scaling according to equation 14, a predicted CLmet of 137 µl/min/kg was calculated. The available data did not allow determination of non-linear kinetics or loss of enzyme activity during the incubation. There appeared to be no binding of FEX to hepatic microsomes. A plot of the concentration-time data including the model fit is shown in Figure 1.

3.2 In vivo results All animals completed surgery successfully and no effects on the physiological status due to the administered drugs were observed. Blood status and circulation were monitored throughout the

19

experiment and remained normal at all times. The concentration-time profiles for FEX from the plasma and bile sampling sites are presented in Figures 2 and 3, respectively. The concentration-time profiles in V.P. and bile for verapamil are shown in Figure 4. The bile flow was 0.6 ± 0.2 ml/min and 0.4 ± 0.2 ml/min in the control and treatment groups, respectively, which are similar to previously published values (Bergman et al., 2009; Petri et al., 2006).

3.3 Pharmacokinetics – non-compartmental analysis The plasma and bile PK parameters for FEX and verapamil are displayed in Table 1. There were no significant differences in PK parameters between the three plasma sites for FEX and hence only parameters related to the V.P. are reported. Plasma CL, t½ and EH values for FEX were 1.7 ± 0.3 h, 217 ± 91 ml/min, 0.03 ± 0.06 in the control group and were not significantly different in the treatment group (2.1 ± 0.6 h, 125 ± 30 ml/min, 0.02 ± 0.04). The observed plasma Cmax was 1440 ± 507 ng/ml and 1910 ± 407 ng/ml in the control and treatment groups, respectively, as measured for all pigs at the first time point of sampling (tmax = 0.33 h). A significant (P<0.05) increase in the plasma AUC for FEX in the V.P. and V.H., but not in the V.F., was seen as a consequence of co-administration of verapamil. The amount FEX excreted into the bile was 24.7 ± 7.4 % and 19.2 ± 10 % of the iv dose in the control and treatment groups, respectively, and the corresponding CLbile values were 51.2 ± 25 and 26.5 ± 15 ml/min. The biliary AUC0-6 was 71 (treatment) to 106 (control) times higher than the AUC0-6 in the V.P, and the Cmax, was 74 (treatment) to 133 (control) times higher in the bile compared to that observed in the V.P. No significant differences in biliary parameters between the groups were detected in this NCA analysis. No difference in fe.urine was observed between the two groups (31.5 % and 21.4 %, respectively). However, CLR was significantly (P<0.05) lower in the treatment group (26.8 ± 9.6 ml/min) than in the control group (68.4 ± 36 ml/min). The intestinal perfusate AUC0-6 for FEX was 13 to 28 times lower than the femoral plasma AUC0-6 for FEX. The fe.int was significantly (P<0.05) lower in the treatment group (0.0045 ± 0.003 %) than in the control group (0.01 ± 0.003 %). The intestinal clearances were 0.05 ± 0.04 ml/min and 0.013 ± 0.008 ml/min in the control and treatment groups, respectively. The recovery of the nonabsorbable volume

20

marker 14C-PEG 4000 was 107 ± 27 % and 115 ± 16 % in the control and treatment groups, respectively. Cmax, AUC0-6 and t½ values for verapamil were 147 ± 43 ng/ml, 267 ± 56 h ng/ml and 1.9 ± 0.6 h, respectively.

Table 1: Fexofenadine pharmacokinetic parameters (means ± SD, n=5) calculated from the portal vein plasma concentration-time profiles, and the bile and urine measurements for fexofenadine alone (control) or with co-administration of verapamil (treatment) using noncompartmental analysis.

bile / urine

plasma

Parameter Control AUC0-6 (h ng/ml) 2800 ± 1100 AUC∞ (h ng/ml) 3060 ± 1280 EH 0.03 ± 0.06 t½ (h) 1.7 ± 0.3 Cmax (ng/ml) 1440 ± 510 Tmax (h) 0.33 ± 0 CL (ml/min) 217 ± 91 AUC0-6 (h µg/ml) 298 ± 110 Cmax (µg/ml) 192 ± 95 Atot.bile (mg) 8.60 ± 2.6 fe.bile (%) 24.7 ± 7.4 CLbile (ml/min) 51.2 ± 25 Atot.urine (mg) 10.9 ± 3.6 fe.urine (%) 31.5 ± 10 68.4 CLR (ml/min) ± 36 * Statistically different from control (P < 0.05)

