The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect

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Abstract

The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors related to their anatomical and pathophysiological differences from normal tissues. For example, angiogenesis leads to high vascular density in solid tumors, large gaps exist between endothelial cells in tumor blood vessels, and tumor tissues show selective extravasation and retention of macromolecular drugs. This EPR effect served as a basis for development of macromolecular anticancer therapy. We demonstrated methods to enhance this effect artificially in clinical settings. Of great importance was increasing systolic blood pressure via slow angiotensin II infusion. Another strategy involved utilization of NO-releasing agents such as topical nitroglycerin, which releases nitrite. Nitrite is converted to NO more selectively in the tumor tissues, which leads to a significantly increased EPR effect and enhanced antitumor drug effects as well. This review discusses molecular mechanisms of factors related to the EPR effect, the unique anatomy of tumor vessels, limitations and techniques to avoid such limitations, augmenting tumor drug delivery, and experimental and clinical findings.

Introduction

Cancer remains the major cause of death in most advanced countries in the world, and the incidence of cancer increases as populations age. The best treatment of malignancies such as gastric, colonic, and cervical cancers is surgical removal of early-stage tumors that are small and confined to a limited area, without metastasis. Chemotherapy, and to a limited extent radiotherapy, have been the last resort to control cancer. However, conventional chemotherapy, which utilizes small molecular drugs, is far from successful, similar to the situation that we have experienced with antibiotics given for microbial infections. This problem derives mostly from the lack of tumor selectivity, or so-called selective toxicity, of these agents, so that severe adverse effects limit usage. Thus, an urgent need exists to develop drugs with high selectivity to target tumors, which may greatly reduce drug toxicity and enhance the therapeutic efficacy of chemotherapeutics.

Another so far unsuccessful direction of recent cancer treatment is so-called molecular target therapy, which usually focuses on specific kinases or receptors that are overexpressed in cancer cells or tissues. Recent clinical results for those molecular target drugs have been disappointing [1], [2], [3]. The benefit for patients undergoing these treatments is a 1–2 month extension of the usual 3- to 5-year overall survival [1], [3]. In another study, a combination of two different types of molecular target drugs resulted in shorter overall survival [2]. Adverse side effects were not easily overcome, and the frequency of medical emergencies was not reduced. Among these drugs, one exception was imatinib (Gleevec), which is used for chronic myelogenous leukemia. However, most cases demonstrated drug resistance after several months of its use. The problems associated with molecular target drugs probably relate to the intrinsic genetic diversity of human solid tumors, which these drugs do not account for [4], [5]. Usually, multiple genes or their product proteins, which make up sophisticated networks, support or promote the growth, cell regulation, invasion and metastasis of tumor cells. These genes are now known to undergo extensive mutations. Findings for 11 patients with breast cancer and 11 patients with colorectal cancer showed that individual tumors demonstrated an average of approximately 90 mutated genes, and 189 genes mutated at a significantly high frequency [5]. These data mean that the patients had only a small (few percent) chance of the likelihood of a positive response to the molecular target drugs. In addition to the high frequency of occurrence of mutant genes, redundant genetic and molecular or metabolic pathways, which constitute the backup system of vital molecular pathways, may invalidate the single gene or receptor concept and single pathway assumption. Thus, such a highly specific molecular approach, targeted to even a single epitopic antigen, receptor, or kinase, seems to be an imperfect if not an unwise approach.

Another problem may reside in the in vitro screening method for cancer chemotherapeutics. This method utilizes the individual cancer cell type panel model, and a drug is thus screened on the basis of tumor cell type. However, even after more than 30 years of screening at least 50 cell types, such as glioblastoma, malignant melanoma, hepatoma, pancreatic cancer and cervical cancer, no revolutionary discovery of new useful drugs has been reported. One problem with this screening system is probably related to a lack of considering pharmacokinetics and the vascular phenomenon named the enhanced permeability and retention (EPR) effect, so that only cytotoxic compounds were identified.

The greatest breakthrough leading to more general targeted antitumor therapy was the discovery of the EPR effect, as commented by Torchilin [6], (in this issue of ADDR).

The EPR effect was first reported by Matsumura and Maeda in 1986 [7] and was described in greater detail and validated by Maeda et al. [8], [9], [10], [11], [12], [13], [14]. Their investigations showed that most solid tumors have blood vessels with defective architecture and usually produce extensive amounts of various vascular permeability factors. Most solid tumors therefore exhibit enhanced vascular permeability, which will ensure a sufficient supply of nutrients and oxygen to tumor tissues for rapid growth. The EPR effect considers this unique anatomical–pathophysiological nature of tumor blood vessels that facilitates transport of macromolecules into tumor tissues. Macromolecules larger than 40 kDa selectively leak out from tumor vessels and accumulate in tumor tissues. In contrast, this EPR effect-driven drug delivery does not occur in normal tissues [7], [8], [9], [10], [11], [12], [13], [14]. This unique phenomenon in solid tumors—the EPR effect—is thus considered to be a landmark principle in tumor-targeting chemotherapy and is becoming an increasingly promising paradigm for anticancer drug development. For example, Doxil, which is a PEGylated (polyethylene glycol-coated) liposome-encapsulated formulation of doxorubicin, was approved for treatment of Kaposi sarcoma and other cancers. Many other polymeric or micellar drugs are in clinical stage development (phases I and II) [15], [16], of which only a few are reviewed in this special issue. Compared with conventional anticancer drugs, most of which are small molecular drugs, these macromolecular drugs have superior in vivo pharmacokinetics (e.g., a prolonged plasma half-life) and, more important, greater tumor selectivity, so that they produce improved antitumor effects with no or less adverse reactions [15], [16], [17].

