Introduction

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It is generally recognized that treatment of solid, peripheral tumors is impeded because the abnormal nature of the tumor vasculature limits delivery of chemotherapeutic agents to the tumor interstitium (Jain, 1990, 1991; Less et al., 1991). Recent studies in rat tumor models demonstrate that stimulation of endogenous B₂ bradykinin receptors with the selective agonist Cereport¹ leads to enhanced delivery of chemotherapeutic agents into solid peripheral tumors (Emerich et al., 2001). This effect is relatively selective for tumor tissue, in that little or no effect is achieved in healthy tissue or organs. Moreover, the enhanced delivery is manifested as suppression of tumor growth and increased survival in tumor-bearing rats. Interestingly, because the plasma levels of Cereport required to achieve this effect are relatively low (i.e., well below the K_i established for Cereport at the B₂ receptor (Bartus et al., 1996b, 2000), this phenomenon may reflect a natural role of bradykinin in mediating vascular flow of blood and nutrients to solid tumors.

Given the novelty of this phenomenon, as well as its potential application for improving the treatment of cancer patients, the present paper attempts to further increase our understanding of the underlying physical and biochemical mechanisms of action. A series of studies was performed to characterize the physical changes of the tumor vasculature induced by Cereport and to identify those that may be responsible for the enhanced delivery. Interstitial fluid pressure within the tumor, tumor blood flow and systemic blood pressure were independently measured during Cereport infusion. Separate measurements of the physical properties of the tumor vasculature included areas of hypoperfusion within the tumor, intravascular pore size and surface area of the tumor vessels. Finally, a parallel series of pharmacological studies was performed to define the roles played by bradykinin's two major signaling pathways.

Bradykinin uses a G-coupled receptor capable of inducing a variety of different effector responses, including activation of phospholipases, channels, and protein kinases (Burch et al., 1993). Two major pathways have been shown to be activated by B₂ receptor stimulation in nearly all tissues (Burch et al., 1993) and they are the focus of our studies here. The first involves the release of arachidonic acid by activation of phospholipase A_2. Arachidonic acid, in turn, is metabolized into a variety of eicosanoids, including prostaglandin E₂ through the action of cyclooxygenase. In this article, we pharmacologically stimulate one component of this pathway by administering exogenous prostaglandin E₂ and suppress the pathway by inhibiting cyclooxygenase activity.

The second major pathway involves the activation of phospholipase C and phosphatidylinositol to release diacylglycerol and inositol phosphates (Burch et al., 1993). Inositol phosphates have been shown to be instrumental in the release of calcium from the endoplasmic reticulum (Fisher, 1995). The elevation in cytosolic calcium has, in turn, been linked to numerous calcium-dependent events, including protein kinase C and calmodulin-dependent protein kinases (Fisher, 1995). Recently, an important signaling role of nitric oxide (NO) has been linked to the bradykinin-induced calcium fluxes (McGehee et al., 1992). NO, in turn, stimulates cGMP activity as an important modulating event (Murad, 1994). We focused our attention on the NO-cGMP component of this pathway, stimulating it by administering NO donors as well as inhibiting the degradation of cGMP. Furthermore, we dampened the effects of this pathway by inhibiting nitric-oxide synthetase activity.

Collectively, the studies we report reveal a complex chain of events that occurs following B₂ receptor stimulation within solid tumor vessels. This involves at least two parallel signaling pathways and multiple, transient physiological and morphological changes within the tumor that can be linked to the increased delivery of chemotherapeutic agents following Cereport infusion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals. Male Fischer rats (N = 569; 170-220 g; Taconic Farms, Germantown, NY) were housed in polypropylene cages with free access to food and water. The vivarium was maintained on a 12-h light/dark cycle with a room temperature of 22 ± 1°C and relative humidity levels of 50 ± 5%. All studies were approved in advance by Alkermes' Institutional Animal Care and Use Committee and were conducted in a manner that met or exceeded National Institutes of Health guidelines.

Cell Maintenance and Implantation. A rat ascites mammary adenocarcinoma cell line (MATB-III; ATCC CRL-1666) was used in the following studies. Cells were grown and maintained as previously described (Emerich et al., 2001). Before implantation, cells were harvested and suspended in serum-free media containing 1.2% methyl cellulose at a density of 5 × 10⁶ cells/ml and 200 µl of the suspension (10⁶ cells) was injected subcutaneously into the rear flank, using a 22-gauge needle. All tumors were palpitated and measured daily until they reached a size of 1 cm² (7-10 days), at which time the animals were used in dosing studies.

