Background technology
Gemcitabine is a kind of Difluoronucleosides class antimetabolite anticarcinogen that destroys cellular replication, it is the water-soluble analogues of cytosine deoxyriboside, it is a kind of substitute of substrate of inhibition to ribonucleotide reductase, this kind of enzyme is at DNA in synthetic and repair process, is vital to the generation of needed Deoxydization nucleotide.Have that antitumor spectra is wide, mechanism of action is unique, with other chemotherapeutics without crossing drug resistant and toxic reaction without characteristics such as stacks.
Gemcitabine is the good substrates of thymine deoxyriboside kinase phosphorylation in cell as a kind of prodrug, changes into following metabolite under the effect of enzyme: gemcitabine monophosphate (dFdCMP), gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP) wherein dFdCDP and dFdCTP are activated product.DFdCDP suppresses ribonucleotide reductase, thereby has reduced the amount (especially dCTP) of the required Deoxydization nucleotide of the synthetic reparation of DNA, and low-level dCTP has reversed the normal negative feedback inhibition of deoxidation glycosides kinases, cause dFdCTP more to gather.simultaneously dFdCDP has suppressed deoxidation born of the same parents ammonia enzyme that dCTP induces to the deamination of dFdCMP, and dFdCTP directly suppresses deoxycytidine and takes off enzyme, thereby make more dFdCMP change into the deamination of active metabolite dFdCMP, and dFdCTP directly suppresses deoxycytidine deaminase, thereby make more dFdCMP change into active metabolite dFdCDP, dFd-CTP dFdCTP enters the DNA chain with dCTP competition combination, be inserted into the site of deoxycytidine in the DNA chain, and permission guanosine and its pairing, the gemcitabine molecule just " is sheltered " by this guanosine and is made it avoid the reparation that removes of exoribonuclease, then the DNA chain is synthetic stops, and then DNA break, cell death.
Now confirm, gemcitabine is remarkable to multiple treatment of solid tumors effect, especially to nonsmall-cell lung cancer, cancer of pancreas and breast carcinoma, and single drug or unite other cancer therapy drug and all obtain very high curative effect.Gemcitabine is shorter (between 32~94min) in the intracellular half-life, must heavy dose of (recommended dose be 1000mg/m2), the during continuous intravenous infusion administration keeps its effective medicinal concentration and to the toxicity of cancerous cell, but this toxicity normal tissue simultaneously also works, and this dose-limiting toxicity affects clinical efficacy.In addition, the inorganizable specificity of gemcitabine, the whole body toxic and side effects is large; Internal metabolism is rapid, and plasma half-life is short; The shortcomings such as drug resistance of tumor.
Therefore, seek a kind of target-oriented drug that can make stronger, the medication that toxic and side effects is less is emphasis and the difficult point of current research.The medicine-carried nano particles that contains active drug is a kind of novel slow-released system, can change conventional administering mode, and extremely wide prospect is arranged, it is the study hotspot in medicine and pharmacology field in recent years, the nanometer medicine in entering body after, medicine slowly discharges in the target area, to alleviate the untoward reaction of medicine.
Huang Lesong, Chunxia WANG, Chen Zhiliang etc. " targeting of gemcitabine poly-alkyl-alfa-cyanoacrylate nanoparticles in mouse brain distributes " (Journal of Chinese Hospital Pharmacy, 2008,28 (16): 1332), and Huang Lesong, Chunxia WANG, Chen Zhiliang etc. " the gemcitabine poly-alkyl-alfa-cyanoacrylate nanoparticles is on the impact of mouse brain Glioma Model research " (China Dispensary, 2009,20 (19): introduced a kind of nanoparticle of gemcitabine 1457) and on the impact of animal.Huang Junqin, Kong Liwen " the mensuration character of the preparation of gemcitabine hydrochloride liposome and content and envelop rate " (Chinese antibiotic magazine, 2010,35 (1): preparation and the content assaying method of 30) having introduced a kind of gemcitabine liposome.CN101444485B discloses a kind of gemcitabine liposome, and this liposome comprises gemcitabine, phospholipid, cholesterol, but the envelop rate of this liposome and dispersibility all perform poor, and stability and the bioavailability of medicine all had adverse effect.CN101926779A discloses a kind of gemcitabine solid lipid nanospheres and preparation method thereof, and said preparation still exists entrapment efficiency not high, and the not good problem of stability, stores about 3 months medicines Partial digestion namely occurs.Although the research of the liposome of some drugs or nanometer medicine has obtained greater advance and even obtained application in clinical.But due to the difference of physical and chemical properties of drugs and pharmacological action, the technology of gemcitabine field application liposome or nanometer medicine does not obtain to break through yet.
