CN102631341B - New application of ampelopsin sodium in treating bladder cancer - Google Patents
New application of ampelopsin sodium in treating bladder cancer Download PDFInfo
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- CN102631341B CN102631341B CN201110036702.1A CN201110036702A CN102631341B CN 102631341 B CN102631341 B CN 102631341B CN 201110036702 A CN201110036702 A CN 201110036702A CN 102631341 B CN102631341 B CN 102631341B
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a new application of ampelopsin sodium in treating bladder cancer. Particularly, the invention provides a pharmaceutical composition containing ampelopsin or its pharmaceutically acceptable salts. The medicine is administrated intravenously, can effectively treat cancers of the urinary system, especially bladder cancer.
Description
Technical field
The present invention relates to drug world, relate more specifically to the purposes of AMP-Na in treatment urinary system cancer, especially AMP-Na treats the novelty teabag of bladder cancer through modes such as intravenously administrables.
Background technology
The threat of malignant tumor to human health and life is very large, and it and cardiovascular disease have become two large difficulties medically.Bladder cancer is the modal malignant tumor of urinary system, and sickness rate occupies the first place of urinary system malignant tumor, and at present, bladder cancer is the ninth-largest cancer in the world.Divide by histological type, bladder cancer can be divided into epithelial tissue and non-epithelial tissue tumor, and epithelial tissue tumor accounts for 95%, and wherein 90% is transitional epithelium tumor (transientcell carcer, TCC).Abroad, the sickness rate of bladder cancer is only second to carcinoma of prostate in male urogenital organs tumor, and occupy the 2nd, then account for first place at home, sickness rate has growth trend in recent years.Male's sickness rate of bladder cancer is about the 3-4 of women doubly, the age with 50-70 year be many.In urologic neoplasms, tumor of bladder is compared with other tumor, and relapse rate is high, and after recurrence, then aggressivity is stronger.
For the treatment of bladder cancer, the past often adopts the methods such as operation, chemotherapy by the malignant tumor tissue formed excision or kills, but has the patient of 50%-70% can recur within a certain period of time, has a strong impact on the prognosis of patient.At present, bladder cancer is adopted clinically to the method for Comprehensive Treatment, comprise surgical operation, radiotherapy, chemotherapy, immunization therapy etc.Chemoprophylaxis in recent years more and more attracts widespread attention, and the chemoprophylaxis of cancer refers to the process utilizing chemicals intervention or block canceration, Tumor Differentiation is reversed, thus reaches the object of prophylaxis of tumours.
Superficial bladder cancer chemotherapeutic medicine when intravenous administration systemic chemotherapy is difficult to arrive tumor tissues, and weak curative effect, side effect is large.To the Drug therapy of superficial bladder cancer and prevention of recurrence mainly with per urethra intubate irrigation bladder treatments such as chemotherapeutic thio-tepa, amycin, cisplatin, ametycin, camptothecines, achieve good curative effect, but this scheme will per urethra intubate continually, during treatment, patient is very painful, the compliance of patient to treatment is low, the Proportion of patients that can accept full course for the treatment of is not high, and, these chemotherapeutic per urethra intravesical against the toxic action that also can produce whole body during property perfusion therapy, as bone marrow depression, cardiac toxicity and nephrotoxicity etc.
In order to improve the effectiveness of chemotherapeutic drug therapy bladder cancer, improve the compliance of patient for treatment, desirable chemotherapeutics reply transitional cell bladder carcinoma cell line lethal effect is strong, and after intravenously administrable, in intravesical urine and bladder body, reach active drug concentration rapidly and the long period can be maintained, but there is no the chemotherapeutics that can reach ideal effect like this after intravenously administrable at present.
Dihydromyricetin (3,5,7,3,4 ', 5 '-hexahydroxy 2,3 flavanonols, ampelopsin, AMP, dihydromyricetin) has another name called ampelopsin, belongs to flavone compound.AMP is present in vitaceae in a large number, and especially in ampelopsis, content is up to 27% ~ 28%.The molecular formula of AMP dihydrate is C
15h
12o
82H
2o, structural formula is as follows:
AMP sterling is white to pale yellow powder, and less stable, easily oxidation reaction occurs, and is insoluble to chloroform, ether, is soluble in ethanol, methanol, dichloromethane and dimethyl sulfoxide (DMSO).AMP is slightly soluble in water, and dissolubility is in aqueous 0.2mg/ml (25 DEG C), due to its water solublity low (0.2mg/ml), is difficult to patent medicine.
AMP-Na (ampelopsin-sodium, AMP-Na) be that the present inventor is in order to improve dissolubility and the stability of ampelopsin, become the pharmaceutical dosage form being more suitable for Clinical practice, and the highly-water-soluble sodium salt (Chinese Patent Application No.: 200610025335.4) of preparation.A kind of preferred sodium salt is AMP tetrasodium salt.This tetrasodium salt often exists with pentahydrate form, and molecular formula is C
15h
8o
8na
45H
2o, relative molecular mass 498.03.The structural formula of the pentahydrate of AMP-Na (tetrasodium salt) is as follows:
AMP-Na lyophilized formulations is brownish red, and stability is better, and be insoluble to chloroform, ether, ethanol, methanol, dichloromethane and dimethyl sulfoxide (DMSO), soluble in water, saturation solubility is in aqueous 50mg/ml (25 DEG C).
Find when studying the antitumor action of AMP-Na: although 1. AMP-Na has certain antitumor action to tumor cells such as people's hepatocarcinoma, people's pulmonary carcinoma in vitro, but when being used alone AMP-Na in vivo, effect is very poor, such as, in body alone AMP-Na to human lung adenocarcinoma SPC-A-1 and the effect of GLC-82 cell Xenografts in nude mice growth inhibited very weak.But AMP-Na vivo medicine-feeding is to agnogenio almost without obvious inhibition of in-vivo tumour.2. in AMP-Na body with some anticarcinogen (as carboplatin) coupling time, obviously can strengthen the tumor-inhibiting action of some anticarcinogen (as carboplatin).Because the alone in vivo cancer resistant effect of AMP-Na is very poor, therefore think at present, for the treatment of general in-vivo tumour, AMP-Na is not suitable as real tumor inhibitor, is more not suitable for being used alone.
In sum, at present for the urologic neoplasms such as bladder cancer, still lack gratifying medicine and means.Therefore, this area is in the urgent need to developing the novel drugs effectively suppressing and treat urologic neoplasms.
Summary of the invention
The object of the invention just provides a kind of novel drugs that can effectively suppress and treat urologic neoplasms.
In a first aspect of the present invention, provide the purposes of a kind of ampelopsin or its pharmaceutically acceptable salt, it is used to the medicine preparing treatment urologic neoplasms.
In another preference, described urologic neoplasms comprises: bladder cancer, carcinoma of prostate and renal carcinoma.
In another preference, the dosage form of described medicine is injection.
In another preference, described medicine contains concentration >=2mg/ml, preferably concentration >=5mg/ml, more preferably the ampelopsin sodium salt of concentration >=10mg/ml or its hydrate.
In another preference, described medicine contains AMP-Na or its hydrate.
In another preference, described medicine is applied to mammalian object in the following manner: intravenous injection and/or intraperitoneal injection.
