CZ2009725A3 - Cholestane derivatives for use as antitumor and antiangiogenic medicaments and pharmaceutical com,positions containing thereof - Google Patents

Abstract

The present invention relates to cholestane derivatives and pharmaceutical compositions thereof for use as antitumor and antiangiogenic drugs, including a method for reducing the growth or complete and selective proliferation capacity of tumor cells. The invention also encompasses methods for arresting the cell cycle using cholestane derivatives resulting from apoptotic changes in the tumor cell. The invention also provides the use of these agents to inhibit angiogenesis through inhibition of migration and tubule formation of human endothelial cells. The invention encompasses their use in the control of the adverse effects of hyperpoliferation and angiogenesis of mammalian cells in vitro and in vivo, in particular the treatment of hyperproliferative diseases in a mammal by means of a composition comprising cholestane derivatives. The present invention also discloses novel therapeutic methods for modifying proliferation and invasion of breast and prostate tumor cells.

Description

Technical field

The invention relates to cholestane derivatives for use in inhibiting hyperproliferation and angiogenesis of mammalian cells, for use in treating proliferative and angiogenic diseases in mammals, for use in treating cancer, and for preventing, inhibiting and treating angiogenesis.

BACKGROUND OF THE INVENTION

The present invention relates to cholestane derivatives, pharmaceutical compositions containing these compounds, and their use in particular in anticancer therapy.

Angiogenesis, the growth of new blood capillaries in animals, is essential for organ growth (Folkman et al., 1974, Adv Cancer Res., 19 (0): 331-358; Folkman et al., 1992, J Biol Chem., 267 ( 16): 10931-10934; Carmeliet, 2005, Nature, 438 (7070): 932-936) and also for the growth of compact tumors and metastases (Bhat and Singh, 2008, Food Chem Toxicol, 46: 1334-1345; Kesisis et al. , 2007, Curr Pharm Des., 13 (27): 2795-2809, Risau, 1997, Nature, 386 (6626): 671-674). Endothelial cells are essential for angiogenesis (Bagley et al., 2003, Cancer Res., 63 (18): 5866-5873) and may be targets for anti-angiogenic therapies as they are untransformed and can be easily treated with attainable doses of anti-angiogenic agents in which drug resistance is unlikely to develop (Bhat and Singh, 2008, Food Chem Toxicol, 46: 1334-1345). Blood supply is crucial for cell nutrition and oxygen distribution. Tumor angiogenesis is a target for discovering new potential drugs. It involves inhibition of proteolytic enzymes that disrupt the extracellular matrix surrounding existing capillaries, inhibition of proliferation and migration, as well as an increase in the number of apoptotic endothelial cells. Effective angiogenic inhibitors capable of blocking tumor growth are well placed to develop new generations of anticancer drugs. (Gourley and Williamson, 2000, Curr Pharm Des., 6 (4): 417-39; Ranieri and Gasparini, 2001; Curr Drug Targets Immune Endocr Metabol Disord., 1 (3): 241-253; Singh and Agarwal, 2003 Curr Cancer Drug Targets, 3 (3): 205-217.

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Many small molecules associated with growth factors have proteins anti-angiogenic: proteins and antibodies. Some steroids were among the first molecules in which anti-angiogenic activity was discovered. These include progestin, medroxyprogesterone acetate, and glucocorticoids such as dexamethasone and cortisone (Folkman et al., 1992, Sem Cancer Biol, 3: 89-96; Folkman et al., 1992, J Biol Chem, 267: 10931-4 ). These angiostatic steroids appear to have a direct antitumor effect on some cancers, as well as a variety of tumor angiogenesis effects, including inhibition of endothelial cell proliferation, inhibition of collagen dissolution, and activation of plasminogen production. Primarily, the activities of these agents were characterized by their ability to prevent the formation of new blood capillaries in the chick embryo allantoic membrane assay (CAM test) system for angiogenesis (Pietras and Weinberg, 2005, Evid Based Complement Altemat Med., 2 (1): 4957). The most effective of these anti-angiogenic steroids, 1α-hydrocortisone and tetrahydrocortisol, lacks mineralocorticoid or glucocorticoid activity and capillary regression in the CAM test system (Teicher et al., 1998, Anticancer Res, 18: 2567-74).

Some anti-angiogenic steroids have measurable activity in animal models, which is often elevated in combination with heparin and related molecules (Teicher et al., 1998, Anticancer Res, 18: 2567-74; Folkman et al., 1983, Science, 221: Sakamoto et al., 1987, J Natl Cancer Inst, 78: 581-5.). Of these anti-angiogenic steroids, it is only medroxyprogesterone that has been clinically tested in patients with drive-treated breast cancer (Jakobsen et al., 1986, Eur J Cancer Clin Oncol, 22: 1067-72.). The study ended because medroxyprogesterone showed minimal clinical activity and did not prolong survival.

The endogenous estrogen metabolite 2-methoxyestradiol has been shown to have anti-angiogenic effects in vitro: inhibition of proliferation, migration and invasion as well as in vivo models (Mooberry et al., 2003, Drug Resist Updates, 6: 355-61). The mechanism of action of this compound is not yet fully understood, but does not appear to be transmitted via classical steroid receptors. Recent evidence suggests that 2-methoxyestradiol inhibits HIF-Ια, which is a key angiogenic transcription factor and is therefore capable of inducing a broad spectrum of cellular effects. This activity of 2-methoxyestradiol correlates with the depolymerization of microtubules. These steroid compounds also initiate apoptosis in vascular endothelial cells and compact tumor cells. In preclinical models, 2-methoxyestradiol reduced tumor size and blood supply. This compound was well tolerated in the initial clinical trials

4 • Patients with different types of cancer (Mooberry et al., 2003, Drug Resist Updates, 6: 355-61).

Squalamin, a natural steroid found in several tissues of Squalus acanthias, causes changes in vascular endothelial cell shape and has significant anti-angiogenic activity in lung, breast, brain and ovarian tumors (Teicher et al., 1998, Anticancer Res, 18: 2567 -74; Eckhardt, 1999, Hospital Pract., 1: 63-78; Sills et al., 1998, Cancer Res, 58: 2784-92). Squalalamine has been first discovered in bile synthesis sites such as the liver and gallbladder, but in minor amounts this aminosterol occurs in the spleen, testes, stomach and intestines (Moore et al., 1993, Proc Natl Acad Sci USA, 90 : 13548). Later, squalalamine has been shown to selectively inhibit the formation of new blood capillaries at relatively low doses. Unlike the aforementioned steroids, squalalamine has significant structural differences and does not act on glucocorticoid and mineralocorticoid receptors (Bruss, 2000, American Cancer Society Guide to Complementary and Alternative Cancer Methods. Atlanta, GA: American Cancer Society). Squalalamine is sperm-conjugated 7,24-dihydroxy-24-sulphate of cholestane at position C-3.

Squalalamine is an angiostatic steroid due to its inhibition of vascular endothelial cell growth in culture, activity in CAM test systems and rabbit comeal micropocket assay in gliomas and lung tumors in vivo (Teicher et al., 1998, Anticancer Res, 18: 2567-74 Sills et al., 1998, Cancer Res 58: 2784-92; Williams, 1999, In Teicher: Antiangiogenic Agents. Totowa, NJ: Humana Press). Squalalamine is somewhat unique among current anti-angiogenic compounds because it inhibits endothelial cell proliferation and migration induced by various types of growth factors, including fibroblast core growth factor (bFGF) and VEGF (Eckhardt, 1999, Hospital Pract, 1: 63-78; Sills et al. , 1998, Cancer Res, 58: 2784-92; Williams, 1999, In Teicher, Antiangiogenic Agents. Totowa, NJ: Humana Press).

