Patented nanomedicines for the treatment of brain ...

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Patented nanomedicines for the treatment of brain tumors Gerardo Caruso*, Giuseppe Raudino & Maria Caffo Patients affected by malignant brain tumors present an extremely poor prognosis, notwithstanding improvements in surgery techniques and therapeutic protocols. Brain tumor treatment has been principally hampered by limited drug delivery across the blood–brain barrier (BBB). An efficacious chemotherapeutic treatment requires a pharmacological agent that can penetrate the BBB and target neoplastic cells. Nanotechnology involves the design, synthesis and characterization of materials that have a functional organization in at least one dimension on the nanometer scale. Nanoparticle systems can represent optimal devices for delivery of various drugs into the brain across the BBB. Nanoparticle drug-delivery systems can also be used to provide targeted delivery of drugs, improve bioavailability and sustain release of drugs for systemic delivery. In this patent review, the recent studies of certain nanoparticle systems in treatment of brain tumors are summarized. Common nanoparticles systems include polymeric nanoparticles, lipid nanoparticles and inorganic nanoparticles. Various patents of nanoparticle systems able to across the BBB to target brain tumors are also reported and discussed.

Gliomas are CNS tumors that can originate from neural stem cells, progenitor cells or from de-differentiated mature neural cells transformed into cancer stem cells [1] . Representing 45–55% of all primary cerebral tumors, gliomas are the most common of the CNS tumors [2] . Conventional treatment of brain tumors include surgery, radiation therapy and chemotherapy [3] . The objectives of the surgery are to prolong patient survival and improve quality of life. Radiation therapy represents the primary adjuvant treatment after surgery but without substantial difference in overall survival [4] . The effectiveness of chemotherapeutic agents is limited by toxic effects on healthy cells and also by the presence of the blood–brain barrier (BBB), which limits the passage of therapeutic compounds [5] . Nowadays, nanomedicine is applied in many fields of biology and medicine, such as drug and gene delivery, probing of DNA structure, tissue engineering, tumor diagnosis and treatment [6] . The sub-micron size of nanoparticle (NP) systems confers considerable advantages, including targeted delivery, higher- and deepertissue penetrability, greater cellular uptake, and ability to cross the BBB [7] . NP drug-delivery systems own the ability to encapsulate various therapeutic agents such as small molecules, peptides and protein-based drugs [8] . NP systems permit the use of a lower dose of drug and selective drug delivery to target tumor cells, both into the tumor central core and the distal foci of tumor cells, within areas characterized from integrity of the BBB [9,10] . This aspect is crucial in early diagnosis, in recurrences, in preoperative histological and grade diagnosis, and in preoperative treatment planning. NPs can be engineered to be multifunctional showing the ability to target diseased tissue, carry imaging agents for detection, and deliver multiple therapeutic agents for combination therapy [11] . NP drug-delivery systems

10.4155/PPA.13.56 © 2013 Future Science Ltd

Pharm. Pat. Analyst (2013) 2(6), 1–10

Neurosurgical Clinic, Department of Neuroscience, University of Messina, School of Medicine, 98125, Messina, Italy *Author for correspondence: Tel.: +39 090 2217167 Fax: +39 090 693714 E-mail: [email protected]

ISSN 2046-8954

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Caruso, Raudino & Caffo

Key terms Polymeric nanoparticles: Characterized by a matrix system in which the drug is dispersed. These carriers show a higher stability in biological fluids. Glioblastoma multiforme: Most frequent primary brain tumor in the adult age. Glioblastoma multiforme represents a major cause of morbidity and mortality in neurologic practice. In patients affected by glioblastoma multiforme the mortality is near to 100% and only 35% of patients have a median survival of one year. Theranostic nanoparticle: Nanosystems capable of diagnosis, drug delivery and monitoring of therapeutic response. Inorganic nanoparticles: Extremely interesting in modern technologies due to their material- and size-dependent physiochemical properties, which are not possible with traditional lipid- or polymer-based nanoparticles. Inorganic nanoparticles, such as magnetic, gold and metal nanoparticles, show vast potential in modern biomedical applications. Lipidic nanoparticles: Spherical lipid particle matrix dispersed in water or in aqueous surfactant solution. These nanocarriers have the potential capacity to carry lipophilic or hydrophilic drugs.

have been shown to enhance the intracellular concentration of drugs in cancer cells avoiding toxicity in normal cells [8,12–13] . In this article, various studies on the application of different types of NPs in the treatment of brain tumors are reported. A recent collection of interesting patents on NP systems in brain tumor treatment are also reported. NP-based approaches in brain tumor treatment

