Technical field

The present invention relates to biomedicine field, particularly, the present invention relates to effect and the application thereof in suppressing platelet derived growth factor receptor A of arteannuin and derivant thereof.

Background technology

Herba Artemisiae Annuae has another name called Herba Artemisiae annuae (Artemisia anaua L), belongs to Compositae, is the annual herb plant.Arteannuin is the sesquiterpene lactones class antimalarial agent that China pharmacy worker extracted from the Chinese medicine Herba Artemisiae Annuae in early 1970s.In view of traditional quinine class medicine generally runs into the Drug resistance problem, arteannuin and its derivant have replaced them at present, become antimalarial main flow medicine in the world.An endoperoxide bridge is arranged on the macro ring of arteannuin, under the catalysis of ferrous ion or heme, can emit the free radical centered by carbon.Intracellular protein and nucleic acid are dead after by these free radical alkanisations.Plasmodium colonizes in the erythrocyte, contains in a large number the hemes from hemoglobin, thereby arteannuin easy being activated very, and then plasmodium is killed.Arteannuin and its derivant, as dihydroartemisinine, Artemether, arteether and artesunate etc. has been widely used in malaria treatment, and more artemisinin derivative is developed.

The antimalarial active of arteannuin and derivant thereof has obtained universally acknowledged, have rapid-action, drug effect is high, less toxic side effect and other advantages.Except being widely used in malaria treatment, artemisinin-based drug also has other multiple pharmacological effect, as anti-schistosome function, arrhythmia, relieving asthma, effects such as antiendotoxin, antiallergic action, anti-lupus erythematosus, immunosuppressant.

Along with deepening continuously to arteannuin and derivatives active research thereof, it is found that, this compounds is inhibited and excellent curative to the growth of kinds of tumor cells, yet its concrete action target spot and coherent signal path and mechanism of action are also indeterminate.Therefore this area presses for and understands the effect in tumor always of arteannuin and derivant thereof.

Summary of the invention

The purpose of this invention is to provide the effect in suppressing platelet derived growth factor receptor A of arteannuin and derivant thereof.

Another object of the present invention provides arteannuin and derivant thereof as the application of platelet derived growth factor receptor A inhibitor/antagonist.

Another object of the present invention provides the method for external treatment tumor.

In a first aspect of the present invention, provide a kind of arteannuin and derivant thereof for the preparation of suppressing to be selected from down the medicine of group disease or the purposes of medicine sensitizer: PDGFR α positive tumor, vascular conditions or fibrous lesions.

In another preference, described PDGFR α positive tumor cell is the positive ovarian cancer cell of PDGFR α or PDGFR α masculine liver cancer cell.

At second portion of the present invention, provide the purposes of a kind of arteannuin and derivant thereof, for the preparation of inhibitor or the antagonist of platelet derived growth factor receptor A (PDGFR α).

In another preference, described platelet derived growth factor receptor A (PDGFR α) comes from the people.

In another preference, described inhibitor or antagonist also are used for:

(i) activation of inhibition PDGFR α tyrosine kinase; And/or

The protein stability that (ii) suppresses PDGFR α; And/or

(iii) promote the degraded of the proteasome mediation that PDGFR α ubiquitin relies on; And/or

(vi) suppress the activation of PDGFR α positive tumor cell signal path PI3K/AKT and MAPK/EPK.

In another preference, described artemisinin derivative is selected from down group: Artemether, dihydroartemisinine, artesunate, two hydrogen Artemether, arteether etc.

In another preference, described artemisinin derivative is dihydroartemisinine.

In a third aspect of the present invention, provide a kind of external non-therapeutic ground to suppress the active method of platelet derived growth factor receptor A (PDGFR α), comprise step: with arteannuin and/or artemisinin derivative and cells contacting, thus platelet derived growth factor receptor A (PDGFR α) in inhibition or the antagonism cell.

In another preference, described contact is in the presence of arteannuin and/or artemisinin derivative, cultivates described cell.

In another preference, described cell comprises tumor cell, preferably comprise PDGFR α positive tumor cell, more preferably comprise the positive ovarian cancer cell of PDGFR α or hepatoma carcinoma cell, as ovarian cancer cell A2780, ovarian cancer cell OVCAR3, hepatoma carcinoma cell Hep3B.

In another preference, described method also is used for following at least a kind of application:

External non-therapeutic ground suppresses the activation of PDGFR α tyrosine kinase;

External non-therapeutic ground suppresses the protein stability of PDGFR α;

External non-therapeutic ground promotes the degraded of the proteasome mediation that PDGFR α ubiquitin relies on;

External non-therapeutic ground suppresses the activation of signal path PI3K/AKT and MAPK/EPK; With

External non-therapeutic suppresses the growth of PDGFR α positive tumor cell.

In a fourth aspect of the present invention, a kind of purposes of preparation combination is provided, described preparation combination comprises:

(I) contain the preparation of arteannuin and/or artemisinin derivative; With

(II) contain the preparation of gemcitabine or carboplatin,

The combination of described preparation for the preparation of:

(a) pharmaceutical composition or the medicine box of inhibition platelet derived growth factor receptor A; Or

(b) prevent and/or treat pharmaceutical composition or the medicine box of tumor.

In another preference, described preparation comprises: tablet, capsule, suppository, injection.

In another preference, described tumor is PDGFR α positive tumor.

In a fifth aspect of the present invention, a kind of method of the PDGFR of preventing and/or treating alpha associated disorders is provided, wherein said disease is relevant with expression excessively or the hyperactivity of PDGFR α, described method comprises step: use arteannuin and/or artemisinin derivative for the object of needs, or use the pharmaceutical composition that contains arteannuin and/or artemisinin derivative.

In another preference, described PDGFR alpha associated disorders comprises tumor (PDGFR α positive tumor), as ovarian cancer or hepatocarcinoma.

In another preference, PDGFR α positive tumor is the positive ovarian cancer of PDGFR α or PDGFR α masculine liver cancer.

In another preference, described using comprises: in the tumor, intravenous injection, oral tablet, lumbar injection etc.

In another preference, described object is mammal.

In a sixth aspect of the present invention, a kind of method is provided, described method is used in the body or growth, migration and the invasion and attack of the activation of vitro inhibition PDGFR α tyrosine kinase, the protein stability of inhibition PDGFR α, the degraded that promotes the proteasome mediation that PDGFR α ubiquitin relies on, the activation that suppresses signal path PI3K/AKT and MAPK/EPK and/or inhibition PDGFR α positive tumor cell, and described method comprises step: use arteannuin and/or artemisinin derivative for the object of needs.

In a seventh aspect of the present invention, a kind of test kit for the ovarian cancer pathological grading is provided, described test kit comprises the reagent that detects in vitro tissue PDGFR alpha expression amount.

In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can making up mutually between specifically described each technical characterictic in below (eg embodiment), thus constitute new or optimized technical scheme.As space is limited, this tired stating no longer one by one.

Description of drawings

Following accompanying drawing is used for explanation specific embodiments of the present invention, and is not used in the scope of the invention that restriction is defined by claims.

Fig. 1 shows that PDGFR α can be used as the direct action target of arteannuin and derivant thereof; Wherein, Fig. 1 a is the vitro kinase experiment, the result shows that arteannuin and derivant arteannuin (ART) thereof, dihydroartemisinine (DHA), artesunate (ARM), Artemether (ARS) can suppress the activity of PDGFR α born of the same parents the inner (aa550-end) tyrosine kinase; Fig. 1 b is surface plasma resonance (SPR) experiment, the result shows, dihydroarteannuin (DHA) can mutually combine with PDGFR α bag outer end (aa24-524) albumen, the positive contrast of Suo Lafeini (Sorafenib), known its can be in conjunction with the activity that suppresses PDGFR α; Fig. 1 c is the experiment of biotin-avidin affinity purification, and the result shows that the DHA of labelling biotin can mutually combine with PDGFR α in the A2780 cell, thereby is come out by affinity purification; Fig. 1 d is that area of computer aided is built with pattern and oppositely butt joint experiment (Computer Assisted Homology modeling and docking), the result shows, dihydroarteannuin (DHA) can be fitted to PDGFR α bag outer end PDGF binding site, simultaneously also can be fitted to it and wrap inner ATP-binding site, block its kinase activator; Fig. 1 e-Fig. 1 f shows that dihydroarteannuin can effectively suppress activity and the protein expression of PDGFR α among PDGFR α positive cell A2780 and the OVCAR3, and little to phosphorylation and the protein level influence of PDGFR β; Fig. 1 g-Fig. 1 i shows that dihydroarteannuin can accelerate the protein degradation of PDGFR α, and this process is the proteasome mediation that ubiquitin relies on.

Fig. 2 has shown that arteannuin and derivant thereof have growth and the transfer ability of the ovarian cancer cell that suppresses the PDGFRa positive; Wherein, Fig. 2 a shows, after giving medicine irritation 48h, arteannuin and derivant thereof do not have obvious growth to people's normal ovarian epithelial cell IOSE144 and suppress ability, less for the SK-OV3 cytotoxicity, but A2780 and OVCAR3 for the PDGFRa positive have the ability that suppresses more by force, and wherein the effect of arteannuin and dihydroartemisinine is the most remarkable; Fig. 2 b is the cell migration experiment, the result shows, the low concentration short time is handled (12h) ovarian cancer cell, and arteannuin and derivant thereof can suppress the transfer ability of PDGFRa positive cell A2780 effectively, and do not have obvious inhibition ability for the cell SK-OV3 of PDGFRa feminine gender; Fig. 2 c has shown that the dihydroarteannuin of variable concentrations is to the cyto-inhibition of 5 kinds of cell lines (IOSE144, A2780, OVCAR3, OVCAR5, SK-OV3); Fig. 2 d has shown that the dihydroarteannuin of variable concentrations is to the influence of the cell migration of 3 kinds of cell lines (A2780, OVCAR3, SK-OV3); Fig. 2 e has shown that the dihydroarteannuin of variable concentrations is to the influence of the cell invasion of 2 kinds of cell lines (A2780, OVCAR3).

Fig. 3 shows that arteannuin and derivant thereof have growth and the transfer ability of the hepatoma carcinoma cell that suppresses the PDGFRa positive; Wherein, Fig. 3 a shows, in the normal hepatic cell line of people (7702 and LO2) and hepatoma cell line (Hep3B, HepG2,7721,7404, LM3,97L, 97M, Huh-7), has only Hep3B high expressed PDGFRa; Fig. 3 b shows that low concentration dihydroarteannuin (3 μ M) (12h) is in short-term handled the Hep3B cell, can significantly suppress its migration and invasive ability; Fig. 3 c is the cell growth experiment, and the result shows that the Hep3B cell of the PDGFRa positive is more responsive for dihydroarteannuin, and other cells are lower for the dihydroarteannuin sensitivity.

