The Effects of Lipiodol and Cyclosporin A on the Hepatobiliary

Article pubs.acs.org/molecularpharmaceutics

The Effects of Lipiodol and Cyclosporin A on the Hepatobiliary Disposition of Doxorubicin in Pigs Ilse R. Dubbelboer,†,‡ Elsa Lilienberg,†,‡ Mikael Hedeland,§,∥ Ulf Bondesson,§,∥ Micheline Piquette-Miller,⊥ Erik Sjögren,† and Hans Lennernas̈ *,† †

Department of Pharmacy, Uppsala University, Box 580, 751 23 Uppsala, Sweden Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry, Uppsala University, Box 574, 751 23 Uppsala, Sweden ∥ Department of Chemistry, Environment and Feed Hygiene, National Veterinary Institute (SVA), 751 89 Uppsala, Sweden ⊥ Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Canada §

ABSTRACT: Doxorubicin (DOX) emulsified in Lipiodol (LIP) is used as local palliative treatment for unresectable intermediate stage hepatocellular carcinoma. The objective of this study was to examine the poorly understood effects of the main excipient in the drug delivery system, LIP, alone or together with cyclosporin A (CsA), on the in vivo liver disposition of DOX and its active metabolite doxorubicinol (DOXol). The advanced, multi-sampling-site, acute pig model was used; samples were collected from three blood vessels (v. portae, v. hepatica and v. femoralis), bile and urine. The four treatment groups (TI− TIV) all received two intravenous 5 min infusions of DOX into an ear vein: at 0 and 200 min. Before the second dose, the pigs received a portal vein infusion of saline (TI), LIP (TII), CsA (TIII) or LIP and CsA (TIV). Concentrations of DOX and DOXol were analyzed using UPLC−MS/MS. The developed multicompartment model described the distribution of DOX and DOXol in plasma, bile and urine. LIP did not affect the pharmacokinetics of DOX or DOXol. CsA (TIII and TIV) had no effect on the plasma pharmacokinetics of DOX, but a 2-fold increase in exposure to DOXol and a significant decrease in hepatobiliary clearance of DOX and DOXol were observed. Model simulations supported that CsA inhibits 99% of canalicular biliary secretion of both DOX and DOXol, but does not affect the metabolism of DOX to DOXol. In conclusion, LIP did not directly interact with transporters, enzymes and/or biological membranes important for the hepatobiliary disposition of DOX. KEYWORDS: doxorubicin, doxorubicinol, lipiodol, liver disposition, pharmacokinetics, cyclosporin A

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palmitate and stearate,3 and the DDS is often formulated as an emulsion of LIP and doxorubicin (DOX) solution as the single chemotherapeutic agent. The recommended treatment approach is based on a regional imaging-guided infusion of the DDS via the hepatic artery (HA) to the tumor-feeding arteries.4 The regional infusion and use of the DDS is intended to increase exposure of the tumor to DOX while reducing systemic exposure, thus reducing drug-associated side effects.5 Following administration, the affected blood vessels are partially

INTRODUCTION

A drug delivery system (DDS) used for palliative treatment of unresectable intermediate stage hepatocellular carcinoma (HCC) is various chemotherapeutics drugs emulsified in the excipient Lipiodol (LIP), an iodized and ethylated derivate of poppy seed oil. Although this DDS has been used for several decades, a standardized operating procedure of the formulation preparation and its clinical use is not available.1 Together with limitations in its positive effect and safety profile, it shows that there is a medical need for improvements, especially as the burden of primary liver cancer, such as HCC, is an expanding problem in the western world, particularly in the US.2 The excipient LIP consists of iodized and ethylated esters of long chain fatty acids (C16 and C18) such as linoleate, oleate, © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1301

December 19, 2013 February 12, 2014 February 21, 2014 February 21, 2014 dx.doi.org/10.1021/mp4007612 | Mol. Pharmaceutics 2014, 11, 1301−1313

Molecular Pharmaceutics

Article

embolized by the DDS,6,7 and it appears that the ethylated and iodinated long chain fatty acids (i.e., LIP) then are endocytosed into the cells to be partly eliminated through bile.8,9 Long chain fatty acids are thought to be transported across biological membranes via a flip-flop mechanism through passive and facilitated carrier-mediated (CM) transport processes.10 In addition, the excipient LIP is retained in tumor tissue where it is visible on X-ray for months.9,11 DOX enters cells by both passive diffusion and CM transport, via the OCT6 (SLC22A16) transporter.12,13 DOX is predominantly eliminated by metabolism (>50%) and excretion into the bile by CM transport.14 It is metabolized primarily to its 13-OH derivate doxorubicinol (DOXol; 35− 45% of the administered dose in humans) by aldo-keto and carbonyl reductases.14−17 Aglycon metabolites are also formed to various extents, mediated by NADPH dependent cytochrome P450 (CYP) reductases.14 At the canalicular membrane of hepatocytes DOX is effluxed into the bile by the BCRP (ABCG2), P-glycoprotein (Pgp, MDR1, ABCB1) and MRP2 (ABCC2) transporters.18,19 Several in vitro studies have suggested that lipid excipients in DDSs increase the transport of drugs across biological membranes.20−23 A number of hypotheses regarding the mechanisms for this increase have been proposed; these include increased membrane fluidity, inhibition of CM efflux transporters, increased aqueous solubility and/or decreased enzymatic hydrolysis.20−23 However, it is difficult to accurately predict the in vivo interactions for locally administered targeted drug products based on in vitro data, since the lack of complex tissue biotransport processes in vitro prolongs contact time between the formulation and the cell model, and thus interaction with the excipients is more likely to occur.24 Recent in vivo reports have indicated that the distribution of LIP in the liver tumor tissue does not correlate with that of DOX and accordingly challenges the indirect tumor drug targeting potential of this drug product.25,26 On the other hand, earlier studies reported higher DOX concentrations in the tumor than in the surrounding liver tissue after administration with LIP.6,27 In addition, a recent study in our lab indicated an interesting trend for the apparent intracellular availability of DOX to be higher when DOX was emulsified in LIP than when it was administered in a particle based DDS, up to 6 h after dosing.28 This might be clinically important, as cellular peak concentrations are thought to give a better indication of the antitumor efficacy of DOX than total plasma exposure (AUC).29 The exact mechanisms behind the effects of LIP on the local disposition of DOX and its active metabolite DOXol are still unknown. The objective of this study was to examine the effect(s) of the excipient LIP (i.e., not emulsified with DOX) on the local and systemic disposition of DOX and DOXol, and to investigate the mechanisms involved. Cyclosporin A (CsA) was used as a positive control, as CsA is a known inhibitor of the CM transport of DOX with a broad inhibitory effect on, for example, Pgp, BCRP, MRP2 and organic anion-transporting polypeptide (OATP)-family transporters, and several CYP-enzymes that are important for drug metabolism.30,31 CsA appears not to affect the activity of the DOX influx transporter SLC22A16. The effects of the inhibitor CsA, which has known effects on the disposition of DOX, were compared with those of a clinical dose of LIP on the disposition of DOX and DOXol. The advanced multi-sampling-site acute (aMSA) pig model, which allows the collection of rich data, has been shown to be of value in a previous

study on DOX containing DDSs.28 This study is part of the liver cancer treatment optimization project at Uppsala University, and is fundamental to improve the knowledge about pharmaceutical excipients’ role in vivo as the long-term goal is to optimize the performance of advanced multifunctional DDSs for the treatment of HCC.