In another preference, described medicine contains cosolvent.
In another preference, described cosolvent is selected from lower group: arginine, lysine or its combination.
In a second aspect of the present invention, provide a kind of pharmaceutical composition being used for the treatment of urologic neoplasms, it is characterized in that, it contains ampelopsin or its pharmaceutically acceptable salt of pharmaceutically acceptable carrier and safe and effective amount.
In another preference, the dosage form of described pharmaceutical composition is injection (injection).
In a third aspect of the present invention, provide a kind of method for the treatment of urologic neoplasms, comprise step: ampelopsin or its pharmaceutically acceptable salt of using safe and effective amount to the object of needs treatment.
In another preference, described using comprises: intravenous injection and/or intraperitoneal injection.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and in below (eg embodiment) specifically described each technical characteristic can combine mutually, thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows AMP-Na (6.7 ~ 7533 μ g/ml) standard curve in NS (normal saline, normal saline).
Fig. 2 shows AMP-Na (6.7 ~ 7533 μ g/ml) standard curve in mouse retention.
Fig. 3 show AMP-Na different action time to the inhibitory action of EJ cell proliferation (24,48, point four administrations in 72h and 48h, n=3-4) note: compare with negative group, * * P < 0.01.
Fig. 4 show AMP-Na suppress the IC50 value of EJ cell proliferation different action time (24,48, divide four administrations in 72h and 48h,
n=3-4) note: a: compare with 24h, P < 0.01; B: compare with 48h, P < 0.01; C: compare with 72h, P < 0.01; D: compare with point four administrations in 48h, P < 0.01.
Fig. 5 show AMP-Na different action time to the inhibitory action of 5637 cell proliferation (24,48, point four administrations in 72h and 48h,
n=3-4).Note: compare with negative group, * * P < 0.01
Fig. 6 show AMP-Na suppress the IC50 value of 5637 cell proliferation different action times (24,48, divide four administrations in 72h and 48h,
n=3-4).Note: a: compared with 24h, P < 0.01; B: compared with 48h, P < 0.01; C: compared with in 48h point of four administration, P < 0.01; D: compared with 24h, P < 0.05
Fig. 7 show AMP-Na different action time to the inhibitory action of BIU-87 cell proliferation (24,48h and 72h,
n=3) note: compare with negative group, * * P < 0.01.
Fig. 8 show AMP-Na suppress BIU-87 cell proliferation different action time IC50 value (24,48,72h,
n=3-4) note: 24h administration group has significant difference compared with 72h administration group, (aP < 0.05)
Fig. 9 shows the impact of AMP-Na intravenous administration on S180 sarcoma kunming mice intravesical transplanted tumor tumor weight.
Detailed description of the invention
The present inventor is through extensive and deep research, be surprised to find that, AMP-Na is very high at the Organ and tissue drug concentration of the urinary systems such as mammiferous prostate, bladder after intravenously administrable, urine drug concentration is high simultaneously, the persistent period is long, and mainly exists with activated proto-drug.Therefore, though ampelopsin or its pharmaceutically acceptable salt are not suitable for treating other tumors mammiferous, the tumor for the treatment of urinary system is applicable to very much.Complete the present invention on this basis.
Particularly, the present inventor studies discovery, and AMP-Na vivo medicine-feeding is almost relevant for characteristic with the medicine of AMP-Na without the reason of obvious inhibition to in-vivo tumour (except urologic neoplasms), blood plasma t after AMP-Na mice i.v.
1/2all very short, be on average only 11.48 ± 3.30min.Because its half-life is too short, the time of contact of tumor cell and medicine is too short, and therefore can not play lasting antitumor action very well to subcutaneous transplantation tumor, thus it is also very poor to the curative effect at other positions of health.
In contrast, AMP-Na is very high at the Organ and tissue drug concentration of the urinary systems such as mammiferous prostate, bladder after intravenously administrable, and the high concentration state of AMP-Na in urine (200 μ g/ml) can tie up continuous 2 hours.Antitumor activity in vitro proves, AMP-Na suppresses the valid density of proliferation of human bladder cancer cells to be about 40 μ g/ml, and therefore this prompting AMP-Na has good therapeutical effect in the characteristics of pharmacokinetics of urine middle and high concentration, long-time excretion to bladder cancer.Animals iv administration experiment further demonstrates the therapeutical effect of AMP-Na to urologic neoplasms.
As used herein, term " the compounds of this invention ", " formula I ", " ampelopsin " or " AMP " are used interchangeably, and all refer to compound or its pharmaceutically acceptable salt with structural formula I.
The compounds of this invention also comprises by pharmaceutically or the salt form of AMP that derives of the acceptable alkali of physiology and hydrate thereof.
These salt include, but is not limited to the salt formed with alkali metal or alkaline-earth metal (as sodium, potassium, calcium or magnesium), and especially the alkali metal salt that significantly improves of water solublity is as sodium salt, potassium salt.Particularly preferably be tetrasodium salt and the pentahydrate thereof of AMP.
Term " AMP-Na " refers to the various sodium salts of ampelopsin, especially the ampelopsin sodium salt of saturation solubility >=2mg/ml in water, preferably >=5mg/ml, more preferably >=10mg/ml.
Preferred the compounds of this invention is a compound for the tetrasodium salt form of AMP, and this compound is called AMP-Na.This compound can synthesize according to a conventional method or buy acquisition.
The compounds of this invention may be used for various urologic neoplasms and treats and/or prevents.Representational example comprises (but being not limited to): bladder cancer, carcinoma of prostate and renal carcinoma, be particularly useful for bladder cancer.
The present invention also comprises the method for pharmaceutical composition and treatment urologic neoplasms, and it comprises to the formula I of administration medicine effective quantity.
When the compounds of this invention is used for such use, can with one or more pharmaceutically acceptable carrier or mixed with excipients, as solvent, diluent etc., and sterile injectable solution or form of suspension (containing 0.05-5% suspending agent of having an appointment in isotonic medium) parenteral routes can be carried out.Such as, these pharmaceutical preparatioies containing the about 0.01-99% mixed with carrier, can more preferably be about the active component of 0.1%-90% (weight).
The effective dose of active component used can change with the order of severity of the pattern of compound used, administration and disease to be treated.But, usually when compound of the present invention gives with the dosage of about 0.5-1000mg/kg the weight of animals every day, gratifying effect can be obtained, preferably give with the dosage of 1-4 time every day.For most of large mammal, the accumulated dose of every day is about 1-100mg/kg, is preferably about 2-100mg/kg.This dosage of scalable is replied to provide optimal treatment.Such as, by an urgent demand for the treatment of situation, dosage can be increased pari passu or reduces every day.
The compounds of this invention or pharmaceutical composition are preferably by administrations such as intravenouss.Liquid carrier comprises: sterilized water, Polyethylene Glycol, propylene glycol, ethanol, dimethyl sulfoxide, nonionic surfactant etc., as long as be applicable to the characteristic of active component and required specific administration mode.In pharmaceutical compositions, normally used adjuvant also can advantageously be included, and such as antiseptic and antioxidant are as vitamin E, vitamin C, citric acid, BHT and BHA.
