CN1723019B - Compounds having anti-proliferative properties - Google Patents
Compounds having anti-proliferative properties Download PDFInfo
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- CN1723019B CN1723019B CN200480001734.6A CN200480001734A CN1723019B CN 1723019 B CN1723019 B CN 1723019B CN 200480001734 A CN200480001734 A CN 200480001734A CN 1723019 B CN1723019 B CN 1723019B
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Abstract
There is provided a method of inhibiting the occurrence of one of more of the following conditions: - the proliferation of monocytes/macrophages; or - the proliferation of smooth muscle cells; or - the expression of CD36 receptors; or - the uptake of oxidized LDL, the method comprising the step of administering an effective amount of one or more phosphate derivatives of one or more electron transfer agents.
Description
Invention field
Electron transfer agents after the present invention relates to modify suppresses the ability that one or more following situations take place: the propagation of monocyte/macrophage, the propagation of smooth muscle cell, the expression of scavenger receptor (scavenger receptor) or to the absorption of oxidized ldl.
Background of invention
In this manual, if quote or discuss document, regulations or clause, this to quote or discuss not be to admit that these documents, regulations or clause or their combination in any are the part of common practise at priority date; Or it is known with to attempt to solve any problem that this description relates to relevant.
When following discussion relates to tocopherol phosphate ester (TP), need should be appreciated that this discussion is just illustrative, this invention is not limited to TP, also other phosphate derivatives with electron transfer agents are relevant similarly in discussion, and electron transfer agents includes but not limited to other tocol, retinol and K1.
Atherosclerosis is a kind of endarterium disease that causes fiber (tremulous pulse medicated porridge sample) speckle formation and luminal stenosis or obstruction.The tremulous pulse that is subjected to atherosclerosis influence has lost elasticity, and along with the growth of medicated porridge sample artery plaque, tremulous pulse narrows down and As time goes on may break.Blood enters medicated porridge sample artery plaque subsequently and makes its increase, makes tremulous pulse narrower thus.Disruptive medicated porridge sample artery plaque discharges the formation that its lipid content is brought out blood clot (thrombosis), blood vessel is narrowed down or strip off sometimes to cause obstruction (thromboembolism).Atherosclerosis can influence the blood vessel of brain, heart, kidney, other vitals and arm and lower limb.When in leading to the tremulous pulse (carotid artery) of brain, generating thromboembolism, can cause apoplexy; When in leading to the tremulous pulse (coronary artery) of heart, forming thromboembolism, can bring out heart disease.
Risk factor includes but not limited to circulation high density lipoprotein (HDL) level of senescence, hypertension, smoking, obesity, diabetes, decline, hdl particle [Lp (a)] level that raises, oxidized low-density lipoprotein (LDL) level that raises, increasing of the iatrogenic oxidized ldl level that causes, and do not get enough athletic exercise, all these all increase the probability of tunica intima generation physical damnification and atheroma formation.
Current a lot of scientist believes that atherosclerotic is because the innermost layer of tremulous pulse is that endothelium has been subjected to damage.Sustain damage equally with other parts of health, tremulous pulse subsequently can inflammation.This inflammation can be brought out following natural process:
Monocytic propagation, monocyte maturation generates macrophage;
Scavenger receptor on the monocyte/macrophage such as CD36 receptor expression; And
Repair the expression of the smooth muscle cell (SMCs) of damage.
Meanwhile, various molecules (lymphocyte, oxidized low-density lipoprotein (LDL), fibrin, platelet, cell debris and calcium) enter the arterial wall inner membrance and heap by the endothelium of damage.This heaps the mRNA that further stimulates inflammatory mediator, this medium to modify signal protein (protein kinase C-alpha (PKC-α), VCAM etc.) and expresses.This inflammation causes above-mentioned molecule in the further heap of inner membrance and the growth of speckle.
Mononuclear cell is a class leukocyte cell.When mononuclear cell enters the ripe macrophage that forms when organizing, macrophage is the leukocyte cell of functionating.Macrophage absorbs and kills the microorganism that causes disease and remove damaged cell.Accumulative macrophage obtains monocytic help and removes the oxidized ldl of piling up.CD36 receptor on the cell surface of macrophage plays a role therein by adhering to the oxidized ldl molecule.When this reset procedure was not regulated, foam cell formed.These foam cells are also heaped in speckle.
The CD36 receptor is the multiple glycoprotein of cell surface, and known its is a big part of organizing in the scavenger receptor that comprises SR-A, MARCO, CD68, LOX-1 and SR-BI.It is believed that the scavenger receptor that comprises CD36 is very important in the formation of macrophage to the absorption of oxidized ldl and foam cell.The absorption of the lipoprotein after modifying is had contribution to known CD36 receptor and they are receptors of thrombostondin, I and II Collagen Type VI, fatty acid and polyanion phospholipid.
If smooth muscle cell will be because to the growth continuous inflammatory reaction of speckle and excessive propagation, these smooth muscle cell also will impel speckle to form.