Fig. 4 shows that dihydroarteannuin can suppress growth and the transfer of mice original position ovarian cancer model; Wherein, Fig. 4 a demonstration dihydroarteannuin is treated mice, and the malignant progression that suppresses ovarian cancer effectively can be arranged; Fig. 4 b shows, compares with matched group, and the situation of the lung cancer metastasis of dihydroarteannuin treatment group mice reduces greatly, and along with the increase of dose, the treatment situation is more good; The two blue and green artemisin of Fig. 4 c demonstration variable concentrations is treated mice, and the situation that send out in the tumor abdominal cavity (lungs, liver, intestinal) obtains obvious suppression; Fig. 4 d is immunohistochemical experiment, and the result shows that its modality process (EMT) of cell that dihydroarteannuin is handled is inhibited; Fig. 4 e is immunoblot experiment, and the result shows that dihydroarteannuin can effectively suppress expression and the phosphorylation of PDGFRa, and downstream signal path PI3K/AKT and MAPK-ERK are suppressed.

Fig. 5 has shown the sensitization of arteannuin and derivant and a line chemotherapeutic; Wherein, to be HepG2 and Hep3B cell handle experiment with arteannuin or dihydroarteannuin individual processing or associating gemcitabine to Fig. 1 a, the result shows, gemcitabine acts on HepG2 and Hep3B cell separately, has certain apoptosis facilitation, arteannuin associating gemcitabine all has the effect of remarkable apoptosis facilitation to HepG2 and Hep3B cell, and two medicine association lists reveal synergism; Fig. 5 b shows, DHA and CBP drug combination had the potentiation facilitation to the OVCAR-3 apoptosis after 24 hours, unite the apoptosis rate that apoptosis rate after 500 μ M CBP (1155%) effect is significantly higher than independent usefulness 1 μ M DHA (266%) or 500 μ M CBP (528%) with the DHA of 1 μ M, and surpassed the apoptosis additive effect of two kinds of chemical compounds, be a kind of potentiation, the A2780 cell has shown additive effect to therapeutic alliance; Fig. 5 c shows, when with the DHA coupling, CBP has significantly suppressed the vigor of ovarian cancer cell, when cellular exposure during in 1 μ M CBP and 1 μ M DHA, the survival rate of A2780 cell has just reduced by 69%, OVCAR-3 cell survival and has then reduced 72%, by contrast, it is more insensitive to treatment that the IOSE144 cell then seems, survival rate had only reduced about 28% when two kinds of medicines all share with 1 μ M; Fig. 5 d is hepatoma carcinoma cell Hep3B and the experiment of HepG2 transplanted tumor in nude mice, the result shows, all has antitumor action in arteannuin and the dihydroarteannuin list medicine body, the cancer suppressing action of dihydroarteannuin is better than arteannuin, arteannuin and dihydroarteannuin all can increase the cancer suppressing action of gemcitabine, associating gemcitabine therapeutic effect is better than single medicine, and especially dihydroarteannuin shows significant chemotherapy sensitization; Fig. 5 e is in ovarian cancer cell A2780 and the experiment of OVCAR3 transplanted tumor in nude mice, the result shows, DHA (dosage be 10 with 25mg/kg) causes A2780 heteroplastic transplantation tumor 24% and 41% growth inhibited (comparing with the matched group of giving normal saline) (P＜0.05) respectively, and OVCAR-3 model 14% and 37% tumor growth suppress (P＜0.05); Be suppressed 56% (A2780) and 46% (OVCAR-3) only for the treatment group tumor growth of single CBP, drug combination (25mg/kgDHA) group then all produces 70% tumor growth and suppresses (P＜0.05) in A2780 and OVCAR-3 animal tumor.

Fig. 6 shows the expression of PDGFR α and the relation of ovarian cancer pathology rank and transfering state; Wherein, Fig. 6 a shows, compares with the low level ovarian cancer, and in the high-level ovarian cancer case, the expression of tumor cell and Interstitial cell PDGFR α on every side significantly increases; Fig. 6 b is PDGFR α coloration result, and the result shows that in high-level ovarian cancer case, the expression of PDGFR α significantly increases.

The specific embodiment

The inventor is through extensive and deep research, find the purposes of arteannuin and derivant thereof, inhibitor or antagonist for the preparation of platelet derived growth factor receptor A (PDGFR α), in addition, arteannuin and derivant thereof can also suppress the activation of PDGFR α tyrosine kinase, the protein stability that suppresses PDGFR α, promote the degraded of the proteasome mediation that PDGFR α ubiquitin relies on, suppress the activation of signal path PI3K/AKT and MAPK/EPK, suppress the growth of PDGFR α positive tumor cell, migration and invasion and attack, the sensitizer that can also be used for the treatment of PDGFR α positive tumor.Finished the present invention on this basis.

Term

Ovarian cancer

Ovarian cancer is grave danger of global WomanHealth.In the U.S., ovarian cancer has become women's cancer of the 4th lethal.Though diagnosed 5 annual survival rates of sending out ovarian cancer patients early stage in disease can reach 80%-90%, be diagnosed as the ovarian cancer patients survival rate in late period but only less than 25%, in addition, most of ovarian cancer patients are that ovarian cancer is to late period diagnosing out.The mortality rate many decades in the past of ovarian cancer patients does not have to take place too big change at present, therefore in oncotherapy and prognosis, seek effective therapeutic scheme, strengthening tumor cell is research worker and clinical practice personnel target for a long time to the sensitivity of chemotherapeutics and the drug resistance that overcomes tumor.

Hepatocarcinoma

Hepatocarcinoma is one of modal malignant tumor, and China is the district occurred frequently of hepatocarcinoma, and its sickness rate has risen to second of malignant tumor.Because the early hepatocarcinoma symptom is not obvious, during therefore the most patient of making a definite diagnosis has been in, late period.The first-selection treatment of primary hepatocarcinoma is excision, and along with the raising for the treatment of technology, 5 years survival rates of postoperative have been increased to 50%-60% at present, but because an excision application diameter is no more than 5cm, the cancer number is no more than 3 small liver cancer.In addition, whether be fit to blood vessel enrichment degree and the individual patient difference that direct excision also depends on the cancer position, these restrictions cause the hepatocarcinoma about 90% to be not suitable for operative treatment.Even can excise hepatocarcinoma, it is that main comprehensive therapy also is necessary that postoperative implements to get involved chemotherapy.Yet at present, rarely have medicine can effectively suppress the growth of hepatocarcinoma and further deterioration, hepatocarcinoma is low to the response rate of traditional chemotherapeutics, the toxic and side effects highly significant, so the slow development of chemotherapy is the main cause of restriction antitumor drug clinical practice.Therefore for chemotherapy of tumors, press for and seek better chemical-therapy synergistic agent and better therapeutic scheme, thereby improve tumor to the sensitivity of chemotherapy, reduce it to the drug resistance of chemotherapy.

Platelet derived growth factor and receptor thereof

Platelet derived growth factor (Platelet derived growth factor, PDGF) be that a class can be synthesized the somatomedin of justacrine in the extracellular matrix, have cell division, propagation, the migration of multiple tissues such as promoting fibrocyte, smooth muscle cell, the ability of sticking of increase cell, in people's fetal development and normal physiological activity, bringing into play important effect.PDGF activates platelet derived growth factor receptor (PDGFR) with autocrine and paracrine form and plays a role.

PDGFR comprises tyrosine kinase receptor PDGFR α and the PDGFR β of two kinds of similar, and wherein the abnormal activation of PDGFR α and multiple disease are closely related, and particularly, the PDGFR relevant disease can be divided three classes:

1. tumor

Because PDGFR (particularly PDGFR α) is present in the kinds of tumors, it may be as a new treatment target spot.The generation that has been found that kinds of tumors is relevant with the excessive activation of PDGFR (particularly PDGFR α), as ovarian cancer, glioma, carcinoma of prostate; In addition, in some tumors, exist the sudden change of PDGFR to activate, as the chronic myeloid leukemia of dermatofibrosarcoma protuberans, gastrointestinal stromal tumor, Bcr-Abl feminine gender, hypereosinophilic syndrome etc.

2. vascular conditions

Discover, PDGF and PDGFR etc. exist overexpression and excessive activation phenomenon in multiple vascular conditions, as atherosclerosis and restenosis, pulmonary hypertension, retinal vasculopathy etc., the symptom of above-mentioned disease can be effectively alleviated in the activation that suppresses the PDGFR signal path, increases its therapeutic effect.

3. fibrous lesions

The PDGF-PDGFR signal path plays an important role in multiple fibrous lesions process, as pulmonary fibrosis, hepatic fibrosis and liver cirrhosis, skin fiber hypertrophy (scleroderma), renal fibrosis, myocardial fibrosis etc., wherein the propagation of the Interstitial cell of PDGF-PDGFR signal path mediation is the common trait of this chronic inflammatory disease.

In the above-mentioned three class diseases, the PDGF-PDGFR signal path plays an important role, and wherein PDGFR β has mainly mediated the vascular system pathological changes, and PDGFR α has mainly mediated the pathological changes of Interstitial cell and fibroblast driving.(Johanna Andrae, et al.Role of platelet-derived growth factors in physiology and medicine, Genes Dev.2008; 22:1276-1312; Zhang Xiuhua etc., platelet derived growth factor receptor and tumor. life sciences, Vol.18, No.3 Jun., 2006)

Arteannuin and derivant thereof

Artemisinine is the sesquiterpene lactones medicine that peroxy-radical is arranged that extracts from the Chinese medicine Artemisia annua L., because arteannuin is complicated macromole, chemical total man worker is difficult to synthetic, therefore mainly is biosynthesis and artificial the extraction at present.

The biosynthesis of sesquiterpenoidss such as arteannuin carries out in Cytoplasm, approach belongs to Plant Isoprenoid Metabolic Pathway, can be divided into three goes on foot greatly: form FPP by acetic acid, synthetic sesquiterpene, formation arteannuin again lactonizes, be specially: FPP → 4,11-diene sesquiterpene → artelinic acid → dihydroartemisinic acid → dioxy artelinic acid peroxide → arteannuin.

The chemical formula of the artemisinin derivative that arteannuin and this area are several frequently seen is as follows:

The application of arteannuin and derivant thereof

The invention provides arteannuin and derivant thereof for the preparation of the inhibitor of platelet derived growth factor receptor A (PDGFR α) or the purposes of antagonist.

In another preference, arteannuin and derivant thereof also are used for:

(i) activation of inhibition PDGFR α tyrosine kinase; And/or

The protein stability that (ii) suppresses PDGFR α; And/or

(iii) promote the degraded of the proteasome mediation that PDGFR α ubiquitin relies on; And/or

(vi) suppress the activation of PDGFR α positive tumor cell signal path PI3K/AKT and MAPK/EPK; And/or

(v) suppress growth, migration and the invasion and attack of PDGFR α positive tumor cell; And/or

(vi) preparation suppresses medicine or the sensitizer of PDGFR α positive tumor; And/or

(vii) medicine or the sensitizer of vascular conditions and fibrous lesions treated in preparation.

In a preference of the present invention, provide a kind of external non-therapeutic ground to suppress the active method of platelet derived growth factor receptor A (PDGFR α), comprise step: with arteannuin and/or artemisinin derivative and cells contacting, thus platelet derived growth factor receptor A (PDGFR α) in inhibition or the antagonism cell.

