Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
  • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin

Definitions

• the present invention relates to an S-enantiomer of a tri-substituted glycerol compound.
• the present invention also relates to a pharmaceutical composition comprising said compound or a pharmaceutically acceptable salt thereof. Further, the invention relates to methods for separating S- and R-enantiomers of the tri- substituted glycerol compound.
• Tri-substituted glycerol compounds belong to the class of synthetic ether- linked alkyl-lysophospholipids, which are known to have an anti-cancerogenic activity. For this reason they are also collectively named "anti-tumor ether lipids" (reviewed, e.g., by Arthur, G., and Bittman, R. (1998) Biochim. Biophys. Acta 1390, 85-102;
• ether lipids do not directly target cellular DNA but rather affect the plasma membrane lipid composition and/or interfere with various signal transduction pathways. Aside from their anti-tumor activity, these ether lipids are believed to be involved in a variety of other physiological processes such as inflammation, the immune response or allergic reactions. Some ether lipids have been suggested as candidate compounds for the treatment of various immune diseases (cf, for example, the International Patent Applications WO 87/01257 and WO 90/14829, respectively).
• Cancer chemotherapy generally aims to slow the growth of, or destroy, cancer cells while avoiding collateral damage to surrounding cells and tissues. Consequently, the most effective anticancer agents are those that are able to selectively target cancer cells while leaving normal cells relatively unaffected.
• Synthetic ether-lipids have been shown to be effective as tumor agents, for example, in order to decrease or to stop tumor progression, i.e. to stabilize the "status quo" of the condition, or even to reduce the size of tumors in mammals.
• Racemic l-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (also referred to as ET-18-OCH3, AP-121 or edelfosine) is considered to be the prototype of the antitumor ether lipids.
• l-0-octadecyl-2-0-methyl-sn-glycero-3-phosphocholine represents a synthetic analogue of the platelet activating factor (PAF; l-O-alkyl-2- acetyl-sn-glycero-3-phosphocholine), a potent phospholipid activator and mediator of many leukocyte functions, including platelet aggregation, inflammation, and anaphylaxis.
• PAF platelet activating factor
• mediator of many leukocyte functions including platelet aggregation, inflammation, and anaphylaxis.
• l-0-octadecyl-2-0-methyl-sn-glycero-3-phosphocholine has been reported to be suitable for the treatment of particular types of tumors such as brain tumors or mamma carcinomas (cf, for example, the German Patent DE 2619686 as well as the International Patent Applications WO 99/59599 and WO 00/01392, respectively).
• ether lipid action include effects on levels of intracellular protein phosphorylation, and disruption of cellular lipid metabolism. Normal cells typically possess the means to avoid or overcome the potentially toxic effects of ether-lipids, while cancer cells do not.
• the present invention relates to an S-enantiomer of a tri-substituted glycerol compound according to formula (I)
• X is selected from the group consisting of phosphate and sulfate
• Ri is selected from the group consisting of C 16 - C 2 o alkyl
• R 2 is selected from the group consisting of C 1 -C 3 alkyl and C 1 -C 3
• R3 is selected from the group consisting of hydrogen and C 1 -C 3 alkyl
• R4 is selected from the group consisting of C 1 -C 3 alkyl and C 3 -C6 cycloalkyl
• R 5 , 5 and R 7 are independently selected from the group consisting of hydrogen and methyl.
• the term “C 18 alkyl” refers to an alkyl group having 18 carbon atoms.
• the alkyl groups or hydroxyalkyl groups according to the invention may be straight or branched.
• enantiomer denotes a compound having a centre of chirality and being one of two stereoisomers that are non-superposable complete mirror images of each other.
• enantiomers differ from each other in their ability to rotate plane-polarized light and may be classified according to the CIP (Cahn-Ingold-Prelog)-convention as S- or R-enantiomer.
• CIP Cert-Ingold-Prelog
• R-enantiomer The S- and Reconfigurations represent the three-dimensional orientation of the four substituents about the chiral center carbon atom.
• an S-enantiomer of a tri-substituted glycerol compound of formula (I) has a centre of chirality at the second or middle carbon atom of the glycerol principal structure which is classified as "S" according to the CIP-convention.
• the tri-substituted glycerol compound has the formula (I), wherein X is phosphate, Ri is -(CH 2 )i7-CH 3 , R 2 is CH 3 , R 3 is H, R4 is -(CH 2 ) 2 -, R 5 is CH 3i 5 is CH 3 and R 7 is CH 3 .
• the tri-substituted glycerol compound of formula (I) is S-edelfosine or S-l-O-octadecyl- 2-0-methyl-sn-glycero-3-phosphocholine or AP-121.
• the tri-substituted glycerol compound may be present in amorphous or in crystalline form.
• amorphous refers to a solid in which there is no long-range order of the positions of the atoms, i.e. a non-crystalline material.
• the tri-substituted glycerol compound is present in crystalline form.
• the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the herein described
• Pharmaceutically acceptable salts may comprise any pharmaceutically acceptable anion neutralizing the positive charge of the nitrogen atom, e.g. chloride, bromide or iodide, and/or any pharmaceutically acceptable cation "neutralizing" the negative charge of the phosphate or sulfate moiety, e.g. sodium or potassium cations, in the phosphocholine or sulfocholine moiety of formula (I).
• a further aspect of the present invention relates to the use of the herein described S-enantiomer of a tri-substituted glycerol compound or the herein described pharmaceutical composition as a medicament.
• Another aspect of the present invention relates to the use of the herein described S-enantiomer of a tri-substituted glycerol compound or the herein described pharmaceutical composition for the treatment of cancer.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a
• cancer denotes any type or form of malignant growth of cells or tissues including inter alia breast cancer, colorectal cancer, prostate cancer, leukemia, lymphomas, melanoma, head cancer, neck cancer, brain tumor, skin cancer, esophageal cancer, Kaposi sarcoma and lung cancer.
• cancer may refer to diseases in which cells are aggressive (i.e. they may grow and divide regardless of normal limits), invasive (i.e. they may invade and destroy adjacent tissues), and/or metastatic (i.e. they may spread to other locations in the body).
• the term “cancer”, as used herein may also cover benign tumors which are self-limited in their growth and do not invade or metastasize.
• tri-substituted glycerol compounds for the treatment of cancer is e.g. shown in WO 2008/055996.
• the compound or the pharmaceutical composition according to the present invention is used for the treatment of skin cancer.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a
• skin cancer refers to any form of malignant growth of skin cells, both of cutaneous (also referred to as dermal) and of subcutaneous (i.e subdermal) cells. Such malignant cell growth may be manifested by various symptoms including inter alia any skin lesions (wounds, sores, abrasions, and the like) or other changes of the skin that do not heal, ulcers in the skin, discoloring in parts of the skin, and changes in existing moles (i.e. melanocytic nevi).