Treatment 4190 ± 780* 4830 ± 1100* 0.02 ± 0.04 2.1 ± 0.6 1910 ± 410 0.33 ± 0 125 ± 30 297 ± 91 142 ± 61 6.67 ± 3.5 19.2 ± 10 26.5 ± 15 7.45 ± 2.0 21.4 ± 5.7 26.8 ± 9.6*

3.4 Pharmacokinetics – physiologically based pharmacokinetic analysis The PBPK model fitted the in vivo data in plasma, bile and urine adequately, as displayed in Figure 5. Table 2 summarizes the mechanistic and PK parameters estimated using the PBPK model. This mechanistic analysis showed that the rate of CM uptake (CLupt = 172 ± 70 ml/min/kg) was about 20 times faster than passive diffusion (CLdif = 8.4 ml/min/kg). CLmet and CLbile were estimated to be 494 ± 270 µl/min/kg and 308 ± 110 µl/min/kg, respectively. Secondary parameters CLH, CLR and Vss were calculated as 4.1 ± 1.5 ml/min/kg, 2.1 ± 1.0 ml/min/kg and 310 ± 52 ml/kg, respectively. Verapamil reduced the CLbile (134 ± 69 µl/min/kg; -56 %) and CLR (1.0 ± 0.21 ml/min/kg; -52%) of FEX, but had no other significant effects on the primary parameters. The reduction in biliary efflux was not reflected in a significant reduction of CLH in verapamil-treated animals (control: 4.1 ± 1.5 ml/min/kg; treatment:

21

3.1 ± 1.1 ml/min/kg). This is a consequence of the multiple sources of variability for the calculation of CLH (equation 19). Similarly, no significant effect of verapamil on CLtot was detected (control: 6.2 ± 1.9 ml/min/kg; treatment: 4.2 ± 1.0 ml/min/kg).

Table 2: Pharmacokinetic parameters (means ± SD; n=5) calculated from the plasma concentrationtime profiles, and the bile and urine measurements for fexofenadine alone (control) or with coadministration of verapamil (treatment) using the physiologically based pharmacokinetic model. Parameter

Control

secondary

primary

CLdif (ml/min/kg)

Treatment 8.4

CLupt (ml/min/kg)

172 ± 70

206 ± 83

CLmet (µl/min/kg)

494 ± 270

379 ± 210

CLbile (µl/min/kg)

308 ± 110

134 ± 69*

CLH (ml/min/kg)

4.1 ± 1.5

3.1 ± 1.1

CLR (ml/min/kg)

2.1 ± 1.0

1.0 ± 0.21*

CLtot (ml/min/kg) Vss (ml/kg)

6.2 ± 1.9 310 ± 52

4.2 ± 1.0 270 ± 44

* Statistically different from control (P < 0.05)

4. Discussion A 50% increase in systemic plasma exposure of FEX following iv administration was observed as a direct consequence of a concomitant single iv dose of verapamil (35mg). Although a significant reduction in renal clearance was observed, no effects on other PK parameters were detected by NCA. Nonetheless, when all available information per animal was used simultaneously in the PBPK analysis, a significant reduction in both biliary and renal clearance was determined. FEX has been reported to be primarily eliminated through biliary and renal excretion in several species (Lippert et al., 1995; Matsushima et al., 2008; Ogasawara et al., 2007; Ujie et al., 2008). As the passive permeability of FEX is poor, the potential importance of CM flux across biological membranes is expected to be high. Inhibition of intestinal P-pg efflux has accordingly been a common hypothesis for the observed alterations in FEX PK after concomitant oral administration of P-gp inhibitors such as verapamil. The increase in FEX plasma exposure caused by verapamil in this study was not observed by Petri et al. after intra-jejunal administration of both FEX and verapamil (Petri et