In the last decade, various studies have demonstrated the potential importance of nanotechnology in brain tumor treatment [7–9,14–15] . NPs can give many ideal devices for delivery of specific compounds to brain tumors, loading them into NP-based carriers via a variety of chemical methods including encapsulation, adsorption and covalent linkage. In this section, we report a brief summary on the application of different kinds of NP systems in the treatment of brain tumors. The potential efficacy of these systems has been evaluated in in vivo and in vitro studies, and in animal models. ■ Polymeric NPs

The most promising application of polymeric NPs is their use as carriers for anticancer drugs in solid tumors. Tian et al. observed in the brain a valid concentrations of temozolomide (3,4-dihydro-3-methyl-4-oxoimidazo [5,1-d]-as-tetrazine-8-carboxamide) (TMZ) bound to polybutylcyanoacrylate coated with polysorbate-80 [16] . Similarly, in another study by Steiniger et al., a long-term remission disease in animal models was demonstrated [17] . In this case, murine glioblastoma multiforme (GBM) models were treated with doxorubicin bound to polysorbate-coated NPs. In a recent study, transferrin (Tf) modified cyclo-[Arg-Gly-Aspd-Phe-Lys] (c[RGDfK])-paclitaxel conjugated (RP) loaded micelle (TRPM) was evaluated. This structural modification increased the TRPM cellular uptake of microvascular endothelial cells in the cerebral parenchyma through Tf receptor-mediated endocytosis. In this study, important drug retention in gliomas and

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peritumoral tissue was also observed [18] . In an in vivo study, of intracranial C6 GBM-bearing mice using real-time fluorescence imaging analysis, the MPEG-NP/ paclitaxel displayed much stronger fluorescence signals in tumor tissues [19] . NanoCurc™ is a polymeric NP of curcumin, a polyphenolic compound, used to treat GBM cells [20] . This formulation caused a growth decrease in multiple brain tumor cell cultures, including the embryonal tumorderived lines DAOY and D283Med, and the GBM neurosphere lines HSR-GBM1 and JHH -GBM14. The reduction in viable cell mass was associated with a combination of G2/M arrest and apoptotic induction. These data suggest that curcumin NPs can inhibit malignant brain tumor growth through the modulation of cell proliferation [20] . Gene therapy involves the delivery of DNA molecules to cancer cells to insert or modify a gene. A biopolymeric gene delivery NP, a cationic albuminconjugated PEGylated NP that incorporates a plasmid encoding proapoptotic Apo2L/TRAIL, has recently demonstrated the ability to delay tumor progression. Following intravenous injection in a C6 murine gliomas model, the growth was inhibited with prolonged survival [21] . In a recent study, targeted gene delivery to C6 glioma cells in a xenograft mouse model using chlorotoxin-labeled NPs was evaluated [22] . The nanovector was structured by an iron oxide NP core, coated with a copolymer of chitosan, PEG and polyethylenimine. Agemy et al. proposed a multifunctional theranostic NP in which the CGKRK peptide provides the targeting function to tumor vascular cells and into their mitochondria [23] . The NP uses the mitochondria-targeted D [KLAKLAK]2 peptide as the drug and iron oxide as a diagnostic component for MRI. In addition, the NP was combined with the tumor-penetrating peptide iRGD, which enhances the NP penetration into the extravascular tumor tissue [23] . ■■ Lipid NPs

Edelfosine is the prototype molecule of a family of anticancer drugs named synthetic alkyllysophospholipids. In a recent study, accumulation of edelfosine was evidenced in brain tissue when administered with lipid NPs [24,25] . Moreover, in vitro studies against the C6 cell line showed that edelfosine-loaded Compritol® and Precirol® lipid NPs reduced the resistance of the cells to the drug, due to the inhibition of P-gp by Tween® 80. The oral administration of edelfosine-loaded lipid NP to mice significantly decreased tumor growth [24] . GBM cells show an IL-13 a2 up-regulated expression. In a recent study, the efficacy of doxorubicinloaded nanoliposomes targeted with conjugated IL-13 in U251 glioma cells was demonstrated [26] . In this

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Patented nanomedicines for the treatment of brain tumors

study, growth inhibition of subcutaneously implanted gliomas was evidenced [26] . The anti-tumor effect of ferrociphenol-loaded lipid nanocapsules, with or without a DSPE-mPEG2000 coating, was evaluated in an orthotopic gliosarcoma model [27,28] . The combination with convention-enhanced delivery significantly increased the survival of tumor‑bearing rats [27,28] . ■■ Inorganic NPs

