Application of edg-receptor binding agent in cancer therapy

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to oncology, and concerns development of a medicinal agent for angiogenesis control. It is ensured by application of sphingosine-1-phosphate receptor agonist representing 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3 diol, or its hydrochloride, or phosphate. The present invention also can include these substances combined with chemotherapeutic agents.

EFFECT: invention provides angiogenesis regulation including inhibition of uncontrolled neoangiogenesis, particularly in solid tumour therapy.

9 cl, 7 dwg

 

The present invention relates to a new use of an agonist of the receptor for sphingosine-1-phosphate (SPH), especially in cancer treatment.

Agonists SF-receptor are the means of accelerating homing lymphocytes (CHL), which cause lymphopenia resulting redistribution, preferably reversible, lymphocytes from circulation to secondary lymphatic tissue, without generalized immunosuppressive actions. Sequestered “untrained” cells; stimulates the migration of T cells CD4 and CD8 and b cells from the blood into lymph nodes (LU) and Peyer's plaques (SP), which leads, for example, to inhibition of infiltration of cells in transplanted organs.

Agonists SF-receptor are typical counterparts sphingosine, such as 2-substituted derivatives of 2-aminopropan-1,3-diol or 2-aminopropanol, for example, a compound including a group of the formula X

where Z denotes H; C1-C6alkyl; C2-C6alkenyl; C2-C6quinil; phenyl; phenyl substituted IT-group; C1-C6alkyl with 1 to 3 substituents selected from the group consisting of halogen, C3-C8cycloalkyl, phenyl and HE-substituted phenyl; or CH2-R4zwhere R4zIT means, alloctype or a residue of formula (a)

p> where Z1means of a simple bond or O, preferably O; each of R5zand R6zindependent means N or C1-C4alkyl, optionally substituted by 1, 2 or 3 halogen atoms;

R1zIT means, alloctype or a residue of formula (a); and each of R2zand R3zindependent means H, C1-C4alkyl or acyl.

A group of the formula X is a functional group that is attached as a terminal group to a part of the molecule, which may be hydrophilic or lipophilic and may contain one or more aliphatic, alicyclic, aromatic and/or heterocyclic residue; this molecule, in which at least one of the groups Z or R1zis or comprises a residue of formula (a), acting as an agonist of one or more receptors for sphingosine-1-phosphate.

Agonists SF-receptor are compounds that signal as agonists at one or more receptors for sphingosine-1-phosphate, for example, with SF on SF. Binding agonist with SF receptor can, for example, lead to the dissociation of intracellular heterotrimeric G-protein Gα-GTP and Gβγ-GTP and/or to increased phosphorylation agonizing receptor and activation of downstream signaling pathways/kinases. The ability to bind receptor agonists SF can is about to be measured, as described in the following paragraph I.

Suitable agonists SF-receptor are, for example:

the compounds described in EP 627406 A1, for example, the compound of formula I

where R1means a straight or branched (C12-C22) carbon chain which may have in the chain a bond or a heteroatom selected from a double bond, a triple bond, O, S, NR6where R6means H, alkyl, aralkyl, acyl or alkoxycarbonyl, and carbonyl, and/or

- which may have as a substituent alkoxygroup, alkenylacyl, alkyloxy, aralkylated, acyl, alkylamino, allylthiourea, allmenalp, alkoxycarbonyl, alkoxycarbonylmethyl, alloctype, allylcarbamate, a nitrogroup, halogen, amino, hydroxyimino, or hydroxy-group-carboxypropyl;

or R1means

- phenylalkyl, in which the alkyl means a straight or branched (C6-C20) carbon chain; or

- phenylalkyl, in which the alkyl means a straight or branched (C1-C30) carbon chain, where the specified phenylalkyl has as Vice

- straight or branched (C6-C20) carbon chain optionally substituted with halogen,

- straight or branched (C6-C20) alkoxy chain, not battelino substituted with halogen,

- straight or branched (C6-C20) alkenylacyl,

- fenilalanina, halogenlampe, generalkonsulat, phenoxyethoxy or phenoxyethyl,

- cycloalkenyl, substituted C6-C20the alkyl,

- heteroaromatic, substituted C6-C20the alkyl,

- heterocyclic6-C20alkyl or

- heterocyclic alkyl, substituted C2-C20the alkyl,

and where

alkyl part may be

- in the carbon chain, a bond or a heteroatom selected from a double bond, a triple bond, O, S, sulfinil, sulfonyl, or NR6where R6matches the above, and

as Deputy alkoxygroup, alkenylacyl, alkyloxy, aralkylated, acyl, alkylamino, allylthiourea, allmenalp, alkoxycarbonyl, alkoxycarbonylmethyl, alloctype, allylcarbamate, the nitro-group, a halogen, an amino group, a hydroxy-group or carboxypropyl, and each of R2, R3, R4and R5independently mean H, C1-C4alkyl or acyl; or a pharmaceutically acceptable salt of this compound;

the compounds described in EP 1002792 A1, for example, the compound of formula II

where m indicates from 1 to 9 and each of R'2, R'3, R'4and R'5independent OSN which denotes H, alkyl or acyl, or a pharmaceutically acceptable salt of these compounds;

the compounds described in EP 0778263 A1, for example, the compound of formula III

where W denotes H; C1-C6alkyl, C2-C6alkenyl or2-C6quinil; unsubstituted or substituted phenyl; R4O(CH2)n; or (C1-C6alkyl having from 1 to 3 substituents selected from the group consisting of halogen, C3-C8cycloalkyl, phenyl and phenyl substituted IT;

X is H or unsubstituted or substituted alkyl straight chain having the number of carbon atoms of R, or unsubstituted or substituted alkoxygroup with a straight chain having a number (p-1) of carbon atoms, for example, with 1-3 substituents selected from the group consisting of C1-C6alkyl, HE, C1-C6alkoxygroup, alloctype, an amino group, a C1-C6alkylamino, allmenalp, oxoprop, Gialos1-C6alkyl, halogen, unsubstituted phenyl and phenyl with 1-3 substituents selected from the group consisting of C1-C6of alkyl, HE, C1-C6alkoxygroup, acyl, alloctype, amino, C1-C6alkylamino, alluminare, Gialos1-C6of alkyl and halogen; Y represents H, C1-C6alkyl, HE

C1-C6alkoxygroup, and the sludge, alloctype, an amino group, a C1-C6alkylamino, allmenalp, Gialos1-C6alkyl or halogen; Z2means a simple link or alkylene with a straight chain having the number of carbon atoms of q;

Each of p and q independently denote an integer from 1 to 20, provided that 6≤p+q≤23, m' is 1, 2 or 3, n represents 2 or 3, each of R1, R2, R3and R4independently mean H, C1-C4alkyl or acyl;

or pharmaceutically acceptable salt of these compounds;

the compounds described in WO 02/18395, for example, compounds of formula IVa or IVb

or

where Xandmeans O, S, NR1sor a group -(CH2)na-, which is unsubstituted or has as substituents of 1 to 4 atoms of halogen; nandmeans 1 or 2, R1smeans H or (C1-C4)alkyl, unsubstituted or substituted with halogen;

R1ameans H, HE, (C1-C4)alkyl or O(C1-C4)alkyl, where the alkyl is unsubstituted or has as substituents 1 to 3 halogen atoms; R1bmeans H, HE, or

(C1-C4)alkyl, which is unsubstituted or substituted with halogen; each of R2aindependently selected from H or (C1-C4)alkyl, unsubstituted or substituted by halogen is m; R3ameans H, HE, halogen or O(C1-C4)alkyl, where the alkyl is unsubstituted or substituted with halogen; and R3bmeans H, HE, halogen,

(C1-C4)alkyl, where the alkyl is unsubstituted or substituted by a hydroxy-group, or-O(C1-C4)alkyl, where the alkyl is unsubstituted or substituted with halogen; Yameans-CH2-, -C(O)-, -CH(OH)-, -C(=NOH)-, O or S, and R4ameans (C4-C14)alkyl or (C4-C14)alkenyl; or pharmaceutically acceptable salts of these compounds;

the compounds described in WO 02/076995, for example, the compound of formula V

where mcmeans 1, 2 or 3; Xcmeans O or a direct bond; R1cmeans N;

C1-C6alkyl, optionally substituted HE, acyl, halogen, C3-C10cycloalkyl, phenyl or hydroxyphenylazo; C2-C6alkenyl; C2-C6quinil; or phenyl, optionally substituted HE; R2cmeans

where R5cmeans H or C1-C4alkyl, optionally substituted by 1, 2 or 3 halogen atoms, and R6smeans N or C1-C4alkyl, optionally substituted with halogen;

each of R3sand R4cindependently mean H, C1-C4alkyl, optionally substituted with halogen, is whether acyl, and Rcmeans13-C20alkyl, which may optionally have in the chain an oxygen atom and which may optionally be substituted by a nitro-group, a halogen, an amino group, a hydroxy-group or carboxypropyl; or a residue of formula (a)

where R7cmeans H, C1-C4alkyl or C1-C4alkoxygroup, and R8cmeans substituted C1-C20alkanoyl, panels1-C14alkyl, where C1-C14alkyl optionally substituted with halogen or HE, cycloalkyl1-C14alkoxygroup or panels1-14alkoxygroup where cycloalkyl or phenyl ring optionally substituted with halogen, C1-C4the alkyl and/or C1-C4alkoxygroup, phenyl-C1-C14alkoxyl1-C14alkyl, venoxis1-C14alkoxygroup or venoxis1-C4alkyl, Rcalso means the residue of formula (a), where R8cmeans C1-C14alkoxygroup, when R1cmeans C1-C4alkyl, C2-C6alkenyl or C2-C6quinil;

or the compound of formula VI

where nxmeans 2, 3 or 4; R1xmeans N; C1-C6alkyl, optionally substituted HE, acyl, halogen, cycloalkyl, phenyl or hydro is sevenroom; With2-C6alkenyl; C2-C6quinil; or phenyl, optionally substituted HE; R2means N; C1-C4alkyl or acyl, each of R3and R4xindependently mean H, C1-C4alkyl optionally substituted by halogen or acyl, R5xmeans H, C1-C4alkyl or C1-C4alkoxygroup, and R6xmeans C1-C20alkanoyl, replaced by cycloalkyl;

cycloalkyl1-C14alkoxygroup where cycloalkyl ring optionally substituted with halogen, C1-C4the alkyl and/or C1-C4alkoxygroup; phenyls1-C14alkoxygroup, where the phenyl ring is optionally substituted with halogen, C1-C4the alkyl and/or C1-C4alkoxygroup, R6xalso means4-C14alkoxygroup, when R6xmeans2-C4alkyl, substituted for IT, or pentyloxy or hexyloxy, when R1xmeans1-C4alkyl, provided that R6xdoes not mean phenylbutyrate, or when R5xmeans H, or R1xmeans methyl; or a pharmaceutically acceptable salt of these compounds;

the compounds described in WO 02/06268 AI, for example, the compound of formula VII

where each of R1dand R2dregardless of the means H or aminosidine group;

R3dmeans hydrogen or hydroxyamino group; R4dmeans lower alkyl;

ndmeans an integer from 1 to 6; Xdmeans ethylene, vinile, ethynylene, a group having the formula-D-CH2(where D is carbonyl, -CH(OH)-, O, S or N), aryl or aryl containing up to three substituents selected from group a below; Ydmeans a simple link, C1-C10alkylene, C1-C10alkylene containing up to three substituents selected from groups a and b, C1-C10alkylene containing atoms O and S in the middle or at the end of the carbon chain, or C1-C10alkylene containing atoms O and S in the middle or at the end of the carbon chain, which contains up to three substituents selected from groups a and b;

R5dmeans hydrogen, cycloalkyl, aryl, heterocycle, cycloalkyl with up to three substituents selected from groups a and b, aryl containing up to three substituents selected from groups a and b, or a heterocycle containing up to three substituents selected from groups a and b; and each of R6dand R7dindependently denotes H or a Deputy selected from the group a;

<group> means halogen, lower alkyl, halogenated lower alkyl, lower alkoxygroup, lower allylthiourea, carboxyl, lower alkoxycarbonyl, the hydroxy-group, a lower aliphatic acyl, amino group, issuu monoalkylamines, the lower dialkylamino, the lower aliphatic allmenalp, cyano or nitro-group;

<b> means cycloalkyl, aryl, heterocycle, each of which optionally has up to three substituents selected from group a;

provided that when R5dmeans hydrogen, Ydmeans either a simple or linear C1-C10alkylene; or pharmaceutically acceptable salts or esters of these compounds;

- compounds described in JP 14316985 (JP 2002316985), for example, the compound of formula VIII

where R1e, R2e, R3E, R4e, R5e, R6th, R7Ene, XeAnd Yedescribed In JP 14316985; or pharmaceutically acceptable salts or esters of these compounds.

