WO2010065329A2 - Nanoparticles for cancer treatment - Google Patents

Abstract

Methods and composition for improving therapeutic potential of isothiocyanate. For example, in certain aspects methods for preparing a composition containing an isothiocyanate nanoparticle formulation and uses thereof are described. Furthermore, the invention provides such a composition.

Description

DESCRIPTION NANOPARTICLES FOR CANCER TREATMENT

[0001] The present application claims benefit of priority to U.S. Provisional Application Serial No. 61/117,696 filed November 25, 2008, the entire contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention [0002] The present invention relates generally to the field of drug delivery. More particularly, it concerns improvement of therapeutic potential of isothiocyanates for cancer treatment.

2. Description of Related Art

[0003] Cancer causes about 13% of all deaths. According to the American Cancer Society, 7.6 million people died from cancer in the world during 2007. Although advances in treatment have been made, there continues to be a need for intervention strategies, including compounds that act as primary protective agents by preventing, delaying, or reversing preneoplastic lesions, as well as those that act on secondary or recurrent cancers as therapeutic agents. There have been many advances in the therapy of cancer following the introduction of cytotoxic chemotherapeutic drugs. However, one of the consequences of chemotherapy is the development/acquisition of drug-resistant phenotypes and the development of multiple drug resistance. The development of drug resistance remains a major obstacle in the treatment of such tumors and therefore, there is an obvious need for alternative approaches. Compounds found in the diet are of particular interest because of their accessibility to the general population, and ongoing research continues to identify novel candidates for use in cancer clinical trials. Isothiocyanates could be potential candidates.

[0004] Isothiocyanates, such as phenethyl isothiocyanate (PEITC) and sulforaphane, have been shown to potentially inhibit carcinogenesis and tumorgenesis and as such are useful chemopreventive agents against the development and proliferation of cancers. They work through multiple mechanisms. Most notably, they have been shown to inhibit

85343306.1 carcinogenesis through inhibition of cytochrome P450 enzymes or selectively kill cancer cells through a reactive oxygen species (ROS)-mediated mechanism.

[0005] However, isothiocyanates such as PEITC have poor solubility and stability, and can be quickly metabolized and eliminated from the human body, and thus have limited therapeutic activity in vivo. Therefore, there is a continued need in the medical arts for more efficient techniques for improving the solubility, stability, and/or the pharmacokinetics of these compounds so that a better in vivo therapeutic activity can be achieved.

SUMMARY OF THE INVENTION

[0006] The inventors recently discovered that phenylethyl isothiocyanate (PEITC) preferentially kills cancer cells through a novel RO S -mediated mechanism with low toxicity to normal cells, kills drug-resistance cancer cells effectively, and that the therapeutic selectivity of PEITC is significantly better than cisplatin. However, the therapeutic potential of PEITC as well as other isothiocyanates is limited by the poor solubility, stability in solution, and quick elimination in vivo.

[0007] Particular aspects of the present invention concerns a novel isothiocyanate (ITC), particularly PEITC, nanoparticle formulation for cancer treatment to overcome these problems, including the method and procedures to prepare the nanoparticles of proper size distribution and defined amount of PEITC suitable for systemic administration in vivo, and characterization of the PEITC nanoparticles. This PEITC nanoparticle formulation has advantageous pharmacological properties including improved solubility, stability, effective anticancer activity, and favorable tolerability in animals.

[0008] In a first embodiment there may be provided a method of a parenteral pharmaceutical composition comprising an isothiocyanate (ITC) formulated in a pharmaceutically acceptable nanoparticle formulation, whereby the nanoparticle formulation solubilizes and stabilizes the ITC.

[0009] Exemplary ITCs may include phenethyl isothiocyanate (PEITC), N- acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC), sulforaphane, benzyl isothiocyanate, methyl isothiocyanate, ethyl isothiocyanate, propyl isothiocyanate, isopropyl isothiocyanate, n-butyl isothiocyanate, t-butyl isothiocyanate, s-butyl isothiocyanate, pentyl isothiocyanate, hexyl isothiocyanate, heptyl isothiocyanate, octyl isothiocyanate, nonyl

85343306.1 isothiocyanate, decyl isothiocyanate, undecane isothiocyanate, phenyl isothiocyanate, o-tolyl isothiocyanate, 2-fluorophenyl isothiocyanate, 3 -fluorophenyl isothiocyanate, 4-fluorophenyl isothiocyanate, 2-nitrophenyl isothiocyanate, 3-nitrophenyl isothiocyanate, 4-nitrophenyl isothiocyanate, 2-chlorophenyl isothiocyanate, 2-bromophenyl isothiocyanate, 3- chlorophenyl isothiocyanate, 3-bromophenyl isothiocyanate, 4-chlorophenyl isothiocyanate, 2,4-dichlorophenyl isothiocyanate, R-(+)-alpha-methylbenzyl isothiocyanate, S-(-)-alpha- methylbenzyl isothiocyanate, 3-isoprenyl-alpha,alpha-dimethylbenzyl isothiocyanate, trans- 2-phenylcyclopropyl isothiocyanate, l,3-bis(isothiocyanatomethyl)-benzene, l,3-bis(l- isothiocyanato-l-methylethyl)benzene, 2-ethylphenyl isothiocyanate, benzoyl isothiocyanate, 1-naphthyl isothiocyanate, benzoyl isothiocyanate, 4-bromophenyl isothiocyanate, 2- methoxyphenyl isothiocyanate, m-tolyl isothiocyanate, alpha, alpha, alpha-trifluoro-m-tolyl isothiocyanate, 3 -fluorophenyl isothiocyanate, 3 -chlorophenyl isothiocyanate, 3-bromophenyl isothiocyanate, 1 ,4-phenylene diisothiocyanate, l-isothiocyanato-4-(trans-4- propylcyclohexyl)benzene, 1 -(trans-4-hexylcyclohexyl)-4-isothiocyanatobenzene, 1 - isothiocyanato-4-(trans-4-octylcyclohexyl) benzene, 2-methylbenzyl isothiocyanate, 2- chlorobenzo isothiocyanate, 3-chlorobenzo isothiocyanate, 4-chlorobenzo isothiocyanate, m- toluyl isothiocyanate, or p-toluyl isothiocyanate. In particular embodiments, the ITC is phenethyl isothiocyanate (PEITC).

[0010] In certain further embodiments, the nanoparticle formulation may comprise a polymer, a monomer, a hydrogel, an emulsion, a liposome, a micelle, a complexing ligand or a hydrotropic agent in order to solubilizes and stabilizes the isothiocyanate. Particularly, the nanoparticle formulation may comprise a DOTAP xholesterol nanoparticle.

[0011] Particularly, the nanoparticle formulation may have a particle diameter of less than 1000 nm, about 10 nm to about 500 nm, about 10 to 200 nm, about 20 to about 200 nm, about 20 to about 100 nm, about 20 to about 30 nm, or any range derivable therein. The particle diameter may be a mean or average diameter.

[0012] Exemplary polymers used in the present invention may include, but not limited to, polyoxyethylene-polyoxypropylene block co-polymer, poly-L-lysine, poly-L- Arginine, albumin, N-(2-hydroxypropyl) methacrylamide (HPMA), polyaspartamide, a dendrimer comprising a polyamido amine and polylysine core, hyaluronic acid, polylactic-co- glycolic acid, heparin, polyacrylic acid, crosslinked polyacrylic acid, carboxymethylcellulose, alginate, alginic acid, propylene glycol alginate, sodium alginate, a polylactide, poly-glutamic

85343306.1 acid, polyerucic-co-sebacic acid or any polymer that may improve ITC solubility and/or stability in an nanoparticle formulation. Particularly, the polymer may be polyoxyethylene- polyoxypropylene block co-polymer, such as Pluronic® or Pluronic® F127.

[0013] The nanoparticle formulation can be a liquid formulation or a solid formulation, such as a powder. Particularly, the composition may be dehydrated or lyophilized for long term storage with improved stability. Alternatively, the composition may be present in a substantially aqueous solution. The composition may be rehydrated or re- suspended in a solution or liquid from the previously lyophilized composition.

[0014] Because the composition may have the potential to treat chemoresistant diseases, the composition may comprise an additional therapeutic agent to exert a synergistic benefit, such as a chemotherapeutic agent as exemplified below.

[0015] The skilled artisan will understand that there may be also provided methods for preparing a composition disclosed above comprising admixing the isothiocyanate into the pharmaceutically acceptable nanoparticle formulation. The method may further comprise homogenization and/or sonication for homogenous dispersion. In order for long term storage, the method may further comprise dehydrating or lyophilizating the formulation. For in vivo administration, the method may further comprise rehydrating or resuspending in a solution or liquid.

