CN102805730A - Ceramide liposome and preparation method and application thereof - Google Patents
Ceramide liposome and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to ceramide liposome and a preparation method and application thereof. The ceramide liposome comprises a therapeutically effective amount of active component and a lipid bilayer film, and is characterized in that the ceramide liposome comprises 0.5 to 20 weight parts of ceramide used as the active component and 10 to 350 weight parts of the lipid bilayer film, wherein the lipid bilayer film comprises the following components in part by weight: 5 to 100 parts of phospholipid substance, 0.5 to 10 parts of cholesterol, 0 to 100 parts of surfactant, 0 to 20 parts of additive and 0.5 to 10 parts of organic solvent. The preparation method comprises the following steps of: fully dissolving the ceramide, the phospholipid substance, the cholesterol and the additive in the organic solvent, dissolving the surfactant in a dispersion medium to prepare the ceramide liposome, and reducing the average particle size of the prepared ceramide liposome to less than 500nm to ensure that the particle sizes are uniformly distributed. The invention also discloses new application of the ceramide liposome to cancer therapy.
Description
Technical field
The invention belongs to technical field of medicine, specifically relate to targeting drug administration preparation short chain ceramide liposome and preparation method thereof, with and new purposes in oncotherapy.
Background technology
Malignant tumor has become the disease that the disease death rate ranks the first, and chemotherapy is one of topmost treatment means.Most late tumor patients all need chemotherapy, and chemotherapy is that whole body cancerous cell diffusion patient is than effective method.But the total effective percentage of chemotherapy is still less than 30% at present clinical, and nearly all chemotherapeutic all can produce the multidrug resistance phenomenon in treatment, thereby further reduced the chemotherapy effective percentage.Therefore, the therapeutic effect that how to improve chemotherapeutics is the key issue of oncotherapy.
Short chain ceramide (C2, C4, C6, C8, first-selected C6) is a kind of lipid molecular that is produced by the hydrolysis of cell membrane sphingomyelin, and it is present in the cell membrane with high concentration, belongs to lipid molecule family, has constituted sphingol and fatty acid.It can bring into play second message,second messenger's in the cell effect discovered in recent years, has mediated the various kinds of cell biological effect, comprises cell differentiation, growth inhibited, apoptosis, immunoreation etc.Ceramide has also been participated in the activation expression regulation of antioncogenes such as JNK, p53, bcl-2, p21.Research recently shows that lonizing radiation, chemotherapeutics (paclitaxel, doxorubicin, daunorubicin etc.), oxidative stress etc. can activate ceramide signaling system in the tumor cell, and synthetic ceramide promotes apoptosis in the inducing cell body.In addition, the stimulation that ceramide gathers in any enhancing cell all can produce apoptosis-promoting effect, and the tumor cell that can not produce enough ceramides can not effectively carry out apoptosis usually.To most tumor cell, endogenic ceramide is inducing apoptosis of tumour cell effectively.Therefore, exogenous interpolation ceramide is as chemotherapeutic sensitizer, thereby effectively increases chemotherapy drug susceptibility, and reducing chemotherapy resistance is an efficacious therapy approach.
However, whole body utilization ceramide still receives its physics, chemistry affect.Because ceramide is hydrophobic fat-soluble, carry in the body and form big aggregation, be difficult to arrive in the tumor cell.Even adding solubilizing agent polyoxy ethyl Oleum Ricini is mixed with injection and also is prone to cause more complication, and is low like anaphylaxis and bioavailability.Liposome is little to the body toxic and side effects, and its lipid bilayer and biomembrane have bigger similarity and tissue intersolubility, are easy to by tissue absorption.Wrapping kmedicine by liposome is a physical process, does not change drug molecular structure, after medicine is wrapped, can reduce drug toxicity, reduces the drug use amount, has slow release and controlled-release function.Therefore, the ceramide liposome of exploitation target tumor position, long circulation slow-release function seems particularly important to satisfy the clinical administration demand.
At present, still do not have the ceramide liposome (comprising injection or peroral dosage form) that can be used for oncotherapy both at home and abroad, therefore, our invention has fabulous novelty.
Summary of the invention
The objective of the invention is to overcome the deficiency that exists in the prior art; But develop the ceramide liposome (comprising injection or peroral dosage form) of a kind of stable in properties and suitability for industrialized production; The method for preparing of ceramide liposome is provided simultaneously, and discloses the new purposes of this ceramide liposome in oncotherapy.
First purpose of the present invention realizes through following technical scheme:
Ceramide liposome comprises a treatment effective amount of actives and a type lipid bilayer thin film, and it is characterized in that: the weight portion of said active component is 0.5-20 part, and active component is a ceramide; The weight portion of said type of lipid bilayer thin film is 10-350 part, comprises following components by part by weight: phospholipid substance 5-100 part, cholesterol 0.5-10 part, surfactant 0-100 part, additives 0-20 part, organic solvent 0.5-10 part.
