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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Res. Author manuscript; available in PMC 2012 April 1.
Published in final edited form as:
PMCID: PMC3070759
NIHMSID: NIHMS272699

Anti-angiogenesis Enhances Intratumoral Drug Retention

Abstract

The tumor vasculature delivers nutrients, oxygen, and therapeutic agents to tumor cells. Unfortunately, the delivery of anti-cancer drugs by tumor blood vessels is often inefficient and can constitute an important barrier for cancer treatment. This barrier can sometimes be circumvented by anti-angiogenesis-induced normalization of tumor vasculature. However, such normalizing effects are transient; moreover, they are not always achieved, as shown here, when 9L gliosarcoma xenografts were treated over a range of doses with the VEGF receptor-selective tyrosine kinase inhibitors axitinib and AG-028262. The suppression of tumor blood perfusion by anti-angiogenesis agents can be turned to therapeutic advantage, however, through their effects on tumor drug retention. In 9L tumors expressing the cyclophosphamide-activating enzyme P450 2B11, neoadjuvant axitinib treatment combined with intratumoral cyclophosphamide administration significantly increased tumor retention of cyclophosphamide and its active metabolite, 4-hydroxycyclophosphamide. Similar increases were achieved using other angiogenesis inhibitors, indicating that increased drug retention is a general response to anti-angiogenesis. This approach can be extended to include systemic delivery of an anti-cancer prodrug that is activated intratumorally, where anti-angiogenesis-enhanced retention of the therapeutic metabolite counterbalances the decrease in drug uptake from systemic circulation, as exemplified for cyclophosphamide. Importantly, the increase in intratumoral drug retention induced by neoadjuvant anti-angiogenic drug treatment is shown to increase tumor cell killing and substantially enhance therapeutic activity in vivo. Thus, anti-angiogenics can be used to increase tumor drug exposure and improve therapeutic activity following intratumoral drug administration, or following systemic drug administration in the case of a therapeutic agent that is activated intratumorally.

Keywords: anti-angiogenesis, cancer therapy, drug retention, drug efflux

Introduction

The tumor vasculature is often characterized by low vascularity, poor organization and abnormal morphology, which results in inefficient transport of oxygen and therapeutic agents into tumors and constitutes a substantial barrier to effective cancer treatment (1). Extensive efforts have been made to improve tumor drug uptake by developing new delivery vehicles or increasing tumor vascular patency (2, 3). The growth and expansion of many tumors is associated with pathological angiogenesis stimulated by vascular endothelial growth factor (VEGF), which is a validated therapeutic target for cancer treatment. Bevacizumab, a neutralizing antibody for human VEGF, is an anti-angiogenic drug that increases the survival of patients with metastatic colorectal cancer and non-small cell lung cancer when given in combination with conventional chemotherapy (4, 5). Small-molecule receptor tyrosine kinase inhibitors (RTKIs) that inhibit VEGF receptors (VEGFR) can also be used to induce anti-angiogenesis. However, despite efficacy observed in clinical trials with several such RTKIs (6, 7), recent phase III trials showed very limited benefits when anti-angiogenic RTKIs were combined with conventional chemotherapy (810). These observations raise the question as to how anti-angiogenesis treatments affect tumor uptake of other chemotherapeutic agents and the overall anti-tumor activity of combination therapies.

