By Julia Y. Ljubimova, MD, PhD
- Laminin-8 is dramatically increased in tumor microvessels of glial brain and breast tumors
- Brain tumors with laminin-8 overexpression recur faster after standard treatment and patients have shorter survival time
- A new non-viral drug delivery system, Polycefin, based on Physarum polycephalum-derived polymalic acid was developed
- In vivo laminin-8 blockage prolongs the animal survival
Glioblastoma multiforme prognoses and responses to therapy, vary greatly even with the same histological diagnosis [1]. It is generally recognized that the improvement of prognosis, prediction of response to treatment, and development of novel effective therapeutic approaches for glial tumors may largely depend upon the introduction into clinical practice of novel specific markers involved in the development gliomas and their subsequent recurrences.
In an earlier study using gene array technology, published in Cancer Research, we found that two genes were consistently up-regulated in all high-grade and low-grade gliomas and in tissues adjacent to GBMs, the most aggressive gliomas. One of the genes was already known to be over-expressed in gliomas. The other was the alpha-4 chain of laminin, a novel gene that had not been known to be overexpressed in any type of tumor. Laminins are heterogenic proteins, each of which are composed of three chains, alpha, beta and gamma, and about 15 laminins are currently known. Laminins are the major component of a thin "basement membrane" that lies beneath the surface layer of blood vessels. We found that during the tumor progression, laminin 9, which is expressed weakly in normal brain and low-grade glial tumors, switched to laminin 8. The data were validated by gene array analysis, semi-quantitative RT-PCR for RNA expression and immunohistochemistry for protein expression [2]. However, before new biopolymer drug-delivery method was recently developed, it was impossible to block the complicated proteins such laminins or collagens consisted from three different peptides.
Specific drug delivery is crucial for treating tumors and reducing side effects for normal cells. For direct targeting of cancer cells to treat tumors the drugs, e.g., monoclonal antibodies, antisense oligonucleotides or small molecules, such as Gefitinib (Iressa) or Erlotinib (Tarceva), should be able to penetrate the cell membrane. There are three basic methods for intracellular drug delivery, passive diffusion through aqueous channels or pores, passive diffusion of lipid-soluble drugs dissolved in a membrane, and carrier-mediated active transport (viral vectors, liposome-mediated gene transfer system, special chemicals) [3-5].
High-molecular-mass molecules recently caught special attention because tumors can selectively accumulate these molecules. This enhanced permeability and retention (EPR) effect was observed in cancer tissue for macromolecules and lipids (MW>45 kDa) [6-8] and is used in cancer treatment for specific drug delivery. Unlike currently used small molecule anticancer drugs, macromolecular (or polymeric) drugs can target tumors with high selectivity through the EPR effect [9-11]. Delivery to tumor vasculature was recently demonstrated using a synthetic polymer, polyacrylic acid (HPMA), conjugated with O-(chloracetil-carbomoyl) fumagillol (TNP-470) [9]. However, HPMA is non-biodegradable and does not pass through blood-brain barrier (BBB) [9]. Another promising drug carrier is ?-poly-L-malic acid (PMLA) [12-18]. This polymer is a natural product of the microorganism Physarum polycephalum [12-15]. The attractive properties of PMLA as a carrier matrix for biopharmaceuticals are its lack of toxicity in vitro and in vivo, non-immunogenicity, biodegradability, stability in the blood stream and easy cellular uptake [14] [16-19]. Most importantly, inhibitors of multiple molecular targets can be easily attached chemically to a single PMLA molecule [20]. A combined blocking of several molecular markers and their simultaneous delivery to inhibit tumor growth has always been a tempting approach in cancer therapy. However, it was not previously feasible because of technical difficulties; therefore, genes and proteins were usually blocked individually.
Previously, we have shown that laminin-8, which is an important component of basement membranes in newly formed tumor vessels [21-23], (Figure 1) was overexpressed in stage IV human glioma (glioblastoma multiforme, GBM) and ductal breast carcinoma. In vitro antisense inhibition of two laminin-8 chains (4 and 1) at the same time prevented the synthesis of laminin-8 more effectively that inhibition of each of these chains alone [24]. In this report, using a novel nanoscale PMLA-based drug, Polycefin, we were able to simultaneously deliver into the cells two different antisense oligonucleotides (AON) and block in vivo expression of two laminin-8 chains at the same time. Current formulation of Polycefin contains Morpholino AON conjugated to PMLA scaffold by disulfide bonds that are cleaved in the cytoplasm by glutathione to release the free drug. Additionally, a monoclonal antibody (mAb) to transferrin receptor was attached to PMLA scaffold to ensure AON targeting to endothelial and tumor cells. This receptor is significantly enriched on fast dividing, e.g., tumor cells, and endothelial cells of brain microvessels facilitating receptor-mediated endosomal uptake and blood-brain barrier (BBB) crossing [25-27].
