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Pretreatment with Dexamethasone Increases Antitumor Activity ofCarboplatin and Gemcitabine in Mice Bearing Human CancerXenografts: In Vivo Activity, Pharmacokinetics, and ClinicalImplications for Cancer Chemotherapy

Hui Wang,1,3 Mao Li,1 John J. Rinehart,2,3 andRuiwen Zhang1,3

1Department of Pharmacology and Toxicology, Division of ClinicalPharmacology, 2Department of Medicine, Division of Hematologyand Oncology, and 3Comprehensive Cancer Center, University ofAlabama at Birmingham, Birmingham, Alabama

AbstractPurpose: The present study was undertaken to deter-

mine the effects of dexamethasone (DEX) pretreatment onantitumor activity and pharmacokinetics of the cancer che-motherapeutic agents carboplatin and gemcitabine.

Experimental Design: Antitumor activities of carbopla-tin and gemcitabine with or without DEX pretreatment weredetermined in six murine-human cancer xenograft models,including cancers of colon (LS174T), lung (A549 andH1299), and breast (MCF-7 and MDA-MB-468) and glioma(U87-MG). Effects of DEX on plasma and tissue pharmaco-kinetics of carboplatin and gemcitabine were also deter-mined by using the LS174T, A549, and H1299 models.

Results: Although DEX alone showed minimal antitu-mor activity, DEX pretreatment significantly increased theefficacy of carboplatin, gemcitabine, or a combination ofboth drugs by 2–4-fold in all xenograft models tested. With-out DEX treatment, the tumor exposure to carboplatin,measured by the area under the curve, was markedly lowerthan normal tissues. However, DEX pretreatment signifi-cantly increased tumor carboplatin levels, including 200%increase in area under the curve, 100% increase in maxi-mum concentration, and 160% decrease in clearance. DEXpretreatment similarly increased gemcitabine uptake intumors.

Conclusions: To our knowledge, this is the first reportthat DEX significantly enhances the antitumor activity ofcarboplatin and gemcitabine and increases their accumula-

tion in tumors. These results provide a basis for furtherevaluation of DEX as a chemosensitizer in patients.

IntroductionDNA and RNA interactive chemotherapeutic agents re-

main the most effective and widely used approach in medicalcancer therapy. Two major problems remain to be overcome toimprove the therapeutic effects and safety profiles of cancerchemotherapy. First, most cancer chemotherapeutic agents havesevere side effects (1), including bone marrow suppression, themajor dose-limiting toxicity of many chemotherapeutic agents(2). To reverse chemotherapy-induced hematotoxicity, post-chemotherapy administration of hematopoietic growth factorssuch as granulocyte colony stimulating factor and granulocytemacrophage colony stimulating factor is frequently used (3).However, this approach has several important limitations: rela-tive ineffectiveness; expense; and failure to prevent genomicalterations and hematopoietic progenitor depletion. In contrast,pretreatment of hematopoietic growth factors may offer pre-ventive benefits. For instance, pretreatment strategies to pro-tect hematopoietic progenitors from chemotherapeutic agent-induced toxicity have been tested in animal models, includingthe use of corticosteroids (4–8) and cytokines (9–14). At leastthree agents to our knowledge have demonstrated hematopro-tective effects in cancer patients receiving chemotherapy: (a)dexamethasone (DEX) (15); (b) granulocyte macrophage colonystimulating factor (16–19); and (c) amifostine (20). However,the mechanisms responsible for the hematoprotective effects arenot fully understood. In addition, there are concerns that pre-treatment with corticosteroids may compromise therapeutic ef-ficiency of cancer chemotherapeutic agents (21).

The second major problem associated with cancer chemo-therapy is drug resistance. The investigations of tumor resist-ance to these agents have nearly always focused on cellular andmolecular mechanisms. However, some evidence has suggestedthat physiological mechanisms may also play an important rolein tumor resistance to chemotherapeutic agents. For example,Teicher et al. (22) demonstrated that resistance of murine tu-mors to chemotherapeutic agents, which was developed in vivo,was not associated with in vitro resistance but with a decreasedaccumulation of drug in tumor in vivo. Therefore, modulatingthe tumor microenvironment may increase drug uptake, revers-ing tumor resistance and increasing therapeutic effects. It hasbeen demonstrated that DEX reduces the tumor interstitial fluidpressure (TIFP) that is elevated in many human solid tumors andis associated with decreased drug uptake and development ofdrug resistance (23–25). We hypothesized that (a) pretreatmentwith DEX may increase the antitumor activity of cytotoxicagents and (b) DEX modulation of pharmacokinetics may play

Received 6/3/03; revised 11/13/03; accepted 11/19/03.Grant support: Funds for Cancer Pharmacology Laboratory from Uni-versity of Alabama at Birmingham Comprehensive Cancer Center.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.Requests for reprints: Ruiwen Zhang, Department of Pharmacologyand Toxicology, University of Alabama at Birmingham, VH 113, Box600, 1670 University Boulevard, Birmingham, Alabama 35294. Phone:(205) 934-8558; Fax: (205) 975-9330; E-mail: [email protected].

1633Vol. 10, 1633–1644, March 1, 2004 Clinical Cancer Research

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a role in increasing antitumor activity and decreasing hosthematotoxicity of cytotoxic agents. To test this hypothesis, thepresent study was undertaken to determine the effects of DEXpretreatment on antitumor activity and pharmacokinetics of car-boplatin and gemcitabine in tumor-bearing animals.

