CN101305990B

CN101305990B - Use of EDG receptor binding agents in cancer

Info

Publication number: CN101305990B
Application number: CN 200810125629
Authority: CN
Inventors: T·鲍姆鲁克尔; V·布林克曼; K·R·拉蒙塔涅; P·T·拉索塔; D·梅希特谢里安科娃; J·M·伍德
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2002-05-16
Filing date: 2003-05-15
Publication date: 2013-06-19
Anticipated expiration: 2023-05-15
Also published as: CN101305990A; GB0211261D0

Abstract

The invention relates to applications of an EDG receptor agonist in treatment of cancers, more particularly, the invention provides using 1-Sphingosine phosphate receptor agonist, optionally in combination with a chemotherapeutic agent, for treating a solid tumor, such as tumor invasion, in particular a method for inhibiting or controlling runaway angiogenesis. The invention also comprises a combination of a 1-sphingosine-phosphate receptor agonist with a chemotherapeutic agent.

Description

The application of EDG receptor-binding agents in cancer

The application is that the PCT that on May 15th, 2003 submits to, that denomination of invention is " application of EDG receptor-binding agents in cancer " applies for dividing an application of PCT/EP03/05125, it is on November 16th, 2004 that described PCT application enters the date in China national stage, and application number is 03811194.2.

Technical field

The present invention relates to the new purposes of S1P (S1P) receptor stimulating agent, particularly the new purposes in the treatment cancer.

Background technology

The S1P receptor stimulating agent is the material that accelerates lymphocyte homing (LH), and it can cause lymphopenia, and reason is that lymphocyte distributes from blood circulation to secondary lymphoid tissue again, preferably reversibility distributes again, but does not cause the general immunosuppressant.Naive cell is isolated; Come the CD4 of autoblood and CD8T-cell and B-cell to be migrated to lymph node (LN) and aggregated lymphoid nodules (PP) by stimulation, and therefore for example suppress the infiltration of cell to transplant organ.

The S1P receptor stimulating agent is the sphingol analog normally, and as 2-amino-propane-1 that 2-replaces, 3-glycol or 2-amino-propanol derivatives for example comprise the compound of formula X group:

Wherein:

Z is H; C _1-6alkyl; C _2-6alkenyl; C _2-6alkynyl; Phenyl; The phenyl replaced by OH; Be selected from halogen, C by 1 to 3 _3-8the C that the substituent group of the phenyl that cycloalkyl, phenyl and OH replace replaces _1-6alkyl; Or CH ₂-R _4z, R wherein _4zresidue for OH, acyloxy or formula (a)

Z wherein ₁for direct bond or O, preferred O; R _5zand R _6zbe H independently of each other, or the C optionally replaced by 1,2 or 3 halogen atom _1-4alkyl;

R _1zresidue for OH, acyloxy or formula (a); And R _2zand R _3zbe H, C independently of each other _1-4alkyl or acyl group.

The group of formula X is to be connected in the functional group of hydrophilic or lipophilic portion as end group, and comprises one or more aliphatic series, alicyclic, aromatics and/or heterocycle residue, and the result reached is formed Z and R wherein _1zhave at least the molecule of a residue that is or comprises formula (a) to carry out signal transduction as agonist on one of a plurality of S1P receptors.

The S1P receptor stimulating agent is to carry out the compound of signal transduction on S1P8 as agonist at one or more S1P receptors, for example S1P1.With the agonist of S1P receptors bind, can for example cause intracellular assorted trimer G-albumen to be dissociated into G α-GTP and G β γ-GTP, and/or the receptor phosphorylation effect of being captured by agonist increase, and downstream signal transduction pathway/kinase activation.The binding affinity of S1P receptor stimulating agent can be measured as described in following paragraph I.

The example of suitable S1P receptor stimulating agent for example has:

Disclosed compound in-EP627406A1, for example compound of formula I

R wherein ₁for straight or branched (C _12-22) carbochain

-it can contain and be selected from following key or hetero atom in carbochain: two keys, triple bond, O, S, NR ₆and carbonyl, wherein R ₆for H, alkyl, aralkyl, acyl group or alkoxy carbonyl,

And/or

-it can contain as substituent alkoxyl, alkenyloxy, alkynyloxy group, aralkyl oxy, acyl group, alkyl amino, alkylthio group, acylamino-, alkoxy carbonyl, alkoxycarbonyl amino, acyloxy, alkyl-carbamoyl, nitro, halogen, amino, oxyimino, hydroxyl or carboxyl; Or

R ₁for

-phenylalkyl, wherein alkyl is straight or branched (C _6-20) carbochain; Or

-phenylalkyl, wherein alkyl is straight or branched (C _1-30) carbochain, wherein said phenylalkyl is replaced by following group:

-straight or branched (the C that optionally replaced by halogen _6-20) carbochain,

-straight or branched (the C that optionally replaced by halogen _6-20) oxyalkyl chain,

-straight or branched (C _6-20) alkenyloxy,

-phenyl alkoxyl, halogenophenyl alkoxyl, phenyl alkoxyalkyl, phenoxy alkoxy or phenoxyalkyl,

-by C _6-20the cycloalkyl-alkyl that alkyl replaces,

-by C _6-20the heteroaryl alkyl that alkyl replaces,

-heterocycle C _6-20alkyl or

-by C _2-20the Heterocyclylalkyl that alkyl replaces,

And wherein:

Moieties can contain:

-in carbochain, contain and be selected from following key or hetero atom: two keys, triple bond, O, S, sulfinyl, sulfonyl or NR ₆, R wherein ₆as defined above, and

-as substituent alkoxyl, alkenyloxy, alkynyloxy group, aralkyl oxy, acyl group, alkyl amino, alkylthio group, acylamino-, alkoxy carbonyl, alkoxycarbonyl amino, acyloxy, alkyl-carbamoyl, nitro, halogen, amino, hydroxyl or carboxyl, and

R ₂, R ₃, R ₄and R ₅be H, C independently of each other _1-4alkyl or acyl group,

Or its pharmaceutically acceptable its salt;

Disclosed compound in-EP 1002792A1, for example compound of formula II

Wherein m is 1 to 9, and R ' ₂, R ' ₃, R ' ₄and R ' ₅be H, alkyl or acyl group independently of each other, or its pharmaceutically acceptable its salt;

Disclosed compound in-EP 0778263A1, for example compound of formula III

Wherein W is H; C _1-6alkyl, C _2-6alkenyl or C _2-6alkynyl; Unsubstituted or replaced by OH phenyl; R " ₄o (CH ₂) _n; Or be selected from halogen, C by 1 to 3 _3-8the C that the substituent group of cycloalkyl, phenyl and the phenyl that replaced by OH replaces _1-6alkyl;

X is H or the carbon number that the contains straight chain alkoxyl that does not replace or replace that to be p unsubstituted or the straight chained alkyl that replaces or the carbon number that contains are p-1, for example is selected from 1 to 3 following substituent group and replaces: C _1-6alkyl, OH, C _1-6alkoxyl, acyloxy, amino, C _1-6alkyl amino, acylamino-, oxo, halo C _1-6alkyl, halogen, unsubstituted phenyl, by 1 to 3, be selected from C _1-6alkyl, OH, C _1-6alkoxyl, acyl group, acyloxy, amino, C _1-6alkyl amino, acylamino-, halo C _1-6the phenyl that the substituent group of alkyl and halogen replaces; Y is H, C _1-6alkyl, OH, C _1-6alkoxyl, acyl group, acyloxy, amino, C _1-6alkyl amino, acylamino-, halo C _1-6alkyl or halogen, Z ₂for singly-bound or the carbon number that the contains straight-chain alkyl-sub-that is q,

P and q are one of integer in 1 to 20 independently of each other, and condition is 6≤p+q≤23, m ' be 1,2 or 3, n be 2 or 3,

R " ₁, R " ₂, R " ₃and R " ₄be H, C independently of each other _1-4alkyl or acyl group, or its pharmaceutically acceptable its salt,

Disclosed compound in-WO02/18395, for example compound of formula IVa or Ivb

or

X wherein _afor O, S, NR _1sor group-(CH ₂) _na-, this group is unsubstituted or is replaced by 1 to 4 halogen; n _abe 1 or 2, R _1sfor H or (C _1-4) alkyl, this alkyl is unsubstituted or is replaced by halogen; R _1afor H, OH, (C _1-4) alkyl or O (C _1-4) alkyl, wherein alkyl is unsubstituted or is replaced by 1 to 3 halogen; R _1bfor H, OH or (C _1-4) alkyl, wherein alkyl is unsubstituted or is replaced by halogen; Each R _2abe selected from independently of each other H or (C _1-4) alkyl, this alkyl is unsubstituted or is replaced by halogen; R _3afor H, OH, halogen or O (C _1-4) alkyl, wherein alkyl is unsubstituted or is replaced by halogen; And R _3bfor H, OH, halogen, (C _1-4) alkyl, wherein alkyl is unsubstituted or is replaced or O (C by hydroxyl _1-4) alkyl, wherein alkyl is unsubstituted or is replaced by halogen; Y _afor-CH ₂-,-C (O)-,-CH (OH)-,-C (=NOH)-, O or S, and R _4afor (C _4-14) alkyl or (C _4-14) alkenyl;

Or its pharmaceutically acceptable its salt;

Disclosed compound in-WO 02/076995, for example compound of formula V:

Wherein

M _cbe 1,2 or 3;

X _cfor O or direct bond;

R _1cfor H; Optionally by OH, acyl group, halogen, C _3-10the C that cycloalkyl, phenyl or hydroxyl phenylene replace _1-6alkyl; C _2-6alkenyl; C _2-6alkynyl; Or the phenyl optionally replaced by OH;

R _2cfor

R wherein _5cfor H or optionally by the C of 1,2 or 3 halogen atom replacement _1-4alkyl, and R _6cfor H or the C that optionally replaced by halogen _1-4alkyl;

R _3cand R _4cthe C that is H independently of each other, is optionally replaced by halogen _1-4alkyl or acyl group, and

R _cfor C _13-20alkyl, it optionally contains oxygen atom in chain, and it is optionally by nitro, halogen, amino, hydroxyl or carboxyl substituted; Or be the residue of formula (a)

R wherein _7cfor H, C _1-4alkyl or C _1-4alkoxyl, and R _8cfor the C replaced _1-20alkanoyl, phenyl C _1-14alkyl, wherein C _1-14alkyl optionally by halogen or OH replace, cycloalkyl C _1-14alkoxyl or phenyl C _1-14alkoxyl, wherein cycloalkyl or phenyl ring are optionally by halogen, C _1-4alkyl and/or C _1-4alkoxyl replaces, phenyl C _1-14alkoxy C _1-14alkyl, phenoxy group C _1-14alkoxyl or phenoxy group C _1-14alkyl,