NP graphene oxide was conjugated with polyacrylic acid to improve the aqueous solubility and increase cell-penetration efficacy, and used as nanocarrier for 1,3-bis(2-chloroethyl)-1-nitrosourea. This nanocarrier significantly prolonged the half-life of bound 1,3-bis(2-chloroethyl)-1-nitrosourea and showed efficient intracellular uptake by GL261 cancer cells [29] . A more recent study combined the chemo-photothermal targeted therapy of glioma within one novel multifunctional drug-delivery system using a targeting peptide (IP)-modified mesoporous silica-coated graphene nanosheet (GSPI) [30] . Doxorubicin was conjugated with the GSPI-based system, showing synergistic chemo-photothermal properties. Cytotoxicity experiments demonstrated a higher rate of death of glioma cells compared with that of single chemotherapy or photothermal therapy. In a recent study, molecular targeting was performed with carbon nanotubes conjugated to monoclonal antibodies (mABs) specific to CD133. The authors demonstrated a photothermal selective destruction of over 80% of A172 glioma cells with high levels of IL-13R expression [31] . In an in vitro study, the efficacy of gold-silica nanoshells was evaluated. In this case, gold-silica nanoshells coated with an antibody direct to IL-13Ra2 characteristically expressed in glioma cells, caused selective cellular destruction [32] . Clear evidence of localization of AUNPs within the brain parenchyma has recently been demonstrated. These results suggest a potential validity in the treatment of brain tumors [33] . Patents review

For the last 10 years, there has been a big move towards the generation and protection of new inventions on NP systems in brain tumor treatment. NP systems of different compositions such as polymeric NPs, inorganic NPs and lipidic NPs have been investigated (Table 1) . ■■ Polymeric NPs

Polymeric NPs are extensively used for the nano encapsulation of various bioactive molecules and drugs. These carriers show a higher stability in biological fluids and against the enzymatic metabolism [8] . A recent application [101] is composed by a polymeric


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structure for the delivery of pharmaceutical agents in a rate-modulated and site-specific manner. The polymer or polymers making up the scaffold degrade in a human or animal body in response to or in the absence of specific biological stimuli and, on degradation, release of the pharmaceutical compound in an area where specific stimuli are encountered [101] . Block co-polymers such as polycaprolactone were found to be suitable for obtaining functional NPs. The shell of the structured NPs is hydrophobic in nature to enable their passage through lipophilic physiological membranes such as the BBB [102] . In a recent patent, the polymerization of butyl-cyanoacrylate monomers into polybutyl-cyanoacrylate was performed. The NPs formed as such were less toxic and used as stabilizers to form BBB‑permeable NPs [103] . Polymer micelles are particularly interesting due to their ability to deliver a large variety of drugs, their improved in vivo stability and their nanoscopic size, which allows a passive accumulation in diseased tissues. Using appropriate surface functionality, polymer micelles are further structured with cell targeting groups and permeation enhancers that can actively target diseased cells and aid in cellular entry, resulting in improved cell-specific delivery [104] . The peptide sequence: H2N-Gly-PheD-Thr-Gly-Phe-Leu-Ser-CONH2 can be opportunely used to carry through the BBB drugs conjugated directly or via a linear or branched poly valent spacer [105] . Examples of polymers that can be advantageously used to prepare the conjugates according to the invention include polymers or copolymers of biodegradable aliphatic hy[alpha]” oxyacids, preferably lactic acid and/or glycolic acid. The drugs carried include anti-tumoral agents, antibacterial, antiviral, analgesics, antagonists or agonists of receptors present in the CNS, antibodies, antisense oligonucleotides, and diagnostic agents. The drugs can be included, adsorbed or absorbed into the NPs, or conjugated directly with the peptide or copolymer. TMZ is an alkylating agent that shows severe side effects. A multifunctional targetable nanoconjugates of TMZ hydrazide has recently been synthesized, using a poly(-l-malic acid) platform, which contained a targeting mAB to Tf receptor, trileucine for pHdependent endosomal membrane disruption and PEG for protection. The strongest reduction of human brain cancer cell viability was obtained by a combination of TMZ nanoconjugates containing treileucine and anti-Tf receptor antibody [106] . The release of chemotherapeutic agents from implantable drug-polymer carrier systems can be further delayed and modulated by embedding drug-loaded NPs within a polymer matrix in the place of pure drug. An interesting patent is characterized by the combination of more anticancer agents. In particular formulations, the composition

Pharm. Pat. Analyst (2013) 2(6)

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Table 1. Patented nanoparticle systems of varying compositions for brain tumors treatment. Patent No.

Title of patent

Authors

Ref.

Polymeric nanoparticles EP20112370055A

Polymeric pharmaceutical dosage form in sustained release

Choonara YE

[101]

US20110020227

Polysaccharide-containing block copolmer particles and uses thereof

McCarthy SP

[102]

US20067025991

Drug targeting system, method of its preparation and its use

Sabel BA et al.

[103]

US20128263665

Polymeric micelles for drug delivery

Sill KN et al.

[104]

EP20071819723A

Drug delivery peptides for crossing blood–brain barrier

Forni F et al.

