Richard W. Ahn

Folate-mediated intracellular drug delivery increases the anticancer efficacy of nanoparticulate formulation of arsenic trioxide

Arsenic trioxide (As2O3) is a frontline drug for treatment of acute promyelocytic leukemia and is in clinical trials for treatment of other malignancies, including multiple myeloma; however, efforts to expand clinical utility to solid tumors have been limited by toxicity. Nanoparticulate forms of As2O3 encapsulated in 100-nm-scale, folate-targeted liposomes have been developed to lower systematic toxicity and provide a platform for targeting this agent. The resultant arsenic “nanobins” are stable under physiologic conditions but undergo triggered drug release when the pH is lowered to endosomal/lysosomal levels. Cellular uptake and antitumor efficacy of these arsenic liposomes have been evaluated in folate receptor (FR)–positive human nasopharyngeal (KB) and cervix (HeLa) cells, as well as FR-negative human breast (MCF-7) tumor cells through confocal microscopy, inductively coupled plasma mass spectroscopy, and cytotoxicity studies. Uptake of folate-targeted liposomal arsenic by KB cells was three to six times higher than that of free As2O3 or nontargeted liposomal arsenic; the enhanced uptake occurs through folate-mediated endocytosis, leading to a 28-fold increase in cytotoxicity. In contrast, tumor cells with lower FR density on the surface (HeLa and MCF-7) showed much less uptake of the folate-targeted drug and lower efficacy. In cocultures of KB and MCF-7 cells, the folate-targeted arsenic liposomes were exclusively internalized by KB cells, showing high targeting specificity. Our studies further indicate that folate-targeted delivery of As2O3 with coencapsulated nickel(II) ions (as a nontoxic adjuvant) potentiates the As2O3 efficacy in relatively insensitive solid tumor–derived cells and holds the promise of improving drug therapeutic index. [Mol Cancer Ther 2009;8(7):1955–63]

Zinc sparks are triggered by fertilization and facilitate cell cycle resumption in mammalian eggs

In last few hours of maturation, the mouse oocyte takes up over twenty billion zinc atoms and arrests after the first meiotic division, until fertilization or pharmacological intervention stimulates cell cycle progression toward a new embryo. Using chemical and physical probes, we show that fertilization of the mature, zinc-enriched egg triggers the ejection of zinc into the extracellular milieu in a series of coordinated events termed zinc sparks. These events immediately follow the well-established series of calcium oscillations within the activated egg and are evolutionarily conserved in several mammalian species, including rodents and nonhuman primates. Functionally, the zinc sparks mediate a decrease in intracellular zinc content that is necessary for continued cell cycle progression, as increasing zinc levels within the activated egg results in the reestablishment of cell cycle arrest at metaphase. The mammalian egg thus uses a zinc-dependent switch mechanism to toggle between metaphase arrest and resumption of the meiotic cell cycle at the initiation of embryonic development.

A Novel Nanoparticulate Formulation of Arsenic Trioxide with Enhanced Therapeutic Efficacy in a Murine Model of Breast Cancer

Purpose: The clinical success of arsenic trioxide (As2O3) in hematologic malignancies has not been replicated in solid tumors due to poor pharmacokinetics and dose-limiting toxicity. We have developed a novel nanoparticulate formulation of As2O3 encapsulated in liposomal vesicles or “nanobins” [(NB(Ni,As)] to overcome these hurdles. We postulated that nanobin encapsulation of As2O3 would improve its therapeutic index against clinically aggressive solid tumors, such as triple-negative breast carcinomas. Experimental Design: The cytotoxicity of NB(Ni,As), the empty nanobin, and free As2O3 was evaluated against a panel of human breast cancer cell lines. The plasma pharmacokinetics of NB(Ni,As) and free As2O3 were compared in rats to measure drug exposure. In addition, the antitumor activity of these agents was evaluated in an orthotopic model of human triple-negative breast cancer. Results: The NB(Ni,As) agent was much less cytotoxic in vitro than free As2O3 against a panel of human breast cancer cell lines. In contrast, NB(Ni,As) dramatically potentiated the therapeutic efficacy of As2O3 in vivo in an orthotopic model of triple-negative breast cancer. Reduced plasma clearance, enhanced tumor uptake, and induction of tumor cell apoptosis were observed for NB(Ni,As). Conclusions: Nanobin encapsulation of As2O3 improves the pharmacokinetics and antitumor efficacy of this cytotoxic agent in vivo. Our findings demonstrate the therapeutic potential of this nanoscale agent and provide a foundation for future clinical studies in breast cancer and other solid tumors. Clin Cancer Res; 16(14); 3607–17. ©2010 AACR.

