Anti-nucleolin agent-conjugated nanoparticles

Abstract

A composition comprises an anti-nucleolin agent conjugated to nanoparticles. The nanoparticles are non-magnetic, not iron oxide and not polyacrylamide. Furthermore, a pharmaceutical composition for treating cancer comprises a composition including an anti-nucleolin agent conjugated to nanoparticles, and a pharmaceutically acceptable carrier.

Description

BACKGROUND

Nucleolin [8] is an abundant, non-ribosomal protein of the nucleolus, the site of ribosomal gene transcription and packaging of pre-ribosomal RNA. This 710 amino acid phosphoprotein has a multi-domain structure consisting of a histone-like N-terminus, a central domain containing four RNA recognition motifs and a glycine/arginine-rich C-terminus, and has an apparent molecular weight of 110 kD. While nucleolin is found in every nucleated cell, the expression of nucleolin on the cell surface has been correlated with the presence and aggressiveness of neoplastic cells [3].

The correlation of the presence of cell surface nucleolin with neoplastic cells has been used for methods of determining the neoplastic state of cells by detecting the presence of nucleolin on the plasma membranes [3]. This observation has also provided new cancer treatment strategies based on administering compounds that specifically targets nucleolin [4].

Nucleic acid aptamers are short synthetic oligonucleotides that fold into unique three-dimensional structures that can be recognized by specific target proteins. Thus, their targeting mechanism is similar to monoclonal antibodies, but they may have substantial advantages over these, including more rapid clearance in vivo, better tumor penetration, non-immunogenicity, and easier synthesis and storage.

Guanosine-rich oligonucleotides (GROs) designed for triple helix formation are known for binding to nucleolin [5]. This ability to bind nucleolin has been suggested to cause their unexpected ability to effect antiproliferation of cultured prostate carcinoma cells [6]. The antiproliferative effects are not consistent with a triplex-mediated or an antisense mechanism, and it is apparent that GROs inhibit proliferation by an alternative mode of action. It has been surmised that GROs, which display the propensity to form higher order structures containing G-quartets, work by an aptamer mechanism that entails binding to nucleolin due to a shape-specific recognition of the GRO structure; the binding to cell surface nucleolin then induces apoptosis. The antiproliferative effects of GROs have been demonstrated in cell lines derived from prostate (DU145), breast (MDA-MB-231, MCF-7), or cervical (HeLa) carcinomas and correlates with the ability of GROs to bind cell surface nucleolin [6].

AS1411, a GRO nucleolin-binding DNA aptamer that has antiproliferative activity against cancer cells with little effect on non-malignant cells, was previously developed. AS1411 uptake appears to occur by macropinocytosis in cancer cells, but by a nonmacropinocytic pathway in nonmalignant cells, resulting in the selective killing of cancer cells, without affecting the viability of nonmalignant cells [9]. AS1411 was the first anticancer aptamer tested in humans and results from clinical trials of AS1411 (including Phase II studies in patients with renal cell carcinoma or acute myeloid leukemia) indicate promising clinical activity with no evidence of serious side effects. Despite the promising clinical results from Phase II studies, AS1411 did not perform as expected in Phase IIB studies, possibly due to the low potency of AS1411.

SUMMARY

In a first aspect, the present invention is a composition, comprising an anti-nucleolin agent conjugated to nanoparticles. The nanoparticles are non-magnetic.

In a second aspect, the present invention is a pharmaceutical composition for treating cancer, comprising a composition, comprising an anti-nucleolin agent conjugated to nanoparticles, and a pharmaceutically acceptable carrier.

In a third aspect, the present invention is a method of treating cancer, comprising administering an effective amount of the pharmaceutical composition of any of the preceding claims, to a patient in need thereof.

In a fourth aspect, the present invention is an agent for imaging, comprising the composition of any of the preceding claims, and a pharmaceutically acceptable carrier.

In a fifth aspect, the present invention is a method of imaging cancer in vivo, comprising administering the imaging agent of any of the preceding claims, to a subject and forming an image of the imaging agent present in the subject.

DEFINITIONS

The term “conjugated” means “chemically bonded to”.

The term “anti-nucleolin oligonucleotides” refers to an oligonucleotide that binds to nucleolin.

The term “GI50” refers to the concentrations required to achieve 50% of cell growth inhibition. GI50 values may be determined by a cell proliferation/cytotoxicity assay (MTT) assay, described by Morgan [23], Girvan et al. [19] and Mosmann [20].

The term “equivalent aptamer concentration” refers to the concentration of anti-nucleolin oligonucleotide present in the conjugate.

Tumors and cancers include solid, dysproliferative tissue changes and diffuse tumors. Examples of tumors and cancers include melanoma, lymphoma, plasmocytoma, sarcoma, glioma, thymoma, leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, esophageal cancer, brain cancer, lung cancer, ovary cancer, endometrial cancer, bladder cancer, kidney cancer, cervical cancer, hepatoma, and other neoplasms. For more examples of tumors and cancers, see, for example Stedman [1].

“Treating a tumor” or “treating a cancer” means to significantly inhibit growth and/or metastasis of the tumor or cancer. Growth inhibition can be indicated by reduced tumor volume or reduced occurrences of metastasis. Tumor growth can be determined, for example, by examining the tumor volume via routine procedures (such as obtaining two-dimensional measurements with a dial caliper). Metastasis can be determined by inspecting for tumor cells in secondary sites or examining the metastatic potential of biopsied tumor cells in vitro.

A “chemotherapeutic agent” is a chemical compound that can be used effectively to treat cancer in humans.

A “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents which are compatible with pharmaceutical administration. Preferred examples of such carriers or diluents include water, saline, Ringer's solutions and dextrose solution. Supplementary active compounds can also be incorporated into the compositions.

“Medicament,” “therapeutic composition” and “pharmaceutical composition” are used interchangeably to indicate a compound, matter, mixture or preparation that exerts a therapeutic effect in a subject.

