WO2007134118A2

WO2007134118A2 - Protein based composition and methods of using same

Info

Publication number: WO2007134118A2
Application number: PCT/US2007/068585
Authority: WO
Inventors: Chulso Moon
Original assignee: Chulso Moon
Priority date: 2006-05-09
Filing date: 2007-05-09
Publication date: 2007-11-22
Also published as: WO2007134118A3

Abstract

In various embodiments, the present invention relates to pharmaceutical compositions comprising one or more cross-linked proteins and one or more active ingredients, to methods of making such compositions, and to methods of using such compositions in treatment of various diseases and disorders.

Description

TITLE PROTEIN BASED COMPOSITION AND METHODS OF USING SAME

[0001] This application claims priority to U.S. Provisional Patent Application No. 60/798,717 filed May 9, 2006, the entirety of which is hereby incorporated by reference herein.

FIELD OF THE INVENTION

[0002] In various embodiments, the present invention relates to pharmaceutical compositions comprising one or more cross-linked proteins and one or more active ingredients, to methods of making such compositions, and to methods of using such compositions in treatment of various diseases and disorders.

BACKGROUND OF THE INVENTION

[0003] Fifty percent of newly diagnosed cancer patients have advanced disease beyond hope of surgical or radiation cure. Patients with advanced disease and those with recurrent disease are candidates for systemic therapy, typically in the form of chemotherapy.

[0004] The annual cost of cancer treatment in the United States is estimated to be $56 billion. The majority of the cost relates to chemotherapy, particularly in stage 4 disease, and the indirect costs associated with ameliorating side effects of chemotherapy. Unfortunately, despite this cost, response rates to various forms of chemotherapy are typically not higher than about 25% or 30%.

[0005] Clearly, much work needs to be accomplished in order to improve current treatment for life threatening diseases such as cancer. If these and other limitations associated with chemotherapy treatment could be improved, a significant advance in the art would result. SUMMARY OF THE INVENTION

[0006] In various embodiments, the invention provides pharmaceutical compositions comprising one or more cross-linked proteins and one or more active ingredients. In one embodiment, the protein comprises recombinant gelatin and the active ingredient comprises an antineoplastic agent.

[0007] In another embodiment, the present invention provides methods of preparing such compositions. In one such embodiment, the protein is cross-linked and combined with the active ingredient. The step of combining the protein with the active ingredient can be performed prior to, during or after the cross-linking step. In another embodiment, the compositions are prepared by enzymatically cross-linking the protein without employing a chemical cross-linker. In still other embodiments, the cross-linking is performed by electrostatic means, by microwaves or radiation (e.g. gamma-ray and electron beam radiation) or in any other suitable manner.

[0008] In additional embodiments, the present invention provides compositions prepared by any of the methods disclosed herein.

[0009] In still further embodiments, the present invention provides methods of treating subjects in need of therapy, for example antineoplastic therapy, with any of the compositions provided by or prepared according to various embodiments of the present invention. In one embodiment, the subject has been diagnosed with cancer, for example cancer of the head or neck, prior to initiation of treatment.

[0010] These and other embodiments of the present invention are described in further detail herein below.

DETAILED DESCRIPTION OF THE INVENTION

[0011] While the present invention is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present disclosure is to be considered as an exemplification of the invention, and is not intended to limit the invention to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the invention in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

[0012] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word "about." In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. As used herein, the terms "about" and "approximately" when referring to a numerical value shall have their plain and ordinary meanings to one skilled in the pertinent art at issue. Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values recited as well as any ranges that can be formed by such values. This includes ranges that can be formed that do or do not include a finite upper and/or lower boundary. This also includes ratios that are derivable by dividing a given disclosed numeral into another disclosed numeral. Accordingly, the skilled person will appreciate that many such ratios, ranges, and ranges of ratios can be unambiguously derived from the data and numbers presented herein and all represent various embodiments of the present invention.

Protein

[0013] In one embodiment, compositions of the invention comprise a protein or mixture of proteins. Any suitable protein(s) can be used. In one embodiment, the protein is a temperature-sensitive, gel-forming protein. A "temperature-sensitive, gel- forming protein" herein is a protein that gels in response to a change in temperature. Non-limiting examples of suitable proteins include gelatin, collagen, casein, whey protein, synthetic polymers containing transglutaminase substrate sites and mixtures thereof. In one embodiment, the protein is derived from bovine or porcine material, for example skin, bone or tissue. In another embodiment, the protein is prepared using recombinant technology. In a related embodiment, the protein contains substantially no human or animal component (e.g. skin, bone or tissue, etc). In still another embodiment, the protein comprises recombinant human gelatin (rhGelatin). Recombinant human gelatin may be prepared according to any suitable process, for example those processes described in U.S. Patent Nos. 6,413,742, 6,428,978 and 6,992,172, each of which is hereby incorporated herein by reference in its entirety. Where the composition comprises rhGelatin, the rhGelatin molecules can be synthesized from human sequences of collagen, for example Type I (e.g. Type Iαl) procollagen chains or fragments thereof.

[0014] In one embodiment, where the protein comprises recombinant gelatin, the recombinant gelatin has a molecular weight range or average of about 1 to about 300 kDa, about 10 to about 250 kDa, or about 20 to about 200 kDa. In another embodiment, the recombinant gelatin has a bloom strength of about 25 to about 500, for example about 50, 100, 150, 200, 250, or 300. The Bloom gel strength is a measure of the ability of a material to form a gel and is measured on an apparatus known as the "Bloom-gel-o-meter" in Bloom grams. The Bloom gel strength is the weight in grams required to depress the gel a distance of 4 mm with a piston having a cross-sectional area of 1 cm², the gel having first been cooled for a defined time under defined conditions. Thus the higher the Bloom value of a material the greater the ability of that material to form a gel.

