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Experimental Therapeutics, Preclinical Pharmacology |
1 Department of Pharmacology and Toxicology, Division of Clinical Pharmacology,
2 Department of Medicine, Division of Hematology and Oncology, and
3 Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama
ABSTRACT
Purpose: The present study was undertaken to determine the effects of dexamethasone (DEX) pretreatment on antitumor activity and pharmacokinetics of the cancer chemotherapeutic agents carboplatin and gemcitabine.
Experimental Design: Antitumor activities of carboplatin and gemcitabine with or without DEX pretreatment were determined in six murine-human cancer xenograft models, including cancers of colon (LS174T), lung (A549 and H1299), and breast (MCF-7 and MDA-MB-468) and glioma (U87-MG). Effects of DEX on plasma and tissue pharmacokinetics of carboplatin and gemcitabine were also determined by using the LS174T, A549, and H1299 models.
Results: Although DEX alone showed minimal antitumor activity, DEX pretreatment significantly increased the efficacy of carboplatin, gemcitabine, or a combination of both drugs by 2–4-fold in all xenograft models tested. Without DEX treatment, the tumor exposure to carboplatin, measured by the area under the curve, was markedly lower than normal tissues. However, DEX pretreatment significantly increased tumor carboplatin levels, including 200% increase in area under the curve, 100% increase in maximum concentration, and 160% decrease in clearance. DEX pretreatment similarly increased gemcitabine uptake in tumors.
Conclusions: To our knowledge, this is the first report that DEX significantly enhances the antitumor activity of carboplatin and gemcitabine and increases their accumulation in tumors. These results provide a basis for further evaluation of DEX as a chemosensitizer in patients.
Introduction
DNA and RNA interactive chemotherapeutic agents remain the most effective and widely used approach in medical cancer therapy. Two major problems remain to be overcome to improve the therapeutic effects and safety profiles of cancer chemotherapy. First, most cancer chemotherapeutic agents have severe side effects (1) , including bone marrow suppression, the major dose-limiting toxicity of many chemotherapeutic agents (2) . To reverse chemotherapy-induced hematotoxicity, post-chemotherapy administration of hematopoietic growth factors such as granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor is frequently used (3) . However, this approach has several important limitations: relative ineffectiveness; expense; and failure to prevent genomic alterations and hematopoietic progenitor depletion. In contrast, pretreatment of hematopoietic growth factors may offer preventive benefits. For instance, pretreatment strategies to protect hematopoietic progenitors from chemotherapeutic agent-induced toxicity have been tested in animal models, including the use of corticosteroids (4, 5, 6, 7, 8) and cytokines (9, 10, 11, 12, 13, 14) . At least three agents to our knowledge have demonstrated hematoprotective effects in cancer patients receiving chemotherapy: (a) dexamethasone (DEX) (15) ; (b) granulocyte macrophage colony stimulating factor (16, 17, 18, 19) ; and (c) amifostine (20) . However, the mechanisms responsible for the hematoprotective effects are not fully understood. In addition, there are concerns that pretreatment with corticosteroids may compromise therapeutic efficiency of cancer chemotherapeutic agents (21) .
The second major problem associated with cancer chemotherapy is drug resistance. The investigations of tumor resistance to these agents have nearly always focused on cellular and molecular mechanisms. However, some evidence has suggested that physiological mechanisms may also play an important role in tumor resistance to chemotherapeutic agents. For example, Teicher et al. (22) demonstrated that resistance of murine tumors to chemotherapeutic agents, which was developed in vivo, was not associated with in vitro resistance but with a decreased accumulation of drug in tumor in vivo. Therefore, modulating the tumor microenvironment may increase drug uptake, reversing tumor resistance and increasing therapeutic effects. It has been demonstrated that DEX reduces the tumor interstitial fluid pressure (TIFP) that is elevated in many human solid tumors and is associated with decreased drug uptake and development of drug resistance (23, 24, 25) . We hypothesized that (a) pretreatment with DEX may increase the antitumor activity of cytotoxic agents and (b) DEX modulation of pharmacokinetics may play a role in increasing antitumor activity and decreasing host hematotoxicity of cytotoxic agents. To test this hypothesis, the present study was undertaken to determine the effects of DEX pretreatment on antitumor activity and pharmacokinetics of carboplatin and gemcitabine in tumor-bearing animals.
