CN101355929A - Means and methods for the treatment and prevention of allergic diseases - Google Patents

Abstract

The present invention relates of the use of certain inner ionic (zwitter ionic) phospholipids, phosphonolipids and phosphate derivatives for the preparation of a pharmaceutical composition for the treatment, prevention and/or amelioration of an immunological disorder related to mast cell sensitization and/or activation. Preferred in this context are Edelfosine, Miltefosine and Perifosine. In a particularly preferred embodiment, the present invention relates to the use of Miltefosine for the preparation of a pharmaceutical composition for the treatment, prevention and/or amelioration of allergic diseases, in particular acute hyperallergic diseases, like asthma, atopic dermatitis and mastocytosis.

Description

The mode and the method for treatment and prevention of allergic diseases

The present invention relates to that ion-type (amphoteric ion type) phospholipid in some, phosphoric acid fat (phosphonolipids) are used for the treatment of in preparation with phosphate derivative, prevention and/or improvement and mastocyte sensitization and/or activate purposes in the pharmaceutical composition of relevant immunology disorder.Preferred in this article edelfosine, miltefosine and perifosine.In particularly preferred embodiments, the present invention relates to miltefosine is used for the treatment of, prevents in preparation and/or improve purposes in the pharmaceutical composition of allergic disease, particularly acute high allergic disease such as asthma, atopic dermatitis and mast cell disease.

Atopic diseases accounts for most of medical expense in industrialized country because these diseases be common, persistent and current be incurable.Current treatment to asthma and rhinitis more or less is effectively in most of patient, and the treatment of atopic dermatitis is demonstrated very little effect usually.Anaphylactic shock is considered to available epinephrine and treats, but still at the prophylactic treatment of seeking the susceptible patient.Therefore, need new medicine and get involved and deal with the mastocyte sensitization and/or activate relevant disorder, as sucking the more serious asthma that corticosteroid can not better be controlled by high dose.In addition, also wish to need to all effectively safe dose regimen of all atopic diseaseses, because atopic diseases often takes place together.

About 10% world population is got involved in allergy.In the U.S., in 4,000 ten thousand allergy patients, have an appointment and 9.9 hundred ten thousand suffer from asthma.In addition, 35% population suffers from allergic rhinitis (" pollinosis "), and 15% suffers from urticaria, and 15% suffers from eczema, and 1% suffers from anaphylaxis.Asthma is the modal chronic disease of those people of under-18s.These disorders can cause unemployment and quality of life to be reduced, and can be fatal for asthma and anaphylaxis.

Clinically, asthma is discerned by air flue hyperkinesia and reversibility airway obstruction.Organize that the pathology disorder of level comprises that airway smooth muscle shrinks, vascular permeability increases that (causing the air flue edema), mucus flow out from goblet cell and mucous gland, the parasympathetic nervous system activation, the airway epithelia confluent monolayer cells strips off with inflammatory cell in flow.Early stage asthma reaction is subjected to histamine and other to induce the labrocyte medium to the rapid effect of target organ, particularly smooth muscle to mediate.

Mast cell disease is to be one group of very different disorder of feature with mastocyte abnormal accumulation in different tissues; wherein mastocyte mainly gathers in skin (skin mast cell disease or urticaria such as urticaria pigmentosa) and bone marrow; also gather in spleen, liver, lymph node and gastrointestinal tract, this depends on the character of disease (systemic mast cell disease).They can influence any age arbitrary property others.Mast cell disease is acquired disease normally, but has also described some rare familial cases.

Typical atopic reaction or " anaphylactic type is super quick " (I type) are reflected at the generation in back 15 minutes that interacts of soluble antigen and mastocyte.Pathology relate to mast cell degranulation, and this reaction is by labrocyte medium such as histamine and leukotrienes C4 driving.The example of copy is the urticaria reaction behind the injection penicillin in the penicillin anaphylaxis patient in the body.

Therefore, mast cells activation is the core event in the alterative inflammation, and most of allergic disease is caused by IgE (IgE) mediated hypersensitivity.The anaphylaxis individuality has synthesized IgE in response to foreign substance (being called allergen).IgE antibody is specific to the allergen that causes them.In tissue in IgE antibody and IgE receptors bind on the surface at basophil on the surface of mastocyte and in blood.The multivalence allergen can make the IgE of surface combination crosslinked, causes cell degranulation.This process can cause that pharmacological active substance such as histamine and prostaglandin discharge, and this causes allergic symptoms again in target organ, for example bronchospasm in asthma or the edema in local atopic reaction.Therefore, in the common allergy of major part, the core cell is a mastocyte, and core element is IgE.

Mastocyte is the special hematopoietic cell that originates from ancestral stem cell in the bone marrow, and they are by secreting a large amount of different chemical media by the stores location in its granule after the stimulation and playing an important role in anaphylactic type (I type) allergy and inflammatory reaction.Mastocyte be characterized as them not only on tissue site and the structure but also the heterogeneity on function and histochemistry level.Inflammatory disorderly as allergy and asthma in, mastocyte number and allergia respond positive correlation between the serious symptom verified mastocyte number increase in illing tissue.In having the systemic mast cell disease that multiple organ participates in,, in tissue, formed mast cells infiltration by around the folliculus of the portal area of liver, spleen, around the blood vessel of skin or the mastocyte group in the lymph gland sinus.Suffered among the patient of systemic mast cell disease and observed hepatomegaly and splenomegaly 50%.

The mastocyte effect of knowing most is the atopic reaction that mediation IgE triggers.Mastocyte and circulation homologue basophilic leukocyte thereof have the high-affinity receptor for IgE that is called Fc ε RI, and it can be crosslinked after in conjunction with IgE and stimulate and discharge multiple biologically active medium such as histamine, Dan Baijutang, protease, 5-hydroxy tryptamine.The incident that causes immediate hypersensitivity in mastocyte and basophilic leukocyte is that antigen combines at cell surface with the receptor that is combined with IgE.In addition, the crosslinked of IgE receptor also can cause the synthetic of prostaglandin, leukotriene and cytokine and discharge.Fc ε RI γ-chain contains immunity receptor tyrosine and activates motif (ITAM), and the signal transduction path that originates from Fc ε RI is similar to by activation and contains those that other immunity receptor of ITAM produces.In brief, after Fc ε RI was crosslinked, src family kinase lyn made the syk kinases to its ITAM phosphorylation of raising.Syk is activated, and makes multiple downstream, particularly kytoplasm target protein phosphorylation.

Mastocyte can also be activated by the mechanism except that crosslinked Fc ε RI, for example in response to the deutero-chemical cytokine of mononuclear phagocyte, the cell-derived cytokine of T and the deutero-anaphylatoxin of complement.Mastocyte can also be by other inflammatory cell or the neurotransmitter that is used for being connected with nervous system replenish and activate.After the activation, mastocyte discharges multiple medium, and these media can cause that blood vessel infiltration increase, vasodilation, bronchus and visceral smooth muscle shrink and local inflammation.In being called the extreme form of anaphylactoid immediate hypersensitivity, the medium that is discharged by mastocyte can suffocate airway limitation a little.The so-called atopic individuals that is easy to take place strong immediate hypersensitivity response can suffer from asthma, pollinosis or chronic eczema.The blood plasma IgE level that these individualities have is higher than average blood plasma IgE level.The cell-stimulating of antigen induction can stimulate by the anti-IgE of multivalence or by anti-Fc ε RI antibody.This antibody-like can activate mastocyte in atopy and ergotropy individuality, and allergen only activates mastocyte in the atopy people.

In mast cell disease, the symptomatic release of mast cell granule can be triggered by following factor: emotionally disturbed, heating, fatigue, physical stimulation (hot, cold, friction, motion, daylight), contact ethanol, medicine such as aspirin, opiate, anticholinergic, NSAID (non-steroidal anti-inflammatory drug) (aspirin), anesthetis, anesthetics, antibiotic, bacteriotoxin, insecticide, virus, antibacterial or fungal infection, mycete, venom, biological polypeptide (Lobster, crayfish, Jellyfish), some food, food coloring agent or correctives, antiseptic or spice.

Allergy medicine should stop in grain fraction such as the histamine release from mast cells discharge or the blocking-up tissue to the response of histamine.Most of current available medicine is to reduce the antihistaminic (histamine H-1 receptor antagonist) of histamine to the effect of tissue by the blocking histamine receptor.They do not stop the generation or the release of the histamine that can cause multiple but not all allergy symptoms.

Multiple inflammatory mediator except that histamine such as leukotriene and multiple vasoactive cytokine are also discharged by mastocyte and basophilic leukocyte.These pro-inflammatory mediators are not influenced by antihistaminic, and they play an important role to the pathophysiology of allergy and asthma.Sometimes the combination of anti-allergic drug and anti-inflammatory agent can be worked better, but these combinations simultaneously can cause bad side effect.In more serious atopic reaction (anaphylaxis), antihistaminic does not have therapeutical effect.In mast cell disease, the urticarial conventional therapy of the disposition of checking colors is relative nullity.Antihistaminic can alleviate some symptoms, but aspirin and codeine can make mast cell degranulation and can increase the weight of symptom.