Described contact is in the presence of arteannuin and/or artemisinin derivative, cultivates described cell.Cell comprises tumor cell, preferably comprises PDGFR α positive tumor cell, more preferably comprises the positive ovarian cancer cell of PDGFR α or hepatoma carcinoma cell, as ovarian cancer cell A2780, ovarian cancer cell OVCAR3, hepatoma carcinoma cell Hep3B.In another preference, described method also is used for following at least a kind of application: external non-therapeutic ground suppresses the activation of PDGFR α tyrosine kinase; External non-therapeutic ground suppresses the protein stability of PDGFR α; External non-therapeutic ground promotes the degraded of the proteasome mediation that PDGFR α ubiquitin relies on; External non-therapeutic ground suppresses the activation of signal path PI3K/AKT and MAPK/EPK; Growth with external non-therapeutic inhibition PDGFR α positive tumor cell.

The present invention also provides a kind of method of the PDGFR of preventing and/or treating alpha associated disorders, wherein said disease is relevant with expression excessively or the hyperactivity of PDGFR α, described method comprises step: use arteannuin and/or artemisinin derivative for the object of needs, or use the pharmaceutical composition that contains arteannuin and/or artemisinin derivative.The PDGFR alpha associated disorders comprises tumor (PDGFR α positive tumor), as ovarian cancer or hepatocarcinoma.In another preference, described using comprises: in the tumor, intravenous injection, oral tablet, lumbar injection etc.Described object is mammal (as the people).

Medicament composition and application thereof

The present invention also provides medicament composition, and in a preference, described preparation combination comprises:

(I) contain the preparation of arteannuin and/or artemisinin derivative; (II) contain the preparation of gemcitabine or carboplatin,

The combination of described preparation for the preparation of: (a) suppress pharmaceutical composition or the medicine box of platelet derived growth factor receptor A; Or (b) prevent and/or treat pharmaceutical composition or the medicine box of tumor.

Described preparation comprises tablet, capsule, suppository, injection; Described pharmaceutical preparation is used for prevention and treatment PDGFR α positive tumor.

Medicament composition of the present invention can be solid or liquid.But solid preparation comprises powder, tablet, pill, capsule, cachet, suppository and dispersible granule, and solid carrier can be one or more materials, and they can be used as diluent, correctives, adhesive, antiseptic, tablet disintegrant or coating material.In powder, carrier is the solid of fine branch, and the active constituent of chemical compound of the present invention and fine branch is present in the mixture.In tablet, this chemical compound mixes with proper proportion with required bonding carrier, and is pressed into required shape and size.Preferentially contain 5 to 70% reactive compounds in powder and the tablet, suitable carrier is magnesium carbonate, magnesium stearate, Talcum, sugar, lactose, pectin, dextrin, starch, gelatin, carboxymethyl cellulose, sodium carboxymethyl cellulose, low melting point is cured and cocoa butter etc.Equally, cachet or lozenge, tablet, powder, capsule, pill, cachet and lozenge can be the solid dosage formss that is suitable for oral administration.In order to prepare suppository, can be with the mixture melt of the cured butter oil acid glyceride of low melting point or cocoa butter, and by stirring this reactive compound component is evenly dispersed in wherein, the homogeneous mixture of fusing is poured into allowed its cooling in the sizeable mould then, and solidify thus.The preparation of solution form comprises solution, suspending agent and Emulsion, for example water or aqueous propylene glycol solution.For non-intestinal injecting fluid preparation, can in moisture polyglycol solution, prepare.Being suitable for oral aqueous solution can be by being dissolved in the water active constituent, and adds suitable coloring agent, correctives, emulsifying agent and thickening agent preparation as required.Be suitable for oral aqueous suspensions and can be scattered in by the active constituent with fine branch in the moisture stickum and prepare, as natural or rubber polymer, resin, methylcellulose, Carboxymethyl cellulose sodium and other suspensoids of knowing.These liquid forms comprise solution, suspensoid and Emulsion, and except active constituent, these preparations can contain coloring agent, correctives, stabilizing agent, buffer agent, synthetic or natural sweetener, dispersant, thickening agent and cosolvent etc.This pharmaceutical preparation preferentially is unit dosage forms, in this dosage form, this preparation is subdivided into the unit dose that contains an amount of active constituent, and this unit dose can be the preparation of packing, this packing contains a certain amount of preparation, as tablet, capsule and the powder in bottle or capsule of packing.This unit dosage forms can also be capsule, tablet, cachet or lozenge itself, or can there be any of these powder of the right quantity in the packaged form in it.

The amount of active constituent can change according to the effectiveness of concrete application and active constituent in the unit dose formulations, treat for non-human mammal (as mice), can be with 0.01mg to about 0.5g, preferably 0.1 to about 3mg capsule is administered three times every day, and said composition can also contain other compatible therapeutic agents in case of necessity.Treatment for the people, can be according to patient's needs, changed by the chemical compound of sanatory seriousness and use, preferably, beginning is with the smaller dose treatment less than this chemical compound optimal dose, after this, increasing this dosage reaches optimum efficiency in a small amount, for the purpose of making things convenient for, total daily dose can be subdivided into again gradation administration in a day if desired.In a preference of the present invention, the dosage of administration of human is approximately between the 0.1/kg-0.5g/kg.

Major advantage of the present invention:

(1) arteannuin and derivant thereof can be for the preparation of inhibitor or the antagonisies of platelet derived growth factor receptor A (PDGFR α);

(2) arteannuin and derivant thereof can suppress growth, migration and the invasion and attack of the activation of PDGFR α tyrosine kinase, the protein stability of PDGFR α, the activation that suppresses signal path PI3K/AKT and MAPK/EPK, inhibition PDGFR α positive tumor cell;

(3) arteannuin and derivant thereof can promote the degraded of the proteasome mediation that PDGFR α ubiquitin relies on;

(4) arteannuin and derivant thereof can be used for the sensitizer of PDGFR α positive tumor treatment.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Experimental technique and material

1. surface plasma resonance

Surface plasma resonance (SPR) is a kind of optical phenomena, can be used to real-time tracking interaction between biomolecule under native state.In the experiment, with recombined human PDGFR α born of the same parents outer ends (Met1-Glu524) albumen, be bonded in biosensor surface, with the dihydroarteannuin solution of variable concentrations with have interactional chemical compound Suo Lafeini and SU11248 solution to inject and the biosensor surface of flowing through with PDGFR α always.Combination between biomolecule causes the increase of biosensor surface quality, causes refractive index to strengthen in same ratio, and the variation of reacting between biomolecule namely is observed, and this reaction is weighed with reacton (RU).

2. cell culture

The non-tumorigenic epithelial cell IOSE-144 of people's ovary immortalization and ovarian cancer cell (A2780, OVCAR-3, SK-OV3, OVCAR-5) available from U.S. ATCC (American Type Culture Collection, Manassas, VA).All cells requires (RPMI-1640 or DMEM culture fluid add 10% hyclone (GIBCO), 100IU/ml penicillin, 100IU/ml streptomycin) to cultivate in 37 ℃ of incubators of 5%CO2 according to the cultivation of ATCC.

3. cell growth inhibition test

Cell growth and propagation inhibition test are by the CCK-8 measuring.Cell with the density cover plant in 3 * 103 every holes of cell in 96 orifice plates.After 12～18 hours, after handling 48 hours with the artemisine compounds of finite concentration gradient (0,1,5,10,25 μ M), add the cells growth activity evaluation after 10 μ L CCK-8 solution carry out drug treating in the every hole of 96 orifice plates, experiment is last by microplate reader (SpectraMax190microplate reader; Molecular Devices, the USA) light absorption (OD value) at measurement 450nm place (every experimental concentration arranges three multiple holes at least, tests triplicate at least at every turn).

4. cell migration, invasion and attack experiment

The migration of ovarian cancer cell and invasive ability are by the transwell measuring, the low concentration artemisinin derivative short time (12h) is handled ovarian cancer cell, cell (5 * 104) with similar number is resuspended in the culture medium of serum-free then, plantation is in transwell (when measuring the invasive ability of cell, transwell spreads matrigel in the upper strata) upper strata culturing room.With 10% hyclone culture medium as chemical chemoattractant.Approximately behind the 8-12h, Qian Yi cell is not wiped with cotton swab, the cell of migration is fixed dyeing, and carry out statistical analysis.

5.SDS-polyacrylamide gel electrophoresis and protein immunoblot analysis

DHA with variable concentrations handled cultured cell 24 hours, and collecting cell is also used cell pyrolysis liquid (RIPA buffer #9806, Cell Signaling) cracking.Change in 4 ℃ of centrifugal 15min with 13,000 afterwards, get supernatant and carry out follow-up analyzing and testing.Protein concentration is by test kit (the protein assay kit of Bio-Rad company; Hercules, CA) quantitative Tot Prot.Equal protein is carried out the separation of SDS-PAGE gel electrophoresis on every hole afterwards.Electrophoresis finishes, with the electrophoresis protein band be transferred to the pvdf membrane that methanol activated (Millipore, Bedford, MA) on.After the commentaries on classics film finishes, film seals with 5% skim milk (PBS dissolving), add primary antibodie and two anti-hatching (the BSA configuration with 5%) then, change with albumen development test kit (ECL plus system, Amersham Pharmacia Biotech) development by the protein band differential expression afterwards and analyze.

6. mice original position ovarian cancer heteroplastic transplantation tumor model body internal therapy experiment

The female BALB/c (nu/nu) in four to six ages in week and raises in SPF level Animal House in accordance with regulations available from Shanghai Slac Experimental Animal Co., Ltd. (Shanghai Experimental Animal Center).All zoopery operations all obtain the approval of animal Ethics Committee of nutrition institute of the Chinese Academy of Sciences.。General steps is as follows: with the A2780 cell of luciferase labelling, be resuspended in RPMI 1640 culture medium of serum-free.Lumbar injection equivalent cell (～3 * 106 cells/0.2ml) is in the inside, abdominal cavity of mice then.Utilize IVIS Lumina bioluminescence system to monitor the growth of tumor situation in real time, and the body weight of record mice.Inject after 5 days, during according to the fluorescence intensity of tumor mice is divided into treatment group and matched group (every group of 8 mouse).Treatment group medicine DHA be dissolved in Oleum Ricini, ethanol and normal saline mixture (Cremophor EL: Ethanol: Saline=5: 5: 90, v/v/v) in, all medicines all pass through intraperitoneal administration.The dosage of DHA is 10 or the 25mg/kg body weight, and administration frequency is administration every day, rests weekly two days.The matched group injecting normal saline.In therapeutic process, utilize the living imaging system to monitor the growth of tumor situation in real time.After treatment finished, the careful taking-up moved abdominal tumor (removing fat and connective tissue), with PIPA solution (the 100mg tissue adds 1ml RIPA) homogenate.The tumor tissues homogenate liquid that obtains is analyzed requirement of experiment by above-mentioned protein immunoblot and is handled, and carries out protein immunoblot afterwards and detects; Or tumor tissues fixed and immunostaining.

Embodiment 1

Arteannuin and derivant thereof suppress its phosphorylation activity directly in conjunction with PDGFR α, promote its degraded

Utilize the experiment of PDGFR α vitro kinase, surface plasma resonance, area of computer aided are built with pattern and oppositely advanced persons such as equimolecular pharmacology and biophysics are tested in butt joint biology techniques and means, confirm that artemisinin derivatives can be directly in conjunction with PDGFR α, the receptor tyrosine kinase activity that suppresses PDGFR α, discover that simultaneously dihydroarteannuin can suppress the protein stability of PDGFR α in the PDGFR α positive cell, promote the degradation process that its ubiquitin relies on.