The oxidized ldl level that raises is considered to cause the inflammation of arterial wall equally, causes the formation of above-mentioned reaction and atheromatous plaque.
Therefore, can suppress smooth muscle cell proliferation, suppress mononuclear cell propagation, reducing that oxidized ldl absorbs or suppress the active material of scavenger receptor may be effectively in to atherosclerosis therapy.
Symptom and treatment
Current do not have direct therapy for atherosclerotic related symptoms.Therefore fitness guru is devoted to remove controlled risk factor, as high blood cholesterol levels.Also encourage the high-risk individual kinds of protect food of considering connoisseur in recent years, comprise various phytochemistry medicines and antioxidation nutrient such as vitamin E.
Because cause atheromatous most of serum cholesterol by the carrying of LDL component, it is the atherosclerotic main method of clinical treatment that the LDL level that raises is reduced.But because the method can not directly be ended seizure of disease, so a kind of indirect method of atheromatosis process is just treated in this treatment.Also not can be used at present directly treatment and reduce the active drug that atheromatous plaque forms.
High fat (Hyperlipidaemic) chemical compound suppresses the propagation of large artery trunks (tremulous pulse that leads to heart) parietal cell to a certain extent indirectly, but needs long-term treatment just can tell on.The a large amount of cholesterol of long-term removing have its risk.Cholesterol is the substrate of synthetic a lot of important compound, comprises steroid hormone, vitamin D, ubiquinone, bile acid, dolichol, method protein, haemachrome A and tRNA.Therefore removing cholesterol in aggressiveness ground may cause problem in some individualities.Same, high fat chemical compound can not directly be treated the LDL of the atherosclerotic basic cause of disease such as oxidative modification and excessive smooth muscle cell proliferation, is not ideal selection therefore.Some chemical compounds such as cancer therapy drug can suppress excessive smooth muscle cell proliferation, but they cause serious adverse simultaneously so can not use.
A trial drug is carried out clinical research at present, this medicine is considered to have and reduces the proteic effect of VCAM-1, and this albumen can reduce the absorption of inflammation site leukocyte cell (monocyte/macrophage).
Also there is not expression that medicine can directly regulate CD36 receptor or other scavenger receptor with the treatment atherosclerosis according to known.
At present not can be used for directly treating effective selection of the excessive propagation of smooth muscle cell.
Tocopherol
Low-level alpha-tocopherol (vitamin E) is relevant with the evidence of coronary heart diseases that increases.On the contrary, increase picked-up to alpha-tocopherol and demonstrate protective effect at heart disease.Because vitamin E is an antioxidant, so thereby it is believed that it can act on atherosclerotic inducement by the oxidation that prevents LDL.The research of checking vitamin E to suppress the potential non-antioxidation mechanism of atheromatous plaque formation is simultaneously also being carried out.This type of reaction comprises and suppresses smooth muscle cell proliferation, preserves endothelial function, suppresses the mononuclear cell adhesion, suppresses monocytic activity oxygen class material and release of cytokines and suppress platelet adhesion and gathering.
But the clinical trial of vitamin E is indeterminate to the atherosclerosis therapy result.Therefore not effective selection of clinical to the additional of vitamin E for the treatment atherosclerosis at present.
Other diseases and situation
The expression of the propagation of monocyte/macrophage, the propagation of smooth muscle cell, scavenger receptor or also be the problem that exists in other diseases and the situation to the absorption of oxidized ldl.The example of other diseases comprises alzheimer's disease and diabetes.Conditions associated example has the oxidized ldl level rising (iatrogenic disease) that is caused by certain drug such as ritonovir.
Need have minimal side effect and can help under low dosage to reduce that monocyte/macrophage propagation, smooth muscle cell proliferation, scavenger receptor are expressed or to the therapy of one or more situations of the absorption of oxidized ldl.
Summary of the invention
The phosphate derivative of having found at present electron transfer agents is more effective than non-phosphorylating electron transfer agents in the generation that suppresses following one or more situations:
Smooth muscle cell proliferation;
Monocyte/macrophage propagation;
The expression of scavenger receptor; Or
Absorption to oxidized ldl.
The phosphate derivative of electron transfer agents can slow down or suppresses the propagation of smooth muscle cell or monocyte/macrophage fully by the dose response pattern, reduces or the expression of restriction street cleaner cell or to the absorption of oxidized ldl.
According to the present invention, provide and suppressed the method that one or more following situations take place:
Monocyte/macrophage propagation; Or
Smooth muscle cell proliferation; Or
The expression of scavenger receptor; Or
Absorption to oxidized ldl.
Described method comprises the step with one or more phosphate derivative administrations of one or more electron transfer agents of effective dose.
Those skilled in the art will recognize that method of the present invention can be used for and the propagation of monocyte/macrophage, the propagation of smooth muscle cell, the expression of scavenger receptor or the treatment of diseases relevant to the absorption of oxidized ldl.The example of these diseases is including, but not limited to diabetes, Alzheimer and atherosclerosis.