These general anti-angiogenic effects of squalalamine may result from inhibition of the surface sodium-proton pump (hence, it changes intracellular pH and thus limits intracellular signaling by some growth factors) and other outgoing signal to endothelial cells (Eckhardt, 1999, Hospital Pract, 1: 63-78; Moore et al., 1993, Proc Natl Acad Sci USA, 90: 1354-8). There are various theories about the mechanism of action of squalalamine that remains to be explored.

It is an object of the present invention to provide cholestane derivatives useful for inhibiting the growth of many different tumor cell lines at micromolar concentrations with minimal effects on normal cells. These antiproliferative effects may at least partially depend on the interaction with steroid receptors. Cholestane derivatives are a new group of substances that are not fully investigated at the molecular level of the mechanism of action and our aim here is to provide an explanation of the molecular mechanisms of action. We show that treatment with cholestane derivatives also induces cytotoxicity and growth inhibition of breast and prostate tumor cells. The cholestane derivatives described herein are also capable of inhibiting angiogenesis in tumors and can therefore be used as antimitotic and proapoptotic agents, and thus as antitumor drugs. It is also an object of the present invention to provide anti-angiogenic compounds based on cholestane derivatives having a high selectivity and efficiency index, i. they are less toxic but more effective than the previously known analogs.

SUMMARY OF THE INVENTION

The invention relates to cholestane derivatives of the formula I

(a) and pharmaceutically acceptable salts thereof, in which:

X is CH or O,

R ¹ represents hydrogen, hydroxyl or, if X is oxygen atom, then R ^< nothing,

R ² represents hydrogen, hydroxyl or keto group, or together with R ³ may represent oxido group,

R ³ is hydrogen, hydroxyl, keto, acetoxy, halogen, EtOCOO that when R ³ represents a group EtOCOO, then between carbons CI and C2 double bond,

R ⁴ represents hydrogen, deuterium, hydroxy, halogen, H2P, or in the case or delta5 ~ 4-double bond or 3,5-cyklovazby nothing,

R ⁵ denotes hydrogen or hydroxyl,

R ⁶ represents a hydrogen atom or a hydroxyl group -YZ- means one of the following structures:

or there is no bond between Y and Z, in which case both Y and Z are COOH, and

...... means single or double bonds, provided that when it is a double bond R ^{4 is} nothing, with the proviso that all R ¹ to R ⁶ may independently have an R or S configuration for use as a medicament.

In particular, the present invention includes said derivatives for use in inhibiting proliferation and inducing apoptosis of mammalian cells, in particular for selective prevention, inhibition and / or treatment

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Abnormal proliferation of tumor cells and preferably for the prevention, inhibition and / or treatment of tumors and angiogenesis.

The invention also provides cholestane derivatives of formula I for use in inhibiting cell proliferation and angiogenesis and inducing apoptosis.

The invention also provides cholestane derivatives of formula I for use in inhibiting cell proliferation and angiogenesis and for inducing apoptosis in mammalian cells. These cholestane derivatives are particularly useful in the treatment of diseases that include cell proliferation and angiogenesis, such as tumors, restenosis, rheumatoid arthritis, lupus, multiple sclerosis, transplant rejection (host versus transplant, transplant versus host). The treatment of the disease is carried out by administering to the mammal a therapeutically effective amount of a cholestane derivative of formula I or a mixture of cholestane derivatives of formula I.

The invention also provides cholestane derivatives of formula I for use in the treatment of cancer and angiogenesis in tumors.

The invention further provides cholestane derivatives of formula I for use as growth regulators in animal and human tissue cultures for regulating proliferation and angiogenesis.

The present invention also provides pharmaceutical compositions comprising the cholestane derivatives of this invention and pharmaceutically acceptable carriers.

The invention further provides pharmaceutical compositions comprising one or more excipients in admixture.

The invention also encompasses cholestane derivatives in the form of the free compound of Formula I or in the form of pharmaceutically acceptable salts. The pharmaceutically acceptable salt may be an alkali metal, ammonia or amine salt of such a compound, an acid addition salt, optionally a racemate, or an optically active isomer.

··

Particularly preferred compounds of the invention are cholestane derivatives of formula I selected from the group consisting of:

o o

Therapeutic applications

Suitable routes of administration are oral, rectal, vascular topical (including ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravitrous, intravenous, intradermal, intrathecal and epidural). The preferred route of administration depends on the condition of the patient, the toxicity of the compound and the site of infection, among other considerations known to the clinician.

The therapeutic composition contains from 1 to 95% active ingredient, the single doses preferably containing from 20 to 90% active ingredient and preferably from 5 to 20% active ingredient in non-single use routes. Unit dosage forms are, for example, film-coated tablets, tablets, ampoules, vials, suppositories or capsules. Other forms of application are, for example, ointments, creams, pastes, foams, elixirs, lipsticks, drops, sprays, dispersions, etc. Examples are capsules containing from 0.05 g to 1.0 g of active ingredient.

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The pharmaceutical compositions of the invention are prepared in a known manner, e.g. by conventional mixing, granulating, coating, dissolving or lyophilizing processes.

Preferably, solutions of the active substances are used, as well as suspensions or dispersions, in particular isotonic aqueous solutions, suspensions and dispersions, which can be prepared before use, for example in the case of lyophilized preparations containing the active substance alone or with a carrier such as mannitol. The pharmaceutical preparations may be sterilized or contain substances of a neutral nature, such as preservatives, stabilizers, humectants or emulsifiers, solubilizers, salts for regulating the osmotic pressure or buffers. They are prepared in a known manner, e.g. by conventional dissolution or lyophilization. Said solutions or suspensions may contain viscosity enhancers such as sodium carboxymethylcellulose, dextran, polyvinyl pyrrolidone or gelatin.

The oily suspensions contain as the oil component the vegetable, synthetic or semisynthetic oils customary for injection purposes. The oils which may be mentioned herein are in particular liquid fatty acid esters which contain, as the acid component, a long chain fatty acid having 8-22, preferably 12-22 carbon atoms, e.g. lauric, tridecanoic, myristic, pentadecanoic, palmitic, margaric, stearic, arachidonic and behenic acids, or the corresponding unsaturated acids such as oleic, alaidic, euricic, brasidic and linoleic acids, optionally with the addition of antioxidants such as vitamin E, β-carotene or 3,5-di- butyl-4-hydroxytoluene. The alcohol component of these fatty acid esters has no more than 6 carbon atoms and is a mono or polyhydroxy alcohol, such as mono-, di- or trihydroxy alcohols such as methanol, ethanol, propanol, butanol or pentanol and their isomers, but mainly glycol and glycerol. Fatty acid esters are preferably, for example, ethyl oleate, isopropyl myristate, isopropyl palmitate, "Labrafil M 2375" (polyoxyethylene glycerol trioleate, Gattefoseé, Paris), "Labrafil M 1944 CS" (unsaturated polyglycolized glycerides prepared by alcoholysis of apricot kernel oil) and composed of glycerides and esters of polyethylene glycol; Gattefoseé, Paris), "Labrasol" (saturated polyglycolized glycerides prepared by TCM alcoholysis and composed of glycerides and esters of polyethylene glycol; Gattefoseé, Paris), "Miglyol 812" (triglyceride of saturated fatty acids with chain length (Cg to C12 from Hols AG, Germany) and in particular vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and peanut oil.

The preparation of the injectable preparation is carried out under sterile conditions in the usual manner, for example by filling into ampoules or vials and sealing the containers.

E.g. pharmaceutical preparations for oral use can be obtained by mixing the active ingredient with one or more solid carriers, optionally granulating the resulting mixture, and if desired by processing the mixture or granules into tablets or film-coated tablets by the addition of other neutral substances.

Suitable carriers are, in particular, fillers such as sugars, e.g. lactose, sucrose, mannitol or sorbitol, cellulose preparations and / or calcium phosphates, preferably calcium phosphate or dicalcium phosphate, further binding agents such as starches, preferably corn, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, and if desired disintegrants such as the aforementioned starches, and further carboxymethyl starch, cross-linked polyvinylpyrrolidine, alginic acid and salts thereof, preferably sodium alginate. Other neutral substances are flow regulators and lubricants, preferably salicylic acid, talc, stearic acid and salts thereof such as magnesium or calcium stearate, polyethylene glycol or derivatives thereof.

