Doxorubicin pharmacokinetics: Macromolecule binding, metabolism, and excretion in the context of a physiologic model
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Doxorubicin Pharmacokinetics: Macromolecule Binding,Metabolism, and Excretion in the Context of aPhysiologic Model
DANIEL L. GUSTAFSON,1 JEFFREY C. RASTATTER,1 TINA COLOMBO,2 MICHAEL E. LONG1
1Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center,4200 East 9th Avenue, Denver, Colorado 80262
2Laboratory of Cancer Chemotherapy, Mario Negri Institute for Pharmacological Research, Via Eritrea 62, 20157 Milan, Italy
Received 19 June 2001; revised 25 September 2001; accepted 25 February 2002
ABSTRACT: The studies described herein were designed to determine whether doxo-rubicin (DOX) pharmacokinetics (PKs) could be described by a physiologically based PKmodel that incorporated macromolecule-specific binding and organ-specific metabolismand excretion. Model parameters were determined experimentally, or were gatheredfrom the literature, in a species-specific manner, and were incorporated into a phy-siologically based description of DOX blood and tissue distribution for mice, dogs, andhumans. The resulting model simulation data were compared with experimentallydetermined data using PK parameters calculated using compartmental or noncompart-mental analysis to assess the predictability of the models. The resulting physiologicallybased PK model that was developed could accurately predict blood and tissue PKs ofDOX in mice. When this model was interspecies extrapolated to predict DOX levels indogs and humans undergoing treatment for cancer, predictions in dog plasma or humanserumwere also consistent with the actual clinical data. This model has potential utilityfor predicting the magnitude of PK interactions of DOX with other drugs, and for pre-dicting changes in DOX PKs in any number of clinical situations. 2002 Wiley-Liss, Inc.and the American Pharmaceutical Association J Pharm Sci 91:14881501, 2002
Keywords: doxorubicin; pharmacokinetics; physiologically based modeling; simula-tion; PBPK
Doxorubicin (DOX) is a naturally occurring an-thracycline that has a broad spectrum of activityfor the treatment of cancer. The five anthracy-clines currently in clinical use worldwide areDOX, daunorubicin, idarubicin, epirubicin, andpirarubicin. Standard combination chemotherapyregimens for the treatment of solid tumors,lymphomas, and leukemias usually contain ananthracycline component.1 The dose-limiting toxi-
cities of the two commonly used anthracyclines,DOX and daunorubicin, are myelosuppression,mucositis, and cardiac toxicity.2
The anthracyclines can react with numerouscellular components to induce a number of effectsthat are thought to have a role in the antineo-plastic and toxic effects of these compounds. An-thracyclines are capable of DNA intercalation andinhibition of RNA and DNA polymerases,3 inter-action with topoisomerase II,4 and alkylation ofDNA.5 DOX is also capable of generating reactiveoxygen species through quinone redox cycling,6,7
and perturbing cellular Ca2 homeostasis throughboth receptor-mediated8,9 and redox-mediated10
mechanisms. Other mechanisms of action havealso been investigated, including inhibition of
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Correspondence to: Daniel L. Gustafson (Telephone: 303-315-0755; Fax: 303-315-6281;E-mail: firstname.lastname@example.org)
Journal of Pharmaceutical Sciences, Vol. 91, 14881501 (2002) 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
thioredoxin reductase11 and interaction with com-ponents of the plasma membrane.12 The role ofeach of these processes in the causation of anti-tumor and side effects is still debated, although itis reasonable to assume that all have some role inthe pharmacology of these compounds.
The pharmacokinetics (PKs) of DOX has beenwell studied in many species. After intravenousdosing, DOX blood levels fall dramatically as thedrug distributes into tissues, followed by a slowelimination phase due to renal and biliary clear-ance and metabolism. DOXmetabolism occurs viareduction of a side chain carbonyl group by aldo-keto reductases13 yielding doxorubicinol, and byreductive cleavage of the sugar moiety to form the7-hydroxy aglycone.14 DOX partitioning fromblood to tissues has been shown to correlate withDNA concentration,15 and DOX is also known tobind to anionic lipids, particularly cardiolipin.16,17
To relate drug dosage to therapeutic and/ortoxic effects, physiologically based PK (PBPK)models are useful tools that allow for simulationand prediction of target tissue concentrations ofactive drug or metabolites. PBPK models are amathematical representation of a biological sys-tem constructed using known physiologic andbiochemical constants. The body is divided intocompartments that represent individual organsand tissue groupings, and the transport, clearance,andmetabolism of xenobiotics between these com-partments or within these compartments is de-scribed using mass balance ordinary differentialequations.18 Physiologically based modeling hasmany advantages which include: (a) utilization ofa large body of physiologic and physiochemicaldata; (b) extrapolation of results both across spe-cies and routes of administration; and (c) predic-tion of PKs and target tissue dosimetry over awiderange of doses.19
Previous PBPK models for DOX have beendeveloped,20,21 but these models have used tissuepartitioning as defined by plasma/tissue concen-tration ratios to describe DOX tissue distribution,and have not included specific terms for thevarious metabolic and elimination pathways thathave a role in DOX disposition. The model de-veloped with these studies uses experimentallydefined biochemical parameters for DOX metabo-lism and macromolecule binding, and places themin the context of a physiologic model representingorgans that are targets for toxicity or have a rolein DOX metabolism and excretion. The resultingmodel output describesDOXdistribution inmousetissues and calculatedDOXPKparameters.When
the model is scaled to canine or human physiologyand biochemical parameters, the resulting outputagain correlates with DOX serum PKs of patientsreceivingDOX therapy. These results suggest thatthe DOX PBPK model developed herein incorpo-rated the major factors that dictate DOX PK inmammalian species.
