Enhanced permeability and retention (EPR) effect-based nanomedicine is a promising strategy for s... more Enhanced permeability and retention (EPR) effect-based nanomedicine is a promising strategy for successful anticancer therapy. The EPR effect is based on tumor blood flow. Because advanced large tumors, as frequently seen in clinical settings, are heterogeneous, with regions of defective vasculature and blood flow, achieving the desired tumor drug delivery is difficult. Here, we utilized the EPR effect to increase drug delivery. To augment the EPR effect for improved therapeutic effects of nanomedicine, we exploited vascular mediators-the nitric oxide (NO) generators nitroglycerin (NG), hydroxyurea, and L-arginine. These compounds generate NO in tumors with relatively high selectivity. Using different nano-sized drugs in our protocol significantly increased (1.5-2 times) delivery of nanomedicines to different solid tumor models, along with markedly improving (2-3 fold) the antitumor effects of these drugs. Also, in 7,12-dimethylbenz[a]anthracene-induced advanced end-stage breast can...
In vascular system, superoxide anion (O 2 − ) generated by xanthine oxidase (XO) is known to regu... more In vascular system, superoxide anion (O 2 − ) generated by xanthine oxidase (XO) is known to regulate vascular tonus by reacting with, and thus consuming nitric oxide (NO), which determines vasorelaxation. We previously reported the remarkable antihypertensive effect of a potent XO inhibitor, 4-amino -6hydroxypyrazolo[3,4-d]pyrimidine (AHPP). However, AHPP is insoluble in water, which hamper its in vivo application. Therefore, in this study we prepared a water soluble polymeric conjugate of AHPP, by using a styrene maleic acid copolymer (SMA, SMA-AHPP). SMA-AHPP showed similar inhibitory activity against XO (K i = 0.25 µM) comparable to native AHPP (K i = 0.17 µM), while exhibiting good water-solubility, which now made it possible for systemic injection. In vivo experiments were carried out to examine the antihypertensive effect of SMA-AHPP using the spontaneously hypertensive rats (SHR) by i.v. injection (15, 30 mg/kg) or by oral administration (100 mg/kg) of SMA-AHPP. The results showed significantly reduced blood pressures (up to 30% reduction) of SHR rats; this antihypertensive effect continued for at least 24 h after SMA-AHPP administration. These findings strongly suggest the potential value of SMA-AHPP as an antihypertensive agent with sustained in vivo activity, which warrants further investigations.
The detrimental role of superoxide anion (O(2)(-)) has been well documented in the pathogenesis o... more The detrimental role of superoxide anion (O(2)(-)) has been well documented in the pathogenesis of ischemia-reperfusion (I/R) injury. Our and other studies suggested that one critical source of O(2)(-) generation may be xanthine oxidase (XO). We thus hypothesized that I/R injury could be protected by inhibiting XO activity, which would reduce the amount of O(2)(-) and hence reduce pathogenic consequences. Among various XO inhibitors, we previously found 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP) exhibited potent XO inhibitory activity. Here, we report that the covalent conjugate of AHPP with amphipathic styrene-maleic acid copolymer (SMA-AHPP) showed protective effect against I/R-induced injury in a rat hepatic I/R model. Liver ischemia was induced by occluding both the portal vein and the hepatic artery for 30 min, and followed by reperfusion. SMA-AHPP was administered via the tail vein two hours before ischemia was initiated. A remarkable increase of liver enzymes in plasma (aspartate aminotransferase, AST; alanine aminotransferase, ALT and lactate dehydrogenase, LDH) was detected three hours after reperfusion, whereas prior injection of SMA-AHPP greatly suppressed this increase of AST, ALT and LDH. Moreover, induction of inflammatory cytokines, i.e. tumor necrosis factor-alpha (TNF-alpha), interleukin-12 (IL-12) and monocyte chemotactic protein-1 (MCP-1) by I/R were significantly inhibited by SMA-AHPP treatment. Accordingly, cytotoxic effect or apoptosis in the liver caused by I/R was clearly reduced by SMA-AHPP pretreatment. Furthermore, thiobarbituric acid-reactive substance assay showed a significant decrease of lipid peroxidation in rat liver after the administration of SMA-AHPP, which is parallel with the decreased XO activity after SMA-AHPP treatment, indicating the involvement of reactive oxygen species generated by XO. In addition, SMA-AHPP was found to bind to albumin, thus to exhibit prolonged in vivo (plasma) half-life. These results suggest that SMA-AHPP exerted a potent cytoprotective effect against I/R injury in rat liver, by inhibiting XO activity and the subsequent generation of O(2)(-).
