CN103054858A - Application of oxibendazole in preparing anti-angiogenesis medicine - Google Patents
Application of oxibendazole in preparing anti-angiogenesis medicine Download PDFInfo
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Abstract
The invention relates to an application of oxibendazole in preparing anti-angiogenesis medicines. Until now, relevant reports about the anti-angiogenesis activity of oxibendazole are not found yet. The invention provides the application of oxibendazole in preparing anti-angiogenesis medicines, anti-tumor medicines and humidity resistance elderly macular pigment degeneration medicines. In-vivo pharmacodynamic experiments in which a zebra fish angiogenesis model is utilized show that oxibendazole can remarkably inhibit zebra fish angiogenesis and remarkably inhibit transplanted human being tumor cell growth and has therapeutic effects on humidity elderly macular pigment degeneration. Therefore, oxibendazole can be used in preparation of angiogenesis, anti-tumor or humidity elderly macular pigment degeneration medicines.
Description
Technical field
The present invention relates to medical technical field, the specifically application of oxibendazole in preparing angiogenesis inhibitor class medicine.
Background technology
Angiogenesis (angiogenesis) and the multiple major disease height correlation of the mankind, as malignant tumor, look degeneration of macula (Age-related macular degeneration old age, AMD), atherosclerosis (Atherosclerosis), rheumatic arthritis (Rheumatoid arthritis), diabetic retinopathy (Diabetic retinopathy) and neoplasm metastasis (Tumor metastasis) etc.
[1].Along with the Chinese population aging aggravates gradually, these major diseases life and health of serious harm China people at present.
1971, Judah professor Folkman of Harvard University proposed the antineoplastic vascular therapy first, and he thinks that the growth of entity tumor and diffusion depend on the formation of neovascularity in tumor, and obtains nutrient by new vessels; The formation of neovascularity and growth, promoted the transfer of tumor cell
[2 – 4].Through a large amount of fundamental research in 40 years, at present based on this therapy, the listing of existing a plurality of heavy pound patent new drug, Bevacizumab (trade name Avastin as Roche, within 2010, the global marketing volume is 6,700,000,000 dollars), the Sorafenib of Bayer (trade name Nexavar, the global marketing volume was 9.94 hundred million dollars in 2010), the Sunitinib of Pfizer (trade name Sutent, the global marketing volume was 10.7 hundred million dollars in 2010)
[5 – 6] .but these drug prices are very expensive, be the medicine giant of foreign enterprise monopolization.Domestic only have a kind of anti-tumor angiogenesis drug of real meaning to ratify listing (rhEndostatin, first sign Pharmaceutical, listing in 2005) by SFDA at present
[7], but annual sales amount seldom (2010 annual sales amounts are 2.5 hundred million RMB only
), the method that there is no participates in global competition.Secondly, due to rhEndostatin (recombinant human vascular endothelial inhibin injection, Recombinant Human Endostatin Injection) be macro-molecular protein class medicine, produce the safe preparation of this class high-purity and need very high technical threshold, quality control is more difficult, and the medicine of this type needs injection, long-term taking inconvenience, and the general protein medicaments half-life in vivo is shorter, has limited the clinical practice of activated protein medicine
[7].
Looking degeneration of macula (Age-related macular degeneration is called for short AMD) old age is a kind of degeneration ophthalmic of involving macula retinae district, optical fundus.It produces the macular area degeneration because of age growth, can cause that central vision sharply descends.According to statistics, this disease is suffered from over 3,000 ten thousand people in the whole world.Degeneration of macula is divided into dryness and moist two kinds.Look degeneration of macula moist old age main because choroidal artery generates extremely, seepage appears in newborn invalid blood capillary, and the liquid of vascular leakage and then destruction macula lutea, cause central vision significantly to descend, and affects quality of life, even cecutiency.Look degeneration of macula (AMD) moist old age and become the blind arch-criminal of over-65s old people
[8-9].