The compounds described in WO 03/29184 and WO 03/29205, for example, the compounds of formula IX

where Xfmeans O or S; R1f, R2f, R3fand nfas described in WO 03/29184 and 03/29205, for example, 2-amino-2-[4-(3-benzyloxyphenyl)-2-chlorophenyl]propyl-1,3-propandiol or 2-amino-2-[4-(benzyloxyphenyl)-2-chlorophenyl]propyl-1,3-propandiol.

In each case, where the following quotes are taken from applications for a patent, they are included in the present invention by reference.

Acyl may be a remainder Ry-CO-, where Rymeans1-C6al the sludge, With3-C6cycloalkyl, phenyl or phenyl-C1-C4alkyl. Unless otherwise specified, alkyl, alkoxygroup, alkenyl or quinil can be straight or branched.

When in the compounds of formula I, the carbon chain, such as R1substituted, preferred substituents are halogen, the nitro-group, amino group, the hydroxy-group or carboxypropyl. When the carbon chain is interrupted by the phenylene which optionally has substituents, the carbon chain is preferably unsubstituted. When fenelonov part is substituted, it is preferably substituted with halogen, a nitro-group, amino group, methoxy group, hydroxy-group or carboxypropyl.

Preferred compounds of formula I are those compounds in which R1means13-C20alkyl, optionally substituted by a nitro-group, a halogen, an amino group, a hydroxy-group or carboxypropyl. More preferred are compounds in which R1means phenylalkyl, substituted C6-C14alkyl chain, optionally substituted with halogen, and the alkyl portion of the means C1-C6alkyl, optionally substituted hydroxy-group. More preferably, when R1means phenyl-C1-C6alkyl substituted by phenyl, straight or razwell is authorized, preferably a straight line With a6-C14alkyl chain. With6-C14the alkyl chain may be in ortho-, meta - or para-, preferably in the para-position.

Preferably, when each of R2to R5means N.

The preferred compound of formula I is 2-amino-2-tetradecyl-1,3-propandiol. Especially preferred agonist SP-receptor formula I is a compound FTY720, i.e. the 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol in free form or in the form of pharmaceutically acceptable salts (hereinafter referred to as “compound A”), for example, in the form of a hydrochloride

The preferred compound of formula II is such a connection, where each R'2to R'5mean N and m means 4, i.e. the 2-amino-2-{2-[4-(1-oxo-5-fenilpentil)phenyl]ethyl} propane-1,3-diol in free form or in the form of pharmaceutically acceptable salts (hereinafter referred to as “compound B”), for example, in the form of hydrochloride.

The preferred compound of formula III is a compound where W stands for CH3; each of R1to R3means N; Z2means ethylene; X means heptyloxy and Y represents N; i.e., 2-amino-4-(4-heptyloxybiphenyl)2-methylbutanol. This connection can be in free form or in the form of pharmaceutically acceptable salts (hereinafter referred to as the connection”), for example, in the form of hydrochloride. Particularly preferred R-enantiomer.

The preferred compound of formula IVa is a compound FTY720-phosphate (R2ameans N, R3AIT means, Ha means Of, R1aand R1bmean IT). The preferred compound of formula IVb is connection-phosphate (R2ameans N, R3AIT means, Xandmeans Of, R1aand R1bmean IT, Yameans Of, R4ameans heptyl). The preferred compound of formula V is a compound B-phosphate.

The preferred compound of formula V is an ester of phosphoric acid and mono[(R)-2-amino-2-methyl-4-(4-pentyloxide)butyl].

The preferred compound of formula VIII is (2R)-2-amino-4-[3-(4-cyclohexylmethyl)benzo[b]Tien-6-yl]-2-methylbutane-1-ol.

In cases where compounds of formulas I through IX are in the molecule one or more asymmetric centers, the subject of the present invention are also various optical isomers, racemates, diastereoisomers and mixtures thereof. Compounds of formula III or IVb in those cases where the carbon atom bearing the amino group is asymmetric, preferably have the R-configuration at this carbon atom.

As examples of pharmaceutically acceptable salts of compounds of formulas I to IX can bring their salt reorganizes the x acids, such as hydrochloride, hydrobromide and sulfate; organic acid salts such as acetate, fumarate, maleate, benzoate, citrate, malate, methanesulfonate, bansilalpet; or, in some cases, salts containing metals such as, for example, sodium, potassium, calcium and aluminium, salts of amines, such as triethylamine; and salts of dibasic amino acids, such as lysine. In the methods of the present invention included hydrate and solvate forms of the compounds and salts.

Found that agonists SF-receptor thanks to the detected activity, for example, homing lymphocytes, for example as described in EP 627406 A1 or US 6004565, efficient, such as immunosuppressants, for example, in the treatment of acute allograft rejection. It is now known that agonists SF-receptor have valuable properties, thanks to which they can be used in chemotherapy of cancer, particularly solid tumors, especially progressive solid tumors. However, there is still a need to expand the Arsenal of treatment of solid cancers, especially in those cases where treatment of anti-cancer compounds does not lead to regression or stabilization of the disease.

On the basis of the results obtained in the present invention are presented:

1.1. A method of treating solid tumors in a subject in need of such cured and, which is mentioned in the introduction to a subject a therapeutically effective amount of the agonist SP-receptor containing a group of formula X, or its pharmaceutically acceptable salt.

1.2. The method of suppressing the growth of solid tumors in a subject in need of such treatment, which comprises introducing said subject a therapeutically effective amount of the agonist SP-receptor containing a group of formula X, or its pharmaceutically acceptable salt.

1.3. Method of inducing regression of the tumor, such as reduction of tumor formation, the subject in need of such a method; the method comprises introducing said subject a therapeutically effective amount of the agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt.

1.4. A method of treating a solid tumor invasiveness or symptoms associated with such tumor growth in a subject in need of such treatment, which comprises introducing said subject a therapeutically effective amount of the agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt.

1.5. A method of preventing metastasis of tumors, or preventing, or inhibiting the growth micrometastases in relation to the subject in need of such a method, the method is wvedenia.patienta to the subject a therapeutically effective amount of the agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt.

1.6. Method of inhibiting or regulation uncontrolled angiogenesis, for example, angiogenesis, mediated by sphingosine-1-phosphate (SPH), in relation to the subject in need of such a method; the method consists in the introduction referred to the subject a therapeutically effective amount of the agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt.

1.7. The method of prevention or treatment of diseases associated with the process of neoangiogenesis or uncontrolled angiogenesis, in relation to a person who needs it; the method consists in the introduction referred to the subject a therapeutically effective amount of the agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt.

The term “solid tumor” refers to a tumor and/or metastases (any location), not related to lymphatic cancer, for example, brain tumors and other tumors of the Central nervous system (e.g., tumors of the meninges, spinal cord, cranial nerves and other parts of Central nervous system, for example, glioblastoma or blastoma bone marrow); cancers of the head and/or neck; breast tumors; tumors of the circulatory system (e.g., tumors of the heart, cf is dostine and pleura, and other intrathoracic organs, tumor vessels and tumor tissue associated with blood vessels); tumors of the urinary system (e.g., tumors of the kidney, renal pelvis, ureter, bladder, as well as tumors of other and unspecified localization of urinary system); tumors of the gastrointestinal tract (e.g., tumors of the esophagus, stomach, small intestine, colon, sigmoid colon, rectum, anus and anal canal), tumors involving the liver and intrahepatic bile ducts, gall bladder, tumors of other and unspecified localization departments biliary tract, pancreatic tumor other tumours of the digestive system); tumor affecting the oral cavity (lip, tongue, gum, floor of mouth, palate and other parts of mouth parotid gland and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform pocket, hypopharynx, and other sites of lip, oral cavity and pharynx); tumors of the reproductive system (e.g., vulva, vagina, cervix, uterine body, uterine, ovarian and other parts associated with female genital organs, placenta, penis, prostate, testicles and other parts connected with male sexual organs); tumors of the respiratory tract (e.g., nasal cavity and middle ear, paranasal sinuses, larynx, trachea, bronchus and easy is th, for example, small cell lung cancer and non-small cell lung cancer); tumors of the skeletal system (e.g., bone and articular cartilage of limbs, bone articular cartilage and other parts); skin tumor (eg, malignant melanoma, non-melanoma skin cancer, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi's sarcoma); and tumors involving other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneal space and peritoneum, eye and adnexa, thyroid, adrenal and other endocrine glands and related structures, secondary and uncertain malignant neoplasm lymph nodes, secondary malignant neoplasm respiratory and digestive systems and secondary malignant neoplasm another location.

In the description of the present invention at the mention of the tumor, tumor diseases, carcinoma or cancer also meant as an alternative or as an addition metastasis in the affected organ or tissue regardless of the localization of the tumor and/or metastasis.

In the case when the agonist SP-receptor is a compound of formula I, e.g. compound a, or compound of formula IVa or IVb, in one of the embodiments of the present invention, it uses methods 1., 1.2, 1.3 or 1.4 for the treatment of a solid tumor, non-tumor breast, prostate, bladder, kidney and lung.

In a series of further specific or alternative embodiments of the present invention also provides:

1.8. The method of increasing the activity of a chemotherapeutic drug or overcome resistance to chemotherapeutic agent that is applied to the subject in need of it, and consists in the introduction of a specified subject a therapeutically effective amount of the agonist SP-receptor, for example, agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt in parallel or sequentially with said chemotherapeutic agent.

1.9. The method according to p in which the chemotherapeutic agent is an inhibitor of the metabolic pathways of signal transduction, directed either against the host cells, or against the processes involved in the formation of a tumor and/or metastasis process, or used by tumor cells to proliferation, survival, differentiation, or development of drug resistance.

1.10. The method corresponding to the method specified above, in which the agonist SP-receptor introduced at intervals.

In further specific series or alternative the x of embodiments of the present invention also provides:

2.1. Agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt, for use in one of the methods mentioned above under paragraph 1.1 to 1.4, preferably a solid tumor, except for tumors of the breast, prostate, bladder, kidney, or lung, when the agonist SP-receptor is a compound of formula I, e.g. compound a, or compound of formula IVa or IVb.

2.2. Agonist SP-receptor, for example, agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt, for use by one of the methods mentioned above under PP-1.10 or below under item 7.

3.1. Agonist SP-receptor containing a group of formula X, or its pharmaceutically acceptable salt for use in the preparation of pharmaceutical compositions for use in one of the methods mentioned above under paragraph 1.1-1.4; use of the composition preferably is in the case of solid tumors, but not in tumors of the breast, prostate, bladder, kidney, or lung, when the agonist SP-receptor is a compound of formula I, e.g. compound a, or compound of formula IVa or IVb.

3.2. Agonist SP-receptor, for example, agonist SP-receptor containing a group of formula X, or its pharmaceutically acceptable salt, for use in you need a kitchen is making pharmaceutical compositions for use in the same way, above under PP-1.10 or below under item 7.

4.1. Pharmaceutical composition for use in one of the methods mentioned above under paragraph 1.1-1.4, containing agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt together with one or more pharmaceutically acceptable diluents or carriers; preferably the composition is intended for solid tumors, but not in tumors of the breast, prostate, bladder, kidney, or lung, when the agonist SP-receptor is a compound of formula I, e.g. compound a, or compound of formula IVa or IVb.