Work as R _1cfor C _1-4alkyl, C _2-6alkenyl or C _2-6during alkynyl, R _ccan be also the residue of formula (a), wherein R _8cfor C _1-14alkoxyl,

Or the compound of formula VI

Wherein

N _xbe 2,3 or 4

R _1xfor H; The C optionally replaced by OH, acyl group, halogen, cycloalkyl, phenyl or hydroxyl phenylene _1-6alkyl; C _2-6alkenyl; C _2-6alkynyl; Or the phenyl optionally replaced by OH;

R _2xfor H, C _1-4alkyl or acyl group

R _3xand R _4xbe H independently of each other, optionally by the C of halogen or acyl substituted _1-4alkyl,

R _5xfor H, C _1-4alkyl or C _1-4alkoxyl, and

R _6xfor the C be substituted by cycloalkyl _1-20alkanoyl; Cycloalkyl C _1-14alkoxyl, wherein cycloalkyl ring is optionally by halogen, C _1-4alkyl and/or C _1-4alkoxyl replaces; Phenyl C _1-14alkoxyl, wherein phenyl ring is optionally by halogen, C _1-4alkyl and/or C _1-4alkoxyl replaces,

Work as R _1xfor the C replaced by OH _2-4during alkyl, R _6xcan be also C _4-14alkoxyl, or work as R _1xfor C _1-4during alkyl, R _6xfor amoxy or hexyloxy,

Condition is: work as R _5xfor H or R _1xduring for methyl, R _6xnot phenyl-butenyloxy, or its officinal salt;

Disclosed compound in-WO 02/06268A1, for example compound of formula VII

R wherein _1dand R _2dbe H or amino protecting group independently of each other;

R _3dfor hydrogen or hydroxyl protecting group;

R _4dfor low alkyl group;

N _dit is one of integer of 1 to 6;

X _dfor ethylidene, ethenylidene, ethynylene, formula-D-CH ₂-group (wherein D be carbonyl ,-CH (OH)-, O, S or N), aryl or be no more than three and be selected from the aryl that hereinafter substituent group of defined group replaces;

Y _dfor singly-bound, C _1-10alkylidene, quilt are no more than three C that are selected from the substituent group replacement of group a and b _1-10alkylidene, the C that middle in carbochain or end contains O or S _1-10alkylidene or in the middle of the carbochain or the end C that contain O or S, be no more than three substituent groups that are selected from group a and b and replace _1-10alkylidene;

R _5dfor hydrogen, cycloalkyl, aryl, heterocycle, be no more than cycloalkyl that three substituent groups that are selected from group a and b replace, be no more than aryl that three substituent groups that are selected from group a and b replace or be no more than three substituent groups that are selected from group a and b the heterocycles that replace; And

R _6dand R _7dbe H or the substituent group that is selected from group a independently of each other;

<group a > be halogen, low alkyl group, junior alkyl halides, lower alkoxy, lower alkylthio, carboxyl, elementary alkoxy carbonyl, hydroxyl, rudimentary aliphatic acidyl, amino, single low-grade alkyl amino, two elementary alkyl amido, lower aliphatic acylamino-, cyano group or nitro;

<group b > be cycloalkyl, acyl group, heterocycle, it can optionally be no more than three substituent groups that are selected from group a respectively and be replaced;

Condition is: work as R _5dduring for hydrogen, Y _dfor singly-bound or straight chain C _1-10alkylidene, or its pharmaceutically useful salt or ester.

Disclosed compound in-JP-14316985 (JP2002316985), for example compound of formula VIII:

R wherein _1e, R _2e, R _3e, R _4e, R _5e, R _6e, R _7e, n _e, X _eand Y _edescribed in JP-14316985;

Or its pharmaceutically useful salt or ester.

Disclosed compound in-WO 03/29184 and WO 03/29205, for example compound of formula IX:

X wherein _ffor O or S, and R _1f, R _2f, R _3fand n _fdescribed in WO 03/29184 and O3/29205, for example, 2-amino-2-[4-(3-benzyloxy phenoxy group)-2-chlorphenyl] propyl group-1,3-PD or 2-amino-2-[4-(benzyloxy thiophenyl)-2-chlorphenyl] propyl group-1,3-PD.

Summary of the invention

When carrying out patent application and quote, theme that will be relevant to described compound is introduced the application as a reference.

Acyl group can be R _y-CO-residue, wherein R _yfor C _1-6alkyl, C _3-6cycloalkyl, phenyl or phenyl-C _1-4alkyl.Unless otherwise indicated, otherwise alkyl, alkoxyl, alkenyl or alkynyl all can be for straight or brancheds.

When the carbochain of the R1 in formula I compound is substituted, it is preferably by halogen, nitro, amino, hydroxyl or carboxyl substituted.When the phenylene be optionally substituted when carbochain interrupts, carbochain is preferably unsubstituted.When phenylen moiety is substituted, it is preferably by halogen, nitro, amino, methoxyl group, hydroxyl or carboxyl substituted.

The preferred compound of formula I is those wherein R ₁for optionally by the C of nitro, halogen, amino, hydroxyl or carboxyl substituted _13-20the compound of alkyl; And, more preferably those R wherein ₁for by C _6-14the compound of the phenylalkyl that alkyl chain (alkyl chain is optionally replaced by halogen and moieties is optionally replaced by hydroxyl) replaces.More preferably, R ₁for on phenyl ring by straight or branched, the preferred C of straight chain _6-14phenyl-C that alkyl chain replaces _1-6alkyl.C _6-14alkyl chain can be at ortho position, a position or para-position, preferably in para-position.

Preferred R ₂to R ₅h respectively does for oneself.

Preferred formula I compound is 2-amino-2-myristyl-1,3-PD.The S1P receptor stimulating agent of particularly preferred formula I is FTY720, i.e. the 2-amino-2-[2-of free form or pharmaceutical acceptable salt (4-octyl phenyl) ethyl] propane-1,3-glycol (hereinafter referred to as compd A), for example hydrochlorate as follows:

The preferred compound of formula II is R ' wherein ₂to R ' ₅respectively the do for oneself compound that H and m be 4, i.e. the 2-amino-2-{2-[4-of free form or pharmaceutical acceptable salt (1-oxo-5-phenylpentyl) phenyl] ethyl) propane-1,3-glycol (hereinafter referred to as compd B), for example hydrochlorate.

The preferred compound of formula III is CH for W wherein ₃, R " ₁to R " ₃h, Z respectively do for oneself ₂for example, for ethylidene, X are oxygen base and Y be H compound, i.e. the 2-amino-4-of free form or pharmaceutical acceptable salt (4-heptyloxybenzene base)-2-methyl-butanols (hereinafter referred to as Compound C), hydrochlorate in heptan.R-enantiomer particularly preferably.

The preferred compound of formula IVa is FTY720-phosphate (R _2afor H, R _3afor OH, X _afor O, R _1aand R _1bfor OH).Formula IV _bpreferred compound be Compound C-phosphate (R _2afor H, R _3bfor OH, X _afor O, R _1aand R _1bfor OH, Y _afor O and R _4afor heptyl).The preferred compound of formula V is compd B-phosphate.

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

The preferred compound of formula VIII is (2R)-2-amino-4-[3-(4-cyclohexyl oxygen Ji Dingji) benzo [b] thiophene-6-yl]-2-methyl fourth-1-alcohol.

When the compound of formula I to IX has one or more asymmetric center in molecule, the present invention is understood to include various optical isomers and racemate, diastereoisomer and composition thereof.When the carbon atom with amino is asymmetric carbon atom, the compound of formula III or IVb preferably has the R-configuration on this carbon atom.

The example of the officinal salt of the compound of formula I to IX comprises the salt with mineral acid, example hydrochloric acid salt, hydrobromate and sulfate, with organic acid salt, as acetate, fumarate, maleate, benzoate, citrate, malate, mesylate and benzene sulfonate, when perhaps suitable, with metal as the salt of sodium, potassium, calcium and aluminum, with amine as the salt of triethylamine and with the salt of dibasic aminoacid as lysine.The compound of method of the present invention and salt comprise hydrate and solvate form thereof.

According to observed activity for example EP627406A1 or the described lymphocyte homing activity of USP 6,004,565, found the S1P receptor stimulating agent can be for example in the acute allograft refection for the treatment of for example as immunosuppressant.Have now found that: the S1P receptor stimulating agent has the characteristic attracted people's attention, and described characteristic makes it can be used for cancer chemotherapy, particularly solid tumor, especially advanced solid tumor.Still need to expand the equipment of solid tumor cancer treatment, especially in the situation that the anticancer compound treatment can not make disease disappear or be stable.

According to special discovery of the present invention, the invention provides:

1.1 the method for a treatment solid tumor in the individuality needed is arranged, comprise the S1P receptor stimulating agent or its officinal salt that give described individual treatment effective dose, the group that wherein said agonist contains formula X.

1.2 a method that suppresses implanted solid tumor growth in the individuality needed is arranged, comprise the S1P receptor stimulating agent or its officinal salt that give described individual treatment effective dose, the group that wherein said agonist contains formula X.

1.3 the method that induced tumor disappears, for example tumor quality alleviates in the individuality needed is arranged, comprise the S1P receptor stimulating agent or its officinal salt that give described individual treatment effective dose, the group that wherein said agonist contains formula X.

1.4 one kind is treated in the individuality needed is arranged, and solid tumor is invaded or the method for the symptom that tumor growth is relevant therewith, comprises the S1P receptor stimulating agent or its officinal salt that give described individual treatment effective dose, the group that wherein said agonist contains formula X.

1.5 one kind in the individuality needed is arranged prophylaxis of tumours shift diffusion or prevention or suppress the method for micrometastasis kitchen range growth, comprise the S1P receptor stimulating agent or its officinal salt that give described individual treatment effective dose, the group that wherein said agonist contains formula X.

A 1.6 method that suppresses or control angiogenesis out of control (for example angiogenesis of S1P (S1P) mediation) in the individuality needed is arranged, comprise the S1P receptor stimulating agent or its officinal salt that give described individual treatment effective dose, the group that wherein said agonist contains formula X.

1.7 the method for the disease that prevention or treatment neovascularity generative process mediate in the individuality needed is arranged or the disease relevant to angiogenesis out of control, comprise the S1P receptor stimulating agent or its officinal salt that give described individual treatment effective dose, the group that wherein said agonist contains formula X.