22

al., 2006). Accordingly, the results of this study support the conclusion reached by Petri et al. that the effect of verapamil is most likely due to its disposition in the liver and kidney rather than a result of intestinal absorption processes (Petri et al., 2006). The results of the PBPK analysis suggest that sinusoidal uptake of FEX by CM processes in the liver is approximately 20 times higher than by passive permeation. This suggests that carriers mediate efficient uptake of FEX from the blood into the hepatocytes, making metabolic and biliary clearances the rate-limiting processes for hepatic elimination. The suggestion that uptake of FEX is not rate limiting for hepatic elimination is in agreement with the results of a mechanistic analysis of the hepatic disposition of FEX in rats (Poirier et al., 2009). FEX is a substrate for several uptake transporters that are expressed in the pig liver, such as OATP1A2, OATP1B1 and OATP1B3 (Goh et al., 2002; Niemi et al., 2005; Ponsuksili et al., 2005; Yu et al., 2013). Verapamil is a potent inhibitor (IC50 = 2.6 µM) of OATP1A2, which is expressed in the cholangiocytes in the human liver (Bailey et al., 2007; Lee et al., 2005). The exact location of OATP1A2 in the pig liver is not known. However, as the CM uptake in this study was not affected by verapamil, it appears that there is only minor involvement of this specific carrier, even if cholangiocytes were not specifically specified in the PBPK model. The result is thus in accordance with the suggestion that OATP1B1 and OATP1B3 are important for the sinusoidal uptake of FEX into the hepatocytes (Shimizu et al., 2005). As the model only permitted unidirectional flow from the intravascular space to bile, no comparison of passive and CM transport across the canalicular membrane was made in this study. However, the FEX concentrations in bile were approximately 100 times higher than those in plasma. As CM uptake at the sinusoidal membrane alone is not sufficient to create this concentration gradient, involvement of CM efflux processes at the canalicular membrane is strongly indicated. Also, the reduction in the biliary excretion rate of FEX by verapamil is in accordance with a previous hypothesis of inhibition of P-gp-mediated efflux of FEX by verapamil (Petri et al., 2006). FEX and verapamil have previously been shown to be P-gp substrate and inhibitor, respectively, and P-gp is expressed in the pig liver (Petri et al., 2004; Schrickx, 2006). Involvement of canalicular efflux of FEX mediated by MRP2 is also possible, as FEX and verapamil are potential substrate and inhibitor, respectively, of this transporter (Goh et al., 2002; Konno et al., 2003; Matsushima et al., 2008; Ming et al., 2011; Schrickx, 2006; Wong et al., 2009). However, without PBPK analysis, the

23

effect of verapamil on the biliary excretion of FEX could not have been detected in this study. This emphasizes the important contribution that a PBPK analysis makes to the mechanistic investigation of in vivo drug disposition processes. The results showed that the metabolic clearance of FEX (494 µl/min/kg) was approximately equal to the biliary excretion clearance (308 µl/min/kg). This is in contrast to the common consensus that metabolism is of negligible importance in the overall elimination of FEX (Cvetkovic et al., 1999; Poirier et al., 2009; Yamazaki et al., 2010). However, there is a lack of unambiguous evidence of this hypothesis in humans. For example, the commonly cited radioactive mass balance study after oral administration reported by Lippert and co-workers does not provide information to exclude metabolic elimination (Lippert et al., 1995). In addition, the fraction of total AUC that was represented by circulating metabolites in plasma was higher after oral (32%) than after iv (16%) administration, indicating that FEX is susceptible to biotransformation in the intestine (Lappin et al., 2010). Furthermore, as FEX is a molecule with poor passive permeation, any prospective metabolite will probably permeate cell membranes even less well. Hence, metabolites formed inside the hepatocytes are not expected to progress easily to the plasma and plasma exposure will thus not reflect the true level of FEX biotransformation. In this study, however, comprehensive data analysis and the adopted in vivo model facilitated estimation of the involved mechanisms in the disposition of FEX in the liver. The in vitro results obtained in this study supported these findings, although the calculation of CLmet using direct scaling from the measured in vitro CLint was approximately 4 times lower than estimated in the PBPK analysis. Interestingly, this level of underestimation is similar to the 4.8-fold underestimation that was reported from an investigation of the hepatic disposition of repaglinide using the same model (Sjögren et al., 2012). Moreover, the fact that verapamil did not significantly affect the metabolism of FEX suggests that enzymes other than the pig CYP3A29/3A39/3A46 enzymes, analogues to CYP3A4, are central for the biotransformation of FEX (Thörn et al., 2011). The contribution of intestinal excretion to the total elimination was negligible, similar to observations made in previous studies performed in our lab (Bergman et al., 2009). The values for t½ and EH reported in this study were not consistent with the results of an intestinal perfusion study performed in pigs by Petri et al. (Petri et al., 2006). The t½ was significantly shorter in