As further improvement of the present invention, described phospholipid substance is selected from one or more in two Laurel phosphatidyl cholines, DSPC, distearyl phosphatidyl glycerol, Phosphatidylserine, phosphatidylinositols, two palmityl phosphatidic acid, dioleoyl phospholipid phatidylcholine, dimyristoyl phosphatidyl choline, phosphatidyl glycerol, phosphatidic acid, phosphatidylcholine, dipalmitoyl phosphatidyl choline, two palmityl phosphatidyls, DOPE, soybean lecithin, Ovum Gallus domesticus Flavus lecithin, cephalin, synthetic phospholipid, the hydrogenated soy phosphatidyl choline.
As further improvement of the present invention, it is the natural cholesterol of raw material production that said cholesterol is selected for use with egg yolk, animal brains, lanoline.
As further improvement of the present invention, said surfactant is selected from one or more the compositions in dexycholate, cholate, deoxycholic acid, cholic acid, tween 80, taurocholate, the poloxamer.
As further improvement of the present invention, said organic solvent is selected from one or more the mixture in ethanol, acetone, methanol, chloroform, ether, the isopropyl alcohol.
As further improvement of the present invention, said additives are selected from one or more among L-isoleucine, malic acid, sodium metabisulfite, sodium sulfite, vitamin C, vitamin E, propyl gallate, fumaric acid, L-cysteine or PH buffer agent phosphate, the EDTA.
As further improvement of the present invention, said ceramide liposome adds long circulation adjuvant, carries out finishing and processes long circulating liposomes, and the mol ratio of long circulation adjuvant of said long circulation adjuvant and phospholipid substance is 1:10-1:200; Said long circulation adjuvant is selected from Polyethylene Glycol-phospholipid derivative, Polyethylene Glycol, polyvinylpyrrolidone, methyl and gathers azoles quinoline, Polyethylene Glycol-dipalmitoyl phosphatidyl choline, Polyethylene Glycol-distearyl acyl group PHOSPHATIDYL ETHANOLAMINE, chitosan or polyvinyl alcohol.
As further improvement of the present invention, said ceramide liposome adds the magnetic decorative material, carries out finishing and processes magnetic liposome; Said magnetic decorative material is a super-fine magnetic powder.
As further improvement of the present invention, said ceramide liposome adds the temperature-sensitive auxiliary material, carries out finishing and processes thermal sensitive liposome; Said temperature-sensitive auxiliary material are the phospholipid that itself has thermal sensitivity, comprise dipalmitoyl phosphatidyl choline (DPPC), DSPC (DSPC); Or the macromolecule thermally sensitive polymeric, comprise polyacrylic acid, gather phosphatidic acid; Or a kind of phase transition temperature that can make liposome is suitable for the oiliness medicine of tumor thermotherapy temperature, comprises elemene or wild duck seed oil.
Traditional liposome particle diameter is bigger, and low, the poor stability of envelop rate is removed it by the phagocyte of reticuloendothelial system especially easily from blood circulation, thereby reduces the dose that arrives tumor tissues.The present invention carries out finishing to liposome; The long circulating liposomes of processing, magnetic liposome, thermal sensitive liposome etc. can further prolong the blood circulation time of ceramide liposome; Improve the targeting property and the bioavailability of ceramide liposome, reduce toxic and side effects.
As further improvement of the present invention, the mean diameter of said ceramide liposome decentralized photo is less than 500nm.
As further improvement of the present invention, said ceramide liposome exists with the form of injection or solid powder.The form of solid powder can prolong the resting period of ceramide liposome greatly, is convenient to transportation.
Ceramide liposome of the present invention can be with injection or oral way administration, and wherein injection system can be intravenous drip, intravenous injection.Subcutaneous injection, intramuscular injection, lumbar injection etc., preferred intravenous injection.
As further improvement of the present invention, said ceramide liposome envelop rate is greater than 80%.
Second purpose of the present invention realizes through following technical scheme:
The method for preparing of ceramide liposome; It is characterized in that; Comprise the steps: fully to be dissolved in ceramide, phospholipid substance, cholesterol and additives in the organic solvent; In disperse medium, adopt film dispersion method or organic solvent injection method to make ceramide liposome surfactant dissolves, adopt ultrasonic, high pressure homogenize then or the method extruded is reduced to below the 500nm mean diameter of the ceramide liposome that makes and uniform distribution.
The detail operations of said film dispersion method is: fully be dissolved in ceramide, phospholipid substance, cholesterol and additives in the organic solvent; This solution is placed in the rotary evaporator; At 40 ℃ of removal organic solvents that reduce pressure down; Form thin film, add the pH value that contains surfactant then and be 3.0 ~ 10.0 disperse medium, make ceramide liposome.
The detail operations of said organic solvent injection method is: fully be dissolved in ceramide, phospholipid substance, cholesterol and additives in the organic solvent; It is 3.0 ~ 10.0 disperse medium that this solution is slowly injected 50-60 ℃ the pH value that contains surfactant; Being stirred to organic solvent with the electronics constant speed stirrer eliminates; The described disperse medium of reuse is diluted to and requires concentration, makes ceramide liposome.
As the further improvement of said method for preparing, said disperse medium is Tris buffer, water, phosphate buffer, carbonate buffer solution, 5% D/W or 0.9%NaCl solution; The weight concentration of the surfactant in the said disperse medium is 1%-30%.