The medicament forms being adapted to inject comprises: aseptic aqueous solution or dispersion liquid and aseptic powder (for extemporaneous preparation of sterile injection solution or dispersion liquid).In all situations, these forms must be aseptic and must be that fluid is to be easy to syringe displacement fluids.Must be stable under conditions of manufacture and storage, and must can prevent the pollution effect of microorganism (as antibacterial and fungus).Carrier can be solvent or disperse medium, wherein containing, for example water, alcohol (as glycerol, propylene glycol and liquid polyethylene glycol), their suitable mixture and vegetable oil.
In addition, the compounds of this invention also can with the medicine of other treatment urologic neoplasms (as cisplatin, carboplatin, amycin, ametycin etc.) coupling.
In examples more of the present invention, the transitional cell bladder carcinoma cell line EJ cell of the present inventor's In vitro culture, 5637 cells, investigate the external lethal effect to transitional cell bladder carcinoma cell line of AMP-Na, when found that AMP-Na is alone, all have very strong lethal effect to human bladder cancer cell line EJ, 5637 cells.
Due to AMP-Na blood plasma t
1/2too short, in subcutaneous tissue, the holdup time is short, the curative effect investigating medicine can not be gone with traditional bladder cancer subcutaneous transplantation tumor model, and the bladder carcinoma in situ cell of direct inoculation in mouse bladder will touch AMP-Na for a long time, more likely observe the Anticancer effect in vivo curative effect of AMP-Na.For this reason, the present inventor is through intraperitoneal injection (this approach is the conventional approach substituting intravenous administration) AMP-Na, find that it alonely all has extremely strong inhibitory action to transplanted tumor in S180 sarcoma mouse bladder and the growth of human bladder cancer nude mice bladder orthotopic transplantation tumor, prompting AMP-Na has good therapeutic effect to bladder cancer through intravenously administrable is alone.
The present inventor is through the alone experiment of intravenous injection, and demonstrating bladder cancer further has good therapeutic effect.
Rat and beasle dog long term toxicity test show, after the quiet note of AMP-Na, toxicity is very low, damage kidney without classic chemotherapy medicine, suppress the toxic action of bone marrow hematogenesis; Observe the mucous membrane of urinary bladder of this medicine to rat and dog simultaneously and do not have damaging action, this illustrates that it has higher selectivity to normal bladder tissue.
As can be seen from these result of the tests, AMP-Na is a relative bladder " targeted drug ", without intraurethral cannula intravesical retroperfusion through venoclysis, the therapeutic effect that can reach irrigation bladder.AMP-Na, to the normal bladder mucosa repetitively administered nonirritant of animal, can avoid as other chemotherapeutics when treating bladder cancer, the mucous membrane of urinary bladder irritation caused by per urethra intubate intravesical retroperfusion; In addition due to AMP-Na intravenous administration, the misery that frequent per urethra intubate intravesical retroperfusion brings to patient can be avoided, improve the compliance of patient for treatment, thus improve the therapeutic effect to bladder cancer.Visible, the clinical treatment for bladder cancer is provided new method through intravenously administrable and/or irrigation bladder by AMP-Na.The inventive method is suitable for the treatment shallow tumor of bladder table and postoperative prevention bladder tumor recurrence; Be applicable to the treatment of patient with invasive bladder tumor and postoperative prevention of recurrence; And and radiotherapy combined, for treatment and the postoperative prevention of recurrence of patient with invasive bladder tumor.
To sum up, the present invention has following major advantages:
A () provides a kind of effective ways for the treatment of urologic neoplasms especially bladder cancer.
B after the quiet note of () AMP-Na, toxicity is very low, damage kidney without classic chemotherapy medicine, suppress the toxic action of bone marrow hematogenesis.
C () AMP-Na has higher selectivity to normal bladder tissue, be a kind of " targeted drug ".
D (), by intravenously administrable, can improve the compliance of patient for treatment, thus improve the therapeutic effect to bladder cancer.
E (), by mode administrations such as intravenously administrables, not only curative effect improves, and does not substantially occur general toxic reaction, also to mucous membrane of urinary bladder nonirritant, overcomes the untoward reaction of classic chemotherapy medicine.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise percentage ratio and number be weight percentage and parts by weight.
Experiment material
1. medicine and reagent
AMP-Na (AMP-Na) 500mg/ props up and props up with 1000mg/, freeze-dried formulation, lot number: 051115 (500mg/ props up) and 061218 (1000mg/ props up), PBS (pH 6.0 and 5.0), by Guangdong, Tai He Pharmaceutical Technology Co., Ltd provides.
Ampelopsin (AMP) 0.5g/ props up lot number: 040513, AMP reference substance 100mg/ props up lot number: 040524 purity >=98%, and by Guangdong, Tai He Pharmaceutical Technology Co., Ltd provides.
Pentobarbital sodium (Chinese Medicine Solution on Chemical Reagents in Shanghai company, lot number: F20020915).
Heparin sodium injection (Jiangsu Wanbang Biological Pharmaceutical Co., Ltd., the accurate word H32020612 of traditional Chinese medicines, lot number: 0607114).
2. instrument
Shimadzu Corporation high performance liquid chromatograph LC-10AT, SPD-M10AvP diode array detector, SIL-HT automatic sampler, the online degasser of DGU-HA, LC-10ATvP infusion pump, CTO-10AvP column oven, Solution chromatographic work station.XW-80A turbine mixer, kylin medical apparatus factory of Jiangsu Haimen City product.TGL-16 type refrigerated centrifuge, Anting Scientific Instrument Factory, Shanghai's product.Animals iv constant-rate administration system, An Lai software Science and Technology Ltd. of Ningbo City product.Stepless speed regulation electric blender, Jiangyin, Jiangsu scientific research apparatus factory.
Chromatographic column: Shimadzu VP-ODS C
18post (150 × 4.6mm, 5 μm), mobile phase: acetonitrile: 2% acetic acid (V/V, 1: 9), flow velocity: 1.0ml/min, determined wavelength: 290nm, column temperature: 25 DEG C of room temperature: 20-23 DEG C.Guard column: Scienhome Scientific, inc product.
3. animal
Mice, Kunming kind, cleaning grade, body weight 20 ~ 29g, is provided by Lanzhou Institute of Biological Products, the quality certification number: the dynamic word 14-001 of doctor.
Embodiment 1
The distribution of AMP-Na in Mice Body and through homaluria feature
The present embodiment drug level of high effective liquid chromatography for measuring AMP-Na after mice i.v. in serum, urine and tissue, thus research AMP-Na (AMP-Na) is in mouse tissue distribution and the excretion feature in mouse retention.
1. the making of standard curve
First standard substance (AMP or AMP-Na) are dissolved in PBS (pH6.0), the standard solution of obtained concentration known, then mixes with serum, urine, organ-tissue (as liver homogenate), with biased sample production standard curve.
