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Background of the Invention Drug delivery techniques are continually being developed in drug therapy to control, regulate, and target the release of drugs in the body. Goals include augmentation of drug availability, maintenance of constant and continuous therapeutic levels of a drug in the systemic circulation or at a specific target organ site, reduction of dosages and/or frequency of administration required to realize the desired therapeutic benefit, and consequent reduction of drug-induced side effects. Drug delivery systems currently include, for example, carriers based on proteins, polysaccharides, synthetic polymers, and liposomes.

Gas filled microbubbles have been conventionally used as contrast agents for diagnostic ultrasound. They have also been described for therapeutic applications, such

as enhancement of drug penetration (Tachibana et al., U. S. Patent No. 5,315, 998), as thrombolytics (e. g. Porter, U. S. Patent No. 5,648, 098), and for drug delivery. Reports of use of microbubbles for drug delivery have generally described the use of some external method of releasing the drug from the microbubbles at the site of delivery, by, for example, raising the temperature to induce a phase change (Unger, U. S. Patent No.

6,143, 276) or exposing the microbubbles to ultrasound (Unger, U. S. Patent No.

6,143, 276; Klaveness et al., U. S. Patent No. 6,261, 537; Lindler et al., Echocardiography 18 (4): 329, May 2001; Unger et al., Echocardiography 18 (4): 355, May 2001; Porter et al., U. S. Patent No. 6,117, 858).

As described in co-owned U. S. Patent No. 5,849, 727, the applicant showed that gas filled, protein-encapsulated microbubbles, conventionally employed as contrast agents in ultrasonic imaging, could be conjugated to therapeutic agents. As described therein, while release of the agent at a target site may comprise the use of ultrasound, the use of ultrasound is not a requirement.

Summary of the Invention In one aspect, the invention is directed to the use of a composition comprising (i) an antiproliferative therapeutic agent and (ii) a suspension of microbubbles which are encapsulated with a filmogenic protein and contain a gas selected from a perfluorocarbon and SF6 for preparation of a medicament for delivering said antiproliferative therapeutic agent to the site of a tumor in a subject, wherein said composition is administered parenterally to said subject.

Preferably, the gas is a perfluorocarbon, such perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, or perfluoropentane, and the protein is albumin, preferably human serum albumin. The composition is typically formed by incubating the agent, generally in solution or suspension, with the suspension of microbubbles. The composition is preferably administered to the subject without application of ultrasound to the composition during or following administration.

In various embodiments, the antiproliferative therapeutic agent is selected from the group consisting of paclitaxel, docetaxel, cisplatin, carboplatin, etoposide, tamoxifen, 5-fluorouracil, adriamycin, daunorubicin, doxorubicin, vincristine, and vinblastine.

Preferred agents include paclitaxel and docetaxel ; other preferred agents include

cisplatin, carboplatin, etoposide, tamoxifen, 5-fluorouracil, vincristine, and vinblastine.

In other embodiments, the therapeutic agent can be selected from the group consisting of amsacrine, mitotane, topotecan, tretinoin, hydroxyurea, procarbazine, carmustine, mechlorethamine hydrochloride, cyclophosphamide, ifosfamide, chlorambucil, melphalan, busulfan, thiotepa, estramustine, dacarbazine, omustine, streptozocin, vinorelbine, vindesine, fludarabine, fluorodeoxyuridine, cytosine arabinoside, cytarabine, azidothymidine, cysteine arabinoside, azacytidine, mercaptopurine, thioguanine, cladribine, pentostatin, arabinosyl adenine, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitoxantrone, bleomycin, plicamycin, ansamitomycin, mitomycin, aminoglutethimide, and flutamide.