As the further improvement of said method for preparing, in the ceramide liposome that makes, add the lyophilized powder protective agent, process lyophilized powder through spray drying or freeze drying process; Said lyophilized powder protective agent is selected from one or more in mannitol, sucrose, trehalose, sodium chloride, glucose, dextran, glycine, lactose, sorbitol, xylitol, fructose, glycerol, arabic gum and the lysine; The lyophilized powder protective agent: the parts by weight proportioning of ceramide liposome is (10-20): (0.5-6.0).
The 3rd purpose of the present invention is:
The purposes of described ceramide liposome in the preparation antitumor drug is provided.
The present invention compared with prior art, advantage is:
The ceramide liposome of the present invention's preparation can have certain targeting property to tumor; Long circulating liposomes can significantly improve liposome circulation time in vivo; Make medicine more long-term effectively targeting in tumor locus; And chemotherapeutics (as: taxanes and gemcitabine class medicine etc.) unite utilization can be more effective inducing apoptosis of tumour cell, reduce chemotherapeutics concentration and avoid chemotherapy resistance, prolonged the biological time-to-live.
Description of drawings
Fig. 1 is to the situation that the influences curve chart of mean tumour volume of the transplanted tumor in nude mice of inoculation pancreatic cancer cell after different drug or the drug regimen administration.
Fig. 2 is to the inhibition situation pictorial diagram of final size of the transplanted tumor in nude mice of inoculation pancreatic cancer cell after different drug or the drug regimen administration.
Fig. 3 is to the situation that the influences curve chart of mean survival time of the nude mice of inoculation pancreatic cancer cell after different drug or the drug regimen administration.
Fig. 4 is to the situation that the influences curve chart of mean tumour volume of the transplanted tumor in nude mice of inoculation pancreatic cancer cell after different drug or the drug regimen administration.
Fig. 5 is to the situation that the influences curve chart of survival rate of the nude mice of inoculation pancreatic cancer cell after different drug or the drug regimen administration.
Fig. 6 is the situation that the influences bar diagram to the cell survival rate of people MIA pancreas cancer cell strain of the different drug that records of mtt assay or drug regimen.
Fig. 7 is the situation that the influences bar diagram to the cell survival rate of people CaOV3 ovarian cancer cell strain of the different drug that records of mtt assay or drug regimen.
Fig. 8 is the situation that the influences bar diagram to the cell survival rate of L3.6 pancreas cancer cell strain of the different drug that records of mtt assay or drug regimen.
The specific embodiment
Below in conjunction with concrete accompanying drawing and embodiment the present invention is described further.
Ceramide 20mg
Ovum Gallus domesticus Flavus lecithin 20mg
Cholesterol 200mg
Ether 10ml
Taurocholate 30mg
Method for making: the 30mg taurocholate is dissolved in the phosphate buffer (pH 7.4), and the weight concentration of taurocholate in phosphate buffer is 15%; 20mg ceramide, 20mg Ovum Gallus domesticus Flavus lecithin and 200mg cholesterol are dissolved in the ether; Then this solution is slowly injected 50-60 ℃ phosphate buffer; Being stirred to ether with the electronics constant speed stirrer with the 600r/min rotating speed eliminates; The above-mentioned phosphate buffer of reuse is diluted to 100ml, promptly gets ceramide liposome, puts 4 ℃ of preservations.
Soybean lecithin 120mg
Cholesterol 250mg
Methanol 10ml
Ethanol 10ml
NaTDC 40mg
Method for making: the 40mg NaTDC is dissolved in the Tris-HCl that an amount of molar concentration is 20mmol/L (PH7.5 the contains 0.15mol/LNaCl) buffer, and the weight concentration of NaTDC in the Tris-HCl buffer is 10%; The 30mg ceramide is dissolved in the 10ml methanol, and 120mg lecithin, 250mg cholesterol are dissolved in the 10ml ethanol, place these two kinds of solution in the rotary evaporator jointly; Remove solvent 40 ℃ of decompressions, form thin film, add above-mentioned then; Add the above-mentioned Tris-HCl buffer that is dissolved with NaTDC then; Supersound process 30min promptly gets ceramide liposome, the spray-dried lyophilized formulations that makes ceramide liposome.Only need abundant aquation to shake in short-term before lyophilized formulations uses, just can form ceramide liposome again.
Ceramide 100mg
Soybean lecithin 1200mg
Hydrogenation saturated phospholipid 300mg
Cholesterol 100mg
Ganglioside 50mg
Dehydrated alcohol 50ml
Vitamin E 50mg
Method for making: with 100mg ceramide, 1200mg soybean lecithin, 300mg hydrogenation saturated phospholipid, 100mg cholesterol, 50mg ganglioside and 6.5mg vitamin E ultrasonic dissolution in the 50ml dehydrated alcohol; Solvent is removed in the 40 ℃ of decompressions in rotary evaporator of this solution, form thin film, add 20mmol/L Tris-HCl (PH7.5 then; Contain 0.15mol/LNaCl) buffer; Supersound process 30min, high pressure homogenizer circulation homogenizing 5-6 time is crossed 0.22 μ m microporous filter membrane then; The packing embedding gets ceramide liposome, through the dry lyophilized formulations that gets ceramide liposome.Only need abundant aquation to shake in short-term before lyophilized formulations uses, just can form liposome again.