Fig. 1 shows that arteannuin and derivant thereof can be directly in conjunction with PDGFR α, and PDGFR α is as the direct target of arteannuin and derivant effect thereof, and can suppress its phosphorylation activity, promotes its degraded.Vitro kinase experiment shows arteannuin and derivant thereof: arteannuin (ART), dihydroartemisinine (DHA), artesunate (ARM), Artemether (ARS) can suppress the activity of born of the same parents the inner (aa550-end) tyrosine kinase of PDGFR α (Fig. 1 a).Surface plasma resonance (SPR) experiment shows that dihydroarteannuin (DHA) can mutually combine with PDGFR α bag outer end (aa24-524) albumen, and Suo Lafeini (Sorafenib) is as positive control, known can be in conjunction with the activity (Fig. 1 b) that suppresses PDGFR α.The DHA of biotin-avidin affinity purification experiment show tags biotin can mutually combine with PDGFR α in the A2780 cell, thereby is come out (Fig. 1 c) by affinity purification.Area of computer aided is built and is oppositely docked experiment (Computer Assisted Homology modeling and docking) experiment and shows that dihydroarteannuin (DHA) can be fitted to PDGFR α bag outer end PDGF binding site with pattern, simultaneously also can be fitted to it and wrap inner ATP-binding site, block its kinase activator (Fig. 1 d).Fig. 1 e-Fig. 1 f has shown that dihydroarteannuin can effectively suppress activity and the protein expression of PDGFR α among PDGFR α positive cell A2780 and the OVCAR3, and little to phosphorylation and the protein level influence of PDGFR β.Fig. 1 g-Fig. 1 i experiment shows that dihydroarteannuin can accelerate the protein degradation of PDGFR α, and this process is the proteasome mediation that ubiquitin relies on.

Embodiment 2

Arteannuin and derivant thereof suppress growth and the transfer of the tumor cell of the PDGFR α positive

Utilize nude mice original position ovarian cancer heteroplastic transplantation tumor model, research dihydroarteannuin ovarian cancer resistance activity in animal body.The Proliferation of Human Ovarian Cell A2780 of luciferase labelling is injected into the abdominal cavity of Balb-c nude mice, sets up the former bit model of nude mice ovarian cancer, give the dihydroarteannuin treatment then: matched group (normal saline), low dose group (DHA 10mg/kg).High dose group (DHA 25mg/kg) is treated weekly five times, rests two days, gives for 6 treatment times in week altogether.Utilize growth and the transfer case of living imaging systematic observation mouse ovarian cancer during this time.

A series of researchs by cellular level and animal level, show that artemisinin derivatives can suppress growth, migration and the invasion and attack (ovarian cancer and hepatocarcinoma) of the tumor cell of the PDGFR α positive effectively, and less for the inhibition ability of the growth of tumour cell of PDGF-B expression R α not and transfer ability.Concrete outcome is as follows:

Fig. 2 shows that arteannuin and derivant thereof suppress growth and the transfer ability of the ovarian cancer cell of the PDGFRa positive.Particularly, shown in Fig. 1 e and Fig. 2 a, PDGFRa is at ovary normal cell epithelial cell IOSE144, and ovarian cancer cell SK-OV3 does not have among the OVCAR5 and expresses high expressed in ovarian cancer cell A2780 and OVCAR3; After giving medicine irritation 48h, the arteannuin of variable concentrations and derivant people normal ovarian epithelial cell IOSE144 thereof do not have obvious growth and suppress ability, less for the SK-OV3 cytotoxicity, but A2780 and OVCAR3 for the PDGFRa positive have the ability that suppresses more by force, and wherein the effect of arteannuin and dihydroartemisinine is the most remarkable.The cell migration experiment show lower concentration short time is handled (12h) ovarian cancer cell, arteannuin and derivant thereof can suppress the transfer ability of PDGFRa positive cell A2780 effectively, and do not have obvious inhibition ability (Fig. 2 b) for the cell SK-OV3 of PDGFRa feminine gender.Dihydroarteannuin suppresses the most obviously (Fig. 2 c-Fig. 2 e) for growth and the migration invasive ability of PDGFRa positive cells in the artemisinin derivative.

Also obtained similar result in the hepatoma carcinoma cell, Fig. 3 shows that arteannuin and derivant thereof can suppress growth and the transfer ability of the hepatoma carcinoma cell of the PDGFRa positive.Particularly, numerous people's normal liver cells (7702 and LO2) and hepatoma cell line (Hep3B, HepG2,7721,7404, LM3,97L, 97M, Huh-7) in, (Fig. 3 is a) for Hep3B high expressed PDGFRa; Low concentration dihydroarteannuin (3 μ M) (12h) processing Hep3B cell in short-term can significantly suppress its migration and invasive ability (Fig. 3 b); The cell growth experiment shows that simultaneously the Hep3B cell of the PDGFRa positive is more responsive for dihydroarteannuin, and express on other PDGFRa ground and the hepatoma carcinoma cell of not having an expression for dihydroarteannuin sensitivity lower (Fig. 3 c).

Zoopery assessment result (Fig. 4) shows, dihydroarteannuin has an inhibition ability for the growth of PDGFRa positive cell A2780 and transfer.Particularly, (Fig. 4 a) after two blue and green artemisins are treated the malignant progression that suppresses ovarian cancer effectively; Compare with the contrast group, the situation for shifting that gives dihydroarteannuin treatment group mice reduces greatly, and the situation that send out in the tumor abdominal cavity also obtains obvious suppression (Fig. 4 b, Fig. 4 c); SABC and immunoblot experiment show simultaneously, dihydroarteannuin can suppress expression and the phosphorylation of PDGFRa effectively, downstream signal path PI3K/AKT and MAPK-ERK are suppressed, matter modality process (EMT) be inhibited (Fig. 4 d, Fig. 4 e) between epithelium.

Embodiment 3

The sensitization of arteannuin and derivant thereof and a line chemotherapeutic

The external independent application of arteannuin and dihydroarteannuin all can significantly suppress growth, propagation and the migration invasive ability of ovarian cancer and hepatoma carcinoma cell.Whether present embodiment has the chemotherapy sensitizing effect to it is detected, arteannuin and dihydroarteannuin are united gemcitabine respectively and are acted on human liver cancer cell HepG2 and Hep3B, and the associating carboplatin acts on the inhibitory action of ovarian cancer A2780 and OVCAR3 observation drug combination cell growth.Result (Fig. 5) is as follows:

HepG2, the Hep3B cell is with 10 μ mol/L arteannuin or dihydroarteannuin individual processing or unite 10 μ g/L gemcitabines processing 48 hours, carries out the apoptosis detection by quantitative as stated above.Particularly, gemcitabine list medicine acts on HepG2, and the Hep3B cell demonstrates certain apoptosis facilitation, and (Fig. 5 a), arteannuin associating gemcitabine is to HepG2, the Hep3B cell has the effect of remarkable apoptosis facilitation, and two medicine association lists reveal synergism, and (Fig. 5 a).DHA and CBP drug combination had potentiation facilitation (Fig. 5 b) to the OVCAR-3 apoptosis after 24 hours; Unite the apoptosis rate that apoptosis rate after 500 μ M CBP (1155%) effect is significantly higher than independent usefulness 1 μ M DHA (266%) or 500 μ M CBP (528%) with 1 μ M DHA, and surpassed the apoptosis additive effect of two kinds of chemical compounds, what demonstrate is a kind of potential potentiation.Yet what the A2780 cell showed therapeutic alliance is additive effect rather than synergy.This may be because their causes lower to the sensitivity of DHA chemical compound (Fig. 5 b, P＜0.05).The effect of longer time may produce better effect.Aspect the inhibition of cell proliferation vigor, when with the DHA coupling, CBP has suppressed the vigor of ovarian cancer cell very significantly.In fact, when cellular exposure during in the DHA of 1 μ M CBP and 1 μ M, the survival rate of A2780 cell has reduced by 69%, OVCAR-3 cell survival and has then reduced 72%.By contrast, it is more insensitive to treatment that the IOSE144 cell then seems, survival rate had only reduced about 28% (Fig. 5 c) when two kinds of medicines all share with 1 μ M.

Cell experiment proof arteannuin and dihydroarteannuin all demonstrate for gemcitabine external, and carboplatin is handled killing and wounding and apoptosis-induced sensitization of cancerous cell.In addition, the transplanted tumor in nude mice experiment proves that further they are effective too for the chemotherapy of in-vivo tumour.Particularly, show in hepatoma carcinoma cell Hep3B and the experiment of HepG2 transplanted tumor in nude mice, all to have antitumor action in arteannuin and the dihydroarteannuin list medicine body as Fig. 5 d, the cancer suppressing action of dihydroarteannuin is better than arteannuin.Arteannuin and dihydroarteannuin all can increase the cancer suppressing action of gemcitabine, and associating gemcitabine therapeutic effect is better than single medicine, and especially dihydroarteannuin shows significant chemotherapy sensitization.In ovarian cancer cell A2780 and the experiment of OVCAR3 transplanted tumor in nude mice (Fig. 5 e), DHA (dosage be 10 with 25mg/kg) causes A2780 heteroplastic transplantation tumor 24% and 41% growth inhibited (comparing with the matched group of giving normal saline) (P＜0.05) respectively, and OVCAR-3 model 14% and 37% tumor growth suppress (P＜0.05); Be suppressed 56% (A2780) and 46% (OVCAR-3) only for the treatment group tumor growth of single CBP.Drug combination (25mg/kg DHA) group then all produces 70% tumor growth and suppresses (P＜0.05) in A2780 and OVCAR-3 animal tumor.

Above cell and the experiment of animal level show, arteannuin and derivant thereof have the sensitization of good independent chemotherapy and associating one linearize treatment medicine, in conjunction with the artemisine derivative compound directly targeting PDGFRa bring into play this result of its anti-tumor activity, the prompting artemisinin-based drug can be used for clinical associating multiclass chemotherapeutics and treat tumour patient, and especially pathological diagnosis is the tumour patient of the PDGFRa positive

Embodiment 4

PDGFR α is with pathological grading and shift relevant

Present embodiment according to the clinical pathology diagnostic message strictness chosen 45 routine ovarian cancer patients' pathology sample, comprising 21 routine low level ovarian cancers (the clinical case rank is I-II, and moderate or highly differentiation do not have tangible lymph node, transfers such as abdominal cavity and nethike embrane); 24 routine high-level ovarian cancer patients (the clinical case rank is III-IV, and low differentiation has large-area lymphatic metastasis, abdominal cavity, nethike embrane, uterus, adnexa and other organ metastasis).

The result shows (see figure 6), compares with the low level ovarian cancer, and in the high-level ovarian cancer case, the expression of tumor cell and Interstitial cell PDGFR α on every side significantly increases.Cell experiment shows, utilizes the gene perturbation technique of slow virus mediation that silence is carried out in the expression of PDGFR α among ovarian cancer cell A2780 and the OVCAR3, finds that the growth of tumor cell and migration invasive ability significantly reduce.