Materials and Methods
Chemicals and Reagents.
All chemicals and solvents were high-performance liquid chromatography (HPLC) grade or of the highest analytical grade available. Methanol, acetonitrile, and acetic acid were purchased from Fisher Chemicals (Atlanta, GA). DEX (analytical grade), carboplatin (analytical grade), and triethylamine were purchased from Sigma (St. Louis, MO). Perchloric acid was purchased from J. T. Baker Inc. (Phillipsburg, NJ). Centrifree micropartition system (catalog no. 4104) was purchased from Millipore Corp. (Bedford, MA). Cell culture media, fetal bovine serum, sodium pyruvate, nonessential amino acids, penicillin-streptomycin, and other cell culture supplies were provided by the Comprehensive Cancer Center Media Preparation Shared Facility, University of Alabama at Birmingham. Carboplatin (clinical grade) was purchased from Bristol-Myers Squibb Company (Princeton, NJ), and gemcitabine (clinical grade) was purchased from Eli Lilly and Co. (Indianapolis, IN). DEX (clinical grade) was purchased from American Regent Laboratories (Shirley, NJ). Matrigel basement membrane matrix was obtained from Becton Dickinson Labware (Bedford, MA). [3H]Gemcitabine was obtained from 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 approved by the Institutional Animal Use and Care Committee of the University of Alabama at Birmingham. Female athymic nude mice (nu/nu; 4–6 weeks of age) were obtained from Frederick Cancer Research Facility (Frederick, MD). All animals were fed with commercial diet and water ad libitum for 1 week before the study.
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 Type Culture Collection (Manassas, VA) and cultured according to the manufacturer’s instructions. All culture media contained 10% fetal bovine serum and 1% penicillin-streptomycin. LS174T cells were cultured in modified Earle’s medium with 0.1 mM nonessential amino acids and Earle’s balanced salt solution. MCF-7 cells were grown in modified Earle’s medium containing 1 mM nonessential amino acids and Earle’s balanced salt solution, 1 mM sodium pyruvate, and 10 mg/liter bovine insulin. MDA-MB-468 cells were grown in DMEM/Ham’s F-12 medium (1:1 mixture). U87-MG cells were cultured in Eagle minimal essential medium supplemented with 1% sodium pyruvate and 1% nonessential amino acids. A549 cells were cultured in Ham’s F-12K medium. The lung cancer cell line H1299 was kindly provided by Dr. J. Chen (Moffit Cancer Center, Tampa, FL) and grown in DMEM.
Animal Tumor Models.
Human cancer xenograft models were established using the methods reported previously (26, 27, 28, 29)
. When confluence reached 80%, cultured cells in monolayer were trypsinized, harvested by centrifugation, washed with the serum-free medium indicated above, and then resuspended in the same medium with Matrigel basement membrane matrix at a 3:1 ratio. The cell suspension was then injected s.c. (5 x 106 cells; total volume, 0.2 ml) into the left inguinal area of the nude mice. The animals were monitored for activity, physical condition, determination of body weight, and measurement of tumor growth. Tumor growth was determined by caliper measurement in two perpendicular diameters of the implant. As reported previously (26, 27, 28, 29)
, tumor mass (in g) was calculated by the formula 1/2a x b2, where a is the long diameter, and b is the short diameter (in cm).
In Vivo Chemotherapy.
Nude mice bearing human cancer xenografts, with an average body weight of 22 ± 2 g, were randomly divided into various treatment and control groups (5 mice/group). Animals were treated with DEX by s.c. injection at the dose of 0.1 mg/day for 5 days or with saline (as controls) before chemotherapy (day -4 to 0). Carboplatin was administered 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 (480 mg/m2) for all models except the U87-MG model. In the U87-MG model, a combination therapy of carboplatin and gemcitabine with or without DEX pretreatment was given. To avoid potential severe toxicity, only one dose of gemcitabine (160 mg/kg) was administered.