Systemic Blood Pressure. Two separate and mutually corroborating studies examined the relationship between alterations in blood pressure and the enhanced delivery of [¹⁴C]carboplatin produced by Cereport. In the first studies, continuous on-line measures of mean arterial blood pressure were recorded from an intrafemoral cannula over the course of the Cereport (0.1, 0.2, 0.5, or 1.0 µg/kg/min) infusion using a MacLab 8 physiology recording system (ADInstruments, Milford, MA). In the second series of studies, the adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) (Research Biochemicals International, Natick, MA) was infused i.v. to decrease blood pressure in a manner that mimicked both the magnitude and time course produced by Cereport. In these experiments, animals received a 15-min i.v. infusion of [¹⁴C]carboplatin (mol. wt. = 371, specific activity = 144 µCi/mg; Amersham, Arlington Heights, IL) followed by a 10-min i.v. infusion of either saline (N = 8) or Cereport (2 µg/kg) (N = 6). Parallel groups of animals received [¹⁴C]carboplatin infusions together with a 10-min i.v. infusion of NECA at a dose of either 0.2 µg/kg/min (N = 8) or 2.0 (N = 8) µg/kg/min.

Regional Blood Flow and Autoradiography. To determine whether significant changes in regional blood flow occur in peripheral tumors following Cereport administration, 4-iodo-N-methyl-[¹⁴C]antipyrine (IAP) autoradiography was used. IAP (25 µCi; Amersham) was administered 30 s before the completion of the Cereport infusion (0.15 µg/kg/min) using a progressively increasing infusion rate (from 0.8 to 3.3 ml/min over 30 s). Timed arterial blood samples were collected from a catheterized femoral artery directly into heparinized 60-µl glass capillary tubes (Ciba Corning Diagnostics Ltd., Suffolk, England) every 5 s over the 30-s tracer infusion period for subsequent analysis of whole-blood radioactivity levels. At the end of the IAP infusion, the tumor was rapidly removed and frozen in 2-methylbutane at 30°C. Blood samples were analyzed using scintillation counts.

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Effect of Cereport on Perfusion of Tumor Blood Vessels. A double fluorescent dye technique was used to determine whether Cereport altered the pattern of perfusion within the tumor vasculature. Animals received an i.v. bolus injection of the fluorescent dye Hoechst 33342 (30 mg/kg; Molecular Probes, Eugene, OR). Five minutes later, the same animals received a 10-min i.v. infusion of saline or Cereport (0.15 µg/kg/min) together with a bolus injection of a second, different fluorescent dye (DiOC₇, 1 mg/kg; Molecular Probes), 2 min into the Cereport infusion.

Effect of Cereport on Interstitial Fluid Pressure. Interstitial fluid pressure (IFP) of subcutaneous MATB-III tumors was measured using the wick-in-needle technique (Scholander et al., 1968). A recording needle was constructed by removing the hub of a 23-gauge × 1-inch needle and drilling a 0.8-mm hole 4 mm from the needle tip. Five 2.5-cm lengths of 6-0 nylon monofilament suture were inserted into the needle (extending 0.5 mm beyond the needle tip) to permit transmission of tumor IFP through the individual channels formed by the suture to the pressure transducer. The recording needle was connected to an ultralow compliance pressure transducer (TXC-310; Micro-Med, Lexington, KY) using 10 cm of PE50 tubing filled with heparinized saline (50 U/ml). Interstitial fluid pressure (mm Hg) was continuously measured six times per second using a Digi-Med Low Pressure analyser and DMSI-200 software (Micro-Med) and the results were averaged across each minute.

Effect of Cereport on Transvascular Pore Size. Specific-size fluorescent polymer microspheres (Duke Scientific Corporation, Palo Alto, CA) were infused i.v. as pairs of different diameter microspheres (0.025 and 0.05 µm or 0.1 and 0.2 µm) containing different fluorescent dyes (red or green) just before an i.v. infusion of Cereport (0.15 µg/kg/min) or vehicle. Animals were sacrificed 2 or 24 h later (to allow the microspheres to clear from the circulation, ensuring that any remaining microspheres within the tumor had extravasated from the vasculature into the interstitial space). The tumor tissue was frozen and sectioned with a cryostat at 20-µm intervals. Using a florescence microscope, the two different-sized microspheres within a single section representing the center of the tumor were independently visualized using fluorescein isothiocyanate and rhodamine filters. Beginning at the edge of the tumor, the section was scanned sequentially across the entire tumor. The first 12 fields (200 µm² each) containing microspheres were digitalized using a color charge-coupled device camera interfaced with an image analysis system (Image Pro-Plus; Media Cybernetics, Silver Spring, MD).