Embodiment 5

Preparation of drug combination

Compositions 1

Compositions 2

Discuss

Existing discovery shows, PDGFR gene mutation or excessive activation can promote the transformation ability and then cause the generation of malignant tumor, as gastrointestinal stromal tumors (GISTs), and pulmonary carcinoma, carcinoma of prostate and breast carcinoma.Somatomedin such as PDGFR is combined the degraded that improves the iuntercellular adhesion molecule with its part, promote invasion by tumor cells and transfer, can also activate the VEGF in the microenvironment simultaneously, FGF and then promote angiogenesis have accelerated the malignant progression process of tumor.Zoopery and Clinical pathological study find that PDGFR α overexpression and activation have participated in generation and the evolution of ovarian cancer, with patient's pathology rank and poor prognosis significant correlation.

Content of the present invention shows, compare with ovarian cancer patient's pathological tissue that low level shifts, the expression of PDGFR α significantly increases in extensive its cancerous cell of ovarian cancer patient that shifts of high-level generation and the mesenchyma stroma of tumors cell, this just shows that PDGFR α can be used as a target spot of ovarian cancer treatment.

Find artemisinin derivatives among the present invention, especially arteannuin and dihydroarteannuin can directly be attached in the PDGFRa molecule, have both suppressed the receptor tyrosine kinase activity of PDGFRa, suppress the protein stability of PDGFRa again, promote its ubiquitin degraded.

Molecule and zoopery show that the medicine of artemisine can be by suppressing multiple growth of tumor, migration and the invasive ability that PDGFRa and then inhibition comprise ovarian cancer and hepatocarcinoma.This result shows that artemisinin-based drug can be used as a kind of special inhibitor of PDGFR, is used for the tumour patient of PDGFRa sudden change clinically or excessive activation, comprises ovarian cancer, hepatocarcinoma, gastrointestinal stromal tumors (GISTs), acute leukemia etc.Artemisinin derivatives also has good anticancer sensitizing activity, for example artemisinin-based drug can significantly improve a line chemotherapeutic carboplatin, gemcitabines etc. are for the therapeutic effect of ovarian cancer and hepatocarcinoma, especially dihydroarteannuin has stronger chemotherapy sensitization to carboplatin and gemcitabine, its characteristics of high efficiency and low toxicity makes it to be expected to as chemotherapeutic sensitizer, is applied to the clinical combined chemotherapy of tumor.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

技术领域

本发明涉及生物医药领域，具体地，本发明涉及青蒿素及其衍生物在抑制血小板衍生生长因子受体A中的作用及其应用。

背景技术

青蒿又名黄花蒿(Artemisia anaua L)，属菊科，是一年生草本植物。青蒿素是我国药学工作者于20世纪70年代初从中药青蒿中提取的倍半萜内酯类抗疟药物。鉴于传统的奎宁类药物普遍遇到抗药性问题，青蒿素和它的衍生物目前已取代它们，成为世界上抗疟疾的主流药物。青蒿素的大环上有一个内过氧化物桥，在亚铁离子或亚铁血红素的催化下，会放出以碳为中心的自由基。细胞内的蛋白质和核酸被这些自由基烷化后死亡。疟原虫寄生在红血球内，含有大量来自血红蛋白的亚铁血红素，因而青蒿素很易被活化，进而将疟原虫杀死。青蒿素和它的衍生物，如二氢青蒿素，蒿甲醚，蒿乙醚及青蒿琥酯等已广泛应用于疟疾治疗，更多的青蒿素衍生物正在研制中。

青蒿素及其衍生物的抗疟活性已得到世界公认，具有起效快、药效高、毒副作用低等优点。除广泛应用于抗疟治疗外，青蒿素类药物还具有其它多种药理作用，如抗血吸虫作用、抗心律失常、平喘、抗内毒素、抗变态反应、抗红斑狼疮、免疫抑制等作用。

随着对青蒿素及其衍生物活性研究的不断深入，人们发现，该类化合物对多种肿瘤细胞的生长具有抑制作用和良好的治疗效果，然而，其具体作用靶点及相关信号通路及作用机制还不明确。因此本领域迫切需要了解青蒿素及其衍生物在一直肿瘤中的作用。

发明内容

本发明的目的是提供青蒿素及其衍生物在抑制血小板衍生生长因子受体A中的作用。

本发明的另一目的是提供青蒿素及其衍生物作为血小板衍生生长因子受体A抑制剂/拮抗剂的应用。

本发明的另一目的是提供体外治疗肿瘤的方法。

在本发明的第一方面，提供了一种青蒿素及其衍生物用于制备抑制选自下组疾病的药物或药物增敏剂的用途：PDGFRα阳性肿瘤、血管性疾病、或纤维性病变。

在另一优选例中，所述的PDGFRα阳性肿瘤细胞为PDGFRα阳性卵巢癌细胞或PDGFRα阳性肝癌细胞。

在本发明的第二部分，提供了一种青蒿素及其衍生物的用途，用于制备血小板衍生生长因子受体A(PDGFRα)的抑制剂或拮抗剂。

在另一优选例中，所述的血小板衍生生长因子受体A(PDGFRα)来自于人。

在另一优选例中，所述的抑制剂或拮抗剂还用于：

(i)抑制PDGFRα酪氨酸激酶的活化；和/或

(ii)抑制PDGFRα的蛋白稳定性；和/或

(iii)促进PDGFRα泛素依赖的蛋白酶体介导的降解；和/或

(vi)抑制PDGFRα阳性肿瘤细胞信号通路PI3K/AKT和MAPK/EPK的活化。

在另一优选例中，所述的青蒿素衍生物选自下组：蒿甲醚、二氢青蒿素、青蒿琥酯、双氢蒿甲醚、蒿乙醚等。

在另一优选例中，所述的青蒿素衍生物为二氢青蒿素。

在本发明的第三方面，提供了一种体外非治疗性地抑制血小板衍生生长因子受体A(PDGFRα)活性的方法，包括步骤：将青蒿素和/或青蒿素衍生物与细胞接触，从而抑制或拮抗细胞中血小板衍生生长因子受体A(PDGFRα)。

在另一优选例中，所述的接触是在青蒿素和/或青蒿素衍生物存在下，培养所述细胞。

在另一优选例中，所述的细胞包括肿瘤细胞，较佳地包括PDGFRα阳性肿瘤细胞，更佳地包括PDGFRα阳性卵巢癌细胞或肝癌细胞，如卵巢癌细胞A2780、卵巢癌细胞OVCAR3、肝癌细胞Hep3B。

在另一优选例中，所述方法还用于至少以下一种应用：

体外非治疗性地抑制PDGFRα酪氨酸激酶的活化；

体外非治疗性地抑制PDGFRα的蛋白稳定性；

体外非治疗性地促进PDGFRα泛素依赖的蛋白酶体介导的降解；

体外非治疗性地抑制信号通路PI3K/AKT和MAPK/EPK的活化；和

体外非治疗性抑制PDGFRα阳性肿瘤细胞的生长。

在本发明的第四方面，提供了一种制剂组合的用途，所述制剂组合包括：

(I)含青蒿素和/或青蒿素衍生物的制剂；和

(II)含吉西他宾或卡铂的制剂，

所述制剂组合用于制备：

(a)抑制血小板衍生生长因子受体A的药物组合物或药盒；或

(b)预防和/或治疗肿瘤的药物组合物或药盒。

在另一优选例中，所述制剂包括：片剂、胶囊、栓剂、注射剂。

在另一优选例中，所述肿瘤为PDGFRα阳性肿瘤。

在本发明的第五方面，提供了一种预防和/或治疗PDGFRα相关疾病的方法，其中所述疾病与PDGFRα的过表达或活性过高有关，所述方法包括步骤：给需要的对象施用青蒿素和/或青蒿素衍生物，或施用含青蒿素和/或青蒿素衍生物的药物组合物。

在另一优选例中，所述的PDGFRα相关疾病包括肿瘤(PDGFRα阳性肿瘤)，如卵巢癌或肝癌。

在另一优选例中，PDGFRα阳性肿瘤为PDGFRα阳性卵巢癌或PDGFRα阳性肝癌。

在另一优选例中，所述的施用包括：瘤内、静脉注射、口服片剂、腹腔注射等。

在另一优选例中，所述的对象为哺乳动物。

在本发明的第六方面，提供了一种方法，所述方法用于体内或体外抑制PDGFRα酪氨酸激酶的活化、抑制PDGFRα的蛋白稳定性、促进PDGFRα泛素依赖的蛋白酶体介导的降解、抑制信号通路PI3K/AKT和MAPK/EPK的活化、和/或抑制PDGFRα阳性肿瘤细胞的生长、迁移和侵袭，所述方法包括步骤：给需要的对象施用青蒿素和/或青蒿素衍生物。

在本发明的第七方面，提供了一种用于卵巢癌病理分级的试剂盒，所述试剂盒包含检测离体组织PDGFRα表达量的试剂。

应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。

附图说明

下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。

图1显示PDGFRα可以作为青蒿素及其衍生物的直接作用靶标；其中，图1a为体外激酶实验，结果显示，青蒿素及其衍生物青蒿素(ART)、二氢青蒿素(DHA)、青蒿琥酯(ARM)、蒿甲醚(ARS)可以抑制PDGFRα胞内端(aa550-末端)酪氨酸激酶的活性；图1b为表面等离子共振(SPR)实验，结果显示，双氢青蒿素(DHA)可以与PDGFRα包外端(aa24-524)蛋白相互结合，索拉菲尼(Sorafenib)为阳性对照，已知其可以结合抑制PDGFRα的活性；图1c为生物素-亲和素亲和纯化实验，结果显示，标记生物素的DHA可以与A2780细胞内PDGFRα相互结合，从而被亲和纯化出来；图1d为计算机辅助同型模建及反向对接实验(ComputerAssisted Homology modeling and docking)，结果显示，双氢青蒿素(DHA)可以嵌合到PDGFRα包外端PDGF结合位点，同时也可嵌合到其包内端ATP结合位点，阻断其激酶激活；图1e-图1f显示双氢青蒿素能够有效抑制PDGFRα阳性细胞A2780和OVCAR3中PDGFRα的活性和蛋白表达，而对PDGFRβ的磷酸化和蛋白水平影响不大；图1g-图1i显示双氢青蒿素可以加速PDGFRα的蛋白降解，这一过程是泛素依赖的蛋白酶体介导的。

图2显示了青蒿素及其衍生物具有抑制PDGFRa阳性的卵巢癌细胞的生长和迁移能力；其中，图2a显示，给予药物刺激48h后，青蒿素及其衍生物对人正常卵巢上皮细胞IOSE144无明显的生长抑制能力，对于SK-OV3细胞毒性较小，但对于PDGFRa阳性的A2780和OVCAR3具有较强抑制能力，其中青蒿素和二氢青蒿素的作用最为显著；图2b为细胞迁移实验，结果显示，低浓度短时间处理(12h)卵巢癌细胞，青蒿素及其衍生物能够有效地抑制PDGFRa阳性细胞A2780的迁移能力，而对于PDGFRa阴性的细胞SK-OV3无明显抑制能力；图2c显示了不同浓度的双氢青蒿素对5种细胞系(IOSE144、A2780、OVCAR3、OVCAR5、SK-OV3)的细胞抑制作用；图2d显示了不同浓度的双氢青蒿素对3种细胞系(A2780、OVCAR3、SK-OV3)的细胞迁移的影响；图2e显示了不同浓度的双氢青蒿素对2种细胞系(A2780、OVCAR3)的细胞侵袭的影响。

