CN102218140B - Tumor resistance effect of medicine combination of salvianolic acid B and coxib medicaments - Google Patents
Tumor resistance effect of medicine combination of salvianolic acid B and coxib medicaments Download PDFInfo
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Abstract
The invention provides a medicinal composition which comprises 1% to 99% of salvianolic acid B and 1 to 99% of coxib medicine by weight percent as well as an pharmacologically acceptable carrier, and application of the medicinal composition to preparation of anti-tumor medicines.
Description
Technical field
The present invention relates to pharmaceutical field, specifically salvianolic acid B and chemotherapeutic are united the purposes of the medicine of preparation treatment tumor.More specifically, be the purposes of the medicine of salvianolic acid B and the medication combined preparation treatment of former times dry goods tumor
Background technology
Labiate Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bge) is herbaceos perennial, is the Chinese medicine of China, has stasis-dispelling and pain-killing, promoting blood circulation to restore menstrual flow, an effect of the relieving restlessness that clears away heart-fire.In the modern clinic, extensively use it for prevention and Cardiovarscular, nephropathy, hepatopathy etc.Research is in recent years found, water-soluble components is important active ingredient in the Radix Salviae Miltiorrhizae, because salvianolic acid is difficult to obtain sterling, and danshensu and protocatechualdehyde are more easily separated and synthetic, so most of pharmaceutical factory is danshensu and the protocatechualdehyde quality control index as Danshen root injection.Also have part pharmaceutical factory that salvianolic acid B salt is used for prevention cardiovascular and cerebrovascular disease, prevention alzheimer disease, treatment renal failure is improved in the medicines such as uremic symptom, antioxidation, antithrombotic, anti-cardiac-cerebral ischemia, the liver protecting and adjusting blood fat.Salvianolic acid B is one of aqueous soluble active constituent of Radix Salviae Miltiorrhizae.Salvianolic acid B magnesium (Magnesium Lithospermate B) is to separate a kind of water-soluble phenolic acids material that obtains from Radix Salviae Miltiorrhizae, and its molecular formula is C
36H
28O
16Mg, structural formula is:
In Chinese patent application 02136970.4 " application of salvianolic acid B in preparation medicine for treating tumor thing ", 200510026457.0 " application of salvianolic acid B in the medicine that the preparation prophylaxis of tumours occurs ", 200510026456.6 " application of Radix Salviae Miltiorrhizae in the medicine that preparation prevention carcinoma of prostate occurs ", disclose salvianolic acid B in vitro study in the inhibition lethal effect of kinds of tumor cells and the animal vivo test to the prophylactic treatment effect of tumor, and the therapeutical effect of salvianolic acid B to hepatocarcinoma disclosed.But, also need further research for the effect of salvianolic acid B and other medicines administering drug combinations.
Celecoxib, have another name called celecoxib, commodity are called celecoxib, belong to NSAID (non-steroidal anti-inflammatory drug) (non-steroidalanti-inflammatory drugs, NSAIDs), the activity of inhibition Cycloxygenase-2 that can high selectivity and do not suppress the activity of Cycloxygenase-1,1999 through the drugs approved by FDA listing, as the curative of rheumatoid arthritis and osteoarthritis.This medical instrument has good antiinflammatory, pain relieving and refrigeration function, and that has reduced obviously simultaneously that traditional NSAIDs has causes the serious gastrointestinal side effects such as gastric ulcer, hemorrhage and perforation.
