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Tumor-Selective Delivery of Macromolecular Drugs via the EPR Effect: Background and Future Prospects Hiroshi Maeda* Laboratory of Microbiology and Oncology, Faculty of Pharmaceutical Sciences, and Division of Applied Chemistry, Graduate School of Engineering, Sojo University, Kumamoto, 860-0082 Japan. Received February 5, 2010; Revised Manuscript Received March 29, 2010
This paper briefly documents the history of the discovery of the EPR (enhanced permeability and retention) effect and elucidates an analogy between bacterial infection involving proteases that trigger kinin generation and cancer. The EPR effect of macromolecules in cancer tissues is defined, and the distinction between the EPR effect (with reference to clearance of macromolecules from the interstitial space of tumor tissues) and the simple passive targeting of drugs to tumors is described. Additional points of discussion include the uniqueness of tumor vessels, the influence of kinin and other vascular mediators such as nitric oxide (NO) and prostaglandins, and the heterogeneity of the EPR effect. Two different strategies to augment the EPR effect that were discovered are elevating blood pressure artificially via slow infusion of angiotensin II and applying nitroglycerin or other NO donors. Use of the nitroagent increased not only the blood flow of the tumor, but also the delivery of drug to the tumor and the drug’s therapeutic effect. This finding shows an intriguing analogy to hypoxic cardiac infarct tissue, in that both are improved by NO. These two methods were applied to treatment of rodents and human cancers, in combination with other anticancer agents, with successful results achieved in rodents as well as humans. These data suggest very appealing prospects for utilization of the EPR effect in future development of cancer therapeutics.
NATURE OF THE EPR EFFECT AND FACTORS AFFECTING THE EPR EFFECT In 1964, I had started to work on a proteinaceous anticancer agent called neocarzinostatin (NCS1; MW ) 12 kDa) (1). My initial research on NCS was extended to the development of an NCS conjugate with a polymer (styrene-maleic acid, or SMA), which I named SMANCS (MW of about 16 kDa) (2, 3). SMANCS could bind to plasma albumin (67 kDa), so that it would become a larger molecule, about 83 kDa (3, 4). This development of the albumin-bound SMANCS formed the basis for my continuing work on what would be now called the EPR (enhanced permeability and retention) effect (see later). Another focus of my research was the molecular mechanism of bacterial infection involving bacterial proteases, because of their pathogenic potential. During this work, we demonstrated that bacterial proteases could activate the bradykinin-generating cascade called the kallikrein-kinin cascade (5–10), which facilitated vascular leakage. Human blood plasma contains no effective inhibitors of bacterial proteases. As a result, a number of precursor proteases (proenzymes) could be activated by limited proteolysis such as that occurring in the coagulation system (which forms fibrin gel or blood clots) (7–9). We found that bacterial proteases in blood plasma could generate the endogenous nonapeptide bradykinin (a member of the kinin family); the bradykinin was liberated from its precursor kininogen in the plasma via limited proteolysis. This kinin causes very potent pain and induces vascular permeability (or edema) in vivo at the site of its generation (5–10) (Figure 1A). This vascular permeability, or * Corresponding author. Tel.: +81 96-326-4114; Fax: +81 96-3263185. E-mail address:
[email protected]. 1 Abbreviations: NCS, neocarzinostatin; SMA, styrene-maleic acid copolymer; EPR, enhanced permeability and retention; NO, nitric oxide; CT, computed tomography; AT-II, angiotensin II.