The coated tablet cores may be provided with suitable coatings which may be resistant to gastric juice, the coatings used being, among others, concentrated sugar solutions which may contain gum arabic, talc, polyvinylpyrrolidine, polyethylene glycol and / or titanium dioxide, and coating solutions in suitable organic solvents. or mixtures of solvents, or for the preparation of gastric juice-resistant coatings, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments are admixed into tablets or coated tablets, eg, to identify or characterize different doses of the active ingredient.

Pharmaceutical compositions which can be taken orally are also hard gelatine capsules or soft sealed gelatine capsules and a plasticizer such as glycerol or sorbitol. The hard capsules may contain the active ingredient in the form of granules, mixed with, for example, fillers such as corn starch, binders or lubricants such as talc or magnesium stearate, and stabilizers. In soft capsules, the active agent is preferably dissolved or suspended in suitable liquid substances of a neutral nature such as a lubricating grease, paraffin oil or liquid polyethylene glycol or fatty acid esters and ethylene; or

propylene glycol, and it is also possible to add stabilizers and detergents, e.g. of the polyethylene sorbitan fatty acid ester type.

Other forms of oral administration are, for example, syrups formulated in a conventional manner, containing the active ingredient, e.g., in suspended form and at a concentration of about 5 to 20%, preferably about 10% or a similar concentration that allows a suitable individual dose, e.g. or 10 ml. Other forms are eg powder or liquid concentrates for making cocktails, eg in milk. Such concentrates may also be packaged in an amount corresponding to a unit dose.

Pharmaceutical preparations which can be used rectally are, for example, suppositories which contain a combination of the active ingredient with a base. Suitable bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alcohols.

Formulations suitable for parenteral administration are aqueous solutions of the active ingredient in a water-soluble form, eg, a water-soluble salt or aqueous suspension for injection, which contains viscosity enhancers, eg, sodium carboxymethylcellulose, sorbitol or dextran, and stabilizers where appropriate . The active agent may also be present in the form of a lyophilisate together with neutral agents where appropriate and may be dissolved prior to parenteral administration by addition of suitable solvents. Solutions which are used for parenteral administration can be used, for example, for infusion solutions. Preferred preservatives are preferably antioxidants such as ascorbic acid or microbicides sorbic or benzoic acid.

Ointments are oil-in-water emulsions containing not more than 70% but preferably 20 to 50% water or an aqueous phase. The fatty phase consists of hydrocarbons, such as petrolatum, paraffin oil or hard paraffins, which preferably contain suitable hydroxy compounds such as fatty alcohols and esters thereof, such as cetyl alcohol or lanolin alcohols, preferably lanolin to improve the water-binding capacity. Emulsifiers are the corresponding lipophilic compounds such as sorbitan fatty acid esters (Span), preferably sorbitan oleate or sorbitan isostearate. Additives to the aqueous phase are, for example, wetting agents such as polyalcohols such as glycerol, propylene glycol, sorbitol and polyethylene glycol, or preservatives or pleasant fragrances.

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Fatty ointments are non-aqueous and contain mainly hydrocarbons, such as paraffin, petrolatum or paraffin oil, as well as natural or semisynthetic fats, such as hydrogenated coconut fatty acid triglycerides or hydrogenated oils, such as hydrogenated castor oil or groundnuts, and partial glycerol fatty acid esters such as glycerol mono- and distearate. They also contain, for example, fatty alcohols, emulsifiers and additives mentioned in connection with ointments which increase water intake.

Creams are oil-in-water emulsions that contain more than 50% water. The oily bases used are fatty alcohols such as isopropyl myristate, lanolin, beeswax, or hydrocarbons, preferably petrolatum and paraffin oil. Emulsifiers are surface-active compounds with predominantly hydrophilic properties, such as the corresponding nonionic emulsifiers, e.g. polyalcohol fatty acid esters or ethyl enoxyaducts thereof, e.g. polyglyceric fatty acids or polyethylene sorbitan esters or polyglyceric fatty acid esters (Tween), and polyoxyethylene fatty alcohol ethers or polyoxyethylene fatty acid esters, or the corresponding ionic emulsifiers, such as alkali salts of fatty alcohol sulphates, preferably sodium lauryl sulphate, sodium cetyl sulphate or sodium stearyl sulphate, which are usually used in the presence of fatty alcohols such as cetyl stearyl alcohol or stearyl alcohol. Additives to the aqueous phase include, but are not limited to, agents that protect creams from drying out, such as polyalcohols such as glycerol, sorbitol, propylene glycol, and polyethylene glycol, as well as preservatives and pleasant fragrances.

Pastes are creams or ointments containing secretion-absorbing powder components such as metal oxides, such as titanium oxides or zinc oxide, as well as talc or aluminum silicates, which are designed to bind the moisture or secretion present.

The foams are applied from pressurized containers and are liquid oil-in-water emulsions in aerosol form using halogenated hydrocarbons such as chlorofluoro- (lower) alkanes, e.g. dichlorofluoromethane and dichlorotetrafluoroethane, or preferably non-halogenated gaseous hydrocarbons, air, N 2 O as propellants. or carbon dioxide. The oil phases used are the same as for ointments and the additives mentioned therein are also used.

Tinctures and solutions usually contain a water-ethanolic base to which are added humectants to reduce evaporation such as polyalcohols, e.g., glycerol, lower. Glycols and polyethylene glycol, as well as lubricants such as fatty acid esters of polyethylene glycols, ie lipophilic substances soluble in an aqueous mixture replacing fatty substances removed from the skin with ethanol and, if necessary, other neutral substances and additives.

This patent further provides veterinary compositions comprising at least one active ingredient together with a veterinary carrier. Veterinary carriers are materials for administration of the preparation and can be solid, liquid or gaseous substances which are inert or acceptable in veterinary medicine and are compatible with the active ingredient. These veterinary preparations may be administered orally, parenterally or by any other desired route.

The invention also relates to processes or methods for treating the diseases mentioned above. The compounds may be administered prophylactically or therapeutically as such or in the form of pharmaceutical preparations, preferably in an amount effective against said diseases, wherein in warm-blooded animals, eg, a human, in need of such treatment, the substance is mainly used in the form of a pharmaceutical preparation. A daily dose of about 0.1 to 50 g, preferably 0.5 to 10 g, is applied to a body weight of about 70 kg.

List of illustrations

Giant. 1 shows the inhibition of the effect of 39 and 42 on the cell viability of the MCF-7 and MDA-MB-468 tumor cell lines. The data represent the results of three independent experiments from which the standard deviation was determined.

Giant. 2 presents the results of the MTT assay where the cytotoxic effect of selected cholestane derivatives (39 and 42) was found in hormone-dependent and hormone-independent breast cancer-derived cell lines (MCF-7 and MDA-MB-468). Both cholestane derivatives inhibited cell growth of both cell lines in a concentration-dependent manner. MDA-MB-468 cells were more sensitive to the administration of cholestane derivatives compared to MCF-7 cells. Table 2 summarizes the IC 50 values (concentration of compounds resulting in 50% inhibition of cell viability after 24h incubation of cells with test substance) calculated from Figure 2.

Giant. 3 shows the results of cell cycle analysis including the subGi phase of the MCF-7 (A) and MDA-MB-468 (B) lines by flow cytometry. Control untreated cells were compared to those treated with 39 and 42. The graphs represent cells in subGi (apoptotic cells with reduced DNA content), Go / Gj, S and G2 / M. Histograms of treated cells were compared to control untreated cells. The data show the number of cells in percent (%) at each stage of the cell cycle.

