Investigation of Hepatobiliary Disposition of ... - ACS Publications

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Investigation of Hepatobiliary Disposition of Doxorubicin Following Intrahepatic Delivery of Different Dosage Forms Elsa Lilienberg,† Charlotte Ebeling Barbier,‡ Rickard Nyman,‡ Mikael Hedeland,§,∥ Ulf Bondesson,§,∥ Niklas Axén,† and Hans Lennernas̈ *,† †

Department Department § Department ∥ Department ‡

of of of of

Pharmacy, Uppsala University, Box 580, 751 23 Uppsala, Sweden Radiology, Uppsala University Hospital, Uppsala University, 751 85 Uppsala, Sweden Medicinal Chemistry, Analytical Pharmaceutical Chemistry, Uppsala University, Box 574, 751 23 Uppsala, Sweden Chemistry, National Veterinary Institute (SVA), 751 89 Uppsala, Sweden

ABSTRACT: Unresectable, intermediate stage hepatocellular carcinoma (HCC) is often treated palliatively in humans by doxorubicin (DOX). The drug is administered either as a drug-emulsified-in-Lipiodol (DLIP) or as drug loaded into drug eluting beads (DEB), and both formulations are administered intrahepatically. However, several aspects of their in vivo performance in the liver are still not well-understood. In this study, DLIP and DEB were investigated regarding the local and systemic pharmacokinetics (PK) of DOX and its primary metabolite doxorubicinol (DOXol). An advanced PK-multisampling site acute in vivo pig model was used for simultaneous sampling in the portal, hepatic, and femoral veins and the bile duct. The study had a randomized, parallel design with four treatment groups (TI−TIV). TI (n = 4) was used as control and received an intravenous (i.v.) infusion of DOX as a solution. TII and TIII were given a local injection in the hepatic artery with DLIP (n = 4) or DEB (n = 4), respectively. TIV (n = 2) received local injections of DLIP in the hepatic artery and bile duct simultaneously. All samples were analyzed for concentrations of DOX and DOXol with UPLC-MS/MS. Compared to DLIP, the systemic exposure for DOX with DEB was reduced (p < 0.05), in agreement with a slower in vivo release. The approximated intracellular bioavailability of DOX during 6 h appeared to be lower for DEB than DLIP. Following i.v. infusion (55 min), DOX had a liver extraction of 41 (28−53)%, and the fraction of the dose eliminated in bile of DOX and DOXol was 20 (15−22)% and 4.2 (3.2−5.2)%, respectively. The AUCbile/AUCVP for DOX and DOXol was 640 (580−660) and 5000 (3900−5400), respectively. In conclusion, DLIP might initially deliver a higher hepatocellular concentration of DOX than DEB as a consequence of its higher in vivo release rate. Thus, DLIP delivery results in higher intracellular peak concentrations that might correlate with better anticancer effects, but also higher systemic drug exposure and safety issues. KEYWORDS: transarterial chemoembolization, drug eluting beads, DC bead, Lipiodol, hepatocellular carcinoma, doxorubicin, doxorubicinol, pharmacokinetics, hepatic drug delivery

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INTRODUCTION Hepatocellular carcinoma (HCC) is a primary liver cancer with a poor prognosis. Despite current therapies HCC is globally ranked as the third most common death-related cancer, and its incidence is increasing, especially in industrial countries.1,2 HCC is typically highly vascularized by the hepatic artery (HA).3 If the tumor is unresectable and classified as intermediate stage, palliative treatment is given via the HA. HA delivery is supposed to allow higher tumoral drug concentrations to be attained while reducing the systemic exposure, thereby increasing tumor response and reducing morbidity.3 In addition, a partial or full occlusion of the tumoral blood flow restricts the oxygen supply and is assumed to increase both concentration and residence time of the drug(s) in the target tumor area.3 Several different formulations of various chemotherapeutics and pharmaceutical excipients have been developed and clinically evaluated for transarterial chemoinfusion (TAI) and chemoembolization (TACE).4 Patients with untreated intermediate HCC, without © 2013 American Chemical Society

portal invasion or extrahepatic spread, have an expected median survival of about 16 months, and with TACE median survival is prolonged with about 4 months.5 To further increase survival rates and reduce morbidity, it is essential to create new, targeted drug-delivery approaches and dosing strategies. Doxorubicin (DOX) has a broad, potent cytotoxicity. It is frequently used against HCC as well as several other solid tumors and leukemias.6 Physicochemical and biopharmaceutical properties important for its drug delivery and liver disposition are summarized in Table 1. Intravenous (i.v.) dosing of DOX is mainly limited by its cardiotoxicity, which is believed to be mediated by an accumulation of the primary active metabolite, doxorubicinol (DOXol) in the heart.7 The risk of cardiotoxicity Received: Revised: Accepted: Published: 131

April 29, 2013 October 3, 2013 October 30, 2013 October 30, 2013 dx.doi.org/10.1021/mp4002574 | Mol. Pharmaceutics 2014, 11, 131−144

Molecular Pharmaceutics

Article

Table 1. Literature Values of Physicochemical Properties of DOX and DOXol Relevant for Drug Delivery and Pharmacokinetics

a slow release of DOX via mechanisms such as diffusion of ions, ion-exchange, and diffusion of DOX.18,19 DEB simultaneously functions as a drug-delivery system and causes a full and permanent embolization in targeted vessels. Even so, a patient can get repeated DEB treatment with reduced dose into other target vessels adjacent to the tumor.18,20 Recent clinical reports have shown that the antitumor effect is similar for DEB and DLIP, but the reported systemic DOX-related adverse events are fewer for DEB than for DLIP.21,22 On the other hand, DEB is associated with a 6.6 times higher risk for liver/biliary injuries than Lipiodol TACE.23 In vivo models are essential for evaluating the performance and characterization of the in vivo drug release and liver disposition of these formulations.24 The main objective of this study was to investigate the local and systemic pharmacokinetics (PK) of DOX and DOXol following an intrahepatic single dose of two different TAI/TACE formulations in an advanced PK multisampling-site acute (MSA) pig model developed in our lab.25−30 Theoretically, this in vivo model would be suitable for characterization of TAI/TACE formulations as the pig liver has a similar size, physiology, and blood supply as the human liver.31−33

can be reduced by decreasing the systemic peak concentrations of DOX and DOXol, by giving DOX as an i.v. infusion over a longer time.8 On the other hand, a mechanistic modeling of in vitro data suggests that the cellular peak concentrations of DOX may be of more importance for its antitumor effect than total exposure.9 In this study, DOX was administered via two formulations: as an aqueous solution emulsified in Lipiodol (DLIP) or loaded into drug-eluting beads (DEB). The lipid phase of DLIP is a poppyseed oil derivate consisting of iodinated, ethylated linoleate (70%), oleate (15%), palmitate (10%), and stearate (5%)10 and is suggested to reach the cells by endocytosis.11 If given alone as TAI, DLIP provides a partial embolization, is completely resorbable and allows repeated HA administration.12,13 Full embolization (TACE) can be achieved if DLIP infusion is followed by injection of an embolizer such as gel foam or gelatin sponge.3 It has been reported that radiolabeled Lipiodol distributes preferentially to tumor tissue, where it is retained longer than in nontumor tissue.14 In addition, the iodine components of Lipiodol can be visualized by computer tomography in tumor tissue months after administration.15 Mechanistically, the retention might be explained by the created partial embolization, tumor-enhanced vascular permeability, and/or reduced capacity of the tumor cells to eliminate lipids.16 It is still unclear whether DOX is retained within the tumor when dosed with DLIP.17 DEB is a nondegradable microparticle formulation based on polyvinyl alcohol hydrogel and chains of 2-acrylamido-2methylpropane sulfonic acid sodium salt (AMPS) that allows

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EXPERIMENTAL SECTION Multisampling-Site Acute Pig Model. Animals. In total, 14 male pigs were randomly assigned into four separate groups in this acute experiment. The pigs were of mixed breed (Yorkshire and Swedish Landrace), 10−12 weeks old, and with a mean (±SD) body weight of 26.0 (±1.3) kg. Feeding was

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dx.doi.org/10.1021/mp4002574 | Mol. Pharmaceutics 2014, 11, 131−144


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Table 2. Study Design sampling sites group TI TII TIII TIV

formulation solution Lipiodol emulsion drug eluting beads Lipiodol emulsion

no. of animals

dose (mg)

dose (μmol)

animal weight (kg)

4 4 4 2

± ± ± ±

± ± ± ±

± ± ± ±

47 3.1 50 22

0.3 0.8 0.0 3.5

86 5.7 92 40

0.9 2.8 0.0 9.0

27 26 25 29

0.3 0.8 0.3 0.9

administration route a

intravenous intrahepatic HAb intrahepatic HAb intrahepatic HAb and BDc

plasma (VP,VH,VF)d

bile

yes yes yes yes

yes yes yes no

a Constant infusion of 55 min given through the ear vein. bHepatic artery. cBile duct. dPortal vein, hepatic vein and femoral vein. Doses and weight are presented as mean ± SE.