Therefore the present invention includes the atherosis method of mitigation symptoms, treatment or prevention of arterial, this method comprises suffering from atherosclerosis or the object administration of ill risk being arranged, and pharmaceutical formulation comprises one or more phosphate derivatives of one or more electron transfer agents of effective dose.
The present invention also comprises the method for mitigation symptoms, treatment or prevent diabetes, and this method comprises suffering from diabetes or the object administration of ill risk being arranged, and pharmaceutical formulation comprises one or more phosphate derivatives of one or more electron transfer agents of effective dose.
The present invention also comprises the method for mitigation symptoms, treatment or prevention Alzheimer, this method comprises suffering from Alzheimer or the object administration of ill risk being arranged, and pharmaceutical formulation comprises one or more phosphate derivatives of one or more electron transfer agents of effective dose.
The invention still further relates to and suppress the method that the vascular system speckle forms.
Further, the invention provides and be used to suppress the pharmaceutical composition that following one or more situations take place: the expression of the propagation of monocyte/macrophage, the propagation of smooth muscle cell, scavenger receptor or to the absorption of oxidized ldl, described compositions comprises one or more phosphate derivatives of one or more electron transfer agents of effective dose.
Further, the invention provides the purposes that one or more phosphate derivatives of one or more electron transfer agents of effective dose and suitable carriers or diluent one is used from the preparation medicine, this medicine can suppress the generation of following one or more situations: the expression of the propagation of monocyte/macrophage, the propagation of smooth muscle cell, scavenger receptor or to the absorption of oxidized ldl.
Another aspect of the present invention, the invention provides and suppress the method that following one or more situations take place: the propagation of monocyte/macrophage, the propagation of smooth muscle cell, CD36 receptor expression or to the absorption of oxidized ldl, described method comprises the step with one or more phosphate derivative administrations of one or more electron transfer agents of effective dose.In the specific embodiments in this regard, one or more phosphate derivatives of one or more electron transfer agents of effective dose are as the prodrug administration.Preferably, the administration object is an animal.More preferably, this animal is behaved.
" effective dose " speech is used herein to expression is enough to suppress the dosage that following one or more situations take place: the expression of the propagation of monocyte/macrophage, the propagation of smooth muscle cell, scavenger receptor or to the absorption of oxidized ldl.It will be appreciated by those skilled in the art that this dosage will vary with each individual.
Usually, the effective dose of one or more phosphate derivatives of one or more electron transfer agents 0.1-10 that is meant the blood plasma of alpha-tocopherol or the organizes average level amount of (the blood plasma mean concentration of alpha-tocopherol is between 30-50 μ M) doubly.More preferably, this effective dose is the blood plasma of alpha-tocopherol or the 2-3 that organizes average level amount doubly.
The general treatment of mitigation symptoms, treatment or prevention such as atherosclerotic disease comprise one or more phosphate derivatives of the electron transfer agents that gives administration object 50-1000mg every day until the blood plasma of electron transfer agents/organize mean concentration be alpha-tocopherol the blood plasma mean concentration 2-10 doubly.The intake of one or more phosphate derivatives of adjusting electron transfer agents subsequently is to keep ideal blood plasma/tissue concentration.
" electron transfer agents " speech is used herein to expression one class and can be accepted an electronics by phosphorylation and (non-phosphorylating form) and generate a kind of metastable molecular radical or accept two electronics and make chemical compound can participate in the chemical compound of reversible oxidation-reduction system.Can be comprised that hydroxychroman comprises the enantiomer and the racemic form of α, β, γ and δ tocol by the example of the kind of the electron transfer agents chemical compound of phosphorylation; Reduction form hydroquinone and the ubiquinone of electron transfer agents K1; Hydroxy kind carotene comprises retinol; Calciferol and ascorbic acid.Preferably, electron transfer agents is selected from tocopherol and other tocols, retinol, electron transfer agents K1 and their mixture.
More preferably, electron transfer agents is selected from tocol and their mixture.Tocol comprises all isomers of the derivant of 6-hydroxy-2-methyl benzodihydropyran (structure is as follows), wherein R
1, R
2And R
3Can be hydrogen or methyl, i.e. α-5,7,8 trimethyls; β-5,8 dimethyl; γ-7,8 dimethyl; And δ-8 methyl-derivatives.In tocopherol, R
4By 4,8,12-trimethyl tridecane replaces, and 2,4 and 8 positions (are seen
*) can be R or S or racemic stereoisomer.In triolefin fertility alcohol, R
4By 4,8,12-trimethyl tridecyl-3,7, the 11-triolefin replaces, and wherein position 2 can be three-dimensional active R or S stereoisomer or racemic modification.More preferably, electron transfer agents is an alpha-tocopherol.
Term " phosphate derivative " comprises slaine such as sodium, magnesium, potassium and calcium salt and other derivants at this acid form, phosphate as expression phosphorylation electron transfer agents; wherein the proton of phosphate radical can be replaced by other substituents, as ethyl or methyl or phosphatidyl group.This term comprises the mixture of phosphate derivative, the particularly derivant that is generated by phosphorylation reaction, and every kind of independent phosphate derivative.For example, this noun comprises single tocopherol phosphate ester (TP) and the mixture of two tocopherol phosphate esters (T2P) and independent TP and T2P.Suitable mixture is described in international patent application no PCT/AU01/01475 to some extent.