After deliberation the influence of some ether lipid and ether phospholipid to the release of histamine from isolating rat hypertrophy cell.According to application conditions, find that in these chemical compounds some can promote or suppress the histamine release that antigen causes.Carrying out these researchs is in order to stimulate the activated hypothesis of macrophage function to study the toxic mechanism of antineoplastic ether phospholipid to cancerous cell according to ether phospholipid.Therefore, these chemical compounds may use as treatment of cancer has been discussed.Referring to: Grosman, Immunopharmacology1990,20,143-149; Grosman, Immunopharmacology 1999,44,211-221; Grosman, Inflamm.Res., supplementary issue I 2002, S05-S06.But the data that provided by Grosman are not conclusive, have described the height variation of immunomodulating in possible treatment of cancer, and this depends on experiment setting.

The chemical compound that specificity reduces mast cell degranulation is considered to hypotoxic, because the exocytosis of being regulated is not the extremely important function of mastocyte, this makes them can be used for participating in the others of immunologic function.In the people, the exocytosis of mastocyte has very little effect or does not have effect the people who lives in the developed country.The immunity of IgE mediation demonstrates to develop into mainly works in resisting parasite infestation.It is rare that this class infects in Western society, has observed the part that mast cell degranulation almost exclusively reacts as the dysfunction allergic now.Therefore, blocking-up mast cell degranulation itself can stop alterative inflammation effectively and seldom undesired consequences is arranged.

Tested of the effect of a limited number of pharmacology material to mast cells activation.Now this class " mast cell stabilizers " of using comprise sodium cromoglicate (

) or ketotifen (

-ketotifen,

), nedocromil and lodoxamide.Sodium cromoglicate is to find by experience to reduce the excretory broad-based medicine of mastocyte, think that now it works by making the chloride channel inactivation, but the usefulness that it has is low.Tested of the inhibition of several novel substances in recent years to mast cell degranulation.Example comprises tryptase inhibitors (people such as He, J.Pharmacol.Exp.Ther.2004,309 (1), 119-126), chymase inhibitor (people such as He, J.Pharmacol.Exp.Ther.1999,291,517-523) and idandones (Yi Daen ketone) (people such as Frankish, J.Pharm.Pharmacol.2004,56,1423-1427).Though some have demonstrated inspirer result, do not have in these materials a kind ofly to be approved for clinically, whether they will make it enter clinical stage actually is quite unclear.

Because current available treatment has above-mentioned shortcoming, resists disorder relevant with mastocyte such as allergy so need therapeutic agent in this field always with method.

According to the present invention, by the chemical compound that following formula I is provided be used for the treatment of in preparation, prevention and/or improvement and mastocyte sensitization and/or activate the solution that purposes in the pharmaceutical composition of relevant immunology disorder can obtain this technical problem:

Wherein:

R ¹Be the C that comprises quaternary nitrogen atoms _4-13Alkyl;

X is O or direct bond;

R ²Be C _10-20Alkyl, wherein one or more hydrogen are optional to be replaced and wherein one or more CH by fluorine ₂Group is optional to be replaced by oxygen, perhaps R ²Be the group of Formula Il:

Y is O, O (CO), S or S (CO);

R ³Be OH, C _1-4Alkyl, O-C _1-3Alkyl, O (CO) NH-C _1-3Alkyl, O (CO)-C _1-6Alkyl, S (CO)-C _1-6Alkyl, O (CO)-C _2-3Alkenyl or CH ₂O-C _1-3Alkyl;

R ³' be H or C _1-4Alkyl; And

R ⁴Be C _10-20Alkyl, wherein one or more hydrogen are optional to be replaced by fluorine.

The lipid bilayer that forms cell membrane is two-dimentional liquid, and it is organized is the object that biochemist and biophysicist many decades are furtherd investigate.Though thought that double-deck integral body is homogeneous liquid, but attempted repeatedly with horizontal heterogeneous lipid micro structure territory be incorporated into we the structure of double-deck liquid and kinetic model in (Glaser, Curr.Opin.Struct.Biol.3 (1993), 475-481; Jacobson, Comments Mol.Cell Biophys.8 (1992), 1-144; Jain, Adv.LipidRes.15 (1977), 1-60; Winchil, Curr.Opin.Struct.Biol.3 (1993), 482-488).

Epithelial cell is polarised to its cell surface in the top and substrate outboard structure territory that has different protein and lipid composition in each these domain, this understanding has caused (lipidraft) new development (Simons of notion of generation " Lipid Rafts ", Biochemistry 27 (1988), 6197-6202; Simons, Nature 387 (1997), 569-572).Withstood detergent and extract and be suggested by observing these assemblages as the notion of the assemblage (assemblies) of the sphingolipid of memebrane protein platform and cholesterol, they are called anti-detergent film (DRM), and (Brown, Cell 68 (1992), 533-544).This is wherein to cut down contact to be equal to 4 ℃ of operability breakthroughs to the opposing of Triton-X100 extraction.The introducing of second criterion promptly uses methyl-beta-schardinger dextrin-to make cholesterol exhaust (Ilangumaran, Biochem.J.335 (1998), 433-440; Scheiffele, EMBO be (1997) J.16,5501-5508) caused losing of anti-detergent character, impel several groups in this field to probe into the effect of lipid micro structure territory in the biological respinse of vast scope.Now the lipid assemblage there is increasing support in adjusting various kinds of cell process, the effect in cell polarity, protein transportation and the signal transduction that comprises.

Cell membrane is two-dimentional liquid.Therefore, horizontal heterogeneity means the liquid-liquid unmixability in membrane plane.Well-known, the function experience that the lipid bilayer of hydration can be used as temperature changes mutually.These transformations that various lipids take place under set point of temperature always comprise some variations of system order.The most important thing is during these change that so-called " master " or " chain is molten " changes, double-deck accurate two-dimentional crystalline solid from high-sequential transforms the two-dimentional liquid that is as the criterion in this transformation.It comprises in the acute variation of system order, particularly double layer planar displacement (position) order and on planar direction perpendicular to this acute variation of the conformation order of lipid chain.The displacement order is relevant with the transverse diffusion coeficient in the membrane plane, and the conformation order is relevant with the trans/gauche form ratio in the acyl chain.The main transformation has been described in order to unordered transformation mutually, so that this biphase orderly solid (s that can be called as below the transition temperature _o) and the above unordered liquid (l of this temperature _d).Cholesterol and phospholipid can form the unordered liquid (l that can lack with cholesterol _d) mutually the coexistence orderly liquid (l _o) phase, make thus and coexist as (Ipsen, Biochem.Biophys.Acta 905 (1987) 162-172 in the whole liquid phase film mutually; Ipsen, Biophys.J.56 (1989), 661-667).Because sterol has flat rigid molecule structure, so that's how things stand for they, when sterol during contiguous chain, this structure can be given contiguous aliphatic chain with conformation ordering (Sankaram, Biochemistry 29 (1990), 10676-10684), and can not make the displacement mobility of lipid that the corresponding (Nielsen that significantly reduces is arranged, Phys.Rev.E.Stat.Phys.Plasmas FluidsRelat.Interdiscip.Topics 59 (1999), 5790-5803).Because sterol is not in strict conformity with s _oThe fact of the lattice of (gel) lipid bilayer phase, so if it this mutually in dissolving, then it will destroy crystallization displacement order, still can significantly not disturb the conformation order.Therefore, suitably the cholesterol of molfraction can be with l _dOr s _oThe lipid bilayer inversion of phases is orderly liquid (l _o) phase.

Lipid Rafts is the lipid platform (being rich in sphingomyelins and cholesterol in the outer little page or leaf of cell membrane) of special chemical composition, and its effect is that the membrane component in the cell membrane is separated.Raft can be understood that it is relatively little (diameter is 30-50nm, and according to used probe and the cell type of being analyzed, its big or small estimated value alters a great deal), but under certain conditions they can in conjunction with.They have shown the behavior that is separated in heterogeneous model membrane systems at the particularity aspect the lipid composition.In fact, they are at many character aspect chemical composition and the detergent dissolubility and similar (the de Almeida of viewed situation in the model system of being made up of the ternary mixture of unsaturated phosphatidylcholine, sphingomyelins (or long-chain saturated phospholipid phatidylcholine) and cholesterol, Biophys.J.85 (2003), 2406-2416).Raft can be considered to be in the l in the heterogeneous l phase lipid bilayer that comprises plasma membrane _oThe domain of phase.Other coexisting phase is what is not clear at present.Consensus is that biomembrane is a liquid, so for most applications, such s _oCoexistence can be left in the basket mutually.Other is l mutually _dOr l _oTo depend on the chemical characteristic of the phospholipid of forming this phase (these phases) and the molfraction of cholesterol wherein mutually.Raft can be counted as orderly liquid phase, and the remainder of film is called non-raft liquid phase.In thermodynamic (al) framework, be by a large amount of molecular macroscopic systems all the time mutually.But, in lipid bilayer, trending towards usually mutually by cracked to minor structure territory (only being several thousand molecules usually), they itself do not have the molecule of enough numbers strictly to meet the thermodynamic definitions of phase separately.Therefore, orderly liquid raft comprises all domains (little or accumulative domain) of raft phase in the film mutually.The remainder (liquid phase) that surrounds the film of raft can be homogeneous percolate bulk phase or can further be subdivided into the liquid structure territory that is not also characterized.