Nearly two during the last ten years, massive epidemiology, Clinical and experimental study show both at home and abroad, non-steroidal anti-inflammatory drugs has prevention and suppresses the effect of human tumor, although its mechanism is still among inquiring into, but mainly think at present relevant with the activity that suppresses Cycloxygenase-2 (cyclooxygenase-2, COX-2).Former times, the dry goods medicine was as a kind of high selectivity cox 2 inhibitor, because its targeting is strong, side effect is little, in the prevention of the tumors such as gastroenteric tumor, hepatocarcinoma, pulmonary carcinoma, breast carcinoma and oral cancer and treatment, will play a significant role, relevant vivo and vitro has been studied Partial Proof its anti-tumor activity, thus in recent years study hotspot become.At present research is thought, former times the dry goods medicine may breed by inhibition tumor cell, inducing cell transfer die, suppress that the tumor neovascularity generates, the metabolism of blocking-up carcinogen and weaken the COX-2 such as immunosuppressant expression dependence or the non-Dependent of tumor mediation, reach the antineoplastic purpose.Research in recent years finds that also it has gamma-rays and chemotherapeutics to the sensitization of tumor.It should be noted that and studies show that in recent years comprise that the former times dry goods medicine of celecoxib may increase patient's adverse cardiac events as osteoarthritis and treating rheumatoid arthritis medicine life-time service the time.Current, its consumption is higher than it as the consumption of antirheumatic when former times, the dry goods medicine was used as antitumor drug, therefore, exists too the risk of adverse cardiac events increase as the former times dry goods medicine of anticancer drug therapy.The method that reduces former times dry goods adverse drug side effect is still being sought in this area.
According to WHO statistics, malignant tumor is one of principal disease that threatens human health, and the annual tumour patient of newly making a definite diagnosis in the whole world is more than 1,000 ten thousand, dead about more than 700 ten thousand people; The annual newly-increased tumor patient of China has more than 200 ten thousand, dead more than 130 ten thousand people approximately.Operation, chemotherapy and radiotherapy are three great tradition therapies for the treatment of malignant tumor.Yet relapse rate is but always high behind the malignant tumor operation, and chemotherapy and radiotherapy not only bring the side effect of destruction property to the patient, and often owing to the problems such as chemical sproof generation and sensitivity are not strong make curative effect limited.Therefore, but this area in the urgent need to new life-time service being provided, having no side effect and the safely and effectively associating for the treatment of malignant tumor medicine or medicine.Have antitumor or potentially have the material of antitumor action often may produce interaction in synergy, therefore not all administering drug combinations can both reach and two kinds of individually dosed suitable antitumous effects of medicine and/or lower toxicity.The report that any salvianolic acid B and the medication combined drug treatment malignant tumor of former times dry goods are not yet arranged at present.
This area needs safety and reliability and effectively treats the medicine of tumor.
Summary of the invention
Therefore, an object of the present invention is to provide the drug regimen of safer and more effective treatment tumor.
In one aspect of the invention, provide a kind of pharmaceutical composition, contained the salvianolic acid B of 1-99 % by weight and the chemotherapeutic of 1-99% % by weight, and acceptable carrier on the materia medica.Chemotherapeutic dry goods medicine of preferred former times, more preferably the dry goods medicine is selected from celecoxib, rofecoxib, Valdecoxib, etoricoxib and lumiracoxib former times.Preferred, former times the dry goods medicine be celecoxib.
In a preference aspect this, salvianolic acid B is the salt that forms with cation, cation chosen from Fe ion, sodium ion, potassium ion, calcium ion and magnesium ion.
In another preference aspect this, this pharmaceutical composition contains salvianolic acid B 1-300 μ M, celecoxib 1-300 μ M.
Also having in the preference aspect this, the concentration ratio of salvianolic acid B and celecoxib is 1: 10-10: 1.The concentration ratio of preferred salvianolic acid B and celecoxib is 1: 1.
Aspect second of the present invention, provide the purposes of aforementioned pharmaceutical compositions for the preparation of antineoplastic agent.The dosage form of preferred antineoplastic agent is selected from liquid dosage form, injection type, powder type, eye dosage form, transfusion dosage form, tablet form, pill-type, ointment dosage form, plaster dosage form, suppository, aerosol, soft capsule, hard capsule, leaching agent, durative action preparation, membrane, sponge agent, microencapsulation, liposome, depot formulation and radiosiotope preparation.
In the preference aspect this, tumor is selected from epithelial cancer, mesenchymal tissue cancer, nervous tissue's cancer, lymph and hemopoietic tissue cancer.