Morphological normalization of surviving tumor blood vessels has been observed following treatment with a variety of anti-angiogenic drugs (11). It has been proposed that anti-angiogenesis may transiently normalize the tumor vasculature, leading to an increase in tumor drug uptake (12). Like the anti-angiogenic antibodies bevacizumab and DC101, the VEGF receptor-selective RTKI axitinib (13) induces morphological normalization of the tumor vasculature and can increase the transport efficiency of individual blood vessels that survive anti-angiogenesis treatment (14, 15). However, the total number of surviving blood vessels decrease and overall tumor blood perfusion is not improved by axitinib treatment, as indicated by the increase in tumor hypoxia and the decrease in tumor uptake of small molecules, including 4-OH-CPA, the active metabolite of anti-cancer prodrug cyclophosphamide (CPA) (14, 16). This lack of improved tumor vascular functionality (“normalization”) by axitinib and certain other anti-angiogenic drugs (1720) could be the result of doses that are optimized for maximal angiogenesis inhibition with acceptable host toxicity but are not optimal for vascular normalization. Supporting this possibility, increases in chemotherapeutic drug uptake are seen in some tumor models at low but not standard doses of the anti-angiogenic agent sunitinib (21, 22), and improved anti-tumor responses are reported when bevacizumab is given to patients at low dose, but not high dose, in combination with conventional chemotherapy (23). An alternative reason why certain VEGF receptor RTKIs might not induce normalization may due to their co-inhibition of PDGFR-β, which promotes the close association between pericytes and endothelial cells that characterizes normal blood vessels (24), thereby destabilizing tumor blood vessels in a way that interferes with tumor vascular normalization induced by VEGF deprivation. These two possibilities are examined in the present study, where the impact of anti-angiogenesis on tumor drug uptake is investigated over a range of doses for both axitinib and AG-028262, an anti-angiogenic RTKI whose specificity for VEGFR-2 as compared to PDGFR-β is ~50-fold greater than that of axitinib (25).

As blood flow to the tumor decreases in response to anti-angiogenesis, blood flow out of the tumor may also decrease. This could potentially increase the retention of therapeutic agents in tumors. Furthermore, for drugs that successfully extravasate into the tumor extracellular matrix, a decrease in tumor interstitial fluid pressure following anti-angiogenesis treatment may slow the leakage of drugs from the tumor periphery to the peritumoral space and could also lead to longer tumor drug retention. However, the inhibition of VEGF signaling reduces the permeability of blood vessels and can adversely affect the extravasation of drugs after they are delivered into the tumor through blood circulation (26). Thus, even if anti-angiogenesis increase the retention of therapeutic agents in tumor vasculature, it is uncertain whether this can be translated into increased interstitial drug concentration and higher tumor cell drug exposure. These questions are presently investigated in a 9L gliosarcoma model that expresses cytochrome P450 2B11, which converts the anticancer prodrug CPA to its active, 4-hydroxy metabolite (27). Our findings demonstrate that pretreatment of P450 2B11-expressing tumors with angiogenesis inhibitors significantly increases tumor retention of CPA, as well as 4-OH-CPA, following intratumoral CPA delivery, leading to increases in tumor cell apoptosis and anti-tumor activity. Furthermore, we show that for a prodrug that can be administered systemically and activated intratumorally, as exemplified by CPA, the decrease in tumor drug uptake following angiogenesis inhibition can be fully reversed by the tumor drug retention effect induced by the same anti-angiogenesis treatment.

Materials and Methods

Additional details on chemicals and analytical procedures, including drug treatments, 4-OH-CPA and CPA analysis, pharmacokinetic data analysis, endothelial cell chemosensitivity to 4-OH-CPA, and analysis of CPA-induced apoptosis are provided in Supplementary Materials

Tumor cell lines and xenograft models

9L rat gliosarcoma cells infected with a retroviral vector encoding P450 2B11 in combination with P450 reductase (9L/2B11 cells), and 9L tumor cells infected with the empty retroviral vector pBabe (9L cells) were described previously (28). Cells were grown in DMEM culture medium containing 10% FBS at 37°C in a humidified, 5% CO2 atmosphere. Immune-deficient male Fox Chase ICR scid mice were purchased from Taconic, Inc. (Germantown, NY) and housed in the Boston University Laboratory of Animal Care Facility in accordance with approved protocols and federal guidelines. Autoclaved cages containing food and water were changed once a week. Mouse body weight was monitored every 3–4 days. On the day of tumor cell inoculation, 9L or 9L/2B11 cells at 70–80% confluence were trypsinized and resuspended in FBS-free DMEM medium. Cells (4 × 106) in a volume of 0.2 ml were injected s.c. into each flank of a 6-wk-old male scid mouse. Tumor sizes were measured every 3–4 days using digital calipers (VWR International, West Chester, PA) and volumes were calculated as (3.14/6) × (L × W)3/2.