For the standard curve of AMP-Na in mouse retention, weigh AMP-Na preparation 0.0231g (containing AMP-Na 7.2mg), with PBS (pH6.0) 0.125ml dissolved substance, make drug level be 57.6mg/ml, getting 30 μ l medicines joins in 200 μ l NS (normal saline) and mice blank diaper, drug level is made to be 7533 μ g/ml, with PBS, doubly or three times are diluted successively to the medicinal liquid of 57.6mg/ml again, getting 10 μ l medicines joins in 200 μ l NS and mice blank diaper, drug level is made to be followed successively by 2880, 1440, 720, 360, 180, 60, 20, 6.7 μ g/ml, vortex mixes, add 600 μ l precipitant acetonitriles: 8% acetic acid (V/V, 8: 2), vortex 3min, 16000rpm/10min, 20 ~ 25 DEG C of room temperatures are centrifugal, supernatant 0.45 μm of frit, continuous sample introduction 2 times.The meansigma methods of the peak area recorded with 2 times is vertical coordinate, and drug level is that abscissa sets up the standard curve of AMP-Na in NS and mouse retention.
2. the distribution test after AMP-Na i.v. in Mice Body
Mice 170, before experiment, water is can't help in 12h fasting, i.v.AMP-Na 400mg/kg in mouse tail vein, respectively at after i.v. 5,15 and 30min orbital vein blood sampling, 4 DEG C solidify 1.5h, core, liver, spleen, lung, kidney, brain, muscle, thymus, pancreas, fat, testis, uterus, ovary, prostate, bladder (non-flushing wash or rinse with water), Stomach duodenum, jejunum, ileum and colon, prepare tissue homogenate.Wherein uterus, ovary, prostate and bladder are little because of tissue weight, merge as a sample, repeat preparation 4 ~ 5 parts and measure with multiple sample.Sample thief doubly adds NS by 1: 3 (W/V, g/ml) and prepares homogenate, gets serum and homogenate 200 μ l, add 600 μ l precipitant immediately, vortex 3min, 16000rpm/10min, 20 ~ 25 DEG C centrifugal, get supernatant frozen, before detecting, room temperature is dissolved, vortex 1min, 16000rpm/5min, 20 ~ 25 DEG C centrifugal, 0.45 μm of frit, measures the AMP-Na concentration in tissue and serum by HPLC method.
Result:
AMP-Na to after mice i.v.400mg/kg during 5min nephridial tissue drug concentration the highest, liver takes second place, and all the other internal organs all have extensive distribution, have in brain trace distribute (lower than lowest detectable limit 2.5 μ g/g).After AMP-Na i.v., during 15min, nephridial tissue drug level is still the highest, and jejunum takes second place.After AMP-Na i.v., during 30min, jejunum drug concentration is the highest, bladder takes second place, the blood drug level content that can detect in brain is 15.2 μ g/g, shows that AMP-Na dosage increases rear post-drug period and can pass through blood brain barrier, but ratio extremely low (for 10.7% of blood concentration).All metabolite is found that there is in kidney, liver, lung, bladder, prostate, uterus, muscle, Stomach duodenum, jejunum, ileum and colon, but be all significantly less than original shape medicine peak area (during 30min, liver and kidney are 22%, the equal < 15% of each time point of remaining tissue).After quiet note 5,15,30min tri-time points, the drug level in bladder body, all higher than 200 μ g/ml, is observed in the test of pharmacodynamics cell in vitro poison, and this concentration can the growth of inhibition tumor cell completely.
Three time period each organ distribution data, in table 1, table 2 and table 3, respectively organize the ratio of drug concentration and blood drug level in table 4 after mice i.v..
The concentration of Organs of Mice Chinese medicine during table 1AMP-Na 400mg/kg i.v. administration 5min
(drug level/μ g/g organizes)
*: concentration unit μ g/ml
The concentration of Organs of Mice Chinese medicine during table 2AMP-Na 400mg/kg i.v. administration 15min
(drug level/μ g/g)
*: concentration unit μ g/ml
The concentration of Organs of Mice Chinese medicine during table 3AMP-Na 400mg/kg i.v. administration 30min
(drug level/μ g/g)
*: concentration unit μ g/ml
The ratio (%) of drug concentration and blood drug level is respectively organized after table 4AMP-Na 400mg/kg i.v.
3. mice 24h homaluria test:
Mice, ♀ ♂ half and half, collects urine with metabolic cage by totally 30.6 mices in each metabolic cage, share 5 metabolic cage parallel laboratory tests.I.v.AMP-Na 200mg/kg after mouse stomach 1ml NS, respectively at after i.v. 0.17,0.33,1,2,4,6,8,12,18 and 24h collect urine, with the urine of cage 6 mices as 1 sample, during collection, in time urine is put 4 DEG C of cold preservations, record volume of urine.Get 200 μ l urines, add 600 μ l precipitant immediately, vortex 3min, 16000rpm/10min, 20 ~ 25 DEG C centrifugal, gets supernatant frozen.Before detecting, room temperature is dissolved, vortex 1min, 16000rpm/5min, and 20 ~ 25 DEG C centrifugal, 0.45 μm of frit, and HPLC method measures drug level, calculates the cumulative amount of thanking arranged by medicine percentage composition by urine.
As a result, AMP-Na 200mg/kg is to after mice i.v., and the accumulative excretion of 24h urine is 19.97% of dosage, and excretion (18.25%) in 1h after concentrating on i.v., urinate drug concentration during 2h still at 200 more than μ g/ml.Any metabolite is found no in urine.In urine, original shape drug level delta data is in table 5.
Table 5AMP-Na 200mg/kg is to original shape drug level change in urine in 24h after mice i.v.
4. mice 12 ~ 156h homaluria test:
Mice, ♀ ♂ half and half, totally 30,6 mices in each metabolic cage, totally 5 metabolic cages, i.v.AMP-Na 200mg/kg, respectively at after i.v. 12,18,24,30,36,42,48,54,60,66,72,78,84,90,96,102,108,114,120,126,132,138,144,150,156h collects urine, with first time same treatment sample.
As a result, original shape medicine output data in the mouse retention of each time point, in table 6.
Original shape medicine output in table 6 urine
5. conclusion
AMP-Na i.v. enters and distributes more after in Mice Body in liver, kidney, lung, pancreas, bladder, prostate and small intestine, points out these organ-tissues may be the target organ of drug action or toxic action.Drug level in bladder and prostata tissue is far above valid density, but in rat with dog long term toxication, all do not observe mucous membrane of urinary bladder have any pathological change relevant to medicine, AMP-Na is selective to bladder body in prompting, and its treatment bladder cancer and carcinoma of prostate etc. have very important clinical meaning and application prospect.
Drug level after the drug level that bladder body is non-flushing when washing rinses apparently higher than bladder body water, illustrates that bladder body mucosa has stronger absorbability to the medicine in urine.
AMP-Na accounts for 20% of dosage to the accumulative output in urine of its original shape after mice i.v., and concentrate the interior excretion (18.25%) of 1h upon administration, drug concentration is urinated still at 200 more than μ g/ml during 2h, far above valid density, it terminates in about 24h excretion from urine, finds no any metabolite in urine.Concentration in urine is high, and prompting medicine can be used for treating urinary system cancer.
Embodiment 2
The external inhibitory action to human bladder cancer cell's propagation of AMP-Na
1. cell strain and medicine
Human bladder infiltrating carcinoma cell strain EJ, human bladder transitional epithelium JEG-3 5637, human bladder shallow table transitional carcinoma cells strain BIU-87, purchased from Chinese Academy of Sciences's Shanghai cell biological institute cell bank.Cell line all derives from people's primary bladder cancer.
