CN108324942B - Application in Osthole and toltrazuril joint preparation treatment gastric cancer medicament - Google Patents
Application in Osthole and toltrazuril joint preparation treatment gastric cancer medicament Download PDFInfo
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- CN108324942B CN108324942B CN201810257586.8A CN201810257586A CN108324942B CN 108324942 B CN108324942 B CN 108324942B CN 201810257586 A CN201810257586 A CN 201810257586A CN 108324942 B CN108324942 B CN 108324942B
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- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
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Abstract
The present invention provides the application in Osthole and toltrazuril joint preparation treatment gastric cancer medicament.The present invention, which for the first time combines Osthole and toltrazuril, is used to treat gastric cancer, and especially HER2 high expresses gastric cancer.In vitro test shows that the combination therapy of Osthole and toltrazuril shows strong tumor killing effect to HER2 high expression gastric cancer N87 cell, and therapeutic effect has been more than that treatment group is used alone in Osthole or Herceptin, and shows collaboration depression effect.In vivo studies shows that Herceptin joint Osthole administration is administered alone compared with Osthole or Herceptin, and the significantly more efficient effect for inhibiting tumour progression is shown in the highly expressed gastric cancer N87 bearing mouse model of HER2.
Description
Technical field
The invention belongs to field of medicaments, specifically, being related to Osthole and toltrazuril joint preparation treatment gastric cancer medicament
In application.
Background technique
Malignant tumour is to seriously endanger the disease of human health, is in second in the disease of various lethals.In recent years
Coming, the disease incidence of malignant tumour obviously rises, meanwhile, therapeutic effect is poor, and advanced stage easily shifts, and prognosis is bad.At present
Clinically, used conventional treatments such as chemicotherapy and operative treatment largely alleviates pain, but these therapies
Side effect and limitation are still very big, and curative effect is difficult to further increase.
The proliferation of normal cell is strictly to activate its growth factor receptors to control by respective ligand, for example grow
Factor receptor tyrosine kinases.Cancer cell is also to be proliferated under the activation of growth factor receptors, but when losing normal proliferative
Strict control.It is this it is out of control may be such as overexpression or the growth factor receptor of growth factor as caused by several factors
The overexpression of body, and the Spontaneous Activation of the biochemical route by growth factor regulation.Participating in carcinogenic receptor includes epidermis
Growth factor acceptor 2 (HER2), from the growth factor receptors (PDGFR) of blood platelet, insulin-like growth factor receptor
(IGFR), trk C (NGFR) and fibroblast growth factor receptor (FGF) etc..
Epidermal growth factor acceptor 2 (i.e. HER2) is also referred to as c-erbB 2/Her2, and family member is growth factor receptor
Body tyrosine kinase, they interact in cell surface and special growth factor or native ligand, such as with EGF or TGFα
It interacts, thus activated receptor tyrosine kinase.HER2 is adjusting growth, reparation and the existence of tumour cell, new vessels
It generates, played an important role in invasion and transfer, while having expression in significant component of human tumor.Much disliking
Property tumour in, HER2 expression it is often related to poor prognosis and lower survival rate.The monoclonal antibody medicine of targeting HER2 can hinder
The activity of disconnected HER2, can be effectively suppressed its phosphorylation and signal transduction, so that the antitumor action of multiple pathways is played, while
It can increase the antitumor curative effect of chemotherapy and radiation.
Herceptin (Trastuzumab, Herceptin) is the humanization list of targeting epidermal growth factor receptor HER2
Clonal antibody has obtained treatment of the U.S. FDA approval for HER2 high expression metastatic breast cancer in 1998.But make in clinic
Only have 30% with the effective percentage of discovery Trastuzumab in the process.The activation of HER2 receptor depends on two approach: (1)
HER2 overexpression.HER2 can form HER2 homodimer when being overexpressed and activated receptor, this process only depend on HER2
Height expression of the molecule on cell.(2) the HER2 heterodimer of ligand-dependent is formed.Firstly, HER1, HER3, HER4 with
Respective native ligand combines and occurs conformational change, allows them to form heterodimer and activated receptor with HER2, this
Overexpression of a process independent of HER2.The main Anticancer Effect and Mechanism of toltrazuril is to lower HER2 receptor to subtract
Its few cell-surface density, to inhibit the formation of homodimer caused by over-expressing due to HER2.