Immunocytochemistry. MATB-III tumors were removed and quickly frozen in chilled isopentane (30°C). Tumors were then sectioned on a cryostat (20 µm), thaw-mounted onto microscope slides, and processed for the selective visualization of tumor blood vessels using CD-31 immunocytochemistry (St. Croix et al., 2000) as follows: 1) slides washed 6 × 10 min in dilution media (Triton X-100 and Tris buffer) followed by 0.1 M sodium periodate for 1 h; 2) slides washed 6 × 10 min in dilution media followed by 0.1 M sodium 3 × 10 min in dilution media, followed by normal horse serum and bovine serum albumin for 1 h; 3) slides incubated for 48 h (24 h at 22°C and 24 h at 4°C) in the primary monoclonal antibody to CD-31 (1:300; Chemicon, Tempecula, CA); 4) slides washed 6 × 10 min in dilution media followed by a 1-h incubation in the appropriate biotinylated secondary IgG antibody (1:200; Vector, Burlingame, CA); 5) slides washed 6 × 10 min in dilution media, rinse slides; 6) slides incubated with "Elite" avidin-biotin complex (1:1000; Vector) for 1.25 h; 7) slides rinsed 3 × 10 min in imidazole/acetate buffer; 8) slides incubated in a solution containing 3,3'-diaminobenzidine and nickel ammonium sulfate dissolved in imidazole/acetate buffer with hydrogen peroxide for 8 min; and 9) reaction terminated by rinsing 3 × 10 min in imidazole/acetate buffer. Sections were stored in phosphate-buffered saline and mounted, dehydrated in alcohol, and cover slipped. Control sections were processed in an identical manner except the primary antibody solvent was substituted for the primary antibody.

Effect of Cereport on Tumor Blood Vessel Size. Cereport (0.15 µg/kg/min) or saline was infused i.v. into tumor-bearing animals. At 2, 5, or 8 min into the infusion, the animals were sacrificed and the tumor was rapidly removed and quickly frozen in chilled isopentane (30°C). Tumors were sectioned on a cryostat (20 µm), thaw-mounted onto microscope slides, and processed for visualization of blood vessels using CD-31 immunohistochemistry as described above.

Quantitation of Drug Delivery to Peripheral Tumors. Studies examining delivery of [¹⁴C]carboplatin to peripheral tumors were conducted using previously published protocols (Bartus et al., 1996a, 2000). Briefly, animals were anesthetized with urethane (1.8 g/kg i.p.) and a cannula was placed in the jugular vein for infusions of Cereport (RMP-7; Alkermes, Inc., Cambridge, MA) and [¹⁴C]carboplatin. Immediately after drug administration, rats were killed and the peripheral tumors were rapidly removed. A 1- to 2-mm-thick slice from the center of tumor was removed, weighed, and placed into a scintillation vial and the amount of radioactivity (nCi/g) was computed using scintillation counts.

Pharmacological Interactions between Bradykinin and Nitric Oxide and Prostaglandins. A series of studies examined the relationship between stimulation of bradykinin B₂ receptors, alterations in the endogenous NO activity, and delivery of [¹⁴C]carboplatin to peripheral tumors. In the first experiment, rats received a 15-min i.v. infusion of [¹⁴C]carboplatin followed by a 10-min i.v. infusion of either saline or Cereport (0.1 µg/kg/min). L-NAME was administered as a 30-min infusion (0.2 µg/kg/min) beginning 15 min before the initiation of the carboplatin infusion and continuing until the end of the Cereport infusion. Tumors were removed at the end of the infusion and processed for scintillation as described above.

Modulation of Cereport's Effects with PDE-V Inhibition. Rats bearing MATB-III tumors were used to determine whether prolonging the action of the NO-mediated second messenger cGMP could modulate the ability of Cereport to enhance delivery of [¹⁴C]carboplatin to the tumor. Animals received i.v. infusions of either saline or Cereport (0.05, 0.2, or 0.5 µg/kg/min) alone or together with the PDE-V inhibitor zaprinast (20 mg/kg, Research Biochemicals International). Zaprinast is a selective, competitive inhibitor of phosphodiesterase V, which enhances the elevations of cGMP by inhibiting its hydrolysis by that enzyme (Thompson, 1991). This dose of zaprinast was determined to be the maximally tolerated dose based on pilot studies demonstrating that significant hypotension occurred with higher doses. All zaprinast infusions were given i.v., to ensure distribution to the tumor vasculature, and continuously with the 15-min [¹⁴C]carboplatin infusion and the 10-min Cereport infusion.