[105]

EP20122509421A

Drug delivery of temozolomide for systemic based treatment of cancer

Black KL

[106]

EP20051883407A

Compositions and methods for treatment for neoplasms

Johanson LM et al.

[107]

US20100015050

Peg and targeting ligands on nanoparticle surface

Panyam J et al.

[108]

US20110077204

Agent for targeted drug delivery to cerebral neurons

Kuchiiwa S et al.

[109]

US20100076092

Lipid-derived nanoparticles for brain-targeted drug delivery

Panyam J et al.

[110]

US20110064821

Encapsulation of biologically active agents

Catchpole IR et al.

[111]

US20020025313

Targeting of liposomes to the blood–brain barrier

Micklus MJ et al.

[112]

US20070110798

Liposomes useful for drug delivery to the brain

Drummond D et al.

[113]

US20120239000

Drug delivery method via brain extracellular space and a device thereof

Hongbin H

[114]

EP20051581186

Artificial low-density lipoprotein carriers for transport of substances across the blood–brain barrier

Alkon LD et al.

[115]

US20108252338

Synthetic LDL as targeted drug delivery vehicle

Forte TM et al.

[116]

Lipid nanoparticles

Inorganic nanoparticles US20070154397

Thermosensitive nanostructure for hyperthermia treatment

Chang WH et al.

[117]

US20070264199

Magnetic nanoparticle composition and methods for using the same

Vinod DL et al.

[118]

US20080206146

Functionalized magnetic nanoparticles and methods of use thereof

Akhtari M et al.

[119]

US20110044911

Use of functionalized magnetic nanoparticles in cancer detection and treatment

Akhtari M et al.

[120]

US20110054236

Compositions and methods for targeting tumors

Yang VC et al.

[121]

EP20110212029

Detection of cancer

Bayford RH et al.

[122]

US20097530940

Methods of enhancing radiation effects with metal nanoparticles

Hainfeld JF et al.

[123]

Theranostic nanoparticles EP20112547329

Theranostic delivery systems with modified surfaces

Ferrari M et al.

[124]

WO2012135592

Theranostic imaging agents and methods of use

Bhujwalla Z et al.

[125]

can include cerivastatin, irinotecan, lovastatin, topotecan, simvastatin conjugated with adefovir dipivoxil, auranofin, epirubicin and idebenone [107] . A limitation for any NP system used in systemic drug delivery is their rapid clearance from the circulation by the reticuloendothelial system. In order to overcome this limitation, a novel compound provides a new technique to anchor PEG and PEG-folate conjugate on the surface of NPs [108] . The purpose of PEG chains is to create a barrier to the adhesion of opsonins present in the blood, so that delivery systems can remain longer in circulation, invisible to phagocytic cells. This technique relies on the interfacial activity of PEG-X block copolymer conjugate, where X is any

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hydrophobic polymer. Poly(lacticide)-PEG is a surface active block copolymer, composed of hydrophobic poly(lacticide) chains and hydrophilic PEG chains. The therapeutic agent can comprise paclitaxel, dexamethasone, heat-shock protein 70, Bcl-2, Bcl-xl or folic acid. A patent that focusses on targeting of drugs to brain neurons has recently been proposed [109] . The new method has been applied to certain neural cells and/or brain neurons, selected from the group consisting of cerebellar Purkinje cells, raphe nucleus neurons, cerebral cortex neurons, hypothalamus neurons, thalamus neurons and brain stem neurons. In this conventional drug-delivery system, after processing




(e.g., encapsulation of a small amount of a drug in small capsules [drug carriers]) the drug is selectively delivered to the target cells. Here, a mechanism of interaction, such as the antigen–antibody reaction, is utilized for selective delivery to target cells. Examples of carriers used in such drug-delivery systems include water-soluble polymers, nanospheres, liposomes, and polymeric micelles where polymers with heterogeneous structure are aggregated. This patent may also be used in the brain tumors treatment overcoming the limits of systemic chemotherapy, BBB overcoming, selective cancer cells drug delivery and cell targeting. ■■ Lipid NPs

