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This Special Issue on the enhanced permeability and retention (EPR) effect commemorates the 35th anniversary of its discovery, the original 1986 Matsumura and Maeda finding being published in Cancer Research as a new concept in cancer chemotherapy. My review here describes the history and heterogeneity of the EPR effect, which involves defective tumor blood vessels and blood flow. We reported that restoring obstructed tumor blood flow overcomes impaired drug delivery, leading to improved EPR effects. I also discuss gaps between small animal cancers used in experimental models and large clinical cancers in humans, which usually involve heterogeneous EPR effects, vascular abnormalities in multiple necrotic foci, and tumor emboli. Here, I emphasize arterial infusion of oily formulations of nanodrugs into tumor-feeding arteries, which is the most tumor-selective drug delivery method, with tumor/blood ratios of 100-fold. This method is literally the most personalized medicine because arterial infusions differ for each patient, and drug doses infused depend on tumor size and anatomy in each patient. Future developments in EPR effect-based treatment will range from chemotherapy to photodynamic therapy, boron neutron capture therapy, and therapies for free radical diseases. This review focuses on our own work, which stimulated numerous scientists to perform research in nanotechnology and drug delivery systems, thereby spawning a new cancer treatment era.

This review focuses primarily on my own research, including pathogenic mechanisms of microbial infection, vascular permeability in infection and tumors, and effects of nitric oxide (NO), superoxide anion radical (O2 −), and 8‐nitroguanosine in the enhanced permeability and retention (EPR) effect for the tumor‐selective delivery of macromolecular agents (nanomedicines). Infection‐induced vascular permeability is mediated by activation of the kinin‐generating protease cascade (kallikrein–kinin) triggered by exogenous microbial proteases. A similar mechanism operates in cancer tissues and in carcinomatosis of the pleural and peritoneal cavities. Infection also stimulates O2 − generation via activation of xanthine oxidase while generating NO by inducing NO synthase. These chemicals function in mutation and carcinogenesis and promote inflammation, in which peroxynitrite (a product of O2 − and NO) activates MMP, damages DNA and RNA, and regenerates 8‐nitroguanosine and 8‐oxoguanosine. We showed vascular permeability by using macromolecular drugs, which are not simply extravasated through the vascular wall into the tumor interstitium but remain there for prolonged periods. We thus discovered the EPR effect, which led to the rational development of tumor‐selective delivery of polymer conjugates, micellar and liposomal drugs, and genes. Our styrene–maleic acid copolymer conjugated with neocarzinostatin was the first agent of its kind used to treat hepatoma. The EPR effect occurs not only because of defective vascular architecture but also through the generation of various vascular mediators such as kinin, NO, and vascular endothelial growth factor. Although most solid tumors, including human tumors, show the EPR effect, heterogeneity of tumor tissue may impede drug delivery. This review describes the barriers and countermeasures for improved drug delivery to tumors by using nanomedicines.

Tumor and inflammation have many common features. One hallmark of both is enhanced vascular permeability, which is mediated by various factors including bradykinin, nitric oxide (NO), peroxynitrite, prostaglandins etc. A unique characteristic of tumors, however, is defective vascular anatomy. The enhanced vascular permeability in tumors is also distinctive in that extravasated macromolecules are not readily cleared. We utilized the enhanced permeability and retention (EPR) effect of tumors for tumor selective delivery of macromolecular drugs. Consequently, such drugs, nanoparticles or lipid particles, when injected intravenously, selectively accumulate in tumor tissues and remain there for long periods. The EPR effect of tumor tissue is frequently inhomogeneous and the heterogeneity of the EPR effect may reduce the tumor delivery of macromolecular drugs. Therefore, we developed methods to augment the EPR effect without inducing adverse effects for instance raising the systemic blood pressure by infusing angiotensin II during arterial injection of SMANCS/Lipiodol. This method was validated in clinical setting. Further, benefits of utilization of NO-releasing agent such as nitroglycerin or angiotensin-converting enzyme (ACE) inhibitors were demonstrated. The EPR effect is thus now widely accepted as the most basic mechanism for tumor-selective targeting of macromolecular drugs, or so-called nanomedicine. (Communicated by Takashi SUGIMURA, M.J.A.)