4.2. Pharmaceutical composition for use in the same way as described above under PP-1.10 or below under item 7, contains agonist SP-receptor, for example, agonist SP-receptor containing a group of formula X, or its pharmaceutically acceptable salt together with one or more pharmaceutically acceptable diluents or carriers.

5.1. Pharmaceutical combination comprising a) a first agent that is an agonist SP-receptor, for example, agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt, and (b) associated tool, which is a chemotherapeutic agent such as described below.

5.2. Farmaceuticas the ia combination, comprising a) a first agent that is an agonist SP-receptor, for example, agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt, and (b) associated tool, which is a chemotherapeutic agent selected from the compounds mentioned below in section XI, to create energichnogo therapeutic effect.

6. The method as described above comprising co-administration, e.g., simultaneous or sequential, a therapeutically effective amount of the agonist SP-receptor, for example, agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt, and a second drug substance, which is a chemotherapeutic agent, such as described in this patent.

7. A method for the treatment of lymphoproliferative or myeloproliferative diseases, for example, treatment of tumor invasiveness or symptoms associated with such tumor growth, the subject who needs it; the method comprises co-administration mentioned the subject, for example, simultaneously or sequentially, agonist SP-receptor, for example, agonist SP-receptor, including a group of the formula X, or its pharmaceutically acceptable salt, and a second drug substance, which is a chemotherapeutic agent, for example, that them as indicated in this patent.

Under “lymphatic cancer” refers to, for example, tumors of blood and lymphatic systems (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-associated lymphomas, malignant immunoproliferative disease, multiple myeloma and malignant ploskokletochnyi neoplasm, lymphoid leukemia, acute or chronic myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of a particular cell type, leukemia unspecified cell type other and unspecified malignant neoplasm lymphoid, haematopoietic and related tissues, for example, both diffuse lymphoma, T-cell lymphoma or cutaneous T-cell lymphoma). Myeloid cancer includes, for example, acute or chronic myeloid leukemia.

The term “chemotherapeutic agent” refers mainly any chemotherapeutic agent other than the agonist SP-receptor. The term includes, but is not limited to:

i. an aromatase inhibitor

ii. antiestrogen, antiandrogen (especially in the case of prostate cancer) or agonist of gonadorelin,

iii. inhibitor of topoisomerase I or topoisomerase II inhibitor,

iv. the tool that is active against microtubules, an alkylating agent, antitumor intimate is of Olite or a platinum compound,

v. the connection linking/down the activity of the protein or lietkynes or activity of the protein or lepidocrocite, another angiogenic compound or a compound which induces cellular differentiation,

vi. the bradykinin receptor 1 or antagonist of angiotensin II,

vii. inhibitor of cyclooxygenase, diphosphonate, inhibitor discontinuties, an inhibitor of heparanase (prevents heparansulfate degradation), for example, the product of PI-88, biological response modifier, preferably lymphokine or interferons, such as interferon-γ, an inhibitor of many targets, or inhibitor, which blocks metabolic pathways antiapoptosis processes,

viii. inhibitor of Ras oncogenic isoforms, for example, H-Ras, K-Ras or N-Ras, or inhibitor farnesyltransferase, for example, L-744832 or DK8G557,

ix. the telomerase inhibitor, for example, teamstation,

H. protease inhibitor, an inhibitor of matrix metalloproteinases, inhibitors methioninamide, for example, benhamed or its derivative, or the proteosome inhibitor, for example, PS-341, and/or

xi. The mTOR inhibitor.

The term “aromatase inhibitor”, as used in the present invention refers to a compound that inhibits the formation of estrogen, for example, the conversion of substrates Androstenedione and testosterone into estrone and estradiol, respectively. The term includes, n is not limited to, steroids, especially atamestane, exemestane and formestane and, in particular, non-steroidal compounds, especially aminoglutetimid, Rogatkin, firedoglake, trilostane, testolactone, ketoconazole, vorozole, fadrozole, anastrozole and letrozole. Exemestane can be entered, for example, in the form of a commercial product AROMASINTM. Formestane you can enter, for example, in the form of a commercial product LENTARONTM. Fadrozole you can enter, for example, in the form of a commercial product AFEMATM. Anastrozole can be entered, for example, in the form of a commercial product ARIMIDEXTM. Letrozole can be entered, for example, in the form of a commercial product FEMARATMor

FEMARTM. Aminoglutetimid you can enter, for example, in the form of a commercial product ORIMETENTM. The combination of the present invention, including chemotherapeutic agent, which is an aromatase inhibitor, is particularly useful in the treatment of tumors that have hormone receptors, such as tumors of the breast.

The concept of “antiestrogen”, as used in the present invention refers to a compound that counteracts the effect of estrogen on the level of estrogen receptors. Includes tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride, but is not limited to these compounds. Tamoxifen can be entered, for example, in fo the IU commercial product NOLVADEX TM. Hydrochloride raloxifene can be entered, for example, in the form of a commercial product EVISTATM. Fulvestrant can be prepared as described in US 4659516, or you can enter, for example, in the form of a commercial product FASLODEXTM. The combination of the present invention, including as a chemotherapeutic drug antiestrogen, are particularly useful in the treatment of, for example, tumors with estrogen receptors, such as tumors of the breast.

In the present invention the term “antiandrogen” refers to compounds capable of inhibiting the biological action of androgenic hormones; anti-androgens include, but are not limited to, bikalutamid (product

CASODEXTM), which can be prepared, for example as described in US 4636505.

The term “agonist of gonadorelin”as it is used in the present invention, includes abarelix, goserelin and goserelin acetate, but is not limited to these compounds. Goserelin is described in US 4100274, you can enter, for example, in the form of a commercial product under the trade name ZOLADEXTM. Abarelix can be prepared, for example as described in US 5843901.

The term “inhibitor of topoisomerase I in the present invention include, but are not limited to, topotecan, irinotecan, 9-nitrocamptothecin and macromolecular camptothecin conjugate PNU-166148 (connect the tion A1, WO 99/17804). Irinotecan can be entered, for example, in the form of a commercial product CAMPTOSARTM. Topotecan can be entered, for example, in the form of a commercial product HYCAMTINTM.

The term “topoisomerase II inhibitor", as used in the present invention include, but are not limited to, anthracyclines, such as doxorubicin (including liposomal form, for example, the product CAELYXTM), daunorubicin, epirubicin, idarubitsin and nemorubicin, the anthraquinones mitoxantrone and losoxantrone, podophyllotoxin etoposide and teniposide. Etoposide can be entered, for example, in the form of a commercial product ETOPOPHOSTM. Teniposide can be entered, for example, in the form of a commercial product VM 26-BRISTOLTM. Doxorubicin can be entered, for example, in the form of a commercial product ADRIBLASTINTM. Epirubicin can be entered, for example, in the form of a commercial product FARMORUBICINTM. Idarubitsin you can enter, for example, in the form of a commercial product ZAVEDOSTM. Mitoxantrone can be entered, for example, in the form of a commercial product NOVANTRONTM.

The term “agent, active against microtubules” relates to stabilizing and destabilizing microtubules microtubules; the term includes, but is not limited to, taxanes, such as paclitaxel and docetaxel, Vinca alkaloids such as vinblastine, the person is but of vinblastine sulfate, vincristine especially vincristine sulfate, and vinorelbine, discodermolide and epothilone and their derivatives, for example, epothilone or its derivative. Paclitaxel can be entered, for example, in the form of a commercial product TAXOLTM. Docetaxel can be entered, for example, in the form of a commercial product

TAXOTERETM. Vinblastine sulfate can be entered, for example, in the form of a commercial product VINBLASTIN R.P.TM. The vincristine sulfate can be entered, for example, in the form of a commercial product FARMISTINTM. Discodermolide can be obtained, for example, as described in US 5010099.

The term “alkylating agent”, as used in the present invention include, but are not limited to, busulfan, chlorambucil, cyclophosphamide, ifosfamide, melphalan or nitrosoanatabine (BCNU or product GliadelTM). Cyclophosphamide can be entered, for example, in the form of a commercial product CYCLOSTINTM. Ifosfamide can be entered, for example, in the form of a commercial product HOLOXANTM.

The term “antineoplastic antimetabolite” includes, but is not limited to, 5-fluorouracil, capecitabine, gemcitabine, cytarabine, fludarabine, tioguanin, methotrexate and edatrexate you can enter, for example, in the form of a commercial product XELODATM. Gemcitabine can be entered, for example, in the form of a commercial product GEMZARTM.

P is the adoption of the platinum compound”, as it is used in the present invention include, but are not limited to, carboplatin, cisplatin and oxaliplatin. Carboplatin can be entered, for example, in the form of a commercial product CARBOPLATTM. Oxaliplatin can be entered, for example, in the form of a commercial product ELOXATINTM.

The concept of “connections linking/decreasing the activity of the protein or lietkynes, or other antiangiogenic compounds”, as used in the present invention include, but are not limited to, inhibitors proteincontaining and/or serine - or trionychinae or inhibitors lietkynes, for example, connection, connecting, decreasing or inhibiting the activity of members of the family of growth factors epidermal receptor tyrosinekinase (EGFR, ErbB2, ErbB3, ErbB4, in the form of Homo - or heterodimers); representatives of the family of growth factors vascular endothelial receptor tyrosinekinase (VEGFR); receptors for platelet-derived growth factor (DERIVED); receptors of fibroblast growth factors (FGFR); receptor 1 insulin-like growth factor (IGF-1R); representatives of the family of Trk-receptor tyrosinekinase; representatives of the family Ah-receptor tyrosinekinase; Ret-receptor tyrosinekinase; Kit/scfr start-receptor tyrosinekinase; representatives of Ab1-family and the products of the merging their genes (for example, BCR-Ab1); representatives of protein kinase C (CSWs) and af family of serine/treoninove kinase; representatives of the MEK, SRC, JAK, FAK, PDK or PI(3) family of kinases or family kinases, close the kinase PI(3); and/or members of the family of cyclin-dependent kinase (CDK). The concept also includes angiogenic compounds having another mechanism of activity, for example, is not associated with the inhibition of protein - or libidinis.

Compounds that bind, decrease or inhibit the activity of VEGFR are mainly compounds, proteins or antibodies which inhibit VEGF-receptor tyrosinekinase, inhibit VEGF receptor or associated with VEGF. In particular, these compounds, proteins or monoclonal antibodies, which in General terms or in detail are described in WO 98/35958, for example, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or its pharmaceutically acceptable salt, for example, succinate, WO 00/27820, for example, derived amide 7U-aryl(thio)Anthranilic acid, for example, 2-[(4-pyridyl)methyl]amino-N-[3-methoxy-5-trifluoromethyl)phenyl]benzamide or 2-[(1 oxido-4-pyridyl)methyl]amino-N-[3 - triptoreline]benzamide, or in WO 00/09495, WO 00/59509, WO 98/11223, WO 00/27819 and in EP 0769947; compounds described M. Prewett, etc. in Cancer Research, 59, 1999, SS-5218; F.Yuan and others in Proc. Nail. Acad. Sci. USA, 93, 1996, SS-14770; Z. Zhu and others, Cancer Res., 58, 1998, cc.3209-3214; J. Mordenti, etc. in Toxicologic Pathology, 27, 1999, SS-21; in WO 00/37502 and WO 94/10202; product AngiostatinTMdescribed M.S. O'reilly and others in the Cell, 79, 1994, SS-328; product EndostatinTMdescribed .S. O'reilly and others in the Cell, 88, 1997, cc.277-285; amide derivatives of Anthranilic acid; ZD4190; ZD6474; SU5416; SU6668; or anti-VEGF antibody, or antibody to the VEGF receptor, for example, RhuMab.

Under the antibodies imply intact monoclonal antibodies, polyclonal antibodies, polyspecific antibodies formed, at least 2 of intact antibodies, and antibody fragments such length that provides the desired biological activity.