(J.Cell.Biol. (2000) 148 for Pralle, 997-1008) adopt photon force microscope (photonicforce microscopy) to measure the size of Lipid Rafts, find that the raft in fibroblastic plasma membrane is diffused as the assemblage of 50nm diameter, being equivalent to surface area is covered by about 3,000 sphingolipids.Data according to from young hamster kidney (BHK) cell of cultivating (its lipid is formed and the organelle surface area is studied in great detail) show that the surface area that individual cells has is about 2,000 μ m ²The lipid composition of cytoplasma membrane contains 26% phosphatidylcholine, 24% sphingomyelins and 12% glycosyl sphingolipid.Because the asymmetric character of lipid tissue in the plasma membrane, most of sphingolipid occupy double-deck outer little page or leaf, and the phosphatidylcholine of having estimated fewer than half is in this little page.

Suppose that most of sphingolipid is that raft is bonded, then raft can cover cell surface over half.The density of having estimated memebrane protein is about 20,000 molecules/μ m ²Therefore, plasma membrane can correspondingly contain and has an appointment 40 * 10 ⁶Individual protein molecule.The number of 50-nm raft will be about 10 ⁶Individual, if the protein density in the raft is identical with protein density in the bilayer on every side, then raft can have about 20 protein molecules separately.If bhk cell is representational, then thereby conclude that the density of buoyant raft in the fibroblast plasma membrane is high.If 20 * 10 ⁶Individual raft protein molecule is more or less by random distribution, and then raft may contain proteinic different subgroup separately.Therefore, the kinases that is connected with the kytoplasm lobule of raft can not be met its substrate in same single raft.The small size of single raft can be important for the signal transduction protein with the raft load remains on " closing " state.Therefore, in order to activate with generation, many rafts have to flock together, and form bigger platform, and in this platform, the protein partner in the signal transduction process can meet and do not disturbed by the outside activity that takes place of platform.Therefore, raft is little, and when being activated, they assemble the bigger platform of formation, and relevant albumen can interact on the function in this platform.

Analyzing raft combination and accumulative a kind of method is by specific antibody raft and non-raft component to be mended (Harder, J Cell Biol.141 (1998), 929-942 on the surface that is connected on living cells; Janes, Semin.Immunol.12 (2000), 23-34).If two kinds of raft components are by antibody linked, then they will form eclipsed patch (patches) in plasma membrane.But the benefit of raft protein and non-raft label such as TfR connects and can cause forming isolating patch.Two kinds of raft components are mended to be connected together to depend on simultaneously two kinds of antibody are joined in the cell.If add antibody successively, then isolating patch is in the great majority.It should be noted that it is cholesterol-dependent that benefit connects behavior.Because the small size of single raft and heterogeneous the composition, these structures must flock together in special mode, if the signal conduction takes place subsequently.

An example of the such raft accumulation process that runs in daily clinical practice is the conduction of IgE signal (Sheets, Curr.Opin.Chem.Biol.3 (1999), the 95-99 in allergia immune response process; Holowka, Semin.Immunol.13 (2001), 99-105).By stimulating mastocyte or basophil to take off allergen that granule causes atopic reaction is multivalent in conjunction with several IgE antibody molecules.Two or more IgE receptors (Fc ε RI) crosslinked increases itself and the combining of raft, as measuring by the anti-detergent that increases.In raft, crosslinked Fc ε RI becomes by the tyrosine of the bonded Lyn of raft (two acidylate Src associated kinase) institute's phosphorylation.Fc ε RI phosphorylation can be recruited tyrosine kinase Syk, and the latter is activated, thereby makes downstream signal conduction and the acidify of support molecular phosphorus, finally causes forming signal conduction platform.This structure comprises raft protein LAT (junctional complex of t cell activation), it the gathering of other raft can be directed in the platform of expansion (Rivera, Int.Arch.AllergyImmunol.124 (2001), 137-141).Signal conduction can cause the calcium mobilization, and the latter can trigger preformed medium such as histamine and store the storehouse in cell and discharge.The partner that is gathered in the raft platform is many more, and signal conduction response is high more.The amplification possibility triggered activity of assembling the out-of-control signal cascade amplification of carrying out by raft is strong excessively, produces life-threatening consequence such as elder brother gram (Quinke) edema and allergic shock.Whole signal conduction assemblage can be dissociated by dephosphorylation or by component regulated downwards by endocytosis institute's internalization (Xu, J.Cell Sci.111 (1998), 2385-2396).Therefore, in IgE receptor signal conduction, Lipid Rafts is used for by concentrate liquid micro structure territory and limit it and otherly increase effectiveness to diffusion so that protein remains on the position of signal conduction of protein that will participate in and lipid.Even deleterious toning (overshoot) be amplified or be caused to the little variation that is assigned in the Lipid Rafts just can the priming signal cascade by amplification, as (Kholodenko taking place in the atopic reaction, Trends CellBiol.10 (2000), 173-178).

In the context of the present invention, unexpectedly find: ion-type phospholipid, phosphoric acid fat and phosphate derivative such as edelfosine, miltefosine, Perisfosine (perifosine), Ilmeofosine (ilmofosine), 1-O-palmityl-2-O-methyl-sn-glyceryl-3-phosphocholine and 1-O-palmityl-2-O-ethyl-sn-glyceryl-3-phosphocholine are effective inhibitor of mast cell degranulation or are used as mast cell stabilizers in some.Especially, unexpectedly find as chemical compound disclosed herein can be used for the treatment of in treatment, prevention and/or improvement and mastocyte sensitization and/or activate relevant disorder, the particularly relevant immunology disorder with mast cell degranulation.

Therefore, the chemical compound that the invention provides following formula I be used for the treatment of in preparation, prevention and/or improvement and mastocyte sensitization and/or activate purposes in the pharmaceutical composition of relevant immunology disorder:

R ¹Be the C that comprises quaternary nitrogen atoms _4-13Alkyl.Preferred R ¹Be the group one of among the Formula Il Ia to IIIc:

n ¹Be integer 1 to 7, preferred 2 to 6, more preferably n ¹Be 2.n ²Be integer 1 or 2.

X is O or direct bond.Preferred X is O.

In an embodiment, R ²Be C _10-20Alkyl, wherein one or more hydrogen are optional to be replaced by fluorine and wherein one or more (preferably one or two) CH ₂Group is optional to be replaced by oxygen.Preferred R ²Be the C that comprises the two keys of one or more (for example two, three or four) _10-20Alkyl or C _10-20Alkyl.In preferred embodiments, R ²Be C _10-20Alkylidene or C _10-20Alkyl.More preferably R ²Be C _12-18Alkyl.

In another embodiment, R ²Be the group of Formula Il:

Y is O, O (CO), S, S (CO).Preferred Y is O.

R ³Be OH, C _1-4Alkyl, O-C _1-3Alkyl, O (CO) NH-C _1-3Alkyl, O (CO)-C _1-6Alkyl, S (CO)-C _1-6Alkyl, O (CO)-C _2-3Alkenyl or CH ₂O-C _1-3Alkyl.In an embodiment, R ³Be OH, O-C _1-3Alkyl, O (CO) NH-C _1-3Alkyl, O (CO)-C _1-6Alkyl, S (CO)-C _1-6Alkyl, O (CO)-C _2-3Alkenyl or CH ₂O-C _1-3Alkyl.Preferred R ³Be O (C _1-2Alkyl) or OCONHCH ₃O (CO)-C _1-6Alkyl is another preferred R ³Group.More preferably R ³Be O (C _1-2Alkyl).

R ³' be H or C _1-4Alkyl, preferred H or CH ₃In an embodiment, R ³' be H.

R ⁴Be C _10-20Alkyl, wherein one or more hydrogen are optional to be replaced by fluorine.Preferred R ⁴Be the C that comprises the two keys of one or more (for example two, three or four) _10-20Alkyl or C _10-20Alkyl.More preferably R ⁴Be C _12-18Alkyl.

In another embodiment, R ²Be the group of following formula I V or following formula V:

Be used to represent singly-bound or two key.

R ⁵Be selected from H and C independently of one another _1-3Alkyl.Preferably in the group of formula IV or V, a R ⁵Be H, and another R ⁵Be CH ₃

R ⁶Be C _9-15Alkyl, wherein one or more hydrogen are optional to be replaced by fluorine.Preferred R ⁶Be C _11-13Alkyl.

The all possible stereoisomer and the diastereomer of chemical compound shown in the general formula that provides among the present invention is intended to contain.In the chemical compound that comprises formula II group, the preferred spatial chemistry that in naturally occurring phosphoglyceride, extensively exists.In the chemical compound of the group that comprises general formula I V or V, the preferred spatial chemistry that in naturally occurring sphingol, exists.