Statistics. The effects of Cereport on blood pressure and IFP were compared using a repeated measures ANOVA. The ranked data in the transvascular pore size studies were compared using a Mann-Whitney nonparametric analysis. The effects of Cereport on transvascular pore size, perfusion of blood vessels, and blood flow were also compared using a one-way ANOVA. Finally, all the effects of pharmacologically modifying delivery of [¹⁴C]carboplatin to tumors were evaluated in rats using a one-way ANOVA (JMP; SAS Institute, Inc., Cary, NC).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Systemic Blood Pressure. Cereport produced a transient, dose-related decrease in blood pressure (Fig. 1). The hypotensive effects were maximal within 1 min and in all cases recovered to control levels within 3 min of beginning the Cereport infusion. The dose-response curve revealed that the hypotension observed with Cereport was not correlated with the shape of Cereport's dose-response curve for enhancing drug delivery to peripheral tumors. For example, although 0.1 and 0.2 µg/kg/min Cereport produced significant and equivalent effects on delivery of [¹⁴C]carboplatin (Emerich et al., 2001), the effects on blood pressure differed, for 0.1 µg/kg/min was without reliable effects, whereas 0.2 µg/kg/min produced a reliable mean decrease of about 10% from baseline. Moreover, although the 0.2-µg/kg/min dose significantly enhanced [¹⁴C]carboplatin delivery, its effects on blood pressure were equivalent to 0.5 µg/kg/min, which produced much less effect on delivery into tumor (Emerich et al., 2001). Finally, the greatest effect on blood pressure was seen with a Cereport dose of 1.0 µg/kg/min, and this dose is beyond the active range of the inverted U dose response for enhanced drug delivery (determined in initial pilot dose range test; data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Transient, dose-related effects of Cereport on blood pressure. The decrease in blood pressure was maximal within 1 min of the Cereport infusion and in all cases recovered to control levels within 3 min of the initiation of the Cereport infusion. These data indicate that 1) the hypotensive effects of Cereport are not closely related to its effects on drug delivery to solid tumors (because the dose-response data related to hypotension and drug delivery do not coincide), and 2) the emergence of hypotensive symptoms (e.g., at 0.2 and 0.5 µg/kg/min) identifies the portion of Cereport's nonmonotonic dose response where its efficacy in enhancing drug delivery to the tumor is just beginning to decline (i.e., between 0.2 and 0.5 µg/kg/min). Group sizes were saline (N = 27), Cereport 0.1 (N = 19), Cereport 0.2 (N = 27), Cereport 0.5 (N = 16), and Cereport 1.0 (N = 17). When considered with other data (see text), the hypotensive effects of Cereport are neither necessary nor sufficient to produce the increased delivery of drugs to solid tumors.

Regional Blood Flow and Autoradiography. Using iodinated antipyrine, in conjunction with quantitative autoradiography, a significant decrease in blood flow to the tumor was observed following Cereport infusions (Fig. 2). This effect began within the 1st min of the infusion and persisted throughout the duration of a 15-min Cereport infusion (decreased 86%, p < 0.001). Thus, although profound changes in blood flow were observed, its seems unlikely that these paradoxical decreases can directly account the increased drug delivery observed.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Significant decrease in blood flow to tumor induced by Cereport. These data are pooled from several time points (i.e., within 1 min of Cereport infusion through the end of the 15-min infusion) for both saline (N = 23) and Cereport-treated (N = 25) animals, which showed substantial, similar, and persistent decreases. ***p < 0.001 versus saline.

Effect of Cereport on Perfusion of Tumor Blood Vessels. The ability of bradykinin B₂ receptor stimulation to alter areas of hypoperfusion within the tumor was evaluated by injecting different fluorescent dyes before and following Cereport administration. This enabled direct comparisons to be made between the patency of vessels within the vascular bed of the tumor under Cereport versus vehicle conditions. Although a trend for decreased areas of perfusion was observed under saline conditions (decreased 24% relative to pretreatment), this effect did not reach statistical significance (p > 0.1). However, perfusion to subregions of the tumor was significantly decreased (66% relative to pretreatment, p < 0.001) during the Cereport infusion (Fig. 3). Thus, these studies did not support the hypothesis that stimulation of B₂ receptors enhances drug delivery to tumors by improving blood flow to regions of the tumor that are normally hypoperfused.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Cereport's effects on perfusion of vasculature within the tumor. The left-hand pair of columns demonstrates that an equivalent number of vessels per field was labeled when fluorescent dye was injected before treatment with either Cereport (N = 6) or saline (N = 7). The right-hand pair of columns depicts the vessels labeled immediately after a 10-min infusion of Cereport or saline. Following Cereport infusion, significantly fewer vessels were labeled, relative to saline. These data corroborate other data using iodinated antipyrine autoradiography that revealed a significant decrease in blood flow within the tumor during infusion of Cereport (Fig. 2). Together, these data indicate that improved blood flow to the tumor cannot be responsible for the increased uptake observed with Cereport. ***p < 0.001 versus pretreatment.