Materials and MethodsChemicals and Reagents. All chemicals and solvents

were high-performance liquid chromatography (HPLC) grade or ofthe highest analytical grade available. Methanol, acetonitrile, andacetic acid were purchased from Fisher Chemicals (Atlanta, GA).DEX (analytical grade), carboplatin (analytical grade), and trieth-ylamine were purchased from Sigma (St. Louis, MO). Perchloricacid was purchased from J. T. Baker Inc. (Phillipsburg, NJ). Cen-trifree micropartition system (catalog no. 4104) was purchasedfrom Millipore Corp. (Bedford, MA). Cell culture media, fetalbovine serum, sodium pyruvate, nonessential amino acids, penicil-lin-streptomycin, and other cell culture supplies were provided bythe Comprehensive Cancer Center Media Preparation Shared Fa-cility, University of Alabama at Birmingham. Carboplatin (clinicalgrade) was purchased from Bristol-Myers Squibb Company (Prin-

ceton, NJ), and gemcitabine (clinical grade) was purchased fromEli Lilly and Co. (Indianapolis, IN). DEX (clinical grade) waspurchased from American Regent Laboratories (Shirley, NJ). Ma-trigel basement membrane matrix was obtained from Becton Dick-inson Labware (Bedford, MA). [3H]Gemcitabine was obtainedfrom Moravek Biochemicals (Brea, CA). Tissue solubilizer (TS-2)was purchased from Research Products Inc (Mount Prospect, IL).

Animals. The animal use and care protocol was approvedby the Institutional Animal Use and Care Committee of theUniversity of Alabama at Birmingham. Female athymic nudemice (nu/nu; 4–6 weeks of age) were obtained from FrederickCancer Research Facility (Frederick, MD). All animals were fedwith commercial diet and water ad libitum for 1 week before thestudy.

Cell Culture. The cell lines of human cancers [LS174T(colon), MCF-7 and MDA-MB-468 (breast), and A549 (lung)]and glioma (U87-MG) were obtained from American TypeCulture Collection (Manassas, VA) and cultured according tothe manufacturer’s instructions. All culture media contained10% fetal bovine serum and 1% penicillin-streptomycin.LS174T cells were cultured in modified Earle’s medium with

Fig. 1 Effects of dexamethsone (DEX) on antitumor activity of carboplatin (A) and gemcitabine (B) chemotherapy in nude mice bearing human coloncancer LS174T xenografts. Animals randomly divided into various treatment and control groups (5 mice/group) were pretreated with DEX (s.c., 0.1mg/day for 5 days, days �4 to 0) or saline (as control). Carboplatin was administered i.p. at a single dose of 120 mg/kg (360 mg/m2) on day 0.Gemcitabine was given twice at an i.p. dose of 160 mg/kg (480 mg/m2) on days 0 and 4. C, representative tumors removed on day 12. Random tumors(3 tumors/group) from each group are shown.

1634 Dexamethasone as a Chemosensitizer in Cancer Therapy



0.1 mM nonessential amino acids and Earle’s balanced saltsolution. MCF-7 cells were grown in modified Earle’s mediumcontaining 1 mM nonessential amino acids and Earle’s balancedsalt solution, 1 mM sodium pyruvate, and 10 mg/liter bovineinsulin. MDA-MB-468 cells were grown in DMEM/Ham’s F-12medium (1:1 mixture). U87-MG cells were cultured in Eagleminimal essential medium supplemented with 1% sodium pyru-vate and 1% nonessential amino acids. A549 cells were culturedin Ham’s F-12K medium. The lung cancer cell line H1299 waskindly provided by Dr. J. Chen (Moffit Cancer Center, Tampa,FL) and grown in DMEM.

Animal Tumor Models. Human cancer xenograft mod-els were established using the methods reported previously(26–29). When confluence reached 80%, cultured cells inmonolayer were trypsinized, harvested by centrifugation,washed with the serum-free medium indicated above, and thenresuspended in the same medium with Matrigel basement mem-brane matrix at a 3:1 ratio. The cell suspension was then injecteds.c. (5 � 106 cells; total volume, 0.2 ml) into the left inguinalarea of the nude mice. The animals were monitored for activity,physical condition, determination of body weight, and measure-ment of tumor growth. Tumor growth was determined by calipermeasurement in two perpendicular diameters of the implant. Asreported previously (26–29), tumor mass (in g) was calculatedby the formula 1/2a � b2, where a is the long diameter, and bis the short diameter (in cm).

In Vivo Chemotherapy. Nude mice bearing human can-cer xenografts, with an average body weight of 22 � 2 g, wererandomly divided into various treatment and control groups (5mice/group). Animals were treated with DEX by s.c. injection atthe dose of 0.1 mg/day for 5 days or with saline (as controls)before chemotherapy (day �4 to 0). Carboplatin was adminis-tered i.p. at a single dose of 120 mg/kg (360 mg/m2) on day 0.Gemcitabine was given as two i.p. doses of 160 mg/kg (480mg/m2) for all models except the U87-MG model. In theU87-MG model, a combination therapy of carboplatin and gem-citabine with or without DEX pretreatment was given. To avoid

potential severe toxicity, only one dose of gemcitabine (160mg/kg) was administered.

