PT1944026E - Use of edg receptor binding agents in cancer - Google Patents

Abstract

Provided is a method for treating solid tumors, e. g tumor invasiveness, and particularly inhibiting or controlling deregulated angiogenesis, using a sphingosine-1-phosphate receptor agonist, optionally in combination with a chemotherapeutic agent. The invention also comprises a combination of a sphingosine-1-phosphate receptor agonist with a chemotherapeutic agent.

Description

ΡΕ1944026 1

DESCRIPTION

" USING AGENTS THAT CONNECT TO AN EDG RECEIVER IN THE CANCER " The present invention relates to the use of a sphingosine-1-phosphate (S1P) receptor agonist, in particular in the treatment of cancer.

S1P receptor agonists are agents that accelerate the shift of lymphocytes to their targets (LH) leading to lymphopenia resulting from a, preferably reversible, lymphocyte redistribution of the circulation to secondary lymphatic tissue without evoking generalized immunosuppression. Negative cells are sequestered; CD4 and CD8 T cells and blood B cells are stimulated to migrate to the lymph nodes (LN) and Peyer's (PP) plugs, and thus, for example, the infiltration of cells into transplanted organs is inhibited.

S1P receptor agonists are typically sphingosine analogs, such as 2-aminopropane-1,3-diol derivatives substituted with 2 or 2-amino-propanol, for example a compound including a group having the formula

formula X wherein Z is H; C1-6 alkyl; C2-6 alkenyl; C2-6 alkynyl; phenyl; OH-substituted phenyl; C1-6 alkyl substituted with 1 to 3 substituents selected from the group consisting of halogen, C3-8 cycloalkyl, phenyl and OH substituted phenyl; or CH2 -R4z wherein R4z is OH, acyloxy or a residue of formula (a)

-ZHL 1

> OR «t la) in which

Z 1 is a direct bond or O, preferably 0; R 5z and R 6z, independently, are H, or C 1-4 alkyl optionally substituted with 1, 2 or 3 halogen atoms;

Riz is OH, acyloxy or a residue of formula (a); and R2z and R3Z, independently, are H, C1-4 alkyl or acyl. The group of formula X is a terminal group attached functional group to a species which may be hydrophilic or lipophilic and include one or more aliphatic, alicyclic, aromatic and / or heterocyclic residues, provided that the resulting molecule in which one at least one less than Z and Riz is or includes a residue of formula (a), signals as an agonist for one or more sphingosine-1-phosphate receptors. 3 ΡΕ1944026

S1P receptor agonists are compounds which signal to one or more sphingosine-1-phosphate receptors, for example S1P1 to S1P8. Binding of an agonist to an S1P receptor may for example lead to the dissociation of heterotrimeric G-proteins in Ga-GTP and 6β-6ΤΡ, and / or an increase in agonist-occupied receptor phosphorylation and activation of signaling pathways / kinases downstream. The binding affinity of the S1P receptor agonists can be measured as described in paragraph I, below. Examples of suitable S1P receptor agonists are, for example the following (only compounds of formula IX as part of the invention):

Compounds such as those described in EP 627,406 A1, for example a compound of formula I wherein R 1 is a straight or branched carbon chain (C 12-22) in which there may be a bond or a heteroatom selected from a double bond, a triple bond, O, S, NR 6, wherein R 6 is H, alkyl, aralkyl, acyl or alkoxycarbonyl, and / or carbonyl, and / or which may have the substituent alkylthio, acylamino, alkoxycarbonyl, alkoxycarbonylamino, acyloxy, alkylcarbamoyl, nitro, halogen, amino, hydroxyimino, hydroxyl or carboxyl; or • R 1 is a phenyl group in which the alkyl is a straight or branched C 6-20 chain carbon chain; or a phenylacyl in which the alkyl is a straight or branched C 1-30 branched carbon chain, wherein said phenylalkyl is substituted with a straight or branched C 6-20 branched carbon chain, optionally substituted with halogen, a straight or branched alkoxy chain in Ce-20, optionally substituted with halogen, a straight or branched alkenyloxy chain in Ce-20r, phenylalkoxy, halophenylalkoxy, phenylalkoxyalkyl, phenoxyalkoxy or phenoxyalkyl, C6-20 alkyl-substituted cycloalkylalkyl, or C6-20 alkyl-substituted heteroarylalkyl C6-20 heterocyclic alkyl or C2-20 alkyl substituted heterocyclic alkyl, and in which the alkyl moiety may have in the carbon chain a bond or a heteroatom selected from a double bond, a triple bond, O, S sulfinyl, sulphonyl, or NR 6, wherein R e is as defined above, and • as a substituent, alkoxy, alkenyloxy alkoxycarbonyl, alkoxycarbonylamino, acyloxy, alkylcarbamoyl, nitro, halogen, amino, hydroxyl or carboxyl, and R2, R3, R4 and R5 are independently H, C1-C4 alkyl, -4 or acyl or a pharmaceutically acceptable salt thereof; 6 ΡΕ1944026 1.002.792 Al,

Compounds such as those described in for example a compound of formula II

EP

W

Wherein m is 1 to 9 and R '2, R' 3, R '4 and R' 5 are independently H, alkyl or acyl, or a pharmaceutically acceptable salt thereof;

Compounds such as those described in EP 0 778 263 A1, for example a compound of formula III in which W is H; C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl; unsubstituted or substituted with OH-substituted phenyl; R &quot; 40 (CH 2) n; or C 1-6 alkyl substituted with 1 to 3 substituents selected from the group consisting of halogen, C 3-8 cycloalkyl, phenyl and OH substituted phenyl; X is H or straight chain alkyl, not substituted or substituted, containing a number of carbon atoms or linear, unsubstituted or substituted alkoxy containing a number of carbon atoms p-1, for example substituted with 1 to 3 substituents selected from the group consisting of C 1-6 alkyl, OH, C 1-6 alkoxy, acyloxy, amino, C 1-6 alkylamino, acylamino, oxo, C 1-6 haloalkyl, halogen, unsubstituted phenyl and phenyl substituted with 1 to 3 substituents selected from C1-6 alkyl, C1-6 alkoxy, acyl, acyloxy, amino, C1-6 alkylamino, acylamino, C1-6 haloalkyl and halogen; Y is H, C 1-6 alkyl, OH, C 1-6 alkoxy, acyl, acyloxy, amino, C 1-6 alkylamino, acylamino, C 1-6 haloalkyl or halogen, Z 2 is a single bond or straight chain alkylene having a number of carbon atoms of q, peq, independently, are integers from 1 to 20, with the proviso that 6p p + q 23, m 'is 1, 2 or 3, n is 2 or 3, R &quot; R &quot; 2, R &quot; 3 and R &quot; 4, independently, are H, C1-4 alkyl or acyl, or a pharmaceutically acceptable salt thereof, Compounds such as those described in WO 02/18395, for example compound of formula IVa or IVb ΡΕ1944026 «·» Λ * if »{?; λ | ™ α <κ5 κ * Φ * ί

Wherein Xa is O, S, NRis or a - (CH2) n - group which is unsubstituted or substituted with 1 to 4 halogens; n is 1 or 2, R 1 is H or C 1-4 alkyl, which is unsubstituted or substituted by halogen; R 1a is H, OH, C 1-4 alkyl or O- (C 1-4) alkyl, wherein the alkyl is unsubstituted or substituted with 1 to 3 halogens; R 1 is H, OH is C 1-4 alkyl, wherein the alkyl is unsubstituted or substituted with halogen; R2a are independently selected from H or (1-4C) alkyl, which is unsubstituted or substituted by halogen; R3a is H, OH, halogen or O- (1-4C) alkyl, wherein the alkyl is unsubstituted or substituted with halogen; and R 3b is H, OH, halogen, C 1-4 alkyl, wherein the alkyl is unsubstituted or is substituted with hydroxy, or O- (C 1-4) alkyl, wherein the alkyl is unsubstituted or substituted with halogen; Y is -O2-, -C (O) -, -CH (OH) -, -C (= NOH) -, O or S, and R4a is (4-4C) alkyl or (4-4C) alkenyl; or a pharmaceutically acceptable salt thereof; ΡΕ1944026 9 02 / 076,995,

Compounds such as for example a compound having the formula

described in formula V

in WO

x;

V wherein mc is 1, 2 or 3;

Xc is O or a direct bond;

R1 is H; C 1-6 alkyl optionally substituted with OH, acyl, halogen, C 3-10 cycloalkyl phenyl or hydroxyphenylene; C2-6 alkenyl; C2-6 alkynyl; or phenyl optionally substituted with OH; R2c is

in which R5c is H or C1-4alkyl optionally substituted with 1, 2 or 3 halogen atoms, and R6c is H or C1-4alkyl optionally substituted with halogen; R3c and R4c are independently H, C1-4alkyl optionally substituted with halogen, or acyl, and ΡΕ1944026 10

Rc is C 1-3 alkyl which may optionally contain in the chain an oxygen atom and which may be optionally substituted by nitro, halogen, amino, hydroxyl, or carboxy; or a residue of formula (a)

in which R 7c is H, C 1-4 alkylalkyl or

C1-4 alkyl, and R.8c is substituted C1-20 alkanoyl, phenyl C1-4 alkyl wherein the C1-4 alkyl is optionally substituted with halogen or OH, cycloalkyl-C1-4 alkoxy or phenyl C1-4 alkoxy, wherein the cycloalkyl or phenyl ring is optionally substituted with halogen, C1-4 alkyl and / or C1-4 alkoxy, phenyl C1-4 alkoxy C1-4 alkyl, phenoxy C1-4 alkoxy or phenoxy C1-4 alkyl,

Rc being also a residue of formula (a) wherein Rc is C1-4 alkoxy when Ric is C1-4 alkyl, C2-6 alkenyl or C2-6 alkynyl,

or a compound of formula VI

in which 11 ΡΕ1944026 nx is 2, 3 or 4

Rix is H; C1-6 alkyl optionally substituted with OH, acyl, halogen, cycloalkyl, phenyl or hydroxy-phenylene; C2-6 alkenyl; C2-6 alkynyl; or phenyl optionally substituted with OH; R 2x is H, C 1-4 alkyl or acyl R 3x and R 4X are independently H, C 1-4 alkyl optionally substituted with halogen or acyl, R 5x is H, C 1-4 alkyl or C 1-4 alkoxy and R 6x is C 1-2 substituted alkanoyl with cycloalkyl; cycloalkyl-C 1-4 alkoxy wherein the cycloalkyl ring is optionally substituted with halogen, C 1-4 alkyl and / or C 1-4 alkoxy; phenyl-C 1-4 alkoxy wherein the phenyl ring is optionally substituted with halogen, C 1-4 alkyl and / or C 1-4 alkoxy, R 6 is also C 4-4 alkoxy when R 9 is C 2-4 alkyl substituted with OH, or pentyloxy or hexyloxy when Rix is C1-4 alkyl, provided that R6 is not phenylbutyleneoxy when either R5x is H, or Rx is methyl, or a pharmaceutically acceptable salt thereof; 12 ΡΕ1944026

Compounds such as those described in WO 02/06688 A1, for example a compound of formula VII '.