[0016] Furthermore, a method of treating a hyperproliferative disease or a quiescent malignant disease comprising administering a therapeutically effective amount of the composition described above to a subject in need of such treatment may be disclosed. The composition used in the methods may be previously dehydrated, lyophilized or in some other aspects, an aqueous solution or liquid formulation of previously lyophilized or dehydrated composition, an effective amount of which are administered to the subject. The present invention also provides, in certain aspects, previously lyophilized or dried composition after being stored at 4 degree for at least 1 week, for at least 3 weeks, for up to 4 weeks, or any period derivable therein, for treating the disease with retained activity after resuspension or rehydration.

[0017] The hyperproliferative disease that may be treated by the present methods may be pre-cancer or cancer, such as melanoma, leukemia, ovarian cancer, colon cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, bladder cancer, breast cancer, gastric

85343306.1 cancer, colon cancer, head and neck cancer, esophagus cancer, synovium cancer, brain cancer, or bronchus cancer, in particular, chronic myelogenous leukemia (CML) or chronic lymphocytic leukemia (CLL). In particular, the disease may be chemo-resistant. The subject to be treated in the present invention may be a mammal, such as a human.

[0018] With improved pharmacokinetics from the nanoparticle formulation, the composition may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, intrathecally, locally, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage. For example, the composition may be administered by injection or infusion.

[0019] To have a better therapeutic benefit, the composition may be administered in combination with at least an additional agent selected from the group consisting of a radiotherapeutic agent, a hormonal therapy agent, an immunotherapeutic agent, a chemotherapeutic agent, a cryotherapeutic agent and a gene therapy agent.

[0020] In particular aspects, the additional agent may be a chemotherapeutic agent because ITC-based composition may be effective for chemo-resistant cancer. Examples of chemotherapeutic agents include, but not limited to, cetuximab (erbitux), herceptin (trastuzumab), fludarabine, cyclophosphamide, rituximab, imatinib, Dasatinib (BMS0354825), cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin, methotrexate, an analog or derivative thereof. Particularly, the chemotherapeutic agent may be fludarabine, cyclophosphamide, rituximab, imatinib or Dasatinib. In a certain aspect, the cancer may be resistant to chemotherapy, such as fludarabine, cyclophosphamide, rituximab, imatinib or Dasatinib.

85343306.1 [0021] For a safe and effective dosage, the composition may be administered at a dose of about 10 to about 500 mg/m² (body surface)/day, about 20 to about 300 mg/m²/day, 20 to about 200 mg/m²/day, about 30 to about 200 mg/m²/day , about 40 to about 100 mg/m /day, about 50 to about 100 mg/m /day or any range derivable therein to a subject such as a human. In certain aspects, the composition may be administered at a dose of about 1 to about 200 mg/kg body weight, about 1 to about 100 mg/kg body weight, 1 to about 50 mg/kg body weight, about 1 to about 20 mg/kg body weight, about 3 to about 10 mg/kg body weight, about 3 to about 6 mg/kg body weight or any range derivable therein to a subject such as a mouse.

[0022] Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.

[0023] As used herein the terms "encode" or "encoding" with reference to a nucleic acid are used to make the invention readily understandable by the skilled artisan however these terms may be used interchangeably with "comprise" or "comprising" respectively.

[0024] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0025] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0026] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0027] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications

85343306.1 within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0029] FIG. 1: A flow chart to illustrate certain aspects of the invention. Non- limiting exemplary steps of preparation and characterization of PEITC nanoparticles.

[0030] FIG. 2: Characterization of PEITC nanoparticles with 2 mg PEITC in 2 ml

5% F127 (particle size 25.7 nm - a good loading condition). These data indicate that a drug loading of 2 mg PEITC in 2 ml of 5% F127 can produce nanomolecules with average size of 25.7 nm and the zeta potential of -7.25 mV, and therefore are good conditions for preparation of PEITC nanoparticles.

[0031] FIG. 3: Characterization of PEITC nanoparticles with 6 mg PEITC in 2 ml

5% F127 (particle size 23.9 nm - a good loading condition). These data indicate that a drug loading of 6 mg PEITC in 2 ml of 5% F 127 can produce nanomolecules with average size of 23.9 nm and the zeta potential of -13.33 mV, and therefore are good conditions for preparation of PEITC nanoparticles.

[0032] FIG. 4: Characterization of PEITC nanoparticles with 12 mg PEITC in 2 ml

5% F127 (particle size 29.0 nm - a good loading condition). These data indicate that a drug loading of 12 mg PEITC in 2 ml of 5% F 127 can produce nanomolecules with average size of 29.0 nm and the zeta potential of -23.77 mV, and therefore are good conditions for preparation of PEITC nanoparticles.

[0033] FIG. 5: Characterization of PEITC nanoparticles with 24 mg PEITC in 2 ml

5% F127 (particle size 617.8 nm - not a good loading condition). These data indicate that a drug loading of 24 mg PEITC in 2 ml of 5% F 127 can not consistently produce

85343306.1 nanomolecules with average size less than 200 nm and this is not a good conditions to prepare PEITC nanoparticles.

[0034] FIG. 6: Characterization of another batch of PEITC nanoparticles with 2 mg

PEITC in 2 ml 5% F127 (particle size 25.9 nm - a good loading condition). These data indicate that a drug loading of 2 mg PEITC in 2 ml of 5% F127 can consistently produce nanomolecules with average size of 25-26 nm and the zeta potential of -7 mV to -17 mV, and therefore are good conditions for preparation of PEITC nanoparticles.

[0035] FIG. 7: Characterization of another batch of PEITC nanoparticles with 6 mg

PEITC in 2 ml 5% F127 (particle size 26.8 nm - a good loading condition). These data indicate that a drug loading of 6 mg PEITC in 2 ml of 5% F127 can consistently produce nanomolecules with average size of 24-26 nm and the zeta potential of -13 mV to -15 mV, and therefore are good conditions for preparation of PEITC nanoparticles.

[0036] FIG. 8: Characterization of another batch of PEITC nanoparticles with 12 mg

PEITC in 2 ml 5% F127 (particle size 30.3 nm - a good loading condition). These data indicate that a drug loading of 12 mg PEITC in 2 ml of 5% F 127 can consistently produce nanomolecules with average size of 29-30 nm and the zeta potential of -10 mV to -20 mV, and therefore are good conditions for preparation of PEITC nanoparticles.

[0037] FIG. 9: Characterization of another batch of PEITC nanoparticles with 24 mg

PEITC in 2 ml 5% F127 (particle size 246.3 nm - not a good loading condition). These data indicate that a drug loading of 24 mg PEITC in 2 ml of 5% F 127 can not consistently produce nanomolecules with average size less than 200 nm and this is not a good conditions to prepare PEITC nanoparticles.

[0038] FIG. 10: Comparison of in vitro cytotoxicity of PEITC nanoparticles and regular PEITC in HL-60 cells. Samples #1 to #4 indicate 4 separate preparations of PEITC nanoparticles. The regular PEITC was dissolve in DMSO and used in parallel experiment for comparison. These data show that the PEITC nanoparticles are potent against leukemia cells in vitro.

[0039] FIG. 11: PEITC nanoparticle solution remained fully active after 3 weeks of storage at 4°C. The cytotoxicity assay was performed in HL-60 cells with 18 h drug incubation. Samples #1 to #4 indicate 4 separate preparations of PEITC nanoparticles.

85343306.1 [0040] FIG. 12: PEITC nanoparticle solution remained fully active after one month of storage at 4°C. The cytotoxicity assay was performed in HL-60 cells with 14 h drug incubation. Samples #1 to #4 indicate 4 separate preparations of PEITC nanoparticles.

[0041] FIG. 13: Regular PEITC solution lost its activity after storage at 4°C for 3-4 weeks. The cytotoxicity assay was performed in HL-60 and Raji cells (14 h drug incubation).

[0042] FIG. 14: PEITC nanoparticles exhibited therapeutic activity as a single agent. PEITC nanoparticles induced regression of established tumor mass.

[0043] FIG. 15: PEITC nanoparticles are well tolerated in mice at the dose range of 30-50 mg/kg (iv). The mice exhibited normal activity without any weight loss.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. The Present Invention

[0044] The instant invention overcomes several major problems with current drug isothiocyanate (ITC) delivery technologies by providing therapeutic compositions and methods about parenteral pharmaceutical composition comprising an isothiocyanate (ITC) formulated in a pharmaceutically acceptable nanoparticle formulation, whereby the nanoparticle formulation solubilizes and stabilizes the ITC. In certain exemplary aspects, the major applications include: (a) use the PEITC nanoparticles for clinical treatments of human leukemia and solid tumors, and (b) use the new PEITC formulations in combinations with other anticancer agents or modalities to enhance therapeutic activity and selectivity against cancer and overcome drug resistance. Because reactive oxygen species (ROS) stress is prevalent in human cancers and drug resistance is a significant clinical problem, this new technology will have broad therapeutic applications in cancer treatment.

[0045] Certain advantages of using ITC (e.g., PEITC) nanoparticle formulation include:

[0046] (1) ITC was considered an antioxidant and a chemopreventive agent. Its ability to preferentially kill cancer cells through ROS-mediated mechanism, and the advantage of ITC nanoparticles for cancer treatment are also discovered as this compound can be used for cancer treatment at the pharmacologically achievable concentrations.