Pharmacokinetics and Tissue Distribution of Carbopla-tin. Pharmacokinetic studies were carried out using a protocolsimilar to that described previously (26), using glass metabolismcages that allowed individual animals to access food and diet adlibitum and to be closely monitored after treatment. Female nudemice bearing human colon cancer LS174T xenografts or humanlung cancer A549 xenografts were used (three animals for eachtime point). Animals were pretreated with DEX (s.c., 0.1 mg/day/mouse for 5 days) or saline (as controls) and, at 1 h after thefifth dose of DEX, were given a single i.v. bolus administrationof carboplatin (60 mg/kg) via a tail vein. After carboplatintreatment, each animal was placed in a glass metabolism cageand fed with commercial diet and water ad libitum. At varioustimes (5, 15, and 30 min and 1, 2, 4, 8, and 24 h after carboplatindosing), blood samples were collected in heparinized tubes, andtissue samples were removed. Plasma was separated by centrif-ugation at 20,000 � g for 5 min. Tissues including liver,kidneys, spleen, and tumor were taken at various times andimmediately blotted on Whatman No. 1 filter paper, trimmed ofextraneous fat or connective tissue, weighed, and homogenizedin 0.9% NaCl physiological saline (5 ml/g wet weight). Theresultant homogenates were stored at �70°C until further anal-ysis. Bone marrow cells were harvested by flushing the femurswith sterile physiological saline as reported previously (30).Briefly, after removing the distal and proximal ends of femur,each end of the bone was punctured with a 20-gauge needlefitted with a 5-ml syringe containing 1 ml of sterile physiolog-ical saline (preweighed). The bone was then held with a forcepsover a test tube, and the needle was inserted into the hole in theproximal end. The 1 ml of saline was flushed through the boneinto the tube by applying gentle, steady pressure to the plunger.The syringe was then filled with air (5 ml) that was gentlyforced through the bone to remove the remaining saline andbone marrow in the shaft. The resultant bone marrow cellsuspension was weighed and lysed by sonicating five times for

Table 1 Effects of DEXa pretreatment on therapeutic effects of carboplatin and gemcitabine

Tumor model Chemotherapy

Tumor mass (% of saline control)b

Relative ratioc

[2]/[1] (%)Without DEX [1] With DEX [2]

LS174T Carboplatin 49 28 57d

Gemcitabine 8 2 25e

A549 Carboplatin 61 41 67e

Gemcitabine 58 37 64e

H1299 Carboplatin 69 41 59e

MCF-7 Carboplatin 67 40 60d

Gemcitabine 52 32 62d

MDA-MB-468 Carboplatin 85 53 62d

U87-MG Carboplatin 55 50 91Gemcitabine 30 32 107Carboplatin � gemcitabine 23 10 43e

a DEX, dexamethasone.b Tumor mass measured at the end of experiments.c The relative ratio illustrates the effects of DEX on therapeutic effects of chemotherapeutic agents. If the ratio is less than 100 with statistical

significance, a positive effect of DEX is indicated.d P � 0.01.e P � 0.05.

1635Clinical Cancer Research



periods of 10 s. After centrifugation at 20,000 � g for 30 min at4°C, the supernatant was removed and stored at �70°C untilfurther analysis.

HPLC Analysis of Carboplatin in Plasma and Tissues.Carboplatin in biological samples was analyzed by an analyticalprocedure involving microfiltration and reversed-phase HPLC(31). Two hundred �l of plasma or tissue homogenates or bonemarrow suspension were added to the reservoir of a Centrifreemicropartition system and then centrifuged at 2000 � g for 5min. All filtrates were transferred into a new microcentrifugetube, and 6 �l of the filtrates were injected onto the HPLCcolumn. The HPLC system consisted of a Hewlett Packard 1050ChemStation with a UV detector (Agilent 1050 series). Deter-mination of carboplatin was achieved using a LiChrosorb diol(10 �m; 250 � 4.6-mm) analytical column with a LiChroCART100 RP-18 guard column. The mobile phase for plasma andtissue samples was composed of 98:2 acetonitrile: H2O (v/v)and 89:11 acetonitrile:0.015% H3PO4 (v/v) for urine. The flowrates were 2 ml/min (for plasma and tissue samples) and 1.1ml/min (for urine). The column elute was monitored by UV at229 nm. Quantitation of plasma or tissue carboplatin was carriedout by using an external standard curve (0–4000.0 �g/ml) thatwas freshly prepared on a daily basis. Linear regression andcorrelation analysis were carried out to establish the standardpeak-area/concentration curves for carboplatin.

Pharmacokinetics and Tissue Distribution of Gemcit-abine. Female nude mice bearing human lung cancer H1299xenograft were used (three mice for each time point). Animalswere pretreated with DEX (s.c., 0.1 mg/day/mouse for 5 days)or saline (as controls) and, at 1 h after the fifth dose of DEX,given a single i.v. bolus administration of [3H]gemcitabine (160mg/kg) via a tail vein. At various times (5, 15, and 30 min and1, 2, 4, 8, and 24 h after drug dosing), blood and tissuesincluding liver, kidneys, heart, lungs, spleen, brain, and tumorwere collected. Plasma was separated by centrifugation, andtissue samples were immediately weighed and homogenized in0.9% NaCl physiological saline (5 ml/g wet weight). Bonemarrow cells were harvested by using the same method de-scribed above. Gemcitabine concentrations in biological sam-ples were analyzed by quantitation of radioactivity.

Quantitation of Gemcitabine by Radioactivity Meas-urements. The total radioactivities of gemcitabine in tissues andbody fluids were determined by liquid scintillation spectrometry(LS 6000T A; Beckman, Irvine, CA), using a method describedpreviously (26, 32). In brief, plasma samples (50 �l) were mixedwith 5 ml of scintillation solvent (Beckman) to determine totalradioactivity. Tissue homogenates (50–200 �l) were mixed with200 �l of solubilizer (TS-2) overnight, neutralized with 400 �l of0.3% acetic acid, and then mixed with scintillation solvent (5 ml) topermit quantitation of total radioactivity.

Histology and Immunohistochemistry of A549 Xe-nografts. A549 xenografts from treated nude mice were re-moved on day 35, fixed and stained with H&E, or snap-frozen inliquid N2. Immunohistochemical staining was undertaken with ratantimouse CD45 (leukocyte common antigen; Ly-5; PharMingen,BD Biosciences) using a DAKO Immunohistochemistry kit onfrozen tumor tissue. Controls, including antimouse CD45 withoutsecondary antibody and secondary antibody alone, were shownnegative in the control CD-1 mouse spleens.