Amphiphilic styrene-maleic acid (SMA) copolymer efficiently formed micelles with a potent heme ox... more Amphiphilic styrene-maleic acid (SMA) copolymer efficiently formed micelles with a potent heme oxygenase inhibitor-zinc protoporphyrin (ZnPP). The micelles were constructed by subtle pH adjustments to form non-covalent interaction between the hydrophobic ZnPP and amphiphilic SMA. The micelles (SMA-ZnPP) thus formed were nanoparticles with narrow size distribution in water (mean diameter 176.5 nm), having tunable loading (from 15% to 60% w/w of ZnPP) with remarkable aqueous solubility. SMA-ZnPP had an average molecular size of 144 kDa as determined by size-exclusion chromatography (SEC), this size is a marked increase from the molecular weight of free ZnPP (626.03 Da), suggesting the formation of micellar structure. The micelles showed a constant ZnPP release rate of about 0.5%/day in vitro at neutral pH. SMA-ZnPP micelles inhibited splenic microsomal HO-1 activity, in a competitive and dose-dependent manner, with an apparent inhibitory constant (K i ) of 0.12 mM, comparable to free ZnPP and also exhibited marked cytotoxic effect on KYSE-510 human esophageal cancer cells. The unique features of SMA-ZnPP micelles are that they are nanoparticles in aqueous solution having high water solubility and loading, yet macromolecular in nature, which can be beneficial in targeted release of a potent HO-1 inhibitor. r
Effective cancer therapy remains one of the most challenging tasks to the scientific community, w... more Effective cancer therapy remains one of the most challenging tasks to the scientific community, with little advancement on overall cancer survival landscape during the last two decades. A major limitation inherent to most conventional anticancer chemotherapeutic agents is their lack of tumor selectivity. One way to achieve selective drug targeting to solid tumors is to exploit abnormalities of tumor vasculature, namely hypervascularization, aberrant vascular architecture, extensive production of vascular permeability factors stimulating extravasation within tumor tissues, and lack of lymphatic drainage. Due to their large size, nano-sized macromolecular anticancer drugs administered intravenously (i.v.) escape renal clearance. Being unable to penetrate through tight endothelial junctions of normal blood vessels, their concentration builds up in the plasma rendering them long plasma half-life. More importantly, they can selectively extravasate in tumor tissues due to its abnormal vascular nature. Overtime the tumor concentration will build up reaching several folds higher than that of the plasma due to lack of efficient lymphatic drainage in solid tumor, an ideal application for EPR-based selective anticancer nanotherapy. Indeed, this selective high local concentration of nano-sized anticancer drugs in tumor tissues has proven superior in therapeutic effect with minimal side effects in both preclinical and clinical settings.
Of the tumor targeting strategies, the enhanced permeability and retention (EPR) effect of macrom... more Of the tumor targeting strategies, the enhanced permeability and retention (EPR) effect of macromolecules is a key mechanism for solid tumor targeting, and considered a gold standard for novel drug design. In this review, we discuss various endogenous factors that can positively impact the EPR effect in tumor tissues. Further, we discuss ways to augment the EPR effect by use of exogenous agents, as well as practical methods available in the clinical setting. Some innovative examples developed by researchers to combat cancer by the EPR mechanism are also discussed.
We had developed a H 2 O 2 generating enzyme, polyethylene glycol conjugated D-amino acid oxidase... more We had developed a H 2 O 2 generating enzyme, polyethylene glycol conjugated D-amino acid oxidase (PEG-DAO), which exhibited potent antitumor activity by generating toxic reactive oxygen species, namely oxidation therapy, subsequently showed remarkable antitumor effect on murine Sarcoma 180 solid tumor, by taking advantage of the enhanced permeability and retention effect. Along this line, we report here the preparation of PEG-DAO by use of recombinant DAO and its antitumor activity by using various tumor cell lines and tumor models. Recombinant DAO (rDAO) was obtained from E. coli BL21 (DE3) carrying the porcine DAO expression vector with high yield (20 mg/l) and high enzyme activity (5.3 U/mg). Pegylated rDAO (PEG-rDAO) showed high stability against sonication, repeated freezing/thawing, lyophilization and exhibited superior in vivo pharmacokinetics. PEG-rDAO had a molecular size of 65 kDa and existed as nanoparticles in aqueous solution with mean particle diameter of 119 nm.
Microcirculation (New York, N.Y. : 1994), Jan 3, 2015
Solid-tumor has unique vascular architecture, excessive production of vascular mediators, and ext... more Solid-tumor has unique vascular architecture, excessive production of vascular mediators, and extravasation of macromolecules from blood vessels into the tumor tissue interstitium. These features comprise the phenomenon named the EPR (enhanced permeability and retention) effect of solid tumors, described in 1986. Our investigations on the EPR revealed that many mediators, such as bradykinin, nitric oxide, and prostaglandins, are involved in the EPR effect, which is now believed to be the most important element for cancer-selective drug delivery. However, tumors in vivo manifest great diversity, and some demonstrate a poor EPR effect, for example, because of impaired vascular flow involving thrombosis, with poor drug delivery and therapeutic failure. Another important element of this effect is that it operates in metastatic cancers. Because few drugs are currently effective against metastases, the EPR effect offers a great advantage in nanomedicine therapy. The EPR effect can also be...
Previous studies indicated the potential of zinc protoporphyrin (ZnPP) as an antitumor agent targ... more Previous studies indicated the potential of zinc protoporphyrin (ZnPP) as an antitumor agent targeting to the tumor survival factor heme oxygenase-1, and/or for photodynamic therapy (PDT). In this study, to achieve tumor-targeted delivery, styrene-maleic acid-copolymer conjugated ZnPP (SMA-ZnPP) was synthesized via amide bond, which showed good water solubility, having ZnPP loading of 15%. More importantly, it forms micelles in aqueous solution with a mean particle size of 111.6 nm, whereas it has an apparent Mw of 65 kDa. This micelle formation was not detracted by serum albumin, suggesting it is stable in circulation. Further SMA-ZnPP conjugate will behave as an albumin complex in blood with much larger size (235 kDa) by virtue of the albumin binding property of SMA. Consequently, SMA-ZnPP conjugate exhibited prolonged circulating retention and preferential tumor accumulation by taking advantage of enhanced permeability and retention (EPR) effect. Clear tumor imaging was thus achi...