Giant. 4 shows a Western blot analysis of proteins (pbcl-2, bcl-2, pRb, Rb, mcl-1, p53) involved in the regulation of apoptosis and cell cycle in mammalian tumor line (MDAMB-468). Protein expression in cells treated with 39 and 42 (5, 15, 30, 40 μΜ) for 24 (A) and 48 hours (B) was compared to protein expression of untreated control cells. Expression of PCNA or α-tubulin proteins was used as an internal control of sample protein levels.

Giant. 5 represents the effect of cholestane derivatives on the expression of steroid receptor proteins (AR, ER-α, ER-β) and cell cycle proteins (CDK-4) in the MCF-7 cell line. The protein expression of cells treated with test substances 39 and 42 (5, 15, 30, 40 μ 40) for 24 and 48 h was compared with that of control cells. Internal control of protein levels was the detection of mcm-7 or α-tubulin.

Giant. 6 shows caspas-3/7 activity in MDA-MB-468 breast cancer cells. Cells treated with cholestane derivatives 39 and 42 were compared to control untreated cells after 24 and 48 h. Data show an increase in relative caspas-3/7 activity.

Giant. 7 shows the inhibitory effect of cholestane derivatives 39 and 42 on the viability of HMEC-1 cells. Cytotoxicity was measured by the Crystal violet assay. Cell viability is presented as a percentage of viable cells (100% control) after 72 h. The results were obtained from three independent experiments performed in hexaplicates from which the standard deviation was determined.

Giant. 8 depicts the results of cell cycle analysis of human microvascular endothelial HMEC-1 cells treated with compound 42 after 24 and 48 h by flow cytometry. The graph represents the number of cells in the G0 / G1, S, and G2 / M phase of the cell cycle compared to control untreated cells. The results were obtained from three independent experiments performed in triplicate.

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Giant. 9 shows the results of quantification of apoptosis (subGi peak) in microvascular endothelial HMEC-1 cells treated with compound 42 after 24 and 48 h. Three concentrations of compound were compared to control untreated cells. Data presented are averages of three independent experiments from which the standard deviation was determined.

Giant. 10 shows inhibition of migration of human HUVEC endothelial cells treated with 30 μ 30 42 (A). Data presented are averages of three independent experiments from which the standard deviation was determined. Scoring test images: control untreated cells (B) and 30 μΜ 42 (C).

Giant. 11 shows the effect of 30 μΜ 42 on tubule formation of human endothelial HMEC-1 cells (A). Data presented are averages of three independent experiments from which the standard deviation was determined. The tubule formation images are control untreated cells (B) and 30 μΜ 42 (C).

The following examples serve to illustrate the use of cholestane derivatives without limiting it in any way.

DETAILED DESCRIPTION OF THE INVENTION

General Protocol

Stocks of test substances (10 mM) were prepared by dissolving an equivalent amount of the substance in dimethylsulfoxide. Culture media (DMEM, RPMI 1640, F-12), fetal calf serum, L-glutamine, penicillin and streptomycin were purchased from Sigma (MO, USA), 3- (4,5-dimethylthiazol-2-yl) -2 _J 5-diphenyltetrazolium bromide (MTT) was from Serva Electrophoresis in (Heidelberg, Germany) and Calcein AM from Molecular Probes (Invitrogen Corporation, CA, USA).

All cell lines used (T-lymphoblastic leukemia cell line CEM, mammary carcinoma-derived cell lines MCF-7 (estrogen-sensitive) and MDAMB-468 (estrogen-non-sensitive), LNCaP (androgen-sensitive) and DU-145 (androgen-insensitive) derived from prostate cancer, HeLa cervical cancer cells, human multiple myeloma cells RPMI 8226, human cell line

glioblastoma T 98 and normal human fibroblasts (BJ) were obtained from the registry of the American Type Culture Collection (Manassas, VA, USA). MCF-7 cells were cultured in F-12 medium (Sigma, MO, USA). LNCaP cells were cultured in RPMI 1640 medium (GIBCO, USA). All other cells were cultured in DMEM medium (Sigma, MO, USA). All media used contains 5 g / l glucose, 2 mM glutamine, 100 U / ml penicillin, 100 µg / ml streptomycin, 10% fetal calf serum and sodium bicarbonate. The cells were cultured at 37 ° C, 100% humidity and 5% CO 2. Cell lines were passaged with trypsin (Sigma, USA) two to three times per week.

Human microvascular endothelial cells HMEC-1 were a kind gift F.J. Candala (CDC, Atlanta, GA). Human HUVEC umbilical vein endothelial cells were isolated from the umbilical cord by collagenase digestion and used in two to three consecutive passages as previously described (McGregor et al., 1994, J Pharmacol Toxicol Methods, 32 (2): 73-7). These cells were cultured in cell endothelial growth medium (Provitro, Berlin, Germany) supplemented with 10% serum under standard conditions at 37 ° C and 5% CO 2 in a humid atmosphere (100%). Cells were passaged twice a week with trypsin. Matrigel was purchased from BD Biosciences (NJ, USA).

Statistical analysis

All experiments were performed in at least three independent replicates in triplicates. All quantitative data are presented as mean standard error (SEM) or as mean ± standard deviation (SD).

EXAMPLE 1

Testing of in vitro cytotoxic activity in cell lines of various types

A cell suspension of approximately 1.25 χ 10 ⁵ cells / ml was transferred to 96-well microtiter plates and, after 12 h stabilization, test cholestane derivatives of various concentrations were added. Compounds were dissolved in DMSO before addition to the plates. Control cells were treated with DMSO only. The final DMSO concentration in the test solution did not exceed 1%. Individual concentrations of test substances were added at time zero as a 20μ1 aliquot to the wells of the microtiter plates. Usually

compounds were diluted to six concentrations in a four-fold dilution series. In routine testing, the highest concentration in the well was 50μΜ, and changes in this concentration depend on the substance. Incubation of cells with test derivatives lasted 72 hours at 37 ° C, 100% humidity and 5% CO 2. At the end of the incubation period, cells were analyzed after addition of Calcein AM solution (Molecular Probes) and incubation continued for an additional 1 hour. Fluorescence (OD) was measured using a Fluoroskan Ascent Labsystem FIA reader (Microsystems). Tumor cell survival (IC 50) was calculated according to the following formula:

IC5 () - (OD exposed wells / control OD wells) 100%.

The IC 50 value, which corresponds to the concentration of the substance where 50% of the tumor cells are killed, was calculated from the obtained dose curves.

Cell lines of different histogenetic origin were used to monitor the cytotoxic effect of cholestane derivatives and related steroids. The human Tlymphoblastic leukemia cell line CEM, cervical carcinoma cells HeLa, the multiple myeloma cell line RPMI 8226, the human glioblastoma cell line T98 and normal human fibroblasts BJ were used for routine screening of the cytotoxic properties of cholestane derivatives. Cells were exposed to 6 concentrations in quadruplicate dilutions for each agent for 72 h to determine the number of surviving cells. IC 50 values obtained with the Calcein AM assay are presented in Table 1.

Tab. 1: Cytotoxicity of cholestane derivatives for different cell line types (IC50: μΜ).

The results were obtained from three independent triplicate experiments.