• the skin cancer is selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, and malignant melanoma, with the latter one being particularly preferred.
• basal cell carcinoma a group consisting of basal cell carcinoma, squamous cell carcinoma, and malignant melanoma, with the latter one being particularly preferred.
• the compound or pharmaceutical composition of the present invention is used for the treatment of breast cancer, in particular for the treatment of subcutaneous metastases from breast cancer.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of breast cancer.
• breast cancer refers to any form of malignant growth of breast tissue including ductal carcinome, lobular carcinoma, inflammatory breast cancer, medullary carcinoma, colloid carcinoma, papillary carcinome, and metaplastic carcinoma. These types of cancer are reviewed, e.g., in Loffeld, A., and Marsden, J.R. (2005) Br. J. Dermatol. 152, 1206-1210; Karakuzu, A. et al. (2006) J. Am. Acad. Dermatol. 55, 1 101-1102; and Seidman, A.D. (2006) Oncology 20, 983- 990.
• composition of the present invention is used in the prevention of breast cancer in a patient with an elevated or even high risk of developing breast cancer.
• this embodiment relates to S-edelfosine or a pharmaceutical
• composition comprising a pharmaceutically effective amount of S-edelfosine for use in the prevention of breast cancer in a patient with an elevated or even high risk of developing breast cancer.
• Accurately defining a patient's risk of developing breast cancer may be performed using population models, such as the Gael Model, as well as the patient's personal and family history and genetic testing.
• population models such as the Gael Model
• the skilled person is familiar with these components and is able to identify those patients at elevated or even high risk.
• Patients with an elevated risk of developing breast cancer may include those with a family history of breast cancer, personal history of breast cancer, significantly dense breast tissue, hormone replacement therapy longer than ten years, and/or a history of atypical ductal hyperplasia or lobular carcinoma in situ without family history of breast cancer.
• Dense breast tissue as used herein, can be seen on a mammogram and may be characterized by more glandular and fibrous tissue and less fatty tissue.
• Patients who are even at a high risk of developing breast cancer may include BRCA1 or BRCA2 gene mutation carriers, those with a personal history of atypical ductal hyperplasia or lobular carcinoma in situ with associated family history, those who have undergone therapeutic or similarly significant radiation exposure, and/or those with a history of a BRCA1 or BRCA2 gene mutation in the family of an untested individual.
• breast cancer risk may be higher among women whose close blood relatives have this disease.
• a woman with cancer in one breast has a three- to fourfold increased risk of developing a new cancer in the other breast or in another part of the same breast.
• composition of the present invention is used for the treatment of brain cancer, in particular for the treatment of glioblastoma multiforme.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of brain cancer.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of glioblastoma multiforme.
• brain tumor refers to any abnormal growth of cells within the brain or inside the skull, which can be malignant or benign. This includes any intracranial tumor created by abnormal and uncontrolled cell division, normally either in the brain itself, in the cranial nerves, in the brain envelopes, skull, pituitary and pineal gland, or spread from cancers primarily located in other organs.
• Glioblastoma multiforme is characterized by the presence of small areas of necrotizing tissue that is surrounded by anaplastic cells and/or the presence of hyperplastic blood vessels.
• the compound or the pharmaceutical composition according to the present invention is used for the treatment of lung cancer, in particular for the treatment of non-small cell lung carcinoma (NSCLC).
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of lung cancer.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of non- small cell lung carcinoma (NSCLC).
• lung cancer refers to any disease of uncontrolled cell growth in tissues of the lung. This includes the main types of lung cancer, i.e. small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) including subtypes thereof, as well as other types of lung cancer such as pleuropulmonary blastoma, carcinoid tumors and secondary cancer.
• the compound or the pharmaceutical composition according to the present invention is used for the treatment of Kaposi's sarcoma.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of Kaposi's sarcoma.
• Kaposi's sarcoma is a cancer that may cause patches of abnormal tissue to grow under the skin, in the lining of the mouth, nose and throat or in other organs. In HIV/ AIDS patients, the disease may move quickly.
• Another aspect of the present invention relates to the use of the herein described S-enantiomer of a tri-substituted glycerol compound of formula (I) or the herein described pharmaceutical composition for treatment of inflammatory conditions.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of inflammatory conditions.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, inflammatory bowel disease, rheumatoid arthritis or vasculitis.
• inflammatory condition refers to any condition involving the response of vascular tissues to harmful stimuli such as pathogens, damaged cells or irritants.
• an "inflammatory condition" may be acute or chronic.
• Disorders or conditions associated with inflammation comprise a large unrelated group of disorders which underlie a wide variety of human diseases.
• the immune system is often involved in inflammatory diseases, demonstrated in both allergic reactions and myopathies, with many immune system disorders resulting in abnormal inflammation.
• inflammatory processes may include cancer, artherosclerosis and ischaemic heart disease.
• conditions or disorders associated with inflammation are asthma, autoimmune diseases, chronic inflammation, chronic prostatitis,
• a further aspect of the present invention relates to the use of the herein described S-enantiomer of a tri-substiuted glycerol compound of formula (I) or the herein described pharmaceutical composition for the treatment of hematological
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of hematological malignancies.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a
• S-edelfosine for use in the treatment of leukemia, lymphoma, multiple myeloma, or myelodysplastic syndrome.
• hematological malignancies also referred to as “hematological neoplasms”
• hematological neoplasms denotes any type of cancer that affects blood, bone marrow or lymphatic organs.
• Hematological malignancies include inter alia leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome.
• leukemia refers to any cancer of the blood or bone marrow characterized by an abnormal proliferation of blood cells, usually of white blood cells (i.e. leukocytes). Within the scope of the present invention, the term includes any acute and chronic forms of leukemia as well as any form of lymphoid or myeloid leukemia.
• lymphoid leukemia forms of lymphoid (or lymphocytic) leukemia are characterized in that lymphoid cells (that is, agranulocytes) such as lymphocytes and monocytes are affected, whereas when myeloid cells (that is, granulocytes) such as eosinophils, neutrophils, and basophils are affected, the disease is referred to as myeloid (or myelogenous) leukemia.
• the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphoid leukemia (CLL).
• lymphoma refers to any form of cancer originating from lymphocytes, i.e. it includes Hodgkin lymphoma which may be characterized by the orderly spread of the disease from one lymph node and the presence of Reed- Sternberg cells as well as to any type of non-Hodgkin lymphoma.
• the hematological malignancy is selected from the group consisting of Hodgkin lymphoma and non-Hodgkin lymphoma.
• S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine may be used in the treatment of non-Hodgkin lymphoma.
• multiple myeloma (also referred to as “myeloma” or “plasmacytoma”), as used herein, denotes a type of cancer of plasma cells, i.e. the immune cells in bone marrow that produce antibodies.
