CN100430054C

CN100430054C - Use of green chiretta diterpene lactone in inhibiting vascularization

Info

Publication number: CN100430054C
Application number: CNB02159421XA
Authority: CN
Inventors: 王振军; 赵博
Original assignee: Peking University First Hospital
Current assignee: Peking University First Hospital
Priority date: 2002-12-31
Filing date: 2002-12-31
Publication date: 2008-11-05
Anticipated expiration: 2022-12-31
Also published as: CN1511522A

Abstract

The present invention discloses a new purpose of a creat diterpene lactone compound on inhibiting angiogenesis. The research indicates that the creat diterpene lactone compound has an effect on inhibiting angiogenesis. The present invention has the technical scheme of the application of the creat diterpene lactone compound on the preparation of treating and/or preventing medicines for inhibiting angiogenesis. The preferable compounds comprise creat lactone, dehydrated creat lactone, deoxidated creat lactone and new creat lactone, and the effective dose ranges are orderly 3.0 to 50.0 mg/kg body weight every day, 5.0 to 50 mg/kg body weight every day, 3.5 to 45 mg/kg body weight every day and 5.0 to 60 mg/kg body weight every day. The creat diterpene lactone compound skillfully uses the angiogenesis as the target of treating diseases, and has the advantages that the treatment aims at started newly formed vessels, so the creat diterpene lactone compound has specificity; because endothelial cells in blood vessels are exposed in blood, and the medicines can directly take effect, the creat diterpene lactone compound has the characteristics of small dose, significant curative effect and little side effect; because the gene expression of endothelial cells is relatively stable, the creat diterpene lactone compound is difficult to generate medicine resistance.

Description

The application of Herba Andrographis diterpenoid-lactone in preparation inhibition angiogenesis drug

Technical field

The present invention relates to novel application of compound, particularly relate to the new purposes of Herba Andrographis diterpenoid-lactone.

Background technology

Studies show that tumor self possesses and starts and promote the ability that new vessels generates, with the metabolism that satisfies self and the needs of nutrient substance supply.Tumor neovasculature generation (angiogenesis) is from existing vascular bed.If there is not angiogenesis, the growth of primary tumo(u)r can not surpass 1～2mm, can not occur soaking into and shifting yet.Suppress tumor-blood-vessel growth, closely related with generation, development, infiltration and the transfer of blocking-up tumor.Suppress tumor-blood-vessel growth, also can stop the pernicious transformation of precancerous lesion to cancer.The not only growth of entity tumor, infiltration and transfer rely on new vessels and generate, and the growth of blood system malignant tumor (as malignant lymphoma, Lymphocytic leukemia etc.) and transfer are also closely related with angiogenesis.Growth of tumor, transfer, recurrence, prognosis and angiogenesis are closely related, angiogenesis with tumor is a target spot, the exploitation angiogenesis inhibitor, not only can be used for the treatment of most of entity tumors, also can be used for the prevention of cancer and the treatment of blood system malignant tumor, have important theory and realistic meaning.

Herba Andrographis is the dry aerial parts of acanthaceous plant Herba Andrographis [Andrographis paniculata (Burm.f.) Nees].Has detoxifcation, detumescence, the effect of removing heat from blood, heat clearing away.Tcm clinical practice is used it for diseases such as treatment cold, fever, furuncle carbuncle, the puckery pain of pyretic stranguria, laryngopharynx swelling and pain, pertussis chronic cough, venom.

Contain a large amount of diterpenic lactones in Folium Andrographis and the root, as andrographolide, desoxyandrographolide, neoandrographolide etc.In research and application in the past, Herba Andrographis is mainly used in treatment acute infectious diseases, infectious disease and influenza etc., the experiment report is arranged, and desoxyandrographolide has certain inhibitory action to W256 transplanted tumor and breast cancer cell, but mechanism is also indeterminate.Though Herba Andrographis has been used for clinical treatment tumour, and certain curative effect is arranged, because the antitumaous effect mechanism of its active component is indeterminate, dosage uses unreasonable, has limited the performance of its antitumaous effect.

Summary of the invention

A kind of new purposes that the purpose of this invention is to provide the Herba Andrographis diterpenoid-lactone.

The inventor studies show that, the Herba Andrographis diterpenoid-lactone has the effect that suppresses angiogenesis.Therefore technical scheme of the present invention is: the Herba Andrographis diterpenic lactone suppresses the application in the medicine for the treatment of and/or preventing of angiogenesis in preparation.

Wherein, preferred Herba Andrographis diterpenic lactone is andrographolide (formula I), dehydrorographolide (formula II), deoxyandrographolide (formula III) and neoandrographolide (formula IV).

(formula I)

(formula II)

(formula III)

(formula IV)

When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc., can also add flavouring agent, sweeting agent etc. in case of necessity.