Cholestane derivatives no. Cell line CEM RPMI 8226 T98 HeLa BJ cholesterol > 50 38.4 > 50 > 50 > 50 1 9.6 12.9 49.9 > 50 2 15.5 > 50 3 4.3 1.7 17.2 14.7 > 50 4 19.7 > ++ 50 5 9.7 42.3 6 10.4 9.0 38.3 21.5 44.6 7 13.9 48.0 8 14.7 47.5 9 14.4 > 50

· V · *

10 11.6 14.4 > 50 11 11.0 47.0 12 16.2 32.2 13 11.8 10.7 14 17.8 16.7 > 50 15 Dec 7.7 16.0 44.1 35.0 45.4 16 8.4 42.7 17 4,5 4.7 46.3 9.0 42.8 18 12.8 48.2 19 Dec 20.5 5.2 41.1 41.2 20 May 10.2 39.8 21 44.5 > 50 22nd 18.5 41.0 23 17.6 > 50 24 19.1 > 50 25 15.5 > 50 26 8.8 3.9 44.9 19.9 35.9 27 Mar: 15.0 > 50 > 50 26 6.3 6.4 16.0 8.7 8.2 29 9.9 12.7 30 12.1 15.0 46.6 15.9 12.7 31 13.1 28.0 47.9 40.6 22.0 32 10.0 19.7 46.6 34.4 44.8 33 18.1 24.5 42.2 40.7 28.5 34 15.3 8.6 44.7 19.0 44.4 35 31.4 36.3 36 17.5 16.1 45.1 33.1 31.5 37 6.9 6.7 46.2 8.7 41.4 38 27.4 8.8 39.9 8.5 7.1 39 6.8 8.5 21.4 13.2 44.6 40 8.3 12.5 43.0 9.8 10.5 41 11.2 16.0 42 5.2 10.5 15.2 11.9 18.4

• 9 ·· ϊ ··· ···

Treatment with cholestane derivatives resulted in a dose-dependent decrease in cell viability of CEM, RPMI 8226 and HeLa cells at various levels. (Table 1). Only active cholestane derivatives are disclosed. The inhibitory effect of 39 and 42 on the breast tumor lines MCF-7 and MDA-MB-468 is shown in Figure 1. Cholestane derivative 3 was the most potent substance on the lymphoblastic leukemia line CEM (IC50: 4.3 μπιοί / ΐ) and on cells multiple myeloma RPMI 8226 (IC50 1.7 μπιοί / ΐ). In addition to compound 3, high cytotoxic efficacy has been reported after administration of compounds 17, 39 and 42. Cholesterol, a non-hairent derivative of plant sterol, is inactive or has almost no cytotoxic activity. Cholesterol also has minimal antitumor activity (IC 50> 35 gmol / L) on cells of the RPMI 8226 line. It is surprising that most cholestane derivatives have little or no effect on the viability of normal human BJ fibroblasts. These results indicate a different effect of cholestane derivatives on tumor cells compared to normal cells. Currently, only a few compounds are known which possess the potential ability to selectively eliminate tumor cells without affecting normal cell growth. This work also provides the first evidence of cholestane derivatives as antitumor compounds with antiproliferative properties.

As shown by studying the relationships between chemical structure and biological activity, the most active cholestane derivatives in the cytotoxicity assay were 3, 39 and 42. Changing the functionality of 6oxo-7-oxalactone to 6-oxo dramatically increases growth inhibition by cholestane derivatives. Thus, compound 42 has three times more potent activity than 36. The 24R side chain is also critical to increase the cytotoxicity of cholestane derivatives. Results from Tab. 1 point to the use of cholestane derivatives as a drug for inhibiting hyperproliferation in tumors.

EXAMPLE 2

Influence of substances on mammalian and prostatic tumor cell lines

Cholestane derivatives analogues were selected for further experiments from the range of tested substances. Their effect was studied in MCF-7 (estrogen-sensitive) and MDA-MB-468 (estrogen-non-sensitive) cell lines derived from mammary gland carcinoma and LNCaP (androgen) cell lines. -sensitive) and DU-145 (androgen-insensitive) derived from prostate cancer.

• 4 *

445 «« *

Prostate cancer in humans is a multistep process that initially is androgen-dependent in most prostate tumors and most patients respond successfully to therapeutic androgen blockade. In some patients, tumors may, over a period of time, pass to the androgen-independent growth stage. These androgen-independent prostate tumors are then resistant to conventional therapy. While androgen-dependent tumor cells undergo apoptotic cell death during androgen ablation, androgen-dependent cells have the ability to bypass apoptotic pathways during androgen deprivation without disrupting this pathway. The development and progression of prostate cancer is related to the accumulation of changes in genes involved in the maintenance of normal cell proliferation and homeostasis. Estrogen-sensitive or estrogen-sensitive mammary tumors represent a similar model of hormonal responsiveness. Nowadays, intensive research efforts are focused on the study of the origin and development of these tumors, which would bring new approaches in cancer therapy.

The effect of substances on cell viability in this case was monitored by an MTT cytotoxicity assay using 3- (4,5-dimethylthiozol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to determine the IC50 concentration of the study compounds in tumor cell lines . 96-well microtiter plates were seeded, depending on cell line type, in 100 μΐ of appropriate medium of 4.5 * 10 ³ (MCF-7, DU-145) or 5 * 10 ³ (MDA-MB-468, LNCaP) cells per well. After 24 h (MCF-7, MDA-MB-468, DU-145) or 48 h (LNCaP) latency during which cells adhered to the bottom of the plate, the cells were affected with selected cholestane derivatives for 24 and 48 h in culture. media. The cells used as controls were incubated with the maximum amount of DMSO solvent only. The concentration of substances (IC50) that resulted in 50% inhibition of cell growth (IC50) was determined from this assay. The measured absorbance, which correlated directly with the number of vital cells, was measured using a Labsystem Multiscan RC ELISA reader at 570 nm. The viability of the affected cells by the test derivatives was related to control cells representing 100% viability. The experiment was performed in triplicate and repeated at least four times independently.

Table 2

Final IC50 (pmol / l) concentrations of cholestane derivatives determined in mammalian and prostate tumor cell lines by MTT cytotoxicity assay. The results were obtained from three independent experiments performed in triplicate.

Test substance MCF-7 Cell line: IC ₅₀ (μπιοί / ΐ) MDA-MB-468 LNCaP DU-145 26 27.8 27.8 13.0 26.0 37 27.4 17.3 16.5 26.0 39 25.9 18.6 29.0 45.0 42 29.5 14.8 27.6 24.0

To determine the effect of selected cholestane derivatives on cell viability, the MTT cytotoxicity assay of hormone sensitive and hormone non-sensitive tumor cell lines (MCF-7, MDA-MB-468, LNCaP and DU-145) was used. Cytotoxic concentrations of derivatives were determined in all cell lines. Cholestane derivatives inhibited cell growth depending on their concentration in all lines. Derivative 42 showed cytotoxic effect at lower concentrations (Fig. 2). Based on the MTT assay, we found concentrations of cholestane derivatives that resulted in a 50% inhibition of cell growth (IC 50) after 24 h (Table 2). Interestingly, there were small differences between the efficacy of cholestane derivatives on different cell lines, with IC50 concentrations ranging at a micromolar level. Only the cell lines MDA-MB-468 and DU-145 showed greater cell resistance to the application of tested cholestane derivatives.

EXAMPLE 3

Effect of cholestane derivatives on caspas-3/7 activity in mammalian cell line tumor cells

The treated cells were harvested by centrifugation and homogenized in extraction buffer (10 mM KCl, 5 mM HEPES, 1 mM EDTA, 1 mM EGTA, 0.2% CHAPS, protease inhibitors, pH 7.4) on ice for 20 min. The homogenates were centrifuged ( 10,000 xg, 20 min, 4 ° C), proteins were quantified by the Bradford method and diluted to the same concentration. Lysates were incubated for 1 h with 100 mM Ac-DEVD-AMC substrate (Sigma-Aldrich) in assay buffer (25 mM PIPES, 2 mM EGTA, 2 mM MgCl ₂ , 5 mM DTT, pH 7.3). For negative

OQ Control Lysates were incubated with 100 mM Ac-DEVD-CHO inhibitor caspas-3/7 (Sigma-Aldrich). Product fluorescence was measured using a Fluoroskan Ascent microplate reader (Labsystems) at 346/442 nm (ex / em).