[0015] In another embodiment, the recombinant gelatin has a percentage hydroxylation of about 1% to about 100%, for example about 1% to about 20%, about 20% to about 80%, or about 80% to about 100%. In other embodiments, the recombinant gelatin is non-hydroxylated, is fully hydroxylated, or is partially hydroxylated. In still other embodiments, the recombinant gelatin is fully hydrolyzed, partially hydrolyzed, or non-hydrolyzed.

[0016] In one embodiment, the protein has a degree of cross-linking of about 5% to about 100%, for example at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. Degree of cross-linking can be determined according to any suitable method. In one embodiment, degree of cross-linking is determined by swelling behavior as a function of pH, temperature, and time.

[0017] In another embodiment, the present invention provides a composition comprising recombinant gelatin, wherein the recombinant gelatin comprises a homogeneous mixture of recombinant gelatin polypeptides. In another aspect, the recombinant gelatin comprises a heterogeneous mixture of recombinant gelatin polypeptides.

[0018] In another embodiment, the present invention comprises recombinant gelatin wherein the recombinant gelatin is derived from collagen. In various embodiments, the collagen is selected from the group consisting of type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX collagen and mixtures thereof. In another embodiment, the recombinant gelatin has endotoxin levels of below about 1.000 EU/mg, below about 0.500 EU/mg, below about 0.050 EU/mg, or below about 0.005 EU/mg.

[0019] In another embodiment, recombinant gelatin suitable for use in compositions of the invention are prepared by method comprising providing recombinant collagen or procollagen or fragments or variants thereof; and processing the recombinant collagen or procollagen or fragments or variants thereof to produce recombinant gelatin. In one embodiment, the recombinant collagen processed to recombinant gelatin is recombinant human collagen. In a further embodiment, the recombinant collagen is produced by co-expressing at least one polynucleotide encoding a collagen or procollagen and at least one polynucleotide encoding a collagen post-translational enzyme or subunit thereof. In one embodiment, the post- translational enzyme is prolyl hydroxylase.

[0020] A composition of the invention can comprises any desired amount of protein, for example at least about 5%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 99%, by weight. In other embodiments, the protein itself constitutes about 30% to about 99%, about 40% to about 95%, or about 50% to about 85% of the total weight of the composition.

Active Ingredient

[0021] In one embodiment, compositions of the invention comprise an active ingredient. In another embodiment, the active ingredient is capable of being transdermally or transmucosally absorbed by a mammal, for example a human, to achieve systemic blood levels of the active ingredient or a metabolite thereof.

[0022] In another embodiment, a composition of the invention comprises the active ingredient(s) in an amount of about 0.001 mg to about 1000 mg, 0.01 mg to about 800 mg, about 0.1 mg to about 500 mg, or about 1 mg to about 250 mg.

[0023] In another embodiment, the active ingredient comprises an antineoplastic agent. The phrase "antineoplastic agent" includes agents that exert antineoplastic effects, i.e., that prevent the development, maturation, or spread of neoplastic cells, directly on the tumor cell, e.g., by cytostatic or cytocidal effects or indirectly through mechanisms such as biological response modification.

[0024] In one embodiment, suitable antineoplastic agents for use in accordance with the present invention include ACE inhibitors, alkylating agents, angiogenesis inhibitors, anthracyclines/DNA intercalators, anti-cancer antibiotics or antibiotic-type agents, antimetabolites, antimetastatic compounds, asparaginases, bisphosphonates, cGMP phosphodiesterase inhibitors, camptothecins, DHA derivatives, DNA topoisomerase, endostatin, epipodophyllotoxins, hormonal anticancer agents, hydrophilic bile acids (URSO), immunomodulators or immunological agents, integrin antagonists, interferon antagonists or agents, MMP inhibitors, miscellaneous antineoplastic agents, monoclonal antibodies, nitrosoureas, ornithine decarboxylase inhibitors, pBATTS, platinum analogs, radio/chemo sensitizers/protectors, retinoids, selective inhibitors of proliferation and migration of endothelial cells, stromelysin inhibitors, taxanes, vaccines, vinca alkaloids and mixtures thereof. [0025] In another embodiment, suitable antineoplastic agents are selected from the group consisting of antimetabolite agents, alkylating agents, antibiotic-type agents, hormonal anticancer agents, immunological agents, interferon-type agents and combinations thereof.

[0026] The phrase "taxane" includes a family of diterpene alkaloids all of which contain a particular eight (8) member "taxane" ring structure. Taxanes such as paclitaxel prevent the normal post division breakdown of microtubules which form to pull and separate the newly duplicated chromosome pairs to opposite poles of the cell prior to cell division. In cancer cells which are rapidly dividing, taxane therapy causes the microtubules to accumulate which ultimately prevents further division of the cancer cell. Taxane therapy also affects other cell processes dependant on microtubules such as cell motility, cell shape and intracellular transport.

