Introduction

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Paclitaxel, one of the most important anticancer drugs developed in the past two decades, is active against multiple types of human solid tumors (Rowinsky, 1993). Paclitaxel enhances tubulin polymerization, promotes microtubule assembly, binds to microtubules, stabilizes microtubule dynamics, induces mitotic block at the metaphase/anaphase transition, and induces apoptosis (Parness and Horwitz, 1981; Manfredi et al., 1982; Jordan et al., 1993, 1996; Derry et al., 1995). The intracellular concentration of paclitaxel is critical for its pharmacological effect; drug resistance in several resistant sublines is correlated with reduced intracellular drug accumulation compared with the sensitive parent cell lines (Lopes et al., 1993; Bhalla et al., 1994; Jekunen et al., 1994; Riou et al., 1994; Speicher et al., 1994).

It has been proposed that drug delivery to the tumor core is necessary to prevent tumor regrowth and is important for treatment efficacy (Durand, 1990; Erlanson et al., 1992; Baguley and Finlay, 1995; Jain, 1996). Drug delivery to the tumor core is governed by several factors, which differ for systemic and regional treatments. Following a systemic intravenous injection, drug delivery to cells in solid tumors involves three processes, i.e., distribution through vascular space, transport across microvascular walls, and diffusion through interstitial space in tumor tissue (Jain, 1987). When the drug is directly injected into a tumor, e.g., by intratumoral injection or by direct instillation into peritumoral space such as in intravesical therapy of superficial bladder cancer and in intraperitoneal therapy of ovarian cancer, drug delivery to cells in solid tumors is primarily achieved by diffusion through interstitial space (Markman et al., 1995; Nativ et al., 1997; Song et al., 1997; Markman, 1998).

The diffusion characteristics of paclitaxel, despite its relatively low molecular mass (853 daltons), are likely to be similar to those of a protein because of its extensive binding to proteins in interstitial fluid (Baguley and Finlay, 1995). We have shown that resistance to paclitaxel penetration into tumor tissue is highly dependent on the cell density; paclitaxel penetration in human tumor histocultures is restricted to the periphery until the cell density is reduced due to drug-induced apoptosis, at which time paclitaxel distributes evenly throughout the tumor (Kuh et al., 1999). The slow appearance of apoptosis in tumor histocultures (i.e., at about 24 h) is in agreement with our observations in multiple human tumor cell lines (Au et al., 1998). Based on these findings, we hypothesize that the delivery of paclitaxel to solid tumors can be enhanced by an apoptosis-inducing pretreatment. This hypothesis was evaluated in the present study. In vitro studies were performed using histocultures of human pharynx FaDu tumor xenograft maintained in immunodeficient mice, and in vivo studies were performed using a syngeneic tumor model, i.e., rats bearing subcutaneously implanted prostate MAT-LyLu tumors.

	Materials and Methods

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Chemicals and Reagents. Paclitaxel was obtained from the Bristol Myers Squibb Co. (Wallingford, CT) and the National Cancer Institute (Bethesda, MD); 3"-[³H]paclitaxel (specific activity, 19.3 Ci/mmol) from the National Cancer Institute; cefotaxime sodium from Hoechst-Roussel Inc. (Somerville, NJ); gentamicin from Solo Pak Laboratories (Franklin Park, IL); other tissue culture supplies were obtained from Life Technologies, Inc. (Grand Island, NY). Solvable tissue gel solubilizer and Atomlight scintillation fluid were obtained from DuPont Biotechnology Systems (Boston, MA), Hyperfilm ³H from Amersham Pharmacia Biotech (Arlington Heights, IL), autoradiographic supplies from Kodak (Rochester, NY), and Cremophor EL from Sigma Chemical Co. (St. Louis, MO).

Animals. Male nu/nu BALB/c mice, weighing 18 to 21 g, were purchased from the National Cancer Institute; male Copenhagen rats, weighing 190 to 210 g, were obtained from Harlan Biomedicals (Dawely, OH). Animal care was provided by the Laboratory Animal Resources in our institution.

