CN1511533A - Use of bufadienolide in inhibiting vascularization - Google Patents
Use of bufadienolide in inhibiting vascularization Download PDFInfo
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- CN1511533A CN1511533A CNA021596875A CN02159687A CN1511533A CN 1511533 A CN1511533 A CN 1511533A CN A021596875 A CNA021596875 A CN A021596875A CN 02159687 A CN02159687 A CN 02159687A CN 1511533 A CN1511533 A CN 1511533A
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Abstract
The present invention discloses the new use of bufadienolide in inhibiting vasculariztion. Research probes the vasculariztion inhibiting effect of bufadienolide. The technological scheme is that bufadienolide is used in preparing medicine for prevention and/or treatment of inhibiting vasculariztion. The present invention uses the vasculariztion as the target of treating diseases and has the advantages of treatment specificity of pointing newly formed vessel; small dosage, high treating effect and less side effect owing to the direct action of medicine to vascular endothelial cell; and less medicine resistance owing to relatively stable endothelial cell gene expression.
Description
Technical field
The present invention relates to novel application of compound, particularly relate to the new purposes of bufadienolide.
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.
The Chinese medicine Venenum Bufonis is combined section animal Bufo siccus or the ear rear gland of Bufo melanostictus and the secretions of glandular integumentaria by toad and is formed through dry processing, has detoxifcation, detumescence, pain relieving, cardiac stimulant effect.Tcm clinical practice is used it for diseases such as anesthesia, furuncle carbuncle, heatstroke vomiting and diarrhoea, laryngopharynx swelling and pain, toothache, heart failure, cancerous protuberance.
Contain a large amount of bufotoxin class materials in the Venenum Bufonis, this type of material all has cardiac activity, belongs to steroid, at C
17On connect a pyrans ketone group again, be bufadienolide (bufadienolide), be the main effective ingredient of Venenum Bufonis and Bufo siccus serosity.In application and research in the past, bufadienolide is mainly used in the disease of treatment cardiovascular system aspect, has Folium Digitalis Purpureae sample cardiotonic, can obviously strengthen the contractility of animal isolated heart, and can increase the blood flow of ischemic myocardium.Though Venenum Bufonis 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 bufadienolide.
The inventor studies show that, bufadienolide has the effect that suppresses angiogenesis.Therefore technical scheme of the present invention is:
Formula I chemical compound suppresses the application in the medicine for the treatment of and/or preventing of angiogenesis in preparation, and wherein, the R in the described formula I chemical compound is H, OCOCH
3, CH
3, OH or COOH.
(formula I)
When the R in the formula I chemical compound was H, the chemical compound that obtains was a formula II chemical compound, was resibufogenin (Resibufogenin); R in formula I chemical compound is OCOCH
3The time, the chemical compound that obtains is the formula III chemical compound, is cinobufagin (Cinobufagin).
(formula II)
(formula III)
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 cinobufagin 0.0143-0.0857mg/kg body weight/day, resibufogenin 0.2857-2.8571mg/kg body weight/day.
Of the present invention studies show that, except treating and/or preventing cancer, with the bufadienolide 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, the spot in the wound healing is formed also 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 the dose-dependent inhibition effect curves of two kinds of bufadienolides to human vascular endothelial cell proliferation.
Fig. 2 handles the optical microscope photograph that the human vascular endothelial cell pipe in back forms for the variable concentrations cinobufagin.
Fig. 3 handles the optical microscope photograph that the human vascular endothelial cell pipe in back forms for the variable concentrations resibufogenin.
Fig. 4 handles the human migration of vascular endothelial cells result's in back optical microscope photograph for the variable concentrations cinobufagin.
Fig. 5 handles the human migration of vascular endothelial cells result's in back optical microscope photograph for the variable concentrations resibufogenin.
The specific embodiment
Embodiment 1: cinobufagin and resibufogenin are 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%, the cinobufagin and the resibufogenin (with the DMSO dissolving) that add each Concentraton gradient of 5ul respectively, 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, stopped cultivating, and abandoned supernatant, every hole adds 150ulDMSO, vibration was measured each hole light absorption value at 490nm wavelength place with BIO-RAD Model 3500 microplate reader after 1 hour gently, represented the quantity of living cells in each hole with light absorption value.Above-mentioned experiment repeats 3 times.The result as shown in Figure 1, as can be seen from the figure, cinobufagin and resibufogenin are the dose-dependent inhibition effect to human umbilical vein endothelial cell's propagation, its half amount of suppression is respectively 1uM and 5uM.