Tumor growth delay study

Mice bearing bilateral 9L/2B11 tumors were randomized on the day of initial drug treatment, when the average tumor volume reached 800–900 mm3 (5–8 mice per group), and treated as follows: 1) vehicle (5 μl per g body weight, i.p., sid for 4 days) followed by 120 μl PBS (3 intratumoral injections per tumor at 20 μl per injection and 2 tumors/mouse, sid for 2 days) beginning 24 hr after the last vehicle injection; 2) axitinib (25 mg/kg body weight, i.p., sid for 4 days); 3) CPA (150 mg CPA/kg body weight, 3 intratumoral injections per tumor at ~ 20 μl per injection and 2 tumors/mouse, sid for 2 days); 4) CPA, followed by axitinib given 24 hr after the second CPA injection; and 5) axitinib, followed by CPA given 24 hr after the last axitinib injection. For groups 1 and 2, the mice were terminated when the tumor volume reached the limit set by institutional guidelines. For groups 3, 4, and 5, drug treatments were repeated every 21 days for 2 cycles and the mice were subsequently maintained drug-free. Tumor volume and mouse body weight were monitored throughout the study.

Statistical analysis

Results are expressed as mean ± SE based on the indicated number of tumors or tissue samples per group. Statistical significance of differences was assessed by two-tailed Student’s t test using GraphPad Prism software, and is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001. All drug-treated samples were compared to controls, unless indicated otherwise.

Results

VEGFR-selective inhibitors suppress 9L tumor uptake of CPA and 4-OH-CPA without vascular normalization

The anti-cancer prodrug CPA is activated by hepatic P450 enzymes and then delivered to the tumor via the bloodstream (29). Axitinib treatment for 4 days (25 mg/kg/day, i.p.) significantly reduced the number of blood vessels in 9L tumors grown subcutanenously in scid mice (Fig 1A), consistent with our earlier findings (14). Axitinib also decreased tumor uptake of CPA and of liver-derived 4-OH-CPA when assayed at the Tmax, 15 min after i.p. injection of CPA (Figs. 1B, 1C), indicating that axitinib suppresses tumor vascular patency, in agreement with earlier studies (14). To ascertain whether lower doses of axitinib might induce functional normalization of tumor blood vessels leading to an increase in tumor drug uptake, mice were treated with axitinib at doses ranging from 2 to 25 mg/kg (p.o., bid). No axitinib-dependent increase in tumor uptake of 4-OH-CPA was seen at any of the doses tested. A dose of 2 mg/kg axitinib was without effect, while 5 mg/kg axitinib resulted in a maximal reduction in the intratumoral 4-OH-CPA concentration (Fig. 1D). Next, we investigated whether an increase in tumor vascular patency and drug uptake could be achieved using AG-208262, a more selective VEGFR-2 RTKI than axitinib (25). AG-028262 induced a progressive, dose-dependent decrease in intratumoral 4-OH-CPA over the full dose range tested (Fig. 1D). At 40 mg/kg, AG-208262 suppressed tumor 4-OH-CPA level by 48%, consistent with its strong anti-angiogenic activity. Similar decreases in tumor uptake of CPA (Supplementary Fig. S1) and 4-OH-CPA (30) were seen in PC-3 tumors pretreated with axitinib at 25 mg/kg/day for times ranging from 0.5 to 6 days. Thus, VEGFR inhibition by either axitinib or AG-208262 is not associated with functional normalization of the tumor vasculature.