Tumor Culture and Drug Treatment: In Vitro Studies. FaDu cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in minimum essential medium supplemented with 9% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 90 µg/ml gentamicin, and 90 µg/ml cefotaxime. Cells were harvested from subconfluent cultures using trypsin and resuspended in fresh medium before plating. Cells with greater than 90% viability, as determined by Trypan Blue exclusion, were used for tumor implantation. Cells were centrifuged and resuspended in culture medium mixed with equal volume of Matrigel (Collaborative Biomedical Products, Bedford, MA). Matrigel is a solubilized tissue basement membrane preparation extracted from the Engelbreth-Holmswarm mouse tumor and supports the growth of human tumors in immunodeficient mice (Kleinman, 1990). Tumor cells (10⁶ cells in 0.1 ml) were injected subcutaneously in the left and right sides of the upper back in a mouse, with a 21-gauge needle. Tumors were removed when they reached a size of about 0.5 g and dissected into 1-mm³ pieces under sterile conditions within 2 h after removal from the host. Five to six tumor pieces were placed on a 1-cm² collagen gel (presoaked with drug-free medium) in a six-well plate and incubated with 4 ml of culture medium at 37°C in a humidified atmosphere of 95% air and 5% CO₂, for 3 to 4 days. Drug treatment was done as previously described (Kuh et al., 1999). Briefly, the histocultures were transferred to a collagen gel presoaked with drug-containing medium for at least 8 h. The presoaking was to eliminate the delay in drug transport from medium through the collagen gel (Kuh et al., 1999). Drug treatment was terminated by carefully transferring histocultures to a collagen gel presoaked with drug-free medium.

Drug Penetration and Accumulation: In Vitro Studies. The spatial distribution of [³H]paclitaxel in histocultures was studied using autoradiography and imaging techniques as described previously (Kuh et al., 1999). Briefly, frozen cross sections (10 µm) taken from the middle of the histocultures were placed on glass slides, followed by exposure to ³H-sensitive film (Hyperfilm ³H) for a week. The film was developed, and the slide was stained with H&E. Image analysis was used to capture the autoradiographic image (where the grains indicated the location of the radiolabeled drug) as well as the histological image of the tissue section stained with H&E (which showed the tissue structure and distribution of tumor cells), and to overlay the two images to visualize the distribution of [³H]paclitaxel in the tumor tissue.

Effect of Apoptosis-Inducing Pretreatment on Drug Penetration and Accumulation in Tissues: In Vitro Studies. Three in vitro studies were performed to examine the effect of pretreatment. The study protocols were as described under Results. Tumor cell density and the fraction of apoptotic cells were determined by counting the number of total tumor cells and apoptotic cells in non-necrotic regions at 400× magnification. Apoptotic cells were determined based on morphological changes in tumor cells such as chromatin condensation and margination, disappearance of nucleoli, formation of membrane blebs, formation of apoptotic bodies, and/or cell shrinkage (Kerr et al., 1994). Our laboratory and others have shown that this method yields the same results as the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method (Gold et al., 1994; Gan et al., 1996).

Effect of Apoptosis-Inducing Pretreatment on Drug Accumulation in Tumors: In Vivo Studies. The in vivo studies were performed using a rat tumor model because of the ease of administering an intravenous infusion and sampling blood repeatedly. The rat MAT-LyLu tumor cells were originally obtained from Dr. J. Isaacs (Johns Hopkins University, Baltimore, MD), and have been maintained in our laboratory in RPMI-1640 medium supplemented with 9% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 90 µg/ml gentamicin, and 90 µg/ml cefotaxime sodium. Tumor cells (5 × 10⁶ cells in 0.1 ml medium, >90% viability as determined by Trypan Blue exclusion) were injected subcutaneously in the right and left upper backs of a male Copenhagen rat with a 21-gauge needle. Experiments were initiated when the tumor was between 0.3 to 0.5 g in size.