24

this study, 1.7 h in comparison to 5.1 h, and the hepatic extraction was significantly lower, only 3% compared to 15% (Petri et al., 2006). These observations suggest that hepatic extraction does not necessarily only reflect hepatic elimination but also partly reflects the distribution. Analysis (not shown) of the plasma profiles presented by Petri et al. indicated that hepatic extraction during the perfusion was significantly higher (21%) than at the end of the perfusion (4%) (Petri et al., 2006). A time-dependent EH has previously been reported by our group for several drugs; this is hypothesized to be caused by distribution processes in the liver and the gut mucosa (Thörn et al., 2012). However, further research is required to fully understand these reversible tissue binding mechanisms in the case of FEX. High variability in t½ (2.6 – 17.6 h) has previously been reported in humans (Smith and Gums, 2009). It has been speculated that this could be a consequence of an undefined terminal phase and intestinal absorption rate-limited kinetics. However, the terminal phase was well characterized in this study and the intestinal segment in the previous perfusion study (Petri et al., 2006) was completely rinsed after perfusion, which excludes the possibility of further absorption. These circumstances are not compatible with the proposed theories for the variable t½ of FEX, suggesting that further investigations may be necessary to find an explanation for this observation. The renal route is important for elimination of FEX in both humans and rats (Lappin et al., 2010; Matsushima et al., 2008). As with humans (Lappin et al., 2010), renal clearance of FEX in this study using pigs accounted for approximately 1/3 of the total plasma clearance. However, in contrast to observations in humans (Yasui-Furukori et al., 2005), concomitant administration of verapamil reduced the CLR of FEX in this study by 50%. This difference does not correspond with the expression of P-gp in the kidney tubule, as it is expressed in humans but is potentially expressed less in pigs (Schrickx, 2006; Thiebaut et al., 1987). However, the expression of P-gp in the pig kidney is yet to be fully investigated (Schrickx, 2006). The mechanism of this interaction could also possibly be related to inhibition of MRP1 and MRP2, which are readily expressed in the pig kidney (Goh et al., 2002; Konno et al., 2003; Matsushima et al., 2008; Ming et al., 2011; Schrickx, 2006; Wong et al., 2009). However, as no unambiguous conclusions can be made either from this study or from previous publications, additional investigations are required to elucidate the mechanisms of the reduced CM renal clearance.

25

In conclusion, the in vivo multiple-sampling-site technique and PBPK analysis used in this study showed that verapamil reduces the biliary and urinary excretion of FEX in pigs. The results indicated that metabolism is probably equally as important as excretion to the bile and urine in the total elimination of the drug. Significant CM uptake from blood into the hepatocytes was also demonstrated. This knowledge was not obtainable by conventional NCA, but was gained through PBPK analysis, demonstrating the potential value of this method when used in combination with a multiple-sampling-site, in vivo model.

Acknowledgements. We thank Martin Herrström, Patrik Forsell and Anders Nordgren for excellent technical assistance.