Figure 1
Axitinib and AG-028262 inhibit tumor uptake of CPA and 4-OH-CPA

Anti-angiogenesis increases exposure of 9L/2B11 tumors to CPA and 4-OH-CPA by increasing drug retention

Next, we investigated whether anti-angiogenic drugs might be used to increase intratumoral drug retention. CPA was injected intratumorally into 9L/2B11 tumors, where CYP2B11, a cytochrome P450 enzyme, catalyzes intratumoral metabolism of CPA to its active metabolite, 4-OH-CPA. CPA that exits the tumor intact can be converted by liver P450 enzymes to 4-OH-CPA, a portion of which may then re-enter the tumor (27). Five min after CPA injection, intratumoral CPA levels were 580 μM in untreated tumors as compared to 1840 μM in axitinib-pretreated tumors (Fig. 2A). However, the initial tumor level of 4-OH-CPA was not significantly higher in the axitinib-pretreated tumors (Fig. 2B), indicating that axitinib has little impact on the intrinsic rate of intratumoral CPA 4-hydroxylation, despite the 3.2-fold increase in initial intratumoral CPA concentration. This finding indicates that CYP2B11 is already saturated at the concentration of 580 μM CPA reached when CPA is injected i.t. without prior axitinib treatment (c.f., Km (CPA) = 70 μM in 9L/2B11 cells (28)). As the intratumoral CPA levels declined with time due to a combination of drug efflux and intratumoral conversion to 4-OH-CPA, we observed an increase in overall intratumoral 4-OH-CPA exposure with axitinib treatment (Fig. 2B). Total intratumoral exposure to CPA, and 4-OH-CPA was 76–77% higher in axitinib-pretreated mice, as indicated by area under the curve (AUC), (Table 1). This finding is consistent with axitinib slowing drug efflux from the tumors. After 30 min, intratumoral 4-OH-CPA levels declined both with and without axitinib-pretreatment, reflecting depletion of the intratumoral pool of CPA available for metabolism to 4-OH-CPA. The Cmax of tumor exposure to 4-OH-CPA was also increased with axitinib pretreatment, by 42% (Table 1B). CPA and 4-OH-CPA levels were substantially lower in plasma and liver than in the tumor (Fig. 2C–2F), as is expected for an intratumoral route of drug delivery. Moreover, at the 5 min time point, plasma and liver 4-OH-CPA levels were significantly lower in axitinib-pretreated mice than in controls (p < 0.05), indicating that the greater retention of CPA and 4-OH-CPA by the axitinib-pretreated tumor renders CPA less available for hepatic metabolism and also reduces the efflux of tumor-derived 4-OH-CPA during this initial time period. By 30 min, however, plasma and liver levels of CPA and 4-OH-CPA were higher in the axitinib-pretreated mice, reflecting the delay in drug release from the tumors.

Figure 2
Impact of axitinib on CPA and 4-OH-CPA pharmacokinetics following intratumoral CPA injection
Table 1
Axitinib pretreatment increases tumor retention of CPA and 4-OH-CPA

To confirm the role of intratumoral P450 metabolism in the observed retention of 4-OH-CPA, we used mice bearing wild-type 9L tumors to investigate the impact of axitinib pretreatment on the intratumoral pharmacokinetics of CPA and 4-OH- following i.t. CPA injection. Wild-type 9L tumors do not express significant levels of P450 enzymes and cannot metabolize CPA to 4-OH-CPA. As anticipated, we observed higher levels of intratumoral CPA in the axitinib-pretreated tumors at both 6 and 15 min, reflecting increased tumor drug retention (Fig. 2G), however, intratumoral 4-OH-CPA levels were lower through 30 min, reflecting the inhibition of tumor uptake of 4-OH-CPA formed in the liver (Fig. 2H).