Liposomes have been used or proposed for use in a variety of applications in research, industry and medicine, particularly for the use as carriers of diagnostic or therapeutic compounds in vivo. In a recent patent, NP composition was characterized by brain lipids, supplemental lipids, PEG-conjugated lipids and drugs [110] . In general brain lipid is referred to as phospholipids. Examples of brain lipids include phosphatidylethanolamine, phosphatidylserine and phosphatic acid [110] . In an experimental application, NPs containing brain-derived lipids were formulated encapsulating 6-coumarin. Encapsulation of the drug in brain lipid-coated NPs resulted in an increase in drug levels. There is great interest in studying the possibility of increasing the cell permeability of liposomes by using membrane-permeable peptides and proteins as carriers. Furthermore, hydrophobic ion pairing agents were found to be efficient for inclusion of BBB-impermeable proteins into the lipophilic core of NPs, hence rendering them BBB-permeable. Anionic proteins could bind cationic hydrophobic ion pairing agents such as cetrimonium bromide leading to efficient encapsulation of the protein into lipophilic poly-butylcyanoacrylate NPs [111] . Recently, an immunoliposome, capable of targeting pharmacological compounds to the brain was investigated. Liposomes are coupled to a mAB binding fragment, such as Fab, F(ab´).sub.2, Fab´ or a single chain polypeptide antibody, which binds to a receptor molecule present on the vascular endothelial cells of the mammalian BBB. The receptor is preferably of the brain peptide transport system, such as the transferring receptor, or insulin receptor, IGF-I or IGF-2 receptors [112] . A novel invention identifies a liposome composition useful for delivery of a variety of therapeutic entities or diagnostic agents that are utilized in diagnosis, prognosis, testing, treatment or prevention of pathological conditions. In this case, substituted ammonium and polyanions are useful for loading and retaining entities inside liposomes [113] .


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An interesting application provides a method of convection-enhanced delivery of drugs in order to cross the BBB [114] . In particular, it related to a method using self-diffusion delivery of drugs in the extracellular space of the brain to enable the drugs to reach the target brain tissue and produce the desired effects. Cytidine-5´-diphosphate choline is a micromolecule, and it’s difficult for cytidine-5´-diphosphate choline to cross the BBB because of its strong polarity. The intake of citicoline is improved by enhancing the permeability of BBB via liposome. This application can reduce delivery time and dose of drugs, relieve injection pressure, decrease damages on normal brain tissue, and reduce the cost of treatment observably. A recent patent discusses a highly efficient artificial LDL carrier system for targeted delivery of therapeutic agents across the BBB [115] . In particular, this invention discusses artificial LDL particles comprised of three lipid elements: phosphatidyl choline, fatty-acylcholesterol esters and at least one apolipoprotein. The invention provides a process for conjugating hydrophilic therapeutic agents with cholesterol to facilitate incorporation of the conjugated therapeutic agent into an artificial LDL particles. In a preferred embodiment, the present invention provides cholesterol-conjugated adriamycin and tetracycline. This interesting invention provides a novel synthetic LDL NP comprising a lipid moiety and a synthetic chimeric peptide, wherein the lipid moiety forms a particle of approximately 10–30 nm in size and the synthetic chimeric peptide comprises an amphipathic a-helix and an LDL receptor [116] . The increased import of LDL into cancerous cells is thought to be due to elevated LDL receptors (LDLR) in these tumors [34] . Comparative studies of normal and malignant brain tissues have shown a high propensity of LDLRs to be associated with malignant and/or rapidly growing brain cells and tissues [35] . These findings suggest that malignant brain tumors exhibit increased expression of LDLRs due to their increased requirement for cholesterol. ■■ Inorganic NPs

Magnetic NPs, used mainly in biomedical applications, have an inorganic NP core and, in most cases, are coated by a suitable coating material. Thermosensitive magnetic NPs were prepared with the magnetic NPs covered by a thermosensitive polymer with a critical temperature of 40–45°C. These systems were suitable for hyperthermia treatment of cancers, such as brain cancer [117] . The NanoTherm® therapy, also termed magnetic fluid hyperthermia, combined with fractionated stereotactic radiotherapy, is a new local heat treatment of solid tumors (such as GBM and prostate carcinoma). Three major components are required