C6 ceramide 250mg
Soybean lecithin 1500mg
Hydrogenation saturated phospholipid 500mg
Cholesterol 150mg
Vitamin E 7.2mg
Oleic acid 50mg
Poloxamer 50mg
Dehydrated alcohol 10ml
Method for making: the 50mg poloxamer is dissolved in the Tris-HCl that an amount of molar concentration is 20mmol/L (PH7.5 the contains 0.15mol/LNaCl) buffer, and the weight concentration of poloxamer in the Tris-HCl buffer is 15%; With 250mg C6 ceramide, 1500mg soybean lecithin, 500mg hydrogenation saturated phospholipid, 150mg cholesterol, 7.2mg vitamin E and 50mg oleic acid and ultrasonic dissolution in the 10ml dehydrated alcohol; Solvent is removed in the 40 ℃ of decompressions in rotary evaporator of this solution, form thin film, add then; Add the above-mentioned Tris-HCl buffer that is dissolved with poloxamer then; Supersound process 30min, high pressure homogenizer circulation homogenizing 5-6 time is crossed the 0.22um microporous filter membrane then; The packing embedding gets ceramide liposome, through the dry lyophilized formulations that gets ceramide liposome.Only need abundant aquation to shake in short-term before lyophilized formulations uses, just can form ceramide liposome again.
Embodiment 5: the ceramide liposome of dual-target tumor
C6 ceramide 250mg
Soybean lecithin 1500mg
Hydrogenation saturated phospholipid 500mg
Cholesterol 150mg
Vitamin E 7.2mg
Oleic acid 50mg
Poloxamer 50mg
Dehydrated alcohol 10ml
The polypeptide of dual-target tumor: adopt the 9-FMOC solid-phase synthesis synthetic, adopt the HPLC purification, adopt mass spectrum to identify.
Method for making: the 50mg poloxamer is dissolved in the Tris-HCl that an amount of molar concentration is 20mmol/L (PH7.5 the contains 0.15mol/LNaCl) buffer, and the weight concentration of poloxamer in the Tris-HCl buffer is 15%; With 250mg C6 ceramide, 1500mg soybean lecithin, 500mg hydrogenation saturated phospholipid, 150mg cholesterol, 7.2mg vitamin E, 50mg oleic acid ultrasonic dissolution in the 10ml dehydrated alcohol; Solvent is removed in the 40 ℃ of decompressions in rotary evaporator of this solution; Form thin film, add the above-mentioned Tris-HCl buffer that is dissolved with poloxamer then, supersound process 30min; High pressure homogenizer circulation homogenizing 5-6 time; Cross the 0.22um microporous filter membrane then, make ceramide liposome, in this ceramide liposome, add the polypeptide of dual-target tumor; Ceramide liposome: the mol ratio of the polypeptide of dual-target tumor is 1:50, through the dry lyophilized formulations that gets the ceramide liposome of dual-target tumor.Only need abundant aquation to shake in short-term before this lyophilized formulations uses, just can form ceramide liposome again.
Embodiment 6: the preparation of ceramide long circulating liposomes
Ceramide 20mg
Hydrogenated soy phosphatidyl choline 200mg
Cholesterol 100mg
PEG-DSPE 50mg
Chloroform 30ml
Alpha-tocopherol 20mg
Method for making: 20mg ceramide, 50mg PEG-DSPE, 200mg hydrogenated soy phosphatidyl choline, 100mg cholesterol and 20mg alpha-tocopherol are dissolved in the 30ml chloroform, put in the pyriform bottle rotary evaporation under nitrogen current; Solvent is removed in decompression, vacuum drying 2-3 days then, gets the constant weight thin film; Tris-HCl buffer (the PH7.0 that adds an amount of 20mmol/L again; Contain 0.15mol/L NaCl), the pyriform bottle is shaken in rotation, obtains the liposome suspension.Behind nitrogen wash, sealed under the room temperature balance 1 day, probe sonication 15min, then under 500Kpa pressure through little fluidization phase mixing chamber, handle through little fluidization of 60-80 stroke, make the liposome particle diameter less than 100nm.With 5.4% glucose solution dialysis desalination, get neural amide long circulating liposomes then, process lyophilized formulations through lyophilized preparation again.Only need abundant aquation to shake in short-term before this lyophilized formulations uses, just can form ceramide liposome again.
Embodiment 7: the preparation of ceramide thermal sensitive liposome
Ceramide 0.4g
Injection fabaceous lecithin 20g
Cholesterol 2.4g
Dipalmitoyl phosphatidyl choline (DPPC) 5.0g
18-amine. 0.1g
Vitamin E 0.2g
Ether 200ml
Method for making: the vitamin E of the dipalmitoyl phosphatidyl choline of the cholesterol of the injection fabaceous lecithin of the ceramide of 0.4g, 20g, 2.4g, 5.0g, 0.2g is dissolved in the diethyl ether solution and pours the pyriform bottle into; Add a small amount of PH buffer agent phosphate solution contain 18-amine. again, ultrasonicly make into even breast, decompression eliminates organic solvent on the Rotary Evaporators; Make into gel; Whirlpool concussion distilling under reduced pressure at interval gets white even liposome solutions, and the reuse buffer dissolves to 200ml surely.Remove thermal source through the film that pressurizeed (0.22um) granulate, divide to be filled to lyophilization in the cillin bottle, repeat 3 times, get neural amide thermal sensitive liposome.