85343306.1 [0047] (2) The new formulation based on nanotechnology described in this invention significantly improves the solubility, stability, and anticancer activity of the ITC. For example, this formulation makes it possible to administer ITC by intravenous injection or infusion so that proper blood drug concentrations and drug exposure time can be achieved as desired. This is important to achieve therapeutic selectivity against cancer cells and not to harm normal cells.

[0048] (3) ITC in its original chemical form is an oily liquid with poor solubility and unstable when diluted in saline. The new formulation of ITC nanoparticles can be prepared in powder form for long-term storage and can be readily reconstituted into solution for in vivo administration.

[0049] (4) The ITC nanoparticles described in this invention are relatively simple to prepare and can be scaled up to produce large amount for clinical use.

[0050] (5) Low manufacturing costs.

[0051] (6) Because ITC is a normal metabolite of naturally occurring compound found in consumable vegetables with low toxicity to normal cells, the inventors expect that this new formulation has high feasibility to be approved by appropriate regulatory agencies for use in clinical treatment of cancer.

[0052] Further embodiments and advantages of the invention are described below.

II. Nanoparticle formulation [0053] In certain aspects, the present invention affords compositions and methods involving a nanoparticle formulation to improve the solubility, stability, and/or pharmacokinetics of isothiocyanate for a better in vivo therapeutic activity. ITC, such as PEITC, is a natural compound found which can be quickly metabolized and eliminated from the human body, and thus has limited therapeutic activity in vivo. The disclosed formulations, e.g., PEITC-nanoparticles, are designed to improve the solubility, stability, as well as the pharmacokinetics of this compound so that a better in vivo therapeutic activity can be achieved. For example, the nanoparticle formulation may prolong the drug retention in blood circulation and increase the drug distribution to tumor tissues and uptake by cancer cells and thus enhance anticancer activity.

85343306.1 [0054] As used herein, the term "nanoparticle" refers to any particles having dimensions in the 1-1,000 nm range which solubilize and stabilize ITC. In some embodiments, nanoparticles have dimensions in the 2-200 nm range, preferably in the 2-150 nm range, and even more preferably in the 2-100 nm range. Nanoparticles used in the present invention include such nanoscale materials as a polymer-based nanoparticle, lipid-based nanoparticle, an emulsion, a hydrogel, a micelle, and the like. In certain aspects, the nanoparticles may be conjugated to isothiocyanate to provide structures with potential application for targeted delivery, controlled release, enhanced cellular uptake and intracellular trafficking in vitro and in vivo.

A. Emulsion and Polymer-based Nanoparticles

[0055] An "emulsion" is a mixture of two immiscible (unblendable) liquids. One liquid (the dispersed phase) is dispersed in the other (the continuous phase). Many emulsions are oil/water emulsions. Surfactants which may be used for making an isothiocyanate nanoparticle emulsion for the present invention have preferably HLB (hydrophilic-lipophilic balance) value of about 8 to about 20. Examples of them are as follows:

[0056] (a) Polyoxyethylene-polyoxypropylene co-polymers: e.g. of the type known and commercially available under the trade names "Pluronic" and "Emkalyx." For example, polymer has a general formula HO(C2H4O)a(C3H6O)b(C2H4O)aH, wherein a is an integer such that the molecular weight represented by the polyoxyethylene portion of the respective block copolymer constitutes between 5% and 95% of the respective block copolymer, and b is an integer such that the molecular weight represented by the polyoxypropylene portion is between 900 Daltons and 15, 000 Daltons. Pluronic® or Pluronic® F 127 may be preferred.

[0057] (b) Reaction products of natural or hydrogenated vegetable oils, and ethylene glycol; i.e., polyoxyethylene glycolated natural or hydrogenated vegetable oils: for example polyoxyethylene glycolated natural or hydrogenated castor oils. Surfactants commercialized under the trade names Cremophor RH-40, Cremophor RH60, Cremophor EL, Nikkol HCO-

40 and Nikkol HCo-60 may be used in the composition according to the present invention.

[0058] (c) Polyoxyethylene sorbitan fatty acid esters: e.g., mono- and tri-lauryl, palmityl, stearyl and oleyl esters; e.g. products of the trade name "T ween," which includes polyoxyethylene sorbitan mono-laurate (T ween), polyoxyethylene sorbitan mono-palmitate

85343306.1 (T ween 40), polyoxyethylene sorbitan mono-oleate (T ween 80), etc. depending on the kind of fatty acid.

[0059] (d) Polyoxyethylene fatty acid esters: for example, polyoxyethylene stearic acid esters of the type known and commercially available under the trade name Myrj as well as polyoxyethylene fatty acid esters known and commercially available under the trade name "Cetiol HE."

[0060] (e) Polyoxyethylene-polyoxypropylene block co-polymers: e.g. of the type known and commercially available under the trade name "Poloxamer."

[0061] (f) Dioctylsuccinate, dioctylsodiumsulfosuccinate, di-[2-ethylhexyl]-succinate or sodium lauryl sulfate.

[0062] (g) Phospholipids, in particular lecithins: especially, soybean lecithin.

[0063] (h) Surfactants such as non-ionic polyoxyethylene fatty acid derivatives, in particular, polyoxyethylene sorbitan fatty acid esters (spans) such as sorbitan sesquiolate are preferred for use as emulsifϊers.

[0064] Additional emulsion systems include triethyl citrate -water, dimethylsulphoxide-triglyceryl cabroate and ethyl citrate-water.

[0065] In an exemplary form for using this method for producing ITC nanoparticles, the emulsion system may contain a polyoxyethylene-polyoxypropylene co-polymer. The polyoxyethylene-polyoxypropylene co-polymer may be present in a concentration (weight/weight) in water of about 1% to about 20%, about 3% to about 15%, any range derivable therein, and preferably from about 5% to 10%. The weight ration between a polyoxyethylene-polyoxypropylene co-polymer and an isothiocyanate may be from about 20:1 to about 500:1 or any range derivable therein.

[0066] The choice of a suitable emulsifier or a combination of emulsifiers can readily be made by those in the field.

[0067] Emulsification is usually performed by applying mechanical force to break down the internal phase liquid into small globules, in the range of 10 to 200 nm, more preferably less than 200 nm, and even more preferably less than 50 nm in diameter, and

85343306.1 molecules of surfactant molecules forming a barrier between the globules and the bulk of the external liquid. Such mechanical force can be applied by mechanical stirring, ultrasonic probes, or by passing the emulsion components through narrow space, as in the case of colloidal mills, or through narrow tubes, valves or orifices. The preferred emulsifϊcation technique is sonication.

[0068] To obtain solid nanoparticles, the emulsion may be diluted to allow total miscibility of the liquid dispersed phase inside the continuous phase. The miscibility of the liquid dispersed phase is accompanied by the formation of the nanoparticles from the resulting one liquid phase system. The dilution step can be carried out with either an additional portion of the continuous phase solution or a liquid that is miscible with both the dispersed and continuous phases. Diluting with the same continuous phase solution is preferred.

[0069] Separation of nanoparticles can be performed using dialysis, filtration, centrifugation, or other known techniques. Nanoparticles can then be formulated into a suspension dosage form according to standard procedures in the art for injection or non- injection use. Nanoparticles can also be dried and reconstituted prior to use.

B. Lipid-based Nanoparticles

[0070] Lipid-based nanoparticles include liposomes, lipid preparations and lipid- based vesicles (e.g., DOTAP: cholesterol vesicles). Lipid-based nanoparticles may be positively charged, negatively charged or neutral. In certain embodiments, the lipid-based nanoparticle is neutrally charged (e.g., a DOPC liposome).

1. Liposomes

[0071] A "liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition. Liposomes of the present invention include unilamellar liposomes, multilamellar liposomes and multivesicular liposomes. Liposomes of the present invention may be positively charged, negatively charged or neutrally charged. In certain embodiments, the liposomes are neutral in charge.

85343306.1 [0072] A multilamellar liposome has multiple lipid layers separated by aqueous medium. They form spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.

[0073] In specific aspects, isothiocyanate may be, for example, embedded in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the isothiocyanate, entrapped in a liposome, complexed with a liposome, or the like.

[0074] A liposome used according to the present invention can be made by different methods, as would be known to one of ordinary skill in the art. For example, a phospholipid, such as the neutral phospholipid dioleoylphosphatidylcholine (DOPC), may be dissolved in tert-butanol. The lipid(s) is then mixed with isothiocyanate, and/or other component(s). Tween 20 is added to the lipid mixture such that Tween 20 is about 5% of the composition's weight. Excess tert-butanol is added to this mixture such that the volume of tert-butanol is at least 95%. The mixture is vortexed, frozen in a dry ice/acetone bath and lyophilized overnight. The lyophilized preparation is stored at -20⁰C. and can be used up to three months. When required the lyophilized liposomes are reconstituted in 0.9% saline.