extravasation of this dye out of the vessels and into interstitial tissue space, can be quantified by determining the amount of dye extracted from the tissue. The Evans blue is so tightly bound to albumin that the extravasation can be regarded as macromolecular extravasation from the vasculature. We also found that similar extravasation occurred in most experimental solid tumors via bradykinin generation (10–15) (Figure 1B). Both normal inflammatory and tumor tissues exhibit similar enhanced vascular permeability (Figure 1). The difference between these tissues is related to the clearance rates of the Evans blue-albumin complex from each tissue, which are quite dissimilar. Clearance of Evans blue-albumin complex from normal tissue is much faster (usually within 1 wk) than clearance from tumor tissue, which is much slower and takes more than 3-4 wk (11–20). I named the enhanced permeability and retention (EPR) effect of macromolecules in tumor tissues (11). The EPR effect was more marked when the lipid contrast agent Lipiodol (with SMANCS dissolved in it) was injected via the tumor-feeding artery (16–20). In the clinical setting, this EPR effect has been observed with gamma ray-emitting gallium in radioscintigraphy, in which gallium-67 bound to the plasma protein transferrin (MW ) 90 kDa) demonstrated the EPR effect, as shown in radioscintigraphic images of tumors. During the past 25 years, considerable evidence supporting the EPR effect has been accumulated. The EPR effect has been 2 Biocompatible in this case means being inert with reference to the immune system, that is, free from removal by macrophages or the reticuloendothelial system. Polycations are readily captured by the negatively charged laminar surface of blood vessels or by the liver or kidney. Conformational changes (denaturation) of plasma proteins or other biocompatible molecules are detected, and they can be rapidly cleared. For instance, R2-macrogloblin has a t1/2 in mice of more than 144 h, but when it is complexed with a protease, its t1/2 is only 2.5 min (47).
10.1021/bc100070g 2010 American Chemical Society Published on Web 04/16/2010
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Figure 1. (A) Extravasation of Evans blue-albumin induced by 1 µg of bacterial protease (serratial 56 kDa) as a result of kinin generation or 1 µg of protease plus the kinin antagonist SQ 20881 (50 µmol), injected at the site of protease injection. The protease-induced vascular permeability was inhibited by the kinin antagonist. (B) Extravasation in murine S-180 solid tumor. (a) Macroscopic tumor (T) under the skin, after intravenous (i.v.) Evans blue injection. (b) Horizontally cut surface of the same tumor, also after i.v. Evans blue injection. In (b), the tumor tissue shows heterogeneous staining of Evans blue as inhomogeneous extravasation of the blue dye-albumin complex. (c) Another example of a horizontally cut surface of an S-180 tumor. The center of the tumor shows no EPR effect, but the intense permeability of tumor vessels is clear at the periphery, which has a pattern similar to that found in human metastatic liver cancer (see Figure 4 Case 2B and ref 19). This type of peripheral uptake of SMANCS/Lipiodol is seen via computed tomography (CT) in metastatic human tumors and is classified as B-type staining (see ref 19 and Figure 4 Case 2B). Arrows in B point to areas in which the EPR effect also occurred in normal tissue as a result of the generation of vascular mediators such as bradykinin. Figure 1B is adapted with permission from ref 14.
Figure 2. Scanning electron micrographs of vascular casts of plastic resin in the liver. (A) Normal capillary structure. (B) Vascular structure of the liver with a metastatic microtumor nodule, as indicated by T. Polymer resin extravasated only into the vascular bed of the tumor, whereas the normal vasculature (N) did not show such polymer leakage. (C) In the same murine tumor model as that used for B, a macromolecular drug (SMA-pirarubicin micelles) (23) had been injected i.v. 1 week earlier, and that caused selective disintegration of the tumor blood vessel bed (seen as an empty void, equivalent to the circled area containing the T in B). The tumor had been chemically induced by dimethylhydrazine in the colon of CBA mice, and a metastasis model was generated (45). Tumor-selective drug delivery and damage could be achieved in the mouse even with a tumor size as small as 200 µm, i.e., micronodules. Tumor nodules as small as 200 µm already have unique blood vasculature, which is evidence of tumor angiogenesis. These pictures are courtesy of Prof. C. Christophi and Ms. J. Daruwalla, University of Melbourne. Parts A and B are adapted with permission from ref 45.