From the achievement of present research, the problems such as the gemcitabine liposome of existing method preparation or the Nano microsphere existence and stability is poor, easily assemble in aqueous solution, whole corrosion or superficial degradation can't obtain practical application in clinical.
Summary of the invention
The object of the invention is to solve the problems of the technologies described above, a kind of envelop rate and stability, minimizing gemcitabine or the side effect of its salt in clinical practice that can strengthen target-oriented drug, raising medicine is provided, and is suitable for gemcitabine or its salt nano-emulsion injection of wide clinical application.
The object of the present invention is to provide the nano-emulsion injection of a kind of gemcitabine or its salt, described injection contains gemcitabine or its salt, oil for injection, surfactant and water for injection, wherein the weight ratio of gemcitabine or its salt, oil for injection, surfactant is 1: 10~100: 10~100,, in Emulsion, the mean diameter of emulsion droplet is 1~100nm.
Described nano-emulsion injection is oil-in-water type, and wherein the weight ratio of gemcitabine or its salt, oil for injection, surfactant is 1: 20~50: 10~20.
The salt of described gemcitabine is hydrochlorate.
Described oil for injection is selected from one or more in mineral oil, vegetable oil, animal oil or artificial oil; Described vegetable oil is selected from one or more in soybean oil, Oleum Arachidis hypogaeae semen, Semen Maydis oil, Oleum sesami, safflower oil, Oleum Ricini, Petiolus Trachycarpi oil, Oleum Gossypii semen, Oleum Cocois, medium-chain fatty acid triglyceride, one or more in preferred soybean oil, Oleum Arachidis hypogaeae semen, Oleum sesami or medium-chain fatty acid triglyceride; Described animal oil is selected from fish oil, sperm oil or its mixture.
Described surfactant is selected from phospholipid, nonionic surfactant or its mixture.
Described phospholipid is selected from lecithin, fabaceous lecithin or its mixture, preferably lecithin or fabaceous lecithin; Described non-ionic surface active agent is selected from polyoxyethylene and polyethylene glycols non-ionic surface active agent, described polyoxyethylene non-ionic surface active agent is selected from polysorbas20, polysorbate40, polysorbate60, Tween 80, polysorbate85, polyoxyethylene castor oil, polyoxyethylene hydrogenated Oleum Ricini, PLURONICS F87 or its mixture, preferred Tween 80 or PLURONICS F87, described polyethylene glycols non-ionic surface active agent are selected from the poly-hexadecane cyanoacrylate of Pegylation, Polyethylene Glycol stearic acid, TPGS or its mixture.
In described Emulsion, the mean diameter of emulsion droplet is 1~70nm, preferred 1~50nm, more preferably 1~20nm.
The injection of described gemcitabine or its salt also comprises one or more in antioxidant or stabilizing agent.
Described antioxidant is selected from water solublity antioxidant or oil-soluble antioxidant, and wherein water solublity antioxidant is selected from sodium sulfite, sodium sulfite, sodium pyrosulfite, sodium ascorbate, Cys or its mixture; Oil-soluble antioxidant is selected from BHA (butylated hydroxyarisol), BHT (2,6-di-t-butyl hydroxy-methylbenzene), vitamin E, ascorbyl palmitate or its mixture, preferred ascorbyl palmitate, wherein said antioxidant accounts for 0%~1w/v% of injection.
Described stabilizing agent is selected from oleic acid, enuatrol, cholic acid, sodium cholate, dextran-70, deoxycholic acid, sodium deoxycholate or its mixture, and wherein said stabilizing agent accounts for 0%~1.5w/v% of injection.