Effect of Cereport on Interstitial Fluid Pressure. Cereport produced a progressive decline in IFP in the tumor that began within 2 to 3 min of its initiation and continued throughout the infusion (Fig. 4). The reduced IFP was most robust within the most effective Cereport dose range (e.g., 0.10 µg/kg/min, p < 0.01), and was only marginal at the high end of the inverted U dose response, where Cereport's uptake effects are greatly diminished (e.g., 1.0 µg/kg/min, p > 0.1). Given the role that elevations in IFP are believed to play in limiting drug delivery to solid tumors, it seems probable that the reduction in IFP observed only within the active dose range of Cereport may provide one mechanism for its ability to enhance drug delivery into tumor.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effects of Cereport on IFP in solid peripheral tumors. Two dosing regimens were compared: 0.1 µg/kg/min for 10 min () (N = 43), which effectively enhances [14C]carboplatin delivery (Fig. 8) and 1.0 µg/kg/min for 5 min () (N = 35), which is at the extreme end of the inverted U dose response and does not reliably enhance delivery (data not shown). A third group received a 10-min saline infusion () (N = 16). IFP was continuously measured in the center of the tumor using the wick-in-needle method, beginning with a pre-Cereport baseline measurement and concluding with a postinfusion period. Note the progressive decline in tumor IFP with the effective dose of Cereport, but not higher, ineffective dose. These data support the hypothesis that one mechanism of Cereport's enhanced drug delivery to solid tumors involves a decline in IFP within the tumor.

Effect of Cereport on Transvascular Pore Size. The ability of Cereport to increase the pore size of the vasculature supplying solid peripheral tumors was evaluated by comparing the extravasation of different-sized fluorescent microspheres from the tumor vasculature to the tumor interstitium. As shown in Table 1, semiquantitative analysis revealed that the extravasation of two consecutive-sized microspheres (0.05 and 0.1 µm) was significantly increased (p < 0.05) by Cereport infusions. In contrast, no effect of Cereport was seen at either the smallest (0.025 µm) or largest (0.2 µm) diameter microspheres (p values >0.1). Collectively, these data indicate that one of the consequences of Cereport infusions is to increase the transvascular pore size of the blood vessels supplying solid peripheral tumors, thereby increasing the opportunity for diffusion of compounds into the tumor tissue.

Effect of Cereport on Tumor Blood Vessel Size. Using the endothelial cell-specific marker CD-31 to visualize individual blood vessels within the tumor, the circumference of vessels within the tumor was measured following saline and Cereport infusions (Figs. 5 and 6). As shown in Fig. 6A, Cereport infusions significantly increased the diameter and circumference of vessel size. This effect was manifest within 2 min (p < 0.001) (the earliest time point examined), peaked at about 5 min (p < 0.01), and persisted throughout the 8-min (p < 0.05) infusion. Thus, another likely mechanism by which Cereport enhances delivery to peripheral tumors is a transient increase in the surface area of the tumor vessels at the precise time the vessels contain high concentrations of drug within their luminal space.

View larger version (138K): [in this window] [in a new window]   Fig. 5.   Representative sections of tumor immunostained for the endothelial cell marker CD-31 to visualize blood vessels. A, low-power photomicrograph of a dense torturous network of vessels. Solid arrows in A and B depict cross-sections of blood vessels to illustrate examples used for quantitative determinations of vessel diameter. B and C, higher power photomicrographs of selected immunopositive vessels depicting cross-sectional areas. Staining was eliminated in sections processed in an identical manner except that the primary antibody was deleted. Bar, 50 µm for A and 20 µm for B and C.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   A, time-related effects of Cereport on vessel size within the tumor. Mean circumference of all vessels with a single section, computed by calculating the minimum and maximum diameter for each vessel. Note that very soon after the Cereport infusion, the size of the tumor vessels increases, reaching a peak at about 5 min. These data indicate that, in addition to decreasing the IFP of tumors, Cereport also increases the surface area of the tumor vasculature, providing an additional, complementary mechanism for Cereport's ability to increase delivery of drugs to solid tumors. Group sizes were 2 min (saline = 8, Cereport = 11), 5 min (saline = 6, Cereport = 6), and 8 min (saline = 6, Cereport = 6). ***p < 0.001 versus saline, **p < 0.01 versus saline, *p < 0.05 versus saline. B, replication of Cereport's effects on tumor vascular circumference and complete inhibition of effect by coadministration of the NO inhibitor L-NAME. These data link the increased NO activity associated with Cereport and the increased vessel size seen with Cereport administration, establishing that the former contributes to the latter, thus demonstrating that both are important mechanistic variables in Cereport's ability to enhance drug delivery to solid peripheral tumors. Groups sizes were eight per group. ***p < 0.001 versus saline.