Data and Statistical Analysis. The antitumor activity(tumor mass) was expressed as mean and SDs, and the signif-icance of differences was analyzed by ANOVA. The pharma-cokinetic parameters of carboplatin and gemcitabine were esti-mated by using WinNonlin programs (Version 2.1; Pharsight,Mountain View, CA): the area under the drug concentration-time curve (AUC); the maximal concentration (Cmax); the elim-ination half-life (T1/2); clearance (CL); and the volume of dis-tribution at steady state (Vss).

Fig. 2 Effects of dexamethsone (DEX) on antitumor activity of carbo-platin and gemcitabine chemotherapy in nude mice bearing human lungcancer A549 or H1299 xenografts. Animals randomly divided intovarious treatment and control groups (5 mice/group) were treated usingthe same protocol as described in the Fig. 1 legend. A and B, A549model treated with carboplatin (A) or gemcitabine (B); C, H1299 modeltreated with carboplatin.




ResultsPretreatment with DEX Enhances Therapeutic Effects ofCarboplatin and Gemcitabine in Vivo

The effects of DEX pretreatment on antitumor activity ofcarboplatin and/or gemcitabine were studied in six murine xe-nograft models of human cancers. When mean tumor massreached 44–68 mg, animals (mean body weight, 22 � 2 g) weretreated with s.c. DEX at a dose of 0.1 mg/day for 5 days (days�4 to 0), followed by chemotherapy (on day 0).

Colon Cancer Model. As illustrated in Fig. 1, the effectof DEX pretreatment on carboplatin or gemcitabine antitumoractivity was demonstrated in nude mice bearing human coloncancer LS174T (p53 wild type) xenografts. DEX alone showed

slight inhibitory effects on tumor growth. Carboplatin was giveni.p. on day 0, at a clinically relevant dose (120 mg/kg or 360mg/m2). Pretreatment with DEX markedly increased the thera-peutic effect of carboplatin (P � 0.01; Fig. 1A; Table 1). DEXpretreatment also increased therapeutic effects of gemcitabine(P � 0.05; Fig. 1B; Table 1). Representative xenograft tumorsremoved from various treatment groups are shown in Fig. 1C.

Lung Cancer Models. The effects of DEX pretreatmenton carboplatin chemotherapy were further investigated in humanlung cancer A549 (p53 wild type) and H1299 (p53 null) models. In

Fig. 3 Effects of dexamethsone (DEX) on antitumor activity of carbo-platin and gemcitabine chemotherapy in nude mice bearing humanbreast cancer MCF-7 or MDA-MB-468 xenografts. Animals randomlydivided into various treatment and control groups (5 mice/group) weretreated using the same protocol as described in the Fig. 1 legend. A andB, MCF-7 model treated with carboplatin (A) or gemcitabine (B); C,MDA-MB-468 model treated with carboplatin.

Fig. 4 Effects of dexamethasone (DEX) on antitumor activity of car-boplatin (A) or gemcitabine (B) chemotherapy and combination ofcarboplatin and gemcitabine (C) chemotherapy in nude mouse xenograftmodel of human glioma U87-MG. Animals randomly divided intovarious treatment and control groups (5 mice/group) were pretreatedwith DEX (s.c., 0.1 mg/day for 5 days) or saline (as controls). Carbo-platin was administered i.p. at a dose of 120 mg/kg or 360 mg/m2 on day0. Gemcitabine was given at a single i.p. dose of 160 mg/kg (480mg/m2) on day 0.




the A549 model, pretreatment with DEX significantly increased thetherapeutic effects of carboplatin (P � 0.05; Fig. 2A; Table 1) andgemcitabine (P � 0.05; Fig. 2B; Table 1). In the H1299 model,DEX pretreatment also significantly increased the therapeutic ef-fects of carboplatin (P � 0.05; Fig. 2C; Table 1).

Breast Cancer Models. In two human breast cancermodels, MCF-7 (p53 wild type) and MDA-MB-468 (p53 mu-tant), similar effects of DEX pretreatment on carboplatin andgemcitabine chemotherapy were demonstrated (Fig. 3; Table 1).In the MCF-7 model, both DEX and carboplatin alone hadlimited effects on tumor growth. However, pretreatment withDEX significantly increased therapeutic effects of carboplatin(P � 0.01; Fig. 3A; Table 1). Pretreatment with DEX alsoincreased therapeutic effects of gemcitabine (P � 0.01; Fig. 3B;Table 1). In the MDA-MB-468 model, DEX or carboplatinalone had minimal effects on tumor growth, but pretreatmentwith DEX followed by carboplatin significantly inhibited tumorgrowth (P � 0.01; Fig. 3C; Table 1).

Glioma Model. Pretreatment with DEX had no remark-able effects on the therapeutic effects of carboplatin (Fig. 4A;Table 1) or gemcitabine therapy (Fig. 4B; Table 1) in the humanglioma model U87-MG (p53 wild type) but significantly in-creased the therapeutic effects of combination therapy of car-boplatin and gemcitabine (P � 0.05; Fig. 4C; Table 1).

Toxicity of Carboplatin, Gemcitabine, and DEX. DEXalone had no sign of toxicity. Effective, clinically relevant doses ofcarboplatin and gemcitabine were chosen that would not inducemortality or morbidity for these studies. As expected, no deathoccurred. By clinical inspection and measuring animal weights asindices of toxicity or side effects, carboplatin and gemcitabine werewell tolerated. In addition, DEX pretreatment had no effect ontoxicity profiles of the two chemotherapeutic agents.