Figure 3. Diagrammatic representation of blood vasculature in tumor tissue under normotensive (A) and hypertensive (B) conditions (adapted from ref 20 with permission). Angiotensin II (AT-II)-induced enhanced extravasation of macromolecular drugs (dark dots) is shown in B. A greater EPR effect occurred in tumor tissue after AT-II-induced systemic hypertension, with tumor vessels opening wider, whereas normal blood vessels constricted and showed small openings of endothelial cell-cell gaps.
observed with biocompatible2 macromolecules or nanoparticles with a molecular size of more than 40 kDa and up to the size of bacteria (approximately 1 µm) in most experimental solid
tumor (see Figure 2). Professor Folkman first observed neoangiogenesis in tumor tissue when a tumor cell cluster (nodule) reached a size of about 2-3 mm (21). More detailed
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visible for only a short time, and no retention as seen in the case of the EPR effect. One of our review articles in Bioconjugate Chemistry with good numbers of citations on this matter appeared in 1992 (24). The EPR effect was observed not only with plasma proteins, but also with micelles composed of block copolymers of n(PEGpoly Asp), as reported by Yokoyama et al. (25); with HPMA (hydroxypropyl methacrylate) copolymer conjugated with drugs (26); and with our micelles composed of SMA copolymer and various encapsulated drugs (23, 27). In addition, similar results have been obtained with liposomes (28), IgG and other plasma proteins (11), DNA-complexes, PEGylated proteins (ref 29 and others), and bacteria with diameters of 1-2 µm (30, 31). In additional studies, we identified nitric oxide (NO), prostaglandins, peroxynitrite, collagenase, and bradykinin, among others, as factors facilitating the EPR effect (33–36).
INDUCED ARTIFICIAL AUGMENTATION OF THE EPR EFFECT, OVERCOMING OF THE HETEROGENEITY, AND CLINICAL OUTLOOK
Figure 4. CT scans showing metastatic liver cancer in humans. Case 1. Metastatic tumor originating from pancreatic cancer. Arrows indicate dark low-density areas or hypovascular tumor areas; Tp points to the site of the primary pancreatic tumor, and Tm, the metastatic tumor in the liver. Case 2. CT scans demonstrating colon cancer metastasized to the liver. Arrows at the periphery of Case 2A are ribs (bones). (A) SMANCS/Lipiodol was infused via the hepatic artery under normotension. (B) SMANCS/Lipiodol was infused via the hepatic artery but under AT-II-induced hypertension (20). The arrows point to the main metastatic tumor in the liver. Note the more effective drug delivery only in the periphery of the tumor mass, which was defined as type B staining (19) (see Figure 1Bc). The intensity of the SMANCS/Lipiodol staining (white area) at the periphery of the tumor depended on the dose, the pressure applied, and the duration of the induced hypertension during arterial infusion, among other factors. Case 3. CT scans of stomach cancer metastatic to the liver. Note the white high-density areas (arrows) in which accumulation of SMANCS/Lipiodol was tumor-selective. This SMANCS/Lipiodol infusion was also performed under AT-IIinduced hypertension (20) (see text). The CT scan in B was obtained 8 months after the scan in A. Note the considerable tumor size reduction (white areas), even in the liver metastatic lesion. The staining seen in A somewhat resembles the B-type staining seen in Case 2B (19), that is, circumferential low-density staining after ATII infusion induced hypertension. As in Case 2, more complete filling of tumor with drug was achieved with AT-II-induced hypertension. Figure 4 is adapted with permission from ref 20.