26

References

Bailey, D.G., Dresser, G.K., Leake, B.F., Kim, R.B., 2007. Naringin is a major and selective clinical inhibitor of organic anion-transporting polypeptide 1A2 (OATP1A2) in grapefruit juice. Clin Pharmacol Ther 81, 495-502. Bergman, E., Lundahl, A., Fridblom, P., Hedeland, M., Bondesson, U., Knutson, L., Lennernäs, H., 2009. Enterohepatic disposition of rosuvastatin in pigs and the impact of concomitant dosing with cyclosporine and gemfibrozil. Drug Metab Dispos 37, 2349-2358. Cvetkovic, M., Leake, B., Fromm, M.F., Wilkinson, G.R., Kim, R.B., 1999. OATP and Pglycoprotein transporters mediate the cellular uptake and excretion of fexofenadine. Drug Metab Dispos 27, 866-871. Dresser, G.K., Bailey, D.G., Leake, B.F., Schwarz, U.I., Dawson, P.A., Freeman, D.J., Kim, R.B., 2002. Fruit juices inhibit organic anion transporting polypeptide-mediated drug uptake to decrease the oral availability of fexofenadine. Clin Pharmacol Ther 71, 11-20. FDA, 2011. Drug Development and Drug Interactions: Table of Substrates, Inhibitors and Inducers, Development & Approval Process (Drugs). U.S. Department of Health & Human services, U.S. Food and Drug Administration (FDA). Fisher, M.B., Campanale, K., Ackermann, B.L., VandenBranden, M., Wrighton, S.A., 2000. In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin. Drug Metab Dispos 28, 560-566. Funk, C., 2008. The role of hepatic transporters in drug elimination. Expert Opin Drug Metab Toxicol 4, 363-379. Giacomini, K.M., Huang, S.M., Tweedie, D.J., Benet, L.Z., Brouwer, K.L., Chu, X., Dahlin, A., Evers, R., Fischer, V., Hillgren, K.M., Hoffmaster, K.A., Ishikawa, T., Keppler, D., Kim, R.B., Lee, C.A., Niemi, M., Polli, J.W., Sugiyama, Y., Swaan, P.W., Ware, J.A., Wright, S.H., Yee, S.W., Zamek-Gliszczynski, M.J., Zhang, L., 2010. Membrane transporters in drug development. Nat Rev Drug Discov 9, 215-236. Goh, L.B., Spears, K.J., Yao, D., Ayrton, A., Morgan, P., Roland Wolf, C., Friedberg, T., 2002. Endogenous drug transporters in in vitro and in vivo models for the prediction of drug disposition in man. Biochem Pharmacol 64, 1569-1578. Hamman, M.A., Bruce, M.A., Haehner-Daniels, B.D., Hall, S.D., 2001. The effect of rifampin administration on the disposition of fexofenadine. Clin Pharmacol Ther 69, 114-121. Hedman, A., Meijer, D.K., 1998. The stereoisomers quinine and quinidine exhibit a marked stereoselectivity in the inhibition of hepatobiliary transport of cardiac glycosides. J Hepatol 28, 240-249. Kobayashi, D., Nozawa, T., Imai, K., Nezu, J., Tsuji, A., Tamai, I., 2003. Involvement of human organic anion transporting polypeptide OATP-B (SLC21A9) in pH-dependent transport across intestinal apical membrane. J Pharmacol Exp Ther 306, 703-708. Konno, T., Ebihara, T., Hisaeda, K., Uchiumi, T., Nakamura, T., Shirakusa, T., Kuwano, M., Wada, M., 2003. Identification of domains participating in the substrate specificity and subcellular localization of the multidrug resistance proteins MRP1 and MRP2. J Biol Chem 278, 22908-22917. Kruijtzer, C.M., Beijnen, J.H., Rosing, H., ten Bokkel Huinink, W.W., Schot, M., Jewell, R.C., Paul, E.M., Schellens, J.H., 2002. Increased oral bioavailability of topotecan in combination with the breast cancer resistance protein and P-glycoprotein inhibitor GF120918. J Clin Oncol 20, 2943-2950. Lappin, G., Shishikura, Y., Jochemsen, R., Weaver, R.J., Gesson, C., Houston, B., Oosterhuis, B., Bjerrum, O.J., Rowland, M., Garner, C., 2010. Pharmacokinetics of fexofenadine: 27

