HK1259723A1 - Echinomycin formulation, method of making and method of use thereof - Google Patents

Description

Echinomycin preparation and preparation method and use method thereof

This application claims priority from U.S. provisional patent application serial No. 62/253,257 filed on day 11/10 of 2015. The entire contents of all of the aforementioned applications are hereby incorporated by reference.

Technical Field

The present application relates generally to compositions and methods for preparing and delivering liposomal echinomycin formulations for the treatment of proliferative disorders, autoimmune diseases, and alloimmune responses.

Background

Hypoxia Inducible Factor (HIF) is a transcription factor and mediates cellular responses to hypoxia. HIF is known to be upregulated in a number of cancers, autoimmune diseases, and allogenic immune responses. In particular, HIF is involved in tumor metabolism, angiogenesis and metastasis (Semenza, G.L., et al, Nature Rev. cancer.2003; 3 (10): 721-32).

Echinomycin (NSC526417) is a member of the quinoxaline family originally isolated in 1957 from Streptomyces echinocandis (Streptomyces echinatus). echinomycin is a small molecule that inhibits the DNA binding activity of HIF1 α. echinomycin has been shown to exhibit anti-tumor activity against B16 melanoma and P388 leukemia engrafted murine tumors, and to exhibit growth of anti-human tumor cells in vitro and in vivo.

Echinomycin was incorporated into clinical trials by the National Cancer Institute. However, echinomycin used in these studies did not exhibit significant anti-tumor activity in previously treated patients. Multiple stage I (7-11) and stage II (12-19) solid tumors have been tested for many years. However, clinical development of echinomycin has been stopped because the drug is not always effective in patients with solid tumors that are refractory to all current therapies. Since these studies have been performed for a long time before echinomycin is known to be a HIF inhibitor (Kong D. et al, Cancer Res.2005; 65 (19): 9047-55), no effect was designedForce studies were conducted to assess the potential benefits of inhibiting HIF. Although echinomycin at 1200. mu.g/m²The dose of (c) was used in some phase II clinical trials in humans, but since no method was available to measure drug concentration from 1985 to 1995, no Pharmacokinetic (PK) data was presented. However, new approaches have recently emerged, revealing that echinomycin has a short half-life in vivo, thereby limiting its clinical use.

Echinomycin is highly insoluble in water, which complicates the means by which it can be formulated into a suitable dosage form. Echinomycin precipitates out of solution rapidly when dissolved in water, and therefore any formulation that relies on a mixture of the free drug with an aqueous solvent does not yield significant bioavailability in the recipient. Furthermore, the available solvents in which echinomycin is soluble, such as DMSO, are not clinically acceptable because these solvents have harsh properties for patients. Clinical trials with echinomycin also revealed significant reported side effects such as severe nausea and vomiting, further limiting its clinical utility.

In view of the above limitations, there is a need for new and effective echinomycin formulations that are non-toxic and effective against proliferative disorders, autoimmune diseases, graft versus host disease or any other disease requiring inhibition of HIF-1.

Disclosure of Invention

One aspect of the present application relates to a liposomal pharmaceutical formulation for treating a disease in a patient characterized by overexpression of HIF-1 α and/or HIF-2 α the liposomal pharmaceutical formulation comprising a plurality of liposomes in a pharmaceutically acceptable carrier the liposomes encapsulating echinomycin and being made from a pegylated phospholipid, a neutral phosphoglyceride and a sterol.

In one embodiment, the pegylated phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dimyristoylphosphatidylethanolamine-polyethylene glycol (DMPE-PEG), dipalmitoyl glyceryl succinate polyethylene glycol (DPGS-PEG), cholesteryl-polyethylene glycol, or a ceramide-pegylated lipid.

In another embodiment, the neutral phosphoglyceride is selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol.

In another embodiment, the molar ratio of the pegylated phospholipid to total lipid in the formulation is from 3% to 6%; (ii) the molar ratio of said neutral phosphoglycerides to total lipid in said formulation is from 45% to 60%; and the molar ratio of said sterol to total lipid in said formulation is from 30% to 50%.

In one embodiment, the pegylated phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), the neutral phosphoglyceride is phosphatidylcholine, and the sterol is cholesterol.

In a specific embodiment, the liposome comprises 5.3% DSPE-PEG-2000, 56.3% Hydrogenated Soy Phosphatidylcholine (HSPC), and 38.4% cholesterol.

In certain embodiments, the mass ratio of echinomycin to total lipid is 2% to 10%. In a specific embodiment, the mass ratio of echinomycin to total lipid is 5%.

In some embodiments, at least 90% of the liposomes in the formulation have a diameter of 80nm to 120 nm.

In some embodiments, the liposomes have an average polydispersity index of less than 0.1 and are sufficiently stable to achieve a lifetime of at least 12 months at 4 ℃.

In other embodiments, the liposome is formulated as a lyophilized powder.

In another aspect, a method of treating a disease in a patient characterized by overexpression of HIF-1 α and/or HIF-2 α using a pegylated liposomal formulation according to the present application comprises administering the pegylated formulation to a patient in need thereof, wherein the liposomal formulation comprises echinomycin in an amount sufficient to treat the disease.

In one embodiment, the disease is a proliferative disorder. In a specific embodiment, the proliferative disorder is leukemia. In another embodiment, the proliferative disorder is breast cancer.

In another embodiment, the disease is an autoimmune disease.

In another embodiment, the disease is graft versus host disease.

In another aspect, a method for preparing a pegylated liposomal formulation according to the present application comprises forming a mixture comprising echinomycin and a lipid component comprising a pegylated phospholipid, a neutral phospholipid, and a sterol in a polar solvent; drying the mixture to remove the polar solvent, thereby forming a dried lipid film; dissolving the dried lipid film in a buffer to form a lipid suspension; extruding the lipid suspension through a polycarbonate filter to obtain liposomes having a desired size range; and sterilizing the liposomes by filtration.

Drawings

Figure 1 physical properties of liposomal echinomycin. (A) Size distribution of a typical formulation measured by Dynamic Light Scattering (DLS) on Malvern Zetasizer software. (B) Summary of mean size and zeta potential of liposomal echinomycin. Data is a summary of +/-s.d of 6 independent formulations. Measurements were made using Malvern Zetasizer software.

Figure 2. in vitro release of echinomycin from liposomal echinomycin. (FIG. A) targeting ddH at 21 deg.C₂Drug release profile of O-dialyzed liposomal echinomycin. Data points represent the mean +/-s.d. of three replicate measurements by HPLC. (panel B) indicates echinomycin peaks at the corresponding time points plotted in (panel A)Representative HPLC chromatograms of (a).

Figure 3 in vitro liposomal echinomycin storage and stability. (panels a and B) liposomal echinomycin was stored at 4 ℃ and sampled after 1 and 3 months of storage to obtain stability parameters. 0 month means that the measurement was directly performed after the preparation. Measurement of mean size and zeta potential (panel a) and mean polydispersity index (PdI) (panel B) of liposomal echinomycin during storage at 4 ℃. Data were generated using Malvern Zetasizer software with error bars representing the s.d. of three replicate measurements. (panel C) HPLC analysis of echinomycin content loss during storage at 4 ℃ expressed as a percentage of the initial measurement (0 months). Error bars represent s.d. of three replicate measurements.

Figure 4 comparison of toxicity and efficacy of liposomal echinomycin with free echinomycin. (panel a) body weight changes in female NSG mice receiving treatment cycles of liposomal echinomycin, PBS echinomycin (20% DMSO in PBS) or equivalent doses of empty liposomal vehicle (vehicle). Mice were treated with 250 μ g/kg of liposomal echinomycin (n-7) or free (n-7) echinomycin or equivalent vehicle (n-4) by intravenous injection (i.v.) once every other day for 3 doses. (panel B) survival of NSG mice administered 1mg/kg liposomal echinomycin or free echinomycin or equivalent dose of empty liposomal vehicle by a single intravenous injection.

FIG. 5 pharmacokinetics of liposomal echinomycin compared to free echinomycin. Serum plasma levels of echinomycin detected by MS following single dose intravenous injection administration of 0.1mg/kg liposomal echinomycin or free echinomycin.

FIG. 6. accumulation of echinomycin in tumors. Echinomycin concentration in breast tumor SUM159 after single dose intravenous injection (0.1mg/kg) of liposomal echinomycin and conventionally formulated echinomycin. n is 3/time point.

FIG. 7 accumulation of HIF-1 α in ETP-ALL (FIG. A) HIF-1 α protein levels in spleen of ETP ALL xenograft models as measured by Western blotting (FIG. B) ETP-ALL-1 cells from spleen of xenografted mice were stained with anti-hCD 45 and CD117 surface markers and stained intracellularly with APC-conjugated HIF-1 α antibody prior to FACS analysis.

Figure 8. Liposomal echinomycin abrogates human ETP-ALL cells in a xenograft mouse model. (panel a) data for the administration regimen of liposomal echinomycin treatment for ETP-ALL humanized mice is in panel B. Day 0 represents the day of transplantation with human ETP-ALL-1 cells. The green (up) arrow indicates a single injection of liposomal echinomycin (intravenous 0.35 mg/kg/injection). Red (down) arrows indicate blood detection. (FIG. B) NSG mice irradiated to 1.3Gy were injected intravenously at 1X 10⁶Human ETP-ALL-1 cells. Detection of human CD45 in recipient PBMCs by FACS analysis on day 34 post-transplant⁺A cell. Human CD45 in PBMCs of recipient mice (vehicle and liposomal echinomycin treatment) before (day 34) and after (days 42, 48, 56 and 64) treatment by FACS analysis⁺The percentages were analyzed. Human CD45 in PBMCs of all recipient mice treated with vehicle or liposomal echinomycin⁺Summary of percentages (n 10 per group). (panel C) liposomal echinomycin was more effective than maximally free echinomycin at well tolerated doses. NSG mice were irradiated to 1.3Gy and injected intravenously at 1X 10⁶Human ETP-ALL-1 cells. FACS analysis was performed on day 21 before treatment. On day 22, mice received the first dose of treatment. One group of recipient mice (PBS-EM, n-5) received 3 cycles of echinomycin in PBS (15 doses total). In each cycle, mice received echinomycin in PBS at an intravenous dose of 0.1mg/kg, injected once daily for 5 doses, and then rested for 5 days before the next cycle was initiated. In another group of recipients (Lipo-EM, n ═ 5), mice received 0.35mg/kg of liposomal echinomycin by intravenous injection. Starting on day 22, liposomal echinomycin was administered every 4 days for the first 2 doses, followed by a rest of 7 days. The mice then received injections every other day, 4 doses, followed by rest for 7 days, and again every other day, 4 doses (10 doses total) to the end. Another group of mice received an empty liposome vehicle (n-5) according to the same protocol in which liposomal echinomycin was administered. Mouse spines by FACS analysisHuman CD45 in recipient PBMCs after mycin treatment (days 34, 50 and 65)⁺The percentage of (c) was analyzed.

FIG. 9. echinomycin reduces the number of viable breast cancer cells in vitro Breast cancer cells with high (SUM 159) or low (MCF7) levels of HIF-1 α were treated with echinomycin at different concentrations in vitro.

FIG. 10. echinomycin accumulation in xenografted breast cancer cells. Human breast cancer cell line SUM-159 was transfected with luciferase and xenografted in situ in the mammary fat pad of female NSG mice. Free DiR or DiR-labeled liposomes in water were administered to xenograft mice according to the bond at the bottom of the figure. Luciferase expression in xenograft cells was assessed using bioluminescent imaging (upper panel) and the presence of liposomal echinomycin was assessed by bioluminescent imaging (lower panel).

Figure 11. in vivo therapeutic effect of liposomal echinomycin on breast cancer. (panel a) female NSG mice were xenografted with human SUM159 breast cancer cells and treated with free echinomycin or vehicle (n ═ 10 per group). Mice received 0.1mg/kg free echinomycin or vehicle by intravenous injection starting on day 28, once every 3 days for 6 doses. The growth kinetics of SUM159 are shown. (panel B) female NSG mice were xenografted with SUM159 breast cancer cells and treated with liposomal echinomycin or vehicle (n ═ 5 per group). The growth kinetics of SUM159 in recipient mice treated with vehicle and liposomal echinomycin are shown. Mice received 0.35mg/kg of liposomal echinomycin or vehicle by intravenous injection on days 9, 11, 13, 25 and 27. The mean tumor volume was recorded throughout the experiment. Error bars represent ± SEM. The individual treatments and doses are indicated by asterisks. (panel C) mice were euthanized, tumors dissected and photographed. (panel D) summary of tumor weights from breast cancer of vehicle and liposomal echinomycin treated mice. Weights are expressed as mean weights ± s.d., and P values are calculated by t-test.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes a plurality of such peptides, reference to "the peptide" refers to one or more peptides known to those skilled in the art, equivalents thereof, and so forth.