The present invention also aims to provide a kind of method for preparing above-mentioned gemcitabine or its salt nano-emulsion injection, comprise the steps:
Under inert gas shielding, oil for injection is stirred into oil phase;
Surfactant is added appropriate water for injection, be stirred into water;
Under high-speed stirred, oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize;
Add gemcitabine or its salt to first Ruzhong;
Regulating pH value is 4~9, continues to add water and is settled to full dose, fully stirs or makes the emulsion droplet mean diameter meet the requirements of nanorize through the high pressure homogenizer homogenize.
The present invention also provides the method for another above-mentioned gemcitabine of preparation or its salt nano-emulsion injection, comprises the steps:
Under inert gas shielding, oil for injection, oil-soluble antioxidant are stirred into oil phase;
Water solublity antioxidant stabilizers and surfactant are added in appropriate water for injection, be stirred into water;
Under high-speed stirred, oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize;
Add gemcitabine or its salt to first Ruzhong;
Adjusting pH value 4~9. continue to add water to be settled to full dose fully stirs or meets the requirements of nanorize through high pressure homogenizer homogenize to emulsion droplet mean diameter.
According to studies show that of inventor, pH value is especially good at 4~7 o'clock.
Above-mentioned preparation has been opened up the new way of gemcitabine or its salt administration, screening by each constituent content proportioning in compositions, and the processing of emulsion droplet grain size nanometer grade improved envelop rate and the stability of medicine greatly, reduced medicine toxic and side effects in use, alleviated patient's medication painful.
The specific embodiment
In order more specifically to describe this invention, further illustrate this invention below in conjunction with specific embodiment, but content of the present invention is not limited to specific embodiment.
Embodiment one
Preparation method: under inert gas shielding, injection soybean oil 200g is stirred evenly into oil phase, separately soybean phospholipid 200g is added in 500ml water for injection, stir evenly into water, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulating pH value is 5.0, adds water and is settled to 1000ml, change in high pressure homogenizer fully and stir, to mean diameter below 40nm; Fill, the inflated with nitrogen encapsulation.
Embodiment two
Preparation method: under inert gas shielding, injection Oleum Arachidis hypogaeae semen 200g is stirred evenly into oil phase, separately soybean phospholipid 200g is added in 500ml water for injection, stir evenly into water, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulating pH value is 5.0, adds water and is settled to 1000ml, change in high pressure homogenizer fully and stir, to mean diameter below 40nm; Fill, the inflated with nitrogen encapsulation.
Embodiment three
Preparation method: under inert gas shielding, injection Oleum sesami 200g is stirred evenly into oil phase, separately soybean phospholipid 200g is added in 500ml water for injection, stir evenly into water, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulating pH value is 5.0, adds water and is settled to 1000ml, change in high pressure homogenizer fully and stir, to mean diameter below 40nm; Fill, the inflated with nitrogen encapsulation.
Embodiment four
Preparation method: under inert gas shielding, injection soybean oil 200g is stirred evenly into oil phase, separately soybean phospholipid 200g is added in 500ml water for injection, stir evenly into water, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulating pH value is 5.0, adds water and is settled to 1000ml, change in high pressure homogenizer fully and stir, to mean diameter below 20nm; Fill, the inflated with nitrogen encapsulation.
Embodiment five
Preparation method: under inert gas shielding, injection soybean oil 225g is stirred into oil phase, be preheated to 60 ℃, soybean phospholipid 125g, sodium sulfite 4g are joined in 600ml water for injection, be stirred into water, be preheated to 60 ℃, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulate pH value to 6.0, add water and be settled to 1000ml, with its transfer to continue in high pressure homogenizer emulsifying to mean diameter in the 20nm left and right; Fill, the inflated with nitrogen sealing by fusing.
Embodiment six
Preparation method: under inert gas shielding, injection soybean oil 225g and oleic acid 6g are mixed into oil phase, be preheated to 60 ℃, soybean phospholipid 125g, sodium sulfite 4g are joined in 600ml water for injection, be stirred into water, be preheated to 60 ℃, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulate pH value to 6.0, add water and be settled to 1000ml, with its transfer to continue in high pressure homogenizer emulsifying to mean diameter in the 15nm left and right; Fill, the inflated with nitrogen sealing by fusing.