Table 1 has shown chemical compound 1 to 21 and 23 to 28, and they are preferred examples of formula I chemical compound.

Table 1

In chemical compound 1 to 21 and 23 to 28, chemical compound 1,2,3,4,5 and 6 is preferred one group of chemical compound.Chemical compound 9,17 and 23 to 27 also is preferred.Chemical compound the 1,2,3,4, the 5th, even preferred one group of chemical compound.Chemical compound 9,23 and 24 also is even preferred one group of chemical compound.In particularly preferred embodiments, formula I chemical compound is miltefosine (chemical compound 3).

Can commercially available acquisition as shown in table 1 or can prepare according to chemical compound used in the present invention by standard method known in the art.

(X is O and R to belong to the chemical compound of the general formula I of phospholipid ²Be formula II group), for example alkoxyl phospholipid (Y is O) and corresponding alkylthio group derivant (Y is S) can be according to document (Bittman, R.; J.Med.Chem.1997,40,1391-1395; Reddy, K.C.; Tetrahedron Lett.1994,35,2679-2682; Guivisdalsky, P.N.; J.Med.Chem.1990,33,2614-2621 and the list of references of wherein quoting) described in method prepare or the standard alternative by wherein said method prepares.The synthetic of corresponding ester and thioesters analog (Y is respectively OCO and SCO) can be finished by the standard acidylate of hydroxyl or sulfenyl precursor substance.

(X is direct bond and R to belong to the chemical compound of the general formula I of phosphoric acid lipid ²Be formula II group), (Y is O and R to for example alkoxyl phosphoric acid fat ²Be formula II group) and alkylthio group derivant (Y is S) can be according to people such as Bittman (Bittman, R. accordingly; J.Med.Chem.1993,36,297-299; Bittman, R.; J.Med.Chem.1994,37,425-430 and the list of references of wherein quoting) disclosed method prepares or the synthetic alternative by wherein said method prepares.The synthetic of corresponding ester and thioesters analog (Y is OCO or SCO) can be finished by the standard acidylate of hydroxyl or sulfenyl precursor substance.

Introduce the different substituent R that contains quaternary nitrogen ¹So that phosphocholine part (R to be provided ¹Be formula III a group and n ¹Being 2) method of or derivatives thereof is at document (Reddy, K.C.; Tetrahedron Lett.1994,35,2679-2682 and the list of references of wherein quoting) in extensively described and can be begun to finish by suitable glycerol derivatives.

Using alcohol to replace to obtain corresponding amphoteric ion type phosphate derivative (R in the similar synthesis strategy of glycerol derivatives ²Be C _10-20Hydrocarbon).

Other substituent R of Cai Yonging in the context of the present invention ¹, particularly have a group (n of different phosphate-nitrogen distance ¹Be not 2) or have nitrogenous heterocyclic group (R ¹Be formula III b or IIIc group) can use document (Eibl, H.; Chem.Phys.Lipids 1988,47,63-68; Pajouhesh, H.; J.LipidRes.1984,25,294-303; Diembeck, W.; Chem.Phys.Lipids 1979,24,237-244; Duclos, R.; J.Med.Chem.1994,37,4147-4154; Ohno, M.; Chem.Pharm.Bull.1985,33,572-582; Krise, J.P.; J.Med.Chem.1999,42,3094-3100) middle scheme and the strategy of describing provides.

R wherein ²The chemical compound that is the general formula I of formula IV or V group can be by above to introducing different substituent R ¹And R ²Scheme of mentioning and document (Merrill, A.H.; Method in the zymetology (Methodsin Enzymology), the 311st volume, academic press (Academic Press), 1999; Koskinen, P.M.; Synthesis 1998,1075; Yamanori, T.; The synthetic combination of the scheme of the synthetic sphingolipid of describing Chem.Lett.1989,335) obtains.

Without being limited by theory, can use interior ion-type phospholipid, phosphoric acid fat and phosphate derivative as described herein destroy raft and: 1) disturb transhipment and the gathering of Fc ε RI at cell surface, 2) transhipment and the gathering of disturbing raft to be undertaken by LAT (junctional complex of t cell activation) at cell surface.Chemical compound as herein described provides positive findings in the test (taking off the granule test) based on cell, this test is the test that test can be used for the material of immunity and autoimmune sexual disorder.

Purposes in the pharmaceutical composition of the immunology disorder of therefore, the invention provides that interior ion-type phospholipid, phosphoric acid fat and phosphate derivative as described herein are used for the treatment of in preparation, prevention and/or improvement are relevant with mastocyte sensitization and/or activation, particularly mast cell degranulation.

In the context of the present invention, the mastocyte sensitization comprises that IgE combines with mastocyte and/or undertaken crosslinkedly by bonded IgE by antigen, and it is sometimes referred to as mast cells activation.The immunology disorder that purposes of the present invention is particularly suitable for method treating, prevent and/or improvement is relevant with mast cells activation.

The disorder for the treatment of, preventing or improve comprises acute allergic disease, allergic disorder and/or alterative inflammation especially in the context of the present invention.Autoimmune disease and the response of high allergia also should be treated.Particularly asthma and other immune disease can be by using as compounds for treating disclosed herein.

The example of the allergic disorder of being treated has graft versus host disease or transplant rejection in the context of the present invention.

Graft versus host disease in the context of the present invention is to cause the syndrome that antagonism takes place in the time of can not repelling its host's (because this host is jejune or immunocompromised host or repressed (for example by radiate or medicine)) immune response in immunity when the allogeneic mortifier that particularly contains immunologically competent cell.But purposes of the present invention and method are not limited to improve allograft rejection, and are usually directed to improve in transplanting/the medical science intervention.

Term " transplanting " preferably relates to autograft, allograft, syngenetic graft, isograft or xenotransplantation as used herein, also relates to homotransplantation.These transplanting are well-known in the art, and they not only relate to the transplanting of cell, and relate to the transplanting of tissue and organ.The transplanting of cell also comprises the transplanting of stem cell.As used herein term " homotransplantation " relate to by with the receiver with kind but have the transplanting of the tissue that the another kind of animal health of different genotype obtains, and/or from the tissue transplantation of a kind of genotypic donor to the genotypic host of another kind, wherein host and donor are the members with kind.Host and donor are called as " allochthonous " in this respect.Term " transplanting " also comprises " isograft " or " isograft ", promptly from body identical twins/compatriot's on individuality of the same race identical on the hereditism such as hereditism transplanting one by one.In the non-human animal, these " isografts " also can be carried out on transgenic animal.

The example of the allergic disease of being treated has asthma, allergic rhinitis (pollinosis), erythema damage, atopic eczema and systemic anaphylaxis such as anaphylactic shock in the context of the present invention.Urticaria and mast cell disease are included in the erythema damage of being treated.Urticarial instantiation comprises cholinergic urticaria, dermatographia (dermagraphism), cold urticaria, solar urticaria, aquagenic urticaria, the urticaria relevant with medicine and the relevant urticaria with toxin.

Therefore, the present invention and purposes provided herein and the method medical science that can be used in particular for mast cell disease and/or the symptom relevant with mast cell disease gets involved and urticaria.Mast cell disease and urticaria can be triggered or based on following factor by following factor: emotionally disturbed (for example " Blushing "), heating, fatigue and the physical stimulation that defines as mentioned.Therefore, material provided herein and chemical compound; edelfosine particularly; miltefosine; ilmofosine; 1-O-palmityl-2-O-methyl-sn-glyceryl-3-phosphocholine can also be used for preventative medical science intervention or be used for curative therapy with 1-O-palmityl-2-O-ethyl-sn-glyceryl-3-phosphocholine: contact described physical stimulation or contact toxin; medicine and/or aggressive species such as ethanol; medicine such as aspirin; opiate; anticholinergic; NSAID (non-steroidal anti-inflammatory drug) (aspirin); anesthetis; anesthetics; antibiotic; bacteriotoxin; insecticide; virus; antibacterial or fungal infection; mycete; venom; biological polypeptide (Lobster; crayfish; Jellyfish); some food; food coloring agent or correctives; antiseptic; spice and (radiation) contrast agent.Chemical compound as herein described, promptly in ion-type phospholipid, phosphoric acid fat and phosphate derivative also can be used for the disorder of IgE mediation, for example atopy antigen sensibilization such as pollen, food, medicine, anthelmintic etc.Therefore, chemical compound provided herein can be used in particular for prevention, improve and/or treatment allergy, allergic disorder and atopic reaction.

Ion-type phospholipid, phosphoric acid fat and phosphate derivative can be used as chemical compound itself in as the purposes of pharmacophore or pharmaceutical composition at it in as herein described, perhaps can be formulated into medicine.Pharmaceutical composition can be chosen wantonly and comprise pharmaceutically acceptable excipient, for example carrier, diluent, filler, disintegrating agent, lubricant, binding agent, coloring agent, pigment, stabilizing agent, antiseptic or antioxidant.