“Antibody” is used in the broadest sense and refers to monoclonal antibodies, polyclonal antibodies, multispecific antibodies, antibody fragments and chemically modified antibodies, where the chemical modification does not substantially interfere with the selectivity and specificity of the antibody or antibody fragment.

An “anti-nucleolin agent” includes any molecule or compound that interacts with nucleolin. Such agents include for example anti-nucleolin antibodies, aptamers such GROs and nucleolin targeting proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of tumor volume of in vivo xenograft tumors in nude mice treated with various agents, while FIG. 1B is a graph of the mean body weight of the same mice during the treatment.

FIGS. 2A and 2B are images of two MDA231 xenograft mice injected retro-orbitally with about 200 ng/100 uL of AS1411-Cy5-GNP or CRO-Cy5-GNP. Image was acquired using a PHOTON IMAGER™ after 2 hrs (A) and 6 hrs (B) post injection.

FIGS. 3A-D show the effect on cell proliferation of different concentrations of one of the composition indicated, or without treatment (NT), using an MTT assay. Cells were seeded in 96 well plate at density of 1000 cells/well. Cells were treated with the different concentration of AS1411 and AS1411-GNP for five days.

FIGS. 4A-D are optical micrographs of MCF7 cells treated by silver enhancement staining for gold nanoparticles, illustrating comparative accumulation of gold nanoparticles after administration of the indicated composition, or without administration.

FIG. 5 is electron micrographs of MDA231 cells after administration of a control oligonucleotide (FIG. 5A) or control oligonucleotide-conjugated gold particles (FIG. 5B), or AS1411 (FIG. 5C), or AS1411-conjugated gold nanoparticles (FIG. 5D).

FIGS. 6A-D are confocal microscope images of MCF7 cells after administration of a control oligonucleotide (FIG. 6A) or control oligonucleotide-conjugated gold particles (FIG. 6C), or AS1411 (FIG. 6B), or AS1411-conjugated gold nanoparticles (FIG. 6D).

FIG. 7 is fluorescent microscopy images of MCF7 cells after 5 days of administration of a control oligonucleotide, AS1411-Cy5, unconjugated GNP, control oligonucleotide-conjugated gold particles, AS1411-Cy5 conjugated gold nanoparticles and non-treated cells.

FIG. 8 is fluorescent microscopy images of MDA231 cells after 5 days of administration of a control oligonucleotide, unconjugated GNP, control oligonucleotide-conjugated gold particles, AS1411-Cy5, AS1411-Cy5 conjugated gold nanoparticles and non-treated cells.

FIG. 9A is a sketch of different linkers used to conjugate AS1411/CRO to gold nanoparticles, and FIG. 9B is a sketch of attaching and detaching aptamers to gold nanoparticles.

FIG. 10 shows the result of a cell proliferation/cytotoxicity (MTT) assay: MDA-231 cells were treated with the various agents for 72 hours at 200 nM equivalent aptamer concentration. For comparison, AS1411 and CRO alone were used at a concentration of 10 μM.

FIGS. 11A-G are graphs of flow cytometric assays of MDA-231 cells stained with both PI and Annexin V: (A) cells not treated (control sample); (B) cells treated with AS1411 (at a concentration of 10 μM) for 72 hours; (C) cells treated with CRO control oligonucleotide; (D) cells treated with unconjugated gold nanoparticles; (E) cells treated with GNP-AS1411 (at 200 nM equivalent aptamer concentration); (F) cells treated with the control GNP-CRO (at 200 nM equivalent aptamer concentration); (G) positive control cells treated with camptothecin (at a concentration of 6 ug/ml for 20 hours).

FIG. 12 shows images of mice treated by intraperitoneal injection with GNP-AS1411-Cy5 (1 mg/kg equivalent aptamer concentration), AS1411-Cy5 (10 mg/Kg) and GNP-CRO-Cy5 (1 mg/kg equivalent aptamer concentration), and GNP-Cy5, and imaged after 96 hours.

FIGS. 13A and B show the biodistribution of AS1411-GNP-Cy5: the mice were treated, euthanized and organs were photographed (A) and examined for fluorescence (B).

DETAILED DESCRIPTION

The present invention makes use of the discovery that anti-nucleolin agents, conjugated to particles, such as aptamer conjugated to gold nanoparticles, have an antiproliferative effect on cancer and tumors. Furthermore, aptamer conjugated gold nanoparticels in particular have a similar or greater antiproliferative effect than the aptamer (anti-nucleolin oligonucleotide) alone, demonstrating similar effects at only 1/10 to 1/100 the dosage. Furthermore, these same agents, preferably having a fluorescent dye conjugated to the particle or attached to the anti-nucleolin agent, may also be used as imaging agents, both in vivo and ex vivo.

Anti-nucleolin agents include (i) aptamers, such as GROs; (ii) anti-nucleolin antibodies; and (iii) nucleolin targeting proteins. Examples of aptamers include guanosine-rich oligonucleotides (GROs). Examples of suitable oligonucleotides and assays are also given in Miller et al. [7]. Characteristics of GROs include:

(1) having at least 1 GGT motif,

(2) preferably having 4-100 nucleotides, although GROs having many more nucleotides are possible,

(3) optionally having chemical modifications to improve stability.

Especially useful GROs form G-quartet structures, as indicated by a reversible thermal denaturation/renaturation profile at 295 nm [6]. Preferred GROs also compete with a telomere oligonucleotide for binding to a target cellular protein in an electrophoretic mobility shift assay [6]. In some cases, incorporating the GRO nucleotides into larger nucleic acid sequences may be advantageous; for example, to facilitate binding of a GRO nucleic acid to a substrate without denaturing the nucleolin-binding site. Examples of oligonucleotides are shown in Table 1; preferred oligonucleotides include SEQ IDs NOs: 1-7; 9-16; 19-30 and 31 from Table 1.