[0027] Antimetabolites are typically reversible or irreversible enzyme inhibitors, or compounds that otherwise interfere with the replication, translation or transcription of nucleic acids. Suitable antimetabolite antineoplastic agents that may be used in the present invention include, but are not limited to acanthifolic acid, aminothiadiazole, anastrozole, bicalutamide, brequinar sodium, capecitabine, carmofur, cladribine, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, cytarabine ocfosfate, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, doxifluridine, fazarabine, floxuridine, fludarabine and fludarabine phosphate, N-(240 -furanidyl)-5- fluorouracil, fluorouracil (5-FU), 5-FU-fibrinogen, gemcitabnine, isopropyl 6- mercaptopurine, methobenzaprim, methotrexate, nafarelin, norspermidine, nolvadex, pemetrexed, pyrrolizine, pentostatin, piritrexim, plicamycin, stearate, thioguanine or 6-thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosine kinase inhibitors, tyrosine protein kinase inhibitors, toremifene, and uricytin.

[0028] Alkylating agents are believed to act by alkylating and cross-linking guanine and possibly other bases in DNA, arresting cell division. Typical alkylating agents include nitrogen mustards, ethyleneimine compounds, alkyl sulfates, platinum analogues (cisplatin, carboplatin, etc.), and various nitrosoureas. Suitable alkylating- type antineoplastic agents that may be used in the present invention include, but are not limited to, aldo-phosphamide analogues, altretamine, anaxirone, bestrabucil, budotitane, busulfan, carboplatin, carmustine (BiCNU), chlorambucil, cisplatin, cyclophosphamide, cyplatate, dacarbazine, diphenylspiromustine, diplatinum cytostatic, Erba distamycin derivatives, elmustine, estramustine phosphate sodium, etoposide phosphate, fotemustine, hepsul-fam, ifosfamide, iproplatin, lomustine (CCNU), mafosfamide, mechlorethamine, melphalan, mitolactol, mycophenolate, oxaliplatin, procarbazine prednimustine, ranimustine, semustine, thiotepa, semustine (methyl-CCNU), spiromus-tine, tauromustine, temozolomide, teroxirone, tetraplatin, triethylenemelamine, and trimelamol.

[0029] Suitable antibiotic-type antineoplastic agents include, but are not limited to, aclarubicin, actinomycin D, actinoplanone, , aeroplysinin derivative, , anthracycline, azino-mycin-A, bisucaberin, bleomycin sulfate, bryostatin-1, , calichemycin, chromoximycin, dactinomycin, daunorubicin, ditrisarubicin B, doxorubicin, doxorubicin-fibrinogen, elsamicin-A, epirubicin, erbstatin, esorubicin, esperamicin- Al, esperamicin-Alb, Erbamont FCE-21954, fostriecin, glidobactin, gregatin-A, grincamycin, herbimycin, idarubicin, illudins, kazusamycin, kesarirhodins, menogaril, mitomycin, mitoxantrone, S, neoenactin, oxalysine, oxaunomycin, peplomycin, pilatin, pirarubicin, porothramycin, pyrindamycin A, rapamycin, rhizoxin, rodorubicin, sibanomicin, siwenmycin, sorangicin-A, sparsomycin, steffimycin B, talisomycin, terpentecin, thrazine, tricrozarin A, and zorubicin.

[0030] Several synthetic nucleosides have been identified that exhibit anticancer activity. A well known nucleoside derivative with strong anticancer activity is 5- fluorouracil (5-FU). 5-Fluorouracil has been used clinically in the treatment of malignant tumors, including, for example, carcinomas, sarcomas, skin cancer, cancer of the digestive organs, and breast cancer. Derivatives of 5-FU with anti-cancer activity have been also been described in U.S. Pat. No. 4,336,381, hereby incorporated by reference herein. [0031] Suitable hormonal agents that may be used as antineoplastic agents in accordance with in the present invention include, but are not limited to abarelix; abiraterone acetate; aminoglutethimide; anastrozole; antide; avorelin; aseranox; bicalutamide; buserelin; cetrolix; clastroban; clodronate disodium; cosudex; cytadren; crinone; deslorelin; droloxifene; dutasteride; elimina; epitiostanol; epristeride; exemestane; fadrozole;; flutamide; formestane; ganirelix; goserelin; idoxifene; isocordoin; ketanserin; lanreotide; letrozol; leuprolide; leuprorelin; liarozole; lisuride hydrogen maleate; loxiglumide; mepitiostane; leuprorelin; orimeten; orimetene; orimetine; ormeloxifene; osaterone; quinagolide; ramorelix; somavert; somatostatin; tamoxifen; methiodide; teverelix; toremifene; triptorelin; vapreotide; and vorozole.

[0032] In another embodiment, the antineoplastic agent is selected from anastrozole, capecitabine, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, etoposide, fluorouracil (5-FU), fiuoxymestrine, gemcitabine, goserelin, irinotecan, ketoconazole, letrozol, leucovorin, levamisole, megestrol, mitoxantrone, paclitaxel, retinoic acid, tamoxifen, thiotepa, topotecan, toremifene, vinorelbine, vinblastine, vincristine, selenium (selenomethionine), ursodeoxycholic acid, sulindac sulfone and eflornithine (DFMO).

[0033] Compositions of the invention may comprise one or more of any of the foregoing antineoplastic agents. In one embodiment, compositions of the invention comprise one or more of 5-FU, cisplatin and/or paclitaxel. In another embodiment, compositions of the invention comprise one or more of methotrexate, bleomycin, hydroxyurea, doxorubicin, vincristine and/or vinorelbine.