Compounds that bind, decrease or inhibit the activity of a family of receptors for growth factors of the epidermis, are mainly compounds, proteins or antibodies which inhibit members of the family of EGF-receptor tyrosinekinase, for example, EGF receptor, ErbB2, ErbB3 and ErbB4, bind to EGF or EGF related ligands, or which have a dual inhibitory effect against ErbB - and VEGF-receptor the kinase. In particular, such compounds, proteins or monoclonal antibodies, which in General terms or in detail are described in WO 97/02266, for example, the compound of example 39, or in EP 0564409, WO 99/03854, EP 0520722, EP 0566226, EP 0787722, EP 0837063, US 5747498, WO 98/10767, WO 97/30034, WO 97/49688, WO 97/38983 and, especially, WO 96/30347 (for example, a compound known as CF 358774), WO 96/33980 (e.g. compound ZD 1839) and WO 95/03283 (for example, the connection ZM105180) or PCT/EP 02/08780; for example, trastuzumab (product HerpetinR), Zeuxis is b, Iressa, OSI-774, CI-1033, HRH-569, GW-2016, E, E, E, E. E, E, E or E.

Compounds that bind, decrease or inhibit the activity DERIVED, are mainly compounds that inhibit PDGF-receptor, for example, a derivative of N-phenyl-2-pyrimidinamine, such as imatinib.

Compounds that bind, decrease or inhibit the activity of members of the family of C-Ab1 and products merge their genes are, for example, a derivative of N-phenyl-2-pyrimidinamine, e.g. imatinib; PD180970; AG957; or NSC 680410.

Compounds that bind, decrease or inhibit the activity of representatives of families of protein kinase C, Raf, MEK, SRC, JAK, FAK and PDK, or representatives of the family of PI(3)kinase or related PI(3)kinase, and/or representatives of the family of cyclin-dependent kinase (CDK)are mainly such compounds as derivatives of staurosporine, which are described in EP 0296110, for example, midostaurin. Examples of other compounds include, for example, UCN-01, safingol, BAY 43-9006, Bryostatin 1, Perifosine; UO 126; Ilmofosine; RO 318220 and RO 320432; GO 6976, ISIS 3521; or LY333531/LY379196.

Other antiangiogenic compounds are, for example, thalidomide (THALOMID) and TNP-470.

Compounds that bind, decrease or inhibit the activity of the protein or lepidocrocite is, for example, inhibitors of phosphatase 1, phosphatase 2A, PTEN or CDC25, for example, Okada is the first acid or its derivative.

Compounds that induce cellular differentiation, are, for example, retinoic acid, α-, γ - or δ-tocopherol or α-, γ - or δ-tocotrienol.

The term “inhibitor of cyclooxygenase,” as it is used in the present invention include, but are not limited to, for example, celecoxib (Celebrex productR, rofecoksib (product VioxxR), etoricoxib, valdecoxib or 5-alkyl-2-arylaminovinylketones acid, for example, 5-methyl-2-(2'-chloro-6'-foronline)phenylacetic acid.

The term “inhibitor discontinuties”as it is used in the present invention, includes MS-27-275, SAHA, pyroxene, FR-901228 and valproate acid, but is not limited to them.

The concept of “diphosphonates”as it is used in the present invention, includes acridology, clodronate, tiludronate, pamidronate, alendronate, ibandronate, risedronate and zoledronic acid, but is not limited to these compounds. Trigonomy acid can be entered, for example, in the form of a commercial product DIDRONELTM. Clodronate acid can be entered, for example, in the form of a commercial product BONEFOSTM. Tiludronate acid can be entered, for example, in the form of a commercial product SKELIDTM. Pamidronovu acid can be entered, for example, in the form of a commercial product AREDIATM. Alendronate acid m is tenderly to enter, for example, in the form of a commercial product FOSAMAXTM. Ibandronate acid can be entered, for example, in the form of a commercial product BONDRANATTM. Risedronate acid can be entered, for example, in the form of a commercial product ACTONELTM. Zoledronic acid can be entered, for example, in the form of a commercial product ZOMETATM.

The term “inhibitor of metalloproteinases matrix”, as used in the present invention include, but are not limited to, collagen and coworkers peptide peptidomimetics inhibitors, derivatives of tetracycline, for example, hydroxamic coworkers peptide inhibitor of batimastat and bioavailable when administered orally similar marimastat, prinomastat, BMS-279251, BAY 12-9566, TEA or AAJ996.

The term “mTOR inhibitor”, as used in the present invention, means rapamycin (sirolimus) or its derivative, but is not limited to these compounds. Rapamycin is a known macrolide antibiotic, formed by Streptomyces hygroscopicus. Acceptable derivatives of rapamycin include, for example, compounds of formula

where

R1aameans of CH3or3-C6quinil

R2aameans H or-CH2-CH2-HE, 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoyl or tetrazolyl, and

XAAmeans =O, (N, is) or (N, IT)

provided that R2aais not H when XAAmean =O and R1aameans of CH3;

or its prodrug, when R2aameans-CH2-CH2HE, for example, physiologically hydrolyzable simple ether.

Compounds of the formula And are described, for example, in WO 94/09010, WO 95/16691, WO 96/41807, US 5362718 or WO 99/15530, in the present invention, they are included as links. They can be prepared as described or similar to the methods described in these references.

Preferred derivatives of rapamycin are 32-desoxidation, 16-Penta-2-ynyloxy-32-desoxidation, 16-Penta-2-ynyloxy-32(S)dihydrocapsaicin, 16-Penta-2-ynyloxy-32(S)-dihydro-40-ortho-(2-hydroxyethyl)rapamycin and, more preferably, 40-ortho-(2-hydroxyethyl)rapamycin. Other examples of rapamycin derivatives include, for example, CCI779 or 40-[3-hydroxymethyl)-2-methylpropanoate]rapamycin or its pharmaceutically acceptable salt, as described in US 5362718, AWT or 40-(tetrazolyl)rapamycin, especially 40-EPI-(tetrazolyl)-rapamycin, for example, described in WO 99/15530, or rapology described in WO 98/02441 and W001/14387, for example, AR.

In each case, when referred to patent applications or scientific publications concerning the nature of the compounds, they are included in the present invention in the form of links. This includes also pharmaceutically acceptable the e salts of these compounds, the corresponding racemates, diastereoisomers, enantiomers, tautomers and the corresponding crystalline modifications of the above described compounds, if any, represented, for example, solvate, hydrates and polymorphs, which are described in the cited publications. Compounds that, in the present invention are used as active ingredients of the combinations, can be obtained and entered, as described in the cited documents. Also within the present invention presents a combination containing more than two different active ingredients, as described above; for example, the pharmaceutical combination in the framework of the present invention could include three or more of the active ingredient. Moreover, the first means and associated means are not the same ingredient.

The use of agonists SF, for example, agonists SF, including a group of the formula X, in the treatment of solid tumors, which are detailed in the present invention above, can be demonstrated in animal tests and in clinical trials, for example, in accordance with the methods described below.

A. In vitro

A.1. Antitumor activity

Use a line of breast cancer cells mouse, originally isolated from breast carcinoma, for example, line JygMC(A). Before the study of quantities of the cells adjusted to a titer of 5×10 5for planting in the tablet with fresh nutrient medium. Cells incubated in fresh medium containing 2.5 mm thymidine, without fetal calf serum (FCS) for 12 h and then washed twice with saline phosphate buffer (FSB) with the subsequent addition of fresh medium containing 10% FCS and further incubated for another 12 hours After that, cells incubated in fresh medium without FCS containing 2.5 mm thymidine for 12 hours To separate the cells washed them twice the FSB and the newly sown in tablets with fresh medium containing 10% FCS. After synchronization, cells incubated with or without various concentrations of the compounds of formula I in 3, 6, 9, 12, 18 or 24 h After treatment with 0.2% EDTA, the cells are harvested, fixed with 70% solution of ethanol, cooled to the temperature of melting ice, hydrolyzing with RNase A (type 1-a: firm Sigma Chem. Co.), taken in the amount of 250 μg/ml, at 37°C for 30 min and stained with iodide propidium at a concentration of 10 mg/ml for 20 minutes After the incubation period, the number of cells determined in two ways: by counting cells in a Coulter counter and SRB colorimetric analysis. Under these conditions, the agonist SF, such as compound B in the form of hydrochloride, inhibits proliferation of tumor cells in the concentration range 10-12- 10-6M

A.2. Analysis SF-mediated about the education of the tubes in the endothelial cells of the umbilical vein of a person (ACPWC)

To analyze the formation of tubules using cells ACPWC from 2-8 passage; however, prior to collection of cell density never exceeds 70%. Cells are prepared for analysis by washing balanced for herpes saline solution (HBSS, firm Clonetics) with subsequent treatment with a solution of trypsin/EDTA (0.25 mg/ml, the firm Clonetics). After separation of about 90% of the cells from the tablet, add an equal volume of trypsin neutralizing solution (TNS, firm Clonetics), then the cells are collected in conical tubes containing at least 10 ml of media computers-2, Clonetics)+0.1% of bovine serum albumin (BSA) (firm Sigma). Cells are centrifuged at 1000 rpm./min for 5 min, remove supernatant and add 5 ml of fresh medium computers-2+0.1% BSA. Cells are counted using hemocytometer and the volume of the cell suspension was adjusted to a final concentration of 500,000 cells/ml conical tubes placed at 100 nmol of the studied compounds and 1 ml pertussis toxin (CT) at a concentration of 10 ng/ml, then make 1 ml of cell suspension in each tube. After this the tubes incubated for an hour at 37°C in an atmosphere of 5% CO2. Migration analysis is performed using the 24-advance plug-tablets Fluoro-Blok, coated with fibronectin (pore size 8 μm, the firm Falcon #351147), instead of separate inserts in 24-well plate. Cells and investigated the connection is through the prepared and pre-incubated, as described above, then 100 μl added to each appropriate well plug the tablet. 300 µl of the medium computers-2+2% desorbed charcoal, without SF, making holes marked “no stimulation (-)and 300 μl of medium containing SF (500 nmol), contribute to the wells marked stimulation (+)”. After that, the tablet incubated for 4 h at 37 C in an atmosphere of 5% CO2.

Calcein AM, 50 mg/vial (company Molecular Probes #C3100) is prepared as follows: first, add in the vial 20 ml of DMSO, then 12.5 ml HBSS (on the tablet) heated to 37°C and 150 μl added to the vial; the contents of the ampoules are transferred back into the remaining HBSS to a final concentration of 4 μg/ml of calcein AM.

Tablet Fluoro-blok removed from thermostat, separating the upper plug the tablet and tap on it to remove excess medium adhering to the inserts. Then plug the tablet is transferred to a fresh 24-well plate containing 500 μl in the wells calcein AM, at a concentration of 4 µg/ml After the tablet incubated for 11/2h at 37°C in an atmosphere of 5% CO2.

After incubation, the tablet reads on the device Cytofluor II when the wave excitation 485 nm and the wave emission 530 nm. Floor Fluoro-Blok in boxes allows you to count only those cells that migrate to the bottom. To analyze the data entered in Excel, charting carry out the BL is using the program SigmaPlot, for significance tests (checking on student test) using the program SigmaStat (Fig.7).

The formation of tubes is determined by counting the number of branching points (two independent connecting strand) in three independent fields of view with a 4-fold increase. The results describe as follows:

ProcessingBranch point
FSB8±5
SF42±13
The compound FTY720-phosphate48±15
The compound FTY720-phosphate + SF14±7
Connection-phosphate44±16
Connection-phosphate + SF18±6

These results demonstrate the unique ability of the compound FTY720-phosphate or connection-phosphate to act as an agonist of angiogenesis as such and, surprisingly, as antagonist CP-mediated angiogenesis. The preferred form of connection-phosphate is the racemate or the R-enantiomer. CT is used as a control for the inhibition of Giα (EDG-1)-Oper Dowanol activity.

B. In vivo

B.1. Antitumor activity

Antitumor activity is expressed by the ratio O/A% (average increase of tumor mass in treated animals to an average increase of tumor mass control animals multiplied by 100).

Aliquot samples of cancer cells (1×107), for example, human melanoma cells A, vaccinated mice of BALB/c-PI/PI. When the tumors reached a size of approximately 10×10 mm, animals randomly divided into four subgroups and start treating the compound of formula I. After two weeks of treatment the animals were slaughtered, and the tumor and the tissue is prepared for morphological and molecular analysis. The size of the tumor is determined with the aid of a compass. In this analysis, the agonist SF, for example connection B or C (in the form of hydrochloride), slows the growth of tumors when introduced in a dose of from 0.5 to 5 mg/kg; control is a physiological solution: for example, the connection-HCl with 5-fold introduction during the week leads to a value of About/ - 30%.