No matter wherein " solid tumor " refer to tumor and/or the metastasis () except lymphatic cancer, for example brain or other central nerve neuroma (as the tumor of meningioma, cerebroma, spinal cord tumor, cranial nerve tumor and central nervous system's other parts, as glioblastoma or marrow blastoma); Head and/or cervical region cancer; Mammary neoplasms; Blood circulation tumor (for example tumor, hemangioma and the tumor relevant with vascular tissue of other organ in heart, mediastinum and pleura and thorax); Excretory system tumor (for example kidney, renal pelvis, ureter, bladder, other and unspecified urinary organs); Gastroenteric tumor (for example esophagus, stomach, small intestinal, colon, colorectum, proctosigmoid binding site, rectum, anus, anal canal), relate to biliary pore in liver and liver, gallbladder, biliary tract other and do not particularly point out part, pancreas and other Alimentary tumor; Head and neck; Oral cavity (other position of other parts, the parotid gland and salivary gland other parts, tonsil, oropharynx, nasopharynx, pyriform hole, hypopharynx and lip, oral cavity and the pharynx in lip, tongue, gingiva, bottom, oral cavity, palate and oral cavity); Genital system tumor (for example other position of other position, Placenta Hominis, penis, prostate, testis and the genital orgnas,male of pudendum, vagina, cervix uteri, body of uterus, uterus, ovary and female sex organ); Respiratory tract neoplasms (for example nasal cavity and middle ear, paranasal sinus, larynx, trachea, bronchus and lung, for example small cell lung cancer or nonsmall-cell lung cancer); Skeletal system tumor (for example bone of extremity and articular cartilage, bone articular cartilage and other position); Cutaneous tumor (for example malignant melanoma of skin, nonmelanoma skin cancer, rodent ulcer, cutaneous squamous cell carcinoma, mesothelioma, Kaposi ' s sarcoma); With relate to other tissue, comprise tumor, Secondary cases and the second malignant neoplasm of unspecified malignant lymphatic dross, breathing and digestive system and the second malignant neoplasm at other position of peripheral nervous and autonomic nervous system, connective tissue and soft tissue, retroperitoneum and peritoneum, eye and adnexa, thyroid, adrenal gland and other endocrine gland and related structure.

Above and below while mentioning tumor, tumor disease, cancer or malignant tumor, also infer individually or simultaneously the metastatic tumor at former organ or tissue and/or other arbitrary position, no matter tumor and/or metastatic tumor are wherein.

For example, when compound that the S1P receptor stimulating agent is formula I compound (compd A) or formula IVa or IVb, in an embodiment, it is used in the treatment of solid tumors method 1.1,1.2,1.3 or 1.4 except mammary gland, prostate, bladder, kidney or lung tumor.

In a series of other concrete or alternative embodiments, the present invention also provides:

A 1.8 method that strengthens the active of chemotherapeutics or overcome the chemotherapeutics drug resistance in the individuality needed is arranged, it comprises together with described chemotherapeutics, simultaneously or give successively the S1P receptor stimulating agent of described individual treatment effective dose, the S1P receptor stimulating agent that for example contains the group of formula X, or its officinal salt.

1.9 according to 1.8 described methods, wherein chemotherapeutics is signal conducting path inhibitor, this signal conducting path is for host cell or tumor forms and/or metastasis forms related process, or can be bred, survive, break up or produce drug resistance by tumor cell utilization.

1.10 method as above, wherein S1P receptor stimulating agent intermittent administration.

2.1 S1P receptor stimulating agent or its officinal salt for the group that comprises formula X of above-mentioned 1.1 to 1.4 defined any methods, when the compound of the S1P receptor stimulating agent compound (as compd A) that is formula I or formula IVa or IVb, it is preferred for the solid tumor beyond mammary gland, prostate, bladder, kidney or lung.

2.2, for the S1P receptor stimulating agent in above 1.5 to 1.10 or hereinafter 7 defined any methods, for example contain the S1P receptor stimulating agent of the group of formula X, or its officinal salt.

3.1 S1P receptor stimulating agent or its officinal salt for the preparation of the group that comprises formula X that is used in the pharmaceutical composition in above-mentioned 1.1 to 1.4 defined any methods, when the compound of the S1P receptor stimulating agent compound (as compd A) that is formula I or formula IVa or IVb, it is preferred for the solid tumor beyond mammary gland, prostate, bladder, kidney or lung.

3.2, for the preparation of being used in the S1P receptor stimulating agent in defined any method in above 1.5 to 1.10 or hereinafter 7, for example contain the S1P receptor stimulating agent of the group of formula X, or its officinal salt.

4.1 the pharmaceutical composition for above-mentioned 1.1 to 1.4 defined any methods, the S1P receptor stimulating agent that it contains the group that comprises formula X or its officinal salt, and one or more pharmaceutically useful diluent or carriers, when the compound of the S1P receptor stimulating agent compound (as compd A) that is formula I or formula IVa or IVb, it is preferred for the solid tumor beyond mammary gland, prostate, bladder, kidney or lung.

4.2 for above 1.5 to 1.10 or the pharmaceutical composition of hereinafter 7 defined any methods, it contains the S1P receptor stimulating agent, the S1P receptor stimulating agent that for example contains the group of formula X, or its officinal salt, and one or more pharmaceutically useful diluent or carriers.

5.1 a drug regimen, it contains a) is the first medicine of S1P receptor stimulating agent, for example contains S1P receptor stimulating agent or its officinal salt of the group of formula X, and b) be the accessory drugs of chemotherapeutics, the chemotherapeutics that for example hereinafter described in detail.

A 5.2 drug regimen, it contains a certain amount of a) for the first medicine of S1P receptor stimulating agent, the S1P receptor stimulating agent or its officinal salt that for example contain the group of formula X, and b) be the accessory drugs of chemotherapeutics, it is selected from hereinafter xi) the defined compound of part, to produce synergistic therapeutic effect.

6. a method as defined above, comprise the S1P receptor stimulating agent for the treatment of effective dose, the S1P receptor stimulating agent or its officinal salt and the second medicine co-administered that for example comprise the group of formula X, for example while or administration in succession, described the second medicine is chemotherapeutics hereinafter described.

One kind in the individuality needed is arranged the treatment lymphocytic hyperplasia or bone marrow proliferation abnormal, for example treat the method for tumor intrusion or the symptom that tumor growth is relevant therewith, it comprises to described individual co-administered, for example while or administration S1P receptor stimulating agent in succession, the S1P receptor stimulating agent or its officinal salt and the second medicine that for example comprise the group of formula X, described the second medicine is chemotherapeutics hereinafter described for example.

" lymphatic cancer " for example refers to blood and lymphsystem tumor (Hodgkin for example, non-Hodgkin lymphoma, Burkitt lymphoma, the lymphoma relevant with AIDS, pernicious immunoproliferative disease, multiple myeloma and malignant plasma cell tumor, leukemic lymphoblastoid, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, the leukemia of other designated cell type, do not particularly point out the leukemia of cell type, other and unspecified lymph, the malignant tumor of hemopoietic and related organization is the dispersivity large celllymphoma for example, T-cell lymphoma or skin T-cell lymphoma).Bone marrow cancer comprises as acute or chronic myelogenous leukemia.

Noun " chemotherapeutics " is any chemotherapeutics of refer to except the S1P receptor stimulating agent especially.It comprises but is not only limited to:

I. aromatase inhibitor,

Ii. antiestrogen, antiandrogen (while particularly suffering from carcinoma of prostate) or gonadorelin agonist,

Iii. topoisomerase I inhibitor or Topoisomerase II inhibitors,

Iv. microtubule activator, alkylating agent, anti-tumor metabolic drug or platinum complex,

V. the compound of targeting/reduction albumen or lipid kinase activity or albumen or lipid phosphatase activity, but the compound of other anti-angiogenic compounds or Cell differentiation inducing activity process,

Vi. Kallidin I 1 receptor or angiotensin-ii antagonist,

Vii. cyclooxygenase-2 inhibitor, diphosphonic acids, histone deacylase inhibitor, heparinase (heparinase) inhibitor (preventing the Heparan sulfate degraded) are as PI-88, biological answer-reply dressing agent, preferred lymphokine or interferon, for example interferon gamma, ubiquitination inhibitor maybe can be blocked the inhibitor of anti-apoptotic approach.

The carcinogenic isotype inhibitor of viii.Ras, for example H-Ras, K-Ras or N-Ras, or farnesyl transferase inhibitor, L-744 for example, 832 or DK8G557,

Ix. telomerase inhibitor, telomestatin for example,

X. protease inhibitor, matrix metallo-proteinase inhibitor, methionine aminopeptidase inhibitor, for example bengamide or derivatives thereof, or albuminous body inhibitor, for example PS-341.

The xi.mTOR inhibitor.

Term used herein " aromatase inhibitor " relates to can suppress estrogen production, is about to the compound that ANDROSTENEDIONE and testosterone substrate are separately converted to estrone and estradiol.This term is including, but not limited to the steroid class, especially atamestane, exemestane and formestane, non-steroid class, especially aminoglutethimide, Rogletimide, pyridine radicals glutethimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, Anastrozole and letrozole especially.Exemestane can be AROMASIN as trade mark as commercial form ^tMform administration.Formestane can be LENTARON as trade mark as commercial form ^tMform administration.Fadrozole can be AFEMA as trade mark as commercial form ^tMform administration.Anastrozole can be ARIMIDEX as trade mark as commercial form ^tMform administration.Letrozole can be FEMARA as trade mark as commercial form ^tMor FEMAR ^tMform administration.Aminoglutethimide can be ORIMETEN as trade mark as commercial form ^tMform administration.Administering drug combinations containing the aromatase inhibitor chemotherapeutics in the present invention is as effective especially as lacteal tumor to treatment hormone receptor positive tumor.

Term used herein " antiestrogen " relates to can be at the compound of Estrogen Receptor antagonism estrogen effect.This term is including, but not limited to tamoxifen, fulvestrant, raloxifene and RALOXIFENE HCL.Tamoxifen can be as commercial form as trade mark NOLVADEX ^tMform administration.RALOXIFENE HCL can be EVISTA as trade mark as commercial form ^tMform administration.Fulvestrant can be by US 4,659, in 516 disclosed content make or take as commercial form as trade mark, be FASLODEX ^tMform administration.The administering drug combinations that contains the antiestrogen chemotherapeutics in the present invention is as effective especially as lacteal tumor to the treatment estrogen receptor positive tumors.

Term used herein " antiandrogen " relates to arbitrary material that can suppress the androgen biological effect, including, but not limited to bicalutamide (CASODEX ^tM), it can be according to US 4,636, disclosed content preparation in 505.

Term used herein " gonadorelin agonist " is including, but not limited to 1: PN: WO02056903 PAGE: 25 claimed protein, goserelin and goserelin acetate.Goserelin is at US 4,100, open in 274, can as trade mark, be ZOLADEX as commercial form ^tMform administration.1: PN: WO02056903 PAGE: 25 claimed protein can be according to US 5,843, disclosed content preparation in 901.

Term used herein " topoisomerase I inhibitor " is including, but not limited to hycamtin, irinotecan, 9-nitrocamptothecin and macromole camptothecine conjugates PNU-166148 (compd A 1 in WO99/17804).Irinotecan for example commercial form is CAMPTOSAR as trade mark ^tMform administration.Hycamtin can be HYCAMTIN as trade mark as commercial form ^tMform administration.