n = 4) were given an intra-arterial injection of DLIP or DEB into the hepatic artery. The administration of these two formulations lasted for approximately 5 min. In TIV (n = 2), DLIP was administered simultaneously through the hepatic artery and the bile duct, in an attempt to increase the local availability of the drug. The intended dose in all groups was 2 mg/kg chosen on the basis of clinical dosing with Lipiodol in human patients13 and that it is a well-tolerated intra-arterial dose of DOX in pigs.34 Preparation and Administration of Investigated Formulations. The investigated formulations were prepared by the hospital pharmacy, Apoteket Farmaci, Uppsala University Hospital. The TAI/TACE procedure was performed under radiological guidance by an experienced interventional radiologist, for introducing a catheter into the common hepatic artery via the inserted femoral artery introducer. The catheter was placed in the left hepatic artery to ensure local administration of DLIP or DEB. Intravenous Solution (TI). A volume of 25 mL of Doxorubicin Teva, 2 mg/mL (Teva Parenteral Medicines Inc., Irvine, CA) was infused through an ear vein with a rate of 0.42 mL/min during 55 min. DOX/Lipiodol Emulsion (TII and TIV). A total of 50 mg of DOX (Adriamycin; Pfizer Inc., New York, NY) was mixed with 2.56 mL of omnipaque 300 mgI/mL (GE Healthcare, Stockholm, Sweden) and 0.44 mL of sterile water to obtain the same specific weight as Lipiodol. Prior to dosing, this prepared solution of DOX was mixed with 10 mL of Lipiodol UltraFluide (Guerbet, Aulnay-sous-Bois, France) by syringe mixing to form a w/o emulsion. DOX in Drug Eluting Beads (TIII). A total of 2 mL of DCbead (100−300 μm) dispersed in sodium solution (Biocompatibles UK Ltd., Farnham, UK) was transferred to a syringe, and the sodium solution was removed. Thereafter, 25 mL of DOX solution (Doxorubicin Teva, 2 mg/mL) was added to the syringe and the loading of DOX into DEB was permitted for a minimum of 5 h. According to product instructions, this loading time will correspond to a 100% drug load. After loading, excess solution was removed until the beads and fluid had a total volume of 4 mL. Additionally, 4 mL of Omnipaque 300 mgI/mL was added to the syringe before administration to the pig. Sampling of Bile and Plasma. Blood (4 mL fractions each) was sampled from the portal (VP), hepatic (VH), and femoral veins (VF) and transferred to vacutainers containing EDTA (3 mL, BD Biosciences, Franklin Lakes, NJ). The total blood volume sampled from each animal was 64 mL. The samples were collected at predose, and then 10, 20, 30, 45, 60, 70, 80, 90, 120, 150, 180, 210, 240, 300, and 360 min after the i.v. dose. The sampling times of the transarterial treatments (TII−TIV) were predose, 5, 10, 20, 30, 45, 60, 75, 90, 120, 150, 180, 210, 240, 300, and 360 min. The plasma was collected after

withdrawn the night before surgery, but they had free access to water. The Animal Ethics Committee in Uppsala (C40/11) approved the study protocol and animal care. Anesthesia. The pigs were premedicated during transport to the hospital, where they were sedated with an intramuscular neck injection of tiletamine 3 mg/kg and zolazepam 3 mg/kg (Zoletil, Virbac S.A., Carros, France), xylazin 2.2 mg/kg (Rompun Vet, 20 ng/mL, Bayer AG, Leverkusen, Germany), and atropine 0.04 mg/kg (Atropin NM pharma 0.5 mg/mL, Merck NM AB, Stockholm, Sweden). General anesthesia was induced with a bolus injection of morphine 1 mg/kg (Morfin Meda 10 mg/mL, Meda AB, Solna, Sweden) and 100 mg of ketamine (Ketaminol Vet, 100 mg/mL, Intervet, Stockholm, Sweden), followed thereafter with a continuous intravenous infusion of morphine 0.5 mg/kg, ketamine 20 mg/kg/h, and pancuronium bromide 0.125 mg/kg/h (Pavulon 2 mg/mL, Organon AB, Gothenburg, Sweden) to ensure that the pigs were pain-free and fully anesthetized during the experiment. Approximately 250 mL of Rehydrex 10 mL/kg/h (Fresenius Kabi AB, Uppsala, Sweden), Ringer Acetate 8 mL/kg/h (Fresenius Kabi AB, Uppsala, Sweden), Macrodex (dextran 70) 60 mg/mL, and NaCl (Meda AB, Solna, Sweden) were administered to maintain normal body fluid and osmotic pressure. A Servo 900C ventilator (Siemens-Elema, Solna, Sweden), containing an oxygen-air mix for respiration, was inserted into an incision in the throat of the pigs. Previous studies have established that the anesthesia has no or limited effect on transport and metabolism of drugs in the liver.25,27−30 Surgery. The surgery was similar to previously reported procedures for the MSA pig model.25,27−30 In brief, the abdominal cavity was opened with a midline incision. Catheters for blood sampling were introduced in the portal, hepatic, and femoral veins. The bile duct was cannulated for bile collection and a tube was inserted in the bladder to divert urine. In two animals, the bile duct was catheterized, instead of cannulated, to allow local injection of drug via the bile duct. In addition, to facilitate the TAI or TACE procedure, 10 pigs had an introducer surgically placed in the femoral artery. The pre-experimental surgery took 90−120 min, followed by a stabilization period for 20−30 min before administration of the drug formulations. The experiment lasted 6 h, and thereafter, each animal was euthanized with a bolus dose of 20−30 nmol potassium chloride in the superior caval vein. During the experiment the pig was continuously monitored for electrocardiograms, heart rate, blood gases, arterial and central venous pressure, and body temperature to maintain normal physiological values. Study Design. The study was a randomized parallel design of four separate groups (TI−TIV); see Table 2. Each group was exposed to different treatments with regard to formulations or routes of administration. The first reference group (TI; n = 4) received an intravenous infusion of DOX solution in an ear vein for 55 min. The second (TII; n = 4) and third groups (TIII; 133



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Stock solutions of DOX, DOXol, and the internal standards were prepared in methanol at concentrations of 0.1−0.3 mg/mL. These solutions were diluted and used to spike blank matrices (plasma and bile) to obtain calibration samples. The calibration was done with a linear curve fit (weighting factor of 1/x for both analytes in plasma and 1/x2 for both analytes in bile) of the peak area ratio (analyte/internal standard) as a function of the analyte concentration in the respective matrix. For plasma, the calibration curve interval for DOX was 0.5−603 ng/mL and for DOXol 0.5−50 ng/mL. For bile, the calibration curve interval for DOX was 0.52−62 μg/mL and for DOXol 0.25−53 μg/mL. The precision expressed as the relative standard deviation (RSD) in the results of quality control samples in plasma was in the interval 1.6−14% (DOX) and 2.0−15% (DOXol). For the bile samples, it was 1.6−8.5% (DOX) and 0.1−6.0% (DOXol). Pharmacokinetic (PK) Data Analysis. Noncompartmental Analysis (NCA). NCA was performed on the plasma and bile concentration−time profiles for DOX and its active metabolite DOXol using the WinNonLin software version 5.2 (Pharsight Corporation, Mountain View, CA). The observed maximum concentration (Cmax) and the time to reach Cmax (tmax) were directly derived from the plasma concentration−time profile. The area under the concentration−time curve (AUC0−6, AUC0−∞) was calculated with the linear and logarithmic trapezoidal method for the ascending and descending parts to the last data point (Clast), respectively, for AUC0−6. The total AUC0−∞ was calculated by adding the residual area (Clast, predicted/λz) where λz is the terminal rate constant estimated from at least three of the last measurable concentrations after Cmax. The terminal halflife was calculated by ln2/λz. When determining the AUCs, concentrations below the lower limit of quantification were set to zero before tmax and excluded after tmax. The total clearance (CLtot) after i.v. infusion (TI) was calculated according to eq 1:

centrifugation of the blood samples for 10 min (3500 rpm, 4 °C, Universal 16R; Hettich, Tuttlingen, Germany). Bile samples were collected on ice in 20 min intervals and thereafter weighed. All samples were distributed to polypropylene tubes and thereafter stored at −60 °C pending analysis. Analytical Method. DOX hydrochloride was purchased from LC Laboratories (Woburn, MA) and DOXol citrate from Toronto Research Chemicals (North York, ON, Canada). The internal standards [13C, 2H3]-DOX trifluoroacetate and [13C,2H3]-DOXol formiate were bought from Alsachim (IllkirchGraffenstaden, France). The water was purified using a Milli-Q water purification system (Millipore, Bedford, MA). All other chemicals were of analytical grade or better and used without further purification. Sample Pretreatment. To 500 μL of plasma, 100 μL of 0.1% formic acid in water and 50 μL each of the two internal standard solutions ([13C,2H3]-DOX 0.50 μg/mL and [13C,2H3]DOXol 0.51 μg/mL) were added, followed by 800 μL of acetonitrile. The samples were then vortexed for 30 s whereafter they were placed in an ice bath for 10 min. They were subsequently centrifuged for 10 min (11 000g). The supernatants were transferred to new tubes and evaporated under a stream of nitrogen gas at 50 °C until about half of the volume remained. Thereafter, the samples were transferred to vials for injection on the ultra performance liquid chromatography− tandem mass spectrometry (UPLC-MS/MS) system. To 50 μL of bile, 100 μL of 0.1% formic acid in water and 50 μL of each internal standard solution ([13C,2H3]-DOX 2.25 μg/mL and [13C,2H3]-DOXol 2.22 μg/mL) were added. The samples were then diluted with 4.0 mL of 0.1% formic acid in water whereafter they were vortexed for 3 min and centrifuged for 10 min (11 000g) and transferred to vials for UPLC-MS/MS analysis. Bile samples with responses above the highest concentration of the calibration curve were diluted before analysis. UPLC-MS/MS. The samples (plasma and bile) were analyzed with UPLC-MS/MS. A Waters Acquity UPLC system was coupled to a Quattro Ultima Pt tandem quadrupole mass spectrometer with an electrospray interface operating in the positive mode (Waters Corporation, Milford, MA). The column was an Acquity UPLC BEH Phenyl (length 50 mm, I.D. 2.1 mm, particle size 1.7 μm) from Waters Corporation kept at 60 °C. The mobile phase consisted of (A) 5.0 mM ammonium formate in water (pH 3.0) and (B) methanol. A gradient was run as follows: initially 25% B, 25−75% B for 4 min, 75−25% B for 0.1 min, and 25% B for 2 min. The total run time was 6.1 min, the flow rate was 200 μL/min, and the injection volume was 20 μL for plasma and 75 μL for bile. The two analytes were analyzed simultaneously in the same chromatographic run using a positive capillary voltage of 4.00 kV. The desolvation and source block temperatures were 300 and 120 °C; the cone and desolvation gas flows were 132 and 871 L/h, respectively. The quantifications were performed in the selected reaction monitoring (SRM) mode with the collision cell filled with argon gas at a pressure of 1.14 × 10−3 mbar. The mass transitions used in SRM were m/z 544 → 397 for doxorubicin (collision energy 12 eV, cone voltage 43 V), m/z 548 → 401 for [13C, 2H3]-DOX (collision energy 12 eV, cone voltage 43 V), m/z 546 → 399 for DOXol (collision energy 12 eV, cone voltage 43 V), and m/z 550 → 403 for [13C,2H3]-DOXol (collision energy 12 eV, cone voltage 43 V). The dwell time was 0.10 s.

CL tot(i.v.) =

dose i.v. AUC0 −∞ ,i.v.(VF)

(1)

where dosei.v. is the total amount infused during 55 min and AUC0−∞, i.v.(VF) is the area under the concentration−time curve for DOX in the VF compartment. The apparent hepatic extraction ratio (EH) was calculated by comparing AUC0−6 from the portal and hepatic vein plasma compartments following an intravenous dose (TI): EH =

(AUC0 − 6(VP) − AUC0 − 6(VH)) AUC0 − 6(VP)

(2)

The ratio was also calculated with another approach where portal and hepatic vein plasma concentrations, at certain time points, were compared to generate the time-dependency of the EH of DOX. In turn, EH from eq 2 was used to calculate the apparent hepatic clearance, CLH, using the following equation:

⎛C ⎞ CL H = E HQ H⎜ B ⎟ ⎝ CP ⎠

(3)

where QH is the hepatic blood flow determined to be 52 mL min−1 kg−1 in pigs35 and CB/CP is the blood/plasma ratio of DOX given as 2.01 in rats.36 The total clearance (CLtot) is the sum of all eliminating pathways in the body according to eq 4: 134



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Figure 1. Plasma concentration−time profiles in VF, VP, and VH of doxorubicin (A) and doxorubicinol (B) after a constant 55 min intravenous infusion (TI, n = 4). The inset displays the same profile on a semilogarithmic scale, and the striped line represents the last data point measured during infusion. All data are compensated for dose and presented as median (interquartile range).

CL tot = CL H,met + CL bile + CL R + CLother

concentration−time profiles of DOX. The models were compared regarding inspection of curve fits, residual sums of squares, and Akaike’s criteria. TI had the best fit with a twocompartment model (constant infusion and first-order output) and multiplicative error model. It was therefore used to estimate the parameters from the initial (C1, λ1) and terminal slope of the curve (C2, λ2) according to eq 10:

(4)

where the hepatic clearance CLH (further divided into clearance dependent on hepatic metabolism (CLH,met) and excretion (CLbile)), renal clearance (CLR), and metabolism in other organs (CLother). The systemic bioavailability (F) after release from the drug delivery systems was calculated as follows: F=

C(t ) = C1(e−λ1t − e−λ1t *) + C2(e−λ2t − e−λ2t *)

CL tot(i.v.) × AUC0 − 6(i.h.) dose(i.h.)

where t is the time in min and ti is the duration of infusion. When t > ti, t* = t − ti, and when t ≤ ti, t* = 0. TII−TIV did not fit well to any of the tested compartment models. Deconvolution was done with WinNonLin software and the data used to estimate the in vivo release rate from the two formulations.39 The release rate was set to be the input function, i(t). The input function was obtained by deconvolving the response function, r(t) (from plasma concentration−time data from DLIP or DEB), with the weight function, w(t) (derived from i.v. plasma concentration−time data) according to i(t) = r(t)//w(t). The parameters “C1” and “C2” were obtained from the two-compartmental analysis of the i.v. data and normalized for dose. The analysis was set to automatic smoothing, and the number of output data was set to measured time points. The initial rate was set to zero. Statistical analysis. Statistical analyses were calculated using Prism 5 (GraphPad Software Inc., La Jolla, CA); the level of statistical significance was set at p < 0.05. Nonparametric Mann−Whitney tests were performed to compare parameters from DLIP with DEB. When the reference (TI) was included in the analysis, nonparametric Kruzkal−Wallis tests were used with Dunn’s multiple comparison tests as post hoc (e.g., for bile flow). The Kruzkal−Wallis test was also applied to look for differences between blood compartments within the same group. The data are given as the median (interquartile range; Q1−Q3) unless otherwise specified.

(5)

where CLtot (i.v.) is the total clearance following i.v. infusion (eq 1) and AUC0−6h(i.h.) and dose(i.h.) are the area under the plasma concentration−time curve and administered dose after intrahepatic (i.h.) administration, respectively. The cumulative amount of DOX or DOXol in the bile (Ae;bile0−6) was used for further calculation of dose-fraction excreted into the bile ( fe;bile0−6) and apparent hepatic biliary clearance (CLbile) according to eqs 6−8: Ae;bile0 − 6 = fe;bile0 − 6 =

CL bile =

∑ C bileVbile

(6)

Ae;bile0 − 6 dose

(7)

Ae;bile0 − 6 AUC VP,DOX,0 − 6

(8)

where Cbile is the drug concentration (μmol/L) obtained in the collected volume, Vbile (L). DOX metabolism into DOXol is predominantly catalyzed in the liver by cytosolic carbonyl reductases (mainly CBR1) and aldo-keto reductases (AKRs).37,38 With the assumption of a linear distribution, limited reabsorption from bile duct, and no saturation of efflux transporters and/or metabolizing enzymes, the apparent intracellular availability (Fi;app) of DOX can be approximated using the AUCbile (min·μM) of metabolite(s) and parent drug according to eq 9: Fi;app =

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RESULTS The surgery, the TAI/TACE procedure, and the experiment were successfully performed on all 14 animals, without any acute adverse events. Pharmacokinetics (PK) of Doxorubicin (DOX) and Doxorubicinol (DOXol) in Pigs Following Intravenous Constant Infusion of DOX (TI). The three plasma

AUC0 − 6,DOXOL,bile (AUC0 − 6,DOX,bile + AUC0 − 6,DOXol,bile )

(10)

(9)

Compartmental PK Analysis. One-, two- and threecompartment PK models were tested to describe the plasma 135



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a

VF, femoral vein; VP, portal vein; VH, hepatic vein; N.A., not applicable. bNCA. ctmax is presented as median (range). dTwo-compartment analysis. eBased on eq 2.

290 (190−390) 11 (7.1−15) Vss (L) Vss (L/kg) 41 (28−53) 59 (53−67) 35 (21−44)

VH VP

1.4 (1.2−2.5) 3.8 (2.8−5.2) 5.4 (5.3−10) 90 (88−98) N.A. N.A. N.A. 1.3 (0.97−2.4) 4.4 (2.8−5.9) 4.9 (4.7−8.7) 140 (110−160) N.A. N.A. N.A.