ABSTRACT Blood vessels in tumors are different to normal blood vessels because they have abnormal... more ABSTRACT Blood vessels in tumors are different to normal blood vessels because they have abnormal architectures and impaired functional regulation. We have studied these abnormalities, in particular vascular permeability in tumors, and found greatly enhanced permeability for macromolecules, which are retained in tumors for extended periods. We named this phenomenon the “enhanced permeability and retention(EPR) effect”. This effect, related to the transport of macromolecular drugs composed of liposomes, micelles, proteinaceous or polymer-conjugated macromolecules, lipid particles, and nanoparticles into the tumor, is the hallmark of solid tumor vasculature. These macromolecular species are therefore ideal for selective delivery to tumor. The EPR effect has facilitated the development of macromolecular drugs consisting of various polymer-drug conjugates (pendant type), polymeric micelles, and liposomes that exhibit far better therapeutic efficacy and far fewer side effects than the parent low-molecular-weight compounds. Here, we discuss various aspects of the EPR effect via examples, including the use of polymeric drugs such as SMANCS [poly(styrene-co-maleic acid-half-n-butylate) (SMA)-conjugated neocarzinostatin (NCS)]. In addition, we review our new macromolecular drug candidates that generate reactive oxygen species via anovel mode of action. Because solid tumors frequently lack antioxystress enzymes, generating oxystress in tumor tissue may be another unique anticancer strategy. Most tumor cells have aweak or limited defense system against reactive oxygen species, and the oxygen radical-generating techniques that we have developed are primarily endogenous. Consequently, an approach to cancer therapy based on the EPR effect and oxyradical induction in order to produce apoptosis appears promising.
High expression of the inducible isoform of heme oxygenase (HO-1) is now well known in solid tumo... more High expression of the inducible isoform of heme oxygenase (HO-1) is now well known in solid tumors in humans and experimental animal models. We reported previously that HO-1 may be involved in tumor growth (Tanaka et al., Br. J. Cancer, 88: 902-909, 2003), in that inhibition of HO activity in tumors by using zinc protoporphyrin (ZnPP) significantly reduced tumor growth in a rat model. We demonstrate here that poly(ethylene glycol)-conjugated ZnPP (PEG-ZnPP), a water-soluble derivative of ZnPP, exhibited potent HO inhibitory activity and had an antitumor effect in vivo. In vitro studies with cultured SW480 cells, which express HO-1, showed that PEG-ZnPP induced oxidative stress, and consequently apoptotic death, of these cells. Pharmacokinetic analysis revealed that PEG-ZnPP-administered i.v. had a circulation time in blood that was 40 times longer than that for nonpegylated ZnPP. More important, PEG-ZnPP preferentially accumulated in solid tumor tissue in a murine model. In vivo tr...
The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors, and ... more The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors, and it can serve as a basis for the development of macromolecular anticancer therapy. We have previously found that recombinant human serum albumin dimer, and especially its S-nitrosated form (SNO-HSA-Dimer), is an enhancer of the EPR effect. In this study, we investigated the influence of SNO-HSA-Dimer on the anti-tumor effect of two types of macromolecular anti-tumor drugs, namely N-(2-hydroxypropyl)methacrylamide polymer conjugated with zinc protoporphyrin, which forms micelles and can be used for fluorescence studies. The other was PEGylated liposomal doxorubicin (Doxil), a typical example of a stealth liposome approved for medical usage. In mice having C26 tumors with highly permeable vasculature, SNO-HSA-Dimer increases tumor accumulation of the drugs by a factor 3-4 and thereby their anti-tumor effects. Experiments with Evans blue revealed increased EPR effect in all parts of the tumor. Furthermore, SNO-HSA-Dimer improves the anti-metastatic effects of Doxil and reduces its minor uptake in non-tumorous organs such as liver and kidney. Tumor accumulation of Doxil in B16 tumors, which are characterized by a low permeable vasculature, increased even more (6-fold) in the presence of SNO-HSA-Dimer, and the improved accumulation lead to decreased tumor volume and increased survival of the animals. The administration of SNO-HSA-Dimer itself is safe, because it has no effect on blood pressure, heart rate or on several biochemical parameters. The present findings indicate that SNO-HSA-Dimer is promising for enhancing the EPR effect and consequently the specific, therapeutic effects of macromolecular anticancer drugs.
Cancer treatment alters microRNA (miRNA) expression, revealing potential therapeutic targets (onc... more Cancer treatment alters microRNA (miRNA) expression, revealing potential therapeutic targets (oncotarget). Here we treated pancreatic cancer (ASPC-1) cells with either recombinant human endostatin (rh-endostatin) or gemcitabine. Then high-throughput sequencing assay was performed to screen for altered miRNAs. Both treatments decreased levels of MiR-19a. We found that miR-19a stimulated cell proliferation, migration, invasion in vitro and tumor growth in vivo. High levels of miR-19a correlated with poor prognosis in patients. Ras homolog family member B (RHOB) was identified as a direct target of miR-19a. Furthermore, RHOB was down-regulated in human pancreatic cancer samples. Restoration of RHOB induced apoptosis, inhibited proliferation and migration of ASPC-1 cells. SP-1 was identified as an upstream transcription factor of miR-19a gene, promoting miR-19a transcription. Rh-endostatin decreased miR-19a expression by down-regulating SP-1. These findings suggest that miR-19a is a pot...
Although the thiol click reaction is an attractive tool for post-polymerization modification of th... more Although the thiol click reaction is an attractive tool for post-polymerization modification of thiolmers, thiol groups are easily oxidized, limiting the potential for covalent immobilization of bioactive molecules. In this study, a series of biodegradable polyurethane elastomers incorporating stable cyclic disulfide groups was developed and characterized. These poly(ester urethane)urea (PEUU-SS) polymers were based on polycaprolactone diol (PCL), oxidized DL-dithiothreitol (O-DTT), lysine diisocyanate (LDI) or butyl diisocyanate (BDI), with chain extension by putrescine. The ratio of O-DTT:PCL was altered to investigate different levels of potential functionalization. PEG acrylate was employed to study the mechanism and availability of both bulk and surface click modification of PEUU-SS polymers. All synthesized PEUU-SS polymers were elastic with breaking strengths of 38-45 MPa, while the PEUU-SS(LDI) polymers were more amorphous, possessing lower moduli and relatively small permane...