In a related aspect, the invention is directed to a composition comprising (i) a suspension of microbubbles which are encapsulated with a filmogenic protein and contain a gas selected from a perfluorocarbon and SF6, and (ii) an antiproliferative therapeutic agent. Preferably, the agent is selected from the group consisting of cisplatin, carboplatin, etoposide, tamoxifen, 5-fluorouracil, amsacrine, mitotane, topotecan, tretinoin, hydroxyurea, procarbazine, carmustine, mechlorethamine hydrochloride, cyclophosphamide, ifosfamide, chlorambucil, melphalan, busulfan, thiotepa, estramustine, dacarbazine, omustine, streptozocin, vincristine, vinblastine, vinorelbine, vindesine, fludarabine, fluorodeoxyuridine, cytosine arabinoside, cytarabine, azidothymidine, cysteine arabinoside, azacytidine, mercaptopurine, thioguanine, cladribine, pentostatin, arabinosyl adenine, idarubicin, mitoxantrone, aminoglutethimide, and flutamide.

The agent may also be an antiproliferative antisense oligomer, preferably a morpholino oligomer having phosphoramidate or phosphorodiamidate linkages.

As above, the gas is preferably a perfluorocarbon, such perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, or perfluoropentane, and the protein is preferably albumin, more preferably human serum albumin.

In selected embodiments, the therapeutic agent is selected from the group consisting of cisplatin, carboplatin, etoposide, tamoxifen, 5-fluorouracil, vincristine, and vinblastine. In other selected embodiments, the therapeutic agent is selected from the group consisting of cisplatin, carboplatin, and etoposide, or it is selected from the group consisting of tamoxifen, 5-fluorouracil, vincristine, and vinblastine.

In a further related aspect, the invention is directed to a method for delivering an antiproliferative therapeutic agent to the site of a tumor in a subject, comprising: administering parenterally to a subject having said tumor a composition comprising the therapeutic agent and a suspension of microbubbles which are encapsulated with a filmogenic protein and contain a gas selected from a perfluorocarbon and SF6.

Preferably, the administration is carried out without application of ultrasound to the composition during or following administration.

The subject is preferably a mammalian subject, such as a human subject or patient.

The composition of suspended microbubble/agent conjugate is administered internally to the subject, preferably parenterally, e. g. intravenously, percutaneously, intraperitoneally, intramuscularly, or intrathecally. The microbubble carrier delivers the agent or agents to the target site, where, in a preferred embodiment, the agent is released without the use of external stimulation. However, if desired, release of the agent may be modulated by application of a stimulus such as radiation, heat, or ultrasound. Application of such a stimulus may also be used to convert a prodrug to the active form of the drug, which is then released.

As above, the gas is preferably a perfluorocarbon, such perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, or perfluoropentane, and the protein is preferably albumin, more preferably human serum albumin. The therapeutic agent is preferably a non-oligonucleotide agent.

In preferred embodiments, the therapeutic agent is selected from the group consisting of paclitaxel, docetaxel, cisplatin, carboplatin, etoposide, tamoxifen, 5-fluorouracil, adriamycin, daunorubicin, doxorubicin, vincristine, and vinblastine. In particular embodiments, it is selected from paclitaxel and docetaxel, or it is selected from the group consisting of cisplatin, carboplatin, etoposide, tamoxifen, 5-fluorouracil, vincristine, and vinblastine.

In other embodiments, the agent can be selected from the group consisting of amsacrine, mitotane, topotecan, tretinoin, hydroxyurea, procarbazine, carmustine, mechlorethamine hydrochloride, cyclophosphamide, ifosfamide, chlorambucil, melphalan, busulfan, thiotepa, estramustine, dacarbazine, omustine, streptozocin, vinorelbine, vindesine, fludarabine, fluorodeoxyuridine, cytosine arabinoside, cytarabine, azidothymidine, cysteine arabinoside, azacytidine, mercaptopurine, thioguanine,

cladribine, pentostatin, arabinosyl adenine, dactinomycin, daunorubicin, doxorubicin, amsacrine, idarubicin, mitoxantrone, bleomycin, plicamycin, ansamitomycin, mitomycin, aminoglutethimide, and flutamide; and preferably from the group consisting of amsacrine, mitotane, topotecan, tretinoin, hydroxyurea, procarbazine, carmustine, mechlorethamine hydrochloride, cyclophosphamide, ifosfamide, chlorambucil, melphalan, busulfan, thiotepa, estramustine, dacarbazine, omustine, streptozocin, vinorelbine, vindesine, fludarabine, fluorodeoxyuridine, cytosine arabinoside, cytarabine, azidothymidine, cysteine arabinoside, azacytidine, mercaptopurine, thioguanine, cladribine, pentostatin, arabinosyl adenine, idarubicin, mitoxantrone, aminoglutethimide, and flutamide.