As used herein, the term "cell proliferative disorder" refers to a disorder characterized by abnormal proliferation of cells. Proliferative disorders do not imply any limitation on the rate of cell growth, but merely indicate a loss of normal control affecting growth and cell division. Thus, in some embodiments, cells of a proliferative disorder may have the same rate of cell division as normal cells, but not respond to signals that limit such growth. Within the scope of a "cell proliferative disorder" is a neoplasm or tumor, which is an abnormal growth of a tissue. "cancer" refers to any of a variety of malignant neoplasms characterized by the ability to proliferate cells that invade surrounding tissues and/or metastasize to new sites of colonization, and includes leukemias, lymphomas, carcinomas, melanomas, sarcomas, germ cell tumors, and blastomas. Exemplary cancers to be treated with the methods of the present disclosure include brain, bladder, breast, cervical, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovarian, prostate, stomach and uterine cancers, leukemia and medulloblastoma.

The term "leukemia" refers to a progressive malignant disease of the blood-forming organs and is generally characterized by abnormal proliferation and development of leukocytes and their precursors in the blood and bone marrow. Exemplary leukemias for treatment include, e.g., acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, leukemia with no increase in leukocytes (aleukemickemia), leukemia with increase in leukocytes (leukacythemic leukemia), basophilic myelocytic leukemia, blastic leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic leukemia, Geross leukemia, hairy cell leukemia, hematopoietic leukemia, hemablastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphoid leukemia, lymphoblastic leukemia, acute myeloblastic leukemia, lymphoblastic leukemia, chronic myeloblastic, Lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micro-myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid myeloblastic leukemia, myelomonocytic leukemia, endogenous leukemia, plasma cell leukemia (plasma cell leukemia), plasma cell leukemia (plasma cytic leukemia), promyelocytic leukemia, polymorphonuclear leukemia (Rieder cell leukemia), acute monocytic leukemia (Schilling's leukemia), stem cell leukemia, sub-leukemia, and undifferentiated leukemia.

The term "cancer" refers to a malignant growth of epithelial cells that tend to penetrate surrounding tissues and cause metastases. Exemplary cancers include, for example, acinar cancer, cystic adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma, adrenocortical carcinoma, alveolar cell carcinoma, basal cell carcinoma (carcinoma basocellulare), basal-like carcinoma, basal squamous cell carcinoma (basosigmamous cell carcinoma), bronchoalveolar carcinoma, bronchiolar carcinoma, cerebroid carcinoma, cholangiocellular carcinoma, choriocarcinoma, glioma, acne carcinoma, corpus uteri carcinoma, ethmoid carcinoma, thyroid carcinoma, skin carcinoma, cylindrical cell carcinoma, ductal carcinoma, scleroid carcinoma, embryonic carcinoma, cerebroid carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma, genitalia carcinoma, precancerous lesion, fibroma, gelatin-like carcinoma, giant cell carcinoma (carcinoma), giant cell carcinoma (carcinomagiomegakaryocyte), giant cell carcinoma, stromal cell carcinoma, hair cell carcinoma (trichocyte carcinoma), and squamous cell carcinoma, Clear cell carcinoma, hypocotyl carcinoma, infantile embryo carcinoma, carcinoma in situ, carcinoma in the epidermis, carcinoma in the epithelium, cobber (Krompecher's carcinosoma), Kulchitzky-cellcarcinoma (Kulchitzky-cellclarconema), large cell carcinoma, lenticular carcinoma (lenticulars), lipomatous carcinoma, lymphatic carcinoma, medullary carcinoma (carcinosoma), medullary carcinoma (medullaria), melanoma, cancerous nevi, mucinous carcinoma, mucinous cell carcinoma, mucinous epidermoid carcinoma, mucosal carcinoma, mucinous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossified carcinoma, bone carcinoma, papillary carcinoma, portal vein carcinoma, precancerous lesion, echinocytic carcinoma, lung adenocarcinoma, renal cell carcinoma, prepared cell carcinoma, vulval carcinoma, squamous cell carcinoma, sarcoma, squamous cell carcinoma, simple cell carcinoma, sarcoid carcinoma, phoma cell carcinoma, simple eggplant cell carcinoma, sarcoid carcinoma, and eggplant-like carcinoma Spongiform carcinoma, squamous cell carcinoma, roping carcinoma, telangiectatic carcinoma (carcinoma telangiectatic), telangiectatic carcinoma (carcinoma telangiectatics), transitional cell carcinoma, nodular carcinoma, verrucous carcinoma, and choriocarcinoma.

The term "sarcoma" refers to a tumor that is composed of a substance such as embryonic connective tissue, and is typically composed of densely packed cells embedded in a fibrous or homogeneous substance. Exemplary sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanoma, myxosarcoma, osteosarcoma, Abemeth's sarcoma, liposarcoma (adipose sarcoma), liposarcoma (liposarcoma), alveolar soft tissue sarcoma, amelogenic sarcoma, botryoid sarcoma, chloroma, choriocarcinoma, embryonal sarcoma, Winteric sarcoma (Wilns ' tulor sarcoma), interstitial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblast sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic polymorphous hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma (e.g., non-Hodgkin's lymphoma), T-cell immunoblastic sarcoma, Yansen sarcoma, Kaposi's sarcoma, Kupffer's sarcoma, angiosarcoma, leukocytoma, malignant metaplastic sarcoma, extraperiosteal sarcoma, extramembranous sarcoma, and angiosarcoma, Reticulosarcoma, rous sarcoma, serous sarcoma, synovial sarcoma, and telangiectatic sarcoma.

The term "melanoma" refers to tumors produced by the melanocytic system of the skin and other organs. Melanoma includes, for example, acral lentigo melanoma, leucoma, benign juvenile melanoma, claudman melanoma, S91 melanoma, Harding Passey melanoma, juvenile melanoma, freckle malignant melanoma, nodular melanoma, angular melanoma (subhealthy melanoma), and superficial diffuse melanoma.

Other cancers include, for example, hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, small cell lung tumor, primary brain tumor, stomach cancer, colon cancer, malignant pancreatic islet tumor, malignant carcinoid cancer, premalignant skin lesion, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortex cancer.

As used herein, the term "autoimmune disease" refers to a condition in which an individual's immune system (e.g., activated T cells) attacks the individual's own tissues and cells. The term "alloimmune response" refers to a condition in which an individual's immune system attacks implanted tissues or cells, such as in a graft or transplant.

Exemplary autoimmune diseases to be treated by the methods of the present disclosure include arthritis, alopecia areata, ankylosing spondylitis, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, Crohn's disease, dermatomyositis, diabetes (type I), glomerulonephritis, Graves ' disease, Guillain-Barre syndrome, Inflammatory Bowel Disease (IBD), lupus nephritis, multiple sclerosis, myasthenia gravis, myocarditis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, sjogren's syndrome, Systemic Lupus Erythematosus (SLE), thyroiditis (such as hashimoto's thyroiditis and alder's thyroiditis), ulcerative colitis, uveitis, vitiligo and wegener's granulomatosis. Exemplary allogenic immune responses for treatment with the methods of the present disclosure include Graft Versus Host Disease (GVHD) and graft rejection.

As used herein, "treatment" refers to any improvement in a proliferative disorder, an autoimmune disease, or an alloimmune response.

The terms "treat" and "treating" refer to ameliorating one or more symptoms associated with a cell proliferative disorder, an autoimmune disease, or an allogeneic immune response; preventing or delaying the onset of one or more symptoms of a cell proliferative disorder, an autoimmune disease, or an alloimmune response; and/or reducing the severity or frequency of one or more symptoms of a cell proliferative disorder, an autoimmune disease, or an alloimmune response.

The terms "improve," "increase," or "decrease" as used herein indicate a value or parameter measured relative to a baseline, e.g., in the same individual prior to initiation of a treatment described herein or in a control individual (or control individuals) in the absence of a treatment described herein.

A "control individual" is an individual having the same disease or disorder as the individual receiving treatment, at about the same age as the individual receiving treatment (to ensure that the disease stage in the individual and the control individual are comparable). The individual being treated (also referred to as "patient" or "subject") may be a mammalian subject, preferably a human subject, such as a fetus, infant, child, adolescent or adult.

Microemulsion echinomycin drug delivery system

The present application provides a microemulsion echinomycin drug delivery system for the treatment of proliferative disorders, autoimmune diseases and alloimmune responses in which HIF-1 α or HIF-2 α is elevated, emulsions are a mixture of two or more liquids that are generally immiscible (immiscible or unblendable). the microemulsion echinomycin drug delivery system can comprise liposomes, micelles, or a mixture of liposomes and micelles.

The present application provides liposome compositions encapsulating echinomycin or echinomycin analogs, and methods of using such compositions for treating proliferative disorders, autoimmune diseases, and alloimmune responses in which HIF-1 α or HIF-2 α is elevated.

In some preferred embodiments, the microemulsion drug delivery system used is a liposomal drug delivery system. In other embodiments, the microemulsion drug delivery system used consists of microparticles (or microspheres), nanoparticles (or nanospheres), nanocapsules, block copolymer micelles, or other polymeric drug delivery systems. In other embodiments, the drug delivery system used is a polymer-based non-microemulsion drug delivery system, such as a hydrogel, film, or other type of polymeric drug delivery system. In still other embodiments, the echinomycin or echinomycin analog is administered parenterally in a lipid-based solvent.

The methods of the invention are useful for treating proliferative disorders, autoimmune diseases and alloimmune responses in all mammalian subjects, particularly human patients. As used herein, a "patient" is a human patient.

Echinomycin (NSC526417) is a member of the quinoxaline family originally isolated from Streptomyces echinata (Streptomyces echinata) a small molecule that inhibits the DNA binding activity of HIF-1 α.

Echinomycin analogs include compounds that exhibit an effect on reducing HIF-1 α or HIF-2 α activity (similar to the effect of echinomycin) due to their structural and functional similarities to echinomycin exemplary echinomycin analogs include YK2000 and YK2005(Kim, j.b. et al, int.j.antimicrob.Agents, 12.2004; 24 (6): 613), quinomycin G (Zhen x. et al, mar.drugs, 2015 11.18/2015; 13 (11): 6947-61), 2QN (baily, c. et al, Anticancer drug. des., 1999 6.6/1999; 14 (3): 291) 303), and quinomycin (KhaN, a.w. et al, indianj.biochem., 19612, (1969) 220-1).

Microemulsion drug delivery vehicles, including liposomes, may be used to deliver echinomycin or echinomycin analogs in cells or patients with proliferative disorders or autoimmune diseases or in patients exhibiting allogeneic immune responses, such as in GVHD. Echinomycin or echinomycin analogs can be encapsulated in (or incorporated into) any suitable microemulsion drug delivery vehicle capable of delivering a drug to a target cell in vitro or in vivo.

As used herein, a microemulsion drug delivery vehicle is a vehicle comprising particles capable of being suspended in a pharmaceutically acceptable liquid medium, wherein the particles range in size from a few nanometers to a few micrometers in diameter. Microemulsion drug delivery systems contemplated in the present application include those that substantially retain their microemulsion properties upon in vivo administration. Microemulsion drug delivery systems include, but are not limited to, lipid-based particles and polymer-based particles. Examples of microemulsion drug delivery systems include liposomes, nanoparticles (or nanospheres), nanocapsules, microparticles (or microspheres), and block copolymer micelles.

Liposomes share many similarities with cell membranes and are contemplated for use in conjunction with the present invention as carriers for echinomycin and echinomycin analogs. They are widely applicable because both aqueous and fat-soluble substances can be encapsulated separately, i.e. within the aqueous space and the bilayer itself. Based on their hydrophobicity, the liposome formulation of the liposomes can be modified by those skilled in the art to maximize the solubility of echinomycin or any analog thereof.

Liposomes suitable for delivery of echinomycin or echinomycin analogs include those liposomes consisting essentially of vesicle-forming lipids. Suitable vesicle-forming lipids for use in the present invention include those that can spontaneously form bilayer vesicles in water, such as those exemplified by phospholipids.

The choice of lipids suitable for use in liposomes depends on the following factors: (1) liposome stability, (2) phase transition temperature, (3) charge, (4) non-toxicity to mammalian systems, (5) encapsulation efficiency, and (6) lipid mixture properties. It is contemplated that one skilled in the art, having the benefit of this disclosure, may formulate liposomes that optimize these factors in accordance with the present invention. Vesicle-forming lipids of this type are preferably those having two hydrocarbon chains (typically acyl chains) and a polar or non-polar head group. The hydrocarbon chain may be saturated or have varying degrees of unsaturation. There are a variety of synthetic vesicle-forming lipids and naturally occurring vesicle-forming lipids including phospholipids, phosphoglycerides, glycolipids such as cerebrosides and gangliosides, sphingolipids, ether lipids, sterols, and caged phospholipids.

Liposomes comprise a liposome shell consisting of one or more concentric lipid monolayers or lipid bilayers. Thus, the lipid shell may be formed of a single lipid bilayer (i.e., the shell may be a monolayer) or several concentric lipid bilayers (i.e., the shell may be multilayers). The lipids may be synthetic, semi-synthetic or naturally occurring lipids including phospholipids, tocopherols, steroids, fatty acids, glycoproteins such as albumin, anionic lipids and cationic lipids. The lipids may have anionic, cationic or zwitterionic hydrophilic head groups and may be anionic, cationic lipid or neutral at physiological pH.