Preferably, one or more phosphate derivatives of one or more electron transfer agents are selected from single tocopherol phosphate ester, two tocopherol phosphate ester and their mixture.Optimally, one or more phosphate derivatives of one or more electron transfer agents are mixture of single tocopherol phosphate ester and two tocopherol phosphate esters.
In some cases, when the extra character of needs such as needs increase water solublity, must use phosphate derivative such as phospholipid.Phosphatidyl derivant is the aminoalkyl derivant of organic phosphate.These derivants can be by having structure R
1R
2N (CH
2)
nThe amine preparation of OH, wherein n is the integer between the 1-6, R
1And R
2Can be hydrogen or contain 3 or the short alkyl chain of carbon still less.R
1And R
2Can be identical also can be different.Replace the preparation phosphatidyl derivant by the hydroxyl proton with electron transfer agents with the phosphate radical entity, the phosphate radical entity reacts with amine subsequently, as ethanolamine or N, and N '-dimethylethanolamine, the phosphatidyl derivant of generation electron transfer agents.A kind of method of preparation phosphatidyl derivant is to use basic solvent such as pyrimidine or triethylamine and phosphorous oxychloride to prepare intermediate, and this intermediate generates the corresponding phospholipids acyl derivative with the oh group reaction of amine subsequently, as P cholyl P biphosphate tocopherol.
In some cases, when the increase of the extra character of needs such as stability or transfer capability, also can use the complex of the phosphate derivative of electron transfer agents." complex of phosphate derivative " refers to one or more phosphate derivatives of electron transfer agents and the product of one or more complexometric reagents.As what in this international patent application no PCT/AU01/01476 as a reference, put down in writing, complexometric reagent is selected from amphoteric surfactant, cationic surfactant, contain nitrogen functional group aminoacid and be rich in these amino acid whose protein.
Preferred complexometric reagent is selected from the amine of arginine, lysine and triple replacements, as meets the amine of following structural:
NR
1R
2R
3
R wherein
1Be selected from C6 to the straight or branched mixed alkyl group of C22 and their carbonyl derivative;
R
2And R
3Be independently selected from H, CH
2COOX, CH
2CHOHCH
2SO
3X, CH
2CHOHCH
2OPO
3X, CH
2CH
2COOX, CH
2CH
2CHOHCH
2SO
3X, CH
2COOX or CH
2CH
2CHOHCH
2OPO
3X, wherein X is H, Na, K or alkanolamine, R
2And R
3Be not H simultaneously; And
Wherein work as R
1During for RCO, R
2Can be CH
3And R
3Can be (CH
2CH
2) N (C
2H
4OH)-H
2CHOPO
3Or R
2And R
3All be N (CH
2)
2N (C
2H
4OH) CH
2COO-.
Preferred complexometric reagent comprises arginine, lysine or Laurel imino-diacetic propanoic acid (lauryliminodipropionic acid), complexation takes place between basic nitrogen atom center and phosphate ester form stable complex.The phosphate derivative of electron transfer agents can comprise ointment and cream as supplement, intestinal canal administration, non-parenteral dosage forms, suppository, intranasal administration dosage form, percutaneous drug delivery by various dosage forms to human or animal's administration.
For example, the phosphate derivative of electron transfer agents can be by the oral or outer dosage form administration of intestinal.These dosage forms comprise tablet, powder, chewable tablet, capsule, oral suspension agent, Emulsion or fluid, administration in child's prescription, the intestinal, nutrient supplement food and health food.
Above-mentioned dosage form can also comprise any additives such as starch or poly binding agent, sweeting agent, coloring agent, emulsifying agent, bag quilt and the analog that is generally used for preparing these dosage forms.Other additive that is fit to is conspicuous for a person skilled in the art.
In a specific embodiments, as what put down in writing in this International Patent Application PCT/AU02/01206 as a reference, above-mentioned dosage form has the intestinal thin film exterior layer.
In another embodiment, as what put down in writing in this International Patent Application PCT/AU02/01003 as a reference, above-mentioned dosage form is local with prescription.
Can comprise in the above-mentioned dosage form that other activity to the phosphate derivative of electron transfer agents does not have the medical compounds of antagonism.This other medicines chemical compound can be before one or more phosphate derivatives of above-mentioned one or more electron transfer agents, simultaneously or administration afterwards.Preferably, this other medicines chemical compound is hypercholesterolemia (hypercholesterolaemic) chemical compound.More preferably, this other medicines chemical compound is selected from scholar's Statins (statins), the phosphate derivative of Shi Tating and their mixture.The example in his spit of fland of suitable taxi comprises pravastatin (provastatin), lovastatin (lovastatin) and atropic Fa Sitating (atorvastatin) and their phosphate derivative.