图3显示青蒿素及其衍生物具有抑制PDGFRa阳性的肝癌细胞的生长和迁移能力；其中，图3a显示，在人正常的肝细胞系(7702和LO2)和肝癌细胞系(Hep3B、HepG2、7721、7404、LM3、97L、97M、Huh-7)中，只有Hep3B高表达PDGFRa；图3b显示，低浓度双氢青蒿素(3μM)短时(12h)处理Hep3B细胞，能够显著抑制其迁移和侵袭能力；图3c为细胞生长实验，结果显示，PDGFRa阳性的Hep3B细胞对于双氢青蒿素更敏感，而其他细胞对于双氢青蒿素敏感程度较低。

图4显示双氢青蒿素能抑制小鼠原位卵巢癌模型的生长和转移；其中，图4a显示双氢青蒿素给予治疗小鼠，能有有效地抑制卵巢癌的恶性进展；图4b显示，与对照组相比，双氢青蒿素治疗组小鼠的肺癌转移的情况大大降低，且随着药量的增加，治疗情况越好；图4c显示不同浓度的双青青蒿素给予治疗小鼠，肿瘤腹腔播散的情况(肺脏、肝脏、肠)得到明显的抑制；图4d为免疫组化实验，结果显示，双氢青蒿素处理的细胞其形态转换过程(EMT)得到抑制；图4e为免疫印迹实验，结果显示，双氢青蒿素能够有效抑制PDGFRa的表达和磷酸化，下游信号通路PI3K/AKT和MAPK-ERK得以抑制。

图5显示了青蒿素及其衍生物与一线化疗药的增敏作用；其中，图1a为HepG2和Hep3B细胞用青蒿素或双氢青蒿素单独处理或联合吉西他宾处理实验，结果显示，吉西他宾单独作用于HepG2和Hep3B细胞，具有一定的凋亡促进作用，青蒿素联合吉西他宾对HepG2和Hep3B细胞都具有显著凋亡促进作用作用，两药联合表现出协同作用；图5b显示，DHA和CBP联合用药24小时后对OVCAR-3细胞凋亡有增效促进作用，用1μM的DHA联合500μM CBP(1155％)作用后的细胞凋亡率显著高于单独用1μM DHA(266％)或500μM CBP(528％)的细胞凋亡率，而且超过了两种化合物的凋亡累加效应，是一种增效作用，A2780细胞对联合治疗显示了累加效应；图5c显示，当与DHA联用时，CBP显著抑制了卵巢癌细胞的活力，当细胞暴露于1μM CBP和1μM DHA时，A2780细胞的存活率就降低了69％，而OVCAR-3细胞存活则减少了72％，相比之下，IOSE144细胞则显得对治疗较不敏感，两种药物均以1μM合用时存活率只降低了约28％；图5d为肝癌细胞Hep3B和HepG2裸鼠移植瘤实验，结果显示，青蒿素和双氢青蒿素单药体内均具有抗肿瘤作用，双氢青蒿素的抑癌作用强于青蒿素，青蒿素及双氢青蒿素均能增加吉西他宾的抑癌作用，联合吉西他宾治疗效果优于单药，尤其是双氢青蒿素表现出显著的化疗致敏作用；图5e为在卵巢癌细胞A2780和OVCAR3裸鼠移植瘤实验中，结果显示，DHA(剂量为10和25mg/kg)分别导致A2780异体移植肿瘤24％和41％的生长抑制(与给生理盐水的对照组相比)(P＜0.05)，OVCAR-3模型14％和37％肿瘤生长抑制(P＜0.05)；只给单一CBP的治疗组肿瘤生长被抑制了56％(A2780)和46％(OVCAR-3)，联合用药(25mg/kgDHA)组则在A2780和OVCAR-3动物肿瘤中均产生70％肿瘤生长抑制(P＜0.05)。

图6显示PDGFRα的表达与卵巢癌病理级别和转移状态的关系；其中，图6a显示，与低级别卵巢癌相比，高级别卵巢癌病例中，肿瘤细胞及周围的间质细胞PDGFRα的表达量显著增加；图6b为PDGFRα染色结果，结果显示，在高级别卵巢癌病例中，PDGFRα的表达显著增加。

具体实施方式

本发明人经过广泛而深入的研究，发现青蒿素及其衍生物的用途，用于制备血小板衍生生长因子受体A(PDGFRα)的抑制剂或拮抗剂，此外，青蒿素及其衍生物还可以抑制PDGFRα酪氨酸激酶的活化、抑制PDGFRα的蛋白稳定性、促进PDGFRα泛素依赖的蛋白酶体介导的降解、抑制信号通路PI3K/AKT和MAPK/EPK的活化、抑制PDGFRα阳性肿瘤细胞的生长、迁移和侵袭、还可以用于PDGFRα阳性肿瘤治疗的增敏剂。在此基础上完成了本发明。

术语

卵巢癌

卵巢癌是全世界的妇女健康的巨大威胁。在美国，卵巢癌已成为第四位致死性的女性癌症。虽然被诊断于病发早期的卵巢癌患者的5年存活率可以达到80％-90％，但是被诊断为晚期的卵巢癌患者存活率却只有不到25％，此外，大多数卵巢癌患者在诊断出是卵巢癌是已至晚期。目前卵巢癌患者的死亡率在过去的数十年没有发生太大的改变，因此在肿瘤治疗和预后中，寻找有效的治疗方案，增强肿瘤细胞对化疗药物的敏感性，和克服肿瘤的耐药性是研究人员和临床实践人员长期以来的目标。

肝癌

肝癌是最常见的恶性肿瘤之一，我国是肝癌的高发区，其发病率已上升到恶性肿瘤的第二位。由于肝癌早期症状不明显，因此确诊的大多数病人已处于中、晚期。原发性肝癌的首选治疗是手术切除，随着治疗技术的提高，目前术后5年生存率已提高至50％-60％，但由于手术切除只适用直径不超过5cm、癌灶数目不超过3个的小肝癌。此外，是否适合直接手术切除还取决于癌灶部位的血管富集程度及病人个体差异，这些限制导致90％左右的肝癌不适合手术治疗。即使是可切除肝癌，术后实施以介入化疗为主的综合性治疗也是必要的。然而目前，鲜有药物能够有效抑制肝癌的生长和进一步恶化，肝癌对传统化疗药物的反应率低，毒副作用非常显著，因此化疗的缓慢发展是制约抗肿瘤药物临床应用的主要原因。因此对于肿瘤化疗来说，迫切需要寻求更好的化疗增效剂和更好的治疗方案，从而提高肿瘤对化疗的敏感度，降低其对化疗的耐药性。

血小板衍生生长因子及其受体

血小板衍生生长因子(Platelet derived growth factor，PDGF)是一类能够合成并分泌到细胞外基质中的生长因子，具有促进纤维细胞、平滑肌细胞等多重组织的细胞分裂、增殖、迁移、增加细胞的黏附能力，在人胚胎发育和正常生理活动中发挥着重要作用。PDGF以自分泌和旁分泌形式激活血小板衍生生长因子受体(PDGFR)而发挥作用。

PDGFR包含两种结构类似的酪氨酸激酶受体PDGFRα和PDGFRβ，其中PDGFRα的异常激活与多重疾病密切相关，具体地，PDGFR相关疾病可以分为三类：

1.肿瘤

由于PDGFR(特别是PDGFRα)存在于多种肿瘤中，它可能作为一个新的治疗靶点。已经发现，多种肿瘤的发生与PDGFR(特别是PDGFRα)的过度激活相关，如卵巢癌、神经胶质瘤、前列腺癌；此外，在一些肿瘤中存在PDGFR的突变激活，如隆突性皮肤纤维肉瘤、胃肠道间质瘤、Bcr-Abl阴性的慢性髓样白血病、嗜酸细胞增多综合征等等。

2.血管性疾病

研究发现，PDGF及PDGFR等在多种血管性疾病中存在过量表达和过度激活现象，如动脉粥样硬化和再狭窄、肺动脉高压、视网膜血管病变等，抑制PDGFR信号通路的激活可以有效缓解上述疾病的症状，增加其治疗效果。

3.纤维性病变

PDGF-PDGFR信号通路在多种纤维性病变过程中发挥重要的作用，如肺纤维化、肝纤维化和肝硬化、皮肤纤维增生(硬皮病)、肾纤维化、心肌纤维化等等，其中PDGF-PDGFR信号通路介导的间质细胞的增殖是该慢性炎症的共同特征。

上述三类疾病中，PDGF-PDGFR信号通路发挥重要的作用，其中PDGFRβ主要介导了血管系统病变，而PDGFRα主要介导了间质细胞和成纤维细胞驱动的病变。(Johanna Andrae，et al.Role of platelet-derived growth factors inphysiology and medicine，Genes Dev.2008；22：1276-1312；张秀华等，血小板源生长因子受体与肿瘤.生命科学，Vol.18，No.3 Jun.，2006)

青蒿素及其衍生物

青篙素是从中药青篙中提取的有过氧基团的倍半萜内酯药物，由于青蒿素是复杂的大分子，化学全人工很难合成，因此目前主要是生物合成和人工提取。

青蒿素等倍半萜类的生物合成在细胞质中进行，途径属于植物类异戊二烯代谢途径，可分为三大步：由乙酸形成FPP，合成倍半萜，再内酯化形成青蒿素，具体为：FPP→4，11-二烯倍半萜→青蒿酸→二氢青蒿酸→二氧青蒿酸过氧化物→青蒿素。

青蒿素和本领域几种常见的青蒿素衍生物的化学式见下：

青蒿素及其衍生物的应用

本发明提供了青蒿素及其衍生物用于制备血小板衍生生长因子受体A(PDGFRα)的抑制剂或拮抗剂的用途。

在另一优选例中，青蒿素及其衍生物还用于：

(i)抑制PDGFRα酪氨酸激酶的活化；和/或

(ii)抑制PDGFRα的蛋白稳定性；和/或

(iii)促进PDGFRα泛素依赖的蛋白酶体介导的降解；和/或

(vi)抑制PDGFRα阳性肿瘤细胞信号通路PI3K/AKT和MAPK/EPK的活化；和/或

(v)抑制PDGFRα阳性肿瘤细胞的生长、迁移和侵袭；和/或

(vi)制备抑制PDGFRα阳性肿瘤的药物或增敏剂；和/或

(vii)制备治疗血管性疾病和纤维性病变的药物或增敏剂。

在本发明的一个优选例中，提供了一种体外非治疗性地抑制血小板衍生生长因子受体A(PDGFRα)活性的方法，包括步骤：将青蒿素和/或青蒿素衍生物与细胞接触，从而抑制或拮抗细胞中血小板衍生生长因子受体A(PDGFRα)。