The method for the treatment of moist AMD mainly contains photodynamic therapy and anti-angiogenic pharmacotherapy
[8-9].Photodynamic therapy is mainly injected photosensitive drug by vein, then adopts the non-thermal energy laser irradiation choroidal neovascularization focus of specific wavelength, and photosensitive drug is activated.Treating moist AMD by photodynamic therapy, can only stablize or reduce the risk of moist AMD visual deterioration, is not etiological treatment, can not stop the possibility of recurrence.Generally need to repeatedly treat.And want lucifuge 48 hours after treatment, and to avoid photosensitivity reaction, cause skin burn, therefore, to the patient, bring a lot of miseries.Treat at present moist AMD, the medicine gone on the market mainly contains: the Macugen (Pegaptanib of Pfizer, trade name macugen), the ranibizumab of Novartis (Ranibizumab, trade name Lucentis), the Ai Liya (VEGF-Trap-eye of Bayer, trade name Eylea), the price of these medicines is very expensive, generally needs the administration of every menstruation intravitreal injection, and this tediously long administration process is difficult to be accepted by the patient.Therefore, to treat age-related macular degeneration be following development trend to the eye drop of development of new cheapness.
In sum, seek new angiogenesis inhibitor and effectively treat above-mentioned disease and become the research and development focus in micromolecular compound, it is extremely urgent that exploitation has the patent targeting angiogenesis inhibitor small-molecule drug of independent intellectual property right.
Oxibendazole, English common name: Oxibendazole; Generic name: oxibendazole, Oxibendazole, Oxibendazole, Equitac, Loditac, Oxibendazolum; Chemical name: 5-propoxyl group-benzimidazolyl-2 radicals-An Ji methyl formate; Molecular weight: 249.27.For broad-spectrum de-worming medicine, can affect the multiple biochemical metabolism approach of polypide.Ascarid, ancylostome and whipworm are all had to obvious effect.Drive the ancylostome medicine relatively with other, oxibendazole is not only better to the Ancylostoma duodenale curative effect, and American hookworm is also had to good therapeutic effect, and the Negative rate on the egg of 2 days and 3 days therapies can reach 56%～100%.General anthelmintic drug is poor to the whipworm curative effect, though when oxantel drives whipworm, Negative rate on the egg can reach 70%, invalid to ancylostome and ascarid, and oxibendazole is not only effective to ancylostome and ascarid, and the curative effect of driving whipworm also can reach 70% left and right
[10].
The chemical structural formula of oxibendazole is as follows:
So far there is not yet the report active about oxibendazole angiogenesis inhibitor (anti-angiogenesis).
Summary of the invention
The present inventor is unexpected to be found, oxibendazole has the effect of angiogenesis inhibitor.
Therefore, the invention provides the application of oxibendazole in preparing angiogenesis inhibitor class medicine.
The present invention also provides the application of oxibendazole in preparing antitumor drug, and described oxibendazole prevents or treat tumor by the generation that suppresses neovascularity in tumor.
The present invention also provides oxibendazole preparing the application of moisture resistance in looking the degeneration of macula medicine old age, and described oxibendazole prevents or treats and look degeneration of macula moist old age by suppressing the choroidal artery paraplasm.
Said medicine is oral administered dosage form, injecting medicine-feeding form, mucosa delivery dosage form or percutaneous dosing dosage form, more specifically, and as tablet, capsule, granule, oral liquid, injection, patch or gel form.Can pass through the methods known in the art useful in preparing drug formulations.
The present invention utilizes the Brachydanio rerio angiogenesis model to carry out the anti-angiogenic pharmacodynamic experiment of oxibendazole.With traditional blood vessel study model (mouse of Rodents and chick embryo allantois mould), compare, a large amount of studies confirm that at present, Brachydanio rerio is optimal Vascular Biology and anti-angiogenic medicaments evaluation model
[11-21].There are shortcoming separately in the mouse of Rodents and chick embryo allantois mould
[12 – 13].By utilizing the Brachydanio rerio angiogenesis model to carry out pharmacodynamic evaluation and the checking of medicine novel targets, existing branched cancer therapy drug enters clinical front experiment (Pre-clinical Trial) or clinical trial (Clinical Trial) stage, as Vatalanib (Novartis)
[14], Compound 6 (TargeGen)
[15], Rosuvastatin
[16], Solenopsin (Eli Lilly)
[17]deng; A kind of old medicine for the treatment of malignant tumor of mainly finding based on Brachydanio rerio angiogenesis inhibitor model is newly used medicine---and reaction stops (Thalidomide) and has obtained FDA approval listing
[18].
