Title:  Sphingomyelin enhancement of tumor therapy

United States Patent:  6,541,462

Issued:  April 1, 2003

Inventors:  Modrak; David (Nutley, NJ)

Assignee:  Center for Molecular Medicine and Immunology (Belleville, NJ)

Appl. No.:  533799

Filed:  March 24, 2000

Cytotoxic tumor therapy in a patient is enhanced by co-administration of sphingomyelin. The invention most likely enhances a tumor cell's ability to undergo ceramide-induced apoptosis by increasing the levels of sphingomyelin in all cellular compartments, thereby providing sufficient substrate for activated sphingomyelinase. A method of treating rheumatoid arthritis also is provided.

DETAILED DESCRIPTION OF THE INVENTION

The present invention enhances tumor therapy. The invention is believed to enhance a tumor cell's ability to undergo ceramide-induced apoptosis by increasing the levels of sphingomyelin in all cellular compartments, thereby providing sufficient substrate for activated sphingomyelinase. Tumor cells typically have altered lipid metabolism, including abnormal sphingomyelin composition and compartmentalization. Most studies suggest that tumor tissues have increased concentrations of sphingomyelin. While most tumor cells may have abnormally high levels of sphingomyelin, it may be unavailable to its hydrolyzing enzyme, sphingomyelinase, due to abnormal, subcellular compartmentalization of sphingomyelin. The alteration of sphingomyelin metabolism can impair a tumor cell's ability to generate ceramide and can lead to reduced sensitivity to certain therapies. Surprisingly and unexpectedly, the present invention demonstrates that administration of additional sphingomyelin increases the tumoricidal activity of tumor therapy.

In accordance with one aspect of the present invention, the tumoricidal activity of tumor therapy is increased by administering to the patient a therapeutically effective amount of sphingomyelin along with the therapy. While the invention is not limited to the proposed mechanism, the administration of sphingomyelin is likely to enhance any therapy which utilizes the sphingomyelin signal transduction pathway for induction of apoptosis. This includes, but is not limited to, therapies which seek to control or inhibit rapid, abnormal growth. Examples include, but are not limited to, tumor therapies, such as chemotherapy, ionizing radiation, immunotherapy and radioimmunotherapy, and cell-mediated therapy of viral infection.

In a preferred embodiment of the present invention, a therapeutically effective amount of sphingomyelin is administered to a patient undergoing tumor therapy with chemotherapy. Sphingomyelin can be co-administered with a variety of chemotherapies. Examples include, but are not limited to, epipodophyllotoxins (e.g., etoposide, tenoposide) anthracyclines (e.g., doxorubicin/adriamycin, daunorubicin, idarubicin), Vinca alkoloids (e.g., vincristine, vinblastine), camptothecins, taxanes (e.g., Taxol) and metabolic inhibitors (e.g., 5FU, gemcitabine).

In a further embodiment, the chemotherapy may be targeted to the tumor cells using an antibody or antibody fragment. Use of antibodies, antibody fragments, or receptor binding peptides to specifically target tumor cells increases the delivery of tumoricidal doses of chemotherapy while causing a significant reduction of toxicity to normal tissues.

In another preferred embodiment of the present invention, a therapeutically effective amount of sphingomyelin is administered to a patient undergoing tumor treatment with ionizing radiation. A variety of sources may be used to generate ionizing radiation for the purpose of tumor therapy. Examples include, but are not limited to, external beam radiation and surgical implantation of radioactive particles or strings of particles.

In still another preferred embodiment of the present invention, a therapeutically effective amount of sphingomyelin is administered to a patient undergoing tumor therapy with immunotherapy. Such treatment, utilizing unconjugated antibodies and antibody fragments, effectively induces cells to undergo apoptosis by cross-linking selected surface receptors, for example the TNF receptor.

In yet another preferred embodiment of the present invention, a therapeutically effective amount of sphingomyelin is administered to a patient undergoing tumor treatment with radioimmunotherapy. Radioimmunotherapy is an attractive therapeutic concept which offers advantages over more traditional forms of cancer treatment. The strategy seeks to deliver tumoricidal doses of radiation to tumor cells with reduced radiation toxicity to normal tissues. Radioimmunotherapy utilizes antibodies, antibody fragments, or receptor binding peptides to specifically target tumor cells. The antibodies, etc., are conjugated to radioisotopes which ideally provide sufficient irradiation to kill tumor cells. Such radiolabeled antibodies, as well as receptor-binding peptides (e.g., somatostatin analogs) have been shown to target cancer cells in animal models and in humans. See Goldenberg, D. M. (editor), Cancer imaging with radiolabeled antibodies. Kluwer Academic Publishers, Boston (1990); Goldenberg, D. M. (editor), Cancer Therapy with Radiolabeled Antibodies. CRC Press: Boca Raton (1995); Krenning et al., J. Nucl. Med., 33: 652-658 (1992). As discussed above, ionizing radiation can initiate apoptosis using the sphingomyelin transduction pathway. Therefore, administering sphingomyelin with radioimmunotherapy will increase the efficacy of such treatment.