"PHA wherein R 1 and R 2d are independently H or an amino protecting group; R3d is hydrogen or a hydroxyl protecting group; R4d is lower alkyl; nd is an integer from 1 to 6;

(Wherein D is carbonyl, -CH (OH) -, O, S or N), aryl or aryl substituted with up to three substituents selected from the set as hereinafter defined;

Y is a single bond, C 1-10 alkylene, C 1-10 alkylene which is substituted with up to three substituents selected from the groups a and b, C 1-10 alkylene containing one O or S atom at the middle or at the end of the carbon chain, or C 1-10 alkylene containing O or 13 ΡΕ1944026 S is at the middle or at the end of the carbon chain which is substituted with up to three substituents selected from the sets a and b; R5d is hydrogen, cycloalkyl, aryl, heterocycle, cycloalkyl substituted with up to three substituents selected from the groups a and b, aryl substituted with up to three substituents selected from the groups a and b, or heterocycle substituted with up to three substituents selected from the groups aeb; and R6c and R7d, independently, are H or a substituent selected from the group a;

Set to &gt; lower alkoxy, lower alkylthio, carboxyl, lower alkoxycarbonyl, hydroxy, lower aliphatic acyl, amino, mono-lower alkylamino, di-lower alkylamino, lower aliphatic acylamino, cyano or nitro;

Set b &gt; cycloalkyl, aryl, heterocycle, any of which is optionally substituted with up to three substituents selected from the group a; with the proviso that when Rsd is hydrogen, Yd is either a single bond or a linear C1-4 alkylene 1944026 derivative, or a pharmacologically acceptable salt or ester thereof. Compounds such as those described in JP-14,316,985 (JP 2002 / 316,985), for example a compound of formula VIII:

in which R1a, R2e, P-3e, P-4e, P-5e, P-6e, P-7e,, Xe and Xe are as described in JP-14,316,985; or a pharmacologically acceptable salt or ester thereof. Compounds such as those described in WO 03/29184 and WO 03/29205, for example compounds of formula IX, which form part of the present invention:

in which Xf is O or S, and R 1a, R 2f, R 3f and nf are as described in WO 03 / 29,184 and WO 03 / 29,205, for example 2-amino-2- [4- (3-benzyloxyphenoxy) -2- propane 1,3-propane diol or 2-amino-2- [4- (benzyloxyphenylthio) -2-chlorophenyl] propyl-1,3-propane-diol.

Acyl may be a residue Ry-CO- in which Ry is 15 ΡΕ1944026 C1-6 alkyl, C3-6 cycloalkyl, phenyl or phenyl C1-4 alkyl. Unless otherwise stated, alkyl, alkoxy, alkenyl and alkynyl may be straight chain or branched.

When in the compounds of formula I the carbon chain such as R1 is substituted it may be substituted with halogen, nitro, amino, hydroxyl or carboxyl. When the carbon chain is interrupted by an optionally substituted phenylene, the carbon chain may be unsubstituted. When the phenylene species is substituted, it may be substituted with halogen, nitro, amino, methoxy, hydroxy or carboxyl.

Compounds of formula I are those in which R1 is C13-20 alkyl, optionally substituted with nitro, halogen, amino, hydroxyl or carboxyl, and more preferably those wherein R1 is phenylalkyl substituted with an optionally substituted C6-14 alkyl chain with halogen and the alkyl moiety is C 1-6 alkyl optionally substituted with hydroxy. R 1 may be phenyl substituted C 1-6 alkyl with a linear or branched, preferably linear, C 6-1 alkyl chain. The C6-14 alkyl chain may be in the ortho, meta or para positions, preferably in para. R2 to R5 may all be H.

A compound of formula I is 2-amino-2β-19β 1944026 tetradecyl-1,3-propanediol. An S1P receptor agonist of formula I is FTY720, ie β-2-amino-2- [2- (4-octylphenyl) ethyl] propane-1,3-diol in free form or as a salt thereof (hereinafter referred to as Compound A), for example the hydrochloride, as shown:

HO

-HCS:

A compound of formula II is one in which R 2 to R 15 are H in is 4, i.e. 2-amino-2- {2- [4- (1-oxo-5-phenylpentyl) phenyl] ethyl} propane-1,3-diol in free form or in the form of a pharmaceutically acceptable salt thereof (hereinafter referred to as Compound B), for example the hydrochloride.

A compound of formula III is one wherein W is CH 3, R 3 is H, Z 2 is ethylene, X is heptyloxy and Y is H, i.e. 2-amino-4- (4-heptyloxyphenyl) -2-methylbutanol in free form or in the form of a pharmaceutically acceptable salt thereof (hereinafter referred to as Compound C), for example the hydrochloride. The R-enantiomer is especially preferred.

A compound of formula IVa is FTY720 phosphate (R2a is H, R3a is OH, Xa is O, R a and Rb are OH). A compound of formula IVb is the phosphate of Compound C (R2a is H, R3b is OH, Xa is O, R a and Rb are OH, Ya is O and R4a is heptyl). A compound of formula V is the phosphate of Compound B. 17 ΡΕ1944026

A compound of formula V is phosphoric acid mono- [(R) -2-amino-2-methyl-4- (4-pentyl-phenyl) -butyl] ester.

A compound of formula VIII is (2R) -2-amino-4- [3- (4-cyclohexyloxybutyl) benzo [b] thien-6-yl] -2-methylbutan-1-ol.

When the compounds of formula IX have one or more asymmetric centers in the molecule, it is to be understood that the present invention includes all of the various optical isomers, as well as the racemates, diastereomers and mixtures of these compounds. The described compounds of formula III or IVb, when the carbon atom bearing the amino group is asymmetric, may have the R configuration on this carbon atom.

Examples of the pharmaceutically acceptable salts of the compounds of the formulas I to IX, salts with inorganic acids, such as the hydrochloride, the hydrobromide and the sulfate, salts with organic acids, such as acetate, fumarate , maleate, benzoate, citrate, malate, methanesulfonate and benzenesulfonate, or, where appropriate, the metal salts, such as those of sodium, potassium, calcium and aluminum, salts with amines, such as triethylamine and the salts as dibasic amino acids, such as lysine. The compounds and salts of the methods of the present invention include their hydrate and solvate forms. 18 ΡΕ1944026

S1P receptor agonists have, based on their observed activity, for example the displacement of lymphocytes to their destination, for example as described in EP 627 406 AI or in USP 6,004,565, utility for example as immunosuppressants, for example in the treatment of acute rejection of allografts. S1P receptor agonists have now been found to have interesting properties that make them useful for cancer chemotherapy, especially solid tumors, in particular advanced solid tumors. There is still a need to expand the armamentarium of cancer treatments for solid tumors, in particular where treatment with anti-cancer compounds is not associated with regression or stabilization of the disease.

In the present invention, there is provided:

A use in inhibiting and controlling dysregulated angiogenesis, for example sphingosine-1-phosphate (S1P) mediated angiogenesis, in a subject in need thereof, comprising administering to said subject a therapeutically active amount of an S1P receptor agonist comprising a group of formula IX, or a therapeutically acceptable salt thereof, and

A use for preventing or treating diseases mediated by a neo-angiogenic process or associated with a deregulated angiogenesis in a subject in need thereof, the method of the present invention comprising administering to said subject an active amount from the A therapeutic view of an S1P receptor agonist comprising a group of formula IX, or a therapeutically acceptable salt thereof. The term &quot; solid tumors &quot; means tumors and / or metastasis (wherever located) other than lymphatic cancer, for example brain and other central nervous system tumors (eg meninges, cerebrum, spinal cord, cranial nerves, and other parts of the central nervous system, for example glioblastomas or blastomas of the spinal cord); cancer of the head and / or neck; breast tumors; tumors of the circulatory system (for example the heart, mediastinum and pleura, and other intrathoracic organs, vascular tumors and vascular tissue associated with tumors); tumors of the excretory system (eg kidney, renal pelvis, ureter, bladder, other unspecified urinary organs); tumors of the gastrointestinal tract (eg, esophagus, stomach, small intestine, colon, rectal, recto-sigmoid, rectum, anal and anal canal), tumors involving the liver and intra- hepatic, gallbladder, other unspecified parts of the biliary tract, pancreas, other digestive organs); of the oral cavity (lip, tongue, gingiva, sublingual, buccal palate, and other parts of the mouth, parotid gland, and other parts of the salivary glands, the amygdala, the oropharynx, the nasopharynx, the piriform bells, of the hypopharynx, and other sites on the lips, oral cavity and pharynx); tumors of the reproductive system (eg the vulva, vagina, cervix uteri, corpus uteri, uterus, ovary, and other sites associated with the female genitalia, placenta, penis, prostate, testis, and other sites associated with the male genital organs); tumors of the respiratory tract (for example the nasal cavity and middle ear, accessory bells, larynx, trachea, bronchi and lung, for example small cell lung cancer or non-small cell lung cancer); tumors of the skeletal system (by cancer of the bones and the articular cartilage of the limbs, articular cartilage of bones and other sites); skin tumors (e.g., malignant skin cancer, nonmelanoma skin cancer, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, mesothelioma, Kaposi's sarcoma); and tumors involving other tissues including peripheral nerves and the autonomic nervous system, connective tissue and soft tissues, retroperitoneum and peritoneum, eye and adnexa, thyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified neoplasm of lymph nodes, secondary malignant neoplasm of the respiratory and digestive systems, and secondary malignant neoplasm of other sites. 21 ΡΕ1944026

When a tumor is mentioned hereinbefore or hereinafter, or a tumoral disease, a carcinoma or a cancer, alternatively or additionally metastases are also implicated in the organ of origin or in another site or tissue and / or in any other alternative location, any which is the location of the tumor and / or metastasis.

In a set of particular or alternative embodiments, the present invention also provides

A use for enhancing the activity of a chemotherapeutic agent or for overcoming resistance to a chemotherapeutic agent in a subject in need thereof, including administering to the subject subject a therapeutically effective amount of an S1P receptor agonist, for example an agonist of the S1P receptor. S1P receptor comprising a group of formula IX, or a pharmaceutically acceptable salt thereof, either concomitantly or sequentially to said chemotherapeutic agent.