In Brachydanio rerio two bodies, vascular pattern confirms, oxibendazole is to (the intersegm-ental vessel of blood vessel between the Brachydanio rerio body segment, ISV) and subintestinal vein blood vessel (subintestinal vessel, SIV) function of remarkable inhibition is all arranged, and present certain dosage according to patience, therefore, can be used for preparing the angiogenesis inhibitor inhibitor.
As cancer transplantation model and moist looking aspect the degeneration of macula model, large quantity research is also arranged Brachydanio rerio both at home and abroad
[22-27].Through Brachydanio rerio human hela (HeLa) transplantation model, confirm, oxibendazole can significantly suppress the growth of human hela (HeLa); Look the degeneration of macula model validation through Brachydanio rerio, oxibendazole has significant therapeutic effect to looking degeneration of macula moist old age.Therefore, oxibendazole can be used for antitumor and treats and look degeneration of macula moist old age.
Oxibendazole of the present invention is cheap, safe, raw material sources are extensive, is aided with pharmaceutically acceptable adjuvant, adopts the conventional formulation technology to can be made into various oral, injections, external preparation, has good DEVELOPMENT PROSPECT.
The accompanying drawing explanation
fig. 1for blood vessel (ISV) model between after fertilization 48h of the present invention (48hpf) blood vessel transgenic fluorescence Brachydanio rerio body segment.Confine the position of zone for the local amplifying observation of Brachydanio rerio body segment blood vessel network.Arrow indication body intersegmental blood vessel (ISV, intersegmental vessel).
fig. 2for the inhibition of qualitative observation oxibendazole of the present invention to angiogenesis (angiogenesis) between the Brachydanio rerio body segment.Figure a-c, the Brachydanio rerio of after fertilization 23hpf, through drug treating 25h, is 48hpf during observation mutually.The figure negative contrast of a (0.1%DMSO), figure b is the oxibendazole processed group, the figure positive contrast of c (10 μ M lovastatin).With negative control, compare, 0.5 μ M oxibendazole can suppress the generation of blood vessel between the Brachydanio rerio body segment (ISV) fully, back of the body major axis blood vessel (DLAV) lacks fully, only at the dorsal aorta position, sees that a little fragmentary vascular endothelial cell sprouts (Sprouts), sees the asterisk indication.
fig. 3for the inhibition of quantitative assessment oxibendazole of the present invention to blood vessel (ISV) generation model between the Brachydanio rerio body segment.With negative control, compare, oxibendazole suppresses blood vessel (ISV) generation between the Brachydanio rerio body segment and presents significant dosage according to patience.* * P<0.001, difference is extremely remarkable.
fig. 4for suppression ratio and the IC of oxibendazole of the present invention to blood vessel (ISV) generation between the Brachydanio rerio body segment
50.The suppression ratio that oxibendazole generates blood vessel (ISV) between the Brachydanio rerio body segment presents step increase along with the rising of concentration, the suppression ratio that each concentration oxibendazole group generates blood vessel (ISV) between the Brachydanio rerio body segment is respectively: 0.1 μ M(0%), 0.25 μ M(18.96%), 0.3 μ M(57.80%), 0.4 μ M(84.71%), 0.5 μ M(100%), 1 μ M(100%).Utilize GraphPad Prism computed in software IC
50≈ 0.29 μ M.
fig. 5for 72 hours (72hpf) blood vessel transgenic fluorescence Brachydanio rerio subintestinal vein blood vessel (SIV) models of after fertilization of the present invention.Confine the position of zone for the local amplifying observation of subintestinal vein blood vessel (SIV) network.Arrow indication subintestinal vein blood vessel (SIV, subintestinal vessel).