Plants produce a diverse array of lineage-specific specialized (secondary) metabolites, which are synthesized from primary metabolites. Plant specialized metabolites play crucial roles in plant adaptation as well as in human nutrition and medicine. Unlike well-documented diversification of plant specialized metabolic enzymes, primary metabolism that provides essential compounds for cellular homeostasis is under strong selection pressure and generally assumed to be conserved across the plant kingdom. Yet, some alterations in primary metabolic pathways have been reported in plants. The biosynthetic pathways of certain amino acids and lipids have been altered in specific plant lineages. Also, two alternative pathways exist in plants for synthesizing primary precursors of the two major classes of plant specialized metabolites, terpenoids and phenylpropanoids. Such primary metabolic diversities likely underlie major evolutionary changes in plant metabolism and chemical diversity by acting as enabling or associated traits for the evolution of specialized metabolic pathways.

The plant shikimate pathway directs a significant portion of photosynthetically assimilated carbon into the downstream biosynthetic pathways of aromatic amino acids (AAA) and aromatic natural products. 3‐Deoxy‐d‐arabino‐heptulosonate 7‐phosphate (DAHP) synthase (hereafter DHS) catalyzes the first step of the shikimate pathway, playing a critical role in controlling the carbon flux from central carbon metabolism into the AAA biosynthesis. Previous biochemical studies suggested the presence of manganese‐ and cobalt‐dependent DHS enzymes (DHS‐Mn and DHS‐Co, respectively) in various plant species. Unlike well‐studied DHS‐Mn, however, the identity of DHS‐Co is still unknown. Here, we show that all three DHS isoforms of Arabidopsis thaliana exhibit both DHS‐Mn and DHS‐Co activities in vitro. A phylogenetic analysis of various DHS orthologs and related sequences showed that Arabidopsis 3‐deoxy‐D‐manno‐octulosonate‐8‐phosphate synthase (KDOPS) proteins were closely related to microbial Type I DHSs. Despite their sequence similarity, these Arabidopsis KDOPS proteins showed no DHS activity. Meanwhile, optimization of the DHS assay conditions led to the successful detection of DHS‐Co activity from Arabidopsis DHS recombinant proteins. Compared with DHS‐Mn, DHS‐Co activity displayed the same redox dependency but distinct optimal pH and cofactor sensitivity. Our work provides biochemical evidence that the DHS isoforms of Arabidopsis possess DHS‐Co activity.

Dose regimens of anticancer agents are usually designed on the basis of the maximum tolerable drug doses, and toxicity prevents drug usage at higher doses, even though the drugs may be more effective at the higher doses. We previously studied macromolecular anticancer drugs, i.e. those larger than 40 kDa, and observed their accelerated accumulation in tumors. Their concentration in tumors was more than 5–100‐fold their blood concentration because of the enhanced permeability and retention (EPR) effect. Here, we report that the EPR effect was enhanced by applying nitroglycerin (NG) ointment on the skin of tumor‐bearing animals. Tumors studied included breast cancer, which was induced in Sprague–Dawley rats by the chemical carcinogen 7,12‐dimethylbenz[a]anthracene, and three different transplanted tumor models in mice. NG was applied on tumor or nontumorous normal skin as well. Two to three times more putative macromolecular drug (an Evans blue/albumin complex) was delivered to solid tumors with NG than without NG. We also demonstrated that NG enhanced tumor delivery with another macromolecular drug candidate, PZP, i.e. polyethylene glycol‐conjugated zinc protoporphyrin IX, which inhibits heme oxygenase‐1. In addition, we investigated the therapeutic effect of NG using a combination with low molecular weight anthracycline or high molecular weight PZP in mouse tumor models. NG had no apparent toxicity at the doses used, and showed significantly increased therapeutic effects in both cases. Regardless of its site of application, NG thus enhanced the delivery of the drug to tumors, and enhanced therapeutic effects. (Cancer Sci 2009; 100: 2426–2430)

Vascular plants direct large amounts of carbon to produce the aromatic amino acid phenylalanine to support the production of lignin and other phenylpropanoids. Uniquely, grasses, which include many major crops, can synthesize lignin and phenylpropanoids from both phenylalanine and tyrosine. However, how grasses regulate aromatic amino acid biosynthesis to feed this dual lignin pathway is unknown. Here we show, by stable-isotope labeling, that grasses produce tyrosine >10-times faster than Arabidopsis without compromising phenylalanine biosynthesis. Detailed in vitro enzyme characterization and combinatorial in planta expression uncovered that coordinated expression of specific enzyme isoforms at the entry and exit steps of the aromatic amino acid pathway enables grasses to maintain high production of both tyrosine and phenylalanine, the precursors of the dual lignin pathway. These findings highlight the complex regulation of plant aromatic amino acid biosynthesis and provide novel genetic tools to engineer the interface of primary and specialized metabolism in plants. The study by El-Azaz et al. uncovers how grasses fine-tune tyrosine and phenylalanine production to support their unique dual entry pathway to lignin and phenylpropanoids. The findings help improve sustainable production of aromatic chemicals in crops.