Term used herein " Topoisomerase II inhibitors " (comprises Lipidosome, for example CAELYX including, but not limited to anthracyclines as amycin ^tM), daunorubicin, epirubicin, idarubicin and Nemorubicin (nemorubicin), anthraquinone mitoxantrone and losoxantrone, and Podophyllinic Acid Lactone (podophyllotoxines) etoposide and teniposide.Etoposide for example commercial form is ETOPOPHOS as trade mark ^tMform administration.Teniposide take as commercial form as trade mark, be VM26-BRISTOL ^tMform administration.Amycin take as commercial form as trade mark, be ADRIBLASTIN ^tMform administration.Epirubicin take as commercial form as trade mark, be FARMORUBICIN ^tMform administration.Idarubicin take as commercial form as trade mark, be ZAVEDOS ^tMform administration.Mitoxantrone take as commercial form as trade mark, be NOVANTRON ^tMform administration.

Term used herein " microtubule activation medicine " relates to that microtubule is stablized medicine and microtubule removes to stablize medicine, including, but not limited to taxanes as paclitaxel and docetaxel, Vinca alkaloids is as vincaleucoblastine, particularly Vinblastine Sulfate, vincristine, particularly vincristine sulfate and vinorelbine, discodermolide (discodermolide) and Epothilones and derivant thereof, as the epothilone B or derivatives thereof.Paclitaxel take as commercial form as trade mark, be TAXOL ^tMform administration.Docetaxel take as commercial form as trade mark, be TAXOTERE ^tMform administration.It is VINBLASTIN R.P. as trade mark that Vinblastine Sulfate be take for example commercial form ^tMform administration.It is FARMISTIN as trade mark that vincristine sulfate be take for example commercial form ^tMform administration.Discodermolide can, as US 5,010, obtain in 099 disclosed content.

Term used herein " alkylating agent " is including, but not limited to cyclophosphamide, ifosfamide, melphalan or nitroso ureas (BCNU or Gliadel ^tM).It is CYCLOSTIN as trade mark that cyclophosphamide be take for example commercial form ^tMform administration.It is HOLOXAN as trade mark that ifosfamide be take for example commercial form ^tMform administration.

Term " anti-tumor metabolic drug " is including, but not limited to 5-fluorouracil, capecitabine, gemcitabine, methotrexate and edatrexate.It is XELODA as trade mark that capecitabine be take for example commercial form ^tMform administration.It is GEMZAR as trade mark that gemcitabine be take for example commercial form ^tMform administration.

Term used herein " platinum complex " is including, but not limited to carboplatin, cisplatin and oxaliplatin.It is CARBOPLAT as trade mark that carboplatin be take for example commercial form ^tMform administration.It is ELOXATIN as trade mark that oxaliplatin be take for example commercial form ^tMform administration.

Targeting, reduce or the compound that suppresses the VEGFR activity especially can suppress the VEGF tyrosine kinase receptor, suppress vegf receptor or the compound of being combined with VEGF, protein or antibody, particularly in those WO 98/35958 or as in WO 00/09495, WO 00/27820, WO00/59509, WO 98/11223, WO 00/27819 and EP 0769947 and concrete disclosed compound, protein or monoclonal antibody, 1-(4-chlorobenzene amino)-4-(4-pyridylmethyl) phthalazines or officinal salt for example, as succinate; Those as people such as M.Prewett in Cancer Research 59 (1999) 5209-5218, the people such as F.Yuan is at Proc.Natl.Acad.Sci.USA, vol.93, pp.14765-14770, in Dec.1996, the people such as Z.Zhu is at Cancer Res.58, in 1998,3209-3214 and the people such as J.Mordenti at Toxicologic Pathology, Vol.27, no.1, pp 14-21, the compound of describing in 1999; Compound in WO 00/37502 and WO 94/10202; The people such as M.S.O ' Reilly are at Cell 79,1994, the Angiostatin described in 315-328 ^tM; The people such as M.S.O ' Reilly are at Cell 88,1997, the Endostatin described in 277-285 ^tM; The ortho-aminobenzoic acid amide; ZD4190; ZD6474; SU5416; SU6668; Or VEGF antibody or anti-VEGF receptor antibody, for example RhuMab.

Antibody refers to complete monoclonal antibody, polyclonal antibody, the multiple types antibody and the antibody fragments that are formed by least two kinds of complete antibody, as long as this antibody fragments can show the biologic activity of hope.

Targeting, the compound that reduces or suppress the Epidermal Growth Factor Receptor Family activity especially can suppress EGF tyrosine kinase receptor family member as the EGF receptor, ErbB2, ErbB3 and ErbB4 or in conjunction with the compound of EGF or the relevant part of EGF, protein or antibody, general in those WO97/02266 and concrete disclosed compound particularly, protein or monoclonal antibody, compound as embodiment 39, perhaps at EP 0 564 409, WO 99/03854, EP 0520722, EP 0 566 226, EP 0 787 722, EP 0 837 063, US 5, 747, 498, WO 98/10767, WO 97/30034, WO 97/49688, WO 97/38983 neutralization is especially at WO 96/30347 (as known compound CP358774), disclosed compound in WO 96/33980 (as known compound ZD 1839) and WO 95/03283 (as compound ZM105180), protein or monoclonal antibody, trastuzumab (Herpetin for example ^r), Cetuximab, Iressa, OSI-774, CI-1033, EKB-569, GW-2016, E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3.

Targeting, reduce or the compound that suppresses the PDGFR activity especially can suppress the compound of pdgf receptor, N-phenyl-2-pyrimidine-amine derivatives for example, as imatinib.

Targeting, reduce or the compound that suppresses c-AbI family member and gene fusion its lytic activity thereof is for example the N-phenyl-2-pyrimidine-amine derivatives, as imatinib; PD180970; AG957 or NSC680410.

Targeting, reduce or Profilin kinase c, Raf, MEK, SRC, JAK, FAK and PDK family member or PI (3) kinases or with disclosed staurosporine derivatives in compound, especially those EP 0 296 110 of the relevant family member of PI (3) kinases and/or the member of cell cycle protein dependent kinase family (CDK) activity as midostaurin; The embodiment of other compound comprises for example UCN-01, Safingol, BAY 43-9006, bryostatin 1, perifosine; Ilmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521; Or LY333531/LY379196.

Other anti-angiogenic compounds is for example Sa Li polyamines (THALOMID) and TNP-470.

Targeting, reduce or the compound of Profilin or lipid phosphatase activity is for example phosphatase 1, phosphatase 2A, PTEN or the CDC25 inhibitor as the okadaic acid or derivatives thereof.

Can inducing cell the compound of mutation process be for example tretinoin, α-, γ-or Delta-Tocopherol or α-, γ-or δ-tocotrienol.

Term cyclooxygenase-2 inhibitor used herein is including, but not limited to for example celecoxib (Celebrex ^r), rofecoxib (Vioxx ^r), etoricoxib, valdecoxib or the amino aminophenyl acetic acid of 5-alkyl-2-is as 5-methyl-2-(2 '-chloro-6 '-fluoroanilino) phenylacetic acid.

Term used herein " histone deacylase inhibitor " is including, but not limited to MS-27-275, SAHA, pyroxamide, FR-901228 or valproic acid.

Term used herein " diphosphonic acids " is including, but not limited to etidronic acid, clodronic acid, tiludronic acid, pamidronic acid, alendronic Acid, ibandronic acid, risedronic acid and zoledronic acid." etidronic acid " take for example commercial form is DIDRONEL as trade mark ^tMform administration." clodronic acid " take for example commercial form is BONEFOS as trade mark ^tMform administration." tiludronic acid " take for example commercial form is SKELID as trade mark ^tMform administration." pamidronic acid " take for example commercial form is AREDIA as trade mark ^tMform administration." alendronic Acid " take for example commercial form is FOSAMAX as trade mark ^tMform administration." ibandronic acid " take for example commercial form is BONDRANAT as trade mark ^tMform administration." risedronic acid " take for example commercial form is ACTONEL as trade mark ^tMform administration." zoledronic acid " take for example commercial form is ZOMETA as trade mark ^tMform administration.

Including, but not limited to collagen simulating peptide and non-simulating peptide inhibitor, tetracycline derivant, for example hydroxamic acid simulating peptide inhibitor batimastat and oral biology thereof can be accepted analog Marimastat, prinomastat, BMS-279251, BAY12-9566, TAA211 or AAJ996 to term used herein " matrix metallo-proteinase inhibitor ".

Term used herein " mTOR inhibitors " includes but are not limited to the rapamycin or derivatives thereof.Rapamycin is the known macrolide antibiotics produced by streptomyces hygroscopicus.Suitable rapamycin derivative comprises for example compound of formula A

Wherein

R _1aafor CH ₃or C _3-6alkynyl,

R _2aafor H or-CH ₂-CH ₂-OH, 3-hydroxyl-2-(methylol)-2-methyl-propiono or tetrazole radical, and

X _aafor=O, (H, H) or (H, OH)

Its condition is to work as X _aafor=O and R _1aafor CH ₃the time, R _2aabe not H,

Perhaps work as R _2aafor-CH ₂-CH ₂during-OH, its prodrug, for example hydrolyzable ester of its physiology.

Formula I compound is at for example WO 94/09010, WO 95/16691, WO 96/41807, USP5,362,718 or WO 99/15530 in open, these documents are hereby incorporated by.Can prepare these compounds according to method disclosed in these documents or similarly.

Preferred rapamycin derivative is 32-deoxidation rapamycin, 16-penta-2-alkynyloxy base-32-deoxidation rapamycin, 16-penta-2-alkynyloxy base-32 (S)-dihydro-rapamycin, 16-penta-2-alkynyloxy base-32 (S)-dihydro-40-O-(2-ethoxy)-rapamycin, and preferred 40-O-(2-ethoxy)-rapamycin.Other examples of rapamycin derivative comprise as USP 5,362, disclosed CCI779 or 40-[3-hydroxyl-2-(methylol)-2 Methylpropionic acid ester in 718]-rapamycin or its officinal salt, as disclosed ABT578 or 40-(tetrazole radical)-rapamycin in WO99/15530,40-table-(tetrazole radical)-rapamycin particularly, perhaps for example, as disclosed forms of rapamycin analogs, AP23573 in WO 98/02441 and WO 01/14387.

In the situation that has provided patent application or science publication quoted passage, the content relevant with compound introduced the application as a reference.These compounds can be also its officinal salt, corresponding racemoid, diastereomer, enantiomer, tautomer and, if present, the corresponding crystal modification of above-claimed cpd, for example solvate, hydrate and polymorphic.The compound that is used as active ingredient in combination of the present invention can be prepared and administration described in institute's citing document.Simultaneously, scope of the present invention also comprises the combination more than the above-mentioned various active ingredients of two kinds, i.e. the combination of pharmacy in the scope of the invention can comprise active ingredient more than three kinds or three kinds.In addition, the first medicine and accessory drugs are not all composition of the same race.