Cancer remains the first or second main cause of death in developed countries. In the world, 7.6 ... more Cancer remains the first or second main cause of death in developed countries. In the world, 7.6 million people died of cancer in 2005 [1]. However, the cure for advanced cancer in major cancers has not improved in the past 50 years, although chemotherapy is supposed to be a last resort, if not all [2,3]. One of the recent successful stories in cancer chemotherapy is imatinib (Gleevec®), a drug for chronic myeloid leukemia (CML) which is an inhibitor of BCR/ABL tyrosine kinase, a product of oncogene. Imatinib shows a remarkable therapeutic effect against CML while a natural course of life span of CML patients is about 5 years. However, upon blastic period when the leukemic cell growth becomes exponential, majority of patients developed drug resistance within 6 months. Therefore, one can conclude that imatinib contributes only 10% prolongation of the life span.
Induction of haem oxygenase-1 (HO-1) may provide an important protective effect for cells against... more Induction of haem oxygenase-1 (HO-1) may provide an important protective effect for cells against oxidative stress. Here, we investigated the mechanism of cytoprotection of HO-1 in solid tumour with a focus on the antiapoptotic activity of HO-1. Treatment of rat hepatoma AH136B cells with the HO inhibitor zinc protoporphyrin IX (ZnPP IX) or tin protoporphyrin IX resulted in extensive apoptotic changes of tumour cells both in vivo and in vitro. Caspase-3 activity of the ZnPP IX-treated hepatoma cells increased significantly. Moreover, ZnPP IX-induced apoptosis was completely inhibited by simultaneous incubation with a specific caspase-3 inhibitor and was partially abrogated by bilirubin, a reaction product of HO. In vivo ZnPP IX treatment did not affect nitric oxide (NO) production and tumour blood flow. Western blot analyses showed that HO-1 expression in AH136B cells was strongly upregulated by NO donors, for example, S-nitroso-N-acetyl penicillamine and propylamine NONOate in vitr...
Hydrogen peroxide (H(2)O(2)) is a strong oxidant that induces apoptosis of tumor cells in vitro. ... more Hydrogen peroxide (H(2)O(2)) is a strong oxidant that induces apoptosis of tumor cells in vitro. Here, we investigated the antitumor activity of an H(2)O(2)-generating enzyme, D-amino acid oxidase (DAO), and its conjugate with polyethylene glycol (PEG; PEG-DAO). Compared with DAO, PEG-DAO showed improved pharmacokinetic parameters in mice after i.v. injection. PEG-DAO administered i.v. accumulated selectively in tumor tissue with insignificant accumulation in normal organs and tissues. To generate cytotoxic H(2)O(2) at the tumor site, PEG-DAO was first administrated i.v. to tumor-bearing mice. After an adequate lag time, the substrate of DAO, D-proline, was injected i.p. This treatment resulted in significant suppression of tumor growth compared with tumor growth in control animals (not given treatment; P < 0.001). Similar treatment with native DAO showed no effect under the same conditions. Oxidative metabolites were significantly increased in solid tumors by administration of P...
Advances in experimental medicine and biology, 2003
Both enhanced vascular permeability and angiogenesis of tumor sustain rapid growth of tumor invol... more Both enhanced vascular permeability and angiogenesis of tumor sustain rapid growth of tumor involving many vascular mediators and high vascular density. On the contrary, however, they can be utilized for macromolecular drug delivery to tumor. Impaired reticuloendothelial/lymphatic clearance of macromolecules from the tumor, or lack of such clearance, is another unique characteristic of tumor tissue, which results intratumor retention of macromolecular drugs thus delivered (Figure 1). Consequently, enhanced permeability and retention (EPR) effect is the basis for the selective targeting of macromolecular drugs to tumor, and the EPR concept is now utilized for selective delivery of many macromolecular anticancer agents in aqueous formation for i.v. or i.a. as well as oily formation for i.a. dosing, which is not possible for low-molecular-weight drugs because of rapid washout by capillary vascular blood flow. This EPR concept has been validated in clinical settings with hepatoma and ot...
High expression of the inducible isoform of heme oxygenase (HO-1) is now well known in solid tumo... more High expression of the inducible isoform of heme oxygenase (HO-1) is now well known in solid tumors in humans and experimental animal models. We reported previously that HO-1 may be involved in tumor growth (Tanaka et al., Br. J. Cancer, 88: 902-909, 2003), in that inhibition of HO activity in tumors by using zinc protoporphyrin (ZnPP) significantly reduced tumor growth in a rat model. We demonstrate here that poly(ethylene glycol)-conjugated ZnPP (PEG-ZnPP), a water-soluble derivative of ZnPP, exhibited potent HO inhibitory activity and had an antitumor effect in vivo. In vitro studies with cultured SW480 cells, which express HO-1, showed that PEG-ZnPP induced oxidative stress, and consequently apoptotic death, of these cells. Pharmacokinetic analysis revealed that PEG-ZnPP-administered i.v. had a circulation time in blood that was 40 times longer than that for nonpegylated ZnPP. More important, PEG-ZnPP preferentially accumulated in solid tumor tissue in a murine model. In vivo tr...
