Permeability In Solid Tumors Research Articles

Photoactivated Organic Nanomachines for Programmable Enhancement of Antitumor Efficacy.

Limited permeability in solid tumors significantly restricts the anticancer efficacy of nanomedicines. Light-driven nanomotors powered by photothermal converting engines are appealing carriers for directional drug delivery and simultaneous phototherapy. Nowadays, it is still a great challenge to construct metal-free photothermal nanomotors for a programmable anticancer treatment. Herein, one kind of photoactivated organic nanomachines is reported with asymmetric geometry assembled by light-to-heat converting semiconducting polymer engine and macromolecular anticancer payload through a straightforward nanoprecipitation process. The NIR-fueled polymer engine can be remotely controlled to power the nanomachines for light-driven thermophoresis in the liquid media and simultaneously thermal ablating the cancer cells. The great manipulability of the nanomachines allows for programming of their self-propulsion in the tumor microenvironment for effectively improving cellular uptake and tumor penetration of the anticancer payload. Taking the benefit from this behavior, a programmed treatment process is established at a low drug dose and a low photothermal temperature for significantly enhancing the antitumor efficacy.

PEG Linker Improves Antitumor Efficacy and Safety of Affibody-Based Drug Conjugates.

Antibody drug conjugates (ADCs) have become an important modality of clinical cancer treatment. However, traditional ADCs have some limitations, such as reduced permeability in solid tumors due to the high molecular weight of monoclonal antibodies, difficulty in preparation and heterogeneity of products due to the high drug/antibody ratio (4–8 small molecules per antibody). Miniaturized ADCs may be a potential solution, although their short circulation half-life may lead to new problems. In this study, we propose a novel design strategy for miniaturized ADCs in which drug molecules and small ligand proteins are site-specifically coupled via a bifunctional poly(ethylene glycol) (PEG) chain. The results showed that the inserted PEG chains significantly prolonged the circulation half-life but also obviously reduced the cytotoxicity of the conjugates. Compared with the conjugate ZHER2-SMCC-MMAE (HM), which has no PEG insertion, ZHER2-PEG4K-MMAE (HP4KM) and ZHER2-PEG10K-MMAE (HP10KM) with 4 or 10 kDa PEG insertions have 2.5- and 11.2-fold half-life extensions and 4.5- and 22-fold in vitro cytotoxicity reductions, respectively. The combined effect leads to HP10KM having the most ideal tumor therapeutic ability at the same dosages in the animal model, and its off-target toxicity was also reduced by more than 4 times compared with that of HM. These results may indicate that prolonging the half-life is very helpful in improving the therapeutic capacity of miniaturized ADCs. In the future, the design of better strategies that can prolong half-life without affecting cytotoxicity may be useful for further improving the therapeutic potential of these molecules.

278 - Treatment of Tumor Hypoxia by Anticancer Prodrug Possessing High Permeability in Solid Tumor

Hypoxia is a feature of solid tumors and tumor hypoxia is known to promote mutagenesis, invasiveness. A strategy for targeting the hypoxic region of tumors is highly required; however, several issues such as adverse effects and poor penetration property of drugs have hampered developments. In this study, we developed a novel system in which antitumor prodrug is inactivated by the modification with the functional group and reduces the adverse effect to normal tissues which are generally present under normoxia condition but releases active anticancer drug under hypoxic condition. We have applied this technology to several anticancer drugs. In vivo experiments, doxorubicin prodrug (pro-Dox) was evaluated using tumor bearing mice and significant anti-tumor effect of pro-Dox was observed. Importantly, administration of pro-Dox did not show any apparent adverse effect and drastically improved survival rate of mice. Immunofluorescence analyses confirmed that the designed prodrug localized at the hypoxic region, in contrast to conventional anticancer drugs, which localized only at the normoxic region. These results indicate that the developed prodrug drastically improved the therapeutic effect by reducing adverse effects, improving drug penetration, and specifically activating the drug at hypoxic regions

Targeting and Treatment of Tumor Hypoxia by Newly Designed Prodrug Possessing High Permeability in Solid Tumors.