fig. 6for the inhibition of qualitative observation oxibendazole of the present invention to Brachydanio rerio subintestinal vein blood vessel (SIV).Figure a-c, the Brachydanio rerio of after fertilization 48hpf, through drug treating 24h, is 72hpf during observation mutually.The figure negative contrast of a (0.1%DMSO), figure b is the oxibendazole processed group, the figure positive contrast of c (10 μ M lovastatin).Confine the position of zone for the local amplifying observation of subintestinal vein blood vessel (SIV) network, arrow indication subintestinal vein blood vessel (SIV, subintestinal vessel).With negative control, compare, 0.5 μ M oxibendazole can suppress Brachydanio rerio subintestinal vein blood vessel (SIV fully, subintestinal vessel) generation, only see at the yolk sac position that fragmentary vascular endothelial cell sprouts (Sprouts), sees the asterisk indication.
fig. 7for the inhibition of quantitative assessment oxibendazole of the present invention to Brachydanio rerio subintestinal vein blood vessel (SIV) model.With negative control, compare, oxibendazole suppresses Brachydanio rerio subintestinal vein blood vessel (SIV) generation and presents significant dosage according to patience.* * P<0.001, difference is extremely remarkable.
fig. 8for suppression ratio and the IC of oxibendazole of the present invention to Brachydanio rerio subintestinal vein blood vessel (SIV) generation
50.The suppression ratio that oxibendazole generates Brachydanio rerio subintestinal vein blood vessel (SIV) presents step increase along with the rising of concentration, the suppression ratio that each concentration oxibendazole group generates Brachydanio rerio subintestinal vein blood vessel (SIV) is respectively: 0.25 μ M(18.04%), 0.3 μ M(49.57%), 0.4 μ M(92.83%), 0.5 μ M(96.02%), 1 μ M(99.62%).Utilize GraphPad Prism computed in software IC
50≈ 0.30 μ M.
fig. 9antitumor drug effect for Brachydanio rerio human hela of the present invention (HeLa) transplantation model evaluation oxibendazole.Figure a-f, transplant the Brachydanio rerio of the rear 2dpf of human hela (HeLa) through drug treating 4d, during observation, is 6dpf mutually.Figure a is blank, the figure negative contrast of b (0.1%DMSO), the oxibendazole processed group that figure d-f is variable concentrations, the figure positive contrast of c (1000 μ M5-FU).
figure 10for the growth inhibition ratio of oxibendazole of the present invention to the carcinoma transplanted cell.Oxibendazole presents step increase to the suppression ratio of mankind's carcinoma transplanted Growth of Cells along with the rising of concentration, three concentration oxibendazole group suppression ratio are respectively: 1 μ M(12.3%), 2.5 μ M(26.8%), 10 μ M(42.6%).
figure 11for quantitative assessment oxibendazole of the present invention to looking the therapeutical effect of degeneration of macula.Figure a-e, the Brachydanio rerio of after fertilization 1dpf, through drug treating 4d, is 5dpf during observation mutually.In border circular areas shown in dotted line, it is choroidal artery.The figure negative contrast of a (0.1%DMSO), figure b is model group (1mg/ml cobaltous chloride), the oxibendazole processed group that figure c-e is various dose.
figure 12for oxibendazole of the present invention to the paraplasm suppression ratio of choroidal artery.Oxibendazole presents step increase to the paraplasm suppression ratio of choroidal artery along with the rising of injected dose, three oxibendazole dosage group suppression ratio are respectively: 0.25 μ g(6.9%), 0.83 μ g(23.8%), 2.49 μ g(35.3%).
The specific embodiment
Below in conjunction with Figure of description and embodiment, the present invention is further elaborated, but protection scope of the present invention is not limited to this.
The Brachydanio rerio initialism of being correlated with
After fertilization hourage: hpf-hours postfertilization
Back of the body major axis blood vessel: DLAV-dorsal longitudinal anastomotic vessel
Blood vessel between body segment: ISV-intersegmental vessel
Dorsal aorta: DA-dorsal aorta
Posterior cardinal vein: PCV-posterior cardinal vessel
Subintestinal vein blood vessel: SIV-subintestinal vessel
Green fluorescent protein: GFP-green fluorescent protein
embodiment 1the inhibition of qualitative observation oxibendazole to blood vessel (ISV) generation model between the Brachydanio rerio body segment
Brachydanio rerio:
The Brachydanio rerio that the present embodiment is used is blood vessel transgenic green fluorescence Brachydanio rerio (a kind of gene of being expressed by the Brachydanio rerio endothelial-cell specific drives green fluorescent protein at Brachydanio rerio vascular endothelial cell specifically expressing as driven element), and raising and Application standard strictly carry out with reference to U.S.'s management of laboratory animal and the requirement of using committee (IACUC).