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EXPERIMENTAL SECTION This acute animal study was performed at the Clinical Research Department, Uppsala University Hospital, Uppsala, Sweden, and was approved by the local ethics committee for the use of laboratory animals in Uppsala, Sweden (C40/11). Study Design. Twelve male pigs of mixed breed (Yorkshire and Swedish landrace), weighing 24.2−30.3 kg (mean 26.7 kg) and aged 10−12 weeks, were investigated. Before the study, food was withheld from all the animals overnight, but all had access to water ad libitum. In this acute, parallel-group study (see outline of study design in Table 1), the pigs were assigned to one of four treatment Table 1. Summary of the Parallel-Group Study Design Showing the Treatment Groups and the Infusion Schedulesa 0−5 minb

165−185 minc

Treatment Group doxorubicin 1.7 mg/mL 19.1 mL Treatment Group doxorubicin 1.7 mg/mL 19.1 mL Treatment Group doxorubicin 1.7 mg/mL 19.1 mL Treatment Group doxorubicin 1.7 mg/mL 19.1 mL

I saline 0.9%, pH 7.4 100 mL II saline 0.9%, pH 7.4 100 mL III cyclosporin A 2.5 mg/mL 100 mL IV cyclosporin A 2.5 mg/mL 100 mL

190−195 minc

200−205 minb

saline 0.9%, pH 7.4 7.8 mL

doxorubicin 1.7 mg/mL 19.1 mL

LIP emulsion 6:1.8 LIP:H2O 7.8 mL


saline 0.9%, pH 7.4 7.8 mL


LIP emulsion 6:1.8 LIP:H2O 7.8 mL


a

Each treatment group included three pigs and data are presented as substance, concentration and volume. bAdministration site: ear vein. c Administration site: portal vein. LIP = Lipiodol, Saline = sodium chloride.

groups (TI−TIV). The study was divided into two treatment phases: 0−160 min was the reference phase (P1), and 200−360 min was the test phase (P2). All pigs received two intravenous (iv) 5 min infusions of DOX (57.8 μmol, 1.7 mg/mL) into an ear vein: one dose at 0 min (i.e., reference phase; TI:P1−TIV:P1) and one dose at 200 min (i.e., test phase; TI:P2−TIV:P2). All pigs thus received two doses of DOX, which allowed each animal to serve as its own control. Before the second dose of DOX, the pigs received an additional treatment. The first treatment group (TI, n = 3), the control group, received only saline as additional treatment before the second dose. The second treatment group (TII, n = 3) received a 4.5−6 min infusion of LIP emulsion (7.8 mL) starting at 190 min. Treatment group three (TIII, n = 3) received a 20 min CsA infusion (2.5 mg/mL, 250 mg in total) starting at 165 min. The fourth treatment group (TIV, n = 3) was given both CsA and LIP successively at 165 and 190 min. If no other infusion was scheduled, a saline solution was administered at 165 and/or 190 min. All the other infusions (saline, LIP and/or CsA) were infused into the portal vein (VP) (Table 1). Clinically, LIP is 1302

dx.doi.org/10.1021/mp4007612 | Mol. Pharmaceutics 2014, 11, 1301−1313


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and pCO2 throughout the experiment with a Solar 8000 (Marquette Medical Systems Inc., Milwaukee, USA). Blood gases were checked every second hour after the start of the experiments until the end of the study, using an ABL5 Radiometer (Diamond Diagnostics, Holliston, MA, USA). After the surgery was completed, usually after 1 h and 20 min, all pigs were allowed to stabilize for 30 min before the experiment started. Sampling of Blood, Urine and Bile. Blood, urine and bile were collected during the experiment. Blood samples were collected from the VP, VH and VF into vacutainers containing K3EDTA (BD Vacutainer; Becton Dickinson AB, Stockholm, Sweden). The blood was collected in 4 mL fractions 5, 10, 20, 30, 45, 65, 95, 125, 160, 205, 210, 215, 225, 245, 265, 295, 330 and 360 min after the start of the first DOX infusion. The blood samples were centrifuged (10 min, 3600 rpm, 4 °C) within 20 min of collection (Universal 16R; Hettic, Tuttlingen, Germany). Urine was collected quantitatively from the start of the first DOX infusion and sampled at the end of the experiment. Bile was continuously quantitatively collected on ice at 20 min intervals, starting at the end of the first dose of DOX and continuing until the end of the experiment. Plasma, urine and bile samples were aliquoted into brown polypropylene vials (2 mL, Sarstedts) to protect DOX from light and stored at −20 °C pending analysis. Plasma, Bile and Urine Analysis. DOX hydrochloride was purchased from LC Laboratories (MA, USA), and DOXol citrate was purchased from Toronto Research Chemicals (North York, ON, Canada). The internal standards [13C,2H3]-DOX trifluoroacetate and [13C,2H3]-DOXol formiate were bought from Alsachim (Illkirch-Graffenstaden, France). Water was purified using a Milli-Q water purification system (Millipore, Bedford, MA, USA). All other chemicals were of analytical grade or better and were used without further purification. The quantification of DOX and DOXol was carried out using methods slightly modified from those described in Lilienberg et al.28 For the plasma analysis, 250 μL of plasma was mixed with 100 μL of water and 50 μL each of the two internal standard solutions ([13C,2H3]-DOX 0.15 μg/mL and [13C,2H3]-DOXol 0.15 μg/mL). The proteins were precipitated by addition of 400 μL of aqueous trichloroacetic acid solution (20% v/v). After vortex mixing and centrifugation at 11500g for 10 min, the supernatants were filtered through a 0.2 μm filter by centrifugation at 11500g for 5 min. The filtrates were transferred to vials for injection onto an ultraperformance liquid chromatography− tandem mass spectrometry (UPLC−MS/MS) system. For the urine analysis, 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 to 50 μL of urine. The samples were then diluted with 2.0 mL of 0.1% formic acid in water, vortex mixed, and transferred to vials for UPLC−MS/MS analysis. Bile was analyzed using the same pretreatment method as in Lilienberg et al.28 The UPLC−MS/MS methods for all matrices were the same as those described in Lilienberg et al.28 For DOX, the lower limits of quantification (LLOQ) were 0.5, 50 and 100 ng/mL in plasma, bile and urine, respectively. The LLOQs for DOXol were 0.5, 50 and 80 ng/mL in plasma, bile and urine, respectively. The precision expressed as the relative standard deviation (RSD) for the results of quality control samples in plasma was in the interval 1.1−13.6% (DOX) and 1.9−11.7% (DOXol). For the bile samples, the precision was 2.6−11.8%