Enhanced permeability and retention (EPR) effect-based nanomedicine is a promising strategy for s... more Enhanced permeability and retention (EPR) effect-based nanomedicine is a promising strategy for successful anticancer therapy. The EPR effect is based on tumor blood flow. Because advanced large tumors, as frequently seen in clinical settings, are heterogeneous, with regions of defective vasculature and blood flow, achieving the desired tumor drug delivery is difficult. Here, we utilized the EPR effect to increase drug delivery. To augment the EPR effect for improved therapeutic effects of nanomedicine, we exploited vascular mediators-the nitric oxide (NO) generators nitroglycerin (NG), hydroxyurea, and L-arginine. These compounds generate NO in tumors with relatively high selectivity. Using different nano-sized drugs in our protocol significantly increased (1.5-2 times) delivery of nanomedicines to different solid tumor models, along with markedly improving (2-3 fold) the antitumor effects of these drugs. Also, in 7,12-dimethylbenz[a]anthracene-induced advanced end-stage breast can...
In vascular system, superoxide anion (O 2 − ) generated by xanthine oxidase (XO) is known to regu... more In vascular system, superoxide anion (O 2 − ) generated by xanthine oxidase (XO) is known to regulate vascular tonus by reacting with, and thus consuming nitric oxide (NO), which determines vasorelaxation. We previously reported the remarkable antihypertensive effect of a potent XO inhibitor, 4-amino -6hydroxypyrazolo[3,4-d]pyrimidine (AHPP). However, AHPP is insoluble in water, which hamper its in vivo application. Therefore, in this study we prepared a water soluble polymeric conjugate of AHPP, by using a styrene maleic acid copolymer (SMA, SMA-AHPP). SMA-AHPP showed similar inhibitory activity against XO (K i = 0.25 µM) comparable to native AHPP (K i = 0.17 µM), while exhibiting good water-solubility, which now made it possible for systemic injection. In vivo experiments were carried out to examine the antihypertensive effect of SMA-AHPP using the spontaneously hypertensive rats (SHR) by i.v. injection (15, 30 mg/kg) or by oral administration (100 mg/kg) of SMA-AHPP. The results showed significantly reduced blood pressures (up to 30% reduction) of SHR rats; this antihypertensive effect continued for at least 24 h after SMA-AHPP administration. These findings strongly suggest the potential value of SMA-AHPP as an antihypertensive agent with sustained in vivo activity, which warrants further investigations.
The detrimental role of superoxide anion (O(2)(-)) has been well documented in the pathogenesis o... more The detrimental role of superoxide anion (O(2)(-)) has been well documented in the pathogenesis of ischemia-reperfusion (I/R) injury. Our and other studies suggested that one critical source of O(2)(-) generation may be xanthine oxidase (XO). We thus hypothesized that I/R injury could be protected by inhibiting XO activity, which would reduce the amount of O(2)(-) and hence reduce pathogenic consequences. Among various XO inhibitors, we previously found 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP) exhibited potent XO inhibitory activity. Here, we report that the covalent conjugate of AHPP with amphipathic styrene-maleic acid copolymer (SMA-AHPP) showed protective effect against I/R-induced injury in a rat hepatic I/R model. Liver ischemia was induced by occluding both the portal vein and the hepatic artery for 30 min, and followed by reperfusion. SMA-AHPP was administered via the tail vein two hours before ischemia was initiated. A remarkable increase of liver enzymes in plasma (aspartate aminotransferase, AST; alanine aminotransferase, ALT and lactate dehydrogenase, LDH) was detected three hours after reperfusion, whereas prior injection of SMA-AHPP greatly suppressed this increase of AST, ALT and LDH. Moreover, induction of inflammatory cytokines, i.e. tumor necrosis factor-alpha (TNF-alpha), interleukin-12 (IL-12) and monocyte chemotactic protein-1 (MCP-1) by I/R were significantly inhibited by SMA-AHPP treatment. Accordingly, cytotoxic effect or apoptosis in the liver caused by I/R was clearly reduced by SMA-AHPP pretreatment. Furthermore, thiobarbituric acid-reactive substance assay showed a significant decrease of lipid peroxidation in rat liver after the administration of SMA-AHPP, which is parallel with the decreased XO activity after SMA-AHPP treatment, indicating the involvement of reactive oxygen species generated by XO. In addition, SMA-AHPP was found to bind to albumin, thus to exhibit prolonged in vivo (plasma) half-life. These results suggest that SMA-AHPP exerted a potent cytoprotective effect against I/R injury in rat liver, by inhibiting XO activity and the subsequent generation of O(2)(-).