Pharmaceutical composition can be by technology well known by persons skilled in the art, for example disclosed technology is prepared in " Lei Shi pharmaceutical science " (Remington ' s Pharmaceutical Sciences, the 20th edition).Pharmaceutical composition can be formulated into and be used for the outer dosage form as intramuscular, intravenous, subcutaneous, intradermal (infradermal), intra-arterial, rectum, nose, part or vaginal application of oral, gastrointestinal tract.Be used for Orally administered dosage form and comprise coating and not tablet, Gelseal, hard-gelatin capsules, lozenge, lozenge, solution, Emulsion, suspensoid, syrup, elixir, the powder that is used for reconstruct and granule, dispersible powder and granule, pastille natural gum, chewable tablet and the effervescent tablet of coating.Be used for powder and granule that dosage form that gastrointestinal tract uses comprises solution, Emulsion, suspensoid, dispersion liquid and is used for reconstruct outward.Emulsion is preferably to be used for the dosage form that gastrointestinal tract is used outward.The dosage form that is used for rectum and vaginal application comprises suppository and avette dose (ovula).Being used for dosage form that nose uses can be by sucking and being blown into, for example using by metered-dose inhaler.The dosage form that is used for local application comprises ointment, gel, ointment, ointment agent (salves), paster and transdermal delivery.

The officinal salt of the chemical compound that can use in the present invention can form with different organic acid and mineral acid and alkali.The exemplary acids addition salts comprises acetate, adipate, alginate, Ascorbate, benzoate, benzene sulfonate, disulfate, borate, butyrate, citrate, camphorate, camsilate, cyclopentane propionate, digluconate, lauryl sulfate, esilate, fumarate, gluceptate, glycerophosphate, Hemisulphate, enanthate, caproate, hydrochlorate, hydrobromate, hydriodate, the 2-isethionate, lactate, maleate, mesylate, the 2-naphthalene sulfonate, nicotinate, nitrate, oxalates, pectate, persulfate, 3-phenylbenzimidazole sulfonic acid salt, phosphate, picate (pik hydrochlorate), Pivalate, propionate, Salicylate, sulfate, sulfonate, tartrate, rhodanate, toluene fulfonate such as toluene fulfonate, hendecane hydrochlorate etc.The exemplary alkali addition salts comprises: ammonium salt, alkali metal salt such as sodium, lithium and potassium salt; Alkali salt, for example calcium and magnesium salt; With the salt of organic base (for example organic amine) as benzazethine (benzyl star), dicyclohexylamine, hydrabine (Ha Bin), N-methyl D-glycosamine, N-methyl D-glucamide (glucamide), tert-butylamine; Salt with aminoacid such as arginine, lysine etc.

The acceptable solvent thing of the chemical compound that can use in the present invention can be to exist with the solvate of water formation such as the form of hydrate, perhaps the solvate forms with organic solvent such as methanol, ethanol or acetonitrile formation exists, promptly respectively as methylate, alcoholate or second nitrile compound.

The pharmaceutically acceptable prodrug of the chemical compound that can use in the present invention be have chemically or in the metabolism cleavable group and by solvolysis or under physiological condition, become the derivant of the chemical compound of the present invention that has pharmaceutical active in vivo.The prodrug of the chemical compound that can use in the present invention can be according to conventional method with the functional group of chemical compound, for example form with amino or hydroxyl.Solubility, histocompatibility can be provided in mammalian organism the prodrug derivant form usually or the advantage that postpones to discharge (referring to Bundgaard, H., the design of prodrug (Design of Prodrugs), the 7-9 page or leaf, the 21-24 page or leaf, like to think only your (Elsevier) company, Amsterdam, 1985).

These pharmaceutical compositions as herein described can be applied to the curee with appropriate dosage.Dosage will be determined by participating in doctor and clinical factor.Well-known in medical domain, arbitrary patient's dosage depends on multiple factor, comprises patient's volume, body surface area, age, the particular compound of being used, sex, the time of using and approach, general health and the other medicines of using simultaneously.Usually, should be in the scope of 0,1 μ g to 5000mg unit/day as the scheme of conventional drug administration compositions, in some embodiments, this scheme was 0.1 μ g to 1000mg unit/day.If scheme is a continuous infusion, then it can also be respectively 0.1ng to 10 μ g unit/kg body weight/minute scope in.Can monitor progress by periodic evaluation.

In the context of the present invention, preferred following local and systemic administration mode.

The topical application anticipation of chemical compound as herein described is used to alleviate indication such as skin mast cell disease, psoriasis, atopic dermatitis, eczema and other dermatosis and the skin disorder relevant with mastocyte, and suitable topical application is the form of other preparation of ointment, ointment, gel, foam, solution, lotion, Emulsion, spray, liposome or micelle suspensoid or penetrable skin outer layer.The exemplary suitable concentration of active component in the topical application preparation is 0.1% to 2%w/w, so that per 100 gram ointments, ointment, gel, foam, solution, lotion, Emulsion, spray, liposome or micelle suspensoid or other preparation contain 0.1g to 2g active component.The other suitable concentration of active component in the topical application preparation is 3% to 6%w/w, and more preferred but more suitable preparation in addition is 7 to 15%w/w.But it is within the technical ability of person skilled that this class concentration is changed.The exemplary dosage regimen that is applicable to the patient of experience treatment can be by participating in the doctor according to determining such as the factor of patient's age, sex, body weight and general health.The selection of treatment concentration and dosage regimen will depend on indication.Be applicable to that the dosage regimen anticipation of topical formulations is used for the treatment of any zone of skin, preferably treats 1 to 2 time every day, also suits but treat 3 to 6 every day.For each application, should cover affected areas fully by applied film.The treatment persistent period is that each treatment cycle is between 1 day to 6 weeks.

For alleviating indication such as systemic mast cell disease, having urticaria, allergic rhinitis, asthma, chronic obstructive pulmonary disease (COPD), irritable bowel syndrome and other general relevant with mastocyte of resistance disorderly for histamine, suitable general application also is considered in this article and describes.It can be the form of rapid release or slow releasing preparation that such general is used, for example capsule, tablet, coated pellets, matrix type preparation (matrix), liposome or micelle or be suitable for other preparation of oral, intranasal, subcutaneous or intramuscular administration.The exemplary suitable concentration that active component is used in the preparation in general is 1mg to 200mg/ tablet, capsule, piller etc.In addition, be applicable to that the dosage regimen that experiences the patient who treats can be by participating in the doctor according to determining such as the factor of patient's age, sex, body weight and general health.The selection of treatment concentration and dosage regimen will depend on indication.The optimal dose of active component in extensive range, preferably about 1 between about 250 microgram/kilograms (μ g/kg) receiver body weight/treatment.Another kind of appropriate dosage can be about 1 to the scope of about 100 μ g/kg body weight, more preferably about 10 to the scope of about 50 μ g/kg body weight.Dosage can be used to reach 1 day to 6 months or reached and be considered to the time necessary and safety every day, and this can come easily to determine by code test according to the disorderly character of treat by participating in the doctor.In addition, the technical staff can change scheme provided herein.

The present invention also provides treatment, improvement or prevention with the mastocyte sensitization and/or activate relevant disorder or the method for disease.Corresponding disease/disorder above is provided, and corresponding, useful interior ion-type phospholipid, phosphoric acid fat and the phosphate derivative that are applied to the patient who needs described improvement, treats and/or prevents also above are being disclosed and are being characterized in appended examples and claim.In most preferred setting, chemical compound as herein described is by being used to these Therapeutic Method with described compound administration in the curee of this treatment of needs, particularly people curee.

Term " treatment " is used for ordinary representation in this article and obtains required pharmacology and/or physiological action.Effect can be preventative according to prevent disease wholly or in part or its symptom, and/or the ill effect that causes according to cure diseases partially or completely and/or by this disease and can be curative.Term " treatment " is encompassed in any treatment of the disease of mammal, particularly philtrum as used herein, comprising: but (a) prevent disease generation in the curee of susceptible disease; (b) suppress disease, promptly stop its development; Or (c) palliate a disease, even disease disappears.

" patient " or " curee " that be used for purpose of the present invention comprises people and other animal, particularly mammal and other organism.Therefore, method can be applicable to human therapy and veterinary applications.In preferred embodiments, the patient is a mammal, and in the most preferred embodiment, the patient is the people.

The present invention is illustrated by following non-limiting figure and embodiment.

Fig. 1 to 8 has shown that the dose dependent of 1 to 6 pair of mast cell degranulation of chemical compound of comparing with cromoglicic acid with the ketotifen fumarate suppresses.

Fig. 9 and 10 has shown the inhibitory action of miltefosine to the histamine release that caused by anti-people-IgE and ionophore A23187 respectively in the C57 cell.

Figure 11 and 12 has shown the inhibitory action of miltefosine to the histamine release that caused by anti-people-IgE and P material respectively in the former generation people mastocyte.

Figure 13 has shown the inhibitory action of miltefosine to the histamine release that caused by C5a or anti-people-IgE in people's basophilic leukocyte.

Figure 14 shown in miltefosine and the pretreated skin of placebo carry out prick test with histamine or patient-specific allergen after at the welt diameter of different time points.