VF VH

29 (28−-31) 37 (34−40) 400 (360−470) 38 (25−45) 5.3 (4.6−5.6) 3.8 (3.1−6.8) 3.2 (2.6−4.0)

VP

54 (47−60) 62 (52−70) 790 (610−970) 45 (45−45) 4.1 (3.0−5.0) 3.4 (3.2−3.5) 2.8 (2.5−3.1)

VF

45 (41−48) 54 (47−61) 660 (560−790) 20 (18−26) 5.4 (4.0−6.6) 2.9 (2.8−3.2) 3.3 (2.2−3.9)

AUC0−6 (min·μM)b AUC0−∞ (min·μM)b Cmax (nM)b tmax (min)b,c t1/2,λ2 (h)b t1/2,λ1 (min)d t1/2,λ2 (h)d DOX parameters EH (%)e CL (mL/min/kg)b CLH,met (mL/min/kg)

DOXol DOX

Table 3. Pharmacokinetic Parameters (median (interquartile range)) of DOX and DOXol in VF, VP, and VH Following Intravenous Infusion during 55 min (TI)a

concentration−time profiles for DOX in the portal (VP), hepatic (VH), and femoral veins (VF) were best described by a two-compartment model after a constant i.v. infusion during 55 min (Figure 1, Table 3). The residual areas of the AUCs estimated from the terminal half-lives in this experimental 6 h long study were less than 25% in all cases. There were no differences between AUC0−6, AUC0−∞ (NCA), and AUC0−∞ from the two-compartmental analysis for each of the specific sampling sites. The CLtot and EH of DOX were 59 (53−68) mL/min/kg and 0.41 (0.28−0.53), respectively (Table 3). Initially, during infusion, hepatic extraction was about 60−70%, but it declined after the infusion ended (Figure 5). Within the 6 h study period, the systemic plasma exposure (AUC0−6h, VF (min·μM)) of the active metabolite DOXol was 2.5 (2.3−5.0)% that of DOX. The Cmax of DOXol occurred within a range of 80−135 min in all blood compartments (Figure 1B, Table 3). The plasma concentration−time profiles of DOXol in all three sampling sites declined in parallel with DOX, indicating that the metabolite PK was controlled by the elimination rate of DOX (Figure 1). The fraction of the i.v. dose excreted in bile as DOX and DOXol was 20 (15−22)% and 4.2 (3.2−5.2)%, respectively (Table 5). The metabolite/parent drug ratio was higher in bile than in plasma (p < 0.05, Figure 4). The CLapp,bile was 11 (8.6− 14) and 2.3 (1.8−3.2) mL/min/kg for DOX and DOXol, respectively, and CLH,met for DOX was estimated to be 35 (21− 44) mL/min/kg. The bile duct exposure of DOX and DOXol was extensive compared to plasma exposure (AUC0−6h,VP) as the AUC bile-to-plasma ratios were 640 (580−660) and 5000 (3900−5400), respectively (Figure 3A). The apparent availability of DOX in the hepatocyte (Fi;app) was approximated to 17 (15−21)% following i.v. administration (Table 5). The biliary flow after an i.v. dose was 19 (15−24) μL/min/kg. DOX/Lipiodol Emulsion (DLIP) Administered in the Hepatic Artery (TII). Before administration, the contents of the syringe were extensively mixed by syringe mixing to prevent phase separation. The median (Q1−Q3) dose of DOX injected as DLIP into the HA was 0.092 (0.089−0.12) mg/kg although the intended dose was 2 mg/kg. The reason for this was that the HA-injection was followed by an unexpected stasis. The plasma concentrations of DOX and DOXol in all compartments are given in Figure 2A, B. The plasma concentrations of the metabolite, DOXol, were below the lower limit of quantification for three of the four animals (Figure 2A, B). The plasma PK parameters in all measured blood compartments are given in Table 4. The bile/plasmaVP concentration ratios for DOX and DOXol are described in Figure 3B. The bioavailability of DOX in VH and VF was 85 (70−94)% and 29 (22−44)%, respectively, when using eq 5, and 110 (92−120)% and 38 (28−56)%, respectively, when deconvolution was applied (Table 4). In Figure 6, the release rates of DOX from DLIP into VH and VF are presented, both as absolute values (A, B) and as cumulative release normalized to dose (C, D). Based on these data, it was approximated that 50% of the administered dose was released into VF after 200 min and into VH after 20 min.The Fi;app of DOX was estimated to be 32 (22−43)% (Table 5), and the DOXol/DOX ratios in bile and plasma (VF) are displayed in Figure 4. The fraction of the dose excreted into the bile as DOX and DOXol was 17 (13−32) and 13 (11−16)%, respectively (Table 5). The biliary flow was similar to that in TI with 18 (17−18) μL/min/kg and, hence, unaffected by the formulation.

1.3 (1.2−1.6) 2.0 (1.4−2.8) 8.9 (4.6−13) 80 (80−90) N.A. N.A. N.A.

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Figure 2. Plasma concentration−time profiles of doxorubicin in the VF, VP, and VH, after intrahepatic injection with the TAI/TACE procedure. Concentrations of doxorubicin (A) and doxorubicinol (B) following injection of Lipiodol emulsion (TII), and doxorubicin (C) and doxorubicinol (D) following injection of drug eluting beads (TIII) are presented. The inset displays the plasma concentration−time profile for doxorubicin on a semilogarithmic scale. All data is normalized to dose and presented as the median (interquartile range).

DLIP Administered Simultaneously via Bile Duct and Hepatic Artery (TIV). DLIP was administered simultaneously in both the bile duct and the HA, to investigate the effect of administration route on plasma PK of DOX and DOXol (n = 2). The total administered dose of DOX was 0.75 (0.61− 0.89) mg/kg; no stasis effect was observed during infusion in the bile duct. No bile was collected in this group, as the administration route required occlusion of the bile flow. The AUC and Cmax of DOX in VP, VH, and VF are presented in Table 4. The VF exposure of DOXol (AUC 0−6h, VF) was 16 (16−17)% compared to that of the parent drug (Figure 4B). The bioavailability of DOX (based on eq 5) in VH and VF was 94 (82−100)% and 36 (30−40)%, respectively. The same parameter for VH and VF derived from deconvolution was 130 (110−140)% and 44 (38−48)%, respectively (Table 4). The release rates of DOX into VH and VF from DLIP administered in HA and bile duct were similar to TII as visualized in Figure 6. The cumulative dose fraction released during the study time did not reach 50% in the VF. In VH, on the other hand, 50% of the dose was released after 30 min.

Drug Eluting Beads (DEB) Administered in the Hepatic Artery (TIII). The intended dose of DOX was successfully administered without any apparent effect on blood vessel stasis. The plasma concentration−time curves of DOX and DOXol in all blood compartments and the plasma PK parameters are displayed in Figure 2C, D and Table 4, respectively. DOXol was quantified in plasma in two animals, and biliary concentrations of DOXol were quantified in all four animals. The bioavailability in VH and VF was 24 (18−29)% and 14 (9.4−18)%, respectively, and similar numbers were obtained with deconvolution (Table 4). The bioavailability was lower than for DLIP (TII; p < 0.05). The DEB release rate of DOX into VH and VF are displayed in Figure 6 where the absolute release rate appears faster than that of DLIP. However, the cumulative released fractions of the administered dose show that DEB never reaches 50% during the study time and hence has a slower release than DLIP (VF, p < 0.05). Biliary excretion parameters of DOX and DOXol are presented in Table 5. The bile/plasma concentration−time curves for DOX and DOXol are presented in Figure 3B. The Fi;app of DOX was 18 (17−21)%, and the DOXol/DOX ratios in the bile and VF compartment are presented in Figure 4. The biliary dose fraction excreted of DOX and DOXol during the experiment was lower than that for DLIP (TII; p < 0.05), with values of 3.8 (3.6−4.4) and 1.1 (0.8−1.5)%, respectively (Table 5). The biliary flow was unaffected by the formulation as the flow was 14 (11−19) μL/min/kg with no significant difference from the other groups.

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DISCUSSION In this study, the local and systemic PK of DOX and DOXol was investigated in three blood compartments (VP,VH, VF) and in bile duct in a MSA pig model. DOX was administered in either of two drug delivery systems (DLIP or DEB) as an intrahepatic single dose. To our knowledge, this is the first in vivo study quantifying the local and systemic exposure and 137


VF, femoral vein; VP, portal vein; VH, hepatic vein. bData are corrected for dose differences. ctmax are presented as median (range). dNCA. ecumulative input from deconvolution. fp < 0.05.