Histology of A549 Xenografts. H&E stains of all fourtumors demonstrated similar areas of necrosis, fibrosis, and vascu-lar development (data not shown). Rare inflammatory cells wereobserved. This was confirmed with CD45 immunohistochemistrystaining, which demonstrated infrequent CD45 cells (data notshown). There were no differences in CD45 staining among treat-ment groups, suggesting that the effects of DEX on antitumoreffects of chemotherapeutic agents as observed above are notrelated to DEX reduction in the number of infiltrating CD45 cells.

DEX Alters Pharmacokinetics of CarboplatinChemotherapy in Murine-Xenograft Models ofHuman Cancers

Nude Mice Bearing Human Colon Cancer LS174TXenografts. The first carboplatin pharmacokinetic studywas performed in nude mice bearing LS174T xenografts(tumor mass, 500-1000 mg). The time-concentration curvesare illustrated in Fig. 5. No significant differences in plasmapharmacokinetics of carboplatin were observed between con-trol and mice pretreated with DEX (Fig. 5A). However, DEXsignificantly increased tumor carboplatin concentrations(Fig. 5B). Without DEX treatment, the tumor exposure tocarboplatin, measured by AUC, was markedly lower thannormal tissues (4% of spleen, 3% of bone barrow, and 23%of liver AUCs). However, DEX significantly increased tumorcarboplatin uptake, including 162% increase in AUC, 103%increase in Cmax, and 160% decrease in CL (Table 2). Incontrast, pretreatment with DEX decreased carboplatin up-take in spleen (Fig. 5C). Pharmacokinetic analysis indicatedthat there were significant decreases in splenic AUC, T1/2,and Cmax and an increase in CL in mice pretreated with DEX(P � 0.001; Table 2). Decreases in bone marrow carboplatin

Fig. 5 Pharmacokinetics of car-boplatin in nude mice bearinghuman colon cancer LS174Txenografts. Animals were pre-treated with DEX (s.c., 0.1 mg/day/mouse for 5 days) or saline (ascontrols) and given carboplatin at asingle i.v. dose of 60 mg/kg (180mg/m2). Plasma and tissue sam-ples were collected at varioustimes up to 24 h. Carboplatin wasanalyzed by high-performance liq-uid chromatography. Pharmacoki-netic parameters are presented inTable 2.




Fig. 6 Pharmacokinetics of car-boplatin in nude mice bearinghuman lung cancer A549 xe-nografts. Animals were treatedwith the same protocol describedin the Fig. 5 legend. Pharmaco-kinetic parameters are presentedin Table 3.

Table 2 Pharmacokinetic parameters of carboplatin in nude mice bearing LS174T xenografts after pretreatment with DEXa or saline

Tissue Parametersb

Without DEX With DEXRatioc

[2]/[1] (%)Mean (SE) [1] Mean (SE) [2]

Plasma AUC [(�g/ml)�h] 44.51 (2.55) 43.16 (2.24) 97.0T1/2 (h) 0.08 (0.01) 0.11 (0.01) 137.5Cmax (�g/ml) 375.27 (57.17) 280.80 (25.67) 74.8CL (ml/g/h) 1.35 (0.08) 1.39 (0.07) 103.0VSS (ml/g) 0.16 (0.02) 0.21 (0.02) 131.3

Spleen AUC [(�g/ml)�h] 353.36 (78.42) 0.48 (0.09) 0.1d

T1/2 (h) 9.91 (5.55) 0.04 (0.01) 0.4d

Cmax (�g/ml) 24.20 (2.61) 9.44 (3.91) 39.0d

CL (ml/g/h) 0.17 (0.09) 1.26 (0.23) 741.2d

VSS (ml/g) 2.43 (0.00) 6.36 (2.64) 261.7e

Bone marrow AUC [(�g/ml)�h] 411.27 (32.47) 269.71 (22.74) 65.6f

T1/2 (h) 11.16 (2.38) 6.84 (1.83) 61.3f

Cmax (�g/ml) 23.36 (5.19) 22.56 (4.91) 96.6CL (ml/g/h) 0.15 (0.05) 0.22 (0.08) 146.7f

VSS (ml/g) 2.42 (0.00) 2.17 (1.78) 89.7Liver AUC [(�g/ml)�h] 51.52 (3.89) 42.45 (3.84) 82.4f

T1/2 (h) 0.49 (0.07) 0.56 (0.12) 114.3Cmax (�g/ml) 26.97 (2.18) 19.41 (2.08) 72.0f

CL (ml/g/h) 1.16 (0.74) 1.41 (1.09) 121.6VSS (ml/g) 0.82 (0.00) 1.14 (0.00) 139.0

Tumor AUC [(�g/ml)�h] 12.79 (1.91) 33.54 (4.62) 262.2e

T1/2 (h) 0.20 (0.10) 0.26 (0.03) 130.0Cmax (�g/ml) 44.16 (3.31) 89.80 (8.82) 203.4e

CL (ml/g/h) 4.69 (1.43) 1.79 (0.25) 38.2e

VSS (ml/g) 1.36 (0.35) 0.67 (0.07) 49.3e

a DEX, dexamethasone.b Pharmacokinetic parameters are as follows: AUC, the area under the drug concentration-time curve; T1/2, the elimination half-life; Cmax, the

maximal concentration; CL, clearance; and VSS, volume of distribution at steady state.c The relative ratio illustrates the effects of DEX on pharmacokinetics of chemotherapeutic agents. If the ratio is less or greater than 100 with

statistical significance, a positive or negative effect of DEX is indicated.d P � 0.001.e P � 0.01.f P � 0.05.




concentrations were also observed (Fig. 5D; Table 2). Therewere slight but significant decreases in AUC and Cmax ofliver carboplatin in mice pretreated with DEX (Table 2). Nodifferences in kidney pharmacokinetics of carboplatin wereobserved (data not shown).