AMP-Na lyophilized preparation: 180mg/ props up, lot number: 081031, purity 27.8%; The special diluent of AMP-Na lyophilized preparation (0.1mol/L, pH=6.8 phosphate buffer): 5ml/ props up, lot number: 081031, and Tai He Pharmaceutical Technology Co., Ltd provides by Guangdong.
Cisplatin (DDP): 10mg/ props up, lot number: 8030031 DB, Jiangsu Qilu Pharmaceutical Co., Ltd. produces.
2. experimental technique
MTT colorimetric method for determining AMP-Na to human bladder cancer EJ, 5637, the inhibitory action of BIU-87 cell proliferation.Negative control group (cell suspension+special diluent), blank group (RPMI RPMI-1640+special diluent), AMP-Na group (cell suspension+AMP-Na), AMP-Na color comparator group (RPMI RPMI-1640+AMP-Na) and positive controls (cell suspension+chemotherapeutics) are established in experiment, if positive drug has color, then establish positive drug color comparator group (RPMI RPMI-1640+chemotherapeutics).Each concentration establishes hole, multiple hole 3, and the average of every 3 hole OD values is as once testing.Exploitative experiment at least carries out 1 time, and effect confirms that experiment at least repeats more than 3 times.
Specific experiment step is as follows:
(1) take the logarithm trophophase EJ, 5637, BIU-87 cell, with 0.25% trypsinization collecting cell, with RPMI RPMI-1640 adjustment cell concentration to 1 × 10
5(BIU-87 cell concentration is 5 × 10 to individual/ml
4individual/ml), be inoculated in 96 well culture plates, every hole 90 μ l, cultivate 24h in immigration incubator and make cell attachment.
(2) to adherent EJ, 5637, BIU-87 cell, add relative medicine once with 10 μ l/ holes, each concentration establishes 3 multiple holes, cultivates 24,48 and 72h respectively.EJ, 5637 cell strains do point four dosings experiment in 48h in addition, namely every 12h dosing once, 2.5 μ l/ time, dosing four times altogether, dosing total amount is still 10 μ l, owing to measuring through prerun, during 1h no longer medicine detected after adding AMP-Na in cell culture fluid, medicine concentration in cell culture fluid can not be accumulated or accumulate, therefore during AMP-Na many dosings, the final concentration of the concentration of medicine in cell culture plate well after each dosing represents.
(3) before experiment stops, the every hole of 4h adds MTT (5mg/ml) 10 μ l, 37 DEG C, continue to hatch 4h, stop cultivating, every hole adds 10%SDS 100 μ l, and 37 DEG C are spent the night, morning next day takes out 96 well culture plates from incubator, concussion 10min, makes crystal fully dissolve, and measures light absorption value in wavelength 570nm place.Suppression ratio (IR) is calculated according to following formula:
Note: negative OD average=negative control group OD average-blank group OD average; By reagent OD average=respectively by reagent group OD average-medicine color comparator group OD average.
3. cell growth inhibition assay result
(A) AMP-Na inhibitory action that human bladder cancer cell line EJ is bred
I inhibitory action that () AMP-Na effect 24h breeds human bladder cancer cell line EJ
After AMP-Na handler effect of EJ cell line 24h, to the propagation of EJ cell, there is obvious inhibitory action, in the scope that concentration is 25 ~ 100 μ g/ml, in obvious concentration dependent.The suppression ratio (IR) of AMP-Na 25,33,43.5,57.4,75.8 and 100 μ g/ml to human bladder cancer cell line EJ is respectively-4.30% ± 3.14%, 7.52% ± 0.71%, 22.14% ± 2.86%, 47.80% ± 1.16%, 65.39% ± 1.16% and 77.42% ± 2.22%, compared with negative group, the suppression ratio of Amp-Na 43.5 ~ 100 μ g/ml group to EJ cell has significant difference (P < 0.01), IC
50value is 64.18 ± 1.27 μ g/ml.
After DDP handler effect of EJ cell line 24h, to the propagation of EJ cell, there is obvious inhibitory action, in obvious concentration dependent in the scope that concentration is 0.25 ~ 8 μ g/ml, DDP 0.25, 0.5, 1, 2, 4 and 8 IRs of μ g/ml to human bladder cancer cell line EJ are respectively 5.75% ± 0.91%, 9.87% ± 1.05%, 30.70% ± 1.34%, 38.49% ± 0.61%, 48.17% ± 3.53% and 65.21% ± 1.80%, compared with negative group, the suppression ratio of DDP 1 ~ 8 μ g/ml group to EJ cell has significant difference (P < 0.01), IC
50be 3.71 ± 0.12 μ g/ml.
(ii) AMP-Na effect 48h inhibitory action that human bladder cancer cell line EJ is bred
After AMP-Na handler effect of EJ cell line 48h, to the propagation of EJ cell, there is obvious inhibitory action, in the scope that concentration is 25 ~ 57.4 μ g/ml, in obvious concentration dependent.Under these three concentration of 57.4,75.8,100 μ g/ml, suppression ratio is very close, does not have obvious concentration dependent.AMP-Na25,33,43.5,57.4,75.8 and 100 IRs of μ g/ml to human bladder cancer cell line EJ are respectively 5.24% ± 1.65%, 12.93% ± 3.04%, 41.13% ± 1.27%, 80.49% ± 4.91%, 84.01% ± 1.66% and 86.29% ± 0.49%
Compared with negative group, the suppression ratio of AMP-Na 43.5 ~ 100 μ g/ml group to EJ cell has significant difference (P < 0.01), IC
50value is 50.20 ± 1.96 μ g/ml.
After DDP handler effect of EJ cell line 48h, to the propagation of EJ cell, there is obvious inhibitory action, in obvious concentration dependent in the scope that concentration is 0.25 ~ 8 μ g/ml, DDP 0.25, 0.5, 1, 2, 4 and 8 IRs of μ g/ml to human bladder cancer cell line EJ are respectively 7.59% ± 3.79%, 32.41% ± 3.83%, 43.53% ± 3.37%, 55.93% ± 3.22%, 65.24% ± 2.32% and 79.69% ± 1.57%, compared with negative group, the suppression ratio of DDP 0.5 ~ 8 μ g/ml group to EJ cell has significant difference (P < 0.01), IC
50be 1.75 ± 0.24 μ g/ml.
(iii) AMP-Na effect 72h inhibitory action that human bladder cancer cell line EJ is bred
After AMP-Na handler effect of EJ cell line 72h, to the propagation of EJ cell, there is obvious inhibitory action, in obvious concentration dependent in the scope that concentration is 25 ~ 57.4 μ g/ml.Under these three concentration of 57.4,75.8,100 μ g/ml, suppression ratio is tending towards close, does not have obvious concentration dependent.The IR of AMP-Na 25,33,43.5,57.4,75.8 and 100 μ g/ml effect 72h to human bladder cancer cell line EJ is respectively 14.58% ± 0.65%, 24.08% ± 1.63%, 53.20% ± 4.88%, 88.29% ± 4.43%, 91.53% ± 1.93% and 94.20% ± 1.46%, compared with negative group, the suppression ratio of AMP-Na 33 ~ 100 μ g/ml group to EJ cell has significant difference (P < 0.01), IC
50value is 40.71 ± 0.40 μ g/ml.