The liposome formulation can comprise a mixture of lipids. The mixture may comprise (a) a mixture of neutral and/or zwitterionic lipids; (b) a mixture of anionic lipids; (c) a mixture of cationic lipids; (d) a mixture of anionic and cationic lipids; (e) a mixture of neutral or zwitterionic lipids and at least one anionic lipid; (f) a mixture of neutral or zwitterionic lipids and at least one cationic lipid; or (g) a mixture of neutral or zwitterionic lipids, anionic lipids and cationic lipids. Further, the mixture can comprise saturated lipids, unsaturated lipids, or a combination thereof. If the unsaturated lipid has two tails, both tails may be unsaturated, or may have one saturated tail and one unsaturated tail. In some embodiments, the mixture of lipids does not contain any unsaturated lipids.

In one embodiment, the lipid formulation is substantially free of anionic lipids, substantially free of cationic lipids, or both. In another embodiment, the lipid formulation is free of anionic lipids or cationic lipids or both. In one embodiment, the lipid formulation comprises only neutral lipids. Typically, the neutral lipid component is a lipid having two acyl groups (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipids having various acyl chain groups of varying chain length and saturation are commercially available or can be isolated or synthesized by well-known techniques.

Exemplary neutral or zwitterionic phospholipids include, but are not limited to, Egg Phosphatidylcholine (EPC), Egg Phosphatidylglycerol (EPG), Egg Phosphatidylinositol (EPI), Egg Phosphatidylserine (EPS), phosphatidylethanolamine (EPE), phosphatidic acid (EPA), Soy Phosphatidylglycerol (SPG), Soy Phosphatidylserine (SPS), Soy Phosphatidylinositol (SPI), Soy Phosphatidylethanolamine (SPE), Soy Phosphatidic Acid (SPA), Hydrogenated Egg Phosphatidylcholine (HEPC), Hydrogenated Egg Phosphatidylglycerol (HEPG), Hydrogenated Egg Phosphatidylinositol (HEPI), Hydrogenated Egg Phosphatidylserine (HEPS), Hydrogenated Egg Phosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA), Hydrogenated Soy Phosphatidylcholine (HSPC), Hydrogenated Soybean Phosphatidylglycerol (HSPG), Hydrogenated Soybean Phosphatidylserine (HSPS), Hydrogenated Soybean Phosphatidylinositol (HSPI), Hydrogenated Soybean Phosphatidylethanolamine (HSPE), Hydrogenated Soybean Phosphatidic Acid (HSPA), Dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), Dioleoylphosphatidylcholine (DOPC), Dimyristoylphosphatidylcholine (DMPC), Distearoylphosphatidylcholine (DSPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1, 2-diarachioyl-sn-glycero-3-phosphatidylcholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1, 2-docosadienoyl-sn-glycero-3-phosphocholine (DEPC), Palmitoyl Oleoyl Phosphatidylcholine (POPC), dilauroyl phosphatidylcholine (DLPC), Palmitoyl Stearoyl Phosphatidylcholine (PSPC), Lysophosphatidylcholine (LPC), dilinoleoyl phosphatidylcholine (DLPC), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylethanolamine (dioledylethanolamine, DOPE), dioleoyl phosphatidylethanolamine (dioleoylphosphatidylethanolamine, DOPE), Dioleoylphosphatidylethanolamine (DOPE), Palmitoyl Oleoyl Phosphatidylethanolamine (POPE), and Palmitoyl Stearoyl Phosphatidylglycerol (PSPG), sterols such as cholesterol and sterols; choline esters, ceramides, cerebrosides, diacylglycerols, sphingosines, sphingomyelins such as cephalin, lecithin, dipalmitoyl sphingomyelin, and distearoyl sphingomyelin dihydrosphingomyelin; and monoacylated phospholipids, such as monooleoyl-phosphatidylethanolamine (MOPE).

Zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic lipids are 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) and dodecyl phosphocholine.

Exemplary anionic lipids include di (hexadecyl) phosphate (DhP), phosphatidylinositol, phosphatidylserine, including diacylphosphatidylserine, such as dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine; phosphatidylglycerols, such as Dimyristoylphosphatidylglycerol (DMPG), Dipalmitoylphosphatidylglycerol (DPPG), Distearoylphosphatidylglycerol (DSPG), Dioleoylphosphatidylglycerol (DOPG), Dilauroylphosphatidylglycerol (DLPG), Distearoylphosphatidylglycerol (DSPG), and Lysophosphatidylglycerol (LPG); phosphatidylethanolamines, such as N-lauroyl phosphatidylethanolamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine; phosphatidic acids, including diphosphatidyl glycerol and diacylphosphatidic acids, such as dimyristoyl phosphate and dipalmitoyl phosphate; cardiolipin and Cholesterol Hemisuccinate (CHEMS).

Exemplary cationic lipids include N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium salt, also known as TAP lipids, e.g., methyl sulfate. suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). other suitable cationic lipids include dimethyldioctadecyl ammonium bromide (DDAB), 1, 2-diacyloxy-3-trimethylammonium propane, N- [1- (2, 3-dioleoyloxy) propyl ] -N, N-dimethylamine (N- [1- (2, 3-dioleoyloxy) propyl ] -N, N-dimethylammonium bromide, N- [1- (2, 3-dioleoyloxy) propyl ] -N, N-dimehyl ] amine, DAP, 1, 2-diacyloxy-3-dimethylammonium bromide, N- [1- (2, 3-dioleoyloxy) propyl ] -N-isopropyl ] -N, N-bis- (3-dioleoyloxy) propyl) -N-propyl ] -N, N-bis- (dodecyloxy) -N-2-propyl) -N, N-bis- (3-diethylaminoethyl-propyl) -N- (3-bis (DL-palmitoyl) propyl) -amide (DDMA) -N-bis (DDN-2-palmitoyl) propyl) -N, N-bis (DL-2-palmitoyl-ethyl-3-ethyl-isopropyl) -amide (DL-ethyl-amide (DDM-N-isopropyl) -amide (DDM-N-bis- (1-bis- (D-bis- (3-isopropyl) -N-bis- (D-ethyl-isopropyl) -N-isopropyl) -amide (DDM-ethyl-N-ethyl-propyl) -amide (DDM-N-propyl) -N-isopropyl-N-propyl) -N-propyl) -N-propyl-N-propyl-N-propyl-N-propyl-N-isopropyl-N-bis (D-propyl-N-isopropyl-N-propyl-N-propyl-N-propyl-N-propyl-N-propyl-N-propyl-N-propyl-N-propyl-N-propyl-.

Typically, liposomal formulations according to the present application comprise at least one pegylated lipid within the liposome, i.e. the lipid comprises a polyethylene glycol moiety. A liposome comprising a pegylated lipid will have a PEG orientation such that it is present at least on the exterior of the liposome (although some PEG may also be exposed to the interior of the liposome, i.e., to the aqueous core). This orientation can be achieved by attaching PEG to the appropriate portion of the lipid. For example, in an amphiphilic lipid, PEG will be attached to a hydrophilic head because it is this head that orients itself to the aqueous-facing exterior of the lipid bilayer. Pegylation in this manner can be achieved by covalently attaching PEG to a lipid using techniques known in the art.

Exemplary pegylated lipids include, but are not limited to, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), including DSPE PEG (1000MW), DSPE PEG (2000MW), and DSPE PEG (5000 MW); myristoylphosphatidylethanolamine-polyethylene glycol (DMPE-PEG), including DMPE PEG (1000MW), DMPE PEG (2000MW), and DMPEPEG (5000 MW); dipalmitoyl glyceryl succinate polyethylene glycol (DPGS-PEG), including DPGS-PEG (1000MW), DPGS (2000MW), and DPGS (5000 MW); stearoyl-polyethylene glycol, cholesteryl-polyethylene glycol and ceramide-based pegylated lipids, such as N-octanoyl sphingosine-1- { succinyl [ methoxy (polyethylene glycol) MW ] }, referred to as C8PEG (MW) ceramide, where MW is 750, 2000 or 5000, or N-palmitoyl sphingosine-1- { succinyl [ methoxy (polyethylene glycol) MW ] }, or referred to as C16PEG (MW) ceramide, where MW is 750, 2000 or 5000. Other pegylated Lipids may be available from Avanti Polar Lipids, Inc.

Liposomes of the invention will generally comprise a plurality of PEG moieties which may be the same or different. The PEG in the liposome of the present invention has an average molecular weight of greater than 350Da but less than 5kDa, such as 0.35-5 kDa, 1-3 kDa, 1-2-6 kDa, 2-3 kDa or 4-5 kDa, or preferably 2kDa (PEG 2000). PEG typically comprises linear polymer chains, but in some embodiments, PEG may comprise branched polymer chains.

In some embodiments, the PEG may be a substituted PEG, for example, wherein one or more carbon atoms in the polymer are substituted with one or more alkyl, alkoxy, acyl, or aryl groups. In other embodiments, the PEG may comprise a copolymer group, such as one or more propylene monomers to form a PEG polypropylene polymer.

In certain embodiments, the liposomes are formed from a mixture of one or more pegylated phospholipids and one or more additional neutral lipids. The mole percentage of the pegylated lipid may be between 0.1 and 20%. In some embodiments, the mole percentage of pegylated lipid is 1 to 9%, 2 to 8%, preferably 5 to 6% of the total amount of lipid in the composition.

As used herein, the "mole percent" of lipid a in a mixture containing lipids A, B and C is defined as:

the molar amount of A/(molar amount of A + molar amount of B + molar amount of C) × 100%

In another embodiment, the liposome is formed from a lipid mixture comprising a pegylated phospholipid, a neutral phosphoglyceride (e.g., phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, or phosphatidylinositol), and a neutral sterol (e.g., cholesterol or ergosterol). In this embodiment, the mole percentage of pegylated phospholipids may be 1 to 10% or 3 to 6% of the total lipid; the amount of neutral phosphoglycerides (based on total lipid) may be 20-60%, or 30-50%, or 33-43%; and the molar ratio of the neutral sterol can be 35-75%, or 45-65%, or 50-60%.

In a specific embodiment, the liposome is formed from a mixture of DSPE-PEG (2000), HSPC and cholesterol. In this embodiment, the mole percent of DSPE-PEG (2000) is about 5.3%, the mole percent of HSPC is about 56.3%, and the mole percent of cholesterol is about 38.4%.

As an alternative to pegylation, lipids can be modified by covalently attaching a moiety other than PEG. For example, in some embodiments, the lipid may comprise polyphosphazene. In some embodiments, the lipid may comprise poly (vinyl pyrrolidone). In some embodiments, the lipid may comprise poly (acrylamide). In some embodiments, the lipid may comprise poly (2-methyl-2-oxazoline). In some embodiments, the lipid may comprise poly (2-ethyl-2-oxazoline). In some embodiments, the lipid may comprise a phosphatidylpolyglycerol. In some embodiments, the lipid may comprise poly [ N- (2-hydroxypropyl) methacrylamide ]. In some embodiments, the lipid may comprise a polyalkylene ether polymer other than PEG.

The homing and distribution of intravenously injected liposomes depends on their physical properties, such as size, fluidity and surface charge. They may last hours or days in tissue, depending on their composition, and have a half-life in blood of minutes to hours. Liposomes are generally divided into three groups: multilamellar vesicles (MLVs); small Unilamellar Vesicles (SUVs); and Large Unilamellar Vesicles (LUVs). MLVs have multiple bilayers in each vesicle, forming several separate aqueous compartments. SUVs and LUVs have a single bilayer encapsulating an aqueous core. MLVs typically have a diameter of 0.5 to 4 μm. Sonication of MLVs results in the formation of Large Unilamellar Vesicles (LUVs) with diameters in the range of 50-500 nm, or with diameters less than 50nm, typically in the range of 50nmSmall Unilamellar Vesicles (SUV) within the scope, comprising an aqueous solution in the core.

Larger liposomes such as MLV and LUV can be rapidly taken up by phagocytes of the reticuloendothelial system, but the physiology of the circulatory system limits the excretion of this large species at most sites. They can only be drained where large openings or pores exist in the capillary endothelium, such as the sinuses of the liver or spleen. Thus, these organs are the primary uptake sites. SUVs, on the other hand, exhibit a more extensive tissue distribution, but are still highly sequestered in the liver and spleen. In general, this in vivo behavior limits the potential targeting of liposomes to organs and tissues that allow their large size to enter and exit. These organs and tissues include blood, liver, spleen, bone marrow, and lymphoid organs.

The liposomes of the present application are preferably SUV having a diameter in the range of 60 to 180nm, 80 to 160nm, or 90 to 120 nm. The liposomes of the present application can be part of a liposome preparation comprising a plurality of liposomes, wherein the liposomes in the plurality of liposomes can have a range of diameters. In some embodiments, the liposome preparation comprises at least 80%, at least 90% or at least 95% of liposomes having an average diameter in the range of 60-180 nm, 80-160 nm, 90-120 nm. Also, the diameters of the various liposomes can have a polydispersity index of <0.2, <0.1, or < 0.05. In some embodiments, the mean diameter of the liposomes is determined using the malvern zetasizer method.