[0034] In one embodiment, a composition of the invention comprises a therapeutically effective amount of the active ingredient. The related terms "therapeutically effective amount," "prophylactically effective amount," "effective amount" or "amount effective to treat" as used herein refer to an amount of active ingredient or agent that is sufficient to elicit the required or desired therapeutic and/or prophylactic response, as the particular treatment context may require. [0035] In one embodiment, a therapeutically effective amount of the active ingredient is an amount sufficient to treat the disease or disorder in question or to provide a therapeutic effect. The terms "treat" or "treatment" in relation a given disease or disorder, includes, but is not limited to, inhibiting the disease or disorder, for example, arresting the development of the disease or disorder; relieving the disease or disorder, for example, causing regression of the disease or disorder; or relieving a condition caused by or resulting from the disease or disorder, for example, relieving, preventing or treating symptoms of the disease or disorder.

[0036] In another embodiment, a therapeutically effective amount of the active ingredient is an amount sufficient to prevent the disease or disorder in question. The terms "prevent" or "prevention" in relation a given disease or disorder means preventing the onset of disease development if none had occurred, preventing the disease or disorder from occurring in a subject that may be predisposed to the disorder or disease but has not yet been diagnosed as having the disorder or disease, and/or preventing further disease/disorder development if already present.

[0037] A "therapeutic effect" in reference to the treatment of a cancer, refers to one or more of the following: 1) reduction in the number of cancer cells; 2) reduction in tumor size; 3) inhibition (i.e., slowing to some extent, or stopping) of cancer cell infiltration into peripheral organs; 4) inhibition (i.e., slowing to some extent, or stopping) of tumor metastasis; 5) inhibition, to some extent, of tumor growth; 6) relieving or reducing to some extent one or more of the symptoms associated with the disorder; and/or 7) relieving or reducing the side effects associated with the administration of antineoplastic agents.

Dosage Forms, Dosage Units and Excipients

[0038] In various embodiments, a composition of the invention is in the form of a dose unit or dosage unit. The synonymous terms "dose unit" and/or "dosage unit" herein refer to a portion of a pharmaceutical composition that contains an amount of a active ingredient suitable for a single administration to provide a therapeutic or prophylactic effect. Such dosage units may be administered one to a small plurality of times per day, or as many times as needed to elicit a therapeutic response. The term "small plurality" herein means more than one but less than about 10. For example, a small plurality in the present context could illustratively represent about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 dosage units or administrations.

[0039] Compositions of the invention can be prepared in any suitable dosage form. In one embodiment, the composition is topically administrable. The term "topically administrable" herein refers to a pharmaceutical composition suitable for administration to the skin of a subject, for example a human subject, to result in topical (e.g. local) or transdermal (systemic) effect. Without limitation, such dosage forms include pastes, liquids, gels, ointments, lotions creams, topical solutions, etc. In one embodiment, topically administrable compositions of the invention comprise more than one active ingredient and provides timed release of the active ingredients. The term "timed release" herein means that the dosage form delivers the active ingredients to the blood of the subject at a predetermined time after administration.

[0040] In another embodiment, a topically administrable composition of the invention comprises an active ingredient substantially dispersed within a protein matrix, for example a protein film. The dispersion can be homogeneous or heterogeneous. In one embodiment, upon contacting the composition to the skin of a subject, at least a portion of the active ingredient migrates from the protein matrix to the skin of the subject to provide topical or transdermal effect.

[0041] In another embodiment, a topically administrable composition of the invention comprises two or more active ingredients dispersed within a protein matrix. Illustratively, degree of cross-linking of the protein and depth of penetration of the active ingredient(s) within the matrix can be predetermined (e.g. by specifying incubation time, type of cross-linker, and other process parameters, etc) to provide desired time release profiles of each of the active ingredients. [0042] In another embodiment, compositions of the invention comprise protein microcapsules which encapsulate at least a portion of the active ingredient(s). The microcapsules can be of a predetermined size dispersion so as to control timing of release of the active ingredient(s).

[0043] Topically administrable compositions of the invention can also be combined with a drug delivery system that helps deliver uniform quantities of drug to the skin over a period of time. Such systems can include adhesive or non-adhesive patches, bandages, pads, etc. The system may be monolithic (incorporating an active ingredient matrix layer between backing and frontal layers) or membrane-controlled (where the composition is in a reservoir sandwiched together with one or more of a rate controlling membrane, backing, adhesive and/or protecting layers). In one embodiment, the membrane comprises a cross-linked protein as described herein. In another embodiment, a dosage form of the invention contains substantially no, or no amount of microcapsules or microspheres.

[0044] In another embodiment, compositions of the invention are suitable for parenteral administration (injection into any site of the body), including but not limited to intra articular, intraspinal, intra-arterial, intracardiac, intravenous, intradermal, intramuscular, subcutaneous delivery. Such compositions can further comprise one or more vehicles for injection such as water for injection, USP (WFI), purified water, USP, sterile water for injection , USP, bacteriostatic water for injection, USP, sodium chloride injection, USP, bacteriostatic sodium chloride injection, USP, Ringer's injection, USP, lactated Ringer's injection, USP or non-aqueous vehicles such as vegetable oils, glycerin, polyethylene glycols, propylene glycol, alcohol, ethyl oleate, isopropyl myristate and dimethyletamide. Such compositions can be sterilized in any suitable manner, for example by steam sterilization, sterilization by filtration, autoclave, gas sterilization, sterilization by ionizing radiation, etc.