Two other anti-angiogenic drugs, AG-028262 and SU5416, were investigated for their effects on intratumoral drug retention in mice bearing 9L/2B11 tumors (Fig. 3). Four days of pretreatment with AG-028262 or SU5416 had no significant effect on intratumoral CPA or 4-OH-CPA levels 15 min after intratumoral CPA injection, although a trend of increasing drug retention was apparent for CPA (Fig. 3A). However, 30 min after CPA injection, when intratumoral CPA concentrations decreased well below the Km for metabolism by CYP2B11 (c.f., Fig. 2A), both anti-angiogenic drugs significantly increased intratumoral 4-OH-CPA compared to controls (p < 0.05) (Fig. 3B). Thus, prolonged tumor drug retention may represent a common response to anti-angiogenesis treatments involving these RTKIs.

Figure 3
Impact of other anti-angiogenic drugs on tumor drug retention following intratumoral CPA administration

Although axitinib decreased tumor levels of both CPA and 4-OH-CPA in mice bearing wild-type 9L tumors following i.p. CPA administration (Fig. 4A and 4B, left set of bars; also see Fig. 1B, 1C) (11, 14), axitinib effected no such decrease in tumor 4-OH-CPA levels in mice bearing 9L/2B11 tumors either 15 min or 30 min after i.p. injection of CPA (Fig. 4B). This can be explained by the increased retention of CPA that enters the axitinib-pretreated tumors, which makes CPA more available for metabolism by the tumor cell-expressed P450 2B11 enzyme, and by the increased retention of tumor cell-derived 4-OH-CPA. This increased retention of CPA and 4-OH-CPA in part compensates for the decreased tumor uptake 4-OH-CPA that is formed in the liver. Thus, anti-angiogenesis not only increases the exposure of tumor cells to drugs delivered intratumorally, but can also increase intratumoral metabolism, and exposure, to the activated form of a prodrug that is administered systemically.

Figure 4
Impact of axitinib on tumor drug retention following systemic CPA administration

In control experiments, we observed that the CPA 4-hydroxylase activity of 9L/2B11 tumor-derived microsomes was not altered by axitinib pretreatment (Supplementary Fig. S2). Hepatic cytochrome P450-catalyzed CPA 4-hydroxylation is also unaffected by axitinib treatment (14). Thus, the increase in intratumoral concentration of 4-OH-CPA seen following axitinib treatment cannot be explained by an increase in the intrinsic CPA 4-hydroxylase activity of either the liver or tumor.

Axitinib enhances CPA-induced tumor cell apoptosis

The impact of the enhanced drug retention following axitinib treatment on tumor cell apoptosis was investigated by measuring caspase activity in tumor samples excised 24 hr after the last drug treatment. Basal 9L/2B11 tumor caspase activity was unaffected by axitinib treatment (Fig. 5A). Intratumoral administration of CPA (50 mg/kg, two injections 24 hr apart) significantly increased caspase activity. However, the highest caspase activity was observed when mice bearing the intratumoral CPA-treated 9L/2B11 tumors were pretreated with axitinib (Fig. 5A). Thus, the prolonged exposure of tumor cells to 4-OH-CPA translates into a significant increase in tumor cell apoptosis. Axitinib had no effect on the intrinsic sensitivity of endothelial cells to activated CPA, as determined using cultured HUVEC cells (Fig. 5B).

Figure 5
Effects of axitinib on CPA-induced tumor cell apoptosis and endothelial cell chemosensitivity