One method of increasing liposome circulation time is to use liposomes derivatized with hydrophilic polymer chains or polyalkyl ethers such as polyethylene glycol (PEG) (see, e.g., U.S. Pat. nos. 5,013,556, 5,213,804, 5,225,212, and 5,395,619). The polymer coating reduces the rate of uptake of the liposomes by macrophages, thereby prolonging the presence of the liposomes in the bloodstream. This can also be used as a mechanism for the prolonged release of liposome-borne drugs. Thus, the liposomal echinomycin formulation according to the present application preferably comprises one or more pegylated lipids.

One skilled in the art can select vesicle-forming lipids that are fluid or rigid to a specified degree. The fluidity or rigidity of liposomes can be used to control factors such as the stability of the liposomes in serum or the release rate of the encapsulating agent in the liposomes. Liposomes with more rigid lipid bilayers or liquid crystal bilayers are achieved by incorporating relatively rigid lipids. The rigidity of the lipid bilayer is related to the phase transition temperature of the lipids present in the bilayer. The phase transition temperature is the temperature at which the lipid changes physical state and changes from an ordered gel phase to a disordered liquid crystal phase. There are several factors that influence the phase transition temperature of lipids, including the hydrocarbon chain length and unsaturation of the lipid, charge, and head group species. Lipids with relatively high phase transition temperatures will produce more rigid bilayers. Other lipid components such as cholesterol are also known to contribute to membrane rigidity in lipid bilayer structures. Cholesterol can be used to manipulate the fluidity, elasticity and permeability of lipid bilayers. It is thought to act by filling the voids in the lipid bilayer. In contrast, lipid fluidity is achieved by incorporating a relatively more fluid lipid, which is typically a lipid with a lower phase transition temperature. The phase transition temperatures of many Lipids are listed in various sources such as the Avanti Polar Lipids catalog and lipidate by Martin Caffrey, CRCPress.

Depending on the molar ratio of lipid to water, phospholipids can form a variety of structures other than liposomes when dispersed in water. In low ratios, liposomes are the preferred structure. The physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can exhibit low permeability to ionic and polar substances, but undergo a phase transition at high temperatures that significantly changes their permeability. Phase transition involves a transition from a tightly packed ordered structure (known as the gel state) to a loosely packed less ordered structure (known as the fluid). This occurs at the characteristic phase transition temperature and results in increased permeability to ions, sugars and drugs.

Liposomes of the present application can be prepared to have a substantially uniform size within a selected size range. One effective size control method for REV and MLV involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes having a selected uniform pore size of 0.03 to 0.2 μm, typically 0.05, 0.08, 0.1 or 0.2 μm. The pore size of the membrane corresponds approximately to the maximum size of the liposomes produced by extrusion of the membrane, particularly when the formulation is extruded two or more times through the same membrane. Homogenization methods can also be used to reduce liposome size to sizes of 100nm or less (Martin, F.J., in Specialized Drug Delivery Systems-Manufacturing and Production Technology, (P.Tyle eds.) Marcel Dekker, New York, p.267 and 316 (1990)). Homogenization relies on shear energy to separate large liposomes into smaller liposomes. Other suitable methods of reducing liposome size include reducing liposome size by vigorously agitating the liposomes in the presence of a suitable solubilizing detergent, such as deoxycholate.

Liposomes that have been controlled to a size in the range of about 0.2-0.4 μm can be sterilized by high throughput-based filtration of the liposomes through conventional sterile filters, typically 0.22 μm filters. Other suitable sterilization methods will be apparent to those skilled in the art.

Non-toxicity of lipids is also an important consideration in this application. Lipids approved for clinical use are well known to those skilled in the art. In certain embodiments, for example, synthetic lipids are preferred over lipids derived from biological sources because the risk of contamination by viruses or proteins from biological sources is reduced.

The original method of forming liposomes involves first suspending phospholipids in an organic solvent and then evaporating to dryness until a dry lipid cake or membrane is formed. An appropriate amount of aqueous medium is added and the lipids spontaneously form multilamellar concentric bilayer vesicles (also known as multilamellar vesicles (MLVs)). These MLVs can be dispersed and reduced in size by mechanical means.

Although echinomycin is water insoluble, the inventors of the present application have found that stable liposomes can be formed by combining echinomycin and a lipid in a polar solvent such as ethanol, drying these components to form a film and then dispersing the liposomes in an aqueous medium. Thus, in one embodiment, after echinomycin and lipids are thoroughly mixed in an organic solvent, the solvent is removed using, for example, a rotary evaporator, thereby obtaining a dried lipid film. The dried lipid membrane is hydrated and dissolved in an appropriate buffer (e.g., PBS, ph7.4), thereby obtaining a lipid suspension. The lipid suspension was then repeatedly extruded through a polycarbonate filter using an Avanti micro-Extruder (AvantiMini-Extruder) to obtain liposomes with the desired size range. The liposomes were then sterilized by filtration (0.45 μm or 0.2 μm sterile filter). The water-soluble echinomycin analogs can be passively encapsulated by hydrating the lipid film with an aqueous solution containing the water-soluble echinomycin analogs.

Echinomycin can be positioned in the liposome bilayer, between two leaflets of the liposome bilayer, within the interior core space, on either side of the bilayer, within or on the PEG portion of the liposome, or a combination thereof. Another method for forming Large Unilamellar Vesicles (LUVs) is reverse phase evaporation, described, for example, in U.S. patent No.4,235,871. This method produces reverse phase evaporation vesicles (REV), which are mostly unilamellar, but usually also contain some oligo-lamellar vesicles. In this process, a mixture of polar lipids in an organic solvent is mixed with a suitable aqueous medium. A homogeneous water-in-oil emulsion is formed and the organic solvent is evaporated until a gel is formed. The gel is then converted to a suspension by dispersing the gelatinous mixture in an aqueous medium.

In an alternative embodiment, echinomycin or echinomycin analogs can be coupled to the surface of the liposome bilayer. In one embodiment, echinomycin is covalently attached to the liposome by amide coupling. For example, a phospholipid having a hydroxyl functional group may be coupled to one of the amino groups present in echinomycin or one of its analogues.

The liposome formulation according to the present invention will have sufficient long term stability to achieve a shelf life of at least 3 months, at least 6 months, at least 12 months, at least 24 months or at least 48 months at room or refrigerated temperature (e.g. 4 ℃).

In some alternative embodiments, echinomycin or echinomycin analogs can be encapsulated in a protective wall material that is polymer based in nature rather than lipid based. The polymers used to encapsulate the bioactive agent are typically single copolymers or homopolymers. The polymeric drug delivery system may be microemulsion or non-microemulsion in nature.

The microemulsion polymer encapsulation structure comprises micro-particles, micro-capsules, micro-spheres, nano-particles, nano-capsules, nano-spheres, block copolymer micelles and the like. Both synthetic polymers, which are artificial, and biopolymers, including proteins and polysaccharides, can be used in the present invention. The polymeric drug delivery system may be composed of biodegradable or non-biodegradable polymeric materials or any combination thereof.

As used herein, "microemulsion" refers to an emulsion comprising microspheres having a regular or semi-regular shape and having a diameter of about 10nm to 500 μm. In some embodiments, the microemulsions of the present application comprise liposomes having a diameter of 20 to 400nm, 30 to 300nm, 50 to 200nm, 60 to 150nm or 80 to 120 nm.

In some embodiments, the microemulsions of the present application comprise micelles having a shell composed of a monolayer of amphiphilic molecules. The core of the micelle creates a hydrophobic microenvironment for the non-polar drug, while the hydrophilic shell provides a stable interface between the micelle core and the aqueous medium. The nature of the hydrophilic shell can be tuned to maximize biocompatibility and avoid reticuloendothelial system uptake and renal filtration. The size of the micelles is typically between 10nm and 100 nm.

The present invention also encompasses non-microemulsion polymeric drug delivery systems including membrane, hydrogel, and "depot" type drug delivery systems. Such non-microemulsion polymer systems may also be used in conjunction with parenteral injection in the present invention, particularly when the non-microemulsion drug delivery system is placed proximal to a targeted cancerous tissue. As used herein, "hydrogel" refers to a solution of a polymer (sometimes referred to as a sol) that is converted to the gel state by small ions or polymers of opposite charge or by chemical crosslinking. "polymeric film" refers to a polymeric base film, typically having a thickness of about 0.5 to 5mm, which is sometimes used as a coating.

In certain embodiments, liposomes, microparticles, nanoparticles, microcapsules, block copolymer micelles, or other polymeric drug delivery vehicles comprising echinomycin or echinomycin analogs can be coated, coupled, or modified with cell-specific targeting ligands. By linking the delivery vehicle to the cell-targeting ligand, echinomycin delivery can be directed to the target cell population bound to the cell-targeting ligand or targeting ligand. As used herein, "targeting ligand" includes any ligand that results in association of the liposome with the target cell type to a greater extent than non-targeted tissue.

Targeting ligands such as antibodies or antibody fragments can be used to bind to the liposome surface and direct the antibody and its drug contents to specific antigenic receptors located on the surface of specific cell types (see, e.g., Mastrobattista et al, 1999). Carbohydrate determinants (glycoproteins, lectins, and glycolipid cell surface components that play a role in cell-to-cell recognition, interaction, and adhesion) can also be used as targeting ligands, as they have the potential to direct liposomes to specific cell types. Certain proteins may be used as targeting ligands, typically those recognized by autologous surface receptors of the targeted tissue. For example, ligands that bind to cell surface receptors that are overexpressed in specific cancer cells can be used to increase uptake of liposomes by the target tissue. In certain embodiments, endocytosed cell surface receptors are preferred. When combined with pegylated liposomes, the targeting ligand is typically attached to the end of a hydrophilic polymer that is exposed to an aqueous medium. Alternatively, the liposome may incorporate a fusion protein, such as a fusogenic protein derived from a virus, which induces fusion of the liposome to the cell membrane.

In certain embodiments, the targeting ligand is a cell surface receptor that is endocytosed by the target cell. Suitable targeting ligands for use in the present application include any ligand that results in increased (relative to non-target cells) binding or association of the liposome to the cell surface of the target cell. The targeting ligand may be a small molecule, peptide, ligand, antibody fragment, aptamer, or synthetic antibody (synbody). Synthetic antibodies are synthetic antibodies generated from a library consisting of random peptide strings screened for binding to a target protein of interest and are described in US 2011/0143953. Aptamers are nucleic acid forms of antibodies comprising a class of oligonucleotides that can form specific three-dimensional structures that exhibit high affinity binding to a variety of cell surface molecules/proteins and/or macromolecular structures. Exemplary cell targeting ligands include, but are not limited to, small molecules (e.g., folate, adenosine, purines) and large molecules (e.g., peptides or antibodies) that bind to (and target) e.g., epidermal dendritic cells as described further below.

Exemplary antibodies or antibody-derived fragments can include any member of the group consisting of: IgG, antibody variable region; an isolated CDR region; a single chain Fv molecule (scFv) comprising VH and VL domains connected by a peptide linker that allows association between the two domains to form an antigen binding site; a bispecific scFv dimer; a minibody comprising an scFv linked to a CH3 domain; a diabody (dAb) fragment; a single chain dAb fragment consisting of a VH or VL domain; a Fab fragment consisting of the VL, VH, CL and CH1 domains; fab' fragments which differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region; fab '-SH fragments, Fab' fragments in which the cysteine residues of the constant domains carry a free thiol group; f (ab')₂A bivalent fragment comprising two linked Fab fragments; an Fd fragment consisting of the VH and CH1 domains; derivatives thereof; and any other antibody fragment that retains antigen binding function. Fv, scFv or diabody molecules can be stabilized by incorporating a disulfide bond that links the VH and VL domains. When antibody-derived fragments are used, any or all of the targeting domains and/or Fc regions therein may be "humanized" using methods well known to those skilled in the art. In some embodiments, the antibody may be modified to remove the Fc region.

Antibody-echinomycin drug conjugates

On the other hand, echinomycin or one of its analogues can be conjugated to a cell binding agent or an antibody, such as the anti-cancer antibodies described above. As used herein, the phrase "antibody conjugate" or "Antibody Drug Conjugate (ADC)" refers to an antibody conjugated to echinomycin or an analog thereof via a linker or bifunctional crosslinker. The high specificity of monoclonal antibodies to tumor associated antigens is combined with the pharmacological potency of echinomycin or analogs thereof using echinomycin/echinomycin analog-antibody conjugates. Examples of ADCs include gemtuzumab ozogamicin (gemtuzumab ozogamicin) (Mylotarg; anti-CD 33 monoclonal antibody conjugated to calicheamicin (calicheamicin), Pfizer/Wyeth); butoximab vunditin (brentuximab vedotin) (SGN-35, Adcetris, CD30 targeting ADC consisting of butoximab covalently linked to MMAE (monomethylorlistatin), Seattle Genetics); and trastuzumab (trastuzumab) -DM1 conjugate (T-DM 1).