Embodiment 8: the preparation of ceramide magnetic liposome
Ceramide 20mg
Soybean lecithin 100mg
Cholesterol 100mg
Two spermaceti phosphatidic acid 20mg
Alpha-tocopherol 20mg
Ether 20ml
Method for making: super-fine magnetic powder is dissolved in the phosphate buffer (pH 7.4), and the weight concentration of super-fine magnetic powder in phosphate buffer is 0.5%; 100mg lecithin, 100mg cholesterol, the two spermaceti phosphatidic acid of 20mg, 20mg alpha-tocopherol and 20mg ceramide are dissolved in the 20ml ether; Form one deck lipid membrane on evaporation under reduced pressure removed organic solvent to the bottle inwall, add the above-mentioned phosphate buffer that contains super-fine magnetic powder, ultrasonic to mixture formation single_phase system on water-soluble type Ultrasound Instrument; Reduction vaporization is to gel formation again; Continue reduction vaporization to forming the muddy liquid of aqueous, inflated with nitrogen, ice-bath ultrasonic promptly gets neural amide magnetic liposome; Add 8% lactose and do caffolding agent, process lyophilized formulations through lyophilized preparation again.
Experimental example 9: the anti-cancer of pancreas effect of ceramide liposome and chemotherapeutics paclitaxel drug combination.
Laboratory animal: the BALB/c male nude mouse at 2 monthly ages (available from Shanghai Si Kelai laboratory animal company), totally 40.
Test method:
The ceramide liposome that we select embodiment 4 and embodiment 5 to make is defined as ceramide liposome 1 and ceramide liposome 2 respectively.
Be based upon body nude mice cancer of pancreas model by following method: nude mice is near right thigh subcutaneous injection 1 * 10
6The L3.6 pancreas cancer cell strain, after 2 weeks, treat visible 50mm
3Behind the entity tumor,, nude mice is divided into 4 groups by 10 every group, is respectively 1 group of matched group, paclitaxel group, paclitaxel+ceramide liposome, 2 groups of the paclitaxel+ceramide liposomes of not administration according to the difference of intravenous administration amount.
Be administered once by preceding 6 day every day, per mode that was administered once in 2 days was carried out administration in back 6 days, carried out altogether 12 days.2 record tumor sizes, nude mice survival rate and body weight write down for 6 weeks altogether weekly, and the record result sees Fig. 1 ~ Fig. 3.
Among Fig. 2: a. contrast, b. paclitaxel, c. paclitaxel+ceramide liposome 1, d. paclitaxel+ceramide liposome 2.
By finding out among Fig. 1 ~ Fig. 3: the exogenous paclitaxel that singly adds, the matched group of tumor size, nude mice survival rate and not administration is obviously difference not; But associating or combination give paclitaxel and ceramide liposome, but can significantly suppress tumor growth, reduce gross tumor volume, prolong the nude mice time-to-live, increase the nude mice survival rate.This conjoint therapy rings not quite the nude mouse ghost image in addition, points out it limited to normal structure and cytotoxic effect.
Above experimentation shows, short chain ceramide paclitaxel and the transplanted tumor in nude mice of inoculation pancreatic cancer cell all had certain antitumor activity, but the tumor killing effect that singly adds ectogenic short chain ceramide or paclitaxel is limited.Short chain ceramide and pharmaceutical preparation thereof just have significant antitumor activity to the transplanted tumor in nude mice of inoculating pancreatic cancer cell after need uniting paclitaxel.
Embodiment 10: the anti-cancer of pancreas effect of ceramide liposome and chemotherapeutics gemcitabine drug combination.
Laboratory animal: the BALB/c male nude mouse at 2 monthly ages (available from Shanghai Si Kelai laboratory animal company), totally 30.
Test method:
Gemcitabine is the goldstandard medicine of pancreatic cancer chemotherapy.The ceramide liposome that we select embodiment 6 to make.
Be based upon body nude mice cancer of pancreas model by following method: nude mice is near right thigh subcutaneous injection 1 * 10
6The L3.6 pancreas cancer cell strain, after 2 weeks, treat visible 50mm
3Behind the entity tumor,, nude mice is divided into 3 groups by 10 every group, is respectively matched group, gemcitabine group, the gemcitabine+ceramide liposome group of not administration according to the difference of intravenous administration amount.
Be administered once by preceding 6 day every day, per mode that was administered once in 2 days was carried out administration in back 6 days, carried out altogether 12 days.Write down gross tumor volume and nude mice survival rate 2 times weekly, write down for 6 weeks altogether, the record result sees Fig. 4, Fig. 5.
Among Fig. 4: A. matched group (tumor DT Doubling Time 5.6 days); B. gemcitabine (tumor DT Doubling Time 5.4 days); C. gemcitabine+ceramide liposome (the tumor DT Doubling Time is 9.1 days); Heteroplastic transplantation cancer of pancreas nude mice growth curve-gemcitabine 5 mg/kg that swell and ache.