Amphiphilic styrene-maleic acid (SMA) copolymer efficiently formed micelles with a potent heme ox... more Amphiphilic styrene-maleic acid (SMA) copolymer efficiently formed micelles with a potent heme oxygenase inhibitor-zinc protoporphyrin (ZnPP). The micelles were constructed by subtle pH adjustments to form non-covalent interaction between the hydrophobic ZnPP and amphiphilic SMA. The micelles (SMA-ZnPP) thus formed were nanoparticles with narrow size distribution in water (mean diameter 176.5 nm), having tunable loading (from 15% to 60% w/w of ZnPP) with remarkable aqueous solubility. SMA-ZnPP had an average molecular size of 144 kDa as determined by size-exclusion chromatography (SEC), this size is a marked increase from the molecular weight of free ZnPP (626.03 Da), suggesting the formation of micellar structure. The micelles showed a constant ZnPP release rate of about 0.5%/day in vitro at neutral pH. SMA-ZnPP micelles inhibited splenic microsomal HO-1 activity, in a competitive and dose-dependent manner, with an apparent inhibitory constant (K i ) of 0.12 mM, comparable to free ZnPP and also exhibited marked cytotoxic effect on KYSE-510 human esophageal cancer cells. The unique features of SMA-ZnPP micelles are that they are nanoparticles in aqueous solution having high water solubility and loading, yet macromolecular in nature, which can be beneficial in targeted release of a potent HO-1 inhibitor. r
Effective cancer therapy remains one of the most challenging tasks to the scientific community, w... more Effective cancer therapy remains one of the most challenging tasks to the scientific community, with little advancement on overall cancer survival landscape during the last two decades. A major limitation inherent to most conventional anticancer chemotherapeutic agents is their lack of tumor selectivity. One way to achieve selective drug targeting to solid tumors is to exploit abnormalities of tumor vasculature, namely hypervascularization, aberrant vascular architecture, extensive production of vascular permeability factors stimulating extravasation within tumor tissues, and lack of lymphatic drainage. Due to their large size, nano-sized macromolecular anticancer drugs administered intravenously (i.v.) escape renal clearance. Being unable to penetrate through tight endothelial junctions of normal blood vessels, their concentration builds up in the plasma rendering them long plasma half-life. More importantly, they can selectively extravasate in tumor tissues due to its abnormal vascular nature. Overtime the tumor concentration will build up reaching several folds higher than that of the plasma due to lack of efficient lymphatic drainage in solid tumor, an ideal application for EPR-based selective anticancer nanotherapy. Indeed, this selective high local concentration of nano-sized anticancer drugs in tumor tissues has proven superior in therapeutic effect with minimal side effects in both preclinical and clinical settings.
Of the tumor targeting strategies, the enhanced permeability and retention (EPR) effect of macrom... more Of the tumor targeting strategies, the enhanced permeability and retention (EPR) effect of macromolecules is a key mechanism for solid tumor targeting, and considered a gold standard for novel drug design. In this review, we discuss various endogenous factors that can positively impact the EPR effect in tumor tissues. Further, we discuss ways to augment the EPR effect by use of exogenous agents, as well as practical methods available in the clinical setting. Some innovative examples developed by researchers to combat cancer by the EPR mechanism are also discussed.
We had developed a H 2 O 2 generating enzyme, polyethylene glycol conjugated D-amino acid oxidase... more We had developed a H 2 O 2 generating enzyme, polyethylene glycol conjugated D-amino acid oxidase (PEG-DAO), which exhibited potent antitumor activity by generating toxic reactive oxygen species, namely oxidation therapy, subsequently showed remarkable antitumor effect on murine Sarcoma 180 solid tumor, by taking advantage of the enhanced permeability and retention effect. Along this line, we report here the preparation of PEG-DAO by use of recombinant DAO and its antitumor activity by using various tumor cell lines and tumor models. Recombinant DAO (rDAO) was obtained from E. coli BL21 (DE3) carrying the porcine DAO expression vector with high yield (20 mg/l) and high enzyme activity (5.3 U/mg). Pegylated rDAO (PEG-rDAO) showed high stability against sonication, repeated freezing/thawing, lyophilization and exhibited superior in vivo pharmacokinetics. PEG-rDAO had a molecular size of 65 kDa and existed as nanoparticles in aqueous solution with mean particle diameter of 119 nm.
Microcirculation (New York, N.Y. : 1994), Jan 3, 2015
Solid-tumor has unique vascular architecture, excessive production of vascular mediators, and ext... more Solid-tumor has unique vascular architecture, excessive production of vascular mediators, and extravasation of macromolecules from blood vessels into the tumor tissue interstitium. These features comprise the phenomenon named the EPR (enhanced permeability and retention) effect of solid tumors, described in 1986. Our investigations on the EPR revealed that many mediators, such as bradykinin, nitric oxide, and prostaglandins, are involved in the EPR effect, which is now believed to be the most important element for cancer-selective drug delivery. However, tumors in vivo manifest great diversity, and some demonstrate a poor EPR effect, for example, because of impaired vascular flow involving thrombosis, with poor drug delivery and therapeutic failure. Another important element of this effect is that it operates in metastatic cancers. Because few drugs are currently effective against metastases, the EPR effect offers a great advantage in nanomedicine therapy. The EPR effect can also be...
Previous studies indicated the potential of zinc protoporphyrin (ZnPP) as an antitumor agent targ... more Previous studies indicated the potential of zinc protoporphyrin (ZnPP) as an antitumor agent targeting to the tumor survival factor heme oxygenase-1, and/or for photodynamic therapy (PDT). In this study, to achieve tumor-targeted delivery, styrene-maleic acid-copolymer conjugated ZnPP (SMA-ZnPP) was synthesized via amide bond, which showed good water solubility, having ZnPP loading of 15%. More importantly, it forms micelles in aqueous solution with a mean particle size of 111.6 nm, whereas it has an apparent Mw of 65 kDa. This micelle formation was not detracted by serum albumin, suggesting it is stable in circulation. Further SMA-ZnPP conjugate will behave as an albumin complex in blood with much larger size (235 kDa) by virtue of the albumin binding property of SMA. Consequently, SMA-ZnPP conjugate exhibited prolonged circulating retention and preferential tumor accumulation by taking advantage of enhanced permeability and retention (EPR) effect. Clear tumor imaging was thus achi...
