Ronit Satchi-Fainaro

Targeting angiogenesis with a conjugate of HPMA copolymer and TNP-470.

Angiogenesis is crucial for tumor growth. Angiogenesis inhibitors, such as O-(chloracetyl-carbamoyl) fumagillol (TNP-470), are thus emerging as a new class of anticancer drugs. In clinical trials, TNP-470 slowed tumor growth in patients with metastatic cancer. However, at higher doses necessary for tumor regression, many patients experienced neurotoxicity. We therefore synthesized and characterized a water-soluble conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, Gly-Phe-Leu-Gly linker and TNP-470. This conjugate accumulated selectively in tumor vessels because of the enhanced permeability and retention (EPR) effect. HPMA copolymer–TNP-470 substantially enhanced and prolonged the activity of TNP-470 in vivo in tumor and hepatectomy models. Polymer conjugation prevented TNP-470 from crossing the blood-brain barrier (BBB) and decreased its accumulation in normal organs, thereby avoiding drug-related toxicities. Treatment with TNP-470 caused weight loss and neurotoxic effects in mice, whereas treatment with the conjugate did not. This new approach for targeting angiogenesis inhibitors specifically to the tumor vasculature may provide a new strategy for the rational design of cancer therapies.

A Unique Paradigm for a Turn-ON Near-Infrared Cyanine-Based Probe: Noninvasive Intravital Optical Imaging of Hydrogen Peroxide

The development of highly sensitive fluorescent probes in combination with innovative optical techniques is a promising strategy for intravital noninvasive quantitative imaging. Cyanine fluorochromes belong to a superfamily of dyes that have attracted substantial attention in probe design for molecular imaging. We have developed a novel paradigm to introduce a Turn-ON mechanism in cyanine molecules, based on a distinctive change in their π-electrons system. Our new cyanine fluorochrome is synthesized through a simple two-step procedure and has an unprecedented high fluorescence quantum yield of 16% and large extinction coefficient of 52,000 M(-1)cm(-1). The synthetic strategy allows one to prepare probes for various analytes by introducing a specific triggering group on the probe molecule. The probe was equipped with a corresponding trigger and demonstrated efficient imaging of endogenous hydrogen peroxide, produced in an acute lipopolysaccharide-induced inflammation model in mice. This approach provides, for the first time, an available methodology to prepare modular molecular Turn-ON probes that can release an active cyanine fluorophore upon reaction with specific analyte.

Administration, distribution, metabolism and elimination of polymer therapeutics.

Polymer conjugation is an efficient approach to improve the delivery of drugs and biological agents, both by protecting the body from the drug (by improving biodistribution and reducing toxicity) and by protecting the drug from the body (by preventing degradation and enhancing cellular uptake). This review discusses the journey that polymer therapeutics make through the body, following the ADME (absorption, distribution, metabolism, excretion) concept. The biological factors and delivery system parameters that influence each stage of the process will be described, with examples illustrating the different solutions to the challenges of drug delivery systems in vivo.

Targeting Bone Metastases with a Bispecific Anticancer and Antiangiogenic Polymer–Alendronate–Taxane Conjugate†

A polymer therapeutic designed for combination anticancer and antiangiogenic therapy inhibited the proliferation of prostate carcinoma cells and the proliferation, migration, and tube-formation of endothelial cells. The nanoconjugate was formed from an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, the bisphosphonate alendronate (for bone targeting), and the chemotherapy agent paclitaxel (PTX), which is cleaved by cathepsin B (see scheme).

In vivo delivery of small interfering RNA to tumors and their vasculature by novel dendritic nanocarriers