• myelodysplastic syndrome (formerly known as "pre-leukemia”), as used herein, denotes a diverse collection of hematological conditions united by ineffective production of blood cells and varying risks of transformation to acute myelogenous leukemia. Anemia requiring chronic blood transfusion is frequently present.
• Another aspect of the invention relates to the use of the herein defined S-enantiomer of a tri-substituted glycerol compound of formula (I) or pharmaceutical composition for the treatment of immune diseases such as autoimmune diseases.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of immune diseases such as autoimmune diseases.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment of multiple sclerosis.
• immune disease refers to any disorder of the immune system.
• immune diseases include inter alia immunodeficiencies (i.e. congenital or acquired conditions in which the immune system's ability to fight infectious diseases is compromised or entirely absent such as AIDS or SCID), hypersensitivity (such as and forms of allergies or asthma), and autoimmune diseases.
• autoimmune disease is to be understood to denote any disorder arising from an overactive immune response of the body against endogenic substances and tissues, wherein the body attacks its own cells.
• autoimmune diseases include inter alia multiple sclerosis, Crohn's disease, lupus erythematosus, myasthenia gravis, rheumatoid arthritis, and polyarthritis.
• a further aspect of the present invention relates to the use of the herein described S-enantiomer of a tri-substituted glycerol compound of formula (I) or the herein described pharmaceutical composition for the treatment and/or prevention of radiation damage or injury.
• this embodiment relates to S-edelfosine or a pharmaceutical composition comprising a pharmaceutically effective amount of S-edelfosine for use in the treatment and/or prevention of radiation damage or injury.
• radiation damage or injury refers to any negative or adverse effect of an exposure to radiation - independent of the radiation dose applied and the time of exposure, respectively - may exert on cells, tissues, organs or organisms resulting in uncontrolled cell proliferation and/or differentiation and as a consequence to the development and progression of tumors.
• radiation damages or injuries include inter alia genetic changes in the cell (e.g., DNA and/or R A mutations, DNA and/or R A decay, chromosomal aberrations) as well as cell death (e.g., programmed cell death/apoptosis). Examples for the use of a tri- substituted glycerol compound for the prevention and/or treatment of radiation damage or injury are shown in WO 2008/055997.
• the compound or pharmaceutical composition according to the present invention may be administered via any parenteral or non-parenteral route.
• Parenteral application methods comprise, for example, intracutaneous, subcutaneous, intramuscular or intravenous injection and infusion techniques.
• Non-parenteral delivery modes include, for instance, oral or topical administration.
• the compound or pharmaceutical composition according to the present invention may be administered locally or systemically. In case a combination of compounds or active agents is used, the individual pharmaceuticals may be administered via the same or via different routes.
• the compound such as S-edelfosine or the pharmaceutical composition of the present invention is administered orally or topically.
• the compound or pharmaceutical composition according to the present invention is a pharmaceutical dosage form suitable for oral application.
• the dosage form may be a solid dosage form.
• dosage forms include inter alia tablets, pills, capsules, granulates, pellets, powders, multi- particulate formulations (e.g., beads, granules or crystals), and dragees.
• the unit doses of multi-particulates may be incorporated into a pharmaceutical solid dosage form, e.g. via compression or shaping into tablets or by placing a requisite amount inside a gelatin capsule.
• the pharmaceutical solid dosage form is selected from the group consisting of tablets, pills, capsules, granulates, pellets, powders, multi-particulate formulations, dragees and granules.
• the pharmaceutical solid dosage form may be selected from the group consisting of tablets, pills, capsules and granules, with tablets being preferred.
• one embodiment refers to pharmaceutical solid dosage form selected from the group consisting of tablets, pills, capsules and granules, with tablets being preferred, wherein the dosage form comprises a pharmaceutically effective amount of
• compositions comprising tri-substituted glycerol compounds for oral administration are exemplified in WO 2008/055996.
• the compound or the pharmaceutical composition according to the present invention is a pharmaceutical dosage form for topical administration.
• the pharmaceutical dosage form according to the present invention may be any therapeutically effective pharmaceutical dosage form for topical administration. Examples of such pharmaceutical dosage forms include inter alia solutions, suspensions, dispersions, tinctures, gels, topical sprays, topical foams, gels, water-in- oil emulsions such as ointments, and oil-in water emulsions such as creams, lotions, and balms.
• the pharmaceutical dosage form for topical administration may be selected from gels and oil-in-water emulsions.
• one embodiment refers to pharmaceutical solid dosage form selected from gels and oil-in-water emulsions, wherein the dosage form comprises a pharmaceutically effective amount of
• gel refers to a colloidal system in which a porous network of interconnected nanoparticles spans the volume of a liquid medium.
• gels are apparently solid, jelly-like materials. Both by weight and volume, gels are mostly liquid in composition and thus exhibit densities similar to liquids, however have the structural coherence of a solid.
• the pharmaceutical dosage form is a hydrogel.
• the gel-forming polymers may be naturally occurring polymers, synthetic polymers or mixtures thereof.
• Hydrogels may comprise more than 99% water. When applied to the skin the water bound in such a hydrogel does not evaporate as fast as from a solution. Due to the thus prolonged contact period the skin becomes moistened which, in turn, results in an improved susceptibility for the uptake of active ingredients present in the hydrogel (i.e. an increased penetration through the skin). This phenomenon is also referred to as "occlusion effect".
• the pharmaceutical dosage form is an oil-in- water emulsion.
• oil- in- water emulsion refers to formulations which are composed of small droplets of a lipid phase (e.g., an oil) dispersed in a continuous aqueous phase.
• An "emulsion” is a mixture of two immiscible (i.e. not mixable) substances. One substance (the dispersed phase) is dispersed (i.e. distributed) in the other (the continuous phase) by the presence of one or more emulsifying agents.
• oil- in- water emulsions are more comfortable and pharmaceutically/cosmetically acceptable as compared to water-in-oil emulsions (such as an ointment) as they are less greasy when applied on the skin and more easily washed off when using water.
• water-in-oil emulsions such as an ointment
• amphiphilic compounds such as the tri- substituted glycerol compounds of the invention
• formulations having only an aqueous phase since the presence of a lipid phase is assumed to aid in crossing the hydrophobic core of biological membranes.
• Exemplary oil-in-water-emulsions of the invention may be selected from the group consisting of creams, lotions, and balms. These formulations primarily differ with regard to their respective viscosities.
• a cream is a semi-solid emulsion, that is it has a medium viscosity.
• a lotion is a low- to -medium- viscosity preparation intended for application to unbroken skin.
• a balm also referred to as liniment
• a balm has a similar viscosity as a lotion (i.e. being significantly less viscous than a cream) but unlike a lotion a balm is applied with friction, that is a liniment is always rubbed in.
• compositions for topical administration comprising a tri- substituted glycerol compound are shown in WO 2008/074573.