PK of both DOX and DOXol following HA dosing of any of these two drug delivery systems. This allowed us to make a direct comparison of the safety profiles for DLIP and DEB. There was a higher systemic exposure (AUC and Cmax) and tendencies toward a higher intracellular exposure (i.e., Fi;app) of DOX after DLIP than after DEB administration. This is probably because of an initially higher dose-normalized release in vivo for DOX from DLIP. In addition, the extensive bile-toplasma (VP) exposure ratios of 640 and 5000 for DOX and its major active metabolite DOXol, respectively, were a strong confirmation that the hepatobiliary transport across the basolateral and/or canalicular membranes is mediated by both carrier-mediated (CM) and passive transcellular transport processes. The implication of these membrane transport mechanisms for the intracellular availability of DOX and the dosing strategies will be investigated in future studies. The MSA in vivo pig model, together with metabolite data, provided insight into the hepatic disposition of DOX and DOXol for two formulations with different release mechanisms. Three different processes may explain our observation that the bile/plasma ratio was about 10 times higher for DOXol than for DOX. First, passive diffusion is slower for the more hydrophilic metabolite DOXol (Table 1). Second, the CM route across the canalicular membrane plays a proportionally greater role for DOXol than for DOX. Finally, the metabolite excreted into bile is formed intracellularly and the canalicular efflux proteins directly transport DOXol into bile rather than into plasma. A high fraction of DOX is effluxed unchanged into bile, as indicated by a DOXol-to-DOX AUC ratio in bile of about 20%. Hydrophilic properties and BCS class III (Table 1) additionally predict that CM process(es) are involved in the membrane transport of DOX.40 To date, the only CM influx transporter associated with DOX is SLC22A16 (OCT6).41 It is expressed in HCC but not to any high degree in normal adult liver.42 The expression of OCT6 in pigs is, to our knowledge, not yet characterized. DOX is a well-known substrate for the CM efflux transporter ABCB1 (P-gp), and it has also been associated with others such as ABCC1 (MRP1) and ABCG2 (BCRP).43 The plasma concentration−time profile following i.v. infusion indicates that DOX was rapidly and extensively distributed in peripheral tissue, as to be expected for BDDCS class I drugs (Table 1).44 DOX has been classified as a BDDCS class I drug which indicates that intracellular metabolism plays a major part (>70%) in the elimination of DOX. Therefore passive diffusion must have a significant role in its transport. Indeed, the amphiphatic DOX undergoes a complex diffusion process consisting of a slow flip-flop mechanism with a fast and strong association to the outer and inner acidic cell membrane.45 However after DOX administration 15−50% of the given dose is eliminated through bile within a week.46,47 Of the eliminated fraction, 50% is eliminated as parent drug and 50% as metabolites (mainly DOXol). This suggests that DOX rather should be classified as a BDDCS class III/IV compound than a class I compound. One additional reason for the apparent misclassification of DOX in BDDCS might be because DOX metabolism also is localized to extrahepatic extracellular space.48 The pig Vss was similar to the corresponding human values (11 vs 22 ± 13 L/kg49) and suggests extensive tissue binding. In this relatively short study duration, the calculated CLtot was higher in pigs than in humans (59 vs 13 ± 4.2 mL/min/kg50). The most plausible explanations for the higher CL are underestimation of the residual AUC and extrahepatic metabolism. This is argued by the short sampling period (6 h) in relation to

a

44 (38−48) 130 (110−140) 17 (11−21)f 32 (24−38) 38 (28−56)f 110 (92−120) Fdeconv (%)e VF VH 7.5 (5−30) 5 (5−30) 7.5 (5−30) 5 (5−10) 5 (5−10) 5 (5−5) tmax (min)c VF VP VH

7.5 (5−10) 10 (10−10) 10 (10−10)

36 (30−40) 37 (30−42) 94 (82−100) 14 (9.4−18)f 18 (13-21) 24 (18−29) 29 (22−44)f 48 (31−68) 85 (70−94) F (%) VF VP VH 0.68 (0.6−0.77)f 0.88 (0.74−1.2) 0.67 (0.65−0.82)f 7.2 (3.8−11)f 13 (5.8−27) 7.7 (4.6−10)f Cmax (nM)b VF VP VH

230 (190−250) 260 (210−300) 400 (350−430) 88 (58−110)f 130 (92−150) 100 (76−120)f

2.6 (2.4−2.8) 2.8 (2.6−2.9) 5.8 (5.3−6.3)

2.8 (2.7−2.9) 2.7 (2.8−2.8) 2.8 (2.6−3.0) 2.8 (2.2−4.6) 3.2 (2.8−3.3) 2.7 (2.4−7.8) 6.6 (6.2−9.4) 4.1 (3.9−27) 4.1 (3.9−27)

TIV TIII TII

t1/2,λ2 (h)d VF VP VH TIV TIII TII

Article

AUC0−6 (min·nM)b VF 180 (140−280)f VP 340 (220−490) VH 350 (290−390)f

Table 4. Pharmacokinetic Plasma Parameters (median (interquartile range)) of Doxorubicin after Three Different TAI/TACE Administrations; Lipiodol Emulsion in the Hepatic Artery (TII), Drug Eluting Beads in the Hepatic Artery (TIII), and Lipiodol Emulsion Administered via the Hepatic Artery and Bile Duct (TIV)a


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Figure 3. Bile-to-plasma concentration ratios in the portal vein for doxorubicin and doxorubicinol are presented for intravenous infusion of doxorubicin, TI (A), for intrahepatic injection of Lipiodol emulsion, TII and for intrahepatic injection of drug eluting beads, TIII (B). When applicable, data are presented as the median (interquartile range).

during the first 6 h. Lipiodol alone is distributed to some extent to bile,14 but the direct influence of the lipid components on the hepatic transport of DOX and DOXol has not yet been investigated in vivo. The different doses given in this study might have affected the Fi;app. However, the effect is difficult to predict because an encompassing picture of the cellular disposition mechanisms involved and their capacity is not known. In addition, the different doses of DLIP and DEB are partly compensated by the difference in release rate, resulting in reduced dissimilarities in the exposure of DOX from the delivery system at the target tissue. The unexpected limitation in the administered dose of DLIP in this study (0.9 mg/kg instead of 2 mg/kg) was presumably a consequence of the phase separation of the emulsion. This was despite vigorous mixing before administration, as the embolic capacity of Lipiodol is dependent on the droplet size.12 Moreover, differences in blood vessel size and anatomy between pigs and humans may also have played a role in the increased resistance and formed stasis. For instance, it has been reported that 3 month old pigs have a narrower VP with thinner vessel wall than humans.60 Thus, differences in other vessels could be expected as well. The pigs that received DLIP through the bile duct and HA (TIV) showed a higher fraction of DOXol in plasma compared to TII (Figure 4B). This might be explainable by increased substrate availability and prohibited biliary efflux that redirected the diffusion of DOXol into the plasma. DEB had a low F in VH (24%) and VF (14%) in addition to a low biliary exposure of DOX and DOXol. This is in accordance with the calculated slower in vivo release of DOX. Accordingly, the extended and possibly incomplete release for several weeks has previously been demonstrated in vitro and in vivo and implies a strong interaction between DEB and DOX.21,58,61−65 The lower and slower release of DOX from DEB compared to DLIP also reduces the effect of the rapid redistribution of DOX on F in VH. The low systemic concentrations with DEB delivery are consistent with a better systemic safety profile than for patients receiving DLIP.21,22 To date, the optimal concentration of drug for local tumor treatment is not established. Therefore, it is not possible to recommend any rational drug delivery strategy yet. Nonetheless, these two investigated formulations represent two very different release rates and most likely different drug-target residence times. In vivo characterization of performance of DLIP and DEB is poorly understood, and more mechanistic in vivo studies of physiological factors of importance are

the long terminal half-life reported in humans (30 ± 14 h).51 The differences are not a consequence of the bile collection procedure that interrupted the enterohepatic recirculation, because DOX has a low intestinal permeability (BCS class III) and accordingly insignificant enterohepatic recycling.52,53 During 6 h nearly 25% of the dose was biliarly eliminated in pigs, unlike in humans where 15−45% of the DOX dose was recovered in faeces and 23% in urine over a week.47 This indicates that pigs eliminate DOX more rapidly than humans and metabolic rate differences in these species have certainly been observed previously.54,55 In pigs, DOX had an intermediary EH of 0.41, in agreement with previous reports for domestic pigs and humans.34,56 Interestingly, our study found a time-dependent EH for DOX. The immediate increased EH following infusion and the subsequent reduced EH may be explained by an initial hepatic accumulation of DOX where the liver acts as a reservoir.34 This is followed by an extensive redistribution to plasma from intrahepatic binding sites. A timeand route-dependency of EH has been reported for other drugs such as finasteride, verapamil, and raloxifene.28,30,57 In future studies, EH will be used in modeling strategies to elucidate the mechanisms of hepatic disposition of DOX and DOXol. Knowledge of these mechanisms will facilitate optimization and development of TAI/TACE drug delivery systems and/or dosing strategies to thereby improve the clinical benefit/safety ratio. The investigated formulations had an impact on the local and systemic disposition processes to varying degrees. The higher bioavailability in VH than in VF after cessation of the infusion was most likely a consequence of redistribution of the intraand extracellularly bound DOX in the liver. The higher systemic exposure (VF) of DOX following DLIP delivery compared to that of DEB (p < 0.05), together with a high bioavailability directly after the liver (VH), indicated that in vivo release from the emulsion was more rapid and extensive than from DEB. This was also observed in the dose-normalized data derived from deconvolution of plasmaconcentration in VH and VF compartment after DLIP administration at two different sites (TII and TIV). In addition, results from an in vitro study supported a faster release from DLIP compared to DEB.58 The higher release rate from DLIP is most probably related to the release mechanism of DOX from the w/o emulsion that involves a partitioning, followed by diffusion through the oil phase.59 DLIP appeared to distribute DOX to the extravascular space of the liver rapidly where it established a higher apparent intracellular availability (Fi;app) of DOX compared to DEB 139


Data are presented as the median (interquartile range). N.A, not applicable. bp < 0.05.