In the solid tumor that as above described in detail for the treatment of, the effectiveness of S1P agonist (as S1P agonist of the group that comprises formula X) can be confirmed in animal experiment or clinical trial method, for example according to test method hereinafter described, confirms.

A. in vitro tests

A.1 anti-tumor activity

The mouse mammary carcinoma cell line of initial separation from breast carcinoma, for example JygMC (A) have been used in test.Before test operation starts, cell number is adjusted to 5 * 10 ⁵with bed board in fresh medium.By cell with contain the 2.5mM thymidine containing not hatching 12 hours together with the fresh medium of FCS, then with PBS, clean twice, add again subsequently the fresh medium containing 10%FCS, and again hatch 12 hours.And then with contain the 2.5mM thymidine containing not hatching 12 hours together with the fresh medium of FCS.For discharging the cell mass that cell is become from institute's adhesion, cell can be cleaned to twice with PBS, and again be laid in the fresh medium that contains 10%FCS.After synchronization, by cell together with the formula I of variable concentrations compound or hatch separately 3,6,9,12,18 or 24 hours.After processing with 0.2%EDTA, harvesting, and fix with 70% ice-cold alcoholic solution, then in 37 ℃ of RnaseA with 250 μ g/ml (1-A type: Sigma Chem.Co.) be hydrolyzed 30 minutes, and dye 20 minutes with the propidium iodide of 10mg/ml.After incubation period, measure cell number by Coulter rolling counters forward cell and SRB colorimetric determination simultaneously.Under these experimental conditions, S1P agonist (for example compd B of hydrochloride form) can be 10 ^-12-10 ^-6the propagation of inhibition tumor cell in the M concentration range.

A.2 the HUVEC tubing string of S1P mediation forms test

The go down to posterity HUVEC in 2-8 generation of use forms test for tubing string, and before results, the cell fusion degree is sure not to surpass 70% simultaneously.Using Herpes balanced salt solution (HBSS is from Clonetics) cleaning cell then with trypsin/EDTA (0.25mg/ml, from Clonetics), it to be carried out to enzymolysis, digestion comes pretreatment cell to be tested.After about 90% cell detachment flat board, add isopyknic trypsin neutralizer (TNS is from Clonetics) and by cell harvesting to the conical tube that at least contains 10ml EBM-2 (Clonetics)+0.1%BSA (Sigma) culture fluid.In 1000rpm centrifuge cell 5 minutes, remove supernatant and supplement the fresh EBM-2+0.1%BSA with 5ml.Use the blood cell calculator counting cells and the cell suspension adjustment is reached to the concentration of 500,000 cells/ml.Use 100nM detection compound and every by all means in the bacillus pertussis extracellular toxin (PTx) of 10ng/ml conical tube is carried out to pretreatment, then to every cell suspension that adds 1ml in by all means.Then in 37 ℃, 5%CO ₂in hatch conical tube half an hour.Use is coated with the Fluoro-Blok 24-Mulitwell insert plate of fibronectin, and (8 μ m pipe diameter sizes Falcon#351147) replace the single insert plate in 24 orifice plates to carry out the cell migration test.Prepare as mentioned above cell and detection compound and carry out preincubate, then in each suitable hole of insert plate, add 100 μ l.300 μ l EBM-2+2% are added to the bottom that is labeled as non-stimulated thing (-) hole through the culture fluid that does not contain S1P of activated carbon adsorption, and the culture fluid that simultaneously 300 μ l is contained to S1P (500nM) adds the bottom that is labeled as stimulus object (+) hole.Then flat board is placed in to 37 ℃, 5%CO ₂in hatch 4 hours.

First in bottle, add 20 μ l DMSO to prepare calcein AM, 50 μ g/ bottles (Molecular Probes#C3100).Then heat the HBSS (every flat board) of 12.5ml to 37 ℃, add 150 μ l in every bottle simultaneously.Then vial content is transferred back in remaining HBSS so that calcein AM ultimate density is 4 μ g/ml.

The Fluoro-Blok flat board is shifted out to couveuse, and separately the top insert plate also " flicks " to remove and is bonded at culture fluid unnecessary on insert plate.Then insert plate is transferred on the 24 new orifice plates of calcein AM of the 4 μ g/ml that contain 500 μ l/ holes.Again by plate in 37 ℃, 5%CO ₂in hatch 1.5 hours.

After hatching, plate is placed on Cytofluor II and carries out reading with the excitation wavelength of 485nm and the emission wavelength of 530nm.Fluoro-Blok coating in insert plate only allows the cell that travels to bottom to be counted.Transfer of data is calculated to Excel, and uses SigmaPlot to generate chart, uses SigmaStat to carry out significance test (t-check) simultaneously.(Fig. 7).

Under 4 times of enlargement ratios, by the number of counting branch point in 3 independent visuals field (junction points of two free bands), come the quantity tube post to form.The result report is as follows:

Process	Branch point
		PBS
	8±5
		S1P	42±13
FTY720-phosphate	48±15
		FTY720-phosphate+S1P	14±7
Compound C-phosphate	44±16
		Compound C-phosphate+S1P	18±6

These results have confirmed FTY720-phosphate or Compound C-phosphate itself unique ability as the angiogenesis agonist, but the antagonist of angiogenesis that the wonderful S1P of can be used as mediates.Compound C phosphate is preferably racemoid or R-enantiomer.PTx is used as suppressing Gi α (EDG-1) the active contrast that mediates.

B. in vivo test

B.1 anti-tumor activity

Anti-tumor activity means (increase of treated animal mean tumour volume is multiplied by 100 again divided by the increase of control animal mean tumour volume) with T/C%.

By the cancer cell of aliquot (1 * 10 ⁷) (for example mankind A375 melanoma cell) migrate to the BALB/c-nu/nu mice.Grow to 10 * 10mm when size in tumor, by animal random assortment to four subgroup, and by formula I compound treatment.After 2 weeks process, put to death animal, gather in the crops tumor and tissue simultaneously and carry out morphology and the analysis of molecules processing.Use slide calliper rule to measure tumor size.In this test, when the dosed administration with 0.5 to 5mg/kg, with contrasting saline, to compare, S1P agonist (for example compd B or C (with hydrochloride form)) can slow down tumor growth; For example, the Compound C hydrochlorate, when the dosed administration with 5 times/week of 2.5mg/kg, can produce the result that final T/C value is 30%.

B.2 with the VEGF-R protein tyrosine kinase inhibitor, combine

The nude mice of transplanting mankind MDA-MB-435 breast tumor for example, for example, is processed two weeks with the VEGF-R protein tyrosine kinase inhibitor (1-(4-chloroanilino)-4-(4-pyridylmethyl) phthalazines succinate) of 5 times/all oral dosage of 100mg/kg, S1P receptor stimulating agent (Compound C (hydrochlorate)) or both drug combinations of 5 times/all vein dosage of 2.5mg/kg.As mentioned above, mean anti-tumor activity with T/C%.With any medicament of independent use, compare, the drug combination of Compound C hydrochlorate and 1-(4-chloroanilino)-4-(4-pyridylmethyl) phthalazines succinate can produce more significant antitumous effect (T/C%27) (Compound C hydrochlorate T/C66%; 1-(4-chloroanilino)-4-(4-pyridylmethyl) phthalazines succinate T/C91%).In the nude mice of transplanting mankind's A375 melanoma cell and being processed with similarity method, identical drug combination has obtained good antitumor reaction too: the T/C% that drug combination produces is 15, and the T/C% that uses separately each medicament to produce is respectively 35 and 44.

B.3 anti-angiogenesis activity

To at 0.5ml 0.8%w/v agar, (contain heparin, contain (i) S1P (5 μ M/ chamber) in 20U/ml) or (ii) to implant the nude mice flank subcutaneous in the porous chamber of human body VEGF (1 μ g/ chamber).S1P or VEGF can induce the growth of the blood vessel tissue around chamber.What this reaction was dose dependent also can come quantitatively by measuring tissue weight and blood volume.4-6 hour from transplanting starts, with compd A (0.3,3,30 or 50mg/kg) oral (i) or R-enantiomer (2.5mg/kg) vein (ii) of Compound C or S enantiomer (2.5mg/kg) vein (iii) or carrier (5% glucose of Compound C, 10ml/kg) oral or vein (iv) is processed nude mice, once a day, continue 4 days.Give last dosage after 24 hours, putting to death animal to carry out the mensuration of blood vessel tissue.Measure weight and the blood volume of chamber peripheral vessels tissue.

With the animal by vehicle treated only, compare, the animal of processing through R or the S enantiomer of compd A or Compound C shows that the weight of its blood vessel tissue and/or blood volume lower.

C. clinical trial

C.1 the clinical Benefit of S1P receptor stimulating agent (for example the compound of formula I, II or III, as compd A, B or C) investigation

20 progressive stage late tumor patients to standard treatments drug resistance or difficult control, accepted described compounds for treating to test determined dosage by dosage escalation.Weekly the patient is checked UP with lab testing to investigate its whole body clinical situation.Within every two months, by radioscopy, evaluate the transfer that its tumor changes and loads.The initial treatment of accepting 2 months of patient.After this, as long as not progress and the drug resistance satisfaction of its disease, the patient can continue this treatment.

The major variable of assessment: safety (adverse events), standard serum biochemistry and hematology, computed tomography (CT) or the determined tumor size of NMR (Nuclear Magnetic Resonance)-imaging (MRI).

C.2 therapeutic alliance

Suitable clinical trial is that for example advanced solid tumor patient's open-label derandominzation dosage escalation is tested.Such test has proved the synergism of the active ingredient of combination of the present invention especially.By these result of the tests or by the variation in the known EXPERIMENTAL DESIGN of the technical staff in the technical field, can directly determine its beneficial effect to proliferative disease.Such test is particularly suitable for the curative effect that active ingredient list medication and the present invention's conjoint therapy is used in contrast.Preferably the dosage of the first medicine (a) increases progressively gradually until reach maximum tolerated dose, and accessory drugs (b) is with the fixed dosage administration simultaneously.Perhaps also can select, the first medicine (a) is with the fixed dosage administration, simultaneously accessory drugs (b) dosage escalation.Equal every day of each patient or interruption are accepted medicament (a) treatment.Can determine the treatment curative effect in these trials, for example after 12,18 or 24 weeks, within every 6 weeks, carry out the radiology assessment.

Perhaps also can select, confirm the therapeutic alliance benefit of invention described herein with the double-blind trial of placebo.