The water (Fish water) of breeding fish:
Collocation method: 1L reverse osmosis water (reverse osmosis (RO) water) adds 0.3g sea salt (Instant Ocean salts).
Dimethyl sulfoxide (DMSO, analytical pure):
Buy in Shanghai Jing Chun Industrial Co., Ltd. (article No. #1095515, lot number #30573).0.1% DMSO solution (negative control) configuration: during use, with the water of breeding fish, be configured to the working solution that concentration is 0.1%, now with the current.
Lovastatin (positive control):
Buy the U.S. logical sequence in Dalian, purity is greater than 98% (HPLC method).During use, by 0.1% DMSO solution allocation, become the required concentration of experiment, in this experiment, the working concentration of positive control drug is 10 μ M.
Oxibendazole (Oxibendazole):
Buy in Sigma-Aldrich company (article No. #20559-55-1, lot number #022M4069), become the oxibendazole solution of variable concentrations during use by 0.1% DMSO solution allocation, working concentration is respectively 0.1 μ M, 0.25 μ M, 0.3 μ M, 0.4 μ M, 0.5 μ M, 1 μ M.
experimental technique:
(1) experiment grouping and embryo process: get 45 well-developed zebrafish embryos, during fetal development, be after fertilization 23hpf (hour-postfertilization mutually, hpf), be divided at random 3 groups of (negative control group, the medicine group, positive controls), every group of embryo's quantity is 15.During operation by embryo's uniform distribution to 48 porocyte culture plate (Greiner, Germany), 15, every hole embryo, every hole embryo raises water 1ml.
(2) drug treating: with pipettor (range 100～1000 μ l, Eppendorf), rapidly pre-configured medicinal liquid is added to Zhong，Mei hole, the hole 1ml that 48 porocyte culture plates are corresponding.Before adding medicinal liquid, with pipettor (range 10～1000 μ l, Eppendorf), the raising water of hatching the embryo in 48 orifice plates is shifted out as possible, this operation needs to complete in advance at short notice, to prevent embryo's drying.The experimental situation temperature is controlled at 28.5 ℃ of left and right, relative humidity 40～70%.Then with masking foil, 48 orifice plates are wrapped, carry out the experiment labelling, be positioned over rapidly in the Brachydanio rerio incubator and continue to cultivate 24h (the incubator temperature is controlled at 28.5 ± 0.5 ℃).
(3) Phenotypic Observation and statistics: observe each hole embryo's phenotype under Stereo microscope, observation index: observe medicine to fetal development, blood circulation, the impact of the aspects such as heartbeat.Then, the affected embryo of blood circulation being placed under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope) and further observing and take pictures, is 48hpf mutually while taking pictures, and to confirm angiogenesis, suppresses phenotype.
Experimental result is shown in
fig. 2.
embodiment 2the inhibition of quantitative assessment oxibendazole to blood vessel (ISV) generation model between the Brachydanio rerio body segment
The Brachydanio rerio vascular endothelial cell sprouts from after fertilization 20hpf, and the 30-31hpf left and right forms blood vessel network between main body segment, and as blood vessel (ISV) between back of the body major axis blood vessel (DLAV) and body segment, 48hpf forms complete axon blood vessel network basically
[28], blood vessel (ISV) between high-visible complete body segment now.Between complete body segment, blood vessel mainly refers to connect that section blood vessel between (DLAV) between dorsal aorta (DA) and back of the body major axis blood vessel, sees
fig. 1(vascular pattern between 48hpf blood vessel transgenic fluorescence Brachydanio rerio body segment).The Brachydanio rerio one of 48hpf has blood vessel (ISVs) between 28 pairs of complete body segments.Experimental technique is as follows:
(1) experiment grouping and embryo process: getting 240 well-developed zebrafish embryos, is after fertilization 23hpf (hour-postfertilization, hpf) mutually during fetal development, is divided at random 8 groups, sees the following form:
30 of every group of zebrafish embryo quantity.During operation by embryo's uniform distribution to 48 porocyte culture plate (Greiner, Germany), 15, every hole embryo, each drug level is processed 30 embryos, every hole embryo raises water 1ml.