Medicine of the present invention can be made various ways such as injection, tablet, powder, granule, capsule, oral liquid, unguentum, cream.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.

The consumption of said medicine is generally andrographolide 3.0-50.0mg/kg body weight/day, dehydrorographolide 5.0-50mg/kg body weight/day, deoxyandrographolide 3.5-45mg/kg body weight/day, neoandrographolide 5.0-60mg/kg body weight/day.

Of the present invention studies show that, except treating and/or preventing cancer, with the Herba Andrographis diterpenoid-lactone be active component the inhibition angiogenesis treat and/or prevent medicine, for having definite treatment and preventive effect with diseases such as the disease of associated angiogenesis such as diabetes, retinopathy, rheumatic arthritis, psoriasis, hemangioma, atherosclerosiss, in wound healing spot being formed simultaneously also has significant inhibitory effect.

The present invention with the target spot of angiogenesis as the treatment disease, has the following advantages dexterously: 1, treatment is carried out at the new vessels that has started, and has specificity; 2, because vascular endothelial cell is exposed in the blood flow, and medicine directly plays a role, so dosage is little, the curative effect height, side effect is little; 3, because the endothelial cell gene expression is relatively stable, so be difficult for producing drug resistance.

Description of drawings

Fig. 1 is andrographolide and the dehydrorographolide dose-dependent inhibition effect curves to human vascular endothelial cell proliferation.

Fig. 2 is deoxyandrographolide and the neoandrographolide dose-dependent inhibition effect curves to human vascular endothelial cell proliferation.

Fig. 3 handles the optical microscope photograph that the human vascular endothelial cell pipe in back forms for the variable concentrations andrographolide.

Fig. 4 handles the optical microscope photograph that the human vascular endothelial cell pipe in back forms for the variable concentrations dehydrorographolide.

Fig. 5 handles the optical microscope photograph that the human vascular endothelial cell pipe in back forms for the variable concentrations deoxyandrographolide.

Fig. 6 handles the optical microscope photograph that the human vascular endothelial cell pipe in back forms for the variable concentrations neoandrographolide.

Fig. 7 handles the human migration of vascular endothelial cells result's in back optical microscope photograph for the variable concentrations andrographolide.

Fig. 8 handles the human migration of vascular endothelial cells result's in back optical microscope photograph for the variable concentrations dehydrorographolide.

Fig. 9 handles the human migration of vascular endothelial cells result's in back optical microscope photograph for the variable concentrations deoxyandrographolide.

Figure 10 handles the human migration of vascular endothelial cells result's in back optical microscope photograph for the variable concentrations neoandrographolide.

The specific embodiment

Embodiment 1: the Herba Andrographis diterpenoid-lactone is to the inhibitory action of human vascular endothelial cell proliferation.

(HUVEC available from Cascade Biologics company, Cot:C-003-5C) places the Medium 200 (available from Cascade Biologics company) that contains 10% hyclone (FBS) to cultivate (37 ℃, 5%CO with the human umbilical vein endothelial cell ₂, 95% humidity) cultivate, get the 4th generation cell be inoculated in 96 well culture plates by density 4000/250ul, set the medication group and the blank of matched group, each Concentraton gradient, all do 3 multiple holes for every group.Treat that the cell stand density reaches 80%, four kinds of Herba Andrographis diterpenic lactones (that is: the andrographolide that adds each Concentraton gradient of 5ul respectively, dehydrorographolide, deoxyandrographolide, neoandrographolide, all with the DMSO dissolving), make final concentration be respectively 1000uM, 100uM, 10uM, 5uM and 1uM, matched group adds 5ulDMSO, continue to cultivate (M200 culture medium, 2%FBS) after 48 hours, Thiazolyl blue tetrazolium bromide salt (MTT) 20ul that adds 5mg/ml, 37 ℃ were continued to hatch 4 hours, stop cultivating, abandon supernatant, every hole adds 150ulDMSO, gently vibration, measure each hole light absorption value at 490nm wavelength place with BIO-RAD Model 3500 microplate reader after 1 hour, represent the quantity of living cells in each hole with light absorption value.Above-mentioned experiment repeats 3 times.The result shows that the Herba Andrographis diterpenoid-lactone is the dose-dependent inhibition effect to human umbilical vein endothelial cell's propagation as shown in Figure 1 and Figure 2, and its half amount of suppression is respectively 150uM, 200uM, 100uM and 150uM.

Embodiment 2: the Herba Andrographis diterpenoid-lactone forms the inhibitory action of ability to human vascular endothelial cell pipe.