When using the S1P receptor stimulating agent separately, in the inventive method enforcement, required every daily dose will change the order of severity of the compound that for example used, host, administering mode and the state of an illness for the treatment of according to following factor.Preferred every day dosage range for approximately from 0.1 to 100mg, single-dose or gradation administration.The suitable every daily dose of patient is for for example 0.1 to 50mg oral.The S1P receptor stimulating agent can be by any conventional route administration, particularly intestinal (for example oral, as the form with tablet, capsule, oral liquid), nasal cavity, lung (by sucking) or the parenteral form of injection or injection suspension (for example with).Suitable oral administration unit dosage form comprises approximately S1P receptor stimulating agent and one or more pharmaceutically useful diluent or its carrier of 0.1 to 30mg (common 0.25 to 30mg).For suppressing angiogenesis, select the S1P receptor stimulating agent of enough high doses very important, because the S1P receptor stimulating agent of low concentration can promote angiogenesis.When giving patient S1P agonist, can test to select to provide by the as above described concentration of A, B and C and dosage escalation the appropriate dose of blood vessel formation against function.

Combination of the present invention also can be combined with operative treatment, the whole body temperature of slight long-time rising and/or irradiation treatment.

Use pharmaceutical composition of the present invention not only produced synergistic therapeutic effect for example (for example with regard to slow down, stop the formation of reversing tumor or extend the response phase of tumor or suppress with regard to angiogenesis) such beneficial effect, but also produced other surprising beneficial effect, for example with only use combination of the present invention in the independent treatment of one of the active constituents of medicine that uses compare, particularly treatment with other as the chemotherapeutics of anticarcinogen during invalid tumor, side effect reduces, quality of life raising or mortality rate and sickness rate reduce.

Beneficial effect on the other hand is the active component that can use than in the combination of the present invention of low dosage, for example dosage not only can be less and also frequency of utilization can be lower, or can be used for reducing the incidence rate of side effect when controlling swollen neoplastic growth.This and the hope that is treated the patient and need to be consistent.

According to one embodiment of the invention, a kind of preferred drug regimen contains

A) compound of formula I, II, III, IVa, IVb, V or VI, for example compd A, B or C, and

B) as one or more above-mentioned paragraphs (ii) of accessory drugs, (iii), (iv), (v), (vii) or the compound (xi), carboplatin for example, cisplatin, paclitaxel, docetaxel, gemcitabine, amycin, compound, bisphosphonates or the mTOR inhibitors of the activity of targeting, reduction or inhibition vascular endothelial growth factor receptor (VEGFR) family's tyrosine kinase or platelet derived growth factor receptor (PDGFR).

Another embodiment of the present invention relates to S1P receptor stimulating agent (a) is combined in treatment lymph or bone marrow cancer (for example as implied above) purposes with chemotherapeutics (b).This combination also can comprise a kind of other accessory drugs (b), for example busulfan, cytosine arabinoside, 6-sulfenyl guanine, fludarabine, hydroxyurea, procarbazine, bleomycin or methotrexate.Topoisomerase II inhibitors (for example daunorubicin), perhaps particularly the compound (for example imatinib) of the activity of targeting, reduction or inhibition PDGFR or c-Abl family member and gene fusion product thereof is better as accessory drugs (b), for example is used for the treatment of lymphatic cancer.

Term used herein " co-administered " or " administering drug combinations " or similar term comprise that the therapeutic agent that will select is to the single patient administration, comprise that medicine wherein is not necessarily with the therapeutic scheme of same route of administration administration or administration simultaneously.

One of purpose of the present invention is to provide a kind of pharmaceutical composition of the combination of the present invention that contains therapeutic alliance proliferative malignant disease effective dose.In said composition, the first medicine a) and accessory drugs b) can be with the unit dosage form of an associating or two independent unit dosage form administrations simultaneously, administration or administration respectively successively.Unit dosage form can be also fixing compositions.

Can prepare in a manner known way by pharmaceutical composition of the present invention, and be those be suitable for enteral (as oral or rectum) and parenteral to mammal (homoiothermic animal), comprise the compositions of people's administration, the coupling component that it contains at least one pharmacological activity as noted above for the treatment of effective dose itself, or contain one or more pharmaceutically useful carrier or diluent simultaneously, particularly be suitable for carrier or diluent that enteral or parenteral are used.

Applicable pharmaceutical composition contains, for example approximately 0.1% to approximately 99.9%, preferred about 1% to 60% active component.For the pharmaceutical preparation of carrying out therapeutic alliance by enteral or parenteral, be the preparation of unit dosage form for example, as sugar coated tablet, tablet, capsule or suppository, or ampoule.If not explanation in addition, prepared by these dosage forms, for example conventional mixing, granulation, sweet tablet, dissolving or freeze-drying in a manner known way.Should be understood that, since can reach required effective dose by a plurality of dosage units of administration, the not pattern of wants of unit content itself effective dose of contained coupling component in the single dosage of each dosage form.

Specifically, the treatment effective dose of each coupling component of the present composition can be simultaneously or with any order administration successively, and each component can be respectively or the combination medicine-feeding to fix.For example, according to the present invention, postponing the development of proliferative malignant disease or the method that it is treated comprises: for example, with therapeutic alliance effective dose, preferred cooperative effective quantity (every daily dose consistent with the amount of describing in literary composition or be interrupted dosage), simultaneously or a) and (ii) the accessory drugs b of free or pharmaceutical acceptable salt of the first medicine of using successively (i) free or pharmaceutical acceptable salt with any order).Each coupling component of combination of the present invention can be in therapeutic process different time administration respectively, or with separately or the administration simultaneously of the form of single compositions.In addition, the term administration also comprises that use can be converted into the prodrug of these coupling components in vivo.Therefore, the present invention is understood to include all these simultaneously or the scheme of alternating treatment, and term " administration " also should be done corresponding explanation.

The effective dose of each coupling component of using in combination of the present invention changes with the order of severity of the specific compound used or pharmaceutical composition, administering mode, the state of an illness for the treatment of, the treatment state of an illness.Therefore, select the dosage of combination of the present invention according to many factors, the kidney liver function that comprises route of administration and patient.Attending doctor, clinicist or veterinary with common skill can promptly determine and specify prevention, stop or control the effective dose of the needed single active component of PD.The concentration of active component the most accurately reached can tell on and do not have in virose scope that need to take active component be basic dosage regimen in the availability kinetics of target spot.

Certainly, the first medicine every daily dose a) is with many factors, the compound of for example selecting, the specific condition for the treatment of and required effect and change.Yet, using and approximately 0.1 to 100mg as every daily dose of single dose or fractionated dose prescription, give the S1P receptor stimulating agent, for example compd A, B or C, can obtain promising result usually.The S1P receptor stimulating agent can be by any conventional route administration, particularly enteral administration, and for example, with the form oral administration of tablet, capsule, oral liquid, or with the form parenteral of for example injection or injection suspension.The unit dosage form that is suitable for oral administration comprises approximately 0.1 to 30mg composition (a) (for example 0.1 to 25mg) and one or more pharmaceutically useful diluent or carriers.

Fadrozole is approximately 0.5 to 10mg/ days for people's oral dose scope, preferably approximately 1 to about 2.5mg/ days.Exemestane is approximately 5 to 200mg/ days for people's oral dose scope, and preferably approximately 10 to about 25mg/ days, or the dosage range of parenteral is approximately 50 to 500mg/ days, preferably approximately 100 to about 250mg/ days.If medicine is with independent pharmaceutical composition administration, can be according to GB2, disclosed mode administration in 177,700.Formestane is approximately 100 to 500mg/ days for people's parenteral dosage range, preferably approximately 250 to about 300mg/ days.Anastrozole is approximately 0.25 to 20mg/ days for people's oral dose scope, preferably approximately 0.5 to about 2.5mg/ days.Aminoglutethimide is approximately 200 to 500mg/ days to the dosage range of people's administration.

TAMOXIFEN CITRATE is approximately 10 to 40mg/ days to the dosage range of people's administration.

Vincaleucoblastine is approximately 1.5 to 10mg/m to the dosage range of people's administration ²my god.Vincristine sulfate is about 0.025 to 0.05mg/kg body weight * jede Woche to the dosage range of people's parenteral.Vinorelbine is approximately 10 to 50mg/m to the dosage range of people's administration ²my god.

Etoposide phosphate is approximately 25 to 115mg/m to the dosage range of people's administration ²my god, as 56.8 or 113.6mg/m ²my god.

Teniposide is that per fortnight is approximately 75 to 150mg to the dosage range of people's administration.Amycin is approximately 10 to 100mg/m to the dosage range of people's administration ²my god, as 25 or 50mg/m ²my god.Epirubicin is approximately 10 to 200mg/m to the dosage range of people's administration ²my god.Idarubicin is approximately 0.5 to 50mg/m to the dosage range of people's administration ²my god.Mitoxantrone is approximately 2.5 to 25mg/m to the dosage range of people's administration ²my god.

The dosage range of Paclitaxel on Human administration is approximately 50 to 300mg/m ²my god.Docetaxel is approximately 25 to 100mg/m to the dosage range of people's administration ²my god.

Cyclophosphamide is approximately 20 to 1500mg/m to the dosage range of people's administration ²my god.Melphalan is approximately 0.5 to 10mg/m to the dosage range of people's administration ²my god.

5-fluorouracil is approximately 50 to 1000mg/m to the dosage range of people's administration ²my god, as 500mg/m ²my god.Capecitabine is approximately 10 to 1000mg/m to the dosage range of people's administration ²my god.Gemcitabine hydrochloride is about 1000mg/m to the dosage range of people's administration ²/ week.

Methotrexate is approximately 5 to 500mg/m to the dosage range of people's administration ²my god.

Hycamtin is approximately 1 to 5mg/m to the dosage range of people's administration ²my god.Irinotecan is approximately 50 to 350mg/m to the dosage range of people's administration ²my god.

Carboplatin is that every four week is approximately 200 to 400mg/m to the dosage range of people's administration ².The dosage range of cisplatin on human administration is that every three week is approximately 25 to 75mg/m ².Oxaliplatin is that per fortnight is approximately 50 to 85mg/m to the dosage range of people's administration ².

Imatinib is approximately 2.5 to 850mg/ days to the dosage range of people's administration, and more preferably 5 to 600mg/ days, and most preferably 20 to 300mg/ days.

Alendronic Acid is approximately 5 to 10mg/m to the dosage range of people's administration ²my god.Clodronic acid is approximately 750 to 1500mg/ days to the dosage range of people's administration.Etidronic acid is approximately 200 to 400mg/ days to the dosage range of people's administration.Ibandronic acid is every three to four stars phase 1-4mg to the dosage range of people's administration.Risedronic acid is approximately 20 to 30mg/ days to the dosage range of people's administration.Pamidronic acid is that every three to four week is approximately 15 to 90mg to the dosage range of people's administration.Tiludronic acid is approximately 200 to 400mg/ days to the dosage range of people's administration.

Trastuzumab is approximately 1 to 4mg/m to the dosage range of people's administration ²/ week.

Bicalutamide is approximately 25 to 50mg/m to the dosage range of people's administration ²/ day.

1-(4-chloroanilino)-4-(4-pyridylmethyl) phthalazines or its salt as succinate to the dosage range of people's administration be approximately 50 to 1500mg/ days, preferably 100 to 750mg/ days, most preferably 250 to 500mg/ days.