Detailed Description of the Invention I. Carrier Compositions The present therapeutic compositions comprise a drug which is conjugated to a microparticle carrier, such as a gaseous microbubble in a fluid medium or a polymeric microparticle, with sufficient stability that the drug can be carried to and released at a target site in a subject. Such conjugation typically refers to noncovalent binding or other association of the drug with the particle, and may be brought about by incubation with a microbubble suspension, as described further below, or intimate mixing of the drug with a polymeric microparticle carrier.

In one embodiment, the pharmaceutical composition comprises a liquid suspension, preferably an aqueous suspension, of microbubbles containing a blood-insoluble gas.

The microbubbles are preferably about 0.1 to 10 u in diameter. Generally, any blood- insoluble gas which is nontoxic and gaseous at body temperature can be used. The insoluble gas should have a diffusion coefficient and blood solubility lower than nitrogen or oxygen, which diffuse in the internal atmosphere of the blood vessel. Examples of useful gases are the noble gases, e. g. helium or argon, as well as fluorocarbon gases and sulfur hexafluoride. Generally, perfluorocarbon gases, such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, and perfluoropentane, are preferred.

It is believed that the cell membrane fluidizing feature of the perfluorobutane gas enhances cell entry for drugs on the surface of bubbles that come into contact with denuded vessel surfaces, as described further below.

The gaseous microbubbles are stabilized by a fluid filmogenic coating, to prevent coalescence and to provide an interface for binding of molecules to the microbubbles.

The fluid is preferably an aqueous solution or suspension of one or more components selected from proteins, surfactants, and polysaccharides. In preferred embodiments, the components are selected from proteins, surfactant compounds, and polysaccharides.

Suitable proteins include, for example, albumin, gamma globulin, apotransferrin, hemoglobin, collagen, and urease. Human proteins, e. g. human serum albumin (HSA), are preferred. In one embodiment, as described below, a mixture of HSA and dextrose is used.

Conventional surfactants include compounds such as alkyl polyether alcohols, alkylphenol polyether alcohols, and alcohol ethoxylates, having higher alkyl (e. g. 6-20 carbon atom) groups, fatty acid alkanolamides or alkylene oxide adducts thereof, and fatty acid glycerol monoesters. Surfactants particularly intended for use in microbubble contrast agent compositions are disclosed, for example, in Nycomed Imaging patents US 6,274, 120 (fatty acids, polyhydroxyalkyl esters such as esters of pentaerythritol, ethylene glycol or glycerol, fatty alcohols and amines, and esters or amides thereof, lipophilic aldehydes and ketones ; lipophilic derivatives of sugars, etc. ), US 5,990, 263 (methoxy- terminated PEG acylated with e. g. 6-hexadecanoyloxyhexadecanoyl), and US 5,919, 434.

Other filmogenic synthetic polymers may also be used; see, for example, U. S. Patent Nos. 6,068, 857 (Weitschies) and 6,143, 276 (Unger), which describe microbubbles having a biodegradable polymer shell, where the polymer is selected from e. g. polylactic acid, an acrylate polymer, polyacrylamide, polycyanoacrylate, a polyester, polyether, polyamide, polysiloxane, polycarbonate, or polyphosphazene, and various combinations of copolymers thereof, such as a lactic acid-glycolic acid copolymer.

Such compositions have been used as contrast agents for diagnostic ultrasound, and have also been described for therapeutic applications, such as enhancement of drug penetration (Tachibana et al., U. S. Patent No. 5,315, 998), as thrombolytics (Porter, U. S.

Patent No. 5,648, 098), and for drug delivery (see below). The latter reports require some external method of releasing the drug at the site of delivery, typically by raising the temperature to induce a phase change (Unger, U. S. Patent No. 6,143, 276) or by exposing the microbubbles to ultrasound (Unger, U. S. Patent No. 6,143, 276; Klaveness et al., U. S.