Chinese medicine is an important component in the traditional medicine and combined therapy of tumour measure in China, is being improved
Patient clinical symptom, raising life quality, extension life cycle, control progression of the disease etc. play an important role.Past
In decades, a large amount of some molecular compounds from Chinese herbal medicine of studies have shown that for preventing and treating cancer have been found to have
There is antitumor effect.Coumarin kind compound is exactly one type.Wherein, Osthole is linear furocoumarins class
(furanocoumarins) representation compound, the entitled osthole (7-methoxy-8- of chemistry
(3-methyl-2-butenyl) coumarin), also known as osthole is from umbelliferae cnidium monnieri (Cnidium
Monnieri (L.) Cusson) dry mature fruit frutus cnidii, Angelica pubescens a variety of Chinese herbal medicines such as root and rhizome extract
A kind of obtained biologically active cumarin.Not only there is domestic and international studies have shown that Osthole anti-hepatitis, anti-sclerotin to dredge
The effects of pine, antiallergic action, anti-mutagenesis, and it is anti-swollen by inhibiting growth of tumour cell and inducing cell apoptosis to play it
Tumor effect.External anticancer experimental study shows that Osthole shows certain antiproliferative effect to tumour cell；To human cervical carcinoma
The cell lines such as passage cell HeLa, human umbilical vein endothelial cell have apparent inhibitory activity and cause the effect of apoptosis.Internal animal
Experimental result shows that Osthole has certain inhibition to make the knurl growth of the BALB/C nude mice model of lung squamous cancer and adenocarcinoma of lung
With, and on the liver of mouse, spleen index and thymus index almost without influence, the characteristics of prompting frutus cnidii to be known as high-efficiency low-toxicity.To sum up
Described, Osthole is as a kind of low toxicity, natural extract or lead compound with anti-tumor effect, in anti-tumor drug
Research in have extensive prospect.
It can be seen that Osthole is a kind of very promising antitumoral compounds.However, Osthole and toltrazuril list
The research of anti-combination therapy HER2 high expression gastric cancer not yet has been reported that.
Summary of the invention
The object of the present invention is to provide the applications in Osthole and toltrazuril joint preparation treatment gastric cancer medicament.
In order to achieve the object of the present invention, the present invention is provided in Osthole and toltrazuril joint preparation treatment gastric cancer medicament
Using.
In the present invention, the gastric cancer refers to that HER2 high expresses gastric cancer.For example, the gastric cancer is and the highly expressed N87 of HER2
Caused by the similar clinical phenotypes of cell line.
The present invention also provides a kind of for treating the composition of gastric cancer, and effective component is Osthole and toltrazuril.
The composition is made of the Osthole of therapeutically effective amount and the toltrazuril of therapeutically effective amount.
The present invention further provides Ostholes and toltrazuril to express the application in gastric cancer in combination therapy HER2 high,
In, the Osthole of therapeutically effective amount and the toltrazuril of therapeutically effective amount be successively administered alone regardless of order or Osthole with
Toltrazuril is administered together simultaneously.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
The present invention, which for the first time combines Osthole and toltrazuril, is used to treat gastric cancer, and especially HER2 high expresses gastric cancer.Body
Experiments have shown that, the combination therapy of Osthole and toltrazuril shows strong suppression to HER2 high expression gastric cancer N87 cell outside
Tumor effect, therapeutic effect have been more than that treatment group is used alone in Osthole or Herceptin, and shows that collaboration inhibits
Effect.In vivo studies shows that Herceptin joint Osthole administration is administered alone compared with Osthole or Herceptin,
The significantly more efficient effect for inhibiting tumour progression is shown in the highly expressed gastric cancer N87 bearing mouse model of HER2.
Detailed description of the invention
Fig. 1 is the external inhibition of Osthole and Herceptin in the embodiment of the present invention 1 combined to gastric cancer N87 cell
Effect.
Fig. 2 is to be used in combination in the embodiment of the present invention 1 using CompuSyn software calculating Osthole and Herceptin
When index of cooperation (CI).Wherein, three groups of various concentrations of toltrazuril and Osthole in embodiment 1 are 1. 2. 3. corresponded respectively to
Combination.
Fig. 3 is that Osthole and Herceptin are combined to the external of breast cancer SK-BR-3 cell in inventive embodiments 1
Depression effect.