The EPR effect has thus now become the “gold standard” in anticancer drug design and anticancer strategies using macromolecular agents, including gene delivery, molecular imaging, antibody therapy, micelles, liposomes, and protein–polymer conjugates (see Torchilin [6] in this issue of ADDR). As evidence of this status, the numbers of citations related to the EPR effect have been progressively increasing in recent years (Fig. 1).

Regardless of the popularity of EPR effect-based drug delivery, many problems with that strategy still exist. We know that large tumors show great pathophysiological heterogeneity. Although we have identified many factors that affect vascular permeability in tumors, as described in the later sections of this article, some parts of tumors, particularly the central area of metastatic cancers, do not exhibit the EPR effect and show less accumulation of macromolecules than other parts [18], [19]. Most of the in vivo experimental studies used mouse tumors that were usually 0.5–1 cm in diameter at least; larger tumors (e.g., 1–2 cm in diameter) tend to contain more necrotic tissues or highly hypovascular areas (with thus less chance of vascular leakage and less tumor growth because of the lack of blood vessels).

In an effort to solve some of these problems, we recently developed methods to intensify the EPR effect and to achieve a more homogeneous drug delivery to tumors, either by elevating blood pressure or by applying nitric oxide (NO)-releasing agents, as described later. The former method utilizes hydrodynamic enhancement of drug delivery; the latter is via generation of NO in tumor tissues. These developments allow one to envision improved cancer chemotherapy via macromolecular drugs in clinical situations.

This review summarizes and discusses the principle of the EPR effect, especially factors influencing this effect, its limitations and methods of avoiding such limitations. In addition, augmenting the EPR effect (tumor drug delivery) and reducing the heterogeneous consequences of the EPR effect to improve clinical outcome are also discussed.

Section snippets

The EPR effect: history and principle

In 1979, Maeda et al. [20] reported the first synthesis of the anticancer protein neocarzinostatin (NCS) conjugated with a polymer (styrene maleic acid copolymer, SMA), which named SMANCS. Later studies found SMANCS to accumulate to a greater degree than NCS in tumor tissues [7], [8], [9], [10], [11], [12], [13], [14], [21], [22]. In addition, this biocompatible polymer conjugation of the protein prolonged the plasma half-life, often up to 200 times longer compared with unmodified free NCS or

Abnormality of tumor blood vasculature: morphology

In contrast to normal tissues and organs, most solid tumors show a higher vascular density (hypervasculature), especially when tumors are small, some exceptions being pancreatic and prostate cancers and large metastatic liver cancers. This finding may relate to the heterogeneity of the EPR effect, as discussed above. Tumor angiogenesis is now well known to be one of the most important features that sustains rapid tumor growth. Folkman [56], [57], [58] first demonstrated that tumors generate an

Factors involved in the EPR effect

Vascular mediators involved in the EPR effect include the following: (a) bradykinin (kinin), which is produced via activation of the kallikrein–kinin system involving a proteolytic cascade [9], [28], [73], [74], [75], [76]; (b) NO generated from l-arginine by the inducible form of NO synthase (iNOS) in leukocytes and tumor cells [76], [77], [78], as well as peroxynitrite (ONOO), an oxidative derivative of NO [79]; (c) prostaglandins (PGs) [76], [80]; (d) angiogensin-converting enzyme (ACE)

Augmentation of the EPR effect

Many macromolecular anticancer drugs are being developed on the basis of the EPR effect. To improve the therapeutic efficacy of these drugs, we focused on the unique pathophysiological features associated with the EPR effect, as described above. We first used AT-II, which produces systemic hypertension. Under these hypertensive conditions, a macromolecular drug is pushed out by hydrodynamic forces into the interstitial space or matrix of tumor tissues. A second method utilizes NO-releasing

Other issues related to the EPR effect

The discussion just presented indicates that the EPR effect is clearly understood to improve targeting of drugs to tumors. However, the EPR effect has other consequences: for example, it facilitates the transport to tumors of nutrients and oxygen that sustain rapid tumor growth. Tumor growth can thus be suppressed by inhibiting or blocking the EPR effect in tumor tissues, i.e., by reducing angiogenesis and extravasation. Certain drugs in clinical use may suppress the EPR effect, for example,

Conclusions

Macromolecular anticancer drugs are receiving more attention than ever in cancer chemotherapy, because the most important mechanism for targeting of drugs to tumors—the EPR effect—would improve therapeutic efficacy and reduce adverse effects, compared with conventional chemotherapy with low-molecular-weight drugs. The EPR effect is the unique and most crucial phenomenon occurring in tumor tissues, in that it accounts for the anatomical and pathophysiological characteristics of tumor blood

Acknowledgements

The work included in this paper was supported in part by the Cancer Priority Grant and other grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 20590049, no. 20015045 and no. S0801085). We thank Ms. J. Daruwalla and Professor C. Christophi of the University of Melbourne, Australia, for their courtesy in allowing us to use Fig. 4, and Dr. Y. Ishima of the Graduate School of Pharmaceutical Sciences, Kumamoto University, Japan for his excellent technical

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