A use according to the above wherein the chemotherapeutic agent is an inhibitor of the signal transduction pathways directed either against host cells or against processes involved in the formation of tumors ΡΕ1944026 and / or metastases, or further used by tumor cells for the proliferation, survival, differentiation or development of drug resistance.

A use as indicated above, wherein the S1P receptor agonist is administered intermittently.

By &quot; lymphatic cancer &quot; for example, tumors of the blood and lymphatic system (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-related lymphomas, immunoproliferative malignancies, multiple myeloma and malignant cell plasma neoplasms, lymphoid leukemia , acute or chronic myelogenous leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other cell leukemias of a specified type, unspecified cell leukemia, other unspecified malignant neoplasms of lymphoid, hematopoietic, and related tissue tissues, for example diffuse large cell lymphoma, T-cell lymphoma or cutaneous T-cell lymphoma). It is included in myeloid cancer for example acute or chronic myeloid leukemia. The term &quot; chemotherapeutic agent &quot; means any chemotherapeutic agent in addition to the S1P receptor agonists according to the following list: ΡΕ1944026 23 i. an aromatase inhibitor, ii. an anti-estrogen, an anti-androgen (in particular in the case of prostate cancer) or a gonadorelin agonist, iii. a topoisomerase I inhibitor or a topoisomerase II inhibitor, iv. An active agent in the microtubules, an alkylating agent, an antineoplastic antimetabolite or a platinum compound, e.g. a targeting / decreasing compound of a protein or a lipid kinase or a lipid protein or phosphatase, an anti-angiogenic compound or a compound that induces cellular differentiation processes, vi. a bradykinin 1 receptor or an angiotensin II antagonist, vii. a cyclooxygenase inhibitor, a bisphosphonate, a histone deacetylase inhibitor, a heparanase inhibitor (prevents degradation of heparan sulfate), for example Pl-88, a biological reaction modifier, preferably a lymphokine or interferon, for example, or an inhibitor which blocks the anti-apoptotic pathways, viii. an inhibitor of the oncogenic isoforms of Ras, for example H-Ras, K-Ras or N-Ras, or a farnesyl transferase inhibitor, for example L-744,832 or DK8G557, i.e. a telomerase inhibitor, for example telomestatin, x. a protease inhibitor, a matrix metalloproteinase inhibitor, an inhibitor of methionine aminopeptidase, for example bengamide or a derivative thereof, or a proteasome inhibitor, for example PS-341, and / or xi) an mTOR inhibitor. The term &quot; aromatase inhibitor &quot; as used herein refers to a compound which inhibits the production of estrogen, i.e. the transformation of the substrates androstenedione and testosterone respectively into estrone and estradiol. The term includes, but is not limited to steroids, in particular atamestane, exemestane and formomestane, and in particular non-steroids, in particular ami-noglutetimide, rogletimide, pyridoglutethimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, zole and letrozole. For example, exemestane can be administered as in the form in which it is marketed, for example under the trademark AROMASINâ "¢. Formestane can be administered, for example, in the form in which it is marketed, for example under the trademark LENTARON ™. Fadrozole may be administered, for example, in the form in which it is marketed, for example under the trademark AFEMA ™. Anastrozole can be administered in the form in which it is marketed, for example under the trademark ARIMIDEX ™. Letrozole may be administered in the form in which it is marketed, for example under the trademark FEMARA ™ or FEMAR ™. Aminoglutethimide can be administered in the form in which it is marketed, for example under the trademark ORIMETEN ™. A combination of the invention including a chemotherapeutic agent which is an aromatase inhibitor is especially useful for the treatment of hormone receptor positive tumors, for example breast tumors. The term &quot; antiestrogen &quot; as used herein refers to a compound which antagonizes the effect of estrogens at the level of the estrogen receptor. The term includes tamoxifen, fulvestrant, raloxifene, and raloxifene hydrochloride. Tamoxifen may be administered, for example, in the form in which it is marketed, for example under the trademark NOLVADEX ™. Raloxifene hydrochloride may be administered, for example, in the form in which it is marketed, for example under the trademark EVISTA ™. The fulvestrant can be formulated as described in US 4,659,516 or may be administered, for example, as in the form in which it is marketed, for example under the trademark FASLODEX ™. A combination of the invention which includes a chemotherapeutic agent which is an antiestrogen is especially useful for the treatment of estrogen receptor positive tumors, for example breast tumors. The term &quot; anti-androgen &quot; as used herein refers to any substance which is capable of inhibiting the biological effects of androgenic hormones and includes bicalutamide (CASODEX ™), which may be formulated, for example as described in US 4,636,505. The term &quot; gonadorelin agonist &quot; as used herein includes abarelix, goserelin and goserelin acetate. Goserelin is disclosed in US 4,100,274 and may be administered, for example, in the form in which it is marketed, for example under the trademark ZOLADEX ™. Abarelix can be formulated, for example as described in US 5,843,901. The term &quot; topoisomerase I inhibitor &quot; as used herein includes topotecan, irinotecan, 9-nitrocamptothecin and the macromolecular camptothecin conjugate PNU-166148 (compound AI in WO 99/17804). Irinotecan may be administered, for example in the form in which it is marketed, for example under the trade mark CAMPTOSAR ™. Topotecan, for example, 27 ΡΕ1944026 may be administered in the form in which it is marketed, for example under the trademark HYCAMTIN ™. The term &quot; topoisomerase II inhibitor &quot; as used herein includes anthracyclines such as doxorubicin (including formulation in liposomes, for example CAELYX ™), daunorubicin, epirubicin, idar-rubicin and nemorubicin, the anthraquinones mitoxantrone and losoxantrone, and the podophyllotoxins etoposide and teniposide. Etoposide can be administered, for example in the form in which it is marketed, for example under the trademark ET0P0PH0S ™. Teniposide can be administered, for example in the form in which it is marketed, for example under the trademark VM 26-BRISTOL ™. Doxorubicin may be administered, for example in the form in which it is marketed, for example under the trademark ADRIBLASTIN ™. Epirubicin may be administered, for example in the form in which it is marketed, for example under the trademark FARMORUBICIN ™. Idarubicin can be administered, for example in the form in which it is marketed, for example under the trademark ZAVEDOS ™. Mitoxantrone can be administered, for example in the form in which it is marketed, for example under the trademark NOVANTRON ™. The term &quot; active agent in microtubules &quot; refers to agents which stabilize or destabilize microtubules including taxanes, for example paclitaxel and docetaxel, vinca alkaloids, for example vinblastine, in particular vinblastine sulfate, vincristine, in particular vincristine sulfate, and vinorelbine, discodermolides and epothilones and their derivatives, for example epothilone B or a derivative thereof. Paclitaxel can be administered for example in the form in which it is marketed, for example under the trademark TAXOL ™. For example, docetaxel may be administered in the form in which it is marketed, for example under the trademark TAXOTERE ™. For example, vinblastine sulfate may be administered in the form in which it is marketed, for example under the trademark VINBLASTIN R.P. ™. Vincristine sulfate may be administered, for example, in the form in which it is marketed, for example under the trademark FARMISTIN ™. Discodermolide can be obtained, for example, as described in US 5,010,099. The term &quot; alkylating agent &quot; as used herein includes, but is not limited to, busulfan, chlorambucil, cyclophosphamide, ifosfamide, melphalan or nitroso-urea (BCNU or Gliadel ™). Cyclophosphamide can be administered, for example, in the form in which it is marketed, for example under the trademark CYCLOSTIN ™. Ifosfamide can be administered, for example, in the form in which it is marketed, for example under the trademark HOLOXAN ™. The term &quot; antineoplastic antimetabolite &quot; includes, but is not limited to, 5-fluorouracil, capecitabine, gemcitabine, cytarabine, fludarabine, thioguanine, methotrexate and edatrexate. Capecitabine may be administered for example 29æ1944026 in the form in which it is marketed, for example under the trademark XELODAâ "¢. Gemcitabine can be administered, for example, in the form in which it is marketed, for example under the trademark GEMZAR ™. The term &quot; platinum compound &quot; as used herein includes, but is not limited to, carboplatin, cisplatin and oxaliplatin. Carboplatin can be administered, for example, in the form in which it is marketed, e.g. under the trademark CARBOPLAT ™. Oxaliplatin can be administered, for example, in the form in which it is marketed, for example under the trademark ELOXATIN ™. The term &quot; targeting / decreasing compound activity of a lipid protein or kinase or other anti-angiogenic compounds &quot; as used herein includes, but is not limited to, tyrosine kinase and / or serine kinase inhibitor proteins and / or threonine kinase inhibitors or to lipid kinase inhibitors, for example targeting compounds, by decreasing activity or inhibiting the epidermal growth factor family receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4 in the form of homodimers or heterodimers), vascular endothelial growth factor (VEGFR) family receptor tyrosine kinases, platelet-derived growth factor (PDGFR) receptors, fibroblast growth factor (FGFR) receptors, factor-1 receptor 1 insulin-like growth 30 ΡΕ1944026 (IGF-1R), the Trk receptor family of tyrosine kinases, the Axl receptor family of tyrosine kinases, the tyrosine tyrosine kinase Retin receptor inase inhibitors, Kit / SCFR receptor tyrosine kinases, members of the c-Abl family and their gene fusion products (for example BCR-Abl), members of the protein kinase C (PKC) and Raf serine / threonine kinases, members of the family of MEK, SRC, JAK, FAK, PDK or PI kinases (3, or the family of PI (3) -kinase related kinases and / or members of the cyclin dependent kinase (CDK) family) and anti-angiogenic compounds having another mechanism for their activity, for example unrelated to the inhibition of protein or lipid kinases.

Compounds which target, decrease activity or inhibit VEGFR are in particular compounds, proteins or antibodies that inhibit the VEGF receptor tyrosine kinase, inhibit a VEGF receptor or bind to VEGF, and are in particular those generic compounds, proteins or monoclonal antibodies and specifically described in WO 98/35958, for example 1- (4-chloroanilino) -4- (4-pyridylmethyl) phthalazine or a pharmaceutically acceptable salt thereof, for example succinate, WO 00/27820 , for example an amide derivative of N-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-trifluoromethylphenyl] benzamide, or WO 00/09496, WO 00/59509, WO 98/11233, WO 00/27819 and EP 0 769 947 ; those such as those described by M. Prewett et al., 19, 19404026, in Cancer Research 59 (1999) 5209-5218, by F. Yuan et al., Proc. Natl. Acad. Know. USA, 93, 14765-14770, de

December 1996, by Z. Zhu et al., Cancer Res. 58, 1998, 3209-3214, and by J. Mordenti et al., In Toxicologic

Pathology, 27 (1), 14-21, 1999; WO 00/37502 and WO 94/10202; Angiostatin ", described by M. S. O'Reilly et al., Cell 79, 1994, 315-328; Endostatin ", described by M. S. O'Reilly et al., Cell 88, 1997, 277-285; anthranilic acid amides; ZD4190; ZD6474; SU5416; SU6668; or anti-VEGF antibodies or anti-VEGF receptor antibodies, for example RhuMab.