Tumor hypoxia, which is associated with poor prognosis in cancer, is known to lead to resistance to radiotherapy and anticancer chemotherapy. Impaired drug penetration in hypoxic regions has been recognized as an essential barrier to drug development in solid tumors. Here, we propose novel hypoxia-activated prodrugs, which drastically improved the penetration property of commonly used anticancer drugs in the hypoxic region. In this design, conventional anticancer drugs were modified with 2-nitroimidazole derivatives. The most important point of this study was that the prodrug designed formed a 6-membered cyclic structure to allow liberation of the active drug in the hypoxic region. This design markedly increased the selectivity of the hypoxia-targeted prodrug, resulting in significant reduction of adverse effects in the normoxic region. In vitro studies confirmed the selective activation under hypoxic conditions. In vivo studies showed drastic reduction of adverse effects associated with conventional anticancer drugs and improvement of the survival rate of mice. Immunofluorescence analyses confirmed that the designed prodrug had a tendency to localize at the hypoxic region, in contrast to conventional anticancer drugs, which localize only at the normoxic region.

Abstract 2307: Small GTPase R-Ras promotes normalization of tumor vasculature: a potential oxygen-sensing independent mechanism.

Abstract Newly formed blood vessels in tumors fail to mature into fully functional vessels due to the chronically angiogenic microenvironment. The functional impairment of these vessels hampers drug delivery, thereby diminishing the efficacy of anti-tumor therapies. Excessive vessel permeability associated with tumors also causes clinical complications such as cerebral edema in brain cancer patients. The ability to control vessel maturity in tumors, therefore, provides a potential therapeutic opportunity. We show that R-Ras is essential for the establishment of mature, functional blood vessels in tumors. R-Ras is a small GTPase of the Ras family with antiangiogenic activity. We found that malformation and malfunction of tumor vessels are exacerbated by genetic disruption of R-Ras. R-Ras-deficient vessels exhibited severely impaired pericyte and basement membrane supports, disrupted adherens junctions, and excessive blood leakage in tumors. Tumor cell penetration into the blood circulation increased in R-Ras-deficient mice, supporting a role for R-Ras in controlling tumor vessel permeability. Decreased blood perfusion of tumor vessels in R-Ras-deficient mice elevated tumor hypoxia, which in turn led to decreased efficacy of radiotherapy in these mice. In contrast to R-Ras disruption, R-Ras gain-of-function improved vessel structure and blood perfusion and blocked plasma leakage by enhancing endothelial barrier function and pericyte association with nascent blood vessels. The studies of cell type-specific expression of R-Ras revealed that R-Ras in endothelial cells and in pericytes both contribute individually to the vessel regulation. For instance, endothelial R-Ras limits vascular permeability by inhibiting VE-cadherin internalization induced by VEGF without affecting VE-cadherin gene expression level. These findings indicate that R-Ras is critical for controlling blood circulation and vascular permeability in solid tumors. It has been shown that haplodeficiency of the oxygen-sensor PHD2 leads to normalization of tumor vasculature through stabilization of endothelial HIF-2 and consequent upregulation of soluble VEGFR1 and VE-cadherin gene expression in endothelial cells. Our studies of endothelial cells in hypoxia and normoxia indicate that R-Ras is not involved in oxygen-sensing mechanisms of vessel regulations mediated by the PHD2/HIF-2 pathway. There are important differences between the vessel regulations by PHD2/HIF-2 and by R-Ras. PHD2 haplodeficiency does not affect vessel density, area, or vessel dilation while it normalizes the endothelial barrier and stability. In comparison, R-Ras does normalize all of these vessel parameters while halting angiogenic sprouting. Our observations suggest that an alternative, oxygen sensing-independent vascular normalization phenomenon. Citation Format: Masanobu Komatsu, Junko Sawada, Takeo Urakami, Fangfei Li, Akane Urakami. Small GTPase R-Ras promotes normalization of tumor vasculature: a potential oxygen-sensing independent mechanism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2307. doi:10.1158/1538-7445.AM2013-2307