[0045] In another embodiment, compositions of the invention are suitable for delivery by inhalation, for example as an aerosol, inhalation or spray. In one embodiment, such a composition further comprises a propellant. In still another embodiment, such a composition comprises an active ingredient in solution or having a mean particle size (by volume or weight) less than about 100, less than about 75, less than about 50, or less than about 40 μm.

[0046] In another embodiment, compositions of the invention are suitable for transmucosal delivery. The term "transmucosal delivery" herein refers to delivery of an active agent across a mucous membrane, for example in the mouth (sublingual), nasal passage or eye.

[0047] Compositions of the invention optionally comprise one or more pharmaceutically acceptable excipients. The term "excipient" herein means any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition or absorption of the active ingredient, and that does not produce unacceptable toxicity or interaction with other components in the composition. Typically, a given excipient, if desired, will be present in a composition of the invention in an amount of about 0.001% to about 70%, about 0.1% to about 50%, or about 1% to about 25%, by weight of the composition. The term "pharmaceutically acceptable" herein means that the substance in question does not produce unacceptable toxicity or interaction with other components of the composition.

[0048] In one embodiment, a composition of the invention comprises a penetration enhancer. A penetration enhancer is a composition that enhances or optimizes percutaneous absorption of the active ingredient by any mechanism including reduction of resistance of the stratum corneum, alteration of hydration of the stratum corneum, imparting a change in the lipid or lipoprotein structure in intracellular channels, through solvent action, through carrier mechanisms, etc. Non-limiting examples of suitable penetration enhances include surfactants, azone, dimethylsulfoxide (DMSO), dimethylacetamide, dimethylformamide, alcohol, acetone, propylene glycol, polyethylene glycol, etc. Suitable wetting agents that can be used as penetration enhancers include quaternary ammonium compounds, for example benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride, dioctyl sodium sulfosuccinate, polyoxyethylene alkylphenyl ethers, for example nonoxynol 9, nonoxynol 10, and octoxynol 9, poloxamers (polyoxyethylene and polyoxypropylene block copolymers), polyoxyethylene fatty acid glycerides and oils, for example polyoxyethylene (8) caprylic/capric mono- and diglycerides (e.g., Labrasol™ of Gattefosse), polyoxyethylene (35) castor oil and polyoxyethylene (40) hydrogenated castor oil; polyoxyethylene alkyl ethers, for example polyoxyethylene (20) cetostearyl ether, polyoxyethylene fatty acid esters, for example polyoxyethylene (40) stearate, polyoxyethylene sorbitan esters, for example polysorbate 20 and polysorbate 80 (e.g., Tween™ 80 of ICI), propylene glycol fatty acid esters, for example propylene glycol laurate (e.g., Lauroglycol™ of Gattefosse), sodium lauryl sulfate, fatty acids and salts thereof, for example oleic acid, sodium oleate and triethanolamine oleate, glyceryl fatty acid esters, for example glyceryl monostearate, sorbitan esters, for example sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate and sorbitan monostearate, tyloxapol, and mixtures thereof.

[0049] Compositions of the invention optionally comprise one or more pharmaceutically acceptable diluents as excipients. Suitable diluents illustratively include, either individually or in combination, lactose, including anhydrous lactose and lactose monohydrate; starches, including directly compressible starch and hydrolyzed starches (e.g., Celutab™ and Emdex™); mannitol; sorbitol; xylitol; dextrose (e.g. Cerelose™ 2000) and dextrose monohydrate; dibasic calcium phosphate dihydrate; sucrose-based diluents; confectioner's sugar; monobasic calcium sulfate monohydrate; calcium sulfate dihydrate; granular calcium lactate trihydrate; dextrates; inositol; hydrolyzed cereal solids; amylose; celluloses including microcrystalline cellulose, food grade sources of α- and amorphous cellulose (e.g., Rexcel™) and powdered cellulose; calcium carbonate; glycine; bentonite; polyvinylpyrrolidone; and the like. Such diluents, if present, typically constitute in total about 5% to about 99%, about 10% to about 85%, or about 20% to about 80%, of the total weight of the composition. [0050] The foregoing excipients can have multiple roles as is known in the art. The classification of excipients above is not to be construed as limiting in any manner. Excipients categorized in any way may also operate under various different categories of excipients as will be readily appreciated by one of ordinary skill in the art.

[0051] In one embodiment, a composition of the invention, upon storage in a closed container maintained at room temperature, refrigerated (e.g. about 5 °C to about 10 °C) temperature, or frozen for a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, still exhibit at least about 90%, at least about 92.5%, at least about 95%, or at least about 97.5% of the active ingredient(s) originally present therein when the composition was prepared or formulated.

Methods of Manufacture

[0052] Compositions of the invention can be prepared in any suitable manner. In one embodiment, the invention provides a method of preparing a composition comprising the steps of combining, in any order, an enzymatic cross-linker, a protein and an active ingredient, and incubating the resulting composition under time and temperature conditions sufficient to form a cross-linked gel composition.

[0053] In another embodiment, a composition of the invention is prepared by cross-linking a protein according to any suitable method (for example chemically or enzymatically, electrostically, with radiation, etc.) t and combining the protein with the active ingredient. The step of combining the protein with the active ingredient can be performed prior to, during or after the cross-linking step. In one embodiment, such compositions are prepared by enzymatically cross-linking the protein substantially in the absence of any chemical cross-linker, for example glutaraldehyde or carbodiimide. In another embodiment, the enzyme used in the cross-linking step comprises a transglutaminase or mushroom tyrosinase.