Pharmacological Interactions between Bradykinin, Nitric Oxide, and Prostaglandins. As shown in Fig. 7, top, i.v. administration of the bradykinin B₂ agonist Cereport significantly enhanced the delivery of [¹⁴C]carboplatin to solid peripheral tumors. Using a dose previously shown to optimally enhance delivery (Emerich et al., 2001), levels of [¹⁴C]carboplatin were increased 140% (p < 0.01). To gain insight into the biochemical pathways involved with the increased drug delivery to peripheral tumors produced by Cereport infusions, a series of pharmacological studies was performed. An initial study revealed that the enhanced delivery produced by Cereport was completely suppressed by infusing the NO synthetase inhibitor L-NAME (Fig. 7, top). A second study replicated the enhanced delivery of [¹⁴C]carboplatin produced by Cereport, demonstrating a 164% increase relative to vehicle (p < 0.05). Administration of the cyclooxygenase inhibitor indomethacin produced a significant, but incomplete dose-related suppression of Cereport's effects (Fig. 7, bottom). Cereport plus indomethacin, at a dose of 0.4 µg/kg/min, increased [¹⁴C]carboplatin levels in tumor by 100% (64% less than the maximal effect obtained with Cereport alone). Increasing the dose of indomethacin (0.8 µg/kg/min) further dampened the effects of Cereport, with the delivery of [¹⁴C]carboplatin to the tumor being increased by only 59% (p > 0.05) relative to vehicle-infused animals. Administration of indomethacin alone, at either dose, did not affect [¹⁴C]carboplatin levels in tumor (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Top, Cereport's effects are completely blocked by pharmacologically inhibiting NO activity. Although Cereport enhanced delivery of [14C]carboplatin into MATB-III tumors, coadministration of the NO inhibitor L-NAME completely blocked Cereport's effects. These data demonstrate that the increased delivery to tumors produced by Cereport is mediated by NO. Group sizes were vehicle (N = 8), Cereport (N = 10), and Cereport + L-NAME (N = 9). Bottom, suppression of Cereport's effects by pharmacologically inhibiting phospholipase A2/prostaglandin signaling via the cyclooxygenase inhibitor indomethacin. Note the dose-related suppression of Cereport's delivery effects to the tumor. Neither dose of indomethacin, given alone, affected [14C]carboplatin levels in the tumor (data not shown). Higher indomethacin doses could not be tested due to significant hypotension. Group sizes were vehicle (N = 6), Cereport (N = 11), 0.4 mg/kg indomethacin (N = 8), and 0.8 mg/kg indomethacin (N = 11). These data reveal an important and contributing role of the phospholipaseA2/prostaglandin signaling pathway in the enhanced tumor delivery observed by bradykinin B2 receptor stimulation. Data are presented as mean ± S.E.M. nanocuries per gram of tissue. *p < 0.05 versus vehicle, **p < 0.01 versus vehicle.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Effect of the NO donor SNAP on [14C]carboplatin delivery to MATB-III tumors. Note that a modest trend toward increased concentrations is achieved with SNAP, but that even the highest dose failed to produce statistically significant effects (p > 0.05). It was not possible to test higher doses of SNAP, because even at the high dose tested, significant hypotension and other cardiovascular side effects were induced. Note that the best effect achieved with the maximum tolerated dose of SNAP is substantially less than that achieved with Cereport (Fig. 7, top). Group sizes were vehicle (N = 4) and SNAP (N = 7/group). Data are presented as mean ± S.E.M. nanocuries per gram of tissue.

View larger version (36K): [in this window] [in a new window]   Fig. 9.   Effect of SNAP and PGE2, alone or in combination, on delivery of [14C]carboplatin to peripheral tumors. Note that although neither agent alone produced significant effects, the two combined nearly doubled the levels of [14C]carboplatin in tumor, approaching the effects achieved by stimulation of the bradykinin B2 receptor. These data support the idea that both the NO and PL/PG signaling pathways are necessary and they work in tandem to achieve the activation of increased drug delivery to tumors seen with Cereport. Group sizes were vehicle (N = 10), 0.025 µg/kg/min SNAP (N = 10), 1.0 µg/kg/min PGE2 (N = 10), 0.01 SNAP + PGE2 (N = 10), and 0.025 SNAP + PGE2 (N = 10). Data presented are mean ± S.E.M. nanocuries per gram of tissue. *p < 0.05 versus saline.

Modulation of Cereport's Effects with PDE-V Inhibition. Consistent with the above-described studies, i.v. Cereport significantly (p < 0.01) enhanced delivery of [¹⁴C]carboplatin to MATB-III tumors. As before, this effect was maximal with 0.2 µg/kg/min Cereport with lesser but still significant (p < 0.05) effects at 0.05 and 0.5 µg/kg/min Cereport. Zaprinast (selective PDE-V inhibitor) given alone (data not shown) did not alter delivery of carboplatin to the tumor. Moreover, administration of zaprinast (20 mg/kg) did not alter the ability of Cereport to enhance delivery of [¹⁴C]carboplatin at any dose tested (Fig. 10).