After DDP handler effect of EJ cell line 72h, to the propagation of EJ cell, there is obvious inhibitory action, in obvious concentration dependent in the scope that concentration is 0.25 ~ 8 μ g/ml, DDP 0.25, 0.5, 1, 2, 4 and 8 IRs of μ g/ml to human bladder cancer cell line EJ are respectively 10.93% ± 2.39%, 39.05% ± 0.58%, 51.50% ± 2.93%, 64.53% ± 2.65%, 76.03% ± 2.73% and 76.95% ± 4.37%, compared with negative group, the suppression ratio of DDP 0.5 ~ 8 μ g/ml group to EJ cell has significant difference (P < 0.01), IC
50be 1.30 ± 0.08 μ g/ml.
(iv) inhibitory action that in AMP-Na 48h, point four administrations are bred human bladder cancer cell line EJ
AMP-Na handler effect of EJ cell line, point four administrations in 48h, namely every 12h is administered once, and 2.5 μ l/ time, total dosage is 10 μ l, has obvious inhibitory action to the propagation of EJ cell.In the scope that concentration is 25 ~ 100 μ g/ml, AMP-Na is obvious concentration dependent.The IR of AMP-Na 25,33,43.5,57.4,75.8 and 100 μ g/ml to human bladder cancer cell line EJ is respectively 17.50% ± 4.51%, 38.98% ± 1.27%, 55.91 ± 1.31%, 83.07% ± 1.39%, 86.87% ± 3.11% and 91.95% ± 0.49%, compared with negative group, AMP-Na 25 ~ 100 μ g/ml group all has significant difference (P < 0.01) to the suppression ratio of EJ cell, IC
50value is 39.43 ± 1.76 μ g/ml.
DDP handler effect of EJ cell line, in 48h, point four administrations, have obvious inhibitory action to the propagation of EJ cell.In the scope that concentration is 0.25 ~ 8 μ g/ml, DDP is obvious concentration dependent.The IR of DDP 0.25,0.5,1,2,4 and 8 μ g/ml to human bladder cancer cell line EJ is respectively 13.36% ± 1.51%, 28.38% ± 1.55%, 44.61% ± 1.58%, 54.77% ± 2.43%, 76.05% ± 2.40% and 89.77% ± 0.59%, compared with negative group, the suppression ratio of DDP 0.25 ~ 8 μ g/ml group to EJ cell has significant difference (P < 0.01), IC
50be 1.31 ± 0.04 μ g/ml.
V () AMP-Na sums up the inhibitory action that human bladder cancer cell line EJ is bred different action time
AMP-Na process EJ cell 24,48, in 72h and 48h point four administrations to the propagation of EJ cell, all there is obvious inhibitory action.24h administration group compared with point four administration groups in 48h, 72h administration group and 48h, IC
50value all has significant difference (P < 0.01); 48h administration group compared with point four administration groups in 72h administration group and 48h, IC
50value all has significant difference (P < 0.01); Point four administration groups are compared with 48h administration group in 48h, and suppression ratio has had and significantly improves, IC
50value has significant difference (P < 0.01); In 48h, point four administration groups are compared with 72h administration group, during AMP-Na low concentration in 48h points of four times administration groups to the suppression ratio of EJ cell higher than 72h administration group, during AMP-Na high concentration, in 48h points of four times administration groups to the suppression ratio of EJ cell lower than 72h administration group (see Fig. 3), but both medium effective concentrations are suitable, IC
50value does not have significant difference (see Fig. 4).
(B) AMP-Na different action time is to the inhibitory action of human bladder cancer 5637 cell proliferation
Similarly, AMP-Na handler bladder cancer 5637 cell 24,48, in 72h and 48h point four administrations to the propagation of 5637 cells, all there is obvious inhibitory action.24h administration group compared with point four administration groups in 48h, IC
50value has significant difference (P < 0.01); 24h administration group compared with 72h administration group, IC
50value has significant difference (P < 0.05); 48h administration group compared with point four administration groups in 48h, IC
50value has significant difference (P < 0.01); In 48h, point four administration groups are compared with 72h administration group, and both medium effective concentrations are suitable, IC
50value does not have significant difference (see Fig. 5, Fig. 6).
(C) the AMP-Na inhibitory action of different action time Human Bladder Cancer Cell Line BIU to be bred
More weak to the inhibitory action of BIU-87 cell proliferation after AMP-Na process Human Bladder Cancer Cell Line BIU 24,48 and 72h, the medium effective concentration (IC of AMP-Na
50) value is all higher.24h administration group compared with 72h administration group, IC
50value has significant difference (P < 0.05) (see Fig. 7, Fig. 8).
(D) AMP-Na sums up the inhibitory action of human bladder cancer three strain cell proliferation different action time
AMP-Na process respectively bladder cancer EJ, 5637, BIU-87 cell strain 24,48 and 72h, result shows, and AMP-Na is the most obvious to the inhibitory action of 5637 cell strains, IC
50be worth minimum; The inhibitory action of AMP-Na to EJ cell strain is only second to 5637, IC
50be worth less; AMP-Na is to the IC of the suppression of BIU-87 cell strain
50be worth larger.
AMP-Na process respectively bladder cancer EJ, 5637, after BIU-87 cell strain 24h, the IC of three strain cells
50value all has significant difference (P < 0.01); AMP-Na process respectively bladder cancer EJ, 5637, after BIU-87 cell strain 48h, the IC of three strain cells
50value all has significant difference (P < 0.01); AMP-Na process respectively bladder cancer EJ, 5637, after BIU-87 cell strain 72h, compared with BIU-87 cell strain, the IC of EJ, 5637 cell strains
50value has significant difference (P < 0.01), and the IC of EJ and 5637 cell strains
50value there was no significant difference (the results are shown in Table 7).
Table 7AMP-Na suppresses human bladder cancer cell to breed the IC of different action time
50value compares (
n=3)
IC
50: medium effective concentration.
Note: allogenic cell IC different action time
50compare, * * p < 0.01;
Different cell, identical action time IC
50compare,
ap < 0.01
To sum up, the propagation of AMP-Na to EJ, 5637 cell strains has significant inhibitory action, and with the increase of concentration, inhibitory action strengthens, and has obvious concentration dependent, and AMP-Na is different, and action time has regular hour dependency between point; The propagation of AMP-Na to BIU-87 cell strain does not have significant inhibitory action.The inhibitory action that AMP-Na breeds three strain bladder cancer cell lines is different, and this may be relevant with the feature of three strain cells, and EJ and 5637 cell strains are invasive bladder carcinoma, and BIU-87 cell strain is shallow phenotype bladder cancer.In AMP-Na effect 48h point four administrations with act on compared with 48h, suppression ratio has had and has significantly improved, this may with the AMP-Na added in cell degrade slow down relevant.
(E) mechanism of action
Carry out antitumor machanism research by methods such as Flow Cytometry (FCM) detection methods of routine, result is as follows.