• the S-enantiomer of a tri-substituted glycerol compound according to formula (I) may be present in any amount being effective to achieve the desired pharmacological effect when administered to a patient. Effective amounts are generally chosen in accordance with a number of factors, e.g., the age, size and general condition of the patient and the medical condition being treated, and determined by a variety of means, for example, dose ranging trials, well known to, and readily practiced by persons of ordinary skill in art given the teachings of this invention.
• S-enantiomer of a tri-substituted glycerol compound according to formula (I) such as S-edelfosine is less than 400 mg, e.g. in the range of 30 to 250 mg, or in the range of 50 to 150 mg. In some embodiments of the invention, the amount of the
• S-enantiomer of a tri-substituted glycerol compound according to formula (I) such as S-edelfosine in an oral dosage form is 75 mg and 100 mg, respectively.
• the amount of the S-enantiomer of a tri-substituted glycerol compound according to formula (I) such as S-edelfosine is at least 2% (w/w), i.e. is at least 2% by weight based on the total weight of the dosage form. This amount corresponds to a concentration of at least 20 mg/g of the dosage form.
• the amount of the S-enantiomer of a tri-substituted glycerol compound according to formula (I) in the pharmaceutical dosage form is at least 5% (w/w), more preferably at least 10% (w/w), and most preferably at least 15% (w/w).
• topical dosage forms comprising at least 20%> (w/w) or at least 25% (w/w) of the tri-substituted glycerol compound according to formula (I) or even higher amounts.
• Gennaro, A.L. and Gennaro, A.R. (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA; Niazi, S.K. (2004) Handbook of Pharmaceutical Manufacturing Formulations, CRC Press, Boca Raton, FL).
• the daily dosage of the S-enantiomer of a tri-substituted glycerol compound according to formula (I) such as S-edelfosine administered to a patient may be less than 1200 mg, typically less than 900 mg, e.g. in the range of 30 to 600 mg, or in the range of 40 to 400 mg, or in the range of 50 to 350 mg. In specific embodiments, the daily dosage is 75, 100, 150, 200, 225, or 300 mg.
• the daily dosage of the S-enantiomer of a tri-substituted glycerol compound according to formula (I) is administered as a single dose such as in form of one or more, e.g. up to four tablets or capsules.
• a single dose such as in form of one or more, e.g. up to four tablets or capsules.
• the compound in multiple doses such as two or three individual doses administered during the day, e.g. in the morning, at noon, and at night.
• the individual pharmaceuticals i.e. the medicament comprising the tri- substituted glycerol compound and the at least one other pharmaceutical
• the pharmaceutical composition of the present invention may comprise at least one pharmaceutically acceptable excipient.
• pharmaceutically acceptable excipient denotes any substance used for the preparation of pharmaceutical dosage forms such as coating materials, film- forming materials, fillers, disintegrating agents, release modifying materials, diluents, binding agents, carrier materials, wetting agents, preservatives, buffers, solvents or solubilizers, agents for achieving a depot effect, lubricants, flowability controlling agents and other adjuvants, all of them well known in the art.
• the compound or pharmaceutical composition according to the invention may be used for the treatment of the herein described diseases individually or in combination with at least one other pharmaceutical comprising at least one additional active ingredient such as chemotherapeutics, monoclonal antibodies, anti-neoplastic agents or antibodies directed against one or more epitopes on the cell surface of
• haemato logical (hematopoetic) cells haemato logical (hematopoetic) cells.
• an adjunction is also within the scope of the present invention, wherein an S-enantiomer of a tri-substituted glycerol compound as described herein or a pharmaceutical composition according to the present invention is used together with at least one other pharmaceutical comprising one or more additional active ingredients, e.g. compounds or active ingredients different from the tri-substituted glycerol compounds as defined in claim 1 or 2.
• S-enantiomers of a tri-substituted glycerol compound according to Formula (I) such as S-edelfosine may be synthesized by a direct enantioselective synthesis.
• the tri-substituted glycerol compounds according to Formula (II) below such as edelfosine may be synthesized as mixtures (racemic or otherwise) of their enantiomers. Mixtures of S- and R-enantiomers may be separated by enantioselective separation methods such as chromatography or capillary
• the goal of these enantioselective purification methods is to produce the more desired enantiomer in high (such as at least 80% or at least 90% or at least 95% or at least 99%) enantiomeric excess
• e.e. which is the relative percent of one enantiomer in excess of its antipode and ignoring any other impurities (e.g. a mixture containing 99.5% of an enantiomer and 0.5% of its antipode has an e.e. of 99% and a mixture containing 90% of an enantiomer and 10% of its antipode has an e.e. of 80%).
• a mixture containing 99.5% of an enantiomer and 0.5% of its antipode has an e.e. of 99% and a mixture containing 90% of an enantiomer and 10% of its antipode has an e.e. of 80%.
• the present invention relates to a method for separating S- and R-enantiomers of a tri- substituted glycerol compound according to Formula (II) such as edelfosine comprising (a) introducing a mixture of these enantiomers to a chiral stationary phase and (b) eluting at least one of the enantiomers with a mobile phase.
• Formula (II) such as edelfosine
• the invention may concern a method for separating S- and R- enantiomers of a tri-substituted glycerol compound according to Formula (II),
• X is selected from the group consisting of phosphate and sulfate
• Ri is selected from the group consisting of C 16 - C 2 o alkyl
• R 2 is selected from the group consisting of C 1 -C 3 alkyl and C 1 -C 3
• R3 is selected from the group consisting of hydrogen and C 1 -C 3 alkyl
• R4 is selected from the group consisting of C 1 -C 3 alkyl and C 3 -C6 cycloalkyl
• R 5 , 5 and R 7 are independently selected from the group consisting of hydrogen and methyl, wherein the method comprises the following steps:
• the tri-substituted glycerol compound of formula (II) has a centre of chirality at the second or middle carbon atom of the glycerol principal structure which is classified as “S" or “R” according to the CIP-convention.
• the term “C 18 alkyl” refers to an alkyl group having 18 carbon atoms.
• the alkyl groups or hydroxyalkyl groups according to the invention may be straight or branched.
• the tri- substituted glycerol compound may have the formula (II) wherein X is phosphate, Ri is -(CH 2 )i7-CH 3 , R 2 is CH 3 , R 3 is H, R4 is -(CH 2 ) 2 -, R 5 is CH 3 Rg is CH 3 and R 7 is CH 3 .
• the tri-substituted glycerol compound of formula (II) is S- or R-edelfosine or S- or R-l-0-octadecyl-2-0-methyl-sn-glycero-3- phosphocholine or S- or R-AP-121.
• the tri-substituted glycerol compound may be present in amorphous or in crystalline form.
• amorphous refers to a solid in which there is no long-range order of the positions of the atoms, i.e. a non-crystalline material.
• the tri-substituted glycerol compound such as edelfosine is present in crystalline form.