Figure 4. Metabolite-to-parent drug amount ratios in bile (A) and concentration ratio in the femoral vein plasma compartment (B) following administration of DOX. DOX was administered as an intravenous infusion (TI), an intrahepatic injection of a Lipiodol emulsion (TII) or drug eluting beads (TIII), or as a simultaneous injection of Lipiodol emulsion into the bile duct and hepatic artery (TIV). Data are presented as the median (interquartile range).

Figure 5. Hepatic extraction ratio (%) of doxorubicin against time, following an i.v infusion of 55 min.

necessary. The advanced MSA pig model allowed detailed, regional, and systemic PK investigation of the formulations. The model-specific drawbacks were the short duration of the acute experiment, the absence of tumor tissue, and the sensitivity of the blood vessels to lipid-based formulations. However, despite the complexity of the in vivo model, such complexity is required for the development of novel parenteral formulations since drug exposure to cells and tissue of interest is highly affected by the availability and exposure time of the formulation components in vivo.24 To be able to improve the treatment of unresectable intermediate stage HCC, there is a need to better understand which factors that affect the in vivo release of DOX from these two investigated formulations. Unfortunately, there are no good in vitro release methods available at the moment in order to establish IVIVCs for parenteral depot formulations.66 The rapid in vivo release of DOX from DLIP probably occurs because the emulsion breaks down when it is mixed with blood following injection, just as it does during in vitro conditions in contact with elution media.58,59,67 Accordingly, physicochemical factors of the formulation (such as physical stability), pH, and hemodynamics would have a major influence on in vivo release of

a

TIII

Article

1.0 (0.76−1.3) 1.1 (0.83−1.5) 4.6 (3.7−4.9) 29 (28−30) 0.13 (0.12−0.13) N.A.

TII

0.57 (0.45−0.99) 13 (11−16) 18 (11−24) 290 (230−320) 1.1 (0.94−1.6) N.A.

TI

3.7 (2.8−4.4) 4.2 (3.2−5.2) 2.3 (1.8−3.2) 81 (72−90) 0.38 (0.35−0.48) 5000 (3900−5400)

TIII

3.5 (3.3−4.0) 3.8 (3.6−4.4)b 9.3 (9.3−9.4) 130 (120−130)b 75 (73−77)b 1100 (750−1800) 22 (20−28) 18 (17−21)

TII

0.74 (0.56−2.3) 17 (13−32)b 29 (18−40) 410 (360−660)b 17 (11−68)b 1700 (1100−2300) 49 (29−77) 32 (22−43)

TI

17 (13−19) 20 (15−22) 11 (8.6−14) 350 (270−440) 3.1 (2.2−4.5) 640 (580−660) 21 (18−27) 17 (15−21) Ae;bile 0−6 (μmol) fe;bile (%) CLapp, bile (mL/min/kg) AUCbile 0−6 ((μM·min)/dose) Cmax (μM/dose) AUCbile/AUCVP AUCDOXol,bile/AUCDOX,bile Fi;app (%)

DOXol DOX

Table 5. Pharmacokinetic Parameters of DOX and DOXol Derived from Bile Data Following Intravenous Constant Infusion During 55 min (TI) or via TAI with Lipiodol (TII) or TACE with Drug Eluting Beads (TIII)a


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Figure 6. Absolute release rate (μmol/min) of DOX following an intrahepatic injection of Lipiodol emulsion (DLIP, TII) or drug eluting beads (DEB, TIII), or following a simultaneous intrahepatic and intrabiliary injection of DLIP (TIV) into the femoral vein (A) and into the hepatic vein (B) is presented. The cumulative fraction DOX released of the administered dose following TII, TIII, and TIV into the femoral vein (C) and into the hepatic vein (D) demonstrates the relative release rate (%). The release rate of DOX was more rapid from DLIP in both TII and TIV compared to DEB (TIII). The data were derived from deconvolution, using intravenous data (T1) as weighting function. The data are presented as the median (interquartile range). Data marked with ¤ indicate a reduced number of animals (n = 2) in the median.

in vitro release methods and in silico models, and to establish IVIVCs for these formulations. In conclusion, there was a difference in systemic and apparent intracellular availability of DOX for the two formulations, DLIP and DEB. The higher drug and metabolite exposure of DOX from DLIP compared to DEB in both local (Fi;app) and peripheral (Cmax, AUC) compartments was suggested to depend on a higher initial in vivo release rate of DOX from DLIP. The local liver disposition of both DOX and its active metabolite was affected by passive and CM membrane transport processes, as indicated by the more than 500−5000-fold higher concentrations in bile than that in plasma. Finally, our MSA pig model seems to be promising for the evaluation of other formulations for transarterial treatments against HCC. This work will be the basis for ongoing experimental, preclinical, clinical, and simulation studies that together with pharmaceutical innovation will optimize the antitumor effect and safety profile of treatment against HCC.

DOX from DLIP. Enhancing the stability with densifiers, emulsifiers, and so forth has been reported to prolong the release in vitro.68 The in vitro release of DOX from DEB exerting ion-exchange mechanisms can be described by five critical steps: (i) diffusion of counterion through elution media toward the DEB, (ii) diffusion of counterion through the interior of the DEB, (iii) chemical exchange between counterions and cationic DOX at the exchanging site within the DEB, (iv) diffusion of DOX through the matrix of the DEB, (v) diffusion of DOX through the elution media away from the DEB.69 Biondi et al. studied the in vitro release of DOX from DEB with a free flow method and managed to describe the release mathematically by simple Boyd models. It was concluded from those models that the release was controlled by the diffusion of DOX through the film layer surrounding the bead.19 However, their analysis is too simplified and inaccurate for this highly dynamic system, and one of the major problems is that the increase of bead volume during release is neglected. In addition, it has been established that flow rate and ionic strength of elution medium are important for the in vitro release from DEB, suggesting that local biological factors such as hemodynamics and pH might have a high influence on the in vivo release.58,62 It is clear that development of more complex theoretical and experimental models is needed to describe these release processes better. The knowledge obtained in this in vivo study (i.e, in this paper) will be used in such development of

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +46 (0)18 471 4317. Fax: +46 (0)18 471 4223. Notes

The authors declare no competing financial interest. 141



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(15) Park, C.; Choi, S. I.; Kim, H.; Yoo, H. S.; Lee, Y. B. Distribution of Lipiodol in hepatocellular carcinoma. Liver 1990, 10 (2), 72−78. (16) Bhattacharya, S.; Novell, J. R.; Winslet, M. C.; Hobbs, K. E. Iodized oil in the treatment of hepatocellular carcinoma. Br. J. Surg. 1994, 81 (11), 1563−1571. (17) Gaba, R. C.; Baumgarten, S.; Omene, B. O.; van Breemen, R. B.; Garcia, K. D.; Larson, A. C.; Omary, R. A. Ethiodized oil uptake does not predict doxorubicin drug delivery after chemoembolization in VX2 liver tumors. J. Vasc. Intervent. Radiol. 2012, 23 (2), 265−273. (18) Lewis, A. L.; Holden, R. R. DC Bead embolic drug-eluting bead: clinical application in the locoregional treatment of tumours. Expert Opin. Drug Delivery 2011, 8 (2), 153−169. (19) Biondi, M.; Fusco, S.; Lewis, A. L.; Netti, P. A. Investigation of the mechanisms governing doxorubicin and irinotecan release from drug-eluting beads: mathematical modeling and experimental verification. J. Mater. Sci.: Mater. Med. 2013, 24 (10), 2359−2370. (20) Malagari, K.; Alexopoulou, E.; Chatzimichail, K.; Hall, B.; Koskinas, J.; Ryan, S.; Gallardo, E.; Kelekis, A.; Gouliamos, A.; Kelekis, D. Transcatheter chemoembolization in the treatment of HCC in patients not eligible for curative treatments: midterm results of doxorubicin-loaded DC bead. Abdom. Imaging 2008, 33 (5), 512−519. (21) Lammer, J.; Malagari, K.; Vogl, T.; Pilleul, F.; Denys, A.; Watkinson, A.; Pitton, M.; Sergent, G.; Pfammatter, T.; Terraz, S.; Benhamou, Y.; Avajon, Y.; Gruenberger, T.; Pomoni, M.; Langenberger, H.; Schuchmann, M.; Dumortier, J.; Mueller, C.; Chevallier, P.; Lencioni, R.; Investigators, P. V. Prospective randomized study of doxorubicin-eluting-bead embolization in the treatment of hepatocellular carcinoma: results of the PRECISION V study. Cardiovasc. Intervent. Radiol. 2010, 33 (1), 41−52. (22) Varela, M.; Real, M. I.; Burrel, M.; Forner, A.; Sala, M.; Brunet, M.; Ayuso, C.; Castells, L.; Montana, X.; Llovet, J. M.; Bruix, J. Chemoembolization of hepatocellular carcinoma with drug eluting beads: efficacy and doxorubicin pharmacokinetics. J. Hepatol. 2007, 46 (3), 474−481. (23) Guiu, B.; Deschamps, F.; Aho, S.; Munck, F.; Dromain, C.; Boige, V.; Malka, D.; Leboulleux, S.; Ducreux, M.; Schlumberger, M.; Baudin, E.; de Baere, T. Liver/biliary injuries following chemoembolisation of endocrine tumours and hepatocellular carcinoma: Lipiodol vs. drug-eluting beads. J. Hepatol. 2012, 56 (3), 609−617. (24) Kwon, I. K.; Lee, S. C.; Han, B.; Park, K. Analysis on the current status of targeted drug delivery to tumors. J. Controlled Release 2012, 164 (2), 108−114. (25) Petri, N.; Bergman, E.; Forsell, P.; Hedeland, M.; Bondesson, U.; Knutson, L.; Lennernas, H. First-pass effects of verapamil on the intestinal absorption and liver disposition of fexofenadine in the porcine model. Drug Metab. Dispos. 2006, 34 (7), 1182−1189. (26) Thorn, H. A.; Hedeland, M.; Bondesson, U.; Knutson, L.; Yasin, M.; Dickinson, P.; Lennernas, H. 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. 2009, 37 (11), 2186−2196. (27) Bergman, E.; Lundahl, A.; Fridblom, P.; Hedeland, M.; Bondesson, U.; Knutson, L.; Lennernas, H. Enterohepatic disposition of rosuvastatin in pigs and the impact of concomitant dosing with cyclosporine and gemfibrozil. Drug Metab. Dispos. 2009, 37 (12), 2349−2358. (28) Lundahl, A.; Hedeland, M.; Bondesson, U.; Lennernas, H. 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. 2011, 39 (5), 847−857. (29) Sjodin, E.; Fritsch, H.; Eriksson, U. G.; Logren, U.; Nordgren, A.; Forsell, P.; Knutson, L.; Lennernas, H. Intestinal and hepatobiliary transport of ximelagatran and its metabolites in pigs. Drug Metab. Dispos. 2008, 36 (8), 1519−1528. (30) Thorn, H. A.; Yasin, M.; Dickinson, P. A.; Lennernas, H. Extensive intestinal glucuronidation of raloxifene in vivo in pigs and impact for oral drug delivery. Xenobiotica 2012, 42 (9), 917−928.