HPLC Analysis. The concentration of nonradiolabeled paclitaxel in plasma and tumor was analyzed using our previously reported column switching HPLC method (Song and Au, 1995). Briefly, preweighed tumors were homogenized in 4 ml of ethyl acetate, and mixed with the internal standard, cephalomanine (1 µg/100 µl in methanol). The extraction yield was 92 ± 4 and 98 ± 8% in plasma (n = 4) and tumor tissue (n = 4), respectively. The HPLC stationary phase consisted of a cleaning column (NovaPak C₈, 75- × 3.9-mm i.d., 4-µm particle size, Waters Associates, Milford, MA) and an analytical column (Bakerbond Octadecyl, 250- × 4.6-mm i.d., 5-µm particle size, J. T. Baker, Phillipsburg, NJ). Samples were injected onto the clean-up column and eluted with the clean-up mobile phase (37.5% acetonitrile in water) at 1 ml/min. Concurrently, the analytical mobile phase (49% acetonitrile in water) was directed through the analytical column at a flow rate of 1.2 ml/min. The fraction from 5 to 12 min, which contained paclitaxel and cephalomanine, was transferred from the clean-up column onto the analytical column. Detection of paclitaxel and cephalomanine was at 229 nm, with a detection limit of 1 ng of paclitaxel per injection.

Statistical Analysis. The statistical significance of the differences between treatment groups was analyzed using the two-tailed unpaired Student's t test.

	Results

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Effect of Apoptosis-Inducing Pretreatment on Drug Delivery in Tumor: In Vitro Studies. Three in vitro studies were performed using the FaDu tumor histocultures. The first study evaluated the importance of apoptosis on paclitaxel penetration. Figure 1 shows the apoptotic cells induced by paclitaxel. This study used two concentrations of [³H]paclitaxel that differed in their ability to induce apoptosis and reduce tumor cell density, with the 50 nM concentration being more effective than the 10 nM concentration (Fig. 2). Tumor penetration of [³H]paclitaxel at the 10 nM concentration was limited to the periphery of the histocultures throughout 72 h. In comparison, drug penetration at the 50 nM concentration was limited to the periphery in the first 24 h but subsequently increased, along with an increase in the apoptotic fraction and a reduction of the cell density, such that the radiolabeled drug was evenly distributed throughout the tumor by 72 h (Figs. 2 and 3). These data confirm our previous finding that apoptosis enhances the penetration of paclitaxel in solid tumors (Kuh et al., 1999).

View larger version (88K): [in this window] [in a new window]   Fig. 1.   Morphological changes in paclitaxel-induced apoptosis. FaDu tumor histocultures were treated with 0 and 1 µM paclitaxel for 3 h. At 21 h after drug treatment, tumors were frozen and cut into 10-µm sections using a cryotome. The sections were evaluated for drug-induced apoptosis. Apoptotic cells were indicated by morphological changes, i.e., formation of apoptotic bodies and/or cell shrinkage (arrow 1) and chromatin condensation and margination (arrow 2); 400× magnification.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effect of pretreatment and paclitaxel concentration on the drug-induced apoptosis and cell density. FaDu tumor histocultures were treated with 10 and 50 nM [3H]paclitaxel continuously, without (closed symbols) or with (open symbols) pretreatment by 1 µM nonradiolabeled paclitaxel for 3 h. The pretreatment was initiated 24 h before the [3H]paclitaxel treatment. Fraction of apoptotic cells (circles) and tumor cell density (squares) were determined by counting the number of total and apoptotic tumor cells per 400× microscopic field (three random fields per tumor). Mean ± S.D. (n = 3 histocultures).

View larger version (114K): [in this window] [in a new window]   Fig. 3.   Effect of pretreatment and drug concentration on the rate of paclitaxel penetration and tissue morphology in tumor histocultures. FaDu tumor histocultures were treated with 10 and 50 nM [3H]paclitaxel continuously, without (A) or with (B) pretreatment by 1 µM nonradiolabeled paclitaxel for 3 h initiated 24 h before the [3H]paclitaxel treatment. In each figure, the top panels are autoradiographic images overlaid on histological image (25× magnification), and the bottom panels are histological images of the indicated boxed region in the autoradiographic images, at 400× magnification. The indicated times refer to the times after initiation of the [3H]paclitaxel treatment.

View larger version (74K): [in this window] [in a new window]   Fig. 4.   Effect of treatment schedule on the rate of paclitaxel penetration and tissue morphology in tumor histocultures. Two groups of FaDu tumor histocultures were treated with the same drug exposure, i.e., 1200 nM · h, but by different schedules. One group was treated with 600 nM for 1 h and, 23 h later, 50 nM for 12 h. The second group was treated continuously with 50 nM for 24 h. The top panels show autoradiographic images overlaid on histological images, at 25× magnification. The bottom panels show histological images of the indicated boxed region in the autoradiographic images, at 400× magnification. The indicated times refer to the times after initiation of treatment.