evaluation of a microdose and assessment of absolute oral bioavailability. Eur J Pharm Sci 40, 125-131. Lee, W., Glaeser, H., Smith, L.H., Roberts, R.L., Moeckel, G.W., Gervasini, G., Leake, B.F., Kim, R.B., 2005. Polymorphisms in human organic anion-transporting polypeptide 1A2 (OATP1A2): implications for altered drug disposition and central nervous system drug entry. J Biol Chem 280, 9610-9617. Lennernas, H., Ahrenstedt, O., Hallgren, R., Knutson, L., Ryde, M., Paalzow, L.K., 1992. Regional jejunal perfusion, a new in vivo approach to study oral drug absorption in man. Pharm Res 9, 1243-1251. Lippert, C., Ling, J., Brown, P., Burmaster, S., Eller, M., Cheng, L., Thompson, R., Weir, S., 1995. Mass balance and pharmacokinetics of MDL 16,455A in healthy male volunteers. Pharm Res, s390. Liu, L., Pang, K.S., 2005. The roles of transporters and enzymes in hepatic drug processing. Drug Metab Dispos 33, 1-9. Lundahl, A., Hedeland, M., Bondesson, U., Lennernas, H., 2011. In vivo investigation in pigs of intestinal absorption, hepatobiliary disposition, and metabolism of the 5alpha-reductase inhibitor finasteride and the effects of coadministered ketoconazole. Drug Metab Dispos 39, 847-857. Mahar Doan, K.M., Wring, S.A., Shampine, L.J., Jordan, K.H., Bishop, J.P., Kratz, J., Yang, E., Serabjit-Singh, C.J., Adkison, K.K., Polli, J.W., 2004. Steady-state brain concentrations of antihistamines in rats: interplay of membrane permeability, P-glycoprotein efflux and plasma protein binding. Pharmacology 72, 92-98. Matsushima, S., Maeda, K., Hayashi, H., Debori, Y., Schinkel, A.H., Schuetz, J.D., Kusuhara, H., Sugiyama, Y., 2008. Involvement of multiple efflux transporters in hepatic disposition of fexofenadine. Mol Pharmacol 73, 1474-1483. Meijer, D.K., Bos, E.S., van der Laan, K.J., 1970. Hepatic transport of mono bisquaternary ammonium compounds. Eur J Pharmacol 11, 371-377. Ming, X., Knight, B.M., Thakker, D.R., 2011. Vectorial Transport of Fexofenadine across Caco-2 Cells: Involvement of Apical Uptake and Basolateral Efflux Transporters. Mol Pharm 8, 1677-1686. Niemi, M., Kivisto, K.T., Hofmann, U., Schwab, M., Eichelbaum, M., Fromm, M.F., 2005. Fexofenadine pharmacokinetics are associated with a polymorphism of the SLCO1B1 gene (encoding OATP1B1). Br J Clin Pharmacol 59, 602-604. Ogasawara, A., Kume, T., Kazama, E., 2007. Effect of oral ketoconazole on intestinal firstpass effect of midazolam and fexofenadine in cynomolgus monkeys. Drug Metab Dispos 35, 410-418. Perloff, M.D., von Moltke, L.L., Greenblatt, D.J., 2002. Fexofenadine transport in Caco-2 cells: inhibition with verapamil and ritonavir. J Clin Pharmacol 42, 1269-1274. Petri, N., Bergman, E., Forsell, P., Hedeland, M., Bondesson, U., Knutson, L., Lennernäs, H., 2006. First-pass effects of verapamil on the intestinal absorption and liver disposition of fexofenadine in the porcine model. Drug Metab Dispos 34, 1182-1189. Petri, N., Tannergren, C., Rungstad, D., Lennernäs, H., 2004. Transport characteristics of fexofenadine in the Caco-2 cell model. Pharm Res 21, 1398-1404. Poirier, A., Funk, C., Scherrmann, J.M., Lave, T., 2009. Mechanistic modeling of hepatic transport from cells to whole body: application to napsagatran and fexofenadine. Mol Pharm 6, 1716-1733. Poirier, A., Lave, T., Portmann, R., Brun, M.E., Senner, F., Kansy, M., Grimm, H.P., Funk, C., 2008. Design, data analysis, and simulation of in vitro drug transport kinetic experiments using a mechanistic in vitro model. Drug Metab Dispos 36, 2434-2444.