TABLE 1
Non-antisense GROs that bind nucleolin and
non-binding controls1,2,3.
		SEQ
		ID
GRO	Sequence	NO:
GRO29A1	tttggtggtg gtggttgtgg tggtggtgg	1
GRO29-2	tttggtggtg gtggttttgg tggtggtgg	2
GRO29-3	tttggtggtg gtggtggtgg tggtggtgg	3
GRO29-5	tttggtggtg gtggtttggg tggtggtgg	4
GRO29-13	tggtggtggt ggt	5
GRO14C	ggtggttgtg gtgg	6
GRO15A	gttgtttggg gtggt	7
GRO15B2	ttgggggggg tgggt	8
GRO25A	ggttggggtg ggtggggtgg gtggg	9
GRO26B1	ggtggtggtg gttgtggtgg tggtgg	10
GRO28A	tttggtggtg gtggttgtgg tggtggtg	11
GRO28B	tttggtggtg gtggtgtggt ggtggtgg	12
GRO29-6	ggtggtggtg gttgtggtgg tggtggttt	13
GRO32A	ggtggttgtg gtggttgtgg tggttgtggt gg	14
GRO32B	tttggtggtg gtggttgtgg tggtggtggt tt	15
GRO56A	ggtggtggtg gttgtggtgg tggtggttgt	16
	ggtggtggtg gttgtggtgg tggtgg
CRO	tttcctcctc ctccttctcc tcctcctcc	18
GRO A	ttagggttag ggttagggtt aggg	19
GRO B	ggtggtggtg g	20
GRO C	ggtggttgtg gtgg	21
GRO D	ggttggtgtg gttgg	22
GRO E	gggttttggg	23
GRO F	ggttttggtt ttggttttgg	24
GRO G1	ggttggtgtg gttgg	25
GRO H1	ggggttttgg gg	26
GRO I1	gggttttggg	27
GRO J1	ggggttttgg ggttttgggg ttttgggg	28
GRO K1	ttggggttgg ggttggggtt gggg	29
GRO L1	gggtgggtgg gtgggt	30
GRO M1	ggttttggtt ttggttttgg ttttgg	31
GRO N2	tttcctcctc ctccttctcc tcctcctcc	32
GRO O2	cctcctcctc cttctcctcc tcctcc	33
GRO P2	tggggt	34
GRO Q2	gcatgct	35
GRO R2	gcggtttgcg g	36
GRO S2	tagg	37
GRO T2	ggggttgggg tgtggggttg ggg	38
1Indicates a good plasma membrane nucleolin-binding GRO.
2Indicates a nucleolin control (non-plasma membrane nucleolin binding).
3GRO sequence without 1 or 2 designations have some anti-proliferative activity.

Any antibody that binds nucleolin may also be used. In certain instances, monoclonal antibodies are preferred as they bind single, specific and defined epitopes. In other instances, however, polyclonal antibodies capable of interacting with more than one epitope on nucleolin may be used. Many anti-nucleolin antibodies are commercially available, and are otherwise easily made. Table 2 list a few commercially available anti-nucleolin antibodies.

TABLE 2
commercially available anti-nucleolin antibodies
Antibody	Source	Antigen source
p7-1A4 Mouse monoclonal antibody	Developmental Studies	Xenopus
(mAb)	Hybridoma Bank	laevis oocytes
Sc-8031 mouse mAb	Santa Cruz Biotech	human
Sc-9893 goat polyclonal Ab (pAb)	Santa Cruz Biotech	human
Sc-9892 goat pAb	Santa Cruz Biotech	human
Clone 4E2 mouse mAb	MBL International	human
Clone 3G4B2 mouse mAb	Upstate Biotechnology	dog (MDCK
		cells)
Nucleolin, Human (mouse mAb)	MyBioSource	human
Purified anti-Nucleolin-Phospho,	BioLegend	human
Thr76/Thr84 (mouse mAb)
Rabbit Polyclonal Nucleolin Antibody	Novus Biologicals	human
Nucleolin (NCL, C23, FLJ45706,	US Biological	human
FLJ59041, Protein C23) Mab Mo xHu
Nucleolin (NCL, Nucl, C23, FLJ45706,	US Biological	human
Protein C23) Pab Rb xHu
Mouse Anti-Human Nucleolin Phospho-	Cell Sciences	human
Thr76/Thr84 Clone 10C7 mAb
Anti-NCL/Nucleolin (pAb)	LifeSpan Biosciences	human
NCL purified MaxPab mouse polyclonal	Abnova	human
antibody (B02P)
NCL purified MaxPab rabbit polyclonal	Abnova	human
antibody (D01P)
NCL monoclonal antibody, clone 10C7	Abnova	human
(mouse mAb)
Nucleolin Monoclonal Antibody (4E2)	Enzo Life Sciences	human
(mouse mAb)
Nucleolin, Mouse Monoclonal Antibody	Life Technologies	human
	Corporation
NCL Antibody (Center E443) (rabbit	Abgent	human
pAb)
Anti-Nucleolin, clone 3G4B2 (mouse	EMD Millipore	human
mAb)
NCL (rabbit pAb)	Proteintech Group	human
Mouse Anti-Nucleolin Monoclonal	Active Motif	human
Antibody, Unconjugated, Clone
3G4B20
Nsr1p - mouse monoclonal	EnCor Biotechnology	human
Nucleolin (mouse mAb)	Thermo Scientific Pierce	human
	Products
Nucleolin [4E2] antibody (mouse mAb)	GeneTex	human

Nucleolin targeting proteins are proteins, other than antibodies, that specifically and selectively bind nucleolin. Examples include ribosomal protein S3, tumor-homing F3 peptides [26, 27] and myosin H9 (a non-muscle myosin that binds cell surface nucleolin of endothelial cells in angiogenic vessels during tumorigenesis).