[0075] Alternatively, a liposome can be prepared by mixing lipids in a solvent in a container, e.g., a glass, pear-shaped flask. The container should have a volume ten-times greater than the volume of the expected suspension of liposomes. Using a rotary evaporator, the solvent is removed at approximately 40⁰C. under negative pressure. The solvent normally is removed within about 5 min. to 2 hours, depending on the desired volume of the liposomes. The composition can be dried further in a desiccator under vacuum. The dried lipids generally are discarded after about 1 week because of a tendency to deteriorate with time.

[0076] Dried lipids can be hydrated at approximately 25-50 mM phospholipid in sterile, pyrogen-free water by shaking until all the lipid film is resuspended. The aqueous liposomes can be then separated into aliquots, each placed in a vial, lyophilized and sealed under vacuum.

85343306.1 [0077] The dried lipids or lyophilized liposomes prepared as described above may be rehydrated and reconstituted in a solution of a protein or peptide and diluted to an appropriate concentration with an suitable solvent, e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer. Unencapsulated additional materials, such as agents including but not limited to hormones, drugs, nucleic acid constructs and the like, are removed by centrifugation at 29,000xg and the liposomal pellets washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., about 50- 200 mM. The amount of additional material or active agent encapsulated can be determined in accordance with standard methods. After determination of the amount of additional material or active agent encapsulated in the liposome preparation, the liposomes may be diluted to appropriate concentrations and stored at 4°C until use. A pharmaceutical composition comprising the liposomes will usually include a sterile, pharmaceutically acceptable carrier or diluent, such as water or saline solution.

[0078] In other alternative methods, liposomes can be prepared in accordance with other known laboratory procedures (e.g., see Bangham et ah, 1965; Gregoriadis, 1979;

Deamer and Uster, 1983; Szoka and Papahadjopoulos, 1978, each incorporated herein by reference in relevant part). Additional liposomes which may be useful with the present invention include cationic liposomes, for example, as described in WO02/100435 Al, U.S.

Pat. No. 5,962,016, U.S. Application 2004/0208921, WO03/015757A1, WO04/029213A2, U.S. Pat. No. 5,030,453, and U.S. Pat. No. 6,680,068, all of which are hereby incorporated by reference in their entirety without disclaimer. A process of making liposomes is also described in WO04/002453A1. Neutral lipids can be incorporated into cationic liposomes

{e.g., Farhood et al., 1995). Various neutral liposomes which may be used in certain embodiments are disclosed in U.S. Pat. No. 5,855,911, which is incorporated herein by reference. These methods differ in their respective abilities to entrap aqueous material and their respective aqueous space-to-lipid ratios.

[0079] The size of a liposome varies depending on the method of synthesis. Liposomes in the present invention can be a variety of sizes. In certain embodiments, the liposomes are small, e.g., less than about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or less than about 50 nm in external diameter. In preparing such liposomes, any protocol described herein, or as would be known to one of ordinary skill in the art may be used. Additional non- limiting examples of preparing liposomes are described in U.S. Pat.

85343306.1 Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; International Applications PCT/US85/01161 and PCT/US89/05040; U.K. Patent Application GB 2193095 A; Mayer et al, 1986; Hope et al., 1985; Mayhew et al. 1987; Mayhew et al, 1984; Cheng et al, 1987; and Liposome Technology, 1984, each incorporated herein by reference).

2. Neutral Liposomes

[0080] In certain embodiments, the lipid based nanoparticle is a neutral liposome {e.g., a DOPC liposome). "Neutral liposomes" or "non-charged liposomes", as used herein, are defined as liposomes having one or more lipid components that yield an essentially- neutral, net charge (substantially non-charged). By "essentially neutral" or "essentially non- charged", it is meant that few, if any, lipid components within a given population {e.g., a population of liposomes) include a charge that is not canceled by an opposite charge of another component {i.e., fewer than 10% of components include a non-canceled charge, more preferably fewer than 5%, and most preferably fewer than 1%). In certain embodiments, neutral liposomes may include mostly lipids and/or phospholipids that are themselves neutral under physiological conditions {i.e., at about pH 7).

[0081] Liposomes and/or lipid-based nanoparticles of the present invention may comprise a phospholipid. In certain embodiments, a single kind of phospholipid may be used in the creation of liposomes {e.g., a neutral phospholipid, such as DOPC, may be used to generate neutral liposomes). In other embodiments, more than one kind of phospholipid may be used to create liposomes.

[0082] Phospholipids include, for example, phosphatidylcholines, phosphatidylglycerols, and phosphatidylethanolamines; because phosphatidylethanolamines and phosphatidyl cholines are non-charged under physiological conditions (i.e., at about pH 7), these compounds may be particularly useful for generating neutral liposomes. In certain embodiments, the phospholipid DOPC is used to produce non-charged liposomes. In certain embodiments, a lipid that is not a phospholipid (e.g. , a cholesterol) may be used.

[0083] Phospholipids include glycerophospholipids and certain sphingo lipids.

Phospholipids include, but are not limited to, dioleoylphosphatidylycholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"),

85343306.1 distearoylphosphatidylcholine ("DSPC"), l-myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"), l-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"), l-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"), l-stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), distearoyl sphingomyelin ("DSSP"), distearoylphophatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), dimyristyl phosphatidylcholine ("DMPC"), l,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2- diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1 ,2-dieicosenoyl-sn-glycero-3- phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoyl phosphatidylcholine ("POPC"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

[0084] Phospholipids may be from natural or synthetic sources. However, phospholipids from natural sources, such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin and plant or bacterial phosphatidylethanolamine are not used, in certain embodiments, as the primary phosphatide (i.e., constituting 50% or more of the total phosphatide composition) because this may result in instability and leakiness of the resulting liposomes.

3. Lipid-Based Vesicles

[0085] Another type of lipid-based nanoparticle contemplated for use in the present invention is a lipid-based vesicle. Lipid-based vesicles may be generated using one or more of the lipids listed above.

[0086] In certain embodiments, the lipid-based vesicle is a DOTAPxholesterol vesicle. DOTAPxholesterol vesicles are prepared by mixing the cationic lipid DOTAP (1,2- bis(oleoyloxy)-3-(trimethylammonio)-propane) with cholesterol.

85343306.1 C. Additional Therapeutic Agents

[0087] The compositions of the present invention may optionally include one or more additional therapeutic agents. For example, the therapeutic agent may be a chemotherapeutic agent.

[0088] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBl-TMl); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfϊromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone,

85343306.1 dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan

(e.g., CPT-I l); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0089] Also included in the compositions may be anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, A- hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides,

85343306.1 particularly those which inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, RaIf and H-Ras; ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor; vaccines such as gene therapy vaccines and pharmaceutically acceptable salts, acids or derivatives of any of the above.

D. Administration of Nanoparticle Formulation

[0090] The composition of this invention enables sustained, continuous delivery of

ITC to tissues adjacent to or distant from an administration site. The biologically-active agent is capable of providing a local or systemic biological, physiological or therapeutic effect. For example, the ITC may act to kill cancer cells, or to control or suppress tumor growth, among other functions.

[0091] The ITC nanoparticles are administered in an amount effective to provide the desired level of biological, physiological, pharmacological and/or therapeutic effect. The ITC may stimulate or inhibit a biological or physiological activity. The concentration of the ITC agent should not be so high that the composition has a consistency that inhibits its delivery to the administration site by the desired method. The lower limit of the amount of the ITC will depend on its activity and the period of time desired for treatment. The ITC may be gradually released by dissolution of the nanoparticles.

E. Targeting of Nanoparticles [0092] Targeted delivery is achieved by the addition of ligands without compromising the ability of nanoparticles to deliver their isothiocyanate loads. It is contemplated that this will enable delivery to specific cells, tissues and organs. The targeting specificity of the ligand-based delivery systems is based on the distribution of the ligand receptors on different cell types. It is preferable that the ligand to be conjugated to the nanoparticles will bind to the receptors that specifically or predominantly express in tumor cells so that the ITC will be preferentially delivered to the tumor cells. For example, specific antibodies such as anti- CD20 (Rituximab) may be conjugated to the nanoparticles to deliver ITC to malignant B- cells such as those of chronic lymphocytic leukemia and B-cell lymphoma.

[0093] The targeting ligand may either be non-covalently or covalently associated with a nanoparticle, and can be conjugated to the nanoparticles by a variety of methods as discussed herein.

85343306.1 [0094] Examples of proteins or peptides that can be used to target nanoparticles include transferin, lactoferrin, TGF-α, nerve growth factor, albumin, HIV Tat peptide, RGD peptide, and insulin, as well as others (Gupta et ah, 2005; Ferrari, 2005).