infused through the HA to direct the treatment to the tumor. However, the aMSA pig model lacks a tumor, and limitations in this model were previously encountered with respect to dose administration of LIP (≤0.9 mg LIP/kg) when LIP was administered via the HA.28 Furthermore the blood flow in VP is 2.4-fold increased compared to HA and the VP contributes with 66% of the total liver blood supply.32−34 As a higher dose and high hepatic exposure were needed, the VP was used as the administration site for both LIP and CsA, rather than the HA. Investigational Drugs and Rationale for Dose Selection. The iv solution of DOX (1.7 mg/mL) was prepared from doxorubicin HCl powder (LC laboratories, Woburn, MA, USA) and saline (9 mg/mL NaCl, Fresenius Kabi, Halden, Norway), adjusted with 1 M HCl to pH 3 and stored at 4−8 °C before the start of the experiment. The total dose of DOX administered was set at 62.5 mg per animal (2.0−2.6 mg/kg), because 3 mg/kg has been reported to be the highest dose tolerated by pigs.35 In this specific pig model, up to 1.8 mg/kg DOX has been previously successfully administered.28 The clinical doses of DOX emulsified with LIP are about 50 mg per treatment (0.7−0.8 mg/kg).36,37 The 250 mg CsA dose (100 mL of 2.5 mg/mL solution) was prepared from 5 mL of a 50 mg/mL CsA solution (Sandimmun, Novartis, Täby, Sweden) and 95 mL of saline shortly before infusion (8.3−10.3 mg/kg). A 2 h iv infusion of 300 mg of CsA has previously been shown to provide effective inhibition of transporters and enzymes and is well tolerated in the aMSA pig model.38,39 Six milliliters of LIP (Lipiodol Ultra-fluide, Guerbet, Aulnay-sous-Bois, France; 0.2−0.25 mL LIP/kg) was vortex-mixed with 1.8 mL of sterile water for 30 s prior to administration, with the intention of creating a water-in-oil emulsion similar to those used in the clinic.37 However, the resulting emulsion was relatively unstable, and phase separation started within minutes. The clinical dose of LIP given to HCC patients is approximately 10 mL (800 mg/mL lipids, 480 mg/mL iodine; approximately 0.1−0.125 mL LIP/kg), but varies within a range of 2−25 mL.36,40,41 Surgical Procedure and Anesthesia. The advanced multi-sampling-site acute (aMSA) pig model was established in our laboratory a decade ago.42 The surgical procedure and sampling methods were conducted as described in Lilienberg et al.28 Briefly, after insertion of a catheter into the right ear vein general anesthesia was induced. A solution of morphine 0.5 mg/kg/h (Morfin Meda 10 mg/mL, Meda AB, Solna, Sweden), ketamine 20 mg/kg/h (Ketaminol Vet, 100 mg/mL, Intervet, Stockholm, Sweden) and rocuronium 2 mg/kg/h (Esmeron, 10 mg/mL, Merck Sharp & Dohme, Sollentuna, Sweden) was administered by continuous iv infusion during the whole experiment to ensure that the pigs were completely pain free and fully anesthetized. To keep the body fluid balance normal and retain osmotic pressure, the pigs received Rehydrex 10 mL/kg/h (Fresenius Kabi AB, Uppsala, Sweden), Ringer’s acetate 8 mL/kg/h (Fresenius Kabi AB, Uppsala, Sweden) and 250 mL of Rheomacrodex (dextran 40) 100 mg/mL in saline. A bolus iv injection of 10 mL of Promiten 60 mg/mL (dextran 40) in saline was given before the Rheomacrodex to prevent allergic reactions. The pigs were ventilated with an oxygen−air mix. A central vein catheter was introduced into the right external jugular vein to monitor blood pressure and heart rate. Catheters for blood sampling were introduced into the VP, hepatic vein (VH) and femoral vein (VF), and catheters for bile and urine collection were introduced into the bile duct and the bladder, respectively. The pigs were constantly monitored for blood pressure, heart rate, central venous pressure, temperature 1303



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This approximation of Fi;app was based on the assumption that the main enzymes metabolizing DOX to DOXol (1 to 1 conversion) are located intracellularly in the cytosol. It was also assumed that there was a linear excretion into the bile, no saturation of metabolizing enzymes and no reabsorption from the bile duct.28 Compartmental Analysis of the iv Dose. The plasma concentration−time curves for DOX from each sampling site (VH, VP and VF) for P1 and P2 were analyzed using one-, twoand three- compartment models, adopting a 1/ŷ2 weighting scheme. The models were compared by visual inspection of the fit of the curves and by comparison of the Akaike Information Criterion (AIC) and Schwarz Bayesian Criterion (SBC).

(DOX) and 0.3−9.2% (DOXol), and 3.9−5.9% (DOX) and 1.5−11.3% (DOXol) for the urine samples. Data Analysis and Pharmacokinetics. Plasma and bile concentration−time profiles for DOX and DOXol in treatment phases P1 and P2 were analyzed separately using Phoenix WinNonLin software version 6.3 (Certara, L.P., St. Louis, MO, USA). Both noncompartmental analyses (NCA) and compartmental analyses were performed on the pharmacokinetics (PK) results for DOX and DOXol as described below. Noncompartmental Analysis. The plasma concentration− time curves for DOX and DOXol were analyzed after administration of DOX to each animal for treatment phases P1 and P2. The maximum concentration (Cmax) and the time to reach Cmax (tmax) were derived from the plasma concentration− time profiles. The area under the concentration−time curve (AUC) was calculated for both treatment phases (AUCP1 and AUCP2) using the linear trapezoidal (ascending part of the curve to Cmax) and logarithmic (descending part to the last data point at Clast) method. Extrapolations to calculate AUCP1−∞ and AUCP2−∞ were based on the terminal rate constant, λz, for each treatment phase. λz was estimated from at least four of the last measurable sample points in P1 or P2, and the terminal half-life (t1/2) for each treatment phase was calculated from ln 2/λz. The apparent hepatic extraction ratio (EH) was calculated by comparing the exposure (AUC) of DOX before the liver (VP samples) with that after the liver (VH samples) per treatment phase: EH =

(AUC VP − AUC VH ) AUC VP

(1)

The clearance (CL) for each treatment phase was calculated according to eq 2, using the AUCP1−∞ or AUCP2−∞ from VP (eq 2): dose CL = AUC

Figure 1. Schematic overview of the multicompartment model describing the distribution and elimination of doxorubicin (DOX) and doxorubicinol (DOXol). Oval shapes represent the distribution compartments reached by DOX or DOXol, and in the experimental design, samples were collected from the plasma, urine and bile (bold text and black outline). The syringe indicates the site of DOX infusion (the ear vein). The distribution of DOX and DOXol to and from peripheral tissue (Tissue) and liver and the net excretion of DOX and DOXol to bile and urine are represented by solid arrows. Dotted arrows represent the metabolism of either DOX to DOXol or DOXol to other metabolites. Sensitivity analyses were performed by reducing the estimated rate constants (i.e., the three metabolism rate constants and the excretion rate constants to bile for DOX and DOXol) by 99% per parameter.