The tumoricidal activity of a variety of tumor therapies can be increased by co-administering to the patient a therapeutically effective amount of sphingomyelin along with the therapy. Examples of such therapies include, but are not limited to, oxygen radicals (e.g., O₂, NO), cytokines (e.g., FAS, TNF.alpha., TRAIL), protein phosphatase inhibitors (e.g., okadaic acid), retinoids (e.g., fenretinide), steroids (e.g., .beta.-Sitosterol), dimethylsphingosine, .DELTA.9-Tetrahydrocannabinol, suramin, sodium butyrate, platinum compounds (e.g., cis-platin, carboplatin), immunomodulators (e.g., cyclosporin, FK506), toxins (e.g., higa-, vero-, Pseudomonas endo-) and phthalocyanine 4-photodynamic therapy. Sphingomyelin also can be used in conjunction with multidrug resistance modulators which increase ceramide levels and potentiate apoptosis (e.g., SDZ PSC 833, VX710).

In another embodiment of the present invention, a therapeutically effective amount of sphingomyelin is administered to a patient suffering from rheumatoid arthritis. The disease is characterized by a proliferation of synovial cells and an infiltration of inflammatory cells that leads to cartilage and bone destruction. Abnormal events within the apoptotic process can result in the proliferation of rheumatoid synovial fibroblasts. C2-ceramide has been shown to induce apoptosis in rheumatoid synovial fibroblasts in vitro and in vivo. See Ichinose et al., J. Lab. Clin. Med., 131: 410-416 (1998). Administration of sphingomyelin is believed to increase ceramide production and, therefore, can provide an effective treatment for rheumatoid arthritis by promoting apoptosis in proliferating synovial fibroblasts. Similarly, sphingomyelin administration can effectively treat other autoimmune diseases which result from ineffective utilization of the sphingomyelin signal transduction pathway for induction of apoptosis.

In one embodiment of the present invention, naturally occurring sphingomyelin is administered to a patient to enhance the tumoricidal activity of tumor therapy. Naturally occurring sphingomyelin typically contains long, side chain derivatives (C₁₆ -C₃₀ N-acyl groups). Such sphingomyelin can be obtained from commercial sources and is usually derived from egg yolk and contains primarily palmitoyl chains. See Sigma Chemicals (St. Louis, Mo.), Catalog #S0756.

The de novo biosynthesis of sphingomyelin is initiated by the condensation of serine and palmitoyl-CoA resulting in the formation of 3-ketosphinganine (3-ketodihydrosphingosine), which is subsequently reduced to dihydrosphingosine. See Hannun, Y. A., J. Biol. Chem., 269: 3125-3218 (1994). Dihydroceramide is formed by the amide linkage of fatty acyl groups to dihydrosphingosine. Ceramide is formed from dihydroceramide by the introduction of the trans-4,5-double bond and serves as a precursor for all other complex sphingolipids. Sphingomyelin is formed by the addition of a phosphorylcholine head group to ceramide primarily through the transfer of choline phosphate from phosphatidylcholine through the action of phosphatidylcholine:ceramide choline phosphotransferase.

In another embodiment of the present invention, sphingomyelin with modified side chains can be administered to a patient to enhance the tumoricidal activity of tumor therapy. For example, sphingomyelin analogs with shorter-than-normal side chains, including C₂ -C₁₅ side chains, can be utilized. Apoptotic studies have shown that ceramide analogs with short side chains (C₂, C₈) effectively induce apoptosis and may act more rapidly than normal length molecules. See Bose et al., Cell, 82: 405-414 (1995); Haimovitz-Friedman et al., J. Exp. Med., 180: 525-535 (1994). Similarly, sphingomyelin analogs with shorter-than-normal side chains offer a further enhancement of the tumoricidal activity of tumor therapy agents. Alternatively, longer-than-normal side chains, including C₂4, also can be effective.