(2) drug treating: see the experimental technique operating procedure (2) in embodiment 1.
(3) Phenotypic Observation and quantitative statistics: the embryo after each drug level is processed observes and takes pictures under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope), while taking pictures, be 48hpf mutually, impact blood vessel (ISV) between the Brachydanio rerio body segment generated to analyze each drug level.Get at random 12 embryos from each experimental group and carry out quantitative statistics, statistical indicator is as follows:
1. blood vessel (ISVs) quantity between complete body segment: connect between dorsal aorta (DA) and back of the body major axis blood vessel (DLAV)
Between blood vessel
Utilize GraphPad Prism software to add up mapping, and calculate oxibendazole and suppress the IC that between the Brachydanio rerio body segment, blood vessel (ISV) generates
50, experimental result is shown in
fig. 3～Fig. 4: the suppression ratio that oxibendazole generates blood vessel (ISV) between the Brachydanio rerio body segment presents step increase along with the rising of concentration, the suppression ratio that each concentration oxibendazole group generates blood vessel (ISV) between the Brachydanio rerio body segment is respectively: 0.1 μ M(0%), 0.25 μ M(18.96%), 0.3 μ M(57.80%), 0.4 μ M(84.71%), 0.5 μ M(100%), 1 μ M(100%), IC
50be 0.29 μ M.
embodiment 3the inhibition of qualitative observation oxibendazole to Brachydanio rerio subintestinal vein blood vessel (SIV) generation model
Brachydanio rerio subintestinal vein blood vessel (SIV, subintestinal vessel) is grown in the yolk sac both sides, and its shape is like one basket, and subintestinal vein blood vessel (SIV) is about 50～100 μ m by the body segment veutro to the length of downward-extension
[20-21].See
fig. 5(72hpf blood vessel transgenic fluorescence Brachydanio rerio subintestinal vein vascular pattern).Experimental technique is as follows:
(1) experiment grouping and embryo process: get 45 well-developed zebrafish embryos, during fetal development, be after fertilization 48hpf (hour-postfertilization mutually, hpf), be divided at random 3 groups of (negative control group, the medicine group, positive controls), every group of embryo's quantity is 15.During operation by embryo's uniform distribution to 48 porocyte culture plate (Greiner, Germany), 15, every hole embryo, every hole embryo raises water 1ml.
(2) drug treating: see the experimental technique operating procedure (2) in embodiment 1.
(3) Phenotypic Observation and statistics: observe each hole embryo's phenotype under Stereo microscope, observation index: observe medicine to fetal development, blood circulation, the impact of the aspects such as heartbeat.Then, the affected embryo of blood circulation being placed under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope) and further observing and take pictures, is 72hpf mutually while taking pictures, and to confirm angiogenesis, suppresses phenotype.
Experimental result is shown in
fig. 6.
embodiment 4the inhibition of quantitative assessment oxibendazole to Brachydanio rerio subintestinal vein blood vessel (SIV) generation model
experimental technique:
(1) experiment grouping and embryo process: getting 240 well-developed zebrafish embryos, is after fertilization 48hpf (hour-postfertilization, hpf) mutually during fetal development, is divided at random 7 groups, sees the following form:
30 of every group of zebrafish embryo quantity.During operation by embryo's uniform distribution to 48 porocyte culture plate (Greiner, Germany), 15, every hole embryo, each drug level is processed 30 embryos, every hole embryo raises water 1ml.
(2) drug treating: see the experimental technique operating procedure (2) in embodiment 1.