FCM detects and finds that typical apoptotic peak appears in AMP-Na processed group, and illustrating that AMP-Na can induce the apoptosis of effect of EJ cell line, is one of its Antitumor Mechanism.
FCM detects and also finds that AMP-Na processed group makes the period profile generation significant change of EJ cell, G0/G1 phase cell increases relatively, S phase cell reduces relatively, G2 phase cell also has certain minimizing, illustrate that AMP-Na can by EJ cell block in the G0/G1 phase, reduce the synthesis of DNA, antiproliferative effect, thus reach antineoplastic effect.AMP-Na retardance the EJ cell cycle G0/G1 phase effect, in time m-concentration dependent.Along with the increase of ampelopsin na concn and the prolongation of drug treating time, the retardance G0/G1 phase acts on more obvious.These results of study are pointed out, and tumor cell can be blocked in the G0/G1 phase by AMP-Na, and this also may be one of its antineoplastic important function mechanism.
RT-PCR detects discovery, and the expression of cell cyclin D1-mRNA is lowered, and in regular hour-concentration dependent.Along with the increase of ampelopsin na concn and the prolongation of administration time, cyclin D
1it is more obvious that the expression of-mRNA is lowered, and has significant difference (P < 0.05, P < 0.01) compared with matched group.
The above results is pointed out, and the mechanism of action of AMP-Na may have the following aspects:
1. cell death inducing;
2. make cyclin D
1the expression of-mRNA is lowered, thus suppresses the transformation of G1 phase to S phase, and then the propagation of inhibition tumor cell;
3. by cell cycle arrest in the G1 phase, slow down its growth rate.
Embodiment 3
The Effect study of transplanted tumor and xenografts in nude mice bladder carcinoma in situ transplanted tumor in AMP-Na intraperitoneal injection drug treatment mouse bladder
1. medicine and reagent
AMP-Na, lot number: 100604, specification: 50mg/ props up (freeze-dried type), and Tai He Pharmaceutical Technology Co., Ltd provides by Guangdong ,-20 DEG C of preservations, dilute with PBS before use.Carboplatin for inj (Carboplatin, CBP) (freeze-dried type), 0.1g/ props up, batch number: 0040031.DA.
2. animal and cell strain
Kunming mice (KM), ♀, body weight: 18 ~ 22g, is provided by Lanzhou University's medical experiment animal center.Nude mouse (BALB/c nu/nu), ♀, 6 ~ 7w, by Hunan, Si Laike Jing Da laboratory animal company limited provides.Human bladder cancer cell line EJ, purchased from Shanghai cell biological institute cell bank, is incubated in RPMI 1640 culture medium (containing penicillin 100U/mL and streptomycin 100 μ g/mL) containing 10% calf serum, is placed in 37 DEG C, 5%CO
2incubator is cultivated.Mouse sarcoma S180 cells, purchased from pharmaceutical research institute of preclinical medicine institute of Lanzhou University.
3. the foundation of the preparation of human bladder cancer EJ single cell suspension and bladder carcinoma in situ Transplanted tumor model
Prepared by EJ single cell suspension: trophophase human bladder cancer cell line EJ of taking the logarithm, with 1% trypsinization 4 minutes, add RPMI 1640 culture medium (10% calf serum+90%1640) the termination digestion of 5mL containing serum, with the rearmounted 50mL centrifuge tube of 10mL suction pipe suspendible, 1500rpm, centrifugal 5min, abandon supernatant, with the RPMI 1640 culture medium lotion 1 time of serum-free, then use RPMI 1640 culture medium of serum-free (containing S180 sarcoma ascites supernatant) that cell concentration is diluted to 2 × 10
6use at once after individual/0.1mL.
Prepared by S180 single cell suspension: extract ascites, with brine, diluting cells to 2 × 10 from tumor-bearing mice intraperitoneal
6use at once after individual/0.1mL.
Intravesical tumor cell inoculation: anaesthetize nude mice with pentobarbital sodium (30mg/kg), under aseptic condition, plastic catheter (external diameter 1.0mm) inserts bladder by urethra, through conduit, 0.1mol/L HCl 100 μ l is injected intravesical, with the neutralization of equal-volume 0.1mol/L KOH solution after 15s, rinse with phosphate buffer, then injecting cell concentration is the RPMI RPMI-1640s of every 100 μ l containing 2.0 × 10 cells, 1h is retained at intravesical, nude mice is made to face upward abdomen and left and right sides changing position totally 4 times respectively, each 15min, fully contact with whole bladder inner chamber to make the tumor cell in RPMI RPMI-1640, tube drawing during 1h after injection cell.
4. administration:
Mice bearing S180 starts administration next day after tumor cell inoculation, and lotus EJ cell nude mice starts administration when the 8th day after tumor cell inoculation, every day 1 time, intraperitoneal injection (ip), and totally 14 times, after last administration, next day puts to death mice, gets tumor block and weighs.
5. observation index and effective criterion
The calculating of tumor weight mensuration and tumour inhibiting rate (%), after last administration, next day weighs, and puts to death animal, and tumor block is peeled off in complete whole dissection, claims tumor heavy (g), and takes pictures.Be calculated as follows tumor weight tumour inhibiting rate (InhibitionRate, IR):
IR＝(C-T)/C×100％
In formula, T is the average tumor weight of administration group, and C is the average tumor weight of negative control group.
Effective criterion: tumour inhibiting rate < 40% is invalid, tumour inhibiting rate >=40% be effective through statistical procedures P < 0.05.
6. statistical analysis
Each group of data are measurement data, Using statistics analysis software SPSS 17.0, and adopt one factor analysis of variance and t inspection to carry out statistical analysis, P < 0.05 represents that difference has statistical significance.
7. result
A.AMP-Na intraperitoneal injection administration is on the impact of S180 sarcoma kunming mice intravesical growth of xenografted:
In S180 sarcoma, transplanted to kunming mice intravesical next day, mice is divided at random negative control, carboplatin and AMP-Na 200mg/kg totally 3 groups, grouping drug treatment on the same day.Carboplatin: 25mg/kg, ip, 1 time on the 2nd; AMP-Na:ip, 1 time on the 1st, totally 14 times.Result, AMP-Na 200mg/kg obviously can suppress S180 sarcoma kunming mice intravesical growth of xenografted, inhibitory rate 56.04%, with the tumor weight ratio of matched group, the tumor weight of AMP-Na 200mg/kg group obviously alleviates, and difference has pole significance (P < 0.01).(table 8).The tumour inhibiting rate (56.04%) of AMP-Na and (58.19%) of carboplatin are closely.
Table 8AMP-Na is on the impact of S180 sarcoma kunming mice intravesical transplanted tumor
*p < 0.01 compared with PBS matched group
The impact that B.AMP-Na intraperitoneal injection administration grows human bladder cancer cell line EJ nude mouse bladder orthotopic transplantation tumor
When human bladder cancer cell line EJ transplants latter 8th day to BALB/c nu/nu nude mouse intravesical, nude mouse is divided at random negative control, carboplatin, AMP-Na 160,200 and 260mg/kg totally 5 groups, divides into groups to start drug treatment the same day.Carboplatin 40mg/kg, ip, 1 time on the 2nd; AMP-Na ip, 1 time on the 1st, totally 14 times.Result, AMP-Na is low, neutralize the growth that high each dosage all can suppress human bladder cancer cell line EJ BALB/c nu/nu mouse bladder orthotopic transplantation tumor, tumour inhibiting rate is respectively 29.71%, 42.83% and 46.15%, and with matched group tumor weight ratio, the tumor weight of each dosage group of AMP-Na all obviously alleviates, and difference has pole significance (P < 0.01), wherein with AMP-Na 260mg/kg effect the strongest (table 9).