New targets for RNA interference (RNAi)‐based cancer therapy are constantly emerging from the increasing knowledge on key molecular pathways that are paramount for carcinogenesis. Nevertheless, in vivo delivery of small interfering RNA (siRNA) remains a crucial challenge for therapeutic success. siRNAs on their own are not taken up by most mammalian cells in a way that preserves their activity. Moreover, when applied in vivo, siRNA‐based approaches are all limited by poor penetration into the target tissue and low silencing efficiency. To circumvent these limitations, we have developed novel polymerized polyglycerol‐based dendrimer core shell structures to deliver siRNA to tumors in vivo. These cationic dendrimers can strongly improve the stability of the siRNA, its intracellular trafficking, its silencing efficacy, and its accumulation in the tumor environment owing to the enhanced permeability and retention effect. Here, we show that our dendritic nanocarriers exhibited low cytotoxicity and high efficacy in delivering active siRNA into cells. With use of human glioblastoma and murine mammary adenocarcinoma cell lines as model systems, these siRNA‐dendrimer polyplexes silenced the luciferase gene, ectopically overexpressed in these cells. Importantly, significant gene silencing was accomplished in vivo within 24 h of treatment with our luciferase siRNA‐nanocarrier polyplexes, as measured by noninvasive intravital bioluminescence imaging. Moreover, our siRNA‐nanocarriers show very low levels of toxicity as no significant weight loss was observed after intravenous administration of the polyplexes. We show a proof of concept for siRNA delivery in vivo using a luciferase‐based model. We predict that in vivo silencing of important cell growth and angiogenesis regulator genes in a selective manner will justify this approach as a successful anticancer therapy.—Ofek, P., Fischer, W., Calderón, M., Haag, R., Satchi‐Fainaro, R. In vivo deliveryof small interfering RNAto tumors and their vasculature by novel dendritic nanocarriers. FASEB J. 24, 3122–3134 (2010). www.fasebj.org

Integrin-assisted drug delivery of nano-scaled polymer therapeutics bearing paclitaxel

Angiogenesis plays a prominent role in cancer progression. Anti-angiogenic therapy therefore, either alone or in combination with conventional cytotoxic therapy, offers a promising therapeutic approach. Paclitaxel (PTX) is a widely-used potent cytotoxic drug that also exhibits anti-angiogenic effects at low doses. However, its use, at its full potential, is limited by severe side effects. Here we designed and synthesized a targeted conjugate of PTX, a polymer and an integrin-targeted moiety resulting in a polyglutamic acid (PGA)-PTX-E-[c(RGDfK)(2)] nano-scaled conjugate. Polymer conjugation converted PTX to a macromolecule, which passively targets the tumor tissue exploiting the enhanced permeability and retention effect, while extravasating via the leaky tumor neovasculature. The cyclic RGD peptidomimetic enhanced the effects previously seen for PGA-PTX alone, utilizing the additional active targeting to the α(v)β(3) integrin overexpressed on tumor endothelial and epithelial cells. This strategy is particularly valuable when tumors are well-vascularized, but they present poor vascular permeability. We show that PGA is enzymatically-degradable leading to PTX release under lysosomal acidic pH. PGA-PTX-E-[c(RGDfK)(2)] inhibited the growth of proliferating α(v)β(3)-expressing endothelial cells and several cancer cells. We also showed that PGA-PTX-E-[c(RGDfK)(2)] blocked endothelial cells migration towards vascular endothelial growth factor; blocked capillary-like tube formation; and inhibited endothelial cells attachment to fibrinogen. Orthotopic studies in mice demonstrated preferential tumor accumulation of the RGD-bearing conjugate, leading to enhanced anti-tumor efficacy and a marked decrease in toxicity as compared with free PTX-treated mice.

A 27-Amino-Acid Synthetic Peptide Corresponding to the NH2-Terminal Zinc-Binding Domain of Endostatin Is Responsible for Its Antitumor Activity

The first recombinant endostatin that elicited strong antitumor activity was expressed in Escherichia coli and administered as a suspension. Under these conditions, the protein retained its full antiangiogenic activity. Lack of requirement for a folded structure prompted us to investigate antitumor properties of synthetic peptides corresponding to different regions of endostatin. Here, we show that the entire antitumor, antimigration, and antipermeability activities of endostatin are mimicked by a 27-amino-acid peptide corresponding to the NH2-terminal domain of endostatin. This peptide contains three histidines that are responsible for zinc binding. Mutations of the zinc-binding histidines abolished its antitumor and antimigration activities, but not antipermeability properties.

Polymer therapeutics—polymers as drugs, drug and protein conjugates and gene delivery systems: Past, present and future opportunities

Welsh School of Pharmacy, Centre for Polymer Therapeutics, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK, University of Mainz, Institute of Organic Chemistry, Duesbergweg 10-14 D-55099, Mainz, Germany, and Vascular Biology Program, Department of Surgery, Children’s Hospital Boston and Harvard Medical School, 1 Blackfan Circle, Karp Family Research Laboratories, Floor 12, Boston, MA 02115, USA

Targeting angiogenesis-dependent calcified neoplasms using combined polymer therapeutics.