MATERIALS AND METHODS
DOX hydrochloride, daunorubicin hydrochloride,calf-thymus DNA, and Hoechst 33258 were pur-chased from Sigma Chemical Co. (St. Louis, MO).All other reagents were of analytical grade.
DOX PK Studies in Mice
Female, Balb/c mice (45 weeks old) were pur-chased from Harlan Sprague Dawley (Indiana-polis, IN) and allowed to acclimate for 7 days.Animals were housed (three per cage) in poly-carbonate cages and kept on a 12-h light/darkcycle. Food andwater were given ad libitum. Afteracclimation, mice were randomly assigned to thetime-point groups, with three mice per group. Allstudies were conducted in accordance with theNational Institutes of Health guidelines for thecare and use of laboratory animals, and animalswere housed in a facility accredited by theAmerican Association of Laboratory Animal Care.
DOX was administered by intravenous (iv) in-jection into the tail vein at a dose of 6 mg/kg in avolume of 50 mL. Animals were killed by cervicaldislocation after methoxyflurane anesthesia, andtissue and blood samples collected at 5, 10, 30, 60,120, and 480 min after injection. Heart, liver,kidney, small intestine, large intestine, skeletalmuscle, fat, spleen, and bone marrow were collec-ted, rinsed with phosphate buffered saline (pH7.6), and stored at808C until assayed. Blood wascollected by cardiac puncture, placed in hepar-inized vials, and stored at 208C until analyzed.
DOX PKs in Dogs
Serum samples were obtained from client animalsbeing treated for lymphoma with DOX (30 mg/m2)every 3 weeks for five cycles at the VeterinaryTeaching Hospital at Colorado State University(Fort Collins, CO). All samples were obtained fromdogs whose owners had signed a consent agree-ment regarding the inclusion of the patient in aclinical PK study.
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DOX High-Pressure Liquid Chromatography(HPLC) Analysis
An extraction protocol modified from De Jonget al.22 was used for blood, serum, and tissuesamples. Briefly, tissue was homogenized in waterto give a final concentration of 10 mg/mL. Analiquot of 100 mL homogenate, 50 mL whole blood,or 100 mL serum was transferred to a 1.5-mL poly-propylene microfuge tube; 50 mL 1 mM daunor-ubicin was added as an internal standard, and0.6 mL methanol was added. The samples werevortex mixed for 15 min, 0.25 mL 12 mM phos-phoric acid was added, and then centrifuged at10,000g for 8 min. The resulting supernatant wascollected, the volume adjusted to 1.0 mL, andanalyzed by HPLC with fluorescence detection aspreviously described.22
The HPLC system used was a Beckman GoldSystem (Beckman-Coulter, Fullerton, CA) con-sisting of a 126 pump module, 508 autosampler,and a Jasco FP-920 fluorescence detector (JascoCorp., Tokyo, Japan) with excitation and emissionwavelengths set at 480 and 580 nm, respectively.The mobile phase consisted of 15 mM NaH2PO4(pH 4)/acetonitrile in a 2:1 volume-to-volumeratio at a flow rate of 1 mL/min. Separationwas done on an Alltima C18 (5 mm) 250 4.6 mmcolumn (Alltech Associates Inc., Deerfield, IL)fitted with an Alltima C18 guard cartridge (AlltechAssociates).
DNA Content Analysis
Tissue preparation and DNA analysis weredone using a protocol modified from Downs andWilfinger.23 Mouse tissues were collected as de-scribed above from untreated animals. Dog tis-sues were collected upon necropsy from clientanimals at the Veterinary Teaching Hospital(Colorado State University) and stored at 808C.Human tissues, either surgical or autopsy speci-mens, were acquired through the CooperativeHuman Tissue Network Western Division (CaseWestern Reserve University, Cleveland, OH).Tissues (10 mg per 0.75 mL) were homogenizedin AT extraction solution (1 N NH4OH, 0.2%Triton X-100) and incubated at 378C for 10 min.An aliquot of 100 mL homogenate was then dilutedto 1 mL with Assay Buffer (100 mM NaCl, 10 mMEDTA, 10 mM Tris, pH 7.0) and centrifugedat 2500g for 30 min at room temperature. Thesupernatant was collected and stored on ice untilassayed.