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Ponsuksili, S., Murani, E., Schellander, K., Schwerin, M., Wimmers, K., 2005. Identification of functional candidate genes for body composition by expression analyses and evidencing impact by association analysis and mapping. Biochim Biophys Acta 1730, 31-40. Poulin, P., Theil, F.P., 2002. Prediction of pharmacokinetics prior to in vivo studies. 1. Mechanism-based prediction of volume of distribution. J Pharm Sci 91, 129-156. Rodgers, T., Leahy, D., Rowland, M., 2005. Physiologically based pharmacokinetic modeling 1: predicting the tissue distribution of moderate-to-strong bases. J Pharm Sci 94, 1259-1276. Rodgers, T., Rowland, M., 2006. Physiologically based pharmacokinetic modelling 2: predicting the tissue distribution of acids, very weak bases, neutrals and zwitterions. J Pharm Sci 95, 1238-1257. Schinkel, A.H., Smit, J.J., van Tellingen, O., Beijnen, J.H., Wagenaar, E., van Deemter, L., Mol, C.A., van der Valk, M.A., Robanus-Maandag, E.C., te Riele, H.P., et al., 1994. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77, 491-502. Schrickx, J., 2006. title. PhD, Universiteit Utrecht, Utrecht. Shimizu, M., Fuse, K., Okudaira, K., Nishigaki, R., Maeda, K., Kusuhara, H., Sugiyama, Y., 2005. Contribution of OATP (organic anion-transporting polypeptide) family transporters to the hepatic uptake of fexofenadine in humans. Drug Metab Dispos 33, 1477-1481. Shitara, Y., Horie, T., Sugiyama, Y., 2006. Transporters as a determinant of drug clearance and tissue distribution. European Journal of Pharmaceutical Sciences 27, 425-446. Simonson, S.G., Raza, A., Martin, P.D., Mitchell, P.D., Jarcho, J.A., Brown, C.D., Windass, A.S., Schneck, D.W., 2004. Rosuvastatin pharmacokinetics in heart transplant recipients administered an antirejection regimen including cyclosporine. Clin Pharmacol Ther 76, 167177. Sjödin, E., Fritsch, H., Eriksson, U.G., Logren, U., Nordgren, A., Forsell, P., Knutson, L., Lennernäs, H., 2008. Intestinal and hepatobiliary transport of ximelagatran and its metabolites in pigs. Drug Metab Dispos 36, 1519-1528. Sjögren, E., Bredberg, U., Lennernäs, H., 2012. The pharmacokinetics and hepatic disposition of repaglinide in pigs: mechanistic modeling of metabolism and transport. Mol Pharm 9, 823841. Sjögren, E., Lennernäs, H., Andersson, T.B., Gråsjo, J., Bredberg, U., 2009. The multiple depletion curves method provides accurate estimates of intrinsic clearance (CLint), maximum velocity of the metabolic reaction (Vmax), and Michaelis constant (Km): accuracy and robustness evaluated through experimental data and Monte Carlo simulations. Drug Metab Dispos 37, 47-58. Smith, S.M., Gums, J.G., 2009. Fexofenadine: biochemical, pharmacokinetic and pharmacodynamic properties and its unique role in allergic disorders. Expert Opin Drug Metab Toxicol 5, 813-822. Solomon, H.M., Schanker, L.S., 1963. Hepatic transport of organic cations: active uptake of a quaternary ammonium compound, procaine amide ethobromide. by rat liver slices. Biochem Pharmacol 12, 621-626. Tannergren, C., Knutson, T., Knutson, L., Lennernäs, H., 2003a. The effect of ketoconazole on the in vivo intestinal permeability of fexofenadine using a regional perfusion technique. Br J Clin Pharmacol 55, 182-190. Tannergren, C., Petri, N., Knutson, L., Hedeland, M., Bondesson, U., Lennernäs, H., 2003b. Multiple transport mechanisms involved in the intestinal absorption and first-pass extraction of fexofenadine. Clin Pharmacol Ther 74, 423-436. Thiebaut, F., Tsuruo, T., Hamada, H., Gottesman, M.M., Pastan, I., Willingham, M.C., 1987. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci U S A 84, 7735-7738. 29