Antibody-drug conjugates have been prepared using a wide range of linker techniques, as described in U.S. patent 9,090,629. Any of the methods and reagents described herein can be used to prepare the antibody-echinomycin conjugates of the present application. As used herein, "bifunctional crosslinker" refers to an agent having two reactive groups; one of which is capable of reacting with a cell-binding agent or antibody and the other of which is capable of reacting with echinomycin, thereby linking the cell-binding agent or antibody to echinomycin, thereby forming a conjugate.

Preferably, the linker molecule binds echinomycin and/or an echinomycin analog to a cell-binding agent or antibody via a chemical bond (as described above) such that the echinomycin and the cell-binding agent are chemically coupled (e.g., covalently bound) to each other.

The linker may be a "cleavable" linker or a "non-cleavable" linker. The cleavable linker may be designed to release the drug when subjected to certain environmental factors, such as when internalized into a target cell. Cleavable linkers include acid labile linkers, protease sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers. The non-cleavable linker tends to remain covalently linked to at least one amino acid of the antibody and drug upon internalization and degradation in the target cell.

In one embodiment, the bifunctional crosslinking agent comprises a non-cleavable linker. The non-cleavable linker is any chemical moiety capable of linking echinomycin to a cell binding agent or antibody in a stable covalent manner. Thus, the non-cleavable linker is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions in which the cytotoxic agent or cell-binding agent remains active.

Suitable crosslinkers that form a non-cleavable linker between echinomycin and a cell binding agent or antibody are well known in the art, in one embodiment, echinomycin and/or echinomycin analogs are linked to a cell binding agent or antibody via a thioether bond, examples of non-cleavable linkers include linkers having a maleimide or haloacyl moiety for reaction with a cytotoxic agent, such bifunctional crosslinkers are well known in the art (see, e.g., U.S. patent application publication No. 2010/0129314), and those bifunctional crosslinkers available from Pierce Biotechnology Inc (Rockland, Ill) including, but not limited to, N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-aminocaproate) which is the "long chain" analog of SMCC (LC-SMCC), N-maleimidoundecanoate (KMUA), gamma-maleimidobutyrate-N-succinimidyl butyrate (GMBS), delta-maleimide-bis-succinimidyl-4- (mpcn-butyl-ethyl-sulfonyl), maleimide-1-carboxy- (lmono-ethyl-4- (bmpc-butyl-ethyl-maleimide), maleimide-4- (bmpc-butyl-ethyl-4- (spc), bis-butyl-maleimide-ethyl-4- (spc-butyl-ethyl-4-maleimide-ethyl-4-maleimide-4- (spc), bis-butyl-maleimide-4-maleimide-butyl-4- (spc-4-ethyl-4-maleimide-ethyl-4-maleimide-butyl-4- (spc-4-ethyl-maleimide-ethyl-4-maleimide-bis-maleimide-bis-ethyl-4-butyl-4- (spc-4-ethyl-bis-maleimide-4-bis-butyl-4-maleimide-bis-maleimide-ethyl-4- (sp-bis-butyl-maleimide-ethyl-bis-4-bis-maleimide-ethyl-4-bis-succinimide-bis-succinimide-bis-succinimide-bis-ethyl-succinimide-bis-succinimide-4- (spc-ethyl-bis-maleimide-4- (spc-bis-maleimide-bis-maleimide-bis-succinimide-bis-maleimide-succinimide-4- (spc-4-bis-maleimide-bis-succinimide-bis-succinimide-maleimide (dmpc-maleimide-succinimide-bis-succinimide-bis-succinimide-bis-succinimide-4- (spc-4-bis-succinimide-.

The antibody solution in aqueous buffer can be incubated with a molar excess of an antibody modifying agent, such as N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC), to introduce maleimide groups, or with N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB) to introduce iodoacetyl groups. The modified antibody is then reacted with a thiol-containing echinomycin derivative to produce a thioether-linked antibody-echinomycin conjugate. The antibody-cytotoxic conjugate is then purified by gel filtration or other methods described above or by methods known to those skilled in the art. Other cross-linking agents that introduce maleimide groups or haloacetyl groups onto cell-binding agents are well known in the art and include the above-described linkers.

The antibody conjugate may comprise an average of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 echinomycin molecules (and/or analogues)/antibody.

In particular, HIF-1 appears to mediate resistance to imatinib by metabolic reprogramming, by activating transketolase expression and thereby increasing glucose flux of the non-oxidizing arm via the pentose phosphate pathway, transition from oxidative metabolism to reductive metabolism has the effect of reducing cellular ROS levels, which may increase resistance to cytotoxic chemotherapy (Semenza, HIF).

In certain embodiments, echinomycin or an analog thereof can be administered in synergistic combination with one or more other chemotherapeutic or anti-cancer agents.

As used herein, the phrase "anti-cancer agent" refers to a "small molecule drug" or protein or antibody that can reduce the growth rate of cancer cells or induce or mediate death (e.g., necrosis or apoptosis) of cancer cells in a subject (e.g., a human). The phrase "small molecule drug" refers to a molecular entity, typically organic or organometallic (rather than a polymer), that is pharmaceutically active and has a molecular weight of less than about 2kDa, less than about 1kDa, less than about 900Da, less than about 800Da, or less than about 700 Da. In addition to proteins or nucleic acids, the term also includes most pharmaceutical compounds referred to as "drugs," although small peptides or nucleic acid analogs may be considered small molecule drugs. Examples include chemotherapeutic anti-cancer drugs and enzyme inhibitors. Small molecule drugs can be obtained synthetically, semisynthetically (i.e., from naturally occurring precursors), or by biological methods.

The anti-cancer agent may be an alkylating agent; anthracycline antibiotics; an antimetabolite; an antidote; an interferon; polyclonal or monoclonal antibodies; an EGFR inhibitor; a HER2 inhibitor; (ii) a histone deacetylase inhibitor; a hormone or anti-hormone agent; a mitotic inhibitor; phosphatidylinositol-3-kinase (PI3K) inhibitors; an Akt inhibitor; mammalian target of rapamycin (mTOR) inhibitors; a proteasome inhibitor; poly (ADP-ribose) polymerase (PARP) inhibitors; Ras/MAPK pathway inhibitors; a centerbody declustering agent; (ii) a multi-kinase inhibitor; serine/threonine kinase inhibitors; tyrosine kinase inhibitors; VEGF/VEGFR inhibitors; a taxane or taxane derivative, an aromatase inhibitor, an anthracycline, a microtubule-targeting drug, a topoisomerase poison drug, a molecular target or enzyme (e.g., a kinase or protein methyltransferase) inhibitor, a cytidine analog, or a combination thereof.

Exemplary alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfex (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); mechlorodiethylamine (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin (Zanosar).

Exemplary anthracycline antibiotics include, but are not limited to, doxorubicin (adriamycin); liposomal doxorubicin (Doxil); mitoxantrone (Novantrone); bleomycin (Blenoxane); daunorubicin (Cerubidine); daunorubicin liposomes (daunoxomes); actinomycin (Cosmegen); epirubicin (elence); idarubicin (idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin (Nipent); or valrubicin (valrubicin) (Valstar).

Exemplary antimetabolites include, but are not limited to, fluorouracil (Adrucil); capecitabine (hiloda); hydroxyurea (hydra); mercaptopurine (purinols); pemetrexed (Alimta); fludarabine (Fludara); nelarabine (araron); cladribine (cladripine Novaplus); clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomes (DepoCyt); hydroxyurea (drosia); pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall); thioguanine (tabloid); TS-1 or cytarabine (Tarabine PFS).

Exemplary antidotes include, but are not limited to, amifostine (ethyl) or mesna (Mesnex).

Exemplary interferons include, but are not limited to, interferon α -2b (Intron A) or interferon α -2a (Roferon-A).

Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab (Vectibix); tositumomab/iodine 131 tositumomab (Bexxar); alemtuzumab (Campath); ibritumoman (Zevalin; In-111; Y-90 Zevalin); gemumab (Mylotarg); eculizumab (Soliris) and ocdenomab (odenosumab).

Exemplary EGFR inhibitors include, but are not limited to, gefitinib (iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab (Emd7200) or EKB-569.

Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.

Exemplary histone deacetylase inhibitors include, but are not limited to, vorinostat (zoinuza), valproic acid, romidepsin, entinostat, abensistat, gifenostat, and moxidectin.

Exemplary hormonal or anti-hormonal agents include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol (Megace); leuprorelin (Lupron; Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoromethyltestosterone (Androxy; Halotestin); megestrol (medroxyprogesterone) (Provera; Depo-Provera); abiraterone acetate (Zytiga); leuprorelin (Lupron); estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron); alburex (Plenaxis); or testolactone (Teslac).

Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onoxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole; an epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amsacrine or lamellarin (lamellarin) D (LAM-D).

Exemplary phosphatidylinositol-3 kinase (PI3K) inhibitors include wortmannin, a reversible inhibitor of wortmannin derivatives demethoxypyridine LY294002, PI3K as irreversible inhibitors of PI3K, BKM120(Buparlisib), Idelalisib (idellalisib) (PI3K delta inhibitor), duflurisib (IPI-145, an inhibitor of PI3K delta and gamma), apelixb (BYL719), a α specific PI3K inhibitor, TGR 1202 (formerly RP5264), an oral PI3K delta inhibitor, and a gulpan lixib (BAY 80-6946), inhibitors of PI3K α, delta predominates.

Exemplary Akt inhibitors include, but are not limited to, miltefosine, AZD5363, GDC-0068, MK2206, piperacillin, RX-0201, PBI-05204, GSK2141795, and SR 13668.

Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torsiel); lapamonie (rapamune), delevous (ridaforolimus); deformous (DEFORMOLIMUS) (AP23573), AZD8055(AstraZeneca), OSI-027(OSI), INK-128, BEZ235, PI-103, Torin1, PP242, PP30, Ku-0063794, WAY-600, WYE-687, WYE-354, and CC-223.

Exemplary proteasome inhibitors include, but are not limited to, bortezomib (PS-341), ixazoib (MLN 2238), MLN 9708, delanzomib (CEP-18770), carfilzomib (PR-171), YU101, olozomib (oprozomib) (ONX-0912), marizomib (marizomib) (NPI-0052), and disufirum (disifiram).

Exemplary PARP inhibitors include, but are not limited to Olaparib, Iniparib, verapamil (velaparib), BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338, and combinations thereof.

Exemplary Ras/MAPK pathway inhibitors include, but are not limited to, trametinib (trametinib), semetinib (selmetiniib), cubimetinib (cobimetinib), CI-1040, PD0325901, AS703026, RO4987655, RO5068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162, and GDC-0973.

Exemplary centrosome declustering agents include, but are not limited to, griseofulvin; noscapine, noscapine derivatives, such as noscapine bromide (e.g., 9-bromobenzlicapine), Reduced Bromoscastine (RBN), N- (3-bromobenzyl) noscapine, sulfamethoxine and its water-soluble derivatives; CW 069; phenanthrene-derived poly (ADP-ribose) polymerase inhibitors, PJ-34; n2- (3-pyridylmethyl) -5-nitro-2-furoamide, N2- (2-thienylmethyl) -5-nitro-2-furoamide and N2-benzyl-5-nitro-2-furoamide.

Exemplary multi-kinase inhibitors include, but are not limited to regorafenib; sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; e7080; ZD 6474; PKC-412; motesanib; or AP 245634.

Exemplary serine/threonine kinase inhibitors include, but are not limited to: lubostatine (ruboxistaurin); ericel/isoidil hydrochloride (eril/easodil hydrochloride); flavonoid antineoplastic agents; celecoxib (seliciclib) (CYC 202; roscovitine); SNS-032 (BMS-387032); PKC 412; bryostatins; KAI-9803; SF 1126; VX-680; AZD 1152; arry-142886 (AZD-6244); SCIO-469; GW 681323; CC-401; CEP-1347 or PD 332991.

Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath); gemumab (Mylotarg); temsirolimus (temsirolimus) (Torisel); pazopanib (Votrient); dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (vatalanib) (Ptk 787; ZK 222584); CEP-701; SU 5614; MLN 518; XL 999; VX-322; azd 0530; BMS-354825; SKI-606 CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG 888.

Exemplary VEGF/VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab; pegaptanib; or vandetanib (vandetanib).

Exemplary microtubule-targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilones, and navelbine.

Exemplary topoisomerase poison drugs include, but are not limited to, teniposide, etoposide, doxorubicin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxel.

Exemplary general chemotherapeutic agents, antineoplastic agents, antiproliferative agents include, but are not limited to, altretamine (Hexalen); isotretinoin (Accutane; Amnesterem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine (matrilone); asparase (Oncaspar); denileukin bifertitox (Ontak); porfield (Photofrin); aldesleukin (Proleukin); lenalidomide (revlimd); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (temsirolimus) (Torsiel); arsenic trioxide (Trisenox); verteporfin (Visudyne); and imidazole-containing (Leucenol).