所述的接触是在青蒿素和/或青蒿素衍生物存在下，培养所述细胞。细胞包括肿瘤细胞，较佳地包括PDGFRα阳性肿瘤细胞，更佳地包括PDGFRα阳性卵巢癌细胞或肝癌细胞，如卵巢癌细胞A2780、卵巢癌细胞OVCAR3、肝癌细胞Hep3B。在另一优选例中，所述方法还用于至少以下一种应用：体外非治疗性地抑制PDGFRα酪氨酸激酶的活化；体外非治疗性地抑制PDGFRα的蛋白稳定性；体外非治疗性地促进PDGFRα泛素依赖的蛋白酶体介导的降解；体外非治疗性地抑制信号通路PI3K/AKT和MAPK/EPK的活化；和体外非治疗性抑制PDGFRα阳性肿瘤细胞的生长。

本发明还提供了一种预防和/或治疗PDGFRα相关疾病的方法，其中所述疾病与PDGFRα的过表达或活性过高有关，所述方法包括步骤：给需要的对象施用青蒿素和/或青蒿素衍生物，或施用含青蒿素和/或青蒿素衍生物的药物组合物。PDGFRα相关疾病包括肿瘤(PDGFRα阳性肿瘤)，如卵巢癌或肝癌。在另一优选例中，所述的施用包括：瘤内、静脉注射、口服片剂、腹腔注射等。所述的对象为哺乳动物(如人)。

药剂组合物及其应用

本发明还提供了药剂组合物，在一个优选例中，所述制剂组合包括：

(I)含青蒿素和/或青蒿素衍生物的制剂；和(II)含吉西他宾或卡铂的制剂，

所述制剂组合用于制备：(a)抑制血小板衍生生长因子受体A的药物组合物或药盒；或(b)预防和/或治疗肿瘤的药物组合物或药盒。

所述制剂包括片剂、胶囊、栓剂、注射剂；所述药物制剂用于预防和治疗PDGFRα阳性肿瘤。

本发明的药剂组合物可以是固体或液体。固体制剂包括散剂、片剂、丸剂、胶囊、扁囊剂、栓剂和可分散颗粒剂，固体载体可以是一种或多种物质，它们可以作为稀释剂、矫味剂、黏合剂、防腐剂、片剂崩解剂或包囊材料。在散剂中，载体是微细分的固体，本发明的化合物与微细分的活性组份存在于混合物中。在片剂中，此化合物与所需的粘合载体以适当比例混合，并压制成所需的形状和大小。散剂和片剂中优先含有5至70％活性化合物，适宜的载体是碳酸镁、硬脂酸镁、滑石、糖、乳糖、果胶、糊精、淀粉、明胶、羧甲基纤维素、羧甲基纤维素钠、低熔点腊和可可脂等。同样，扁囊剂或锭剂、片剂、散剂、胶囊、丸剂、扁囊剂和锭剂可以是适合于口服给药的固体剂型。为了制备栓剂，可以将低熔点腊乳脂肪酸甘油脂或可可脂的混合物熔化，并通过搅拌将此活性化合物组份均匀地分散在其中，然后将熔化的均匀混合物倒入大小合适的模中让其冷却，并由此固化。溶液形式的制剂包括溶液、悬浮剂和乳剂，例如水或含水丙二醇溶液。对于非肠道注射液体制剂，可以在含水聚乙二醇溶液中制备。适于口服的含水溶液可以通过将活性组份溶解于水中，并按照需要加入适宜的着色剂、矫味剂、乳化剂和增稠剂制备。适于口服的含水混悬剂可以通过将微细分的活性组份分散于含水粘性物质中制备，如天然或合成胶、树脂、甲基纤维素、羟甲基纤维素钠及其他熟知的混悬剂。这些液体形式包括溶液、混悬剂和乳剂，除活性组份外，这些制剂可含有着色剂、矫味剂、稳定剂、缓冲剂、合成或天然甜味剂、分散剂、增稠剂和助溶剂等。此药物制剂优先是单位剂型，在此剂型中，此制剂被再分为含适量活性组份的单位剂量，此单位剂量可以是包装的制剂，该包装含有一定量的制剂，如包装的片剂、胶囊和在小瓶或胶囊中的散剂。此单位剂型还可以是胶囊、片剂、扁囊剂或锭剂本身，或其可以存在包装形式中的适当数量的任何这些散剂。

单位剂量制剂中活性组份的量可根据具体的应用和活性组份的效力而变化，对于非人哺乳动物(如小鼠)治疗，可以将0.01mg至约0.5g，较佳地0.1至约3mg的胶囊每天给药三次，必要时该组合物还可以含其他相容的治疗剂。对于人的治疗，可以根据患者的需要、被治疗病症的严重性以及使用的化合物而变化，较佳地，开始以小于该化合物最佳剂量的较小剂量治疗，此后，小量增加此剂量达到最佳效果，方便起见，如果需要可将总日剂量再细分为一天内分次给药。在本发明的一个优选例中，给予人的剂量大约为0.1/kg-0.5g/kg之间。

本发明的主要优点：

(1)青蒿素及其衍生物可以用于制备血小板衍生生长因子受体A(PDGFRα)的抑制剂或拮抗剂；

(2)青蒿素及其衍生物可以抑制PDGFRα酪氨酸激酶的活化、PDGFRα的蛋白稳定性、抑制信号通路PI3K/AKT和MAPK/EPK的活化、抑制PDGFRα阳性肿瘤细胞的生长、迁移和侵袭；

(3)青蒿素及其衍生物可以促进PDGFRα泛素依赖的蛋白酶体介导的降解；

(4)青蒿素及其衍生物可以用于PDGFRα阳性肿瘤治疗的增敏剂。

下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York：ColdSpring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。

实验方法和材料

1.表面等离子共振

表面等离子共振(SPR)是一种光学现象，可被用来实时跟踪在天然状态下生物分子间的相互作用。实验中，将重组人PDGFRα胞外端(Met1-Glu524)蛋白，键合在生物传感器表面，将不同浓度的双氢青蒿素溶液和一直与PDGFRα有相互作用的化合物索拉菲尼及SU11248溶液注入并流经生物传感器表面。生物分子间的结合引起生物传感器表面质量的增加，导致折射指数按同样的比例增强，生物分子间反应的变化即被观察到，这种反应用反应单位(RU)来衡量。

2.细胞培养

人卵巢永生化的非致瘤性上皮细胞IOSE-144和卵巢癌细胞(A2780，OVCAR-3，SK-OV3，OVCAR-5)购自美国ATCC(American Type CultureCollection，Manassas，VA)。所有细胞依据ATCC的培养要求(RPMI-1640或DMEM培养液加入10％胎牛血清(GIBCO)、100IU/ml青霉素、100IU/ml链霉素)于5％CO2的37℃培养箱中培养。

3.细胞生长抑制实验

细胞生长及增殖抑制试验通过CCK-8实验测定。细胞以3×103个细胞每孔的密度铺种于96孔板中。12～18小时后，以一定浓度梯度(0、1、5、10、25μM)的青蒿素类化合物处理48小时之后，在96孔板每孔中加10μL CCK-8溶液进行药物处理后的细胞生长活性评价，实验最后通过酶标仪(SpectraMax190microplate reader；Molecular Devices，USA)测量450nm处的光吸收(OD值)(每实验浓度至少设置三个复孔，每次实验至少重复三次)。

4.细胞迁移、侵袭实验

卵巢癌细胞的迁移和侵袭能力通过transwell实验测定，低浓度青蒿素衍生物短时间(12h)处理卵巢癌细胞，然后将相同数目的细胞(5×104个)重悬在无血清的培养基中，种植到transwell(测定细胞的侵袭能力时，transwell上层铺上基质胶)上层培养室中。以10％胎牛血清培养基作为化学趋化剂。大约8-12h后，未迁移的细胞用棉签擦去，将迁移的细胞进行固定染色，并进行统计分析。

5.SDS-聚丙烯酰胺凝胶电泳和蛋白免疫印迹分析

以不同浓度的DHA处理培养细胞24小时，收集细胞并用细胞裂解液(RIPAbuffer #9806，Cell Signaling)裂解。之后以13,000转于4℃离心15min，取上清进行后续的分析检测。蛋白浓度通过Bio-Rad公司的试剂盒(protein assay kit；Hercules，CA)定量蛋白总量。之后每孔上等量蛋白进行SDS-PAGE胶电泳分离。电泳完毕，将电泳蛋白条带转移至甲醇活化过的PVDF膜(Millipore，Bedford，MA)上。转膜完毕后，膜以5％的脱脂牛奶(PBS溶解)封闭，然后后加一抗和二抗孵育(以5％的BSA配置)，之后通过蛋白条带表达差异变化以蛋白显影试剂盒(ECL plus system，Amersham Pharmacia Biotech)显影分析。

6.小鼠原位卵巢癌异体移植瘤模型体内治疗实验

四到六周龄的雌性BALB/c(nu/nu)购自上海斯莱克实验动物有限公司(Shanghai Experimental Animal Center)，并按规定饲养于SPF级动物房。所有动物实验操作均得到中科院营养所动物伦理委员会的批准。。大体步骤如下：将荧光素酶标记的A2780细胞，重悬于无血清的RPMI 1640培养基中。然后腹腔注射等量细胞(～3×106 cells/0.2ml)于小鼠的腹腔内部。利用IVIS Lumina生物荧光系统实时监测肿瘤的生长情况，并记录小鼠的体重。注射5天后，根据肿瘤的荧光强度时将小鼠平均分成治疗组和对照组(每组8只老鼠)。治疗组药物DHA溶解在蓖麻油、乙醇和生理盐水的混合物(CremophorEL∶Ethanol∶Saline＝5∶5∶90，v/v/v)中，所有药物均通过腹腔给药。DHA的给药剂量为10或25mg/kg体重，给药频率为每天给药，每周停歇两天。对照组注射生理盐水。在治疗过程中利用活体成像系统实时监测肿瘤的生长状况。治疗结束后，小心取出移腹部肿瘤(除掉脂肪和结缔组织)，以PIPA溶液(100mg组织加入1ml RIPA)匀浆。得到的肿瘤组织匀浆液按上述的蛋白免疫印迹分析实验要求进行处理，之后进行蛋白免疫印迹检测；或将肿瘤组织进行固定和免疫染色。

实施例1

青蒿素及其衍生物直接结合PDGFRα，抑制其磷酸化活性，促进其降解

利用PDGFRα体外激酶实验、表面等离子共振、计算机辅助同型模建及反向对接实验等分子药理学以及生物物理学等先进的生物学技术和手段，证实青蒿素类衍生物可以直接结合PDGFRα，抑制PDGFRα的受体酪氨酸激酶活性，同时研究发现双氢青蒿素可以抑制PDGFRα阳性细胞中PDGFRα的蛋白稳定性，促进其泛素依赖的降解过程。