Caspas-3/7 activity was measured in MDA-MB-468 cells treated with cholestane derivatives 39 and 42 using the fluorogenic substrate Ac-DEVD-AMC and / or the caspas-3/7 inhibitor Ac-DEVD-CHO. Cells were treated with agents in dependence on time and their concentration that exceeded their IC50 values (5, 15, 30, 40 μ po) after 24 and 48 h. Compound 42 strongly induced caspas-3/7 activity; after 24 h treatment of 30 μΜ 42 cells, effector caspas activity increased five-fold compared to untreated control cells. Caspas activity decreased after treatment with 40 μΜ of this substance and / or after prolonged treatment with the substance (48 h) (Fig. 7). In contrast to compound 42, compound 39 only slightly activated caspase-3/7: caspase activity increased twice after 24 hours of treatment with the compound, but this activity decreased after higher dose and / or longer incubation with the substance (data not shown here).

EXAMPLE 4

Cholestane derivatives regulate cell cycle and apoptosis in tumor cells of mammalian lineages

Breast adenocarcinoma MCF-7 cells, which are a frequent experimental model, decrease the proportion of DNA (S phase) synthesizing cells after antiestrogen treatment, causing a typical inhibitory effect. This decrease in the S phase of the cell cycle is accompanied by an increase in the number of cells in the G0 / G1 phase. Changes in cellular signaling also regulate androgen receptor (AR) activity. Various growth factors are capable of activating AR independent of ligand in prostate cancer cells. Although AR may be activated in the absence of hormone, it appears that conditions may be more limited than those observed in ER. The use of physiological doses of ligand (dihydrotestosterone, DHT) is significant in LNCaP cells that only proliferate at a concentration of 10 ^-9 M DHT or less. Higher doses of ligand inhibit proliferation. In contrast to proliferation, activation of specific AR target genes (eg PSA) continues with increasing doses of DHT ^{10 10} M and higher. This could be the reason why cholestane derivatives 39 and 42, due to estrogen or androgen receptors, have potent cytotoxic activity on the hormone-sensitive (MCF-7) cell line compared to hormone-sensitive breast adenocarcinoma cells MDA-MB-468. Therefore, we have verified the possibility that cholestane derivatives could cause cell cycle disorders in breast tumor cells. Flow cytometry was used to evaluate the number of cells in the respective phases of the cell cycle, including cells in the subGi phase. Control and treated cells after 24 and 48 h incubation were washed twice in PBS and centrifuged (1000 xg, 10 min, 4 ° C), washed twice in cold PBS and fixed with ice-cold ethanol (70%; v / v) to Slow stirring. DNA content was determined after staining of the cell nuclei with propidium iodide. Cells were analyzed on a Cell Lab Quanta flow cytometer (Beckman Coulter, CA, USA).

Table 3

Cell cycle phase distribution in breast adenocarcinoma cells MCF-7 and MDA-MB-468 by flow cytometry. Histograms of cells treated with cholestane derivatives were compared with histograms of control cells. The data represent the percentage of cells of the subGi fraction and in the individual phases of the cell cycle Gq / Gi, S, G2 / M.

Cell line Control / Compound (40 μΜ; 24 h) Apoptosis subG / Phases of the cell cycle Gr / Gi WITH G ^ M MCF-7 Control 2% 54% 17% 27% 42 2% 64% 11% 22% 39 20% 41% 13% 27% MDA-MB-468 Control 3% 68% 15% 13% 42 11% 60% 13% 16% 39 15% 57% 9% 10%

Cell cycle analysis by flow cytometry shows cell growth in the subGi phase (apoptotic cells) in the MCF-7 and MDA-MB-468 cell lines after treatment with cholestane derivatives 39 or 42 (Table 3). After treatment with 42, the number of cells in the subGi and Go / G1 phases increased, with a concomitant decrease in the number of S-phase cells in MCF-7 cells. Compound 39 caused a strong increase in the number of apoptotic cells (subGi phase) with a concomitant decrease in the number of cells in the other phases of the cell cycle in the breast tumor cell line MCF-7 (Fig. 3A). In MDA-MB-468 breast adenocarcinoma cells, treatment with 42 caused an increased proportion of cells in the subGi phase and a decrease in the S phase. Compound 39 induced only a slight increase in the number of subGi cells in MDA-MB-468 cells (Fig. 3B). Both cholestane derivatives cause a decrease in the number of cells in the S phase of the cell cycle, but only a stronger effect was seen in estrogen-sensitive MCF-7 cells (Fig. 3). It appears that cholestane derivatives could affect steroid receptors.

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QC · · «C C C C C C

EXAMPLE 5

Effect of 39 and 42 on the expression of steroid receptor proteins and cell cycle proteins in mammalian cell line tumor cells

For Western blot analysis, fifteen to thirty micrograms of proteins were separated on a 10% or 12% polyacrylamide gel, respectively. Electrophoretically separated proteins were transferred to a nitrocellulose membrane (Amersham Biosciences, Austria) using the semidry method. A color molecular weight standard (Rainbow ™, Amersham Biosciences, Austria) was used to determine the molecular weight. Non-specific binding sites were blocked for a minimum of 2 hours in a 5% solution of low fat milk powder with 0.1% Tween 20 in PBS. After incubating the membrane with the appropriate diluted primary antibody for 12-14 h at 4 ° C, the membrane was washed with cold wash buffer (PBS solution with 0.1% Tween 20) for 10 min for one hour. Diluted horseradish peroxidase-conjugated secondary antibody was then applied to the membranes. A secondary rabbit antibody against mouse immunoglobulins (1: 6,000 dilution, Santa Cruz, USA) was used for mouse primary antibodies and a secondary goat anti-rabbit antibody (1: 2,000 dilution, Dako, Denmark) was used. The incubation time of the membrane with the secondary antibody was 45 min at 4 ° C. This was followed by washing of the membrane with cold wash buffer, which was changed for 10 min for one hour. Proteins were detected by a chemiluminescent system (Amersham Biosciences, Austria) according to the manufacturer's protocol. Ponceau S staining (Sigma, USA) and α-tubulin detection (Sigma, USA) or mcm-7 (Santa Cruz, USA) were used to control the quality and quantity of proteins transferred to the membrane. The experiments were repeated three times. Protein expression in cells treated with test substances was compared to protein expression of control cells.

Western blot analysis revealed that MCF-7 control cells showed high expression of both types of estrogen receptors ER-α and ER-β, while MDA-MB468 cells expressed very low levels of ER-β and did not express ER-α. Treatment of cells with 39 in MCF-7 cells resulted in a decrease in ER-α expression in a concentration- and time-dependent manner. ER-β expression was reduced only after 48 hours of exposure to 39 (30 and 40 μΜ). In MDA-MB-468 cells, the ER-β level was at the limit of detection. MCF-7 cells showed a decrease in the level of androgen receptors (AR) after 48 hours of exposure to substance 39 at 40 μΜ. After treatment of MCF-7 cells with substance 42, a decrease in ER-α levels was found both in concentration and in time (Fig. 5).

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A decrease in the cdk-4 cell cycle protein level was found in MCF-7 cells treated with substance 39 (40 μ po) for 48 hours, whereas treatment of cells with substance 42 resulted in a decrease in protein levels depending on the concentration and duration of treatment.

EXAMPLE 6

Detection of apoptosis in breast cancer-derived cells

Apoptosis detection by flow cytometry

According to some studies, apoptosis may be cell cycle dependent. Therefore, in the following experiments we tested whether cholestane derivatives 39 and 42 cause apoptosis of breast tumor cells by blocking the cell cycle. The late phase of apoptosis was detected by fragmentation of low molecular weight DNA by cell cycle analysis by flow cytometry. This technique makes it possible to detect specific forms of cell death based on the cell fraction of the cell cycle subGi. The analysis showed that both cholestane derivatives 39 and 42 increased the percentage of subGi cells in breast cancer cells derived from MCF-7 and MDA-MB-468 compared to control cells (Table 3). Cholestane derivatives mediated apoptotic cell death is an important finding because molecular analysis of human cancers reveals frequent mutation of cell cycle regulators in the most common malignancies.