Fig. 5: heteroplastic transplantation cancer of pancreas nude mice survival rate
A. contrast; B. gemcitabine; C. add gemcitabine+ceramide liposome.
By finding out among Fig. 4, Fig. 5: the exogenous gemcitabine that singly adds, the matched group of gross tumor volume, nude mice survival rate and not administration be obviously difference not; But associating or combination give gemcitabine and ceramide liposome, but can significantly suppress tumor growth, reduce gross tumor volume, increase the nude mice survival rate.
Embodiment 11: ceramide liposome is to the propagation inhibition test of the human pancreas cancer cell strain of In vitro culture.
The test cell: people MIA pancreas cancer cell strain derives from US mode culture collection warehousing (ATCC).
Test drug and reagent: ceramide (Avanti, USA), 3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazole bromine salt (MTT) (sigma, USA); Trypsin sigma, USA); Dimethyl sulfoxine (DMSO) (sigma, USA); The DMEM cell culture medium (GIBCO, Invitragen, USA); The RPMI1640 cell culture fluid (GIBCO, Invitragen, USA); Hyclone (GIBCO, Invitragen, USA); Superfine hyclone Fetal Bovine Serum (Hyclone).
Instrument: ultra cold storage freezer (Nature, USA), inverted microscope (NIKON, Japan), ELIASA (Bio-Tek Instruments, USA); Cell culture incubator (SANYO, Japan), superclean bench (FLC-Harbin Dong Lian instrument plant), the PH meter (Thermo orion, USA).
Experimental technique:
Adopt mtt assay by routine: with people MIA pancreas cancer cell strain by conventional method cultivations of going down to posterity, the cell of collection exponential phase, the adjusting concentration of cell suspension is 5*10
4About individual/ml.
Cell suspension is added in 96 well culture plates, and each hole adds 180 μ l; Place 37 ℃ of constant incubators to cultivate 24h.
Gemcitabine and ceramide liposome (ceramide liposome that embodiment 6 makes) are made into the solution of gradient concentration (0.75 μ g/ml, 1.5 μ g/ml, 3 μ g/ml) respectively with the DMEM cell culture fluid.Experimental group adds Jixitabin solution (A), the ceramide liposome+gemcitabine mixed solution (B) of each concentration; They add by above-mentioned concentration simultaneously; Every hole adds 20 μ l, establishes 4 parallel holes for every group, and establishes blank well (only adding the DMEM cell culture fluid) with zeroing.
Cultivate 48h at 37 ℃ of incubators, with the exhaustion of the liquid in 96 well culture plates, it is the MTT solution of 1mg/ml that every then hole adds 30 μ l concentration, puts 37 ℃, 5%CO with liquid-transfering gun
2Incubator continue to cultivate 4h.
Abandon supernatant, every hole adds dimethyl sulfoxine (DMSO) 150 μ l, puts shaking table and shakes up 30min, under 492nm, detects every hole absorbance (A) value with ELIASA, uses SPSS software statistics experimental result then, and is specifically as shown in Figure 6.
Can find out that by Fig. 6 associating or combination give the survival rate that gemcitabine and ceramide liposome significantly reduce people MIA pancreas cancer cell strain, cause people MIA pancreas cancer cell strain significantly dead, obviously be superior to the anti-cancer of pancreas effect of list with gemcitabine.
Embodiment 12: ceramide liposome is to the propagation inhibition test of the Proliferation of Human Ovarian Cell of In vitro culture.
The test cell: the strain of people CaOV3 ovarian cancer cell derives from US mode culture collection warehousing (ATCC).
Test drug and reagent: ceramide (Avanti, USA), 3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazole bromine salt (MTT) (sigma, USA); Trypsin sigma, USA); Dimethyl sulfoxine (DMSO) (sigma, USA); The DMEM cell culture medium (GIBCO, Invitragen, USA); The RPMI1640 cell culture fluid (GIBCO, Invitragen, USA); Hyclone (GIBCO, Invitragen, USA); Superfine hyclone Fetal Bovine Serum (Hyclone).
Instrument: ultra cold storage freezer (Nature, USA), inverted microscope (NIKON, Japan), ELIASA (Bio-Tek Instruments, USA); Cell culture incubator (SANYO, Japan), superclean bench (FLC-Harbin Dong Lian instrument plant), the PH meter (Thermo orion, USA).
Experimental technique:
Adopt mtt assay by routine: with the ovarian cancer cell strain by conventional method cultivations of going down to posterity, the cell of collection exponential phase, the adjusting concentration of cell suspension is 5*10
4About individual/ml.
Cell suspension is added in 96 well culture plates, and each hole adds 180 μ l; Place 37 ℃ of constant incubators to cultivate 24h.