By "antibody" is meant intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least 2 intact antibodies, and antibody fragments which still exhibit the desired biological activity.

Compounds which target, decrease activity or inhibit the epidermal growth factor receptor family are in particular compounds, proteins or antibodies that inhibit EGF family members of receptor tyrosine kinases, for example the EGF receptor, the ErbB2 receptor, the receptor ErbB3 and the ErbB4 receptor, or which bind EGF or EGF-related ligands, or which have a dual inhibitory effect on ErbB and VEGF receptor kinases and are in particular those compounds, proteins or monoclonal antibodies generally and specifically described in WO 97 / 02266, for example the compound of Ex. 39, or EP 32 ΡΕ 1944026 0 564 409, WO 99/03454, EP 0 520 722, EP 0 566 226, EP 0 787 722, EP 0 837 063, US 5,747,498, WO 98/10767, WO WO 97/38838, WO 97/38983, and in particular WO 96/30347 (for example the compound known as CP 358774), WO 96/33980 (for example ZD 1839) and in WO 95/03333 (for example compound ZM105180) or in PCT / EP02 / 08,780; for example trastuzumab (Herpetin®), cetuximab, Iressa, OSI-774, CI-1033, EKB-569, GW-2016, El.l, E2.4, E2.5, E6.2, E6.4, E2.ll , E6.3 or E7.6.3.

Compounds which target, decrease the activity or inhibit PDGFR are particularly compounds which inhibit the PDGF receptor, for example an N-phenyl-2-pyrimidine-amine derivative, for example imatinib.

Compounds which target, decrease activity or inhibit the members of the c-Abl family and their gene fusion products, for example an N-phenyl-2-pyrimidine-amine derivative, for example imatinib; PD180970; AG957; or NSC 680410.

Compounds which target, decrease activity or inhibit the protein kinase C, Raf, MEK, SRC, JAK, FAK and members of the PDK family, or PI (3) kinase or related members of the PI (3) kinase family, and / or members of the family of cyclin-dependent kinases (CDK) are especially the staurosporine derivatives described in EP 0 296 110, for example midostaurin; Examples of further compounds for example UCN-01, safingol, BAY 43-9006, Briostatin 1, Perifosin are included in 33 ΡΕ1944026; U0126; Ilmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521; or LY333531 / LY379196. Additional anti-angiogenic compounds are for example thalidomide (THALOMID) and TNP-470.

Compounds which target, decrease activity or inhibit a protein or lipid phosphatase are for example inhibitors of phosphatase 1, phosphatase 2A, PTEN or CDC25, for example, ochaic acid or a derivative thereof.

Compounds which induce differentiation processes are for example retinoic acid, tocopherols (a, γ or δ) or tocotrienols (a, γ or δ). The term cyclooxygenase inhibitor as used herein includes, but is not limited to, for example, celecoxib (CelebrexR), rofecoxib (VioxxR), etori-coxib, valdecoxib or a 5-alkyl-2- arylamino-phenylacetic acid, for example 5-methyl-2- (2'-chloro-6'-fluoroanilino) phenylacetic acid. The term &quot; histone deacetylase inhibitor &quot; as used herein includes MS-27-275, SAHA, pyroxamide, FR-901228 or valproic acid. The term &quot; bisphosphonates &quot; as used herein includes, but is not limited to, etridonic, clodronic, tiludronic, pamidronic, alendronic, ibandronic, risedronic and zoledronic acids. The &quot; etridonic acid &quot; can be administered, for example, in the form in which it is marketed, for example under the trade mark DIDRONEL ™. The &quot; clodronic acid &quot; can be administered, for example, in the form in which it is marketed, for example under the trademark BONEFOS ™. The "tiludronic acid" can be administered, for example, in the form in which it is marketed, for example under the trademark SKELID ™. The &quot; pamidronic acid &quot; can be administered, for example in the form in which it is marketed, for example under the trademark AREDIA ™. For example, alendronate can be administered in the form in which it is marketed, for example under the trademark FOSAMAX ™. The term &quot; ibandronic acid &quot; can be administered, for example, in the form in which it is marketed, for example under the trademark BONDRANAT ™. The "risedronic acid" can be administered, for example, in the form in which it is marketed, for example under the trademark ACTONEL ™. The term &quot; zoledronic acid &quot; can be administered, for example in the form in which it is marketed, e.g. under the trademark ZOMETA ™. The term &quot; metalloproteinase inhibitor &quot; as used herein includes, but is not limited to, is not limited to peptidomimetic and non-enzymatic collagenase inhibitors, tetracycline derivatives, for example peptidomimetic hydroxamate inhibitors, batimastat and the 35 ΡΕ1944026 its orally bioavailable analogue marimastat, prino-mastat, BMS-279251, BAY 12-9566, TAA211 or AAJ996 . The term &quot; mTOR inhibitor &quot; as used

in this document includes, but is not limited to, rapamycin (sirolimus) or a derivative thereof. Rapamycin is a known macrolide antibiotic produced by Streptomyces hygroscopicus. Suitable derivatives of rapamycin are included for example compounds of formula A

in which

R 3a is CH 3 or C 3-6 alkynyl R 2aa is H or -CH 2 -CH 2 -OH, 3-hydroxy-2- (hydroxymethyl) -2-methylpropanoyl or tetrazolyl, and

Xaa is = 0, (Η, H) or (H, OH) provided that R2aa is not H when Xaa is = O and Rlaa is CH3. or a precursor thereof when R 2aa is -CH 2 -CH 2 -OH, for example a physiologically hydrolyzable ether thereof. 36 ΡΕ1944026

Compounds of formula A are described for example in WO 94/0910, WO 95/16691, WO 96/41807, USP 5 362 718 or WO 99/15530. They may be prepared as described or by analogy with the processes described in this reference.

Preferred derivatives of rapamycin are 32-deoxorapamycin, 16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S) -dihydro-rapamycin, 16-pent-2-ynyloxy -32 (S) -dihydro-40- (2-hydroxyethyl) -rapamycin, and more preferably 40-0- (2-hydroxyethyl) -rapamycin. Further examples of rapamycin derivatives are for example CC1779 or 40- [3-hydroxy-2- (hydroxymethyl) -2-methylpropanoate] -rapamycin or a pharmaceutically acceptable salt thereof, as described in USP 5,362,718, ABT578 or 40- (tetrazolyl) -rapamycin, especially 40-epi- (tetrazolyl) -rapamycin, for example as described in WO 99/15530, or rapalogues such as those described for example in WO 98 / 02,441 and WO 01 / 14,387, for example AP23573.

Where citations of patent applications or scientific publications are included, matters relating thereto to the compounds are thus incorporated in the present application by subpoena. Also included are the pharmaceutically acceptable salts thereof, the corresponding racemates, diastereomers, enantiomers, tautomers, as well as the corresponding crystalline modifications of any of the said compounds, when present, for example solvates, hydrates and polymorphs, described in these documents. The compounds used as active ingredients in the combinations of the invention may be prepared and administered, respectively, as described in the cited documents. Also within the scope of this invention is the combination of more than two distinct active ingredients, as enumerated above, i.e., a pharmaceutical combination within the scope of this invention may include three or more active ingredients. In addition, both the first agent and the co-agent are not the identical ingredient.

The utility of S1P agonists, for example S1P agonists comprising a group of formula X according to the claims, may be demonstrated to treat solid tumors as specified hereinbefore in methods involving animal testing, as well as in clinical trials, for example according to the methods described hereinbelow.

All examples are reference examples. A. In Vitro A. Antitumor Activity

A breast cancer line in mice, originally isolated from mammary carcinomas, is used, for example JygMC (A). The number of cells is adjusted to 5x105 for plating in fresh medium prior to the procedure. The cells are incubated in fresh medium containing 2.5 mM thymidine without FCS for 12 h and then washed twice with PBS, then fresh medium is added with 10% FCS and incubated for a further 12 h. The cells are then incubated with fresh medium containing 2.5 mM thymidine without FCS for 12 h. To release the cells from the block, the cells are washed twice with PBS and plated in fresh medium containing 10% FCS. After being synchronized, cells are incubated with or without various concentrations of a compound of formula I for 3, 6, 9, 12, 18 or 24 h. The cells are harvested after treatment with 0.2% EDTA, fixed with a 70% ice-cold ethanol solution, hydrolyzed with 250æg / ml RNase A (Type 1-A: Sigma Chem Co.) at 37 ° C for 30 minutes and contrasted with propidium iodide at 10 mg / ml for 20 minutes. After the incubation period, the number of cells is determined either by counting in a Coulter counter or by a SRB colorimetric assay. Under these conditions an S1P agonist, for example Compound B in the form of its hydrochloride salt, inhibits the proliferation of tumor cells at concentrations between 10-12 and 10-6 M. A.2 HUVEC Tube Formation Test Mediated by

S1P are used

For the tube formation test, HUVEC from passages 2-8 and never more than 70 39 ΡΕ1944026 confluent before harvest. The cells are prepared for assay by lavage with Balanced Saline Herpes Solution (Clonetics HBSS) and then trypsinized with Trypsin / EDTA (0.25 mg / mL, Clonetics). After about 90% of the cells have been released from the plate, an equal volume of Trypsin Neutralizing Solution (TNS from Clonetics) is added and the cells are obtained in a conical tube containing at least 10 ml of EBM-2 medium (Clonetics ) + 0.1% BSA (Sigma). The cells are centrifuged at 1000 rpm for 5 minutes and the supernatant is removed, which is replaced with 5 ml of fresh EBM-2 + 0.1% BSA medium. The cells are packed using a hemocytometer and the volume of the cell suspension is adjusted to achieve a concentration of 500,000 cells / mL. Conical tubes are prepared with 100 nM test compounds and 10 ng / ml pertussis toxin (PTx) in each, then 1 ml of the cell suspension is added to each tube. Tubes are then incubated for 4 hours at 37øC under 5% CO2. The migration assay is performed using 24-well fibronectin-coated Fluoro-Blok Plates (8-μm pore size, Falcon # 351147) instead of the individual inserts in a 24-well plate. Test cells and compounds are prepared and preincubated as described above, then 100 μl is added to each of the appropriate wells in the Insertion Plate. 300 Âμl of EBM-2 medium + 2% of medium cleaned on charcoal and without S1P, and 300 Âμl of the S1P-containing media (500 nM) are added to the bottom of the labeled wells for stimulation (+). The plate is then incubated for 4 hours at 37øC under 5% CO 2. 40 ΡΕ1944026

AM calcein is prepared at 50æg / vial (Molecular Probes # C3100) by first adding 20æl DMSO to the vial. Then 12.5 ml HBSS (per plate) is added and heated to 37 ° C, adding 150 μl to the flask. The contents of the vials are then transferred back to the remaining HBSS to give the final concentration of 4 pg / mL in calcein AM.