View larger version (26K): [in this window] [in a new window]   Fig. 10.   Effect of i.v. zaprinast, a selective PDE-V competitive inhibitor, on Cereport's ability to enhance delivery of [14C]carboplatin to peripheral MATB-III tumors. Animals received infusions of saline or zaprinast concurrent with a range of Cereport doses. Note the nonmonotonic dose-response curve with maximal effects at 0.2 µg/kg/min Cereport and lesser effects with 0.05 and 0.5 µg/kg/min. Zaprinast alone (data not shown) did not impact delivery of [14C]carboplatin to the tumors. Zaprinast (20 mg/kg) combined with Cereport did not affect delivery of [14C]carboplatin, relative to Cereport alone, at any dose treated. Group sizes were vehicle (N = 21), 0.05 Cereport (N = 7), 0.05 Cereport plus zaprinast (N = 8), 0.2 Cereport (N = 10), 0.2 Cereport plus zaprinast (N = 8), 0.5 Cereport (N = 9), and 0.5 Cereport plus zaprinast (N = 8). Data are presented as mean ± S.E.M. nanocuries per gram of tissue. *p < 0.05 versus saline, **p < 0.01 versus saline.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The experiments described in this manuscript explored the physical and biochemical mechanisms that may be responsible for the novel finding that increased delivery of chemotherapeutic agents to solid peripheral tumors can be achieved by stimulating B₂ bradykinin receptors with the selective agonist Cereport. These experiments used a MATB-III cell line derived from F-344 rats that forms solid peripheral tumors with an infiltrating vasculature that bears considerable homology to the vasculature in human solid tumors [see Emerich et al. (2001) for a detailed discussion].

Consistent with the general vasoactive properties established for both bradykinin (Wahl et al., 1983; Unterberg et al., 1984; Yong et al., 1992) and Cereport (Elliott et al., 1996a,b), a number of vascular responses occurred during Cereport infusion. Three of these seem sufficiently linked to the increased drug delivery to the tumor to suggest important roles in the physical mechanism of action. First, a steady decrease in IFP occurred during the infusion of an effective concentration of Cereport (but not during infusion of a higher, ineffective concentration). Second, the diameter and surface area of the tumor vessels was markedly increased during the Cereport infusion. Importantly, just as inhibiting NO activity (with L-NAME) completely blocked Cereport's ability to enhance delivery to tumors, it also completely blocked Cereport's effect on tumor vessel size. Both the decrease in IFP and the increase in vessel surface area were empirically linked to the increased delivery afforded by Cereport, providing at least partial explanations for how Cereport achieves the enhanced delivery. Although the vasculature feeding many solid tumors is known to be leaky (Jain, 1987), increased fluid pressure within the tumor interstitium contributes to the inability of drugs to transit from the lumen of the tumor vessel to the interstitial fluid of the tumor (most likely by reducing convection currents in that direction) (Jain, 1990, 1991). By reducing IFP, Cereport shifts the pressure gradient between the tumor vasculature and tumor interstitium to favor enhanced flow from the lumen into the tumor. By simultaneously increasing vessel surface area, Cereport provides an even greater opportunity for drugs within the vascular lumen to flow to the tumor interstitium. Finally, evidence for increased pore size was also observed, which would further facilitate flow of agents from the vascular lumen to the tumor interstitium (Jain, 1989; Monsky et al., 1999).

Several other physiological effects of Cereport were observed but were not linked to the increased drug delivery to the tumors. For example, the well known hypotensive effects of Cereport and bradykinin were observed (Wahl et al., 1983; Unterberg et al., 1984; Yong et al., 1992; Elliott et al., 1996a,b), but could not have produced the enhanced delivery described here. First, the timing of the hypotensive effects were not linked to the enhanced delivery, because studies manipulating the timing of Cereport and carboplatin demonstrate that peak hypotension occurred at the worst time point for delivery (Emerich et al., 2001). Second, the dose-response curve for hypotension did not correlate with the respective shape of the dose-response curve for enhancing delivery. Finally, dropping blood pressure in the same temporal pattern and overall magnitude, as seen with Cereport, using the adenosine agonist NECA, did not improve drug delivery. Together, these mutually corroborating data suggest that hypotension is neither necessary nor sufficient for Cereport to enhance delivery to tumors and it therefore does not contribute, in any simple way, to this phenomenon.

Interestingly, and in contrast to what might have been expected, Cereport also caused a dramatic decrease in blood flow to the tumor and a corresponding increase in hypoperfusion within the tumor (i.e., increase in the number of nonperfused vessels). Thus, the increased drug delivery to the tumor occurs despite changes in tumor perfusion that, if they were to occur in isolation, would likely impede delivery. These observations provide a likely explanation for the need to administer the chemotherapeutic agent before initiating the Cereport infusion. That is, the concentration of the chemotherapeutic agent within the tumor vessel must be elevated prior to the onset of the physiological changes induced by Cereport (Emerich et al., 2001). Otherwise, the decreased perfusion of the tumor caused by Cereport would limit, rather than enhance, delivery. For this reason, as long as chemotherapeutic drug concentrations within the lumen of the tumor vessels are sufficiently high before the Cereport infusion, the decrease in blood flow to the tumor does not obviate the benefits induced by the reduction in IFP, increase in vessel surface area and increase in pore size.