ABSTRACT Blood vessels in tumors are different to normal blood vessels because they have abnormal... more ABSTRACT Blood vessels in tumors are different to normal blood vessels because they have abnormal architectures and impaired functional regulation. We have studied these abnormalities, in particular vascular permeability in tumors, and found greatly enhanced permeability for macromolecules, which are retained in tumors for extended periods. We named this phenomenon the “enhanced permeability and retention(EPR) effect”. This effect, related to the transport of macromolecular drugs composed of liposomes, micelles, proteinaceous or polymer-conjugated macromolecules, lipid particles, and nanoparticles into the tumor, is the hallmark of solid tumor vasculature. These macromolecular species are therefore ideal for selective delivery to tumor. The EPR effect has facilitated the development of macromolecular drugs consisting of various polymer-drug conjugates (pendant type), polymeric micelles, and liposomes that exhibit far better therapeutic efficacy and far fewer side effects than the parent low-molecular-weight compounds. Here, we discuss various aspects of the EPR effect via examples, including the use of polymeric drugs such as SMANCS [poly(styrene-co-maleic acid-half-n-butylate) (SMA)-conjugated neocarzinostatin (NCS)]. In addition, we review our new macromolecular drug candidates that generate reactive oxygen species via anovel mode of action. Because solid tumors frequently lack antioxystress enzymes, generating oxystress in tumor tissue may be another unique anticancer strategy. Most tumor cells have aweak or limited defense system against reactive oxygen species, and the oxygen radical-generating techniques that we have developed are primarily endogenous. Consequently, an approach to cancer therapy based on the EPR effect and oxyradical induction in order to produce apoptosis appears promising.
High expression of the inducible isoform of heme oxygenase (HO-1) is now well known in solid tumo... more High expression of the inducible isoform of heme oxygenase (HO-1) is now well known in solid tumors in humans and experimental animal models. We reported previously that HO-1 may be involved in tumor growth (Tanaka et al., Br. J. Cancer, 88: 902-909, 2003), in that inhibition of HO activity in tumors by using zinc protoporphyrin (ZnPP) significantly reduced tumor growth in a rat model. We demonstrate here that poly(ethylene glycol)-conjugated ZnPP (PEG-ZnPP), a water-soluble derivative of ZnPP, exhibited potent HO inhibitory activity and had an antitumor effect in vivo. In vitro studies with cultured SW480 cells, which express HO-1, showed that PEG-ZnPP induced oxidative stress, and consequently apoptotic death, of these cells. Pharmacokinetic analysis revealed that PEG-ZnPP-administered i.v. had a circulation time in blood that was 40 times longer than that for nonpegylated ZnPP. More important, PEG-ZnPP preferentially accumulated in solid tumor tissue in a murine model. In vivo tr...
The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors, and ... more The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors, and it can serve as a basis for the development of macromolecular anticancer therapy. We have previously found that recombinant human serum albumin dimer, and especially its S-nitrosated form (SNO-HSA-Dimer), is an enhancer of the EPR effect. In this study, we investigated the influence of SNO-HSA-Dimer on the anti-tumor effect of two types of macromolecular anti-tumor drugs, namely N-(2-hydroxypropyl)methacrylamide polymer conjugated with zinc protoporphyrin, which forms micelles and can be used for fluorescence studies. The other was PEGylated liposomal doxorubicin (Doxil), a typical example of a stealth liposome approved for medical usage. In mice having C26 tumors with highly permeable vasculature, SNO-HSA-Dimer increases tumor accumulation of the drugs by a factor 3-4 and thereby their anti-tumor effects. Experiments with Evans blue revealed increased EPR effect in all parts of the tumor. Furthermore, SNO-HSA-Dimer improves the anti-metastatic effects of Doxil and reduces its minor uptake in non-tumorous organs such as liver and kidney. Tumor accumulation of Doxil in B16 tumors, which are characterized by a low permeable vasculature, increased even more (6-fold) in the presence of SNO-HSA-Dimer, and the improved accumulation lead to decreased tumor volume and increased survival of the animals. The administration of SNO-HSA-Dimer itself is safe, because it has no effect on blood pressure, heart rate or on several biochemical parameters. The present findings indicate that SNO-HSA-Dimer is promising for enhancing the EPR effect and consequently the specific, therapeutic effects of macromolecular anticancer drugs.
Cancer treatment alters microRNA (miRNA) expression, revealing potential therapeutic targets (onc... more Cancer treatment alters microRNA (miRNA) expression, revealing potential therapeutic targets (oncotarget). Here we treated pancreatic cancer (ASPC-1) cells with either recombinant human endostatin (rh-endostatin) or gemcitabine. Then high-throughput sequencing assay was performed to screen for altered miRNAs. Both treatments decreased levels of MiR-19a. We found that miR-19a stimulated cell proliferation, migration, invasion in vitro and tumor growth in vivo. High levels of miR-19a correlated with poor prognosis in patients. Ras homolog family member B (RHOB) was identified as a direct target of miR-19a. Furthermore, RHOB was down-regulated in human pancreatic cancer samples. Restoration of RHOB induced apoptosis, inhibited proliferation and migration of ASPC-1 cells. SP-1 was identified as an upstream transcription factor of miR-19a gene, promoting miR-19a transcription. Rh-endostatin decreased miR-19a expression by down-regulating SP-1. These findings suggest that miR-19a is a pot...
Although the thiol click reaction is an attractive tool for post-polymerization modification of th... more Although the thiol click reaction is an attractive tool for post-polymerization modification of thiolmers, thiol groups are easily oxidized, limiting the potential for covalent immobilization of bioactive molecules. In this study, a series of biodegradable polyurethane elastomers incorporating stable cyclic disulfide groups was developed and characterized. These poly(ester urethane)urea (PEUU-SS) polymers were based on polycaprolactone diol (PCL), oxidized DL-dithiothreitol (O-DTT), lysine diisocyanate (LDI) or butyl diisocyanate (BDI), with chain extension by putrescine. The ratio of O-DTT:PCL was altered to investigate different levels of potential functionalization. PEG acrylate was employed to study the mechanism and availability of both bulk and surface click modification of PEUU-SS polymers. All synthesized PEUU-SS polymers were elastic with breaking strengths of 38-45 MPa, while the PEUU-SS(LDI) polymers were more amorphous, possessing lower moduli and relatively small permane...