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for NanoTherm therapy: NanoTherm, Nanoplan® and NanoActivator™ F100 (MagForce Nanotechnologies AG, Berlin, Germany). NanoTherm is a magnetofluid consisting of superparamagnetic iron oxide NPs, which are colloidally dispersed in water with a high iron concentration. Once inside the alternating magnetic field applicator, NanoActivator, the NPs begin to oscillate and warmth is produced directly from within the tumor tissue [117] . Depending on the temperature reached and length of treatment, the tumor cells are either directly destroyed or sensitized for the accompanying chemo therapy or radiation. NanoTherm therapy has been used successfully to treat GBM in a Phase II clinical trial [36] . In this trial, combined treatment of fractionated stereotactic radiotherapy and NanoTherm therapy was applied to 59 patients with recurrent GBM. The magnetic fluid, an aqueous dispersion of superparamagnetic nanoparticles, was injected directly into the tumor with the help of a neuro-navigational guide. The median temperature measured within the tumor area during hyperthermia was 51.2°C. The median overall survival from diagnosis of the first tumor recurrence among the 59 patients with recurrent GBM was 13.4 months. Median overall survival after primary tumor diagnosis was 23.2 months while the median time interval between primary diagnosis and first tumor recurrence was 8.0 months [36] . Magnetic NPs that are dispersible in lipid and aqueous phases were also structured. These new NPs own a core surrounded by oleic acid and stabilized by a block copolymer based on ethylene oxide and propylene oxide. These lipophilic magnetic NPs (containing doxorubicin) were capable of efficient drug targeting deeply into the cancerous brain tissue [118] . A recent patent is characterized by an internal magnet to which the drug could be attached. The internal layer is coated by a polymeric layer of dextran or silicon; this in turn is coated with an extra layer of polysorbate 80. This approach shows that the nano-magnets, surrounded by a layer of dextran or silicon, could attached the drug in a highly stable system covered by outer polysorbate 80 layer and that it enables BBB penetration due to the effect of polysorbates [119] . Newly, functionalized magnetic NPs were produced to contain a functional group having selectivity to some types of brain cancer such as gliomas. Additionally, it was found that the ratio of diameter of the magnetic core to the whole functionalized NP could affect the permeation through the BBB and various tissues [120] . Recently, novel magnetic iron oxides NPs were designed for brain tumor targeting. The coating molecule was attached noncovalently to polyethyleneimene or low-molecular-weight protamine to which the anti-tumor agent is attached. This patent

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allows that the anti-tumor agent to pass from the carotid arteries through BBB by the assistance of the penetrating peptide and then to target the tumor tissue by the effect of an external magnetic field [121] . Gold NPs show the ability to bind amine and thiol groups, allowing surface modification and use in biomedical applications. Gold NPs are used to prepare nanoshells composed of gold and copper, or gold and silver to function as contrast agents in MRI, and goldsilica for photothermal ablation of tumor-cells. A new invention [122] relates to a novel method for imaging tumors by detecting tumor biomarkers. Electrical impedance tomography is an imaging technique in which an image of the conductivity of a part of a body is inferred from surface electrical measurements. Electrical impedance tomography can be used for detecting NPs to which ligands are attached, such as antibodies or fragments thereof, which are capable of attaching to a biomarker on a tumor. Examples of specific brain tumor biomarkers are represented by endothelial growth factor receptor and mesenchymal-epithelial transition factor. The core material can be gold, iron, silver, copper or platinum, or combinations thereof. In addition to antibody or antibody fragment ligands, the NPs may have anticancer drugs; for example, doxorubicin and/or cytotoxic compounds attached to them in order to treat the tumor to which they become attached or entrapped within. A recent invention provides a method of using metal NPs to enhance the dose and effectiveness of x-rays or of other kinds of radiation in therapeutic regimes of ablating a target tissue, such as tumors [123] . The metal NPs can also be linked to chemical and/or biochemical moieties, which bind specifically to the target tissue. Tumors that can be treated by the present methods include any solid tumors such as carcinomas, brain tumors, melanomas, lymphomas, plasmocytomas, sarcomas, gliomas and thymomas. Metal NPs suitable for use in radiation therapy are composed of a metal core and typically a surface layer surrounding the metal core. Metals that can be used to form NPs suitable for enhancing radiation effects are heavy metals, including gold, silver, platinum, palladium, cobalt, iron, copper, tin, tantalum, vanadium, molybdenum, tungsten, osmium, iridium, rhenium, hafnium, thallium, lead, bismuth, gadolinium, dysprosium, holmium and uranium. The shell or surface-layer material typically surrounding the metal can be molecules containing sulfur, phosphorus or amines, such as phosphines, phenanthrolines, silanes and organo-thiols. ■■ Theranostic NPs

An interesting attribute of NP delivery systems is their multifunctionality, characterized by multiple




components, which include, imaging agents, therapeutic agents and targeting ligands. The multifunctionality of these nanosystems offers various advantages over conventional agents. These include targeting to a diseased site thereby minimizing systemic toxicity, the ability to solubilize hydrophobic or labile drugs leading to improved pharmacokinetics and their potential to image, treat and predict therapeutic response [37] . In a new patent, the NP surface is modified or functionalized with at least a portion of an isolated cellular membrane [124] . In various compounds, the NP is a lipid particle or a liposome that contains a lipid layer. In addition, the NP contains at least one active agent, such as a therapeutic and/or imaging agent, and may be used for targeted delivery of an active agent. A novel invention is characterized by targeted nanoplex molecules able to carry imaging and contemporaneously to target enzyme inhibitors [125] . This technique can be useful to down-regulate multidrug resistance pathways, or repair enzymes with the goal of increasing the efficacy, safety and efficiency of chemotherapeutic or irradiation therapies. Discussion