B.2. The combination with an inhibitor of VEGF-R-proteincontaining

Naked mice with transplanted tumors of the mammary glands of human MDA-MB-435 treated within two weeks inhibitor of VEGF-R-proteincontaining, for example, succinate and 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine, at a dose of 100 mg/kg orally at 5-fold introduction during the week, agonist SP-receptor, for example,compound (hydrochloride), at a dose of 2.5 mg/kg intravenously for 5-fold introduction in the course of a week, or a combination of both compounds. Antitumor effect of expressed attitude About/To%, as described above. The combination of connection-HCl with succinate and 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine has a greater anti-tumor effect (/% 27) than one means (connection-HCl, About 66%; succinate 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine, O/K% 91). Good antitumor responses get when naked mice vaccinated melanoma cells human A3 75 and treated in a similar manner using the same combination: combined treatment leads to indicator O/To% 15, while treatment with only one of the means to indicators O/To% 35 and 44, respectively.

B.3. Antiangiogenic activity

Porous chamber containing (i) sphingosine-1-phosphate (5 µmol/camera) or (ii) VEGF (1 μg/camera) in 0.5 ml of 0.8% (wt./volume) agar (containing heparin, 20 units/ml) injected mice subcutaneously in the flank. SF or VEGF induce the growth of vascularized tissue around the camera. This response is dose-dependent and is characterized quantitatively by measuring weight and blood content of the tissue. Mice treated once a day (i) oral connection And at 0.3, 3, 30, or 50 mg/kg), or (ii) intravenous R-enantiomer of compound (2.5 mg/kg), or (iii) intravenous S-enantio the rum connection (2.5 mg/kg), or (iv) oral or intravenous solution (5% glucose, 10 ml/kg), starting at 4-6 h prior to the introduction of cameras and continuing for 4 days. Animals slaughtered for measuring vascularized tissues 24 h after the last dose. Determine the weight and blood filling vascularized tissues around the camera. Animals that were treated with compound a, or R - or S-enantiomer of the compound, shows a decrease in the weight and/or blood filling vascularized tissues compared with similar results in animals that received only one solvent.

C. Clinical study

B.1. The study of clinical efficacy of agonists SF-receptor, for example, compounds of formulas I, II or III, for example, compounds a, B or C

The study involved 20 patients with advanced solid tumors in advanced stage, resistant or difficult-to standard therapy; they get referred to the connection portion, which is selected by a gradual increase in dose. Weekly medical and laboratory examination General clinical condition of patients. The status of the tumor and metastasis appreciate every 2 months by x-ray studies. Initially, patients receive treatment within 2 months. After this treatment continued until on the, while the disease does not cease to progress and until the sick satisfactorily tolerate the medication.

The main variables for the evaluation of treatment outcome: safety (side effects), standard biochemical parameters of serum and blood cells, the tumor size assessment method computer tomography (CT) or magnetic resonance imaging (MRI).

B.2. Combined treatment

Suitable clinical studies are, for example, open non-randomized with increasing doses of the study patients with advanced solid tumors. These studies show, in particular, the synergy of active ingredients in combination proposed in the present invention. The beneficial effects on proliferative diseases can be installed directly on the results of these studies or by changes in the course of the study, which is essentially known to any person skilled in the art. Such studies, in particular, applicable to compare the effects of monotherapy, which use active ingredients and effects from the use of the combination of the present invention. Preferably the dose means (a) increase until, until it reaches the tolerable upper intake; concomitant agent (b) is administered at a fixed dose. Alternative cf is a rotary (a) is administered in a fixed dose, and the dose of the concomitant means (b) increase. Each patient receives a dose of means (a) either daily or intermittently. The effectiveness of treatment in these studies may be installed, for example, after 12, 18 or 24 weeks by x-ray evaluation of tumors with an interval of 6 weeks.

The alternative is a double-blind trials with placebo control; it can be used in order to confirm the advantages referred to in the present invention combination.

Daily dose required for the embodiment of the method of the present invention, when there is only one agonist SP receptors vary depending on, for example, from a compound that is used, organism, route of administration and the severity of the disease, whose treatment. The preferred daily dose is in the range from 0.1 to 100 mg; it is administered as a single dose or divided into several doses. Acceptable daily doses to patients are dose, for example, about 0.1-50 mg, oral. Agonist SP-receptor may be entered by any standard method, especially enterline, for example, orally, for example in the form of tablets, capsules or medicines through the nose, by inhalation, or parenterally, for example in the form of solutions or suspensions for injection. Acceptable forms uniform is Siroki for oral administration comprise from about 0.1 to 30 mg, usually from 0.25 to 30 mg agonist receptor SF, together with one or more pharmaceutically acceptable diluents or carriers. In order to inhibit angiogenesis, it is important to choose a high enough dose of the agonist receptor SF, because low concentrations of agonists SF-receptor stimulate angiogenesis. Acceptable dose to ensure antiangiogenic actions when a patient is entered SF agonist can pick up by increasing the concentration and dose, as described above for a, B and C.

The combination of the present invention can also be used in combination with surgery, mild prolonged hyperthermia of the whole organism and/or radiation therapy.

Introduction the pharmaceutical combination of the present invention leads to a positive effect, e.g. a synergistic therapeutic effect, for example, in respect of slowing, arresting or reversing neoplasms, distribution or growth, metastasis, or longer duration of tumor response, or suppression of angiogenesis; the use of the pharmaceutical combination may also lead to other valuable effects, such as reduced side effects, improved quality of life or reduced mortality and morbidity compared with monotherapy, which applies only the one pharmaceutically active ingredients, used in combination with the present invention. This especially applies to the treatment of cancer that is not amenable to treatment with other chemotherapeutics, known as cancer.

Another advantage is that the combination of the present invention it is possible to use lower doses of the active ingredients, dosages can be smaller and used less frequently or in lower doses can be used to reduce the degree of side effects in the course of controlling the growth of tumor formation. This is in accordance with the wishes and needs of patients.

In accordance with one embodiments of the present invention it is preferred pharmaceutical combination includes:

a) compounds of formulas I, II, III, IVa, IVb, V or VI, for example, compound a, B or C, and

b) one or more compounds as related tools such as referred to in paragraphs (ii), (iii), (iv), (v), (vii) or (xi), for example, carboplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, doxorubicin, a compound that binds, reduces or inhibits the activity of representatives of the family of growth factors vascular endothelial receptor tyrosinekinase (VEGFR) or receptor platelet-derived growth factor (DERIVED), or diphosphonates or mTOR inhibitor.

Another option is implemented the I the present invention relates to the use of agonist SF receptor (a) in combination with a chemotherapeutic agent (b) in the treatment of lymphatic or myeloid cancer, for example, as described above. The combination can include as additional collateral means (b), for example, busulfan, cytarabine, 6-tioguanin, fludarabine, hydroxyurea, procarbazine, bleomycin, or methotrexate. As related funds (b), for example, for use in the treatment of lymphatic cancer, are preferred inhibitors of topoisomerase II, such as daunorubicin or, especially, compounds that bind, decrease or inhibit the activity DERIVED or representatives of the family of C-Ab1 and products merge their genes, such as imatinib.

The concept of “co-administration”or “combined introduction”, or similar terms as they are used in the present invention, means the introduction of selected therapeutic agents to one patient and also refers to the use of such regimens, when funds are not necessarily entered in the same way or at the same time.

One of the objectives of the present invention was to provide a pharmaceutical composition that includes a number of combinations of the present invention, which in a joint application therapeutically effective against proliferating malignant disease. In this composition the first tool (a) and associated tool (b) can be administered together, one after the other, or Adelino in the form of one combined uniform dose, or in the form of two uniform dose forms. Form a uniform dose can also be fixed combination.

The pharmaceutical compositions according to the present invention can be prepared in a manner known per se. They are suitable for enteral, for example oral or rectal, and parenteral administration mammals (warm-blooded animals), including humans. The compositions contain a therapeutically effective amount of at least one pharmacologically active ingredient combinations, for example, as described above, or in combination with one or more pharmaceutically acceptable carriers or solvents, especially suitable for enteral or parenteral application.

Acceptable pharmaceutical compositions contain, for example, the active ingredient (or active ingredients) in the amount of approximately from 0.1% to 99.9%, preferably from 1% to 60%. Pharmaceutical drugs for combination therapy for enteral or parenteral administration are, for example, forms a uniform doses, such as tablets, sugar coated tablets, capsules, suppositories or ampoules. Unless otherwise indicated, are prepared by a known per se manner, for example, using such conventional processes as mixing, granulating, coating sugar shell, rest the drilling or lyophilization.

Valuable is the fact that the content of the units of the mixed pharmaceutical composition consists of separate doses of each component of the combination, taken in an amount which alone is a sufficient basis for the activity; the necessary effective amount can be achieved by the introduction of multiple dose units.

In particular, a therapeutically effective amount of each component of the combination of the present invention can be administered simultaneously or sequentially in any order, the components can be entered separately or as a fixed combination. For example, the method of containment of progression or treatment of proliferating malignant diseases according to the present invention may include: (i) the introduction of the first means (a) in free form or in the form of a pharmaceutically acceptable salt and (ii) the introduction associated means (b) in free form or in the form of pharmaceutically acceptable salts; the introduction is carried out simultaneously or sequentially in any order, in amounts therapeutically effective in case of joint application, preferably in amounts confer a synergistic effect, for example, daily or intermittent dosages corresponding to the amounts described in this invention. The individual components of the combination the present invention can be entered separately at different times throughout the course of treatment, or both as separate forms or as a single combined form. The notion of “introduction” also includes the use of prodrugs as a component of combination, which in vivo is transformed into a component of the combination. The present invention therefore should be understood as encompassing all of these regimes of simultaneous or alternating treatment and the term “introduction” should be interpreted accordingly.

The effective dosage of each of the components of the combination of the present invention may vary depending on whether an individual compound or pharmaceutical composition, and depending on the method of administration, disease, whose treatment, and its severity. Therefore, the dosage of the combination of the present invention may be chosen based on various factors, including route of administration, the kidneys and the liver of the patient. Private physician or veterinarian can readily determine and prescribe the effective amount of the individual active ingredients required to prevent, counter or deter the development of the disease. The optimal choice is such a concentration of active ingredients, which falls within the range of concentrations that ensure the effectiveness of treatment in the absence of toxicity; this choice is based on the kinetic ability of the active ingredients to reach m is I added.

The daily dosage of the first tool or the component (a) will, of course, vary depending on various factors, for example, selected from compounds of the features of the disease, whose treatment, and the desired effect. In General, satisfactory results are achieved with the introduction of agonist SP-receptor, for example, compounds a, B or C in daily doses of the order of approximately 0.1-100 mg as a single dose or as several separate doses. Agonist SP-receptor can be entered in any traditional way, especially enterline, for example, orally, for example in the form of tablets, capsules, mixtures, or parenterally, for example in the form of solutions or suspensions for injection. Acceptable forms of single doses for oral administration comprise from about 0, 1 to 30 mg of the component (a), for example, from 0.1 to 25 mg, together with one or more pharmaceutically acceptable diluents or carriers.

Fadrozole you can enter the person orally in a dosage range varying from about 0.5 to 10 mg/day, preferably from 1 to 2.5 mg/day. Exemestane you can enter the person orally in a dosage range varying from about 5 to 200 mg/day, preferably from 10 to 25 mg/day. This drug can also enter parenteral, from about 50 to 500 mg/day, prefer the Ino from 100 to 250 mg/day. If this medication will be administered in separate pharmaceutical compositions, it can be entered in the form described in GB 2177700. Formestane you can enter the person parenterally in a dosage range varying from about 100 to 500 mg/day, preferably from 250 to 300 mg/day. Anastrozole can enter the person orally in a dosage range varying from about 0.25 to 20 mg/day, preferably from 0.5 to 2.5 mg/day. Aminoglutaric you can enter a man in a dosage range varying from about 200 to 500 mg/day.