Background There is an immense clinical need for novel therapeutics for the treatment of angiogenesis-dependent calcified neoplasms such as osteosarcomas and bone metastases. We developed a new therapeutic strategy to target bone metastases and calcified neoplasms using combined polymer-bound angiogenesis inhibitors. Using an advanced “living polymerization” technique, the reversible addition-fragmentation chain transfer (RAFT), we conjugated the aminobisphosphonate alendronate (ALN), and the potent anti-angiogenic agent TNP-470 with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer through a Glycine-Glycine-Proline-Norleucine linker, cleaved by cathepsin K, a cysteine protease overexpressed at resorption sites in bone tissues. In this approach, dual targeting is achieved. Passive accumulation is possible due to the increase in molecular weight following polymer conjugation of the drugs, thus extravasating from the tumor leaky vessels and not from normal healthy vessels. Active targeting to the calcified tissues is achieved by ALNs affinity to bone mineral. Methods and Finding The anti-angiogenic and antitumor potency of HPMA copolymer-ALN-TNP-470 conjugate was evaluated both in vitro and in vivo. We show that free and conjugated ALN-TNP-470 have synergistic anti-angiogenic and antitumor activity by inhibiting proliferation, migration and capillary-like tube formation of endothelial and human osteosarcoma cells in vitro. Evaluation of anti-angiogenic, antitumor activity and body distribution of HPMA copolymer-ALN-TNP-470 conjugate was performed on severe combined immunodeficiency (SCID) male mice inoculated with mCherry-labeled MG-63-Ras human osteosarcoma and by modified Miles permeability assay. Our targeted bi-specific conjugate reduced VEGF-induced vascular hyperpermeability by 92% and remarkably inhibited osteosarcoma growth in mice by 96%. Conclusions This is the first report to describe a new concept of a narrowly-dispersed combined polymer therapeutic designed to target both tumor and endothelial compartments of bone metastases and calcified neoplasms at a single administration. This new approach of co-delivery of two synergistic drugs may have clinical utility as a potential therapy for angiogenesis-dependent cancers such as osteosarcoma and bone metastases.

Dendritic poly(ethylene glycol) bearing paclitaxel and alendronate for targeting bone neoplasms.

Poly(ethylene glycol) (PEG) is the most popular polymer for protein conjugation, but its potential as carrier of low molecular weight drugs has been limited by the intrinsic low loading, owing to its chemical structure. In fact, only the two end chain groups of PEG can be modified and exploited for drug coupling. We have demonstrated that by synthesizing a dendrimer structure at the polymer end chains, it is possible to increase the drug payload and overcome this limitation. Furthermore, this approach can be improved by using heterobifunctional PEG. These polymers allow the precise linking of two different drugs, or a drug and a targeting agent, on the same polymeric chain. Heterobifunctional PEG-dendrimers have been obtained with defined chemical structures leading to their attractive use as drug delivery systems. In fact, they offer a double benefit; first, the possibility to choose the best drug/targeting agent ratio, and second, the separation of the two functions, activity and targeting, which are coupled at the opposite polymer end chains. In this study, we investigated the role of a PEG-dendrimer, H(2)N-PEG-dendrimer-(COOH)(4), as carrier for a combination of paclitaxel (PTX) and alendronate (ALN). PTX is a potent anticancer drug that is affected by severe side effects originating from both the drug itself and its solubilizing formulation, Cremophor EL. ALN is an aminobiphosphonate used for the treatment of osteoporosis and bone metastases as well as a bone-targeting moiety. The PTX-PEG-ALN conjugate was designed to exploit active targeting by the ALN molecule and passive targeting through the enhanced permeability and retention (EPR) effect. Our conjugate demonstrated a great binding affinity to the bone mineral hydroxyapatite in vitro and an IC(50) comparable to that of the free drugs combination in human adenocarcinoma of the prostate (PC3) cells. The PTX-PEG-ALN conjugate exhibited an improved pharmacokinetic profile compared with the free drugs owed to the marked increase in their half-life. In addition, PTX-PEG-ALN could be solubilized directly in physiological solutions without the need for Cremophor EL. The data presented in this manuscript encourage further investigations on the potential of PTX-PEG-ALN as treatment for cancer bone metastases.