[0054] In another embodiment, the invention provides a method of preparing a pharmaceutical composition, the method comprising (a) providing a solution of a protein, for example and aqueous solution of rhGelatin; (b) adding a cross-linker and an active ingredient to the solution to form a mixture or second solution; and (c) incubating the resulting mixture or second solution under time and temperature conditions under which an otherwise similar mixture or second solution lacking the cross-linker would gel.

[0055] The term "cross-linking" herein means formation of covalent bonds between polymer chains to make a single network with increased strength and resistance to solvents. The term "enzymatic cross-linker" herein refers to any enzyme capable of cross-linking a temperature-sensitive, gel-forming protein. Chemical cross- linkers, on the other hand, tend to produce amorphous, non-uniform gels and can be toxic, immunogenic or inflammatory. In one embodiment, the enzymatic cross-linker comprises a transglutaminase, for example factor XIII. Factor XIII includes complete factor XIII zymogen tetramer and factor XIIIa, as well as activation intermediates and subunits thereof. In another embodiment, the enzymatic cross-linker comprises mushroom tyrosinase.

[0056] In one embodiment, transglutaminases useful within the present invention include those produced by microorganisms and higher organism, for example factor XIII, keratinocyte transglutaminase, tissue transglutaminase, prostate transglutaminase, and epidermal transglutaminase. The transglutaminase can be recombinant (i.e., production using genetically engineered host cells) or naturally derived.

[0057] In one embodiment, the step of providing a solution of a protein is performed by dissolving the protein (e.g. recombinant gelatin) in an aqueous solvent buffered at pH of about 5 to about 9 and of low to moderate ionic strength (equivalent to about 1 to 1000 mM NaCl or about 50 to 300 mM NaCl). While salt is not required in the buffer, certain proteins are more soluble in the presence of salt, and near- physiological concentrations of salt can be beneficial. In one embodiment, pH of the solution is about 5.5 to about 8.5 or about 6 to about 8. [0058] An illustrative aqueous solvent is phosphate buffered saline (PBS; 10 mM sodium phosphate pH 7.4, 120 mM NaCl, 2.7 mM KCl). Other suitable buffers include borate, phosphate, HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2- ethanesulfonic acid]), etc. The concentration of protein in the solution will be determined according to intended dosage unit properties, but will generally be about 50% or less by weight, for example about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 10% or about 1% to about 5% by weight. Optionally, active ingredient can be added to the solution at this point as well.

[0059] In one embodiment, the protein is allowed to swell, typically for about 15 minutes to about three hours, and is then melted by raising the temperature to a temperature above the gel point of the solution, for example about 32 °C to about 100 °C. The solution can then be gelled, for example, by allowing it to stand overnight at room temperature, and stored under refrigerated conditions until needed. Optionally, an active ingredient can be added prior, during or after gelling is complete. Next, an enzymatic cross-linker and optionally an active ingredient can be added to the protein solution to form a mixture or second solution, for example while the first solution is in a sol state. If a transglutaminase is used, the solution may also contain calcium (e.g. about 1 to about 18 mM or about 3 to about 10 mM, for example in the form of CaCl₂) for transglutaminase activity. Mammalian transglutaminases, including factor XIII, tissue transglutaminase, keratinocyte transglutaminase, epidermal transglutaminase, and prostate transglutaminase typically require Ca⁺⁺ for activity, while others, including certain bacterial transglutaminases do not require calcium ions.

[0060] In one embodiment, the ratio of protein:transglutaminase in the solution will generally be about 1500:1 to 1 :100, about 1000:1 to about 50:1, about 500:1 to about 25:1, about 100:1 to about 10:1, or about 10:1 to about 5:1, by weight. In another embodiment, the concentration of transglutaminase within the solution will typically be about 0.1 mg/ml to about 5.0 mg/ml. The solution can then be incubated under conditions of time and temperature in which an otherwise similar solution lacking cross-linking enzyme (e.g. transglutaminase) will gel, for example about 4 °C up to the gel point, about 4 °C to about 33 °C, or about 10 °C up to about 32 °C and for about 15 minutes or more, for example about 30 minutes to about 10 hours. Those skilled in the art will recognize that time to gel formation is a factor of, inter alia, temperature and protein concentration, and the actual time and temperature of incubation will be determined with consideration of all relevant parameters.

[0061] In various embodiments, the incubation time conditions comprise about 10 minutes to about 10 hours, about 10 minutes to about 8 hours, about 10 minutes to about 5 hours or about 10 minutes to about 2 hours, and the incubation temperature conditions comprise about 1 °C to about 90 ⁰C, about 5 °C to about 70 ⁰C, about 10 ⁰C to about 50 °C, or about 15 ⁰C to about 30 ⁰C.

[0062] As indicated above, in an alternate embodiment of the invention, cross- linking enzyme can be added to a composition of a temperature-sensitive gel-forming protein during or subsequent to gelation, in the latter case allowing the enzyme to diffuse into the gel. For example, an aqueous solution of enzyme {e.g. transglutaminase) can be applied to the surface of a previously-formed recombinant gelatin composition (optionally containing an active ingredient) and allowed to diffuse into the gel. The gel and enzyme can then be incubated under conditions conducive to gel formation for the protein used within the gel. Diffusion time, and amount and concentration of enzyme can be adjusted to provide the desired depth of penetration and degree of cross-linking.