The Fluoro-Blok plate is removed from the incubator and the top insert plate is separated, &quot; to remove the excess medium still clinging to the inserts. The insert plate is then transferred to a fresh 24-well plate containing 500 μl / well of the 4 μg / ml AM calcein solution. The plate is then incubated for 1½ hours at 37øC under 5% CO 2.

After incubation, the plate is read on a Cytofluor II, with excitation at 485 nm and emission at 530 nm. The Fluoro-Blok coating on the inserts allows you to count only the cells that have migrated to the bottom. Data are transferred to an Excel program for calculations, graphs are created using SigmaPlot, and SigmaStat is used for significance tests (t-test). (Figure 7).

Tubing is counted by counting the number of branching points (bonding between two independent strands) in 3 independent fields under an increase of 4x. The results are reported as follows: 41 ΡΕ1944026

Treatment Branching Point PBS 8 + 5 S1P 42 ± 13 FTY720 Phosphate 48 ± 15 FTY720 + S1P Phosphate 14 ± 7 Compound C Phosphate 44 ± 16 Compound C + S1P Phosphate 18 ± 6

These results demonstrate the unique ability of FTY720 Phosphate or Compound C Phosphate to act as angiogenesis agonists by themselves, but surprisingly, as antagonists of S1P-mediated angiogenesis. The phosphate of Compound C is preferably the racemate or the R-enantiomer. PTx is used as a control to inhibit Gia-mediated activity (EDG-1). B. In Vivo B.l Antitumor Activity Antitumor activity is expressed as T / C% (mean increase in tumor volumes of treated animals divided by the mean increase in control tumor volumes, multiplied by 100).

Aliquots of cancer cells (1x10 7), for example human A375 melanoma cells, are transplanted into BALB / c-nu / nu mice. When tumors reach dimension 42 ΡΕ1944026 from ca. 10x10 mm, the animals are randomized into four subgroups and treatment is started with a compound of formula I. The animals are sacrificed after 2 weeks of treatment, at which point tumors and tissues are harvested, for morphological and molecular analysis. Tumor dimensions are determined with a notch. In this assay, an S1P agonist, for example Compound B or C (as its hydrochloride salt), slows tumor growth when administered at a dose of 0.5-5 mg / kg, compared to that of a saline control: for example, by administering a dose of 2.5 mg / kg of Compound C-HCl 5x / week a final T / C value of 30% is obtained.

B.2 Combination with a VEGF-R protein tyrosine kinase inhibitor

Bovine mice transplanted with human breast tumors MDA-MB-435 for 2 weeks with a VEGF-R protein tyrosine kinase inhibitor, for example 1- (4-chloroanilino) -4- (4-pyridylmethyl) phthalazine succinate , at a dose of 100 mg / kg po 5x / week, an S1P receptor agonist, for example Compound C (hydrochloride salt), at a dose of 2.5 mg / kg i.v. 5x / week, or a combination of both. The antitumor activity is expressed as T / C% as indicated above. A combination of Compound C-HCl with 1- (4-chloroanilino) -4- (4-pyridylmethyl) -phthalazine succinate gives a greater antitumor effect (T / C% 27) compared to any of the agents per se (Compound C-HCl, T / C 66%, 1- (4-chloroanilino) -4- (4-pyridylmethyl) phthalazine, T / C% 91) succinate. good anti-tumor reactions are also obtained when nude mice are transplanted with A375 human melanoma cells and treated in a similar manner with the same combination: the combined treatment gives a T / C% 15 while treatment with only one agent alone respectively gives T / C% values of 35 and 44. B.3 Antiangiogenic Activity

Porcine chamber mice are implanted subcutaneously on the flank of (i) sphingosine-1-phosphate (5 μΜ / chamber) or (ii) human VEGF (1æg / chamber) in 0.5 mL agar 0.8 % w / v (containing heparin, 20 U / ml). S1P or VEGF induce the growth of vascularized tissue around the chamber, this dose-dependent reaction can be quantified by measuring the weight and blood content of the tissue. The mice are treated daily (i) orally with Compound A (at 0.3, 3, 30 or 50 mg / kg) or (ii) intravenously with the R enantiomer of Compound C (2.5 mg / kg) or (iv) intravenously with the S-enantiomer of Compound C (2.5 mg / kg) or (iv) orally or intravenously with vehicle (5% glucose, 10 ml / kg) 4-6 hours prior to the implantation of the chambers and continuing for 4 days. The animals are sacrificed to measure the vascularized tissues 24 h after the last dose. The weight of the vascularized tissues around the chamber and its content in blood is determined. 44 ΡΕ1944026

Animals treated with Compound A or the R or S enantiomers of Compound C denote lower weight and / or blood content in vascularized tissues, as compared to animals treated with vehicle alone. C. Clinical Trial

C. Investigation of the clinical benefit of an S1P receptor agonist, for example of a compound of formula I, II or III, for example Compound A, B or C

Twenty patients with solid progressing, advanced stage tumors, resistant or refractory to standard therapies, are screened to receive the referred compound at a dosage as determined in the study of increasing doses. The general clinical status of each patient is investigated weekly by physical and laboratory examination. The changes of each tumor and the load of metastases are evaluated every 2 months, by radiological examination. Initially patients receive treatment for 2 months. Then they remain under treatment for the period of time during which their disease does not progress and the drug is tolerated satisfactorily.

Key variables for evaluation: Safety (adverse events), blood biochemistry standard and hematology, tumor dimensions by computerized tomography (CT) scan or magnetic resonance imaging (MRI). 45 ΡΕ1944026 C.2 Combined Treatment

Appropriate clinical studies are, for example, non-randomized, open label, dose-escalation studies in patients with advanced solid tumors. These studies particularly prove the synergy between the active ingredients of the combination of the invention. The beneficial effects on proliferative diseases can be determined directly through the results of these studies, or by changes in study design known to those skilled in the art. These studies are in particular suitable for comparing the effects of a monotherapy using the active ingredients and those of the combination of the invention. Preferably, the dose of agent (a) is escalated to the Tolerated Maximum Dosage, and the co-agent (b) is administered at a fixed dose. Alternatively, the agent (a) is administered at a fixed dose and the dose of the co-agent (b) is scaled. Each patient receives doses of the agent (a) either daily or intermittent. The efficacy of treatment in these studies, for example at the end of 12, 18 or 24 weeks, can be determined by radiological evaluation of the tumors every 6 weeks.

Alternatively, a double-blind, placebo-controlled study may be used to prove the benefits of the combination of the invention mentioned herein. 46 ΡΕ1944026

The daily dosages required in practicing the method of the present invention when using only an S1P receptor agonist will vary depending on, for example, which compound is used, the host, the mode of administration and the severity of the condition being treated . A preferred daily dosage range is between about 0.1 and 100 mg in the form of a single dose or in divided doses. Suitable daily dosages for patients are in the range of, for example, 0.1 to 50 mg p.o. The S1P receptor agonist may be administered by any of the conventional, especially enteral, routes, for example orally, in the form of tablets, capsules, nasal, pulmonary (inhalation) or parenteral solutions, for example in the form of injectable solutions or suspensions. Dosage unit forms suitable for oral administration contain between ca. from 0.1 to 30 mg, usually 0.25 to 30 mg of S1P receptor agonist, together with one or more pharmaceutically acceptable diluents or carriers therefor. In order to inhibit angiogenesis it is important to select a sufficiently high dose of the S1P receptor agonist, since small concentrations of the S1P receptor agonist promote angiogenesis. A suitable dose to provide an anti-angiogenic effect when administering an S1P receptor agonist to a patient may be selected by concentration and increasing dose studies as described in A, B, and C above. the combination of the invention 47 ΡΕ1944026 may also be applied in conjunction with a surgical intervention, with prolonged mild hyperthermia of the whole body and / or with irradiation therapy. Administration of a pharmaceutical combination of the invention results in a beneficial effect, for example a synergistic therapeutic effect, for example in order to slow down, stop or reverse the formation of neoplasm, the spread or growth of metastases or a longer duration of the reaction of the tumor or an inhibition of angiogenesis; may also result in other beneficial effects, for example fewer side effects, a better quality of life or a lower mortality and morbidity compared to a monotherapy applying only one of the active pharmaceutical ingredients of the invention used in the combination of the invention, treatment of a tumor that is refractory to the other chemotherapeutics known as anti-cancer agents.

An additional benefit is that smaller doses of the active ingredients of the combination of the invention may be used, for example, and that the required dosages are not only minor but are also applied at a lower frequency or can be used to decrease the incidence of effects collaterals while controlling the rate of neoplasm formation. This goes against how much they want and need.

According to one embodiment of the invention, a preferred pharmaceutical combination includes 48 Å 19440266 a) a compound of formula I, II, III, IVa, IVb, V or VI, for example Compound A, B or C, and b) by way of (iii), (iv), (v), (vii) or (xi) above, for example carboplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, doxorubicin, a targeting compound, by decreasing activity or inhibiting the vascular endothelial growth factor family of receptor tyrosine kinases (VEGFR) or platelet-derived growth factor (PDGFR) receptors, a bisphosphonate or an mTOR inhibitor.

A further embodiment of the invention relates to the use of an S1P receptor agonist (a) in combination with a chemotherapeutic agent (in the treatment of a lymphatic or myeloid cancer, for example as described above. -Additional agent b) for example busulfan, cytarabine, 6-thioguanine, fludarabine, hydroxyurea, procarbazine, bile-micin or methotrexate. Preferred co-agents are (b) topoisomerase II inhibitors, for example daunorubicin or, in particular, compounds which target, decrease the activity or inhibit PDGFR or members of the c-Abl family and their gene fusion products , for example imatinib, for example for use in the treatment of a lymphatic cancer. 49 ΡΕ1944026

The terms &quot; co-administration &quot; or &quot; combined administration &quot; or the like as used herein are intended to include administration of the selected therapeutic agents to the same patient and are intended to include treatment regimens in which the agents are not necessarily administered either by the same route of administration or simultaneously . It is an object of this invention to provide a pharmaceutical composition comprising an amount that is therapeutically effective against a malignant proliferative disease comprising a combination of the invention. In this composition, the first agent a) and the co-agent (b) may be administered together, one after the other, or separately, in unit dosage form or in two separate dosage unit forms. Dosage unit forms may also constitute a fixed combination.