Polycefin is a nanoscale drug delivery system containing poly-L-malic acid (the scaffold), and different functional units for target inhibition (AON in our case), endosomal uptake, escape from endosomes by membrane disruption, AON release into the cytoplasm by disulfide cleavage and drug imaging using a fluorescent reporter. The complete drug is shown schematically on The prototype (Polycefin, see below) and variant drug delivery devices are directed towards brain tumors. The device is built from biologically degradable and non-toxic molecular blocks [20]. Polycefin modules as shown in (Figure 2) are (1) Morpholino AON1 (to laminin a4 chain) and Morpholino AON2 (to laminin B1 chain) conjugated to the scaffold by disulfide bonds, which are cleaved in the cytoplasm to release the free drug, (2) antibody to transferrin receptor for cancer cell targeting and receptor-mediated endocytosis, (3) polyethylene glycol (PEG) for protection and accessibility of the tissue targeting moiety to bind to the receptor on the target cell surface, (4) L-valine to provide pH-dependent lipophilicity to disrupt endosomal membranes, and (5) optional fluorescent reporter dye for drug detection. The drug-releasing unit contains a disulfide bond, which is stable in blood circulation and in endosomes/lysosomes, but is cleaved naturally by cytoplasmic glutathione [20]. Blood-tumor barrier (BTB) and BBB are targeted by the conjugated mAb to the transferrin receptor on the endothelial cells [26-27]. Morpholino AON are used in Polycefin to specifically block the expression of vascular component, laminin-8, overexpressed in glial tumors. These AONs did not have any toxic effects on glioma cell lines U87MG and M059K in vitro [24].
This was performed for Polycefin and Polycefin(-mAb), without any tumor inoculation. Three systemic intravenous treatments each with Polycefin at 2.5 mg/kg and 5 mg/kg, respectively, were performed in 3 rats each (days 1, 4, and 7). After sacrificing animals in 40 days, all major organs such as heart, lungs, liver, kidneys, skin and brain were analyzed histologically for pathological changes. Gross and micro-pathological analysis revealed no abnormal changes in any tissues or organ. Animals did not develop any neurological abnormalities and their appetite was normal throughout the experiment. Similar results were obtained in separate experiments using naked Morpholino AON or PMLA or intracranial administration; all showed complete lack of toxicity.
The vessels in tumors and surrounding tissues were visualized by immunostaining for panendothelial markers, von Willebrand factor and CD31 [28] , with very similar results. Two markers were used because the existing data on the accuracy of a given endothelial antigen for vessel counting are contradictory and show significant differences in various tumor types [28]. Microvascular density in the xenotransplanted U87MG human tumors without treatment was significantly higher than in normal brain (Figure 3). After four intracranial Polycefin treatments tumor vessel density was significantly reduced compared to untreated tumors (p<0.001) and was similar to vessel density in normal adjacent brain tissue. The mean number of vessels per field was 25.7 in normal rat brain, 37.2 in tumor xenografts and 29.1 in tumors after drug treatment. Similar data were obtained regarding vessel area. It significantly increased in tumors vs. normal brain and then significantly decreased after treatment (not shown).
Human glioblastoma U87MG cells were injected intracranially into rats using a stereotactic device. The treatments with Polycefin or with Polycefin+sense/mock (Polycefin with AON substituted by sense oligonucleotides) started three days after inoculation, on days 3, 7, 10 and 14 (four treatments, Figure 4). Drug doses of 0.5 and 2.5 mg/kg gave similar results in the survival study. After intracranial administration of four doses of Polycefin, the animal survival time was significantly increased compared to mock-treated rats. The mean survival time was 32.0±1.5 days for mock-treated rats, and it was increased to 40.0±1.3 days for Polycefin-treated rats, p<0.0004 by Kaplan-Meier survival test (Figure 4). The tendency for prolonged survival was already observed after two treatments but the data were not significant. Polycefin(-mAb) did not affect animal survival (not shown), again suggesting that transferrin receptor-mediated endocytosis was responsible for AON delivery into glioma cells in vivo.