Anti-nucleolin agents may be conjugated to particles made of a variety of materials solid materials, including (1) metals and elements; (2) oxides; (3) semiconductors; and (4) polymers. Metals and elements, preferbly non-magnetic metals and elements, include gold, silver, palladium, iridium, platinum and alloys thereof; elements include silicon, boron and carbon (such as diamond, graphene and carbon nanotubes), and solid compounds thereof. Oxides include titanium dioxide, silicon dioxide, zinc oxide, iron oxide, zirconium oxide, magnesium oxide, aluminum oxide and complex oxides thereof, such as barium titanate. Semiconductors include quantum dots, zinc sulfide, silicon/germanium alloys, boron nitride, aluminum nitride, and solid solutions thereof. Polymers include polyethylenes, polystyrenes, polyacrylamide, polyacrylates and polymethacrylates, and polysiloxanes. Preferably, the particles are non-toxic. The particles are preferably nanoparticles having an average particle diameter of 1-100 nm, more preferably 1-50 nm, including 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 nm.

Oligonucleotides and proteins have been attached to solid materials, such metals and elements, oxides, semiconductors and polymers, by a variety of techniques. These same techniques may be used to attached anti-nucleolin agents to particles. Further attachment of dyes to the anti-nucleolin agent conjugated nanoparticles (conjugates), such as cyanine dyes, allows the conjugates to be used as imaging agents, both in vivo and ex vivo.

Anti-nucleolin agent conjugated nanoparticles may be used to formulate a pharmaceutical composition for treating cancer and tumors, and targeting cancer cells expressing cell surface nucleolin, by forming mixtures off the anti-nucleolin agent conjugated nanoparticles and a pharmaceutically acceptable carrier, such as a pharmaceutical composition. Methods of treating cancer in a subject include administering a therapeutically effective amount of an anti-nucleolin agent conjugated nanoparticles.

Particularly preferred compositions are aptamers conjugated to gold nanoparticles. Gold nanoparticles (GNPs) exhibit low toxicity, versatile surface chemistry, light absorbing/scattering properties, and tunable size. Aptamers effectively cap gold particles and prevent aggregation, are safe, stable, easy to synthesize, and non-immunogenic. Aptamer conjugated GNPs offer many advantages over alternative approaches, such as enhanced antiproliferative activity in cancer cells over AS1411 alone and improved efficacy in vivo, causing durable regression of established breast cancer xenograft in mice, without evidence of side effects. Aptamer conjugated GNP are highly selective for cancer cells over normal cells, and when attached to cyanine dyes are excellent imaging agents, for example Cy2, Cy3, Cy5, Cy®5.5, Cy7, Alexa Fluor® 680, Alexa Fluor 750, IRDye® 680, and IRDye® 800CW (LI-COR Biosciences, Lincoln, Nebr.). Aptamer conjugated GNP may be used as an imaging agent, and may be administered as compositions which further contain a pharmaceutically acceptable carrier. The imaging agent may be administered to a subject in a method of imaging cancer in vivo, to form an image of the imaging agent present in the subject.

The amounts and ratios of compositions described herein are all by weight, unless otherwise stated. Accordingly, the number of anti-nucleolin agents per nanoparticle may vary when the weight of the nanoparitcle varies, even when the equivalent anti-nucleolin agent concentration (or equivalent aptamer concentration) is otherwise the same. For example, the number of anti-nucleolin agent molecules per nanoparticle may vary from 2 to 10,000, or 10 to 1000, including 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800 and 900.

A pharmaceutical composition is formulated to be compatible with its intended route of administration, including intravenous, intradermal, subcutaneous, oral, inhalation, transdermal, transmucosal, and rectal administration. Solutions and suspensions used for parenteral, intradermal or subcutaneous application can include a sterile diluent, such as water for injection, saline solution, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL® (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid so as to be administered using a syringe. Such compositions should be stable during manufacture and storage and are preferably preserved against contamination from microorganisms such as bacteria and fungi. The carrier can be a dispersion medium containing, for example, water, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and other compatible, suitable mixtures. Various antibacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain microorganism contamination. Isotonic agents such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride can be included in the composition. Compositions that can delay absorption include agents such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active agents, and other therapeutic components, in the required amount in an appropriate solvent with one or a combination of ingredients as required, followed by sterilization. Methods of preparation of sterile solids for the preparation of sterile injectable solutions include vacuum drying and freeze-drying to yield a solid.

The pharmaceutical composition described herein may further comprise other therapeutically active compounds, and/or may be used in conjuction with physical techniques as noted herein which are suitable for the treatment of cancers and tumors. Examples of commonly used therapeutically active compounds include vinorelbine (Navelbine®), mytomycin, camptothecin, cyclyphosphamide (Cytoxin®), methotrexate, tamoxifen citrate, 5-fluorouracil, irinotecan, doxorubicin, flutamide, paclitaxel (Taxol®), docetaxel, vinblastine, imatinib mesylate (Gleevec®), anthracycline, letrozole, arsenic trioxide (Trisenox®), anastrozole, triptorelin pamoate, ozogamicin, irinotecan hydrochloride (Camptosar®), BCG, live (Pacis®), leuprolide acetate implant (Viadur), bexarotene (Targretin®), exemestane (Aromasin®), topotecan hydrochloride (Hycamtin®), gemcitabine HCL (Gemzar®), daunorubicin hydrochloride (Daunorubicin HCL®), gemcitabine HCL (Gemzar®), toremifene citrate (Fareston), carboplatin (Paraplatin®), cisplatin (Platinol® and Platinol-AQ®) oxaliplatin and any other platinum-containing oncology drug, trastuzumab (Herceptin®), lapatinib (Tykerb®), gefitinb (Iressa®), cetuximab (Erbitux®), panitumumab (Vectibix®), temsirolimus (Torisel®), everolimus (Afinitor®), vandetanib (Zactima™), vemurafenib (Zelboraf™), crizotinib (Xalkori®), vorinostat (Zolinza®), bevacizumab (Avastin®), radiation therapy, hyperthermia, gene therapy and photodynamic therapy.

In the treatment of cancer, an appropriate dosage level of the therapeutic agent will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day. A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day. The compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day. Administration by continuous infusion is also preferable. All amounts and concentrations of anti-nucleolin oligonucleotide conjugated gold nanoparticles are based on the amount or concentration of anti-nucleolin oligonucleotide only.