Fig. 4 is N87 Level of Apoptosis measurement chart in the embodiment of the present invention 2.
Fig. 5 is N87 Apoptosis proportion grading figure in the embodiment of the present invention 2.
Fig. 6 is that Osthole and Herceptin are combined to the tumor-bearing mice for being inoculated with N87 cell in the embodiment of the present invention 3
Internal depression effect curve.
It is thin from gastric cancer N87 Fig. 7 is receives Osthole and Herceptin combination treatment in the embodiment of the present invention 3 after
Tumour weighing analysis is taken out in born of the same parents' tumor-bearing mice.
Fig. 8 is the toxotest experimental result of Osthole and Herceptin drug combination in the embodiment of the present invention 4.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
It is defined as follows involved in following embodiment: HER2: epidermal growth factor acceptor 2；CCK-8: viable count examination
Agent box 8；Trastuzumab: Herceptin；Osthole: Osthole.Herceptin is purchased from company, Roche Group；CCK-
8 reagents are purchased from Dojindo company；Osthole is purchased from Mike woods biochemical technology company.
The external depression effect of 1 Osthole of embodiment and Herceptin combined to gastric cancer N87 cell
The good N87 cell of growth conditions is taken, with every hole 1.5 × 103A to be uniformly laid in 96 porocyte culture plates, hole is thin
In born of the same parents' culture plate, 37 DEG C, 8%CO2It is cultivated for 24 hours in incubator；Group is applied alone in setting Herceptin, and group and cnidium monnieri is applied alone in Osthole
Sub- element joint Herceptin group.Every hole be added the gradient dilution since 50 μ g/mL Herceptin (Tra,
Trastuzumab it), and three times repeats；The Osthole (Ost, Osthole) from 100 μM of gradient dilutions is added in every hole；
After drug-treated 48h, CCK-8 reagent colour development is added.The readings under 450 nm wavelength of iMark microplate reader calculates Cell viability.
Cell viability (%)=[(test group light absorption value-blank group light absorption value)/(control group light absorption value-blank group extinction
Value)] × 100
Experimental result is as shown in Figure 1.Ordinate is inhibition rate of tumor growth in Fig. 1, and abscissa is that the song of various concentration is appropriate
The combination of pearl monoclonal antibody and Osthole.The experimental results showed that Herceptin joint Osthole treats the highly expressed stomach of HER2
Cancer N87 cell is applied alone group or Herceptin that group is applied alone to have more significant inhibition growth result compared with Osthole.
Further, association of the toltrazuril with Osthole when various concentration combines is determined using Chou-Talalay method
Same-action.A combination thereof concentration is as follows: 1. 3.125 μ g/mL+6.25 μM of Ostholes of toltrazuril；2. 12.5 μ g/mL toltrazurils+25
μM Osthole；3. 50 μ g/mL+100 μM of Ostholes of toltrazuril.As a result as shown in Fig. 2, being calculated using Compusyn software
Index of cooperation value (CI) under three of the above composition concentration, CI prove there is synergistic effect between drug less than 1.As a result table
Bright, CI value is respectively less than 1 under three kinds of composition concentrations, it was demonstrated that Osthole joint Herceptin can cooperate with inhibition gastric cancer
N87 growth of tumour cell.
Depression effect is cooperateed with to whether other tumour cells have in order to verify toltrazuril with Osthole combination, the present invention
The two combination is further had detected to the growth inhibitory activity of the breast cancer cell SK-BR-3 of the HER2 positive.Using above-mentioned identical
Method, experimental result is as shown in Figure 3.The result shows that group or Herceptin be applied alone that group is applied alone relative to Osthole, it is bent appropriate
Pearl monoclonal antibody combines Osthole and treats the highly expressed breast cancer SK-BR-3 cell of HER2, does not show collaboration depression effect.
As it can be seen that toltrazuril is only shown in stomach cancer cell N87 cell with Osthole combination cooperates with depression effect, and
Other tumour cells are not shown then with significant collaboration depression effect.