The every hole of 24 well culture plates adds BD Matrigel ^TMMatrix virgin rubber 200ul, make it to aggregate into glue, the 4th generation human umbilical vein endothelial cell (HUVEC) suspension is seeded in 24 orifice plates that scribble Matrigel glue by 30000cell/500ul density, set the medication group of matched group, each Concentraton gradient, the medication group adds Herba Andrographis diterpenoid-lactone (200uM, the 1000uM of tool 10ul Concentraton gradient respectively, with the DMSO dissolving), matched group adds aseptic DMSO10ul, all does 3 multiple holes for every group.37 ℃, 5%CO ₂, 95% humidity cultivated 24 hours, OLYMPUS CK40-RFL observation by light microscope vascular endothelial cell pipe forms, 4 low power fields countings are got in every hole, take pictures with OLYMPUS CK40 digital camera.Result such as Fig. 3, Fig. 4, Fig. 5, shown in Figure 6, as can be seen from the figure, four kinds of Herba Andrographis diterpenoid-lactones are dose-dependent inhibition to human vascular endothelial cell pipe formation ability.

Embodiment 3: the Herba Andrographis diterpenoid-lactone is to the inhibitory action of human migration of vascular endothelial cells ability.

1, preparation chemotactic factor

Get the NIH3T3 cell that goes down to posterity and grew fine in back second day.The soft rinsing of serum-free DMEM 2 times.Serum-free DMEM, 5%CO ₂, cultivate 24-48hours for 37 ℃.The collecting cell supernatant.Centrifugal (12000g, 4 ℃, 10min).Supernatant bacteriological filtration (0.22um filter membrane), (20 ℃) are preserved in packing.

2, Matrigel invasion and attack experiment

The Matrigel 25ul (virgin rubber is pressed 1: 2 dilution proportion with DMEM) that gets dilution adds chamber on the Transwell plate, covers whole polyester film surface, hatches 30min for 37 ℃, makes Matrigel aggregate into glue.The 4th generation human umbilical vein endothelial cell (HUVEC) suspension is seeded to the chamber by 30000/250ul density, PBS washing 3 digestion and harvestings from culture bottle.DMEM makes single cell suspension with serum-free, and 5 * 10 ⁵Cells/ml, the medication group adds the medicine (200uM, 1000uM are with the DMSO dissolving) of each Concentraton gradient, and matched group adds the DMSO with volume.The chamber adds the chemotactic factor of 500ul step 1 preparation under the Transwell plate.5%CO ₂, cultivated 24 hours for 37 ℃.Wipe the cell of gel and polyester film upper surface gently with the wet cotton swab of DMEM.The careful taking-up gone up the chamber, and ice-cold methanol is fixed 30 minutes.Haematoxylin dyeing 1 minute.Gradient alcohol dehydration (80%, 95%, 100%, 100%), dimethylbenzene is transparent.Carefully polyester film is cut from last chamber, place resinene mounting on the microscope slide.The cell (400 *) under high power lens that is attached to the polyester film lower surface is got 6 visuals field countings at random and is averaged and take pictures.Repeated experiments 2 times.Result such as Fig. 7, Fig. 8, Fig. 9, shown in Figure 10 show that four kinds of Herba Andrographis diterpenoid-lactones are the dose-dependent inhibition effect to the transfer ability of human vascular endothelial cell.

Embodiment 4: the tumor bearing nude mice in vivo test.

Results human colon cancerous cell line LoVo cell is made cell suspension with PBS, and the right side thigh portion that is seeded to big BALB/cAnN-nu/nu nude mice of 8 weeks with 2,000,000/200ul is subcutaneous.When treating the tumor bulk-growth, be divided into two groups at random, 5 every group to 5mm * 5mm size.Andrographolide medication group dosage is 150mg/kg, dehydrorographolide medication group dosage is 100mg/kg, deoxyandrographolide medication group dosage is 100mg/kg, neoandrographolide medication group dosage is 200mg/kg (medicine is all to contain the PBS solution dissolving of 10%DMSO), matched group is given the PBS solution 200ul that contains 10%DMSO, successive administration 60 days writes down the major diameter and the minor axis of tumor body every day.During the medication, untoward reaction does not appear in mice.After putting to death mice, take out tumor, detect the microvessel density of tumor.The result shows that four medication group tumor bodies dwindle than matched group, and growth is obviously slowed down, and microvessel density is starkly lower than matched group.Illustrate that four kinds of chemical compounds all have the effect that suppresses tumor in the body, and side effect is very little.

Claims

1, the Herba Andrographis diterpenic lactone suppresses the application in the medicine for the treatment of and/or preventing of angiogenesis in preparation; Wherein, described Herba Andrographis diterpenoid-lactone is the neoandrographolide shown in dehydrorographolide shown in the formula II or the formula IV;

(formula II)

(formula IV).

2, application according to claim 1 is characterized in that: described medicine is for treating and/or preventing angiomatous medicine.

3, application according to claim 1 and 2 is characterized in that: also add one or more pharmaceutically acceptable carriers in described medicine.