In another preference aspect this, tumor is selected from nasopharyngeal carcinoma, esophageal carcinoma, hepatocarcinoma, cancer of pancreas, gastric cancer, colorectal cancer, renal carcinoma, pulmonary carcinoma, breast carcinoma, cervical cancer, ovarian cancer, carcinoma of prostate, glioma and leukemia.
Description of drawings
Fig. 1 has shown that salvianolic acid B, celecoxib and the combination thereof of variable concentrations are on the impact of HNSCC cell proliferation.
Fig. 2 A-C has shown respectively the inhibitory action that salvianolic acid B, celecoxib and the combination thereof of variable concentrations are expressed COX-2 in the HNSCC cell.
Fig. 3 A has shown the in time stimulation that LPS expresses PGE2 (PGE2).Fig. 3 B has shown that the salvianolic acid B of various dose for the inhibitory action of the PGE2 expression of LPS stimulation, has wherein used respectively.Fig. 3 C has shown independent use salvianolic acid B, celecoxib, has united the inhibition of using salvianolic acid B and celecoxib that PGE2 is expressed.
Fig. 4 has shown contrast, has used salvianolic acid B, celecoxib and administering drug combinations to the curve of in-vivo tumour inhibition with event separately.
Fig. 5 A and Fig. 5 B have shown respectively the western engram analysis result that COX-2 expresses in the BPH1 that stimulates with LPS and the BPH1C1 cell.Wherein show respectively use LPS positive control, used separately the effect of salvianolic acid B, celecoxib and administering drug combinations.
Fig. 6 has shown the alone or in combination impact expressed of the PGE2 that induces for LPS of administration of salvianolic acid B and/or celecoxib in BPH1 and BPH1C1 cell.
Fig. 7 has shown the impact on its gross tumor volume of untreated fish group, salvianolic acid B processed group, celecoxib processed group and administering drug combinations group treatment tumor-bearing mice.
The specific embodiment
In order to help to understand and implement the present invention, below provide the definition of some term used herein.
As used herein, " chemotherapeutic " refers to affect the medicine that its growth cycle is bred or blocked to growth of tumour cell by chemical action.These medicines comprise cytotoxic drug, such as the alkylating agent class, destroy cell DNA and RNA, enzyme, protein and cause cell death, such as chlormethine, BCNU, busulfan etc.; The anti-metabolism medicine, impact and blocking-up nucleic acid are synthetic, such as fluorouracil, cytosine arabinoside etc.; Antibiotics, such as actinomycin D, mitomycin, mithramycin etc.; Alkaloids is used for the formation of spindle in the interference cell, makes cell rest on mitosis metaphase, such as vincristine, podophyllotoxin, etoposide etc.; Technology type affects tumor growth, such as tamoxifen, Progesterone, dexamethasone etc. for environment in changing; And the chemotherapeutic of other type.
As used herein, " effectively " of the medicine of medicine or pharmacological activity amount or " treatment effective dose " refer to avirulence, but medicine or the medicament that required effect is provided of q.s.In therapeutic alliance of the present invention, a kind of " effective dose " of composition refers to the amount of this chemical compound required effect of effective supply with other composition use in conjunction the time in the associating." effectively " amount can be different because of experimenter's difference, according to age and individual ordinary circumstance, specific active medicine etc.Therefore, can not always specify accurate " effective dose ".Yet suitable " effectively " amount can be used conventional experimental technique by the member of ordinary skill in the art and measures in any individual case.
Term used herein " treatment " refers to that the order of severity of symptom alleviates and/or the minimizing of frequency, symptom and/or cause the elimination of symptom reason, and the generation of symptom and/or they cause the prevention of the reason of symptom, the improvement of infringement or correction.Therefore, for example " treatment " patient comprises physiological event harmful in the specific disease of prevention or the susceptible individual, and processes the individuality that clinical symptoms is arranged.
Term used herein " adverse cardiac events " refers to disease or the disease that cardiovascular system occurs suddenly; It also refers to the unexpected deterioration of this disease or disease.The example of cardiovascular event includes, but are not limited to: heartbeat stops, myocardial infarction, ischemia, apoplexy, angina pectoris deterioration, congestive heart failure.