scanning electron microscopic observations of metastatic tumor nodules in the liver, in which vascular casts of blood vessels were obtained with plastic resin, indicated that tumor nodules of 100 µm have a highly permeable vascular bed (ref 22 and others) that is not seen in normal blood vasculature (Figure 2). Administration of a micellar drug (SMA-pirarubicin) (23) selectively destroyed the tumor micronodules by means of the EPR effect and did not damage normal blood vessels (Figure 2c). The EPR effect with macromolecular drugs or lipid particles is clearly different from the so-called passive targeting of drugs to tumors, in that the drug retention period in tumors showing the EPR effect is more than days to weeks, whereas that of passive targeting may be only a few minutes, or no more than 10 min, as routinely observed during tumor angiography. Radiology clinics normally use low-molecular-weight angiographic contrast agents, which usually do not remain in tumor tissues for more than 10 min, so that the radiographic image is
Our interest is now focused on the artificial augmentation of the EPR effect for more efficient tumor-selective drug delivery. One method that we utilized for this purpose of enhancing drug delivery is elevation of systemic blood pressure by means of angiotensin II (AT-II) (20, 37) (Figure 3). The method was originally described by Suzuki et al. (38). Indeed, we discovered that this method improved drug delivery as well as therapeutic efficacy markedly against highly refractory solid tumors such as metastatic liver cancer, and even cancers of the gallbladder and pancreas (20) (Figures 3 and 4). The second method used to enhance drug delivery involved nitroglycerin and other NO-releasing agents (39, 40). This method is unique because in hypoxic tumor tissue nitroglycerin can be converted into nitrite, which can then be reduced to NO (41) (Figure 5A). As stated above, NO is one of the potent mediators of vascular extravasation. Thus, topical application of nitroglycerin at any skin site resulted in a 2-3-fold increase in drug delivery (Figure 5B,C), which also potentiated the therapeutic effect in experimental tumors (39, 40) as well as in human cancers (42, 43). Both Yasuda et al. (42, 43) and Graham’s group (44), in agreement with our findings, independently reported an advantage of nitroglycerin or nitroagents for improving therapeutic efficacy. It is interesting to note that hypoxia and the low pH of tumor tissues are analogous to conditions in cardiac tissue in the presence of angina pectoris (41). In such cardiac tissues, release of nitrite from nitroglycerin and conversion of nitrite to NO (Figure 5A) facilitated vascular blood flow and hence elevated pO2, with a consequent enhancement of the EPR effect and drug delivery (39, 40). Despite the heterogeneity of drug distribution (on the EPR effect) in tumor tissues (Figure 1B,b and c), the just-described methods of augmenting the EPR effect, i.e., elevation of blood pressure and use of nitroglycerin, did indeed enhance drug delivery and therapeutic efficacy in a more ubiquitous manner, that is, for solid tumors in general. The EPR effect may therefore become increasingly realized as useful for cancer therapy and diagnosis that would have more universal application than the antibody-dependent selectivity of a single molecular target species. Furthermore, this enhanced EPR effect, induced by either elevating blood pressure or utilizing nitroglycerin, appears to be effective in apparently hypovascular tumors, such as metastatic liver cancer and pancreatic cancer; in tumors refractory to radiotherapy and most of chemotherapy (39, 40, 42, 43); and even in minute tumor nodules as small as 100 µm (Figure
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Figure 5. (A) Mechanism of NO generation from nitroglycerin (NG). NO2- is generated from NG, to a greater degree in hypoxic tumor tissues than in normal tissues. NO2- is then reduced to NO · in tumor and hypoxic tissues, which contributes to the EPR effect (14, 33). (B) Time-dependent increase in delivery of Evans blue (EB)-albumin to tumors after topical application of NG ointment to the skin (1 mg/mouse). This effect lasted more than 24 h. (C) Dose-dependent increase in delivery of Evans blue-albumin to tumors in mice. Tumor diameters were 5-8 mm; dye concentration was determined after extraction of the dye and measuring absorption. Adapted with permission from ref 40.
2), so that invisible metastatic tumor foci in the liver may be more selectively targeted (45, 46).
CONCLUSIONS The EPR effect is mediated by various vascular mediators including bradykinin and NO, among others. Some tumors undergo active angiogenesis, which results in high vascular density, whereas others are dormant or hypovascular. Many other tumors exhibit heterogeneous vascular features. However, we can effectively enhance drug delivery to most of these tumors, if not all, via augmenting the EPR effect, by elevating systemic blood pressure with AT-II. Another method of enhancing drug delivery is application of nitroglycerin, which we discovered generates NO in hypoxic tumor tissues and facilitates drug delivery. Both methods have been demonstrated to work in clinical settings.
ACKNOWLEDGMENT The author would like to thank the generosity of Ms. J. Daruwella and Prof. C. Christophi of University of Melbourne, Australia, for providing the electron micrographs of Figure 2, and my colleagues, the late Dr. T. Konno of Kumamoto University for Figure 1, Case 1, and Dr. A. Nagamitsu of Nagamitsu Clinic, Fukuoka, for Figure 4, Cases 2 and 3. I also acknowledge the help and assistance of my previous and present colleagues including Ms. A. Takaki and Ms. J. Gandy for their typing and editing of the manuscript.
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