Citrate tamoxifen you can enter the person in the range of doses ranging from about 10 to 40 mg/day.

Vinblastine you can enter the person in the dosage range varying from about 1.5 to 10 mg/m2/day. Sulfate vincristine, you can enter the person parenterally in a dosage range varying from about 0.025 to 0.05 mg/kg of body weight per week. Vinorelbine you can enter the person in the range of doses ranging from about 10 to 50 mg/m2/day.

Phosphate etoposide you can enter a man in a dosage range varying from about 25 to 115 mg/m2/day, for example, 56,8 or 113,6 mg/m2/day.

Teniposide you can enter the person in the dosage range varying from about 75 to 150 mg, approximately once every two weeks. Doxorubicin you can enter chelovekov dose range, ranging from about 10 to 100 mg/m2/day, for example, 25 or 50 mg/m2/day.

Epirubicin you can enter the person in the range of doses ranging from about 10 to 200 mg/m2/day. Idarubitsin you can enter a man in a dosage range varying from about 0.5 to 50 mg/m2/day.

Mitoxantrone you can enter the person in the dosage range varying from about 2.5 to 25 mg/m2/day.

Paclitaxel you can enter the person in the range of doses ranging from approximately 50 to 300 mg/m2/day. Docetaxel can enter a person in the range of doses ranging from approximately 25 to 100 mg/m2/day.

Cyclophosphamide can be entered to the man in the dosage range varying from about 50 to 1500 mg/m2/day. Melphalan you can enter the person in the dosage range varying from about 0.5 to 10 mg/m2/day.

5-Fluorouracil can be entered to the man in the dosage range varying from about 50 to 1000 mg/m2/day, for example, 500 mg/m2/day.

Capecitabine, you can enter the person in the range of doses ranging from about 10 to 1000 mg/m2/day. Gemcitabine hydrochloride, you can enter the human in a dose approximately equal to or greater than 1000 mg/m2in the week. Methotrexate can enter a person in the range of doses varying bring the flax from 5 to 500 mg/m 2a day.

Topotecan you can enter the person in the range of doses ranging from about 1 to 5 mg/m2/day. Irinotecan you can enter the person in the dosage range varying from about 50 to 350 mg/m2/day.

Carboplatin you can enter the person in the range of doses ranging from approximately 200 to 400 mg/m2approximately every four weeks. Cisplatin can enter a person in the range of doses ranging from approximately 25 to 75 mg/m2approximately every three weeks. Oxaliplatin, you can enter the person in the dosage range varying from about 50 to 85 mg/m2every two weeks.

Imatinib, you can enter the person in the dosage range varying from about 2.5 to 850 mg / day, more preferably from 5 to 600 mg/day and more preferably from 20 to 300 mg/day.

Alendronate acid can enter the person in the range of doses, ranging approximately from 5 to 10 mg/day. Clodronate acid can be introduced, for example, in a dosage range varying from about 750 to 1500 mg/day. Trigonomy acid can enter the person in the dosage range varying from about 20.0 to 400 mg/day. Ibandronate acid can enter the person in the range of doses ranging from about 1 to 4 mg every three to four weeks. Risedronate acid mo is but to enter the person in the dose range, ranging approximately from 20 to 30 mg/day. Pamidronate acid can enter the person in the dosage range varying from about 15 to 90 mg every three to four weeks. Tiludronate acid can enter the person in the range of doses ranging from approximately 200 to 400 mg/day.

Trastuzumab you can enter the person in the range of doses ranging from about 1 to 4 mg/m2a week.

Bikalutamid you can enter a man in a dosage range varying from about 25 to 50 mg/m2/day.

1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or its salt, for example, succinate, you can enter the person in the dose range from approximately 50 to 1500, more preferably from 100 to 750, and most preferably from 250 to 500 mg/day.

Rapamycin or its derivative, for example, 40-ortho-(2-hydroxyethyl)-rapamycin, you can type in a dosage range varying from about 0.1 to 25 mg

Sample composition: soft capsules

The compound of formula I, e.g. compound a, hydrochloride30 mg
Polyethylene glycol 300300 mg
Polysorbate 8020 mg
Only35 mg

Agonists SF-receptor, for example, agonist SP-receptor, including a group of the formula X, is well tolerated by the patients in dosages that are required for use in accordance with the present invention. For example, acute LD50to connect A>10 mg/kg when orally administered to rats and monkeys.

On the other hand, the present invention relates to the use SF-agonists as Pro-angiogenic drugs. Recently, it was generally recognized that the induction of neoangiogenesis is an excellent target for a number of diseases (such as myocardial angiogenesis, wound healing or diabetic vascular dysfunction/ vasculopathies).

As described above, agonists SF-receptor at high concentrations (2 μm or above, for example, 2-5 μm or about 5 μm) exhibit antiangiogenic effects, and agonists SF-receptor can inhibit VEGF-induced angiogenesis. In contrast, low concentrations (0.1 to 1 μm, for example, 0.1-0.5 micron 0.5-1 micron) SF-agonists enhance angiogenesis and can potentiate VEGF-mediated angiogenesis. Thus, SF agonists may exhibit biphasic effects on angiogenesis.

Accordingly, the present invention also provides:

8. Use SF-agonist, for example, SF-agonist, with the group containing a series of formula X, for example, compound a or compound A-phosphate, in the induction process of neoangiogenesis, for example, as a Pro-angiogenic agent, for example, in cases where it is shown stimulation of angiogenesis;

9. Method of preparation of medicines for treating or preventing diseases associated with the inhibition of the process of neoangiogenesis, for example, diseases associated with angiogenic factors, for example, in cases where it is shown stimulation of angiogenesis, for example, in wound healing or in the treatment of myocardial infarction or diabetic vascular dysfunction/vasculopathy; the method includes the use of agonist SP-receptor, for example, SF-agonist comprising a group of formula X, for example, compound a or compound A-phosphate as an active ingredient;

10. A method of treating or preventing diseases associated with the inhibition of the process of neoangiogenesis, for example, diseases associated with angiogenic factors, for example, in cases where it is shown stimulation of angiogenesis, for example, in cases such as wound healing or treatment of myocardial infarction or diabetic vascular dysfunction/vasculopathy; the method comprises the administration to a subject requiring such treatment, an effective amount of agonist SP-receptor, for example, SF-agonist comprising a group which has the formula X, for example, compound a or compound A-phosphate.

SF agonists suitable for stimulation of angiogenesis include compounds described above as a means for treating cancer, for example, SF agonists, including a group of formula X or compounds corresponding to formulas I-IX, or pharmaceutically acceptable salts or esters. Preferred CF-agonist is compound A-phosphate. You can only use one SF agonist, or a combination with one or more other tools that stimulate angiogenesis, for example, VEGF.

In order to stimulate angiogenesis, it is important to choose a sufficiently low dose of agonist SP-receptor, since high concentrations of agonists SF-receptor inhibit angiogenesis. Acceptable dose in order to ensure Pro-angiogenic action when SF agonist is introduced to the patient, you can pick based on the research of increasing concentrations and doses, as described above, p.p, B and C.

Description of figures

Figure 1 shows that the compound a-phosphate significantly stimulates the formation capillarities network bell-shaped dose-dependent manner, with maximum activity at an approximate dose of 0.5 μm.

Figure 2 shows that compound a-phosphate or compound in concentrations of from 0.5 to 1 μm does not attenuate VEGF-mediated remodel is the formation, but better interact with polypeptide growth factor.

Figure 3 shows that the formation of tubules stimulated as compound A-phosphate and SF, almost completely inhibited by pertussis toxin (CT, 50 ng/ml), an inhibitor of heterotrimeric G protein αi/otype. This can be explained by the possible involvement of signaling events mediated EDG-1 (SF1)receptor, stimulated by compound a-phosphate of biological responses.

Figure 4 shows that sphingosine concentration of 1 μm, which, apparently, is less effective than SF, weakens the ability as SF and compound A-phosphate to induce capillaroscopy patterns without inhibitory effect on VEGF-induced formation of tubules. In this regard, sphingosine behaves differently than compound A. These data indicate that the balance between sphingosine and SF, apparently, is critical for the activation of endothelial cells/angiogenesis, most likely through representatives of the family of EDG receptors. It is essential that high concentrations of sphingosine and connection And (2-5 μm) inhibited the formation of tubules initiated by VEGF.

Figure 5 shows that treatment ACPWC compound A-phosphate in a concentration of 0.5 μm may cause temporary activation of ERK1/2 with peak phosphorylation/ is ctively on 10 th minute and return to the original level in the 20th minute.

6. Investigated the ability of compound A, compound A-phosphate, sphingosine and SF also to induce tissue factor in ACPWC. The obtained data show that none of these compounds, applied by itself or in combination with other compounds which increase the activity of tissue factor, as shown in Fig.6. Compound a and compound a-phosphate can increase the content of VEGF-induced tissue factor, but not TNF-α-induced tissue factor.

Fig.7 shows the effect of compounds In the analysis SF-mediated formation of tubules ACPWC.

List of abbreviations:

BSA: bovine serum albumin

ECGS: a set of growth factors, endothelial cells

From: sphingosine

JNK1/2: c-jun-N-terminal kinase 1/2

Equivalents TF: equivalents of tissue factor

EGR-1/NFAT: protein 1 response initial growth / nuclear factor of activated T-cells

And 1 f: compound A-phosphate (FTY720-phosphate)

PHL: enhanced chemiluminescence

FSB: phosphate-saline buffer

The role of agonists SF-receptor, for example SF-agonists containing a group of formula X, in stimulating angiogenesis can be exemplified by the following methods.

, Cell Culture and materials

Endothelial cells of the umbilical vein of a person (ACPWC) were cultured at 37°C in atmospheres is 5% CO 2in medium M199 with the addition of 20% SCS (firm HyClone, Logan, Utah, USA), 1 unit/ml heparin, 50 μg/ml ECGS, 2 mm glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin, In the experiment using cells up to the fifth passage. Use cells incubated under conditions of intermittent fasting, which ACPWC cultured for 5 h in medium M199 with reduced content of SCS (1%). Recombinant VEGF165humans receive from the company PromoCell (Heidelberg, Germany). Policlonal antibodies to phosphospecific ERK1/2, R the kinase, antibodies to Neosho-ERK1/2 and chemiluminescent LumiGLO reagent (company New England Biolabs, Beverly, mA), polyclonal antibodies to 1kV (company Santa Cruz Biotechnology, Santa Cruz, California). The conjugated donkey anti-rabbit immunoglobulin G (IgG) and sheep antimisting IgG with horseradish peroxidase was purchased from a company Amersham LIFE SCIENCE (Amersham Place, UK). The blotting membrane Immobilon P - production company Millipore (Bedford, mA, USA). Sphingosine derived from the company Sigma Chemical Co.; SF from company Biomol. The original solution of compound A-phosphate is prepared as follows. The compound a-phosphate is dissolved in methanol containing traces of concentrated HCl (0.5 mg of compound A-phosphate in 500 μl of methanol containing 2 μl of HCl). From the resulting solution evaporated under vacuum, the solvent and the precipitate dissolved (option 1) in 0.1% solution of skim BSA erased in the school deionized water (500 µl) or (option 2) 0.5% solution of Triton X-100 in deionized water. The initial solutions (2.5 mm) is treated with ultrasound and stored at 4°C.

Analysis of coagulation

Cells were seeded in 6-hole tablets at a density of 80-90% and grown over night. Cells are collected from the tablet and analyze the activity of tissue factor by the method described by M. Clauss in J. Biol. Chem., 271, 1996,. 17629-17634; D. Mechtcheriakova in Blood, 93, 1999, SS-3823. Briefly, the analysis is performed as follows: cells, after induction for 4 h with VEGF (1.5 nm), TNF-α ((100 units/ml), s (0.5-2 µm), SP (0.5 to 2 μm), compound A (0.5 to 2 μm) and compound a-phosphate (0.5 to 2 μm), washed twice, and then harvested in 1 ml of coagulating buffer (12 mm sodium acetate, 7 mm diethylbarbituric and 130 mm sodium chloride; pH 7.4). 50 μl resuspending cells mixed with 50 μl of citrate plasma; coagulation time determined after rekaltsifikatsii in 50 μl of a solution of CaCl3(concentration 20 mm) at 37°C. the Equivalent timeframe determined using a standard curve obtained by thromboplastin from rabbit brain.