(3) Phenotypic Observation and quantitative statistics: the embryo after each drug level is processed observes and takes pictures under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope), while taking pictures, be 72hpf mutually, impact Brachydanio rerio subintestinal vein blood vessel (SIV) generated to analyze each drug level.Get at random 10 embryos from each experimental group and carry out quantitative statistics, statistical indicator is as follows:
1. subintestinal vein blood vessel area (SIV area): utilize Nikon AZ100 body formula fluorescence microscope configuration
NIS-Elements 3.1 softwares are calculated
Utilize GraphPad Prism software to add up mapping, and calculate oxibendazole and suppress the IC that Brachydanio rerio subintestinal vein blood vessel (SIV) generates
50, experimental result is shown in
fig. 7～Fig. 8: the suppression ratio that oxibendazole generates Brachydanio rerio subintestinal vein blood vessel (SIV) presents step increase along with the rising of concentration, the suppression ratio that each concentration oxibendazole group generates Brachydanio rerio subintestinal vein blood vessel (SIV) is respectively: 0.25 μ M(18.04%), 0.3 μ M(49.57%), 0.4 μ M(92.83%), 0.5 μ M(96.02%), 1 μ M(99.62%), IC
50be 0.30 μ M.
embodiment 5brachydanio rerio human hela (HeLa) transplantation model is estimated the antitumor drug effect of oxibendazole
The growth of entity tumor and diffusion depend on the formation of neovascularity in tumor, and obtain nutrient by new vessels; The formation of neovascularity and growth, promoted the transfer of tumor cell.The present embodiment is for illustrating that oxibendazole can suppress growth and the migration of tumor.Experimental technique is as follows:
(1) experiment grouping and embryo process: getting the zebrafish embryo that 150 transplanting have human hela (HeLa) cell, is after fertilization 2dpf (day-postfertilization, dpf) mutually during fetal development, is divided at random 5 groups, sees the following form:
30 of every group of zebrafish embryo quantity.During operation by embryo's uniform distribution to 6 porocyte culture plate (Greiner, Germany), 30, every hole embryo, each drug level is processed 30 embryos, every hole embryo raises water 3ml.
(2) drug treating: with pipettor, rapidly pre-configured medicinal liquid is added to Zhong，Mei hole, the hole 3ml that 6 porocyte culture plates are corresponding.Then with masking foil, 6 orifice plates are wrapped, carry out the experiment labelling, be positioned in the Brachydanio rerio incubator and continue to cultivate 4d (the incubator temperature is controlled at 35.5 ± 0.5 ℃).
(3) Phenotypic Observation and quantitative statistics: the embryo after each concentration drug treating is observed and takes pictures under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope), while taking pictures, be 6dpf mutually, to analyze the inhibitory action of each drug level to Brachydanio rerio human hela (HeLa) transplantation model.Get at random 10 embryos from each experimental group and carry out quantitative statistics, statistical indicator is as follows:
1. the inhibitory action of qualitative evaluation oxibendazole to neoplasm metastasis;
2. the inhibitory action of quantitative assessment oxibendazole to tumor growth: utilize Nikon NIS-Elements 3.1 computed in software tumor cell fluorescence intensities (S), the statistical procedures result means with mean ± SE; Oxibendazole is as follows to the inhibition computing formula of tumor growth:
Utilize GraphPad Prism software to add up mapping, experimental result is shown in
fig. 9～Figure 10: oxibendazole presents step increase to the suppression ratio of mankind's carcinoma transplanted Growth of Cells along with the rising of concentration, three concentration oxibendazole group suppression ratio are respectively: 1 μ M(12.3%), 2.5 μ M(26.8%), 10 μ M(42.6%).
embodiment 6the quantitative assessment oxibendazole is looked the therapeutical effect of degeneration of macula to moist old age
Look degeneration of macula moist old age main because choroidal artery generates extremely, seepage appears in newborn invalid blood capillary, the liquid of vascular leakage and then destruction macula lutea.Cobaltous chloride can be induced Brachydanio rerio retina choroid plexus blood vessel hyperplasia, visual cell degeneration, is similar to the change that the mankind look degeneration of macula moist old age
[29].Embodiment is for illustrating that oxibendazole has therapeutic effect to looking degeneration of macula moist old age.Experimental technique is as follows:
(1) experiment grouping and embryo process: getting 150 well-developed zebrafish embryos, is after fertilization 1dpf (day-postfertilization, dpf) mutually during fetal development, is divided at random 5 groups, sees the following form:
30 of every group of zebrafish embryo quantity.During operation by embryo's uniform distribution to 6 porocyte culture plate (Greiner, Germany), 30, every hole embryo, every hole embryo raises water 3ml.