The pharmaceutical compositions according to the invention can be prepared in a manner which is known per se and is suitable for enteral, oral or rectal, and parenteral administration to mammals (warm-blooded animals), including humans , which comprises a therapeutically effective amount of at least one partner only in a pharmacologically active combination, for example as set forth above, or a combination thereof with one or more 50 ΡΕ1944026 carriers or diluents acceptable from the point of view pharmaceutical composition, in particular suitable for enteral or parenteral application.

Suitable pharmaceutical compositions contain, for example, from about 0.1% to about 99.9%, preferably from about 1% to about 60%, of the active ingredient (s). Pharmaceutical preparations for combination therapy for enteral or parenteral administration are, for example, those in dosage unit forms, such as sugar coated tablets, tablets, capsules or suppositories, or ampoules. Unless otherwise noted, they are prepared in ways known per se, for example including conventional blending, granulating, sugar coating, dissolving or lyophilizing processes. It will be understood that the unit content of a partner of a combination contained in an individual dose of each dosage form need not itself constitute an effective amount, since said effective amount can be achieved by administration of several units of dosage.

In particular, a pharmaceutically effective amount of each of the partners of the combination of the invention may be administered simultaneously, or such administration may be sequential in any order, and the components may be administered separately or in the form of a combination. For example, the method for delaying the progression or for treating a malignant proliferative disease according to the invention may include (i) administering the first agent a) in free form or a pharmaceutically acceptable salt thereof and (ii) administering a co-agent b) in free form or a pharmaceutically acceptable salt thereof simultaneously or sequentially in any order in amounts which together are therapeutically effective, preferably in amounts which exhibit synergistic effectiveness, for example in daily or intermittent dosages corresponding to the amounts described herein. Individual partners of the combination of the invention may be administered separately at different times during the course of therapy or together in divided forms or in a single combination. In addition, the term administering also includes the use of a drug precursor for any of the partners of the combination, which is transformed in vivo into the partner of the combination as such. The present invention is therefore to be understood as including all such treatment regimes simultaneously or alternately, and the term &quot; administering &quot; should be interpreted accordingly. The effective dosage of any of the partners of the combination that is employed in the combination of the invention may vary depending upon the particular compound or pharmaceutical composition to be employed, the mode of administration, the condition being treated, the severity of the condition ΡΕ1944026 if you are treating. Accordingly, the dosage regimen of the combination of the invention is selected and according to a variety of factors including the route of administration and the renal and hepatic function of the patient. A physician, clinician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of each of the single active ingredients necessary to prevent, counteract or halt the progression of the condition. An optimum precision to be attained as to the concentration of the active ingredients, within the range of which results in efficacy without toxicity, requires a regime based on the kinetics of the availability of the active ingredients at the targeted sites.

The daily doses of the first agent or first component (a) will of course vary depending on a variety of factors, for example the compound selected, the state to be specifically treated, and the desired effect. In general, however, satisfactory results can be obtained by administration of an S1P receptor agonist, for example Compound A, B or C, at daily dosage rates in the range of about 0.1 to 100 mg as a dose single or divided doses. The S1P receptor agonist may be administered by any of the conventional routes, especially enterally, for example orally, for example in the form of tablets, capsules, drinkable solutions, or parenotherally, for example under the injectable solutions or suspensions. Suitable dosage unit forms for oral administration include from about 0.1 to 30 mg of component (a), for example 0.1 to 25 mg, together with one or more acceptable diluents or carriers from the view for her.

Fadrozole can be administered orally to a human in a dosage range ranging from about 0.5 to about 10 mg / day, preferably from about 1 to about 2.5 mg / day. Exemestane can be administered orally to humans in a dosage range ranging from about 5 to about 200 mg / day, preferably from about 10 to about 25 mg / day, or parenterally between about 50 and 500 mg / day, preferably between about 100 and about 250 mg / day. When administering the drug in a separate composition, it may take the form described in GB 2,177,700. The formestane can be administered parenterally to a human in a dosage range ranging from about 100 to 500 mg / day, preferably from about 250 to about 300 mg / day. Anastrozole can be administered orally to a human in a dosage range ranging from about 0.25 to 20 mg / day, preferably from about 0.5 to about 2.5 mg / day. Aminoglutemide can be administered to a human in a dosage range ranging from about 200 to 500 mg / day.

Tamoxifen citrate can be administered to a human in a dosage range ranging from about 10 to 40 mg / day. 54 ΡΕ1944026

Vinblastine can be administered to a human in a dosage range ranging from about 1.5 to 10 mg / m2 daily. Vincristine sulfate can be administered parenterally to a human in a dosage range ranging from about 0.025 to 0.05 mg / kg body weight * week. Vinorelbine can be administered to a human in a dosage range ranging from about 10 to 50 mg / m2 * day. Etoposide phosphate can be administered to a human in a dosage range ranging from about 25 to 115 mg / m 2 * day, for example 56.8 or 113.6 mg / m 2 * day.

Teniposide can be administered to a human in a dosage range ranging from about 75 to 150 mg to about every two weeks. Doxorubicin can be administered to a human in a dosage range ranging from about 10 to 100 mg / m 2 * day, for example 25 or 50 mg / m 2 * day. Epirubicin can be administered to a human in a dosage range ranging from about 10 to 200 mg / m2 * day. Idarubicin can be administered to a human in a dosage range ranging from about 0.5 to 50 mg / m2 daily. Mitoxantrone can be administered to a human in a dosage range ranging from about 2.5 to 25 mg / m2 daily.

Paclitaxel can be administered to a human in a dosage range ranging from about 50-300 mg / m2 * day. The docetaxel can be administered to a human in a range of dosages ranging from about 25 to 100 mg / m 2 * day. 55 ΡΕ1944026

Cyclophosphamide may be administered to a human in a dosage range ranging from about 50 to 1500 mg / m 2 * day. Melphalan can be administered to a human in a dosage range ranging from about 0.5 to 10 mg / m2 * day.

The 5-fluorouracil can be administered to a human in a dosage range ranging from about 50 to 1000 mg / m2 * day, for example 500 mg / m2 * day. Capecitabine can be administered to a human in a dosage range ranging from about 10 to 1000 mg / m2 * day. Gemcitabine hydrochloride can be administered to a human in a dosage range ranging from about 1,000 mg / m 2 / week. Methotrexate can be administered to a human in a dosage range ranging from about 5 to 500 mg / m2 * day.

Topotecan can be administered to a human in a dosage range ranging from about 1 to 5 mg / m2 * day. Irinotecan can be administered to a human in a dosage range ranging from about 50-350 mg / m2 * day.

Carboplatin can be administered to a human in a dosage range ranging from about 200-400 mg / m2 every four weeks. Cisplatin can be administered to a human in a dosage range ranging from about 25 to 75 mg / m 2 of about 56 æGe 1944026 every three weeks. Oxaliplatin can be administered to a human in a dosage range ranging from about 50 to 85 mg / m 2 every two weeks.

Imatinib can be administered to a human in a dosage range of about 2.5 to 850 mg / day, more preferably 5 to 600 mg / day and preferably 20 to 300 mg / day.

Alendronic acid can be administered to a human in a dosage range ranging from about 5 to 10 mg / day. Clodronic acid can be administered to a human, for example in the dosage range ranging from about 750 to 1500 mg / day. Etidronic acid can be administered to a human in a dosage range ranging from about 200 to 400 mg / day. Ibandronic acid can be administered to a human in a dosage range ranging from about 1 to 4 mg every three to four weeks.

The risedronic acid can be administered to a human in a dosage range ranging from about 20 to 30 mg / day. The pamidronic acid can be administered to a human in a dosage range ranging from about 15 to 90 mg every three to four weeks. Tiludronic acid can be administered to a human in a dosage range ranging from about 200 to 400 mg / day.

Trastuzumab can be administered to a human 57? 1944026 in a dosage range ranging from about 1 to 4 mg / m 2 / week.

Bicalutamide can be administered to a human in a dosage range ranging from about 25 to 50 mg / m2 * day.

1- (4-Chloroanilino) -4- (4-pyridylmethyl) phthalazine or a salt thereof, for example succinate, can be administered to a human in a dosage range of from about 50 to 1500, more preferably from between about 100 and 750, and preferably between 250 and 500, mg / day.

Rapamycin or a derivative thereof, for example 40-0- (2-hydroxyethyl) -rapamycin, may be administered in a dosage range ranging from about 0.1 to 25 mg.

Formulation Example: soft capsules 30 mg 30 mg 200 mg 350 mg

Compound of formula I, for example Compound A, HCl Polyethylene Glycol 300 Polysorbate 80 Total

S1P receptor agonists, for example an S1P receptor agonist including a group of formula X, are well tolerated at the dosages required for use in accordance with the present invention. For example, 58 ΡΕ1944026 to acute LD50 for Compound A is> 10 mg / kg p.o. in rats and monkeys.

In a further aspect, the present invention relates to the use of S1P agonists as pro-angiogenic drugs. The induction of neo-angiogenesis has been recognized lately as an excellent target in several states (for example in myocardial angiogenesis, wound healing or diabetic vascular dysfunction or vasculopathy).

As described above, elevated concentrations of S1P receptor agonists (2 μg or greater, for example 2-5 μg or about 5 μg) exhibit anti-angiogenic effects, and S1P receptor agonists are able to inhibit angiogenesis induced by VEGF. In contrast, small concentrations (0.1 -1 μ, for example 0.1-0.5 μg or 0.5-1 μg) of S1P agonists have an angiogenesis-promoting effect and are capable of potentiating mediated angiogenesis by VEGF. Thus, S1P agonists may have biphasic effects on angiogenesis.