• the method for separating S- and R-enantiomers of a tri- substituted glycerol compound according to Formula (II) such as edelfosine further comprises a step of monitoring the eluate produced in the eluting step for at least one of the enantiomers.
• the chiral stationary phase is in a liquid chromatography device or in a capillary electrophoresis device.
• the liquid chromatography device may be a high- liquid pressure chromatography device.
• the chiral stationary phase comprises an immobilized cellulose tris(3,5-dichlorophenyl-carbamate) polymer.
• the chiral stationary phase may be immobilized on silica.
• the mobile phase comprises at least one organic water soluble solvent and/or water.
• the at least one organic water soluble solvent may be selected from the group consisting of methanol, acetonitril or a mixture thereof.
• the mobile phase is a mixture of acetonitril and methanol, e.g. acetonitril :methanol 85: 15 (v:v), or acetonitril :methanol 90: 10, or
• acetonitril :methanol 80:20 the ratio of acetonitril to methanol may range from 90: 10 to 80:20.
• the mobile phase may further comprise a salt, wherein the salt is optionally a monovalent salt such as ammonium acetate.
• mixture of the S- and R-enantiomer includes racemic and non-racemic mixtures.
• the mixture of the enantiomers is typically introduced to the chiral stationary phase as a solution. Further, the solution may comprise mobile phase or a component or components thereof.
• separation enantiomers includes all non-racemic mixtures of the enantiomers that are obtained from the separation of a racemic mixture of the enantiomers and all non-racemic mixtures of the enantiomers wherein the
• enantiomeric purity of at least one of the enantiomers is increased by e.g. 1%, 2%, 4% or 5% compared to the enantiomeric purity of the enantiomer before separation.
• Mobile phase may comprise a single solvent or a soluble mixture of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or more solvents.
• Mobile phase may also comprise at least one additive.
• An additive suitable for chromatography of the tri-substituted glycerol compound according to Formula (II) such as edelfosine is typically an amine such as tri-methyl amine, tri-ethyl amine and the like, or an organic salt such as sodium or potassium acetate or an inorganic salt such ammonium acetate or ammonium chloride.
• An additive suitable for chromatography of the tri-substituted glycerol compound according to Formula (II) such as edelfosine is typically an amine such as tri-methyl amine, tri-ethyl amine and the like, or an organic salt such as sodium or potassium acetate or an inorganic salt such ammonium acetate or ammonium chloride.
• An additive suitable for chromatography of the tri-substituted glycerol compound according to Formula (II) such as edelfosine is typically an amine such as tri-methyl amine,
• chiral stational phase is typically an inorganic salt such as those described herein.
• Eluate may or may not contain a tri-substituted glycerol compound according to Formula (II) such as edelfosine dissolved therein.
• Eluate may be collected for analysis of any material dissolved therein or for isolation and recovery of any material dissolved therein by conventional means such as evaporation of mobile phase, optionally with crystallization of the material.
• eluate may be recycled directly by re-introduction to the stationary phase via a recycle stream or indirectly by introduction to an interconverting unit followed by introduction of the resulting mixture of the at least one enantiomer and its antipode to the stationary phase via a recycle stream.
• eluate may be introduced to another stationary phase (chiral or achiral) which may be the same or different than the previous stationary phase and is flowingly connected to the prior stationary phase.
• eluate includes a raffinate stream, wherein the mobile phase contains dissolved therein a majority of one enantiomer of the tri-substituted glycerol compound according to Formula (II), and an extract stream, wherein the mobile phase contains dissolved therein a majority of the other enantiomer of the tri-substituted glycerol compound according to Formula (II) thereof.
• Eluate streams may or may not contain one or both of the enantiomers dissolved therein.
• Eluate can be monitored for the presence or absence of the S- and/or R-enantiomer of the tri-substituted glycerol compound according to Formula (II) such as edelfosine by any conventional means such as, e.g. by passing the eluate, or a portion thereof, through a detector.
• the detector may be compatible with liquid chromatography and/or may be capable of determining chirality.
• detectors compatible with liquid chromatography include photodiode array detectors that may scan ultraviolet light wavelengths from about 210 nm wavelength to about 320 nm wavelength to detect UV-active components, devices that monitor rotation of plane polarized light such as the IBZ CHIRALYSER available from JM Science, Inc., Grand Island, New York, refractive index detectors and evaporative light scattering detectors. Alternatively, eluate may be monitored by timing fractions, e.g.
• an enantiomer such as the S-enantiomer
• by sampling untimed or timed fractions and analyzing the samples by, for example, visual inspection, UV light illumination in conjunction with visual inspection, non- enantio selective or enantioselective HPLC, nuclear magnetic resonance, mass spectrometry, derivatization and analysis of the resulting derivative, and the like; by evaporating fractions and analyzing the resulting residue for the presence of an enantiomer such as the S-enantiomer, such as by visual inspection, UV light illumination in conjunction with visual inspection, melting point, non-enantio- selective or enantioselective HPLC, nuclear magnetic resonance spectrometry, mass spectrometry, and the like; or by adding a derivatizing agent to fractions of the eluate or to the residue therefrom, and analyzing the resulting derivative as described above.
• Monitoring can be done simultaneously with the introducing or eluting step, after an introducing or eluting step, or both simultaneously with an introducing or eluting step and after the introducing or eluting step. For example, in an enantioselective chromatography, monitoring may not be done until after the introducing and eluting steps have been completed and after at least one of the enantiomers in the eluate has been isolated. Monitoring is any process or activity by which one of ordinary skill in the art would know whether any portion of eluate will contain, contains, or did contain at least one of the enantiomers.
• % volume/volume equals (the volume of the liquid component in question divided by the volume of the mixture containing the component) times 100.
• % weight/weight equals (the weight of the component in question divided by the weight of the mixture containing the component) times 100.
• substantially free of mobile phase means the absence of mobile phase or any component thereof, or the presence of less than 0.5% weight/weight of each component thereof.
• solution means a mixture of the enantiomers that consists essentially of a solution, meaning there may be trace amounts of undissolved material (the enantiomers or impurities) that will not interfere with a successful practice of a method described herein, typically because they can be filtered out prior to chromatography.
• a mobile phase useful in the method described herein is a substantially homogeneous solution at the proportions of individual components being used. It is expected that the individual components of the mobile phase will be miscible in any ratio specified by the methods described herein without separation of any phases.
• a typical solution is one wherein the solvent mixture comprises the mobile phase. Also suitable are solutions wherein the solvent comprises a solvent or mixture of two solvents that is a component of the mobile phase.
• feed solution means a solution of the mixture of the enantiomers that is freshly introduced to the chiral stationary phase or reverse phase, chiral stationary phase.
• recycle solution means eluate (e.g. raffinate or extract) containing a mixture of the enantiomers that is being reintroduced to the chiral stationary phase or reverse phase, chiral stationary phase.