As used herein, term " salvianolic acid B " refers to the slaine of the soluble salvianolic acid phenolic acid of labiate Radix Salviae Miltiorrhizae as indicated above (Salviamiltiorrhiza Bge.), and its purity is higher than 90%.Wherein the salify metal ion includes but not limited to magnesium ion, sodium ion, calcium ion, iron ion, potassium ion etc.
As used herein, term " former times dry goods medicine " is a kind of specific C OX-2 inhibitor, and it can be crude drug, also can be acceptable dosage form on any pharmaceutics.Existing former times, the dry goods medicine had common chemical feature, was the chemical compound that three rings form, and was the substantitally planar triangle and arranged, and the para-position of A ring connects sulfamoyl or mesyl phenyl; The B ring is the phenyl of phenyl or replacement; The C ring is five yuan or hexa-atomic aromatic rings, fragrant heterocycle or fat (mixing) ring, connects AB two rings.For ensureing active requirement, A ring and B ring link with the ortho position of C ring, and are cis-configuration.Celecoxib, rofecoxib and support examine the west chemical constitution and former times the dry goods pharmacophore schematically as follows:
Term " administering drug combinations " refers to certain dosage regimen and gives object with drug regimen as used herein, and is as described herein.Salvianolic acid B can give simultaneously with former times dry goods medicine.Perhaps can give to give salvianolic acid B before the former times dry goods medicine.In addition, in the embodiment that gives respectively two kinds of medicines, can postpone to give the second medicine, so that larger conjoint therapy therapeutic effect to be provided.Pharmacology and medical domain technical staff will be familiar with being applicable to determine to give concept, method and the material of the transient factor in the non-while therapy.The example of correlative factor includes but not limited to: patient's physiological rhythm, with the cell cycle characteristics (for example, tumor cell type) of disease association to be treated and the pharmacokinetic parameters of used medicine.
The invention provides pharmaceutical composition, described compositions comprise salvianolic acid B and former times dry goods medicine and pharmaceutically acceptable carrier.Salvianolic acid B and former times the dry goods medicine exist with suitable ratio, with the safely and effectively therapeutic dose that provides two kinds of medicines to use filling into animal joint.
Salvianolic acid B and former times the dry goods medicine can with one or more pharmaceutically acceptable carrier or mixed with excipients, such as solvent, diluent etc., thereby form pharmaceutical composition.
Solid-state carrier comprises: starch, lactose, calcium hydrogen phosphate, microcrystalline Cellulose, sucrose and kaolin, and liquid carrier comprises: sterilized water, Polyethylene Glycol, nonionic surfactant and edible oil (such as Semen Maydis oil, Oleum Arachidis hypogaeae semen and Oleum sesami), as long as be fit to the characteristic of active component and required specific administration mode.Normally used adjuvant also can advantageously be included in pharmaceutical compositions, for example flavoring agent, pigment, antiseptic and antioxidant, such as vitamin E, vitamin C, 2,6 ditertiary butyl p cresol (BHT) and butylhydroxy anisole (BHA).
Usually, pharmaceutical composition of the present invention comprises following dosage form: liquid dosage form, injection type, powder type, eye dosage form, transfusion dosage form, tablet form, pill-type, ointment dosage form, plaster dosage form, aerosol, soft capsule, hard capsule, leaching agent, durative action preparation, membrane, sponge agent, microencapsulation, liposome, depot formulation and radiosiotope preparation etc.The tablet of oral administration, capsule, dispersible powder, granule or suspension (suspensoid) (containing according to appointment 0.05%-5% suspending agent (cosolvent)), syrup (containing according to appointment 10%-50% sugar) and elixir (containing the 20%-50% ethanol of having an appointment); the suppository of rectum or vagina administration perhaps carries out the parenteral administration with sterile injectable solution or suspensoid form (containing the 0.05%-5% cosolvent of having an appointment in the medium waiting to ooze).These pharmaceutical preparatioies can contain the about 0.01%-99.9wt% that mixes with carrier usually, 0.1%-99.5wt% preferably, more preferably the salvianolic acid B of 1%-99wt% (weight) and former times the dry goods medicine, by the gross weight of compositions.