Gliomas are the most common primary CNS tumors and their treatment represents a great challenge to neuro-oncologists. Tumor invasion into eloquent cerebral areas, lack of chemosensitivity and shortcomings of the systemic delivery make these lesions resistant to conventional strategies [8] . Aggressive treatments have extended the median survival from 4 months to 1 year, but survival is often associated with significant impairment in the quality of life [8] . A reduced number of chemotherapeutic agents are partially able to cross the BBB (e.g., nitrosoureas and alkylating agents) but owning to a low-molecular weight, they do not achieve an effective steady state concentrations in neoplastic cells [38,39] . Currently, most therapeutic agents targeting brain tumors are delivered by systemic administrations. The limited effectiveness are also due to tumor cell chemo-resistance, and poor selectivity of the anti-tumor drugs. Due to recent advances in material science and nano-engineering, the nanotechnologies have become very attractive for their applications in medicine. In nanomedicine the NPs have been used in specific applications, such as tissue-engineered scaffolds and devices, site specific drug-delivery systems, cancer therapy, and clinical bioanalytical diagnostics and therapeutics [6,22,40,41] . The nanosized materials provide a mechanism for local- or site-specific targeted delivery of macromolecules to the tissue/organ of interest [8–10,42] . The newer developments in material science and nano-engineering are currently being leveraged


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to formulate therapeutic agents in biocompatible nanocomposites such as NPs, nanocapsules, micellar systems and conjugates. Current research on NPs for drug delivery to the brain is mainly focused on finding new enhancers to be attached to the formulated NPs to increase permeation across the BBB. A large number of patents reported in this review investigate this field [102–104,108–114,117,118,120–122] . Their final objective is represented by a most efficient delivery of the pharmacological compound at the desired site. The mechanisms proposed include receptor-mediated transcytosis, adsorptive transcytosis, a general surfactant effect, the creation of a concentration gradient and inhibition of the efflux system. Moreover, these systems must show a reduced premature excretion associated to a prolonged activity of the therapeutic agent. In order to overcome this limitation, the mechanism adopted in an interesting patent [115] appear to be potentially very efficacious. A particular attribute of NP systems is their multifunctionality, characterized by multiple components including imaging agents, therapeutic agents, targeting ligands and substances that avoid interference with the immune system. Multifunctionality of these systems offers a number of advantages over conventional agents. These include targeting to a diseased site thereby minimizing systemic toxicity, the ability to solubilize hydrophobic or labile drugs leading to improved pharmacokinetics and their potential to image, treat and predict therapeutic response [15,37,43,44] . Targeted NP systems with diagnostic capabilities are referred to as theranostic agents and they form a class of agents that show diagnostic and therapeutic functions simultaneously [15,43,44] . Recently, a theranostic nanoplatform, based on a polyacrylamide NP core, with encapsulated components for synergistic cancer detection, diagnosis and treatment has been developed [45] . This platform is a combination of MRI contrast enhancement, photodynamic therapy and specific targeting to tumor sites using F3 peptide [46] . In this study, the authors have provided sufficient evidence to suggest that significant therapeutic benefit with photodynamic therapy was obtained when an F3-targeted polymeric NP formulation consisting of encapsulated imaging agent (iron oxide) and photosensitizer (Photofrin®) was administered to gliomabearing rats. Using these multifunctional NPs, the authors demonstrated that NPs could be targeted to intracerebral rat 9L gliomas and detected using MRI [46] . In the light of these observations, a reported patent investigates this attractive field and its potential application appears to be very important [125] . However, the principal target of these reported patents is represented by the ideal overcoming of


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Patent Review


the BBB. An efficient overcoming of the BBB could permit a better delivery of pharmacological compounds. It is clear that the methods discussed in these patents are equipped for the passage of various therapeutic and/or diagnostic agents, able to treat various brain diseases including brain tumors. Besides, various applications can also increase the efficacy, safety and efficiency of chemotherapeutic or irradiation therapies. Another piece of evidence that emerges from this review is the lack of specifically structured patents looking at the treatment of brain tumors. The application of inhibitors of mediators of glioma invasion and/or glioma angiogenesis with NP systems can be hypothesized. Although nanomedicine applications have great potential, there are some concerns regarding adverse effects of NPs on human health and the environment. The properties that make NPs so promising are also the properties that are likely to have an impact on ecosystems and organisms. NPs are likely to cause different impacts on human health, occupational health and the environment, depending on

the size, shape and chemical composition of the NP [37] . Moreover, the efficiency of targeting NPs to the tumor is not very high and the targeting is always not perfect. Prior to the use of nano–engineered materials in clinical applications, major concerns, including biocompatibility and biodistribution, side-effects and long-term effects, must be addressed. Future perspective