Table 9AMP-Na is on the impact of human bladder cancer cell line EJ BALB/c nude mice bladder orthotopic transplantation tumor
*p < 0.01 compared with PBS matched group
8. conclusion
AMP-Na 200mg/kg obviously suppresses S180 sarcoma kunming mice intravesical growth of xenografted, and 160-260mg/kg suppresses the growth of human bladder cancer cell line EJ nude mouse bladder orthotopic transplantation tumor, and is dose dependent to the inhibitory action of tumor growth.
AMP-Na has powerful inhibitory action to transplanted tumor in mouse bladder, in urine, drain that concentration is high, the persistent period is long again in conjunction with AMP-Na, and this pharmacodynamic profile of mainly draining with activated proto-drug, therefore can set up and a kind ofly reached the effect of bladder cancer being carried out to irrigation bladder treatment by venoclysis, the clinical treatment for bladder cancer provides a kind of new method.
By bladder orthotopic transplantation tumor modeling method (Chin, J., Kadhim to people such as Chin, S., Garcia, B., Kim, Y.S., Karlik, S., 1991.Magnetic resonance imaging fordetecting and treatment monitoring of orthotopic murine bladder tumorimplants.145, improvement 1297-301), the present inventor makes the tumor formation rate of human bladder cancer's Orthotopic implantation in nude mice tumor model bring up to 100%.In addition, this experiment first by S180 sarcoma successful implantation to kunming mice intravesical, this provides a kind of easy new animal model by for investigating in other urine the basic research of draining the high antitumor drug of concentration.
Embodiment 4
AMP-Na intravenous administration is to the dose-effect relationship of S180 sarcoma kunming mice intravesical transplanted tumor
1. medicine and reagent
AMP-Na, lot number: 100604, specification: 50mg/ props up (freeze-dried type), and Tai He Pharmaceutical Technology Co., Ltd provides by Guangdong ,-20 DEG C of preservations, dilute with PBS before use.Carboplatin for inj (Carboplatin, CBP) (freeze-dried type), 0.1g/ props up, batch number: 0040031.DA.Benzylpenicillin sodium for injection is produced by HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory; ZHUSHEYONG LIUSUAN LIANMEISU is produced by Huabei Pharmaceutic Co., Ltd.
2. animal and cell strain
Kunming mice (KM), ♀, body weight: 18 ~ 22g, is provided by Lanzhou University's medical experiment animal center, and quality certification SCXK (sweet): 2009-0004.S180 sarcoma, by preclinical medicine institute of Lanzhou University conservation that pharmaceutical research goes down to posterity.
3. tumour transplatation in mouse bladder:
With embodiment 3.
4. administration:
Mice bearing S180 starts administration, 2 days 1 time, iv next day after tumor cell inoculation, totally 7 times, at the end of experiment, gets tumor of bladder, weighs, and calculates tumour inhibiting rate.
5. observation index and effective criterion:
With embodiment 3.
6. statistical analysis
Each group of data are measurement data, Using statistics analysis software SPSS 17.0, and adopt one factor analysis of variance and t inspection to carry out statistical analysis, P < 0.05 represents that difference has statistical significance.
7. result
AMP-Na intravenous administration is on the impact of S180 sarcoma kunming mice intravesical growth of xenografted:
Transplant next day in S180 sarcoma kunming mice intravesical, mice is divided at random negative control, carboplatin, AMP-Na 160,200 and 260mg/kg totally 5 groups, often organize 10-12 mice, divide into groups to start drug treatment the same day.Carboplatin: 25mg/kg, ip, 1 time on the 2nd; AMP-Na:iv, 1 time on the 2nd, totally 7 times.Result, AMP-Na is low, neutralize high each dosage all can suppress S180 sarcoma kunming mice intravesical growth of xenografted, tumour inhibiting rate is respectively 37.93%, 45.61% and 48.66%, and with matched group tumor weight ratio, the tumor weight of each dosage group of AMP-Na all obviously alleviates, and difference all has pole significance (P < 0.01), wherein with AMP-Na 260mg/kg effect the strongest (table 10, Fig. 1 and Fig. 2).
Table 10AMP-Na intravenous administration is on the impact of S180 sarcoma kunming mice intravesical transplanted tumor
*p < 0.01 compared with PBS matched group
8. conclusion
After AMP-Na 160-260mg/kg intravenous injection, obviously can suppress S180 sarcoma kunming mice intravesical growth of xenografted, and be dose dependent to the inhibitory action of tumor growth.
Embodiment 5
Prepare the pharmaceutical composition (injection) of AMP-Na
Formula 1:
AMP-Na 50mg
Non-aqueous matchmaker (injection) adds to 1ml
By above-mentioned formula, get AMP-Na and appropriate non-aqueous matchmaker, principal agent sterilizing becomes sterilized powder, packs respectively afterwards.Matching while using during injection.
Formula 2:
AMP-Na 200mg
L-arginine 280mg
PH4.0 citric acid-sodium hydrogen phosphate 0.7ml
Buffer
By above-mentioned formula, get AMP-Na (pentahydrate) and L-arginine, after pH4.0 citric acid-sodium hydrogen phosphate buffer solution, regulate PH to 7.2, degerming through microporous filter, be loaded in the bottle of 5ml specification, lyophilizing, sealing.Add before injection after sterilized water dissolves and directly use.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (6)
1. a purposes for ampelopsin or its pharmaceutically acceptable salt, is characterized in that, for the preparation of the medicine for the treatment of urologic neoplasms; And described urologic neoplasms is bladder cancer, and described medicine is applied to mammalian object by intravenous injection mode.
2. purposes as claimed in claim 1, it is characterized in that, the dosage form of described medicine is injection.
3. purposes as claimed in claim 1, it is characterized in that, described medicine contains ampelopsin sodium salt or its hydrate of concentration >=2mg/ml.
4. purposes as claimed in claim 1, it is characterized in that, described medicine contains ampelopsin sodium salt or its hydrate of concentration >=5mg/ml.
5. purposes as claimed in claim 1, it is characterized in that, described medicine contains ampelopsin sodium salt or its hydrate of concentration >=10mg/ml.
6. the purposes as described in as arbitrary in claim 3-5, it is characterized in that, described medicine contains AMP-Na or its hydrate.
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| CN101347425A (en) * | 2008-09-03 | 2009-01-21 | 福建省中医药研究院 | Uses of ampelopsin and total flavone valid target rich in ampelopsin in preparing medicament for preventing and treating prostate gland cancer |
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| CN101347425A (en) * | 2008-09-03 | 2009-01-21 | 福建省中医药研究院 | Uses of ampelopsin and total flavone valid target rich in ampelopsin in preparing medicament for preventing and treating prostate gland cancer |
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