TRANSLATIONAL IMPACT

In patients with locally advanced solid tumors, the first-line treatment is often neo-adjuvant or pre-operative chemotherapy, which helps shrink tumors before surgery, allowing for more conservative surgical approaches and reducing the potential for developing systemic disease. However, despite aggressive chemotherapy, long-term survival for many patients remains poor, in part owing to limitations with the targeting and accumulation of cytotoxic drugs in tumor tissue.The vasculature of solid tumors is abnormal, both in terms of vessel architecture and the dynamics of blood flow. Permeable heterogeneous vessel walls allow the leakage of proteins and fluid that, coupled with the inefficiency of lymphatic drainage, could be exploited to develop novel, enhanced drug delivery strategies that are therapeutically selective and improve clinical outcome.This work describes a previously unappreciated role for transforming growth factor beta (TGFβ) in regulating vascular stability and vessel permeability in solid tumors. Using mouse models, the authors demonstrate an endogenous pathway that regulates normal vascular permeability, which is controlled by perivascular collagen, the metalloproteinase enzyme MMP14, and TGFβ. In wild-type mice, inhibitors of either MMP14 or TGFβ signaling induce blood vessel permeability. Conversely, enhanced MMP14 or TGFβ activity in the mouse epidermis decreases leakage across cutaneous vessels. This pathway remains functional during tumor progression, as acute blockade of either MMP14 or TGFβ signaling transiently alters vessel stability, ‘opening’ vascular beds and promoting intravenous delivery of high molecular weight compounds to the tumor.The delivery of standard therapeutic agents or diagnostic molecular imaging agents to tumor tissue may be enhanced by transient blockade of the TGFβ pathway. If so, this could advance disease therapy and/or diagnostic imaging, not only in cancer medicine, but also in fibrotic disorders such as scleroderma and kidney failure.

Hyperthermia and Thermosensitive Liposomes for Improved Delivery of Chemotherapeutic Drugs to Solid Tumors

Lipid-based nanocarriers or liposomes have been proven successful in the delivery of chemotherapeutic agents and are currently applied clinically in the treatment of various types of cancer. Liposomes offer the advantage of a high drug payload, decreased drug toxicity and enhanced drug accumulation at tumor sites. Increased accumulation is due to the relatively leaky tumor vasculature that allows liposome extravasation. Between different types of tumors and even within one tumor, vascular permeability and thus liposome extravasation may differ greatly. Furthermore, upon accumulation of liposomes in the tumor area, drug bioavailability is not guaranteed. At present, these are the major issues for clinically used liposomal drugs.Mild hyperthermia (HT), the heating of tumor tissue to temperatures of up to 43°C, has been developed in the past decades as an established and efficacious treatment modality in combination with chemo- and radiotherapy. HT can be used to further improve liposomal chemotherapy in two ways: HT is known to increase vascular permeability in solid tumors and may therefore increase levels of liposome accumulation, and thermosensitive liposomes have been developed that can be triggered to release their contents upon hyperthermia. By applying these two strategies, drug delivery to tumors can be strongly enhanced.

Inhibition of Vascular Endothelial Growth Factor-A Signaling Induces Hypertension: Examining the Effect of Cediranib (Recentin; AZD2171) Treatment on Blood Pressure in Rat and the Use of Concomitant Antihypertensive Therapy