ACKNOWLEDGMENTS The authors would like to thank Anders Nordgren and Monika Hall for excellent skills during the animal surgeries. We are also grateful for Elisabeth Fredriksson’s invaluable help with all analyses of DOX and DOXol.

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ABBREVIATIONS AUC, area under the concentration−time curve; BCS, biopharmaceutical classification system; BDDCS, biopharmaceutical drug disposition classification system; CL, clearance; CM, carrier-mediated; DEB, drug eluting beads; DLIP, doxorubicin-Lipiodol emulsion; DOX, doxorubicin; DOXol, doxorubicinol; EH, hepatic extraction ratio; HA, hepatic artery; HCC, hepatocellular carcinoma; MSA pig model, multisampling-site acute pig model; PK, pharmacokinetics; TACE, transarterial chemotherapy embolization; TAI, transarterial chemoinfusion; VF, femoral vein; VH, hepatic vein; VP, portal vein

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REFERENCES

(1) El-Serag, H. B. Hepatocellular carcinoma. New Engl. J. Med. 2011, 365 (12), 1118−1127. (2) Ferlay, J.; Shin, H. R.; Bray, F.; Forman, D.; Mathers, C.; Parkin, D. M. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int. J. Cancer 2010, 127 (12), 2893−2917. (3) Carr, B. I. Hepatocellular carcinoma: Diagnosis and treatment, 2nd ed.; Humana Press: Philidelphia, 2010; pp 527−568. (4) Tam, K. Y.; Leung, K. C.; Wang, Y. X. Chemoembolization agents for cancer treatment. Eur. J. Pharm. Sci. 2011, 44 (1−2), 1−10. (5) European Association for the Study of the Liver; European Organisation for Research and Treatment of Cancer. EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma. Eur. J. Cancer 2012, 48 (5), 599−641. (6) Martindale: The complete drug reference. http://www. medicinescomplete.com/ (01-18). (7) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol. Rev. 2004, 56 (2), 185−229. (8) Legha, S. S.; Benjamin, R. S.; Mackay, B.; Ewer, M.; Wallace, S.; Valdivieso, M.; Rasmussen, S. L.; Blumenschein, G. R.; Freireich, E. J. Reduction of doxorubicin cardiotoxicity by prolonged continuous intravenous infusion. Ann. Intern. Med. 1982, 96 (2), 133−139. (9) El-Kareh, A. W.; Secomb, T. W. Two-mechanism peak concentration model for cellular pharmacodynamics of Doxorubicin. Neoplasia 2005, 7 (7), 705−713. (10) Untoro, J.; Schultink, W.; West, C. E.; Gross, R.; Hautvast, J. G. Efficacy of oral iodized peanut oil is greater than that of iodized poppy seed oil among Indonesian schoolchildren. Am. J. Clin. Nutr. 2006, 84 (5), 1208−1214. (11) Bhattacharya, S.; Dhillon, A. P.; Winslet, M. C.; Davidson, B. R.; Shukla, N.; Gupta, S. D.; Al-Mufti, R.; Hobbs, K. E. Human liver cancer cells and endothelial cells incorporate iodised oil. Br. J. Cancer 1996, 73 (7), 877−881. (12) de Baere, T.; Denys, A.; Briquet, R.; Chevallier, P.; Dufaux, J.; Roche, A. Modification of arterial and portal hemodynamics after injection of iodized oils and different emulsions of iodized oils in the hepatic artery: an experimental study. J. Vasc. Intervent. Radiol. 1998, 9 (2), 305−310. (13) Noren, A.; Urdzik, J.; Duraj, F.; Barbier, C. E.; Karlson, B. M.; Haglund, U. Longterm follow-up after transarterial chemotherapy for hepatocellular carcinoma in a Scandinavian centre. HPB (Oxford) 2010, 12 (9), 637−643. (14) Iwai, K.; Maeda, H.; Konno, T. Use of oily contrast medium for selective drug targeting to tumor: enhanced therapeutic effect and Xray image. Cancer Res. 1984, 44 (5), 2115−2121. 142



Article

(31) Arvidsson, D.; Svensson, H.; Haglund, U. Laser-Doppler flowmetry for estimating liver blood flow. Am. J. Physiol. 1988, 254 (4 Pt 1), G471−G476. (32) Upton, R. N. Organ weights and blood flows of sheep and pig for physiological pharmacokinetic modelling. J. Pharmacol. Toxicol. Methods 2008, 58 (3), 198−205. (33) Court, F. G.; Wemyss-Holden, S. A.; Morrison, C. P.; Teague, B. D.; Laws, P. E.; Kew, J.; Dennison, A. R.; Maddern, G. J. Segmental nature of the porcine liver and its potential as a model for experimental partial hepatectomy. Br. J. Surg. 2003, 90 (4), 440−444. (34) August, D. A.; Verma, N.; Vaertan, M. A.; Shah, R.; Brenner, D. E. An evaluation of hepatic extraction and clearance of doxorubicin. Br. J. Cancer 1995, 72 (1), 65−71. (35) Nordgren, A.; Karlsson, T.; Wiklund, L. Glutamine concentration and tissue exchange with intravenously administered alphaketoglutaric acid and ammonium: a dose-response study in the pig. Nutrition 2002, 18 (6), 496−504. (36) Lee, H. J.; Chiou, W. L. Erythrocytes as barriers for drug elimination in the isolated rat liver. I. Doxorubicin. Pharm. Res. 1989, 6 (10), 833−839. (37) Kassner, N.; Huse, K.; Martin, H. J.; Godtel-Armbrust, U.; Metzger, A.; Meineke, I.; Brockmoller, J.; Klein, K.; Zanger, U. M.; Maser, E.; Wojnowski, L. Carbonyl reductase 1 is a predominant doxorubicin reductase in the human liver. Drug Metab. Dispos. 2008, 36 (10), 2113−2120. (38) Skarka, A.; Skarydova, L.; Stambergova, H.; Wsol, V. Anthracyclines and their metabolism in human liver microsomes and the participation of the new microsomal carbonyl reductase. Chem.Biol. Interact. 2011, 191 (1−3), 66−74. (39) Langenbucher, F.; Mysicka, J. In vitro and in vivo deconvolution assessment of drug release kinetics from oxprenolol Oros preparations. Br. J. Clin. Pharmacol. 1985, 19 (Suppl 2), 151S−162S. (40) Benet, L. Z. The role of BCS (biopharmaceutics classification system) and BDDCS (biopharmaceutics drug disposition classification system) in drug development. J. Pharm. Sci. 2013, 102 (1), 34−42. (41) Okabe, M.; Unno, M.; Harigae, H.; Kaku, M.; Okitsu, Y.; Sasaki, T.; Mizoi, T.; Shiiba, K.; Takanaga, H.; Terasaki, T.; Matsuno, S.; Sasaki, I.; Ito, S.; Abe, T. Characterization of the organic cation transporter SLC22A16: a doxorubicin importer. Biochem. Biophys. Res. Commun. 2005, 333 (3), 754−762. (42) Lal, S.; Mahajan, A.; Chen, W. N.; Chowbay, B. Pharmacogenetics of target genes across doxorubicin disposition pathway: a review. Curr. Drug Metab. 2010, 11 (1), 115−128. (43) Jamieson, D.; Boddy, A. V. Pharmacogenetics of genes across the doxorubicin pathway. Expert Opin. Drug Metab. Toxicol. 2011, 7 (10), 1201−1210. (44) Benet, L. Z.; Broccatelli, F.; Oprea, T. I. BDDCS applied to over 900 drugs. AAPS J. 2011, 13 (4), 519−547. (45) Regev, R.; Eytan, G. D. Flip-flop of doxorubicin across erythrocyte and lipid membranes. Biochem. Pharmacol. 1997, 54 (10), 1151−1158. (46) Danesi, R.; Fogli, S.; Gennari, A.; Conte, P.; Del Tacca, M. Pharmacokinetic-pharmacodynamic relationships of the anthracycline anticancer drugs. Clin. Pharmacokinet. 2002, 41 (6), 431−444. (47) Di Fronzo, G.; Lenaz, L.; Bonadonna, G. Distribution and excretion of adriamycin in man. Biomedicine 1973, 19 (4), 169−171. (48) Forrest, G. L.; Gonzalez, B. Carbonyl reductase. Chem.-Biol. Interact. 2000, 129 (1−2), 21−40. (49) Mross, K.; Maessen, P.; van der Vijgh, W. J.; Gall, H.; Boven, E.; Pinedo, H. M. Pharmacokinetics and metabolism of epidoxorubicin and doxorubicin in humans. J. Clin. Oncol. 1988, 6 (3), 517−526. (50) Camaggi, C. M.; Comparsi, R.; Strocchi, E.; Testoni, F.; Angelelli, B.; Pannuti, F. Epirubicin and doxorubicin comparative metabolism and pharmacokinetics. A cross-over study. Cancer Chemother. Pharmacol. 1988, 21 (3), 221−228. (51) Benjamin, R. S.; Riggs, C. E., Jr.; Bachur, N. R. Plasma pharmacokinetics of adriamycin and its metabolites in humans with normal hepatic and renal function. Cancer Res. 1977, 37 (5), 1416− 1420.