(2) drug treating: add DMSO in negative control group, making its final concentration is 0.1%; Add cobaltous chloride in model group, making its final concentration is 1 mg/ml; Oxibendazole, by the administration of microinjection mode, is all injected 30 embryos for every group, after injection, the embryo is put into respectively to the raising water that 3ml contains 1 mg/ml cobaltous chloride by group.
(3) Phenotypic Observation and quantitative statistics: the embryo after each dose drug is processed observes and takes pictures under body formula fluorescence microscope (Nikon AZ100 body formula fluorescence microscope), while taking pictures, be 5dpf mutually, to analyze the inhibitory action of each drug dose to Brachydanio rerio eye choroidal abnormalities hypertrophy blood vessel.Get at random 10 embryos from each experimental group and carry out quantitative statistics, statistical indicator is as follows:
1. the inhibitory action of qualitative evaluation oxibendazole to eye choroidal abnormalities hypertrophy blood vessel;
2. the inhibitory action of quantitative assessment oxibendazole to choroidal abnormalities hypertrophy blood vessel: utilize NIS-Elements 3.1 computed in software choroidal abnormalities hypertrophy blood vessel fluorescence intensities (S), the statistical procedures result means with mean ± SE; Oxibendazole is as follows to the inhibition computing formula of choroidal abnormalities hypertrophy blood vessel:
Utilize GraphPad Prism software to add up mapping, experimental result is shown in
figure 11～Figure 12: oxibendazole presents step increase to the paraplasm suppression ratio of choroidal artery along with the rising of dosage, three oxibendazole dosage group suppression ratio are respectively: 0.25 μ g(6.9%), 0.83 μ g(23.8%), 2.49 μ g(35.3%).
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Claims (9)
1. the application of oxibendazole in preparing angiogenesis inhibitor class medicine.
2. application according to claim 1, is characterized in that, described medicine is oral administered dosage form, injecting medicine-feeding form, mucosa delivery dosage form or percutaneous dosing dosage form.
3. application according to claim 1, is characterized in that, described medicine is tablet, capsule, granule, oral liquid, injection, patch or gel form.
4. the application of oxibendazole in preparing antitumor drug.
5. application according to claim 4, is characterized in that, described oxibendazole prevents or treat tumor by the generation that suppresses neovascularity in tumor.
6. application according to claim 4, is characterized in that, described medicine is tablet, capsule, granule, oral liquid, injection, patch or gel form.
7. oxibendazole is preparing the application of moisture resistance in looking the degeneration of macula medicine old age.
8. application according to claim 7, is characterized in that, described oxibendazole prevents or treats and look degeneration of macula moist old age by suppressing the choroidal artery paraplasm.
9. application according to claim 7, is characterized in that, described medicine is tablet, capsule, granule, oral liquid, injection, patch or gel form.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105424666A (en) * | 2015-12-11 | 2016-03-23 | 山东省科学院生物研究所 | Method for quickly and quantitatively evaluating blood vessel generation promotion function of chemical compounds on zebra fish |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101035530A (en) * | 2004-08-31 | 2007-09-12 | 新南部创新有限公司 | VEGF inhibition |
| CN101111248A (en) * | 2004-12-06 | 2008-01-23 | 新南部创新有限公司 | Treatment for cancer |
| WO2009043093A1 (en) * | 2007-10-04 | 2009-04-09 | Newsouth Innovations Pty Limited | Hif inhibition |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101035530A (en) * | 2004-08-31 | 2007-09-12 | 新南部创新有限公司 | VEGF inhibition |
| CN101111248A (en) * | 2004-12-06 | 2008-01-23 | 新南部创新有限公司 | Treatment for cancer |
| WO2009043093A1 (en) * | 2007-10-04 | 2009-04-09 | Newsouth Innovations Pty Limited | Hif inhibition |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105424666A (en) * | 2015-12-11 | 2016-03-23 | 山东省科学院生物研究所 | Method for quickly and quantitatively evaluating blood vessel generation promotion function of chemical compounds on zebra fish |
| CN105424666B (en) * | 2015-12-11 | 2018-06-29 | 山东省科学院生物研究所 | A kind of quick, quantitative assessment compound is to the method for zebra fish angiogenesis facilitation |
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