Inhibition of vascular endothelial growth factor-A (VEGF) signaling is a key therapeutic approach in oncology given the role of VEGF in angiogenesis and vascular permeability in solid tumors. Clinical trials examining VEGF signaling inhibitors commonly report hypertension. We examined the effect of cediranib, a highly potent VEGF signaling inhibitor, on the blood pressure of rats and the ability of standard antihypertensive agents to modulate the consequences of VEGF signaling inhibition. The ability of cediranib to induce hypertensive changes and the effect of giving antihypertensive therapy were investigated in conscious, unrestrained telemetered rats. Two antihypertensive agents were studied: captopril, an angiotensin-converting enzyme inhibitor, and nifedipine, a dihydropyridine calcium channel blocker. The antitumor activity of cediranib, alone and in combination with nifedipine, was also evaluated in a LoVo human colorectal tumor xenograft model in nude rats. All treatments were given orally. Administration of 0.1 to 1.5 mg/kg/d of cediranib for 4 consecutive days induced a relatively mild hypertensive effect, elevating diastolic blood pressure by 10 to 14 mmHg. Dosing 3 mg/kg/d cediranib for 4 days induced a marked hypertension of 35 to 50 mmHg. Captopril (30 mg/kg, qd) was effective at lowering a 10 mmHg increase in blood pressure but not a 35 to 50 mmHg increase. However, the latter was rapidly reversed by administration of nifedipine (10 mg/kg, bd). Coadministration of nifedipine did not negatively affect the antitumor activity of cediranib (1.5 mg/kg/d). Hypertension is a direct consequence of inhibiting VEGF signaling but can be controlled with appropriately selected, standard antihypertensive medication.

Heme oxygenase and nitric oxide synthase on tumor growth

It is well known that heme oxygenase-1 (HO-1) as well as nitric oxide (NO) synthase expressed in tumor cell line and solid tumor. NO may be beneficial to the tumor growth, not only because enhanced vascular permeability in solid tumor is mediated by NO, but because the l-arginine-dependent NO pathway mediates angiogenic activity and vascular endothelial growth factor (VEGF) induces angiogenesis via formation of NO. However, NO has a powerful cytostatic action on tumor cells by inhibiting DNA synthesis and causing oxidative injury. While it has been suggested that induction of HO-1 may provide an important protective response by cells against oxidative damage, HO-1 expression in tumor cells is strongly enhanced by NO donor. Strong induction of HO-1 is observed in solid tumors after occlusion or embolization of the tumor-feeding artery, indicating that ischemic stress which may involve oxidative stress triggers HO-1 induction. In addition, it is of great importance that an HO inhibitor, zinc protoporphyrin IX, suppressed the tumor growth to a great extent. In conclusion, HO-1 expression in the solid tumor may confer resistance of tumor cells to hypoxic stress as well as to NO-mediated cytotoxicity.

Enhanced vascular permeability in solid tumor involving peroxynitrite and matrix metalloproteinases.

Peroxynitrite (ONOO ‐), which is generated from nitric oxide (NO) and superoxide anion (O2‐) under pathological conditions, plays an important role in pathophysiological processes. Activation of matrix metalloproteinases (MMPs) contributes to tumor angiogenesis and metastasis. NO mediates the enhanced vascular permeability and retention (EPR) effect in solid tumors, and ONOO‐ activates proMMP to MMP in vitro. In this study, we examined the role of ONOO‐ in the EPR effect in solid tumors and normal tissues as related to MMP activation. Authentic ONOO‐, at 50 nmol or higher concentrations, induced the enhanced vascular permeability in normal dorsal skin of mice. ONOO‐ scavengers ebselen and uric acid significantly suppressed the EPR effect in mouse sarcoma 180 (S‐180) tumors. Indirect evidence for formation of ONOO‐ in S‐180 and mouse colon adenocarcinoma (C‐38) tumors included strong immunostaining for nitrotyrosine in the tumor tissue, predominantly surrounding the tumor vessels. MMP inhibitor BE16627B (66.6 mg/kg i.v., given 2 tunes) or SI‐27 (10 mg/kg i.p., given 2 times) significantly suppressed the ONOO‐induced EPR effect in S‐180 tumors and in normal skin. Soybean trypsin inhibitor (Kunitz type), broad‐spectrum proteinase inhibitor ovomacroglobulin, and bradykinin receptor antagonist HOE 140 also significantly suppressed the ONOO‐induced EPR effect in normal skin tissues. These data suggest that ONOO‐ may be involved in and promote the EPR effect in tumors, which could be mediated partly through activation of MMPs and a subsequent proteinase cascade to generate potent vasoactive mediators such as bradykinin.