Gemcitabine, ceramide liposome 1 (ceramide liposome that embodiment 4 makes) and ceramide liposome 2 (ceramide liposome that embodiment 5 makes) are made into the solution of gradient concentration (0.75 μ g/ml, 1.5 μ g/ml, 3 μ g/ml) respectively with the DMEM cell culture fluid.Experimental group adds Jixitabin solution (A), ceramide liposome 1+ gemcitabine mixed solution (B), the ceramide liposome 2+ gemcitabine mixed solution (C) of each concentration; They add by above-mentioned concentration simultaneously; Every hole adds 20 μ l; Establish 4 parallel holes for every group, and establish blank well (only adding the DMEM cell culture fluid) with zeroing.
Cultivate 48h at 37 ℃ of incubators, with the exhaustion of the liquid in 96 well culture plates, it is the MTT solution of 1mg/ml that every then hole adds 30 μ l concentration, puts 37 ℃, 5%CO with liquid-transfering gun
2Incubator continue to cultivate 4h.Abandon supernatant, every hole adds dimethyl sulfoxine (DMSO) 150 μ l, puts shaking table and shakes up 30min, under 492nm, detects every hole absorbance (A) value with ELIASA, uses SPSS software statistics experimental result then, and is specifically as shown in Figure 7.
Can find out that by Fig. 7 the cell survival rate that the CaOV3 human oophoroma cell line singly adds ectogenic Jixitabin solution different time has significant difference (cell survival detects with general MTT method).Associating or combination give the survival rate that gemcitabine and ceramide liposome have significantly reduced the CaOV3 Proliferation of Human Ovarian Cell, cause the CaOV3 human oophoroma cell line significantly dead, obviously are superior to the ovarian cancer resistance effect of list with gemcitabine.
Embodiment 13: ceramide liposome and ceramide antitumaous effect are relatively.
The test cell: the L3.6 pancreas cancer cell strain derives from US mode culture collection warehousing (ATCC).
Test drug and reagent: ceramide (Avanti, USA), 3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazole bromine salt (MTT) (sigma, USA); Trypsin sigma, USA); Dimethyl sulfoxine (DMSO) (sigma, USA); The DMEM cell culture medium (GIBCO, Invitragen, USA); The RPMI1640 cell culture fluid (GIBCO, Invitragen, USA); Hyclone (GIBCO, Invitragen, USA); Superfine hyclone Fetal Bovine Serum (Hyclone).
Instrument: ultra cold storage freezer (Nature, USA), inverted microscope (NIKON, Japan), ELIASA (Bio-Tek Instruments, USA); Cell culture incubator (SANYO, Japan), superclean bench (FLC-Harbin Dong Lian instrument plant), the PH meter (Thermo orion, USA).
Experimental technique:
Adopt mtt assay by routine: with the L3.6 pancreas cancer cell strain by conventional method cultivations of going down to posterity, the cell of collection exponential phase, the adjusting concentration of cell suspension is 5*10
4About individual/ml.
Cell suspension is added in 96 well culture plates, and each hole adds 180 μ l; Place 37 ℃ of constant incubators to cultivate 24h.
Experimental group be divided into be 2 greatly the group, first group only adds ceramide or ceramide liposome, second largest group the utilization gemcitabine the basis on add ceramide or ceramide liposome.The corresponding respectively matched group of A, B, C, D, E, add ceramide liposome 1, add ceramide liposome 2, add the C6 ceramide (1 μ g/ml), add C6 ceramide (10 μ g/ml).Be specially: the A group does not add ceramide; The B group adds the ceramide liposome 1 (ceramide liposome that embodiment 4 makes) of 1 μ g/ml; The C group adds the ceramide liposome 2 (ceramide liposome that embodiment 5 makes) of 1 μ g/ml; The D group adds the C6 ceramide of 1 μ g/ml of free state, and the E group adds the C6 ceramide of 10 μ g/ml of free state; Every hole adds 20 μ l, establishes 4 parallel holes for every group, and these five experimental grouies are established matched group (only adding the DMEM cell culture fluid) simultaneously, with zeroing.
Cultivate 48h at 37 ℃ of incubators, with the exhaustion of the liquid in 96 well culture plates, it is the MTT solution of 1mg/ml that every then hole adds 30 μ l concentration, puts 37 ℃, 5%CO with liquid-transfering gun
2Incubator continue to cultivate 4h.Abandon supernatant, every hole adds dimethyl sulfoxine (DMSO) 150 μ l, puts shaking table and shakes up 30min, under 492nm, detects every hole absorbance (A) value with ELIASA, uses SPSS software statistics experimental result then, and is specifically as shown in Figure 8.
Can find out by Fig. 8: can promote pancreatic cancer cell dead behind the ceramide of liposome and the gemcitabine drug combination more efficiently.The ceramide of liposome only needs 1 μ g/ml just can reach the effect of free ceramide 10 μ g/ml, and the free state ceramide of low concentration does not almost have influence (Fig. 8) to the cancer cell death of induced by chemotherapeutic agents on an equal basis.Prompting lipid physical ability effectively promotes ceramide to get in the cell, and low concentration (Cf 10%) liposome ceramide can significantly increase the chemosensitivity of gemcitabine to cancer of pancreas.
Claims (10)
1. ceramide liposome comprises a treatment effective amount of actives and a type lipid bilayer thin film, and it is characterized in that: the weight portion of said active component is 0.5-20 part, and active component is a ceramide; The weight portion of said type of lipid bilayer thin film is 10-350 part, comprises following components by part by weight: phospholipid substance 5-100 part, cholesterol 0.5-10 part, surfactant 0-100 part, additives 0-20 part, organic solvent 0.5-10 part.