Brief description of drawings
Fig. 1. the influence of tocopherol on cell proliferation;
Fig. 2. the influence of tocopherol phosphate mixture on cell proliferation;
Fig. 3. the influence of tocopherol phosphate ester on cell proliferation;
Fig. 4. the influence of various compositions on cell proliferation---adherent cell counting;
Fig. 5. the influence of various compositions on cell proliferation---MTS;
The FACS of Fig. 6 .THP-1 mononuclear cell and anti-CD36-FITC antibody;
Combination and the absorption of ocLDL-DiO in Fig. 7 .THP-1 mononuclear cell;
The monocytic growth inhibited of Fig. 8 .THP-1.
Embodiment
By following indefiniteness embodiment the present invention is further specified and explains.
Present embodiment has been studied the influence to rat large artery trunks smooth muscle cell proliferation of tocopherol and tocopherol phosphate ester.
The rat large artery trunks smooth muscle cell (RASMC) that is used for proliferation research is taken from aortic inner membrance of adult rat and film healthy, no fibrous plaque.This cell strain is the model accepted of studying atherosclerotic, because found the increase of arterial smooth muscle quality in atherosclerotic inner film injury.The second filial generation cell of freezing preservation RASMC, it can breed 16 population doublings at least.RASMC comes the various factors are reacted by cell proliferation and undue growth, and this is the atherosis distinctive marks of angiopathy medium-sized artery.RASMC be suitable for the research of trunk smooth muscle cell growth and differentiation equally and with the rat model of living as external model.
Material
6 orifice plates (cell counting)
96 orifice plates (MTT analysis)
DMEM/F12 culture medium---GIBCO/Life Technologies
Hyclone (serum)
Rat large artery trunks smooth muscle cell (RASMCs) the 4th generation CellApplications company
Gentamycin-GIBCO/LifeTechnologies
·Cell?Titer?96?Aqueous?One?Solution(MTT)-Promega
Ethanol (EtOH), 1/1000
Tocopherol (0.25,0.5,1,5,10,20,50,100 μ M)
Tocopherol phosphate ester (mixture of TP and T2P) (0.25,0.5,1,5,10,20,50,100 μ M)
Smooth muscle cell proliferation---cell counting
Rat large artery trunks smooth muscle cell (RASMC) is inoculated in the growth medium of 6 orifice plates (in the basal medium+10%FBS) (50,000 cells/well).After 24 hours, use Hanks buffer salt solution washed cell 2 times, with the insufficient culture medium of serum (basal medium+0.2%FBS) join in each hole.Make cell lack serum 48 hours.In growth medium, prepare to handle and join subsequently (3ml/ hole) in each hole.Each processing repeats 3 times.Tocopherol and tocopherol phosphate ester are tested under 8 concentration the influence of smooth muscle cell proliferation: 0.25,0.5,1,5,10,20,50,100 μ M.The contrast treatment comprises: growth medium and growth medium+excipient (EtOH, 1/1000).At 37 ℃, 5% CO
2The middle cultivation after 72 hours carried out cell counting.
The result
Estimate with quantitative by the cell counting on cell proliferation.In the basal medium that has replenished 0.2% serum with cell after hungry 48 hours the pair cell counting add testing compound subsequently.The average cell number of three repeating holes is～60,000.This has outnumbered the hungry number (50,000) of inoculating cell before.Therefore determine that cell is alive, will handle as previously mentioned to add in each hole.
Cell is counted after 72 hours with compound treatment.Fig. 1 and Fig. 2 have shown the result of the test of tocopherol and tocopherol phosphate ester respectively.Under this experimental condition, be used to dilute the excipient of testing compound, EtOH (1/1000), on cell proliferation is influence not.Tocopherol only suppresses cell proliferation to a certain extent under the concentration of 1,5,10,20,50 and 100 μ M.But the tocopherol phosphate ester suppresses cell proliferation in dose-dependent mode under the concentration of 1,5,10,20,50 and 100 μ M.The tocopherol phosphate ester is observed on cell proliferation 100% and is suppressed (Fig. 3) under 100 μ M concentration.
Discuss
Result of the test proof tocopherol phosphate mixture can suppress excessive cell proliferation in dosage dependence mode under 5 μ M, 20 μ M, 50 μ M and 100 μ M concentration.Importantly, realized new plastidogenetic inhibition fully at 50 μ M.
Handle the propagation that suppresses cell in 1 μ M, 5 μ M and 10 μ M lower parts with alpha-tocopherol but can not reduce propagation fully at high dose more.Optimal inhibition to propagation reaches stable about 60%, with open source literature is consistent.This makes that alpha-tocopherol is unreliable so is not suitable for atherosclerosis therapy.On this basis and according to nearest disclosed document, it is irrational that alpha-tocopherol is used for atherosclerosis.
Although alpha-tocopherol presents part and suppresses under low concentration, the tocopherol phosphate ester can be realized the inhibition of on cell proliferation 100% and obviously become a kind of more favourable antiproliferative reagent owing to it.In addition, the inhibition to excessive cell proliferation that takes place in dosage dependence mode shows that the tocopherol phosphate mixture is a kind of more reliable and predictable treatment, is suitable for atherosclerosis therapy.