In some embodiments, echinomycin or echinomycin analogs are administered in synergistic combination with one or more SOC MTX/calcineurin inhibitors to treat GVHD.

Additional such chemotherapeutic agents may be loaded into the liposomes with echinomycin or an analog thereof, may be in the form of a separate liposome formulation that is co-administered with the liposome formulations of the present application, or administered by other means (e.g., oral administration, intravenous injection, etc.).

A pharmaceutical formulation. Pharmaceutical compositions of the invention comprising echinomycin or an echinomycin analogue and a microemulsion drug delivery vehicle (carrier) such as a liposome are prepared according to standard techniques. They may further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" refers to a molecular entity or composition that does not produce an adverse reaction, allergic reaction, or other untoward reaction when properly administered to an animal or human. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and/or antifungal agents, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, and the like that can serve as a medium for a pharmaceutically acceptable substance.

Exemplary carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol. Exemplary pharmaceutically acceptable carriers include one or more of water, saline, isotonic aqueous solution, phosphate buffered saline, dextrose, 0.3% glycine aqueous solution, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride, or glycoproteins for enhanced stability, such as albumin, lipoproteins and globulins, in the composition. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the useful life or effectiveness of the therapeutic agent.

These compositions can be sterilized by conventional sterilization techniques well known to those skilled in the art. Sufficiently small liposomes can be sterilized, for example, using sterile filtration techniques.

Formulation characteristics that may be varied include, for example, pH and osmotic pressure. For example, it may be desirable to obtain a formulation with a pH and osmotic pressure similar to human blood or tissue to promote the effectiveness of the formulation when administered parenterally. Alternatively, alternative characteristics may be altered in order to facilitate effectiveness of the disclosed compositions when administered via other routes of administration.

Buffers are useful in the present invention for, among other purposes, controlling the overall pH of a pharmaceutical formulation (particularly where it is desired to use for parenteral administration). Various buffering agents known in the art may be used in the formulations of the present invention, such as various salts of organic or inorganic acids, bases or amino acids, and include various forms of citrate, phosphate, tartrate, succinate, adipate, maleate, lactate, acetate, bicarbonate or carbonate ions. Particularly advantageous buffering agents for use in the parenteral administration forms of the compositions presently disclosed in this invention include sodium or potassium buffering agents, including sodium phosphate, potassium phosphate, sodium succinate, and sodium citrate.

Sodium chloride can be used to modify the toxicity of solutions at concentrations of 0 to 300mM (150 mM being the best for liquid dosage forms). For the lyophilized formulation, a cryoprotectant, mainly 0-10% (preferably 0.5-1.0%) sucrose, may be included. Other suitable cryoprotectants include trehalose and lactose. For the lyophilized formulation, a bulking agent, mainly 1-10% (preferably 2-4%) mannitol may be included. The stabilizer can be used in liquid and freeze-dried dosage forms, and is mainly 1-50 mM (preferably 5-10 mM) L-methionine. Other suitable bulking agents include glycine, arginine, and can be included as 0-0.05% (preferably 0.005-0.01%) polysorbate-80.

In one embodiment, sodium phosphate is used at a concentration of about 20mM to achieve a pH of about 7.0. A particularly effective sodium phosphate buffer system comprises sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate heptahydrate. When such a combination of sodium dihydrogen phosphate and disodium hydrogen phosphate is used, advantageous concentrations of each are about 0.5 to about 1.5mg/ml monobasic base and about 2.0 to about 4.0mg/ml dibasic base, with a preferred concentration of about 0.9mg/ml monobasic base and about 3.4 mg/ml dibasic phosphate. The pH of the formulation varies depending on the amount of buffer used.

Depending on the dosage form and intended route of administration, it may be advantageous to adjust the pH of the composition to encompass other ranges instead using different concentrations of buffer or using other additives. Useful pH ranges for the compositions of the present invention include a pH of about 2.0 to a pH of about 12.0.

In some embodiments, it is also advantageous to use surfactants in the presently disclosed formulations, wherein these surfactants do not disrupt the drug delivery system used. Surfactants or anti-adsorbents which have proven useful include polyoxyethylene sorbitan, polyoxyethylene sorbitan monolaurate, polysorbate-20, e.g., Tween-20^TMPolysorbate-80, polysorbate-20, hydroxycellulose, genapol, and BRIJ surfactants. For example, when any surfactant is used in the present invention to produce a composition for parenteral administration, it is advantageous to use it at a concentration of about 0.01 to about 0.5 mg/ml.

Other useful additives can be readily determined by those skilled in the art, depending on the particular needs or intended use of the composition and formulator. One such particularly useful additional substance is sodium chloride, which can be used to adjust the osmotic pressure of the formulation to achieve the desired resulting osmotic pressure. A particularly preferred osmolality range for parenteral administration of the disclosed compositions is from about 270 to about 330 mOsm/kg. The optimum osmolarity of compositions for parenteral administration, particularly injectable compositions, is about 3000sm/kg and can be achieved by using sodium chloride at a concentration of about 6.5 to about 7.5mg/ml, with a sodium chloride concentration of about 7.0mg/ml being particularly effective.

The echinomycin-containing liposome or echinomycin-containing microemulsion drug delivery vehicle can be stored under sterile conditions in the form of a lyophilized powder and combined with a sterile aqueous solution prior to administration. As mentioned above, the aqueous solution used for resuspending the liposomes may contain pharmaceutically acceptable auxiliary substances as necessary to approximate physical conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and the like.

In other embodiments, echinomycin-containing liposomes or microemulsions drug delivery vehicles containing echinomycin can be stored as a suspension, preferably as an aqueous suspension, prior to administration in some embodiments, the solution used to store the liposome or microemulsion drug carrier suspension will include a lipid protecting agent that protects the lipids from free radical and lipid peroxidation damage upon storage.

As described further below, echinomycin/echinomycin analog doses can be tested in a suitable animal model. As a general recommendation, a therapeutically effective amount of echinomycin, echinomycin analogs, or other anti-cancer agents will be administered in the range of about 10ng/kg body weight/day to about 100mg/kg body weight/day, whether by one or multiple administrations. In a specific embodiment, each fusion protein or expression vector is administered in the following dosage ranges: about 10ng/kg body weight/day to about 10mg/kg body weight/day, about 10ng/kg body weight/day to about 1mg/kg body weight/day, about 10ng/kg body weight/day to about 100 μ g/kg body weight/day, about 10ng/kg body weight/day to about 10 μ g/kg body weight/day, about 10ng/kg body weight/day to about 1 μ g/kg body weight/day, 10ng/kg body weight/day to about 100ng/kg body weight/day, about 100ng/kg body weight/day to about 100mgkg body weight/day, about 100ng/kg body weight/day to about 10mg/kg body weight/day, about 100ng/kg body weight/day to about 1mg/kg body weight/day, about 100ngkg body weight/day to about 100 μ g/kg body weight/day, About 100ng/kg body weight/day to about 10 μ g/kg body weight/day, about 100ng/kg body weight/day to about 1 μ g/kg body weight/day, about 1 μ g/kg body weight/day to about 100mg/kg body weight/day, about 1 μ g/kg body weight/day to about 10mg/kg body weight/day, about 1 μ g/kg body weight/day to about 1mg/kg body weight/day, about 1 μ g/kg body weight/day to about 100 μ g/kg body weight/day, about 1 μ g/kg body weight/day to about 10 μ g/kg body weight/day, about 10 μ g/kg body weight/day to about 100mg/kg body weight/day, about 10 μ g/kg body weight/day to about 10mg/kg body weight/day, or, About 10 μ g/kg body weight/day to about 1mg/kg body weight/day, about 10 μ g/kg body weight/day to about 100 μ g/kg body weight/day, about 100 μ g/kg body weight/day to about 100mg/kg body weight/day, about 100 μ g/kg body weight/day to about 10mg/kg body weight/day, about 100 μ g/kg body weight/day to about 1mg/kg body weight/day, about 1mg/kg body weight/day to about 100mg/kg body weight/day, about 1mg/kg body weight/day to about 10mg/kg body weight/day, about 10mg/kg body weight/day to about 100mg/kg body weight/day.

In some embodiments, echinomycin is administered in the following dosimeters based on body surface area: 10 to 30,000 μ g/m²、100～30,000μg/m²、500～30,000μg/m²、1000～30,000μg/m²、1500～30,000μg/m²、2000～30,000μg/m²、2500～30,000μg/m²、3000～30,000μg/m²、3500～30,000μg/m²、4000～30,000μg/m²、100～20,000μg/m²、500～20,000μg/m²、1000～20,000μg/m²、1500～20,000μg/m²、2000～20,000μg/m²、2500～20,000μg/m²、3000～20,000μg/m²、3500～20,000μg/m²、100～10,000μg/m²、500～10,000μg/m²、1000～10,000μg/m²、1500～10,000μg/m²、2000～10,000μg/m²Or 2500 to 10,000 μ g/m²。

In other embodiments, echinomycin is administered in the following ranges: about 10ng to about 100ng per single administration, about 10ng to about 1 μ g per single administration, about 10ng to about 10 μ g per single administration, about 10ng to about 100 μ g per single administration, about 10ng to about 1mg per single administration, about 10ng to about 10mg per single administration, about 10ng to about 100mg per single administration, about 10ng to about 1000mg per injection, about 10ng to about 10,000mg per single administration, about 100ng to about 1 μ g per single administration, about 100ng to about 10 μ g per single administration, about 100ng to about 100 μ g per single administration, about 100ng to about 1mg per single administration, about 100ng to about 10mg per single administration, about 100ng to about 100mg per single administration, about 100ng to about 1000mg per injection, about 100ng to about 10,000mg per single administration, about 1 μ g to about 10 μ g per single administration, about 1 μ g to about 100 μ g per administration, About 1 μ g to about 100 μ g per single administration, about 1 μ g to about 1mg per single administration, about 1 μ g to about 10mg per single administration, about 1 μ g to about 100mg per single administration, about 1 μ g to about 1000mg per injection, about 1 μ g to about 10,000mg per single administration, about 10 μ g to about 100 μ g per single administration, about 10 μ g to about 1mg per single administration, about 10 μ g to about 10mg per single administration, about 10 μ g to about 100mg per single administration, about 10 μ g to about 1000mg per injection, about 10 μ g to about 10,000mg per single administration, about 100 μ g to about 1mg per single administration, about 100 μ g to about 10mg per single administration, about 100 μ g to about 100mg per single administration, about 100 μ g to about 1000mg per injection, about 100 μ g to about 10,000mg per injection, About 1mg to about 10mg per single administration, about 1mg to about 100mg per single administration, about 1mg to about 1000mg per injection, about 1mg to about 10mg per administration, about 10mg to about 100mg per single administration, about 10mg to about 1000mg per injection, about 10mg to about 10,000mg per single administration, about 100mg to about 1000mg per injection, about 100mg to about 10,000mg per single administration, and about 1000mg to about 10,000mg per single administration. The fusion protein or expression vector may be administered daily, every 2,3, 4, 5,6, or 7 days, or every 1,2, 3, or 4 weeks.

In other specific embodiments, echinomycin can be administered in the following amounts: about 0.0006 mg/day, 0.001 mg/day, 0.003 mg/day, 0.006 mg/day, 0.01 mg/day, 0.03 mg/day, 0.06 mg/day, 0.1 mg/day, 0.3 mg/day, 0.6 mg/day, 1 mg/day, 3 mg/day, 6 mg/day, 10 mg/day, 30 mg/day, 60 mg/day, 100 mg/day, 300 mg/day, 600 mg/day, 1000 mg/day, 2000 mg/day, 5000 mg/day, or 10,000 mg/day. As expected, the dosage will depend on the condition, size, age and condition of the patient.

The dosage may be tested in several art-accepted animal models suitable for the particular proliferative disorder, autoimmune disease or alloimmune response.

The therapeutic agent in the pharmaceutical composition may be formulated in a "therapeutically effective amount". A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic result at the requisite dosage and for a period of time. A therapeutically effective amount of a liposomal formulation or other microemulsion drug delivery vehicle may vary depending on factors such as: the condition to be treated, the severity and course of the condition, the mode of administration, the bioavailability of the particular agent, the ability of the delivery vehicle to elicit a desired response in the individual, previous therapy, the age, weight and sex of the patient, the clinical history and response to the antibody of the patient, the type of fusion protein or expression vector used, the discretion of the attending physician, and the like. A therapeutically effective amount is also one in which the therapeutically beneficial effect outweighs any toxic or deleterious effect of the delivery vehicle.

Methods of administering echinomycin or echinomycin analogs

In one aspect, the microemulsion drug delivery system of the present invention is used in a method of treating a mammalian subject suffering from a proliferative disorder, an autoimmune disease, or exhibiting an alloimmune response.

In one embodiment, a method of treating and/or reducing the severity of a proliferative disorder in a mammalian subject comprises: administering to the subject a pharmaceutical composition comprising a microemulsion drug delivery vehicle comprising echinomycin or an echinomycin analog in an amount effective to treat and/or reduce the severity of a proliferative disorder in the subject.