Pharmacokinetics and Tissue Distribution of Carboplatin.
Pharmacokinetic studies were carried out using a protocol similar to that described previously (26)
, using glass metabolism cages that allowed individual animals to access food and diet ad libitum and to be closely monitored after treatment. Female nude mice bearing human colon cancer LS174T xenografts or human lung cancer A549 xenografts were used (three animals for each time 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 the fifth dose of DEX, were given a single i.v. bolus administration of carboplatin (60 mg/kg) via a tail vein. After carboplatin treatment, each animal was placed in a glass metabolism cage and fed with commercial diet and water ad libitum. At various times (5, 15, and 30 min and 1, 2, 4, 8, and 24 h after carboplatin dosing), blood samples were collected in heparinized tubes, and tissue samples were removed. Plasma was separated by centrifugation at 20,000 x g for 5 min. Tissues including liver, kidneys, spleen, and tumor were taken at various times and immediately blotted on Whatman No. 1 filter paper, trimmed of extraneous fat or connective tissue, weighed, and homogenized in 0.9% NaCl physiological saline (5 ml/g wet weight). The resultant homogenates were stored at -70°C until further analysis. Bone marrow cells were harvested by flushing the femurs with 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 needle fitted with a 5-ml syringe containing 1 ml of sterile physiological saline (preweighed). The bone was then held with a forceps over a test tube, and the needle was inserted into the hole in the proximal end. The 1 ml of saline was flushed through the bone into the tube by applying gentle, steady pressure to the plunger. The syringe was then filled with air (5 ml) that was gently forced through the bone to remove the remaining saline and bone marrow in the shaft. The resultant bone marrow cell suspension was weighed and lysed by sonicating five times for periods of 10 s. After centrifugation at 20,000 x g for 30 min at 4°C, the supernatant was removed and stored at -70°C until further analysis.
HPLC Analysis of Carboplatin in Plasma and Tissues.
Carboplatin in biological samples was analyzed by an analytical procedure involving microfiltration and reversed-phase HPLC (31)
. Two hundred µl of plasma or tissue homogenates or bone marrow suspension were added to the reservoir of a Centrifree micropartition system and then centrifuged at 2000 x g for 5 min. All filtrates were transferred into a new microcentrifuge tube, and 6 µl of the filtrates were injected onto the HPLC column. The HPLC system consisted of a Hewlett Packard 1050 ChemStation with a UV detector (Agilent 1050 series). Determination of carboplatin was achieved using a LiChrosorb diol (10 µm; 250 x 4.6-mm) analytical column with a LiChroCART 100 RP-18 guard column. The mobile phase for plasma and tissue samples was composed of 98:2 acetonitrile: H2O (v/v) and 89:11 acetonitrile:0.015% H3PO4 (v/v) for urine. The flow rates were 2 ml/min (for plasma and tissue samples) and 1.1 ml/min (for urine). The column elute was monitored by UV at 229 nm. Quantitation of plasma or tissue carboplatin was carried out by using an external standard curve (0–4000.0 µg/ml) that was freshly prepared on a daily basis. Linear regression and correlation analysis were carried out to establish the standard peak-area/concentration curves for carboplatin.
Pharmacokinetics and Tissue Distribution of Gemcitabine.
Female nude mice bearing human lung cancer H1299 xenograft were used (three mice for each time 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 the fifth dose of DEX, given a single i.v. bolus administration of [3H]gemcitabine (160 mg/kg) via a tail vein. At various times (5, 15, and 30 min and 1, 2, 4, 8, and 24 h after drug dosing), blood and tissues including liver, kidneys, heart, lungs, spleen, brain, and tumor were collected. Plasma was separated by centrifugation, and tissue samples were immediately weighed and homogenized in 0.9% NaCl physiological saline (5 ml/g wet weight). Bone marrow cells were harvested by using the same method described above. Gemcitabine concentrations in biological samples were analyzed by quantitation of radioactivity.