Embodiment 1: the mast cell degranulation test

Mastocyte is to use high widely atopic reaction or asthmatic model system.On its surface, they express the high-affinity receptor (Fc ε RI) for IgE.After antigen-specific IgE combination, recipient cell becomes to antigen (allergen) sensitivity.When sensitized cell ran into polyvalent antigen, the gathering of IgE-Fc ε RI complex initiation finally caused degranulated cell cascade of events,, discharged inflammation and cell-stimulating medium, for example cytokine, eicosanoid, histamine and enzyme that is.It is that raft is dependent that several steps are arranged in this cascade, and for example the Fc ε RI that triggers of antigen is to the relocating of raft, at the offing normal of the destruction of the signal conduction complex of LAT fitted around and/or phosphoinositide, Ca ²⁺-Nei stream (the raft location of plasma membrane calcium channel), film fluctuation (relating to the cytoskeleton reorganization of Akt/WASP/FAK) and exocytosis.Therefore, this test can be as differentiating the raft modulating compound, particularly can be used for the screening method of chemical compound of the medical response of asthma.

1. foreword

This test determination the release of β-hexosaminidase, β-hexosaminidase is various preformed pharmacology substance responds in high-affinity IgE receptor (Fc ε RI) by gathering that polyvalent antigen-the IgE complex carries out and d/d label.With resisting-sensitization of DNP specific IgE, DNP-BSA excites with multivalence with rat basophilic leukemia (RBL-2H3) cell (mast cell degranulation model commonly used).By fluorogenic substrate 4-methyl umbrella base (=umbelliferyl)-N-acetyl group-β-D-glucosamine glycosides by enzymatic conversion be N-acetyl group-β-D-glycosamine and the height fluorescence methyl umbelliferone measure the release of β-hexosaminidase in the liquid of upper strata, at Tecan Safire ^TMCome quantitatively by fluoroscopic examination in the plate reader.

2. material

Chemical reagent and special reagent

Surfact-Amps X-100 solution is obtained by Pierre Si (Pierce) company; DNP-bovine albumin conjugate (DNP-BSA) and 4-methyl umbrella base-N-acetyl group-β-D-glucosamine glycosides (MUG) are obtained by Ka Er Biochemics Inc.; the single ethylethers of three (ethylene glycol) (TEGME) are obtained by Aldrich (Aldrich) chemical company, and DMSO Hybri-Max and people DNP-albumin are obtained by Sigma company.Rat anti-DNP IgE monoclonal antibody is obtained by Biot company (Biozol).Remove hyclone (FCS) from the PAA laboratory (

Germany) outside, all cells culture medium, buffer agent and supplement are obtained by hero (Invitrogen) company.Other reagent is standard laboratory quality or more high-quality.

If not explanation in addition, other chemical reagent is standard laboratory rank or higher level.

Buffer and solution

Phosphate buffered saline (PBS) (PBS) and 1M HEPES are provided by the indoor service facility.Tai Luodeshi buffer (Tyrode ' s buffer) (TyB) comprises and is supplemented with 2mM GlutaMAX ^TM-I supplement (hero company) and 10mM HEPES, do not contain phenol red minimum essential medium (hero company).The dissolving buffer comprises 25mM TrisHCl (pH7.5), 150mM NaCl, 5mM EDTA and 1% (w/v) Triton X-100.(Millipore water) is dissolved to 1mg/ml with people DNP-BSA with millipore water.The MUG substrate solution is 2.5mM 4-methyl umbrella base-N-acetyl group-β-D-glucosamine glycosides, 0.05M citrate (pH4.5), and stop bath is 0.1M NaHCO ₃/ 0.1M Na ₂CO ₃(pH10).

Cell culture

At 5%CO ₂With 37 ℃ under will be supplemented with 2mM GlutaMAX by the RBL-2H3 cell that Germany microbial preservation center (Braunschweig, Germany) obtains ^TMKeep in 70% minimum essential medium of-I (hyclone that contains E Ershi (Earle) salt/20%RPMI 1640/10% heat-deactivation), and carry out routine examination to confirm no mycoplasma contamination.Will be at 175cm ²The cell of growth is with 0.05% trypsin/EDTA cracking and be resuspended in the 20ml fresh culture in the bottle.100 and 50 μ l cell suspensions are layered on 24 holes troop in every hole of plate (cluster plates) (Ka Xida (Costar) company, Schiphol-Rijk, Holland), cell used behind bed board respectively in 1 or 2 day.

3. the mensuration that discharges of β-hexosaminidase

Method

Hatching preceding 2 to 24 hours with test-compound, remove culture medium, with 0.4 μ g/ml anti--DNPIgE makes cell sensitization in fresh culture.After the sensitization, cell with warm TyB washing 1 time, is the test-compound of 100 μ M or the highest non-toxic concn (the carrier total concentration transfers to 1%) with the Cmax in TyB or hatched 60 minutes with 1% carrier in 37 ℃.Add DNP-HSA (0.1 μ g/ml final concentration) or buffer separately, cell was hatched 15 minutes in 37 ℃.In 4 ℃ under 250 * g with centrifugal 5 minutes of plate, transfer in the ice immediately.Collect upper strata liquid, cell is dissolved with the dissolving buffer.By 25 μ l aliquots and 100 μ l MUG substrate solutions being hatched 30 minutes, measure the hexosaminidase activity in upper strata liquid and the solute in 96 orifice plates in 37 ℃.Add 150 μ l stop baths and make reaction terminating.At Tecan Safire ^TMUnder the setting of 365nm excitation wavelength and 440nm emission wavelength, measured fluorescence in the plate reader.

Result of the test quantitatively

Independently measured each chemical compound in the experiment at least 3 times, duplicate.Use following formula deducting the release that β-hexosaminidase is calculated in non-specific release (not adding the release under the antigen) back:

% takes off granule=100 * RFU _{Upper strata liquid}/ RFU _Solute

Following calculating β-hexosaminidase discharges the inhibition with respect to contrast:

% suppresses=100 * (1-(RFU _{The upper strata liquid of chemical compound}/ RFU _{The upper strata liquid of contrast}))

Will be from the taking off the value that granule suppresses and average of independent experiment, these values can be accepted when standard deviation (SD)≤15%.

Table 2 has shown the result who obtains in the mast cell degranulation test.

The inhibition of table 2. mast cell degranulation

ID ₅₀: the concentration when reaching for 50% maximum the inhibition.

CD ₅₀: the concentration when reaching the release of 50% maximum lactic acid dehydrogenase in the cell toxicity test (Pu Luomaige (Promega) company, Cytotox-One catalog number (Cat.No.) 67891).

N.d.: do not carry out.

Table 2 has been listed usefulness (ID50) and maximum suppress and cytotoxicity (CD50) in film integrality test (Pu Luomaige company, Cytotox-One, catalog number (Cat.No.) 67891) of chemical compound 1 to 28 in the mast cell degranulation activity.Take off granule suppress be 50% or bigger and therapeutic index (CD50/ID50) be 10 or bigger being considered to the exploitation of medicaments derivative is correlated with.Therefore, closely-related chemical compound 1 and 2 (2-O-methyl PAF C-16 and 2-O-ethyl PAF C-16) and chemical compound 9,23 and 24 have similar usefulness and maximum inhibition is active, but the cytotoxicity of chemical compound 9 is less.Chemical compound 3,4 and 5 (miltefosine, ilmofosine and edelfosine) and chemical compound 17,25,26 and 27 have similar maximum activity, but their usefulness is lower, and the cytotoxicity of edelfosine is higher.Chemical compound 6,7,8 (mcPAF C-16, crotonyl PAF C-16 and pyrrolidino PAF C-16) and 28 usefulness have only 15 to 40% of chemical compound 1,2,9,23 and 24.Chemical compound 6 and 9 toxicity are low, so their possible therapeutic index are more than 10.All the other chemical compounds are in the inhibition that receives greatly most all not obtain under the amount of reagent more than 50%.

Chemical compound 1 to 5,9,17 and 23 to 27 demonstrates the inhibition more than 75% under 10 or 25 μ M, therefore they are particularly preferred in the context of the present invention.Chemical compound 6 demonstrates the inhibition more than 75% under 50 μ M, therefore it also is preferred in the context of the present invention.Though chemical compound 7,8,11 to 14,16,20 and 28 does not have the active high of chemical compound 1 to 6,9,17 and 23 to 27, they have still demonstrated activity in the mast cell degranulation test, so they can be used for context of the present invention aptly.In order to compare purpose chemical compound 22 is tested, it has provided relatively poor result in the mast cell degranulation test.

Show that the active Fig. 1 to 8 of the inhibition of chemical compound in mast cell degranulation further illustrates the result of exemplary compounds.This test is an industry standard of measuring the mastocyte stabilizing active of possible antiallergic compounds.As can be seen, chemical compound 1 to 6 has demonstrated good usefulness (ID50) (Fig. 1 to 6) under low micro-molar concentration in this experiment, and two kinds of mast cell stabilizers (ketotifen fumarate and cromoglicic acid) that go through and use clinically do not have activity or low activity (Fig. 7 and 8) is only arranged.