2 Osthole of embodiment and Herceptin drug combination promote N87 apoptosis
Take the good N87 cell of growth conditions with 5 × 106// hole is uniformly taped against in 6 orifice plates.To cell in each hole respectively into
Row following four processing: 1. IgG (Control) (10 μ g/mL)；2. the Herceptin of 10 μ g/mL；3. 40 μM of frutus cnidii
Element；4.+40 μM of Herceptin of the Osthole of 10 μ g/mL；Treatment conditions are 37 DEG C of 8%CO2It is incubated for 30 hours.Then it uses
Annexin (Annexin V) and propidium iodide (PI) are dyed, and measure apoptotic cell ratio using flow cytometer.
Experimental result is as shown in Figure 4 and Figure 5, and Fig. 4 is N87 Level of Apoptosis measurement chart, and Fig. 5 is N87 Apoptosis ratio
Example analysis chart.The ratio of Herceptin and Osthole drug combination group apoptotic cell is apparently higher than Herceptin and group is applied alone
Or group is applied alone in Osthole.Therefore, the promotion of Apoptosis ratio caused by drug combination further illustrates Herceptin
The mechanism of action for inhibiting gastric cancer N87 growth of tumour cell is cooperateed with Osthole.
3 Osthole of embodiment and Herceptin joint for inhibiting growth of tumour cell in vivo
With gastric cancer N87 cell subcutaneous inoculation female NOD-SCID mouse, reach after inoculated tumour cell to tumor tissues
80mm3-120mm3Size is injected intravenously following 4 groups of mouse, every group of 6 mouse: 1. human IgG (Control) (15 mg/kg)；②
Herceptin group (Trastuzumab) (15mg/kg)；3. Osthole group (Osthole) (100 mg/kg)；4. toltrazuril
Monoclonal antibody (15mg/kg)+Osthole (100mg/kg) group (Trastuzumab+Osthole).Wherein Osthole administration mode
For daily all carry out intraperitoneal administration, toltrazuril administration mode be every three days give a medicine, co-injection 2 weeks.In administration process, every
Observation tumour growth situation on the 2nd measures tumor size and weight to evaluate the tumor-inhibiting action of drug.Experimental result such as Fig. 6 and Fig. 7
It is shown.Ordinate is gross tumor volume in Fig. 6, and abscissa is number of days.Ordinate is the weight of tumour after being administered in Fig. 7, horizontal
Coordinate is number of days.
The experimental results showed that Herceptin, which combines Osthole group, is applied alone group or Herceptin list compared with Osthole
The significantly more efficient effect for inhibiting tumour progression is shown in the highly expressed gastric cancer N87 bearing mouse model of HER2 with group,
Difference is all statistically significant.
The experiment of the toxotest of 4 Osthole of embodiment and Herceptin drug combination
During being administered to tumor bearing nude mice, we to the changes of weight situation of the tumor-bearing mice of four processing groups into
Monitoring gone to assess the non-specific toxicity of drug combination, including following four processing group: 1. human IgG (Control)
(15mg/kg)；2. Herceptin group (Trastuzumab) (15mg/kg)；Osthole group 3. (Osthole) (100mg/
kg)；4. Herceptin (15mg/kg)+Osthole (100mg/kg) group (Trastuzumab+Osthole).Experimental result
As shown in Figure 8.The result shows that Herceptin, which combines Osthole group average mice body weight, is applied alone group or song compared with Osthole
It is substantially reduced that trastuzumab is applied alone group not have, i.e., simultaneously genotoxic potential is not present in the drug combination of two kinds drugs.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (3)
1. the application in Osthole and toltrazuril joint preparation treatment gastric cancer medicament, the gastric cancer refer to that HER2 high expresses stomach
Cancer.
2. a kind of for treating the composition of gastric cancer, which is characterized in that effective component is Osthole and toltrazuril, the gastric cancer
Refer to that HER2 high expresses gastric cancer.
3. composition according to claim 2, which is characterized in that by the Osthole and therapeutically effective amount of therapeutically effective amount
Toltrazuril composition.
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Effective date of registration: 20201104 Address after: 453700 comprehensive R & D building V (A-F) (2-3) - 1, torch Park, high tech Zone, 1789 Xinfei Avenue, Xinxiang City, Henan Province Patentee after: HENAN PUNUOYI BIOLOGICAL PRODUCT RESEARCH INSTITUTE Co.,Ltd. Patentee after: XINXIANG MEDICAL University Address before: No. 601 Hongqi District Jinsui road in Xinxiang city of Henan Province in 453003 Patentee before: XINXIANG MEDICAL University |