(2)

The fraction excreted in bile ( fe,bile) per treatment phase was calculated as follows:

Ae,bile

fe,bile =

(3)

dose

where Ae,bile is the cumulative amount of DOX or DOXol recovered in the bile per treatment phase and dose is the administered dose in each treatment phase. In addition, Ae,bile was divided by the total hepatic exposure of DOX or DOXol in the VP (AUCVP,P1−∞ or AUCVP,P2−∞) as in eq 4 to obtain the apparent biliary clearance per treatment phase: CLapp,bile =

Multicompartment Pharmacokinetic Model. A multicompartment PK model (Figure 1) was constructed (i) to describe the plasma concentration−time profiles for DOX and DOXol and the cumulative amounts of these compounds excreted in bile and urine and (ii) to elucidate the mechanisms behind the effects of LIP and CsA on the in vivo disposition of DOX and DOXol. A three compartment physiological model structure, representing plasma (central), tissue and liver, was adopted with first-order reactions describing the disposition processes (distribution/elimination). Equation 7 describes the first-order mass transport in the model:

Ae,bile AUC VP

(4)

The urinary fraction excreted was calculated as follows: fe,urine =

Ae,urine dose tot

(5)

where Ae,urine is the cumulative amount of DOX or DOXol, respectively, in urine at 360 min and dosetot the sum of the two administered doses of DOX. The apparent intracellular availability (Fi;app) of DOX in each phase was approximated using AUCP1 and AUCP2 in eq 6: Fi;app =

dA = CkV dt

where A is the amount of compound, C the concentration of compound in the compartment, k the rate constant and V the volume of the compartment.

AUCDOXol,bile (AUCDOX,bile + AUCDOXol,bile )

(7)

(6) 1304



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the control group (TI). This involved comparison of each PK parameter between the phases; e.g., AUCTI:P1 vs AUCTI:P2. In order to compare the effects of LIP and/or CsA on the PK of DOX, PK parameters were corrected for interindividual differences, by dividing each PK parameter from the test phase (P2) by the corresponding parameter from P1 for the same animal, resulting in a PK parameter ratio. The nonparametric Kruskal−Wallis test was then used to investigate the effect of the different treatments on the calculated PK parameter ratios per treatment group (TI:P2/P1 vs TII:P2/P1, etc.). Because the treatment groups were small, no post hoc analysis was performed, and statistical significance is presented at a group level. As all the animals received the same treatment in P1, the data from P1 are presented as means ± standard deviations (SD) (TI−TIV:P1, n = 12). For P2, where the groups received different treatments before the DOX dose, the data are presented as medians (ranges) (TI:P2−TIV:P2, n = 3 for each group) unless otherwise specified.

The model includes the net biliary and urinary excretion of DOX and DOXol from the liver and central compartment, respectively. The metabolic pathway of DOX to DOXol was included in the tissue and liver compartments, as the aldo-keto and carbonyl reductases are also expressed in tissues other than the liver, although to a lesser extent.43 Other metabolic routes of DOX were not included as other metabolic pathways of DOX are minor compared with metabolism of DOX to DOXol.17 Metabolism of DOXol to unidentified entities was included in the liver compartment. In the absence of specific porcine data, the human plasma protein binding value was used (0.71).44 A total of 11 PK parameters were estimated, including volume of distribution at steady state (Vss), rate constants of excretion and metabolism and the fraction unbound in liver (Table 2). The parameter estimations for DOX and DOXol Table 2. Estimates of the 12 Model Parameters Used in the Multicompartment Model, Predicted by Modeling the Average Concentrations of DOX and DOXol from the First Treatment Phase (TI:P1−TIV:P1, n = 12) parameters volume of distribution (L) excretion rate constants (min−1)

substance(s) DOX and DOXol DOX DOXol DOX DOXol DOX DOXol

metabolism rate constants (min−1)

fraction unbound

DOX to DOXol DOX to DOXol DOXol to unknown metabolites DOX and DOXol

compartment

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RESULTS Surgery was successful in all animals. One acute adverse event, potentially related to high systemic exposure of DOX, was observed during the study period; the heart of one pig arrested 4 min after the second dose of DOX (the dose was preceded by infusions of CsA and LIP). Rescue with injection of 0.1−0.2 mg of adrenaline (Adrenalin Mylan 1 mg/mL, Mylan AB, Stockholm, Sweden) into an ear vein allowed the experiment to continue. No compartmental analysis could be performed for DOXol because of its continuous formation in all 12 pigs and lack of an elimination phase during the 6 h experiment. Hence, only NCA results were used for statistical comparison of DOX and DOXol PK parameters. No significant differences were found between the estimated PK parameters of DOX and DOXol in the VP, VH and VF blood compartments. The PK parameters for the VP plasma concentrations are presented in this report. PK of DOX and DOXol in TI−TIV:P1. The plasma concentration−time curves for DOX from all sampling sites during the first phase were best described by a twocompartment PK model with constant iv infusion and firstorder elimination (n = 12) (Figure 2A, P1). All results for the PK parameters for P1 are presented as mean ± SD. The Cmax of DOX occurred directly after cessation of the iv infusion in all plasma compartments. Both the clearance and the Vss of DOX were high, with values of 1.3 ± 0.27 L/min and 130 ± 98 L, respectively (Table 3). The t1/2 of DOX was 160 ± 69 min, and the EH was 0.065 ± 0.13 (Table 3) in this experimental in vivo model. The slope of the plasma concentration−time curve of DOXol increased rapidly after the end of the iv DOX infusion in all animals, followed by a sudden drop after 10−15 min and then a further increase (Figure 2B, P1). The median tmax was 95 min for DOXol, fluctuating between 15 and 160 min (Figure 2B, P1), while t1/2 could only be determined for 5 animals (Table 3, DOXol). The DOXol/DOX plasma AUC ratio was 0.13 ± 0.036 in the VP, and the DOXol/DOXVP concentration ratio reached a plateau approximately 50−100 min after the dose (Figure 3A, P1). The combined fe,bile for DOX and DOXol was 11.1%, and the AUCbile/AUCVP ratios for DOX and DOXol were 370 ± 110 and 580 ± 210, respectively (Table 4). The excretion rate tended to decline for DOX but remained constant for DOXol (Figure 4). There was no lag time in the biliary excretion of DOX or DOXol (Figure 4). The total bile volume collected during 0−160 min (TI−TIV:P1) was 65 ± 32.5 mL. The bile

estimates

plasma tissue tissue liver to bile liver to bile plasma to urine plasma to urine liver tissue liver

2.2 444 108 0.53 2.1 0.028

liver

0.16

0.11 0.042 0.0048 0.021

were estimated simultaneous fitting of the average plasma (VP), bile and urine data obtained during the reference phase (TI:P1−TIV:P1, n = 12). The differential equations were solved with nonlinear least-squares regression and a weighting scheme of 1/ŷ2 using Phoenix WinNonLin (v 6.3). The multicompartment PK model and the estimated parameters from P1 were then used to simulate the potential effect of inhibition of metabolism and/or biliary excretion of DOX and DOXol. As with the experimental design, two consecutive iv doses of DOX were administered, the first dose at 0 min and the second dose at 200 min. A series of sensitivity analyses were done by reducing the values of metabolic and biliary excretion parameters to 1% of the estimated values in various combinations (Table 6). The simulated inhibition started at 200 min and was constant until the end of the simulation (360 min). These simulations were compared with the observed data to elucidate the mechanisms of the effects of CsA on DOX and DOXol disposition. The AUC for the simulated plasma concentrations was calculated for each treatment phase as described for the experimental results. Statistical Analysis. The statistical analyses were performed using GraphPad Prism 5 with the level of statistical significance set at p < 0.05. Differences in PK parameters between the three plasma compartments (VP, VH, VF) in the reference phase (P1) were analyzed with one-way ANOVA. The Wilcoxon-rank test was performed to test the effect of repetitive iv dosing of DOX on the PK of DOX and DOXol in 1305