Quantitation of Gemcitabine by Radioactivity Measurements.
The total radioactivities of gemcitabine in tissues and body fluids were determined by liquid scintillation spectrometry (LS 6000T A; Beckman, Irvine, CA), using a method described previously (26
, 32)
. In brief, plasma samples (50 µl) were mixed with 5 ml of scintillation solvent (Beckman) to determine total radioactivity. Tissue homogenates (50–200 µl) were mixed with 200 µl of solubilizer (TS-2) overnight, neutralized with 400 µl of 0.3% acetic acid, and then mixed with scintillation solvent (5 ml) to permit quantitation of total radioactivity.
Histology and Immunohistochemistry of A549 Xenografts.
A549 xenografts from treated nude mice were removed on day 35, fixed and stained with H&E, or snap-frozen in liquid N2. Immunohistochemical staining was undertaken with rat antimouse CD45 (leukocyte common antigen; Ly-5; PharMingen, BD Biosciences) using a DAKO Immunohistochemistry kit on frozen tumor tissue. Controls, including antimouse CD45 without secondary antibody and secondary antibody alone, were shown negative in the control CD-1 mouse spleens.
Data and Statistical Analysis.
The antitumor activity (tumor mass) was expressed as mean and SDs, and the significance of differences was analyzed by ANOVA. The pharmacokinetic parameters of carboplatin and gemcitabine were estimated 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 elimination half-life (T1/2); clearance (CL); and the volume of distribution at steady state (Vss).
Results
Pretreatment with DEX Enhances Therapeutic Effects of Carboplatin and Gemcitabine in Vivo
The effects of DEX pretreatment on antitumor activity of carboplatin and/or gemcitabine were studied in six murine xenograft models of human cancers. When mean tumor mass reached 44–68 mg, animals (mean body weight, 22 ± 2 g) were treated 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 effect of DEX pretreatment on carboplatin or gemcitabine antitumor activity was demonstrated in nude mice bearing human colon cancer LS174T (p53 wild type) xenografts. DEX alone showed slight inhibitory effects on tumor growth. Carboplatin was given i.p. on day 0, at a clinically relevant dose (120 mg/kg or 360 mg/m2). Pretreatment with DEX markedly increased the therapeutic effect of carboplatin (P < 0.01; Fig. 1A
; Table 1
). DEX pretreatment also increased therapeutic effects of gemcitabine (P < 0.05; Fig. 1B
; Table 1
). Representative xenograft tumors removed from various treatment groups are shown in Fig. 1C
.
|
|
; Table 1
) and gemcitabine (P < 0.05; Fig. 2B
; Table 1
). In the H1299 model, DEX pretreatment also significantly increased the therapeutic effects of carboplatin (P < 0.05; Fig. 2C
; Table 1
).
|
; Table 1
). In the MCF-7 model, both DEX and carboplatin alone had limited effects on tumor growth. However, pretreatment with DEX significantly increased therapeutic effects of carboplatin (P < 0.01; Fig. 3A
; Table 1
). Pretreatment with DEX also increased therapeutic effects of gemcitabine (P < 0.01; Fig. 3B
; Table 1
). In the MDA-MB-468 model, DEX or carboplatin alone had minimal effects on tumor growth, but pretreatment with DEX followed by carboplatin significantly inhibited tumor growth (P < 0.01; Fig. 3C
; Table 1
).
|
; Table 1
) or gemcitabine therapy (Fig. 4B
; Table 1
) in the human glioma model U87-MG (p53 wild type) but significantly increased the therapeutic effects of combination therapy of carboplatin and gemcitabine (P < 0.05; Fig. 4C
; Table 1
).
|
Histology of A549 Xenografts.
H&E stains of all four tumors demonstrated similar areas of necrosis, fibrosis, and vascular development (data not shown). Rare inflammatory cells were observed. This was confirmed with CD45 immunohistochemistry staining, which demonstrated infrequent CD45 cells (data not shown). There were no differences in CD45 staining among treatment groups, suggesting that the effects of DEX on antitumor effects of chemotherapeutic agents as observed above are not related to DEX reduction in the number of infiltrating CD45 cells.