Pharmaceutical preparation may be pre-packaged in ready-to-administer form, in amounts that correspond with a single dosage, appropriate for a single administration referred to as unit dosage form. Unit dosage forms can be enclosed in ampoules, disposable syringes or vials made of glass or plastic.

However, the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the patient undergoing therapy.

Examples

AS1411-linked gold nanoparticles for treating cancer and for cancer imaging we synthasized. Studies to assess the anticancer activity of AS1411 linked to 5 nm gold nanoparticles indicate that the conjugates have greatly enhanced antiproliferative effects on breast cancer cells compared to AS1411 (SEQ ID NO. 10) alone. Microscopic examination revealed increased uptake in breast cancer cells for GNP-AS1411 compared to GNP alone or GNP conjugated to a control oligonucleotide. In addition, GNP-AS1411 induced breast cancer cell vacuolization and death, similar to that seen at higher concentrations of AS1411. The GI50 values for AS1411 conjugated GNP against breast cancer cells are in the 50-250 nM range, compared to 1-10 uM range for unconjugated AS1411 (equivalent aptamer concentration). Studies indicate that these AS1411-GNPs have selective uptake in tumor tissue following systemic administration in mice. Moreover, AS1411-GNPs retained the cancer-selectivity of AS1411 and had no effect on non-malignant cells.

Pilot in vivo studies in mice with MDA-MB-231 (MDA231) xenografts have shown that intravenous administration of AS1411-GNPs at 1 mg/kg/day for 14 days could almost completely inhibit tumor growth. These activities were aptamer-related because GNPs conjugated to control DNA had little or no activity, in terms of internalization in breast cancer cells, uptake in xenografts, and inhibitory effects on breast cancer cells growing in culture or in vivo.

Preparation of Aptamer Conjugated Gold Nanoparticles (GNP)

The aptamers AS1411 and CRO (the control oligonucleotide) with 5′ prime thiol modification and or 3′ fluorophore Cy5 were purchased from Integrated DNA Technologies (IDT).

AS1411 with thiol link at 5′:
5′-/5ThioMC6-D/TTT TTT GGT GGT GGT GGT
TGT GGT GGT GGT GGT TT/-3′.
CRO with thiol link at 5′:
5′-/5ThioMC6-D/TTT TTT CCT CCT CCT CCT TCT
CCT CCT CCT CCT TT/-3′.
AS1411 with thiol link at 5′ and
fluorophore Cy5 at 3′:
5′-/5ThioMC6-D/TTT TTT GGT GGT GGT GGT TGT
GGT GGT GGT GGT TT/Cy5Sp/-3′.
CRO with thiol link at 5′ fluoro-
phore Cy5 at 3′:
5′-/5ThioMC6-D/TTT TTT CCT CCT CCT CCT TCT
CCT CCT CCT CCT TT/Cy5Sp/-3′.

The thiol ends of aptamers was reduced by tri(2-carboxyethyl) phosphine TECP (50 mM) which is active in slightly acidic pH 6.5 of Tris-EDTA (10 mM) solution for 4-8 hours at room temperature. The solution of aptamers and TECP was purified using NAP-columns sephadex G-25. Accurate Spherical Gold nanoparticles 5 nm was purchased from NANOPARTZ and/or TED PELLA INC. The gold nanoparticles were filtered using 0.5 micron syringe filter. Gold nanoparticles and aptamers were mixed in the molar ratio of 1:40 in 25 ml RNAse and DNAse free water at room temperature overnight. Excess reagents were then removed by centrifugation at 15000 rpm for 20 min, followed by 3× wash with RNAse and DNAse free water and centrifugation to remove any unbound aptamers. To quantify the amount of aptamers conjugated on the nanoparticles surface, the aptamer conjugated GNP was incubated in 0.1M DTT at room temperature followed by the separation from the GNP by centrifugation. The supernatant was diluted and measured either spectrophotometically (A260 nm), then calculating the concentration from the aptamers standard dilution curve or by NanoDrop 2000 UV-VIS spectrophotometer. Similarly, the concentration of gold nanoparticles was calculated using spectrophotometric optical density (OD) at 511 nm and plotting the standard dilution curve to extrapolate the concentration of gold nanoparticles and the standard data provided by vendors.

In Vivo Efficacy of AS1411-GNPs

BALB/c nu/nu female mice (Harlan Laboratories Inc, IN, USA) were purchased and housed in University of Louisville animal care facility under pathogen free conditions. After one week acclimatization, mice were injected subcutaneously in each flank with 5×106 MDA-MB-231 cells in PBS. When the tumors were palpable (6 days), mice were randomized into five groups with five mice in each group as shown in Table 3. Mice then received daily intraperitoneal (i.p.) injection for 12 days. The weight and tumor volume was measured with digital vernier calipers (Fisher Scientific) twice a week and tumor volume was calculated using the formula ½ (width2×height) [17, 18]. Statistical analysis was performed using Sigma Stat 10, to determine the statistical significance by using standard student t-test. Treatment with AS1411 (1 mg/kg of aptamer) reduced tumor growth to 75% of vehicle-treated tumors (p<0.005) and GNP-AS1411 (equivalent to 1 mg/kg of aptamer) reduced growth to 94% of control tumors (p<0.005), whereas GNP and GNP-CRO (control aptamers) had only a modest effect on tumor growth (FIG. 1A). There were no significant changes in body weights of mice in any group (FIG. 1B).

TABLE 3
Treatment Groups
	Group	Number	Dose*
	Vehicle only	5	0
	AS1411	5	1	mg/kg
	GNP	5	200	pg/kg
	GNP-CRO	5	1	mg/kg
	GNP-AS1411	5	1	mg/kg
*equivalent aptamer concentration

GNP indicates the group treated with 200 picograms/kg of unconjugated gold nanoparticles (GNP); CRO-GNP indicates the group treated with 10 mg/kg of a control oligonucleotide-conjugated GNP, based on the amount of oligonucleotide only; AS1411 indicates the group treated with 10 mg/kg of AS1411 (GRO26B; unconjugated nucleotide having SEQ ID NO. 10); AS1411-GNP indicates the group treated with 1 mg/kg of a AS1411-conjugated GNP, based on the amount of oligonucleotide only; and Non Treated indicates the group which was not treated. The values in the lower left corner are relative p-values determined using the T-test; values below 0.05 indicate a statistically significant difference between the groups. The single p-value greater than 0.05, GNP vs. AS1411, indicated that there was no statistically significant difference between these two groups (likely due to the small sample sizes use in this study).