III. Isothiocyanate [0095] As used herein, "isothiocyanate" (ITC) refers to any compound having the formula, R — N=C=S, where R may be saturated or unsaturated, substituted or unsubstituted, or an aliphatic or aromatic group. Non-limiting examples of R include phenethyl, benzyl, methyl; ethyl; propyl; isopropyl; n-butyl; t-butyl; s-butyl; pentyl; hexyl; heptyl; octyl; nonyl; decyl; undecane; phenyl; o-tolyl; 2-fluorophenyl; 3 -fluorophenyl; 4-fluorophenyl; 2- nitrophenyl; 3-nitrophenyl; 4-nitrophenyl; 2-chlorophenyl; 2-bromophenyl; 3-chlorophenyl; 3-bromophenyl; 4-chlorophenyl; 2,4-dichlorophenyl; R-(+)-alpha-methylbenzyl; S-(-)-alpha- methylbenzyl; 3-isoprenyl-alpha,alpha-dimethylbenzyl; trans-2-phenylcyclopropyl; (SCN)CH2C6H4°CH2— ; (SCN)CH(CH3)CH2— C6H4°CH2CH(CH3)CH2— ;

CH3S(O)CH2CH2CH2CH2— ; 2-ethylphenyl; benzoyl; 1-naphthyl; benzoyl; A- bromophenyl; 2-methoxyphenyl; m-tolyl; alpha, alpha, alpha-trifluoro-m-tolyl; 3- fluorophenyl; 3-chlorophenyl; 3-bromophenyl; (SCN)C6H4 — ; (propylcyclohexyl)benzyl; (hexylcyclohexyl)benzyl; (octylcyclohexyl)benzyl; 2-methylbenzyl; 2-chlorobenzo; 3- chlorobenzo; 4-chlorobenzo; m-toluyl; and p-toluyl. Preferably, R is phenethyl or benzyl or CH3 S(O)CH2CH2CH2CH2— .

[0096] The ITC can be either isolated from natural sources or prepared by chemical synthesis. Natural sources of ITC include cruciferous vegetables such as watercress, horseradish, radishes, onions, mustards, alyssum, candytuft, cabbage, and broccoli (U.S. Pat.

Nos. 5,725,895, 5,968,567, and 5,968,505).

[0097] Non-limiting examples of ITCs include phenethyl isothiocyanate, benzyl isothiocyanate, sulforaphane (SFN); methyl isothiocyanate; ethyl isothiocyanate; propyl isothiocyanate; isopropyl isothiocyanate; n-butyl isothiocyanate; t-butyl isothiocyanate; s- butyl isothiocyanate; pentyl isothiocyanate; hexyl isothiocyanate; heptyl isothiocyanate; octyl isothiocyanate; nonyl isothiocyanate; decyl isothiocyanate; undecane isothiocyanate; phenyl isothiocyanate; o-tolyl isothiocyanate; 2-fluorophenyl isothiocyanate; 3 -fluorophenyl isothiocyanate; 4-fluorophenyl isothiocyanate; 2-nitrophenyl isothiocyanate; 3-nitrophenyl isothiocyanate; 4-nitrophenyl isothiocyanate; 2-chlorophenyl isothiocyanate; 2-bromophenyl isothiocyanate; 3-chlorophenyl isothiocyanate; 3-bromophenyl isothiocyanate; A-

85343306.1 chlorophenyl isothiocyanate; 2,4-dichlorophenyl isothiocyanate; R-(+)-alpha-methylbenzyl isothiocyanate; S-(-)-alpha-methylbenzyl isothiocyanate; 3-isoprenyl-alpha,alpha- dimethylbenzyl isothiocyanate; trans-2-phenylcyclopropyl isothiocyanate; 1,3- bis(isothiocyanatomethyl)-benzene; 1 ,3-bis(l -isothiocyanato- 1 -methylethyl)benzene; 2- ethylphenyl isothiocyanate; benzoyl isothiocyanate; 1-naphthyl isothiocyanate; benzoyl isothiocyanate; 4-bromophenyl isothiocyanate; 2-methoxyphenyl isothiocyanate; m-tolyl isothiocyanate; alpha, alpha, alpha-trifluoro-m-tolyl isothiocyanate; 3 -fluorophenyl isothiocyanate; 3 -chlorophenyl isothiocyanate; 3-bromophenyl isothiocyanate; 1 ,4-phenylene diisothiocyanate; 1 -isothiocyanato-4-(trans-4-propylcyclohexyl)benzene; 1 -(trans-4- hexylcyclohexyl)-4-isothiocyanatobenzene; 1 -isothiocyanato-4-(trans-4-octylcyclohexyl) benzene; 2-methylbenzyl isothiocyanate; 2-chlorobenzo isothiocyanate; 3-chlorobenzo isothiocyanate; 4-chlorobenzo isothiocyanate; m-toluyl isothiocyanate; p-toluyl isothiocyanate and the like. Preferably, the isothiocyanate is phenethyl or benzyl isothiocyanate or sulforaphane. A particular example is phenylethyl isothiocyanate in the present invention.

[0098] Phenylethyl isothiocyanate (PEITC) is a naturally occurring compound found in cruciferous vegetables such as broccoli, cauliflower, watercress routinely consumed by human. This compound has been studied previously as a potential cancer preventive agent (Hecht et al, 1999). The cancer preventive effect of PEITC is attributed to its ability to block metabolic activation of the tobacco carcinogen NNK and to enhance the expression of detoxifying enzyme and thus increase the excretion of the detoxified metabolites (Chung et al, 1993; Yu et al 1998). Epidemiologic study revealed an inverse correlation between cruciferous vegetables consumption and ovarian cancer risk (Pan, 2004). Previous studies on PEITC mainly focused on its potential cancer prevention activity, which eventually led to a NIC-sponsored phase I clinical trial using PEITC for prevention of lung cancer in smokers.

[0099] In contrast, the ability of PEITC to effectively kill cancer cells as a potential therapeutic agent has only been recognized in the recent years. PEITC exhibits suppressive activity on tumor cell growth in vitro and in vivo (Conaway et al, 2005; Yang et al, 2005; Kim et al, 2006; Khro et al, 2006). Several studies showed that this compound can cause apoptosis in cancer cell lines, and ROS generation was observed during the PEITC-induced cell death (Xu et al, 2001; Zhang et al, 2003; Xiao et al, 2006). In some cases, phenethyl isothiocyanate (PEITC) has been shown to induce apoptosis in cells that are resistant to some

85343306.1 currently used chemotherapeutic drugs, for example, in drug resistant leukemia cells which produce the powerful apoptosis inhibitor protein BCl-2.

[00100] The inventors recently showed that this compound causes severe ROS accumulation preferentially in cancer cells, due to its potent ability to disable the glutathione antioxidant system by two complementary mechanisms: causing depletion of GSH and inhibiting glutathione peroxidase enzyme activity (Trachootham, 2006). Importantly, the inventors showed that cancer cells transformed by Ras or leukemia cells (CML) carrying Bcr- AbI were metabolically active with increased ROS generation, highly dependent on the glutathione system, and sensitive to inhibition by PEITC, whereas normal cells with lower basal ROS output are significantly less sensitive to PEITC (Zhang, 2008; Tralchootham, 2006). Thus, it is clear that the cytotoxic action of PEITC is largely dependent on the levels of intrinsic cellular ROS stress and the dependency of the cancer cells on the glutathione antioxidant system.

[00101] Because most cancer cells exhibit increased oxidative stress due to oncogenic transformation, active metabolism, and/or mitochondrial dysfunction, these malignant cells are more dependent on the glutathione antioxidant system to maintain redox balance. Thus, abrogation of this antioxidant system by PEITC will cause severe oxidative damage to cancer cells leading to suppression of cancer growth or death of the cancer cells.

In fact, the inventors recently demonstrated that PEITC is effective against a variety of cancer cell types including solid tumors (e.g. ovarian cancer and colon cancer) and leukemia cells, especially the drug-resistant leukemia cells isolated from patients with chronic lymphocytic leukemia (CLL) resistant to fludarabine or chronic myelogenous leukemia (CML) resistant to

Gleevec (Zhang, 2008; Tralchootham, 2008).

[00102] Based on these promising data and the facts that PEITC has poor solubility and is unstable when diluted in standard solvents such as PBS, the inventors reasoned that it is necessary to develop a new formulation that improve the solubility and stability of PEITC for clinical use of this compound in cancer treatment. Furthermore, because PEITC effectively induces cancer cell death within a short period of time (5-20 h) and normal cells can better tolerate such PEITC exposure, the inventors reasoned that a systemic administration of PEITC in proper formulation and dosages to achieve a blood drug concentration range for a proper duration sufficient to kill cancer cells but not normal cells may preferentially eliminate cancer cells with minimum toxicity to the normal tissues. This

85343306.1 novel therapeutic concept has led the inventors to invent a novel formulation in which ITC such as PEITC is imbedded in nanoparticles with the size range of preferably 20-30 nanometers. In certain embodiments, this formulation of PEITC nanoparticles exhibits excellent chemical and pharmacological properties suitable for in vivo administration systematically (i.v. or i.p.), and shows in vivo therapeutic efficacy as described in this invention.