In another embodiment, a method of treating and/or reducing the severity of an autoimmune disease in a mammalian subject comprises: administering to the subject a pharmaceutical composition comprising a microemulsion drug delivery vehicle comprising echinomycin or an echinomycin analog in an amount effective to treat and/or reduce the severity of an autoimmune disease in the subject.

In further embodiments, a method of preventing the development of, or reducing the severity of, GvHD in a mammalian subject receiving an allogeneic Hematopoietic Stem Cell (HSC) transplant comprises: administering to the subject, the transplanted HSCs, or both, a pharmaceutical composition combination comprising a microemulsion drug delivery vehicle comprising echinomycin in an amount effective to prevent or reduce the severity of GvHD in the subject.

When echinomycin or echinomycin analogs are encapsulated in liposomes or other microemulsion drug delivery vehicles, any effective amount of echinomycin or echinomycin analogs can be administered. Preferably, the liposomal formulation or other microemulsion drug delivery vehicle containing echinomycin or echinomycin analog is administered by parenteral injection, including intravenous, intraarterial, intramuscular, subcutaneous, intratissue, intranasal, intradermal, instillation, intracerebral, intrarectal, intravaginal, intraperitoneal, intratumoral injection.

Intravenous administration of liposomal echinomycin has been tolerated by mice at a dose of about 1mg/kg body weightAnd does not reach LD₅₀The value is obtained. In contrast, LD of free echinomycin₅₀The value was 0.629 mg/kg.

Other routes of administration include oral, topical (nasal, transdermal, intradermal, or intraocular), mucosal (e.g., intranasal, sublingual, buccal, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intravesical, intrathecal, enteral, intrapulmonary, intralymphatic, intracavity, intraorbital, intracapsular, and transurethral, as well as local delivery through a catheter or stent.

In certain embodiments, the composition may be formulated as a depot preparation. Such long-acting formulations may be administered by implantation at a suitable site or by parenteral injection, particularly intratumoral injection or injection at a site adjacent to cancerous tissue.

The liposomal formulation or other microemulsion delivery vehicle can be lyophilized (preferably under vacuum) and stored as a sterile powder and then reconstituted in bacteriostatic water (containing, e.g., benzyl alcohol preservative) or sterile water prior to injection. The pharmaceutical compositions may be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion).

The delivery vehicle can be administered to the patient at one time or over a series of treatments, and can be administered to the patient at any time from the start of diagnosis. The delivery vehicle may be administered as monotherapy or in combination with other drugs or therapies that may be used to treat the disease in question.

The invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents, and published patent applications, as well as the figures and tables, cited throughout this application are incorporated herein by reference. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples

Example 1 preparation of liposomal echinomycin formulation. The lipid component and echinomycin were weighed out on an analytical balance and dissolved in the appropriate ratio in a glass scintillation vial in an appropriate solvent such as chloroform/methanol (2:1, v/v) and mixed in a vortex mixer. A slow nitrogen flow was used to evaporate the organic solvent and produce a uniform lipid film on the walls of the glass vial. To prevent gelation, the drying process was carried out at 65 ℃. By adding double distilled water (ddH) preheated to 65 deg.C₂O) 10% sucrose (w/v) hydrated the lipid membrane such that the final concentration of total lipid was 7.1 mg/mL. The hydrated mixture was kept above 65 ℃ and vortexed vigorously until all membranes were dissolved. A hydrated solution of Large Multilamellar Vesicles (LMV) was rapidly extruded through 200nm, 100nm and 50nm stacked Polycarbonate (PC) filters using AvantiMini-Extruder in 1mL increments until the average size of the liposomes in the combined post-extrusion mixture was determined by Dynamic Light Scattering (DLS) to be in the 94-99 nm range with a polydispersity index (PdI) of less than 0.05. To prevent membrane fouling, the extrusion process was performed at 65 ℃ with PC membrane changes between each 1mL increment. The minimum number of extrusions per 1mL increment was 21, but if the DLS quality standard measurement did not meet after analysis of the extruded mixture, an additional number was performed using a 50nmPC film. The resulting suspension of Small Unilamellar Vesicles (SUVs) was allowed to stabilize overnight at room temperature (21 ℃). The product was then sterile filtered once through a 33mm diameter 0.22 μmPES membrane (Millipore) to remove unencapsulated echinomycin, and filtrate samples for HPLC and DLS analysis were taken and stored in sterile glass vials at 2-8 ℃ until use.

Preparation of DSPC-echinomycin preparation. To 1.25mL of 0.2mg/mL echinomycin dissolved in chloroform 1.25mL of 13.75mg/mL Distearoylphosphatidylcholine (DSPC) in chloroform was added to the round bottom flask. Then, 1.25mL of 9.8mg/mL cholesterol in chloroform was added and gently mixed by hand rotation for 5 to 10 seconds. Next, chloroform was evaporated under vacuum in a rotary evaporator at maximum rotation speed for 45 minutes until a lipid film containing echinomycin was formed on the wall of the flask and all solvents had completely evaporated. The membrane was rehydrated with 5mL of lipid buffer and vortexed vigorously for 45 minutes to produce a heterogeneous mixture of liposomes. The mixture was then extruded through a 0.2 μm filter and the resulting mixture was analyzed for encapsulation efficiency by HPLC.

1.2. preparation of mPEG-DSPE-DOPC-echinomycin formulation DOPC can be replaced with DOPC to improve encapsulation efficiency, although mPEG-DSPE can be added to reduce clearance of the reticuloendothelial system (RES) and increase in circulation time in vivo in one embodiment, 1.25mL of 0.2mg/mL echinomycin is dissolved in chloroform, 1.25mL of 13.75mg/mL of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in chloroform is added to a round bottom flask, then 1.25mL of 9.8mg/mL of cholesterol dissolved in chloroform and 1.25mL of 1.25mg/mL of poly (ethylene glycol) - α -distearoylphosphatidylethanolamine (mPEG-DSPE) -2000 in chloroform are added and mixed by hand vortexing for 5 to 10 seconds, then chloroform is evaporated at maximum rpm in a rotary evaporator for 45 minutes until a lipid containing mixture is formed on the wall of mPEG-DSPE and the resulting lipid mixture is vortexed again for 5 minutes and the resulting lipid mixture is vortexed and then vortexed for a further 0.5 minutes of heterogeneous lipid mixture is expressed by a 0.5. HPLC filter.

Preparation of mPEG-DSPE-EPC-HEPC-echinomycin preparation. 0.25mg echinomycin was dissolved in 1.25ml chloroform in a round bottom flask, and a solution of 12.2mg/ml Egg Phosphatidylcholine (EPC), 2.28mg/ml Hydrogenated Egg Phosphatidylcholine (HEPC), 2.28mg/ml cholesterol and 5.4mg/ml methoxypolyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE) in 1.25ml chloroform/methanol was added to the echinomycin solution in the round bottom flask. Once the lipids were well mixed in the solvent, the solvent was evaporated under vacuum to remove the solvent and form a lipid film on the walls of the round bottom flask. The membrane was rehydrated with 5mL lipid buffer and vortexed vigorously for 45 minutes to produce a heterogeneous mixture of liposomes. The mixture was then extruded through a 0.2 μm filter and the resulting mixture was analyzed for encapsulation efficiency by HPLC.

Preparation of mPEG-DSPE-HSPC-echinomycin preparation. In another example, a 14mL batch of liposomal echinomycin was prepared as follows: 60mg HSPC, 20mg DSPE-PEG (2000) and 20mg sheep wool cholesterol were dissolved in a 5mL glass scintillation vial containing 4mL chloroform/methanol (2:1 (v/v)). To the mixture was added 0.5mL of 10mg/mL echinomycin in chloroform/methanol (2:1(v/v)), and the mixture was mixed by vortexing for 30 seconds. The mixture was then heated to 65 ℃ in a water bath and the organic solvent was evaporated using a slow nitrogen stream and the vial was rotated by hand until a homogeneous membrane containing echinomycin and lipid was formed on the vial wall. During evaporation of the organic solvent, it is critical to keep the mixture at 65 ℃ to avoid gelation. Alternatively, a round bottom flask can be used in an automatic evaporator system in a heated water bath and rotated; however, for small lab scale production batches, rotating the vial by hand is sufficient to produce a uniform film layer. In a small oven, a sufficient volume of hydration solution was preheated to 65 ℃. In this example, the hydration solution was 10% sucrose (w/v) in distilled deionized water (sucDDW), but it should be noted that other hydration solutions are also acceptable, such as conventional DDW, 0.9% saline in DDW, or 1x PBS. The lipid-echinomycin membranes were then hydrated at 65 ℃ in 14mL sucDDW and mixed vigorously by vortex shaker (vortex) until all membranes were completely dissolved (about 1 hour) to produce a white suspension of Large Multilamellar Vesicles (LMV). The hydration solution of the large lamellar vesicles (LMV) was then rapidly (extensivelyy) extruded through 200nm, 100nm and 50nm stacked Polycarbonate (PC) filters using Avanti Mini-Extruder in 1mL increments until the mean ring power diameter of the liposomes in the combined extruded mixture was in the 94nm to 99nm range as determined by Dynamic Light Scattering (DLS) and the polydispersity index (PdI) was less than 0.05. To prevent membrane fouling, the extrusion process was performed at 65 ℃ and the PC membrane was replaced between each 1mL increment. The minimum number of extrusions per 1mL increment was 21, but additional times were made for 50nm PC film if the DLS quality standard measurement was not achieved after analysis of the post-extrusion mixture. The resulting suspension of Small Unilamellar Vesicles (SUV) was allowed to stabilize at room temperature (21 ℃) for 12 to 15 hours. The product was then sterile filtered once through a 33mm0.22 μm PES membrane (Millipore) to remove unencapsulated echinomycin and other possible contaminants. Samples of the filtrate for HPLC and DLS analysis were taken and stored in sterile glass bottles at 2 to 8 ℃ until use.

Example 2 size distribution and physical characterization of liposomal echinomycin. The liposomal echinomycin formulation was characterized using Malvern Zetasizer software to determine the average size and zeta potential. As shown by the representative Dynamic Light Scattering (DLS) curve in diagram a of fig. 1, the size distribution was found to be always within a very narrow range with a polydispersity index of less than 0.1. It was reported that for liposomes with a liposome size <100nm, liposomes accumulate in tumor tissue due to the enhanced penetration and retention rate (EPR) and the maximal immunoinvasiveness of pegylated sphingomyelinsomes as a result of the rapid uptake by the reticuloendothelial system (RES). The mean hydrodynamic diameter of the liposomal echinomycin was found to be about 98 d.nm; this is easily reproduced between batches (fig. 1, fig. B).

Measuring the zeta potential of a liposomal formulation can provide a reliable method for predicting particle stability and aggregation tendency. The mean zeta potential of liposomal echinomycin was found to be about-30 mV, indicating that the product was stable and unlikely to aggregate (fig. 1, panel B). A summary of other physical properties (including PdI, mean and standard deviation) of 6 independent liposomal echinomycin formulations is shown in table 1. All data were generated using Malvern Zetasizer software.

TABLE 1

Example 3 in vitro release of echinomycin from echinomycin liposomes. Assessment of liposomal echinomycin in vitro drug release was performed by dialysis by measuring the release rate of echinomycin by HPLC over a period of 240 hours at each time point in water at room temperature. Echinomycin concentrations were calculated at each time point according to an echinomycin standard curve. Percent release was calculated as a function of the initial echinomycin concentration detected in the liposomal echinomycin sample prior to starting dialysis. The in vitro release profile of the liposomes is summarized in the cumulative release percentage as shown in panel a of figure 2. A representative HPLC chromatogram showing an echinomycin peak at the corresponding time point is plotted in panel B of fig. 2.

Example 4 in vitro storage and stability of liposomal echinomycin. To test the stability of liposomal echinomycin under storage conditions, the physical properties of liposomal echinomycin were monitored over a 3 month period at 4 ℃. The size, PdI and zeta potential of the liposomal echinomycin were evaluated at 1 and 3 months of storage using Malvern Zetasizer software. None of these parameters was found to vary significantly from the initial measurements (fig. 3, panel a and B). Drug content loss of liposomal echinomycin was tested by removing accumulated drug precipitate by 0.22 μm PES membrane filtration at various time points of 1 and 3 months. In HPLC analysis of the filtrate, no evidence of any drug leakage or precipitation from liposomes was found under storage conditions.

Example 5 toxicity of liposomal echinomycin in mice. To test the toxicity of liposomal echinomycin in mice, a 250 μ g/kg dose (liposomal formulated, non-liposomal formulated, or equivalent dose of empty liposomal vehicle) was intravenously injected to administer an echinomycin treatment cycle. Injections were given every other day for a total of 3 doses. The body weight of the mice was monitored throughout this period. The results of this analysis showed that mice receiving liposomal echinomycin lost less weight and lost weight recovered faster than mice receiving comparable dose and schedule of free echinomycin (figure 4, panel a). At higher drug doses (1mg/kg), all free echinomycin-treated mice died within one week, while mice receiving echinomycin-loaded liposomes survived 70% over the entire 3 month observation period (fig. 4, panel B).