Approximately 0.1 to 25mg dosage range administration of rapamycin or derivatives thereof (for example 40-O-(2-ethoxy)-rapamycin).

Formula examples: soft capsule

Formula I compound

Compd A hydrochlorate 30mg for example

Liquid Macrogol 300mg

Polyoxyethylene sorbitan monoleate 20mg

----

Total amount 350mg

For example, under S1P receptor stimulating agent (the S1P receptor stimulating agent that comprises the group of the formula X) dosage required in application of the present invention, toleration is good.For example, in rat and monkey, the acute oral LD of compd A ₅₀for>10mg/kg.

On the other hand, the present invention relates to the purposes of S1P agonist as angiogenesis promoting medicine.Recently, induce new vessels to generate the splendid target position that has been recognized as treatment numerous disease (for example angiogenesis of cardiac muscle, wound healing or diabetic vascular dysfunction/vascular lesion).

As mentioned above, the S1P receptor stimulating agent of high concentration (2 μ M or higher, for example 2-5 μ M or approximately 5 μ M) shows blood vessel formation against function, and the S1P receptor stimulating agent can suppress the angiogenesis that VEGF-induces simultaneously.On the contrary, the S1P receptor stimulating agent of low concentration (0.1-1 μ M, for example 0.1-0.5 μ M or 0.5-1 μ M) has the effect of promoting angiogenesis, can strengthen the angiogenesis that VEGF-induces simultaneously.Therefore, the S1P agonist can have biphasic effect in angiogenesis.

Accordingly, the present invention further provides:

For example, 8.S1P agonist, contain the S1P agonist of the group of formula X, as compd A or compd A phosphate, the purposes in inducing the neovascularity generative process, for example, as the Angiogensis agent, for example promote in the indication of angiogenesis at needs;

9. the preparation method of the medicine of (for example mediating through anti-angiogenesis) disease, the indication that for example needs to promote angiogenesis, for example wound healing or treatment myocardial infarction or the diabetic vascular dysfunction/vascular lesion that is used for the treatment of or prevents to be mediated by inhibition neovascularity generative process, it comprises use S1P receptor stimulating agent, the S1P agonist that for example contains the group of formula X, as compd A or compd A phosphate as sexual element.

10. the method for the treatment of or prevention are mediated by inhibition neovascularity generative process (for example mediating through anti-angiogenesis) disease, the indication that for example needs to promote angiogenesis, for example wound healing or treatment myocardial infarction or diabetic vascular dysfunction/vascular lesion, it comprises the S1P receptor stimulating agent of the individual effective dose of giving this treatment of needs, the S1P agonist that for example contains the group of formula X, as compd A or compd A phosphate.

Be applicable to promote the S1P agonist of angiogenesis to comprise as above about defined those compounds for the treatment of of cancer, for example contain the S1P agonist of group of formula X or the compound of formula I to IX, or its officinal salt or ester.Preferred S1P agonist is compd A phosphate.The S1P agonist can be used alone, or other medicaments that promote angiogenesis with one or more for example VEGF combine use.

For promoting angiogenesis, select the S1P receptor stimulating agent of enough low dosages very important, because the S1P receptor stimulating agent of high concentration can suppress angiogenesis.When the S1P agonist is delivered medicine to the patient, can be by as above the described concentration of A, B and C and dosage escalation test are selected for the suitable S1P agonist dosage that angiogenesispromoting effect is provided.

The accompanying drawing explanation

Fig. 1 demonstration, compd A phosphate can rely on form with bell dosage and promote consumingly blood capillary sample network structure to form, and shows that maximum activity is in 0.5 μ M left and right simultaneously.

Fig. 2 shows, when 0.5-1 μ M, compd A phosphate and compd A all do not weaken the reconstruction that VEGF mediates, but with the polypeptide growth factor co-action.

Fig. 3 demonstration, the tubing string that compd A phosphate and S1P stimulate forms in fact can be by bacillus pertussis extracellular toxin (PTX, 50ng/ml) (a kind of α _i/othe inhibitor of the assorted trimer G albumen of type) suppress fully.This may be interpreted as, and in the biological respinse stimulated at compd A phosphate, may relate to EDG-1 (S1P ₁) signal transduction activity that mediates of receptor.

Fig. 4 shows, when 1 μ M, itself seems can weaken than the sphingol of S1P poor efficiency the ability that S1P and compd A phosphate are induced the blood capillary spline structure, and the tubing string of simultaneously VEGF being induced forms does not have inhibitory action.At this on the one hand, the effect of sphingol performance differs from compd A.Data show, it is of crucial importance via the vascular remodeling/angiogenesis of EDG receptor family for most probable that the balance between sphingol and S1P seems.It is also important that, the sphingol of high concentration and compd A (2-5 μ M) can suppress the tubing string formation that VEGF triggers.

Fig. 5 shows, can cause the of short duration activation of ERK1/2 with the compd A phosphate treated HUVEC of 0.5 μ M, and phosphorylation/activation peak occurred in the time of the 10th minute, after 20 minutes, returns back to baseline values.

Fig. 6 whether detected compd A, compd A phosphate, sphingol or S1P also can be on HUVEC the induced tissue factor.The data of finding show, in these compounds, none can combine the raising tissue factor activity separately or with other compounds, specifically as shown in Figure 6.Compd A and compd A phosphate can slightly strengthen VEGF but not tissue factor activity that TNF α induces.

Fig. 7 has shown that the HUVEC tubing string that Compound C mediates at S1P forms the effect in test.

Abbreviation used is as follows:

BSA: bovine serum albumin

ECGS: endothelial cell growth factor (ECGF) group ECL: enhanced chemiluminescence

S: sphingol PBS: phosphate buffered saline (PBS)

JNK1/2:c-jun-N-end kinases 1/2 RT: room temperature

TF equivalent: tissue factor equivalent

EGR-1/NFAT: early growth reactive protein 1/ nuclear factor of activated T cells

F1P: compd A phosphate (FTY720-phosphate)

The effectiveness of S1P agonist (as S1P agonist of the group that contains formula X) in promoting angiogenesis can be confirmed in for example according to test method hereinafter described.

D. cell culture and material

At 37 ℃, 5%CO ₂, in the M199 culture fluid of supplementary 20%SCS (HyClone, Logan, UT), 1U/ml heparin, 50 μ g/ml ECGS, 2mM glutamine, 100U/ml penicillin and 0.1mg/ml streptomycin, cultivate Human umbilical vein endothelial cells (HUVEC).After maximum 5 generations of going down to posterity by cell for the test.Starve cell 5 hours to obtain the of short duration hungry HUVEC cell that is subject to the M199 that contains 1%SCS.Recombinant human VEGF ₁₆₅available from PromoCell (Heideberg, Germany).Phosphoric acid specificity ERK1/2, p38 kinases polyclonal antibody, without phosphoric acid ERK1/2 antibody and LumiGLO chemiluminescence agent from New England BioLabs (Beverly, MA), polyclone I kappa B antibody is from Santa Cruz Biotechnology (Santa Cruz, Calif).The anti-rabbit immunoglobulin G of the donkey of peroxidase-labelling (Ig G) and the anti-mouse IgG of sheep are purchased from AmershamLIFE SCIENCE (Amersham Place, Britain).The product (Bedford, MA) that the Immobilon-P transfer membrane is Millipore.S is available from Sigma Chemical Co.; S1P is from Biomol.By following scheme, prepared by compd A phosphate storing solution.First compd A phosphate is dissolved in to (0.5mg compd A phosphate adds in the methanol of 2 μ l HCl in 500 μ l) in the methanol that contains the dense HCl of trace.The evaporation solvent in solution that forms in vacuum, and in 0.1% defat BSA the solution (variant 1) in sterile deionized water (500 μ l) or again dissolve resulting residue in the solution in deionized water (variant 2) in 0.5%Triton X-100.By produced storing solution (2.5mM) supersound process and in 4 ℃ of preservations.

Agglutination test

When the 80-90% degrees of fusion by the cell kind in 6 orifice plates and grow overnight.According to Clauss, M., J.Biol.Chem.271,17629-17634 (1996), Mechtcheriakova, D., Blood 93, and the method described in 3811-3823 (1999) is stripped off cell from flat board, and analyzes tissue factor activity.Briefly, after together with VEGF (1.5nM), TNF-α (100U/ml), S (0.5-2 μ M), S1P (0.5-2 μ M), compd A (0.5-2 μ M) and compd A phosphate (0.5-2 μ M), hatching 4 hours, cell is cleaned to twice, and then strip off and move to (12mM sodium acetate, 7mM barbiturates diethylester and 130mM sodium chloride in 1ml coagulation buffer; PH7.4).The blood plasma of the cell of 50 μ l suspendible again and 50 μ l Citrateds is mixed, and with 50 μ l 20mM CaCl ₂solution again after calcification, is measured its coagulation time in 37 ℃.Use is available from the standard curve determination TF equivalent of Medulla Leporis seu Oryctolagi Thromboplastin.

E. western blot analysis

After various processing, cell is cleaned to twice with cold PBS, then cracking digestion in 100 μ l Laemmli buffer, finally strip off and heat 5 minutes in 95 ℃.Use SDS-PAGE separate total cell pyrolysis liquid and be transferred on the Immobilon-P film.By the sealing of the PBS containing 0.1%Tween-20 and 3% defatted milk 30 minutes for film, and under RT, with together with primary antibodie in being diluted in the sealing buffer, hatch 1 hour.Resulting film cleans three times with the PBS that contains 0.1%Tween-20 again, and each 5 minutes, and then with two of peroxidase labelling, hatch 1 hour together with anti-under room temperature.After cleaning, then film is hatched 1 minute together with ECL reagent, and be exposed to as requested film.Use other antibody as wish and again survey, need again film be cleaned to twice with PBS, then in 55 ℃ of uses, strip off buffer (62.5mM Tris-HCl, pH6.8,2%SDS, 100mM 2 mercapto ethanol) strip off 30 minutes, and under room temperature, use again PBS to clean 3 times, each 5 minutes.After each immune detection, film wet covering in 4 ℃, SaranWrap preserved.

Angiogenesis test on external Matrigel

According to manufacturing process, carry out endotheliocyte to the form of blood capillary spline structure on the Matrigel substrate of somatomedin (BD Bioscience) and test having lowered.Briefly, first HUVEC is carried out to trypsin digestion, then suspendible again in the M199 containing the serum-free of soybean trypsin inhibitor (1mg/ml, Sigma).After centrifugal, then by cell in the culture fluid of serum-free with 0.5 * 10 ⁵the density of cell/ml is suspendible again, and again by cell suspension kind (Costar in 96 well culture plates, Corning Incorporated), culture plate is coated with the compd A of S, 0.1-2 μ M of S1P, 0.1-2 μ M of VEGF, 0.1-2 μ M of the 50 μ l Matrigel:1.5nM that contain or do not contain following various stimulus object and the compd A phosphate of 0.1-2 μ M in advance.After 8 hours, with containing the fixing cell on Matrigel of the PBS of 3% formaldehyde, and in 4 ℃ of preservations.Finally use the Nikon Diaphot microscope photographing image with cold CCD camera (Kappa GmbH, Gleichen, Germany), and come in the image quantitative result by the number of branch point in two fields of microscope in each test repeating hole of direct counting.