On the other hand, the present invention also provides test kit, use the drug alone form salvianolic acid B and former times the dry goods medicine so that conjoint therapy is more convenient.In one embodiment, this test kit comprises independently pharmaceutical composition of two kinds of medicines, namely comprises first pharmaceutical composition and the second pharmaceutical composition that comprises former times dry goods medicine of salvianolic acid B.These pharmaceutical compositions can be concentrate or lyophilized products, its comprise appropriate amount salvianolic acid B or former times dry goods medicine and required adjuvant, be ready for use on and be injected into before use medium of patient, perhaps they can be to be ready for use on the liplid emulsions that is injected directly into the patient.
The salvianolic acid B that the present invention is used and former times the dry goods medicine, pharmaceutical composition of the present invention can be by oral, intravenous, intramuscular or subcutaneous route administration.The preferred oral administering mode.
But the salvianolic acid B that the present invention is used and former times dry goods medicine parenteral or intraperitoneal administration.Also can in glycerol, liquid, Polyethylene Glycol and the mixture in oil thereof, prepare dispersion liquid.Under conventional storage and service condition, contain antiseptic in these preparations to prevent growth of microorganism.
Using salvianolic acid B of the present invention and former times during the dry goods medicine, also can with means or other medicines (such as the toremifene) coupling of other prophylaxis of tumours.
The effective dose of used active component can change with the order of severity of the pattern of administration and disease to be treated.Yet, usually when medicine of the present invention every day with about 0.1-8000mg/kg the weight of animals (1-1000mg/kg body weight preferably, when dosage more preferably 1-500mg/kg body weight administration) gives, can obtain gratifying effect, preferably give with 1-4 time dosage every day, or with the slow release form administration.For most of large mammal, the accumulated dose of every day is about 1-16000mg or higher, preferably 10-8000mg.The dosage form that is applicable to take orally comprises the medicine of the about 0.1-8000mg that mixes with solid-state or liquid pharmaceutically acceptable carrier.This dosage of scalable is to provide optimal treatment to reply.For example, by an urgent demand for the treatment of situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.
From being easy to prepare the position with administration, preferred pharmaceutical composition is solid-state or fluid composition.
The present invention includes the method for the following cancer for the treatment of or tumor: epithelial cancer, mesenchymal tissue cancer, nervous tissue's cancer, lymph and hemopoietic tissue cancer; In the specific embodiment, tumor is selected from, nasopharyngeal carcinoma, esophageal carcinoma, hepatocarcinoma, cancer of pancreas, gastric cancer, colorectal cancer, renal carcinoma, pulmonary carcinoma, breast carcinoma, cervical cancer, ovarian cancer, carcinoma of prostate, glioma and leukemia.
Clinically, implement treatment of the present invention and use the present composition or medicine box will cause the reduction of the size of cancerous growths or quantity and/or related indication reduction when applicable ().On the pathology, implement the inventive method and use the present composition to produce the pathology correlation effect, for example, anticancer propagation, reduce cancer or tumor size, prevent from further shifting and suppressing Tumor angiogenesis.The method for the treatment of these diseases comprises that the present invention's combination with the treatment effective dose gives object.Can repeat the method when needing.Implement treatment of the present invention and use the present composition or medicine box has also reduced the toxic and side effects of medicine simultaneously, such as the causing danger of adverse cardiac events of former times dry goods medicine, therefore improved safety.
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 normal condition, or the condition of advising according to manufacturer.
1. the clone forms and breeding potential mensuration
Utilize the clone to form and the breeding potential experiment detects salvianolic acid B (Sal-B), celecoxib (Cel) Combined Treatment to the impact of Head and neck squamous cell carcinoma cell (HNSCC is from the former generation HNSCC culture of Johns Hopkins University's head and neck cancer research department) Growth of Cells.