Biological evaluation of pH-responsive polymer-caged nanobins for breast cancer therapy.

A series of doxorubicin-loaded polymer-caged nanobins (PCN(DXR)) were evaluated in vivo in a murine MDA-MB-231 xenograft model of triple-negative breast cancer. The cross-linked polymer cage in PCN(DXR) offers protection for the drug payload while serving as a pH-responsive trigger that enhances drug release in the acidic environments commonly seen in solid tumors and endosomes. Varying the degree of cross-linking in the polymer cage allows the surface potential of PCN(DXR), and thus the in vivo circulation lifetime of the nanocarriers, to be tuned in a facile fashion. Given these design advantages, the present study provides the first in vivo evidence that PCN(DXR) can effectively inhibit tumor growth in a murine model of breast cancer. Importantly, PCN(DXR) was well-tolerated by mice, and drug encapsulation attenuated the toxicity of free doxorubicin. Taken together, this study demonstrates the potential utility of the PCN platform in cancer therapy.

Coencapsulation of Arsenic- and Platinum-based Drugs for Targeted Cancer Treatment

Arsenic- and platinum-based drugs are highly potent but also toxic agents used in cancer therapy.[1, 2] Arsenic trioxide (Trisenox®, As2O3) is a front-line drug for treatment of acute promyelocytic leukemia[3] and is in clinical trials for treatment of other malignancies, including multiple myeloma.[4] However, clinical outcomes of As2O3 in solid tumors have been poor in many cases,[1, 5] mainly due to limited bioavailability of the drug in the tumor site. Clinical application to solid tumors is also impeded by toxicity including neutropenia, liver failure and cardiac toxicity[1, 6] at higher doses.[7] Cisplatin (cis-diamine dichloroplatinumII, cisPt, Figure 1a) is commonly used in the treatment of a variety of solid tumors, including lung, ovarian, bladder, and testicular cancer.[2] The active intracellular species appear to be the hydrolyzed monoaqua- and diaqua-cisplatin (aqua-cisPt, Figure 1a).[8] Broader therapeutic applications of cisPt are limited by serious systemic toxicities, development of drug resistance, and rapid inactivation of the drug due to complexation with plasma and tissue proteins.[2, 8] These problems can be reduced by using a drug delivery system that prevents drug deactivation, extends the circulation time of drug in blood and increases its accumulation at tumor sites.[9] Lipid-based carriers have been successfully applied in clinics for improving the therapeutic efficacy of numerous drugs, such as liposomal doxorubicin (Doxil®),[10] mainly via the enhanced permeability and retention (EPR) effects.[9]

Development of novel therapeutics targeting the urokinase plasminogen activator receptor (uPAR) and their translation toward the clinic.

The urokinase plasminogen activator receptor (uPAR) mediates cell motility and tissue remodeling. Although uPAR may be expressed transiently in many tissues during development and wound healing, its constitutive expression appears to be associated with several pathological conditions, including cancer. uPAR expression has been demonstrated in most solid tumors and several hematologic malignancies including multiple myeloma and acute leukemias.Unlike many tumor antigens, uPAR is present not only in tumor cells but also in a number of tumor-associated cells including angiogenic endothelial cells and macrophages. The expression of uPAR has been shown to be fairly high in tumor compared to normal, quiescent tissues, which has led to uPAR being proposed as a therapeutic target, as well as a targeting agent, for the treatment of cancer. The majority of therapeutic approaches that have been investigated to date have focused on inhibiting the urokinase plasminogen activator (uPA)-uPAR interaction but these have not led to the development of a viable uPAR targeted clinical candidate. Genetic knockdown approaches e.g. siRNA, shRNA focused on decreasing uPAR expression have demonstrated robust antitumor activity in pre-clinical studies but have been hampered by the obstacles of stability and drug delivery that have limited the field of RNA nucleic acid based therapeutics. More recently, novel approaches that target interactions of uPAR that are downstream of uPA binding e.g. with integrins or that exploit observations describing the biology of uPAR such as mediating uPA internalization and signaling have generated novel uPAR targeted candidates that are now advancing towards clinic evaluation. This review will discuss some of the pitfalls that have delayed progress on uPAR-targeted interventions and will summarize recent progress in the development of uPAR-targeted therapeutics.