Patent No. 6,261, 537; Lindler et al., cited below, Unger et al., cited below ; Porter et al.,

U. S. Patent No. 6, 117, 858).

In one embodiment, the carrier is a suspension of perfluorocarbon-containing dextrose/albumin microbubbles known as PESDA (perfluorocarbon-exposed sonicated dextrose/albumin). Human serum albumin (HSA) is easily metabolized within the body and has been widely used as a contrast agent. The composition may be prepared as described in co-owned U. S. Patents 5,849, 727 and 6,117, 858. Briefly, a dextrose/albumin solution is sonicated while being perfused with the perfluorocarbon gas. The microbubbles are preferably formed in an N2-depleted, preferably N2-free, environment, typically by introducing an N2-depleted (in comparison to room air) or N2- free gas into the interface between the sonicating horn and the solution. Microbubbles formed in this way are found to be significantly smaller and stabler than those formed in the presence of room air. (See e. g. Porter et al., U. S. Patent No. 6,245, 747, which is incorporated by reference.) To conjugate the microbubbles with the therapeutic agent, the microbubble suspension is generally incubated, with agitation if necessary, with a liquid formulation of the agent, such that the agent non-covalently binds at the gas/fluid interface of the microbubbles. Preferably, the liquid formulation of the drug (s) is first filtered through a micropore filter and/or sterilized. The incubation may be carried out at room temperature, or at moderately higher temperatures, as long as the stability of the drug or the microbubbles is not compromised. The microbubble/therapeutic agent composition is thus provided in isolated form for administration to a subject.

Drugs with limited aqueous solubility (such as paclitaxel, for example) can be solubilized or intimately dispersed in pharmaceutically acceptable vehicles, such as, for example, alcohol, DMSO, or an oil such as castor oil or CremophorTM, by methods known in the pharmaceutical arts. Other solubilizing formulations are known in the art; see, for example, U. S. Patent No. 6,267, 985 (Chen and Patel, 2001), which discloses formulations containing triglycerides and a combination of surfactants.

Other microbubble-therapeutic compositions are described in, for example, U. S.

Patent Nos. 6,143, 276 (Unger) and 6,261, 537 (Klaveness et al.), which are incorporated herein by reference. These references, as well as Lindler et al., Echocardiography 18 (4): 329, May 2001, and Unger et al., Echocardiography 18 (4): 355, May 2001, describe use of the microbubbles for therapeutic delivery of the conjugated compounds,

in which the compounds are released from the microbubbles by application of ultrasound at the desired point of release.

The applicants have shown that neither ultrasound, nor other external stimulation, such as heat, was required for microbubble-mediated delivery of therapeutically effective amounts of the drug rapamycin to angioplasty-injured coronary vessels (see e. g. PCT Pubn. No. 2003/92741). Accordingly, the compositions are preferably administered without application of external stimulation, such as ultrasound, to the composition during or after administration. However, if desired, release of the agent from the microbubbles may be modulated by application of a stimulus such as light, temperature variation, pressure, ultrasound or ionizing energy or magnetic field. Application of such a stimulus may also be used to convert a prodrug to the active form of the drug, which is then released.

In addition to gas-filled microbubbles, other microparticles, such as biocompatible polymeric particles, may be used for delivery of a conjugated drug to a target site. In this sense, "nanoparticles"refers to polymeric particles in the nanometer size range (e. g. 50 to 750 nm), while"microparticles"refers to particles in the micrometer size range (e. g. 1 to 50 u), but may also include particles in the submicromolar range, down to about 0.1 Il.

For use in the methods described herein, a size range of about 0.1 to 10 R is preferred.

Such polymeric particles have been described for use as drug carriers into which drugs or antigens may be incorporated in the form of solid solutions or solid dispersions, or onto which these materials may be absorbed or chemically bound. See e. g. Kreuter 1996; Ravi Kumar 2000; Kwon 1998. Methods for their preparation include emulsification evaporation, solvent displacement,"salting-out", and emulsification diffusion (Soppimath et al. ; Quintanar-Guerrero et al.), as well as direct polymerization and solvent evaporation processes (Cleland).