Axitinib enhances the anti-tumor activity of intratumoral CPA injection

The therapeutic impact of axitinib-enhanced tumor drug retention was investigated in a tumor growth delay study. Four days of axitinib treatment resulted in a transient (~3 day) delay in 9L/2B11 tumor growth, whereas maximum tolerated dose-scheduled CPA (2 intratumoral CPA administration spaced 24 hr apart) resulted in substantially longer tumor growth delay (Fig. 6A; Table 2). However, robust tumor growth eventually resumed, even when each cycle of CPA treatment was followed by axitinib treatment (CPA/axitinib schedule). In contrast, when axitinib was administered for 4 days prior to intratumoral CPA injection (axitinib/CPA schedule), i.e., conditions under which axitinib increases drug retention and intratumoral exposure to 4-OH-CPA, tumor growth stasis was sustained for at least 22 days after the last cycle of drug treatment (Fig. 6A) (p < 0.05, one-way ANOVA, day 0 to day 49 for axitinib/CPA vs. CPA/axitinib schedule). Similar body weight profiles were observed for the CPA monotherapy and for both axitinib-CPA schedules (Fig. 6B), indicating that no additional toxicities were associated with the axitinib/CPA schedule. A small body weight loss occurred after each CPA treatment, as is typical for this cytotoxic drug. Thus, the extended drug retention associated with neoadjuvant axitinib treatment significantly enhances CPA anti-tumor activity.

Figure 6
Impact of neoadjuvant axitinib treatment on anti-tumor activity of intratumoral CPA treatment
Table 2
Effect of drug schedule on tumor doubling time

Discussion

In the present study, we investigated how VEGF receptor-targeted anti-angiogenic agents modulate chemotherapeutic drug delivery and drug retention by the tumor. Anti-angiogenesis was found to decrease tumor uptake of the anti-cancer prodrug CPA and its active metabolite, 4-OH-CPA, consistent with the established requirement for a functional tumor vasculature and blood flow for effective drug delivery. However, in mice bearing tumors that express the CPA-activating cytochrome P450 enzyme CYP2B11, neoadjuvant axitininb treatment significantly increased the AUC of intratumoral 4-OH-CPA exposure following intratumoral CPA administration, leading to an increase in tumor cell apoptosis and a major increase in anti-tumor activity, as seen in tumor growth delay studies. Increased tumor drug retention was also seen with two other anti-angiogenic agents, indicating that this effect is a general response to tumor anti-angiogenesis. Importantly, the increases in therapeutic activity were achieved despite the transient nature of the tumor drug retention effect of neoadjuvant axitinib treatment, and they cannot be explained by an increase in tumor cell or endothelial cell chemosensitivity following axitinib treatment (14). Furthermore, no increase in anti-tumor activity was seen when CPA treatment preceded axitinib administration, consistent with the proposed mechanism for the improved therapeutic response, namely, anti-angiogenesis-dependent drug retention leading to increased tumor cell exposure to 4-OH-CPA. Finally, when CPA was administered systemically and activated intratumorally, the decreases in tumor uptake of both CPA and 4-OH-CPA due to anti-angiogenesis were counter-balanced, and fully compensated for, by anti-angiogenesis-induced drug retention. Thus, when a tumor-activated prodrug is administered systemically, anti-angiogenesis-induced drug retention can counteract the decrease in drug uptake while at the same time retaining the therapeutic benefits of anti-angiogenesis-induced tumor cell starvation.

Anti-angiogenesis induces morphological normalization of the tumor vasculature, which involves pruning of immature blood vessels, a decrease in blood vessel tortuosity and dilation, and a closer association between pericytes and tumor endothelial cells (12). This can lead to functional improvements, as shown by the increases in tumor vascular patency and drug uptake and decreases in tumor hypoxia reported in preclinical studies with several anti-angiogenic agents (31). However, these effects are short lived and they disappear with continued anti-angiogenic drug treatment (32, 33). Moreover, for axitinib (15, 34) and certain other anti-angiogenic drugs (17, 35, 36), although morphological normalization of the tumor vasculature and improved functionality of individual blood vessels may occur, overall tumor vascular patency and capacity for drug uptake actually decrease. Presently, we investigated the hypothesis that tumor blood vessel normalization leading to increased drug uptake can be achieved by reducing the dose of the anti-angiogenic RTKI, however, we obtained no evidence for such functional normalization of tumor vasculature over a >10-fold dose range of either axitinib or AG-028262. Moreover, our studies with AG-028262 rule out cross-inhibition with PDGFR-β as a reason for the absence of normalization, given the high selectivity of this RTKI for VEGFR inhibition (25).