The assay mixture consisted of 10 mMTris (pH 7.0), 100 mM NaCl, 10 mM EDTA, and100 ng/mL Hoechst 33258. Fifty microliters ofsample (blank, standards, and unknowns) wasadded to 2 mL of the assay mixture, and thefluorescence determined using a Hitachi F-2000fluorescence spectrophotometer (Hitachi Instru-ments Inc., San Jose, CA) with excitation andemission wavelengths set at 350 and 455 nm,respectively. Blanks, samples, and unknownswere all measured in triplicate, and measure-ments were considered to be valid if replicatesamples differed by less than 10%. The concentra-tion of DNA in unknown samples was calculatedusing a standard curve generated by plottingfluorescence units versus DNA concentration instandards made up using calf thymus DNA.MolarDNA concentration was calculated for each tissueusing a value of 330.9 formula weight for DNAbases and a density of 1 g/mL for all tissues.
DOX PBPK Model Development
A PBPK model for DOX was developed incorpor-ating DNA and cardiolipin binding, tissue-specificmetabolism, and biliary and urinary eliminationof DOX into a seven-compartment flow-limitedmodel. A schematic of this model is shown inFigure 1. Physiologic parameters (tissue volumesand blood flows) for mouse, dog, and human aretaken from Brown et al.24 and are presented inTable 1. Bone marrow blood flow parameters wereestimated from studies in rat25 for the mousemodel, and humans26 for the dog and humanmodels. Tissue DNA content was measured inmouse, dog, and human tissues and the resultsare presented in Table 2. Cardiolipin levels inmouse, dog, and human tissues were estimatedfrom measurements made in rat tissues.27 Theuse of the rat cardiolipin values to estimate thelevels in mouse, dog, and human tissues is sup-ported by previous studies that show similartissue levels of cardiolipin, when measured aspercent of total lipid phosphorous, in mouse,human, and other species as compared with therat.28,29
Affinity constants for DOXDNA (KDNA) andDOXcardiolipin (KCAL) binding were set at 200and 400 nM, respectively. These values are con-sistent with the in vitro binding characteristics ofDOX to DNA and cardiolipin.17 The concentrationof DNA and cardiolipin in the tissues was modi-fied by a factor that represents the number ofmolecules of DNA or cardiolipin that bind one
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DOX molecule. From in vitro studies, it has beenapproximated that the binding site in DNA forDOX is made up of two base pairs (four bases)30;however, in the modeling process, this term was
derived as 500 bases. This discrepancy betweenthe in vitro number of bases constituting a DOXbinding site and the model-derived term can beeasily accounted for because of obvious differencesin naked DNA used in in vitro studies andprotein-coated dynamic DNA that is presentin vivo. From in vitro data with cardiolipin, amolar ratio of 2:1 (DOX/cardiolipin) has beenproposed,17 and the number used in the model forcardiolipin molecules per DOX binding site is two.
A generic tissue compartment mass balanceequation is represented by eq. 1:
QTCA CVT 1
where AT is the amount of drug in the compart-ment, QT is blood flow to the compartment(Table 1), and CA and CVT are the arterial andvenous blood concentrations of drug being deliv-ered to and exiting from the compartment. Thearterial blood concentration available to all tis-sues, with the exception of liver, is considered tobe the free drug concentration in the blood, andit is assumed that DOX is 70% bound to plasmaprotein.31 For liver, the model assumes that bothfree and bound drugs are available for tissueuptake.32 To account for binding of DOX to DNAand cardiolipin within the respective tissues, theconcentration of DOX leaving in the tissue venousblood flow is modified by eq. 2:
VT TDNA VTKDNA CVT
TCAL VTKCAL CV
where VT is the volume of the tissue compartment(Table 1), TDNA, and TCAL are the DNA and car-diolipin binding capacity available (DNA or car-diolipin concentration divided by the number ofmolecules per DNA binding site), and KDNA andKCAL are the binding affinities of DOX to DNAand cardiolipin, respectively. This mathematicalrepresentation of saturable chemical-specific bind-ing to macromolecules in tissues is modified fromthose used to describe 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding and PKs using a phy-siologic model.33
Metabolic and Excretory Model Parameters
DOX metabolism to doxorubicinol and the 7-OH-aglycone occurs in hepatic and extrahepatictissues. Km and Vmax values for mice, dogs, andhumans were used as previously reported forDOX metabolism by aldo-keto reductases in liver
Table 1. Physiologic Pa...