Embodiment seven
Preparation method: under inert gas shielding, injection soybean oil 300g and oleic acid 5g are mixed into oil phase, be preheated to 60 ℃, soybean phospholipid 200g, sodium sulfite 5g are joined in 650ml water for injection, be stirred into water, be preheated to 60 ℃, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulate pH value to 5.0, add water and be settled to 1000ml, with its transfer to continue in high pressure homogenizer emulsifying to mean diameter in the 15nm left and right; Fill, the inflated with nitrogen sealing by fusing.
Embodiment eight
Preparation method: under inert gas shielding, injection soybean oil 450g and oleic acid 12g are mixed into oil phase, be preheated to 60 ℃, soybean phospholipid 150g, sodium sulfite 8g are joined in 650ml water for injection, be stirred into water, be preheated to 60 ℃, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulate pH value to 5.0, add water and be settled to 1000ml, with its transfer to continue in high pressure homogenizer emulsifying to mean diameter in the 15nm left and right; Fill, the inflated with nitrogen sealing by fusing.
Embodiment nine
Preparation method: under inert gas shielding, injection soybean oil 300g and oleic acid 10g are mixed into oil phase, be preheated to 60 ℃, Tween 80 135g, sodium ascorbate 5g are joined in 600ml water for injection, be stirred into water, be preheated to 60 ℃, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulate pH value to 5.0, add water and be settled to 1000ml, with its transfer to continue in high pressure homogenizer emulsifying to mean diameter in the 20nm left and right; Fill, the inflated with nitrogen sealing by fusing.
Embodiment ten
Preparation method: under inert gas shielding, injection soybean oil 300g and oleic acid 10g are mixed into oil phase, be preheated to 60 ℃, Tween 80 135g, sodium ascorbate 5g are joined in 600ml water for injection, be stirred into water, be preheated to 60 ℃, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulate pH value to 5.0, add water and be settled to 1000ml, with its transfer to continue in high pressure homogenizer emulsifying to mean diameter in the 50nm left and right; Fill, the inflated with nitrogen sealing by fusing.
Embodiment 11
Preparation method: under inert gas shielding, injection soybean oil 300g is mixed into oil phase, be preheated to 60 ℃, Tween 80 135g, sodium ascorbate 5g and enuatrol 10g are joined in 600ml water for injection, be stirred into water, be preheated to 60 ℃, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulate pH value to 5.7, add water and be settled to 1000ml, with its transfer to continue in high pressure homogenizer emulsifying to mean diameter in the 50nm left and right; Fill, the inflated with nitrogen sealing by fusing.
Embodiment 12
Preparation method: under inert gas shielding, medium-chain fatty acid triglyceride 250g is mixed into oil phase, be preheated to 70 ℃, Tween 80 150g, sodium ascorbate 7g and enuatrol 13g are joined in 650ml water for injection, be stirred into water, be preheated to 70 ℃, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulate pH value to 6.0, add water and be settled to 1000ml, with its transfer to continue in high pressure homogenizer emulsifying to mean diameter in the 20nm left and right; Fill, the inflated with nitrogen sealing by fusing.
Embodiment 13
Preparation method: under inert gas shielding, injection soybean oil 300g and ascorbyl palmitate 5g are mixed into oil phase, be preheated to 60 ℃, Tween 80 135g and enuatrol 10g are joined in 600ml water for injection, be stirred into water, be preheated to 60 ℃, under high-speed stirred, that oil phase is mixed with water evenly, and make colostrum through the high pressure homogenizer homogenize, add gemcitabine hydrochloride 10g, regulate pH value to 5.7, add water and be settled to 1000ml, with its transfer to continue in high pressure homogenizer emulsifying to mean diameter in the 20nm left and right; Fill, the inflated with nitrogen sealing by fusing.