Generally speaking, the tocopherol phosphate mixture relies on the mode effect with dosage, therefore compares with alpha-tocopherol, provides reliable more and effective to excessive cell proliferation and has suppressed.The more important thing is that the tocopherol phosphate mixture has been realized the inhibition of on cell proliferation 100% at 50 and 100 μ M.This shows the early onset thereof step that unexpectedly can prevent smooth muscle cell proliferation in predictable mode owing to the tocopherol phosphate mixture, and it can be used to atherosclerotic direct treatment.
This embodiment adopts two class cell counting analyses: adherent cell counting and MTS analyze, and have estimated the antiproliferative activity of alpha-tocopherol phosphate ester (TP), two tocopherol phosphate ester (T2P), TP/T2P mixture and alpha-tocopherol.
But carry out the MTS proliferation assay and further support and affirm the adherent cell analysis of accounts.It is the very sophisticated method that is used to estimate cell proliferation that MTS analyzes, and this method is to adhering to the living cells (with the adherent cell counting) on the dish and breaking away from process of the test and the cell (can be omitted in the adherent cell counting) that floats in the culture medium is counted.
Material
6 orifice plates (cell counting)
96 orifice plates (MTS analysis)
DMEM/F12 culture medium---GIBCO/Life Technologies
Hyclone (serum)
Rat large artery trunks smooth muscle cell (RASMCs) the 4th generation CellApplications company
Gentamycin---GIBCO/Life Technologies
·Cell?Titer?96?Aqueous?One?Solution(MTT)——Promega
Ethanol (EtOH), 1/1000
Tocopherol SIGMA (0,20,50,100 μ M)
The mixture of TP/T2P (80%: 20%) (0,20,50,100 μ M)
Pure tocopherol phosphate ester (0,20,50,100 μ M)
Pure pair of tocopherol phosphate ester (0,20,50,100 μ M)
The result
Add the difference that does not have between the excipient on the statistics at single culture base and culture medium.All data all derive from the difference % (being that the excipient contrast is 0% in the drawings) of excipient contrast.
In this research to remaining on that the cell of trying to get to the heart of a matter is counted and antiproliferative activity being assessed according to the number that sticks to the living cells on the dish.The result hints that T2P and TP/T2P mixture all are strong antiproliferative reagent, and smooth muscle cell proliferation is produced maximal percentage inhibition (85-90%), yet TP does not suppress (Fig. 4) to smooth muscle cell proliferation in this research.
Research 2.MTS analyzes
Result of study has confirmed that again under the concentration of 100 μ M T2P and TP/T2P mixture can reach 85-90% (Fig. 5) to the inhibition of smooth muscle cell.What is interesting is that pure TP can suppress the propagation (reaching 85%) of smooth muscle cell equally under the concentration of 100 μ M.This result hints that the mechanism of TP anti-proliferative effect is different from independent T2P and TP/T2P mixture.
Conclusion
These discoveries mean that it all is strong antiproliferative reagent that TP, T2P and TP/T2P mixture are compared with alpha-tocopherol.Has identical activity at TP aspect the inhibition smooth muscle cell proliferation with T2P.As if but TP realizes its antiproliferative activity in the mode that is different from T2P, TP/T2P mixture and alpha-tocopherol.
The purpose of this research is the influence of the expression of CD36, the absorption of oxidized ldl (oxLDL) and people THP-1 mononuclear cell being grown at external relatively TP/T2P mixture and tocopherol.
Step
Cell culture: respectively tocopherol and TP/T2P mixture are dissolved in the ethanol, determine the concentration of storing solution with spectrophotography.Mononuclear cell (THP-1) is grown in RPMI/10%FCS.
Labelling oxLDL:OxLDLs (90% to 100% oxygenation efficiency) is available from Intracell company.A spot of LDL CuSO
4(20mmol/L) 37 ℃ of oxidations 18 to 22 hours.The oxidation of LDL confirms by the characteristic hangover band that occurs on the agarose gel.The labelling of oxLDL carries out substantially as stated above.OxLDLs and DiO (molecular probe) cultivated 15 hours in 37 ℃ in the serum that lipoprotein lacks.The oxLDLs of labelling (oxLDL-DiO) repeatedly exchanges dialysis 6 hours with the supercentrifugation purification and to sodium-EDTA (1.5mol/LNaCl-0.01%EDTA) on the KBr gradient.