[0063] Composition prepared according to any of the above described methods represent further embodiments of the present invention.

Methods of Treatment and Prevention

[0064] Compositions of the invention are useful in treatment and prevention of various diseases and disorders. In one embodiment, compositions of the invention are useful in the treatment of cancer or neoplasia. In another embodiment, compositions of the present invention are useful in the treatment or prevention of neoplasia or neoplasia-related disorders including acral lentiginous melanoma, actinic keratoses, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenoid cycstic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectum cancer, astrocytic tumors, bartholin gland carcinoma, basal cell carcinoma, benign cysts, biliary cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, bronchial cancer, bronchial gland carcinomas, carcinoids, carcinoma, carcinosarcoma, cholangiocarcinoma, chondosarcoma, choriod plexus papilloma/carcinomal chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue cancer, cystadenoma, cysts of the female reproductive system, digestive system cancer, digestive tract polyps, duodenum cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endometriosos, endothelial cell cancer, ependymal cancer, epithelial cell cancer, esophagus cancer, Ewing's sarcoma, eye and orbit cancer, female genital cancer, fibroid tumors, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, germ cell tumors, glioblastoma, glucagonoma, heart cancer, hemangiblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileum cancer, insulinoma, intaepithelial neoplasia, interepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunum cancer, joint cancer, Kaposi's sarcoma, kidney and renal pelvic cancer, large cell carcinoma, large intestine cancer, larynx cancer, leiomyosarcoma, lentigo maligna melanomas, leukemia, liver cancer, lung cancer, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, melanoma, meningeal cancer, mesothelial cancer, metastatic carcinoma, mouth cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal tract cancer, nervous system cancer, neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial cancer, oral cavity cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillary serous adenocarcinoma, penile cancer, pharynx cancer, pituitary tumors, plasmacytoma, prostate cancer, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spine cancer, squamous carcinoma, squamous cell carcinoma, stomach cancer, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T cell leukemia, testis cancer, thyroid cancer, tongue cancer, undifferentiated carcinoma, ureter cancer, urethra cancer, urinary bladder cancer, urinary system cancer, uterine cervix cancer, uterine corpus cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, vipoma, vulva cancer, well differentiated carcinoma, and Wilm's tumor.

[0065] In one embodiment, compositions of the invention are administered to a subject in an amount sufficient to provide active ingredient in an amount of about 0.01 mg/kg body weight to about 500 mg/kg body weight per day, for example about 0.1 mg/kg to about 50 mg/kg or about 1 mg/kg to about 25 mg/kg. In another embodiment, the composition is administered to the subject to provide the subject with a daily dose of active ingredient(s) of about 5 mg to about 1000 mg/kg, about 10 mg/kg to about 800 mg/kg, or about 20 mg/kg to about 300 mg/kg, for example about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 10, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 790, about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870, about 880, about 890, about 900, about 910, about 920, about 930, about 940, about 950, about 960, about 970, about 980, about 990 or about 1000 mg/kg body weight per day over a period of 1 to about 100, 1 to about 75, 1 to about 60, 1 to about 40, 1 to about 30, 1 to about 20, 1 to about 10 or 1 to about 5 days.

[0066] In another embodiment, compositions of the invention are administered to a subject in an amount sufficient to provide active ingredient in an amount of about 0.001 mg/m² (BSA; body surface area) to about 5000 mg/kg BSA per day, 0.01 mg/m² to about 2500 mg/m² per day, or about 1 0.001 mg/m² to about 2000 mg/m² per day, for example for example about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 10, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 790, about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870, about 880, about 890, about 900, about 910, about 920, about 930, about 940, about 950, about 960, about 970, about 980, about 990, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3100, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400, about 4500, about 4600 about 4700, about 4800, about 4900 or about 5000 mg/m² per day over a period of 1 to about 100, 1 to about 75, 1 to about 60, 1 to about 40, 1 to about 30, 1 to about 20, 1 to about 10 or 1 to about 5 days. [0067] Compositions of the invention can be administered in combination with or as an adjunct to (e.g. before, during or after) additional cancer therapy including surgery, radiation, photodynamic treatment, laser therapy, angiogenesis inhibitors, bone marrow and peripheral blood stem cell transplantation, gene therapy, hyperthermia, biological therapies (e.g. cetuximab) and antiemetics.

[0068] In one embodiment, topically administrable compositions of the invention can be administered in combination with one or more topical analgesics. The phrase "administered in combination with" herein means that the compositions in question can be administered substantially simultaneously, or as part of a combined regimen in which the two or more compositions are administered in a desired sequence within a period of about 5 hours, 10 hours, 15 hours, 1 day, 2 to 15 days or 1 month. Illustratively, topical analgesics include rubefacients (e.g. traditional formulations based on salicylate and nicotinate esters, capsaicin and capsicum extracts and derivatives), NSAIDs (e.g. diclofenac, felbinac, ibuprofen, ketoprofen, piroxicam, naproxen, flurbiprofen), local anesthetics and a miscellaneous group: including benzydamine, mucopolysaccharide polysulphate, salicylamide and cooling sprays.

[0069] Local anesthetics include, without limitation, amino esters (e.g. benzocaine, chloroprocaine, cocaine, procaine and tetracaine), amino amides (e.g. bupivacaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, articaine and trimecaine.

EXAMPLES

[0070] Example 1. rhGelatin-5-FU: Cream for Head and Neck.