Numerous strategies are well-known in the art for altering the activity of biological molecules by modifying their structure. In general, modifications to a naturally occurring compound can increase its biological activity or facilitate its uptake by appropriate cell machinery. Besides varying the length of a molecule's side chains, incorporating additional elements or functional groups also can enhance the performance of a naturally occurring compound. Examples of such substituents include, but are not limited to, aliphatic groups, e.g., C₁ -C₆ straight or branched chain alkyl or cycloalkyl groups, aromatic groups, functional groups, e.g., cyano-, nitro-, azido-, halo- and epoxy- groups, and other elements, e.g., sulfur, selenium, boron and metals, as well as insertion of, e.g., oxygen or nitrogen atoms in the side chains. Sphingomyelin activity also can be enhanced by adding double or triple bonds to the molecule. See Kishida et al., J. Lipid Mediat. Cell Signal, 16: 127-137 (1997).

In one embodiment of the present invention, sphingomyelin is administered to a patient orally. In another embodiment, it is administered parenterally. Parenteral administration refers to a variety of methods of administrating a compound to a patient including, but not limited to, admninistration intravenously/intra-arterially, intrathecally, subcutaneously and via a transdermal patch.

In another embodiment, gene therapy is used to increase the sphingomyelin concentration within target cells of a patient undergoing cytotoxic tumor therapy. Gene therapy requires a system for introducing a vector containing an enzyme involved in the synthesis of sphingomyelin into target cells. Any enzyme, including those of mammalian, bacterial or fungal origin, which increases the concentration of sphingomyelin in a cell can be used. Examples include, but are not limited to, serinepalmitoyltransferase, ceramide synthase and sphingomyelinase.

The construction of a suitable vector can be achieved by any of the methods well-known in the art for the insertion of exogenous DNA into a vector. See Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y. In addition, the prior art teaches various methods of introducing exogenous genes into cells in vivo. See Rosenberg et al., Science 242:1575-1578 (1988); Wolff et al., PNAS 86:9011-9014 (1989). The routes of delivery include systemic administration and administration in situ. Well-known techniques include systemic administration with cationic liposomes, and administration in situ with viral vectors. See Caplen et at., Nature Med., 1:39-46 (1995); Zhu et al., Science, 261:209-211 (1993); Berkner et al., Biotechniques, 6:616-629 (1988); Trapnell et al., Advanced Drug Delivery Rev., 12:185-199 (1993); Hodgson et al., BioTechnology 13: 222 (1995). Vectors and gene delivery systems which specifically direct the exogenous genes to target cells are most preferred. It is anticipated that future developments in targeted gene delivery will increase the significance of this embodiment.

A "therapeutically effective" amount of sphingomyelin can be determined by prevention or amelioration of adverse conditions or symptoms of diseases, injuries or disorders being treated. Optimization of the timing and dosage of sphingomyelin administered to a patient in conjunction with tumor therapy by convention is adapted to, among other things, the particular characteristics of the patient and the extent of the tumorgenesis. Such adaptations are routine and do not require undue experimentation or skill in the art. Similarly, optimization of the timing and dosage of sphingomyelin administered to a patient as a therapy for rheumatoid arthritis also is adapted to, among other things, the particular characteristics of the patient. The methods and pharmaceutical compositions of the invention can be used to treat a variety of mammals and are used most preferably to treat humans and domesticated animals, such as livestock and pets.

The liposomes of the invention can be combined with inert pharmaceutical excipients such as lactose, oil, mannitol and starch to form pharmaceutical compositions/preparations. Such compositions can be formulated into dosage forms such as elixirs, liquids, ointments, lotions, IV fluids, alcohol, tablets, capsules, and the like. For parenteral, intramuscular, subcutaneous and intravenous administration, the liposomes can be formulated with an inert, parenterally acceptable vehicle such as water, saline, sesame oil, ethanol buffered aqueous medium, propylene glycol and the like. For topical and oral administration, the liposomes can be formulated with waxes, oils, buffered aqueous medium, and the like. These various pharmaceutical dosage forms are prepared by methods well-known to the pharmacist's art.

In another embodiment, there is provided a kit useful for enhancing cytotoxic tumor therapy, comprising sphingomyelin and ancillary reagents to effect administration of the sphingomyelin. Examples of ancillary reagents include, but are not limited to, buffered solutions and application devices, such as syringes. Similarly, there is provided a kit useful for treating rheumatoid arthritis in a patient, comprising sphingomyelin and ancillary reagents to effect administration of the sphingomyelin.

Claim 1 of 10 Claims

What is claimed is:

1. A method of treatment comprising administering a cytotoxic tumor therapy to a patient, and a therapeutically effective amount of sphingomyelin as an adjunct to said cytotoxic tumor therapy, wherein said therapeutically effective amount of sphingomyelin enhances the cytotoxicity of said cytotoxic tumor therapy.
 

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