Excessive production of nitric oxide in rat solid tumor and its implication in rapid tumor growth

Rapid tumor growth is caused by angiogenesis factors, growth factors, etc. We previously reported a possible connection between nitric oxide (NO) and enhanced vascular permeability in solid tumor. In the present experiment, the role of NO in solid tumor pathology was further investigated in animal tumor. To identify NO formed in solid tumor (AH136B) implanted in the feet of rats, electron paramagnetic resonance (EPR) spectroscopy was performed directly on the frozen tumor tissue at 110K by measuring endogenous nitrosyl iron-sulfur complexes, and by using exogenously added NO capturing agents, i.e., diethyldithiocarbamate (DETC)-Fe2+ and N-(dithiocarboxy) sarcosine (DTCS)-Fe2+ complexes. Induction of inducible isoform of nosymthase iNOS mRNA was examined with reverse transcriptase-polymerase chain reaction (RT-PCR) combined with Southern blot analysis. In addition, vascular permeability was assessed by measuring extravasation of 51Cr-labeled bovine serum albumin in solid tumor. Strong EPR signals from NO adducts of DETC-Fe2+ and DTCS-Fe2+ as well as strong signals from NO-hemoglobin and dinitrosyl iron sulfur complex were generated by tumor. The signal height of NO-(DTCS)2-Fe2+ observed in AH136B solid tumor was increased as the tumor gained up to 1.75 g. Induction of iNOS mRNA expression was confirmed by the above methods. Enhanced vascular permeability was suppressed by NOS inhibitors N omega- monomethyl-L-arginine or S-methylisothiourea sulfate and augmented with administration of L-arginine. Excessive NO production by iNOS in solid tumor was identified unequivocally by EPR spectroscopy. NO formed in solid tumor can be involved in enhanced vascular permeability and increased blood flow, and hence sustain tumor growth.

Multiple functions of nitric oxide in pathophysiology and microbiology: analysis by a new nitric oxide scavenger.

A wide range of biological functions of nitric oxide (NO) was analyzed using a newly discovered nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide (PTIO) or its water-soluble derivative carboxy-PTIO. The chemistry is very simple in that NO was oxidized by PTIO, yielding one mole each of NO2 and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl. Based on the potent NO-scavenging activity of PTIO derivatives, the diverse functions of NO under physiological states as well as various pathological conditions such as endotoxin shock and viral diseases are now explicated. It was found that PTIO and carboxy-PTIO showed significant inhibitory activity against a series of biological actions of NO: (1) endothelium-dependent vascular relaxation in an ex vivo system, (2) pathogenicity of NO produced excessively in endotoxin shock in rats and in influenza virus pneumonitis in mice, and (3) enhanced vascular permeability in solid tumors mediated by NO. PTIO directly extinguishes NO generated by NO synthase (NOS) without affecting NOS activity, which is a clear contrast to NOS inhibitors. Therefore, characterization of this unique mode of action of PTIO appears to be helpful not only in understanding of the pathophysiological role of NO but also in the treatment of various diseases caused by excessive production of NO.

Enhanced vascular permeability in solid tumor is mediated by nitric oxide and inhibited by both new nitric oxide scavenger and nitric oxide synthase inhibitor.

A newly discovered nitric oxide radical scavenger, an imidazolineoxyl N‐oxide derivative, was used to investigate the role of nitric oxide radical (*NO) in the vascular permeability enhancement of solid tumor. Sarcoma‐180 solid tumor in ddY mice was used for this experiment. Electron spin resonance spectroscopy was used to quantitate the reacted and unreacted scavenger. The results showed that extensive extravasation, assessed by intravenous injection of Evans blue, could be greatly suppressed by both *NO scavenger administered orally and *NO synthase inhibitor administrated intraperitoneally. This indicates that *NO is responsible for the vascular permeability in solid tumors.