DEX Alters Pharmacokinetics of Carboplatin Chemotherapy in Murine-Xenograft Models of Human Cancers
Nude Mice Bearing Human Colon Cancer LS174T Xenografts.
The first carboplatin pharmacokinetic study was performed in nude mice bearing LS174T xenografts (tumor mass, 500-1000 mg). The time-concentration curves are illustrated in Fig. 5
. No significant differences in plasma pharmacokinetics of carboplatin were observed between control and mice pretreated with DEX (Fig. 5A)
. However, DEX significantly increased tumor carboplatin concentrations (Fig. 5B)
. Without DEX treatment, the tumor exposure to carboplatin, measured by AUC, was markedly lower than normal tissues (4% of spleen, 3% of bone barrow, and 23% of liver AUCs). However, DEX significantly increased tumor carboplatin uptake, including 162% increase in AUC, 103% increase in Cmax, and 160% decrease in CL (Table 2)
. In contrast, pretreatment with DEX decreased carboplatin uptake in spleen (Fig. 5C)
. Pharmacokinetic analysis indicated that 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 concentrations were also observed (Fig. 5D
; Table 2
). There were slight but significant decreases in AUC and Cmax of liver carboplatin in mice pretreated with DEX (Table 2)
. No differences in kidney pharmacokinetics of carboplatin were observed (data not shown).
|
|
. No significant differences in plasma pharmacokinetics of carboplatin were observed between control and mice pretreated with DEX (Fig. 6A
; Table 3
). However, DEX significantly increased tumor carboplatin concentrations (Fig. 6B)
. Pharmacokinetic analysis indicated that there was a 50% increase in tumor AUC in mice pretreated with DEX (Table 3)
. In contrast, pretreatment with DEX decreased carboplatin uptake in spleen (Fig. 6C)
. Pharmacokinetic analysis indicated that 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 were also observed (Fig. 6D
; Table 3
).
|
|
. No significant differences in plasma pharmacokinetics of gemcitabine were observed between controls and mice pretreated with DEX (Fig. 7A)
. However, DEX pretreatment increased tumor gemcitabine concentrations (Fig. 7B)
. Pharmacokinetic analysis indicated that there was a 55% increase in tumor AUC in mice pretreated with DEX (Table 4)
. In contrast, pretreatment with DEX decreased gemcitabine uptake in spleen (Fig. 7C)
. Pharmacokinetic analysis indicated that there were significant decreases in splenic AUC, T1/2, and Cmax and an increase in CL in mice pretreated with DEX (Table 4)
. A slight decrease in bone marrow gemcitabine concentration was also observed (Fig. 7D
; Table 4
). No differences in pharmacokinetics of gemcitabine were observed in other tissues including kidneys, lungs, heart, and brain (data not shown).
|
|
Pretreatment with corticosteroids represents a novel approach to preventing chemotherapy-induced toxicity (4, 5, 6, 7, 8 , 15) . Our interest in administration of corticosteroids before chemotherapy originated from our observations that pretreatment of mice with cortisone acetate reduced hematopoietic toxicity of carboplatin (6, 7, 8) . Our recent studies showed that DEX has hematoprotective activity equal to cortisone acetate in normal CD-1 mice receiving carboplatin (33) . Pharmacokinetic studies in CD-1 mice demonstrated that DEX pretreatment markedly reduced bone marrow and splenic concentrations of carboplatin (33) . Although these studies provide the rationale for the use of corticosteroids including DEX as chemoprotective agents, there is a concern regarding the effects of DEX on antitumor activity of the chemotherapeutic agents (21) . In addition, the mechanisms by which DEX reduces hematotoxicity and enhances antitumor effects of carboplatin-based chemotherapy have not been elucidated.