Example 6 pharmacokinetics of liposomal echinomycin with free echinomycin in mice. Due to the presence of PEG moieties on the liposome surface, "stealth" liposomes are known to exhibit extended circulation times in the blood stream compared to their free drug counterparts. PEG protects liposomes from rapid uptake by RES and provides steric stability to the liposome particles. To measure the prolonged circulation of liposomal echinomycin in the bloodstream compared to free echinomycin, the pharmacokinetics of both formulations were assessed by detecting echinomycin levels in plasma by mass spectrometry after intravenous injection of 0.1mg/kg of liposomal echinomycin or free echinomycin in mice (figure 5). The results of this analysis indicate that echinomycin concentration and circulation time are significantly increased in the blood of mice receiving liposomal echinomycin compared to mice receiving comparable doses of free echinomycin. In particular, the data show that plasma concentrations of >1ng/ml were achieved only 15 minutes after administration of 0.1mg/kg free echinomycin, but the same dose of liposomal echinomycin maintained this concentration for more than 8 hours after administration (fig. 5).

In addition, pharmacokinetics of conventional echinomycin and liposome-formulated echinomycin in breast cancer tissues of NSG mice were evaluated and found to be significantly increased. As shown in FIG. 6, tumor concentrations of >5ng/g were achieved 2 hours after conventional echinomycin administration. In contrast, liposomal echinomycin (Lipo-EM) was administered at the same dose of 0.1mg/kg to achieve tissue concentrations of >5ng/g, which extended more than 10 hours after administration (fig. 6). More importantly, the peak drug levels in the liposomal echinomycin-treated group were about 6-fold higher than in the free echinomycin-treated group.

Example 7 treatment of ALL with echinomycin the basis was used. Early T-cell precursor acute lymphoblastic leukemia (ETPALL) was recently recognized as a form of T-cell ALL (T-ALL) with poor prognosis (Coustan-Smith et al, Lancet Oncol., 2009 Feb; 10 (2): 147-. ETP ALL is characterized by a very early arrest of differentiation and unique genetic and transcriptional characteristics most associated with hematopoietic stem cells and bone marrow progenitor cells. Although the immunophenotype, gene expression profile, and gene mutation profile of ETP ALL have been characterized, the molecular mechanisms of ETP ALL pathology are poorly understood and effective therapeutic targets remain to be identified.

Previously, the spontaneous mouse model for the identification of T-ALL of leukemic stem cells revealed that HIF1 α signaling was selectively activated in mouse T-ALL and human AML stem cells even under normoxic conditions (Wang Y et al, Cell stemcell.2011; 8 (4): 399-.

Example 8 therapeutic efficacy of liposomal echinomycin administration in xenograft ETP-ALL NSG mice as expected from the results in example 7, HIF1 α protein was found to be overproduced in ETP ALL cells (fig. 7, panel a and panel B) compared to PBMC control samples from patients with ETP-ALL hi high accumulation of HIF-1 α was detected in 3 ETP-ALL samples by western blot analysis (fig. 7, panel a) or by intracellular staining of HIF1 α followed by FACS analysis (fig. 7, panel B).

To test whether administration of liposomal echinomycin (Lipo-EM) can eliminate human ETP-ALL cells in vivo, Lipo-EM was administered to xenograft ETP-ALL NSG mice according to the administration protocol outlined in figure a of figure 8. Briefly, 1X 10 were injected intravenously⁶Individual ETP ALL-1 cells were transplanted into 1.3Gy irradiated NSG mice. Reconstitution of human ETP-ALL cells was monitored by detecting hCD45 in peripheral blood of recipient mice. The results of this analysis showed that about 10 to-20% of human CD45 was detected in the blood of recipient mice on day 34 post-transplantation⁺A cell. The 20-day Lipo-EM treatment regimen consisted of two cycles with 4 consecutive treatments every other day as a cycle, with 8 days rest between cycles, beginning on day 35. The percentage of human ETP ALL-1 cells was monitored after treatment. The results of this analysis showed that Lipo-EM significantly reduced ETP-ALL cells in the blood of xenograft mice (fig. 8, panel B). In particular, FACS plots depicting the percentage of human ETP ALL cells before administration and at three time points after Lipo-EM treatment showed that the average percentage of human CD45 in vehicle mice was 10.73% at day 34, increased to 22.35% at day 42, increased to 45.61% at day 48, increased to 57.44% at day 56, and finally reached 80.18% at day 64 in the blood. However, in Lipo-EM treated mice, ETP-ALL-1 cells were almost completely eliminated by this treatment regimen, such that the percentage of human CD45 cells in the recipient mouse blood dropped from 17.47% before treatment to 1.19% after treatment on day 64. These results indicate that liposomal echinomycin is effective in eliminating ETP-ALL cells in xenografted mice.

To compare the efficacy of liposomal echinomycin with free echinomycin in this mouse xenograft model for ETP-ALL, 1x 10 was injected intravenously⁶Individual ETP ALL-1 cells were transplanted into 1.3Gy irradiated NSG mice. The% hCD45 in peripheral blood when Lipo-EM treatment was started on day 22 according to FACS analysis on day 21⁺The cells reached about 1%. The mice were divided into 3 groups of 5 mice each. All treatments were performed by intravenous injection. The first group received a total of 15 doses of echinococcus in PBSElement, divided into 3 identical cycles. In each cycle, mice received 0.1mg/kg echinomycin in PBS once daily for a total of 5 doses, followed by a rest for 5 days before the next cycle started. The second group received a total of 10 doses of Lipo-EM, with the first 2 doses administered every 4 days, followed by 7 days rest, then 4 doses every other day, followed by another 7 days rest, then 4 doses followed by every other day. Another group of mice received an empty liposome vehicle (n-5) according to the same protocol in which Lipo-EM was administered. Peripheral blood at days 34, 50 and 65 was determined by FACS analysis to compare the growth of ETP-ALL-1 cells in recipients during treatment (fig. 8, panel C). The results of this analysis indicate that while treatment with echinomycin in PBS does inhibit the growth rate of ETP-ALL-1 cells, treatment with Lipo-EM provides superior efficacy compared to the former (figure 8, figure C).

Example 9 in vitro role of echinomycin in vitro breast cancer cells to better understand the importance of high HIF-1 α expression in breast cancer cells, echinomycin was tested for its ability to reduce breast cancer cell survival in two breast cancer cell lines, MCF7 and SUM159 more specifically, MCF7 and SUM159 were treated with different concentrations of echinomycin for 48 hours followed by MTT assay to measure cell viability₅₀1 nM). In contrast, MCF7 cells were relatively less sensitive to echinomycin (fig. 9). These data indicate that cancers that overexpress HIF proteins are more sensitive to echinomycin.

Example 10 therapeutic efficacy of liposomal echinomycin in breast cancer cells in vivo. To determine whether Lipo-EM allows for efficient delivery of echinomycin to breast cancer tumor sites, mice bearing luciferase-expressing SUM159 tumor xenografts were administered D-luciferin to visualize tumor masses by bioluminescence. In addition, Lipo-EM is labeled with the fluorescent dye 1,1 '-dioctadecyl tetramethylindotricarbocyanineiodide (1, 1' -diazacytylmethylidene, DiR), thereby enabling in vivo tracking and accumulation of liposomes. Tumor growth was followed by endogenously expressed luciferase activity. The results of this analysis show that Lipo-EM accumulates selectively in human breast cancer SUM159 tumors when imaged 24 hours after administration of fluorescently labeled liposomal echinomycin. In contrast, administration of comparable doses of free DiR dye in water did not produce significant accumulation or fluorescence signal in the fluorescence channel. Importantly, mice without any tumor had no significant accumulation of liposomes in other organs (fig. 10). These data indicate that Lipo-EM can selectively accumulate in xenograft tumors of human breast cancer in mice.

To test the therapeutic efficacy of free echinomycin (in PBS), Lipo-EM and vehicle in breast cancer cells in vivo, SUM159 cells were xenografted into the mammary fat pad of NOD-SCID mice. No significant difference in tumor size was observed between echinomycin-treated and vehicle-controlled groups when 0.1mg/kg free echinomycin was administered (figure 11, panel a). To test the efficacy of Lipo-EM in human breast tumors in vivo, SUM159 breast cancer cells were transplanted into 10 NSG mice, 5 of which were administered Lipo-EM by intravenous injection, at a dose of 0.35mg/kg on days 9, 11, 13, 25 and 27, and vehicle controls (liposomes only) were administered to 5 mice at these same time points. In addition, the growth kinetics of the transplanted tumors were measured by volume (fig. 11, panel B) and weight (fig. 11, panel C and panel D). After tumor size in vehicle mice reached early takeout criteria, 10 mice were sacrificed and tumors were weighed (fig. 11, panel C and panel D). The results of these analyses showed that tumor growth was significantly reduced in mice treated with Lipo-EM that received only a total of 5 injections compared to vehicle treated mice.

The results of these analyses indicate that liposomal echinomycin is a stable formulation that exhibits reduced toxicity, increased circulation time in the bloodstream, and increased ability to accumulate in human solid tumor xenografts in mice compared to free echinomycin or vehicle controls liposomal echinomycin exhibits sufficient anti-tumor effect when administered to mice bearing human xenografts expressing high levels of HIF-1 α for hematopoietic and solid tumor malignancies.

The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and is not intended to detail all such obvious modifications and variations that will become apparent to the skilled worker upon reading this specification. It is intended, however, that all such obvious modifications and variations be included within the scope of the invention which is defined by the following claims. The claims are intended to cover the claimed components and steps in any sequence which is effective to meet the objectives intended, unless the context clearly indicates the contrary.

Claims (19)

1. A pegylated liposomal pharmaceutical formulation for treating a disease in a patient, the formulation comprising:

echinomycin,

Polyethylene glycol phosphatide,

Neutral phosphoglyceride, and

a sterol, and

wherein the composition comprises a plurality of pegylated liposomes encapsulating echinomycin and

wherein the liposomes are suspended in a pharmaceutically acceptable carrier.

2. The formulation of claim 1, wherein the pegylated phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dimyristoylphosphatidylethanolamine-polyethylene glycol (DMPE-PEG), dipalmitoyl glycerosuccinate polyethylene glycol (DPGS-PEG), cholesteryl-polyethylene glycol, or a ceramide pegylated lipid.

3. The formulation of claim 1 or 2, wherein the neutral phosphoglyceride is selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol.

4. The preparation of any one of claims 1 to 3, wherein the molar ratio of the pegylated phospholipid to total lipid in the preparation is from 3% to 6%,

wherein the molar ratio of the neutral phosphoglycerides in the preparation to total lipid is from 45% to 65%, and

wherein the molar ratio of sterol to total lipid in the preparation is from 30% to 50%.

5. The formulation of any one of claims 1 to 4, wherein the pegylated phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), the neutral phosphoglyceride is phosphatidylcholine, and the sterol is cholesterol.

6. The formulation of claim 5, comprising DSPE-PEG-2000, Hydrogenated Soy Phosphatidylcholine (HSPC), and cholesterol.

7. The formulation of claim 6, wherein the molar ratios of DSPE-PEG-2000, HSPC, and cholesterol to total lipid are 5.3%, 56.3%, and 38.4%, respectively.

8. The formulation according to any one of claims 1 to 7, wherein the mass ratio of echinomycin to total lipid is between 2% and 10%.

9. The preparation of any one of claims 1 to 7, wherein the mass ratio of echinomycin to total lipid is 5%.

10. The formulation of any one of claims 1 to 9, wherein at least 90% of the liposomes in the formulation have a diameter of 80nm to 120 nm.

11. The formulation of any one of claims 1 to 10, wherein the liposomes have an average polydispersity index of less than 0.1 and are sufficiently stable to achieve a lifetime of at least 12 months at 4 ℃.

12. The formulation of any one of claims 1 to 11, wherein the liposome is formulated as a lyophilized powder.

13. A method of treating a disease in a patient, the method comprising:

administering the formulation of claim 1 to a patient in need thereof,

wherein the liposomal formulation comprises echinomycin or an echinomycin analog in an amount sufficient to treat a proliferative disorder, an autoimmune disease, or graft-versus-host disease, wherein the disease is characterized by HIF-1 α or HIF-2 α overexpression.

14. The method of claim 13, wherein the disease is a proliferative disorder.

15. The method of claim 14, wherein the proliferative disorder is leukemia.

16. The method of claim 14, wherein the proliferative disorder is breast cancer.

17. The method of claim 13, wherein the disease is an autoimmune disease.

18. The method of claim 13, wherein the disease is graft versus host disease.

19. A process for preparing the pharmaceutical composition of claim 1, the process comprising:

forming a mixture comprising echinomycin and a lipid component in a polar solvent, the lipid component comprising a pegylated phospholipid, a neutral phospholipid and a sterol;

drying the mixture to remove the polar solvent, thereby forming a dried lipid film;

dissolving the dried lipid film in a buffer to form a lipid suspension;

extruding the lipid suspension through a polycarbonate filter to obtain liposomes having a desired size range; and

the liposomes were sterilized by filtration.