Nude Mice Bearing Human Lung Cancer A549Xenografts. Carboplatin pharmacokinetic studies were fur-ther performed in nude mice bearing human lung cancerA549 xenografts (tumor mass, approximately 500 mg). Thetime-concentration curves are illustrated in Fig. 6. No signif-icant differences in plasma pharmacokinetics of carboplatinwere observed between control and mice pretreated withDEX (Fig. 6A; Table 3). However, DEX significantly in-creased tumor carboplatin concentrations (Fig. 6B). Pharma-cokinetic analysis indicated that there was a 50% increase intumor AUC in mice pretreated with DEX (Table 3). Incontrast, pretreatment with DEX decreased carboplatin up-take in spleen (Fig. 6C). Pharmacokinetic analysis indicatedthat there were significant decreases in AUC, T1/2, and Cmax

and an increase in CL in mice pretreated with DEX (Table 3).Decreases in bone marrow carboplatin concentrations werealso observed (Fig. 6D; Table 3).

DEX Alters Pharmacokinetics of GemcitabineChemotherapy in Murine- Xenograft Models ofHuman Cancer

The gemcitabine pharmacokinetic study was accom-plished in nude mice bearing human lung cancer H1299xenografts (tumor mass, approximately 500 mg). The time-concentration curves are illustrated in Fig. 7. No significantdifferences in plasma pharmacokinetics of gemcitabine wereobserved between controls and mice pretreated with DEX(Fig. 7A). However, DEX pretreatment increased tumor gem-citabine concentrations (Fig. 7B). Pharmacokinetic analysisindicated that there was a 55% increase in tumor AUC inmice pretreated with DEX (Table 4). In contrast, pretreatmentwith DEX decreased gemcitabine uptake in spleen (Fig. 7C).Pharmacokinetic analysis indicated that there were signifi-cant decreases in splenic AUC, T1/2, and Cmax and an in-crease in CL in mice pretreated with DEX (Table 4). A slightdecrease in bone marrow gemcitabine concentration wasalso observed (Fig. 7D; Table 4). No differences inpharmacokinetics of gemcitabine were observed in othertissues including kidneys, lungs, heart, and brain (data notshown).

Table 3 Pharmacokinetic parameters of carboplatin in nude mice bearing A549 xenografts after pretreatment with DEXa or saline

Tissue Parametersb


[2]/[1] (%)Mean (SE) [1] Mean (SE) [2]

Plasma AUC [(�g/ml)�h] 55.22 (1.78) 66.08 (2.08) 119.7T1/2 (h) 0.11 (0.01) 0.11 (0.01) 100.0Cmax (�g/ml) 357.34 (20.16) 411.89 (21.43) 115.3CL (ml/g/h) 1.09 (0.04) 0.91 (0.03) 83.5VSS (ml/g) 0.17 (0.01) 0.15 (0.01) 88.2

Spleen AUC [(�g/ml)�h] 359.44 (76.59) 75.59 (0.20) 21.0d

T1/2 (h) 0.73 (0.22) 0.11 (0.00) 15.1d

Cmax (�g/ml) 341.99 (52.10) 182.14 (5.30) 53.3e

CL (ml/g/h) 0.17 (0.03) 0.79 (0.00) 464.7d

VSS (ml/g) 0.18 (0.03) 0.12 (0.00) 66.7e

Bone marrow AUC [(�g/ml)�h] 641.04 (34.21) 517.21 (23.45) 80.7f

T1/2 (h) 0.50 (0.11) 0.50 (0.09) 100.0Cmax (�g/ml) 32.58 (2.01) 27.29 (1.03) 83.8CL (ml/g/h) 0.01 (0.00) 0.01 (0.00) 100.0VSS (ml/g) 1.97 (0.09) 2.72 (0.04) 138.1

Liver AUC [(�g/ml)�h] 26.32 (3.24) 21.68 (1.32) 82.4f


Tumor AUC [(�g/ml)�h] 10.16 (0.18) 15.48 (0.24) 152.4f

T1/2 (h) 0.22 (0.01) 0.17 (0.01) 77.3f

Cmax (�g/ml) 32.15 (0.87) 64.09 (2.04) 199.3f

CL (ml/g/h) 5.90 (0.11) 3.88 (0.06) 65.8f

VSS (ml/g) 1.87 (0.05) 0.94 (0.03) 50.3e

a DEX, dexamethasone.b Pharmacokinetic parameters are as follows: AUC, the area under the drug concentration-time curve; T1/2, the elimination half-life; Cmax, the


statistical significance, a positive or negative effect of DEX is indicated.d P � 0.001.e P � 0.01.f P � 0.05.




DiscussionPretreatment with corticosteroids represents a novel ap-

proach to preventing chemotherapy-induced toxicity (4–8, 15).Our interest in administration of corticosteroids before chemo-therapy originated from our observations that pretreatment ofmice with cortisone acetate reduced hematopoietic toxicity ofcarboplatin (6–8). Our recent studies showed that DEX hashematoprotective activity equal to cortisone acetate in normalCD-1 mice receiving carboplatin (33). Pharmacokinetic studiesin CD-1 mice demonstrated that DEX pretreatment markedlyreduced bone marrow and splenic concentrations of carboplatin(33). Although these studies provide the rationale for the use ofcorticosteroids including DEX as chemoprotective agents, thereis a concern regarding the effects of DEX on antitumor activityof the chemotherapeutic agents (21). In addition, the mecha-nisms by which DEX reduces hematotoxicity and enhancesantitumor effects of carboplatin-based chemotherapy have notbeen elucidated.