Cancer remains the first or second main cause of death in developed countries. In the world, 7.6 ... more Cancer remains the first or second main cause of death in developed countries. In the world, 7.6 million people died of cancer in 2005 [1]. However, the cure for advanced cancer in major cancers has not improved in the past 50 years, although chemotherapy is supposed to be a last resort, if not all [2,3]. One of the recent successful stories in cancer chemotherapy is imatinib (Gleevec®), a drug for chronic myeloid leukemia (CML) which is an inhibitor of BCR/ABL tyrosine kinase, a product of oncogene. Imatinib shows a remarkable therapeutic effect against CML while a natural course of life span of CML patients is about 5 years. However, upon blastic period when the leukemic cell growth becomes exponential, majority of patients developed drug resistance within 6 months. Therefore, one can conclude that imatinib contributes only 10% prolongation of the life span.
Induction of haem oxygenase-1 (HO-1) may provide an important protective effect for cells against... more Induction of haem oxygenase-1 (HO-1) may provide an important protective effect for cells against oxidative stress. Here, we investigated the mechanism of cytoprotection of HO-1 in solid tumour with a focus on the antiapoptotic activity of HO-1. Treatment of rat hepatoma AH136B cells with the HO inhibitor zinc protoporphyrin IX (ZnPP IX) or tin protoporphyrin IX resulted in extensive apoptotic changes of tumour cells both in vivo and in vitro. Caspase-3 activity of the ZnPP IX-treated hepatoma cells increased significantly. Moreover, ZnPP IX-induced apoptosis was completely inhibited by simultaneous incubation with a specific caspase-3 inhibitor and was partially abrogated by bilirubin, a reaction product of HO. In vivo ZnPP IX treatment did not affect nitric oxide (NO) production and tumour blood flow. Western blot analyses showed that HO-1 expression in AH136B cells was strongly upregulated by NO donors, for example, S-nitroso-N-acetyl penicillamine and propylamine NONOate in vitr...
Hydrogen peroxide (H(2)O(2)) is a strong oxidant that induces apoptosis of tumor cells in vitro. ... more Hydrogen peroxide (H(2)O(2)) is a strong oxidant that induces apoptosis of tumor cells in vitro. Here, we investigated the antitumor activity of an H(2)O(2)-generating enzyme, D-amino acid oxidase (DAO), and its conjugate with polyethylene glycol (PEG; PEG-DAO). Compared with DAO, PEG-DAO showed improved pharmacokinetic parameters in mice after i.v. injection. PEG-DAO administered i.v. accumulated selectively in tumor tissue with insignificant accumulation in normal organs and tissues. To generate cytotoxic H(2)O(2) at the tumor site, PEG-DAO was first administrated i.v. to tumor-bearing mice. After an adequate lag time, the substrate of DAO, D-proline, was injected i.p. This treatment resulted in significant suppression of tumor growth compared with tumor growth in control animals (not given treatment; P < 0.001). Similar treatment with native DAO showed no effect under the same conditions. Oxidative metabolites were significantly increased in solid tumors by administration of P...
Advances in experimental medicine and biology, 2003
Both enhanced vascular permeability and angiogenesis of tumor sustain rapid growth of tumor invol... more Both enhanced vascular permeability and angiogenesis of tumor sustain rapid growth of tumor involving many vascular mediators and high vascular density. On the contrary, however, they can be utilized for macromolecular drug delivery to tumor. Impaired reticuloendothelial/lymphatic clearance of macromolecules from the tumor, or lack of such clearance, is another unique characteristic of tumor tissue, which results intratumor retention of macromolecular drugs thus delivered (Figure 1). Consequently, enhanced permeability and retention (EPR) effect is the basis for the selective targeting of macromolecular drugs to tumor, and the EPR concept is now utilized for selective delivery of many macromolecular anticancer agents in aqueous formation for i.v. or i.a. as well as oily formation for i.a. dosing, which is not possible for low-molecular-weight drugs because of rapid washout by capillary vascular blood flow. This EPR concept has been validated in clinical settings with hepatoma and ot...
High expression of the inducible isoform of heme oxygenase (HO-1) is now well known in solid tumo... more High expression of the inducible isoform of heme oxygenase (HO-1) is now well known in solid tumors in humans and experimental animal models. We reported previously that HO-1 may be involved in tumor growth (Tanaka et al., Br. J. Cancer, 88: 902-909, 2003), in that inhibition of HO activity in tumors by using zinc protoporphyrin (ZnPP) significantly reduced tumor growth in a rat model. We demonstrate here that poly(ethylene glycol)-conjugated ZnPP (PEG-ZnPP), a water-soluble derivative of ZnPP, exhibited potent HO inhibitory activity and had an antitumor effect in vivo. In vitro studies with cultured SW480 cells, which express HO-1, showed that PEG-ZnPP induced oxidative stress, and consequently apoptotic death, of these cells. Pharmacokinetic analysis revealed that PEG-ZnPP-administered i.v. had a circulation time in blood that was 40 times longer than that for nonpegylated ZnPP. More important, PEG-ZnPP preferentially accumulated in solid tumor tissue in a murine model. In vivo tr...
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