Apoptotic index detection by TdT-Mediated dUTP nick end labeling (TUNEL)

TUNEL (terminal deoxynucleotide transferase (TdT) -mediated deoxyuridine triphosphate (dUTP) nick end labeling) was also used to detect apoptotic cells. For the analysis were prepared by growing the cells on the cover glasses in 60 mm Petri cell culture dishes in the number l, 4xl0 ⁴ cells / cm ² (MCF-7) or l, 6 × 10 ⁴ cells / cm ² (MDA-MB-468). 24 h followed by incubation of 39 or 42 (5, 15, 30 and 40 μΜ) with the cells for 24 and 48 h. After the treatment period, the cells were subsequently rinsed with PBS and fixed for 10 min with cooled acetone: methanol ( 1: 1, v / v). Apoptosis-induced nuclear DNA fragmentation was detected by terminal deoxynucleotidyltransferase (TdT) binding by TUNEL according to the manufacturer's protocol (In Cell Cell Death Detection Kit; Roche Diagnostics, Mannheim, Germany). After further cell washing in PBS, the nuclei were stained with DAPI (50 µg / ml; Sigma, St. Louis, MO, USA) for 10 min in the dark. This was followed by rapid rinsing of the cells in deionized water and the glass was immersed in Mowiol aqueous medium (Calbiochem, Fremont, CA, USA) diluted in a solution of glycerol and PBS (1: 3, v / v) for fluorescence. The cells affected were analyzed by fluorescence microscopy and compared to control cells.

The TUNEL method was used to detect apoptotic cells after affecting mammalian tumor cell lines with cholestane derivatives 39 and 42. Based on this experiment, both types of derivatives were found to increase the number of TUNEL positive cells depending on their concentration and duration of action.

Table 4

Detection of DNA breaks in nuclei of apoptotic cells determined by TUNEL method in MCF-7 and MDA-MB-468 cells treated with 39 and 42 for 24 and 48 h.

K24 / K48 ..... control cells (24 h, 48 h); % ..... percent of TUNEL positive cells.

MDA-MB-468

MCF-7

24h

48h

39 % 42 % r 39 % 42 ------------ 1 % K24 0 Κ24 0 Κ24 0 Κ24 0 5 μΜ 1 5μΜ 1 5 μΜ 4 5 μΜ 1 15 μΜ 2 15 μΜ 3 15 μΜ 6 15 μΜ 3 30 μΜ 8 30 μΜ 4 30 μΜ 12 30 μΜ 6 L 40 μΜ 12 40 μΜ 10 40 μΜ 20 May 40 μΜ 11 'K48 0 Κ48 0 Κ48 0 Κ48 0 5 μΜ 1 5 μΜ 2 5 μΜ 6 5 μΜ 4 15 μΜ 5 15 μΜ 8 15 μΜ 14 15 μΜ 7 30 μΜ 13 30 μΜ 12 30 μΜ 19 Dec 30 μΜ 15 Dec L 40 μΜ 26 40 μΜ 23 40 μΜ 29 40 μΜ 20 May

24h

48h

EXAMPLE 7

Effect of cholestane derivatives on expression of apoptosis-regulating proteins based on Western blot analysis in mammalian cell line tumor cells

To prepare the samples, cells were seeded in a number of l, 4xl0 ⁴ cells / cm ² (MDA-MB-468 and MCF-7) in the loom culture Petri dishes and after 24 hours at 70-80% confluency were influenced by the test compounds 39 and 42 concentrations of 5, 15, 30 and 40 μΜ. Control cells were prepared by incubation with an appropriate volume of DMSO as vehicle. After 24 and 48 h, cells were washed with cold phosphate buffer (10 mM, pH 7.4) and released from the dishes by scraping into extraction RIPA buffer containing 20 mM Tris-HCl, pH 7.4; 5 mM EDTA; 2 mM EGTA; 100 mM NaCl; 2 mM NaF; 0.2% Nonidet P-40; 30 mM PMSF; 1 mM DTT; 10 mg / ml aprotinin and leupeptin. Cells were incubated in protein extraction buffer for 30 min. with occasional stirring at 4 ° C. After centrifugation (10,000 ^x g, 4 ° C, 30 min), the supernatant was collected, aliquoted, and frozen at -80 ° C. Total protein concentration in the supernatant was measured by the Bradford method according to the manufacturer's protocol (BioRad Laboratories, CA, USA). An appropriate volume of 4 * concentrated lysis buffer (Laemmli buffer: 200 mM TRIS-base, pH 6.8; 400 mM dithiotreitol; 8% SDS, 40% glycerol and bromophenol blue) was added to the protein extracts at a ratio of 3: 1.

Twenty to thirty micrograms of proteins were separated on a 10% or 12% SDS polyacrylamide gel, respectively. Electrophoretically separated proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, CA, USA) and stained with Ponceau S to control the amount of loaded proteins. Membranes were blocked in 5% BSA and 0.1% Tween-20 in TBS buffer for 2 h and incubated with the appropriate primary antibody overnight. After washing in TBS with 0.1% Tween-20 for 30 minutes, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody and visualized with the Super Pico West Pico chemiluminescence system (Thermo Fisher Scientific, Rockford, USA). Monoclonal anti-PCNA or anti-α-tubulin monoclonal antibody immunodetection was used to control the quality and quantity of proteins transferred to the membrane. The experiments were repeated three times. Protein expression in cells treated with cholestane derivatives was compared with protein expression in control cells.

Based on Western blot analysis, changes in the expression of proteins involved in apoptosis were observed in mammary carcinoma-derived cell lines. Cells treated with 39 and 42 for 24 and 48 h at concentrations of 5, 15, 30 and 40 μΜ were used to detect changes during treatment. The changes in expression of individual proteins regulating apoptotic cell death after treatment of cells with cholestane derivatives are shown in FIG. 4. Tumor suppressor p53 was detected in control cells and in cells treated with cholestane derivatives 39 and 42, where it increased expression of this protein after 24 and 48 h. The expression of p53 increased sharply after application of 30 μΜ of 39 and 42 but at 40 μΜ caused a slight decline. Increasing p53 protein expression is associated with a decrease in the anti-apoptotic protein Mcl-1 protein after treatment with 30 and 40 μΜ 39 and 42. A decrease in pRb S780 phosphorylation was found after administration of the same concentrations of test substances at the same times.

Phosphorylation of the antiapoptotic protein pBcl-2 S70 increased after application of 5, 15 and 30 μΜ of test substances 39 and 42 (48 h). (Fig. 4 A, B)

It has been previously reported that activation of the caspase cascade plays an important role in the effector phase of apoptosis (Budihardjo et al., 1999, Annu. Rev. Cell Dev. Biol., 15: 269-290). Caspasa-3 is an execution protease that is involved in the cleavage of proteins involved in cell construction and in the repair of damaged DNA (PARP) during apoptotic cell death (Allen et al., 1998, Cell. Mol. Life Sci. 54: 427-445 Cain et al., 2002, Biochimie 84: 203-214. Our results suggest that cholestane derivatives may promote apoptotic death of breast cancer-derived cell lines through caspase-3 activation (Fig. 6) and modulation of Bcl-2 protein (Fig. 4).

EXAMPLE 8

Antiangiogenic effects of cholestane derivatives on human endothelial cells

Influence of cholestane derivatives on viability of human endothelial cells

Proliferation of endothelial cells was measured using a crystal violet DNA staining assay. HMEC-1 cells were seeded at a density of 1500 cells per well of a 96-well assay plate. After 24 h adheration, one plate was stained with crystal violet (0.5% [v / v] crystal violet, 20% [v / v] methanol) for 10 minutes, rinsed with water and air dried (day 0). The other plates were incubated with the test substances for 72 h. Thereafter, the test compound plates were also stained and dried. The color was then dissolved in ethanol and citrate and the absorbance was measured on a photometer at 540 nm (SPECTRAFluor Plus TECAN, Crailsheim, Germany). Absorption correlates linearly with cell number. Each experiment was performed at least three times in hexaplicates.