Effect of Apoptosis-Inducing Pretreatment on Drug Accumulation in Tumor: In Vivo Studies. The selection of treatment schedules in rats was based on three considerations. First, the dose and treatment duration of the pretreatment should be sufficient to induce apoptosis in tumors. Second, the interval between the pretreatment and the subsequent dose should allow significant apoptosis to occur. Third, the duration of infusion of the second dose should be sufficiently long to allow the drug concentrations in plasma and tumor to reach a steady state, in which case the tumor-to-plasma concentration ratio provides a measurement of the drug partition from plasma to tumor. Pharmacokinetic and tissue morphology studies were conducted to identify the treatment schedules. The results, listed in Figs. 5 and 6 and in Table 2, showed that 1) a pretreatment dose of 5 mg/kg infused over 1 h was sufficient to induce apoptosis and reduce tumor cell density at 24 h, which, as shown in the in vitro studies described above, are sufficient to enhance drug penetration in tumors, and 2) the plasma concentrations approached plateau values after infusion at 0.83 mg/kg/h for 6 and 12 h. These treatment schedules were used in subsequent studies.

View larger version (101K): [in this window] [in a new window]   Fig. 5.   Changes in tumor composition after intravenous infusions of paclitaxel. The dose, treatment schedule, and timing of sample are indicated. Magnification, 400×.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Plasma concentration-time profiles after intravenous infusions of paclitaxel. Rats were given infusions of paclitaxel at the indicated schedule.

	Discussion

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Inadequate delivery of anticancer drugs to the tumor core is an obstacle to successful chemotherapy (Jain, 1987, 1996; Durand, 1990; Erlanson et al., 1992). We have shown an inverse relationship between paclitaxel penetration in solid tumors and tumor cell density (Kuh et al., 1999). The present study was designed to test the hypotheses that an apoptosis-inducing pretreatment with paclitaxel enhances its delivery to solid tumors and that the paclitaxel treatment schedule can affect the drug delivery to solid tumors. The results show that 1) modification of paclitaxel treatment schedules by including an apoptosis-inducing pretreatment reduces cell density and enhances the penetration and accumulation of a properly timed subsequent dose in solid tumors, and 2) dose fractionation into two doses separated by an interval to allow for apoptosis to occur results in greater drug delivery into solid tumors compared with a single treatment. For paclitaxel, the drug-induced apoptosis occurs after a delay of about 16 to 24 h, as shown earlier (Cheng et al., 1995; Au et al., 1998, 1999) and confirmed in the present study.

The apoptosis-induced enhancement of drug delivery to tumors is likely the result of an increased interstitial space. For example, the diffusion coefficient in a gel structure is a function of interstitial space and tortuosity (Schultz and Armstrong, 1978; Fox and Wayland, 1979). Hence, apoptosis, by creating a larger fraction of interstitial space and/or a decrease in tortuosity, would result in a more rapid drug diffusion. Drug-induced apoptosis may also, by decreasing the interstitial fluid pressure and decompressing the tumor microvessel, result in enhanced drug permeability across the tumor microvasculature and drug diffusion in interstitial space (Griffon-Etienne et al., 1999).

In summary, results of the present study indicate that the delivery of paclitaxel to tumor can be enhanced by using a paclitaxel pretreatment that induces apoptosis and reduction in cell density and by using treatment schedules designed to take advantage of these drug-induced changes in tumor tissue composition. The enhancement in paclitaxel delivery was observed in tumor histocultures where drug transport is by diffusion through the interstitial space as well as in tumors grown in animals where blood flow contributes to drug transport. Hence, our approach of using apoptosis-inducing pretreatment to enhance drug delivery to solid tumors applies to regional therapy where the drug is supplied from the extracellular fluid surrounding the tumor as well as systemic therapy where the drug is supplied through the circulation from within the tumor. It is also noteworthy that induction of apoptosis was achieved at paclitaxel concentrations (i.e., 600-6000 nM) that are within the plasma concentration range attained in patients given intravenous paclitaxel therapy (Sonnichsen and Relling, 1994), indicating the potential clinical utility of the pretreatment approach.