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compared to TI (Figure 2B, Table 5). The metabolite/parent ratio (AUCDOXol/AUCDOX) was significantly higher in the VP plasma compartments in TIII and TIV animals as compared to TI (p < 0.05) (Figure 3A). Effects of LIP and CsA on Biliary and Urinary Excretion of DOX and DOXol in TII−TIV. The PK parameters of DOX and DOXol in bile and urine and the PK ratios that differed from control (TI) are presented in Tables 4 and 5, respectively. The excretion of DOX and DOXol into bile was not affected by LIP (TII), but was immediately reduced following iv infusion of CsA in TIII and TIV animals (p < 0.05; Figure 4, Table 5). CsA administered either alone (TIII) or together with LIP (TIV) reduced the median fe,bile value for DOX by over 97% in both treatment groups (Table 5). The fe,bile for DOXol was decreased by 94 and 96% in the TIII and TIV groups, respectively (Table 5). Interestingly, when the full dose of CsA was infused into the VP, the color of the bile changed rapidly and significantly from red, caused by the chromophoric characteristics of DOX and/or DOXol, to light yellow. This color was a somewhat weaker yellow than the original dark yellow color observed in the reference bile sample taken just prior to the start of the study (Figure 6). The reductions in excretion of DOX and DOXol in groups TIII and TIV had a significant impact on all the biliary PK parameters (Figure 4; Table 5). Neither CsA nor LIP affected the bile flow (Figure 5). The biliary DOXol/DOX concentration ratio (Figure 3B) was not significantly affected in any of the treatment groups (TII, TIII and TIV). The metabolite/parent ratio curves from P2 had a similar shape to those in P1, with a slight increase in the slope for all treatment groups (Figure 3B). Fi;app tended to increase in the TIII and TIV groups after the second dose of DOX, by 40 and 50%, respectively (Table 5). The percentages of DOX and DOXol recovered in urine are shown in Table 4. The amount of DOXol excreted into the urine was not significantly affected by CsA and/or LIP, but the amount of DOX excreted tended to decrease in the TIII and TIV groups (Table 4). Multicompartment PK and Sensitivity Analysis. Figure 7 shows the observed data from the reference phase (P1) and the fitted curves using the multicompartment PK model. Parameters estimated from this analysis representing metabolism, biliary and renal excretion and distribution (volumes and flows) are summarized in Table 2. The results of the subsequent sensitivity analyses in the test phase (P2), where the model estimates were reduced by 99% to obtain 1% of control activity of metabolism and biliary excretion for DOX and DOXol from 200 to 360 min, are shown in Table 6 and Figure 8. Simulations demonstrated that a 99% reduction in the biliary excretion of DOX increased the plasma AUC of DOX by 22% and reduced the amount of DOX excreted into bile by 98%. The plasma AUC of DOXol increased by 14%, 54% and 79% after inhibition of the biliary excretion of DOX, DOXol and the combination of DOX and DOXol, respectively. The amount of DOXol excreted in bile decreased by 97% and 96% after inhibition of biliary excretion of DOXol and combined DOX and DOXol, respectively, but increased by only 19% after inhibition of biliary excretion of DOX alone (Table 6, Figure 8). Simulations demonstrated that a 99% reduction in metabolism of DOX to DOXol from 200 to 360 min had a negligible effect on plasma AUC and the amount of DOX excreted to bile (0−12%, Table 6). The 99% reduction of all estimates of metabolism of DOX to DOXol decreased the plasma AUC of DOXol by 86%, while inhibition of the

Figure 2. The plasma concentration−time curves (medians and ranges) for (A) doxorubicin (DOX) and (B) the main metabolite doxorubicinol (DOXol), sampled from the portal vein in pigs. DOX was given at the start of the reference phase (P1; 0−160 min) and at the start of the test phase (P2; 200−360 min). Additional treatment was given before the second dose of DOX. Treatment group TI was the control group, TII received lipiodol (LIP), TIII received cyclosporin A (CsA) and TIV received LIP and CsA. The plasma concentration−time profiles were best described by a two-compartment PK model, based on Akaike’s Information Criterion and the Schwarz Bayesian Criterion.

flow (mL/min) was relatively stable throughout the experiment and was not affected by the administration of DOX (Figure 5). Effect of Second Dose of DOX (P2) on PK of DOX and DOXol in the TI Group. There were no significant changes in the PK parameters for DOX or DOXol between P2 and P1 in the control group (TI). However, there was a trend for increases in DOXol plasma Cmax:P2 and AUCTI:P2 by 50 (20−70)% and 60 (20−90)%, respectively, and increases in DOXol fe,bile (more than 2-fold increase) and DOX Fi;app (nearly 2-fold) after the second DOX dose (Table 5). The bile flow was not significantly affected by the second dose of DOX (Figure 5). Effects of LIP and CsA on Plasma Exposure of DOX and DOXol in TII−TIV. The PK parameters for DOX and DOXol after administration of LIP and/or CsA are presented in Table 3. P2/P1 ratios of plasma and biliary PK parameter values for the treated animals (TII−TIV) and the untreated animals (TI) are presented in Table 5. The plasma concentration−time profiles of DOX in P2 were not significantly affected by any of the treatments (Figure 2A, P2). LIP administration (TII) had no effect on the plasma PK of DOXol (Figure 2B), and there were consequently no changes in the plasma metabolite/parent ratios compared to TI (Figure 3A). The median plasma AUC of DOXol increased in animals receiving CsA, by 47% (TIII) and 76% (TIV), respectively, 1306



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Table 3. Pharmacokinetic (PK) Parameters for Doxorubicin (DOX) and Doxorubicinol (DOXol) in Porcine Blood Samples Taken from the Portal Vein after Infusion of DOX into an Ear Vein at the Beginning of Two Treatment Phases (Reference Phase 0−160 min, P1; and Test Phase 200−360 min, P2)a ref phase (P1) TI−TIV DOX t1/2 (min) tmax (min) Cmax (μM) AUC (min·μM)b CL (L/min) Vss (L) EH DOXol t1/2 (min) tmax (min) Cmax (μM) AUC (min·μM)b CL (L/min) Vss (L)

test phase (P2) control (TI)

LIP (TII)

CsA (TIII)

CsA + LIP (TIV)

160 ± 69 5.9 ± 0.26 4.0 ± 1.1 38 ± 8.8 1.3 ± 0.27 130 ± 98 0.065 ± 0.13c

98 (96−200) 210 (210−210) 3.7 (2.6−4.3) 38 (27−46) 1.3 (1.2−1.4) 87 (65−220) 0.21 (0.03−0.31)