The development of new targeted chemical compounds as well as noninvasive targeted strategies is gaining a greater attention. A better knowledge of the genetic bases of gliomas and of the invasive behavior may suggest new molecular targets to overcome the limits of the actual therapies and, at the same time, target different biological events of gliomagenesis. Basic knowledge of cell biology, tumor biology, immunology and cancer biology are necessary to the rational design of NPs for brain tumor therapy. With the aid of nanomaterials of high specificity and multifunctionality, therapeutic, imaging and diagnostic molecules can be delivered to the brain across

Executive summary Gliomas Cerebral gliomas are the most common primary brain tumors and affected patients present a poor prognosis. Conventional multimodal treatment – surgery, radio and/or chemotherapy – has only extended the median survival from 4 months to one year. Nanotechnology Nanomedicine is used in drug delivery systems, and in cancer diagnosis and therapy. Nanoparticles (NPs) systems commonly adopted in brain tumors treatment include polymeric NPs, lipid NPs, inorganic NPs. Polymeric NPs Steiniger et al. demonstrated a long-term remission, in murine glioblastoma models, after treatment with doxorubicin bound to polysorbate-coated NPs [17]. In a C6 murine gliomas model, a cationic albumin-conjugated pegylated NP that incorporates a plasmid encoding proapoptotic Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand has demonstrated growth inhibition [21]. The NPs formed by the polymerization of butyl-cyanoacrylate monomers into poly-butyl-cyanoacrylate is less toxic and most permeable to the blood–brain barrier. [103]. A reduction of human brain cancer cell viability was obtained by temozolomide nanoconjugates with poly(-l-malic acid) platform,which contained a targeting monoclonal antibody to transferrin receptor, trileucine for pH-dependent endosomal membrane disruption, and PEG for protection [106]. Lipid NPs In vitro studies, edelfosine loaded Compritol® and Precirol® lipid NPs, has reduced the resistance of the cells to the drug. The doxorubicin-loaded nanoliposomes targeted with conjugated IL-13 has showed growth inhibition, in U251 glioma cells [26]. In a recent patent application, NPs containing brain-derived lipids were formulated encapsulating 6-coumarin with increase of drug levels [111]. Inorganic NPs Nanoparticle graphene oxide conjugated with polyacrylic acid to improve the aqueous solubility and increase the cellular penetration was used as nanocarrier for BCNU [29]. Gold-silica nanoshells coated with an antibody direct to interleukin-13 receptor a2, expressed in glioma cells, caused selective cellular destruction [32]. NanoTherm® therapy has been used successfully to treat murine glioblastoma in a Phase II trial [36]. Magnetic NPs, dispersible in lipid and aqueous phases, were capable of efficient drug targeting into the cancer tissue [118]. Theranostic NPs The NPs system contain one active agent such as a therapeutic and/or imaging agent [124] .

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the BBB, enabling considerable progress in the fundamental understanding, diagnosis and treatment of CNS diseases. An optimal system that overcomes the problems associated with these novel strategies requires the identification of specific neoplastic markers, the development of technology for the biomarker-targeted delivery of therapeutic agents, and the simultaneous capability of avoiding biological and biophysical barriers.

Patent Review

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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■■ Patents 101 University of the Witwatersrand: EP-2370055 (2011). 102 University Of Massachusetts: US0020227 (2011).

107 CombinatoRx, Incorporated: EP-1883407 (2005). 108 Wayne State University: US0015050 (2010). 109 Kagoshima University: US0077204 (2011). 110 Wayne State University: US0076092 (2010). 111 Catchpole IR, Gough GW, Papanicolaou I: US0064821 (2011). 112 Micklus MJ, Greig NH, Rapoport SI: US0025313 (2002). 113 Merrimack Pharmaceuticals, Inc.: US0110798 (2007). 114 Hongbin H: US0239000 (2012). 115 Blanchette Rockefeller Neurosciences Institute: EP-1581186 (2005). 116 The Regents of the University of California, Children’s Hospital & Research Center Oakland: US8252338 (2010). 117 Industrial Technology Research Institute: US0154397 (2007). 118 Board of Regents of the University of Nebraska: US0264199 (2007). 119 The Regents of the University of California: US0206146 (2008). 120 The Regents of the University of California: US0044911 (2011). 121 The Regents of the University of Michigan: US0054236 (2011). 122 Middlesex University Higher Education: US0212029 (2011). 123 Nanoprobes, Inc.: US7530940 (2009)

103 Nanodel Technologies Gmbh: US7025991 (2006).

124 Board of Regents of the University of Texas System: EP-2547329 (2013).

104 Intezyne Technologies, Inc.: US8263665 (2012).

125 The Johns Hopkins University: WO135592 (2012).

105 Università degli Studi di Modena e Reggio Emilia: EP-1819723 (2007). 106 Cedars-Sinai Medical Center: EP-2509421 (2012).