图1表明，青蒿素及其衍生物能够直接结合PDGFRα，PDGFRα作为青蒿素及其衍生物作用的直接靶标，且能够抑制其磷酸化活性，促进其降解。体外激酶实验显示青蒿素及其衍生物：青蒿素(ART)、二氢青蒿素(DHA)、青蒿琥酯(ARM)、蒿甲醚(ARS)可以抑制PDGFRα的胞内端(aa550-末端)酪氨酸激酶的活性(图1a)。表面等离子共振(SPR)实验显示，双氢青蒿素(DHA)可以与PDGFRα包外端(aa24-524)蛋白相互结合，索拉菲尼(Sorafenib)做为阳性对照，已知可以结合抑制PDGFRα的活性(图1b)。生物素-亲和素亲和纯化实验显示标记生物素的DHA可以与A2780细胞内PDGFRα相互结合，从而被亲和纯化出来(图1c)。计算机辅助同型模建及反向对接实验(Computer Assisted Homology modeling anddocking)实验显示双氢青蒿素(DHA)可以嵌合到PDGFRα包外端PDGF结合位点，同时也可嵌合到其包内端ATP结合位点，阻断其激酶激活(图1d)。图1e-图1f显示了双氢青蒿素能够有效抑制PDGFRα阳性细胞A2780和OVCAR3中PDGFRα的活性和蛋白表达，而对PDGFRβ的磷酸化和蛋白水平影响不大。图1g-图1i实验显示双氢青蒿素可以加速PDGFRα的蛋白降解，这一过程是泛素依赖的蛋白酶体介导的。

实施例2

青蒿素及其衍生物抑制PDGFRα阳性的肿瘤细胞的生长和转移

利用裸鼠原位卵巢癌异体移植瘤模型，研究双氢青蒿素在动物体内的抗卵巢癌活性。将荧光素酶标记的人卵巢癌细胞A2780注射入Balb-c裸鼠的腹腔，建立裸鼠卵巢癌原位模型，然后给予双氢青蒿素治疗：对照组(生理盐水)，低剂量组(DHA 10mg/kg)。高剂量组(DHA 25mg/kg)每周治疗五次，停歇两天，共给予6周治疗时间。期间利用活体成像系统观察小鼠卵巢癌的生长和转移情况。

通过细胞水平和动物水平的一系列研究，显示青蒿素类衍生物能够有效地抑制PDGFRα阳性的肿瘤细胞的生长、迁移和侵袭(卵巢癌和肝癌)，而对于不表达PDGFRα的肿瘤细胞生长和迁移能力的抑制能力较小。具体结果如下：

图2表明，青蒿素及其衍生物抑制PDGFRa阳性的卵巢癌细胞的生长和迁移能力。具体地，如图1e和图2a所示，PDGFRa在卵巢正常细胞上皮细胞IOSE144，卵巢癌细胞SK-OV3，OVCAR5中无表达，在卵巢癌细胞A2780和OVCAR3中高表达；给予药物刺激48h后，不同浓度的青蒿素及其衍生物人正常卵巢上皮细胞IOSE144无明显的生长抑制能力，对于SK-OV3细胞毒性较小，但对于PDGFRa阳性的A2780和OVCAR3具有较强抑制能力，其中青蒿素和二氢青蒿素的作用最为显著。细胞迁移实验显示低浓度短时间处理(12h)卵巢癌细胞，青蒿素及其衍生物能够有效地抑制PDGFRa阳性细胞A2780的迁移能力，而对于PDGFRa阴性的细胞SK-OV3无明显抑制能力(图2b)。青蒿素衍生物中双氢青蒿素对于PDGFRa阳性的细胞的生长和迁移侵袭能力抑制最为明显(图2c-图2e)。

肝癌细胞中也得到了类似的结果，图3表明，青蒿素及其衍生物能够抑制PDGFRa阳性的肝癌细胞的生长和迁移能力。具体地，在众多人正常肝细胞(7702和LO2)和肝癌细胞系(Hep3B，HepG2，7721，7404，LM3，97L，97M，Huh-7)中，Hep3B高表达PDGFRa(图3a)；低浓度双氢青蒿素(3μM)短时(12h)处理Hep3B细胞能够显著抑制其迁移和侵袭能力(图3b)；细胞生长实验同时显示PDGFRa阳性的Hep3B细胞对于双氢青蒿素更为敏感，而其他PDGFRa地表达和无表达的肝癌细胞对于双氢青蒿素敏感程度较低(图3c)。

动物实验评估结果(图4)表明，双氢青蒿素对于PDGFRa阳性细胞A2780的生长和转移的具有抑制能力。具体地，双青青蒿素给予治疗后能有有效地抑制卵巢癌的恶性进展(图4a)；与对照组别相比，给予双氢青蒿素治疗组小鼠的为转移的情况大大降低，肿瘤腹腔播散的情况也得到明显的抑制(图4b、图4c)；免疫组化和免疫印迹实验同时显示，双氢青蒿素能够有效地抑制PDGFRa的表达和磷酸化，下游信号通路PI3K/AKT和MAPK-ERK得以抑制，上皮间质形态转换过程(EMT)得到抑制(图4d、图4e)。

实施例3

青蒿素及其衍生物与一线化疗药的增敏作用

青蒿素及双氢青蒿素体外单独应用均能够显著抑制卵巢癌和肝癌细胞的生长、增殖和迁移侵袭能力。本实施例对其是否具有化疗增敏作用进行了检测，青蒿素及双氢青蒿素分别联合吉西他宾作用于人肝癌细胞HepG2和Hep3B，以及联合卡铂作用于卵巢癌A2780和OVCAR3观察联合用药对细胞生长的抑制作用。结果(图5)如下所示：

HepG2，Hep3B细胞用10μmol/L青蒿素或双氢青蒿素单独处理或联合10μg/L吉西他宾处理48小时，按上述方法进行细胞凋亡定量检测。具体地，吉西他宾单药作用于HepG2，Hep3B细胞显示出一定的凋亡促进作用(图5a)，青蒿素联合吉西他宾对HepG2，Hep3B细胞具有显著凋亡促进作用作用，两药联合表现出协同作用(图5a)。DHA和CBP联合用药24小时后对OVCAR-3细胞凋亡有增效促进作用(图5b)；用1μM DHA联合500μM CBP(1155％)作用后的细胞凋亡率显著高于单独用1μM DHA(266％)或500μM CBP(528％)的细胞凋亡率，而且超过了两种化合物的凋亡累加效应，显示出的是一种潜在的增效作用。然而，A2780细胞对联合治疗则显示的是累加效应而不是增效效应。这可能是由于它们对DHA化合物的敏感度较低的缘故(图5b，P＜0.05)。更长时间的作用可能产生更好的效果。在细胞增殖活力抑制方面，当与DHA联用时，CBP非常显著地抑制了卵巢癌细胞的活力。事实上，当细胞暴露于1μM CBP和1μM的DHA时，A2780细胞的存活率降低了69％，而OVCAR-3细胞存活则减少了72％。相比之下，IOSE144细胞则显得对治疗较不敏感，两种药物均以1μM合用时存活率只降低了约28％(图5c)。

细胞实验证明青蒿素以及双氢青蒿素在体外都显示出对于吉西他宾，卡铂处理癌细胞的杀伤和诱导凋亡的增敏作用。此外，裸鼠移植瘤实验进一步证明它们对于体内肿瘤的化疗也一样有效。具体地，如图5d显示，在肝癌细胞Hep3B和HepG2裸鼠移植瘤实验中，青蒿素和双氢青蒿素单药体内均具有抗肿瘤作用，双氢青蒿素的抑癌作用强于青蒿素。青蒿素及双氢青蒿素均能增加吉西他宾的抑癌作用，联合吉西他宾治疗效果优于单药，尤其是双氢青蒿素表现出显著的化疗致敏作用。在卵巢癌细胞A2780和OVCAR3裸鼠移植瘤实验中(图5e)，DHA(剂量为10和25mg/kg)分别导致A2780异体移植肿瘤24％和41％的生长抑制(与给生理盐水的对照组相比)(P＜0.05)，OVCAR-3模型14％和37％肿瘤生长抑制(P＜0.05)；只给单一CBP的治疗组肿瘤生长被抑制了56％(A2780)和46％(OVCAR-3)。联合用药(25mg/kg DHA)组则在A2780和OVCAR-3动物肿瘤中均产生70％肿瘤生长抑制(P＜0.05)。

以上细胞和动物水平实验显示，青蒿素及其衍生物具有良好的单独化疗和联合一线化疗药物的增敏作用，结合青蒿素类衍生化合物可以直接靶向PDGFRa发挥其抗肿瘤活性这一结果，提示青蒿素类药物可以用于临床中联合多类化疗药物来治疗肿瘤病人，尤其是病理诊断为PDGFRa阳性的肿瘤病人

实施例4

PDGFRα与病理分级和转移相关

本实施例根据临床病理诊断信息严格选取了45例卵巢癌病人的病理样本，其中包括21例低级别卵巢癌(临床病例级别为I-II，中度或高度分化，无明显的淋巴结，腹腔和网膜等转移)；24例高级别卵巢癌病人(临床病例级别为III-IV，低分化，具有大面积的淋巴结转移，腹腔，网膜，子宫，附件及其他器官转移)。

结果显示(见图6)，与低级别卵巢癌相比，高级别卵巢癌病例中，肿瘤细胞及周围的间质细胞PDGFRα的表达量显著增加。细胞实验显示，利用慢病毒介导的基因干扰技术将卵巢癌细胞A2780和OVCAR3中PDGFRα的表达进行沉默，发现肿瘤细胞的生长和迁移侵袭能力显著降低。

实施例5

药物组合物的制备

组合物1

组合物2

讨论

已有发现表明，PDGFR基因突变或过度激活可以促进细胞的转化能力进而导致恶性肿瘤的发生，如胃肠间质瘤，肺癌，前列腺癌和乳腺癌。PDGFR与其配体结合提高细胞间黏附分子的降解，促进肿瘤细胞的侵袭和转移，同时还能激活微环境中的VEGF，FGF等生长因子进而促进血管生成，加速了肿瘤的恶性进展过程。动物实验和临床病理研究发现，PDGFRα过度表达和激活参与了卵巢癌的发生和发展过程，与病人的病理级别和不良预后显著相关。

本发明内容表明，与低级别未发生转移的卵巢癌病人病理组织相比，高级别发生大规模转移的卵巢癌病人其癌细胞和肿瘤间质细胞中PDGFRα的表达量显著增加，这就表明，PDGFRα可以作为卵巢癌治疗的一个靶点。

本发明中发现青蒿素类衍生物，尤其是青蒿素和双氢青蒿素可以直接结合到PDGFRa分子中，既抑制PDGFRa的受体酪氨酸激酶活性，又抑制PDGFRa的蛋白稳定性，促进其泛素化降解。

分子和动物实验显示，青蒿素类的药物可以通过抑制PDGFRa进而抑制包括卵巢癌和肝癌在内的多重肿瘤的生长、迁移和侵袭转移能力。该结果表明，青蒿素类药物可以作为PDGFR的一种特殊的抑制剂，用于临床上PDGFRa突变或过度激活的肿瘤病人，包括卵巢癌、肝癌、胃肠间质瘤、急性白血病等等。青蒿素类衍生物也具有良好的抗癌增敏活性，例如青蒿素类药物可以显著提高一线化疗药卡铂，吉西他滨等对于卵巢癌和肝癌的治疗效果，尤其是双氢青蒿素对卡铂和吉西他宾具有更强的化疗致敏作用，其高效低毒的特点使之有望作为化疗增敏剂，应用于肿瘤的临床联合化疗。

在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。