Embodiment 2: the activation and the medium that suppress the application on human skin mastocyte in vitro and in vivo discharge

Experimental design

External and isolated experiment:

In order to determine whether miltefosine can discharge at the activation and the medium of vitro inhibition application on human skin mastocyte, and we have used following technology and material and experimental technique:

The preparation of miltefosine storing solution

Prepared miltefosine storing solution (5mM) with DMSO, in-20 ℃ of preservations.For preparation work concentration (2mM), storing solution is diluted with PAG-CM (PAG-CM=contains the PIPES albumin glucose of calcium and magnesium).The 40%DMSO that other dilution of all of miltefosine all is used in the PAG-CM buffer prepares.

The separation of people's basophilic leukocyte and purification

Use Fei Keer Parker (Ficoll Paque) (D=1.077) from whole blood, to separate people's basophilic leukocyte.In brief, the heparinization whole blood is diluted with 1: 3 with PBS (w/o Ca and Mg), at Fei Keer Parker solution higher slice, under 400 * g centrifugal 30 minutes.From the interface between blood plasma and ficoll solution, reclaim low-density particles such as lymphocyte, mononuclear cell, basophilic leukocyte and platelet.Will be under 200 * g centrifugal 10 minutes from the cell suspension at interface, amount to 2 times, from the basophilic leukocyte part, to remove blood platelet.Measured the number of basophilic leukocyte in the cell suspension by Toluidine blue staining.The productive rate of basophilic leukocyte is about 2%.These in advance the basophilic leukocyte of purification be used for histamine release test.For the release of cytokines test, use " basophilic leukocyte separating kit " that basophilic leukocyte has been carried out being further purified to eliminate non-basophilic leukocyte from the cell suspension of interface.For this purpose, cell suspension was hatched 30 minutes with the bonded mixtures of antibodies of hapten (containing CD3, CD7, CD14, CD5, CD16, CD36 and CD45RA), contact with the magnetic bead that is coated with hapten specificity antibody then in the first step.Separate and the bonded cell of magnetic bead with AutoMACS.The negative part of this separation scheme contains purity greater than 95% basophilic leukocyte.

Former generation the skin mastocyte separation and purification

From the application on human skin that the cosmetic surgery process, obtains, isolate tissue mast cell.The use of application on human skin is carried out according to Helsinki criterion statement (Declaration of Helsinki Principles), and by Charit é-

Berlin institutional review board (Institutional ReviewBoard of the Charit é- Berlin) approval.In order to separate mastocyte, by in 4 ℃ of Bacillus polymyxa Neutral proteinase overnight incubation, thereby separated epidermis through enzyme process with 1mg/ml concentration.To remain corium disperses by hatching 1 hour in 37 ℃ of mixture with collagenase I and hyaluronidase.Disperse to repeat 3 times, with the cell washing of collecting.Then, at 37 ℃ and 5%CO ₂In being supplemented with basic Iscar literary composition (Iscove) culture medium of 10% hyclone, glutamine, penicillin, streptomycin and monothioglycerol, cell culture is spent the night under/95% air.By from adherent cell, separate the non-adhesive mastocyte with cold PBS cyclic washing.For the histamine release test, the cell of collecting is suspended among the PAG-CM, use the CD117 microballon (the beautiful day Ni biotech company (Miltenyi Biotech) of Germany) of directed anti-Kit receptor (CD 117) that affinity has been carried out purification.In brief, in 4 ℃ hypodermal cell and CD117 magnetic bead were hatched 30 minutes.By making cell pass through the AutoMACS system from unlabelled cell the cell of separation marking.Record final mastocyte purity＞90% by Toluidine blue staining.The viablity of mastocyte is by trypan blue dyeing (people such as Gr ü tzkau, 2000; People such as Artuc, 2002) be evaluated as＞95%.

Histamine release test (HRA)

Use automatic histamine analyzer system (Burger water technology company (Firm Borgwelt Technik)) quantitative histamine levels.Excitation wavelength is 355-360nm, and fluorimetric emission wavelength is 450-460nm.Fluorescence intensity is directly proportional with the concentration of histamine in the sample.Calculated the amount of the histamine that discharges according to following formula: clean histamine release=(release-blank) * (100/ is whole).Herein, " release " for example is defined as the upper strata liquid by anti--IgE stimulated cells, and the upper strata liquid of unprovoked cell, is that spontaneous release is called " blank "." all " represents total histamine content of mastocyte (behind the high chloro acid dissolution).In the PAG-CM buffer system, carried out the histamine release test.Be used for each the sample mastocyte of histamine release test or the number of basophilic leukocyte and be about 1 * 10 ⁴Individual cell.In order to stimulate, cell is contacted with anti--IgE, P material, Ca-ionophore or C5a.

Hatch in advance and irritation cell with miltefosine

With cell with PBS w/o Ca, Mg washing 2 times, with PAG-CM washing 1 time, under 250 * g centrifugal 10 minutes then.The cell precipitation thing is suspended among the warm in advance PAG-CM (37 ℃).Cell suspension is distributed in 18 Falcon pipes, carries out 6 kinds of different disposal and 3 incubation periods (10 minutes, 30 minutes or 60 minutes) separately:

1-PAG-CM，

2-DMSO/PAG-CM，

3-5 μ M miltefosine,

4-10 μ M miltefosine,

5-20 μ M miltefosine,

6-25 μ M miltefosine.

Be incubated in the tepidarium (37 ℃) and carry out.

The aliquot of the cell suspension of hatching is in advance joined in the pipe that has prepared, hatches 30 minutes to stimulate in 37 ℃:

Pipe 1=2% perchloric acid,

Pipe 2=PAG-CM,

Pipe 3=resists-IgE or P material or Ca-ionophore.

Make reaction terminating with cold PAG-CM buffer, under 4 ℃ and 250 * g with centrifugal 10 minutes of sample.Upper strata liquid is poured into the special cup that is used for measuring histamine.In order to measure cytokine or arachidonic acid metabolite, after hatching in advance with the miltefosine of variable concentrations, cell suspension is divided into 2 parts of aliquots, have or nonreactive-IgE in the presence of hatch 30 minutes (in 37 ℃), under 250 * g centrifugal 10 minutes then.Preserve upper strata liquid to be used in-80 ℃ with the post analysis arachidonic acid metabolite.Cell partly is resuspended in basic Iscar literary composition (Iscove) culture medium (being supplemented with 10% hyclone, glutamine, penicillin, streptomycin and monothioglycerol), cultivated 24 hours in 37 ℃ then.Then, shift out upper strata liquid, in-80 ℃ of preservations (being used to analyze TNF α).

Experiment in the body:

For the evidence of the idea that suppresses IgE-dependency people mastocyte stimulation and medium release in the miltefosine body is provided, we adopt the selected allergia volunteer (n=5) who suffers from known I type sensitization to study.Known allergen with the volunteer has carried out standard prick test (table 3) to them on two forearms and with positive (histamine) and negative (saline) contrast.Use double blinding placebo method, each experimenter's a forearm has been carried out pretreatment with local miltefosine (6% solution).Offside has all carried out pretreatment preceding 2 hours of prick test with placebo solution (saline).Subsequently, with high-quality standardized method, be the development that macroscopical standard prick test evaluation people such as (, 2005) Heinzerling, stereometry imaging, thermograph and digital time lapse photography (DTLP) have been measured skin symptom.The known volunteer who suffers from permanent serious disease, especially influences immune disease gets rid of from research.Begin all not oral any histamine of volunteer or leukotriene antagonist in preceding 7 days in research.And they all do not use the oral or depot formulation of any corticosteroid or other immunosuppressant yet in preceding 21 days of test.

Vertical diameter by measuring welt and erythema zone and quadrature footpath macroscopic evaluation the process of damage (welt and erythema zone) development.Subsequently, with the summation of two kinds of diameters divided by 2.In addition, the official hour point has carried out stereometry imaging and thermograph imaging and DTLP before prick test and behind prick test.

Use special thermograph photographing unit, the thermograph imaging has detected the radiation in the infra-red range of electromagnetic spectrum.All targets all can be sent infrared radiation according to its temperature.The radiating amount of being sent by target increases with temperature.Therefore, but the variation of thermograph imaging detected temperatures.Because the development of prick test damage and disappear and be attended by variations in temperature and the thermograph imaging can be distinguished even little variations in temperature (0.1 ℃) is so the thermograph imaging is the high precision method that the prick test reaction is manifested.Behind prick test, carry out the thermograph imaging and reach 1 hour (FLIRThermaCAM S60, FLIR System Co., Ltd (FLIT Systems GmbH), Frankfurt/Main, Germany) with 5 minutes interval.

Carried out digital time lapse photography (DTLP) and gone up perceptible prick test damage to monitor macroscopic view.Carry out digital photographing with 5 minutes intervals and reach 1 hour, the skin area that influenced by the prick test reaction that used plane determination and analysis system-computed.