190 (130−280) 210 (210−210) 4.8 (2.9−4.8) 43 (30−49) 0.95 (0.73−1.6) 150 (98−180) 0.12 (0.065−0.29)

290 (93−430) 210 (210−210) 6.1 (4.7−7) 54 (44−66) 0.75 (0.66−0.9) 110 (43−280) 0.18 (0.084−0.2)

230 (140−260) 210 (210−210) 6.4 (4.7−7.3) 57 (46−73) 0.76 (0.59−0.93) 120 (39−130) 0.17 (0.05−0.18)

680 ± 770d 91 ± 43 0.037 ± 0.011 4.6 ± 1.3 4.9 ± 4.44 1900 ± 480d

200 (120−480) 250 (210−250) 0.039 (0.037−0.067) 5.0 (4.9−8.7) 4.0 (2.3−5.0) 1500 (730−1600)

730−930e 270 (220−270) 0.055 (0.036−0.058) 6.4 (4.3−8) 1.1−1.6e 1100−2100e

1100f 300 (230−360) 0.093 (0.063−0.097) 13 (8.7−13) 0.40 f 660 f

NA 360 (330−360) 0.094 (0.079−0.1) 11 (10−13) NA NA

a

Before the second dose of DOX (at start of P2), the pigs received saline (control, TI), Lipiodol (LIP, TII), cyclosporin A (CsA, TIII) or LIP + CsA (TIV) as an infusion into the portal vein. Results are shown as means ± standard deviations for P1 (TI−TIV, n = 12) and as medians (ranges) per treatment group for P2 (n = 3 per treatment group). bAUC0−160 min or AUC200−360 min. cn = 11. dn = 5. en = 2. fn = 1. AUC = area under concentration−time curve; Cmax = maximum concentration; CL = clearance; EH = apparent hepatic extraction ratio; NA = not available; t1/2 = elimination half-life; tmax = time to Cmax; Vss = volume of distribution at steady state.

Table 4. Pharmacokinetic (PK) Parameters for Doxorubicin (DOX) and Doxorubicinol (DOXol) in Porcine Biliary and Urinary Samples after Infusion of DOX into an Ear Vein Collected during Two Treatment Phases (Reference Phase 0−160 min, P1; and Test Phase 200−360 min, P2)a ref phase (P1) DOX fe,bile (%) CLapp,bile (L/min) AUCbile/AUCVP Fi;app fe,urine (%) DOXol fe,bile (%) CLapp,bile (L/min) AUCbile/AUCVP fe,urine (%)

test phase (P2)

TI−TIV

control (TI)

LIP (TII)

CsA (TIII)

CsA + LIP (TIV)

9.2 ± 4.1 0.14 ± 0.061 370 ± 110 0.16 ± 0.039 NA

12 (4.8−19) 0.25 (0.076−0.25) 310 (280−610) 0.38 (0.15−0.39) 3.1 (2.7−4.85)

5.8 (5.7−16) 0.12 (0.082−0.16) 190 (150−230) 0.36 (0.35−0.41) 2.8 (2.4−7.5)

0.26 (0.20−0.52) 0.0026 (0.0018−0.0072) 6.1 (5.7−13) 0.38 (0.38−0.44) 1.5 (1.2−1.9)

0.26 (0.18−0.83) 0.0032 (0.0019−0.0057) 14 (4.3−15) 0.46 (0.31−0.48) 1.4 (0.97−1.5)

1.9 ± 1.1 0.26 ± 0.15 580 ± 210 NA

3.6 (3.2−7.7) 0.39 (0.25−0.85) 1000 (780−1000) 1.0 (0.94−1.3)

4.1 (3.0−9.5) 0.42 (0.32−0.73) 830 (600−840) 1.0 (0.93−2.9)

0.22 (0.12−0.33) 0.0091 (0.0058−0.023) 20 (19−41) 1.2 (0.98−1.8)

0.22 (0.17−0.41) 0.01 (0.01−0.018) 41 (23−47) 0.88 (0.64−1.1)

a

Before the second DOX dose (at start of P2), the pigs received saline (control, TI), Lipiodol (LIP, TII), cyclosporin A (CsA, TIII) or LIP + CsA (TIV) as an infusion into the portal vein (VP). Results are shown as means ± standard deviations for P1 (T1−TIV, n = 12) and as medians (ranges) per treatment group for P2 (n = 3 per treatment group). AUC = area under the concentration−time curve in bile or VP plasma; CLapp,bile = apparent biliary clearance; fe,bile = fraction excreted in bile; fe,urine = fraction excreted in urine; Fi;app = apparent intracellular availability; NA = not available.

between the excipient LIP and hepatic transporters/enzymes, and a lack of effect on the fluidity of membranes associated with the disposition of DOX and DOXol. The positive control CsA, alone and together with LIP, had a distinct effect on some aspects of hepatobiliary DOX and DOXol disposition. The stable bile flow seen in most of the pigs suggests that they retained a stable condition after surgery and during the study. None of the treatments affected the bile flow, indicating that DOX, LIP and CsA are not cholestatic substances in the pig. There are no previous reports of DOX and LIP being cholestatic substances, but CsA has been classified as cholestatic in rats.45−47 However, in agreement with the data in this study, other studies have found that CsA is not cholestatic in pigs or liver-transplanted patients.38,48 The discoloration of bile after

metabolism of DOXol to other metabolites increased the plasma AUC of DOXol by 124%. The biliary excretion of DOXol decreased by 87% after the 99% reduction in the metabolism of DOX to DOXol and increased by 109% after metabolism of DOXol to other metabolites was inhibited (Table 6, Figure 8).

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DISCUSSION This study showed that while two consecutive iv doses of DOX had no significant effect on the PK of DOX in any of the three plasma compartments, there was a tendency for the Cmax, AUC, fe,bile and CLapp,bile for DOXol to be increased after the second dose. The excipient LIP had no observed effect on the PK of DOX or DOXol, indicating the lack of a direct interaction 1307



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nonsignificantly by about 50% and 60%, respectively, after the second dose. These increases in DOXol PK can be explained by a combination of two factors: the short dosage interval of 3 h and the long t1/2 of DOXol (30−33 h).17,53 The accumulation of DOXol in plasma was adequately explained by the multicompartment model. Administration of the excipient LIP did not significantly affect the PK parameters and intracellular availability of DOX or its metabolite DOXol in the pigs. In contrast, DOX concentrations have been reported to be significantly higher in tumor tissue than in nontumor tissue after it was administered in a LIP-based DDS (emulsion; water-in-oil) in the VX2 rabbit model.27 The same study indicated that it is important to emulsify DOX in LIP (water-in-oil) to gain the increased intratumor concentrations of DOX.27 In addition, it has recently been reported that DOX penetrated deeper into vascularized tumor tissue when it was emulsified in LIP than when it was given iv in the VX2 rabbit model.54 In a recent study in healthy pigs, the intracellular concentrations (Fi;app) of DOX were also higher when DOX was given in a LIP-based

Figure 3. The ratios of doxorubicinol/doxorubicin (A) concentrations in the portal vein (VP) plasma compartment and (B) amounts in the biliary compartment of treated pigs (medians and ranges). Doxorubicin was given at the start of the reference phase (P1; 0− 160 min) and at the start of the test phase (P2; 200−360 min). Additional treatment was given before the second dose of DOX. The treatment groups received saline (TI; controls), Lipiodol (LIP; TII), cyclosporin A (CsA; TIII) or LIP and CSA (TIV). In panel B, an outlying value (5.4) from one animal in the TIV group at 195 min is not shown.