IV. Pharmaceutical Preparations

[00103] Where clinical application of the particles of the present invention is undertaken, it will generally be beneficial to prepare the particles as a pharmaceutical composition appropriate for the intended application. This will typically entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One may also employ appropriate buffers to render the complex stable and allow for uptake by target cells.

[00104] The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as a human, as appropriate. For animal {e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[00105] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives {e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. A pharmaceutically acceptable carrier is preferably formulated for administration to a human, although in certain embodiments it may be desirable to use a pharmaceutically acceptable carrier that is formulated for administration to a non-human animal but which would not be acceptable {e.g. , due to governmental regulations) for administration to a human. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

85343306.1 [00106] The actual dosage amount of a composition of the present invention administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[00107] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound, such as. In other embodiments, the active compound may comprise between about 1% to about 75% of the weight of the unit, or between about 5% to about 50%, for example, and any range derivable therein. In other non- limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 30 milligram/kg/body weight, about 40 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 microgram /kg/body weight to about 5 milligram /kg/body weight, about 50 microgram/kg/body weight to about 50 milligram/kg/body weight, etc., can be administered.

[00108] An ITC may be administered in a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,

20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more mg of ITC per dose. Each dose may be in a volume of 1, 10, 50, 100, 200, 500, 1000 or more μl or ml.

[00109] Solutions of therapeutic compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[00110] The therapeutic compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection

85343306.1 may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[00111] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.

[00112] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[00113] The therapeutic compositions of the present invention may include classic pharmaceutical preparations. Administration of therapeutic compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal, topical, or aerosol.

[00114] An effective amount of the therapeutic composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[00115] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended

85343306.1 goal of treatment (e.g. , alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.

B. Route of Administration

[00116] In pharmacology and toxicology, a route of administration is the path by which a drug, fluid, poison or other substance is brought into contact with the body. In the present invention, the composition comprising an ITC nanoparticle formulation may be formulated in a topical formulation or an enteral formulation, and preferably, a parenteral formulation. In particular aspects, the compositions may be administered to a subject by injection or infusion for a systematic delivery.

[00117] Obviously, a substance must be transported from the site of entry to the part of the body where its action is desired to take place (even if this only means penetration through the stratum corneum into the skin). However, using the body's transport mechanisms for this purpose can be far from trivial. The pharmacokinetic properties of the ITC (that is, those related to processes of uptake, distribution, and elimination) are critically influenced by the route of administration.

[00118] Routes of administration can broadly be divided into: topical (local effect, substance is applied directly where its action is desired); enteral (desired effect is systemic (non-local), substance is given via the digestive tract); parenteral (desired effect is systemic, substance is given by routes other than the digestive tract). The U.S. Food and Drug Administration recognizes 111 distinct routes of administration. The following is a brief list of some routes of administration.

[00119] Topical form of administration includes epicutaneous (application onto the skin), e.g. allergy testing, typical local anesthesia; inhalational, e.g. asthma medications; enema, e.g. contrast media for imaging of the bowel; eye drops (onto the conjunctiva), e.g. antibiotics for conjunctivitis; ear drops - such as antibiotics and corticosteroids for otitis externa; intranasal route (into the nose), e.g. decongestant nasal sprays; vaginal, e.g. topical estrogens, antibacterials.

[00120] Enteral form of administration involves any part of the gastrointestinal tract: by mouth (orally), many drugs as tablets, capsules, or drops; by gastric feeding tube,

85343306.1 duodenal feeding tube, or gastrostomy, many drugs and enteral nutrition; rectally, various drugs in suppository or enema form;

[00121] Parenteral form of administration by injection or infusion involves intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), subcutaneous (under the skin), intraosseous infusion (into the bone marrow) - in effect, an indirect intravenous access because the bone marrow drains directly into the venous system; intradermal (into the skin itself), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion

(infusion into the urinary bladder). Other parenteral administration include transdermal (diffusion through the intact skin), sublingual, i.e. under the tongue, nitroglycerine, buccal

(absorbed through cheek near gumline), vaginal suppositories, inhalational, epidural (i.e., peridural) (injection or infusion into the epidural space), intravitreal.

[00122] Injection encompasses intravenous (IV), intramuscular (IM), and subcutaneous (sub-Q). In acute situations, in emergency medicine and intensive care medicine, drugs are most often given intravenously. This is the most reliable route, as in acutely ill patients the absorption of substances from the tissues and from the digestive tract can often be unpredictable due to altered blood flow or bowel motility.

V. Hyperproliferative Diseases

[00123] In certain embodiments of the invention, a therapeutically effective amount of the parenteral pharmaceutical composition comprising an isothiocyanate (ITC) formulated in a pharmaceutically acceptable nanoparticle formulation may be used to treat a diseases and/or condition in a subject.

A. Definitions

[00124] "Treatment" and "treating" refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a treatment may include administration of a parenteral pharmaceutical composition comprising an isothiocyanate (ITC) formulated in a pharmaceutically acceptable nanoparticle formulation, whereby the nanoparticle formulation solubilizes and stabilizes the ITC.

85343306.1 [00125] A "subject" refers to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.

[00126] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[00127] A "disease" or "health-related condition" can be any pathological condition of a body part, an organ, or a system resulting from any cause, such as infection, genetic defect, and/or environmental stress. The cause may or may not be known. Specifically, a tissue or cell with the disease or condition may have an increased reactive oxygen species (ROS) compared with a normal tissue or cell. The compositions disclosed may be particularly used to treat such a cell or tissue because ITC may induce oxidative stress and cause severe ROS accumulation in the tissue or cell with increased ROS generation, leading to selective tissue or cell killing.

[00128] A "hyperproliferative disease" includes diseases and conditions that are associated with any sort of abnormal cell growth or abnormal growth regulation, specifically a cancer.

[00129] In some embodiments of the invention, the methods include identifying a patient in need of treatment. A patient may be identified, for example, based on taking a patient history, based on findings on clinical examination, based on health screenings, or by self-referral.

B. Diseases

[00130] The present invention can find application in the treatment of any disease for which delivery of a therapeutic isothiocyanate to a cell or tissue of a subject is believed to be of therapeutic benefit. Examples of such diseases include hyperproliferative diseases and quiescent malignant diseases.. In particular embodiments, the disease is a

85343306.1 hyperproliferative disease, such as cancer of solid tissues or blood cells. Quiescent malignant diseases that can be treated by ITC nanoparticles include, for example, chronic lymphocytic leukemia.

[00131] For example, an isothiocyanate (ITC) formulated in a pharmaceutically acceptable nanoparticle formulation may be administered to treat a hyperproliferative disease.

The hyperproliferative disease may be cancer, leiomyomas, adenomas, lipomas, hemangiomas, fϊbromas, pre-neoplastic lesions (such as adenomatous hyperplasia and prostatic intraepithelial neoplasia), carcinoma in situ, oral hairy leukoplakia, or psoriasis.

[00132] The cancer may be a solid tumor, metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In certain embodiments, the cancer is ovarian cancer. In particular aspects, the cancer may be a chemo-resistant cancer.

[00133] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar

85343306.1 cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

85343306.1 VI. Combination Treatments

[00134] In certain embodiments, the compositions and methods of the present invention involve ITC nanoparticle formulation-based composition as set forth herein with a second or additional therapy. Such therapy can be applied in the treatment of any disease for which treatment with the ITC nanoparticle formulation is contemplated. For example, the disease may be a hyperproliferative disease, such as cancer.

[00135] The methods and compositions including combination therapies enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with a therapeutic nucleic acid, such as an inhibitor of gene expression, and a second therapy. A tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) including one or more of the agents (i.e., inhibitor of gene expression or an anti-cancer agent), or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) an inhibitor of gene expression; 2) an anti-cancer agent, or 3) both an inhibitor of gene expression and an anti-cancer agent. Also, it is contemplated that such a combination therapy can be used in conjunction with a chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

[00136] A therapeutic ITC nanoparticle formulation-containing composition set forth herein may be administered before, during, after or in various combinations relative to an anti-cancer treatment. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the ITC nanoparticle formulation- containing composition is provided to a patient separately from an additional anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two agents would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the inhibitor of gene expression therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more preferably, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly

85343306.1 where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between respective administrations.

[00137] Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, depending on the condition of the patient, such as their prognosis, strength, health, etc.

[00138] Various combinations may be employed. For the example below a therapeutic ITC nanoparticle formulation-containing composition is "A" and an anti-cancer therapy is "B":

[00139] A/B/A BIAJB BIBIA A/A/B A/B/B BIAJA AJBIBIB

B/A/B/B

[00140] B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

[00141] B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A

A/A/B/A

[00142] Administration of any compound or therapy of the present invention to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as radiation and surgical intervention, may be applied in combination with the described therapy.

[00143] In specific aspects, it is contemplated that a standard therapy will include chemotherapy, radiotherapy, immunotherapy, surgical therapy or gene therapy and may be employed in combination with the inhibitor of gene expression therapy, anticancer therapy, or both the therapeutic nucleic acid and the anti-cancer therapy, as described herein.