[0071] rhGelatin-5-FU is used as a topical cream for head and neck cancer. rhGelatin-5-FU is applied directly to the recurrent or refractory tumor nodule itself.

[0072] Example 2. rhGelatin-paclitaxel: Intravenous injection for lung cancer. [0073] rhGelatin-paclitaxel will be tested as a second line therapy for lung cancer and will be compared to existing agents like Taxotere® (docetaxel) or Alamta® (pemetrexid).

[0074] Example 3. Primary, metastatic lung cancer.

[0075] rhGelatin-paclitaxel will be tested as a primary agent for metastatic lung cancer and will be compared to existing agents such as paclitaxel-based chemotherapy or a nanoalbumin paclitaxel-based regimen.

[0076] Example 4. Intravenous injection for primary, metastatic breast cancer.

[0077] rhGelatin-paclitaxel will be tested by intravenous injection as a second line agent for breast cancer and will be compared to existing agents like Taxol® or nanoalbumin-based Taxol®.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising a cross-linked recombinant protein and an active ingredient, wherein the active ingredient is present in a therapeutically or prophylactically effective amount.

2. The composition of claim 1 wherein the cross-linked recombinant protein comprises a temperature-sensitive, gel-forming protein.

3. The composition of claim 2 wherein the temperature-sensitive, gel-forming protein comprises recombinant gelatin.

4. The composition of claim 3 wherein the recombinant gelatin is synthesized from human sequences of collagen or fragments thereof.

5. The composition of claim 3 wherein the recombinant gelatin has an average molecular weight of about 1 to about 300 kDa.

6. The composition of claim 3 wherein the recombinant gelatin has an average molecular weight of about 75 to about 150 kDa.

7. The composition of claim 1 wherein the active ingredient comprises an antineoplastic agent selected from an antimetabolite antineoplastic agent, an alkylating antineoplastic agent, an antibiotic-type antineoplastic agent, a hormonal antineoplastic agent, an immunological antineoplastic agent, an interferon-type agent or combinations thereof.

8. The composition of claim 7 wherein the chemotherapeutic agent is selected from 5-FU, cisplatin paclitaxel, methotrexate, bleomycin, hydroxyurea, doxorubicin, vincristine, vinorelbine or combinations thereof.

9. The composition of claim 8 wherein the composition comprises a topically administrable dosage form.

10. The composition of claim 9 wherein the dosage form is selected from a paste, a gel, an ointment, a lotion, a cream, a solution, or a patch.

11. A topically administrable pharmaceutical composition comprising a cross- linked recombinant protein and an antineoplastic agent.

12. The composition of claim 11 wherein the cross-linked recombinant protein comprises a temperature-sensitive, gel-forming protein.

13. The composition of claim 12 wherein the temperature-sensitive, gel-forming protein comprises recombinant gelatin.

14. The composition of claim 13 wherein the recombinant gelatin is synthesized from human sequences of collagen or fragments thereof.

15. The composition of claim 13 wherein the recombinant gelatin has an average molecular weight of about 1 to about 300 kDa.

16. The composition of claim 13 wherein the recombinant gelatin has an average molecular weight of about 75 to about 150 kDa.

17. The composition of claim 11 wherein the chemotherapeutic agent is selected from 5-FU, cisplatin paclitaxel, methotrexate, bleomycin, hydroxyurea, doxorubicin, vincristine, vinorelbine or combinations thereof.

18. The composition of claim 17 wherein the chemotherapeutic agent is selected from taxol and 5-flourouracil.

19. A method for preparing a pharmaceutical composition comprising a cross- linked recombinant protein and an active ingredient, the method comprising the steps of (a) providing an aqueous solution comprising a recombinant protein; (b) adding to the solution, in any order, an enzymatic cross-linker and an active ingredient to form a mixture; and (c) incubating the mixture under time and temperature conditions under which an otherwise similar mixture lacking the enzymatic cross-linker would gel to form a pharmaceutical composition.

20. The method of claim 19 wherein the recombinant protein comprises recombinant human gelatin.

21. The method of claim 19 wherein the enzymatic cross-linker comprises a tranglutaminase.

22. The method of claim 21 wherein the transglutaminase comprises Factor XIII.

23. The method of claim 19 wherein the active ingredient comprises an antineoplastic agent.

24. The method of claim 19 wherein the time and temperature conditions comprise about 10 minutes to about 8 hours and about 10 ⁰C to about 50 °C.

25. A pharmaceutical composition prepared according to the method of claim 19.

26. Use of a composition of claim 11 in manufacture of a medicament for treatment or prevention of cancer.

27. A method of treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a cross-linked recombinant protein and an antineoplastic agent present in a therapeutically or prophylactically effective amount.

28. The method of claim 27 wherein the cancer is a cancer of the head or neck.

29. The method of claim 27 wherein said administering step comprises topical, parenteral, transmucosal or transdermal administration of the composition to the subject.

30. The method of claim 29 wherein the antineoplastic agent comprises 5-FU, cisplatin paclitaxel, methotrexate, bleomycin, hydroxyurea, doxorubicin, vincristine, vinorelbine.

31. A pharmaceutical composition comprising an active ingredient dispersed within a cross-linked matrix comprising recombinant human gelatin.

32. The pharmaceutical composition of claim 31 wherein the active ingredient is selected from 5-FU, cisplatin paclitaxel, methotrexate, bleomycin, hydroxyurea, doxorubicin, vincristine, vinorelbine and mixtures thereof.