D. Western blot analysis

After various treatments, the cells washed twice with cold FSB, are lysed in 100 ál of buffer laemmli's method, collect and heated for 5 min at 95°C. the resulting lysates of cells separated by denaturing electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-page) is transferred to Immobilon P membrane. The membrane is blocked with the help of the FSB, containing 0.1% tween-20 and 3% skim milk for 30 min and incubated at room temperature for 1 h with primary antibody diluted in blocking buffer. The resulting membrane was washed three times for 5 min FSB, containing 0.1% tween-20, and incubated with conjugated secondary antibodies with peroxidase for 1 h at room temperature. After washing the membrane is incubated for 1 min with ECL reagent (reagent for electrogenerating chemiluminescence) and exhibit a film in a period of time sufficient to produce the result. To re-study with another antibody the membrane washed twice in FSB, desorbed for 30 min at 55°C With desorbers buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 100 mm 2-mercaptoethanol) and washed with FSB three times for 5 min at room temperature. After each immunological analysis of the membrane is kept moist, wrapped in Saran (SaranWrap), at 4°C.

Analysis of angiogenesis on Matrigel in vitro

Morphogenesis of endothelial cells, which in education capillarity structures, is performed on the matrix of the Matrigel with reduced number of growth factor (firm BD Bioscience) according to the instructions for use. Briefly, the analysis is performed as follows: ACPWC treated with trypsin, resuspended in Bess is orotocol medium M199, containing soybean trypsin inhibitor (1 mg/ml, the firm Sigma). After centrifugation cells resuspended in serum-free medium, leading to a density of 0.5×105cells/ml, and the cell suspension is seeded in 96-well plates to cell cultures (firm Costar, Corning Incorporated), in which pre contribute 50 ál of Matrigel without/with various stimulants: VEGF at a concentration of 1.5 nm, SF in a concentration of 0.1-2 μm, in a concentration of 0.5-2 μm, compound a at a concentration of 0.5 to 2 μm and the compound a-phosphate in a concentration of 0.1-2 μm. After 8 h, cells on the Matrigel fixed with 3% formaldehyde solution in FSB and stored at 4°C. the Results were evaluated quantitatively by images taken with a microscope (Nikon Diaphot”, equipped with a cooled camera with repeating unit (firm Kappa GmbH, Gleichen, Germany) by direct calculation of the branching points in the two fields of view of the microscope for each well is two-fold repetition.

E. Analysis of in vitro on Matrigel formation of tubules (capillarity structures) in the morphogenesis of endothelial cells due to induction by compound a-phosphate and possible participation in this process, Gi-mediated signaling pathways (or paths)

The effect of compound a and compound A-phosphate on morphogenetic differentiation of endothelial cells to determine whether IP is by the use of analysis of angiogenesis in vitro on Matrigel. Morphogenesis of endothelial cells is a complex process that requires interactions of cells with extracellular matrix, followed by a reconstruction matrix-induced migration, intercellular interactions, and perivascular by proteolysis. As shown in figure 1, compound a-phosphate to a large extent can activate education capillarities network bell-shaped dose-dependent manner, showing maximum activity near a concentration of 0.5 μm. The number of branching points in the field of view of the microscope, which reflects the induction potency of the stimulus, comparable to compound A-phosphate and SF and may significantly exceed the effects initiated by VEGF. In itself, the compound a at a concentration of 0.5 to 1 μm is weak compared to compound a-phosphate, but consistently rising action. Neither compound a-phosphate or compound in a concentration of 0.5-1 μm does not weaken the reconstruction mediated VEGF, but rather interact with a polypeptide growth factor (see, e.g., figure 2). Moreover, the formation of tubules stimulated by compound a-phosphate or SF completely inhibited by pertussis toxin (CT, 50 ng/ml), an inhibitor of heterotrimeric G protein αi/otype. This can be interpreted as the possible involvement of EDG-1 (SF1) receptorpositive signal is selected actions stimulated by compound a-phosphate of biological responses (see, for example, figure 3). Sphingosine concentration of 1 μm, which, apparently, is less active than SF, weakens the ability and SF, and compound A-phosphate to induce capillaroscopy patterns without showing inhibitory effect on the formation of tubules induced by VEGF (see, for example, figure 4). In this regard, sphingosine behaves differently than compound A. These data indicate that the balance between sphingosine and SF, apparently, is critical for the activation of endothelial cells/angiogenesis, most likely through the EDG family receptors. It is essential that high concentrations of sphingosine and connections And (2-5 μm) inhibited VEGF-initiated the formation of tubules. These data suggest a biphasic dose-dependent effect of compound a and compound A-phosphate on angiogenesis in vitro.

J. Activation of ERK1/2 MAP-kinases compound A-phosphate

Signal transduction through MAR-kinase plays a key role in the implementation of the various functions of endothelial cells. Processing ACPWC compound A-phosphate in a concentration of 0.5 μm may cause temporary activation of ERK1/2, with peak phosphorylation/activation in the 10th minute and return to the original level in the 20th minute (see figure 5). In ACPWC not detect activation by compound a-phosphate R-kinase and JNK1/2. Moreover, the compound a-phosphate may be mandatory UN paid the encoded activation of ERK1/2 dose-dependent manner, showing the greatest activity at a concentration of 2 μm. These data are opposite to the results of the analysis of the formation of tubules, where the compound a-phosphate at a concentration of 2 μm may show less activity than in a concentration of 0.5 μm. Neither the connection nor the sphingosine not able to induce activation of map kinase in endothelial cells in the kinetic processing range from 5 minutes to 60 minutes. To evaluate the possible role of inflammatory/NFκB-dependent program in biological responses of endothelial cells stimulated by compound a-phosphate, re-examines the membrane with antibodies to Iκ. Treatment with compound a-phosphate does not affect the level Iκ. Moreover, treatment of endothelial cells with compound a-phosphate is probably not induces the expression of E-selectin as NFκB-dependent secondary response gene. Thus, these data convincingly indicate that the alarm is compound A-phosphate does not involve NFκB-activation - main cascade in acute inflammatory responses in endothelial cells.

H. Compound, and a phosphate does not induce expression of tissue factor on endothelial cells

An important distinguishing feature of classical inducer of inflammation, TNF-α, and the main angiogenic growth factor VEGF on endothelial cells is their ability to increase danilovavassori. Investigated the ability of compound A, compound A-phosphate, sphingosine and SF also to induce tissue factor in ACPWC. The obtained data show that none of these compounds, either alone or in combination, can increase the activity of tissue factor (see, for example, 6). Compound a and compound a-phosphate can to some extent it can enhance tissue factor-induced VEGF, but not TNF-α. Together these data indicate that compound a, compound a-phosphate, sphingosine and SF mechanism of action different from angiogenic VEGF and induce inflammation TNF-α.

I. the Ability of agonists SF-receptor to bind with separate SF-receptors man can be installed in the following tests:

Transient transfection of SF receptors of human cells NC

Spend cloning EDG receptors and Gi-proteins, mix equal amounts of 4 cDNA: EDG-receptor, Giα, Gi-β and Ci-γ; the resulting mixture was used for transfection of monolayer cell cultures NEC, which use the method of precipitation of calcium phosphate (M.Wigler and others in the Cell., 11, 1977, p.223; DS. Im and others in Mol. PharmacoL, 57, 2000, s). Briefly, the method consists in the following: a mixture of DNA containing 25 μg DNA and 0.25 M CaCl, are added to a solution of 2 mm Na2HPO4in HEPES-buffer. Subconfluent monolayers of cells NC pickle 25 the M chloroquine, and then the precipitated DNA is applied to the cells. After 4 h, the monolayers washed with saline phosphate buffer and add culture medium (90% 1:1 modified by Dulbecco basic medium (DMEM:F-12+10% fetal calf serum). After 48-72 h after addition of DNA, the cells are harvested, washing their NME-buffer (in mm: 20 HEPES, 5MgCl2, 1 EDTA, pH 7.4), containing 10% sucrose, on ice, and homogenized using a homogenizer of the downs. After centrifugation at 800 g supernatant diluted NME without sucrose and centrifuged at 100000 g for 1 h the precipitate is re-homogenized and centrifuged for a further one hour at 100000 g. The obtained coarse sediment membranes resuspended in NME with sucrose, divided into aliquots and rapidly frozen by immersion in liquid nitrogen. Membranes stored at -70°C. the protein Concentration determined by the spectroscopic analysis of proteins by Bradford.

The analysis of binding γS using SF-receptor / NEC membrane preparations

Research associate γS conducted according to the technique described DS. Im and others in Mol. PharmacoL, 57, 2000, s. Liganddependent linking γ8 with G-proteins evaluated in the GTP-binding buffer (in mm: HEPES 50, 100 NaCl, 10 MgCl2, pH 7.5)using 25 μg of the preparation of cell membranes NECK, subjected to transfection. The ligand is added to the membranes in the presence of 10 μm GDF and 0.1 nm [35S] γ (1200 CI/mmol) and incubated at 30°C for 30 minutes Associated γS separated from unbound using a collection company Brandel (Gaithersburg, MD, USA), and consider using a liquid scintillation counter.

1. The use of agonist of the receptor for sphingosine-1-phosphate, which is 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3 diol in free form or in the form of pharmaceutically acceptable salts for the preparation of drugs for the control of angiogenesis.

2. The use of agonist of the receptor for sphingosine-1-phosphate according to claim 1 in the form of its hydrochloride.

3. The use of agonist of the receptor for sphingosine-1-phosphate according to claim 1, where the agonist of the receptor for sphingosine-1-phosphate is a compound of formula IVa

where R2ameans hydrogen, R3aIT means, X is O, each of R1aand R1bmeans IT or its pharmaceutically acceptable salt.

4. The use of agonist of the receptor for sphingosine-1-phosphate according to claims 1, 2 or 3 at a concentration of more than 2 μm for the preparation of pharmaceuticals for inhibition of impaired angiogenesis.

5. The use of agonist of the receptor for sphingosine-1-phosphate according to claim 3, where the concentration is between 2 μm and 5 μm.

6. The use of agonist of the receptor for sphingosine-1-phosphate according to claims 1, 2 or 3 at a concentration of 0.1-1.0 μm to prepare a medicinal product for enhancing angiogenesis.

7. Application of the agonist receptor is sphingosine-1-phosphate according to claims 1, 2 or 3 for the preparation of medicines for the prevention or treatment of diseases mediated by the process of neoangiogenesis or associated with dysregulation of angiogenesis.

8. The use according to claims 1, 2 or 3, where the medicinal product further includes a chemotherapeutic agent.

9. The use according to claim 7, where the chemotherapeutic agent selected from
i) aromatase inhibitor,
ii) antiestrogen, antiandrogen or agonist of gonadorelin,
iii) an inhibitor of topoisomerase I or topoisomerase II inhibitor,
iv) means active against microtubules, alkylating tools, antitumor antimetabolite or platinum compounds,
v) connections linking/down-the activity of the protein or lietkynes or activity of the protein or lepidocrocite, other antiangiogenic compounds or compound that induces cellular differentiation,
vi) bradykinin receptor 1 or an antagonist of angiotensin II,
vii) an inhibitor of cyclooxygenase, diphosphonate, inhibitor discontinuties, inhibitor heparanase, biological response modifier, an inhibitor of many targets, or inhibitor, which blocks anti-apoptotic pathways,
viii) an inhibitor of Ras oncogenic isoforms,
ix) an inhibitor of telomerase,
x) protease inhibitor, an inhibitor of metallopro Ainazi matrix, inhibitor methioninamide, or the proteosome inhibitor, and/or
xi) mTOR inhibitor.