Polymers able to deliver inhibitory agents to tumor cells increasingly gain importance because they are less immunogenic than viral vectors, and therefore, more useful for repetitive treatments [9,10]. To prevent or inhibit tumor growth and progression, simultaneous inhibition of several molecular targets may be highly effective. One of the vehicles that can simultaneously carry drugs to several tumor targets is PMLA, which was successfully used here for the delivery of anti-angiogenic antisense oligonucleotides to glial tumors. The novel PMLA-based polymeric drug, Polycefin, was designed to deliver several biologically active AONs into tumor cells. Antisense oligonucleotides that bind and inactivate specific RNA sequences are efficient tools for inhibiting gene function and validating new therapeutic targets for drug development [29,30]. Synthetic, nuclease resistant AON variants that mimic natural AONs bind complementarily to mRNA and prevent translation of proteins such as tumor markers [35-36]. The most promising synthetic types of AON are Morpholinos and peptide nucleic acid (PNA) oligonucleotides being non-toxic and resistant to nucleases, specific and stable in the blood stream [30,31]. AON including Morpholinos have shown good efficacy in the in vitro studies but in vivo, their direct delivery to tumors and anti-tumor effects were less successful [31,32].
A recent, rapidly evolving other approach that leads to targeted elimination of mRNA is exemplified by small interfering RNAs (siRNAs) that are potent gene expression silencers and potential anticancer drugs [33,34]. However, siRNA are sensitive to cell nucleases in vivo, and resistant synthetic variants are not available. Delivery of naked siRNA appears to be very difficult without at least their encapsulation into liposomes in the form of DNA plasmid [33,34] , with known side effects for cells. The new drug Polycefin used here simultaneously carried two different Morpholino AONs to laminin-8, 4 and, 1 chains together with other moieties. The combination of these AONs was more effective in blocking the synthesis of a complex trimeric protein, laminin-8, than single AONs to either A4 or B1 chain in glioma cultures [24].
Polycefin with these two AONs efficiently prevented laminin-8 synthesis in vitro and in vivo.
Significant reduction of tumor vessel density and area as a result of inhibition of laminin-8, a structural component of newly formed tumor vessels, demonstrated the efficacy of this new drug for in vivo brain tumor treatment. This effect on tumor vasculature may be the underlying mechanism for an increased animal survival observed after a limited number of Polycefin treatments. Recently, treatment with drugs blocking receptors of several angiogenic growth factors was also shown to significantly increase survival of glioma-bearing rats [35].
Brain tumors are particularly difficult to treat with systemically administered drugs. This is due to BBB (also characteristic for brain tumors) that prevents penetration of water-soluble and ionized or polar compounds [33] [36-38]. As a result, local intracranial delivery of drugs including antiangiogenic drugs is widely used to treat brain tumor patients and is often more efficient than systemic delivery [38-42]. It allows one to bypass BBB and dramatically reduce effective drug doses [38-39]. Because of high clinical relevance of intracranial glioma treatment, our studies with Polycefin in glioma-bearing rats were mainly concentrated on this approach. Intracranially administered Polycefin efficiently and significantly increased survival of tumor-bearing animals and decreased tumor vascularity. Therefore, it seems to be a promising prototype drug to treat human gliomas as a single agent or in combination with other treatment methods.
Our data constitute a proof of principle that a non-toxic high-molecular-mass, PMLA-based Polycefin with anti-tumor AON to laminin-8 chains was able to effectively inhibit target vascular protein expression in vivo and in vitro and increase survival of tumor-bearing animals. Adding other anti-tumor drugs and/or other tumor targeting moieties, may increase the efficacy of the drug and achieve clinically significant effect in other tumors than brain as well.
Inhibition of laminin-8 expression using Polycefin interfered with neovascular development and had a beneficial effect on survival of tumor-bearing animals. These results hold promise for an efficient brain tumor treatment using laminin-8 as a therapeutic target alone or in combination with other molecular markers, or conventional chemotherapy. Because laminin-8 is also increased in breast cancer [21]. Polycefin could also be efficient for inhibition of breast cancer growth and invasion. The present data emphasize the feasibility of antisense approach using laminin-8 as a target for treatment of brain gliomas. In combination with other treatment methods or with blocking of other targets as well (EGFR, MMPs) it may prolong disease-free periods and increase survival of glioma patients. Future developments of laminin-8 blocking for therapeutic purposes may also include the use of specific monoclonal antibodies and/or small interfering RNA (siRNA) that is an emerging very promising approach for gene silencing.
In combination with several new well-characterized proteins associated with glioma progression, such as EGFR, tenascin-C, MMP-2 and MMP-9, [4,9] [15-19], or chemotherapy laminin-8 may be an important tool for potential diagnosis or treatment of gliomas.
Julia Y. Ljubimova, MD, is Director of Molecular Oncology at the Maxine Dunitz Neurosurgical Institute at Cedars-Sinai Medical Center in Los Angeles.