Experimental example one entrapment efficiency determination
Medicine assay method: chromatographic column: Diamonsil
TMC
18(4.6mm*250mm, 5 μ m); Mobile phase: with 0.05mol/L ammonium acetate buffer (get ammonium acetate 3.85g, add water 800ml dissolving, transfer pH to 5.7 with glacial acetic acid, add water to 1000ml)-methanol (90: 10); Detect wavelength: 268nm; Flow velocity: 1.0ml/min; 40 ℃ of column temperatures, sample size: 20 μ L.
The entrapment efficiency determination method: 1 of sample thief, the accurate 2.0ml that draws is placed in the 10ml measuring bottle, is diluted with water to scale, with 8010 type ultrafilters (MILLIPORE company) ultrafiltration, discards just filtrate, and getting subsequent filtrate is need testing solution.Draw respectively need testing solution, reference substance solution 20 μ L injection liquid chromatographies, record chromatogram, calculate preparation aqueous phase medicament contg with external standard method, be designated as W; The another medicine total content of pressing method calculating book product under the assay item is designated as W
0Be calculated as follows the envelop rate of sample.
Envelop rate En=(W
0-W)/W
0* 100%
Measuring as stated above averages after the envelop rate of three batch samples is E, and measurement result is as shown in table 1:
Table 1 gemcitabine hydrochloride injection is sealed rate and is measured
The embodiment title |
E(%) |
Embodiment one |
95.8 |
Embodiment two |
93.5 |
Embodiment three |
94.6 |
Embodiment four |
96.9 |
Embodiment five |
97.7 |
Embodiment six |
99.3 |
Embodiment seven |
99.1 |
Embodiment eight |
98.9 |
Embodiment nine |
99.5 |
Embodiment ten |
98.7 |
Embodiment 11 |
99.1 |
Embodiment 12 |
99.6 |
Embodiment 13 |
99.8 |
Conclusion: the injection entrapment efficiency effect that can find out this invention from the above results is fine, meets the national drug prescription, has good clinical value.
Experimental example two stability tests
Measure as follows the stability data of injection of the present invention.
Assay method: the sample of getting respectively each embodiment of 1ml is placed in special centrifuge tube, puts into table model high speed centrifuge centrifugal with the 2000rpm rotating speed, after centrifugal 15 minutes, takes out centrifuge tube.By the bottom, sample is splashed into small beaker appropriate, draw 50.0 μ L with microscale sampler and be added in the 25ml measuring bottle, be diluted to scale with water for injection, mixing.Under the 500nm wavelength, detect its trap value (A) with the water level blank.Get 50.0 μ L raw samples again and be placed in the 25ml measuring bottle, use the water for injection standardize solution, its absorption value (A is detected at the place at Same Wavelength
0).Press mathematical formulae K
E=(| A
0-A|/A
0) * 100%, calculate the stability parameter K of this Emulsion
EK
ELess Emulsion is more stable.Experimental result is as shown in table 2:
Table 2 gemcitabine hydrochloride injection Stability Determination
The embodiment title |
K
EValue
|
Sample appearance |
Embodiment one |
0.451 |
Have no oil droplet |
Embodiment two |
0.469 |
Have no oil droplet |
Embodiment three |
0.416 |
Have no oil droplet |
Embodiment four |
0.446 |
Have no oil droplet |
Embodiment five |
0.421 |
Have no oil droplet |
Embodiment six |
0.403 |
Have no oil droplet |
Embodiment seven |
0.412 |
Have no oil droplet |
Embodiment eight |
0.432 |
Have no oil droplet |
Embodiment nine |
0.451 |
Have no oil droplet |
Embodiment ten |
0.415 |
Have no oil droplet |
Embodiment 11 |
0.406 |
Have no oil droplet |
Embodiment 12 |
0.408 |
Have no oil droplet |
Embodiment 13 |
0.408 |
Have no oil droplet |
In addition, by conventional method working sample long-time stability, after sample stores 6 months at ambient temperature, measure Emulsion percentage of head rice in all embodiment, result shows that the Emulsion percentage of head rice of each embodiment sample all reaches more than 92%.
Conclusion: after measured, sample stability is better, is suitable for clinical practice, but the longer-term storage.