The present study was undertaken to determine whetherpretreatment with DEX can be further developed as a chemo-protectant and chemosensitizer in cancer chemotherapy and toexamine DEX modulation of carboplatin pharmacokinetics andantitumor activity. We have now demonstrated that DEX pre-treatment significantly enhanced antitumor effectiveness of car-boplatin or gemcitabine monotherapy in the majority of thetested human cancer models, regardless of p53 status. We havealso demonstrated that pretreatment with DEX significantlyaltered carboplatin and gemcitabine pharmacokinetics in nudemice bearing human cancer xenografts, which may be associ-ated with its effects on antitumor activity of carboplatin andgemcitabine. Furthermore, we found that DEX simultaneouslydecreased carboplatin and gemcitabine concentrations in spleen

and bone marrow, which may be associated with its hematopro-tective effects. These results provide a basis for further testing ofthe effect of DEX as a potential antitumor sensitizer and che-moprotectant of cancer chemotherapeutics in human clinicaltrials.

Perhaps the most striking finding in the present study isthat DEX pretreatment increased in vivo antitumor activities ofcarboplatin or gemcitabine in five of the six nude mouse modelsof human cancers. DEX administered alone showed limitedeffects on tumor growth. There are reports demonstrating in vivoantitumor activity of DEX in epithelial cell cancers (34, 35). Themechanisms of DEX direct antitumor activity have not beenelucidated, but several DEX activities may explain this obser-vation. DEX may decrease tumor secretion or tumor associated-cell secretion of tumor growth factors. For example, Nishimuraet al. demonstrated that DEX inhibited the growth of humanprostate cancer DU145 xenografts in nude and severe combinedimmunodeficient mice, possibly through the disruption of thenuclear factor-�B-interleukin-6 pathway (36). In addition, DEXmay enhance tumor apoptosis by inhibiting nuclear factor-�Bactivity by at least two mechanisms: DEX induces translation ofI�B and glucocorticoid-induced leucine zipper, which inhibitNF-�B translocation to the nucleus (37) and interaction with tran-scription sites, respectively (38). Nuclear factor-�B induces tran-scription of multiple antiapoptotic proteins in stressed cells (39).

Several other possible mechanisms may explain DEX en-hancement of the antitumor effects of carboplatin and gemcit-abine. Tumors exhibit multiple physiological abnormalities in-cluding markedly abnormal tortuous vasculature characterizedby decreased blood flow and decreased lymphatic and promis-cuous movement of high molecular weight solutes throughabnormal interendothelial pores (40–43). These abnormalities

Fig. 7 Time-concentration profiles ofgemcitabine in nude mice bearing hu-man cancer H1299 xenografts. Ani-mals were pretreated with DEX (0.1mg/day/mouse for 5 days) or saline (ascontrols) and given [3H]gemcitabine ata single i.v. dose of 160 mg/kg. Phar-macokinetic parameters are presentedin Table 4.




result in elevated TIFP (24) and volume (44), which, in turn,paradoxically reduce the movement of agents that are transientlypresent in the plasma into the tumor interstitial fluid space aspredicted by Darcy’s Law (45). The mechanisms of induction of“leaky” interendothelial pores are probably multifactorial. Tu-mor endothelial cells are structurally abnormal (41). Moreover,tumor endothelial cells are exposed to several cytokines such asvascular endothelial growth factor, interleukin-1, interleukin-8,and transforming growth factor �, which alter both normal andtumor vasculature to allow movement of solutes and fluid intothe interstitial space (46, 47). The origin of those cytokines isclearly multiple and may include tumor cells and tumor-infil-trating cells such as macrophages (46–49). Several studies haveshown that glucocorticosteroids reduce interendothelial poresize in pure, normal endothelial cell cultures in vitro (50) andinhibit secretion of inflammatory cytokines from many cells,including macrophages (37, 51). Braunschweiger and Schiffer(41) demonstrated that treatment with DEX of mice bearingautochronous tumors decreased the movement of high molecu-lar weight molecules into the tumors and the tumor interstitialvolume. As predicted by those studies, treatment of murine-human colon cancer LS174T xenografts with DEX reducedelevated TIFP (23). Using the same model (LS174T) in thepresent study, we have now demonstrated that DEX increasesdrug accumulation in tumors and improves therapeutic effects ofcarboplatin and gemcitabine. Taken together, DEX may en-

hance the antitumor effects of carboplatin and gemcitabine bydecreasing in tumors the promiscuous interendothelial loss ofsolutes and water into the interstitial space, thus decreasingelevated interstitial volume and pressure in the interstitial spacein tumors. Decreased TIFP, in turn, allows for increased drugmovement into tumors and improved antitumor effects. Furtherstudy is needed to provide direct evidence for this hypothesis.

Of note, other mechanisms may be associated with DEXantitumor activity. For instance, Yu et al. demonstrated thatDEX enhanced the in vitro and in vivo antitumor effect of1,25-dihydroxycholecalciferol, an active metabolite of vitaminD, probably by increasing vitamin D receptor ligand-bindingactivity (52). The same laboratory further showed that both cellcycle arrest and apoptosis were enhanced by DEX. They alsosuggested the involvement of the extracellular signal-regulatedkinase and Akt signaling pathways in the antiproliferative ef-fects of the combination of 1,25-dihydroxycholecalciferol andDEX (53). In addition, no significant effects of DEX on anti-tumor activity in the glioma model were observed when carbo-platin and gemcitabine administered alone in the current study,suggesting that other unknown molecular mechanisms are in-volved in DEX-associated effects on tumor growth, which maybe tumor type dependent.