CsA infusion suggests, in addition to inhibition of DOX excretion, that CsA also inhibits the excretion of endogenous substances. The difference in color between reference (blank) bile and bile samples after CsA infusion might be explained by an inhibition of MRP2, causing a reduction of excretion of conjugated bilirubin into bile.49 CsA-induced inhibition of other transporters such as the bile salt export pump (BSEP) may also contribute to this color change.30,50 The PK parameters CL, t1/2 and Vss for DOX and DOXol after the first peripheral iv infusion of DOX were consistent with those from an earlier aMSA pig study in our laboratory.28 AUC per dose of DOX in this study was slightly lower than for the previous study (6.4 vs 33 μM/min/dose), which could be explained by the difference in formulations and administration sites.28 Liver extraction of DOX was low or not possible to establish, which agrees with previous pig studies showing low levels of liver extractions after cessation of infusion.28 There were no changes in the plasma PK of DOX and DOXol when consecutive doses were given to cancer patients with a dosing interval of approximately 3 weeks.51,52 In this study, a second iv dose of DOX administered 200 min after the first dose affected neither the plasma PK nor the biliary and urinary excretion of DOX. However, both the Cmax and the AUC of DOXol were affected by consecutive dosing of DOX, increasing

Figure 4. The cumulative amounts of (A) doxorubicin (DOX) and (B) the main metabolite doxorubicinol (DOXol) excreted into pig bile, and (C) the median ratio between the two treatment phases (P2/P1) for DOX and DOXol. DOX was given at the start of the reference phase (P1; 0−160 min) and at the start of the test phase (P2; 200− 360 min). Additional treatment was given before the second dose of DOX. In panels A and B results are shown in medians and ranges from the treatment groups that received saline (TI, controls), Lipiodol (LIP; TII), cyclosporin A (CsA; TIII) or LIP and CSA (TIV). 1308



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Figure 5. The bile flow (mL/min) during the whole study period for each individual pig. All pigs received doxorubicin, and then subsequently received saline (TI), Lipiodol (LIP; TII), cyclosporin (CsA; TIII) or LIP and CsA (TIV) before a second dose of doxorubicin. The timing of each drug administration (see Table 1) is shown as a gray area. A light gray area represents saline infusion, and a dark gray area represents infusion of assigned treatment. For animal TIII:02 no bile was collected during the first hour because of faulty catheter placement in the bile duct. TIV:02’s heart arrested at 204 min, but it was revived with adrenaline.

Table 5. Test Phase/Reference Phase (P2/P1) Ratios of the PK Parameters for Doxorubicin (DOX) and Doxorubicinol (DOXol) in Porcine Plasma and Bile Samples (P1 0−160 min, and P2 200−360 min) after Infusion of DOX via an Ear Veina TI Plasma DOXol Cmax (μM) AUC (min·μM)b Bile DOX fe,bile (%) CLapp,bile (L/min) AUCbile/AUCVP Fi;app DOXol fe,bile (%) CLapp,bile (L/min) AUCbile/AUCVP

TII

TIII

TIV

significancec

1.5 (1.2−1.7) 1.6 (1.2−1.9)

1.2 (1.1−1.7) 1.5 (1.2−1.5)

2.3 (1.5−2.6) 2.3 (1.9−2.6)

3 (1.6−3.2) 2.8 (1.7−2.9)

1.2 (1.1−1.4) 0.98 (0.82−1.4) 0.64 (0.47−1.4) 1.9 (1.9−2)

1.2 (0.78−1.4) 1 (0.52−1.6) 0.73 (0.54−0.77) 2 (1.6−2.4)

0.03 (0.028−0.1) 0.02 (0.015−0.093) 0.015 (0.014−0.074) 2.7 (2.4−3.1)

0.034 (0.017−0.049) 0.024 (0.013−0.041) 0.037 (0.012−0.042) 2.9 (2.3−3.3)

P < 0.05 P < 0.05 P < 0.05

2.7 (2.4−3) 1.6 (1.5−1.9) 1.3 (0.86−1.9)

2.3 (1.7−5.3) 1.6 (1.2−4.4) 1.4 (1.2−1.8)

0.16 (0.11−0.24) 0.07 (0.044−0.13) 0.047 (0.038−0.14)

0.11 (0.079−0.21) 0.039 (0.027−0.12) 0.079 (0.024−0.11)

P < 0.05 P < 0.05 P < 0.05

P < 0.05

a

Before P2, the pigs received infusions of saline (control, TI), Lipiodol (LIP, TII), cyclosporin (CsA, TIII) or LIP + CsA (TIV) into the portal vein (VP) (n = 3 per treatment group). The table shows only ratios that indicated significant differences or a tendency for difference from values after the first dose (P1). bAUC0−160 min or AUC200−360 min. cStatistical significance measured at a group level with Kruskal−Wallis nonparametric test. AUC = area under the concentration−time curve in bile or VP plasma; Cmax = maximum concentration; CLapp,bile = apparent biliary clearance; fe,bile = fraction excreted in bile; Fi;app = apparent intracellular availability.

DDS than when it was given as an iv solution.28 Overall, our in vivo findings in the aMSA pig model indicated that LIP has no direct effect on transporters, enzymes and biological membranes that are of clinical importance for the hepatobiliary disposition of DOX and DOXol. Hence, it is suggested that the formulation properties of the LIP-based DDS affects the in vivo distribution of DOX rather than any direct interactions between the excipient LIP and membrane barriers and/or transport proteins. CsA reduced the biliary excretion of DOX by 97% over the 160 min bile collection period. These results are consistent with previous studies,18,19,31,55,56 and CsA is thought to inhibit the canalicularly located efflux transporters of DOX mediated by Pgp, MRP2 and BCRP. While CsA can inhibit sinusoidal

OATP transporters,30 interaction with canalicular rather than sinusoidal membrane transporters in this study is supported by previous findings with rosuvastatin and pravastatin, which are substrates for sinusoidal uptake. In those studies, CsA-mediated inhibition of sinusoidal transporters was associated with an increased plasma exposure and decreased hepatic clearance of rosuvastatin and pravastatin.38,57 This was in accordance with the known inhibition by CsA of the sinusoidal OATP transporters.30 In our study, the plasma exposure of DOX was unchanged, which suggests that sinusoidal uptake transport was not inhibited by CsA. However, inhibition of canalicular efflux transporters is supported by our bile collection data and the sensitivity analysis simulations with the multicompartment model. The lack of effect of CsA on DOX plasma concentrations 1309



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Figure 6. The figure illustrates the in vivo importance of canalicular transport of doxorubicin (DOX) and the potential of cyclosporin A (CsA) to interact with the transporters of importance for DOX. Bile samples collected at 20 min intervals from pig TIII:03 between 0 and 360 min demonstrate red discoloration after DOX administration (at time 0) compared to bile before DOX administration (blank). Infusion of CsA (at 165− 185 min) before the second dose of DOX (at 200 min) clearly shows that secretion of DOX into the bile was inhibited. The bile collected after CsA administration was also paler than the blank, which indicates that the excretion of (an) endogenous substance(s) was also inhibited.