85343306.1 A. Chemotherapy

[00144] A wide variety of chemotherapeutic agents may be used in accordance with the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas. Examples of these agents have been previously set forth. Specifically, a therapeutic agent for combination with ITC nanoparticles in cancer treatment may also be a compound that specifically targets a specific molecule in a cancer cell or on the cancer cell surface. Such target-specific therapeutic agents include, for example, Imatinib (Gleevec), rituximab, cetuximab (erbitux), and herceptin (trastuzumab).

B. Radiotherapy

[00145] Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287) and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[00146] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing, for example, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

85343306.1 C. Immunotherapy

[00147] In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Trastuzumab (Herceptin™) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.

[00148] Another immunotherapy could also be used as part of a combined therapy with the present invention. In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.

Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-I,

MCP-I, IL-8 and growth factors such as FLT3 ligand. Combining immune stimulating molecules, either as proteins or using gene delivery in combination with a tumor suppressor has been shown to enhance anti-tumor effects (Ju et al., 2000). Moreover, antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein.

[00149] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, e.g., interferons α, β and γ; IL-I, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et

85343306.1 al, 1998); gene therapy, e.g., TNF, IL-I, IL-2, p53 (Qin et al, 1998; Austin- Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-ganglioside GM2, anti-HER-2, anti-pl85 (Pietras et al, 1998; Hanibuchi et al, 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the nanoparticle therapies described herein.

[00150] In active immunotherapy, an antigenic peptide, polypeptide or protein, or an autologous or allogenic tumor cell composition or "vaccine" is administered, generally with a distinct bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al, 1992; Mitchell et al, 1990; Mitchell et al, 1993).

[00151] In adoptive immunotherapy, the patient's circulating lymphocytes, or tumor infiltrated lymphocytes, are isolated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg et al, 1988; 1989).

D. Surgery

[00152] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.

[00153] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery

(Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[00154] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4,

85343306.1 and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

E. Other Agents

[00155] It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents.

[00156] Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP- 1, MIP-lbeta, MCP-I, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL (Apo-2 ligand) would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increasing intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.

[00157] In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.

[00158] Another form of therapy for use in conjunction with chemotherapy, radiation therapy or biological therapy includes hyperthermia, which is a procedure in which a patient's tissue is exposed to high temperatures (up to 106⁰F). External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia. Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body. Internal heat may involve a sterile probe , including thin, heated wires or

85343306.1 hollow tubes filled with warm water, implanted microwave antennae, or radiofrequency electrodes.

[00159] A patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets. Alternatively, some of the patient's blood may be removed and heated before being perfused into an area that will be internally heated. Whole -body heating may also be implemented in cases where cancer has spread throughout the body. Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose.

[00160] Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.

VII. Examples

[00161] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 Method to prepare PEITC nanoparticles

[00162] In certain embodiments of the present invention, PEITC nanoparticles may be prepared for improved stability and function. The following method is a non-limiting example.

[00163] First, the inventors prepared main starting materials such as Pluronic® F127 (F127), β-phenylethyl isothiocyanate (PEITC), Ethyl acetate (EtAc). Further, the

85343306.1 inventors also have the key equipments ready to use: High-Speed TlO homogenizer (IKA, Germany), Ultrasonicator, Rotating evaporator (BUCHI, Switzerland), Lyophilizer, Laser diffraction particle size analyzer (Nano-ZS, Malvern Instrument, UK), Malvern zeta analyzer (Nano-ZS, Malvern Instrument, UK).

[00164] The inventors performed the following key steps to prepare PEITC nanoparticles (all the steps/procedures should avoid direct exposure to light):

[00165] (1) prepared PEITC solution in EtAc at various concentrations.

[00166] (2) prepared Pluronic® F 127 (F 127) at the concentration range of 5-

10% (w/w).

[00167] (3) introduced PEITC solution into F127 aqueous solution with proper ratios as desired.

[00168] (4) made a well-mixed oil-in-water (O/W) emulsion using high-speed

TlO homogenization with intensive sonication. This should be performed in an ice-bath.

[00169] (5) evaporated off EtAc using a rotating evaporator at 4oC for 4-6 hours to obtain a slurry of PEITC nanoparticles

[00170] (6) lyophilized the slurry to obtain PEITC nanoparticles in powder form.

[00171] Pluronic F-127 (CAS#: 9003-11-6, MDL#: MFCD00082049,

PubChem Substance ID: 24897874) is one exemplary polymer that could be used in the present invention. It has an average molecular weight 12.6 kDa, may form gels above 10 ⁰C when used at concentrations of 18-50%. It will re-liquefy when cooled to below 10⁰C. The gel should remain stable during autoclaving.

Example 2 Characterization of PEITC nanoparticles

[00172] The proper size distribution is important for the therapeutic potential of the PEITC nanoparticles. Preferably, the average size of the PEITC nanoparticles may be

85343306.1 about 20 to about 30 nm. To explore the conditions to make particles around the desired size range, the inventors used different amounts of PEITC to prepare nanoparticles with the same amount of polymer formulation and characterized the resulting nanoparticles.

[00173] For characterization of these nanoparticle preparations, the inventors determined article size distribution of the PEITC nanoparticles by laser diffraction particle sizer (Nano-ZS, Malvern Instrument, UK). The experiments were done in triplicates, and the results were expressed as mean +/- SE. They also measured the zeta potential of PEITC nanoparticles by Malvern Zeta analyzer (Nano-ZS, Malvern Instrument, UK) (the temperature was kept at 20⁰C during the measuring process). Each sample was analyzed for 10 runs and the results were expressed as mean +/- SE.

[00174] As shown in FIGs. 2 and 6, two different preparations of a drug loading of 2 mg PEITC in 2 ml of 5% F 127 can consistently produce nanomolecules with average size of 25-26 nm and the zeta potential of -7 mV to -17 mV, and therefore are good conditions for preparation of PEITC nanoparticles. Similarly, a drug loading of 6 mg PEITC in 2 ml of 5% F 127 may consistently produce nanomolecules with average size of 24-26 nm and the zeta potential of -13 mV to -15 mV, and therefore are good conditions for preparation of PEITC nanoparticles (FIGs. 3 and 7). A drug loading of 12 mg PEITC in 2 ml of 5% F 127 may also consistently produce nanomolecules with average size of 29-30 nm and the zeta potential of -10 mV to -20 mV, and therefore are good conditions for preparation of PEITC nanoparticles (FIGs. 4 and 8). However, a drug loading of 24 mg PEITC in 2 ml of 5% F 127 can not consistently produce nanomolecules with average size less than 200 nm and this is not a good conditions to prepare PEITC nanoparticles (FIGs. 5 and 9).

Example 3 Biological activity of PEITC nanoparticles in vitro

[00175] (1) In vitro cytotoxicity against cancer cells:

[00176] A comparison of the in vitro cytotoxicity of PEITC nanoparticles and regular PEITC in HL-60 cells showed that the PEITC nanoparticles are potent against leukemia cells in vitro (FIG. 10). Various concentrations of PEITC nanoparticle solution were made from lyophilized powder. Samples #1 to #4 indicate 4 separate preparations of

85343306.1 PEITC nanoparticles. HL-60 cells were treated with 10 uM PEITC nanoparticle solution in comparison with 10 uM regular PEITC (dissolved in DMSO and further diluted in culture medium). The drug incubation time was 14-18 h. Then annexin V-FITC/PI double staining was performed to evaluate cytotoxicity using flow cytometry analysis.

[00177] (2) Stability of PEITC nanoparticles in comparison with regular

PEITC solution

[00178] To test the stability of PEITC nanoparticles, the cytotoxicity of PEITC nanoparticles were tested after different periods of storage. PEITC nanoparticle solution remained fully active after 3 weeks of storage at 4°C (FIG. 11). The cytotoxicity assay was performed in HL-60 cells with 18 h drug incubation. Samples #1 to #4 indicate 4 separate preparations of PEITC nanoparticles. Similarly, PEITC nanoparticle solution remained fully active after one month of storage at 4°C (FIG. 11). The cytotoxicity assay was performed in HL-60 cells with 14 h drug incubation. Samples #1 to #4 indicate 4 separate preparations of PEITC nanoparticles. In comparison, regular PEITC solution (not a nanoparticle formulation) lost its activity after storage at 4°C for 3-4 weeks (FIG. 13). The cytotoxicity assay was performed in HL-60 and Raji cells (14 h drug incubation).

Example 4 Testing of PEITC nanoparticles in vivo [00179] PEITC nanoparticles were prepared as injectable solution in PBS.

Nude mice (a group of 5) bearing human malignant melanoma xenografts were given the drug by tail-vein injection. Drug dosage: 30 mg/kg body weight, i.v. (intravenously), every other day for two injections. Then the dose was escalated to 40 mg/kg, i.v., every other day for three injections. Current dose has been escalated to 50 mg/kg.