FIGS. 2A and 2B are images of two MDA231 xenograft mice (nude mice injected with MDA231 cancer cells), 2 hours (A) and 6 hours (B) after injection retro-orbitally with AS1411 conjugated with a fluorophor (cyanine dye Cy5) and gold nanoparticles (AS1411-Cy5-GNP), or with a control oligonucleotide conjugated with the fluorophor (cyanine dye Cy5) and gold nanoparticles (CRO-Cy5-GNP). The images were acquired using a PHOTON IMAGER™ at 680 nm. The images show that the tumors accumulate AS1411-Cy5-GNP, while CRO-Cy5-GNP is not accumulated.

AS1411-GNPs Tumor-Selective Antiproliferative Effects on Cells

FIGS. 3A-D show the effect cell proliferation of different concentrations of one of the composition indicated, or without treatment (NT), using an MTT assay. Cells were seeded in 96 well plates at a density of 1000 cells/well, and treated for 5 days. GNP indicates the group treated with unconjugated gold nanoparticles; GNP-CR indicates the group treated a control oligonucleotide-conjugated GNP; AS1411 indicates the group treated with AS1411; GNP-AS indicates the group treated with AS1411-conjugated GNP. FIG. 3A shows the result of treatment of MDA231 cells (estrogen negative breast cancer cell line), FIG. 3B shows the result of treatment of MCF7 cells (estrogen dependent breast cancer cell line), FIG. 3C shows the result of treatment of MCF10A cells (human epithelial cell line) and FIG. 3D shows the result of treatment of Hs27 cells (human fibroblast cell line).

FIGS. 4A-D are optical micrographs of MCF7 cells treated by silver enhancement staining for gold nanoparticles, illustrating comparative accumulation of gold nanoparticles after administration of the indicated composition, or without administration. As shown, no significant accumulation occurs after administration of unconjugated gold nanoparticles (FIG. 4B), and only slight accumulation occurs after administration of control oligonucleotide-conjugated gold particles (FIG. 4C). Administration of AS1411-conjugated gold nanoparticles, however, results in significant accumulation of gold nanoparticles, as indicated by the arrows (FIG. 4D). Non-treated cells are also shown (FIG. 4A).

AS1411-GNPs were synthesized for treating cancer and for cancer imaging. Studies to assess the anticancer activity of AS1411 linked to 5 nm gold nanoparticles indicated that the conjugates had greatly enhanced antiproliferative effects on breast cancer cells compared to AS1411 alone. Microscopic examination revealed increased uptake in breast cancer cells for GNP-AS1411 compared to GNP alone or GNP conjugated to a control oligonucleotide. FIG. 5 is electron micrographs of MDA231 cells after administration of the indicated composition. As shown, no significant effect occurs after administration of a control oligonucleotide or control oligonucleotide-conjugated gold particles. The initial stages of apoptosis are apparent after administration of AS1411. Administration of AS1411-conjugated gold nanoparticles, however, results in formation of vacuoles and what appear to be apoptotic bodies, indicating that the cells are dying off in an apoptosis-like process.

Administration of GNP-AS1411 induced breast cancer cell vacuolization and death, similar to that seen at higher concentrations of AS1411. The GI50 for AS1411 conjugated GNP against breast cancer cells were in the 50-250 nM range, compared to 1-10 uM range for unconjugated AS1411 (equivalent aptamer concentration). Preliminary studies indicated that these AS1411-GNPs had selective uptake in tumor tissue following systemic administration in mice. Moreover, AS1411-GNPs retained the cancer-selectivity of AS1411 and had no effect on non-malignant cells.

FIGS. 6A-D are confocal microscope images of MCF7 cells after administration of the indicated composition; different shading indicates cell membranes and gold nanoparticles. As shown, no significant effect occurs after administration of a control oligonucleotide (FIG. 6A) or control oligonucleotide-conjugated gold particles (FIG. 6C). The initial stages of apoptosis are apparent after administration of AS1411 (FIG. 6B). Administration of AS1411-conjugated gold nanoparticles, however, show a dramatic enlargement of the cells (FIG. 6D).

FIG. 7 is fluorescent microscopy images of MCF7 cells after 5 days of administration of the indicated composition; different shading indicates cell membranes and gold nanoparticles. As shown, no significant effect occurs after administration of a control oligonucleotide, unconjugated GNP or control oligonucleotide-conjugated gold particles. The initial stages of apoptosis are apparent after administration of AS1411-Cy5. Administration of AS1411-Cy5 conjugated gold nanoparticles, however, show a dramatic enlargement of the cells and accumulation of GNP. Non-treated cells are also shown.

FIG. 8 is fluorescent microscopy images of MDA231 cells after 5 days of administration of the indicated composition; different shading indicates cell membranes and gold nanoparticles. As shown, no significant effect occurs after administration of a control oligonucleotide, unconjugated GNP or control oligonucleotide-conjugated gold particles. The initial stages of apoptosis are apparent after administration of AS1411-Cy5. Administration of AS1411-Cy5 conjugated gold nanoparticles, however, show a dramatic enlargement of the cells and accumulation of GNP. Non-treated cells are also shown.