The absorption of the absorption of oxLDL: oxLDL is studied by fluorescent activation cell sorting system (FACS).For FACS, cell was cultivated 6 hours with oxLDL-DiO (5 μ g/mL culture medium) subsequently with 50 μ M tocopherols, tocopherol phosphate ester or alcohol solvent (contrast) pretreatment 16 hours.In competition experiments, cell and anti-CD36 monoclonal antibody (60 μ g/5mL DMEM) (Ancell), non-specificity homotype pairing antibody (mice IgM, Ancell) or unlabelled oxLDL (100 μ g/5mL DMEM) (Intracell company) co-cultivation.Subsequently, cell washs 2 times with PBS-3mg/mL BSA with PBS washing 3 times, back trypsin 0.25% trypsin, 0.03%EDTA) peptic cell.Cell with DMEM/10%FCS results, centrifugal, with the PBS washed twice, be fixed among the PBS with 4% paraformaldehyde subsequently.FACS carries out with FACScan (Becton-Dickinson).From the fluorescence of handling the back sample, deduct cell autofluorescence and calculate data.
Thin layer chromatography: eluent adopts chloroform/normal hexane (1: 1 v/v), carries out 20 minutes.Observe at 254nm by UV.
Result and discussion
The surface expression of CD36 scavenger receptor (Fig. 6):
Fig. 6 has shown the variation of cell number along with the time by the cell number that absorbs fluorescent antibody.TP/T2P mixture peak is compared the less cell of explanation that moves to left and has been absorbed antibody with contrast peak (tocopherol), so the CD36 expression of receptor is less.
The remarkable minimizing that the processing of only using the TP/T2P mixture of 5 μ g/ml that THP-1 mononuclear cell (people source) is carried out causes CD36 to express.Hyperfluorescence explanation control cells (handling with tocopherol) by anti-CD36 fluorescent antibody labelling has been expressed a large amount of CD36 receptors.The TP/T2P mixture that 5 μ g are described that significantly moves to left can suppress the CD36 receptor expression.Notice that its yardstick is logarithmic.
The combination of oxLDL-DiO and absorption (Fig. 7):
Fig. 7 has shown the variation along with the time of cell number that the cell number by the oxLDL that absorbs labelling shows.All curves are all similar, illustrate the TP/T2P mixture under the concentration of 5 and 25 μ g/ml and the tocopherol (contrast) of 22 μ g/ml (50 μ M) have same effect.Arrow has emphasized that the TP/T2P mixture of 25 μ g/ml can reduce the fact that oxLDL absorbs significantly significantly.
Personnel selection THP-1 mononuclear cell test TP/T2P.The 5 μ g/ml that are combined in of oxLDL-DiO slightly suppress, and suppressing when 25 μ g/ml increases.OxLDL-DiO be absorbed in 5 μ g/ml the time be suppressed, when 25 μ g/ml, suppress significantly to increase the weight of.
The absorption signal (intermediate value at peak) of OxLDL-DiO has reduced by 33% in the cell after the tocopherol of 50 μ M is handled.In cell, under the concentration that is lower than 10 μ M, can obtain same effect with the TP/T2P mixture process.The minimizing that inhibition that tocopherol is expressed CD36 has caused the oxLDL of CD36 mediation to absorb can be inferred, and identical effect can be obtained with the TP/T2P mixture of low concentration.The population of cells's (arrow labelling) that oxLDL-DiO is had high absorbent capacity can be suppressed by TP/T2P mixture height.
The TP/T2P mixture is to the monocytic growth inhibited of THP-1: mononuclear cell propagation is the critical event of atherosclerosis outbreak and upgrading.Fig. 8 illustrates that tocopherol (T) and ethanol contrast (E) is compared and can not suppress this propagation.On the other hand, the TP/T2P mixture particularly after handling 48 hours, can suppress propagation at 30 μ M (TP1) and 60 μ M (TP2).
Conclusion
Present embodiment shows that the TP/T2P mixture can significantly suppress the expression of CD36 scavenger receptor.This inhibition that CD36 is expressed has caused minimizing that oxidized ldl is absorbed.In addition, the TP/T2P mixture suppresses monocytic propagation.Seemingly the TP/T2P mixture is exclusive for this phenomenon, and the utilization tocopherol can not be realized.Result relevant and disclosed unanimity as a result before about tocopherol with tocopherol.
The TP/T2P mixture significantly reduces the CD36 receptor expression in the person monocytic cell.All things considered, presentation of results TP/T2P mixture exceeds 5-10 doubly than the ability of tocopherol.
TP/T2P compares with tocopherol and has suppressed combination and the absorption of mononuclear cell to oxidized ldl more significantly.Observed inhibition degree needs 50uM tocopherol (promptly needing to be lower than 5 times mixture) under the 10 μ MTP/T2P.
TP/T2P significantly suppresses person monocytic cell's propagation (TP/T2P have surpass 90% suppression ratio but tocopherol does not suppress propagation).
Studies show that TP/T2P more effectively reduces absorption to cholesterol oxidized forms than alpha-tocopherol, suppress CD 36 expression of receptor and then reduce the absorption of oxLDL and suppress monocytic propagation and transfer.
The more important thing is that TP/T2P has an effect in dose-dependent mode and has more significant reduction effect than tocopherol.
" comprising " speech that uses in description and claims and the various forms of " comprising " speech do not limit the present invention, do not get rid of other variant or additive.