The present study was undertaken to determine whether pretreatment with DEX can be further developed as a chemoprotectant and chemosensitizer in cancer chemotherapy and to examine DEX modulation of carboplatin pharmacokinetics and antitumor activity. We have now demonstrated that DEX pretreatment significantly enhanced antitumor effectiveness of carboplatin or gemcitabine monotherapy in the majority of the tested human cancer models, regardless of p53 status. We have also demonstrated that pretreatment with DEX significantly altered carboplatin and gemcitabine pharmacokinetics in nude mice bearing human cancer xenografts, which may be associated with its effects on antitumor activity of carboplatin and gemcitabine. Furthermore, we found that DEX simultaneously decreased carboplatin and gemcitabine concentrations in spleen and bone marrow, which may be associated with its hematoprotective effects. These results provide a basis for further testing of the effect of DEX as a potential antitumor sensitizer and chemoprotectant of cancer chemotherapeutics in human clinical trials.
Perhaps the most striking finding in the present study is that DEX pretreatment increased in vivo antitumor activities of carboplatin or gemcitabine in five of the six nude mouse models of human cancers. DEX administered alone showed limited effects on tumor growth. There are reports demonstrating in vivo antitumor activity of DEX in epithelial cell cancers (34
, 35)
. The mechanisms of DEX direct antitumor activity have not been elucidated, but several DEX activities may explain this observation. DEX may decrease tumor secretion or tumor associated-cell secretion of tumor growth factors. For example, Nishimura et al. demonstrated that DEX inhibited the growth of human prostate cancer DU145 xenografts in nude and severe combined immunodeficient mice, possibly through the disruption of the nuclear factor-
B-interleukin-6 pathway (36)
. In addition, DEX may enhance tumor apoptosis by inhibiting nuclear factor-B activity by at least two mechanisms: DEX induces translation of IB and glucocorticoid-induced leucine zipper, which inhibit NF-B translocation to the nucleus (37)
and interaction with transcription sites, respectively (38)
. Nuclear factor-B induces transcription of multiple antiapoptotic proteins in stressed cells (39)
.
Several other possible mechanisms may explain DEX enhancement of the antitumor effects of carboplatin and gemcitabine. Tumors exhibit multiple physiological abnormalities including markedly abnormal tortuous vasculature characterized by decreased blood flow and decreased lymphatic and promiscuous movement of high molecular weight solutes through abnormal interendothelial pores (40, 41, 42, 43) . These abnormalities result in elevated TIFP (24) and volume (44) , which, in turn, paradoxically reduce the movement of agents that are transiently present in the plasma into the tumor interstitial fluid space as predicted by Darcy’s Law (45) . The mechanisms of induction of "leaky" interendothelial pores are probably multifactorial. Tumor endothelial cells are structurally abnormal (41) . Moreover, tumor endothelial cells are exposed to several cytokines such as vascular endothelial growth factor, interleukin-1, interleukin-8, and transforming growth factor ß, which alter both normal and tumor vasculature to allow movement of solutes and fluid into the interstitial space (46 , 47) . The origin of those cytokines is clearly multiple and may include tumor cells and tumor-infiltrating cells such as macrophages (46, 47, 48, 49) . Several studies have shown that glucocorticosteroids reduce interendothelial pore size in pure, normal endothelial cell cultures in vitro (50) and inhibit secretion of inflammatory cytokines from many cells, including macrophages (37 , 51) . Braunschweiger and Schiffer (41) demonstrated that treatment with DEX of mice bearing autochronous tumors decreased the movement of high molecular weight molecules into the tumors and the tumor interstitial volume. As predicted by those studies, treatment of murine-human colon cancer LS174T xenografts with DEX reduced elevated TIFP (23) . Using the same model (LS174T) in the present study, we have now demonstrated that DEX increases drug accumulation in tumors and improves therapeutic effects of carboplatin and gemcitabine. Taken together, DEX may enhance the antitumor effects of carboplatin and gemcitabine by decreasing in tumors the promiscuous interendothelial loss of solutes and water into the interstitial space, thus decreasing elevated interstitial volume and pressure in the interstitial space in tumors. Decreased TIFP, in turn, allows for increased drug movement into tumors and improved antitumor effects. Further study is needed to provide direct evidence for this hypothesis.