• the enantiomers may optionally be first subjected to an interconverting step before being reintroduced to the chiral stationary phase or reverse phase, chiral stationary phase.
• a recycle solution embraces a solution recirculated internally to a chromatography pathway and an externally prepared solution that allows recycling of enantiomers that have been previously subject to a chromatography. In circumstances wherein an eluate stream (e.g.
• the eluate stream may be recycled to an interconverting step and then an amount of the mixture of enantiomers (i.e. a mixture that has not yet been introduced to an instant chiral stationary phase or reverse phase, chiral stationary phase) may then be dissolved in the irradiated stream to give a feed stream
• the S-enantiomer of a tri-substituted glycerol compound according to Formula (I) such as S-edelfosine is the antipode of the corresponding R-enantiomer of the tri- substituted glycerol compound, and vice versa.
• Steady state recycling chromatography includes SSRC known by the tradename CYCLOJET(R) (Novasep Societe Par Actions Simplifiee, Pompey, France) and by the trademark "SteadyCycle(TM)" (CYBA Technologies, LLC, Mystic, Connecticut, USA).
• CYCLOJET(R) Novasep Societe Par Actions Simplifiee, Pompey, France
• StepadyCycle(TM) CYBA Technologies, LLC, Mystic, Connecticut, USA.
• k'[pi] [(retention volume of peak n) minus (dead time)] divided by (dead time), k' for Peak n relates to how long Peak n is retained on a column; the longer Peak n is retained, the higher k'.
• chiral stationary phase includes a solid support and a chiral adduct such as a polysaccharide-, (tartaric acid)-, poly-(S)-N-acryloylphenylal amine ethyl ester-, 3, 5-dinitrobenzoyl -phenyl glycine-, L-proline bound to polyacrylamide-, vancomycin- or JI-tris(l,10-phenanthroline)ruthenium sodium magnesium-derived chiral adduct, and the like, wherein the chiral adduct may be covalently bound to the solid support.
• a chiral adduct such as a polysaccharide-, (tartaric acid)-, poly-(S)-N-acryloylphenylal amine ethyl ester-, 3, 5-dinitrobenzoyl -phenyl glycine-, L-proline bound to polyacrylamide-, vancomycin- or JI-tris(
• the chiral polysaccharide stationary phases include silica gel supporting
• the chiral polysaccharide stationary phase may comprise a synthetically made polysaccharide, a naturally occurring
• polysaccharide or a modified version of a naturally occurring polysaccharide, or a polysaccharide selected from amylosic, cellulosic, chitin, chitosan (e.g., beta-1,4- chitosan), xylan (e.g., beta-l,4-xylan), curdan, mannan (e.g., beta-l,4-mannan), dextran, glucan (e.g., alpha- 1,3-glucan and beta-l,3-glucan), and inulin class of polysaccharides.
• the chiral polysaccharide stationary phase may comprise a polysaccharide selected from cellulose tribenzoate, cellulose
• the chiral polysaccharide stationary phase comprising silica gel supporting a microcrystalline cellulose triacetate derivative is MCTA or CTA-I (Merck).
• the chiral polysaccharide stationary phase comprising silica gel supporting a cellulose derivative wherein each glucose monomeric unit thereof is substituted with three benzoyl groups is CHIRALCEL® OBTM (Chiral Technologies, Inc., West Chester, PA).
• the chiral polysaccharide stationary phase comprising silica gel supporting a cellulose derivative wherein each glucose monomeric unit thereof is substituted with three 4-chlorophenylaminocarbonyl groups is CHIRALCEL® OFTM (Chiral Technologies, Inc., West Chester, PA).
• the chiral polysaccharide stationary phase comprising silica gel supporting a cellulose derivative wherein each glucose monomeric unit thereof is substituted with three 3,5-dimethylphenyl carbamate groups is CHIRALCEL® ODTM (Chiral Technologies, Inc., West Chester, PA).
• the chiral polysaccharide stationary phase comprising silica gel supporting a cellulose derivative wherein each glucose monomeric unit thereof is substituted with three 4- methylbenzoyl groups is CHIRALCEL® OJTM (Chiral Technologies, Inc., West Chester, PA).
• the chiral polysaccharide stationary phase comprising silica gel supporting an amylose derivative wherein each glucose monomeric unit thereof is substituted with three 3,5-dimethylphenyl carbamate groups is
• CHIRALPAK® ADTM Chiral Technologies, Inc., West Chester, PA
• the chiral polysaccharide stationary phase comprising silica gel supporting an amylose derivative wherein each glucose monomeric unit thereof is substituted with three (S)-alpha-phenethylcarbamate groups is CHIRALPAK® ASTM or
• CHIRALPAK® AS-VTM Chiral Technologies, Inc., West Chester, PA.
• the chiral polysaccharide stationary phase comprising silica gel supporting a cellulose tris(3,5-dichlorophenylcarbamate) is CHIRALPAK® ICTM (Chiral Technologies, Inc., West Chester, PA).
• CHIRALPAK® ICTM Chiral Technologies, Inc., West Chester, PA.
• polysaccharide stationary phase is from Daicel Chemical Industries Ltd., Japan. These stationary phases are described in US 4,912,205 and US 5,434,299.
• the chiral tartaric acid stationary phases include silica gel supporting ⁇ , ⁇ '-bis (3,5- dimethylbenzoyl)-N,N'-diallyl-L-tartar diamide that is polymerized with a multifunctional hydrosilane to give a covalently bound chiral material or silica gel supporting ⁇ , ⁇ '-bis (4-tert-butylbenzoyl)-N,N'-diallyl-L-tartar diamide that is polymerized with a multifunctional hydrosilane to give a covalently bound chiral material.
• the chiral tartaric acid stationary phase comprising silica gel supporting ⁇ , ⁇ '-bis (3,5-dimethylbenzoyl)-N,N'-diallyl-L-tartar diamide that is polymerized with a multifunctional hydrosilane to give a covalently bound chiral material is KROMASIL® DMB (Eka Nobel AB, Bohus, Sweden).
• the chiral tartaric acid stationary phase comprising silica gel supporting ⁇ , ⁇ '-bis (4-tert- butylbenzoyl)-N,N'-diallyl-L-tartar diamide that is polymerized with a multifunctional hydrosilane to give a covalently bound chiral material is KROMASIL® TBB (Eka Nobel AB, Bohus, Sweden).
• polyacrylamide/silica composite stationary phases include CHIRASPHER® (Merck KGAA, Darmstadt, Germany) available from E. Merck.
• the chiral 3, 5 -dinitrobenzoyl-phenylgly cine-derived stationary phases are ⁇ -acidic and ⁇ -basic (Pirkle-type) phases that include DNBPG available from Regis
• the chiral JI-tris(l,10-phenanthroline)ruthenium sodium magnesium- derived stationary phases are metal complex based phases that include Ceramosphere available from Shiseido Company, Ltd., Japan.