Therefore, the present invention also provides: The use of an S1P agonist, for example an S1P agonist including a group of formula X, for example Compound A or Compound A phosphate, in inducing the neoangiogenesis process , for example as pro-angiogenic agent, for example in indications in which promotion of angiogenesis is indicated; A process for the preparation of a medicament for the treatment or prevention of diseases mediated by inhibition of the neoangiogenesis process, for example mediated by anti-angiogenic factors, for example in indications in which the promotion of angiogenesis is indicated, for example in wound healing or in the treatment of myocardial infarction or diabetic vascular dysfunction or vasculopathy, including using an S1P receptor agonist, for example an S1P agonist which includes a group of formula X, for example Compound A or the phosphate of Compound A, as an active ingredient; A method of treating or preventing diseases mediated by inhibition of the neoangiogenesis process, for example mediated by anti-angiogenic factors, for example in indications in which the promotion of angiogenesis is indicated, such as for example wound healing or in the treatment of myocardial infarction or diabetic vascular dysfunction or vasculopathy, which comprises administering an effective amount of an S1P receptor agonist, for example an S1P agonist comprising 60 ΡΕ1944026 a group of the formula X, for example Compound A or the phosphate of Compound A, to a subject in need of such treatment.

Suitable S1P agonists for promoting angiogenesis include those defined above in connection with the treatment of cancer, for example S1P agonists which include a group of formula X or compounds according to formulas I to IX, or salts or esters thereof acceptable from a pharmaceutical point of view. Preferably the S1P agonist is the Compound A phosphate. The S1P agonist may be used alone, or in combination with one or more additional agents which promote angiogenesis, for example VEGF.

To promote angiogenesis it is important to select a sufficiently small dose of the S1P receptor agonist, since high concentrations of the S1P receptor agonist inhibit angiogenesis. A suitable dose to provide a pro-angiogenic effect when administering an S1P agonist to a patient may be selected by studies of increasing concentrations and doses, as described in sections A, B, and C above.

Description of the Figures Figure 1 shows that Compound A phosphate strongly promotes the formation of capillary networks of 61 ΡΕ1944026 in a dose-dependent manner with a bell-shaped curve, with maximal activity at about 0.5 μΜ. Figure 2 shows that both Compound A phosphate and Compound A at 0.5-1 μΜ do not attenuate VEGF-mediated remodeling but rather cooperate with polypeptide growth factor. Figure 3 shows that Compound A phosphate as well as S1P-stimulated tube formation is substantially completely inhibited by pertussis toxin (PTX, at 50 ng / ml), an inhibitor of the heterotrimeric G-type O 2 proteins /O. This can be interpreted as a possible involvement of EDG-1 (SlPi) receptor mediated signaling of events in Compound A phosphate-stimulated bio-reactions. Figure 4 shows that 1 μ esf sphingosine, which itself appears to be less potent which S1P attenuates the ability of both S1P and Compound A phosphate to induce capillary-like structures without exhibiting an inhibitory effect on VEGF-induced tube formation. In this regard, sphingosine behaves differently from Compound A. The data indicate that the balance between sphingosine and S1P appears to be critically important for the activation of endothelial cells / angiogenesis, with a high probability through the family of EDG receptors. Importantly, high concentrations of sphingosine and Compound A (2-5 μΜ) inhibit the formation of VEGF-triggered tubes. Figure 5 shows that treatment of HUVEC with 0.5 μ Compound A phosphate can result in a temporary activation of ERK1 / 2 with a phosphoylation / activation peak at 10 minutes and returning to the baseline at 20 minutes. Figure 6

It was tested whether Compound A, Compound A phosphate, sphingosine or S1P also induced tissue factor in HUVEC. The data obtained demonstrate that none of these compounds per se or in combination can increase tissue factor activity as shown in Figure 6. Compound A and Compound A phosphate can slightly increase the induced tissue factor by VEGF but not that induced by TNF-α. Figure 7 shows the effect of Compound C on an S1P-mediated tube formation assay in HUVEC. ΡΕ1944026 63

The following abbreviations are used: BSA: bovine serum albumin ECGS: set of endothelial cell growth factors S: sphingosine JNK1 / 2: kinase 1 / N-c-jun terminal

TF equivalents: EGR-1 / NFAT tissue factor equivalents: activated nuclear T-cell nuclear protein ECL: increased chemiluminescence PBS: phosphate buffered saline TA: ambient temperature

. Growth factor / P-factor: Compound A phosphate (FTY720-phosphate)

The utility of S1P receptor agonists, for example S1P agonists including a group of formula X, can be demonstrated in promoting angiogenesis, for example according to the methods described hereinbelow. D. Cell Culture and Materials

Human umbilical cord endothelial cells (HUVEC) were cultured at 37øC under 5% CO2 in M199 medium supplemented with 20% SCS (HyClone, Logan, UT), 1 U / ml heparin, 50æg / ml of ECGS, 2 mM in glutamine, with 100 U / ml penicillin and 0.1 mg / ml streptomycin. Use 64- ΡΕ1944026

the cells obtained up to passage number 5. The unabused HUVECs are obtained in experiments for a short period of time by growing them in M199 containing 1% SCS for 5 h. Recombinant human VEGF165 is obtained from PromoCell (Heidelberg, Germany). Phospho-specific ERK1 / 2 reagents, polyclonal antibodies to p38 kinase, nonphosphorous ERK1 / 2 antibodies and LumiGLO luminescent reagent were obtained from New England BioLabs (Beverly, MA), IkB polyclonal antibodies from Santa Cruz Biotechnology (Santa Cruz , Calif.) Anti-rabbit donkey immunoglobulin G (IgG) and sheep anti-mouse IgG were purchased from Amersham Life Science (Amersham Place, England). Immobilon-P transfer membranes are products of Millipore (Bedford, MA). S was obtained from Sigma Chemical Co.; S1P from Biomol was obtained. A starting solution of Compound A phosphate is prepared using the following protocol. Compound A phosphate is dissolved in concentrated HCl-scanned methanol (0.5 mg Compound A phosphate in 500 æl methanol containing 2 æl HCl). The solvent of the resulting solution is evaporated in vacuo and the obtained residue (variant 1) is redissolved in 0.1% defatted BSA solution in sterile deionized water (500 μl) or (variant 2) in solution at 0 ° C , 5% Triton X-100 in deionized water. The obtained starting solutions are sonicated (2.5 mM) and stored at 4 ° C.

Coagulation Assay

The cells are seeded in 6-well plates at 80-90% confluency and allowed to stand overnight. 65 ΡΕ1944026

The cells of the plaques are scraped and tissue factor activity is analyzed according to the method described in Clauss, M., J. Biol. Chem. 271, 17629-17634 (1996), Mechtcheriakova, D., Blood 93, 3811-3823 (1999). In short,

after being induced for 4 hours with VEGF (1.5 nM), TNF-α (100 U / ml), S (0.5-2 μΜ), S1P (0.5-2 μΜ), Compound A , 5-2 μΜ), and Compound A phosphate (0.5-2 μΜ), the cells are washed twice and then scraped with 1 mL of coagulation buffer (12 mM in sodium acetate, 7 mM in diethyl barbitrate and 130 mM in sodium chloride, pH 7.4). 50 æl of new cell suspension is mixed with 50 æl citrated plasma, and the clotting periods are determined after recalculation with 50 æl of a 20 mM CaCl 2 solution at 37øC. TF equivalents are determined using a standard curve obtained with rabbit brain thromboplastin. E. Western Blot Analysis Following several treatments, the cells were washed twice with cold PBS, lysed in 100æl of Laemmli buffer, scraped and heated for 5 minutes at 95øC. The total lysates were separated by SDS-PAGE and transferred to an Immobilon-P membrane. The membrane is blocked for 30 minutes with PBS containing 0.1% Tween 20 and 3% skim milk and incubated for 1 hour at RT with a primary antibody diluted in blocking buffer. The obtained membrane is washed three times for 5 minutes each time with 0.1% PBS containing Tween 20 and 66 ΡΕ1944026 incubated with a peroxidase conjugated secondary antibody for 1 hour at RT. After a washing step, the membrane is incubated for 1 minute with ECL reagent and exposed as a film, as required. To re-test with another antibody, the membrane is washed twice in PBS, cleaned for 30 minutes at 55øC with cleaning buffer (62.5 mM Tris-HCL, pH 6.8, 2 % SDS, 100 mM in 2-mercaptoethanol) and then washed three times for 5 minutes each time with PBS at RT. The SaranWrap wrapped membrane is stored at 4 ° C after each immunodetection.

In vitro angiogenesis assay on Matrigel

Morphologically transfected endothelial cells are carried out in capillary type structures in a Matrix Factor with Reduced Growth Factor (BD Bioscience) following the manufacturer's process. In summary, HUVEC is trypsinized, resuspended in serum free M199 medium containing trypsin inhibitor, soybean (1 mg / ml, Sigma). After centrifugation the cells are resuspended in serum-free medium at a density of 0.5 x 105 cells / ml, and the cell suspension is seeded into 96-well cell culture dishes (Costar, Corning Incorporated) precoated with 50 æL of Matrigel in the absence or presence of various stimuli: 1.5 nM VEGF, 0.1-2 μM S1P, 0.5-2 μ S S, Compound A at 0.5 -2 μΜ, and 0.1-2 μ Compos Compound A phosphate. Eight hours later, the cells were fixed on Matrigel with 3% formaldehyde 67 ΡΕ1944026 in PBS and held at 4 ° C. The results are quantitated from the Nikon Diaphot microscope images equipped with a cooled CCD camera (Kappa GmbH, Gleichen, Germany) by counting the branching points directly in two microscopic fields of each well, obtained in duplicate. F. Phosphate-induced endothelial cell morphogenesis of Compound A in an in vitro in vitro assay on Matrigel and a possible involvement of Gi mediated signaling pathway (s)

The effect of Compound A and Compound A phosphate on the morphogenic differentiation of endothelial cells is determined using an in vitro angiogenesis assay on Matrigel. Endothelial cell morphogenesis is a complex process that requires extracellular interactions of the cell with the matrix, followed by matrix remodeling, stimulated migration, cell interactions, and perivascular proteolysis. As shown in Figure 1, Compound A phosphate is able to strongly promote the formation of a capillary network in a dose-dependent manner according to a bell curve, denoting maximal activity at about 0.5 μΜ. The number of branching points per microscopic field, which reflects the potency of the induction by the stimulus, is comparable for Compound A phosphate and for S1P, and may be significantly greater than the effects ensued by VEGF. Compound A itself at 68 ΡΕ1944026 0.5 - 1 Μ has a weak enhancing effect, compared to that of Compound A, more consistent phosphate. Both Compound A phosphate and Compound A at 0.5-1 μg do not attenuate VEGF-mediated remodeling but instead collaborate with the polypeptide growth factor (see for example Figure 2). In addition, Compound A-stimulated phosphate-stimulated microtubule formation by SlP is completely inhibited by pertussis toxin (PTX, at 50 ng / ml), an inhibitor of G-type heterotrimeric G proteins. This may be interpreted as due to a possible involvement of EDG-1 receptor mediated signaling events (SlPi) in the phosphate-stimulated bio-reactions of Compound A (see for example Figure 3). Salt μ parece, which by itself appears to be less potent than SlP, attenuates the ability of both SlP and Compound A phosphate to induce capillary-type struttures, without having an inhibitory effect on VEGF tube formation (see, for example, Figure 4). In this regard, S behaves differently from Compound A. The data indicate that the balance between S and SlP appears to be critically important for endothelial cell / angiogenesis activation most likely through the EDG family of receptors. Importantly, high concentrations of S and Compound A (2-5 μΜ) inhibited the formation of VEGF-triggered tubes. These data suggest dose dependent biphasic effects of Compound A and Compound A phosphate on angiogenesis in vitro. 69 ΡΕ1944026