We first investigated the effect of cholestane derivatives on the viability of human endothelial cells (HMEC-1) using a crystal violet staining assay. Cells were exposed to a series of different concentrations of solutions of each substance for 72 h. Control cells were treated with DMSO only. Survival cell numbers were estimated after treatment with cholestane derivatives 39 and 42 (Fig. 7). Compounds 39 and 42 reduced the viable cell count to 50% at a dose of 10μΜ.

• ·

Influence of cholestane derivatives on cell cycle and apoptosis

HMEC-1 cells were typified, seeded into 6-well assay plates and immediately incubated with test substance. After 48 h, cells were peeled off with trypsin, washed and stained overnight at 4 ° C in 0.1% [w / v] sodium citrate, 0.1% [v / v] Triton X100, and 50 µg / ml propidium. iodide in PBS. DNA content was evaluated by flow cytometer (FACSCalibur, Becton Dickinson, Heidelberg, Germany). The cell division in the histogram is in the cell cycle phases of subGi ("apoptotic cells"), Go / Gi, S and G ₂ / M. Peaks were quantified using FlowJo software (Tree Star, Oregon, USA).

Flow cytometry analysis was used to quantify the distribution of HMEC-1 cells into individual phases of the cell cycle including subGi cells, an indicator of the number of apoptotic cells. We observed an increase in the number of cells in S phase after incubation with 30 μΜ 42 (Fig. 8).

We also observed an increased number of apoptotic cells after treatment with compound 42, seven times at 24 h and thirteen times at 48 h compared to untreated (Fig. 9). Test compounds were effective in inducing apoptosis and cell cycle arrest.

Cell migration after treatment with test substance

Confluent monolayer HUVEC cells were scraped off and immediately treated with either starved M199 medium (serum free, negative control; PAN Biotech, Aidenbach, Germany) or complete endothelial cell medium containing test substance. After 16 h, images were taken using a TILLvisiON system (TILL Photonics, Lochham, Germany) connected to an Axiovert 200 microscope (Zeiss, Germany). Migration was expressed as the ratio of the cells covered by the cells to the total number of pixels in the groove area [S.CO LifeScience (Garching, Germany)].

We then evaluated the effect of cholestane derivative on HUVEC cell migration by scoring (Liang et al., 2007, Nat Protoc., 2 (2): 329-333). Control cells migrated 100% compared to cells cultured in starving media where they were unable to migrate (0%). The 30 μΜ 42 treatment inhibited migration to 38% (Fig. 10).

• · *

Inhibition of tubule formation by cholestane derivatives

Ibidi μ-plates (18-well, Ibidi GmbH, Munich, Germany) were coated with Matrigel® (Schubert & Weiss-OMNILAB, Munich, Germany). Medium containing 1x10 ⁴ HMEC cells and test substance was added to the wells. After 16 h, images were taken using a TILLvision-system connected to an Axiovert 200 microscope. This experiment was evaluated as the tubule number and node number of treated cells compared to untreated control cells using specific software [S.CO LifeScience (Garching, Germany)].

The tube formation assay used to determine the anti-angiogenic activity of cholestane derivatives was based on in vitro endothelial cell tubulogenesis analysis. Control HMEC-1 cells readily form junction (tubules) on Matrigel®. Cells treated with 30 μΜ 42 reduce the number of tubules to 66% compared to control cells. In contrast to the decreasing number of tubules, the number of kinks was not reduced after application of substance 42 (Fig. 11). Interestingly, incubation with compound 42 increased knot formation.

EXAMPLE 9

Dry capsules

5000 capsules, each containing as active ingredient 0.25 g of one of the compounds of the formula I mentioned in the preceding or following examples, are prepared as follows:

Ingredients

Active ingredient

1250 g

Talc

180 g

Wheat starch

120 g

Magnesium stearate g

Laktosa g

Preparation procedure: Spreaded substances are passed through a sieve with a mesh size of 0.6 mm. Dose

0.33 g of the mixture is transferred to a gelatin capsule using a capsule filling machine.

9 9 9 9 9 99 99

9 ·· ·

EXAMPLE 10

Soft capsules

5000 soft gelatin capsules, each containing as active ingredient 0.05 g of one of the compounds of the formula I mentioned in the preceding or following examples, are prepared as follows:

Ingredients

Active ingredient

Lauroglykol

250 g liters

Preparation procedure: The powdered active substance is suspended in Lauroglycol® (propylene glycol laurate, Gattefoseé SA, Saint Priest, France) and spread in a wet pulverizer to a particle size of about 1 to 3 mm. A batch of 0.419 g of the mixture is then transferred to soft gelatin capsules using a capsule filling machine.

EXAMPLE 11

Soft capsules

5000 soft gelatin capsules, each containing as active ingredient 0.05 g of one of the compounds of the formula I mentioned in the preceding or following Examples, are prepared as follows:

Ingredients Active ingredient 250g PEG 400 1 liter Tween 80 1 liter

Preparation procedure: The powdered active ingredient is suspended in PEG 400 (polyethylene glycol of Mr between 380 and 420, Sigma, Fluka, Aldrich, USA) and Tween® 80 (polyoxyethylene sorbitan monolaurate, Atlas Chem. Ind., Inc., USA, supplied). Sigma, Fluka, Aldrich, USA) and spread in a wet pulverizer to a particle size of 1 to 3 mm. A batch of 0.43 g of the mixture is then transferred to soft gelatin capsules using a capsule filling machine.

Claims (11)

Patent Claims and pharmaceutically acceptable salts thereof in which: X is CH or O, R ¹ represents hydrogen, hydroxyl, or when X is oxygen, then R ¹ does not, R ² represents hydrogen, hydroxyl or keto group, or together with R ³ may represent oxido R ³ is hydrogen, hydroxyl, keto, acetoxy, halogen, EtOCOO that when R ³ represents a group EtOCOO, then between carbons CI and C2 double bond, R ⁴ represents a hydrogen atom, a deuterium atom, a hydroxyl atom, a halogen atom, a H ₂ P group, or, in the case of a delta 4 or delta 5 -double bond or 3,5-cyclo bond, means nothing, R ⁵ denotes hydrogen or hydroxyl, R ⁶ represents a hydrogen atom or a hydroxyl group -YZ- means one of the following structures: or there is no bond between Y and Z, in which case both Y and Z are COOH, and ...... means single or double bonds, provided that when it is a double bond R ^{4 *} means nothing, for use as medicaments. A compound according to claim 1, wherein R ¹ to R ⁶ may be independently in the R or S configuration. The cholestane derivatives according to claim 1 of the general formula I for use in inhibiting hyperproliferation and angiogenesis of mammalian cells. The cholestane derivatives of claim 1 for use in inducing apoptosis in mammalian tumor cells. • v The cholestane derivatives according to claim 1 of formula I for use as growth regulators in animal and human cell tissue cultures for controlling their proliferation and angiogenesis. The cholestane derivatives according to claim 1 of formula I for use in the treatment of metastatic tumors. 7. The cholestane derivatives of claim 1 for use in inhibiting angiogenesis in tumors. Cholestane derivatives for use according to claim 6 or 7, wherein the tumors are selected from the group consisting of leukemias, breast, uterus or prostate tumors. The cholestane derivatives according to claim 1 of formula I for use in the treatment of diseases caused by excessive cell proliferation, in particular diseases selected from the group consisting of cancer, restenosis, rheumatoid arthritis, psoriasis, multiple sclerosis, transplant rejection. The cholestane derivatives according to claim 1 of formula I for use in the treatment of diseases caused by excessive angiogenesis, in particular diseases selected from the group consisting of rheumatoid arthritis, arthritis of large and small joints, asthma bronchiale, chronic pancreatitis, veno-occlusive liver disease, trophic and inflammatory CNS proliferative diabetic retinopathy, age maculopathy, pterigium, glaucoma, nephropathy, endometriosis, psoriasis. A pharmaceutical composition comprising at least one cholestane derivative of the Formula I according to claim 1 and at least one pharmaceutically acceptable carrier.