The absence of tumor vessel normalization in these preclinical studies is consistent with the limited therapeutic benefit reported in several phase III clinical trials combining anti-angiogenic agents with conventional chemotherapies (810). The poor performance of these combination therapies indicates a need to consider new approaches, such as the anti-angiogenesis-induced tumor drug retention approach reported here. In particular, for cytotoxic agents that can be delivered or activated intratumorally, persistent angiogenesis inhibition may further increase drug retention and therapeutic activity. Other approaches may include optimization of the timing and sequencing of anti-angiogenic agents given in combination with cytotoxic drugs, as discussed elsewhere (11). For anti-angiogenic agents that show limited activity in a monotherapy setting and that transiently induce functional normalization of the tumor vasculature, intermittent neoadjuvant anti-angiogenesis prior to each cycle of cytotoxic drug administration may increase tumor drug uptake. However, for combination therapies that include potent anti-angiogenic drugs that do not induce tumor vascular normalization, a brief period of anti-angiogenic drug treatment at a reduced dose, or temporary dilation of tumor blood vessels (37) prior to chemotherapy administration may be required to minimize the negative impact of angiogenesis inhibition on tumor drug uptake.

Tumor uptake of 4-OH-CPA, the active metabolite of CPA, was inhibited by axitinib and AG-028262, which reduce tumor blood perfusion and total vascular volume and decrease the number of patent tumor blood vessels (16, 34). These responses not only decrease chemotherapeutic drug uptake, but as shown here, they also decrease the rate at which drug molecules exit from the tumor, resulting in prolonged drug retention and increased tumor drug exposure. In the case of 9L/2B11 tumors given a single intratumoral injection of CPA, axitinib increased tumor cell exposure to CPA, and to 4-OH-CPA almost 2-fold, as judged by AUC values. As axitinib also decreases tumor uptake of liver-derived 4-OH-CPA, with no change in the intrinsic CPA 4-hydroxylase activity of 9L/2B11 tumors, these increases in tumor drug exposure likely underestimate the extent to which axitinib inhibits tumor efflux of 4-OH-CPA per se. Indeed, axitinib decreased the initial rate of tumor efflux of CPA by ~3-fold, as judged from the 3.2-fold higher residual intratumoral CPA concentration determined 5 min after drug injection (1840 vs 580 μM). P450 2B11 has a Km (CPA) of 70 μM in 9L/2B11 cells (28), indicating that tumor cell capacity for CPA 4-hydroxylation (drug activation) is saturated during the initial 15–30 min period after intratumoral CPA injection. Thus, the increase in tumor cell apoptosis and therapeutic activity seen in our experiments may very well underestimate the drug retention effect of anti-angiogenesis. A larger increase in therapeutic activity can therefore be anticipated for other, direct-acting drugs, or in the case of CPA, for tumors that express a prodrug activation enzyme less efficient than CYP2B11.