The HNSCC cell is seeded in the BD Falcon 6 hole tissue culturing plates (PaloAlto, CA) with the density of 200 cells/well.With the cell absorption of spending the night.Culture uses Sal-B (1.25-250mM) or Cel (10-40mM) or Sal-B+Cel to process 24 hours.From each hole, remove culture medium, with cell PBS buffer rinsing.With fresh joining in the culture without the medicine culture medium, put back to incubator and hatch, form the clone in 10 days.Any clone who contains above 50 cells has represented a kind of great-hearted clonogenic cell.Being cloned in counting after the methylene blue dyeing in the hole.3 such experiments are carried out in per a kind of processing.
Count results statistical confirmation (data do not provide), Sal-B or Cel have inhibitory action and are dose-effect relationship cell clonal formation.The Sal-B minimum inhibitory concentration is lower than 1.25mM, when concentration is greater than or equal to 250mM, can thoroughly suppresses HNSCC cell formation clone when the processing time is 24hr.The Cel minimum inhibitory concentration is lower than 10mM, can thoroughly suppress the HNSCC cell when being 24hr and forms the clone when concentration is greater than or equal to the 40mM processing time.When 125mM Sal-B and 20mM Cel use in conjunction, effect when its effect that suppresses the cell proliferation rate is obviously used separately greater than the Sal-B of same concentrations or Cel, present synergistic effect, when 250mM Sal-B and 40mM Cel use in conjunction, the effect (seeing Fig. 1) when its effect that suppresses the cell proliferation rate is significantly used separately greater than the Sal-B of same concentrations or Cel.
2. flow cytometry detection Sal-B and/or Cel process the impact of the COX-2 expression that LPS is induced
The HNSCC cell is inoculated into the 25cm that contains 2% hyclone
2In the cell bottle, and be exposed among the Sal-B of 100ng/ml lipopolysaccharide (LPS, a kind of COX-2 stimulant) and various concentration or Cel or the Sal-B+Cel 6 hours, as shown in Figure 2.Then, cell is collected, and rinsing also is suspended among the ice-cold PBS.Cell is fixing in 80% methanol of pre-cooling, then, in the dark hatches 30min under the room temperature with the solution that contains COX-2-FITC (1: 200) (Cayman ChemicalCompany, Ann Arbor, MI).The COX-2 level is by FACStar flow cytometer (Becton Dickinson ﹠amp; Co., San Jose, CA) measure.Analyze 10,000 cells.
Experimental result shows, the effect that Sal-B or Cel can antagonism LPS stimulate COX-2 to express has inhibitory action and is dose-effect relationship (Fig. 2 A and Fig. 2 B) the expression of COX-2.When 100ug/ml Sal-B and 10mM Cel use in conjunction, the effect that it suppresses effect that COX-2 expresses when obviously using separately greater than the Sal-B of same concentrations or Cel presents synergistic effect (Fig. 2 C).
3. enzyme immunoassay detection Sal-B and/or Cel process the impact of PGE2 (PGE2) expression that LPS is induced
The HNSCC cell was processed 2,4,6,8 hours with the 100ng/ml LPS that is in the 2% hyclone culture medium respectively, or with the Sal-B of 100ng/ml LPS and variable concentrations (in μ M) or Cel or its Combined Treatment 6 hours.Collect culture medium, PGE2 concentration is measured (Correlate-EIATMPGE2 Enzymoimmune reagent kit) with the PGE2 Enzymoimmune reagent kit.
Shown in Fig. 3 A-C, the result shows, when not having other interference factors to exist, LPS stimulates can lure PGE2 concentration raise rapidly (such as Fig. 3 A) into.When the Sal-B of LPS and variable concentrations united use, along with increasing of Sal-B concentration, the concentration of PGE2 reduced rapidly, but this shows the effect (Fig. 3 B) of Sal-B antagonism LPS.When the Cel use in conjunction of the Sal-B of 100ug/ml and 10uM, it suppresses effect that PGE2 expresses significantly greater than the effect of using separately Sal-B, shows that the two has obvious synergism (Fig. 3 C).