It will be apparent to those of skill in the art about modification of the present invention or improvement.These modifications or improvements are expected in protection scope of the present invention.
Claims (10)
1. the application in the medicine of one or more phosphate derivatives of one or more electron transfer agents of effective dose formation of speckle in preparation inhibition vascular system;
One or more phosphate derivatives of wherein said one or more electron transfer agents are selected from single tocopherol phosphate ester, two tocopherol phosphate ester or their mixture.
2. the application described in claim 1, one or more phosphate derivatives of one or more electron transfer agents wherein are selected from the mixture of single tocopherol phosphate ester and two tocopherol phosphate esters.
3. the application described in claim 1 or 2, tocopherol wherein is selected from alpha-tocopherol, betatocopherol, Delta-Tocopherol and Gamma-Tocopherol.
4. the application described in claim 3, electron transfer agents wherein is an alpha-tocopherol.
5. application as claimed in claim 1, effective dose wherein are 0.1 to 10 times amounts of the blood plasma mean concentration of alpha-tocopherol.
6. application as claimed in claim 5, effective dose wherein are 2 to 3 times amounts of the blood plasma mean concentration of alpha-tocopherol.
7. application according to claim 1, speckle is formed for preventing and/or treating arteriosclerosis in the wherein said inhibition vascular system.
8. the application in the medicine of one or more phosphate derivatives of one or more electron transfer agents of effective dose formation of speckle in preparation inhibition vascular system;
One or more phosphate derivatives of wherein said one or more electron transfer agents are selected from one or more complex form of single tocopherol phosphate ester and/or two tocopherol phosphate esters.
9. application as claimed in claim 8, wherein said complex is by the preparation of complexometric reagent Laurel imino-diacetic propanoic acid.
10. one or more phosphate derivatives of one or more electron transfer agents of effective dose prevent and/or treat application in the medicine of arteriosclerosis in preparation;
One or more phosphate derivatives of wherein said one or more electron transfers are selected from single tocopherol phosphate ester, two tocopherol phosphate ester or their mixture.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2003900200 | 2003-01-17 | ||
| AU2003900200A AU2003900200A0 (en) | 2003-01-17 | 2003-01-17 | Compounds having antiproliferative properties |
| AU2003901698 | 2003-04-09 | ||
| AU2003901698A AU2003901698A0 (en) | 2003-04-09 | 2003-04-09 | Compounds having antiproliferative properties |
| PCT/AU2004/000056 WO2004064831A1 (en) | 2003-01-17 | 2004-01-16 | Compounds having anti-proliferative properties |
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| CN1723019A CN1723019A (en) | 2006-01-18 |
| CN1723019B true CN1723019B (en) | 2011-04-13 |
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| CN200480001734.6A Expired - Fee Related CN1723019B (en) | 2003-01-17 | 2004-01-16 | Compounds having anti-proliferative properties |
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| AU (1) | AU2003900200A0 (en) |
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| TWI844795B (en) * | 2020-09-17 | 2024-06-11 | 日商力森諾科股份有限公司 | Autophagy activators |
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| EP0436911A2 (en) * | 1990-01-05 | 1991-07-17 | Eisai Co., Ltd. | Novel vitamin E derivatives and process for the production thereof |
| CN1173869A (en) * | 1995-10-17 | 1998-02-18 | 昭和电工株式会社 | High-purity tocopherol phosphates, process for the preparation thereof, method for analysis thereof, and cosmetics |
| WO2000057876A1 (en) * | 1999-03-26 | 2000-10-05 | Lipogenics, Inc. | Novel antioxidant formulations and methods for using them |
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| WO2002036736A2 (en) * | 2000-11-02 | 2002-05-10 | The Regents Of The University Of California | Alpha-tocopherol transfer protein knockout animals |
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2003
- 2003-01-17 AU AU2003900200A patent/AU2003900200A0/en not_active Abandoned
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| EP0436911A2 (en) * | 1990-01-05 | 1991-07-17 | Eisai Co., Ltd. | Novel vitamin E derivatives and process for the production thereof |
| CN1173869A (en) * | 1995-10-17 | 1998-02-18 | 昭和电工株式会社 | High-purity tocopherol phosphates, process for the preparation thereof, method for analysis thereof, and cosmetics |
| EP1053749A1 (en) * | 1998-02-03 | 2000-11-22 | Senju Pharmaceutical Co., Ltd. | Preventives and remedies for neuron digeneration-associated diseaseas |
| CN1325303A (en) * | 1998-09-23 | 2001-12-05 | 研究发展基金会 | Tocopherols, tocotrienols, other chroman and side chain derivatives and uses |
| WO2000057876A1 (en) * | 1999-03-26 | 2000-10-05 | Lipogenics, Inc. | Novel antioxidant formulations and methods for using them |
| WO2001022937A1 (en) * | 1999-09-27 | 2001-04-05 | Sonus Pharmaceuticals, Inc. | Compositions of tocol-soluble therapeutics |
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| CN1723019A (en) | 2006-01-18 |
| AU2003900200A0 (en) | 2003-01-30 |
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