2. ceramide liposome as claimed in claim 1 is characterized in that: described phospholipid substance is selected from one or more in two Laurel phosphatidyl cholines, DSPC, distearyl phosphatidyl glycerol, Phosphatidylserine, phosphatidylinositols, two palmityl phosphatidic acid, dioleoyl phospholipid phatidylcholine, dimyristoyl phosphatidyl choline, phosphatidyl glycerol, phosphatidic acid, phosphatidylcholine, dipalmitoyl phosphatidyl choline, two palmityl phosphatidyls, DOPE, soybean lecithin, Ovum Gallus domesticus Flavus lecithin, cephalin, synthetic phospholipid, the hydrogenated soy phosphatidyl choline; It is the natural cholesterol of raw material production that said cholesterol is selected for use with egg yolk, animal brains, lanoline; Said surfactant is selected from one or more the compositions in dexycholate, cholate, deoxycholic acid, cholic acid, tween 80, taurocholate, the poloxamer; Said organic solvent is selected from one or more the mixture in ethanol, acetone, methanol, chloroform, ether, the isopropyl alcohol; Said additives are selected from one or more among L-isoleucine, malic acid, sodium metabisulfite, sodium sulfite, vitamin C, vitamin E, propyl gallate, fumaric acid, L-cysteine or PH buffer agent phosphate, the EDTA.
3. ceramide liposome as claimed in claim 1; It is characterized in that: said ceramide liposome adds long circulation adjuvant; Carry out finishing and process long circulating liposomes, the mol ratio of long circulation adjuvant of said long circulation adjuvant and phospholipid substance is 1:10-1:200; Said long circulation adjuvant is selected from Polyethylene Glycol-phospholipid derivative, Polyethylene Glycol, polyvinylpyrrolidone, methyl and gathers azoles quinoline, Polyethylene Glycol-dipalmitoyl phosphatidyl choline, Polyethylene Glycol-distearyl acyl group PHOSPHATIDYL ETHANOLAMINE, chitosan or polyvinyl alcohol.
4. ceramide liposome as claimed in claim 1 is characterized in that: said ceramide liposome adds the magnetic decorative material, carries out finishing and processes magnetic liposome; Said magnetic decorative material is a super-fine magnetic powder.
5. ceramide liposome as claimed in claim 1 is characterized in that: said ceramide liposome adds the temperature-sensitive auxiliary material, carries out finishing and processes thermal sensitive liposome; Said temperature-sensitive auxiliary material are the phospholipid that itself has thermal sensitivity, comprise dipalmitoyl phosphatidyl choline (DPPC), DSPC (DSPC); Or the macromolecule thermally sensitive polymeric, comprise polyacrylic acid, gather phosphatidic acid; Or a kind of phase transition temperature that can make liposome is suitable for the oiliness medicine of tumor thermotherapy temperature, comprises elemene or wild duck seed oil.
6. ceramide liposome as claimed in claim 1 is characterized in that: the mean diameter of said ceramide liposome decentralized photo is less than 500nm; Said ceramide liposome exists with the form of injection or solid powder; Said ceramide liposome envelop rate is greater than 80%.
7. the method for preparing of ceramide liposome; It is characterized in that; Comprise the steps: fully to be dissolved in ceramide, phospholipid substance, cholesterol and additives in the organic solvent; In disperse medium, adopt film dispersion method or organic solvent injection method to make ceramide liposome surfactant dissolves, adopt ultrasonic, high pressure homogenize then or the method extruded is reduced to below the 500nm mean diameter of the ceramide liposome that makes and uniform distribution.
8. the method for preparing of ceramide liposome as claimed in claim 7, it is characterized in that: said disperse medium is Tris buffer, water, phosphate buffer, carbonate buffer solution, 5% D/W or 0.9%NaCl solution; The weight concentration of the surfactant in the said disperse medium is 1%-30%.
9. the method for preparing of ceramide liposome as claimed in claim 7 is characterized in that: in the ceramide liposome that makes, add the lyophilized powder protective agent, process lyophilized powder through spray drying or freeze drying process; Said lyophilized powder protective agent is selected from one or more in mannitol, sucrose, trehalose, sodium chloride, glucose, dextran, glycine, lactose, sorbitol, xylitol, fructose, glycerol, arabic gum and the lysine; The lyophilized powder protective agent: the parts by weight proportioning of ceramide liposome is 10-20:0.5-6.0.
10. each described ceramide liposome of claim 1 ~ 6 is in the purposes of preparation in the antitumor drug.
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| CN105651987A (en) * | 2016-01-29 | 2016-06-08 | 苏州联辰生物技术有限公司 | Method for manufacturing novel nano beam concentration material |
| CN106581695A (en) * | 2016-10-27 | 2017-04-26 | 山东省分析测试中心 | Cationic liposome, preparation method and applications thereof |
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| CN109453043B (en) * | 2018-12-13 | 2021-09-28 | 诺斯贝尔化妆品股份有限公司 | Nano emulsion with good stability |
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