Thörn, H.A., Hedeland, M., Bondesson, U., Knutson, L., Yasin, M., Dickinson, P., Lennernäs, H., 2009. Different effects of ketoconazole on the stereoselective first-pass metabolism of R/S-verapamil in the intestine and the liver: important for the mechanistic understanding of first-pass drug-drug interactions. Drug Metab Dispos 37, 2186-2196. Thörn, H.A., Lundahl, A., Schrickx, J.A., Dickinson, P.A., Lennernäs, H., 2011. Drug metabolism of CYP3A4, CYP2C9 and CYP2D6 substrates in pigs and humans. Eur J Pharm Sci 43, 89-98. Thörn, H.A., Sjogren, E., Dickinson, P.A., Lennernäs, H., 2012. Binding processes determine the stereoselective intestinal and hepatic extraction of verapamil in vivo. Mol Pharm 9, 30343045. Ujie, K., Oda, M., Kobayashi, M., Saitoh, H., 2008. Relative contribution of absorptive and secretory transporters to the intestinal absorption of fexofenadine in rats. Int J Pharm 361, 711. Webborn, P.J., Parker, A.J., Denton, R.L., Riley, R.J., 2007. In vitro-in vivo extrapolation of hepatic clearance involving active uptake: theoretical and experimental aspects. Xenobiotica 37, 1090-1109. Wong, I.L., Chan, K.F., Tsang, K.H., Lam, C.Y., Zhao, Y., Chan, T.H., Chow, L.M., 2009. Modulation of multidrug resistance protein 1 (MRP1/ABCC1)-mediated multidrug resistance by bivalent apigenin homodimers and their derivatives. J Med Chem 52, 5311-5322. Yamazaki, A., Kumagai, Y., Yamane, N., Tozuka, Z., Sugiyama, Y., Fujita, T., Yokota, S., Maeda, M., 2010. Microdose study of a P-glycoprotein substrate, fexofenadine, using a nonradioisotope-labelled drug and LC/MS/MS. J Clin Pharm Ther 35, 169-175. Yasui-Furukori, N., Uno, T., Sugawara, K., Tateishi, T., 2005. Different effects of three transporting inhibitors, verapamil, cimetidine, and probenecid, on fexofenadine pharmacokinetics. Clin Pharmacol Ther 77, 17-23. Yu, Y., Liu, X., Zhang, Z., Xiao, Y., Hong, M., 2013. Cloning and functional characterization of the pig (Sus scrofa) organic anion transporting polypeptide 1a2. Xenobiotica.

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Figure captions: Figure 1: The depletion of fexofenadine in pig hepatic microsomes (1.2 mg protein/ml) at 0.2 µM (filled circles) and 20 µM (squares) over 90 min, showing the model fit (dashed lines). The observed data are shown as means ± SD (n=3).

Figure 2: The concentration-time profiles for fexofenadine in plasma (means ± SD, n=5) for the control and treatment groups in (A) portal vein (V.P.), (B) hepatic vein (V.H.) and (C) femoral vein (V.F.).

Figure 3: The concentration-time profiles for fexofenadine in bile (means ± SD, n=5) for the control and treatment groups.

Figure 4: The concentration-time profiles for verapamil (means ± SD, n=5) in the portal vein (V.P.) and bile for the treatment group.

Figure 5: The concentration-time profiles for fexofenadine in plasma (open symbols) and the accumulated amounts of fexofenadine in urine (filled circle) and bile (filled diamond) for a typical animal. Solid lines are the PBPK model fit for each site of observation.

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