F. external Matrigel tubing string forms the endothelial cell morphology generation that in test, compd A phosphate is induced and the G that may relate to _ithe signal conducting path of-mediation

Test to measure the impact of compd A and compd A phosphate Human Umbilical Vein Endothelial Cells morphological differentiation with external Matrigel angiogenesis.Endothelial cell morphology is the process of a complexity, it requires the interaction of cell-extracellular matrix and thing followed matrix rebuilding, stimulate divide a word with a hyphen at the end of a line, cell-cell interaction and the hydrolysis of blood vessel peripheral protein.As shown in fig. 1, compd A phosphate can rely on form with bell dosage and promote consumingly the cancellated formation of blood capillary sample, and shows that its maximum activity is positioned at 0.5 μ M left and right.But it is also suitable for compd A phosphate and S1P that the response stimulus thing is induced the number of branch point in each field of microscope of effect, and significantly surpass the effect that VEGF triggers.With compd A phosphate, compare, the compd A of 0.5-1 μ M itself has the faint but effect of strengthen continuously.0.5-1 the compd A phosphate of μ M and compd A all do not weaken the reconstruction that VEGF induces, but with polypeptide growth factor co-action (referring to Fig. 2).In addition, the tubing string that compd A phosphate and S1P stimulate forms can be by bacillus pertussis extracellular toxin (PTX, 50ng/ml) (a kind of α _i/othe inhibitor of the assorted trimer G albumen of type) suppress fully.This may be interpreted as, and in the biological respinse stimulated at compd A phosphate, may relate to EDG-1 (S1P ₁) signal transduction activity (referring to as Fig. 3) that mediates of receptor.When 1 μ M, itself seem can weaken than the sphingol of S1P poor efficiency the ability that S1P and compd A phosphate are induced the blood capillary spline structure, the tubing string of simultaneously VEGF being induced forms does not have inhibitory action (referring to Fig. 4).At this on the one hand, the effect of sphingol performance differs from compd A.Data show, it is of crucial importance via the vascular remodeling/angiogenesis of EDG receptor family for most probable that the balance between sphingol and S1P seems.It is also important that, the S of high concentration and compd A (2-5 μ M) can suppress the tubing string formation that VEGF triggers.This Notes of Key Data, compd A and compd A phosphate can have the dose-dependent effects of two-phase in vitro to angiogenesis.

G. compd A phosphate is to the kinase whose activation of ERK1/2MAP

Signal conduction via map kinase plays an important role in the various kinds of cell function.Can cause the of short duration activation of ERK1/2 with the compd A phosphate treated HUVEC of 0.5 μ M, and phosphorylation/activation peak occurred in the time of the 10th minute, return back to baseline values (referring to Fig. 5) after 20 minutes.P38 kinases and the combined thing A of JNK1/2 phosphate not detected in HUVEC activates.In addition, compd A phosphate can dose-dependent mode trigger the activation of ERK1/2, and at 2 μ M, shows strong active.The result that this and tubing string form test forms contrast, at tubing string, forms in test, and the effect of compd A phosphate when 2 μ M is low during than 0.5 μ M.In the processing dynamics range of 5 minutes to 60 minutes, compd A and S all can not induce map kinase to activate in endotheliocyte.Struvite/NF κ B-dependency the program of wish assessment may act in the biological respinse that the compd A phosphate of endotheliocyte stimulates, can re-start detection with anti-I kappa B antibody by film.I κ B level is not subject to that compd A is parkerized to be affected.In addition, with compd A phosphate treated endotheliocyte, may, as NF κ B dependency secondary response gene, induce E-Selectin to express.Therefore, data show effectively, and the transmission of compd A phosphate signal does not relate to the activation (this is the main cascade reaction in the endotheliocyte acute inflammatory reaction) of NF κ B.

H. compd A and compd A phosphate inducing endothelial cell tissue factor expression not.

An important characteristic attribute of classical inflammation excimer TNF-α and main angiogenic growth factor VEGF Human Umbilical Vein Endothelial Cells raises the potential of tissue factor for it.Compd A, compd A phosphate, S or S1P all detected its whether also can be on HUVEC the induced tissue factor.The data of finding show, in these compounds, none can separately or be combined with other compounds and improves tissue factor activity (referring to Fig. 6).Compd A and compd A phosphate can slightly strengthen VEGF but not tissue factor activity that TNF α induces.The data that obtain also show simultaneously, and compd A, compd A phosphate, S or S1P work with different mechanism from VEGF and the inflammatory TNF-α of angiogenic.

I. can be by the binding affinity of following test determination S1P receptor stimulating agent and human body S1P receptor.Human body S1P receptor is to the transient transfection in the HEK293 cell

Clone's EDG receptor and G _ialbumen, then the EDG receptor G of mixed equal quantities _i-α, G _i-β and G _ithe cDNA of-γ is also used calcium phosphate precipitation method (people such as M.Wigler, Cell.1977; 11; 223 and the people such as DS.Im, Mol.Pharmacol.2000; 57; 753) by it for transfection monolayer HEK293 cell.Briefly, will contain 25 μ g DNA and 0.25M CaCl ₂the DNA mixture add to the 2mM Na of HEPES buffering ₂hPO ₄in.Use the 25mM chloroquine to poison the inferior monolayer HEK293 cell merged, then the DNA precipitate is added to cell.After 4 hours, with PBS and refed culture fluid (90%1: 1 improved basic culture solutions of Dulbecco ' s (DMEM): the F-12+10% hyclone) clean cell monolayer.After adding DNA 48-72 hour, on ice in the HME buffer (mM:20HEPES of unit, the 5MgCl that contain 10% sucrose ₂, 1EDTA, pH 7.4) in strip off and harvesting, and use Dounce homogenizer cell lysis.After centrifugal with 800xg, use sucrose-free HME dilution supernatant, and with 100,000xg centrifugal 1 hour again.By resulting precipitate homogenate again, and with 100,000xg centrifugal 1 hour again.By this, rough membranaceous precipitate is again in containing suspendible, decile specimen again in the HME of sucrose, and immerses quick-freezing in liquid nitrogen.By film in-70 ℃ of storages.By Bradford Protein Detection spectroscopic assay protein concentration.

Use the GTP γ S of S1P receptor/HEK293 film preparation thing in conjunction with detection

As people Mol.Pharmacol.2000 such as DS.Im; 57; The 753 described GTP γ S that carry out are in conjunction with experiment.Use 25 μ g from the film preparation thing of the HEK293 cell of transient transfection at GTP binding buffer liquid (mM:50HEPES of unit, 100NaCl, 10MgCl ₂, pH7.5) the middle combination of measuring ligand-mediated GTP γ S to G-albumen.At 10 μ M GDP and 0.1nM[ ³⁵s] part is added in film and in 30 ℃ and hatches 30 minutes under the existence of GTP γ S (1200Ci/mmol).Use Brandel harvester (Gaithersburg, MD) that the GTP γ S of combination is separated with unconjugated, and use the liquid scintillation counter counting.

Claims

1.S1P the purposes of receptor stimulating agent in the medicine for the preparation of suppressing implanted solid tumor growth, wherein said S1P receptor stimulating agent is selected from 2-amino-2-[2-(4-octyl phenyl) ethyl of free form or pharmaceutical acceptable salt] propane-1, 2-amino-the 4-of 3-glycol and free form or pharmaceutical acceptable salt (4-heptyloxybenzene base)-2-methyl-butanols, condition is, when S1P receptor stimulating agent 2-amino-2-[2-(4-octyl phenyl) ethyl that is free form or pharmaceutical acceptable salt] propane-1, during the 3-glycol, described tumor is not mammary gland, prostate, bladder, the tumor of kidney or lung.

2.S1P receptor stimulating agent for the preparation of suppress or control the medicine of angiogenesis out of control in purposes, wherein said S1P receptor stimulating agent is selected from 2-amino-2-[2-(4-octyl phenyl) ethyl of free form or pharmaceutical acceptable salt] propane-1, the 2-amino-4-of 3-glycol and free form or pharmaceutical acceptable salt (4-heptyloxybenzene base)-2-methyl-butanols.

3. the purposes of claim 2, wherein said angiogenesis is the angiogenesis by the S1P mediation.

4.S1P the purposes of receptor stimulating agent in the medicine for the preparation of prevention or the disease that mediated by the neovascularity generative process for the treatment of or the disease relevant to angiogenesis out of control, wherein said S1P receptor stimulating agent is selected from 2-amino-2-[2-(4-octyl phenyl) ethyl of free form or pharmaceutical acceptable salt] propane-1, the 2-amino-4-of 3-glycol and free form or pharmaceutical acceptable salt (4-heptyloxybenzene base)-2-methyl-butanols.

5.S1P the purposes of receptor stimulating agent in the medicine of the drug resistance for the preparation of overcoming chemotherapeutics, wherein said S1P receptor stimulating agent is selected from 2-amino-2-[2-(4-octyl phenyl) ethyl of free form or pharmaceutical acceptable salt] propane-1, the 2-amino-4-of 3-glycol and free form or pharmaceutical acceptable salt (4-heptyloxybenzene base)-2-methyl-butanols.

6. according to the purposes of any one in claim 1-5, wherein by S1P receptor stimulating agent intermittent administration.

7. according to the purposes of any one in claim 1-5, wherein, by S1P receptor stimulating agent and the second medicine simultaneously or successively co-administered, described the second medicine is chemotherapeutics.

8. a drug regimen, it comprises is a) the first medicine of S1P receptor stimulating agent, wherein said S1P receptor stimulating agent is defined as claim 1, and b) be the accessory drugs of chemotherapeutics.

9. combination according to claim 8, accessory drugs wherein is selected from:

I. aromatase inhibitor,

Ii. antiestrogen, antiandrogen or gonadorelin agonist,

Iii. topoisomerase I inhibitor or Topoisomerase II inhibitors,

Vi. Kallidin I 1 receptor or angiotensin-ii antagonist,

Vii. cyclooxygenase-2 inhibitor, diphosphonic acids, histone deacylase inhibitor, heparinase inhibitor, biological answer-reply dressing agent, ubiquitination inhibitor maybe can be blocked the inhibitor of anti-apoptotic approach,

The carcinogenic isotype inhibitor of viii.Ras,

Ix. telomerase inhibitor,

X. protease inhibitor, matrix metallo-proteinase inhibitor, methionine aminopeptidase inhibitor or albuminous body inhibitor, and/or

The xi.mTOR inhibitor.