The mechanisms responsible for the findings, i.e., pretreat-ment with DEX decreases the concentrations of carboplatin andgemcitabine in host tissues but increase concentrations of these

Table 4 Pharmacokinetic parameters of gemcitabine in nude mice bearing H1299 xenografts after pretreatment with DEXa or saline

Tissue Parametersb


[2]/[1] (%)Mean (SE) [1] Mean (SE) [2]

Plasma AUC [(�g/ml)�h] 277.38 (58.55) 322.43 (52.65) 116.2T1/2 (h) 0.67 (0.14) 0.94 (0.55) 140.3Cmax (�g/ml) 286.72 (4.18) 237.16 (17.14) 82.7d

CL (ml/g/h) 0.58 (0.12) 0.50 (0.08) 86.2VSS (ml/g) 5.96 (1.3) 9.90 (1.23) 166.1

Spleen AUC [(�g/ml)�h] 909.92 (83.81) 505.4 (50.59) 55.5e

T1/2 (h) 2.84 (0.74) 1.95 (0.32) 68.7e

Cmax (�g/ml) 221.99 (17.51) 179.42 (9.66) 80.8d

CL (ml/g/h) 0.18 (0.04) 0.32 (0.04) 177.8d

VSS (ml/g) 0.72 (0.06) 0.89 (0.05) 123.6Bone marrow AUC [(�g/ml)�h] 21.1 (5.25) 16.41 (3.92) 77.8d


Liver AUC [(�g/ml)�h] 123.27 (24.34) 107.85 (23.24) 87.5T1/2 (h) 0.50 (0.18) 0.47 (0.13) 94.0Cmax (�g/ml) 171.8 (26.36) 157.46 (18.95) 91.7CL (ml/g/h) 1.30 (0.36) 1.48 (0.32) 113.8VSS (ml/g) 0.93 (0.14) 1.02 (0.12) 109.7

Tumor AUC [(�g/ml)�h] 434.55 (35.25) 674.44 (19.73) 155.2d

T1/2 (h) 6.12 (2.06) 10.02 (2.95) 163.7d

Cmax (�g/ml) 49.15 (3.69) 46.66 (5.37) 94.9CL (ml/g/h) 0.37 (0.08) 0.23 (0.07) 62.2d

VSS (ml/g) 9.01 (5.34) 12.72 (1.71) 141.2a DEX, dexamethasone.b Pharmacokinetic parameters are as follows: AUC, the area under the drug concentration-time curve; T1/2, the elimination half-life; Cmax, the


statistical significance, a positive or negative effect of DEX is indicated.d P � 0.05.e P � 0.01.




drugs in tumor tissues, are not fully understood. We hypothesizethat because interstitial fluid pressure is already low (�5 to 0mm Hg versus 5–100 mm Hg in tumors) in normal tissues, thedominant effect of DEX is retardation of solute movement (i.e.,drug) into bone marrow and spleen. However, the role of TIFPin tumor uptake and retention of anticancer drugs may be drugtype-dependent. Large molecule drugs, such as proteins andantibodies, are highly sensitive to the convective barriers estab-lished as a result of increased interstitial fluid pressure in tu-mors, due to their dependence on convection for delivery. How-ever, delivery of small molecules such as carboplatin andgemcitabine involves both convection and diffusion. Conse-quently, decreasing TIFP as a consequence of decreasing trans-vascular movement of fluid may have a much greater impact onthe convective clearance of the drug from the tumor interstitialspace than on its initial delivery because the concentrationgradient is directed from the plasma to the tissue, and thediffusion will drive the process forward until the plasma con-centration drops. Decreasing the vascular permeability is likelyto increase the mean residence time in the tumor by reducing theclearance as seen in our pharmacokinetic studies. In addition,the increase in therapeutic effects by DEX can be explained, inpart, as an additive tumor response to DEX and the chemother-apeutic agent(s) used in combination, although the potentialactivity of DEX alone is relatively weak. Therefore, our obser-vations of increased tumor inhibition are likely to involve bothadditive cytotoxic effects and drug transport effects (increaseduptake and decreased clearance). The contribution of each mayvary with the tumor type.

Our findings in normal tissues suggest that pretreatment ofpatients with DEX may decrease hematotoxicity and increaseantitumor effects of carboplatin or gemcitabine. We have un-dertaken a Phase I pilot trial using DEX pretreatment in patientsreceiving carboplatin-based therapy (15). DEX decreasedcarboplatin-induced hematotoxicity. In patients receiving DEX,the incidence of partial or complete responses was higher thanthat in patients not receiving DEX, although the small patientnumber and heterogeneous patient population precluded conclu-sions regarding tumor response. The results reported here and inour pilot clinical trial have led us to undertake additional clinicaland laboratory studies in this area, providing a potential newavenue for therapeutic use of DEX as a tumor chemosensitizerand chemoprotectant in the treatment of human cancers.

AcknowledgmentsWe thank Jie Hang, Zhenqi Shi, Gautam Prasad, Zhuo Zhang,

Aselle Adaim, Kexuan Wang, Veronika Schachinger, MichaelaHaslinger, Bing Pang, and Lin Lin for excellent technical assistance andDrs. Trenton R. Schoeb and Mitchell Pate for pathology studies. Wealso thank Dr. Al LoBuglio for helpful discussions.

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2004;10:1633-1644. Clin Cancer Res Hui Wang, Mao Li, John J. Rinehart, et al. ChemotherapyPharmacokinetics, and Clinical Implications for Cancer Human Cancer Xenografts: In Vivo Activity,Activity of Carboplatin and Gemcitabine in Mice Bearing Pretreatment with Dexamethasone Increases Antitumor

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