Utilize in vivo test to detect Sal-B or Cel or the two Combined Treatment to the impact of HNSCC Growth of Cells
4 ages in week, female, nude mouse (Nu/Nu) obtain from Beijing dimension tonneau China company.They fed for 1 week with the given rodent diet method that contains 101mg/kg tocopherol and sufficient water before use.Mus is fed in the room of per 12 hours controlled conversion light and shades of temperature (1 ℃ in 23 soil).Mice is divided into 4 groups (5 every group): Sal-B processed group, Cel processed group, Combined Treatment group and untreated control group.Body weight and food/water absorbs to be measured 2 times in 1 week.JHU-013 cancerous cell (2 * 10
6Individual cell/100 μ l/ Mus) is subcutaneously injected into the lower back portion of mice with the syringe needle of 25 gauges, obtains Mus people heteroplastic graft tumor model.Behind injection JHU-013 cell, mice is accepted Sal-B (100mg/kg) or Cel (5mg/kg) or Sal-B+Cel (50mg/2.5mg/kg) by peritoneal injection respectively every day.Process and carry out every day, continue 21d.
Respectively at the 7th, the 14th major diameter and minor axis with the vernier caliper measurement tumor nodule, gross tumor volume V=1/2 major diameter * minor axis
2, employing is slaughtered laboratory animal and is peeled off inoculated tumour in the time of the 21st day at last, and the method for in-vitro measurements gross tumor volume obtains data.
As shown in Figure 4, Sal-B and Cel process the growth that all can suppress in-vivo tumour, and the effect of the two use in conjunction is obvious time-effect relationship greater than the effect of using separately Sal-B or Cel.
1.Western Blot method detection salvianolic acid B and/or celecoxib are processed the inhibitory action of the COX-2 expression that LPS is induced
BPH1 and BPH1C1 cell (from ATCC) are inoculated into the 25cm that contains 2% hyclone
2In the cell bottle, and be exposed among 100ng/ml lipopolysaccharide (LPS) and 125 μ M Sal-B or 20 μ M Cel or the Sal-B+Cel 6 hours.Then, cell is collected, and rinsing also is suspended among the ice-cold PBS.Cell is fixing in 80% methanol of pre-cooling, and then, the COX-2 level utilizes the COX-2 antibody of HRP labelling to carry out qualitative analysis by Western Blot.
Shown in Fig. 5 A and Fig. 5 B, experimental result shows, the effect that Sal-B or Cel energy antagonism LPS stimulate COX-2 to express, when 125 μ M Sal-B and 20 μ M Cel use in conjunction, the effect that it suppresses effect that BPH1 cell COX-2 expresses when obviously using separately greater than the Sal-B of same concentrations or Cel, present synergistic effect, the result of BPH1C1 similarly.
2. enzyme immunoassay detection Sal-B and/or Cel process the impact of the PGE2 expression that LPS is induced
BPH1 and BPH1C1 cell were processed 2,4,6,8 hours with the 100ng/ml LSP that is in the 2% hyclone culture medium respectively, or with processing 6 hours among 100ng/ml LPS and 125 μ M Sal-B or 20 μ M Cel or the Sal-B+Cel.Collect culture medium, PGE2 concentration is measured (Correlate-EIATM PGE2 Enzymoimmune reagent kit) with the PGE2 Enzymoimmune reagent kit.
Fig. 6 shows, when not having other interference factors to exist, LPS stimulates and can lure that PEG2 concentration raises rapidly into, and when LPS and Sal-B unite when using, the concentration of PGE2 is constant, when LPS and Cel unite when using, the concentration of PGE2 is constant, and when Sal-B and Cel use in conjunction, its effect that suppresses BPHl cell PEG2 expression is significantly greater than the effect of using separately Sal-B or Cel, show that the two has obvious synergism, the result of BPH1C1 is not obvious.