• the chiral stationary phase in a method of the present invention may comprise a solid support selected from silica gel, zirconium, magnesia, titanium oxide, glass, kaolin, alumina, a ceramic, and a silica other than silica gel.
• the chiral stationary phase may comprise a solid support selected from polystyrene, polyacrylamide, and polyacrylate.
• the phrase "reverse phase, chiral stationary phase” means an immobile phase suitable for reverse phase enantio selective liquid chromatography, comprising a solid support and a chiral adduct, wherein the chiral adduct may be covalently bound to the solid support.
• the reverse phase, chiral stationary phases useful in a method of the present invention include a chiral a-cyclodextrin stationary phase, a chiral ⁇ -cyclodextrin stationary phase, a chiral ⁇ -cyclodextrin stationary phase, a chiral macrocyclic glycopeptide stationary phase, a chiral D-amine stationary phase, a chiral cd-acid glycopeptide stationary phase, a chiral cellobiohydrolase stationary phase, and a chiral human serum albumin stationary phase.
• Examples include a chiral a-cyclodextrin stationary phase, a chiral ⁇ -cyclodextrin stationary phase, a chiral ⁇ -cyclodextrin stationary phase, a chiral macrocyclic glycopeptide stationary phase, a chiral D-amine stationary phase, a chiral cd-acid glycopeptide stationary phase, a chiral cellobiohydrolase stationary phase, and a
• cyclodextrin stationary phase or a chiral macrocyclic glycopeptide stationary phase.
• the chiral [a] -cyclodextrin stationary phase is CYCLOBOND III (Advanced Separation Technologies, Inc., Whippany, New Jersey).
• the chiral [beta] -cyclodextrin stationary phase is CYCLOBOND I 2000 or CYCLOBOND I 2000 DM (Advanced Separation Technologies, Inc., Whippany, New Jersey).
• the chiral [yj-cyclodextrin stationary phase is
• the chiral macrocyclic glycopeptide (e.g., vancomycin) stationary phase is CHIROBIOTIC® V, CHIROBIOTIC® T, CHIROBIOTIC® TAG, or CHIROBIOTIC® R (all by Advanced Separation Technologies, Inc., Whippany, New Jersey).
• the chiral D-amine stationary phase is ASTEC CLC-D (Advanced Separation Technologies, Inc., Whippany, New Jersey).
• the chiral [alpha]l-acid glycopeptide stationary phase is CHIRAL- AGP® (ChromTech, Ltd., Cheshire, United Kingdom).
• the chiral cellobiohydrolase stationary phase is CHIRAL-CBH (Advanced Separation
• the chiral human serum albumin stationary phase is CHIRAL-HSC (Advanced Separation Technologies, Inc., Whippany, New Jersey).
• the chiral cyclodextrin stationary phase is CYCLOSE® (Chiralsep Corporation, La Frenaye, France) available from ChiralSep, Nucleodex available from MACHEREY-NAGEL Inc., Easton, Pennsylvania, or CHIRASEP® (E. Merck, Darmstadt, Germany) available from YMC, Inc in the United States of America and YMC Europe GmbH, Germany.
• the amount of chiral adduct on solid support in a method of the present invention will typically be from about 1% weight/weight ("wt/wt") to about 99% wt/wt, typically from about 5% wt/wt to about 50% wt/wt, more typically from about 15 > wt/wt to about 30%) wt/wt.
• stationary phases typically have a chiral adduct that is substantially homogeneous.
• Stationary phase particle size in a method of the present invention is from about 1 ⁇ to about 300 ⁇ , typically from about 1 ⁇ to about 100 ⁇ , from about 5 ⁇ to about 75 ⁇ .
• the particle size generally is from about 1 ⁇ ⁇ about 10 ⁇ , typically from about 1 ⁇ to about 300 ⁇ , from about 2 ⁇ to about 100 ⁇ , from about 5 ⁇ to about 75 ⁇ , or from about 10 ⁇ to about 30 ⁇ .
• the particles of the stationary phases useful in a method of the present invention may be poreless or porous. When the particles are porous, the average diameter of the pores typically ranges from about 10 A to about 10,000 A, more typically from about 200 A to about 2,000 A.
• Figure 1 shows the results of a Western Blot analysis as described in Example 1 of the Fas receptor expression in six different glioblastoma multiforme (GBM) cell lines.
• FIG. 2 shows the relative expression of the Fas receptor in six different glioblastoma multiforme (GBM) cell lines, which has been determined according to the method described in example 1.
• GBM glioblastoma multiforme
• Figure 3 shows the separation of racemic AP-121 on Chiralpak IC with
• Figure 4 shows the elution order of single enantiomers of AP-121 on Chiralpak IC (R-enantiomer is first eluting, S-enantiomer is second eluting.)
• Example 1 Fas-Receptor (Fas-R) expression in GBM (glioblastoma multiforme) cell lines
• AP-121 (also known as edelfosine, ET-18-OCH3) is a synthetic analogue of phosphatydilcholine that induces apotosis through activation of Fas signal transduction pathway in human cancer cells. Fas and mitochondria have been involved in the apoptotic response to ET-18-OCH3.
• Bid B cell lymphoma-2-interacting domain
• a Fas mitochondria linker into Fas-containing membrane rafts suggests that Bid could act as a bridge between Fas activation and mitochondria in ET-18-OCH3-induced apoptosis, providing a molecular explanation for the involvement of mitochondria into the Fas-mediated apoptotic response triggered by ET-18-OCH3 (Gajate J. et al, J Exp Med 2004, 200, 353).
• the death inducing signaling complex comprising Fas, Fas-associated death domain (FADD), and caspase-8/10 is assembled via homotypic associations between death domains (DDs) of Fas and FADD and between death effector domains (DEDs) of FADD and caspase-8/10.
• DDs death domains
• DEDs death effector domains
• ET-18-OCH3 acts at the cell membrane and not with DNA. As such, its effects may be independent of the proliferative state of target cells.
• a two-step model for selective action on cancer cells includes uptake into tumor cells, but not in normal cells, and intracellular activation of Fas/CD95 through its translocation and capping into membrane rafts.
• ET-18-OCH3 has recently been shown to target two different subcellular structures in a cell type- dependent manner, namely cell surface lipid rafts in leukemic cells, and endoplasmic reticulum in solid tumor cells as represented by HeLa and A549 (Nieto -Miguel et al, J Biol Chem. 2006).
• Glioblastoma cell lines have differential sensitivity to the ligand of FasR - FasL that may correlate with their ability to undergo increased apoptosis upon treatment with ET-18-OCH3.