Comparison of Different Routes of Injection for Delivery of AS1411-GNP to Target Tissue

Three different routes of injection for delivery of GNP-AS1411 to target tissue were tested: intraperitoneal, intravenous, via tail vein, retro-orbital, injection. Based on pilot studies, it was determined that for long term and repeated injections (as in therapeutic dosing), intraperitoneal injection was preferred for its convenience and because the slower biodistribution (compared to intravenous or retro-orbital) was not a concern. For imaging, either tail vein or retro-orbital injections were used because it delivered the drug directly into the blood, resulting in more rapid systemic distribution and avoiding residual signal in the peritoneum that was observed when delivering through the intraperitoneal route. Pilot in vivo studies in mice with breast cancer cell line MDA-MB-231 xenografts have shown that intravenous administration of AS1411-GNPs at 1 mg/kg/day for 14 days could almost completely inhibit tumor growth. These tumor growth inhibitory activities were aptamer-related because GNPs conjugated to control DNA had little or no activity, in terms of internalization in breast cancer cells, uptake in xenografts, and inhibitory effects on breast cancer cells growing in culture or in vivo.

Effect of GNP Size and Linkers Length on Cell Proliferation

Syntheses and analyses of GNPs and linkers were performed as follows: colloid spherical gold nanoparticles of different size (5, 10, 15 nm) were purchased from Ted Pella Inc. (Redding, Calif.) and Nanopartz (Loveland, Colo.). Size analyses of these gold nanoparticles were confirmed using PARTICLES SIZE ANALYZER 90 PLUS (Brookhaven Instrument), and the sizes of gold nanoparticles were within the ranges as described by the manufacturers. Fluorophore (Cy5)-linked oligonucleotides (AS1411 and CRO), with or without carbon spacers and thiol groups, were purchased from Integrated DNA Technologies (San Diego, Calif.). Cy5, or cyanine-5 phosphoramidite (1-[3-(4-monomethoxytrityloxy)propyl]-1′-[3-[(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidityl]propyl]-3,3,3′,3′-tetramethylindodicarbocyanine chloride) has the structure shown in Formula I:

The linkers, C3-thiol (1-O-dimethoxytrityl-propyl-disulfide,1′-succinyl-Icaa), MC6-D/iSP-9 (9-O-dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]), and MC6-D/iSP-18(18-O-dimethoxytritylhexaethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]), have the structures shown in Formulas II, III and IV, respectively:

FIG. 9A is a cartoon representation of the various agents tested. FIG. 9B is an illustration of the reaction to form conjugates, and to separate the parts of the conjugates. A cell proliferation/cytotoxicity assay (MTT) was performed by treating MDA-231 cells with different sizes of gold nanoparticles (5, 10, 15 nm) and three different linker lengths (C3-thiol, MC6-D/iSP-9, and MC6-D/iSP-18) for 72 hours at concentrations equivalent to 200 nM oligonucleotide. The optimal size and composition for GNP-AS1411 particles was determined in terms of their antiproliferative activity against breast cancer cells. The most profound effects on the cell proliferation occurred using GNP-AS1411 of average diameter 5 nm with C3 thiol linker (64% inhibition), followed by 10 nm with C3 thiol linker (50% inhibition) (FIG. 10 and Table 4).

TABLE 4
Percent cell growth after treatment with GNP-AS1411, with different
GNP sizes and linker length for 72 hours.
	Non-treated				MC6-D/
GNP size	(control)	GNP only	C3-thiol	MC6-D/iSP-9	iSP-18
5 nm	100	92	36	61	78
10 nm	100	90	50	84	83
15 nm	100	93	74	68	65

Gold Nanoparticles (5 nm) Conjugated AS1411 (GNP-AS1411) Cause Late Apoptosis/Necrosis of MDA-MB-231 Breast Cancer Cells

The extent and mechanism of cell death induced by the active GNP-AS1411 was investigated by flow cytometry (FIGS. 11A-G). Flow cytometric analysis of MDA-MB-231 cells stained with FITC-Annexin V and propodium iodide (PI) showed that GNP-AS1411 could potently and specifically induce cell death in breast cancer cells. Flow cytometric assays were performed using FITC Annexin V Apoptosis Detection Kit (BD Bioscience) and data analysis was performed using FLOW JO software (ver. 7.6.5). MDA-MB-231 cells (5×104) were plated in T-25 flask in complete medium and treated as indicated. Staining identified cells that were viable (negative for both PI and Annexin V), early apoptotic (positive for Annexin v, but negative for PI), and late apoptotic or necrotic cells (positive for both PI and Annexin V). (A) In the MDA-MB-231 non-treated control sample, the majority of cells (83%) were viable. (B) In cells treated with AS1411 (10 μM) for 72 hours, there was a decrease in the viable population and an increase in the early apoptotic (12%) and late apoptotic/necrotic population (14%). (C) Cells treated with CRO control oligonucleotides showed a mostly viable population of cells (90%), similar to untreated control. (D) Cells treated with unconjugated gold nanoparticles showed most of the cells were viable (83%). (E) MDA-MB-231 cells treated with GNP-AS1411 (200 nM equivalent aptamer concentration) had a greatly increased population of cells positive for annexin V and/or PI, with most (71%) showing evidence of late apoptosis or necrosis. (F) Cells treated with the control GNP-CRO (200 nM equivalent aptamer concentration) showed comparable results with untreated and GNP-treated controls. The positive control (G) were the cells treated with camptothecin (6 ug/ml for 20 hours) to show the Annexin V and PI positive cells indicative of apoptosis (21%) and late apoptotic (17%) cells.