4 ages in week, female, nude mouse (Nu/Nu) obtain from Beijing dimension tonneau China company.They fed for 1 week with the given rodent diet method that contains 101mg/kg tocopherol and sufficient water before use.Mus is fed in the room of per 12 hours controlled conversion light and shades of temperature (23 ± 1 ℃).Mice is divided into 4 groups (5 every group): Sal-B processed group, Cel processed group, Combined Treatment group and untreated control group.Body weight and food/water absorbs to be measured 2 times in 1 week.BPH1 or BPH1C1 cell (2 * 10
6Individual cell/100 μ l/ Mus) is subcutaneously injected into the lower back portion of mice with the syringe needle of 25 gauges, obtains Mus people heteroplastic graft tumor model.Behind injection BPH1 or BPH1C1 cell, mice is accepted Sal-B (100mg/kg) or Cel (5mg/kg) or Sal-B+Cel (50mg/2.5mg/kg) by peritoneal injection respectively every day.Process and carry out every day, continue 7 days.Employing is slaughtered laboratory animal and is peeled off inoculated tumour in the time of the 7th day, and the method for in-vitro measurements gross tumor volume obtains data.
The result of Fig. 7 shows that Sal-B and Cel process the growth that all can suppress in-vivo tumour, and the effect of the two use in conjunction is obvious cooperative effect greater than the effect of using separately Sal-B or Gel.
Therefore, above-mentioned evidence salvianolic acid B and former times the dry goods medicine in vivo with external coordinate repression for tumor cell proliferation and growth.Disclosed the meaning of this drug regimen in the medicine for the treatment of cancer and tumor.
Claims (4)
1. a pharmaceutical composition is characterized in that, described pharmaceutical composition contains salvianolic acid B 1-300 μ M, celecoxib 1-300 μ M, and the concentration ratio of described salvianolic acid B and celecoxib is 1: 10-10: 1, and acceptable carrier on the materia medica.
2. pharmaceutical composition as claimed in claim 1 is characterized in that, described salvianolic acid B is the salt that forms with cation, described cation chosen from Fe ion, sodium ion, potassium ion, calcium ion and magnesium ion.
3. pharmaceutical composition as claimed in claim 1 is characterized in that, the concentration ratio of described salvianolic acid B and celecoxib is 1: 1.
4. pharmaceutical composition claimed in claim 1 is for the preparation of the purposes of antineoplastic agent, and described tumor is selected from Head and neck squamous cell carcinoma and carcinoma of prostate.
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| CN104666289A (en) * | 2014-09-11 | 2015-06-03 | 南京中医药大学 | Application of danshinolic acid A in preparation of anti-tumor cell epithelial-mesenchymal transition drug |
| CN105983097B (en) * | 2015-01-28 | 2021-06-08 | 华中科技大学同济医学院附属协和医院 | Anti-tumor preparation and preparation method thereof |
| CN105520927A (en) * | 2015-12-08 | 2016-04-27 | 上海交通大学医学院附属第九人民医院 | Purpose of Sal B nanometer preparation |
| CN112358492B (en) * | 2020-11-13 | 2022-04-15 | 兰州大学 | Carboborane celecoxib, preparation thereof and application thereof in boron neutron capture treatment medicine for head and neck cancer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408353A (en) * | 2002-09-13 | 2003-04-09 | 上海天甲生物医药有限公司 | Use of tanshin polyphenolic acid B in preparing tumor curing medicine |
| CN1872043A (en) * | 2005-06-03 | 2006-12-06 | 上海天甲生物医药有限公司 | Application of salvianolic acid B in preparing medication for preventing tumorigenesis |
| CN1872159A (en) * | 2005-06-03 | 2006-12-06 | 上海天甲生物医药有限公司 | Application of red sage root in preparing medication for preventing prostatic csarcinoma from occurring |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408353A (en) * | 2002-09-13 | 2003-04-09 | 上海天甲生物医药有限公司 | Use of tanshin polyphenolic acid B in preparing tumor curing medicine |
| CN1872043A (en) * | 2005-06-03 | 2006-12-06 | 上海天甲生物医药有限公司 | Application of salvianolic acid B in preparing medication for preventing tumorigenesis |
| CN1872159A (en) * | 2005-06-03 | 2006-12-06 | 上海天甲生物医药有限公司 | Application of red sage root in preparing medication for preventing prostatic csarcinoma from occurring |
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