(52) Maniez-Devos, D. M.; Baurain, R.; Lesne, M.; Trouet, A. Doxorubicin and daunorubicin plasmatic, hepatic and renal disposition in the rabbit with or without enterohepatic circulation. J. Pharmacol. 1986, 17 (1), 1−13. (53) Kim, J. E.; Cho, H. J.; Kim, J. S.; Shim, C. K.; Chung, S. J.; Oak, M. H.; Yoon, I. S.; Kim, D. D. The limited intestinal absorption via paracellular pathway is responsible for the low oral bioavailability of doxorubicin. Xenobiotica 2012, 43 (7), 579−591. (54) Thorn, H. A.; Lundahl, A.; Schrickx, J. A.; Dickinson, P. A.; Lennernas, H. Drug metabolism of CYP3A4, CYP2C9 and CYP2D6 substrates in pigs and humans. Eur. J. Pharm. Sci. 2011, 43 (3), 89−98. (55) Anzenbacher, P.; Soucek, P.; Anzenbacherova, E.; Gut, I.; Hruby, K.; Svoboda, Z.; Kvetina, J. Presence and activity of cytochrome P450 isoforms in minipig liver microsomes. Comparison with human liver samples. Drug Metab. Dispos. 1998, 26 (1), 56−59. (56) Garnick, M. B.; Ensminger, W. D.; Israel, M. A clinicalpharmacological evaluation of hepatic arterial infusion of adriamycin. Cancer Res. 1979, 39 (10), 4105−4110. (57) Thorn, H. A.; Sjogren, E.; Dickinson, P. A.; Lennernas, H. Binding processes determine the stereoselective intestinal and hepatic extraction of verapamil in vivo. Mol. Pharmaceutics 2012, 9 (11), 3034−3045. (58) Lewis, A. L.; Gonzalez, M. V.; Lloyd, A. W.; Hall, B.; Tang, Y.; Willis, S. L.; Leppard, S. W.; Wolfenden, L. C.; Palmer, R. R.; Stratford, P. W. DC bead: in vitro characterization of a drug-delivery device for transarterial chemoembolization. J. Vasc. Intervent. Radiol. 2006, 17 (2 Pt 1), 335−342. (59) Bjerregaard, S.; Soderberg, I.; Vermehren, C.; Frokjaer, S. Formulation and evaluation of release and swelling mechanism of a water-in-oil emulsion using factorial design. Int. J. Pharm. 1999, 193 (1), 1−11. (60) Wang, P. J.; Li, W. C.; Xi, G. M.; Wang, H. Q.; Zhang, Z. H.; Yao, B. C.; Tang, W.; Deng, Z. H.; Zhang, X. H. Biomechanical study of hepatic portal vein in humans and pigs and its value in liver transplantation. Transplant. Proc. 2009, 41 (5), 1906−1910. (61) Gonzalez, M. V.; Tang, Y.; Phillips, G. J.; Lloyd, A. W.; Hall, B.; Stratford, P. W.; Lewis, A. L. Doxorubicin eluting beads-2: methods for evaluating drug elution and in-vitro:in-vivo correlation. J. Mater. Sci.: Mater. Med. 2008, 19 (2), 767−775. (62) Jordan, O.; Denys, A.; De Baere, T.; Boulens, N.; Doelker, E. Comparative study of chemoembolization loadable beads: in vitro drug release and physical properties of DC bead and hepasphere loaded with doxorubicin and irinotecan. J. Vasc. Intervent. Radiol. 2010, 21 (7), 1084−1090. (63) Biondi, M.; Fusco, S.; Lewis, A. L.; Netti, P. A. New insights into the mechanisms of the interactions between doxorubicin and the ionexchange hydrogel DC Bead for use in transarterial chemoembolization (TACE). J. Biomater. Sci., Polym. Ed. 2012, 23 (1−4), 333−354. (64) Namur, J.; Citron, S. J.; Sellers, M. T.; Dupuis, M. H.; Wassef, M.; Manfait, M.; Laurent, A. Embolization of hepatocellular carcinoma with drug-eluting beads: doxorubicin tissue concentration and distribution in patient liver explants. J. Hepatol. 2011, 55 (6), 1332− 1338. (65) Lewis, A. L.; Taylor, R. R.; Hall, B.; Gonzalez, M. V.; Willis, S. L.; Stratford, P. W. Pharmacokinetic and safety study of doxorubicineluting beads in a porcine model of hepatic arterial embolization. J. Vasc. Intervent. Radiol. 2006, 17 (8), 1335−1343. (66) Larsen, C.; Larsen, S. W.; Jensen, H.; Yaghmur, A.; Ostergaard, J. Role of in vitro release models in formulation development and quality control of parenteral depots. Expert Opin. Drug Delivery 2009, 6 (12), 1283−1295. (67) Yamashita, Y. T., T.; Fukushima, S.; Nishida, M.; Nakano, M. Experimental Study of Hepatic Artery Administration of Various Formulations of Lipiodol with Anticancer Agents. Kumamoto Med. J. 1987, 40 (2), 53−65. (68) Yi, S. W.; Kim, Y. H.; Kwon, I. C.; Chung, J. W.; Park, J. H.; Choi, Y. W.; Jeong, S. Y. Stable lipiodolized emulsions for hepatoma targeting and treatment by transcatheter arterial chemoembolization. J. Controlled Release 1998, 50 (1−3), 135−143. 143



Article

(69) Boyd, G. E.; Schubert, J.; Adamson, A. W. The exchange adsorption of ions from aqueous solutions by organic zeolites; ionexchange equilibria. J. Am. Chem. Soc. 1947, 69 (11), 2818−2829. (70) Moffat, A. C. Clarke’s analysis of drugs and poisons: In pharmaceuticals, body fluids and postmortem material, 3rd ed.; Pharmaceutical Press: London; Chicago, 2004. (71) Rivory, L. P.; Avent, K. M.; Pond, S. M. Effects of lipophilicity and protein binding on the hepatocellular uptake and hepatic disposition of two anthracyclines, doxorubicin and iododoxorubicin. Cancer Chemother. Pharmacol. 1996, 38 (5), 439−445. (72) Varma, M. V.; Khandavilli, S.; Ashokraj, Y.; Jain, A.; Dhanikula, A.; Sood, A.; Thomas, N. S.; Pillai, O.; Sharma, P.; Gandhi, R.; Agrawal, S.; Nair, V.; Panchagnula, R. Biopharmaceutic classification system: a scientific framework for pharmacokinetic optimization in drug research. Curr. Drug Metab. 2004, 5 (5), 375−388.

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