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
). Moreover, for axitinib (15
) and certain other anti-angiogenic drugs (17
), 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 (8
). 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
). 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 (38
) and can also be used in cases where tumors are not amenable to resection or as an adjuvant following tumor resection (41
). 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
). 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
), indicating that the vasculature in these tissues may also be targeted for anti-angiogenesis-induced drug retention.