Of note, other mechanisms may be associated with DEX antitumor activity. For instance, Yu et al. demonstrated that DEX enhanced the in vitro and in vivo antitumor effect of 1,25-dihydroxycholecalciferol, an active metabolite of vitamin D, probably by increasing vitamin D receptor ligand-binding activity (52) . The same laboratory further showed that both cell cycle arrest and apoptosis were enhanced by DEX. They also suggested the involvement of the extracellular signal-regulated kinase and Akt signaling pathways in the antiproliferative effects of the combination of 1,25-dihydroxycholecalciferol and DEX (53) . In addition, no significant effects of DEX on antitumor activity in the glioma model were observed when carboplatin and gemcitabine administered alone in the current study, suggesting that other unknown molecular mechanisms are involved in DEX-associated effects on tumor growth, which may be tumor type dependent.
The mechanisms responsible for the findings, i.e., pretreatment with DEX decreases the concentrations of carboplatin and gemcitabine in host tissues but increase concentrations of these drugs in tumor tissues, are not fully understood. We hypothesize that because interstitial fluid pressure is already low (-5 to 0 mm Hg versus 5–100 mm Hg in tumors) in normal tissues, the dominant effect of DEX is retardation of solute movement (i.e., drug) into bone marrow and spleen. However, the role of TIFP in tumor uptake and retention of anticancer drugs may be drug type-dependent. Large molecule drugs, such as proteins and antibodies, are highly sensitive to the convective barriers established as a result of increased interstitial fluid pressure in tumors, due to their dependence on convection for delivery. However, delivery of small molecules such as carboplatin and gemcitabine involves both convection and diffusion. Consequently, decreasing TIFP as a consequence of decreasing trans-vascular movement of fluid may have a much greater impact on the convective clearance of the drug from the tumor interstitial space than on its initial delivery because the concentration gradient is directed from the plasma to the tissue, and the diffusion will drive the process forward until the plasma concentration drops. Decreasing the vascular permeability is likely to increase the mean residence time in the tumor by reducing the clearance as seen in our pharmacokinetic studies. In addition, the increase in therapeutic effects by DEX can be explained, in part, as an additive tumor response to DEX and the chemotherapeutic agent(s) used in combination, although the potential activity of DEX alone is relatively weak. Therefore, our observations of increased tumor inhibition are likely to involve both additive cytotoxic effects and drug transport effects (increased uptake and decreased clearance). The contribution of each may vary with the tumor type.
Our findings in normal tissues suggest that pretreatment of patients with DEX may decrease hematotoxicity and increase antitumor effects of carboplatin or gemcitabine. We have undertaken a Phase I pilot trial using DEX pretreatment in patients receiving carboplatin-based therapy (15) . DEX decreased carboplatin-induced hematotoxicity. In patients receiving DEX, the incidence of partial or complete responses was higher than that in patients not receiving DEX, although the small patient number and heterogeneous patient population precluded conclusions regarding tumor response. The results reported here and in our pilot clinical trial have led us to undertake additional clinical and laboratory studies in this area, providing a potential new avenue for therapeutic use of DEX as a tumor chemosensitizer and chemoprotectant in the treatment of human cancers.
ACKNOWLEDGMENTS
We thank Jie Hang, Zhenqi Shi, Gautam Prasad, Zhuo Zhang, Aselle Adaim, Kexuan Wang, Veronika Schachinger, Michaela Haslinger, Bing Pang, and Lin Lin for excellent technical assistance and Drs. Trenton R. Schoeb and Mitchell Pate for pathology studies. We also thank Dr. Al LoBuglio for helpful discussions.
FOOTNOTES
Grant support: Funds for Cancer Pharmacology Laboratory from University of Alabama at Birmingham Comprehensive Cancer Center.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Ruiwen Zhang, Department of Pharmacology and Toxicology, University of Alabama at Birmingham, VH 113, Box 600, 1670 University Boulevard, Birmingham, Alabama 35294. Phone: (205) 934-8558; Fax: (205) 975-9330; E-mail: ruiwen.zhang{at}ccc.uab.edu
Received 6/ 3/03; revised 11/13/03; accepted 11/19/03.
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