Embodiment 2: cinobufagin and resibufogenin form 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 to by 30000cell/500ul density in 24 orifice plates that scribble Matrigel glue, and (cinobufagin is respectively 0.5uM, 1uM, 5uM, 10uM to the medication group of setting matched group, each Concentraton gradient; Resibufogenin is respectively 1uM, 2.5uM, 5uM, 10uM, with the DMSO dissolving), the medication group adds the cinobufagin and the resibufogenin of 10ul tool Concentraton gradient respectively, and 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. 2, shown in Figure 3 show that cinobufagin and resibufogenin are dose-dependent inhibition to human vascular endothelial cell pipe formation ability, and its half amount of suppression is respectively 0.5uM and 2.5uM.
Embodiment 3: cinobufagin and resibufogenin are 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 of each Concentraton gradient, and (cinobufagin is respectively 0.5uM, 1uM, 5uM, 10uM; Resibufogenin is respectively 1uM, 2.5uM, 5uM, 10uM), 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. 4 shown in 5, show that cinobufagin and resibufogenin 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.Cinobufagin medication group dosage is 0.7mg/kg, resibufogenin medication group dosage is 5.1mg/kg (medicine is all to contain the PBS solution dissolving of 10%DMSO), matched group is given the PBS solution 200ul contain 10%DMSO, and 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 two 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 two medicines all have the effect that suppresses tumor in the body, and side effect is very little.
Claims (10)
1, formula I chemical compound suppresses the application in the medicine for the treatment of and/or preventing of angiogenesis in preparation, and wherein, the R in the described formula I chemical compound is H, OCOCH
3, CH
3, OH or COOH.
(formula I)
2, application according to claim 1 is characterized in that: the R in the described formula I chemical compound is H or OCOCH
3
3, application according to claim 2 is characterized in that: in the medicine of described inhibition angiogenesis, the effective dose of cinobufagin is the 0.0143-0.0857mg/kg body weight/day, resibufogenin 0.2857-2.8571mg/kg body weight/day.
4, application according to claim 1 and 2 is characterized in that: also add one or more pharmaceutically acceptable carriers in the medicine of preparation inhibition angiogenesis.
5, application according to claim 1 is characterized in that: described medicine is the medicine that treats and/or prevents cancer.
6, application according to claim 1 is characterized in that: described medicine is to suppress the medicine that spot forms in the wound healing.
7, application according to claim 1 is characterized in that: described medicine is the medicine that treats and/or prevents diabetes, retinopathy, retinopathy of prematurity and retinal vein occlusion.
8, application according to claim 1 is characterized in that: described medicine is for treating and/or preventing the degeneration of old plate-like speckle, rheumatic arthritis and psoriatic medicine.
9, application according to claim 1 is characterized in that: described medicine is for treating and/or preventing angiomatous medicine.
10, application according to claim 1 is characterized in that: described medicine is for treating and/or preventing atherosclerotic medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021596875A CN1511533A (en) | 2002-12-30 | 2002-12-30 | Use of bufadienolide in inhibiting vascularization |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021596875A CN1511533A (en) | 2002-12-30 | 2002-12-30 | Use of bufadienolide in inhibiting vascularization |
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| CN1511533A true CN1511533A (en) | 2004-07-14 |
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| CNA021596875A Pending CN1511533A (en) | 2002-12-30 | 2002-12-30 | Use of bufadienolide in inhibiting vascularization |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104721195A (en) * | 2013-12-20 | 2015-06-24 | 张震东 | New use of cinobufagin in preparation of drug for treatment of diabetic insulin resistance |
-
2002
- 2002-12-30 CN CNA021596875A patent/CN1511533A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104721195A (en) * | 2013-12-20 | 2015-06-24 | 张震东 | New use of cinobufagin in preparation of drug for treatment of diabetic insulin resistance |
| CN104721195B (en) * | 2013-12-20 | 2018-04-27 | 张震东 | Purposes of the cinobufagin in the medicine for preparing treatment diabetes insulin resistance |
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