In Vivo Biodistribution of AS1411-GNP Conjugated to Fluorophore Cy5

The use of multimodal imaging approaches utilizing optical and microCT was useful for detection of primary or disseminated breast cancer tumors. In this experiment a Cy5 fluorophore was linked to the 5′-end of AS1411 and conjugated to the GNP (to give GNP-AS1411-Cy5), in order to evaluate its utility as a complex not only for optical imaging but also as a contrast agent for computed tomography (CT). A similar construct with CRO was synthesized as a control. Nude mice with MDA-MB-231 breast cancer xenografts on each flank were administered a single injection of fluorophore-oligonucleotide-GNP. Images were acquired using IVIS Imaging System/MAESTRO Fluorescence Imaging and preliminary data showed that GNP-AS1411-Cy5 (1 mg/kg) concentration in the tumor is many times more than that using AS1411-Cy5 without GNP (10 mg/kg), or GNP-CRO-Cy5 (FIG. 12). It was noted that all mice exhibited strong signals on their extremities (legs and paws) and tails; these were artifacts from the urine and feces of the mice in cage where they were housed (possibly due to a fluorescent substance in the animal feed). Washing the mice and housing them in new clean new cages before imaging can prevent this problem. Biodistribution analysis also confirmed that, besides liver, kidney and intestine, most of the GNP-AS1411 accumulated in the tumor (FIG. 13). This is a proof of concept that conjugating AS1411 to gold nanoparticles can specifically target the tumors. Mice were treated by intraperitoneal injection with the indicated substances and were imaged after 96 h. Images showed high accumulation of GNP-AS1411-Cy5 (1 mg/kg aptamer concentration) in breast cancer xenograft, as compared to AS1411 (10 mg/Kg) and GNP-CRO alone.

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Claims

1. A composition, comprising an anti-nucleolin agent conjugated to nanoparticles, wherein the nanoparticles comprise goldand wherein the anti-nucleolin agent is AS1411 (SEQ ID NO: 10).

2. The composition of claim 1, further comprising a cyanine dye.

3. The composition of claim 1, wherein the nanoparticles have an average diameter of 1 to 50 nm.

4. The composition of claim 1, wherein the nanoparticles have an average diameter of 1 to 20 nm.

5. A pharmaceutical composition, comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

6. A method of treating cancer, comprising administering an effective amount of the pharmaceutical composition of claim 5, to a patient in need thereof.

7. The method of claim 6, wherein the cancer is selected from the group consisting of melanoma, lymphoma, plasmocytoma, sarcoma, glioma, thymoma, leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, esophageal cancer, brain cancer, lung cancer, ovary cancer, endometrial cancer, bladder cancer, kidney cancer, cervical cancer and hepatoma.

8. The method of claim 7, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, colon cancer and lung cancer.

9. The method of claim 6, further comprising administering a second cancer treatment selected from the group consisting of vinorelbine, mytomycin, camptothecin, cyclyphosphamide, methotrexate, tamoxifen citrate, 5-fluorouracil, irinotecan, doxorubicin, flutamide, paclitaxel, docetaxel, vinblastine, imatinib mesylate, anthracycline, letrozole, arsenic trioxide, anastrozole, triptorelin pamoate, ozogamicin, irinotecan hydrochloride, BCG live, leuprolide acetate implant, bexarotene, exemestane, topotecan hydrochloride, gemcitabine HCL, daunorubicin hydrochloride, toremifene citrate, carboplatin, cisplatin, oxaliplatin and any other platinum-containing oncology drug, trastuzumab, lapatinib, gefitinb, cetuximab, panitumumab, temsirolimus, everolimus, vandetanib, vemurafenib, crizotinib, vorinostat, bevacizumab, radiation therapy, hyperthermia, gene therapy and photodynamic therapy.

10. A pharmaceutical composition, comprising the composition of claim 2 and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition, comprising the composition of claim 3 and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition, comprising the composition of claim 4 and a pharmaceutically acceptable carrier.

13. A method of treating cancer, comprising administering an effective amount of the pharmaceutical composition of claim 10, to a patient in need thereof.

14. A method of treating cancer, comprising administering an effective amount of the pharmaceutical composition of claim 11, to a patient in need thereof.

15. A method of treating cancer, comprising administering an effective amount of the pharmaceutical composition of claim 12, to a patient in need thereof.

16. The method of claim 15, wherein the cancer is selected from the group consisting of melanoma, lymphoma, plasmocytoma, sarcoma, glioma, thymoma, leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, esophageal cancer, brain cancer, lung cancer, ovary cancer, endometrial cancer, bladder cancer, kidney cancer, cervical cancer and hepatoma.

17. The method of claim 16, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, colon cancer and lung cancer.

18. The method of claim 15, further comprising administering a second cancer treatment selected from the group consisting of vinorelbine, mytomycin, camptothecin, cyclyphosphamide, methotrexate, tamoxifen citrate, 5-fluorouracil, irinotecan, doxorubicin, flutamide, paclitaxel, docetaxel, vinblastine, imatinib mesylate, anthracycline, letrozole, arsenic trioxide, anastrozole, triptorelin pamoate, ozogamicin, irinotecan hydrochloride, BCG live, leuprolide acetate implant, bexarotene, exemestane, topotecan hydrochloride, gemcitabine HCL, daunorubicin hydrochloride, toremifene citrate, carboplatin, cisplatin, oxaliplatin and any other platinum-containing oncology drug, trastuzumab, lapatinib, gefitinb, cetuximab, panitumumab, temsirolimus, everolimus, vandetanib, vemurafenib, crizotinib, vorinostat, bevacizumab, radiation therapy, hyperthermia, gene therapy and photodynamic therapy.

19. The method of claim 14, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, colon cancer and lung cancer.

20. The method of claim 14, further comprising administering a second cancer treatment selected from the group consisting of vinorelbine, mytomycin, camptothecin, cyclyphosphamide, methotrexate, tamoxifen citrate, 5-fluorouracil, irinotecan, doxorubicin, flutamide, paclitaxel, docetaxel, vinblastine, imatinib mesylate, anthracycline, letrozole, arsenic trioxide, anastrozole, triptorelin pamoate, ozogamicin, irinotecan hydrochloride, BCG live, leuprolide acetate implant, bexarotene, exemestane, topotecan hydrochloride, gemcitabine HCL, daunorubicin hydrochloride, toremifene citrate, carboplatin, cisplatin, oxaliplatin and any other platinum-containing oncology drug, trastuzumab, lapatinib, gefitinb, cetuximab, panitumumab, temsirolimus, everolimus, vandetanib, vemurafenib, crizotinib, vorinostat, bevacizumab, radiation therapy, hyperthermia, gene therapy and photodynamic therapy.