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

Abstract

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_vβ₃ 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_vβ₃-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.

Introduction

Pharmacological, molecular and genetic evidence demonstrate that tumor progression is angiogenesis-dependent [1], [2]. Cell adhesion mechanisms facilitating migration and invasion through the extracellular matrix are critical to the growth of new blood vessels [3]. Integrins, a class of receptors involved in cell adhesion, play a key role in cell matrix interactions and thus in angiogenesis. Integrin alterations are responsible for a number of pathological manifestations, such as defective embryogenesis, blood coagulation, osteoporosis, acute renal failure, retinopathy and cancer [4], [5].

α_vβ₃ integrin receptors are involved in angiogenesis and tumor invasiveness, essential in cell adhesion, motility, growth and differentiation [6]. α_vβ₃ integrin binds the Arg-Gly-Asp (RGD) sequence, which constitutes the recognition domain of adhesion proteins including laminin, fibronectin and vitronectin [7]. RGD peptidomimetics compete with extracellular matrix proteins to bind to integrin receptors [8]. α_vβ₃ receptors are expressed mainly on the luminal surface of the endothelial cell predominantly during angiogenesis, making it a compelling target for agents within the vascular space. In addition, α_vβ₃ integrin is overexpressed on proliferating tumor endothelial cells, as well as on various tumor cells, such as glioblastoma [9]. It is a marker of poor prognosis for some tumor types [10], [11]. The bis-cyclic peptide E-[c(RGDfK)₂] is a vascular-targeting ligand used in this study to actively and selectively target the chemotherapeutic drug paclitaxel (PTX) to tumor cells and their surrounding tumor endothelial cells via binding to α_vβ₃ integrin receptor.

The microtubule-interfering agent PTX is a clinically well-established and highly-effective anti-neoplastic drug used for the treatment of many carcinomas including prostate, breast, ovarian, and non-small cell lung cancer. It is also the drug of choice for the treatment of metastatic breast carcinoma. In addition to its anti-neoplastic activity, PTX exhibits anti-angiogenic and pro-apoptotic properties [11]. Due to its hydrophobic nature, PTX is solubilized in CremophorEL or ethanol, when used clinically, causing hypersensitivity reactions in addition to the severe side effects associated with PTX itself (such as neurotoxicity). Moreover, PTX’s poor pharmacokinetics (short half-life, low selectivity) lead to the fact that only a small amount of the drug localizes in the tumor. An additional limitation of PTX is that it is a substrate of efflux pumps [12], resulting in multiple drug resistance.

One of the most successful approaches to deliver PTX, has been the development of an albumin-based PTX nanoparticle named Abraxane^® (ABI-007, Celgene Corporation). It was approved by the FDA in 2004 for the treatment of breast cancer after the failure of combination chemotherapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy. PTX is physically entrapped in the Abraxane^® nanoparticle, leading to enhanced solubility of PTX and thus avoids harmful solubilizing agent [13].

A chemical conjugation of PTX to a carrier could offer pharmacological advantages. Conjugation of PTX with the non-degradable polymer, N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer showed improved pharmacokinetics and promising advantages [14]. The use of this strategy failed clinically due to premature release of PTX in the circulation causing a similar toxicity profile as free PTX. Cell Therapeutics Inc. (Seattle) took a different approach conjugating PTX with the biodegradable polyglutamic acid (PGA), OPAXIO™. It showed clinical benefits, such as safety superiority compared to free PTX, in paclitaxel-based cancer treatment alone or in combination with radiotherapy or other small drugs such as cisplatin [15], [16], [17].

PGA is a water-soluble multivalent polymer, which allows the conjugation of more than one compound or targeting residue within the polymer backbone. It is non-immunogenic, non-toxic, and biodegradable by cathepsin B [18], [19], [20], an enzyme that is highly expressed in most tumor tissues [21], [22], [23], [24]. In this manuscript, we evaluated the anti-angiogenic effect on top of the known anti-tumor effect of polymer conjugate of PTX. In addition, we improved PGA–PTX conjugate’s anti-tumor efficiency and widened its clinical applications. We designed and synthesized a PGA-PTX-E-[c(RGDfK)₂] conjugate that targets the tumor both (i) passively due to the enhanced permeability and retention (EPR) effect by virtue of its size and structure as PGA–PTX conjugate [25], and (ii) actively due to integrin binding.

We have characterized an original and superior targeted therapeutic synthetic nanoconjugate named PGA-PTX-E-[c(RGDfK)₂] with the potential to target angiogenesis [26], [27] detected by in vitro assays on endothelial cells. In addition, we evaluated its anti-tumor effect on cancer cell lines both in vitro and in vivo in tumor-bearing mice.