• Fas-receptor FasR
• AP-121 the level of Fas-receptor (FasR) expression in 6 glioblastoma cell lines has been analyzed that have been challenged to grow in presence of various concentrations of AP-121. All these cell lines expressed comparable absolute amounts of Fas-receptor except LN-18 cells. The highest level of relative FasR expression was found in LN229, followed by U-87MG, LN18, Ul 18MG, T98G and A172. The IC50 values of AP-121 among these glioma cells were in the same concentration range except LN18 cell line that was about 5 times more sensitive to AP-121. Thus, these results suggest that AP-121 may achieve its effect in
• glioblastoma cells via modulation of phosphatidylcholine biosynthesis and not through Fas-receptor aggregation.
• Cell lines were purchased from the ATCC collection and expanded in tissue culture to prepare a small cell bank of each cell line. Cells were collected, divided into aliquots for each cell line, and cryopreserved frozen stocks were stored in liquid nitrogen for use in future studies. Cell lines
• T98G glioblastoma multiforme, p53 mut, pTEN mut
• LN- 18 (glioblastoma/glioma p53 mut, pTEN w.t.), ATCC #CRL-2610
• LN-229 (glioblastoma, p53mut,pTEN w.t.), ATCC #CRL-2611
• Beta-Actin mAB mouse monoclonal AB, #AC-15, (Sigma), 1 :1000 final dilution
• total cell lysates were prepared in 1 ml of lysis buffer [lxPBS (pH 7.4), 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 20mM NaF, 1 mM Na 4 P 2 O 7 xl0H 2 O, 1 mM Na 3 V0 4 , 30 mM ⁇ -glycerophosphat , 0.5 mM PMSF, complete mini protease inhibitors (Roche)] for 15 min on ice and were centrifuged at 10,000 x g for 30 min. Equal amounts of lysates (20 ⁇ g) were boiled in SDS sample buffer and separated on SDS-PAGE.
• immunoreactive products were detected by primary anti-FasR rabbit polyclonal antibody C-20 (Santa Cruz) at 1 :500 dilution, restained with secondary goat anti-rabbit IgG (Jackson Labs) at 1 :2500 dilution and visualized with ECL detection system (Amersham Pharmacia, Piscataway, NJ).
• Anti-P-actin mouse monoclonal antibody AC- 15 (Sigma) at 1 : 1000 dilution was used as a control primary antibody, and goat anti-mouse IgG (Jackson Labs) at 1 :2500 dilution as secondary antibody.
• the absolute amount of Fas-R has been determined by scanning the Western blot images by using Alpha Innotec Imager station FluorChem8000 with AlphaEaseFCTM software analysis package. To normalize the amount of FasR protein in each cell line, the total amount of Fas-R has been divided on the amount of ⁇ -actin in the same sample. The smallest ratio was taken as 1 and the ratios for the other cell lines have been calculated proportionally to this ratio.
• Fas-R Fas-receptor
• a representative panel of six glioblastoma cell lines with various mutation status of key tumor suppressor genes p53 and pTEN has been selected (cf. Table 1).
• Table 1 Cell lines and their status of p53, pTEN, FasL and FAS expression
• glioblastoma cells express wild type receptor of Fas and its ligand (FasL).
• FasL FasL
• U-87MG, A172, LN-18, LN-229 and T98G cells contain intact Fas and FasL proteins.
• Substantial literature search revealed that U-l 18MG cells have never been tested on the level of Fas receptor and ligand expression.
• a Western Blot analysis using polyclonal rabbit antibody against human Fas receptor was conducted. The results of this experiment are given in Figure 1.
• the western blot analysis of protein extracts from U-118MG cells revealed significant levels of expression of Fas and FasL proteins (cf. Fig. 1).
• the level of the control protein ⁇ -actin varied significantly between the tested samples. Such difference could be explained by a different level of expression of both ⁇ -actin and FasR proteins in these gliomas cell lines.
• the amount of Fas-R detected by Western blot in each cell line was normalized to its expression of ⁇ -actin by using FluorChem8000 imager station.
• Fas-receptor Fas-R
• Fas-R the levels of expression of Fas-R in these cells were determined and compared to the sensitivity of tested cell lines to AP-121. Based on the Western blot analysis with polyclonal AB C-20 (Santa Cruz) to Fas-R, the absolute expression of Fas-receptor in three glioblastoma cell lines A172, LN299, and U-118MG was in the same range (cf. Table 2) and was half that of U87MG cells. Moreover, the amount of FasR was two times lower in T98G and 4 times lower in LN-18 compared to A 172, LN299 or U-1 18MG. Nevertheless, the sensitivity to AP-121 of five glioblastoma cell lines except LN-18 was also in the same micromolar range (cf. table 3), neglecting the differences in Fas protein expression.
• Table 3 Comparison of Fas expression levels and sensitivity to AP-121 in six GBM cell lines.
• LN-18 cell line with lowest protein level of Fas receptor was the most sensitive to AP-121 treatment.
• the absence in correlation between sensitivity of glioblastoma cells to AP-121 and the level of Fas-receptor expression suggests that similarly to NSCLC and cervical carcinoma, the major target of AP-121 in brain cancer cells is phosphatidylcholine biosynthesis.
• the other possible explanation of the absence of correlation between the level of FasR expression and sensitivity to AP-121 could involve ability to form functional DISC complexes and the induction of apoptosis in the tested cell lines.
• Example 2 Comparison of IC50 values for the S-enantiomer, the R-enantiomer and racemic mixtures
• IC50 values for glioblastoma U87MG cells and NSCLC A-549 cells of each of the compounds were determined. Methods for the determination of IC50 values are well known to the person skilled in the art and are e.g. described in WO 2008/055995, in particular in Example 3 of WO 2008/055995. Table 4: IC50 values for the S-enantiomer (AP-121-S), the R-enantiomer (AP-121- R) and the racemat (AP-121) in U87MG cells and A-549 cells
• Example 3 Separation of racemic AP-121 into the S-enantiomer and the R- enantiomer
• a methanol/water mixture (80:20 v/v) was used to ensure the solubility of the racemate and the elution of the S-enantiomer from the column.
• an Agilent 1200 HPLC-system G-2390 (ELSD SEDEX Model 85) using a Chiralpak IC column (Chiral Technologies, Inc., West Chester, PA 19380, USA) was used.
• the identification of the single enantiomers was demonstrated by using analytical samples of racemic AP-121 and the individual enantiomers using the conditions of Example 4. The identification is shown in Figure 4. The separation of the enantiomers is shown in Figure 3.
• Example 4 Separation of racemic AP-121 into the S-enantiomer and the R- enantiomer under optimized conditions Separation with even shorter retention times than in Example 3 could be achieved by using acetonitril methanol 85: 15 (v/v) using ammonium acetate as modifier.
• any numerical value indicated is typically associated with an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question.
• the deviation from the indicated numerical value is in the range of ⁇ 10% and preferably ⁇ 5%.

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