The mechanistic studies demonstrated that Cereport achieves increased drug delivery by activating NO and phospholipase A₂/prostaglandin E₂ (PL/PG) pathways as essential, synergistically acting intracellular signaling events. Although each of these pathways is known to be stimulated by bradykinin (Burch et al., 1993), the fact that both had to be activated to achieve the enhanced delivery to tumors reported here was not expected. It is well established that both NO and PL/PG are common signaling pathways used by bradykinin receptor systems (Burch et al., 1993). Our pharmacological manipulations demonstrated that inhibiting NO synthetase completely blocked Cereport's ability to enhance drug delivery to the tumor, indicating that this pathway is necessary for the phenomenon reported here. Similarly, blocking PL/PG activity substantially reduced Cereport's effects (from >150% increase to approximately 50%), although the emergence of confounding hypotensive side effects limited our ability to fully test the involvement of this pathway in the tumor delivery phenomenon. Nonetheless, these data do suggest important roles for the PL/PG pathway as well. The fundamentally important role of these biochemical pathways was further highlighted by additional pharmacological studies. Stimulating NO activity with SNAP produced modest, but nonsignificant trends toward enhanced delivery, whereas administration of exogenous PGE produced no measurable effects on drug delivery. However, simultaneous stimulation of both pathways produced a significant, nearly 2-fold increase of carboplatin levels in the tumor, clearly supporting an important role of both of these bradykinin-linked pathways, working in concert.

The fact that simultaneous, pharmacological stimulation of these two pathways did not achieve the same 3-fold effect achieved by Cereport stimulation of the B₂ receptor might be explained in a least three different (but not mutually exclusive) ways: 1) the dose combination and temporal parameters of SNAP and PGE required to maximize the response may not have been optimized, 2) flooding the vascular lumen with NO donors and PGE may never be able to produce as great a response as that which occurs naturally following receptor stimulation and subsequent natural activation of these two signaling pathways, and 3) another, still-undefined signaling pathway may participate in the phenomenon. Nonetheless, the data reported here clearly establish that both pathways are necessary and that neither is sufficient by itself to achieve the effects reported here.

As briefly discussed (Emerich et al., 2001) the increased delivery to peripheral tumors achieved with Cereport and that previously reported for improved drug delivery to brain tumors differ in several phenomenological ways, including the dose level, the shape of the dose-response function, and the optimal dosing protocol required to achieve the desired effect. A number of apparent mechanistic differences are also apparent, for, in contrast to the findings presented here, no evidence of reduced cerebral blood flow or changes in intracranial tumor pressure has been seen in studies with brain tumors following Cereport infusion. Moreover, although a disengagement of the tight junctions comprising the blood-brain tumor barrier has been implicated as playing a major role in Cereport's effects on brain tumors (Sanovich et al., 1995), an analogous effect on vascular pore size (in the present studies) plays a less exclusive role. Finally, although both the increased delivery to brain tumors and solid, peripheral tumors involves activation of bradykinin B₂ receptors and NO as an intracellular second messenger, even here an interesting difference was revealed. In prior studies with brain tumors, enhancing the duration of the NO-linked second messenger cGMP (via the PDE-V inhibitor zaprinast) potentiated the effects of both bradykinin (Sugita and Black, 1998) and Cereport (Dean et al., 1999). However, in the present studies, zaprinast neither enhanced the effects of an optimal dose nor a range of suboptimal doses of Cereport. This suggests that, in contrast to brain tumors, the NO-induction of cGMP in solid peripheral tumors that occurs following B₂ stimulation may achieve near-optimal levels and therefore does not benefit from a pharmacologically aided extension of second messenger duration. Thus, although a number of similarities are shared between Cereport's effects on enhancing delivery to brain tumors and peripheral, solid tumors, several interesting differences exist as well.

In summary, the data presented offer new and detailed information regarding the novel finding that stimulation of bradykinin B₂ receptors can increase delivery of chemotherapeutics to solid, peripheral tumors. This phenomenon appears to require the participation of two parallel bradykinin signaling pathways working in tandem. Additionally, among the many hemodynamic changes observed, a decrease in interstitial fluid pressure, an increase in vascular surface area, and an increase in vascular pore size all appear to contribute to the end effect. On the other hand, significant decreases in systemic blood pressure, tumor blood flow, and increases in areas of hypoperfusion within the tumor are unlikely participants in the phenomenon. Further studies might next determine why the bradykinin-mediated responses are more robust and consistent in tumor vasculature (e.g., higher density of receptors, more efficient signaling pathways, etc.) relative to healthy, nontumor vasculature.