 

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1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the new pyridine and new pyrimidine derivative, their pharmaceutically accepted salt or hydrate of the general formula (I): . The invention also relates to the pharmaceutical composition, which possesses the inhibiting activity with respect to the receptor of the growth factor of hepatocytes; to the inhibitor of the receptor of the growth factor of hepatocytes, the inhibitor of angiogenesis, the antitumor drug, the inhibitor of cancerous metastatic spreading, that contains the pharmacologically effective dose of the said compounds, its pharmaceutically acceptable salt or hydrate.

EFFECT: inhibitory activity.

27 cl, 45 tbl, 540 ex

FIELD: medicine.

SUBSTANCE: invention describes application of 9-oxoacridine-10-acetic acid, it pharmaceutically acceptable salts and its ethers as additional means for treatment of conditions implying androgen action decrease is considered to be favourable, and method of condition treatment implying that androgen action decrease is considered to be favourable, and needy patient in introduced with effective amount of 9-oxoacridine-10-acetic acid it pharmaceutically acceptable salts and its ethers combined with hormonotherapy directed on androgen action decrease. Besides, method implies androgen-dependent tissue sensibility intensifying to antiandrogen hormonotherapy directed on androgen action decrease.

EFFECT: increased efficiency of antiandrogen hormonotherapy.

30 cl, 5 tbl, 9 ex

FIELD: medicine; immunotherapy.

SUBSTANCE: indications for adjuvant immunotherapy of primary and metastatic malignant unresectable liver tumours are determined. For this purpose within surgical process incision biopsy of tumour and healthy liver tissue is performed. Then immediately after operation lymphocytes of tumour tissue, healthy tissue and peripheral blood by markers CD4+, CD8+, CD16+, CD56+ are immunophenotyped. In case lymphocyte marker levels CD4+, CD8+ and NK-cells levels CD16+ and CD56+ are lowered more than 10% for tumour tissue and peripheral blood in comparison with healthy tissue and peripheral blood, postoperative intraportal prolonged introduction of rhoncoleukin is indicated.

EFFECT: provides accurate direct evaluation of local immunity condition; enables to improve treatment results.

12 dwg, 4 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: in compound of formula I , R1 is hydrogen; R2 is phenyl substituted by trifluoromethyl and optionally by other substitute selected out of a group including lower hydroxyl alkyl, lower alkylamino, lower hydroxyl alkylamino, dilower alkylamino, 1H-imidazolyl, lower alkyl-1H-imidazolyl, carbamoyl, lower alkylcarbamoyl, pyrrolidino, piperazino, lower alkylpiperazino, morpholino, lower alkoxy, trilfuoro-lower alkoxy, phenyl, pyridyl and halogenyl; R4 is methyl; where 'lower' prefix denotes radical with up to 7 carbon atoms. Also invention concerns pharmaceutical composition and method of treatment, as well as application of the claimed compounds in obtaining pharmaceutical composition.

EFFECT: improved proteinkinase inhibition properties.

9 cl, 98 ex

FIELD: medicine.

SUBSTANCE: offered is application Aplydine for production of medical product for leukaemia or lymphoma treatment by means of combined therapy using Aplydine and other medical product chosen from group consisting of methotrexate, cytosine arabinoside, mythoxanthrone, vynblastine, methylprennisolone and doxyrubicine, related methods of treatment (versions), pharmaceutical composition and kit. Cancer synergism of listed agents is shown in combination with Aplydine.

EFFECT: provision of effective treatment of fumours.

34 cl, 15 dwg, 6 ex, 11 tbl

FIELD: medicine; oncology.

SUBSTANCE: invention can be used for treatment of an acute myelogenetic leukemia or myelodysplastic syndrome. For this purpose use a combination of preparations hemetuzumab ozohamicin, daunorubicin and cytarabinum in certain doses and regimens.

EFFECT: invention promotes effective treatment of the specified diseases due to synergistic effect at influence of these preparations on an organism.

2 cl, 2 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention concerns application of N-[(9S,10R,11R,13R)-2,3,10,11,12,13-hexahydro-10-methoxy-9-methyl-1-oxo-9,13-epoxy-1H,9N-diindolo [1,2,3-gh:3', 2', 1'-lm] pyrrolo[3,4-j][1,7]benzodiasonine-11-yl]-N-methylbenzamide of formula or its salts for production of pharmaceutical composition intended for treatment of diseases associated with uncontrolled activity of receptor tyrosine kinase FLT3, pharmaceutical preparation and product, containing connection of formula (II).

EFFECT: high efficiency of treatment.

6 cl, 1 tbl, 2 ex

FIELD: medicine, oncology, hematology.

SUBSTANCE: method involves the complex using symptomatic, antibacterial, general tonic agents and nonspecific immunomodulating therapy. For this aim, lysozyme hydrochloride powder is given orally in the daily dose 500-1800 mg, 2 times per a day, every day for 20-30 days in combination with dosing sodium nucleinate in the daily dose 100-1500 mg, 2 times per a day, for 20-30 days by oral or sublingual route, and lactulose given orally in the dose 2.5-5 ml, 1-2 times per a day, every day for 10-30 days. Lysozyme hydrochloride is given 0.5-1 h before eating, sodium nucleinate is given after intake of lysozyme hydrochloride directly and lactulose is given 20-30 min before eating, or before eating immediately, or in 3-4 h after intake of lysozyme hydrochloride and sodium nucleinate. Method provides the complex correction of nonspecific resistance of body in patients suffering from leukosis and involving maintenance of immune homeostasis, plasma proteolysis, intestine microecology and reparative processes and improved tolerance of scheduled polychemotherapy.

EFFECT: improved method of treatment.

FIELD: medicine, oncology.

SUBSTANCE: invention relates to a method for chemotherapy of acute leucosis. Method involves isolation of blast cells and interphase cells from marrow puncture sample leukocyte fraction of blood of a patient subjected for chemotherapy. Then cells are deposited by centrifugation in medium 199 and their concentration is brought about to the level (2-3) x 106 cells/ml. Then isolated cells are incubated with each chemotherapeutic drug chosen from the following group: dexamethasone, cyclophosphanum, vincristine, teniposide, etoposide, citarabinum that are diluted preliminary with isotonic solution to the concentration 1:1000. Then cells treated with chemotherapeutic drugs are centrifuged repeatedly in medium 199 followed by carrying out the annexin test. In the schedule treatment drugs that showed the maximal percent of cells apoptosis are used. Method provides maximal decreasing adverse and toxic effects of chemotherapeutic drugs and to enhance apoptosis of tumor cells based on individual selection of chemotherapeutic drugs for a patient, to prolong remission period and to exclude using additional curative effects.

EFFECT: improved and enhanced method of chemotherapy.

2 ex

FIELD: organic chemistry, medicine, oncology, pharmacy, biochemistry.

SUBSTANCE: invention relates to amide derivative represented by the following formula [1]:

in any of the following cases (A) or (B), or its salt. In the case (A) R1 represents 5-7-membered saturated cyclic group comprising 1-2 nitrogen atoms as atom forming cycle (saturated cyclic amino-group can be substituted with 1-3 similar or different substitutes chosen from group consisting of (C1-C10)-alkyl, (C1-C10)-alkoxycarbonyl), mono-(C1-C10)-alkylamino- or di-(C1-C10)-alkylamino-group; R2 represents (C1-C10)-alkyl, halogen atom, halogen-(C1-C10)-alkyl, (C1-C10)-alkoxy-group, (C1-C10)-alkoxycarbonyl, nitro-group, mono-(C1-C10)-alkylcarbamoyl, di-(C1-C10)-alkylcarbamoyl or cyano-group; R3 represents hydrogen atom, halogen atom or (C1-C10)-alkoxy-group; Het1 represents any of the following formulae: [2] , [3] , [4] , [5] , [6] , [7] and [8] ; Het2 represents pyridyl, pyrimidinyl, pyrazinyl or 1,2-dihydropyridazinyl (wherein Het2 can be substituted with 1-3 similar or different substitutes chosen from halogen atom) but except for compound wherein R1 means (i) pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl and each of them can be substituted with 1-3 similar or different substitutes chosen from group consisting of alkyl, alkoxycarbonyl, halogen atom, halogenalkyl, hydroxyalkyl, amino-, monoalkylamino-, dialkylamino-group, carbamoyl, monoalkylcarbamoyl and dialkylcarbamoyl; (ii) monoalkylamino-group, or (iii) dialkylamino-group; Het1 means group of the formula [6], and Het2 means pyrazinyl or pyridyl and each of them can mean a substituted alkyl. In case the (B) R1 represents 4-methylpiperazin-1-yl, 1-pyrrolidinyl, piperidino-group, 4-ethylpiperazin-1-yl, 4-n-propylpiperazin-1-yl, cis-3,5-dimethylpiperazin-1-yl, morpholino-, dimethylamino- or diethylamino-group; R2 represents methyl, halogen atom, trifluoromethyl, methoxy-group, methoxycarbonyl, nitro-group, dimethylcarbamoyl or cyano-group; R3 represents hydrogen atom, bromine atom or methoxy-group; Het1 represents compound of the formula [6]; Het2 represents 3-pyridyl. Invention relates to a pharmaceutical composition possessing inhibitory activity with respect to BCR-ABL tyrosine kinase comprising amide derivative of the formula (I) or its salt as active component and a pharmaceutically acceptable nontoxic and inert carrier. Also, invention relates to BCR-ABL tyrosine kinase inhibitor, therapeutic agents comprising amide derivative of the formula (I) or its salt and, optionally, a pharmaceutically acceptable nontoxic and inert carrier used in treatment of chronic myelogenous leukemia, acute lymphoblast cell leukemia, acute myelogenous leukemia. Invention provides and proposes amide derivative inhibiting activity of BCR-ABL tyrosine kinase.

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

8 cl, 2 tbl, 83 ex

FIELD: medicine, oncology, pharmacy.

SUBSTANCE: invention proposes a pharmaceutical composition that contains compounds of chlorogenic acid isolated from Piper betel leaves extract or from any other part of plant Piper betel and a pharmaceutically acceptable excipient. Invention provides enhanced effectiveness of treatment of such diseases as acute and chronic myeloid leucosis and lymphoid leucosis and absence of its effect on normal cells. Invention can be used in treatment of patients suffering from acute and chronic myeloid and lymphoid leucosis.

EFFECT: enhanced and valuable medicinal properties of pharmaceutical composition.

32 cl, 4 tbl, 4 dwg, 11 ex

FIELD: organic chemistry, pharmacy, veterinary science.

SUBSTANCE: invention relates to compound comprising 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine or pharmaceutically acceptable salt of this compound and pharmaceutical composition used for stimulation of biosynthesis of cytokine based on abovementioned compound Also, invention claims a method for stimulation of biosynthesis of cytokines in animal body involving administration in animal body of above described compound or its salt. Invention provides preparing a novel compound possessing useful biological properties.

EFFECT: valuable biological properties of compound and pharmaceutical composition.

3 cl, 12 tbl, 213 ex

FIELD: medicine, peptides.

SUBSTANCE: invention relates to osteogenic growth oligopeptides used as stimulators of hemopoiesis. Invention proposes using an oligopeptide of molecular mass in the range from 200 to 1000 Da, comprising one of the following sequence: Tyr-Gly-Phe-Gly-Gly, Met-Tyr-Gly-Phe-Gly-Gly used in preparing a pharmaceutical composition and enhancing mobilization of hemopoietic stem cells from many differentiation line into peripheral blood, in particular, CD34-positive hemopoietic stem cells. Advantage of the invention involves expanding field in using oligopeptides used in stimulation of hemopoiesis.

EFFECT: enhanced and valuable properties of oligopeptides.

34 cl, 2 tbl, 7 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, oncology, and can be used in therapy of epidermoid skin cancer without metastases in regional lymph nodes. Said therapy is ensured by close-focus roentgenotherapy of total focal dose 6000 R. The irradiation course is followed with over- and subfascial introduction of Klein solution in tumour projection in a dose 20 ml. Then tumour-associated fascia is removed by endoscopic or surgical technique at the distance at least 1 cm from the irradiation zone.

EFFECT: method allows ensuring greatest possible therapeutic radicalism, removing lymphatic capillaries and postcapillaries assisting in dissemination of cancer cells, reducing metastasis probability with maintaining the cosmetic effect.

2 ex

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