Nano-Encapsulation of Arsenic Trioxide Enhances Efficacy against Murine Lymphoma Model while Minimizing Its Impact on Ovarian Reserve In Vitro and In Vivo

Advances in cancer therapy have increased the rate of survival of young cancer patients; however, female lymphoma patients frequently face a temporary or permanent loss of fertility when treated with traditional cytotoxic agents. The potential loss of fertility is an important concern that can influence treatment decisions for many premenopausal cancer patients. The negative effect of chemotherapeutic agents and treatment protocols to patients’ fertility–referred to as fertotoxicity–are thus an increasingly important cancer survivorship issue. We have developed a novel nanoscale formulation of arsenic trioxide, a potent drug for treatment of hematological malignancies, and demonstrate that it has significantly better activity in a murine lymphoma model than the free drug. In parallel, we have developed a novel in vitro assay of ovarian follicle function that predicts in vivo ovarian toxicity of therapeutic agents. Our results reveal that the nanotherapeutic agent is not only more active against lymphoma, but is fertoprotective, i.e., it is much less deleterious to ovarian function than the parent drug. Thus, our in vitro assay allows rapid evaluation of both established and experimental anticancer drugs on ovarian reserve and can inform the selection of efficacious and fertility-sparing treatment regimens for reproductive-age women diagnosed with cancer.

MDCT of chest, abdomen, and pelvis using attenuation-based automated tube voltage selection in combination with iterative reconstruction: an intrapatient study of radiation dose and image quality.

OBJECTIVE The purpose of this study was intrapatient comparison of image quality and radiation dose between MDCT scans of the chest, abdomen, and pelvis obtained with attenuation-based automated kilovoltage selection and sinogram-affirmed iterative reconstruction and scans obtained with standard kilovoltage selection and a filtered backprojection image reconstruction algorithm. MATERIALS AND METHODS One hundred one oncology patients who had undergone two chest, abdominal, and pelvis CT scans within 1 year were imaged with standard tube voltage selection of 120 kVp using a filtered backprojection reconstruction algorithm (protocol 1) and with attenuation-based automated tube voltage selection using an iterative reconstruction algorithm (protocol 2). Radiation dose parameters (volumetric CT dose index [CTDIvol], dose-length product, and effective dose) as well as image noise, signal-to-noise ratio, and contrast-to-noise ratio were compared. Two independent radiologists evaluated image quality and sharpness. Student t test, Fisher exact test, and Wilcoxon signed-rank test were used for analysis. A p value less than 0.05 was considered significant. RESULTS Mean ± SD CTDIvol values were 19.9 ± 4.43 mGy and 12.53 ± 4.79 mGy for protocols 1 and 2, respectively (p < 0.0001). Effective dose was 38.2% lower on average using protocol 2 compared with protocol 1 (12.08 vs 19.55 mSv; p < 0.0001). Objective image quality parameters were significantly better in protocol 2 (p < 0.0001). Both radiologists found the overall image quality and sharpness to be similar for both protocols (p > 0.05). CONCLUSION In patients undergoing CT examination of the chest, abdomen, and pelvis, the combination of attenuation-based automated tube voltage selection with iterative reconstruction significantly reduced radiation dose parameters and maintained objective image quality when compared with standard tube voltage selection associated with filtered backprojection reconstruction.

The many spaces of uPAR: delivery of theranostic agents and nanobins to multiple tumor compartments through a single target.

The urokinase plasminogen activator (uPA) system is a proteolytic system comprised of uPA, a cell surface receptor for uPA (uPAR), and an inhibitor of uPA (PAI-1) and is implicated in many aspects of tumor growth and metastasis. The uPA system has been identified in nearly all solid tumors examined to date as well as several hematological malignancies. In adults, transient expression of the uPA system is observed during wound healing and inflammatory processes while only limited expression is identified in healthy, quiescent tissue. Members of the uPA system are expressed not only on cancer cells but also on tumor-associated stromal cells. These factors make the uPA system an ideal therapeutic target for cancer therapies. To date most therapeutics targeted at the uPA system have been inhibitors of either the uPA-uPAR interaction or uPA proteolysis but have not shown robust anti-tumor activity. There is now mounting evidence that uPAR participates in a complex signaling network central to its role in cancer progression, which provides a basis for the hypothesis that uPAR may be a marker for cancer stem cells. Several new uPAR-directed therapies have recently been developed based on this new information. A monoclonal antibody has been developed that disrupts the interactions of uPAR with signaling partners and is poised to enter the clinic. In addition, nanoscale drug delivery vehicles targeted to the uPA system using monoclonal antibodies, without disrupting the normal functioning of the system, are also in development. This review will highlight some of these new discoveries and the new uPA system-based therapeutic approaches that have arisen from them.

Robust Structure and Reactivity of Aqueous Arsenous Acid- Platinum(II) Anticancer Complexes**

The first molecular adducts of platinum and arsenic based anticancer drugs - arsenoplatins - show unanticipated structure, substitution chemistry, and cellular cytotoxicity. The PtII-AsIII bonds in these complexes are stable in aqueous solution and strongly influence the lability of the trans ligand.