Use new volume determination system (Primos contact, close this technology company limited of GFM (GFM Messtechnik GmbH), Teltow, Germany) to carry out the stereometry analysis of prick test damage.This system can measure the swelling of the prick test damage relevant with skin surface.Stereometry has been carried out at interval with 5 minutes in the process of thermograph imaging and digital time lapse photography.

Table 3-allergia volunteer's overview

Volunteer's numbering	Sex	Age	Allergen	The miltefosine pretreatment	Placebo (NaCl) pretreatment
						1	The man	55	The grass mixture	Right arm	Left arm
2	The man	30	The grass mixture	Left arm	Right arm
						3	The man	40	Derm.pteroniss. (Pi Te Nice)	Right arm	Left arm
4	The woman	26	Cat hair	Right arm	Left arm
						5	The woman	33	Derm.pteron. (Pi Te Nice)	Right arm	Left arm

The result

External and isolated experiment:

Miltefosine is to the effect of histamine release in the C57 cell

The C57 mouse hypertrophy cell of the α chain transfection of personnel selection high-affinity IgE receptor ties up to demonstrating after anti-people-IgE stimulation and takes off particle response reliably.The C57 cell is hatched the inhibition that has caused histamine release in advance with the miltefosine of variable concentrations.This inhibition is dosage and time dependence (Fig. 9).After hatching 60 minutes in advance, obtained maximum inhibition (40%) with miltefosine.Miltefosine not only suppresses the histamine release that the IgE receptor relies in the C57 cell, but also suppresses the histamine release (Figure 10) that the Ca-ionophore causes.

Miltefosine dose dependent ground suppresses the histamine release that the Ca-ionophore causes, the maximum inhibition concentration (Figure 10) that 25 μ M representative is tested.Different incubation times has in advance caused remarkable different antagonism-IgE inhibitory action (Fig. 9 and 10) that rely on or the histamine release that the Ca-ionophore causes.

Miltefosine is to the effect of histamine release in people's mastocyte

Miltefosine dose dependent ground suppresses the histamine release that IgE causes in people's mastocyte, and its maximum effect is at 25 μ M places.The inhibitory action of miltefosine in people's mastocyte do not rely on incubation time in advance, promptly in addition short incubation time in advance just can produce maximum inhibitory action (35%) (Figure 11).

The inhibitory action of the histamine release that miltefosine causes P material (SP) is similar to the effect of the release that antagonism-IgE causes.Miltefosine dose dependent ground suppresses histamine release (45%), but for the histamine release that IgE causes, inhibitory action does not rely on incubation time (Figure 12) in advance.

Miltefosine is to the effect of histamine release in people's basophilic leukocyte

That miltefosine dose dependent ground suppresses is anti-in the basophilic leukocyte-histamine release that IgE causes, but should effect very little.Therefore, use 25 μ M miltefosines maximum inhibitory action after hatching 10 minutes in advance just to reach 10%.Compare with the effect of seeing after anti--IgE stimulation, we observe 25 μ M miltefosines and have suppressed the histamine release that C5a causes (75%) strongly and significantly (Figure 13).

Experiment in the body:

In order to estimate the evolution of prick test damage, measure diameter by the different time points behind prick test and estimated the welt size on a macro scale.Interesting is, compares with the pretreated control zone of placebo (saline), and the size of the welt that is caused by allergen in the pretreated zone of 6%-miltefosine solution is significantly less.In addition, compare with the control arm of saline treatment, the contrast welt that histamine causes on the pretreated arm of miltefosine also has been reduced, though the welt that its degree causes less than allergen (table 4 and Fig. 7).

Table 4-carries out the welt analysis of macroscopic evaluation by the different time points behind prick test in miltefosine and the pretreated skin of placebo

Discuss

In fact, with the mastocyte diseases associated in all treatment get involved and all concentrate on by blocking H with hydryllin ₁-histamine receptor suppresses the process of histamine-mediated.Suppressing mast cell degranulation is the another kind of interesting method of the control symptom relevant with other labrocyte medium with histamine.But, up to the present, also do not identify and have special and the effective material of mastocyte stability property.We at first prove herein: miltefosine can suppress the activation of mastocyte and basophilic leukocyte and take off granule, carries out Local treatment with 6% miltefosine solution and can suppress the stimulation of IgE dependency people mastocyte in vivo.It should be noted that most, miltefosine external mastocyte Stabilization demonstrate be not limited to that the IgE dependency activates and body in inhibitory action be intensive, promptly this to act among all other curees except that one be detectable, and be significant, promptly be better than the desired effect of addressing up to now of any mast cell stabilizers (for example cromoglycate).Interesting is, inhibitory action also is detectable in the body of miltefosine in the welt that histamine causes forms, though its degree is less, this shows that miltefosine can block other short scorching approach.

Claims (22)

1. The chemical compound of following formula I be used for the treatment of in preparation, prevention and/or improvement and mastocyte sensitization and/or activate purposes in the pharmaceutical composition of relevant immunology disorder:

   Wherein:

   R ¹Be the C that comprises quaternary nitrogen atoms _4-13Alkyl;

   X is O or direct bond;

   R ²Be C _10-20Alkyl, wherein one or more hydrogen are optional to be replaced and wherein one or more CH by fluorine ₂Group is optional to be replaced by oxygen, perhaps R ²Be the group of Formula Il:

   

   Y is O, O (CO), S or S (CO);

   R ³Be OH, C _1-4Alkyl, O-C _1-3Alkyl, O (CO) NH-C _1-3Alkyl, O (CO)-C _1-6Alkyl, S (CO)-C _1-6Alkyl, O (CO)-C _2-3Alkenyl or CH ₂0-C _1-3Alkyl;

   R ³' be H or C _1-4Alkyl; And

   R ⁴Be C _10-20Alkyl, wherein one or more hydrogen are optional to be replaced by fluorine.
2. 2. according to the purposes of claim 1, R wherein ¹Be the group one of among the Formula Il Ia to IIIc:

   

   N wherein ¹Be integer 1 to 7 and n ²Be integer 1 or 2.
3. 3. according to the purposes of claim 1 or 2, wherein X is O.
4. 4. according to each purposes of claim 1 to 3, wherein R ²Be C _10-20Alkyl, wherein one or more hydrogen are optional to be replaced and wherein one or more CH by fluorine ₂Group is optional to be replaced by oxygen.
5. 5. according to the purposes of claim 4, R wherein ²Be C _12-18Alkyl.
6. 6. according to each purposes of claim 1 to 3, wherein R ²Be formula II group and X, Y, R ³, R ³' and R ⁴As defined in claim 1.
7. 7. according to the purposes of claim 5, wherein Y is O.
8. 8. according to the purposes of claim 6 or 7, R wherein ³Be O-C _1-2Alkyl.
9. 9. according to each purposes of claim 6 to 8, wherein R ⁴Be C _12-18Alkyl.
10. 10. according to the purposes of claim 1, its Chinese style I chemical compound is selected from edelfosine, miltefosine, perifosine, ilmofosine, 1-O-palmityl-2-O-methyl-sn-glyceryl-3-phosphocholine and 1-O-palmityl-2-O-ethyl-sn-glyceryl-3-phosphocholine.
11. 11. according to the purposes of claim 10, its Chinese style I chemical compound is a miltefosine.
12. 12. according to each purposes of claim 1 to 11, wherein immunology is disorderly relevant with mast cell degranulation.
13. 13. according to each purposes of claim 1 to 12, wherein the immunology disorder is acute allergic disease, allergic disorder or alterative inflammation.
14. 14. according to the purposes of claim 13, wherein the immunology disorder is selected from renal fibrosis, hepatic fibrosis and pulmonary fibrosis, scleroderma, multiple neurofibromatosis, interstitial cystitis, irritable bowel syndrome, chronic obstructive pulmonary disease (COPD), food allergy and Cyclical vomiting syndrome.
15. 15. according to the purposes of claim 13, wherein allergic disease is selected from asthma, allergic rhinitis (pollinosis), erythema damage, atopic eczema, systemic anaphylaxis, neurodermatitis and atopic dermatitis.
16. 16. according to the purposes of claim 15, wherein the erythema damage is selected from urticaria, mast cell disease and lupus erythematosus.
17. 17. according to the purposes of claim 16, wherein said urticaria is selected from cholinergic urticaria, dermatographia, cold urticaria, solar urticaria, aquagenic urticaria, the urticaria relevant with medicine and the relevant urticaria with toxin.
18. 18. according to the purposes of claim 13, wherein said allergic disorder is graft versus host disease or transplant rejection.
19. 19. treatment, prevention and/or improvement and mastocyte sensitization and/or activate the method for relevant immunology disorder, wherein said method comprise with pharmaceutically active dosage as claim 1 to 11 in each defined compound administration in the curee of this treatment of needs, prevention and/or improvement.
20. 20. the method for claim 19, wherein said immunology disorder relates to mast cell degranulation.
21. 21. the method for claim 19 or 20, wherein said immunology disorder is acute allergic disease, allergic disorder or alterative inflammation.
22. 22. each method of claim 19 to 21, wherein said curee is people curee.

Images