Table 6. Results of the Sensitivity Analysis Performed Using a Multicompartment Model on Experimental Values for Area under the Plasma Concentration−Time Curve (AUC) and Cumulative Amount Excreted in Bile ( fe,bile) for Doxorubicin (DOX) and Doxorubicinol (DOXol) in Pigs after Administration of DOX plasma AUC (min·μM) inhibition (0.01 × original estimate) no

Figure 7. The average observed (symbols) and predicted (lines) plasma concentration−time curves and cumulative amounts excreted in bile for doxorubicin (DOX) and doxorubicinol (DOXol).

fe,bile (%)

DOXol

DOX

4.4

7.3

1.9

7.3

8.5

3.0

67 55 67

8.3 11.2 13.0

0.13 8.5 0.13

3.6 0.092 0.11

56 60 61 55

7.0 1.3 1.0 16.3

8.7 9.2 9.5 8.5

2.6 0.89 0.38 6.3

Phase 1 (0−160 min) 48 Phase 2 (200−360 min) 55

no biliary excretion DOX DOXol DOX and DOXol metabolism DOX to DOXol liver DOX to DOXol tissue DOX to DOXol liver and tissue DOXol to metabolites liver

in our study is in agreement with earlier reports with short sampling periods (24 h) have demonstrated increased DOX AUC when CsA is coadministered in mice and in human cancer patients.55,58 The results of those studies suggested that DOX is redistributed into plasma when the biliary excretion route is inhibited by CsA. Obviously, the redistribution process is comparatively slow and cannot be observed when the sampling period is short. The decrease in urinary excretion of DOX after addition of CsA may be explained by the inhibition of renal tubular efflux mediated by Pgp and MRP2.59 It has also been reported that DOXol concentrations are affected by Pgp modulators in rats and mice.19,55,56 In our study, CsA inhibited the biliary excretion of both DOX and DOXol to the same extent; the DOXol/DOX biliary excretion ratio in P2 was similar to that in P1. Thus, DOXol may be transported by the same carrier-mediated uptake and efflux mechanisms as DOX, as also supported by predictions based on the sensitivity analysis (see Figure 8). Furthermore, the increase in some plasma PK parameters for DOXol suggested that CsA does not inhibit the metabolism of DOX to DOXol. This was confirmed by the sensitivity analysis, as the unaffected metabolism of DOX to DOXol was necessary in order to fit the observed DOXol plasma concentrations in the model. These results suggested that CsA does not have an inhibitory effect on aldo-keto and carbonyl reductases, as substantiated by a lack of reports in the literature of such an interaction.30 CsA is known to inhibit several metabolic enzymes, including the CYPs. While CYP2D and -3A are thought not to be involved in the metabolism of DOX to DOXol, they could be involved in the conversion of DOX to other metabolites.60 If so, our study suggests that they contribute only minimally to the DOX metabolism and the

DOX

DOXol

total clearance of DOX and DOXol, in comparison with aldoketo and carbonyl reductases and transporters. The sensitivity analysis also showed that the plasma exposure of DOX only changed slightly when DOX metabolism to DOXol was inhibited, which can be explained by three related factors: (i) The sensitivity analysis only lasted for 160 min, and slight differences in terminal elimination half-life, causing differences in AUC, would not have been apparent in such a short period. (ii) The distribution rates from plasma to tissue/liver were faster than the redistribution rates from tissue/liver to plasma. The peripheral tissue compartment will therefore hold a reservoir of DOX, which will be redistributed only slowly from this compartment. (iii) The amount DOX excreted into bile and urine increased slightly with decreased metabolism. The impact of the changes in transporter and metabolic activity on DOX PK and hepatocellular disposition needs further investigation. For instance, development of a physiologically based PK (PBPK) model where physiological factors can be altered might be necessary to identify rate-determining processes involved in the overall hepatobiliary drug transport process, i.e., its impact on plasma and tissue exposure.61 As an example, PBPK modeling has been successfully used to predict the PK and disposition of pravastatin in humans and of repaglinide in pigs.57,62 In conclusion, the excipient LIP, which consists of esters of long chain fatty acids, did not significantly alter the 1310



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Figure 8. Results of the sensitivity analysis for doxorubicin (DOX) in plasma (A) and bile (C) and doxorubicinol (DOXol) in plasma (B) and bile (D); symbols represent observed data, and lines represent simulated values. Squares represent the average concentrations or amounts excreted in animals that did not receive CsA (TI and TII), while circles represent the average concentrations or amounts excreted in animals that did receive CsA (TIII and TIV). Only simulations deviating from curves representing no inhibition are shown in the graphs. 99% inhibition of original estimated parameters was applied to metabolism for DOX to DOXol (met DOX), DOXol to other metabolites (met DOXol), or the combined metabolism of DOX and DOXol, and the combined biliary excretion for DOX and DOXol (biliary excretion).

ratio; fe, fraction of dose excreted; Fi;app, apparent intracellular availability; HA, hepatic artery; LIP, lipiodol; OATP, organic anion-transporting polypeptide; PBPK, physiological based pharmacokinetic; Pgp, P-glycoprotein; PK, pharmacokinetics; t1/2, elimination half-life; TACE, transarterial chemoembolization; tmax, time to maximum concentration; VF, femoral vein; VH, hepatic vein; VP, portal vein; Vss, volume of distribution at steady state

hepatobiliary disposition or plasma PK of DOX and DOXol in this aMSA pig model, ruling out the possibility of direct membrane transport interaction with the excipient. It is the properties of the total LIP-based parenteral DDS rather than those of the excipient itself that are of importance for tumor directed delivery. Administration of CsA reduced the biliary excretion of both DOX and DOXol through inhibition of canalicular CM transport rather than of sinusoidal transport proteins, resulting in a tendency for DOXol concentrations in plasma to increase. Finally, CsA seems to have no inhibiting effect on DOX metabolism mediated by aldo-keto and carbonyl reductases.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +46 18 471 4317. Fax: +46 18 471 4223. Author Contributions

‡ Contributed equally to the execution of the study and writing the article.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to take the opportunity of thanking Anders Nordgren for his help, support and hard work during the actual experiment and Elisabeth Fredriksson for her careful, professional handling of the sample analysis.

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ABBREVIATIONS USED aMSA pig model, advanced multi-sampling-site acute pig model; AUC, area under plasma/bile concentration−time curve; BSEP, bile salt export pump; CL, clearance; CM, carrier mediated; Cmax, maximum concentration; CsA, cyclosporin A; CYP, cytochrome P450; DDS, drug delivery system; DOX, doxorubicin; DOXol, doxorubicinol; EH, hepatic extraction 1311



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The Effects of Lipiodol and Cyclosporin A on the Hepatobiliary

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