G. Activation of ERK1 / 2 MAP kinases by Compound A phosphate Signal transduction by MAP kinases plays a key role in a number of endothelial cell functions. Treating HUVEC with 0.5 μM Compound A phosphate may result in a temporary activation of ERK1 / 2 with a peak of phosphorylation / activation for 10 minutes, returning to the baseline after 20 minutes (see for example Figure 5) . No activation of p38 kinase or JNK1 / 2 can be detected by Compound A phosphate in HUVEC. In addition, Compound A phosphate can trigger ERK1 / 2 activation in a dose-dependent manner, denoting maximal activity at 2 μ. This contrasts with the results of the tube forming assay in which the 2 μm Compound A phosphate may be less potent than 0.5 μΜ. Neither Compound A nor S are capable of inducing MAP kinase activation in endothelial cells with a treatment kinetics ranging from 5 minutes to 60 minutes. In order to estimate the possible role of the NFκB-dependent / inflammatory program in Compound A phosphate-stimulated bio-reactions in endothelial cells, the membranes are rescreened with anti-κκ antibodies. The IkB levels are not affected by Compound A phosphate treatment. In addition, the treatment of endothelial cells with Compound A phosphate may not be able to induce the expression of E-Selectin as an NFκB-dependent reactive secondary gene. Thus, 70 ΡΕ1944026 data strongly indicate that Compound A phosphate signaling does not involve the activation of NFκB-the major cascade in the acute inflammatory reaction in endothelial cells. Η. Compound A and Compound A phosphate do not induce the expression of tissue factor in endothelial cells

An important feature of both the classic inflammatory stimulus TNF-α and the major angiogenic growth factor VEGF in endothelial cells is its potency to upregulate the tissue factor. Compound A, Compound A, S or S1P phosphate are tested for whether they also induce tissue factor in HUVEC. The data obtained demonstrate that none of these compounds alone or in combination can increase tissue factor activity (see for example Figure 6). Compound A or Compound A phosphate may slightly increase tissue factor induced by VEGF but not TNF-α induced factor. The set of data obtained indicates that Compound A, Compound A, S and S1P phosphate function mechanistically in different ways than angiogenic VEGF and inflammatory TNF-α. I. The binding ability of S1P receptor agonists to individual human S1P receptors can be determined by the following procedure: 71 ΡΕ1944026

Transient transfection of human S1P receptors in HEK293 cells

EDG receptors and G + proteins are cloned, and equal amounts of 4 cDNA are mixed for the EDG receptor, for G-α, for Gi-β and for Gi-γ, using to transfect HEK293 cell monolayers using calcium phosphate precipitate method (M. Wigler et al., Cell 1977, 11, 223 and DS Im et al., Mol. Pharmacol., 2000, 57, 753). In summary, a DNA mixture containing 25 .mu.g of DNA and 0.25 M CaCl2 is added to 2 mM Na2HPO4 buffered with HEPES. Subconfluent monolayers of HEK293 cells are pooled with 25 mM chloroquine, and the precipitated DNA is then loaded onto the cells. After 4 h, the monolayers are washed with phosphate buffered saline and returned to medium (90% Dulbecco's modified essential medium (DMEM) 1: 1 with F-12 + 10% fetal serum of bovine). Cells are harvested 48-72 h after addition of the DNA by scraping the HME buffer (in mM: 20 in HEPES, 5 in MgCl 2, 1 in EDTA, pH 7.4) containing 10% sucrose on ice, and break using a Dounce homogenizer. After centrifugation at 800xg, the supernatant is diluted with HME without sucrose and centrifuged at 100,000xg for 1 h. The resulting pellet is homogenized and centrifuged for an additional hour at 100,000xg. This crude membrane pellet is resuspended in HME with sucrose, divided into aliquots, and freeze rapidly by immersion in liquid nitrogen. The membranes are stored at 70 ° C. The 72 ΡΕ1944026 protein concentration is determined spectroscopically by the determination of Bradford proteins.

GTPγS binding assay using HEP293 membrane SlP / membrane preparations

The GTPγS binding assays are run as described in DS. Im et al., Mol. Pharmacol. 2000; 57: 753. GTPγS ligand mediated binding to G proteins in GTP binding buffer (in mM: 50 in HEPES, 100 in NaCl, 10 in MgCl2, pH 7.5) is measured using 25æg of a preparation of membranes from transfected HEK293 cells. It is added by binding to membranes in the presence of 10 μg GDP and 0.1 nM [35S] GTPγS (at 1200 Ci / mmol) and incubated at 30 ° C for 30 minutes. The bound GTPyS is detached from unbound using the Brandel harvest device (Gaithersburg, MD) and counts with a liquid scintillation counter.

Lisbon, September 12, 2013

Claims (11)

An S 1 P receptor agonist which is a compound of formula IX R in which R 1 is halogen, trihalomethyl, hydroxy, lower alkyl containing 1 to 7 carbon atoms, phenyl, aralkyl, lower alkoxy containing 1 to 4 carbon atoms, trifluoromethyloxy, phenoxy, cyclohexylmethyloxy, substituted or unsubstituted aralkyloxy, pyridylmethyloxy, cinnamyloxy, naphthylmethyloxy, phenoxymethyl, hydroxymethyl, hydroxyethyl, lower alkylthio containing 1 to 4 carbon atoms, lower alkylsulfinyl containing 1 to 4 carbon atoms, lower alkylsulfonyl containing 1 to 4 carbon atoms, benzylthio, acetyl, nitro or cyano; R 2f is hydrogen, halogen, trihalomethyl, lower alkoxy containing 1 to 4 carbon atoms, lower alkyl containing 1 to 7 carbon atoms, phenethyl, or benzyloxy; R3f is hydrogen, halogen, trifluoromethyl, lower alkoxy containing 1 to 4 carbon atoms, hydroxyl, benzyloxy, lower alkyl containing 1 to 7 carbon atoms, phenyl or lower alkoxymethyl containing 1 to 4 carbon atoms; Xf is O or S; and n is an integer from 1 to 4, or a pharmacologically acceptable salt thereof, for use in a method of preventing or treating diseases mediated by a neo-angiogenesis process or associated with a deregulated angiogenesis, wherein said diseases are selected from between the group consisting of cancer, myocardial angiogenesis, wound healing and diabetic vascular dysfunction or vasculopathy. An S1P receptor agonist for use according to claim 1, to prevent and to treat solid tumors. An S1P receptor agonist for use according to claim 1 for inhibiting the growth of solid tumors or for treating the invasiveness of solid tumors or the symptoms associated with such tumor growth. 3 ΡΕ1944026 An S1P receptor agonist of formula IX as defined in claim 1, or a pharmaceutically acceptable salt thereof, for use in a method for preventing or treating diseases mediated by a neoangiogenesis process or associated with a deregulated angiogenesis, wherein the said diseases are selected from the group consisting of cancer, myocardial angiogenesis, wound healing, diabetic vascular dysfunction or vasculopathy, lymphoproliferative and myeloproliferative disorders, wherein the S1P receptor agonist is administered concomitantly or sequentially with a chemotherapeutic agent. An S1P receptor agonist for use according to claim 4, wherein the chemotherapeutic agent is selected from i. an aromatase inhibitor, ii. an anti-estrogen, an anti-androgen or a gonadorelin agonist, iii. a topoisomerase I inhibitor or a topoisomerase II inhibitor, iv. an active agent in microtubules, an alkylating agent, an antineoplastic antimetabolite or a platinum compound, ΡΕ1944026 4 v. a targeting / decreasing compound of a protein or lipid kinase or the activity of a protein or lipid phosphatase, an additional anti-angiogenic compound or a compound that induces cellular differentiation processes, vi. a bradykinin receptor or an angiotensin II antagonist, vii. a cyclooxygenase inhibitor, a bisphosphonate, a histone deacetylase inhibitor, a heparanase inhibitor, a biological reaction modifier, a ubiquitin inhibitor, or an inhibitor that blocks anti-apoptotic pathways, viii. an inhibitor of the oncogenic isoforms of Ras, ix. a telomerase inhibitor, e.g. a protease inhibitor, a matrix metalloproteinase inhibitor, a methionine aminopeptidase inhibitor, or a proteasome inhibitor, and / or xi) an mTOR inhibitor. 5 ΡΕ1944026 An S1P receptor agonist for use according to claim 4 or claim 5, for preventing or treating tumors of the blood or lymphatic system or myeloid cancer. An S1P receptor agonist for use according to claim 4, in combination with a chemotherapeutic agent, for treating a lymphatic or myeloid cancer, wherein the chemotherapeutic agent is selected from busulfan, cytarabine, 6-thioguanine, fludarabine, hydroxyurea, procarbazine, bleomycin, and methotrexate. An S1P receptor agonist for use according to claim 4, in combination with a chemotherapeutic agent, for treating a lymphatic cancer, wherein the chemotherapeutic agent is selected from daunorubicin and imatinib. An S1P receptor agonist for use according to any one of the preceding claims, wherein the S1P receptor agonist is administered intermittently. An S1P receptor agonist as defined in claim 1, for use in the prevention or treatment of diseases mediated by inhibition of the neo-angiogenesis process, wherein said diseases are selected from the group consisting of cancer, ΡΕ1944026 myocardial angiogenesis, wound healing and diabetic vascular dysfunction or vasculopathy. An S1P receptor agonist for use according to any one of the preceding claims, wherein the S1P receptor agonist is 2-amino-2- [4- (benzyloxyphenylthio) -2-chlorophenyl] propyl-1,3-propane diol or 2-amino-2- [4- (3-benzyloxyphenoxy) -2-chlorophenyl] propyl-1,3-propane diol in free or a pharmaceutically acceptable salt form. Lisbon, September 12, 2013