There are several important limitations of the anti-angiogenesis-induced tumor drug retention effect reported here. First, anti-angiogenesis slows down but does not completely block tumor drug efflux. Moreover, drug retention becomes less prominent when baseline intratumoral drug concentrations are very low. Presumably, the residual efflux capability of the tumor vasculature is sufficient for export when the chemotherapeutic drug is present at low concentrations. Most important, the same anti-angiogenic mechanisms that increase tumor drug retention also decrease chemotherapeutic drug uptake by the tumor. Thus, in order to take advantage of the increase in tumor drug retention and translate it into a meaningful therapeutic benefit, it needs to be utilized in a way that overcomes or circumvents the associated decrease in drug uptake from systemic circulation. This can be achieved by combining neoadjuvant anti-angiogenic treatment with direct delivery of a chemotherapeutic drug into target tissues. While intratumoral drug delivery is not suitable for all solid tumors and may impose certain practical limitations, it has been used in the clinic for treatment of head and neck cancer, lung cancer, and breast cancer (3840) and can also be used in cases where tumors are not amenable to resection or as an adjuvant following tumor resection (41, 42). The benefits of anti-angiogenesis induced tumor drug retention can also be realized in combination therapies involving systemic administration of a tumor-activated prodrug. This approach was exemplified for the P450 prodrug CPA in mice bearing tumors that express CYP2B11, where the axitinib-dependent decrease in tumor uptake of CPA and 4-OH-CPA from systemic circulation was fully compensated by the increase in tumor retention of 4-OH-CPA generated intratumorally. This drug retention effect helps explain our earlier finding that maximal anti-tumor activity is achieved in mice bearing 9L/2B11 tumors when systemic CPA administration is preceded by axitinib treatment (30). Finally, the potential limitations imposed by the subcutaneous tumor xenografts model used here should be noted. The tumor microenvironment can have a significant impact on angiogenesis (43), and malignant gliomas grown orthotopically may be more hypoxic and less highly vascularized than the subcutaneous 9L tumors used in our studies (14, 44). These differences in tumor microenvironment may impact drug uptake as well as the extent to which anti-angiogenesis increases overall drug exposure via the drug retention effect described here.

The principle of anti-angiogenesis-induced tumor drug retention presented here may be applied to systemic treatments based on other therapeutic agents that can be activated intratumorally, including other P450 prodrugs (29), prodrugs activated by other enzymes (45), and bioreductive drugs, which are activated within hypoxic tumor regions (46). Anti-angiogenesis-induced tumor drug retention may also be extended to increase the retention of tumor cell replicating, oncolytic viral vectors (47) as well as tumor-targeted nanoparticles (48). As tumor-specific delivery of nanoparticles in part depends on the enhanced permeability and leakiness of the tumor vasculature (48), the net impact of anti-angiogenesis on tumor vascular permeability and drug retention is uncertain and will require further study. Increased tumor drug retention can also be expected for agents that transiently normalize tumor vasculature, once they ultimately decrease tumor vascular patency with continued use, and for vascular disrupting agents, which induce an acute interruption of tumor blood perfusion (49). The latter possibility is supported by the increased tumor exposure to the alkylating agent melphalan following pretreatment with the vascular disruption agent 5,6-dimethylxanthenone-4-acetic acid (50), and by the increase in activity when doxorubicin was combined with the vascular disruption agent ICT2588, where maximal anti-tumor activity was achieved when doxorubicin was administered after the collapse of the tumor vasculature induced by ICT2558 (51).

In conclusion, anti-angiogenesis-induced tumor drug retention is an intrinsic action of anti-angiogenic drugs and can be applied to a variety of anti-angiogenesis treatments. This drug retention effect was employed to significantly increase the therapeutic activity of CPA treatment in a 9L xenograft model, and similar benefits can be expected with other tumor types. An even more pronounced drug retention effect can be anticipated for chronic anti-angiogenesis treatment, when the decrease in functional tumor vasculature becomes more substantial, and perhaps for vascular disrupting agents as well. These findings provide a novel perspective and insight into the complex pharmacokinetic and pharmacodynamic effects and interactions between anti-angiogenic drugs and chemotherapeutic agents and may stimulate further research to take advantage of these findings in a way that may circumvent the decrease in drug uptake that is also intrinsic to anti-angiogenic therapies. Finally, certain normal tissues are also sensitive to VEGF/VEGFR inhibition (25, 52), indicating that the vasculature in these tissues may also be targeted for anti-angiogenesis-induced drug retention.

Supplementary Material

Acknowledgments

Supported in part by NIH grant-CA049248. We thank Pfizer Global Research and Development for providing axitinib and AG-028262, and Dr. Dana Hu-Lowe for useful discussions.

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