Preferably, the polymer is bioerodible in vivo. Biocompatible and bioerodible polymers that have been used in the art include poly (lactide-co-glycolide) copolymers, polyanhydrides, and poly (phosphoesters). Poly (orthoester) polymers designed for drug delivery, available from A. P. Pharma, Inc., are described in Heller et al., J. Controlled Release 78 (1-3): 133-141 (2002). In one embodiment, the polymer is a diol-diol monoglycolide-orthoester copolymer. The polymer can be produced in powdered form, e. g. by cryogrinding or spray drying, intimately mixed in powdered form with a

therapeutic compound, and fabricated into various forms, including microspheres and nanospheres.

II. Therapeutic Agents and Treatment For microbubble compositions used for delivery to a tumor site, the antiproliferative therapeutic agent to be delivered is an antineoplastic agent. Known antineoplastic agents include, for example, cisplatin, carboplatin, spiroplatin, iproplatin, paclitaxel, docetaxel, rapamycin, tacrolimus, asparaginase, etoposide, teniposide, methotrexate, tamoxifen, amsacrine, mitotane, topotecan, tretinoin, hydroxyurea, procarbazine, BCNU (carmustine) and other nitrosourea compounds, as well as compounds classified as alkylating agents (e. g. , mechlorethamine hydrochloride, cyclophosphamide, ifosfamide, chlorambucil, melphalan, busulfan, thiotepa, carmustine, estramustine, dacarbazine, omustine, streptozocin), plant alkaloids (e. g., vincristine, vinblastine, vinorelbine, vindesine), antimetabolites (e. g., folic acid analogs, methotrexate, fludarabine), pyrimidine analogs (fluorouracil, fluorodeoxyuridine, cytosine arabinoside, cytarabine, azidothymidine, cysteine arabinoside, and azacytidine), and purine analogs (mercaptopurine, thioguanine, cladribine, pentostatin, arabinosyl adenine). Also included are aminoglutethimide (an aromatase inhibitor), flutamide (an anti-androgen), gemtuzumab ozogamicin (a monoclonal antibody), and oprelvekin (a synthetic interleukin), as well as cell cycle inhibitors and EGF receptor kinase inhibitors in general. Antitumor antibiotics include adriamycin, dactinomycin, daunorubicin, doxorubicin, amsacrine, idarubicin, mitoxantrone, bleomycin, plicamycin, ansamitomycin, and mitomycin.

Antisense oligonucleotides having antiproliferative effects may also be delivered to a tumor site using the compositions described herein. Preferred oligonucleotide analogs include morpholino-based oligomers having uncharged, phosphorus-containing linkages, preferably phosphoramidate or phosphorodiamidate linkages, as described, for example, in U. S. Patent Nos. 5,185, 444 and 5,142, 047 and in Summerton and Weller, Antisense Nucleic Acid Drug Dev. 7: 63-70 (1997). Oligomers antisense to c-myc may be used, and include those described in PCT Pubn. No. WO 00/44897 and U. S. Appn. Pubn. No.

20010024783. In other embodiments, the oligomer is an antiproliferative antisense oligomer which is not targeted to c-myc. Such oligomers include those targeted to other

genes involved in cell transformation, cell survival, metastasis, and angiogenesis, such as, for example, PKC, PKA, p53, bcl-2, c-raf, ras, c-fos, MDR1, M1VII'-9, HER-2/neu, p21, bcr-abl, and MDM2.

In other selected embodiments, the agent is a non-oligonucleotide agent; that is, it is not an oligonucleotide or oligonucleotide analog.

For example, the antiproliferative agent may be selected from the group consisting of paclitaxel, docetaxel, cisplatin, carboplatin, etoposide, tamoxifen, methotrexate, 5-fluorouracil, adriamycin, daunorubicin, doxorubicin, vincristine, and vinblastine. In selected embodiments, the agent is selected from the group consisting of paclitaxel, other taxanes, such as docetaxel, and active analogs, derivatives or prodrugs of these compounds. In one embodiment, the agent is paclitaxel or docetaxel. In still further embodiments, the antiproliferative agent is selected from the group consisting of cisplatin, carboplatin, 5-fluorouracil, etoposide, tamoxifen, vincristine, and vinblastine.