[00180] The inventors obtained the following results:

[00181] (1) PEITC nanoparticles are well tolerated in mice at the dose range of 30-40 mg/kg. No observable toxic side effect was noted after 5 repeat injections. Mice appeared health with normal activity. Further dose escalation may be required to determine MTD (maximum tolerated dose).

85343306.1 [00182] (2) At the dose range of 30-40 mg/kg described above, PEITC nanoparticles exhibited therapeutic activity as a single agent. In all 5 mice tested, the drug either induced shrinkage of the established tumors (2/5) or suppressed tumor growth

(3/5) (FIG. 14). It is worth noting that malignant melanoma is highly resistant to other anticancer agents and grow rapidly once tumor is established without treatment.

[00183] (3) PEITC nanoparticles are well tolerated in mice at the dose range of 30-50 mg/kg (i.v.). The mice exhibited normal activity without any weight loss (FIG. 15).

[00184] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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85343306.1

Claims

1. A parenteral pharmaceutical composition comprising an isothiocyanate (ITC) formulated in a pharmaceutically acceptable nanoparticle formulation, whereby the nanoparticle formulation solubilizes and stabilizes the ITC.

2. The composition of claim 1, wherein the ITC is phenethyl isothiocyanate (PEITC), N- acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC), sulforaphane, benzyl isothiocyanate, methyl isothiocyanate, ethyl isothiocyanate, propyl isothiocyanate, isopropyl isothiocyanate, n-butyl isothiocyanate, t-butyl isothiocyanate, s-butyl isothiocyanate, pentyl isothiocyanate, hexyl isothiocyanate, heptyl isothiocyanate, octyl isothiocyanate, nonyl isothiocyanate, decyl isothiocyanate, undecane isothiocyanate, phenyl isothiocyanate, o-tolyl isothiocyanate, 2-fluorophenyl isothiocyanate, 3 -fluorophenyl isothiocyanate, 4-fluorophenyl isothiocyanate, 2-nitrophenyl isothiocyanate, 3-nitrophenyl isothiocyanate, 4-nitrophenyl isothiocyanate, 2-chlorophenyl isothiocyanate, 2-bromophenyl isothiocyanate, 3- chlorophenyl isothiocyanate, 3-bromophenyl isothiocyanate, 4-chlorophenyl isothiocyanate, 2,4-dichlorophenyl isothiocyanate, R-(+)-alpha-methylbenzyl isothiocyanate, S-(-)-alpha- methylbenzyl isothiocyanate, 3-isoprenyl-alpha,alpha-dimethylbenzyl isothiocyanate, trans- 2-phenylcyclopropyl isothiocyanate, l,3-bis(isothiocyanatomethyl)-benzene, l,3-bis(l- isothiocyanato-l-methylethyl)benzene, 2-ethylphenyl isothiocyanate, benzoyl isothiocyanate, 1-naphthyl isothiocyanate, benzoyl isothiocyanate, 4-bromophenyl isothiocyanate, 2- methoxyphenyl isothiocyanate, m-tolyl isothiocyanate, alpha, alpha, alpha-trifluoro-m-tolyl isothiocyanate, 3 -fluorophenyl isothiocyanate, 3 -chlorophenyl isothiocyanate, 3-bromophenyl isothiocyanate, 1 ,4-phenylene diisothiocyanate, l-isothiocyanato-4-(trans-4- propylcyclohexyl)benzene, 1 -(trans-4-hexylcyclohexyl)-4-isothiocyanatobenzene, 1 - isothiocyanato-4-(trans-4-octylcyclohexyl) benzene, 2-methylbenzyl isothiocyanate, 2- chlorobenzo isothiocyanate, 3-chlorobenzo isothiocyanate, 4-chlorobenzo isothiocyanate, m- toluyl isothiocyanate, or p-toluyl isothiocyanate.

3. The composition of claim 2, wherein the ITC is phenethyl isothiocyanate (PEITC).

4. The composition of claim 1, wherein the nanoparticle formulation comprises a polymer, a monomer, a hydrogel, an emulsion, a liposome, a micelle, a complexing ligand or a hydrotropic agent.

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5. The composition of claim 1, wherein the nanoparticle formulation comprises a DOTAP: cholesterol nanoparticle.

6. The composition of claim 1, wherein the nanoparticle formulation has an average particle diameter of about 10 nm to about 200 nm.

7. The composition of claim 6, wherein the nanoparticle formulation has an average particle diameter of about 20 nm to about 30 nm.

8. The composition of claim 4, wherein the polymer is polyoxyethylene- polyoxypropylene block co-polymer, poly-L-lysine, poly-L-Arginine, albumin, N-(2- hydroxypropyl) methacrylamide (HPMA), polyaspartamide, a dendrimer comprising a polyamido amine and polylysine core, hyaluronic acid, polylactic-co-glycolic acid, heparin, polyacrylic acid, crosslinked polyacrylic acid, carboxymethylcellulose, alginate, alginic acid, propylene glycol alginate, sodium alginate, a polylactide, poly-glutamic acid, or polyerucic- co-sebacic acid.

9. The composition of claim 8, wherein the polymer is polyoxyethylene- polyoxypropylene block co-polymer.

10. The composition of claim 9, wherein the polymer is Pluronic® or Pluronic® F127.

11. The composition of claim 1, wherein the nanoparticle formulation is a liquid formulation.

12. The composition of claim 1, wherein the nanoparticle formulation is a solid formulation.

13. The composition of claim 1, wherein the nanoparticle formulation is a powder.

14. The composition of claim 1, wherein the composition is present in a substantially aqueous solution.

15. The composition of claim 1, wherein the composition is previously lyophilized .

16. The composition of claim 15, wherein the composition is rehydrated in a solution or liquid from the previously lyophilized composition.

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17. The composition of claim 15, wherein the composition is re-suspended in a solution or liquid from the previously lyophilized composition.

18. The composition of claim 1, wherein the composition further comprises an additional therapeutic agent.

19. The composition of claim 18, wherein said additional therapeutic agent is a chemotherapeutic agent.

20. A method of preparing a composition according to claim 1 comprising admixing said isothiocyanate into said pharmaceutically acceptable nanoparticle formulation.

21. The method of claim 20, wherein the method further comprises homogenization and/or sonication.

22. The method of claim 20, wherein the method further comprises dehydrating the formulation.

23. The method of claim 20, wherein the method further comprises lyophilizating the formulation.

24. The method of claim 22, wherein the method further comprises rehydrating or resuspending in a solution or liquid.

25. A method of treating a hyperproliferative disease or a quiescent malignant disease comprising administering a therapeutically effective amount of the composition according to claims 1 to 19 to a subject in need of such treatment.

26. The method of claim 25, wherein the composition is previously dehydrated.

27. The method of claim 26, wherein the composition is previously lyophilized.

28. The method of claim 26, wherein the composition is an aqueous solution or liquid formulation of previously lyophilized or dehydrated composition.

29. The method of claim 28, wherein the previously lyophilized or dried composition is stored at 4 degree for at least 1 week.

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30. The method of claim 29, wherein the previously lyophilized or dried composition is stored at 4 degree for at least 3 weeks.

31. The method of claim 29, wherein the previously lyophilized or dried composition is stored at 4 degree for up to 4 weeks.

32. The method of claim 25, wherein the hyperproliferative disease is cancer.

33. The method of claim 32, wherein the cancer is melanoma, leukemia, ovarian cancer, colon cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, bladder cancer, breast cancer, gastric cancer, colon cancer, head and neck cancer, esophagus cancer, synovium cancer, brain cancer, or bronchus cancer.

34. The method of clam 33, wherein the leukemia is chronic myelogenous leukemia (CML) or chronic lymphocytic leukemia (CLL).

35. The method of claim 25, wherein the subject is human.

36. The method of claim 25, wherein said composition is administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, intrathecally, locally, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage.

37. The method of claim 25, wherein the composition is administered in combination with at least an additional agent selected from the group consisting of a radiotherapeutic agent, a hormonal therapy agent, an immunotherapeutic agent, a chemotherapeutic agent, a cryotherapeutic agent and a gene therapy agent.

38. The method of claim 37, wherein the additional agent is a chemotherapeutic agent.

39. The method of claim 38, wherein the chemotherapeutic agent is cetuximab (erbitux), herceptin (trastuzumab), fludarabine, cyclophosphamide, rituximab, imatinib, Dasatinib (BMS0354825), cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,

85343306.1 daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin, methotrexate, or an analog or derivative thereof.

40. The method of claim 39, wherein the chemotherapeutic agent is fludarabine.

41. The method of claim 39, wherein the chemotherapeutic agent is imatinib.

42. The method of claim 32, wherein the cancer is resistant to chemotherapy.

43. The method of claim 25, wherein the composition is administered at a dose of about 1 to about 20 mg/kg body weight.

44. The method of claim 43, wherein the composition is administered at a dose of about 3 to about 6 mg/kg body weight.

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