Diverse natural products are synthesized in plants by specialized metabolic enzymes, which are often lineage-specific and derived from gene duplication followed by functional divergence. However, little is known about the contribution of primary metabolism to the evolution of specialized metabolic pathways. Betalain pigments, uniquely found in the plant order Caryophyllales, are synthesized from the aromatic amino acid L-tyrosine (Tyr) and replaced the otherwise ubiquitous phenylalanine-derived anthocyanins. This study combined biochemical, molecular and phylogenetic analyses, and uncovered coordinated evolution of Tyr and betalain biosynthetic pathways in Caryophyllales. We found that Beta vulgaris, which produces high concentrations of betalains, synthesizes Tyr via plastidic arogenate dehydrogenases (TyrAa/ADH) encoded by two ADH genes (BvADHα and BvADHβ). Unlike BvADHβ and other plant ADHs that are strongly inhibited by Tyr, BvADHα exhibited relaxed sensitivity to Tyr. Also, Tyr-insensitive BvADHα orthologs arose during the evolution of betalain pigmentation in the core Caryophyllales and later experienced relaxed selection and gene loss in lineages that reverted from betalain to anthocyanin pigmentation, such as Caryophyllaceae. These results suggest that relaxation of Tyr pathway regulation increased Tyr production and contributed to the evolution of betalain pigmentation, highlighting the significance of upstream primary metabolic regulation for the diversification of specialized plant metabolism.

s Aromatic amino acid aminotransferases (AAA-ATs) catalyze the reversible transamination reactions of proteinogenic and non-proteinogenic aromatic amino acids to corresponding keto acids and vice versa . The products of plant AAA-ATs serve as key precursors of many primary and secondary metabolites that are crucial for both plant and human metabolism and physiology. In most microbes, l -tyrosine (Tyr) and l -phenylalanine (Phe) aminotransferases (Tyr and Phe-ATs) catalyze the final steps of Phe and Tyr biosynthesis. On the other hand, plants use different pathways to synthesize Tyr and Phe via arogenate, in which prephenate-specific aminotransferases (PPA-ATs) catalyze the committed step in the plastids. Plant Tyr and Phe-ATs, unlike microbial counterparts, often prefer the reverse reactions and metabolize Tyr and Phe to their respective aromatic keto acids, which serve as precursors of various plant natural products (e.g. benzenoid volatiles, tocochromanols, plastoquinone, and tropane and benzylisoquinoline alkaloids). Unlike plastidic PPA-ATs, plant Tyr/Phe-ATs are localized outside of the plastids, have broad substrate specificity, and interlink Tyr and Phe metabolism. l -Tryptophan (Trp) aminotransferases (Trp-ATs) are involved in biosynthesis of the plant hormone auxin. Although significant advancement has been made on biochemical, molecular, and genetic characterizations of plant AAA-ATs, there are still many critical knowledge gaps, which are highlighted in the current review.

Plants invest large amounts of resources to produce the twenty proteinogenic amino acids that are essential for growth. However, we still lack a comprehensive understanding of the regulation of amino acid metabolism during the plant life cycle. Plants have a highly conserved ACT domain repeats (ACR) family proteins, which are structurally similar to the bacterial sensor protein GlnD that regulates a key enzyme for nitrogen assimilation and amino acid biosynthesis, glutamine synthetase (GS). We investigated the role of the plastidial ACR proteins and in the regulation on amino acid metabolism by quantifying the levels of amino acids and other metabolites in Arabidopsis and knockout mutants grown under varying light and nitrogen fertilization conditions. Unlike plants, which showed only minor growth alterations, mutants exhibited markedly delayed growth and carbon/nitrogen imbalance. At the metabolic level, plants showed overaccumulation of free amino acids and other nitrogen-containing metabolites, particularly when grown under high nitrogen conditions. Further, plants exhibited a marked decrease in the levels of keto acid intermediates from central carbon metabolism that are precursors to amino acid biosynthesis. Quantification of total GS activity, a potential regulatory target for according to previous studies, shows similar levels of GS activity between and Col-0 controls under the growth conditions tested here. Our findings suggest that is a negative regulator of plant nitrogen metabolism that operates through a mechanism different from bacterial GlnD, possibly regulating other molecular targets besides plastidial GS.