In particular, chemotherapeutic agents currently in widespread use include the platinum-containing agents, such as cisplatin and carboplatin, paclitaxel (TaxolO) and related drugs, such as docetaxel (Taxotere@), etoposide, and 5-fluorouracil. Taxol (D (paclitaxel) constitutes one of the most potent drugs in cancer chemotherapy and is widely used in therapy for ovarian, breast and lung cancers. Etoposide is currently used in therapy for a variety of cancers, including testicular cancer, lung cancer, lymphoma, neuroblastoma, non-Hodgkin's lymphoma, Kaposi's Sarcoma, Wilms'Tumor, various types of leukemia, and others. Fluorouracil has been used for chemotherapy for a variety of cancers, including colon cancer, rectal cancer, breast cancer, stomach cancer, pancreatic cancer, ovarian cancer, cervical cancer, and bladder cancer.

The clinical utility of such drugs has often been limited by cost, dose-limiting adverse effects, and, in some case, such as paclitaxel, low aqueous solubility.

Solubilizers such as Cremophor0 (polyethoxylated castor oil) and alcohol have been demonstrated to improve solubility. Dose-limiting side effects of such drugs typically include reduction in white and red blood cell counts, nausea, loss of appetite, hair loss, joint and muscle pain, and diarrhea. By targeting the composition to the tumor site, systemic adverse effects can be reduced.

Other therapeutic agents that may be used beneficially in combination with antineoplastic agents include antiinflammatory compounds, such as dexamethasone and

other steroids, and immunostimulatory compounds.

As described above, the microbubble compositions are generally prepared by incubating an antiproliferative agent of choice with a suspension of microbubbles.

Preferably, the microbubbles are coated with a filmogenic protein, such as albumin (or an albumin/dextrose mixture) and contain a perfluorocarbon gas, preferably perfluoropropane or perfluorobutane.

Tumors to be targeted will generally be solid tumors, which can be located anywhere in the body. Tumors for which the present delivery method is useful, include, for example, solid tumors of the brain, liver, kidney, pancreas, pituitary, colon, breast, lung, ovary, cervix, prostate, testicle, esophagus, stomach, head or neck, bone, or central nervous system. The method is useful to slow the growth of tumors, prevent tumor growth, induce partial regression of tumors, and induce complete regression of tumors, to the point of complete disappearance. The method is also useful in preventing the outgrowth of metastases derived from solid tumors.

The compositions are typically administered parenterally, for example by intravenous injection or slow intravenous infusion. For localized lesions, the compositions can be administered by local injection. Intraperitoneal infusion can also be employed. Dosing regimens are determined by the physician in accordance with standard clinical procedures, taking into consideration the drug administered, the type and extent of disease, and the overall condition of the patient.

Materials and Methods General Procedure for Conjugation of a Therapeutic Agent to Albumin-Encapsulated Microbubbles PESDA (perfluorocarbon-exposed sonicated dextrose/albumin) microbubbles are prepared as described in, for example, U. S. Patent No. 6,245, 747 and PCT Pubn. No.

WO 2000/02588. In a typical procedure, 5% human serum albumin and 5% dextrose, obtained from commercial sources, are drawn into a 35 mL syringe in a 1: 3 ratio, hand agitated with 6-10 mL of decafluorobutane, and sonicated at 20 kilohertz for 75-85 seconds. As described in U. S. 6, 245, 747, the mean size of four consecutive samples of PESDA microbubbles produced in this manner, as measured with hemocytometry, was 4.6~0.4 microns, and mean concentration, as measured by a Coulter counter, was 1.4x109

bubbles/mL.

A solution or suspension of a therapeutic agent in a pharmaceutically acceptable solvent, such as aqueous saline, buffer, alcohol, DMSO, or castor oil, is incubated with agitation with the PESDA microbubble suspension at room temperature. Upon settling, the drug-conjugated microbubbles generally rise to the top of the mixture.